Rhizopus oryzae strain as well as composition and application thereof

Abstract

The invention discloses a novel rhizopus oryzae strain, a brewing microbial inoculum containing the rhizopus oryzae strain and application of the novel rhizopus oryzae strain in brewing of white spirit, yellow wine and rice wine. The rhizopus oryzae disclosed by the invention has the characteristics of strong oxidation resistance, high content of bioactive substances such as total phenols, high esterifying enzyme activity and strong capability of producing flavor substances such as ethyl acetate and amino acid, and the fermented wine has the advantages of high total ester content, high amino acid and organic acid content, aromatic flavor and the like; the wine-making microbial inoculum composition is used for fermentation of rice wine and white spirit, and can improve the product quality and yield of the white spirit, yellow wine and rice wine.

Description

Technical Field

[0001] The present invention belongs to the field of wine brewing technology, and specifically relates to Rhizopus oryzae, a solid bacterial agent containing the fungus, and the application of the Rhizopus oryzae and a composition thereof in the brewing of white wine, rice wine and yellow wine. Background Art

[0002] Rhizopus oryzae, also known as Rhizopus arrhizus (Rhizopus oryzae), is one of the important molds commonly found in various types of koji and mash. It is widely distributed in traditional fermentation processes, especially playing an important catalytic role in the saccharification and fermentation stages. Studies have shown that R. arrhizus has significant advantages in the yeast of multiple brands of rice wine, and has a profound impact on the quality of yeast and fermentation effect ( L. et al., Int. J. Food Microbiol., 2017; Yuan, H. et al., PLoS One, 2024).

[0003] Rhizopus oryzae can secrete a variety of enzymes (such as amylase, saccharifying enzyme and protease, etc.). Due to its excellent enzyme activity, Rhizopus oryzae has been widely used in the production of traditional Chinese fermented wines such as white wine, yellow wine and rice wine. In the brewing of white wine, especially in the production of Xiaoqu white wine, Rhizopus oryzae shows excellent adaptability in medium and low temperature fermentation environments. It promotes the production of alcohol and flavor substances by secreting amylase and pectinase, and synergizes with other microorganisms to enhance the taste and aroma of the wine. In the brewing of rice wine, yellow wine and Xiaoqu white wine, Rhizopus oryzae can not only promote saccharification and fermentation efficiency, but also produce aromatic substances and organic acids through metabolic activities, significantly improving the flavor and quality of the wine (Lei, Yan et al., Lwt, 2024; Liang, J. et al., Front Microbiol, 2024).

[0004] Amino acids are important precursors of higher alcohols in liquor, and higher alcohols and their derivatives are key aromatic substances in liquor, giving liquor a rich aroma layer (Zhou Henggang, Brewing Technology, 1998). Similarly, the quality of rice wine is closely related to its amino acid nitrogen content. The higher the amino acid nitrogen content, the higher the quality grade of rice wine (national standard for rice wine GB / T 13662-2018). In the process of brewing, acid proteases have multiple functions. They can not only dissolve fermentation raw materials and promote microbial growth, but also generate a variety of aromatic substances by decomposing proteins, thereby improving the yield and quality of wine (Tang Shengqiu et al., Brewing Technology, 2005). Adding an appropriate amount of acid protease can significantly increase the amino acid content in rice wine, promote the sensory enhancement and aroma formation of the wine body, and at the same time improve its nutritional value.

[0005] Organic acids, such as acetic acid and lactic acid, are important flavor components in white wine, yellow wine, and rice wine. Rhizopus oryzae can secrete a variety of enzymes and organic acids, such as acetic acid and lactic acid, during the fermentation process. In the fermentation of white wine, the role of Rhizopus oryzae is particularly critical, and the organic acids and higher alcohols produced by its metabolic activities lay the foundation for the flavor of white wine. In the brewing of yellow wine, Rhizopus oryzae not only promotes fermentation by secreting amylase and protease, but also produces a large amount of acetic acid and lactic acid in the process. These acids give yellow wine a mellower taste and improve its overall quality. Rhizopus oryzae produces abundant acetic acid and citric acid during the fermentation of rice wine, significantly increasing the acidity, and improving the flavor and sensory properties through synergy with other microorganisms. The wide application of Rhizopus oryzae in the brewing of white wine, yellow wine, and rice wine has significantly improved the flavor, acidity balance, and overall quality of these traditional fermented wines through its metabolic activity (Xiaowei Zheng et al., Science and Engineering of Chinese Liquor (Baijiu), 2023).

[0006] Esterification enzymes play a key role in the food industry, especially in traditional brewing processes such as rice wine and yellow wine. They effectively improve the aroma and flavor complexity of products by promoting the synthesis of ester compounds (Chen et al., Foods, 2023). In particular, Rhizopus strains with high esterification activity have shown great potential for enhancing the accumulation of flavor compounds (Zhu Hongxia et al., Bioprocessing, 2021). In addition, the synergistic co-fermentation of Rhizopus with other microorganisms can effectively increase the content of ester compounds in fermented wine, making the flavor of rice wine and yellow wine richer and more complex. Esterification enzymes generate esters by reacting with organic acids and alcohol. These esters give fermented products a rich fruity and floral aroma, making the overall flavor of the product more mellow and elegant (Huang Yan et al. 2021).

[0007] During the fermentation process, Rhizopus can significantly increase the content of functional compounds such as polyphenols and flavonoids, and enhance the antioxidant capacity. Studies have shown that solid-state fermentation can significantly improve the antioxidant activity in products. For example, after Rhizopus arrhizus HC-1 was used for solid-state fermentation of soybeans, the total phenol and total flavonoid content increased significantly, thereby improving the ABTS free radical scavenging ability (Jiaxu Chen et al., LWT-Food Sci. Technol., 2023). Similarly, fermentation of chickpeas with Rhizopus oryzae can significantly improve the extraction efficiency of polyphenolic compounds and improve the ABTS free radical scavenging ability (Ren Jiamin et al., China Condiments, 2024). In terms of the manufacture of functional rice wine, the better antioxidant capacity of rice wine fermented with the preferred Rhizopus oryzae indicates that different strains have differences in enhancing antioxidant capacity (Yang, Q. et al., Front. Microbiol., 2021). These research results show that Rhizopus plays an important role in improving the nutritional value and health care function of rice wine by improving the antioxidant activity and content of functional ingredients in the manufacture of functional rice wine.

[0008] Saccharomyces cerevisiae is an important microorganism widely used in a variety of traditional fermented wines such as yellow rice wine, white wine, and rice wine. During the fermentation process, Saccharomyces cerevisiae produces ethanol through the metabolism of sugars, and also produces a variety of flavor compounds, such as esters and alcohols, which contribute greatly to the aroma, flavor, and taste of the wine. The synergistic fermentation of yeast with microorganisms such as molds and lactic acid bacteria can significantly enhance the production of esters and organic acids during the fermentation process, thereby giving the wine a more complex and unique flavor. For example, artificial composite bacterial agents composed of Saccharomyces cerevisiae and Rhizopus oryzae have excellent performance in the brewing of yellow rice wine, significantly increasing the ester content in the wine, making the wine more fragrant and the flavor more coordinated (Xiao Fucai et al., China Brewing, 2024). These studies provide a scientific basis for the improvement of traditional brewing technology, which will help to further improve the quality and market competitiveness of brewing products.

[0009] Chinese patent (CN202010287643.4) reported a strain of Rhizopus, koji and rice wine, and a method for producing the koji. Chinese patent (CN201410676014.5) reported a microbial composite agent for food fermentation, which includes Rhizopus oryzae ZSM-003, Rhizopus oryzae ZSM-004 and Brettanomyces custersii ZSM-001. Chinese patent (CN202110026138.9) reported a strain of Rhizopus oryzae that degrades ethyl carbamate and its application. Chinese patent (CN202211375615.3) reported a process for increasing the content and activity of antioxidant substances in pine pollen by fermentation with Rhizopus oryzae, which increased the polyphenol, flavonoid content and antioxidant activity of pine pollen through fermentation, thereby improving the efficacy of pine pollen. Chinese patent (CN117327588A) reported a strain of Rhizopus oryzae HC-1 and its preparation method and application of solid-state fermented soybeans. Solid-state fermentation of soybeans by Rhizopus oryzae HC-1 can significantly improve the levels of total phenols, total flavonoids, and aglycone isoflavones in soybeans, and has high in vitro free radical scavenging activity and iron ion reduction ability. Chinese patent (CN202410395952.1) reported a method for preparing ultrafine powder of hemp seeds fermented by Rhizopus oryzae. The prepared ultrafine powder of hemp seeds fermented by Rhizopus oryzae can be used to prepare products such as hypolipidemic products, hypoglycemic products, and antioxidant products, and has the advantages of stronger efficacy, better free radical scavenging effect, and more conducive to production and application. Chinese patent (CN112011467A) reported Rhizopus oryzae AC01 and its application in bran koji preparation and liquor brewing. The strain improves the flavor and taste of the liquor by increasing the activity of saccharifying enzymes. Chinese patent (CN115491315A) reported space-bred Rhizopus oryzae CGMCC No.40234, which enhances fruity aroma in liquor production by increasing the content of isoamyl acetate. Chinese patents (CN117757640A) and (CN118165832A) reported the use of Rhizopus oryzae strains NX-2 and NX-1 in improving liquor production efficiency and quality. Patent (CN118308223A) reported the use of Rhizopus oryzae JLR005 in fermented glutinous rice production, aiming to improve product quality by increasing reducing sugar content and complex aroma. Patent (CN118546795A) reported the unique ability of Rhizopus delemar JSY-F002 in producing pyrazine flavor substances. Patent (CN118562627A) reports the application of Rhizopus oryzae SFM-1 in vinegar mash, with the focus on increasing the total acid content and improving the aroma.Chinese patent (CN118667667A) reported the application of Rhizopus americanus X2 in improving the production of Xiaoqu liquor, especially its contribution in reducing fusel oil content and increasing liquor yield.

[0010] The disadvantage of the existing Rhizopus oryzae patents is that the reports mainly focus on the saccharifying enzyme activity and fermentation flavor of the strains. The existing commercial Rhizopus oryzae strains do not produce bioactive substances such as total phenols and have low antioxidant capacity, and the strain functions are relatively single, that is, the existing Rhizopus oryzae strains have low ability to produce bioactive substances such as flavonoids, low ester production capacity, and weak antioxidant capacity. It is difficult to form a unique flavor in the brewing of white wine, yellow wine and rice wine. According to the existing retrieval, there is no report on the Rhizopus oryzae strains with high production of bioactive substances such as flavonoids, strong antioxidant capacity and ester production capacity. Summary of the invention

[0011] The purpose of the present invention is to provide a new Rhizopus oryzae strain, which has high antioxidant performance and high production of biological activities such as flavonoids and the ability to produce high wine-making flavor substances such as ethyl acetate, and can increase the total acid and amino acid content in fermented wine, and is of great value in improving the taste of white wine, yellow wine and other wines and improving the quality of wines.

[0012] Another object is to provide a brewing agent containing Rhizopus oryzae.

[0013] Another object of the present invention is to provide application of Rhizopus oryzae in wine fermentation.

[0014] Another object of the present invention is to provide a method for preparing brewing bacteria agent.

[0015] Another object of the present invention is to provide an application of a wine-making agent in wine-making.

[0016] In order to solve the above problems, the present invention adopts the following technical solutions:

[0017] On the one hand, the present invention discloses a Rhizopus arrhizus strain, which is Rhizopus arrhizus W17, which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20242897 and a deposit date of December 24, 2024.

[0018] The Rhizopus oryzae W17 strain disclosed in the present invention is isolated and screened from a brewing matrix in Guangxi Zhuang Autonomous Region. The strain has the following characteristics:

[0019] The morphological characteristics of Rhizopus oryzae W17 strain are: after culturing on YPD plates for 2-3 days, the colonies are white to grayish white, with a rough, slightly flocculent surface and clear edges. The spores of Rhizopus oryzae W17 are spherical or nearly round, and are distributed singly or in groups.

[0020] PCR amplification and sequencing of ITS rDNA of Rhizopus oryzae W17 strain showed that the ITS rDNA homology of strain W17 and Rhizopus arrhizus CICC 41164 (Rhizopus arrhizus = Rhizopus oryzae) reached 100%, confirming that W17 is a Rhizopus oryzae. The phylogenetic tree of the sequence is shown in Figure 2. Figure 1 shown.

[0021] On the other hand, the present invention discloses a brewing bacteria agent, including the bacteria of Rhizopus oryzae W17 and its metabolites.

[0022] On the other hand, disclosed is the use of a brewing bacteria agent in fermenting rice wine and white wine.

[0023] In another aspect, the present invention discloses a brewing composition comprising a Rhizopus oryzae strain.

[0024] Furthermore, the brewing composition comprises the following components:

[0025] Rhizopus oryzae W17: 1×10 5 cfu / g

[0026] Saccharomyces cerevisiae: 6×10 7 cfu / g

[0027] Among them, Rhizopus oryzae W17 is CCTCC NO:M20242897, and Saccharomyces cerevisiae is CCTCC NO:M2023450.

[0028] The invention also discloses application of the Rhizopus oryzae W17 bacterial agent composition in fermenting rice wine and white wine.

[0029] The reference strain Rhizopus oryzae CICC 41164 used in the present invention is listed in the catalog of the China Industrial Microbiological Culture Collection (CICC) and is in a public state. Researchers can apply to the collection center for access. Saccharomyces cerevisiae CCTCC NO: M2023450 is a patented preserved strain, which has been disclosed in Chinese patent 202310411161.9. Any qualified researcher can obtain it from the China Type Culture Collection.

[0030] The bacterial agent disclosed in the present invention can be in solid or liquid form. Preferably, the W17 Rhizopus bacterial agent in the present invention is solid.

[0031] The present invention also discloses a method for preparing a brewing bacteria agent, comprising the following steps:

[0032] (1) Activation of strains: Rhizopus oryzae strain W17 and Saccharomyces cerevisiae strain SY91 were inoculated into solid PDA medium, respectively. After the culture was completed, Rhizopus oryzae spores or yeast cells on the culture plate were scraped with a sterile inoculation loop, and sterile water was added to prepare a bacterial suspension with a concentration of 10 5 -10 6 cfu / mL.

[0033] (2) Solid culture: The solid culture medium is made of bran and rice flour, which are moistened with water, cooked and cooled to loosen; the activated Rhizopus oryzae W17 suspension and Saccharomyces cerevisiae SY91 are inoculated into the solid culture medium at a ratio of 4-8%, respectively, to obtain a solid culture, and the fermentation temperature is maintained at 25-35° C. for 2-3 days.

[0034] (3) Cold air drying and crushing: the fermented solid culture is dried in a cold air drying system, and after drying, the solid culture is crushed to obtain solid powdered Rhizopus oryzae W17 inoculant and Saccharomyces cerevisiae SY91 inoculant;

[0035] (4) Mixing: Mix the Rhizopus oryzae W17 agent and the Saccharomyces cerevisiae SY91 agent according to a certain ratio to obtain a brewing agent.

[0036] The fermented solid cultures of Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 were dried in a cold air drying system at 10°C, and the moisture content of the samples dropped below 10%. After drying, the cultures were crushed using a universal grinder to prepare solid powdered inoculants, which were then stored in sealed plastic bags. The number of live bacteria in the solid inoculant of Rhizopus oryzae W17 was 1.2×10 5 cfu / g, the bacterial count of Saccharomyces cerevisiae SY91 solid inoculum was 3.6×10 8 cfu / g.

[0037] (4) Mixing and packaging: The solid bacterial agents of Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 were uniformly mixed at a mass ratio of 5:1, packed in a plastic bag and sealed to prepare a brewing bacterial agent composition.

[0038] Furthermore, in step (1), Rhizopus oryzae strain W17 and Saccharomyces cerevisiae strain SY91 were inoculated in solid PDA and cultured at 28° C. for 24 hours.

[0039] Furthermore, in step (2), the solid culture medium has a formula of 70% bran and 30% rice flour, which is moistened with water to a humidity of 50%.

[0040] Furthermore, in step (3), the solid culture is dried in a 10° C. cold air drying system until the moisture content drops below 10%.

[0041] Furthermore, in step (3), after drying, the culture was crushed using a universal crusher to prepare a solid powdered bacterial agent, and then stored in a sealed plastic bag. The number of live bacteria in each sample of Rhizopus oryzae W17 solid bacterial agent was 1.2×10 5 cfu / g, the bacterial count of Saccharomyces cerevisiae SY91 solid inoculum was 3.6×10 8 cfu / g.

[0042] Furthermore, in step (4), the mass ratio of the mixture of Rhizopus oryzae W17 inoculant and Saccharomyces cerevisiae SY91 inoculant is 4-6:1, and the optimal ratio is 5:1.

[0043] Furthermore, in step (4), the mixture is packed into a plastic bag and sealed for storage after mixing.

[0044] Advantages of the present invention:

[0045] The Rhizopus oryzae W17 strain provided by the present invention is a new strain and has the following advantages:

[0046] 1. The strain has strong ability to produce bioactive substances such as total phenols and antioxidants. The total phenol and total flavonoids content of the fermentation product of the W17 strain were 726.76 mg / L and 215 μg / L, respectively, which were 81.8% and 58.2% higher than those of Rhizopus oryzae AGM. The ABTS free radical scavenging rate of the W17 strain reached 94.41%, much higher than the 58.94% of Rhizopus oryzae AGM.

[0047] 2. Rhizopus oryzae W17 strain has high esterase activity and strong ability to produce flavor substances such as ethyl acetate. The esterase activity of W17 strain is as high as 2539.4U / g, which is 4.85 times higher than that of Rhizopus oryzae AGM. The total volatile flavor substance content of W17 strain reaches 42486.09μg / kg, which is 70.36% higher than that of AGM strain. The content of ethyl hexadecanoate in the fermentation product of W17 strain reaches 756.66μg / kg, which is 72.96% higher than that of AGM strain. The content of acetoin in the fermentation product of W17 strain is 13159.35μg / kg, which is 316.8% higher than that of AGM strain.

[0048] 3. Rhizopus oryzae W17 strain has high protease activity, amino acid and total peptide content. The protease activity of W17 strain is 547.7U / g, which is 45.2% higher than that of the standard strain CICC 41164 of Rhizopus oryzae. The total amount of umami amino acids produced by solid-state fermentation of rice wine by W17 strain is 3228.93mg / L, which is 30.3 times the content of Rhizopus oryzae AGM. The total peptide content of the fermentation product of W17 strain is 1590.31mg / L, which is 99.1% higher than that of AGM.

[0049] 4. Rhizopus oryzae W17 strain has a strong ability to produce organic acids. The malic acid content of the W17 strain in solid-state fermented rice wine is 842.58 mg / L, which is 54.15% and 21.46% higher than that of Rhizopus oryzae AGM and the standard strain CICC 41164, respectively. The citric acid content produced by the W17 strain is 1322.49 mg / L, which is 238.6% and 211.1% higher than that of Rhizopus oryzae AGM and the standard strain CICC 41164, respectively.

[0050] 5. Advantages of combination fermentation: When combined with brewer's yeast, the amino acid nitrogen and ethyl acetate content in the rice wine sample fermented by the W17 brewing composition were higher than those in the control group, and the total acid and total ester of the fermented Xiaoqu liquor sample were higher than those in the control group. The fermented rice wine and liquor have good flavor and rich aroma.

[0051] That is, compared with the Rhizopus oryzae AGM strain, the Rhizopus oryzae W17 strain has the characteristics of high content of bioactive substances such as total phenols, strong antioxidant activity, and higher esterase and protease activity. It can significantly increase the content of flavor substances such as ethyl acetate and umami amino acids in wine samples, thereby significantly improving the taste and quality of fermented white wine, rice wine and yellow wine. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 Phylogenetic tree of ITS rDNA sequence of Rhizopus oryzae W17 strain DETAILED DESCRIPTION

[0053] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and cannot limit the scope of protection of the present invention.

[0054] CICC in this article refers to China Industrial Microbiology Culture Collection Center, and CCTCC refers to China Center for Type Culture Collection.

[0055] AGM is the abbreviation of the commercially available Rhizopus ammoniaci

[0056] Example

[0057] Example 1 Isolation and identification of Rhizopus oryzae W17 strain

[0058] 1.1 Isolation of Rhizopus oryzae W17 strain

[0059] Rice wine koji and fermentation matrix were purchased from multiple rice wine producing areas across the country. 10g of sample was weighed and added to 90mL of sterilized acidic PDA liquid medium (pH 3.0). The mixture was then enriched and cultured on a shaker at 30°C and 30rpm for 48 hours. Subsequently, 1mL of bacterial suspension was taken for serial dilution. The dilution was 10 -2 , 10 -3 and 10-4 The samples were spread on Martin-Bengal red medium, and each dilution was repeated 3 times. After 48 hours of incubation in a constant temperature incubator at 30℃, the colony morphology was observed. The colonies with white hyphae on the surface were selected, and after being confirmed as mold hyphae by microscopic examination, they were further separated and purified by spot inoculation. Through the results of colony morphology, microscopic observation of spore production structure and whether rhizoids were formed, 20 Rhizopus strains (numbered W1~W20) were isolated from samples such as wine koji, which were used for fermentation to detect the ability of each strain to produce bioactive substances and antioxidants.

[0060] The 20 isolated Rhizopus strains, 1 Rhizopus oryzae standard strain CICC 41164 and 1 Rhizopus oryzae commercial strain (AGM) were inoculated on PDA plates, and after activation, they were inoculated into 100 mL of glutinous rice culture medium with a sugar content of 30°P and cultured at 30°C for 72 hours. After fermentation, the total phenol and total flavonoid contents in the fermentation broth were determined using a kit as shown in Table 1. The results showed that among all the Rhizopus strains tested, the total phenol content of the fermentation product of the W17 strain was the highest, reaching 726.76 mg / L, which was 63.6% and 81.8% higher than the standard strain CICC 41164 and Rhizopus oryzae AGM, respectively; the total flavonoid content of the W17 strain was 215 μg / L, which was 98.0% and 58.2% higher than CICC 41164 and Rhizopus oryzae AGM, respectively. In addition, the antioxidant capacity of the fermentation broth was determined using a kit as shown in Table 1. The results showed that the ABTS free radical scavenging rate of strain W17 was 94.41%, which was 46.3% and 60.2% higher than those of CICC 41164 and Rhizopus oryzae AGM, respectively.

[0061] Table 1 Results of total phenols, flavonoids and antioxidant capacity of different Rhizopus strains fermented

[0062]

[0063]

[0064] The present invention deposits a new isolated Rhizopus arrhizus W17 strain in the China Center for Type Culture Collection, a depository designated by the State Intellectual Property Office, with a deposit number of CCTCC NO: M20242897 and a deposit date of December 24, 2024. The China Center for Type Culture Collection is referred to as CCTCC, located in Wuhan University, Wuhan, Hubei Province, zip code 430072, telephone: 027-68752319, Email: cctcc@whu.edu.cn.

[0065] 1.2 Identification of Rhizopus oryzae W17 strain

[0066] The W17 strain was identified by morphological observation, physiological and biochemical determination and ITS region gene sequence analysis. The Rhizopus oryzae standard strain CICC 41164 was used as a control in the identification. This strain is a common microbial strain, which is published in the China Industrial Microbial Culture Collection (CICC) and is available to scientists upon request.

[0067] 1.3.1 Observation of morphological characteristics of Rhizopus oryzae W17 strain

[0068] After culturing on PDA plates for 2 days, R. oryzae W17 showed the characteristics of unlimited growth of colonies, with grayish white colonies and fluffy hyphae. Microscopic observation showed that the W17 strain had typical root-like hyphae and branched sporangia, which were spherical with a diameter of 30-100 microns, and spores were mostly round or oval and uniform in size.

[0069] 1.3.2 Phylogenetic status identification by ITS region sequence analysis

[0070] PCR amplification was performed using the genomic DNA of the Rhizopus strain as a template to amplify its ITS region. The primers used were forward primer ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and reverse primer ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR reaction system was 50 μL, including 25 μL Taq DNA polymerase, 22 μL triple distilled water, 1 μL of each primer and 1 μL of extracted DNA template. The PCR cycle program was: pre-denaturation at 95°C for 5 minutes; 35 cycles, each cycle including 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for 1 minute; finally, extension at 72°C for 10 minutes and insulation at 16°C. The PCR product was verified by 0.8% agarose gel electrophoresis, and the samples with clear bands were sent to the sequencing company for sequencing.

[0071] The 500bp fragments obtained by sequencing were manually proofread by DNAStar software, and then homologous sequences were searched in the NCBI nucleic acid sequence database to analyze the relationship and phylogenetic status between the test strain and known Rhizopus strains. According to the results of homologous sequence comparison, the ITS region sequence of the model strain with the closest relationship to the experimental strain was selected, and the sequence was aligned using ClustalX software, and the phylogenetic tree was constructed using MEGA 11 software using the neighbor-joining method and 1000 Bootstrap tests. The results of molecular sequencing and homologous sequence comparison showed that the ITS region of Rhizopus oryzae W17 had a 100% homology with Rhizopus arrhizus, confirming that the W17 strain was Rhizopus arrhizus. Figure 1 A phylogenetic tree constructed based on the ITS region sequences is shown.

[0072] Example 2 Analysis of the ability of Rhizopus oryzae W17 strain to produce organic acids, esters and flavor substances 2.1 Comparison of the ability of Rhizopus oryzae W17 strain to produce organic acids

[0073] The Rhizopus oryzae W17 strain, the Rhizopus oryzae standard strain CICC 41164 and the AGM strain screened in Example 1 were inoculated into glutinous rice for solid-state fermentation. After the fermentation was completed, the organic acid content in the fermentation broth was determined using an Agilent high performance liquid chromatograph. The determination of organic acids was performed using an Agilent Polaris C18-A chromatographic column, using 97.5% of a 0.1% phosphoric acid aqueous solution and a 2.5% methanol solution as the mobile phase, and separation and analysis were performed under isocratic mobile phase conditions. The results show that as shown in Table 2, the malic acid content of the W17 strain is 842.58 mg / L, which is 54.15% and 21.47% higher than that of Rhizopus AGM and the standard strain CICC 41164, respectively. This shows that the W17 strain can produce more malic acid during the fermentation process, making the rice wine flavor more refreshing and refreshing. In addition, the citric acid content of W17 is 1322.49 mg / L, which is 211.1% higher than that of the standard strain CICC 41164. This shows that the W17 strain is able to ferment and produce higher levels of citric acid, significantly enhancing the sourness and antioxidant capacity of rice wine.

[0074] Table 2 Determination results of organic acid content produced by fermenting glutinous rice with different strains of Rhizopus oryzae (unit: mg / L)

[0075]

[0076] 2.2 Comparison of esterase and saccharifying enzyme activities produced by Rhizopus oryzae W17 strain

[0077] The activated Rhizopus oryzae W17 strain, CICC 41164 strain and Rhizopus oryzae AGM strain were inoculated into 100 mL of glutinous rice culture medium with a sugar content of 30°P, and cultured at 30°C for 72 hours. 10 mL of fermentation broth was centrifuged, the supernatant was collected, and the esterase and saccharification enzyme activities were determined by colorimetry. The results showed that as shown in Table 3, the esterase activity of the W17 strain was 2539.4 U / g, which was 385.3% and 423.5% higher than that of Rhizopus oryzae AGM and the standard strain CICC 41164, respectively; the saccharification enzyme activity was 163.8 U / g, which was equivalent to the enzyme activity of Rhizopus oryzae AGM, but 40.1% higher than that of CICC 41164. It shows that the W17 strain can more efficiently decompose starch and catalyze ester substances during the fermentation process.

[0078] Table 3 Results of determination of esterase and saccharifying enzyme activities of different strains of Rhizopus oryzae

[0079]

[0080] 2.3 Comparison of the ability of Rhizopus oryzae W17 strain to produce volatile flavor substances

[0081] The determination method of volatile flavor compounds is as follows: the volatile compounds in the fermentation end samples were extracted by headspace solid phase microextraction (HS-SPME) method and analyzed by Agilent 7890B gas chromatograph-5977B mass spectrometer detector (Agilent, USA). The extraction used SPME fiber (50 / 30μm DVB / CAR / PDMS, Supelco, USA) and separated on Agilent J&W DB-WAX UI capillary column (60m×0.320mm×0.25μm, Agilent, USA). The comparison of volatile flavor products of three strains of Rhizopus oryzae in glutinous rice solid culture medium (see Table 4) showed that the total volatile flavor content of strain W17 in glutinous rice solid culture medium reached 42486.09μg / kg, which was 70.3% and 186.1% higher than that of commercial Rhizopus AGM and standard strain CICC41164, respectively. Among them, the hexadecanoic acid ethyl ester content of the W17 strain was 756.66 μg / kg, which was 72.9% and 225.3% higher than that of the AGM and CICC 41164 strains, respectively; the acetoin content of the W17 strain was 13159.35 μg / kg, which was 316.8% and 764.4% higher than that of the AGM and CICC 41164 strains, respectively. The high levels of these components give the rice wine a rich fruity aroma, a soft wine aroma, and a rich flavor layer. The result of the sensory evaluation was that the aroma score of the W17 strain was 9 points, which was significantly higher than the 7 points of the commercial Rhizopus AGM and the 6 points of CICC 41164, and was the highest score among all strains.

[0082] Table 4 Comparison of the total volatile flavor substance content and aroma production capacity of glutinous rice fermented by different strains of Rhizopus oryzae (unit: μg / kg)

[0083]

[0084] Example 3 Comparison of protease activity and total peptide content produced by fermentation of Rhizopus oryzae W17 strain

[0085] Rhizopus oryzae W17 strain, standard strain CICC 41164 and Rhizopus oryzae AGM strain were inoculated for solid-state fermentation of glutinous rice at 28°C for 7 days. After the fermentation, the umami amino acids (such as glutamic acid and aspartic acid) in the fermentation broth were determined by liquid chromatography. The amino acid determination method is to first precipitate the supernatant with an equal volume of 5% trichloroacetic acid (w / v) at 4°C for 5 hours. The precipitate was separated by centrifugation (12,000rpm, 10 minutes), and the supernatant was filtered through a 0.22μm microporous membrane and analyzed using an Agilent 1260II high-performance liquid chromatograph. Amino acids need to be derivatized with o-phthalaldehyde before analysis, and the method is referred to the relevant literature (Gong et al., 2020; Liang et al., 2020). The results showed (see Table 5) that the total amount of umami amino acids in the W17 strain was the highest, reaching 3228.93 mg / L, which was 2932.7% and 4195.5% higher than that of the AGM and CICC 41164 strains, respectively.

[0086] In addition, the total peptide content in the fermentation broth was determined using a kit (see Table 5). The results showed that the total peptide content of the fermentation product of the W17 strain reached 1590.31 mg / L, which was 99.1% and 78.4% higher than that of the AGM and CICC 41164 strains, respectively. The protease activity of the W17 strain reached 547.7 U / g, which was 4.7% and 45.2% higher than that of the AGM and CICC 41164 strains, respectively.

[0087] Table 5 Determination results of umami amino acids in glutinous rice fermented by different strains of Rhizopus oryzae

[0088]

[0089]

[0090] Example 4 Preparation method of Rhizopus oryzae W17 brewing bacteria agent and composition thereof

[0091] (1) Activation of strains: Rhizopus oryzae strain W17 and Saccharomyces cerevisiae strain SY91 were inoculated into solid PDA medium and cultured at 28°C for 24 hours. After the culture was completed, Rhizopus oryzae spores or yeast cells on the culture plate were scraped with a sterile inoculation loop, and sterile water was added to prepare a bacterial suspension with a concentration of 10 5 -10 6 cfu / mL.

[0092] (2) Solid culture: The solid culture medium is formulated with 70% bran and 30% rice flour, which is moistened with water to a humidity of 50%, and then cooled and loosened after steaming. The activated Rhizopus oryzae W17 suspension and Saccharomyces cerevisiae SY91 are inoculated into the solid culture medium at a ratio of 4-8%, respectively, and the fermentation temperature is maintained at 25-35° C. for 2-3 days.

[0093] (3) Cold air drying and crushing: The fermented solid cultures of Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 were dried in a cold air drying system at 10°C, and the moisture content of the samples was reduced to below 10%. After drying, the cultures were crushed using a universal grinder to prepare solid powdered inoculants, which were then stored in sealed plastic bags. The samples were tested using the dilution plate counting method, and the number of viable bacteria in the solid inoculant of Rhizopus oryzae W17 was 1.2×10 5 cfu / g, the bacterial count of Saccharomyces cerevisiae SY91 solid inoculum was 3.6×10 8 cfu / g.

[0094] (4) Mixing and packaging: The solid bacterial agents of Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 were uniformly mixed at a mass ratio of 5:1, packed in a plastic bag and sealed to prepare a brewing bacterial agent composition.

[0095] Example 5 Application of Rhizopus oryzae W17 winemaking agent in solid-state yellow wine fermentation

[0096] Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 were mixed in a mass ratio (w / w) of 1:1, 2:1, 3:1, 4:1, 5:1 and 6:1, respectively, and inoculated into glutinous rice solid culture medium at 1% of the mass of glutinous rice, and solid-state yellow wine fermentation was carried out for 30 days. After the fermentation, the fermented wine samples were analyzed by gas chromatography. The results showed (Table 6) that when Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 were mixed and fermented in a ratio of 5:1, the ethanol content in the fermented wine sample was moderate, the content of ethyl acetate, phenylethanol, acetic acid, etc. was high, the proportion of flavor substances was coordinated, and the sensory score reached 95 points. This shows that the combination of W17 and SY91 in a ratio of 5:1 performed well in terms of flavor and alcohol content and was the best fermentation combination. This shows that after adding an appropriate proportion of Rhizopus oryzae W17 brewing bacteria to Saccharomyces cerevisiae, the yield of rice wine and the content of flavor substances were increased. The total higher alcohol content of the fermented wine sample was moderate, the taste was mellow, the body was full, and the overall performance was excellent.

[0097] Table 6 Analysis and sensory evaluation results of flavor substances and physicochemical indexes of rice wine fermented with W17 and SY91 in different proportions

[0098]

[0099] Example 6 Effect of combined fermentation of Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 on the quality of glutinous rice wine

[0100] The brewing bacterial agent composition comprising Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 prepared in Example 4 was selected for solid-state fermentation of rice wine, and the AGM strain was used to replace the W17 strain and mix with Saccharomyces cerevisiae SY91 to form a Rhizopus oryzae AGM combined bacterial agent. The inoculation was carried out according to the ratio of 2.5g combined bacterial agent to 250g glutinous rice. The glutinous rice was soaked for 5 hours and then boiled. The initial fermentation temperature was set to 28°C, maintained for 2 days and then dropped to 20°C, and fermented for 3 days. During the entire fermentation process, the material-water ratio was maintained at 1:1. After the fermentation was completed, the fermented wine sample was analyzed by gas chromatography. The results show (Table 7) that the combination of Rhizopus oryzae W17 and Saccharomyces cerevisiae SY91 performed best in sensory evaluation (including aroma and taste), with a sensory score of 95 points, which was higher than the sensory scores of the wine samples of the Rhizopus oryzae AGM group and the traditional Xiaoqu group. This shows that the combination of W17 strain and Saccharomyces cerevisiae SY91 showed good sensory quality in rice wine brewing. In addition, the amino nitrogen content in the rice wine sample fermented by the W17 Rhizopus combination was 0.48 g / L, which was 20.0% and 37.1% higher than that of the Rhizopus oryzae AGM group and the traditional Xiaoqu group, respectively. The ethyl acetate content in the rice wine sample fermented by the W17 Rhizopus combination reached 940.14 mg / L, which was 118.8% higher than that of the Rhizopus oryzae AGM group and the traditional Xiaoqu group, and 150.0% higher than that of the traditional Xiaoqu group. The increase of ethyl acetate gave the rice wine more fruity and refreshing feeling.

[0101] Table 7 Analysis of physical and chemical indicators and sensory evaluation results of rice wine fermented with commercial Rhizopus and W17 Rhizopus combination

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[0103]

[0104] Example 7 Application of Rhizopus oryzae W17 wine brewing combination bacterial agent in improving Xiaoqu liquor yield and quality 7.1 Test method:

[0105] The mash after distillation in the brewing workshop of a certain liquor company was spread out to cool and piled up at 250kg per group. The control group was inoculated with traditional distiller's yeast at a ratio of 6% of the feed amount. The two experimental groups were inoculated with traditional distiller's yeast at a ratio of 3% of the feed amount (reduced by 50%), and the W17 brewing bacteria composition (W17 group) and Rhizopus oryzae AGM brewing bacteria composition prepared in Example 4 were added respectively, and the inoculation ratio was 0.3% (volume percentage) of the feed amount, replacing 50% of the traditional distiller's yeast. After inoculation, the fermentation was carried out in the cellar for 15 days, and samples were taken for distillation. Three parallel samples were set for each treatment, and the alcohol components in the distilled samples were determined using a gas chromatograph. 7.2 Test results:

[0106] The results of gas chromatography analysis and sensory evaluation showed (Table 8) that the alcohol content of the fermented grains in the test group containing the W17 brewing bacteria composition reached 10.89% Vol, which was 4.41% higher than that of the Rhizopus oryzae AGM group. The ethyl acetate content of the W17 group was 591.58 mg / L, which was 60.9% higher than that of the Rhizopus oryzae AGM group, and the total acid and total ester were increased by 13.0% and 35.6% respectively, which had a significant effect on improving the flavor and taste of the original wine.

[0107] In the sensory evaluation, the W17 group received a high score of 95-100 points, with comments such as "mellow and sweet, rich and harmonious aroma, and full body". In contrast, the control group (traditional Xiaoqu) scored 80-85 points, with comments such as "mellow and sweet, light aroma, and strong irritation at the entrance"; the Rhizopus oryzae AGM group scored 85-90 points, with comments such as "mellow and sweet, strong aroma, and mellow body". This shows that the W17 brewing bacteria composition can significantly increase the total acid, total ester and other flavor components of liquor, making the aroma in the liquor more harmonious and the taste better.

[0108] Table 8 Gas chromatography analysis results of various components in fermented mash distilled liquor samples

[0109]

Claims

1. A Rhizopus oryzae strain, which can be used for brewing white wine, yellow wine and rice wine, characterized in that: The strain is Rhizopus arrhizus W17, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M20242897.

2. A brewing agent containing the Rhizopus oryzae strain according to claim 1, characterized in that: The active ingredients of the brewing bacteria agent include the bacteria of Rhizopus oryzae W17 and its metabolites.

3. A brewing composition, characterized in that: Contains the Rhizopus oryzae strain according to claim 1.

4. The brewing composition according to claim 3, characterized in that: The brewing composition comprises Rhizopus oryzae W17 and Saccharomyces cerevisiae; Among them, Rhizopus oryzae W17 is CCTCC NO: M20242897, and Saccharomyces cerevisiae is CCTCC NO:M2023450.

5. The brewing composition according to claim 3, characterized in that: The brewing composition is a solid bacterial agent.

6. A method for preparing a brewing bacteria agent, characterized in that: The following steps are involved: (1) Activation of strains: Rhizopus oryzae strain W17 and Saccharomyces cerevisiae strain SY91 were inoculated into solid PDA medium, respectively. After the culture was completed, Rhizopus oryzae spores or yeast cells on the culture plate were scraped with a sterile inoculation loop, and sterile water was added to prepare a bacterial suspension with a concentration of 10 5 -10 6 cfu / mL; (2) solid culture: the solid culture medium is made of bran and rice flour, which are moistened with water, cooked and cooled to loosen; the activated Rhizopus oryzae W17 suspension and Saccharomyces cerevisiae SY91 are inoculated into the solid culture medium at a ratio of 4-8%, respectively, to obtain a solid culture, and the fermentation temperature is maintained at 25-35° C. for 2-3 days; (3) Cold air drying and crushing: the fermented solid culture is dried in a cold air drying system, and after drying, the solid culture is crushed to obtain solid powdered Rhizopus oryzae W17 inoculant and Saccharomyces cerevisiae SY91 inoculant; (4) Mixing: Mix the Rhizopus oryzae W17 agent and the Saccharomyces cerevisiae SY91 agent according to a certain ratio to obtain a brewing agent.

7. The method for preparing the brewing bacteria agent according to claim 6, characterized in that: In step (3), the solid culture is dried in a cold air drying system until the moisture content drops below 10%.

8. The method for preparing the brewing bacteria agent according to claim 6, characterized in that: In step (4), the mass ratio of the mixture of Rhizopus oryzae W17 inoculant and Saccharomyces cerevisiae SY91 inoculant is 4-6:

1.

9. Use of the Rhizopus oryzae strain according to claim 1 in the brewing of white wine, yellow wine and rice wine.

10. Use of the brewing composition according to claim 3 in the brewing of white wine, yellow wine and rice wine.

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