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OULU 2010

C 370

Sanna Taskila

IMPROVED ENRICHMENT CULTIVATION OF SELECTED FOOD-CONTAMINATING BACTERIA

UNIVERSITY OF OULU,FACULTY OF TECHNOLOGY,DEPARTMENT OF PROCESS AND ENVIRONMENTAL ENGINEERING

C 370

ACTA

Sanna Taskila

C370etukansi.kesken.fm Page 1 Wednesday, October 27, 2010 2:58 PM

A C T A U N I V E R S I T A T I S O U L U E N S I SC Te c h n i c a 3 7 0

SANNA TASKILA

IMPROVED ENRICHMENT CULTIVATION OF SELECTED FOOD-CONTAMINATING BACTERIA

Academic dissertation to be presented, with the assent ofthe Faculty of Technology of the University of Oulu, forpublic defence in Auditorium IT115, Linnanmaa, on 26November 2010, at 12 noon

UNIVERSITY OF OULU, OULU 2010

Copyright © 2010Acta Univ. Oul. C 370, 2010

Supervised byProfessor Heikki OjamoProfessor Peter NeubauerDocent Mika Tuomola

Reviewed byDoctor Tadhg O'SullivanDocent Maria Saarela

ISBN 978-951-42-6356-9 (Paperback)ISBN 978-951-42-6357-6 (PDF)http://herkules.oulu.fi/isbn9789514263576/ISSN 0355-3213 (Printed)ISSN 1796-2226 (Online)http://herkules.oulu.fi/issn03553213/

Cover designRaimo Ahonen

JUVENES PRINTTAMPERE 2010

Taskila, Sanna, Improved enrichment cultivation of selected food-contaminatingbacteria University of Oulu, Faculty of Technology, Department of Process and EnvironmentalEngineering, University of Oulu, P.O.Box 4300 FI-90014 University of Oulu, Finland Acta Univ. Oul. C 370, 2010Oulu, Finland

AbstractThe aim of this work was to assess and improve the enrichment cultivation of food-

contaminating bacteria prior to detection by means of RNA-based sandwich hybridization assay(SHA). The examples of beer-spoiling lactic acid bacteria (LAB) and food-borne SalmonellaTyphimurium were selected based on their relevance in Finnish food industry. Also universalchallenges affecting on the selection of the enrichment cultivation procedure are discussed,including some potential possibilities for improved enrichment cultivation. The results of thisstudy may therefore be used for the assessment of the efficiency of bacterial cultivation in otherapplications.

The evaluation of the enrichment cultivation procedures prior to SHA lead to followingconclusions: i) the enrichment cultivation procedure is necessary prior to rRNA-based SHA, andit directly influences the accuracy of SHA; ii) the improvement of the enrichment cultivation mayallow faster recovery and growth of bacteria; iii) the improved recovery of bacteria can beachieved by reducing environmental stress factors in the enrichment culture; and iv) the growth ofbacteria may be accelerated by assuring the selectivity of medium and allowing accessibility togrowth factors. Several growth factors were studied by means of full factorial design and responsesurface modeling. Measured cell densities, as well as predicted lag-times and maximum growthrates in the bacterial cultures were used as responses.

The results show that small shifts in the cultivation conditions extend the lag-time and decreasethe growth rate of both LAB and Salmonella. Besides adjusting the temperature and pH, thegrowth of LAB was facilitated by reducing osmotic and oxidative stresses in the enrichmentmedium. In this study, a novel enzyme controlled glucose delivery system was used for the firsttime in the enrichment cultivation of food-contaminating bacteria. The glucose delivery systemimproved the growth of LAB in single strain cultures and in actual brewing process samples. Therecovery of injured Salmonella was also enhanced by using the glucose delivery system togetherwith selective siderophore ferrioxamine E, both in terms of reduced lag-times and increasedgrowth rates. Based on the SHA, the adjusted BPW broth enhanced the molecular detection ofheat-injured Salmonella in meat.

Keywords: beer, enrichment cultivation, food microbiology, lactic acid bacteria,Lactobacillus, meat, microbiological methods, Pediococcus, Salmonella, sandwichhybridization, 16S rRNA, 23S rRNA

5

Acknowledgements

This study was conducted at the University of Oulu, Department of Process and

Environmental Engineering. Funding by the Finnish Graduate School in

Environmental Science and Technology (EnSTe, Joensuu, Finland), University of

Oulu Faculty of Technology, TEKES (grants no. 70042/02 and 40254/07), EU

(grant no. 512009), and Hartwall Ltd. is gratefully acknowledged.

My gratitude is due to my supervisors, Professor Heikki Ojamo, Professor

Peter Neubauer and Docent Mika Tuomola. I especially thank Heikki for the

support in the last phases of the thesis process; Peter for arranging funding and

facilities, and allowing networking with both industrial and academic partners;

and Mika for the valuable insight into food diagnostic applications and detection

of food-borne Salmonella. I also thank the pre-examiners Docent Maria Saarela

and Doctor Tadhg O’Sullivan for the careful review of this thesis. Mrs. Regina

Castelejn-Osorno is thanked for the language review.

I am grateful for my industrial cooperators Doctor Jukka Kronlöf, Anna-

Kaisa Vehviläinen, Ritva Alamäki and Terttu Liedes from Hartwall Ltd.; and

Doctor Anu Surakka and Anna Peltola from Valio Ltd. I also thank Doctor Antti

Vasala and Maria Ruottinen from BioSilta Oy, and Doctor Antje Breitenstein from

Scanbec GmbH.

I thank Professor Riitta Keiski for advice during the study process; Liisa

Myllykoski for the patient guiding in both scientific and financial matters; my co-

authors Doctor Beatrix Fahnert, Doctor Timo Nieminen, Ekaterina Osmekhina,

Jari Ruuska and Marika Kurkinen for their contribution; Lilja Kosamo, Kirsi

Ahtinen, Kalle Vähälä, Juho Järvinen and Anne Räsänen for assistance. During

my work I have been accompanied by people that I owe for the pleasant working

atmosphere. I specially thank Johanna for the evening beer/tea sessions, and

Katya for our discussions on hot topics. I also thank Anu, Daniela, Heta, Jaakko,

Mari, Marita, Monika, Rami, Ritva, Sina, and Tiina for making it so nice.

I warmly acknowledge my family and friends: my parents for encouraging

me to study, my brothers for being there, my grandmothers for showing me what

a woman is made of, the godparents of our children for the babysitting and

support, and my parents in law for all the help. Finally, I thank the man of my life

Vesa and our little darlings for giving the best things in my life!

Oulu, October 14, 2010 Sanna Taskila

6

7

Abbreviations

ABD Advanced Beer Detection broth

ANOVA Analysis of variance

BPW Buffered Peptone Water broth

B-MRS De Man-Rogosa-Sharpe broth with 25% beer

E. coli Escherichia coli

FISH Fluorescence in situ -hybridization

L litre

L. Lactobacillus

LAB Lactic acid bacteria

LAMP Loop-mediated isothermal amplification

L-cysteine-HCl L-cysteine hydrochloride

MRS De Man-Rogosa-Sharpe broth

MKTTn Müller-Kauffman tetrathionate novobiocin broth

NASBA Nucleic acid sequence –based amplification

NB Nutrient broth

NBB Nachweismedium für Biershädlich Bacterien

NBB-C Concentrated NBB

OD Optical density

OD490 Optical density at 490 nm

p Probability for regression according to F-test

P. Pediococcus

PCR Polymerase chain reaction

rRNA Ribosomal ribonucleic acid

RSM Response surface modelling

RVS Rappaport-Vassiliadis Soy broth

S. Salmonella

SC Selenite cystine broth

SHA Sandwich hybridization assay

S.Typhimurium S. enterica subspecies enterica serovar Typhimurium

Sensitivity ratio The proportion of true positives from all positive results.

Specificity ratio The proportion of true negatives from all negative results.

TT Tetrathionate broth

U Enzyme activity unit

UPB Universal preenrichment broth

8

9

List of original articles

This thesis is based on the following original articles which are referred to in the

text by their Roman numerals. In addition, some unpublished data are included.

Articles in peer-reviewed journals:

I Huhtamella S, Leinonen M, Nieminen T, Fahnert B, Myllykoski L, Breitenstein A & Neubauer P (2007) RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples. J Microbiol Methods 68: 543–553.

II Taskila S, Neubauer P, Tuomola M, Breitenstein A, Kronlöf J & Hillukkala T (2009) Improved Enrichment Cultivation of Beer Spoiling Lactic Acid Bacteria by Continuous Glucose Addition to the Culture. J Inst Brew 115(3): 177–182.

III Taskila S, Tuomola M, Kronlöf J & Neubauer P (2010) Comparison of enrichment media for routine detection of beer spoiling lactic acid bacteria and development of trouble-shooting medium for Lactobacillus backi. J Inst Brew 116(2): 151–156.

IV Taskila S, Tuomola M, Kronlöf J, Ruuska J & Neubauer P (2010) Note-Preliminary applications of response surface modeling to the evaluation of optimal growth conditions for beer-spoiling Pediococcus damnosus. J Inst Brew 116(3): 211–214.

Supplementary material:

V Taskila S, Osmekhina E, Tuomola M, Ruuska J & Neubauer P Modification of buffered peptone water for improved recovery of heat injured Salmonella Typhimurium. Manuscript submitted to J Food Sci.

The author’s contribution

I S. Taskila (nee Huhtamella) was responsible for the tests at the brewery. She

wrote the article in cooperation with the other authors.

II–IV S. Taskila wrote the article, is the corresponding author and interpreted the

results. She was responsible for all experimental work, except for the detection of

actual samples at the breweries.

V S. Taskila was responsible for the RSM and the enrichment cultivations. She

wrote the article in cooperation with the other authors.

10

11

Contents

Abstract Acknowledgements 5 Abbreviations 7 List of original articles 9 Contents 11 1 Introduction 13

1.1 Background ............................................................................................. 13 1.2 Aims and outline of the study ................................................................. 16

2 Literature review 17 2.1 Detection and enrichment cultivation of beer-spoiling LAB .................. 17

2.1.1 Detection of beer-spoiling LAB ................................................... 18 2.1.2 Enrichment media for beer-spoiling LAB .................................... 20

2.2 Detection and enrichment cultivation of food-borne Salmonella ............ 22 2.2.1 Detection of food-borne Salmonella ............................................. 23 2.2.2 Enrichment media for Salmonella ................................................ 25

2.3 Important factors in the selection of the enrichment procedure .............. 29 2.3.1 Regulations ................................................................................... 29 2.3.2 Sample matrix ............................................................................... 30 2.3.3 Background microbiota ................................................................ 31 2.3.4 Concentration and condition of target cells .................................. 33 2.3.5 Stress factors in enrichment media ............................................... 35 2.3.6 Detection methods ........................................................................ 37

3 Summary of the material and methods 39 3.1 Bacterial strains and preparation of injured cells .................................... 39 3.2 Enzyme controlled glucose delivery system ........................................... 39 3.3 Experimental design and variable analysis ............................................. 39 3.4 Growth curves and definition of lag-times and growth rates .................. 40 3.5 Measurement of rRNA by means of SHA ............................................... 40 3.6 Food and process samples ....................................................................... 40

4 Summary of results and discussion 43 4.1 Improved enrichment cultivation of beer-spoiling LAB (Studies

I-IV) ........................................................................................................ 43 4.1.1 The role of enrichment cultivation in 16S RNA SHA

detection of beer-spoiling LAB .................................................... 43 4.1.2 Reduced thermal and alkaline pH stress ....................................... 46

12

4.1.3 Reduced oxidative stress .............................................................. 47 4.1.4 Reduced osmotic stress ................................................................. 50

4.2 The effect of enzyme controlled glucose delivery .................................. 52 4.3 Improved enrichment cultivation of heat-injured Salmonella

(Study V) ................................................................................................. 55 4.3.1 Enrichment cultivation in 23S rRNA SHA ................................... 55 4.3.2 Improved growth of Salmonella in BPW ..................................... 57 4.3.3 Improved recovery of injured Salmonella in single strain

cultures ......................................................................................... 58 4.3.4 Improved recovery of heat-injured Salmonella in minced

meat .............................................................................................. 60 5 Conclusions 61 References 63 Original articles 81

13

1 Introduction

1.1 Background

Food safety is one of the most important issues with regard to the sustainable

development in global scale. It has been estimated that the growth of global food

production and trade will result in an increase in the burden of food-borne disease

in the next few decades. In order to assure safety, the food industry puts

significant effort into the microbiological testing of foods. Good hygiene in the

process can also directly contribute to the profitability by increasing the

fermentation rates or avoiding recalls of spoiled product batches. In the past, the

detection of microbial contaminants in foods was done mainly by cultivation,

followed by microbiological and biochemical analyses. The contaminating

microbes were identified by means of microscopy, enzymatic tests, and Gram-

staining. These cultivation based assays are still widely in use, but in many cases

together with molecular diagnostics.

The cultivation based detection methods have advantages, such as possibility

for quantification of viable cells and further studies using the isolated strains, and

the generally accepted protocols. The drawbacks of the cultivation based

detection include lengthy procedures, and poor accuracy and selectivity. These

methods are also operator-dependent and therefore, the results can be subjective.

Due to growing efficiency of production and logistics, the management of food

safety in the future relies on rapid microbial analysis methods (von Blankenfeld-

Enkvist & Brännback 2002), also referred to as alternative methods in comparison

to reference methods. Although, novel rapid detection methods and more effective

sample processing techniques are frequently reported, the need for enrichment

cultivation exists. This is mostly due to the low concentration of bacteria (Back

2005, Hage et al. 2005), and the presence of inhibitory components in the samples

(Juvonen et al. 1999, Kanki et al. 2009). For example, in the European Union

(EU), total absence of Salmonella is required in 25 g of minced meat

(Anonymous 2005b). None of the currently available detection methods can meet

the sensitivity demand for the Salmonella detection without enrichment

cultivation.

The cultivation of bacteria in batch cultures, i.e. cultures with limited amount

of nutrients, generally includes the following four main phases: i) lag phase (i.e.

lag-time), during which bacteria adapt themselves to growth conditions; ii)

14

exponential growth phase, during which the cells double; iii) stationary phase,

during which the growth rate slows down as a result of nutrient depletion and

accumulation of toxic products; and iv) death phase, during which bacteria die.

The enrichment cultivation generally comprises the most time-consuming part of

the whole detection procedure of food-contaminating bacteria. The duration of the

cultivation step mostly depends on the length of the bacterial lag-time, which can

extend the detection procedure up to one week. Therefore, the word rapid usually

refers only to the molecular detection method, and in order to speed up the

detection procedure enhanced enrichment cultivation is needed.

The enrichment cultivation procedure depends on the detection methods used.

In this study, the enrichment cultivation of food contaminating bacteria was

evaluated and improved with respect to the rRNA targeting sandwich

hybridization assay (SHA). The case examples of beer-spoiling lactic acid

bacteria and food-borne Salmonella Typhimurium were selected for the study,

based on their relevance in national and international food industries (Back 2005,

Anonymous 2008).

The principle for the SHA-based detection has been described by Rautio and

co-workers (2003). The SHA includes four main steps: the binding of the rRNA

target to magnetic particles by means of specific oligonucleotide probes, referred

to as capture probes; the binding of a second labeled oligonucleotide probe to the

capture probe-RNA forming the sandwich; the signal generation via reaction

between the label and corresponding enzyme; and the measurement of the signal

intensity (fluorescent or electric read-out), or visual evaluation of the color

change (colorimetric assay).

SHA has been applied in the detection of bacteria and their harmful

metabolites in various samples, and in the quantification of genetic markers for

microbial metabolism in bioreactors (Table 1). The most recent applications of

SHA include the quantification of collagen prolyl 4-hydroxylase during

recombinant production in different expression systems (Osmekhina et al. 2010)

and the detection of staphylococcal virulence factor antigens on electric

biosensors (Quiel et al. 2010).

15

Table 1. Applications of SHA in the detection of food contaminants and their

metabolites, and measurement of transcriptional levels in cells.

Application Target molecules References

Detection and identification of microbes or their metabolites

Food-poisoning Bacillus cereus marker DNAs (Gabig-Ciminska et al.

2005, Liu et al. 2007)

Legionella in water 16S rRNA (Leskelä et al. 2005)

Viruses in different samples capsid proteins (Los et al. 2005)

E. coli, Pseudomonas aeruginosa, Enterococcus

faecalis, Staphylococcus aureus, Staphylococcus

epidermidis

16S rRNA (Elsholz et al. 2006)

Mycobacteria in environmental samples 16S rRNA (Nieminen et al. 2006,

Pakarinen et al. 2007)

LAB in brewery samples 16S rRNA This study

Salmonella in minced meat 23S rRNA This study

Staphylococcal virulence factors virulence factor antigens (Quiel et al. 2010)

Quantification of changes in metabolism

Bacillus licheniformis different mRNAs (Pioch et al. 2008)

Bacillus subtilis different mRNAs (Jürgen et al. 2005)

E. coli different mRNAs (Soini et al. 2005,

Kemmer & Neubauer

2006, Thieme et al.

2008)

prolyl 4-hydroxylase (Osmekhina et al. 2010)

Pichia pastoris different mRNAs (Resina et al. 2007)

Saccharomyces cerevisiae 18S rRNA, SUC mRNA (Rautio et al. 2003)

Based on the literature, the use of rRNA instead of other nucleic acids as target

for the detection is advantageous due to several reasons (Amann & Ludwig 2000).

RNA molecules are abundant in growing bacterial cells (Oerther et al. 2000),

which contributes to the sensitivity of detection. It has also been shown that in

non-growing cells precursor rRNA degrades rapidly and therefore the use of

rRNA assures that only viable cells are detected. Additionally, the use of rRNA

allows the differentiation between bacteria in species and genus level (Berthier &

Ehrlich 1998).

16

1.2 Aims and outline of the study

The present study aimed to assess the enrichment cultivation of food

contaminating bacteria prior to RNA-based SHA and to address bottlenecks in the

enrichment cultivation with regard to the accuracy of detection. The final aim of

this study was to improve the enrichment cultivation of beer-spoiling LAB and

food-borne Salmonella Typhimurium in order to improve the applicability of

rRNA SHA and also other alternative detection methods in the microbial quality

control.

During the study rRNA-based SHA was applied in the detection of beer-

spoiling LAB in brewing process samples, and S. Typhimurium in minced meat in

order to assess the enrichment cultivation procedure. The effects of the cultivation

conditions and media supplements on the growth of these bacteria in commonly

used enrichment broths were studied by means of variable analysis and response

surface modeling (RSM). Finally, the relevance of the effects was assessed with

respect to the SHA. The work was done in international and national research

projects and in close cooperation with the Finnish industry.

17

2 Literature review

2.1 Detection and enrichment cultivation of beer-spoiling LAB

The heterogenic group of LAB consists of Gram-positive, aerobic to facultative

anaerobic, non-sporulating rods and cocci. They are oxidase and benzidine

negative, lack cytochromes, and do not reduce nitrates to nitrite (Carr et al. 2002).

LAB are divided into homofermentative and heterofermentative, based on their

products of sugar fermentation. Homofermentative LAB ferment glucose with

lactic acid as the primary product, while heterofermentative LAB produce also

acetic acid and carbon dioxide. Most LAB are anaerobic, but some of them can

shift to oxygen-dependent metabolism in aerobic conditions (Murphy & Condon

1984, Sedewitz et al. 1984).

LAB consist of a number of genera, from which Lactobacillus (L.) and

Pediococcus (P.) are the most relevant with regard to the brewing industry (Priest

2003). Among the LAB, L. brevis, L. lindneri and P. damnosus are the most

commonly encountered beer-spoilers (Hollerova & Kubizniakova 2001, Priest

2003, Thelen et al. 2006, Suzuki et al. 2008a, Menz et al. 2010). These three

species show strong beer-spoiling ability and high tolerance to hop acids, and are

often found in the brewing process samples. L. brevis grows relatively well in

many culture media compared to other generally hard-to-cultivate species of beer-

spoiling LAB, which may partially explain its predominance in incident reports

for microbiologically spoiled beer products (Suzuki et al. 2008a). L. lindneri has

been reported to be heat resistant and may survive through sub-optimal

pasteurization process (Suzuki et al. 2006a). Novel LAB species occasionally

emerge in brewing environments, most recently described including L. backi

(Bohak et al. 2006), L. paracollinoides (Suzuki et al. 2004a), L. paucivorans

(Ehrmann et al. 2009), L. rossiae (Corsetti et al. 2005) and P. claussenii (Chaban

et al. 2002). At least the first two mentioned species are regarded as beer spoilers

(Suzuki et al. 2008a).

The beer-spoiling LAB cause significant losses at the breweries by retarding

the fermentation (Narendranath et al. 1997) and spoiling the final products that

have a relatively long shelf-life (Back 2005). The ability of bacteria to spoil beer

is known to be genetic. Several studies have shown that the hop resistance genes,

such as horA, are essential for LAB to grow in beer (Sakamoto et al. 2001,

Suzuki et al. 2002, Haakensen & Ziola 2008). Besides the hop resistance, the

18

beer-spoiling bacteria need to tolerate low pH, high concentrations of ethanol and

carbon dioxide, and low oxygen tension (Jespersen & Jakobsen 1996).

2.1.1 Detection of beer-spoiling LAB

The main emphasis of microbiological quality assurance at the breweries is on

preventive measures (Back 2005). These include process steps in which the raw

materials or intermediate products are sterilized by means of heating or filtration,

maintenance of hygiene in the process, and increasing the awareness of personnel

on the topic. The filling stage has been recognized as a major place of bacterial

contamination for beer, mostly due to presence of airborne bacteria (Henriksson

& Haikara 1991) and bacterial biofilms (Timke et al. 2005).

In microbiological quality control, breweries mainly rely on classical

cultivation methods, followed by phenotypic characterization of isolates. The

detection of microbial contaminants in beer usually involves collecting cells from

the samples by membrane filtration followed by plating on selective or non-

selective media. The shelf-life test samples are incubated for up to six weeks,

while the use of concentrated media at an elevated temperature (27–30°C) allows

shortening of the incubation time to two weeks. Afterwards, confirmatory tests

are carried out for suspected positive samples.

The major limitation of cultivation of beer-spoiling LAB is the long

incubation time. The results are rarely available early enough to make decisions

about the re-use of the pitching yeast or the release of product. Furthermore, the

analysis of naturally turbid process samples on agar media can be difficult.

Another drawback of the cultivation-based methods is the possibility of bacteria

to enter into a viable but non-culturable state, which may result to false negative

results in standard media (Amann et al. 1995, Suzuki et al. 2006a). Despite these

disadvantages, cultivation-based methods are widely in use due to their simplicity

and sensitivity, their possibility to distinguish between viable and dead cells, and

isolation of bacteria for further analyses.

In order to speed up the detection of LAB, various alternative methods have

been developed and utilized for use at brewery quality control. Due to low

concentrations of LAB in the brewery samples, all of these methods require 24–

48 hours of enrichment cultivation prior to detection. The alternative methods are

mostly based on nucleic acid targets and antigens. The reported nucleic acid –

based methods include hybridization assays, PCR, and other amplification

techniques.

19

Fluorescence in situ hybridization (FISH) has been used for the quantitative

detection of beer-spoiling LAB in several studies. FISH is based on the direct

detection of bacteria with a species-specific fluorescent probe targeted to rRNA.

DNA extraction is not needed since the FISH probes penetrate the target cells,

which reduces the hand-on time and duration of the procedure. Thelen and co-

workers (2002) showed the applicability of FISH for the detection of LAB in

filtrated and non-filtrated brewery process samples, and for the evaluation of the

prevalence of brewery associated LAB (Thelen et al. 2006). The detection limit

for the assay is approximately 1,000 cells per mL. Asano and co-workers (2009)

combined FISH with microcolony cultivation. Compared to the cultivation of

traditional bacterial colonies, the microcolony method could detect considerably

smaller colonies, and therefore the duration of the cultivation could be reduced.

The nucleic acid amplification techniques applied for the detection of beer-

spoiling LAB include various different PCR-based methods and loop-mediated

isothermal amplification (LAMP). The amplification techniques can be linked to

other molecular methods in order to increase the sensitivity of the detection. For

example, the use of PCR in combination with colorimetric hybridization (Satokari

et al. 1998) and electric biosensors (Elsholz et al. 2009) may improve the

detection of bacteria.

The majority of the PCR methods for the detection of beer-spoiling LAB rely

on either specific marker genes for the spoiling ability (Suzuki et al. 2006b,

Haakensen et al. 2007) or on highly conserved 16S rRNA sequences within

species (Nakagawa et al. 1994, Suzuki et al. 2004b, Haakensen et al. 2008a,

Juvonen et al. 2010). Selected examples of the used marker genes are presented in

Table 2. Nowadays traditional end-point PCR detection has been mostly

substituted by faster or more accurate techniques. These include fluorescent real-

time PCR, such as LightCycler® (Roche Diagnostics, Mannheim, Germany),

based on real-time fluorescent quantification of PCR products; and reverse

transcriptase PCR (RT-PCR) which targets RNA instead of DNA and therefore

allows the distinction between viable and dead cells (Juvonen et al. 2010). Loop-

mediated isothermal amplification (LAMP), in which the amplification reaction

takes place in isothermal conditions, has been applied for the detection and

identification of beer spoilage Lactobacillus and Pediococcus (Tsuchiya et al.

2007).

20

Table 2. Selected marker genes for the beer-spoiling ability of LAB.

Marker Description References

bsrA, bsrB Beer-spoiling related genes in Pediococcus

sp. Function not yet known.

(Haakensen et al. 2009a)

gyrB Gene related to isohumulone resistance. (Nakakita et al. 2003)

hitA Gene encoding hop-inducible cation transporter.

Putative divalent cation transporter that

counteracts the hop-induced depletion of

intracellular manganese.

(Back 1980, Hayashi et al. 2001)

horABC Hop-resistance genes. Encode efflux pumps

for transportation of hob bitter acids out of

cells.

(Sami et al. 1997, Sakamoto et al. 2001,

Suzuki et al. 2002, Suzuki et al. 2005,

Iijima et al. 2007, Haakensen et al.

2007)

ORFB1-B5 Open reading frame regions surrounding horA

gene. Generally conserved in beer-spoilage

Lactobacillus strains.

(Suzuki et al. 2004c)

Immunoassays for the detection of beer-spoiling LAB utilize specific antibodies

that bind to marker antigens on bacterial cell surfaces and combination of them

with for example fluorescent or luminescent labels (Whiting et al. 1992, Yasui &

Yoda 1997, Ziola et al. 2000, March et al. 2005). The current trends in the field of

immunoassays include the use of multiple monoclonal antibodies to marker

antigens related with beer-spoiling ability for simultaneous detection of several

beer-spoiling bacteria (Tsuchiya et al. 2000, Nakakita et al. 2002).

2.1.2 Enrichment media for beer-spoiling LAB

The enrichment cultivation of LAB at breweries commonly takes place at the

temperature of 29 ± 1 °C under anaerobic conditions. The enrichment media

include various solid and broth media (Jespersen & Jakobsen 1996, Carr et al.

2002, Suzuki et al. 2008a), from which only a few are nowadays used in

combination with molecular detection methods.

The most frequently used media for enrichment cultivation of beer-spoiling

LAB include De Man-Rogosa-Sharpe broth (MRS)(de Man et al. 1960) and

Nachweismedium für Bierschädliche Bakterien (NBB)(Dachs 1981, Back et al.

1984, Hammes et al. 1992, Holzapfel 1992). Their compositions are presented in

Table 3. Both media have been used in combination with various detection

methods, such as PCR and FISH (Juvonen et al. 1999, Takahashi et al. 2000,

21

Thelen et al. 2002, March et al. 2005, Haakensen et al. 2008a, Haakensen et al.

2008b). The common ingredients in MRS and NBB include i) beef extract, yeast

extract and glucose as sources for vitamins, nitrogen, amino acids, and carbon; ii)

polysorbate 80 as an emulsifier and iii) the inclusion of salts for buffering and

growth factors. Sodium acetate is used in MRS as a growth factor for LAB (de

Man et al. 1960). The pH of MRS is 6.5±0.2. NBB contains casein digest and

maltose as additional sources for energy. It also contains L-cysteine as an

antioxidant, L-malic acid as a growth factor for LAB, and chlorophenol red as a

pH indicator. The pH of NBB is 5.8±0.2. Concentrated NBB (NBB-C) and MRS

broths are recommended for the forcing test in EBC Analytica Microbiologica

(Hage et al. 2005).

The popularity of MRS is based on its ability to support the growth of a large

variety of LAB, and its use in several generally accepted standard protocols

(Crumplen et al. 1991, Brewery Convention of Japan 1999, Hage et al. 2005).

The supplementation of MRS with beer (Beer-MRS, B-MRS) has been shown to

benefit the enrichment of brewery strains (Holzapfel 1992, Storgårds et al. 1998,

Juvonen et al. 1999). In B-MRS the concentration of beer is 20–25% and the pH

is approximately 5.5.

In order to further improve the growth of beer-spoiling LAB, MRS has been

modified by adding fructose (Juvonen et al. 1999), L-cysteine-HCl (Arroyo et al.

1994), and mevalonic acid (Hammes et al. 1992, Difco Laboratories 1995),

substituting glucose with mannose cellobiose or salicin (Simpson & Taguchi

1995), and decreasing pH (Garvie 1984, Raccach 1987, Holzapfel 1992).

Advanced Beer Detection -medium (ABD)(Suzuki et al. 2008b) is based on the

adjustment of the concentrations of components and decreasing the pH of B-MRS

broth. ABD has been successfully used in combination with FISH (Asano et al.

2009).

22

Table 3. Composition of MRS and NBB broths (per L).

MRS NBB

Meat extract 8 g

Casein peptone 10 g

Yeast extract 4 g

Glucose 20 g

Polysorbate 80 1 g

Sodium acetate 5 g

Dipotassium hydrogen phosphate 2 g

Diammonium hydrogen citrate 2 g

Magnesium sulfate 0.2 g

Manganese sulfate 0.05 g

Beef extract 2 g

Pancreatic digest of casein 5 g

Yeast extract 5 g

Glucose 15 g

Polysorbate 80 0.5 g

Potassium acetate, 6 g

Disodium phosphate 2 g

Chlorphenol red 0.07 g

L-cysteine hydrochloride 0.2 g

L-malic acid 0.5 g

Maltose 15 g

Final pH 6.2±0.2 (25 °C) Final pH 5.8±0.2 (25 °C)

2.2 Detection and enrichment cultivation of food-borne Salmonella

Salmonella is a genus of rod-shaped, Gram-negative, non-spore forming,

predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths

from 2 to 5 µm, with peritrichous flagella (Madigan et al. 2009). They obtain

their energy from oxidation and reduction reactions using organic sources, and are

facultative anaerobes. Also, most species produce hydrogen sulfide. The optimum

temperature for growth of Salmonella is between 35 to 37 °C, but growth has

been observed at temperatures ranging from 5 °C to 47 °C. The optimum pH is

near 7.0, but growth may occur between pH 4.0 and pH 9.0.

The genus Salmonella consists of two species, S. bongori and S. enterica,

which currently include some 2,500 serotypes (Popoff et al. 2001). Among

Salmonella, only S. enterica subsp. enterica is considered clinically significant

for humans. The virulence of Salmonella requires the expression of numerous

genes and it has been shown that serotypes that do not possess the invABC genes

are unable to invade mammalian cells (Galan & Curtiss 1991). In Finland, the

majority of salmonellosis incidents are caused by S. enterica serotypes S.

Enteritidis and S. Typhimurium, from which the latter is the most common cause

for the domestic infections (Anonymous 2008). Food borne salmonellosis is an

important public health problem worldwide. The annual cost of salmonellosis in

the USA has been estimated to be over $2,600 million (Economic Research

Service 2010) and accordingly, Salmonella has been considered as a major public

23

health risk (Voetsch et al. 2004). The major sources of salmonellosis outbreaks

include eggs, meats and dairy products (EFSA 2008).

2.2.1 Detection of food-borne Salmonella

The detection of Salmonella in foods faces with several challenges, such as the

low concentration of target bacteria in foods, sub-lethally injured bacteria, and the

high amount of other bacteria and inhibitory food components in samples.

Furthermore, the legislative demand, the absence of Salmonella cells in 10–25 g

of food, sets strict performance requirements for methods of analysis

(Anonymous 2005a). The important characteristics of the detection method

include the speed of analysis, ease of use, the analytical performance, possibility

for automation, and reduction of total cost. In addition, other factors such as

robustness, reliability, through-put, overall convenience, and the level of

validation and standardization affect the applicability of the method.

The traditional confirmation and identification procedures for Salmonella spp.

are usually based on preliminary identification based on the colony appearance on

chromogenic and other selective agar media, followed by confirmation using

classical biochemical and serological testing. The biochemical tests include

fermentation of glucose, negative urease reaction, lysine decarboxylase activity,

H2S production, and fermentation of dulcitol. Serological confirmation tests

typically use antisera for detection of flagellar (H) and somatic (O) antigens.

Isolates with a typical biochemical profile, which agglutinate with both H and O

antisera are identified as Salmonella spp. Positive isolates are often sent for

further serotyping and identification using techniques such a phage typing,

antibiotic susceptibility and pulsed-field gel electrophoresis (PFGE).

In the EU, the reference detection methods are published by the International

Organization for Standardization (ISO). The reference procedure for the detection

of food borne Salmonella includes non-selective enrichment cultivation in

buffered peptone water (BPW) broth for 16–20 hours, selective enrichment

cultivation in two selective media and incubation on two different selective agar

plates for isolation of colonies (Anonymous 2002a). The colonies are then

identified by means of biochemical tests. This procedure takes at least 72 hours to

complete. In the United States (US), the government publishes the Bacteriological

Analytical Manual (BAM) which includes a collection of recommended

procedures for the detection of pathogens and toxins in foods. The BAM

procedure for the detection of food-borne Salmonella includes non-selective

24

enrichment cultivation in nutrient broth (NB) for 16 hours and selective

enrichment cultivation in either Rappaport–Vassiliadis (RV) or tetrathionate

brilliant green (TBG) broth for an additional 16 hours (Andrews & Hammack

2007). The colonies are isolated by using selective agar plates and identified

biochemically.

Also, various rapid confirmation methods have been reported for Salmonella,

and a large number of them are commercially available. Salmonella rapid test and

screening kits range from complex procedures including sophisticated techniques,

such as thin agar underlay method or immunomagnetic separation, to simple

lateral flow assays incorporating immunochromatographic technology. To date

over 30 alternative Salmonella detection methods have been validated by the

European validation and certification bodies such as Association française de

Normalisation (AFNOR), the Nordic system for validation of alternative

microbiological methods (NordVal), and MicroVal. The majority of these tests are

based on either immunoassays or PCR (Anonymous 2010a, Anonymous 2010b,

Anonymous 2010c). The Association of Official Analytical Chemists (AOAC) is

a globally recognized, non-profit association that provides tools for development

of the voluntary consensus standards. Since 2009, MicroVal and AOAC have

worked together in order to combine their requirements for the certification and

performance testing.

The immunoassays for Salmonella are mainly based on the heterogeneous

non-competitive ELISA format where the target cells are bound between

antibodies directed against H or O antigens (Cudjoe et al. 1995, Valdivieso-

Garcia et al. 2001, Magliulo et al. 2007). The detection limits of Salmonella in

foods with immunoassays range between 104 and 106 cells per assay.

Immunoassays are generally robust and do not require extensive sample

preparation. However, antibodies may cross-react with antigens in closely related

bacteria, while showing low reactivity with some Salmonella serotypes. The

commercially available immunoassays for the detection of Salmonella include

both manual lateral flow devices (such as DuPont Lateral Flow System by

Qualicon, US) and fully automated immunoanalyzers (such as VIDAS by

bioMérieux, France).

More than 30 Salmonella specific genes have been utilized for different PCR

applications (Levin 2009). These target genes include rRNA genes, genes coding

for toxins and enzymes, and repetitive elements. Especially rRNA genes are

prominent targets because the widely available information on rRNA sequences,

and the possibility for differentiation at for example species or strain levels. Also,

25

the use of virulence genes, such as invA, allows also the confirmation of

suspected Salmonella isolates. PCR together with enrichment cultivation has been

applied for the detection of Salmonella in various food matrices, such as eggs

(Malorny et al. 2007, Chen et al. 2010) and different meats (O'Regan et al. 2008,

McGuinness et al. 2009). However, in many cases the methods have not been

validated properly against the reference method, and the performances of the

assays can therefore have significant variations. Most of the PCR methods which

are in routine use provide results in approximately 24 hours, and the detection

limits are in the range of 103–104 cells per mL. A current trend in PCR diagnostics

is the use of multiplex assays for simultaneous detection of several specific

targets.

Other nucleic acid –based detection methods for food-borne Salmonella

include the isothermal nucleic acid sequence-based amplification (NASBA),

LAMP, and different hybridization assays. Probably the most widely used of these

methods is fluorescence in situ hybridization (FISH) that has been reported to

allow the detection within less than 24 hours in various food matrices (Fang et al.

2003, Almeida et al. 2010). The combination of hybridization with PCR can

increase the sensitivity of detection (Feder et al. 2001, Thompson et al. 2006).

The bacteriophage assays for detection of Salmonella utilize either the gene

transfer between the bacteriophage and the host cells or the lysis of bacteria by

the bacteriophages. The transfer of luminescent or fluorescent genes from the

bacteriophage to the host cell allows the measurement of appropriate signal

intensity (Chen & Griffiths 1996, Mosier-Boss et al. 2003). The detection using

lytic bacteriophages includes the infection of the host cells and the measurement

of the level of the lysis (Favrin et al. 2001). Bacteriophages can also be used as

probes coupled with biosensors. For example, Olsen and coworkers developed an

acoustic wave biosensor with bacteriophages as probes. This biosensor is claimed

to detect as low as 100 Salmonella cells per mL of sample (Olsen et al. 2006).

However, the use of bacteriophages may also cause false negative results due to

the exclusion of Salmonella serotypes.

2.2.2 Enrichment media for Salmonella

The enrichment media for food-borne Salmonella can be divided into non-

selective and selective media. Since the molecular detection is usually done after

cultivation in broth media, the solid media are not described here. The most

frequently used non-selective enrichment broth is buffered peptone water (BPW).

26

A universal pre-enrichment broth (UPB) and nutrient broth (NB) are also widely

used, although they are not common in the validated alternative methods. The use

of these listed media is based on the maximal recovery of small amounts of sub-

lethally injured cells (de Boer 1998). The drawback of the non-selective broths is

that they also support the growth of various other microbes. The non-selective

broths typically contain only peptones as sources of nitrogen, carbon, vitamin,

and minerals. BPW and UPB also contain sodium chloride for maintaining the

osmotic balance and phosphates for buffering. In addition to those, UPB contains

salts as ion sources and glucose as an energy source. The pH of the non-selective

media is initially adjusted near to 7.0.

A variety of enrichment media have been developed and evaluated for the

selective isolation of Salmonella. These include the Rappaport-Vassiliadis soy

broth (RVS) (Rappaport et al. 1956, van Schothorst & Renaud 1983, van

Schothorst & Renaud 1985), selenite cystine broth (SC) (Leifson 1939),

tetrathionate broth (TT)(Müller 1923), tetrathionate brilliant green broth (TGB),

and the Muller-Kauffmann tetrathionate novobiocin broth (MKTTn)(Müller 1923,

Kauffmann 1930, Kauffmann 1935). Summary of selectivity agents used for the

cultivation of Salmonella is presented in Table 4.

RVS has been shown to be superior in the selective enrichment cultivation of

Salmonella in several studies (van Schothorst & Renaud 1983, Rhodes et al. 1985,

Maijala et al. 1992, June et al. 1995, Schönenbrucher et al. 2008) and it is

included in the ISO standard method (Anonymous 2002a). The selectivity of RVS

is based on the ability of Salmonella to tolerate relatively high osmotic pressure

and low pH, as well as high concentrations of malachite green and magnesium

chloride. However, other enteric bacteria are still able to grow in RVS

(Krascsenicsova et al. 2006). The enrichment media used in some examples of

alternative methods are summarized in Table 5.

The reported performances of Salmonella-selective media are controversial.

However, following conclusions are obtained in the majority of studies: i) none of

the developed media is 100% selective for Salmonella; ii) the selectivity of

medium depends on the characteristics and concentration of background microbes

(Beckers et al. 1987, Chen et al. 1994); iii) a certain minimum concentration of

cells is required for Salmonella to survive in the selective conditions (Chen et al.

1993). Currently, a major trend is to develop enrichment media for the

simultaneous isolation of several pathogenic bacteria; for example selective

enrichment broths SEL and SSL may be used for detection of both Salmonella

and Listeria (Kim & Bhunia 2008, Yu et al. 2010). However, simultaneous

27

detection of pathogens may constitute a risk of overgrowth of certain bacteria

(Besse et al. 2010) which can in the worst case lead to false negative results.

Table 4. Summary of the selectivity agents used in the enrichment cultivation of food-

borne Salmonella and media in which they are used.

Factor Media Target microbes References

Acriflavine SEL Several bacteria (Kim & Bhunia 2008)

Bile salts MKTTn, RVS,

TGB, TT

Gram-positive bacteria (Müller 1923, Kauffmann 1930,

Kauffmann 1935)

Brilliant green MKTTn, RVS,

TGB

Gram-positive bacteria,

Gram-negative bacilli

(Kauffmann 1930, Kauffmann

1935)

Cycloheximide SEL Eukaryotic cells (Kim & Bhunia 2008)

Fosfomycin SEL Several bacteria (Kim & Bhunia 2008)

Lithium chloride SSL Several bacteria (Yu et al. 2010)

Malachite green RVS Several bacteria (Rappaport et al. 1956)

Nalidixic acid Other Enterobacteria, some

Gram-negative bacteria

(Kim & Bhunia 2008, Yu et al.

2010)

Novobiocin MKTTn Proteus (Kauffmann 1930, Kauffmann

1935, Restaino et al. 1977)

Potassium tellurite SSL Coliforms and intestine

bacteria

(Yu et al. 2010)

Sodium selenite SC Gram-positive bacteria,

coliforms

(Leifson 1939)

Tetrathionate MKTTn, TGB,

TT

Coliforms and intestine

bacteria

(Müller 1923)

28

Table 5. Summary of the enrichment cultivation broths included in examples of

validated alternative methods for the detection of food-borne Salmonella. In case that

the enrichment procedure depends on the food type, the procedures for meat/meat

products are presented. A – AFNOR, N – NordVal, BPW – buffered peptone water, RVS

– Rappaport Vassiliadis Soy broth, BHI – brain heart infusion broth, TGB -

tetrathionate brilliant green broth.

Method (manufacturer) Enrichment cultivation procedure

Broth Time [h] Temperature [°C]

Validation

Non-selective Selective A N

Immunoassays

Oxoid Salmonella Rapid Test

(OXOID Thermofisher Scientific)

BPW 18 35-38 SRTEM1 24 40.5-41.5 x

RAPIDCHEK™ SELECT™

(Strategic Diganostics Inc.)

16-22 40.5-42.52 6-8 40.5-42.52

RayAl Salmonella assays

(RayAl)

BPW 16-20 36-38 RVS 18-24 40.5-42.5 x x

RIDASCREEN® Salmonella

(R-Biopharm AG)

BPW 16-20 37 - x3

TAG 24 Salmonella

(BioControl Systems)

BPW4 16-20 36-38 BHI4 4-5 40.5-42.5 x

Tecra Salmonella Ultima/Unique

(TECRA INTERNATIONAL Pty Ltd)

BPW 16-20 36-38 RVS 18-24 40.5-42.5 x

TRANSIA® Plate Salmonella Gold

(BioControl Systems)

BPW 16-20 36-38 RVS 18-24 40.5-42.5 x x

VIDAS Easy Salmonella

(Biomerieux)

BPW 16-22 36-38 SX2 22-26 40.5-42.5 x

PCR-based methods

ADIAFOOD Salmonella

(AES Chemunex)

x

Assurance GDS Salmonella

(BioControl Systems)

BPW 18-24 36-38 BHI 2-4 35-38 x

BAX Salmonella

(DuPont Qualicon)

BPW 16-20 36-38 - x

GeneDisc Salmonella spp.

(Pall GeneSystems)

BPW 16-20 36-38 - x

iQ-Check™ Salmonella II

(BIO-RAD)

BPW 20-22 36-38 x x

TAQMAN Salmonella

(Applied Biosystems SA)

BPW 16-20 36-38 - x

Hybridization-based methods

GeneQuence Salmonella

(Neogen Corporation)

NB or BPW 18-24

35

RVS+TBG5 22-26 42.5 x

Lumiprobe 24 Salmonella

(EUROPROBE SA)

RM 6-8 36-38 RVS 17-19 40.5-42.5 x

1 Salmonella Rapid Test Elective Medium, included in the test kit, 2 Primary and secondary enrichment

broths utilizing Salmonella-specific bacteriophages are included in the test kit, 3 Also validated by

MicroVal, 4 Specific TAG supplement included in the test kit is added, 5 Selective enrichment in parallel in

RVS and TBG.

29

2.3 Important factors in the selection of the enrichment procedure

Although the standard media for the enrichment of food contaminating bacteria

are widely accepted and in some cases also tightly regulated, it has been reported

that the use of case-specific enrichment media could be beneficial (Besse et al.

2010). The factors that should be taken into consideration in selection of the

media include the regulations laid by the authorities, the sample matrix, the

concentration of the background microbiota in the sample, the concentration and

the physiological condition of the target bacteria, and the applied detection

method. Additionally, the applicability and the cost-efficiency of the selected

enrichment procedure should be at an acceptable level.

2.3.1 Regulations

Beer-spoiling LAB are not known to be pathogenic to human, and therefore their

detection is not regulated by the authorities. However, there are associations that

give guidelines for the efficient microbial quality control at the breweries. These

include the European Brewing Convention (EBC), American Society of Brewing

Chemists (ASBC) and the Brewery Convention of Japan (BCOJ). Due to the long

process time and the shelf-life of beer, it is generally regarded that even a low

level of spoilage microbes constitutes a risk (Jespersen & Jakobsen 1996).

Therefore, the microbiological guidelines suggest for unpasteurized beer a range

between 0 and 50 cells in 100–250 mL sample volume (Jespersen & Jakobsen

1996, Back 2005). In pitching yeast and in process samples before beer filtration,

a single spoilage organism in 106–108 cultivation yeast cells should be detected.

The detection of food-borne Salmonella is essential for the safety of the

consumers. Therefore, the use and validation of the monitoring procedures are

regulated by the authorities. In the EU, the regulations are given by the European

Commission (Anonymous 2005a). In Finland, the National Salmonella control

program has been applied since 1995 to protect consumers against Salmonella

infections spread through foods of animal origin (Anonymous 1994). Due to a

low prevalence of Salmonella in Finland and Sweden, each batch of meat, minced

meat and eggs imported to Finland and Sweden must be tested for Salmonella

using either the ISO reference method (Anonymous 2002a) or an alternative

method that has been compared against the ISO method, validated according to

the ISO 16140 method and certified by a third party (Anonymous 2002b).

30

2.3.2 Sample matrix

The composition of the enrichment broth and the cultivation conditions should be

selected based on the sample matrix. Firstly, food components can hinder or slow

down the nutrient transfer in the enrichment culture, and thereby affect the growth

of bacteria. Food particles may also challenge the pre-processing of the samples,

or inhibit the growth of target bacteria. Additionally, the enrichment cultivation

may be needed for the removal or dilution of food components that might hamper

molecular detection.

Effect on the pre-processing methods

Pre-processing of food samples usually aims to increase the concentration of

target bacteria prior to enrichment cultivation (pre-concentration) and change the

structure of the sample (homogenization). The increase in the concentration of

target cells may shorten the lag-time of the cells in the enrichment culture and

fasten the detection, while the removal of food particles allows decreasing the

cultivation volume. Pre-processing may also remove inhibitory components that

could hamper the detection. In the food industry, common applications of pre-

processing techniques include for example filtration of beer samples, removal of

yeast cells by means of differential centrifugation (Whiting et al. 1992), and

immunomagnetic separation (IMS) of Salmonella cells from enrichment cultures

prior to detection (Cudjoe et al. 1995, Hagren et al. 2008, Moreira et al. 2008).

The use of pre-concentration methods is sometimes hampered due to sample

components. Examples include clogging of membranes, co-flocculation of

bacteria with sample components in differential centrifugation, or non-specific

binding of immunomagnetic beads with other than target proteins (Payne & Kroll

1991, Stevens & Jaykus 2004a). According to Asano and co-workers (2007), the

membrane filtration of brewery samples may cause false negative results in

detection due to passing of bacteria that have changed their morphology during

the adaptation in the environment.

When the use of pre-concentration methods is not possible, the concentration

of bacteria in the sample remains low and the enrichment is more challenging. In

such cases the structure of the sample material may be changed by

homogenization. The attachment to the food particles has been shown to improve

the survival of Salmonella under stressful conditions (Kinsella et al. 2007), and

homogenization may be needed for the separation of attached cells from the food

31

particles. However, it may cause excessive elution of PCR inhibiting food

components (Kanki et al. 2009).

Inhibition of growth by food compounds

Several food components are known to be antibacterial. These components can

cause the induction of so called viable but not-culturable status of the target

bacteria and thereby lead to failure in the enrichment cultivation. For example, the

growth of Salmonella has been shown to be inhibited by site metabolites of LAB

(Rubin et al. 1982, Alakomi et al. 2007). Muniesa and co-workers (2005) showed

that the enrichment cultivation of contaminating bacteria can also fail due to

bacteriophages in food samples. The inhibitory effects of certain components may

vary in the sample and in the enrichment broth (Mccann et al. 2005), which can

lead into false negative results in the detection. The effect of inhibitory

compounds can be reduced by sufficient dilution of samples for the enrichment

cultivation (Ramnani et al. 2010).

Inhibition of molecular detection

Molecular detection methods may be inhibited by certain food components.

Especially biosensor-based detection tools have been reported to have a low

tolerance for interference with contaminating food particles (Gomez et al. 2001,

Radke & Alocilja 2005, Terry et al. 2005). Also, several sources report the

inhibition of PCR detection by food components (Rossen et al. 1992, Rådström et

al. 2003, Chen et al. 2010). The use of internal amplification controls in PCR

enables the recognizing of false negative results originating from inhibitory

compounds (Juvonen & Haikara 2009, Chen et al. 2010).

2.3.3 Background microbiota

Enrichment cultivation might be needed in order to reduce background microbiota.

Nonpathogenic microbes present in the foods can overgrow the target bacteria or

hamper their detection. For example, meat products carry LAB that have been

shown to inhibit the growth of enteric bacteria (Vold et al. 2000). Some Listeria

monocytogenes strains have been reported to be outcompeted by other strains

within the species (Besse et al. 2010) or even different lineages of one strain

(Bruhn et al. 2005). Singer and co-workers (2009) showed that the cultivation

32

media can select preferentially for specific Salmonella strains and decrease the

overall sensitivity of Salmonella detection. Furthermore, elevated initial

concentrations of certain serotypes did not necessarily increase the probability for

their detection. Brewery samples often include pitching yeast and wild yeast cells

that may challenge the enrichment cultivation and detection of LAB (Priest 2003).

In order to suppress the growth of background microbiota, the selectivity of

the medium can be increased by the addition of antibiotics or selective growth

factors, or by adjusting the pH and the temperature. In the case of beer-spoiling

LAB, antibiotics are usually added into the medium. Cycloheximide and 2-

phenylethanol are commonly used for the inhibition of yeasts and gram-negative

bacteria, respectively. Supplementing media with beer or its components supports

the growth of beer-spoiling bacteria and suppresses the growth of other bacteria

present (Holzapfel 1992, Suzuki et al. 2008b, Haakensen et al. 2009b). The

bacteriocins produced by some LAB can increase the selectivity of the

enrichment cultivation.

The enrichment cultivation of Salmonella is usually carried out in two steps,

of which the latter takes place in selective media. The reports on the necessity of

the selective cultivation are controversial. Several detection methods allow the

detection of Salmonella directly after cultivation in non-selective media

(Löfström et al. 2009, Tatavarthy et al. 2009, Matias et al. 2010, Chen et al.

2010). However, several case studies have shown that selective enrichment

cultivation enhances the accuracy of molecular detection (Myint et al. 2006,

Upadhyay et al. 2010). The performance of selective media varies depending on

the target strain (Huang et al. 1999, Besse et al. 2010).

The use of selective media is not advisable if the concentration of the cells is

low (Chen et al. 1993). Therefore, usually a non-selective enrichment cultivation

period is preferred at the beginning of analysis. On the other hand, the

introduction of some selectivity in the non-selective enrichment cultivation has

been shown to prevent from the overgrowth of the background microbiota.

Antibiotics, such as novobiocin or malachite green, might significantly reduce the

amount of background bacteria and therefore, benefit the recovery of Salmonella

(van Schothorst & Renaud 1985, Jensen et al. 2003). However, the addition of

antibiotics at the early stage of enrichment cultivation also causes stress to

Salmonella cells and can therefore risk their recovery (Chen et al. 1993). Test kit

manufactured by Strategic Diagnostics Inc. (RAPIDCHEK™ SELECT™)

utilizes selective bacteriophages for the suppression of the background microbiota

33

and thereby improves enrichment cultivation of Salmonella (Stave & Teaney

2009).

Techniques that allow a release of selective supplements in the non-selective

culture have been utilized for the cultivation of Salmonella. Sveun and Harman

(1977) used wax-coated gelatin capsules for a gradual release of iodine and

selenite into a non-selective broth. So called solid-repair methods have been used

for the two-step recovery of injured bacteria (Kang 2002). Initially the recovery

takes place inside or on top of the non-selective agar plate. The selective agents

are then diffused to the non-selective agar, changing the cultivation conditions to

selective. Disadvantages of solid-repair methods include formation of small

colonies that are difficult to pick under the selective medium layer, and the

negative effect of the melted selective overlay agar on the injured cells.

Additionally, the direct pour-plating of the sample also incorporates inhibitors

present in the food sample into the culture, which may negatively influence the

enrichment procedure.

Oxoid Inc. markets a test that incorporates the use of special growth

facilitators for an improved recovery of injured cells, followed by the timed

release of selective agents into the recovery medium. The method has been shown

to improve the rate of detection of low numbers of injured Salmonella cells after

24 hours of enrichment cultivation (Baylis et al. 2000a, Baylis et al. 2000b).

Panula-Perälä and co-workers (2008) developed a method for the controlled

release of glucose into the cultivation medium. The method is based on enzymatic

degradation of glucose from polymeric materials, in either gel or soluble form.

Commercial products based on the method (EnBase®, BioSilta Oy, Finland) have

been used to enhance the protein production in E. coli (Krause et al. 2010,

Glazyrina et al. 2010, Siurkus et al. 2010).

2.3.4 Concentration and condition of target cells

The concentration and physiological condition of the bacterial cells have been

shown to affect their recovery in laboratory media. Harvey and Price (1975)

showed that the performance of different medium brands can vary depending on

the inoculation concentration of Salmonella. The effect of the inoculation

concentration on the lag-time has been shown for example for L. plantarum

(Smelt et al. 2002), Salmonella Typhimurium (Stephens et al. 1997) and Listeria

monocytogenes (Besse et al. 2006).

34

Food-borne bacteria may be injured due to food processing and handling

procedures, such as thermal treatment, refrigeration, freezing, drying, irradiation,

from exposure to preservatives, acidity, and low water activity, or from being

starved. The presence of injured cells needs to be considered when enrichment

cultivation conditions are selected (Shintani 2006). Moreover, different stress

factors can cause large variations in the lag-time between individual cells

(Mackey & Derrick 1984). When the concentration of cells is low the effect of

stress factors on the bacterial lag-time tends to increase (Shida et al. 1975b). It is

known that sub-lethally injured cells become temporarily susceptible to many

selective compounds in the media, and that they do not repair or multiply in the

presence of the selective compounds (Busta 1976). Therefore, the primary

recovery should take place in non-selective conditions.

The enrichment cultivation of Salmonella is usually challenging due to the

poor physiological status of the cells. Sub-lethal injury of Salmonella cells has

been reported to cause changes in the metabolism leading to for example

degradation of ribosomal DNA and RNA (Tomlins & Ordal 1971, Gomez &

Sinskey 1973, Gomez et al. 1976, Chambliss et al. 2006). This can lead in failure

in recovery of the cells, especially on nutrient rich media (Clark & Ordal 1969,

Gomez et al. 1973). The tolerance of S. enterica against different stress factors

varies between serotypes (Sherry et al. 2004).

Solutions for improved recovery

The recovery of injured cells can be improved using repair methods, based on

either solid or liquid media. These methods have been recently reviewed in detail

(Wu 2008). A liquid-repair method for Salmonella cultivation reported by Kang

and Siragusa (2001) includes a two-fold serial dilution of samples in a 96-well

microtiter plate containing BPW, followed by incubation at 37 °C for 3 hours for

resuscitation of sub-lethally injured cells. After that an equal volume of double

strength selective broth is added to each well, and the cultures are further

incubated for 13 hours at 37 °C in the dark. Solid-repair methods that are used in

the release of selective agents in the non-selective culture (see previous chapters)

have been reported to improve the recovery of injured bacteria, including S.

Typhimurium (Kang & Fung 2000, Wu & Fung 2006).

The use of growth factors, such as iron supplements in the medium can

improve the recovery of sub-lethally injured cells (Gast & Holt 1995).

Siderophores, high-affinity iron chelating compounds secreted by bacteria, have

35

been shown to improve the recovery of injured bacteria in an enrichment

cultivation. Ferrioxamines are selective siderophores that have been used for

resuscitation of injured Salmonella (Reissbrodt et al. 1996, Reissbrodt et al.

2000). Some Salmonella cells may even be non-recoverable without the addition

of such sole iron supplement (Thammasuvimol et al. 2006).

Since magnesium is necessary for the recovery of injured bacteria, the

addition of magnesium sulfate may be beneficial in enrichment cultivation (Hurst

1977). Tween 80 is an emulsifier, that has been shown to increase the recovery of

injured bacteria by repairing damaged cells membranes (Murthy & Gaur 1987).

The use of antioxidants (see next chapter) may also contribute to the recovery of

injured bacteria.

2.3.5 Stress factors in enrichment media

The effect of low concentration and poor physiological condition of the target

cells on enrichment cultivations are further increased if the growth conditions in

the sample and the media are different (Gomez & Sinskey 1973, Gomez et al.

1973, Zwietering et al. 1991, Zwietering et al. 1994, Suzuki et al. 2008b,

Haakensen et al. 2009b). The recovery and the lag-time of especially injured

bacteria depends largely on the cultivation conditions. Important factors include

the osmolarity, i.e. the concentration of nutrients and salts (Clark & Ordal 1969,

Shida et al. 1975a, Shida et al. 1975b, Chambliss et al. 2006); pH (Shida et al.

1975b, Chambliss et al. 2006); oxygen concentration (Lushchak 2001); and

temperature (Shida et al. 1975b, Zwietering et al. 1994, Mattick et al. 2001). The

stress responses of bacteria can vary between species and even within the species

(van de Guchte et al. 2002, Alegria et al. 2004, Sherry et al. 2009).

Osmotic stress

Changes of nutrient and salt concentrations in the medium can cause osmotic

stress to bacteria. Osmotic stress can affect both lag-time and growth rate in

bacterial cultures (Shida et al. 1975a, Shida et al. 1975b). Viable Salmonella cells

tolerate high osmolarity relatively well compared to other Enterobacteriaceae.

Therefore, elevated salt concentration may be used to suppress the growth of

competing bacteria in enrichment cultivation of food-borne Salmonella (van

Schothorst & Renaud 1983). However, sub-lethal injury of cells reduces the

tolerance to high osmolarity (Gomez & Sinskey 1973), and therefore the use of

36

media with low osmolarity is recommended for the recovery of Salmonella in

foods (Clark & Ordal 1969, Gomez et al. 1973).

Acidic and alkaline stresses

The stress caused by a sudden decrease or increase in pH is called acidic or

alkaline stress, respectively. Bacteria can tolerate acidic and alkaline stresses by

applying various mechanisms (Humphrey et al. 1993, Flahaut et al. 1997), but in

the case of enrichment cultivation pH shifts can extend the lag-time of the culture

(Cheroutre-Vialette et al. 1998), and even cause the death of individual cells. The

appropriate pH of the enrichment broth depends therefore on the pH of the sample;

in case of the beer-spoiling LAB the inoculation from beer to broth with neutral

pH causes alkaline stress. Besides during inoculation, pH may shift also during

the enrichment cultivation; the metabolites produced by bacteria often decrease

the pH in the bacterial cultures. Therefore, the buffering capacity of enrichment

medium should be sufficient to slow down the acidification.

Oxidative stress

Oxidative stress is a condition in which the production of reactive oxygen species

results in adverse effects on bacteria. Formation of reactive oxygen radicals in the

broth media has been shown to affect the recovery of injured bacteria (Mizunoe et

al. 1999). The major enzymes protecting the bacterial cells against reactive

oxygen species are superoxide dismutase, catalase, and peroxidases (Morris 1977).

Although the majority of LAB are catalase negative, some strains are able to

degrade hydrogen peroxide through the action of specific heme-dependent

catalases (An et al. 2010b) or superoxide dismutases (Götz et al. 1980a). The

activity of enzymes involved in degradation of reactive oxygen species in bacteria

can diminish due to sub-lethal injury, which may explain the toxic effects of

relatively low peroxide levels in enrichment media (Andrews & Martin 1979,

Kobayashi et al. 2005).

The addition of antioxidants into the enrichment medium has been shown to

improve the growth of both LAB and Salmonella. The recovery of beer-spoiling

LAB may be facilitated by addition of L-cysteine-HCl (Nishikawa & Kohgo 1985,

Taguchi et al. 1990, Anonymous 2007) or sodium bicarbonate (Tharmaraj & Shah

2003). Antioxidants used in the cultivation of Salmonella include catalase

37

(Rayman et al. 1978), sodium puryvate (Rayman et al. 1978), and Oxyrase®

(Niroomand & Fung 1992, Reissbrodt et al. 2002).

Thermal stress

Thermal stress is caused by a shift in the cultivation temperature. The temperature

has been shown to influence both lag-time and growth rate of bacteria (Shida et al.

1975b). The cold and heat tolerance of LAB, as well as their ability to adapt after

sudden temperature shifts, varies between species (De Angelis & Gobbetti 2004).

According to the literature, the effect of the cultivation temperature on the lag-

times and growth rates of Salmonella does not significantly vary between

serotypes (Oscar 2000). The recovery from thermal stress may relate to other

stress responses. According to Kobayashi and co-workers (Kobayashi et al. 2005),

the recovery of Salmonella induces the expression of both heat-inducible and

oxidative stress related genes. Therefore, the selection of enrichment medium

requires holistic analysis of possible stress factors and their possible interactions.

2.3.6 Detection methods

The performance of enrichment broths is linked to the selected detection method

in several ways. The medium components can directly interfere with the detection

and therefore the optimal media for the recovery of bacteria might not always be

the optimal choice with respect to following analytical steps. PCR is known to be

relatively sensitive to interference, and inhibition by medium components, such as

agar or antibiotics, has been reported (Gibb & Wong 1998, Faraq et al. 2010).

Enrichment medium may also affect the detection by decreasing the concentration

of marker molecules in the cells. For example, the cultivation of sub-lethally

injured Salmonella in unsuitable medium can cause denaturation of certain

antigens (Hahm & Bhunia 2006) and repression of virulence marker genes

(Cochrane & O'Connor 2002).

The duration and the number of the enrichment cultivation steps depend on

the selectivity and the specificity of the detection method. However, due to the

need for detecting low levels of food-borne Salmonella and the non-equal

distribution of the cells in the samples almost all rapid test protocols include at

least one enrichment cultivation step. For example, PCR methods generally

require 8–24 hours of non-selective enrichment cultivation. Some bacteriophage

assays are claimed to allow the detection already after six hours of cultivation in

38

BPW. In case of some foods even the PCR detection directly from the samples

may be possible (Stevens & Jaykus 2004b). However, nearly all of the alternative

methods validated by AFNOR or NordVal require enrichment cultivation, and

take at least 24 hours to complete (Anonymous 2010a, Anonymous 2010c).

Despite enrichment cultivation steps, the majority of the alternative detection

methods can be completed in less than 48 hours.

In summary, the length of the total detection procedure can be affected by

decreasing the enrichment cultivation periods by allowing faster and more

efficient recovery, and accelerating the growth of the target cells while

suppressing the growth of background microbiota. Various efforts have been

made to validate shorter enrichment cultivation procedures and therefore allow

faster detection of Salmonella in foods. Generally, the use of highly selective

detection method may allow the use of less selective enrichment media. However,

if the analyzed food carries high concentration of background microbes the use of

only non-selective enrichment media creates a risk of outcompeting of the target

bacteria.

Despite active reporting on novel enrichment cultivation media or techniques

for detection of Salmonella, the majority of the reported studies focus on novel

media for the second, selective or indicative enrichment cultivation, while the

non-selective enrichment cultivation is still usually carried out in BPW (Table 5).

This is probably because the use of commonly accepted enrichment procedure is

safe and allows saving research and development resources.

39

3 Summary of the material and methods

An overview of the materials and methods used in this study is given below.

Detailed descriptions are presented in the original articles according to Table 6.

3.1 Bacterial strains and preparation of injured cells

The type strains of LAB were obtained from the German Collection of

Microorganisms (Braunschweig, Germany). Beer-spoiling LAB strains isolated

from a Finnish brewery was purchased from VTT Technical Research Centre of

Finland, Culture Collection (Espoo, Finland). Salmonella Typhimurium was a

food isolate strain kindly provided by Dr. Antje Breitenstein (Scanbec GmbH,

Halle, Germany). The beer-spoiling LAB were adapted to beer for a minimum of

three days prior to the experiments. Injured Salmonella cells were prepared at

temperatures of 55 ºC (Feng et al. 2007), 4 ºC (Yuk & Schneider 2006) or – 20 ºC

(Wu et al. 2001).

3.2 Enzyme controlled glucose delivery system

The glucose delivery system used for the cultivation of LAB was based on a two-

phase gel and glucoamylase controlled release of glucose (Panula-Perälä et al.

2008). For cultivation of Salmonella, the soluble carbon source format of the

same system was used (Krause et al. 2010). The reagents for both EnBase®

applications were purchased from BioSilta Oy, Oulu, Finland.

3.3 Experimental design and variable analysis

The experimental design and variable analysis were done by means of RSM using

MODDE 8 software for Design of Experiments and Optimization (UMETRICS

AB, Umeå, Sweden)(Eriksson et al. 2000). A full factorial design was used for the

screening experiments. The RSM was done with face centered central composite

design (CCF) in case of only quantitative factors. D-optimal design was used for

the RSM with both qualitative and quantitative factors. The data was fitted using

multiple linear regressions (MLR). The analysis of variance (ANOVA) of the

models was done using F-test.

40

3.4 Growth curves and definition of lag-times and growth rates

The growth curves of the cultures based on optical densities at 490 nm (OD490)

were fitted into the bacterial growth model described by Baranyi and Roberts

(1994) using DMFit software (Baranyi & Tamplin 2004). The corresponding lag-

times and maximum growth rates were calculated by the software.

3.5 Measurement of rRNA by means of SHA

Oligonucleotide probes targeting highly conserved 16S rRNA in LAB were

designed using the ARB software package (Ludwig et al. 2004). The specificity

of probes was checked in silico using the program “Probe Match” provided by the

Ribosomal Database Project II (RDP II, http://rpd.cme.msu.edu/index.jsp) (Cole

et al. 2003) and in the EMBL database using the NCBI Blast2 alignment tool

(Altschul et al. 1997). Salmonella enterica 23S rRNA targeting oligonucleotide

probes were designed using the CloneManager5 software (Scientific &

Educational Software, Cary, NC) and their specificity was checked in silico using

BLAST database (Altschul et al. 1990) and in SHA against E. coli cell extracts.

The rRNA for the SHA was constructed from the corresponding PCR amplified

rRNA genes by means of T7 RNA polymerase in vitro transcription using the

MAXIscript™ T7-kit (Ambion, Austin, TX, USA). The rRNA concentrations

were measured by means of SHA described by Rautio et al. (2003) using either

fluorescent or electric signal detection.

3.6 Food and process samples

Brewer’s yeast slurries were aseptically collected from the bottom of fermentation

tanks in 50 mL bottles. In standard cultivation-based detection 200 µL of each

sample were incubated on Schwarz differential agar (SDA) and inoculated into

MRS broth (Oxoid MRS Broth, Fisher Scientific International, Basingstoke, UK)

containing 25% (v/v) of beer and 0.01 g per L of cycloheximide. The samples

from the beer can seamer were taken either by swabbing, or by processing a can

containing sterile Ringer solution through the seaming device. The beer samples

were vacuum filtered through membrane discs with a pore size of 0.45 µm and a

diameter of 47 mm (EZ-Pak Sterile Membrane Filters, Millipore Oy, Espoo,

Finland). Minced meat samples were prepared using Finnish commercial minced

meat and spiked with heat injured Salmonella cells.

41

Table 6. Summary of the methods used in this study. For more detailed information,

see the original articles.

Objective Method Study

Cultivation of LAB Microaerophilic cultivation in B-MRS I-IV

Cultivation of Salmonella Aerobic cultivation in BPW or RVS V

Monitoring of growth OD490 measurement II-V

Calculation of cells per mL Agar plating on selective media II-V

Growth curve fits DMFit software IV-V

RSM MODDE8 software III-V

rRNA detection SHA I-V

42

43

4 Summary of results and discussion

4.1 Improved enrichment cultivation of beer-spoiling LAB (Studies

I-IV)

4.1.1 The role of enrichment cultivation in 16S RNA SHA detection of

beer-spoiling LAB

The first aim of this study was to assess the enrichment cultivation of beer-

spoiling LAB with respect to detection by means of rRNA-based SHA. For this

purpose, 16S rRNA-based SHA was applied for the detection of beer-spoiling

LAB in actual pitching yeast and beer samples in a Finnish lager brewery (52

samples). The detection procedure of SHA included the filtration of beer samples,

inoculation of samples into B-MRS broth, over-night incubation at 29±1 ºC,

harvesting of the cells by means of centrifugation, and SHA. The results of SHA

were compared to simultaneous detection by cultivation (reference method).

Group specific oligonucleotide probes targeting several Lactobacillus and

Pediococcus strains were used in the SHA (Study I, Table 3). In comparison to the

reference method, 10 false negative and 8 false positive results were detected in

SHA (Study I, Fig. 4). The relative specificity and sensitivity of detection of SHA

were 85 and 81%, respectively.

The false results of SHA may originate from any of the steps in the detection

procedure, or from the failure in the reference method. False positive results in

SHA may originate from the detection of slow-growing LAB that were not

recovered during the cultivation (Storgårds et al. 1998, Suzuki et al. 2008b).

Since the used oligonucleotide probes are specific to certain groups of

Lactobacillus and Pediococcus, while several other LAB may be encountered at

the breweries (O'Sullivan et al. 1999), it is possible that some false negative

results were due to detection of other LAB in the reference method. Furthermore,

some of the positive results in the reference method may originate from the poor

selectivity and thereby detection of bacteria other than LAB (Storgårds et al.

1998).

It was also concluded that too short enrichment cultivation could be a major

origin of false negative results. Therefore, 16 more beer samples were analyzed

by means of SHA by using up to 144 hours of enrichment cultivation in B-MRS

broth (unpublished data). The 16S rRNA concentrations in the cultures were

44

measured in 12 hour intervals using oligonucleotide probes targeting a large

group of beer-spoiling LAB (Study III). After 144 hours of enrichment cultivation,

5 false positive results and no false negative results were detected in SHA in

comparison to the reference method (Table 7). Accordingly, the specificity and the

sensitivity of SHA were 29 and 100%. Enrichment cultivation intervals needed

for positive detection in SHA ranged from 36 to 144 hours. The specificity of the

SHA decreased and the sensitivity increased when the enrichment cultivation was

prolonged (Fig. 1). However, this may be due to poor performance of the

reference method. It is possible, that the prolonged enrichment cultivation prior to

SHA allowed recovery and detection of fastidious LAB strains, that were not

recovered in the cultivation-based detection (Jespersen & Jakobsen 1996,

Storgårds et al. 1998, Suzuki et al. 2008a).

Table 7. The results of the detection of LAB in brewery samples by means of SHA and

reference method (unpublished data). Additionally, the enrichment cultivation

intervals needed for positive detection of LAB in brewery samples by means of 16S

rRNA SHA are presented. Pos – positive result; Neg – negative result.

Sample Reference method SHA Enrichment cultivation [h]

1 Pos Pos 96

2 Pos Pos 144

3 Neg Neg 144

4 Pos Pos 120

5 Pos Pos 36

6 Pos Pos 36

7 Neg Pos 144

8 Neg Pos 72

9 Pos Pos 72

10 Pos Pos 72

11 Neg Pos 96

12 Neg Pos 48

13 Pos Pos 48

14 Pos Pos 96

15 Neg Pos 72

16 Neg Neg 144

45

Fig. 1. The changes of relative specificity (■) and sensitivity (∆) of SHA after 36–144

hours of enrichment cultivation of beer-spoiling LAB in brewery samples

(unpublished data).

Based on the present results, the decision concerning the duration of enrichment

cultivation is critical for the 16S rRNA-based detection of beer-spoiling LAB.

Enrichment cultivation for 48 hours or less may be enough for the detection of

LAB by means of bioluminescence measurement (Takahashi et al. 2000),

immunoassay (Yasui & Yoda 1997), PCR (Juvonen et al. 1999) or FISH (Thelen

et al. 2002). However, those methods are more sensitive than SHA; the detection

limit of SHA is approximately 106 cells per assay (Study I), while for example the

detection limit of PCR can be as low as 10 cells per assay (Juvonen & Haikara

2009).

In order to shorten the enrichment cultivation period, the lag-time of LAB

should be minimized, and the growth rate maximized. The concentration of 16S

rRNA in LAB cells has been shown to depend on the growth phase (de Vries et al.

2004). Therefore, the accelerated growth might also increase the concentration of

target molecules per cell. Based on the literature (see chapter 2.3), the

possibilities to improve the recovery are to i) adjust the temperature in order to

reduce thermal stress; ii) decrease the salt and sugar concentrations in order to

reduce osmotic stress; iii) decrease the pH in order to reduce alkaline pH stress;

and iv) add antioxidants in the medium in order to reduce oxidative stress.

Additionally, the growth rate of LAB may be increased by suppressing the

growth of other bacteria in the enrichment culture, and improving the accessibility

of LAB to growth factors (Enfors et al. 2001). In order to improve the

accessibility to nutrients, enzyme controlled glucose delivery described by

Panula-Perälä and co-workers may be useful (2008). Shaking of cultures allows

equal distribution of growth factors in the culture, although it can also increase

0

20

40

60

80

100

36 48 60 72 84 96 108 120 132 144

Rat

io [%

]

Duration of cultivation [h]

46

the diffusion of oxygen to the media and therefore accelerate the production of

reactive oxygen species. The effects of different stress factors on growth of beer-

spoiling LAB, as well as possibilities to reduce them and improve the recovery

and growth rate of beer-adapted cells are discussed in the following chapters.

4.1.2 Reduced thermal and alkaline pH stress

The thermal stress due to elevated cultivation temperature may extend the lag-

time of LAB in enrichment cultures. Therefore, the effect of varying the

cultivation temperature between 25 and 30 °C on the growth of beer-spoiling

LAB was studied by means of RSM. The elevation of the temperature affected

both lag-times and maximum growth rates in the cultures of beer-spoiling P.

damnosus (Study IV, Fig. 2). The elevated temperature extended the lag-time with

approximately 12 hours.

Besides thermal stress, the LAB transferred from brewery samples into B-

MRS may experience alkaline pH stress due to the elevated pH. The effect of

alkaline pH stress on the recovery of P. damnosus was therefore also studied by

means of RSM. Similarly to the elevated temperature, the pH in B-MRS broth

extended the lag-times in the cultures (Study IV, Fig. 2). The effect of pH on the

lag-time of P. damnosus was some hours more compared to the effect of

temperature. However, unlike elevated pH, the elevated temperature affected also

the maximum growth rates in the cultures, and therefore the effect of cultivation

temperature on the OD490 was larger than that of the pH (Study IV, Fig. 1).

The enrichment cultivation of beer-spoiling LAB commonly takes place at

29±1 °C. However, the present results suggest that this temperature is too high for

the efficient recovery of all the relevant strains. The cultivation temperature has

been shown to affect the lag-time of bacteria especially in the low cell

concentrations (Smelt & Haas 1978, Robinson et al. 2001). Therefore, the effect

of the temperature on the recovery of beer-spoiling LAB is likely more prominent

when actual samples with very low cell concentrations are used.

The present results suggest the use of pH 5.2 for the cultivation of P.

damnosus. This is in agreement with the literature, indicating that the pH around

5.5 or even higher in the commonly used B-MRS broth may cause extensive lag-

times of LAB in the enrichment cultivation and therefore affect their detection

(Jespersen & Jakobsen 1996, Satokari et al. 2000, Carr et al. 2002, Suzuki et al.

2008b). Besides reducing the alkaline pH stress, the use of low pH suppresses the

growth of bacteria not able to spoil beer, and thereby acts as a selection step in the

47

enrichment cultivation (Behr et al. 2006). In the future, the actual effect of the

adjusted cultivation conditions will be tested with respect to the relevant

microbiota and preferably using actual brewery samples.

4.1.3 Reduced oxidative stress

The oxidative stress of bacteria is caused by reactive oxygen species. Since most

LAB are catalase-negative, their oxidative stress may be reduced by adding

antioxidants in the enrichment medium. In this study, L-cysteine-HCl and sodium

bicarbonate were used as antioxidants in the cultivation of beer-spoiling LAB. L-

cysteine-HCl was selected, since it is widely used in the cultivation of LAB

(Taguchi et al. 1990) as well as other bacteria (Stark et al. 1994). Sodium

bicarbonate is used in the storage of probiotic LAB (Tharmaraj & Shah 2003),

and it was therefore thought to improve the recovery of beer-spoiling LAB as

well.

As could be expected, the addition of L-cysteine-HCl improved the growth of

LAB (Study III, Fig. 2; Study IV, Fig. 4). The suggested concentration of L-

cysteine-HCl was 0.25 g per L, which is close to those recommended in the

literature (Taguchi et al. 1990, Anonymous 2007). The use of sodium bicarbonate

was beneficial for the growth of L. backi and L. brevis, (Study III, Fig. 2–3) but

did not affect the growth of P. damnosus (Fig. 2). The proposed optimal

concentration of sodium bicarbonate within the studied range is 4 g per L.

48

Fig. 2. The effects of antioxidants on the growth of beer-spoiling P. damnosus

(unpublished data). The scaled effect of each model term with 95% confidence level is

presented. The effect is statistically significant if the confidence intervals do not cross

the x-axis. X-axis: L-cys - L-cysteine-HCl; Na-c - sodium bicarbonate.

The addition of 0.25 g per L of L-cysteine-HCl and 4 g per L of sodium

bicarbonate in B-MRS broth improved the growth of L. backi and L. brevis in

single strain cultures (Study III, Fig. 3). The effect could be seen in the measured

OD both after over-night incubation and at the end of cultivation. The use of

antioxidants did not affect the lag-times of the cultures, but the growth rates were

enhanced for both species (Fig. 3). The growth in the cultures supplemented with

antioxidants continued for longer time than in cultures with plain B-MRS. This

suggests that the benefit from the addition of antioxidants can continue for several

days, and therefore also enhances the growth of slowly recovering strains.

0.0

0.1

0.2

0.3

0.4

0.5

0.6

L-cys*L-cys L-cys Na-c

Eff

ects

on

OD

490

Model term

49

Fig. 3. Effect of supplementing B-MRS with L-cysteine-HCl and sodium bicarbonate on

lag-times (A) and maximum growth rates (B) of L. backi (grey columns) and L. brevis

(white columns). The average values and standard deviations for three independent

cultures are presented. X-axis: 1 – plain B-MRS; 2 – B-MRS supplemented with L-

cysteine-HCl and sodium bicarbonate.

The applicability of B-MRS supplemented with antioxidants was further tested in

the enrichment cultivation of LAB from the actual brewery samples prior to SHA-

based detection (Study III, Fig. 4). In the majority of the samples, the measured

concentrations of 16S rRNA were higher when plain B-MRS was used. However,

for the samples with the highest cell densities, the use of antioxidants improved

the growth. Considering the lag-times of LAB in the single strain cultures (Fig. 3),

as well as the measured 16S rRNA concentrations (Study III, Fig. 4), the use of

antioxidants may not necessarily speed up the adaptation of LAB in B-MRS broth.

However, their use seems to increase the growth rate in cultures, which

contributes to higher concentration of cells after for example 48 hours of

enrichment cultivation in comparison to plain B-MRS. Therefore, also the

accuracy of SHA could be improved by addition of antioxidants. Some LAB are

also known to be able to utilize oxygen and degrade reactive oxygen species

(Götz et al. 1980a, Götz et al. 1980b). The presence of such strains could explain

the variability in the effect of antioxidants.

The beneficial effect of antioxidants onto the cultivation of bacteria has been

reported in several studies. Besides L-cysteine-HCl and sodium bicarbonate,

various other antioxidants may be used for the improved cultivation of beer-

spoiling LAB. These include for example Oxyrase® (Ordonez et al. 2000),

catalase (An et al. 2010a), ascorbic acid (Elli et al. 2002), and thiol compounds

(Elli et al. 2002). Probably the optimal combination of the antioxidants depends

A

Medium

1 2

La

g-tim

e [

h]

0

2

4

6

8

B

Medium

1 2

Max

. gr

owth

rat

e [lo

g cf

u/h]

0.00

0.05

0.10

0.15

0.20

0.25

50

on the bacterial strain; therefore the screening of the most suitable ones with

respect to specific application is advisable.

4.1.4 Reduced osmotic stress

The effect of reduced osmolarity on the recovery of beer-spoiling LAB was

studied by adjusting the composition of enrichment broths. Firstly, the effect of

initial glucose concentration was demonstrated in cultivation of L. backi, L. brevis

and L. lindneri in B-MRS with 0.1- 2.0% glucose (Fig. 4). Based on the growth

curves, the common B-MRS that contains 2.0% glucose would be the most

suitable for the cultivation of these strains. In average, the shortest lag-times were

achieved in B-MRS with 1.0% glucose, and the highest growth rates in 2.0%

glucose. The effects of glucose concentration on the lag-times or maximum

growth rates in the cultures were not statistically significant (in both cases p > 0.1,

unpublished data). However, earlier it has been shown that especially injured

bacteria benefit from the use of medium with low osmolarity for the recovery

(Gomez & Sinskey 1973, Gomez et al. 1973). Therefore, the decreased

concentration of sugars and salts could allow faster recovery of stressed LAB

present in the actual brewery samples (Suzuki et al. 2006a, Suzuki et al. 2008b).

Fig. 4. The growth of beer-spoiling LAB in B-MRS broth containing 0.1–2.0% glucose

(unpublished data). The average growth curves (A), lag-times (B) and maximum

growth rates (C) for the three strains in B-MRS containing 0.1 (□), 0.5 (■), 1.0 (○) or 2.0

(●) % glucose are presented. The cultures were inoculated with ca. 100 cells of L.

backi, L. brevis or L. lindneri. The error bars present the standard deviations between

the three strains.

In order to facilitate the growth of beer-spoiling LAB, the composition of B-MRS

broth was adjusted by means of RSM, using beer-spoiling P. damnosus as the test

Cultivation time [h]

0 16 32 48 64 80 96

OD

490

0.00.20.40.60.81.01.21.41.6

A

Concentration of glucose [%]

0.0 0.5 1.0 1.5 2.0

Lag-

time

[h]

25.0

25.5

26.0

26.5

27.0

27.5

28.0

Concentration of glucose [%]

0.0 0.5 1.0 1.5 2.0

Max

. gro

wth

rate

[log

cfu

/h]

0.04

0.05

0.06

0.07

0.08

0.09

0.10B C

51

strain. The tested concentrations of beer and dehydrated MRS powder (Difco)

were selected between the concentrations that are used in ABD (Suzuki et al.

2008b) and B-MRS (Holzapfel 1992). Accordingly, the concentrations of MRS

powder from 2.6 to 55 g per L and beer from 25 to 100% were used. The

concentration of dehydrated MRS powder (Difco) showed the largest effect on the

measured OD after 96 hours of cultivation (Fig. 5). Based on the RSM, the

optimal concentration of MRS powder is lower than what is recommended by the

manufacturer (Study IV, Fig. 4). Varying the concentration of beer from 25% to

100% (v/v) did not affect the growth of P. damnosus.

Fig. 5. The effects of concentrations of compounds in the B-MRS broth on the growth

of beer-spoiling P. damnosus (unpublished data). The scaled effect of each model

term with 95% confidence level is presented. The effect is statistically significant if the

confidence intervals do not cross the x-axis. X-axis: MRS – Difco dehydrated MRS

powder; L-Cys - L-cysteine-HCl.

The adjusted B-MRS broth containing 25% of beer, and 40 g per L of Difco MRS

powder was tested in the cultivation of P. damnosus (0). The difference between

common and adjusted B-MRS broths was minor in terms of measured OD, lag-

time and maximum growth rate.

-0.4

-0.3

-0.2

-0.1

-0.0

0.1

0.2

MR

S*M

RS

L-cy

s*L-

cys

MR

S

Bee

r*M

RS

Bee

r*L-

cys

MR

S*L

-cys

L-cy

s

Bee

r

Bee

r*B

eer

Eff

ects

on

OD

490

Model term

52

Fig. 6. The growth of beer-spoiling P. damnosus in B-MRS broth containing different

concentrations of beer and MRS powder (unpublished data). The average growth

curves for three independent cultures incubated in adjusted (●) or common (○) B-

MRS broth are presented in A. Average lag-times (grey columns) and maximum

growth rates (white columns) for the three independent cultures are presented in B.

The error bars present the standard deviations for three series. X-axis: 1 – common B-

MRS; 2 – B-MRS with adjusted concentrations of beer and MRS powder.

The effect of reduced osmolarity on the enrichment cultivation of beer-spoiling

LAB in actual brewery samples was demonstrated by comparing the efficiencies

of B-MRS and ABD (Suzuki et al. 2008b) broths prior to 16S rRNA-based SHA.

The use of ABD medium, that contains relatively low concentrations of salts and

sugars, did not allow faster SHA detection of LAB in the actual brewery samples

(Study III, Fig. 1). The specificities and sensitivities of SHA using both media

were 80 and 100%, respectively (unpublished data). Based on the present results,

the effect of osmolarity on the growth of beer-spoiling LAB varies between

different strains. Although, direct effect on the detection procedure was not

achieved, the reduced osmolarity might improve the detection of some fastidious

LAB. The use of media with low osmolarity could therefore be beneficial in for

example trouble-shooting cases, for tracking of certain slowly growing

contaminants.

4.2 The effect of enzyme controlled glucose delivery

The accessibility to nutrients is one of the factors affecting the growth of bacteria.

However, as was discussed earlier, the initially high concentration of nutrients

B

Medium

B-MRS PDM 14 15

Lag-

time

[h]

0

5

10

15

20

25

30

Max

. gr

owth

rat

e [lo

g cf

u/h]

0.000.010.020.030.040.050.060.070.080.09

1 2 1 2

0

0.1

0.2

0.3

0.4

0.5

0.6

0 8 16 24 32 40 48 56 64 72

OD

49

0

Cultivation time [h]

A

53

may suppress the recovery of stressed cells. In order to improve the accessibility

to nutrients without increasing the osmolarity of the medium, enzyme controlled

glucose delivery system developed by Panula-Perälä and co-workers (2008) was

applied for the cultivation of beer-spoiling LAB in B-MRS broth.

The feasibility of the gel-based glucose delivery system was demonstrated in

cultivation of L. brevis type strain in B-MRS (Study II, Fig. 1). The use of glucose

delivery increased the measured OD after 24 hours of incubation, as well as at the

end of cultivation (Study II, Fig. 3). However, the effects on lag-times and growth

rates were not statistically significant (Fig. 7). These results support the findings

of other studies, suggesting that the glucose supply allows increasing the final cell

density by controlled growth rather than accelerates the growth rates in the

bacterial cultures.

Fig. 7. The effect of glucose delivery on lag-times and growth rates of L. brevis type

strain in B-MRS (unpublished data). Average lag-times (grey columns) and maximum

growth rates (white columns) for three independent cultures are presented. The error

bars present the standard deviations of three cultures. X-axis: 1 – B-MRS; 2 – B-MRS

with gel-based glucose delivery and 1,000 units per L enzyme.

Due to the positive effect on growth of the L. brevis type strain, the glucose

delivery system was also applied to cultivation of beer-spoiling LAB strains

(Study II, Fig. 2). With the exception of slow-growing strain L. malefermentans,

the growth of beer-spoiling Lactobacillus increased in proportion to the increased

enzyme concentration. Similarly the growth of P. damnosus was improved by the

use of glucose delivery (Fig. 8). The initial glucose concentration did not affect

the final OD.

Medium

1 2 126 127

Lag-

time [h]

0

2

4

6

8

10

12

14

Max.

gro

wth

rate

[lo

g cf

u/h]

0.00

0.05

0.10

0.15

0.20

0.25

0.30

1 2 1 2

54

Fig. 8. The effect of glucose delivery on the growth of beer-spoiling P. damnosus

(unpublished data). The scaled effect of each model term with 95% confidence level

are presented. The effect is statistically significant if the confidence intervals do not

cross the x-axis. X-axis: Enz – enzyme in glucose delivery system; Glu - glucose.

The glucose delivery system was applied at brewery quality control for

cultivation of LAB in actual samples. The use of B-MRS with glucose delivery

resulted in higher OD in the enrichment cultures (Study II, Fig. 4). In further tests,

the use of glucose delivery also directly contributed to shorter total detection time

by means of SHA (Study III, Fig. 5). The supplementing of the B-MRS broth with

the glucose delivery system increased the sensitivity of SHA from 63 to 88%, and

decreased the specificity from 100 to 67% (unpublished data). The increased

sensitivity and specificity could be due to recovery of contaminants that were not

recovered in plain B-MRS.

The use of the glucose delivery system is also likely to support the growth of

bacteria other than LAB in the samples (Panula-Perälä et al. 2008). Therefore, the

medium that is used with it should be selective enough to suppress the

overgrowth of LAB by fast-growing background microbiota. The concentration of

enzyme applied for the cultivation of LAB was several orders of magnitude

higher than what is recommended for E. coli in the literature (Panula-Perälä et al.

2008). This may be explained by the different applications of the glucose delivery

system; the reported uses for high cell density cultivations of E. coli are based on

avoiding the anaerobic over-flow metabolism, which may lead to accumulation of

side metabolites and decrease the pH of the medium (Panula-Perälä et al. 2008,

Krause et al. 2010). However, especially beer-spoiling LAB are tolerant to low

pH, which allows growth despite the accumulated acidic metabolites. For slow-

growing strains, too high concentration of enzyme might result in the

0.0

0.1

0.2

0.3

0.4

Enz*Enz Enz Glu*Enz Glu

Eff

ects

on

OD

at

490

nm

Model term

55

accumulation of glucose in the culture during the lag-time, and even in the

suppression of growth (Panula-Perälä et al. 2008). Therefore, the enzyme

concentration in the system should be separately optimized with respect to the

studied bacteria.

4.3 Improved enrichment cultivation of heat-injured Salmonella

(Study V)

4.3.1 Enrichment cultivation in 23S rRNA SHA

In order to assess the enrichment cultivation of food-borne Salmonella, SHA with

rRNA-targeting oligonucleotide probes was applied. Due to the similarity of 16S

rRNA sequences within different enterobacteria (Lin & Tsen 1996, Christensen et

al. 1998), the probes were targeted to S. enterica 23S rRNA (Study V, Table 1).

The detection limit of the SHA was approximately 106 cells per mL (unpublished

data). Despite the relatively low sensitivity, SHA was chosen for the study due to

its flexible detection system that allows various different applications with minor

modifications (see chapter 1.1). Minced meat was used as sample matrix due to

its high concentration of background microbiota. The SHA was not affected by

the interference of sample matrix (Study V, Fig. 5).

Due to the low concentration of Salmonella in foods, and the relatively high

detection limit of the SHA, the applied method would not allow the detection of

Salmonella directly in food samples. Even if the cell concentration would be high

enough, the partial degradation of ribosomal DNA and RNA in Salmonella cells

due to sub-lethal heat injury could falsify the results of SHA (Deutscher 2009,

Chen & Deutscher 2010). Therefore, the measurement of Salmonella 23S rRNA

without enrichment cultivation is not feasible.

The detection of Salmonella 23S rRNA directly from the non-selective

enrichment culture may be possible. However, the concentration of 23S rRNA in

the Salmonella cells has been shown to decrease rapidly when the cells reach the

stationary growth phase (Hsu et al. 1994). Since the growth rate of Salmonella

cells depends on a variety of unknown factors, such as the lag-time of the cells or

the growth of background microbiota, it is theoretically possible that Salmonella

cells are already in stationary phase after over-night incubation. Therefore, the use

of a selective enrichment cultivation step prior to SHA is advisable.

56

The duration of the non-selective cultivation step could possibly be shortened

by several hours from the commonly used 18–24 hours by optimizing the growth

conditions. However, the over-night incubation assures the sufficient recovery of

Salmonella cells, and thereby allows them to survive in the selective conditions

(Chen et al. 1993). Moreover, the adaptation time needed for the sufficient

recovery of injured cells cannot be accurately predicted (Beckers et al. 1987), and

therefore shortening of the non-selective enrichment cultivation may lead to a

failure in the detection. Considering the workflow in the industrial laboratories, it

is probably of minor relevance whether the enrichment cultivation takes 18 or 24

hours, since the analysis is not processed during the night. In the future, the use of

automated detection systems that would proceed with the samples during the

night could perhaps change this situation. Generally, the over-night cultivation is

also practical due to its flexibility; the samples taken at different times during the

day are basically ready for the analysis in the morning at the same time. Based on

the discussed reasons, the non-selective over-night incubation followed by short

incubation in selective broth may be the most practical choice for the enrichment

cultivation procedure prior to SHA. However, the efficiency of the enrichment

cultivation steps could possibly be improved by means of medium optimization.

The improved growth of Salmonella in non-selective medium can be

achieved by either facilitating the recovery of injured cells, or by accelerating the

growth after recovery. The addition of nutrients or other growth factors into non-

selective medium might affect both recovery and growth of Salmonella (Bailey &

Cox 1992). However, the selection of suitable initial glucose concentration is

somewhat complex; although Salmonella tolerate high osmolarity better than

many other bacteria, the provided glucose may actually end up accelerating the

growth of fast-recovering background microbiota. Furthermore, the use of

complex medium might suppress or extend the lag-times in the cultures (Clark &

Ordal 1969, Gomez & Sinskey 1973, Gomez et al. 1973). Therefore, the slow

release of nutrients into the culture could be beneficial. The enzyme controlled

glucose delivery system (Panula-Perälä et al. 2008) that was earlier in this study

successfully applied for the cultivation of LAB is a potential candidate for the

nutrient supply.

Besides adding more nutrients to the medium, the growth may be improved

by increasing the selectivity of conditions. Since the addition of selective agents

into the recovery medium is not advisable due to possible inhibitory effect against

stressed Salmonella cells, the suitable possibilities include the suppression of

background microbes by means of inhibitory compounds, such as bacteriophages

57

(Stave & Teaney 2009); the use of Salmonella-specific growth factors (Reissbrodt

& Rabsch 1993); the adjustment of the cultivation temperature (Rhodes et al.

1985); and the increase of the buffering capacity.

4.3.2 Improved growth of Salmonella in BPW

In order to improve the growth of Salmonella, BPW broth was supplemented with

glucose. Instead of adding the glucose batchwise, it was slowly released by means

of enzyme controlled glucose delivery system. The feasibility of glucose delivery

for Salmonella was tested in over-night cultivation of viable S. Typhimurium and

Citrobacter freundii, which belongs to non-pathogenic background microbiota in

meat. Cultures were inoculated with approximately 103 cells of Salmonella or 107

cells of Citrobacter. Gel-based glucose delivery and 3 U per L enzyme were used

based on the literature (Panula-Perälä et al. 2008). As was expected, the use of

glucose delivery improved the growth of both strains. The measured OD after

over-night incubation was doubled when the glucose delivery was used instead of

plain BPW (Fig. 9).

Fig. 9. The growth in single strain cultures of S. Typhimurium and Citrobacter freundii

in BPW with and without glucose delivery (unpublished data). The average OD and

standard deviations for three independent cultures of Salmonella (grey columns) and

Citrobacter (white columns) are presented. X-axis: 1 – plain BPW; 2 – BPW

supplemented with gel-based glucose delivery system and 3 U per L enzyme.

Medium

0.0 0.5 1.0 1.5 2.0 2.5 3.0

OD

490

0.0

0.1

0.2

0.3

0.4

0.5

1 2

58

4.3.3 Improved recovery of injured Salmonella in single strain cultures

After showing the feasibility of glucose delivery in the cultivation of viable

Salmonella, it was applied in the recovery of injured cells. Both gel-based and

soluble carbon source based glucose delivery systems were tested. Injuring of

Salmonella cells was carried out at temperatures of 4, -20 or 55 ºC. In case of

cold-injured cells, the highest OD after 18 hours of cultivation was measured in

cultures where soluble carbon source based glucose delivery was used (Fig. 10).

Heat-injured cells were only recoverable in cultures where glucose delivery

systems were used.

Fig. 10. The recovery of stressed S.Typhimurium cells in different BPW broths

(unpublished data). White columns present the OD after 18 hours of cultivation. The

grey columns present the OD after 24 hours (A and B) or after 48 hours of cultivation.

Average values and standard deviations for three independent cultures are presented.

The cells were injured at temperatures of 4 ºC (A), -20 ºC (B) or 55 ºC (C). X-axis: 1 –

BPW; 2 –BPW + gel-based glucose delivery; 3 – BPW + soluble carbon source-based

glucose delivery.

Due to its positive effect on the recovery of injured cells, the soluble carbon

source –based glucose delivery system was used in cultivation of heat-injured

Salmonella. Based on the literature and this study, the use of glucose delivery is

likely to facilitate also the growth of background microbiota in minced meat

samples. In order to specifically support the recovery of Salmonella, siderophore

ferrioxamine(Reissbrodt et al. 2000) E was added to BPW. Additionally, elevated

cultivation temperatures were tested. The use of glucose delivery improved the

growth of heat-injured Salmonella in single strain cultures by decreasing the lag-

times (Study V, Fig. 2&3) and increasing the maximum growth rates (Fig. 11). As

0.0

0.1

0.2

0.3

0.4

0.5

1 2 3

OD

490

Recovery medium

B

0.0

0.1

0.2

0.3

0.4

0.5

1 2 3

OD

490

Recovery medium

A

0.0

0.2

0.4

0.6

0.8

1 2 3

OD

490

Recovery medium

C

59

could be expected, the use of ferrioxamine E was also beneficial for the recovery

of Salmonella (Study V, Fig. 2).

In order to minimize the lag-times, the proposed optimal levels of the tested

compounds are 15 g per L of carbon source, 6 U per L enzyme, and 0.5 µg per L

ferrioxamine E. The suggested cultivation temperature is 40 ºC. Independent of

the cultivation temperature, the use of BPW with proposed composition resulted

in improved growth of heat-injured S. Typhimurium in single strain cultures

(Study V, Fig. 4).

Fig. 11. The effect of soluble carbon source-based glucose delivery on growth of heat-

injured S. Typhimurium in BPW (unpublished data). The scaled effects of each model

term on the maximum growth rates with 95% confidence levels are presented. The

effect is statistically significant if the confidence intervals do not cross the x-axis. X-

axis: Enz - EnzI’m, Sub – Soluble carbon source; Temp – temperature; FOE -

ferrioxamine E.

The glucose delivery systems have not been used earlier for the cultivation of

Salmonella. However, the reported effects on the growth of E. coli are similar to

the effects on Salmonella presented here (Panula-Perälä et al. 2008, Krause et al.

2010, Osmekhina et al. 2010). The optimal concentration of the enzyme proposed

in this study is also in agreement with earlier studies, suggesting that the use of

concentration of 6 U per L would be suitable in order to facilitate the growth in

the beginning of the cultivation. Although, the use of elevated temperature did not

seem to have an effect in the cultivation of heat-injured Salmonella in single

strain cultures (Study V, Fig. 4), it may increase the selectivity of cultivation in

-0.3

-0.2

-0.1

-0.0

0.1

0.2

Enz

*Enz

Sub

FO

E

Tem

p*T

emp

Tem

p

Sub

*Sub

FO

E*F

OE

Sub

*Tem

p

FO

E*S

ub

FO

E*T

emp

Enz

FO

E*E

nz

Sub

*Enz

Enz

*Tem

p

Eff

ects

on

max

imum

gro

wt

rate

Model term

60

the presence of background microbiota (Rhodes et al. 1985, Krascsenicsova et al.

2006). However, it has to be noted that Salmonella serotypes may respond

differently to the elevated cultivation temperature, and that the heat-tolerance can

also depend on the sample matrix and enrichment medium (Quintavalla et al.

2001).

4.3.4 Improved recovery of heat-injured Salmonella in minced meat

In the previous chapters, the adjustment of BPW was shown to improve the

growth of viable Salmonella cells, as well as to allow faster recovery of injured

cells. Therefore, the effect of supplementing BPW broth with glucose delivery

and ferrioxamine E on the SHA detection of Salmonella in minced meat was

studied. Four different commercial minced meats were used as sample matrices.

The samples were spiked with low concentrations of heat-injured Salmonella

(Study V). Statistically significant difference in the measured 23S rRNA

concentrations between plain and supplemented BPW started to show after six

hours of cultivation (Study V, Fig. 6).

Although, the growth of Salmonella could be facilitated by the use of glucose

delivery, the possibility for shorter enrichment cultivation was not studied. Based

on the measured 23S rRNA concentrations, four hours of enrichment cultivation

was not sufficient for the recovery of heat-injured cells (Study V, Fig. 6). Similar

findings have been reported by Zhao and Doyle (2001), who claimed that an

incubation time of less than 6 h would not assure sufficient recovery of injured

Salmonella in foods.

The present results indicate that the recovery of heat-injured Salmonella, as

well as its detection in food samples may be enhanced by addition of the tested

glucose delivery system. The effect of glucose delivery in case of other

Salmonella strains, or different sample matrices should be tested before the

applicability of the system can be evaluated. Additionally, it is advisable to adjust

the concentrations of soluble carbon source and enzyme separately for different

detection systems. The applied glucose delivery system could also be beneficial in

the selective cultivation of Salmonella, which possibility is in the scope of further

studies.

61

5 Conclusions

This study has shown the effect of reducing different stress factors on the

recovery and growth of food-contaminating bacteria with respect to rRNA-based

detection. The results can be used in the evaluation of enrichment cultivation

procedures in food diagnostics, and in the development of new cultivation media

and techniques. The provided information may be useful in the assessment of

bacterial cultivations also in other applications. The main findings of this thesis

were as follows.

1. The used SHA is comparable to other alternative detection methods in terms

of industrial applicability (i.e. cost-efficiency, hands-on time, and the duration

of the assay). The sensitivity of SHA is relatively low compared to e.g. PCR.

However, due to low concentration and slow growth of LAB in brewery

samples, enrichment cultivation is required also for other alternative detection

methods. Therefore, with respect to the total duration of the procedure, the

difference in sensitivity is not fundamental.

2. The enrichment cultivation influences the rRNA-based SHA detection of

food-contaminating bacteria in several ways. The duration of the cultivation

is especially critical with respect to specificity and sensitivity of the detection.

In practice, the SHA detection without enrichment cultivation is impossible in

case of both beer-spoiling LAB and food-borne Salmonella.

3. In single strain cultures even minor adjustment of the cultivation conditions

may reduce the lag-time and increase the growth rate of beer-spoiling LAB.

However, similar effect is not necessarily seen in the cultivation of LAB in

actual brewery samples. This may be due to complexity of mixed bacterial

cultures. The only solution for actually improving the overall quality of

detection might be to separately optimize the enrichment cultivation with

respect to the relevant sample and bacterial species.

4. Slow release of glucose by means enzyme controlled glucose delivery system

improves the growth of beer-spoiling LAB in single strain cultures, and in

case of actual brewery samples. The duration of the detection procedure can

be decreased by several hours by the use of the glucose delivery.

5. The recovery of injured Salmonella cells was enhanced by using the glucose

delivery system together with selective siderophore ferrioxamine E. This

effect was seen in reduced lag-times and increased growth rates. The adjusted

BPW broth increased the 23S rRNA concentrations also in mixed cultures,

62

and it may therefore improve the rRNA-based detection of Salmonella. The

glucose delivery system is one potential tool in the development of more

efficient media for enrichment cultivation of food-contaminating bacteria.

6. In order to minimize the necessary resources, the possibility for

miniaturization and automation of the enrichment cultivation should be

considered. Other proposals for future studies include i) screening for

components that would inactivate the growth inhibitors present in the cultures;

ii) improved techniques for timed release of medium components by

development of biodegradable capsule materials, or methods for monitoring

the physiological status in the cultures; and iii) the development of basal

media, into which for example selective components could be added in case

of tracking specific contaminant sources at the processes.

63

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Original articles

I Huhtamella S, Leinonen M, Nieminen T, Fahnert B, Myllykoski L, Breitenstein A & Neubauer P (2007) RNA-based sandwich hybridisation method for detection of lactic acid bacteria in brewery samples. J Microbiol Methods 68: 543–553.

II Taskila S, Neubauer P, Tuomola M, Breitenstein A, Kronlöf J & Hillukkala T (2009) Improved Enrichment Cultivation of Beer Spoiling Lactic Acid Bacteria by Continuous Glucose Addition to the Culture. J Inst Brew 115(3): 177–182.

III Taskila S, Tuomola M, Kronlöf J & Neubauer P (2010) Comparison of enrichment media for routine detection of beer spoiling lactic acid bacteria and development of trouble-shooting medium for Lactobacillus backi. J Inst Brew 116(2): 151–156.

IV Taskila S, Tuomola M, Kronlöf J, Ruuska J & Neubauer P (2010) Note-Preliminary applications of response surface modeling to the evaluation of optimal growth conditions for beer-spoiling Pediococcus damnosus. J Inst Brew 116(3): 211–214.

Supplementary material:

V Taskila S, Osmekhina E, Tuomola M, Ruuska J & Neubauer P (2010) Modification of buffered peptone water for improved recovery of heat injured Salmonella Typhimurium. Manuscript.

Reprinted with permission from Elsevier (I) and the Journal of the Institute of

Brewing (II-IV).

Original publications are not included in the electronic version of the dissertation.

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