CN110093285B - Acid-resistant lactobacillus fermentum and application thereof - Google Patents
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Abstract
The invention discloses acid-resistant lactobacillus fermentum and application thereof, and belongs to the technical field of fermentation engineering. The strain is preserved in China general microbiological culture collection center (CGMCC) in 2016, 9 and 5, and the preservation number is 12934. The strain has good tolerance to acid, grows well under the condition that the pH value is 3.85-4.40, and can metabolize saccharides to generate lactic acid through liquid fermentation culture, wherein the total amount of the lactic acid reaches 27.37 g/L; the strain is added in the fermented grain sealing stage in the vinegar brewing process, so that the lactic acid content in vinegar can be increased, and the taste and flavor of the vinegar can be improved.
Description
Technical Field
The invention belongs to the technical field of fermentation engineering, and particularly relates to an acid-resistant lactobacillus fermentum and a method for converting saccharides into lactic acid by using the same.
Background
Lactic acid is an important flavor development substance of many traditional fermented foods, such as vinegar, white spirit, pickle and the like. Lactic acid is the most volatile acid in vinegar, and the content of the nonvolatile acid in some vinegar is used as an important index for grading vinegar. Therefore, increasing the content of lactic acid is important for softening the acidity of vinegar, reducing the irritation of acetic acid, increasing the vinegar grade, and the like, and is one of key substances for adjusting the pH value of vinegar and prolonging the shelf life of vinegar.
Lactic acid in vinegar is mainly metabolized by lactic acid bacteria in the acetic acid fermentation process, and the lactic acid bacteria (Lactobacillus) are the main ones, including Lactobacillus helveticus (l.helveticus), Lactobacillus harderi (l.hamsteri), Lactobacillus ponticus (l.pontis), Lactobacillus bakeri (l.panis) and the like. In the early stage of acetic acid fermentation, lactic acid bacteria are dominant in a vinegar brewing microbial community, but due to accumulation of metabolites such as lactic acid and acetic acid in a system, the total acid content in vinegar grains is increased, most of the lactic acid bacteria gradually die due to intolerance of high acid, the acetic acid bacteria gradually become a dominant bacterial community in the system, and due to insufficient carbon source in the later stage of fermentation, lactic acid is consumed by part of microorganisms, which is one of main reasons for reducing the content of main non-volatile acid in vinegar. Therefore, the screening of the lactobacillus strain which can ferment sugar to produce lactic acid and has good acid resistance (especially acetic acid) has important practical significance for improving the lactic acid content of vinegar in production. At present, the method for enhancing the yield of lactic acid in the production of vinegar by using the acid-resistant lactobacillus fermentum is not reported.
Disclosure of Invention
In view of the technical problems in the production, the invention aims to improve the taste, flavor and quality of vinegar by screening an acid-resistant lactic acid bacterium and utilizing the acid-resistant lactic acid bacterium in the vinegar brewing process to increase the proportion of nonvolatile acid in total acid in the vinegar.
The invention firstly provides an acid-resistant Lactobacillus fermentum which is preserved in the common microorganism center of the China Committee for culture Collection of microorganisms, China institute of microbiology, institute of microbiology and sciences, No.1, north Chen, west, No. 3, located in the sunny region of Beijing, in 2016 (9.5.2016), with the preservation number of CGMCC NO.12934, and is named as Lactobacillus fermentum LFE02 in classification.
Obtaining a 16S rRNA gene sequence of the strain, and determining the species of the strain by BLAST comparison in GenBank, wherein the full length of the 16S rRNA gene sequence of the strain is 1541bp, the strain with the highest homology is Lactobacillus fermentum (GenBank accession number: KF030758.1), and the 16S rRNA gene sequence is shown in SEQ ID NO: l.
The main saccharides available to the strain include D-glucose, D-fructose, L-trehalose, D-galactose, D-mannose, L-rhamnose and sucrose.
The strain is subjected to static culture under an anaerobic condition to metabolize carbohydrate to generate lactic acid.
The process for producing lactic acid by fermentation comprises the following steps:
seed/liquid fermentation medium: 40g/L of glucose, 20g/L of tryptone, 20g/L of beef extract, 10g/L of yeast powder, 10g/L of sodium acetate, 4g/L of diammonium hydrogen citrate, 4g/L of dipotassium hydrogen phosphate, 1.16g/L of magnesium sulfate heptahydrate, 0.38g/L of manganese sulfate monohydrate, 802 mL/L of tween, 3.85-4.40 of pH value and 20min of sterilization at 121 ℃; ② 1g/L of L-cysteine hydrochloride, and sterilizing for 20min at 121 ℃. Mixing the first step and the second step in equal volume under the aseptic condition;
the seed culture method comprises the following steps: the liquid loading capacity of a 50mL triangular flask is 10mL, and anaerobic static culture is carried out for 4 days at 36 ℃;
the fermentation culture method comprises the following steps: inoculating the seed solution into a fermentation culture medium with the inoculation amount of 1-8%, filling the seed solution into a 250mL triangular flask with the liquid volume of 100mL, and performing standing culture at 35-37 ℃ under an anaerobic condition.
The invention provides an application of the strain, and the content of lactic acid produced by the strain can reach 9.04 +/-0.11 g/L by adopting the fermentation process.
In one embodiment of the invention, the yield of lactic acid fermented by the strain can be improved to 27.37 +/-0.13 g/L by increasing the content of glucose in a culture medium.
In one embodiment of the invention, the glucose content is 50 g/L.
The invention also provides application of the strain, the lactobacillus fermentum is added in the fermented grain sealing stage of solid vinegar brewing, and cane sugar with the mass of 2-5% of the fermented grain is added at the same time, so that the content of lactic acid and the proportion of non-volatile acid in vinegar can be improved, and the vinegar is softer in taste.
The invention also provides an application of the strain, and in the process of brewing the liquid white rice vinegar, after the fermentation of the acetic acid bacteria is finished, cane sugar accounting for 2-5% of the mass of the vinegar liquid is added, and the lactobacillus fermentum is added, so that the content of lactic acid in the white rice vinegar can be increased, and the taste and the flavor of the white rice vinegar can be improved.
The lactic acid producing strain with better acid resistance is obtained by screening from the vinegar culture and is identified as Lactobacillus fermentum. The strain can grow well under the condition that the pH value is 3.85-4.40, and simultaneously can metabolize saccharides to generate lactic acid, wherein the lactic acid yield can reach 27.37 +/-0.13 g/L. The strain is added in the unstrained spirits sealing stage of the vinegar brewing process, so that the yield of lactic acid in vinegar can be increased, and the taste and flavor of vinegar can be improved.
Detailed Description
Example 1: screening of lactic acid-producing and acid-resistant strains
Taking 10g of a sample from Zhenjiang aromatic vinegar fermented grains, putting the sample into a 500mL triangular flask mixed with glass beads and 90mL sterile water, oscillating the sample on a shaker at 36 ℃ at 115rpm for 30min, taking 1mL of a bacterial suspension, diluting the suspension with 10-fold gradient of sterile physiological saline, and diluting the suspension with 10-fold gradient of sterile physiological saline-3～10-6Coating a separation culture medium plate on the gradient dilution sample, and performing anaerobic static culture at 36 ℃ for 7-14 days; separating and purifying the abundant bacterial colony on the plate, inoculating the bacterial colony into a slant culture medium, culturing for 7 days at 36 ℃, and preserving at 4 ℃; meanwhile, the bacterial colony is inoculated into a liquid culture medium, after being cultured for 7 days at 36 ℃, the content of lactic acid in fermentation liquor is determined by adopting High Performance Liquid Chromatography (HPLC), and thus the acid-resistant lactic acid-producing strain is obtained by screening.
Isolation/slant medium: 40g/L of glucose, 20g/L of tryptone, 20g/L of beef extract, 10g/L of yeast powder, 10g/L of sodium acetate, 4g/L of diammonium hydrogen citrate, 4g/L of dipotassium phosphate, 1.16g/L of magnesium sulfate heptahydrate, 0.38g/L of manganese sulfate monohydrate, 802 mL/L of tween, 40g/L of agar powder, 3.6-6.6 of pH and 20min of sterilization at 121 ℃; ② 1g/L of L-cysteine hydrochloride, and sterilizing for 20min at 121 ℃. First, and second, under aseptic condition, the separation/slant culture medium is prepared after equal volume mixing.
Liquid culture medium: 40g/L of glucose, 20g/L of tryptone, 20g/L of beef extract, 10g/L of yeast powder, 10g/L of sodium acetate, 4g/L of diammonium hydrogen citrate, 4g/L of dipotassium hydrogen phosphate, 1.16g/L of magnesium sulfate heptahydrate, 0.38g/L of manganese sulfate monohydrate, 802 mL/L of tween, 3.6-6.6 of pH and 20min of sterilization at 121 ℃; ② 1g/L of L-cysteine hydrochloride, and sterilizing for 20min at 121 ℃. The first step and the second step are mixed in equal volume under the aseptic condition to prepare a liquid culture medium.
The specific HPLC method is as follows:
(ii) pretreatment method of sample
2mL of fermentation supernatant was measured accurately, the culture broth was centrifuged (12000rpm, 5min) to obtain a supernatant, 800. mu.L of zinc sulfate (300g/L) and 800. mu.L of potassium ferrocyanide (106g/L) were added, and the mixture was shaken, mixed well and centrifuged. And filtering the supernatant with a 0.22 mu m water-based microporous filter membrane to obtain a sample to be detected. Meanwhile, a standard curve of the lactic acid is drawn by taking a lactic acid standard product as an external standard.
(HPLC measurement conditions are shown in Table 1).
TABLE 1 conditions for the HPLC determination of lactic acid
According to the HPLC results, an acid-tolerant (pH 3.85) and simultaneously lactic acid-producing strain was obtained with a lactic acid yield of 9.04. + -. 0.11g/L, and stored in slant medium at 4 ℃ for further analysis.
Example 2: molecular characterization of lactic acid producing and acid tolerant strains
Purifying and screening the obtained strains, taking fresh bacterial liquid in exponential growth phase, centrifugally collecting thalli, and extracting genome DNA by using a bacterial genome extraction kit. Using a bacterial universal primer P0/P6Amplifying the full-length sequence of the 16S rRNA gene, which comprises the following steps:
P0:5’-GAG AGT TTG ATC CTG GCT CAG-3’
P6:5’-CTA CGG CTA CCT TGT TAC GA-3’
reaction System (50. mu.L)
Reaction procedure
The PCR product was separated and checked by 1.0% agarose gel electrophoresis at a voltage of about 11V/cm for 20 min.
The purification of PCR amplification products is carried out according to the instruction of a small-amount glue recovery PCR product purification kit of Shanghai Biotechnology company, and the sequencing is completed by the Shanghai Biotechnology company.
The obtained 16S rRNA gene sequence of the bacteria is subjected to BLAST comparison in GenBank to determine the species, the full length of the 16S rRNA gene sequence of the strain is 1541bp, the strain with the highest homology is Lactobacillus fermentum (GenBank accession number: KF030758.1), and the 16S rRNA gene sequence is shown in SEQ ID NO: l.
The strain is preserved in China general microbiological culture collection center (CGMCC) in 2016, 9 and 5, and the preservation number is 12934.
Example 3: physiological and biochemical characteristics of strain CGMCC 12934
CGMCC 12934 was inoculated into a separation medium, and after anaerobic culture at 36 ℃ for 4 days, the cell morphology of the strain was observed by a Transmission Electron Microscope (TEM) and a Scanning Electron Microscope (SEM). Inoculating CGMCC 12934 in liquid culture medium, and measuring OD of culture solution600Detecting the temperature suitable for the growth of the strain and the tolerance range of the strain to acetic acid; the carbon source available to the strain was analyzed by the BIOLOG (AN microplate) assay. The above characteristics of strain CGMCC 12934 are shown in Table 2.
TABLE 2 physiological and biochemical characteristics of strain CGMCC 12934
Example 4: experiment for producing lactic acid by strain CGMCC 12934 under different sugar concentrations
Inoculating the strain CGMCC 12934 with the inoculation amount of 1% into a seed culture medium containing 10mL, carrying out standing culture at 36 ℃ for 4 days, then inoculating the strain CGMCC 12934 with the inoculation amount of 4% into a liquid fermentation culture medium, carrying out standing culture under the anaerobic condition for 7 days, and then measuring the content of lactic acid in a fermentation liquid by HPLC.
Seed culture medium: 40g/L of glucose, 20g/L of tryptone, 20g/L of beef extract, 10g/L of yeast powder, 10g/L of sodium acetate, 4g/L of diammonium hydrogen citrate, 4g/L of dipotassium hydrogen phosphate, 1.16g/L of magnesium sulfate heptahydrate, 0.38g/L of manganese sulfate monohydrate, 802 mL/L of tween, 3.85 of pH value and 20min of sterilization at 121 ℃; ② 1g/L of L-cysteine hydrochloride, and sterilizing for 20min at 121 ℃. Mixing the first step and the second step in equal volume under the aseptic condition;
liquid fermentation medium: 40g/L of glucose, 20g/L of tryptone, 20g/L of beef extract, 10g/L of yeast powder, 10g/L of sodium acetate, 4g/L of diammonium hydrogen citrate, 4g/L of dipotassium hydrogen phosphate, 1.16g/L of magnesium sulfate heptahydrate, 0.38g/L of manganese sulfate monohydrate, 802 mL/L of tween, 3.85 of pH value and 20min of sterilization at 121 ℃; ② 1g/L of L-cysteine hydrochloride, and sterilizing for 20min at 121 ℃. First, and second, under aseptic conditions, equal volume mixing. Wherein different glucose contents were set, with final concentrations of 20, 30, 40 and 50g/L, respectively.
The lactic acid production of the strain in the fermentation broth was measured at different sugar concentrations according to the HPLC detection method described in example 1 above, and the results showed that the lactic acid production increased as the sugar concentration increased, and was 9.04. + -. 0.11g/L, 13.41. + -. 0.09g/L, 19.35. + -. 0.06g/L and 27.37. + -. 0.13g/L, respectively.
Example 5: adding CGMCC 12934 to increase lactic acid content of solid vinegar
In order to avoid the utilization of lactic acid by acetic acid bacteria, CGMCC 12934 is added in the fermentation stage of Zhenjiang aromatic vinegar after the acetic acid fermentation stage is finished.
Control group: zhenjiang aromatic vinegar produced by fermentation according to the traditional vinegar brewing process.
Treatment group: culturing CGMCC 12934 to logarithmic phase, wherein the concentration of bacteria reaches about 3 × 108cfu/mL. Inoculating according to the inoculation amount of 5% of the fermented grains, adding sucrose 3% of the fermented grains, sealing, and aging.
The content of lactic acid in Zhenjiang aromatic vinegar was determined by high performance liquid chromatography, and the specific method was the same as in example 1. Analysis results show that compared with a control group, the content of lactic acid in the vinegar of the treatment composition is increased to 1.76g/100mL by 27.5 percent; the proportion of non-volatile acid in the total acid is increased from 26.2% to 29.7%, which is increased by 13.0%, so that the taste of the vinegar is softer, and the overall quality is also improved.
Table 3 physicochemical characteristics of solid vinegar before and after adding strain CGMCC 12934
SEQUENCE LISTING
<110> Jiangnan University
<120> acid-resistant lactobacillus fermentum and application thereof
<130> 2019.02.26
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1541
<212> DNA
<213> Lactobacillus fermentum
<400> 1
gagagtttga tcctggctca ggatgaacgc cggcggtgtg cctaatacat gcaagtcgaa 60
cgcgttggcc caattgattg atggtgcttg cacttgatag attttggtcg ccaacgagtg 120
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
ggatcatcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccat 1440
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ggacagatga ttagggtgaa gtcgtaacaa ggtagccgta g 1541
Claims (1)
1. An acid-resistant Lactobacillus fermentum is characterized in that the strain is preserved in the common microorganism center of the institute of microorganism culture preservation management of China institute of microbiology, No.1, west way of the Ministry of microbiology, No. 3, located in the area of the south facing the Yangtze, Beijing, within 2016 (9 months and 5 days), the preservation number is CGMCC NO.12934, and the strain is classified and named as Lactobacillus fermentum.
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| CN112538450B (en) * | 2020-12-29 | 2022-09-27 | 江南大学 | Application of high-yield flavor acid-resistant lactobacillus in food production |
| CN113699069B (en) * | 2021-08-30 | 2023-07-21 | 江苏恒顺醋业股份有限公司 | Bacterial strain HSCY2073, separation screening thereof and application thereof in improving flavor quality of vinegar |
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Effective date of registration: 20230602 Address after: 214000 1800 Lihu Avenue, Binhu District, Wuxi, Jiangsu Patentee after: Jiangnan University Patentee after: JIANGSU HENGSHUN VINEGAR-INDUSTRY Co.,Ltd. Patentee after: Zhenjiang Hengshun Wine Industry Co.,Ltd. Address before: School of pharmacy, Jiangnan University, 1800 Lihu Avenue, Wuxi City, Jiangsu Province, 214122 Patentee before: Jiangnan University |