POLITEKNIK TUN SYED NASIR SYED ISMAIL

THE EFFECT OF FERMENTATION

TIME AND TEMPERATURE
TOWARDS THE PHYSICOCHEMICAL
CHARACTERISTICS OF SOYBEAN-
BASED YOGURT

ALYAA SYAFIAH BINTI

KAMARUDIN
(33DTM21F1021)
NURSYAFIANA BINTI MOHD FAUZI
(33DTM21F1024)
MOHAMAD AIEMAN BIN JEFFRI
(33DTM21F1025)

JABATAN TEKNOLOGI KIMIA DAN

MAKANAN

SESI II 2022/2023

1
POLITEKNIK TUN SYED NASIR SYED ISMAIL

THE EFFECT OF FERMENTATION

TIME AND TEMPERATURE
TOWARDS THE PHYSICOCHEMICAL
CHARACTERISTICS OF SOYBEAN-
BASED YOGURT

ALYAA SYAFIAH BINTI KAMARUDIN

(33DTM21F1021)
NURSYAFIANA BINTI MOHD FAUZI
(33DTM21F1024)
MOHAMAD AIEMAN BIN JEFFRI
(33DTM21F1025)

Report Submitted to the Department of Chemical & Food

Technology in Partial Fulfilment of the Requirements for the
Diploma of Food Technology

JABATAN TEKNOLOGI KIMIA DAN MAKANAN

SESI II 2022/2023

2
 DECLARATION

THE EFFECT OF FERMENTATION

TIME ANDTEMPERATURE
TOWARDS THE
PHYSICOCHEMICALCHARACTERIS
TICS OF SOYBEAN-BASED YOGURT

1. We, 1. ALYAA SYAFIAH BINTI KAMARUDIN(33DTM21F1021)

2. NURSYAFIANA BINTI MOHD FAUZI (33DTM21F1024)

3. MOHAMAD AIEMAN BIN JEFFRI (33DTM21F1025)

are the student Semester 4 Diploma in Food Technology,

Department ofChemical and Food Technology, Tun Syed
Nasir Syed Ismail Polytechnic,located at Hab Pendidikan
Tinggi Pagoh KM 1 Jalan Panchor 84600 Pagoh, Johor,
Malaysia.

2. We acknowledge that ‘The Effect of Fermentation Time and

Temperature Towards the Physicochemical Characteristics of
Soybean-Based Yogurt ’ and the intellectual property contained there
in are our original work or inventions without taking or copying any
intellectual property from other parties

3. We agree to transfer ownership of the intellectual property of ‘The

Effect of Fermentation Time and Temperature Towards the
Physicochemical Characteristics of Soybean-Based Yogurt’ to Tun Syed
Nasir Syed Ismail Polytechnic to meet the requirements for the award of
the Diploma in Food Technology to us.

3
 Made and duly acknowledged by the said:

a) ALYAA SYAFIAH BINTI

KAMARUDIN ………………………
(ID NUMBER: 030804-04-0188), ALYAA
SYAFIAH
BINTI
KAMARUDI
N

b) NURSYAFIANA BINTI MOHD FAUZI

……………………………….
(ID NUMBER: 030213-01-1094),and

NURSYAFI
ANA BINTI
MOHD
FAUZI

c) MOHAMAD AIEMAN BIN

JEFFRI ……………………………….
(ID NUMBER: 030225-01-0560) MOHAMAD
AIEMAN
BIN
JEFFRI

in……………… on…..…………

In front of me,

………………………………………….

ENCIK MUHAMMAD AKMAL BIN JELANI

As a supervisor on the date ………………….

4
 ACKNOWLEDGEMENT

First and foremost, we would want to thank God for finally allowing us to
complete our Seminar study assignment on time. Although a few issues
among us while working on this proposal, the lecturer's task was completed
with all afford by group members. Fortunately, all of the issues have been
resolved, and we have been able to react appropriately and effectively.

Aside from that, we would like to express our deep and sincere gratitude to
our research supervisor, Sir Muhammad Akmal bin Jelani, for his critical
involvement in completing our research project in this manner. He was
always making exciting recommendations and encouraging us on how to
finish our study project in order to get a positive output from the research
that had been researched.He taught us the methodology for conducting the
research and presenting the findings as clearly as possible.We also
appreciate the effort you spent editing and fixing our errors in this study's
text.

On the other side, we would like to express our gratitude to the Seminar
instructor, Madam Raz Zarinda Binti Mohd Rashid, who constantly teach
and assist us in understanding the research themes that we should be aware
of when studying Seminar.We would also want to show our gratitude to all
of the Lab Assistant at Polytechnic Tun Syed Nasir Syed Ismail, Encik
Hanafi bin Jais, who allowed us to utilise the laboratory's equipment and
machine.

Last but not least, thank our classmates that always work hard to share and
improve our research project,as well as for greatly assisting us in making
this research project success.. Hope that all the effort will give a lot of
benefits to us, and our study project.

5
6
 ABSTRAK

Kacang soya atau nama saintifiknya adalah Glycine max, merupakan

tumbuhan serba boleh dan mempunyai khasiat tersendiri yang tergolong dalam
keluarga Leguminosae. Berdasarkan kajian yang dikaji, terdapat penyataan
masalah yang perlu diselesaikan . Antara penyataan masalah dalam kajian ini
adalah kesan suhu penapaian yang berbeza terhadap tekstur yogurt berasaskan
kacang soya dan ketidakseimbangan dalam keadaan penapaian yang boleh
menyebabkan variasi dalam kandungan dan kualiti protein serta suhu
penapaian ke atas kelikatan dan ketegasan yogurt berasaskan kacang soya,
menilai kesan masa dan suhu penapaian ke atas kandungan protein yogurt
berasaskan kacang soya, serta menyiasat bagaimana pemprosesan yang
berbeza, termasuk suhu dan masa, menjejaskan kestabilan warna yogurt.
Kajian analitikal ini akan melibatkan ujian kelikatan yogurt kacang soya
menggunakan viskometer, menyiasat kandungan protein menggunakan kaedah
KJELDAHL, dan melihat perubahan warna menggunakan spektrofotometer
UV.

7
 ABSTRACT

Soybean (Glycine max) is a versatile and nutritious plant that belongs to the
Leguminosae family. Additionally, the problem statement for this study is the
effect of different fermentation temperatures on the viscosity of soybean-based
yogurt. In addition, inconsistencies in the fermentation conditions of soybean-
based yogurt can cause variations in its protein content and quality,
challenging for production and marketability. In addition, processing
conditions, including fermentation time and temperature can affect the color of
soy protein products. The objective of this product is to investigate the effect
of fermentation time and temperature on the viscosity and firmness of
soybean-based yogurt, to evaluate the effect of fermentation time and
temperature on the protein content of soybean-based yogurt and to investigate
how processing differs. conditions, including temperature and time, affect the
color stability of yogurt. Our analytical study is based on the viscosity test for
soybean yogurt using a viscometer, investigating the protein content using
KJELDAHL method and seeing the color change using a UV
spectrophotometer.

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 TABLE OF CONTENT

CHAPTER CONTENT PAGE

ABSTRACT 6
ABSTRAK 7
TABLE OF CONTENT 8-9
LIST OF TABLES 10
LIST OF FIGURES 11
LIST OF SYMBOLS 12
LIST OF ABBREVIATIONS 13
1 INTRODUCTION 14
1.1 BACKGROUND OF STUDY 14
1.2 PROBLEM STATEMENT 15-16
1.3 RESEARCH OBJECTIVE 16
1.4 RESEARCH SCOPE 17-18
1.5 SIGNIFICANT OF STUDY 19-20
1.6 EXPECTED OUTCOME 20-21
2 LITERATURE REVIEW 22
2.1 DAIRY PLAIN YOGURT 22
2.2 SOYBEAN-BASED YOGURT 23
2.3 SOYMILK 23-24
2.4 LACTIC ACID BACTERIA STRAINS 24
2.5 FERMENTATION 24
2.6 PHYSICAL ATTRIBUTES OF THE 25
SOYBEAN-BASED YOGURT
2.6.1 Viscosity 25
2.6.2 Color 25
2.7 CHEMICAL ATTRIBUTES OF THE 26
SOYBEAN-BASED YOGURT

9
 2.7.1 Protein Content

3 METHODOLOGY 27

3.1 INTRODUCTION 27
3.2 RESEARCH FRAMEWORK 28 - 29
3.3 MATERIALS AND METHOD 30
3.3.1MATERIALS 30
3.3.2PRODUCTION OF SOYBEAN- BASED 30
YOGURT
3.3.3 EQUIPMENT 31

3.3.3.1 Viscosity 31-32

3.3.3.2 Color 33-34
3.3.3.3 Protein Content 35
3.3.4STATISTICAL METHOD 36
APPENDIXES

-GANTT CHART 37
-COST ESTIMATION 38
REFERENCES 39-41

10
 LIST OF TABLES

NO. TABLE TITLE PAGE

1 Gantt Chart 37
2 Cost Estimation 38

11
 LIST OF FIGURES

NO. TITLE PAGE

FIGURES
2.1 Dairy Plain Yogurt 22
3.3.3.1 Brookfield Viscometer 25
3.3.3.2 Spectrophotometer 33
3.3.3.3 Protein Analyzer 1(VELP UDK 159 35

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 LIST OF SYMBOLS

◦ Degree
％ Percentage

13
 LIST OF ABBREVIATIONS

SB Soybeans
SBY Soy-based Yogurt
USDA United States Department of Agriculture
FT Fermentation Time
ͦ
FT Fermentation Temperature
C Celcius
hrs Hours
SP Soy Protein
SM Soy milk
LA Lactic Acid
L. Lactobacillus
H2SO4 Sulphuric Acid
N Nitrogen
rpm Revolution per minute
ml milimeter
SPSS Statistical Packages for the Social Sciences

14
 CHAPTER 1

INTRODUCTION

1.1 BACKGROUND OF STUDY

Soybeans (SB) which scientific name is Glycine max (USDA,2021) are a

legume that have been cultivated for thousands of years for their protein-rich
seeds, which can be used for a variety of food products. SB are rich in proteins,
essential amino acids, minerals, and vitamins, making them a valuable source
of nutrition (Yang et. al, 2018). As per the United Nations Food and
Agriculture Organization (FAO), soybeans rank as the third-largest crop in the
world, preceded only by wheat and maize. Furthermore, by research from
Sarwar et. al, 2020, soy-based yogurt (SBY) can be an excellent alternative for
those who are lactose intolerant or have a milk allergy. It has been identified
that the functional properties of SBY can help develop new food formulations
that can benefit the food industry and consumers. In the production of
SBY, it is important to note that fermentation time (FT) and fermentation

°
temperature (FT ) significantly affect the physicochemical properties of soy
yogurt, including viscosity and protein content, as well as color stability. In

°
this study, it was found that increased FT and FT led to higher levels of
viscosity, acidity, and protein content in the SBY (Kim et. al, 2017). However,
it was also observed that the color stability decreased because

of these conditions. Thus, the authors emphasized the importance of carefully

controlling the fermentation conditions to achieve desired physicochemical
properties in soy yogurt.

15
1.2 PROBLEM STATEMENT

°
The problem that this study addresses is the effect of different FT on the
texture of SBY.SBY is a popular plant-blased alternative to dairy yogurt,
which is rich in protein. and other essential nutrients. However, the quality
°
and texture of SBY can be. significantly impacted by the FT . This can
lead to inconsistencies in the final product, affecting consumer acceptance and
overall profitability. Based on studied by Chen et. al, (2019), the quality and
°
viscosity of SBYare significantly influenced by the FT . The study
determined that the optimal temperature range of SBY production is 42℃ to
44℃ with a FT of 8 to 10 hours(hrs). At these temperatures, the soybean milk
ferments successfully, producing a smooth and creamy texture with a mild sour
ͦ
flavor. However, if the FT exceeds 44℃, the texture of the yogurt becomes
grainy and sandy. due to the heightened activity of proteolytic enzymes that
break down the proteins in the SB milks. Conversely, at temperatures below
42℃, the fermentation process is sluggish, resulting in weakly flavored and
runny-textured yogurt.

Furthermore, inconsistencies in the fermentation conditions of SBY can

lead to variations in its protein content and quality, which pose challenges for
production. The study conducted by (Jin et. al, 2020), found that a longer FT
of 24 hrs at a lower temperature of 37°C resulted in a higher protein content
and quality compared to a shorter FT of 8 hrs at a higher temperature of 45°C.
The extended FT allowed for greater microbial activity and enzymatic
hydrolysis of the soy protein (SP), leading to a higher protein content and
quality. Moreover, the lower temperature of 37°C facilitated the growth of
lactic acid bacteria, which produced organic acids responsible. for the tangy
flavor of yogurt. Conversely, the higher temperature of 45°C during the shorter
FT may have caused protein denaturation, reducing the protein quality.
by altering the protein structure and decreasing its biological activity and
nutritional value.

16
 Next, in SBY production, the color of the final product is affected by
various processing conditions, including temperature and time. According to the
research by Dhanapala et. al, (2018), revealed that the color of SP products can
be significantly affected by processing conditions such as temperature and time.
The studied observed that higher temperatures caused the product to become
darker due to the Maillard reaction which was accelerated. at higher
temperatures, leading to a more intense Browning effect. Similarly, the study
found that as the processing time increased, the color of SP products became
darker due to the prolonged exposure to heat. The heat caused the proteins
to denatured and form aggregates resulting in a darker color. Therefore, produce
SBY with desirable color, it is crucial to carefully control the processing
conditions including the temperature and time.

1.3 RESEARCH OBJECTIVE

In this analysis, the objectives to be executed are :

i. To produce plant based soybean-based yogurt with fermentation time at 6

hours but difference fermentation temperature (30°C, 42° C, 47°C )
ii. To investigate the effect of fermentation time and temperature on the
physicochemical properties in terms of viscosity, color stability and
protein content.

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1.4 RESEARCH SCOPE

The scientific name for soybeans is Glycine max. Soybean (Glycine max)
is a versatile and nutritious plant that belongs to the Leguminosae family. It is
the main ingredient used for the production of soybeans-based yogurt.
Soybeans, pandan leaves and sugar are bought from Family Store which
always focuses on residential areas located in Duyong, Melaka. Probiotic
capsules were purchased from Pharmacy Seri Murni Simpang Renggam. The
chemicals used in this analysis are concentrated sulfuric acid, sodium
hydroxide, copper sulfate, potassium sulfate and boric acid.

The scope of the study describes the extent to which the research area will
be explored in the study and defines the parameters within which the study will
operate. For the study, analysis related to the chemical content and physical
properties of SBY using soy milk as the main ingredient because soy milk
(SM) has a comparable amount of protein to cow’s milk, but very little
saturated fat and no cholesterol. That is able to replace cow’s milk which is the
main ingredient in the production of traditional yogurt. Due to the above, SM
is considered one of them as a healthy alternative to cow’s milk. Researchers
like to identify whether through analysis SBY will have viscosity, protein
content and color in each part that is affected by FT and FT°. The method used
for analysis is DV-1 VISKOMETER to measure the viscosity of yogurt,
SPECTROPHOTOMETER SP-UV 200 to determine the good color of
yogurt and finally using PROTEIN ANALYZER 1 (VELP UDK 159) to
determine the optimal protein content in yogurt. In this research, using a FT for

°
6 hours but different FT (30°C, 42°C and 47°C).

18
 For physical analysis involving viscosity and color. The texture and
viscosity of yogurt are important sensory properties which affects the

°
consumer’s perception of the product. FT is a critical factor in yogurt
production as it affects growth and activity bacteria responsible for

°
fermentation. Research has been conducted to determine the effect of FT
on soy-based physical properties yogurt. In addition, processing
temperature is one of the critical parameters that affect the color stability of
yogurt. During the yogurt making process, milk is heated to a certain
temperature to denature milk proteins and promote the growth of lactic acid
bacteria. The heating temperature can vary depending on the type of yogurt
and the desired texture and taste. Finally, involving chemical analysis which

°
is the protein content. FT and FT can significantly affect the protein content
and quality of SBY.

19
1.5 SIGNIFICANT OF STUDY

The research finding will contribute towards the production of soy-

based yogurt that has gained considerable attention due to its potential health
benefits, especially for those who are lactose or allergic to dairy products. One
°
area of interest research is the effect of different FT on the texture of SBY.
The study to understand the effect of different fermentation temperatures on
the texture of SBY is essential for developing a high-quality product that meets
consumer preferences. Additionally, the slower the fermentation process, it
allows the breakdown of soy protein occurs, resulting in a more homogenous
texture. According to Lee et. al, (2019), the researchers found that lower
fermentation will reduce particle size in soy-based yogurt and result in a
smoother texture. The study to understand the effect of different fermentation
temperatures on the texture of SBY is essential for developing a high-quality
product that meets consumer preferences.

The study on the effect of fermentation time and temperature on the

protein content of soybean-based yogurt is significant as it provides insights
into the optimal conditions for enhancing the nutritional value and quality of
soybean-based yogurt, which is important for production and marketability.
The study can also contribute to the development of new and improved
processing methods for soy-based products, especially for individuals who
cannot consume dairy products. Additionally, the findings of the study can
inform consumers of the importance of selecting SBY that are fermented for a
longer duration and at a lower temperature to obtain the maximum nutritional
benefits.

20
 Moreover, the processing conditions, including temperature time and
temperature can affect the color of SP product. In this study, the impact of
various processing conditions, such as temperature and time, on the color
stability of soy protein isolate was explored (Zhang et. al, 2021). The
researchers discovered that increased processing temperatures and prolonged
processing times led to the production of soy protein products with a darker
color. Additionally, this effect was more noticeable when the pH levels were
alkaline.

1.6 EXPECTED OUTCOME

The fermentation process plays a crucial role in the production of soy-

based yogurt, affecting the physical and chemical properties of the final
product. The temperature of fermentation is a critical factor in determining the
yogurt's viscosity, color stability, and protein content. Fermenting SBY at
30°C for six hrs is expected to result in a yogurt with moderate viscosity, good
color stability, and optimal protein content. The fermentation rate at this
temperature is moderate, resulting in a moderately thick viscosity. The
production of lactic acid during fermentation leads to the denaturation and
coagulation of milk proteins, contributing to the yogurt's thickening and
moderate increase in viscosity. The color stability of the yogurt is expected to
remain stable, as the temperature is relatively low, and the FT is short. The
protein content of the yogurt is also expected to be optimal, as this temperature
range is ideal for the activity of lactic acid bacteria during fermentation.
Therefore, careful temperature control during fermentation is essential to
produce a high-quality SBY with the desired physical and chemical properties.

Fermentation temperature plays a crucial role in the production of soy-

based yogurt, affecting the physical and chemical properties of the final
product. At a high temperature of 42°C, fermentation is expected to occur at a
faster rate, resulting in a thinner viscosity due to a higher production of lactic
acid that denatures and coagulates milk proteins. The concentration of solids

21
and the pH of the yogurt also impact its thickness. The increased production of
lactic acid at 42°C leads to a rapid decrease in pH, which can further influence
the viscosity of the soy-based yogurt. To achieve the desired thickness in the
final product, careful temperature control during fermentation is necessary. It
is recommended to use a lower temperature range to produce SBY with
moderately thick viscosity.
Fermenting soy-based yogurt at 47°C for 6 hrs is expected to produce a
thicker and more viscous product due to the critical role of fermentation
temperature in the activity of lactic acid bacteria. This bacterium converts
lactose into lactic acid during fermentation, causing the milk proteins to form a
gel-like structure that determines the texture and viscosity of the final product.
Previous research (Cruz et. al, 2019) has shown that the optimal temperature
range for soy-based yogurt production is 40-47°C, and higher temperatures can
result in undesirable flavor, texture, and viscosity. However, higher
temperatures are expected to increase microbial activity and lactic acid
production, leading to a thicker and more viscous SBY product

22
 CHAPTER 2

LITERATURE REVIEW

2.1 DAIRY PLAIN YOGURT

Dairy plain yogurt is a well-liked dairy product that is appreciated

globally for its flavor, consistency, and nutritional value. The process of
making it involves heating milk to a specific temperature to break down the
milk proteins, followed by cooling and introducing live lactic acid (LA)
bacteria cultures. These bacteria consume the lactose present in the milk,
producing lactic acid (LA) which causes the proteins to coagulate and form a
thick, smooth consistency. The American Dairy Products Institute (2021)
defines plain yogurt as a fermented milk product containing the characteristic
bacterial cultures of Lactobacillus(L.) Bulgaricus and Streptococcus
Thermophilus, which aligns with the yogurt standard set by the U.S. Food and
Drug Administration.Due to its high protein, calcium, and nutrient content,
plain yogurt is a popular option among health- conscious consumers. It can be
consumed on its own or utilized as an ingredient in various dishes, ranging
from dips and smoothies to dressings and marinades.

Figure 2.1 : Dairy Plain Yogurt

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2.2 SOY-BASED YOGURT

SBY can be produced by fermenting soy milk, which is made from ground
SB and water and can be used as a base for yogurt production. The addition of
bacterial cultures such as L. Bulgaricus and Streptococcus Thermophilus to soy
milk leads to the fermentation process that produces SBY. Another study
(Shokryazdan et. al, 2019), explored the effects of probiotic bacteria on SBY
and found that the inclusion of L. Rhamnosus and Bifidobacterium Lactis
enhanced its nutritional properties and health benefits. While SBY may have a
slightly different taste and texture compared to dairy- based yogurt, research
has shown that it is generally well-received by consumers, particularly those
with dietary restrictions or a focus on health (Khalid et. al, 2021).

2.3 SOYMILK

Soymilk is a heart-healthy alternative to cow's milk as it is low in saturated

fat and does not contain cholesterol. Additionally, it can be fortified with
functional ingredients like probiotics or prebiotics to provide added health
benefits beyond basic nutrition. According to Huang et. al, 2019, incorporation
of soy protein isolates to soymilk led to higher protein content, lower fat
content, and improved sensory properties. Kim et. al, 2021, the fermentation of
soymilk using probiotic strains has the potential to enhance the texture, flavor
and nutritional attributes of the product, offering potential health advantages. It
can be concluded that the good suggestion for soymilk production by
conducting an optimal fermentation and duration process that can increase the
bioavailability of nutrient and may also impact the final product. However, the
limitation of the production of soymilk is the presence of anti-nutritional
factors in soybeans, such as trypsin inhibitors, phytic acid, and lectins, which
can affect the absorption of nutrients and contribute to digestive problems in
some individuals (Kumar and Prasad, 2020). These factors need to be
addressed through processing techniques such as soaking, heating, and
fermentation to improve the nutritional quality and digestibility of the final
product. Additionally, the availability and cost of SB can also be a limiting

24
factor in the production of SM. In addition, it may be beneficial to clearly label
soy-containing ingredients in food products, including SM, to help
individuals

with soy allergies avoid allergen exposure. Further research could also be
conducted to better understand the prevalence and severity of soy allergies and
to develop more effective allergen management strategies in the food industry.

2.4 LACTIC ACID BACTERIA STRAINS

LA bacteria play a vital role in the fermentation process of soymilk

yogurt, contributing to its unique texture and tangy taste. Depending on the
manufacturer, different strains of LA bacteria, such as L. Acidophilus,
Bifidobacterium Bifidum, and Streptococcus Thermophilus, may be used (Chen
et. al, 2019). Research has shown that these strains of LA bacteria have several
health benefits, including enhancing digestion, improving the immune system,
and reducing the risk of certain diseases (Ouwehand et. al, 2020).
Additionally, the fermentation process increases the bioavailability of soybean
nutrients, making them more easily absorbed by the body (Li et. al, 2019).

2.5 FERMENTATION

In the process of producing SBY, fermentation plays a crucial role in

transforming SM into a thick and creamy yogurt-like product. To initiate
fermentation, a starter culture containing LA bacteria like L. Bulgaricus and
Streptococcus thermophilus is typically added. These bacteria break down
lactose in the SM, converting it into lactic acid, which lowers the pH of the
mixture and causes proteins to coagulate, resulting in the thick texture of
yogurt. In recent years, there has been a research emphasis on improving the
fermentation process of SBY to enhance its nutritional and sensory properties.
An example of this is the study conducted by Liu et. al, (2019), which
investigated the impact of incorporating prebiotics in the fermentation process
of SBY. The findings showed that the inclusion of prebiotics could augment
the growth and activity of probiotic bacteria, thereby resulting in an improved

25
sensory quality and probiotic content of the yogurt

26
2.6 PHYSICAL ATTRIBUTES OF THE SOYBASED YOGURT

2.6.1 Viscosity

The thickness or resistance to flow of a liquid is called viscosity, and

SBY typically has lower viscosity than dairy-based yogurt because of its lower
protein content. Additionally, the texture and consistency of SBY can be
influenced by the type and quantity of stabilizers and thickeners utilized during
production, with ingredients such as pectin, carrageenan, and starch being
effective at increasing viscosity and enhancing texture (Ahmed, Anjum,
Randhawa, & Saeed, 2021). Fermentation is a key factor that can affect the
viscosity of SBY. Extending the FT can result in a thicker and creamier yogurt,
but excessive fermentation should be avoided since it can cause the yogurt to
become too thick and possibly develop an unpleasant sour flavor.

2.6.2 Color

Kamal et. al, (2021) reported that the color of SBY can be influenced
by chemical and biochemical changes occurring during fermentation, making
it an important physical characteristic of the product. Specifically, the study
found that variations in the concentration of SP and pH levels can impact the
yogurt's color. During the fermentation of SBY, LA bacteria produce lactic
acid, which leads to a decrease in pH. As a result, the SP in the yogurt can
denature and coagulate, causing a color change from pale yellow to creamy
white. The extent of this color change depends on the concentration of SP in
the product. Additionally, other compounds such as phenolic compounds and
carotenoids can impact the color of SBY and also provide antioxidant
properties. Zeng et. al, (2020) discovered that the inclusion of black SB hull
extract in SBY increased its color intensity due to the presence of
anthocyanins, which are water- soluble pigments. The addition of black SB
hull extract also enhanced the yogurt's antioxidant activity.

27
2.7 CHEMICAL ATTRIBUTES OF THE SOYBASED YOGURT

2.7.1 Protein Content

SB are known for their high protein content, which translates to the
protein content of SBY. However, the amount of protein in soy yogurt can
vary depending on factors such as the type and concentration of SM, the
fermentation process, and the inclusion of other ingredients like stabilizers and
sweeteners. According to the United States Department of Agriculture(USDA)
reports that a cup of plain soy yogurt typically contains around 6 grams of
protein(USDA, 2021). The protein content of soy yogurt can be influenced by

°
chemical attributes like the concentration of SM, FT and FT , and the use of
stabilizers and thickeners. Recent studies suggest that increasing the
concentration of SM, fermenting for longer periods at higher temperatures, and
adding specific stabilizers can significantly boost the protein content of SBY.

28
 CHAPTER 3

METHODOLOGY

3.1 INTRODUCTION

Methodology is the strategy used to undertake the research, and is a pivotal

online for the project. Methodology depends on the nature and purpose of
theory in the pursuit of knowledge, and influences procedures in the
experiment. The analysis and equipment that used in this research project are
Brookfield viscometer to find the viscosity of SBY. Kjeldahl method, which
involves the determination of the nitrogen content of a sample and then
converting it into protein content using a conversion factor and last one are
color using a spectrophotometer. Kjeldahl analysis is a widely used method for
determining the protein content of food products, including SBY. The Kjeldahl
method involves the digestion of the sample with sulfuric acid(H2SO4) and
heating with a catalyst to convert nitrogen(N) to ammonium sulfate. The
amount of N in the sample is then measured and converted to protein content
using a conversion factor. The viscosity of SBY is an important parameter that
affects the product’s texture and mouthfeel. Viscosity can be measured using a
viscometer, which measures the resistance of a fluid to flow. The most
commonly used viscometer for food products is the Brookfield viscometer.
The viscosity of SBY can be affected by various factors, including the type and
amount of stabilizers used, processing conditions, and storage temperature
(Aryana & Olson, 2019). Next, the analysis that was used in this research
project was color. Color is an important sensory characteristic of food products
and can influence consumer acceptance. Color can be measured using a
spectrophotometer, which measures the amount of light absorbed or
transmitted by a sample at different wavelengths. The color of SBY can be
affected by various factors, including the type and amount of coloring agents
used, processing conditions, and storage temperature (Chen et. al, 2020).

29
3.2 RESEARCH FRAMEWORK

The Effect of Fermentation Time and Temperature Towards the

Physicochemical Characteristics on Soybean-Based
Yogurt.

Production of Soybean-based Yogurt

500g soybean was washed and soaked in

water for two hours.

Drained the soybean and soaked well.

The soybeans were ground and pressed well into

bean curd machine with 1350ml water.

The soymilk had been heated to a temperature of

82℃

The heated soymilk was cooled to 45℃.

Probiotic capsule Lactobacillus Acidophiles and

60g of sugar were added to soymilk.

30
 The soymilk was fermented at different temperature
for 6 hours

Formulation 3 (The
Formulation 2 (The soymilk ferment at 42℃ for 6 hours)
Formulation 1 (The
soymilk ferment at soymilk ferment at
30℃ for 6 hours) 47℃ for 6 hours)

Analysis

PHYSICAL CHEMICAL
ANALYSIS ANALYSIS

VISCOSITY PROTEIN
COLOR
CONTENT

DV-1 SPECTROPHOTOMETER PROTEIN

SP-UV 200 ANALYZER 1
VISCOMETER
(VELP UDK 159)

31
3.3 MATERIALS AND METHOD

3.3.1 MATERIALS

Soybeans, pandan leaves and sugar were purchased from Family Store that
always focusses on residential area which located in Duyong, Melaka.
Probiotic capsule was purchased from Pharmacy Seri Murni Simpang
Renggam. Chemical used in the present work were concentrated H2SO4,
sodium hydroxide, copper sulphate, potassium sulphate and boric acid.

3.3.2 PRODUCTION SOYBEAN-BASED YOGURT

About 500g soybean were first washed and soaked in water for two
hours.Then,drainned the soybeans and rinsed them well. The soaked soybeans
were ground and pressed into bean curd with 1350ml water. Auto seperation
by bean curd machine were seperated into soymilk and soybeans dreg. The
soymilk was pour into a pot and added with pandan essence heated to 82℃ for
15 minutes. The heated soymilk was then cooled to room temperature which
below to 55℃. About 1.79g of probiotic capsules which Lactobacillus
Acidophiles strain was added into pasteurized soymilk with also by added
60gram of sugar.

Then it will divided into difference three formulations which the sample
fermented with the same fermentation time for 6 hours but in difference
fermentation temperature such 30℃, 42℃ and 47℃.

32
3.3.3 EQUIPMENT

3.3.3.1 Viscosity

Figure 3.3.3.1 : Brookfield Viscometer

The rotational viscometer principle is used in the Brookfield viscometer.

He states that the torque required to rotate an object such as a spindle in a
liquid indicates the viscosity of the liquid. The viscous drag of the test fluid on
the spindle is measured by applying a torque via a calibrated spring to the
fluid-immersed disk or pendulum spindle. The torque required to turn the
spindle and the resulting viscosity of the Newtonian fluid determine the
amount of viscous drag. For non-Newtonian fluids, compare Brookfield
viscosities obtained with the same test setup (model, spindle, speed,
temperature, test time, vessel and other sample preparation techniques that may
affect fluid behavior) can. When developing new testing techniques, trial and
error is often required to select the correct spindle and speed. An effective test
method yields torque values between 10 and 100. The rheological behavior of
test fluids can be monitored using the same spindle at different speeds, but
accurate rheometry is not possible with this setup. This is because the geometry
of the fluid around a rotating bob or disc spindle in a large vessel prevents a
single shear allocation rate.

33
 A clean, dry beaker was used to pour the required amount of SBY, which
was 50 ml. The viscometer used was calibrated according to the manufacturer's
instructions, using a standard calibration fluid with a known viscosity. The
viscosity of the SBY was then measured using a Brookfield Viscometer, and
spindle number four was determined to be the most suitable choice for the
expected viscosity. The spindle was carefully attached and inserted into the
sample, and a rotational speed of 21rpm was selected to ensure stability. The
viscosity of the sample was measured and recorded, with three measurements
taken for each starter culture and an average calculated.

34
 3.3.3.2 Color

Figure 3.3.3.2 : Spectrophotometer

Color is an important sensory characteristic of food products, and can

significantly influence consumer acceptance (Chen et. al, 2020). Therefore,
accurate measurement of color is crucial for food technologists to ensure that
products meet consumer preferences and expectations. In this essay, we will
outline the procedure for measuring the color of soy-based yogurt using a
spectrophotometer. As a first step in the procedure, the soy-based yogurt
samples are taken and brought to room temperature. They are then

35
mixed thoroughly using a magnetic stirrer, and 1 ml of the sample is
transferred into a glass cuvette using a pipette. Another glass cuvette is filled
with deionized water, which will serve as the blank. Next, the
spectrophotometer is turned on and allowed to warm up for 15 minutes. The
wavelength is adjusted to the desired setting, typically between 400-700 nm for
food products, and the spectrophotometer is set to measure the absorbance of
light. Calibration is then performed by placing the blank cuvette into the
spectrophotometer and setting the absorbance to zero. The blank cuvette is then
replaced with the sample cuvette, and the absorbance is recorded. This process
is repeated for each sample at the desired wavelengths, and the absorbance
readings for each sample are recorded in a data table. The absorbance readings
are then converted to color values using appropriate color measurement
software, and the color data is analyzed using statistical software to identify
any significant differences among the samples. Measuring the color of soy-
based yogurt using a spectrophotometer is a crucial step in product
development and quality control, as factors such as processing conditions,
storage temperature, and the type and amount of coloring agents used can all
influence the color of the final product.

36
 3.3.3.3 Protein Content

Figure 3.3.3.3 : Protein Analyzer 1

In order to ascertain the protein composition of diverse food products, protein

analysis is a critical step in the food industry. A popular method for protein analysis
is the Protein Analyzer 1(VELP UDK 159) method. To guarantee a uniform
distribution of the protein for this study, the food sample is first ground and
homogenised. The protein is then extracted using a suitable solvent, such as sulfuric
acid or sodium hydroxide.The Protein Analyzer 1(VELP UDK 159) instruments,
which are made to measure the amount of protein in the sample, are then used to
quantify the extracted protein.
To determine the protein content of SBY, a representative sample (e.g. 2.3grams)
is first weighed out using a digital balance. The sample is then transferred to a
KJELDAHL digestion flask and a small amount (e.g. 0.1-0.2g) of copper catalyst is
added. Next, a volume (e.g. 20 ml) of concentrated sulphuric acid is added to the
flask, taking care to avoid splashing. The flask is gently heated on a KJELDAHL
digestion apparatus until the sample is completely digested and the solution is clear.
After cooling the digestate, a volume (e.g. 100ml) of distilled water is added along
with a volume (e.g. 20 ml) of 40% sodium hydroxide solution. The contents of the
flask are then transferred to a KJELDAHL distillation apparatus and ammonia is
distilled and collected in a known volume of standardized hydrochloric acid. The
excess hydrochloric acid is titrated using a burette and phenolphthalein indicator.
Finally, the protein content of the sample is calculated using the formula
protein(%)=(Nx6.25)/weight of sample, where N is the amount of nitrogen
measured in the sample. This process enables an accurate determination of the
protein content of the soybean-based yogurt sample.

37
 3.3.4 Statistical Method

Statistical is method of analyzing or representing statistical data; a

procedure for calculating a statistic. There are many categories of statistical
method that can used for analyzing or representing statistical data which is,
descriptive methods, parametric inferential methods, nonparametric inferential
methods, and predictive methods. Analysis of variance, or ANOVA, is a
statistical method that has been used for pegaga ice cream analysis. ANOVA,
is a statistical method that separates observed variance data into different
component to use for additional test. A one-way ANOVA is used for three or
more groups of data, to gain information about the relationship between the
dependent and independent variables. ANAVO also determine if there is a
statistically different between two or more categorical groups by testing for
differences. Statistical Packages for the Social Sciences (SPSS) 20.0 software
was used to analyses all of the data obtained. All the data was gathered with
this (SPSS) 20.0 software was reported as mean and standard deviation.

38
 APPENDIXES

i. GANTT CHART ( PROCESS OF SOYBEAN-BASED YOGURT)

Table 1 : Gantt Chart

39
ii. COST ESTIMATION

INGREDIENTS QUANTITY COST/UNIT COST

USED
Soybean 500g RM2.45/500g RM2.45
Sugar 60g RM3.80/1kg RM0.23
Probiotic capsule 4 capsule RM22/10capsule RM8.80
(Lactobacillus Acidophiles)
Pandan Essence 0.50ml RM2.69/25ml RM0..05
Total RM16.48

Table 2 : Cost Estimation

40
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