CN112063573B

CN112063573B - Lactic acid bacteria isolation medium formula and culture method

Info

Publication number: CN112063573B
Application number: CN202010877453.8A
Authority: CN
Inventors: 陈良强; 尉洪涛; 吴耀领; 韩培杰; 杨帆; 王和玉; 白逢彦; 王莉
Original assignee: Kweichow Moutai Co Ltd
Current assignee: Kweichow Moutai Co Ltd
Priority date: 2020-08-27
Filing date: 2020-08-27
Publication date: 2023-01-20
Anticipated expiration: 2040-08-27
Also published as: CN112063573A

Abstract

The invention belongs to the technical field of liquor brewing, and particularly relates to a lactobacillus separation culture medium formula and a culture method. The lactic acid bacteria separation culture medium provided by the invention contains absolute ethyl alcohol, D-lactic acid, L-lactic acid, acetic acid, serum, nutrient salts and vitamins, and does not contain citric acid diamine. The culture medium has good effect of separating lactic acid bacteria, and particularly can separate and culture lactic acid bacteria which are difficult to separate in the fermentation process of white spirit. The fermented grains have microbial diversity in the fermentation process of the Maotai-flavor liquor, and the fermented grains contain lactic acid bacteria which account for more than 50% of the total amount of the lactic acid bacteria in the fermentation process and are the main lactic acid bacteria in the fermentation of the Maotai-flavor liquor, however, the existing culture medium cannot culture and separate the lactic acid bacteria. The culture medium of the invention can separate the lactic acid bacteria, and provides viable strains for researching the functional characteristics and the genotype of the strains.

Description

Formula and culture method of lactobacillus isolation medium

Technical Field

The invention belongs to the technical field of white spirit brewing, and particularly relates to a lactobacillus separation culture medium formula and a culture method.

Background

It has been found that there are a large number of microorganisms in nature that do not have a suitable culture method. The difficulty in culturing microorganisms is due to several special reasons that prevent culturing, including temperature, pH, oxygen, nutrients, etc. Furthermore, in the face of unfavorable growth conditions, the microorganisms may enter a "viable but non-dividing state" or "dormant state" in order to survive, i.e. the cells are viable but no longer dividing, and the microorganisms can be revived only when external conditions become favorable or provide suitable growth factors and signals. The production of white spirit is an open solid-state fermentation process, and the microbial community composition of fermented grains is very complex. Through the long-term acclimation of the brewing activity, the microorganisms in the fermented grains can better grow and metabolize in the environment with high acidity and various nutrient substances.

Lactic acid bacteria are a general name of a type of microorganisms capable of fermenting saccharides to produce lactic acid, are commonly used in the fermentation process of white spirit, have important influence on the quality of the white spirit, and have important significance on the research of brewing technology by deeply researching the acid production metabolism mechanism of different types of lactic acid bacteria.

At present, the common lactic acid bacteria separation method is that MRS basic culture medium is directly separated, and the lactic acid bacteria in the solid fermentation process are acclimated for a long time and are more suitable for growing in the fermented grain environment, so that the common MRS separation culture medium has low selectivity and lacks of trace substances required by the growth of the lactic acid bacteria in the fermented grain for the separation of the lactic acid bacteria in the solid fermentation fermented grain. Therefore, the number and the kind of the lactic acid bacteria to be separated are small, and the separation effect is easily interfered by microorganisms such as yeast.

Disclosure of Invention

The present invention provides a culture medium which can culture lactic acid bacteria (hereinafter referred to as objective lactic acid bacteria) having a 16S rRNA gene sequence shown in SEQ ID NO. 1.

Lactic acid bacteria with 16S rRNA gene sequence shown in SEQ ID NO:1 are reported to be present in fermented grains of Maotai-flavor liquor ^[1] And Zhenjiang white vinegar and vinegar residue ^[2] And has 100 percent of identity with the 16S rRNA of the lactobacillus cultured by the culture medium.

The invention also provides a method for separating and culturing the target lactic acid bacteria from fermented grains.

In some embodiments, the present invention provides a medium formulation that enables isolation of the lactic acid bacteria to obtain viable strains.

In some embodiments, the present invention provides a culture medium for lactic acid bacteria comprising absolute ethanol, D-lactic acid, L-lactic acid, acetic acid, serum, nutrient salts, and vitamins, and no diamine citrate.

In some embodiments, the medium contains anhydrous ethanol 3-5% v/v, D-lactic acid 0.2-0.4% v/v, L-lactic acid 0.2-0.4% v/v, acetic acid 0.2-0.4% v/v, serum 2-4% v/v, nutrient salts 0.05-0.15% v/v and vitamins 0.05-0.15% v/v.

In some embodiments, the medium contains 3.5-4.5% v/v anhydrous ethanol, 0.25-0.35% v/v D-lactic acid, 0.25-0.35% v/v L-lactic acid, 0.25-0.35% v/v acetic acid, 2.5-3.5% v/v serum, 0.08-0.1% v/v nutrient salts, and 0.08-0.1% vitamins.

In some embodiments, the medium contains anhydrous ethanol 3.5-4%v/v, D-lactic acid 0.3-0.32%.

In some embodiments, the medium further comprises KH ₂ PO ₄ 。

In some embodiments, the KH is ₂ PO ₄ The concentration of (b) is 0.1-6 g/L.

In some embodiments, the medium further comprises FeSO ₄ ·7H ₂ O。

In some embodiments, the FeSO ₄ ·7H ₂ The concentration of O is 0.001-0.01 g/L.

In some embodiments, the medium further comprises sodium acetate 7-25g/L.

In some embodiments, the concentration of sodium acetate is 10-25g/L.

In some embodiments, the composition of the nutrient salt comprises CuSO ₄ ·5H ₂ O 5-8g/L、FeSO ₄ ·7H ₂ O 0.5-2g/L、 MnCl ₂ ·4H ₂ O 3-10g/L、ZnSO ₄ ·7H ₂ O 1-2g/L。

In some embodiments, the vitamin component comprises thiamine hydrochloride in an amount of 45 to 65mg/L, riboflavin in an amount of 45 to 65mg/L, nicotinic acid in an amount of 90 to 150mg/L, pyridoxine hydrochloride in an amount of 45 to 65mg/L, inositol in an amount of 45 to 65mg/L, calcium pantothenate in an amount of 45 to 65mg/L, p-aminobenzoic acid in an amount of 45 to 65mg/L, biotin in an amount of 20 to 30mg/L, and cobalamin in an amount of 45 to 65mg/L.

In some embodiments, the culture medium further comprises 8-12g/L of peptone, 3-10g/L of yeast extract, 1-5 g/L of beef extract, 16-24g/L of glucose, 0.4-3g/L of K2HPO4, 0.1-0.22g/L of MgSO4 & 7H2O, 0.006-0.04 g/L of MnSO4 & H2O, 0.54-1.62g/L of Tween 80, 15-30g/L of agarose, and 300-700mL/L of fermented grain leachate.

In some embodiments, the culture medium further comprises peptone 9-11g/L, yeast extract 3-6g/L, beef extract 1-3 g/L, glucose 17-22g/L, K2HPO4 0.4-1g/L, mgSO4 & 7H2O 0.1-0.2g/L, mnSO4 & H2O 0.006-0.03 g/L, tween 80.54-1.62 g/L, agarose 15-25g/L, and fermented grain leachate 300-600mL/L.

In some embodiments, the medium contains Tween 80 in an amount of 0.08 to 0.11% v/v.

In some embodiments, the medium contains Tween 80 in an amount of 0.085 to 0.11% v/v.

In some embodiments, the peptone is tryptone.

In some embodiments, the preparation method of the fermented grain leachate comprises the following steps: adding water into the fermented grains, shaking for 5-15min at the speed of 150-200 r/min, carrying out ultrasonic treatment for 10-25min, centrifuging for 5-15min at the speed of 10000-15000 rpm, and taking supernatant to obtain fermented grain leachate, wherein the ratio relation of the added fermented grains to the water is 1-3.

In some embodiments, the ratio of the added fermented grains to water is 3-5.

In some embodiments, the ratio of the added fermented grains to water is 4-5.

In some embodiments, the preparation method of the fermented grain leachate comprises the following steps: adding 500mL of water into 100-300g of fermented grains, shaking at 180r/min for 10min, performing ultrasonic treatment for 20min, centrifuging at 12000r for 10min, and collecting supernatant to obtain fermented grain leachate.

In some embodiments, 500mL of water is added into 150-250g of fermented grains, and the mixture is uniformly mixed, subjected to ultrasonic treatment and centrifugation, and the supernatant is taken to obtain fermented grain leachate; .

In some embodiments, 500mL of water is added into 200-250g of fermented grains, and the mixture is uniformly mixed, subjected to ultrasonic treatment and centrifugation, and the supernatant is taken to obtain the fermented grain leachate.

The culture medium of the lactic acid bacteria in the prior art is difficult to culture the lactic acid bacteria with the 16S rRNA gene sequence shown in SEQ ID NO 1.

However, the inventor finds that the culture medium formula of the invention can culture and isolate lactic acid bacteria with 16S rRNA gene sequence shown in SEQ ID NO. 1 besides general lactic acid bacteria. The culture medium has good practical significance because the lactobacillus accounts for more than 50% of the total amount of the lactobacillus in the fermentation process and is the main lactobacillus in the fermentation of the Maotai-flavor liquor.

In some embodiments, the present invention provides a method of preparing the culture medium, comprising the steps of:

(1) Weighing tryptone, yeast extract, beef extract, glucose and KH ₂ PO ₄ Sterilizing K2HPO4, feSO4.7H2O, mgSO4.7H2O, mnSO4.H2O, tween-80, sodium acetate, agarose, vitamins, nutrient salt, fermented grain leachate and water at 121 ℃ for 30 min; (2) And (2) continuously adding absolute ethyl alcohol, D-lactic acid, L-lactic acid, acetic acid and serum into the step (1) to obtain the compound.

In some embodiments, the present invention provides the use of a culture medium as described above for isolating or screening lactic acid bacteria.

In some embodiments, the lactic acid bacteria are dominant lactic acid bacteria in the fermentation process of the Maotai-flavor liquor.

In some embodiments, the 16S rRNA gene sequence of the lactic acid bacterium has at least 90% or at least 91% or at least 92% or at least 93% or at least 94% or at least 95% or at least 96% or at least 97% or at least 98% or at least 99% or at least 99.5% or at least 99.8% or at least 99.9%, or 100% sequence identity to the sequence set forth as SEQ ID No. 1.

In some embodiments, the 16S rRNA gene sequence of the lactic acid bacterium is set forth in SEQ ID NO 1.

Drawings

FIG. 1 shows lactic acid bacteria isolated by the culture medium and culture method of the present invention.

Detailed Description

The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others based on the teachings of the present invention are within the scope of the invention.

In the examples herein, the following ingredients were prepared as follows:

the preparation method of the leachate of the fermented grains comprises the following steps: adding a certain amount of deionized water into 200g of fermented grains, shaking for 10min at 180r/min, performing ultrasonic treatment for 20min, centrifuging for 10min at 12000rpm, collecting supernatant to obtain leaching solution, and diluting to 500 mL.

Yeast extract (B): biometrics (Shanghai) Ltd.

Beef extract: qingdao Haibo Biotechnology, inc.

Tween 80: chemical agents of the national drug group, ltd.

Agarose: national chemical group chemical agents, ltd.

Serum: gibco.

Nutrient salt: cuSO4 & 5H2O 0.64g, feSO4 & 7H2O 0.11g, mnCl2 & 4H2O 0.79g, znSO4 & 7H2O 0.15g, and distilled water was added to the solution to a constant volume of 100mL for use.

Vitamins: thiamine hydrochloride 50mg, riboflavin 50mg, nicotinic acid 100mg, pyridoxine hydrochloride 50mg, inositol 50mg, calcium pantothenate 50mg, p-aminobenzoic acid 50mg, biotin 25mg, cobalamin 50mg, and distilled water to a volume of 1L for use.

Test example 1 high throughput sequencing of lactic acid bacteria

According to the existing literature reports, high-throughput sequencing shows that in the fermentation process of Maotai-flavor liquor, the target lactic acid bacteria account for 56.2% of the total number of lactic acid bacteria, are important dominant bacteria in the fermentation of Maotai-flavor liquor, and play an important role in the production of Maotai-flavor liquor ^[1] The preservation number of the target lactobacillus is CGMCC1.16734.

EXAMPLE 1 culture formulation and preparation method thereof

The preparation method of the culture medium comprises the following steps:

10.0g of tryptone, 5.0g of yeast extract, 2.0g of beef extract, 20.0g of glucose, 0.5g of KH2PO4, 0.5g of K2HPO4, 10.0mg of FeSO4 & 7H2O, 200.0mg of MgSO4 & 7H2O, 7.5mg of MnSO4 & H2O, 1.0mL of Tween 80, 20.0g of sodium acetate, 20.0g of agarose, 0.5mL of nutrient salt, 0.5mL of vitamin, 400 mL of wine mash leachate, supplementing deionized water to 1L,121 ℃ and sterilizing for 30 min. After sterilization, 30mL of absolute ethanol, 2mL of D-lactic acid, 2mL of L-lactic acid and 2mL of acetic acid are added, and 20mL of filter sterilized import serum is added. 15-20mL of the above solution was aseptically poured onto a plate and condensed for use.

TABLE 1 culture Medium formulation

EXAMPLE 2 culture formulation and preparation method thereof

The preparation method of the culture medium comprises the following steps:

10.0g of tryptone, 5.0g of yeast extract, 2.0g of beef extract, 20.0g of glucose, 0.5g of KH2PO4, 0.5g of K2HPO4, 10.0mg of FeSO4 & 7H2O, 200.0mg of MgSO4 & 7H2O, 7.5mg of MnSO4 & H2O, 1.0mL of Tween 80, 20.0g of sodium acetate, 20.0g of agarose, 1mL of nutrient salt, 1mL of vitamin, 500mL of wine mash leachate, 1L of deionized water supplement, 121 ℃, and 30min for sterilization. After sterilization, 35mL of absolute ethyl alcohol, 3mL of D-lactic acid, 3mL of L-lactic acid and 3mL of acetic acid are added, and 25mL of filter sterilized import serum is added. 15-20mL of the above solution was aseptically poured onto a plate and condensed for use.

TABLE 2 media formulations

EXAMPLE 3 culture formulation and preparation method thereof

The preparation method of the culture medium comprises the following steps:

10.0g of tryptone, 5.0g of yeast extract, 2.0g of beef extract, 20.0g of glucose, 0.5g of KH2PO4, 0.5g of K2HPO4, 10.0mg of FeSO4 & 7H2O, 200.0mg of MgSO4 & 7H2O, 7.5mg of MnSO4 & H2O, 1.0mL of Tween 80, 20.0g of sodium acetate, 20.0g of agarose, 1.5mL of nutrient salt, 1.5mL of vitamin, 600mL of wine mash leachate, 1L of deionized water, and 30min for sterilization. After sterilization, 40mL of absolute ethyl alcohol, 4mL of D-lactic acid, 4mL of L-lactic acid and 4mL of acetic acid are added, and 30mL of filter sterilized import serum is added. 15-20mL of the above solution was aseptically poured onto a plate and condensed for use.

TABLE 3 culture Medium formulation

Comparative example 1

TABLE 4

The preparation method of the Daqu-fermented grain leaching liquor comprises the following steps:

(1) Weighing and mixing fermented grains and Daqu according to the ratio and content of Daqu-fermented grains shown in Table 1;

(2) Adding pure water into the mixture of the Daqu and the fermented grains and boiling for 10-20 min;

(3) After cooling, filtering by adopting 4 layers of gauze to obtain filtrate for later use;

(4) Freezing the filtrate at-20 deg.C overnight, thawing, centrifuging at 12000rpm at 4 deg.C for 10min, and collecting supernatant.

The preparation method of the lactobacillus isolation medium comprises the following steps:

(1) Weighing each component according to the formula of table 1, adding peptone, beef extract, yeast powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate, manganese sulfate, agar, tween 80 and a Daqu-fermented grain leaching liquor into a container, adding a lactic acid solution, adjusting the pH to 5.5, and sterilizing at 121 ℃ for 15-20min;

(2) Dissolving amphotericin B in dimethyl sulfoxide, shaking up, filtering and sterilizing to prepare a mother solution of 16g/L for later use;

(3) And (3) cooling the sterilized mixture obtained in the step (1) to below 60 ℃, and uniformly mixing the cooled mixture with the amphotericin B mother liquor obtained in the step (2).

Comparative example 2

Comparative example 2 medium 1: the medium originates from the lactic acid bacteria isolation culture medium (1) of example 1 in patent application CN110066735A (paragraph 0019 (5) of the specification of patent application CN 110066735A).

Comparative example 2 medium 2: the medium originates from the lactic acid bacteria isolation culture medium (2) of example 1 in patent application CN110066735A (paragraph 0019 (6) of the specification of patent application CN 110066735A).

Comparative example 2 medium 3: the culture medium is derived from reference [2 ]]Page 2 right column materials and methods the medium used for the isolation of the lactic acid bacterial strains. Among them, reference [2]Medium lactobacillus HSLZ-75 ^T The 16S rRNA gene sequence of (a) is the same as the lactic acid bacterium sequence of interest herein.

Comparative example 3

TABLE 5 MRS Medium formulation

Comparative example 4

TABLE 6 LBS Medium formulation

Components	Content (c) of
		Casein peptone	10g
Yeast extract	5g
		Glucose	20g
Potassium dihydrogen phosphate	6g
		Ferrous sulfate	0.034
Magnesium sulfate	0.075
		Manganese sulfate	0.12g
Sodium acetate	25g
		Agar-agar	15g
Citric acid diamine	2g

Example 4 lactic acid bacteria isolation Effect test

0.1mL of the diluted fermented grains solution in the cellar was applied to the culture medium of example 1-3, the culture medium of comparative example 1-4, the culture medium of comparative example 2 1-3, the MRS culture medium and the LBS culture medium, and aerobic culture was carried out at 37 ℃ for 168-336 hours. All strains in the plate were picked and subjected to molecular characterization by 16S rRNA full-length sequencing, the results are shown in Table 7.

Meanwhile, the microorganisms in the diluted fermented grain solution in the round of cellar are subjected to 16S rRNA high-throughput sequencing (according to the method of the test example 1), and the determination result shows that the lactic acid bacteria with 16S rRNA gene sequences shown as SEQ ID NO:1 are main strains (called target bacteria in the text) and account for 99.8 percent of the total amount of the lactic acid bacteria.

TABLE 7

The results in table 7 show that the target lactobacillus does not grow in MRS, LBS media and other modified media, but can grow on the inventive media, indicating that the inventive media can isolate the target lactobacillus detected by high-throughput sequencing. In example 1, the lactic acid bacteria isolated and cultured in the medium are shown in FIG. 1.

The results show that the culture medium has good separation effect on the lactic acid bacteria.

Wherein SEQ ID NO 1 is as follows:

GTCGCGCGCACTCCCGTAGATGATTTTGATGCTCTGCATCAAATGATTCTACATTCGA GTGAGCGGCGGATGGGTGAGTAACACGTGGGTAACCTGCCCTTGAGTCTGGGATAGCAT CTGGAAACGGATGATAATACCGGATAACAACTGAAACCACATGGTTTCAGTTTGAAAGG CGCGTTTGCGTCGCTCTTGGAGGGACCCGCGGTCCATTAGTTAGTTGGTAAGATAAAGG CCTACCAAGACGATGATGGATAGCCGAACTGAGAGGTTAATCGGCCACATTGGAACTGA GACACGGTCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTTCACAATGGGCGAAA GCCTGATGAAGCAACACCGCGTGAATGAAGAAGGGTTTCGGCTCGTAAAATTCTGTTGC TAGAGAAGAACGTACCAAGTAGGAAATGGCTTGGTAGTGACGGTATCTGGTTAGAAAGT CACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGAT TTATTGGGCGTAAAGCGAGCGCAGGCGGTTGTTTAAGTCCGATGTGAAAGCCTTCGGCT TAACCGAAGAAGTGCATTGGAAACTGAGCGACTTGAGTACAGAAGAGGACAGTAGAAC ACCATGTGTAGCGGTGAAATGCGTAGATATATGGTAGAATACCAGTGGCGAAAGCGGCT GTCTGGTCTGTCACTGACGCTGAGGCTCGAAAGCATGGGTAGCAAACAGGATTAGATAC CCTGGTAGTCCATGCTGTAAACGTTGAGTGCTAGGTGTTAGAGGATTTCCGTCCTTTAGT GCCGAAGTTAACACATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTC AAAGGAATTGACGGGGACCCGCACAAGTGGTGGAGCATGTGGTTTAATTCGATGCTACG CGAAGAACCTTACCAGGCCTTGACATACTTCTGTTAGGCTAAGAGATTAGCTGTTCCCTT CGGGGACAGATCTACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGG TCAAGTCCCGCAACGAGCACAACCCTTGTGACTAGTTGCCAGCATTCAGTTGGGCACTC TAGTCAGACTGCCGGTGATAAACCGGAGGAAGGTGAGGATGACGTCTGATCATCATGCC CCTTATGGCCTGGGCTACACACGTGCTACAATGGACAGTACAATGGGTTGCGAAACCGC GAGGTCAAGCTAATCCCATAAAGCTGTTCTCAGTTCGGACTGCAGTCTGCAACTCGACT GCACGAAGCTGGAATCACTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCCCG GGTCTTGTACATACCGCCCGTCACACCATGAGAGTCCGTAACACCCAAAGATAGTGCGG CAACCTC

[1]WuYL,Hao F,Lv XB,Chen B,Yang YB,Zeng XL,Yang F,Wang HY, Wang L(2020)Diversity of lactic acid bacteria in Moutai-flavor liquor fermentation process.Food Biotechnology.34(3):212–227.

[2]YuYJ,LiX,Zhang JH,Chai LJ,Lu ZM,Xu ZH(2019)Lactobacillus jinshani sp.nov.,isolated from solid-state vinegar culture of Zhenjiang aromatic vinegar.113(1):43-54。

sequence listing

<110> Guizhou Maotai liquor Ltd

<120> lactic acid bacteria isolation medium formula and culture method

<160> 1

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<212> DNA

<213> Homo sapiens

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gtcgcgcgca ctcccgtaga tgattttgat gctctgcatc aaatgattct acattcgagt 60

gagcggcgga tgggtgagta acacgtgggt aacctgccct tgagtctggg atagcatctg 120

gaaacggatg ataataccgg ataacaactg aaaccacatg gtttcagttt gaaaggcgcg 180

tttgcgtcgc tcttggaggg acccgcggtc cattagttag ttggtaagat aaaggcctac 240

caagacgatg atggatagcc gaactgagag gttaatcggc cacattggaa ctgagacacg 300

gtccagactc ctacgggagg cagcagtagg gaatctttca caatgggcga aagcctgatg 360

aagcaacacc gcgtgaatga agaagggttt cggctcgtaa aattctgttg ctagagaaga 420

acgtaccaag taggaaatgg cttggtagtg acggtatctg gttagaaagt cacggctaac 480

tacgtgccag cagccgcggt aatacgtagg tggcaagcgt tatccggatt tattgggcgt 540

aaagcgagcg caggcggttg tttaagtccg atgtgaaagc cttcggctta accgaagaag 600

tgcattggaa actgagcgac ttgagtacag aagaggacag tagaacacca tgtgtagcgg 660

tgaaatgcgt agatatatgg tagaatacca gtggcgaaag cggctgtctg gtctgtcact 720

gacgctgagg ctcgaaagca tgggtagcaa acaggattag ataccctggt agtccatgct 780

gtaaacgttg agtgctaggt gttagaggat ttccgtcctt tagtgccgaa gttaacacat 840

taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacggggac 900

ccgcacaagt ggtggagcat gtggtttaat tcgatgctac gcgaagaacc ttaccaggcc 960

ttgacatact tctgttaggc taagagatta gctgttccct tcggggacag atctacaggt 1020

ggtgcatggt cgtcgtcagc tcgtgtcgtg agatgttggg tcaagtcccg caacgagcac 1080

aacccttgtg actagttgcc agcattcagt tgggcactct agtcagactg ccggtgataa 1140

accggaggaa ggtgaggatg acgtctgatc atcatgcccc ttatggcctg ggctacacac 1200

gtgctacaat ggacagtaca atgggttgcg aaaccgcgag gtcaagctaa tcccataaag 1260

ctgttctcag ttcggactgc agtctgcaac tcgactgcac gaagctggaa tcactagtaa 1320

tcgtggatca gcatgccacg gtgaatacgt tcccgggtct tgtacatacc gcccgtcaca 1380

ccatgagagt ccgtaacacc caaagatagt gcggcaacct c 1421

Claims

1. A lactic acid bacteria culture medium is characterized by comprising the following components: 3-5% v/v of absolute ethyl alcohol, 0.2-0.4% v/v of D-lactic acid, 0.2-0.4% v/v of L-lactic acid, 0.2-0.4% v/v of acetic acid, 2-4% v/v of serum, 0.05-0.15% v/v of nutrient salt, 0.05-0.15% v/v of vitamin and KH ₂ PO ₄ 0.1～6 g/L、FeSO ₄ ·7H ₂ 0.001 to 0.01g/L of O, 10 to 25g/L of sodium acetate, 8 to 12g/L of peptone, 3 to 10g/L of yeast extract, 1 to 5g/L of beef extract, 16 to 24g/L of glucose and K ₂ HPO ₄ 0.4-3 g/L、MgSO ₄ ·7H ₂ O 0.1-0.22 g/L、MnSO ₄ ·H ₂ 0.006-0.04 g/L of O, 0.54-1.62g/L of Tween 80, 15-30g/L of agarose and 300-700mL/L of fermented grain leachate, wherein the lactobacillus is dominant lactobacillus in the fermentation process of Maotai-flavor liquor, and the nutrient salt is CuSO ₄ ·5H ₂ O 5-8 g/L、FeSO ₄ ·7H ₂ O 0.5-2 g/L、MnCl ₂ ·4H ₂ O 3-10 g/L、ZnSO ₄ ·7H ₂ O 1-2 g/L。

2. The culture medium according to claim 1, wherein the culture medium comprises 3.5-4.5% v/v of anhydrous ethanol, 0.25-0.35% v/v of D-lactic acid, 0.25-0.35% v/v of L-lactic acid, 0.25-0.35% v/v of acetic acid, 2.5-3.5% v/v of serum, 0.08-0.1% v/v of nutritive salt, and 0.08-0.1% v/v of vitamin.

3. The culture medium according to claim 1, wherein the culture medium comprises 3.5-4% v/v of anhydrous ethanol, 0.3-0.32% v/v of D-lactic acid, 0.3-0.32% v/v of L-lactic acid, 0.3-0.32% v/v of acetic acid, 3-3.2% v/v of serum, 0.08-0.1% v/v of nutritive salt, and 0.08-0.1% v/v of vitamin.

4. The culture medium according to claim 1, wherein the vitamin composition comprises thiamine hydrochloride 45-65mg/L, riboflavin 45-65mg/L, nicotinic acid 90-150mg/L, pyridoxine hydrochloride 45-65mg/L, inositol 45-65mg/L, calcium pantothenate 45-65mg/L, p-aminobenzoic acid 45-65mg/L, biotin 20-30mg/L, and cobalamin 45-65mg/L.

5. The culture medium of claim 1, wherein the culture medium comprises peptone 9-11g/L, yeast extract 3-6g/L, beef extract 1-3 g/L, glucose 17-22g/L, K ₂ HPO ₄ 0.4-1 g/L、MgSO ₄ ·7H ₂ O 0.1-0.2 g/L、MnSO ₄ ·H ₂ 0.006-0.03 g/L of O, 0.54-1.62g/L of Tween 80, 15-25g/L of agarose, and 300-600mL/L of fermented grain leachate.

6. The culture medium of claim 1, wherein the peptone is tryptone.

7. The culture medium according to claim 1, wherein the preparation method of the fermented grain leachate comprises the following steps:

adding water into the fermented grains, shaking for 5-15min at the speed of 150-200 r/min, carrying out ultrasonic treatment for 10-25min, centrifuging for 5-15min at the speed of 10000-15000 rpm, and taking supernatant to obtain fermented grain leachate, wherein the ratio of the added fermented grains to the water is 1-3.

8. The culture medium according to claim 7, wherein the ratio of the added fermented grains to water is 3-5 g/mL.

9. The culture medium according to claim 7, wherein the ratio of the added fermented grains to water is 4-5 g/mL.

10. A method of preparing a culture medium according to any one of claims 1 to 9, comprising the steps of:

(1) Weighing tryptone and yeast, extractingMaterial, beef extract, glucose, KH ₂ PO ₄ 、K ₂ HPO ₄ 、FeSO ₄ ·7H ₂ O、MgSO ₄ ·7H ₂ O、MnSO ₄ ·H ₂ O, tween-80, sodium acetate, agarose, vitamins, nutrient salt, fermented grain leachate and water, sterilizing at 121 ℃ for 30 min;

(2) And (2) continuously adding absolute ethyl alcohol, D-lactic acid, L-lactic acid, acetic acid and serum into the step (1) to obtain the compound.

11. The use of the culture medium of any one of claims 1-9 for the isolation or screening of lactic acid bacteria, wherein the lactic acid bacteria are dominant lactic acid bacteria during the fermentation of Maotai-flavor liquor, and the 16S rRNA gene sequence of the lactic acid bacteria is shown in SEQ ID NO 1.