Clostridium coriolis, pit mud and application of clostridium coriolis and pit mud in wine brewing

Abstract

The invention relates to the field of microorganisms, and discloses clostridium coriolis, pit mud and application of the clostridium coriolis and the pit mud in wine brewing. The clostridium coriolis provided by the invention can produce high-yield hexanoic acid under a strict anaerobic condition, and the yield of hexanoic acid is about one time higher than that of existing clostridium coriolis; the viable count of clostridium coriolis in the pit mud prepared from the clostridium coriolis with the preservation number of CGMCC No.32345 reaches 105 CFU / g or above, the content of ethyl hexanoate in a wine body can be increased when the pit mud is used for brewing wine, and the wine body is pure in fragrance and sweet and clean. The clostridium coriolis and pit mud provided by the invention have important application value for high-quality large-scale production of white spirit.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and specifically relates to a Clostridium kluyveri, pit mud, and their applications in brewing. Background Art

[0002] The quality of pit mud greatly affects the flavor and taste of the liquor body. Therefore, effective maintenance of pit mud is required to obtain high-quality and highly active excellent pit mud, thereby improving the quality of the liquor body. The time for pit mud to spontaneously transform from degraded pit mud into excellent pit mud is quite long. Therefore, if artificial and active maintenance of pit mud is not carried out, excellent pit mud cannot be obtained in a short time. Existing means for maintaining pit mud include adding components such as pit mud, bottom pit grains, and koji medicine, or introducing microorganisms. However, simply adding components such as pit mud, bottom pit grains, and koji medicine cannot promptly and efficiently increase the number of core functional microorganisms and the contents of organic acids and esters in the pit mud, and the introduction of microorganisms may also affect the subsequent fermentation of the liquor body.

[0003] Therefore, there is an urgent need to develop a method for preparing and maintaining pit mud, thereby improving the quality of liquor fermented from pit mud. Summary of the Invention

[0004] The purpose of the present invention is to overcome the above problems existing in the prior art, and provide a Clostridium kluyveri, pit mud, and their applications in brewing.

[0005] To achieve the above purpose, on the one hand, the present invention provides a strain of Clostridium kluyveri, and the preservation number of the Clostridium kluyveri is CGMCC No. 32345.

[0006] On the second aspect, the present invention provides a bacterial agent, and the bacterial agent contains the Clostridium kluyveri as described above.

[0007] On the third aspect, the present invention provides a method for preparing a bacterial agent, and the method includes: inoculating the Clostridium kluyveri as described above into a culture medium for fermentation.

[0008] On the fourth aspect, the present invention provides the application of the Clostridium kluyveri or the bacterial agent as described above in preparing or maintaining pit mud.

[0009] On the fifth aspect, the present invention provides the application of the Clostridium kluyveri or the bacterial agent as described above in producing caproic acid.

[0010] On the sixth aspect, the present invention provides the application of the Clostridium kluyveri or the bacterial agent as described above in improving the aroma and / or the content of ethyl caproate in brewing.

[0011] On the seventh aspect, the present invention provides the application of the pit mud as described above in brewing.

[0012] Through the above technical solutions, the beneficial effects obtained by the present invention at least include:

[0013] (1) The Clostridium kluyveri with the preservation number of CGMCC No. 32345 provided by the present invention can rapidly reproduce and produce high yields of caproic acid under strictly anaerobic conditions, and the caproic acid yield is approximately doubled compared to other Clostridium kluyveri strains.

[0014] (2) The viable count of Clostridium kluyveri in the pit mud prepared from the Clostridium kluyveri with the preservation number of CGMCC No. 32345 provided by the present invention reaches 10 5 CFU / g or more.

[0015] (3) The pit mud prepared from the Clostridium kluyveri with the preservation number of CGMCC No. 32345 provided by the present invention can increase the ethyl caproate content in the brewed liquor body, and make the liquor body have a pure, sweet and clean aroma. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a colony morphology diagram of the Clostridium kluyveri with the preservation number of CGMCC No. 32345 provided by the present invention.

[0017] BIOLOGICAL DEPOSIT

[0018] The strain provided by the present invention is classified and named as Clostridium kluyveri, and was deposited at the China General Microbiological Culture Collection Center on October 25, 2024. The address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No. 32345. DETAILED DESCRIPTION OF THE INVENTION

[0019] In the ranges disclosed herein, the endpoints and any value are not limited to the exact range or value, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0020] In the present invention, unless otherwise specified, "Clostridium kluyveri / strain CGMCC No. 32345", "Clostridium kluyveri / strain B-E-3", "the Clostridium kluyveri as described above", "the Clostridium kluyveri provided by the present invention" and the like all refer to the same strain, that is, the Clostridium kluyveri strain with the preservation number of CGMCC No. 32345. The above expressions have the same meaning and can be used interchangeably.

[0021] During the research process, the inventor of the present invention accidentally isolated a strictly anaerobic and high-caproic acid-producing strain from the pit mud of the pit, as Figure 1As shown, the colonies are yellow or yellowish green. The strain identified by 16S rDNA sequencing is Clostridium kluyveri, and its 16S rDNA is shown in SEQ ID No. 1.

[0022] SEQ ID No. 1:

[0023]

[0024] Based on the above findings, in the first aspect of the present invention, a strain of Clostridium kluyveri is provided, and the preservation number of the Clostridium kluyveri is CGMCC No. 32345.

[0025] In the second aspect of the present invention, a bacterial agent is provided, and the bacterial agent contains the Clostridium kluyveri as described above.

[0026] Preferably, the viable count of Clostridium kluyveri in the bacterial agent is 10 4 -10 12 CFU / mL.

[0027] Preferably, the bacterial agent contains excipients, and the excipients can be selected from glycerol and / or corn oil.

[0028] In the third aspect of the present invention, a method for preparing a bacterial agent is provided, and the method includes: inoculating the Clostridium kluyveri as described above into a culture medium for fermentation and amplification culture.

[0029] The culture medium can be a culture medium that can be used for culturing and / or preserving Clostridium kluyveri in the art, and is preferably a liquid culture medium.

[0030] Preferably, the conditions for the fermentation include: the temperature is 30-40 °C, and the time is 9-15 d.

[0031] According to the present invention, the composition of the culture medium used for the fermentation may include:

[0032] Based on 1 L of the culture medium, 3-6 g of sodium acetate, 0.5-1.5 g of yeast extract, 0.2-0.5 g of dipotassium hydrogen phosphate, 4-6 g of ammonium sulfate, 0.1-0.3 g of magnesium sulfate, 8-12 g of calcium carbonate, 90-110 g of fresh distillers grains, 20-40 g of tail wine, 15-30 g of absolute ethanol, and the balance is water.

[0033] According to the present invention, the method for the amplification culture may include: amplifying the Clostridium kluyveri provided by the present invention in an amplification culture medium, and the amplification method may be ten-fold amplification culture. Preferably, the number of times of ten-fold amplification culture is 1-10 times, the temperature is 30-40 °C, and the total time is 9-100 d.

[0034] According to the present invention, the method for the ten-fold amplification culture may be: inoculating the bacterial liquid into a system containing a culture medium at an inoculation amount of 9-11 vol% for fermentation, and the fermentation can be carried out in a fermentation tank commonly used in the art such as a Carlsberg tank, and the volume of the fermentation tank can be adjusted according to actual needs.

[0035] In order to further improve the activity of Clostridium kluyveri, increase the caproic acid yield, and improve the quality of the fermented wine body, the culture medium for the expanded culture preferably contains yellow water, and the content of yellow water is preferably 200-800 g / L of the culture medium.

[0036] According to the present invention, the yellow water can be the yellow leaching water formed by the leakage of the fermented grains in the cellar during the production of Chinese liquor, preferably the yellow leaching water formed by the leakage of the fermented grains in the cellar during the production of the rich and fragrant type of Chinese liquor. The yellow water contains substances such as residual starch, sugars, and alcohol, has a special aroma without odor, is brownish-yellow, slightly thick, and sour with astringency. The Clostridium kluyveri provided by the present invention can grow and reproduce rapidly and produce high yields of caproic acid in the culture medium containing yellow water as described above.

[0037] According to the present invention, the composition of the culture medium used for the expanded culture may include:

[0038] Expanded culture medium: yellow water: sterilized water weight ratio is 1:(3-4). Based on the total weight of yellow water + sterilized water, sodium acetate is 1-3 wt%, dipotassium hydrogen phosphate is 0.1-0.2 wt%, ammonium bicarbonate is 0.3-1.3 wt%, and sodium hydroxide is used to adjust the pH to 6.5-7.

[0039] The fourth aspect of the present invention provides the application of the Clostridium kluyveri or the bacterial agent as described above in the maintenance or preparation of pit mud.

[0040] According to the present invention, the method for maintaining pit mud includes: inoculating the Clostridium kluyveri or the bacterial agent as described above into the pit mud. In order to shorten the maintenance time and improve the performance of the pit mud, preferably, the pit mud is perforated before inoculation, and then the Clostridium kluyveri or the bacterial agent as described above is inoculated into the holes, the holes are leveled, and then the remaining Clostridium kluyveri liquid is evenly smeared.

[0041] According to the preferred embodiment of the present invention, when maintaining the pit mud, the dosage of the Clostridium kluyveri or the bacterial agent as described above is 1-2 kg / m 3 pit, or the dosage of the Clostridium kluyveri or the bacterial agent as described above is 0.005-0.02 kg / per hole, wherein the hole distribution can be 50-110 holes / m 2 pit.

[0042] According to the present invention, the method for preparing pit mud includes:

[0043] Mixing the raw materials containing nutrients, the old pit mud, and the Clostridium kluyveri or the bacterial agent as described above evenly and then adding them to the pit mud pool for fermentation.

[0044] Preferably, the raw materials containing nutrients include at least one of daqu powder, fruits, beef bone powder, ammonium bicarbonate, and compound fertilizer; more preferably, the fruits are apples and / or kiwifruits.

[0045] Preferably, the composition of the raw material containing nutrients is 10-30 wt% of aged pit mud, 2-10 wt% of Daqu powder, 3-5 wt% of apples, 0.2-0.5 wt% of ammonium bicarbonate, 0.2-0.5 wt% of bovine bone meal, and 0.2-0.5 wt% of compound fertilizer.

[0046] According to a preferred embodiment of the present invention, the method for preparing pit mud includes:

[0047] Add 10-30 wt% of aged pit mud, 2-10 wt% of Daqu powder, 3-5 wt% of apples or kiwifruits, 0.3-1 wt% of Clostridium kluyveri liquid, 0.2-0.5 wt% of ammonium bicarbonate, 0.2-0.5 wt% of bovine bone meal, 0.2-0.5 wt% of compound fertilizer (total nutrients ≥ 45 wt%, total nitrogen content ≥ 13.5 wt%, available phosphorus content ≥ 13.5 wt%, potassium oxide content ≥ 13.5 wt%), 1-5 wt% of distiller's grains powder, 3-10 wt% of yellow water, and 3-10 wt% of tail wine to the purified loess. After mixing evenly, beat into a pulp while supplementing yellow water, tail wine, and Clostridium kluyveri liquid, and then send it into the pit mud pool for the first fermentation. During the first fermentation, sprinkle the pit mud with yellow water to make the water content of the pit mud 40% ± 2%. After fermenting for 2-4 months, turn over and beat the pit mud. When turning over and beating, evenly add 20-40 kg of Daqu powder per cubic meter of pit mud, and add sterilized fresh yellow water and tail wine with an alcohol content of 35-45 degrees according to the dryness and wetness of the pit mud (the mass ratio of yellow water to tail wine is 1:(0.5-1.5), and the addition amount of yellow water is 10-30 kg) to control the water content of the pit mud at 38-42%. After the turning over and beating is completed, ferment again according to the above method for 60-120 days to obtain artificial pit mud.

[0048] According to the present invention, the aged pit mud can be the pit mud commonly used by wineries during the wine-making process, can be high-quality pit mud capable of brewing high-quality wine bodies, or can be pit mud that was once used for wine-making but cannot be used for wine-making anymore due to performance degradation or hardening. According to a preferred embodiment of the present invention, the aged pit mud comes from the pit mud commonly used by wineries during the wine-making process.

[0049] The fifth aspect of the present invention provides the application of the Clostridium kluyveri or the microbial agent as described above in the production of caproic acid.

[0050] According to the present invention, the method for producing caproic acid includes: anaerobically culturing the strain with the preservation number of CGMCC No. 32345 provided by the present invention in a fermentation medium for 10-15 d. The composition of the fermentation medium: based on 1 L of the medium, 4-6 g of sodium acetate, 0.5-1.5 g of yeast extract, 0.2-0.6 g of dipotassium hydrogen phosphate, 3-6 g of ammonium sulfate, 0.1-0.3 g of magnesium sulfate, 5-15 g of calcium carbonate, 50-150 g of fresh distiller's grains, 10-50 g of tail wine, 10-30 g of absolute ethanol, and the balance is water.

[0051] The sixth aspect of the present invention provides the application of Clostridium kluyveri or the bacterial agent as described above in improving the aroma and / or ethyl caproate content in wine brewing.

[0052] The seventh aspect of the present invention provides the application of the cellar mud as described above in wine brewing.

[0053] According to the present invention, the method for wine brewing includes:

[0054] (1) Steaming the fully soaked sorghum in a water bath for 4 - 6 h, then mixing it evenly with other raw materials (ground glutinous rice, rice, and corn grains), and then steaming it in a water bath for 10 - 50 min. The steaming temperature is 90 - 110 °C. After the steaming is completed, koji (the addition amount is 1 - 10 g / kg of the steamed raw materials) is added and saccharified for 40 - 55 h, and then daqu powder is added to obtain the fermented grains. Among them, the addition amount of daqu powder is 150 - 250 g / kg of the saccharified raw materials;

[0055] (2) Evenly spreading the cellar mud on the cellar pit, and then punching holes on the cellar mud. The hole distribution is 50 - 110 holes / m 2 cellar pit; inoculating the bacterial agent as described above into the holes, and the inoculation amount is 0.005 - 0.02 kg / each hole;

[0056] (3) Adding the fermented grains obtained in step (1) to the mud cellar, strictly sealing and fermenting. The fermentation temperature is 15 - 35 °C, and the fermentation time is 40 - 120 days.

[0057] In the wine body brewed using the bacterial agent as described above and the method for wine brewing, compared with the wine body brewed without inoculating Clostridium kluyveri or the Clostridium kluyveri bacterial agent, the content of ethyl caproate can be increased by more than 55 wt%, and the sensory aroma of the wine body is pure, sweet, and clean.

[0058] The present invention will be described in detail below through examples. It should be understood that the following examples are only used to further explain and illustrate the content of the present invention by way of example, and are not used to limit the present invention.

[0059] In the following examples, unless otherwise specified, the reagents and materials used are all commercially available products purchased from regular chemical / biological reagent or material suppliers, and the reagents are all of analytical grade.

[0060] Pasteur synthetic medium: sodium acetate 0.5 wt%, ammonium sulfate 0.05 wt%, dipotassium hydrogen phosphate 0.04 wt%, magnesium sulfate 0.02 wt%, yeast extract powder 0.4 wt%, agar 2 wt%, absolute ethanol 2 wt%, and the balance is water;

[0061] Fermentation medium: based on 1L of medium, 5g sodium acetate, 1g yeast extract, 0.4g dipotassium hydrogen phosphate, 5g ammonium sulfate, 0.2g magnesium sulfate, 10g calcium carbonate, 100g fresh distiller's grains, 30g tail wine, 20g anhydrous ethanol, and the balance is water;

[0062] Expansion culture medium: yellow water: sterile water = 1:4, based on the total weight of yellow water + sterile water, sodium acetate 2wt%, dipotassium hydrogen phosphate 0.14wt%, ammonium bicarbonate 0.8wt%, sodium hydroxide is used to adjust the pH to 6.5-7;

[0063] Compound fertilizer: Devol potassium chloride compound fertilizer, purchased from Yingcheng Xindu Chemical Compound Fertilizer Co., Ltd., item number NPK: 15-15-15. Total nutrients ≥ 45wt%, total nitrogen content ≥ 13.5wt%, available phosphorus content ≥ 13.5wt%, potassium oxide content ≥ 13.5wt%;

[0064] Tail wine: the last 20L of wine produced in the cellar;

[0065] Fresh lees: the remaining mash after the wine is produced from the cellar;

[0066] High-quality loess: collected from the Tujia and Miao Autonomous Prefecture of Hunan Province, it is yellow, stone-free, clean and odorless. It contains about 10 grams of organic matter, 120 milligrams of ammonia nitrogen, and 2.0 milligrams of available phosphorus per kilogram. The sand content is less than 3wt%. The content of pesticide residues and heavy metals meets the national standard GB 15618-2018, "Soil Environmental Quality Agricultural Land Soil Pollution Risk Control Standard (Trial)": 666 (hexachlorocyclohexane) ≤ 0.1 mg / kg, DDT (dichlorophenyltrichloroethane) ≤ 0.1 mg / kg; heavy metal (lead) and other contaminated soil, iron and calcium content are less than 10 mg / g dry soil.

[0067] Example 1

[0068] A strain with high caproic acid production was isolated from fresh high-quality pit mud. It was cultured at 35℃ for 5 days in pasteurized synthetic medium, centrifuged at 10000r / min for 1min, and the bacterial precipitate was collected. The bacterial genomic DNA was extracted using a bacterial genomic DNA extraction kit, and the 16S rRNA gene universal primers 27F (5'-AGAGTTTG ATCCTGGCTCA-3') and 1492R (5'-GGTTACCTTGTTA CGACTT-3') were used as templates for PCR amplification (94℃ pre-denaturation for 5min, 95℃ denaturation for 45s, 60℃ renaturation for 45s, 72℃ extension for 1min, 30 cycles). The amplified product was detected by 1wt% agarose electrophoresis, and the amplified product was sent to the company for sequencing (Shanghai Biotech). The measured bacterial 16S rRNA gene sequence was subjected to BLAST comparison analysis in the NCBI database.

[0069] The strain identified by 16S rRNA sequencing has a homology of 99.86% with Clostridium kluyveri. It was named BE-3 and was deposited in the General Microbiology Center of China Microorganism Culture Collection Committee (address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code: 100101) on October 25, 2024 (the abbreviation of the depository unit is CGMCC), and the deposit number is CGMCC No. 32345.

[0070] Example 2

[0071] Reference strain: Clostridium cohnii, with the collection number CICC 8023, was purchased from China Industrial Microbiological Culture Collection Center.

[0072] Single colonies of the reference strain and strain BE-3 were transferred into fermentation medium test tubes and cultured anaerobically for 10 days to mature them. 2 mL of fermentation broth was taken, and then 2 mL of 2 wt% copper sulfate solution and 1 mL of ether were added in sequence. The mixture was shaken thoroughly to allow the reaction to separate into layers. At this time, the ether layer appeared blue, indicating that both strains could produce caproic acid.

[0073] 5 mL of mature fermentation broth sample was taken into a 25 mL stoppered colorimetric tube, 2 wt% sulfuric acid solution was added to adjust the pH to 2, 5 mL of n-hexane was added, vortexed at 3000 r / min for 1 min, and then centrifuged at 3000 r / min for 3 min, the supernatant was taken, and the sample was injected and tested after passing through a 0.22 μm microporous filter membrane. The chromatograph was Agilent 7890B gas chromatograph, the chromatographic column was PEG2000 capillary column (50 m×0.25 mm×0.25 μm), the carrier gas was high-purity nitrogen, and the combustion gas was H2. The column temperature was programmed to rise, with the initial temperature at 60°C, maintained for 1 min, then heated to 100°C at a rate of 10°C / min, and then heated to 210°C at a rate of 6°C / min and maintained for 1 min. The injection temperature was 250°C, and the detection chamber temperature was 250°C. The flow rate of carrier gas (99.999 vol% high purity nitrogen) was 24 mL / min, hydrogen was 30 mL / min, and air was 300 mL / min. The column flow rate was 1 mL / min, the split ratio was 20:1, and the injection volume was 1 μL.

[0074] The caproic acid production of BE-3 was as high as 10.64 g / L, while the caproic acid production of the reference strain was 5.63 g / L.

[0075] Example 3

[0076] Rejuvenate Clostridium coli: Use a sterile inoculation loop to pick Clostridium coli liquid from the cryopreservation tube under sterile conditions and put it into pasteurized synthetic medium. Incubate at 35℃ for 10 days until the viable count of the liquid is 104 CFU / mL.

[0077] Add 0.1 L of reactivated Clostridium kluyveri and 0.85 L of expanded culture medium into a 1-L flat-bottom flask, introduce nitrogen until oxygen is exhausted, seal the flask, and maintain strict anaerobic conditions. Incubate at 35 °C for 10 days, then inoculate into a carboys containing expanded culture medium at an inoculation amount of 10 vol%, and expand the culture at 35 °C for 10 days until maturity for use as seeds. Inoculate the mature seed liquid into a carboys at an inoculation amount of 10 vol% and incubate at 35 °C for 10 days. After the secondary culture is completed, inoculate the mature carboys strain into a small fermenter for tertiary expansion culture at an inoculation amount of 10 vol%. After the small fermenter culture matures, transfer it into a 6-t large fermenter, and the large fermenter culture matures after 15 days.

[0078] Example 4

[0079] The volume of the pit is 8 m 3 , and the surface area is 20 m 2 .

[0080] After all the fermented grains obtained from the previous fermentation are removed from the pit, clean the residual grains on the pit wall and bottom, and carry out wine fermentation in the pit according to the process requirements.

[0081] Brewing steps:

[0082] 1. Raw material cleaning and soaking: Soak sorghum for 20 h, and rinse it with clean water to remove impurities. Crush glutinous rice, rice, and corn and soak them in water. During the soaking process, it is required that the materials are fully soaked, evenly mixed, do not flow, and do not form lumps;

[0083] 2. Raw material steaming and gelatinization: Steam sorghum for 4.5 h in a water bath, then evenly spread the soaked and mixed glutinous rice, rice, and corn flour on the sorghum, mix well, and steam for 25 min to obtain cooked materials;

[0084] 3. Culture bacteria and saccharification: Add starter (the addition amount of starter is 5 g / kg of cooked materials) to the cooked materials, put them into a box for saccharification, and the saccharification time is 48 h until completely gelatinized, requiring a clear fragrance, sweet taste, soft texture, no peculiar smell, no sour spoilage, and no juice leakage;

[0085] 4. Continuous distiller's grains batching: Spread the saccharified raw materials evenly on the fermented grains, mix well, and then add daqu powder. The addition amount of daqu powder is 200 g / kg of saccharified raw materials, and mix again;

[0086] 5. Mud pit fermentation: Add the mixed grain fermented grains into a mud pit for sealed fermentation. The fermentation time is 60 days for natural fermentation.

[0087] In addition, for the cellar walls and cellar mud at the bottom of the cellar that are completely the same in terms of composition and aging condition and have been cleaned, use a rake to punch holes (the hole distribution is about 100 holes per square meter), evenly pour the Clostridium kluyveri liquid prepared in Example 3 into the holes in the cellar mud (0.01 kg per hole), then use a trowel to level the holes, and finally evenly smear the remaining Clostridium kluyveri liquid at the bottom of the cellar with a broom. Then, carry out wine fermentation according to the same method.

[0088] Testing method for the excellent product rate of the wine body: Evaluate the wine according to the standards in Table 1. An evaluation group consisting of 20 professional wine tasters comprehensively scores the wine from four aspects: color, aroma, taste, and off-flavors. The total score is 85 points. When the score reaches 81 points, the wine is considered an excellent product. In addition, use gas chromatography to measure the ethyl caproate content in the wine.

[0089] Table 1

[0090]

[0091]

[0092] Compared with the cellar without pouring in the Clostridium kluyveri liquid, the ethyl caproate content in the wine body brewed in the cellar maintained with the Clostridium kluyveri liquid increased by 60 wt%, the sensory aroma of the wine body was pure, sweeter and cleaner, and the excellent product rate of the wine body increased by 3%.

[0093] Example 5

[0094] Select high-quality loess with strong viscosity, uniform thickness without lumping, clean and odorless, and no weathered stones. Carry out drying, sterilization, impurity removal, and pulverization treatments to obtain a loess raw material that is evenly pulverized without lumping, free of stone particles, wooden sticks, and tree roots. Add the following fermentation raw materials to the loess raw material (calculated based on the total weight of the yellow mud and fermentation raw materials): 20 wt% of old pit mud, 5 wt% of Daqu powder, 4.2 wt% of apples (apples must not be rotten and leaking juice before pulping), 0.45 wt% of Clostridium kluyveri solution, 0.3 wt% of ammonium bicarbonate, 0.3 wt% of bovine bone meal, 0.3 wt% of compound fertilizer, 2 wt% of distiller's grains powder, 5 wt% of yellow water, and 5 wt% of tail liquor (alcohol content is 40 ± 2 degrees). After evenly stirring the fermentation raw materials and the loess, use a mud-making machine to make pulp and send it into the pit mud pool for fermentation. After putting it into the pit, scoop out the accumulated water on the surface of the pit mud, and cover the surface of the pit mud with a film. The film is required to be smooth and flat to prevent water evaporation. During fermentation, the pit mud is maintained by sprinkling yellow water. After 3 months of fermentation of the pit mud, dig out the pit mud and put it into the mud bucket of the mud-making machine for turning and beating. When turning and beating, evenly add 30 kg of Daqu powder per cubic meter of pit mud. In order to control the moisture content of the pit mud at 40% ± 2%, at the same time, evenly add 20 kg of fresh yellow water and tail liquor with a special aroma, no odor, brownish-yellow, slightly thick, sour and astringent, and sterilized to each cubic meter of pit mud (the weight ratio of yellow water to tail liquor is 1:1). After the turning and beating is completed, ferment according to the above method again. After 90 days of fermentation, artificial pit mud is obtained.

[0095] The number of Clostridium kluyveri in the pit mud prepared by the present invention reaches 4×10 5 CFU / g or more, which is higher than the number of Clostridium kluyveri in the existing old pit mud of the winery (1×10 5 CFU / g).

[0096] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A Clostridium kluyveri, characterized in that, The preservation number of the Clostridium kluyveri is CGMCC No. 32345.

2. A bacterial agent, characterized in that, The bacterial agent contains the Clostridium kluyveri described in claim 1; Preferably, the viable count of Clostridium kluyveri in the microbial agent is 10 4 -10 12 CFU / mL.

3. The microbial agent according to claim 2, wherein, The bacterial agent contains excipients, and the excipients are selected from glycerol and / or corn oil.

4. A method for preparing a microbial agent, characterized in that, The method includes: inoculating the Clostridium kluyveri described in claim 1 into a culture medium for fermentation and amplification culture.

5. The method according to claim 4, wherein, The conditions for the fermentation include: the temperature is 30 - 40 °C, and the time is 10 - 15 d.

6. The method according to claim 4, wherein, The conditions for the amplification culture include: the temperature is 30 - 40 °C, and the total time is 9 - 100 d; and / or, the amplification culture medium contains yellow water, and preferably, the content of the yellow water is 200 - 800 g / L of the culture medium.

7. Use of the Clostridium kluyveri described in claim 1 or the bacterial agent described in claim 2 or 3 in maintaining or preparing pit mud.

8. Use of the Clostridium kluyveri described in claim 1 or the bacterial agent described in claim 2 or 3 in producing caproic acid.

9. Use of the Clostridium kluyveri described in claim 1 or the bacterial agent described in claim 2 or 3 in improving the aroma and / or the content of ethyl caproate in wine brewing.

10. Use of the pit mud described in claim 7 in wine brewing.

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