CN110093285A - A strain of acid-resistant fermented Lactobacillus and its application - Google Patents

Abstract

The invention discloses an acid-resistant fermentation lactobacillus and its application, belonging to the technical field of fermentation engineering. The strain was deposited in the China General Microorganism Culture Collection Center (CGMCC) on September 5, 2016, with a preservation number of 12934. The strain has good tolerance to acid and grows well under the condition of pH 3.85-4.40. After liquid fermentation culture, the strain can metabolize carbohydrates to produce lactic acid, and the total amount of lactic acid reaches 27.37g/L; Adding the strain in the sealing stage of vinegar making can increase the content of lactic acid in vinegar and improve the taste and flavor of vinegar.

Description

A strain of acid-resistant fermented Lactobacillus and its application

technical field

The invention belongs to the technical field of fermentation engineering, and in particular relates to a strain of acid-resistant fermenting Lactobacillus and a method for using the strain to convert carbohydrates into lactic acid.

Background technique

Lactic acid is an important taste substance in many traditional fermented foods, such as vinegar, white wine, pickles, etc. For vinegar, lactic acid is the non-volatile acid with the highest content, and some vinegars also use the non-volatile acid content as an important indicator for classifying vinegar. Therefore, increasing the content of lactic acid plays an important role in softening the sour taste of vinegar, reducing the irritation of acetic acid and improving the grade of vinegar. At the same time, it is also one of the key substances to adjust the pH of vinegar and extend its shelf life.

Lactic acid in vinegar is mainly metabolized by lactic acid bacteria in the acetic acid fermentation process, among which Lactobacillus is the main species, including L. helveticus, L. hamsteri, and Lactobacillus bridge (L.pontis) and Lactobacillus bread (L.panis), etc. In the early stage of acetic acid fermentation, lactic acid bacteria dominated the vinegar-making microbial community, but due to the accumulation of metabolites such as lactic acid and acetic acid in the system, the total acid content in the vinegar grains increased, and most lactic acid bacteria gradually died out due to their intolerance to high acidity. Acetic acid bacteria gradually became the dominant flora in the system. Due to the lack of carbon source in the later stage of fermentation, lactic acid will be consumed by some microorganisms, which is one of the main reasons for the reduction of the main non-volatile lactic acid content in vinegar. Therefore, screening for lactic acid bacteria strains that can ferment sugar to produce lactic acid and have good acid resistance (especially acetic acid) has important practical significance for increasing the lactic acid content of vinegar in production. At present, there is no report of using the acid-resistant fermentative Lactobacillus described in the present invention to enhance the production of lactic acid in the production of vinegar.

Contents of the invention

In view of the above-mentioned technical problems in production, the present invention aims to improve the taste of vinegar by screening an acid-resistant lactic acid bacterium and using it in the vinegar brewing process to increase the proportion of non-volatile acid in the total acid in vinegar , flavor and quality.

The present invention first provides a strain of acid-resistant fermenting Lactobacillus (Lactobacillus fermentum), which was preserved on September 5, 2016 at the Chinese Academy of Sciences Institute of Microbiology, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing. General Microbiology Center of the Preservation Management Committee, the deposit number is CGMCC NO.12934, and the classification is named Lactobacillus fermentum (Lactobacillus fermentum) LFE02.

Obtain the 16S rRNA gene sequence of the bacterial strain, and carry out BLAST comparison in GenBank to determine its species. The full-length 16S rRNA gene sequence of this bacterial strain is 1541bp, and the bacterial strain with the highest homology is Lactobacillusfermentum (GenBank accession number: KF030758. 1), the 16S rRNA gene sequence is shown in SEQ ID NO: 1.

The main carbohydrates available to the strain include D-glucose, D-fructose, L-trehalose, D-galactose, D-mannose, L-rhamnose and sucrose.

The bacterial strain can metabolize carbohydrates to produce lactic acid through static culture under anaerobic conditions.

The process for producing lactic acid by fermentation is:

Seed/liquid fermentation medium: ①glucose 40g/L, tryptone 20g/L, beef extract 20g/L, yeast powder 10g/L, sodium acetate 10g/L, diammonium hydrogen citrate 4g/L, dipotassium hydrogen phosphate 4g/L, magnesium sulfate heptahydrate 1.16g/L, manganese sulfate monohydrate 0.38g/L, Tween 80 2mL/L, pH 3.85-4.40, sterilized at 121°C for 20min; ② L-cysteine hydrochloride 1g/L, sterilized at 121°C for 20min. ① and ② are mixed in equal volume under sterile conditions;

Seed culture method: 50mL Erlenmeyer bottle with 10mL liquid volume, anaerobic static culture at 36°C for 4 days;

Fermentation culture method: Inoculate the seed liquid into the fermentation medium with 1-8% inoculum amount, fill a 250-mL Erlenmeyer flask with a liquid volume of 100 mL, and cultivate statically at 35-37°C under anaerobic conditions.

The present invention provides an application of the bacterial strain. By adopting the above-mentioned fermentation process, the content of lactic acid produced by the bacterial strain can reach 9.04±0.11g/L.

In one embodiment of the present invention, increasing the glucose content in the culture medium can increase the lactic acid production of the strain to 27.37±0.13g/L.

In one embodiment of the present invention, the glucose content is 50g/L.

The present invention also provides an application of the strain, adding the Lactobacillus fermentum in the fermented grain stage of solid-state vinegar brewing, and at the same time adding 2-5% sucrose of the mass of the fermented grains of vinegar, which can increase the content of lactic acid in the vinegar The ratio of vinegar and non-volatile acid makes the taste of vinegar softer.

The present invention also provides an application of the strain. In the brewing process of liquid white rice vinegar, after the fermentation of acetic acid bacteria, sucrose with a mass of 2-5% of the vinegar solution is added, and the Lactobacillus fermentum is added to improve the production of white rice vinegar. Medium lactic acid content improves the taste and flavor of white rice vinegar.

The present invention obtains a lactic acid producing bacterium strain with better acid tolerance from the vinegar fermented grains, which is identified as Lactobacillus fermentum. The strain can grow well under the condition of pH 3.85-4.40, and can metabolize carbohydrates to produce lactic acid, and the lactic acid production can reach 27.37±0.13g/L. Adding the strain in the sealing stage of vinegar brewing can increase the production of lactic acid in vinegar and improve the taste and flavor of vinegar.

Detailed ways

Example 1: Screening of lactic acid-producing and acid-resistant bacterial strains

Take 10g sample from Zhenjiang balsamic vinegar unstrained spirits and put it into a 500mL Erlenmeyer flask mixed with glass beads and 90mL sterile water, shake it at 115rpm on a shaker at 36°C for 30min, then take 1mL of the bacterial suspension and add 10 times of sterile saline Gradient dilution, with 10 ^-3 to 10 ^-6 gradient dilutions, spread the separation medium plate, anaerobic static culture at 36°C for 7 to 14 days; after separation and purification, the abundant colonies on the plate are inserted into the slant medium, 36 Cultivate for 7 days at ℃ and store at 4℃; at the same time, put the colonies into the liquid medium, and after culturing at 36℃ for 7 days, use high-performance liquid chromatography (HPLC) to measure the content of lactic acid in the fermentation broth, so as to screen out the acid-resistant lactic acid-producing strains .

Separation/slope medium: ①glucose 40g/L, tryptone 20g/L, beef extract 20g/L, yeast powder 10g/L, sodium acetate 10g/L, diammonium hydrogen citrate 4g/L, dipotassium hydrogen phosphate 4g /L, magnesium sulfate heptahydrate 1.16g/L, manganese sulfate monohydrate 0.38g/L, Tween 80 2mL/L, agar powder 40g/L, pH 3.6～6.6, sterilized at 121℃ for 20min; ②L-cysteine Amino acid hydrochloride 1g/L, sterilized at 121°C for 20min. ① and ② were mixed in equal volumes under sterile conditions to prepare separation/slant medium.

Liquid medium: ①glucose 40g/L, tryptone 20g/L, beef extract 20g/L, yeast powder 10g/L, sodium acetate 10g/L, diammonium hydrogen citrate 4g/L, dipotassium hydrogen phosphate 4g/L , magnesium sulfate heptahydrate 1.16g/L, manganese sulfate monohydrate 0.38 g/L, Tween 80 2mL/L, pH 3.6-6.6, sterilized at 121°C for 20 minutes; ② L-cysteine hydrochloride 1g/L , sterilized at 121°C for 20 minutes. ① and ② were mixed in equal volumes under sterile conditions to prepare a liquid culture medium.

The specific method of HPLC determination is as follows:

① Sample pretreatment method

Accurately measure 2 mL of the fermentation supernatant, centrifuge the culture solution (12000 rpm, 5 min) to obtain the supernatant, add 800 μL of zinc sulfate (300 g/L) and 800 μL of potassium ferrocyanide (106 g/L) respectively, shake and mix, centrifugal. The supernatant was filtered through a 0.22 μm aqueous microporous membrane to be the sample to be tested. At the same time, a standard curve of lactic acid was drawn using the lactic acid standard as the external standard.

②HPLC determination conditions are shown in Table 1.

Table 1 Conditions for determination of lactic acid by HPLC

According to the results of HPLC, an acid-resistant (pH 3.85) and lactic acid-producing strain was obtained. The lactic acid production was 9.04±0.11 g/L. It was stored in slant medium at 4°C for further analysis.

Example 2: Molecular identification of lactic acid producing and acid resistant strains

Purify and screen the strains obtained, take the fresh bacterial liquid in the exponential growth phase, collect the bacterial cells by centrifugation, and extract the genomic DNA using the bacterial genome extraction kit. The full-length sequence of the 16S rRNA gene was amplified using bacterial universal primers P ₀ /P ₆ , as follows:

P ₀ :5'-GAG AGT TTG ATC CTG GCT CAG-3'

P ₆ :5'-CTA CGG CTA CCT TGT TAC GA-3'

①Reaction system (50μL)

②Reaction program

The PCR products were separated and checked by 1.0% agarose gel electrophoresis, the voltage was about 11V/cm, and the electrophoresis time was 20min.

Purification of PCR amplified products was carried out according to the instructions of Shanghai Sangon Biotechnology Company's Small Volume Gel Recovery PCR Product Purification Kit, and the sequencing was completed by Shanghai Sangon Biotechnology Company.

The obtained bacterial 16S rRNA gene sequence was compared with GenBank to determine its species. The full-length 16S rRNA gene sequence of this strain is 1541bp, and the strain with the highest homology is Lactobacillusfermentum (GenBank accession number: KF030758.1) , the 16S rRNA gene sequence is shown in SEQ ID NO: 1.

The strain was deposited in the China General Microorganism Culture Collection Center (CGMCC) on September 5, 2016, with a preservation number of 12934.

Example 3: Physiological and biochemical properties of bacterial strain CGMCC 12934

CGMCC 12934 was inoculated in the isolation medium, and after anaerobic culture at 36°C for 4 days, the cell morphology of the strain was observed by transmission electron microscope (Transmission Electron Microscope, TEM) and scanning electron microscope (Scanning Electron Microscope, SEM). CGMCC 12934 was inoculated in the liquid medium, and the temperature suitable for the growth of the strain and its tolerance to acetic acid were detected by measuring the OD ₆₀₀ of the culture medium; the available carbon source of the strain was analyzed by BIOLOG (AN microplate) test. The above characteristics of strain CGMCC 12934 are shown in Table 2.

Table 2 Physiological and biochemical characteristics of strain CGMCC 12934

Example 4: Lactic acid production experiment of bacterial strain CGMCC 12934 under different sugar concentrations

The strain CGMCC 12934 was inoculated in 10 mL of seed medium with 1% inoculum amount, and after static culture at 36°C for 4 days under anaerobic conditions, it was transferred to liquid fermentation medium with 4% inoculum amount, anaerobic conditions After static culture for 7 days, the lactic acid content in the fermentation broth was determined by HPLC.

Seed medium: ①glucose 40g/L, tryptone 20g/L, beef extract 20g/L, yeast powder 10g/L, sodium acetate 10g/L, diammonium hydrogen citrate 4g/L, dipotassium hydrogen phosphate 4g/L , magnesium sulfate heptahydrate 1.16g/L, manganese sulfate monohydrate 0.38 g/L, Tween 80 2mL/L, pH 3.85, sterilized at 121°C for 20min; ② L-cysteine hydrochloride 1g/L, 121 Sterilize at ℃ for 20min. ① and ② are mixed in equal volume under sterile conditions;

Liquid fermentation medium: ①glucose 40g/L, tryptone 20g/L, beef extract 20g/L, yeast powder 10g/L, sodium acetate 10g/L, diammonium hydrogen citrate 4g/L, dipotassium hydrogen phosphate 4g/L L, magnesium sulfate heptahydrate 1.16g/L, manganese sulfate monohydrate 0.38g/L, Tween 80 2mL/L, pH 3.85, sterilized at 121°C for 20min; ② L-cysteine hydrochloride 1g/L, Sterilize at 121°C for 20 minutes. ① and ② are mixed in equal volume under sterile conditions. Wherein, different glucose contents were set, and the final concentrations were 20, 30, 40 and 50 g/L respectively.

According to the HPLC detection method described in the above-mentioned Example 1, the production of lactic acid in the fermentation broth of the strain was measured at different sugar concentrations, and the results showed that as the sugar concentration increased, the production of lactic acid increased, which were 9.04±0.11g/L, 13.41 ±0.09g/L, 19.35±0.06g/L and 27.37±0.13g/L.

Embodiment 5: Adding CGMCC 12934 improves solid vinegar lactic acid content

In order to prevent lactic acid from being utilized by acetic acid bacteria, we chose to add CGMCC 12934 to the fermented grains after the acetic acid fermentation stage of Zhenjiang balsamic vinegar was completed.

Control group: Zhenjiang aromatic vinegar produced by fermentation according to the traditional vinegar-making process.

Treatment group: CGMCC 12934 was cultured to the logarithmic phase, and the bacterial concentration reached about 3×10 ⁸ cfu/mL. Before sealing the fermented grains, inoculate according to the inoculum amount of 5% of the mass of the vinegar fermented grains, add sucrose of 3% of the mass of the vinegar fermented grains at the same time, and then seal the fermented grains for aging.

The content of lactic acid in Zhenjiang fragrant vinegar was determined by high performance liquid chromatography, and the specific method was the same as in Example 1. The analysis results showed that compared with the control group, the content of lactic acid in the processed vinegar increased to 1.76g/100mL, an increase of 27.5%; the proportion of non-volatile acid in the total acid increased from 26.2% to 29.7%, an increase of 13.0% , making the mouthfeel of vinegar softer and improving the overall quality.

Table 3 Physicochemical characteristics of solid vinegar before and after adding strain CGMCC 12934

SEQUENCE LISTING

<110> Jiangnan University

<120> A strain of acid-resistant fermented Lactobacillus and its application

<130> 2019.02.26

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<170> PatentIn version 3.3

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<211> 1541

<212>DNA

<213> Lactobacillus fermentum

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gagagtttga tcctggctca ggatgaacgc cggcggtgtg cctaatacat gcaagtcgaa 60

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gcggacgggt gagtaacacg taggtaacct gcccagaagc gggggacaac atttggaaac 180

agatgctaat accgcataac aacgttgttc gcatgaacaa cgcttaaaag atggcttctc 240

gctatcactt ctggatggac ctgcggtgca ttagcttgtt ggtggggtaa tggcttacca 300

aggcgatgat gcatagccga gttgagagac tgatcggcca caatgggact gagacacggc 360

ccatactcct acgggaggca gcagtaggga atcttccaca atgggcgcaa gcctgatgga 420

gcaacaccgc gtgagtgaag aagggtttcg gctcgtaaag ctctgttgtt aaagaagaac 480

acgtatgaga gtaactgttc atacgttgac ggtatttaac cagaaagtca cggctaacta 540

cgtgccagca gccgcggtaa tacgtaggtg gcaagcgtta tccggattta ttgggcgtaa 600

agagagtgca ggcggttttc taagtctgat gtgaaagcct tcggcttaac cggagaagtg 660

catcggaaac tggataactt gagtgcagaa gagggtagtg gaactccatg tgtagcggtg 720

gaatgcgtag atatatggag gaacaccagt ggcgaaggcg gctacctggt ctgcaactga 780

cgctgagact cgaaagcatg ggtagcgaac aggattagat accctggtag tccatgccgt 840

aaacgatgag tgctaggtgt tggagggttt ccgcccttca gtgccggagc taacgcatta 900

agcactccgc ctggggagta cgaccgcagg gttaaactca aaggaattga cgggggcccg 960

cacaagcggt ggagcatgtg gtttaattcg aagctacgcg aagaacctta ccaggtcttg 1020

acatcttgcg ccaaccctag agatagggcg tttccttcgg gaacgcaatg acaggtggtg 1080

catggtcgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1140

cttgttacta gttgccagca ttaagttggg cactctagtg agactgccgg tgacaaaccg 1200

gaggaaggtg gggacgacgt cagatcatca tgccccttat gacctgggct acacacgtgc 1260

tacaatggac ggtacaacga gtcgcgaact cgcgagggca agcaaatctc ttaaaaccgt 1320

tctcagttcg gactgcaggc tgcaactcgc ctgcacgaag tcggaatcgc tagtaatcgc 1380

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Claims (8)

1. one plant of acidproof lactobacillus fermenti, it is characterized in that, which was deposited on September 5th, 2016 positioned at Chaoyang District, Beijing City China Committee for Culture Collection of Microorganisms, Institute of Microorganism, Academia Sinica commonly micro- life of the institute 3 of North Star West Road 1 Object

Center, deposit number are CGMCC NO.12934, and classification naming is lactobacillus fermenti ((Lactobacillus fermentum)

LFE02。

2. bacterial strain as described in claim 1, it is characterized in that, lactobacillus fermenti ((Lactobacillus fermentum) CGMCC NO.12934 can tolerate the acidic environment of pH to 3.85.

3. bacterial strain as described in claim 1, it is characterized in that, lactobacillus fermenti ((Lactobacillus fermentum) CGMCC NO.12934 metabolizable sugar substance generates lactic acid；The glucide includes D-Glucose, D-Fructose, L- seaweed Sugar, D- galactolipin, D-MANNOSE, L- rhamnose and sucrose.

4. bacterial strain as described in claim 1, it is characterized in that, lactobacillus fermenti ((Lactobacillus fermentum) CGMCC NO.12934,16S rRNA gene order has sequence shown in SEQ ID NO:l.

5. bacterial strain as described in claim 1, it is characterized in that, lactobacillus fermenti ((Lactobacillus fermentum) The culture medium of CGMCC NO.12934 are as follows: glucose 20g/L, tryptone 10g/L, beef extract 10g/L, yeast powder 5

G/L, sodium acetate 5g/L, diammonium hydrogen citrate 2g/L, dipotassium hydrogen phosphate 2g/L, bitter salt 0.58g/L, a hydration Manganese sulfate 0.19g/L, Tween 80 1ml/L, L-cysteine hydrochloride 0.5g/L, pH3.85~4.40.

6. bacterial strain as described in claim 1, it is characterized in that, lactobacillus fermenti ((Lactobacillus fermentum) CGMCC NO.12934 is metabolized the cultural method that glucide generates lactic acid are as follows: after medium sterilization, accesses the bacterial strain, anaerobism Under the conditions of, 35~37 DEG C of stationary cultures.

7. bacterial strain as described in claim 1, it is characterized in that, lactobacillus fermenti ((Lactobacillus fermentum) When CGMCC NO.12934 fermentation generates lactic acid, increase the content of glucose in culture medium, lactic acid production significantly improves.

8. a kind of application of lactobacillus fermenti described in claim 1, which is characterized in that in the envelope unstrained spirits rank of solid-state fermented vinegar brewing process Lactobacillus fermenti (Lactobacillus fermentum) the CGMCC NO.12934 is added in section, while adding vinegar fermented grain quality 2 ~5% sucrose improves the content of lactic acid and the accounting of fixed acid in vinegar, enhances the mildness of vinegar tart flavour.