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ICoFAB2018 International Conference on Food, Agriculture and Biotechnology

Preface

The integrated ASEAN Economic Community (AEC) was launched in 2015. One of the

main purposes of this integration is for the development of science and technology since

it is a key factor in sustaining economic growth, enhancing community wellbeing and

promoting integration in this region.

In order for ASEAN science to become world class and be globally competitive, it requires

the driving forces from the three main scientific areas of (1) food science and

technology (2) agricultural technology and (3) biotechnology. ASEAN is home to one of the

world’s most precious natural resources, and the most diverse microbial community.

Scientific strength in this region would be significantly enhanced provided that appropriate

collaborative networks amongst member countries are promoted. In addition, education

sectors should focus more on internationalizing their curricula and universities across this

region should find more opportunities to collaborate in research and academic activities.

The Faculty of Technology, Mahasarakham University (MSU) has organized the The 5th

International Conference on Food, Agriculture and Biotechnology (5th ICoFAB 2018) with

the aims to share research experience on food, agriculture and biotechnology amongst

Thai and international postgraduates. The conference will provide a starting stage for

collaborative networks among postgraduates from Thai universities and ASEAN

countries. This will strengthen research community locally and internationally and

provide the international academic medium for postgraduates to benefit from it.

(Assoc. Prof. Dr. Anuchita Moongngarm)

Dean of the Faculty of Technology

Mahasarakham University


MSU Editorial Board

1. Assoc. Prof. Dr. Anuchita Moongngam 2. Asst. Prof. Dr. Sirirat Deeseenthum

3. Assoc. Prof. Dr. Maratree Plainsirichai 4. Assoc. Prof. Dr. Anut Chantiratikul

5. Assoc. Prof. Dr. Thalisa Yuwa-amornpitak 6. Assoc. Prof. Prasit Chutichudech

7. Asst. Prof. Dr. Wantana Sinsiri 8. Asst. Prof. Dr. Pheeraya Chottanom

9. Asst. Prof. Dr. Wasan Duangkhamchan 10. Asst. Prof. Dr. Vijitra Luang-In

11. Dr. Nantaporn Sutthi 12. Dr. Kedsirin Sakwiwatkul

Scientific Committee

1. Prof. He Chaoxing Institute of Vegetable and Flowers Chinese

Academy of Agricultural Sciences, China

2. Prof. Ping Zhang Xishuangbanna Tropical Botanical Garden,

China

3. Prof. Yongqi Shao Zhejiang University, China

4. Prof. C. Hanny Wijaya Bogor Agricultural University, Indonesia

5. Honorary Prof. Colin Wrigley QAAFI, University of Queensland, Australia

6. Prof. Emeritus Ian Warrington Massey University, New Zealand

7. Assoc. Prof. Dr. Ko-Tung Chang National Pingtung University of Science and

Technology, Taiwan

8. Dr. Abdulhadi Albaser University of Sebha, Libya

9. Assoc. Prof. Dr. Khamsah Universiti Sultan Zainal Abidin, Terengganu,

Suryati Mohd Malaysia

10. Asst. Prof. Dr. Gerhard BOKU – University of Natural Resources and

Schleining Life Sciences, Austria

11. Dr. Nurul Huda Abd Kadir Universiti Malaysia Terengganu, Malaysia

12. Asst. Prof. Dr. Bundit Yuangsoi Khon Kaen University, Thailand

13. Assoc. Prof. Dr. Maratree Mahasarakham University, Thailand

Plainsirichai

14. Assoc. Prof. Dr. Thalisa Mahasarakham University, Thailand

Yuwa-amornpitak


Scientific Committee (Cont.)

15. Asst. Prof. Dr. Rumpai Mahasarakham University, Thailand

Gaensakoo

16. Asst. Prof. Dr. Kannika Mahasarakham University, Thailand

Chookietwattana

17. Asst. Prof. Dr. Bussagon Mahasarakham University, Thailand

Thongbai

18. Dr. Sunisa Roi-Doung Mahasarakham University, Thailand

19. Dr. Srinual Jantathai Mahasarakham University, Thailand


Content Full papers: Page 1: Utilization of Sugarcane Syrup and Non-Lysed Dried Spent .…………………..

Brewer’s Yeast for Cost Effective Succinate Production

by Actinobacillus succinogenes

1

Apichai Sawisit

2: Preferences and Factors Influencing the Purchase Intention …………………….

of Healthy Snacks among Millennials in Jakarta

15

Grace Aurelia Kahono

3: Impact of Concentration of Glucono Delta Lactone (GDL), Tempe ……………

Starter and Soaking Time to Physical Characteristics

of Tempe and Overripe Tempe

24

Irvan Setiadi Kartawiria

4: Efficacy of Fresh Herbs Knee Mask Formula to Relieve ………………………..

Knee Pain in Osteoarthritis Elderly Patients 36

Jongkol Poonsawat

5: Influence of Urea on Butanol Production from Sugarcane Juice ………………..

by Clostridium beijerinckii TISTR 1461

48

Kitipong Wechgama

6: Identification of Cellulase-Producing Bacteria from Soil ……………………….

in Nasinuan Forest, Kantarawichai District,

Mahasarakham Province

Manatchanok Yotchaisarn

58

7: Effect of Different Culture Media on Growth of Microalgae……………………

Haematococcus sp. TISTR 9450RE

72

Narisara Wongsing


Content Full papers: Page 8: Applying Lean Thinking to the Horticultural Value Chain in …………………...

New Zealand

80

Nigel P. Grigg

9: Preliminary Groundwater Quality Assessment under Groundwater……………..

Banking Project for Agricultural Uses in

Tung Kula Rong Hai

92

Rawintra Eamrat

10: Chemical Composition and Insecticidal Activities of Essential Oil ……………

of Aegle marmelos (L.) Correa. against Tribolium castaneum

Herbst (Coleoptera: Tenebrionidae)

105

Ruchuon Wanna

11: Extraction of Coffee Silverskin and the Development of ………………………

Antioxidant-Rich Products

117

Samuel P. Kusumocahyo

12: Insect Diversity in Rajamangala University of Technology Isan, ……………...

Roi Et Campus at Tung Kula Rong Hai

132

Sukanya Lapkratok

13: Effect of Thai Jasmine Rice on the Fruiting Body Growth of…………………..

Cordyceps militaris

140

Surachai Rattanasuk

14: Plant Growth Promoting Activity of Two Plant extracts ………………………

Combined with Chitosan on Chilli Seedlings

146

Tarntip Rattana


Content Full papers: Page 15: Indole-3-Acetic Acid Production of Chlorpyrifos Tolerant…………..………..

Bacteria Isolated from Agricultural Soils

156

Thanakorn Saengsanga

16: Establishment of a Logistic Equation for Crop Biomass …………..…………..

and Analysis of Interspecific Interaction in

Wheat and Faba Bean Intercropping

163

Wenlian Bai

Abstracts: 1: Promoting Farmers to Re-forest and Utilize Non-Timber .…………..………….. 179

Products for Value Addition

Krailert Taweekul

2: Medicinal Plants in Asia …………..…………………………………………….. 180

Yek-Cheng Ong

3: Antioxidant and Antimicrobial Activities of Edible ……………….……...…….. 181

Plant Leaf Extracts

Bussagon Thongbai

4: Antioxidant Properties of Soybean Residue from ……………………....……….. 182

Soy Milk Production

Ekkarat Tangkhawanit

5: Effect of Rice Varieties and Germination Time …………………….....….……... 183

on Melatonin and Its Derivative in Rice

Jakkaphan Kaennok


Content Abstracts: Page 6: Phenolic Content and Antioxidant Activity of …………..……………………..

Pasteurized Mulberry Leaf Tea Mixed Soymilk

Powder and Its Correlation with Different

Antioxidant Assay

Jintana Sangsopha

184

7: Butanol Production from Molasses by …………..…………………..

Clostridium beijerinckii TISTR 1461 under

Different Yeast Extract Concentration Conditions

Kanlayani Charoensopharat

185

8: Optimization of Process Variables for Pulsed …………..…………………..

Vacuum Osmotic Dehydration of Model Food

Cubes

Kulab Sitttisuanjik

186

9: The Antioxidant Capacities of Ethanolic Extract …………..…………………

from Traditional Herbs in Sahatsakhan District,

Kalasin Province

Kwanyuen Leamsamrong

187

10: Effects of Different Phosphorus Forms on …………..…………………

Phosphorus Absorption and Utilization of

Maize in Red Soil

Lian-Ya Zhang

188

11: Oleic Acid and Palmitic Acid Induced Non-Alcoholic …..…………………..

Fatty Liver Disease (NAFLD) in HepG2 Model

Nadta Sukkasem

189


Content Abstracts: Page 12: Effect of Paederia Linearis Hook. f. Root on …………..…………………..

the Pasting Behavior of Waxy Rice Flour

Pharita Sampaoton

190

13: Analysis and Comparison of Pumping Concrete ……..…….……………. 191

Plug in Tunnel no. 10 and no. 12 in Longkou

Power Station

Liang Shuang

14: Study on the Microbiology of Intercropping on ……..…….……………. 192

the Prevention and Control of Faba Bean Fusarium

Wilt

Sirui Wang

15: Chemical and Physical Properties of Instant ……..…….…………….. 193

Edible-Swift Let Bird Nest

Wichuda Jitthimol

16: Using of Alcohol-Based and Biomass Pellet on ……..…….…………….. 194

Tobacco Leaves Curing Process

Chen Yanjie

17: Cytotoxicity of the Mallotus Repandus Extract ……..…….…………….. 195

and Its Active Constituent Bergenin on Human

HepG2 Cells

Yollada Sriset

18: Bioconversion of Molasses into Lipids ……..…….……………………….. 196

Accumulation by Oleaginous Yeasts as

Biodiesel Feedstock Kusumawadee Thancharoen


Utilization of Sugarcane Syrup and Non-Lysed Dried Spent Brewer’s

Yeast for Cost Effective Succinate Production by

Actinobacillus succinogenes

Apichai Sawisit1,4, Surawee Jampatesh4, Sirima Suvarnakuta Jantama2,

Thipwarin Rimlumduan3, Sukanya Lapkratok1, Rawintra Eamrat1, Sukanya Mingyai1,

Kaemwich Jantama4*

1Department of Agricultural Technology and Environment, Faculty of Sciences and Liberal Arts,

Rajamangala University of Technology Isan, Nakhon Ratchasima, 30000, Thailand2Division of Biopharmacy, Faculty of Pharmaceutical Sciences, Ubon Ratchathani University,

Ubon Ratchathani, 34190, Thailand 3Department of Applied Biology, Faculty of Sciences and Liberal Arts, Rajamangala University of

Technology Isan, Nakhon Ratchasima, 30000, Thailand4Metabolic Engineering Research Unit, School of Biotechnology, Institute of Agricultural Technology,

Suranaree University of Technology, Nakhon Ratchasima, 30000, Thailand *Corresponding author: [email protected]

Abstract

In this study, non-lysed dried spent brewer’s yeast (DSB) was evaluated as a low-cost

nitrogen source for succinate production by Actinobacillus succinogenes 130ZT using

sugarcane syrup as an inexpensive source of carbon. The results indicated that the DSB

could promote the succinate production efficiency. Succinate concentration at 8.32 g/L

with a yield of 0.82 (g/g glucose) from 15 g/L initial glucose that were supplemented

with 30 g/L of DSB were achieved. This result was comparable to that obtained using

commercial yeast extract at 5 g/L in which succinate concentration at 8.37 g/L was

obtained. The highest yield and productivity of succinate at 0.74 g/g glucose and 0.53

g/L/h in glucose-based medium were obtained, respectively by controlling pH at 6.8 and

continuously supplying external CO2. The optimized concentration of sugarcane syrup

was observed at 10% (w/v) in which the succinate concentration at 35.75 g/L with a

yield of 0.76 (g/g glucose) were attained. The present study suggested that DSB and

sugarcane syrup could be used as nitrogen and carbon sources for industrial succinate

production.

1

DOI:10.14457/MSU.res.2018.44


Keywords: Succinate, sugarcane syrup, dried spent brewer’s yeast,

Actinobacillus succinogenes, CO2

Introduction

Succinate is considered as one of the top 12 building block chemicals that could be

manufactured from renewable feedstocks [1]. It is a potential precursor for the synthesis

high value products of commercial importance including polymers, surfactants, green

solvents, detergents, flavors and fragrances [2]. Up to now, succinate is mainly

produced commercially from n-butane through the hydrogenation of petroleum-derived

maleic anhydride. However, the increase in price of oil and petroleum derivatives has

made the microbial production of succinate from cheap carbon substrates as an

economically attractive option for succinate as a renewable commodity chemical [3]. In

addition, the microbial succinate production incorporates CO2, a primary greenhouse

gas, providing further incentive for production by white biotechnology [4]. Therefore,

the succinate fermentation from low-cost substrates offers the opportunity to be both

greener and more cost effective than petroleum-based alternative products.

It has been reported by the U.S. Department of Agriculture (USDA, 2008) that raw cane

sugar and sugarcane molasses were sold at prices less than 0.50 US$/kg, and therefore

many fermentation industries have used refined sucrose and sugarcane molasses as

target substrates to produce lower price bio-based products. In Thailand, sugarcane is

cultivated in various parts of the country, but the entire crop is used in sugar production.

Up to now, many fermentation industries in Thailand, especially ethanol industries have

used molasses, a by-product in the production of sugar as the substrate [5]. As the

increasing industrial demand for bio-fuel and bio-based chemicals production coupled

with an unstable supply of sugarcane molasses, this resulted in the increasing industrial

demand for bio-based production. Hence, this carbon source is becoming limited.

Moreover, using molasses in fermentation causes serious problems in the downstream

processing and in the final waste treatment. Unlike sugarcane molasses, sugarcane syrup

containing 55% inverted sugar is not only renewable and abundant but also a cheap

source of carbon as compared with another refines carbohydrate like glucose or sucrose

[6]. Utilization of sugarcane syrup in the fermentation industry would reduce the use of

2


molasses. An alternative renewable carbon source is required to sustain the fermentation

industries thus sugarcane syrup seems to be the most promising one.

A few literatures have been reported the replacement of a commercial yeast extract in

succinate production by the low-cost nitrogen source such as corn steep liquor solution

[7] or dried corn steep liquor [8], and spent brewer’s yeast hydrolysate [9]. However,

the different strategies for using spent brewer’s yeast hydrolysate along with various

preparation process have used for succinate production. In a previous study, our

laboratory has accomplished the development of an inexpensive nitrogen source, which

is spent brewer’s yeast extract (SBE) for replacement of commercial yeast extract in

succinate production by A. succinogenes 130ZT [10]. Nevertheless, the preparing of

SBE is quite difficult, and needs to be prepared in the large volume of reaction in order

to make it more economically as an alternative nitrogen source. Sridee et al. [11]

showed that dried spent brewer’s yeast cell (DSB), a by-product from brewery industry,

contained high nitrogen and many essential mineral salts. Therefore, it may be used as a

low-cost nitrogen supplementation for succinate production instead of commercial yeast

extract. To the best of our knowledge, there is no report on directly used DSB for

succinate production. In this report, we investigate the feasibility of using sugarcane

syrup as an alternative carbon source supplemented with DSB as a low-cost nitrogen

source with aim to develop an economical succinate production. We also evaluate the

effect of sugarcane syrup concentrations, pH values as well as CO2 supply on the

production of succinate in titer, yield, and productivities by A. succinogenes 130ZT.

Materials and methods

Materials

Sugarcane syrup containing the total soluble solids of 76.19 ºBrix, 55.79% inverted

sugar kindly supported from Mitr Phol Sugar Corp., Ltd., Thailand. The DSB

(Saccharomyces uvarum) with a moisture content of 10% (dry basis) was kindly

provided by Khon Kaen Brewery Company Ltd., Thailand. The SBE was prepared as

described previously [10].

3


Microorganism and inoculum preparation

A. succinogenes 130ZT (DSM 22257) was purchased from the German Type Culture

Collection. The strain was pre-culture in 100 mL sealed-anaerobic bottles containing 75

mL of the culture medium with the composition (g/L): KHCO3 10.0, NaH2PO4 8.5,

K2HPO4 15.5, MgSO4.7H2O 0.05, NaCl 1.0 and yeast extract 10.0. The pH of the

medium was adjusted at 7.5 with concentrated NaOH before sterilization (15 min at

121ºC). For inoculum preparation, five grams per liter of glucose (autoclaved

separately) was added to the medium after sterilization. Aseptic CO2 was sparged for 2

min to make an anaerobic environment before inoculation. The seed medium was

inoculated with 0.75 mL of stock culture and incubated at 37 ºC for 16-18 h with

intermittent gentle shaking.

Anaerobic fermentation

Fermentation in anaerobic bottles

Fermentation in anaerobic bottles was carried out in sealed 100-mL anaerobic bottles

containing 50 mL culture medium. The fermentation medium was the same as described

above. The initial pH of the medium was adjusted at 7.5 before sterilization. Fifteen

grams per liter of glucose (autoclaved separately) was added to the medium after

sterilization. Aseptic CO2 was sparged for 2 min to make an anaerobic environment.

The medium was incubated at 37 ºC with 150 rpm shaking speed. Succinate production

was examined up to 24 h at a regular interval of 12 h.

Anaerobic fermentation in stirred bioreactors

Batch fermentation was performed in 2-L stirred bioreactor. The different pH controlled

was estimated (pH 6.2, 6.8, and 7.2 using 3M KOH as a neutralizing base). Further, the

effect of carbon dioxide gas was also investigated at different levels (non-limiting CO2

supply, limiting CO2 supply for 1 h at the beginning, and without supply of CO2).

Subsequently, production of succinate was optimized at different concentrations of

sugarcane syrup (1, 5, 10, 15 and 20% w/v) in the fermentation medium maintained at

the optimized pH and performing at the optimized CO2 supply obtaining from the

previous experiment.

4


Analytical methods

Biomass concentration was determined by measuring the optical density at 550 nm

using a spectrophotometer (Spekol-1500, Anatytik Jena, Thailand). The optical

densities were then converted to dry cell weight (OD 1.0 = 0.333 mg of cell dry

weight/L) and defined as biomass concentration. Organic acids (succinate, formate, and

acetate), ethanol, and total sugars were quantified by high performance liquid

chromatography (Agilent technology, Japan) equipped with an ion exclusion column

(BIO RAD, Aminex, HPX-87H, USA) with a column temperature of 45 ºC using 4 mM

H2SO4 as a mobile phase with a flow rate of 0.4 mL/min). Concentrations of the three

sugars (sucrose, glucose and fructose) were combined and reported as total sugars

concentration. The total nitrogen (TN) content in samples was measured by Kjeldahl

method [11].

Statistical analysis

Analysis of variance (ANOVA) was conducted using SPSS software (SPSS 17.0 for

Windows; SPSS Inc., Chicago, IL). The differences among mean values were

established using Duncan’s multiple-ranges test (DMRT) at 95% significance level.

Results and discussions

Effect of yeast concentration on succinate production

In order to improve the efficiency of succinate production DSB was used as nitrogen

source, DSB was dissolved in sterilized water with 1:1 solid-liquid ratio. The yeast

particles of DSB were then removed by membrane filtration. The suspension after

removing of yeast particles was subsequently used as a nitrogen source for succinate

production by A. succinogenes 130ZT. As shown in Fig. 1, succinate production was

increased with increasing of DSB concentration from 5 to 30 g/L. Beyond this

concentration, a constant in succinate production was observed. The succinate

5


production at 8.37 g/L was obtained from 15 g/L glucose supplemented with 5 g/L of

commercial yeast extract. The succinate level obtained here was comparable to those of

succinate formation using 30 g/L DSB or 5 g/L SBE) in which succinate production at

8.32 and 8.14 g/L, respectively. According to the comparison of nitrogen content in

various nitrogen sources, DSB has about 2.5 times lower in total nitrogen content

(31.78) as compared with commercial yeast extract (85.32) or SBE (83.90) (Table 1).

Thus, the higher amount of nitrogen source in succinate production supplemented with

DSB was required. Based on the obtained results, supplementing of 30 g/L of DSB

provide the equivalent amount of succinate production from either 10 g/L YE or 10 g/L

SBE. Sridee et al. [12] compared some nutrients and trace elements in commercial yeast

extract, (HiMedia laboratory, India) and DSB (Beerthip Brewery (1991) Co., Ltd.,

Thailand). They found that DSB contained much higher amount of trace elements such

as calcium, magnesium and iron, whereas sodium and chloride contents were lower than

that of commercial yeast extract. These higher of trace elements content in DSB may

benefit to the growth microorganism and therefore significantly accelerated the rate of

biomass formation and succinate production [12]. As for biomass formation, it was

revealed that the biomass formation was increased by the increased of DSB

concentration from 5 to 50 g/L. This result is acceptable since DSB contained high

amount of protein, defined as total nitrogen content (Table 1). It has been shown that A.

succinogenes is a fastidious microorganism and nitrogen source supplied important

growth factors for succinate production [13]. It is implied that DSB contains various

amino acids, vitamins, minerals, and growth factors thus promoting growth of

microorganisms. Similar result was observed by Jiang et al. [14] who used fresh spent

brewer’s yeast hydrolysate as extra nitrogen source in succinate production by A.

succinogenes NJ113. They found that the fresh spent brewer’s yeast hydrolysate

significantly accelerates the rate of biomass formation and succinate production. In

Thailand, the cost of YE (Bio-basic INC, bacteriological grade, Canada), and DSB

(Khon Kaen Brewery, Co., Ltd., Thailand) was approximately 160.5 and 0.36 US$/kg,

respectively. Moreover, the application of DSB is still limited, being basically used as

animal feed [15]. From this result, it is likely that the high cost of commercial yeast

extract could be reduced when the fermentation medium was supplemented by DSB.

This finding indicated that supplementing of culture medium with DSB as nitrogen

6


source at concentration as 30 g/L was the most promising substrate for commercial

succinate production.

Fig. 1 Effect of various concentration of yeast extract on succinate production by A.

succinogenes in 100-mL anaerobic bottle. Abbreviations: YE, commercial yeast extract,

SBE, spent brewer’s yeast extract, DSB, dried spent brewer’s yeast cell. Different

superscripts are significantly different (p


utilization rate were found in the fermentation without providing of CO2, which was

about 5 times lower when compared to that of the fermentation with non-limiting CO2

supply (Fig. 2A and 2B). Similarly, Samuelov et al. [16] and Lu et al. [17] reported that

succinate production was enhanced by adding of CO2 gas during the fermentation of

rumen bacteria. Also, Xi et al. [18] found that higher succinate production by A.

succinogenes NJ113 was obtained at a higher supply of CO2 level. They suggested that

CO2 is not only a substrate for succinate production, but it is also necessary for cell

growth. On the other hand, external CO2 supply had a negative effect on

Anaerobiospirillum succiniciproducens growth, while it had a somewhat positive effect

on succinate production [19]. Additionally, Vemuri et al. [20] explained that succinate

is principally formed through two pathways: the reductive arm of the TCA cycle and the

glyoxylate shunt. Through the reductive arm of the TCA cycle CO2 is incorporated into

the final product succinate via the enzyme phosphoenolpyruvate carboxylase. This gas

is therefore necessary for succinate production, and CO2 availability impacts substrate

utilization and succinate accumulation rates. Furthermore, CO2 functions as an electron

acceptor and alters the flux of phosphoenolpyruvate, which metabolizes to pyruvate and

lactate/ethanol at low CO2 levels but makes succinate at high CO2 concentration [2].

Fig. 2 Effect of carbon dioxide gas on succinate production by A. succinogenes 130ZT

under pH controlled at 6.8 in 2-L fermenter. Glucose concentration used was 50 g/L.

(A) succinate production; (B) glucose utilization; (C) biomass production.

Effect of different controlled pH on succinate production

Since the culture pH can affect the activity of key enzyme for CO2 fixation in succinate

fermentation by succinate-producing rumen bacteria. Fermentations were performed at

three pH levels (pH 6.2, 6.8, and 7.2) to confirm the optimum pH for succinate

C

0 12 24 36 48 60 720.0

0.5

1.0

1.5

2.0

Partly CO2No CO2Non-limiting CO2

Fermentation time (h)

Bio

mas

s (g

/L)

0 12 24 36 48 60 720

5

10

15

20

25

30

35 APartly CO2Non-limiting CO2No CO2

Suc

cina

te (

g/L)

B

0 12 24 36 48 60 720

10

20

30

40

50

No CO2

Partly CO2Non-limiting CO2G

luco

se (

g/Ll

)

Fermentation time (h) Fermentation time (h) Fermentation time (h)

8


production and to investigate their effect on cell growth. As shown in Table 2, succinate

production was maximized at pH 6.8. The concentration of succinate increased from

23.6 g/L to 31.11 g/L when pH increased from 6.2 to 6.8 and slightly decreased

thereafter when a controlled pH 7.2 was applied. The highest succinate yields as 0.76

(g/g glucose) and productivity as 0.54 g/L/h, were also obtained at the controlled pH

6.8. Although, the succinate yield and productivity at pH 7.2 and 6.8 were not

significantly different (p


concentration of 32.11 g/L was observed as a major fermentative product while 6.11 g/L

acetate, 3.55 g/L formate, and 0.06 g/L ethanol were also detected.

Fig. 3 Time course of glucose fermentation by A. succinogenes 130ZT in 2-L fermenter

under non-limitation CO2 supply (0.5 vvm CO2 sparging) and controlled pH at 6.8.

Glucose and DSB concentration used were 50 g/L and 30 g/L, respectively.

Succinate production from different concentrations of sugarcane syrup

Sugarcane syrup is an inexpensive and readily available carbon source for a next target

bio-conversion of fermentative succinate. The production of succinate increased with

increasing of sugarcane syrup concentration from 1 to 15% (w/v) (Fig. 4). The

maximum values of succinate concentration (35.7 g/L) and biomass formation (2.08

g/L) were obtained in the culture with initial sugarcane syrup at the concentration of

15% (w/v). However, the succinate concentration along with biomass formation was

decreased when initial concentration of sugarcane syrup at 20% (w/v) was used. Similar

phenomenon was observed by Lin et al. [22] who demonstrated that the high level of

glucose concentration over 100 g/L was lower the succinate level. Additionally,

Kotzamanidis et al. [23] discussed that the decreased sugar utilization encountered with

high sugar concentration was due to the osmotic effects during the lactic acid

fermentation from beet molasses by Lactobacillus delbrueckii NCIMB8130. In this

study, it is likely that the cell growth and succinate concentration were also inhibited

when high concentration of sugarcane syrup as 20% (w/v) was used. It might be due to

substrate inhibition. For biomass formation, the maximum biomass was obtained at 24 h

in all fermentations of various sugarcane syrup concentrations. It also found that the

0 12 24 36 48 60 720

10

20

30

40

50

0.0

0.5

1.0

1.5

2.0

2.5

Glucose

SuccinateAcetateFormate

Biomass

Fermentation time (h)

Glu

cose

,or

gani

c ac

ids,

(g/L

) Biom

ass (g/L)

10


titers of succinate at the end of fermentation from 10 to 15% (w/v) sugarcane syrup used

were not significantly different. Even though, higher succinate (35.70 g/L) was

produced from sugarcane syrup at 15%, w/v the high residual sugars (37.5 g/L) was

obtained compared with the residue sugars of 5.8 g/L (from the initial concentration of

10%, w/v sugarcane syrup) (Fig. 4). Since the higher amount of sugars leftover at the

end of fermentation may increase the cost of succinate purification in downstream

processing. Therefore, considering economically, the optimal concentration of

sugarcane syrup would be at 10% (w/v).

Fig. 4 Effect of different concentrations of sugarcane syrup supplemented with 30 g/L

DSB on succinate production by A. succinogenes at 72 h incubation time under non-

limitation CO2 supply (0.5 vvm CO2 sparging) at controlled pH 6.8. Biomass presented

was at 24 h incubation time. *Total sugar residual is consisting of glucose, fructose, and

sucrose.

Considering the price of sugarcane syrup (containing 76% total sugars) and glucose,

they were about 0.53 and 2.67 US$/kg, respectively. Assuming the succinate yield of

80% (w/w) total sugar of sugarcane syrup (or glucose), the raw material cost of the

bioprocess was therefore estimated to be 1.33 and 3.34 US$/kg succinate. According to

the downstream purification cost accounts for 60-70% of the product cost [12], the total

raw material cost of producing succinate from sugarcane syrup (or glucose) would be

about 2.26 US$/kg succinate (or 5.68 US$/kg succinate), indicating the cost saving

around 60.21%. Therefore, it is clear that the cost of succinate production from

sugarcane syrup was much lower than that from glucose.

1 5 10 15 200

10

20

30

40

50

60

70

80

0.0

0.5

1.0

1.5

2.0

2.5

Succinate Biomass Total sugars residual

a a

cb

d

Sugarcane syrup (%, w/v)

Succ

inat

e,to

tal s

ugar

s (g

/L) B

iomass (g/L)

11


Conclusion

This study demonstrated that the by-product from brewery industry, DSB could be

successfully used as a low-cost nutrient supplement instead of commercial yeast extract

for batch succinate fermentation by A. succinogenes 130ZT. The succinate production

by A. succinogenes 130ZT was enhanced by supplying of CO2 during fermentation in

which the succinate fermentation was 5 times higher than that of those without CO2 supplementation. In term of the controlled pH investigation, the highest succinate yield

of 0.76 (g/g glucose) was obtained at controlled pH at 6.8. Further, the optimized

concentrations of sugarcane syrup, was observed at 10% (w/v) in which the succinate

production at 35.75 g/L was obtained. Based on these results, the cost effectiveness of

succinate production could be obtained from sugarcane syrup fermentation

supplemented with the DSB.

Acknowledgements

The authors thank Khon Kaen Brewery Company Ltd., Thailand for generously

supplying the DSB and Mitr Phol Sugar Corp., Ltd., Thailand for providing the

sugarcane syrup. The experimental set-up facilities were kindly provided at the

Metabolic Engineering Research Unit, School of Biotechnology, Suranaree University

of Technology, Thailand. In addition, the authors gratefully acknowledge financial

support from the Faculty of Sciences and Liberal Arts, Rajamangala University of

Technology Isan (Nakhonratchasima), Thailand.

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http://job-search.jobstreet.co.th/thailand/company/mitr-phol-sugar-corp-ltd-jobs/


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organic acids: expanding the markets. Trends Biotechnol. 2008; 26, 100-108.

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[5] Limtong S. Sringiew C. and Yongmanitchai W. Production of fuel ethanol at

high temperature from sugar cane juice by a newly isolated Kluyveromyces

marxianus. Bioresour. Technol. 2007; 98, 3367-3374.

[6] Timbuntam W. Sriroth K. and Tokiwa Y. Lactic acid production from sugar-

cane juice by a newly isolated Lactobacillus sp. Biotechnol. Lett. 2006; 28, 811-

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[7] Agarwal L. Isar J. Meghwanshi G. K. and Saxena R. K. A cost effective

fermentative production of succinic acid from cane molasses and corn steep

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[8] Xi Y. L. Chen K. Q. Dai W. Y. Ma J. F. Zhang M. Jiang M. Wei P. and Ouyang

P. K. Succinic acid production by Actinobacillus succinogenes NJ113 using corn

steep liquor powder as nitrogen source. Bioresour. Technol. 2013; 136, 775-779.

[9] Li J. Zheng X. Y. Fang, X. J. Liu S. W. Chen K. Q. Jiang M. Wei P. and Ouyang

P. K. A complete industrial system for economical succinic acid production by

Actinobacillus succinogenes. Bioresour. Technol. 2011; 102, 6147-6152.

[10] Sawisit A. Seesan S. Chan S. Kanchanatawee S. Jantama S. S. and Jantama K.

Validation of fermentative parameters for efficient succinate production in batch

operation by Actinobacillus succinogenes 130ZT. Adv. Mater. Res. 2012; 550-

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[13] Liu Y. P. Zheng P. Sun Z. H. Ni Y. Dong J. J. and Zhu L. L. Economical

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13


[14] Jiang M. Chen K. Liu Z. Wei P. Ying H. and Chang H. Succinic acid production

by Actinobacillus succinogenes using spent brewer's yeast hydrolysate as a

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14


Preferences and Factors Influencing the Purchase Intention of Healthy

Snacks among Millennials in Jakarta

Grace Aurelia Kahono1, Della Rahmawati1*, I Kadek Putra Yudha Prawira2,

Maria D.P.T. Gunawan Puteri1, Abdullah Muzi Marpaung1

1Department of Food Technology, Faculty of Life Sciences, Swiss German University, Alam Sutera,

Tangerang, Banten 15143 Indonesia 2Department of Food Science and Technology, Faculty of Agricultural Technology, Bogor Agricultural

University, Jl. Raya Dramaga Bogor, 16680 West Java, Indonesia

*Corresponding author: [email protected]

Abstract

Trend of healthy eating has been increasing almost over all region in the world,

including Indonesia. More than half (75%) of urban Indonesians are willing to have

healthier diet. This growing trend will influence food industries in coming years,

including snack industries in Indonesia, since Indonesia is the country with the biggest

snacking habit in Asia Pacific. The snacking habit is popular with all ages, especially

Millennials. Nowadays, Millennials have been shifting their snacking behaviors into a

healthier snack consumption more rapidly than any other groups. The objective of this

study was to know the preferences and the factors influencing the purchase intention of

healthy snack among Millennials in Jakarta, as Jakarta is the capital city of Indonesia

and it covers urban population. The study was carried out by market survey to 475

Millennials in Jakarta who had interest in consuming healthy snack in Jakarta and the

data was analyzed using Friedman Test and Wilcoxon Signed-Rank Test. The research

revealed that yogurt was the preference of healthy snacks among Millennials in Jakarta,

compared to healthy biscuit/ cookies, snack bar, fruit and vegetable (including their

products). In addition, taste became the most influential intrinsic factor, while price and

convenience were the most influential extrinsic factors to the purchase intention of

healthy snacks among them.

Keywords: Healthy Snack, Intrinsic Factors, Extrinsic Factors, Purchase Intention,

Millennials 15

DOI:10.14457/MSU.res.2018.40

mailto:[email protected]


Introduction

Indonesia is the country with the biggest snacking habit in Asia Pacific. Twenty four

percent of Indonesians are snacking on a daily basis, also known as ‘Snack Frenzy’ [1].

A snacking habit is popular with all ages, especially Millennials, people who were born

between 1981-1996 [2]. Consumer research by Mintel (2016) showed that 77% of

Millennials eat snacks every day, a higher proportion than any other age group. The

reasons for snacking are varied; hunger, for an energy boost, to save time, boredom, and

others. The importance of snacking gives opportunity for food companies to develop

snack products, especially for young consumers.

Nowadays, Millennials have been shifting their snacking behaviors into a healthier

snack consumption more rapidly than any other age groups [3]. The shifting pattern in

snack consumption has been driven by the growing trend, ‘healthy eating’, which is also

led by Millennials [4]. The trend is believed to keep growing in the future due to better

level of knowledge and education about health and healthy eating [5]. This growing

trend will influence the food industry in coming years, as more people will be interested

in adopting healthy snacking habits along with the convenience and the availability [6].

The global healthy snack market has been annually rising every year [7].

The trend of healthy snacking is also entering Indonesia as Indonesians start to have

awareness of the importance of a healthy diet. The majority of urban Indonesians (75%)

are willing to have healthier diet, such as avoiding refined sugar, consuming more food

with high protein, and others [8]. According to Snapcart Indonesia, Indonesians

consumers are disappointed by the lack of healthy snacks option in the market.

Therefore, the trend of snacking and healthy diet in Indonesia become a good

opportunity for snack food producers to develop healthy snack products in Indonesia

[9].

However, Nielsen Survey in 60 countries revealed that each country has specific

preferences of healthy snack, and thus food industry needs to consider local tastes when

developing healthy snacks [10]. Besides preferences, today’s consumers also consider

several factors in choosing healthy snacks, such as taste, nutrition, healthiness, and

16


convenience. These influential factors can be divided into two categories; intrinsic and

extrinsic characteristics [11].

Based on previous researches, this research will give information about the preference

and the most influential intrinsic and extrinsic factors for consumers’ purchase intention

towards healthy snack products among Millennials of Jakarta that may help the food

industry to develop healthy snacks.


The method used is by distributing the 5-point Likert scale online questionnaire to 475

respondents. The questionnaire consists of three sections; preferences of healthy snacks,

intrinsic factors, and extrinsic factors. There are 4 questions regarding preferences of

healthy snacks where the respondents are asked to rate their interest towards four types

of healthy snacks; yogurt, healthy cookies, snack bar, fruit and vegetable (including

their products). There are 5 questions regarding intrinsic factors where the respondents

are asked towards the importance of five intrinsic factors; taste, texture, appearance,

ingredients, and aroma. There are 7 questions regarding extrinsic factors where the

respondents are asked towards the importance of seven extrinsic factors; price,

packaging, convenience, advertising, label, packaging, and environment. All

information and questions in the questionnaire are based on in-depth interview to

several experts, literature review, and previous researches.

Slovin Formula was used to determine the number of respondents required in this study

[12]. The validity and reliability of the questionnaire is tested by Pearson Product

Moment Correlation Test and Cronbach’s Alpha. The data from the market survey is

being analyzed using Friedman Test and Wilcoxon Signed-Rank Test.

Results and discussion

Based on Friedman Test, p value preferences of healthy snack, intrinsic factors, and

extrinsic factors are less than 0.05. Therefore, Wilcoxon Test is performed to know in

which group the difference exist.

17


Preferences of Healthy Snacks

Based on Friedman Test, preference of healthy snacks has p value of 0.000, which is

less than 0.05. Therefore, there are preference healthy snack different among

Millennials in Jakarta.

Table 1. Ranks Preferences of Healthy Snacks

Preferences of Healthy Snacks Value*

Yogurt (P1) 2.75

Healthy cookies (P2) 2.60

Snack bar (P3) 2.47

Fruit, vegetable products (P4) 2.18

*the highest value means the most preferred healthy snack

*N= 475, value= mean

Based on the ranks (Table 1), yogurt has the highest rank which means that it is more

preferred than other healthy snacks; healthy cookies, snack bar, and fruit and vegetable

(including their products). Further analysis was conducted by Wilcoxon Test to know

whether there is significant preference different between yogurt and other healthy

snacks (Table 2). The p value which is less than 0.05 indicates that there is significant

preference different between two healthy products. The result is yogurta, healthy

cookiesb, snack barb, fruit and vegetable productsc.

Table 2. Wilcoxon Test of Preferences of Healthy Snacks

Preferences of Healthy Snacks P1 P2 P3 P4

P1 - 0.027* 0.001* 0.000*

P2 0.027* - 0.066 0.000*

P3 0.001* 0.066 - 0.000*

P4 0.000* 0.000* 0.000* -

*p value < 0.05, there was significant different.

The result of the research which indicated that yogurt is more preferred than healthy

biscuit/ cookies, snack bar, and fruit and vegetable (including their products) is

18


supported by fact that demand of dairy products is increasing in Indonesia. Dairy

products, such as yogurt, sour milk, cheese held the second largest market share in

Indonesia in 2013 [8].

Intrinsic Factors

Based on Friedman Test, intrinsic factors have p value of 0.000, which is less than 0.05.

Therefore, there are intrinsic factor different among Millennials in Jakarta.

Table 3. Ranks Intrinsic Factors

Intrinsic Factors Value*

Taste (I1) 3.63

Ingredients (I2) 3.14

Appearance (I3) 2.80

Texture (I4) 2.78

Aroma (I5) 2.66



Table 4. Wilcoxon Test of Intrinsic Factors

Intrinsic Factors I1 I2 I3 I4 I5

I1 - 0.000* 0.000* 0.000* 0.000*

I2 0.000* - 0.001* 0.002* 0.000*

I3 0.000* 0.001* - 0.996 0.015*

I4 0.000* 0.002* 0.996 - 0.022*

I5 0.000* 0.000* 0.015* 0.022* -


Based on ranks (Table 3), taste has the highest rank, followed by ingredients,

appearance, texture, and aroma. Wilcoxon Signed-Rank Test was performed to know

whether there is significant intrinsic factor different between taste and others (Table 4).

The p value which is less than 0.05 indicates that there is significant intrinsic factor

19


different. The result from Wilcoxon Test is tastea, ingredientsb, appearancec, texturec,

and aromad.

The result of this research which indicated that taste is the most influential intrinsic

factor is in accordance with previous researches [11]. The result could be explained by

the fact that human behavior towards food consumption is strongly influenced by the

effects of taste and flavor as it brings pleasure value to them [10, 13].

Extrinsic Factors

Based on Friedman Test, extrinsic factors have p value of 0.000, which is less than 0.05.

Therefore, there are intrinsic factor different among Millennials in Jakarta.

Table 5. Ranks Extrinsic Factors

Extrinsic Factors Value*

Price (E1) 4.69

Convenience (E2) 4.67

Brand (E3) 4.07

Packaging (E4) 3.96

Environment (E5) 3.68

Label (E6) 3.55

Advertising (E7) 3.38



Based on ranks for extrinsic factors (Table 5), price gets the highest rank, followed by

convenience, brand, packaging, environment, label, and advertising. Wilcoxon Signed-

Rank Test was conducted to know whether there is significant extrinsic factor different

between price and others (Table 6). The p value which is less than 0.05 indicates that

there is significant extrinsic factor different. The result is pricea, conveniencea, brandb,

packagingb, environmentc, labelcd and advertisingd.

20


Based on the result, price and convenience are the most influential extrinsic factors are

in accordance with the previous research [11, 14]. Price is more important than brand in

choosing snacks [15], because for most people, price is highly associated with products

quality.

Table 6. Wilcoxon Test of Extrinsic Factors

Extrinsic

Factors E1 E2 E3 E4 E5 E6 E7

E1 - 0.250 0.000* 0.000* 0.000* 0.001* 0.002*

E2 0.240 - 0.000* 0.000* 0.000* 0.000* 0.000*

E3 0.000* 0.000* - 0.096 0.000* 0.000* 0.000*

E4 0.000* 0.000* 0.096 - 0.010* 0.000* 0.000*

E5 0.000* 0.000* 0.000* 0.010* - 0.123 0.044*

E6 0.001* 0.000* 0.000* 0.000* 0.123 - 0.653

E7 0.002* 0.000* 0.000* 0.000* 0.044* 0.653 -


Nowadays, convenience is important, especially among young population of urban

Indonesians. They are adopted to easy-to-eat food due to the changing lifestyle where

people need to reduce preparation and eating time, and thus increased the necessity of

convenience food. Due to price consideration and convenience, smaller package sizes

are often preferred since it has cheaper price and has less weight (lighter) [8].

Conclusions

Yogurt is the most preferred or the favorite healthy snacks among Millennials in Jakarta

compared to healthy biscuit/ cookies, snack bar, and fruit and vegetable (including their

products). Taste becomes the most influential intrinsic factor, while price and

convenience become the most influential extrinsic factors in the purchase intention of

healthy snacks among them.

21


References [1] YouGov. Asian Snacking Habit, Available at: https://my.yougov.com/en-

my/news/2015/04/29/asian-snacking-habit/, accessed November 2017.

[2] Pew Research Center. Defining Generations: Where Millennials End and Post-

Millennials Begin. Available at: http://www.pewresearch.org/, accessed

February 2018

[3] Amplify Snack Brands. Better-For-You Snacks: The New Snacking Reality,

available at:https://amplifysnackbrands.com/documents/Amplify-2017-Snack-

Study.PDF, accessed November 2017

[4] The Nielsen Company. We Are What We Eat Healthy Eating Trends: Around

The World. Available at: http://www.nielsen.com/content/, accessed December

2017

[5] Lappo A, Bjørndal T, Polanco J.F, Lem A. Consumers Concerns and External

Drivers in Food Market. FAO Fisheries and Aquaculture Circular No 1102.

2015, 1-17

[6] Deloitte. Food and beverage 2012: a taste of things to come. Available at:

http://www.deloitte.com/, accessed November 2017

[7] Grand View Research. Industy Analysis of Healthy Snack Market. Available at:

http://www.grandviewresearch.com/industry-analysis/healthy-snack-market,

accessed November 2017

[8] European Union. The Food and Beverage Market Entry Handbook: Indonesia.

2016

[9] Snapcart Indonesia. Indonesian and Their Snacking Habit 2018. Available at:

https:snapcart.global/indonesians-and-their-snacking-habits/, accessed January

2018

[10] The Nielsen Company. Understand The Why Before The Buy of Snacking:

Around The World. Available at: http://www.nielsen.com/content/, accessed

January 2018

[11] Forbes, S. L., Kahlya, E., and Balderstone, C. Analysis of Snack Food

Purchasing and Consumption Behavior. Journal of Food Products Marketing.

2015, DOI: 10.1080/10454446.2014.949992

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https://my.yougov.com/en-https://my.yougov.com/en-http://www.pewresearch.org/https://amplifysnackbrands.com/documents/Amplify-2017-Snack-Study.PDFhttps://amplifysnackbrands.com/documents/Amplify-2017-Snack-Study.PDFhttp://www.nielsen.com/content/http://www.deloitte.com/http://www.grandviewresearch.com/industry-analysis/healthy-snack-markethttp://www.nielsen.com/content/


[12] Stephanie E. Slovin’s Formula Sampling Techniques. Available at: https://

sciencing.com/slovins-formula-sampling-techniques-5475547.html, accessed

February 2018

[13] Brunsø K, Fjord T.A, Grunert K.G. Consumers’ Food Choice and Quality

Perception. Working paper no 77. 2002, 5-54.

[14] Li X.E, Jervis S.M, Drake M.A. Examining Intrinsic Factors That Influence

Product Acceptance: A Review. Journal of Food Science. 2015; 5, 901-909

[15] Mintel. Executive Summary: Chips, Popcorn, Nuts, and Dips. Available at:

http://oxygen.mintel.com/display/680843/, accessed April 2018

23

http://oxygen.mintel.com/display/680843/


Impact of Concentration of Glucono Delta Lactone (GDL), Tempe

Starter and Soaking Time to Physical Characteristics of Tempe and

Overripe Tempe

Irvan Setiadi Kartawiria1, Maria D.P.T. Gunawan-Puteri2*, Chita S. Putrianti2

1Chemical Engineering Department, Faculty of Life Sciences and Technology, Swiss German University,

Tangerang, Indonesia 15143

2Food Technology Department, Faculty of Life Sciences and Technology, Swiss German University,

Tangerang, Indonesia 15143 *Corresponding author: [email protected]

Abstract

Tempe production requires 48 h of fermentation and another 72 h for overripe tempe.

Adding Glucono Delta-Lactone (GDL) in soaking process could reduce the time,

however appropriate concentration of GDL, tempe starter and length of soaking time are

still unknown. In this study, tempe and overripe tempe were produced with variations of

GDL concentration (4 g/l, 12 g/l, 20 g/l, 28 g/l and 36 g/l), tempe starter (2 g/kg, 3 g/kg,

4 g/kg) and soaking time (60 min, 90 min, 120 min, 150 min, 180 min). The best

applications were selected based on the time required for fermentation from fresh tempe

to overripe tempe and from visual, colorimeter evaluation for color and texture

characteristics using penetrometer. As result, soaking with 36 g/l GDL concentration for

180 min with 4 g/kg tempe starter produced firm, full coverage of white mycelium and

requires 54 h – 72 h to be overripe. Tempe firmness texture was observed having value

of 25.53 ± 2.31 N, tempe color index value was determined as: L* 75.76±4.85; a*

7.59±0.93; b* 19.08±1.62. Whereas, soaking with 36 g/l GDL concentration for 120

min with 4 g/kg tempe starter produced firm but visible grains and required 30 – 36 h to

be overripe. Texture observed was 8.96±1.78 N, color L* 50.39±5.61; a* 10.75±0.69;

b* 25.90±1.73. Best application for fresh tempe is 36 g/l GDL for 180 min with 4 g/kg

starter; while for overripe tempe is 36 g/l GDL for 120 min with 4 g/kg starter. The

protein profile compared shown significant different of the protein content, soluble

amino acid and protein digestibility.

24

DOI:10.14457/MSU.res.2018.32


Keywords: Tempe, Overripe Tempe, Glucono Delta-Lactone, Soaking Time, Protein

Introduction

Tempe is a traditional fermented food product originated from Java, Indonesia, that

made from soybean seeds by using Rhizopus spp. into a compact solid form with

slightly gray-ish white color and has distinctive tempe aroma [1]. After inoculation, the

fermentation process for tempe requires up to 48 hours (two days) and 120 hours (five

days) for overripe tempe [2]. The mold fermentation which occur on tempe production

is stop and taken over by the bacteria for further fermentation causing white mycelium

on the fresh tempe into yellowish-brown velvety surface with softer consistency [3]–[5].

During the bacteria fermentation, proteins are hydrolysed which increases the amount of

nitrogen, while fat are also hydrolysed causing pungent odor and softer texture [6].

There are 17 amino acids occurred in overripe tempe, with glutamic acid is the most

abundant amino acid [7]. Overripe tempe has been found that it could be used as food

ingredients and flavour enhancer that has natural ‘umami’ flavour due to the glutamic

acid [8]. Glutamic acid was found to be the important substance for umami taste and has

been used for savory seasonings around the world, although its taste is masked by

flavors from fat or herbs [3].

The problem of long production time, especially in soybean soaking steps before

fermentation could take place, has been of interest in many studies. Addition of

Glucono Delta-Lactone (GDL) as chemically acidifying agent in the soaking process

could reduce total production time up to 12 hours, which took 36 hours of fermentation

with no changes of taste and aroma of tempe due to the odorless and tasteless character

of the GDL solutions [5], [9], [10]. GDL itself had been recognized as safe

(GRAS/Generally Recognized as Safe) and had usually been used in silken tofu

production [11]. GDL could also save water usage during production, decrease water

waste, and produce more environmentally friendly waste [9].

The exact formula for implementation of GDL in tempe production, however, is varied

between studies with broad range. It is important to determine an appropriate range of

25


GDL concentration used for soaking the soybean, especially due to the price of GDL

that might have significant impact to production cost of tempe. It is also important to

understand how the GDL solution is affecting the tempe itself in relation to rate of

fermentation to produce fresh tempe and further fermentation to overripe tempe. This

study objectives were to obtain the appropriate concentration of GDL in tempe and

overripe tempe production, the length of soaking time, and the amount of tempe starter

needed in such production. Characteristics of tempe produced were observed as texture,

color, and the protein profile. By understanding this basic production parameters,

further studies related to implementation of GDL soaking process in tempe making

could be explored.


Raw material of soybean was obtained from local market in Tangerang (Indonesia),

while tempe starter used was Raprima (RTI, Bogor, Indonesia). Soaking of soybean was

done by diluting variable amount of GDL (Tristar Chem, Surabaya, Indonesia), from 4

to 36 g in one liter of water. Standard tempe making was used based on procedure set

by Rumah Tempe Indonesia (RTI, Bogor, Indonesia).

The determination of GDL concentration was done by diluting 4, 12, 20, 28, and 36 g of

GDL in one liter of water and used in soaking of soybean for 120 minutes, followed by

tempe fermentation using 2 g of tempe starter per kg of dry soybean. Observations were

made by measuring the texture using GY-4 Fruit Penetrometer Total (NK HP Series,

China) for firmness, and determining the color using colorimeter colorimeter PCE-

SCM7 (England).

Further step was determining the appropriate amount of tempe starter, by implementing

the result from GDL concentration determination experiment. The amount of tempe

starter was increased from 2 g/kg dry soybean to 3 g/kg and 4 g/kg dry soybean. The

resulted tempe was observed of its texture, color, and time to reach the fresh tempe and

time needed to reach overripe tempe state. Short time of tempe fermentation indicates

that the process is suitable for fresh tempe production, while the time required to reach

overripe tempe indicates it suitability of overripe tempe production. 26


After the amount of GDL concentration and amount of starter were determined, the

process was repeated to determine the appropriate soaking time to produce fresh tempe

and overripe tempe. The soaking time variables were 60, 90, 120, 150, and 180 minutes

in predetermined GDL concentration, followed by fermentation using predetermined

amount of tempe starter. The tempe firmness texture, color, and protein characteristics

were measured. Protein was determined using Lowry method [10] by Biuret and Folin-

Ciocalteu reagent (Merck, Germany). A 0.3 ml of sample extract was mixed

homogeneously with 1.5 ml biuret reagent and was left for 10 minutes in room

temperature. Then, 75 μl Folin-ciocalteu (1:2) reagent was added, homogenized and

incubated in room temperature for 30 minutes. The samples were analyzed using

spectrophotometer (GENESYS 10S UV-Vis, Thermo Scientific, USA) at wavelength of

650 nm. The result was recorded and interpolated with the standard curve using BSA

(Sigma-Aldrich, Germany) as the substrate. Soluble amino acid was determined by

preparing sample added with 500 μl distilled water and with phosphate buffer pH 8. The

solutions were incubated for five min at 37°C. Then 750 μl 10% of TCA (Merck,

Germany) was added and centrifuged at 10.000 rpm for 10 minutes (Rotina 35R

Hettlich, Germany). 300 μl supernatant was collected, followed with the addition of

1000 μl 0.5M Na2CO3 (Merck, Germany) and 200 μl Folin-Ciocalteu reagent. The

mixtures were analysed using UV-Vis spectrophotometer at 578nm against tyrosine

standard curve. Protein digestibility was measured by digesting the protein using

porcine pancreatin solution (Sigma-Aldrich, Germany) for 5 minutes in 37 oC. The

protein after digestion was measured in similar method using spectrophotometer at

578nm against tyrosine standard curve.

Statistical analysis using t-Test (Paired Two Sample for Means, p


in Table 1. This definition is important because the Indonesian National Standard for

tempe only shows qualitative reference of color and texture [1].

(A) (B)

Fig.1 Standard reference tempe produced by natural acidification;

(A) fresh tempe; (B) overripe tempe

Table 1. Texture and color of standard reference tempe

Standard Firmness Texture (N) Color

L* a* b*

Fresh Tempe (FT) 14.79 – 21.39 55.53 – 84.11 4.18 – 9.55 11.03 – 26.74

Overripe Tempe (OT) 10.11 – 24.09 31.76 – 61.92 8.18 – 12.05 17.87 – 33.11

GDL Concentration Determination

Tempe was processed in five different variations of GDL concentration, ranging

between 4 g/l to 40 g/l, which was based on previous researches. GDL was once used

with range concentration 0.4% - 1.4% or 4 g/l – 14 g/l [12], however tempe made with

concentration 4 g/l had low reproducibility. Another study showed successful tempe and

overripe tempe production with GDL concentration 40 g/l [10]. Therefore, in this study

five points were taken, taking 4 g/l as the minimal and 40 g/l as the maximal, resulting 4

g/l, 12 g/l, 20 g/l, 28 g/l and 36g/l as the points. Upon observation, soybean soaked by

GDL concentration less than 28 g/l have a poor reproducibility in tempe fermentation.

In several repetition, the fungal mycelium was not fully covered the soybean or

unpleasant smell was detected, indicating that the fermentation condition was not

reached through the acidification. There are several purposes of soaking process,

namely to penetrate a desirable pH onto the soybean for the growth of mold later in the

mold fermentation process, which is between pH range 3.5-5.2, and to prevent the

growth of undesired microorganism such as pathogenic bacteria [13], [14]. Variations of

GDL concentrations resulting in different of pH value. As the GDL concentration 28


increases, the pH decreases. The pH of the GDL soaking solutions were more acidic

than the soaking water for natural acidification. However, the chemical acidification

was only held for 120 minutes or two hours whereas the natural acidification is usually

conducted for 24 hours. It is suspected that the cotyledons did not have pH within the

range yet, and it had been stated that acidified beans allowed profuse growth of

pathogenic contaminants [15]. The characteristics of tempe produced by 28 g/l and 36

g/l of GDL solution is presented in Table 2.

Table 2. Characteristics of tempe produced in various GDL concentration

Sample

Code pH ∆t Firmness Texture (N)

Color

L* a* b*

FT-28 3.6 48h 18.99±3.21 69.51±1.16 8.19±0.65 20.24±1.32

OT-28 3.6 11.44±0.76 61.46±1.22 9.03±0.81 24.15±3.10

FT-36 3.4 51h 20.74±2.07 70.36±0.50 7.55±0.45 19.02±0.88

OT-36 3.4 6.53 ± 1.37 47.29±6.00 10.31±1.40 23.93±1.17

Sample Code: FT (fresh tempe); OT (overripe tempe) – XX (concentration of GDL in g/l)

Tempe Starter Determination

GDL concentration of 28 g/l and 36 g/l were used in further processing and the amount

of 3 g/kg and 4g/kg of tempe starter were used. The usual concentration of tempe starter

used in natural tempe is 2 g of inoculum, however the chemically acidified tempe

production with GDL reduced the soaking time, whereas the aim of soaking process is

for acidification or acid fermentation. It allows the growth of lactic acid bacteria which

will lowers the pH of soybean, provide favorable environment and eliminate

undesirable contaminant bacteria. Reducing soaking time might cause a not completed

process of elimination of contaminant bacteria and successful tempe might not be

obtained, thus this stage is expected to overcome that problem by increasing the amount

of inoculum or tempe starter. The result of tempe produced using various tempe starter

is presented in Table 3.

As shown in Table 3, from the visual character that all of the tempe surface were

covered by mycelium and the cotyledons were visible very slightly, which all of the 29


samples were considered as successful tempe. All the color index value were within the

standard range. Whereas, from the texture character, all of the sample were firm and

compact and the cotyledons did not move when pressure were given. However,

analysis using penetrometer, the FT-28-4, FT-36-3, FT-36-4, were harder than the

standard. The tempe starter concentration then selected from the greater time differences

needed from fresh tempe to overripe tempe. Thus, tempe starter concentration of 4 g/kg

dried soybean that gives more than 54 hours of time differences was selected.

Table 3. Characteristics of tempe produced in various amount of tempe starter

Sample Code Firmness Texture (N) Color

L* a* b*

FT-28-3 20.20 ± 2.12 70.41 ± 1.57 7.29 ± 0.59 18.50 ± 1.51

FT-28-4 24.28 ± 1.05 73.60 ± 0.48 7.15 ± 0.72 18.75 ± 1.50

FT-36-3 21.57 ± 1.88 72.34 ± 4.93 7.29 ± 0.58 19.85 ± 1.25

FT-36-4 26.21 ± 1.49 75.27 ± 1.30 7.11 ± 0.12 18.15 ± 0.10

OT-28-3 11.75 ± 0.50 52.86 ± 4.35 10.27 ± 1.22 26.10 ± 0.40

OT-28-4 18.42 ± 0.85 55.36 ± 2.61 10.94 ± 2.84 26.17 ± 0.43

OT-36-3 15.98 ± 1.36 48.77 ± 4.48 10.54 ± 0.02 26.50 ± 2.61

OT-36-4 21.65 ± 6.66 56.20 ± 6.73 12.19 ± 2.08 29.53 ± 2.32

Sample Code: FT (fresh tempe); OT (overripe tempe) – XX (concentration of GDL in g/l) – Y (amount of

starter in g/kg)

Soaking Time Determination

Soybean was processed with the same method as before to produce tempe with the

selected GDL concentration of 28 g/l and 36 g/l and selected tempe starter concentration

4 g/kg, with varied of soaking time, from 60, 90, 120, 150 and 180 minutes. The

soaking time written on the patent is the range of 90-240 minutes [12], but the best

length was not determined.

The soybean soaked in 60 minutes was found to be difficult to be dehulled thus further

process could not be conducted. This soaking time is then eliminated. The 90 minutes

soaking time resulting in hard tempe, that is difficult to be cut using knifes. Based on 30


this observation, the 90 minutes soaking time is also deemed not suitable. Among 120,

150 and 150 minutes of soaking time that resulting in acceptable tempe, the best length

for fresh tempe production was the 180 minutes in 36 g/l GDL concentration, which is

selected based on the ripeness time requirement. It shows the longest time to stand as

fresh tempe (60 hours). This might happen due to the long soaking process with

collaboration of more concentrated solution of of GDL resulted a bigger chance to

lower the pH of soaking water due, make an optimum condition for mold growth

development yet also prevent pathogenic contaminants. The long soaking process also

contributes to an easier de-hulling process, which is important due to the hull is

considered as barrier because it blocks mold enzymes to digest the cotyledons and

resulted into a tender tempe, which in fact, the mycelium will penetrate into several

layers of soybean cells up to 25% of the width of cotyledon [16]. In addition, soaking

also increase the moisture content of the beans up to 62-65% and render the beans

edible [17]. Whereas for the best length for overripe tempe was 120 minutes of soaking

time with also the 36 g/l of GDL concentration. Inversely from the long soaking time,

shorter soaking time could gave insufficient time of pH penetration onto the cotyledons

which further have not prevent the bacterial growth. The time required to reach overripe

tempe from fresh tempe was about 51 hours. The characteristics of tempe produced in

different soaking time is presented in Table 4. The appearance of tempe produced is

presented in Figure 2.

(A) (B) (1) (2)

© (D) (3) (4)

Fig. 2. Tempe produced in various soaking time; (A&B) fresh tempe, soaking time 120

min; (C&D) overripe tempe, 120 min; (1&2) fresh tempe, 180 min; (3&4) overripe

tempe, 180 min

31


Protein Analysis

Protein analysis were conducted on fresh tempe produced using 36 g/l GDL

concentration, 4 g/kg starter and 120 min soaking time and overripe tempe produced

using180 min soaking time to understand the profile and potential as umami flavor

source. Table 5 shows the protein profile, and confirming previous studies, the amount

of soluble amino acid and digestibility is increased in overripe tempe on both methods.

The overripe tempe had bigger soluble amino acid due to the further fermentation

process which occurred on overripe tempe. During the growth of mold, the protein is

hydrolyzed to amino acids and peptides by proteolytic enzymes [18]. Overripe tempe

had been identified that there are amino acids present, which there is 17 different of

amino acids were found and glutamic acid is the most abundant amino acid in overripe

tempeh [7].

Table 4. Characteristics of tempe produced in various soaking time (GDL 36 g/l, starter

4 g/kg)

Sample Code Firmness Texture

(N)

Color

L* a* b*

FT-28-4-120 25.39 ± 1.40 75.56 ± 1.57 7.73 ± 0.48 20.28 ± 1.72

FT-28-4-150 25.05 ± 2.45 74.81 ± 3.66 7.03 ± 0.13 18.00 ± 0.11

FT-28-4-180 26.99 ± 1.43 75.87 ± 4.98 7.40 ± 0.25 19.12 ± 0.55

FT-36-4-120 21.36 ± 2.97 72.82 ± 6.29 8.13 ± 1.19 20.41 ± 2.46

FT-36-4-150 25.85 ± 1.03 75.99 ± 4.36 8.54 ± 1.72 22.23 ± 4.15

FT-36-4-180 25.53 ± 2.31 75.76 ± 4.85 7.59 ± 0.93 19.08 ± 1.62

OT-28-4-120 15.79 ± 3.69 54.13 ± 0.02 11.16 ± 0.13 28.55 ± 0.16

OT-28-4-150 12.82 ± 5.26 47.85 ± 0.59 12.00 ± 1.40 28.23 ± 1.17

OT-28-4-180 12.5 ± 1.95 53.74 ± 1.49 10.34 ± 1.67 27.52 ± 2.09

OT-36-4-120 8.96 ± 1.78 50.39 ± 5.61 10.75 ± 0.69 25.90 ± 1.73

OT-36-4-150 15.68 ± 5.80 51.07 ± 4.86 11.48 ± 2.14 29.23 ± 0.24

OT-36-4-180 17.45 ± 0.27 55.63 ± 0.70 10.70 ± 0.09 31.07 ± 2.82

Sample Code: FT (fresh tempe); OT (overripe tempe) – XX (concentration of GDL in g/l) – Y (amount of

starter in g/kg) – ZZZ (soaking time in minutes)

32


Table 5. The protein profile, and confirming previous studies.

Test Criteria Unit FT-36-4-180 OT-36-4-120

Natural

acidification

tempe [14]

Protein Content

(Lowry)

mg BSA eq/g dry base 70.18±0.48a 76.79±2.29b 55.23±4.94

Soluble Amino

Acid

mg Tyrosine eq/g dry base 387.06±23.83aa 459.66±29.13bb 391.51±35.67aa

Protein

Digestibility

mg Tyrosine eq/g dry base 416.00±46.05aaa 515.97±69.85bbb

Different superscript in the same row shows significant different in results (p


References

[1] Badan Standardisasi Nasional, Standard Nasional Indonesia (SNI) 3144 2015

Tempe Kedelai. Badan Standardisasi Nasional RI, 2015.

[2] J. Hedger, “Production of Tempe, an Indonesian Fermented Food,” University

College of Wales, 1982.

[3] M. D. Gunawan-Puteri, T. R. Hassanein, E. K. Prabawati, C. H. Wijaya, and A.

N. Mutukumira, “Sensory Characteristics of Seasoning Powders from Overripe

Tempeh, a Solid State Fermented Soybean,” Procedia Chem., vol. 14, pp. 263–

269, 2015.

[4] E. Mursito, “Developing Vegetarian Paste Condiment With Overripe Tempe

(Tempe Semangit) as Base Ingredients,” Swiss German University Indonesia,

2015.

[5] W. Shurtleff and A. Aoyagi, History of tempe, a fermented soyfood from

Indonesia. Lafayette: Soyfood Center, 1986.

[6] J. Reu, F. Rombouts, and M. Nout, “Influence of acidity and initial substrate

temperature on germination of Rhizopus oligosporus sporangiospores in tempe

fermentation,” J. Appl. Bacteriol., vol. 78, pp. 200–208, 1995.

[7] Y. Witono, S. Widjanarko, M. Mujianto, and D. Rachmawati, “Amino Acids

Identification of Over Fermented Tempeh, The Hydrolysate and The Seasoning

Product Hydrolysed by Calotropin from Crown Flower (Calotropis gigantea),”

Int. J. Adv. Sci. Eng. Inf. Technol., vol. 5, no. 2, 2015.

[8] B. Setiadharma, I. S. Kartawiria, and M. D. Gunawan-Puteri, “Development of

Instant Stock Cube from Overripe Tempe,” in International Conference on

Innovation, Entrepreneurship, and Technology, 2015.

[9] C. H. Wijaya, “Solusi masalah mutu, lingkungan dan ekonomidengan teknologi

tempe cepat,” Risal. Kebijak. Pertan. dan Lingkung., 2014.

[10] S. Djunaidi, “Acceleration of Overripe Tempe Production Process Using

Glucono Delta-Lactone Application and Control of Environment of

Fermentation,” Swiss German University, 2016.

[11] Sarwono, Membuat Tempe dan Oncom. Jakarta: Penebar Swadaya, 2005.

[12] C. H. Wijaya, “Proses Pembuatan Tempe dengan Pengasam Kimiawi

Menggunakan Glukono Delta Lakton,” PATEN NO IDP000035720, 2007. 34


[13] Hermana and M. Karmini, Pengembangan Teknologi Pembuatan Tempe.

Jakarta: Yayasan Tempe Indonesia, 1996.

[14] S. Djunaidi, M. D. Gunawan-Puteri, C. H. Wijaya, and E. K. Prabawati,

“Physicochemical & Microbial Characterization of Overripe Tempeh,” INSIST,

vol. 2, no. 1, pp. 48–51, 2017.

[15] M. Nout and J. Kiers, “A Review Tempe Fermentation, Innovation, and

Functionality: Update into The Third Millenium,” J. Appl. Microbiol., vol. 98,

no. 4, pp. 789–805, 2005.

[16] T. Varzakas, “Rhizopus oligosporus mycelial penetration and enzyme diffusion

in soya bean Tempe,” Process Biochem., vol. 33, pp. 741–747, 1998.

[17] J. Witoyo, “Perubahan Biokimia Selama Proses ‘Tempe,’” Universitas

Brawijaya, 2015.

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fermentation,” J. Agric. Food Chem., vol. 47, no. 10, pp. 4375–4378, 1999.

35

ICoFAB2018

International Conference on Food, Agriculture and Biotechnology

36

Efficacy of Fresh Herbs Knee Mask Formula to Relieve Knee Pain in

Osteoarthritis Elderly Patients

Jongkol Poonsawat1, Wiwat Sriwicha1,2,*, Saowalak Phankaew2, Pornnipa

Rattanaphook1, Ronnachai Poowanna1, Tanawat Nuangsri1, Benjaporn Mayoon2

1Department of Thai Traditional Medicine, Faculty of Natural Resources, Rajamangala University of

Technology Isan, SakonNakhon Campus, SakonNakhon 47160, Thailand

2LuangPu Fab Supatto, SakonNakhon Thai Traditional Medicine Hospital, Rajamangala University

of Technology Isan, SakonNakhon Campus, SakonNakhon 47160, Thailand

*Corresponding author: [email protected]

Abstract

Efficacy of fresh herbs knee mask formula was investigated to relieve knee pain and

degree of motion in 60 osteoarthritis elderly patients at Ban Dong Kham Pho Health

Promoting Hospital, Waritchaphum District, SakonNakhon Province. The samples were

divided into two groups as (1) patients who received fresh herbs knee mask service, and

(2) patients who did not receive fresh herbs knee mask service. Experimental tools

included knee pain assessment and a goniometer for range of motion. Statistical analyses

were conducted using dependent and independent t-tests. The results showed that initial

knee pain at a scale of 4.87 (S.D.= 1.18). After 5 days of treatment with fresh herbs knee

mask service, knee pain reduc

icofab2018 - techno2.msu.ac.th · international conference on food, agriculture and biotechnology...

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