Fermented brown rice and its uses

Abstract

The present invention relates to a brown rice fermentation product and its uses, more specifically, to the use of the brown rice fermentation product to moisten the intestines, improve bowel movements, and contribute to the regulation of the intestinal microbiota. The present invention has found that the brown rice fermentation product can improve constipation, have NO removal effects, and down-regulate the relative expression levels of the nos1, nos2a, and nos2b genes. Treatment with the brown rice fermentation product can regulate the species richness of the intestinal microbiota of constipated zebrafish, with certain effects on species uniformity and diversity, increasing the abundance of beneficial bacteria (Bacteroidetes) and decreasing the abundance of harmful bacteria (Firmicutes, Aeromonas, Enterobacteriaceae). The brown rice fermentation product of the present invention can be widely used in the food industry as a functional ingredient.

Description

[Technical Field]

[0001] The present invention relates to the technical field of functional substance research, specifically to a brown rice fermented product and its uses, and more specifically to the use of the brown rice fermented product in improving constipation and intestinal flora. [Background technology]

[0002] Rice is the third most produced agricultural product in the world (Reference 1). Brown rice is whole grain rice, with the outer tissues remaining, consisting of the bran layer, germ, and endosperm. Brown rice contains nutrients such as starch, protein, fat, minerals, and vitamins, as well as dietary fiber, oryzanol, glutathione, gamma-aminobutyric acid, and rice bran polysaccharides, of which crude fiber content is 1.48% (References 2, 3, 4). Dietary fiber is a non-starch polysaccharide that effectively exerts physiological functions such as preventing gastrointestinal diseases, promoting intestinal peristalsis, improving constipation, improving the diversity of intestinal microflora, and lowering blood sugar and blood lipid levels (Reference 5).

[0003] However, brown rice is covered with a hard seed coat, which means many nutrients are bound and difficult to absorb. In particular, brown rice contains a high amount of cellulose, which makes it difficult to cook during processing and cooking, limiting its texture. Current research has shown that treating brown rice using a fermentation process not only improves its flavor but also imparts new nutritional value to it (Reference 6). Reference 13 discloses a brown rice fermented product that improves the flavor of brown rice, enhances and increases the content of water-soluble dietary fiber, enriches it with probiotic metabolites, and increases the content of VB1, VB2, VE, amylase, protease, and lipase, thereby enriching it with multiple active ingredients that can be used in various products, and a method for producing the same. Reference 14 discloses a method for producing a brown rice fermented concentrate for use in cosmetics, and the resulting product. The method provides a short production cycle, stable product quality, high active ingredient content, and whitening effects, making it suitable for use in cosmetics. Cited Document 15 discloses a method for producing a fermented rice beverage that employs mixed fermentation with lactic acid bacteria, double enzyme treatment, and ultra-high pressure treatment to improve the product's unique taste and aroma. Cited Document 16 discloses a fermented rice extract and its production method and uses, and it has been confirmed that the fermented rice product obtained in this manner has strong anti-inflammatory and allergy effects and skin repair effects.

[0004] Meanwhile, in recent years, zebrafish have been widely used as a novel, cost-effective animal model for evaluating the efficacy and human health and safety of food and pharmaceutical products (Reference 7). Zebrafish have a similar gastrointestinal structure to humans, consisting of endothelial cells, connective tissue, external longitudinal muscle, and circular muscle. Furthermore, the transparent body of larval zebrafish allows for easy observation of the entire gastrointestinal tract (Reference 8). Aluminum sulfate can be used to induce a constipation model in zebrafish. Aluminum sulfate absorbs water in the intestine, forming crystal water, which not only leads to stool drying but also inhibits intestinal peristalsis and propulsion, ultimately causing constipation (Reference 17).

[0005] Furthermore, neurotransmitters in the large intestine control colonic motility, and nitric oxide (NO), as the primary neurotransmitter affecting intestinal motility, plays an important role in the functional activity of the gastrointestinal tract (Reference 9). Some current studies have shown that NO regulates intracellular Ca2+ levels. 2+ Decreasing the concentration of Ca 2+ It has been shown that oxidative stress relaxes smooth muscle by reducing sensitivity, potentially inhibiting intestinal motility and causing constipation (References 10 and 11). Reference 12 discloses compositions and methods for treating constipation and other gastrointestinal disorders, in which intestinal NO concentration is used as an indicator of a drug's ability to improve constipation and other gastrointestinal disorders. Nitric oxide synthase (NOS) is a key enzyme in endogenous NO production. Currently, three types of NOS are known: endothelial (eNOS or NOS3), neuronal (nNOS or NOS1), and cytokine-induced (iNOS or NOS2) (Reference 18). Research has shown that increasing the expression of glial cell-derived neurotrophic factor (GDNF) mRNA and decreasing NOS mRNA expression can promote intestinal motility (Reference 19).

[0006] Other studies have shown that dysbiosis of the gut microbiota may influence the occurrence of constipation. In patients with constipation, the gut microbiota is disrupted, often manifesting as a decrease in the abundance of bacterial flora, a significant decrease in the abundance of beneficial bacteria, and a significant increase in the abundance of pathogenic bacteria (Reference 20). Dysbiosis of the gut microbiota causes abnormalities in the gut microbiota and its metabolites, which in turn leads to abnormal intestinal motility (Reference 21).

[0007] Currently, there is no verifiable public information regarding the gastrointestinal environment-improving effects and mechanisms of fermented brown rice, and there are no reports on the improvement and adjustment of the intestinal flora by fermented brown rice. [Prior art documents] [Patent documents]

[0008] Citation 1: Punia S, Sandhu KS, Grasso S, Singh Purewal S, Kaur M, Kumar Siroha A, Kumar K, Kumar V, Kumar M. Aspergillus oryzae Fermented Rice Bran: A Byproduct with Enhanced Bioactive Compounds and Antioxidant Potential. Foods. 2020 Dec 31;10(1):70. Citation 2: Yang Y, Guo M, Sun S, et al. Natural variation of OsGluA2 is involved in grain protein content regulation in rice[J]. Nature Communications, 2019, 10(1). Citation 3: OECD, Revised consensus document on compositional considerations for new varieties of rice (oryza sativa): key food and feed nutrients, anti-nutrients and other constituents, 2016, Organisation for Economic Co-operation and Development, Paris Citation 4: Lü Chengwei, Yue Yulan, Wang Zheng, Li Zhuolin, Li Tiezhu, Hu Jimei. "Research Progress on the Nutritional Value and Processing Technology of Brown Rice" [J]. Science and Technology of Cereals, Oils and Foods, 2020, 28(6):5. Citation 5: Huang Suya, Qian Bingjun, Dou Yun. "Research Progress on the Function of Dietary Fiber" [J]. Food Industry, 2016(1):5. Citation 6: Gallo M, Nigro F, Passannanti F, et al. Rice Fermentation by Lactobacillus Paracasei CBA L74[J]. 2018. Reference 7: Mcgrath P. Zebrafish: Methods for Assessing Drug Safety and Toxicity[M]. John Wiley & Sons, 2011. Reference 8: Rich A. A new high-content model system for studies of gastrointestinal transit: the zebrafish[J]. Neurogastroenterol Motil, 2009(3). Cited document 9: Idrizaj E, Traini C, Vannucchi M, et al.Nitric Oxide: From Gastric Motility to Gastric Dysmotility.[J].International journal of molecular sciences, 2021, 22(18). Reference 10: Fan Yufeng, Jiang Ming, Huang Xueqin et al. "Nitrogen monooxygenate and slow-transit constipation" [J]. Chinese Journal of Proctology, 2016(2):3. Reference 11: Dang Getsu, Tian Mengyuan, Wang Chengxiang et al. "Study on the Intestinal Moisturizing and Constipation-Improving Effects and Mechanisms of Moringa oleifera Leaf Extract in Constipated Mice" [J]. Chinese Herbal Medicine, 2021, 52(14):7. Reference 12: Borody, Thomas Julius. "Laxative compositions and methods for treating constipation and related gastrointestinal disorders and symptoms": CN201380056438.5[P][2023-08-28]. Cited document 13:CN202111559673.7; Cited document 14:CN201810171483.X; Cited document 15:CN201410261208.9; Cited document 16:CN202010254708.5; Reference 17: Talley NJ, Jones M, Nuyts G, et al.Risk factors for chronic constipation based on a general practice sample[J].American Journal of Gastroenterology, 2003, 98(5):1107-1111. Reference 18: Knyushko TV, Sharov VS, Williams TD, et al.3-Nitrotyrosine Modification of SERCA2a in the Aging Heart: A Distinct Signature of the Cellular Redox Environment[J].Biochemistry, 2005, 44(39):13071-81. Reference 19: Fan Yihong, Xu Guoping, Feng Wen et al. "Increasing colonic ink propulsion velocity in slow-transit constipated rats by elimination of fecal matter," and its effect on GDNF and NOS mRNA expression [J]. Chinese Journal of Integrated Traditional Chinese and Western Medicine, 2012, 32(4):4. Reference 20: Shi Min, Liu Fulin, Xia Xuting, Liao Chenmin. "Research progress of traditional Chinese medicine in the treatment of slow-transit constipation by adjusting intestinal flora." [J]. China Medical Guide, 2022, 19(32):47-50. Reference 21: Choi CH, Chang SK. Alteration of Gut Microbiota and Efficacy of Probiotics in Functional Constipation[J]. Journal of Neurogastroenterology & Motility, 2015, 21(1):4-7. Summary of the Invention [Problem to be solved by the invention]

[0009] Currently, there is a great deal of research being conducted on fermented brown rice products, but most of the research focuses on production methods. A few existing techniques have investigated the skin-improving effects of fermented brown rice products and applied them to the cosmetics field. However, research on fermented brown rice products still has room for development.

[0010] Based on the above-mentioned research on the functions of brown rice fermentation products in conventional technology, the present invention has investigated the possible biological functions of brown rice fermentation products and newly discovered that brown rice fermentation products have a clear effect of improving constipation, and also unexpectedly found that brown rice fermentation products have a clear regulating effect on the disruption of intestinal flora that causes or is caused by constipation. [Means for solving the problem]

[0011] The present invention has found that the above problems can be solved by the following inventions.

[0012] [1]. Use of a brown rice fermentation product in the preparation of a food or health food that moistens the intestines, improves bowel movements, and / or contributes to the regulation of intestinal flora, wherein the method for preparing the brown rice fermentation product comprises: an inoculation step of placing brown rice in a fermentation vessel and adding 0.1 to 1% of fermentation bacteria based on the mass of the dried brown rice; and a fermentation step of sealing the fermentation vessel and then carrying out fermentation at a fermentation temperature of 22 to 37°C for a fermentation time of 30 to 45 hours. [2] The use according to [1], characterized in that the brown rice has been steamed and cooked until the grains are plump, the outside is hard, the inside is soft, and there is no core left. [3] The use according to [1] or [2], characterized in that the fermentation vessel has a vessel volume of 10 to 50 L per 1 kg of dried brown rice and the thickness of the brown rice in the vessel is 20 cm or less. [4] The use according to any one of [1] to [3], characterized in that the fermentation bacteria include Rhizopus. [5] The use according to any one of [1] to [4], wherein the moistening of the intestines and improvement of bowel movements and / or contribution to the regulation of the intestinal flora includes one or more of the following: improvement of constipation, removal of intestinal NO, downregulation of NOS gene expression, increase in the abundance of beneficial intestinal bacteria, and decrease in the abundance of harmful intestinal bacteria. [6] The use according to [5], characterized in that the NOS gene includes one or more of the nos1 gene, the nos2a gene, and the nos2b gene. [7] The use according to [5] or [6], characterized in that the beneficial intestinal bacteria include probiotics of the Bacteroidetes phylum. [8] The use according to any one of [5] to [7], characterized in that the harmful intestinal bacteria include one or more of bacteria from the phylum Firmicutes, Aeromonas, and Enterobacteriaceae. [9]. Use of an edible product that moisturizes the intestines, improves bowel movements, and / or contributes to regulating the intestinal flora, characterized in that the edible product contains the brown rice fermented product described in any one of [1] to [8] or is prepared from the brown rice fermented product described in any one of [1] to [8].

[10] . The use according to [9], characterized in that the content of the brown rice fermented product is 1 to 90 mass % based on the total mass of the edible product. [Effects of the Invention]

[0013] The present invention can achieve the following effects based on the implementation of the above aspects. Experimental data from the present invention show that the brown rice fermented product provided by the present invention effectively prevents intestinal constipation (i.e., moistens the intestines and improves bowel movements) and improves the composition and abundance of intestinal microbiota (i.e., contributes to the regulation of intestinal microbiota). Specific effects include: alleviating constipation, having an NO scavenging effect, and down-regulating the relative expression levels of nos1, nos2a, and nos2b genes. Treatment with brown rice fermented product can improve intestinal microbiota disorders and regulate the species richness of the intestinal microbiota of constipated zebrafish, with certain effects on species uniformity and diversity. Treatment with brown rice fermented product can change the composition of the intestinal microbiota of constipated zebrafish, increasing the abundance of beneficial bacteria (Bacteroidetes) and decreasing the abundance of harmful bacteria (Firmicutes, Aeromonas, Enterobacteriaceae). Overall, fermented brown rice products (such as freeze-dried fermented brown rice powder) have the function of preventing constipation by reducing NOS gene expression and can improve intestinal bacterial flora disorders caused by constipation.

[0014] The fermented brown rice product of the present invention has a wide range of uses as a functional ingredient, and can be used in a variety of carriers, including foods, health foods, pharmaceuticals, and cosmetics, and in dosage forms such as powders, tablets, granules, oral liquids, capsules, gummy candies, beverages, dairy products, soy milk, etc. Oral administration of the product can fill the gap in products with constipation prevention efficacy. [Brief explanation of the drawings]

[0015] [Figure 1] Representative images of the fluorescence intensity in the intestinal tract of zebrafish after treatment with freeze-dried powder of fermented brown rice. The dashed box indicates the analyzed area of ​​the intestine of zebrafish. [Figure 2] Analysis of the fluorescence intensity in the intestinal tract of zebrafish after treatment with freeze-dried powder of fermented brown rice. *** indicates p<0.001 compared with the model control group. [Figure 3] Representative images of NO fluorescence intensity in the intestine of zebrafish after treatment with freeze-dried powder of fermented brown rice. The dashed box indicates the analyzed area of ​​the intestine of zebrafish. [Figure 4] Analysis of NO fluorescence intensity in the intestines of zebrafish after treatment with freeze-dried powder of fermented brown rice. *** indicates p<0.001 compared with the model control group. [Figure 5] 1 is an analytical graph of the relative expression level of the nos1 gene. [Figure 6] 1 is an analytical graph of the relative expression level of the nos2a gene. [Figure 7] 1 is an analytical graph of the relative expression level of the nos2b gene. [Figure 8A] This is an analysis of the species diversity of the gut microbiota. The vertical axis is the Shannon index. In the box plot, the lines at both ends of the box represent the upper and lower quartiles (interquartile range, IQR), the center line is the median, the upper and lower ends represent the maximum and minimum values ​​of the range (1.5 times the IQR), and points outside the upper and lower ends represent outliers. The numbers on the lines between the columns are the P values ​​of the T-test (if the P value is greater than 0.05, it is not displayed by default). [Figure 8B] This is an analysis of the species diversity of the gut microbiota. The vertical axis is Simpson's index. In the box plot, the lines at both ends of the box represent the upper and lower quartiles (interquartile range, IQR), the center line represents the median, the upper and lower ends represent the maximum and minimum values ​​of the range (1.5 times the IQR), and points outside the upper and lower ends represent outliers. The numbers on the lines between the columns are the P values ​​of the T-test (if the P value is greater than 0.05, it is not displayed by default). [Figure 9] PCA analysis diagram. The horizontal axis represents the first principal component, and the percentage represents the contribution of the first principal component to the sample difference. The vertical axis represents the second principal component, and the percentage represents the contribution of the second principal component to the sample difference. [Figure 10] PCoA analysis diagram. The horizontal axis represents the first principal component, and the percentage represents the contribution of the first principal component to the sample difference. The vertical axis represents the second principal component, and the percentage represents the contribution of the second principal component to the sample difference. [Figure 11]NMDS analysis diagram. The horizontal axis represents the first principal component, and the percentage represents the contribution of the first principal component to the sample difference. The vertical axis represents the second principal component, and the percentage represents the contribution of the second principal component to the sample difference. Each point in the diagram represents a sample, and different colors represent different groups. A stress of less than 0.2 indicates a certain degree of reliability in the NMDS analysis, and the closer samples are to each other on the coordinate graph, the higher the similarity. [Figure 12] Figure 1 shows the gut microbial compositional changes at the phylum level for each sample. The horizontal axis shows the sample name (1, 2, and 3 represent three replicates each), and the vertical axis shows the percentage of relative abundance. Different colors represent different species, and stacked columns show the top 10 taxa by relative abundance at each taxonomic level. [Figure 13] Figure 1 shows the compositional changes of gut microbes at the order level for each sample. The horizontal axis shows the sample name (1, 2, and 3 represent three replicates each), and the vertical axis shows the percentage of relative abundance. Different colors represent different species, and stacked columns show the top 10 taxa by relative abundance at each taxonomic level. [Figure 14] Figure 1 shows the gut microbial compositional changes at the genus level for each sample. The horizontal axis shows the sample name (1, 2, and 3 represent three replicates each), and the vertical axis shows the percentage relative abundance. Different colors represent different species, and stacked columns show the top 10 taxa by relative abundance at each taxonomic level. [Figure 15] Phylogenetic tree of species from phylum to genus (LEfSe analysis). The circles radiating from the inside to the outside of the cladogram represent the taxonomic ranks from phylum to species. Each small circle at a different taxonomic rank represents a classification at that rank, and the diameter of the small circle is proportional to the relative abundance. Different colors represent different groups, and nodes of different colors represent microbial groups that play important roles within the group represented by that color. [Figure 16] This is a bar graph of the LDA value distribution (LEfSe analysis). The horizontal axis shows the LDA value obtained by LEfSe analysis, and the vertical axis shows the microbial groups that play important roles. [Figure 17A]This is an analysis of the characteristic bacterial flora (Methylobacterium genus and Beijerinchiaceae family) of the blank control group selected in Figure 16. The vertical axis is the LDA value of relative abundance. [Figure 17B] This is an analysis of the characteristic bacterial flora (Bacteroidetes and Rhizobiales) of the sample group selected in Figure 16. The vertical axis is the LDA value of relative abundance. [Figure 17C] This is an analysis of the characteristic bacterial flora (Gammaproteobacteria, Enterobacteriaceae, and Aeromonas) of the model control group selected in Figure 16. The vertical axis is the LDA value of relative abundance. DETAILED DESCRIPTION OF THE INVENTION

[0016] Although the present invention will be described below by way of example, it should be understood that the present invention is not limited to these examples. Furthermore, the present invention is not limited to the configurations described below, and various modifications are possible within the scope of the present invention. Examples obtained by appropriately combining the technical means described in different embodiments and examples are also included within the scope of the present invention.

[0017] In this specification, a numerical range expressed as "numerical value A to numerical value B" means a range including the limit values ​​A and B. In this specification, a range of values ​​expressed as "greater than or equal to" or "less than or equal to" refers to a range of values ​​that includes the value. In this specification, the term "may" includes both cases where some processing is performed and cases where some processing is not performed. As used herein, the terms "optionally" or "optional" refer to the use or non-use of a certain substance, component, step, application condition, or other element. Unless otherwise specified, "room temperature" as used herein generally means a temperature of 23±2°C. All unit names used in this specification are international standard unit names, and unless otherwise specified, "%" refers to the content by weight or mass %. As used herein, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "embodiments," etc., mean that the particular elements (e.g., features, structures, properties, and / or characteristics) described in connection with an embodiment are included in at least one embodiment described herein and may or may not be present in other embodiments. Furthermore, it is to be understood that such elements may be combined in any suitable manner in the various embodiments. Furthermore, unless otherwise defined, other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0018] The present invention aims to discover new functions of fermented brown rice through biological research and to expand the existing applications of fermented brown rice based on this research. The present invention was made mainly based on the following findings.

[0019] In this study, zebrafish were used as the research subject, and water containing a certain concentration of fermented brown rice was consumed. By analyzing the zebrafish's intestinal Nile red fluorescence intensity, intestinal NO fluorescence intensity, relative expression of NOS genes, intestinal microbiota 16S rRNA, and species composition at different taxonomic levels, it was found that fermented brown rice (freeze-dried powder) has the effect of preventing and improving constipation, as well as regulating the composition and abundance of the intestinal microbiota.

[0020] (fermented brown rice) In the present invention, the term "fermented brown rice" refers to a product obtained by fermenting brown rice. The specific type of brown rice is not particularly limited, but for example, one or more of Japonica rice, Indica rice, and glutinous rice can be used as the brown rice. In some specific embodiments, the present invention preferably uses Japonica brown rice.

[0021] In some embodiments, the method for preparing a brown rice fermented product includes: an inoculation step of placing brown rice in a fermentation vessel and adding 0.1 to 1% of fermentation bacteria based on the mass of the dried brown rice; After sealing the fermentation vessel, the fermentation process includes a fermentation step in which fermentation is carried out at a fermentation temperature of 22 to 37°C for a fermentation time of 30 to 45 hours.

[0022] In some embodiments, the brown rice is The rice is cooked in a steaming process until the grains are plump, firm on the outside and soft on the inside, with no core remaining.

[0023] I. Inoculation process: In some specific embodiments, the inoculation step involves placing cooked brown rice in a fermentation vessel and adding fermenting bacteria. Any fermentation vessel available in the food industry can be used. From the perspectives of optimizing the alcohol content, aroma, texture, and taste of the fermented product, moistening the intestines, further improving bowel movements, and regulating the intestinal flora, the fermentation vessel preferably has a volume of 10 to 50 L per kg of dried brown rice (i.e., a volume ratio of dried brown rice to air in the vessel of 1:19 to 1:49), preferably 15 to 45 L, and the thickness of the brown rice in the vessel is 20 cm or less, preferably 18 cm or less. Without being bound by theory, it is believed that a volume less than 10 L results in the production of a large amount of alcohol, while a volume greater than 50 L is thought to result in excessive oxygen supply, which is detrimental to fermentation. Furthermore, if the thickness of the brown rice in the vessel is too thick, the breathability will be reduced and a large amount of alcohol will be produced. On the other hand, while there is no particular lower limit for the thickness of the brown rice, a thickness that is too thin is undesirable because it increases production costs. Therefore, in the present invention, a thickness of 2 cm or more is preferable. In one embodiment, the fermentation vessel used has a vessel volume of 30 L per 1 kg of dried brown rice (i.e., the volume ratio of dried brown rice to air in the vessel is 1:29), and the thickness of the brown rice in the vessel is 15 cm.

[0024] The fermenting bacteria that can be used in the present invention are not particularly limited, and can be fermenting bacteria commonly used in food processing, such as Rhizopus and Aspergillus. From the perspective of reducing the alcohol content, Rhizopus is preferred. Without being bound by theory, Rhizopus can produce small amounts of alcohol-producing enzymes and has a certain alcohol-producing ability, allowing fermentation to occur simultaneously with saccharification. However, because its alcohol-producing ability is not high, it imparts a unique wine aroma to the sweet mash in addition to the rice aroma. The amount of fermenting bacteria added is preferably 0.1 to 1% of the dried brown rice mass, for example, 0.1%, 0.3%, 0.5%, 0.8%, or 1.0% of the dried brown rice mass. Addition of less than 0.1% of the dried brown rice mass is disadvantageous to fermentation, while addition of more than 1.0% of the dried brown rice mass increases the alcohol content and decreases the soluble solids content. In a specific embodiment, 0.5% of the mass of dried brown rice is added with Rhizopus, and in another specific embodiment, 1.0% of the mass of dried brown rice is added with Rhizopus.

[0025] II. Fermentation process: In some specific embodiments, the fermentation step involves sealing the inoculated fermentation vessel (e.g., by covering it with a film) and then carrying out fermentation. From the viewpoints of achieving a high soluble solids content, a low alcohol content, excellent aroma, texture, and taste, and of the fermented product moistening the intestines, further improving bowel movements, and regulating the intestinal flora, the fermentation temperature can be 22 to 37°C, preferably 25 to 35°C, and the fermentation time can be 30 to 45 hours, preferably 32 to 43 hours. In a specific embodiment, the fermentation temperature can be 28°C, and the fermentation time can be 36 hours. In another specific embodiment, the fermentation temperature can be 26°C, and the fermentation time can be 34 hours.

[0026] III. Rice cooking process: The rice cooking process increases the soluble solids in the fermentation product to a certain extent, moistening the intestines, further improving bowel movements, and more effectively regulating the intestinal flora. In some specific embodiments, the rice cooking process is a process of steaming brown rice. The rice cooking method is not particularly limited as long as the raw brown rice grains are plump and swelled, and the rice is steamed until the rice is hard on the outside and soft on the inside, with no core remaining. For example, rice can be cooked by adding water to brown rice and steaming it. The amount of water used during steaming is usually 0.5 to 2 times the mass of the dried brown rice. If the amount of water used is less than 0.5 times the mass of the dried brown rice, the brown rice will not be steamed sufficiently. If the amount of water used is more than 2 times the mass of the dried brown rice, not only will water resources be wasted, but the rice grains will become too soft and lose stickiness, making it difficult to maintain whole grains. The cooking temperature is typically 90 to 130°C, preferably 95 to 120°C, and may be, for example, 95°C, 100°C, 105°C, 110°C, 115°C, or 120°C. If the temperature is too low, the brown rice will not steam properly, resulting in a longer cooking time and wasting energy. If the temperature is too high, the actual production cost will increase and this is not preferred. The cooking time is not particularly limited as long as the rice grains are plump and swelled, with a hard outside and a soft inside, and the rice can be steamed until there is no remaining core, but it is typically 5 to 70 minutes, preferably 10 to 60 minutes. In one embodiment, the amount of water used during steaming is 1.5 times the mass of dried brown rice, the cooking temperature is 110°C, and the cooking time is 20 minutes.

[0027] Furthermore, from the viewpoint of making rice grains easier to steam and obtaining a fermentation product that is excellent in aroma, texture, and taste, it is preferable to use brown rice that has been treated in a pretreatment step as the brown rice used in the rice cooking step.

[0028] In some specific embodiments, the pretreatment step is a step of softening the brown rice to be fermented by absorbing water. The pretreatment method is not particularly limited as long as it allows the brown rice to be softened by absorbing water. For example, this pretreatment can be carried out by soaking rice in water at 15 to 25°C. The amount of water used for soaking is 1 to 5 times the mass of the dried brown rice, preferably 1 to 4 times the mass of the dried brown rice, and the soaking time is 1 to 12 hours, preferably 2 to 10 hours. In one embodiment, the amount of water used for soaking can be twice the mass of the dried brown rice, and the soaking time can be 6 hours.

[0029] Furthermore, from the viewpoint of improving the quality and texture of the product, the pretreatment may further include a step of removing impurities from the brown rice and washing it. Removing impurities refers to a step of removing impurities that affect the quality and texture of the product, such as straw, rice bran, and stones that are mixed in the brown rice. Washing refers to a step of washing with water, and can be carried out by removal and washing methods commonly used in food processing.

[0030] IV. Sterilization: Furthermore, the brown rice treated in the fermentation step is preferably sterilized in the sterilization step described below. In some specific embodiments, sterilization is a step of sterilizing the fermented brown rice. Sterilization can be performed by a method known in the food industry. A known sterilization method includes, for example, filling the fermented brown rice into a heat-resistant package and retorting it at 95 to 100°C for 10 to 30 minutes. In a specific embodiment, it is preferable to fill the fermented brown rice into a heat-resistant package and retort it at 100°C for 20 minutes. The heat-resistant package used in the present invention is not particularly limited as long as it can package the fermented brown rice and can withstand high-temperature heating without introducing impurities or unpleasant odors into the fermented brown rice, and heat-resistant packages commonly used in the food industry can be used.

[0031] The method for preparing the brown rice fermented product of the present invention may include other steps as necessary, as long as the effects of the present invention are not affected.

[0032] In the brown rice fermented product prepared by the above-described preparation method of the present invention, the soluble solids content is preferably 30 Brix or more, more preferably 35 Brix or more, and the alcohol content is preferably less than 0.5%.

[0033] In the present invention, the form of the brown rice fermentation product is not particularly limited, but examples include liquid (liquid obtained by filtering the brown rice fermentation product), semi-solid (aqueous slurry), powder (freeze-dried powder), granules, or block (freeze-dried block, or one obtained by pressing).

[0034] (edible products) The edible products according to the present invention comprise or are prepared from the brown rice fermentation product.

[0035] In some specific embodiments, the fermented brown rice product can be used in the edible product as a dry product, a solution, or a slurry, preferably as a dry product. Such a dry product typically has a moisture content of 3% by weight or less, preferably 2% by weight or less, and more preferably 1% by weight or less, and has a block or powdery appearance.

[0036] In addition to the above essential ingredients, the edible product according to the present invention may contain other optional food ingredients such as other plants or plant extracts (e.g., vegetables, fruits, grains, nuts, legumes, etc., and extracts thereof), animal ingredients (e.g., animal meat products, animal dairy products, etc.), microbial ingredients (e.g., probiotics, prebiotics, etc.), functional additive ingredients (e.g., vitamin supplements, mineral supplements, unsaturated fatty acid supplements, etc.), and optional food-acceptable additives (e.g., stabilizers, thickeners, sweeteners, emulsifiers, antioxidants, coloring agents, etc.), depending on the needs of the final product, and these ingredients may be used in liquid, solid, or semi-solid form.

[0037] The present invention is not particularly limited by the specific type of edible product, and examples include oral preparations (e.g., tablets, powders, granules, capsules, oral liquids, etc.), animal milk-derived products (e.g., liquid milk, milk powder, block milk, milk-containing beverages, etc.), plant milk-derived products (plant protein beverages such as soy milk and soy milk yogurt, etc.), candies (e.g., gummy candies, candy tablets, etc.), pasta products (e.g., bread, cakes, biscuits, noodles, steamed buns, steamed buns, dumplings, wontons, etc.), beverages (instant coffee, grain powders, nut powders, lotus root powders, fruit and vegetable powders, etc.), and the like. In principle, the content of the fermented brown rice product in the edible product is not particularly limited. From the viewpoints of satisfying nutritional needs, complying with relevant laws and regulations, and having the desired effect of moistening the intestines, improving bowel movements, and / or contributing to the regulation of the intestinal flora, the content of the fermented brown rice product is preferably 1 to 90% by mass based on the total mass (dry weight) of the edible product.

[0038] (Use to moisten the intestines, improve bowel movements and / or contribute to the regulation of intestinal flora) The present invention is the first to propose that suitable fermented brown rice can be used to moisten the intestines, improve bowel movements, and regulate the intestinal flora, and therefore the edible product also has the effects of moistening the intestines, improving bowel movements, and regulating the intestinal flora.

[0039] In some embodiments, moistening the intestines and improving bowel movements and / or contributing to the regulation of the intestinal microbiota as described in the present invention includes any one or more of the following: improving constipation, removing intestinal NO, downregulating the expression of NOS genes, increasing the abundance of beneficial intestinal bacteria, and decreasing the abundance of harmful intestinal bacteria.

[0040] In some specific embodiments, the NOS genes include any one or more of the nos1 gene, the nos2a gene, and the nos2b gene.

[0041] In some specific embodiments, the beneficial gut bacteria comprise probiotics of the Bacteroidetes phylum.

[0042] In some specific embodiments, the bad gut bacteria include any one or more of bacteria from the phylum Firmicutes, Aeromonas, and Enterobacteriaceae.

[0043] The moistening of the intestines to improve bowel movements and / or contribution to the regulation of the intestinal flora or improvement of constipation described in the present invention is not intended to prevent and / or treat diseases.

[0044] Furthermore, the edible product of the present invention is suitable in principle for everyone, but is particularly suitable for people who need to moisten the intestines, improve bowel movements, and / or regulate the intestinal flora, including, but not limited to, people who suffer from constipation, and the ingredients of the edible product can be adjusted to suit people with various characteristics. [Example]

[0045] The present invention will be further described below with reference to examples in order to more clearly explain the present invention, but the following examples are only a partial example of the present invention and are not intended to limit the present invention. Unless otherwise specified, all of the instruments, reagents, materials, experimental animals, etc. used in the present invention can be obtained by ordinary commercial means.

[0046] Example 1: Constipation prevention effect of freeze-dried powder of fermented brown rice 1. Test materials 1.1 Sample preparation Freeze-dried powder sample of fermented brown rice: After thoroughly soaking brown rice in water, the rice grains were steamed until they were plump, the outside was hard, the inside was soft, and there was no core remaining. After placing the rice in a fermentation vessel, 0.2% of Rhizopus powder (Angel Yeast Co., Ltd.) was added to the dried brown rice mass and fermented (fermentation temperature 28°C, fermentation time 36 hours). The resulting brown rice fermentation liquid was retort sterilized (temperature 100°C, retort time 20 minutes). Finally, the sterilized brown rice fermentation liquid was freeze-dried to obtain a freeze-dried powder of fermented brown rice. Unfermented brown rice flour sample: After threshing, the raw whole grain rice was repeatedly ground into powder in a rice mill and then filtered through a 120-mesh sieve to recover a powder with a uniform texture. Freeze-dried powder solution of fermented brown rice: Prepared with standard dilution water to a 20.0 mg / mL stock solution and used immediately after preparation. Unfermented brown rice flour solution: Prepared with standard dilution water to a 20.0 mg / mL mother solution and used immediately after preparation. Positive control sample: Motilium® Domperidone Tablets (hereinafter referred to as Domperidone), white tablets, Lot No. 190104499, Xian-Janssen Pharmaceutical Ltd., stored in a cool, dark place. Prepared with DMSO to a 10.0 mg / mL mother solution and stored at -20°C.

[0047] 1.2 Experimental animals All zebrafish were kept in culture water at 28°C (water quality: 200 mg of instant sea salt added per liter of reverse osmosis water; conductivity: 450–550 μS / cm; pH: 6.5–8.5; hardness: 50–100 mg / L CaCO3). They were bred and provided by the Aquaculture Center of Hangzhou Huante Biotechnology Co., Ltd., under the Experimental Animal Use Permit Number: SYXK (Zhejiang) 2022-0004, and their husbandry management met the requirements of the international AAALAC certification (certification number: 001458). Wild-type AB strain zebrafish were bred by natural mating. Using 5-day-postfertilization (5 dpf) zebrafish, the maximum detectable concentration (MTC) of freeze-dried powder of fermented brown rice was measured to assess its effects on preventing and alleviating constipation (NO removal), its effects on constipation-related genes, and its regulatory mechanism for intestinal health.

[0048] 1.3 Equipment, Consumables and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bioland Biotechnology Co., Ltd., China); conventional PCR amplification device (T100, BIO-RAD, Singapore); fluorescent quantitative PCR device (CFX Connect, BIO-RAD, Singapore); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); UV-visible spectrophotometer (Nanodrop 2000, Thermo, USA); microplate mini centrifuge (BE-6100, Haimen QiLinbeier Instruments Company, China); low-profile skirted 96-well plate (transparent) (HSP9601, Bio-rad, USA). Methylcellulose (Lot No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd, China); aluminum sulfate (Lot No. RH424739, Shanghai Yien Chemical Technology Co., Ltd, China); dimethyl sulfoxide (DMSO, Lot No. BCCD8942, Sigma, Switzerland); Nile Red (Lot No. SLBP9326V, Sigma, India); iTaq Universal SYBR Green Supermix (Cat. No. 1725124, Bio-Rad, USA).

[0049] 2. Method for evaluating the effect of preventing constipation Five dpf wild-type AB strain zebrafish were randomly selected and randomly divided into six-well plates, with 30 zebrafish per well (experimental groups). Lyophilized powder of fermented brown rice (concentrations shown in Table 1), unfermented brown rice flour at 1000.0 μg / mL, and domperidone (positive control) at 50.0 μg / mL dissolved in water were administered. A normal control group and a model control group were also established. Each well contained 3 mL of lyophilized powder. After 24 hours of incubation at 28°C, the lyophilized powder of fermented brown rice and unfermented brown rice flour were removed, and each experimental group was administered Nile Red in water to stain the intestines. After staining, all experimental groups except the normal control group were administered aluminum sulfate in water to establish a constipation model. After 6 hours of aluminum sulfate treatment, 10 zebrafish were randomly selected from each group and photographed under a fluorescence microscope. The images were then saved and analyzed using NIS-Elements D 3.20 advanced image processing software. The fluorescence intensity of the zebrafish intestine was analyzed, and statistical analysis of this index was used to evaluate the constipation prevention effect of freeze-dried fermented brown rice powder. Statistical results were expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, with p<0.05 indicating statistical significance.

[0050] 3. Evaluation results of constipation prevention effect Under these experimental conditions, freeze-dried powder of fermented brown rice was effective in preventing constipation. Analysis of the fluorescence intensity in the zebrafish intestine (Table 1, Figures 1 and 2) revealed that as the sample concentration increased from 250 to 1000 μg / mL, the fluorescence intensity signal in the intestine gradually weakened, demonstrating a favorable dose-effect relationship. After the sample concentration reached 655 μg / mL, the constipation prevention effect was significantly improved compared to the constipation model group (p<0.001), indicating that freeze-dried powder of fermented brown rice was effective in preventing constipation, with the lowest effective concentration being 655 μg / mL. No significant difference was observed between unfermented brown rice powder and the constipation model group.

[0051] [Table 1]

[0052] Example 2: Constipation prevention and NO removal effects of freeze-dried powder of fermented brown rice 1. Test materials 1.1 Sample preparation Freeze-dried powder sample of fermented brown rice: Prepared in the same manner as in Example 1. Unfermented brown rice flour sample: Prepared as in Example 1. Freeze-dried powder solution of fermented brown rice: Prepared in the same manner as in Example 1. Unfermented brown rice flour solution: Prepared as in Example 1. Positive control sample: Prepared as in Example 1.

[0053] 1.2 Experimental animals Same as Example 1.

[0054] 1.3 Equipment, Consumables and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bioland Biotechnology Co., Ltd., China); conventional PCR amplification device (T100, BIO-RAD, Singapore); fluorescent quantitative PCR device (CFX Connect, BIO-RAD, Singapore); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); UV-visible spectrophotometer (Nanodrop 2000, Thermo, USA); microplate mini-centrifuge (BE-6100, Haimen QiLinbeier Instruments) Company, China); low-profile skirted 96-well plate (transparent) (HSP9601, Bio-rad, USA); optical adhesive sealing film B (MSB1001, Bio-rad, USA). Methylcellulose (Lot No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd, China); aluminum sulfate (Lot No. RH424739, Shanghai Yien Chemical Technology Co., Ltd, China); dimethyl sulfoxide (DMSO, Lot No. BCCD8942, Sigma, Switzerland); nitric oxide detection probe (Lot No. D3308010, Yeasen Biotechnology (Shanghai) Co., Ltd, China); Nile red (Lot No. SLBP9326V, Sigma, India).

[0055] 2. Evaluation method for NO removal effect Five dpf wild-type AB strain zebrafish were randomly selected and randomly divided into six-well plates, with 30 zebrafish per well (experimental group). Lyophilized powder of fermented brown rice (concentrations of 250–1000 μg / mL, see Table 2 for details), unfermented brown rice flour at 1000 μg / mL, and domperidone (positive control) at 50 μg / mL were administered in water. A normal control group and a model control group were also administered. Each well contained 3 mL of lyophilized powder. After 24 hours of treatment at 28°C, the lyophilized powder of fermented brown rice and unfermented brown rice flour were removed. All experimental groups, except the normal control group, were administered aluminum sulfate in water to establish a constipation model. After 6 hours of treatment at 28°C, the samples were stained with a NO fluorescent kit. After staining, 10 zebrafish were randomly selected from each group and photographed under a fluorescent microscope. The images were then saved and analyzed using NIS-Elements D 3.20 advanced image processing software. The NO fluorescence intensity in the zebrafish intestine was analyzed, and the statistical analysis of this index was used to evaluate the constipation prevention (NO removal) effect of freeze-dried fermented brown rice powder. Statistical results were expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, with p<0.05 indicating statistical significance.

[0056] 3. Evaluation results of NO removal effect The NO fluorescence intensity in the zebrafish intestine was found to be weaker at sample concentrations between 500 and 1000 μg / mL compared to the model control, and the signal intensity gradually weakened as the sample concentration increased. This indicated that the freeze-dried powder of fermented brown rice had an NO removal effect and a favorable dose-response relationship compared to the model control. No significant differences were observed between unfermented brown rice flour and fermented brown rice powder compared to the model control, indicating that fermented brown rice was better able to remove NO fluorescence signal intensity. See Table 2 and Figures 3 and 4 for details.

[0057] [Table 2]

[0058] Example 3: Effect of freeze-dried powder of fermented brown rice on constipation-related genes 1. Test materials 1.1 Sample preparation Freeze-dried powder sample of fermented brown rice: Prepared in the same manner as in Example 1. Unfermented brown rice flour sample: Prepared as in Example 1. Freeze-dried powder solution of fermented brown rice: Prepared in the same manner as in Example 1. Unfermented brown rice flour solution: Prepared as in Example 1. Positive control sample: Prepared as in Example 1.

[0059] 1.2 Experimental animals Same as Example 1.

[0060] 1.3 Equipment, Consumables and Reagents Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); motorized focus continuous zoom fluorescence microscope (AZ100, Nikon, Japan); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Bioland Biotechnology Co., Ltd., China); conventional PCR amplification device (T100, BIO-RAD, Singapore); fluorescent quantitative PCR device (CFX Connect, BIO-RAD, Singapore); high-speed refrigerated centrifuge (Heraeus Fresco17, ThermoFisher, Germany); UV-visible spectrophotometer (Nanodrop 2000, Thermo, USA); microplate mini-centrifuge (BE-6100, Haimen QiLinbeier Instruments) Company, China); low-profile skirted 96-well plate (transparent) (HSP9601, Bio-rad, USA); optical adhesive sealing film B (MSB1001, Bio-rad, USA); fully automatic sample high-speed grinding device (JXFSTPRP-24L, Shanghai Allsheng Laboratory Equipment Science and Technology Department, China); fully automatic nucleic acid extraction device (Auto-Pure32A, Hangzhou Allsheng Instruments Co., Ltd., China). Methylcellulose (Lot No. C2004046, Shanghai Aladdin Biochemical Technology Co., Ltd., China); aluminum sulfate (Lot No. RH424739, Shanghai Yien Chemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, Lot No. BCCD8942, Sigma, Switzerland); nitric oxide detection probe (Lot No. D3308010, Yeasen Biotechnology (Shanghai) Co., Ltd., China); Nile Red (Lot No. SLBP9326V, Sigma, India); iTaq Universal SYBR Green Supermix (Cat. No. 1725124, Bio-Rad, USA); FastKing cDNA First-Strand Synthesis Kit (Genomic DNA-Free) (Lot No. X0320, Tiangen Biotech (Beijing) Co., Ltd., China); Universal RNA Extraction TL Kit C (Cat. No. TL2204001643C, Foshan Aowei Biotechnology Co., Ltd., China).

[0061] 2. Method for evaluating the effect on constipation-related genes First, after sample processing, zebrafish total RNA was extracted and the RNA concentration and A260 / A280 ratio were measured using a UV-visible spectrophotometer (Table 3) to assess the quality of zebrafish total RNA. Primer sequence information was then measured. Five dpf wild-type AB strain zebrafish were randomly selected and randomly divided into 6-well plates, with 30 zebrafish per well (experimental group). Lyophilized powder of fermented brown rice and unfermented brown rice flour (both at 1000 μg / mL) were administered, along with domperidone (50.0 μg / mL) dissolved in water as a positive control. A normal control group and a model control group were also established, with each well containing 3 mL of fluid. After 24 hours of treatment at 28°C, the lyophilized powder of fermented brown rice and unfermented brown rice flour were removed, and all experimental groups except the normal control group were administered aluminum sulfate dissolved in water to establish a constipation model. After 6 hours of incubation at 28°C, total RNA from each group of zebrafish was extracted using an automated nucleic acid extraction system. Total RNA concentration and purity were measured using a UV-visible spectrophotometer. 2.00 μg of total RNA from each zebrafish sample was used to synthesize 20.0 μL of cDNA according to the instructions of the cDNA first-strand synthesis kit. Expression of β-actin, nos1, nos2a, and nos2b genes was then detected by q-PCR. Relative RNA expression levels of nos1, nos2a, and nos2b genes were calculated using β-actin as an internal reference for gene expression. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS 26.0 software, with p<0.05 indicating statistical significance.

[0062] 3. Evaluation of effects on constipation-related genes 3.1 RNA extraction results and primer sequence information After sample processing was completed, zebrafish total RNA was extracted and the RNA concentration and A260 / A280 ratio were measured using a UV-visible spectrophotometer. The results are shown in Table 3. The A260 / A280 ratios were all in the range of 1.8-2.2, indicating that the extracted zebrafish total RNA was of good quality and could be used for subsequent q-PCR experiments. Primer sequences are listed in Table 4.

[0063] [Table 3]

[0064] [Table 4]

[0065] 3.2 Effects on constipation-related genes Total RNA from zebrafish samples was analyzed by q-PCR using a cDNA first-strand synthesis kit to detect the expression of the constipation-related genes nos1, nos2a, and nos2b. The results showed that freeze-dried powder of fermented brown rice could downregulate the relative expression levels of nos1, nos2a, and nos2b. In contrast, no significant difference was observed between unfermented brown rice flour and fermented brown rice compared to the model control group, indicating that fermented brown rice significantly reduces the relative expression levels of nos1 and nos2b. See Table 5 and Figures 5–7 for details.

[0066] [Table 5]

[0067] Example 4: Evaluation of intestinal flora 16S rRNA using freeze-dried powder of fermented brown rice 1. Test materials 1.1. Sample preparation Freeze-dried powder sample of fermented brown rice: Prepared in the same manner as in Example 1. Freeze-dried powder solution of fermented brown rice: Prepared in the same manner as in Example 1.

[0068] 1.2. Experimental animals All zebrafish were raised in culture water at 28°C (water quality: 200 mg of instant sea salt added per liter of reverse osmosis water, conductivity: 450-550 μS / cm; pH: 6.5-8.5; hardness: 50-100 mg / L CaCO3), and were provided by our aquaculture center. Experimental Animal Use Permit Number: SYXK (Zhejiang) 2022-0004, and breeding management met the requirements of the international AAALAC certification (certification number: 001458). Wild-type AB strain zebrafish were bred by natural mating at 5 dpf to study and evaluate the mechanism by which freeze-dried powder of fermented brown rice regulates gut health.

[0069] 1.3. Equipment, Consumables and Reagents A dissecting microscope (SZX7, OLYMPUS, Japan); a precision electronic balance (CP214, OHAUS, USA); a 6-well plate (Zhejiang Bioland Biotechnology Co., Ltd., China); a microplate reader (synergy HTX, GeneCompang Limited, China); an instant centrifuge (OSE-MC8, Tiangen Biotech (Beijing) Co., Ltd., China); a vortex mixer (vortex-2G560E, SCIENTIFICINDUSTRIES.INC, USA); and a gradient PCR apparatus (veriti96well9902, Appliedbiosystem, USA). Aluminum sulfate (Lot No. RH424739, Shanghai Yien Chemical Technology Co., Ltd., China); TGuide S96 Magnetic Soil / Fecal DNA Kit (DP812, Tiangen Biotechnology Co. Ltd., China); KOD FX Neo (TOYOBO) (KFX-201S, Beijing Biolink Biotechnology Co., Ltd., China); TransStart FastPfu Fly DNA Polymerase (AP231-12, TransGen Biotech Co., Ltd., China); Phusion HF MM (M0544L, Beijing Biolink Biotechnology Co., Ltd., China); VAHTS™ DNA Clean Beads (N411-03, Vazyme Biotech Co., Ltd., China).

[0070] 2. Evaluation method of intestinal microbiota 16S rRNA Five dpf wild-type AB strain zebrafish were randomly selected and randomly divided into six-well plates, with 30 zebrafish per well (experimental group). Each well had a volume of 3 mL. Ten replicates were set up in parallel. Lyophilized powder of fermented brown rice was dissolved in water and administered at a concentration of 1000 μg / mL. A normal control group (blank control group) and a model control group were also set up. Each well had a volume of 3 mL. After 24 hours of treatment at 28°C, the lyophilized powder of fermented brown rice was removed, and aluminum sulfate was dissolved in water and administered to all experimental groups except the normal control group to establish a constipation model. After treatment with aluminum sulfate for 6 hours, each experimental group was washed with ultrapure water to remove the chemical solution and then immediately transferred to a 1.5 mL EP (Eppendorf) tube (100 cells / tube). After all the liquid was sucked out, the tubes were immediately poured into liquid nitrogen for 3 minutes and stored at -80°C for subsequent detection of intestinal flora 16S rRNA.

[0071] The main steps for 16S rRNA detection are as follows: 1) Sequencing Data Quality Assessment (ASV Analysis): DNA was extracted from the collected samples and subjected to quality testing and sequence quality assessment. First, Trimmomatic was used to filter the raw data quality, then Cutadapt was used to identify and remove primer sequences. USEARCH was then used to assemble paired-end reads and remove chimeras to obtain high-quality sequences for subsequent analysis. Quality assessment was based on the Labchip Touch platform. Through an automated analysis and judgment process, the Labchip automatically identified main peaks, performed peak smear analysis, and determined whether the sequencing data quality was acceptable. If acceptable, the data could be used for subsequent library construction. 2) Species diversity analysis of gut microbiota (α diversity analysis): The Shannon index was calculated using mothur software, and the Simpson index was calculated using the Simpson diversity index formula. Given the same species richness, the greater the evenness of each species in the community, the higher the community diversity. The higher the Shannon and Simpson index values, the higher the species diversity of the sample. 3) Analysis of differences in microbial communities in each group (β diversity analysis): Principal component analysis (PCA), principal coordinate analysis (PCoA), correlation analysis between environmental factors and sample composition (RDA / CCA) were plotted based on the R language platform.

[0072] 3. Evaluation results of intestinal flora 16S rRNA 3.1 Analysis of gut microbiota species diversity As shown in Figures 8A-8B, the Simpson index and Shannoneven index were lower in the model control group compared to the normal control group (p<0.05), indicating a decrease in the species diversity of the gut microbiota of constipated zebrafish and an impact on species richness and evenness. The Simpson index and Shannoneven index were higher in the sample group compared to the model control group (p<0.05), indicating an increase in the species diversity of the gut microbiota of zebrafish after sample treatment, restoring species richness and evenness to those of the normal control group. These results demonstrate that sample treatment restores the species richness of the gut microbiota of constipated zebrafish and has a certain effect on species evenness and diversity.

[0073] 3.2 Analysis of differences in microbial communities in each group As shown in Figures 9, 10, and 11, we evaluated the differences between the microbial communities of each group using OTU-based principal component analysis (PCA), principal coordinate analysis (PCoA), and non-metric multidimensional scaling (NMDS). Each point in the figure represents a sample, and different colors represent different groups. The ellipses indicate the 95% confidence ellipses (i.e., if there are 100 samples in this sample group, 95 samples are distributed within the ellipse). The results showed that the samples in each group were significantly clustered, indicating that the gut microbiota of each group was consistent; there were differences in the microbiota composition between the normal control group and the model control group, indicating that the gut microbiota composition of the constipated zebrafish was disrupted; and the gut microbiota composition of the sample group differed from that of the model control group, indicating that the sample treatment altered the gut microbiota composition of the constipated zebrafish.

[0074] 3.3 Analysis of species composition at different taxonomic levels As shown in Figure 12, at the phylum level, the abundance of Proteobacteria and Bacteroidota decreased, and the abundance of Firmicutes and Actinobacteria increased in the samples of the model control group compared to the normal control group. Sample treatment had no recovery effect on Proteobacteria, but increased the abundance of Bacteroidetes and decreased the abundance of Firmicutes and Actinobacteria.

[0075] As shown in Figure 13, at the order level, the abundance of Enterobacterales was increased in the samples of the model control group compared to the normal control group, and treatment of the samples could reduce the abundance of Enterobacterales.

[0076] As shown in Figure 14, at the genus level, the abundance of Aeromonas was increased in the samples of the model control group compared to the normal control group, and sample treatment could reduce the abundance of Aeromonas.

[0077] Changes in the composition of gut microbiota at the phylum, order, and genus levels in each group indicated that the composition of the gut microbiota was specific among groups. Sample treatments were able to modulate the abundance of beneficial bacteria (Bacteroidetes) and harmful bacteria (Firmicutes, Enterobacteriaceae, and Aeromonas) in the zebrafish gut microbiota, which manifested as an increase in the abundance of Bacteroidetes and a decrease in the abundance of Firmicutes, Aeromonas, and Enterobacteriaceae.

[0078] 3.4 Analysis of differences in species composition within each group A linear discriminant analysis effect size (LEfSe) was used to compare groups and identify species with significantly different abundances between groups based on the rank order of linear discriminant analysis values ​​(LDAscore ≥ 2). A characteristic bacterial flora is defined as the bacterial flora with the highest LDA value among three groups: a normal control (blank control), a model control, and a sample. The characteristic bacterial flora of the normal control group included Methylolbacterium and Beijerinckiaceae. The characteristic bacterial flora of the model control group included Gammaproteobacteria, Enterobacteriales, and Aeromonas. The characteristic bacterial flora of the sample group included Bacteroidetes and Rhizobiales. The results are shown in Figures 15–17C. <Application example>

[0079] The following application examples provide methods for producing several food and health products using fermented brown rice. However, these are not intended to limit the scope of the present invention and are merely examples of the application of the present invention. Unless otherwise specified, all equipment, reagents, materials, edible ingredients, etc. used in the present invention are available through conventional commercial means. Here, the method for preparing the freeze-dried powder of fermented brown rice product is the same as in the above examples, and all "parts" are parts by weight, and all component contents (%) are percentages by weight.

[0080] <Application example 1> The soy milk beverage containing freeze-dried powder of fermented brown rice is prepared from the following components in the amounts listed below per 1000 parts of soy milk beverage. The ingredients used in the soy milk beverage of the present invention were 670 parts purified water, 200 parts soybeans, 80 parts freeze-dried powder of brown rice fermentation product, 5 parts machinine, 30 parts xylitol, and 15 parts grain powder. 200 parts soybeans were washed and retorted at high temperature. Then, 80 parts freeze-dried powder of brown rice fermentation product and 5 parts machinine were added and ground together. The mixture was homogenized (600 / 120 bar), 30 parts xylitol, and 15 parts grain powder were added to adjust the flavor. 670 parts purified water was added for standardization, thoroughly mixed, and homogenized (600 / 120 bar). After cooling, the mixture was UHT sterilized at 140-145°C for 4-6 seconds and aseptically filled to obtain brown rice enzyme soy milk. The content of the freeze-dried powder of brown rice fermentation product in the product was 8%.

[0081] <Application example 2> The freeze-dried milk block containing freeze-dried powder of fermented brown rice was prepared from the following components in the weight parts per 1000 parts of freeze-dried milk block: The ingredients used in the freeze-dried milk block of the present invention were 500 parts purified water, 150 parts milk powder, 250 parts freeze-dried powder of brown rice fermentation product, 90 parts granulated sugar, and 10 parts citric acid. The ingredients were thoroughly mixed and stirred for 25 minutes. The mixture was sterilized by ultra-high pressure treatment and cooled. After cooling, the mixture was poured into a mold and the solution was evenly spread into the mold with a spatula. The mixture was quickly frozen at -80°C for 12 hours, and the solid of the quickly frozen mixture was placed in a vacuum freeze dryer and freeze-dried for 24 hours. The final product was obtained after packaging. The freeze-dried powder of brown rice fermentation product in the product accounted for 25%.

[0082] <Application example 3> The triangular milk block containing freeze-dried powder of fermented brown rice was prepared from the following ingredients in the following weight parts per 1000 parts of the triangular milk block: The ingredients used in the triangular milk block of the present invention were 250 parts whole milk powder, 200 parts isomaltooligosaccharides, 200 parts cheese, 160 parts freeze-dried powder of fermented brown rice, 80 parts butter, 50 parts purified water, 40 parts cream, and 20 parts emulsifying salt. 250 parts whole milk powder, 200 parts isomaltooligosaccharides, 200 parts cheese, 160 parts freeze-dried powder of fermented brown rice, and 20 parts emulsifying salt were homogeneously premixed. The resulting premixed ingredients were homogeneously mixed with 80 parts butter, 50 parts purified water, and 40 parts cream, stirred and heated to 85-95°C, emulsified while keeping warm, and then rapidly cooled, formed into a sheet, sterilized by microwave sterilization, packaged, and stored refrigerated to obtain the final product. The freeze-dried powder of fermented brown rice in the product accounted for 16%.

[0083] <Application Example 4> The milk-containing beverage containing freeze-dried powder of fermented brown rice was prepared from the following weight parts of ingredients per 1000 parts of the milk-containing beverage. The ingredients used in the milk-containing beverage of the present invention are 600 parts purified water, 200 parts lactic acid bacteria fermentation liquid, 100 parts fruit and vegetable juice, 60 parts steviol glycoside, 20 parts freeze-dried powder of brown rice fermentation product, 10 parts pectin, and 10 parts L-ascorbic acid. The ingredients are thoroughly mixed and homogenized, and the homogenized raw material liquid is sterilized in a sterilizer. The sterilized raw material liquid is aseptically filled into packages to obtain the final product. The freeze-dried powder of brown rice fermentation product in the product accounts for 2%.

[0084] <Application example 5> The formula containing freeze-dried powder of fermented brown rice is prepared from the following ingredients in the following weight parts per 1000 parts of formula. The ingredients used in the formula of this invention are 350 parts skim milk powder, 260 parts whole milk powder, 300 parts desalted whey powder, 50 parts freeze-dried powder of brown rice fermentation product, 6 parts lecithin, 4 parts docosahexaenoic acid, 5 parts arachidonic acid, 5 parts taurine, 6 parts phosphatidylserine, 4 parts tryptophan, 5 parts lysine, 2 parts complex prebiotics, 1 part complex vitamin, and 2 parts complex mineral. The above ingredients were mixed uniformly, and the thoroughly mixed ingredients in a sterile environment were filled with nitrogen to obtain the final product. The freeze-dried powder of brown rice fermentation product in the product accounted for 5%.

[0085] <Application Example 6> Hard capsules containing freeze-dried powder of fermented brown rice are prepared from the following ingredients in the following weight parts per 1000 hard capsules: The raw materials used in the hard capsules of the present invention are 800 parts freeze-dried powder of fermented brown rice, 160 parts microcrystalline cellulose, and 20 parts magnesium stearate. After uniformly mixing the above raw materials, they were filled into gelatin capsule shells (the capsule shells are mainly gelatin, with gelatin accounting for 20 parts), packaged, and the final product was obtained. The freeze-dried powder of fermented brown rice accounted for 80% of the product.

[0086] <Application Example 7> A dry powder supplement or solid beverage containing a freeze-dried powder of fermented brown rice is prepared from the following parts by weight of ingredients per 1000 parts of the dry powder supplement or solid beverage: The ingredients used in the dry powder supplement or solid beverage of the present invention are 345 parts isomaltooligosaccharide, 160 parts indigestible dextrin, 80 parts fructooligosaccharide, 400 parts freeze-dried powder of brown rice fermentation product, 5 parts Bifidobacterium lactis (BB-12), 4 parts Bifidobacterium lactis (HN019), and 6 parts Lactobacillus rhamnosus (HN001). The above ingredients were homogeneously mixed and thoroughly dry-blended in a sterile environment. The homogeneously mixed solid beverage powder was then packaged under nitrogen to obtain the final product. The freeze-dried powder of brown rice fermentation product in the product accounted for 40%.

[0087] <Application Example 8> Supplement tablets or candy tablets containing freeze-dried powder of fermented brown rice are prepared from the following weight parts of ingredients per 1000 parts of supplement tablets or candy tablets. The ingredients used in the supplement tablets or candy tablets of the present invention are 220 parts isomaltooligosaccharide, 100 parts total fruit and vegetable powder, 500 parts freeze-dried powder of brown rice fermentation product, 50 parts mixed fruit and vegetable enzymes, 40 parts cassia seed powder, 30 parts lotus leaf powder, 25 parts aloe powder, and 15 parts steviol glycoside. The ingredients are homogeneously mixed, granulated, dried, sieved, and mixed with 20 parts of additives (a mixture of microcrystalline cellulose, magnesium stearate, and silicon dioxide), with or without flavoring, and compressed into semi-finished products. The semi-finished granules are coated to obtain finished products, which are then packaged to obtain the final product. The freeze-dried powder of brown rice fermentation product in the product accounts for 50%.

[0088] <Application Example 9> The complex fruit and vegetable powder containing freeze-dried powder of fermented brown rice is prepared from the following ingredients in the following weight parts per 1000 parts of complex fruit and vegetable powder: The raw materials used in the composite fruit and vegetable powder of the present invention were 300 parts freeze-dried powder of fermented brown rice, 160 parts pear, 140 parts candied orange, 100 parts corn, 100 parts apple, 50 parts spinach, 50 parts carrot, 50 parts melon, and 50 parts broccoli. After washing, the raw fruits and vegetables were shredded. The 140 parts candied orange, 100 parts corn, 50 parts spinach, 50 parts carrot, and 50 parts broccoli were first steamed in a steamer for 10 minutes, and then mixed with 160 parts pear, 100 parts apple, and 50 parts melon to produce a vegetable juice. 300 parts freeze-dried powder of fermented brown rice was uniformly mixed with the vegetable juice and steamed for 10 minutes to obtain a concentrate, which was then spray-dried to obtain the final product. The freeze-dried powder of fermented brown rice in the product accounted for 30%. [Industrial Applicability]

[0089] The fermented brown rice products provided by the present invention can be widely used in the fields of food and health foods.

Claims

1. Use of fermented brown rice in the preparation of a food product that moistens the intestines, improves bowel movements, and / or contributes to the regulation of intestinal flora, The method for preparing the brown rice fermented product comprises: an inoculation step of placing brown rice in a fermentation vessel and adding 0.1 to 1% of fermentation bacteria based on the mass of the dried brown rice; and a fermentation step of sealing the fermentation vessel and then carrying out fermentation at a fermentation temperature of 22 to 37°C for a fermentation time of 30 to 45 hours.

2. The use according to claim 1, wherein the brown rice has been processed by a rice cooking process in which the grains are steamed until they are plump, the outside is hard, the inside is soft, and there is no core left.

3. The use according to claim 1 or 2, wherein the fermentation vessel has a vessel volume of 10 to 50 L per 1 kg of dried brown rice and the thickness of the brown rice in the vessel is 20 cm or less.

4. The use according to any one of claims 1 to 3, characterized in that the fermenting bacteria comprises Rhizopus.

5. The use according to any one of claims 1 to 4, wherein the moistening of the intestines and improving bowel movements and / or contributing to the regulation of the intestinal flora includes one or more of the following: improvement of constipation, removal of intestinal NO, downregulation of NOS gene expression, increase in the abundance of beneficial intestinal bacteria, and decrease in the abundance of harmful intestinal bacteria.

6. The use according to claim 5, characterized in that the NOS gene includes any one or more of the nos1 gene, the nos2a gene and the nos2b gene.

7. 7. The use according to claim 5 or 6, characterized in that the beneficial intestinal bacteria include probiotics of the Bacteroidetes phylum.

8. The use according to any one of claims 5 to 7, wherein the harmful intestinal bacteria include one or more of bacteria belonging to the phylum Firmicutes, Aeromonas, and Enterobacteriaceae.

9. Use of an edible product that moistens the intestines, improves bowel movements, and / or contributes to regulating the intestinal flora, characterized in that the edible product comprises the brown rice fermented product according to any one of claims 1 to 8 or is prepared from the brown rice fermented product according to any one of claims 1 to 8.

10. The use according to claim 9, characterized in that the content of the fermented brown rice product is 1 to 90 mass % based on the total mass of the edible product.

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