**3. Soybean as a protein source**

According to the *Poverty and Shared Prosperity Report*, after the pandemic, the estimative world percentage of people in extreme poverty (characterized by a daily income up to U\$1,90) will reach about 9.1% to 9.4% of the world population. This estimate is alarming because, before the pandemic, it was estimated that poverty would fall to 7.9% in 2020. Unfortunately, people in poverty have a low caloric intake and nutritional deficiencies, especially regarding access to micronutrients and essential amino acids [73]. Food proteins, from animal or vegetable sources, supply the essential amino acids important for the construction and maintenance of the basic body structures therefore, they are fundamental for the right physical and mental development of children. Vegetable proteins represent a low-cost source of nutrients and energy, but, in many cases, they are poor in highly digestible essential amino acids. Experts predict a large increase in the world population. Consequently, the

demand for plant proteins will increase in the same proportion due to the low cost and lower environmental impact of their production [74].

For centuries, humankind has cultivated seed crops as a protein source, legumes and cereals, as the principal cultivated crops. These cultures currently provide more than 70% of the protein for human consumption. Nonetheless, legumes accumulate higher quantities of protein in contrast to cereal, and among the legume crops, soybean is the one with the highest percentage of proteins [75]. Soybean seeds are a rich source of high-quality digestible proteins and contain all the essential amino acids found in animal proteins, without cholesterol and with a low level of saturated fatty acids [76]. Most soybean seed components, including proteins and peptides, isoflavones, saponins, and protease inhibitors, have been shown to have biological activity [77, 78].

The principal advantages of soybean as a protein source are (1) good balance in amino acids composition, containing all the essential amino acids; (2) presence of components physiologically beneficial to human health, which are shown to lower the cholesterol and reduce the risk of hyperlipidemia and cardiovascular diseases; (3) excellent processing ability, as emulsification, gelling, water- and oil- holding capacity; and (4) excellent nutritional and functional properties of their proteins, for example, solubility, emulsifying, film-forming and foaming properties[79, 80]. Their composition varies according to the variety, location, and conditions of its planting, such as climate and farming practices [76] and besides that, stimuli, like genetic modification, can also modify protein profile, expression, and accumulation rates [81].

Soybean proteins can be classified into four main groups: albumins, globulins, prolamins, and glutelins [82]. Based on solubility patterns, soybean proteins can be classified into two categories, albumins (water-soluble) and globulins (salt solution-soluble), which represent the primary protein type [83]. When separated with ultracentrifugation, two major storage proteins can be identified, glycinin (11S) and β-conglycinin (βCG, 7S), corresponding, respectively, to ~40% and ~ 50% of soy proteins amount [81] corresponding to the largest mass of the soy seed. The sedimentation coefficient values are a more precise pattern for identifying soybean proteins, where larger S (Svedberg units) numbers correspond to a larger protein. By ultracentrifugation under appropriate buffer conditions (0.5 ionic strength and pH 7.6), soybean proteins can be separated into four main groups, 2S, 7S, 11S, and 15S fractions [84]. While the 2S fraction (20% of total proteins) contains the most albumins, such as 2S globulin, cytochrome C, Kunitz trypsin inhibitor, and Bowman-Birk trypsin inhibitor, both the inhibitors are associated with delayed growth in children [84, 85]. The globulins are mainly present in 7S, 11S, and 15S fractions of soybean proteins. Like the 2S fraction, the 7S fraction (40% of total protein) is also highly heterogeneous, containing β-conglycinin, α-amylase, lipoxygenase, and hemagglutinin [86], and the 11S fraction (30% of total protein) consists of only the glycinin, which is the major protein in soybean seeds. The β-conglycinin and the glycinin correspond to the major component of storage proteins. The minor component, typically about 10% of the total proteins, the 15S fraction, maybe be a polymer (possibly a dimer of glycinin) [84].

### **3.1 Soybean-based meat**

The plant-based meat industry is focused on developing burgers, patties, mince, and sausages. Besides that, at the moment, the production of the primary meat cuts, such as steak, is not the primary search worry, due to their structure composition complexity, many groups have progressed in the research of synthetic beef. Between the commercially available plant-based meat, the Beyond Burger (BB) and the Impossible Burger (IB) can be highlighted. The main ingredients of this burger can vary, but usually, it contains soy protein, wheat gluten, egg protein, or milk proteins. In the specific case of BB, non-genetically engineered ingredients such as beetroot are incorporated to give red color to meat analogs and promote the feeling of a "bleeding" meat when cooked [87, 88].

At the beginning of the 21st century, meat analogs entered the mainstream due to the demand for healthy foods, and the worry about the sustainability implications of the consumer's diet continued to increase. Also called meat substitute, meat alternatives, fake or mock meat, and imitation meat, the meat analog is, for definition, the product of replacing the main ingredient with meat [89]. The search for sustainable, healthy, and tasty meat analogs started in the early 1960s [90].

Usually, the meat analogs found in the markets are advanced plant-based meat. The texture and taste are like the conventional meat, which uses plant-derived ingredients that have attributed almost exactly to animal-derived meat and can be indistinguishable from their animal-based equivalents. The biggest challenge for food producers is developing acceptable quality meat analogs because their characteristics depend on the ingredients used. Plant-based meat needs to be unfolded, cross-linked, and realigned to form microscopic and macroscopic fibers [89]. Different techniques are applied to plant-based meat proteins to improve their "meat qualities" to texture, processes such as extrusion, spinning, and simple shear flow have been used [91]. To solidify the structure, following the previous treatment, heating, cooling, drying, or coagulation can be applied [92].

Among the vegetables used to produce plant-based meat, the soybean stands out due to the presence of the leghemoglobin protein that mimics animal myoglobin. The soybean-based meat was the first kind of plant-based meat; in the early 1960s, traditionally, soybean proteins were used as ingredients for food analogs such as tofu and tempeh (fermented soybean cake). These products were made basically by simple processing/fermentation techniques and have been highly consumed in southeast Asia countries for centuries since 965 BCE [93]. In addition to these traditional Asian products, in the mid to late 20th century, the Texturized Vegetable Protein (TVP) was introduced as meat alternative, obtained from the extruded defatted soybean meal soybean proteins concentrates or wheat gluten, made most from soybeans [94, 95].

Meanwhile, Soybean Leghemoglobin (SLH) is used in IB, whose function is not only to provide red-colored liquid mimicking the 'bleeding' of minced meat but can also impart a meat flavor profile in plant-based products of meat [96]. SLH is a close structural ortholog of animal myoglobin that plays a crucial role in the consumption of animal-based meat because, during the cooking, this especially abundant heme protein unfolds and exposes the heme cofactor, responsible for the catalyzes the transformation of the amino acids, nucleotides, vitamins, and sugars naturally present in animal muscle tissue, into a mainly specific and diverse set of flavor and aroma compounds, which combination creates the distinctive and unmistakable meat flavor [96]. SLH, in your turn, acts the parallel role of unfolding under cooking, releasing their heme cofactor to catalyze the transformation of the same ubiquitous biomolecules, isolated from plant sources, into a wide range of compounds that mimic the unique meat flavor and aroma [97].

Besides all these advantages, in many cases, the plant-based meat can present insufficient rates of essential amino acids and trace elements, which can become more *Soybean Functional Proteins and the Synthetic Biology DOI: http://dx.doi.org/10.5772/intechopen.104602*

challenging to produce plant-based products that perfectly mimic the meat's nutritional values, such as the meat flavor and aroma [89]. The SynBio rises like an efficient approach that will allow the perfect plant-based meat production. By engineering the plant's existing compounds, like SLH, into more "animal-like" compounds and by creating and introducing artificial and synthetic compounds that can improve the meat quality. This development can be achieved by protein and metabolic engineering with the aim to produce the needed ingredients to create a synthetic mimic of plantbased meat.

### **3.2 Proteomics studies in soybean**

Proteomics is a useful tool for examining changes in protein profile generated by the response to various external or internal stimuli such as salt concentration, drought, desiccation, cold, heat, mineral toxicity, mineral deficiency, mutations, and gene introduction or silencing. In addition, proteomics can analyze differences in nutrition-relevant food proteomes, such as identifying marks for the quality of processed foods [98].

It is safe to work with soybean because several materials about their genetic information are available in the literature. A high-quality soybean genome (Wm82) is currently available in databases and is used as a reference for several studies involving omics [12]. Genomes of wild and cultivated soybeans are also available. Recently, the genome and proteome of a highly productive tropical Brazilian species (BRS 537) (EMBRAPA, 2021) are deposited in NCBI in accession GCA\_012273815.2. In addition, data from several soybean proteomes under several conditions are also available for prospecting proteins. All these data provide solid information about the genetics and behavior of the crop, facilitating the identification of targets of interest with precision. Species proteomic sets are one of the richest materials for finding these targets, as they indicate that genes present in the genome are being translated into functional proteins.

The fact that soybean is naturally rich in protein content (40%) demonstrates a wide range of protein material to be explored, which is why soybean proteomes are widely studied worldwide. The search for new targets and potential uses is getting bigger every day. Currently, with large-scale proteomics, the isolation of a greater number of proteins is made possible, and much remains to be explored in this crop [99]. In addition to the genetic variability and availability of data on the culture, its importance in producing recombinant proteins for industrial purposes is also due to several other factors. How reducing production costs, easy cultivation in the greenhouse, high protein/biomass ratio, production safety, dosage accuracy allow generating of marketable formulations that may not require purification, low technology sustainability for the production line, reducing the risk of contamination, scalability, and minimal waste production. No other protein expression technology is as efficient as the soybean system [7].

The state-of-the-art studies involving proteomic analysis of soybean seeds in the last 16 years are described in **Table 2**. Since 2005, 50 articles that evaluated the set of proteins in soybean seeds were computed, these studies involve a wide range of proteomic scenarios that come helping in the screening for proteins that can play roles in metabolic pathways for the synthesis of essential amino acids, bioactive proteins, tolerance to various environmental factors, production of sustainable fuels, and others [139], besides providing increasingly solid knowledge about the behavior of the crop under different conditions and stages of development.


**Table 2.**

*Studies involving proteomics analysis in soybean seeds.*

### **Figure 2.**

*Examples of conventional soybean seeds are characterized by functional annotation.* **Label***: BR16; BRS 257, BRS 258, Embrapa 48, BRS 267; Mindou 6; Maverick. Numbers represent the percentage of protein in each category.*

Proteomes for studies of nutritional factors are also widely observed in the literature, as they directly influence protein digestibility. Natarajan and team [107] investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (*Glycine soja*), ancestor of cultivated soybean. They identified that KTI exists as multiple isoforms in soybean. The authors noted that the number and intensity of proteins between wild and cultivated genotypes varied. These data suggest that the greatest variation in protein profiles occurred between wild and cultivated soybean genotypes rather than between genotypes in the same group. However, genetic variation of genes related to KTI1, KTI2, and KTI3 was detected within and between groups (**Figure 2**).

A proteomic study on developing soybean seeds (*G. max* var. Mindou 6) showed 48 differentially expressed proteins [109]. Among these proteins, 25% were related to protein destination and storage, 42% to energy and metabolism, 15% to disease/ defense, 6% to transporters, 4% to secondary metabolism, 4% to transcription, 2% to the synthesis of proteins and 2% for cell growth/division. It was observed that with the maturity of the seeds, the number of proteins varied, some decreased, and others increased their concentrations. The sucrose-binding protein (SBP) 2 precursors, which can contribute to improving the digestibility, nutritional value, and food quality of seeds, were increased with maturity (**Figure 2**).

### **4. Soybean functional proteins**

The function proprieties of proteins are physicochemical aspects that influence the behavior of proteins in food preparations, for example. Based on epidemiological studies, soybean consumption has been associated, for many years ago, with several potential health benefits in reducing chronic diseases such as insulin resistance/type II diabetes, cardiovascular disease, obesity, certain types of cancer, and immune disorders [76]. Nowadays, it is already proven that soybeans are a rich source of

phytochemicals, and many of these compounds have important benefits to human and animal health. Among these phytochemicals, phytoestrogens, mainly isoflavones (genistein and daidzein) and lignans, usually get more attention [140]. Nonetheless, in recent years, those physiological functions have been attributed to soybean proteins intact or more commonly bioactive and functional peptides derivate from soybean processing. These bioactive peptides are small protein fragments produced by enzymatic hydrolysis, fermentation, food processing, and gastrointestinal digestion of larger soybean proteins [141], showing multiple beneficial metabolic effects [78, 142].

The soybean seeds contain ~38% protein, ~18% oil, ~30% carbohydrates, ~14% moisture, ash, and secondary metabolites, are a considerable source of vitamins (A, thiamin, riboflavin, pyridoxine, and folic acid) and minerals (Fe, Zn, Mg, K, Ca, Mn, and Se), phytoestrogens and fibers, as well as a widely important source of protein [143]. More important than quantity is the quality of the proteins found in soybean, all eight essential amino acids, which are necessary to human nutrition, but are not produced by the human body, are found in soybean. While the sulfur-containing amino acids (methionine and cysteine) are a limiting factor with a chemical score of 47, compared to 100, as ideal protein for human nutrition [144], soybean proteins are an extraordinary source of lysine.

The two major storage proteins, glycinin (11S) and β-conglycinin (βCG, 7S), are considered naturally bio-inactive, but different ratios of βCG and glycinin may have other nutritional and physiological effects. However, many bioactive peptides are inert while still constituting a larger protein but become activated when released from the original structure by gastrointestinal digestion, enzyme and food processing, or fermentation. These peptides common are 2 to 20 amino acids in length and can be absorbed by the human intestine, falling into the bloodstream, where they can exercise systemic or local physiological effects in target tissues [76]. It has shown a difference in the human intestinal absorption of 11S peptides compared to 11S globulin or amino acids mixture, being that the 11S peptides take to a significantly greater increase in venous blood amino acids concentration. This difference is more notable for aromatic and branched-chain amino acids, which could indicate that hydrolyzed soybean proteins are faster and more efficiently absorbed in the human intestine [145].

Thus, in the last decade, the focus of research on the functionality of soy-based foods has shifted from proteins to bioactive peptides. Moreover, numerous bioactive soybean peptides have been identified with widespread beneficial physiological effects, such as anti-diabetic, anti-cancer, hypotensive, anti-inflammatory, antioxidant, and lipid-lowering (hypocholesterolemic, hypotriglyceridemic, anti-obesity) (**Table 3**) [76]. Among these is the lunasin, one chemopreventive peptide that consists of 43 amino acids residues with a C-terminal of nine aspartic acid and cell adhesion motif, enabling the binding to non-acetylated H3 and H4 histones, preventing their acetylation, which gives they the anti-carcinogenic activity [160, 169].

Lectin (hemagglutinin or agglutinin), a highly specific carbohydrate-binding protein with an important role in biological recognition, can be founded in soybean seed, ~0.2–1% of total protein [171]. Trypsin and protease inhibitors encompass several proteins and peptides, such as the Bowman-Birk protease inhibitor (BBI), the Kunitz trypsin inhibitor (KTI), and lunasin. BBI is a small protein of ~10 kDa, belonging to the serine protease inhibitor family, that mightily interacts with trypsin and/or chymotrypsin and strongly inhibits their enzymatic function [172]. The soybean KTI consists of a protein of ~20 kDa, with a single polypeptide chain cross-linked by two disulfide bridges, which inhibits trypsin and, at a lesser rate, chymotrypsin [171].

**Soybean Protein Source Bioactive Peptide Properties Ref. βCG** YVVNPDNDEN Hypocholesterolemic [146, 147] YVVNPDNNEN LAIPVNKP ACE inhibition [77, 148] LPHF **Glycinin** IAVPGEVA Hypocholesterolemic Anti-diabetic [147, 149–151] IAVPTGVA [147, 149, 152, 153] LPYP [146, 147, 150, 154, 155] VLIVP ACE inhibition [77] SPYP WL SFGVAE Hypocholesterolemic [150] HCQRPR Phagocytosis stimulatory peptide [155–157] QRPR **Lunasin** SKWQHQQDSCRKQKQ GVNLTPCEKHIMEKIQ GRGDDDDDDDDD Antioxidative Anti-inflammatory Anti-cancer Hypocholesterolemic [77, 141, 157–159] **Bowman-Birk Inhibitor** Anti-cancer Proteinase inhibition Chemoprevention [160–168] **Soybean Protein** YVVFK; IPPGVPYWT; PNNKFPQ; NWGLPV; TPRVF Hypotensive [157, 169, 170] WGAPSL; VAWWMY; FVVNATSN Hypocholesterolemic [77, 155, 157]

### *Soybean Functional Proteins and the Synthetic Biology DOI: http://dx.doi.org/10.5772/intechopen.104602*

### **Table 3.**

*Some examples of the principal soybean bioactive peptides and their properties. Adapted from [76].*

Both soybean lectins and protease inhibitors usually have been classified as antinutrients because they may lower the nutritional value of soybean. Therefore, their consumption has shown preventing effects against many diseases, such as cancer. The BBI already demonstrated an anti-inflammatory function that prevents the development of cancer and coronary diseases [171, 173].

In addition to these proteins with known functions and characteristics, several aspects of human health are attributed to soybean proteins. The proteins of many animal species show a high-fat content that can be implicated in increasing blood cholesterol, triglycerides, and Low-Density Lipoprotein (LDL-c), which stimulated the search for other protein sources [143]. Studies about the soybean protein consumption effect on subjects with hypercholesterolemia concluded that it could reduce total blood cholesterol, triglycerides, and LDL-c levels [174]. In 1999, the US Food and Drug Administration (FDA) approved the label for foods containing soybean proteins as protection against coronary heart disease. Its potential role in reducing risk factors for cardiovascular disease is one of the highest causes of death worldwide [143].

Soybean proteins can positively impact the angiotensin-converting enzyme (ACE) activity, acting as ACE inhibitor peptides that can be released enzymatically from a larger protein precursor *in vivo* during gastrointestinal digestion and *in vitro* by food processing. These peptides can reduce blood pressure by limiting the effects of ACE II in vasoconstriction and improving the vasodilatory effects of bradykinin, a potent endothelium-dependent vasodilator and mild diuretic [175, 176].

### **4.1 Synthetic biology applied to functional proteins**

Being a wide broad domain with many new and emerging fields, SynBio can give the necessary tools to face many of the challenges of the modern world. The inherent complexity and redundancy of the plant genome represent a problem to be solved by SynBio, too, for this, it applies the most important engineering principles: decoupling, abstraction, and standardization [177]. Decoupling is the simplification of complex problems into smaller ones that can be solved individually. Abstraction divides the topology of information into hierarchical levels, allowing limited and selected data to be exchanged between levels. Standardization is used to determine and characterize orthogonal parts and standardized conditions for testing. Engineering a biological system is one method to manipulate information, process chemicals, provide food, constructing materials, and help to maintain or enhance human health and our environment [178].

Plants are the great chemist of nature, being a perfect platform for SynBio approaches. The rise of SynBio broadened the horizons of plant engineering. However, as SynBio is still dependent on existing transformation techniques, the major challenge to implementing SynBio in the production of modified interest plants is the time and expense involved in the propagation, transformation, and screening of higher plants [39]. But with, the application of the SynBio approach in other fields of functional, protein production, like food production systems, will save water resources, improve land-use efficiency, and avoid the use of pesticides and fertilizers [179]. In addition, the SynBio based functional proteins and food manufacturing systems are less affected by uncontrollable environmental factors and are easier to carry out according to high-quality standards and scale at an industrial level. By constructing cell-based food factories, foods such as plant-based meat analogs, animal-free bioengineered milk, and sugar substitutes can be created from completely renewable resources [179].

Soybean proteins have been widely used to produce many protein-based food formulations due to their excellent nutritional and functional properties. Physical modification, chemical modification, and enzymatic modification have been applied to improve the functional aspects of soybean proteins [80]. But to overcome the future food and global climate challenges, just improvement in processing techniques is not enough, it is a critical step in the development of new soybean varieties that can be able to meet both the demands of the consumer market and the producers. In many cases, the traditional plant breeding cannot attend to this demand, proving to be necessary classical and, mainly, new genetic engineering techniques to add value to the soybean crop, such as reduction of allergens and antinutrients factors along with the increase of quantity and quality proteins, oil, and carbohydrates [98].

The first option for the improved soybean crops in a "functional way" is the generation of genetically modified varieties. For example, the genetic engineering techniques, such as CRISPR-Cas9, represents a great opportunity to improve the

*Soybean Functional Proteins and the Synthetic Biology DOI: http://dx.doi.org/10.5772/intechopen.104602*

nutritional value of soybean-based foods, for instance, by developing carotenoidenriched functional crops and oilseeds crops with elevated levels of omega 3 fatty acid [39]. Besides the increment/silencing of the expression of target genes, soybean can also represent an important vehicle for the creation of bio fabric, to produce a wide range of bioactive compounds by the heterology expression of desirable genes or by the metabolic engineering of the plant. For example, based on classical genetic transformation techniques, soybean has already been used to produce functional human growth hormone and coagulation factor IX [180]; and anti-HIV Cyanovirin-N [181].

### **5. Conclusion**

Soybean seeds are an excellent source of proteins, as they provide all essential amino acids and a promising source of biologically active proteins/peptides with a wide range of effects such as anti-diabetic, anti-hypertensive, anti-cancer, antioxidant, anti-inflammatory, hypolipidemic, immunostimulatory, and neuromodulatory properties. However, soybean has a low content of sulfur amino acids, and many consumers may exhibit allergenic and antinutritional reactions due to the presence of certain proteins and peptides, such as protease inhibitors. But the same inhibitors, like KTI and BBI, show anti-cancer and anti-inflammatory activity, respectively. Thus, the future of soybean-based foods is not just about the classic plant breeding and/ or new processing techniques to remove undesirable characters because they may be interesting for other applications. SynBio rises as a modern solution to create a more "consumable" soybean, through protein and metabolic engineering, to remove just the exact allergenic and antinutritional factors. In addition, soybean can be a great platform to create biofabrics combined with SynBio techniques.

### **Author details**

Lilian Hasegawa Florentino1 \*, Rayane Nunes Lima1 and Mayla D.C. Molinari<sup>2</sup>

1 Embrapa Genetic Resources and Biotechnology, Brasilia, DF, Brazil

2 Arthur Bernardes Foundation, Embrapa Soybean, Londrina, PR, Brazil

\*Address all correspondence to: lilian.florentino@embrapa.br

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
