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

INTRODUCTION

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

2 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

3 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.

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.

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.

Subjects and scope of research

Lima bean (Supplied by Hai Duong Xanh Agricultural Food Joint Stock Company, Ham Ech – Thong Cong, Cong Hoa Ward, Chi Linh City, Hai Duong Province, Vietnam)

Modified protein from lima bean using Transglutaminase

The research was conducted at the laboratory of the Food Technology Department of Chemical and Food Technology, Ho Chi Minh City University of Technology and Education

Scientific and practical significance

The research results of the project contribute to providing a process for obtaining modified lima bean protein

It offers information on the physicochemical properties and functional characteristics of lima bean protein modified by the TGs This serves as a reference for students, postgraduate learners, and individuals interested in the field of food technology

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

LITERATURE REVIEW

Overview of lima beans and protein

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

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

6 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)

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

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

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)

2.1.2 Definition and classification of protein

Proteins are complex linear hetero polymers composed of more than 20 amino acid monomers They exhibit a distinctive primary sequence with side chains arranged at regular intervals (Caetano-Anollés, 2009)

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)

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):

- 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

- 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

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

- Pseudocereal Proteins: Obtained from amaranth, chia

- Edible Seeds Proteins: Present in quinoa, buckwheat

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

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

10 in the world can be found Examples include faba bean or peas (Viciaes), mung beans or lima beans (Phaseoleae), lupines (Lupinae), soybeans, and peanuts (Glycinae) (Gueguen, 1983)

As a result, several varieties of beans are now processed into components such as protein concentrate and protein isolate for use in commercial food production Soybeans and peas are the most prevalent, followed by mung beans Proteins from other bean seeds, such as lima beans, are frequently researched at the laboratory scale rather than the commercial size (Phillips

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)

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

13 examples of using ethanol precipitation in protein experiments, such as a study demonstrating ethanol precipitation in human plasma (Fu, Garnham, et al., 2005) While methanol as a solvent produced a protein product less soluble than protein precipitated from ethanol, the use of n-butanol created a protein containing more fat and lactose, making it nearly insoluble in water

Therefore, in this study, we chose to use ethanol (70°) as it was a safer solvent than the others and had the ability to produce more readily soluble proteins, making it suitable for use in food technology

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

Applications of protein in food technology based on each characteristic

The solubility is one of the commonly measured properties of proteins, which results from the hydrophilic groups on the protein's surface interacting with water The pH is an important factor in determining solubility, as solubility decreases around the isoelectric point of the protein Protein with good solubility can easily interact with various types of food over a wide pH range This is an important functional property that determines the protein's application in food technology, especially in the production of protein-enriched beverages (dietary supplements), yogurt, and salad dressings

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 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,

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

15 on the protein structure, decreases The emulsifying capacity of proteins plays an important role in products such as mayonnaise, yogurt, butter, margarine,

The foaming capacity is the ability to create a stable foam system with air using the protein This function is crucial for various food products such as sponge cakes, meringues, pastries, ice cream, nougat, These properties are measured through the expansion of the foam, the volume of the foam, or any attributes related to the increase in volume of a protein in a foam system by incorporating air through whipping, stirring, or aeration.

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)

Objective: Increase the heat resistance of the protein

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

Objective: Alter the isoelectric point, enhance solubility

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

Objective: Modify protein structure, enhance functional properties

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

Objective: Stimulate disulfide bond formation, improve gel-forming properties

Result: Increased gel-forming ability, improved elasticity

Objective: Disrupt protein structure, create products with specific properties

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

Objective: Reduce or eliminate microorganisms and increase protein stability

Result: Extended shelf life, reduced risk of bacterial contamination

Objective: Improve solubility, foaming, and viscosity

Result: Enhanced viscosity, foaming ability, and water solubility

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.

Overview of Transglutaminase

Transglutaminase (protein-glutamine-glutamyltransferase, EC 2.3.2.13) facilitates an acyl- 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)

Table 2.3 Reactions catalyzed by TGs: (a), acyltransfer reaction; (b), crosslinking reaction between Gln and Lys residues of proteins or peptides; (c), deamidation

There are three general approaches to creating industrially viable TGs The first method includes extracting and purifying the enzyme from the tissues or bodily fluids of food- producing animals such as cattle, pigs, and fish such as cod, salmon, and flounder The second method is to obtain the enzyme by genetic modification of host microbes such E coli, Bacillus, yeast, and Aspergillus The third technique is to look for bacteria that produce TGs by classic fermentation techniques may be used to mass manufacture TGs (Motoki & Seguro, 1998) The microbe was later classified as a variety of Streptoverticillium mobaraense (Washizu et al., 1994) Low-cost production of microbial transglutaminase enables the catalysis of crosslinking in various food proteins through the creation of an ɛ-(γ-glutamyl) lysine bond Numerous studies indicate that microbial transglutaminase holds significant potential for applications in food processing (Motoki & Seguro, 1998)

Application of TGs in food processing (Yokoyama, Nio, & Kikuchi, 2004): Many food proteins gel after being incubated with TGs

 Meat products: TGs can create restructured meat by binding small meat pieces together Meat pieces, including minced meat, can be bound together without the need for sodium chloride or phosphates, resulting in 'healthy' meat products

 Dairy products: Milk casein, which does not gel even when heated, serves as an excellent substrate for TGs These enzymes convert it into a heat-resistant, firm gel Yogurt, a milk gel formed by acidic fermentation with lactic starter, faces challenges such as serum separation upon changes in temperature or physical impact The addition

18 of TGs can address this issue by improving the water-holding capacity of the gel TGs also enables the production of dairy products, including ice cream and cheese, with low fat content or reduced levels of non-fat solids

 Soybean products: The inclusion of TGs allows the prolonged maintenance of the smooth texture in retorted tofu

 Wheat products: Sakamoto et al (1996) discovered that the application of TGs to noodles and pasta prevented texture degradation during cooking, enhancing the strength of the products, especially when using lower-grade flours They also proposed that the addition of TGs could potentially increase or maintain the loaf volume of various breads, especially when certain ingredients were substituted or reduced during dough mixing (Sakamoto, 1996).

Research situations of the topics

During the synthesis of various sources of literature, we have not yet found any research results or documents related to the topic “Production and evaluation of physicochemical and functional properties of modified protein from lima bean (Phaseolus lunatus) using Transglutaminase” However, there are also studies related to proteins from other legume plant varieties

Nguyen Thi Hien (2017) studied the influence of technological factors in the ultrasonic and/or enzyme treatment process on the efficiency of protein extraction from defatted peanut flour, on the composition and properties of peanut protein The study also investigated the purification process of peanut protein extract using ultrafiltration techniques The research results showed a significant increase in protein recovery efficiency (up to 94.7%) when combining ultrasound and enzyme treatment The peanut protein product studied exhibited better solubility and foam stability than soybean protein products but showed lower fat absorption and foam formation abilities (Hiền, 2017)

Bui Thanh Binh et al (2011) conducted a study on establishing the production process of protein isolate from soybeans In this research, the authors investigated factors such as pH, solvents, temperature, time, and washing conditions in the protein extraction process The results indicated an efficiency of approximately 72% for the production process of protein isolate compared to the protein content in the raw material (Bùi Thanh Bình, 2011)

Regarding the overseas situation, we have not found any literature or research specifically addressing the modification of lima bean protein However, there are studies on the modification of other bean proteins, including research on improving the technological functional properties of isolated mung bean proteins treated with TGs by Moon et al (2023) The study demonstrated enhanced water holding capacity, gelling properties, emulsification capacity, and stability after TGs treatment Additionally, a decrease in protein solubility and surface hydrophobicity was observed TGs treatment increased the hardness, elasticity, toughness, and adhesiveness of the heat-induced gels (Moon & Cho, 2023)

The potential use of isolated faba bean protein treated with TGs as an emulsifying agent to maintain the physical stability and oxidation resistance of oil-in-water emulsions has been investigated by Liu et al (2019) The TGs treatment method creates cross-links in isolated faba bean protein, increasing the surface charge of the protein by 5% – 8% and enlarging the emulsion droplet size by 19% – 135% Treating with TGs for 60 minutes turns isolated faba bean protein into a potential emulsifier to maintain physical stability while improving the oxidative stability of lipids in the emulsion, possibly due to a thicker interfacial layer, larger droplet size, and protective action of the protein (Liu, Damodaran, & Heinonen, 2019)

Ali et al (2010) also studied the effect of cross-linking between isolated proteins from three types of beans with TGs enzyme at different NaCl concentrations TGs treatment significantly improved the solubility of the polymerized proteins of each bean type at high NaCl concentrations The cross-linked proteins were less turbid when heated at a higher temperature compared to natural proteins, and the temperature at which the proteins turned turbid also increased in the polymerized proteins The emulsification and foaming properties of the polymerized proteins were significantly enhanced at all NaCl concentrations for all bean types (Ali, Ahmed, Mohamed, Ahmed, & Babiker, 2010)

Tang et al (2006) investigated the impact of TGs covalent cross-linking on the thermal properties of soybean protein isolates, including heat modification and glass transition, using both modulated differential scanning calorimetry and traditional methods TGs treatment also improved the resistance of soy protein isolate to modification caused by urea The enhanced hydration ability of the protein and the formation of high-molecular-weight biopolymer

20 structures may account for the changes in the thermal properties of soybean protein induced by TGs cross – linking (Tang, Chen, Li, & Yang, 2006)

Therefore, the overall results of the research situation both domestically and overseas indicate that the study of recovering modified protein from lima beans has not been extensively explored Therefore, we have undertaken the research project "Production and evaluation of physicochemical and functional properties of modified protein from lima bean (Phaseolus lunatus) using Transglutaminase” with the aim of studying the potential application of this type of protein in the field of food technology

MATERIALS AND RESEARCH METHODS

Materials, chemical, and equipment

Lima bean (figure 3.1) supplied by Hai Duong Xanh Agricultural Food Joint Stock Company, Ham Ech – Thong Cong, Cong Hoa Ward, Chi Linh City, Hai Duong Province, Vietnam, and purchased at Bach Hoa Xanh store

Transglutaminase was provided from My Uc Science and Technology Development Joint Stock Company, address 115A Trinh Dinh Trung, Phu Trung Ward, Tan Phu District, Ho Chi Minh City

The technical specifications of the Transglutaminase are shown in the Table 3.1

Table 3.1 The technical specifications of transglutaminase

Description Conformed White or slight yellow powder

As (mg/kg) Not detected ≤ 3.0

Salmonella Not detected Negative in 25g

Antibacterial activity Not detected Negative

Source: Adapted from the product certificate of analysis (CoA) (My Uc company)

Sodium hydroxide (NaOH), Sodium chloride (NaCl), Ethanol (C₂H5OH), Sodium carbonate (Na2CO3), Copper sulfate pentahydrate (CuSO4.5H2O), Sodium kali tartrate (NaC4H4O6), Sodium Dodecyl Sulphate (SDS), Sulphuric acid concentrated (H₂SO4), Hydrochloric acid 0.1N standard solution (HCl), Boric acid (H3BO3), Potassium sulfate (K2SO4), Copper(II) sulfate (CuSO4), Phenolphthalein (C20H14O4), purchased at Bach Khoa Chemical Equipment Joint Stock Company, address 270 Ly Thuong Kiet Street, Ward 14, District 10, Ho Chi Minh City

- 2 – digit and 4 – digit analytical balance (Sartorius, Germany)

- Automatic Kjeldahl Distillers (Buchi B-324, Germany) and Soxhlet (Germany)

- Incubator shaker (Jeiotech IST-4075R, Korea)

- Particle size analyzer (Zetasizer, UK)

- Fourier Transform Infrarer with dual beams (Jato, Japan)

- Constant temperature oven (DKM 600 Yamato, Japan)

- Necessary tools such as beaker, Erlenmeyer flasks, volumetric flask, petri dish, pipette, pipette micro, pipette tip, test tube, centrifuge tube, glass cuvette, drying tray, glass rod, thermometer, sieve (ỉ 100 mesh), desiccator, crucibles, tongs, cuvettes, filter paper, mortar and pestle, …

Research methods

Research diagram for protein modification with the Transglutaminase is shown below:

Figure 3.2 Research diagram for lima protein modification using Transglutaminase

Determine the water and oil – holding capacity ean powder Isolation of lima bean protein Isolation of lima bean protein

Modification of lima bean protein using Transglutaminase

Investigate pH value (4 and 7), enzyme concentration (0 –10 U/g, step of 2.5U/g), duration time (0–4 hours, step of 1 hour) in protein modification process

Determine the particle size distribution, molecular weight of the protein and recovery efficiency

Determine the secondary structure of the protein Determine the nitrogen solubility of protein Determine the solubility of protein

Determine the water and oil absorption capacity Determine the emulsifying ability and stability Determine the foaming ability and stability Evaluation of the properties of modified proteins

The protein recovery process from Lima beans in this study was based on the method researched in author Nguyen Thi Bac's graduation thesis in 2021, and was presented in Figure 3.3 below:

Figure 3.3 The process of Lima bean protein extraction procedure

Explain the isolation process of Lima bean protein

First of all, after soaking the lima beans for about 6 hours in water (1:2 ratio of lima bean to water), the shells were removed and ensure the uniform quality of the beans, facilitating the milling process to become more efficient Then, the lima beans were convectively dried at a temperature of 55°C until they reached a dry state (moisture content < 10%) within approximately 12 hours After drying, lima beans were ground into a fine powder (ỉ = 100 mesh) in a milling machine for five minutes This step helped reduce the size of lima beans and enhanced extraction efficiency, creating favorable conditions for the preservation process Next, lima bean powder was de – fatted by soaking in 96% ethanol (ratio 1:6) for 24 hours Subsequently, the fatty portion and the solvent on the surface were removed The separated powder was then conveniently dried at a temperature of 55°C to obtain the de – fatted powder Mixed the de – fatted powder with 0.15N NaCl in a ratio of 1:15 (w/v), and adjust the pH of the solution to a pH = 11 using a 1N NaOH solution This step helped in extracting soluble proteins while eliminating insoluble components, such as carbohydrates and fiber After stirring for 1 hour in a magnetic stirrer, perform centrifugation twice at a speed of 3000 rpm for 20 minutes to obtain the protein extract Adjust the pH of the suspension solution to the isoelectric point of the solution (pH = 4.5) using 1N HCl and let the solution settle for 2 hours at a temperature of 4℃ Subsequently, centrifuge at 3000 rpm for 20 minutes to obtain the protein precipitate Finally, perform convective drying of the protein precipitate at 50℃ until dry state (moisture content 118 kDa were synthesized at the top of the separating and stacking gels, which appeared to be polymers from cross-linking between monomers and intermediate subunits of 11S and 7S (Liu et al., 2019) Thus, it can be seen that TGs have an impact on lima bean protein at pH7, which was also compatible with previous research results (Ando et al, 1989) Therefore, we decided to choose pH 7 as the optimal environment for TGs to function and use modification in this study y = -0.0112x + 2.1569 R² = 0.9813

The choice of TGs concentration for protein modification

To choose the appropriate TGs concentration for the modification process, we conducted a survey on the modification of lima bean protein with different TGs concentrations, respectively 0 U/g protein; 2.5 U/g protein; 5 U/g protein; 7.5 U/g protein and 10 U/g protein were available at 45ºC for 3 hours, at pH 7 with incubating speed of 250 rpm/mins After modification, the samples were inactivated at 92C for 5 minutes, and the particle size measurement method using DLS was used to measure these samples the results are presented in Table 4.1 Dynamic light scattering (DLS), also known as photon correlation spectroscopy or semi-elastic light scattering, is a spectroscopic method used in the fields of chemistry, biochemistry, and physics to determine the size distribution of particles (polymers, proteins, colloids, etc.) in a solution or suspension (Falke & Betzel, 2019) The sample preparation process for DLS measurement method was carried out according to strict procedures regarding time and sampling operations The sampling time was fixed and the interval between sampling times was the same After inactivation, the samples were allowed to settle naturally for 4 hours, then decant the supernatant, dilute the solution to 5%, and measure DLS immediately The measurement results are presented in Table 4.1

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

TG’s concentration (U/g protein) Z - average (nm) Recovery efficiency

Note: The values in the same column with different letters (a-b) indicate significant differences (p < 0.05)

Results in Table 4.1 show that the particle sizes of samples treated with different TGs concentrations respectively MPP 0U (395.65 nm), MPP 2.5U (362.65 nm), MPP 5U (224.7 nm), and MPP 7.5U (261.15 nm), MPP 10U (266.80 nm) When the enzyme concentration increased from 0 U/g protein to 2.5 U/g protein, the particle size of these samples did not change, but from 2.5 U/g protein to 10 U/g protein, the particle size in these samples tended to

43 decrease gradually This difference can be explained by the fact that during the treatment of protein solutions with TGs, the process of linking protein fragments together occurred, leading to changes in molecular weight Molecules with larger weights were likely to settle, while molecules with medium and small masses would remain suspended in the solution in suspension form At the enzyme treatment concentration of 2.5 U/g protein (accounting for 1.7% of dry matter content), in a fixed time of 3 hours, the level of bond formation and change in particle size was not significant (reduced from 395.65 nm down to 362.65 nm) When the concentration of the enzyme was increased from 5 U/g protein to 10 U/g protein (accounting for approximately 3.33 – 6.67% of the dry matter content), the particle size of proteins in the suspension phase decreased

Besides, the protein recovery efficiency after modification also changed and the analysis results are presented in Table 4.1 above The recovery efficiency of the samples respectively MPP 0U (61.40%), MPP 2.5U (61.97%), MPP 5U (68.99%), MPP 7.5U (69.53%), MPP 10U (70.12%) Thus, when treated with TGs of 2.5 U/g protein, the recovery efficiency of this sample did not increase significantly When increasing the TGs concentration from 2.5 to 5 U/g protein, the recovery efficiency also increased from 61.97 to 68.99% However, when continuing to increase the TGs concentration from 5 to 10 U/g protein, the recovery efficiency did not change This result can be explained because at low TGs concentrations (2.5 U/g protein, equivalent to about 1.7% dry matter content), the reactions occur at low intensity, and many new bonds had not yet been formed, small-sized particles tend to dissolve in the liquid phase and were lost during centrifugation, leading to low recovery efficiency When increasing the enzyme concentration to 5, 7.5, and 10 U/g protein, there was an increase in recovery efficiency, but the difference in these samples was not significant The explanation for this result may be that when the concentration was increased, there was enough enzyme to interact with protein molecules thereby increasing the ability to form bonds, the large particles were settled, and then, when the sediment was centrifuged and dried the recovery efficiency was also increased

Consequently, with the two results obtained in Tables 4.1, we proposed to choose the concentration level of 5U/g protein as the concentration level used for the process of modification of protein from lima beans with TGs in this study Further more, this result was also consistent with the results in some previous studies of performing protein modification (Liu et al., 2019) (Moon & Cho, 2023)

Appropriate incubation time for protein modification

Besides, investigated factors such as pH value and TGs concentration mentioned above, incubated time with TGs can also be a factor so it needed to be investigated Protein samples were modified with TGs 5 U/g at pH 7.0, incubated temperature 45ºC, incubated speed 250 rmp/min with different time from 0 – 4 hours Modified protein samples were inactivated at 92ºC for 5 minutes and then analyzed with the SDS – PAGE electrophoresis method and the results were presented in Figure 4.3 and Figure 4.4 below Samples MPP7_0 to MPP7_4 were modified lima bean protein samples with time intervals from 0 hours to 4 hours respectively

Figure 4.3SDS – PAGE measurement results of modified protein samples with different incubation time

Figure 4.4The calibration curve between the logarithm of molecular mass and protein segments travel distance Images of SDS – PAGE gel samples and ratios of protein subunits after different TGs reaction times were shown in Figure 4.3, respectively In general, there has been a decrease in the color intensity of the protein bands at a molecular size of 25 kDa since the first hour and a considerable decrease after the third hour of modification From the second hour onwards, a new line appeared Specifically, quantification results through a calibration curve between the logarithm of molecular mass and protein segments' travel distance (Figure 4.4) It showed that the lima bean protein samples all contain main bands with low molecular weight from 10 – 15 kDa, average 25 – 35 kDa, and high molecular weight ~ 70kDa corresponding to 2S albumin (10 – 18 kDa), globulin 11S (20 – 37 kDa) and globulin 7S (55 – 75 kDa) similar to legume (Abdel-Shafi et al., 2022) (González-Pérez & Arellano, 2009)

Figure 4.3 showed that the initial lima bean sample and the modified lima protein sample at 0 hours displayed similar gel images, indicating that time was not effective and the addition of TGs did not change the fractions nor protein molecular weight The reason for this result may be due to the preparation process of TGs – treated samples with an incubation time of 0 hours including dissolved protein powder with water, then adjusted pH to 7, added TGs, and heated the solution at high temperatures immediately aimed to block enzyme activity, so the y = -0.0173x + 2.2163 R² = 0.9912

MPP7_1MPP7_0MPP7_2MPP7_3MPP7_4Standard marker

46 enzyme has not had time to bind to proteins After 1 hour of treatment with TGs, there was a difference with some segments of the MPP7_1 sample that faded and disappeared at a molecular size of about 45 – 70 kDa corresponding to 11S globulin protein subunits When the TGs treatment time to 2 hours, in general, there was the formation and appearance of segments having molecular size above 75 kDa, and at the same time some segments below 15 kDa began to fade However, when the treatment time was increased to 3 and 4 hours, the gel electrophoresis results in Figure 4.3 showed no difference between these two samples, some lost segments have molecular sizes of about 8.9 kDa, 12.5 kDa, and 45 – 50 kDa and no new segments were appearing compared to the 2 – hour sample The reduction of some segments and the formation of new bands have shown that protein subunits, such as 7S globulin and 11S globulin, were cross – linked by MTG, increasing their molecular weight (Moon & Cho, 2023)

Additionally, some new segments were formed at around the molecular weight of 75 kDa and this was probably related to the TGs – treated polymer molecules that formed some high molecular weight fractions (Basman, Kửksel, & Ng, 2002) To sum up, there were significant differences in protein subunit fractions between samples treated with TGs for 0, 1, 2, and 3 hours; but there was no significant difference between the two samples treated at 3 and 4 hours

Thus, we decided to choose a sample with an incubation time of 3 hours for the modification process to form new bonds between TGs and lima bean protein.

The complete process for Lima bean protein modification using Transglutaminase 46

With the results of experiments 1,2 and 3 above, we decided to syntonize the procedure according to section (4.4) to obtain a modified protein with a pH value of 7 and the TGs concentration of was used 5U/g and modification time was 3 hours and presented in Figure 4.5

Figure 4.5 The complete process for Lima bean protein modification using Transglutaminase

The recovery efficiency of the modified protein

The protein sample after modification was enzymatically inactivated at a temperature of 92℃ for five minutes After that, the modified protein was centrifuged and combined with convection drying at a temperature of 50℃ to recover the product The recovery efficiency of modified proteins is shown in Table 4.2

Table 4.2 The recovery efficiency of the protein after modification process

Based on data Table 4.2 on the recovery efficiency of modified proteins, we find that the efficiency recovered after modification by centrifugation combined with convection drying is at a relative level, reaching around 70% at the 3 – hour modification time point (68.99% ± 0.6) One of the possible causes of sample loss during the recovery process of modified proteins was that after the incubation and centrifugation period, the sample in the Erlenmeyer flask or centrifuge tube was not thoroughly collected or was lost during the weighing process and drying.

Chemical composition of the modified protein

The chemical composition of lima bean protein before (PP) and after modification (MPP7_3) recovered by centrifugation combined with convection drying is shown and analyzed in Table 4.3

Table 4.3Chemical composition of PP and MPP7_3 samples

Sample Protein (%) Fat (%) Carbohydrate (%) Moisture (%) Ash (%)

Note: The values in the same column with different letters (a-b) indicate significant differences (p < 0.05)

Analysis results in Table 4.3 indicated significant differences in protein, fat, carbohydrate, and ash content between PP and MPP7_3 samples The protein, fat, carbohydrate, moisture, and ash content in MPP7_3 were 68.54%, 0.73%, 17.09%, 10.22%, and 3.42%, respectively The moisture content of both MMP7_3 and PP samples was about 10.20–10.22%, the difference was not significant (p > 0.05) However, the protein content of MPP7_3 (68.54%) was higher

49 than that of PP sample (61.19%) Meanwhile, the fat, carbohydrate, and ash content in MPP7_3 (0.73%, 17.09%, and 3.42%) were lower than that in the PP sample (0.82%, 21.78%, and 6.01%) The main reason for the decrease in fat, carbohydrate, and ash content can be due to some losses that occur after modification Centrifugation was one of the steps in the modification process that can cause the loss of some water – soluble components such as monosaccharides and carbohydrates that dissolve well in water, leading to dissolution in the liquid phase and were remove, leading to a reduction in carbohydrate content Besides, the protein content of MPP7_3 increased slightly compared to the PP sample which can be predicted due to the contribution of TGs enzyme added during the modification process (accounting for about 3.33%), because it is also naturally a type of protein.

Secondary structure of modified protein

The initial lima bean protein sample (PP) and modified protein samples (MPP7_3) were analyzed by infrared spectroscopy in the region 4000 – 400 cm -1 and the results are depicted in the graph of Figure 4.6

Figure 4.6 FTIR infrared spectra of samples PP and MPP7_3

The result of Fourier-transform infrared (FTIR) analysis results in Figure 4.6 of both samples showed some characteristic bands ranging from 1800 to 1300 cm -1 and these bands

50 were very useful for protein matrix analysis (Van der Ven et al., 2002) The stretching of C=O bonds was the main source of the amide I band (1600 – 1700 cm −1 ), whereas the stretching of

C – N bonds and the deformation of N – H bonds result in the amide II band (1600 – 1500 cm −1 ) The amide III band, which arises from N – H bending and C – N stretching, was located in the region of 1220 to 1330 cm −1 (Krunić, Obradović, & Rakin, 2019) Figure 4.6 additionally shows values at specific peaks within the amide A range in addition to the amide I, II, and III ranges Amide A range that mostly represents the stretching vibrations of hydroxyl O – H or the bending vibrations of H – N is 3300-3500 cm -1 (Ji and others, 2020)

FTIR infrared spectra of samples PP and MPP7_3 showed that the amide I band had peaks at wave number 1631 cm -1 , primarily related to C=O stretching bonds In the amide II band, the samples also had a wave number of 1529 cm -1 , these were mainly N – H deformation bonds and C=O stretching bonds Meanwhile, the amide III bands of samples with the appearance of wave number 1233 cm -1 were mainly N – H bent bonds and C=O stretching bonds In addition, the samples also had a peak at 1393 cm -1 , these were mainly COO- stretching bonds (Barth, 2007) Overall, the unmodified and TGs – modified protein samples showed similar characteristic fingerprint regions of protein functional groups This demonstrates that different structural changes occur in the same group and that the secondary structure of the protein was not destroyed (Akbari et al, 2020) Additionally, this can be explained by the Beer-Lambert law which shows that the signal intensity is proportional to the sample concentration so the reason for the insignificant difference could be the error when using the amount of sample to perform FTIR measurements (Swinehart, 1962).

Nitrogen solubility in modified protein

Water solubility, a crucial functional property in the food industry, was influenced by the formation of high – molecular – weight proteins through TGs treatment These alterations in protein solubility could potentially impact other functional properties, including gelling, emulsifying, and foaming properties The results depicting the influence of pH on the solubility of the MPP7_3 sample compared to the PP sample are presented in Figure 4.7

Figure 4.7Nitrogen solubility between PP and MPP7_3 samples The graph (Figure 4.7) illustrates the solubility of both samples with a change in pH value from 2 to 10 The results in Figure 4.7 indicated that samples PP and MPP7_3 had the lowest solubility at pH = 4, 40.88%, and 12.14%, respectively, and the highest solubility at pH = 10, 98.19%, and 70.17%, respectively

The nitrogen solubility of the PP sample showed a typical “V” shape according to the pH change At pH = 4, the PP sample had the lowest solubility (40.88%) due to near protein isoelectric pH (pH = 4.5) At isoelectric pH, proteins can aggregate because protein-protein interactions dominate over protein – water interactions (Belit et al, 2009) At values outside the isoelectric pH point, it showed that the solubility of the PP sample was improved due to the appearance of electrostatic repulsion from positive and negative charges on the surface of the protein (Hall, 1996) Therefore, the solubility of PP increased significantly from pH = 6 and reached its maximum at pH = 10 (98.18%)

About the MPP7_3 sample, the solubility was also “V” shaped, however, the nitrogen solubility property of MPP7_3 changed after exposure to TGs, especially significantly decreased This can be explained by the effect of cross-linking, which changes the tertiary structure of proteins and forms insoluble proteins with high molecular weight (Moon & Cho, 2023) In addition, the change in solubility may also be due to the change in overall charge and

Nitrogen solubility (% E BSA/àg) pH

52 surface hydrophobicity due to the incubation of TGs (Moon & Cho, 2023) In general, proteins exhibited high solubility when their molecular mass was low, the total net charge was high, and the exposure of hydrophobic groups was low (Walsh et al, 2003) This result of reduced solubility is consistent with previous results observed for strains isolated from walnuts treated with TGs (Tang et al, 2008) (Shi et al, 2018) (Moon & Cho, 2023).

Water absorption capacity and oil absorption capacity

Take 1g of the protein sample mixed with 10 mL of distilled water or olive oil, and shaken at the highest speed for 2 minutes The mixture was allowed to stabilize at room temperature for 30 minutes before being centrifuged at a speed of 3000 rpm/min, and the measurement results were recorded in Table 4.4

Table 4.4 Water and oil absorption capacities of protein samples

Note: Means in the same column with different letters (a-b) indicate a significant difference (p < 0.05)

WAC was the ability to absorb and retain water molecules in a protein matrix, determined by various physicochemical factors, such as molecular structure and surface charge of amino acids (Moon & Cho, 2023) Table 4.4 illutrated that the lima bean protein sample modified with TGs (5.34 g/g) has a higher water absorption capacity and was almost twice that of the unmodified lima bean protein sample (2.46 g/g)

This can be attributed to the fact that the cross – linking formed by TGs treatment changed the tertiary structure of the protein, which can physically retain water in the protein matrix (Moon & Cho, 2023) These enhancements in WAC align with findings from previous studies on TGs – treated wheat, barley, soybean, and whey protein (Moon & Cho, 2023) (Ahn et al, 2005) The increase in the WAC of MPP7_3 with TGs might be attributed to the formation of large clusters of protein molecules (Ahn et al, 2005) (Liu et al., 2019) The covalent bonds between ε – (γ – glutamine) lysyl, catalyzed by TGs, were approximately 20 times stronger than the hydrophobic and hydrogen bonds, resulting in the enhancement of textural attributes in crosslinked products (Liu et al., 2019)

Through analysis results, the water absorption ability of the post – modified sample is superior to that of the pre – modified sample, opening up many applications of this product in the food Water absorption is an important property to consider when adding MPP7_3 to many foods such as soups, sausages, dough, etc In these foods, proteins with high water absorption capacity will absorb water, but not completely dissolve They will swell and create properties such as shape, consistency viscosity, etc for the food

Specifically, in cake dough, adding MPP7_3 not only increases protein content but also forms intermolecular interactions with each other and with gluten protein molecules In addition, MPP7_3 has a high water absorption ability, so it can improve the consistency of dough, leading to an increase in the water-binding ability of the ingredients and a decrease in the amount of free water (Dogan et al., 2005) From there, it brings better stability to the protein network and helps retain air bubbles formed during kneading and CO2 created by sodium bicarbonate during baking, so cake volume and height increase significantly (Gómez et al events, 2010)

OAC was influenced by various physicochemical factors, mainly surface hydrophobicity, which affects the absorption and retention of oil molecules in the protein matrix (Moon & Cho, 2023) Analysis of data in Table 4.4 revealed that sample MPP7_3 had a higher oil absorption capacity, significantly superior to sample PP (increased by 0.73 mL/g)

This indicates that TGs treatment affects OAC and the reason for the increase in OAC can be attributed to its enhancement of surface hydrophobicity (Moon & Cho, 2023) Fats and proteins interact through the association of fatty lipid chains with nonpolar side chains of amino acids, so proteins with higher surface hydrophobicity tend to absorb more oil (Lin & Zayas, 1987) Besides, increased OAC can also be partly attributed to a significant decrease in bulk density, as fat absorption depends on physical oil trapping (Siu et al., 2002) Fat molecules are retained through hydrocarbon bonds of the oil with nonpolar amino acids (Wang et al., 2020) From there, the modification process helps increase the water – binding area of the protein, enhancing the oil-absorbing ability of the modified product

The significantly improved oil absorption ability of the modified sample can be applied to products in the food industry The ability of proteins to bind fat plays a very important role in

54 cases where plant proteins are used to replace animal proteins such as eggs (Garcia-Vaquero et al., 2017), and this characteristic was considered a necessary condition to consider adding protein to meat foods such as sausages or cakes, chiffon, mayonnaise, and salad dressing (Lam et al., 2018) In sausage products, the modified protein can be applied to help improve texture and enhance product flavor through their ability to retain water and fat by binding to liquid ingredients and forming a gel network that increases hardness, helping the sausage achieve the desired structure Besides, the sausage structure is more stable with MPP7_3 because water and oil are retained inside the gel network and avoid loss during processing and storage, thereby bringing economic efficiency better (García-Vaquero et al, 2017) In addition, fat has the effect of preserving flavor, and increasing appetite when eating, so proteins with high fat absorption ability have good emulsifying ability, helping to retain flavor and improve sensation.

Emulsifying activity and stability

The EAI and ESI results of sample PP and sample MPP7_3 were analyzed and presented in Table 4.5

Table 4.5 Emulsifying activity and stability of PP and MPP7_3

Note: The values within the same pH column with different letters (a-b) indicate a significant difference (p < 0.05)

Proteins have hydrophobic and hydrophilic areas that operate as emulsifiers The emulsifying ability of a protein was primarily determined by its molecular structure, the ratio of hydrophilicity to hydrophobicity of its amino acids, and its molecular weight (Ghribi et al., 2015), (Zhu et al., 2020) In general, the increase in the surface hydrophobicity of the protein enhanced its emulsifying properties because this is a key factor in protein absorption at the oil/water interface (Yan, Xu, Zhang, & Li, 2021)

We could see that sample MPP7_3 had EAI (42.25 m 2 /g) and ESI (164.42 min), about twice as high as sample PP with EAI (22.17 m 2 /g) and ESI (80.22 min) A reduction in protein solubility also led to the creation of larger protein aggregates or complexes, contributing to a thicker and more stable oil/water interface, thereby enhancing their emulsifying properties (Nivala, et al, 2021) Furthermore, the TGs treatment formed cross – linkages, increasing the

55 molecular weight of MPP7_3 This network trapped and stabilized oil droplets within the emulsion, preventing them from coalescing or separating from the aqueous phase It also increased the viscosity and elasticity of the emulsion, leading to improved stability and reduced coalescence of oil droplets (Glusac, Isaschar – Ovdat, & Fishman, 2020) These enhancements in EAI were consistent with prior findings observed in TGs – treated fava bean protein isolates and soy protein (Liu, Damodaran, & Heinonen, 2019), (Babiker, 2000)

The improved ESI of MPP7_3 (164.42 min) compared to PP (80.22 min) may be attributed to a higher net charge on the oil droplets, sufficient to overcome various attractive forces and stabilize the electrostatic repulsion force between the oil droplets (Dickinson, 2009) Additionally, there was a possibility that the amide reduction of glutamine to glutamic acid, promoted by TGs, leads to an increase in negative charge and the overall negative properties of the protein (Gaspar & de Góes-Favoni, 2015).

Foam ability

Protein foam is a crucial component in the food industry, as it serves as the key to producing various food products with unique textures and sensory properties Protein foam is typically formed when a protein solution is agitated or whipped in the presence of water and air This type of foam can be used as a base for many food items, including sponge cakes, pastries, mousses, and fresh cream (Moon & Cho, 2023) Figure 4.8 and 4.9 illustrated that pH significantly influences the foaming ability of PP and MPP7_3 suspension solutions

Figure 4.8 Influence of pH of MPP7_3 samples on foam ability

Time (minutes) pH2 pH4 pH6 pH8 pH10

Figure 4.9Influence of pH of PP samples on foam ability

At t = 0, the FA of PP and MPP7_3 suspensions followed the order of pH: 6 < 4 < 2 ≈ 8 ≈

10 This result was consistent with the findings reported by Betancur-Ancona et al (Betancur‐ Ancona, et al., 2004), among them, the foaming ability of PP is highest under acidic and alkaline conditions Under these conditions, proteins were electrically charged and repulsed each other away As a result, protein molecules become more mobile and diffuse more rapidly to the air- liquid interface to envelop air pockets, thereby increasing their foaming ability (Adebowale & Lawal, 2003) Conversely, the low FA at pH 6 and 4 might be due to the low solubility and mobility of PP near its isoelectric point (Wani, Sogi, Shivhare, & Gill, 2015) There was no statistically significant difference in the FA between MPP7_3 and PP These minor differences in the FA of MPP7_3 are consistent with previous observations for TGs-treated wheat gluten (Agyare, Addo, & Xiong, 2009)

After 60 minutes, the order of foam stability differs from the order of FA for the pH of the

PP and MPP7_3 samples, which is 10 < 8 < 2< 6 < 4 (p < 0.05) This result indicated that although FA at pH 4 and 6 is the lowest, the foam was relatively stable compared to the foam formed at pH 10 and 8 The reason was that at pH near the isoelectric point, electrostatic repulsion between protein molecules is minimal, thus creating conditions for the formation of a thicker protein layer at the air-liquid interface (Pham, Nguyen, & Trinh, 2022) When treated

Time (minutes) pH2 pH4 pH6 pH8 pH10

57 with TGs, it had the potential to increase the foam stability of MPP7_3 compared to PP TGs treatment significantly increased the molecular weight of MPP7_3, which might improve foam stability Protein molecular weight might impact foaming properties because larger proteins have more surface-active amino acid residues, including lysine, arginine, and histidine, which can reduce the surface tension at the air-liquid interface and foaming stability (Moon & Cho, 2023)

CONCLUSIONS AND RECOMMENDATIONS

During the research process, we reached the following conclusions:

Recommended technical parameters (pH value 7, enzyme concentration 5U/g protein, and incubation time 3 hours) to obtain the modified lima bean protein sample MPP7_3 with a recovery efficiency of nearly 70% The modified lima bean protein content was 68.54%, slightly increased compared to the PP sample (61.19%)

Electrophoresis results indicated that the MPP sample after undergoing the process of modification with the enzyme Transglutaminase, appeared segments with a large molecular weight > 118 kDa and lost some molecular weight segments (10 – 12 kDa and 40 – 45 kDa) Meanwhile, the particle size of the modified bean protein was reduced compared to the original bean protein

The properties of MPP samples such as oil absorption, water absorption, and emulsifying ability were significantly improved However, properties such as foaming ability and solubility of MPP samples are less effective than PP samples due to the cross – linking of protein molecules

With the difference in properties (good ability to emulsify, absorb water, and absorb oil) of MPP when compared to initial lima bean protein, it contained all the original special nutrients and essential amino acids Therefore, MPP – modified protein can be very suitable for adding to soups, sausages, dough cakes PP samples were very suitable for application in meat processing technology and bread products as an additive and stabilize structures

During the implementation of the research project, we observed that there were still many issues that needed further investigation to refine the process of modifying lima bean protein using the transglutaminase enzyme Additionally, we recognized that the protein modification process using TGs from lima bean sources presents numerous potential research directions, particularly concerning health-related issues and applications in food technology Therefore, we hope that future projects can delve into the following aspects:

The study suggests exploring alternative processing methods beyond TGs like treating enzymes with ultrasonic waves to investigate and assess how different modified levels will affect the functional properties and their applications in specific products Utilizing modern techniques such as X-rays to determine particle size distribution

Research investigates the influence of TGs-modified lima bean protein on the properties, structure, sensory characteristics, and quality of various products such as bread, dairy products like ice cream and yogurt, and beverages containing protein, …

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Certificate of analysis of transglutaminase enzyme product

Appendix 1: Certificate of analysis of transglutaminase enzyme product

Methods for determining the chemical composition of materials and product70

 Method for determining moisture content

Dried the petri dish to constant mass in an oven preheated to 105°C (don't forget to mark the dish and lid!) Then, it was cooled in a desiccator and weighed with an analytical balance with an accuracy of 0.0001g

To have enough grinding material and enable repeated determinations, mix the test sample and grind it to the proper amount Ensured that the sample was not too coarse or too fine and passed through a 1.0 mm sieve Subsequently, 2g of the sample was added to the petri dish and dried at 105°C until the weight remained constant After drying, the sample was cooled and the petri dish in a desiccator was weighed using an analytical balance, the experiment three times, and the results The moisture content of the sample was calculated as the following formula (I)

In which, m0 is the weight of the initial petri dish; m1 is the weight of the sample and petri dish before drying; m2 is the weight of the sample and petri dish after drying

 Method for determining ash content

Heated in a clean, washed ceramic or metal crucible in a furnace at 550 – 600℃ until a constant weight was achieved Cooled in a desiccator and weighed on an analytical balance accurate to 0.0001g Then, placed 2g of the sample in a porcelain crucible, and accurately weighed each one with an analytical balance as described above Placed it all in the furnace and gradually increased the temperature to 550 – 600℃ Calcined until white ash was formed, which usually takes 6 – 7 hours and indicates that all biological stuff has been removed Weighed precisely after cooling in a desiccator Once another thirty minutes of calcined were up, cooled the mixture in a desiccator and weighed the mixture until it reached a steady weight For every 1g of the test sample, there should be no more than 0.0005g of difference in the findings between two successive calcined and weighed Repeat the experiment three times and analyze the data The ash content of sample was calculated as a formula (II)

In which, W0 was the weight of the initial porcelain crucible (g); W1 was the weight of the sample and porcelain crucible after calcining (g); W2 was the weight of the sample and porcelain crucible before calcining (g)

 Method for determining total protein content

Nitrogen in food is mostly contained in protein molecules (protein nitrogen), and there is also a small amount of nitrogen in other ingredients called non-protein nitrogen The sum of these two types of nitrogen is called total nitrogen In biological materials, because the non- protein nitrogen content is small and the separation is very complicated, by convention people calculate the protein content according to the total nitrogen content and call it crude protein or total protein Currently, total nitrogen content is often determined by the Kjeldahl or Dumas method In the study, we used the Kjeldahl method for analyzing total nitrogen content

Sample digestion: First, 0.2g of sample was accurately weighed (measured) and then placed into a Kjeldahl tube Then, the Kjeldahl tubes containing the sample were inserted into the sample, Kjehdahl digester, and then slowly added 10 mL of concentrated H2SO4, 0.03g CuSO4, and 1g K2SO4 to the Kjeldahl tube K2SO4 increases the boiling temperature while CuSO4 catalyzes the reaction Boiled until the solution in the jar was clear and there were no black carbon stains left, then stopped

Distillation: After digesting the sample, added 30 mL of H3BO3 into the refrigerator and installed it into the machine Ensured that the outlet of the condensation tube was submerged in the H3BO3 solution Installed the program for the machine according to the parameters: steam rate: 30%, volume of 40% NaOH: 25 – 30mL, distillation time: 15 minutes) Transferred the entire sample solution after inorganicization in the Kjeldahl flask into the Erlenmeyer flask for titration and determination of residual H3BO3

Titration: Removed the Erlenmeyer flask from the system, added 3 drops of phenolphthalein to the flask, and determined with 0.1N HCl When the solution turned pale pink, readed the buret to the nearest 0.05 mL

Total nitrogen content in the sample was determined according to formula (III.a and III.b):

In which, M (N) was the standard HCl concentration used for titration; V1 (ml) was the volume of HCl used for titration; V2 (ml) was the volume of blank water; 14 was the atomic weight of nitrogen; m (g) was the weight of the sample

 Method for determining fat content

Method used: Soxhlet method 4.5.01 with some changes

Weighed 2g of sample (wrapped in a paper bag) into the extraction cylinder, noting that the mass of the wrapping paper had been determined Then poured the solvent consisting of diethyl ether and petroleum ether (1:1) from the top of the condenser tube through the extraction cylinder and finally down to the solvent tank Turned on the water source to cool the condenser tube and turned on the heat source to evaporate the solvent The evaporated solvent was cooled by the condenser and condenser tube system, flowing down the extraction cylinder and then the solvent tank

This circulation process helped extract all fat in the sample after about 8 – 12 hours In addition, we can determine the stopping point of the experiment by taking a few drops of solvent from the extraction cylinder onto filter paper When the solvent evaporated without leaving an oil slick, the experiment stopped The fat content was determined following the formula (IV):

In which, m1 (g) was the weight of paper wrapped and initial sample; m2 (g) was the weight of paper wrapped and sample after lipid extraction and drying; m was the initial sample.

SDS-PAGE Separating Gel and Stacking Gel Preparation Procedure

Prepared the Resolving Gel (12.5%) at the Positive Pole

Prepared the stacking gel (4%) at the negative pole

Staining Solution: 10% (v/v) Acetic acid, 0.25% CBB-R250 or G250, and 50% (v/v) Methanol

De-staining Solution: 10% (v/v) Acetic acid, 25% (v/v) Methanol, and 65% water.

Determination of soluble proteins by the Lowry method

Method used: Lowry et al (1954) method

Prepare the following chemical solutions

 Solution A: 0.4g NaOH (0.1N) and 2g Na2CO3 (2%) mixed in 100ml distilled water

 Solution B: 0.5g CuSO4.5H2O (0.5%) mixed in 1% sodium and potassium tartratesolution

 Solution C: Mixture of two solutions A and B in a ratio of 100:2

 Folin reagent diluted twice with distilled water before use

 Bovine serum albumin 1mg/mL

Experimental sample: Used a micropipette to accurately take 0.5ml of protein solution with the appropriate concentration into a test tube, added 2 ml of solution C, shaken well, and let stand for 1 minute Then 0.25ml of Folin reagent was added to the mixture in the test tube, shaken vigorously and evenly immediately, and left for 20 minutes until the mixture changed to blue and reached maximum color intensity Compared the color of the absorption mixture on a UV-VIS machine at a wavelength of 750nm Measured on the machine 3 times and take the average value

Control sample: 0.5ml of distilled water was put into a test tube and 2ml of solution C, the next step was carried out as the experimental sample

The soluble protein content in the solution was also determined by the Lowry method, using bovine serum albumin (BSA) as the standard

Experimental sample: Used a micropipette to take exactly 0.5ml of BSA solution at the above dilution concentration into a test tube After that, 2ml of solution C was added to each tube and left at room temperature for 10 minutes, then 0.25ml of Folin reagent was added and put into the test tube Colorimetric with 750nm wavelength

Control sample: the test tube included 0.5 ml of distilled water and 2 ml of solution C and the next steps are carried out for the experimental sample.