Soybean is the second largest acreage crop in the United States (29%), right after corn (35%) according to the American Soybean Association . Soybean is widely consumed in the world, particularly in Asian countries. The various soybean products could be separated into non-fermented and fermented soybean products. The non-fermented soybean products include soymilk, tofu, yuba, soybean sprouts, okara, roasted soybeans, soynuts and soy flour, immature soybeans, cooked whole soybeans, and the fermented oriental soybean products include soy paste (Jiang and Miso), soy sauce, Tempeh, Natto, soy nuggets (Douchi), sufu. In the United States, soy oil is often used for food and biodiesel production. The soybean processing process is shown in Figure 1. After the oil extraction, the residue – flaked soy meal, is usually produced into four products (textured soy flour, soy protein concentrate and soy protein isolate, 48% soy meal, soluble soy carbohydrate). The textured soy flour could be used in bakery products, meat products, infant food etc. Soy protein concentrate and isolate could be used in baby food, bakery products, cereals, lunch meat etc. SSPS (soluble soybean polysaccharides) functions as a dispersing agent, stabilizer, emulsifier, and has good adhesion properties . The 48% soy meal is used for animal feed. The portions of different animal usages are poultry (48%), swine (26%), beef (12%), dairy (9%), pets (2%), others (3%) . Poultry and swine usages account for74%.
The popular usage of soybean for food and feed is due to its nutritional profile. Soybean is a good protein source and the only dietary isoflavone source together with other legumes. Anti-nutritional factors in soybean, such as phytic acid, oligosaccharides, trypsin inhibitor etc, limit its usage. Fermentation with GRAS (generally recognized as safe) microorganisms has been used to help degrade these anti-nutritional factors. The nutritional value of fermented soybean and soy meal products with additional nutritional factors is then largely enhanced.
2. Nutritional enhancement of soybean and soy meal via fermentation
2.1. Soy Protein
Protein content in soybean and soy meal are around 40%- 50% respectively. The high protein content makes soybean and soy meal a rich protein source for food and feed. As food source, the Protein Digestibility Corrected Amino Acid Score (PDCAAS), which is adopted by FDA and FAO/WHO, for isolated soy protein is 0.92, soy protein concentrate is 0.99, comparing with beef (0.92) and egg white (1.00). The human subject studies show that well-processed soy protein could serve as the sole source of protein intake for human beings . FDA claims that diets containing 25 g of soy protein can reduce levels of low-density lipoproteins by as much as 10 percent and have considerable value to heart health. The specific reason for the heart protection function is unclear, for there are hundreds of protective compounds in soybean. As feed source, soy protein is high in lysine, but low in sulfur-containing amino acids, with methionine being the most limiting amino acid, followed by threonine . The complementation of soy and corn for lysine and methionine makes them a valuable feed when combined.
2.1.1. Fermentation increases protein and amino acid content, and degrades protein into small functional peptides.
During fermentation, microorganisms digest the carbohydrates in soybean or soy meal and use for their own growth. The decreased dry matter and increased microorganisms weight ratio result in enhanced protein content[5-7]. In reference , fermented soy meal with
Four parameters have been often used to evaluate the protein degradation of fermented soybean products. They are trichloroacetic acid (TCA) soluble nitrogen, degree of protein hydrolysis, SDS-PAGE profile, and amino acid content. Usually peptides having 10 or fewer amino acids would dissolve in TCA. During fermentation, the degree of protein hydrolysis increases because of protease hydrolysis [13-14]. Meanwhile, TCA soluble nitrogen and peptide contents could also be enhanced [6, 9, 13-14]. SDS-PAGE analysis shows less large (>70 kDa) and medium (20-60 kDa) peptides and more small (<10 kDa) peptides in soy meal after fermentation of
2.1.2. Functional biopeptide
Fermentation degrades large protein molecules into small peptides and amino acids. The biologically active peptides from soybean play an important role as angiotenisin converting enzyme (ACE) inhibitor  and as antioxidants . In this section, we will discuss the ACE inhibitor. The antioxidant activity of biopeptides will be discussed in the antioxidant section.
Angiotensin I-converting enzyme (ACE, EC 220.127.116.11) is a dipeptidylcarboxypeptidase associated with the regulation of blood pressure as well as cardiovascular functions . ACE-inhibitory substances are used to lower the blood pressure of hypertensive patients. Various ACE inhibitory peptides have been isolated from traditional fermented soybean foods, like natto, doujiang, soy sauce, and miso paste [20-23].
ACE inhibitory activity of peptides generated by protease is greatly dependent upon fermentation time. ACE inhibitory activity in Textured Vegetable Protein (TVP) fermented by
2.1.3. Fermentation decreases soy immunoreactivity
Soybean is defined as one of the “big 8” food allergens in the United States .The estimated prevalence of soybean allergies is about 0.5% of the total U.S. population . Patients with soy allergy could react subjectively and objectively with 0.21 and 37.2 mg of soy protein, respectively . The principle for food allergy is that epitopes in allergenic protein bind to the immunoglobulin E (IgE) molecules residing in the mast cells and basophils, causing them to release inflammatory mediators, including histamine. Alpha- (72 kDa) and beta- (53 kDa) conglycinin subunits, P34 fraction, and glycinin basic (33 kDa) and acidic (22 kDa) subunits, and trypsin inhibitor (20 kDa) are the main protein components causing plasma immunoreactivity . Glycine was found to stimulate local and systemic immune responses in allergic piglets and had negative effects on piglet performance . The severity of the immune reactions depends on the dose of glycinin; higher doses cause more severe symptoms. The effect of purified beta-conglycinin on the growth and immune responses of rats were investigated . Results showed that puriﬁed beta-conglycinin possesses intrinsic immune-stimulating capacity and can induce an allergic reaction. Also, newly weaned pigs with limited stomach acid and enzymatic secretions in the small intestine can have difficulty digesting proteins with complex structures and large molecular weights .
Studies have confirmed degradation of soybean allergens during fermentation by microbial proteolytic enzymes in fermented soybean products, such as soy sauce, miso and tempeh [5, 29]. In the fermented soy products, soy protein has been hydrolyzed into smaller peptides and amino acids;therefore the structure of antigen epitopes might be altered, becoming less reactive. The IgE binding potential is reduced and therefore the immunoreactivity is lowered. In soy sauce, proteins are completely degraded into peptides and amino acids after fermentation and allergens are no longer present . The reduction of immunoreactivity by nature and induced fermentation of soy meal with
Animal experiments have also confirmed the hypoallergenic properties of fermented soybean or soy meal products. With regard to the soybean allergy, fermentation of soy meal decreased the immune response to soy protein in piglets and the level of serum IgG decreased by 27.2% . Antigenic soybean proteins in the diet of early weaned pigs provoke a transient hypersensitivity associated with morphological changes including villi atrophy and crypt hyperplasia in the small intestine . All of these morphological changes can cause a malabsorption syndrome [26, 33], growth depression, and diarrhea [34, 35]. Differences of the villi condition in such pigs fed soy meal and fermented soy meal were investigated by using scanning electron microscopy . Piglets fed soy meal had shorter, disordered, and broader villi, whereas piglets fed fermented soy meal had long, round, regular, and tapering villi that could better digest and absorb nutrients.
One of the acknowledged bioactive compounds in soybean is isolavone. Isoflavones generally exist in soybeans and soy foods as aglycones (daidzein, genistein, and glycitein), beta-glycosides (daidzin, genistin, and glycitin), acetylglycosides (6’’-O-acetyldaidzin, 6’’-O-acetylgenistin, and 6’’-O-acetylglycitin), and malonylglycosides (6’’-O-malonyldaidzin, 6’’-O—malonylgenistin, and 6’’-O-mlonylglycitin). The structures of the 12 isomers are shown in figure 2.Isoflavones physiological effects include their estrogenic activity, antioxidant and antifungal activity, and more importantly, to act as anti-carcinogens. Isoflavones may also help to reduce blood serum cholesterol levels .
Among soy isoflavones, the relative abundance of genistein including respective derivatives, is the highest – about 50% of isolavone content, followed by daidzein (40%) and glycitein (10%). However, glycitein has been shown to be more bio-available than other isoflavones, followed by genistein . Most of the isoflavones are associated with proteins in soy, with very little present in the lipid fraction. In their natural state, the majority of isoflavones exists as inactive glycosides (genistin, daidzin, and glycitin) and the remaining as their active aglycone forms (genistein, daidzein, and glycitein, respectively). Glycoside forms are heat sensitive, being converted into malonyl-beta-glycosylated isoflavone when heated. The aglycone forms are quite stable at high temperature .
Isoflavones when ingested are metabolized extensively in the intestinal tract, absorbed, transported to the liver, and undergo enterohepatic recycling. Intestinal bacterial glucosidases cleave the sugar moieties and release the biologically active isoflavones as aglycones. Aglycones could be directly absorbed in the adult and these can be further biotransformed by microorganisms to the specific metabolites, equol, desmethylangolensin, and p-ethylphenol. All of these phyto-oestrogens are then eliminated, mainly by the kidney, and therefore share the physiological features and behavior of endogenous estrogens. Among the glycones, beta-glucosides are easier to be hydrolyzed than 6’’-O-malonylglucosides and 6’’-O-acetylglucosides.
Some microorganisms have been reported to secrete beta-glucosidase, which could convert isoflavones to aglycones. The quantities of melonyl, acetyl, and glycosidyl isoflavonoids decrease during fermentation but those of isoflavonoid aglycones, daidzein, and genistein increase by over 10 to 100 fold. In Meju (long term fermented soybeans), compared with unfermented samples, the total glycosides in 60 hr fermented samples decreased from 1827 ug/g to 487 ug/g, while total aglycones increased from 22 ug/g to 329 ug/g, with daidzein increasing from trace to 152 ug/g, genistein from 16 ug/g to 170 ug/g. However, the quantity of glycitein was not increased . In another study about meju fermented by
Aglycones could alleviate the symptoms of type2 diabetes, which the beta-glycosides, acetylglycosides and malonylglycosides forms are not able to do. Type 2 diabetes mellitus emerges from uncompensated peripheral insulin resistance that is associated with unregulated nutrient homeostasis, obesity, peripheral insulin resistance and progressive beta-cell failure. The effect of isoflavones in Meju on alleviating the symptoms of the type2 diabetes was investigated and four mechanismswere found . Isoflavonoid aglycones could improve insulin-stimulated glucose intake. Also, they could induce PPAR-γ activation to increase insulin-stimulated glucose uptake. The PPAR-γ is the central regulator of insulin and glucose metabolism. It could help improve insulin sensitivity in type 2 diabetic patients and in diabetic rodent models. Besides, aglycones have strong effects for insulin/IGF – 1 signaling through IRS2, which plays a crucial role in beta-cell growth and survival. In this study, aglycones in meju increased GLP-1 secretion. GLP-1 is one of the incretins secreted from enteroendocrine L-cells that augments insulin secretion after the oral intake of glucose and free fatty acids. The induction of its secretion can prevent and/or relieve diabetic symptoms.
2.3. Antioxidant activity
Oxidative stress has been found to be the primary cause of many chronic diseases as well as the aging process itself. Antioxidants could help to delay or prevent oxidative stress. Epidemiological studies show that antioxidants could lower the risk of cardiovascular disease, cancer (overall risk reduction between 30 – 50%), diabetes, neurological diseases, immune diseases, eye diseases et al. . Antioxidant compounds play an important role as a health protecting factor. It is beneficial to eat antioxidant enriched food. Products prepared through the fermentation of soybean including various traditional oriental fermented products of soybean such as natto, tempeh, miso and other fermented products, have been found to exhibit a significantly higher antioxidant activity than their respective non-fermented soybean substrate.
Di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium(DPPH) radical scavenging activity, Fe2+ chelating activity and reducing property have been used to quantify antioxidant activity. DPPH is a stable nitrogen-centered, lipophilic free radical that is used in evaluating the antioxidant activities in a short time. Ferrous ionsare the most effective pro-oxidants in food system. Metal chelation agents prevent metal-assisted homolysis of hydroperoxides and block the formation of chain initiators. IC50 and the relative scavenging effect are the parameters to describe the DPPH radical scavenging and Fe2+ chelating abilities. IC50 is the inhibition concentration of extracts required to decrease initial DPPH radical or Fe2+ concentration by 50%. The relative scavenging effect is calculated by dividing the extract content with the IC50 of the respective extract, and then compare with the scavenging effect of control samples. The reducing property indicates that these compounds are electron donors and can reduce the oxidized intermediates, and therefore, can act as primary or secondary antioxidants. The reduction of Fe3+/ferricyanide complex to ferrous form in presence of antioxidants has been used to test the reducing activity of samples .
Antioxidant activity of methanol extracts of soybean koji fermented with
The enhanced antioxidant activities in fermented soybean products may be due to the increased phenolic compounds contents. Phenolic compounds have been demonstrated to exhibit a scavenging effect for free radicals and metal-chelating ability . Most phenolic acids in cereals primarily occur in the bound form, as conjugates with sugars, fatty acids, or proteins .Isoflavones are the predominant phenolics in soybean, and the glucoside form of isoflavones represents 99% of the total isoflavones in soybean .This condition lowers the antioxidant activity since the availability of free hydroxyl groups in the phenolic structure is an important characteristic for the resonance stabilization of free radicals. The enhancement of bioactive phenolic compounds by enzymatic hydrolysis from different cereals was reported by Wojdylo  and Yuan . Different enzymes during bacteria or fungi fermentation, such as alpha-amylase, alpha and beta-glucosidase, beta-glucuronidase, cellulase et al., have been reported to be involved in the lignin remobilization and phenolic compounds contents enhancement . Fermented soybean products have higher amount of phenolic compounds [42, 44]. In Cheonggukjang fermented by
The other reason is the short chain peptides generated by fungi or bacterial protease. Antioxidant activities of peptides have been reported [50-52]. In , five different proteases were used to hydrolyze soybean β-conglycinin and the hydrolysates from three of them had significant enhanced antioxidative activities. Peptide antioxidant activity is related, but not limited, to the amino acid composition and its sequence. In reference , peptides isolated from the antioxidative beta-conglycinin hydrolysate contain histidine, proline or tyrosine residue in their sequences and hydrophobic amino acids, valine or leucine at the N terminus. The constituent amino acids had no antioxidant activity when mixed at the same concentration as the peptides. Anti-oxidative activities of peptides with different amino acid composition or sequences have different antioxidant mechanisms. Reference  studied the anti-oxidative properties of combinatorial tri-peptides. Tri-peptides Tyr-His-Tyr, Xaa-Xaa-Trp/Tyr, and Xaa-Xaa-Cys (SH) had a strong synergistic effect with phenolic antioxidants, a high radical scavenging activity, and a high peroxynitrite scavenging activity, respectively.
2.4. Phytic acid
Phytate is the calcium-magnesium-potassium salt of inositol hexaphosphoric acid commonly known as phytic acid [Figure 3]. Phytate and phytic acid are also referred to as phytin in some literature.
Phytate is the main storage form of phosphorous in soybean. Its content in soybean ranged from 1.00 to 1.47% on a dry matter basis .Phytate is known to be located in the protein bodies, mainly within their globoid inclusions. Phosphorous in the phytate form could not be absorbed by monogastric animals, because they lack phytase, the digestive enzyme required to release phosphorus from the phytate molecule. Phytic acid could form protein-phytate or protein-phytate-protein complexes; these have more resistance to digestion by proteolytic enzymes; thus, utilization of dietary protein is reduced. Also, phytic acid has a strong binding affinity to important minerals such as calcium, magnesium, iron, and zinc. When a mineral binds to phytic acid, it becomes insoluble, precipitates, and is not absorbable in the intestines. In food industry, the presence of phytic acid in high concentration is undesirable. In feed industry, the unabsorbed phytate passes through the gastrointestinal tract of monogastric animals, elevating the amount of phosphorus in the manure. Excess phosphorus excretion can lead to environmental problems such as eutrophication. With the pressure on the swine industry to reduce the environmental impact of pork production, it is important to use feed ingredients that can minimize this influence.
The ability of the molds for oriental fermented soybean food to produce phytase has been investigated. Phytase is the enzyme hydrolyzing phytic acid to inositol and phosphoric acid and thereby removing the metal chelating property of phytic acid. There are two strains of
Galacto-oligosacchrides (GOS) generally represent approximately 4 to 6% of soybean dry matter. In soy meal produced at 10 commercial processing plants in the United States, concentrations of stachyose, raffinose, and verbascose ranged from 41.0 to 57.2, 4.3 to 9.8, and 1.6 to 2.4 mg/g DM, respectively . Oligosaccharides in the carbohydrate fraction, particularly raffinose and stachyose [figure 4], could lead to flatulence and abdominal discomfort for monogastric animals [62-63] because of the deficiency of alpha-galactosidase.
Galacto-oligosacchrides are digested to some extent in the small intestine (76 to 88% for stachyose, 31 to 65% for raffinose, and 32 to 55% for verbascose), resulting in the production of carbon dioxide and hydrogen . In some cases, the accumulation of flatulent rectal gas provokes gastrointestinal distress such as abdominal pain, nausea, and diarrhea. Weanling pigs fed a GOS-free diet supplemented with 2% stachyose or fed a diet containing soy mealhad increased incidence of diarrhea compared with pigs fed a control diet . Additionally, fermentation of GOS has been implicated to have negative effects on nutrient digestibilities and energy availability of soy meal. Roosters fed soy meal with low oligosaccharide concentrations had higher total net metabolizable energy values (2931 kcal/kg dm) than those fed conventional soy meal (2739 kcal/kg DM) . The removal of polysaccharides from soy foods and feed is, therefore, a major factor in improving their nutritive value. To reduce non-digestible oligosaccharides, fermentation with fungi, yeast, and bacteria with alpha-galactosidase secreting ability has been attempted over the years.
The enzyme alpha-D-Gal (alpha-D-galactoside galactohydrolase, EC 18.104.22.168) is of interest for hydrolyzing the raffinose-type sugars found in soybeans.
Alpha-galactosidase has been isolated from plant and microbial sources, and its properties have been documented. In general, alpha-galactosidase acts upon gal-gal bonds in the tetrasaccharide stachyose, releasing galactose and raffinose, and also acts upon gal-glu bonds with the release of sucrose. Sucrose is, in turn, split by invertase, producing fructose and glucose . So, α-galatosidase activity is noticeably dependent on the type of sugar. The type and concentration of the carbon source are known to be nutritional factors that regulate the synthesis of bacterial galactosidase. Reference  found that the existence of glucose and fructose inhibit the alpha-galatosidase expression both in
2.6. Trypsin inhibitor
Protease inhibitors constitute around 6% of soybean protein . Two protein protease inhibitors have been isolated from soybeans. The Kunitz trypsin inhibitor has a specificity directed primarily toward trypsin and a molecular weight of about 21.5 kDa. The Bowman-Birk (BB) inhibitor is capable of inhibiting both trypsin and chymotrypsin at independent reactive sites and has a molecular weight of 7.8 kDa . Trypsin and chymotrypsin, the two major proteolytic enzymes produced in the pancreas, belong to the serine protease class.
Trypsin inhibitors present in soybeans are responsible for growth depression by reducing proteolysis and by an excessive fecal loss of pancreatic enzymes rich in sulfur-containing amino acid which can not be compensated by dietary soy protein . Trypsin inhibitors account for 30-50% of the growth inhibition effect . Rats fed a raw soybean extract from which trypsin inhibitors had been inactivated showed improved growth performance when compared with control rats fed diets containing raw soybeans from which inhibitors had not been inactivated .
Diets with a trypsin inhibitor concentration of 0.77 mg/g and less did not reduce the growth of pigs according to reference . And, research showed that after fermentation with
The increased content of some vitamins or provitamins, both water-soluble and fat-soluble vitamins, such as riboflavin, niacin, vitamin B6, β-carotene et al., which are due to fungal metabolic activities,is one of the healthy and nutritional advantages of fermented soybean products and has been extensively examined.
Vitamin or provitamin formation during tempeh fermentation by
Vitamin K is an essential cofactor for the posttranslational conversion of glutamic acid residues of specific proteins in the blood and bone into γ-carboxyglutamic acid (Gla). There are two naturally occurring forms of vitamin K, vitamins K1 and K2. Vitamin K1 (phylloquinone) is formed in plants. Vitamin K2 (menaquinone, MK) is primarily synthesized by bacteria .Menaquinone (MK) plays an important role in blood coagulation and bone metabolism . Japanese fermented soybean product, Natto, has been regarded as a high content of MK source (about 6- 9 ug/g) and is found in everyday products. Natto produced by a mutated
After fermentation by GRAS microorganisms, the anti-nutritional factors in soybean or soy meal are totally degraded, including oligosaccharides, trypsin inhibitor and phytic acid. Fermentation could also degrade large soy protein into peptides and amino acids, therefore, removing the allergenic effect of soy protein. Nutritional factors are formed during fermentation along with removal of undesirable factors. Functional peptides, such as peptides with ACE inhibitory activity are created by protein degradation. Isoflavones are converted to their functional forms, the aglycones. Antioxidant activity is enhanced, contributed by the increase of short chain peptides and phenolic compounds. Certain vitamins or provitamins are formed such as riboflavin, β-carotene, vitamin K2 and ergosterol. Total nutritional profiles of soybean and soy meal are greatly enhanced by fermentation.