Immobilized compound caproic acid bacteria, and preparation method and application thereof

Abstract

The application discloses immobilized compound caproic acid bacteria and a preparation method and application thereof. The preparation method is as follows: (1) preparation of pure caproic acid bacteria liquid; (2) preparation of yeast bacteria liquid; (3) preparation of compound caproic acid bacteria liquid: the pure caproic acid bacteria liquid is mixed with the yeast bacteria liquid; (4) preparation of an adsorption carrier: pleurotus eryngii is taken, chopped, washed, sterilized and then obtained; (5) adsorption of the compound caproic acid bacteria: the treated adsorption carrier is immersed in the prepared compound caproic acid bacteria liquid and fully adsorbed; (6) preparation of an embedding liquid: agar is dissolved in water, heated, melted, sterilized and then obtained; (7) the pleurotus eryngii carrier with the adsorbed caproic acid bacteria is uniformly immersed in the embedding liquid, and the immobilized compound caproic acid bacteria is obtained after solidification. The compound immobilized caproic acid bacteria provided by the application has high caproic acid production and stable acid production effect. The immobilized caproic acid bacteria is mainly applied to the field of liquor brewing, can significantly increase the content of caproic acid and ethyl caproate in liquor and significantly improve the quality of the liquor.

Description

Technical Field

[0001] This invention belongs to the field of brewing microorganisms and brewing technology, specifically relating to an immobilized compound hexanoic acid bacteria, its preparation method, and its application. Background Technology

[0002] Hexanoic acid bacteria refer to a class of microorganisms that can produce hexanoic acid when cultured, and are important functional bacteria in cellar mud. The characteristic aroma compound of strong-aroma baijiu is ethyl hexanoate, mainly produced by the esterification reaction between hexanoic acid produced by hexanoic acid bacteria in the cellar mud and ethanol produced by yeast metabolism. The content and hexanoic acid production capacity of hexanoic acid bacteria in the cellar mud determine the quality of the cellar mud and thus affect the quality of strong-aroma baijiu. Applying hexanoic acid bacteria solution for cellar maintenance or artificially cultivating cellar mud can replenish the cellar mud with highly active hexanoic acid bacteria, improve the biological activity of the cellar mud, and further enhance the quality of baijiu.

[0003] Traditional liquid culture of caproic acid bacteria has a long cultivation cycle, requiring 5-6 days to enter the acid-producing phase, which lasts approximately 3-4 days. Furthermore, the reaction time is short and production costs are high. Immobilized microbial culture technology, which emerged in the 1980s, offers advantages such as rapid cultivation, short cycle, low cost, simple process, and reusability, and has been widely adopted by many factories for producing caproic acid bacteria solutions.

[0004] Currently, most methods for immobilizing caproic acid bacteria use substances such as agar, polyvinyl alcohol, and sodium alginate as immobilization materials, and the immobilized caproic acid bacteria are mostly seed liquid from pit mud or pure caproic acid bacteria. For example, Xie Guopai et al. ("Research on Agar-Immobilized Cellar Mud Hexanoic Acid Bacteria Cultivation Technology", Brewing, December 2016) disclosed a method for producing hexanoic acid using agar-immobilized Clostridium kleinemannii; Zhong Guohui et al. ("Research Report on New Materials of Immobilized Hexanoic Acid Bacteria", Brewing, March 2009) disclosed a method for preparing immobilized hexanoic acid bacteria using polyvinyl alcohol (PVA) encapsulation and activated carbon adsorption, with the immobilized strain being Clostridium kleinemannii; Patent CN1033644 (A New Method for the Preparation of Immobilized Hexanoic Acid Bacteria and Its Application in the Brewing of Strong-Aroma Baijiu) disclosed a method for immobilizing hexanoic acid bacteria using SiO2, Al2O3, and Fe2O3 as carriers; Zhang Jie ("Application of Hexanoic Acid Bacteria Cell Immobilization Technology in the Production of Strong-Aroma Baijiu", Brewing, January 1999) disclosed a method for preparing immobilized hexanoic acid bacteria using sodium alginate as a carrier. However, the microbial community structure of immobilized pit mud seed liquid is unclear and easily affected by environmental factors, leading to imbalances in the microbial community structure. Furthermore, the inconsistent microbial communities contained in different batches of pit mud result in varying effects between different batches of immobilized pit mud seed liquid. The hexanoic acid production capacity of immobilized pure-culture hexanoic acid bacteria is also easily affected by environmental factors during its application in pits. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a method for preparing and applying immobilized composite solid hexanoic acid bacteria.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] In a first aspect, the present invention provides a method for preparing immobilized compound caproic acid bacteria, comprising the following steps:

[0008] (1) Preparation of pure culture of caproic acid bacteria: Inoculate the seed culture of caproic acid bacteria into sodium ethanol acetate medium at an inoculation rate of 5%-10% (v / v) of the medium, with a liquid volume of 80%-95%, and incubate at 33℃-35℃ for 5-7 days.

[0009] (2) Preparation of yeast culture: Add thermoresistant active dry yeast to YPD medium, culture in a shaker at constant temperature, and then transfer to YPD medium for scale-up culture under the same conditions;

[0010] (3) Preparation of compound caproic acid bacteria solution: Mix pure caproic acid bacteria solution with yeast solution;

[0011] (4) Preparation of adsorption carrier: Take king oyster mushroom, chop it, wash it and sterilize it.

[0012] (5) Adsorption of compound caproic acid bacteria: The treated adsorption carrier is immersed in the prepared compound caproic acid bacteria solution for full adsorption;

[0013] (6) Preparation of embedding solution: Dissolve agar in water, heat to melt, and sterilize to obtain the solution;

[0014] (7) The Pleurotus ostreatus carrier that has adsorbed caproic acid bacteria is evenly immersed in the embedding solution, and after solidification, immobilized composite caproic acid bacteria are obtained.

[0015] Furthermore, in step (1), the hexanoic acid bacterium is the fast-growing Clostridium JSJ-1CCTCC M 2020881. This glucose-inhibiting aerobic hexanoic acid bacterium is the strain disclosed in patent ZL202110311823.6, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2020881, and its classification name is Clostridium celerecrescens JSJ-01 (deposit date: December 9, 2020, deposit address: Wuhan University, Wuhan, China).

[0016] Further, in step (1), the composition of the sodium ethanolacetate (ES) culture medium used to cultivate hexanoic acid bacteria is as follows: 1-1.5 parts yeast extract, 0.3-0.6 parts anhydrous sodium acetate, 0.02-0.04 parts MgSO4·7H2O, 0.03-0.06 parts K2HPO4, 0.03-0.06 parts (NH4)2SO4, 100 parts distilled water, and then aseptically added 2-2.5 parts anhydrous ethanol and 0-1.5 parts dry heat sterilized CaCO3.

[0017] Furthermore, in step (2), the culture is scaled up at an inoculum of 5% (v / v).

[0018] Furthermore, in step (2), the mass composition of the YPD culture medium is as follows: 1 part yeast extract, 2 parts peptone, 2 parts glucose, and 100 parts distilled water.

[0019] Furthermore, in step (2), the amount of dry yeast added is 0.05%-0.1% of the mass of YPD culture medium; the culture temperature is 34℃, the shaking speed is 200r / min, and the culture time is 16h.

[0020] Furthermore, in step (2), the dry yeast is Angel high-temperature resistant active dry yeast.

[0021] Furthermore, in step (3), the acetic acid bacteria solution and the yeast solution are mixed at a volume ratio of 1:1.5-2.

[0022] Furthermore, in step (4), the king oyster mushrooms are cut into 1-2cm pieces. 3 The square.

[0023] Furthermore, in step (4), sterilization is performed by dry heat sterilization at 130-135℃ for 1-2 hours.

[0024] Furthermore, in step (5), the ratio of the mass of the adsorption carrier to the volume of the hexanoic acid bacteria liquid is 0.3-0.5g:10-12mL.

[0025] Furthermore, in step (5), the adsorption temperature is 30-35℃ and the adsorption time is 24-48h.

[0026] Furthermore, in step (6), the mass ratio of agar to water is 1:14-16; the sterilization temperature is 115℃ and the sterilization time is 15-20min.

[0027] Furthermore, in step (7), the solidification temperature is 20-25℃.

[0028] In a second aspect, the present invention provides immobilized composite hexanoic acid bacteria prepared using the method of the first aspect.

[0029] Thirdly, the present invention provides the application of immobilized compound hexanoic acid bacteria in the fermentation production of hexanoic acid.

[0030] Furthermore, the application of immobilized compound caproic acid bacteria in the fermentation of caproic acid involves adding immobilized compound caproic acid bacteria to a fermentation system containing ethanol and organic acids to produce caproic acid.

[0031] Fourthly, the present invention provides the application of immobilized compound caproic acid bacteria in the fermentation production of strong-aroma baijiu.

[0032] Furthermore, the application of immobilized compound caproic acid bacteria in the field of baijiu brewing refers to the application of immobilized compound caproic acid bacteria or immobilized compound caproic acid bacteria fermentation broth to cultivate artificial cellar mud, improve the quality of cellar mud, or produce strong-aroma baijiu without cellar mud.

[0033] Furthermore, the production and cultivation of artificial pit mud using immobilized compound caproic acid bacteria is achieved through the following steps: Select high-quality soil, steam it, and add a certain amount of water. Then add ES medium, 3%-6% of the soil mass of immobilized compound caproic acid bacteria or immobilized compound caproic acid bacteria fermentation broth, stir evenly, seal with plastic wrap, and incubate at 33-35℃ for 30-50 days.

[0034] Furthermore, the application of immobilized compound caproic acid bacteria in the production of strong-aroma baijiu without fermentation pit mud is achieved through the following steps: After washing, soaking, steaming, and cooling the grains, add Daqu (a type of starter culture) and steamed rice husks. Add the prepared immobilized compound caproic acid bacteria (or immobilized compound caproic acid bacteria fermentation liquid) at 3%-8% of the raw grain weight. Seal and ferment in a solid-state environment for 30-45 days. Distill the fermented mash to obtain strong-aroma baijiu without fermentation pit mud.

[0035] The present invention has the following beneficial effects:

[0036] (1) This invention employs a composite immobilization method of adsorption-embedding to immobilize Hexanoic acid bacteria. During adsorption, *Pleurotus eryngii* is used as the immobilization material, providing a novel immobilization carrier for the cell immobilization of *Hexanoic acid bacteria*. *Pleurotus eryngii* has strong water absorption and retention capacity, enabling it to effectively adsorb bacterial solutions. Furthermore, the dense structure of *Pleurotus eryngii* and its porous internal structure provide space for microbial growth. Through adsorption, *Hexanoic acid bacteria* can be immobilized on *Pleurotus eryngii*. Compared to free cells, immobilized *Hexanoic acid bacteria* also protects the strain from external environmental influences. Both *Pleurotus eryngii* and *Hexanoic acid bacteria* are fungi; *Hexanoic acid bacteria* exhibits better binding force on the *Pleurotus eryngii* carrier, making it easier to adsorb and less susceptible to degradation. In contrast, the single adsorption method easily leads to strain loss. This invention further utilizes agar for embedding on top of the adsorption method, resulting in better immobilization. It is non-toxic to microorganisms, has a simple immobilization process, and provides excellent immobilization results.

[0037] (2) This invention uses a compound caproic acid bacterial solution with a well-defined bacterial community structure, which has stronger adaptability to the environment. The compound caproic acid bacterial solution is a mixture of fast-growing Clostridium celerecrescens JSJ-1 and Angel thermostable yeast. Fast-growing Clostridium celerecrescens JSJ-1 is a high-yielding, oxygen-tolerant caproic acid bacterium that inhibits caproic acid production by glucose. When used in combination with Angel thermostable yeast, it can alleviate the inhibitory effect of glucose on caproic acid production by C. celerecrescens JSJ-1, and the ethanol produced by the yeast from glucose can provide ethanol, the substrate for caproic acid production by C. celerecrescens JSJ-1. Yeast is a facultative anaerobic microorganism, while C. celerecrescens JSJ-1 is an anaerobic bacterium that can tolerate a certain amount of oxygen. When yeast and C. celerecrescens JSJ-1 coexist, the yeast can create a more favorable living environment for C. celerecrescens JSJ-1 by utilizing oxygen.

[0038] (3) The immobilized hexanoic acid bacteria prepared by the present invention can be used for fermentation to produce hexanoic acid, and can also be used in the brewing of liquor. It can significantly increase the content of hexanoic acid and ethyl hexanoate in liquor, and the effect is stable. Detailed Implementation

[0039] The present invention will be further described in detail below through embodiments. These embodiments are only used to illustrate the present invention and do not limit the scope of the present invention.

[0040] Unless otherwise specified, all quantities mentioned in the following examples are parts by weight. The glucose-inhibiting aerobic caproate-tolerant bacteria described in the examples are the strain disclosed in patent ZL202110311823.6, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC M 2020881, and its classification name is Clostridium celerecrescens JSJ-01 (deposit date: December 9, 2020, deposit address: Wuhan University, Wuhan, China).

[0041] Example 1

[0042] Preparation of immobilized compound caproic acid bacteria

[0043] (1) Preparation of pure hexanoic acid bacteria culture: Take one C. celerecrescens JSJ-1 glycerol tube and inoculate it into ES medium at 5% (v / v) (1 part yeast extract, 0.3 parts anhydrous sodium acetate, 0.02 parts MgSO4·7H2O, 0.04 parts K2HPO4, 0.05 parts (NH4)2SO4, 100 parts distilled water, then aseptically add 2 parts anhydrous ethanol and 0.8 parts dry heat sterilized CaCO3), filling the medium to 90% capacity, and incubate statically at 34℃ for 5 days. Then inoculate it into 1000 parts of ES medium at 5% (v / v) inoculation, fill the medium to 90% capacity, and incubate statically at 34℃ for 6 days until the hexanoic acid bacteria concentration reaches 10. 5 CFU / mL.

[0044] (2) Preparation of yeast culture: Add 0.05%-0.1% of Angel thermotolerant active dry yeast (1 part yeast extract, 2 parts peptone, 2 parts glucose, 100 parts water) to YPD medium and culture for 16 h at 34℃ and 200 r / min. Then, transfer the culture to YPD medium at an inoculation rate of 5% (v / v) for scale-up culture under the same conditions. At this time, the yeast concentration is approximately 10. 7 CFU / mL.

[0045] (3) Preparation of compound caproic acid bacteria solution: Mix pure caproic acid bacteria solution and yeast solution at a volume ratio of 1:2.

[0046] (4) Preparation of adsorption carrier: Take 40 portions of king oyster mushrooms and cut them into 1cm pieces. 3 Wash the left and right squares, sterilize them at 130℃ for 1.5 hours, and set aside.

[0047] (5) Adsorption of compound caproic acid bacteria: The treated adsorption carrier was immersed in the prepared compound caproic acid bacteria solution and allowed to stand at 35°C for 24 hours for adsorption.

[0048] (6) Preparation of embedding solution: Add 15 parts water to 1 part agar, heat until melted, and then place in a high-pressure steam sterilizer and sterilize at 115°C for 20 min.

[0049] (7) After the sterilized agar solution is taken out, it is cooled to 70°C. 1 / 3 of the agar solution is poured into a shallow dish that has been disinfected with medical alcohol. The king oyster mushroom carrier that has adsorbed caproic acid bacteria is evenly sprinkled in. Then the remaining agar solution is poured into the shallow dish and left to stand at 20°C for 25 minutes until solidified. It is then cut into small pieces with a knife that has been disinfected with alcohol to obtain immobilized composite caproic acid bacteria.

[0050] Example 2

[0051] Production of caproic acid bacteria liquid using immobilized compound caproic acid bacteria

[0052] Immobilized compound hexanoic acid bacteria were prepared according to Example 1. They were added to sterilized ES medium at a mass of 0.5% to fill 90% of the medium. The medium was then incubated at 34°C for 10 days. The hexanoic acid content in the fermentation broth was measured to be 14.86 g / L.

[0053] Example 3

[0054] Application of immobilized compound caproic acid bacteria in the production of strong-aroma baijiu without cellar mud

[0055] (1) Preparation of immobilized compound caproic acid bacteria: Immobilized compound caproic acid bacteria were prepared according to the method of Example 1, except that the amount of yeast extract added to the ES medium was 1.5 parts, anhydrous sodium acetate was 0.5 parts, and anhydrous ethanol was 2.5 parts.

[0056] (2) Steaming and cooking sorghum: Take 1000 portions of sorghum, wash it with water 6-7 times, then soak it in hot water at about 70℃ for 18 hours, and then wash it with cold water 4-5 times. Steam the washed sorghum at 127℃ for 30 minutes, then soak it in hot water for about 30 minutes, and then steam it again at 121℃ for 20 minutes.

[0057] (3) Mixing the starter: Spread the steamed sorghum to cool to 30°C, add about 20% of the raw grain weight of medium-high temperature starter and 5% of the steamed rice husks, and mix well.

[0058] (4) Add immobilized compound caproic acid bacteria: Take 600 parts of the mixed raw material and add 30 parts of immobilized compound caproic acid bacteria, mix well. Spread 20 parts of immobilized compound caproic acid bacteria at the bottom of the tank truck, then add about 200 parts of the mixed raw material and level it. Then add the raw material mixed with immobilized compound caproic acid bacteria, level it, and then load the remaining mixed raw material.

[0059] (5) Tank truck fermentation: Seal the top of the tank truck with a layer of plastic film and place it at 26℃ for 35 days to ferment.

[0060] (6) Distillation in a still: The fermented mash is loaded into a still for distillation. When loading the still, the material on both sides of the still should be about 1-2 cm higher than the middle. When collecting the liquor, the beginning and end should be removed. The collection should be stopped when the original liquor alcohol content is lower than 50% vol.

[0061] (7) Detection of aroma components in baijiu: Ethyl hexanoate, ethyl butyrate, ethyl acetate and ethyl lactate in baijiu were detected by gas chromatography.

[0062] Table 1. Detection of the content of four major esters in Baijiu (Chinese liquor)

[0063]

[0064] As shown in Table 1, the ethyl hexanoate content in the strong-aroma baijiu produced using immobilized compound hexanoic acid bacteria without cellar mud can reach 1082.39 mg / L.

[0065] Comparative Example 1

[0066] (1) Steaming and cooking sorghum: Take 1000 portions of sorghum, wash it with water 6-7 times, then soak it in hot water at about 70℃ for 18 hours, and then wash it with cold water 4-5 times. Steam the washed sorghum at 127℃ for 30 minutes, then soak it in hot water for about 30 minutes, and then steam it again at 127℃ for 30 minutes.

[0067] (2) Mixing the starter culture: Spread the steamed sorghum to cool to 30°C, add about 20% of the raw grain weight of medium-high temperature starter culture and 5% of the steamed rice husks, and mix well.

[0068] (3) Tanker fermentation: The mixture of yeast and rice husks is loaded into a tanker, the top of the tanker is sealed with a layer of plastic film, and fermented at 26℃ for 35 days.

[0069] (4) Distillation in a still: The fermented mash is loaded into a still for distillation. When loading the still, the material on both sides of the still should be about 1-2 cm higher than the middle. When collecting the liquor, the beginning and end should be removed. The collection should be stopped when the original liquor alcohol content is lower than 50% vol.

[0070] (5) Detection of aroma components in baijiu: Ethyl hexanoate, ethyl butyrate, ethyl acetate and ethyl lactate in baijiu were detected by gas chromatography.

[0071] Table 2. Detection of the content of the four major esters in the control group of Baijiu.

[0072]

[0073] As shown in Table 2, in Comparative Example 1, without the addition of immobilized compound caproic acid bacteria, the ethyl caproate content was only 16.88 mg / L. Using immobilized compound caproic acid bacteria to produce strong-aroma baijiu without cellar mud resulted in a significant increase in ethyl caproate content compared to the control group, with an increase of 1065.51 mg / L.

[0074] Example 4

[0075] Artificial pit mud prepared by immobilized compound caproic acid bacteria

[0076] Immobilized compound hexanoic acid bacteria were prepared according to the method described in Example 1.

[0077] 1000 portions of lotus pond mud were selected, and water was added to a moisture content of approximately 40%. The mud was then steamed for 120 minutes. Next, ES culture medium (10 parts yeast extract, 5 parts anhydrous sodium acetate, 0.2 parts MgSO4·7H2O, 0.5 parts K2HPO4, 0.5 parts (NH4)2SO4, 20 parts anhydrous ethanol, and 10 parts CaCO3) and 50 parts immobilized compound hexanoic acid bacteria were added, mixed thoroughly, and sealed with sealing film. The mixture was then incubated statically at 34℃ for 30 days, with samples taken and tested every 10 days.

[0078] Table 3. Trends in pH and butyric acid and hexanoic acid content during the cultivation of pit mud.

[0079]

[0080] As shown in Table 3, when immobilized compound hexanoic acid bacteria were used to cultivate pit mud, butyric acid and hexanoic acid were synthesized starting on the 10th day, and the hexanoic acid content reached 4.23 g / L on the 30th day of cultivation.

[0081] The cultured cellar mud was applied to the bottom and sides of the tank truck, and the remaining steps were the same as those in Comparative Example 1. The obtained liquor was then analyzed using gas chromatography.

[0082] Table 4. Content of four major esters in Baijiu fermented in artificial cellar mud carts

[0083]

[0084] As shown in Table 4, the ethyl hexanoate content in the liquor obtained after the artificial pit mud was cultured and coated onto the tank truck for fermentation was 2135.14 mg / L, while the ethyl hexanoate content in the liquor obtained under the same conditions in Comparative Example 1, which was fermented in a tank truck without pit mud coating, was only 16.88 mg / L.

[0085] Example 5

[0086] The hexanoic acid production effect of the immobilized compound hexanoic acid bacteria in Example 1 was compared with the hexanoic acid production effect of the free culture of compound hexanoic acid bacteria and the compound hexanoic acid bacteria prepared by the agar method.

[0087] Free culture: Following the method in Example 1, the compound hexanoic acid bacteria solution was directly inoculated into ES medium at an inoculation rate of 5% (v / v) and cultured for 10 days. Samples were taken periodically to detect hexanoic acid.

[0088] Agar fixation of compound caproic acid bacteria: The compound caproic acid bacteria solution was prepared according to Example 1. 15 parts water were added to 1 part agar, heated until melted, and then sterilized in an autoclave at 115°C for 20 minutes. After sterilization, the agar solution was removed and cooled to 70°C. The prepared compound caproic acid bacteria solution was mixed thoroughly with the agar solution and poured into a shallow dish sterilized with medical alcohol. The mixture was allowed to stand at 20°C for 25 minutes until solidified. The solidified bacteria were then cut into small pieces with an alcohol-sterilized knife to obtain the agar-fixed compound caproic acid bacteria. The agar-fixed compound caproic acid bacteria were added to ES medium at a concentration of 0.5% by weight and cultured for 10 days. Samples were taken periodically to detect caproic acid.

[0089] The effects of hexanoic acid production under the three conditions are shown in the table below.

[0090] Table 1 Comparison of Hexanoic Acid Production Effects

[0091]

[0092] Compared to free-cultured hexanoic acid bacteria, immobilized hexanoic acid production is significantly better, with a much earlier formation time. The immobilized composite hexanoic acid bacteria of this invention achieves hexanoic acid production in just 5 days, approaching the level of free culture after 10 days. Furthermore, the maximum hexanoic acid yield during immobilized culture is significantly higher than in free culture. In free culture, free cells are in direct contact with metabolites, leading to a large accumulation of hexanoic acid and a strong inhibitory effect on cell production. In this invention, the immobilized composite hexanoic acid bacteria, when culturing to produce hexanoic acid, have cells fixed within a carrier, which to some extent protects the bacteria from external environmental interference.

[0093] Compared with immobilized composite caproic acid bacteria prepared by direct encapsulation using the agar method, the immobilized caproic acid bacteria prepared by combining Pleurotus eryngii adsorption and agar encapsulation in this invention have better acid production effect and higher caproic acid yield.

[0094] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the invention.

Claims

1. A method for preparing immobilized compound caproic acid bacteria, characterized in that, Includes the following steps: (1) Preparation of pure hexanoic acid bacteria culture: The hexanoic acid bacteria seed culture was inoculated into sodium ethanol acetate culture medium at an inoculation rate of 5%-10% (v / v) of the culture medium, with a liquid volume of 80%-95%, and then statically cultured at 33℃-35℃ for 5-7 days; the hexanoic acid bacteria is Clostridium JSJ-1 CCTCC M 2020881, which is the strain disclosed in patent ZL202110311823.6; (2) Preparation of yeast culture: Add thermostable active dry yeast to YPD medium, culture in a shaker at constant temperature, and then transfer to YPD medium for scale-up culture under the same conditions; (3) Preparation of compound caproic acid bacteria solution: Mix pure caproic acid bacteria solution with yeast solution; mix caproic acid bacteria solution and yeast solution at a volume ratio of 1:1.5-2; (4) Preparation of adsorption carrier: Take king oyster mushroom, chop, wash and sterilize to obtain the adsorption carrier; (5) Adsorption of compound caproic acid bacteria: The treated adsorption carrier is immersed in the prepared compound caproic acid bacteria solution for full adsorption; the ratio of the mass of the adsorption carrier to the volume of the caproic acid bacteria solution is 0.3-0.5 g: 10-12 mL; (6) Preparation of embedding solution: Dissolve agar in water, heat to melt, and sterilize to obtain the solution; (7) The Pleurotus ostreatus carrier that has adsorbed caproic acid bacteria is evenly immersed in the embedding solution, and after solidification, immobilized composite caproic acid bacteria are obtained.

2. The method according to claim 1, characterized in that: In step (2), amplification culture is carried out at an inoculation amount of 5% of the culture medium mass.

3. The method according to claim 1, characterized in that: In step (4), the king oyster mushrooms are cut into cubes of 1-2 cm³.

4. The method according to claim 1, characterized in that: In step (4), sterilization is performed by dry heat sterilization at 130-135℃ for 1-2 hours.

5. An immobilized composite hexanoic acid bacteria, characterized in that: Prepared by the method described in any one of claims 1-4.

6. The application of the immobilized composite hexanoic acid bacteria as described in claim 5 in the fermentation production of hexanoic acid.

7. The application of the immobilized compound caproic acid bacteria as described in claim 5 in the fermentation production of strong-aroma baijiu.

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