Rhizopus arrhizus strain for brewing delicate flavor liquor and application thereof

Abstract

The application provides a rhizopus arrhizus strain for brewing delicate flavor liquor and application thereof, and belongs to the technical field of liquor-making microorganisms. The application provides a rhizopus arrhizus strain with excellent performance, which significantly improves the saccharification and aroma-producing double functions of the prepared white koji. Brewing is carried out by using the strain or the koji prepared from the strain, so that the raw material utilization efficiency can be effectively improved, and the generation and coordination of the complex aroma in the liquor can be significantly enhanced, so that the final liquor body presents a more elegant, rich and long-lasting typical style, and the quality and value of high-end liquor are improved.

Description

Technical Field

[0001] This invention relates to the field of brewing microbial technology, and in particular to a Rhizopus spp. strain for the brewing of elegant-aroma baijiu and its application. Background Technology

[0002] Elegant aroma baijiu is one of the innovative aroma types of Chinese baijiu, pursuing a harmonious unity of elegance, comfort, mellow sweetness, and a pleasant cellar aroma. The brewing process of this type of baijiu typically integrates features of various traditional techniques, such as the combined use of small and large koji (fermentation starters). In this process, the small koji (often white koji) first undertakes the crucial saccharification task, converting the starch in the grain raw materials into fermentable sugars, laying the foundation for subsequent alcoholic fermentation.

[0003] White koji is a saccharification and fermentation agent made primarily from grains such as rice, through artificial inoculation or natural cultivation of specific microbial communities. Among these, Rhizopus is one of the most crucial microbial groups in white koji. Rhizopus secretes a rich system of amylases, especially saccharifying enzymes, which efficiently catalyze the hydrolysis of starch. The strength of its saccharification ability directly affects the alcohol yield. Therefore, for a long time, the selection of industrial strains has primarily focused on saccharifying enzyme activity as a core indicator.

[0004] However, the flavor of baijiu is a complex system. Besides alcohol, various trace aroma components, especially esters, play a decisive role in shaping the aroma, taste, and style of the liquor. The formation of these aroma substances occurs not only during fermentation and distillation but is also closely related to the metabolic activities of microorganisms during the saccharification stage. Traditional single high-saccharification-capacity microbial strains, while ensuring high distillation efficiency, have limitations in shaping a rich and multi-layered flavor profile. Therefore, developing novel microbial strains that combine high saccharification capacity with aroma-promoting functions is of great significance for enhancing the quality and uniqueness of specific baijiu types. Summary of the Invention

[0005] The purpose of this invention is to provide a Rhizopus spp. strain for the brewing of elegant-aroma baijiu and its application, which solves the technical problem that the existing white koji core strains used in baijiu brewing have a single function, resulting in simple aroma layers and an insufficiently rich and full-bodied style of the baijiu.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a Rhizopus spp. strain, which is deposited at the China General Microbiological Culture Collection Center with accession number CGMCC No. 41926. The yeast prepared by this strain alone has dual functions of saccharification and aroma production.

[0007] Preferably, the ITS gene sequence of the above-mentioned Rhizopus spp. strain is shown in SEQ ID NO.1.

[0008] The present invention also provides a white koji containing the above-mentioned Rhizopus spp. strain as the core fermentation strain.

[0009] Preferably, the raw materials for preparing the above-mentioned white koji include rice.

[0010] The present invention also provides a method for preparing the above-mentioned white koji, which includes the step of inoculating the above-mentioned Rhizopus oryzae strain into a raw material containing rice for solid-state fermentation.

[0011] The present invention also provides a bran starter containing the above-mentioned Rhizopus oryzae strain.

[0012] The present invention also provides a method for preparing the above-mentioned bran koji, which includes the step of inoculating the above-mentioned Rhizopus oryzae strain into a bran culture medium and culturing it at 30°C to 45°C.

[0013] The present invention also provides a saccharifying agent for baijiu brewing, which comprises the above-mentioned Rhizopus oryzae strain.

[0014] The present invention also provides a method for brewing elegant-aroma baijiu, which includes the following steps: saccharifying the raw materials with a saccharifying agent containing the above-mentioned Rhizopus spp. strain, and then adding medium-high temperature koji to the saccharified material for fermentation.

[0015] The present invention also provides an elegant aroma type of baijiu, which is prepared by the above-described method for brewing elegant aroma type baijiu.

[0016] The beneficial effects of this invention are: This invention provides a high-performance Rhizopus spp. strain that significantly enhances the saccharification and aroma production functions of the prepared koji. Using this strain or its koji preparation for brewing can effectively improve the utilization efficiency of raw materials and significantly enhance the generation and coordination of complex aromas in baijiu, thereby making the final liquor present a more elegant, rich and long-lasting style, thus improving the quality and value of high-end baijiu.

[0017] Compared with similar strains in the prior art, the Rhizopus spp. strain of the present invention exhibits significant technical differences in both application mode and flavor contribution: For example, the existing technology 1 (Wanbo, Research on the Isolation, Fermentation and Application of Rhizopus spp. in Small Qu, 2025) discloses that the Rhizopus spp. W17 strain needs to be combined with three kinds of yeast to obtain significant flavor substances. However, the strain of the present invention can be used to prepare white qu or bran qu with only a single strain. Without the addition of aroma-producing yeast, it can independently produce high content of key flavor substances such as ethyl acetate and β-phenylethanol during the saccharification stage, realizing the dual function integration of saccharification and aroma production.

[0018] For example, prior art 2 (Chinese patent CN118726105A) discloses a strain of Rhizopus oligospermum, which can improve saccharification power and alcohol yield. In contrast, the Rhizopus oligospermum JLFM-Q of this invention is specifically designed for brewing elegant-aroma baijiu, aiming for a harmonious and elegant blend of floral, honey, and cellar aromas; however, this strain cannot achieve this brewing objective. A comparison of the content of key esters and floral aroma substances shows that, under similar fermentation conditions (both using bran koji + daqu), the elegant-aroma baijiu brewed by this invention has a more harmonious content and ratio of ethyl acetate and ethyl hexanoate, and typical floral aroma substances such as phenylethanol accumulate in large quantities during the saccharification stage (96.05 mg / L), while the content of phenylethanol in the final base liquor of the strain in prior art 2 is only 0.23–0.73 mg / L, a difference of two orders of magnitude.

[0019] Therefore, the strain of the present invention is significantly superior to similar strains in the prior art in terms of aroma compatibility, content of key flavor substances, and ability to produce koji independently. Attached Figure Description

[0020] Figure 1 The image shows the physicochemical test results of the wine sample. Information on the preservation of biological materials

[0021] The biological material involved in this invention is Rhizopus oligosporus (…). Rhizopus arrhizus The strain JLFM-DQ has been deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 41926, on April 24, 2025. The address of the depository is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, 100101, China. Detailed Implementation

[0022] This invention provides a novel Rhizopus spp. ( Rhizopus arrhizus The strain provided by this invention, when used alone to prepare the starter culture, has dual functions of saccharification and aroma production. It can directly impart rich fruit and floral aromas to the mash without adding exogenous aroma-producing microorganisms. In particular, it significantly increases the content and harmony of key flavor substances such as ethyl acetate, ethyl hexanoate, and phenylethanol, thereby simplifying the brewing process, reducing production costs, and providing a unique flavor foundation for elegant-aroma baijiu.

[0023] The specific strain provided in this invention has been deposited at the China General Microbiological Culture Collection Center (CGMCC) prior to the application date, ensuring its reproducibility and sufficiency of disclosure. The accession number is CGMCC No. 41926. This strain was obtained from traditional high-temperature Daqu (a type of starter culture) through a specific isolation, purification, and functional screening process. Its core characteristic, which distinguishes it from ordinary industrial strains, is that it simultaneously possesses the ability to produce high levels of both saccharifying enzymes and esterases.

[0024] Preferably, to accurately identify and define this strain molecularly, the internal transcribed spacer region of its ribosomal DNA was sequenced. The ITS gene sequence is shown in SEQ ID NO: 1. The ITS sequence is a standard barcode region for fungal species identification, and its sequence determination and alignment provide conclusive molecular evidence for the taxonomic status of the strain. After comparison with authoritative fungal databases, this sequence is similar to *Rhizopus oligosporus* (…). Rhizopus arrhizus The standard sequence of the genus showed a high degree of consistency (99%), thus confirming its species at the molecular level.

[0025] This invention also provides a white koji, which uses the aforementioned Rhizopus spp. strain as the core fermentation strain. White koji, also known as "white medicine," is an important type of traditional Chinese koji, mainly used in the brewing of rice-aroma, light-aroma, and some mixed-aroma baijiu. Traditional white koji uses rice, rice bran, or wheat bran as the main raw materials, inoculated with pure Rhizopus spp., yeast, and other microorganisms, and is cultured in a solid state under suitable temperature and humidity. The finished product is often covered with white mycelium, hence the name "white koji."

[0026] In the white koji of this invention, the Rhizopus oryzae strain CGMCC No. 41926 is inoculated into the raw materials as a core functional strain for fermentation. The inoculation amount can be adjusted according to the production scale and process requirements. For example, based on the dry weight of the material, the inoculation amount can be 0.5% to 5%, more preferably 1% to 3%, and most preferably 2%.

[0027] Preferably, the raw material for preparing the white koji includes rice. Rice, as a raw material, provides abundant carbohydrates (mainly amylopectin) and contains appropriate amounts of protein and trace elements, providing balanced nutrition for the growth and enzyme secretion of Rhizopus. The rice used can be japonica rice or indica rice, and the particle size can be 20 to 60 mesh, more preferably 30 to 50 mesh. Appropriate amounts of auxiliary nitrogen sources (such as soybean meal powder) and inorganic salts (such as magnesium sulfate and potassium dihydrogen phosphate) can also be added to the raw material according to conventional techniques in the art to promote cell growth.

[0028] The present invention also provides a method for preparing the above-mentioned white koji, which belongs to the category of solid-state fermentation. Its core steps include inoculating the aforementioned Rhizopus spp. strain into raw materials containing rice for solid-state fermentation.

[0029] Specifically, solid-state fermentation refers to the process of growing microorganisms on a moist solid substrate with little or no free-flowing water. This method includes the following conventional steps: Raw material processing: The rice raw material is crushed, moistened, and the moisture content is adjusted. Moisture content is a key parameter affecting fermentation, generally controlled between 18% and 38%, with a further optimal range of 22% to 32%.

[0030] Sterilization and cooling: Sterilize the raw materials to eliminate interference from other microorganisms. The sterilization conditions can be 105℃~121℃, maintained for 15~60 minutes. Then cool to the inoculation temperature.

[0031] Inoculation: The cultured Rhizopus spore suspension or activated mycelium is aseptically mixed into the cooled raw material.

[0032] Fermentation: The inoculated material is placed in a fermentation container and cultured under controlled temperature and humidity conditions. The culture temperature can be in the range of 28℃ to 37℃, and more preferably 30℃ to 35℃. The fermentation time is generally 24 to 72 hours. During this period, necessary stirring can be performed to regulate temperature, humidity, and oxygen supply.

[0033] Drying and storage: After fermentation, the koji is dried at a low temperature below 40℃ to a safe moisture content (usually below 12%) to obtain the finished white koji.

[0034] This invention also provides a bran koji containing the aforementioned Rhizopus spp. Bran koji is an enzyme preparation or fermentation agent made by inoculating pure microorganisms into wheat bran as the main raw material. It is an improved form of traditional large and small koji produced by modern fermentation industry to simplify processes and improve efficiency. Wheat bran is loose and porous, with moderate nutrients, making it particularly suitable for the mycelial growth and enzyme production of molds.

[0035] The preparation of the bran koji is also achieved through solid-state fermentation. The main raw material is wheat bran, and the mass ratio of wheat bran to water is crucial to the physical state of the culture medium. This ratio can be 100:45 to 100:65, more preferably 100:50 to 100:60, and most preferably 100:55. The prepared bran koji can be used as a highly active saccharifying agent and a source of flavor precursors, and can be directly applied to baijiu brewing.

[0036] The present invention also provides a method for preparing the above-mentioned bran koji, which includes the step of inoculating a Rhizopus spp. strain into a bran culture medium and culturing it at a controlled temperature.

[0037] The specific process parameters of this method can be conventionally selected: the culture temperature is an important factor affecting the growth and enzyme activity expression of Rhizopus, and the culture temperature range is 30℃ to 45℃, more preferably 35℃ to 40℃. The culture time is usually 36 hours to 96 hours. After the culture is completed, the bran koji can be dried at 40~50℃ until the moisture content is below 10%, and then pulverized for later use. The saccharifying enzyme activity and esterase activity of the obtained bran koji are significantly higher than those of commercially available ordinary Rhizopus bran koji.

[0038] This invention also provides a saccharifying agent for baijiu brewing, comprising the aforementioned Rhizopus oryzae strain. This saccharifying agent is a direct manifestation and application of the aforementioned white koji or bran koji in brewing. In baijiu brewing, the core function of the saccharifying agent is in the "saccharification" stage, converting the starch in the raw materials (sorghum, rice, etc.) into fermentable sugars (such as glucose and maltose), providing a substrate for subsequent yeast alcoholic fermentation.

[0039] The saccharifying agent of this invention has both high saccharifying and high esterifying power due to the fact that its core functional strains not only ensure a high starch conversion rate, but also catalyze the generation of aroma precursors such as ethyl acetate and ethyl hexanoate in advance during the saccharification stage, laying the foundation for the formation of the unique complex aroma of "elegant aroma" baijiu.

[0040] This invention also provides a method for brewing elegant-aroma baijiu. Elegant-aroma baijiu is an innovative style of baijiu characterized by its mellow sweetness, refreshing taste, elegant and comfortable texture, and moderate cellar aroma. Its brewing process typically incorporates features from multiple types of yeast.

[0041] The method includes the following core steps: First, the brewing raw materials (such as sorghum, rice, wheat, etc.) are saccharified using a saccharifying agent containing the Rhizopus oryzae strain of the present invention (i.e., the aforementioned white koji or bran koji). This saccharification process can be carried out at 25℃~40℃ for 20~48 hours. After saccharification, "saccharified grains" rich in reducing sugars and flavor precursors are obtained.

[0042] Subsequently, medium- and high-temperature koji is added to the saccharified materials for fermentation. Medium- and high-temperature koji is a type of koji cultivated at a relatively high temperature (typically above 50°C), rich in bacteria, yeast, and heat-resistant molds, which produces abundant protein-degrading enzymes and aroma-producing microbial communities. This step constitutes a typical "combined fermentation of large and small koji" or "compound fermentation" process. Fermentation can be carried out in pits, earthenware jars, or other containers, and the fermentation cycle can be designed according to the target product style, for example, from 15 to 60 days. After fermentation, the mash is distilled, aged, and blended to obtain the finished elegant-aroma baijiu.

[0043] Finally, this invention provides an elegant-aroma baijiu, prepared using the aforementioned brewing method. Because the specific bacterial strains and saccharifying agents of this invention are used during the saccharification stage, the content and proportion of key aroma esters such as ethyl acetate and ethyl hexanoate in this baijiu are more harmonious. Sensoryly, while maintaining the inherent style of the elegant aroma, this baijiu exhibits a richer aroma profile, with a better fusion of floral, fruity, and honey sweetness with a pleasant cellar aroma. The taste is mellow, sweet, and elegant, with a long finish, demonstrating the targeted enhancement of the final product's style by the functional bacterial strains.

[0044] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0045] Example 1 Isolation, purification and identification of molds in medium and high temperature koji Isolation medium: PDA medium: Wash, peel, and cut potatoes into pieces. Add an appropriate amount of distilled water (about 500 mL) and cook until soft. Filter with gauze to collect the potato pulp. Add 20 g of glucose and 20 g of agar, and add water to 1000 mL. Sterilize at 121℃ for 20 min, cool to about 40℃, and pour into plates.

[0046] Sample preparation: Take out the Daqu powder and crush it. Add 5 g of Daqu powder to 45 mL of sterile water and stir thoroughly. Dilution and plating: The bacterial suspension was serially diluted, and the diluted solution was plated onto PDA plates. Three plates were plated for each dilution and incubated at 32℃, 37℃, and 42℃ respectively.

[0047] Isolation and purification: After the colonies grow, pick the mold hyphae and streak them onto potato dextrose agar (PDA) plates. Keep the incubation temperature constant and perform 3-5 isolation and purification operations until no other bacteria are present.

[0048] Isolation and purification results: A total of 30 strains of mold were obtained after isolation and purification.

[0049] Functional screening Functional identification culture medium: ① Starch culture medium: soluble starch 2 g / L, beef extract 5 g / L, peptone 10 g / L, agar powder 20 g / L, sterilized at 115℃ for 20 min.

[0050] ②Ester production medium: glucose 20 g / L, yeast extract 10 g / L, peptone 20 g / L, glyceryl tartrate 4 mL / L, agar 20 g / L, sterilized at 115 ℃ for 20 min.

[0051] Principle: Starch medium is used to detect the ability of strains to produce saccharifying enzymes; the appearance of a clear zone around the colony indicates that the starch has been hydrolyzed. In esterification medium, tributyric acid glyceride is used as the substrate; if the strain produces esterase, it will decompose the glyceride to produce butyric acid, which turns yellow around the colony under the action of a pH indicator. Initial functional screening is performed by the ratio of the diameter of the clear zone to the colony diameter (HC value) and the degree of color change, thus providing a preliminary assessment of the enzyme production activity of the strain.

[0052] 1) After isolation and purification, the mold was washed with sterile water to remove the spores, and a spore suspension was prepared and then serially diluted. 2) Spread the diluted solution onto the functional identification medium and incubate at the incubation temperature specified in step 1; 3) After 48 h of incubation, the HC values ​​were measured. The measurement results for each mold are shown in Table 1 below: Table 1 Results of initial screening for functional fungi JLFM-DQ HC=2.6 HC=1.8 MFG-156 0 0 JLFM-XS HC=1.22 0 MFG-157 0 0 JLFM-003 HC=1.3 HC=1.37 MFG-158 0 0 MFG-096 0 HC=1.22 MEN-164 HC=1.10 0 MHD-097 0 HC=1.13 MEN-165 0 0 MFG-098 0 HC=1.10 MEN-166 0 0 MHD-099 0 0 MEN-167 HC=1.67 0 MHD-119 0 0 MEN-168 0 0 MHD-120 HC=1.85 HC=1.47 MEN-169 0 HC=1.5 MHD-121 0 HC=4 MEN-170 HC=2.00 0 MHD-122 0 HC=2.5 MEN-172 0 0 MHD-123 HC=2.5 0 MEN-173 0 0 MHD-124 0 HC=1.7 MPM-177 0 0 MHD-125 0 HC=4 MPM-178 HC=1.40 0 MFG-154 none 0 MFG-155 HC=2.78 0 Based on the screening results, a total of 3 strains possess both saccharifying enzyme and ester mold, among which the JLFM-DQ strain showed the most significant effect and will be used as the strain for subsequent experiments.

[0053] Enzyme activity assay Wheat bran culture medium: 37 g wheat bran, moisture content 55% (water weight / dry weight), sterilized at 121℃ for 60 min.

[0054] Spore suspensions were prepared using the JLFM-DQ strain, inoculated into bran culture medium, and cultured for 2 days. The bran koji was then collected, dried, and stored for later use.

[0055] Commercially available Rhizopus syrup bran koji was used as a control group to determine the activities of saccharifying enzymes and esterases. The detection methods were based on Shen Yifang's "Complete Book of Baijiu Production Technology".

[0056] The sources of the yeast used for comparison in the following experiments are as follows: Commercially available Rhizopus koji test group 1: Huaxi Qingxiang Xiaoqu (a type of koji) from Pengzhou Huaxi Winery; Commercially available Rhizopus koji test group 2: Q303 Traditional Xiaoqu from Chongqing Hechuan Xinxing Quyao Factory. The enzyme activity detection results are shown in Tables 2 and 3: Table 2 Results of saccharifying enzyme activity assay JLFM-DQ test group 117.5 Commercially available Rhizopus test group 1 70.4 Commercially available Rhizopus test group 2 96.4 Table 3. Results of esterase activity assay JLFM-DQ test group 105.4 Commercially available Rhizopus test group 1 78.3 Commercially available Rhizopus test group 2 79.6 According to the experimental results, the JLFM-DQ Rhizopus strain exhibited significantly better esterification and saccharification enzyme activities than the control strain.

[0057] Genomic DNA was extracted from the selected strain JLFM-DQ using a fungal genomic DNA extraction kit (Sangon Biotech (Shanghai) Co., Ltd.). PCR amplification was performed using primers ITS1 and ITS4 to obtain the products. The PCR amplification products were sequenced by Sangon Biotech (Shanghai) Co., Ltd. The sequencing results are shown in Table 4. Table 4 Sequence Information JLFM-DQ As shown in SEQ ID NO.1 ITS1 TCCGTAGGTGAACCTGCGG as shown in SEQ ID NO.2 ITS4 TCCTCCGCTTATTGATATGC is shown in SEQ ID NO.3. Sequencing results were compared with the NCBI database using BLAST. The strain with the highest alignment score was selected for display, and sequence length, score, E value, identity, and gaps were provided for reference. The UNITE database (fungi) was also used for verification. The results were: sequence alignment score of 1122; sequence identity of 99%; and 2 gaps. Based on the alignment results, JLFM-DQ was identified as *Rhizopus arrhizus*, and it was deposited (accession number CGMCC No. 41926).

[0058] Rhizopus jLFM-DQ was cultured on a PDA slant, and spores were collected. Then, the spores were expanded through four stages of culture (slant, Erlenmeyer flask, and secondary shallow dish koji making) to produce mold rice koji. Saccharification and fermentation were carried out according to the brewing method of elegant aroma type baijiu in Chinese patent CN107365665A. Commercially available Rhizopus koji was used as a control group.

[0059] Methods for detecting reducing sugar content in saccharified grains: Solution preparation Solution A: Dissolve 15 g of copper sulfate pentahydrate and 0.05 g of methylene blue in distilled water and bring the volume to 1000 mL; Solution B: Dissolve 50 g of potassium sodium tartrate and 75 g of sodium hydroxide in distilled water, add 4 g of potassium ferrocyanide, and bring the volume to 1000 mL after complete dissolution.

[0060] Glucose standard solution (1 g / L): After drying, take 1 g of glucose, dissolve it in distilled water, add 5 mL of hydrochloric acid, and make up to 1000 mL.

[0061] 2) Preparation of saccharified grain test solution: Take 10.00 g of saccharified grain, add 40 mL of distilled water, boil in a water bath for 1 h, cool and then make up to 500 mL for later use. 3) Standardization: Take 5 mL of each of solution A and solution B, heat the solution to boiling, and titrate it with glucose standard solution until the blue color disappears. Record the volume of glucose standard solution V0 (mL). 4) Pre-titration: Take V1 (mL) of saccharified grain test solution, add 5 mL each of solution A and B, heat to boiling, and then titrate with glucose standard solution. Record the volume of glucose standard solution V3 (mL). 5) Titration of saccharified grain test solution: Take V1 (mL) of saccharified grain test solution, add 5 mL each of solution A and B, add (V0-V3) mL of distilled water, add V3-1 mL of glucose standard solution, heat to boiling, and titrate with glucose standard solution. Record the volume of glucose standard solution V2 (mL).

[0062] 6) Result Calculation: X=[(V0-V2)*C / (m*V1*1000)]*500*100 In the formula: X — reducing sugar content of saccharified grain, in g / 100g; V0—Volume of glucose solution consumed during calibration, in mL; V1—Saccharified grain test solution, unit: mL; V2—Volume of glucose standard solution consumed in the titration of saccharified grain test solution, in mL; 500 — Volume of the sample after treatment, in mL; C — Concentration of glucose standard solution, in g / L; m — mass of saccharified grain, in grams.

[0063] Methods for detecting the acidity of saccharified grains: 1) Solution preparation: Sodium hydroxide standard solution (0.05 mol / L): Prepared according to GB / T 601, then diluted and standardized; 10 g / L phenolphthalein indicator: Weigh 0.1 g of phenolphthalein, dissolve it in 95% ethanol solution and dilute to 100 mL.

[0064] Preparation of saccharified grain test solution: Refer to preparation method 5.1. Take 50 mL of saccharified grain test solution, add 2 drops of phenolphthalein indicator, and titrate with sodium hydroxide standard solution until a faint red color appears and does not fade after 30 seconds. Record the volume of sodium hydroxide consumed, V mL.

[0065] Result Calculation X = (C * V * 500) / 50 In the formula: X — the amount of sodium hydroxide solution consumed to neutralize 10 g of saccharified sample, in mmol / 10g; C – Concentration of sodium hydroxide solution, in mol / L; V—The volume of sodium hydroxide consumed in the titration, in mL; 50 — Volume of saccharified grain test solution taken, in mL; 500 — Total volume of saccharified grain test solution, in mL.

[0066] Yeast counting Take 10 g of saccharified grains, add 90 mL of distilled water, stir thoroughly, and count under a microscope.

[0067] Improved glycation effect The analysis results are shown in Table 5. Compared with commercially available Rhizopus bran koji (Chongqing Hechuan Xinxing Koji Factory, Q303 traditional koji), the reducing sugar content of the saccharification mash during the saccharification stage was significantly increased. The reducing sugar content of commercially available Rhizopus bran koji after saccharification was 19.8 g / 100g, while the Rhizopus spp. strain provided by this invention achieved a reducing sugar content of 22.6 g / 100g under the same conditions, an increase of 14.14%. The acidity of the saccharified grain showed a certain degree of increase, which is related to the higher saccharifying enzyme activity. The Rhizopus spp. strain selected in this study has stronger saccharifying enzyme activity, which can more efficiently catalyze starch hydrolysis to generate usable sugars and organic acids.

[0068] This strain also provided a richer substrate for yeast metabolism, significantly promoting yeast proliferation and metabolic activity. After saccharification, the yeast colony count in the Rhizopus spp. experimental group reached 3.0 × 10⁻⁶. 7 CFU / g, which is equivalent to commercially available Rhizopus bran koji (1.2×10⁻⁶). 7 The concentration of CFU / g was 2.5 times higher. Under the synergistic effect of yeast and Rhizopus, the accumulation of flavor compounds increased significantly, and the sensory quality of the saccharified grains was also more harmonious.

[0069] Table 5. Physicochemical test results and sensory evaluation of saccharified grains in the experimental and control groups. Reducing sugar content of saccharified grains (g / 100g) 22.6 19.8 Acidity of saccharified grains (mmol / 10g) 0.65 0.49 Yeast count (cfu / g) <![CDATA[3.0×10 7 ]]> <![CDATA[1.2×10 7 ]]> Sensory evaluation of saccharified grain It has a pleasant grain aroma, a rich fruity aroma, a slight acidity, a hint of alcohol, and a harmonious blend of aromas. It has a pleasant grain aroma and a sweet taste, without any obvious fragrance or alcoholic flavor. Results of volatile aroma compound test in saccharified grains: As shown in Table 6, tests revealed that *Rhizopus septemlobus* (…) Rhizopus arrhizus The total amount of volatile aroma compounds in the saccharified grain of the *Rhizopus spp.* strain was 30, while 28 were detected in commercially available *Rhizopus* bran koji saccharified grain, indicating that the *Rhizopus spp.* strain produces a richer variety of aroma compounds. Notably, the *Rhizopus spp.* strain JLFM-DQ showed significantly higher levels of most key aroma compounds than commercially available *Rhizopus spp.* bran koji. Specifically, the following four aroma compounds showed significant differences in content: 1. Ethyl acetate (exhibits a sweet, fruity aroma); 2. 2,3-Butanediol (exhibits a fruity sweet aroma); 3. β-Phenylacetyl alcohol (exhibits a typical rose fragrance); 4. 3-Hydroxy-2-butanone (exhibits a creamy, mild aroma with a slightly sweet taste).

[0070] Table 6. Detection results of volatile aroma substances in saccharified grains in the experimental and control groups. n-Propanol 15.74 16.02 Ethyl lauryl ester 1.29 6.05 Isobutanol 196.46 157.72 Ethyl phenylpropionate 1.55 1.15 n-Butanol 4.52 0 Acetaldehyde 79.94 78.8 Isoamyl alcohol 300.74 275.02 n-Propionaldehyde 29.53 68.92 2-Heptanol 5.79 4.39 furfural 52.37 41.35 Heptanol 13.45 14.6 3-Hydroxy-2-Butanone 876.19 648.71 Octyl alcohol 0.91 1.5 Acetic acid 522.63 472.32 2,3-Butanediol 613.87 567.99 propionic acid 10.61 11.53 1,2-Propanediol 41.08 73.3 Isobutyric acid 25.63 20.14 β-Phenylenol 96.05 54.16 butyric acid 4.83 4.94 Ethyl acetate 591.78 116.75 Isovalerate 6.9 4.73 Butyl hexanoate 18.19 18.12 valeric acid 1.57 0 Hexyl butyrate 5.68 5.91 hexanoic acid 80.58 71.31 Ethyl nonanoate 1.98 6.93 bitter 6.57 6.68 Physicochemical test data of the test wine samples are as follows Figure 1As shown: In the aroma compounds of the elegant-aroma baijiu, the content of the four major esters, particularly in the Rhizopus spp. JLFM-DQ white koji test group, showed significantly higher levels of ethyl acetate and ethyl hexanoate than in commercially available Rhizopus koji, indicating that the Rhizopus spp. in this invention possesses stronger esterase activity. Furthermore, the ratio of ethyl acetate to ethyl hexanoate is more harmonious, resulting in a more typical baijiu style. The sensory evaluation of the baijiu is shown in Table 7. Table 7 Sensory evaluation of baijiu in the experimental and control groups JLFM-DQ wine sample with less Rhizopus The aroma is harmonious, with prominent floral and honey notes and a moderate cellar aroma. The aftertaste is long, mellow, sweet, and elegant. Commercially available Rhizopus bran koji control group Sweet and mild, with a pleasant fruity and honey aroma, a slight hint of fermentation, and a short flavor profile. As demonstrated by the above embodiments, this invention provides a unique Rhizopus strain with low Rhizopus content. This strain exhibits significant advantages in both saccharifying enzyme and esterase activity. Its application in the preparation of white koji and the brewing of elegant-aroma baijiu effectively enhances the accumulation of reducing sugars and yeast activity during the saccharification stage, promoting the formation of a more diverse and abundant range of volatile aroma compounds in the saccharified grains. Ultimately, baijiu brewed using this strain has a higher and more harmonious content of key esters, resulting in significant improvements in aroma intensity, complexity, and stylistic typicality.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A Rhizopus spp. strain, characterized in that, It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 41926. The yeast prepared by this strain alone has dual functions of saccharification and aroma production.

2. The Rhizopus oryzae strain according to claim 1, characterized in that, Its ITS gene sequence is shown in SEQ ID NO.

1.

3. A type of white koji, characterized in that, It contains the Rhizopus spp. strain described in claim 1 or 2 as the core fermentation strain.

4. The white koji according to claim 3, characterized in that, Its raw materials include rice.

5. The method for preparing white koji according to claim 3, characterized in that, The step includes inoculating the Rhizopus oryzae strain of claim 1 or 2 into a raw material containing rice for solid-state fermentation.

6. A type of bran koji, characterized in that, It contains the Rhizopus oryzae strain as described in claim 1 or 2.

7. The method for preparing bran koji according to claim 6, characterized in that, The method includes the step of inoculating the Rhizopus oryzae strain of claim 1 or 2 into a bran culture medium and culturing it at 30°C to 45°C.

8. A saccharifying agent for Baijiu brewing, characterized in that, It contains the Rhizopus oryzae strain as described in claim 1 or 2.

9. A method for brewing elegant-aroma baijiu, characterized in that, Includes the following steps: The raw material is saccharified using a saccharifying agent containing the Rhizopus oryzae strain described in claim 1 or 2, and then medium-high temperature koji is added to the saccharified material for fermentation.

10. The elegant-aroma type of baijiu prepared by the method of claim 9.

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