Medical Application of Substances Derived from Non-Pathogenic Fungi Aspergillus oryzae and A. luchuensis-Containing Koji

Although most fungi cause pathogenicity toward human beings, dynasties of the East Asian region have domesticated and utilized specific fungi for medical applications. The Japanese dynasty and nation have domesticated and utilized koji fermented with non-pathogenic fungus Aspergillus oryzae for more than 1300 years. Recent research has elucidated that koji contains medicinal substances such as Taka-diastase, acid protease, koji glycosylceramide, kojic acid, oligosaccharides, ethyl-α-d-glucoside, ferulic acid, ergothioneine, pyroglutamyl leucine, pyranonigrin A, resistant proteins, deferriferrichrysin, polyamines, Bifidobacterium-stimulating peptides, angiotensin I-converting enzyme inhibitor peptides, 14-dehydroergosterol, beta-glucan, biotin, and citric acid. This review introduces potential medical applications of such medicinal substances to hyperlipidemia, diabetes, hypertension, cardiovascular and cognitive diseases, chronic inflammation, epidermal permeability barrier disruption, coronavirus disease 2019 (COVID-19), and anti-cancer therapy.


Introduction
Fungi in general cause many infectious pathogenicities in human beings, including Aspergillus, Candida, Cryptococcus, and Mucor species [1]. However, several fungi have been utilized as medicines across the world since the prehistoric ages. Fungi are eukaryotic microbes that have complex biosynthesis pathways and various morphologies. They have unique biosynthesis pathways and thus produce unique substances that are not contained in mammals, plants, or prokaryotic bacteria [2].
Within fungi, mushrooms have attracted attention in terms of their medical potential since the prehistoric ages [3], possibly because of their visibility without microscopes. For example, hoelen, which is a Wolfiporia cocos mushroom proliferating on the roots of pine trees, has been used in East Asian countries, including China and Japan, as a medicine for more than 1800 years [4]. Moreover, a Ganoderma lucidum mushroom is described in Shennong Ben Cao Jing, which was written in 25-220 A.D. in China, as being able to increase the lifespan [5].
The beta-glucan contained in Schizophyllum commune, Schizophyllan, and Lentinula edodes, Lentinan, has been registered and utilized as immunoactivating medicines to treat cervical, stomach, and colon cancer [6]. However, medical applications of fungi which do not form mushrooms had not been described until the modern age, possibly because they are invisible without microscopes and microscopes only prevailed across the world in the 18-19th centuries.
The use of a red fungus Monascus in China is one of these examples [7]. The health benefits of red rice koji has been recognized in China for more than approximately 700 years. Its medical use was first described in Compendium of Materia Medica, which was written in 1498 A.D., indicating that it clears the blood and supports digestion of foods in the intestine. Dr. Endo elucidated, in 1979, that monacolin K produced by Monascus ruber has

Koji Glycosylceramide
Glycosylceramide is a sphingolipid consisting of a sphingoid base, fatty acid, and a sugar moiety ( Figure 2). It is contained in various species, including plants and fungi. Sphingolipids have versatile biological functions, such as signal transduction [19], enhancement of the immune system [20], and raft formation [21]. Their structures have great variations, depending on the structure and type of the sphingoid base, fatty acid, and sugar moiety. The characteristic structure of A. oryzae is N- 8-sphingadienine, and the corresponding molecules in which sugar moiety is substituted for galactose instead of glucose [22][23][24][25][26]. It is contained in koji and Japanese fermented foods at 1-4 mg/serving [27]. Most of the glycosylceramide in koji is considered not to be degraded by enzymes in the small intestine, and passes through to the large intestine [28]. The dietary intake of the glycosylceramide purified from koji at a concentration of 1% w/w of diet in mice increases intestinal Blautia coccoides, Bacteroides sartorii, and Hathewaya histolytica in mice. Since B. coccoides is considered to be a beneficial microbe, koji glycosylceramide could be considered as a new type of prebiotics. Also, feeding of glycosylceramide at a concentration of 1% w/w of diet to obese mice increases bile acids and lowers liver cholesterol in obese mice [29]. Since liver cholesterol is one of the causes of hyperlipidemia, koji glycosylceramide could potentially be medically utilized to treat hyperlipidemia. Furthermore, glycosylceramide purified from koji increases the expression of the genes involved in tight junctions and ceramide delivery in normal human epidermal keratinocytes [30]. The dietary intake of the glucosylceramide derived from konjac extract improves transepidermal water loss (TEWL) in hairless mice with sodium dodecyl sulfate (SDS)-induced skin roughness [31]. Since the glycosylceramide contained in koji contains the same species of glucosylceramide contained in konjac [23], the dietary intake of koji might improve TEWL. Therefore, the dietary intake of koji or the topical administration of Furthermore, glycosylceramide purified from koji increases the expression of the genes involved in tight junctions and ceramide delivery in normal human epidermal keratinocytes [30]. The dietary intake of the glucosylceramide derived from konjac extract improves transepidermal water loss (TEWL) in hairless mice with sodium dodecyl sulfate (SDS)-induced skin roughness [31]. Since the glycosylceramide contained in koji contains the same species of glucosylceramide contained in konjac [23], the dietary intake of koji might improve TEWL. Therefore, the dietary intake of koji or the topical administration of koji to the skin might be beneficial for skin disorders.

Kojic Acid
A. oryzae produces kojic acid (Figure 3), which functions as a competitive and reversible inhibitor of animal and plant polyphenol oxidases, xanthine oxidase, and some amino acid oxidases, and is used as an food additive for preventing enzymatic browning or in cosmetic preparations as a skin-lightening agent [32]. Therefore, koji contains kojic acid at the concentration of 0-9.5 mg/g, and the topical administration of kojic acid at 50 µg/mL inhibits browning of the skin [33]. Although some researchers have reported the occurrence of thyroid adenomas when continuously administered, it is generally recognized that the consumption of kojic acid at levels normally found in food is not a concern for safety.

Oligosaccharides
Rice, barley, wheat, and soy contain various oligosaccharides consisting of glucose, fructose, xylulose, galactose, maltose, and arabinose [34]. For example, starch consists of α-1,4-linked glucose polymers, with α-1,6-linked branches [35]. Xylan is a polymer consisting of β-1,4-linked xylulose [36]. The genome of A. oryzae contains abundant genes of glycolytic enzymes [37]. For example, α-amylase degrades starch and generates various lengths of α-1,4-linked glucose polymers randomly with α-1,6-linked branches [38]. These glucose oligomers are degraded by pancreatic amylases to glucose, and the resultant glucose is absorbed in the small intestine [39]. However, since pancreatic enzymes do not degrade oligosaccharides which contain unique structures derived from starch or includes xylulose or arabinose, such oligosaccharides are not degraded in the small intestine, instead passing through to the large intestine and, as a result, are assimilated by intestinal microbes. Indeed, koji manufactured from rice and sake manufactured using koji contain various oligosaccharides including isomalto-pligosaccharide containing α-1,2-bond [40] and glucooligosaccharide with adjacent α-1,6 branches at the non-reducing end derived from starch ( Figure 4) [41] and shoyu manufactured from rice koji and soy contains oligosaccharides consisting of galactose, arabinose, and galacturonic acid [42]. It has been reported that the dietary ingestion of oligosaccharides increases intestinal Bifidobacterium [43]. Moreover, the dietary ingestion of oligosaccharide is beneficial for ulcerative colitis and minimal hepatic encephalopathy [44,45]. Furthermore, Shoyu polysaccharide produced by koji enzymes from soy alleviates perennial allergic rhinitis in humans [43] and promotes iron absorption in rats and humans [46]. Koji-fermented barley also contains oligosaccharides consisting of glucose, xylose, and arabinose that stimulate the growth of lactic acid bacteria and Bifidobacterium [47]. Since Bifidobacterium infantis ameliorates intestinal mucositis in rats [48] and Bifidobacterium adolescentis extract has an anti-proliferative effect on human colon cancer cell lines [49], it is considered that the dietary ingestion of koji-fermented foods and drinks (especially those manufactured from raw rice, barley, wheat, and soy) has potential medical applications to treat such patients.

Ethyl-α-D-Glucoside
Ethyl-α-D-glucoside ( Figure 5), as well as α-glyceryl glucoside, ethyl-α-maltoside, and ethyl-α-isomaltoside, are formed from ethanol or glycerol and glucose, maltose, or isomaltose by the action of the transglycosidase produced by koji [50,51]. For example, the concentration of ethyl-α-d-glucoside is reported to be 0.33% w/w [50]. The dietary intake of the ethyl-α-D-glucoside (1% w/w) contained in sake and sake lees, the remnant of sake production, improves epidermal permeability barrier disruption by UVB irradiation in hairless mice [52]. It also upregulates collagen I and fibroblast growth factor I and VII in cultured human dermal fibroblasts [53]. Moreover, the topical application of ethylα-D-glucoside increases the intercellular lipid content, accelerates the differentiation of corneocytes, and reduces the thickness of the epidermis, thus improving the functions of the stratum corneum in murine epidermis [54]. Therefore, the dietary intake of sake or sake lees leads to the intake of ethyl-α-D-glucoside, thus resulting in improvement of epidermal permeability barrier disruption.

Ferulic Acid
Ferulic acid is ester-bound to carbohydrates ( Figure 6) in cereals [55], and is formed by koji enzymes during sake brewing [56]. Ferulic acid is contained in sake at a concentration of 1-6 mg/L [57]. The dietary intake of ferulic acid lowers the lipid levels in hyperlipidemic diabetic rats [58]. The oral administration of 30 mg/kg/day ferulic acid or ethyl ferulate provides protective effects toward retinal degeneration [59]. Furthermore, the dietary intake of ferulic acid is protective toward the toxicity of the β-amyloid peptide, which causes Alzheimer's disease [60]. Therefore, the dietary intake of koji leads to the intake of ferulic acids, thus resulting in amelioration of dyslipidemia, retinal degeneration, and Alzheimer's disease.

Ergothioneine
Ergothioneine is a derivative of amino acid that has a strong antioxidative activity (Figure 7) [61,62]. Koji fermented with A. oryzae is reported to contain 59.8 ± 20.4 µg/g ergothioneine [62]. Feeding of ergothioneine at a concentration of 8 mg/kg body weight to mice protected against cisplatin-induced neuronal injury and enhances cognition [63]. Since skin cells and tissue can absorb ergothioneine, and it is accumulated in peripheral skin cells, it is considered to contribute to the antioxidant activity of skin cells and tissue [64].
Functional variants of the ergothioneine transporter, OCTN cation transporter genes, are associated with ulcerative colitis, Crohn's disease, and autoimmune thyroid disease [65]. Therefore, it was hypothesized that the dietary intake of koji leads to the intake of ergothioneine and may contribute to the amelioration of these diseases.

Pyroglutamyl-Leucine
Peptides generated by proteases in koji are further modified during sake brewing ( Figure 8). The pyroglutamyl-leucine contained in sake is one such example of a peptide, and is contained in sake at a concetration of 10-15 mg/L [66]. It has been shown to attenuate hepatitis and colitis in animal models [66].

Pyranonigrin A
It has been reported that koji contains some antioxidant substances [67,68]. Later, one such antioxidant substance was identified as pyranonigrin A (Figure 9) [69]. Pyranonigrin A is a potential main protease inhibitor of SARS-CoV-2 and might be utilized to prevent infection of coronavirus disease 2019 (COVID-19) [70]. Therefore, the dietary intake of koji and related fermented products leads to the intake of pyranonigrin A, resulting in the prevention of cardiovascular diseases and COVID-19.

Resistant Proteins
Most proteins in rice are degraded during sake brewing by the proteases contained in koji. However, nondigestable proteins, especially prolamin accumulated in the protein bodies of the endosperm of rice [71], remain in the sake lees, the remnant of sake brewing. Nondigestable proteins are called resistant proteins. As a result, sake lees contain a high content of resistant proteins. These resistant proteins are also resistant to human pancreatic proteases, and thus resistant proteins pass through the stomach and the small intestine and reaches the large intestine. As a result, they inhibit the absorption of lipids in the intestine, and improve intestinal microbial flora [72]. Therefore, the dietary intake of sake lees leads to the intake of resistant proteins, thus resulting in improvement of hyperlipidemia.

Deferriferrichrysin
Koji and its fungus A. oryzae contain a low-molecular-weight iron chelating peptide, deferriferrichrysin. Since it functions as a natural antioxidant [73], it might be beneficial for skin protection and prevention of inflammation.

Polyamines
During the manufacturing of koji, one of polyamines, agmatine (Figure 10), is produced at 3.1-8.7 mmol/L [74,75]. Polyamines have anti-inflammatory effects through the regulation of aberrant DNA methylation and decreased incidence of colon tumors, although accelerated the growth of established tumors [76]. Therefore, dietary intake of koji may lead to intake of polyamines, thus resulting in preventing the effects of colon tumors at a certain concentration.

Bifidobacterium-Stimulating Peptides
Proteases contained in koji acts on rice proteins and gives rise to various peptides. Certain peptides containing glutamate, serine, and alanine in koji were shown to increase Bifidobacteria, B. longum, B. adolescentis, and B. breve [77]. Therefore, koji is considered to improve the intestinal microbe.

Angiotensin I-Converting Enzyme Inhibitor Peptides
The renin-angiotensin system increases blood pressure. Angoitensin I-converting enzyme catalyzes the conversion of Ang I to Ang II, which eventually generates AT1R and causes proinflammatory effects [78,79]. Sake utilizing koji contains the angiotensin I-converting enzyme inhibitor peptides Val-Tyr, His-Tyr, Arg-Phe, Val-Trp, and Tyr-Trp. These peptides have been shown to be antihypertensive in spontaneously hypertensive rats [80,81]. Therefore, the dietary intake of sake and koji-fermented products leads to the intake of these peptides, thus resulting in anti-hypertensive effects.

Beta-Glucan
Beta-glucan ( Figure 12) was first identified as a medical component of Agaricus blazeii from the observations of lower incidence of cancers and infections of virus and bacteria, as well as the increased life span within native Americans [84]. Later, it was found that beta-glucan contained in the fungus is an immunostimulant and activates macrophages through dectin1 and CR3 (CD11b/CD18) [85,86]. It also ameliorates allergies and reduces the triglyceride and cholesterol levels in the blood [87]. Aspergillus spp. contains β-(1→3)glucan as its cell wall component (Figure 11), and β-(1→3)-glucan of Aspergillus fumigatus, a close relative of A. oryzae, activates dectin-1 [88]. Since koji is manufactured using A. oryzae, it is considered to contain beta-glucan. The administration of beta-glucan is considered to improve the quality of life of cancer patients and is useful as a complementary or adjuvant therapy and immunomodulatory agent in cancer patients in combination with other cancer treatments [89,90]. Therefore, the content of beta-glucan in koji is desired to be investigated.

Biotin
Lack of biotin ( Figure 13) is known to causes skin disorders such as dermatitis, hair loss, neuritis, and susceptibility to infections [91]. It is also severely teratogenic in rodents. Since Aspergillus oryzae used for production of koji has a biosynthetic pathway of biotin [92] and koji contains 1.01 mg/100 g biotin [93], dietary intake of koji might contribute to the prevention of these disorders.

Citric Acid
A. luchuensis, which has been used for manufacturing awamori and shochu in Okinawa prefecture and Kyusyu island, produces citric acid ( Figure 14) and koji fermented with A. luchuensis contains citric acid at approximately 1800 mg/L [94]. Therefore, koji manufactured using A. luchuensis contains citric acid ( Figure 12). The dietary intake of citric acid (2 g/L) in drinking water inhibits the development of cataracts, proteinuria, and ketosis in diabetic rats [95]. Moreover, in a mouse model of systemic inflammation, dietary intake of citric acid (1-2 g/kg) has been shown to decrease brain lipid peroxidation and inflammation, liver damage, and DNA fragmentation in lipopolysaccharide-treated mice [96]. Therefore, fermented foods containing koji manufactured with A. luchuensis might have beneficial health effects toward diabetic patients and chronic inflammation, which should be a target of future study.

Potential Risk of Koji
A. oryzae in general produces aflatoxin and cyclopiazonic acid [97]. Ingestion of aflatoxin causes an acute hepatic necrosis [98]. Cyclopiazonic acid shows immunosuppressive activity at low doses and causes necrosis of various tissues [99]. However, since A. oryzae used for manufacture of koji has been long succeeded in Japan, it has lost functional genes to produce aflatoxin [100] or acquired a new gene to produce less toxic cyclopiazonic acid [101]. Also, A. luchuensis has lost the functional gene to synthesize ochratoxin A [102].
It should be noted that some strains produce trace amounts of certain mycotoxins including aspirochlorin and csypyrones [97] and it is necessary to monitor the concentrations of these substances.

Conclusions
In conclusion, koji fermented with A. oryzae or A. luchuensis and koji-fermented products contain abundant substances that have the potential for use in medical applications ( Figure 15). It is desired that the medical benefits of these substances be verified in human studies and applied to practical medical use. More research is desired to medically apply koji to cure diseases across the world in the future.

Conclusions
In conclusion, koji fermented with A. oryzae or A. luchuensis and koji-fermented products contain abundant substances that have the potential for use in medical applications ( Figure 15). It is desired that the medical benefits of these substances be verified in human studies and applied to practical medical use. More research is desired to medically apply koji to cure diseases across the world in the future.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Conflicts of Interest:
The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.