A strain of Oceanobacillus oncorhynchi producing caproic acid and its application

Abstract

The present invention belongs to the technical field of microbial fermentation, specifically a strain of Oceanobacillus caeni producing caproic acid and its application. This strain was deposited in the Guangdong Provincial Microbial Culture Collection Center on May 27, 2024, with the deposit number GDMCC NO: 64695, and the deposit address is the 5th floor of Building 59, Institute of Microbiology, Guangdong Academy of Sciences, 100 Xianlie Middle Road, Guangzhou. This bacterium was screened from the old cellar mud of Luzhou-flavor liquor; the 16S rDNA sequence is as shown in SEQ ID N0.1. The Oceanobacillus caeni in the present invention was screened from the cellar mud of Luzhou-flavor liquor. When the culture temperature is 35 °C, the pH is 6.0, and the culture medium is sodium acetate medium, the highest caproic acid production is 708 mg / L. At the same time, this bacterium can assimilate C2-C6 and polysaccharides to produce caproic acid under anaerobic conditions.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and specifically relates to an Oceanobacillus caeni producing caproic acid and its application. Background Art

[0002] As a unique traditional solid-state distilled liquor in China, Chinese liquor has formed its unique brewing process and liquor body style during its long development. Luzhou-flavor Chinese liquor, as an important representative of Chinese liquor, occupies a dominant position in the Chinese liquor industry in terms of the sensory flavor recognition and market share (more than 70%).

[0003] Ethyl caproate, as the main flavor component of Luzhou-flavor Chinese liquor, determines the typicality and quality of the Luzhou-flavor Chinese liquor style. And caproic acid, the precursor substance for the synthesis of ethyl caproate, is mainly synthesized by caproic acid bacteria in the pit mud. Caproic acid bacteria, as the key functional bacteria in Luzhou-flavor pit mud, the amount of them in the pit mud determines the quality of the pit mud.

[0004] At present, the caproic acid-producing bacteria screened from pit mud mainly include Clostridium, Bacillus, Ruminnococcaceae, etc. However, these strains still have the problem of single strain, so more caproic acid-producing bacteria genera need to be obtained. Summary of the Invention

[0005] Based on the problems existing in the prior art, the present invention provides an Oceanobacillus caeni SD2023JN-117. Oceanobacillus caeni belongs to the genus Oceanobacillus; when Oceanobacillus caeni is used in the production of Luzhou-flavor Chinese liquor, due to its strong caproic acid production ability, it has good application prospects in the development of pit mud functional bacteria liquid, pit mud maintenance and the preparation of artificial pit mud.

[0006] In order to achieve the above object of the invention, the technical solution of the present invention is as follows:

[0007] An Oceanobacillus caeni SD2023JN-117 has been deposited with the Guangdong Provincial Microbial Culture Collection Center on May 27, 2024, with the deposit number GDMCC NO: 64695, and the deposit address is the 5th floor of Building 59, Institute of Microbiology, Guangdong Academy of Sciences, 100 Xianlie Middle Road, Guangzhou.

[0008] Furthermore, the Oceanobacillus caeni SD2023JN-117 was screened from the aged pit mud of Luzhou-flavor liquor; its 16S rDNA sequence is as shown in SEQ ID N0.1.

[0009] As a preferred embodiment of the present application, the Oceanobacillus caeni SD2023JN-117 has a high ability to produce caproic acid, up to 708 mg / L.

[0010] As a preferred embodiment of the present application, the Oceanobacillus caeni SD2023JN-117 has a relatively stable ability to produce caproic acid, that is, with the extension of the fermentation time to 12 d, it still maintains a high level of caproic acid production.

[0011] As a preferred embodiment of the present application, under anaerobic conditions, the Oceanobacillus caeni SD2023JN-117 can utilize different carbon sources to produce caproic acid and can maintain a certain caproic acid yield, which is 125 mg / L to 230 mg / L.

[0012] As a preferred embodiment of the present application, the different carbon sources include, but are not limited to, any one or a mixture of several of starch, sodium acetate, glycerol, succinic acid, glucose, sucrose, and xylose.

[0013] As a preferred embodiment of the present application, when the carbon source is starch, the Oceanobacillus caeni SD2023JN-117 has the highest caproic acid yield, which is 230 mg / L.

[0014] As a preferred embodiment of the present application, the present application protects the application of the above-mentioned Oceanobacillus caeni SD2023JN-117 in the microbial fermentation production of caproic acid.

[0015] As a preferred embodiment of the present application, the present application protects the application of the above-mentioned Oceanobacillus caeni SD2023JN-117 in the production of Luzhou-flavor liquor.

[0016] As a preferred embodiment of the present application, the present application protects the application of the above-mentioned Oceanobacillus caeni SD2023JN-117 in the preparation of pit mud functional bacteria liquid.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) Oceanobacillus caeni SD2023JN-117 was screened from high-quality aged pit mud in liquor production, and has good adaptability in the pit mud environment. It can be further applied to the development of pit mud functional bacterial liquid, pit mud maintenance, and the preparation of artificial pit mud, etc., and has good application prospects.

[0019] (2) Oceanobacillus caeni SD2023JN-117 can ferment a variety of carbon sources (assimilating C2 to C6 and polysaccharides) to produce caproic acid under anaerobic conditions, and has a strong ability to produce caproic acid, which is 125 mg / L to 230 mg / L. Especially under the condition that the carbon source is starch, the caproic acid yield is the highest, which is 230 mg / L, and it is the best carbon source for producing caproic acid. This strain has the ability to utilize a wide range of substrates to produce caproic acid, and has a wide application range.

[0020] (3) When the culture temperature of Oceanobacillus caeni SD2023JN-117 is 35 °C, the pH is 6.0, and the culture medium is sodium acetate medium, the caproic acid yield is the highest, and the caproic acid content can reach 708 mg / L. And as the fermentation time extends to 12 d, it still maintains a relatively high level of caproic acid production, indicating that the SD2023JN-117 strain has a relatively stable ability to produce caproic acid. Description of the Drawings

[0021] Figure 1 It is the peak chromatogram of standard products of acetic acid, butyric acid and caproic acid under the gas chromatography conditions in Example 1;

[0022] Figure 2 It is the colony map of Oceanobacillus caeni SD2023JN-117 strain anaerobically cultured on a solid separation medium;

[0023] Figure 3 It is the microscopic morphology map of Oceanobacillus caeni SD2023JN-117 strain;

[0024] Figure 4 It is the electrophoresis map of the 16S rDNA PCR product of Oceanobacillus caeni SD2023JN-117 strain; among them, band 1 is the 16S rDNA PCR product, and band 2 is the DNA Marker;

[0025] Figure 5Phylogenetic tree of 16S rDNA of Oceanobacillus caeni strain SD2023JN-117;

[0026] Figure 6 Acid production curve of Oceanobacillus caeni strain SD2023JN-117 when fermenting with starch as carbon source for 6 days. Detailed implementation mode

[0027] In order to make the content of the present invention easier to understand, the following will further elaborate on the solutions described in the present invention in combination with specific embodiments. However, this should not be understood as limiting the scope of the above-mentioned subject matter of the present invention to the following embodiments.

[0028] Example 1:

[0029] Screening of Oceanobacillus caeni strain SD2023JN-117

[0030] The caproic acid-producing Oceanobacillus caeni was isolated from high-quality old cellar mud in a Luzhou-flavor liquor cellar in Sichuan. The composition of the sodium acetate fermentation liquid medium used is: sodium acetate 5.0 g / L, anhydrous magnesium sulfate 0.2 g / L, ammonium sulfate 0.5 g / L, dipotassium hydrogen phosphate 0.4 g / L, yeast extract 2.0 g / L, agar powder 20.0 g / L, calcium carbonate 10.0 g / L, absolute ethanol 25 mL / L. After adjusting the pH to 6.0, it was sterilized at 121 °C for 20 min. Calcium carbonate was sterilized by dry heat alone at 160 °C for 2 h, and absolute ethanol and calcium carbonate were added after the medium was cooled to 40 °C - 50 °C. Absolute ethanol was added after passing through a 0.22 μm sterile filter membrane. The sodium acetate solid separation medium was added with 20 g / L of agar on the basis of the liquid medium.

[0031] 1. Enrichment of cellar mud

[0032] The cellar wall mud and cellar bottom mud at 20 cm from the cellar bottom were taken and mixed evenly to obtain a cellar mud mixed sample. 5.0 g of the cellar mud mixed sample was added to 20 mL of sterile normal saline, stirred, and then shaken at 180 r / min on a shaker for 30 min to make a cellar mud mixed sample suspension. 10 mL of the cellar mud mixed sample suspension was added to 200 mL of the liquid enrichment medium, and cultured statically at 35 °C until the bacterial liquid was significantly turbid and accompanied by a large amount of bubbles. The enriched culture bacterial liquid was detected by the copper sulfate colorimetric method every 24 h until obvious color development occurred, and then used for the separation and purification of the strain.

[0033] 2. Isolation and purification of the strain

[0034] After thoroughly mixing the enriched fermentation broth, use a pipette to aspirate 1 mL, add 100 μL of the bacterial solution to a centrifuge tube containing 900 μL of sterile water, and vortex it three times for 5 s each time to mix thoroughly. Then, aspirate 100 μL of the mixed solution and add it to another centrifuge tube containing 900 μL of sterile water. Repeat the above steps to mix evenly. And so on, prepare solutions with different dilution concentrations of the original solution, 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 From each dilution tube, aspirate 100 μL of the diluted solution onto a separation medium plate, spread it evenly, and place the plate in a constant temperature incubator at 35 °C for anaerobic culture for 5 - 7 d. Streak and purify the colonies with different morphological characteristics at least three times to obtain single colonies.

[0035] 3. Screening of caproic acid-producing bacteria

[0036] Select the single colonies grown in Step 2, use an inoculation loop to inoculate them into a test tube containing 15 mL of sodium acetate fermentation liquid medium under sterile operating conditions, and then culture them in an anaerobic incubator at 35 °C until the bacterial solution becomes turbid. Conduct a preliminary screening by the copper sulfate color reaction method, and select the strains with obvious color reaction. Detect the caproic acid content in their fermentation broth by gas chromatography. Finally, a caproic acid-producing strain was screened out and named SD2023JN-117.

[0037] Example 2:

[0038] Identification of Oceanobacillus caeni SD2023JN-117

[0039] 1. Observation of strain morphology and culture characteristics

[0040] Inoculate SD2023JN-117 with obvious copper sulfate color reaction obtained from the rescreening in Example 1 on sodium acetate solid separation medium, and culture it at 35 °C until colonies grow. Observe the morphological characteristics of the colonies, and the characteristics are as follows: The colonies are round, with regular edges, small, and light yellow in color. Observe its cell morphology under a microscope as follows: The cells are rod-shaped, with round and straight ends, and produce spores. The colony morphology and cell microscopic morphology are as shown in Figure 2 、 Figure 3 .

[0041] 2. 16S rDNA sequence determination and construction of its phylogenetic tree

[0042] DNA extraction: Use the TSINGKE plant DNA extraction kit (general type) to extract the bacterial DNA. Since the structures of plant cells and bacterial microorganisms are similar, this kit can be used for the extraction of general microorganisms.

[0043] PCR amplification: The 16S rDNA of a single strain was amplified using primers 27F / 1492R. The specific amplification primers and amplification conditions are shown in Tables 1 - 3.

[0044] Table 1 Primer sequences for PCR amplification

[0045]

[0046] Table 2 Composition of the PCR reaction system

[0047] Reagent Dosage / μL 1×TSE101 Gold Mix 45 27F(10P) 2 1492R(10P) 2 Genomic DNA Template 1

[0048] Table 3 PCR amplification conditions

[0049] Stage Temperature Time Number of Cycles Pre-denaturation 98℃ 3 min --

[0050] Cycle stage

[0051]

[0052] The amplified PCR product was subjected to agarose gel electrophoresis (2 μL sample + 6 μL bromophenol blue) at 300 V for 12 min to obtain an identification gel image. The PCR amplification results are shown in Figure 4 , the size of the PCR product is 1363 bp. After sequencing and identification, the 16S rDNA sequence of strain SD2023JN - 117 is shown in SEQ ID N0.1.

[0053] SEQ ID N0.1:

[0054] aacacgtgggc aacctaccta taagactggg ataactcgcg gaaacgtgag ctaataccgg60ataatacttt ttattgcata ataagaagtt tgaaaggcgg cgtaagctgt cacttataga120tgggcccgcg gcgcattagc tagttggtga ggtaaaggct caccaaggca acgatgcgta180gccgacctga gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg240aggcagcagt agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag300tgaagaaggt tttcggatcg taaaactctg ttgttaggga agaacaagtc aggtagtaac360tgacctgacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc420ggtaatacgt aggtggcaag cgttgtccgg atttattggg cgtaaagcgc tcgcaggcgg480tcttttaagt ctgatgtgaa agcccacggc ttaaccgtgg agggtcattg gaaactggag540gacttgagtg cagaagagga gagtggaatt ccatgtgtag cggtgaaatg cgtagagata600tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa660gcgtgggtag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta720ggtgttaggg ggtttccgcc ccttagtgct gaagttaacg cattaagcac tccgcctggg780gagtacggcc gcaaggctga aactcaaaag aattgacggg gacccgcaca agcggtggag840catgtggttt aattcgaagc aacgcgaaga accttaccaggtcttgacat cctttcgacc900tccctagaga tagggatttc ccttcgggga cgaaagtgac aggtggtgca tggttgtcgt960cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct tgatcttagt1020tgccagcatt aagttgggca ctctaaggtg actgccggtg acaaaccgga ggaaggtggg1080gatgacgtca aatcatcatg ccccttatga cctgggctac acacgtgcta caatggatgg1140tacaaagggc agcgaaaccg caaggtcaag caaatcccat aaaaccattc tcagttcgga1200ttgtaggctg caactcgcct acatgaagcc ggaatcgcta gtaatcgcgg atcagcatgc1260cgcggtgaat acgttcccgg gtcttgtaca caccgcccgt cacaccacga gagttggtaa 1320

[0055] cacccgaagt cggtgaggta accttttgga gccagccgcc gaa 1363。

[0056] Phylogenetic tree construction: After the sequencing sequences were trimmed by BioEdit, the reliable sequence fragments were uploaded to the NCBI nucleic acid alignment website for alignment. The sequence with the highest sequence homology was selected, and the MEGA 11.0 software was used to construct the phylogenetic tree. The phylogenetic tree based on 16S rDNA is as Figure 5 shown.

[0057] After morphological characteristics and 16S rDNA sequencing of strain SD2023JN-117, it was identified as Oceanobacillus caeni. The growth temperature of this bacterium is 35 °C and the growth pH is 6.0.

[0058] Example 3: Detection of caproic acid production and fermentation culture of Oceanobacillus caeni SD2023JN-117

[0059] 1. Fermentation culture of SD2023JN-117 and detection of caproic acid production

[0060] Gas chromatography conditions: DB-FFAP (30×320×0.25) chromatographic column; the carrier gas is high-purity nitrogen, the initial temperature is 60°C, it is heated to 100°C at a rate of 10°C / min, and then heated to 180°C at a rate of 5°C / min; the split ratio is 20:1; the detector temperature is 250°C; the injection port temperature is 250°C; the injection volume is 0.5 μL; the carrier gas: high-purity nitrogen, the flow rate is 1.5 mL / min; the hydrogen flow rate is 30 mL / min; the air flow rate is 400 mL / min. Under these conditions, the peak emergence situation of the standard sample is as Figure 1 shown, and the peak emergence times of acetic acid, butyric acid, and caproic acid are 7.285 min, 10.233 min, and 14.287 min respectively.

[0061] The SD2023JN-117 strain obtained in Example 1 was aseptically inoculated into a test tube containing a fermentation liquid medium with an inoculation loop and cultured in an anaerobic incubator at 35°C for 3 - 5 d to obtain a seed liquid; at 12 d of fermentation, 2 mL of the fermentation broth was taken with a disposable syringe, centrifuged at 5000 r / min for 5 min, and then an appropriate amount of the supernatant was aspirated and passed through a 0.22 μm microporous filter membrane. Then, 0.5 μL was taken and placed in an injection vial for gas chromatography qualitative and quantitative analysis by the external standard method. The yields of caproic acid, butyric acid, and acetic acid of SD2023JN-117 using sodium acetate as the carbon source were measured, and three replicates were made for each sample. The detection results are shown in Table 4. At this time, the yields of caproic acid, butyric acid, and acetic acid in the fermentation broth are 708 mg / L, 389 mg / L, and 1180 mg / L respectively.

[0062] Table 4 Acid production of SD2023JN-117 when using sodium acetate as the carbon source for 12 d of culture

[0063] Sample Name Caproic Acid (mg / L) Butyric Acid (mg / L) Acetic Acid (mg / L) SD2023JN-117 708 389 1180

[0064] 2. Caproic acid production by SD2023JN-117 fermentation with different carbon sources

[0065] The seed liquid obtained in Example 1 was inoculated into 150 mL of each medium with a pH of 6.8, where the carbon sources were starch (20.0 g / L), sodium acetate (20.0 g / L), glycerol (20.0 g / L), succinic acid (20.0 g / L), glucose (20.0 g / L), sucrose (20.0 g / L), and xylose (20.0 g / L), and the other component ratios were the same (sodium chloride 5.0 g / L, peptone 10.0 g / L, yeast powder 5.0 g / L, beef extract 10.0 g / L, soluble starch 1.0 g / L, L-cysteine salt 0.5 g / L). At 35 °C, the cultures were sealed and statically incubated. At 0 d, 6 d, 8 d, and 10 d, an appropriate amount of the fermentation broth was aseptically taken, filtered through a 0.22 μm filter membrane, and then analyzed by gas chromatography to determine the yields of acetic acid, butyric acid, and caproic acid. When sodium acetate was used as the carbon source, the fermentation time was plotted on the abscissa and the acid production concentration was plotted on the ordinate to draw the fermentation acid production curve. The results are as Figure 6 shown. The highest acid production was 230 mg / L at 6 d of culture. The acid production test results of each carbon source medium at 6 d of fermentation are shown in Table 5. As can be seen from Table 5, under the condition that the carbon source was starch, the yields of acetic acid, butyric acid, and caproic acid were the highest, making it the optimal carbon source for acid production. Secondly, the strain also showed relatively high acid production ability when using glycerol and sodium acetate.

[0066] Table 5 Acid production of samples fermented for 6 d in media with different carbon sources

[0067] Concentration (mg / L) Starch Sodium Acetate Glycerol Succinic Acid Glucose Sucrose Xylose Caproic Acid 230 209 219 170 160 125 191 Butyric Acid 130 112 108 80 80 70 72 Acetic Acid 650 618 585 378 399 243 237

[0068] Although the present invention has been described in detail through specific embodiments, those of ordinary skill in the art should understand that any changes in form and detail made on this basis without exceeding the scope of the claims are within the scope of protection of the present invention.

Claims

1. A strain of Bacillus oceanisediminis ( Oceanobacillus caeni Oceanobacillus caeni ) SD2023JN-117 was deposited at the Guangdong Provincial Culture Collection of Microorganisms on May 27, 2024, with the deposit number GDMCC NO: 64695, and the deposit address is the 5th floor of Building 59, Institute of Microbiology, Guangdong Academy of Sciences, 100 Xianlie Middle Road, Guangzhou.

2. Use of Bacillus oceanisediminis ( Oceanobacillus caeni ) SD2023JN-117 in the microbial fermentation production of caproic acid.

3. Use of Bacillus oceanisediminis ( Oceanobacillus caeni ) SD2023JN-117 in the production of Luzhou-flavor Baijiu.

4. Use of Bacillus oceanisediminis ( Oceanobacillus caeni ) SD2023JN-117 in the preparation of pit mud functional bacteria liquid.

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