key: cord-0018119-9aqjgbok authors: Kim, Il-Sup; Hwang, Cher-Won; Yang, Woong-Suk; Kim, Cheorl-Ho title: Current Perspectives on the Physiological Activities of Fermented Soybean-Derived Cheonggukjang date: 2021-05-27 journal: Int J Mol Sci DOI: 10.3390/ijms22115746 sha: e242ce17ca187ad23c32bdd6e68efe7fb29c765e doc_id: 18119 cord_uid: 9aqjgbok Cheonggukjang (CGJ, fermented soybean paste), a traditional Korean fermented dish, has recently emerged as a functional food that improves blood circulation and intestinal regulation. Considering that excessive consumption of refined salt is associated with increased incidence of gastric cancer, high blood pressure, and stroke in Koreans, consuming CGJ may be desirable, as it can be made without salt, unlike other pastes. Soybeans in CGJ are fermented by Bacillus strains (B. subtilis or B. licheniformis), Lactobacillus spp., Leuconostoc spp., and Enterococcus faecium, which weaken the activity of putrefactive bacteria in the intestines, act as antibacterial agents against pathogens, and facilitate the excretion of harmful substances. Studies on CGJ have either focused on improving product quality or evaluating the bioactive substances contained in CGJ. The fermentation process of CGJ results in the production of enzymes and various physiologically active substances that are not found in raw soybeans, including dietary fiber, phospholipids, isoflavones (e.g., genistein and daidzein), phenolic acids, saponins, trypsin inhibitors, and phytic acids. These components prevent atherosclerosis, oxidative stress-mediated heart disease and inflammation, obesity, diabetes, senile dementia, cancer (e.g., breast and lung), and osteoporosis. They have also been shown to have thrombolytic, blood pressure-lowering, lipid-lowering, antimutagenic, immunostimulatory, anti-allergic, antibacterial, anti-atopic dermatitis, anti-androgenetic alopecia, and anti-asthmatic activities, as well as skin improvement properties. In this review, we examined the physiological activities of CGJ and confirmed its potential as a functional food. Cheonggukjang (CGJ) is a traditional Korean dish produced by fermenting boiled soybeans rice straw, which naturally contains Bacillus subtilis. Fresh CGJ is prepared by spreading rice straw on boiled soybeans and keeping them warm at 40-50 • C for 2-3 days ( Figure 1A ) [1] [2] [3] [4] [5] . During the fermentation process of CGJ, soy protein is decomposed into amino acids by potent proteolytic enzymes produced by B. subtilis ( Figure 1B) , thus improving digestibility and increasing the vitamin B 2 and calcium contents of the final product [6] . The sticky mucous substance produced during fermentation contains poly-γ-glutamic acid (γ-PGA), the main component, and altered isoflavone compounds. Polyglutamic acid is beneficial to health, as it aids in the absorption of calcium [7, 8] . Isoflavone, the main physiologically active substance in soybeans, improves the absorption and bioavailability of nutrients from the fermented soybeans [2, 9, 10] . [2] and the broad type of traditional fermented soybean products in Asia (B) [5] . found in CGJ [2, 4, 5, 23] . In addition, CGJ reportedly contains higher antioxidant levels than non-fermented soybeans [24] . CGJ also contains peptides that can lower blood pressure by acting as angiotensin I-converting enzyme (ACE) inhibitors [4, 5, 10, 25] . ACE converts angiotensin I to angiotensin II, increasing blood pressure in vivo [26] . Thus, CGJ contains physiologically active substances that have anticancer, antioxidant, and blood pressurelowering activities, as well as hypertension and osteoporosis prevention properties. uous efforts have been directed toward researching fermented foods in Korea, a unified support system for such research has yet to be established at the national level [10, 27, 28] . At present, research is performed sporadically without an integrated database to converge study findings and there is a general lack of comprehensive discussion regarding the health functionality of traditional fermented foods [10, 30] . In this review, we examine recent information on the novel physiological activities of CGJ, including anti-obesity, anti-diabetes, anti-inflammatory, and antimicrobial activities as well as immunostimulatory, neuroprotective, and skin improvement effects based on the following aspects: (1) nutritional properties of CGJ, (2) identification and characterization of microorganisms used in fermentation, and (3) health functionality assessment. Furthermore, this review aims to suggest future directions for study through the examination of CGJ characteristics, with a special focus on health functionality assessment. CGJ is a well-known fermented food mainly produced between fall and early spring in Korea, as shown in Figure 1 . Soybeans, the base ingredient of CGJ, comprises approximately 40% proteins and 20% lipids, resembling meat rather than grains in terms of nutritional values. Soybeans also contain 12% dietary fibers (2.3% soluble dietary fibers and 9.7% insoluble dietary fibers), which is not present in beef, and lacks cholesterol ( Figure 3A ) [1, 2, 10] , which is associated with various diseases [31] . Aside from its unique flavor, CGJ ensures the most effective intake of soybeans, easily providing protein that may be deficient in Korean diets that are mainly composed of cereal grains [4] . Additionally, CGJ has higher protein and fat contents than doenjang (soybean paste) or gochujang (red chili paste) [24, 31] . Hence, it has been an essential source of protein for a long time, along with other paste-based foods, for Koreans with a relatively low protein intake. Different enzymes secreted by CGJ-producing bacteria during fermentation mediate degradation of the soybean outer coat, cell membrane fibers, and intracellular sugars and proteins to improve digestibility and increase the free amino acids contents [32] . During fermentation, proteins are degraded into peptone, polypeptides, dipeptides, and amino acids through protease activities ( Figure 3B ) [33] . β-amylase mediates the degradation of carbohydrates into glucose [34] . The sticky substance generated during CGJ fermentation is the combined product of the polysaccharide fructan and the amino acid polyglutamic acid [4, 5, 33] . There is a substantial increase in the vitamin B 2 level during fermentation; the vitamin B 2 content is 5-to 10-fold higher in steamed than in raw soybeans. Among the substances produced by CGJ bacteria, vitamin K (menaquinone; types K 1 and K 2 ) ( Figure 3C ) is required for the synthesis of proteins involved in blood coagulation [2, 6, 35, 36] . In raw soybeans, vitamin K 1 is present in trace amounts and the vitamin K 2 level is negligible. However, CGJ contains low levels of vitamin K 1 but 5-to 10-fold higher levels of vitamin K 2 than other vegetables [1] [2] [3] [4] 10] . As vitamin K 2 is directly engaged in osteogenesis, vitamin K deficiency may increase the risk of fracture [6, 24, 31, 35, 36] . In a study in Japan, the use of B. subtilis for natto fermentation was reportedly the most effective method of improving vitamin K 2 intake [2, 10, 37] . Thus, the bioactive effects of CGJ can be predicted from the main microbial strain (Bacillus) used for fermentation. The functionality of food products is dictated not only by their nutritional content but also by their ability to prevent and treat diseases [24, 31] . Recent studies have highlighted the functionality of CGJ associated with enhanced blood circulation and intestinal regulation [3] [4] [5] , thus encouraging research more than at any other time in the past [4, 5, 33] . Including CGJ in the diet is ideal to prevent excessive salt intake, as CGJ can be produced without salt and studies have demonstrated the association between excessive intake of refined salt and high incidence of gastric cancer, hypertension, and stroke among Koreans [4, 5, 24, 31] . Soybean fermentation by Bacillus results in the production of various physiologically active substances, microorganisms, and enzymes [38] . Further, fermentation carried out by B. subtilis or B. licheniformis can also exert antimicrobial effects on pathogenic bacteria in the gut, reduce the activities of intestinal bacteria, and mediate the release of harmful substances through adsorption [2, 10] . Novel nutritional, sensory, and bioregulatory functions produced during the microbial fermentation of CGJ, which were initially absent in the base ingredient, have been reported in previous studies [39] [40] [41] [42] [43] [44] [45] [46] [47] [48] [49] . These novel bioactive substances mediate the functions of CGJ, such as thrombolysis [43] , hypertension prevention [41] , improved lipid metabolism [44] , antimutagenic and anticancer activities [24] , and antimicrobial effects [45] . Studies on CGJ may be broadly divided into two categories: (1) those focusing on enhancing product quality based on changes in nitrogen and aromatic components and the proximate composition of CGJ upon treatment with different bacterial strains or modifications to the fermentation process, and (2) those related to the functions of physiologically active substances in CGJ [3, [19] [20] [21] [22] . In addition to the nutritional values of the base ingredient, CGJ contains substances with known beneficial health-promoting properties, such as dietary fiber, phospholipids, isoflavones (genistein, daidzein, etc.), phenolic acids, saponins, trypsin inhibitor, and phytic acid, as noted above [2, 4, 5, 10] . These bioactive agents produced during microbial fermentation are involved in mediating anticancer effects (against breast, colorectal, lung cancer, etc.) [39] , as well as preventing osteoporosis [35] , geriatric dementia [40] , diabetes [41] , heart diseases [42] , and arteriosclerosis [42] . The following section focus on studies reporting bioactivities of CGJ to determine the potential applicability of CGJ as a healthy functional food. The antithrombotic effect of CGJ was mostly verified in studies using Sprague-Dawley (SD) rats or in vitro experiments. During wound repair, thrombin is activated in the blood by a complex cascade mechanism in vivo, facilitating the conversion of fibrinogen to fibrin and subsequently forming a three-dimensional lattice polymer with platelets and insoluble stereoscopic structures [50] . Fibrinogen is composed of three different pairs of polypeptide chains linked by disulfide bonds. The molecular weights of the α, β, and γ polypeptide chains are 64,500 Da, 55,000 Da, and 47,000 Da, respectively. Thrombin hydrolyzes Arg-Gly bonds at the N-terminus of the α and β chains, and fibrin monomers and fibrinopeptides A and B, which consist of 16 and 14 amino acids, respectively, are cleaved. Subsequently, the binding site where fibrin can horizontally aggregate is exposed, allowing fibrin monomers to aggregate by hydrogen and hydrophobic bonding, forming a soft clot. This soft clot is converted into a hard clot by an enzymatic reaction. Blood clots that are generated to repair small wounds in vivo are not broken down but remain in circulation after wound repair and are dissolved by thrombolytic agents that cause fibrinolysis and clot retraction [51] [52] [53] [54] [55] . The clotting and dissolution of blood clots are always in equilibrium in vivo [50] . However, homeostatic imbalances due to various triggers result in the accumulation of blood clots in small blood vessels, including cerebral blood vessels, which block blood circulation and impede the supply of nutrients and oxygen to cells and tissues [56, 57] . In such cases, blood pressure increases. Thus, when blood clots accumulate in cerebral blood vessels, cerebral venous thrombosis can occur, leading to hemiplegia [58] . Moreover, when cerebral blood vessels rupture, cerebral hemorrhage can occur [58] . Bleeding in the space between the brain and skull (subarachnoid hemorrhage) is fatal [59] . Impaired blood flow in the cardiovascular system can lead to cardiac failure or arrest, causing death [60] . According to a report on current mortality rates, intravascular disorders are the leading cause of death, accounting for approximately 40% of all deaths [61] . Therefore, many studies have investigated strategies of thrombus removal. After consumption of CGJ, daidzein is partly converted to equol by human gut bacteria, including various bifidobacteria (Lactobacillus sp., Leuconostoc sp., etc.) [62] . Isoflavones (aglycones, genistein, daidzein, and equol; Figure 2 ) can permeate the blood-brain barrier and exert neuroprotective effects by regulating tolllike receptor (TLR) and nuclear factor-kappa B (NF-κB) signaling pathways, which lead to the release of proinflammatory cytokines and brain insulin ( Figure 4 ) [4, 63] . . Potential mechanism of CGJ in memory function. The boxed panel contains a schematic diagram representing the metabolism of isoflavones in the intestine. Soybean-fermented CGJ components actively influence cellular metabolism in the liver and brain, exerting positive effects through the gut-intestine-microbiome-liver-brain axis. Improved cellular metabolism in the hippocampus decreases β-amyloid accumulation, insulin resistance, neuroinflammation, and memory impairment in the brain. This result suggests that a diet containing CGJ, in part, protects against type 2 diabetes, Alzheimer's disease, and post-stroke symptoms [3, 63] . ISO, isoflavone; ISO-A, isoflavone aglycone; ISO-G, isoflavone glycoside; ISO-g, isoflavone glucuronide; ISO-s, isoflavone sulfate; sHPA-axis, short hypothalamic-pituitary-adrenal axis; LPH, membranebound lactase phlorizin hydrolase; MRP; (multi-drug resistance-related protein); γ-PGA, poly-γ-glutamic acid; SCFA, short-chain fatty acids. Figure adapted Streptokinase, urokinase, and tissue plasminogen activator (TPA)-which are widely used to treat thrombosis-can lead to systemic bleeding and other side effects, are expensive, and may not be amenable to oral administration, except for urokinase [64, 65] . Recently, several studies have reported the use of oral agents to increase thrombolysis, alone or in combination with intravascular drugs. In particular, thrombolytic enzymes have been produced and isolated from CGJ and reportedly possess thrombolytic activi-ties that are three to four times stronger than that of natto. According to a recent report, the alkaline thrombophilic serine protease produced by B. licheniformis and isolated from CGJ demonstrates similarities and differences from known enzymes, including nattokinase [66, 67] . Interestingly, this protease exhibited excellent thrombolytic activity and a plasminogen activator effect [68] . In addition, approximately 90% and 45% of the enzyme's thrombolytic activity was maintained after heat treatment at 100 • C for 5 min and 30 min, respectively [67, 69] . Therefore, CGJ has potential applications as a thrombolytic agent. Furthermore, CGJ prepared by fermentation with Bacillus spp. also produces different molecules (Tables 1 and 2) , including fibrinolytic enzymes, bioactive peptides (VE, VL, VT, and LE), amino acids (Tyr, Arg, and Thr), γ-PGA, conjugated phospholipids (i.e., CLA), isoflavones, oligosaccharides, phytic acid, lignan, and saponin [4, 5, 68, [70] [71] [72] [73] [74] [75] [76] , which can enhance the functionality of CGJ. Hypertension is a prevalent condition that affects an estimated 15-20% of middle-aged and elderly individuals and influences the entire cardiovascular system, causing heart and kidney diseases [77, 78] . Cerebral hemorrhage, which is a complication of hypertension, is a chronic degenerative disease with a high mortality rate [77, 78] . The World Health Organization defines hypertension as a condition in which the maximum and minimum blood pressures are ≥160 mmHg and ≥95 mmHg, respectively [79] . Essential hypertension, which accounts for ≥80% of hypertensive cases, has unknown causes, whereas secondary hypertension is due to underlying diseases [80] . Hypertension mainly involves the physiological and biochemical mechanisms associated with the renin-angiotensin-aldosterone system (RAAS) [80, 81] . Renin, which is secreted by the kidney in response to a decrease in blood pressure, converts angiotensinogen to angiotensin I [81] . This decapeptide is then converted by ACE to angiotensin II, which has the highest blood pressure-elevating activity among all components of the RAAS. As a result, the smooth muscles of blood vessels contract, and blood pressure increases [81] . Moreover, ACE inactivates bradykinin, a vasodilator, further contributing to increased blood pressure [81] . When blood pressure decreases, angiotensin II stimulates the adrenal glands to secrete a hormone called aldosterone. This hormone acts on the kidneys to promote sodium absorption, resulting in increased blood pressure [81] [82] [83] . ACE inhibitors, such as captopril and enalapril, are used to treat hypertension; however, these are chemically synthesized products with safety issues and several side effects [84] . Thus, ACE inhibitors based on peptides derived from natural products are continuously and vigorously being investigated. In particular, casein from animal products and plant proteins from soybeans inhibit ACE activity in vitro [85, 86] . Studies investigating functional foods with positive effects on hypertension are actively being conducted. Among these, CGJ is known to contain amino acids with blood pressurelowering effects ( Figure 5 ) [33, 87] . CGJ also contains polyglutamic acid, which has been shown to enhance the absorption of calcium and drugs in the small intestines of rats, particularly the anticancer drug paclitaxel [87, 88] . The effects of steamed soybeans and CGJ powder on blood pressure and lipid metabolism in spontaneously hypertensive rats (SHRs) and the anti-atherosclerotic effects of soy products subjected to heat treatment and fermentation have been investigated [48] . The consumption of steamed soybeans and CGJ, in which casein is replaced as the protein source, contributes to the prevention of cardiovascular diseases by managing hypertension and hyperlipidemia, which are risk factors for atherosclerosis [4, 5, 48] . In another study, the effect on systolic blood pressure was investigated after consumption of a diet supplemented with soy protein hydrolysate prepared by ultrafiltration. SHRs fed such a diet exhibited significantly decreased blood pressures after 5 weeks, indicating that the soy protein may have lowered the activity of local ACE in the thoracic aorta [89, 90] . . Soybean-protein-mediated blood pressure regulation via angiotensin-converting enzyme (ACE). Bradykinin, a peptide, a component of the kallikrein-kinin system associated with a blood pressure-lowering effect, is degraded by ACE.ACE inhibition has been postulated as a strategy for treating hypertension, which is an important factor associated with numerous diseases conditions, such as ischemic heart-diseases and cerebrovascular events [33] . ↑, increase; ↓, decrease. Figure adapted from Chattet, L. et al. [33] . In addition to hypertension, a high plasma cholesterol level, especially low-density lipoprotein (LDL) cholesterol, is a major risk factor for atherosclerosis and myocardial infarction [91] . Cholesterol in the blood serves as a source of lipids for blood clot formation [91] . Thus, maintaining normal plasma cholesterol levels is crucial. Studies examining dietary components that lower serum cholesterol levels are in progress. In a previous study, CGJ and freeze-dried steamed soybean powder were added to the diet of the experimental group, whereas casein or another source was included in the diet of the control group. After several weeks, the total serum cholesterol concentration of the experimental group was significantly lower than that of the control group. In addition, the experimental group exhibited a significant decrease in serum and hepatic cholesterol concentrations [40, 49, 92, 93] . Enhanced excretion of bile acids in feces likely contributed to lowering serum cholesterol, triglyceride, and hepatic cholesterol concentrations (Table 3 ) [49, 92, 93] . These results are consistent with those of several studies in which soy protein decreased serum cholesterol concentrations in rats fed a high-cholesterol diet [4, 5, 94, 95] . It is assumed that differences in the effects of plant and animal proteins on lipid metabolism are due to respective differences in protein composition and amino acid ratio, particularly the arginine-to-lysine ratio. Lowering total cholesterol and triglyceride levels by controlling lipid metabolism, such as lysophosphatidylcholines and phosphatidylcholines [49] The anticancer properties of fermented soy-based foods have also been observed for raw soybeans and decomposed or newly synthesized compounds during soybean fermentation [68] . Such properties have been attributed to protease inhibitors, phytic acids, and isoflavones derived from soybeans [33, 96] . Phytic acid is a natural compound consisting of six phosphate groups bound to inositol. It mainly acts as storage for phosphorus in plants [33, 97] . Phytic acid is known as a harmful substance that suppresses the absorption of cations in the intestine by chelation, especially divalent cations in the following order of decreasing affinity: copper, zinc, cobalt, manganese, iron, and calcium [98, 99] . However, the chelating activity of phytic acid also contributes to its anticancer effects and other physiological activities that prevent free radical-mediated diseases, such as inflammation [98, [100] [101] [102] [103] . Isoflavone, which contributes to the astringency of soybeans, has already been verified to have anticancer effects in several epidemiological, animal, and in vitro studies. It is known to be particularly effective in preventing breast, prostate, and colon cancers (Table 4; Figure 6 ) [104] [105] [106] [107] [108] [109] [110] [111] . In addition, isoflavones were confirmed to inhibit the enzyme system involved in the conversion of xenobiotics into carcinogens [109] [110] [111] . For example, Bacillus sp.-derived CGJ enhances anticancer activity by promoting γ-PGA and isoflavone derivate (genistein (GS) and daidzein(DZ))-mediated apoptosis; inhibiting pro-proliferative functions leading to DNA fragmentation and inactivation of poly (ADPribose) polymerase (PARP), caspase 3, cyclooxygenase 2 (COX-2), and inducible nitric oxide synthase (iNOS); and activating γ-PGAand isoflavones (GS and DZ)-mediated 5 -adenosine monophosphate-activated protein kinase (AMPK) in HT-29 human colorectal cancer cells (Table 4 ; Figure 6 ) [9, [104] [105] [106] [107] [108] [109] [110] [111] . Table 4 . Anticancer effects of soybean-derived CGJ. Raw soybean B. subtilis MYCO10001 B. subtilis ATCC 21228 Enhancing levels of isoflavones (daidzein and genistein), their derivatives (isoflavone-β-glucosides and isoflavone-aglycones), or succinyl derivatives (succinyl-β-daidzin and succinyl-β-genistin), and β-glucosidase [104, 105] Raw soybean L. acidophilus KCTC 3925 L. rhamnosus KCTC 3929 Producing aglycone-formed isoflavones and exopolysaccharide [106] Raw soybean B. subtilis Enhancing isoflavone levels by controlling CGJ fermentation conditions, including temperature, time, germination, and osmolarity [107] Healthy Korean men (age range 21-29 years) Bacillus sp. Improving human health function of isoflavones following CGJ ingestion [108] HT-29 human colorectal cancer cells Bacillus sp. FBL-2 Promoting poly-γ-glutamic acid (γ-PGA)-mediated apoptosis inhibiting pro-proliferative functions leading to DNA fragmentation, and inactivation of poly (ADP-ribose) polymerase (PARP), cyclooxygenase (COX-2), and inducible nitric oxide synthase (iNOS); activating 5'-adenosine monophosphate-activated protein kinase (AMPK) [109, 110] Raw soybean B. subtilis 168 Producing arginase, an arginine-degrading and ornithine-producing enzyme, used to treat arginine-dependent cancer [111] Figure 6. The mechanism of daidzein (DZ) and genistein (GS) regulation at transcriptional and translational levels in HT29 colon cancer cells. GS represses expression of phosphorylated p38 (p-p38), and matrix metalloproteinases (MMP2 and MMP9) to inhibit HT29 cell proliferation, upregulates Bax/Bcl-2, caspase-8, and caspase-3 activity to enhance HT29 cell apoptosis, reverses epithelial-mesenchymal transition (EMT) through regulation of EMT markers and regulates Wnt signaling pathways by increasing Wnt inhibitory factor 1(WIF1) to block HT29 cell migration and invasion. Additionally, DZ and GS inhibit expression of phosphatidylinositol 3-kinase (PI3K) and forkhead box O3 (FOXO3) to suppress HT29 cell proliferation and decrease lipid droplet accumulation to provoke HT29 cells apoptosis [9] . ADRP, adipose differentiation-related protein; Bax2, apoptosis regulator belonging to Bcl2 family; Bcl2, anti-or pro-apoptosis regulator; c-Myc, multifunctional transcriptional factor; FARP1, RhoGEF and pleckstrin domain-containing protein 1; Fas, fatty acid synthetase; FOXC1, forkhead box C1; MMP2; matrix metalloproteinase 2; MTTP, microsomal triglyceride transfer protein; Notch-1, member of type 1 transmembrane protein family; p65 (RELA), transcription factor 65; p-NF-κB, phosphorylated nuclear factor kappa B; PPARγ, peroxisome proliferator-activated receptor-gamma; Snail/Slug, master regulatory transcription factor; Tip-47, lipid droplets-associated protein; TWIST, time without symptoms of diseases and subjective toxic effects of treatment; UCP2, uncoupling protein 2; ZEB, zinc-finger E-box-binding homeobox protein as a transcription factor; ↑, induction; ↓, repression; →, activation; , inhibition. Soybeans are recognized by the National Cancer Institute as an anticancer product [112] . Compared with non-fermented soybeans, CGJ has additional beneficial substances, such as proteolytic enzymes, polymeric nucleic acids, browning substances, and polyglutamic acid [4, 5, 39] . Particularly, trypsin inhibitors present in CGJ are known to have anticancer effects [5] . The results of the Ames test comparing the antimutagenic effects of raw or steamed soybeans and CGJ demonstrated that CGJ exerted stronger antimutagenic effects than raw or steamed soybeans [39, 113, 114] . The fermented products effectively showed antimutagenicity as a result of the fermentation process of CGJ. Soybeans are also known to have antioxidant effects. The representative antioxidants present in soybeans and CGJ include chlorogenic acid, isochlorogenic acid, caffeic acid, isoflavones, phenolic acids, tocopherols, amino acids, peptides, and nitrogen-containing compounds such as aromatic amines, phospholipids, and saponins [4, [115] [116] [117] [118] . Genistein, the major isoflavone in soybeans, has been shown to have anticancer effects on breast and genital cancers associated with genistein's antioxidant activity [119] . Specifically, genistein was found to inhibit the formation of superoxide anion and exert antioxidant effects by removing the tumor-promoting factor hydrogen peroxide, which remarkably prevented the production of 8-hydroxy-2 -deoxyguanosine from oxidative damage to DNA under ultraviolet (UV) irradiation or in the Fenton reaction system. The antioxidant properties of soybeans confer resistance to oxidative stress in vivo and are expected to decrease or prevent the occurrence of various diseases or aging caused by oxidation in vivo ( Figure 7 ) [4, 20, 24, 31, [115] [116] [117] [118] [120] [121] [122] [123] [124] . In addition, powdered CGJ extracted with alcohol was shown to possess stronger antioxidant properties than butylated hydroxyanisole (BHA) ( Table 5 ) [31, 124] . Using a comet assay and lipid peroxidation measurement in NIH/3T3 cells and mice, an ethanol extract of CGJ prevented oxidative stress. Other in vitro experiments evaluating radical and nitrite scavenging activities and peroxide levels indicated that CGJ could potentially induce antioxidant effects. In addition to antioxidant effects, CGJ has been shown to have anti-inflammatory effects. As shown Table 5 and Figure 7 , CGJ improves anti-inflammatory activity by regulating the expression of genes associated with NF-κB-mediated inflammation and by increasing the production of hyaluronic acid in mice [20, 44] , RAW264.7 macrophage, HaCaT cells [125] , and SD rats [4, 120] . Validation of the safety of CGJ extracts through biological experiments can render the use of such extracts possible as natural antioxidant and anti-inflammation agents in food products such as edible oils. Protective mechanism against apoptosis, inflammation, and oxidative stress, and the membrane potential (ψ) of genistein induced by soybean-fermented CGJ to reduce cell death. Nuclear signaling through estrogen receptors (ERs) α and β, peroxisome proliferator-activated receptor gamma (PPARγ), and aryl hydrocarbon receptor (AhR) upregulates the expression of antiapoptotic genes, including growth factors and their receptors and antiapoptotic members of the B-cell lymphoma 2 (Bcl2) family. Conversely, the proapoptotic Bcl2 members, including Bad and Bax, are downregulated. Nuclear action mode also inhibits inflammation by suppressing nuclear factor kappa B (NF-κB) and activating NF-κB inhibitor (IκB). This, in turn, decreases the expression of genes associated with several inflammation mediators. Additionally, nuclear signaling also stimulates mitochondrial transcription factor A (TFAM) to produce mitochondrial energy and maintain membrane potential. Extranuclear signaling through membrane association (ERs and G protein-coupled ER (GPER)) and interaction with GFr stimulates multiple intracellular signaling pathways to prevent inflammation, apoptosis, and oxidative stress through NF-κB, c-Jun N-terminal kinase (JNK), and NF-κB repressing factor (Nrf), respectively. In addition, genistein acts as an electron acceptor to reduce levels of reactive oxygen and neutralize reactive oxygen species in the cell. [20] . Aβ, beta-amyloid; AhR, aryl hydrocarbon receptor; AIF, apoptosis-inducing factor; Akt, protein kinase B; ATP, adenosine triphosphate; Bad, bipolar affective disorder; Bax, Bcl2-associated X; Bcl2 and BclXL, anti-apoptotic multi-domain protein; BDNF, brain-derived neurotrophic factor; c-Jun, transcription factor involved in extensive pathophysiological process; Cox2, cyclooxygenase 2; eNOS, endothelial nitric oxide synthase; ERK1/2, extracellular regulated kinases 1 and 2; ERs, estrogen receptors; GDNF, glial cell line-derived neurotrophic factor; GFr, growth factor receptor; GPX, glutathione peroxidase; GPER, G protein-coupled ER; ERK, extracellular signal-regulated kinase; GSH, glutathione (reduced form); HO1, heme oxygenase 1; IκB, inhibitory-κB; IL-1β, interleukin 1 beta; iNOS, inducible NOS; JNK, c-Jun N-terminal kinase; Keap1, Kelch-like ECH-associated protein 1; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinase; MEK, MAPK-kinase; MMP, matrix metalloproteinase; NF-κB, nuclear factor-κB; NGF, nerve growth factor; nNOS, neuronal NOS; NOX, nitric oxide; Nrf2, nuclear factor E2-related factor 2; PI3K, phosphoinositide 3-kinase; PKA, protein kinase A; PPARγ, peroxisomeproliferator-activated receptor gamma; SOD, superoxide dismutase; TFAM, mitochondrial transcription factor A; TNF-α, tumor necrosis factor alpha; Trk, tropomyosin receptor kinase; VEGF, vascular endothelial growth factor; ↑, upregulation; ↓, downregulation; →, activation; , inhibition. Figure adapted from Schreihofer, D.A. and Oppong-Gyebi, A. [20] . Increasing levels of total phenolics, isoflavones (isoflavone-malonylglycoside, isoflavone-acetylglycoside, and isoflavone-aglycone), and amino acids; exhibiting highest amounts of palmitic acid, stearic acid, and linoleic acid; elevating DPPH, ABST, FRAP, and superoxide dismutase (SOD)-like activity; augmenting levels of calcium, iron, sodium, and zinc [24, 122, 123] Male Sprague-Dawley rats Bacillus sp. Inducing antioxidative mechanism by redox homeostasis via inactivation of nuclear factor kappa B (NF-κB) and modulating the expression of genes involved in NF-κB-related inflammation [3, 120] C57BL/6J mice B. subtilis HCD02 B. amyloliquefaciens EMD17 B. amyloliquefaciens MJ1-4 Producing total phenolics and flavonoids, including quercetin (quercetin glycoside) Enhancing Nrf2-mediated transcription of gene encoding antioxidant molecules (GSH) and enzymes (i.e., NQQ1, GSH reductase, HO-1, γ-glutamylcysteine ligase) → improving DPPH, FRAP, ABST, and TBARS activity [3, 121] Raw soybean B. subtilis Enhancing tyrosinase inhibitory activity through arbutin production [124] Anti-inflammatory activity Mice L. acidophilusKCTC3925 Suppressing serum triglyceride levels; increasing serum HDL-C, GOT, GPT, and leptin levels; downregulating expression of COX-2, IL-4, and ICAM1-1 genes [44] RAW 264.7 macrophage and HaCaT cells B. subtilis Inhibiting the expression of lipopolysaccharide-induced iNOS and COX-2 protein; suppressing TNF-α, IL-6, and prostaglandin E2 secretion; inducing the production of hyaluronic acid and the expression of filaggrin and serine palmitoyltransferase [125] Male Sprague-Dawley rats Bacillus sp. Suppressing NF-κB-related activities of genes associated with inflammatory proteins, including iNOS, COX-2, and vascular cell adhesion molecule-1 (VCAM-1) [3, 120] 3. The anti-inflammatory effect of CGJ was confirmed by measuring the levels of inflammatory cytokines in RAW264.7 macrophages after treatment with CGJ, revealing upregulated. NF-κB activity and relevant gene expression [126, 127] . Additionally, an anti-allergic effect of CGJ on asthma and atopic dermatitis was demonstrated through assessment of ear edema and passive cutaneous anaphylaxis in mast cells [128] . CGJ is also known to exert an inhibitory effect on apoptosis, based on the measured activities of thymocytes and splenocytes in BALB/cByJ mice [126] . Further, CGJ was shown to protect pancreatic beta and hippocampal cells from apoptosis due to temporary arterial occlusion that reduced levels of proinflammatory cytokines. In studies involving immunosuppressed male C57BL/6 mice or human primary immune cells, a positive effect on immune activity was demonstrated upon treatment with raw materials (i.e., isoflavones) extracted from CGJ [129, 130] . Furthermore, treatment of MCF7 breast cancer cells with an ethanol extract of CGJ exerted a potential preventive effect on breast cancer via inhibition of the inflammatory gene expression (Table 6 ) [131] . Moreover, CGJ did not induce cytotoxicity in Institute for Cancer Research (ICR) mice [132] . Table 6 . Immunostimulatory activity of soybean-derived CGJ. Strain Used in Fermentation Action Mode Reference Increasing levels of functional polysaccharides Activating mRNA expression of nitric oxide (NO) synthase and tumor necrosis factor-α (TNF-α) by inducing nuclear factor-kappa B (NF-κB) → increased production of NO and TNF-α [126, 127] Mast cells - inhibiting degranulation of mast cells [128] Male C57BL/6 mice B. amyloliquefaciens CJ3-27 B. amyloliquefaciens CJ1526 B. subtilis CJ1553 Enhancing T helper type-1-mediated immune response by upregulating production ratio of IFN-γ vs. IL-4 and IgG2a vs. IgG1 in T and B cells; improving enhanced splenic natural killer cell activity → activating humoral and cellular immunity to Th1 response [129] Human primary immune cells -Modulating isoflavone-derived immunostimulatory activity by enhancing cell proliferation, nitrite, and transcriptional activation of TNF-α, IL-6, iNOS, and COX-2 gene [130] Male BALB/cByJ mice B. subtilis Improving lymphocyte proliferation, natural killer cell activity, and white blood cell population through activation of NO and immunostimulatory cytokines (IL-6 and IL-12) [126] Decreasing growth of breast cancer MCF7 cells via upregulation of TGFβ1 and SMAD3, downregulation of inflammation-related genes (CSF2, CSF2RA, and CSF3), and differential expression of the genes encoding chemokines (CCL2, CCL3, CCL3L3, CXCL1, and CXCL2) → preventing breast cancer by TGFβ-dependent signaling mechanism and inhibiting inflammation [131] ICR mice B. subtilis MC31 L. sakei 383 Not inducing any specific toxicity in liver and kidney organs ICR at dose 100 mg/kg body weight/day as no observed effect level [132] 3. Among the studies investigating the health benefits of CGJ, most studies have focused on anti-obesity and anti-diabetic effects. An anti-obesity effect was reported in studies using high-fat diet-fed C57BL/6J mice, as well as SD and Wistar rats. CGJ intake was found to improve blood lipid patterns, while increasing and decreasing the expression of genes related to lipid oxidation and accumulation, respectively (Table 7) [9, 41, 42, 44] . Studies using diabetes-induced SD rats, as well as C57BL/KsJ-db/db and KK-Ay/TaJcl mice, reported that CGJ reduced hyperlipidemia diabetic rats, while increasing levels of enzymes involved in liver glucose metabolism, and improving insulin sensitivity in peripheral tissues (Table 7 ; Figure 8 ) [4, 9, 133, 134 ]. An extract of CGJ was shown to inhibit lipid accumulation in preadipocytes (3T3-L1 cells), exert a positive effect on glucose-induced insulin secretion in beta cells (Min6 cells), and increase cell survival [135] [136] [137] . The key CGJ substances associated with these health benefits are likely isoflavone and isoflavonoid aglycone [133, [138] [139] [140] [141] [142] . Anti-obesity and anti-diabetes activity of daidzein (DZ), genistein (GS), and its metabolite (6-hydroxydaidzein; 6-HD) through lower lipogenesis, liver oxidative stress, hyperglycemia, urinary glucose secretion, insulin tolerance, and weight; and decreased levels of triacylglycerol (TG), low-density lipoprotein (LDL), free fatty acids (FFAs), fasting blood glucose (FBG), and plasma insulin [9] . ACC, acetyl-CoA carboxylase; ADRP, adipose differentiation-related protein activator protein 1; AMPK, adenosine 5 -monophosphate-activated protein kinase; Akt, protein kinase B (serine/threoninespecific protein kinase); BMI, body mass index; C/EBP-α, CCAAT/enhancer-binding protein-alpha; ERK1/2, extracellular signal-regulated kinase 1 and 2; FAS, fatty acid synthetase; GLUT4, glucose transporter type 4; gp91 (NOX2), NADPH oxidase subunit; ICAM-1, intracellular adhesion molecule 1; IL-1, interleukin 1; ISN1, inosine 5 -monophosphate (IMP)specific 5 -nucleotidase; MCP1, monocyte chemoattractant protein 1; MDA, malondialdehyde; MGO, methylglyoxal; NFIL3, nuclear factor interleukin-3-regulated protein; NF-κB, nuclear factor-kappa B; p-AMPK, phosphorylated adenosine 5 -monophosphate-activated protein kinase; p-ERK, phosphorylated extracellular signal-regulated kinase; PI3K, phosphatidylinositol 3-kinase; PKA, protein kinase A; PPARα, peroxisome proliferator-activated receptor-alpha; PPARγ, peroxisome proliferator-activated receptor-gamma; SCD1, stearoyl-CoA desaturase 1; TBARs, thiobarbituric acid reactive substances; UCP1, uncoupling protein 1; TNF-α, tumor necrosis factor-alpha; ↑, enhancing; ↓, lowering; →, activation; , inhibition. Figure adapted Enhancing decrease in glycosylated hemoglobin level, improvement in insulin sensitivity, and decrease in serum glycerides and low-density lipoprotein-cholesterol levels Activating peroxisome proliferator-activated receptor-γ (PPARγ); regulating glucose uptake via AMP-activated kinase stimulation [3, 134] C57BL/KsJ-db/db mice B. subtilis Improving insulin resistance and hyperglycemia in type 2 diabetic animals that are partly medicated by regulating hepatic glucose enzymes, including glucokinase, glucose-6-phosphatase, and phosphoenolpyruvate carboxykinase), hepatic glycogen content, and insulin sensitivity in peripheral tissues [133] Type 2 diabetic male rats B. lichemiformis Enhancing glucose-stimulated insulin secretion during hyperglycemic clamp through regulation of hepatic insulin signaling pathway (i.e., Akt and AMPK) [136, 137] 3T3-L1 cell B. subtilis Stimulating translocation of glucose transporter-4 into the plasma membrane through phosphorylation of insulin receptor substrate-1 and Akt → enhancing glucose utilization via activating insulin signaling and stimulating peroxisome proliferator-activated receptor (PPAR-γ) activity by increasing isoflavonoid aglycones (especially daidzein) in adipocytes Improving glucose-stimulated insulin secretion by increasing levels of small peptides with low polarity in insulinoma cells [145] With respect to the anti-obesity effects of CGJ, plasma apoB levels were significantly reduced in comparison to the placebo group when overweight or obese individuals were administered 26 g per day of freeze-dried CGJ powder for 12 consecutive weeks [143] . The visceral fat area and apo B/apo A1 ratio also displayed a decreasing trend, indicating that CGJ exerted an anti-atherogenic effect. Additionally, when overweight or obese individuals were administered 35 g per day of freeze-dried CGJ powder for 12 consecutive weeks in a crossover study, body fat ratio, waist circumference, and waist-to-hip ratio in females were significantly reduced, as well as the level of high-sensitivity C-reactive protein (hs-CRP), a risk factor of arteriosclerosis [144] . Moreover, in both male and female subjects, the apo B/apo A1 ratio was a significantly decreased during the CGJ-intake period, suggesting that long-term consumption of CGJ could improve body composition and cardiovascular risk factors in overweight or obese individuals. In another study conducted with adults with mild hyperglycemia, consumption of 20 g of freeze-dried CGJ and red ginseng CGJ for 8 weeks led to significantly decreased levels of fasting blood glucose, total cholesterol, and LDL-cholesterol, indicating that CGJ positively impacted hyperglycemia by lowering blood glucose levels and improving blood lipids in individuals with impaired glucose tolerance ( Figure 8 ) [9, 93, 135, 145] . ICR mice administrated CGJ exhibited a beneficial effect on neurodegenerative disease assessed by passive avoidance and cognitive tests, which was associated with induced secretion of nerve growth factors [4, 146] . CGJ also exerted a neuroprotective effect in C57BL/6J mice by improving abnormal cognitive function [4, 121] , as shown in Table 8 . Moreover, CGJ improved neuroprotection by enhancing tyrosine inhibitory activity via arbutin production [124] . Figure 4 illustrates the predicted brain mechanism of CGJ in memory function [4, 63] . Thus, dietary CGJ (approximately 20-30 g/day) protected against type 2 diabetes and brain diseases, including Alzheimer's disease and post-stroke symptoms [4, 10] . The results of studies using SD rats [147] [148] [149] , C57BL/6J mice [150] , senescenceaccelerated mouse prone 6 [35, 40] , SD rats [35, 147] , OVX mice [151] , and MC3T3-E1 subclone 4 (CRL-2593) cells [36, 152] suggest that CGJ helps increase bone mineral content and growth hormone secretion, and facilitates growth by activating various metabolites, including vitamin K, isoflavones, flavonoids, and phenolics, and by regulating signaling pathways, such as the inactivation of NF-κB and dephosphorylation of mitogen-activated protein kinase (MAPK). Additionally, the administration of CGJ in hysterectomized C57BL/6 mice and SD rats was shown to enhance bone health and prevent postmenopausal osteoporosis (Table 8 ). Based on these results, Figure 9 shows a predicted mechanism for the anti-osteoporotic activity of CGJ in cells (MG-63, MC3T3-E1, Saos-2, and bone marrow cells), and rats/mice [9] . Decreasing the number of dead cells in the granule cell layer of the dentate gyrus; suppressing acetylcholinesterase activity; activating nerve growth factor (NGF) and its receptor signaling pathway, including TrkA high affinity receptor and p75 low affinity receptor; downregulating Bax/Bcl2 and caspase 3 expression; promoting increase in superoxide dismutase activity and decrease in lipid peroxidation [146] ICR mice B. subtilis B. licheniformis Modulating the gut-microbiome-brain-axis, brain insulin sensitivity, and neuroinflammation by producing bioactive peptides, dietary fiber, poly-γ-glutamic acid (γ-PGA), and isoflavone aglycones [3] C57BL/6J mice B. subtilis HCD02 B. amyloliquefaciens EMD17 B. amyloliquefaciens MJ1-4 Inhibiting growth of mouse hippocampal HT22 and human neuroblast-like SHSY5Y cells Reducing the frequency of behavioral dysfunction induced by D-galactose; improving cognitive abnormal function [3, 121] Raw soybean B. subtilis Enhancing tyrosinase inhibitory activity through arbutin production [124] Anti-osteoporotic effect Senescenceaccelerated mouse prone 6 Increasing bone mineral density (BMD) and relative bone length; enhancing osteopontin reactivity; upregulating expression of Alp, Col1a1, Fak, Bmp2/4, Smad1/5/8, and Runx2; downregulating expression of Rnakl and Nfatc1; decreasing Cathepsin K level; increasing osteoprotegerin/Rankl ratio [35, 40] MC3T3-E1 subclone 4 (CRL-2593) cells B. subtilis KCTC 12392BP Suppressing osteoclast formation and increasing cell proliferation, cellular alkaline phosphatase activity, osteocalcin, and calcium deposition by upregulating the ratio of osteoprotegerin/receptor activator of NF-κB ligand; promoting bone health [36] Female/male Sprague-Dawley rat B. subtilis Preventing bone loss; increasing BMD, bone mineral content (BMC), and trabecular number [35, 147] Female sham-operated and OVX mice B. subtilis Enhancing various isoflavone metabolites, including intact isoflavones, 3-hydroxygenistein, genistein 4 -sulfate, and equol 7-glucuronide → promoting bone health of postmenopausal women [151] Male/female Sprague-Dawley rat B. subtilis Accumulating total flavonoids, phenolics, and isoflavones; increasing spine BMD and femur BMC; stimulating growth hormone (GH) secretion, which leads to activation of the GH receptor downstream signaling pathway via induction of phosphorylation of Akt and Erk, but not STAT5 [147] [148] [149] C57BL/6J mice B. amyloliquefaciens KCTC11712BP Increasing BMD through upregulation of bone morphogenic protein 2 (Bmp2) and osteopontin in bone tissues Increasing isoflavone-mediated osteogenesis via the Bmp2 signaling pathway; reducing receptor activator of NF-κB induced by NF-κB inactivation and MAPK dephosphorylation [150] Osteoblastic cell line MC3T3-E1 -Producing vitamin K 1 and K 2 Stimulating osteoblast mineralization mechanism by elevating transcriptional ratio of osteoprotegerin and the receptor activator of NF-κB ligand [36, 152] Comparative studies on the effects of CGJ intake and massage on skin improvement have been conducted since 2016. The effects of CGJ diet and back massage on the skin and body shape of middle-aged women in their 40s and 50s were assessed, revealing that the individuals in the CGJ diet group experienced decreased melanin levels and red spots on facial skin, as well as reduced body weight and body fat ratio and increased muscle mass [153, 154] . The effects increased even further upon combining the CGJ diet with back massage [153, 154] . In another study, consumption of CGJ after grinding and fermenting at 40 • C for 24 h was found to increase facial sebum and moisture levels with decreases melanin levels and red spots, while the skin pH was lower and skin color was enhanced (Table 9 ) [153, 155] . Therefore, these results suggest a positive effect of CGJ on the skin of middle-aged women. Consumption of CGJ was also shown to reduce body weight, body fat, and body mass index, while increasing skeletal muscle mass, exerting a positive effect on the body shape and weight of middle-aged women [153] [154] [155] . The effects were enhanced upon combining the treatment with meridian back massage. To date, the skin brightening effect or antimicrobial activity of CGJ has been mostly associated with its tyrosinase inhibitory activity [124] . More recently, CGJ induced an anti-dry skin effect by downregulating the expression of gene associated with iNOS and COX-2, tumor necrosis factor-alpha (TNF-α), interleukin 6 (IL-6), and prostaglandin E2 (PGE2) secretion, and upregulating the expression of genes involved in filaggrin and serine palmitoyltransferase (Table 9 ) [125, 155] . Furthermore, CGJ plays a critical role in skin-lightening by activating genes associated with tyrosine and tyrosinase ( Figure 10A ) [156] and by stimulating diverse signaling pathways, including Wnt, Frizzled (Fzd), stem cell factor (SCF)-Kit as a tyrosine kinase receptor, and α-melanocyte-stimulating hormone (MSH)/adrenocorticotropic hormone (ACTH)/agonist stimulating protein (ASP) ( Figure 10B ) [157] . These results suggest that CGJ could be a potential natural skin-lightening agent. [156] and core molecular pathways associated with regulation of melanin production in melanocytes (B) [157] . Genes encoding specific melanogenic enzymes, including tyrosinase precursor protein (TYR), and tyrosinase-related protein 1 and 2 (TRP1 and TRP2), are regulated by the master regulator-microphthalmia-associated transcription factor (MITF), which is in turn regulated by a number of important signaling pathways, including α-melanocyte-stimulating hormone (α-MSH)/adrenocorticotropic hormone (ACTH)/agonist stimulating protein (ASP), tyrosine kinase receptor (KIT)/stem cell factor (SCF), and wingless-related integration site (Wnt)/Frizzled (Fzd). Signal transduction is mediated by cyclic adenosine monophosphate (cAMP)/cAMP-dependent protein kinase (PKA), renin-angiotensin-system (RAS)/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), and β-catenin pathways [157] . APC, adenomatous polyposis coli; AXIN, axis inhibitor; CREB, cAMP response element binding protein; GSK-3β, glycogen synthase kinase-3β; LRP5/6, low-density lipoprotein receptor-related protein 5 and 6; MC1R, melanocyte-specific melanocortin-1 receptor; PKA, protein kinase A; RAF, rapidly accelerated fibrosarcoma; →, activation; , inhibition; , indirect activation. Figure adapted from Hanif, N. et al. [156] and Qian, W. et al. [157] . Table 9 . Skin improvement effect of soybean-derived CGJ. Korean middle-aged women Bacillus sp. Decreasing melanin level and red spots on facial skin; reducing body weight and body fat ratio; increasing muscle mass [153, 154] RAW 264.7 macrophage and HaCaT cell lines B. subtilis Anti-dry skin activity: inhibiting the expression of lipopolysaccharide-induced iNOS and COX-2 protein; suppressing TNF-α, IL-6, and prostaglandin E2 secretion; inducing the production of hyaluronic acid and expression of filaggrin and serine palmitoyltransferase [125, 155] Raw soybean B. subtilis Skin lightening activity: enhancing tyrosinase inhibitory activity through arbutin production [124] 3.9. Antimicrobial Activity and Improved Probiotic Efficacy CGJ fermented by various Bacillus spp. or Enterococcus spp. also modulates the regulation of microbial growth. As shown in Table 10 , CGJ inhibited the growth of a wide range of microorganisms, including B. cereus, Listeria monocytogenes, and Penicillium spp., by activating the genes encoding surfacing synthetase A, fengycin, and iturin [45, 158] , producing Bac W42 as a bacteriocin [159] and an antibiotic-like lipopeptidal compound (BSAP-254) as an antagonistic effector [158] , and introducing B. subtilis-infecting bacteriophages [45, 160] . B. licheniformis-dependent CGJ regulated the growth of Xantomonas oryzae causing rice bacterial blight by enhancing the production of daidzein, glycitein, genistein, and surfactins A and B [161] . CGJ fermented by Lactobacillus curvatus and E. faecium inhibited bacterial growth by controlling tyramine content [162] for B. cereus, Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella enterica [163] , and inhibited the growth of B. subtilis ATCC 15245 by activating γ-PGA hydrolase [164, 165] . CGJ fermented by B. amyloliquefaciens also showed antimicrobial activity for Aspergillus spp. and Penicillium spp. by producing iturin and bioactive amino acids [70] . In addition, Bacillus spp.-dependent CGJ improved the activity of beneficial human bacterial strains, including lactic acid bacteria (i.e., Lactobacillus sp. and Bacillus sp.), Faeclaibacterium prausnitzi, Adlercreutzia equolifaciens, Slackia isoflavoniconvertens, Coprococcus sp., Ruminococcus sp., and Bifidobacterium sp., through the production of isoflavone-derived metabolites, including O-desmethylangolensin (O-DMA), S(-)-equol, daidzein (DZ), and genistein (GS) (Figure 11 ) [9] as well as S-adenosyl-L-methionine [12] , short-chain fatty acid, and methionine biosynthesis [77] (Table 10) . Thus, these results suggest that CGJ could play an important role as an antimicrobial effector with antibacterial and antifungal activities without affecting the growth of soybean-fermenting bacteria and may enhance the population of probiotic bacteria associated with beneficial effects for human health [70, 158] . In addition to the various activities mentioned above, CGJ also demonstrates antityrosinase, anti-proliferative, syringic acid-mediated estrogen [166] , anti-atopic dermatitis [167, 168] , anti-androgenetic alopecia [169] , and anti-asthmatic activities (Table 11 ) [170] . Additionally, CGJ decreased toxic effects in the liver and kidneys by regulating various enzymes and reducing blood urea nitrogen and serum creatinine levels in ICR mice [132] . Lactobacillus spp.-and B. subtilis-derived CGJ enhanced the activity of bioactive compounds via the production of exopolysaccharide [106, 107] , γ-PGA [109, 171] , fatty acid and volatile compounds [172] , xylanase enzyme [173] , and γ-glutamyl transpeptidase (Table 11 ) [174] . Especially, consumption of CGJ-derived isoflavones improved the health conditions of healthy Korean men (Table 4 ) [108] . Table 11 . Other effects of soybean-derived CGJ. Raw soybean -Anti-tyrosinase, anti-proliferative, and syringic acid-mediated estrogenic activities [166] Mice B. amyloliquefaciens SCGB1 Amelioration of atopic dermatitis-like skin lesion: suppressing mast cell infiltration, immunoglobulin-E expression, and TH2 IL-4 and itch-related IL-31 cytokine; alleviating collagen fiber deposition; activating dephosphorylation of NF-κB and MAPK [167] IL4/Luc/CNS-1 Tg mice B. subtilis MC31 Therapeutic effect on atopic dermatitis: producing high concentration of gamma-aminobutyric acid (GABA); improving common allergic responses, including decreased thickness of ear and dermis, and reduction of auricular lymph node (ALN) weight and infiltrating mast cells, while decreasing IgE levels in epidermis [168] Men with stage II to V patterns of hair loss Leuconostoc holzapfelii, Leuconostoc mesenteroides L. sake Anti-androgenetic alopecia: enhancing hair count and thickness; promoting hair growth without reverse hair loss and side effects such as diarrhea [169] C57BL/6J mice B. subtilis Anti-asthmatic activity: suppressing histamine release of rat peritoneal mast cells by inhibiting calcium uptake as well as ear swelling by permeation of inflammatory cells; downregulating the population of eosinophil and monocytes in the lungs of mice; repressing histopathological changes, such as eosinophil infiltration, mucus accumulation, goblet cell hyperplasia, and collagen fiber deposits [170] ICR mice B. subtilis MC31 L. sakei 383 Reduction of toxic effects in liver and kidney; regulating levels of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase; reducing blood urea nitrogen and serum creatinine levels [132] Enhanced nutritional value: increasing the amounts of most amino acids including proline, sugars (sucrose, fructose, glucose, mannose, and xylose), and organic acids (glutamic acid, succinic acid, and malonic acid) [175] Raw soybean Enterococcusfaecium Establishment of fermentation culture platform for high accumulation of tyramine: reducing the safety risks that may arise when consuming CGJ with high tyramine amount by lowering fermentation temperature and shortening duration period; controlling tyrosine decarboxylase (TDC) activity using nicotinic acid as a TDC inhibitor to produce high amount of tyramine [162, 177, 178] Raw soybean Bacillus spp. Bacillus taxonomy: developing recQ as a genetic marker for the classification of Bacillus taxa [179] Raw soybean -Phenotype characterization of osmotic (~8% NaCl) tolerant B. glycinifermentans sp. [180] From a nutritional perspective, consumption of CGJ increases the amounts of most amino acids, sugars, organic acids [175] , and isoflavone and its metabolites [104, 105] . CGJ also enhances purine metabolism by increasing levels of uracil, thymine, xanthine, adenine, hypoxanthine, and guanine [43, 176] . Recently, a culture platform based on Enterococcus faecium-derived CGJ with high accumulation of tyramine was shown to reduce the safety risks that may arise when consuming CGJ [162, 177, 178] . Heo et al. [179] developed recQ as a genetic marker for the classification of Bacillus taxa. Kim et al. [180] exhibited phenotype characterization of osmotic tolerant B. glycinifermentans (Table 11 ). All results analyzed above are summarized in Figure 12 . In recent years, CGJ has been of interest to researchers and consumers owing to its nutritional health benefits, broad physiological activity, and exceptional sensory characteristics. The biological activities of CGJ have been extensively studied and include the following effects: (1) Proteolytic enzymes produced during the fermentation process dissolve blood clots, thereby preventing stroke and atherosclerosis, and peptides, which are decomposition products of CGJ and have a blood pressure-lowering effect. (2) Soybean isoflavones act similarly to female hormones, in that they are effective in preventing and inhibiting the progression of osteoporosis caused by female hormone deficiency in postmenopausal women. They also help prevent senile dementia by increasing the amount of acetylcholine, a neurotransmitter that is deficient in patients with dementia. (3) Soybeans, the raw material of CGJ, contain various physiologically active substances, such as isoflavones, phenolic compounds, phytic acid, and saponins, which have antioxidant and anticancer effects. In particular, isoflavones effectively prevent breast, colon, and prostate cancers by removing harmful free radicals. The isoflavone content of CGJ is higher than that of doenjang, and isoflavones in CGJ have excellent bioavailability. (4) B. subtilis present in CGJ promotes the growth of beneficial bacteria in the large intestine, and the dietary fiber and saponin from CGJ help relieve constipation. With increased public awareness and lifestyle improvements, consumers pay more attention not only to the nutritional value and flavor of CGJ, but also to its safety. Most traditional soybean products, such as CGJ, are manufactured using a production process that may introduce pathogenic microorganisms, and harmful by-products or metabolites are still the main barrier in fermented soybean production. In order to produce CGJ with high quality, nutritional value, and safety, we must constantly improve the traditional fermented soybean system and production parameters, discovery strains suitable for industrial application, upgrade for fermentation process, and fully understand the mechanism of fermentation. Furthermore, we need to elucidate the composition of the microbial community, as well as the identity and characteristics of metabolites produced during the fermentation process [3] . Further study investigating changes in beneficial bioactive molecules during CGJ fermentation will be the future trend in research. In the 21st century, it is rare to find a more rustic, but advanced, food than CGJ. The use of CGJ is limited by seasonal constraints, as it is only available from fall to early spring during which it is used as a seasoning for cooking stews or vegetables. Volatile substances such as butyric acid, valeric acid, tetra-methylpyrazine, and ammonia generated during the fermentation of CGJ impart a unique odor, which may pose a challenge to increasing the consumption of CGJ among children and younger adults who prefer Westernized and simplified diets. While a certain group of Koreans in their 40s or above may be nostalgic about the unique odor of CGJ from eating stews in their childhood, improving the odor and nutri-tional value of CGJ is crucial to appeal to the younger generation so that it continues to thrive as a traditional Korean food. Therefore, methods to preserve and reproduce traditional CGJ should be developed. Differentiating the quality of CGJ products, diversifying uses of CGJ, and standardizing the production process will invigorate the CGJ industry and increase the consumer base. For example, CGJ quality can be enhanced for consumer appeal, automatic processes can be established for mass production, and packaging and hygiene can be improved for better marketability. The current efforts to differentiate CGJ ingredients may capture the attention of those reluctant to try CGJ, thus enhancing its commercialization and consumption. The authors declare no conflict of interest. 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Front The basics of brain aneurysms: A guide for patients Brain-heart interaction: Cardiac complications after stroke Preventing deaths due to the hypertensive disorders of pregnancy Equol: A bacterial metabolite from the daidzein isoflavone and its presumed beneficial health effects The key importance of soy isoflavone bioavailability to understanding health benefits Comparison of streptokinase, urokinase, and recombinant tissue plasminogen activator in an in vitro model of venous thrombolysis Fibrinolysis inhibitors: Potential drugs for the treatment and prevention of bleeding Recent advances in seafood bioactive peptides and their potential for managing osteoporosis Fibrinolytic and antiplatelet aggregation properties of a recombinant Cheonggukjang kinase Bioactivity of soy-based fermented foods: A review Biocontrol of Fusarium wilt disease in strawberries using bioorganic fertilizer fortified with Bacillus licheniformis X-1 and Bacillus methylotrophicus Z-1 Isolation of Bacillus amyloliquefaciens strains with antifungal activities from Meju Characterization of a 27 kDa fibrinolytic enzyme from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang Improvement of fibrinolytic activity of Bacillus subtilis 168 by integration of a fibrinolytic gene into the chromosome Characterization of AprE176, a fibrinolytic enzyme from Bacillus subtilis HK176 Cloning of fibrinolytic enzyme gene from Bacillus subtilis isolated from Cheonggukjang and its expression in protease-deficient Bacillus subtilis strains Cloning and expression of a bpr gene encoding Bacillopeptidase F from Bacillus amyloliquefaciens CH86-1 Characterization of a fibrinolytic enzyme secreted by Bacillus amyloliquefaciens CB1 and its gene cloning Effects of digested Cheonggukjang on human microbiota assessed by in vitro fecal fermentation Haemodynamic definitions and updated clinical classification of pulmonary hypertension The goal of blood pressure in the hypertensive patient with diabetes is defined: Now the challenge is go from recommendations to practice Role of the renin-angiotensin-aldosterone system beyond blood pressure regulation: Molecular and cellular mechanisms involved in end-organ damage during arterial hypertension Metastin stimulates aldosterone synthesis in human adrenal cells Atrial natriuretic factor inhibits the stimulation of aldosterone secretion by angiotensin II, ACTH and potassium in vitro and angiotensin II-induced steroidogenesis in vivo Angiotensin-I-converting enzyme (ACE) inhibitors from marine resources: Prospects in the pharmaceutical industry Anti-hypertensive effects of peptides derived from rice bran protein ACE inhibitory properties and phenolics profile of fermented flours and of baked and digested biscuits from buckwheat Natto mucilage containing poly-gamma-glutamic acid increases soluble calcium in the rat small intestine Conjugation of camptothecins to poly-(L-glutamic acid) Development of a soy protein hydrolysate with an antihypertensive effect Local generation and release of angiotensin II in peripheral vascular tissue Lipoprotein (a) as a predictor of coronary heart disease: The PRIME study Effects of spice added natto supplementation on the lipid metabolism in rats Supplementation of cheonggukjang and red ginseng cheonggukjang can improve plasma lipid profile and fasting blood glucose concentration in subjects with impaired fasting glucose Soybean protein suppresses hepatic lipogenic enzyme gene expression in Wistar fatty rats Effects of soy protein and saponins on serum, tissue and feces steroids in rat Soy, soy foods and their role in vegetarian diets Phytic acid interactions in food systems Diverse role of phytic acid in plants and approaches to develop low-phytate grains to enhance bioavailability of micronutrients A comparative transcriptome and proteomics analysis reveals the positive effect of supplementary Ca 2+ on soybean sprout yield and nutritional qualities Prostate cancer and inositol hexaphosphate: Efficacy and mechanisms Complex formation of phytic acid with selected monovalent and divalent metals Nutrient and nonnutrient components of legumes, and its chemopreventive activity: A review Reduction of antiproliferative capacities, cell-based antioxidant capacities and phytochemical contents of common beans and soybeans upon thermal processing Modification of isoflavone profiles in a fermented soy food with almond powder Formation of succinyl genistin and succinyl daidzin by Bacillus species Aglycone isoflavones and exopolysaccharides produced by Lactobacillus acidophilus in fermented soybean paste Effects of germination and osmopriming treatment on enhancement of isoflavone contents in various soybean cultivars and cheonggukjang (fermented unsalted soybean paste) Nutrikinetic study of fermented soybean paste (Cheonggukjang) isoflavones according to the Sasang typology Poly-(gamma-glutamic acid) production and optimization from agroindustrial bioresources as renewable substrates by Bacillus sp. FBL-2 through response surface methodology Poly-gamma-glutamic acid induces apoptosis via reduction of COX-2 expression in TPA-induced HT-29 human colorectal cancer cells Expression, purification, and biochemical properties of arginase from Bacillus subtilis 168 The continuing search for antitumor agents from higher plants Effects of ripening process on the mutagenicity and antimutagenicity of Sufu, a Chinese traditional fermented product of soybean Antimutagenic activity of soybeans fermented with basidiomycetes in Ames/Salmonella test Physicochemical characteristics and isoflavones content during manufacture of short-time fermented soybean product (cheonggukjang) Correlation between antioxidative activities and metabolite changes during Cheonggukjang fermentation Simultaneous analysis and measurement of uncertainty estimation of six isoflavones in Cheonggukjang by liquid chromatography-electrospray tandem mass spectrometry Comparative evaluation of six traditional fermented soybean products in East Asia: A metabolomics approach Genistein: Its role in metabolic diseases and cancer The anti-inflammatory action of fermented soybean products in kidney of high-fat-fed rats Soy products fermented with sprouted garlic have increased neuroprotective activities and restore cognitive functions Effects of bacterial fermentation on the biochemical constituents and antioxidant potential of fermented and unfermented soybeans using probiotic Bacillus subtilis (KCTC 13241) Characteristics and antioxidant effect of garlic in the fermentation of Cheonggukjang by Bacillus amyloliquefaciens MJ1-4 Tyrosinase inhibitory activity of soybeans fermented with Bacillus subtilis capable of producing a phenolic glycoside Fermented dried Citrus unshiu peel extracts exert anti-inflammatory activities in LPS-induced RAW264.7 macrophages and improve skin moisturizing efficacy in immortalized human HaCaT keratinocytes Cheonggukjang polysaccharides enhance immune activities and prevent cyclophosphamide-induced immunosuppression Immunostimulatory activity of polysaccharides from Cheonggukjang Food and natural materials target mechanisms to effectively regulate allergic responses Effect of fermented soybean products intake on the overall immune safety and function in mice Immunostimulatory activity of isoflavone-glycosides and ethanol extract from a fermented soybean product in human primary immune cells Inflammation-related signaling pathways implicating TGFβ are revealed in the expression profiling of MCF7 cell treated with fermented soybean, chungkookjang Toxicity of fermented soybean product (cheonggukjang) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on liver and kidney of ICR mice Beneficial effect of chungkukjang on regulating blood glucose and pancreatic beta-cell functions in C75BL/KsJ-db/db mice Antidiabetic effect of Morinda citrifolia (Noni) fermented by cheonggukjang in KK-A(y) diabetic mice The inhibitory effect of saponin derived from Cheonggukjang on adipocyte differentiation In vitro Soybean fermentation with Bacillus licheniformis increases insulin sensitizing and insulinotropic activity Standardized chungkookjang, short-term fermented soybeans with Bacillus lichemiformis, improves glucose homeostasis as much as traditionally made chungkookjang in diabetic rats Antioxidant and antigenotoxic activities of Korean fermented soybean Long-term consumption of fermented soybean-derived Chungkookjang attenuates hepatic insulin resistance in 90% pancreatectomized diabetic rats Prevalence, genetic diversity, and antibiotic resistance of Bacillus cereus isolated from Korean fermented soybean products Comparison of the antioxidant activities of small-black-bean-Chungkukjang-added black food and soybean Chungkukjang extracts Fermented soybeans, Chungkookjang, prevent hippocampal cell death and β-cell apoptosis by decreasing pro-inflammatory cytokines in gerbils with transient artery occlusion Effects of Chungkookjang supplementation on obesity and atherosclerotic indices in overweight/obese subjects: A 12-week, randomized, double-blind, placebo-controlled clinical trial Korean traditional Chungkookjang improves body composition, lipid profiles and atherogenic indices in overweight/obese subjects: A double-blind, randomized, crossover, placebo-controlled clinical trial Long-term consumption of fermented soybean-derived Chungkookjang enhances insulinotropic action unlike soybeans in 90% pancreatectomized diabetic rats Neuroprotective effects of fermented soybean products (Cheonggukjang) manufactured by mixed culture of Bacillus subtilis MC31 and Lactobacillus sakei 383 on trimethyltin-induced cognitive defects mice Effects of Rubus coreanus-Cheonggukjang on bone mineral density and bone mineral content in growing rats Growth sensitivity in the epiphyseal growth plate, liver and muscle of SD rats is significantly enhanced by treatment with a fermented soybean product (cheonggukjang) through stimulation of growth hormone secretion Fermented soybean product (Cheonggukjang) improved some attributes of protein and growth hormone measurements in Sprague-Dawley rats Dihydrodaidzein and 6-hydroxydaidzein mediate the fermentation-induced increase of antiosteoporotic effect of soybeans in ovariectomized mice Nutrikinetics of isoflavone metabolites after fermented soybean product (cheonggukjang) ingestion in ovariectomized mice Improved menaquinone (Vitamin K 2 ) production in cheonggukjang by optimization of the fermentation conditions Effects of soybean diet and back massage on 40's∼50's women's facial skin Effects of soybean diet and back massage on 40's∼50's women's body Impact of ferment premilling Chung-guk diet and back meridian massage on the facial skin of middle-aged women Plant-based skin lightening agents: A review Natural skin-whitening compounds for the treatment of melanogenesis (Review) Isolation and characterization of a novel analyte from Bacillus subtilis SC-8 antagonistic to Bacillus cereus Properties of Bac W42, a bacteriocin produced by Bacillus subtilis W42 isolated from Cheonggukjang Bacteriophages BCP1-1 and BCP8-2 require divalent cations for efficient control of Bacillus cereus in fermented foods Antimicrobial compounds profile during Cheonggukjang fermentation against Xanthomonas oryzae pv. oryzae (Xoo) Reduction of tyramine by addition of Schizandra chinensis Baillon in Cheonggukjang Selection and characterization of broad-spectrum antibacterial substance-producing Lactobacillus curvatus PA40 as a potential probiotic for feed additives High prevalence of Bacillus subtilis-infecting bacteriophages in soybean-based fermented foods and its detrimental effects on the process and quality of Cheonggukjang Complete nucleotide sequence analysis of a novel Bacillus subtilis-infecting bacteriophage BSP10 and its effect on poly-gamma-glutamic acid degradation Determination of bioactive compounds in fermented soybean products using GC/MS and further investigation of correlation of their bioactivities Soybean fermented with Bacillus amyloliquefaciens (Cheonggukjang) ameliorates atopic dermatitis-like skin lesion in mice by suppressing infiltration of mast cells and production of IL-31 cytokine Quantitative evaluation of the therapeutic effect of fermented soybean products containing a high concentration of GABA on phthalic anhydride-induced atopic dermatitis in IL-4/Luc/CNS-1 Tg mice Do kimchi and cheonggukjang probiotics as a functional food improve androgenetic alopecia? A clinical pilot study Cheonggukjang ethanol extracts inhibit a murine allergic asthma via suppression of mast cell-dependent anaphylactic reactions Factors increasing poly-γ-glutamic acid content of cheongguk-jang fermented by Bacillus subtilis 168 Time course effects of fermentation on fatty acid and volatile compound profiles of Cheonggukjang using new soybean cultivars Cloning of a family 11 xylanase gene from Bacillus amyloliquefaciens CH51 isolated from cheonggukjang Improvement of viscous substance production during Cheonggukjang fermentation added with glycine Metabolite profiling of Cheonggukjang, a fermented soybean paste, inoculated with various Bacillus strains during fermentation based metabolic profiling of cheonggukjang (fast-fermented bean paste) during fermentation and its correlation with metabolic pathways The role of Enterococcus faecium as a key producer and fermentation condition as an influencing factor in tyramine accumulation in cheonggukjang Tyramine reduction by tyrosine decarboxylase inhibitor in Enterococcus faecium for tyramine controlled cheonggukjang Genetic marker gene, recQ, differentiating Bacillus subtilis and the closely related Bacillus species Bacillus glycinifermentans sp. nov., isolated from fermented soybean paste