Rhizopus strain, koji and production method of rice wine

Abstract

The application belongs to the technical field of wine starter, and particularly relates to a Rhizopus strain, wine starter and a production method of rice wine; the Latin name of the Rhizopus strain is Rhizopus delemar b21, and the preservation number is CCTCC NO:2026399; the wine starter prepared by using the above Rhizopus b21 can significantly improve the aroma and taste of fermented products such as rice wine and sweet wine, and has stronger environmental adaptability and is more suitable for traditional wine starter and fermented food production; the most suitable inoculation amount of the wine starter brewed by using the above Rhizopus b21 or wine starter to brew rice wine is 0.5% to 1%.

Description

Technical Field

[0001] This invention belongs to the field of brewing starter technology, specifically relating to a Rhizopus strain, brewing starter, and a method for producing rice wine. Background Technology

[0002] Rice wine, also known as fermented rice, is mainly made from glutinous rice and yeast. Currently, various rice wine products are sold in major supermarkets across the country, but the quality varies greatly. Most rice wine companies use local traditional yeast, which has a simple and extensive process, resulting in a rich microbial community structure that gives rice wines from different regions their unique flavors. However, traditional yeast has drawbacks such as difficulty in controlling unwanted bacteria, a tendency for harmful bacteria to exceed standards, poor stability, and a higher content of impurities (CN202010287643).

[0003] The yeast starter (qu) is the soul of Chinese winemaking. China has a long history of yeast starter making and brewing techniques, evolving from early loose yeast starters to cake yeast starters, and finally to large yeast starters and small yeast starters. The main strains in traditional yeast starters are Rhizopus and yeast. Rhizopus belongs to the genus Rhizopus of the family Mucoraceae in the order Mucorales of the subphylum Zygomycetes. Rhizopus fungi play an absolutely central role in rice wine fermentation. They secrete highly active amylase and saccharifying enzymes, which efficiently break down the starch in rice that cannot be directly utilized into small sugar molecules such as glucose. Yeast, as an alcohol-producing functional strain, works synergistically to enhance the flavor of rice wine.

[0004] Currently, the excessive proliferation, gas production, and acid production of yeast during rice wine brewing are significant reasons for the yellowing of the fermented gluten surface, cloudiness of the liquid, excessive sediment at the bottom, and a sour taste. Therefore, screening for Rhizopus strains with strong alcohol-producing capabilities is of great importance for improving the overall sensory characteristics of rice wine. Summary of the Invention

[0005] This invention provides a method for producing Rhizopus strains, yeast, and rice wine, which can directly utilize raw starch as raw material, resulting in faster fermentation, higher raw material utilization, and richer flavor compounds produced by metabolism.

[0006] To achieve the above objectives, according to one aspect of the present invention, a Rhizopus strain is provided, the Latin name of which is Rhizopus delemar b21, and the accession number is CCTCC NO:2026399.

[0007] Furthermore, this strain can produce a variety of hydrolytic enzymes during fermentation, forming a complex enzyme system that can simultaneously produce starch-degrading enzymes, protein-degrading enzymes, and plant cell wall-degrading enzymes.

[0008] A method for culturing Rhizopus strains as described above is as follows: Dilute honeycomb yeast and spread it on PDA solid medium, incubate at 28-30℃ for 2-3 days, pick out single colonies after they grow on the plate, purify them, and place them in A medium for Rhizopus sporulation.

[0009] Furthermore, the PDA solid culture medium is prepared as follows: 200g of peeled potatoes are cut into small pieces, boiled in water until soft, filtered through gauze to obtain the filtrate, 20g of glucose and 15-20g of agar are added, stirred and heated to dissolve, and then diluted to 1L with distilled water. The pH is 5.6-6.0. The medium is then autoclaved at 121℃ for 15min, cooled to 50℃, and poured into plates for later use.

[0010] Furthermore, the A culture medium consists of 1% yeast extract and 0.5% wheat bran extract, and is prepared as follows: boil wheat bran, filter to obtain wheat bran juice, then add yeast powder, mix evenly, and adjust the pH to 6.5 for later use.

[0011] According to another aspect of the present invention, a brewing starter is provided containing a Rhizopus strain, which is the Rhizopus strain described above.

[0012] A method for producing yeast as described above is as follows: S1: Select the purified Rhizopus strain and prepare a spore suspension by rinsing the slant with sterile water; S2: Use a mixture of wheat bran and rice flour as the base material for the starter culture, adjust the moisture content to 50% to 55%, inoculate with spore suspension and stir thoroughly, spread loosely and flat, cultivate in a natural aerobic environment at 28 to 30℃, and then dry to obtain pure starter culture.

[0013] Furthermore, the spore suspension described in S1 has a spore concentration of up to 10. 6 ~10 8 CFU / mL.

[0014] Furthermore, in the koji base material described in S2, the mass ratio of wheat bran to rice flour is 8:2.

[0015] The spore suspension described in S2 is added at a rate of 5% to 10% of the mass of the koji material.

[0016] According to another aspect of the present invention, a rice wine is provided containing a Rhizopus strain, which is the Rhizopus strain described above.

[0017] According to another aspect of the present invention, a rice wine is provided, which is brewed using yeast. The yeast is the aforementioned yeast, and the preparation method is as follows: glutinous rice is weighed, soaked, steamed, and cooled to 30-35°C. Crushed Rhizopus yeast is added at a ratio of 0.5%-1% of the raw material mass, and stirred until evenly mixed. Saccharification and fermentation are carried out at 25-30°C for 48-72 hours. The pre-fermentation is completed when the rice liquefies and produces juice with a rich aroma. Subsequent low-temperature post-fermentation can be carried out for 3-7 days.

[0018] Preservation information of the strain of this invention: A strain of Rhizopus, with the Latin name Rhizopus delemar b21, is deposited at the China Center for Type Culture Collection (CCTCC) on March 9, 2026, with accession number CCTCC NO:2026399. Attached Figure Description

[0019] Figure 1 This is a colony diagram of strain b21 growing on PDA solid medium.

[0020] Figure 2 This is the phylogenetic tree of strain b21.

[0021] Figure 3 This is a microscopic image of strain b21 stained with lactophenol cotton blue.

[0022] Figure 4 This is a comparison of the sequence similarity between strain b21 and existing strains. Detailed Implementation

[0023] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0024] As analyzed in the background section of this application, there is a need for a Rhizopus strain that can simultaneously produce starch-degrading enzymes, protein-degrading enzymes, and plant cell wall-degrading enzymes, enabling its deep application in fields such as starch deep processing, bio-fermentation, food industry, and resource utilization of agricultural by-products. This application provides a Rhizopus strain, a yeast starter, and a method for producing rice wine.

[0025] In one typical embodiment of this application, a Rhizopus strain is provided, the Latin name of which is Rhizopus delemar b21, and the accession number is CCTCC NO:2026399 (hereinafter referred to as Rhizopus b21).

[0026] This invention investigated the physiological and biochemical characteristics of the aforementioned Rhizopus b21. The strain showed a sequence similarity of 99.33% and a coverage of 95% with *Rhizopus sarrhizus* (type strain CBS 112.07, GenBank accession number NR_103595.1), with an E value of 0.0. Based on morphological characteristics, it was ultimately identified as *Rhizopus sarrhizus*, a known species. This Rhizopus was deposited on March 9, 2026, at the China Center for Type Culture Collection (CCTCC), Wuhan University, Wuhan, with accession number CCTCC NO:2026399.

[0027] The fasta sequence of the ITS rDNA gene obtained by ITS primer amplification and sequencing of Rhizopus with accession number CCTCC NO:2026399 is as follows:

[0028] The above-mentioned Rhizopus b21 was cultured as follows: The honeycomb yeast was diluted and spread onto PDA solid medium, then incubated at 30℃ for 3 days. Once single colonies appeared on the plate, the colonies were identified based on typical Rhizopus colony morphology (e.g., Figure 1As shown, a single colony (white, flocculent, with well-developed hyphae) was picked, purified, and placed in medium A for Rhizopus sporulation. PDA solid medium was prepared as follows: 200g of peeled potatoes were cut into small pieces, boiled in water until soft, filtered through gauze, and the filtrate was added to 20g of glucose and 17g of agar. After stirring and heating to dissolve, the mixture was brought to a final volume of 1L with distilled water, adjusting the pH to 5.8. The medium was then autoclaved at 121℃ for 15 minutes, cooled to 50℃, and poured into plates for later use. Medium A consisted of 1% yeast extract and 0.5% wheat bran extract, prepared as follows: Wheat bran was boiled, filtered to obtain the wheat bran extract, and then yeast powder was added, mixed thoroughly, and the pH was adjusted to 6.5.

[0029] Table 1. Mycological properties of Rhizopus b21 The Rhizopus of the present invention possesses the aforementioned mycological properties, such as Figure 3 The image shows *Rhizopus* b21 stained with lactophenol blue. The mycelium is broad and unseptate or sparsely septate. Vegetative hyphae differentiate into stolons that extend in all directions. Rhizoids grow from the stolons. The pleiophores are solitary and clustered, opposite the rhizoids, often forming bundles. Each pleiophore bears a spherical sporangium at its apex. The sporangia are spherical, oval, or irregular, colorless or light brown. The rachis is distinct, spherical or hemispherical, and forms a receptacle at the base where it connects to the sporangium. There is no collar, consistent with the characteristics of the *Rhizopus* genus.

[0030] This application provides a brewing starter containing a Rhizopus strain, which is the Rhizopus strain described above. Its preparation method is as follows: Purified Rhizopus strains were selected, and spore suspensions were prepared by rinsing the agar slant with sterile water. The spore concentration was then adjusted to 10⁻⁶. 6 ~10 8 CFU / mL, using a mixture of wheat bran and rice flour in an 8:2 ratio as the base material for the starter culture, adjusting the moisture content to 50%–55%, and adding spore suspension at a ratio of 5%–10% of the starter culture mass and stirring until evenly mixed, then filling the culture container and spreading it loosely, and culturing it at 28–30℃ in a natural aerobic environment, maintaining ventilation and oxygen supply in the early stage to promote mycelial growth, and controlling the temperature and allowing it to clump together in the later stage, with a total culture time of 48–72 hours. After the surface of the starter culture material is covered with white mycelium and a large number of spores are produced, it is dried to obtain pure starter culture.

[0031] This application provides a rice wine containing the aforementioned Rhizopus strain b21, or the rice wine is brewed using a yeast starter, the aforementioned yeast starter, and the preparation method is as follows: Weigh 1 kg of glutinous rice, soak it, steam it, and cool it to 30-35℃. Add crushed Rhizopus yeast at a ratio of 0.5%-1% of the raw material weight, stir until even, and put it into a clean container with holes in the middle for ventilation. Saccharify and ferment at 25-30℃ for 48-72 hours. When the rice liquefies and produces juice with a strong aroma of wine, the pre-fermentation is complete. The subsequent low-temperature post-fermentation can be sealed for 3-7 days.

[0032] Production of yeast Example 1 The koji base material consists of 80 parts wheat bran and 20 parts rice flour by weight, with the moisture content adjusted to 55%, and the inoculation concentration is 1×10⁻⁶. 7 A suspension of Rhizopus spores at CFU / mL was inoculated at 8% of the substrate mass and cultured at 28–30°C under aerobic conditions for 60 h.

[0033] Example 2 The koji base material consisted of 70 parts by weight of wheat bran and 30 parts by weight of rice flour, with the other conditions being the same as in Example 1.

[0034] Example 3 Keep the substrate unchanged, raise the culture temperature to 33-35℃, and keep all other conditions the same.

[0035] Example 4 The concentration of the spore suspension was changed to 1×10 6 With the inoculum concentration remaining constant at CFU / mL, the bacterial growth cycle is extended, requiring 72 hours of cultivation to reach maturity, thus extending the rice wine fermentation cycle by 24 hours.

[0036] Comparative Example 1 Rhizopus M101 was used instead of Rhizopus b21 in the previous examples to produce rice wine starter products.

[0037] Comparative Example 2 Rhizopus 3.866 was used to replace Rhizopus b21 in the previous examples to produce rice wine starter products.

[0038] Comparative Example 3 Rhizopus spp. was used to replace Rhizopus b21 in the previous examples to produce rice wine starter products.

[0039] Rice wine brewing 1. Ingredients: glutinous rice, yeast.

[0040] 2. Process Flow: Weigh 1 kg of glutinous rice and soak it for 4 hours, keeping the grains intact and the rice soft. The rice absorbs about 25% of the water. Then wash the soaked glutinous rice, drain it, steam it until cooked, and let it cool to 30-35℃. Then mix the cooled glutinous rice with yeast, adding 0.8% of the dry weight of the glutinous rice. Finally, carry out saccharification and fermentation at 25℃-30℃ for 48-72 hours.

[0041] Rice wine was prepared using different types of yeast through the above process, and the sugar content, acidity, and alcohol content of the rice wine were tested. The reference standard was QB / T 8152-2025 "Fermented Rice Wine".

[0042] Table 2 As shown in Table 2, among the rice wines made with the above-mentioned yeast, the Rhizopus b21 yeast in Example 1 showed the best enzyme production capacity and rice wine fermentation performance. The resulting rice wine had sugar content, alcohol content, and acidity that met the requirements of QB / T 8152-2025 and related standards.

Claims

1. A Rhizopus strain, characterized in that, The Latin name is Rhizopus delemar b21, and the accession number is CCTCC NO:2026399.

2. The method for culturing the Rhizopus strain according to claim 1, characterized in that, The process includes the following steps: dilute the honeycomb yeast and spread it on PDA solid medium, incubate at 28-30℃ for 2-3 days, pick out single colonies after they grow on the plate, purify them, and place them in A medium for Rhizopus sporulation.

3. The method for culturing Rhizopus strains according to claim 2, characterized in that: The PDA solid culture medium is prepared as follows: 200g of peeled potatoes are cut into small pieces, boiled in water, filtered through gauze to obtain the filtrate, 20g of glucose and 15-20g of agar are added, stirred and heated to dissolve, and then diluted to 1L with distilled water. The pH is 5.6-6.

0. The medium is then autoclaved at 121℃ for 15min, cooled to 50℃, and poured into plates for later use.

4. The method for culturing Rhizopus strains according to claim 2, characterized in that: The A culture medium consists of 1% yeast extract and 0.5% wheat bran extract. The preparation method is as follows: boil wheat bran, filter to obtain wheat bran juice, add yeast powder, mix evenly, and adjust pH to 6.5 for later use.

5. A brewing starter containing a Rhizopus strain, characterized in that, The Rhizopus strain is the Rhizopus strain described in claim 1.

6. The method for producing yeast according to claim 5, characterized in that, Includes the following steps: S1: Select the purified Rhizopus strain and prepare a spore suspension by rinsing the slant with sterile water; S2: Use a mixture of wheat bran and rice flour as the base material for the starter culture, adjust the moisture content to 50% to 55%, inoculate with spore suspension and stir, spread loosely and evenly, cultivate in a natural aerobic environment at 28 to 30℃, and dry to obtain pure starter culture.

7. The method for producing yeast according to claim 5, characterized in that: The spore suspension described in S1 has a spore concentration of up to 10. 6 ~10 8 CFU / mL.

8. The method for producing yeast according to claim 4, characterized in that: The koji base material described in S2, wherein the mass ratio of wheat bran to rice flour is 8:2; The spore suspension described in S2 is added at a rate of 5% to 10% of the mass of the koji material.

9. A type of rice wine, comprising glutinous rice and yeast, characterized in that, The yeast starter is the yeast starter described in claim 5.

10. A method for producing rice wine according to claim 9, characterized in that, The process includes the following steps: Weigh glutinous rice, soak it, steam it, and cool it to 30-35℃. Then, add crushed Rhizopus yeast at a ratio of 0.5%-1% of the raw material mass, stir, and saccharify and ferment at 25-30℃ for 48-72 hours. Seal and ferment at low temperature for 3-7 days.

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