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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
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
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
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
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
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
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
),(
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
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.
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)
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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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four commercial pig crossbreeds in China. Genet. Mol. Res. 2012
[3] Bennett G. L., Tess M. W., Dickerson G. E. and Johnson R. K. Simulation of heterosis
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[6] Poldvere A., Tanavots A., Saar R., Torga1 T., Kaart T., Soidla1 R., Mahla1 T., Andreson
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[8] Johnson Z. B., Chewning J. J. and Nugent R. A. Maternal effects on traits measured during
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[9] Goran KUŠEC, Gordana KRALIK, Antun PETRIČEVIĆ, Vladimir MARGETA, Zlata
GAJČEVIĆ, Draženka GUTZMIRTL and Mario PEŠO. Different in slaughtering
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[12] Hoque M. A., Kadowaki H., Shibata T., Oikawa T. and Suzuki K. Genetic parameters for
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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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[18] Bressan M. C., Almeida J., Santos Silva J., Bettencourt C., Francisco A. and Gama L. T.
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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).
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
11
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
12
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.
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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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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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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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:
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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
[] a[] a
[] a
[] a
[] a [] a[] a
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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).
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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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34
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
39
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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40
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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41
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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
42
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.
References
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[2] Leitzmann C. Vegetarian Diets: What Are the Advantages? In: Elmadfa I (ed). Diet
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[6] Marsh AG, Sanchez TV, Michelsen O, Chaffee FL, Fagal SM. Vegetarian Lifestyle and
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calcium from milk and tempeh consumed by postmenopausal Malay women using the dual
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pneumoniae During Tempeh Fermentation and Proof of Enterotoxin Absence by PCR. Appl
Environ Microbiol 1994; 60(5): 1495-1499.
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[11] Liem IT, Steinkraus KH, and Cronk TC. Production of Vitamin B-12 in Tempeh, a
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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,
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 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].
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
51
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
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Functions (review article). Vet. Med. International. 2011, DOI:10.4061/2011/686137
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Selected Medicinal Plants in Western Region of India. Advances in Biological Research, 2010:
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[3] Eko suharton, Ella Viani, Mustaqim Apriyansa Rahmadhan, Imam Syahuri Gultom,
Muhammad Farid Rakhman, and Danny Indrawardhana. Total flavonoid and antioxidant
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[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
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-
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[6] Dai, J. & Mumper, R. Plant Phenolics: Extraction, analysis and their antioxidant and
anticancer properties. Molecules. 2010, DOI: 10.3390/molecules15107313
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properties of S. palustris, an edible medicinal fern. Bot Studies. 2012: 53, 439-446.
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Phenolic,Flavonoid, Carotenoid Content. International Journal Research of Pharmaceutical
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53
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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 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-
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
55
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
57
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
58
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%
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
59
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
60
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
61
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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
62
References
[1] Rattanapanone N, Chongsawat C, Chaiteep S, Fresh-cut Fruits in Thailand,
HORTSCIENCE. 2000; 35, 1-4.
[2] Consumers and Ready-to-Eat Meals, Available at:
https://www.scribd.com/document/239399788/ Consumer-and-Ready-to-Eat-Meals, accessed
May 2017.
[3] Microbiological quality guide for ready-to-eat foods, Available at:
http://www.foodauthority.nsw.gov.au/_Documents/scienceandtechnical/microbiological_qual
ity_guide _for_ RTE_food.pdf, accessed on May 2017.
[4] Atsuka M, Chaicumpa W, Chongsa-Nguan M, Samosornsuk S, Monden S, Kouichi T.
Prevalence of foodborne pathogens in open markets and supermarkets in Thailand, Food
Control. 2009; 21, 221–226.
[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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63
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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64
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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66
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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68
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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69
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.
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solutions and fruit juices. J. Food Microbial. 2011; 146, 111–117.
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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
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[2] Hardeland R, Pandi-Perumal SR, Cardinali DP.Melatonin".The International Journal of
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doi:10.016/j.biocel.2005.08.020.
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[7] Reiter RJ, Acuña-Castroviejo D, Tan DX, Burkhardt S. Free radical-mediated molecular
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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
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[10] Chen, G., Huo, Y., Tan, DX., Liang, Z., Zhang, W. and Zhang, Y. (2003). Melatonin in
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[11] AOAC. (2000). Official Methods of Analysis of international (17th ed.). Washington
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[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
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[18] Marin F, Luquet G, Marie B, Medakovic D. Molluscan shell proteins: primary structure,
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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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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
[1] V. Panyakul, “Palm sugar: The ingenous sweetnees. ILEIA Newsletter,” vol. 13, pp. 19,
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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
of Biological Sciences, vol. 11, pp. 989-995, 2008.
[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
heating at relatively high temperatures”, Journal of Food Engineering, vol. 39, pp. 415- 422,
1999.
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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,
2005.
[10] S. I. F. S. Martins, W. M. F., Jongen, M. A. J. S., van Boekel, “A review of Maillard
reaction in food and implications to kinetic modeling”, Trends in Food Science and
Technology, vol. 11, pp. 364–373, 2001.
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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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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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
99
[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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100
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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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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
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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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
107
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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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.
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
109
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
IPSFAB-2017 International Postgraduate Symposium on Food, Agriculture and Biotechnology 2017
110
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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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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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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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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