Variation of gaba content and physical properties of sprouted mung bean fortified yoghurt

The effect of germinated mung beans and storage time on the properties of yoghurt are determined.. Germinated mung bean-fortified yoghurt having high gamma-aminobutyric acid GABA and low

THAI NGUYEN

UNIVERSITY OF AGRICULTURAL AND FORESTRY

KIMBERLY ANNE A BAUTISTA

GERMINATED MUNG BEAN-FORTIFIED YOGHURT

BACHELOR THESIS

Study Mode: Full-time Major : Food Technology Faculty : Advanced Education Program Office Batch : 2017-2021

DOCUMENTATION PAGE WITH ABSTRACT

Thai Nguyen University of Agriculture and Forestry

Degree Program Bachelor of Food Technology

Student name Kimberly Anne A Bautista

Yoghurt and mung beans each have their benefits in the aspect of health The effect

of germinated mung beans and storage time on the properties of yoghurt are determined Germinated mung bean-fortified yoghurt having high gamma-aminobutyric acid (GABA) and low phytic acid (PA) content with competent physical and sensory qualities is produced in aiding the need to improve health

This project followed certain analytical methods for the determination of total dry matter content, protein content, GABA content, PA content, acidity, pH level, and water holding capacity 2-factor ANOVA analysis and Tukey comparison were used for data processing The results concluded that the protein and GABA content rises with the temperature On the contrary, PA and total dry matter content lower when the germination time and temperature increase Further results state that storage time modifies the viscosity, acidity, pH, water holding capacity, protein, PA, GABA, and total dry matter of yoghurt However, it does not exceptionally affect the physical

properties of the yoghurt The project then provides recommendations for future researches regarding this topic It is suggested to expand the germination temperature range to more specifically monitor the change of components in mung bean seeds and, to study some other lactic acid bacteria that are capable of synthesizing GABA

My gratitude also goes to the Advance Education Program of Thai Nguyen University

of Agriculture and Forestry for the support they have given to all of the students in their final year Great thanks to Vietnam National University of Agriculture for allowing me

to use their laboratory and equipment which are necessary for the completion of this dissertation

I would also like to thank Nguyen Thi Thu Thuy for helping with the usage of the laboratory and its equipment The next ones I would like to thank are my friends who gave simple advice and discussions during mental blockage I would also like to gratify

my family for their continuous encouragement

Lastly and most importantly, I would like to state my deepest gratitude to God who made everything in life a possibility

TABLE OF CONTENT

List of Figures 2

List of Tables 3

List of Abbreviations 4

PART I INTRODUCTION 5

1.1 Research rationale 5

1.2 Research’s objectives 6

1.3 Research questions and hypotheses 6

1.4 Limitations 7

PART II LITERATURE REVIEW 8

PART III METHODS 11

3.1 Material and chemicals 11

3.2 Methods 12

3.2.1 Production process of germinated mung bean yoghurt 12

3.2.2 Analytical method 13

3.2.2.1 Determination of total dry matter content 13

3.2.2.2 Determination of protein content 14

3.2.2.3 Determination of GABA content 15

3.2.2.4 Determination of phytic acid content 16

3.2.2.5 Determination of the PH of yoghurt 17

3.2.2.6 Determination of the acidity of yoghurt 17

3.2.2.7 Determination of the viscosity of yoghurt 18

3.2.2.8 Determination of water holding capacity (WHC) 18

3.2.2.9 Sensory assessment 19

3.2.3 Data processing method 19

PART IV RESULTS 21

4.1 Chemical composition of raw green beans 21

4.2 Total dry matter content in germinated mung beans 21

4.3 Content of gamma-aminobutyric acid in mung bean seeds germination 22

4.4 Phytic acid content in germinated mung beans 22

4.5 Protein content in germinated green beans 23

4.6 Total dry matter content of germinated mung bean yoghurt during cold storage ………23

4.7 Gamma-aminobutyric acid content of germinated mung bean yoghurt during cold storage 24

4.8 Phytic acid content of germinated mung bean yoghurt during cold storage 24 4.9 Protein content of germinated mung bean yoghurt during cold storage 25

4.10 Effect of storage time on pH of germinated green bean yoghurt 25

4.11 Effect of storage time on acidity of mung bean yoghurt germination 26

4.12 Effect of storage time on the viscosity of mung bean yoghurt germination……… … 26

4.13 Effect of storage time on water holding capacity of mung bean yoghurt

germination 27

4.14 Sensory quality of mung bean yoghurt 27

PART V DISCUSSION AND CONCLUSION 29

5.1 Discussion 29

5.2 Conclusion 35

REFERENCES 37

APPENDICES 42

LIST OF FIGURES

Figure 1 Effect of time and temperature on total dry matter content of mung beans

……….……… ……… 21 Figure 2 Effect of time and temperature on GABA content of mung beans …… 21 Figure 3 Effect of time and temperature on PA content of mung beans……… …22 Figure 4 Effect of time and temperature on protein content of mung beans.…… 22 Figure 5 Effect of cold storage time on total dry matter content of mung bean yoghurt ……… … 23

Figure 6 Effect of cold storage time on GABA content of mung bean yoghurt… 23

Figure 7 Effect of cold storage time on PA content of mung bean yoghurt…… 24

Figure 8 Effect of cold storage time on protein content of mung bean yoghurt… 24

Figure 9 Effect of cold storage time on the pH level of mung bean yoghurt…… 25

Figure 10 Effect of cold storage time on the acidity of mung bean yoghurt…… 25

Figure 11 Effect of cold storage time on the viscosity of mung bean yoghurt……26

Figure 12 Effect of cold storage time on WHC of mung bean yoghurt……… …26

Figure 13 Mung bean yoghurt final product……… …….26

Figure 14 Sensory evaluation results of mung bean yoghurt……… ………27

LIST OF TABLES

Table 1 The 9-Point Hedonic Scale 19

Table 2 Chemical parameters of raw green peas 20

Table 3 Statistical processing table of effects of temperature and germination time

on GABA, PA and chemical composition of mung bean seeds 20

LIST OF ABBREVIATIONS

FeCl3 iron (III) chloride

H2NaO+ sodium hydrate

H2SO4 sulfuric acid

HClO4 perchloric acid

TUAF Thai Nguyen University of Agriculture and Forestry

VNUA Vietnam National University of Agriculture

WHC water holding capacity

PART I INTRODUCTION

1.1 Research rationale

Recently, people tend to acquire much awareness when it comes to their overall health condition including the physical and mental aspects In aiding the need to improve health, yoghurt containing compounds with human health benefits is a product we can consider

Yoghurt contains vitamins, minerals, and even probiotics which are why it is recognized to have great health benefits to human beings such as in improving bone health and aiding digestion (Ware, 2018) Studies show that those who consume yoghurt obtained a “better overall diet quality” (Yoghurt Nutrition, 2017) There have been a number of yoghurt products fortified with beans in the market such as soybean yoghurt

Mung bean-fortified yoghurt is a possible alternative to be able to surpass the limitations of soy yoghurt Mung bean has an extensive history of being used as a traditional medicine It is recognized as a functional food since it contains a lot of nutrients, vitamins, minerals, and bioactive compounds (Hou et al., 2019) In addition, it also contains gamma-aminobutyric acid (GABA) GABA is a non-protein amino acid It is produced by plants, animals, and microorganisms When

it comes to food industries, GABA-rich products are developed for its health benefits which include gut modulation, neurostimulation, and cardioprotection (Diez-Gutiérrez et al., 2020) Nevertheless, mung bean also has a certain amount

recognized as an anti-nutrient since it interferes with mineral absorption (Butner, 2021) In cereals, PA is one of the main storage forms of phosphorous Phytase which is an enzyme is responsible for lowering the PA content (Kumar & Anand, 2021) Moreover, the germination of mung beans enhanced the nutritional value Higher concentration of nutrients, lower PA content, and higher protein content and ascorbic acid is obtained when sprouting (Shah et al., 2011)

All things considered, yoghurt and mung beans each has their own benefits in the aspect of health Germinated mung bean is suitable in producing fortified yoghurt having high GABA content and low PA maximizing the benefits that can be acquired

1.2 Research’s objectives

This project aimed to produce germinated mung bean-fortified yoghurt having high GABA and low PA content enhancing its health benefits with competent physical and sensory qualities

1.3 Research questions and hypotheses

The stated research questions have been answered to successfully accomplish the objective

1 How does the germination process affect GABA, PA content, and composition of mung beans?

2 How does cold storage affect GABA, PA content, and the properties of yoghurt?

A series of analytical tests are conducted to test the following: GABA, PA, and composition in mung beans

Ha: Storage times directly affect the GABA, PA content and properties of mung bean fortified yoghurt

PART II LITERATURE REVIEW

It is defined that nutrition is a biological process that involves the consumption of food (Piccardi & Manissier, 2009) In this modern generation, nutrition will be so much different compared to how it was back in the previous century To address certain health concerns, the development of functional foods is emerging in the market place (Hasler, 2000)

Fermented products are associated to the improvement of human health (Savaiano&Hutkins, 2020) and yoghurt is recognized to be a healthy food due to its numerous nutrients (Fazilah et al., 2018) “Reduced risk of type 2 diabetes, metabolic syndrome, and heart disease, along with improved weight management” are some health benefits that a person can obtain from the consumption of yoghurt

or some other fermented foods In addition, consuming these products has been studied to also improve lactose malabsorption, reduced respiratory infections, and enhanced immune and anti-inflammatory responses Based on the demand, fortification of yoghurt by enhancing some of its properties or qualities that is beneficial to health is continuously investigated It is possible to add ingredients such as plant extract to develop functional yoghurt with added beneficial properties

which is much better than conventional yoghurt (Fazilah et al., 2018) Streptococcus thermophilus, Lactobacillus delbrueckiisubsp bulgaricus, Bifidobacterium, and Lactobacillus strains are yoghurt starter cultures that are

manipulated due to their probiotic properties (Kok and Hutkins, 2018)

Gamma-aminobutyric acid is studied for its physiological benefits considering it

as an essential bioactive compound There is a wide range of GABA-enriched food products due to its wide health benefits without affecting the product’s sensory qualities (Park & Oh, 2007) The main function it is valued and why is it suitable for the development of functional foods is because it acts as a major inhibitor of neurotransmitter in the brain (Al-Taher&Nemzer, 2019) Furthermore, it acts as an antihypertensive agent that is considered as a possible treatment for disorders related to sleep and neurological issues (Chua, Koh and Liu, 2018) Not only for neurostimulation, it has also helped when it comes to gut modulation and cardioprotection (Diez-Gutiérrez et al., 2020) For the development of functional foods, directly adding pure GABA to products is not permissible In this case, adding GABA to enhance food health properties is done through seed germination

or microbial fermentation (Chua, Koh and Liu, 2018)

Mung beans are seeds that contain GABA Mung beans have been used as a traditional medicine way back in the past for containing numerous nutrients, vitamins, minerals, and bioactive compounds which is why it is also recognized as

a functional food (Hou et al., 2019) The process of germination is associated to obtaining higher GABA Materials that are germinated such as barley, soy bean, and grains are identified to have higher contents of GABA than those not undergoing the process of germination (Tiansawang et al., 2016) Another thing to consider when using mung bean is its PA content Fewer nutrients are absorbed into the body since PA blocks the absorption of specific minerals such as iron and zinc (Butner, 2021) As a solution, the best things to use are beans that went

through the process of germination Germinated seeds are associated to acquiring less PA content which is better and healthier for human consumption (Shah et al., 2011)

Previous literatures regarding mung bean-fortified yoghurt were limited; however, functional yoghurt with soy bean is gaining popularity in the market Materials that undergoes germination is best to use for it has higher concentration of GABA (Tiansawang et al., 2016) and has lower content of PA (Shah et al., 2011) which makes it greatly suitable for the production of mung bean-fortified yoghurt

PART III METHODS

The project took place in the Faculty of Food Technology Laboratory and Central Food Technology Center Laboratory - Vietnam National University of Agriculture (VNUA), Hanoi, Vietnam The stated laboratories for this project are used from April to May 2021

There are 2 parts for this project:

Part 1: Influence of germination (time and temperature) on GABA, PA content, and composition of mung bean

Part 2: Influence of cold storage time on GABA content, PA content, and physical and sensory qualities of yoghurt Resulting yoghurts were stored at 4-6˚C

3.1 Material and chemicals

Mung bean seeds were supplied by Tan Tuong An Manufacturing and Trading Co., Ltd They were bought as whole seeds with their olive-green colored skin Lactic acid bacteria XPL-20 from CHR-Hansen, Denmark was provided by Brenntag

Vietnam Company, containing strains of Lactococcus lactis subsp cremoris, Lactococcus lactis ssp lactis and Streptococcus thermophilus Sugar with the

brand name Lam Sơn was purchased at Big C Long Bien – Gia Thuy – Long Bien – Hanoi, Vietnam Whole milk powder or whey, skimmed milk was supplied by

The equipment and tools needed for the experiment and assessments are provided

by the stated laboratories The project used the following: analytical balance, centrifuge, centrifuge tube, Brookfield DV-I PRIME viscometer, Orion model 230A+ portable PH meter, refrigerator, and drying oven The following is used for measurements and storage: petri dish, measuring cups, pipette, and triangular flask where each tools have different capacity Other necessary equipment and tools were also used

3.2 Methods

3.2.1 Preparation of germinated mung bean yoghurt

Mung beans were soaked in fresh water at 36 ° C for 10 hours and water was replaced at 2-hour intervals by freshwater The beans were taken out and germinated under the following conditions: 28⁰C, 30⁰C, 32⁰C in 12, 18, and 24 hours Afterward, mung bean milk was extracted by removing the peel, grinding it with water, and filtering the residues Following that, the appropriate amount of the following ingredients were mixed: sugar (7%), powdered milk (4%), whey powder (2%), and avocado oil (2%) and it was allowed to rest for 6⁰C / 2 hours

Furthermore, the mung bean milk was added to the mixture and stirred at 20MPa / 55⁰C This process reduced the fat globules, which improved the product's viscosity Pasteurization is also performed at 90 °C for 5 minutes to kill microorganisms and increase the enzyme's water-holding capacity It was then cooled to 43 ° C to facilitate microorganism inoculation before being poured into containers and fermented at 43 ° C with a pH of 4.5 Finally, it was refrigerated for

21 days at a temperature of 4–6 °C

3.2.2 Analytical method

3.2.2.1 Determination of total dry matter content

The dry matter content was determined using the drying method specified in TCVN 4295:2009 5g of sample was weighed and placed in a petri dish with an identified mass before being dried to constant mass The dish containing the sample was placed in the oven and dried for 4 hours, until it reached a temperature

of 102 ± 2° C The dish was removed and allowed to cool for about 30 minutes The mass of the sample-containing dish was then weighed and recorded to the nearest 0.001 g After another 60 minutes of drying, the mass was weighed and recorded for the second time The sample was considered dried when the difference between the two recorded masses did not exceed 0.001 The water content, in percentage by mass (X) was calculated by:

𝑋 = 𝑀2− 𝑀1

𝑀1 × 100

The formula is presented by the percentage of dry matter content (X), final mass

in grams of the dish containing the sample after drying (M2), and the initial mass

in grams of the dish containing the sample before drying (M1)

3.2.2.2 Determination of protein content

TCVN 8125:2009 was used to determine the protein content using the Kjeldahl method

1 mL of yoghurt (approximately 1.03 g 0.001) and 10 mL of concentrated H2SO4 were weighed, placed in a Kjeldahl flask, and left for 30 minutes A stove was used

in the experiment to slowly heat the sample When white smoke appeared, 4–5 drops of HClO4 catalyst were added, yielding a clear colorless solution After cooling, the sample was transferred to a 100 mL volumetric flask and used as the solution for nitrogen distillation Furthermore, 30 mL of sample was aspirated into

an Erlenmeyer flask, a few drops of taxiro indicator were added, and the solution was neutralized with 30% NaOH until it turned green The flask was heated using

an electric heating system The NH3 vapor was condensed through the cooling system and collected in the receiver during this operation The solution was then made in an Erlenmeyer flask to collect NH3, then pipetted into the receiver with around 25 mL of boric acid and a few drops of tashiro indicator solution (the solution was pink at this stage) It was used in a system where the tip of the condenser was immersed in a borate acid solution The nitrogen distillation process was repeated until the solution in the collecting tank turned green, after which it was left for another 10–15 minutes to capture all of the NH3 In the collection vessel, the sample was titrated with H2SO4 0.1N, and the solution changed from

green to red The same procedure was applied to the test flask that does not contain any sample

The protein content (%) by mass was determined by the following formula:

𝑋 = (𝑉1− 𝑉2) ∗ 𝑇 ∗ 𝑉 ∗ 0.014 ∗ 100 ∗ 6.25

𝑣 ∗ 𝑀 ∗ 1000

The amount of nitrogen is 0.014 which is equivalent to using 1 ml of 0.1N (g) sulfuric acid solution The formula is presented by the number of ml of 0.1N H22SO4 used for blank titration (V2), number of ml of 0.1N H2SO4 used for sample titration (V1), conversion factor of nitrogen content to protein content (6.25), mass in grams of sample being analyzed (M), gram-equivalent concentration of H2SO4 solution (T), number of mL of sample solution used for ammonia distillation (v), and total volume of diluted sample solution (V)

3.2.2.3 Determination of GABA content

In the Eppendorf tube, 1 g of sample was mixed with 5 ml of deionized water and shaken for an hour before decanting the clear solution 0.5 mL of the solution, 0.2

mL of borate buffer with pH = 9.0, 1 mL of phenol, and 0.4 mL of sodium hydroxide were placed in a glass tube, and the mixture was thoroughly shook After that, it was placed in a boiling water bath for 10 minutes, followed by a cold water bath for 20 minutes, and then the mixture was shaken repeatedly until the color blue appeared 2 mL of 60% ethanol was added to the mixture Furthermore, the sample was analyzed at 645 nm A standard curve graph with concentrations of 0;

0.04; 0.08; 0.12; 0.16; 0.2 mg / l was used to determine the GABA content by light absorbance using an ultraviolet-visible spectrophotometer (Zhang et al 2014)

3.2.2.4 Determination of phytic acid content

Various colorimetric methods existed to determine the PA content of such products, including AOAC, Wade, Chen, and Haug-Lantzsch (H-L) (Kahrman et al., 2020) The PA content in this project was analyzed using the colorimetric method developed by Wade (Gao et al., 2007)

The standard PA solution was made by diluting standard sodium phytate with distilled water to achieve concentrations of 0; 5; 10; 20; 40; and 80 g/ml A 14ml eppendorf tube was filled with 0.5g (0.001g) of the homogenized sample and 10ml

of 2.4 % hydrochloric acid It was shaken at 220 rpm for 16 hours before being centrifuged at 1,000 rpm for 20 minutes at 100 ° C The clear solution was gradually poured into a centrifuge tube, and 0.5 g of 99.5 % pure NaCl was added The salt was dissolved by shaking the vortex at 350 rpm for 20 minutes For another 20 minutes, the sample was incubated at -200°C After that, it was centrifuged at 1,000 rpm for 20 minutes at 100°C to separate the clear fluid 1 mL

of clear solution was diluted in 24 mL of distilled water Shake 3 ml of the diluted solution and 1 ml of the Wade solution (0.03 % FeCl3 6H2O + 0.3 % sulfosalicylic acid) well in the eppendorf tube At 100 ° C, samples were centrifuged at 1000 rpm for 10 minutes Using a molecular absorption spectrometer, the absorbance of analytical samples and calibrators was measured at 500nm (Shimazu UV 1800, Japan) Three replicates of the experimental samples were analyzed The PA content was calculated and expressed by the formula:

𝑃𝐴 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 (𝑚𝑔/𝑔𝐶𝐾) = 𝐶∗𝑉∗100

𝑚∗𝐶𝐾∗1000

The formula is presented by volume of extract (V), mass of sample in mg (m), percentage of dry matter content in sample (CK), and PA concentration in mg / ml (C)

3.2.2.5 Determination of the pH of yoghurt

The pH of yoghurt was measured using a Singapore Eutechbenchtop pH meter after fermentation and 1, 7 14 and 21 days of cold storage

3.2.2.6 Determination of the acidity of yoghurt

The acidity of yoghurt was determined using Fermented milks - Determination of titratable acidity - Potentiometric method TCVN 6509: 2013 (ISO 11869: 2012)

The sample was prepared and allowed to reach 22 °C ± 2 °C before being mixed evenly Approximately 10 g of the test sample and 10 ml of water were weighed and mixed in a 50-ml beaker The electrode for the pH meter was prepared and placed in the test sample The mixture was then titrated in the beaker with sodium hydroxide solution while stirring for 4–5 seconds until it reached a stable pH of 8.30 ± 0.01 The amount of sodium hydroxide used was measured in milliliters with a measurement error of less than 0.05 ml Calculate the titrable acidity as a percentage of the sample weight in grams (Advanced Acid and Ester Titration Basics, 2019) Use the following formula:

% 𝑎𝑐𝑖𝑑 = 𝑁 𝑥 𝑉 𝑥 𝑀

𝑆 𝑥 10

(N), volume of standard NaOH used for titration in milliliters (V), molecular weight of the predominant acid in the sample divided by the number of hydrogen ions in the acid molecule that are titrated (M), and sample size in milliliters or grams (S)

3.2.2.7 Determination of the viscosity of yoghurt

Viscosity was measured at 6˚C using Brook-field DV2T The speed was 12 rpm and the measurement time was 25 seconds using the LV4-64 measuring axis It was determined after fermentation, as well as after 1, 7, 14, and 21 days of cryopreservation The viscosity units were shown as Pa.s

3.2.2.8 Determination of water holding capacity (WHC)

The water holding capacity of yoghurt was determined according to the method stated by Alline et al (2018)

A centrifuge tube was filled with approximately 2 g (0.001 g) of yoghurt samples and spun Centrifuged for 10 minutes at 6 °C at a rotation speed of 3000 rpm The whey was carefully separated after centrifugation, and the remainder was weighed Water retention was determined at the end of fermentation as well as after 1, 7, 14, and 21 days of cold storage WHC was calculated with the given formula:

%𝑋 = 𝐺1− 𝐺2

𝐺 × 100

The formula is presented by mass in grams of yoghurt and the centrifuge tube after dehydration (G1), mass of the centrifuge tube (G2), and mass of yoghurt before centrifuging (G)

3.2.2.9 Sensory assessment

A sensory assessment was conducted to evaluate the color, taste, texture, and overall sensory qualities of yoghurt based on the perceptions of the consumers In this case, we are able to specify the preferences of the consumers and manipulate the qualities, adapting to the public's liking using the 9-point hedonic scale

Table 1 The 9-Point Hedonic Scale

3.2.3 Statistical data analysis

The difference between the experimental formulas was compared using a 2-factor ANOVA analysis of variance on Minitab 16 software The difference in mean

values between formulas was compared using the Tukey comparison method with

a confidence level of 95%