msc. baoyu zhang - ghent university · the author and the promoter give the authorization to...

MSc. Baoyu Zhang

Promoters: Prof. dr. ir. Frank Devlieghere

Dr. Simbarashe Samapundo

Laboratory of Food Microbiology and Food Preservation

Department of Food Safety and Food Quality

Faculty of Bioscience Engineering

Ghent University, Belgium

Dean: Prof. dr. ir. Guido Van Huylenbroeck

Rector: Prof. dr. Anne De Paepe

Examination committee:

Chairman: Prof. dr. ir. Marc Van Meirvenne

Secretary: Prof. dr. ir. Herman Van Langenhove

Prof. dr. ir. Frank Devlieghere (UGent)

Dr. Simbarashe Samapundo (UGent)

Prof. dr. ir. Peter Ragaert (UGent)

Prof. dr. ir. Monica Höfte (UGent)

Prof. dr. Bart Nicolai (K.U. Leuven)

MSc. Baoyu Zhang

EVALUATION OF MODIFIED ATMOSPHERES AS A

PRESERVATION TOOL FOR FRESH-CUT FRUIT

Thesis submitted in fulfilment of the requirements for the degree of

Doctor (PhD) in Applied Biological Sciences

Thesis title in Dutch:

Evaluatie van gemodificeerde atmosferen als beschermingsmiddel voor vers

gesneden fruit

Illustration on cover:

Spoiled pineapple cubes

Printer: University Press

To refer to this thesis:

Zhang, B.-Y. (2013). Evaluation of modified atmospheres as a preservation tool for

fresh-cut fruit. PhD dissertation, Faculty of Bioscience Engineering, Ghent

University, Ghent.

ISBN: 978-90-5989-667-3

The author and the promoter give the authorization to consult and copy parts of this work for

personal use only. Every other use is subject to copyright laws. Permission to reproduce any

material contained in this work should be obtained from the author.

ACKNOWLEDGEMENTS

I would like to express my deep gratitude to my promoters Prof. dr. ir. Frank Devlieghere and Dr.

Simbarashe Samapundo, for accepting my request to pursue my doctoral studies in the laboratory

and the enthusiast teaching, guidance and support they have provided throughout this time. I

would like to sincerely thank the China Scholarship Council, Flanders’FOOD, Ghent University

and Chiquita for their financial, instrumental and experimental material support, which enabled

this study to be done. I also extend my sincere gratitude to the members of the Examination

Committee (Prof. dr. ir. Marc Van Meirvenne, Prof. dr. ir. Herman Van Langenhove, Prof. dr. ir.

Peter Ragaert, Prof. dr. ir. Monica Höfte and Prof. dr. Bart Nicolai) for their time, evaluation and

constructive comments that helped to improve this thesis.

I would like to thank Dr. Bert Noseda and Evy for giving me a lot of help in the laboratory

academically and socially. Thanks Vasileios for helping me identify lactic acid bacteria and your

advices for my experiment. To all the colleges from my office (Nanou, Aga, Anh Ngoc, Ramize,

Ilse Van Bree and Irena), thank you for your support. Everyone amongst you had a role to play

during my study. I would also like to sincerely thank Danny who picked me up at the Gent

Station when I arrived in Belgium at the first time and also gave me great support throughout my

study in the lab. To Quenten, Ilse de Baenst, Markus, Marzia, Dr. Winnok De Vos and Lore,

thank you for your help in the lab and technical support of the packaging machines, HPLC, GC-

MS, SIFT-MS and confocal microscope. Trang, I appreciate your help when I started my PhD

study. Thanks Michael for helping statistical analysis. I would like to thank my student Göknur

who also contributed to this thesis.

To all the members of the Laboratory of Food Microbiology and Food Preservation, thank you

for all the support. Thank you for joining the sensory panel of fresh-cut fruit. I wish you and your

families happy, success and good health. Special thanks to Prof. Mieke Uyttendaele, Prof.

Benedikt Sas, Prof. Liesbeth Jacxsens, An Vermeulen, Ann Dirckx, Ariane, Dieter, Paco, Ihab,

Elien Verguldt, Steffi, Leentje, Anja, Inge Van Wassenhove, An De Coen, Katty, Andreja,

Ambroos, Siele, Melanie, Stefanie, Ann De Keuckelaere, Varvara and Jessica.

I would like to thank all my Chinese friends in Ghent (especially, Dan, Xiang, Wanzhao,

Donghong, Xiaoyun, Wenwen, Shengjing, Xu Jing, Jisheng, Yun, Liming, Lizi, Lili, Yuanyuan,

Shangshang, Renfei, Liuyi, Nana, Zhiming, Jiang, Baoyi, Ningning, Qiaoyan, Rongduo and

Jianyun). Thank you for your great help, kindness and friendly talk.

I wish to show my great appreciation to my parents, parents-in-law, brother, sister and nephews,

especially my beloved wife Haiqing and dearest son Hanbo for your love, encouragement and

support.

To all those that I have not mentioned, your kindness and assistance is appreciated. Wish you and

your families all the best.

Thank you -谢谢！

Baoyu - 保玉

5 December 2013, Ghent

TABLE OF CONTENTS

ABBREVIATIONS .......................................................................................................................... i

INTRODUCTION AND OBJECTIVES ....................................................................................... iii

Chapter 1: Literature Review: Spoilage of Fresh-cut Fruit during Refrigerated Storage ............... 1

Chapter 2: Spoilage Organisms of Fresh-cut Pineapple, Honeydew Melon and Apple ............... 47

Chapter 3: Effect of Reduced Headspace Oxygen Level on Growth and Volatile Metabolite

Production by the Spoilage Microorganisms of Fresh-cut Pineapple and Honeydew Melon ...... 57

Chapter 4: Effect of Atmospheres Combining High Oxygen and Carbon Dioxide Levels on the

Microbial Spoilage and Sensory Quality of Fresh-cut Pineapple and Honeydew Melon ........... 101

Chapter 5: Effect of Modified Atmospheres (Incl. High Oxygen and Carbon Dioxide) on the

Shelf-life of Fresh-cut Apple ....................................................................................................... 141

Chapter 6: General Discussion, Conclusions and Perspectives .................................................. 159

REFERENCE LIST ..................................................................................................................... 169

SUMMARY ................................................................................................................................ 204

SAMENVATTING ..................................................................................................................... 207

CURRICULUM VITAE ............................................................................................................. 211

i

ABBREVIATIONS

AFLP Amplified fragment length polymorphism

ANOVA Analysis of variance

a* Green chromaticity

aw Water activity

b Branching ratio

b* Yellow chromaticity

CO2 Carbon dioxide

CFU Colony forming units

Fe Iron

EVOH Ethylene vinyl alcohol

GC-MS Gas chromatography - mass spectrometry

GMP Good manufacturing practice

HPLC High performance liquid chromatography

IH Initial headspace

k Reaction rate coefficients

L* Lightness

LAB Lactic acid bacteria

MA Modified atmosphere

MAP Modified atmosphere packaging

ABBREVIATIONS

ii

N2 Nitrogen

NaCl Sodium chloride

NADH Reduced nicotinamide adenine dinucleotide

MRS de Man Rogosa Sharpe

O2 Oxygen

OPA Oriented polyamide

OT Human olfactory threshold

OTR Oxygen transmission rate

PCA Plate count agar

PE Polyethylene

PP Polypropylene

PPS Physiological peptone saline solution

RCA Reinforced clostridial agar

ROS Reactive oxygen species

RH Relative humidity

SB Sabouraud broth

SIFT-MS Selective ion flow tube - mass spectrometry

TAC Total aerobic counts

TAnC Total anaerobic counts

VOC Volatile organic compound

YGC Yeast glucose chloramphenicol

INTRODUCTION AND OBJECTIVES

iii


Fresh-cut fruit sales have considerably increased in the United States and European Union over

the last two decades (Gorny, 2005); Rabobank (2010). Although the marketing of fresh-cut fruit

is still in an early stage of development in Asian countries, these products are increasingly being

sold in supermarkets (James and Ngarmsak, 2010). The increase in consumption of fresh-cut fruit

is mostly because people are more aware of the importance and benefits of healthy eating habits

and have less time for food preparation. Fresh-cut produce fit the many needs of a modern

lifestyle as they serve colourful, flavourful and nutritional (energy, vitamins, minerals, and

dietary fibre) compounds and are also convenient to use and consume (Kader and Barrett, 2005;

Olivas and Barbosa-Canovas, 2005).

However, fresh-cut fruit are highly perishable products and have a very short shelf-life, even at

chill temperatures. The fast deterioration of fresh-cut fruit results mostly from the damage caused

to the cells and tissues by cutting and trimming and the removal of their natural protective

barriers (Kays, 1991; Olivas et al., 2005). Cutting and/or peeling may induce ripening, cause

senescence and aid the rapid growth of contaminating microorganisms due to the release of

nutrients from the damaged cells and tissues. Therefore, the spoilage phenomena on fresh-cut

fruit are related to biochemical processes, both in microorganisms and in the fruit tissue itself.

Additionally, consumers demand natural (additive-free) fresh-cut fruit. Hence, it is still a

challenge to maintain a high quality of fresh-cut fruit throughout storage.

Modified atmosphere packaging (MAP) is suitable for the preservation of fresh-cut fruit as it fits

the consumers’ requirements for more natural food. In literature, some research already focused

on the modified atmospheres (MAs) with reduced oxygen (O2) levels combined with slightly

elevated carbon dioxide (CO2) levels on the quality of fresh-cut fruit. These type of MAs may

benefit the quality of fresh-cut fruit by reducing the fruits respiration rate, delaying senescence,

and inhibiting the growth of spoilage organisms (Al-Ati and Hotchkiss, 2003). However, the

volatile organic metabolites produced by the spoilage organisms of fresh-cut fruit during storage,

which may greatly influence the odour and flavour of cut fruit, have not yet been evaluated.

Additionally, MAs combining high O2 and CO2 levels can be evaluated to extend the shelf-life of


iv

fresh-cut fruit. To date, only a handful of studies have been performed in which the positive

effect of atmospheres with high O2 and CO2 levels on the shelf-life of fresh-cut carrots and

peppers has been reported (Amanatidou et al., 2000; Conesa et al., 2007). No studies have yet

been published regarding the effect of such types of atmospheres on the quality of fresh-cut fruit.

The general aim of this thesis is to extend the shelf-stability of fresh-cut pineapple, honeydew

melon and apple by means of MAs. The effect of MAs on the microbial spoilage and

physiological decay of fresh-cut fruit was evaluated. Therefore, different analysis methods were

used in this thesis, resulting in multidisciplinary approach (microbiology, volatile compounds,

sensory analysis, sugar analysis, etc.). The effect of various MAs on the growth and volatile

metabolite production of spoilage isolates of fresh-cut fruit was first evaluated on simulation agar.

On the basis of the results of the experiments performed on simulation agar, selected MAs were

applied on fresh-cut honeydew melon, apple and pineapple with the intention of determining

their effect on the growth of the native microorganisms, physical parameters, volatile organic

compounds (VOCs), sensory quality and ultimately the shelf-life of these fresh-cut fruit.

Chapter 1 is an overview of the available literature on the trends in the marketing, causes of loss

of quality (including microbial growth, the changes of cut surface colour, off-odour and off-

flavour development, texture softening and nutrition losses), methods for maintaining and

evaluating the quality and shelf-life of fresh-cut fruit.

In Chapter 2, the ecology of spoiled fresh-cut pineapple, honeydew melon and apple was

examined. Additionally, spoilage microorganisms for fresh-cut fruit were isolated and identified

by API system. The most frequently occurring species based on the results determined by API

were further identified by gene sequence analysis.

The effect of initial headspace (IH) O2 level (0 - 21%, balance N2) on the shelf-life of fresh-cut

pineapple and honeydew melon was evaluated in Chapter 3. In the first part of this chapter, the

effect of IH O2 levels on growth and volatile metabolite production of different spoilage

microorganisms on pineapple agar and honeydew melon agar was evaluated at 7°C. In the second

part of this chapter, the effect of selected IH O2 level(s) on the microbiological, physical and

sensory quality of commercial pineapple cubes and honeydew melon cubes was performed

separately at 7°C.


v

In Chapter 4, the experimental methodology developed in Chapter 3 was used for evaluating the

effect of novel MAs combining high O2 (21 - 70%) and CO2 (21 - 50%) levels on the microbial

spoilage and sensory quality of fresh-cut pineapple and honeydew melon.

On the basis of the previous two chapters, the microbial profile, sensory quality and VOCs of

fresh-cut apple packaged in air and selected MAs were evaluated in Chapter 5.

In Chapter 6 the results obtained are discussed, leading to the general conclusions of this thesis

and the perspective for future studies.

An outline of the PhD study is given in Figure 0.1.

Figure 0.1: Outline of thesis.

Chapter 1

Literature review: Spoilage of fresh-cut fruit during refrigerated storage

Chapter 6

General discussion, conclusion and perspectives

Chapter 2

Spoilage microorganisms of fresh-cut pineapple, honeydew melon and apple

Chapter 4

Effect of modified atmospheres

combining high oxygen and carbon

dioxide levels on the microbial

spoilage and sensorial quality of

fresh-cut pineapple and honeydew

melon

Chapter 3

Effect of reduced headspace oxygen

levels on growth and volatile

metabolite production by spoilage

microorganisms of fresh-cut

pineapple and honeydew melon

Chapter 5

Effect of modified atmospheres

(including high oxygen and

carbon dioxide) on the shelf-life

of fresh-cut apple


vi

CHAPTER 1

LITERATURE REVIEW: SPOILAGE OF FRESH-CUT FRUIT

DURING REFRIGERATED STORAGE

Chapter 1: Literature Review: Spoilage of Fresh-cut Fruit during Refrigerated Storage

CHAPTER 1

3

Chapter 1

Literature Review: Spoilage of Fresh-cut Fruit during Refrigerated

Storage

1.1 Trends in the marketing of fresh-cut produce

Nowadays, consumers are more aware of the importance of healthy eating habits and have less

time for food preparation. Fresh-cut produce fit the many needs of a modern lifestyle as they

serve colourful, flavourful and nutritional (energy, vitamins, minerals, and dietary fibre)

compounds and are also convenient to use and consume (Kader et al., 2005; Olivas et al., 2005).

The United Fresh Produce Association defines fresh-cut fruit or vegetables (lightly or minimally

processed or ready to eat fruit or vegetable) as products made from fruit or vegetable which have

been trimmed and/or peeled and/or cut into 100% usable product that is bagged or pre-packaged

to offer consumers high nutrition, convenience, and flavour while still maintaining its freshness

(Lamikanra, 2002b). According to the US FDA “fresh” and “minimally- processed” fruit and

vegetables are fresh-cut (pre-cut) products that have been freshly-cut, washed, packaged and

maintained by means of refrigeration. Fresh-cut products are in a raw state and even though

processed (physically altered from the original form), they remain in a fresh state, ready to eat or

cook, without freezing, thermal processing, or treatments with additives or preservatives

(Beaulieu and Gorny, 2001; FDA, 2013). However, treatments of fresh-cut produce such as a

mild chlorine or acid wash and treatment of raw foods with ionizing radiation which do not

exceed 1 kiloGray are allowed (FDA, 2013).

In the last two decades, fresh-cut fruit and vegetable sales have considerably increased, with

double digit growth rates in the United States and European Union (Gorny, 2005; Rabobank,

2010). Figure 1.1 shows that fresh-cut produce sales in the US increased in value from US $ 3.3

billion in 1994 to 28 billion in 2012 (Cook, 2008, 2011; Cook, 2012). van Rijswick (2010)

reported that the total EU fresh-cut produce market is estimated to grow around 4 per cent per

year on average from 2011, both in value and volume terms (see Figure 1.2).

LITERATURE REVIEW

4

In Asian countries, fresh-cut produce is still in an early stage of development (James et al., 2010).

Fresh-cut produce, particularly fresh-cut fruit, is sold in open-air markets and food stands in

many Asian countries and without chill chain so their shelf-life is frequently not extended beyond

the day of display (James et al., 2010). However, Asian fresh-cut produce is increasing being sold

in supermarket (James et al., 2010).

Figure1.1: Estimated fresh-cut produce sales in the US, all marketing channels. Source: Cook, 2008, 2011

and 2012.

Figure 1.2: EU fresh-cut fruit and vegetables market compared to other fruit and vegetables segments,

2005-2014f volume growth (f=forecast). Source: van Rijswick, 2010.

3.36.0

8.911.8

15.5

23.4

28.0

0

5

10

15

20

25

30

1994 1999 2003 2005 2007 2011 2012

$ billion

YOY growth

CHAPTER 1

5

Fresh-cut fruit are the newest and fastest growing segment in the fresh-cut produce industry

(Gorny, 2005). Buzby et al. (2009) reported that the estimated average food loss for fruit at the

supermarket level decreased 2.3 percentage points from 10.7% in 2005 to 8.4% in 2006. One

possible theory for this reduced loss could be the increased popularity of fresh-cut fruit (Buzby et

al., 2009). As can be seen in Figures 1.3 and 1.4, the volume of fresh-cut fruit is around 10% in

both EU and US market in fresh-cut segment (Cook, 2012; van Rijswick, 2010). Although the

fresh-cut fruit market has been growing considerably (van Rijswick, 2010), processors of fresh-

cut fruit products have faced numerous challenges not commonly encountered during fresh-cut

vegetable processing and during storage of intact fruit (Beaulieu et al., 2001; Olivas et al., 2005).

The difficulties encountered with fresh-cut fruit, while not insurmountable, require a new and

higher level of technical and operational sophistication.

Figure 1.3: Estimated market volumes of fresh-cut produce in the EU in 2010. Source: van Rijswick, 2010.

Figure 1.4: Estimated market volumes of value-added produce in select supermarkets in the US in 2012.

Source: Cook, 2012.

Fresh-cut salads

50% Other fresh-cut

vegetables (stir-

fry, crudites,

etc.) 40%

Fresh-cut fruits

10%

Bagged salads

60.6%

Value-added

vegetables

27.9%

Fresh-cut fruits

10.6%

Other value-

added fruits

0.9%

LITERATURE REVIEW

6

1.2 Quality losses of fresh-cut fruit

Fresh-cut fruit must have an attractive appearance, acceptable flavour, appropriate texture, and a

positive nutritional image to attract initial and continued purchases by consumers (Barrett et al.,

2010). However, fresh-cut fruit have a very limited shelf-life even at chill temperatures

(Ahvenainen, 1996; Olivas et al., 2005; Oms-Oliu et al., 2009; Rico et al., 2007; Rojas-Grau et al.,

2009; Watada et al., 1996; Watada and Qi, 1999), mainly due to water loss, translucency,

browning, softening, surface dehydration, off-flavour and off-odour development, and microbial

spoilage (Rojas-Grau et al., 2009). Because fresh-cut products are spoiled easily, more than US

$1 billion may be lost each year after harvest (Delheimer, 2012). The fast deterioration of fresh-

cut fruit results from the damage caused to cells and tissues by cutting and trimming and the

removal of their natural protective skin peel (Kays, 1991; Olivas et al., 2005). When fruit are cut,

peeled or in any other way wounded, their tissue responds with a steep rise in respiration rate,

causing accelerated consumption of sugars, lipids, and organic acids, and increased ethylene

production, which induces ripening and causes senescence (Kays, 1991; Olivas et al., 2005). On

the other hand, microbial spoilage can occur due to the removal of their natural protective skin

and the release of nutrient components from cells. Hence, the quality losses of fresh-cut fruit are

both of a physiological and microbiological nature. In this section, the phenomenon and

mechanisms of quality loss aspects of fresh-cut fruit such as microbial contamination, browning,

softening, off-odour and off-favour developments and nutritional loss, are reviewed. The control

of these quality losses is discussed in § 1.3.

1.2.1 Microbial contamination

Microbial contamination can occur at every step in the production chain of fresh-cut fruit from

the farm until packaging process at the processors (see Figure 1.5). Notably, no treatments during

the production of fresh-cut fruit could ensure the total elimination of microorganisms that might

be present on the surface of fresh-cut fruit. It is well known that fresh-cut fruit create a favourable

environment for proliferation of spoilage organisms which can be a major cause of spoilage of

CHAPTER 1

7

fresh-cut fruit and shelf-life limiter (Barrett et al., 2005; Beaulieu et al., 2001; Fleet, 2003; Heard,

2002). The microbial decay of fresh-cut fruit may occur much more rapidly than in vegetable

products due to high levels of sugars found in most fruit (Beaulieu et al., 2001).

The normal micro flora found on the cut surface of fresh-cut fruit is diverse, but it generally

includes a variety of fungi (yeasts and moulds) and mostly harmless bacteria (Garcia and Barrett,

2005). Firstly, the acidity of fruit tissue usually helps suppress bacterial growth, but not growth of

yeasts and moulds (Beaulieu et al., 2001). Secondly, unlike on whole fruit, growth of moulds on

fresh-cut fruit does not appear to be a major problem as in the fresh-cut environment, faster-

growing yeasts tend to outgrow moulds to cause spoilage (Heard, 2002). Yeast spoilage is

therefore a major problem to industries that process fruit (Fleet, 1992), due to the low pH (see

Table 1.1), high sugar content of most fresh-cut fruit and high humidity environment during

storage. In fresh-cut melons with a higher pH than most acidic fresh-cut fruit products, such as

honeydew melon (pH 5.2 - 5.6) and cantaloupe (pH 6.2 - 6.5), bacterial spoilage is also a relevant

with regards to their spoilage (Beaulieu et al., 2001; Garcia et al., 2005). Moreover, pathogens

Figure 1.5: The simplified production procedures of fresh-cut fruit.

Whole fruit

Pretreatment

Cutting process

Coating or dipping process

Packaging

Chill storage

LITERATURE REVIEW

8

such as Listeria monocytogenes, Escherichia coli O157:H7 and Salmonella spp. may potentially

exist and grow on this type of fresh-cut fruit (Garcia et al., 2005; Nguyenthe and Carlin, 1994).

However, the safety of fresh-cut fruit is beyond the scope of this literature study. Therefore, the

following sections mainly discuss the spoilage of fresh-cut fruit by yeasts, soft rot and lactic acid

bacteria.

Table 1.1: Approximate pH values of some fresh fruit. Source: Garcia et al. (2005).

Fruit pH Fruit pH

Apple 2.9-3.9 Grape 3.0-4.0

Apricot 3.3-4.4 Grapefruit 2.9-4.5

Banana 4.5-4.7 Lime 1.8-2.4

Blackberry 3.0-4.2 Lemon 2.2-2.6

Cantaloupe 6.2-6.5 Melon 6.3-6.7

Cactus pear 5.8-6.0 Orange 3.3-4.3

Cherry 3.2-4.0 Peach 3.3-4.2

Cranberry 2.5-2.7 Plum 2.8-4.6

Coconut 6.0 Strawberry 3.0-3.9

Fig 4.6 Watermelon 5.2-5.6

1.2.1.1 Spoilage by yeasts

Spoilage by yeasts is mostly characterized by the fermentation of carbohydrates to produce CO2,

alcohol, flavour and off-flavour compounds (such as acids, esters and higher alcohols) or the

formation of films and off-odours (Heard, 2002). These metabolites are mainly from the

fermentation of glucose and the catabolism of amino acid. Visual evidence of yeast spoilage of

fresh-cut fruit occurred in the form of yeast colonies and the swelling of the packages due to CO2

production (see Figure 1.6).

Glucose catabolic metabolism affected by O2 and glucose is briefly discussed in the following

sections. Figure 1.7 shows a simplified scheme of glucose metabolism in Saccharomyces

CHAPTER 1

9

cerevisiae. Glycolysis is a metabolic process for conversion of intracellular glucose to pyruvate

in the cytosol, whereby production of energy in form of ATP is coupled to the generation of

intermediates and reducing power in form of NADH for biosynthetic pathways. After glucose

uptake, the first step of glycolysis is that glucose is phosphorylated by kinases to glucose 6-

phosphate and then isomerized to fructose 6-phosphate by phosphoglucose isomerase. The

subsequently acting enzymes are shown in Figure 1.7. On the glucose 6-phosphate level, glucose

6-phosphate may go through the pentose phosphate pathway (PP pathway) reentered glycolysis at

the level of fructose 6-phosphate or glyceraldehyde 3-phosphate. PP pathway is a process that

generates cytosolic NADPH and pentose sugars for biosynthetic reactions, such as the production

of fatty acids, amino acids and sugar alcohols (Kruger and von Schaewen, 2003).

Figure 1.6: (A) Swelling of package cut pineapple at the end of their shelf-life. (B) yeast colony formation

on fresh-cut pineapple.

B

A

LITERATURE REVIEW

10

Figure 1.7: Scheme of glucose catabolic metabolism in Saccharomyces cerevisiae, modified from

Taherzadeh et al. (2002). NADH, NADPH, NAD(P)+, ATP, ADP, GTP, H

+, Pi, and CoA are not included

in the Picture.

Glucose

Glucose-6P

Fructose-6P

Fructose-1,6BP

Glyceraldehyde-3P

1,3-Bisphosphoglycerate

3-Phosphoglycerate

2-Phosphoglycerate

Phosphoenolpyruvate

Pyruvate

Acetyl-CoA

Oxaloacetate

Citrate

Isocitrate

2-Oxoglutarate

Succinyl-CoA

Succinate

Malate

Fumarate

Pentose phosphate

pathway

Glycerol

Acetaldehyde Ethanol

Acetate

Ethyl acetate

1

2

3

4

5

6

7

8

9

16 10 11

12

13

14

15

17

18

19

20 21

22

24

25

1 Glucokinase (GLK) 2 Phosphoglucose isomerase (PGI)

3 Phosphofructokinase (PFK)

4 Aldolase (ALD)

5 Triosephosphate dehydrogenase (TDH)

6 Phosphoglycerate kinase (PGK)

7 Phosphoglycerate mutase (GPM) 8 Enolase (ENO)

9 Pyruvate kinase (PYK)

10 Pyruvate decarboxylase (PDC) 11 Alcohol dehydrogenase (ADH)

12 Alcohol acetyltransferase (AAT)

13 Aldehyde dehydrogenase (AlDH) 14 Acetyl-CoA synthetase (ACS)

15 Pyruvate dehydrogenase (PDH)

16 Pyruvate carboxylase (PYC) 17 Citrate synthase (CS)

18 Aconitase

19 Isocitrate dehydrogenase (IDH, IDP1, IDP2) 20 2-Oxoglutarate dehydrogenase (OGDH)

21 Succinyl-CoA synthetase

22 Succinate dehydrogenase 23 Fumarate reductase (cytosolic, FRDS)

24 Fumarase

25 Malate dehydrogenase

23

PDH-bypass

Acetyl-CoA Mitochondria

Cytosol

Oxaloacetate

TCA Cycle

CHAPTER 1

11

At the pyruvate (the end product of glycolysis) branching point, the carbon flux may be

distributed between the respiratory and fermentation pathways depending on the yeast species

and the environmental conditions such as O2 (Rodrigues et al., 2006). In general, when yeasts

such as S. cerevisiae and its closely related species in aerobic condition and low content of

external sugars, pyruvate is metabolized to CO2 and H2O through Krebs cycle (also named

tricarboxylic acid cycle, TCA cycle) in the mitochondrion of the cell. Pyruvate can be firstly

converted to oxaloacetate and acetyl-cofactor A (CoA) which are intermediates of the TCA cycle

(see Figure 1.7). The role of the enzyme pyruvate carboxylase is to catalyse the reaction of

pyruvate to oxaloacetate at the expenses of one molecule of ATP and carbon dioxide. This

anaplerotic reaction functions to further replenish the TCA pathway for biosynthetic purposes or

organic acid production. Pyruvate may be converted to acetyl-CoA through the pyruvate

dehydrogenase (PDH) complex (pyruvate might be directly converted to acetyl–cofactor A by the

mitochondrial multienzyme complex PDH after its transport into the mitochondria by the

mitochondrial pyruvate carrier) or the so-called PDH-bypass pathway (pyruvate may be

converted to acetyl-CoA in the cytosol via acetaldehyde and acetate) (Rodrigues et al., 2006). It

is still matter of debate if the flux distribution during pyruvate metabolism determines whether

the pyruvate flows inward through the PDH complex or through the PDH-bypass pathway and

thereby the split between respiration and fermentation (Rodrigues et al., 2006). The reduction of

NAD+ occurs both in the cytosol by glycolysis and in the mitochondria by the PDH complex and

dehydrogenases of the TCA cycle. O2 can be the terminal electron acceptor under aerobic

condition for oxidizing both pools of NADP by the mitochondrial respiratory chain. Whilst under

anaerobic conditions, fermentation occurs resulting in the production of ethanol, ethyl acetate and

other flavour and off- flavour compounds. In this case, cytosolic redox balance is restored by

ethanol and glycerol formation (Rodrigues et al., 2006). Taherzadeh et al. (2002) concluded that

the production of glycerol in yeasts is known i) as a sink for the excess NADH which is produced

by anabolic reactions during anaerobic conditions and ii) as an osmolyte balancing a high

external osmotic pressure during e.g. salt stress.

However, some yeasts produce ethanol even under aerobic conditions and are known as Crabtree-

positive yeasts, whereas others i.e. Crabtree-negative yeasts do not (Ragaert et al., 2006a; Ragaert

et al., 2006b; Vanurk et al., 1990). Fiechter and Seghezzi (1992) stated that the Crabtree effect

LITERATURE REVIEW

12

study was criticized on the basis of its ill-defined experimental determination, leading it to be

applicable only under specific conditions. For example, S. cerevisiae, the Crabtree-positive

prototype, shows a complete respiratory (aerobic) metabolism in low-glucose conditions

(Gonzalez-Siso et al., 2000), whilst in the presence of surplus glucose (> 0.15 g per litre) it

displays a respiro-fermentative glucose metabolism, where respiration and fermentation occur

simultaneously (Fiechter et al., 1992; Fredlund, 2004; Jouhten et al., 2012; Kappeli, 1986;

Verduyn et al., 1984). A species-specific glucose flux is degraded oxidatively. When the

respiratory capacity is saturated, ethanol formation begins. Respiro-fermentative metabolism

affects synthesis of enzymes by unknown mechanisms. Hence, the content of glucose in the

media also plays an important role in determining the type of metabolism (Kappeli, 1986).

Rodrigues et al. (2006) reported that at increasing glucose concentrations, the glycolytic rate will

increase and more pyruvate is formed, saturating the PDH-bypass and shifting the carbon flux

through ethanol production and beginning the fermentation (see Figure 1.7). In addition to the

different enzyme affinities for pyruvate, the enzymatic activities may also play a role in the

regulation of pyruvate flux. In S. cerevisiae, high glucose concentrations induce an increase of 3

– 4 times the pyruvate decarboxylase activity and a decrease of aldehyde dehydrogenase (AlDH)

activity, favouring the alcoholic fermentation. On the other hand, at low glucose concentrations,

pyruvate is mainly converted to acetyl–CoA by the PDH complex (Rodrigues et al., 2006). Hence,

the external glucose may also control the distribution of pyruvate flux between respiration and

alcoholic fermentation.

The intermediates of the Ehrlich pathway (known as fusel alcohols, fusel acids, fusel aldehydes)

(see Figure 1.8,), formed by the successive deamination, decarboxylation and hydrogenation

(with conversion of NADH to NAD+) of the amino acids (leucine, valine, isoleucine,

phenylalanine, tyrosine, and tryptophan and methionine), may also affect the flavour of fruit

(Boumba et al., 2008; Hazelwood et al., 2008; Ragaert et al., 2006b). Respiro-fermentation may

also impact the amino acid catabolism via the Ehrlich pathway. According to Hazelwood et al.

(2008), the Ehrlich pathway may provide an alternative, energy-efficient means for NADH

regeneration under anaerobic conditions for yeasts. The explanation of the correlation could be

that pyruvate can be converted to isoleucine, valine and leucine by pyruvate acetohydroxy-acid

synthase (Boumba et al., 2008; Gottschalk, 1986; Ouchi et al., 1980). Hence, volatile metabolites

CHAPTER 1

13

are produced by oxidizing NADH to NAD+, which is needed for glycolysis in anaerobic

conditions for yeasts, during the ethanol formation and Ehrlich pathway.

It may be concluded that when respiro-fermentation or fermentation occurs in yeasts (as a result

of low O2 levels or high levels of external sugars i.e. glucose, sucrose and fructose), the

formation of ethanol, ethyl acetate, fusel compounds and other related volatile metabolites occurs.

On one hand, some of the volatile metabolites produced by yeasts have the potential to enhance

the flavour of fresh-cut fruit. On the other hand, they can also be off-flavours and off-odours

which limit the shelf-life of fresh-cut fruit. To date the effect of the volatile organic compounds

produced by yeasts and fruit tissue itself on the quality of fresh-cut fruit during storage has not

yet been reported in literature.

Figure 1.8: The Ehrlich pathway. Catabolism of branched-chain amino acids (leucine, valine, and

isoleucine), aromatic amino acids (phenylalanine, tyrosine, and trytophan), and the sulfur-containing

amino acid (methionine) leads to the formation of fusel acids and fusel alcohols. Source: Hazelwood, 2008.

Amino acids

α-keto acid

2-oxoglutarate

glutamate

‘fusel aldehyde’

‘fusel acid’ in ‘fusel alcohol’

CO2

NAD+

NADH, H+

NAD+

NADH, H+

ATP

ADP

‘fusel acid’ out

Transaminatio

Decarboxylation

Oxidation

Export

Reduction

LITERATURE REVIEW

14

1.2.1.2 Soft rot

Soft rot, which is characterized by water-soaking and formation of a slimy surface on plant

tissues, has been identified as the leading cause of storage disorders in many types of whole fruit

and has been observed in fresh-cut cantaloupe (Barth et al., 2010; Liao, 2005; Oconnor-Shaw et

al., 1996; Ukuku and Fett, 2002a). Barth et al. (2010) reported that soft rot results from

degradation of plant cell walls by pectolytic enzymes produced by a variety of microorganisms,

including Erwinia carotovora, Pseudomonas marginalis, Botrytis, Clostridium, Alternaria,

Geotrichum, and Fusarium. The pectolytic enzymes, including pectin methyl esterase,

polygalacturonase, pectin lyase, and pectate lyase, can degrade pectin in the middle lamella of the

cell, thereby resulting in liquefaction of the plant tissue which lead to conditions such as soft rot

(Barth et al., 2010). Those microorganisms can grow and are active over a wide range of

temperatures, 5 - 35°C (Baldwin, 2002)

1.2.1.3 Spoilage by lactic acid bacteria

Lactic acid bacteria (i.e. Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, and

Enterococcus) have been associated with the spoilage of fresh-cut fruit (especially fresh-cut

melons) that are packaged in modified atmospheres with <2% O2 and >10% CO2 and stored at

7°C (Barth et al., 2010). Lactic acid bacteria can only obtain ATP by fermentation, usually of

sugars (Barth et al., 2010). Since they do not use oxygen in their energy production, lactic acid

bacteria can also grow well under anaerobic conditions. Fermentation by lactic acid bacteria

lowers the pH due to the production of lactic acid and results in an off-flavour similar to

buttermilk as a result of the production of acetylmethylcarbinol and diacetyl. Other fermentation

products include acetic acid, ethanol, formic acid, and CO2. Lactic acid bacteria may be

associated with the microbial spoilage of many fresh-cut fruit such as honeydew melon,

cantaloupe melon (Selma et al., 2008) and grapes (Nobile et al., 2008).

CHAPTER 1

15

1.2.2 Cut surface colour changes

The colour of the flesh of fruit can be the first factor influencing the customer perception about

the quality of fresh-cut fruit. Enzymatic browning and translucency are the most common quality

defects of fresh-cut fruit. Other discolorations (such as developing pink, red, green, blue-green or

blue tonalities as a consequence of cell disruption) are not discussed in this section since it is a

particular problem in vegetable products rather than fresh-cut fruit (Toivonen and Brummell,

2008).

1.2.2.1 Enzymatic browning

During the peeling and cutting of fresh-cut fruit, fruit cells on the surface are broken. Their

contents (including previously compartmentalized enzymes) are suddenly freed and come in

contact with their substrates (Garcia et al., 2005). A group of enzymes called polyphenol oxidases

(PPO), which occur in particularly high amounts in fruit such as apples and pears, are responsible

for the discoloration referred to as enzymatic browning (Garcia et al., 2005). The PPO, in the

presence of O2, converts phenolic compounds in fruit and vegetables into dark coloured pigments

(Beaulieu et al., 2001). Enzymatic browning which is a serious quality defect in fresh-cut fruit

(Barrett et al., 2010), increases with temperature, water stress and oxygen (Beaulieu et al., 2001;

Kader, 2002). Modified atmosphere packaging and coating may be used to reduce PPO-mediated

cut surface discoloration by reduced O2, acidification and reducing agents (Barrett et al., 2010).

In addition, browning is also highly related to fruit cultivars (see § 1.3.6.1).

1.2.2.2 Translucency

Translucency is a common quality defect of fresh-cut fruit, especially fresh-cut melons and

pineapples (Aguayo et al., 2003; Aguayo et al., 2004; Bai et al., 2003; Chen and Paull, 2001;

Montero-Calderon et al., 2008; Portela and Cantwell, 1998, 2001; Saftner et al., 2003).

LITERATURE REVIEW

16

Translucency is a physiological disorder, also termed “glassiness” or “watercore” and is

characterized by the alteration of flesh texture to become dark and glassy (Aguayo et al., 2004).

Chen et al. (2001) found an increase in electrolyte leakage in ‘Smooth Cayenne’ pineapple

translucent tissue and considered that its development was related to the rate of diffusion of water

and solutes across the fruits cell membranes, changes in membrane permeability, sugar content

and differences in internal osmotic pressure which could promote the removal of water from the

phloem into the apoplast. Translucency of honeydew melon cubes was greater with summer

melons than winter melons (Bai et al., 2003). Montero-Calderon et al. (2008) observed that

colour parameters of the lightness (L*) and blue-yellowness (b*) of pineapple flesh significantly

decreased during storage and were directly attributed to the translucency phenomenon. Calcium

(Ca) salt and Ca amino acid chelate treatments more than doubled tissue calcium content and

inhibited changes in melon firmness, surface colour, and the development of tissue translucency

during storage (Saftner et al., 2003). Aguayo et al. (2004) concluded that translucency is a

symptom of senescence, other than a result of calcium deficiency.

1.2.3 Texture softening

The texture of fruit is derived from their turgor pressure, the composition of individual plant cell

walls, and the middle lamella ‘glue’ that holds individual cell together (Barrett et al., 2010). Thus,

texture softening may result from cell softening enzymes present in the fruit tissue and decreasing

turgor due to water loss (Beaulieu et al., 2001). Although some degree of softening is required for

optimal quality in fruit, over-softening is undesirable and is a sign of senescence or internal decay

(Aked, 2002). Tissue softening and associated loss of integrity and leakage of juice from some

fresh-cut products can be the primary cause of poor quality and unmarketability (Kader, 2002).

Firmness of fresh-cut fruit products can be maintained by treatment with calcium salts (Beaulieu

et al., 2001). Also, initial firmness, temperature, and vibration influence the rate of softening and

juice leakage from fresh-cut fruit (Kader, 2002).

CHAPTER 1

17

1.2.4 Off-flavour and off-odour development

The production of off-flavours and off-odours may be stimulated by mechanical wounding during

peeling and cutting. Generally, peeling and cutting increase the respiration rate of fresh-cut fruit

compared to the same fresh whole product (Chung and Moon, 2009a; Toivonen and DeEll, 2002;

Wu et al., 2012b). Higher respiration rates indicate a faster production of ethylene and other

volatiles and deterioration (Wu et al., 2012b). Off-flavours associated with acetaldehydes can be

greatly increased in strawberries when acute tissue injuries occurred (Ke et al., 1991a; Toivonen,

1997).

Off-flavours of fresh-cut fruit may be produced through the action of enzymes such as

lipoxygenase (LOX) or peroxidase (POD), which form reactive free radicals and hydroperoxides

that may catalyse the oxidation of lipids (Barrett et al., 2010; Oms-Oliu et al., 2008c). Wounding

and physiological stress may stimulate LOX and POD (Lamikanra and Watson, 2001; Oms-Oliu

et al., 2008c). Increase in activity of LOX and POD has also been related to the senescence of

fruit tissues (Lamikanra, 2002a). When these reactions occur, the result may be the development

of undesirable flavours described as rancid, cardboard, oxidized, or wet dog (Barrett et al., 2010).

The development of off-odours and off-flavours of fresh-cut fruit as a result of microbial spoilage

is discussed in § 1.2.1. However, the interaction of biochemical processes between the

physiological processes and microbiological processes is scarce. Ragaert et al. (2006c) reported

that ethanol (which mostly produced by yeasts at counts ˃ 5.0 log10 CFU/g) can be converted to

ethyl acetate by the living tissues of strawberries. Additionally, the accumulation of ethyl acetate

resulted in sensory unacceptable of strawberries at 7°C.

The off-odours and off-flavours of fresh-cut fruit may also be produced by the fermentation of

fruit tissues due to unsuitable packaging i.e. reduced O2 modified atmosphere (see § 1.3.4.1).

Generally these defects result from the accumulation of acetaldehyde, ethanol, and/or ethyl

acetate (Barrett et al., 2010; Forney et al., 2009).

LITERATURE REVIEW

18

1.2.5 Nutritional losses

During storage of fresh-cut fruit little change occurs in the dietary fibre and mineral content, but

the vitamins are lost (Barrett et al., 2010). Cutting stimulates ethylene production which in turn

increases respiration and senescence leading to even more rapid loss of certain vitamins (Barrett

et al., 2010). Vitamin C content decreased in most fruit during storage (Soliva-Fortuny and

Martin-Belloso, 2003b). Therefore, it can be used as an index of freshness (Barrett et al., 2010).

The phenolic content may change from a nutritional point of view in some fresh-cut fruit during

storage because of the participation of phenols in enzymatic browning reactions (Soliva-Fortuny

et al., 2003b). Soluble solid content (SSC) and titratable acidity (TA) can change (decrease or

increase) during storage of fresh-cut fruit. The SSC and TA content generally correlate with

sweetness, flesh taste and flavour, which are very important characteristics for fresh-cut quality

(Beaulieu et al., 2001). Therefore, the SSC and TA content have been evaluated to assess the

quality of some fresh-cut fruit in many studies (Gomes et al., 2012; Lima et al., 2010; Montero-

Calderon et al., 2010b; Rossle et al., 2011a; Russo et al., 2012; Saftner and Lester, 2009).

1.3 The quality of fresh-cut fruit improvement and maintenance

Several studies focused on improving and maintaining the quality of fresh-cut fruit during

production and storage have been reported in literature. These include pre-treatments (i.e.

washing, decontamination), physical decontamination techniques, edible coatings, modified

atmosphere packaging, chilling storage, etc.

1.3.1 Pre-treatments of whole fresh fruit

Pre-treatment before the cutting/peeling process is one of the most important processes in the

production of fresh-cut fruit where a reduction of the number of spoilage microorganism and

safety improvement can be achieved (see Table 1.2). Currently, the most common treatment is

CHAPTER 1

19

sodium hypochlorite solution (NaOCl). Additionally, researchers are looking for new solutions

due to the environmental and health risks related to the use of chlorine. Hot water and hydrogen

peroxide (H2O2) treatments may be potential alternatives.

NaOCl is the most common disinfectant used in the fresh-cut industry. The concentrations used

range from 50 to 200 mg/L with contact times of 1 - 2 minutes (Graca et al., 2011). Manuwong et

al. (2007) found that NaOCl treatment at 200 mg/L was the best treatment to reduce Salmonella

spp. on the peels of pineapple. This treatment resulted in a 2.72 log10 CFU/cm2 reduction.

Rodrigues et al. (2007) reported that the use of sanitizers (50 and 100 ppm NaOCl, 4 and 6%

H2O2) resulted in greater solubilization of pectic substances in fresh-cut pequi fruit (Caryocar

brasiliense Camb.), during storage. However, the sanitizers did not influence on the variable

firmness, loss of mass and activity of polygalacturonase (Rodrigues et al., 2007). Antoniolli et al.

(2005) reported that H2O2 (200 or 1000 mg/L) and NaOCl (200 mg/L) were inefficient at

reducing endophytic mesophilic aerobes, moulds and yeasts on fresh-cut pineapple.

Carvacrol, calcium lactate and calcium chloride (CaCl2) were also studied as sanitizers for fresh-

cut fruit. According to Roller and Seedhar (2002), treatment with 1 mM of carvacrol or cinnamic

acid delayed spoilage of fresh-cut kiwi and honeydew melon at chill temperatures without

adverse sensory consequences. Fresh-cut papaya pretreated with 1% calcium lactate solution

were firmer than control fruit, but translucency limited the shelf-life of cubes dipped in either

water or calcium lactate (Wall et al., 2010). Besides, the addition of 0.5, 1.0 and 1.5% of CaCl2 or

0.5% of polyethylene glycol in the washing water did not ensure the maintenance of the texture

of fresh-cut strawberry at the end of the period of storage at 5°C. (Ponce et al., 2010). Likewise,

the use of CaCl2 in fresh-cut 'Perola' pineapple did not provide beneficial effects on the texture

and interfered negatively on the flesh color of fruit (Antoniolli et al., 2003).

Hot water rinsing and brushing (HWRB) is an environmentally friendly technology that is very

useful in cleaning and disinfecting whole melon destined for processing without affecting the

quality, if the product is stored at reasonably low temperatures after processing (Fallik et al.,

2007). Fan et al. (2008a) reported that hot water pasteurization of whole cantaloupes frequently

resulted in lower total plate counts of fresh-cut fruit during storage and did not negatively affect

the quality of fresh-cut cantaloupes. Additionally, mild heat pre-treatments, when applied to firm

ripe kiwi at temperatures below 45°C during less than 25 min improve the quality, mainly the

LITERATURE REVIEW

20

firmness (Beirao-da-Costa et al., 2006). Colour is only marginally affected. However, Manuwong

et al. (2007) reported that NaOCl treatment was more effective than hot water treatment at 50 and

60°C for reducing Salmonella spp. on fresh-cut pineapple.

The use of heated H2O2 (70°C) to reduce Salmonella counts levels on contaminated whole

cantaloupes will enhance the microbial safety of the fresh-cut product (Ukuku et al., 2004).

Likewise, H2O2 treatments before processing also significantly reduces the transfer of native

micro-flora from melon rind to fresh-cut pieces (Ukuku, 2004). H2O2 applied at 50°C is superior

to other whole-melon treatments, yielding a fresh-cut shelf-life of > 2 weeks (Sapers et al., 2001).

1.3.2 Physical decontamination techniques

1.3.2.1 Ultraviolet light - C

Ultraviolet light – C (UV-C) has been approved for use as a food surface disinfectant in the USA

(US-FDA, 2002). Mild intensity (˂ 1.2 kJ/m2) of UV-C light exposure can be an effective

technology for surface decontamination and can also enhance the health promoting compounds

and reduce tissue softening and browning of fresh-cut fruit (Alothman et al., 2009; Chisari et al.,

2011; Manzocco et al., 2011a). Manzocco et al. (2011a) reported that mild UV-C light treatments

resulted in apple slices which were much more stable than the untreated controls in terms of

microbial growth and the development of browning and off-flavours. Similarly, exposure of

fresh-cut melon to UV-C light lead to 2 log10 CFU/g reductions of the total viable count and

Enterobacteriaceae during storage (Manzocco et al., 2011b). Fonseca and Rushing (2006)

suggested that UV-C light could potentially be used for sanitizing fresh-cut watermelon.

Additionally, UV-C treatment improved the polyphenolic profile of honey pineapple, banana, and

guava (Alothman et al., 2009; Karim et al., 2009) and reduced both tissue softening and browning

of fresh-cut melon (Cucumis melo L. var. reticulatus) (Chisari et al., 2011). On the other hand,

UV-C light may decrease vitamin C content and the concentration of esters of fresh-cut fruit

(Alothman et al., 2009; Karim et al., 2009; Lamikanra and Richard, 2004).

CHAPTER 1

21

1.3.2.2 Low-dose gamma irradiation

Fan et al. (2006) reported that the combination of hot-water pasteurization of whole cantaloupe

and low-dose irradiation of packaged fresh-cut melon can reduce the population of native micro

flora while maintaining the quality of this product. Fan et al. (2005) also found that the

combination of calcium ascorbate (CaA) and gamma (γ) irradiation enhanced microbial food

safety while maintaining the quality of fresh-cut apple slices. Boynton et al. (2005) stated that

irradiation of fresh-cut cantaloupe has potential for shelf-life extension and for integration with

modified atmosphere packaging systems. However, low levels of furan (C4H4O) can be induced

by irradiation in some fruit, especially those fruit that have a high amount of simple sugars and

low pH (Fan and Sokorai, 2008b). Furan is regarded as a possible carcinogen by the U.S. Dept. of

Health and Human Services and the Intl. Agency for Research on Cancer. Moreover, irradiation

alone can soften the texture and increase colour change during the processing of fresh-cut apple

(Fan et al., 2005).

1.3.2.3 Ozone

Karim et al. (2010) found that the total phenol and flavonoid contents of fresh-cut honey

pineapple and banana 'pisang mas' increased significantly when exposed to ozone (O3) at a flow

rate of 8 ± 0.2 mL/s for up to 20 min, with a concomitant increase in ferric reducing/antioxidant

power and 1,1-diphenyl-2-picrylhydrazyl values. The opposite was observed for fresh-cut guava

(Karim et al., 2010). Ozone treatment significantly decreased the vitamin C content of all three

fruit (Karim et al., 2010). Gaseous ozone is an effective option to risk reduction and spoilage

control of fresh and fresh-cut melon. Moreover, depending on the timing of contamination and

post-contamination conditions, rapid drying combined with gaseous ozone exposure may be

successful as combined or sequential disinfection steps to minimize the persistence of Salmonella

on the surface of cantaloupe melons and their transfer during processing of home preparations

(Selma et al., 2008).

LITERATURE REVIEW

22

Table 1.2: Effect of pre-treatments of whole fruit before cutting/peeling process or treatments of fresh-cut fruit on the quality attributes

of fresh-cut fruit.

Conditions Treatments Microbial

contamination Colour Texture SSC Flavour References

Cantaloupe

4°C, 20d

submerged whole cantaloupes into

76°C water for 3 min

lower total plate counts NE NE NE NE Fan et al. (2008a)

Kiwifruit

4°C, 10d

Immerse firm ripe kiwi at < 45°C

water for < 25 min

ND marginally

affected

improved better

retention

ND Beirao-da-Costa et al.

(2006)

Pineapple dipped pineapple in hot NaOCl

treatment at 200 mg/L

Salmonella spp.

reduced 2.15 log10

cfu/cm2

NE NE negative

effected

ND Manuwong et al. (2007)

Cantaloupe

4°C, > 14d

washed with H2O2 at 50°C reducing endogenous

microbial populations

delay

changes

ND ND off-odour

delayed

Sapers et al. (2001)

Cantaloupe dipped in water (97°C) or hydrogen

peroxide (70°C) for 1min

reduce the population

of Salmonella

ND ND ND ND Ukuku et al. (2004)

Pequi fruit

6°C, 15d

sanitized with NaClO 50ppm and

100ppm, H2O2 4% and 6%

reduce the population

of Salmonella

NE NE ND NE Rodrigues et al. (2007)

Pineapple

4°C, 10d

Disinfected with H2O2 (200 or 1000

mg/L) and NaOCl (200 mg/L)

inefficient at reducing

mesophile aerobic,

mould and yeast

endophytic population

NE NE NE NE Antoniolli et al. (2005)

Cantaloupe and

honeydew melons

5°C, 15d

washed with nisin (10 mµg/mL)-

EDTA (0.02 M) for 5 min

educing populations of

yeast and mould and

Pseudomonas spp

positive

effect

positive

effect

ND positive

effect

Ukuku and Fett (2002b)

‘Galia’ fruit

5°C, 10d

Galia fruit treated with 1 µL/L

1-Methylcyclopropene at 20°C for 1d.

slightly reduced

populations of both

total aerobic organisms

and Enterobacterium

reduced

water

soaking

delayed

softening

ND positive

effect

Ergun et al. (2007)

NE: no significant (P˃0.05) effect (positive or negative) on the quality parameter; ND: not determined; SSC: soluble solid contents.

CHAPTER 1

23

Table 1.3: Effect of physical decontamination of fresh-cut fruit on the quality attributes of fresh-cut fruit.

Conditions Treatments Microbial

contamination Colour Texture SSC Flavour references

Apple UV-C light treatments at 1.2,6.0,12.0

and 24.0 kJ/m2

1-2 log reduction in

total viable counts

positive

effect

positive

effect

ND positive

effect

Manzocco et al. (2011a)

‘Galia’ melon

5°C, 10d

UV-C, 254 nm, 0.04 kJ/s·m2, radiation

for different times (30, 60, 120 s)

ND positive

effect

positive

effect

ND ND Chisari et al. (2011)

Cantaloupe

6°C, 14d

UV-C, 0, 1.2, 6 and 12 kJ/m2 for up to

10min

2 log reductions for

both total viable count

and Enterobacteriaceae

NE NE ND positive

effect

Manzocco et al. (2011b)

Watermelon

3°C, 16d

UV-C, <1.4 kJ/m2 at 254 nm Positive effect ND ND ND ND Fonseca et al. (2006)

Cantaloupe

4°C, 7d

gamma radiation, 0.5 kGy low microbial load NE NE ND ND Fan et al. (2006)

Apple

10°C, 21d

gamma radiation, 0.5 and 1 kGy positive effect slightly

increased

browning

reduced

fruit

firmness

ND ND Fan et al. (2005)

Apple

4 or 10°C, 12d

ultrasound in combination with

ascorbic acid

ND anti-

browning

ND ND ND Jang et al. (2009); Jang and

Moon (2011)

Cantaloupe

5°C, 7d

gaseous ozone reduced total coliforms,

Pseudomonas

fluorescens, yeast and

lactic acid bacteria

positive

effect

positive

effect

ND positive

effect

Selma et al. (2008)

Apple

4°C, 14d

high pressure argon, 150 MPa,

10min

positive effect positive

effect

negative

effect

NE positive

effect

Wu et al. (2012b)

NE: no significant (P˃0.05) effect (positive or negative) on the quality parameter; ND: not determined; SSC: soluble solid contents.

LITERATURE REVIEW

24

The ozonation of strawberries for 60 minutes was significantly more effective in reducing (p <

0.05) the count of mesophilic aerobic microorganisms, yeasts and moulds and coliforms, when

compared to the results obtained with strawberries ozonizated for 30 minutes and immersed in a

solution of organic chlorine (Ponce et al., 2010). However, it should be note that ripe tissues

reduce the efficacy of these gaseous ozone treatments on fresh-cut fruit, potentially by oxidative

reaction with soluble refractive solids (Selma et al., 2008).

1.3.2.4 High pressure argon and ultrasound

High pressure (HP) argon (Ar) processing has recently been studied for extending the shelf-life of

fresh-cut apple and pineapple by reducing respiration, ethylene production, browning, microbial

growth and maintaining the sensory quality (Wu et al., 2012a; Wu et al., 2012b). A shelf-life

extension of 6 days was achieved by applying HP Ar treatment (0.5-4.5 MPa) on fresh-cut

pineapples during cold storage in air or in modified atmosphere packaging (Wu et al., 2012a).

It was reported that the use of ultrasound in combination with ascorbic acid had a positive effect

on quality maintenance of fresh-cut apples, because several enzymes related to the browning of

fresh-cut ‘Fuji’ apples were inhibited by the combined treatment with ultrasound and ascorbic

acid during storage (Jang et al., 2009; Jang et al., 2011).

1.3.3 Edible coatings

The application of edible coatings is mainly aimed at delaying the physiological decay of fruit. In

addition, the spoilage microorganisms present at the moment of application can be reduced in

numbers. Recently, some studies have reported the use of multilayered edible coating for fresh-

cut fruit (Vargas et al., 2008). Generally, the coatings contain several ingredients or are applied as

a series dipping treatments (see Figure 1.9). In this section, we review the most commen coating

solutions studied to maintain the quality for different fresh-cut fruit, especilly pineapples, melons,

apples, pears, and other fruits.

CHAPTER 1

25

Figure 1.9: Schematic representation of coating a minimally processed (MP) fruit (e.g. a fresh-cut apple slice) with a

multilayered edible coating by using the layer-by-layer assembly with three dipping and washing steps. Source:

Vargas et al., 2008.

1.3.3.1 Apple

Since browning is the most important factor limiting the shelf-life of fresh-cut apples, the

application of coatings which could significantly improve their colour maintenance during

storage has been the most studied aspect (see Table 1.4). Some coating solutions successfully

reduced browning and softening during chill storage. Alandes et al. (2011) reported that a

combined treatment using natural additives, which included calcium lactate, cysteine, glutathione

and malic acid, was appropriate to maintain the quality of fresh-cut ‘Fuji’ apple by maintaining

texture and colour. Moreover, honey could also be used as a natural antioxidant to prevent the

enzymatic browning of fresh-cut apples. Jeon and Zhao (2005) found that honey in combination

with vacuum impregnating (vacuum at 75 mmHg for 15 min followed with 30 min restoration at

atmospheric pressure) may have great potential for developing high-quality fresh-cut fruit.

Chitosan-coating treatments effectively retarded enzymatic browning on minimally processed

apples during storage and they effectively retarded or avoided tissue softening (Qi et al., 2011).

Chitosan treated apple slices underwent a little loss of firmness. Sliced apples processed at an

earlier stage of ripeness, but dipped in a solution of ascorbic acid (AA) and CaCl2 and stored

under 100% N2, preserved their original colour with minor changes over the shelf-life (Soliva-

LITERATURE REVIEW

26

Fortuny et al., 2002c). The dual effectiveness of sodium chlorite to inhibit enzymatic browning

and inactivate Escherichia coli may allow this compound to achieve a prominent role in

improving the quality and safety of fresh-cut apple and other food products (Luo et al., 2011).

Some calcium related coating solutions may be beneficial for the texture of fresh-cut apple during

storage. Calcium propionate treatment has consolidating and structuring effects on the

parenchyma and partially minimizes the degradation of fresh-cut ‘Fuji’ apples (Quiles et al.,

2007). Seo et al. (2006) reported that the calcium content of apples dipped in a CaCl2 solution at

45°C was higher than that of the control and when dipping was done in a non-heated CaCl2

solution. The fruit dipped in CaCl2 solution at 45°C were also firmer than those dipped in only

calcium or these given only a heat treatment. Dipping treatment in an aqueous solution of 1% AA

and 1% citric acid for 3 min resulted in an increase in the ‘Golden Delicious’ apple slices

antioxidant activity (Cocci et al., 2006). Furthermore, the combination of antioxidant solution (1%

AA and 1% citric acid) reduced both the initial oxygen respiration rate (for about 10 h of storage)

and the inhibitory effect that CO2 had on the O2 consumption of packed ‘Golden Delicious’ apple

slices (Rocculi et al., 2006).

In contrast, some coating solutions achieved only minor effects on browning or inhibited

browning but also had negative effect on texture, microbiology and other quality attributes.

Worakeeratikul et al. (2007) and Perez-Gago et al. (2005) reported that whey protein concentrate

(WPC) coating had no significant effect (P ˃ 0.05) on fresh-cut apple during storage. Soliva-

Fortuny et al. (2002c) found the solution of 1% (w/v) AA and 0.5% (w/v) CaCl2 and 100% N2

packaging could not effectively prevent the apple slices from softening, and the apple flesh

underwent a progressive loss of firmness from 8.0 to 4.8 Newton (N).

1.3.3.2 Pineapple

A handful of studies also evaluated coating solutions for fresh-cut pineapple as shows in Table

1.5. Mohammed and Wickham (2005) found that the combined treatment of 300ppm AA +

200ppm 4-hexylresorcinol (4-HR) on pineapple was most effective in the inhibition of browning

and microbial spoilage during storage. Gonzalez-Aguilar et al. (2005) reported that a higher

CHAPTER 1

27

content of AA and isoascorbic acid (IAA) was associated with better compositional quality

parameters and appearance of the pineapple slices during storage. Starch-based coatings were

found to efficiently reduce the respiration rate and promoted better preservation of texture and

colour characteristics on fresh-cut pineapple (Bierhals et al., 2011). However, the shelf-life of

fresh-cut pineapple treated by starch-based coatings were not extended as a result of higher

microbial growth in coated samples than in control samples (Bierhals et al., 2011).

1.3.3.3 Melon

Table 1.6 shows coating solutions that have been evaluated for fresh-cut melons. Barrett and

LunaGuzman (1996) reported that postharvest treatment with calcium salts (chloride or lactate)

results in a significant improvement in texture and longer shelf-life of fresh-cut melon. 1% (w/w)

dips in either calcium chloride or calcium lactate maintained the texture of fresh-cut melon over a

12 day period (Barrett et al., 1996). Alandes et al. (2009b) found that the combined treatment

with N-acetyl-L-cysteine/glutathione, calcium lactate, and malic acid preserved the quality

(colour, texture and structure) of fresh-cut Piel de Sapo melon during storage. In addition, 1-

methylcyclopropene (1-MCP) treatment deferred the physical deterioration (softening and

translucency) of fresh-cut 'Galia' melon cubes stored at 5°C by 2-3 days compared to the controls

(Ergun et al., 2007).

1.3.3.4 Pear

Edible coatings that have been used for preventing the quality depreciation of fresh-cut pears are

listed in Table 1.7. Xiao et al. (2010) found that chitosan incorporated with rosemary suppressed

polyphenol oxidase activity, reduced the loss of phenolic content and improved sensory qualities

of fresh-cut pears, providing a great advantage in the reduction of browning.

LITERATURE REVIEW

28

Table 1.4: Coating solutions for fresh-cut apples.

Composition References

10% honey solution Jeon et al. (2005)

Calcium ascorbate Karaibrahimoglu et al. (2004)

Calcium lactate Alandes et al. (2006)

1-methylcyclopropene Rupasinghe et al. (2005)

Whey protein- and hydroxypropyl methylcellulose-based edible composite coatings Perez-Gago et al. (2005)

Ascorbate + cysteine Larrigaudiere et al. (2007)

3% CaCl2 solution Seo et al. (2006)

vanillin Rupasinghe et al. (2006)

Peroxyacetic acid, acidic electrolyzed water and chlorine Wang et al. (2006)

Calcium propionate Quiles et al. (2007)

Natural waxes, and additives Aloe vera (4%), ellagic acid (0.01%) and gallic acid (0.01%) Saucedo-Pompa et al. (2007)

0 (control), 2.5, 5.0 and 7.5% (w/v), wrapped with PVC film Worakeeratikul et al. (2007)

1% chitosan; 2% ascorbic acid + 0.5% CaCl2; 2% ascorbic acid + 0.5% CaCl2 + 1% chitosan Qi et al. (2011)

Sodium chlorite, acidified sodium chlorite, citric acid, calcium chloride, calcium ascorbate Luo et al. (2011)

1% ascorbic acid Jang et al. (2011)

calcium lactate Alandes et al. (2011)

1% ascorbic acid Jang et al. (2009)

3% sodium acid sulfate Fan et al. (2009)

1% (w/v) N-acetylcysteine, or 1% (w/v) ascorbic acid (control) Rojas-Grau et al. (2007)

Sodium chlorite (0.5-1.0 g L-1

), sodium bisulfite (0.5 g/L) and calcium L-ascorbate (10 g/L) Lu et al. (2007)

1% ascorbic acid and 1% citric acid Cocci et al. (2006)

Ascorbic acid and CaCl2 Soliva-Fortuny et al. (2002c)

0.5 g/100 mL carrageenan, 5 g/100 mL whey protein concentrate Lee et al. (2003)

Ascorbic acid solution (0.1 and 0.5%, w/v), citric acid solution (0. 1 and 0.5%, w/v) Zuo and Lee (2004)

4-hexylresorcinol, isoascorbic acid, a sulfur-containing amino acid, and calcium propionate. Buta et al. (1999)

Calcium lactate, N-acetyl-L-cysteine, glutathione, and malic acid Alandes et al. (2009b)

Calcium ascorbate (0, 2, 6, 12 and 20%, w/w) Aguayo et al. (2010)

Cassava starch concentration (2-4% w/w), glycerol content (1-3% w/w) and carnauba wax: stearic acid ratio (0.0:0.0-0.4:0.6% w/w) Chiumarelli and Hubinger (2012)

CHAPTER 1

29

Table 1.5: Coating solutions for fresh-cut pineapples.

Table 1.6: Coating solutions for fresh-cut melons.


300ppm ascorbic acid and/or 200ppm 4-hexylresorcinol Mohammed et al. (2005)

1% and 3% calcium salts (chloride, sulphate and lactate) Eduardo et al. (2008)

100 and 200 mg/L NaOCl Manuwong et al. (2007)

0.25% ascorbic acid and 10% sucrose Liu et al. (2007)

0.1 mol/L isoascorbic acid, 0.05 mol/L ascorbic acid or 0.05 mol/L acetyl cysteine Gonzalez-Aguilar et al. (2004)

Syrup (340 mg of potassium metabisulphite/kg and 413 mg sodium benzoate/kg) Vijayanand et al. (2001)

CaCl2 solutions (1% and 2%) Antoniolli et al. (2003)

10-4

Mmethyl jasmonate Martinez-Ferrer and Harper (2005)

0.5% ascorbic acid and 1% of citric acid, 2% of calcium lactate and 2% of cassava starch suspensions Bierhals et al. (2011)

Ascorbic acid, isoascorbic acid and N-acetyl-cysteine Gonzalez-Aguilar et al. (2005)



Polysaccharide-based edible coatings Oms-Oliu et al. (2008f)

0.5% or 1% calcium Machado et al. (2008b)

1-methylcyclopropene and two calcium salts Machado et al. (2008a)

L-cysteine (0.125. 0.25 and 0.5%) Campos-Vargas et al. (2008)

Ascorbic acid (1.25, 2.5, 2.2 mM) + EDTA (10 mM); ascorbic acid (2.5 mM) + MnCl2 (2.5 mM) Lamikanra et al. (2001)

50 ppm hypochlorite solution (pH 6) Ayhan et al. (1998)

Osmotic dehydration and pectin edible coating (Ferrari et al., 2013)

LITERATURE REVIEW

30

Table 1.7: Coating solutions for fresh-cut pears.


2% chitosan solution with 0.03% rosemary; 0.5% ascorbic acid + 2% chitosan solution Xiao et al. (2010)


Calcium lactate Alandes et al. (2009a)

2% ascorbic acid + 1% CC (or 0.01% 4-hexylresorcinol) + 1% CaCl2 Arias et al. (2008)

0.75% N-acetylcysteine + 0.75% glutathione solution Oms-Oliu et al. (2008d)

2% ascorbic acid , 1% calcium lactate and 0.5% cysteine Gorny et al. (2002)

10 g/L ascorbic acid and 5 g/L CaCl2 Soliva-Fortuny et al. (2004)

0.05% benzoic acid solution Pilizota et al. (2004)

1 % ascorbic acid, 0.25% calcium chloride and 0.1% potassium sorbate Olivas et al. (2003)

1% ascorbic acid + 1.% calcium lactate (or + 0.01% 4-hexylresorcinol) Dong et al. (2000)

4-hexylresorcinol, isoascorbic acid, N-acetylcysteine, and potassium sorbate Buta and Abbott (2000)

Sodium erythorbate, CaCl2 and 4-hexylresorcinol Sapers and Miller (1998)

20% honey (or + 0.4% alpha-tocopherol-acetate/0.4% free alpha-tocopheral/0.8 % water-soluble alpha-tocopherol-acetate) Lin et al. (2006)

CHAPTER 1

31

Treatments consisting of dips in 10 g/L AA and 5 g/L CaCl2 preserved the initial appearance of

pear cubes for at least 14 days (Soliva-Fortuny et al., 2004). Calcium lactate was used to improve

the structural integrity of fresh-cut "Flor de Invierno" pears (Alandes et al., 2009a). This was due

to the fact that calcium lactate reinforced the structure of the fruit maintaining the fibrillar

packing in the cell walls and the cell-to-cell contacts. Lin et al. (2006) reported that the vitamin E

content of pears dipped with honey had significantly higher lightness, lower microbial counts,

lower browning index, and higher consumer acceptance rating than the controls (unfortified).

1.3.3.5 Mangoes, grape and banana

Robles-Sanchez et al. (2009) reported that dipping treatments with 1% AA + 2% citric acid (CA)

+ 2% CaCl2 positively affected the delay of quality deterioration of fresh-cut mango in

comparison to the whole fruit. However, the treatment had a negative effect on the sensory

quality of fresh-cut mangoes judged by the panellists (Robles-Sanchez et al., 2009). Djioua et al.

(2010) showed that chitosan coating, either alone or combined, did not affect the taste and the

flavour of mangoes slices. Calcium ascorbate with CA and N-acetyl-L-cysteine maintained cut

mango slices attractiveness in storage by keeping light colour (Plotto et al., 2010).

Honey solutions significantly maintained the general quality of minimally processed grape by

delaying quality loss and berry decay (Sabir et al., 2011). Saisung and Theerakulkait (2011)

indicated that pineapple shell extract (PSE) and its permeate could be potentially used as a natural

anti-browning agent for controlling enzymatic browning in fresh-cut banana slices.

1.3.4 Modified atmosphere packaging

Modified atmosphere packaging (MAP) is one of the most successful preservation techniques for

agricultural and food products due to the increase in consumer demand for natural antimicrobial

compounds, i.e. molecules of natural origin, not toxic for humans, environmentally safe and not

expensive (Mastromatteo et al., 2010). The basic principles of MAP are a modified atmosphere

LITERATURE REVIEW

32

which can be created either passively by using suitably permeable packaging materials, or

actively by using a specified gas mixture. The aim of both principles is to create an optimal gas

balance inside the package, where the respiration activity of a product is as low as possible, but

the levels of oxygen (O2) and carbon dioxide (CO2) are not detrimental to the product

(Ahvenainen, 1996). Three main gases used in MAP are O2, CO2 and nitrogen (N2). Although

other gases such as nitrous and nitric oxides, sulphur dioxide, ethylene, chlorine, ozone and

propylene oxide have been investigated, they are not applied commercially due to safety,

regulatory and cost considerations (Sandhya, 2010).

MAP is used to increase the shelf-life of packaged produce by reducing fresh-cut fruit respiration

rate, delaying senescence, and inhibiting the growth of many spoilage organisms, ultimately

increasing product shelf-life (Al-Ati et al., 2003). Currently, the most studied MAPs for

minimally processed fruit are i) those with reduced O2 levels (< 20%) and/or combined with

slightly elevated CO2 levels, and ii) high O2 atmosphere (>21%). Although some researchers

reported that MAP did not prevent quality changes of fresh-cut pears (Gomes et al., 2010; Gorny

et al., 2002; Soliva-Fortuny and Martin-Belloso, 2003a), most studies have reported that MAP

(both reduced O2 MAP and high O2 MAP) greatly improves the quality of fresh-cut fruit

(Chonhenchob et al., 2007; Odriozola-Serrano et al., 2010; Oms-Oliu et al., 2009; Oms-Oliu et al.,

2008b; Oms-Oliu et al., 2008c; Oms-Oliu et al., 2008e; Rojas-Grau et al., 2007; Saxena et al.,

2008).

1.3.4.1 Effect of reduced O2 MAP on the quality of fresh-cut fruit

The benefits of reduced O2 MAP on fresh-cut fruit may include inhibiting aerobic microbial

growth, preventing browning, delaying softening, maintaining vitamin C and phenolic contents

and reducing ethylene production, thus, increasing the shelf-life of fresh-cut fruit (see Table 1.8).

The most commonly studied reduced O2 MAP for fresh-cut fruit is 2.5 kPa O2 + 7 kPa CO2 (≈ 2.5%

O2 + 7% CO2), rest N2. For instance, Odriozola-Serrano et al. (2010) proposed 2.5 kPa O2 + 7

kPa CO2 atmospheres to prevent oxidation of the main antioxidant compounds in fresh-cut

strawberries. This agrees with what Oms-Oliu et al. (2007b) reported, which is that low initial O2

concentrations could prevent the loss of vitamin C. Oms-Oliu et al. (2009) also stated that a shelf-

CHAPTER 1

33

life of 10 days was possible for partially ripe fresh-cut pears packaged under a 2.5 kPa O2 + 7 kPa

CO2 atmosphere. Rojas-Grau et al. (2007) demonstrated that the same atmosphere could extend

the shelf-life of apple slices because of a significant inhibition of ethylene production. However,

Gorny et al. (2002), Soliva-Fortuny et al. (2003a) and Oms-Oliu et al. (2008c) found that reduced

O2 MAP did not effectively inhibit the microbial growth and resulted in the development of

undesirable odours and increased peroxidase activity during storage of fresh-cut fruit.

1.3.4.2 Effect of high O2 MAP on the quality of fresh-cut fruit

Recently, Oms-Oliu et al. (2008b; 2008c; 2008e) reported that high O2 MAP was better

compared to reduced O2 MAP for maintaining the quality of fresh-cut melons during storage. The

use of 70 kPa O2 was proposed for the preservation of microbial quality, avoidance of

fermentative reactions, reduction of CO2 production rates, maintenance of firmness and

chewiness of fresh-cut ‘Piel de Sapo’ melon over 2 weeks of storage (Oms-Oliu et al., 2008b;

Oms-Oliu et al., 2008c; Oms-Oliu et al., 2008e). Li et al. (2012b) also observed that high O2

MAP (O2/CO2: 30/5 and 80/0) could be used to inhibit browning and prolong the shelf-life of

fresh-cut 'Yaoshan' pears at 4°C. Van der Steen et al. (2002) reported that applying a high

O2 atmosphere in a high-barrier film, had a beneficial effect on the microbial and sensory shelf-

life of raspberries and strawberries, by inhibiting the development of moulds and by maintaining

fresher sensory properties, as long as atmospheric conditions did not induce fermentation.

However, the application of high O2 MAP may decrease phenolic, vitamin C and soluble solid

content, accelerate browning and stimulate the growth of some mesophille aerobic bacteria (Li et

al., 2012a; Li et al., 2012b; Odriozola-Serrano et al., 2010; Oms-Oliu et al., 2008e; Poubol and

Izumi, 2005a, 2005b). Additionally, O2 levels >25% is considered to be explosive and the

practical safety issues that need to be employed may not be feasible (Werner et al., 2006).

To date only a handful of studies have been performed in which the positive effect of

atmospheres combining high O2 and CO2 on the shelf-life of fresh-cut vegetables has been

reported (Amanatidou et al., 2000; Conesa et al., 2007; Van der Steen et al., 2002). No studies

have yet been published regarding the effect of atmospheres combining high O2 and CO2 levels

on the shelf-life of fresh-cut fruit.

Table 1.8: Effect of MAP on the quality of fresh-cut fruit

Conditions MAP/Combined with other techniques Microbial

contamination Colour Texture Nutrition Flavour Reference

apple

4°C, >1 month

2.5% O2 +7% CO2 combined with a dip

in N-acetylcysteine

ND prevented

browning

Positive ND reduce ethylene

production

Rojas-Grau et al.

(2007)

apple

4°C, 21d

dipped in a solution of ascorbic acid and

calcium chloride and stored in 100% N2

ND not

effectively

not

effectively

ND anaerobic

fermentation

Soliva-Fortuny et al.

(2002c)

apple

4°C, 21-28d

MA condition (most of the air inside the

bags was extracted)

Not significant positive positive positive positive Aguayo et al. (2010)

pear

4°C, 14d

2.5 kPa O2 + 7 kPa CO2 (N2 balanced) inhibited microbial

growth in mature-

green pears

not

effectively

Positive ND reduce ethylene

production

Oms-Oliu et al.

(2009)

pear

4°C, 14d

dipped into 0.75% w/v N-acetylcysteine

+ 0.75% w/v glutathione solution and

packaged under 2.5% O2 +7% CO2

ND Positive ND Positive reduce ethylene

production

Oms-Oliu et al.

(2008d)

pear

4°C, 60d

100% N2 ND ND Positive ND ND Soliva-Fortuny et al.

(2002b)

conference pears

4°C, 75d

100% N2 or 2.5% O2 +7% CO2 ND Positive ND ND ND Soliva-Fortuny et al.

(2002a)

pear

4°C, 10d

low O2 (0.25 or 0.5 kPa) elevated CO2

(air enriched with 5, 10 or 20 kPa CO2),

or superatmospheric O2 (40, 60,

or 80 kPa)

ND did not

effectively

did not

effectively

ND did not

effectively

Gorny et al. (2002)

pear

4°C, >21d

2.5 kPa O2+7 kPa CO2 (N2 balanced) did not effectively ND ND positive Developed

undesirable

odours

Soliva-Fortuny et al.

(2003a)

pineapple

4°C, 10d

1-MCP treatment and MAP in a

N2O(86.13 kPa N2O, 10.13 kPa O2 and

5.07 kPa CO2)

increased the lag

phase

did not

effectively

softening

delay

ND reduce ethylene

production

Rocculi et al. (2009)

pineapple

4°C, 7d

Immersed in solution containing 0.25%

ascorbic acid and 10% sucrose. MAP

contained 4% O2+10% CO2+86% N2

positive positive positive ND positive Liu et al. (2007)

pineapple and

mango

5°C, 25d

4% O2+10% CO2+86% N2 positive positive positive ND positive Martinez-Ferrer et al.

(2002)

Table 1.8 (continued)

35



pineapple

5°C, 14d

12% O2 + 1% CO2; 38% O2 ND ND ND did not

effectively

ND Montero-Calderon et

al. (2010a)

pineapple

4.5°C

5 kPa O2+15 kPa CO2;

80 kPa O2 +15 kPa CO2

Positive Positive Positive ND Positive Budu et al. (2007)

pineapple

10°C, 13d

6% O2 +14% CO2 Positive Positive Positive Positive Positive Singh et al. (2007)

cantaloupe

5°C, 9d

4 kPa O2 + 10 kPa CO2 Positive Positive NE NE NE Bai et al. (2001)

kiwiftuit

4°C, 12d

90% N2O +5% O2 + 5%CO2 ND Positive Positive NE ND Rocculi et al. (2005)

mango

13°C

60 kPa O2 stimulated the

growth of

mesophille aerobic

bacteria

accelerated ND NE ND Poubol et al. (2005a)

melon

5°C, 35d

2.5 kPa O2 +7 kPa CO2 packaging

atmosphere and a dip of 1% ascorbic

acid and 0.5% calcium

slowing down maintaining Reducing NE ND Oms-Oliu et al.

(2007a)

melon

4°C, 14d

70 kPa O2 atmospheres ND ND maintain

firmness

decrease in

the soluble

solid

content

avoid

fermentative

reactions

Oms-Oliu et al.

(2008e)

melon

4°C, 14d

2.5 kPa O2 + 7 kPa CO2 ND ND ND maintained

vitamin C

and

phenolic

content

increase of

peroxidase

activity

Oms-Oliu et al.

(2008c)

melon

4°C, 14d

70 kPa O2 ND ND ND NE prevent

anaerobic

conditions

Oms-Oliu et al.

(2008c)

melon

5°C, 14d

2.5 kPa O2 + 7 kPa CO2 significantly

reduced the growth

Positive NE ND reduce ethylene

production

(Oms-Oliu et al.,

2008b)

melon

5°C, 14d

70 kPa O2 significantly

reduced the growth

Negative Positive ND prevented

fermentation

(Oms-Oliu et al.,

2008b)

Table 1.8 (continued)

36



strawberry 2.5 kPa O2 + 7 kPa CO2, ND ND ND Positive ND Odriozola-Serrano et

al. (2010)

strawberry

wedges

60 kPa O2 and 80 kPa O2 ND ND ND Negative ND Odriozola-Serrano et

al. (2010)

honey pomelo

4°C, 15d

3.0 kPa O2 + 5.0 kPa CO2 inhibited Positive did not

effectively

Positive ND Li et al. (2012a)

honey pomelo

4°C, 15d

75 kPa O2 inhibited Negative Positive Total

phenolic

content

decreased

significantly

ND Li et al. (2012a)

pear

5°C, 8d

18.2 kPa O2 + 1.0 kPa CO2, 2.0 kPa O2 +

5.1 kPa CO2, and 0.5 kPa O2 + 5.1 kPa

CO2 (balance N2)

ND higher

browning in

18.2 kPa O2

did not

effectively

did not

effectively

ND Gomes et al. (2012)

NE: no significant (P˃0.05) effect (positive or negative) on the quality attribute; ND: not determined.

CHAPTER 1

37

1.3.5 Temperature

Generally, the shelf-life of fresh-cut fruit can be extended at chilling temperatures compared with

storage at ambient temperatures as chilling temperature decrease the respiration rate, enzymatic

browning, nutritional degradation of fresh-cut fruit and the growth of spoilage microorganisms.

Temperature management during the processing, distribution and marketing is therefore the most

significant factor in shelf-life extension of fresh-cut fruit.

The benefits of chill storage for fresh-cut fruit have been reported by many researchers.

Minimally processed fruit (honeydew melon, kiwifruit, papaya, pineapple, and cantaloupe) have

longer shelf-life at 4°C than at temperatures recommended for whole fruit when these were

greater than 4°C (O’Connor-Shaw et al., 1994). The quality of peeled cactus pear fruit can be

maintained at 4°C for 8 days (Piga et al., 2000). Giacalone et al. (2010) found that the shelf-life

of the fruit salad kept at 4°C was longer than that kept at 10°C. Damiani et al. (2008b) reported

that 0°C and 5°C are the most effective temperatures for the maintenance of the quality of fresh-

cut pequi fruit, 10°C and 22°C are less effective. The microbiological quality and potentially

health-promoting attributes of minimally processed oranges were preserved for 12 days at 4°C

(Plaza et al., 2011).

Lightness (L* value) and whiteness index (WI = L* - 3b*) of the fresh-cut fruit flesh stored at 12

± 2°C were significantly lower than those on fruit stored at 4 ± 2°C which was a result of

significant increase in the browning index (Supapvanich et al., 2011a). Pinto et al. (2007)

observed that the temperature of 5°C was indicated to the storage of fresh-cut 'Ponca' tangerine

based in minor changes in colour, titratable acidity, soluble solids, mass loss and juiciness

although losses in vitamin C (65.93%) and β-carotene (42.75%). Supapvanich et al. (2011a) also

found that storage at 4 ± 2°C could reduce change in flesh colour and maintained the peel colour

and nutritional values of fresh-cut wax apple fruit during storage. Storage at 10°C resulted in

accelerated vitamin C degradation, lower phenolic content and lower total antioxidant capacity

than storage at 4°C at the end of the storage period (Giacalone et al., 2010). Those results indicate

that the lower temperature during the storage, the smaller the change in colour and nutritional

values of fresh-cut fruit. Moreover, the larger the temperature and time of storage, the greater the

quality loss for the fresh-cut fruit (Damiani et al., 2008a; Giacalone et al., 2010).

LITERATURE REVIEW

38

1.3.6 Other factors effecting the quality of fresh-cut fruit

1.3.6.1 Cultivar

Fruit cultivars have varied ripening behaviours which differ in such characteristics as external

colour, flesh colour, firmness, seed cavity tissue, soluble solid content, flavour, aroma, and

patterns of carbon dioxide and ethylene production (Miccolis and Saltveit, 1995). The shelf-life

of fresh-cut fruit can therefore differ according to the cultivar. Saftner et al. (2005) reported that

among Fuji, Granny Smith, Pink Lady, and Gold Rush apples, Gold Rush and Pink Lady were

promising new, high-quality apple cultivars for fresh cutting on the basis of flavour, texture,

sensory quality, etc. Likewise, 'Malasia' fruit were the better than Hart and Nota 10 cultivars for

fresh-cut star fruit according to the evaluation of colour, soluble solid content, ascorbic acid

content, etc. (Ogassavara et al., 2009). Hybrid muskmelon is a promising new melon type for

fresh-cut processing and marketing; fresh-cut hybrid muskmelon maintained good quality for 14

days in air at 5°C (Saftner et al., 2009). Ergun et al. (2008) illustrated that among 13 grape

cultivars Big Perlon, Hatun Parmagi and Ribol were found more desirable attributes for minimal

processing and storage at 4°C.

Additionally, the content of vitamins can differ in cultivars resulting in different shelf-life of

fresh-cut fruit. Gonzalez-Aguilar et al. (2008) demonstrated that there is a correlation between

carotene and vitamin C content of 'Ataulfo' mango and its longer shelf-life compared with the

other cultivars ('Keitt' and 'Kent'). Wall et al. (2010) reported that vitamin A content was greatest

in fresh-cut "Laie Gold" and "Rainbow" fruit and averaged 35.1 µg retinol activity equivalents

per 100 g for all cultivars. Among the cultivars resistant to internal yellowing, "Rainbow" had the

highest vitamin A and sugar contents, and did not develop translucency (Wall et al., 2010).

Cultivars differ in their browning and the enzyme activity. As mentioned earlier, browning is one

of the major defects on fresh-cut apples. Kim et al. (1993) reported that among 12 apple cultivars

slices of Cortland, Empire, Golden Delicious, New York 674, and Delicious showed the least

browning after 3 days at 2°C. The main limitation of using fresh-cut oranges is their

susceptibility to juice loss and ascorbic acid degradation because of enzymatic alterations. In a

CHAPTER 1

39

study comparing the enzymatic activity (pectinmethylesterase, polyphenol oxidase and ascorbate

oxidase) and sensory quality of blood-orange cultivars (Moro nucellare, Sanguinello nucellare,

Tarocco arcimusa, Tarocco gallo and Tarocco meli), Tarocco meli was determined to be the

most suitable clone for the production of fresh-cut blood-orange (Catalano et al., 2009).

1.3.6.2 Maturity

Maturity is an important quality attribute of fresh-cut fruit because immature fruit lack good

sensory quality and over-mature fruit (which deteriorate rapidly) have limited shelf-life (Watada

et al., 1999). The effect of the state maturity of pear, apple, honeydew melon and guavas on their

shelf-life as fresh-cut fruit products has been evaluated in several studies.

Bai et al. (2009) reported that about 80% of a sensory panel liked slices from delayed-harvest

pears over commercially harvested pears, especially in terms of visual quality (65 - 85%),

sweetness (75 - 95%), taste (70 - 80%) and overall quality (75 - 80%) during 21 days storage at

1°C. Slightly under-ripe pears exhibited less browning and firmness degradation (Soliva-Fortuny

et al., 2004). Overall, the pears processed at partially ripe maturity can be the most suitable for

fresh-cut.

According to Soliva-Fortuny et al. (2002c), mature-green apples (because of their lower

respiration and ethanol formation) were the most suitable to process for extending post-cutting

shelf-life among the categories of mature-green (77 ± 2.1 N), partially ripe (68 ± 2.3 N), and ripe

(53 ± 3.4 N). However, 'Granny Smith' apple slices that were harvested two weeks early or earlier

than the optimal maturity, as determined by internal ethylene and starch clearing indices, had

significant levels of cut-edge browning despite the post-cutting application of anti-browning dip

( 7 g/L NatureSeal ®; Mantrose-Haueser Co., Inc., Westport, Conn.) and then packaged in zip-loc

bags and held at 5°C (Toivonen, 2008).

Watada et al. (1999) found that the quality of young honeydew melon cubes with 8.8% soluble

solids was lower than that of more mature fruit with 13% soluble solids after a few days of

storage. In contrast, honeydew cubes with 13% soluble solids had good taste/aroma, but

deteriorated more rapidly than those with 11% soluble solids at 10°C (Watada et al., 1999).

LITERATURE REVIEW

40

The harvest point is one of the most important factors for guavas destined to minimal processing

(Pinto et al., 2010). 'Kumagai' and 'Pedro Sato' guavas were harvested at three stages of maturity

stages defined by the skin colour green, light-green and yellowish-green. The light-green and

yellowish-green maturity stages are the most suitable for minimal processing. (Pinto et al., 2010).

1.3.6.3 Effect of shape of cut fruit on the quality of fresh-cut fruit

A handful of studies also investigated the effects of different cut types (e.g. wedge, slice, cube,

rectangular parallelepiped, cylinder and sphere) on the quality of fresh-cut fruit, especially

papaya and guava. Rivera-Lopez et al. (2005) stated that papaya slices stored at 10°C and 5°C

had a shelf-life of 1 day and 2 days longer than papaya cubes, respectively. Similarly, Morais and

Arganosa (2010) also investigated the effect of the cut type (cube, rectangular parallelepiped,

cylinder and sphere) on the quality and shelf-life of fresh-cut papaya (Carica papaya L. cv.

Sunrise Solo) stored at 10°C. They found that the most favourable physico-chemical and

microbiological results were observed in sphere-shaped (1.55 cm radius) papaya. These included

smaller changes in L*, a* (hue angle and chroma colour coordinates), firmer texture, lower rise in

pH, higher values and lower decrease of titratable acidity; reduced weight loss; lowest decrease in

ascorbic acid content; and lower microbial loads (Morais et al., 2010). Consumer preferences

regarding the shape of the cut fruit and packaging of minimally processed guava in a specific area

of the supermarket was investigated by Lima et al. (2010). They reported that the consumers

preferred guava that was cut in halves with pulp and packed in polyethylene terephthalate (PET),

although this packaging promoted condensation of water vapour on the inner surface of the lid,

compromising the appearance of the product. Yellow melon slices were reported to maintain

larger firmness values compared to cubes (Russo et al., 2012).

CHAPTER 1

41

1.4 Instrumental methods for assessing volatile organic compounds of fresh-

cut fruit

Flavour of fresh-cut produce is critical for consumer satisfaction. As it has been described in §

1.2.4, flavour may be influenced by microbial spoilage and the enzymatic reactions of living

tissues of fresh-cut fruit during storage. However, little sensory research has been published on

the flavour quality of fresh-cut fruit or other fresh-cut produce. In contrast extensive research has

been conducted on whole fruit flavour (Soliva-Fortuny et al., 2003b). Therefore, more research is

needed to evaluate the flavour quality of fresh-cut fruit during storage. This section firstly

describes the traditional methods used for the identification and quantification of volatiles of

fresh-cut fruit and thereafter the more recently introduced methods.

1.4.1 Traditional methods for identifying and quantifying volatile organic compounds of fresh-

cut produce

Gas chromatography (GC) is the most used method with several identification and quantification

methods (detectors) possible. The most commonly used detectors are the flame ionization

detector (FID) and the thermal conductivity detector (TCD) (Abdulra'uf et al., 2012; Del Nobile

et al., 2009; Gomes et al., 2012; Nobile et al., 2008). Determining the contribution of individual

volatile compounds to the flavour of fruit requires sensory evaluation of individual compounds to

determine their odour activity, which has been achieved by gas chromatography-olfactometry

(GC-O) (Forney et al., 2009). GC-O combines chemical and sensory analyses. This method

employs a GC with a split column with some flow going to the chemical detector and some to a

sniff port and a ‘‘human’’ sensor. The panellist at the sniff port determines which peaks from the

chemical detector have odour activity and can also rank their intensity. Spanier et al. (1998)

examined the volatiles of fresh-cut pineapple during storage by means of GC-O which showed an

increase of unpleasant odours and volatiles such as fermented, cheesy, sour dough, alcohol, oily,

etc. These volatiles were associated to the growth of yeasts in fresh-cut pineapple during storage.

However, the drawback to this method is that the interactive effects of the volatile compounds

LITERATURE REVIEW

42

with each other and with sugars and acids, both chemically and in terms of human perception,

cannot be determined (Forney et al., 2009).

There are multiple methods for the isolation of volatiles from the headspace (HS) of a food for

identification with gas chromatography–mass spectrometry (GC–MS) (Noseda, 2012). The

analyte(s) is isolated from the food matrix without using a liquid solvent in headspace sampling

techniques (Bicchi et al., 2008). The main aim of HS sampling is to sample the gaseous or vapour

phase in equilibrium with the solid or liquid matrix, in order to characterise its composition.

Traditionally, HS sampling operates either in static (S-HS) or in dynamic mode (D-HS). In the

last two decades, high concentration capacity headspace techniques (HCC-HS), which acts as a

bridge between S-HS and D-HS, have been developed. The immediate success of HCC-HS based

techniques was mainly because they are as simple, fast, and reliable as S-HS, and, at the same

time, show analyte concentration factors comparable to those of D-HS. Several successful

techniques based on the HCC-HS approach are nowadays widely used in addition to conventional

S-HS and D-HS sampling including HS solid-phase micro-extraction (HS-SPME), in-tube

sorptive extraction (INCAT, HS-SPDE), and headspace sorptive extraction (HSSE). Among these

techniques, SPME GC-MS has been frequently applied to extract and identify the volatiles in

some fruit and fresh-cut fruit such as melons (Perry et al., 2009a; Shan et al., 2012; Verzera et al.,

2011) and apples (Rossle et al., 2010). Generally, a homogenate of the fruit flesh is placed in a

vial and a SPME fibre is exposed to the headspace for a considerable period of time before

injection into the GC. The addition of inorganic salts can enhance the activity coefficients of

volatile components in aqueous solutions, increasing their concentration in the headspace. The

selectivity of the volatile extraction is dependent on the choice of the fibre.

Although SPME is a simple and rapid technique, the applicability of SPME is limited by the

small amount of polydimethylsiloxane (PDMS) on the needle (typically less than 0.5 mL) which

results in low extraction efficiencies (Baltussen et al., 1999). Baltussen et al. (1999) suggested

that stir bar sorptive extraction (SBSE) can be a rapid and sensitive alternative to SPME. SBSE

has been applied to extract volatiles from fresh-cut cantaloupe and honeydew melons with

subsequent quantification via GC–MS (Amaro et al., 2012). Amaro et al. (2012) reported that

storage time significantly alters the balance of individual volatiles and volatile classes in

flavourful cantaloupe and in an inodorous honeydew cultivar.

CHAPTER 1

43

Although the methods discussed above have been successfully used for studying the shelf-life of

fresh-cut fruit and vegetables, they are expensive, time-consuming and require considerable

analytical skill (Barrett et al., 2010; Torri et al., 2010). Torri et al. (2010) reported use of the

electronic nose as a fast and reliable alternative method to evaluate the volatiles of fresh-cut fruit.

According to Gardner and Bartlett (1994), the electronic nose is an instrument which comprises

an array of electronic chemical sensors with partial specificity and an appropriate pattern-

recognition system, capable of recognising simple or complex odours. The several advantages of

the application of the electronic nose may include simple and fast approach, non-damaging

analysis, no need for sample preparation, and automatic data collection (Torri et al., 2010). In

their study Torri et al. (2010) showed that the electronic nose was able to discriminate between

several samples and to monitor changes in volatile compounds correlated with quality decay of

fresh-cut pineapple stored at different temperatures.

1.4.2 New technique

Volatiles related to the spoilage of fish and fishery products have been successfully monitored by

means of Selective Ion Flow Tube Mass Spectrometry (SIFT-MS) (Noseda, 2012). SIFT-MS is a

direct mass spectrometric technique based on the chemical ionization of a gas sample (excluding

the major air gases) using specific, selected precursor (reagent) positive ions (Olivares et al.,

2010). Using SIFT-MS, real time quantification of a large number of volatile compounds in

humid air can in principle be achieved without external calibration. The schematic diagram of the

SIFT-MS (Voice 100®) is presented in Figure 1.11. Three reagent ions of most utility for SIFT-

MS measurements (H3O+, NO

+, and O2

+) are generated in a microwave discharge ion source.

These ions are extracted into the upstream chamber and pass through an array of electrostatic

lenses and the upstream quadrupole mass filter and then the selected ionic species are passed into

the flow tube where they are carried along in a stream of helium. The sample is introduced by

means of heated inlet into the flow tube. The chemical ionization of volatile organic compounds

of samples by selected positive precursor ions (H3O+, NO

+, and O2

+) takes place in the flow tube

during a well-defined time period. H3O

+ almost always reacts with the analyte by proton transfer,

O2+ usually reacts by charge transfer or dissociative charge transfer and NO

+ reacts by a number

LITERATURE REVIEW

44

of mechanisms including charge transfer, adduct formation and hydride abstraction. The selected

product ions of these reactions between precursors and volatile compounds are monitored by a

mass spectrometer located at the downstream end of the flow tube. The ion detection optics is

physically separated. The amplitude of the product ion signal provides a quantitative measure for

the amount of volatile compounds in the headspace using the reaction rate coefficients of the

reaction between the precursor ions and the volatiles (Noseda, 2012; Prince et al., 2010). This is

shown in Eq. 1.1 (Syft, 2009).

Figure 1.11: Schematic diagram of the Voice 100® SIFT-MS of precursor production, sample

inlet and ionization and detection. Source: Prince et al., 2010.

CHAPTER 1

45

[ ] [ ]

[ ]

(Eq. 1.1)

Where:

[A] is the analyte concentration in the flow tube

[P+] is the concentration of product ions, measured as counts per second at the detector

[I+] is the concentration of reagent ions, measured as counts per second at the detector

k is the reaction rate co-efficient (a physical constant)

Df is a diffusion and discrimination correction determined for the instrument at the factory

tr is the reaction time

The advantages of SIFT-MS compared to GC-MS are that it avoids the chromatographic

separation of the volatiles which makes it a very fast technique (Noseda, 2012), and volatiles in

the headspace of the samples are directly quantified according to Eq. 1.1. Additionally, for most

applications no sample pre-treatment or pre-concentration is required (Syft, 2009).

Although the quantification of volatiles by means of SIFT-MS has been reported in plants

(Amadei and Ross, 2011), sausage (Olivares et al., 2010), olive oil (Davis and McEwan, 2007;

Davis et al., 2005), fish and fishery products (Noseda et al., 2010a; Noseda et al., 2012; Noseda

et al., 2010b), SIFT-MS cannot always distinguish between isomeric species, such as 1,2- and

1,3-dimethylbenzene (Prince et al., 2010).

LITERATURE REVIEW

46

CHAPTER 2

SPOILAGE ORGANISMS OF FRESH-CUT PINEAPPLE,

HONEYDEW MELON AND APPLE

Chapter 2: Spoilage Organisms of Fresh-cut Pineapple, Honeydew Melon and Apple

48

Summary

The microbial ecology of spoiled fresh-cut pineapple, honeydew melon and apple was evaluated

in this chapter. Primary identification of the spoilage microorganisms were analysed by API

system. Furthermore, a few selected isolates were identified by gene sequence analysis. The

results showed that yeasts were the major contaminants of spoiled pineapple cubes and apple

slices, whilst both yeasts and lactic acid bacteria were dominant microbiota on spoiled honeydew

melon cubes. Primary identification showed that amongst the yeasts the most frequently

occurring species were the Candida spp., more specifically Candida sake, Candida lusitaniae

and Candida parapsilosis. These three were found on all the fresh-cut fruit evaluated in this

study. All lactic acid bacteria identified on spoiled honeydew melon cubes were determined to be

Leuconostoc mesenteroides. The definitive identification of the most frequently occurring

species by gene sequence analysis showed that the spoilage isolates on pineapple cubes were

Meyerozyma caribbica, Candida argentea and C. sake. C. sake, Candida fragi and L.

mesenteroides were found on honeydew melon cubes. The most frequently occurring species on

fresh-cut apple was C. sake.

CHAPTER 2

49

2.1 Introduction

The quality of fresh-cut fruit can be decreased due to the growth and metabolite production of

spoilage microorganisms during storage. However, the predominant spoilage microbiota in fresh-

cut fruit has been investigated by only few studies. Corbo et al. (2004) reported that the

packaging of processed cactus pear fruit in modified atmospheres (65% N2, 30% CO2, 5% O2)

resulted in a homogeneous bacterial population compared to that isolated from fruit stored in air.

Additionally modified atmosphere favoured the growth of Leuconostoc mesenteroides on cactus

pear fruit (Corbo et al., 2004). Poubol et al. (2005b) found that the bacterial flora of 'Nam

Dokmai' mango cubes consisted mostly of Gram-negative rods assigned primarily to

phytopathogenic bacteria such as Pantoea agglomerans and Burkholderia cepacia. The

collection of spoilage isolates from fresh-cut fruit may be useful for the study of the role of

microbial growth on the quality of fresh-cut fruit during storage. The general microbial analysis

has been evaluated on fresh-cut pineapple (Abadias et al., 2008; Di Egidio et al., 2009; Fleet,

1992; Iversen et al., 1989; Liu et al., 2007; Montero-Calderon et al., 2008; Rocculi et al., 2009;

Spanier et al., 1998), honeydew melon (Bai et al., 2003; O’Connor-Shaw et al., 1994; Saftner et

al., 2006; Saftner et al., 2003) and apple (Abadias et al., 2008; Aguayo et al., 2010; Anese et al.,

1997).

The aim of this study was to investigate the microbial ecology of spoiled fresh-cut pineapple,

honeydew melon and apple, to ascertain strains highly associated with spoilage of fresh-cut fruit.

2.2 Materials and methods

2.2.1 Microbiological analysis

Samples of fresh-cut fruit were collected from one of the largest international fresh-cut producer.

2 - 3 samples from different batches of pineapple cubes, honeydew melon cubes and apple slices,

respectively were analysed.

SPOILAGE ORGANISMS OF FRESH-CUT FRUIT

50

The samples were received at the lab and stored at 7°C for at least 120% of their normal shelf-

life. This ensured the products were spoiled at the time of analysis. Thereafter, the

microbiological analysis of the samples was performed. In brief, the samples were opened

aseptically and 10 - 15 g of each sample were collected and placed in a sterile stomacher bag.

Primary decimal dilutions of each sample were prepared by adding an appropriate volume of

physiological peptone saline solution [PPS, 8.5 g NaCl; 1 g peptone per litre, Oxoid (Hampshire,

UK)] and then a serial decimal dilution was prepared in PPS. To determine the yeasts the

decimal dilutions were spread plated on Yeast Glucose Chloramphenicol agar [YGC, Bio-Rad

(Marnes-la-Coquette, France)], whilst the total aerobes and total anaerobes were determined by

pour plating the dilution on Plate Count Agar [PCA, Oxoid (Hampshire, UK)] and Reinforced

Clostridial Agar [RCA, Oxoid (Hampshire, UK)] (with an over-layer) respectively. Lactic acid

bacteria (LAB) were determined by pour plating the decimal dilutions on de Man Rogosa Sharpe

agar [MRS agar, Oxoid (Hampshire, UK)] (with an over-layer). To inhibit the growth of yeasts

on MRS agar, 1.4 g sorbic acid (Sigma–Aldrich, St. Louis, MO, USA) were added per litre of

agar before boiling to adjust the pH of agar to 5.7 (Björkroth and Holzapfel, 2006). The RCA

plates were placed in sealed jars together with Anaerogen sachets (Oxoid, Hampshire, UK) to

create an anaerobic environment. After plating the plates were placed in an incubator at 22°C and

incubated for up to 3 - 5 days.

2.2.2 Identification of spoilage microorganisms

After enumeration was performed, five colonies were randomly and aseptically picked from the

plates for identification per sample. The plates i.e. MRS or YGC agar from which the colonies

were picked were determined i) by their contribution to the total counts and ii) the need to have a

broader idea of what could be present on the products (microbial ecology of products). The

colonies picked from the plates of YGC agar were cultured in 10 mL of sterile Sabouraud Broth

[SB, Oxoid (Hampshire, UK)] whilst the colonies picked from MRS agar plates were cultured in

10 mL of de Man Rogosa Sharpe broth [MRS broth, Oxoid (Hampshire, UK)] at 22 ± 1°C for 2

days. Subsequently, the purity of the isolates was checked through 4×4 looping out on YGC agar

CHAPTER 2

51

or MRS Agar. 0.5mL of each purified isolate liquid culture (SB broth or MRS broth) was

transferred into the sterile cryo tube (1.8mL, Sigma–Aldrich, St. Louis, MO, USA) with sterile

double concentrated medium (SB broth or MRS broth), 30% glycerol solution (Sigma–Aldrich,

St. Louis, MO, USA) and glass beads. After removing the well mixed liquid culture from the

cryo tubes, the cryo tubes of the isolates are maintained in the culture collection of the

Laboratory of Food Microbiology and Food Preservation (Ghent University, Ghent, Belgium).

The primary identification was done by means of the API kits (BioMérieux SA, Marcy l’Etoile,

France). Definitive identification of the selected yeasts was performed at BCCM/MUCL

(BCCM/MUCL Agro (Industrial) Fungi and Yeasts Collection, Louvain-la-Neuve, Belgium)

based on morphological, physiological and molecular (sequencing of the large-subunit rDNA

D1/D2 domain and the internal transcribed spacer, or ITS rDNA) analyses. The definitively

identification of selected LAB was done at BCCM/LMG (Laboratory for Microbiology, Faculty

of Sciences, Ghent University, Ghent, Belgium) used the amplified fragment length

polymorphism (AFLP) analysis. AFLP analysis was based on fingerprint comparison generated

by digestion of the bacterial DNA with the restriction enzymes (TaqI) and (EcoRI) and

subsequent amplification of the fragments with specific primers.

2.3 Results and discussion

It can be seen from Tables 2.1 and 2.2 that the yeasts dominated the ecology of spoiled pineapple

cubes and apple cubes. LAB were not found in spoiled pineapples cubes (<1 × 102 CFU/g) but

were found at low levels of 4 × 10² CFU/g in one of the two samples of spoiled apple slices

analysed. It can be seen in Table 2.3 that in honeydew melon cubes, LAB were the most

dominant part of the spoilage microbiota in two of the three samples evaluated, occurring at high

counts of ca. 108 CFU/g. However, yeasts still occurred at high counts of ca. 10

7 CFU/g and are

therefore also presumed to play an important role in the spoilage of honeydew melon cubes.


52

Table 2.1: Microbial quality of spoiled pineapple cubes and identification of the spoilage isolates of each sample by API.

Samples CFU g

-1

Identified species by API TAC

a TAnC

b Yeasts LAB

c

Batch 1 3.2 x 106 - 3.1 x 10

6

(5)d

<1×102 Candida parapsilosis (2)

e

Kloeckera sp. (doubtful profile)

Candida lusitaniae

Candida parapsilosis (unacceptable

profile)

Batch 2 1.8 x 106 - 1.6 x 10

6

(5)

<1×102 Candida sake (2)

Candida guilliermondii

Kloeckera apsis/apiculata

Candida sake/Candida intermedia

(unacceptable profile)

Batch 3 7.2 x 107 - 5.2 x 10

7

(5)

<1×102 Candida parapsilosis (2)

Candida lusitaniae (2)

Candida guilliermondii (unacceptable

profile)

a TAC = total aerobic counts,

b TAnC = total anaerobic counts,

c LAB = lactic acid bacteria,

d indicates number of colonies selected for

identification from these plates, e indicates the number of colonies identified as a particular species if it occurred 2 times.

CHAPTER 2

53

Table 2.2: Microbial quality of spoiled apple slices and identification of the spoilage isolates of each sample by API.

Samples CFU g

-1


a TAnC

b Yeasts LAB

c

Batch 1 2.4 x 107 - 2.0 x 10

7

(5)

4.0 x 102 Candida sake

Kloeckera spp.

Debaryomyces etchellsii/carsonii

Candida lusitaniae/Candida

tropicalis/Candida parapsilosis (2)

Batch 2 1.6 x 106 - 1.7 x 10

6

(5)d

<1 x 102 Candida parapsilosis (2)

e

Kloeckera japonica

Candida sake

Kodamaea ohmeri/Candida albicans 1

(low discrimination)


b TAnC = total anaerobic counts, ,





54

Table 2.3: Microbial quality of spoiled honeydew melon cubes and identification of the spoilage isolates of each sample by API.

Samples CFU g

-1


a TAnC

b Yeasts LAB

c

Batch 1 1.6 x 108 - 1.2 x 10

6

(2)d

9.2 x 107

(3)

Leuconostoc mesenteroides ssp.

mesenteroides/dextranicum 1 (3) e

Candida sake (2)

Batch 2 2.2 x 107 - 1.1 x 10

7

(3)

3.4 x 106

(2) Candida sake (2)

Cryptococcus albidicus (unacceptable

profile)


mesenteroides/dextranicum 1 (2)

Batch 3 4.7 x 108 - 6.1 x 10

7

(2)

2.8 x 108

(3) Candida sake (2)


mesenteroides/dextranicum 2 (3)


b TAnC = total anaerobic counts, ,




CHAPTER 2

55

Table 2.4: Results of sequence analysis of the most frequently isolated species on the basis of the results determined by API system.

FF code Isolates identified by API system Isolates identified by Sequence analysis

Pineapple cubes

FF 639 Candida parapsilosis Candida sake

FF 604 Candida guilliermondii Meyerozyma caribbica

FF 581 Candida sake Candida argentea

FF 641 Candida lusitaniae Candida sake

Apple slices

FF 649 Candida sake Candida sake

FF 647 Kloeckera spp. Hanseniaspora (Kloeckera) sp.

FF 648 Candida spp. Candida sake

FF 635 Candida parapsilosis Candida sake


Honeydew melon cubes


FF 652 Candida sake Candida fragi

FF 653 Leuconostoc mesenteroides ssp. mesenteroides/dextranicum 1 Leuconostoc mesenteroides

FF654 Leuconostoc mesenteroides ssp. mesenteroides/dextranicum 1 Leuconostoc mesenteroides

FF 657 Leuconostoc mesenteroides ssp. mesenteroides/dextranicum 1 Leuconostoc mesenteroides


56

The primary identification of the isolates according to the results of API system showed that

amongst the yeasts the most frequently occurring species were the Candida spp. More

specifically, Candida sake was the most frequently occurring strain and was found on all three

types of fruit analysed (see Tables 2.1 - 2.3); Candida lusitaniae and Candida parapsilosis were

mostly identified on pineapple cubes and apple slices. All LAB identified on honeydew melon

cubes were determined to be Leuconostoc mesenteroides.

On the basis of the identification by API system, the most frequently isolated species of fresh-cut

pineapple, apple and honeydew melon were further identified by gene sequence analysis. It can

be seen in Table 2.4 that four isolates on spoiled pineapple cubes were determined to be

Meyerozyma caribbica, Candida argentea and C. sake while five spoilage isolates of C. sake,

Candida fragi and L. mesenteroides were found on spoiled honeydew melon cubes. The most

frequently occurring species on fresh-cut apple were C. sake. These isolates could be used to

study the relationship between microbial spoilage and the shelf-life of fresh-cut fruit which has

been described in Chapters 3 and 4.

Generally, our results of the microbial quality are in agreement with the previous findings as

discussed in §1.2.1 of Chapter 1. The microbiota found on the cut surface of fresh-cut fruit is

diverse, but it generally includes a variety of fungi (yeasts and moulds) and mostly harmless

bacteria (Garcia et al., 2005). The growth of yeasts is generally a major problem for companies

that process fruit (Fleet, 1992), due to their low pH, high sugar content and high humidity

environment of most fresh-cut fruit. Our results are in agreement with Iversen et al. (1989), Fleet

(1992) and Spanier et al. (1998) who reported that yeasts are the predominant factor causing the

spoilage of fresh-cut pineapple. In contrast, large populations of bacteria as well as yeasts and

moulds have been also found on fresh-cut pineapple during refrigerated storage previously (Di

Egidio et al., 2009; Liu et al., 2007; Montero-Calderon et al., 2008; Rocculi et al., 2009). Both

yeasts and LAB have been found on fresh-cut honeydew melon during storage (Bai et al., 2003;

O’Connor-Shaw et al., 1994; Saftner et al., 2006; Saftner et al., 2003). However, moulds were not

found on fresh-cut fruit evaluated in this study. This could have been due to the fact that yeasts

grow faster than moulds on fresh-cut fruit (Heard, 2002).

CHAPTER 3

EFFECT OF REDUCED HEADSPACE OXYGEN LEVEL ON

GROWTH AND VOLATILE METABOLITE PRODUCTION BY

THE SPOILAGE MICROORGANISMS OF FRESH-CUT

PINEAPPLE AND HONEYDEW MELON

Chapter 3: Effect of Reduced Headspace Oxygen Level on Growth and Volatile Metabolite

Production by the Spoilage Microorganisms of Fresh-cut Pineapple and Honeydew Melon

Parts of this chapter have been redrafted from:

Zhang, B.-Y., Samapundo, S., Rademaker, M., Noseda, B., Denon, Q., de Baenst, I., Sürengil, G.,

De Baets, B., Devlieghere, F. 2014. LWT-Food Science and Technology 55, 224-231.

REDUCED OXYGEN LEVELS ON FRESH-CUT FRUIT

58

Summary

The effect of initial headspace (IH) O2 level (0 - 21%, balance N2) on the shelf-life of fresh-cut

pineapple and honeydew melon was evaluated in this chapter. In the first part, the effect of IH O2

levels on growth and volatile metabolite production of different spoilage organisms on pineapple

agar and honeydew melon agar was evaluated at 7°C. In the second part, the effect of selected IH

O2 level(s) on the microbiological, physical and sensory quality of commercial fresh-cut

pineapple and honeydew melon was evaluated (separately) at 7°C. The results showed that the

IH O2 level had a minor effect on the growth of yeasts and lactic acid bacteria on fruit simulation

agar. The quantities of volatile metabolites in the headspace produced by yeasts during

incubation were determined to be generally smaller the lower the IH O2 level on both pineapple

agar and honeydew melon agar. However, the trend was not clear for the volatile metabolites

produced by lactic acid bacteria on honeydew melon agar. The only exception was that the final

quantity of butane-2.3-dione produced by lactic acid bacteria was higher in air than in other

atmospheres. Pineapple cubes packaged in IH 5% O2 were significantly different in sensory

characteristics to those packaged in IH 21% O2 (P < 0.05) on day 5 of storage. The sensory panel

generally preferred the samples packaged in IH 5% O2. This was probably due to better visual

quality and lower amounts of the volatile metabolites in the headspace. Although honeydew

melon cubes packaged in IH 5% O2 also had considerable lower headspace volatile organic

compounds and slightly better colour retention than those in air, no significant difference in

sensory characteristics was found between honeydew melon cubes packaged in IH 5% O2 and air.

The results imply that packaging in an IH O2 level of 5% in high gas barrier films can be used to

extend the shelf-life of fresh-cut pineapple, but this extension is very limited for fresh-cut

honeydew melon.

CHAPTER 3

59

3.1 Introduction

As it has been described in Chapter 1 (§ 1.2.1.1), yeast spoilage is characterized by the

fermentation of carbohydrates to produce CO2, alcohol, flavour and off-flavour compounds, such

as acids, esters and higher alcohols (Heard, 1999; Heard, 2002). Nevertheless, different fresh-cut

produce seem to undergo different spoilage patterns in relation to the characteristics of the raw

materials (Ahvenainen, 2002; Huxsoll and Bolin, 1989). With the exception of Spanier et al.

(1998) who reported that alcohols were derived from native flora yeast fermentation during the

storage of fresh-cut pineapple, no further literature could be found about the volatile organic

compounds (VOCs) produced by spoilage microorganisms during the storage of fresh-cut

pineapple. Amaro et al. (2012) evaluated the aroma volatiles of fresh-cut honeydew melon in

passive modified atmosphere package (MAP) and low O2 controlled atmospheres with 5kPa O2 +

10kPa CO2, while the effect of microorganism growth and their volatile metabolites was not

considered in this study.

Spoilage microorganisms cannot be eliminated during the processing and storage of fresh-cut

fruit. Modified atmospheres (MAs), which could be considered as supplementary to temperature

control and disinfection processes (Varoquaux and Ozdemir, 2005), have been studied recently at

lowered levels of O2 (2 - 6%) with regards to their effect on microbial growth, shelf-life, physical

and physiological aspects (such as colour, texture, respiration rate, etc.) on fresh-cut pineapple

(Liu et al., 2007; Martinez-Ferrer et al., 2002; Singh et al., 2007) and fresh-cut honeydew melon

(Amaro et al., 2012; Bai et al., 2003; Oms-Oliu et al., 2008a; Portela et al., 1998; Qi et al., 1999;

Saftner et al., 2006; Saftner et al., 2003). As it has been discussed in § 1.3.4.1 of Chapter 1, a

lowered O2 MA may increase the shelf-life of packaged produce by reducing respiration rates,

delaying senescence of living tissues, and inhibiting the growth of spoilage microorganisms (Al-

Ati et al., 2003).

To date no studies have been performed to evaluate the growth and volatile metabolite

production of spoilage microorganisms of fresh-cut pineapple and fresh-cut honeydew melon.

Volatile organic compounds (VOCs) may have a strong effect on the unique flavour

characteristics of fruit which determine their desirability to the consumer (Amaro et al., 2012;


60

Forney et al., 2009). Only Ragaert et al. (2006b) have evaluated the volatile metabolite

production of spoilage yeasts on strawberry agar.

The aim of this study was to investigate the effect of initial headspace O2 (IH O2) level on

growth and volatile organic metabolite production of spoilage organisms responsible for the

spoilage of fresh-cut pineapple and honeydew melon on sterile pineapple agar and honeydew

melon agar separately. A shelf-life study on modified atmosphere packaged fresh-cut pineapple

cubes and honeydew melon cubes was performed in order to evaluate the effect of the headspace

O2 level on growth and volatile metabolite production of real fruit parts with their natural flora.

Additionally, the effect of the headspace O2 level on the sensory quality and physical parameters

was evaluated.

3.2 Materials and Methods

3.2.1 Effect of IH O2 level on the growth of spoilage isolates

3.2.1.1 Isolates

To investigate the effect of headspace O2 level on growth and volatile metabolite production of

spoilage isolates on fresh-cut pineapple and honeydew melon, the spoilage organisms isolated in

Chapter 2 from spoiled commercial fresh-cut pineapple [Candida argentea (FF 581), Candida

sake (FF 641) and Meyerozyma caribbica (FF 604)] and fresh-cut honeydew melon [Candida

sake (FF 655) and Leuconostoc mesenteroides (FF 653)] were used in this study. These isolates

are maintained in the culture collection of the Laboratory of Food Microbiology and Food

Preservation (Ghent University, Ghent, Belgium).

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61

3.2.1.2 Preparation and inoculation of fruit agar

Pineapple agar and honeydew melon agar were used as a simulation medium for fresh-cut

pineapple and fresh-cut honeydew melon, respectively. Pure pineapple juice (Materne, Belgium)

supplemented with 1.5% Bacteriological Agar [Oxoid (Hampshire, UK)] was boiled in Schott

bottles over a Bunsen burner flame for two minutes. Thereafter it was cooled in a water bath at

48°C. Subsequently, 20 ± 0.2 g of pineapple agar was then poured into petri plates. While

honeydew melon juice was made by blending fresh-cut honeydew melon pieces (Braun

Mr530multi Quick Heavy Duty Hand Blender, Braun, Kronberg, Germany) and squeeze-filtering

the juice through a towel. The filtered honeydew melon juice was then added with 1.5%

Bacteriological Agar and honeydew melon agar plates were made by using the same procedure

as pineapple agar plates. The initial water activity (aw) and pH of the fruit agar were then

measured in triplicate by means of aw-kryometer (NAGY, Gaeufelden, Germany) and a

SevenEasy pH meter (Mettler Toledo GmbH, Schwerzenbach, Switzerland), respectively. The aw

and pH of the simulation agar did not differ significantly (P < 0.05) from that of the fruit juices.

Spoilage organisms isolated from commercial fresh-cut pineapple and honeydew melon were

individually inoculated on pineapple agar and honeydew melon agar, respectively. To prepare

the inoculum for the growth experiments, yeasts were sub-cultured in 10 mL of sterile Sabouraud

Broth [SB, Oxoid (Hampshire, UK)] whilst the lactic acid bacteria were sub-cultured in 10mL of

de Man Rogosa Sharpe broth [MRS broth, Oxoid (Hampshire, UK)] at 22 ± 1°C for 2 days.

Second sub-cultures were prepared in the same type of culture as the first and incubated for the

same duration at 22 ± 1°C before being transferred to a refrigerator at 7 ± 1°C for 7 hours to

adapt the spoilage isolates to the final incubation temperature used in the experiments. 100 µL

aliquots of an appropriate dilution of the temperature adapted isolates were separately inoculated

and spread on the simulation medium resulting in an initial inoculation level of 102-3

CFU/cm² of

agar. Inoculated plates were singly packaged in a high O2 barrier film (O2 transmission rate = 2

cm3/m

2.d.bar at 23°C, 90% relative humidity (RH), NX90, Euralpack, Wommelgem, Belgium)

under the following initial atmosphere conditions: 0% O2, 1% O2, 3% O2, 5% O2 and IH 21 % O2

(air), balanced with N2 and stored at 7 ± 1°C. The packaging machine consisted of a


62

MULTIVAC A300/42 packaging machine (Sepp. Haggenmüller KG, Wolfertschwenden,

Germany) combined with a gas mixing unit (WITT, Vilvoorde, Belgium). For each packaged

plate, a fruit agar to gas volume ratio of 1/4 (v/v) was used.

Based on trial experiments (data not shown), sample analysis was planned on the day of

inoculation itself (day 0) and on days 2, 4, 6 and 8 for most isolates and days 2, 4, 6, 9, 12 and 15

for M. caribbica. During packaging, six plates were packaged per spoilage isolates per condition

per analysis day. Two plates were used for identification of volatile organic compounds (VOCs)

by GC-MS and a further two were for quantification of the VOCs by Selected Ion Flow Tube -

Mass Spectrometry (SIFT-MS, Voice 200, Syft Technologies Ltd, New Zealand). The other two

plates were used for measurement of the pH, headspace O2 and CO2 levels and assessment of the

number of yeasts. The headspace O2 and CO2 levels were measured by a headspace analyser

(PBI - Dansensor, CheckMate 9900 O2, O2/CO2 Headspace Analyzer, Denmark).

3.2.1.3 Identification of volatile metabolites by GC-MS

The identification of the headspace volatile organic compounds (VOCs) was carried out by

headspace GC-MS (Agilent 7890A Gas Chromatograph and an Agilent 5975C - Inert XL Mass

Selective Detector with CombiPAL autosampler). Separation of the VOCs occurred on a DB -

WAX 122 - 7062 capillary column (60 m × 250 µm × 0.25 µm, J&W Scientific, Agilent

Technologies, USA). The identification of the VOCs was carried out on the basis of the retention

time and by comparison of the resultant mass spectra from each sample with the mass spectra

from the library database (NIST 2005). On each day of sample analysis, 7 g of agar were

aseptically collected from each plate and put in a 20 mL glass vial closed with a PTFE-faced

septum screw cap (Agilent Technologies, Diegem, Belgium) and then stored in a freezer at -18°C

until analysis. On the day the VOCs were actually identified, the frozen samples were first

thawed by placing them in a refrigerator at 4°C for at least 2 hours. The thawed samples were

then incubated at 80°C for 10 min before 1000 µl of headspace was injected into the GC. The

GC oven temperature program used was as follows: 30-90°C at 4°C min-1

, hold at 90°C for 2

min, 90-130°C at 3°C min-1

, 130-150°C at 5°C min-1

, 150-220°C at 40°C min-1

, hold at 220°C

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63

for 5 min, increase to a final temperature of 250°C for 2 min as a post run. The identified VOCs

were used to develop a method for their quantification using the Selected Ion Flow Tube - Mass

Spectrometry (SIFT-MS, Voice 200, Syft Technologies Ltd, New Zealand).

3.2.1.4 Quantification of volatile metabolites by SIFT-MS

The VOCs were quantified by SIFT-MS, based on the method developed by Noseda (2012).

Samples (20 ± 0.2g) intended for the quantification of the VOCs were stored in a freezer at -

18°C until the analysis date. Duplicate samples were collected per condition evaluated. On the

day of analysis each sample was initially repackaged in 0.9L of N2 by the MULTIVAC

packaging machine and stored at 4°C for at least 2 hours, to thaw and allow the liquid and gas

phases to equilibrate (Noseda et al., 2010b). Repackaging was done in bags made from a high O2

barrier film (O2 transmission rate = 2 cm3/m

2.d.bar at 23°C, 90% relative humidity (RH), NX90,

Euralpack, Wommelgem, Belgium).

The multiple ion monitoring (MIM) mode was used to target VOCs. The scanning time was set

at 60 seconds. Thereafter, the VOCs were selected from the data base of the software and scan

masses were calculated for each VOC. The ionized masses used for quantification of the VOCs

produced by yeasts and lactic acid bacteria are shown in Table 3.1 and Table 3.2, respectively.

The VOCs were selected based on the headspace static GC-MS results (see § 3.2.1.3), in

combination with other compounds reported in literature to be related to the spoilage of fresh-cut

fruit (Bai et al., 2003; Spanier et al., 1998).

Although an m/z signal (43) was used for quantification of both propan-1-ol and ethyl acetate,

the characteristic product ions for these two compounds were detected separately due to these

two compounds ionized by different precursors. A possible overestimation of the concentration

of 2-methylbutanal was possible as its characteristic product ion has a similar m/z (87) as the

characteristic production ion of 3-methylbutanal. The latter was quantified with another non-

overlapping characteristic product ion. However, the product masses (i.e. 132) of 2-


64

methylbutanoic acid and 3-methylbutanoic acid completely overlapped. Hence, the reported

concentrations are a sum of the concentrations of these two compounds.

At the beginning of each day on which analytical work was performed with the SIFT-MS, an

automated check test was executed to ensure that the instrument is performing correctly. The test

comprises evaluation of the flow, temperature, and quadrupole performance, and single-point

accuracy and precision determinations using a certified standard containing benzene,

ethylbenzene, ethylene, hexafluorobenzene, isobutene, octafluorotoluene, perfluorohexane,

perfluoro-2-methyl-2-pentene, perfluoroheptane, and octadecafluorooctane. Thereafter, the

method developed above was transferred to the instruments database and applied to quantify the

targeted VOCs. VOCs are introduced through the heated inlet into the flow tube, where reactions

with precursor ions - H3O+, NO

+, O2

+ - result in ionized masses. The ionized masses are then

monitored by a mass spectrometer, located at the downstream end of the flow tube. The ratio of

the product ion count to the precursor ion count provides a quantitative measure for the amount

of VOCs in the headspace as shown in Eq. 1.1 (see § 1.4 of Chapter 1). A headspace sample

volume of 125 ± 50mL is drawn up during a period of 60 seconds by the SIFT-MS through a

septum placed on the sampling bag.

During analysis, 10 empty sample bags filled with 0.9L of N2 were analyzed randomly between

the other samples and were considered as blank samples. The data was exported by using

LabSyft Data Viewer (Syft Technologies Ltd, New Zealand). Quantification limits (LOQ) for

every compound evaluated was calculated based on the average of 10 blank samples (xbl) plus six

times the standard deviation (SDbl) (see Eq. 3.1).

LOQ = xbl + 6SDbl (Eq. 3.1)

The reported concentrations were adjusted for the background concentrations. The reported

concentrations are concentrations > LOQ minus the xbl.

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65

Table 3.1: Volatile organic compounds identified by GC-MS in the headspace of fruit agar inoculated

with spoilage yeasts and spoiled commercial fresh-cut fruit and quantified by SIFT-MS, with

characteristic product ions, the coinciding precursor ions, mass-to-charge ratio (m/z), branching ratio (b)

and reaction rate coefficient (k). The corresponding human olfactory threshold (OT) was reported by

Devos (1990).

VOC’s Precursor m/z b (%) k Characteristic

product ion

OT

(mg/m3)

Alcohols

ethanol O2+ 45 75 2.30×10

-09 C2H5O

+ 54.95

3-methyl-1-butanol O2+ 59 85 2.10×10

-09 C3H7O

+ 0.16

butan-1-ol O2+ 56 80 2.50×10

-09 C4H8

+ 1.51

2-methyl-1-butanol O2+ 28 5 2.30×10

-09 C2H4

+ 0.85

pentan-1-ol NO+ 87 85 2.50×10

-09 C5H11O

+ 1.70

propan-1-ol H3O+ 43 90 2.70×10

-09 C3H7

+ 6.03

NO+ 59 100 2.30×10

-09 C3H7O

+

Esters

ethyl acetate NO+ 43 10 2.10×10

-09 CH3CO

+ 9.77

ethyl octanoate H3O+ 173 90 3.00×10

-09 C10H21O2

+ 0.004

ethyl butanoate O2+ 116 10 2.50×10

-09 C6H12O2

+ 0.11

3-methylbut-1-yl

ethanoate NO

+ 160 100 2.50×10

-09 C7H14O2.NO

+ -

ethyl hexanoate H3O+ 145 100 3.00×10

-09 C8H16O2.H

+ 0.01

NO+ 174 95 2.50×10

-09 C8H16O2.NO

+

Acids

phenylacetic acid NO+ 91 50 2.50×10

-09 C7H7

+ 0.59

hexanoic acid H3O+ 99 25 3.00×10

-09 C6H11O

+ 0.06

propanoic acid NO+ 104 70 1.50×10

-09 NO

+.C2H5COOH.H2O 0.11

2-methylbutanoic acid NO+ 132 65 2.50×10

-09 C5H10O2.NO

+ 0.01

3-methylbutanoic acid NO+ 132 70 2.50×10

-09 C5H10O2.NO

+ 0.01

pentanoic acid O2+ 60 80 2.40×10

-09 CH3COOH

+ 0.02

Aldehydes

nonanal O2+ 69 10 3.20×10

-09 C5H9

+ 0.01

2-methylpropanal O2+ 72 70 3.00×10

-09 C4H8O

+ 0.12

3-methylbutanal O2+ 44 35 2.40×10

-09 C2H4O

+ 0.01

2-methylbutanal H3O+ 87 94 3.70×10

-09 C5H10O.H

+ -

3.2.1.5 Growth assessment

As mentioned earlier, the growth of the spoilage isolates was assessed in duplicate. The

headspace O2 and CO2 levels in two packaged plates were first measured. Subsequently, the


66

packages were opened and the fruit agar was divided aseptically in two. One half was used for

the measurement of the pH and the other half was used for assessment of the counts of spoilage

isolates. The piece of agar that was used for assessment of the counts of spoilage isolates was

aseptically transferred into a sterile stomacher bag and primary decimal dilutions of each sample

were prepared by adding an appropriate volume of physiological peptone saline solution [PPS,

8.5g NaCl; 1g peptone per litre, Oxoid (Hampshire, UK)]. The samples were homogenized for

30 seconds in a stomacher (Stomacher Lab-Blender 400, Led Techno, Eksel, Belgium).

Subsequent decimal dilutions were then prepared from the primary decimal dilution in test tubes

containing 9mL of sterile PPS. The decimal dilutions were then spread plated on Yeast Glucose

Chloramphenicol agar [YGC, Bio-Rad (Marnes-la-Coquette, France)] plates for the yeast or pour

plated on de Man Rogosa Sharpe agar [MRS agar, Oxoid (Hampshire, UK)] for the LAB. The

plates were then incubated at 22 ± 1°C until the colonies were sufficiently large enough for

enumeration (ca. 2 days). The counts (n) of the isolates on fruit agar were expressed in log10

CFU/cm2. Based on the surface area (57.6 cm²) of the agar petri dishes on which the inoculation

was done and the weight of the agar on the plates (20 ± 0.1g), conversion between log10

CFU/cm2 and log10 CFU/g could be done according Eq. (3.2):

n Log10 CFU/cm2 = (n + 0.45) Log10 CFU/g (Eq. 3.2)

Table 3.2: Volatile organic compounds identified by GC-MS in the headspace of fruit agar inoculated

with lactic acid bacteria and quantified by SIFT-MS, with characteristic product ions, the coinciding

precursor ions, mass-to-charge ratio (m/z), branching ratio (b) and reaction rate coefficient (k). The

corresponding human olfactory threshold (OT) was reported by Devos (1990).

VOC’s Precursor m/z b (%) k Characteristic

product ion OT (mg/m

3)

butane-2,3-diol H3O+ 91 100 3.00×10

-09 C4H10O2.H

+

NO+ 89 100 2.30×10

-09 C4H9O2

+

butane-2,3-dione H3O+ 87 100 1.70×10

-09 C4H7O2

+ 0.02

acetic acid NO+ 90 100 9.00×10

-10 NO

+.CH3CHOOH 0.36

O2+ 60 50 2.30×10

-09 CH3COOH

+

acetoin H3O+ 89 100 3.00×10

-09 C4H8O2.H

+

NO+ 118 100 2.50×10

-09 C4H8O2.NO

+

ethanol H3O+ 47 100 2.70×10

-09 C2H7O

+ 54.95

NO+ 45 100 1.20×10

-09 C2H5O

+

CHAPTER 3

67

3.2.1.6 Sugar analysis

The concentrations of sucrose, glucose, fructose, lactic acid and acetic acid in fruit agar samples

were determined according to the HPLC method developed by Ragaert et al. (2006b). For the

extraction, 10g of fruit agar was homogenized for 30 seconds with 10g of distilled water. After

filtration (Ø125 mm, Schleicher & Schwell, Dassel, Germany), the filtrate was held at 80°C for

15 min to denature proteins including enzymes. After centrifugation for 15 min at 8050 g

(Centrifuge 5415C, Eppendorf, Hamburg, Germany) and filtration through a 0.2 µm filter

(HPLC Syringe filter; GRACE, Deerfield IL, USA), 200 µl of the filtrate was injected in the

HPLC coupled to a RI detector (Refractive Index Detector 133, Gilson). The HPLC consisted of

a pump (Pump 307, Gilson, Wisconsin, USA), an injector (Rheodyne 9096, Bensheim,

Germany), a pre-column (MetaCarb 87H Guard cartridge, Varian, USA), a column (MetaCarb

87H, length 300 mm, ID 7.8 mm, Varian, USA) with column oven (35°C) (Model 7990, Jones

Chromatography, California, USA) and a RI detector. 0.005 M H2SO4 (VWR, Leuven, Belgium)

was used as mobile phase at a flow rate of 0.6 mL/min.

The concentrations of the sugars and acids were calculated using standard curves for sucrose

(UCB, Leuven, Belgium), glucose (Sigma–Aldrich, St. Louis, MO, USA), fructose (Sigma–

Aldrich, St. Louis, MO, USA), lactic acid (Sigma–Aldrich, St. Louis, MO, USA) and acetic acid

(Sigma–Aldrich, St. Louis, MO, USA) which were made by adding each of these compounds in

different known concentrations to distilled water.

3.2.2 Effect of IH O2 level on the quality of fresh-cut fruit

The growth and VOC production of the natural spoilage flora of commercial fresh-cut pineapple

was evaluated in IH O2 levels of 21% (the atmosphere currently used by the commercial

processor who supplied fresh-cut pineapple and the reference condition for the statistical tests), 5%

and 1%, balance N2. Commercial honeydew melon cubes in IH O2 levels of 21% and 5% were


68

evaluated. Triangle tests were performed to determine the effect of the IH O2 level on the

sensory quality. Additionally, colour, juicy leakage, firmness and juiciness were also evaluated.

Fresh-cut pineapple cubes or honeydew melon cubes (1 ± 0.2 cm thick, 7 - 9g, each) used in the

experiment were delivered to our laboratory within three hours of processing by a commercial

processor. 120 ± 1g of fruit cubes were aseptically transferred into trays (volume = 443 mL,

OTR = 0.5 - 13 cm3/m

2.d.bar at 23°C, 0% RH, polypropylene (PP)/ethylene vinyl alcohol

(EVOH), DECAPAC NV, Herentals, Belgium) before being sealed in the desired atmospheres.

A high O2 barrier film (OTR = 5 cm3/m

2.d.bar at 23°C, 50% RH. OPA (oriented

polyamide)/EVOH/PE (polyethylene)/PP, BEMIS EUROPE Flexible Packaging, Monceau-sur-

Sambre, Belgium) was used to seal the trays by means of a Tray sealer (MECA 900, DECAPAC

NV, Belgium). The trays were then stored at 7°C for up to five days. Two trays per headspace

condition were randomly selected at each sampling date for headspace gas composition analysis,

VOCs measurement, pH and microbiological analysis. 12 trays of air packed fresh-cut pineapple

cubes or honeydew melon cubes and six trays of each modified atmosphere packed pineapple

cubes or honeydew melon cubes were randomly chosen for sensory evaluation on each analysis

date.

3.2.2.1 Microbial quality of fresh-cut fruit

The microbiological quality (total aerobic counts, total anaerobic counts, yeasts and lactic acid

bacteria) of the fresh-cut fruit samples packaged in the different IH O2 levels was determined in

duplicate on days 0, 1, 3, 5 of storage. As done in the experiments on fruit agar, the headspace O2

and CO2 levels in two packages per conditions were first measured. Subsequently, the packages

were opened and samples were aseptically collected for measurement of the pH (ca. 15g) and the

microbiological quality (ca. 30g). The samples for assessment of the microbiological quality

were transferred to a sterile stomacher bag and primary and subsequent decimal dilutions were

prepared in PPS as described above for the experiments on fruit agar. To determine the yeasts the

decimal dilutions were spread plated on YGC, whilst the total aerobes and total anaerobes were

determined by pour plating the dilution on Plate Count Agar [PCA, Oxoid (Hampshire, UK)] and

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69

Reinforced Clostridial Agar [RCA, Oxoid (Hampshire, UK)] respectively. LAB were determined

by pour plating the dilution on MRS agar by adding 1.4g/L Sorbic Acid (Sigma–Aldrich, St.

Louis, MO, USA) to adjust the pH of media to 5.7 before boiling. The plates were then incubated

at 22 ± 1°C until the colonies were sufficiently large for enumeration (ca. 3 days).

3.2.2.2 Volatile organic compound and sugar contents of fresh-cut fruit

On each day of sample analysis, 20g of fresh-cut fruit cubes were aseptically collected from each

tray and put in a 60 mL plastic container and then stored in a freezer at -18°C until analysis.

VOCs quantification by SIFT-MS was described previously (see § 3.2.1.4).

The samples used for sugars (ca. 20 g each) were thoroughly homogenized in a Stomacher bag

with an equal amount of distilled water for 2 min. Further preparation of the samples as well as

the analysis with HPLC was performed as is described in § 3.2.1.6.

3.2.2.3 Assessment of colour and juice leakage

Juice leakage from pineapple or honeydew melon cubes was measured according to the method

of Montero-Calderon et al. (2008). In brief, the packages were tilted at a 20° angle for 5 min after

which the accumulated liquid was recovered with a 5-mL syringe. Results were reported as

liquid volume (mL) recovered per 100g of fresh-cut fruit in the package.

The colour of the fruit cubes was determined on 5 randomly chosen points on the surface of each

randomly chosen cube with a Spectrophotometer Minolta Model CM-2500d (Konica Minolta

Sensing Inc., Osaka, Japan). Zero calibration and white calibration were performed before the

measurements were taken. Each reading consisted of L* (lightness), a* (green chromaticity) and

b* (yellow chromaticity) coordinates. 6 pieces of pineapple cubes were measured from each pair

of trays for each MA condition. For the reference (day 0), the concept of MA is not applicable,

meaning the same two replicates will be used to compare to the different MAs.


70

3.2.2.4 Sensory quality of fresh-cut fruit

As mentioned above, triangle tests were used to determine the effect of the headspace O2 level

on the sensory quality of fresh-cut fruit cubes. The procedures followed were according to the

BS ISO 4120:2004 (Sensory analysis – Methodology – Triangle test) standard method (ISO,

2004). The tests evaluated potential differences between samples packaged in IH O2 of 21%

(reference condition) to those packaged in IH O2 levels of either 1 or 5%. In particular, the visual

appearance, flavour and odour were evaluated. The sensory evaluation of fresh-cut pineapple

was performed on the same sampling days at Lavetan NV (Turnhout, Belgium) by a test panel

with 11 - 13 panellists who have experience in the sensory evaluation of fresh-cut pineapple

from the same processor who provided our samples. The sensory evaluation of fresh-cut

honeydew melon was performed in a purpose built sensory analysis room with isolated booths

(UGent Sensolab, Ghent University) by 15 untrained assessors (age range: 24 -– 44 years,

average age: 29 years, 8 female assessors and 7 male assessors). Honeydew melon cubes (ca.

25g) were transferred into plastic containers labelled with three-digit number codes. The plastic

containers were closed and kept at 7°C for one hour before the triangle tests were performed. For

each test three coded samples, of which one was different from the other two, were presented to

the assessors. Additionally, water was provided for cleansing the palate and the assessors

recorded their responses on paper scorecards. Each assessor was asked to identify the different

sample based on colour, odour and taste. The assessor were instructed to only assess the taste of

the samples if they thought the colour or the odour were still acceptable. When the assessors

finished the triangle test, they were asked to select the sample(s) they preferred and to indicate

the reasons of why they considered a sample to be unacceptable or preferable. The test was

always performed between 10.30am-11.30am in a single repetition.

Firmness and juiciness of fresh-cut pineapple and honeydew melon were evaluated on day 0

(initial condition), day 3 and day 5 by 15 assessors who were the same group assessors as

triangle test in fresh-cut honeydew melon. Samples were coded with three-digit number and

presented to assessors in randomized order. The samples were presented under red-light to mask

the colour the samples. Water was used to cleanse the palate between samples. Firmness and

juiciness were scored separately on a 5 point scale where 1 = very soft (mushy) / very dry, 2 =

CHAPTER 3

71

soft / dry, 3 = neither firm nor soft / neither juicy nor dry, 4 = firm / juicy, 5 = very firm / very

juicy. Firmness and juiciness were always evaluated between 10.30am-11.30am in single

repetition. The assessors recorded their responses on paper scorecard.

3.2.3 Statistical analysis

An individual package tray was used as one replicate on each sampling day, per treatment. The

results of triangle tests were analysed according to BS ISO 4120:2004 (ISO, 2004). For 11, 13

and 16 assessors, the critical number of correct responses required to obtain a statistically

significant difference (P < 0.05) are 7, 8 and 9 respectively.

The significance of the effect of IH O2 level on the growth characteristics of the yeasts was

assessed by a permutation test based on concordant pair counts, as in a Kendall’s rank correlation

(Kendall, 1938). We constructed all possible CFU count rankings for 10 experiments and

computed the corresponding rank correlation between each of these rankings and the O2 ranking.

Remark that if there is no effect of O2, each of these rankings is equally likely to occur. In this

way, we were able to compute that the probability of randomly obtaining rank correlations

equally strong as or stronger than those observed in our experiments is well below 0.05 for each

of the yeasts. To determine the effect of storage time and MAs on colour and juice leakage,

proper ANOVA contrasts were used, with significant differences being established at P < 0.05.

Significant differences in juiciness and firmness were analysed by Kruskal-Wallis test due to the

ordinal nature of these data. The permutation test was performed in MatLab (MatLab R2012b,

MathWorks, USA), the other statistical analyses were performed in SPSS version 21(SPSS,

Chicago, IL, USA). Where applicable, the 21% O2 packages functioned as the reference

condition by employing suitable contrasts in the statistical analyses.

http://en.wikipedia.org/wiki/MathWorks


72

3.3 Results and Discussion

3.3.1 Effect of initial headspace O2 level on growth of spoilage isolates

As it can be seen in Figure 3.1, all the spoilage yeasts of fresh-cut pineapple grew immediately

on pineapple agar (no observable lag phase), irrespective of the IH O2 level. According to the

permutation test, the IH O2 level appears to have a small but significant effect (P < 0.05) on the

growth rate of the yeasts, with all yeasts most likely growing faster in higher IH O2 levels from

day four onwards. As an example, M. caribbica reached 5 log10 CFU/cm², the level at which

yeasts are generally known to start causing spoilage (Fleet, 1992), after 10 and 7 days of

incubation in IH O2 levels of 0 and 21%, respectively. Although the growth of C. argentea and C.

sake was less influenced by the IH O2 level than that of M. caribbica, slightly higher maximum

population densities of both C. argentea and C. sake were achieved at higher IH O2 levels than

those at lower IH O2 levels after 6 days incubation. Hence, the IH O2 levels slightly influenced

the growth of C. argentea and C. sake. In contrast, although the growth of M. caribbica was

slower than that of C. argentea and C. sake, it was more influenced by the headspace O2 level.

The growth of C. sake and L. mesenteroides on honeydew melon agar showed the same trend as

that on pineapple agar. The permutation test showed that the IH O2 level has no effect on the

growth of L. mesenteroides. However, a small but significant effect was also observed on the

growth of C. sake from day six onwards with higher population density occurring in higher IH

O2 level (see Figure 3.2).

These results are generally in agreement with those of some studies reported in literature for

fresh-cut fruit. Ragaert et al. (2006b) reported that the maximum counts of Debaryomyces

melissophilus and Rhodotorula glutinis grown on strawberry agar at 7°C were significantly

higher in air (7.5 - 8.5 log10 CFU/cm2) than they were in 3% O2 + 5% CO2, balance N2 (5.6 - 6.4

log10 CFU/cm²). In contrast, Varoquaux et al. (2005) reported that lowering the headspace O2

level from 10 to 1.5% did not significantly reduce the total yeast count on slices of kiwi stored at

10°C during 10 days. Yeasts represent a very divergent group of yeasts with respect to their

carbon metabolism and oxygen requirements (Rozpedowska et al., 2011).

CHAPTER 3

73

Figure 3.1: Growth curves of C. argentea (A), C. sake (B) and M. caribbica (C) during incubation on

pineapple agar in different IH O2 levels (() 0% O2, () 1% O2, () 3% O2, () 5% O2 and () 21% O2,

rest N2) at 7°C. Data points are the means, with bars representing two measurement values.

2

3

4

5

6

7

8

9

0 2 4 6 8

log

10

CF

U/c

m2

Time (days)

A

2

3

4

5

6

7

8

9

0 2 4 6 8

log

10

CF

U/c

m2

Time (days)

B

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14 16

log

10

CF

U/c

m2

Time (days)

C


74

Figure 3.2: Growth curves of C. sake (A) and L. mesenteroides (B) during incubation on honeydew melon

agar in different initial headspace O2 levels (air (), 5% O2 (), 3% O2 (), 1% O2 () and 0% O2 ())

at 7°C. Data points are the means, with bars representing two measurement values.

The yeasts evaluated in this study exhibited almost the same growth rate in low O2 levels as in

air. The reason can be that yeast possess a good fermentative potential and can still proliferate in

the absence of O2 (Merico et al., 2009). Additionally, yeasts, including Saccharomyces

cerevisiae and its close relatives, have a cytoplasmic version of dihydroorotate dehydrogenase

(DHODase) which are independent from the respiratory chain (Gojkovic et al., 2004; Hall et al.,

2005). Only the cytoplasmic DHODase promotes growth of yeasts in the absence of oxygen.

2

3

4

5

6

7

8

9

0 2 4 6 8

Lo

g10

CF

U/c

m2

Time (days)

A

2

3

4

5

6

7

8

9

0 2 4 6 8

Lo

g1

0C

FU

/cm

2

Time (days)

B

CHAPTER 3

75

3.3.2 The evolution of headspace O2 and CO2 during growth of spoilage isolates

The evolution of the headspace concentrations of O2 and CO2 of the inoculated and packaged

pineapple agar is shown in Figure 3.3. During the first four days of incubation, the headspace O2

and CO2 levels did not change appreciably in all conditions and for all evaluated yeasts. From

day four onwards, the headspace O2 and CO2 levels in the packages containing pineapple agar

inoculated with C. argentea and C. sake, rapidly decreased and increased, respectively, in all

conditions. This rapid consumption of O2 and production of CO2 coincided with yeast counts of

6.0 – 7.0 log10 CFU/cm2. A sharp increase in CO2 production has been reported to be a signal of

the end of the shelf-life of fresh-cut pineapple (Marrero and Kader, 2006). Babic et al. (1992)

also reported that the large amounts of CO2 produced by C. lambica could have caused spoilage

of shredded carrots at the end of storage. In our study, the production of CO2 by C. argentea and

C. sake was found to increase with growth at all IH O2 levels investigated and it could therefore

be part of the spoilage phenomena of these isolates. The IH O2 and CO2 concentrations did not

change appreciably in the packages with agar inoculated with M. caribbica. This was most likely

a result of its low population densities during incubation (< 5.5 log10 CFU/cm2 after 9 days).

Figure 3.4 shows that C. sake quickly consumed the headspace O2 from day four and depleted on

day six, irrespective of the IH O2 levels, whilst a large amount of CO2 was produced with higher

amount being produced in higher IH O2 level. In contrast, the headspace O2 only slightly

decreased towards the end of incubation of L. mesenteroides in all MAs, especially in air. A final

concentration of 8 -10 % of CO2 was produced by L. mesenteroides, irrespective of IH O2 levels.


76

Figure 3.3: The evolution of headspace O2 (┄) and CO2 (—) of C. argentea (A), C. sake (B) and M.

caribbica (C) during incubation on pineapple agar in different IH O2 levels (() 0% O2, () 1% O2, ()

3% O2, () 5% O2 and () 21% O2, rest N2) at 7°C. Data points are the means, with bars representing

two measurement values.

0

10

20

30

40

50

60

70

0

5

10

15

20

25

0 2 4 6 8

CO

2(%

)

O2

(%)

Time (days)

A

0

10

20

30

40

50

60

70

0

5

10

15

20

25

0 2 4 6 8

CO

2(%

)

O2

(%)

Time (days)

B

0

10

20

30

40

50

60

70

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16

CO

2(%

)

O2

(%)

Time (days)

C

CHAPTER 3

77

Figure 3.4: The evolution of headspace O2 (┄) and CO2 (—) of C. sake (A) and L. mesenteroides (B)

during incubation on honeydew melon agar in different initial headspace O2 levels (air (), 5% O2 (), 3%

O2 (), 1% O2 () and 0% O2 ()) at 7°C. Data points are the means, with bars representing two

measurement values.

3.3.3 Effect of headspace O2 level on sugar consumption, pH and VOC production

3.3.3.1 Sugar consumption

Sugar (sucrose, glucose and fructose) consumption by C. sake, L. mesenteroides on honeydew

melon agar and C. argentea on pineapple agar generally followed the same trend as that

0

10

20

30

40

50

60

70

0

5

10

15

20

25

0 2 4 6 8

CO

2(%

)

O2

(%)

Time (days)

A

0

10

20

30

40

50

60

70

0

5

10

15

20

25

0 2 4 6 8

CO

2(%

)

O2

(%)

Time (days)

B


78

observed in air (see Table 3.3) and in other MAs (data not shown). As it can be seen in Table

3.3A, sucrose, glucose and fructose were all steadily metabolized by C. argentea on pineapple

agar from day 4 onwards. Almost half of the sucrose present in honeydew melon agar was

consumed by C. sake at the end of incubation, while glucose and fructose were almost depleted

(see Table 3.3B). Small amounts of lactic acid were also detected. Unlike C. sake,

L. mesenteroides consumed most of the sucrose, which was almost depleted at the end of

incubation and at a rate faster than it consumed either glucose or fructose. The concentration of

glucose decreased gradually, but amount of fructose first decreased then slightly increased

during incubation. The fructose released from sucrose as a result of dextransucrase transferring

glucose residues to the reducing end of a growing dextran chain may be the reason why the

amounts of fructose were increased slightly (Raemaekers and Vandamme, 1997).

3.3.3.2 pH

The pH of pineapple agar inoculated with C. argentea, C. sake and M. caribbica did not change

in all initial headspace O2 levels during incubation (data not shown). The pH of honeydew melon

agar inoculated with C. sake and L. mesenteroides decreased during incubation in all the

atmospheres evaluated (see Figure 3.5). However, the pH was slightly higher in lower IH O2

level during growth of C. sake. The decrease in pH of honeydew melon agar was mainly due to

the production of lactic acid and acetic acid by both C. sake and L. mesenteroides as shown in

Table 3.3.

3.3.3.3 VOC production by yeasts

Various esters, acids, aldehydes and alcohols were produced by C. argentea and C. sake on

pineapple agar and C. sake on honeydew melon agar (see Table 3.1). These VOCs could

potentially influence the sensory quality of fresh-cut fruit during storage.

CHAPTER 3

79

Table 3.3

(A) Amounts of sucrose, glucose, fructose, lactic acid and acetic acid (g/100g) on pineapple agar during

incubation of C. argentea in air at 7°C.

Isolates Time

(days) Sucrose Glucose Fructose Lactic acid Acetic acid

0 6.14

a

(5.71 – 6.57)

2.96

(2.77 - 3.15)

2.98

(2.77 – 3.19) 0.00 0.00

C. argentea

4 5.96

(5.79- 6.12)

2.87

(2.78 - 2.95)

2.88

(2.86 - 2.91) 0.00 0.00

8 4.53

(3.54 – 5.52)

1.86

(1.46 – 2.26)

2.21

(1.74 – 2.69) 0.00 0.00

(B) Amounts of sucrose, glucose, fructose, lactic acid and acetic acid (g/100g) on honeydew melon agar

during incubation of C. sake and L. mesenteroides in air at 7°C.

Isolates Time

(days) Sucrose Glucose Fructose Lactic acid Acetic acid

0 2.66

a

(2.66 - 2.67)

3.01

(2.95 - 3.07)

3.91

(3.75 - 4.08) 0.00 0.00

C. sake

4 1.57

(1.47 - 1.67)

1.92

(1.83 - 2.01)

2.31

(2.15 - 2.48) 0.00 0.00

8 1.29

(1.24 - 1.34)

0.21

(0.20 - 0.23)

0.19

(0.05 - 0.32)

0.14

(0.13-0.15) 0.00

L. mesenteroides

4 1.43

(1.40 - 1.45)

1.95

(1.93 - 1.97)

2.27

(2.20 - 2.34) 0.00 0.00

8 0.07

(0.06 - 0.08)

1.44

(1.37 - 1.51)

2.55

(2.40 - 2.70)

0.49

(0.40-0.58)

0.23

(0.17 - 0.29)

a values are mean of two measurements


80

Figure 3.5: pH changes of C. sake (A) and L. mesenteroides (B) during incubation on honeydew melon

agar in different initial headspace O2 levels (air (), 5% O2 (), 3% O2 (), 1% O2 () and 0% O2 ())


Among the VOCs evaluated, ethyl acetate and 3-methylbutanal were the only compounds which

appreciably increased during incubation of M. caribbica (results not shown). To illustrate the

effect of IH O2 level on the production of VOCs by the yeasts evaluated (except M. caribbica),

the results obtained for some alcohols (ethanol, 3-methyl-1-butanol and butan-1-ol), esters (ethyl

acetate and ethyl octanoate), acids (phenylacetic acid and hexanoic acid) and aldehydes (nonanal

and 2-methylpropanal) are shown in Figures 3.6, - 3.8. The concentration of the VOCs produced

by C. argentea and C. sake on pineapple agar and C. sake on honeydew melon increased during

incubation in all IH O2 levels investigated and the quantity of any particular VOC produced at

any given time during incubation was smaller the lower the IH O2 level. As an example, 124.5

(103.7 – 145.2), 55.8 (35.3 – 76.3), 46.7 (46.4 – 47.0), 50.0 (40.4 – 59.6), and 44.3 (42.9 – 45.8)

4.0

4.5

5.0

5.5

6.0

6.5

0 2 4 6 8

pH

Time (days)

A

4.0

4.5

5.0

5.5

6.0

6.5

0 2 4 6 8

pH

Time (days)

B

CHAPTER 3

81

mg/m3 of ethanol was measured in packages with pineapple agar inoculated with C. argentea

which had IH O2 levels of 21, 5, 3, 1 and 0%, respectively, after 8 days. This was due to the fact

that the yeasts grew better the higher the IH O2 level. The only exception to this general trend

was for the effect of headspace O2 level on the production of ethyl acetate and 3-methylbutanal.

It can be seen in Figure 3.6D that C. argentea produced higher levels of ethyl acetate in the low

IH O2 level. The trends are not clear with regard to the effect of IH O2 level on the production of

ethyl acetate by C. sake. However, it seems that C. sake produced higher quantities of ethyl

acetate at lower headspace O2 levels from on day 4 of incubation on pineapple agar (see Figure

3.7D). This was also observed for C. sake on honeydew melon agar (see Figure 3.8D).

Nevertheless, the production of ethyl acetate by C. sake on honeydew melon agar was greater at

high IH O2 level from day 6 onwards.

M. caribbica produced lower amounts of 3-methylbutanal on pineapple agar in an IH O2 level of

21% compared to the amounts produced in IH O2 levels of 0, 1, 3 and 5% (results not shown). It

can be seen from Figures 3.6 through 3.8 that C. argentea and C. sake on pineapple agar and C.

sake on fruit agar only began producing large quantities of most volatile metabolites after 4 days

of incubation, at which time their counts were between 6.0 – 7.0 log10 CFU/cm2. There were a

few exceptions, including butan-1-ol which was produced almost immediately by C. sake on

pineapple agar during incubation and ethyl acetate, which was produced after just two days of

incubation on pineapple agar. M. caribbica, which grew the slowest, actually started producing

ethyl acetate and 3-methylbutanal at lower population densities of 4.2 and 5.3 log10 CFU/cm²,

respectively. Moreover, metabolite production was dependent on the yeast species. As it can be

seen from Figures 3.6 - 3.7, C. sake produced higher amounts of ethanol and phenylacetic acid

on pineapple agar than C. argentea in an IH 21% O2 at the end of incubation. However, for the

other VOCs, the opposite was true for IH 21% O2 (see Figures 3.6 and 3.7). Furthermore,

metabolite production was strain dependent as seen by C. sake FF 641 and FF 655 which were

isolated from fresh-cut pineapple and honeydew melon, respectively. C. sake (FF 655) produced

higher quantities of 3-methyl-1-butanol, ethyl acetate, ethyl butanoate, nonanal and 3-

methylbutanal on honeydew melon than C. sake (FF 641) produced on pineapple agar in an IH

21% O2, whereas other VOCs followed an opposite trend in IH 21% O2.


82

Moller et al. (2002) reported that the ethyl acetate concentration in a bioreactor headspace

produced by Saccharomyces kluyveri under aerobic condition at 30°C increased throughout the

glucose-consumption phase, and the maximum volumetric productivity was achieved at the time

of glucose depletion. However, the volumetric productivity of ethyl acetate decreased after

depletion of glucose (Moller et al., 2002). Ethyl acetate can be synthesized via two pathways;

both pathways use ethanol as one of the substrates, either together with acetyl co-enzyme A,

catalysed by the enzyme alcohol acetyl transferase or with acetate, by the reverse reaction of

enzyme-catalysed hydrolysis (esterase activity) (Fredlund, 2004). Therefore, increased ethanol

production may lead to increased ethyl acetate production (Ragaert et al., 2006b). Our results

showed that the quantity of ethanol produced by C. argentea on pineapple agar were lower in an

IH 21% O2 level whilst those of ethyl acetate were higher (see Figure 3.6A and D). Additionally,

we found that C. sake on honeydew melon in an IH 21% O2 level produced much lower quantity

of ethanol than the quantity of ethyl acetate (see Figure 3.8A and D). Therefore, ethyl acetate

may probably be synthesized via other pathways in addition to the two described above or the

synthesis of ethyl acetate may be affected by other factors, such as the level of trace elements in

the media (Loser et al., 2012). Loser et al. (2012) found that at a high iron (Fe) dosage (400 to

12000 µg/L in a fed medium), yeast growth was not limited by this metal. Higher formation rates

were found for ethyl acetate, pyruvate, and glycerol while ethanol and acetate were formed in

small amounts (Loser et al., 2012). Lester (2008) determined that the concentration (fresh weight

basis) of Fe was 2.4 - 4.1 µg/g in tissues of mature orange-fleshed honeydew melon.

Manimegalai et al. (1998) investigated that the concentration (dry weight basis) of iron (Fe) was

0.99 - 4.82 mg/100g in pineapple juices. Hence, compared to the Fe dosage reported by Loser et

al. (2012), both fresh-cut pineapple and honeydew melon contain high Fe dosage.

Among the VOCs detected in this study, ethanol, ethyl acetate and ethyl butanoate are pyruvate

metabolites obtained via pyruvate decarboxylase (Pronk et al., 1996; Ragaert et al., 2006b) and

3-methyl-1-butanol, 2-methyl-1-butanol, 2-methylbutanoic acid, 3-methylbutanoic acid, 2-

methylpropanal, 3-methylbutanal and 2-methylbutanal are intermediates of the Ehrlich pathway

(Hazelwood et al., 2008). Those compounds are also known as fusel alcohols, fusel acids and

fusel aldehydes. To our knowledge, the effect of these metabolites on the sensory quality of

fresh-cut fruit is still unknown.

CHAPTER 3

83

Figure 3.6: VOCs produced by C. argentea in MAP on pineapple agar during storage at 7°C.

0

40

80

120

160

0 2 4 6 8

mg

/m3

Time (days)

A

0

15

30

45

60

0 2 4 6 8

mg

/m3

Time (days)

B

0

7

14

21

28

35

0 2 4 6 8

mg

/m3

Time (days)

C

0

30

60

90

120

0 2 4 6 8

mg

/m3

Time (days)

D

0

10

20

30

40

0 2 4 6 8

mg

/m3

Time (days)

E

0

115

230

345

460

0 2 4 6 8

mg

/m3

Time (days)

F

0

40

80

120

0 2 4 6 8

mg

/m3

Time (days)

G

0

10

20

30

40

50

0 2 4 6 8

mg

/m3

Time (days)

H

0

1

2

3

4

0 2 4 6 8

mg

/m3

Time (days)

I

VOCs:

(A) Ethanol

(B) 3-Methyl-1-butanol

(C) Butan-1-ol

(D) Ethyl acetate

(E) Ethyl butanoate

(F) Phenylacetic acid

(G) Hexanoic acid

(H) Nonanal

(I) 3-Methylbutanal

MAP:

() 0% O2

() 1% O2

() 3% O2

() 5% O2

() 21% O2

Data points are the

means, with bars

representing two

measurement values.

t

t t •

Ij)

~+mt • • m t + c em. 1!1

coem+ 0. ++ • + • 0

t t t


84

Figure 3.7: VOCs produced by C. sake in MAP on pineapple agar during storage at 7°C.

0

50

100

150

200

250

0 2 4 6 8

mg

/m3

Time (days)

A

0

6

12

18

24

30

0 2 4 6 8

mg

/m3

Time (days)

B

0

3

6

9

12

15

0 2 4 6 8

mg

/m3

Time (days)

C

0

10

20

30

40

0 2 4 6 8

mg

/m3

Time (days)

D

0

1

2

3

4

5

0 2 4 6 8

mg

/m3

Time (days)

E

0

300

600

900

1200

0 2 4 6 8

mg

/m3

Time (days)

F

0

10

20

30

40

50

0 2 4 6 8

mg

/m3

Time (days)

G

0

4

8

12

0 2 4 6 8

mg

/m3

Time (days)

H

0.0

0.4

0.8

1.2

1.6

0 2 4 6 8

mg

/m3

Time (days)

I

VOCs:

(A) Ethanol


(C) Butan-1-ol

(D) Ethyl acetate

(E) Ethyl butanoate


(G) Hexanoic acid

(H) Nonanal

(I) 3-Methylbutanal

MAP:

() 0% O2

() 1% O2

() 3% O2

() 5% O2

() 21% O2

Data points are the

means, with bars

representing two

measurement values.

CHAPTER 3

85

Figure 3.8: VOCs produced by C. sake in MAP on honeydew melon agar during storage at 7°C.

0

45

90

135

0 2 4 6 8

mg

/m3

Time (days)

A

0

10

20

30

40

50

0 2 4 6 8

mg

/m3

Time (days)

B

0

2

4

6

8

10

0 2 4 6 8

mg

/m3

Time (days)

C

0

130

260

390

520

650

0 2 4 6 8

mg

/m3

Time (days)

D

0

2

4

6

8

0 2 4 6 8

mg

/m3

Time (days)

E

0

100

200

300

400

500

0 2 4 6 8

mg

/m3

Time (days)

F

0

5

10

15

20

25

0 2 4 6 8

mg

/m3

Time (days)

G

0

6

12

18

24

30

0 2 4 6 8

mg

/m3

Time (days)

H

0

7

14

21

28

35

0 2 4 6 8

mg

/m3

Time (days)

I

VOCs:

(A) Ethanol


(C) Butan-1-ol

(D) Ethyl acetate

(E) Ethyl butanoate


(G) Hexanoic acid

(H) Nonanal

(I) 3-Methylbutanal

MAP:

() 0% O2

() 1% O2

() 3% O2

() 5% O2

() 21% O2

Data points are the

means, with bars

representing two

measurement values.

+ 1!1 • •

t +

t

I +

I

t t +


86

In addition, phenylacetic acid is related to amino acid catabolism (Hammer et al., 1996). As

described in § 1.2.1.1, the Ehrlich pathway may provide an alternative, energy-efficient means

for NADH regeneration under anaerobic conditions for yeasts. As it can be seen in Figures 3.3

and 3.4, headspace O2 depletion occurred on day 6 during the incubation of C. argentea and

C. sake on pineapple agar and C. sake on honeydew melon agar in lowered IH O2 levels (0-5%).

Large amounts of ethanol, ethyl acetate and fusel compounds (see Figure 3.6 - 3.8) produced by

these spoilage isolates on day 6 may be due to fermentative metabolism and amino acid

catabolism via the Ehrlich pathway. However, ethanol, ethyl acetate and fusel compounds were

also produced significant quantities even though the headspace O2 had not yet been completely

depleted on day 6 in the packages with an IH 21% O2. Two likely explanations are the Crabtree

effect and the glucose effect. As explained in in § 1.2.1.1, the growth of these yeasts showed

respiro-fermentative glucose metabolism on pineapple agar and honeydew melon agar when O2

is present in the headspace of packages, due to the presence of sufficiently high concentrations of

glucose (see Table 3.3).

3.3.3.4 VOC production by lactic acid bacteria on honeydew melon agar

Butane-2,3-diol, butane-2,3-dione, acetic acid, acetoin and ethanol which originate from the

heterofermentation of sugar by Leuconostoc (Axelsson, 2004), were detected in the headspace

during the incubation of honeydew melon agar inoculated with L. mesenteroides in air and

lowered IH O2 levels (see Table 3.2). The amounts of butane-2,3-diol and ethanol were only

slightly increased in air and MAs (data not shown). Whilst the quantities of butane-2,3-dione,

acetic acid and acetoin increased appreciably during incubation (see Figure 3.9), the effect of IH

O2 level was not clear, except for butane-2,3-dione which produced at slightly higher amounts in

air than other in IH O2 levels. During aerobic growth, acetate is a major end-product of hexose

metabolism by Leuconostoc spp., since the ethanol branch of this pathway wastes high energy

phosphate (Ito et al., 1983; Lucey and Condon, 1986). According to Raemaekers et al. (1997), a

higher energy efficiency, due to a lower mannitol and a higher acetic acid production, might

result in a higher protein and enzyme production, explaining higher growth and

CHAPTER 3

87

glucosyltransferase production in aerated condition. In contrast to the yeast, L. mesenteroides

produced fewer types and quantities of VOCs on honeydew melon agar.

Figure 3.9: Butane-2,3-dione (A), acetic acid (B) and acetoin (C) production in the headspace during

incubation of L. mesenteroides on honeydew melon agar in different initial headspace O2 levels (air (),

5% O2 (), 3% O2 (), 1% O2 () and 0% O2 ()) at 7°C. Data points are the means, with bars

representing two measurement values.

3.3.4 Effect of headspace O2 level on the growth of the native flora of fresh-cut fruit

The counts of total aerobes and yeasts occurring naturally on fresh-cut pineapple during storage

in different IH O2 levels at 7°C are shown in Table 3.4A. The growth of the yeasts took place

immediately on the pineapple cubes without an apparent lag phase, irrespective of the IH O2

level.

0.00

0.02

0.04

0.06

0.08

0.10

0 2 4 6 8

mg

/m3

Time (days)

A

0.00

0.05

0.10

0.15

0.20

0 2 4 6 8

mg

/m3

Time (days)

B

0.00

0.03

0.06

0.09

0.12

0 2 4 6 8

mg

/m3

Time (days)

C


88

Table 3.4

(A) Total aerobe counts and yeast (log10 CFU/g) on pineapple cubes packaged in different initial

headspace (IH) O2 levels during storage at 7°C.

Packaging

condition

Storage time (days)

0 1 3 5

Total aerobe count

IH 21% O2

4.55

(4.41 - 4.69)

- 6.75 (6.08 -7.42) 10.85 (9.83 – 11.87)

IH 5% O2 - 7.55 (7.42 – 7.68) 9.42 (9.29 - 9.55)

IH 1% O2 - 6.93 (9.18 – 7.68) 8.46 (7.04 – 9.88)

Yeast count

IH 21% O2 3.22

(3.04 – 3.41)

4.84 (4.61 – 5.06) 6.48 (6.45 – 6.52) 10.96 (10.84 – 11.09)

IH 5% O2 5.98 (4.81 – 7.16) 8.04 (7.51 – 8.57) 8.69 (8.66 – 8.72)

IH 1% O2 5.72 (4.84 – 6.61) 8.74 (8.52 – 8.96) 7.88 (7.61 – 8.16)

(B) Total aerobe, total anaerobe, yeast and lactic acid bacteria (LAB) counts (log10 CFU/g) on honeydew

melon cubes packaged in different IH O2 levels during storage at 7°C.

Packaging condition Storage time (days)

0 1 3 5

Total aerobe count

IH 21% O2 4.6 (4.3 - 4.9)

6.0 (5.2 - 6.8) 8.0 (7.9-8.1) 8.5 (8.5 – 8.5)

IH 5% O2 5.7 (5.5 - 5.9) 7.8 (7.8 -7.8) 8.6 (8.5 - 8.7)

Total anaerobe count

IH 21% O2 4.0 (3.8 – 4.2)

5.4 (4.6 – 6.2) 7.0 (7.0 – 7.0) 8.4 (8.2 – 8.6)

IH 5% O2 5.0 (5.0 - 5.0) 6.7 (6.5 - 6.9) 7.4 (7.4 – 7.4)

Yeast count

IH 21% O2 4.9 (4.6 – 5.2)

5.5 (5.3 – 5.7) 7.6 (7.4 - 7.8) 7.7 (7.6 – 7.8)

IH 5% O2 5.8 (5.7 – 5.9) 7.2 (7.0 – 7.4) 7.4 (7.4 – 7.4)

LAB count

IH 21% O2 3.1 (2.4 – 3.9)

4.0 (3.5 – 4.5) 6.5 (6.0 – 7.0) 8.1 (8.1 – 8.1)

IH 5% O2 4.1 (4.1 – 4.1) 6.2 (5.9 – 6.5) 6.5 (6.4 – 6.6) a values are mean with two measurement values

CHAPTER 3

89

At the end of storage (day 5) the yeast counts were highest on pineapple cubes packaged in an IH

O2 level of 21% (11.0 log10 CFU/g) and were 7.9 and 8.7 log10 CFU/g on the pineapple cubes

packaged in IH O2 levels of 1 and 5%, respectively. The trend observed here was in agreement

with that observed for the yeasts on pineapple agar.

The total anaerobe and lactic acid bacteria counts were both < 3 log10 CFU/g on fresh-cut

pineapple in all initial atmospheres during storage. The total aerobe counts followed the same

trend as that of the growth of the native yeasts on fresh-cut pineapple during storage. This is

assumed that most of the total aerobes of fresh-cut pineapple are yeasts. However, large

populations of bacteria as well as yeasts and moulds have been also found on fresh-cut pineapple

during refrigerated storage previously (Di Egidio et al., 2009; Liu et al., 2007; Montero-Calderon

et al., 2008; Rocculi et al., 2009). Micro flora of fresh-cut fruit can be affected by many factors

such as farm location, handling on the farm and conditions during processing. Additionally, in

the fresh-cut environment, faster-growing yeasts tend to outgrow moulds to cause spoilage

(Heard, 2002). Hence, yeasts were the dominant microbial flora on the pineapple cubes (Spanier

et al., 1998).

The native flora grew immediately during storage of fresh-cut honeydew melon (see Table 3.4B).

Although the aerobes were not affected by lowered O2 levels, yeast counts were slightly higher

in air than those in IH 5% O2 during storage which was in agreement with the results obtained

for C. sake growth on honeydew melon agar. However, the amounts of anaerobes and lactic acid

bacteria were appreciably lowered on day 5 of storage in IH 5% O2 compared to those on

honeydew melon packaged in air. This was contradicted with the results of L. mesenteroides

growth on honeydew melon agar, probably due to the variability of oxygen sensitivity among

L. mesenteroides species and other lactic acid bacteria. It can be seen in Table 3.4B that the

initial yeast count on fresh-cut honeydew melon was higher than the tolerance value (4 log10

CFU/g) on the day of production. However, total aerobic, total anaerobic and lactic acid bacteria

counts were lower than the tolerance values, proposed by Uyttendaele et al. (2010). This resulted

in population densities of the native yeasts that were greater than 5 log10 CFU/g, which is

recommended for fresh-cut fruit at the end of shelf-life. However the final population density of

LAB of fresh-cut honeydew melon may have still been acceptable, as the end of shelf-life value

of LAB may up to 8 log10 CFU/g (Jacxsens et al., 2003; Kakiomenou et al., 1996). Overall, the


90

effect of low O2 MA on the growth of the native flora of fresh-cut honeydew melon was very

limited. It has been reported that atmospheres containing 3% O2 with or without 10% CO2 had

little or no effect on the spoilage microorganisms on cut lettuce (Barriga et al., 1991; King et al.,

1991).

The pH and sugars (sucrose, glucose and fructose) of the pineapple cubes and honeydew melon

cubes (results not shown) did not change significantly throughout storage in all IH O2 levels. The

evolution of the headspace O2 and CO2 concentrations observed in the packages with pineapple

cubes was similar to that observed in the studies performed on pineapple agar (see Figure 3.10A).

However, headspace O2 and CO2 levels in the packages containing pineapple cubes decreased

and increased more rapidly, respectively, as from day 1 of storage compared to day 4 of

incubation on inoculated pineapple agar. The quicker change in the headspace O2 and CO2 of

packaged fresh-cut pineapple during the storage period was most likely a result of a combination

of the respiration of the living tissues of pineapple (Rocculi et al., 2009) and microbial growth

(Marrero et al., 2006), especially at the end of the shelf-life of the fresh-cut pineapple, with rapid

consumption of O2 and sharp increase of CO2 considered as a typical signal of microbial spoilage.

The headspace O2 and CO2 concentrations decreased and increased, respectively in the

headspace of the packaged honeydew melon cube during storage (Figure 3.10B). It can be seen

in Figure 3.10 that O2 concentrations decreased at a lightly faster rate during storage of fresh-cut

pineapple than fresh-cut honeydew melon in IH 21% O2 level. The quicker O2 change during

storage of pineapple cubes than honeydew melon cubes could be due to (1) different O2

consumption properties of native aerobes on pineapple cubes and honeydew melon cubes and (2)

different respiration properties of the fruit themselves.

3.3.5 Effect of initial headspace O2 level on VOC production on fresh-cut fruit

It should be noted that some of the VOCs shown in Table 3.1 have also been identified on fresh

pineapple and pineapple products i.e. ethyl acetate (Tokitonio et al., 2005), ethyl butanoate (Elss

et al., 2005; Tokitonio et al., 2005; Wei et al., 2011), ethyl hexanoate (Elss et al., 2005;

Kaewtathip and Charoenrein, 2012; Montero-Calderon et al., 2010b; Tokitonio et al., 2005; Wei

CHAPTER 3

91

et al., 2011), ethyl octanoate (Elss et al., 2005; Kaewtathip et al., 2012; Montero-Calderon et al.,

2010b), phenylacetic acid (Tokitonio et al., 2005), nonanal (Montero-Calderon et al., 2010b; Wei

et al., 2011), pentan-1-ol and butan-1-ol (Elss et al., 2005). Additionally, ethyl octanoate, ethyl

butanoate and ethyl hexanoate are important contributors of pineapple aroma (Takeoka et al.,

1989). Similarly, ethanol, butan-1-ol, pentan-1-ol, ethyl acetate, ethyl butanoate, ethyl hexanoate

and nonanal associated to spoilage metabolites can also be produced by the living tissues of

honeydew (Amaro et al., 2012; Bai et al., 2003; Guler et al., 2013; Perry et al., 2009b; Saftner et

al., 2006). Hence, whilst some VOCs may enhance the flavour of fresh-cut fruit during storage,

others can be off-flavours.

Figure 3.10: The evolution of headspace O2 (┄) and CO2 (―) of fresh-cut pineapple (A) and fresh-cut

honeydew melon (B) during storage in different initial headspace O2 levels (air (), 5% O2 (), and 1%

O2 ()) at 7°C. Data points are the means, with bars representing two measurement values.

A few studies have reported the VOCs present on fresh-cut pineapple during storage (Elss et al.,

2005; Montero-Calderon et al., 2010a). As mentioned earlier, the production of volatile

metabolites (alcohols, esters, acids, aldehydes etc.) by spoilage microorganisms that may

influence the quality of fresh-cut fruit has received little attention so far. With regards to fresh-

cut pineapple, only Spanier et al. (1998) observed that during storage, some of the pineapple and

fruity flavoured volatiles increased slightly (1-methylethyl acetate, propyl acetate and 1-butanol

3-methyl acetate) while others either decreased slightly (ethyl acetate, methyl butanoate, 1-

0

10

20

30

40

50

60

70

0

5

10

15

20

25

0 1 2 3 4 5

CO

2(%

)

O2

(%)

Time (days)

A

0

10

20

30

40

50

60

70

0

5

10

15

20

25

0 1 2 3 4 5

CO

2(%

)

O2

(%)

Time (days)

B


92

butanol 3-methyl, 2-methyl ethyl butanoate, and methyl hexanoate) or showed a variable

response (2-methyl methyl butanoate and methyl pentanoate). Nevertheless, our results showed

that the quantity of VOCs related to spoilage yeasts in the headspace (see Table 3.1) increased

sharply from day 3 of storage of fresh-cut pineapple cubes, irrespective of the IH O2 level (see

Table 3.5). As observed on pineapple agar, the quantity of VOCs at the end of storage of

pineapple cubes was determined to be the highest on pineapple cubes packaged in air, followed

by those packaged in an IH O2 level of 1%. Pineapple cubes packaged in IH 5% O2 were

determined to have the lowest concentrations of VOCs on day 5 of storage.

As it has been shown in Table 3.6, the quantities of VOCs (with the exception of butan-1-ol and

hexanoic acid) in the headspace of packaged honeydew melon cubes were slightly higher in MA

than they were in air on day 3 of storage. However, the VOCs increased appreciably at the end of

shelf-life with lower quantities being produced in IH 5% O2 than in air. Although the quantities

of some VOCs were higher than the human olfactory threshold (OT) on the initial storage (day 0),

the quantities of the VOCs evaluated (except ethanol) in air at the end of storage were several

times higher than their corresponding OT values (see Tables 3.1, 3.2 and 3.6). Portela et al.

(1998) reported that the typical aroma of honeydew melon generally decreased during storage

but the development of off - odours were associated with macroscopic decay. On the contrast,

Amaro et al. (2012) found that the summed volatile concentration of 25 integrated volatile

compounds was 1.4 times higher than the initial value for fresh-cut cantaloupe and honeydew

melon by the end of storage time.

As it has been discussed above, the VOCs evaluated in this study can be produced by both the

spoilage yeasts and the living tissues of fresh-cut fruit during storage. The VOCs detected at the

start of storage (day 0) are mostly produced by the living tissues due to the cutting and peeling

processes which can accelerate the production and release of VOCs. This could be the reason

why a high quantity of ethyl acetate (ca. 63 mg/m3) was detected on day 0 on fresh-cut

honeydew melon (see Table 3.6). During storage of the fruit stress factors such as cutting and

peeling, atmospheres with reduced O2 levels and microbial infection may also stimulate

fermentative metabolism (Chung et al., 2009a; Purvis, 1997; Toivonen et al., 2002; Wu et al.,

2012b). Ragaert et al. (2006c) reported that ethanol produced by yeasts (from ˃ 5.0 log10 CFU/g)

can be converted to ethyl acetate by the living tissues of strawberries.

CHAPTER 3

93

Table 3.5: VOC levels (mg/m3) in the headspace of pineapple cubes packaged in air, IH 5% O2 and IH 1% O2 at 7°C.

Day of

storage Day 0 Day 1 Day 3 Day 5

VOC’s Air/MAs Air 5% O2 1% O2 Air 5% O2 1% O2 Air 5% O2 1% O2

Ethanol 0.15 a

(0.10-0.20)

1.85

(1.41-2.28)

3.64

(2.30-4.97)

2.08

(1.18-2.99)

9.49

(6.17-12.81)

19.83

(16.99-22.67)

40.01

(37.22-42.80)

181.41

(166.32-196.50)

106.46

(76.60-136.31)

139.32

(132.91-145.74)

3-Methyl-1-

butanol

0.53

(0.0.4-0.56)

1.10

(0.97-1.23)

1.36

(0.83-1.88)

0.70

(0.64-0.76)

0.99

(0.74-1.24)

2.43

(1.98-2.88)

5.38

(5.32-5.44)

215.74

(185.98-245.50)

63.11

(7.13-119.09)

73.91

(52.79-95.40)

Butan-1-ol 2.60

2.41-2.78

3.24

(2.82-3.66)

3.62

(2.91-4.33)

3.42

(3.30-3.54)

4.12

(4.08-4.17)

4.65

(4.14 -5.17)

7.25

(6.58-7.92)

142.11

(132.08-152.13)

40.27

(10.61-69.94)

65.41

(45.31-85.51)

2-Methyl-1-

butanol

0.00 0.00 0.00 0.00 0.00 0.00 0.00 2.94

(2.55-3.33)

0.16

(0.06-0.25)

0.55

(0.53-0.58)

Ethyl acetate 4.58

(4.56-4.60)

12.93

(12.89-12.96)

27.72

(18.55-36.89)

11.70

(6.83-16.56)

33.22

(22.95-43.48)

56.62

(52.51-60.73)

87.09

(86.65-87.54)

372.61

(340.29-404.92)

154.21

(71.54-236.87)

189.98

(165.37-214.60)

Ethyl

octanoate

0.25

(0.21-0.29)

0.36

(0.30-0.42)

0.35

(0.0.30-0.41)

0.31

(0.27-0.35)

0.57

(0.49-0.65)

0.66

(0.56-0.0.76)

1.52

(1.35-1.69)

78.86

(77.44-80.28)

22.43

(3.414-41.44)

44.95

(32.98-56.93)

Phenylacetic

acid

0.06

(0.06-0.06)

1.87

(1.02-2.72)

7.31

(2.22-12.41)

2.32

(0.82-3.81)

36.90

(14.10-59.70)

108.46

(73.12-

143.80)

281.99

(233.74-

330.24)

1109.26

(1095.30-

1123.22)

779.94

(516.26-

1043.26)

979.23

(921.702-

1036.76)

Hexanoic acid 2.30

2.17-2.44

4.10

(3.93-4.26)

3.38

(3.30-3.45)

3.66

(2.65-4.68)

5.94

(5.70-6.19)

6.49

6.47-6.51

12.24

(11.57-12.91)

164.85

(153.49-176.22)

64.61

(20.78-108.44)

131.55

(103.74-159.35)

Nonanal 1.47

(1.29-1.64)

2.33

(1.81-2.85)

2.32

(1.94-2.69)

2.05

(1.99-2.11)

3.02

(2.84-3.20)

2.85

(2.22-3.48)

7.11

(6.12-8.10)

195.96

(189.67-202.64)

49.96

(11.16-88.76)

88.44

(60.63-116.24)

3-

Methylbutanal

0.00 0.21

(0.18-0.24)

0.20

(0.12-0.28)

0.00 0.34

(0.21-0.47)

0.40

(0.39-0.41)

0.69

(0.67-0.70)

19.19

(16.34-22.04)

3.37

(0.75-5.99)

4.58

(2.99-6.18)

a values are mean with two measurement values


94

Table 3.6: VOC levels (mg/m3) in the headspace of honeydew melon cubes packaged in air, IH 5% O2 and IH 1% O2 at 7°C.

Day of storage Day 0 Day 1 Day 3 Day 5

VOC’s Air/5% O2 Air 5% O2 Air 5% O2 Air 5% O2

Ethanol 3.16 a

(2.49 - 3.83)

3.38

(3.35 - 3.71)

3.74

(3.26 - 4.22)

8.90

(4.54 - 13.26)

13.38

(13.11 - 13.65)

50.60

(50.36 - 50.84)

22.89

(21.55 - 24.23)

3-Methyl-1-butanol 0.07

(0.11 - 0.03)

0.11

(0.07 - 0.15)

0.07

(0.04 - 0.10)

0.23

(0.05 - 0.41)

0.31

(0.22 - 0.40)

3.08

(2.48 - 3.68)

0.75

(0.72 - 0.78)

Butan-1-ol 0.00 0.00 0.00 0.32

(0.22 - 0.42)

0.24

(0.17 - 0.31)

2.54

(2.26 - 2.82)

0.85

(0.63 - 1.07)

2-Methyl-1-butanol 3.20

(2.81 - 3.59)

4.86

(4.66 - 5.05)

4.05

(3.20 - 4.90)

7.45

(6.27 - 8.63)

9.42

(8.79 - 10.05)

8.42

(8.02 - 8.82)

8.02

(7.45 - 8.59)

Ethyl acetate 63.03

(49.61 - 76.44)

52.56

(46.62 - 58.50)

62.54

(55.79 - 69.29)

67.29

(35.25 - 99.33)

109.97

(104.86 - 115.08)

166.98

(150.57 - 183.39)

69.14

(53.79 - 84.49)

Ethyl octanoate 0.03

(0.02 - 0.04)

0.02

(0.01 - 0.03)

0.03

(0.01 - 0.05)

0.05

(0.07 - 0.03)

0.06

(0.05 - 0.07)

0.38

(0.33 - 0.43)

0.12

(0.07 - 0.17)

Phenylacetic acid 1.35

(0.92 - 1.78)

0.88

(0.69 - 1.07)

1.86

(1.48 - 2.24)

18.43

(8.46 - 28.40)

31.00

(28.38 - 33.62)

181.47

(177.77 - 184.70)

89.48

(82.07 - 96.89)

Hexanoic acid 0.00 0.00 0.00 0.00 0.00 2.55

(2.03 - 3.07)

0.57

(0.34 - 0.80)

Nonanal 0.37

(0.33 - 0.41)

0.42

(0.21 - 0.63)

0.45

(0.34 - 0.56)

0.57

(0.29 - 0.85)

0.74

(0.57 - 0.91)

3.49

(2.06 - 4.92)

1.23

(0.85 - 1.61)

3-Methylbutanal 2.03

(2.47 - 2.59)

2.01

(1.79 - 2.23)

2.21

(1.64 - 2.78)

2.82

(1.83 - 3.81)

4.78

(4.39 - 5.17)

7.43

(7.34 - 7.52)

2.82

(2.29 - 3.35)

2,3-Butanediol 2.41

(1.86 - 2.96)

2.23

(2.10 - 2.36)

2.53

(2.29 - 2.77)

7.64

(3.23 - 12.05)

15.20

(14.15 - 16.25)

40.81

(39.51 - 41.11)

17.51

(15.41 - 19.61)

2,3-Butanedione 0.04

(0.03 - 0.05)

0.03

(0.02 - 0.04)

0.01

(0.00 - 0.02)

0.03

(0.01 - 0.05)

0.09

(0.05 - 0.13)

0.13

(0.10 - 0.16)

0.04

(0.03 - 0.05)

Acetic acid 0.22

(0.18 - 0.26)

0.23

(0.22 - 0.24)

0.21

(0.13 - 0.29)

0.30

(0.10 - 0.50)

0.38

(0.37 - 0.39)

1.15

(1.12 - 1.18)

0.68

(0.50 - 0.86)

Acetoin 16.48

(14.86 -18.10)

17.60

(17.54 - 17.66)

16.89

(14.09 - 19.69)

26.76

(19.34 – 34.18)

45.59

(44.96 - 46.22)

88.80

(84.30 – 93.30)

44.02

(32.42 - 53.62) a values are mean with two measurement value

CHAPTER 3

95

Respiration of the living tissues of pineapple cubes in low O2 MAs could have also accelerated

the shift from a respiro-fermentative to a fully fermentative metabolism of the yeasts on

pineapple cubes in IH 1% O2. As it shown in Table 3.5, production of volatiles (ethanol and ethyl

acetate) related to microbial fermentation in IH 1% O2 was slightly higher than in IH 5% and 21%

O2 on day 3. Therefore, the amounts of VOCs during storage of fresh-cut pineapple and

honeydew melon could be produced by living tissues in combination with yeasts. However, the

counts of yeasts on fresh-cut pineapple and honeydew melon generally reached 6.5 log10 CFU/g

on day 3 of storage, irrespective of the atmosphere conditions and had reached 7.7 – 11.0 log10

CFU/g on day 5 of storage (see Table 3.4). As it has been discussed in § 3.3.3.3, yeasts evaluated

on fruit simulation agar began producing large quantities of most VOCs when their counts were

between 6.0 – 7.0 log10 CFU/cm2 (ca. 6.4 – 7.4 log10 CFU/g). Hence, at the end of storage (day 5)

the higher quantities of VOCs detected on fresh-cut pineapple and honeydew melon packaged at

the high O2 levels were most likely produced by spoilage microorganisms.

3.3.6 Effect of the initial headspace O2 level on the physical parameters of fresh-cut fruit

Table 3.7 shown the results of colour parameter assessment performed at different storage times

and MAs. As it can be seen in Table 3.7, the values of the colour parameters L* and b* of

pineapple cubes significantly decreased (P < 0.05) in all atmospheres during storage. Significant

differences (P < 0.05) were established between the colour parameters of pineapple cubes

packaged in low O2 MAs and those packaged in IH O2 level of 21%. The colour parameter of a*

did not change significantly (P > 0.05) during storage. However, a significant difference was

found between pineapple cubes packaged in IH O2 levels 21% and 5% O2 at the end of storage.

Juiciness significantly (P < 0.05) decreased during storage in all atmospheres evaluated, but no

significant differences were observed between the different atmospheres evaluated. Firmness and

juice leakage did not show significant differences during storage and among all atmospheres.

No significant differences (P < 0.05) were observed between the honeydew melon cubes

packaged in air and IH 5% O2 in terms of the colour parameters (L*, a* and b*), juice leakage,

firmness and juiciness (see Table 3.7).


96

Table 3.7: Sensorial quantity attributes on pineapple cubes and honeydew melon cubes packaged in

different initial headspace (IH) O2 levels during storage at 7°C.

Packaging

condition

Storage time (days)

0 1 3 5

Fresh-cut pineapple

L* β

IH 21% O2 72.9 ± 2.9 A, a

69.8 ± 5.4 A, ab

69.4 ± 5.0 A,b

61.0 ± 5.4 A, c

IH 5% O2 72.9 ± 2.9 A, a

70.9 ± 3.6 A, ab

69.9 ± 4.6 A,b

68.8 ± 3.8 B, b

IH 1% O2 72.9 ± 2.9 A,a

72.3 ± 3.2 A, a

71.5 ± 3.7 A, a

67.9 ± 3.4 B, b

a* β

IH 21% O2 1.5 ± 1.3 A, a

1.1 ± 1.5 A, a

1.3 ± 1.1 A, a

1.0 ± 0.9 A, a

IH 5% O2 1.5 ± 1.3 A, ab

1.9 ± 1.5 A, ab

1.1 ± 0.8 A, a

2.1 ± 1.6 B, b

IH 1% O2 1.5 ± 1.3 A, ab

1.7 ± 1.2 A, a

1.3 ± 1.3 A, ab

0.9 ± 1.1 A, b

b* β

IH 21% O2 43.9 ± 3.6 A, a

38.6 ± 4.9 A, b

37.2 ± 4.7 A, b

31.3 ± 4.8 A, c

IH 5% O2 43.9 ± 3.6 A, a

41.8 ± 4.6 B, ab

40.3 ± 2.9 B, b

41.4 ± 4.6 B, ab

IH 1% O2 43.9 ± 3.6 A, a

42.6 ± 4.4 B, ab

41.1 ± 4.4 B, bc

39.5 ± 2.9 B, c

Juice leakage γ

IH 21% O2

4.0 ± 1.1 A, a

5.1 ± 1.4 A, a

6.3 ± 0.4 A, a

IH 5% O2 3.6 ± 1.0 A, a

4.8 ± 2.0 A, a

4.6 ± 0.8 A, a

IH 1% O2 2.8 ± 0.7 A, a

6.0 ± 2.2 A, a

6.9 ± 2.2 A, a

Firmness δ

IH 21% O2 3 (2 – 4) A, a

3 (2 – 3) A, a

2.5 (2 – 3) A, a

IH 5% O2 3 (2 – 4) A, a

3 (2 – 4) A, a

3 (2.5 – 3) A, a

IH 1% O2 3 (2 – 4) A, a

3 (2 – 4) A, a

2 (2 – 3.5) A, a

Juiciness δ

IH 21% O2 5 (4 – 5) A, a

4 (4 – 5) A, ab

4 (4 – 5) A, b

IH 5% O2 5 (4 – 5) A, a

4 (3.5 – 5) A, ab

4 (4 – 4) A, b

IH 1% O2 5 (4 – 5) A, a

4 (4 – 5) A, ab

4 (3 – 4) A, b

Fresh-cut honeydew melon

L* β

air 74.0 ± 2.2 A, a

73.3 ± 3.3 A, a

70.3 ± 5.7 A, b

71.6 ± 4.0 A, ab

IH 5% O2 74.0 ± 2.2 A, a

72.3 ± 3.1 A, a

72.2 ± 3.5 A, a

73.4 ± 3.5 A, a

a* β

air -2.5 ± 1.0 A, a

-2.6 ± 0.9 A, a

-2.4 ± 0.8 A, a

-2.2 ± 0.9 A, a

IH 5% O2 -2.5 ± 1.0 A, a

-2.4 ± 0.6 A, a

-2.2 ± 0.9 A, a

-2.4 ± 0.9 A, a

b* β

air 14.4 ± 2.6 A, a

13.3 ± 2.8 A, ab

13.7 ± 2.2 A, ab

12.5 ± 2.9 A, b

IH 5% O2 14.4 ± 2.6 A, a

13.1 ± 1.7 A, ab

12.0 ± 2.4 B, b

13.2 ± 2.7 A, ab

Juice leakage γ

air

1.8 ± 1.1 A, a

0.8 ± 0.4 A, a

1.0 ± 0.6 A, a

IH 5% O2 1.6 ± 0.5 A, a

2.2 ± 1.2 A, a

0.8 ± 0.8 A, a

Firmness δ

air 4 (3 - 4) A, a

3 (3 - 4) A, a

3 (2 - 3) A, b

IH 5% O2 4 (3 - 4) A, a

3 (3 - 4) A, a

3 (3 - 4) A, a

Juiciness δ

air 4 (4 - 4) A, a

4 (3 - 4) A, a

4 (3 - 5) A, a

IH 5% O2 4 (4 - 4) A, a

4 (3 - 4) A, a

4 (3 - 4) A, a

Values followed by the different letters within columns (capital letters) and within lines (small letters) are

significantly different (P < 0.05) according to Tukey’s test and Kruskal-Wallis test.

CHAPTER 3

97

β mean ± standard deviation, n= 30

γ mean ± standard deviation, n= 3

δ The median value (50

th percentile) with values of 25

th percentile and 75

th percentile. Respectively 50, 75 and 25

percent (rounded down) of the assessors gave at least this value or greater. Fractional values were indicated by

assessors as “in between two classes”.

The L* and b* colour parameters and firmness slightly decreased but statistical significant (P <

0.05) in the packages in air during storage, whilst IH 5% O2 only had a small but significant (P <

0.05) effect on b* during storage. The decrease of colour (L* and b*) of fresh-cut pineapple

during storage observed in our study agrees with those obtained by previous studies (Bierhals et

al., 2011; Gonzalez-Aguilar et al., 2004; Marrero et al., 2006; Montero-Calderon et al., 2008).

According to Montero-Calderon et al. (2008) the decreases of L* and b* were due to translucency

development, rather than tissue browning. Low O2 MAs could improve the retention of colour

(Marrero et al., 2006) whilst headspace gas composition did not affect the other physiological

parameters, such as juiciness and firmness, during storage of fresh-cut pineapple (Montero-

Calderon et al., 2008; Santos et al., 2005). Although lowered O2 level (3% O2 + 10% CO2)

maintained acceptable visual quality of lettuce (Barriga et al., 1991), Amaro et al. (2012) reported

that surface colour of fresh-cut honeydew cubes was not affected by packaging atmospheres.

3.3.7 Effect of the initial headspace O2 level on the sensory quality of fresh-cut fruit

The results of the sensory evaluation tests (see Table 3.8) showed that no significant difference

occurred between the samples packaged in different IH O2 levels over the first 3 days of storage.

However, a significant difference (P < 0.05) was found between samples packaged in IH O2 level

of 5% and 21%. Samples packaged in IH O2 levels of 21% and 1% did not differ yet on day 5. In

addition, VOCs related to spoilage microorganisms were found to strongly affect the sensory

quality of pineapple cubes, as most of the assessors rejected pineapple cubes packaged with IH

21% O2 because of the fermented odour, with some assessors even reporting a strong banana

odour at the end of shelf-life. This was probably due to the production by yeasts of 3-methylbut-

1-yl ethanoate, which has a strong banana odour (Fahlbusch et al., 2003).


98

Although some panellists observed that the colour retention of honeydew melon cubes in IH 5%

O2 level was slightly better than those in air, no significant difference (P < 0.05) was found on

honeydew melon cubes packaged in air and IH 5% O2 level during storage (see Table 3.8).

Table 3.8: The results of sensory evaluation of fresh-cut pineapple cubes and honeydew melon cubes

packaged in different initial headspace O2 levels during storage at 7°C. For 11, 13 and 16 assessors, the

critical number of correct responses required to obtain a statistically significant difference (P < 0.05) are 7,

8 and 9 respectively.

Initial headspace O2 levels Day of storage

3 5

Pineapple cubes

21%a - 1% 6

b/11

c 6/13

21% - 5% 4/11 8/13 (P < 0.05)

Honeydew melon cubes

21% - 5% 5/16 5/16

a 21% O2 is the control in triangle tests

b Number of people who correctly identified the odd sample

c Total number of panel

Generally, samples of fresh-cut pineapple and honeydew melon on day 3 of storage were all

acceptable according to the sensory panel, irrespective of the atmospheres they were packaged in.

However, only fresh-cut pineapple packaged in IH 5% O2 was still acceptable on day 5 of storage.

Hence, the sensory shelf-life for fresh-cut pineapple packaged in IH 5% O2 with high gas barrier

film may be extended by 1 – 2 days. However, according to the microbiological criteria for fresh-

cut fruit proposed by Uyttendaele et al. (2010) of a end of shelf-life limit for yeasts of 5 log10

CFU/g, the microbiological shelf-life of fresh-cut pineapple and honeydew melon was exceeded

after only one day of storage, irrespective of initial atmospheres (see Table 3.4). This was

probably due to the fact that the volatile metabolites produced by yeasts may not always have

negative effects on the sensory quality of fresh-cut fruit, even when the amounts produced are

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99

above the human olfactory thresholds. However, the VOCs evaluated in this thesis surely affected

the sensory quality of fresh-cut fruit at the end of sensory shelf-life.

3.4 Conclusions

Effects of headspace O2 levels were observed on the maximum population densities, with

generally higher densities at higher IH O2 levels for all spoilage yeasts investigated on pineapple

agar and honeydew melon agar. The production of most VOCs by these yeasts was significantly

lower in low O2 atmospheres (0 - 5% O2) than in air on fruit agar, results which are in agreement

with the validation study performed on fresh-cut fruit cubes. Unlike yeasts, L. mesenteroides

produced much less quantity and quality of VOCs on honeydew melon agar. The sensory quality

(shelf-life) of fresh-cut pineapples was also determined to be dependent on the IH O2 level, with a

longer shelf-life being observed on samples packaged in an IH O2 level of 5% compared to 21%.

Therefore, the application of low O2 MAP (5% O2, rest N2) could be used to extend the shelf-life

(1 – 2 days) of fresh-cut pineapples by reducing the quantity of volatile metabolites formed and

improving the retention of colour during storage. Fresh-cut honeydew melon in low O2 level had

slightly better colour retention. However, no significance was found between those in air and low

O2 level during storage in sensory quality by triangle tests and firmness and juiciness assessment.

As use of different types of packaging films could have resulted in different results, it should be

stressed that the results observed in this chapter are pertinent to high gas barrier films. After the

evaluation of reduced headspace O2 level on the growth and the production of VOCs of

microorganisms of fresh-cut fruit, the focus of the study shifted in Chapter 4 to determining the

effect of MAs combining high O2 and CO2 levels on the microbial spoilage and the shelf-life of

fresh-cut fruit.


100

CHAPTER 4

EFFECT OF ATMOSPHERES COMBINING HIGH OXYGEN

AND CARBON DIOXIDE LEVELS ON THE MICROBIAL

SPOILAGE AND SENSORY QUALITY OF FRESH-CUT

PINEAPPLE AND HONEYDEW MELON

Chapter 4: Effect of Atmospheres Combining High Oxygen and Carbon Dioxide Levels on the

Microbial Spoilage and Sensory Quality of Fresh-cut Pineapple and Honeydew Melon

Redrafted from:

Zhang, B.-Y., Samapundo, S., Pothakos, V., de Baenst, I., Sürengil, G., Noseda, B., Devlieghere,

F. 2013. Postharvest Biology and Technology 86, 73-84.

Zhang, B.-Y., Samapundo, S., Pothakos, V., Sürengil, G., Devlieghere, F. 2013. International

Journal of Food Microbiology 166, 378-390.

HIGH OXYGEN AND CARBON DIOXIDE ATMOSPHERES ON FRUIT

102

Summary

Fresh-cut fruit such as pineapple and honeydew melon have a very limited shelf-life. The study

had the major objective of prolonging the shelf-life of fresh-cut pineapple and honeydew melon

by means of modified atmospheres (MAs). The effect of MAs combining high O2 (21 - 70%) and

CO2 (21 - 50%) levels on the microbial spoilage and sensory quality of fresh-cut pineapple and

honeydew melon was therefore evaluated. In the first part of the study, the behaviour of spoilage

microorganisms of fresh-cut pineapple (Candida sake, Candida argentea and Leuconostoc

citreum) and honeydew melon (Candida sake, Leuconostoc mesenteroides and Leuconostoc

gelidum) was monitored on pineapple agar and honeydew melon agar separately. In the second

part of the study, the shelf-life of commercial fresh-cut pineapple cubes and honeydew melon

cubes packaged in selected MAs was evaluated at 7°C. The results showed that MAs combining

high O2 and high CO2 levels had a large inhibitory effect on the growth and volatile metabolite

production of spoilage yeasts on both pineapple agar and honeydew melon agar, especially MAs

consisting of 50% O2 + 50% CO2 and 70% O2 + 30% CO2. In difference, the MAs evaluated

only had a minor effect on the growth and volatile metabolite production of lactic acid bacteria.

Overall, the effect of the MAs evaluated on colour, juice leakage, juiciness, firmness of fresh-cut

honeydew melon and pineapple was small during storage. Sensory preference was generally for

fresh-cut fruit packaged in MA of 50% O2 + 50% CO2. Fresh-cut honeydew melon and pineapple

packaged in air had expanded and were not acceptable any more by day 5 and day 7, respectively,

while those packaged in 50% O2 and 50% CO2, which also retarded the growth of aerobes and

yeasts during storage, were still acceptable. Therefore, 50% O2 + 50% CO2 is a potential MA

solution for extending the shelf-life of fresh-cut pineapple and honeydew melon.

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103

4.1 Introduction

MAP with high O2 levels has recently been suggested as an alternative to the use of low O2

levels to extend the shelf-life by inhibiting the growth of naturally occurring spoilage

microorganisms, preventing fermentation and decay of fresh and fresh-cut produce (Day, 2001;

Jacxsens et al., 2001; Oms-Oliu et al., 2008e; Wszelaki and Mitcham, 2000). However, the

sensitivity of different organisms to O2 may vary greatly. Additionally, high O2 MAP with even

ca. 99% O2 cannot prevent the growth of Pseudomonas fragi, Aeromonas hydrophila, Yersinia

enterocolitica, and Listeria monocytogenes (Kader and Ben-Yehoshua, 2000). To date only a

handful of studies have been performed in which the positive effect of atmospheres combining

high O2 and CO2 on the shelf-life of fresh-cut vegetables has been reported (Amanatidou et al.,

2000; Conesa et al., 2007). No studies have yet been published regarding the effect of such types

of atmospheres on the shelf-life of fresh-cut fruit.

The objective of this study was to investigate the effect of modified atmospheres (MAs)

consisting of elevated levels of O2 (21-70%) and CO2 (21-50%) on the growth and volatile

organic compound (VOC) production of the spoilage organisms of fresh-cut pineapple and

honeydew melon on simulation agar. Additionally, the effect of the headspace O2 and CO2 levels

on the shelf-life of fresh-cut pineapple cubes was evaluated in order to evaluation the effect of

these atmospheres on the evolution of the microbiological, physical and sensory quality.


The experiments performed in this study were performed in two stages. The experimental

methodology developed in Chapter 3 was used in this chapter. In the first part of the study, the

spoilage isolates of fresh-cut fruit were individually inoculated on fruit agar which was then

packaged in MAs with different combinations of initial headspace (IH) O2 (21-70%) and CO2 (0-

50%) levels and stored at 7°C. In the second part of the study, commercial fresh-cut pineapple

and honeydew melon cubes were packaged in air and selected MA(s) and stored at 7°C. The


104

atmospheres evaluated in this part were selected on the basis of the results of the experiments

performed on fruit agar. The methods used in each part of this study are described in detail below.

4.2.1 Effect of MA on growth and volatile metabolite production of spoilage isolates

4.2.1.1 Isolates

The experiments on pineapple simulation agar were performed using Candida argentea (FF 581),

Candida sake (FF 641) and Leuconostoc citreum (PM 42) previously isolated from spoiled

commercial pineapple cubes (see Chapter 2). The experiments on honeydew simulation agar

were performed using Candida sake (FF 655), Leuconostoc mesenteroides (FF653) and

Leuconostoc gelidum (HM45) previously isolated from spoiled commercial honeydew melon

cubes (see Chapter 2). All the isolates are maintained in the culture collection of the Laboratory

of Food Microbiology and Food Preservation (Ghent University, Ghent, Belgium).

4.2.1.2 Preparation and inoculation of fruit agar

The method used to prepare pineapple agar and honeydew melon agar is descripted in Chapter 3

(see § 3.2.1.2). After the agar was prepared and had cooled to 48°C, 71 ± 0.2 g of pineapple agar

or honeydew melon agar were poured into trays (volume = 269 mL, O2 transmission rate (OTR)

= 0.5 - 13 cm3/m

2.d.bar at 23°C, 0% relative humidity (RH), polypropylene (PP)/ethylene vinyl

alcohol (EVOH), DECAPAC NV, Herentals, Belgium). For safety reasons, the packaging

machine used in Chapter 3 (MULTIVAC A300/42 packaging machine) could not be used to

package samples in high O2 concentrations. Therefore, in this chapter a tray sealer (MECA 900,

DECAPAC NV, Herentals, Belgium) was used. This entailed the use of trays in this chapter as

opposed to Petri plates in Chapter 3, which in turn resulted in a different gas-to-product ratio (ca.

1/3).

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105

To prepare the inoculum, the yeasts were individually sub-cultured in 10 mL of sterile

Sabouraud Broth [SB; Oxoid (Hampshire, UK)] whilst the LAB was sub-cultured in 10 mL of de

Man Rogosa Sharpe broth [MRS broth, Oxoid (Hampshire, UK)] at 22 ± 1°C for 2 days. Second

sub-cultures were prepared in the same media as the first sub-cultures and incubated for the same

duration at the same temperature. Thereafter, the second sub-cultures were transferred to a

refrigerator at 7 ± 1°C for 7 hours to adapt the spoilage isolates to the final incubation

temperature used in the experiments. 100 µl aliquots of appropriate dilutions (ca. 105-6

CFU/mL)

of the temperature adapted isolates were separately inoculated and spread on fruit agar in trays

resulting in an initial inoculation level of 102-3

CFU/cm² of agar. Inoculated trays were

individually sealed by the tray sealer using a high O2 barrier film (OTR = 5 cm3/m

2.d.bar at 23°C,

50 % RH. OPA(oriented polyamide)/EVOH/PE (Polyethylene)/PP, BEMIS EUROPE Flexible

Packaging, Monceau-sur-Sambre, Belgium) in the following initial conditions: 21% O2 + 21%

CO2, 50% O2 + 30% CO2, 50% O2 + 50% CO2, 70% O2 + 30% CO2 and 21 % O2 (air), all

balanced with N2 and stored at 7°C ± 1.

Two trays were prepared per atmosphere per isolate per sampling moment (days 0, 2, 4, 6, 8, 10

and in some cases day 12 or day 14) for the purpose of assessing the microbial growth,

headspace gas composition, VOCs, pH and sugar levels. The headspace O2 and CO2 levels in the

tray were first measured by a headspace analyser (CheckMate 9900 O2, O2/CO2 Headspace

Analyser, PBI – Dansensor, Denmark). Subsequently, the packages were opened aseptically and

20 0.1g of honeydew melon agar was immediately transferred to a sterile plastic container (60

mL) and closed quickly. This sample was used for quantification of VOCs by means of SIFT-MS

(Selected Ion Flow Tube Mass Spectrometer, Voice 200, Syft Technologies). The rest of the agar

in the tray was used for the measurement of the pH, sugars and assessment the growth of the

spoilage isolates.

4.2.1.3 Microbial analysis

On each day of analysis a piece of inoculated agar (ca. 10g) was aseptically transferred to a

sterile stomacher bag. Primary decimal dilutions of each sample were prepared by adding an


106

appropriate volume of physiological peptone saline solution [PSS, 8.5g NaCl; 1g peptone per

litter, Oxoid (Hampshire, UK)]. The samples were homogenized for 30 seconds in a stomacher

(Stomacher Lab-Blender 400, Led Techno, Eksel, Belgium). Subsequent decimal dilutions were

then prepared from the primary decimal dilution in test-tubes containing 9 mL of sterile PSS.

The decimal dilutions were then spread plated on Yeast Glucose Chloramphenicol agar [YGC,

Bio-Rad (Marnes-la-Coquette, France)] for the spoilage yeasts whilst the LAB was determined

by pour plating on de Man Rogosa Sharpe agar [MRS agar, Oxoid (Hampshire, UK)]. The plates

were then incubated at 22 ± 1°C until the colonies were sufficiently large for enumeration (2-3

days). The counts (n) of the isolates on fruit agar were expressed in log10 CFU/cm2. Based on the

surface area (256.2 cm²) of the agar plastic trays on which the inoculation was done and the

weight of the agar on the trays (71 ± 0.1g), conversion between log10 CFU/cm2 and log10 CFU/g

could be done according Eq. (4.1):

n Log10 CFU/cm2 = (n + 0.41) Log10 CFU/g (Eq. 4.1)

4.2.1.4 Quantification of volatile organic compounds

The VOCs were identified by GC-MS in § 3.2.1.3 in Chapter 3. The quantification of the VOCs

by SIFT-MS was performed as describes in § 3.2.1.4.

4.2.1.5 Sugar and acid analysis

The concentrations of sucrose, glucose, fructose, lactic acid and acetic acid in fruit agar samples

were determined as described in § 3.2.1.6.

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107

4.2.2 Effect of MAs on the shelf-life of fresh-cut pineapple and honeydew melon

4.2.2.1 Packaging of fresh-cut pineapple and honeydew melon

Fresh-cut fruit were received and packaged in plastic trays as described in § 3.2.2 of Chapter 3.

Compared to Chapter 3, different atmospheres were evaluated (air, IH 50% O2 + 30% CO2, IH

50% O2 + 50% CO2). The trays were then stored at 7 ± 1°C for up to 7 days for fresh-cut

pineapple and 5 days for fresh-cut honeydew melon. Two trays per MA were randomly selected

at each sampling date (fresh-cut pineapple sampling days 0, 1, 3, 5 and 7; fresh-cut honeydew

melon sampling days 0, 1, 3 and 5) for headspace gas composition analysis, VOCs measurement,

pH; sugars and microbial analysis. Besides, 10 trays packaged in both air and MA were chosen

for sensory evaluation for every analysis date. As the evaluation of the MAs on honeydew melon

cubes was performed at the same time in this chapter and in Chapter 3, the results of the

parameters evaluated on honeydew melon cubes packaged in 21% O2 (control sample) reported

in this chapter are the same as that in Chapter 3.

4.2.2.2 Effect of MAs on the microbial quality of fresh-cut fruit

The microbiological quality (total aerobic, total anaerobic, LAB and yeast counts) of the cut

pineapple and honeydew melon samples packaged in the different IH O2 and CO2 levels was

determined in duplicate on days 0, 1, 3, 5 or even up to day 7 in fresh-cut pineapple of storage at

7°C. As done in the experiments on the simulation agar, the headspace O2 and CO2 levels in two

packages per condition were first measured before the packages were opened. Subsequently, the

packages were opened and samples were aseptically collected for measurement of the pH (ca.

15g) and the microbiological quality (ca. 30g). The samples for assessment of the

microbiological quality were transferred to a sterile stomacher bag and primary and subsequent

decimal dilutions were prepared in PSS as described above for the experiments on pineapple agar.


108

To determine the yeasts the decimal dilutions were spread plated on YGC, whilst the total

aerobes and total anaerobes were determined by pour plating the dilution on Plate Count Agar

[PCA, Oxoid (Hampshire, UK)] and Reinforced Clostridial Agar [RCA, Oxoid (Hampshire, UK)]

respectively. LAB were determined by pour plating the dilution on MRS agar by adding 1.4g/L

Sorbic Acid (Sigma–Aldrich, St. Louis, MO, USA) to adjust the pH of media to 5.7 before

boiling. The plates were then incubated at 22 ± 1°C until the colonies were sufficiently large for

enumeration (2 - 3 days).

4.2.2.3 Volatile organic compounds and sugars of fresh-cut honeydew melon

On each day of sample analysis, 20g of pineapple cubes or honeydew melon cubes were

aseptically collected from each tray and put in a 60mL plastic container and then stored in a

freezer at -18 ± 1°C until analysis. VOCs quantification by SYFT-MS was performed as is

described in § 4.2.1.4.

The samples used for sugars (ca. 20g each) were thoroughly homogenized in a Stomacher bag

with an equal amount of distilled water for 2 min. Further preparation of the samples as well as

the analysis with HPLC was performed as is described in § 4.2.1.5.

4.2.2.4 Assessment of colour and juice leakage

Assessment of colour and juice leakage was performed as described in § 3.2.2.3 in Chapter 3.

4.2.2.5 Sensory quality of fresh-cut fruit

Triangle tests were used to determine if the MAs combining high O2 and high CO2 levels had an

impact on the sensory quality of fresh-cut pineapple cubes and honeydew melon cubes. The tests

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109

evaluated potential differences between samples packaged in air (reference condition) to those

packaged in IH 50% O2 + 50% CO2 for the cut honeydew melon and IH 50% O2 + 30% CO2 and

IH 50% O2 + 50% CO2 for fresh-cut pineapple. The sensory evaluation was performed in a

purpose built sensory analysis room (UGent Sensolab, Ghent University) by 16 assessors (8

female assessors and 8 male assessors). The procedures followed are described in ISO 4120:2004

(Sensory analysis – Methodology – Triangle test) (ISO, 2004). The detailed procedure of the

triangle tests is described in § 3.2.2.4.

Firmness and juiciness of fresh-cut pineapple and honeydew melon were also evaluated as

described in § 3.2.2.4 in Chapter 3.


An individual tray was used as one replicate on each sampling day, per treatment. The results of

triangle tests were analysed according to ISO 4120:2004 (ISO, 2004). For 16 assessors, the

critical number of correct responses required to obtain a statistically significant (α = 0.05)

difference are 9.

The maximum growth rates (µMAX, log CFU/cm2/day) of the spoilage isolates were estimated by

fitting the Baranyi growth model to the data (Baranyi et al., 1993) using the non-linear regression

function of SPSS version 21 (IBM, SPSS, Chicago, IL, USA). To determine the effect of storage

time and MAs on colour and juice leakage, proper ANOVA contrasts were used, with significant

differences being established at P < 0.05. Significant differences in juiciness and firmness were

analysed by Kruskal-Wallis test due to the ordinal nature of these data. These statistical analyses

were also performed in SPSS. Where applicable, the air packages functioned as the reference

condition by employing suitable contrasts in the statistical analyses.


110


4.3.1 Effect of MA on the growth of spoilage isolates on fruit agar

The effect of modified atmospheres combining high O2 and high CO2 on the growth of

C. argentea and C. sake on pineapple agar was large, but it was minor for the growth of

L. citreum at 7°C (see Table 4.1A and Figure 4.1). For instance, the initial population density of

C. argentea and C. sake on pineapple agar was 2.4 - 3.2 log10 CFU/cm2 and the population of

these yeasts increased to over 5 log10 CFU/cm2, the level at which yeasts are generally known to

start causing spoilage (Fleet, 1992) after ca. two days in air, whilst it took 3 - 10 days in MAs

with high O2 and CO2 levels. The longest time to reach 5 log10 CFU/cm² occurred in an initial

atmosphere with 50% O2 + 50% CO2. However, L. citreum only reached 5.9 - 6.2 log10 CFU/cm2

after 14 days on pineapple agar, irrespective in air and all MAs evaluated. The final population

density did not exceed the end of shelf-life values for LAB (7 - 8 log10 CFU/g) recommended by

Uyttendaele et al. (2010) for fresh-cut fruit.

It can be seen in Table 4.1A and Figure 4.1 that the fastest growth of C. argentea and C. sake on

pineapple agar occurred in air at which the stationary phase (ca. 8 log10 CFU/cm²) was reached

after 6 days incubation at 7°C. Retardation of the growth of both C. sake and C. argentea

occurred when the IH O2 and CO2 levels were increased. The inhibitory effect on growth was

greatest in IH 50% O2 + 50% CO2 for both C. sake and C. argentea. The µMAX estimated for

growth in IH 50% O2 + 50% CO2 were ca. 1/3 than those in air. However, the growth of the

spoilage yeasts was inhibited when the IH O2 at high CO2 levels increased, combining with high

IH CO2, as can be seen when growth in IH 70% O2 + 30% CO2 and IH 50% O2 + 30% CO2 are

compared. The inhibitory effect of CO2 on the yeasts increased when the IH CO2 level was

increased. This was observed when the growth in IH 50% O2 + 50% CO2 was compared to

growth in IH 50% O2 + 30% CO2 (see Table 4.1A and Figure 4.1). Additionally, the λ estimated

for yeasts in IH 50% O2 + 50% CO2 were the largest compared to other atmospheres. However,

both λ and µMAX estimated for L. citreum on pineapple agar had no appreciable differences

among the atmospheres evaluated.

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111

Figure 4.1: Growth curves of C. argentea, C. sake and L. citreum during incubation on pineapple agar in

different initial headspace O2 and CO2 levels (() air, () IH 21% O2 + 21% CO2, () IH 50% O2 + 30%

CO2, () IH 50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2) at 7°C. Data points are the means,

with bars representing the two measured values.

It can be seen in Table 4.1B and Figure 4.2 that the MAs with high O2 and CO2 levels also had a

large inhibitory effect on the growth of C. sake on honeydew melon agar. On the other hand, the

effect of the MAs evaluated was negligible on growth of the LAB on honeydew melon agar (see

Table 4.1B and Figure 4.2).

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12

log

10

CF

U/c

m2

Time (days)

C. argentea

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12

log

10

CF

U/c

m2

Time (days)

C. sake

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12 14

log

10

CF

U/c

m2

Time (days)

L. citreum


112

Table 4.1: Estimated growth parameters (lag phase λ (day), maximum specific growth rate µmax (log10

(CFU/cm2)/day) and initial count N0 (log10 CFU/cm

2) of the yeasts and lactic acid bacteria on fruit agar,

stored under different high O2 combined with high CO2 at 7°C for 8-14 days, with standard deviation.

Isolates Package condition λ µmax R2

(A) Pineapple agar

C. argentea

21% O2 0.3 ± 0.2 1.2 ± 0.1 0.999

21% O2 + 21% CO2 0.5 ± 0.9 0.6 ± 0.1 0.994

50% O2 + 30% CO2 1.1 ± 1.6 0.5 ± 0.1 0.987

50% O2 + 50% CO2 2.4 ± 0.18 0.3 ± 0.2 0.987

70% O2 + 30% CO2 0 0.3 ± 0.0 0.999

C. sakes

21% O2 1.2 ± 0.1 1.4 ± 0.1 0.999

21% O2 + 21% CO2 1.2 ± 0.4 0.8 ± 0.1 0.997

50% O2 + 30% CO2 1.4 ± 0.5 0.6 ± 0.1 0.997

50% O2 + 50% CO2 2.7 ± 0.3 0.5 ± 0.0 0.999

70% O2 + 30% CO2 1.2 ± 0.6 0.5 ± 0.1 0.997

L.citreum

21% O2 0 0.1 ± 0.2 0.981

21% O2 + 21% CO2 0 0.1 ± 0.2 0.980

50% O2 + 30% CO2 0 0.1 ± 0.2 0.983

50% O2 + 50% CO2 0 0.1 ± 1.2 0.978

70% O2 + 30% CO2 0 0.1 ± 1.6 0.998

(B) Honeydew melon agar

C. sake

21% O2 0.5 ± 0.7 1.2 ± 0.3 0.996

21% O2 + 21% CO2 1.0 ± 0.6 1.0 ± 0.2 0.995

50% O2 + 30% CO2 0.7 ± 0.6 0.7 ± 0.1 0.998

50% O2 + 50% CO2 0 0.4 ± 0.1 0.994

70% O2 + 30% CO2 0 0.4 ± 0.1 0.997

L. mesenteroides

21% O2 1.4 ± 0.5 1.4 ± 0.2 0.997

21% O2 + 21% CO2 0.4 ± 0.7 1.3 ± 0.2 0.994

50% O2 + 30% CO2 0 1.0 ± 0.1 0.999

50% O2 + 50% CO2 0 0.8 ± 0.1 0.997

70% O2 + 30% CO2 0.2 ± 0.8 1.0 ± 0.2 0.994

L. gelidum

21% O2 0.7 ± 0.7 1.2 ± 0.3 0.992

21% O2 + 21% CO2 0.9 ± 0.4 1.3 ± 0.2 0.996

50% O2 + 30% CO2 1.2 ± 1.4 1.2 ± 0.5 0.953

50% O2 + 50% CO2 1.4 ± 1.0 1.1 ± 0.4 0.973

70% O2 + 30% CO2 1.1 ± 1.4 1.2 ± 0.7 0.936

The growth of L. mesenteroides and L. gelidum in all MAs reached the stationary phase after 6 -

8 days of incubation. The µMAX and λ of L. mesenteroides was slightly higher in air than in any

of the MAs evaluated. Unlike L. mesenteroides, MA had no appreciable effect on the µMAX and λ

of L. gelidum.

Generally, the effect of MAs on the growth of LAB on pineapple agar and honeydew melon agar

was small, compared to the appreciable retardation observed on the growth of yeasts. This is

CHAPTER 4

113

mostly due to the fact that LAB are not sensitive to high O2 and CO2 levels (Amanatidou et al.,

1999; Lyhs et al., 2004). As it has been discussed in § 1.2.1.3 of Chapter 1, lactic acid bacteria

grow well under anaerobic conditions and they can also grow in the presence of oxygen due to

that they do not use oxygen in their energy production.

Figure 4.2: Growth curves of C. sake, L. mesenteroides and L. gelidum during incubation on honeydew

melon agar in different initial headspace O2 and CO2 levels (() air, () IH 21% O2 + 21% CO2, () IH

50% O2 + 30% CO2, () IH 50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2) at 7°C. Data points are

the means, with bars representing the two measured values.

It has been previously reported that 21% O2 + 15% CO2 greatly retarded the macroscopic decay

of fresh-cut honeydew melon (Portela et al., 1998) and microbial growth for fresh-cut ‘Amarillo’

melon (Aguayo et al., 2007). In contrast, Amanatidou et al. (1999) reported that high O2 levels

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10

Lo

g10

CF

U/c

m2

Time (days)

L. mesenteroides

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10 12

Lo

g10

CF

U/c

m2

Time (days)

L. gelidum

2

3

4

5

6

7

8

9

10

0 2 4 6 8 10 12

Lo

g10

CF

U/c

m2

Time (days)

C. sake


114

(80%) considerably stimulated the rate of growth of C. sake and Candida guilliermondii on an

agar-surface model system. Although the inhibitory effects of high O2 atmospheres to yeasts

have been reported in some studies (Conesa et al., 2007; Zheng et al., 2008), the mechanism(s)

are yet unclear. According to Moradas-Ferreira et al. (1996), reactive oxygen species (ROS),

notably superoxide (O2-) and hydroxyl (OH

-) radicals, hydrogen peroxide (H2O2) and singlet

oxygen (1O2), can originate in high O2 partial pressures resulting in damage to cellular

components (via oxidation of lipids, proteins and nucleic acids) when ROS levels exceed the

antioxidant capacity of yeast cells (oxidative stress). On the other hand, higher CO2 levels in the

headspace result in higher concentrations of dissolved CO2 (dCO2) in the media, thereby

increasing the inhibition (= decreasing the growth rate) of spoilage microorganisms (Devlieghere

and Debevere, 2000; Jones and Greenfield, 1982). The proposed mechanisms for the inhibitory

effect of CO2 include alterations in membrane fluidity, direct inhibition of certain enzyme

activities and internal acidification by the hydration of CO2 into carbonic acid (H2CO3). However,

all of these are still essentially hypothetical (Aguilera et al., 2005; Dixon and Kell, 1989; Garcia-

Gonzalez et al., 2007). Aguilera et al. (2005) reported that the growth of Saccharomyces

cerevisiae was more sensitive to CO2 in the presence of O2 than it was to the absence of O2 in

glucose-limited chemostat cultures. Therefore, it may be inferred that high O2 combined with

high CO2 can achieve stronger inhibition to yeasts than high O2 or high CO2 used alone.

L. mesenteroides and L. gelidum have been determined to be the predominant LAB in vegetables

and fruit packaged in high O2 and CO2 MAs (Amanatidou et al., 1999; Lyhs et al., 2004).

Although LAB cannot synthesize heme and cytochrome oxidases, for most LAB, NADH oxidase

activity plays an important role for protection against ROS during aerobic respiration or during

encounters with oxidative stress (Axelsson, 2004; Higuchi et al., 2000). However, LAB

generally do not have the same potential to protect themselves against the toxic effect of oxygen

as genuine aerobic organisms (Axelsson, 2004). Leuconostocs may possess manganese-

accumulating systems, which are based on the specific accumulation of Mn2+

to high

intracellular concentrations (30 - 35 mM) and have a scavenging effect on superoxide (Axelsson,

2004; Whittaker, 2010).

As it can be seen in Figures 4.3-4.4, differences were observed between the evolution of

headspace CO2 and O2 levels in the packages containing agar inoculated with yeasts on one hand

CHAPTER 4

115

and those inoculated with the LAB on the other hand. The most notable difference was that

whilst the evolution of the headspace CO2 and O2 levels was dependent on the initial atmosphere

in the packages inoculated with yeasts, this was not apparent for the LAB. Yeasts consumed O2

and produced CO2 after 4 days of incubation in the packages with air and after 4 - 8 days in IH

20% O2 + 20% CO2 and IH 50% O2 + 30% CO2.

Figure 4.3: The evolution of headspace O2 and CO2 concentrations in the packages with pineapple agar

inoculated with C. argentea (A and B) and C. sake (C and D) during incubation in different initial

headspace O2 and CO2 levels (() air, () IH 21% O2 + 21% CO2, () IH 50% O2 + 30% CO2, () IH

50% O2 + 50% CO2 and () IH 70% O2 + 30% CO2) at 7°C. Data points are the means, with bars

representing two measured values.

0

20

40

60

80

100

0 2 4 6 8 10 12

O2

(%)

Time (days)

A

0

20

40

60

80

100

0 2 4 6 8 10 12

CO

2(%

)

Time (days)

B

0

20

40

60

80

100

0 2 4 6 8 10 12

O2

(%)

Time (days)

C

0

20

40

60

80

100

0 2 4 6 8 10 12

CO

2(%

)

Time (days)

D


116

Figure 4.4: The evolution of O2 and CO2 concentrations in the headspace during incubation of C. sake,

L. mesenteroides and L. gelidum on honeydew melon agar in different initial modified atmospheres (()

21% O2, () 21% O2 + 21% CO2, () 50% O2 + 30% CO2, () 50% O2 + 50% CO2 and () 70% O2 +

30% CO2) at 7°C. Data points are the means, with bars representing two measurement values.

0

20

40

60

80

100

0 2 4 6 8 10

O2

(%)

Time (days)

C. sake

0

20

40

60

80

100

0 2 4 6 8 10

CO

2(%

)

Time (days)

C. sake

0

20

40

60

80

100

0 2 4 6 8 10

O2

(%)

Time (days)

L. mesenteroides

0

20

40

60

80

100

0 2 4 6 8 10

CO

2(%

)

Time (days)

L. mesenteroides

0

20

40

60

80

100

0 2 4 6 8 10 12

O2

(%)

Time (days)

L. gelidum

0

20

40

60

80

100

0 2 4 6 8 10 12

CO

2(%

)

Time (days)

L. gelidum

CHAPTER 4

117

With the exception of the slight increase and decrease in headspace O2 and CO2 levels at the

beginning of incubation, due to the dissolution of CO2 in the water-phase of the fruit agar, the

yeasts did not consume O2 nor produce CO2 in the packages with IH 50% O2 + 50% CO2 and 70%

O2 + 30% CO2. This was most likely due to growth retardation in these two MAs. The headspace

gas composition in the packages containing pineapple agar inoculated with L. citreum did not

change during the incubation, except for a slight increase in the headspace O2 levels and a slight

decrease of the headspace CO2 levels at the beginning of incubation (data not shown). As

observed for the yeasts and L. citreum, the headspace O2 and CO2 levels in the packages

containing honeydew melon agar inoculated with L. mesenteroides and L. gelidum (and

packaged with initially at least 20% CO2) slightly increased and decreased, respectively, at the

beginning of incubation. Thereafter, the headspace O2 and CO2 levels remained stable until day 6

of incubation. From day 6 onwards the headspace CO2 levels in the packages with honeydew

melon agar inoculated with LAB appreciably increased, whilst the O2 levels decreased slightly.

The slight decrease in headspace O2 levels was mostly likely a result of compensation for the

CO2 produced as opposed to actual consumption by the LAB.

The pH of pineapple agar (3.7 ± 0.1) inoculated with either yeasts or LAB did not change

appreciably during incubation. However, the pH of honeydew melon agar inoculated with C.

sake and packaged in IH 50% O2 + 50% CO2 and 70% O2 + 30% CO2 decreased from 5.8 to 5.5

during incubation. A larger pH reduction from ca. 6 to lower than 5 occurred on the agar

inoculated with C. sake and packaged in air and the other MAs evaluated in this study (see

Figure 4.5). This was mostly due to the production of lactic acid (data not shown). The pH of

honeydew melon agar inoculated with L. mesenteroides and L. gelidum decreased to ca. 4 and 5 -

5.5, respectively (see Figure 4), as a result of the production of lactic acid and acetic acid (ca.

0.23 and 0.19, respectively) towards the end of incubation in air and other MAs. The pH of

honeydew melon agar was generally lower at the end of incubation on the samples packaged in

air and 21% O2 + 21% CO2 than it was on the other MAs that were evaluated. Our results

showed that the pH of honeydew melon agar usually started to decrease when the population

densities of the yeast or LAB were higher than ca. 6 log10 CFU/cm2.


118

Figure 4.5: The pH changes of C. sake, L. mesenteroides and L. gelidum during the incubation on

honeydew melon agar in different initial modified atmospheres (() 21% O2, () 21% O2 + 21% CO2,

() 50% O2 + 30% CO2, () 50% O2 + 50% CO2 and () 70% O2 + 30% CO2) at 7°C. Data points are

the means, with bars representing two measurement values.

4.3.2 Effect of MAs on volatile metabolite production and sugar consumption of spoilage

isolates

4.3.2.1 VOCs produced by yeasts

Volatile organic compounds produced by the yeasts evaluated in this study (see Table 3.1 of

Chapter 3) were quantified during the incubation of inoculated pineapple agar and honeydew

4.5

5.0

5.5

6.0

6.5

0 2 4 6 8 10 12

pH

Time (days)

C. sake

3.5

4.0

4.5

5.0

5.5

6.0

6.5

0 2 4 6 8 10

pH

Time (days)

L. mesenteroides

4.5

5.0

5.5

6.0

6.5

0 2 4 6 8 10 12

pH

Time (days)

L. gelidum

CHAPTER 4

119

melon agar packaged in the MAs investigated. The mechanism of these VOCs produced by

yeasts has been discussed previously (see § 3.3.3.3 of Chapter 3). A similar pattern was observed

with regards to the effect of MA on the production of VOCs by the yeasts. VOC production by

the yeasts in air took place after ca. only 4 days while it was retarded for 12 days in the other

MAs evaluated as shown for some VOCs in Figures 4.6 - 4.8. The lowest quantities of VOCs

were produced in IH 50% O2 + 50% CO2, followed by IH 70% O2 + 30% CO2 and IH 50% O2 +

30% CO2. The highest quantities of VOCs were produced in air and IH 21% O2 + 21% CO2. As

an example, whilst the quantity of ethanol produced by C. sake and C. argentea on pineapple

agar increased to 138 - 200 mg/m3 after 8 days incubation in air, it was only 4.8 - 7.6 mg/m

3 in

MA with IH 50% O2 + 50% CO2 after 12 days incubation.

4.3.2.2 VOCs produced by LAB

Ethanol, acetoin, acetic acid, butane-2,3-dione (diacetyl) and butane-2,3-diol detected originate

from the heterofermentation of sugar by Leuconostoc spp. (Axelsson, 2004). However, no

volatile metabolites in the headspace changed appreciably throughout incubation of L. citreum

on pineapple in all atmospheres (data not shown) which could mostly due to its final low

population density on pineapple agar and the low pervasive character of the metabolites of the

LAB (Jacxsens et al., 2003). Even though the LAB grew at a faster rate and to higher population

densities than the yeasts, L. mesenteroides and L. gelidum produced fewer types and lower

amounts of VOCs on honeydew melon agar. Of the VOCs detected on the honeydew melon agar

inoculated with the LAB, only the butane-2,3-dione, acetic acid and acetoin increased during

incubation (see Figure 4.9). Unlike the results observed for the yeasts, the effect of the MAs

evaluated on the production of these VOCs by the LAB was minor. Butane-2,3-dione, acetic acid

and acetoin production by L. gelidum was generally higher in air than in the other MAs evaluated

whereas acetic acid production by L. mesenteroides was slightly higher in 70% O2 + 30% CO2

than other MAs at the end of incubation.


120

Figure 4.6: VOCs produced by C. argentea in MAP on pineapple agar during storage at 7°C.

0

40

80

120

160

0 2 4 6 8 10 12

mg

/m3

Time (days)

A

0

15

30

45

60

0 2 4 6 8 10 12

mg

/m3

Time (days)

B

0

10

20

30

40

50

0 2 4 6 8 10 12

mg

/m3

Time (days)

C

0

50

100

150

200

250

0 2 4 6 8 10 12

mg

/m3

Time (days)

D

0

10

20

30

40

50

0 2 4 6 8 10 12

mg

/m3

Time (days)

E

0

250

500

750

1000

1250

0 2 4 6 8 10 12

mg

/m3

Time (days)

F

0

30

60

90

120

0 2 4 6 8 10 12

mg

/m3

Time (days)

G

0

20

40

60

80

100

0 2 4 6 8 10 12

mg

/m3

Time (days)

H

0

3

6

9

12

15

0 2 4 6 8 10 12m

g/m

3

Time (days)

I

VOCs:

(A) Ethanol


(C) Butan-1-ol

(D) Ethyl acetate

(E) Ethyl butanoate


(G) Hexanoic acid

(H) Nonanal

(I) 3-Methylbutanal

MAP:

() 21% O2

() 21% O2 + 21% CO2

() 50% O2 + 30% CO2

() 50% O2 + 50% CO2

() 70% O2 + 30% CO2

Data points are the

means, with bars

representing two

measurement values.

CHAPTER 4

121

Figure 4.7: VOCs produced by C. sake in MAP on pineapple agar during storage at 7°C.

0

60

120

180

240

0 2 4 6 8 10 12

mg

/m3

Time (days)

A

0

30

60

90

120

0 2 4 6 8 10 12

mg

/m3

Time (days)

B

0

30

60

90

120

0 2 4 6 8 10 12

mg

/m3

Time (days)

C

0

200

400

600

800

0 2 4 6 8 10 12

mg

/m3

Time (days)

D

0

30

60

90

120

0 2 4 6 8 10 12

mg

/m3

Time (days)

E

0

250

500

750

1000

1250

0 2 4 6 8 10 12

mg

/m3

Time (days)

F

0

20

40

60

80

100

0 2 4 6 8 10 12

mg

/m3

Time (days)

G

0

45

90

135

180

0 2 4 6 8 10 12

mg

/m3

Time (days)

H

0

14

28

42

56

70

0 2 4 6 8 10 12m

g/m

3

Time (days)

I

VOCs:

(A) Ethanol


(C) Butan-1-ol

(D) Ethyl acetate

(E) Ethyl butanoate


(G) Hexanoic acid

(H) Nonanal

(I) 3-Methylbutanal

MAP:

() 21% O2

() 21% O2 + 21% CO2

() 50% O2 + 30% CO2

() 50% O2 + 50% CO2

() 70% O2 + 30% CO2

Data points are the

means, with bars

representing two

measurement values.


122

Figure 4.8: VOCs produced by C. sake in MAP on honeydew melon agar during storage at 7°C.

0

50

100

150

200

0 2 4 6 8 10 12

mg

/m3

Time (days)

A

0

15

30

45

60

75

90

0 2 4 6 8 10 12

mg

/m3

Time (days)

B

0

10

20

30

40

50

0 2 4 6 8 10 12

mg

/m3

Time (days)

C

0

200

400

600

800

1000

0 2 4 6 8 10 12

mg

/m3

Time (days)

D

0

5

10

15

20

25

0 2 4 6 8 10 12

mg

/m3

Time (days)

E

0

150

300

450

600

0 2 4 6 8 10 12

mg

/m3

Time (days)

F

0

5

10

15

20

25

30

0 2 4 6 8 10 12

mg

/m3

Time (days)

G

0

10

20

30

40

50

0 2 4 6 8 10 12

mg

/m3

Time (days)

H

0

15

30

45

60

0 2 4 6 8 10 12m

g/m

3

Time (days)

I

VOCs:

(A) Ethanol


(C) Butan-1-ol

(D) Ethyl acetate

(E) Ethyl butanoate


(G) Hexanoic acid

(H) Nonanal

(I) 3-Methylbutanal

MAP:

() 21% O2

() 21% O2 + 21% CO2

() 50% O2 + 30% CO2

() 50% O2 + 50% CO2

() 70% O2 + 30% CO2

Data points are the

means, with bars

representing two

measurement values.

CHAPTER 4

123

Figure 4.9: Butane-2,3-dione, acetic acid and acetoin production in the headspace during incubation of

L. mesenteroides (A, C and E) and L. gelidum (B, D and F) on honeydew melon agar in different initial

modified atmospheres (() 21% O2, () 21% O2 + 21% CO2, () 50% O2 + 30% CO2, () 50% O2 + 50%

CO2 and () 70% O2 + 30% CO2) at 7°C. Data points are the means, with bars representing two

measurement values.

0.0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 10

mg

/m3

Time (days)

Butane-2,3-dioneA

0.0

0.1

0.2

0.3

0.4

0.5

0 2 4 6 8 10 12

mg

/m3

Time (days)

Butane-2,3-dioneB

0.0

0.1

0.2

0.3

0 2 4 6 8 10

mg

/m3

Time (days)

Acetic acidC

0.0

0.1

0.2

0.3

0 2 4 6 8 10 12

mg

/m3

Time (days)

Acetic acidD

0.0

0.2

0.4

0.6

0 2 4 6 8 10

mg

/m3

Time (days)

AcetoinE

0

2

4

6

0 2 4 6 8 10 12

mg

/m3

Time (days)

Acetoin F


124

4.3.2.3 Sugars

The content of glucose, fructose and sucrose in pineapple agar inoculated with C. argentea

decreased gradually during incubation in air at 7°C (see Figure 4.10A). However, these sugars

did not change appreciably in the other atmospheres evaluated (see Figure 4.10B, C, D and E).

For C. sake, the concentrations of glucose and fructose decreased throughout incubation in air

(data not shown) and unlike C. argentea, also in the other atmospheres evaluated (data not

shown). Moreover, the production of lactic acid coincided with the onset of the depletion of

glucose during incubation of C. sake in air on pineapple agar. In difference to C. argentea,

sucrose levels in the pineapple agar inoculated with C. sake did not change during incubation in

all the atmospheres evaluated at 7°C. C. argentea and C. sake consumed glucose and fructose

simultaneous with an apparently higher rate of glucose uptake than fructose. Damore et al. (1989)

reported that glucose and fructose utilization by yeast share the same membrane transport

components. However, Emmerich and Radler (1983) observed that two different mechanisms or

carrier systems exist for the uptake of glucose and fructose in S. bailii, one resistant and the other

very sensitive to uranyl ions.

It can be seen in Figure 4.11A that sucrose, glucose and fructose were all catabolized by C. sake

on the honeydew melon agar packaged in air. Glucose and fructose were in particular almost

depleted after eight days incubation. Whilst these sugars were consumed as rapidly over the first

four days in the samples packaged in an IH of 21% O2 + 21% CO2 (see Figure 4.11B) as they

were in air, the levels of fructose and glucose remained more or less stable up to day 10 of

incubation, whilst the levels of glucose further decreased at a reduced rate. It can be seen in

Figure 4.11C in an IH of 50% O2 + 30% CO2 that the content of these sugars only decreased

appreciably after 8 days incubation of C. sake. The sugar levels in honeydew melon agar

packaged in MAs of 50% O2 + 50% CO2 and 70% O2 + 30% CO2 remained almost constant

during the growth of C. sake for 10 days (see Figure 4.11D and E).

CHAPTER 4

125

Figure 4.10: The evolution of glucose (), fructose () and sucrose () in pineapple agar inoculated with C. argentea in different initial

modified atmospheres ((A) 21% O2, (B) 21% O2 + 21% CO2, (C) 50% O2 + 30% CO2, (D) 50% O2 + 50% CO2 and (E) 70% O2 + 30% CO2) at

7°C. Data points are the means, with bars representing two measurement values.

0

1

2

3

4

5

6

7

0 2 4 6 8 10 12

g/1

00

g

Time (days)

A

2

3

4

5

6

7

0 2 4 6 8 10 12

g/1

00

g

Time (days)

B

2

3

4

5

6

7

0 2 4 6 8 10 12

g/1

00

g

Time (days)

C

2

3

4

5

6

7

0 2 4 6 8 10 12

g/1

00

g

Time (days)

D

1

2

3

4

5

6

7

0 2 4 6 8 10 12

g/1

00g

Time (days)

E


126

Figure 4.11: The evolution of glucose (), fructose () and sucrose () in honeydew melon agar inoculated with C. sake in different initial

modified atmospheres ((A) 21% O2, (B) 21% O2 + 21% CO2, (C) 50% O2 + 30% CO2, (D) 50% O2 + 50% CO2 and (E) 70% O2 + 30% CO2) at

7°C. Data points are the means, with bars representing two measurement values.

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

A

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

B

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

C

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

D

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

E

CHAPTER 4

127

Figure 4.12: The evolution of glucose (), fructose () and sucrose () in honeydew melon agar inoculated with L. mesenteroides in different

initial modified atmospheres ((A) 21% O2, (B) 21% O2 + 21% CO2, (C) 50% O2 + 30% CO2, (D) 50% O2 + 50% CO2 and (E) 70% O2 + 30% CO2)


0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

A

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

B

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

C

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

D

0

1

2

3

4

5

0 2 4 6 8 10

g/1

00

g

Time (days)

E


128

The consumption of sugars by L. mesenteroides (see Figure 4.12) and L. gelidum (data not

shown) in honeydew melon agar packaged in air and other MAs followed the same trend. It is

interesting to note that sucrose, which was almost depleted by L. mesenteroides after 8 days

incubation in air (see Figure 4.12A), was metabolized faster than glucose by both L.

mesenteroides and L. gelidum. Additionally, the sucrose and glucose decreased slower during

incubation in MAs of high O2 and CO2 levels than that in air. In contrast, fructose appeared to

even appreciably increase at the end of incubation in all MAs (see Figure 4.12). In some

lactococci, sucrose is transported by sucrose sugar phosphotransferase system (PTS) and a

specific sucrose-6-phosphate hydrolase cleaves the sucrose-6-phosphate to glucose-6-phosphate

and fructose (Axelsson, 2004; Thompson and Chassy, 1981). Therefore, the apparent increase in

fructose levels was most likely a result of sucrose-6-phosphate being metabolized during

heterofermentation.

4.3.3 Effect of high O2 and CO2 MAs on microbial growth on commercial fresh-cut fruit

In addition to air (the reference atmosphere), 50% O2 + 50% CO2 and 50% O2 + 30% CO2 were

selected for evaluation in the part of the study based on the experiments performed on pineapple

agar. While commercial fresh-cut honeydew melon cubes were evaluated in air and 50% O2 + 50%

CO2.

Fresh-cut pineapple

In agreement with the trends observed on pineapple agar, the growth of yeasts and aerobes

contaminating the pineapple cubes was markedly retarded by IH high O2 and CO2 levels (see

Table 4.2). The initial total aerobic counts on pineapple cubes was 4.0 - 4.2 log10 CFU/g. Growth

of aerobes on the pineapple cubes took place after a lag phase of one day, irrespective of the

packaging condition. At the end of storage (day 7), pineapple cubes packaged in IH 50% O2 + 50%

CO2 had the lowest total aerobic counts (4.0 - 4.4 log10 CFU/g), followed by those packaged in

CHAPTER 4

129

IH 50% O2 + 30% CO2 (5.6 - 5.8 log10 CFU/g). Pineapple cubes packaged in air had the highest

total aerobic counts (6.9 - 7.3 log10 CFU/g) on packaged pineapple cubes (see Table 4.2). The

same trend was observed for the yeasts, with counts of ca. 7.5, 6.0 and 5.3 log10 CFU/g on

pineapple cubes packaged in air, IH 50% O2 + 30% CO2 and IH 50% O2 + 50% CO2,

respectively, on day 7 of storage, (see Table 4.2). Although high numbers of bacteria as well as

yeasts and moulds have been previously found on fresh-cut pineapple during refrigerated storage

(Di Egidio et al., 2009; Liu et al., 2007; Montero-Calderon et al., 2008; Rocculi et al., 2009), the

population density of LAB in our study remained < 3 log10 CFU/g of pineapple in all the

atmospheres evaluated during storage which is consistent with the quite slow growth rate of L.

citreum on pineapple agar. Generally, LAB may affect the organoleptic qualities of fresh-cut

produce when the count is reached 7 or even up to 8 log10 CFU/g due to the low pervasive

character of the metabolites as discussed above (Jacxsens et al., 2003). Overall, the results in

present study are in agreement with Iversen et al. (1989) and Fleet (1992) who reported that

yeasts are the predominant factor causing the spoilage of fresh-cut pineapple.


Table 4.2B shows that total aerobic and total anaerobic counts on fresh-cut honeydew melon

were slightly higher on the honeydew melon packaged in air than on the melon packaged in 50%

O2 + 50% CO2 over the first three days of storage. Thereafter the total aerobic and anaerobic

counts reached the same levels (8 log10 CFU/g) at the end of storage (day 5). The yeast counts on

honeydew melon were also much lower on samples packaged in 50% O2 + 50% CO2 than those

packaged in air throughout storage. The population density of LAB was slightly lower on the

honeydew melon packaged in 50% O2 + 50% CO2 than in melon packaged in air throughout

storage. It could be concluded from Table 4.2B that at the beginning of storage (on days 0 and 1)

of honeydew melon cubes in air and MA, yeasts were dominated the microbiota.

Thereafter, the growth of yeasts in melon packaged in MA of 50% O2 + 50% CO2 was inhibited

throughout storage. Other micro flora (such as LAB) grew rapidly and became the dominant

flora of fresh-cut honeydew melon in 50% O2 + 50% CO2 from day 3 of storage onwards.


130

Table 4.2

(A) Total aerobe and yeast counts (log10 CFU/g) on pineapple cubes packaged in different initial

headspace (IH) modified atmospheres during storage at 7°C.


0 3 5 7

Total aerobe count

IH 21% O2

4.1 a (4.0 - 4.2)

5.0 (4.9 - 5.2) 6.6 (6.4 - 6.8) 7.1 (6.9 - 7.3)

IH 50% O2 + 30%CO2 4.8 (4.6 - 5.0) 4.9 (4.7 - 5.1) 5.7 (5.6 - 5.8)

IH 50% O2 + 50%CO2 4.7 (4.6 - 4.8) 4.1 (4.1 - 4.1) 4.2 (4.0 - 4.4)

Yeast count

IH 21% O2

4.0 (3.9 - 4.1)

5.4 (5.3 - 5.4) 6.9 (6.9 - 6.9) 7.5 (7.3 - 7.7)

IH 50% O2 + 30%CO2 4.9 (4.7 - 5.1) 4.9 (4.8 - 5.0) 6.0 (5.9 - 6.1)

IH 50% O2 + 50%CO2 4.6 (4.6 - 4.6) 4.4 (4.2 - 4.6) 5.3 (4.5 - 6.1)

(B) Total aerobe, total anaerobe, yeast and lactic acid bacteria (LAB) counts (log10 CFU/g) on honeydew

melon cubes packaged in different IH modified atmospheres during storage at 7°C.


0 1 3 5

Total aerobe count

IH 21% O2 4.6 (4.3 - 4.9)

6.0 (5.2 - 6.8) 8.0 (7.9-8.1) 8.5 (8.5 – 8.5)

IH 50% O2 + 50%CO2 4.6 (4.5 – 4.7) 4.7 (4.6 – 4.8) 8.4 (8.3 – 8.5)

Total anaerobe count

IH 21% O2 4.0 (3.8 – 4.2)

5.4 (4.6 – 6.2) 7.0 (7.0 – 7.0) 8.4 (8.2 – 8.6)

IH 50% O2 + 50%CO2 4.3 (4.2 – 4.4) 6.7 (6.6 – 6.8 ) 8.5 (8.5 – 8.5)

Yeast count

IH 21% O2 4.9 (4.6 – 5.2)

5.5 (5.3 – 5.7) 7.6 (7.4 - 7.8) 7.7 (7.6 – 7.8)

IH 50% O2 + 50%CO2 5.2 (5.0 – 5.4) 6.0 (6.0 – 6.0) 6.6 (6.6-6.6)

LAB count

IH 21% O2 3.1 (2.4 – 3.9)

4.0 (3.5 – 4.5) 6.5 (6.0 – 7.0) 8.1 (8.1 – 8.1)

IH 50% O2 + 50%CO2 3.7 (3.4 – 4.0) 5.8 (5.7 – 5.9) 7.5 (7.4 – 7.6)

a values are mean with two measurement values

CHAPTER 4

131

The results observed on honeydew melon are in agreement with those observed on honeydew

melon agar. Additionally, the number of both yeasts and LAB on honeydew melon cubes at the

end of storage exceeded the end of shelf-life values for yeasts (5 log10 CFU/g) and LAB (7 - 8

log10 CFU/g), respectively, in fresh-cut fruit (Uyttendaele et al., 2010).

4.3.4 Evaluation of the headspace O2 and CO2, pH and sugar levels of fresh-cut pineapple and

honeydew melon during storage

Headspace CO2 and O2 were steadily produced and consumed, respectively, during storage of

fresh-cut pineapple in all the atmospheres evaluated (see Figure 4.13 A), due to respiration of the

living tissues of the cut pineapple (Rocculi et al., 2009) and microbial growth (Marrero et al.,

2006). In contrast, the headspace O2 was depleted by the end of storage (day 5) of honeydew

melon cubes in air. This was accompanied by the production of a large amount of CO2 (see

Figure 4.13 B). Previous studies (Bai et al., 2003; Larson and Johnson, 1999) also reported the

consumption of O2 and production of CO2 during storage as a result of the respiration of living

tissues and microbial activity. In contrast to the trends observed in air, the headspace O2 and CO2

of the packages with honeydew melon cubes in IH 50% O2 + 50% CO2 only slightly decreased

and increased, respectively, during storage. This indicates that the respiration of both the living

tissues and microbial activity were inhibited in this atmosphere.

The pH of the pineapple cubes (data not shown) did not change significantly throughout storage

from an initial value of 3.3 ± 0.1, irrespective of the packaging condition. Montero-Calderon et

al. (2008) found similar results for fresh-cut pineapple (pH 3.58 ± 0.04) stored in high (40%) or

low (11.4%) oxygen atmospheres. The pH of the packaged honeydew melon did not change

appreciably in both air and 50% O2 + 50% CO2 throughout storage. In agreement with our results,

Larson et al. (1999) found the pH of fresh-cut honeydew melon during storage remained constant

or decreased slightly at 7°C. In difference to our results, Roller et al. (2002) reported that the pH

of fresh-cut honeydew melon decreased from 5.4 – 5.5 to 4.4 - 4.9 during storage as a result of

microbial activity.


132

The sugar levels in pineapple cubes packaged in the atmospheres evaluated in this study did not

change appreciably during storage (data not shown). The levels of glucose, fructose and sucrose

in the honeydew melon also remained constant during storage in both conditions (ca. 2.6, 1.6 and

2.0 respectively). Supapvanich and Tucker (2011b) and Portela et al. (1998) reported that soluble

solid content (SSC) remained constant throughout storage of fresh-cut honeydew melon.

Figure 4.13: The evolution of headspace O2 (―) and CO2 (┄) during storage of fresh-cut

pineapple (A) and honeydew melon (B) in different initial headspace O2 and CO2 levels (() air,

() IH 50% O2 + 30% CO2 and () IH 50% O2 + 50% CO2) at 7°C.

4.3.5 Volatile metabolite production of fresh-cut fruit

Most of the VOCs detected in the headspace of the trays containing the pineapple cubes,

including butan-1-ol, 3-methyl-1-butanol, ethyl octanoate, 2-methylpropanal, hexanoic acid,

increased slightly during storage in all the atmospheres evaluated (data not shown). The

concentrations of ethanol, ethyl acetate, phenylacetic acid and 3-methylbut-1-yl ethanoate (=

isopentyl acetate) during storage are shown in Table 4.3. It should be noted that some of the

VOCs were produced not only by the spoilage yeasts but also by the living tissues of the

pineapple cubes as it has been discussed in § 3.3.5. The lower quantities of VOCs in the

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

0 1 2 3 4 5 6 7

CO

2(%

)

O2

(%)

Time (days)

A

0

10

20

30

40

50

60

70

0

10

20

30

40

50

60

70

0 1 2 3 4 5

CO

2(%

)

O2

(%)

Time (days)

B

CHAPTER 4

133

headspace of trays with pineapple cubes compared to those detected in pineapple agar could be

attributed to the lower population densities of native yeasts (ca. 7.5. log10 CFU/g) at the end of

storage of the packaged pineapple cubes in air compared to the final population density of

spoilage yeasts on pineapple agar (ca. 8.0 log10 CFU/cm2 = ca. 8.4 log10 CFU/g). The sugar

levels in pineapple cubes packaged in these atmospheres did not change appreciably during

storage. Hence, the ethyl acetate detected in the headspace could have been largely produced by

the living tissues of pineapple cubes and to a lesser extent by the native yeasts. Furthermore, as

observed on pineapple agar, the amount of ethyl acetate detected was higher in IH 50% O2 +

50% CO2 than in air. However, the amount of ethanol and phenylacetic acid were higher in air

than in IH 50% O2 + 30% CO2 or IH 50% O2 + 50% CO2, resulting from the growth of native

spoilage yeasts.

Table 4.3: Volatile metabolites during storage of fresh-cut pineapple cubes in IH air (A), IH 50% O2 + 30%

CO2 (B) and IH 50% O2 + 50% CO2 (C) at 7°C.

IH Day

Ethanol

(mg/m3)

Ethyl acetate

(mg/m3)

Phenylacetic acid

(mg/m3)

3-methylbut-1-yl ethanoate

(mg/m3)

0 0.25 (0.22 - 0.28) 2.11 (1.49-2.74) 0.01 (0.00 - 0.02) 2.36 (3.32 - 2.40)

A

3 0.74 (0.49 - 0.99) 3.75 (2.55 - 4.95) 0.20 (0.06 - 0.34) 1.93 (1.82 - 2.05)

5 5.40 (5.14 - 5.66) 31.41 (31.15 - 31.67) 10.93 (8.71 - 13.15) 2.86 (2.78 - 2.93)

7 10.80 (7.41 - 14.20) 52.71 (39.98-65.45) 37.64 (14.54 - 60.74) 3.07 (2.83 - 3.32)

B

3 1.72 (1.45 - 1.99) 11.30 (9.39 - 13.22) 0.60 (0.27 - 0.94) 1.95 (1.57 - 2.34)

5 2.60 (2.21 - 3.00) 10.73 (8.55 - 12.91) 1.61 (1.04 - 2.17) 1.60 (1.59 - 1.61)

7 4.32 (3.95 - 4.68) 20.59(16.50 - 24.67) 5.34 (3.95 - 6.74) 2.11 (1.65 - 2.56)

C

3 3.16 (3.14 - 3.18) 37.78 (32.71 - 42.84) 3.76 (3.26 - 4.27) 2.09 (1.95 - 2.22)

5 4.03 (3.87 - 4.19) 33.31 (30.33 - 36.30) 5.91 (4.93 - 6.90) 2.41 (2.09 - 2.73)

7 6.79 (6.48 - 7.10) 61.79 (56.35 - 67.23) 11.55 (9.55 - 13.54) 2.40 (2.12 - 2.68)


134

Table 4.4: VOC production on honeydew melon cubes (mg/m3) during storage packaged in IH 21% O2 (air) and IH 50% O2 + 50% CO2 (MA) at 7°C.

Day of storage Day 0 Day 1 Day 3 Day 5

VOCs Air MA Air MA Air MA

Ethanol 3.16

a

(2.49-3.83)

3.38

(3.35 - 3.71)

4.95

(4.39 - 5.51)

8.90

(4.54 - 13.26)

7.06

(6.36 – 7.76)

50.60

(50.36 – 50.84)

9.96

(9.09 - 10.83)

3-methyl-1-butanol 0.07

(0.11 - 0.03)

0.11

(0.07 - 0.15)

0.13

(0.06 - 0.20)

0.23

(0.05 - 0.41)

0.16

(0.10 - 0.22)

3.08

(2.48 - 3.68)

0.35

(0.33 - 0.37)

Butan-1-ol 0 0 0 0.32

(0.22 - 0.42) 0

2.54

(2.26 - 2.82)

0.11

(0.09 - 0.13)

2-methyl-1-butanol 3.20

(2.81 - 3.59)

4.86

(4.66 - 5.05)

4.86

(4.50 - 5.22)

7.45

(6.27 - 8.63)

6.35

(6.30 - 6.40)

8.42

(8.02 - 8.82)

6.27

(4.30 - 8.24)

Ethyl acetate 63.03

(49.61 - 76.44)

52.56

(46.62 - 58.50)

86.03

(73.20 - 98.86)

67.29

(35.25 - 99.33)

73.11

(65.85 - 80.37)

166.98

(150.57 - 183.39)

70.50

(47.50 - 93.50)

Ethyl octanoate 0.03

(0.02 - 0.04)

0.02

(0.01 - 0.03)

0.03

(0.01 - 0.05)

0.05

(0.07 - 0.03)

0.04

(0.02- 0.06)

0.38

(0.33 - 0.43)

0.06

(0.05 - 0.07)

Phenylacetic acid 1.35

(0.92 - 1.78)

0.88

(0.69 - 1.07)

2.80

(2.27 - 3.33)

18.43

(8.46 - 28.40)

7.14

(6.02 - 8.26)

181.47

(177.77 - 184.70)

21.24

(20.26 - 22.22)

Hexanoic acid 0 0 0 0 0 2.55

(2.03 - 3.07)

0.14

(0.06 - 0.22)

Nonanal 0.37

(0.33 - 0.41)

0.42

(0.21 - 0.63)

0.58

(0.56 - 0.60)

0.57

(0.29 - 0.85)

0.47

(0.46 - 0.48)

3.49

(2.06 - 4.92)

0.67

(0.61 - 0.73)

3-methylbutanal 2.03

(2.47 - 2.59)

2.01

(1.79 - 2.23)

3.22

(2.71 – 3.73)

2.82

(1.83 - 3.81)

3.02

(2.56 - 3.48)

7.43

(7.34 - 7.52)

2.74

(1.75 - 3.73)

butane-2,3-diol 2.41

(1.86 - 2.96)

2.23

(2.10 - 2.36)

4.25

(3.22 - 5.28)

7.64

(3.23 - 12.05)

5.88

(4.62 - 7.14)

40.81

(39.51 - 41.11)

7.72

(4.93 -10.51)

Butane-2,3-dione 0.04

(0.03 - 0.05)

0.03

(0.02 - 0.04)

0.01

(0.01 - 0.01)

0.03

(0.01 - 0.05)

0.02

(0.01 - 0.03)

0.13

(0.10 - 0.16)

0.04

(0.03 -0.05)

Acetic acid 0.22

(0.18 - 0.26)

0.23

(0.22 - 0.24)

0.23

(0.20 - 0.26)

0.30

(0.10 - 0.50)

0.26

(0.25 - 0.27)

1.15

(1.12 - 1.18)

0.41

(0.38 - 0.44)

Acetoin 16.48

(14.86 -18.10)

17.60

(17.54 - 17.66)

23.94

(20.48 – 27.40)

26.76

(19.34 – 34.18)

25.35

(24.84 - 25.86)

88.80

(84.30 – 93.30)

25.38

(17.74 - 33.02)

a values are mean of two measurements.

CHAPTER 4

135

As can be seen in Table 4.4, all the VOCs increased in the headspace of fresh-cut honeydew

melon packaged in both air and MA during storage. In agreement with the results observed on

honeydew melon agar, the quantities of VOCs were much lower on the melon packaged in IH 50%

O2 + 50% CO2 than on the samples in packaged in air at the end of storage. Of the VOCs

detected, ethanol, butan-1-ol, pentan-1-ol, ethyl acetate, ethyl butanoate, ethyl hexanoate and

nonanal can also be produced by the living tissues of honeydew melon as it has been described §

3.3.5 of Chapter 3. Portela et al. (1998) reported that the typical aroma of honeydew melon

generally decreased during storage but the development of off-odours were associated with

macroscopic decay. In contrast, Amaro et al. (2012) found that the summed volatile concentration

of 25 integrated volatile compounds was 1.4 times higher than the initial value for fresh-cut

cantaloupe and honeydew melon by the end of storage time. However integrated volatile

abundance was more effected by storage time than the atmospheric composition (≈ 5% O2 + 10%

CO2, balanced N2) (Amaro et al., 2012). Likewise, Saftner et al. (2003) observed that the total

volatile abundance of melon chunks increased during storage at 10°C and could be influenced by

the temperature. Bai et al. (2003) indicated that translucency and off-odour were the main factors

in deterioration of honeydew melon cubes which had better colour retention, reduced respiration

rate and microbial population, and longer shelf-life in active MAP (≈5% O2 + 5% CO2) than

those in passive MAP. Therefore, some VOCs may be produced by the living tissues of

honeydew melon themselves, but VOCs detected in present study are mainly produced by

spoilage yeasts on fresh-cut honeydew melon during storage due to the high number of yeasts

(see Table 4.2B) and the low pervasive character of the metabolites of the LAB (Jacxsens et al.,

2003).

4.3.6 Physical parameters of fresh-cut pineapple and honeydew melon

Although all physical parameters of fresh-cut pineapple cubes evaluated did not differ

significantly between the atmospheres investigated, the colour parameters (L* (lightness), b*

(yellow chromaticity) and a* (green chromaticity)) and juiciness significantly decreased (P <

0.05) during storage in all the atmospheres (see Table 4.5A).


136

Table 4.5: Changes in the colour, juice leakage (mL/100g), firmness and juiciness of pineapple cubes and

honeydew melon cubes packaged under in IH air (A), IH 50% O2 + 30% CO2 (B) and IH 50% O2 + 50%

CO2 (C) at 7°C.

Packaging

conditions

Storage time (days)

0 1 3 5 7

(A) Fresh-cut pineapple

L* β

A 69.8 ± 5.4 A, a

69.8 ± 5.4 A, ab

69.4 ± 5.0 A, b

61.0 ± 5.4 A, c

60.6 ± 5.9 A, c

B 69.8 ± 5.4 A, a

62.9 ± 7.7 A, b

68.8 ± 3.8 A, a

61.5 ± 8.2 A, b

60.7 ± 8.7 A, b

C 69.8 ± 5.4 A, a

61.9 ± 6.7 A, b

64.4 ± 4.8 A, b

63.7 ± 4.8 A, b

62.9 ± 6.6 A, b

a* β

A 1.6 ± 1.4 A, a

1.1 ± 1.5 A, a

1.3 ± 1.1 A, a

1.0 ± 0.9 A, a

-0.3 ± 0.9 A, b

B 1.6 ± 1.4 A, a

0.3± 1.3 A, b

0.9 ± 0.8 A, ab

0.3 ± 0.8 A, b

0.03 ± 0.89 A, b

C 1.6 ± 1.4 A, a

0.8 ± 0.8 A, bc

1.2 ± 1.0 A, ab

0.7 ± 0.9 A, bc

0.3 ± 0.9 A, c

b* β

A 43.9 ± 3.6 A, a

38.6 ± 4.9 A, b

37.2 ± 4.7 A, b

31.3 ± 4.8 A, c

32.9 ± 3.5 A, c

B 43.9 ± 3.6 A, a

32.0 ± 6.2 A, bc

35.6 ± 3.4 A, b

29.5 ± 6.8 A, c

30.1 ± 7.1 A, c

C 43.9 ± 3.6 A, a

33.2 ± 6.2 A, bc

35.9 ± 4.0 A, b

31.4 ± 4.7 A, c

30.4 ± 4.4 A, c

Juice leakage γ

A

4.0 ± 1.1 A, a

5.1 ± 1.4 A, a

6.3 ± 0.4 A, a

6.1 ± 1.2 A, a

B 3.2 ± 1.0 A, a

5.5 ± 1.7 A, a

5.6 ± 1.9 A, a

6.1 ± 2.1 A, a

C 3.9 ± 0.9 A, a

5.2 ± 1.8 A, a

4.8 ± 1.8 A, a

5.7 ± 2.7 A, a

Firmness δ

A 3 (2 – 4) A, a

3 (2 – 3) A, a

2.5 (2 – 3) A, a

2.5 (2 – 3) A, a

B 3 (2 – 4) A, a

3 (2 – 4) A, a

2 (2 – 3.5) A, a

3 (2 – 4) A, a

C 3 (2 – 4) A, a

4 (3 – 4) A, a

3 (2 – 4) A, a

3 (2.5 – 4) A, a

Juiciness δ

A 5 (4 – 5)) A, a

4 (4 – 5)) A, a

4 (4 – 5)) A, b

3.5 (3 – 4)) A, b

B 5 (4 – 5) A, a

4 (4 – 5) A, b

4 (3.5 – 4) A, bc

4 (3.5 – 4) A, c

C 5 (4 – 5) A, a

4 (4 – 5) A, b

4 (3.5 – 5) A, b

4 (3 – 4) A, b

(B) Honeydew melon cubes

L* β

A 74.0 ± 2.2 A, a

73.3 ± 3.3 A, a

70.3 ± 5.7 A, b

71.6 ± 4.0 A, ab

C 74.0 ± 2.2 A, a

69.3 ± 4.0 B, b

71.1 ± 4.5 A, b

68.6 ± 5.1 B, b

a* β

A -2.5 ± 1.0 A, a

-2.6 ± 0.9 A, a

-2.4 ± 0.8 A, a

-2.2 ± 0.9 A, a

C -2.5 ± 1.0 A, a

-2.2 ± 1.1 A, a

-2.3 ± 0.7 A, a

-2.1 ± 1.2 A, a

b* β

A 14.4 ± 2.6 A, a

13.3 ± 2.8 A, ab

13.7 ± 2.2 A, ab

12.5 ± 2.9 A, b

C 14.4 ± 2.6 A, a

12.3 ± 3.5 A, b

12.4 ± 3.1 A, ab

11.4 ± 2.6 A, b

Juice leakage γ

A 1.8 ± 1.1 A, a

0.8 ± 0.4 A, a

1.0 ± 0.6 A, a

C 0.6 ± 0.2 A, a

1.3 ± 0.9 A, a

1.6 ± 0.6 A, a

Firmness δ

A 4 (3 – 4) A, a

3 (3 – 4) A, a

3 (2 – 3) A, b

C 4 (3 – 4) A, a

3 (2 – 4) A, a

3 (2 – 3) A, a

Juiciness δ

A 4 (4 – 4) A, a

4 (3 – 4) A, a

4 (3 – 5) A, a

C 4 (4 – 4) A, a

4 (4 – 4) A, a

3.5 (3 – 4) A, a

Values followed by the different letters within columns (capital letters) and within lines (small letters) are

significantly different (P < 0.05) according to ANOVA contrasts and Kruskal-Wallis test.

CHAPTER 4

137


γ mean ± standard deviation, n= 3

δ The median value (50

th percentile) with values of 25

th percentile and 75

th percentile. Respectively 50, 75 and 25

percent (rounded down) of the assessors gave at least this value or greater. Fractional values were indicated by

assessors as “in between two classes”.

Additionally, the difference seemed to occur earlier on pineapple cubes packaged in IH 50% O2 +

30% CO2 and IH 50% O2 + 50% CO2 than on cubes packaged in air. Juice leakage slightly

increased during storage, whilst firmness did not change significantly (P > 0.05) during storage.

The colour parameter a*, juice leakage, firmness, juiciness of honeydew melon cubes were not

significantly affected (P > 0.05) by storage time and composition of the atmosphere (see Table

4.5B). The colour parameters L* and b* decreased slightly but significantly (P < 0.05) during

storage (see Table 4.5B). 50% O2 + 50% CO2 seems to have a small but significant effect on L*

(data not shown) which is lower on honeydew melon packaged in 50% O2 + 50% CO2 than on

melon packaged in air at the end of storage. This was in part due to oxidation in the presence of

high O2 levels. Amaro et al. (2012) also found that small, but significant, colour changes were

observed during storage while firmness of honeydew melon cubes decreased during storage of

the first 4 - 7 days. Supapvanich et al. (2011b) observed that flesh colour remained constant

whilst firmness decreased significantly throughout storage. The loss of firmness was associated

with increases in polygalacturonase and galactanase activities. Additionally, colour and firmness

of fresh-cut honeydew melon can have a significant variation among varieties (Portela et al.,

1998).

4.3.7 Sensory quality of fresh-cut pineapple and honeydew melon

As it can be seen in Table 4.6, the results of the triangle test performed showed that no significant

sensory difference occurred between fresh-cut pineapple cube packaged in air and those packaged

in IH 50% O2 + 30% CO2. However, a significant difference (P < 0.05) was found between

samples packaged in air and those packaged in IH 50% O2 + 50% CO2 on day 7 of storage at 7°C


138

(see Table 4.6). Sensory preference in this case was for the samples packaged in an IH 50% O2 +

50% CO2. During the sampling for microbiological and sensory evaluation it was apparent that

differences in gas production by native flora and the tissues were evident as most of the packages

in air were blown on day 7, whilst those packaged in IH 50% O2 + 50% CO2 and IH 50% O2 + 30%

CO2 were not yet in this condition. Our results are in agreement with those of Torri et al. (2010)

who observed the same trend with regards to the increase in the levels of volatile compounds

during incubation as a result of the metabolism of the micro-flora of fresh-cut pineapple stored in

air at 7 - 8°C. Additionally, the panellists indicated that differences between pineapple cubes

packaged in different MAs were mainly based on the odour, indicating the importance of volatile

metabolite production (and storage atmosphere) on the shelf-life of fresh-cut pineapple products.

Although lower quantities of VOCs were detected in the headspace at the end of storage in

pineapple cubes packaged in air, they were described as being less fresh and having a fermented

odour, especially banana-like odour. This was probably due to the slight increase in the

concentration of 3-methylbut-1-yl ethanoate in the headspace (see Table 4.4) and the low human

olfactory thresholds (OT) for other volatile metabolites (Devos, 1990; Renger et al., 1992). 3-

methylbut-1-yl ethanoate has a typically strong banana odour (Fahlbusch et al., 2003).

Significant sensory differences (P < 0.05) were found between honeydew melon cubes packaged

in air and 50% O2 + 50% CO2 on days 3 and 5 of storage (see Table 4.6). Honeydew melon cubes

packaged with IH 50% O2 + 50% CO2 had a longer shelf-life (5 days) than those packaged in air

(3 days). Additionally, as it had been found on fresh-cut pineapple during storage, MA of 50% O2

+ 50% CO2 also inhibited the gas production of fresh-cut honeydew melon during storage.

However, three in 16 panellists reported a lightly sparkling test in fresh-cut honeydew melon

packaged in IH 50% O2 + 50% CO2; most likely a result of the high level of dissolved CO2.

Overall, sensory preference was generally for the fresh-cut honeydew melon packaged in IH 50%

O2 + 50% CO2.

The evaluation of sensory quality showed that the sensory shelf-life of fresh-cut pineapple and

honeydew melon can be extended by 1 – 2 days by packaging in 50% O2 + 50% CO2 in place of

air. As it can be seen in Table 4.2, counts of native yeasts on fresh-cut pineapple packaged in IH

21% O2 and 50% O2 + 50% CO2 exceeded on day 3 and 5, respectively, the suggested end of

shelf-life limit value of 5 log10 CFU/g (Uyttendaele et al., 2010). However, counts of native

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139

yeasts on fresh-cut honeydew melon packaged in air and MA exceeded the shelf-life limit value

at day 1 of storage. This was due to a higher initial yeast population on fresh-cut honeydew

melon compared to fresh-cut pineapple. Hence, the microbiological shelf-life of fresh-cut fruit in

this study did not coincide with the sensory shelf-life. As it has been discussed in § 3.3.7 of

Chapter 3, the reason could be that volatile metabolites produced by yeasts may influence the

flavour of fresh-cut fruit when the amounts were much higher than the OT values.

Table 4.6: The results of sensorial evaluation of fresh-cut pineapple cubes and honeydew melon cubes

packaged indifferent initial headspace high O2 combined with high CO2 levels during the storage at 7°C.

For 16 panellists, the critical number of correct responses required to obtain a statistically significant (P <

0.05) difference are at least 9.

Initial Headspace O2 and CO2 levels Day of storage

3 5 7

Fresh-cut Pineapple

21% O2a - 50% O2 + 30% CO2 6

b/16

c 5/16 7/16

21% O2 - 50% O2 + 50% CO2 6/16 6/16 10/16


21% O2 - 50% O2 + 50% CO2 10 /16 (P < 0.05) 10/16 (P < 0.05)

a 21% O2 is the control in tests.

b Number of people who correctly identified the odd sample.

c Total number of panel.

4.4 Conclusions

The microbial spoilage of fresh-cut pineapple is mainly due to the growth and volatile metabolite

production of yeasts under respiro-fermentative and fermentative metabolism during storage.

However, both yeasts and lactic acid bacteria could play a role in the spoilage (shelf-life) of

honeydew melon. MAs combining high O2 and CO2 levels in high gas barrier films are shown to

strongly retard the growth of yeasts and their production of volatile organic compounds. In


140

contrast, the inhibitory effect was minor on the growth and VOC production of LAB. The effect

of the MAs evaluated was minor on colour retention, firmness, juiciness and juice leakage of

fresh-cut pineapple cubes and honeydew melon during storage at 7°C. Overall, the application of

MAP with high O2 and high CO2 (e.g. 50% O2 + 50% CO2) could be used to maintain the quality

of fresh-cut pineapple and honeydew melon. After the evaluation of modified atmospheres

combining high O2 and high CO2 levels on the quality of fresh-cut pineapple and honeydew

melon, the study now shifted its focus in chapter 5 to the application of the MAs evaluated in

present study together with the MAs evaluated in chapter 3 on fresh-cut apple.

CHAPTER 5

EFFECT OF MODIFIED ATMOSPHERES (INCL. HIGH

OXYGEN AND HIGH CARBON DIOXIDE) ON THE SHELF-

LIFE OF FRESH-CUT APPLE

Chapter 5: Effect of Modified Atmospheres (Incl. High Oxygen and Carbon Dioxide) on the

Shelf-life of Fresh-cut Apple

Redrafted from:

Zhang, B.-Y., Samapundo, S., Sürengil, G., Devlieghere, F. 2013. Journal of Food Science and

Technology. Under review.

MODIFIED ATMOSPHERES ON FRESH-CUT APPLE

142

Summary

The microbial profile, sensory quality and volatile organic compounds (VOCs) of fresh-cut apple

packaged in air, 1/0/99, 5/0/95, 50/30/20 and 50/50/0 (% O2/CO2/N2) in a high gas barrier film

were evaluated over seven days at 7°C. The modified atmosphere (MA) with 50% O2 and 50%

CO2 had the greatest inhibitory effect on the growth of aerobes and yeasts on the apple slices.

However, the production of most of VOCs (i.e. ethanol, ethyl acetate, 3-methylbutanal, etc.) by

the cut apple was stimulated by the atmospheres with high O2 and high CO2 levels. In contrast,

the production of phenylacetic acid and 2-methyl-1-butanol was appreciably higher in the

packages which had low initial O2 levels. In general the lowest quantities of VOCs were

produced in the samples packaged in air. Atmospheres with high CO2 concentrations also caused

an effervescent mouth-feel which was negatively judged by the sensory panel. The MAs

evaluated had no appreciably effect on the colour of apple slices as ascorbic acid had been

applied to them before packaging. It can be concluded that the application of MA in a high gas

barrier film to extend the shelf-life of fresh-cut apple is not recommended. Additionally, the

results clearly illustrate that VOCs should not be ignored when the shelf-life of fresh-cut fruit is

evaluated.

CHAPTER 5

143

5.1 Introduction

Fresh-cut apple is one of the most popular fresh-cut fruit nowadays, not only because of its

freshness, convenience of use and low calorific value, but also due to its unique flavour

characteristics (Elss et al., 2006). The characterization of the VOCs of apples, apple juices and

ciders has been extensively studied over the past 50 years (Aaby et al., 2002; Echeverria et al.,

2004a; Echeverria et al., 2004b; Elss et al., 2006; Guedes et al., 2004; Moya-Leon et al., 2007;

Peng et al., 2009; Saquet et al., 2003). Current technology (mostly gas chromatography - mass

spectroscopy) has allowed the recognition of about 400 volatile organic compounds (VOCs)

present in apples (Salas Salazar and Olivas Orozco, 2011).

Fresh-cut apple is a very perishable product with a very short shelf-life even at chill temperatures.

This is a result of the active metabolism of the plant tissues, damage during peeling, grating,

shredding and increased microbial spoilage of fruit through transfer of skin (surface) micro-flora

to the fruit flesh where they can grow rapidly upon exposure to nutrient juices (O’Connor-Shaw

et al., 1994; Raybaudi-Massilia et al., 2007). VOC production may interfere in the unique aroma

of fresh-cut fruit during storage. As an example, Amaro et al. (2012) have reported that volatile

changes during storage are likely to have a stronger impact on the sensory appraisal of fresh-cut

melons than other quality attributes which are known to affect acceptance by consumers such as

colour, firmness or soluble solid content.

A wide range of treatments have been applied to maintain the quality of fresh-cut apples

including high pressure argon (Wu et al., 2012b), edible coating (Chiumarelli et al., 2012; Qi et

al., 2011; Rojas-Grau et al., 2008), anti-browning agents (Chung and Moon, 2009b; Jang et al.,

2009; Jeon et al., 2005; Lee et al., 2003; Luo et al., 2011; Soliva-Fortuny et al., 2001), heat

(Barrancos et al., 2003) and disinfection (Graca et al., 2011). In addition, modified atmosphere

packaging (MAP) has also been used to extend the shelf-life of fresh-cut apple (Aguayo et al.,

2010; Rocculi et al., 2004; Soliva-Fortuny et al., 2001). Reduced O2 storage atmospheres (≤ 5%

O2) were found to be beneficial in maintaining quality and extending the shelf-life of apple slices

(Gil et al., 1998; Rojas-Grau et al., 2007). Zhou et al. (2011) reported that fresh-cut 'Fuji' apples

stored in MAs of 60% O2 (balance N2) and 100% O2 at 4°C significantly inhibited cut-surface


144

browning, in particular the MA with 60% O2. MAs combining high O2 and CO2 levels may retard

the growth of aerobic and anaerobic microorganisms (Amanatidou et al., 2000). A combination

of 50% O2 + 30% CO2 prolonged the shelf-life of sliced carrots compared to storage in air by 2 to

3 days (Amanatidou et al., 2000). Nevertheless, the effect of modified atmospheres of high O2

and high CO2 has not yet been studied for fresh-cut apple.

Despite several studies which have been performed on apples to date, very little is actually

known about the relationship between modified atmospheres (MAs), non-ethylene volatile

organic compound (VOC) production and sensory quality of fresh-cut apple during storage. The

main objective of this study was to evaluate microbial growth, VOC production, physical

characteristics, sensory quality and the shelf-life of fresh-cut apple in low O2 atmospheres (1 and

5% O2, rest N2) and atmospheres combining high O2 (50%) and high CO2 (30% and 50%) levels.

Air (21% O2, rest N2) was used as the reference atmosphere in this study as it is currently used by

the company which supplied us with fresh-cut apple.


5.2.1 Raw materials and packaging

Fresh-cut Malus x domestica Borkh (Jonagold) apple slices (6 - 8 g/slice) dipped in an ascorbic

acid solution (1%, w/v) were delivered to our laboratory within three hours of processing by a

commercial processor. 120 ± 2g of apple slices were aseptically transferred into trays (volume =

443 mL, O2 transmission rate (OTR) = 0.5 - 13 cm3/m

2.d.bar at 23°C, 0% relative humidity (RH),

oriented polyamide/ethylene vinyl alcohol, DECAPAC NV, Herentals, Belgium) before they

were sealed in the desired atmospheres (21% O2 (rest N2), 1% O2 (rest N2), 5% O2 (rest N2), 50%

O2 + 30% CO2 (rest N2), 50% O2 + 50% CO2). A high O2 barrier film (OTR = 5 cm3/m

2.d.bar at

23°C, 50 % RH, polyamide/ethylene vinyl alcohol/polyethylene/polypropylene, BEMIS

EUROPE Flexible Packaging, Monceau-sur-Sambre, Belgium) was used to seal the trays by

means of a Traysealer (MECA 900, DECAPAC NV, Belgium). The trays were then stored at 7 ±

1°C for up to seven days.

CHAPTER 5

145

5.2.2 Quantification of headspace O2, CO2 and volatile organic compound levels

The headspace O2 and CO2 levels in the trays were first measured by a headspace analyser

(O2/CO2 Headspace Analyser, CheckMate 9900 O2, PBI - Dansensor, Denmark). Thereafter, the

trays were aseptically opened and 20 ± 0.1g of apple slices were transferred within 10s into

plastic containers which were then closed quickly. Two replicates were collected per atmosphere

per sampling moment (days 0, 1, 3, 5 and 7) for VOC analysis. These samples were kept at -

18°C until quantification of VOCs was done by means of SIFT-MS (Selected Ion Flow Tube

Mass Spectrometer, Voice 200, Syft Technologies).

The quantification of the VOCs by SIFT-MS was performed as describes in § 3.2.1.4 of Chapter

3. The target VOCs are shown in Table 3.1 in Chapter 3.

5.2.3 Microbial quality of fresh-cut apple slices

Approximately 20g of apple slices were aseptically transferred from each tray (after measurement

of the headspace O2 and CO2 levels and collection of the samples for quantification of the VOCs)

to a sterile stomacher bag after which primary and subsequent decimal dilutions were prepared in

physiological peptone saline solution [PPS, 8.5g NaCl; 1 g peptone per litre, Oxoid (Hampshire,

UK)]. The yeasts were enumerated by spread plating the decimal dilutions on Yeast Glucose

Chloramphenicol agar [YGC, Bio-Rad (Marnes-la-Coquette, France)], whilst the total aerobes

and total anaerobes were enumerated by pour plating the decimal dilutions on Plate Count Agar

[PCA, Oxoid (Hampshire, UK)] and Reinforced Clostridial Agar [RCA, Oxoid (Hampshire, UK)],

respectively. Lactic acid bacteria were enumerated by pour plating the decimal dilutions on de

Man Rogosa Sharpe agar [MRS agar, Oxoid (Hampshire, UK)] whose pH had been adjusted to

pH 5.7 with sorbic acid (Sigma–Aldrich, St. Louis, MO, USA) before boiling. The plates were

then incubated at 22 ± 1°C until the colonies were sufficiently large for enumeration (ca. 3 days).


146

5.2.4 Quantification of sugars during storage

HPLC was used to determine the concentration of sucrose, glucose and fructose in the apple

slices based on the method developed by Ragaert et al. (2006b). The concentrations of the sugars

were calculated using standard curves for sucrose (UCB, Leuven, Belgium), glucose (Sigma–

Aldrich, St. Louis, MO, USA) and fructose (Sigma–Aldrich, St. Louis, MO, USA). The solutions

of the sugars used to construct the standard curves were made by adding each of these

compounds in different known concentrations to distilled water.

5.2.5 Colour and pH evaluation

The surface colour of the apple flesh was determined on five randomly chosen points on the cut

surface of apple slices with a Spectrophotometer Minolta Model CM-2500d (Konica Minolta

Sensing Inc., Osaka, Japan). 8 pieces of apple slices were evaluated from each pair of trays per

modified atmosphere condition. Zero calibration and white calibration had been performed before

measuring the apple slice surface. Five readings of L* (lightness), a* (green chromaticity) and b*

(yellow chromaticity) coordinates were recorded for each apple slices.

Apple slices for pH measurement were first cut into small pieces then measured in duplicate by

means of a SevenEasy pH meter (Mettler Toledo GmbH, Schwerzenbach, Switzerland).

5.2.6 Sensory quality of apple slices

Triangle tests were used to determine if the modified atmospheres could have an impact on the

sensory quality of fresh-cut apple slices. The tests evaluated potential differences between

samples packaged in initial headspace (IH) O2 of 21% (reference condition) to those packaged in

IH 1% O2, 5% O2 50% O2 + 30% CO2 and 50% O2 + 50% CO2. In particular, the visual

appearance, flavour and odour were evaluated. The sensory evaluation was performed in a

purpose built sensory analysis room with isolated booths (UGent Sensolab, Ghent University) by

CHAPTER 5

147

16 untrained assessors (age range: 24 -– 44 years, average age: 29 years, 8 female assessors and 8

male assessors). The procedures followed were according to those described in ISO 4120:2004

(Sensory analysis – Methodology – Triangle test) (ISO, 2004) which has been described in §

3.2.2.4 of Chapter 3.

Firmness and juiciness of fresh-cut apple were also evaluated as described in Chapter 3.


An individual package tray was used as one replicate on each sampling day, per treatment. The

results of triangle tests were analysed according to ISO 4120:2004 (ISO, 2004). In the case of 16

assessors, the critical number of correct response to obtain a statistically significant (α = 0.05)

difference is 9. To determine the effect of storage time and MAs on colour, proper ANOVA

contrasts were used, with significant differences being established at P < 0.05. Significant

differences in juiciness and firmness were analysed by Kruskal-Wallis test due to the ordinal

nature of these data. These statistical analyses were performed in SPSS software (version 21.0,

SPSS, Chicago, IL, USA). Where applicable, the samples packaged in air functioned as the

reference condition by employing suitable contrasts in the statistical analyses.


5.3.1 Effect of MA on the microbial quality of fresh-cut apple slices during storage

It can be seen in Figure 5.1 that alteration of the atmosphere (from air) appreciably retarded the

growth of the aerobes and yeasts on apple slices during storage at 7°C. The total anaerobic and

lactic acid bacteria counts did not exceed 3 log10 CFU/g throughout storage, irrespective of the

initial atmosphere the apple slices were packaged in (data now shown). Growth of the aerobic

microorganisms and yeasts was characterized by an initial lag phase of ca. 1 day in air, IH 1% O2


148

and 5% O2, which increased to ca. 3 – 5 days in IH 50% O2 + 30% CO2. Subsequently, the counts

of the aerobic microorganisms and yeasts increased on the apple slices packaged in all

atmospheres with the exception of those on the apple slices packaged in 50% O2 + 50% CO2,

which remained constant over the seven day storage. Fastest growth of the aerobic

microorganisms and yeasts took place in air (21% O2, rest N2), followed by the low O2

atmospheres evaluated (1% O2 and 5% O2, rest N2) and then IH 50% O2 + 30% CO2, in

decreasing order of growth rate.

Figure 5.1: The evolution of total aerobes and yeasts on fresh-cut apple slices during storage in different

initial modified atmospheres (() 1% O2, () 5% O2, () 21% O2, () 50% O2 + 30% CO2 and () IH

50% O2 + 50% CO2) at 7°C. Data points are the means, with bars representing the two measurement

values.

The headspace O2 and CO2 levels decreased and increased, respectively, irrespective of the initial

atmosphere in which the apple slices were packaged (see Figure 5.2). Concentration of headspace

2

3

4

5

6

7

0 1 2 3 4 5 6 7

log

10

CF

U/g

Time (Days)

Total aerobic counts

2

3

4

5

6

7

0 1 2 3 4 5 6 7

log

10

CF

U/g

Time (Days)

Yeasts

CHAPTER 5

149

CO2 in the packages with IH 50% O2 + 30% CO2 and IH 50% O2 + 50% CO2 kept stable or

slightly decreased during the first day of storage, probably due to a balance between the

dissolution of CO2 in the water-phase of apple slices after the packaging and the production of

CO2 by living tissue of apple slices during this period.

Figure 5.2: The evolution of headspace O2 (A) and CO2 (B) during storage of apple slices in different

initial modified atmospheres (() 1% O2, () 5% O2, () 21% O2, () 50% O2 + 30% CO2 and () IH

50% O2 + 50% CO2) at 7°C. Data points are the means, with bars representing two the measurement

values.

The MAs evaluated in this study appreciably retarded microbial growth on fresh-cut apples.

Growth retardation was largest on the apple slices packaged in atmospheres combining high O2

and high CO2 levels. Although several studies can be found in literature which have investigated

0

10

20

30

40

50

60

0 1 2 3 4 5 6 7

O2 (

%)

Time (Days)

A

0

10

20

30

40

50

60

70

80

0 1 2 3 4 5 6 7

CO

2 (%

)

Time (Days)

B


150

the shelf-life of fresh-cut apple, very few has evaluated this from a microbial point of view.

Anese et al. (1997) reported that CO2/N2 (20/80, v/v) and vacuum packaging were the most

effective atmospheres for inhibiting microbial growth and retaining the colour of apple slices

during storage. Aguayo et al. (2010) found that microbial counts for fresh-cut apple treated with a

calcium ascorbate solution (6, 12 and 20%) and stored in air or MA (extraction of most of the air

inside the bags) remained relative low over 28 days. The microbial quality of apple slices has not

been intensively evaluated probably because of the fact that physical and physiological

characteristics (such as colour) are considered to be more shelf-life limiting. In our study, the

total aerobic counts did not exceed the end of shelf-life value for fresh-cut fruit (7 log10 CFU/g),

proposed by Uyttendaele et al. (2010), irrespective of the atmosphere in which the apple slices

were packaged in. However, it can be seen in Figure 5.1 that the population density of the yeasts

on the apple slices packaged in air, IH 5% and IH 1% O2 had on day 3 almost reached 5 log10

CFU/g, the level at which yeasts are generally known to start causing spoilage (Fleet, 1992). This

was in contrast with the findings under high O2 and high CO2 atmosphere where the growth of

aerobes and yeasts were clearly suppressed.

5.3.2 VOC production of fresh-cut apple

Analysis of the headspace VOCs showed different behaviours during storage of fresh-cut apple

slices as a result of the different initial atmosphere conditions. Of the VOCs quantified (see Table

3.1 of Chapter 3), the concentrations of propan-1-ol, ethyl hexanoate, 3-methylbut-1yl ethanoate

and propanoic acid did not change appreciably during storage. On the other hand, although the

concentrations of 3- and 2-methylbutanoic acid increased during storage, their concentrations

were not affected by the composition of the atmospheres the apple slices were packaged in (data

not shown). The production of the majority of the VOCs evaluated (ethanol, butan-1-ol, pentan-

1-ol, ethyl acetate, ethyl butanoate, ethyl octanoate, hexanoic acid, nonanal, 2-methylpropanal, 3-

methylbutanal and 2-methylbutanal) followed the same trend as the examples shown in Figure

5.3. As it can be seen in Figure 5.3, the production of the VOCs over the first three days was

generally limited and was not affected by the composition of the atmosphere in which the apple

slices were packaged.

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151

Figure 5.3: Ethanol, ethyl acetate, ethyl butanoate, hexanoic acid, nonanal and 3-methylbutanal

concentrations during storage of fresh-cut apple in different initial modified atmospheres (() 1% O2, ()

5% O2, () 21% O2, () 50% O2 + 30% CO2 and () IH 50% O2 + 50% CO2) at 7°C. Data points are the

means, with bars representing the two measurement values.

0

20

40

60

80

100

0 1 3 5 7

mg

/m3

Time (days)

Ethanol

0

300

600

900

0 1 3 5 7

mg

/m3

Time (days)

Ethyl acetate

0

5

10

15

20

25

30

0 1 3 5 7

mg

/m3

Time (days)

Ethyl butanoate

0

6

12

18

0 1 3 5 7

mg

/m3

Time (days)

Hexanoic acid

0

3

6

9

12

0 1 3 5 7

mg

/m3

Time (days)

Nonanal

0

15

30

45

60

75

0 1 3 5 7

mg

/m3

Time (days)

3-Methylbutanal


152

However, from day 3 onwards, the quantities of VOCs increased most rapidly in the packages

with an IH of 50% O2 + 50% CO2. Consequently the highest concentrations of these VOCs were

found in these packages, and these (excepted ethyl acetate) were at the end of storage (day 7) at

least twice as high as those found in the headspace of the samples packaged in the other

atmospheres evaluated. The second highest concentrations of VOCs were generally found in the

packages with an IH of 50% O2 and 30% CO2. Very small differences were found between the

quantities of VOCs produced in air, IH 1% O2 and IH 5% O2.

In difference to the general trend observed, the highest quantities of phenylacetic acid and 2-

methyl-1-butanol were produced in IH 1% O2, followed by IH 5% O2 (see Figure 5.4), whilst the

lowest were produced in the packages with high initial O2 and CO2 levels.

Figure 5.4: Phenylacetic acid and 2-methyl-1-butanol concentrations during storage of fresh-cut apple in

different initial modified atmospheres (() 1% O2, () 5% O2, () 21% O2, () 50% O2 + 30% CO2 and

() IH 50% O2 + 50% CO2) at 7°C. Data points are the means, with bars representing the two

measurement values.

Despite the fact that growth of yeasts was effectively inhibited by the combination of high O2 and

high CO2 concentrations, high VOC quantities were observed in the headspaces of apple slices

packaged in these atmospheres. Yeasts are generally known to start causing spoilage when the

population density reaches 5 log10 CFU/cm2 (Fleet, 1992). Additionally, as it has been described

0

20

40

60

80

100

0 1 3 5 7

mg

/m3

Time (days)

Phenylacetic acid

0

4

8

12

16

0 1 3 5 7

mg

/m3

Time (days)

2-Methyl-1-butanol

CHAPTER 5

153

in Chapters 3 and 4, that yeasts on fruit agar only begin to produce large quantities of most

volatile metabolites at counts between 6.0 – 7.0 log10 CFU/cm2 (ca. 6.4 – 7.4 log10 CFU/g).

Ragaert et al. (2006b) also reported that detection of ethanol occurred when the yeast counts on

strawberry agar were above ca. 5 log10 CFU/cm2 in both air and MA (3% O2 and 5% CO2,

balanced N2). The highest concentrations of most VOCs (see Figure 5.3) were found in the

packages with an IH of 50% O2 + 50% CO2 where the growth of yeasts was suppressed and their

counts remained too low (ca. 4 log10 CFU/g on day 7) to produce large quantities of the VOCs.

On the other hand, all the VOCs that were detected and evaluated in this study (except for

phenylacetic acid) have also been detected in apples (Dimick and Hoskin, 1983). Therefore, one

can conclude that the high amounts of VOCs present in the headspaces of the apple slices

packaged in high O2 and high CO2 atmospheres can attributed to the apple tissue and not to

microbial proliferation.

It is indeed known that VOC production can be induced by MAP during storage of fresh-cut fruit,

mainly due to the shift of the tissue metabolism towards fermentation in the presence of low O2

levels and/or too high CO2 levels. High CO2 atmospheres and low O2 atmospheres may both have

a profound effect on various respiratory enzymes in fruit (Mathooko, 1996). CO2 inhibits

mitochondrial activity, thereby diverting pyruvate to acetaldehyde and ethanol formation even

though ample O2 is present (Mathooko, 1996), as was the case in our study. Ke et al. (1991b)

reported that fruit packaged in a low O2 atmosphere combined with or without a slightly elevated

level of CO2 may look good, it would taste bad due to the occurrence of alcoholic off-flavours.

From Figures 5.3 and 5.4 it is clear that the effect of the MAs evaluated on the production of

ethanol, ethyl acetate, ethyl butanoate, hexanoic acid, nonanal and 3-methylbutanal is similar,

whilst on the other hand the production of phenylacetic acid and 2-methyl-1-butanol follow a

different pattern. While the high O2 levels were not able to compensate for the effect of the high

CO2 levels (resulting in the production of ethanol), high O2 levels suppressed the production of

phenylacetic acid. However, this effect of high O2 levels on the production of phenylacetic acid

was reduced in the packages with IH CO2 level of 50%. Phenylacetic acid has been correlated

with the aroma amino acid metabolism of yeasts (Hammer et al., 1996) when they suffer carbon

or nitrogen starvation but has also been found to be a flavour compound in other fruits, such as

pineapple (Tokitonio et al., 2005), strawberry (Zabetakis and Holden, 1997) and rose apple fruit


154

(Guedes et al., 2004). Strangely, 2-methyl-1-butanol followed the same pattern as phenylacetic

acid and not that of 3-methylbutanal which are both related with Ehrlich pathway (Hazelwood et

al., 2008).

5.3.3 Effect of MA on the colour, firmness and juiciness of fresh-cut apple

In general the MAs evaluated in this study had a minor effect on the colour retention of fresh-cut

apple slices (see Table 5.1). The colour parameters evaluated [L* (lightness), a* (green

chromaticity) and b* (yellow chromaticity)] significantly (P < 0.05) decreased during storage. In

addition, significance differences (P < 0.05) also occurred between the samples according to the

initial atmosphere they were packaged in. Firmness and juiciness of the apple slices decreased

during storage in all the atmospheres evaluated. According to the Kruskal-Wallis test, firmness

significantly (P < 0.05) decreased from days 3 and 5 of storage, while juiciness significantly (P <

0.05) declined from day 3 of storage. On day 5 of storage, the juiciness of the apple slices

packaged in air was significantly better than that of the apple slices in packaged in IH 50% O2 +

50% CO2.

The pH of the apple slices increased slightly from 3.2 ± 0.1 to 3.4 ± 0.1, whilst the levels of

sucrose, glucose and fructose (2.9 ± 1.2, 0.8 ± 0.4 and 3.8 ± 1.6 g/100g, respectively) did not

change appreciably during storage, irrespective of the initial atmosphere in which the apples

where packaged (data not shown).

Although significant differences were found between the colour parameters (L*, a* and b*) of the

apple slices packaged in the atmospheres evaluated in this study, the panellists did not observe

any visual differences during the sensory analysis. In addition, none of the products showed

browning defects during storage. It is most likely that MAP had little effect on the visual

appearance (colour) of the apple slices due to the fact that the apples were dipped in ascorbic acid

solution. Rossle et al. (2011b) found that the degree of browning in apple wages is negatively

correlated to O2 levels (-0.69) and is positively (0.70) correlated to CO2 levels. Gil et al. (1998)

stated that although low O2 atmospheres can reduce cut-surface browning by inhibiting

polyphenol oxidase (PPO), when applied alone it is not as effective in preventing browning as

ascorbic acid.

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155

Table 5.1: Changes in the colour, firmness and juiciness of apple slices packaged in in air (21% O2) and

modified atmospheres during storage at 7°C.


0 1 3 5 7

L* β

1% O2 83.49 ± 1.41A, a 81.32 ± 1.70 A, cd 82.56 ± 0.59 A, ab 80.77 ± 1.63 A, d 82.34 ± 1.73 B, bc

5% O2 83.49 ± 1.41A, a 83.20 ± 1.25 BC, a 84.15 ± 1.41 B, a 81.74 ± 1.82 A, b 82.69 ± 2.32 B, b

21% O2 83.49 ± 1.41A, a 82.22 ± 0.98 AB, bc 82.62 ± 0.99 A, abc 82.35 ± 1.86 A, c 83.30 ± 1.15 A, a

50% O2 + 30% CO2 83.49 ± 1.41A, a 83.38 ± 1.37 C, a 83.52 ± 1.20 AB, a 81.22 ± 2.99 A, b 81.02 ± 3.22 B, b

50% O2 + 50% CO2 83.49 ± 1.41A, a 80.20 ± 1.19 A, ab 81.28 ± 1.21 C, b 80.82 ± 1.66 A, b 80.55 ± 4.61 B, b

a* β

1% O2 -0.50 ± 0.28 A, a -0.49 ± 0.31 A, a -0.58 ± 0.40 AB, a -0.27 ± 0.45 A, a -0.99 ± 0.66 AB, a

5% O2 -0.50 ± 0.28 A, a -0.64 ± 0.32 AB, ab -0.56 ± 0.36 A, ab -0.51 ± 0.89 A, a -0.96 ± 0.33 AB, b

21% O2 -0.50 ± 0.28 A, a -0.65 ± 0.42 AB, a -0.39 ± 0.45 A, a -1.08 ± 0.30 BC, b -1.24 ± 0.23 A, b

50% O2 + 30% CO2 -0.50 ± 0.28 A, a -0.83 ± 0.27 B, abc -0.91 ± 0.37 B, bc -0.62 ± 0.48 AB, ab -1.01 ± 0.64 AB, c

50% O2 + 50% CO2 -0.50 ± 0.28 A, a -0.84 ± 0.17 B, bc -0.51 ± 0.33 A, a -1.11 ± 0.27 C, c -0.78 ± 0.60 B, ab

b* β

1% O2 19.91 ± 2.80 A,a 20.91 ± 1.72 A, a 20.18 ± 1.30 AB, a 19.61 ± 2.63 A, a 18.88 ± 2.71 A, a

5% O2 19.91 ± 2.80 A,ab 20.35 ± 1.47 A, ab 21.25 ± 1.95 A, a 21.08 ± 2.74 A, a 18.45 ± 2.11 A, b

21% O2 19.91 ± 2.80 A,a 19.76 ± 1.83 A, a 20.39 ± 1.65 AB, a 20.92 ± 2.50 A, a 19.42 ± 1.89 A, a

50% O2 + 30% CO2 19.91 ± 2.80 A,a 20.62 ± 1.56 A, a 19.64 ± 1.60 B, a 19.45 ± 3.94 A, a 20.02 ± 2.67 A, a

50% O2 + 50% CO2 19.91 ± 2.80 A,a 19.72 ± 1.58 A, a 21.03 ± 1.67 AB, a 21.05 ± 1.42 A, a 20.17 ± 2.15 A, a

Firmness γ

1% O2 4 (4 - 5) A,a 4 (3 - 4) A, ab 3 (3 - 4) A, b 3 (2.5 - 4) A, b

5% O2 3 (2 - 4) A, ab 3 (2 - 3) B, b 2 (2 - 3) A, b

21% O2 3 (3 - 4) A, b 3 (2 - 4) AB, bc 2 (2 - 3) A, c

50% O2 + 30% CO2 4 (2 - 4) A, ab 3 (2 - 4) AB, b 3 (2 - 3) A, b

50% O2 + 50% CO2 3 (2 - 4) A, b 3 (2 - 3) AB, ab -

Juiciness γ

1% O2 4 (4 - 5) A,a 4 (3 - 4) A, b 4 (3 - 4) AB, b 4 (3 - 4) A, b

5% O2 3 (3 - 4) A, b 4 (3 - 4) AB, b 3 (3 - 4) A, b

21% O2 3 (3 - 4) A, b 4 (3 - 4) A, b 3 (3 - 4) A, b

50% O2 + 30% CO2 3.5 (3 - 4) A, b 4 (3 - 4) AB, b 3 (2.5 - 4) A, b

50% O2 + 50% CO2 4 (3 - 4) A, b 3 (3 - 4) B, b -

Values followed by different letters within columns (capital letters) and within rows (small letters) are significantly

different (P < 0.05) according to ANOVA contrasts test and the Kruskal-Wallis test.


γ The median value (50th percentile) with values of 25th percentile and 75th percentile. 50, 75 and 25 percent

(rounded down) of the assessors, respectively, gave at least this value. Fractional values were indicated by assessors

as “in between two classes”.


156

5.3.4 Sensory quality of fresh-cut apple

Four triangle tests were performed to evaluate if any differences occurred during storage between

the sensory quality of apples packaged in air (the reference condition) and those packaged in the

MAs evaluated in this study. As shown in Table 5.2, no significant differences (P > 0.05)

occurred between the sensory qualities of fresh-cut apple slices packaged in air compared to

those packaged in 5% O2 (rest N2) and 50% O2 + 30% CO2 (rest N2). However, significant

differences (P < 0.05) were found between the sensory qualities of fresh-cut apple slices

packaged in air and those packaged in 50% O2 + 50% CO2 from the third day of storage.

According to the responses of the panellists who correctly identified the odd samples, the apples

packaged in 50% O2 + 50% CO2 had more offensive off-odours and had an effervescent mouth-

feel. Apple slices packaged in 1% O2 were also determined to be significantly different (P < 0.05)

in sensory quality from those packaged in air only on day 3 of storage. Overall, the apple slices

were considered unacceptable for consumption on day 7 of storage irrespective of the atmosphere

in which they were packaged. This was due in most part to the development of fermentation type

off-flavours in the headspace. It is also evident in Figures 5.3 and 5.4 that although the VOCs

were detected in the headspace of samples repackaged in 0.9L of N2, the concentrations of VOCs

(except ethanol) at day five of storage were already higher than their human olfactory threshold

(OT) values (see Table 3.1 of Chapter 3). Among these VOCs, the concentration of ethyl acetate

increased more rapidly than others. The sensory shelf-life of fresh-cut apple packaged in air and

other MAs (except 50% O2 + 50% CO2) in our study was 5 days. Although the microbiological

end of shelf-life criteria were not exceed on fresh-cut apple packaged in 50% O2 + 50% CO2

during storage for seven days (see Figure 5.1), the apples were sensory unacceptable on day three

of storage.

Although Gunes et al. (2001) reported that ‘Delicious’ apple slices can tolerate up to ca. 30%

CO2 and high CO2 levels (ca. 30%) resulted in an approximately 30% reduction in accumulation

of ethanol in apple slices under anaerobic condition, they did not evaluate the sensory quality of

the apple slices. Our study showed that high CO2 (≥ 30%) modified atmospheres have a negative

effect on the sensory quality of apple slices. High CO2 level stimulated off-flavour production in

apple slices and had a sparkling mouth-feel. The accumulation of CO2 in the headspace of the

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157

packages resulted in even higher CO2 concentrations than the initial concentrations due to the

respiration of apples and microorganisms in combination with the use of high gas barrier films

(see Figure 5.2). Therefore, packaging in high CO2 (≥ 30%) modified atmospheres and high CO2

barrier bags are not recommended for the storage of fresh-cut apple slices.

Table 5.2: Results of the triangle tests in which the sensory quality of fresh-cut apple slices packaged in 21%

O2 (control) was compared to that of apple slices packaged in various modified atmosphere packaging. For

16 panellists, the critical number of correct response to obtain a statistically significant (P < 0.05)

difference is 9.

Control and MAP

Day of storage

3 5 7

21% O2 - 1% O2 9 a/16

b (P < 0.05) 8/16 5/16

21% O2 - 5% O2 4/16 6/16 5/16

21% O2 - 50% O2 + 30% CO2 7/16 8/16 8/16

21% O2 - 50% O2 + 50% CO2 9/16 (P < 0.05) 9/16 (P < 0.05) -

a Number of people who correctly identified the odd sample,

b Total number of panellists.

5.4 Conclusions

Although the growth of the spoilage organisms of fresh-cut apple was effectively suppressed in

atmospheres combining high O2 and high CO2 levels, compared to their growth in air, VOC

production was stimulated in these atmospheres. Thus, VOC production during storage of fresh-

cut apple is a major factor limiting the shelf-life and sensory quality and should therefore not be


158

neglected when MAP alone or combined with other preservation techniques such as edible

coating are applied to maintain the quality of fresh-cut fruit.

CHAPTER 6

GENERAL DISCUSSION, CONCLUSIONS AND

PERSPECTIVES

Chapter 6: General Discussion, Conclusions and Perspectives

GENERAL DISCUSSION, CONCLUSIONS AND PERSPECTIVES

160

CHAPTER 6

161

Chapter 6

General discussion, conclusions and perspectives

Quality deterioration of fresh-cut fruit is generally attributed to the combined effect of

endogenous enzymes, enhanced respiration, microbial growth, etc. (Al-Ati and Hotchkiss, 2002).

No single treatment is known to limit overall quality deterioration. Modified atmospheres (MAs)

have been considered as supplementary to temperature control and disinfection processes to

maintain the quality of fresh-cut fruit (Jeffrey and Devon, 2009; Varoquaux et al., 2005).

Recently, MAs with reduced O2 levels and/or slightly elevated CO2 levels have been found to be

able to inhibit aerobic microbial growth, prevent browning, delay softening, maintain vitamin C

and phenolic contents and reduce ethylene production, thus, increase the shelf-life of fresh-cut

fruit (Bai et al., 2001; Odriozola-Serrano et al., 2010; Oms-Oliu et al., 2009; Rojas-Grau et al.,

2007; Soliva-Fortuny et al., 2002a). However, atmospheres combining high O2 and CO2 levels

have not yet been evaluated on the quality of fresh-cut fruit. Additionally, only a few studies can

be found in literature which report experiments performed to determine the predominant spoilage

microorganisms of fresh-cut fruit products (Heard, 2002).

Identification of predominant spoilage microorganisms of fresh-cut fruit was partly addressed in

Chapter 2 of this thesis for fresh-cut honey dew melon, pineapple and apple. Yeasts were the

dominant microorganisms on spoiled pineapple cubes and apple slices while both yeasts and

lactic acid bacteria (LAB) were dominant on spoiled honeydew melon cubes. Amongst the yeasts,

the most frequently occurring species on all the fresh-cut fruit evaluated were Candida spp.,

especially Candida sake. The most frequently occurring species of LAB on honeydew melon

cubes was Leuconostoc mesenteroides. As it has been described in § 1.2.1 of Chapter 1, spoilage

by yeasts is a major problem in the fruit processing industry (Fleet, 1992), due in part to the low

pH, high sugar content and high humidity environment of most fresh-cut fruit products. However,

as illustrated in this thesis, the growth of LAB could also play an important role in their spoilage

for fresh-cut fruit with pH values that are higher than most acidic fresh-cut fruit products such as

honeydew melon (pH 5.2 - 5.6) (Beaulieu et al., 2001; Garcia et al., 2005). Therefore, the pH of


162

the fruit (raw material) can greatly affect the microbiota and therefore the spoilage mechanisms

of fresh-cut fruit.

In addition, the microbiota of fresh-cut fruit can also be influenced by the processing

environment. It is clear that in most published literature, fresh-cut fruit was prepared in

laboratories (Liu et al., 2007; Montero-Calderon et al., 2008; Rocculi et al., 2009). However, the

microbiota and their initial numbers on fresh-cut fruit produced under industrial conditions are

potentially very different from those on fresh-cut fruit produced in the laboratory. The

microorganisms on commercially produced fresh-cut fruit may arise from the equipment used,

food handling employees, cross contamination, etc. However, these sources of microbial

contamination are easily controlled and/or avoided in the laboratory. Moreover, the quantity of

fresh-cut fruit produced in laboratories for research purposes is much smaller than that produced

commercially. Importantly, it has to be taken into consideration that due to the fact that the initial

microbial population density on fresh-cut fruit processed in laboratories can be very low,

microbial spoilage may play a less prominent role in their spoilage compare to its role in

commercially processed products. Therefore, when researchers evaluate the effect of various

treatments (such as disinfection, dipping/coating, MAP) on the quality of microorganisms of

fresh-cut fruit, we suggest using fresh-cut fruit produced by commercial processers rather than

that produced in a laboratory. Additionally, producers of fresh-cut fruit must follow good

manufacturing practice (GMP) to reduce the initial microorganism density, avoid cross

contamination and therefore ensure the microbial quality and shelf-life of fresh-cut fruit.

In comparison to the recommended initial yeast count on fresh-cut fruit by Uyttendaele et al.

(2010) (3 log10 CFU/g), the initial population densities of yeasts on the commercial fresh-cut

products that were evaluated were high. The yeast counts on fresh-cut honeydew melon on the

packaging day (day 0) were even ca. 5 log10 CFU/g (see Tables 3.4 and 4.2), which is equivalent

to the end of shelf-life value proposed by Uyttendaele et al. (2010). Hence, in this case, the

growth of microorganisms can be the predominant factor that limiting the shelf-life of fresh-cut

fruit. In order to obtain a high initial quality of fresh-cut fruit, decontamination techniques for

fresh-cut fruit should be developed to reduce the initial microbial contamination level. Although

some decontamination techniques have been developed (see Chapter 1), it is still a challenge to

develop a method for fresh-cut fruit which has no negative effect on the physical parameters

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163

(such as firmness) and the sensory quality, whilst at the same time fitting the consumers’

increasing requirements for natural foods.

With regards to the application of MAs to inhibit microbial spoilage of fresh-cut fruit, the major

part of this thesis focused on building up knowledge through the analysis of microbial growth and

their volatile production and their effect on the sensory quality of fresh-cut fruit in MA condition.

MAs with lowered O2 levels only have a minor effect on the growth of yeasts and LAB on fruit

simulation agar (Chapter 3). However, lowered O2 levels appreciably retarded the production of

volatile organic compounds (VOCs) by yeasts on both honeydew melon and pineapple agar. The

trend was less clear for LAB on honeydew melon agar. It is generally known that low O2 levels

may result in fermentation of both fresh-cut fruit tissues and yeasts. This is mostly due to the fact

that reduced O2 level may limit the function of the TCA cycle of the yeasts as O2 is generally the

terminal electron acceptor. However, fresh-cut fruit usually have a high level of glucose and

fructose resulting in a respiro-fermentative metabolism of yeasts even O2 is present. Hence, the

MAs with reduced O2 level had a minor effect on the growth of yeasts. However, MAs with

reduced O2 levels can retard the production of spoilage associated volatile metabolites and CO2

on fresh-cut honeydew melon and pineapple.

Atmospheres with high O2 level (balanced with N2) have been evaluated towards their effect on

the quality attributes (microbial growth, colour, texture, etc.) of fresh-cut fruit by several studies.

These atmospheres have recently been recommended as an alternative to low O2 MAs to extend

the shelf-life by inhibiting the growth of naturally occurring spoilage microorganisms, preventing

undesired anoxic respiratory processes and decay of fresh and fresh-cut produce (Day, 2001;

Jacxsens et al., 2001; Oms-Oliu et al., 2008e; Wszelaki et al., 2000). However, high O2 levels

may stimulate the browning of fresh-cut fruit and may also stimulate the growth of some yeast

species (Amanatidou et al., 1999).

Until the studies reported in this thesis, the effect of high CO2 atmospheres on the shelf-life and

quality of fresh-cut fruit products had not yet been studied. This is probably due to the fact that

high CO2 atmospheres are known to cause CO2 injury to fruit tissue (Forney et al., 2010). CO2

injury is a physiological disorder directly caused by CO2 (Meheriuk et al., 1994). It frequently

occurs on whole fruit, especially in apples and pears, during storage (Meheriuk et al., 1994;

Watkins et al., 1997). CO2 injury on whole fruit includes both external and internal injuries. For


164

example, the symptoms of external CO2 injury on apples are similar to those of superficial

scalding but in difference the affected areas are rough (Watkins et al., 1997), whilst the internal

symptoms in apples are mainly browning of the cortex and core tissues (Meheriuk et al., 1994).

Franck et al. (2007) concluded that browning of the flesh is caused by an imbalance between

oxidative and reductive processes due to metabolic gas gradients inside the fruit. This may lead to

an accumulation of reactive oxygen species (ROS) which, in turn, may induce loss of membrane

integrity which becomes macroscopically visible through the enzymatic oxidation of phenolic

compounds to brown coloured polymers (Franck et al., 2007). Hence, it can be deduced that high

CO2 atmospheres may result in i) anaerobic conditions which may stimulate the production of

off-flavours by fruit tissues and ii) browning of the flesh of fresh-cut fruit.

However, different fruit may have different responses to high CO2 level. High CO2 levels can

either slow down, stimulate or have no effect on respiratory rates depending on the commodity

and the concentration of gas employed (Mathooko, 1996). The results from Chapters 4

illustrated that CO2 injury does not occur or only occurs slightly on fresh-cut pineapple and

honeydew melon over the shelf-life, respectively. However, it was determined in Chapter 5 that

CO2 injury occurred on fresh-cut apple during storage, resulting which stimulated the production

of VOCs resulting in bad sensory quality.

The application of modified atmospheres combining high O2 and high CO2 levels can potentially

maintain a good microbial quality of fresh-cut fruit as the growth of aerobes and anaerobes can

be negatively affected by the high CO2 level and high O2 level, respectively. In contrast with

whole fruit, modified atmospheres combining high O2 levels and high CO2 levels may avoid CO2

injury on fresh-cut fruit products due to:

a) The tolerance limits to high CO2 atmosphere are known to increase with an increase in O2

level (Kader et al., 1989).

b) Minimally processed (cut, sliced, or otherwise prepared) fruits and vegetables have fewer

barriers to gas diffusion, and consequently they tolerate higher concentrations of CO2 and

lower O2 levels than intact commodities (Kader et al., 1989).

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165

c) Fresh-cut fruit stored at low temperatures generally have a short shelf-life of 3 - 7 days.

Hence, fresh-cut fruit may tolerate higher levels of CO2 or lower levels of O2 than whole

fruit which are stored for long duration (Kader et al., 1989).

d) As discussed in § 6.2, the potential browning in atmospheres with high O2 levels and/or

high CO2 levels can be effectively inhibited by treatment with ascorbic acid before

packaging.

In Chapter 4, it was determined that MAs combining a high O2 and high CO2 levels greatly

retarded the growth of spoilage yeasts and their production of VOCs on fruit simulation agar. The

MA of 50% O2 and 50% CO2 showed the greatest inhibitory effect on the growth of spoilage

yeasts, resulting in growth rates which were ca. three times slower than those observed in air. In

difference, the MAs with high O2 and high CO2 levels only had a minor effect on the growth and

production of volatile metabolites of LAB.

The TCA cycle of yeasts may be affected by the external concentration of sugars and

atmospheres with reduced O2 levels resulted in fermentation and consequently the production of

volatile metabolites. Atmospheres with high O2 levels and/or high CO2 levels may also disturb

the TCA cycle. Additionally, high CO2 levels may change the fluidity of the membranes of the

yeasts (Aguilera et al., 2005; Dixon et al., 1989; Garcia-Gonzalez et al., 2007). However, MAs

with reduced O2 levels had only a minor effect on the growth of yeasts evaluated. In contrast, the

effect of atmospheres combining high O2 and CO2 levels on the growth of yeasts investigated was

large. It could be concluded from the studies performed on fruit simulation agar that the

retardation of the growth of yeasts by MAs combining high O2 and CO2 levels is most likely a

result of the effect of CO2 on the membrane rather than the effect of high CO2 level and/or high

O2 levels on the TCA cycle of yeasts. However, yeasts may be more sensitive to CO2 in aerobic

conditions than in anaerobic conditions in glucose-limited chemostat cultures (Aguilera et al.,

2005). Therefore, MAs combining high O2 and CO2 levels can achieve stronger inhibition to

yeasts than high O2 or high CO2 used alone.

Whilst MAs combining high O2 and CO2 levels can retard the growth of yeasts they can also

stimulate their production of VOCs. For instance, when the population densities of C. sake on


166

honeydew melon agar packaged in an initial headspace (IH) of air and in an IH of 50% O2 + 30%

CO2 reached ca. 8 log10 CFU/cm2 on days 6 and 10 of incubation, respectively, the

concentrations of ethanol in the packages were ca. 52 and 109 mg/m3, respectively. The

explanation could be that atmospheres combining high O2 and high CO2 levels disturbed the TCA

cycle of the yeasts and increased the flux of pyruvate that entered the fermentation pathways

which are responsible for ethanol production and its related compounds. Nevertheless, the

quantity of VOCs in the headspace of samples packaged in the MAs at the moment of evaluation

was much lower than that in air partly due to the fact that the population densities of yeasts on

simulation agar packed in air were generally higher than in MAs (see Figures 4.2 and 4.8).

However, microbiological specifications for fresh-cut fruit packaged under MAs at the end of the

shelf-life should be lower than for fresh-cut fruit stored in air as spoilage yeasts produce higher

quantities of VOCs in MAP than in air.

Fresh-cut fruit packaged in MAs with high O2 and CO2 levels generally resulted in a larger

decrease of the colour parameters at the beginning of storage. The changes of the flesh colour of

fresh-cut fruit during storage are due to the action of polyphenol oxidases, which convert

phenolic compounds into dark coloured pigments in the presence of O2. Nevertheless, the colour

of fresh-cut fruit in packaged in air or MAs with reduced O2 levels or high O2 levels combined

with high CO2 levels was acceptable during the shelf-life judged by the sensory panellists.

Generally, the MAs evaluated in this thesis have a small effect on the cut surface colour of fresh-

cut fruit mostly due to the fact that fresh-cut fruit were dipped in ascorbic acid solution before

packaging.

It could be concluded that since an atmosphere combining high O2 and CO2 levels can greatly

retard the growth of aerobes and yeasts on fresh-cut fruit, it may extend the shelf-life of fresh-cut

pineapple and honeydew melon by 1 – 2 days. Even 1 – 2 days extension of the shelf-life may

result in important logistic benefits to the producers of fresh-cut fruit. However, physiological

disorders attributed to high CO2 levels can occur during the storage of fresh-cut apple, which

could be impact the shelf-life negatively.

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167

Recommendations for further study

i) Nowadays, commercial fresh-cut fruit are mostly packaged in air. Commercial processers

can also consider applying the MAs evaluated in this thesis, however, the suitability of

these MAs still need to be specifically assessed for the intended products before they are

applied. Despite the fact that various MAs in high gas barrier films have been evaluated in

this thesis, a broader range of MAs (with regards to the combination of O2 and CO2

levels) in both high gas barrier films and gas permeable films should be further examined

and this for several different types of fresh-cut fruit. Additionally, more research on the

mechanisms of the responses of different fresh-cut fruit to these MAs is needed,

especially why the CO2 injury is more obvious in fresh-cut apple than in others.

ii) The VOCs produced by the yeasts evaluated in this thesis were produced in the presence

of high external sugar concentrations. The effect of MAs evaluated in this study on the

growth and VOC production of yeasts may further studied on media with low external

sugar content. In this way, more knowledge would be obtained regarding the effect of

MAs on the metabolism of yeasts. Moreover, the VOCs measured by SIFT-MS in this

thesis surely affected the sensory quality of fresh-cut fruit during storage. However, some

of these VOCs may have a greater effect on the sensory quality attributes than others.

Hence, further studies should also focus on the effect of specific VOCs on the sensory

quality of fresh-cut fruit. These key VOCs can be used as indicators for spoilage. Since

the amounts of ethanol, ethyl acetate and phenylacetic acid increased more rapidly than

other VOCs during storage of fresh-cut pineapple, honeydew melon and apple, the

potential indicators for spoilage of fresh-cut fruit could be ethanol, ethyl acetate and

phenylacetic acid.

iii) Although the translucency of fresh-cut fruit during storage was not measured in this thesis,

it did occur in fresh-cut pineapple and honeydew melon. Translucency was also noted to

increase during storage, irrespective of the initial atmospheres. Hence, translucency, being

a quality defect of fresh-cut melon and pineapple should be taken into consideration in the


168

future studies.

iv) Although the safety of fresh-cut fruit was not evaluated in this thesis, fresh-cut fruit

tissues represent a better substrate for both spoilage microorganisms and foodborne

pathogens. In order to ensure the safety of fresh-cut fruit, the effect of MAs, especially

atmospheres with high O2 and high CO2 levels, on pathogens such as Listeria

monocytogenes, Escherichia coli O157:H7 and Salmonella spp. should be studied,

especially for high pH fruits such as honeydew melon.

REFERENCE LIST

Aaby, K., Haffner, K., Skrede, G. 2002. Aroma quality of gravenstein apples influenced by

regular and controlled atmosphere storage. Food Science and Technology/Lebensmittel-

Wissenschaft und Technologie 35, 254-259.

Abadias, M., Usall, J., Anguera, M., Solson, C., Vinas, I. 2008. Microbiological quality of fresh,

minimally-processed fruit and vegetables, and sprouts from retail establishments. International

Journal of Food Microbiology 123, 121-129.

Abdulra'uf, L.B., Chai, M.K., Tan, G.H. 2012. Applications of solid-phase microextraction for

the analysis of pesticide residues in fruits and vegetables: A review. Journal of AOAC

International 95, 1272-1290.

Aguayo, E., Allende, A., Artes, F. 2003. Keeping quality and safety of minimally fresh processed

melon. European Food Research and Technology 216, 494-499.

Aguayo, E., Escalona, V.H., Artes, F. 2004. Metabolic behavior and quality changes of whole

and fresh processed melon. Journal of Food Science 69, S148-S155.

Aguayo, E., Escalona, V.H., Artes, F. 2007. Quality of minimally processed cucumis melo var.

Saccharinus as improved by controlled atmosphere. European Journal of Horticultural Science

72, 39-45.

Aguayo, E., Requejo-Jackman, C., Stanley, R., Woolf, A. 2010. Effects of calcium ascorbate

treatments and storage atmosphere on antioxidant activity and quality of fresh-cut apple slices.

Postharvest Biology and Technology 57, 52-60.

Aguilera, J., Petit, T., de Winde, J.H., Pronk, J.T. 2005. Physiological and genome-wide

transcriptional responses of saccharomyees cerevisiae to high carbon dioxide concentrations.

FEMS Yeast Research 5, 579-593.

Ahvenainen, E.L.a.R. 2002. Minimal processing of fresh fruits and vegetables. In: Jongen,

W.M.F., (Ed.), Fruit and vegetable processing: Improving quality. CRC Press; Woodhead Pub.,

Boca Raton, FL Cambridge, England, 288-308.

REFERENCE LIST

170

Ahvenainen, R. 1996. New approaches in improving the shelf life of minimally processed fruit

and vegetables. Trends in Food Science & Technology 7, 179-187.

Aked, J. 2002. Maintaining the post-harvest quality of fruits and vegetables. In: Jongen, W.M.F.,

(Ed.), Fruit and vegetable processing: Improving quality. CRC Press, pp 119-149.

Al-Ati, T., Hotchkiss, J.H. 2002. Application of packaging and modified atmosphere to fresh-cut

fruits and vegetables. In: Lamikanra, O., (Ed.), Fresh-cut fruits and vegetables: Science,

technology, and market. Taylor & Francis.

Al-Ati, T., Hotchkiss, J.H. 2003. The role of packaging film permselectivity in modified

atmosphere packaging. Journal of Agricultural and Food Chemistry 51, 4133-4138.

Alandes, L., Hernando, I., Quiles, A., Perez-Munuera, I., Lluch, M.A. 2006. Cell wall stability of

fresh-cut fuji apples treated with calcium lactate. Journal of Food Science 71, S615-S620.

Alandes, L., Perez-Munuera, I., Llorca, E., Quiles, A., Hernando, I. 2009a. Use of calcium lactate

to improve structure of "flor de invierno" fresh-cut pears. Postharvest Biology and Technology

53, 145-151.

Alandes, L., Quiles, A., Perez-Munuera, I., Hernando, I. 2009b. Improving the quality of fresh-

cut apples, pears, and melons using natural additives. Journal of Food Science 74, S90-S96.

Alandes, L., Quiles, A., Perez-Munuera, I., Hernando, I. 2011. Manzana fresca cortada tratada

con aditivos naturales: Calidad y aspectos estructurales fresh-cut apple treated with natural

additives: Quality and structural aspects. Cyta-Journal of Food 9, 17-24.

Alothman, M., Bhat, R., Karim, A.A. 2009. Uv radiation-induced changes of antioxidant capacity

of fresh-cut tropical fruits. Innovative Food Science & Emerging Technologies 10, 512-516.

Amadei, G., Ross, B.M. 2011. The reactions of a series of terpenoids with h3o+, no+ and o-2(+)

studied using selected ion flow tube mass spectrometry. Rapid Communications in Mass

Spectrometry 25, 162-168.

Amanatidou, A., Slump, R.A., Gorris, L.G.M., Smid, E.J. 2000. High oxygen and high carbon

dioxide modified atmospheres for shelf-life extension of minimally processed carrots. Journal

of Food Science 65, 61-66.

REFERENCE LIST

171

Amanatidou, A., Smid, E.J., Gorris, L.G.M. 1999. Effect of elevated oxygen and carbon dioxide

on the surface growth of vegetable-associated micro-organisms. Journal of Applied

Microbiology 86, 429-438.

Amaro, A.L., Beaulieu, J.C., Grimm, C.C., Stein, R.E., Almeida, D.P.F. 2012. Effect of oxygen

on aroma volatiles and quality of fresh-cut cantaloupe and honeydew melons. Food Chemistry

130, 49-57.

Anese, M., Manzano, M., Nicoli, M.C. 1997. Quality of minimally processed apple slices using

different modified atmosphere conditions. Journal of Food Quality 20, 359-370.

Antoniolli, L.R., Benedetti, B.C., de Souza, M.D.M. 2003. Effect of calcium chloride on quality

of fresh-cut 'perola' pineapple. Pesquisa Agropecuaria Brasileira 38, 1105-1110.

Antoniolli, L.R., Benedetti, B.C., Filho, M., Borges, M.F., Garruti, D.S. 2005. Evaluation of

hydrogen peroxide as alternative to the use of sodium hypochlorite in fresh-cut 'perola'

pineapple. Proceedings of the 5th International Postharvest Symposium, Vols 1-3, 1859-1864.

Arias, E., Gonzalez, J., Lopez-Buesa, P., Oria, R. 2008. Optimization of processing of fresh-cut

pear. Journal of the Science of Food and Agriculture 88, 1755-1763.

Axelsson, L. 2004. Lactic acid bacteria: Classification and physiology. In: Salminen, S., Wright,

A.v., Ouwehand, A., (Eds.), Lactic acid bacteria : Microbiology and functional aspects, 3rd ed.

Marcel Dekker, New York, pp. 1-67.

Ayhan, Z., Chism, G.W., Richter, E.R. 1998. The shelf-life of minimally processed fresh cut

melons. Journal of Food Quality 21, 29-40.

Babic, I., Hilbert, G., Nguyenthe, C., Guiraud, J. 1992. The yeast flora of stored ready-to-use

carrots and their role in spoilage. International Journal of Food Science and Technology 27,

473-484.

Bai, J.H., Saftner, R.A., Watada, A.E. 2003. Characteristics of fresh-cut honeydew (cucumis

xmelo l.) available to processors in winter and summer and its quality maintenance by

modified atmosphere packaging. Postharvest Biology and Technology 28, 349-359.

Bai, J.H., Saftner, R.A., Watada, A.E., Lee, Y.S. 2001. Modified atmosphere maintains quality of

fresh-cut cantaloupe (cucumis melo l.). Journal of Food Science 66, 1207-1211.

REFERENCE LIST

172

Bai, J.H., Wu, P., Manthey, J., Goodner, K., Baldwin, E. 2009. Effect of harvest maturity on

quality of fresh-cut pear salad. Postharvest Biology and Technology 51, 250-256.

Baldwin, J.C.B.a.E.A. 2002. Flavor and aroma of fresh-cut fruits and vegetables. In: Lamikanra,

O., (Ed.), Fresh-cut fruits and vegetables: Science, technology, and market. CRC Press, Boca

Raton, 391-426.

Baltussen, E., Sandra, P., David, F., Cramers, C. 1999. Stir bar sorptive extraction (sbse), a novel

extraction technique for aqueous samples: Theory and principles. Journal of Microcolumn

Separations 11, 737-747.

Baranyi, J., Roberts, T.A., McClure, P. 1993. A nonautonomous differential-equation to model

bacterial-growth. Food Microbiology 10, 43-59.

Barrancos, S., Beirao-da-costa, M.L., Moldao-Martins, M., Abreu, M., Goncalves, E.M., Costa,

S.B.D. 2003. The effect of heat pre-treatment on quality and shelf-life of fresh-cut apples. In:

Verlinden, B.E., Nicolai, B.M., DeBaerdemaeker, J., (Eds.), Proceedings of the international

conference postharvest unlimited. International Society Horticultural Science, Leuven,

Belgium, 595-601.

Barrett, D.M., Beaulieu, J.C., Shewfelt, R. 2010. Color, flavor, texture, and nutritional quality of

fresh-cut fruits and vegetables: Desirable levels, instrumental and sensory measurement, and

the effects of processing. Critical Reviews in Food Science and Nutrition 50, 369-389.

Barrett, D.M., LunaGuzman, I. 1996. Fresh cut melons: Use of refrigeration and postharvest

treatments to extend shelf life. New Developments in Refrigeration for Food Safety and

Quality, 285-289.

Barrett, D.M., Somogyi, L.P., Ramaswamy, H.S. 2005. Processing fruits science and technology,

2nd ed. CRC Press, Boca Raton.

Barriga, M.I., Trachy, G., Willemot, C., Simard, R.E. 1991. Microbial changes in shredded

iceberg lettuce stored under controlled atmospheres. Journal of Food Science 56, 1586-&.

Barth, M., Hankinson, T.R., Zhuang, H., Breidt, F. 2010. Microbiological spoilage of fruits and

vegetables, Compendium of the microbiological spoilage of foods and beverages. Springer

New York, 135-183.

REFERENCE LIST

173

Beaulieu, J.C., Gorny, J.R. 2001. Fresh-cut fruits, The commercial storage of fruits, vegetables,

and florist and nursery stocks. Usda handbook, vol. 66, 1-49.

Beirao-da-Costa, S., Steiner, A., Correia, L., Empis, J., Moldao-Martins, M. 2006. Effects of

maturity stage and mild heat treatments on quality of minimally processed kiwifruit. Journal of

Food Engineering 76, 616-625.

Bicchi, C., Cordero, C., Liberto, E., Sgorbini, B., Rubiolo, P. 2008. Headspace sampling of the

volatile fraction of vegetable matrices. Journal of Chromatography A 1184, 220-233.

Bierhals, V.S., Chiumarelli, M., Hubinger, M.D. 2011. Effect of cassava starch coating on quality

and shelf life of fresh-cut pineapple (ananas comosus l. Merril cv "perola"). Journal of Food

Science 76, E62-E72.

Björkroth, J., Holzapfel, W. 2006. Genera leuconostoc, oenococcus and weissella. In: Dworkin,

M., Falkow, S., Rosenberg, E., Schleifer, K.-H., Stackebrandt, E., (Eds.), The prokaryotes, vol.

Volume 4: Bacteria: Firmicutes, Cyanobacteria. Springer, pp. 267-319.

Boumba, V.A., Ziavrou, K.S., Vougiouklakis, T. 2008. Biochemical pathways generating post-

mortem volatile compounds co-detected during forensic ethanol analyses. Forensic Science


Boynton, B.B., Welt, B.A., Sims, C.A., Brecht, J.K., Balaban, M.O., Marshall, M.R. 2005.

Effects of low-dose electron beam irradiation on respiration, microbiology, color, and texture

of fresh-cut cantaloupe. Horttechnology 15, 802-807.

Budu, A.S., Joyce, D.C., Terry, L.A. 2007. Quality changes in sliced pineapple under controlled

atmosphere storage. Journal of Horticultural Science & Biotechnology 82, 934-940.

Buta, J.G., Abbott, J.A. 2000. Browning inhibition of fresh-cut 'anjou', 'bartlett', and 'bosc' pears.

HortScience 35, 1111-1113.

Buta, J.G., Moline, H.E., Spaulding, D.W., Wang, C.Y. 1999. Extending storage life of fresh-cut

apples using natural products and their derivatives. Journal of Agricultural and Food

Chemistry 47, 1-6.

REFERENCE LIST

174

Buzby, J.C., Wells, H.F., Axtman, B., Mickey, J. 2009. Supermarket loss estimates for fresh fruit,

vegetables, meat, poultry, and seafood and their use in the ers loss-adjusted food availability

data. United States Department of Agriculture, Economic Research Service.

Campos-Vargas, R., Defilippi, B.G., Romero, P., Valdes, H., Robledo, P., Prieto, H. 2008. Effect

of harvest time and l-cysteine as an antioxidant on flesh browning of fresh-cut cherimoya

(annona cherimola mill.). Chilean Journal of Agricultural Research 68, 217-227.

Catalano, A.E., Ingallinera, B., Todaro, A., Rapisarda, P., Spagna, G. 2009. Degradative

enzymatic activities in fresh-cut blood-orange slices during chilled-storage. International

Journal of Food Science and Technology 44, 1041-1049.

Chen, C.-C., Paull, R.E. 2001. Fruit temperature and crown removal on the occurrence of

pineapple fruit translucency. Scientia Horticulturae 88, 85-95.

Chisari, M., Barbagallo, R.N., Spagna, G., Artes, F. 2011. Improving the quality of fresh-cut

melon through inactivation of degradative oxidase and pectinase enzymatic activities by uv-c

treatment. International Journal of Food Science and Technology 46, 463-468.

Chiumarelli, M., Hubinger, M.D. 2012. Stability, solubility, mechanical and barrier properties of

cassava starch - carnauba wax edible coatings to preserve fresh-cut apples. Food

Hydrocolloids 28, 59-67.

Chonhenchob, V., Chantarasomboon, Y., Singh, S.P. 2007. Quality changes of treated fresh-cut

tropical fruits in rigid modified atmosphere packaging containers. Packaging Technology and

Science 20, 27-37.

Chung, H.-S., Moon, K.-D. 2009a. Browning characteristics of fresh-cut ‘tsugaru’apples as

affected by pre-slicing storage atmospheres. Food Chemistry 114, 1433-1437.

Chung, H.S., Moon, K.D. 2009b. Browning characteristics of fresh-cut 'tsugaru' apples as

affected by pre-slicing storage atmospheres. Food Chemistry 114, 1433-1437.

Cocci, E., Rocculi, P., Romani, S., Dalla Rosa, M. 2006. Changes in nutritional properties of

minimally processed apples during storage. Postharvest Biology and Technology 39, 265-271.

REFERENCE LIST

175

Conesa, A., Artes-Hernandez, F., Geysen, S., Nicolai, B., Artes, F. 2007. High oxygen combined

with high carbon dioxide improvesmicrobial and sensory quality of fresh-cut peppers.


Cook, R. 2008. Trends in the marketing of fresh produce and fresh-cut products [Online].

Available at http://agecon.ucdavis.edu/extension/presentations/files/cook/trends-in-the-

marketing-of-fresh-produce-and-fresh-cut-products.pdf.

COOK, R. 2011. Trends in the marketing of fresh produce and fresh-cut products [Online].

Available at http://ucce.ucdavis.edu/files/datastore/234-2115.pdf.

Cook, R. 2012. Trends in the marketing of fresh produce and fresh-cut/value-added produce

[Online]. Available at http://agecon.ucdavis.edu/people/faculty/roberta-

cook/docs/Articles/Freshcut2012Cook120927Final.pdf.

Corbo, M.R., Altieri, C., D'Amato, D., Campaniello, D., Del Nobile, M.A., Sinigaglia, M. 2004.

Effect of temperature on shelf life and microbial population of lightly processed cactus pear

fruit. Postharvest Biology and Technology 31, 93-104.

Damiani, C., Boas, E., Pinto, D.M. 2008a. Fresh-cut tangerine stored under two temperatures.

Ciencia E Agrotecnologia 32, 308-313.

Damiani, C., Boas, E., Pinto, D.M., Rodrigues, L.J. 2008b. Influence of different temperatures in

maintenance of quality of fresh-cut pequi. Ciencia E Agrotecnologia 32, 203-212.

Damore, T., Russell, I., Stewart, G.G. 1989. Sugar utilization by yeast during fermentation.

Journal of Industrial Microbiology 4, 315-324.

Davis, B.M., McEwan, M.J. 2007. Determination of olive oil oxidative status by selected ion

flow tube mass spectrometry. Journal of Agricultural and Food Chemistry 55, 3334-3338.

Davis, B.M., Senthilmohan, S.T., Wilson, P.F., McEwan, M.J. 2005. Major volatile compounds

in head-space above olive oil analysed by selected ion flow tube mass spectrometry. Rapid

Communications in Mass Spectrometry 19, 2272-2278.

Day, B.P.F. 2001. Modified atmosphere packaging of fresh fruit and vegetables - an overview. In:

BenArie, R., PhilosophHadas, S., (Eds.), Proceedings of the 4th international conference on

postharvest science, vols 1 and 2, 585-590.

http://agecon.ucdavis.edu/extension/presentations/files/cook/trends-in-the-marketing-of-fresh-produce-and-fresh-cut-products.pdf

http://agecon.ucdavis.edu/extension/presentations/files/cook/trends-in-the-marketing-of-fresh-produce-and-fresh-cut-products.pdf

http://ucce.ucdavis.edu/files/datastore/234-2115.pdf

http://agecon.ucdavis.edu/people/faculty/roberta-cook/docs/Articles/Freshcut2012Cook120927Final.pdf

http://agecon.ucdavis.edu/people/faculty/roberta-cook/docs/Articles/Freshcut2012Cook120927Final.pdf

REFERENCE LIST

176

Del Nobile, M.A., Conte, A., Scrocco, C., Laverse, J., Brescia, I., Conversa, G., Elia, A. 2009.

New packaging strategies to preserve fresh-cut artichoke quality during refrigerated storage.

Innovative Food Science & Emerging Technologies 10, 128-133.

Delheimer, S. 2012. S-294 (2005-2011), quality and safety of freshcut produce [Online].

Available at http://saaesd.ncsu.edu/docs/S294Impact%20StatementFINAL20120920.pdf.

Devlieghere, F., Debevere, J. 2000. Influence of dissolved carbon dioxide on the growth of

spoilage bacteria. Food Science and Technology/Lebensmittel-Wissenschaft und Technologie

33, 531-537.

Devos, M. 1990. Standardized human olfactory thresholds IRL Press at Oxford University Press,

Oxford.

Di Egidio, V., Sinelli, N., Limbo, S., Torri, L., Franzetti, L., Casiraghi, E. 2009. Evaluation of

shelf-life of fresh-cut pineapple using ft-nir and ft-ir spectroscopy. Postharvest Biology and

Technology 54, 87-92.

Dimick, P.S., Hoskin, J.C. 1983. Review of apple flavor - state of the art. CRC Critical Reviews

in Food Science and Nutrition 18, 387-409.

Dixon, N.M., Kell, D.B. 1989. The inhibition by co2 of the growth and metabolism of micro-

organisms. Journal of Applied Bacteriology 67, 109-136.

Djioua, T., Charles, F., Freire, M., Filgueiras, H., Ducamp-Collin, M.N., Sallanon, H. 2010.

Combined effects of postharvest heat treatment and chitosan coating on quality of fresh-cut

mangoes (mangifera indica l.). International Journal of Food Science and Technology 45, 849-

855.

Dong, X., Wrolstad, R.E., Sugar, D. 2000. Extending shelf life of fresh-cut pears. Journal of Food

Science 65, 181-186.

Echeverria, G., Fuentes, T., Graell, J., Lara, I., Lopez, M.L. 2004a. Aroma volatile compounds of

'fuji' apples in relation to harvest date and cold storage technology - a comparison of two

seasons. Postharvest Biology and Technology 32, 29-44.

http://saaesd.ncsu.edu/docs/S294Impact%20StatementFINAL20120920.pdf

REFERENCE LIST

177

Echeverria, G., Graell, J., Lopez, M.L., Lara, I. 2004b. Volatile production, quality and aroma-

related enzyme activities during maturation of 'fuji' apples. Postharvest Biology and


Eduardo, M.D.P., Benedetti, B.C., Ferraz, A.C.O. 2008. Firmness indexes evaluation for fresh-

cut sliced pineapple treated with calcium salts solutions. Engenharia Agricola 28, 154-163.

Elss, S., Preston, C., Appel, M., Heckel, F., Schreier, P. 2006. Influence of technological

processing on apple aroma analysed by high resolution gas chromatography-mass

spectrometry and on-line gas chromatography-combustion/pyrolysis-isotope ratio mass

spectrometry. Food Chemistry 98, 269-276.

Elss, S., Preston, C., Hertzig, C., Heckel, F., Richling, E., Schreier, P. 2005. Aroma profiles of

pineapple fruit (ananas comosus l. Merr.) and pineapple products. Lwt-Food Science and


Emmerich, W., Radler, F. 1983. The anaerobic metabolism of glucose and fructose by

saccharomyces-bailii. Journal of General Microbiology 129, 3311-3318.

Ergun, M., Akkaya, O., Ergun, N. 2008. Suitability of some mid-season table grape cultivars and

types for minimally processed produce. Journal International Des Sciences De La Vigne Et Du

Vin 42, 99-105.

Ergun, M., Jeong, J., Huber, D.J., Cantliffe, D.J. 2007. Physiology of fresh-cut 'galia' (cucumis

melo var. Reticulatus) from ripe fruit treated with 1-methylcyclopropene. Postharvest Biology

and Technology 44, 286-292.

Fahlbusch, K.G., Hammerschmidt, F.J., Panten, J., Pickenhagen, W., Schatkowski, D., Bauer, K.,

Garbe, D., Surburg, H. 2003. Flavors and fragrances, ullmann's encyclopedia of industrial

chemistry Wiley–VCH, Weinheim.

Fallik, E., Rodov, V., Horev, B., Sela, S., Alkalai-Tuvia, S., Vinokur, Y. 2007. Hot water rinsing

and brushing technology for the fresh-cut industry. Proceedings of the International

Conference on Quality Management of Fresh Cut Produce, 229-235.

REFERENCE LIST

178

Fan, X., Annous, B.A., Beaulieu, J.C., Sites, J.E. 2008a. Effect of hot water surface

pasteurization of whole fruit on shelf life and quality of fresh-cut cantaloupe. Journal of Food

Science 73, M91-M98.

Fan, X., Sokorai, K.J.B. 2008b. Effect of ionizing radiation on furan formation in fresh-cut fruits

and vegetables. Journal of Food Science 73, C79-C83.

Fan, X.T., Annous, B.A., Sokorai, K.J.B., Burke, A., Mattheis, J.P. 2006. Combination of hot-

water surface pasteurization of whole fruit and low-dose gamma irradiation of fresh-cut

cantaloupe. Journal of Food Protection 69, 912-919.

Fan, X.T., Niemera, B.A., Mattheis, J.P., Zhuang, H., Olson, D.W. 2005. Quality of fresh-cut

apple slices as affected by low-dose ionizing radiation and calcium ascorbate treatment.

Journal of Food Science 70, S143-S148.

Fan, X.T., Sokorai, K.J.B., Liao, C.H., Cooke, P., Zhang, H.Q. 2009. Antibrowning and

antimicrobial properties of sodium acid sulfate in apple slices. Journal of Food Science 74,

M485-M492.

FDA. 2013. Code of federal regulations title 21, Sec. 101.95. U.S. Food and Drug Administration.

Ferrari, C.C., Sarantopoulos, C., Carmello-Guerreiro, S.M., Hubinger, M.D. 2013. Effect of

osmotic dehydration and pectin edible coatings on quality and shelf life of fresh-cut melon.

Food and Bioprocess Technology 6, 80-91.

Fiechter, A., Seghezzi, W. 1992. Regulation of glucose-metabolism in growing yeast-cells.

Journal of Biotechnology 27, 27-45.

Fleet, G. 1992. Spoilage yeasts. Critical Reviews in Biotechnology 12, 1-44.

Fleet, G. 2003. Yeasts in fruit and fruit products. In: Boekhout, T., Robert, V., Phaff, H., (Eds.),

Yeasts in food : Beneficial and detrimental aspects. Behr's Verlag, Hamburg, pp. 267-287.

Fonseca, J.M., Rushing, J.W. 2006. Effect of ultraviolet-c light on quality and microbial

population of fresh-cut watermelon. Postharvest Biology and Technology 40, 256-261.

Forney, C.F., Mattheis, J.P., Baldwin, E.A. 2009. Effects on flavor. In: Yahia, E.M., (Ed.),

Modified and controlled atmospheres for the storage, transportation, and packaging of

horticultural commodities. CRC Press/Taylor & Francis, Boca Raton, pp. 119-158.

REFERENCE LIST

179

Forney, C.F., Mattheis, J.P., Baldwin, E.A. 2010. Effects on physiological disorders. In: Yahia,

E.M., (Ed.), Modified and controlled atmospheres for the storage, transportation, and

packaging of horticultural commodities. CRC Press, 159-193.

Franck, C., Lammertyn, J., Ho, Q.T., Verboven, P., Verlinden, B., Nicolai, B.A. 2007. Browning

disorders in pear fruit. Postharvest Biology and Technology 43, 1-13.

Fredlund, E. 2004. Central carbon metabolism of the biocontrol yeast pichia anomala: Influence

of oxygen limitation. Doctoral, Dept. of Microbiology, Swedish Univ. of Agricultural

Sciences, Uppsala.

Garcia-Gonzalez, L., Geeraerd, A.H., Spilimbergo, S., Elst, K., Van Ginneken, L., Debevere, J.,

Van Impe, J.F., Devlieghere, F. 2007. High pressure carbon dioxide inactivation of

microorganisms in foods: The past, the present and the future. International Journal of Food


Garcia, E., Barrett, D.M. 2005. Fresh-cut fruits. In: Barrett, D.M., Somogyi, L.P., Ramaswamy,

H.S., (Eds.), Processing fruits: Science and technology, second edition. CRC PressINC, pp.

53-72.

Gardner, J.W., Bartlett, P.N. 1994. A brief history of electronic noses. Sensors and Actuators B:

Chemical 18, 210-211.

Giacalone, G., Chiabrando, V., Bardi, L. 2010. Changes in nutritional properties of minimally

processed fresh fruit during storage. Italian Journal of Food Science 22, 305-312.

Gil, M.I., Gorny, J.R., Kader, A.A. 1998. Responses of 'fuji' apple slices to ascorbic acid

treatments and low-oxygen atmospheres. HortScience 33, 305-309.

Gojkovic, Z., Knecht, W., Zameitat, E., Warneboldt, J., Coutelis, J.B., Pynyaha, Y., Neuveglise,

C., Moller, K., Loffler, M., Piskur, J. 2004. Horizontal gene transfer promoted evolution of the

ability to propagate under anaerobic conditions in yeasts. Molecular Genetics and Genomics

271, 387-393.

Gomes, M.H., Beaudry, R.M., Almeida, D.P.F., Malcata, F.X. 2010. Modelling respiration of

packaged fresh-cut 'rocha' pear as affected by oxygen concentration and temperature. Journal

of Food Engineering 96, 74-79.

REFERENCE LIST

180

Gomes, M.H., Fundo, J.F., Pocas, M.F., Almeida, D.P.F. 2012. Quality changes in fresh-cut

'rocha' pear as affected by oxygen levels in modified atmosphere packaging and the ph of

antibrowning additive. Postharvest Biology and Technology 74, 62-70.

Gonzalez-Aguilar, G.A., Celis, J., Sotelo-Mundo, R.R., de la Rosa, L.A., Rodrigo-Garcia, J.,

Alvarez-Parrilla, E. 2008. Physiological and biochemical changes of different fresh-cut mango

cultivars stored at 5 degrees c. International Journal of Food Science and Technology 43, 91-

101.

Gonzalez-Aguilar, G.A., Ruiz-Cruz, S., Cruz-Valenzuela, R., Rodriguez-Felix, A., Wang, C.Y.

2004. Physiological and quality changes of fresh-cut pineapple treated with antibrowning

agents. Food Science and Technology/Lebensmittel-Wissenschaft und Technologie 37, 369-

376.

Gonzalez-Aguilar, G.A., Ruiz-Cruz, S., Soto-Valdez, H., Vazquez-Ortiz, F., Pacheco-Aguilar, R.,

Wang, C.Y. 2005. Biochemical changes of fresh-cut pineapple slices treated with

antibrowning agents. International Journal of Food Science and Technology 40, 377-383.

Gonzalez-Siso, M.I., Freire-Picos, M.A., Ramil, E., Gonzalez-Dominguez, M., Torres, A.R.,

Cerdan, M.E. 2000. Respirofermentative metabolism in kluyveromyces lactis: Insights and

perspectives. Enzyme and Microbial Technology 26, 699-705.

Gorny, J.R. 2005. Leveraging innovative fresh-cut technologies for competitive advantage.

Proceedings of the International Conference Postharvest Unlimited Downunder 2004, 141-147.

Gorny, J.R., Hess-Pierce, B., Cifuentes, R.A., Kader, A.A. 2002. Quality changes in fresh-cut

pear slices as affected by controlled atmospheres and chemical preservatives. Postharvest

Biology and Technology 24, 271-278.

Gottschalk, G. 1986. Bacterial metabolism Springer, New York.

Graca, A., Abadias, M., Salazar, M., Nunes, C. 2011. The use of electrolyzed water as a

disinfectant for minimally processed apples. Postharvest Biology and Technology 61, 172-177.

Guedes, C.M., Pinto, A.B., Moreira, R.F.A., De Maria, C.A.B. 2004. Study of the aroma

compounds of rose apple (syzygium jambos alston) fruit from brazil. European Food Research


REFERENCE LIST

181

Guler, Z., Karaca, F., Yetisir, H. 2013. Volatile compounds and sensory properties in various

melons, which were chosen from different species and different locations, grown in turkey.

International Journal of Food Properties 16, 168-179.

Gunes, G., Watkins, C.B., Hotchkiss, J.H. 2001. Physiological responses of fresh-cut apple slices

under high co2 and low o-2 partial pressures. Postharvest Biology and Technology 22, 197-

204.

Hall, C., Brachat, S., Dietrich, F.S. 2005. Contribution of horizontal gene transfer to the evolution

of saccharomyces cerevisiae. Eukaryotic Cell 4, 1102-1115.

Hammer, E., Kneifel, H., Hofmann, K., Schauer, F. 1996. Enhanced excretion of intermediates of

aromatic amino acid catabolism during chlorophenol degradation due to nutrient limitation in

the yeast candida maltosa. Journal of Basic Microbiology 36, 239-243.

Hazelwood, L.A., Daran, J.-M., van Maris, A.J.A., Pronk, J.T., Dickinson, J.R. 2008. The ehrlich

pathway for fusel alcohol production: A century of research on saccharomyces cerevisiae

metabolism. Applied and Environmental Microbiology 74, 2259-2266.

Heard, G. 1999. Microbial safety of ready-to-eat salads and minimally processed vegetables and

fruits. Food Australia 51, 414-420.

Heard, G.M. 2002. Microbiology of fresh-cut produce. In: Lamikanra, O., (Ed.), Fresh-cut fruits

and vegetables : Science, technology, and market. CRC Press, Boca Raton, Fla., pp. 187-248.

Higuchi, M., Yamamoto, Y., Kamio, Y. 2000. Molecular biology of oxygen tolerance in lactic

acid bacteria: Functions of nadh oxidases and dpr in oxidative stress. Journal of Bioscience

and Bioengineering 90, 484-493.

Huxsoll, C.C., Bolin, H.R. 1989. Processing and distribution alternatives for minimally processed

fruits and vegetables. Food Technology 43, 124-128.

ISO. 2004. Sensory analysis–methodology–triangle test, BS ISO 4120. BSI, London, UK.

Ito, S., Kobayashi, T., Ohta, Y., Akiyama, Y. 1983. Studies on lactic-acid bacteria .3. Inhibition

of glucose catabolism by aeration in leuconostoc-mesenteroides. Journal of Fermentation


REFERENCE LIST

182

Iversen, E., Wilhelmsen, E., Criddle, R.S. 1989. Calorimetric examination of cut fresh pineapple

metabolism. Journal of Food Science 54, 1246-1249.

Jacxsens, L., Devlieghere, F., Ragaert, P., Vanneste, E., Debevere, J. 2003. Relation between

microbiological quality, metabolite production and sensory quality of equilibrium modified

atmosphere packaged fresh-cut produce. International Journal of Food Microbiology 83, 263-

280.

Jacxsens, L., Devlieghere, F., Van der Steen, C., Debevere, J. 2001. Effect of high oxygen

modified atmosphere packaging on microbial growth and sensorial qualities of fresh-cut

produce. International Journal of Food Microbiology 71, 197-210.

James, J., Ngarmsak, T. 2010. Processing of fresh-cut tropical fruits and vegetables: A technical

guide. Food and Agriculture Organization of the United Nations Regional Office for Asia and

the PacificRap Publication 16.

Jang, J.H., Kim, S.T., Moon, K.D. 2009. Inhibitory effects of ultrasound in combination with

ascorbic acid on browning and polyphenol oxidase activity of fresh-cut apples. Food Science

and Biotechnology 18, 1417-1422.

Jang, J.H., Moon, K.D. 2011. Inhibition of polyphenol oxidase and peroxidase activities on fresh-

cut apple by simultaneous treatment of ultrasound and ascorbic acid. Food Chemistry 124,

444-449.

Jeffrey, S.B., Devon, Z. 2009. Modified and controlled atmosphere packaging technology and

applications, Modified and controlled atmospheres for the storage, transportation, and

packaging of horticultural commodities. CRC Press.

Jeon, M., Zhao, Y. 2005. Honey in combination with vacuum impregnation to prevent enzymatic

browning of fresh-cut apples. International Journal of Food Sciences and Nutrition 56, 165-

176.

Jones, R.P., Greenfield, P.F. 1982. Effect of carbon-dioxide on yeast growth and fermentation.

Enzyme and Microbial Technology 4, 210-222.

REFERENCE LIST

183

Jouhten, P., Wiebe, M., Penttila, M. 2012. Dynamic flux balance analysis of the metabolism of

saccharomyces cerevisiae during the shift from fully respirative or respirofermentative

metabolic states to anaerobiosis. FEBS Journal 279, 3338-3354.

Kader, A.A. 2002. Quality parameters of fresh-cut fruit and vegetable products. In: Lamikanra,

O., (Ed.), Fresh-cut fruits and vegetables : Science, technology, and market. CRC Press, Boca

Raton, Fla., pp 11-20.

Kader, A.A., Barrett, D.M. 2005. Classification, composition of fruits, and postharvest

maintenance of quality. In: Barrett, D.M., Somogyi, L.P., Ramaswamy, H.S., (Eds.),

Processing fruits: Science and technology, second edition. CRC Press Florida, 3-22.

Kader, A.A., Ben-Yehoshua, S. 2000. Effects of superatmospheric oxygen levels on postharvest

physiology and quality of fresh fruits and vegetables. Postharvest Biology and Technology 20,

1-13.

Kader, A.A., Zagory, D., Kerbel, E.L. 1989. Modified atmosphere packaging of fruits and

vegetables. Critical Reviews in Food Science and Nutrition 28, 1-30.

Kaewtathip, T., Charoenrein, S. 2012. Changes in volatile aroma compounds of pineapple

(ananas comosus) during freezing and thawing. International Journal of Food Science and


Kakiomenou, K., Tassou, C., Nychas, G.J. 1996. Microbiological, physicochemical and

organoleptic changes of shredded carrots stored under modified storage. International Journal

of Food Science and Technology 31, 359-366.

Kappeli, O. 1986. Regulation of carbon metabolism in saccharomyces-cerevisiae and related

yeasts. Advances in Microbial Physiology 28, 181-209.

Karaibrahimoglu, Y., Fan, X.T., Sapers, G.M., Sokorai, K. 2004. Effect of ph on the survival of

listeria innocua in calcium ascorbate solutions and on quality of fresh-cut apples. Journal of

Food Protection 67, 751-757.

Karim, A.A., Alothman, M., Bhat, R. 2009. Uv radiation-induced changes of antioxidant capacity

of fresh-cut tropical fruits. Innovative Food Science & Emerging Technologies 10, 512-516.

REFERENCE LIST

184

Karim, A.A., Alothman, M., Kaur, B., Fazilah, A., Bhat, R. 2010. Ozone-induced changes of

antioxidant capacity of fresh-cut tropical fruits. Innovative Food Science & Emerging

Technologies 11, 666-671.

Kays, S.J. 1991. Metabolic processes in harvested products, Postharvest physiology of perishable

plant products. Springer, 75-142.

Ke, D., Goldstein, L., O'MAHONY, M., Kader, A.A. 1991a. Effects of short‐term exposure to

low o2 and high co2 atmospheres on quality attributes of strawberries. Journal of Food

Science 56, 50-54.

Ke, D.Y., Rodriguezsinobas, L., Kader, A.A. 1991b. Physiology and prediction of fruit tolerance

to low-oxygen atmospheres. Journal of the American Society for Horticultural Science 116,

253-260.

Kendall, M.G. 1938. A new measure of rank correlation. Biometrika 30, 81-93.

Kim, D.M., Smith, N.L., Lee, C.Y. 1993. Quality of minimally processed apple slices from

selected cultivars. Journal of Food Science 58, 1115-&.

King, A.D., Magnuson, J.A., Torok, T., Goodman, N. 1991. Microbial-flora and storage quality

of partially processed lettuce. Journal of Food Science 56, 459-461.

Kruger, N.J., von Schaewen, A. 2003. The oxidative pentose phosphate pathway: Structure and

organisation. Current Opinion in Plant Biology 6, 236-246.

Lamikanra, O. 2002a. Enzymatic effects on flavor and texture of fresh-cut fruits and vegetables.

In: Lamikanra, O., (Ed.), Fresh-cut fruits and vegetables: Science, technology, and market.

CRC Press, Boca Raton, Fla., 125.

Lamikanra, O. 2002b. Preface In: Lamikanra, O., (Ed.), Fresh-cut fruits and vegetables : Science,

technology, and market. CRC Press, Boca Raton, Fla.

Lamikanra, O., Richard, O.A. 2004. Storage and ultraviolet-induced tissue stress effects on fresh-

cut pineapple. Journal of the Science of Food and Agriculture 84, 1812-1816.

REFERENCE LIST

185

Lamikanra, O., Watson, M.A. 2001. Effects of ascorbic acid on peroxidase and

polyphenoloxidase activities in fresh-cut cantaloupe melon. Journal of Food Science 66, 1283-

1286.

Larrigaudiere, C., Ubach, D., Oms-Oliu, G., Martin-Belloso, O. 2007. Oxidative behaviour of

fresh-cut 'fuji' apples treated with chemical stabilizers. Proceedings of the International

Conference on Quality Management of Fresh Cut Produce, 315-322.

Larson, A.E., Johnson, E.A. 1999. Evaluation of botulinal toxin production in packaged fresh-cut

cantaloupe and honeydew melons. Journal of Food Protection 62, 948-952.

Lee, J.Y., Park, H.J., Lee, C.Y., Choi, W.Y. 2003. Extending shelf-life of minimally processed

apples with edible coatings and antibrowning agents. Lebensmittel-Wissenschaft Und-

Technologie-Food Science and Technology 36, 323-329.

Lester, G.E. 2008. Antioxidant, sugar, mineral, and phytonutrient concentrations across edible

fruit tissues of orange-fleshed honeydew melon (cucumis melo l.). Journal of Agricultural and

Food Chemistry 56, 3694-3698.

Li, L., Ban, Z.J., Li, X.H., Wang, X.L., Guan, J.F. 2012a. Phytochemical and microbiological

changes of honey pomelo (citrus grandis l.) slices stored under super atmospheric oxygen,

low-oxygen and passive modified atmospheres. International Journal of Food Science and


Li, W.L., Li, X.H., Fan, X., Tang, Y., Yun, J. 2012b. Response of antioxidant activity and

sensory quality in fresh-cut pear as affected by high o-2 active packaging in comparison with

low o-2 packaging. Food Science and Technology International 18, 197-205.

Liao, C.-H. 2005. Bacterial soft rot. In: Sapers, G.M., Gorny, J.R., Yousef, A.E., (Eds.),

Microbiology of fruits and vegetables. Taylor & Francis, 117-134.

Lima, M.S., Pires, E.M.F., Maciel, M.I.S., Oliveira, V.A. 2010. Quality of minimally processed

guava with different types of cut, sanification and packing. Ciencia E Tecnologia De

Alimentos 30, 79-87.

REFERENCE LIST

186

Lin, D.S., Leonard, S.W., Lederer, C., Traber, M.G., Zhao, Y. 2006. Retention of fortified

vitamin e and sensory quality of fresh-cut pears by vacuum impregnation with honey. Journal

of Food Science 71, S553-S559.

Liu, C.L., Hsu, C.K., Hsu, M.M. 2007. Improving the quality of fresh-cut pineapples with

ascorbic acid/sucrose pretreatment and modified atmosphere packaging. Packaging

Technology and Science 20, 337-343.

Loser, C., Urit, T., Forster, S., Stukert, A., Bley, T. 2012. Formation of ethyl acetate by

kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron

limitation. Applied Microbiology and Biotechnology 96, 685-696.

Lu, S.M., Luo, Y.G., Turner, E., Feng, H. 2007. Efficacy of sodium chlorite as an inhibitor of

enzymatic browning in apple slices. Food Chemistry 104, 824-829.

Lucey, C.A., Condon, S. 1986. Active-role of oxygen and nadh oxidase in growth and energy-

metabolism of leuconostoc. Journal of General Microbiology 132, 1789-1796.

Luo, Y.G., Lu, S.M., Zhou, B., Feng, H. 2011. Dual effectiveness of sodium chlorite for

enzymatic browning inhibition and microbial inactivation on fresh-cut apples. Lwt-Food

Science and Technology 44, 1621-1625.

Lyhs, U., Koort, J.M.K., Lundstrom, H.S., Bjorkroth, K.J. 2004. Leuconostoc gelidum and

leuconostoc gasicomitatum strains dominated the lactic acid bacterium population associated

with strong slime formation in an acetic-acid herring preserve. International Journal of Food


Machado, F.L.D., Alves, R.E., Figueiredo, R.W. 2008a. Application of 1-methylcyclopropene,

calcium chloride and calcium amino acid chelate on fresh-cut cantaloupe muskmelon.

Pesquisa Agropecuaria Brasileira 43, 569-574.

Machado, F.L.D., Alves, R.E., Silva, E.D. 2008b. Fresh-cut of cantaloupe melon using doses of

calcium chloride and calcium amino acid chelate. Horticultura Brasileira 26, 56-60.

Manimegalai, G., Neelakantan, S., Vennila, P. 1998. Changes in the trace elements content

during pulping of fruits in different mixies. Journal of Food Science and Technology (Mysore)

35, 262-264.

REFERENCE LIST

187

Manuwong, S., Uthairatanakij, A., Jitareerat, P. 2007. Effects of hot water and sodium

hypochlorite treatments on survival of salmonella spp. And qualities of fresh-cut pineapple.

Proceedings of the International Conference on Quality Management of Fresh Cut Produce,

401-408.

Manzocco, L., Da Pieve, S., Bertolini, A., Bartolomeoli, I., Maifreni, M., Vianello, A., Nicoli,

M.C. 2011a. Surface decontamination of fresh-cut apple by uv-c light exposure: Effects on

structure, colour and sensory properties. Postharvest Biology and Technology 61, 165-171.

Manzocco, L., Da Pieve, S., Maifreni, M. 2011b. Impact of uv-c light on safety and quality of

fresh-cut melon. Innovative Food Science & Emerging Technologies 12, 13-17.

Marrero, A., Kader, A.A. 2006. Optimal temperature and modified atmosphere for keeping

quality of fresh-cut pineapples. Postharvest Biology and Technology 39, 163-168.

Martinez-Ferrer, M., Harper, C. 2005. Reduction in microbial growth and improvement of

storage quality in fresh-cut pineapple after methyl jasmonate treatment. Journal of Food

Quality 28, 3-12.

Martinez-Ferrer, M., Harper, C., Perez-Muroz, F., Chaparro, M. 2002. Modified atmosphere

packaging of minimally processed mango and pineapple fruits. Journal of Food Science 67,

3365-3371.

Mastromatteo, M., Conte, A., Del Nobile, M.A. 2010. Combined use of modified atmosphere

packaging and natural compounds for food preservation. Food Engineering Reviews 2, 28-38.

Mathooko, F.M. 1996. Regulation of respiratory metabolism in fruits and vegetables by carbon

dioxide. Postharvest Biology and Technology 9, 247-264.

Meheriuk, M., Prange, R., Lidster, P., Porritt, S. 1994. Postharvest disorders of apples and pears

Agriculture and Agri-Food Canada.

Merico, A., Galafassi, S., Piskur, J., Compagno, C. 2009. The oxygen level determines the

fermentation pattern in kluyveromyces lactis. FEMS Yeast Research 9, 749-756.

Miccolis, V., Saltveit, M.E. 1995. Influence of storage period and temperature on the postharvest

characteristics of 6 melon (cucumis-melo l, inodorus group) cultivars. Postharvest Biology and


REFERENCE LIST

188

Mohammed, M., Wickham, L.D. 2005. Effect of antioxidants on postharvest quality attributes of

fresh-cut pineapples. Proceedings of the IVth International Pineapple Symposium, 309-314.

Moller, K., Christensen, B., Forster, J., Piskur, J., Nielsen, J., Olsson, L. 2002. Aerobic glucose

metabolism of saccharomyces kluyveri: Growth, metabolite production, and quantification of

metabolic fluxes. Biotechnology and Bioengineering 77, 186-193.

Montero-Calderon, M., Rojas-Grau, M.A., Aguilo-Aguayo, I., Soliva-Fortuny, R., Martin-

Belloso, O. 2010a. Influence of modified atmosphere packaging on volatile compounds and

physicochemical and antioxidant attributes of fresh-cut pineapple (ananas comosus). Journal

of Agricultural and Food Chemistry 58, 5042-5049.

Montero-Calderon, M., Rojas-Grau, M.A., Martin-Belloso, O. 2008. Effect of packaging

conditions on quality and shelf-life of fresh-cut pineapple (ananas comosus). Postharvest

Biology and Technology 50, 182-189.

Montero-Calderon, M., Rojas-Grau, M.A., Martin-Belloso, O. 2010b. Aroma profile and volatiles

odor activity along gold cultivar pineapple flesh. Journal of Food Science 75, S506-512.

Moradas-Ferreira, P., Costa, V., Piper, P., Mager, W. 1996. The molecular defences against

reactive oxygen species in yeast. Molecular Microbiology 19, 651-658.

Morais, A., Arganosa, A. 2010. Quality during storage of fresh-cut papaya (carica papaya l.) in

various shapes. Philippine Agricultural Scientist 93, 88-96.

Moya-Leon, M.A., Vergara, M., Bravo, C., Pereira, M., Moggia, C. 2007. Development of aroma

compounds and sensory quality of 'royal gala' apples during storage. Journal of Horticultural

Science & Biotechnology 82, 403-413.

Nguyenthe, C., Carlin, F. 1994. The microbiology of minimally processed fresh fruits and

vegetables. Critical Reviews in Food Science and Nutrition 34, 371-401.

Nobile, M.A., Sinigaglia, M., Conte, A., Speranza, B., Scrocco, C., Brescia, I., Bevilacqua, A.,

Laverse, J., La Notte, E., Antonacci, D. 2008. Influence of postharvest treatments and film

permeability on quality decay kinetics of minimally processed grapes. Postharvest Biology


REFERENCE LIST

189

Noseda, B. 2012. Volatile spoilage markers indicating bacterial spoilage in modified atmosphere

packaged fish and fishery products. PhD dissertation, Ghent University, Ghent.

Noseda, B., Dewulf, J., Goethals, J., Ragaert, P., Van Bree, I., Pauwels, D., Van Langenhove, H.,

Devlieghere, F. 2010a. Effect of food matrix and ph on the volatilization of bases (tvb) in

packed north atlantic gray shrimp (crangon crangon): Volatile bases in map fishery products.

Journal of Agricultural and Food Chemistry 58, 11864-11869.

Noseda, B., Islam, M.T., Eriksson, M., Heyndrickx, M., De Reu, K., Van Langenhove, H.,

Devlieghere, F. 2012. Microbiological spoilage of vacuum and modified atmosphere packaged

vietnamese pangasius hypophthalmus fillets. Food Microbiology 30, 408-419.

Noseda, B., Ragaert, P., Pauwels, D., Anthierens, T., Van Langenhove, H., Dewulf, J.,

Devlieghere, F. 2010b. Validation of selective ion flow tube mass spectrometry for fast

quantification of volatile bases produced on atlantic cod (gadus morhua). Journal of

Agricultural and Food Chemistry 58, 5213-5219.

O’Connor-Shaw, R., Roberts, R., Ford, A., Nottingham, S. 1994. Shelf-life of minimally

processed honeydew, kiwifruit, papaya, pineapple and cantaloupe. Journal of Food Science 59,

1202-1207.

Oconnor-Shaw, R.E., Roberts, R., Ford, A.L., Nottingham, S.M. 1996. Changes in sensory

quality of sterile cantaloupe dice stored in controlled atmospheres. Journal of Food Science 61,

847-851.

Odriozola-Serrano, I., Soliva-Fortuny, R., Martin-Belloso, O. 2010. Changes in bioactive

composition of fresh-cut strawberries stored under superatmospheric oxygen, low-oxygen or

passive atmospheres. Journal of Food Composition and Analysis 23, 37-43.

Ogassavara, F.O., Durigan, J.F., Teixeira, G.H.D., Cunha, L.C. 2009. Comparison among

cultivars of star fruits for production of fresh-cut. Revista Brasileira De Fruticultura 31, 544-

551.

Olivares, A., Dryahina, K., Navarro, J.L., Flores, M., Smith, D., Spanel, P. 2010. Selected ion

flow tube-mass spectrometry for absolute quantification of aroma compounds in the headspace

of dry fermented sausages. Analytical Chemistry 82, 5819-5829.

REFERENCE LIST

190

Olivas, G.I., Barbosa-Canovas, G.V. 2005. Edible coatings for fresh-cut fruits. Critical Reviews

in Food Science and Nutrition 45, 657-670.

Olivas, G.I., Rodriguez, J.J., Barbosa-Canovas, G.V. 2003. Edible coatings composed of

methylcellulose, stearic acid, and additives to preserve quality of pear wedges. Journal of Food

Processing and Preservation 27, 299-320.

Oms-Oliu, G., Aguilo-Aguayo, I., Soliva-Fortuny, R., Martin-Belloso, O. 2009. Effect of ripeness

at processing on fresh-cut 'flor de invierno' pears packaged under modified atmosphere

conditions. International Journal of Food Science and Technology 44, 900-909.

Oms-Oliu, G., Martinez, R., Soliva-Fortuny, R., Martin-Belloso, O. 2008a. Effect of

superatmospheric and low oxygen modified atmospheres on shelf-life extension of fresh-cut

melon. Food Control 19, 191-199.

Oms-Oliu, G., Martinez, R., Soliva-Fortuny, R., Martin-Belloso, O. 2008b. Effect of

superatmospheric and low oxygen modified atmospheres on shelf-life extension of fresh-cut

melon. Food Control 19, 191-199.

Oms-Oliu, G., Odriozola-Serrano, I., Soliva-Fortuny, R., Martin-Belloso, O. 2008c. The role of

peroxidase on the antioxidant potential of fresh-cut 'piel de sapo' melon packaged under

different modified atmospheres. Food Chemistry 106, 1085-1092.

Oms-Oliu, G., Odrjozola-Serrano, I., Soliva-Fortuny, R., Martin-Belloso, O. 2008d. Antioxidant

content of fresh-cut pears stored in high-o-2 active packages compared with conventional low-

o-2 active and passive modified atmosphere packaging. Journal of Agricultural and Food

Chemistry 56, 932-940.

Oms-Oliu, G., Soliva-Fortuny, R., Martin-Belloso, O. 2007a. Effect of ripeness on the shelf-life

of fresh-cut melon preserved by modified atmosphere packaging. European Food Research


Oms-Oliu, G., Soliva-Fortuny, R., Martin-Belloso, O. 2008e. Modeling changes of headspace gas

concentrations to describe the respiration of fresh-cut melon under low or superatmospheric

oxygen atmospheres. Journal of Food Engineering 85, 401-409.

REFERENCE LIST

191

Oms-Oliu, G., Soliva-Fortuny, R., Martin-Belloso, O. 2008f. Using poly saccharide-based edible

coatings to enhance quality and antioxidant properties of fresh-cut melon. Lwt-Food Science


Oms-Oliu, G., Souva-Fortuny, R., Martin-Belloso, O. 2007b. Respiratory rate and quality

changes in fresh-cut pears as affected by superatmospheric oxygen. Journal of Food Science

72, E456-E463.

Ouchi, K., Yamamoto, Y., Takagishi, M., Akiyama, H. 1980. Regulation of isoamyl alcohol

formation via ehrlich pathway in saccharomyces-cerevisiae. Journal of Fermentation


Peng, B., Yue, T., Yuan, Y. 2009. Analysis of key aroma components in cider from shaanxi

(china) fuji apple. International Journal of Food Science and Technology 44, 610-615.

Perez-Gago, M.B., Serra, M., Alonso, M., Mateos, M., del Rio, M.A. 2005. Effect of whey

protein- and hydroxypropyl methylcellulose-based edible composite coatings on color change

of fresh-cut apples. Postharvest Biology and Technology 36, 77-85.

Perry, P.L., Wang, Y., Lin, J. 2009a. Analysis of honeydew melon (cucumis melo var. Inodorus)

flavour and gc-ms/ms identification of (e,z)-2,6-nonadienyl acetate. Flavour and Fragrance

Journal 24, 341-347.

Perry, P.L., Wang, Y., Lin, J.M. 2009b. Analysis of honeydew melon (cucumis melo var.

Inodorus) flavour and gc-ms/ms identification of (e,z)-2,6-nonadienyl acetate. Flavour and

Fragrance Journal 24, 341-347.

Piga, A., D'Aquino, S., Agabbio, M., Emonti, G., Farris, G.A. 2000. Influence of storage

temperature on shelf-life of minimally processed cactus pear fruits. Lebensmittel-Wissenschaft

Und-Technologie-Food Science and Technology 33, 15-20.

Pilizota, V., Subaric, D., Tiban, N.N., Kopjar, M., Babic, J. 2004. Influence of browning

inhibitors on colour and polyphenols in fresh-cut pears and pear juice. Actual Tasks on

Agricultural Engineering 32, 405-414.

Pinto, D.M., Boas, E., Damiani, C. 2007. Quality of fresh-cut 'ponca' tangerine, stored at 5 c.

Ciencia E Agrotecnologia 31, 1131-1135.

REFERENCE LIST

192

Pinto, P.M., Jacomino, A.P., Cavalini, F.C., Cunha, L.C., Inoue, K.N. 2010. Maturity stages of

'kumagai' and 'pedro sato' guava fruits for fresh cut. Ciencia Rural 40, 37-43.

Plaza, L., Crespo, I., de Pascual-Teresa, S., de Ancos, B., Sanchez-Moreno, C., Munoz, M., Cano,

M.P. 2011. Impact of minimal processing on orange bioactive compounds during refrigerated

storage. Food Chemistry 124, 646-651.

Plotto, A., Narciso, J.A., Rattanapanone, N., Baldwin, E.A. 2010. Surface treatments and

coatings to maintain fresh-cutmango quality in storage. Journal of the Science of Food and

Agriculture 90, 2333-2341.

Ponce, A.D., Bastiani, M.I.D., Minim, V.P., Vanetti, M.C.D. 2010. Physicochemical and

microbiological characteristics of fresh-cut strawberry. Ciencia E Tecnologia De Alimentos 30,

113-118.

Portela, S.I., Cantwell, M.I. 1998. Quality changes of minimally processed honeydew melons

stored in air or controlled atmosphere. Postharvest Biology and Technology 14, 351-357.

Portela, S.I., Cantwell, M.I. 2001. Cutting blade sharpness affects appearance and other quality

attributes of fresh-cut cantaloupe melon. Journal of Food Science 66, 1265-1270.

Poubol, J., Izumi, H. 2005a. Physiology and microbiological quality of fresh-cut mango cubes as

affected by high-o-2 controlled atmospheres. Journal of Food Science 70, M286-M291.

Poubol, J., Izumi, H. 2005b. Shelf life and microbial quality of fresh-cut mango cubes stored in

high co2 atmospheres. Journal of Food Science 70, M69-M74.

Prince, B.J., Milligan, D.B., McEwan, M.J. 2010. Application of selected ion flow tube mass

spectrometry to real‐time atmospheric monitoring. Rapid Communications in Mass

Spectrometry 24, 1763-1769.

Pronk, J.T., Steensma, H.Y., vanDijken, J.P. 1996. Pyruvate metabolism in saccharomyces

cerevisiae. Yeast 12, 1607-1633.

Purvis, A.C. 1997. The role of adaptive enzymes in carbohydrate oxidation by stressed and

senescing plant tissues. HortScience 32, 1165-1168.

REFERENCE LIST

193

Qi, H.P., Hu, W.Z., Jiang, A.L., Tian, M.X., Li, Y.Q. 2011. Extending shelf-life of fresh-cut 'fuji'

apples with chitosan-coatings. Innovative Food Science & Emerging Technologies 12, 62-66.

Qi, L., Wu, T.Z., Watada, A.E. 1999. Quality changes of fresh-cut honeydew melons during

controlled atmosphere storage. Journal of Food Quality 22, 513-521.

Quiles, A., Hernando, I., Perez-Munuera, I., Lluch, M.A. 2007. Effect of calcium propionate on

the microstructure and pectin methy-lesterase activity in the parenchyma of fresh-cut fuji

apples. Journal of the Science of Food and Agriculture 87, 511-519.

Rabobank. 2010. Fresh-cut fruit and vegetables doing well despite downturn [Online]. Available

at https://www.rabobank.com/en/press/news/fresh-

cut_fruit_and_vegetables_doing_well_despite_downturn.html.

Raemaekers, M.H.M., Vandamme, E.J. 1997. Production of alternansucrase by leuconostoc

mesenteroides nrrl b-1355 in batch fermentation with controlled ph and dissolved oxygen.

Journal of Chemical Technology and Biotechnology 69, 470-478.

Ragaert, P., Devlieghere, F., Devuyst, E., Dewulf, J., Van Langenhove, H., Debevere, J. 2006a.

Volatile metabolite production of spoilage micro-organisms on a mixed-lettuce agar during

storage at 7 °c in air and low oxygen atmosphere. International Journal of Food Microbiology

112, 162-170.

Ragaert, P., Devlieghere, F., Loos, S., Dewulf, J., Van Langenhove, H., Debevere, J. 2006b.

Metabolite production of yeasts on a strawberry-agar during storage at 7 °c in air and low

oxygen atmosphere. Food Microbiology 23, 154-161.

Ragaert, P., Devlieghere, F., Loos, S., Dewulf, J., Van Langenhove, H., Foubert, I.,

Vanrolleghem, P.A., Debevere, J. 2006c. Role of yeast proliferation in the quality degradation

of strawberries during refrigerated storage. International Journal of Food Microbiology 108,

42-50.

Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Sobrino-Lopez, A., Soliva-Fortuny, R., Martin-

Belloso, O. 2007. Shelf-life extension of fresh-cut "fuji" apples at different ripeness stages

using natural substances. Postharvest Biology and Technology 45, 265-275.

http://www.rabobank.com/en/press/news/fresh-cut_fruit_and_vegetables_doing_well_despite_downturn.html

http://www.rabobank.com/en/press/news/fresh-cut_fruit_and_vegetables_doing_well_despite_downturn.html

REFERENCE LIST

194

Renger, R.S., Vanhateren, S.H., Luyben, K. 1992. The formation of esters and higher alcohols

during brewery fermentation - the effect of carbon-dioxide pressure. Journal of the Institute of

Brewing 98, 509-513.

Rico, D., Martin-Diana, A.B., Barat, J.M., Barry-Ryan, C. 2007. Extending and measuring the

quality of fresh-cut fruit and vegetables: A review. Trends in Food Science & Technology 18,

373-386.

Rivera-Lopez, J., Vazquez-Ortiz, F.A., Ayala-Zavala, J.F., Sotelo-Mundo, R.R., Gonzalez-

Aguilar, G.A. 2005. Cutting shape and storage temperature affect overall quality of fresh-cut

papaya cv. 'Maradol'. Journal of Food Science 70, S482-S489.

Robles-Sanchez, R.M., Islas-Osuna, M.A., Astiazaran-Garcia, H., Vazquez-Ortiz, F.A., Martin-

Belloso, O., Gorinstein, S., Gonzalez-Aguilar, A. 2009. Quality index, consumer acceptability,

bioactive compounds, and antioxidant activity of fresh-cut "ataulfo" mangoes (mangifera

indica l.) as affected by low-temperature storage. Journal of Food Science 74, S126-S134.

Rocculi, P., Del Nobile, M.A., Romani, S., Baiano, A., Rosa, M.D. 2006. Use of a simple

mathematical model to evaluate dipping and map effects on aerobic respiration of minimally

processed apples. Journal of Food Engineering 76, 334-340.

Rocculi, P., Emiliano, C., Romani, S., Sacchetti, G., Rosa, M.D. 2009. Effect of 1-mcp treatment

and n2o map on physiological and quality changes of fresh-cut pineapple. Postharvest Biology


Rocculi, P., Romani, S., Dalla Rosa, M. 2004. Evaluation of physico-chemical parameters of

minimally processed apples packed in non-conventional modified atmosphere. Food Research


Rocculi, P., Romani, S., Dalla Rosa, M. 2005. Effect of map with argon and nitrous oxide on

quality maintenance of minimally processed kiwifruit. Postharvest Biology and Technology 35,

319-328.

Rodrigues, F., Ludovico, P., Leão, C. 2006. Sugar metabolism in yeasts: An overview of aerobic

and anaerobic glucose catabolism, Biodiversity and ecophysiology of yeasts. Springer, 101-

121.

REFERENCE LIST

195

Rodrigues, L.J., Boas, E., Piccoli, R.H., de Paula, N.R.F., Pinto, D.M., Boas, B.M.V. 2007.

Effect of the cut type and sanitizers on the softening of fresh cut pequi fruit (caryocar

brasiliense camb.). Ciencia E Agrotecnologia 31, 1793-1799.

Rojas-Grau, M.A., Grasa-Guillem, R., Martin-Belloso, O. 2007. Quality changes in fresh-cut fuji

apple as affected by ripeness stage, antibrowning agents, and storage atmosphere. Journal of

Food Science 72, S36-S43.

Rojas-Grau, M.A., Oms-Oliu, G., Soliva-Fortuny, R., Martin-Belloso, O. 2009. The use of

packaging techniques to maintain freshness in fresh-cut fruits and vegetables: A review.

International Journal of Food Science and Technology 44, 875-889.

Rojas-Grau, M.A., Tapia, M.S., Martin-Belloso, O. 2008. Using polysaccharide-based edible

coatings to maintain quality of fresh-cut fuji apples. Lwt-Food Science and Technology 41,

139-147.

Roller, S., Seedhar, P. 2002. Carvacrol and cinnamic acid inhibit microbial growth in fresh-cut

melon and kiwifruit at 4 degrees and 8 degrees c. Letters in Applied Microbiology 35, 390-394.

Rossle, C., Brunton, N., Gormley, R.T., Ross, P.R., Butler, F. 2010. Development of potentially

synbiotic fresh-cut apple slices. Journal of Functional Foods 2, 245-254.

Rossle, C., Brunton, N., Gormley, T.R., Butler, F. 2011a. Quality and antioxidant capacity of

fresh-cut apple wedges enriched with honey by vacuum impregnation. International Journal of

Food Science and Technology 46, 626-634.

Rossle, C., Gormley, T.R., Brunton, N., Butler, F. 2011b. Quality and antioxidant properties of

fresh-cut apple wedges from 10 cultivars during modified atmosphere packaging storage. Food

Science and Technology International 17, 267-276.

Rozpedowska, E., Galafassi, S., Johansson, L., Hagman, A., Piskur, J., Compagno, C. 2011.

Candida albicans - a pre-whole genome duplication yeast - is predominantly aerobic and a

poor ethanol producer. FEMS Yeast Research 11, 285-291.

Rupasinghe, H.P.V., Boulter-Bitzer, J., Ahn, T., Odumeru, J.A. 2006. Vanillin inhibits

pathogenic and spoilage microorganisms in vitro and aerobic microbial growth in fresh-cut

apples. Food Research International 39, 575-580.

REFERENCE LIST

196

Rupasinghe, H.P.V., Murr, D.P., DeEll, J.R., Odumeru, J. 2005. Influence of 1-

methylcyclopropene and natureseal on the quality of fresh-cut "empire" and "crispin" apples.

Journal of Food Quality 28, 289-307.

Russo, V.C., Daiuto, E.R., Vieites, R.L. 2012. Fresh cut yellow melon (cac) submitted to

different type cuts and concentrations of calcion chloride stored under modified passive

atmosphere. Semina-Ciencias Agrarias 33, 227-236.

Sabir, A., Sabir, F.K., Kara, Z. 2011. Effects of modified atmosphere packing and honey dip

treatments on quality maintenance of minimally processed grape cv. Razaki (v. Vinifera l.)

during cold storage. Journal of Food Science and Technology-Mysore 48, 312-318.

Saftner, R., Abbott, J.A., Lester, G., Vinyard, B. 2006. Sensory and analytical comparison of

orange-fleshed honeydew to cantaloupe and green-fleshed honeydew for fresh-cut chunks.


Saftner, R.A., Abbott, J.A., Bhagwat, A.A., Vinyard, B.T. 2005. Quality measurement of intact

and fresh-cut slices of fuji, granny smith, pink lady, and goldrush apples. Journal of Food

Science 70, S317-S324.

Saftner, R.A., Bai, J.H., Abbott, J.A., Lee, Y.S. 2003. Sanitary dips with calcium propionate,

calcium chloride, or a calcium amino acid chelate maintain quality and shelf stability of fresh-

cut honeydew chunks. Postharvest Biology and Technology 29, 257-269.

Saftner, R.A., Lester, G.E. 2009. Sensory and analytical characteristics of a novel hybrid

muskmelon fruit intended for the fresh-cut industry. Postharvest Biology and Technology 51,

327-333.

Saisung, P., Theerakulkait, C. 2011. Inhibitory effect of pineapple shell extract and its

ultrafiltered fractions on polyphenol oxidase activity and browning in fresh-cut banana slices

efecto inhibidor de extracto de cascara de pina y sus fracciones ultrafiltradas en la actividad

polifenoloxidasa y pardeamiento de rodajas de platano recien cortadas. Cyta-Journal of Food 9,

37-42.

Salas Salazar, N., Olivas Orozco, G. 2011. The aroma of apples. Interciencia 36, 265-271.

REFERENCE LIST

197

Sandhya. 2010. Modified atmosphere packaging of fresh produce: Current status and future needs.

Lwt-Food Science and Technology 43, 381-392.

Santos, J.C.B., Boas, V., de Barros, E.V., Prado, M.E.T., Pinheiro, A.C.M. 2005. Evaluation of

quality in fresh-cut'pérola'pineapple stored under modified atmosphere. Ciência e

Agrotecnologia 29, 353-361.

Sapers, G.M., Miller, R.L. 1998. Browning inhibition in fresh-cut pears. Journal of Food Science

63, 342-346.

Sapers, G.M., Miller, R.L., Pilizota, V., Mattrazzo, A.M. 2001. Antimicrobial treatments for

minimally processed cantaloupe melon. Journal of Food Science 66, 345-349.

Saquet, A.A., Streif, J., Bangerth, F. 2003. Impaired aroma production of ca-stored 'jonagold'

apples as affected by adenine and pyridine nucleotide levels and fatty acid concentrations.

Journal of Horticultural Science & Biotechnology 78, 695-705.

Saucedo-Pompa, S., Jasso-Cantu, D., Ventura-Sobrevilla, J., Saenz-Galindo, A., Rodriguez-

Herrera, R., Aguilar, C.N. 2007. Effect of candelilla wax with natural antioxidants on the shelf

life quality of fresh-cut fruits. Journal of Food Quality 30, 823-836.

Saxena, A., Bawa, A.S., Raju, P.S. 2008. Use of modified atmosphere packaging to extend shelf-

life of minimally processed jackfruit (artocarpus heterophyllus l.) bulbs. Journal of Food

Engineering 87, 455-466.

Selma, M.V., Ibanez, A.M., Cantwell, M., Suslow, T. 2008. Reduction by gaseous ozone of

salmonella and microbial flora associated with fresh-cut cantaloupe. Food Microbiology 25,

558-565.

Seo, J.Y., Kim, E.J., Hong, S.I., Yoon, H.H., Kim, D. 2006. Combined effects of postharvest

calcium chloride and heat treatment on the quality characteristics of fresh-cut tsugaru apple.

Food Science and Biotechnology 15, 529-533.

Shan, W., Zhao, C., Fan, J., Cong, H., Liang, S., Yu, X. 2012. Antisense suppression of alcohol

acetyltransferase gene in ripening melon fruit alters volatile composition. Scientia

Horticulturae 139, 96-101.

REFERENCE LIST

198

Singh, S.P., Chonhenchob, V., Chantarasomboon, Y., Singh, J. 2007. Testing and evaluation of

quality changes of treated fresh-cut tropical fruits packaged in thermoformed plastic containers.

Journal of Testing and Evaluation 35, 522-528.

Soliva-Fortuny, R.C., Alos-Saiz, N., Espachs-Barroso, A., Martin-Belloso, O. 2004. Influence of

maturity at processing on quality attributes of fresh-cut conference pears. Journal of Food

Science 69, S290-S294.

Soliva-Fortuny, R.C., Biosca-Biosca, M., Grigelmo-Miguel, N., Martin-Belloso, O. 2002a.

Browning, polyphenol oxidase activity and headspace gas composition during storage of

minimally processed pears using modified atmosphere packaging. Journal of the Science of

Food and Agriculture 82, 1490-1496.

Soliva-Fortuny, R.C., Grigelmo-Miguel, N., Hernando, I., Lluch, M.A., Martin-Belloso, O.

2002b. Effect of minimal processing on the textural and structural properties of fresh-cut pears.

Journal of the Science of Food and Agriculture 82, 1682-1688.

Soliva-Fortuny, R.C., Grigelmo-Miguel, N., Odriozola-Serrano, I., Gorinstein, S., Martin-Belloso,

O. 2001. Browning evaluation of ready-to-eat apples as affected by modified atmosphere

packaging. Journal of Agricultural and Food Chemistry 49, 3685-3690.

Soliva-Fortuny, R.C., Martin-Belloso, O. 2003a. Microbiological and biochemical changes in

minimally processed fresh-cut conference pears. European Food Research and Technology

217, 4-9.

Soliva-Fortuny, R.C., Martin-Belloso, O. 2003b. New advances in extending the shelf-life of

fresh-cut fruits: A review. Trends in Food Science & Technology 14, 341-353.

Soliva-Fortuny, R.C., Oms-Oliu, G., Martin-Belloso, O. 2002c. Effects of ripeness stages on the

storage atmosphere, color, and textural properties of minimally processed apple slices. Journal

of Food Science 67, 1958-1963.

Spanier, A.M., Flores, M., James, C., Lasater, J., Lloyd, S., Miller, J.A. 1998. Fresh-cut

pineapple (ananas sp.) flavor. Effect of storage. Food Flavors: Formation, Analysis, and

Packaging Influences 40, 331-343.

REFERENCE LIST

199

Supapvanich, S., Pimsaga, J., Srisujan, P. 2011a. Physicochemical changes in fresh-cut wax apple

(syzygium samarangenese blume merrill & l.M. Perry) during storage. Food Chemistry 127,

912-917.

Supapvanich, S., Tucker, G.A. 2011b. Physicochemical changes in fresh-cut honeydew melon

fruit during storage. African Journal of Agricultural Research 6, 2737-2742.

Syft. 2009. Sift-ms primer an introduction to the chemistry and methodology of sift-ms Syft

Technologies Ltd, Christchurch, New Zealand.

Taherzadeh, M.J., Adler, L., Lidén, G. 2002. Strategies for enhancing fermentative production of

glycerol—a review. Enzyme and Microbial Technology 31, 53-66.

Takeoka, G., Buttery, R.G., Flath, R.A., Teranishi, R., Wheeler, E., Wieczorek, R., Guentert, M.

1989. Volatile constituents of pineapple (ananas comosus [l.] merr.), ACS Symposium Series

American Chemical Society (USA), vol. 388. ACS Publications. 223-237.

Thompson, J., Chassy, B.M. 1981. Uptake and metabolism of sucrose by streptococcus-lactis.

Journal of Bacteriology 147, 543-551.

Toivonen, P.M., DeEll, J. 2002. Physiology of fresh-cut fruits and vegetables. In: Lamikanra, O.,

(Ed.), Fresh-cut fruits and vegetables. Crc press, boca raton, fl. CRC Press, Boca Raton, Fla.,

91-123.

Toivonen, P.M.A. 1997. Non-ethylene, non-respiratory volatiles in harvested fruits and

vegetables: Their occurrence, biological activity and control. Postharvest Biology and


Toivonen, P.M.A. 2008. Influence of harvest maturity on cut-edge browning of 'granny smith'

fresh apple slices treated with anti-browning solution after cutting. Lwt-Food Science and


Toivonen, P.M.A., Brummell, D.A. 2008. Biochemical bases of appearance and texture changes

in fresh-cut fruit and vegetables. Postharvest Biology and Technology 48, 1-14.

Tokitonio, Y., Steinhaus, M., Buttner, A., Schieberle, P. 2005. Odor-active constituents in fresh

pineapple (ananas comosus l. Merr.) by quantitative and sensory evaluation. Bioscience

Biotechnology and Biochemistry 69, 1323-1330.

REFERENCE LIST

200

Torri, L., Sinelli, N., Limbo, S. 2010. Shelf life evaluation of fresh-cut pineapple by using an

electronic nose. Postharvest Biology and Technology 56, 239-245.

Ukuku, D.O. 2004. Effect of hydrogen peroxide treatment on microbial quality and appearance of

whole and fresh-cut melons contaminated with salmonella spp. International Journal of Food


Ukuku, D.O., Fett, W. 2002a. Behavior of listeria monocytogenes inoculated on cantaloupe

surfaces and efficacy of washing treatments to reduce transfer from rind to fresh-cut pieces.

Journal of Food Protection 65, 924-930.

Ukuku, D.O., Fett, W.F. 2002b. Effectiveness of chlorine and nisin-edta treatments of whole

melons and fresh-cut pieces for reducing native microflora and extending shelf-life. Journal of

Food Safety 22, 231-253.

Ukuku, D.O., Pilizota, V., Sapers, G.M. 2004. Effect of hot water and hydrogen peroxide

treatments on survival of salmonella and microbial quality of whole and fresh-cut cantaloupe.

Journal of Food Protection 67, 432-437.

US-FDA. 2002. Ultraviolet radiation for the processing and treatment of food., Code of Federal

Regulations, 21 Part 179.39, United States Food and Drug Administration.

Uyttendaele, M., Jacxsens, L., De Loy-Hendrickx, A., Devlieghere, F., Debevere, J. 2010.

Microbiologische richtwaarden & wettelijke microbiologische criteria Laboratory of Food

Microbiology and Food Preservation, Ghent University, Ghent, Belgium.

Van der Steen, C., Jacxsens, L., Devlieghere, F., Debevere, J. 2002. Combining high oxygen

atmospheres with low oxygen modified atmosphere packaging to improve the keeping quality

of strawberries and raspberries. Postharvest Biology and Technology 26, 49-58.

van Rijswick, C. 2010. The eu fresh-cut fruits and vegetables market [Online]. Available at

http://www.freshconveniencecongress.com/resources/documents/1308561709cindyvanrijswic

k.pdf.

Vanurk, H., Voll, W.S.L., Scheffers, W.A., Vandijken, J.P. 1990. Transient-state analysis of

metabolic fluxes in crabtree-positive and crabtree-negative yeasts. Applied and Environmental


http://www.freshconveniencecongress.com/resources/documents/1308561709cindyvanrijswick.pdf

http://www.freshconveniencecongress.com/resources/documents/1308561709cindyvanrijswick.pdf

REFERENCE LIST

201

Vargas, M., Pastor, C., Chiralt, A., McClements, D.J., Gonzalez-Martinez, C. 2008. Recent

advances in edible coatings for fresh and minimally processed fruits. Critical Reviews in Food

Science and Nutrition 48, 496-511.

Varoquaux, P., Ozdemir, I.S. 2005. Packaging and produce degradation. In: Lamikanra, O., Imam,

S.H., Ukuku, D., (Eds.), Produce degradation : Pathways and prevention. Taylor & Francis,

Boca Raton, 117-154.

Verduyn, C., Zomerdijk, T.P.L., Vandijken, J.P., Scheffers, W.A. 1984. Continuous measurement

of ethanol-production by aerobic yeast suspensions with an enzyme electrode. Applied

Microbiology and Biotechnology 19, 181-185.

Verzera, A., Dima, G., Tripodi, G., Ziino, M., Lanza, C.M., Mazzaglia, A. 2011. Fast quantitative

determination of aroma volatile constituents in melon fruits by headspace-solid-phase

microextraction and gas chromatography-mass spectrometry. Food Analytical Methods 4, 141-

149.

Vijayanand, P., Nair, K.K.S., Narasimham, P. 2001. Preservation of pineapple, mango and

papaya chunks by hurdle technology. Journal of Food Science and Technology-Mysore 38,

26-31.

Wall, M.M., Nishijima, K.A., Fitch, M.M., Nishijima, W.T. 2010. Physicochemical, nutritional

and microbial quality of fresh-cut and frozen papaya prepared from cultivars with varying

resistance to internal yellowing disease. Journal of Food Quality 33, 131-149.

Wang, H., Feng, H., Luo, Y.G. 2006. Dual-phasic inactivation of escherichia coli o157 : H7 with

peroxyacetic acid, acidic electrolyzed water and chlorine on cantaloupes and fresh-cut apples.

Journal of Food Safety 26, 335-347.

Watada, A.E., Ko, N.P., Minott, D.A. 1996. Factors affecting quality of fresh-cut horticultural

products. Postharvest Biology and Technology 9, 115-125.

Watada, A.E., Qi, L. 1999. Quality of fresh-cut produce. Postharvest Biology and Technology 15,

201-205.

Watkins, C.B., Silsby, K.J., Goffinet, M.C. 1997. Controlled atmosphere and antioxidant effects

on external co2 injury of 'empire' apples. HortScience 32, 1242-1246.

REFERENCE LIST

202

Wei, C.B., Liu, S.H., Liu, Y.G., Lv, L.L., Yang, W.X., Sun, G.M. 2011. Characteristic aroma

compounds from different pineapple parts. Molecules 16, 5104-5112.

Werner, B., Hotchkiss, J., Sapers, G., Gorny, J., Yousef, A. 2006. Modified atmosphere

packaging. In: Sapers, G.M., Gorny, J.R., Yousef, A.E., (Eds.), Microbiology of fruits and

vegetables, 437-459.

Whittaker, J.W. 2010. Metal uptake by manganese superoxide dismutase. Biochimica Et

Biophysica Acta-Proteins and Proteomics 1804, 298-307.

Worakeeratikul, W., Srilaong, V., Uthairatanakij, A., Jitareerat, P. 2007. Effect of whey protein

concentrate on quality and biochemical changes in fresh-cut rose apple. Proceedings of the

International Conference on Quality Management of Fresh Cut Produce, 435-441.

Wszelaki, A.L., Mitcham, E.J. 2000. Effects of superatmospheric oxygen on strawberry fruit

quality and decay. Postharvest Biology and Technology 20, 125-133.

Wu, Z.S., Zhang, M., Adhikari, B. 2012a. Application of high pressure argon treatment to

maintain quality of fresh-cut pineapples during cold storage. Journal of Food Engineering 110,

395-404.

Wu, Z.S., Zhang, M., Wang, S. 2012b. Effects of high pressure argon treatments on the quality of

fresh-cut apples at cold storage. Food Control 23, 120-127.

Xiao, C.L., Luo, W., Liu, M.Y., Zhu, L.W., Li, M., Yang, H.S., Deng, Y. 2010. Quality of fresh-

cut pears (pyrus bretschneideri rehd cv. Huangguan) coated with chitosan combined with

ascorbic acid and rosemary extracts. Philippine Agricultural Scientist 93, 66-75.

Zabetakis, I., Holden, M.A. 1997. Strawberry flavour: Analysis and biosynthesis. Journal of the

Science of Food and Agriculture 74, 421-434.

Zheng, Y., Yang, Z., Chen, X. 2008. Effect of high oxygen atmospheres on fruit decay and

quality in chinese bayberries, strawberries and blueberries. Food Control 19, 470-474.

Zhou, Y., Wan, S., Li, X., Xue, T. 2011. Effect of high oxygen modified atmosphere packaging

on browning characteristics of fresh-cut 'fuji' apple. In: Cao, Z., He, Y.H., Sun, L., Cao, X.Q.,

(Eds.), Application of chemical engineering, pts 1-3, vol. 236-238. Trans Tech Publications,

Switzerland, 2391-2394.

REFERENCE LIST

203

Zuo, L., Lee, J.H. 2004. Effects of anti-browning agents on the quality of minimally processed

apple cubes. Food Science and Biotechnology 13, 40-45.

SUMMARY

204

SUMMARY

In Chapter 1 a literature study is presented in four sections. In the first section, a brief overview

of the trends in the marketing of fresh-cut produce, especially for fruit, is given. In the last two

decades, fresh-cut produce sales have increased rapidly by double digit rates in the United States

and the European Union. However, the fresh-cut produce market in Asian countries is still in an

early stage of development. Although the volume of fresh-cut fruit is around 10% of the fresh-cut

sector in both the EU and US markets, fresh-cut fruit are currently the newest and fastest growing

segment of the fresh-cut sector. In the second section of the literature study, the causes of quality

losses in fresh-cut fruit were discussed. The quality losses of fresh-cut fruit are generally due to

the actions of microbial contaminants, cut surface colour changes, texture softening, off-odour

and off-flavour development and nutritional losses. The quality of fresh-cut fruit can decrease

rapidly during storage due to these reasons. The third section of this chapter discussed the factors

affecting the quality of fresh-cut fruit and techniques that are used to extend their shelf-life.

These factors are including the type of cultivar, the level of maturity, pre-treatments applied to

the whole fruit before cutting, physical decontamination techniques, application of dipping and

edible coatings, modified atmosphere packaging (MAP), temperature, etc. Ideally, all these

factors should be optimized to obtain a high quality fresh-cut product. In last section of the

literature study, methods for assessing the quality of fresh-cut fruit were briefly introduced.

The microbiota of spoiled fresh-cut pineapple, honeydew melon and apple was examined in

Chapter 2. Spoilage isolates were firstly identified by the API system while the definitive

identification of the most frequently occurring species was done by gene sequence analysis. The

gene sequence identification of selected yeasts was based on morphological, physiological and

molecular (sequencing of the large-subunit rDNA D1/D2 domain and the internal transcribed

spacer, or ITS rDNA) analyses. The amplified fragment length polymorphism (AFLP) analysis

was used for identification of lactic acid bacteria. The results showed that yeasts were

predominant on spoiled pineapple cubes and apple slices. However, in addition to yeasts, lactic

acid bacteria were also equally predominant on spoiled honeydew melon cubes. Primary

identification showed that the most frequently occurring yeast species were Candida. More

SUMMARY

205

specifically, Candida sake, Candida lusitaniae and Candida parapsilosis were found on all the

fresh-cut fruit evaluated. All the lactic acid bacteria colonies that were isolated from spoiled

honeydew melon and selected for identification were determined to be Leuconostoc

mesenteroides. The definitive identification showed that spoilage isolates on spoiled pineapple

cubes were Meyerozyma caribbica, Candida argentea and C. sake. C. sake, Candida fragi and L.

mesenteroides occurred on spoiled honeydew melon cubes whilst the most predominant yeast

species on fresh-cut apple were C. sake.

In Chapter 3, the effect of the initial headspace (IH) O2 level (0 - 21%, balance N2) on the shelf-

life of fresh-cut pineapple and honeydew melon was evaluated. In the first part of the chapter, the

effect of IH O2 levels on the growth and volatile organic compound (VOC) production of

different spoilage organisms on pineapple agar and honeydew melon agar was evaluated

separately at 7°C. In the second part of the chapter, the effect of selected IH O2 levels on the

microbiological, physical and sensory quality of commercial pineapple and honeydew melon

cubes was performed separately at 7°C. The results showed that the IH O2 level had a minor

effect on the growth of yeasts and lactic acid bacteria on fruit simulating agar. The quantities of

the VOCs in the headspace produced by the yeasts during incubation were determined to be

generally smaller the lower the IH O2 level on both pineapple agar and honeydew melon agar.

However, the trend was not clear for VOCs produced by lactic acid bacteria on honeydew melon

agar. Pineapple cubes packaged in IH 5% O2 were significantly different in sensory

characteristics to those packaged in IH 21% O2 (P < 0.05) on day 5 of storage. Sensory

preference was for the samples packaged in IH 5% O2. However, no significant difference in

sensory characteristics was found to honeydew melon cubes packaged in IH 5% O2 and air.

In Chapter 4, the effect of MAs combining high O2 (21 - 70%) and CO2 (21 - 50%) levels on the

microbial spoilage and sensory quality of fresh-cut pineapple and honeydew melon was evaluated.

The experimental methodology developed in Chapter 3 was used in this chapter. The results

showed that MAs combining high O2 and CO2 levels had a large inhibitory effect on the growth

and VOC production of spoilage yeasts on both pineapple agar and honeydew melon agar,

especially MAs consisting of 50% O2 + 50% CO2 and 70% O2 + 30% CO2. In difference, the

MAs evaluated only had a minor effect on the growth and VOC production of lactic acid bacteria

mostly because lactic acid bacteria were not sensitive to high O2 and CO2 levels. Overall, the

SUMMARY

206

effect of MAs on colour, juice leakage, juiciness, firmness of fresh-cut honeydew melon and

pineapple was small during storage. Sensory preference was generally for fresh-cut fruit

packaged in MA of 50% O2 + 50% CO2. Therefore, 50% O2 + 50% CO2 is a potential MA

solution for extending the shelf-life of fresh-cut pineapple and honeydew melon.

In Chapter 5, the microbial profile, sensory quality and VOCs of fresh-cut apple packaged in air,

1/0/99, 5/0/95, 50/30/20 and 50/50/0 (% O2/CO2/N2) were evaluated over seven days at 7°C. The

modified atmosphere (MA) with 50% O2 and 50% CO2 had the greatest inhibitory effect on the

growth of aerobes and yeasts of the apple slices. However, the production of most of VOCs (i.e.

ethanol, ethyl acetate, 3-methylbutanal, etc.) by the cut apple was stimulated by the atmospheres

with high O2 and high CO2 levels. In contrast, the production of phenylacetic acid and 2-methyl-

1-butanol was appreciably higher in the packages which had low initial O2 levels. In general the

lowest quantities of VOCs were produced in the samples packaged in air. Atmospheres with high

CO2 concentrations also caused an effervescent mouth-feel which was negatively judged by the

sensory panel. The MAs evaluated had no appreciable effect on the colour of apple slices as

ascorbic acid had been applied to them before packaging. It can be concluded that the application

of MA to extend the shelf-life of fresh-cut apple is not recommended. Additionally, the results

clearly illustrate that VOCs should not be ignored when the shelf-life of fresh-cut fruits is

evaluated.

A general discussion of the most significant findings of the studies performed in this thesis is

given in Chapter 6. Additionally, some future perspectives are given to address important aspects

that were not addressed in this thesis.

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SAMENVATTING

De literatuurstudie wordt in het eerste hoofdstuk in vier secties opgedeeld. In het eerste gedeelte

wordt een kort overzicht gegeven van de ontwikkelingen van vers gesneden producten, en in het

bijzonder voor fruit. In de laatste twee decennia steeg de verkoop van verse, versneden groenten

en fruit bijzonder vlug in de U.S.A en E.U.. In de Aziatische landen bevindt deze markt zich nog

in een vroeg stadium van ontwikkeling. Het volume van het vers gesneden fruit bedraagt

ongeveer 10% van vers gesneden sector in de Europese en de Amerikaanse markten, dit segment

is het snelst groeiende in de sector.

In het tweede deel van de literatuurstudie worden de oorzaken van het verlies van de kwaliteit

van vers gesneden fruit besproken. Het kwaliteitsverlies van vers gesneden fruit is te wijten aan

bacteriologische verontreinigingen, kleurveranderingen, verzachten van de textuur, verlies van

voedingswaarde, etc… Hierdoor kan de kwaliteit snel afnemen tijdens de opslag.

In het derde deel van dit hoofdstuk worden de factoren die de kwaliteit van het vers gesneden

fruit beïnvloeden en de technieken, die toegepast worden om de houdbaarheid te vergroten,

besproken. Deze factoren zijn: het type cultivar, de graad van rijpheid, voorbehandeling toegepast

op de hele vrucht vóór het snijden, fysieke ontsmettingstechnieken, gemodificeerde atmosfeer

verpakking (MAP), temperatuur, etc…). Idealiter zouden al deze factoren moeten worden

geoptimaliseerd om een hoge kwaliteit van het product te verkrijgen.

Tot slot werden inschattingsmethodes van de kwaliteit van vers gesneden fruit in het kort

overlopen.

In hoofdstuk 2 worden de microbiota van bedorven vers gesneden ananas, meloen en appel

onderzocht. Isolaten die het bederf veroorzaken werden eerst geïdentificeerd met het API-

systeem terwijl de definitieve identificatie van de meest voorkomende soorten werd gedaan door

gensequentieanalyse. De gensequentie identificatie van geselecteerde gisten is gebaseerd op

morfologische, fysiologische en moleculaire analyses (opeenvolging van de grote-subunit rDNA

D1/D2 domein en de interne getranscribeerde spacer, of ITS rDNA). AFLP (amplified fragment

length polymorphism) analyse werd gebruikt voor identificatie van melkzuurbacteriën. De

resultaten tonen aan dat gisten overheersten op de bedorven stukjes ananas en appel. Naast gisten

waren ook melkzuurbacteriën aanwezig op bedorven meloen blokjes. De meest voorkomende

soort gist was Candida. Meer in het bijzonder werden Candida sake, Candida lusitaniae en

SAMENVATTING

208

Candida parapsilosis op al het geëvalueerde vers gesneden fruit gevonden. Alle

melkzuurbacteriën die uit bedorven meloen werden geïsoleerd en geselecteerd, werden

geïdentificeerd als Leuconostoc mesenteroides. Op bedorven ananas blokjes werden de isolaten

geïdentificeerd als Meyerozyma caribbica, Candida argentea en C. sake. Op blokjes van meloen

trof men C. sake, Candida fragi en L. mesenteroides aan, terwijl de overheersende gist op vers

gesneden appel C.sake was.

In hoofdstuk 3, wordt het effect van de initiële kopruimte (IH) O2 -niveau (0-21%, balans N2) op

de houdbaarheidsdatum van vers gesneden ananas en meloen geëvalueerd. In het eerste gedeelte

van dit hoofdstuk wordt het effect van IH O2 niveau op de groei en de aanmaak van vluchtige

organische stoffen (VOCs), door de verschillende normale bederforganismen op ananas- en

meloenagar bij 7°C, afzonderlijk geëvalueerd.

In het tweede gedeelte van dit hoofdstuk, wordt het effect van de geselecteerde IH O2 niveaus op

de microbiologische, fysische en sensorische kwaliteit van commerciële ananas en meloen

blokjes afzonderlijk uitgevoerd bij 7°C. Het onderzoek wees uit dat het IH O2-niveau een gering

effect had op de groei van gisten, melkzuurbacteriën en op fruit agar simulant. De hoeveelheid

VOCs (volatile organic compound) in de hoofdruimte, geproduceerd door gisten gedurende de

incubatie, bleek lager te zijn naargelang het IH O2 niveau lager was op ananas agar zowel als

meloen agar. Voor VOC’s geproduceerd door melkzuurbacteriën op meloen daarentegen was dit

niet zo duidelijk het geval. Op dag vijf van de opslag was er een duidelijk verschil te merken in

de sensorische kenmerken bij ananasblokjes verpakt in IH 5% O2 pakjes ten opzichte van die

verpakt in IH 21% O2 (P <0,05) pakjes. De zintuiglijke voorkeur ging uit naar die verpakt in IH 5%

O2 pakjes. Bij blokjes honigmeloen, verpakt in IH 5% O2 en in lucht, werd geen beduidend

verschil gevonden in de sensorische kenmerken.

Hoofdstuk 4 behandelt het effect van MAs (Modified Atmospheres) die hoge O2 (21 - 70%)

combineren met CO2 (21-50%) niveaus, op het microbieel bederf en sensorische kwaliteit van

vers gesneden ananas en meloen. De experimentele methodes ontwikkeld en besproken in

hoofdstuk 3 werden toegepast in dit hoofdstuk. Het onderzoek toonde aan dat MAs die hoge O2

en CO2 niveaus combineren de groei en VOC productie van gisten die bederf veroorzaken, zowel

bij ananas agar als bij meloen agar, in hoge mate verhinderden. In het bijzonder de MAs

bestaande uit 50% O2 + 50% CO2 en 70% O2 + 30% CO2 hadden dit effect. Daarentegen hadden

de gebruikte MA’s, slechts een gering effect op de groei en VOC productie van de

melkzuurbacteriën gezien melkzuurbacteriën niet gevoelig waren voor de hoge O2 en CO2

SAMENVATTING

209

niveaus. Er werd vastgesteld dat het effect van de MA’s op de kleur, het vochtverlies, de

sappigheid en de stevigheid van vers gesneden meloen en ananas tijdens de bewaartijd gering is.

Hier ging de zintuigelijke voorkeur uit naar vers gesneden fruit verpakt in MA van 50% O2 + 50%

CO2. Bijgevolg is 50% O2 + 50% CO2 een mogelijke oplossing voor het verlengen van de

houdbaarheidsduur van vers gesneden ananas en meloen.

Hoofdstuk 5 bespreekt de evaluatie van het microbiële profiel, zintuigelijk kwaliteit en VOCs

van vers gesneden appel verpakt in lucht gedurende 7 dagen bij 7°C, 1/0/99, 5/0/95, 50/30/20 en

50/50/0 (% O2/CO2/N2). De gemodificeerde atmosfeer (MA) met 50% O2 en 50% CO2 had het

grootste remmende effect op de groei van aerobe bacteriën en de gisten bij appelschijfjes.

Nochtans werd de productie van de meeste VOC’s (bijvoorbeeld ethanol, ethylacetaat, 3-

methylbutanal, etc.) bij gesneden appel gestimuleerd door de atmosfeer met hoge O2 en CO2

niveau. Dit in tegenstelling tot de productie van fenylazijnzuur en 2-methyl-1-butanol die

aanzienlijk hoger was in de packages die lage O2 niveaus hadden. Er werd vastgesteld dat de

laagste hoeveelheden VOC’s tot stand komen in de monsters verpakt in lucht. Atmosferen met

een hoge CO2 concentraties veroorzaakten ook een bruisend mondgevoel dat negatief werd

beoordeeld door het sensorische panel. De geëvalueerde MA had geen belangrijk effect op de

kleur van de appelschijfjes doordat ascorbinezuur werd toegepast vóór het verpakken. Hieruit kan

men vaststellen dat de toepassing van MA, voor wat betreft het verlengen van de

houdbaarheidsduur van vers gesneden appel, niet aanbevolen is. Daarbovenop geven de

resultaten duidelijk aan dat rekening dient gehouden te worden met VOC’s bij evaluatie/bepaling

van houdbaarheidsduur/houdbaarheidsdatum van vers gesneden fruit.

Een algemene bespreking van de belangrijkste bevindingen van de studies uitgevoerd in dit

proefschrift wordt gegeven in hoofdstuk 6. Daarnaast worden een aantal toekomstige

onderzoeksperspectieven gegeven voor aspecten die niet in dit proefschrift werden onderzocht.

SAMENVATTING

210

CURRICULUM VITAE

211

CURRICULUM VITAE

Baoyu Zhang was born on November the 27th

in1984 in Shaanxi, China. In 2007, he obtained his

Bachelor's degree in viticulture and wine at Northwest A&F University in China. In 2009, he

obtained his Master's degree in Enology at Northwest A&F University.

In 2009, he obtained a scholarship for PhD study abroad from China Scholarship Council (CSC)

and started the PhD at Laboratory of Food Microbiology and Food Preservation, Department of

Food Safety and Food Quality, Faculty of Bioscience Engineering, Ghent University. His

promoters are Prof. dr. ir. Frank Devlieghere and Dr. Simbarashe Samapundo.

Publications in A1 peer-reviewed journals

Zhang, B.-Y., Samapundo, S., Pothakos, V., de Baenst, I., Sürengil, G., Noseda, B., Devlieghere,

F. 2013. Effect of atmospheres combining high oxygen and carbon dioxide levels on microbial

spoilage and sensory quality of fresh-cut pineapple. Postharvest Biology and Technology 86, 73-

84.

Zhang, B.-Y., Samapundo, S., Pothakos, V., Sürengil, G., Devlieghere, F. 2013. Effect of high

oxygen and high carbon dioxide atmosphere packaging on the microbial spoilage and shelf-life of

fresh-cut honeydew melon. International Journal of Food Microbiology 166, 378-390.

Zhang, B.-Y., Samapundo, S., Rademaker, M., Noseda, B., Denon, Q., de Baenst, I., Sürengil, G.,

De Baets, B., Devlieghere, F. 2014. Effect of headspace oxygen level on growth and volatile

metabolite production by the spoilage microorganisms of fresh-cut pineapple. LWT-Food

Science and Technology 55, 224-231.

CURRICULUM VITAE

212

Zhang, B.-Y., Samapundo, S., Sürengil, G., Devlieghere, F. 2013. Effect of modified

atmospheres (incl. high oxygen and high carbon dioxide) on the shelf-life of fresh-cut apple.

Journal of Food Science and Technology. Under review.

Dang, T.D.T., De Maeseneire, S.L., Zhang, B.-Y., De Vos, W.H., Rajkovic, A., Vermeulen, A.,

Van Impe, J.F., Devlieghere, F. 2012. Monitoring the intracellular pH of zygosaccharomyces

bailii by green fluorescent protein. International Journal of Food Microbiology 156, 290-295.

Abstracts of symposia and workshops

Zhang, B.-Y., Samapundo, S., de Baenst, I., Devlieghere, F. 2012. Effect of modified

atmospheres on the microbial spoilage and sensorial quality of fresh-cut pineapple. The

symposium of Microbiological research of foods: ‘own work’ and ‘heterogeneity in food safety’.

Oral presentation. 19th

June, Wageningen, Nederland.

Zhang, B.-Y., Samapundo, S., Pothakos, V., de Baenst, I., Devlieghere, F. 2013. Effect of

modified atmospheres combining high oxygen and high carbon dioxide on the microbial spoilage

and the shelf-life of fresh-cut pineapple and honeydew melon. International Congress of Spoilers

in Food, p. 99. Flash poster presentation. 1st – 3

rd July, Quimper, France.

Pothakos, V., Nyambi, C., Zhang, B.-Y., Papastergiadis, A., De Meulenaer, B., Devlieghere, F.

2013. Spoilage potential of psychrotrophic lactic acid bacteria (LAB) Leuconostoc gasicomitatum

and Lactococcus piscium under different packaging conditions. International Congress of

Spoilers in Food, p. 82. Flash poster presentation. 1st – 3

rd July, Quimper, France.

Zhang, B.-Y., Samapundo, S., de Baenst, I., Denon, Q., Devlieghere, F. 2012. Effect of

modified atmosphere packaging on the growth and volatile metabolite production by specific

spoilage organisms of fresh-cut pineapple. 5th

International Symposium on Food Packaging -

Scientific Developments supporting Safety and Innovation. Poster presentation. 14th

– 16th

November, Berlin, Germany.

Zhang, B.-Y., Samapundo, S., Denon, Q., Pauwels, D., Eriksson, M., Devlieghere, F. 2010.

Influence of residual O2 level on growth and volatile organic metabolite production of Candida

sake on pineapple agar. 16th

PhD symposium on Applied Biological Sciences. Poster presentation.

20th

December, Ghent, Belgium.

msc. baoyu zhang - ghent university · the author and the promoter give the authorization to...

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