High-antioxidant Shaoxing wine, its preparation method and the bacteria used

Abstract

This invention belongs to the field of food fermentation, specifically relating to a type of rice wine with high antioxidant capacity, its preparation method, and the bacteria used. This invention provides a rope-shaped basket-shaped fungus (…). Talaromyces funiculosus The specimen, 7-2-B3-1, has the accession number CGMCC No. 41921. This invention also provides a method for preparing a high-antioxidant rice wine, in which *Bombyx mori* (a type of fungus) is used during the saccharification process. Talaromyces funiculosu s) 7-2-B3-1 culture medium. Using the method of this invention, rice wine with excellent antioxidant properties can be obtained; and the fermentation time is short.

Description

Technical Field

[0001] This invention belongs to the field of food fermentation, specifically relating to a rice wine with high antioxidant capacity, its preparation method, and the bacteria used. Background Technology

[0002] Currently, the Shaoxing rice wine industry has achieved certain results in the breeding of specialized glutinous rice varieties, the cultivation of high-quality rice, and the optimization of traditional brewing techniques. However, research on functional rice wine development is insufficient, limiting the further development of the industry. The current product structure of Shaoxing rice wine is too simple to meet the needs of different consumer groups. Substances with antioxidant activity in rice wine include polysaccharides, phenolic compounds, and Maillard reaction products. However, the antioxidant capacity of ordinary rice wine is relatively limited. Adding exogenous ingredients such as Codonopsis pilosula, Polygonatum sibiricum, Citrus aurantium, and Matsutake mushrooms is one effective method to improve the antioxidant activity of rice wine, but these substances can affect the taste and increase production costs. Optimizing the fermentation process of rice wine is another effective method to improve its antioxidant activity. The isolation and purification of various active polysaccharides and polypeptides in rice wine has been a research hotspot in recent years, but research on preparing rice wine with high antioxidant polysaccharide content using suitable strains to optimize the fermentation process is still limited.

[0003] The invention No. 201911306635.3, entitled "A strain producing Hubei Ophiopogon japonicus polysaccharide and its application," provides a *Rhizoctonia solani* strain (…). Talaromyces funiculosus strain EF048 was used to prepare polysaccharides from Ophiopogon japonicus in Hubei Province. The polysaccharides produced by this fungus are effective against four free radicals (DPPH, O2). - OH, ABTS + All of them have certain in vitro scavenging activity.

[0004] The invention "A Cellulase-Producing Rope Basket Fungus and Its Application" (202410245885.5) provides a cellulase-producing rope basket fungus (CGMCC NO. 41032) that has a good cellulose degradation effect on woody plants such as mulberry and caragana, and can be co-fermented with brewer's yeast for the production of woody protein feed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a rice wine with high antioxidant capacity, a method for its preparation, and the bacteria used.

[0006] To solve the above-mentioned technical problems, the present invention provides a rope-shaped basket-shaped fungus ( Talaromyces funiculosus )7-2-B3-1, with accession number CGMCC No. 41921, deposited by China General Microbiological Culture Collection Center, deposited on April 22, 2025.

[0007] This invention also provides a method for preparing high-antioxidant rice wine, in which *Bombyx mori* (a type of fungus) is used during the saccharification process. Talaromyces funiculosu s)7-2-B3-1.

[0008] As an improvement to the preparation method of the high antioxidant rice wine of the present invention, the following steps are included:

[0009] 1) Raw material pretreatment:

[0010] Weigh and wash the glutinous rice used as raw material, and then soak it in water (add 1.5 to 2 times the weight of the glutinous rice and soak at room temperature for 36 to 48 hours) as a pretreatment to obtain soaked glutinous rice;

[0011] 2) Steaming, boiling, and drizzling rice:

[0012] Steam the soaked glutinous rice obtained in step 1) until cooked (steam for 30±2 min in a pot according to the conventional method), and then cool it (rinse the cooked rice with purified water and cool it to 37±2 ℃) to obtain cooled rice;

[0013] 3) Inoculation and fermentation:

[0014] Mix the cooled rice obtained in step 2) with yeast and basketweed (Fragariae rubra). Talaromyces funiculosu s) 7-2-B3-1 culture medium is placed in a ceramic jar; saccharification is carried out at 20±2 ℃ (with the ceramic jar open) for 5~7 days, then wheat koji is added, and pure water equal in weight to glutinous rice is added, and then sealed and fermented at room temperature for 60±1 days (with the ceramic jar sealed); fermentation mash is obtained.

[0015] The yeast starter is 0.3-0.4% of the weight of the glutinous rice;

[0016] Glutinous rice: The culture medium of *Basilella fragilis* 7-2-B3-1 is (110~140) g / 2 mL, preferably (125±5) g / 2 mL;

[0017] The koji (rice malt) is 10-20% of the weight of the glutinous rice, preferably 15%;

[0018] 4) Separation of residue and liquid:

[0019] The fermentation mash obtained in step 3) is subjected to residue-liquid separation (by pressing the residue-liquid separation), and the separated liquid is allowed to stand (for sedimentation for 2±0.5 days) to obtain the supernatant.

[0020] 5) Refrigerating wine:

[0021] The supernatant obtained in step 4) is directly put into a pot and boiled (80~100 ℃ for 10 minutes) to obtain high antioxidant rice wine.

[0022] As a further improvement to the preparation method of the high antioxidant rice wine of the present invention, the preparation method of the *Bacillus ligustus* 7-2-B3-1 culture medium is as follows:

[0023] ① Wash the rice and soak it in water (add 1.5 to 2 times the weight of the rice in purified water and soak at room temperature for 12 to 24 hours); then steam the soaked rice (steam for 30 ± 5 minutes in a pot as usual) to get cooked rice;

[0024] Place cooked rice in a container and add water at 10-20 times its weight. Grind the rice into a paste and sterilize (121℃, 0.103 MPa for 20 min) to obtain rice culture medium.

[0025] ② Inoculate *Basilaria flavescens* 7-2-B3-1 into rice culture medium and culture at a constant temperature of 30±1℃ until the concentration of *Basilaria flavescens* 7-2-B3-1 reaches 10. 6 per mL.

[0026] The present invention also provides a high-antioxidant rice wine prepared using any of the above methods.

[0027] The method of this invention can produce rice wine with excellent antioxidant properties and a short fermentation time. In summary, this invention will have significant scientific and economic implications for promoting innovation and development in the rice wine industry. Attached Figure Description

[0028] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0029] Figure 1 Morphological characteristics of strain 7-2-B3-1;

[0030] Figure 1 middle:

[0031] A: Front view of colony morphology of strain 7-2-B3-1 on PDA medium;

[0032] B: Reverse view of colony morphology of strain 7-2-B3-1 on PDA medium;

[0033] C: Colony diameter of strain 7-2-B3-1 after 5 days of culture.

[0034] Figure 2 A comparison chart showing the improvement of DPPH free radical scavenging rate in rice wine by strain 7-2-B3-1;

[0035] Figure 3 Comparison of strain 7-2-B3-1 enhancing the iron ion reducing power (FRAP) of rice wine;

[0036] Figure 4Comparison chart showing the improvement of ABTS cationic free radical scavenging rate in rice wine by strain 7-2-B3-1;

[0037] illustrate:

[0038] Figures 2 to 4 middle:

[0039] Taihu glutinous rice + 7-2-B3-1 represents the high antioxidant rice wine obtained in Example 3 (using Taihu glutinous rice as raw material and adding 7-2-B3-1 bacteria during fermentation), and Taihu glutinous rice represents the blank control group of Taihu glutinous rice;

[0040] Jianhu Nuo No. 1 + 7-2-B3-1 represents the high antioxidant rice wine obtained in Example 4 (using Jianhu Nuo No. 1 as raw material and adding 7-2-B3-1 bacteria during fermentation), and Jianhu Nuo No. 1 represents the blank control group of Jianhu Nuo No. 1.

[0041] Shaonuo 9714+7-2-B3-1 represents the high antioxidant rice wine obtained in Example 5 (using Shaonuo 9714 as raw material and adding 7-2-B3-1 bacteria during fermentation), and Shaonuo 9714 represents the blank control group of Shaonuo 9714.

[0042] Figures 2 to 4 In this context, a, b, and c represent statistical significance markers; that is, different letters (a, b, c) indicate that the results are significantly different at the P<0.05 level. Detailed Implementation

[0043] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0044] Example 1: Acquisition of the strain

[0045] (1) After grinding the yeast sample from Shaoxing, Zhejiang Province, weigh 30g and put it into an Erlenmeyer flask containing 270 mL of sterile physiological saline and a stir bar. Stir at 500 rpm for 30 min on a magnetic stirrer to prepare a sample homogenate (1:10 sample homogenate).

[0046] (2) Take 1 mL of the sample homogenate and add it to a test tube containing 9 mL of sterile physiological saline. Mix it thoroughly to prepare a 1:100 sample homogenate. Repeat the above steps to prepare a series of ten-fold dilutions of the sample homogenate, up to a total of 6 dilutions.

[0047] (3) Take 100 μL from the homogenate of each dilution and spread it on a PDA plate. Incubate at 30°C for about 72 h.

[0048] (4) After the culture is completed, observe the growth of bacteria on the PDA plate. Use an inoculation needle to inoculate the spores of the bacteria onto a new PDA plate. Repeat this operation 3 to 5 times to obtain a pure culture of bacteria.

[0049] (5) The obtained strain 7-2-B3-1 is blue on the front, and the color gradually lightens from the inside to the edge. The core of the colony on the bottom is orange, and the color is slightly darker than the surrounding area. When it grows for 7 days after inoculation, the edge is irregular, serrated, and has small protrusions. The surface is irregular, and there are fluffy mycelia in the middle.

[0050] The comparison results from NCBI showed that... Talaromyces funiculosus, Talaromyces verruculosus, Talaromyces sp. They all showed high similarity. Comparison was made based on the characteristic features of strain 7-2-B3-1. Talaromyces funiculosus, Talaromyces verruculosus, Talaromyces sp. The characteristic description reveals its similarity to Talaromyces funiculosus Their characteristics are quite similar, so they are initially judged to be Talaromyces funiculosus .

[0051] The preservation information for strain 7-2-B3-1 of this invention is as follows:

[0052] Preservation name: Cord-shaped basket fungus Talaromyces funiculosu s, depositary institution: China General Microbiological Culture Collection Center, depositary address: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCC No. 41921, deposit date: April 22, 2025.

[0053] Example 2, Cordless Basket Fungus ( Talaromyces funiculosu The preparation of s)7-2-B3-1 culture medium is carried out by following these steps in sequence:

[0054] ① Wash the rice (any type of rice is fine), add water (purified water) twice the weight of the rice, and soak at room temperature for 12 hours; then steam for 30 minutes as usual.

[0055] Next, the cooked rice was placed in a culture container and water (purified water) was added in a volume 10 times the weight of the rice. The mixture was then homogenized into a paste using a homogenizer and sterilized (121℃, 0.103 MPa for 20 min) to obtain the rice culture medium.

[0056] ②, the rope-shaped basket-shaped bacteria ( Talaromyces funiculosu s)7-2-B3-1 was inoculated into 250 mL of rice culture medium using a sterile inoculation loop and gently stirred until homogeneous. It was then incubated at 30℃ for 7 days to obtain the initial bacterial culture.

[0057] The initial bacterial culture was inoculated into rice culture medium at an inoculation rate of 10% (volume %) and incubated at 30°C for approximately 7 days to obtain a concentration of 10. 6 String-shaped basket bacteria per mL ( Talaromyces funiculosu s)7-2-B3-1 culture medium.

[0058] Example 3: A method for preparing a high-antioxidant Shaoxing rice wine (Shaoxing Yuanhong rice wine), comprising the following steps:

[0059] 1. Raw material pretreatment

[0060] Weigh and rinse the glutinous rice, add twice its weight of purified water, and soak at room temperature for 48 hours.

[0061] The glutinous rice may be, for example, Taihu glutinous rice.

[0062] 2. Steaming, boiling, and pouring rice over rice

[0063] Steam the soaked glutinous rice from step 1 for 30 minutes, then rinse the cooked rice with purified water and cool it to about 37°C to obtain cooled rice.

[0064] 3. Inoculation and fermentation

[0065] Set the following dosage ratio:

[0066] Fermentation starter: 0.3% by mass of glutinous rice from step 1;

[0067] For every 125g of glutinous rice from step 1, use 2mL of solution with a bacterial colony concentration of 10. 6 1 / mL of rope-shaped basket bacteria ( Talaromyces funiculosu s) 7-2-B3-1 culture medium; that is, the ratio of glutinous rice to 7-2-B3-1 culture medium is 125g / 2mL.

[0068] Wheat koji: 15% of the glutinous rice from step 1 by mass;

[0069] After cooling, the rice obtained in step 2 is mixed with yeast and 7-2-B3-1 culture medium of Basilella ovalis, and then placed in a ceramic jar for saccharification at 20°C for 7 days.

[0070] Then mix in wheat koji and add purified water of equal weight to the glutinous rice, seal and ferment at room temperature for 60 days to obtain fermented mash.

[0071] The yeast starter is, for example, AN01, a rice wine yeast starter purchased from Angel Yeast Co., Ltd.

[0072] The preparation method of wheat koji can be a conventional preparation method in this field. For example, you can refer to the preparation method of wheat koji for rice wine on pages 76-77 of "Production Technology and Process of Rice Wine" published by Chemical Industry Press.

[0073] 4. Separation of residue and liquid

[0074] The fermentation mash obtained in step 3 is separated into residue and liquid by a press. The separated liquid is allowed to stand and settle for 2 days to obtain the supernatant.

[0075] 5. Frying wine

[0076] The supernatant obtained in step 4 was directly boiled in a pot (80~100℃ for 10 minutes) to obtain high antioxidant rice wine. According to GB / T 13662-2018 "Rice Wine", its alcohol content was 9.3% vol.

[0077] Taihu glutinous rice blank control group: Compared with Example 3, the fungus *Cladosporium fasciatus* was removed. Talaromyces funiculosu The use of 7-2-B3-1 (i.e., the use of the 7-2-B3-1 culture medium of *Bacillus ligustus* is omitted), and the rest is the same as in Example 3.

[0078] Example 4: Compared to Example 3, the following changes are made:

[0079] Taihu glutinous rice was replaced with Jianhu glutinous rice No. 1; the rest was the same as in Example 3. The alcohol content was tested and found to be 9.8% vol.

[0080] Jianhu Nuo No. 1 blank control group: Compared with Example 4, the fungus *Cladophora flavescens* was removed. Talaromyces funiculosu The use of s)7-2-B3-1 is the same as in Example 4.

[0081] Example 5: Compared to Example 3, the following changes are made:

[0082] Taihu glutinous rice was replaced with Shaoxing glutinous rice 9714; the rest was the same as in Example 3. The alcohol content was tested and found to be 10.6% vol.

[0083] Shaonuo 9714 blank control group: Compared with Example 5, *Bacillus ligustus* was removed. Talaromyces funiculosu The use of s)7-2-B3-1 is the same as in Example 5.

[0084] Experiment 1: Scavenging rate of DPPH free radicals by rice wine

[0085] 1. The experimental procedure and calculation method are as follows:

[0086] (1) Experimental procedure

[0087] Take 1.0 mL of rice wine sample and put it into a 5.0 mL EP tube. Then add 1.0 mL of 0.2 mmol / L DPPH solution, mix well, and react at 37 ℃ in the dark for 30 min. The absorbance value A1 is measured at 517 nm. Anhydrous ethanol is used instead of DPPH solution as the control group, and the absorbance value As is measured. Distilled water is used instead of rice wine sample as the blank group, and the absorbance value A0 is measured.

[0088] (2) Calculation method

[0089] The DPPH free radical scavenging rate of the sample to be tested is calculated using the following formula:

[0090]

[0091] In the formula, A1 represents the absorbance value of the measuring tube, A s A0 represents the absorbance value of the control tube, and A0 represents the absorbance value of the blank tube.

[0092] The results are as follows Figure 2 As shown:

[0093] In Example 3, the DPPH free radical scavenging rate of the rice wine made from Taihu glutinous rice (with added 7-2-B3-1) was 49.9%, while the DPPH free radical scavenging rate of the rice wine in the Taihu glutinous rice blank control group (without added 7-2-B3-1) was 37.1%.

[0094] In Example 4, the DPPH free radical scavenging rate of the rice wine made with Jianhu Nuo No. 1 (with 7-2-B3-1 added) was 56.1%, while the DPPH free radical scavenging rate of the rice wine corresponding to the blank control group of Jianhu Nuo No. 1 (without 7-2-B3-1 added) was 35.8%.

[0095] In Example 5, the DPPH free radical scavenging rate of the rice wine prepared with Shaonuo 9714 (with 7-2-B3-1 added) was 48.5%, while the DPPH free radical scavenging rate of the rice wine corresponding to the blank control group of Shaonuo 9714 (without 7-2-B3-1 added) was 36.7%.

[0096] That is, compared with the corresponding blank control group, the addition of bacteria 7-2-B3-1 significantly improved the DPPH free radical scavenging rate of rice wine.

[0097] Experiment 2: Determination of the reducing power (FRAP) of ferric ions in rice wine

[0098] 1. The experimental procedure and calculation method are as follows:

[0099] (1) Experimental procedure

[0100] Take 1 mL of rice wine solution into the test tube, add 3 mL of FRAP working solution, mix well, and react in a water bath at 37 ℃ in the dark for 10 min. Measure the absorbance at 593 nm. Prepare standard curves by preparing sample solutions of FeSO4 with concentrations of 0, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 mmol / L. The antioxidant capacity of the samples is expressed as the number of millimoles of FeSO4 required to achieve the same absorbance. Calculate the reducing power based on the standard curves.

[0101] (2) Calculation method

[0102] A standard curve was constructed with the concentration of FeSO4 solution standard as the x-axis and the absorbance values ​​corresponding to different concentrations of FeSO4 solution as the y-axis. The curve formula was obtained using an Excel spreadsheet: Y = 1.9549X + 0.0698(R²). 2 =0.997), where X is the concentration of FeSO4 (mmol / L) and Y is the absorbance value of the corresponding sample.

[0103] Substitute the absorbance values ​​measured in the sample wells into the calculation formula to obtain the results. See Table 1 for details. Figure 3 :

[0104] Table 1

[0105]

[0106] Note: The data on the reducing power of iron ions in Table 1 are the average values ​​of three measurements.

[0107] Experiment 3: Determination of ABTS cationic free radical scavenging rate in rice wine

[0108] 1. The experimental procedure and calculation method are as follows:

[0109] (1) Experimental procedure

[0110] Take 0.2 mL of rice wine sample and add 4 mL of ABTS solution to mix. React at room temperature for 10 min. The absorbance measured at 734 nm is A1. If distilled water is used instead of ABTS solution, the absorbance measured at 734 nm is A2. If distilled water is used instead of rice wine sample, the absorbance measured at 734 nm is A0.

[0111] (2) Calculation method

[0112]

[0113] The ABTS cationic radical scavenging rates obtained in Examples 3 (Taihu glutinous rice, with added 7-2-B3-1), the blank group of Example 3 (without added 7-2-B3-1), Example 4 (Jianhu glutinous rice No. 1, with added 7-2-B3-1), the blank group of Example 4 (without added 7-2-B3-1), and Example 5 (Shaonuo 9714, with added 7-2-B3-1), and the blank group of Example 5 (without added 7-2-B3-1) are as follows: Figure 4 As shown.

[0114] Comparative Example 1-1, compared to Example 3, the following changes were made:

[0115] The ratio of Taihu glutinous rice to 7-2-B3-1 culture medium was changed from "125g / 2mL" to "125g / 1mL", and the rest was the same as in Example 3.

[0116] The DPPH free radical scavenging rate was 43.8%, the iron ion reducing power was 0.692 mmol / L, and the ABTS cationic free radical scavenging rate was 59.6%.

[0117] Comparative Examples 1-2, compared to Example 3, are modified as follows:

[0118] The ratio of Taihu glutinous rice to 7-2-B3-1 culture medium was changed from "125g / 2mL" to "125g / 3mL", and the rest was the same as in Example 3.

[0119] The DPPH free radical scavenging rate was 48.6%, the iron ion reducing power was 0.763 mmol / L, and the ABTS cationic free radical scavenging rate was 62.6%.

[0120] Comparative Example 2, relative to Example 4

[0121] The ratio of Jianhu Nuo No. 1 to 7-2-B3-1 culture medium was changed from "125g / 2mL" to "125g / 1mL", and the rest was the same as in Example 4.

[0122] The DPPH radical scavenging rate was 47.8%, and the ABTS cationic radical scavenging rate was 68.2%.

[0123] The present invention also conducted the following experiments:

[0124] The strain EF048 and the strain 7-2-B3-1 of the present invention, which is a ligusticum var. truncatum in 202410245885.5, were used to prepare rice wine according to Example 3. The DPPH free radical scavenging rate was not significantly different from that of the blank group.

[0125] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. *Talaromyces funiculosus* 7-2-B3-1, characterized by: The accession number is CGMCCNo. 41921, the depositary institution is the China General Microbiological Culture Collection Center, and the deposit date is April 22, 2025.

2. A method for preparing high-antioxidant rice wine, characterized in that: Talamyces funiculosus 7-2-B3-1 was used in the saccharification process.

3. The method for preparing high-antioxidant rice wine according to claim 2, characterized in that... Includes the following steps: 1) Pre-treat the glutinous rice used as raw material to obtain soaked glutinous rice; 2) Steam and cook the soaked glutinous rice obtained in step 1) to obtain cooled cooked rice; 3) Inoculation and fermentation: After cooling, the rice obtained in step 2) is mixed with yeast and 7-2-B3-1 culture medium of Basilella ovalis and then placed in a jar. After saccharification at 20±2°C for 5-7 days, wheat yeast is added and pure water of equal weight to the glutinous rice is added. Then, the mixture is sealed and fermented at room temperature for 60±1 days to obtain fermented mash. The yeast starter is 0.3-0.4% of the weight of the glutinous rice; Glutinous rice: Fungus 7-2-B3-1 culture medium was (110~140) g / 2 mL; The amount of wheat koji is 10-20% of the weight of glutinous rice; 4) Separate the fermentation mash obtained in step 3) to obtain the supernatant; 5) Refrigerating wine: Boil the supernatant obtained in step 4) to obtain high antioxidant rice wine.

4. The method for preparing high-antioxidant rice wine according to claim 3, characterized in that: The preparation method of the culture medium for *Basilaria cordifolia* 7-2-B3-1 is as follows: ① Cook rice into rice, add water in a ratio of 10 to 20 times the weight of the rice, grind it evenly into a paste, sterilize it, and obtain rice culture medium; ② Inoculate *Basilaria flavescens* 7-2-B3-1 into rice culture medium and culture at a constant temperature of 30±1 °C until the concentration of *Basilaria flavescens* 7-2-B3-1 reaches 10. 6 per mL.

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