A strain producing octanoic acid and its screening and cultivation method and application

Abstract

The invention discloses a strain producing octanoic acid and a screening and culturing method and application thereof, belonging to the technical field of brewing microorganisms. The strain is a true bacterium ( Eubacterium sp.) strain JSY12851, which is deposited in Guangdong Microbiological Culture Collection Center, with a deposit number of GDMCC No: 64552. The strain provided by the present invention is isolated from the Luzhou-flavor liquor cellar mud of Jiangsu Jinshiyuan Liquor Co., Ltd. (longitude: 119.04 latitude: 33.6), can produce caproic acid and octanoic acid at the same time, and has good application value in the field of liquor brewing.

Description

Technical Field

[0001] The invention belongs to the technical field of brewing microorganisms, and specifically relates to a strain producing octanoic acid, a screening and culturing method and an application thereof. Background Art

[0002] The key process of Luzhou-flavor liquor is fermentation in mud pits. Mud pits provide many flavor substances and their precursors for Luzhou-flavor liquor, among which caproic acid and octanoic acid are the most representative, which can be esterified with ethanol to form ethyl caproate and ethyl octanoate. Ethyl caproate is considered to be the main aroma of Luzhou-flavor liquor and determines the quality of Luzhou-flavor liquor. Ethyl octanoate is considered to be a component of the flavor of the pit, especially the flavor of the old pit, and is widely present in liquor.

[0003] As the main aroma of Luzhou-flavor liquor, ethyl caproate directly affects the quality of Luzhou-flavor liquor. GB / T10781.1-2021 stipulates that the implementation standard of the product is ≤ one year from the production date. The ethyl caproate of the high-quality Luzhou-flavor liquor is above 1.5g / L. [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] Therefore, it is an urgent problem for Luzhou-flavor liquor companies to increase the content of ethyl caproate in liquor through liquor brewing technology.

[0004] The aroma of ethyl caprylate in liquor is fruity. [3] , widely found in Luzhou-flavor liquor, Maotai-flavor liquor, and sesame-flavor liquor [4] And other fermented liquor [5, 6] Among the aroma components of Luzhou-flavor liquor, the absolute content of ethyl caprylate is not high, but its aroma intensity contribution value is greater than that of ethyl acetate, ethyl lactate, and ethyl butyrate among the four major esters in traditional view, and is second only to ethyl caproate. [7] Ethyl caprylate is expressed as cellar aroma in Luzhou-flavor liquor, especially as a component of old cellar aroma, and its aroma is more elegant.

[0005] Hexanoic acid is the precursor of ethyl hexanoate, which mainly comes from hexanoic acid bacteria in pit mud. [8]. Caproic acid bacteria is a hot topic in the research of cellar mud microorganisms. The industry and academia have been keen on screening caproic acid bacteria from nature. It is known that caproic acid bacteria isolated from the natural environment are distributed in: Clostridium, Caproiciproducens, Megasphaera, Caproiciproducens, Eubacterium, Rhodospirillum, Pseudoramibacter, Caproicibacterium, Rummeliibacillus and Enterococcus. At present, the caproic acid bacteria isolated from cellar mud are concentrated in the Clostridiaceae and Oscillospiraceae families, and related caproic acid bacteria have not yet been isolated from the Eubacteriaceae family. According to high-throughput results, microorganisms of Eubacteriaceae have a considerable abundance in the cellar mud flora, about 1.75%-4.41%. [9] The microbial research of Eubacteriaceae focuses on intestinal microbes, which are part of the core microbial community in the human gut.

[10] .

[0006] Caprylic acid is the precursor of ethyl caprylate and is a medium-chain fatty acid. It has the functions of improving intestinal morphology, improving animal growth performance, preventing and treating diseases, regulating immunity, and antibacterial.

[11] In wine fermented by a single bacteria, medium-chain fatty acids mainly come from the process of yeast decomposing fatty acids or synthesizing fatty acids.

[12] However, there is no relevant report on the production mechanism of liquor in multi-strain fermentation system.

[0007] Eubacterium Eubacterium sp.) is an important functional bacteria species in liquor cellar mud, belonging to the Eubacteriaceae family. It can use the residual sugar and short-chain fatty acids in yellow water and cellar mud to synthesize medium-chain fatty acids such as caproic acid and octanoic acid.

[13] A strain of Pyruvic acid Eubacterium microorganism ( Eubacterium pyruvativorans ), and its metabolic characteristics, that is, it has similar metabolism to Clostridium kluyveri to produce butyric acid and caproic acid, but it is not seen to produce octanoic acid

[13] . Eubacterium is a common microorganism in old cellar mud. Taking the Eubacteriaceae family as an example, our laboratory data has been analyzed through high-throughput data of old cellar mud over 20 years old in the company and cellar mud samples from external units. The abundance of Eubacterium family microorganisms in old cellar mud is between 0.1% and 1%. This shows that Eubacterium is common in old cellar mud. Microorganisms of this family adapt to the cellar environment and are one of the components of old cellar mud microorganisms. The microbial growth of Eubacterium requires strict anaerobic conditions, has a long growth cycle, harsh nutritional requirements, and unclear culture conditions. So far, it has not been isolated and screened from the cellar mud. Eubacterium microorganisms still need to be further developed for the improvement of the microecological composition of the cellar mud and the flavor of liquor. Summary of the invention

[0008] The purpose of the present invention is to provide a strain of octanoic acid-producing bacteria and a screening and cultivation method and application thereof, to screen true bacterium from the mud of a Luzhou-flavor cellar, to simultaneously produce caproic acid and octanoic acid, to improve the flavor of liquor during the liquor brewing process, and to have a very important significance for improving the quality of liquor.

[0009] To achieve the above object, the present invention provides the following technical solution: a strain of octanoic acid-producing bacteria, which is a true bacterium ( Eubacterium sp.) strain JSY12851, which was deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCCNo:64552.

[0010] The 16S rDNA nucleotide sequence of the bacterial species is shown in SEQ ID NO: 1.

[0011] The colony morphology of the Eubacterium species JSY12851 is as follows: the colonies are white, round, dry and opaque.

[0012] Among them, the bacterial morphology of the Eubacterium species JSY12851 is as follows: the bacterial body is short rod-shaped, 0.3-0.5 μm×1.0-1.5 μm, single, Gram-positive, and no spores are found.

[0013] The growth characteristics of the Eubacterium species JSY12851 are as follows: Ph is between 22 and 47, with 32 being the best; Ph is between 4.5 and 10, with 6.5 being the best; and it can tolerate 0.5% to 2% NaCl, with the best growth at 1%.

[0014] A method for screening and culturing octanoic acid-producing bacteria, the method for screening and culturing the Eubacterium species JSY12851 comprising: enriching and culturing cellar mud, applying a suspension dilution on a culture medium, and anaerobic culturing at 37°C for 72 hours; applying glass beads, picking a single colony, and purifying more than 3 times to obtain a pure culture; amplifying 16S rDNA of the pure culture, and comparing and analyzing the sequence with NCBI to obtain the Eubacterium species JSY12851.

[0015] Wherein, the specific steps of the screening and culturing method are as follows:

[0016] (1) Culture medium 1 (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;

[0017] Vitamin B solution stock solution (g / L): niacin 0.02, cobalamin 0.02, thiamine hydrochloride 0.01, p-aminobenzoic acid 0.01, pyridoxine hydrochloride 0.05, pantothenic acid 0.005. After preparation, pack according to the actual usage and store at -20℃.

[0018] (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 in advance for deoxygenation; boil ultrapure water for 5-10 minutes on an induction cooker, pour it into a 500 mL blue-capped bottle, stir and blow 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 the anaerobic box; loosen the lid for further deoxygenation; wait for it to cool and add vitamins;

[0019] (3) Anaerobic chamber status detection: Take out the target particles in the anaerobic chamber, dry them at 80°C, 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 2 MPa;

[0020] (4) Strain enrichment: Take 1 g of cellar mud, put it into a 15 mm × 150 mm test tube, transfer it into an anaerobic box, add 10 mL of culture medium; plug it with a rubber stopper, and culture it anaerobically at 37°C 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 use liquid-liquid extraction chromatography to test the amount of caproic acid and octanoic acid. Select the experimental group with high octanoic acid / caproic acid and enter the next round of culture. Repeat 5 times;

[0021] (5) Microbial screening: The enriched solution from step (4) was diluted with deoxygenated culture medium for 10 -5 -10 -7Glass beads were spread on a solid plate of culture medium, and cultured anaerobically at 37°C for 7 days, and colonies were picked; the colonies were streaked and purified more than 3 times to obtain pure cultured bacteria, which were named JSY12851.

[0022] In step (1), the mother solution of B vitamins (g / L) includes: 0.02 g of niacin, 0.02 g of cobalamin, 0.01 g of thiamine hydrochloride, 0.01 g of p-aminobenzoic acid, 0.05 g of pyridoxine hydrochloride, 0.005 g of pantothenic acid, and the remainder is water; the solution is packaged according to the actual usage amount and stored at -20°C.

[0023] The invention discloses an application of a strain of caprylic acid-producing bacteria, wherein the strain can produce caproic acid and caprylic acid simultaneously and is applied in the field of liquor brewing.

[0024] The beneficial effects of the present invention are as follows: a Eubacterium strain JSY12851 is screened out from the cellar mud of Luzhou-flavor liquor, and the strain can produce caproic acid and octanoic acid at the same time, with the octanoic acid production capacity reaching 5.92 g / L and the caproic acid production capacity reaching 3.71 g / L, which can improve the flavor of Luzhou-flavor liquor and has broad application prospects in the field of liquor brewing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the morphological characteristics of strain JSY12851;

[0026] Figure 2 This is a scanning electron microscope photo of strain JSY12851;

[0027] Figure 3 This is the evolutionary tree of strain JSY12851. DETAILED DESCRIPTION

[0028] The technical solution of the present invention is further described below in conjunction with specific embodiments, but it should not be understood as limiting the technical solution; wherein, concentrations not specified refer to mass percentages.

[0029] Example 1: Strain screening, identification and growth characteristics

[0030] (1) Strain screening

[0031] Culture medium 1 (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 B vitamin solution stock solution, and the rest water; adjust pH to 7.0±0.2 before sterilization, sterilize at 115℃ for 15 min; B vitamin solution stock solution (g / L): 0.02 niacin, 0.02 cobalamin, 0.01 thiamine hydrochloride, 0.01 p-aminobenzoic acid, 0.05 pyridoxine hydrochloride, 0.005 pantothenic acid, and the rest water, package according to actual usage, and store at -20℃;

[0032] Deoxygenation: Prepare and weigh the dry sample of the culture medium and put it into the anaerobic box; put the wet sample of the culture medium into the anaerobic box in advance for deoxygenation; boil ultrapure water for 5-10 minutes on an induction cooker, pour it into a 500mL blue-capped bottle, stir and blow nitrogen for 20 minutes, wait for it to cool, seal the bottle mouth, and transfer it to the anaerobic box; prepare the culture medium in the anaerobic box and put it into 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; put the prepared culture medium into the sterilizing pot, wait for it to cool to 95℃ after sterilization, and quickly transfer it into the anaerobic box; loosen the lid for further deoxygenation; wait for it to cool and add vitamins;

[0033] Anaerobic box status detection: take out the target particles in the anaerobic box, dry them at 80℃, and put them back into the anaerobic box; add resazurin to sterile water, sterilize it and put it into the anaerobic box as an anaerobic indicator; check the remaining amount of mixed gas every day, replace the mixed gas in time, and ensure that the mixed gas pressure is above 2mpa;

[0034] Strain enrichment: Take 1g of pit mud, put it into a 15 mm × 150 mm 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 cultivation, and detect the amount of caproic acid and octanoic acid by liquid-liquid extraction chromatography, select the experimental group with high caprylic acid / caproic acid, and enter the next round of cultivation, repeat 5 times;

[0035] Microbial screening: The above enriched solution was diluted with deoxygenated culture medium for 10 -5 -10 -7 Glass beads were spread on a solid plate of culture medium, and cultured anaerobically at 37°C for 7 days, and colonies were picked; the colonies were streaked and purified more than 3 times to obtain pure cultured bacteria, which were named JSY12851.

[0036] (2) Identification

[0037] Morphological characteristics of the strain: The isolated Eubacterium strain JSY12851 was cultured anaerobically at 37°C in a culture medium for 7 days to obtain a bacterial suspension; after fixation, the bacterial body was observed under an electron microscope, which showed that the bacterial body was short rod-shaped, 0.3-0.5μm×1.0-1.5μm, single, and no spores were found; Gram staining of the bacterial suspension showed that it was Gram-positive under an optical microscope; the bacterial suspension was spread on a solid culture medium plate by four-zone marking, and the colony morphology was white, round, dry on the surface, and opaque; Figure 1 , Figure 2 , Figure 3 shown.

[0038] 16SrRNA sequence comparison results: The phylogenetic tree was constructed after PCR amplification using bacterial 16S rDNA universal primers (27F, 1492R). The 16S rDNA of the measured strain was compared with the sequence of the model strain (published in international authoritative or relevant journals as a representative strain of this type of bacteria) in the NCBI database. The phylogenetic tree was constructed using the neighbor-joining method in the MEGA software. The 16S rDNA similarity with the model strain was less than 95%. According to the conventional standards for new species of bacterial classification (16SrDNA < 97%), JSY12851 was proved to be a species of the genus Eubacterium or a new family.

[0039] Table 1: Comparison of the genetic relationship between strain JSY12851 and published model strains

[0040]

[0041] (3) Strain growth characteristics

[0042] Using the above-mentioned culture medium 1 as the basic culture medium, different growth conditions were set up, and 3 parallel cultures were inoculated with equal amounts of strain JSY12851 at a 10% (v / v) inoculation rate. After anaerobic culture for 7 days, the OD600 value in the fermentation broth was measured. The results showed that the optimum temperature and pH value of strain JSY12851 were 32°C and 6.5, and it could tolerate 0.5%-2% NaCl and grew best at 1%.

[0043] Table 2: OD of strain JSY12851 at different temperatures 600 Value (20 times dilution)

[0044]

[0045] Table 3: OD of strain JSY12851 at different pH 600 Value (20 times dilution)

[0046]

[0047] Table 4: OD of strain JSY12851 under different NaCl conditions 600 value

[0048]

[0049] (4) Physiology and biochemistry

[0050] Table 5: Fatty acid composition of JSY12851

[0051]

[0052] Table 6: JSY12851 API 50CH test results

[0053]

[0054] 1. Culture Conditions

[0055] (1) Culture medium 2 (g / L): glucose 20 (other carbon sources: sodium acetate 27.3; sodium butyrate 25; sodium lactate 24.89; starch 20; ethanol 25.35 mL; 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, the rest water; adjust pH to 7.0 ± 0.2 before sterilization, sterilize at 115 °C for 15 min;

[0056] (2) Culture conditions: 37°C, anaerobic;

[0057] (3) Cultivation period: 7-10 days;

[0058] (4) Inoculation amount: 10%;

[0059] 2. Detection Method

[0060] (1) Liquid-liquid extraction: Pipette 2 mL of sample solution into a mL centrifuge tube, centrifuge at 10,000 rpm for 10 min, pipette 800 μL of sample solution, add 200 μL of internal standard, mix well, add 1 mL of dichloromethane, centrifuge at 6,000 rpm for 5 min, and pipette 500 μL of the lower organic phase for determination; internal standard: 1.25 g / L of tert-pentanoic acid (1.25 g solid), 15 mL of concentrated hydrochloric acid, add water to make up to 100 mL;

[0061] (2) GC conditions:

[0062] Chromatographic column: Agilent CP-wax 57 CB;

[0063] Inlet: temperature 220°C, pressure 27 psi;

[0064] Column oven: 60°C, hold for 0.5 min; heat up to 190°C at 20°C per minute, hold for 4.5 min, total running time 12 min;

[0065] Detector: temperature 220°C, air flow 450 ml / min, hydrogen 40 ml / min, N2 total flow 65 ml / min (septum purge 3 ml / min; split flow: 60 ml / min; column flow: 2 ml / min), tail nitrogen: 45 ml / min;

[0066] Split ratio: 30:1;

[0067] 3. Results

[0068] Three parallel groups were set up, and the 0-hour culture solution was taken as the blank control. The blank control was removed from the final data, and the average value was taken. The results are summarized in Table 7 below;

[0069] Table 7: Comparison of organic acid production by strain JSY12851 using different carbon sources

[0070]

[0071] The results showed that in the culture medium with hexanoic acid as the main carbon source, the octanoic acid production of strain JSY12851 reached an average of 4.81 g / L, with a maximum of 5.46 g / L; in the culture medium with butyric acid as the main carbon source, the caproic acid production of strain JSY12851 reached an average of 2.53, with a maximum of 3.71 g / L; From the above results, it can be seen that strain JSY12851 can use butyric acid to produce hexanoic acid, and use hexanoic acid to produce octanoic acid.

[0072] 1. Culture Conditions

[0073] (1) Yellow water culture medium: dilute yellow water at a ratio of 50%, adjust the pH to 5.5-6.0 with sodium carbonate or sodium hydroxide, sterilize at 121°C for 30 min, seal and cool with nitrogen;

[0074] (2) JSY12851 and JSY12841 seed solutions: inoculate with 10% JSY12851, 10% JSY12841, and 5% JSY12851 + 5% JSY12841, respectively, to ensure that the inoculation amount of the functional bacterial solution is 10%;

[0075] (3) Anaerobic culture at 37°C for 15 days;

[0076] 2. Detection Method

[0077] (1) Liquid-liquid extraction: Pipette 2 mL of sample solution into a mL centrifuge tube, centrifuge at 10,000 rpm for 10 min, pipette 800 μL of sample solution, add 200 μL of internal standard, mix well, add 1 mL of dichloromethane, centrifuge at 6,000 rpm for 5 min, and pipette 500 μL of the lower organic phase for determination; internal standard: 1.25 g / L of tert-pentanoic acid (1.25 g solid), 15 mL of concentrated hydrochloric acid, add water to make up to 100 mL;

[0078] (2) GC conditions:

[0079] Chromatographic column: Agilent CP-wax 57 CB;

[0080] Inlet: temperature 220°C, pressure 27 psi;

[0081] Column oven: 60°C, hold for 0.5 min; heat up to 190°C at 20°C per minute, hold for 4.5 min, total running time 12 min;

[0082] Detector: temperature 220°C, air flow 450 ml / min, hydrogen 40 ml / min, N2 total flow 65 ml / min (septum purge 3 ml / min; split flow: 60 ml / min; column flow: 2 ml / min), tail nitrogen: 45 ml / min;

[0083] Split ratio: 30:1;

[0084] 3. Results

[0085] Table 8: Organic acid content in yellow water from mixed fermentation of strains JSY12851 and JSY12841

[0086]

[0087] The results showed that the mixed fermentation of caproic acid-producing functional bacteria JSY12841 and strain JSY12851 could effectively increase the octanoic acid content in the fermentation broth; considering the cost of butyric acid and caproic acid, in order to reduce costs and increase efficiency, strain JSY12851 was mixed with butyric acid and caproic acid-producing functional bacteria to ferment caproic acid and octanoic acid.

[0088] [References] are as follows:

[0089] [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:

[0090] [2] State Administration for Market Regulation, National Administration of Standardization. GBT17204-2021: Terminology and classification of alcoholic beverages[S]. 2021:

[0091] [3] Fan Wenlai, Xu Yan. Wine Flavor Chemistry[M]. Beijing: China Light Industry Press, 2014.

[0092] [4] Sun Baoguo, Huang Mingquan. Research progress on flavor chemistry and health effects of liquor[J]. Journal of Chinese Institute of Food Science and Technology, 2021.

[0093] [5] Li Yunzhe, Chen Rui, Jiang Cheng, et al. Study on volatile components of pomegranate wine from different origins based on HS-SPME-GC-MS and rapid gas phase electronic nose technology[J]. 2024.

[0094] [6] Zhou Hailong, Jia Zhilong, Qiao Haijun, et al. Analysis of characteristic flavor compounds in Tibetan-flavor liquor series[J]. 2024.

[0095] [7] Li Zhibin, Jing Li. Analysis of the content and contribution of ethyl octanoate, an important substance in Luzhou-flavor liquor[J]. 2013.

[0096] [8] WANG X, DU H, XU Y. Source tracking of prokaryoticcommunities infermented grain of Chinese strong-flavor liquor [J]. InternationalJournal ofFood Microbiology, 2017, 244: 27-35.

[0097] [9] Zhang Huimin, Xing Xinhui, Wang Yue, et al. Research progress on caproic acid bacteria in the fermentation system of Luzhou-flavor liquor[J]. 2023.

[0098]

[10] MUKHERJEE A, LORDAN C, ROSS RP, et al. Gut microbes from thephylogenetically diverse genus Eubacterium and their various contributions to gut health [J]. Gut Microbes, 2020, 12(1).

[0099]

[11] Chen Qianqian, Wang Jinrong, Peng Wang, et al. Research progress on the biological functions of medium-chain fatty acids and their application in animal production[J]. 2022.

[0100]

[12] Hu Wenxiao, Yao Binbin, Chen Mingguang, et al. Study on the production mechanism and regulation strategy of medium-chain fatty acid ethyl esters in wine[J]. 2023.

[0101]

[13] WALLACE RJ, CHAUDHARY LC, MIYAGAWA E, et al. Metabolicproperties of Eubacterium pyruvativorans, a ruminal 'hyper-ammonia-producing'anaerobe with metabolic properties analogous to those of Clostridium kluyveri[J]. Microbiology, 2004, 150(9): 2921-30.

[0102] Sequence Listing

[0103] <110> Jiangsu Jinshiyuan Wine Co., Ltd.

[0104] <120> A strain producing octanoic acid and its screening and cultivation method and application

[0105] <130> none

[0106] <160> 1

[0107] <170> JSY12851

[0108] <212> rDNA sequences

[0109] ttcttggtcg ggcctaccat gcagtcgagc 30

[0110] gggaatgaag ttcagatgct tcggttgacg 60

[0111] aacttcatgg agagcggcgg acgggtgagt 90

[0112] aacgcgtggg caacctgccc cttacaggga 120

[0113] gatagccgag ggaaacttcg agtaatatcc 150

[0114] cataaagcct attgatcaca tgatcgaaag 180

[0115] gccaaagatt ttatcggtaa gggatgggcc 210

[0116] cgcgtccgat tagccagttg gcagggtagc 240

[0117] ggcctaccaa agcgacgatc ggtagccggc 270

[0118] ctgagagggc ggacggccac attggaactg 300

[0119] agacacggtc cagactccta cgggaggcag 330

[0120] cagtggggga tattgcacaa tggaggaaac 360

[0121] tctgatgcag caacgccgcg tgaaggagga 390

[0122] aggccttcgg gtcgtaaact tctgtcctca 420

[0123] gggaagaaca aaatgacggt acctgaggag 450

[0124] gaagccccgg ctaactacgt gccagcagcc 480

[0125] gcggtaacac gtagggggcg agcgttatcc 510

[0126] ggaattactg ggcgtaaaga gtgcgtaggt 540

[0127] ggctaagcaa gcgcggggtt taatttcacg 570

[0128] gcccaaccgt gaaccgccct gcgaactgtt 600

[0129] aagcttgagt acaggagagg aaggcggaat 630

[0130] tcctagtgta gcggtgaaat gcgtagatat 660

[0131] taggaggaac accggtggcg aaggcggcct 690

[0132] tctggactga aactgacact gaggcacgaa 720

[0133] agcgtgggta gcaaacagga ttagataccc 750

[0134] tggtagtcca cgccgtaaac gatgagcact 780

[0135] aggtgtcggg ctcgagagag ttcggtgccg 810

[0136] gagcaaacgc attaagtgct ccgcctgggg 840

[0137] agtacgcacg caagtgtaaa actcaaagga 870

[0138] attgacgggg acccgcacaa gcagcggagc 900

[0139] atgtggttta attcgaagca acgcgaagaa 930

[0140] ccttaccaga gcttgacatc ctcctgaccg 960

[0141] gggaagtaat gttccccttc tcttcggagc 990

[0142] aggagtgaca ggtggtgcat ggttgtcgtc 1020

[0143] agctcgtgtc gtgagatgtt gggtcaagtc 1050

[0144] ccgcaacgag cgcaaccctt gctgtcagtt 1080

[0145] gccatcatta agttgggcac tctgacggga 1110

[0146] ctgccgggga taacccggag gaaggcgggg 1140

[0147] atgacgtcaa atcatcatgc cccttatgtt 1170

[0148] ctgggcgaca cacgtgctac aatggccggc 1200

[0149] acagagggat gcaaaggcgc gagcccaagc 1230

[0150] gaacccctaa agccggtccc agttcggact 1260

[0151] gcaggctgaa acccgcctgc acgaagtcgg 1290

[0152] agttgctagt aatcgcggat cagaatgccg 1320

[0153] cggtgaatgc gttcccgggt cttgtacaca 1350

[0154] ccgcccgtca caccatggaa gccgggggtg 1380

[0155] cccgaagtcg gacaaagtct cctaagatct 1410

[0156] atgggg 1416

Claims

1. A strain producing octanoic acid, characterized in that: The bacterium is Eubacterium sp. strain JSY12851, which is deposited in Guangdong Microbiological Culture Collection Center with the deposit number GDMCC No: 64552.

2. The octanoic acid-producing strain according to claim 1, characterized in that: The 16S rDNA nucleotide sequence of the bacterial species is shown in SEQ ID NO:

1.

3. A strain of octanoic acid producing bacteria according to claim 1, characterized in that: The colony morphology of the Eubacterium strain JSY12851 is as follows: the colonies are white, round, dry and opaque.

4. A strain of octanoic acid producing bacteria according to claim 1, characterized in that: The bacterial morphology of the Eubacterium strain JSY12851 is as follows: the bacterial body is short rod-shaped, 0.3-0.5 μm×1.0-1.5 μm, single, Gram-positive, and no spores are found.

5. The octanoic acid-producing strain according to claim 1, characterized in that: The growth characteristics of the Eubacterium strain JSY12851 are as follows: 22-47°C, with 32°C being the best; pH 4.5-10, with 6.5 being the best; and being able to tolerate 0.5%-2% NaCl, with the best growth being at 1%.

6. A strain of octanoic acid producing bacteria according to claim 5, characterized in that: The growth characteristics of the Eubacterium strain JSY12851 are as follows: the best growth temperature is 32°C; the best growth temperature is pH 6.5; and the best growth temperature is 1% NaCl.

7. The use of a strain producing octanoic acid according to claim 1, characterized in that: The strain can produce caproic acid and octanoic acid simultaneously and is applied in the field of liquor brewing.

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