Current Perspectives on the Physiological Activities of Fermented Soybean-Derived Cheonggukjang

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.


Introduction
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.

Nutritional Properties of CGJ
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.

Bioactivities and Human Health Benefits of CGJ
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.

Thrombotic Effects
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.

Blood Pressure-Lowering and Lipid-Lowering Effects
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]

Antioxidant Effects
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.

Immunostimulatory Effects
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.

Model
Strain Used in Fermentation Action Mode Reference

MCF7 cells B. licheniformis B1
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.

Anti-Obesity and Anti-Diabetic Effects
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-Osteoporotic Effect and Cognitive Functional Enhancement
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].

Skin Improvement
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.

Model Strain Used in Fermentation Action Mode Reference
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].

Model Strain Used in Fermentation Action Mode Reference
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.

Conclusions and Perspectives
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.

Conflicts of Interest:
The authors declare no conflict of interest.