Compound microbial agent for producing hexanoic acid by utilizing wine brewing waste as well as preparation method and application of compound microbial agent

Abstract

The invention discloses a compound microbial agent for producing hexanoic acid by utilizing wine-making waste as well as a preparation method and application of the compound microbial agent, and aims to solve the problems that the reaction activity is difficult to control and the reaction effect is unstable when the wine-making waste is converted into the hexanoic acid at present. The viable count ratio of bacillus coagulans to pediococcus ethanolidurans to clostridium wellfuensis to clostridium rumen to clostridium coriolis in the compound microbial agent for producing hexanoic acid by utilizing the wine brewing wastes is (6-8): (7-9): (3-5): (5-7): (6-8). According to the invention, the caproic acid-producing complex microbial inoculant with stable performance is scientifically proportioned, the microbial inoculant can adapt to a high-ethanol and high-lactic-acid environment of bacteria-containing wine-making wastes, macromolecular substances such as proteins and saccharides and small molecular compounds such as acetic acid, lactic acid and ethanol in the wine-making wastes are fully utilized, and the microbial inoculant has the advantages of shorter caproic acid production cycle, higher yield and higher yield. The caproic acid yield is higher, the batch error is lower, and the brewing waste can be continuously and stably treated.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and specifically relates to a composite microbial agent for producing caproic acid by utilizing winemaking waste, and a preparation method and application thereof. Background Art

[0002] my country is a major producer and consumer of liquor, producing more than 11 million kiloliters of liquor each year. However, every liter of liquor produced will produce 3 to 4 tons of lees and discharge 12 to 20 tons of brewing wastewater. The liquor industry produces more than 40 million tons of lees and more than 600 million tons of wastewater annually. The characteristics of lees are high water content and high acidity. A large amount of sewage is easily leaked during the stacking process. In addition to water, lees also contain a large amount of organic matter and nutrients such as nitrogen, phosphorus and potassium. At present, lees are mainly treated by combustion, but this method has many problems. First, if the combustion process is not properly controlled, harmful substances such as nitrogen oxides will be released, which will pollute the environment. Secondly, since the lees itself has a high water content, in order to make it burnable, it needs to be dried at a high cost. Brewing wastewater has the characteristics of many suspended particles, high organic matter concentration, and large B / C value. Its treatment method is mainly based on standard discharge. Conventional treatment methods include physical and chemical technology, biotechnology, and combined technology. In addition, many researchers are also exploring new treatment technologies such as electrolysis and catalysis. At present, except for some wine companies that make use of a small amount of spent lees as resources, a large amount of existing winemaking waste (including spent lees and winemaking wastewater) has not been reasonably and effectively utilized. If it can be recycled and utilized as resources, it can not only solve environmental problems, but also reduce the burden on enterprises and improve economic benefits.

[0003] At present, some studies have reported on the resource utilization forms of waste lees or brewing wastewater. In terms of conventional resource utilization, it is mainly converted into fertilizer or feed. Patent CN116969783A discloses a method for preparing organic fertilizer using brewing wastewater and straw. Patent CN118452319A discloses a feed additive prepared from brewing waste and its preparation method. Feeding laying hens with this additive can increase the egg production rate of laying hens and reduce the feed-egg ratio. Patent CN110540462A discloses an organic fertilizer binder based on brewing waste and its preparation method. Through the comprehensive utilization of brewing waste cellar mud, lees, koji waste straw and lees leachate, an organic fertilizer granulation binder is prepared. These methods can convert brewing waste into products of certain value, but the products of these methods are all primary mixed substances, which are suitable for agriculture and animal husbandry. They cannot form high-purity and high-value chemical products, and the economic benefits are low.

[0004] Caproic acid is a high-value, multifunctional, easy-to-separate high-quality chemical product and has attracted much attention. In terms of converting brewing waste into caproic acid, patent CN118620787A discloses a caproic acid-producing strain and its screening method and a method for preparing high-ester flavored wine by fermenting yellow water. The caproic acid-producing strain can utilize lactic acid, residual sugar and starch in yellow water to produce caproic acid. Patent CN110372490A discloses a method for continuously producing medium-chain carboxylic acids using brewing wastewater. However, these methods face a common problem, that is, the inoculant is either an unknown microorganism such as cellar mud and sludge, and its reaction activity is difficult to avoid huge differences between batches; or a single species is used as the inoculant, and it is difficult to face the influence of self-generated microorganisms of complex substrates on functional bacteria. Generally speaking, the reaction effect is not stable.

[0005] Therefore, how to stably and continuously convert brewing waste into high value-added chemical products, enable the reaction system to have the ability to resist the bacterial environment of the substrate, make full use of the sugar and protein macromolecules in the brewing waste, and be suitable for the special environment of high ethanol and high lactic acid in brewing waste is the problem that the present invention needs to solve. Summary of the invention

[0006] The purpose of the present invention is to solve the problem that the reaction activity of the existing brewing waste converted into caproic acid is difficult to control and the reaction effect is unstable. The present invention provides a composite microbial agent for producing caproic acid using brewing waste. The composite agent can make full use of sugar and protein macromolecules in the brewing waste and can be suitable for the special environment of high ethanol and high lactic acid in the brewing waste. It can effectively convert brewing waste of various proportions into high-value caproic acid products, and has the dual environmental significance of environmental protection and waste resource utilization.

[0007] The composite microbial agent for producing caproic acid from brewing waste of the present invention comprises Bacillus coagulans, ethanol-resistant Pediococcus, Clostridium suijingfang, Clostridium rumenii and Clostridium cohnii, and the ratio of the number of live bacteria of Bacillus coagulans, ethanol-resistant Pediococcus, Clostridium suijingfang, Clostridium rumenii and Clostridium cohnii in the composite microbial agent is (6-8):(7-9):(3-5):(5-7):(6-8).

[0008] The bacterial species in the composite microbial agent of the present invention are all anaerobic or facultative anaerobic functional bacteria, all have high ethanol and high lactic acid tolerance, can adapt well to the special environment of brewing waste and perform functions. Among them, Bacillus coagulans can convert macromolecular substances in brewing waste into lactic acid and acetic acid; ethanol-resistant Pediococcus can convert macromolecular substances in brewing waste into lactic acid; Clostridium shuijingfang can use lactic acid and macromolecular substances in brewing waste to produce acetic acid, ethanol and caproic acid; Clostridium rumenii can convert lactic acid in brewing waste into caproic acid; Clostridium cohnii can convert ethanol and acetic acid in brewing waste into butyric acid, and convert ethanol and butyric acid into caproic acid.

[0009] The preparation method of the composite microbial agent for producing caproic acid by using brewing waste of the present invention is achieved by the following steps:

[0010] 1. inoculating Bacillus coagulans into an activation medium for anaerobic activation to obtain an activated mother solution of Bacillus coagulans;

[0011] 2. inoculating the ethanol-resistant Pediococcus into an activation medium for anaerobic activation treatment to obtain an ethanol-resistant Pediococcus activation mother solution;

[0012] 3. Inoculating the Clostridium suijingfang into the activation medium for anaerobic activation treatment to obtain the Clostridium suijingfang activated mother solution;

[0013] 4. inoculating the rumen Clostridium into the activation medium for anaerobic activation treatment to obtain the rumen Clostridium activation mother solution;

[0014] 5. inoculating Clostridium cohnii into an activation medium for anaerobic activation treatment to obtain Clostridium cohnii activation mother solution;

[0015] 6. The activated mother liquor of Bacillus coagulans, the activated mother liquor of ethanol-resistant Pediococcus, the activated mother liquor of Clostridium shuijingfang, the activated mother liquor of Clostridium rumenii and the activated mother liquor of Clostridium cohnii are inoculated into the composite culture medium for anaerobic composite culture to obtain the composite microbial agent for producing caproic acid using brewing waste.

[0016] The application of the composite microbial agent for producing caproic acid from brewing waste of the present invention is to use the composite microbial agent as an inoculant, inoculate it into brewing waste for anaerobic culture to produce caproic acid.

[0017] The composite microbial agent for producing caproic acid from brewing waste and its preparation method and application include the following beneficial effects:

[0018] 1. The present invention scientifically proportions a composite caproic acid-producing bacterial agent with stable performance, and the cross-division of labor between bacterial species is clear, which can adapt to the high-ethanol and high-lactic acid environment of bacterial brewing waste, and make full use of macromolecular substances such as proteins and sugars and small molecular compounds such as acetic acid, lactic acid, and ethanol in the brewing waste.

[0019] 2. Compared with conventional single strains or unknown microbial inocula such as sludge, cellar mud, and soil, the caproic acid-producing composite microbial agent configured in the present invention has a shorter caproic acid production cycle, a greater caproic acid yield, and a lower batch error. It can continuously and stably treat brewing waste and meets the requirements of industrial production.

[0020] 3. The composite microbial agent obtained by the present invention has the best treatment effect for various types and ratios of brewing waste, and can be widely applied to different brewing industry by-products or wastes, and has a broad prospect of application. DETAILED DESCRIPTION

[0021] Specific implementation method 1: In this implementation method, the composite microbial agent for producing caproic acid from brewing waste includes Bacillus coagulans, ethanol-resistant Pediococcus, Clostridium suijingfang, Clostridium rumenii and Clostridium cohnii. The ratio of the number of live bacteria of Bacillus coagulans, ethanol-resistant Pediococcus suijingfang, Clostridium rumenii and Clostridium cohnii in the composite microbial agent is (6-8):(7-9):(3-5):(5-7):(6-8).

[0022] The ratio of the number of live bacteria of Bacillus coagulans, ethanol-resistant Pediococcus, Clostridium suifangensis, Clostridium rumenii and Clostridium cohnii in the composite microbial agent of this embodiment is preferably 7.3:8.2:4.5:6.4:7.8.

[0023] The composite microbial agent of this embodiment includes Bacillus coagulans ATCC 23498, Pediococcus ethanolidurans CICC 22725, Clostridium swellfunianum CICC 10730, Clostridium sp. ATCC TSD-31, and Clostridium kluyveri ATCC 12489. The above strains are all from laboratory preservation and can be purchased from the American Type Culture Collection (ATCC) or the China Industrial Microbiological Culture Collection Center.

[0024] Specific implementation method 2: The preparation method of the composite microbial agent for producing caproic acid using brewing waste in this implementation method is implemented according to the following steps:

[0025] 1. inoculating Bacillus coagulans into an activation medium for anaerobic activation to obtain an activated mother solution of Bacillus coagulans;

[0026] 2. inoculating the ethanol-resistant Pediococcus into an activation medium for anaerobic activation treatment to obtain an ethanol-resistant Pediococcus activation mother solution;

[0027] 3. Inoculating the Clostridium suijingfang into the activation medium for anaerobic activation treatment to obtain the Clostridium suijingfang activated mother solution;

[0028] 4. inoculating the rumen Clostridium into the activation medium for anaerobic activation treatment to obtain the rumen Clostridium activation mother solution;

[0029] 5. inoculating Clostridium cohnii into an activation medium for anaerobic activation treatment to obtain Clostridium cohnii activation mother solution;

[0030] 6. The activated mother liquor of Bacillus coagulans, the activated mother liquor of ethanol-resistant Pediococcus, the activated mother liquor of Clostridium shuijingfang, the activated mother liquor of Clostridium rumenii and the activated mother liquor of Clostridium cohnii are inoculated into the composite culture medium for anaerobic composite culture to obtain the composite microbial agent for producing caproic acid using brewing waste.

[0031] In step six of this embodiment, the anaerobic composite culture time is controlled to be 6 to 10 days.

[0032] Specific implementation method three: The difference between this implementation method and specific implementation method two is that the anaerobic activation treatment in steps one to five is cultured at 37° C. and 180 rpm for 72 hours.

[0033] Specific embodiment 4: This embodiment is different from specific embodiment 2 or 3 in that the activation culture medium in step 1 is mixed with 10.0 g of casein peptone, 10.0 g of beef extract powder, 5.0 g of yeast extract powder, 1.0 g of Tween 80, 5.0 g of sodium acetate, 2.0 g of triammonium citrate, 2.0 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·H2O and 1000.0 mL of distilled water; then sterilized at 121°C under high pressure for 20 min and allowed to stand at room temperature, 20.0 g of glucose is added in the clean bench, and the pH is adjusted to 6.2-6.5.

[0034] Specific embodiment five: This embodiment is different from specific embodiments two to four in that the activation culture medium in step two is mixed with 10.0 g of casein peptone, 10.0 g of beef extract powder, 5.0 g of yeast extract powder, 1.0 g of Tween 80, 5.0 g of sodium acetate, 2.0 g of triammonium citrate, 2.0 g of K2HPO4, 0.2 g of MgSO4·7H2O, 0.05 g of MnSO4·H2O and 1000.0 mL of distilled water; then sterilized at 121°C under high pressure for 20 min and allowed to stand at room temperature, 20.0 g of glucose is added in the clean bench, and the pH is adjusted to 6.2-6.5.

[0035] Specific embodiment six: This embodiment is different from specific embodiments two to five in that the activation culture medium in step three is prepared by mixing 0.5 g of yeast extract powder, 3.0 g of trypsin, 0.33 g of NH4Cl, 0.33 g of KCl, 0.33 g of KH2PO4, 1.0 mL of trace salt solution SL10, 1.0 mg of resazurin, 0.33 g of CaCl2·2H2O, 0.33 g of MgCl2·6H2O, 4 g of glucose and 1000.0 mL of distilled water; then, the mixture is sterilized at 121°C under high pressure for 20 min and allowed to stand at room temperature, and 0.5 g of cysteine ​​hydrochloride and 10.0 mL of vitamin solution are added in an ultra-clean workbench, and the pH is adjusted to 7.0-7.3.

[0036] Specific embodiment seven: This embodiment is different from any of specific embodiments two to six in that the activation culture medium in step four is prepared by mixing 10.0 g of casein peptone, 10.0 g of beef extract powder, 5.0 g of yeast extract powder, 0.45 g of K2HPO4, 0.9 g of KH2PO4, 0.1 g of MgSO4·7H2O, 0.05 g of MnSO4·H2O, 1.0 mg of resazurin, 0.1 g of CaCl2·2H2O, 1.0 mL of trace salt solution SL10, and 1000.0 mL of distilled water; then, the mixture is sterilized at 121° C. under high pressure for 20 min and allowed to stand at room temperature, and 5 mL of lactic acid, 1 g of cysteine ​​hydrochloride, and 10.0 mL of vitamin solution are added in an ultra-clean workbench, and the pH is adjusted to 6.2 to 6.5.

[0037] Specific embodiment eight: This embodiment is different from specific embodiments two to seven in that the activation culture medium in step five is prepared by mixing 5 g of yeast extract powder, 5.0 g of sodium acetate, 0.41 g of K2HPO4, 0.23 g of KH2PO4, 0.45 g of MgSO4·7H2O, 0.25 g of NH4Cl, 0.1 g of CaCl2·2H2O, 1.0 mL of trace salt solution SL10, and 1000.0 mL of distilled water; then, the mixture is sterilized at 121° C. under high pressure for 20 min and allowed to stand at room temperature, and 20 mL of ethanol, 0.4 g of cysteine, and 10.0 mL of vitamin solution are added in an ultra-clean workbench, and the pH is adjusted to 6.2-6.5.

[0038] Specific embodiment nine: This embodiment is different from any of specific embodiments two to eight in that the composite culture medium in step six is ​​prepared by mixing 5 g of casein peptone, 5 g of yeast extract powder, 5.0 g of sodium acetate, 0.41 g of K2HPO4, 0.23 g of KH2PO4, 0.45 g of MgSO4·7H2O, 0.25 g of NH4Cl, 0.1 g of CaCl2·2H2O, 1.0 mL of trace salt solution SL10 and 1000.0 mL of distilled water; then sterilized at 121°C under high pressure for 20 min and allowed to stand at room temperature, 10 mL of ethanol, 10 mL of lactic acid, 0.4 g of cysteine ​​and 10.0 mL of vitamin solution are added in an ultra-clean workbench, and the pH is adjusted to 6.2-6.5.

[0039] Specific embodiment ten: This embodiment is different from any of specific embodiments six, seven or nine in that the composition of the trace salt solution SL10 is as follows: HCl (25%; 7.7M) 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O2 4.0 mg, Na2MoO4·2H2O 36.0 mg, and distilled water 990.0 mL.

[0040] Specific embodiment eleven: This embodiment is different from any one of specific embodiments six to nine in that the composition of the vitamin solution is as follows: 2.0 mg biotin, 2.0 mg folic acid, 10.0 mg pyridoxine hydrochloride, 5.0 mg thiamine hydrochloride·2H2O, 5.0 mg riboflavin, 5.0 mg niacin, 5.0 mg DCA calcium pantothenate, 0.1 mg vitamin B12, 5.0 mg p-aminobenzoic acid, 5.0 mg lipoic acid, and 1000.0 mL distilled water.

[0041] Specific embodiment 12: In this embodiment, the application of the composite microbial agent for producing caproic acid from brewing waste is to use the composite microbial agent as an inoculum, inoculate it into brewing waste for anaerobic culture to produce caproic acid.

[0042] The brewing waste used in this embodiment is not sterilized.

[0043] Specific embodiment 13: This embodiment is different from specific embodiment 12 in that the bacterial concentration in the composite microbial agent is 1×10 9 ~2×10 9 CFU / mL.

[0044] Specific implementation mode fourteen: This implementation mode is different from specific implementation mode twelve in that the brewing waste contains spent grains and brewing wastewater.

[0045] Embodiment: The preparation method of the composite microbial agent for producing caproic acid using brewing waste in this embodiment is implemented according to the following steps:

[0046] 1. Activation of Bacillus coagulans:

[0047] Bacillus coagulans was pre-cultured in a 120 mL anaerobic bottle containing 50 mL of Bacillus coagulans activation medium: Bacillus coagulans was inoculated, nitrogen was purged for 10 minutes, and the culture was activated at 37°C and 180 rpm for 72 hours. The bacterial concentration of the activated mother solution of Bacillus coagulans was 7.3 × 10 8 CFU / mL;

[0048] The configuration of the activation medium for Bacillus coagulans is as follows: casein peptone 10.0 g, beef extract powder 10.0 g, yeast extract powder 5.0 g, Tween 80 1.0 g, sodium acetate 5.0 g, ammonium citrate tribasic 2.0 g, K2HPO4 2.0 g, MgSO4·7H2O 0.2 g, MnSO4·H2O 0.05 g, and distilled water 1000.0 mL; sterilize at 121°C for 20 min and then let stand at room temperature, add 20.0 g of glucose in the clean bench, and adjust the pH to 6.5.

[0049] 2. Activation of ethanol-resistant Pediococcus:

[0050] Use a 120 mL anaerobic bottle filled with 50 mL of ethanol-resistant Pediococcus activation medium to pre-culture ethanol-resistant Pediococcus: inoculate ethanol-resistant Pediococcus, purge with nitrogen for 10 minutes, and activate by culturing at 37 ° C and 180 rpm for 72 hours. The bacterial concentration of the activated mother solution of ethanol-resistant Pediococcus is 4.1×10 8 CFU / mL;

[0051] Preparation of ethanol-resistant Pediococcus activation medium: casein peptone 10.0g, beef extract powder 10.0g, yeast extract powder 5.0g, Tween 80 1.0g, sodium acetate 5.0g, ammonium citrate trihydrate 2.0g, K2HPO4 2.0g, MgSO4·7H2O 0.2g, MnSO4·H2O 0.05g, distilled water 1000.0mL. After high-pressure sterilization at 121℃ for 20min, let it stand at room temperature, add 20.0g of glucose in the clean bench, and adjust the pH to 6.5.

[0052] 3. Activation of Clostridium difficile in Shuijingfang:

[0053] Use a 120 mL anaerobic bottle filled with 50 mL of Clostridium suifen activation medium to pre-culture Clostridium suifen: inoculate Clostridium suifen, purge with nitrogen for 10 minutes, and activate by culturing at 37 °C and 180 rpm for 72 hours. The bacterial concentration of the Clostridium suifen activation mother solution is 4.5 × 10 8 CFU / mL;

[0054] The configuration of Clostridium shuijingfang activation medium: yeast extract powder 0.5g, tryptone 3.0g, NH4Cl 0.33g, KCl0.33g, KH2PO40.33g, trace salt solution SL101.0mL, resazurin 1.0mg, CaCl2·2H2O 0.33g, MgCl2·6H2O0.33g, glucose 4g, distilled water 1000.0mL; sterilize at 121℃ for 20min and let stand at room temperature, add cysteine ​​hydrochloride 0.5g and vitamin solution 10.0mL in the clean bench, and adjust the pH to 7.0;

[0055] The composition of the vitamin solution is as follows: biotin 2.0 mg, folic acid 2.0 mg, pyridoxine hydrochloride 10.0 mg, thiamine hydrochloride·2H2O 5.0 mg, riboflavin 5.0 mg, niacin 5.0 mg, DCA calcium pantothenate 5.0 mg, vitamin B12 0.1 mg, p-aminobenzoic acid 5.0 mg, lipoic acid 5.0 mg, distilled water 1000.0 mL; trace salt solution SL10: HCl (25%; 7.7 M) 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0mg, distilled water 990.0mL.

[0056] 4. Activation of Rumen Clostridium:

[0057] A 120 mL anaerobic bottle containing 50 mL of ruminal Clostridium activation medium was used to pre-culture ruminal Clostridium: inoculated with ruminal Clostridium, purged with nitrogen for 10 minutes, and cultured for 72 hours at 37°C and 180 rpm for activation. The bacterial concentration of the ruminal Clostridium activation mother solution was 3.2 × 10 8 CFU / mL;

[0058] Configuration of ruminal Clostridium activation medium: casein peptone 10.0g, beef extract powder 10.0g, yeast extract powder 5.0g, K2HPO4 0.45g, KH2PO4 0.9g, MgSO4·7H2O 0.1g, MnSO4·H2O 0.05g, resazurin 1.0mg, CaCl2·2H2O 0.1g, trace salt solution SL10 1.0mL, distilled water 1000.0mL; sterilize at 121℃ for 20min and let stand at room temperature, add lactic acid 5mL, cysteine ​​hydrochloride 1g, vitamin solution 10.0mL in the clean bench, and adjust pH to 6.5;

[0059] The composition of the vitamin solution is as follows: biotin 2.0 mg, folic acid 2.0 mg, pyridoxine hydrochloride 10.0 mg, thiamine hydrochloride·2H2O 5.0 mg, riboflavin 5.0 mg, niacin 5.0 mg, DCA calcium pantothenate 5.0 mg, vitamin B12 0.1 mg, p-aminobenzoic acid 5.0 mg, lipoic acid 5.0 mg, distilled water 1000.0 mL; trace salt solution SL10: HCl (25%; 7.7 M) 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0mg, distilled water 990.0mL.

[0060] 5. Activation of Clostridium coli:

[0061] Use 120 mL anaerobic bottles filled with 50 mL of Clostridium coli activation medium to pre-culture Clostridium coli: inoculate Clostridium coli, purge with nitrogen for 10 minutes, and activate by culturing at 37 °C and 180 rpm for 72 h. The concentration of Clostridium coli activated mother solution was 3.9 × 10 8 CFU / mL;

[0062] Configuration of Clostridium cohnii activation medium: 5g yeast extract powder, 5.0g sodium acetate, 0.41g K2HPO4, 0.23g KH2PO4, 0.45g MgSO4·7H2O, 0.25g NH4Cl, 0.1g CaCl2·2H2O, 1.0mL trace salt solution SL10, 1000.0mL distilled water; sterilize at 121℃ for 20min and let stand at room temperature, add 20ml ethanol, 0.4g cysteine, 10.0mL vitamin solution in the clean bench, and adjust the pH to 6.5;

[0063] Trace salt solution SL10: HCl (25%; 7.7 M) 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O2 4.0 mg, Na2MoO4·2H2O 36.0 mg, distilled water 990.0 mL.

[0064] 6. Optimize the ratio of Bacillus coagulans, ethanol-resistant Pediococcus, Clostridium difficile, Clostridium difficile and Clostridium cohnii.

[0065] A 250mL anaerobic bottle containing 100mL of composite culture medium was inoculated with activated mother liquor of Bacillus coagulans, activated mother liquor of ethanol-resistant Pediococcus, activated mother liquor of Clostridium shuijingfang, activated mother liquor of Clostridium rumenii and activated mother liquor of Clostridium cohnii in different proportions for composite culture.

[0066] The composite culture medium was prepared by: casein peptone 5 g, yeast extract powder 5 g, sodium acetate 5.0 g, K2HPO4 0.41 g, KH2PO4 0.23 g, MgSO4·7H2O 0.45 g, NH4Cl 0.25 g, CaCl2·2H2O 0.1 g, trace salt solution SL10 1.0 mL, distilled water 1000.0 mL, autoclave at 121°C for 20 min and let stand at room temperature, add ethanol 10 mL, lactic acid 10 mL, cysteine ​​0.4 g, and vitamin solution 10.0 mL in a clean bench, and adjust the pH to 6.5;

[0067] The composition of the vitamin solution is as follows: biotin 2.0 mg, folic acid 2.0 mg, pyridoxine hydrochloride 10.0 mg, thiamine hydrochloride·2H2O 5.0 mg, riboflavin 5.0 mg, niacin 5.0 mg, DCA calcium pantothenate 5.0 mg, vitamin B12 0.1 mg, p-aminobenzoic acid 5.0 mg, lipoic acid 5.0 mg, distilled water 1000.0 mL; trace salt solution SL10: HCl (25%; 7.7 M) 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0mg, distilled water 990.0mL.

[0068] The volume ratios of the activated mother liquor of Bacillus coagulans, the activated mother liquor of Pediococcus ethanol-resistant, the activated mother liquor of Clostridium shuijingfang, the activated mother liquor of Clostridium rumenii and the activated mother liquor of Clostridium cohnii are as follows: 1:1:1:1:1, 2:1:1:1:1, 1:2:1:1:1, 1:1:2:1:1, 1:1:1:2:1, 1:1:1:1:2, 2:2:1:1:1, 2:1:2:1:1, 2:1:1:2:1, 2:1:1:1:2, 1:2:2:1:1, 1:2:1: The inoculum size of each component was 2% (v / v). Nitrogen was purged for 20 minutes. The culture conditions were 37°C and 180 rpm. The caproic acid concentration was detected by gas chromatography on the 10th and 20th days of culture. The results are shown in Table 1.

[0069] Table 1 Hexanoic acid yield of composite activated mother liquor with different inoculation ratios

[0070]

[0071] It can be seen from Table 1 that in the group with a large inoculation score, a greater caproic acid production can be achieved on the 10th day, but the ratio corresponding to the maximum caproic acid production on the 10th day is not necessarily the optimal ratio value. When the volume ratios of activated mother liquor of Bacillus coagulans, activated mother liquor of ethanol-resistant Pediococcus, activated mother liquor of Clostridium shuijingfang, activated mother liquor of Clostridium rumenii and activated mother liquor of Clostridium cohnii were 1:2:1:2:2 respectively (when the ratio of live bacteria was 7.3:8.2:4.5:6.4:7.8) as inoculants, the mixed fermentation system showed the best caproic acid production capacity.

[0072] Application Example: The application of the composite microbial agent producing caproic acid in brewing waste in this embodiment includes the following steps:

[0073] 1. Preparation of composite microbial agents

[0074] The activated mother liquor of Bacillus coagulans, the activated mother liquor of Pediococcus ethanol-resistant, the activated mother liquor of Clostridium shuijingfangensis, the activated mother liquor of Clostridium rumenicola and the activated mother liquor of Clostridium cohnii were inoculated into a 250mL anaerobic bottle containing 100mL of composite culture medium at a ratio of 1:2:1:2:2 for composite culture. Nitrogen was purged for 20 minutes at the beginning of the culture. The culture conditions were 37°C, 180rpm, and 7 days to form a liquid composite microbial agent. At this time, the bacterial concentration of the liquid composite microbial agent was 1.76×10 9 CFU / mL.

[0075] Configuration of composite culture medium: casein peptone 5g, yeast extract powder 5g, sodium acetate 5.0g, K2HPO4 0.41g, KH2PO4 0.23g, MgSO4·7H2O 0.45g, NH4Cl 0.25g, CaCl2·2H2O 0.1g, trace salt solution SL101.0mL, distilled water 1000.0mL, autoclave at 121℃ for 20min and let stand at room temperature, add ethanol 10mL, lactic acid 10mL, cysteine ​​0.4g, vitamin solution 10.0mL in the clean bench, and adjust the pH to 6.2-6.5.

[0076] The composition of the vitamin solution is as follows: biotin 2.0 mg, folic acid 2.0 mg, pyridoxine hydrochloride 10.0 mg, thiamine hydrochloride·2H2O 5.0 mg, riboflavin 5.0 mg, niacin 5.0 mg, DCA calcium pantothenate 5.0 mg, vitamin B12 0.1 mg, p-aminobenzoic acid 5.0 mg, lipoic acid 5.0 mg, distilled water 1000.0 mL; trace salt solution SL10: HCl (25%; 7.7 M) 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0mg, distilled water 990.0mL.

[0077] 2. Application of composite microbial agents in brewing waste

[0078] Take 20g of liquid bacterial agent as inoculum, inoculate it into 500mL anaerobic bottles containing 300g of non-sterilized brewing waste in different proportions for cultivation, adjust the pH of the brewing waste to 6.3-6.5, use nitrogen to purge for 20 minutes at the beginning of cultivation, and the culture conditions are 37℃ and 180rpm. Use gas chromatography to determine the daily production of caproic acid, and determine the production cycle and maximum production of caproic acid.

[0079] Among them, when the caproic acid concentration has not increased or decreased for 3 consecutive days, the first day without increase or decrease is considered to be the caproic acid production cycle. The mass ratio of brewing waste is spent lees: brewing wastewater = 1:0, 2:1, 1:1, 1:2 or 0:1. When spent lees: brewing wastewater = 2:1, 1:1, 1:2, 0:1, take the original solution and filter it through a 0.22μm filter membrane, and directly use gas chromatography for determination. When spent lees: brewing wastewater = 1:0, take the mixture and mix it with pure water in a 1:1 ratio for 10 minutes, take the supernatant and filter it through a 0.22μm filter membrane, and use the filtrate for gas chromatography determination.

[0080] Comparative Example 1:

[0081] The difference between Comparative Example 1 and the application example is that in Comparative Example 1, 20 g of sludge is taken as inoculum, and the sludge is taken from a sewage plant in Harbin.

[0082] Comparative Example 2:

[0083] The difference between Comparative Example 2 and the application example is that in Comparative Example 2, 20 g of cellar mud is taken as inoculum, and the cellar mud is taken from a winery in Harbin.

[0084] Comparative Example 3:

[0085] The difference between Comparative Example 3 and the application example is that in Comparative Example 3, 20 g of soil is taken as inoculum, and the soil is taken from a wetland park in Harbin.

[0086] Table 2 Maximum caproic acid production and caproic acid production cycle in brewing wastewater with different proportions of composite bacterial agent, sludge, cellar mud and soil.

[0087]

[0088] Table 2 shows the maximum caproic acid production and caproic acid production cycle of composite bacteria, sludge, cellar mud, and soil in different ratios of lees: brewery wastewater. It can be seen that the caproic acid production capacity of composite bacteria in different ratios of brewery waste is the best. When the substrate is lees: brewery wastewater = 1:0, compared with the mean of other groups, the maximum caproic acid production of composite bacteria increased by 71.08%, the caproic acid production cycle was shortened by 30.49%, and the batch error was reduced by 67.19%. When the substrate is lees: brewery wastewater = 2:1, compared with the mean of other groups, the maximum caproic acid production of composite bacteria increased by 70.34%, the caproic acid production cycle was shortened by 30.56%, and the batch error was reduced by 40%. When the substrate is lees: brewery wastewater = 1:1, compared with the mean of other groups, the maximum caproic acid production of composite bacteria increased by 85.06%, the caproic acid production cycle was shortened by 29.03%, and the batch error was reduced by 51.61%. When the substrate ratio was lees: brewing wastewater = 1:2, compared with the mean values ​​of other groups, the maximum caproic acid yield of the composite bacterial agent increased by 87.42%, the caproic acid production cycle was shortened by 21.25%, and the batch error was reduced by 37.84%. When the substrate ratio was lees: brewing wastewater = 0:1, compared with the mean values ​​of other groups, the maximum caproic acid yield of the composite bacterial agent increased by 55.58%, the caproic acid production cycle was shortened by 28.57%, and the batch error was reduced by 53.66%.

[0089] Compared with conventional unknown biological inoculants such as sludge, cellar mud, and soil, the composite microbial inoculant of the present invention has the shortest production cycle, the largest output, and the lowest batch error, and can be well applied to winemaking waste of various components. The inoculant is simple to prepare, with clear division of labor and complementary functions between strains, and can form a stable functional system, which has broad market application prospects.

[0090] Although the specific embodiments of the present invention have been described in detail, they should not be construed as limiting the scope of protection of this patent. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A composite microbial agent for producing caproic acid from brewing waste, characterized in that The composite microbial agent for producing caproic acid from brewing waste includes Bacillus coagulans, ethanol-resistant Pediococcus, Clostridium suijingfang, Clostridium rumenii and Clostridium cohnii. The ratio of the number of live bacteria of Bacillus coagulans, ethanol-resistant Pediococcus, Clostridium suijingfang, Clostridium rumenii and Clostridium cohnii in the composite microbial agent is (6-8):(7-9):(3-5):(5-7):(6-8).

2. The method for preparing a composite microbial agent for producing caproic acid using brewing waste according to claim 1, characterized in that The preparation method of the composite microbial agent for producing caproic acid using brewing waste is achieved by the following steps:

1. inoculating Bacillus coagulans into an activation medium for anaerobic activation to obtain an activated mother solution of Bacillus coagulans; 2. inoculating the ethanol-resistant Pediococcus into an activation medium for anaerobic activation treatment to obtain an ethanol-resistant Pediococcus activation mother solution; 3. Inoculating the Clostridium suijingfang into the activation medium for anaerobic activation treatment to obtain the Clostridium suijingfang activated mother solution; 4. inoculating the rumen Clostridium into the activation medium for anaerobic activation treatment to obtain the rumen Clostridium activation mother solution; 5. inoculating Clostridium cohnii into an activation medium for anaerobic activation treatment to obtain Clostridium cohnii activation mother solution; 6. The activated mother liquor of Bacillus coagulans, the activated mother liquor of ethanol-resistant Pediococcus, the activated mother liquor of Clostridium shuijingfang, the activated mother liquor of Clostridium rumenii and the activated mother liquor of Clostridium cohnii are inoculated into the composite culture medium for anaerobic composite culture to obtain the composite microbial agent for producing caproic acid using brewing waste.

3. The method for preparing a composite microbial agent for producing caproic acid from brewing waste according to claim 2, characterized in that The anaerobic activation treatment in step 1 to step 5 is cultured at 37° C. and 180 rpm for 72 hours.

4. The method for preparing a composite microbial agent for producing caproic acid using brewing waste according to claim 2, characterized in that The activation medium in step 1 is composed of 10.0 g casein peptone, 10.0 g beef extract powder, 5.0 g yeast extract powder, 1.0 g Tween 80, 5.0 g sodium acetate, 2.0 g triammonium citrate, 2.0 g K2HPO4, 0.2 g MgSO4·7H2O, and 0.2 g MnSO4·H2O. 0.05g of the solution was mixed with 1000.0mL of distilled water; then, it was sterilized at 121°C under high pressure for 20min and allowed to stand at room temperature. 20.0g of glucose was added in a clean bench and the pH was adjusted to 6.2-6.

5.

5. The method for preparing a composite microbial agent for producing caproic acid using brewing waste according to claim 2, characterized in that The activation medium in step 2 is composed of 10.0 g casein peptone, 10.0 g beef extract powder, 5.0 g yeast extract powder, 1.0 g Tween 80, 5.0 g sodium acetate, 2.0 g triammonium citrate, 2.0 g K2HPO4, 0.2 g MgSO4·7H2O, and 0.2 g MnSO4·H2O. 0.05g of the solution was mixed with 1000.0mL of distilled water; then, it was sterilized at 121°C under high pressure for 20min and allowed to stand at room temperature. 20.0g of glucose was added in a clean bench and the pH was adjusted to 6.2-6.

5.

6. The method for preparing a composite microbial agent for producing caproic acid using brewing waste according to claim 2, characterized in that In step 3, the activation medium is prepared by mixing 0.5 g of yeast extract powder, 3.0 g of tryptone, 0.33 g of NH4Cl, 0.33 g of KCl, 0.33 g of KH2PO4, 1.0 mL of trace salt solution SL10, 1.0 mg of resazurin, 0.33 g of CaCl2·2H2O, 0.33 g of MgCl2·6H2O, 4 g of glucose and 1000.0 mL of distilled water; then sterilized at 121° C. for 20 min and allowed to stand at room temperature, 0.5 g of cysteine ​​hydrochloride and 10.0 mL of vitamin solution are added in the clean bench, and the pH is adjusted to 7.0-7.3; The composition of the trace salt solution SL10 is as follows: 7.7 mol / L HCl 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0 mg, and distilled water 990.0 mL.

7. The method for preparing a composite microbial agent for producing caproic acid from brewing waste according to claim 2, characterized in that in step 4, the activation culture medium is mixed with 10.0 g of casein peptone, 10.0 g of beef extract powder, 5.0 g of yeast extract powder, 0.45 g of K2HPO4, 0.9 g of KH2PO4, 0.1 g of MgSO4·7H2O, 0.05 g of MnSO4·H2O, 1.0 mg of resazurin, 0.1 g of CaCl2·2H2O, 1.0 mL of trace salt solution SL10, and 1000.0 mL of distilled water; then sterilized at 121° C. for 20 min and allowed to stand at room temperature, 5 mL of lactic acid, 1 g of cysteine ​​hydrochloride, and 10.0 mL of vitamin solution are added in a clean bench, and the pH is adjusted to 6.2-6.5; The composition of the trace salt solution SL10 is as follows: 7.7 mol / L HCl 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0 mg, and distilled water 990.0 mL.

8. The method for preparing a composite microbial agent for producing caproic acid from brewing waste according to claim 2, characterized in that In step 5, the activation medium is prepared by mixing 5 g of yeast extract powder, 5.0 g of sodium acetate, 0.41 g of K2HPO4, 0.23 g of KH2PO4, 0.45 g of MgSO4·7H2O, 0.25 g of NH4Cl, 0.1 g of CaCl2·2H2O, 1.0 mL of trace salt solution SL10, and 1000.0 mL of distilled water; then sterilized at 121° C. for 20 min and allowed to stand at room temperature, 20 mL of ethanol, 0.4 g of cysteine, and 10.0 mL of vitamin solution are added in a clean bench, and the pH is adjusted to 6.2-6.5; The composition of the trace salt solution SL10 is as follows: 7.7 mol / L HCl 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0 mg, and distilled water 990.0 mL.

9. The method for preparing a composite microbial agent for producing caproic acid from brewing waste according to claim 2, characterized in that In step 6, the composite culture medium is prepared by mixing 5 g of casein peptone, 5 g of yeast extract powder, 5.0 g of sodium acetate, 0.41 g of K2HPO4, 0.23 g of KH2PO4, 0.45 g of MgSO4·7H2O, 0.25 g of NH4Cl, 0.1 g of CaCl2·2H2O, 1.0 mL of trace salt solution SL10 and 1000.0 mL of distilled water; then sterilized at 121° C. for 20 min and allowed to stand at room temperature, 10 mL of ethanol, 10 mL of lactic acid, 0.4 g of cysteine ​​and 10.0 mL of vitamin solution are added in a clean bench, and the pH is adjusted to 6.2-6.5; The composition of the trace salt solution SL10 is as follows: 7.7 mol / L HCl 10.0 mL, FeCl2·4H2O 1.5 g, ZnCl2 70.0 mg, MnCl2·4H2O 100.0 mg, H3BO3 6.0 mg, CoCl2·6H2O 190.0 mg, CuCl2·2H2O 2.0 mg, NiCl2·6H2O 24.0 mg, Na2MoO4·2H2O 36.0 mg, and distilled water 990.0 mL.

10. The use of the composite microbial agent for producing caproic acid from brewing waste according to claim 1, characterized in that The composite microbial agent is used as an inoculum and inoculated into brewing waste for anaerobic culture to produce caproic acid.

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