Screening culture and application of bacillus paranthracis capable of producing caproic acid
By screening and breeding the Bacillus paranthracis strain LLMJ-JS-1, the problem of insufficient acid production and stability of the existing strains was solved, efficient production of caproic acid was achieved, and the quality of liquor and cellar mud was improved.
Patent Information
- Application Number
- CN202510023297.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-07
- Publication Date
- 2025-04-11
AI Technical Summary
The existing caproic acid-producing strains have shortcomings in acid production and acid production stability, which affect the flavor of liquor and the activity of cellar mud.
A strain of Bacillus paranthracis, named LLMJ-JS-1, was screened and selected to improve its acid production ability and stability through carefully designed screening and culture methods.
This strain showed high acid yield under anaerobic conditions, with an acid yield of 10.9 g/L, which significantly improved the microbial activity of the cellar mud and the quality of the liquor.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of brewing microorganisms, and particularly relates to the screening, cultivation and application of caproic acid-producing Bacillus paranthracis. Background Art
[0002] Caproic acid-producing bacteria play a core role in the brewing process of Luzhou-flavor liquor and are key functional microorganisms that affect the flavor of liquor. These microorganisms synthesize caproic acid through metabolic pathways and then react with ethanol to produce ethyl caproate, which plays a decisive role in improving the sensory quality of Luzhou-flavor liquor. There is a positive correlation between the synthesis efficiency of ethyl caproate and the cumulative concentration of caproic acid during the fermentation process, that is, the increase in caproic acid concentration will promote the synthesis of ethyl caproate, thereby enhancing the aroma of liquor.
[0003] In the process of cellar mud maintenance, caproic acid-producing bacteria also play a vital role. As a microbial carrier for liquor fermentation, the quality of cellar mud directly affects the quality of liquor. Cellar mud used for a long time may degrade, such as hardening, which will inhibit the activity of caproic acid-producing bacteria, resulting in a decrease in acid production, which in turn affects the flavor of liquor. In order to maintain the activity and acid production capacity of cellar mud, bioremediation of cellar mud can be carried out by applying high-concentration caproic acid bacteria fermentation liquid to stabilize and improve the overall quality of the original liquor.
[0004] However, the current hexanoic acid-producing strains have limited acid-producing capacity. Therefore, breeding high-caproic acid-producing strains and optimizing the acid-producing performance of strains based on their acid-producing mechanisms are of great significance for improving the microbial activity of cellar mud and the quality of base liquor. This not only involves the construction and regulation of functional microbial communities in cellar mud, but also the synthesis and regulation of flavor substances in the entire liquor brewing process. Summary of the invention
[0005] In view of the deficiencies of the above-mentioned prior art, the present invention provides a screening, cultivation and application of caproic acid-producing Bacillus paranthracis. The strain has been carefully selected and bred to effectively solve the deficiencies of existing strains in terms of acid production and acid production stability. Caproic acid-producing Bacillus paranthracis has shown significant potential in cellar mud production, cellar mud curing liquid preparation and quality improvement of Luzhou-flavor liquor with its excellent metabolic activity and environmental adaptability. This innovative achievement has important theoretical and practical significance for the brewing process of Luzhou-flavor liquor.
[0006] The first technical solution provided by the present invention is a caproic acid-producing Bacillus paranthracis, named LLMJ-JS-1, which was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) on October 31, 2024, with a deposit number of CGMCC No: 32446.
[0007]
[0008] Among them, the caproic acid-producing Bacillus paranthracis LLMJ-JS-1 has a strong acid production capacity under anaerobic conditions, with an acid production of 10.9 g / L.
[0009] The colony morphology of the caproic acid-producing Bacillus paranthracis LLMJ-JS-1 is as follows: the colonies are light yellow, with irregular edges, a dry surface, and are opaque.
[0010] The fermentation performance of the caproic acid-producing Bacillus paranthracis LLMJ-JS-1 is as follows: the temperature is 34-39° C., with 37° C. being the best; it can tolerate 1%-2.5% ethanol, with the best performance at 2%.
[0011] The screening and culturing method of the caproic acid-producing Bacillus paranthracis LLMJ-JS-1 comprises the following steps: using a liquid culture medium to enrich the pit mud for 4-7 days, performing a copper sulfate colorimetric test on the enriched solution for preliminary screening, selecting a darker blue-green enriched solution for dilution and applying it to a solid culture medium, selecting a single strain and inoculating it in the liquid culture medium for 4-7 days, and performing gas chromatography quantitative analysis on the culture solution after extraction.
[0012] Among them, the specific steps of the screening and cultivation method are as follows:
[0013] (1) Culture medium: Liquid culture medium: 3 g yeast extract, 1.5 g anhydrous sodium acetate, 0.06 g MgSO4, 0.12 g K2HPO4, 0.15 g (NH4)2SO4, 300 mL distilled water, sterilized at 121°C for 20 min. Add 2% ethanol and 3 g dry heat sterilized CaCO3 before use. Solid culture medium: Liquid culture medium plus 6 g agar, sterilized at 121°C for 20 min. Add 2% ethanol before use.
[0014] (2) Enrichment culture-primary screening: Take 10g of pit mud sample, place it in a sterile bag containing 90mL of sterile water, and shake it at 180r / min for 30min to make the pit mud sample evenly distributed in the sterile water. Place the shaken pit mud mixture in a constant temperature water bath at 85℃ for 10min to kill the bacteria. After the water bath, inoculate it into the liquid culture medium at a 10% inoculation rate, seal it with a sealing film, place it in an anaerobic culture bag containing an anaerobic gas production bag, and place it in an incubator at 34-39℃ for enrichment culture for 4-7d, and enrich it three times in a row. After the enrichment culture is completed, the enrichment solution is subjected to a copper sulfate color development test, and the culture solution with a darker blue-green color is selected for the next step of screening and separation.
[0015] (3) Isolation and culture of microorganisms: The enriched solution with obvious color development is diluted in a gradient manner and then spread on a solid culture medium. Anaerobically culture it at 37°C for 4-7 days, and a single colony is picked and purified by streaking more than 3 times.
[0016] (4) Rescreening of caproic acid-producing bacteria: The purified strains were inoculated into liquid culture medium and cultured anaerobically at 34-39°C for 4-7 days. The culture fluid was extracted and subjected to gas chromatography quantitative analysis. The strain with the highest caproic acid production was selected for whole genome sequencing. After gene sequence alignment and combined with the construction of a phylogenetic tree, the strain was identified as Bacillus paranthracis.
[0017] The second technical solution provided by the present invention is a microbial preparation containing the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution.
[0018] In certain embodiments, the concentration of the caproic acid producing Bacillus LLMJ-JS-1 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.
[0019] Furthermore, the bacterial concentration of the caproic acid-producing Bacillus LLMJ-JS-1 in the microbial preparation is at least 1×10 8 CFU / mL or 1×10 8 CFU / g.
[0020] The third technical solution provided by the present invention is a method for producing caproic acid, wherein the method is to produce caproic acid by fermentation using the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution or the microbial preparation described in the second technical solution.
[0021] In certain embodiments, the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution or the microbial preparation described in the second technical solution is introduced into the fermentation system at an inoculation rate of 1%-10%, and anaerobically cultured at 33-39° C. for 4-7 days.
[0022] Preferably, the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution or the microbial preparation described in the second technical solution is introduced into the fermentation system at an inoculation rate of 1%, and anaerobically cultured at 33-39° C. for 4-7 days.
[0023] In certain embodiments, the fermentation system contains 0.5%-1% yeast extract, 0.5%-1% anhydrous sodium acetate, 0.02%-0.05% MgSO4, 0.02%-0.05% K2HPO4, and 0.05%-0.1% (NH4)2SO4.
[0024] Preferably, the fermentation system contains 0.01 g / mL of yeast extract, 5 μg / mL of anhydrous sodium acetate, 0.2 μg / mL of MgSO4, 0.4 μg / mL of K2HPO4, and 0.5 μg / mL of (NH4)2SO4.
[0025] The fourth technical solution provided by the present invention is the use of the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution or the microbial preparation described in the second technical solution in the preparation of caproic acid or a product containing caproic acid.
[0026] The fifth technical solution provided by the present invention is a cellar mud curing liquid, wherein the cellar mud contains a fermentation liquid produced by the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution or the microbial preparation described in the second technical solution.
[0027] The sixth technical solution provided by the present invention is a cellar mud, which contains the fermentation liquid of the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution or the microbial preparation described in the second technical solution.
[0028] The seventh technical solution provided by the present invention is the use of the caproic acid-producing Bacillus LLMJ-JS-1 described in the first technical solution or the microbial preparation described in the second technical solution in cellar mud production and / or cellar mud maintenance.
[0029] The technical effects of the present invention are as follows:
[0030] 1. The caproic acid-producing Bacillus paranthracis LLMJ-JS-1 strain of the present invention has an acid production level of up to 10.9 g / L in a pasteurized synthetic liquid culture medium, and its caproic acid production capacity is significantly better than that of traditional strains, which effectively solves the problem of low acid production and insufficient acid production stability of existing strains.
[0031] 2. The strain is derived from the bottom mud of Luzhou-flavor liquor cellars and is a wild strain with stable genome and phenotypic characteristics. It has good adaptability in the cellar environment, providing a solid scientific basis for its production and application in the production of cellar mud and the preparation of cellar curing liquid, and has significant potential and broad application prospects in improving the quality of base liquor.
[0032] Biomaterial Deposit
[0033] A strain of Bacillus LLMJ-JS-1, taxonomically named Bacillus paranthracis, was deposited in the General Microbiology Center of China Microorganism Culture Collection (CGMCC) on October 31, 2024. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC No: 32446. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the peak diagram of the standard sample of caproic acid under the gas chromatography conditions in Example 1 of the present invention.
[0035] Figure 2 This is a morphological characteristic diagram of hexanoic acid-producing Bacillus paranthracis LLMJ-JS-1.
[0036] Figure 3 This is the evolutionary tree of Bacillus paranthracis LLMJ-JS-1.
[0037] Figure 4 This is the effect of fermentation temperature on the acid production of strain LLMJ-JS-1.
[0038] Figure 5 This is the effect of ethanol addition on the acid production of strain LLMJ-JS-1.
[0039] Figure 6 This is the effect of fermentation time on the acid production of strain LLMJ-JS-1. DETAILED DESCRIPTION
[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.
[0041] Test method:
[0042] Hexanoic acid detection method:
[0043] (1) Sample pretreatment: Take 10 mL of fermentation broth, add 10 mL of ultrapure water, acidify with 2 M sulfuric acid, and dilute to 50 mL. Take 4 mL of liquid sample, mix evenly with ether at a ratio of 1:1, add 400 μl of internal standard, centrifuge or static stratification, filter the supernatant through a 0.22 μm microporous filter membrane, and take 1 mL into a gas phase injection vial for gas chromatography analysis.
[0044] (2) Chromatographic conditions: Agilent 7820A gas chromatograph (Agilent Technologies, Inc.) was used, equipped with an Agilent DB-WAX UI chromatographic column (30m*0.25mm*0.5μm; Agilent Technologies, Inc.); the initial column temperature of the chromatographic analysis was set at 40°C, then increased to 80°C at a rate of 3°C / min, then increased to 200°C at a rate of 5°C / min, and maintained at this temperature for 30 minutes; the initial column pressure was set to 8psi, maintained for 5 minutes, then increased to 17psi at a rate of 3psi / min, and maintained at this pressure for 57 minutes. The detector used was a flame ionization detector (FID), and its temperature was maintained at 250°C; the injection port temperature was set to 250°C and the pressure was maintained at 8psi.
[0045] The raw materials used in the embodiment:
[0046] Liquid culture medium: 1% yeast extract, 0.5% anhydrous sodium acetate, 0.02% MgSO4, 0.04% K2HPO4, 0.05% (NH4)2SO4, sterilized at 121°C for 20 min. Add 2% ethanol and 1% dry heat sterilized CaCO3 before use.
[0047] Solid culture medium: Add 2% agar powder to liquid culture medium before sterilization.
[0048] The raw materials used in the culture medium were purchased from Sinopharm Chemical Reagent Co., Ltd.
[0049] Example 1 Screening, identification and preservation of strains
[0050] 1.1 Strain screening
[0051] (1) Enrichment culture-preliminary screening: Take 10g of pit mud sample, place it in a sterile bag containing 90mL of sterile water, and shake it at 180r / min for 30min to make the pit mud sample evenly distributed in the sterile water. Place the shaken pit mud mixture in a constant temperature water bath at 85℃ for 10min to kill the bacteria. After the water bath, inoculate it into the liquid culture medium at a 10% inoculation rate, seal it with a sealing film, place it in an anaerobic culture bag containing an anaerobic gas production bag, and place it in a 37℃ incubator for enrichment culture for 4-7d, and enrich it three times in a row. After the enrichment culture is completed, perform a copper sulfate color development test on the enrichment solution, and select the culture solution with a darker blue-green color for the next step of screening and separation.
[0052] (2) Isolation and culture of microorganisms: The enriched solution with obvious color development is diluted in a gradient manner and then spread on a solid culture medium. Anaerobically culture it at 37°C for 4-7 days, and a single colony is picked and purified by streaking more than 3 times.
[0053] (3) Rescreening of caproic acid-producing bacteria: The purified strains were inoculated into liquid culture medium and cultured anaerobically at 37°C for 4-7 days. The culture medium was extracted and quantitatively analyzed by gas chromatography ( Figure 1 ).
[0054] 1.2 Strain identification
[0055] Select the strain with the highest hexanoic acid production and inoculate it into the pasteurized solid medium. Cultivate until colonies grow and observe their morphological characteristics, such as Figure 2 As shown, the colonies of this strain are light yellow, with irregular edges, dry surface and opaque. Whole genome sequencing was performed, and after gene sequence alignment, combined with the construction of a phylogenetic tree ( Figure 3 ) and determined that the strain was Bacillus paranthracis (Bacillus paraanthracis) and was named LLMJ-JS-1.
[0056] 1.3 Strain preservation
[0057] This strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 31, 2024, with the deposit number: CGMCC No: 32446.
[0058] Example 2 Study on the fermentation performance of strains
[0059] (1) Effect of fermentation temperature on acid production of strain LLMJ-JS-1
[0060] The caproic acid-producing Bacillus LLMJ-JS-1 in Example 1 was inoculated into a liquid culture medium at an inoculation rate of 1%, and anaerobically cultured at 33-39° C. for 7 days.
[0061] like Figure 4 As shown in the figure, the fermentation broth of the LLMJ-JS-1 strain can produce caproic acid in the range of 33-39℃. The production of caproic acid shows a trend of first increasing and then decreasing with the increase of temperature. The caproic acid production is the highest at 37℃, which is 9.43g / L. After exceeding 37℃, the acid production is inhibited with the increase of temperature. Therefore, the optimal fermentation temperature of this strain is 37℃.
[0062] (2) Effect of ethanol addition on acid production of strain LLMJ-JS-1
[0063] The caproic acid-producing Bacillus LLMJ-JS-1 in Example 1 was inoculated into a liquid culture medium at an inoculation rate of 1%, and the amount of ethanol added to the liquid culture medium was 1%-3%. The culture was anaerobically cultured at 37° C. for 7 days.
[0064] The results are as follows Figure 5As shown, the production of caproic acid first increases and then decreases with the increase of ethanol addition, and the production of caproic acid is the highest when the ethanol addition is 2%. Ethanol is an essential carbon source in the growth and metabolism of the strain. When the addition amount is 1-2%, the ethanol content is low and is insufficient to meet the substrate requirements for the synthesis of caproic acid. Therefore, with the increase of ethanol addition, the production of caproic acid increases accordingly. However, when the ethanol addition amount exceeds 2% ml, the concentration of ethanol is too high, which inhibits the growth and metabolic activity of the strain, resulting in a decrease in the production of caproic acid.
[0065] (3) Effect of fermentation time on acid production of strain LLMJ-JS-1
[0066] The caproic acid-producing Bacillus LLMJ-JS-1 in Example 1 was inoculated into a liquid culture medium at an inoculation rate of 1%, and 2% ethanol was added to the culture medium before inoculation. The culture medium was anaerobically cultured at 37° C. for 3-6 days.
[0067] The results are as follows Figure 6 As shown in the figure, at the beginning of fermentation (day 3), the production of caproic acid was low, at 4.8 g / L. As the fermentation process progressed, the production of caproic acid showed a clear growth trend. On day 5 of fermentation, the production of caproic acid reached a peak of 10.9 g / L.
[0068] Example 3 Application of Hexanoic Acid-Producing Bacillus LLMJ-JS-1 in Pit Mud Maintenance
[0069] The LLMJ-JS-1 strain was inoculated into a liquid culture medium and cultured anaerobically at 37°C for 5 days to obtain a fermentation broth, which could be used only after the hexanoic acid production reached the standard as determined by gas chromatography. 100 g of low-quality pit mud was placed in a conical flask and inoculated at 5% (the fermentation broth concentration reached 2.5 × 10 8 CFU / mL) was inoculated into the pit mud as the curing group, and the pit mud without fermentation liquid was used as the control group (before curing). After sealing, it was cultured at 37℃ for 7 days. The caproic acid content in the pit mud before and after curing was determined, and the results are shown in Table 1.
[0070] Table 1 Caproic acid content before and after cellar mud curing
[0071]
[0072] By analyzing the cellar mud before and after curing with gas chromatography (Table 1), it was found that the caproic acid production of the cellar mud after curing was significantly increased compared with that before curing. The application of LLMJ-JS-1 strain fermentation liquid in cellar mud curing has a positive effect on increasing the production of caproic acid in cellar mud.
[0073] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.
Claims
1. A strain of Bacillus paranthracis LLMJ-JS-1, characterized in that: It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on October 31, 2024, with the deposit number CGMCC No: 32446.
2. A microbial preparation containing the Bacillus LLMJ-JS-1 according to claim 1.
3. The microbial preparation according to claim 2, characterized in that The bacterial concentration of Bacillus LLMJ-JS-1 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.
4. A method for producing caproic acid, characterized in that The method is to produce caproic acid by fermentation using the Bacillus LLMJ-JS-1 described in claim 1 or the microbial preparation described in claim 2 or 3.
5. The method according to claim 4, characterized in that The Bacillus LLMJ-JS-1 according to claim 1 or the microbial preparation according to claim 2 or 3 is introduced into the fermentation system at an inoculation rate of 1%-10%, and anaerobically cultured at 33-39° C. for 4-7 days.
6. The method according to claim 5, characterized in that The fermentation system contains 0.5%-1% yeast extract, 0.5%-1% anhydrous sodium acetate, 0.02%-0.05% MgSO4, 0.02%-0.05% K2HPO4, and 0.05%-0.1% (NH4)2SO4.
7. Use of the Bacillus LLMJ-JS-1 according to claim 1 or the microbial preparation according to claim 2 or 3 in the preparation of caproic acid or a product containing caproic acid.
8. A pit mud curing liquid, characterized in that: The pit mud contains the fermentation liquid of the Bacillus LLMJ-JS-1 according to claim 1 or the microbial preparation according to claim 2 or 3.
9. A pit mud, characterized in that: The pit mud contains the fermentation liquid of the Bacillus LLMJ-JS-1 according to claim 1 or the microbial preparation according to claim 2 or 3.
10. Use of the Bacillus LLMJ-JS-1 according to claim 1 or the microbial preparation according to claim 2 or 3 in pit mud production and / or pit mud maintenance.