Caproic acid-producing strain and screening method thereof, and method for preparing high-ester flavoring wine by fermenting yellow water thereof

Abstract

The invention discloses a caproic acid-producing strain and a screening method thereof, and a method for preparing high-ester flavored wine by fermenting yellow water with the strain, belonging to the technical field of brewing microorganisms. The strain of the strain is named ( Caproicibacterium lactatifermentans )JSY12841, deposited in Guangdong Microbiological Culture Collection Center, with the deposit number of GDMCC.No 64551. The strain provided by the present invention is separated from the mud of the Luzhou-flavor liquor cellar of Jiangsu Jinshiyuan Liquor Co., Ltd. (longitude: 119.04 latitude: 33.6), and lactic acid, residual sugar and starch in yellow water are fermented to produce caproic acid. The fermented yellow water is then esterified and distilled to prepare high-ester flavoring wine, which has good application value in the field of liquor brewing.

Description

Technical Field

[0001] The present invention belongs to the technical field of brewing microorganisms, and relates to a caproic acid-producing strain and a screening method thereof, and a method for preparing high-ester flavored wine by fermenting yellow water thereof. Background Technology

[0002] The flavor components in liquor are mainly alcohol, aldehyde, acid, and ester substances, among which ester substances are important volatile aroma components produced during the fermentation process. There are many types and quantities of ester substances in liquor, and different types and quantities of ester substances give liquor different styles. Ethyl caproate, as the main aroma of Luzhou-flavor liquor, directly affects the quality of Luzhou-flavor liquor. GB / T10781.1-2021 stipulates that the product shall be implemented for ≤ one year from the date of production. The ethyl caproate of Luzhou-flavor liquor of superior grade reaches 1.5g / L or more [1] . GB / T 17204-2021 stipulates that the definition of Luzhou-flavor liquor emphasizes "no direct or indirect addition of edible alcohol and color, aroma and flavor substances not produced by self-fermentation" [2] , so it is an urgent problem for Luzhou-flavor liquor enterprises to improve the content of ethyl hexanoate in liquor through liquor brewing technology.

[0003] Yellow water is a by-product of liquor fermentation, which contains a variety of organic acids, alcohols, sugars, starches and other substances, and has a high utilization value [3] . In the traditional brewing process, yellow water is used as the bottom pot water for continuous distillation to further extract ethanol and some organic acids in the yellow water. However, most of the organic acids, sugars, and starches in the yellow water are not used efficiently.

[0004] The present invention provides a caproic acid bacteria, which can utilize lactic acid, residual sugar and starch in yellow water to produce caproic acid. After the conditions are optimized, the caproic acid yield can reach up to 14g / L. The fermented yellow water is converted into a high-ester flavoring wine through esterification and distillation. It not only improves the reuse efficiency of yellow water, a byproduct of winemaking, but also realizes green production. The application of high-ester flavoring wine is of great significance for improving the quality of liquor. SUMMARY OF THE INVENTION

[0005] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a strain of caproic acid-producing bacteria and a screening method thereof, and a method for using the strain to ferment yellow water to prepare high-ester flavored wine. The strain is separated from the mud of the Luzhou-flavor liquor cellar of Jiangsu Jinshiyuan Liquor Co., Ltd. (longitude: 119.04 latitude: 33.6), and the lactic acid, residual sugar and starch in the yellow water are fermented to produce caproic acid. The fermented yellow water is then esterified and distilled to prepare high-ester flavored wine, thereby improving the reuse efficiency of yellow water, a by-product of winemaking, realizing green production, and improving the quality of liquor. It has good application value in the field of liquor brewing.

[0006] To achieve the purpose of the invention, the present invention adopts the following technical scheme: a strain of caproic acid-producing bacteria, the strain of which is named (Caproicibacterium lactatifermentans) JSY12841, and is deposited in the Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC No: 64551.

[0007] Furthermore, the strain was isolated from the cellar mud of the Luzhou-flavor liquor of Jiangsu Jinshiyuan Liquor Co., Ltd. (longitude: 119.04 latitude: 33.6).

[0008] Wherein, the method for screening caproic acid-producing strains comprises the following steps:

[0009] (1) Culture medium (g / L): 3.0 sodium acetate, 20.0 glucose, 2.0 ammonium sulfate, 1.0 potassium dihydrogen phosphate, 0.5 potassium dihydrogen phosphate, 10.0 peptone, 10.0 yeast powder, 0.1 magnesium sulfate heptahydrate, 0.15 ferrous sulfate heptahydrate, 0.01 calcium chloride, 0.01 manganese sulfate, 0.002 cobalt chloride heptahydrate, 0.002 zinc sulfate, 1 mL 1% (w / v) resazurin solution, 1 mL vitamin B complex solution stock solution, and the rest water. Adjust pH to 7.0±0.2 before sterilization and sterilize at 115℃ for 15 min.

[0010] (2) Deoxygenation: Prepare and weigh the dry sample of the culture medium and place it in an anaerobic box; prepare the wet sample of the culture medium and place it in an anaerobic box for deoxygenation in advance; boil ultrapure water for 5-10 minutes on an induction cooker, pour it into a 500mL blue-capped bottle, stir and blow with nitrogen for 20 minutes, wait for it to cool, seal the bottle mouth, and transfer it to an anaerobic box; prepare the culture medium in the anaerobic box and place it in the blue-capped bottle, ensuring that the amount of culture medium reaches 90% of the capacity of the blue-capped bottle, seal the blue-capped bottle, and move it out of the anaerobic box; place the prepared culture medium in the sterilizing pot, wait for it to cool to 95°C after sterilization, and quickly transfer it to an anaerobic box; loosen the lid for further deoxygenation; wait for it to cool, and add the mother solution of B vitamins solution;

[0011] (3) Anaerobic chamber status detection: Take out the target particles in the anaerobic chamber, dry them at 80℃, and put them back into the anaerobic chamber; add resazurin to sterile water, sterilize it and put it into the anaerobic chamber as an anaerobic indicator; check the remaining amount of the mixed gas every day, replace the mixed gas in time, and ensure that the mixed gas pressure is above 2mpa;

[0012] (4) Strain enrichment: Take 1g of cellar mud, put it into a 15mm×150mm test tube, transfer it into an anaerobic box, add 10mL of culture medium; plug it with a rubber stopper, and culture it anaerobically at 37℃ for 7 days. After culturing for 2 days, test the pH and add calcium carbonate to adjust the pH; take samples after 5, 6, and 7 days of culture, and test the amount of caproic acid and octanoic acid by liquid-liquid extraction chromatography, select the experimental group with high octanoic acid / caproic acid, and enter the next round of culture, repeat 5 times;

[0013] (5) Microbial screening: The enrichment solution in step (4) is gradient diluted with the deoxygenated culture medium, and 10 -5 -10 -7 is spread on the solid plate of the culture medium with glass beads and anaerobically statically cultured at 37°C for 7 days, and colonies are picked; after the colonies are purified by streaking more than 3 times, pure cultured bacteria are obtained and named JSY12841.

[0014] In step (1), the stock solution of B vitamin solution (g / L): nicotinic acid 0.02, cobalamin 0.02, thiamine hydrochloride 0.01, p-aminobenzoic acid 0.01, pyridoxine hydrochloride 0.05, calcium pantothenate 0.005, and the rest is water; after preparation, it is sub-packed according to the actual usage amount and stored at -20°C.

[0015] Among them, the method for fermenting yellow water with caproic acid-producing strains to prepare high-ester flavoring wine includes the following steps: The strains utilize lactic acid, residual sugar and starch in the yellow water to ferment and produce caproic acid, and the fermented yellow water is further subjected to esterification distillation to prepare high-ester flavoring wine.

[0016] Furthermore, the preparation method of high-ester flavoring wine includes the following specific steps:

[0017] (1) Preparation of functional bacterial liquid: The glycerol tube or anaerobic slant of JSY12841 is taken into the anaerobic workstation from the refrigerator. After warming up, it is streaked and spread on the solid plate of the culture medium, anaerobically cultured at 37°C for 7 days to obtain a colony plate; single colonies are picked and inoculated into a test tube containing the culture medium, anaerobically cultured at 37°C for 7 days to obtain a seed liquid; the seed liquid is amplified cultured with an inoculation amount of 5-10%, and a functional bacterial liquid is obtained; the culture medium (g·L): glucose 20.0, ammonium sulfate 2.0, dipotassium hydrogen phosphate 1.0, potassium dihydrogen phosphate 0.5, peptone 10.0, yeast powder 10.0, magnesium sulfate heptahydrate 0.1, ferrous sulfate heptahydrate 0.15, calcium chloride 0.01, manganese sulfate 0.01, cobalt chloride heptahydrate 0.002, zinc sulfate 0.002, 1 mL of 1% (w / v) resazurin solution, 1.5-2.0% agar, and the rest is water. The pH is adjusted to 7.0±0.2 before sterilization and sterilized at 115°C for 15 min;

[0018] (2) Preparation of yellow water fermentation liquid: Dilute the yellow water according to a ratio of 80%, adjust the pH to 5.5-6.5 with sodium carbonate or sodium hydroxide, sterilize at 121°C for 30 min, seal and cool with nitrogen blowing; the JSY12841 functional bacterial liquid in step (1) is inoculated into the yellow water culture medium with an inoculation amount of 10%, and anaerobically cultured at 30°C for 7 days;

[0019] (3) Preparation of flavored wine: adjust the pH of the fermented liquid in step (2) to 3.5, add 10% corn alcohol and 1% lipase, intermittently introduce air and stir at 25°C, react for 4 hours, and sieve with an 80-mesh gauze bag to obtain the esterified liquid; put 400 parts of the esterified liquid into the distillation kettle, set the distillation negative pressure to -90kPa, and gradually reduce the vacuum degree with reduced pressure distillation to -20kPa, collect 30% of the distillate from the first stage, and obtain the strong-flavored flavored wine.

[0020] The present invention has the following advantages:

[0021] 1. The strain comes from the mud in the liquor cellar. It is a wild strain with stable genome and phenotype. It also belongs to the liquor brewing strain. It is used as a functional bacteria to produce flavored liquor, and the product is safe and reliable.

[0022] 2. Use single bacteria to anaerobic ferment yellow water to make high-ester flavored wine. The fermentation conditions are clear and easy to control. The results after fermentation are stable and it is not easy to have secondary deterioration.

[0023] 3. Use caproic acid-producing bacteria to ferment yellow water to prepare high-ester flavoring wine. The preparation method is simple to operate and suitable for industrial scale-up.

[0024] 4. After the conditions are optimized, the maximum caproic acid yield is 13.8g / L, which is used to make high-ester flavoring wine with higher ester content, higher production efficiency and better product quality.

[0025] 5 Using yellow water, a byproduct of winemaking, as the main raw material has a higher utilization rate, conforms to the energy-saving and green production concept, and is conducive to the protection of the ecological environment. Brief Description of the Figures

[0026] Figure 1 :Colonial morphology of JSY12841 strain;

[0027] Figure 2 :Phylogenetic tree of JSY12841 strain;

[0028] Figure 3 : Radar chart of sensory evaluation of high-ester flavored wine;

[0029] Figure 4 :Chromatogram of flavor substances in high-ester flavoring wine. Specific implementation method

[0030] The technical scheme of the present invention is further illustrated by specific examples. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention. The strain was isolated from the cellar mud of the Luzhou-flavor liquor of Jiangsu Jinshiyuan Liquor Co., Ltd. (longitude: 119.04 latitude: 33.6).

[0031] Example 1: Screening, Identification and Preservation of Functional Bacteria

[0032] 1.1 Strain Screening

[0033] Medium (g / L): Sodium acetate 3.0, Glucose 20.0, Ammonium sulfate 2.0, Dipotassium hydrogen phosphate 1.0, Potassium dihydrogen phosphate 0.5, Peptone 10.0, Yeast extract 10.0, Magnesium sulfate heptahydrate 0.1, Ferrous sulfate heptahydrate 0.15, Calcium chloride 0.01, Manganese sulfate 0.01, Cobalt chloride heptahydrate 0.002, Zinc sulfate 0.002, 1% (w / v) Resazurin solution 1 mL, Mother liquor of B vitamin solution 1 mL, the rest is water, adjust the pH to 7.0 ± 0.2 before sterilization, sterilize at 115 °C for 15 min; Mother liquor of B vitamin solution (g / L): Nicotinic acid 0.02, Cobalamin 0.02, Thiamine hydrochloride 0.01, p-Aminobenzoic acid 0.01, Pyridoxol hydrochloride 0.05, Calcium pantothenate 0.005, the rest is water, dispense according to the actual usage amount after preparation, store at -20 °C;

[0034] Deoxygenation: Weigh the dry sample of the medium and put it into the anaerobic chamber; Put the wet sample of the medium into the anaerobic chamber in advance for deoxygenation; Boil the ultrapure water with an induction cooker for 5 - 10 minutes, pour it into a 500 mL blue-capped bottle, stir and blow with nitrogen for 20 minutes, wait for it to cool, seal the bottle mouth, and transfer it to the anaerobic chamber; Prepare the medium in the anaerobic chamber, put it into a blue-capped bottle, ensure that the amount of the medium reaches 90% of the capacity of the blue-capped bottle, seal the blue-capped bottle, and take it out of the anaerobic chamber; Put the prepared medium into the autoclave, after sterilization, wait for it to cool to 95 °C, and quickly transfer it to the anaerobic chamber; Loosen the lid to further deoxygenate; Wait for it to cool and add the mother liquor of B vitamin solution;

[0035] Anaerobic chamber status detection: Take out the target particles in the anaerobic chamber, dry them at 80 °C, and reload them into the anaerobic chamber; Add resazurin to sterile water, sterilize and put it into the anaerobic chamber for use as an anaerobic indicator; Check the remaining amount of the mixed gas every day and replace the mixed gas in time to ensure that the pressure of the mixed gas is above 2 mpa;

[0036] Strain enrichment: Take 1 g of pit mud, put it into a 15 mm × 150 mm test tube, transfer it to the anaerobic chamber, and add 10 mL of the medium. Plug the rubber stopper and culture anaerobically and statically at 37 °C for 7 days. After culturing for 2 days, detect the pH and add calcium carbonate to adjust the pH; Take samples on the 5th, 6th, and 7th days of culture, and detect the amounts of caproic acid and caprylic acid by liquid-liquid extraction chromatography. Screen the experimental groups with a high caprylic acid / caproic acid ratio and enter the next round of culture, repeat 5 times;

[0037] Microorganism screening: Gradient dilute the above enrichment solution with the deoxygenated medium, 10 -5 -10 -7Glass beads were coated on the solid medium plate and anaerobically statically cultured at 37°C for 7 days, and colonies were picked; after the colonies were purified by streaking more than 3 times, pure cultured bacteria were obtained and named JSY12841.

[0038] 1.2 Strain identification: After PCR amplification and sequencing using the bacterial 16S rDNA universal primers (27F, 1492R), a result with a length of 1411 bp (SEQ ID NO.1) was obtained. The result was compared in the NCBI database, and the result showed that the similarity of this strain to Caproicibacterium lactatifermentans LBM19010 was 99.58%, and it was considered to be the same microorganism.

[0039] 1.3 Strain preservation

[0040] Name of the strain: Caproicibacterium lactatifermentans

[0041] Latin name: Caproicibacterium lactatifermentans

[0042] Strain number: JSY12841

[0043] Taxonomic name: Caproicibacterium lactatifermentans

[0044] Preservation institution: Guangdong Microbial Culture Collection Center

[0045] Abbreviation of the preservation institution: GDMCC

[0046] Address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangdong City

[0047] Preservation date: May 8, 2024, Registration number of the preservation center: GDMCC NO: 64551

[0048] Example 2: The following specific steps are used to prepare high-ester flavor liquor

[0049] (1) Preparation of functional bacterial liquid: The JSY12841 glycerol tube or anaerobic slant is taken from the refrigerator into the anaerobic workstation. After warming up, it is streaked and spread on the solid plate of the medium, and anaerobically cultured at 37°C for 7 days to obtain a colony plate; single colonies are picked and inoculated into a test tube containing the medium, and anaerobically cultured at 37°C for 7 days to obtain a seed liquid; the seed liquid is expanded and cultured with an inoculation amount of 5-10%, and a functional bacterial liquid is obtained; the medium (g·L-1): glucose 20.0, ammonium sulfate 2.0, dipotassium hydrogen phosphate 1.0, potassium dihydrogen phosphate 0.5, peptone 10.0, yeast powder 10.0, magnesium sulfate heptahydrate 0.1, ferrous sulfate heptahydrate 0.15, calcium chloride 0.01, manganese sulfate 0.01, cobalt chloride heptahydrate 0.002, zinc sulfate 0.002, 1% (w / v) resazurin solution 1 mL, 1.5-2.0% agar, and the rest is water. Adjust the pH to 7.0±0.2 before sterilization and sterilize at 115°C for 15 min;

[0050] (2) Preparation of yellow water fermentation liquid: Dilute the yellow water according to a ratio of 80%, adjust the pH to 5.5-6.5 with sodium carbonate or sodium hydroxide, sterilize at 121°C for 30 min, seal and cool with nitrogen blowing; inoculate the JSY12841 functional bacterial liquid in step (1) into the yellow water medium with an inoculation amount of 10%, and anaerobically culture at 30°C for 7 days;

[0051] (3) Preparation of flavoring wine: Adjust the pH of the fermentation liquid in step (2) to 3.5, add 10% corn alcohol and 1% lipase, intermittently introduce air and stir at 25°C, after reacting for 4 h, sieve with an 80-mesh gauze bag to obtain the esterification liquid; load 400 parts of the esterification liquid into the distillation kettle, set the distillation negative pressure to -90 kPa, and the vacuum degree gradually decreases with vacuum distillation. When it reaches -20 kPa, collect the first 30% of the distillate to obtain the strong-flavor flavoring wine.

[0052] Example 3: Strain JSY12841 produces organic acids using different carbon sources

[0053] 1. Culture conditions

[0054] (1) Medium (g / L): glucose 20 (other carbon sources: sodium acetate 27.3; sodium butyrate 25; sodium lactate 24.89; starch 20; ethanol 25.35Ml; sodium hexanoate 24; cellobiose 16.5; sucrose 20; D-fructose 20; D-(+)xylose 20), ammonium sulfate 2.0, dipotassium hydrogen phosphate 1.0, potassium dihydrogen phosphate 0.5, peptone 10.0, yeast powder 10.0, magnesium sulfate heptahydrate 0.1, ferrous sulfate heptahydrate 0.15, calcium chloride 0.01, manganese sulfate 0.01, cobalt chloride heptahydrate 0.002, zinc sulfate 0.002, 1% (w / v) resazurin solution 1 mL, and the rest is water; adjust the pH to 7.0±0.2 before sterilization and sterilize at 115°C for 15 min.

[0055] (2) Culture conditions: 37°C, anaerobic

[0056] (3) Culture period: 7 - 10 days

[0057] (4) Inoculation amount: 10%

[0058] 2. Detection method

[0059] (1) Liquid - liquid extraction: Pipette 2 mL of the sample solution into a mL centrifuge tube, centrifuge at 10000 rpm for 10 min, pipette 800 μL of the sample solution, add 200 μL of the internal standard, mix well, then add 1 mL of dichloromethane, centrifuge at 6000 rpm for 5 min, and pipette 500 μL of the lower - layer organic phase for determination. Internal standard: 1.25 g / L of tert - pentanoic acid (1.25 g of solid), 15 mL of concentrated hydrochloric acid, make up the volume to 100 mL with water.

[0060] (2) GC conditions:

[0061] Chromatographic column: Agilent CP - wax 57CB

[0062] Injector: Temperature 220°C, pressure 27 psi

[0063] Oven: 60°C, hold for 0.5 min; increase the temperature by 20°C per minute to 190°C, hold for 4.5 min, total running time 12 min.

[0064] Detector: Temperature: 220°C, air flow rate 450 ml / min, hydrogen 40 ml / min, total N2 flow rate 65 ml / min (septum purge 3 ml / min; split flow rate: 60 ml / min; column flow rate: 2 ml / min), tail - blow nitrogen: 45 ml / min.

[0065] Split ratio: 30:1

[0066] 3. Results:

[0067] Set up 3 parallel groups, and at the same time take the culture solution at 0 h as the blank control. Finally, remove the blank control from the data, take the average value, and organize the results in the following table

[0068] Table 1: Comparison table of the amounts of organic acids produced by strain JSY12841 using different carbon sources

[0069] Carbon source Acetic acid g / L Butyric acid g / L Caproic acid g / L Caprylic acid g / L Ethanol 0.11 0.03 0.36 0.67 Acetic acid 0.87 0.25 0.48 0.00 Butyric acid 0.62 -1.03 1.99 0.00 Caproic acid 0.04 0.01 -0.59 0.14 Lactic acid 0.00 0.05 2.16 0.28 Glucose 0.51 0.09 1.96 0.18 Fructose 0.52 0.00 0.02 0.56 Xylose 0.02 0.05 0.40 0.10 Sucrose 0.03 0.09 0.74 0.17 Cellobiose 0.17 0.07 0.63 0.16 Starch 0.34 0.21 2.90 0.22

[0070] The results showed that JSY12841 could produce caproic acid from butyric acid and octanoic acid from caproic acid. From the perspective of caproic acid production, the strain produced high caproic acid in the culture medium with lactic acid, glucose, starch and butyric acid as the main carbon sources; however, the maximum amount of caproic acid produced by JSY12841 in the culture medium with a single carbon source did not exceed 2.16, which was far from the process requirements; in the experiment, we found that the caproic acid production of JSY12841 strain in mixed carbon sources was higher than that in a single carbon source. The above carbon sources all exist in yellow water. The following will use yellow water as the culture medium to conduct the caproic acid production experiment of JSY12841 strain.

[0071] Example 4: Fermentation of strain JSY12841 in yellow water

[0072] The yellow water used is the yellow water from Jinshiyuan Luzhou Fragrance Workshop. The physical and chemical properties of the yellow water are as follows:

[0073] Table 2: Physical and chemical properties of yellow water from a certain Luzhou-flavor workshop of Jiangsu Jinshiyuan Wine Co., Ltd.:

[0074] Acetic acid g / L 11.0-12.0 pH 3.2-3.6 Lactic acid g / L 46.0-55.0 Acidity 5.2-6.9 Caproic acid g / L 0.5-0.9 Alcohol content % VoL 4.2-5.4 Butyric acid g / L 0-1 Residual sugar % 0.4-0.6 Ethyl acetate g / L 0.2-0.5 Residual starch % 2.0-2.7 Ethyl butyrate g / L 0.0-0.1 COD mg / L 185000-207000 Ethyl caproate g / L 0.0-0.1 Total nitrogen 2460-3935 Ethyl lactate g / L 0.6-1.3

[0075] Yellow water is the main by-product of the brewing process. It is rich in organic acids, higher alcohols and lower fatty acid ethyl esters, as well as various nutrients such as protein, starch, and reducing sugar that seep out of the mash. It is an ideal medium for JSY12841 fermentation. However, the conditions for JSY12841 to produce caproic acid in yellow water fermentation need further exploration.

[0076] Exploration of different concentrations of yellow water conditions: The yellow water was adjusted to pH 7.0 with sodium hydroxide solution, and diluted by 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. Sterilize at 121℃ for 30 minutes, and after cooling, obtain yellow water culture medium of different concentrations for use; inoculate JSY12841 functional bacterial liquid with an inoculation rate of 10% in the yellow water culture medium of different concentrations, and set up three parallels for each group; anaerobic culture was carried out at 35℃ for 7 days, and the organic acids in the culture medium were detected.

[0077] Exploration of yellow water conditions with different pH values: The yellow water was diluted by 50% and the pH was adjusted to natural (3.28), 4, 5, 6, 7, 8, 9, 10, 11, and 12 with sodium hydroxide solution; due to the complex buffer substances in the yellow water, the pH value fluctuated before and after sterilization. The solution was sterilized at 121°C for 30 minutes and the pH was measured again after cooling. The results were 3.23, 4.6, 5.01, 5.75, 6.39, 6.82, 7.58, 8.52, 9.06, and 9.25, respectively, to obtain yellow water culture media with different concentrations; the JSY12841 functional bacterial solution was inoculated at an inoculation rate of 10% into the yellow water culture media with different pH values, and three parallel cultures were set up for each group; the culture was anaerobically cultured at 35°C for 7 days to detect organic acids in the culture medium.

[0078] Fermentation of Huangshui at different temperatures: Dilute the above Huangshui by 50%, adjust the pH to 7 with sodium hydroxide solution, sterilize at 121°C for 30 minutes, and cool for later use. Inoculate the JSY12841 functional bacterial liquid into the above Huangshui culture medium at an inoculation amount of 10%, and set three parallels for each group. Conduct anaerobic culture, set the temperatures at 20°C, 25°C, 30°C, 35°C, and 40°C, and culture for 7 days. Detect the organic acids in the culture solution

[0079] Detection method:

[0080] (1) Liquid-liquid extraction: Pipette 2 mL of the sample solution into a mL centrifuge tube, centrifuge at 10000 rpm for 10 min, pipette 800 μL of the sample solution, add 200 μL of the internal standard, mix well, add 1 mL of dichloromethane, centrifuge at 6000 rpm for 5 min, and pipette 500 μL of the lower organic phase for determination. Internal standard: tert-Amyl acid 1.25 g / L (1.25 g of solid), 15 mL of concentrated hydrochloric acid, and make up the volume to 100 mL with water.

[0081] (2) GC conditions:

[0082] Chromatographic column: Agilent CP-wax 57 CB

[0083] Injection port: Temperature 220°C, pressure 27 psi

[0084] Column oven: 60°C, hold for 0.5 min; increase the temperature by 20°C per minute to 190°C, hold for 4.5 min, and the total running time is 12 min.

[0085] Detector: Temperature: 220°C, air flow rate 450 ml / min, hydrogen 40 ml / min, total N2 flow rate 65 ml / min (septum purge 3 ml / min; split flow rate: 60 ml / min; column flow rate: 2 ml / min), tail gas purge nitrogen: 45 ml / min.

[0086] Split ratio: 30:1

[0087] Table 3: Contents of organic acids in the Huangshui culture medium fermented by JSY12841 at different concentrations

[0088] Yellow water concentration Acetic acid g / L Butyric acid g / L Caproic acid g / L Caprylic acid g / L 20％ 0.76 0.45 3.40 0.19 30％ 0.93 0.51 3.72 0.19 40％ 1.20 0.74 5.82 0.24 50％ 1.50 0.88 7.21 0.25 60％ 1.84 1.00 8.38 0.27 70％ 2.23 1.14 9.45 0.27 80％ 2.54 1.21 9.58 0.25 90％ 3.09 1.26 8.46 0.21 100％ 3.44 1.25 7.70 0.19

[0089] Table 4: Contents of organic acids in the Huangshui culture medium fermented by JSY12841 at different pH values

[0090] Yellow water pH Acetic acid g / L Butyric acid g / L Caproic acid g / L Caprylic acid g / L 3.28 2.08 0.28 0.53 0.05 4 2.09 0.27 0.53 0 5 2.08 0.29 0.65 0 6 1.27 0.91 9.76 0.35 7 1.43 0.95 9.25 0.33 8 1.68 1.02 7.83 0.27 9 1.73 0.93 6.85 0.24 10 2.24 0.64 3.41 0.16 11 2.18 0.29 0.68 0 12 2.16 0.29 0.56 0

[0091] Table 5: Contents of organic acids in the Huangshui culture medium fermented by JSY12841 at different temperatures

[0092]

[0093]

[0094] It can be concluded from the results in Table 3 that the optimal yellow water concentration for JSY12841 to ferment yellow water to produce caproic acid is 80%; it can be seen from the results in Table 4 that for JSY12841 to ferment yellow water to produce caproic acid, the most suitable pH is 6 - 7, and the actual value is about pH 5.75 - 6.39; it can be concluded from the results in Table 5 that for JSY12841 to ferment yellow water to produce caproic acid, the most suitable temperature is 30°C.

[0095] Example 5: It can be obtained from Example 4 that the optimal conditions for strain JSY12841 to ferment yellow water are as follows: yellow water concentration 80%, culture temperature 30°C, and medium pH 5.75 - 6.39. During the experiment, we found that during the fermentation of yellow water by JSY12841, the pH changed continuously, showing an initial decrease, then an increase in the middle, and the pH range changed between 5.0 - 9, exceeding the optimal caproic acid production conditions. Sodium hydroxide solution and sulfuric acid were used to control the pH of the solution during the fermentation process to keep strain JSY12841 under the optimal caproic acid production conditions.

[0096] The experimental operation is as follows: Cultivate the yellow water medium: yellow water concentration 80%, pH 7, sterilize at 121°C for 30 minutes, and cool for later use; inoculate the JSY12841 functional bacterial liquid into the above yellow water medium at an inoculation amount of 10%, adjust the pH of the medium at 24h, 48h, 72h, 96h, 120h, 144h, and 168h respectively, and maintain the pH of the fermentation liquid process between 4.5 - 5.5, 5.5 - 6.5, and 6.5 - 7.5. After culturing for 7 days, detect the organic acids.

[0097] Detection method:

[0098] (1) Liquid - liquid extraction: Pipette 2Ml of the sample solution into an mL centrifuge tube, centrifuge at 10000rpm for 10min, pipette 800μL of the sample solution, add 200μL of the internal standard, mix well, then add 1mL of dichloromethane, centrifuge at 6000rpm for 5min, and pipette 500μL of the lower - layer organic phase for determination. Internal standard: Tert - pentanoic acid 1.25g / L (1.25g solid), 15mL of concentrated hydrochloric acid, and make up the volume to 100mL with water.

[0099] (2) GC conditions:

[0100] Chromatographic column: Agilent CP - wax 57CB

[0101] Injection port: Temperature 220°C, pressure 27psi

[0102] Column oven: 60 °C, hold for 0.5 min; increase the temperature by 20 °C per minute to 190 °C, hold for 4.5 min, total running time 12 min.

[0103] Detector: Temperature: 220 °C, air flow rate 450 ml / min, hydrogen 40 ml / min, total N2 flow rate 65 ml / min (septum purge 3 ml / min; split flow rate: 60 ml / min; column flow rate: 2 ml / min), tail gas purge nitrogen: 45 ml / min.

[0104] Split ratio: 30:1

[0105] Table 6: Contents of organic acids in the yellow water medium in different pH ranges during the fermentation process of JSY12841

[0106] PH section Acetic acid g / L Butyric acid g / L Caproic acid g / L Caprylic acid g / L 4.5-5.5 1.99 0.48 3.58 0.15 5.5-6.5 1.07 0.82 12.12 0.41 6.5-7.5 2.48 3.70 11.17 0.32

[0107] From the results in Table 6, it can be concluded that controlling the pH during the fermentation process of JSY12841 can increase the concentration of caproic acid in the fermentation broth.

[0108] Example 6: Use an anaerobic fermenter in production, monitor the pH and temperature in real time, increase the content of caproic acid in the fermentation, and produce high-ester flavoring wine

[0109] In production use, through the anaerobic fermenter, the anaerobic fermenter has nitrogen pressure maintenance, which can meet the anaerobic requirements of the strain JSY12841. The anaerobic fermenter has temperature and pH sensors, which can monitor and adjust the temperature and pH of the fermentation broth during specific production operations; the yellow water medium with a concentration of 80% and a pH of 7 is configured in the fermenter, and the whole tank is sterilized at 121 °C for 30 minutes. After sterilization, it is cooled to 30 °C by nitrogen blowing and reserved; the JSY12841 functional bacterial liquid is inoculated into the above-mentioned yellow water medium in the anaerobic fermenter with an inoculation amount of 10% by the fire ring inoculation method; the fermenter is monitored, and the pH of the solution during the fermentation process is controlled at 6.2 ± 0.2 using sodium hydroxide solution and sulfuric acid. After 7 days, the fermentation broth is taken to measure the concentration of organic acids.

[0110] Detection method:

[0111] (1) Liquid-liquid extraction: Pipette 2Ml of the sample solution into an mL centrifuge tube, centrifuge at 10000 rpm for 10 min, pipette 800 μL of the sample solution, add 200 μL of the internal standard, mix well, then add 1 mL of dichloromethane, centrifuge at 6000 rpm for 5 min, and pipette 500 μL of the lower organic phase for determination. Internal standard: tert-amyl acid 1.25 g / L (1.25 g of solid), 15 mL of concentrated hydrochloric acid, and make up to 100 mL with water.

[0112] (2) GC conditions:

[0113] Chromatographic column: Agilent CP-wax 57CB

[0114] Inlet: Temperature 220℃ Pressure 27psi

[0115] Column oven: 60℃, hold for 0.5min; heat up to 190℃ at 20℃ per minute, hold for 4.5min, total running time 12min.

[0116] Detector: Temperature: 220℃, air flow 450ml / min, hydrogen 40ml / min, N2 total flow 65ml / min (septum purge 3ml / min; split flow: 60ml / min; column flow rate: 2ml / min), tail nitrogen: 45ml / min.

[0117] Split ratio: 30:1

[0118] Table 7: Organic acid content of JSY12841 in yellow water medium of anaerobic fermentation tank

[0119] Sample Acetic acid g / L Butyric acid g / L Caproic acid g / L Caprylic acid g / L Fermenter 1.84 1.56 13.84 0.26

[0120] The fermentation liquid in the fermentation tank is adjusted to pH 3.5 with lactic acid, 10% corn alcohol and 1% lipase are added, air is intermittently introduced at 25°C for stirring, and after reacting for 4 hours, it is sieved with an 80-mesh yarn bag to obtain the esterified liquid; 400 portions of the esterified liquid are loaded into the distillation kettle, and the negative pressure of distillation is set to -90kPa. The vacuum degree is gradually reduced with the reduced pressure distillation to -20kPa, and the first 30% of the distillate is collected to obtain the Luzhou-flavored flavoring wine.

[0121] Table 8: Flavored Wine Data

[0122]

[0123]

[0124] Use caproic acid-producing bacteria to ferment yellow water to prepare high-ester flavoring wine, and the caproic acid yield can reach up to 13.8g / L. The ester content of high-ester flavoring wine produced in this way is higher and the product quality is good. The preparation method of high-caproic acid fermentation broth in anaerobic fermentation tank is simple to operate and suitable for industrial scale-up

[0125] [1] State Administration for Market Regulation, National Administration of Standardization. GBT10781.1-2021: Quality requirements for liquor Part 1: Luzhou-flavor liquor: [S]. Beijing: 2021:

[0126] [2] State Administration for Market Regulation, National Standardization Administration. GBT17204-2021 Terminology and Classification of Alcoholic Beverages: [S]. 2021: ​​

[0127] [3] Wang Zihao, Ping Huang, Jia Zheng, et al. Research Status and Comprehensive Utilization Progress of Huangshui, a By-Product of Baijiu Brewing [J]. China Brewing, 2023.

Claims

1. A strain producing caproic acid, characterized in that: The strain of the bacteria was named Caproicibacterium lactatifermentans JSY12841, and was deposited in the Guangdong Microbiological Culture Collection Center with the collection number GDMCC No: 64551. The strain was isolated from the cellar mud of the Luzhou-flavor liquor of Jiangsu Jinshiyuan Liquor Co., Ltd.

2. The use of the caproic acid-producing strain according to claim 1, characterized in that: The strain is used for fermenting yellow water to prepare high-ester flavoring wine; the preparation method of the high-ester flavoring wine comprises the following steps: the strain utilizes lactic acid, residual sugar and starch in the yellow water to ferment to produce caproic acid, and the fermented yellow water is then esterified and distilled to prepare the high-ester flavoring wine.

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