Lactobacillus brevis and application thereof in promoting production of flavor substances

Abstract

The invention discloses lactobacillus brevis and application thereof in promoting production of flavor substances, and belongs to the technical field of food microorganisms. The lactobacillus brevis CCTCC NO: M 20242019 not only has excellent acid resistance and ethanol resistance, but also has good high temperature resistance; in addition, the lactobacillus brevis CCTCC NO: M 20242019 can also produce various flavor substances in high yield in the fermentation process of the sorghum juice culture medium. When the bacterial strain is prepared into a microbial agent to be applied to the liquor brewing production process, the quality and flavor of liquor can be remarkably improved. The strain disclosed by the invention is good in practical application effect and has a good application prospect in Baijiu brewing production.

Description

Technical Field

[0001] The invention relates to a lactobacillus brevis strain and application thereof in promoting the production of flavor substances, belonging to the technical field of food microorganisms. Background Art

[0002] The brewing process of liquor is a complex microbial activity, which includes the metabolism of multiple microorganisms. The microorganisms in the brewing process play a vital role in the quality and flavor of the liquor. In the process of microbial screening, the adaptability of microorganisms screened in different systems is also different. Therefore, screening beneficial microorganisms in the liquor brewing system to improve the quality of liquor and add flavor has practical application value.

[0003] However, the liquor brewing environment has extremely harsh requirements on microorganisms, including high temperature, high concentration of alcohol, acidic environment and other harsh conditions, which have posed great challenges to the growth and metabolism of microorganisms. Therefore, in the continuous optimization and innovation of liquor fermentation technology, screening a strain that is acid-resistant, ethanol-resistant, high-temperature-resistant and can produce rich flavor substances is of vital importance to improving the overall quality and flavor level of liquor, as well as improving the liquor brewing process and product quality.

[0004] CN109370932A discloses a strain of Lactobacillus brevis isolated from silage that can produce ferulic acid esterase and can be used in the preparation of silage additives. Although it can still grow at a pH of 8.5, it hardly grows in an acidic environment of pH < 3. Similarly, its optimal growth temperature is 37°C and it hardly grows at 50°C. However, since it was screened from silage, and there is a large difference between silage and the liquor brewing environment, it is impossible to determine whether this strain of Lactobacillus brevis can adapt to the liquor brewing environment; CN107418912B discloses a strain of Lactobacillus brevis screened from fermented dough that can improve the fermented flavor of old dough steamed bread, which can produce up to 61 volatile substances, adding flavor to old dough steamed bread. The strain has a malt flavor, but due to the different screening systems, the adaptability of the strain in the brewing environment of liquor is still questionable; CN102660477B discloses a Lactobacillus brevis screened from the gastrointestinal mucosa of healthy piglets, which has the properties of acid resistance, bile salt resistance, high temperature resistance and good antibacterial properties, but due to the different screening systems, the adaptability of the strain in the brewing environment of liquor is still unclear; CN108034599B discloses a Lactobacillus brevis derived from a liquor brewing system, which can efficiently synthesize γ-aminobutyric acid (GABA), adapt to the high acid and high alcohol environments of the liquor fermentation system, and can be used for the preparation of high-purity GABA and feed-grade GABA, but does not mention the ability of the strain to produce flavor substances.

[0005] Therefore, in order to overcome the problems existing in the prior art and improve the overall quality and flavor level of liquor, the present invention is committed to screening a strain that is acid-resistant, ethanol-resistant, high-temperature-resistant and can produce rich flavor substances, providing useful reference and reference for the liquor brewing industry and the food fermentation industry. Summary of the invention

[0006] In order to solve the above problems, the present invention screened a strain of Lactobacillus brevis W2024090802 from the liquor brewing system. The strain has excellent acid resistance, ethanol resistance and high temperature resistance, can grow stably and metabolize efficiently under extreme fermentation environments, and produce a variety of flavor precursors and aroma components, thereby greatly enriching the taste and aroma of liquor, and has broad application prospects in liquor brewing production.

[0007] The first object of the present invention is to provide a strain of Lactobacillus brevis, which has been deposited in the China Center for Type Culture Collection on September 20, 2024, with the deposit address being No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number being CCTCC NO: M 20242019.

[0008] The Lactobacillus brevis W2024090802 has the following characteristics:

[0009] (1) Growth characteristics: The strain reached the stable phase after 24 hours of growth in sorghum juice medium; the optimal growth temperature in sorghum juice medium was 30°C; the strain had the best activity in sorghum juice medium containing 3% (v / v) ethanol; the strain had the best activity in sorghum juice medium with a pH of 5.55;

[0010] (2) Ethanol resistance: The strain grew best when cultured in sorghum juice medium with 3% (v / v) ethanol content for 36 h. After being cultured in sorghum juice medium with 12% (v / v) ethanol content for 36 h, the strain still maintained its vitality, which was 38.6% of that in 3% (v / v) ethanol content.

[0011] (3) High temperature resistance: It can withstand a high temperature of 50°C. Compared with the growth in sorghum juice medium at the optimum growth temperature of 30°C, the activity of the plant after growing in sorghum juice medium at 50°C for 36 hours was 66.3% of the optimum growth temperature, and it was still able to grow well.

[0012] (4) Acid resistance: The optimal growth pH of Lactobacillus brevis in sorghum juice medium is 5.55. After culturing in sorghum juice medium with a pH of 2 for 36 h, the strain still maintains good vitality, and its vitality is 29.9% of that at pH 5.55;

[0013] (5) Flavor production performance: During the fermentation process of sorghum juice medium, Lactobacillus brevis can produce up to 50 flavor substances, including 13 alcohols, 10 esters, 14 acids, 6 aldehydes, 1 ketone, 4 phenols, 1 furan, and 1 terpene; the volatile compounds with a content of more than 50 mg / L include 4-vinylphenol (473.58 mg / L), 2,3-dihydrobenzofuran (236.93 mg / L), heptanediol (231.67 mg / L), tridecanoic acid (75.98 mg / L), and ethyl butyrate (69.93 mg / L);

[0014] (6) It can be used in liquor brewing to enhance the flavor of liquor.

[0015] The second object of the present invention is to provide a microbial agent containing the Lactobacillus brevis CCTCC NO: M20242019 of the present invention.

[0016] In one embodiment of the present invention, the microbial agent uses Lactobacillus brevis CCTCC NO: M 20242019 as the main microorganism.

[0017] In one embodiment of the present invention, the number of viable bacteria of Lactobacillus brevis CCTCC NO: M 20242019 in the microbial agent is 10 5 ~10 8 CFU / mL.

[0018] In one embodiment of the present invention, the bacterial concentration of Lactobacillus brevis CCTCC NO: M 20242019 in the microbial agent is in the range of 10 5 ~10 8 CFU / mL.

[0019] In one embodiment of the present invention, the microbial agent is obtained by preparing the seed solution of Lactobacillus brevis CCTCC NO: M20242019 and then expanding the culture.

[0020] In one embodiment of the present invention, the microbial agent is a liquid agent or a solid agent containing Lactobacillus brevis CCTCC NO: M20242019.

[0021] In one embodiment of the present invention, the microbial agent contains living cells of Lactobacillus brevis CCTCC NO: M20242019, dry cells of Lactobacillus brevis CCTCC NO: M 20242019 obtained by freeze-drying, immobilized cells of Lactobacillus brevis CCTCC NO: M 20242019 of the present invention, or Lactobacillus brevis CCTCC NO: M 20242019 of the present invention in any other form.

[0022] In one embodiment of the present invention, the microbial agent also contains any strain of any species that can be used for fermenting food, such as Saccharomyces cerevisiae, Bacillus subtilis, Bacillus licheniformis, etc.

[0023] In one embodiment of the present invention, the microbial agent further contains any carrier that can be used for fermenting food.

[0024] The third object of the present invention is to provide the application of the Lactobacillus brevis CCTCC NO: M 20242019 or a bacterial agent containing the Lactobacillus brevis CCTCC NO: M 20242019 in liquor brewing.

[0025] In one embodiment of the present invention, the liquor includes Luzhou-flavor liquor, Qing-flavor liquor, Maotai-flavor liquor, and fruit-flavor liquor.

[0026] The fourth object of the present invention is to provide the use of the Lactobacillus brevis CCTCC NO: M 20242019 or a bacterial agent containing the Lactobacillus brevis CCTCC NO: M 20242019 in the preparation of fermented food.

[0027] In one embodiment of the present invention, the fermented food is an alcoholic beverage, a soy product, a fermented vegetable, a fermented grain product, a fermented meat product, a dairy product or a condiment.

[0028] The fifth object of the present invention is to provide the use of the Lactobacillus brevis CCTCC NO: M 20242019 or a bacterial agent containing the Lactobacillus brevis CCTCC NO: M 20242019 in producing flavor substances in fermented foods.

[0029] In one embodiment of the present invention, the flavor substances include one or more of the following:

[0030] Including alcohols such as cis-3-nonen-1-ol, 2-ethylhexanol, 4-methyl-2-pentanol, 3-methyl-1-butanol, 1-octen-3-ol, heptanediol, isopropanol, 1-decanol, 1-nonanol, 2-octen-1-ol, 2-nonen-1-ol, 1-hexanol, 2-heptanol; esters such as ethyl butyrate, ethyl heptanoate, ethyl hexanoate, ethyl tetradecanoate, ethyl hexadecanoate, ethyl nonanoate, n-hexadecanoic acid, ethyl octanoate, ethyl acetate, ethyl p-hydroxybenzoate; acids such as heptanoic acid, Nonanoic acid, undecanoic acid, octanoic acid, hexanoic acid, dodecanoic acid, decanoic acid, tetradecanoic acid, tridecanoic acid, acetic acid, benzoic acid, 2,4-dimethyl-2-pentenoic acid, 2-methylhexanoic acid, 3-decenoic acid, aldehydes such as heptanal, decanal, octanal, nonanal, 3,4-dimethylbenzaldehyde, hexanal, ketones such as 1-(1H-pyrrol-2-yl)ethanone, phenols such as phenol, vanillin, 4-vinylphenol, 2-methoxy-4-vinylphenol, furans such as 2,3-dihydrobenzofuran, and terpenes such as linalool.

[0031] The sixth object of the present invention is to provide a method for producing liquor.

[0032] In one embodiment of the present invention, the method is to add the Lactobacillus brevis CCTCC NO: M 20242019 or a bacterial agent containing the Lactobacillus brevis CCTCC NO: M 20242019 during the fermentation process of liquor.

[0033] In one embodiment of the present invention, the liquor includes Luzhou-flavor liquor, Qing-flavor liquor, Maotai-flavor liquor, and fruit-flavor liquor.

[0034] Beneficial Effects

[0035] The Lactobacillus brevis W2024090802 of the present invention is obtained by screening from liquor. This strain shows excellent acid resistance, ethanol resistance and high temperature resistance. At the same time, Lactobacillus brevis W2024090802 has a strong ability to produce alcohol, acid and ester flavor substances, which play a key role in enriching the taste and aroma of liquor. Therefore, Lactobacillus brevis W2024090802 is a multifunctional strain that integrates acid resistance, ethanol resistance, high temperature resistance and flavor production ability, which is of great significance for improving the quality and flavor characteristics of liquor.

[0036] Biomaterial Deposit

[0037] Lactobacillus brevis W2024090802, taxonomic name: Lactobacillus brevis, was deposited in the China Center for Type Culture Collection on September 20, 2024, the collection address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the collection number is CCTCCNO: M 20242019. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the growth curve of Lactobacillus brevis W2024090802.

[0039] Figure 2 This is a graph showing the high temperature resistance performance of Lactobacillus brevis W2024090802.

[0040] Figure 3 This is a graph showing the ethanol resistance results of Lactobacillus brevis W2024090802.

[0041] Figure 4 This is a graph showing the acid resistance results of Lactobacillus brevis W2024090802.

[0042] Figure 5 This is a graph showing the yield of volatile compounds produced by fermentation of Lactobacillus brevis W2024090802 in sorghum juice medium. DETAILED DESCRIPTION

[0043] (I) The culture medium involved in the following embodiments:

[0044] MRS broth medium (g / L): 10.0 g of peptone, 8.0 g of beef extract powder, 4.0 g of yeast extract powder, 20.0 g of glucose, 2.0 g of dipotassium hydrogen phosphate, 2.0 g of diammonium hydrogen citrate, 5.0 g of sodium acetate, 0.2 g of magnesium sulfate, 0.04 g of manganese sulfate, and 1.0 g of Tween 80. Dissolve in 1 L of water, adjust the pH to 5.7, and sterilize at 118°C for 18 min.

[0045] MRS solid culture medium: 10.0 g peptone, 8.0 g beef extract powder, 4.0 g yeast extract powder, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween 80, 20 g agar, dissolved in 1 L water, adjusted to pH 5.7, and sterilized at 118 ° C for 18 min.

[0046] Sorghum juice medium: Use japonica sorghum to prepare sorghum juice medium. The specific steps are as follows:

[0047] (1) Crush the raw sorghum into granules for about 10 seconds. After crushing, add water twice the amount of raw sorghum added and soak in a refrigerator at 4°C for 12 hours to obtain raw sorghum soaking liquid.

[0048] (2) Add 30 u / g of α-high temperature amylase (purchased from Jiangsu Ruiyang Biotechnology Co., Ltd., enzyme activity: 40000 U / g) to the raw sorghum soaking liquid obtained in step (1), place in a high temperature sterilizer at 105°C for 45 min, and obtain enzymolysis solution 1.

[0049] (3) Add the same amount of water as in step (1) to the enzymatic hydrolyzate 1 obtained in step (2), add 30 U / g of α-high temperature amylase (purchased from: Jiangsu Ruiyang Biotechnology Co., Ltd., enzyme activity: 40000 U / g), and cook on an induction cooker at 100°C until the sugar content reaches 8°Bx.

[0050] (4) When the temperature is cooled to 60°C, 259 u / g of saccharifying enzyme (purchased from Jiangsu Ruiyang Biotechnology Co., Ltd., enzyme activity: 100,000 U / g) is added, and the mixture is placed in an oven at 60°C for 4 h to obtain enzymatic solution 2.

[0051] (5) Filter the enzymatic hydrolyzate 2 obtained in step (4) using absorbent cotton gauze to obtain the filtrate, and dispense it into centrifuge bottles, with each bottle being filled with 1 / 2 (250 mL).

[0052] (II) Strain growth curve determination method

[0053] (1) Lactobacillus brevis W2024090802 was inoculated into MRS broth medium and cultured at 30°C for 28 h to obtain seed solution. The concentration of the seed solution was 6.41×10 8 CFU / mL.

[0054] (2) 7.80×10 -3 mL of seed solution from step (1) to make the final bacterial concentration of the culture medium 1×10 6 CFU / mL, and incubate at 30°C for 60 h.

[0055] (3) After culturing, samples were taken every 6 hours, 200 μL was added to a 96-well plate, and the absorbance at 600 nm was measured using an ELISA reader to record the growth of the strain. Three replicates were set for each strain at each time point.

[0056] (III) Experiment on high temperature resistance of strains

[0057] (1) Lactobacillus brevis W2024090802 was inoculated into MRS broth medium and cultured at 30°C for 28 h to obtain seed solution. The concentration of the seed solution was 7.26×10 8 CFU / mL.

[0058] (2) 5 mL of sorghum juice medium was used to inoculate 6.89 × 10 -3 mL of seed solution from step (1) to make the final bacterial concentration of the culture medium 1×10 6 CFU / mL, and cultured at 25, 30, 37, 45, and 50 °C for 36 h, with three replicates at each temperature.

[0059] (3) After incubation, 200 μL was taken out and placed in a 96-well plate, and its absorbance at 600 nm was measured using an ELISA reader.

[0060] (IV) Ethanol resistance test of strains

[0061] (1) Lactobacillus brevis W2024090802 was inoculated into MRS broth medium and cultured at 30°C for 28 h to obtain seed solution. The concentration of the seed solution was 4.89×10 8 CFU / mL.

[0062] (2) Before inoculation, ethanol was added to make the ethanol concentration of sorghum juice medium 3, 6, 9, 12 (%, v / v), and three replicates were set for each ethanol concentration.

[0063] (3) 1.02×10 -2 mL of seed solution from step (1) to make the final bacterial concentration of the culture medium 1×10 6 CFU / mL, incubate at 30°C for 36 h, take 200 μL into a 96-well plate, and measure the absorbance at 600 nm using an ELISA reader.

[0064] (V) Strain acid resistance test

[0065] (1) Lactobacillus brevis W2024090802 was inoculated into MRS broth medium and cultured at 30°C for 28 h to obtain seed solution. The concentration of the seed solution was 9.76×10 8 CFU / mL.

[0066] (2) Use lactic acid or hydrochloric acid to adjust the pH of the sorghum juice medium to 5.0, 4.0, 3.0, and 2.0, respectively, and set up three parallel groups for each pH.

[0067] (3) 5.12×10 -3 mL of seed solution from step (1) to make the final bacterial concentration of the culture medium 1×10 6 CFU / mL, incubate at 30°C for 36 h, take 200 μL into a 96-well plate, and measure the absorbance at 600 nm using an ELISA reader.

[0068] (VI) Composition analysis method of volatile compounds produced by bacterial fermentation

[0069] (1) Lactic acid bacteria were inoculated into MRS broth medium and cultured at 30°C for 28 h to obtain seed solution. The concentration of the seed solution was 6.57×10 8 CFU / mL.

[0070] (2) 20 mL of sorghum juice medium was used to inoculate 3.04 × 10 -2 mL of seed solution from step (1) to make the final bacterial concentration of the culture medium 1×10 6 CFU / mL, and incubate them at 30°C for 36 h in triplicate.

[0071] (3) Determination of volatile compounds by GC-MS. The specific operation steps are as follows:

[0072] Take 5 mL of the fermentation broth at the end of the fermentation, centrifuge it, take 5 mL of the supernatant and put it in a brown headspace bottle, and add 1.5 g NaCl and 10 μL menthol internal standard (concentration is 22 mg / L). Use headspace solid phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) to detect the types and contents of volatile compounds in the fermentation broth at the end of the fermentation of the strain.

[0073] GC conditions: injection port temperature 250 °C, carrier gas high purity He, flow rate 1 mL / min. Injection mode is splitless injection. Chromatographic column is DB-FFAP (60m×0.25mm id×0.25μm, Agilent, CA, USA) capillary column. Heating program: start temperature 55 °C for 2 min, increase to 230 °C at 5 °C / min, and hold for 15 min.

[0074] MS conditions: electron impact (EI) ion source, electron energy 70 e V, ion source temperature 230 °C, mass scanning range m / z 35-500 amu.

[0075] Example 1: Screening of strains

[0076] A strain of Lactobacillus brevis that is resistant to high temperature, acid, and ethanol and produces a variety of flavor substances was screened from the liquor fermentation process. The screening process is as follows:

[0077] Accurately weigh 2.0 g of the fermented grains of liquor, add 40 mL of 0.9% sterile saline and 10 glass beads, and culture on a shaker at 30°C and 200 rpm for 1 h. After the culture, take the supernatant of the sample and dilute it to 10 1 times, 10 2 times, 10 3 times, 10 4 times, 10 5Times, take the last three dilution gradients, evenly spread them on the MRS solid culture medium plate for primary screening, and place them in a constant temperature and humidity incubator at 30℃ for 24h. Then observe the colony morphology on the plate, select the colonies that may be lactic acid bacteria, and use the three-zone streak method to spread them on the re-screening solid culture medium plate, and place them in a constant temperature incubator at 30℃ for 24h. Select colonies with obvious lactic acid bacteria characteristics with obvious calcium dissolution rings around, good growth, milky white and moist colony surface, and neat edges, and use the three-zone streak method to spread them on MRS solid culture medium, place them in a constant temperature and humidity incubator at 30℃ for 24h, and repeat the streak until a single bacterium is isolated. Pick a single colony into MRS broth culture medium, place it in a constant temperature and humidity incubator at 30℃ for 24h, use 50% glycerol for strain preservation, and name it W2024090802.

[0078] Example 2: Bacterial species identification

[0079] A single colony of the strain was inoculated into MRS broth medium, and after culturing for 28 hours, the total DNA of the strain was extracted, and the 16S rDNA fragment of the strain was amplified using primers for bacterial classification and identification, and gel electrophoresis was performed. The amplified product was sequenced and the sequence was compared. The strain obtained by the above screening was Lactobacillus brevis, and its 16S rDNA sequence was shown in SEQ ID NO.1.

[0080] This strain was deposited in the China Center for Type Culture Collection on September 20, 2024. The deposit address is No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 20242019.

[0081] SEQ ID NO.1:

[0082] GCGGCATGCTATACATGCAGTCGAACGAGCTTCCGTTGAATGACGTGCTTGCACTGATTT

[0083] CAACAATGAAGCGAGTGGCGAACTGGTGAGTAACACGTGGGGAATCTGCCCAGAAGCA

[0084] GGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACAACAAAATCCGCATGGATTTT

[0085] GTTTGAAAGGTGGCTTCGGCTATCACTTCTGGATGATCCCGCGGCGTATTAGTTAGTTGG

[0086] TGAGGTAAAGGCCCACCAAGACGATGATACGTAGCCGACCTGAGAGGGTAATCGGCCA

[0087] CATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCA

[0088] CAATGGACGAAAGTCTGATGGAGCAATGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGT

[0089] AAAACTCTGTTGTTAAAGAAGAACACCTTTGAGAGTAACTGTTCAAGGGTTGACGGTAT

[0090] TTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAA

[0091] GCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGA

[0092] AAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGGAGACTTGAGTGCAGAAGA

[0093] GGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTG

[0094] GCGAAGGCGGCTGTCTAGTCTGTAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAA

[0095] CAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTT

[0096] TCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCA

[0097] AGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTA

[0098] ATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATCTTCTGCCAATCTTAGAGATA

[0099] GACGTTCCCTTCGGGGACAGATGACAGTGTGCATGTGTCGTCAGCTCGTGTCGTGAGAT

[0100] GTGGGTAGTCCGCACGAGCGCACCTATATCAGTGCAGCATCAGTGGCACTCTGTGAGAC

[0101] TGCGTGACAACGGAGGAAGGTGGGATGACGTCATCATCATGCCTTATGACTGGCTACAC

[0102] ACTGCTCATGACGTTCACGATTCGAGATCTAGGCTAGCATTCTTAAGCGTC

[0103] Example 3: Growth of Lactobacillus brevis W2024090802 in sorghum juice medium

[0104] The growth curve determination method is shown in the specific implementation method (II). Figure 1 It can be seen that after 24h of inoculation of Lactobacillus brevis W2024090802 into sorghum juice medium, the strain entered the stable phase. 600 was 0.639, and the strain entered the decay phase after 60 h of culture (Table 1).

[0105] Table 1: Growth curve OD of Lactobacillus brevis W2024090802 600 Value (n=3)

[0106] Growth time (h) <![CDATA[OD 600 Value]]> 0 0.100 6 0.226 12 0.420 18 0.586 24 0.639 30 0.663 36 0.689 42 0.687 48 0.673 54 0.633 60 0.625

[0107] Example 4: Analysis of the high temperature resistance of Lactobacillus brevis W2024090802

[0108] The experimental method is shown in the above specific implementation method (III). Figure 2 It can be seen that the optimal growth temperature of Lactobacillus brevis W2024090802 is 30℃, at which OD 600 It was 0.704. After being cultured at 50℃ for 36h, it still grew well. 600 It was 0.467, indicating that Lactobacillus brevis W2024090802 has the potential to be used in high temperature fermentation (Table 2).

[0109] Table 2: Growth of Lactobacillus brevis W2024090802 at different temperatures (n=3)

[0110]

[0111]

[0112] Example 5: Analysis of ethanol resistance of Lactobacillus brevis W2024090802

[0113] The experimental method is shown in the specific implementation method (IV). Figure 3 It can be seen that after culturing Lactobacillus brevis W2024090802 in sorghum juice medium with an ethanol concentration of 3% (v / v) for 36 hours, Lactobacillus brevis W2024090802 grew best at this time, OD 600 0.690; after culturing in sorghum juice medium with an ethanol concentration of 12% (v / v) for 36 hours, Lactobacillus brevis W2024090802 still maintained its vitality, OD 600 It was 0.266, indicating that Lactobacillus brevis W2024090802 had good tolerance to ethanol (Table 3).

[0114] Table 3: Growth of Lactobacillus brevis W2024090802 at different ethanol concentrations (n=3)

[0115]

[0116] Example 6: Acid resistance analysis of Lactobacillus brevis W2024090802

[0117] The experimental method is shown in the specific implementation method (V). Figure 4 It can be seen that the optimal growth pH of Lactobacillus brevis W2024090802 is 5.55, which is the pH condition of sorghum juice medium. Lactobacillus brevis grows better under extremely acidic conditions. After culturing in sorghum juice medium with pH = 2 for 36 hours, OD 600 After culturing in sorghum juice medium at pH = 3 for 36 h, OD 600 It is 0.216, indicating that Lactobacillus brevis W2024090802 has the potential to be used in acidic food fermentation (Table 4).

[0118] Table 4: Growth of Lactobacillus brevis W2024090802 at different pH values ​​(n=3)

[0119]

[0120]

[0121] Example 7: Ability of Lactobacillus brevis W2024090802 to produce volatile compounds in sorghum juice medium fermentation

[0122] The experimental method is shown in the specific implementation method (VI), and the test substance is analyzed, such as Figure 5 As shown, after Lactobacillus brevis W2024090802 was cultured in sorghum juice medium for 36 hours, a total of 50 volatile compounds were detected, including: 13 alcohols, 10 esters, 14 acids, 6 aldehydes, 1 ketone, 4 phenols, 1 furan, and 1 terpene; among them, the production of alcohols reached 372.53 mg / L, the production of esters reached 186.40 mg / L, the production of acids reached 113.12 mg / L, the production of aldehydes reached 55.04 mg / L, the production of ketones reached 29.77 mg / L, the production of phenols reached 495.32 mg / L, the production of furans reached 236.93 mg / L, and the production of terpenes reached 5.4 mg / L (Table 5).

[0123] Table 5: Substances and contents of volatile compounds produced in fermentation of Lactobacillus brevis W2024090802 (n=3)

[0124]

[0125]

[0126] 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 Lactobacillus brevis, characterized in that: The Lactobacillus brevis was deposited in the China Center for Type Culture Collection on September 20, 2024, with the deposit number being CCTCC NO: M 20242019.

2. A microbial agent, characterized in that: The microbial agent contains the Lactobacillus brevis CCTCC NO: M 20242019 according to claim 1, or contains the dry bacteria of the Lactobacillus brevis CCTCC NO: M 20242019 obtained by freeze-drying, or contains the cells of the Lactobacillus brevis CCTCC NO: M 20242019 obtained by solidification technology.

3. The microbial agent according to claim 2, characterized in that: The number of viable bacteria of Lactobacillus brevis CCTCCNO:M 20242019 in the microbial agent is 10 5 ~10 8 CFU / mL.

4. The microbial agent according to claim 2, characterized in that: The bacterial concentration range of Lactobacillus brevis CCTCCNO:M 20242019 in the microbial agent is 10 5 ~10 8 CFU / mL.

5. The microbial agent according to claim 2, characterized in that: The microbial agent is a liquid agent or a solid agent containing Lactobacillus brevis CCTCC NO: M 20242019.

6. Use of the Lactobacillus brevis CCTCC NO: M 20242019 according to claim 1 or the microbial agent according to claim 2 in the preparation of fermented food.

7. The use according to claim 6, characterized in that: The fermented food is an alcoholic beverage, a soy product, a fermented vegetable or grain product, a fermented meat product, a dairy product or a condiment.

8. Use of the Lactobacillus brevis CCTCC NO: M 20242019 according to claim 1 or the microbial agent according to claim 2 in producing flavor substances in fermented foods.

9. The use according to claim 8, characterized in that: The flavor substances include one or more of the following: alcohols such as cis-3-nonene-1-ol, 2-ethylhexanol, 4-methyl-2-pentanol, 3-methyl-1-butanol, 1-octen-3-ol, heptanediol, isopropanol, 1-decanol, 1-nonanol, 2-octen-1-ol, 2-nonene-1-ol, 1-hexanol, and 2-heptanol; esters such as ethyl butyrate, ethyl heptanoate, ethyl hexanoate, ethyl tetradecanoate, ethyl hexadecanoate, ethyl nonanoate, hexadecanoic acid, ethyl octanoate, ethyl acetate, and p-hydroxybenzoic acid. Ethyl ester; acids including heptanoic acid, nonanoic acid, undecanoic acid, octanoic acid, hexanoic acid, dodecanoic acid, decanoic acid, tetradecanoic acid, tridecanoic acid, acetic acid, benzoic acid, 2,4-dimethyl-2-pentenoic acid, 2-methylhexanoic acid, 3-decenoic acid; aldehydes including heptaldehyde, decanal, octanal, nonanal, 3,4-dimethylbenzaldehyde, hexanal; ketones including 1-(1H-pyrrol-2-yl)ethanone; phenols including phenol, vanillin, 4-vinylphenol, 2-methoxy-4-vinylphenol; furans including 2,3-dihydrobenzofuran; terpenes including linalool.

10. A method for producing liquor, characterized in that: During the fermentation process of liquor, add the Lactobacillus brevis CCTCC NO: M 20242019 described in claim 1 or the microbial agent described in claim 2.

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