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Page 1: Preface - Mahasarakham University...10. Assoc. Prof. Dr. Ko-Tung Chang National Pingtung University of Science and Technology, Taiwan 11. Dr. Nicolas Joly Institut Jacques Monod, Universite
Page 2: Preface - Mahasarakham University...10. Assoc. Prof. Dr. Ko-Tung Chang National Pingtung University of Science and Technology, Taiwan 11. Dr. Nicolas Joly Institut Jacques Monod, Universite

IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

Preface

In 2016, the integrated ASEAN Economic Community (AEC) has been officially launched.

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 4th

International Postgraduate Symposium on Food, Agriculture and Biotechnology (IPSFAB

2017) 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

Page 3: Preface - Mahasarakham University...10. Assoc. Prof. Dr. Ko-Tung Chang National Pingtung University of Science and Technology, Taiwan 11. Dr. Nicolas Joly Institut Jacques Monod, Universite

IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

MSU Editorial Board

1. Assoc. Prof. Dr. Anuchita Moongngam 2. Assoc. Prof. Dr. Anut Chantiratikul

3. Asst. Prof. Dr. Sirirat Deeseenthum 4. Asst. Prof. Dr. Pheeraya Chottanom

5. Asst. Prof. Dr. Wasan Duangkhamchan 6. Asst. Prof. Dr. Waranyoo Kaewduangta

7. Asst. Prof. Dr. Wipavee Thaimuangphol 8. Dr. Vijitra Luang-In

9. Dr. Nantaporn Sutthi 10. Dr. Surasak Boontang

11. Dr. Kedsirin Sakwiwatkul

Scientific Committee

1. Prof. He Chaoxing Institute of vegetable and flowers. Chinese

Academy of Agricultural Sciences, China

2. Dr. Xiaoming Song Hangzhou Normal University, China

3. Prof. Dr. ir. Jan Pieters Ghent University, Belgium

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

5. Dr. John Rossiter Imperial College London, UK

6. Prof. Satoru Kondo Chiba University, Japan

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

Life Sciences, Austria

8. Honorary Professor Colin Wrigley QAAFI, University of Queensland, Australia

9. Professor Emeritus Ian Warrington Massey University, New Zealand

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

Technology, Taiwan

11. Dr. Nicolas Joly Institut Jacques Monod, Universite Paris, France

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

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

14. Asst. Prof. Dr. Kitiya Vongkamjan Prince of Songkhla University, Thailand

15. Assoc. Prof. Dr. Maratree Plainsirichai Mahasarakham University, Thailand

16. Assoc. Prof. Dr. Thalisa Yuwa-amornpitak Mahasarakham University, Thailand

17. Assoc. Prof. Dr. Sirithon Siriamornpun Mahasarakham University, Thailand

Page 4: Preface - Mahasarakham University...10. Assoc. Prof. Dr. Ko-Tung Chang National Pingtung University of Science and Technology, Taiwan 11. Dr. Nicolas Joly Institut Jacques Monod, Universite

IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

Content

Full papers: Page

1: Effect of breeds on growth performance in swine ………………………………... 1

Sagesan Techepenrattanakul

2: Effect of pre-treatment processes and stability testing of lemongrass …………….

(Cymbopogon citratus) extract on α-glucosidase inhibitor (AGI)

and α-amylase inhibitor (AAI) activities

10

Diah Widiputri

3: Evaluation of cookies quality enriched with resistant starch type 2 (RS2) and …

resistant starch type 3 (RS3) from banana (Musa paradisiaca formatypica)

21

Mutiara Pratiwi

4: Potential application of overripe tempe dried powder as plant-based instant …...

stock

34

Maria Dewi Puspitasari Tirtaningtyas Gunawan-Puteri

5: Extraction and stability analysis of antioxidant activity from Stenochlaena ……

palustris

45

Della Rahmawati

6: Prevalence of foodborne pathogens in ready-to-eat foods in the markets in …….

Khon Kaen, Thailand

53

Pitchayapa Pholkaw

7: Survival of probiotic bacteria in fruit juice jelly products ………………………. 63

Warangkanang Ampornpat

8: Chemical composition, physical properties, and sensory evaluation of …………

an instant powder beverage containing melatonin prepared from vegetables

71

Wariya Hochin

9: Effect of thermal processings on physical, chemical properties and volatile ……

compounds of coconut (Cocos nucifera L.) sugar

80

Araya Rakphon

10: Species composition of fish in rice fields of That Phanom District, Nakhon ….

Phanom Province, Northeast Thailand

92

Nattanan Tiengtam

Page 5: Preface - Mahasarakham University...10. Assoc. Prof. Dr. Ko-Tung Chang National Pingtung University of Science and Technology, Taiwan 11. Dr. Nicolas Joly Institut Jacques Monod, Universite

IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

Content

Full papers: Page

11: Effect of hydrolyzed Cordyceps militarys on probiotic growth ……………… 100

Surachai Rattanasuk

12: Isolation of protein and nutrient characteristics analysis from lentil …………. 107

Zar Zar Oo

Page 6: Preface - Mahasarakham University...10. Assoc. Prof. Dr. Ko-Tung Chang National Pingtung University of Science and Technology, Taiwan 11. Dr. Nicolas Joly Institut Jacques Monod, Universite

IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

1

Effect of breeds on growth performance and meat quality in swine

Sagesan Techepenrattanakul1, Doungnapa Promket2,*, Songsak Chumpawadee2

1 Graduate student, Animal Science, Department of Agricultural Technology, Faculty of Technology,

Mahasalakham University, Mahasakham, Thailand, 44000.

2 Animal Science, Department of Agricultural Technology, Faculty of Technology, Mahasalakham University,

Mahasakham, Thailand, 44000.

*Corresponding author: [email protected]

Abstract:

The objective of this research was to evaluate the effect of different breeds on growth

performance and meat quality in swine. Swine is another important economic animal. Growth

performance and meat quality traits are economic traits in swine production. If pigs thrive can

sell quickly, reduce production costs. The breed is one of the important factors for growth

performance and meat quality. This study used growth performance and meat quality data from

the commercial farm in different 3 breed pigs (Duroc, Pietrain and Crossbreed). The analysis

effect of breeds on growth performance and meat quality using PROC GLM, predicted

regression linear model using PROC STEPWISE and correlation among growth performance

and meat quality traits using PROC CORR by SAS (1998). The result found that, means of

percent lean (PL, %) and average daily gain (ADG, g/d) were 55.92% and 143.58 g/d,

respectively. The effect of different breeds on growth performance and meat quality was found

for PL, ADG, back fat (BF, cm), loin eye area (LEA, cm2), live weight (LW; kg), and average

daily gain at 104 days (ADG 104 d, g/d) (P<0.01). Breed of Pietrain and Crossbreed pigs were

PL, BF and LEA higher more than Duroc pig. Moreover, Pietrain pig was higher ADG (147.91

g/d) more than Duroc (143.26 g/d) and crossbreed (143.24 g/d). The LW and ADG 104 d found

that Duroc and Crossbreed pigs were higher than Pietrain pig. The result of regression linear

model address that, LW, BF, ADG 104 d and LEA accounted for the greatest amount of

variation of PL (R2 = 0.93). The correlation between ADG 104 d and LW was higher (r = 0.82,

P<0.001). Moreover, the correlation of LEA and PL was (r = 0.81, P<0.01). The conclusion of

this research showed that crossbreed pig was high growth performance.

Keywords: breeds, growth performance, swine

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doi: 10.10.14457/MSU.res.2017.25
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

2

Introduction

The swine is another important economic animals. Growth traits and meat quality are the

importance economic trait in swine production. If pigs thrive can sell quickly, reduce

production costs. The breeds is one of factor important for the growth. Cause crossbreeding is

extensively used in pig production to increase the total efficiency of pig production.

Accordingly, when choosing the best animal crossbreeding strategy, it is important to recognize

that growth and meat quality traits depend on the crossbreed [1, 2, 3]. A number of research

has the objective for improving growth performance and meat quality in swine. [2], study

carcass and meat quality traits of for commercial pig in China show that the DLY (Duroc x

(Landrace x Yorkshire)) and PIC (foreign five-way crossbreed) had heavier live weights more

than LM (Landrace x Meishan) and DLM (Duroc x (Landrace x Meishan)). Evaluation of

Duroc and Pietrain pigs on carcass and meat quality, the result found that Pietrain progeny had

a higher percentage of lean at slaughter more than Duroc pig (52.6 vs. 50.7, P < 0.05) [4].

Moreover, Pietrain progeny had more loin muscle area when compared with the crossbreed pig

(Duroc x Pietrain) [5]. Duroc boars appropriate with a valuable source of genetic material for

improving the carcass and meat quality of finisher pigs [6]. Therefore, our objective of this

study was analysis the effect of breed on growth performance and meat quality in swine.

Materials and methods

Animals

For this study, 3,007 pigs (855 Duroc, 217 Pietrain and 1,935 Crossbreed pigs) from

the commercial farm in Thailand were used in this study. Pig was standard managed according

to commercial conditions until achieved a body weight of approximately 104 kg. All pig was

fed and water ad libitum until slaughtered at the commercial slaughter house.

Growth performance and meat quality traits

The individual pig (year of birth between 2012 - 2016) was weighted (LW) before

slaughter, average daily gain (ADG) and average daily gain at 104 days (ADG 104 d, g/d) were

calculated. Within 45 min post – mortem, back fat thickness (BF, cm) were a measurement of

the first rib and percent lean (PL, %). After chilling at 4 oC, loin eye area (LEA, cm2) were

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

3

place a plastic grid over the loin eye and count the dots or square the fall within the boundaries

of the longissimus muscle convert to square inches by dividing the number of dots or squares

by the appropriate conversion factor on the grid.

Statistical analysis

The means of growth performance and meat quality traits were analyzed using PROC

MEANS [7]. Multiple linear regressions using to predict the equation model by PROC

Stepwise. The prediction model selected was the most right best fit model with a maximum R2

and minimum mean square error (MSE).The correlation among traits used PROC CORR. The

effect of breeds on growth performance and meat quality using the GLM procedure [7]. The

means between variables were considered significantly different at p < 0.05

where:

Results and discussion

Table 1 shows the means standard deviation (SD) minimum and maximum for growth

performance and meat quality in three breeds pig. This study showed the means of PL in three

breeds pig (Duroc, Pietrain and Crossbreed) were 55.44%, 56.15% and 56.12%, respectively.

Means of ADG in all pig was 143.58 g/d. Moreover, means of BF, LEA, LW and ADG 104 d

were 0.88 cm, 35.80 cm2, 105.15 kg and 736.04 g/d, respectively. Mean of ADG 100 d in

Canada Duroc pig was 880 g/d. [8]. [5, 9] report mean of PL of Pietrain and Duroc pig was

55.20% and 56.86 %, which similarly with this study. [10, 11] showed mean of LEA in Duroc

pig were 37.00 cm2 and 36.99 cm2, respectively. Contradictory [4] report mean of LEA in

Pietrain and Duroc pig was 53.2 cm2, 50.2 cm2 respectively. The LEM higher more than

referent [10, 11] because of pig high LW (150 kg). Moreover [12] report mean of ADG 105d,

g/d in Duroc pig was 870 ± 110 g/d. But [13] report mean of BF in Duroc pig was 2.249 cm.

The effect of breeds shown the follows Pietrain and Crossbreed pig were PL, BF and

LEA higher than Duroc pig. The Pietrain breed is known for its high of lean meat [14].

ijiij ebreedy

effectresidualrandomise

CrossbreedandPietrainDurocbreedofeffectfixedtheisbreed

meantheis

qualitymeatandepreformancGrowththeisyij

),(

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

4

Moreover, Pietrain pig was higher ADG more than Duroc and Crossbreed pigs (147.91

g/d, 143.26 g/d and 143.24 g/d, respectively). Duroc and Crossbreed pig were LW and ADG

104 d higher than Pietrain pig (Table2). [9] reported a similar result for carcasses of Pietrain

group had significantly higher percent lean than the Duroc (P<0.001). Duroc pigs had more

back fat than Pietrain pigs. Furthermore, Pietrain had more loin muscle area when compare

with Duroc pig, similar to results from this study [4].

Table 1 The descriptive data of growth performance and meat quality in three breeds pig

Breed Number Variable Mean SD Minimum Maximum

Duroc 855

LW; kg 105.28 5.36 92.00 125.00

ADG, g/d 143.26 8.08 123.28 172.92

ADG 104 d, g/d 738.02 66.5 531.95 968.03

BF, cm 0.91 0.14 0.58 1.47

PL, % 55.44 0.99 52.10 58.46

LEA, cm2 34.96 1.84 30.12 42.52

Pietrain 217

LW; kg 103.43 4.49 90.00 118.00

ADG, g/d 147.92 9.46 125.41 173.20

ADG 104 d, g/d 702.90 63.81 563.17 887.15

BF, cm 0.87 0.15 0.52 1.39

PL, % 56.15 1.03 51.52 59.22

LEA, cm2 36.36 2.20 30.12 43.61

Crossbreed 1935

LW, kg 105.29 5.43 90.00 125.00

ADG, g/d 143.24 8.90 109.92 177.43

ADG 104 d, g/d 738.89 71.50 525.06 994.93

BF, cm 0.87 0.14 0.58 1.57

PL, % 56.12 0.95 52.52 59.60

LEA, cm2 36.12 2.04 30.48 43.51

Total 3,007

LW; kg 105.15 5.36 90.00 125.00

ADG, g/d 143.58 8.79 109.92 177.43

ADG 104 d, g/d 736.04 70.17 525.06 994.93

BF, cm 0.88 0.14 0.52 1.57

PL, % 55.92 1.01 51.52 59.60

LEA, cm2 35.80 2.06 30.12 43.61

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

5

Table 2 The effect of breeds on growth performance and meat quality in swine

Traits Breeds

P-

Value Duroc Pietrain Crossbreed

Mean SE Mean SE Mean SE

LW; kg 105.27a 0.18 103.43b 0.36 105.28a 0.12 **

ADG, g/d 143.26b 0.29 147.91a 0.59 143.24b 0.19 **

ADG 104 d, g/d 738.02a 2.37 702.90b 4.72 738.9a 1.58 **

BF, cm 0.91a 0.004 0.87b 0.00 0.86b 0.003 **

PL, % 55.44b 0.03 56.14a 0.06 56.11a 0.02 **

LEA, cm2 34.95b 0.06 36.36a 0.13 36.12a 0.04 **

** significant different the 0.01 level of probability (P<0.01)

a,b row means with common superscripts do not differ

Multiple linear regression analysis was performed to predict the PL using the data of growth

performance and meat quality in three breeds pig. The highly significant model (R2 = 0.94)

were 2 models (model 3 and model 4), could be obtained by a combination of LW, BF, ADG

104 d and LEA. While, model 2 prediction equations of PL were shown as the dependent

variable and independent variables were BF and LEA (R2 = 0.93). The lowest R2 was model 1

and showed the independent variables was LEA (Table3). [15], predicted live and carcass lean

weight in pig, the result showed the greatest accountability model length R2 was 0.844.

Moreover, in the presence of [16] investigation multiple regression models using theses

parameter resulted showed good predictability of commercial lean cuts weight (R2 = 0.62).

Table 4 shows the correlation of growth performance and meat quality in swine. The correlation

on growth performance and meat quality between -0.86 to 0.82. The result showed the

correlation between LW and ADG 104 d was found the highest correlation (0.82). The highest

correlation of this study showed that, the swine high LW and high ADG 104 d also. In addition,

to high relationship correlation between PL and LEA was 0.81. [17], report 10th-rib back fat

was negative correlated with loin muscle area (-0.23). Moreover, the correlations between

percentage composition in lean and Back fat was negatively relationship (-0.20) [18]. [19],

report percentage lean was negatively correlated with back fat depth and positively correlated

with loin eye depth.

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

6

Table 3 The regression linear model of growth performance and meat quality in swine

Model Dependent

variables

Independent

Variables β P- Value R2 MSE

1 PL, % Intercept = 41.60 0.66 0.34

LEA, cm2 0.39 **

2 Intercept = 44.52 0.93 0.06

BF, cm -3.72 **

LEA, cm2 0.41 **

3 Intercept = 45.65 0.94 0.06

LW; kg -0.008 **

BF, cm -3.76 **

LEA, cm2 0.40 **

4 Intercept = 45.81 0.94 0.06

LW; kg -0.01 **

ADG 104 d, g/d 0.0001 **

BF, cm -3.76 **

LEA, cm2 0.40 **

** Significant different the 0.01 level of probability (P<0.01)

Table 4 The correlation of growth performance and meat quality in swine

Traits LW;

kg ADG, g/d

ADG 104 d,

g/d BF, cm PL, %

LEA,

cm2

LW; kg 1.00 -0.43** 0.82** -0.11** -0.24** -0.31**

ADG, g/d 1.00 -0.86** 0.06* -0.43** 0.01

ADG 104 d, g/d 1.00 -0.10** -0.12** -0.17**

BF, cm 1.00 -0.48** 0.04*

PL, % 1.00 0.81**

LEA, cm2 1.00

* significant different the 0.05 level of probability (P<0.05)

** significant different the 0.01 level of probability (P<0.01)

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

7

Conclusions

Results of this study indicate that breed was affected on growth performance and meat quality

in swine. Duroc and Crossbreed pig are appropriate for growth performance, such as LW and

ADG 104 d. Pietrain and Crossbreed pig were high meat quality (BF, PL and LEA). Crossbreed

pigs showed good of the growth performance and meat quality.

Acknowledgements

I would like to thank the faculty of technology Mahasalakham University, Mahasakham,

Thailand for financial support. Betagro Company Limited, Thailand, for data of growth

performance and meat quality, which were the main information of this research.

References

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

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[9] Goran KUŠEC, Gordana KRALIK, Antun PETRIČEVIĆ, Vladimir MARGETA, Zlata

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[13] Jung-Seok Choi, Hyun-Jin Lee, Sang-Keun Jin, Yang-Il Choi, and Jae-Joon Lee.

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

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[19] Smith R. M., Gabler N. K., Young J. M., Cai W., Boddicker N. J., Anderson M. J., Huff-

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

10

Effect of pre-treatment processes and stability testing of lemongrass

(Cymbopogon citratus) extract on α-glucosidase inhibitor (AGI) and α-

amylase inhibitor (AAI) activities

Diah Widiputri1,*, Nadya Mariana1, Blandina Josopandojo, Maria Gunawan-Puteri2,

Irvan Kartawiria1

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

Tangerang, Indonesia

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

Tangerang, Indonesia

*Corresponding author: [email protected]

Abstract:

Lemongrass was proved in the previous studies to be one of Indonesian local plants with

relatively high activity in inhibiting α-glucosidase and α-amylase enzymes and thus it can be

useful to lower blood glucose level in diabetic patients. This health benefit of lemongrass has

unfortunately not been widely explored in the herbal industries. Even though lemongrass has

become one of the main raw materials in such industries, the use of lemongrass has been

purposed mostly to obtain its aroma and taste. Commercialization of lemongrass as herbal

medicine or functional food ingredients with α-glucosidase inhibitor (AGI) and α-amylase

inhibitor (AAI) activities requires a closer study on how these activities can be affected by

different pre-treatment processes of fresh lemongrass. In this work, the effect of different

washing and drying scenarios during the pre-treatment process of lemongrass extraction on

both AGI and AAI activities was studied. The result showed that a combination between 1 time

washing and oven drying at 40°C offered the optimum AGI activity. The AAI level was found

to be significantly decreased as lemongrass had gone through drying process. However, when

compared among different drying methods and different sequences of washing process, the

AAI level was found to be relatively unaffected. Stability testing of powdered lemongrass

extract was additionally conducted in real time and accelerated conditions to make an

estimation of the shelf-life. The shelf-life of powdered lemongrass extract was found to be ± 8

months (at 5°C), ± 3 months (at 25°C) and ± 1.5 months (at 30°C).

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doi: 10.10.14457/MSU.res.2017.22
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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Keywords: lemongrass, AGI, AAI, diabetes

Introduction

Various Indonesian medicinal plants have been studied in previous researches to find the

presence of α-glucosidase and α-amylase inhibitory activities [1], [2]. Among these plants,

Cymbogon citratus, commonly known as lemongrass, had been found to show high anti-

diabetic potencies, while showing good stability when undergone heat challenges. Lemongrass

extract obtained through an extraction from fresh plant using water at 70°C for 40 minutes

resulted in a sucrase inhibitory activity that ranged from ±70-100% [2]. This ability of

lemongrass extract to inhibit the breaking down of disaccharide into monosaccharide shows a

promising potential to help diabetic patients in maintaining their blood glucose level. Thus, an

effort to commercialize lemongrass extract as a functional food or herbal medicine ingredient

is necessary to be made.

Unfortunately, the use of lemongrass in herbal industries has not been focused on taking

benefit of its α-glucosidase inhibitor (AGI) and α-amylase inhibitor (AAI) activities yet.

Indonesian herbal industries commonly use lemongrass as one of their main raw materials with

a main purpose to obtain its taste and aroma, rather than its bioactive materials content. An

industrial observation conducted in this work revealed the need to firstly study the impact of

different pre-treatment processes of fresh lemongrass prior to extraction process, on the level

of AGI and AAI activities of the resulting lemongrass extract. Moreover, since it is found to

be more favorable to have the extract in powder form for further use as an ingredient,

pulverization of the lemon grass extract and a stability test on this powdered extract become

very essential to be conducted.

Materials and methods

Materials

Materials used in this research were Cymbopogon citratus or lemongrass plants and

chemicals for analysis purposes. The fresh lemongrass used was obtained from a farm in Bogor,

Indonesia. The chemicals listed were used for sucrase inhibition assay, porcine pancreatic

amylase inhibition assay, and filler used in spray drying process. Rat intestinal acetone powders

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needed for sucrase inhibition assay was provided by Sigma-Aldrich. Potassium phosphate

buffer, ethylenediaminetetraacetic acid (EDTA), analytical grade sucrose, and other chemicals

used for this purpose were purchased from Merck, Germany, whereas the glucose kit was

purchased from Wako, Japan. For porcine pancreatic amylase inhibition assay the chemicals

needed were citric acid, di sodium hydrogen phosphate dodecahydrate, and analytical grade

starch obtained from Merck, Germany. Maltodextrin and Arabic gum used in the pulverization

process were technical grade and purchased from PT Bratachem Indonesia.

Research methodology

This study was divided into 4 stages, started with an industrial observation in 3

Indonesian reputable herbal industries. The result of this benchmarking study was then taken

into consideration in the second stage, which was purposed to study the effect of different pre-

treatment processes on the AGI and AAI activities of the lemongrass extract.

This stage was broken down further into 2 steps, which included an observation of

washing and drying process. There were two options of washing sequences conducted; the first

one is to perform the washing process one time, prior to drying. The second washing scenario

was to perform it two times, prior to and subsequent to drying process (shown in Figure 1).

After being had undergone the washing process observation, both lemongrass samples from

each washing option were then dried by using an oven at 40°C until the moisture content was

less than 10%. The resulted dried lemongrass were then milled and extracted with water, then

the extract was analyzed to compare their AGI and AAI activities. The washing scenario that

offered best result was then chosen to be used in the next observation, which was the drying

process optimization (Figure 2). The effect of drying methods on AGI and AAI activities was

observed using three different drying methods; these were sun drying, oven drying at 40°C,

and oven drying at 60°C. Similarly with the previous step, the extract obtained from each

drying variation was then analyzed to determine the most optimum drying method.

After having determined the effect of pre-treatment processes on AGI and AAI

activities of lemongrass extract, pulverization of the liquid extract through spray drying was

conducted. In a previous study by [2] an optimization of fresh lemongrass extraction process

to yield the highest percentage of total soluble solids (TSS) content had been done. It was

recommended that fresh lemongrass is extracted with water at 70°C for 40 minutes, with plants

to water ratio of 3:10 under continuous stirring, where a TSS content up to 2.00% can be

yielded.

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In this work, the selection of the best filling agent (filler) to be used in spray drying

process was conducted. Addition of filler might help protect the stability of the sample and

even further improve their shelf life. The use of two types of filler were compared; the first

type was 100% maltodextrin, and the other was mixing between Arabic-gum and maltodextrin

with a ratio of 40:60. The result between these fillers were then compared through AGI and

AAI assay.

In the last stage, stability testing of the pulverized lemongrass extract was performed.

In food and drug industries, principle of kinetic degradation can be used to analyze the stability

of certain compound or the shelf life of the product in certain condition. This can later be used

to determine the best condition to store the product [3]. The lemongrass powder extract

produced in the previous stage was tested in 2 different conditions; at real time for climate zone

II (± 25°C ± 2 / 60 ± 5 RH) [4] and in an accelerated condition at ± 40°C ± 2 / 75 ± 5 RH. A

prediction of the shelf life of this powder extract in other climate zones that represents possible

storing conditions in Indonesia was then made based on the stability testing.

Analysis techniques

The sucrase and α-amylase inhibitory activity was analyzed and determined using the

method previously described in [5] with slight adjustments. For sucrase inhibition assay, a total

of 100 μL extract in 50% dimethyl sulfoxide (DMSO) was placed in a tube containing a pre-

incubated sucrose. The solution was then reacted by 200 μL enzyme and incubated at 37°C for

20 minutes. The reaction was stopped by adding Tris-HCl 2 M at pH 9, and the solution was

then passed through aluminum oxide column. Afterwards, 50 μL of this solution was mixed

with 200 μL of glucose kit. The mixture was incubated at 37°C for 10 minutes, and was then

measured using micro-plate reader at the wavelength of 490 nm.

The α-amylase inhibitory activity was analyzed through porcine pancreatic amylase (PPA)

assay. For this assay, 200 μL lemongrass extract in 50% DMSO was mixed with a pre-

incubated starch azure solution (2 mg/700 μL) at 100°C, then reduced to 37°C, for 5 minutes

at each temperature. Afterwards, it was reacted with PPA enzyme and incubated at 37°C for

10 minutes. The reaction was stopped using 50% acetic acid. The solution was then centrifuged

at 4°C, 3000 rpm for 5 minutes and was read using micro-plate reader at the wavelength of 630

nm. The control was analyzed using the same method in each assay, only replacing the extract

in 50% DMSO with 50% DMSO only.

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Figure 1 Optimization of washing process Figure 2 Optimization of drying process

Results and discussion

Effect of pre-treatment process on AGI and AAI activity

This study started with an industrial observation in 3 reputable Indonesian herbal

industries, who produce different types of herbal products and medicines. The visit to these

industries was purposed to learn, whether lemongrass was utilized as a raw material in their

production processes and the reason for this utilization. The result of this observation showed

that lemongrass was indeed one of the main raw materials used in producing herbal products,

with the main purpose to obtain the typical aroma and taste of this plant. Several health benefits

of lemongrass were also targeted through the use of this plant in herbal products, as it is

believed that lemon grass can help in treating anemia, it can act as anti-inflammatory remedy

as well as anti-microbial agent [6]. However, there has not been any attempt made to

industrially produce lemongrass extract for the purpose of diabetes therapy. Complete

information resulted from this industrial observation is summarized in Table 1.

Based on the results of this industrial observation, it was concluded that processing

lemongrass in industrial scale requires the plants to be dried after harvesting, since in dried

condition, this material can be transported in a more efficient, practical and safer way.

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Moreover, it was learned that once the dried plants was delivered to an industry, it would be

possible that a standardization of quality must take place. This pre-treatment of raw materials

can involve washing, drying and grinding processes. Additionally, it was also found out, that

the most common drying methods used in the herbal industry to pre-treat their raw materials

are sun drying and oven at 40°C - 60°C. These information was taken into account in the next

stages of this experiment, in order to study further how the AGI and AAI activity of lemongrass

could be affected after being pre-treated before even going through the extraction process.

Table 1 Result of benchmarking study to Indonesian herbal industries

INDUSTRY 1 INDUSTRY 2 INDUSTRY 3

Plants as raw

material

Most of the raw materials

were bought from

distributors in dried

condition. Lemongrass is

utilized in fresh condition,

to obtain scent and flavors.

All raw materials were

provided by distributors in

dried condition and were

ready to be used in

production.

All raw materials were

bought from farmers in

fresh condition.

Standardization

of raw

materials

Re-standardization:

Sorting, Washing, Drying,

Grinding, Sieving

Sampling to conduct

several quality assurance

tests in laboratory.

All plants were ensured to

be fresh at delivery by

giving farmers trainings

on necessary SOPs

(standard operating

procedures) previously.

Pre-treatment

process of raw

materials on-

site

Washing to remove soil

and dirt

Drying using hot air

conveyor

Drying using oven at 60°C

Grinding

No pre-treatment process

necessary, sun drying had

been conducted by farmers

before delivering to the

industry.

Washing

Drying using spinner

Chopping

Drying using air dryer

Drying using hot air dryer

at 40°C

Understanding the possibility for the lemongrass to undergo 2 times of washing process; first

washing is done by the farmer directly after harvesting and prior to sun drying, and the second

one is on-site done by the herbal industry, it became necessary to study whether there could be

a reduction of AGI and AAI activity due to these washing processes. An observation on the

enzymes inhibition (%) is shown in Figure 3, which depicts a significant reduction of AGI

activity when the lemongrass was washed two times (p-value = 0.002). The reduction of α-

glucosidase inhibition in 2 times washing was expected to happen due to the leaching of some

water soluble contributing bio-active compounds during the process, which are suspected to be

mostly phenolic compounds [7]. This was also proven through an analysis of AGI activity in

the rinsing water, which resulted in ± 9% inhibition of α-glucosidase enzyme. However, the

inhibition of α-amylase enzyme was not significantly affected by doing multiple washing (with

a p-value of 0.08).

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In the next step, an observation on the drying methods’ effect on AGI and AAI activity level

was conducted. Figure 4 showed a comparison of the inhibition level of both α-glucosidase and

α-amylase enzymes between different drying methods. The results were also compared to the

AGI and AAI level of lemongrass extract obtained from its fresh condition (undried). From

this figure, it can be seen that the highest inhibition activity was actually obtained when the

extract came from fresh (undried) lemongrass. However, when obtaining lemongrass in fresh

condition in a large scale is not an option, it can be seen from Figure 4 that oven drying at 40°C

will deliver the highest AGI activity. Meanwhile, the activity of AAI was found to be

insignificantly affected by the observed drying methods. Despite having the lower activity of

AGI and AAI when compared to fresh lemongrass extract, dried materials are still more

preferable in industries because it can facilitate a long term storage of raw materials, and can

enable more effective and efficient processing, such as the need of a smaller amount of solvent

for extraction [8].

Figure 3 Comparison of AGI and AAI activity between 1 times and 2 times washed

lemongrass

Figure 4 Effect of drying processes of lemongrass on AGI and AAI activity

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Stability testing of lemongrass powder extract

In the subsequent stage to studying the effect of different pre-treatment processes on

the AGI and AAI activity level, a stability testing of lemongrass powder extract was performed

in this work. The method used to pulverize the liquid extract is as previously discussed in the

research methodology part of this paper. However, since heat treatment can cause denaturation

of organic compounds, the application of spray drying as one of the most widely used methods

for pulverization in the food and pharmaceutical industries is concerned to lower the AGI and

AAI activity of lemongrass extract. Thus, an addition of filling agent (filler) is required, which

is expected to act as an encapsulation agent that can enhance product stability even when

exposed to high temperature.

As can be seen in Table 2, the α-glucosidase (sucrase) and α-amylase inhibition of

powder extracts using two types of filler were analyzed, and compared with the inhibition of

liquid extract (prior to spray drying). The extract used in this part of experiment was obtained

using fresh (undried) lemongrass plants. The results showed that there is no significant

difference between the use of 100% maltodextrin and mixture of maltodextrin-Arabic gum

(60:40). The reduction of AGI activity after spray dried was ± 38.5% and of AAI was ± 40%.

However, due to practicability and economical reason, 100% maltodextrin was selected to be

used in producing the powder extract. The stability test was then performed on this powder

extract.

Table 2 Filler selection

Sample Filler Inhibition

AGI (%) AAI (%)

Pre evaporation Without filler 100.00 0.00 81.77 0.25

Post evaporation 100% Maltodextrin 61.22 0.23 48.53 0.55

Post evaporation (60:40) Matrodextrin-

Arabic gum

61.69 0.62 49.49 1.52

The stability or shelf-life testing was conducted in 30 consecutive days under 2 different

conditions. The first condition was labelled Real Life (RL) which represented a sample storing

at 25°C and the second condition represented the accelerated condition at 40°C. The results of

AGI and AAI activity level observation were plotted using Arrhenius kinetics principles, to

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find out that the inhibition activity degradation of the lemongrass powder extract followed a

first order reaction. Equation 1 below was used to find the activation energy of the degradation

reaction. By using the same equation, different rate constants (k) at different temperatures can

be determined.

211

2

T

1

T

1

R

E

k

kln (1)

where k is constant rate at temperature (K), E is the activation energy (J/mol), and R is

universal/ideal gas constant (8.314 J/mol.K).

The shelf-life of the lemongrass powder extract in different temperatures was

determined using equation for calculating the half-life of a product (equation 2) with an

adjustment that the final concentration should be 90% of the initial condition instead of 50%.

Table 3 shows the α-glucosidase (sucrase) and α-amylase inhibition degradation down to its

90% and 50% of its initial inhibition at different storage temperature. Based on its climate

condition, Indonesia was grouped into zone 4 with a condition of 30°C/ 70% RH [4]. Other

possible storing condition of the lemon grass extract would be under refrigerated condition,

which can be taken as an average of ± 5°C. At these two temperatures, based on the AGI

activity only, the shelf-life of lemongrass powder extract is ± 1.5 months and ± 8 months

respectively.

k

C50

C

t A

A

21

.ln

(2)

where T1/2 or tC=0.5 denotes the half-life or t-half (time) and C is the concentration.

Conclusions

Pre-treatment processes of fresh lemongrass affect the α-glucosidase and α-amylase inhibitory

activity of its extract. Results of this study showed that conducting multiple washing sequence

and drying process can decrease the AGI activity significantly, whereas the AAI is only slightly

affected. The pulverization of lemongrass extract can be done through spray drying with the

addition of maltodextrin as a filler, where a reduction in the inhibition activity of around 40%

will be resulted. The shelf life based on AGI activity of the lemongrass powder extract at room

temperature 25°C, in Indonesian climate zone (30°C) and under refrigerated condition (5°C)

was found to be around 3, 1.5 and 8 months respectively.

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Table 3 Shelf life testing at different temperatures

Inhibitor Temperature

(°C)

Rate constant

(k) %inh. Day-1

tC=0.5

(Months)

Shelf-life

(Months)

α-glucosidase (sucrase)

40 1.20.10-02 1.92 0.29

30 2.80. 10-02 8.25 1.25

27 1.80. 10-03 12.83 1.95

25 1.30. 10-03 17.77 2.70

5 4.63.10-04 49.94 7.59

α-amylase

40 8.60. 10-03 2.68 0.41

30 1.70. 10-03 13.59 2.06

27 1.00. 10-03 23.10 3.51

25 7.00. 10-04 33.00 5.02

5 1.62.10-04 142.53 21.67

tC=0.5: the time required to achieve 50% inhibition activity of initial

Shelf life : the time required to achieve 90% inhibition activity of initial

References

[1] Arsiningtyas IS, Gunawan-Puteri MDPT, Kato E., and Kawabata J. Identification of α-

glucosidase inhibitors from the leaves of Pulchea indica (L.) Less., a traditional Indonesian

herb: promotion of natural product use. Natural Product Research: Formerly Natural Product

Letters. 2014; 28(17), p. 1350-1353.

[2] Gunawan-Puteri, Josopandojo, Adiyoga, Kartawiria, and Widiputri. Aqueous Extraction

Optimization of C. Citratus for Development of Food Ingredients with Alpha Glucosidase

Inhibitory Activities. In: Integrated Sci-Tech: The Interdisciplinary Research Approach

Volume 2. Research Institute and Community Service of Universitas Lampung, 2016, p. 55 –

61, ISBN: 978-602-0860-14-5

[3] Oliveira, M.A. et al., Degradation kinetics of atorvastatin under stress conditions and

chemical analysis by HPLC. Molecules 2013; 18(2), pp.1447–1456.

[4] Patgiri, B., Soni, H. & Bhatt, S., Evaluation of stability study of Ayurvedic formulation –

Rasayana Churna. Journal of Pharmacognosy and Phytochemistry 2014; 2 (5). 126-130, 2(5),

pp.126–130.

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

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[5] Gunawan-Puteri MDPT, and Kawabata J. Novel α-glucosidase inhibitors from Macaranga

tanarius leaves. Food Chemistry 123. 2010, p. 384-389.

[6] Ranade, Shruti Sunil and Padma Thiagarajan. 2015. Lemongrass. International Journal of

Pharmaceutical Sciences Review and Research 35(2):162–67.

[7] Fontana Pereira, Danielle et al. Effects of Flavonoids on α-Glucosidase Activity: Potential

Targets for Glucose Homeostasis. Nutrition 2011; 27(11–12): 1161–67. DOI:

10.1016/j.nut.2011.01.008.

[8] Sukrasno, Irda Fidriany, Kusnandar Anggadiredja, Wafiq Auliana Handayani, and Khairul

Anam. Influence of Drying Method-Flavonoid Content of Cosmos caudatus (Kunth) Leaves.

Research Journal of Medicinal Plant 2011; 5(2); 189-195, DOI: 10.3923/rjmp.2011.189.195.

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Evaluation of cookies quality enriched with resistant starch type 2 (RS2)

and resistant starch type 3 (RS3) from banana (Musa paradisiaca

formatypica)

Mutiara Pratiwi1,*, Evelyne Dermawan1, Nila Kusumawaty2

1Department of Food Technology, Faculty of Life Science and Technology, Swiss German University, Alam

Sutera, Tangerang 15143 Indonesia

2Center of Agroindustrial Technology-BPPT Laptiab building, Puspiptek, Serpong, Tangerang Selatan 15314

Indonesia

*Corresponding author: [email protected]

Abstract:

This research was aimed to evaluate cookies quality with the enrichment of resistant starch

type 2 (RS2) and resistant starch type 3 (RS3) from unripe banana. Cookies were formulated

with the addition of RS2 and RS3 with three levels of substitution: 10%, 20%, and 30% of the

wheat flour basis. RS2 was obtained through water-alkaline extraction, while RS3 was obtained

through starch modification by using autoclaving-cooling cycle method. Quality of cookies

was evaluated on in vitro digestibility, hardness, color, and sensory acceptance. Both RS2 and

RS3 was found to decrease the digestibility of cookies. However, effect of RS3 was more

obvious compared to RS2 even at 10% of substitution level. In both type of RS, the higher

substitution level resulted in the lower digestibility. Hardness of RS2 and RS3-enriched cookies

at all substitution levels did not show any significant difference compared to control. Color

measurement showed that both type of RS resulted in the significantly darker color of cookies,

though the effect was more intense in RS3 substitution. The darker color of cookies was

observed along with the increasing substitution level. Sensory acceptance test conducted for

aroma, taste, hardness, and overall acceptance attributes showed that panelists rated all the RS2

and RS3-enriched cookies at all substitution levels equally with the control but lower for color

attribute. The addition of RS3 at 20% level of substitution was suggested as it was found to be

the most acceptable in all attributes tested, while having the significantly lower digestibility

compared to control cookies.

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doi: 10.10.14457/MSU.res.2017.21
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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Keywords: autoclaving-cooling, banana starch, cookies, in vitro digestibility, resistant starch

Introduction

Cookies are commonly consumed by people worldwide in a wide range of ages, starting from

children until the elderly people. Cookies are easily served, having a long shelf-life, and

generally come with convenient taste and texture which make it suitable to be consumed as

snacks [1]. However, cookies contribute to a high intake of calories particularly due to its high

carbohydrate content. The carbohydrate content may come from the use of wheat flour,

sucrose, glucose, fructose syrup, hydrolyzed starch, or even corn syrup as its ingredient [2].

Cookies can contain up to 70% of sugar that can be easily digested resulting in the raise of

blood glucose [3]. It may then create some health problems for certain people particularly those

with the risk of diabetes or having an overweight problem.

Now that people are getting more health-conscious, a considerable interest has been

given to the healthier food alternative yet with convenient sensory properties. Some attempts

have been conducted to produce healthier cookies with low-intake of calories, such as by using

a natural non-caloric sweetener [4], replacement of wheat flour with other flour such as pea

and soybean flour [5], enrichment with fiber [6], and enrichment with resistant starch [7,8].

The use of resistant starch (RS) is however getting more attention as it performs similar

functional properties with fiber in our digestive system but with minor influence on the sensory

properties when applied to food product [9]. Traditional fiber was reported to affect texture,

taste, and flavor of food enriched with the component, and thus becoming a shortcoming on its

application [10]. On the other hand, RS offers some excellences over the fiber such as colorless,

bland flavor, low water-holding capacity, and small particle size, which favorable for its

application in varying food products including cookies [11]. Thus, RS with its low caloric

content was hypothesized to give functional value of food by reducing the digestibility of

product, while maintaining the sensory properties.

RS refers to the portion of starch and starch products that resist digestion as they pass

through the gastrointestinal tract and may be fermented by microbiota in the large intestine,

affecting some physiological functions such as reduction of glycemic response,

hypocholesterolemic effect, and protective effect against colorectal cancer [12]. It should be

noted that different sources of starch may affect the nutritional and functional properties of RS

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[8]. Among the available sources of starch, banana has been known as a potential source of

resistant starch type II (RS2) which renowned for its health benefit [13] where its nutritional

quality has been pointed out by several authors [14,15]. It also contains observable amount of

starch particularly on its unripe state, which is up to 70-80% on a dry weight basis [13].

However, RS2 exhibits a shortage in terms of its stability upon heat treatment, while resistant

starch type III (RS3) is found to be more heat-stable [9]. In this study, each RS2 and RS3 from

banana source was tried to be incorporated in cookies formulation with three levels of

substitution: 10%, 20%, and 30% of the wheat flour basis. RS3 was obtained through physical

modification of autoclaving-cooling cycles treated to the banana starch. Effect of RS type and

its concentration in the cookies formulation was evaluated on the following parameters: in vitro

digestibility of cookies, texture, color, and sensory acceptance.

Materials and methods

Materials

The raw material used in this study was Indonesian local white kepok banana (Musa

paradisiaca formatypica) of the plantain group Musa AAB (triploid cultivar), purchased from

a local supplier in BSD, Tangerang, Banten. The selected bananas were green, hard, aged 90-

120 days, and with no molds. The other materials used for production of cookies were eggs,

wheat flour, margarine and sugar. The reagents used were sodium hydroxide technical grade,

α-amylase enzyme (Sigma Aldrich, USA), maltose standard (Sigma-Aldrich, USA), phosphate

buffer solution, acetic acid, ethanol 95%, iodine, potassium iodide, glucose standard (Merck,

Darmstadt, Germany), amylose standard (Sigma-Aldrich, USA), starch (BDH Laboratory

Supplies, England), acetate buffer, DNS solution and distilled water.

Extraction of Banana Starch [13, 16]

Banana was cut into thin slices and crushed and stirred in 0.1 N NaOH solution for 3 h,

using blender machine. The mixture was then filtered with muslin cloth. The filtrate was

collected, added with distilled water, and allowed for 2 h. The mixture was sieved with 120 µ

siever and the supernatant was removed from the starch portion. The starch portion was stirred

again in 0.1 N NaOH solution for 2 h, sieved with 120 µ, added with distilled water, and

allowed overnight. The supernatant underwent further extraction with the addition of distilled

and allowing it for 2 h to obtain another remaining starch portion. Supernatant was separated

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from the starch, and the starch was oven-dried at 40°C for 12 h. The dried starch was sieved

using 100 µ siever and stored in a sealed container. The banana starch obtained in this

extraction would be labeled later as RS2.

Preparation of RS3 through Autoclaving-cooling Cycles

Starch was added with distilled water to obtain 20% of starch concentration. The starch

suspension was heated at 80°C for 5 min with constant stirring. The suspension was autoclaved

at 121°C for 15 min and allowed at room temperature until it reached approximately 40°C. It

was cooled at 4 °C and allowed for 24 h. The autoclaving-cooling was repeated for another two

cycles. The suspension was dried in oven at 40°C, then ground into 100 µ particle size. The

starch was sieved with and stored in a sealed container.

Formulation and Preparation of cookies

Cookies were formulated with the addition of resistant starch (RS2 and RS3) at 10%

(F1), 20% (F2), and 30% (F3) substitution level of wheat flour basis. Control cookies were

made with 100% wheat flour, without incorporation of RS. The detail formulation of cookies

was presented in Table 1.

In vitro digestibility tests [17]

Sample (1 g) was suspended with 100 mL distilled water, stirred and heated until 90°C,

then cooled down to room temperature. As much as 2 mL sample solution was put into test

tubes quantitatively using micropipette, then 3 mL distilled water and 5 mL phosphate buffer

pH 7 solution were added into the test tubes. Each sample was made in duplicate, one as the

sample and the other was used as blank. Each tube was then covered and incubated at 37°C for

15 min. The sample was added with 5 mL α-amylase solution in 1 mg/mL phosphate buffer pH

7 solution and the blank solution was added with 5 mL phosphate buffer pH 7. Both tubes were

incubated for 30 min and then both solution in test tubes were transferred to lidded test tubes

containing 2 mL DNS. The solutions were then heated in boiled water for 12 min and cooled

down. After that, as much as 8 mL distilled water was added into the solutions and stirred until

homogenized using vortex. As much as 260 µL for each solution was pipetted into microplate

and the absorbance was measured using microplate reader at wavenumber of 520 nm. The

starch digestibility (in percentage) is calculated using this formula:

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Table 1 Formulation of cookies

Ingredients Control F1 F2 F3

Wheat Flour (g) 125 112.5 100 87.5

Resistant Starch / RS (g) 0 12.5 25 37.5

Margarine (g) 75 75 75 75

White egg (g) 19 19 19 19

Egg Yolk 19 19 19 19

Sugar (g) 50 50 50 50

Baking powder (g) 2 2 2 2

Texture analysis of cookies

Texture of cookies was measured with Texture Analyzer (Stable Micro Systems Ltd,

UK) using three point bending rig as a probe. The textural parameter measured in this test was

hardness, as an important cooking quality of cookies. First, the individual sample of cookies

was placed on the platform and the blade was attached to the crosshead of the instrument. The

cutting knife would move downward until the cookie was broken. Hardness of the cookie was

reported in gram unit. The texture analysis was performed five times for every sample.

Color analysis of cookies

Color of the cookies were quantitatively measured by using chromameter (Konica

Minolta CR-40) and recorded in the L*a*b* color system. The L*a*b* color system consists

With,

A = maltose in sample (mg)

a = maltose in blank sample (mg)

B = maltose in starch (mg)

b = maltose in blank starch (mg)

In vitro starch digestibility (%) = (A-a) x 100

(B-b)

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of a luminance or lightness component (L*) and chromatic components: the (a*) component

for green (-a) to red (+a) and the (b*) component from blue (-b) to yellow (+b). The colorimeter

was calibrated using standard white plate. The measurement was repeated thrice for each

sample.

Sensory analysis of cookies

Thirty-five untrained panelists were asked to evaluate the sensory attributes of the

cookies with the hedonic test. Samples which consisted of control and resistant starch-enriched

cookies (F1, F2 and F3) were randomly coded and given to the panelists simultaneously.

Panelists were provided with drinking water to cleanse the palate between samples. Panelists

were asked to rate each sample by giving a rating score, with the hedonic scale ranging from 1

(dislike extremely) to 9 (like extremely). The sensory attributes being evaluated were color,

aroma, taste, texture and overall acceptability.

Statistical Analysis

Statistical analysis was conducted using data analysis ToolPak in Microsoft Office

Excel. The data for sensory hedonic test was analyzed by using Friedman test and continued

with the Wilcoxon post-hoc test. Other statistical analysis in this study was carried out using

One-way analysis of variance (ANOVA) with single factor and followed with Tukey HSD

post-hoc test to evaluate significance of differences between the mean values of measured

parameters. A p-value of ≤ 0.05 was considered as statistically significant.

Results and discussion

In vitro digestibility tests

Effect of resistant starch (RS2 and RS3) addition to the in vitro digestibility of cookies

was presented in the Table 2. As hypothesized, the addition of both types of RS had led to the

decrease of in vitro starch digestibility. The higher substitution level of RS resulted in the

greater reduction of in vitro digestibility.

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Table 2. Result of in vitro digestibility test in formulated cookies compared to control

Type of RS Starch Digestibility (%)

Control F1 F2 F3

RS 2 56.68 ± 2.64a 51.40 ± 1.53ab 46.82 ± 3.40b 42.93 ± 0.98b

RS 3 56.68 ± 2.64a 46.82 ± 1.87b 40.22 ± 2.42bc 32.22 ± 1.87c

* Different superscript letter in the same row indicates significant difference

RS3 was found to be more effective in reducing the starch digestibility, showing the lowest

digestibility value at 30% level of substitution, which was significantly lower (p<0.05)

compared to control. At the same level of substitution, RS2 showed higher digestibility than

RS3. RS3 was formed due to the autoclaving-cooling treatment applied, through retrogradation

mechanism. During retrogradation, the amylose molecules and amylopectin molecules realign

between amylose-amylose and amylose-amylopectin which make the hydrogen bond stronger,

forming double helix structure. The double helix structure will bind with another double helix

structure to from crystallite which finally can increase the resistance of starch [18]. The higher

effectivity of RS3 in reducing starch digestibility compared to RS2 was due to the better

stability of RS3 [9]. It was also stated in [19], that RS3 exhibited substantially higher

thermostability, compared to RS2.

Texture analysis of cookies

Hardness is analyzed by measuring the force values needed to reach a certain

deformation [7]. The hardness values of cookies enriched with RS2 and RS3 compared to

control were presented in Figure 1. As seen in the chart, both RS2 and RS3 did not considerably

affect hardness of cookies at all levels of substitution. However, generally RS resulted in the

lower value for hardness compared to control, except for RS2 at 30% substitution level. The

lowest hardness value was showed in cookies with RS3 enrichment at 20% substitution level.

In similar research carried out in cookies enriched with different types of RS and fiber at 5%,

15%, and 25% substitution level, it was shown that generally fiber led to a harder texture of

cookies [7]. The similar trend was also shown in other research conducted in tortillas product,

where the addition of RS resulted in tortillas with less strength, that was easier to tear, and less

dense compared to the flour-based tortilla [20]. However, a too low hardness value for cookies

is also unexpected as it may be difficult in transportation and storage.

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Figure 1. Hardness of RS-enriched cookies compared to control

Color analysis of cookies

Color of cookies enriched with RS2 and RS3 were presented in Table 3 and 4,

respectively. As seen in the Table 3, the L* value of cookies at the level of 10% was higher

than control, meaning that it was less dark compared to control. However, as the substitution

level was increasing, the L* value was getting lower. The a* value was found lower at the level

of 10%, compared to control. While as the substitution level was increasing, the a* value was

getting higher. The b* value was higher at 10% level compared to control and the value was

getting lower as the substitution level was increasing. The increase in a* value and the decrease

in L* and b* value could be translated into a more intense golden-brown color that could be

seen visually along with the increase of RS substitution level.

Table 3. Result of color measurement in RS2-enriched cookies

Control Formula

F1 F2 F3

L* 68.31 ± 0.12b 70.56 ± 0.06a 68.72 ± 0.20b 62.64 ± 0.22c

a* 7.68 ± 0.10b 6.17 ± 0.05c 7.70 ± 0.03b 10.13 ± 0.10a

b* 31.07 ± 0.06c 32.06 ± 0.08a 31.53 ± 0.06b 28.72 ± 0.06d

* Different superscript letter in the same row indicates significant difference

In the substitution with RS3, the trend was similar with RS2, indicating the formation of a more

intense golden-brown color in cookies enriched with resistant starch. Comparing to RS2

substitution, RS3 showed a more obvious effect of darkening in cookies. The significantly

darker color in RS3 substitution, compared to RS2, was also observed in batter preparation

with RS3 (Novelose 330 and C*Actistar) and RS2 (Hi-maize 260) addition [21]. The

[] a

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850.00

900.00

950.00

1000.00

1050.00

1100.00

1150.00

Control F1 F2 F3

Ha

rdn

ess

(g)

RS2 RS3

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phenomenon happens in both types of RS substitution is due to the higher amount of reducing

sugars that contribute to Maillard reactions and caramelization [22].

Table 4. Result of color measurement in RS3-enriched cookies

* Different superscript letter in the same row indicates significant difference

Sensory analysis

The addition of resistant starch in cookies formulation was expected to improve the

functional value of cookies without impairing the sensory attributes. Thus, the sensory analysis

is an important parameter for the cookies evaluation. Result of the hedonic sensory test for RS2

and RS3-enriched cookies was displayed in Table 5 and 6, respectively.

The addition of RS2 at all levels of substitution did not seem to have significant effect

in almost all the sensory attributes tested, except in color attribute. In the color attribute, the

RS2 addition did not lower the acceptance score significantly at 10% and 20% level of

substitution. However, the score given at the level of 30% was found to decrease significantly.

In texture attribute, the score for RS2-enriched cookies at the level of 20% was the highest

among all samples tested, including control, however this highest score was not statistically

significant. Correlating this result with the result of hardness analysis, it was assumed that the

higher score given to the 20% RS2-substituted cookies might be due to softer texture of the

cookies, compared to other samples. The overall acceptance also showed that 20% RS2-

subsituted cookies was rated with the highest score compared to control and other samples.

While for aroma and taste, all the samples were rated equally, showing that the addition of RS2

did not contribute an obvious effect on the two attributes.

Control

Formula

F1 F2 F3

L* 68.31 ± 0.12a 67.26 ± 0.21b 62.99 ± 0.24c 60.54 ± 0.26d

a* 7.68 ± 0.10c 7.93 ± 0.12c 9.07 ± 0.11b 9.60 ± 0.26a

b* 31.07 ± 0.06a 30.73 ± 0.12b 28.70 ± 0.10c 26.49 ± 0.14d

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Table 5. Result of Hedonic Sensory Test of Cookies Formulated with RS2

Attributes Control F1 F2 F3

Color 7.03 ± 1.18 a 6.60 ± 1.82ab 7.37 ± 1.14a 6.03 ± 1.67b

Aroma 7.29 ± 1.02a 6.54 ± 1.60a 6.97 ± 1.07a 7.14 ± 1.24a

Taste 7.03 ± 1.38a 6.91 ± 1.17a 7.09 ± 1.04a 7.17 ± 1.10a

Texture 7.26 ± 1.40 ab 6.83 ± 1.25b 7.83 ± 0.71a 7.00 ± 1.21ab

Overall

Acceptance

7.31 ± 1.08 ab 6.77 ± 0.97b 7.51 ± 0.89a 7.14 ± 0.97ab

* Different superscript letter in the same row indicates significant difference

Table 6. Result of Hedonic Sensory Test of Cookies Formulated with RS3

Attributes Control F1 F2 F3

Color 7.17 ± 1.20 a 7.34 ± 1.08a 7.09 ± 1.20a 5.77 ± 1.82b

Aroma 6.89 ± 1.13 a 6.43 ± 1.40a 6.63 ± 1.44a 6.74 ± 1.77a

Taste 7.00 ± 1.31 a 6.63 ± 1.21a 6.77 ± 1.44a 6.23 ± 2.00a

Texture 6.83 ± 1.40 ab 6.71 ± 1.38ab 7,40 ± 0.74a 6.26 ± 2.13b

Overall

Acceptance

7.09 ± 1.34 ab 6.60 ± 1.42b 7.40 ± 0.74ab 6.86 ± 0.94ab

* Different superscript letter in the same row indicates significant difference

The addition of RS3 was shown to exhibit a similar result in sensory evaluation, especially in

the color attribute. Comparing to control, the cookies enriched with RS3 was observed with

lower score in color attribute. A significant decrease of the score was found at 30% level of

substitution. This was in line with the result of color measurement showing that cookies showed

the significantly darkest color at that level of substitution. Similar to RS2, RS3-enriched

cookies at 20% level was rated with highest score, compared to control and other samples,

though it was not statistically significant. This might be related to the softer texture of this

sample, which was also in a good accordance with the result of hardness analysis. In the aroma,

taste, and overall acceptance, the scores given to the RS-enriched cookies were not significantly

different from the control.

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Conclusions

Incorporation of both types of RS in cookies formulation had been found effective in reducing

the in vitro digestibility of cookies, suggesting the reduced caloric value of cookies. However,

the effect was more obvious in RS3 addition. The increasing concentration of RS2 and RS3

applied in the formula had led to a lower digestibility value, but it was not significantly different

between 20% and 30% level of substitution. Both types of RS in all substitution levels were

not found to affect hardness of cookies significantly. In terms of color, RS2 and RS3 showed

a darkening effect in cookies, with the higher intensity found in RS3. In the sensory acceptance,

RS-enriched cookies were overall accepted equally with the control, except in color attribute

at 30% substitution level, with the lower score for RS-enriched cookies. Finally, the use of RS3

at 20% substitution level was suggested as it was accepted better by the panelists while having

the significantly lower in vitro digestibility value compared to control.

Acknowledgements

The study was carried out with financial support from Research Center of Food and Health

Development, Academic Research and Community Service (ARCS), Swiss German University

(SGU), Indonesia, through Central Research Fund (CRF).

References

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

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[5] Amin T, Bashir A, Dar BN, Naik HR. Development of high protein and sugar-free cookies

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[19] Lehmann U, Jacobasch G, Schmiedl D. Characterization of resistant starch type III from

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Potential application of overripe tempe dried powder as plant-based

instant stock

Maria D.P.T. Gunawan-Puteri1*, Kevin Samuel1, Felicya1, Irvan S Kartawiria2,

Christofora Hanny Wijaya3

1Department of Food Technology, Faculty of Life Sciences and Technology, Swiss German University, The

Prominence Tower, Jalan Jalur Sutera Barat No. 15, Alam Sutera, Tangerang, Banten 15143 Indonesia

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

The Prominence Tower, Jalan Jalur Sutera Barat No. 15, Alam Sutera, Tangerang, Banten 15143 Indonesia

3Department of Food Science and Technology, Faculty of Agricultural Engineering and Technology, Bogor

Agricultural University, Kampus IPB Dramaga, Jl Lingkar Akademik, Darmaga Campus, Bogor, 16680

Indonesia

*Corresponding author: [email protected]

Abstract:

Recent studies along with the local wisdom support the usage overripe tempe (OT) as plant-

based umami source ingredients (USI). A market survey was done in this study in order to

select target commercial USI product. Subsequently, OT was processed and formulated to

compete the selected target product. The selected survey area was defined as modern market

located in Tangerang City, Indonesia. Thirty validated respondents were taken into this survey.

The selection of target USI commercial product was done based on the criteria as follow: (1)

sold in selected survey area; (2) used by validated respondents; and (3) used in Indonesian

savory dishes. Among five selected USI, instant chicken stock was defined as target

commercial product in OT powder application. Subsequent market survey showed that in case

of instant stock, taste property is the most important selection factor. Dried powders of OT

were prepared by using oven- and freeze-drying method. Intensity scaling of attribute was

conducted by employing 30 validated panelists to evaluate the umami intensity of the OT

powder. Oven dried OT had highest umami intensity and thus was selected for further

formulation of OT stock. OT stock was developed from chicken stock formula, using Design

Expert software, to meet national standard requirement of stock and consommé. Taste

acceptance and umami intensity of selected OT stock formula (6.71 + 1.27, 5.65 + 1.64) was

significantly below the commercial target products (7.24 + 1.24, 7.68 + 1.78). Further

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doi: 10.10.14457/MSU.res.2017.20
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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development of OT powder as plant-based instant stock must to take into account improvement

in the umami intensity and reduction of insoluble solid residue.

Keywords: overripe tempe, instant stock, formulation, umami

Introduction

Plant-based protein have been an increasing lifestyle in food consumption [1] due to published

benefits in health, and well-being, include among others economical, ecological and social

concern [2-4]. However calcium and vitamin B12 intake in vegetarian diet remained concern

[5-6] that required diet diversification in health promotion. Tempe had been know as potential

source of protein in Indonesia as well as in other countries [7] as it was reported to promote the

calcium absorption [8-9] and to provide vitamin B-12 that was not exist in it original beans

[10-11].

Further solid-state fermentation as in overripe tempe increased the total soluble amino

acid [12-13], increasing its value as protein source. Further sensory evaluation also indicating

the potency of overripe tempe to provide umami-flavor and enhance other taste [13], which

make it a potential to be developed as plant-based umami source ingredients (USI). Attempt

has been made to create standard of physical and chemical characteristic of overripe tempe

based on its potential use as USI [14]. However, the direction of product development is

required for the formulation of overripe tempe ingredient. In this study, the most potential

utilization of overripe tempe powder as plant-based USI was defined using structurized market

survey and formulation in consumer-led product development.

Materials and methods

Market survey

Survey area and respondents were first defined as tools in the market survey. Selected

survey area is modern market meeting these criteria: (1) located inside the shopping mall; (2)

place at least five variance of USI from different brands. Respondents of defined survey area

were validated using below criteria: (1) visited at least one of the supermarket in target market

area with minimum shopping frequency 4 times a month; (2) prepared their food themselves at

home minimum 5 times a week; (3) have ever used USI in their cooking. Candidates of target

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commercial USI from the defined market area matched following criteria: (1) sold in selected

survey area; (2) used by validated respondents; and (3) used in Indonesian savory dishes. The

respondents were subsequently requested to select important attributes of the selected umami

source ingredients that affect their decision of purchase on consumption. Furthermore the

respondents were also requested to select the Indonesian savory dishes that match the

application of selected umami source ingredients.

Overripe tempe powder formulation

Overipe tempe was grinded and exposed to oven drying (60 °C, 6 h), and freeze drying

(-80 °C, 24 h) to form powder with moisture content below 10% as described in previous study

[12]. In the market survey target commercial umami source ingredient was defined along with

its important attributes that affect consumer preference. The most important attribute in

combination with Indonesian national standard (SNI) of the product were then used as

guidelines for the umami source ingredient formulation development using overripe tempe.

The formulation used Design Expert® software version 6.0.8 using limitation derived from

SNI. And the selected formula was then compared to the commercial target using hedonic and

umami intensity rating sensory evaluation. And further improvement was acquired from

physical chemical characteristic and naive consumer response to the umami source ingredients

formulated from overripe tempe powder.

Sensory evaluation

Two types of sensory evaluation were performed to the selected formula of umami

source ingredients from overripe tempe powder, which were hedonic evaluation and umami

intensity rating test. In hedonic sensory evaluation, 30 naive respondents with no aversion to

soy and soy derivative products were employed to evaluate the acceptance of the product.

Responses were measured using nine ordinal responses from extremely dislike to extremely

like and were the analyzed statistically using Wilcoxon test. The umami intensity-rating test

was conducted using 30 panelists that have followed screening on basic tastes and 42 h training

on sensory attributes, sensory evaluation protocol, basic tastes introduction and recognition,

and also basic tastes ranking and rating for pure solution and products. Responses for the

umami intensity-rating test were measured using nine numerical responses with 1 defined as

not detected and 9 defined as very strong intensity and were analyzed statistically using

Friedman test.

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Results and discussion

Epidemiological study showed that socioeconomic status (SES) is highly related with diet

preference and quality. Higher nutrition knowledge and awareness on well-being might

contribute to the higher diet quality in people of higher SES [15]. The higher SES group tends

to have more consumption of lean-protein, whole grains, and plant-based products [16].

Therefore in this study, survey area was defined as commercial market in Tangerang City,

Indonesia, where higher SES group tend to shop for their groceries and those that provide

higher alternative protein or umami-source source ingredients. Six modern supermarkets were

then validated as the survey area, which were then used as criteria in the selection of

respondents and target commercial umami-source ingredient. Respondents taken were those

having habit to shop in the survey area with minimum frequency 4 times a month, and have

been using umami-source ingredients in their daily cooking. Thirty validated respondents were

then chosen to conduct the survey.

Overripe tempe (OT) higher glutamic acid contents shown to contribute to the umami-

taste of OT and was able to enhance the intensity of basic tastes [13]. Processing of OT into

powder was made as an attempt to increase the shelf life, availability, and practicality in the

overripe tempe further development as umami-source ingredients [12-13], and thus powder

form was taken into account in the selection of target commercial umami source ingredients.

Long history of OT usage in Indonesian savory dishes [17] was also taken into account as one

criterion in the selection. Based on the criteria, five varieties of USI were chosen as target

commercial product. Trends of usage and applications of the selected USI, showed instant

chicken stock as the most preferred USI and soup or soup like dishes as the most preferred

application (Table 1).

Subsequent survey to select attributes of instant stock that contribute to consumer

preference showed that taste was the most important attributes, followed by aroma, and natural

source of ingredients (Table 2). National standard of Indonesia for stock and consommé [18]

requires commercial chicken stock to meet following requirements: (1) total nitrogen minimum

100 mg/l; (2) total fat minimum 3 g/l; (3) maximum salt 12.5 g/l; and (4) negative

microorganism and limited metal contamination. And therefore the OT stock formula was

developed to meet total criteria (1) and (2) using commercial chicken stock formula as starting

formula.

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Table 1. Trend of usage and application of umami source ingredients selected in the market

survey

Umami Source

Ingredients

(USI) Varieties

Usage

trend

(%)1

Application trend of each USI in Indonesian savory dishes (%)2

Curry

(-like)

dishes

Grilled

dishes

Mari-

nated

dishes

Noodle

-based

dishes

Porridge

-based

dishes

Rice-

based

dishes

Soup

(-like)

dishes

Vegetable-

based

dishes

Instant chicken

stock

96.67 46.67 20.00 40.00 30.00 40.00 10.00 93.33 10.00

Monosodium

glutamate (MSG)

66.67 36.67 10.00 20.00 43.33 16.67 3.33 53.33 3.33

Chicken-flavored

flavor enhancer

93.33 63.33 53.33 50.00 43.33 36.67 20.00 86.67 16.67

Mushroom- and

plant-based stock

73.33 30.00 33.33 23.33 20.00 13.33 10.00 46.67 23.33

Mixed spices and

herbs seasoning

76.67 66.67 43.33 16.67 26.67 3.33 10.00 16.67 6.67

1Values were expressed as percentage of respondents that used the USI in comparison to total response

2Values were expressed as percentage of respondents that chose a certain application of the USI in comparison to total response

Table 2. Attributes that affect instant stock preference

Attributes of instant stock Impact on preference (%)1

Aroma 60.61

Natural ingredients 51.52

Price 18.18

Taste 87.88

Other 27.27

1Values were expressed as percentage of respondents that considered the attributes are important factor in affecting

their decision to consume or purchase instant stock in comparison to total response

Freeze and oven-dried OT powder was prepared as 2.0 % w/v solution and compared for its

sensory attributes intensity, except umami intensity that was compared in four different

concentration (0.5, 1.0, 1.5, 2.0%). Statistically, significant difference was only found in the

color attributes where oven-dried OT powder in solution has darker color compare to the

freeze-dried (Figure 1). Heating may induce non-enzymatic browning reaction such as Maillard

[19], especially due to presence of free amino acid and simple carbohydrates as product of

mould and bacteria activities during OT fermentation [20]. Maillard reaction was also known

to contribute in the production of chemical components correlated with savory flavors [19].

The umami intensity between oven-dried and freeze-dried OT powder in solution has no

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significant different statistically, however in all four concentration tested, umami intensity of

oven-dried OT in solutions were constantly higher in value (Table 3). In agreement with

previous study aroma of freeze-dried OT powder was significantly stronger than the oven-dried

OT powder [12], however, upon the dilution in water, difference in the intensity was not

detected. As taste was considered as the most important attribute in stock, further OT

formulation was done using oven-dried OT powder.

Table 3. Sensory attributes intensity of oven-dried and freeze-dried overripe tempe powder in

solution

Attribute intensity

evaluated

Overripe tempe (OT)

dissolution

concentration (% w/v)

Oven-dried OT Freeze-dried OT

Aroma 4.0 6.15 + 1.58a 5.94 + 1.58 a

Color 4.0 5.45 + 1.52 a 1.94 + 1.39 b

Taste:

Bitterness 2.0 2.75 + 1.55 a 2.35 + 1.57 a

Saltiness 2.0 3.54 + 2.57 a 3.66 + 2.82 a

Sourness 2.0 2.63 + 1.31 a 2.70 + 1.34 a

Sweetness 2.0 1.25 + 0.98 a 1.80 + 1.42 a

Umami 0.5 4.36 + 2.03 a 3.55 + 1.99 a

Umami 1.0 4.70 + 1.91 a 4.30 + 1.93 a

Umami 1.5 5.48 + 2.25 a 4.58 + 1.93 a

Umami 2.0 7.15 + 1.16 a 6.85 +1.91 a

1Values were expressed as mean (n=30) + standard deviation. Numbers with similar alphabet indicated no

significant differences (p > 0.05) along the same row.

Commercial chicken stock formula was defined as starting formula in the development of OT

stock. Major ingredients used were oven-dried OT powder in replacement of chicken extract

and flavor enhancer, and other ingredients added were salt, oil, caramel syrup, garlic powder,

and pepper (Table 4). Formulation was made in correlation to Indonesian standard (SNI)

minimum requirement of nitrogen and fat (100 mg/l; 3 g/l) and maximum limit of salt (12.5

g/l) [18]. Formula optimization was conducted using maximum total nitrogen, minimum

dissolution time, and minimum cost as decision factor. The selected formula (26.5% of OT

powder, 20.7% salt, 20.0% oil, 20.0% of caramel syrup, 6.5% garlic powder, and 6.3% of

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pepper) was subsequently tested for its conformity to SNI (Table 5) and compared to the

commercial products in term of sensory intensity and acceptance solubility properties, color,

and protein content (Table 6).

Table 4. Ingredients of commercial chicken stock and overripe tempe stock formula

Ingredients

Composition (%) in

Referred

commercial

chicken stock

formula

Various OT

Stock

Formula

Selected OT

Stock formula

Chicken flavor, chicken

extract, yeast extract,

MSG, sugar, other

additives

32.71 -

-

OT Powder - 24.62-43.20 26.5

Salt 38.98 20.70-25.00 20.7

Liquid chicken fat 1.69 -

Vegetable oil 15.25 10.00-20.00 20.0

Sugar 10.17 -

Caramel syrup 0.17 10.00-20.00 20.0

Garlic powder - 6.48 - 10.48 6.5

White pepper - 2.00-6.30 6.3

Xanthan gun 0.17 0.00-2.34 0.0

Table 5. Comparison of overripe tempe stock conformity with requirement in Indonesian

National Standard (SNI)

Evaluation Criteria

Value in

Overripe

Tempe Stock

Requirement

in National

Standard

Total nitrogen (mg/L)

Total fat (g/L)

Sodium chloride (g/L)

228

5.44

3.31

min. 100

min. 3

max. 12.5

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The selected formula was shown to meet the Indonesian national standard (SNI) requirement

[18]. However OT stock had significantly lower umami and saltiness intensity (5.64 + 1.64;

5.35 + 1.64) that might contribute to its lower taste and overall acceptance (4.84 + 1.75; 5.48

+ 1.55) compared to the commercial products. Analysis of solubility properties and protein

content showed that OT stock had significantly higher insoluble solid and solubility time (1.71

+ 0.17%; 74.75 + 0.71 s) and significantly lower total protein content (8.99 + 0.56 mg BSA

eq/ml). The higher fiber content of OT stock as plant-based USI might contribute to above

situation. Color analysis showed that OT stock already has the same range of color with the

commercial stocks (Table 6).

Further market survey of OT stock as guideline for further improvement showed that

70% of the respondent dislike or neither like nor dislike the product. Most of the respondents

(83%) suggested "taste improvement and a small group of the respondents (10%) also

suggested consistency improvement. More specific consistency improvement might refer to

reduce the insoluble solid as indicated in 70% response stating "dislike" or "dislike very much"

the high amount of residue in the OT stock (Table 7). It is at best, that further development of

OT stock will work to improve the taste acceptance by increasing the umami intensity and

apply such processing to reduce the amount of insoluble solid residue after dissolution and also

the dissolution time.

Conclusions

Preliminary market study define chicken stock in soup or soup like-dishes as target commercial

product in the development of overripe tempe (OT) powder as plant-based umami source

ingredients. Oven-dried OT powder, were chosen as basic ingredients of OT stock, as it

constantly showed higher umami intensity value in various concentration compared to the

freeze-dried. The formula of OT stock were developed using emphasize to meet requirement

of the Indonesian national standard (SNI) and taste acceptance. The selected formula (26.5%

of OT powder, 20.7% salt, 20.0% oil, 20.0% of caramel syrup, 6.5% garlic powder, and 6.3%

of pepper) successfully met the national standard requirement. However, further development

is required to improve taste, as well as to reduce the amount of insoluble solid residue.

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Table 6. Comparison of sensory intensity and acceptance, solubility properties, color, and

protein content of overripe tempe (OT) stock with commercial stock products.

Evaluation items OT Stock Commercial 1 Commercial 2

Sensory intensity1:

Aroma

Umami

Saltiness

5.52 + 2.50 a

5.65 +1.64 a

5.35 + 1.64 a

6.55 + 1.73 a

7.68 + 1.78 c

8.16 + 1.07 b

5.55 + 2.00 a

6.81 + 1.35 b

5.58 + 1.71 a

Sensory acceptance1:

Color

Aroma

Taste

Overall

5.06 + 1.95 a

6.16 + 2.44 a

4.84 + 1.75 a

5.48 + 1.55 a

4.94 + 2.02 a

7.42 + 1.31 a

6.13 + 2.45 b

6.29 + 1.75 b

5.58 + 1.80 a

6.84 +1.49 a

7.10 + 1.87 c

7.10 + 1.75 c

Solubility properties1:

Solubility time (s)

Soluble solid (% Brix)

Insoluble solid (% w/w)

74.75 + 0.71 a

1.15 + 0.07 a

42.75 + 1.75 a

18.00 + 0.71 b

1.55 + 0.07 b

20.00 + 0.75 b

16.75 + 1.06 c

2.05 + 0.07 c

6.00 + 0.50 c

Color (L*, a*, b*)2 27.22; 1.95; 2.44 29.48; 1.06;

4.00

28.06; 0.85;

3.70

Protein content (mg BSA eq/ml) 1 8.99 + 0.56 a 11.42 +0.43 c 10.14 + 0.27 b

1Values were expressed as mean (n=3) + standard deviation. Numbers with similar alphabet indicated no

significant differences (p > 0.05) along the same row.

2L* (Lightness): dark (0) to white (100); a*: green (-80) to red (+80); b*: blue (-80) to yellow (+80)

Table 7. Trend of suggestion for improvement area and acceptance level in the suggested area

Suggestion on Improvement Acceptance on Suggested Area of Improvement

Area of

improvement

Suggestion

Trends1 Acceptance Level

Trend on

Taste2

Trend of High

Insoluble Residue2

Taste

Consistency

Other

83%

10%

7%

Like or like very much

Neither like nor dislike

Dislike or dislike very much

30%

40%

30%

0%

30%

70%

1Values were expressed, as percentage of respondents that considered the attributes requires improvement in

presented OT stock to create better sensory acceptance in comparison to total response

2Values were expressed, as percentage of respondents that chose a certain level of acceptance of presented OT

stock in comparison to total response

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Acknowledgements

This research project was supported by a grant from the Directorate General of Resources for

Science, Technology and Higher Education of the Republic of Indonesia with contract number

SP DIPA-042.06.1.401516/2017, 6 December 2016.

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pneumoniae During Tempeh Fermentation and Proof of Enterotoxin Absence by PCR. Appl

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

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[11] Liem IT, Steinkraus KH, and Cronk TC. Production of Vitamin B-12 in Tempeh, a

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Microbial Changes During Storage of Overripe Tempeh Powder as Seasoning Material. Int. J.

Sci. Eng. 2015; 8(2): 131-134.

[13] Gunawan-Puteri MDPT, Hassanein TR, Prabawati EK, Wijaya CH, Mutukumira AN.

Sensory Characteristics of Seasoning Powders from Overripe Tempeh, a Solid State Fermented

Soybean, Pro. Chem. 2015; 14: 263-269.

[14] Djunaidi S, Gunawan-Puteri MDPT, Wijaya CH, Prabawati EK, Physicochemical &

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Extraction and stability analysis of antioxidant activity from

Stenochlaena palustris

Della Rahmawati*, Nadia Amanda Rifky, Abdullah Muzi Marpaung

Department of Food Technology, Faculty of Life Science and Technology, Swiss German University, Indonesia

*Corresponding author: [email protected]

Abstract:

Stenochlaena palustris is an edible fern from the Blechnaceae family and is a known natural

antioxidant sources. Antioxidants are compounds with a molecular structure that is able to

donate its electrons to free radical molecules and terminate the chain reaction. Antioxidants

have a function to prevent various diseases caused by oxidative stress. The human body will

be healthier if vegetables and fruits with high antioxidant content are sufficiently consumed.

The aims of this research was to determine the proper maceration solvent to obtain an S.

palustris extract with the highest antioxidant activity and to analyze the antioxidant stability

through different temperatures, pH, light condition, and time of the extract S. palustris from

the selected solvent. The solvents used were ethanol 70% and distilled water with three

different extraction times, 12, 24, and 48 hours at room temperature. It is revealed that

maceration using distilled water for 48 hours had the highest antioxidant activity compared to

other extraction samples using DPPH assay. The extract with highest antioxidant activity was

observed through stability test with different temperatures (50C, 300C, 500C, 700C, 900C), pH

(4, 5, 6, 7), and light conditions (dark and bright). The stability test revealed that the antioxidant

activity, total phenolic and flavonoids content of the selected extract on pH 4 and 5 stored at

700C was more stable than the other conditions.

Keywords: S. palustris, antioxidant activity, extraction, stability.

Introduction

Free radicals trigger cell damage by pairing the unpaired electron with other molecule, which

this unification will lead to oxidative stress process in cells and molecules. Furthermore,

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doi: 10.10.14457/MSU.res.2017.19
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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oxidative stress resulted from free radicals will contribute to several chronic diseases, for

instance, diabetes mellitus, heart disease, cancer, stroke, and Alzheimer [1]. Therefore, in

order to neutralize free radicals, a human body definitely needs antioxidant. Antioxidants are

abundant in some plants are present in seeds, vegetables, and fruits [2]. One of the sources of

natural antioxidant is from Kalakai plant or Stenochlaena palustris that is arises in tropical

district especially Central Kalimantan [3]. As supported by Ho et al (2010), bioactive

components of ferns mainly belong to phenolic, flavonoids, alkaloid, and terpenoid families.

Dai & Mumper (2010) added that phenolic compounds and flavonoids have been demonstrated

to be potent antioxidant. According to the experiment done by Chai et al (2012), S. palustris

had total of 51.69 mg/g dry matter polyphenol content and 58.05 mg/g dry matter flavonoids.

In previous experiments, the antioxidant extract of S. palustris plant was obtained through

maceration. Fidrianny et al (2013) reported that antioxidant activity is influenced by the

polarity of solvent and length of extraction time. Thus, extraction of S. palustris in different

choices of solvent and length of times needs to be done in order to determine which condition

will provide maximum extraction based on antioxidant activity.

Furthermore, the utilization of antioxidant compounds in food industries is getting

better along with the growing of free radical awareness [8]. Some of studies have showed the

relationship between antioxidant-rich foods with prevention of human diseases [9].

Antioxidant as one of functional characteristic that is had by S. palustris is the potential for its

utilization as food ingredient. Additionally, S. palustris could be harvested in the wild and sold

in food markets [6]. However, S. palustris plant has not been utilized on food products

industrially. Means, the proper condition to maintain its antioxidant activity during food

processing is still unknown. In this research, analysis of S. palustris plant as a source of

antioxidant in foods and beverages and its stability condition will be performed.

Materials and methods

The materials which were used in this research are the Kalakai plant or S. palustris, which were

obtained from Central Kalimantan, Indonesia. The used chemical substances were ethanol

70%, distilled water, DPPH (1,1-diphenyl-2-picrylhydrazyl), methanol, Folin-Ciocalteu

reagent, galic acid, deionized water, quercetin, aluminum chloride dehydrate, ethanol 70%,

ethanol 96%, sodium carbonate, sodium nitrite, sodium hydroxide, pH buffer solution 4-7.

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DPPH radical scavenging activity for antioxidant activity analysis, total phenolic

content, and total flavonoids content of S. palustris extract was determined using the method

which previously modified by Chai et.al (2012).

Sample preparation

S. palustris plant from Kalimantan was used as the raw material. The plant was cleaned

from dirt, washed, drained, and weighed as wet weight. Then, the plant was oven dried for 24

hours at 40℃ to totally remove the moisture content and weighed again as a dry weight. Next,

the dried sample was blended into powder, sieved and put to plastic bag in order to avoid

humidity and filth

Extraction

After the powder of S. palustris plant was obtained from prepared sample, the powder

and solvent solutions (ethanol and water) were combined with ratio 1:20 [6]. Using water bath

shaker, the extraction of antioxidant from S. palustris was started at two different types of

solvent which are ethanol and water and three different times which are 12, 24, 48 (hours).

Further, the maceration process was stopped and the samples were filtered with filter paper and

vacuum filtration. Then, the samples were centrifuged at 6000 rpm at room temperature for 20

minutes. The most proper maceration method was next determined after the data analysis was

taken from UV-Visible Spectrophotometer on purpose of obtaining antioxidant activity, total

phenolic content, and total flavonoid content of the sample extract.

Antioxidant activity assay

DPPH radical scavenging activity of S. palustris extract was determined using the

method which previously modified by [6]. In preparing the DPPH assay, 39.4 mg of DPPH

were diluted with 1 L of analytical grade methanol to make 0.10 mM DPPH solution. In

undertaking the analysis, 1 ml of S. palustris extract was mixed with 1 ml of 0.10 mM DPPH

solution. Next, the mixture was left for 30 minutes in dark condition. Afterwards, the

absorbance of the mixture was measured at 517 nm using UV-Vis spectrophotometer.

Likewise, the blank sample was prepared by replacing DPPH solution with methanol. DPPH

scavenging ability (%) was estimated as follows:

DPPH radical scavenging ability (%) = (A_control-A_sample)/A_control x 100 (1)

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The results are showed as IC50 number, which represents the capability of the

concentrations of sample extract to inhibit 50% of DPPH radicals [6].

Total phenolic content analysis

The concentration of total phenolics in S. palustris was completed using a Folin-

Ciocalteu colorimetric assay [6]. Gallic acid was used as a standard. Zero to 100 mg/l gallic

acid were prepared to make a standard curve. In conducting the analysis, 0.2 ml of S. palustris

extract was added with 0.8 ml of deionized water and 0.1 of Folin-Ciocalteu reagent. Then, the

mixture was incubated at room temperature for 3 minutes. Afterwards, the previous mixture

was added with 0.3 ml of sodium carbonate (Na2CO3) (20% w/v). Next, the mixture was

incubated again at room temperature but for 120 minutes. After that, the absorbance of the

mixture was measured at 765 nm using UV-Vis spectrophotometer. Total phenolic content was

represented in mg gallic acid equivalents (GAE)/g dry matter.

Total flavonoid content analysis

The concentration of total flavonoids in S. palustris extract was accomplished using a

method adapted from [6]. Zero to 500 µg/ml quercetin were dissolved in 80% analytical grade

ethanol and next used as a standard calibration curve. In undertaking the analysis, 0.2 ml of

S.palustris plant extract was added to 0.15 ml of NaNO2 (5% w/v). Then, the mixture was

incubated for 6 min at room temperature. Next, 0.15 of AlCl3.6H2O (10% w/v) was added to

the mixture and abandoned again at room temperature for 6 minutes. Afterwards, the mixture

was mixed together with 0.8 ml of sodium hydroxide (10% w/v). After that, the mixture was

left again for 15 minutes at room temperature. The mixture was then read at 510 nm using UV-

Vis spectrophotometer. For the blank, the sample of S.palustris extract was replaced with

water. Total flavonoid content was expressed in mg quercetin equivalents/g dry matter.

Stability Test

The stability test during storage was done by firstly conducting an extraction of

antioxidant by using the obtained solvent and time for S. palustris dried plant. Afterwards, the

extract will be then divided into four different food pH levels; 4, 5, 6, 7 with the addition of pH

buffer solution 4-7. The stability during storage were observed at 5oC, 30oC, 70oC, and 900C.

Next, the samples also will be tested through light exposures; in dark and transparent vials. The

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stability test resulted the data analysis of antioxidant activity (DPPH), total phenolic content,

and total flavonoid content.

Results and discussion

Stenochlaena palustris was extracted through various treatments in purpose of determining the

most proper condition of extraction. The treatments included two factors. The factors are three

different extraction times (12, 24, 48 hours), and 2 different solvents (ethanol and water).

Extraction with water as the solvent and 48 hours as the extraction time was found to be the

most proper condition to obtain high antioxidant activity, total phenolic and flavonoids content

of S. palustris.

Table 1. Antioxidant activity, Total phenolic content and Total flavonoid content of S. palustris

extract extracted by water for 48h.

Response Content

Antioxidant Activity (IC50), µg/ml 4.20 0.01

Total Phenolic Content, mg GAE/g 4.85 0.07

Total Flavonoids Content, mg QE/g 4.24 0.04

The stability test was completed with four different pH levels, which were pH 4, 5, 6, and 7

storage for 6 days showed in Figure 1. It is presented that pH 4 had lowest decreasing value of

percent antioxidant activity during 6 days storage followed by pH 5. This result was supported

by previous research that is stated that plant aqueous extracts were able to inhibit H2O2 as free

radicals more effectively at acidic pH [11]. Additionally, S. palustris extract was proven to

have high average amount of antioxidant activity compare with vitamin C [12]. Vitamin C is

an electron donor and it is also a powerful water-soluble antioxidant in humans [13]. Moreover,

Vitamin C is more potent as antioxidant at lower pH [14].

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Figure 1. Antioxidant activity at pH 4-7 during stability

During storage, total phenolic content of S. palustris extract was also observed in pH 4, 5, 6,

and 7. As the result, during storage phenolic content in pH 5 showed higher value than other

pH condition (Figure 2). Some naturally occurring polyphenolic compounds are damaged when

exposed to higher pH [15]. High amount of hydroxycinnamic acids and their proportion in total

phenolic content was found to increase in the extract of S. palustris [6]. Total flavonoids

content of S. palustris extract was also observed in pH 4, 5, 6, and 7 during storage. It is

presented that total flavonoid in pH 5 had higher value compared to other pH treatment.

Anthocyanins were likely the key of flavonoids compounds responsible for radical scavenging

activity in the extract of young fronds of S. palustris [6]. Further, anthocyanins are considered

as flavylium cation, which is stable in acidic pH.

Figure 2. Total phenolic content at pH 4-7 during stability

The stability test on different temperatures was observed to find out the effect of thermal

coverage on antioxidant from S. palustris. The stability test was completed with five different

temperatures, which were 5oC, 30oC, 50oC, 70oC, and 90oC. pH 4 and 5 were not too influenced

by heat treatment since the data indicates a tendency of higher antioxidant activity, phenolic

0

20

40

60

80

0 2 4 6

% A

nti

oxi

dan

t A

ctiv

ity

Day

pH 4

pH 5

pH 6

pH 7

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and flavonoids content. In contrary, pH 6 and 7 were affected by heat treatment where there

was a decrease in antioxidant activity, phenolic and flavonoids as well. Many phenolic

compounds are easily hydrolyzed and oxidized when temperature is increased [5] and some

naturally occurring polyphenolic compounds are damaged when exposed to high pH [15]. The

temperature of 70oC was found to be the suitable temperature storage condition for antioxidant

activity and total flavonoids content, while 50oC is the suitable temperature storage condition

for total phenolic content.

Conclusions

Extraction with distilled water and for 48 hours were found to be the suitable condition for

antioxidant properties in S. palustris extract. During stability, pH 4 showed higher antioxidant

activity and pH 5 showed both higher phenolic and flavonoids content. 70oC was found to be

suitable for antioxidant activity and flavonoids content, while 50oC was suitable for phenolic

content.

Acknowledgements

We thank for Swiss German University to grant competitive research fund (to D.R).

References

[1] Bansal, A. K and Bilaspuri G.S. Impact of Oxidative Stress and Antioxidants on Semen

Functions (review article). Vet. Med. International. 2011, DOI:10.4061/2011/686137

[2] Vinay R Patel, Prakash R Patel and Sushil S. Kajal. 2010. Antioxidant Activity of Some

Selected Medicinal Plants in Western Region of India. Advances in Biological Research, 2010:

4(1), 23-26.

[3] Eko suharton, Ella Viani, Mustaqim Apriyansa Rahmadhan, Imam Syahuri Gultom,

Muhammad Farid Rakhman, and Danny Indrawardhana. Total flavonoid and antioxidant

activity of some selected medicinal plants in South Kalimantan of Indonesian. APCBEE

Procedia, 2012 : 4, 235-239.

[4] Ho, R., Teai T, Bianchini JP., Lafont R and Raharivelomanana P. Ferns: From traditional

uses to pharmaceutical development, chemical identification of active principles. In: Kumar

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

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[5] K., Fernandez H., Revilla M. (eds) Working with ferns. Springer, New York, 2011, p. 321-

346.

[6] Dai, J. & Mumper, R. Plant Phenolics: Extraction, analysis and their antioxidant and

anticancer properties. Molecules. 2010, DOI: 10.3390/molecules15107313

[7] Chai, T., Panirchellvum, E., Ong, H. and Wong, F. Phenolic contents and antioxidant

properties of S. palustris, an edible medicinal fern. Bot Studies. 2012: 53, 439-446.

[8] Fidrianny, I., Listya, P. & Komar, R. W., Antioxidant Activities from Various Bulbs

Extracts of Three Kinds Allium Using DPPH, ABTS assays and Correlation with Total

Phenolic,Flavonoid, Carotenoid Content. International Journal Research of Pharmaceutical

Science. 2016: 5 (Issue 4), 189-194

[9] Stephanie, C. 2015. Karakterisasi Simplisia dan Skrining Fitokimia serta Uji Aktivitas

Antioksidan Ekstrak Etanol Herba Kelakai (S. palustris (Burm.f.) Bedd). Universitas Sumatera

Utara.

[10] Rathore, G., M. Suthar, A. P. & R.N., G., 2011. Nutritional antioxidants: A battle for better

health. J. Nat. Pharm. 2011: II, 2-14.

[11] Chai, T.-T. & Wong, F.-C. Antioxidant properties of aqueous extracts of Selaginella

willdenowii. Journal of Medicinal Plants Research. 2012, DOI: 10.5897/JMPR11.1376

[12] Bayliak, M. M., Burdyliuk, N. I. & Lushchak, V. I., 2016. Effects of pH on antioxidant

and prooxidant properties of common medicinal herbs. Open Life Science Journal. 2016 : DOI:

https://doi.org/10.1515/biol-2016-0040

[13] Maharani, D., Haidah, S. & Haiyinah. Studi Potensi Kelakai (S. palustris (Burm.f.) Bedd)

Sebagai Pangan Fungsional. Program Kreativitas Mahasiswa Penelitia. 2005, p. 1-13

[14] Sebastian J. Padayatty, Katz A, Wang Y, Eck P, Kwon O, Lee JH, Chen S, Corpe C, Dutta

A, Dutta SK, Levine M. Vitamin C as an Antioxidnt: Evaluation of Its Role in Disease

Prevention. Journal of the American College of Nutrition. 2003: XXII(1), 18-35.

[15] Gramlich, G., Zhang, J. & M. Nau, W., 2002. Increased Antioxidant Reactivity of Vitamin

C at Low pH in Model Membranes. J. Am. Chem. Soc. 2002: DOI: 10.1021/ja026927b

[16] Settharaksa, S.,Jongjareonrak, A., Hmadhlu, P., Chansuwan, W. and Siripongvutikorn, S.

Flavonoid, Phenolic Contents and Antioxidant Properties of Thai Hot Curry Paste Extract and

Its Ingredients as Affected of pH, Solvent Types and High Temperature. International Food

Research Journal. 2012: IV(19), 1581-1587.

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Prevalence of foodborne pathogens in ready-to-eat foods in the markets in

Khon Kaen, Thailand

Pitchayapa Pholkaw, Patimakorn Pasuwan*

Department of Food Technology, Faculty of Technology, Khon Kaen University, Khon Kaen, Thailand, 40000

*Corresponding author: [email protected]

Abstract:

The aims of this research were to evaluate consumer behavior regarding the consumption of

ready-to-eat foods and to evaluate the prevalence of pathogens in ready-to-eat food products

obtained from the markets of Khon Kaen, Thailand. As to the consumer behavior survey, top

three highly consumed ready-to-eat foods had been reported as fresh vegetable salads, followed

by sandwiches and Thai fresh spring rolls, respectively. Subsequently, the microbial

contamination in the selected ready-to-eat food products was collected, analyzed and reported

as the prevalence of this study. The 54 samples were collected from two types of markets; open

markets and supermarkets located in Khon Kaen. The presence of contaminated pathogenic

bacteria; Salmonella spp., Listeria monocytogenes, Staphylococcus aureus was investigated by

selective media. Poor hygiene was indicated by total coliforms and the presence of Escherichia

coli. The results revealed that Salmonella spp. was detected in 5 out of 27 (19%) samples in

open markets and 5 out of 27 (19%) samples in supermarkets. L. monocytogenes was detected

in 4 out of 27 (15%) samples in open markets and 3 out of 27 (11%) samples in supermarkets.

S. aureus was detected in 8 out of 27 (30%) samples in open markets and 9 out of 27 (33%)

samples in supermarkets. Total coliforms were contaminated in 27 out of 27 (100%) samples

in open markets and 23 out of 27 (85%) samples in supermarkets. E. coli was contaminated in

2 out of 27 (7%) samples of open markets and 1 out of 27 (4%) samples of supermarkets. A

contamination of L. monocytogenes in open markets was higher than that in supermarkets. A

contamination of S. aureus in supermarkets was higher than that in open markets; however, a

contamination of Salmonella spp. in supermarkets was equal to that in open markets. This data

provided food safety information of ready-to-eat foods for consumption of both markets in

Khon Kaen.

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doi: 10.10.14457/MSU.res.2017.18
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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Keywords: Prevalence, ready-to-eat, foodborne pathogens, Thailand

Introduction

Ready-to-eat foods are very popular in Thailand [1]. As to SCRIBD website, Thailand is in the

first ranking of top 10 markets which claim to purchase ready-to-eat foods frequently [2]. Thai

consumers are the most frequent buyers of ready-to-eat foods in the world. Due to these foods

are convenient, inexpensive and readily available in the markets.

Salmonella spp., Listeria monocytogenes and Staphylococcus aureus are commonly

contaminated bacteria in ready-to-eat foods and caused foodborne diseases. Moreover, the

presence of total coliforms and Escherichia coli in these foods are confirmed of poor hygienic

practices [3]. The presence of these pathogens in ready-to-eat foods is also likely to be either

post contamination or cross contamination in food products. If the level of contamination of

foodborne pathogens is high, it may lead to public health issues. Salmonella spp., Listeria

monocytogenes, Staphylococcus aureus and Escherichia coli are normally found in soil, water,

air as well as animal and human intestine. Therefore these pathogens can easily contaminate in

foods. Moreover, the production of ready-to-eat foods regarding preparation, storage and

distribution; some steps have been performed without any heating step that can involve the

contamination of both pathogens. Thus, the aims of this study were to evaluate the frequency

of ready-to-eat foods consumption and to estimate risk assessment or the probability of

foodborne illness for the sample collections of ready-to-eat food products obtained in markets

of Khon Kaen, Thailand.

Materials and methods

Sample Collection

According to consumer behavior survey, the questionnaire survey was performed to

achieve the data. The questionnaire survey was collected, analyzed and interpreted of the

experiences and behaviors of a group of people from a target population through the asking of

questions. The questionnaire survey was divided into two parts to obtain the general

information (gender, age, education, and occupation) and consumer behavior (types of ready-

to-eat foods, frequency of consumption, places to buy and health issues when consuming ready-

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to-eat foods). A total of 100 persons participated in the questionnaire. The results from

consumer behavior survey were used as criteria of sample collections for the second part of the

study. This consisted of 54 samples of ready-to-eat foods composing of 18 samples each of

fresh vegetable salads, sandwiches, and Thai fresh spring rolls that were purchased in local

markets and analyzed in the laboratory within 3 h [4].

Consumer Behavior Survey towards the Consumption of ready-to-eat Foods

Part 1 General Information

1. Gender

Male

Female

2. Age

16-25 years old

26-40 years old

41-65 years old

3. Education Level

Undergraduate less than bachelor/ high school

Bachelor degree

Master degree

Doctoral degree

4. Occupation

Government officer

Private company employee

State enterprise employee

Student

Business owner

Others

Part 2 Consumer behavior

5. How often do you eat ready-to-eat foods per week?

Less than twice a week

2-4 times per week

6-8 times per week

10-12 times per week

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More than 12 times per week

6. Which of the following ready-to-eat food do you often eat?

Hamburger

Sandwich

Salad

Thai fresh spring roll

Other

7. Where do you usually buy ready-to-eat foods?

Open market

Supermarket

Convenience store

8. Do you have any health issues when eating ready-to-eat foods? (e. g. diarrhea,

vomiting, headache, fever)

Yes

Never

Not sure

Microbiological Analysis

Salmonella spp.

A sample of 25 g was added to 225 ml of lactose broth [5] and incubated at 35 °C for

24 h ± 2 h [4]. Then, the 0.1 ml of an overnight sample was transferred to 10 ml of an

enrichment broth and broth was incubated for 24 ± 2 h at 42 ± 0.2°C. After incubation, RV

medium was streaked on xylose lysine deoxycholate (XLD) agar and incubated 24 ± 2 h at

35°C. Suspected colonies were submitted to Triple Sugar Iron (TSI), lysine iron agar (LIA)

slants and biochemical test.

Listeria monocytogenes

A 25 g sample was added to 225 ml of Half Fraser broth and incubated at 37°C for 48

h. A 0.1 ml portion of broth was transferred to 10 ml of Fraser Broth and incubated for 24 h at

35°C. A loopful of overnight culture was streaked on Oxford agar (OXA) and PALCAM and

incubated at 35°C for 24-48 h [5]. Suspected colonies from each selective agar were streaked

on purity to Trypticase soy agar with 0.6% yeast extract (TSAYE) and incubated at 30° C for

24-48 h [5]. All the isolates were subjected to biochemical tests and β-hemolytic activity.

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Staphylococcus aureus

A 25 g sample was added to 225 ml Butterfield's phosphate-buffered water. Decimal

dilutions were prepared by 10 ml from previous dilution plus 90 ml sterile dilution water. Each

1 ml of dilution was transferred to sample suspension and each dilution was spread onto three

plates of Baird-Parker agar and incubated at 35-37°C for 45-48 h [5]. Suspected colonies were

confirmed by the coagulase test with Rabbit plasma.

Total coliforms and Escherichia coli

A 25 g sample was added to 225 ml Butterfield's phosphate-buffered water. Decimal

dilutions were prepared by 10 ml from previous dilution plus 90 ml of sterile dilution water.

Each 1 ml portions of each dilution were transferred to three Lauryl Tryptose Broth (LST)

tubes and incubated at 35°C ± 0.5°C for 24 ± 2 h. The positive result was a gas production. For

negative result, tubes were re-incubated and examined again at 48 ± 2 h. The test was confirmed

on all presumptive positive (gas) tubes [5]. A loopful of each gassing LST tube was transferred

to EC broth and incubated at 45.5 °C for 24 ± 2 h. The positive result was gas production. As

to the negative result, broth was re-incubated and examined again at 48 ± 2 h. Results of this

test were calculated the numbers of fecal coliform as MPN/g [5]. A loopful of EC tube was

transferred and culture was streaked for isolation on L-EMB agar plate and incubated at

35°C±0.5°C for 18-24 h.

Statistical Analysis

The data was analyzed using descriptive analysis. The chi-square test was used to test

for differences. All statistical analysis was performed using SPSS program Version 19 or

Microsoft Excel 2010.

Results and discussion

Consumer Behavior Surveys

According to the consumer behavior survey questionnaires, the questionnaires survey

was focusing on the consumer’s behavior in Khon Kaen. The results of 100 questionnaires have

revealed 6-8 times of ready-to-eat foods consumption per week (32%). The open market was

the most favorite location to purchase ready-to-eat foods (65%) followed by supermarket

(28%) and convenience store (7%), respectively. These results are similar to the findings of

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other authors that showed the 76.29% of consumers in Nigeria, they would rather buy ready-

to-eat food products from open markets than supermarkets. Their reasons would be open

markets were close to their resident [7]. Ready-to-eat fresh vegetable salad was the most

consumed, followed by sandwiches and Thai fresh spring rolls, respectively. As to the

questions regarding health issues, 60% of consumers say they never experienced any health

issues when consuming ready-to-eat foods. However, the results revealed that 29% of the

consumers in Khon Kaen felt sick after food consumption and 11% of the consumers probably

felt sick when consuming the ready-to-eat food products.

Table 1 Socio-Demographic Characteristics of the Sample (n=100)

General information % of Total

Gender

Male 32%

Female 68%

Age group

16-25 years old 33%

26-40 years old 33%

40-65 years old 34%

Educational level

Undergraduate less than

bachelor/high school 23%

Bachelor’s degree 67%

Master’s degree 9%

Professional degree or PhD. 1%

Occupation

Government officer 30%

Private company employee 8%

State enterprise employee 9%

Student 36%

Business owner 9%

Others 8%

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Prevalence of Foodborne Pathogens

Open markets

From Table 2, total coliforms were the majority of the contamination in ready-to-eat

foods (100%), followed by S. aureus (30%), Salmonella spp. (19%), L. monocytogenes (15%)

and E. coli (7%), respectively. The number of total coliforms and E. coli was ranged from 110

to >11,000 and 30 to >750 MPN/g, respectively. S. aureus was detected in fresh vegetable

salads (44%), Thai fresh spring rolls (33%) and sandwiches (11%). Salmonella spp. was

detected in Thai fresh spring rolls (44%), fresh vegetable salads (11%) but not detected in

sandwiches. L. monocytogenes was detected in Thai fresh spring rolls (33%), fresh vegetable

salads (11%) and not detected in sandwiches. E. coli was detected in fresh vegetable salads

(22%), but not detected in Thai fresh spring rolls and sandwiches. Total coliforms were

detected in fresh vegetable salads, Thai fresh spring rolls and sandwiches (100%).

Supermarkets

From Table 2, the most contamination bacterium was total coliforms (85%), followed

by S. aureus (33%), Salmonella spp. (19%), L. monocytogenes (11%) and E. coli (4%),

respectively. The number of total coliforms was ranged from <30 to >11,000 MPN/g. E. coli

was reported as 30 MPN/g. S. aureus was detected in sandwiches (67%) and Thai fresh spring

rolls (33%) but not detected in fresh vegetable salads. Salmonella spp. was detected in fresh

vegetable salads (33%) and Thai fresh spring rolls (22%) but not detected in sandwiches. L.

monocytogenes was detected in sandwiches (33%) but not detected in both of Thai fresh spring

rolls and fresh vegetable salads. E. coli was detected in sandwiches (11%) but not detected in

both of Thai fresh spring rolls and fresh vegetable salads. Total coliforms were detected in Thai

fresh spring rolls (100%), sandwiches and fresh vegetable salads (78%), respectively.

The study compared products from two types of markets (n=54); S. aureus was the most

contaminated pathogen in ready-to-eat foods (31%), followed by Salmonella spp. (19%) and

L. monocytogenes (13%), respectively. According to poor hygiene indicators, total coliforms

were detected in ready-to-eat foods (93%) and E. coli was detected in ready-to-eat foods (6%).

From these results, Salmonella spp. and S. aureus were reported as two out of top 3 pathogens

that caused the illness in Thailand [6]. S. aureus was positively found in all ready-to-eat food

products. Because its resources from human body. It can present in the hair, nasal and skin of

vendors. Moreover, environmental conditions as humid weather in Thailand and products were

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stored under room temperature due to lack of refrigeration in open markets. Furthermore,

ready-to-eat foods were handled extensively during preparation and there was no further

processing such as cooking to inactivate potential pathogens [8]. As for the fresh vegetable

salads tested in this study, S. aureus was detected in 22%. This result was higher than those of

a similar study conducted by Hasan et al., which detected S. aureus in 12% of vegetable salads.

Salmonella spp. was detected in 22% of the tested salads, which is higher than the results of

the study of Mediterranean salads by Zeki et al., which detected 14% [9], but lower than the

41.8% found in the study of salads by Anderson et al. [10]. L. monocytogenes was detected in

6%, which is not detected in Mediterranean salads [9]. Moreover, L. monocytogenes in

sandwiches was detected in 17%. While the findings of a study by Moustafa et al. showed that

L. monocytogenes was detected in 14% [11]. In a comparison between the two types of markets;

the prevalence of L. monocytogenes in open markets was higher than in supermarkets. The

contamination of S. aureus in supermarkets was higher than the open markets, and Salmonella

spp. was equally found in both markets. These results differ from another study that showed a

prevalence of contamination of Salmonella and L. monocytogenes in supermarkets was higher

than in open markets [4]. The results of the consumer behavior survey showed the highest

frequency of ready-to-eat foods consumption as 6-8 times per week (32%), and contaminated

foodborne pathogens were found in all of ready-to-eat foods samples. It might be assumed that

if one serving of ready-to-eat food products is weighed about 200 g, the consumer who had

consumed the ready-to-eat food very often (6-8times/week) had much more chances to have

foodborne illness than the other group of consumers.

Conclusions

According to consumer behavior survey questionnaires, the results showed that the most

favorite location to purchase ready-to-eat food products of consumers in Khon Kaen was an

open market. Consumers in Khon Kaen were purchased ready-to-eat food products 6-8 times

per week. The results also revealed that fresh vegetable salad, followed by sandwich and Thai

fresh spring roll were top three of ready-to-eat foods consuming in Khon Kaen, Thailand. As

to the prevalence of foodborne pathogens in ready-to-eat foods of the markets in Khon Kaen,

the results showed that Salmonella spp., L. monocytogenes, and S. aureus had been found in

these food products. The indicators of poor hygienic practices; total coliforms and E. coli in

ready-to-eat foods also had been positive. The contamination of foodborne pathogenic bacteria

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in ready-to-eat foods in Khon Kaen possibly implied that the ready-to-eat food products in

open market and supermarket possibly caused foodborne illness for the Khon Kaen consumers.

The results in this study can be provided the awareness of ready-to-eat food consumption in

Khon Kaen for the consumers and also the guidance for local producers to apply safety

standards such as GMP, HACCP and personal hygiene for preparing foods.

Table 2 Prevalence of Foodborne Pathogens and Poor Hygiene Indicators in Ready-to-Eat

Foods in Markets in Khon Kaen, Thailand

Sample Isolation OM* Prevalence

(%) SM*

Prevalence

(%)

p-Value

**

Salad Salmonella spp. 1/9 11% 3/9 33% 0.169

L. monocytogenes 1/9 11% 0/9 0% 0.347

S. aureus 4/9 44% 0/9 0% 0.035

E. coli 2/9 22% 0/9 0% 0.169

Total coliforms 9/9 100% 7/9 78% 0.169

Sandwich Salmonella spp. 0/9 0% 0/9 0%

L. monocytogenes 0/9 0% 3/9 33% 0.081

S. aureus 1/9 11% 6/9 67% 0.013

E. coli 0/9 0% 1/9 11% 0.347

Total coliforms 9/9 100% 7/9 78%

Thai fresh

spring roll

Salmonella spp. 4/9 44% 2/9 22% 0.169

L. monocytogenes 3/9 33% 0/9 0% 0.081

S. aureus 3/9 33% 3/9 33%

E. coli 0/9 0% 0/9 0%

Total coliforms 9/9 100% 9/9 100%

*Number of positive samples/total of samples in the open market (OM) and the supermarket (SM)

** Significant at p < 0.05 (2-sided) based on comparison between supermarket and open market

Acknowledgements

This study was granted by the Graduate School of Khon Kaen University and by the new

researcher program of Khon Kaen University 2015, Khon Kaen, Thailand.

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[5] AOAC international. Bacteriological Analytical Manual. 8th ed, Gaithersburg, USA,

1998, p. 1-202.

[6] Food Poisoning, Available at: http://www.boe.moph.go.th/, accessed on May 2017.

[7] S. Ohen, G. E. Umeze, and E. O. Inyang, “Consumer Purchasing Behaviour for Fruits and

Vegetables among Civil Servants in Essien Udim Local Government Area, Akwa Ibom State,

Nigeria”, Food Science and Quality Management, vol. 23, 2014.

[8] Hasan A, Serdar C, Timothy HS. Incidence of Staphylococcus aureus in ready-to-eat

meals from military cafeterias in Ankara, Turkey. Food Control. 2004; 16, 531–534.

[9] Zeki G, Sebnem P, Yeliz Y, Nurhan E. The microbiological quality of ready-to-eat salads

in Turkey: A focus on Salmonella spp. and Listeria monocytogenes”, International Journal of

Food Microbiology. 2014; 196, 79–83.

[10] Anderson SS, Mariza L, Maria TD, Bernadette DGMF. Prevalence and counts of

Salmonella spp. in minimally processed vegetables in São Paulo, Brazil. Food Microbiology.

2011; 28, 1235-1237.

[11] Moustafa ES, Mohamed ES, Jordi M, Jose MS. Listeria spp. in Street-Vended Ready-to-

Eat Foods. Hindawi Publishing Corporation Interdisciplinary Perspectives on Infectious

Diseases. 2011; 2011, 1-6.

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Survival of probiotic bacteria in fruit juice jelly products

Warangkanang Ampornpat*, Borwonsak Leenanon

1Department of Food Technology Faculty of Technology Khon Kaen University

Nai Mueang, Mueang, Khon Kaen 40000 Thailand

*Corresponding author: [email protected]

Abstract:

The aims of this work were to determine the sensory evaluation (in terms of color, odor, flavor,

textureand overall liking) of three types of fruit juice jelly products, growth curves of

Lactobacillus acidophilus TISTR 1338 and L. casei TISTR 390 and their survivals in fruit juice

jelly products during storage at 5°C for 9 days. The sensory scores showed that there was no

significant difference of overall liking scores among jelly products. According to their growth

curves, both probiotic bacteria had entered the stationary phase with an average of 9 log cfu/ml

after incubation for 26 h. Probiotic bacteria were cultured for 26 h for studying the survival of

probiotic bacteria in fruit juice jelly products. It was found that the numbers of L. acidophilus

TISTR 1338 and L. casei TISTR 390 decreased from the initial numbers of 9.23 log cfu/g and

8.97 log cfu/g to 6.80 log cfu/g and 7.73 log cfu/g, respectively, after 9 days of refrigerate

storage. However, there was no significant difference (p > 0.05) between viabilities of L.

acidophilus TISTR 1338 and L. casei TISTR 390 on day 9. The survival of bacteria reduced

during storage; however the final cell concentrations were still higher than the minimum

therapeutic levels (≥5 Log cfu/g) for probiotics in the products.

Keywords: Probiotic bacteria, Survivals, Fruit juice jelly

Introduction

Probiotics are living microorganisms that provide a beneficial effect on human health, such as

a decrease in serum cholesterol level, reduction of lactose intolerance, stimulation of immune

system, improving the digestive system (Sanders et al., 2013), inhibiting the growth of

pathogenic bacteria as well as synthesizing vitamins and antimicrobial agents. The efficiency

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of added probiotics depends on the dose level and their viability throughout storage, product

shelf life and their survival in the gut environment (Kailasapathy & Chin, 2000). Therefore,

the concentration of probiotics in the product should be at least 105cfu g-1 or mL-1 at the moment

of consumption (FAO/WHO, 2001). Probiotic bacteria are usually added to dairy products,

such as yogurt and milk because there is no need for great changes in the manufacturing

processes. (Boza-Mendéz et al., 2012) but the use of dairy products may also be limited by

lactose-intolerance, allergies and vegetarian (Perricone et al., 2014).

Recently, there has been an interest in the development of fruit and vegetable juices as

functional beverages with probiotic (Mortazavian et al., 2006). Fruit juices may represent the

alternative means of providing probiotic cultures to consumers because they are considered

healthy products and are regularly consumed (Sheehan et al., 2007). Moreover, they are rich in

sugars, minerals and vitamins, which can be used by probiotics and they contain no starter

cultures to compete with the probiotic cultures for substrates (Ding & shah, 2008). However,

juice supplementation with probiotics is more complicated than in dairy products, since the

juices present insufficient quantities of peptides and free amino acid, required for the

metabolism of probiotic cultures and the strains are usually sensitive to more acidic conditions

(Sheehan et al., 2007). Moreover, probiotic cultures may change juice sensory characteristics

(Granto et al., 2010).

Few studies have evaluated the effect of supplementation of fruit juice jelly products

with probiotics. This can be seen as a further way to protectand preserve the probiotic cultures,

since the product is classified as moderately moist and stable on storage, which is expected to

reduce the growth rate of other microorganisms and also help stabilize the probiotic bacteria

that are added to the product to achieve the required standard level of survival during storage.

Therefore, the objectives of this study were to evaluate the sensory characteristics of

different fruit juice jelly products, growth curves of probiotic bacteria and their survivals in

those products during storage at refrigeration temperature.

Materials and methods

Preparation of fruit juice jelly products

Three different commercial fruit juices were purchased from a super market: orange

juice (pH, 4.22; soluble solids, 17 °Bx), Lychee juice (pH, 4.05; soluble solids, 15 °Bx) and

grape juice (pH, 3.49; soluble solids, 17 °Bx). Jelly preparation, drinking water (160 mL/L)

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was heated to 80 °C, mixed with 80 g/L of sucrose and 6 g/L of carrageenan, then stirred for 3

min and mixed with 752 mL/L of juices. The prepared jelly was adjusted to pH 3.5-3.7 with 2

g/L citric acid. Samples of different juices were poured in a plastic cup and left at room

temperature for gelling and stored at 5 °C.

Activated probiotic cultures

Freeze-dried cultures of L. acidophilus TISTR 1338 and L. casei TISTR 390 were

obtained from Bangkok MIRCEN, TISTR, Thailand. The strain were inoculated into MRS (de

Man, Rogosa, Sharpe) broth (HiMedia®) and incubated at 37 °C for 24 h, checked for purity

and maintained on MRS Agar (HiMedia®). The strain was reactivated for three consecutive

before use.

Growth curves determination of probiotic bacteria

The cultures were grown in MRS (de Man, Rogosa, Sharpe) (HiMedia®) medium 10%

(v/v) inoculum and incubated at 37 °C for 48 h. A 3 mL sample of the culture was taken every

2 h to determine the absorbance at OD600 via spectrophotometer and viable cell count using

pour plate method.

Sensory evaluation

The sensory properties such as color, odor, flavor, texture and overall liking of three

fruit juice jelly (orange, lychee and grape) were evaluated by 30 untrained panelists (11 women

and 9 men) through 9 point hedonic scale test (9 = like extremely, 1 = dislike). Data from each

panelist for each attribute were analyzed by two-way ANOVA.

Survival of probiotic bacteria in grape juice jelly

L. acidophilus TISTR 1338 and L. casei TISTR 390 were inoculated in MRS (de Man,

Rogosa, Sharpe) (HiMedia®) medium and incubated at 37 °C for 26 h. Cultures were harvested

by centrifugation at 2000xg at 25 °C for 15 min and washed twice with 0.85% sterile saline

solution. The washed bacterial cells were re-suspended in 0.85% sterile saline solution and then

added to the grape juices jelly solution. The grape juices jelly were then stored at 5 °C for 9

days.

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Statistical analysis

The data were submitted to ANOVA by using SPSS version 17 for windows.The

determination of significant differences among treatment means ± standard deviation was done

by Duncan’s new multiple range tests.

Results and discussion

Growth curves

The bacterial growth curves were presented in Fig. 1 for L. acidophilus TISTR 1338 and L.

casei TISTR 390. The relation between OD600 and log cfu/mL were used to estimate the

concentration of viable cells in MRS medium under the same growth conditions. As seen in

Fig. 1and Fig.2, it was found that both strains were in lag phase at 0 -2 h, with the numbers of

viable cells about 2- 2.5 log cfu/mL and then entered the log phase at 3-24 h, with an increase

in numbers. After that, they entered the stationary phase after 26 h, with an average number of

9 log cfu/mL.

Figure 1 OD 600 and viable count (log cfu/mL) of L. acidophilus TISTR 1338 and L. casei

TISTR 390over time in MRS medium incubated at 37 °C for 48 hrs.

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Sensory evaluation

The liking scores in term of color, odor, flavor, texture and overall liking of the jelly

products are shown in Table 1. The results revealed that all three types of fruit juice jelly

products had the liking scores in the range of 6-7 on a 9–point hedonic scale, indicating that

the panelists had evaluated the products liking scores from “slightly” to “moderately”. In

addition, attributes of color, odor, texture and overall liking of all products were not

significantly different (p>0.05). However, the flavor scores of lychee and grape jelly products

were significantly higher than orange jelly product (p≤0.05). Eventually, grape jelly product

was selected for further experiment.

Table 1 Sensory scores of three types of fruit juice jelly products

Means ± standard deviation in the same low followed by the same superscripts are not significantly different (p >

0.05).

Survival of probiotic bacteria in grape juice jelly.

The survivals of L. acidophilus TISTR 1338 and L. casei TISTR 390 in the grape juice

jelly during refrigeration storage (5 °C) for 9 days were determined. The numbers of surviving

bacterial cells on day 0, 3, 5, 7 and 9 are presented in Fig. 3. The numbers of L. acidophilus

TISTR 1338 and L. casei TISTR 390 decreased from the initial ones of 9.23 log cfu/g and 8.97

log cfu/g to 6.80 log cfu/g and 7.73 log cfu/g respectively, after 9 days of refrigeration storage.

However, there was no significant difference (p > 0.05) between viabilities of L. acidophilus

TISTR 1338 and L. casei TISTR 390 on day 9. The results were similar to the studies of Mousavi

et al., (2011), who reported that the microbial population of L. paracasei and L.

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acidophilus in pomegranate juice decreased approximately three logarithmic during the first

week of cold storage (4 °C) and lost their viability after 2 weeks. The reason could be related

to the lack of their ability to survive in the low pH and high acidity of the pomegranate juice.

Also Sheehan et al., (2007) reported that low pH fruit juices, with a range of pH typically

between 2.5 and 3.7, caused the bacterial sensitivity to stressful conditions to increase.

A loss of probiotic viability could be related to acidity and the presence of oxygen in

the product. When probiotic cells are present in low pH environments (<4.5), increased energy

is required to maintain the intracellular pH, causing a lack of ATP for other critical functions

and thereby causing cell death (Nualkaekul et al., 2011). In addition, the presence of oxygen

can cause formation and accumulation of toxic metabolites in cells, which can lead to cell death

by oxidative damage (Boza-Mendéz et al., 2012).

In order to enhance the survival of probiotic bacteria in low pH products, Sohail et al.,

(2012) improved the viability of L. rhamnosus and L. acidophilus using microencapsulation

method, which reduced the acidification and assured the survival of probiotics at 25 °C for at

least 9 days in orange juice. Ding and shah (2008) also reported that fruit juices containing

microencapsulated probiotic bacteria were more stable than those containing free probiotic.

Moreover, encapsulated probiotics (L. rhamnosus, L. salivarius, L. plantarum, L. acidophilus,

L. paracasei, B. longum, B. lactis type Bi-o4 and Bi-07) were protected from the acidic

environment of orange juice.

Although in the present study, the survival of probiotic bacteria reduced during storage,

the final cell count was still higher than the minimum therapeutic dose (FAO/WHO, 2001).

Figure 3. Survival of L. acidophilus TISTR 1338 and L. casei TISTR 390 in the grape juice

jelly during refrigeration storage (5 °C) for 9 days.

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Conclusion

Grape juice could successfully be used to deliver probiotic bacteria to consumers since the

probiotic numbers were higher than the minimum therapeutic dose after refrigeration storage.

Thus, this product could be a challenging one for alternative choice of functional foods in the

market.

Reference

[1] Sanders ME, Guarner F, Guerrant R, Holt PR, Quigley EM, Sartor RB, Sherman PM and

Mayer EA. An update on the use and investigation of probiotics in health and disease. Gut,

2013;62(5), 787-796.

[2] K Kailasapathy and JC Chin. Survival and therapeutic potential of probiotics organisms

with reference to Lactobacillus acidophilus and Bifidobacterium spp. Immunology and Cell

Biology.vol 78.2000,p.80-88.

[3] FAO/WHO. Health and nutritional properties of probiotics in food including powder milk

with live lactic,: Food and Agriculture Organization of the united Nations and World health

organization Expert Consultation Report. Cordoba, Argentina, 2001, p. 1-5.

[4] Boza-Mendéz E, López-Calvo R and CortésMuñoz M. Innovative dairy products

development using probiotics: Challenges and limitations. In E.C. Rigobelo (Ed), probiotics.

InTech. Available at http://www.intechopen.com/books/probiotics/innovative-dairy-products-

development-using-probiotics-chall-enges-and-limitations. accessed 3 October 2012.

[5] Perricone M, Bevilacqua A, Corbo MR and SiNgaglia M. Technological characterization

and probiotic traits of yeasts isolated from Altamura to sourdough select promising

microorganisms as functional starter cultures for cereal-based products. Food

Microbiology.vol38, 2014, p. 26-35.

[6] Mortazavian AM, Ehsani MR, Mousavi SM, Sohrabvandi S and Reinheimer JA. Combined

effects of temperature-related variables on the viability of probiotic microorganisms in yogurt.

J. Dairy Technol 2006; 61, 248-52.

[7] Sheehan VM, Ross P and Fitzgerald GF. Assessing the acid tolerance and the technological

robustness of probiotic cultures for fortification in fruit juices. Innovative Food Science and

Emerging Technologies. 2007; 8, 279-284.

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[8] Ding WK and shah NP. Survival of free and encapsulated probiotic bacteria in orange and

apple juices. International food research journal. 2008; 15, 219-232.

[9] Granto D, Branco GF, Nazzaro F, Cruz AG And Faria JA. Functional food and nondairy

probiotic food development : Trends, concepts, and products. Compr Rev Food sci Food saf.

2010; 9, 292-302.

[10] Nualkaekul S and Charalampopoulos D. Survival of Lactobacillus plantarum in model

solutions and fruit juices. J. Food Microbial. 2011; 146, 111–117.

[11] Mousavi ZE, Mousavi SM, Razavi SH, Emamdjomeh Z and Kiani H. Fermentation of

pomegranate juice by probiotic lactic acid bacteria. J. Microbiol Biotechnol, 2011; 27, 123–

128.

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Chemical composition, physical properties, and sensory evaluation of an

instant powder beverage containing melatonin prepared from vegetables

Wariya Hochin, Anuchita Moongngarm*

Department of Food Technology and Nutrition, Faculty of Technology, Mahasarakham University,

Mahasarakham 44150, Thailand

*Corresponding author: [email protected]

Abstract:

Melatonin is a naturally occurring compound found in plants, animals, and microorganism. It

plays an important role in maintaining the body's circadian rhythm in animals and it has

antioxidant properties. This study was carried out to develop an instant powder beverage using

germinated red bean, lemon glass, and onion as sources of melatonin. The proportions of the

components used in the beverage were determined using the Mixture Design Method, which

generated instant beverages for 6 formulas including germinated red bean (X1 = 30-50%),

lemon grass (X2 = 10-35%) and onion (X3 = 15-42%). The effects of different proportions of

raw materials on the appearance, color, texture and overall acceptability of the products were

evaluated using a 9-Point hedonic scale. The results indicated that the proportions of

germinated red bean: lemon grass: onion of 50:40:10 gained the highest score of acceptance.

Six beverage powder formulas were also determined for physical properties and chemical

composition. It was found that the beverage powder containing melatonin had suitable

physicochemical properties, with respect to water absorption index (1.11 - 2.44 g / g), water

soluble index (19.57 - 20.79%) and solubility (18.73 - 20.83 % residue). The study suggested

that a melatonin-rich beverage powder could be produced as a functional food.

Keywords: Antioxidant, Functional food, Instant beverage, Biological rhythm,

Body's circadian rhythm

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Introduction

Currently, consumer awareness about health has greatly increased. As a result, there is

increasing consumption of healthy food products and a great demand of functional foods in the

food market. A number of bioactive compounds from plants have been studied; however, there

are only a few studies of melatonin. Melatonin (N-acetyl-5-methoxy tryptamine) [1] is

a hormone produced by the pineal gland in animals. It regulates sleep and wakefulness [2].

Melatonin is also produced in plants to function as a defense against oxidative stress [3]. In

animals, melatonin is involved in the synchronization of the circadian rhythms including sleep-

wake timing, blood pressure regulation, seasonal reproduction, and other functions [4]. Many

of its biological effects in animals are produced through the activation of melatonin receptors

[5], its role as an antioxidant [6], with a particular role in the protection of nuclear and mitochondrial

DNA [7]. Nowadays, more and more people of working-age want to rest for a limited time.

Moreover, people who travel across time zones, as well as other consumers in general, need

specific functional foods for better relaxation. Melatonin is a key bioactive compound to

provide in functional foods for relaxation. From our previous study, we found that some

vegetables contain high amounts of melatonin [1, 2, 8, 9]. Since vegetables are available

throughout the year and inexpensive, they have the potential to use as a source of melatonin as

a functional food for relaxation. However, consuming vegetables before sleeping may be

inconvenient for consumers. Therefore, this study was conducted to develop an instant powder

beverage as a functional drink product high in melatonin.

Materials and methods

Raw materials

Red beans (Phaseolus vulgaris Linn.), lemon grass (Cymbopogon citratus DC.Stapf.)

and onions (Allium cepa Linn.) were purchased from local market in Mahasarakham province,

Thailand. Red bean sprouts were prepared by germination of red bean seed for 3 days

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Preparation of instant powder beverage

Germinated red beans, lemon grass, and onions were selected and used for developing

an instant beverage. The vegetables were cleaned and cut into small sized pieces approximately

1x1 cm prior to boiling in hot water (80-90°C) until the texture of raw materials became

soft. After that, the cooked vegetables were finely ground with water added (50% v/w) using a

blender and filtered through cheese cloth. The samples were then reduced to dryness using

freeze dryer. The dried samples were ground and used to develop the beverage. A total of 6

formulas of instant beverage were obtained using the mixture expert design program. These

beverages were used for sensory evaluation, chemical composition analysis, and physical

property determination. The sensory evaluation of this study was approved by the Human

Ethics Committee, Mahasarakham University. The 9-Point Hedonic Scale technique was

applied for sensory evaluation.

Determination of melatonin

Sample extraction for melatonin determination

Vegetable samples were freeze dried and finely ground before use. Samples of 5.0 g

were extracted with aqueous methanol (MeOH, 80%), 50 ml. The mixture was shaken in a

shaking incubator for 30 min., then centrifuged for 16-22 hours at 2500 rpm. The supernatant

was evaporated to dryness in a rotary evaporation machine, then re-suspended in MeOH (80%),

10 ml prior to analysis by HPLC-RP [10].

Melatonin determination

The extract was filtered through a nylon filter syringe (0.45 µm). The HPLC system

used to determine melatonin was a Shimadzu (Prominance HPLC, Shimadzu, Japan) with a

fluorescent detector, 10A version and Series 20A, excitation wavelength at 290 nm and the

emission wavelength at 330 nm, pumping system using an RF-20Axs. An analytical column

used was RP-C18 column (4.6 x 250 mm, 5µm), (GL Science, Japan). The mobile phase

consisted of solvent A (50 mM phosphate buffer at pH 7.2) and Solvent B (acetonitrile). The

mobile phase was computer program-controlled for gradient elution as follows: (time, solvent

B), (0.0 min, 0%), (5.00 min, 35%), (12.00 min, 40%), (20.00 min, 45 %), (25.00 min, 50%),

(30.00 min, 0%), stopped at 40 min, with a flow rate of 1.0 mL/min. The injection volume was

20 μL. The concentration of melatonin in each sample was calculated using the area under the

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graph obtained from the analysis comparing with a melatonin standard and the concentration

was expressed as ng/g dry sample [10].

Determination of chemical composition

The proximate composition of the instant beverage samples was analyzed by the

method of [11] including moisture content, ash, protein, fat, fiber, and carbohydrate.

Determination of physical properties

The physical properties measured in this study included water absorption index, water

dissolution index, and the solubility (%).

Results and discussion

Melatonin content

The melatonin content of the instant powder beverages and raw materials are indicated

in Table 1.

It was found that all instant powder drinks were not significantly different in melatonin

content which ranged from 15.18 to 19.18 ng/ g (dry sample). The level of melatonin found in

this study was higher than those reported by Arnao et.al (2014) [12] and Padumanonda et.al

(2014) [1]. The content of melatonin in plants can vary depending on several factors such as

varieties [13], growing conditions, harvesting periods [14] and growth environment [15].

According to the report of a study of turnips [16], a melatonin dose of 0.5-5 μg could reduce

jet lag. A melatonin dose of 0.1 µg per day has been applied to treat insomnia before a bedtime.

This amount was enough for helping a person with insomnia to sleep if it provided a level of

about 200pg in the blood [17]. If approximately 100 g of the instant beverage was taken, it

could contribute a melatonin amount of about 1.6 μg. Therefore, this instant powder beverage

is likely to be useful and has sufficient potential to warrant further development.

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Table 1. Melatonin content in raw materials and instant powder beverages (ng/g dry sample)

Formula Germinated red bean

(%)

Lemon Glass

(%)

Onion

(%) Melatonin

1 48 10 42 16.57±0.19

2 30 28 42 19.18±0.23

3 41.6 23.6 34.8 17.21±0.15

4 50 35 15 15.18±0.08

5 30 35 35 18.89±0.14

6 50 10 40 16.21±0.21

Raw material 100 7.30±0.26

Raw material 100 21.82±0.49

Raw material 100 25.92±0.86

Sensory evaluation test of beverage powder with melatonin in 6 formulas

The results of a sensory evaluation of the finished products of instant powder beverage,

expressed as liking score (sensorial score), are shown in Table 2. It was found that all attributes

including color, sweetness, saltiness, bitterness, smell and overall liking were significantly

different in liking score (P<0.05), which the liking scores varied between 4.60 and 8.58. The

formula 6 ingredient containing 50% red bean sprouts, 10% lemon grass and 40% onions

obtained the best liking score in, sweetness, saltiness, bitterness, and smell, while liking scores

of the powder beverage containing higher level of onion gained lower liking scores. This may

due to the strong smell of onion itself.

Analysis of Chemical compositions

The results of chemical composition, including moisture content, ash, protein, fat, fiber,

and carbohydrate of the instant powder beverage are shown in Table 3. The results indicated

that the proportion of raw materials added to each formula significantly affected the moisture,

ash, protein and fiber contents of the six powders (P < 0.05), whereas the powders did not differ

significantly in fat content (P > 0.05). The moisture content, ash, fat, fiber, carbohydrate, and

protein of the products ranged from 6.09 to 6.91%, 4.10 to 4.77%, 0.08 to 0.13%, 38.15 to

41.57%, 15.09 to 20.89% and 28.086-30.112%, respectively. The red bean is high in protein

and it was added in high proportion in each formula resulting in the high protein content in

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some of the beverages. Fiber content was high in lemon grass; as a result, this ingredient

contributed high fiber content to the relevant beverages.

Table 2. Liking scores of instant powder beverages obtained using 9-point hedonic scale for

evaluation

Formula

Germinated

red bean

(%)

Lemon

Glass

(%)

Onion

(%) Color Sweetness Bitterness Saltiness Odor

Overall

liking

1 48 10 42 6.47±0.82b 6.87±0.63b 6.63±0.67b 6.73±0.74b 6.73±0.74b 7.06±0.43b

2 30 28 42 4.17±0.59c 5.13±0.82d 5.43±0.63c 4.33±0.76e 5.40±0.56e 4.60±0.61d

3 41.6 23.6 34.8 6.80±0.85b 5.93±0.69c 6.10±0.88b 6.03±0.72c 6.13±0.57c 6.93±0.61c

4 50 35 15 8.03±1.00a 8.00±0.83a 8.07±0.83a 8.03±0.56b 8.03±0.56b 8.23±0.79a

5 30 35 35 6.13±0.97b 5.77±0.82c 5.10±0.76c 5.33±0.66d 6.03±0.56d 5.50±0.72c

6 50 10 40 8.06±0.81a 8.23±0.80a 8.06±0.81a 8.13±0.85a 8.19±0.75a 8.58±0.70a

Means within a column with different superscript letters (a, b, c,…) are different (p<0.05)

Table 3. Chemical composition of instant powder beverages

Formula

Germinated

red bean

(%)

Lemon

Glass

(%)

Onion

(%)

Moisture

content

(%)

Ash

(%)

Protein

(%)

Fat ns

(%)

Fiber

(%)

Carbohydrate

(%)

1

2

3

4

5

6

48

30

41.6

50

30

50

10

28

23.6

35

35

10

42

42

34.8

15

35

40

6.31±0.07c

6.09±0.07d

6.30±0.06c

6.52±0.17b

6.91±0.04a

6.55±0.03b

4.22±0.05d

4.10±0.01e

4.19±0.06d

4.73±0.06a

4.77±0.06a

4.28±0.14c

18.36±0.09b

15.09±0.04c

18.42±0.09b

20.77±0.10a

15.85±0.24a

20.89±0.09a

0.08±0.04

0.12±0.07

0.13±0.03

0.10±0.03

0.11±0.03

0.13±0.03

38.96±0.54bc

38.15±1.50bc

38.05±1.09c

40.44±1.42b

38.64±1.61bc

41.57±0.66a

28±1.15bc

28±0.96bc

28±1.08bc

30±1.12b

28±0.97bc

30±0.16a

Means within a column with different superscript letters (a, b, c,…) are different (p<0.05)

Table 4. Physical properties of instant powder beverages

Formula

Germinated

red bean

(%)

Lemon

Glass

(%)

Onion

(%)

Water absorption

index

(g/g)

Water soluble

index

(%)

Water soluble

ability

(%residue)

1

2

3

4

5

6

48

30

41.6

50

30

50

10

28

23.6

35

35

10

42

42

34.8

15

35

40

2.44±0.07a

1.11±0.06d

1.81±0.03c

1.76±0.07c

1.83±0.06c

2.26±0.05b

20.79±0.10a

19.57±0.06b

19.92±0.07b

19.60±0.17b

19.70±0.34b

20.58±0.50a

18.73±0.41c

20.83±0.10a

19.73±0.09b

20.49±0.31a

19.57±0.40b

19.53±0.45b

Means within a column with different superscript letters (a, b, c,…) are different (p<0.05)

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Analysis of Physical compositions

The water absorption index (WAI), solubility index (WSI) and solubility (WSA) of

instant beverage powder are presented in Table 4. The results indicated that the WAI, WSI,

and WSA of products were significantly affected by the proportions of raw materials (P < 0.05).

The WAI, WSI, and WSA varied from 1.11 to 2.44 g/g, 19.57 to 20.79% and 18.73 to 20.83%,

respectively. The WAI of the food product depends on the amounts of soluble dietary fiber

[18], protein, and fat. If products contain high levels of fiber and protein but low fat content, a

high WAI is expected [19]. Formulas 1 and 6 contain higher proportions of red bean than others

(except Formula 4!!); as a result in higher level of protein, therefore, the WAI was higher than

other formulas.

Conclusions

This study has demonstrated the feasibility of developing an instant powder beverage as a

functional drink product containing melatonin. The six trial products obtained were acceptable

by panelists with generally high liking scores. The products all contained melatonin; however,

its concentration in the products still need to be increased to ensure effectiveness. the amount

of melatonin which this may be achieved by extracting the melatonin from the raw materials

prior to added it back to the finished product.

Acknowledements

Acknowledgement the financial support of Faculty of Technology, Mahasarakham University,

Thailand.

References

[1] Padumanonda, T, Johns, J, Sangkasat, A and Tiyaworanant, S. Determination of melatonin

content in traditional Thai herbal remedies used as sleeping aids. DARU Journal of

Pharmaceutical Sciences, 2014; 22, 1-5.

[2] Hardeland R, Pandi-Perumal SR, Cardinali DP.Melatonin".The International Journal of

Biochemistry and Cell Biology.2006; 38 (3): 3136.PMID16219483.

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78

doi:10.016/j.biocel.2005.08.020.

[3] Tan DX, Hardeland R, Manchester LC, Korkmaz A, Ma S, Rosales-Corral S, Reiter RJ

(2012). Functional roles of melatonin in plants, and perspectives in nutritional and

agricultural science.Journal of Experimental Botany. 2012;63(2): 577–97. PMID 22016420.

doi:10.1093/jxb/err256.

[4] Altun A, Ugur-Altun B. Melatonin: therapeutic and clinical utilization. Int. J. Clin. Pract.

2007; 61 (5): 835–45. PMID 17298593. doi:10.1111/j.1742-1241.2006.01191.x

[5] Boutin JA, Audinot V, Ferry G, Delagrange P. Molecular tools to study melatonin

pathways and actions". Trends Pharmacol. 2005; Sci. 26 (8):

412419. PMID 15992934. doi:10.1016/j.tips.2005.06.006.

[6] Hardel and R . Antioxidative protection by melatonin: multiplicity of mechanisms from

radical detoxification to radical avoidance. Endocrine. 2005;27(2):119–30.PMID16217125.

doi:10.1385/ENDO:27:2:119.

[7] Reiter RJ, Acuña-Castroviejo D, Tan DX, Burkhardt S. Free radical-mediated molecular

damage. Mechanisms for the protective actions of melatonin in the central nervous

system". Ann. N. Y. Acad. Sci. 2001; 939: 200–15. PMID 11462772. doi:10.1111/j.1749-

6632.2001.tb03627.x.

[8] Hochin W, Moongngarm A. Melatonin content and antioxidant activity in local vegetables.

Khon Kaen Agr. J. 2017; 45 SUPPL. 1174-1179.

[9] Hattori A, Migitaka M, Ligo M, Itho K, Amamoto K, Ohtani-Kaneko R, Reiter R.

Identification of melatonin in plants and its effects on plasma melatonin level and binding to

melatonin receptors in vertibrates. Biochemistry and Molecular Biology International. 1995;

35, 627-634.

[10] Chen, G., Huo, Y., Tan, DX., Liang, Z., Zhang, W. and Zhang, Y. (2003). Melatonin in

Chinese medicinal herbs. Life sciences, 73, 19-26.

[11] AOAC. (2000). Official Methods of Analysis of international (17th ed.). Washington

D.C.,USA: Association of Official Analytical Chemists.

[12] Arnao, MB. and Hernández-Ruiz, J. (2014). Melatonin: plant growth regulator and/or

biostimulator during

[13] Korkmaz, A., Değer, Ö. and Cuci, Y. (2014) .Profiling the melatonin content in organs of

the pepper plant during different growth stages. Scientia horticulturae, 172, 242-247.

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[14] Stürtz, M., Cerezo, A.B., Cantos-Villar, E., Garcia-Parrilla, M.C. (2011). Determination

of the melatonin content of different varieties of tomatoes (Lycopersicon esculentum) and

strawberries (Fragariaananassa). Food chemistry, 127, 1329-1334.

[15] Riga, P., Medina, S., García-Flores, LA. and Gil-Izquierdo, Á. (2014). Melatonin content

of pepper and tomato fruits: effects of cultivar and solar radiation. Food chemistry, 156, 347-

352.

[16] Herxheimer A and Petrie K. Melatonin for the prevention and treatment of jet lag. 2002;

Available from: https://www.ncbi.nlm.nih.gov/pubmed/12076414. [accesses June 2016].

[17] Zhdanova, IV, Wurtman,RJ, Meredith M. Regan,MM, Taylor, JA, Shi, JP, Leclair, OU.

Melatonin Treatment for Age-Related Insomnia. J Clin Endocrinol Metab. 2001; 86 (10):

4727-4730.DOI:https://doi.org/10.1210/jcem.86.10.7901

[18] Marin F, Luquet G, Marie B, Medakovic D. Molluscan shell proteins: primary structure,

origin, and evolution. Current topics in developmental biology. 2007; 80, 209-276.

[19] Yamazaki, Y. Learning styles and typologies of cultural differences: A theoretical and

empirical comparison. International Journal of Intercultural Relations. 2005; 29, 521-548.

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Effect of thermal processing on physical, chemical properties and volatile

compounds of coconut (Cocos nucifera L.) sugar

Araya Rakphon*, Voranuch Srijesadaruk

Department of Food Technology, Khon Kaen University, Khon Kaen, Thailand

*Corresponding author: [email protected]

Abstract:

The objectives of this research were to study the physical, chemical properties and volatile

compounds of both coconut sap and sugar and investigate changes in the physical, chemical

properties and volatile compounds of coconut sugar during heating processes. The physical,

chemical properties including moisture content (MC), water activity (aw), total soluble solids

(TSS), color parameters, intimidated browning product (IBP) and browning index (BI) values

and volatile compounds were determined. The coconut sap was heated at 110 and 120oC for 6

and 4.5 hours, respectively. The physical, chemical properties and volatile compounds of

coconut sugar were investigated. The results found that the MC and aw decreased with

increasing heating times while TSS increased with the times for both of heating temperatures

of 110 and 120oC. The color parameters for both heating temperatures of 110 and 120oC fell in

the same trend which was the L* decreased, a*, b*, IBP and BI values increased with heating

times. The major volatile compounds found in coconut sugar for both temperatures were 2, 3-

dimethyl pyrazine, 2, 5-dimethyl pyrazine, 2-methyl pyrazine and 5-methyl furfural. Most of

volatile compounds increased with heating times. Long heating time could be concerned to

develop the aroma of coconut sugar.

Keywords: Coconut sugar, Heating conditions, Physical and chemical properties, Volatile

compounds.

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doi: 10.10.14457/MSU.res.2017.9
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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Introduction

Cocos nucifera L. is a scientific name of coconut. It belongs to Arecaceae family. The coconuts

are applied by many traditional uses, ranging from food to cosmetics. For example, the fruit

contains a number of water (also called “juice”) which provides health benefits and also gives

oil and milk that are used in cooking and frying as well as in soaps and cosmetics. The fluid,

chiefly water with dissolved sugars and mineral salts derived from coconut, it is called “sap”.

The coconut sap is commonly used to produce palm sugar.

Palm sugar is one of the local sweeteners which are widely consumed in Thailand or

Asian countries. Normally, the sugar is used for making cakes, desserts, food or drinks because

it provides better taste and aroma when compare with sugar cane. So, palm sugar is alternative

sugar in the present. The sugar is made by heating the sap obtained from the coconut tree

(Coconut nucifera L.) or palm tree (Arenga pinnata) [1]. For the traditional production, the sap

is filtered and then transferred into a big wok. The filtrated is heated at 100-120oC for a few

hours to obtain a concentrated sap which provides a typical aroma. The sap is poured into the

bamboo moulds to form a solid palm sugar.

Normally, quality attributes of food products including color, flavor and texture are

very important for consumers. Then both color and flavor play a vital role in customer

perceptions. In the case of palm sugar production, the color and flavor is developed during

heating processes. It means heating conditions have an effect on the product qualities. The

major reaction occurs during heating process that is Maillard reaction and caramelisation. Both

reactions are caused of color and aroma formation [2]. The degree of aroma formation and

color intensity depend on the structure of sugar present in the sap and the degree of interaction

between the amino groups [3]. Ho and others [4] had studied the changes in volatile compounds

of palm sap during heating process and found that the important volatiles compound were 2,

3-diethyl-5-methyl pyrazine (2,3 D-5MP) and 2-ethyl-3,5-dimethyl pyrazine (2E-3,5 DP). It

was found that most of volatile compounds in N-Heterocyclic, O-Heterocyclic, aldehyde and

ketones groups increased with heating times.

Nowadays, the study of volatile compounds in coconut sap or coconut sugar is still

limited. Therefore, the aims of this research were to study the effect of heating conditions and

observed changes during heating processes on the physical, chemical properties as well as

volatile compounds of coconut sugar.

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Materials and methods

Materials

Coconut saps obtained from the Ampawa-Chaiputtananuruk Conservation Project

which located in Ampawa District, Samut Songkram Province, Thailand. Each 20 L of the

coconut sap was collected in plastic bottles and kept under -18oC during transportation. The

frozen samples were stored at 5oC for 3 days before analysis. Physical and chemical properties

including moisture content [2] water activity, pH [5], total soluble solid, color, intimidated

browning product (IBP) [2] and browning index (BI) [2], as well as volatile compounds [4] of

the coconut sap were determined.

The chemical standards were purchased from Sigma-Aldrich, Switzerland. The

standards used in the experiment were 2-methyl pyrazine, 2, 5-dimethyl pyrazine, 2, 3-dimethyl

pyrazine, ethyl pyrazine, 2-ethyl-3, 5-dimethyl pyrazine, 2, 3-diethyl-5-methyl pyrazine,

furfural, 5-methyl furfural and tridecane.

Coconut sugar production

The methods of Ho and others [5] and Naknean [2] were modified by using 2.5 L of

coconut sap and 2 heating temperatures (110oC and 120oC). Each coconut sap samples was

heated at 110oC and 120oC and stirred with a rotational speed of 150 rpm. Approximately 60

mL of the heated samples were collected at intervals of 30 min. The samples temperatures were

determined. Heating process was terminated when the total soluble solid of the samples was

equal to 80oBrix. The samples were cooled immediately and store at -18oC until the time of

analysis.

Moisture content

Moisture contents of coconut sugars were determined by a modified method of

Naknean [2]. Approximately 2-5 g of sample were weighted and placed into the pre-dried

empty moisture can. The moisture can containing samples was dried in the vacuum oven

(VD53, Binder, Germany) at 60oC under pressure lower than 9.33 kPa for 6 hr. The moisture

cans were cooled down by transferring to a desiccator for 30 min. The moisture cans were

weighed and the moisture contents of the samples were calculated.

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Water activity

Water activity of coconut sugars was determined at room temperature (30oC) by using

a water activity meter (Series 3 TE, Aqualab, USA). The sample was placed into a sample cup

and inserted into a water activity meter. The water activity values obtained recorded.

Total soluble solids

Total soluble solids of each coconut sugars for 10 µL was measured using refractometer

(0 – 82%) (Atago, Japan).

Color

Color of coconut sugars was measured using a colorimeter (HunterLab, Reston, VA,

USA) following the CIELAB space. Seven milliliter of each sample was applied and

transferred to the colorimeter. The colorimeter was adjusted for reflectance, illuminant D 65,

and angle of 10o. Color parameters obtained were in terms of L* (lightness or darkness), a*

(redness or greenness), and b* (yellowness or blueness) values.

Intimidated and Browning index

The IBP and BI of coconut sugar were investigated at the absorbance of 280 and 420

nm, respectively following the method of Naknean [2]. The sample was diluted with distilled

water. The appropriate dilution was 8 and 4-fold for both IBP and BI, respectively to obtain

reliable absorbance readings. The spectrophotometer (Lampda 25, PerkinElmer, USA) was

used to measure the absorbance of samples.

Volatile compounds

The method of Ho and others [4] was modified to apply in both sample preparation and

volatile compounds analysis. Solid phase micro extraction (SPME) was used to extract the

volatile compounds from coconut sugars. Two gram of each sample was taken into a 12 mL

vial and mixed with 1 ppm of Tridecane. The vial was covered with PTFE/silicone septum.

Then, the vial was equilibrated in a water bath at 60oC for 20 min. A 50/30 µm Divinylbenzene/

Carboxen/ Polydimethysioxane SPME fiber was inserted into the vial to trap N- and O-

heterocyclic group volatile compounds and was exposed to the sample headspace at 60oC for

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10 min. The fiber was transferred directly to the injector port of gas chromatography-mass

spectrophotometry (GC-MS) system.

The volatile compounds of coconut sugars were identified using GC-MS (Scion 436-

GC, Bruker, Germany) with DB-625 capillary column (30 m x 0.25 mm) (Agilent

Technologies, USA). The injector and detector temperature were 240 and 280oC, respectively.

The helium gas was used to be a carrier gas. The flow rate of 2 mL/min and constant pressure

of 117.5 kPa were applied. The column temperature was programmed from 50oC (held for 2

min), at 20oC/ min to 80oC (held for 1 min), at 20oC/ min to 100oC (held for 1 min), then at

30oC/ min to 230oC (held for 2 min). Other conditions were as follows: scanning mass range

(m/z) 50 – 550 a.m.u at a rate of 1.5 scan/ s; electron ionization energy at 70eV.

Results and discussion

Physical and chemical properties of coconut sugar during heating process

Heating was applied to concentrate the coconut sap at 110 and 120oC, for 6 and 4.5

hours, respectively. Changes during heating process in physical and chemical properties of

coconut sugar for 110 and 120oC are presented in Table 1 and 2, respectively. The results

revealed that heating time had a significant effect on MC and aw (p<0.05). Heating temperature

at 120oC had a shorter heating time than those of 110oC because performing at higher

temperature led to increasing driving force that accelerated moisture movement faster. The MC

of coconut sugars for both 110 and 120oC decreased from 83.07 to 6.85% w.b. and 83.07 to

11.17% w.b., respectively, aw decreased from 0.986 to 0.576 and 0.677, respectively with

increasing of heating time. Increasing sample temperature led to removal of water from coconut

sap by evaporation. According to the decreasing of MC and aw of both heating temperatures

with heating times, the TSS increased with increasing heating time. The results were supported

by Akochi-K and others [6] and Rao and others [7] who exhibited that the increasing of TSS

and decreasing of MC and aw could be related with rapid evaporation of water from coconut

sap during heating process.

For color parameters, both of heating temperatures for 110oC and 120oC were in the

same trend. L* values of both of heating temperature for 110oC and 120oC were in the ranges

of 82.25 - 50.12 and 82.25 – 67.73, respectively. Increasing heating times led to decrease

lightness. The results indicated that the coconut became darker with heating time when

compared with the control at 0 hr. The a* values of both heating temperature increased from -

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0.01 – 6.21 for heating temperature of 110oC and 0.01 – 6.99 for 120oC with increasing heating

time. In addition, the b*, IBP and BI values increased with heating time for both temperature.

Ibarz and others [8], Naknean [2] and Rattanathanalerk and others [9] had studied the effect of

thermal processing on the color changes of food products. The results demonstrated that

increasing heating times had an effect to decrease L* and increase a*, b* IBP and BI value.

The changes of color were described by Martins and others [10] who reported that cooking

temperature and heating time were important to both Maillard and caramelisation reaction that

affected color changing.

Changes in volatile compounds of coconut sugar during heating process

The volatile compounds can be divided into 2 groups which were N-heterocyclic

including 2, 3-diethyl-5-methyl pyrazine (2,3 D-5MP), 2,3-dimethyl pyrazine (2,3 DP), 2,5-

dimethyl pyrazine (2,5 DP), 2-ethyl-3,5-dimethyl pyrazine (2E-3,5 DP), 2-methyl pyrazine

(2MP) and ethyl pyrazine (EP) and O-heterocyclic such as 5-methyl furfural (5MF) and

furfural (F). The volatile compounds of coconut sugars for both 110oC and 120oC heating

temperatures are demonstrated in the Table 3 and 4. The results found that 2,3 DP, 2,5 DP,

2MP and 5MF were the major compounds of coconut sugar for both 110 and 120oC heating

temperatures. At the end of heating times of 6 and 4.5 hours for heating temperatures of 110

and 120oC, respectively, most of volatile compounds increased after the heating process in both

heating temperatures. The results were agreed with Ho and others [4] who revealed that high

temperature was necessary for the formation of various volatile compounds and heating time

also required for the aroma developments.

Conclusions

The results of this research revealed that coconut sap contained 83.07% w.b. of moisture

content, 0.986 of water activity, 15.25 oBrix of total soluble solids, 0.580 of IBP and 0.039 of

BI values. The content of 2,3 D5MP was found to be the highest (0.390 ppb) followed by 5MF,

2,3 DP, 2E-3,5 DP, 2,5 DP, 2MP, EP and F, respectively. The moisture content and water

activity of coconut sugar during heating process decreased with increasing heating time while

the total soluble solids increased with the times. In the end of heating process of 6 and 4.5 hours

for heating temperature of 110 and 120oC, respectively, the L* value decreased, a*, b*, IBP

and BI values increased with heating times. The major volatile compounds were 2,3 DP, 2,5

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DP, 2MP and 5MF. Most of volatile compounds increased with heating times. Long heating

time could be required to produce aromatic coconut sugar.

Acknowledgements

We would like to thank the Ampawa-Chaiputtananuruk Conservation Project for their raw

materials support.

References

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[2] P. Naknean, “Factors affecting browning and crystallisation of palm sugar syrup and palm

sugar cake,” Ph.D. Thesis, Prince of Songkla University, Songkla, Thailand, 2010.

[3] L. W. Kroh, “Caramelisation in food and beverages,” Food Chemistry, vol. 92, pp. 371-

379, 1994.

[4] C. W. Ho, W. M. Wan Aida, M. Y. Maskat, and H. Osman, “Changes in volatile

compounds of plam sap (Arenga pinnata) during the heating process for production of palm

sugar,” Food Chemistry, vol. 102, pp. 1156-1162, 2007.

[5] C. W. Ho, W. M. Wan Aida, M. Y. Masat, and H. Osman, “Effect of thermal processing

of palm sap on the physic-chemical composition of traditional palm sugar,” Pakistan Journal

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[6] E. Akochi-K, I. Alli, S. Kermasha, and V. Yaylayan, “Quantitation of alkylpyrazines in

maple syrup, maple flavors and non-maple syrups,” Food Research International, vol. 27, pp.

451-457, 1994.

[7] P.V.K. Jagannadha Rao, M. Das, and S.K. Das, “Changes in physical and thermo-physical

properties of sugarcane, palmyra-palm and date-palm juices at different concentration of

sugar”, Journal of Food Engineering, vol. 90, no. 4, pp. 559-566, 2009.

[8] A. Ibarz, J. Pagán, and S. Garza, “Kinetic models for colour changes in pear puree during

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[9] M. Rattanathanalerk, N. Chiewchan, and W. Srichumpoung, “Effect of thermal processing

on the quality loss of pineapple juice”, Journal of Food Engineering, vol. 66, pp. 259–265,

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[10] S. I. F. S. Martins, W. M. F., Jongen, M. A. J. S., van Boekel, “A review of Maillard

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Table 1: Physical, chemical properties of coconut sugar during heating process at 110oC

Heating times

(hr.)

Sample temperatures

(ºC)

Moisture Content

(% w.b.) aw

TSS

(oBrix)

Color parameters IBP BI

L* a* b*

0 30.88 83.07±0.37a 0.986±0.06a 15.25±0.10m 82.25±0.81 a 0.01±0.21d 12.03±0.78j 0.58±0.32ef 0.039±0.01g

0.5 72.00 82.64±0.22a 0.986±0.01a 16.07±0.10l 85.03±0.84a -0.10±0.14d 11.86±0.33j 0.438±0.04f 0.027±0.00i

1.0 72.50 81.35±0.15b 0.984±0.00a 17.43±0.08k 84.63±0.70a -0.08±0.09d 12.57±0.49ij 0.391±0.07f 0.021±0.00m

1.5 72.50 79.42±0.96c 0.987±0.00a 18.83±0.34j 83.58±0.95ab -0.05±0.10d 13.80±0.76i 0.507±0.04f 0.023±0.00k

2.0 73.50 77.69±0.51d 0.986±0.00a 21.10±0.41i 82.46±0.27ab -0.04±0.03d 15.16±0.20h 0.608±0.05ef 0.021±0.00l

2.5 73.50 74.44±0.86e 0.980±0.00b 23.53±0.59h 80.86±1.46ab -0.06±0.09d 16.10±0.58h 0.684±0.03ef 0.025±0.00j

3.0 73.00 71.24±0.77f 0.973±0.00c 26.70±0.93g 79.37±1.19 ab 0.08±0.10d 18.65±0.82g 0.884±0.01de 0.031±0.00h

3.5 73.50 66.65±1.23i 0.970±0.00c 30.57±0.48f 78.24±1.51 ab 0.05±0.12d 20.49±0.91f 1.122±0.04d 0.069±0.03e

4.0 73.25 60.15±2.50j 0.957±0.01d 36.03±2.23e 74.36±1.51 ab 0.38±0.16d 24.98±1.36e 1.880±0.46bc 0.053±0.00f

4.5 73.00 51.69±1.46k 0.936±0.00e 43.30±0.85d 73.42±1.44 ab 0.47±0.19d 28.33±0.89d 1.650±0.05c 0.071±0.01d

5.0 73.50 41.79±2.39l 0.897±0.01f 52.57±1.72c 71.75±0.18b 1.14±0.07c 33.41±0.93c 2.063±0.29b 0.083±0.01c

5.5 73.50 19.72±0.95m 0.755±0.01g 73.80±0.52b 74.48±0.04ab 2.56±0.41b 45.77±1.97a 2.411±0.00b 0.103±0.01b

6.0 76.50 6.85±1.34n 0.576±0.05h 80.57±0.48a 50.12±9.98c 6.21±1.96a 40.20±3.72b 2.414±0.01a 0.223±0.03a

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Table 2: Physical, chemical properties of coconut sugar during heating process at 120oC

Heating times

(hr.)

Sample temperatures

(ºC)

Moisture Content

(% w.b.) aw

TSS

(oBrix)

Color parameters IBP BI

L* a* b*

0 30.88 83.07±0.37a 0.986±0.06a 15.25±0.10j 82.25±0.81 a 0.01±0.21h 12.03±0.78j 0.58±0.32e 0.039±0.01i

0.5 75.5 81.86±0.30b 0.986±0.00a 17.03±0.15i 81.34±2.01b 0.23±0.32g 12.73±1.89i 0.518±0.03e 0.022±0.00j

1.0 76.25 79.15±0.19c 0.985±0.00a 19.23±0.23h 80.37±2.59c 0.21±0.27g 15.15±0.59h 0.547±0.07e 0.023±0.00j

1.5 75.75 75.98±1.06d 0.983±0.00a 21.63±0.50g 77.49±3.52d 0.30±0.25f 16.25±0.31g 0.678±0.03e 0.039±0.01i

2.0 76.25 72.58±0.56e 0.975±0.00b 25.67±0.68f 76.48±2.95e 0.28±0.29f 18.98±0.28f 1.069±0.12d 0.063±0.03e

2.5 76.75 66.43±0.95f 0.968±0.00c 30.13±0.16e 73.19±2.80f 0.48±0.28e 21.98±0.11e 1.387±0.04cd 0.051±0.01f

3.0 77.00 59.37±1.10g 0.955±0.00d 36.10±0.99d 71.15±3.96g 0.65±0.39d 25.43±0.06d 1.822±0.20b 0.075±0.01d

3.5 76.50 47.66±2.80h 0.933±0.01e 47.13±5.55c 69.51±3.43h 1.04±0.26c 29.79±1.43c 2.075±0.28bc 0.099±0.03c

4.0 77.25 30.02±7.23i 0.842±0.05f 65.40±5.92b 68.66±4.53i 2.56±0.22b 39.42±3.92b 2.379±0.03a 0.186±0.08b

4.5 78.00 11.17±0.97j 0.677±0.03g 80.37±0.34a 67.73±0.11j 6.99±1.45a 58.00±5.74a 2.387±0.01a 0.297±0.06a

aw: water activity, TSS: total soluble solids (oBrix)

IBP: Intermediate Browning Product

BI: Browning Intensity

Mean values in the same column with different superscripts are significantly different at P ≤0.05 by Duncan’s Multiple Range Test.

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Table 3: Volatile compounds of coconut sugar during heating process at 110oC

Heating

time

(hr.)

Sample

temperatures

(ºC)

Volatile compounds content

(ppb)

N-heterocyclic O-heterocyclic

2,3 D-5MP 2,3 DP 2,5 DP 2E-3,5 DP 2MP EP 5MF F

0 30.88 0.390±0.78 ns 0.023±0.04 ns 0.014±0.00 ns 0.015±0.01 ns 0.014±0.00 bc 0.003±0.01 ns 0.024±0.01 ns 0.002±0.00 a

0.5 72.00 0.144±0.20 ns 0.005±0.01 ns 0.019±0.01 ns 0.015±0.00 ns 0.013±0.00c 0.006±0.01 ns 0.023±0.00 ns 0.002±0.00 a

1.0 72.50 0.084±0.12 ns 0.008±0.00 ns 0.018±0.01 ns 0.015±0.00 ns 0.015±0.00bc 0.006±0.01 ns 0.024±0.01 ns 0.002±0.00 a

1.5 72.50 0.000±0.00 *ns 0.008±0.00 ns 0.020±0.01 ns 0.013±0.00 ns 0.012±0.00 c 0.006±0.01 ns 0.022±0.00 ns 0.002±0.00 a

2.0 73.50 0.000±0.00 *ns 0.008±0.00 ns 0.017±0.01 ns 0.012±0.00 ns 0.012±0.00 c 0.006±0.01 ns 0.021±0.00 ns 0.002±0.00 a

2.5 73.50 0.000±0.00 *ns 0.004±0.01 ns 0.012±0.00 ns 0.013±0.00 ns 0.013±0.00 c 0.000±0.00 *ns 0.022±0.00 ns 0.002±0.00 a

3.0 73.00 0.000±0.00 *ns 0.004±0.01 ns 0.012±0.00 ns 0.011±0.00 ns 0.013±0.00 c 0.006±0.01 ns 0.022±0.00 ns 0.002±0.00 a

3.5 73.50 0.000±0.00 *ns 0.008±0.00 ns 0.032±0.03 ns 0.016±0.01 ns 0.012±0.00 c 0.000±0.00 *ns 0.021±0.00 ns 0.002±0.00 a

4.0 73.25 0.000±0.00 *ns 0.004±0.01 ns 0.012±0.00 ns 0.014±0.00 ns 0.013±0.00 c 0.000±0.00 *ns 0.022±0.00 ns 0.002±0.00 a

4.5 73.00 0.000±0.00 *ns 0.004±0.01ns 0.022±0.01 ns 0.012±0.00 ns 0.013±0.00 c 0.000±0.00 *ns 0.021±0.00 ns 0.002±0.00 a

5.0 73.50 0.000±0.00 *ns 0.000±0.00 *ns 0.022±0.00 ns 0.011±0.00 ns 0.013±0.00 c 0.000±0.00 *ns 0.022±0.00 ns 0.000±0.00*b

5.5 73.50 0.000±0.00 *ns 0.009±0.00 ns 0.016±0.00 ns 0.010±0.00 ns 0.019±0.01 b 0.011±0.00 ns 0.022±0.00 ns 0.000±0.00*b

6.0 76.50 0.000±0.00 *ns 0.008±0.00 ns 0.025±0.00 ns 0.010±0.00 ns 0.031±0.00 a 0.011±0.00 ns 0.022±0.00 ns 0.000±0.00*b

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Table4: Volatile compounds of coconut sugar during heating process at 120oC

Heating

time

(hr.)

Sample

temperatures

(ºC)

Volatile compounds content

(ppb)

N-heterocyclic O-heterocyclic

2,3 D-5MP 2,3 DP 2,5 DP 2E-3,5 DP 2MP EP 5MF F

0 30.88 0.390±0.78 ns 0.023±0.04 ns 0.014±0.00b 0.015±0.01ab 0.014±0.00b 0.003±0.01bc 0.024±0.01 ns 0.002±0.00a

0.5 72.00 0.000±0.00 *ns 0.040±0.06 ns 0.040±0.01a 0.022±0.01a 0.014±0.00 b 0.000±0.00 *c 0.040±0.03 ns 0.001±0.00ab

1.0 72.50 0.000±0.00 *ns 0.040±0.06 ns 0.019±0.01 b 0.012±0.00 b 0.013±0.00 b 0.000±0.00 *c 0.023±0.00 ns 0.002±0.00a

1.5 72.50 0.000±0.00 *ns 0.000±0.00 *ns 0.018±0.00b 0.011±0.00 b 0.012±0.00 b 0.000±0.00 *c 0.021±0.00 ns 0.001±0.00ab

2.0 73.50 0.000±0.00 *ns 0.040±0.06 ns 0.017±0.01b 0.011±0.00 b 0.013±0.00 b 0.007±0.01abc 0.022±0.00 ns 0.002±0.00a

2.5 73.50 0.595±0.29 ns 0.000±0.00 *ns 0.016±0.00b 0.011±0.00 b 0.016±0.00 b 0.006±0.01 abc 0.021±0.00 ns 0.002±0.00a

3.0 73.00 0.600±0.00 ns 0.040±0.06 ns 0.016±0.01b 0.011±0.00 b 0.017±0.00 b 0.000±0.00 *c 0.021±0.00 ns 0.002±0.00a

3.5 73.50 0.000±0.00 *ns 0.080±0.00 ns 0.016±0.00b 0.011±0.001 b 0.016±0.00 b 0.006±0.01abc 0.021±0.00 ns 0.002±0.00a

4.0 73.25 0.053±0.08 ns 0.080±0.00 ns 0.014±0.00 b 0.010±0.00 b 0.021±0.001 b 0.014±0.00 ab 0.020±0.00 ns 0.000±0.00*b

4.5 73.00 0.000±0.00ns 0.047±0.05 ns 0.048±0.03a 0.010±0.00 b 0.051±0.02 a 0.016±0.00a 0.021±0.00 ns 0.000±0.00*b

2,3 D-5MP: 2, 3-diethyl-5-methyl pyrazine, 2,3 DP: 2,3-dimethyl pyrazine, 2,5 DP: 2,5-dimethyl pyrazine, 2E-3,5 DP: 2-ethyl-3,5-dimethyl pyrazine, 2MP: 2-methyl

pyrazine, 5MF: 5-methyl furfural, EP: ethyl pyrazine, F: furfural. Mean values in the same column with different superscripts are significantly different at P ≤0.05 by Duncan’s

Multiple Range Test. ns is not significant different.*The a number of the compounds cannot detect but the peak was found in GC-MS chromatogram

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Species composition of fish in rice fields of That Phanom District,

Nakhon Phanom Province, Northeast Thailand

Nattanan Tiengtam*, Adithepchaikarn Pachanawan, Jirawan Khamtorn

Division of Fisheries, Faculty of Agriculture and Technology, Nakhon Phanom University,

Nakhon Phanom 48000 Thailand

*Corresponding author: [email protected]

Abstract:

Species composition of fish in rice fields of That Phanom District, Nakhon Phanom Province,

Northeast Thailand were studied during May - September 2016. From 1,018 collected

specimens in total 7 orders, 18 families, 31 genera and 38 species of fishes were recognized.

The most dominant order was Cypriniformes (18 species, 47.37% of all species) and the next

was Perciformes (10 species, 26.32% of all species) followed by Siluriformes (4 species,

10.53% of all species). The nine species of air-breathing fish (Notopterus notopterus, Clarias

batrachus, Monopterus albus, Anabas testudineus, Betta smaragdina, Trichopodus

trichopterus, Trichopsis pumila, T. vittata, and Channa striata) were found and accounted for

23.68% of all species. Only one species of alien species Oreochromis niloticus has been

collected from the area. This study indicated that the rice fields play an important role in

maintenance of biodiversity of a local area.

Keywords: Rice fields, Species, Fish, That Phanom District, Nakhon Phanom Province

Introduction

Northeast Thailand is the largest region of the country, representing approximately one-third

of the total 16.8 million ha with 22 million people. Most agricultural lands (53%) are used for

rice production [1]. Rice production takes many forms, but most rice is grown under flooded

conditions [2]. The rice field can be described as a “temporary aquatic environment” or “a

special type of wetland” that can be considered “a successor of shallow marshes or swamps”,

which is influenced and maintained by farmers activities [3]. The rice field ecosystem consists

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doi: 10.10.14457/MSU.res.2017.4
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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of two physically and morphologically distinct habitats namely, the rectangular or similar

shaped flooded fields comprising mainly of the rice plants, and the surrounding bunds (levees),

which harbour weeds or cultivated plants. Under the irrigated conditions, this mosaic system

is connected with irrigation canals and ditches, while sump ponds, marshes and tanks serve as

contiguous aquatic habitats [4]. Although being a monoculture agro-ecosystem, a rice field

undergoes three major ecological phases; aquatic, semi-aquatic and a terrestrial dry phase,

during a single paddy cultivation cycle [5]. Physically, the aquatic phase has a shallow

fluctuating water depth of 5–30 cm depending on the availability of water and the type of water

management followed, which are used as necessary temporary habitats for spawning and

nursery grounds by many fish species [6,7]. In addition, rice fields provide habitats for wildlife

species that include plants, amphibians, reptiles, molluscs, crustaceans and insects, many of

which can be captured, collected or farmed as sources of food and medicine [8].

The important role of rice field environment for local aquatic organisms has been

recognized, however only little information is known and most of documented information is

that of rice fields in temperate countries such as Japan [6,7,9]. As we know, only few reports

in literature exist on this subject in Southeast Asian countries so far and none of them deal with

freshwater fishes in detail [5]. The purpose of this study was to accumulate knowledge about

species diversity of fish in rice field environment of That Phanom District, Nakhon Phanom

Province, Northeast Thailand.

Materials and methods

The specimens were collected by a fine-mesh drug nets (3 m x 1 m, mesh size 1 mm x 1 mm)

and scoop nets (mesh size 1 mm x 1 mm) in the rice field area (4 stations, 48,000 m²) at Tambon

Na Thon, That Phanom District, Nakhon Phanom Province, Northeast Thailand (Figure 1)

during May - September 2016. All specimens were preserved in 10 % of formalin solution.

Thereafter the specimens were sent for examination in a laboratory. The specimens were sorted

and identified using the taxonomical documents [10-12]. In this study the authors held the

morphological character for identifying the specimens. Standard length (SL) was measured

between a tip of snout to the end of the hypural plate. Taxonomic arrangements follow Nelson

[13]. All specimens of freshwater fishes in this study are deposited at Research Laboratory of

Ichthyology, Division of Fisheries, Nakhon Phanom University (RLINPU), Nakhon Phanom,

Thailand.

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Figure 1. Study area for collect fish in rice fields in That Phanom District,

Nakhon Phanom Province, Northeast Thailand.

Results and discussion

Totally, 7 orders, 18 families, 31 genera and 38 species of fishes have been recognized from

1,018 specimens collected from rice fields of That Phanom District, Nakhon Phanom Province,

Northeast Thailand (Table 1). The most dominant order is Cypriniformes (18 species, 47.37%

of all species) and the next is Perciformes (10 species, 26.32% of all species) and then

Siluriformes (4 species, 10.53% of all species. Dominant families are Cyprinidae (9 genera, 15

species [39.47% of all species]), Osphronemidae (3 genera, 4 species [10.53% of all species])

and Cobitidae (2 genera, 3 species [7.89% of all species]). In all recognized fish species,

47.37% belonged to the order Cypriniformes which is usually known to be diverse in Southeast

Asia [11, 13-15]. Members of fish in the suborder Anabantoidei occupied 60.00% of the order

Perciformes which was the next dominant group and also known to be diverse in tropical Asia

especially in stagnant water habitats [16]. According to the rice field area is stagnant water

which is not directly connected with rivers or their tributaries. It is noteworthy that species

diversity of fish in the order Siluriformes or catfish was relatively low (4 species, 10.53%) in

the study area although this group has usually been ranked as the next dominant fish group to

order Cypriniformes in many fish surveys [11,17]. This probably was because many catfish

species inhabit in rivers and the study area of the present study was not directly connected with

N

That Phanom District 4

3

2 1

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surrounding canals or rivers. Esomus metallicus has been known as species found widely in

temporal waters including floodplains and rice fields in Indochina [11,18]. In this study, E.

metallicus was the most dominant species in quantitity (14.15% of all specimens). The nine

species of air-breathing fish (Notopterus notopterus, Clarias batrachus, Monopterus albus,

Anabas testudineus, Betta smaragdina, Trichopodus trichopterus, Trichopsis pumila, T.

vittata, and Channa striata) were found and accounted for 23.68% of all species (Figure 2).

Only one species of alien species Oreochromis niloticus has been collected from the area. The

result of this study showed the importance of rice fields in That Phanom District, Nakhon

Phanom Province as artificial flood plains for freshwater fishes which helps in the maintenance

of biodiversity of a local area.

Conclusions

Totally, 7 orders, 18 families, 31 genera and 38 species of fishes have been recognized from

1,018 specimens collected from rice fields of That Phanom District, Nakhon Phanom Province,

Northeast Thailand. Members of fish in the suborder Anabantoidei were found as the next

dominant fish group to order Cypriniformes. The nine species of air-breathing fish have been

recognized. Only one species of alien species or introduced species, Oreochromis niloticus,

has been collected from the area. This study indicated that the rice fields in That Phanom

District, Nakhon Phanom Province act as artificial flood plains for many fish species and play

an important role in the maintenance of biodiversity of a local area.

Acknowledgements

We are grateful to the Faculty of Agriculture and Technology, Nakhon Phanom University for

partly financial support. We would like to thanks Miss Suchitta Champa, Miss Ubonrat

Doungya and Mr. Phiboon Kunsornnan for their help in field sampling.

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Notopterus notopterus 37.50 mm SL Clarias batrachus 92.70 mm SL

Monopterus albus 126.75 mm SL Anabas testudineus 31.90 mm SL

Betta smaragdina 30.15 mm SL

Trichopodus trichopterus 53.20 mm SL

Trichopsis pumila 30.50 mm SL

Trichopsis vittata 31.35 mm SL

Channa striata 45.70 mm SL

Figure 2. Air-breathing fish found on this exploration.

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Table 1 Checklist of fish found from sampling stations in rice fields in That Phanom District, Nakhon Phanom

Province, Northeast Thailand.

Order/Family/Scientific name Number of fish species in each stations Total

1 2 3 4

Osteoglossiformes

Notopteridae

1. Notopterus notopterus (Pallas, 1780)

Clupeiformes

Clupeidae

2. Clupeichthys aesarnensis Wongratana, 1983

Cypriniformes

Cyprinidae

3. Parachela siamensis (Günther, 1868)

4. Parachela williaminae Fowler, 1934

5. Amblypharyngodon chulabhornae Vidthayanon &

Kottelat, 1990

6. Esomus metallicus Ahl, 1924

7. Rasbora aurotaenia Tirant, 1885

8. Rasbora borapetensis Smith, 1934

9. Rasbora rubrodorsalis Donoso-Büchner & Schmidt,

1997

10. Rasbora spilocerca (Rainboth & Kottelat, 1987)

11. Anematichthys armatus (Valenciennes, 1842)

12. Anematichthys repasson (Bleeker, 1853)

13. Hampala dispar Smith, 1934

14. Barbodes aurotaeniatus (Tirant, 1885)

15. Puntigrus partipentazona (Fowler, 1934)

16. Henicorhynchus siamensis (Sauvage, 1881)

17. Labiobarbus leptocheilus (Valenciennes, 1842)

Cobitidae

18. Acanthopsoides gracilentus (Smith, 1945)

19. Acanthopsoides hapalias Siebert, 1991

20. Lepidocephalichthys hasselti (Valenciennes, 1846)

Siluriformes

Bagridae

21. Hemibagrus nemurus (Valenciennes, 1840)

Siluridae

22. Kryptopterus cheveyi Durand, 1940

23. Ompok bimaculatus (Bloch, 1794)

Clariidae

24. Clarias batrachus (Linnaeus, 1758)

Beloniformes

Adrianichthyidae

25. Oryzias mekongensis Uwa & Magtoon, 1986

Belonidae

26. Xenentodon cancila (Hamilton, 1822)

Synbranchiformes

Synbranchidae

27. Monopterus albus (Zuiew, 1793)

Mastacembelidae

28. Macrognathus siamensis (Günther, 1861)

Perciformes

2

6

4

5

51

45

14

15

25

14

4

1

5

2

3

2

2

2

5

3

11

1

1

3

35

3

2

1

5

3

1

31

32

1

34

27

8

1

1

1

3

4

1

1

2

2

2

28

2

7

16

18

3

12

8

2

4

1

21

1

4

8

5

7

44

49

10

35

29

6

3

2

6

2

3

2

3

2

4

9

1

3

47

1

2

2

7

26

12

13

142

144

28

96

89

30

8

3

12

3

8

5

8

6

8

13

22

2

1

9

131

4

7

2

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Table 1 Checklist of fish found from sampling stations in rice fields in That Phanom District, Nakhon Phanom

Province, Northeast Thailand (cont.).

Order/Family/Scientific name Number of fish species in each stations Total

1 2 3 4

Ambassidae

29. Parambassis siamensis (Fowler, 1937)

Cichlidae

30. Oreochromis niloticus (Linnaeus, 1758)

Eleotridae

31. Oxyeleotris marmorata (Bleeker, 1852)

Gobiidae

32. Brachygobius mekongensis Larson & Vidthayanon,

2000

Anabantidae

33. Anabas testudineus (Bloch, 1792)

Osphronemidae

34. Betta smaragdina Ladiges, 1972

35. Trichopodus trichopterus (Pallas, 1770)

36. Trichopsis pumila (Arnold, 1936)

37. Trichopsis vittata (Cuvier, 1831)

Channidae

38. Channa striata (Bloch, 1793)

Total

4

3

5

3

4

1

5

4

6

10

311

1

5

2

4

1

2

2

5

3

216

2

6

3

3

12

8

5

132

5

8

2

5

12

3

4

10

11

10

359

10

16

9

10

26

8

14

28

30

28

1018

References

[1] M Halwart and MV Gupta (eds.). Culture of fish in rice fields. FAO and The WorldFish

Center, Rome, Italy, 2004, 83 p.

[2] JL Maclean, DC Dawe, B Hardy and GP Hettel. Rice Almanac: Source Book for the Most

Important Economic Activity of Earth. CABI Publishing, Wallingford, UK, 2002, 257 p.

[3] PA Roger. Biology and management of the floodwater ecosystem in rice fields. International

Rice Research Institute, Los Banos, Laguna, Philippines, 1996, 250 p.

[4] CNB Bambaradeniya, JP Edirisinghe, DND Silva, CVS Gunatilleke, KB Ranawana and S

Wijekoon. Bidiversity associated with an irrigated rice agro-ecosystem in Sri lanka.

Biodiversity and Conservation. 2004; 13, 1715-1753.

[5] CH Fernando. Rice fields are aquatic, semi-aquatic, terrestrial and agricultural: A complex

and questionable limnology. In: KH Timotius and F Goltenboth (eds.). Tropical Limnology 1,

1995, p. 121-148.

[6] O Katano, K Hosoya, KI Iguchi, M Yamaguchi, Y Aonuma and S Kitano. Species diversity

and abundance of freshwater fishes in irrigation ditches around rice fields. Environmental

Biology of Fishes. 2003; 66(2), 107-121.

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

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[7] M Yamazaki, Y Hamada, N Kamimoto, T Momii and M Kimura. Composition and

structure of aquatic organism communities in various water conditions of a paddy field.

Ecological research. 2004; 19(6), 645-653.

[8] M Halwart. Valuing aquatic biodiversity in agricultural landscapes. Diversifying Food and

Diets: Using Agricultural Biodiversity to Improve Nutrition and Food Security.

Routledge/Earthscan, 2013, p. 88-108.

[9] Y Natuhara. Ecosystem services by paddy fields as substitutes of natural wetlands in Japan.

Ecological engineering. 2013; 56, 97-106.

[10] HM Smith. The Freshwater Fish of Siam or Thailand. United States Government Printing

Office, Smithosonion Institution, Washinhton, 1945, Bull. No. 188, 622 p.

[11] WJ Rainboth. FAO species identification field guide for fishery purposes, Fish of the

Cambodian Mekong. Food and Agriculture Organization of the United Nations (FAO), Rome,

1996, 265 p, XXVII pls.

[12] M Kottelat. Fishes of Laos. Gunaratne Offset Ltd., Sri Lanka, 2001, 198 p.

[13] JS Nelson. Fishes of the world. 4th ed. John Wiley and Son, Inc., New York, 2006, 601 p.

[14] TM Berra. Freshwater fish distribution. Academic press, California, 2001, 604 p.

[15] J Bohlen, V Šlechtová, H Ta and R Britz. Phylogeny of the southeast Asian freshwater

fish genus Pangio (Cypriniformes; Cobitidae). Molecular phylogenetics and evolution. 2011;

61(3), 854-865.

[16] TR Robert. The Freshwater Fishes of Western Borneo (Kalimantan Barat, Indonesia).

Calif. Acad. Sci. Mem. 1989; 14, 1-210.

[17] Y Taki. An Analytical Study of the Fish Fauna of the Mekong Basin as a Biological

Production System in Nature. Res. Inst. Evol. Biol. 1978; Spec. Publ. 1, 1-77.

[18] A Iwata, N Ohnishi and Y Kiguchi. Habitat Use of Fishes and Fishing Activity in Plain

Area of Southern Laos. Asian and African Studies. 2003; 3, 51-86.

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Effect of hydrolyzed Cordyceps militaris on probiotic growth

Surachai Rattanasuk* and Tanyarat Pruemprom

Major of Biology, Department of Science and Technology

Faculty of Liberal Arts and Science, Roi Et Rajabhat University, Roi Et, Thailand

*Corresponding author: [email protected]

Abstract:

Galactomannan is polysaccharide that found in Cordyceps militaris which useful for

mannooligosaccharide production. This research aimed to study the effect of hydrolyzed C.

militaris on probiotic growth. C. militaris were hydrolyzed by Bacillus subtilis KS1, at 37 oC,

150 rpm for 7 days and reducing sugar was determined. The supernatant of C. militaris

hydrolysis was added into de Man, Rogosa and Sharpe (MRS) medium containing each

probiotic (Lactobacillus plantarum TISTR 543 or Lactobacillus casei TISTR 390 or

Lactobacillus acidophilus TISTR 1338) in 96-well culture plate and hydrolyzed copra meal

was used as positive control in triplicate experiments. Culture plates were incubated for 48 hr.

The optical density (600 nm) of probiotic was measured at before and after incubation. The

result demonstrated that hydrolyzed C. militaris can be promoted all probiotics but the

maximum promoting was observed in L. acidophilus TISTR 1338.

Keywords: Lactobacillus acidophilus TISTR 1338, Cordyceps militaris, probiotic growth

Introduction

Cordyceps militaris is medical fungus that belonging to Ascomycota, Sordariomycetidae,

Hypocreales and Codycipitaceae and well known for their pharmacological activities including

anti-liver fibrosis, immunomodulatory, anti-inflammatory, lowering blood glucose, antitumor,

and antibacterial activities and other favorable effects [1]. It contains biologically active

substance such as nucleosides (cordycepin; 3’-deoxyadenosine, and adenosine),

polysaccharides (galactomannan) and ergosterol [2, 3].

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doi: 10.14457/MSU.res.2017.1
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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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Galactomannan is mainly polysaccharide that found in C. militaris which is a non-

starch polysaccharide that consists of a mannose in main chain with galactose side chains [4].

Mannooligosaccharides (MOS) are prebiotics that obtained from galactomannan hydrolysis

using mannanase. MOS were applied as a feed additive to promote the growth of probiotics,

prevent pathogen colonization and modulate the immune system of host animals [5].

Probiotics are live microorganisms that confer a health benefit on the host [6]. Many

functional roles of probiotics such as restoration of the gut microbiome, reduction of the

concentration of cancer-promoting in the gut, pathogen exclusion, epithelial barrier,

bacteriocin production, prevention of gut inflammation and other intestinal or systemic disease

phenotypes [7,8]. From previous research [3] found C. militaris polysaccharide composed of

mannose and galactose. [3] The propose of this research was to study the effect of hydrolyzed

C. militaris on probiotic growth for new alternative way of food additive as prebiotics.

Materials and methods

C. militaris culture

C. militaris inoculum broth was purchased from kasetbuddy farm, Saraburi. C.

militaris inoculum was inoculated into bottle containing rice medium. The bottles were

incubated in dark at 18 oC for 15 days before continued culturing under light 18 oC for 45 days.

C. militaris was collected and dried at 60 °C for 18 hrs. Dried C. militaris were grinded to fine

powder and stored for later use.

Mannanase producing-bacteria

Bacillus subtilis KS1 was obtained from previous research [9]. B. subtilis KS1 was

cultured using sterilized Luria-Bertani (LB) and incubated at 37 oC, 150 rpm for 48 hrs prior

hydrolysis.

C. militaris hydrolysis

One milliliter of Bacillus subtilis KS1 cultured broth were added into 100 ml LB broth

containing 1% C. militaris incubated at 37oC, 150 rpm for 7 days. Copra meal hydrolysis was

used as positive control with the same culture condition. One milliliter of cultured broth was

collected every 24 hrs for sugar determination using dinitrosalicylic acid (DNS) method.

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Effect of hydrolyzed C. militaris on probiotic growth

Cultured broth was sterile by filtration and used as crude mannooligosaccharide (MOS).

Probiotics, Lactobacillus plantarum TISTR 543, Lactobacillus casei TISTR 390 and

Lactobacillus acidophilus TISTR 1338 were cultured using de Man, Rogosa and Sharpe (MRS)

medium for 24 hrs and adjusted the concentration at optical density 600 = 0.1. Crude MOS was

added into MRS medium containing each probiotic in 96-well culture plate and hydrolyzed

copra meal was used as positive control in triplicate experiments. The optical density (600 nm)

of each well were measured at before and after incubation at 37 oC for 48 hrs.

Results and discussion

C. militaris culture

After incubated in dark at 18 oC for 15 days and continued culturing under light at 18

oC for 45. C. militaris was grown (Fig. 1) and collected before dried at 60 °C. Dried C. militaris

were grinded to fine powder (Fig. 2) and used as substrate for mannanase hydrolysis using

Bacillus subtilis KS1. The culture condition is not similar to Kang et al. (2014) which used at

25 oC for liquid state culture.

Figure 1. Cordyceps militaris growth under culture condition.

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Figure 2. Cordyceps militaris powder.

C. militaris hydrolysis

C. militaris was hydrolyzed using Bacillus subtilis KS1 for 7 days. Reducing sugar on

each day was determined using DNS method. The results indicated that at day 7 demonstrated

highest amount of sugar concentration at 0.936 mg/ml for C. militaris and 0.137 mg/ml for

copra meal (Table 1.).

Table 1. Reducing sugar of C. militaris and copra meal hydrolysis

Day

Reducing sugar

concentration (mg/ml)

C. militaris Copra meal

1 0.204 0.057

2 0.314 0.058

3 0.338 0.060

4 0.436 0.065

5 0.715 0.084

6 0.719 0.107

7 0.936 0.137

Effect of hydrolyzed C. militaris on probiotic growth

Hydrolyzed C. militaris was obtained from hydrolysis using Bacillus subtilis KS1 for

7 days and was filter sterile before added into 96-well culture plate containing each probiotic.

Three strains of probiotic bacteria, Lactobacillus plantarum TISTR 543, Lactobacillus casei

TISTR 390 and Lactobacillus acidophilus TISTR 1338 showed similar growth curves and took

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approximately 18 hr and 36 hr to reach mid-exponential phase and stationary phase,

respectively [10]. The results indicated that 60 µl hydrolyzed C. militaris can be promoted all

probiotics, but the maximum promoting was observed in L. acidophilus TISTR 1338 when

supplemented with 10 µl hydrolyzed C. militaris (Table 2-4). In addition, hydrolyzed copra

meal can be promoted all selected probiotics but lower than hydrolyzed C. militaris.

Table 2. The average optical density at 600 nm of Lactobacillus plantarum TISTR 543.

Volume

(µl)

Hydrolyzed C. militaris Hydrolyzed copra meal

Before After (x̄±SD) Before After (x̄±SD)

10 0.11 0.67±0.02 0.11 0.14±0.02

20 0.11 0.67±0.04 0.11 0.14±0.03

30 0.11 0.70±0.03 0.11 0.16±0.03

40 0.11 0.71±0.02 0.11 0.16±0.03

50 0.11 0.72±0.03 0.11 0.17±0.02

60 0.11 0.89±0.01 0.11 0.17±0.04

70 0.11 0.75±0.02 0.11 0.19±0.02

Table 3. The average optical density at 600 nm of Lactobacillus casei TISTR 390.

Volume

(µl)

Hydrolyzed C. militaris Hydrolyzed copra meal

Before After (x̄±SD) Before After (x̄±SD)

10 0.11 1.27±0.03 0.11 0.73±0.03

20 0.11 1.34±0.02 0.11 0.65±0.02

30 0.11 1.38±0.03 0.11 0.48±0.04

40 0.11 1.39±0.02 0.11 0.42±0.04

50 0.11 1.50±0.03 0.11 0.38±0.03

60 0.11 1.56±0.03 0.11 0.30±0.04

70 0.11 1.43±0.02 0.11 0.21±0.03

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Table 4. The average optical density at 600 nm of Lactobacillus acidophilus TISTR 1338.

Volume

(µl)

Hydrolyzed C. militaris Hydrolyzed copra meal

Before After (x̄±SD) Before After (x̄±SD)

10 0.11 1.24±0.02 0.11 0.13±0.03

20 0.11 1.96±0.03 0.11 0.16±0.02

30 0.11 1.58±0.03 0.11 0.17±0.03

40 0.11 1.50±0.02 0.11 0.17±0.04

50 0.11 1.47±0.03 0.11 0.19±0.02

60 0.11 1.34±0.01 0.11 0.24±0.03

70 0.11 1.30±0.02 0.11 0.27±0.02

Conclusions

C. militaris was cultured in bottle containing rice medium under dark condition for 15 days

and continued culturing under light condition for 45 days at 18 oC. C. militaris was collected,

dried and grinded to fine powder. C. militaris powder and copra meal were hydrolyzed for 7

days using B. subtilis KS1. Cultured broth was collected for sugar determination using DNS

method. The results found that at day 7 showed highest amount of sugar concentration at 0.936

mg/ml for C. militaris and 0.137 mg/ml for copra meal. Hydrolyzed C. militaris was added

into 96-well plate containing each probiotic and incubate for 48 hr. Results presented that

hydrolyzed C. militaris can be enhanced all probiotics, but the maximum stimulating was

observed in L. acidophilus TISTR 1338 when supplemented with 10 µl hydrolyzed C. militaris.

C. militaris is alternative way for prebiotics production and feed additive for human and

livestock. Although the C. militaris is expensive but only small amount can be promoted the

probiotic.

Acknowledgements

This work was supported by Roi Et Rajabhat University.

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References

[1] Kang C, Wen T-C, Kang J-C, Meng Z-B, Li G-R, Hyde KD. Optimization of large-

scale culture conditions for the production of cordycepin with Cordyceps militaris by liquid

static culture. The Scientific World Journal. 2014, 1-15.

[2] Yeo JM, Lee SJ, Lee SM, Shin SH, Lee SH, Ha JK, et al. Effects of Cordyceps militaris

mycelia on in vitro rumen microbial fermentation. Asian-Australasian journal of animal

sciences. 2009;22(2):201-5.

[3] Yan H, Zhu D, Xu D, Wu J, Bian X. A study on Cordyceps militaris polysaccharide

purification, composition and activity analysis. African Journal of Biotechnology. 2008;7(22)

[4] LA Schalinske. 2015, Impact of β-Galactomannan on health status and immune

function in rats. M.Sc. Thesis. Iowa State University, Ames, Iowa.

[5] Rungrassamee W, Kingcha Y, Srimarut Y, Maibunkaew S, Karoonuthaisiri N,

Visessanguan W. Mannooligosaccharides from copra meal improves survival of the Pacific

white shrimp (Litopenaeus vannamei) after exposure to Vibrio harveyi. Aquaculture.

2014;434:403-10.

[6] Hotel ACP, Cordoba A. Health and nutritional properties of probiotics in food including

powder milk with live lactic acid bacteria. Prevention. 2001;5(1).

[7] Hemarajata P, Versalovic J. Effects of probiotics on gut microbiota: mechanisms of

intestinal immunomodulation and neuromodulation. Therapeutic advances in gastroenterology.

2013;6(1):39-51.

[8] De Vrese M, Schrezenmeir J. Probiotics, prebiotics, and synbiotics. Food

biotechnology: Springer; 2008. p. 1-66.

[9] Rattanasuk S, Prasertsang K, and Phiwphech S. Isolation of thermophilic mannanase-

producing bacteria useful for mannooligosaccharide (MOS) production. Science and

Technology (TICST), 2015 International Conference on; 2015: IEEE.

[10] Nguyen T. and Leenanon B. Effects of Various Stresses on Survivability of Probiotic

Bacteria in Acidic Condition 2016. The 18th Food Innovation Asia Conference, 2016 16 -18

June.

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Isolation of protein and nutrient characteristics analysis from lentil

Zar Zar Oo1*, Thwe Linn Ko2, Soe Soe Than3

1Industrial Chemistry Department, Yadanabon University, Mandalay, Myanmar

2Industrial Chemistry Department, University of Mandalay, Mandalay, Myanmar

3Industrial Chemistry Department, University of Yangon, Yangon, Myanmar

*Corresponding author: [email protected]

Abstract:

The main purpose of this research work was to isolate the most refined form of protein from

lentil bean for food processing. In this research work, lentils (Lens culinaris L.) was collected

from Monywa Township, Sagaing Region and nutritional characteristics such as moisture

content, ash content, fat content, carbohydrate content, protein content, fiber content and

energy value were determined. The fat of raw bean flour was removed by bulk soaking in

ethanol and also by soxhlet extraction using ethanol as solvent before isolating the protein. In

addition, the fiber and starch from defatted lentil flour was removed by alkaline extraction and

acid precipitation method to isolate the protein (isoelectric precipitation). Protein solubility,

water and oil absorption capacity, emulsifying capacity and stability, foaming capacity and

stability of lentil protein isolate have been determined. The solubility curve corresponding to

the lentil protein isolate indicated the minimum solubility at pH 4 (protein solubility of 20 %)

and maximum solubility at pH 12 (protein solubility of 85 %) respectively. The lentil protein

isolate had water absorption capacity of 1.82 ml H2O/g. protein and oil absorption capacity of

1.95 ml oil/g. protein. It was found that emulsion stability of isolated lentil protein was

41%with foaming capacity was 22.67 %., The foam stability was preserved up to 150 min.

Isolated lentil protein improved texture appearance and taste than the lentil flour and thus it

can better be used as nutrition and functional ingredients in many food products.

Keywords: Lentil, Defatted flour, Soxhlet extraction, Isoelectric precipitation, Lentil protein

isolate

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Received: 20 Aug 2017 Revised: 15 Sep 2017 Accepted: 20 Sep 2017
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Introduction

Plant proteins play significant roles in human nutrition, particularly in developing countries

where average protein intake deficient. Because of inadequate supplies of food proteins, there

has been a constant search for unconventional protein sources for use as both functional food

ingredients and nutritional supplements. Legumes such as lentil contain a high concentration

of proteins, carbohydrates and dietary fiber and make an important contribution to human diet

in many countries [7].

Lentil is a protein calorie crop, its protein content is 22% to 35%. Lentil is deficient in

the amino acids methionine and cysteine; it is an excellent supplement to cereal grain diets

because of its good protein and carbohydrate content. Plant protein products are gaining interest

as ingredients in food systems throughout many parts of the world; the final success of utilizing

plant protein additives depends greatly upon the favorable characteristics that they impart to

foods [7].

Beans are one of the most consumed legume worldwide. Beans reported to contain

17.6-23.62 % proteins, 1.27-3.62 % fat 2.86-5.00 % ash and 56.53-61.56 % carbohydrate [8]

.They have a balanced amino acid composition while they are low in sulfur-containing amino

acids (methionine and tryptophan) [9].

Isolates are the most refined form of protein products containing the greatest

concentration of protein but unlike flour and concentrates contains no dietary fiber. They are

very digestible and easily incorporated into different food products. Protein isolates are

nowadays believed to have played a major role in the development of new class of formulated

foods. It is high concentration of protein with the advantage of color, flavor and functional

properties make it an ideal raw ingredient for used in beverages, infant foods and children milk

food, textured protein products and certain types of specialty foods [6]. The objectives of this

research were to remove the fat, fiber and starch from lentil flour for enhancement of protein

isolation and to determine the characteristics of lentil protein isolate.

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Materials and Methods

Materials

Lentil was collected from Monywa Township, Saging Region, Myanmar. Ethanol from

(BDH Chemicals Ltd), Sodium hydroxide and hydrochloric acid of analar grades were used.

Methods

Preparation of Lentil Flour

About 300g of lentil seeds. were washed with water to remove foreign materials and

then the seeds were soaked in 1000 ml. of distilled water for 12 hr. and dehulled. After that,

the seeds were crushed to smaller fragments with a blender and dried in an oven at 60 ̊C for 12

hr. They were powdered and sieved with 80 mesh screen and then stored in an air tight

container.

Defatting the Lentil Flour

Lentil flour 100 g. was soaked in 600 ml. of 95 % ethanol for 16 hr. and followed by

soxhlet extraction (material to solvent ratio were 1:5 )at extraction temperature 60 ̊C. In order

to remove all ethanol, defatted lentil flour was dried in an oven at 60 ̊C for 12 hr. After that, it

was ground in the grinder and sieved with 200 mesh screen. Then, defatted flour powder was

packed with air- tight plastic bags.

Preparation of Lentil Protein Isolate

The protein isolate was obtained from defatted flour. Because the lentil proteins display

a higher solubility for pH>10, the pH of the defatted flour dispersion prepared in water was

adjusted, by using 2N NaOH, to 11.3. Fiber and starch fractions were removed from the

alkaline dispersion by centrifugation at 3000 rpm, for 30 min. Solubilized proteins were

collected as supernatant which subsequently was used for the protein fraction recovery by

isoelectric precipitation (pH 4.7). For pH adjustment, 2N HCl solution was used. After

precipitation, the proteins were separated by centrifugation at 3500 rpm, for 40 min. The

precipitate was washed with distilled water (pH 7.0) for three times, to achieve a complete

removal of any existing contaminant. The precipitate was allowed to dry at room temperature

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for 10 hr and then milled to pass 200 mesh screen. The isolated protein powder was stored in

air- tight plastic bags.

Methods of Analysis

Physico-chemical properties of lentil flour, defatted flour and lentil protein isolate such

as protein content, moisture, ash, fiber ,carbohydrate, fat content (AOAC- Method, 2000 ) [1]

and also protein solubility, water absorption capacity, oil absorption capacity, emulsion

capacity and stability, foaming capacity and stability of protein isolate were determined. The

ED-XRF, Energy Dispersive X-ray Fluorescence Spectrometer (SPETRO XEPOS, Benchtop

XRF Spectrometer) was used for the determination of elemental composition and FT-

IR,Fourier Transform Infrared Spectroscopy(FT-IR, Perkin Elmer, 8400, Shimadzu) was

examined the various functional groups of lentil protein isolate.

Results and Discussion

Physico-chemical properties of lentil flour were determined and presented in Table 1. It was

observed that the protein content utilized local lentil flour (22.58 %) of local lentil flour was

lower than that of the literature value, (31.12%) [5]. Fat content of utilized local lentil flour

(1.17 %) was also lower than that of the literature value (1.81%) [5].The moisture content of

local lentil flour was 9.62% to protect the greater danger of bacteria action and mould growth

which produce undesirable changes. However, the crude fiber of local lentil flour, 0.68 % was

significantly different from the literature value, 3.68 %. The high fiber content in literature may

be due to bean’s hulls. Thus, dehulling can reduce the fiber. The proximate composition of

bean flour can be varied depending on the weather and soil conditions, cultivation area, and

species of lentil, harvesting time and storage condition. High fat content may interfere protein

isolation and protein may be denatured. Therefore, fat should firstly be removed to isolate the

protein. In the preparation of lentil protein isolate, the highest isolation of protein was related

to the highest fiber removal and starch removal percentages from defatted lentil flour by using

isoelectric precipitation method. The best defatted lentil flour was obtained by soaking it in

ethanol solution for 16 hr, followed by soxhlet extraction (meal to solvent ratio were 1:5) at 60

˚C. By combining the two processes, the highest fat removal of 29.03 % was achieved with

relatively high protein content of 56.35 %. Isolation of protein from lentil was interrelated to

the fat removal percentage.

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Table 1. Physico-chemical Properties of Lentil Flour and Defatted Flour

Sr.

No. Composition (Dry Basis) Lentil Flour

*Literature

Values Defatted Flour **Literature Values

1 Protein content

(%w/w)

22.58 31.12 56.35 53.5

2 Moisture content

(% w/w)

9.62 9.14 10.14 8.7

3 Ash content

(% w/w)

2.42 2.62 8.81 4.6

4 Fiber content

(% w/w)

0.68 3.68 0.53 1.0

5 Carbohydrate content

(% w/w)

63.53 51.63 23.73 30.0

6 Fat content

(% w/w)

1.17 1.81 0.44 2.2

7 Energy value

Kcal /100 g

357 353 -

*Qayyum, et al., 2012 [5]

**Mehmet, 2010 [4]

Table 2. Characteristics of Lentil Protein Isolate

* Suliman, et al, 2006 [7]

**Note : The highest fiber removal, 69.81 % and starch removal, 66.89 % were achieved with relatively highest

protein content, 84.12% from defatted flour at pH 4.7 ( isoelectric point) by centrifugation at 3500 rpm, for

40min .

Characteristics of lentil protein isolate are described in Table 2. Lentil protein isolate was

characterized by protein content 84.12 % and low content in fiber, 0.16 % and in ash, 2.23 %.

By refinement, the carbohydrate level was substantially diminished to 7.87 %. Lentil protein

isolate showed a water absorption capacity (WAC) of 1.82 mlH2O/g protein. Water binding

properties of protein is determined by their degree of interaction with water. Lentil protein

Sr. No. Characteristics Lentil Protein

Isolate

*Literature

Values

1 Protein content (% w/w) 84.12 84.46

2 Moisture content (% w/w) 5.20 4.03

3 Ash content (% w/w) 2.23 2.85

4 Fiber content (% w/w) 0.16 0.18

5 Carbohydrate content (% w/w) 7.87 7.88

6 Fat content (% w/w) 0.42 0.60

7 Water absorption capacity (ml.H2O/g.) 1.82 1.90

8 Oil absorption capacity (ml.oil/g.) 1.95 1.9

9 Emulsion stability (%) 41 40-46

10 Foaming capacity (%) 22.67 23.45

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isolate has a higher capacity of swelling, distortion and separation, that allows additional

exposure of binding sites of water and increases water absorption [7].The oil absorption

capacity (OAC) of lentil protein isolate was 1.95 ml oil/g. proteins. Lentil protein isolate

showed higher oil absorption capacity than chickpea[7].The mechanism of absorption as a

physical entrapment of oil; several authors have related the oil absorption capacity to

interaction of nonpolar side chain of the protein as well as to the conformation features of the

proteins. High values of OAC are convenient in the protein isolates that are used as ingredients

in the cold meat industry, particularly for sausages [7]. The WAC and OAC are determinants

properties to develop a food of acceptable quality. The OAC is an important functional property

because it improves mouth feel and flavor retention [7].Emulsion stability of isolated protein

was 41 %. It was within the literature value [7].They [7] also reported that the emulsion stability

depends primarily upon the water and oil absorption capacity. The foaming capacity of lentil

protein isolate was 22.67% similar with the literature value [7]. Lentil protein foam had a lower

capacity but highly stable compared to soy protein that studied [7].

The elemental compositions of lentil protein isolate was analyzed by ED-XRF. The

data are shown in Table 3. It shows a rich source of chlorine. Chlorine is a component of all

body secretions and excretions resulting from processes of building (anabolism) and

breakdown (catabolism) body tissues.

Table 3. Elemental Compositions of Lentil Protein Isolate Analyzed by ED-XRF Method

Sr. No. Elements Compositions

(%w/w)

1 Chlorine (Cl) 3.437

2 Phosphorus (P) 0.6384

3 Calcium (Ca) 0.3932

4 Potassium (K) 0.2090

5 Silicon (Si) 0.1647

6 Aluminum (Al) 0.1260

7 Sulfur (S) 0.09842

8 Iron (Fe) 0.05680

9 Manganese (Mn) 0.01418

10 Titanium (Ti) 0.00986

11 Vanadium (V) 0.00273

12 Chromium (Cr) 0.00142

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113

Figure 1 showed the minimum solubility was observed at pH 4 to 6 and maximum solubility

occurred at the extreme pH. Therefore, the lack of electrical charge for pH 4.7, influenced

negatively the water binding and the solubility of protein. For extreme pH values, the net

electrical charges are high, and allow rejection forces between the protein chains and thus the

protein solubility increases. At pH 10, the lentil protein isolate solubility was 70% while at pH

2, the lentil protein isolate solubility was 53%. Also at pH 12, protein solubility was 85 % while

at pH 4, the protein solubility was 20%.The reduction in solubility, at very low pH values could

be due to the protein denaturation and insolubilization processes [2]. Lentil protein showed

good solubility in both acid and alkaline pH region, which is an important characteristic for

food formulation [7].

Figure 1. Effect of pH on the Protein Solubility of Lentil Protein Isolate

Emulsion capacity of lentil protein isolate at different pH are shown in Figure 2. Emulsion

plays an important role in the manufacture of food products such as ice cream, mayonnaise,

dressings and emulsified sausages [2]. The emulsifying capacity is an indicator used to evaluate

the emulsion stabilizing properties of the lentil protein isolate. The ability of proteins to form

stable emulsions depends on the size, charge, hydrophobic surface and flexibility of protein

molecules. It was observed that emulsion capacity of protein was affected by environmental

factor like pH [2].

Most vegetable proteins are globular proteins with low foaming properties [2].By contrast, the

foam developed by lentil protein isolate still stable after keeping the foam for 150 minutes at

room temperature, and the foam volume still retained 50.11% as show at Figure 3.

0

20

40

60

80

100

Pro

tein

So

lub

ilit

y (

%)

pH

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

114

Figure 2. Effect of pH on the Emulsion Capacity of Lentil Protein Isolate

Figure 3. Effect of Time on the Foaming Stability of Lentil Protein Isolate

Various functional groups of lentil protein isolate was determined by FT-IR and the respective

spectrum are shown in Figure 4 .The main absorption bands of peptide linkages are related to

C=O stretching at 1635.69 cm-1 for lentil protein isolate represent amide primary, N-H bending

and C-N stretching at 1528.64 cm-1 for lentil protein isolate represent amide secondary .With

regard to the presence of primary amine group, the spectrum indicated by the vibrational

frequencies for amine were at 3469.09 cm-1 for lentil protein isolate. In addition, the bands

observed at 2864.39 cm-1 for lentil protein isolate is due to the presence of OH stretching. Thus,

it was a normal lentil protein isolate consisting of, amide, carboxylic acids and carbonyl groups

[3].

0

20

40

60

80

100

30 60 90 120 150

Fo

amin

g

Sta

bil

ity

(%)

Time (min.)

0

10

20

30

40

50

60

70

80

2 4 6 8 10 12E

muls

ion C

apac

ity (

%).

pH

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

115

Figure 4. FT-IR Spectrum of Lentil Protein Isolate

(a) (b) (c) (d)

Figure 5. (a) Lentil (b) Lentil flour (c) Defatted flour (d) Lentil protein isolate

Conclusions

Lentil flour could be effectively defatted by using the combination of soaking in ethanol

solution followed by soxhlet extraction. It was found that the highest fat removal percentage

29.03 % was achieved with the highest protein content 56.35 %. The highest isolation of protein

was related to the highest fiber removal and starch removal percentages from defatted flour by

using isoelectric precipitation. The highest protein content 84.12 % was achieved at pH 4.7. At

pH 12, protein solubility was 85 % while at pH 4, the solubility was 20 %.The lentil protein

isolate had water absorption capacity, 1.82 ml H2O/g. protein and oil absorption capacity of

1.95 ml oil/g. protein. The foam developed by lentil protein isolate was found to be still stable

up to 150 minutes at room temperature, and of foam volume retained 50.11 %. Having their

excellent functional properties, lentil protein isolate can be further utilized for the

supplementation of various food products.

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Acknowledgements

I would like to express my gratitude to Faculty of Technology, Mahasarakam University for

giving the chance and kind permission to present the research paper at IPSFAB 2017 and Dr.Yi

Yi Myint,Professor and Head (Retd), Industrial Chemistry Department, Yadanabon University,

Myanmar for her encouragement and editing the manuscript. Finally, I also wish to

acknowledge to my supervisor Dr.Soe Soe Than, Professor, Industrial Chemistry Department

, University of Yangon and co-supervisor Dr.Thwe Linn Ko, Professor, Industrial Chemistry

Department, University of Mandalay, Myanmar, for their invaluable guidance, and kind advice

throughout the research period.

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[2] Aurelia, I, et al., 2009,“Chemical and Functional Characterization of Chickpea Protein

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[3] Kudre TG., Benjakula S, & Kishimura H, 2013,“Comparative Study on Chemical

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IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017

117

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