**2.3 Biologically active components in** *Aspergillus oryzae***-mediated fermentation products of brown rice**

Many kinds of biologically active compounds in rice bran have a potential to improve diseased condition and maintain human health, while rice bran itself is not easy for daily intake. However, plant-derived solid substances are often decomposed and utilized by various environmental lives. *Aspergillus* fungi are industrially important for food fermentation and production of biological/bioactive compounds, and *Aspergillus oryzae* is the most common mold for the fermentation of soybeans, rice, grains, and potatoes [27]. *A. oryzae* has also been used for Japanese traditional food, miso, sake, say sauce. During the fermentation process, *A. oryzae* produces amylase, protease, and β-glucosidase, and changes rice constituents to amino acids, fatty acids, organic acids, sugar and sugar alcohol, nucleotides, and various secondary metabolites [28]. Antioxidants and phenolic acid timedependently increased during the fermentation [28, 29]. Production of natural iron chelator deferriferrichrysin from *A. oryzae* has been reported as a candidate of novel food-grade antioxidant [30]. *A. oryzae*-derived protease preparation showed beneficial effects on colonic environment in high-fat diet fed rats [31]. Neutral polysaccharide produced in fermented Korean brown rice vinegar was reported to have immunostimulatory activity [32]. *A. oryzae*-mediated fermentation of other grains wheat germ or sorghum also produced anti-adipogenic activity in cultured adipocytes [33] and anti-inflammatory effect in atherosclerotic mice model [34].

"Brown rice and rice bran fermented with *Aspergillus oryzae* (FBRA)" described in the following subsection is a processed food with *A. oryzae*-mediated fermentation (**Figure 1**). General constituents of raw powder FBRA used for our studies is shown in **Table 1** (excerpted from Kataoka et al. [36]). After the fermentation process, FBRA becomes to be taken more easily than the original material bran. Dried fermented

#### **Figure 1.**

*Raw powder of FBRA. Upper panel shows photo of brown rice and rice bran as starting material, and raw powder of FBRA as fermented product (kindly provided by Dr. Hideyuki Nemoto, Koken Co., Ltd., Japan). Lower panel illustrates the fermentation procedure previously described in Horie et al [35].*

product is packaged and is commercially available and has been accumulating eating experiences. While eating quality of FBRA is not directly estimated by comparing with cooked brown rice, 36 healthy adult participants in our clinical study could consume 21.0 g/day FBRA for 2 weeks without dropout [37]. Dietary fibers are partially digested, and free vitamins and phenolic compounds have increased in FBRA [38, 39]. Increase of polyamines, phenolic acids, and ergothioneine have been demonstrated by LC/ESI-MS/MS [35, 40, 41]. Polyamines such as putrescine and spermidine are essential for cell growth, proliferation and tissue regeneration, but the expression of polyamine synthetic enzymes have been declining with aging [35]. Ergothioneine is an amino acid derivative which has a strong antioxidant activity as a scavenger of reactive oxygen species, and also has the potential to prevent central neurological disorders [40]. Interestingly, Takusagawa et al. [42] recently reported that *A. oryzae* can synthesize ergothioneine from histidine contained in the food material such as rice.


*Listed items were analyzed by Japan Food Research Laboratories (Tokyo, Japan). (excerpeted from Kataoka K et al. [36], Journal of Functional Foods. 2021; 78: 104356, supplemental Table 1)*

#### **Table 1.**

*Ingredient of "Brown rice and rice bran fermented with* Aspergillus oryzae *(FBRA)".*

Bioactive components produced through *A. oryzae*-mediated fermentation could bring us more beneficial effects and will contribute to preventing or ameliorating various diseased conditions. On the other hand, we should clarify what elements are effective and how the elements works against diseased conditions, including optimal dose and adverse effects. In manufacturers, management of fermentation process with using adequate control compounds described by Lee et al. [28] is essential to keep the products quality.

## **2.4 Biological activity of raw powder FBRA in** *in vitro* **and** *in vivo* **disease models**

In parallel with the above component analysis, many research groups have been conducting *in vitro* and *in vivo* studies to estimate the functions of FBRA. A raw powder FBRA used in our study is provided by the manufacturer (Genmai Koso Co. Ltd., Sapporo, Japan). In *in vitro* studies, FBRA extract induced apoptosis of tumor cells by activating mitochondrial pathway in human colorectal tumor cells [43] and via death receptor pathway in human lymphoblastic leukemia cells [44]. These results are consistent with the previous results with those of nonfermented brown rice, while the results should be carefully interpreted because of direct addition of an aqueous extract to cultured cell lines.

Antitumorigenic *in vivo* effects of dietary administration of FBRA have been examined at 5 or 10% dietary concentration. In chemically induced tumorigenesis models, preventive effects of FBRA were demonstrated in colon [45], liver [46], esophagus [47], urinary bladder [48], oral cavity [49], stomach [50], lung [51], pancreas [52]. Preventive effects were also demonstrated in prostate carcinogenesis in TRAP rats [53] and spontaneous lymphomagenesis in AKR/NSlc female mice [54]. Sakurai et al. [55] reported inhibitory effect of oral FBRA on metastasis of colon tumor cells to the liver through a mechanism leading to a Th1-dominant immune state and activation of macrophages via anti-oxidative properties. Chemoprevention mechanisms associated with dietary brown rice components have been reviewed by Henderson et al. [11]. They depicted that rice bran constituents act through anti-oxidative protection against free radicals in initiation stage, anti-proliferative/pro-apoptotic action on malignant cells, modulation of immunity and inflammation in the early or late stage, and mucosal protection through altered microbiota and intestinal environment, and that complex mixture of rice branderived bioactive compounds cooperatively suppress many stages of carcinogenic process. Antitumorigenic components in FBRA might be basically the same as those in brown rice and rice bran, while fermentation process possibly influence the activity of FBRA.

Anti-inflammatory effects of FBRA have been observed against the development of hereditary hepatitis in Long-Evans Cinnamon rats [56], DSSinduced colitis in rats [57], and inflammation-related tumor models [58, 59]. Phutthaphadoong et al. [58] presented that DSS-induced inflammation promoted the colorectal carcinogenesis in *Apc*Min/+ mice, but the increased severity was ameliorated by FBRA feeding. Onuma et al. [59] have demonstrated that FBRA prevents inflammation-related carcinogenesis in mice through inhibition of inflammatory cell infiltration.

Modifying effects of FBRA feeding on intestinal environment was investigated in rats and healthy human adults. Dietary FBRA increased resident *Lactobacillus* species in rat [60]. In healthy adults, significant effect of FBRA intake was not detected, but no adverse phenomenon was found in this clinical study at the used dose [37].
