production and evaluation of physicochemical and functional properties of modified protein from lima bean phaseolus lunatus using transglutaminase

MINISTRY OF EDUCATION AND TRAINING HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING Ho Chi Minh City, January 2024GRADUATION PROJECT FOOD TECHNO

MINISTRY OF EDUCATION AND TRAINING

HO CHI MINH CITY UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY FOR HIGH QUALITY TRAINING

Ho Chi Minh City, January 2024GRADUATION PROJECT

FOOD TECHNOLOGY

PRODUCTION AND EVALUATION OF

PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF MODIFIED PROTEIN FROM LIMA BEAN

(Phaseolus lunatus) USING TRANSGLUTAMINASE

S K L 0 1 2 5 3 3

LECTURER: PHAM THI HOA

NGUYEN HUU KHANG

STUDENT: HO NGOC TRAM NGUYEN TRAN GIA VUI

HO CHI MINH UNIVERSITY OF TECHNOLOGY AND EDUCATION FACULTY OF INTERNATIONAL EDUCATION

GRADUATION PROJECT Thesis code: 2023 – 19116032

Students: Ho Ngoc Tram, ID: 19116032

Nguyen Tran Gia Vui, ID: 19116038 Major: Food Technology

Supervisors: Pham Thi Hoan, Ph.D

Ho Chi Minh City, January 2024

PRODUCTION AND EVALUATION OF PHYSICOCHEMICAL AND FUNCTIONAL PROPERTIES OF MODIFIED PROTEIN FROM

LIMA BEAN (Phaseolus lunatus) USING

TRANSGLUTAMINASE

THE SOCIALIST REPUBLIC OF VIETNAM

Independence – Freedom– Happiness

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GRADUATION THESIS ASSIGNMENT

Student name: Ho Ngoc Tram Student ID: 19116032Student name: Nguyen Tran Gia Vui Student ID: 19116038Major: Food Technology Class: 19116 CLASupervisor 1: Pham Thi Hoan, Ph.D

Supervisor 2: Nguyen Huu Khang, B.E

Email: hoanpt@hcmute.edu.vn Email: khang.nhk213@gmail.comDate of assignment: 21/08/2023 Date of submission: 22/01/2024

1 Thesis title: Production and evaluation of physicochemical and functional properties of

modified protein from Lima bean (Phaseolus lunatus) using Transglutaminase

The content and requirements of the graduation thesis have been approved by the Chair of the Food Technology program

CHAIR OF THE PROGRAM

(Sign with full name)

Ho Chi Minh City, 22nd January, 2024

SUPERVISOR

(Sign with full name)

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ACKNOWLEDGEMENT

First of all, we would like to express our sincere gratitude to all the professors, and lecturers at the Ho Chi Minh City University of Technology and Education and with special thanks to those in the Department of Food Technology, Faculty of Chemical and Food Technology They enthusiastically helped us as we conducted research and studies Thanks to the care, assistance, and dedicated guidance from our teachers, they have equipped us with a solid foundation to confidently step forward on our academic and professional journey in the future

We especially, would like to extend our profound gratitude to the PhD Pham Thi Hoan is a lecturer in the Faculty of Chemical and Food Technology at Ho Chi Minh University of Technology and Education and B.E Nguyen Huu Khang carefully advised, instructed, and assisted us in completing the thesis We have received considerable support, assistance, and guidance from them, including the usage of equipment and machinery Throughout the project, they consistently offered guidance and encouragement when we ran into problems

In addition, we would like to express our gratitude to Ms Ho Thi Thu Trang, the specialist in charge of laboratories and practical workshops in the Food Technology Department, Faculty of Chemical and Food Technology Her significant support in terms of equipment, machinery, and guidance on their usage has been invaluable to us We would also like to express our appreciation to the teachers in charge of the workshops and laboratories for providing the necessary space and time to assist us in completing this thesis

Finally, we would like to extend our gratitude to our families, friends, and the entire class of our Food Technology major, Class of 2019, for the High-quality training program Throughout our academic journey and the preparation of this thesis, your continuous support, encouragement, and aid have been essential

It is possible that mistakes were created in the thesis because of our inexperience, lack of information, and time constraints Please pardon us and provide helpful criticism so we can get better We wish everyone good health and success in your life

Sincerely thank,

Ho Ngoc Tram and Nguyen Tran Gia Vui

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DECLARATION OF AUTHORSHIP

We hereby declare that all content presented in the graduation thesis is our work We took research ethics very seriously when we were implementing the study All the data and results presented in the graduation thesis are entirely truthful, and all reference materials are cited accurately and thoroughly following regulations

Ho Chi Minh City, 22nd January, 2024

STUDENTS

(Sign with full name)

Nguyen Tran Gia Vui Ho Ngoc Tram

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TABLE OF CONTENT

ACKNOWLEDGEMENT i

DECLARATION OF AUTHORSHIP ii

GRADUATION THESIS ASSESSMENT FORM iii

GRADUATION THESIS ASSESSMENT FORM Error! Bookmark not defined.GRADUATION THESIS ASSESSMENT FORM Error! Bookmark not defined.TABLE OF CONTENT xiv

LIST OF ABBREVIATIONS xvii

LIST OF FIGURES xviii

LIST OF TABLES xviii

1.4 Subjects and scope of research 3

1.5 Scientific and practical significance 4

1.5.1 Scientific significance 4

1.5.2 Practical significance 4

Chapter 2: LITERATURE REVIEW 5

2.1 Overview of lima beans and protein 5

2.1.1 Introduction of lima beans 5

2.1.2 Definition and classification of protein 8

2.1.3 Legume protein 9

2.1.4 Lima protein 11

2.2 Methods for recovery lima bean protein 12

2.3 Applications of protein in food technology based on each characteristic 14

2.4 Overview of protein modification 15

2.5 Overview of Transglutaminase 16

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2.6 Research situations of the topics 18

2.6.1 Domestic situation 18

2.6.2 Overseas situation 19

Chapter 3: MATERIALS AND RESEARCH METHODS 21

3.1 Materials, chemical, and equipment 21

3.2.2 Manufacturing of lima beans 25

3.2.3 Procedure of Lima bean protein modification using Transglutaminase 26

3.3 Experimental design 29

3.3.1 Experimental 1: Investigate the pH value of the protein modification process 29

3.3.2 Experimental 2: Influence of TGs concentrations on the efficiency of protein modification process 31

3.3.3 Experiment 3: Influence of incubation time on the efficiency of protein modification process 33

3.4 Analytical methods 35

3.4.1 Determine the recovery efficiency of modified protein 35

3.4.2 Methods for determining chemical compositions 35

3.4.3 Particle size distribution of modified protein 35

3.4.4 Molecular weight of modified protein 35

3.4.5 Secondary structure of modified protein 36

3.4.6 Nitrogen solubility in modified protein 37

3.4.7 Water absorption capacity 37

3.4.8 Oil absorption capacity 38

3.4.9 Emulsifying activity and stability 38

3.4.10 Foaming capacity and stability 39

3.4.11 Data analysis 39

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Chapter 4: RESULTS AND DISCUSSION 40

4.1 The choice of pH value used for the modification process 40

4.2 The choice of TGs concentration for protein modification 42

4.3 Appropriate incubation time for protein modification 44

4.4 The complete process for Lima bean protein modification using Transglutaminase 464.5 The recovery efficiency of the modified protein 48

4.6 Chemical composition of the modified protein 48

4.7 Secondary structure of modified protein 49

4.8 Nitrogen solubility in modified protein 50

4.9 Water absorption capacity and oil absorption capacity 52

4.10 Emulsifying activity and stability 54

4.11 Foam ability 55

Chapter 5: CONCLUSIONS AND RECOMMENDATIONS 58

REFERENCES 60

APPENDIX 69

Appendix 1: Certificate of analysis of transglutaminase enzyme product 69

Appendix 2: Methods for determining the chemical composition of materials and product70Appendix 3: SDS-PAGE Separating Gel and Stacking Gel Preparation Procedure 72

Appendix 4: Determination of soluble proteins by the Lowry method 73

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LIST OF ABBREVIATIONS

TGs: Transglutaminase

MPP: Modified Phaseolus lunatus protein with Transglutaminase

PP: Phaseolus lunatus protein

DLS: Dynamic Light Scattering

SDS – PAGE: Sodium Dodecyl Sulfate – Polyacrylamide Gel Electrophoresis

FTIR: Fourier Transform Infrared Spectroscope

WAC: Water absorption capacity

OAC: Oil absorption capacity

EAI: Emulsifying activity index

ESI: Emulsifying stability index

FA: Foaming ability

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LIST OF FIGURES

Figure 2.1 Lima bean 5Figure 2.2 Baby lima bean (small – seeded group) 6Figure 2.3 Carolina silva lima bean (large – seeded group) 6Figure 2.4 Christmas lima bean (speckled group) 6

Figure 3.1 Lima bean 21Figure 3.2 Research diagram for lima protein modification using Transglutaminase 24Figure 3.3 The process of Lima bean protein extraction procedure 25Figure 3.4 The process of lima bean protein modification using Transglutaminase 27Figure 3.5 Procedure for investigate the pH value 30Figure 3.6 Procedure for investigating the TGs concentration 32Figure 3.7 Procedure for investigating the time incubation 34

Figure 4.1 SDS – PAGE results of modified protein samples with different pH values 40Figure 4.2 The calibration curve between the logarithm of molecular mass and protein

segments travel distance 41

Figure 4.3 SDS – PAGE results of modified protein samples with different incubation time 44Figure 4.4 The calibration curve between the logarithm of molecular mass and protein

segments travel distance 45

Figure 4.5 The complete process for Lima bean protein modification using Transglutamina 47Figure 4.6 FTIR infrared spectra of samples PP and MPP7_3 49Figure 4.7 Nitrogen solubility between PP and MPP7_3 samples 51Figure 4.8 Influence of pH of MPP7_3 samples on foam ability 55Figure 4.9 Influence of pH of PP samples on foam ability 56

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LIST OF TABLES

Table 2.1 Chemical composition of lima beans 7Table 2.2 Amino acid content in lima bean protein 12Table 2.3 Reactions catalyzed by TGs: (a), acyltransfer reaction; (b), crosslinking reaction

between Gln and Lys residues of proteins or peptides; (c), deamidation 17

Table 3.1 The technical specifications of transglutaminase 22

Table 4.1 Results of DLS measurements and recovery efficiency with protein samples of

different concentrations 42

Table 4.2 The recovery efficiency of the protein after modification process 48Table 4.3 Chemical composition of PP and MPP7_3 samples 48Table 4.4 Water and oil absorption capacities of protein samples 52Table 4.5 Emulsifying activity and stability of PP and MPP7_3 54

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ABSTRACT

The purpose of this study was to a) determine the technological parameters of protein modification process using Transglutaminase (TGs) and b) evaluate some physicochemical properties as well as functional properties of modified protein At the beginning, we isolated

protein from lima beans (Phaseolus lunatus), then, we treated protein solution (5%, w/v) with

TGs at different pH (pH4 and pH7), different concentrations of enzyme (0 –10 U/g protein) and

different time (0 – 4 hours) The analytical results showed that, the modified Phaseolus lunatus

protein (MPP) sample was incubated with TGs at concentration of 5 U/g protein, pH7, temperature of 45C in 3 hours with a fixed shaking speed of 250 rpm/min (sample MPP7_3) had the higher recovery effiecency (68.99%) The research results showed that the MPP7_3

sample had a protein content of 68.54%, higher than Phaseolus lunatus protein sample (PP)

befor modification (61.19%) The functional properties of MPP7_3, such as oil absorption capacity (2.28 mL/g) and water absorption capacity (5.34 g/g), were improved compared to the PP sample with OAC (1.55 mL/g) and WAC (2.46 g/g) Additionally, it was observed that the protein solubility of MPP7_3 decreased compared to PP due to TGs treatment forming cross-links and increasing the molecular weight The emulsifying activity and emulsifying stability of MPP7_3 were 42.25 m2/g and 164.42 mins, respectively, more than twice as high as the PP sample with EAI (22.17 m2/g) and ESI (80.22 min) This investigation further demonstrated that TGs – catalyzed cross-linking can alter the technical functional characteristics of PP, permitting its application in food items including bread, dairy products, and processed meat

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Chapter 1: INTRODUCTION 1.1 Pose the problem

For human nutrition, proteins are vital Meat has historically been the most readily available and important source of protein but due to several factors, including animal diseases, a worldwide scarcity of animal protein, and the growing desire for religious and healthful food, the amount of meat consumed has been declining while the amount of vegetable proteins consumed is rising over time (Joshi & Kumar, 2015) Besides, the majority of health organizations advise regular consumption of vegetable protein because it is known to lower blood cholesterol levels, the risk of coronary heart disease, and diabetes, and because animal protein sources sometimes include high amounts of saturated fat and cholesterol (Martínez-Villaluenga et al, 2006) The prevalent sources of vegetable protein from legumes (such as lupini, peas, beans, soybeans, etc.) which play a significant role in human nutrition because of their high protein content (Klupšaitė et al, 2015) In addition, legumes also provide energy, dietary fiber, protein, minerals, and vitamins required for human health (Klupšaitė et al, 2015)

Soybean protein is one of the least expensive and popular vegetable proteins which has a wealth of healthy nutrients, including fats and necessary amino acids (Liu et al, 1997) Millions of people rely on soybeans as a source of oil and protein in their diets, and countless industrial items are made from them It is arguably the most valuable crop in the world Furthermore, it is the gold standard by which other vegetable food elements are measured because it is a remarkably abundant amount of fat and protein as well as a good source of energy, vitamins, and minerals It is, however, trypsin inhibitors are anti-nutrients present in soybean protein that lower its nutritious value by preventing the digestive enzyme trypsin from acting (Cabrera-Orozco et al, 2013) About 0.5% of people worldwide suffer from an allergy to soybean products, and soybeans are one of the eight main allergenic foods that cause 90% of food allergies (Pi et al, 2019) For this reason, there has to be another source of protein, and lima beans are a good one since, according to several studies, they are yet another naturally occurring plant-based protein source that includes the essential amino acids that are easily absorbed and digested (Thrane et al, 2017) It is also a viable protein source for the food industry because of its high protein content (26%) when compared to other species and its protein isolates (from isoelectric precipitation), which contain about 72% protein (Chel-Guerrero et al, 2011) This protein has numerous potential uses as a functional ingredient in food systems, such

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as bread goods, spices, and sausages, among others, because of its functional and nutritional qualities (Chel-Guerrero & al, 2008) However, since legume proteins exhibit lower solubility and functional properties than frequently used animal-based proteins, some modifications may be necessary before they can be used as an alternative (Karaca, 2021) To modify the structural, physicochemical, and functional characteristics of legume proteins, many approaches for adjustment are required such as physical, chemical, or biological methods can be applied It has been shown that every modification technique has pros and cons specific to how well it works and how well it applies to certain food matrices Enzymatic cross-linking with transglutaminase is the biological approach employed among modification approaches to enhance protein functionality

Transglutaminase (TGs) (EC:2.3.2.13) is an enzyme that catalyzes the acyl-transfer reaction between the γ-carboxamide group of glutamine residues and various primary amines, such as the "-amino group of lysine residues; this reaction forms a "-(γ-glutamyl) lysine isopeptide bond, changing the protein's molecular weight, molecular structure, and surface hydrophobicity (Motoki & Seguro, 1998) These modifications are frequently used in a variety of food industries, including dairy and bread products as well as plant-based meat because they can improve the techno-functional qualities of proteins (Motoki & Seguro, 1998) According to Babiker (2000), this enzyme can effectively improve the functional characteristics of milk and soy proteins such as hydration, gelation, surface association (emulsification, foaming), and heat stability Furthermore, it can be applied to a variety of processes, including film and encapsulation, in addition to the food sectors (Moon & Cho, 2023) Consequently, TGs may be applied to improve Lima bean protein's subpar techno-functional qualities

The aforementioned arguments lead us to focus our thesis on the use of the TGs to modify proteins belonging to the legume family In this instance, we use protein that comes

from Lima beans (Phaseolus lunatus) Except for cysteine and methionine, lima bean protein

is regarded as a promising protein since it is complete in terms of necessary amino acids (both in quantity and composition) (Betancur‐Ancona et al., 2009) Furthermore, low in fat and high in minerals and fiber, lima beans are a good fit for diets aimed at managing weight However, the techno-functional characteristics of microbial transglutaminase-treated Lima bean protein have not been thoroughly investigated In Vietnam research on the modification of Lima bean proteins is relatively limited, mostly concentrating on plant proteins produced from soybeans Meanwhile, studies conducted abroad have addressed the process of modifying protein using

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the TGs, evaluating functional properties as well as the biological activity of modified proteins from other beans, such as mung bean, faba beans, and kidney beans (Moon & Cho, 2023) (Liu et al, 2019) (Tang et al, 2008), but there is no information available regarding Lima beans In general, research and reports on modified Lima bean protein, both in general and specifically with TGs, are still very limited in both Vietnam and abroad From these perspectives, the research topic “Production and evaluation of physicochemical and functional properties of

modified protein from Lima bean (Phaseolus lunatus) using Transglutaminase” is considered

necessary to be implemented

1.2 Research objective

The research project “Production and evaluation of physicochemical and functional

properties of modified protein from Lima bean (Phaseolus lunatus) using Transglutaminase” is

conducted with the following objectives:

 Applying appropriate techniques to recover modified protein from Lima beans using TGs

 Analyzing the chemical composition and some properties of the modified protein

1.3 Research content

This research topic includes the following specific contents:

- Synthesizing literature and determining the process, and suitable parameters to modified protein from lima beans using TGs

- Investigating pH value, enzyme concentration and incubation time to protein modification capacity

- Evaluating the physicochemical properties (chemical composition, molecular weight, particle size of protein) and functional properties (solubility, water and oil holding capacity, emulsifying ability and stability, foaming ability and stability) of modified Lima bean protein using Transglutaminase

1.4 Subjects and scope of research

1.5.2 Practical significance

The project's results help to diversify the sources of modified plant-derived protein The process for obtaining modified protein from lima beans can be applied on a pilot and an industrial scale

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Chapter 2: LITERATURE REVIEW 2.1 Overview of lima beans and protein

2.1.1 Introduction of lima beans

Phaseolus lunatus, commonly called Lima bean is a member of the family Leguminosae

(or Fabales), genus Phaseolus, tribe Phaseoleae, and subgenus Phaseolinae (Baudoin, 1993)

Lima beans have their origins in Guatemala, Mexico, and Peru After domestication, they proliferated across the Americas, with the Spaniards introducing them to the Pacific Islands and the Philippines Subsequently, the lima bean found its way to Southeast Asia, and through the slave trade, it reached Western and Central Africa Today, lima beans have become extensively naturalized in tropical regions in the world In Vietnam, Lima beans have been introduced and cultivated in various provinces and cities including Lam Dong province, where the average yield reaches 2100 kg/ha (Lam Dong province, 2016) This type of bean is known for its high nutritional content and thrives well in the climatic conditions of Vietnam However, currently, it is only sporadically featured in dishes such as Vietnamese sweet soup, and stewed, and globally there are not many processed products made from lima beans, with most available in canned or frozen forms that do not fully reflect its developmental potential Therefore, researching to uncover and fully exploit its potential is a value attempt, as lima beans have yet to be fully utilized in line with their promising development prospects

Figure 2.1 Lima bean

Classification of lima beans

Currently, lima beans varieties exhibit distinct differences in the number of days to harvest, size, and color of the pods As a result, lima bean varieties are broadly categorized into three main groups: small-seeded (Baby lima bean), large-seeded (Large-seed lima bean), and

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speckled (Speckled lima bean) The small-seeded group includes varieties such as Eastland and Parkers, … About the large-seeded group comprises common varieties like Carolina Silva, Jersey, Bliss ; and the speckled group includes varieties such as Christmas, Jackson Wonders (Bailey, 1896)

Figure 2.2 Baby lima bean (small – seeded group)

Figure 2.3 Carolina silva lima bean (large – seeded group)

Figure 2.4 Christmas lima bean (speckled group)

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Chemical composition of lima bean

The composition of lima beans is shown in Table 2.1 In general, lima beans are a rich source of food with high protein content (21–26 g/100 g) and low fat (Oshodi & Adeladun, 1993) Lima bean protein contains more phenylalanine and tyrosine protein (10.67 g/100 g) (Betancur‐Ancona et al, 2004) making lima beans a viable source of amino acids for food applications

Table 2.1 Chemical composition of lima beans Chemical composition % Weight

Moisture 10 – 14 Protein 21 – 26 Carbohydrate 55.1 – 60.7

Crude fat 3.0 – 4.9 Crude fiber 5 – 8

Ash 3.4 – 6.4

Sources: (Chel-Guerrero & et al, 2002), (Giami, 2001) and (Oshodi & Adeladun, 1993)

Lima beans have a carbohydrate content ranging from 55.1% to 60.7% (Oshodi & Adeladun, 1993) In particular, the relatively high fiber content in lima beans helps maintain the intestinal microbiota to protect human health (Yellavila et al, 2015) Lima beans have a crystalline form β (less digestible than the α form found in cereals), which helps to feel full for a longer time, making them suitable for diet or starch-restricted diets (Adeparusi, 2001)

Lima beans contain very little fat, ranging from 3.0 to 4.9% by weight, making them suitable for individuals with cardiovascular issues, diabetes, and trying to lose weight (Oshodi & Adeladun, 1993)

The fiber content of lima beans is approximately 5 – 8% (Chel et al., 2002) Fiber present in lima beans also helps reduce cholesterol levels in the blood, prevent cancer, lower the risk of developing diabetes, increase blood pressure, and prevent arterial stiffness (Yellavila et al., 2015)

Protein molecules play a crucial role in life, serving as essential components in various biological activities within the cell, along with functional Ribonucleic Acid They are instrumental in defining enzymatic chemistries, facilitating transport processes in metabolic pathways, regulating gene expression, participating in signal transduction, and constituting the molecular and cellular machinery essential for life Proteins exhibit a high degree of diversity and are characterized by multiple layers of molecular organization within a hierarchical framework Their evolution is intricate and governed by factors related to molecular structure, thermodynamics, and function (Caetano-Anollés, 2009)

2.1.2.2 Classification

Classification by origin

Food proteins are valuable components in terms of their safety, nutritional richness, easy digestibility, agricultural sustainability, and cost–effectiveness They can be broadly categorized into three main types based on their sources (Cao, 2019):

Animal Proteins

- Milk Proteins: Found in dairy products

- Egg Proteins: Present in eggs

- Blood Proteins: Extracted from blood

- Meat Proteins: Obtained from various meats

- Insect Proteins: Derived from insects

Plant Proteins

- Cereal Proteins: Found in wheat, corn, barley, oats, and rice

- Legumes and Pulses Proteins: Present in peas, soybeans, lupins, lentils

- Tubers Proteins: Derived from potatoes

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- Oil Seeds Proteins: Found in rapeseed, cottonseed, peanut, sunflower, hemp seed

- Pseudocereal Proteins: Obtained from amaranth, chia

- Edible Seeds Proteins: Present in quinoa, buckwheat

- Algae Proteins

Fungi Proteins

- Mycoproteins: Derived from fungi

These diverse sources of food proteins offer a wide range of options for meeting nutritional needs, and their classification helps in understanding their origins and potential applications in the food industry

Classification based on protein extraction method and protein concentration

Protein Concentrate: Typically produced through methods such as solvent extraction, filtration, or concentration from natural protein sources and has a lower protein concentration compared to protein isolate Typically contains around 60 – 80% protein (Wright, 2009), with the remainder being fats, carbohydrates, and other minerals

Protein Isolate: Requires more complex processing methods to eliminate unwanted components, often involving additional extraction, filtration, and refinement processes Protein isolate has a higher protein concentration, usually above 90% (Wright, 2009), with fats, carbohydrates, and other components either removed or significantly reduced

2.1.3 Legume protein

Nowadays, plant-based proteins have played a crucial role as a valuable nutritional source, and some proteins are industrially processed to serve various fields Plant-based proteins can be obtained from diverse sources, each with its distinctive protein composition The most common protein extracted from leguminous plants is soy protein, which contains a high content of globulin (D Fukushima, 2011)

From an economic perspective, plants of the Fabaceae (Leguminosae) family, including

leguminous plants, are among the most important plant families They serve as an abundant

source of protein for both humans and animals Among various tribes including Viciae,

Phaseoleae, Lupinae, Glycineae, Diocleae, and Trifoliae, the majority of protein – rich seeds

About protein segments, globulins are major storage proteins in legume seeds, accounting for around 70% of total protein content The remaining 30% is split evenly between albumins (20%) and prolamins (10%) Legumin and vicilin are the principal reserve globulins in most legumes, and the globulins are deficient in sulfur-containing amino acids (methionine and cysteine) In comparison, albumin includes more sulfur-containing amino acids and lysine (Hahn & al, 1982)

2.1.3.1 Globulin

According to the sedimentation coefficient, the principal storage globulins in beans are 7S vicilin – type globulins and 11S legumin-type globulins These globulins dissolve in 0.5–1.0 M NaCl solution (Casey, 1999) 11S globulins are also known as legumins since they are proteins found only in legumes Legumins are generally hexameric proteins with molecular masses ranging from 300.000 to 450.000 Da, composed of six subunits with molecular masses ranging from 50.000 to 60.000 Da and connected together by non – covalent connections (Mills, Jenkins, & Bannon, 2003) These subunits are made up of single polypeptide chains that are broken at the N – G peptide link by an asparaginyl endopeptidase post-translationally Following cleavage, a large polypeptide chain (called acidic or α) and a tiny polypeptide chain (called basic or β) with approximate molecular weights of 40.000 Da and 20.000 Da are formed They are joined by an intramolecular disulphide bond (Mills et al., 2003)

In contrast, 7S globulins in legumes are commonly referred to vicilins since they are found in the seeds of the legume tribe Viciae They are trimeric proteins with molecular weights ranging from 150.000 to 200.000 Da and three subunits with masses ranging from 40.000 to 80.000 Da (Mills et al., 2003)

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2.1.3.2 Albumin

Albumins are water – soluble globular storage proteins with a low molecular weight, with 2S albumin being prevalent in legume seeds 2S albumin has a high nitrogen concentration and a molecular mass ranging from 10.000 to 18.000 Da (Moreno & Clemente, 2008) Furthermore, considerable levels of sulfur-containing amino acids, especially cysteine, have been discovered in the amino acid composition of the 2S albumin protein group (Youle & Huang, 1981) 2S albumin is made up of two polypeptide chains that are generated as precursor proteins and then broken by proteolysis to yield small subunits (Mr 4.000 – 5.000 Da) and tiny subunits Disulphide bonds connect big molecules (Mr 9.000 –10.000 Da) (Monsalve, Villalba, Rico, Shewry, & Rodríguez, 2003)

2.1.3.3 Prolamin

Prolamin glutenin is a protein found in small amounts in legume seeds It is characterized by its high content of proline and amide nitrogen, as well as its solubility in alcohol/water mixtures (60 – 70% v/v ethanol) Glutenin has a mass ranging from 95.000 to 145.000 Da and consists of small low molecular weight subunits and large high molecular weight subunits connected by disulfide bonds (Fukushima, 1991)

2.1.4 Lima protein

Lima bean protein content ranged from 21.8% to 26.2% (Oshodi & Adeladun, 1993) Furthermore, lima bean protein, like soy protein, was a complete protein source, including all essential amino acids for the human body in both quality and quantity (Polanco-Lugo et al., 2014)

Most proteins in lima beans were storage proteins, predominantly globulins (35–72%) These included legumin (11S), a four-helix bundle protein with 4 subunits and a molecular weight of about 43 – 75 kDa, and vicilin (7S), a four-helix bundle protein with 3 subunits and a molecular weight of 22 – 39 kDa Currently, most functional properties of the entire globulin 7S and 11S fractions of lima beans have been identified, with the 11S component accounting for 58.3% and the 7S component accounting for 41.7% of the total globulin content (Kadam & Salunkhe, 1989)

Globulin and albumin molecules were the primary storage proteins in lima bean seeds Globulins had the largest content, accounting for 70 – 80% of the mass (with 11S globulin

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accounting for 58.3% and 7S globulin accounting for 41.7% of the total globulin content) Guerrero et al., 2011) The remaining portion included 2S albumins, and depending on the variety and cultivation conditions, the content may vary

(Chel-The amino acid compositions of the protein are presented in Table 2.2 based on Ancona et al., 2004

Betancur-Table 2.2 Amino acid content in lima bean protein Amino acids

Methionine + Cysteine 2.05 Glycine 4.8 Threonine 4.87 Arginine 7.01

Leucine 8.54 Alanine 5.01 Isoleucine 4.29

Valine 5.12 Histidine 3.2

2.2 Methods for recovery lima bean protein

There are various methods for protein separating, but they generally rely on systematically altering factors such as temperature, ionic strength, pH, to precipitate the protein

Protein precipitation using organic solvents

Methods of precipitating with organic solvents were among the most common and present in many production processes because they helped produce proteins with a bland taste and higher nutritional value than other methods (Peng et al., 2021) This process relied on the non-reversible modification of proteins by the solvent

Many water – miscible organic solvents, such as methanol, ethanol, and n-butanol, have been studied for protein extraction The solubility layer around the protein decreased as the organic solvent gradually removed water from the protein surface and bound it in hydration layers around organic solvent molecules With smaller hydration layers, proteins could aggregate through electrostatic attractive forces (Novák & Havlíček, 2016) There were

Protein precipitation at the isoelectric point

The isoelectric point (pI) is the pH at which the net charge of a protein is zero At pH levels higher than the pI, the protein surface is predominantly negatively charged, leading to electrostatic repulsion between negatively charged molecules Similarly, at pH levels lower than the pI, the protein surface is predominantly positively charged, resulting in repulsion between positively charged proteins However, at the pI, the negative and positive charges balance each other, reducing electrostatic repulsion and allowing attractive forces to dominate, leading to aggregation and precipitation The pI values for most proteins typically fall within the pH range of 4 to 7 (Novák & Havlíček, 2016) We applied this method to the protein extract of lima beans to induce precipitation at its isoelectric point at pH = 4.5

Proteins precipitation with neutral salts

The salting – out is a spontaneous process when reaching the appropriate concentration of salt in the solution Hydrophobic patches on the protein surface create highly ordered water shell layers The addition of neutral salts compressed the solvation layer around proteins and increased protein-protein interactions As the salt concentration in the solution increases, most water molecules bind more with salt ions As a result, fewer water molecules were available for the solvation layer around protein molecules, exposing hydrophobic patches on the protein surface The protein could then exhibit hydrophobic interactions, coalesce, and precipitate from the solution (Novák & Havlíček, 2016) The essence of this process is to induce protein precipitation by adding neutral salt solution to the protein solution Various salts are used to precipitate proteins, but commonly used ones include sulfate, citrate, phosphate, and chloride (Shih, Prausnitz, & Blanch, 1992) In this study, we used sodium chloride 0.1N

Water absorption capacity

Water Absorption Capacity is the amount of water (moisture) absorbed by dry protein powder, after subtracting the evaporated water and the relative humidity of the powder This is an important functional characteristic in many food products, especially those derived from milk, undergoing heat treatment or extrusion such as soups, sausages, In these foods, proteins with high water absorption capacity will absorb water but may not completely dissolve Therefore, they will swell and create properties such as thickness, viscosity, in the food

Oil absorption capacity

Oil absorption capability of protein is defined as the amount of oil that can be absorbed per gram of protein (Lin & Zayas, 1987) The oil absorption capacity of protein is influenced by particle size, protein content, as well as the ratio of non-polar side chains of amino acids on the surface of the protein molecule (Chavan, McKenzie, & Shahidi, 2001) Proteins with high oil absorption capacity are used as functional ingredients in food products such as meat, sausages, coatings on ice cream surfaces, sponge cake, chiffon,

Emulsifying capacity

The emulsifying capacity of food is related to the amount of oil and nonpolar amino acids on the surface of proteins, water, and other components in the food As the amount of nonpolar amino acids on the protein surface increases, the energy barrier for adsorption, which depends

2.4 Overview of protein modification

There are various methods for protein modification, and each method yields different results depending on the specific goals of the modification process Below are some common methods and the main outcomes they can achieve (Karaca, 2021)

Heat treatment

Objective: Increase the heat resistance of the protein

Result: Improved heat stability, reduced protein modification, enhanced heat tolerance

Acid or alkali modification

Objective: Alter the isoelectric point, enhance solubility

Result: Proteins become soluble in acidic or alkaline environments, suitable for various food applications

Enzymatic modification

Objective: Modify protein structure, enhance functional properties

Result: Increased binding ability, gel formation, or alteration of chemical properties

Ultraviolet radiation modification

Objective: Stimulate disulfide bond formation, improve gel-forming properties Result: Increased gel-forming ability, improved elasticity

Mechanical treatment

Objective: Disrupt protein structure, create products with specific properties

Result: Products with fine structures, gel-like properties, or foaming characteristics can be achieved

Irradiation modification

Objective: Reduce or eliminate microorganisms and increase protein stability

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Result: Extended shelf life, reduced risk of bacterial contamination

Surfactant treatment

Objective: Improve solubility, foaming, and viscosity

Result: Enhanced viscosity, foaming ability, and water solubility

Transglutaminase enzyme treatment

Objective: Create bonds between polypeptide chains Result: Improved viscosity, elasticity, and water retention

While chemical modifications have been widely explored to enhance the functional properties of food proteins (Feeney & Whitaker, 1982) (Kinsella & Shetty, 1979), concerns regarding safety and nutritional effects have hindered their widespread application Therefore, the systematic investigation of enzyme usage is warranted as it provides specificity and control that are often lacking in most chemical methods (Tanimoto & Kinsella, 1988)

Transglutaminase has potential applications in modulating the functional properties of food proteins However, current research on TGs is still limited (Tanimoto & Kinsella, 1988) Therefore, in this study, we focus on the evaluation of physicochemical and functional properties of modified protein from lima bean using TGs

2.5 Overview of Transglutaminase

Transglutaminase (protein-glutamine-glutamyltransferase, EC 2.3.2.13) facilitates an transfer reaction between the carboxyamide group of peptide-bound glutamine residues (acyl donors) and various primary amines (acyl acceptors), including the amino group of lysine residues in specific proteins In the absence of amine substrates, TGs triggers the deamidation of glutamine residues, utilizing water molecules as acyl acceptors TGs has the capability to modify proteins through amine incorporation, crosslinking, and deamidation (Figure 2.3) (Motoki & Seguro, 1998)