CN110093285A - One plant of acidproof lactobacillus fermenti and its application - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12J—VINEGAR; PREPARATION OR PURIFICATION THEREOF
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Abstract
The invention discloses one plant of acidproof lactobacillus fermenti and its applications, belong to fermentation engineering field.The bacterial strain was preserved in China General Microbiological culture presevation administrative center (CGMCC), deposit number 12934 on September 5th, 2016.The bacterial strain has good tolerance to acid, well-grown under conditions of pH is 3.85~4.40, and through liquid state fermentation culture, which generates lactic acid, and lactic acid total amount reaches 27.37g/L；The bacterial strain is added in the envelope unstrained spirits stage for making vinegar process, lactic acid content in vinegar can be improved, improve the taste and flavor of vinegar.
Description
Technical field
The invention belongs to fermentation engineering fields, and in particular to one plant of acidproof lactobacillus fermenti and use bacterial strain general
The method that glucide is converted into lactic acid.
Background technique
Lactic acid is the important taste compound of many traditional fermented foods, such as vinegar, white wine, pickles.For vinegar,
Lactic acid is the wherein highest fixed acid of content, and part vinegar is also using fixed acid content as dividing one of vinegar grade
Important indicator.Therefore, tart flavour of the content for soft vinegar of lactic acid is improved, the excitement of acetic acid is reduced and improves vinegar grade
Etc. playing a significant role, while it is also to adjust one of vinegar pH, the key substance for extending its shelf-life.
Lactic acid in vinegar, which is mainly metabolized by the lactic acid bacteria during acetic fermentation, to be generated, wherein with Lactobacillus
(Lactobacillus) based on, including Lactobacillus helveticus (L.helveticus), Kazakhstan Bacillus acidi lactici (L.hamsteri),
Bridge Bacillus acidi lactici (L.pontis) and bread Bacillus acidi lactici (L.panis) etc..In acetic fermentation early period, lactic acid bacteria is making the micro- life of vinegar
Account for leading in object group, but due to the accumulation of the metabolites such as lactic acid, acetic acid in system, total acid content is increased in vinegar fermented grain, mostly
Number lactic acid bacteria is gradually withered away due to not tolerating peracid, and acetic acid bacteria is increasingly becoming the dominant microflora in system, due to the later period of fermenting
Carbon source is insufficient, and lactic acid can be fallen by part microbial consumption, this causes main fixed acid lactic acid content in vinegar to reduce
One of the main reasons.Therefore, screening can be with sugar fermentation lactic acid producing, and has the lactic acid bacteria of good acid resistance (especially acetic acid)
Bacterial strain has important practical significance for improving vinegar lactic acid content in production.It is had not been reported at present using of the present invention
Acidproof lactobacillus fermenti for vinegar production in strengthen lactic acid production.
Summary of the invention
In view of the technical problem in above-mentioned production, the present invention is directed to pass through one plant of acidproof lactic acid bacteria of screening, and utilized
During vinegar brewing, accounting of the fixed acid in total acid in vinegar is improved, mouthfeel, flavor and the quality of vinegar are improved.
Present invention firstly provides one plant of acidproof lactobacillus fermenti (Lactobacillus fermentum), the bacterial strain in
On September 5th, 2016 is deposited in the Institute of Microorganism, Academia Sinica of Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC NO.12934, and classification naming is
Lactobacillus fermenti (Lactobacillus fermentum) LFE02.
The 16S rRNA gene order for obtaining the bacterial strain carries out BLAST in GenBank and compares its determining kind, should
Bacterial strain 16S rRNA gene order overall length is 1541bp, is Lactobacillus with its bacterial strain with highest homology
Fermentum (GenBank accession number: KF030758.1), 16S rRNA gene order are shown in SEQ ID NO:l.
The available main sugar substance of bacterial strain includes D-Glucose, D-Fructose, L- trehalose, D- galactolipin, D-
Mannose, L- rhamnose and sucrose.
Under anaerobic, stationary culture metabolizable sugar substance generates lactic acid to the bacterial strain.
The technique of the fermenting lactic acid are as follows:
Seed/liquid state fermentation culture medium: 1. glucose 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, bitter salt 1.16g/L, a hydration
Sterilize 20min at 3.85~4.40,121 DEG C of manganese sulfate 0.38g/L, Tween 80 2mL/L, pH；2. L-cysteine hydrochloride
1g/L, sterilize at 121 DEG C 20min.1. with 2. aseptically mixing in equal volume；
Seed culture method: 50mL triangular flask liquid amount 10mL, 36 DEG C anaerobism stationary culture 4 days；
Fermentation culture method: seed liquor is accessed by fermentation medium, 250mL triangular flask liquid amount with 1~8% inoculum concentration
100mL, under anaerobic condition, 35~37 DEG C of stationary cultures.
The present invention provides a kind of applications of bacterial strain, using above-mentioned zymotechnique, the lactic acid content of bacterial strain production
Up to 9.04 ± 0.11g/L.
In one embodiment of the present invention, increase glucose content in culture medium, the lactic acid production of the strain fermentation can
It improves to 27.37 ± 0.13g/L.
In one embodiment of the present invention, the glucose content is 50g/L.
The present invention also provides a kind of applications of bacterial strain, and the fermentation is added in the envelope unstrained spirits stage of solid-state fermented vinegar brewing
Lactobacillus, while the sucrose of vinegar fermented grain quality 2~5% is added, the content of lactic acid and fixed acid ratio in vinegar can be improved, make to eat
Vinegar mouthfeel is softer.
The present invention also provides a kind of applications of bacterial strain, in liquid white rice vinegar brewing process, acetic acid bacteria fermentation knot
Shu Hou adds the sucrose of vinegar liquid quality 2~5%, and the lactobacillus fermenti is added, and lactic acid content in white rice vinegar can be improved, and improves
The taste and flavor of white rice vinegar.
The present invention is screened from vinegar fermented grain obtains one plant of lactic acid producing bacterial strain with preferable acid-fast ability, is identified as acidified milk
Bacillus (Lactobacillus fermentum).The bacterial strain can well be grown under conditions of 3.85~4.40 pH, while can
It is metabolized glucide and generates lactic acid, lactic acid production can reach 27.37 ± 0.13g/L.In the envelope unstrained spirits stage of vinegar brewing process
The bacterial strain is added, lactic acid production in vinegar can be improved, improves the taste and flavor of vinegar.
Specific embodiment
Embodiment 1: the screening of lactic acid producing and acidproof bacterial strain
From taking 10g sample to be fitted into the 500mL triangular flask for being mixed with bead and 90mL sterile water in zhenjiang vinegar vinegar fermented grain,
30min is vibrated with 115rpm on 36 DEG C of shaking tables, takes bacteria suspension 1mL 10 times of gradient dilutions of sterile saline, then with 10-3~
10-6Gradient dilution sample be coated with isolation medium plate, 36 DEG C anaerobism stationary culture 7~14 days；By bacterium colony warp abundant on plate
Slant medium is accessed after isolating and purifying, 36 DEG C are cultivated 7 days, 4 DEG C of preservations；Bacterium colony is accessed in fluid nutrient medium simultaneously, 36 DEG C
After culture 7 days, using the content of high performance liquid chromatography (HPLC) measurement Lactic Acid from Fermentation Broth, so that screening obtains acidproof production
Lactic bacteria strain.
Separation/slant medium: 1. glucose 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, bitter salt 1.16g/L, Manganous sulfate monohydrate
0.38g/L, Tween 80 2mL/L, sterilize at 3.6~6.6,121 DEG C of agar powder 40g/L, pH 20min；2. L-cysteine salt
Hydrochlorate 1g/L, sterilize at 121 DEG C 20min.1. separating/slant medium with being 2. aseptically made after isometric mixing.
Fluid nutrient medium: 1. glucose 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, bitter salt 1.16g/L, 0.38 g/ of Manganous sulfate monohydrate
Sterilize 20min at 3.6~6.6,121 DEG C of L, Tween 80 2mL/L, pH；2. L-cysteine hydrochloride 1g/L goes out at 121 DEG C
Bacterium 20min.1. with 2. aseptically mix in equal volume after obtained fluid nutrient medium.
The specific method is as follows for HPLC measurement:
1. the preprocess method of sample
It is accurate to measure 2mL fermented supernatant fluid, medium centrifugal (12000rpm, 5min) is obtained into supernatant, is separately added into
800 μ L zinc sulfate (300g/L) and 800 μ L potassium ferrocyanides (106g/L), oscillation mix, centrifugation.Take supernatant with 0.22 μm
It is sample to be tested after water system miillpore filter filtering.At the same time, the standard curve of lactic acid is drawn using lactate standard product as external standard.
2. HPLC determination condition is as shown in table 1.
The condition of 1 HPLC of table measurement lactic acid
According to HPLC as a result, obtaining one plant acidproof (pH 3.85) and the simultaneously bacterial strain of lactic acid producing, lactic acid production be 9.04 ±
0.11 g/L is placed in preservation in slant medium in 4 DEG C in case further analysis.
Embodiment 2: the Molecular Identification of lactic acid producing and acidproof bacterial strain
The bacterial strain that purifying screening obtains, takes exponential phase of growth fresh bacterium solution, thalline were collected by centrifugation, takes out using bacterial genomes
Extraction reagent kit extracts genomic DNA.Using bacterial universal primers P0/P6Its 16S rRNA full length gene sequence is expanded, specifically such as
Under:
P0:5’-GAG AGT TTG ATC CTG GCT CAG-3’
P6:5’-CTA CGG CTA CCT TGT TAC GA-3’
1. reaction system (50 μ L)
2. response procedures
PCR product is separated with 1.0% agarose gel electrophoresis and is examined, voltage about 11V/cm, electrophoresis time 20min.
The purifying of pcr amplification product recycles PCR product purification kit by a small amount of glue of Shanghai Sheng Gong biotech company
Illustrate to carry out, sequencing is completed by Shanghai Sheng Gong biotech company.
The bacterial 16 S rRNA gene order of acquisition carries out BLAST in GenBank and compares its determining kind, the bacterial strain
16S rRNA gene order overall length is 1541bp, is Lactobacillus with its bacterial strain with highest homology
Fermentum (GenBank accession number: KF030758.1), 16S rRNA gene order are shown in SEQ ID NO:l.
The bacterial strain was preserved in China General Microbiological culture presevation administrative center (CGMCC), preservation on September 5th, 2016
Number is 12934.
Embodiment 3: the physio-biochemical characteristics of bacterial strain CGMCC 12934
CGMCC 12934 is inoculated in isolation medium, 36 DEG C after Anaerobic culturel 4 days, pass through transmission electron microscope
(Transmission Electron Microscope, TEM) and scanning electron microscope (Scanning Electron
Microscope, SEM) observe the cellular morphology of the bacterial strain.CGMCC 12934 is inoculated in liquid culture medium, is trained by measurement
Nutrient solution OD600, detect the temperature and its tolerance range to acetic acid of the bacterial strain suitable growth；Pass through BIOLOG (AN microwell plate)
The available carbon source of the analysis of experiments bacterial strain.The features described above of bacterial strain CGMCC 12934 is as shown in table 2.
The physio-biochemical characteristics of 2 bacterial strain CGMCC 12934 of table
Embodiment 4: 12934 lactic acid producing of bacterial strain CGMCC is tested under different sugar concentration
Bacterial strain CGMCC 12934 is inoculated in the seed culture medium containing 10mL with 1% inoculum concentration, 36 DEG C of anaerobic conditions
Lower stationary culture is transferred in liquid state fermentation culture medium after 4 days, by it with 4% inoculum concentration, stationary culture 7 days under anaerobic condition
Afterwards, its Lactic Acid from Fermentation Broth content is measured by HPLC.
Seed culture medium: 1. glucose 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, bitter salt 1.16g/L, 0.38 g/ of Manganous sulfate monohydrate
Sterilize 20min at 3.85,121 DEG C of L, Tween 80 2mL/L, pH；2. L-cysteine hydrochloride 1g/L sterilizes at 121 DEG C
20min.1. with 2. aseptically mixing in equal volume；
Liquid state fermentation culture medium: 1. glucose 40g/L, tryptone 20g/L, beef extract 20g/L, yeast powder 10g/L, second
Sour sodium 10g/L, diammonium hydrogen citrate 4g/L, dipotassium hydrogen phosphate 4g/L, bitter salt 1.16g/L, Manganous sulfate monohydrate
Sterilize 20min at 3.85,121 DEG C of 0.38g/L, Tween 80 2mL/L, pH；2. L-cysteine hydrochloride 1g/L, at 121 DEG C
Sterilize 20min.1. with 2. aseptically mixing in equal volume.Wherein, different glucose contents, final concentration difference are set
For 20,30,40 and 50g/L.
HPLC detection method described in accordance with the above-mentioned embodiment 1 measures the bacterial strain under different sugar concentration in fermentation liquid
The yield of lactic acid, as the result is shown with sugared concentration increase, lactic acid production increase, be followed successively by 9.04 ± 0.11g/L, 13.41 ±
0.09g/L, 19.35 ± 0.06g/L and 27.37 ± 0.13g/L.
Embodiment 5: addition CGMCC 12934 improves solid-state fermented vinegar lactic acid content
To avoid lactic acid from being utilized by acetic acid bacteria, after we select the zhenjiang vinegar acetic fermentation stage, in its envelope unstrained spirits
Stage adds CGMCC 12934.
Control group: according to the zhenjiang vinegar of Traditional Vinegar Manufacturing technique fermenting and producing.
Processing group: CGMCC 12934 is cultivated to logarithmic phase, bacterium is dense to reach about 3 × 108cfu/mL.It seals before unstrained spirits according to vinegar
The inoculum concentration of unstrained spirits quality 5% is inoculated with, while adding the sucrose of vinegar fermented grain quality 3%, then seals unstrained spirits ageing.
Using the content of lactic acid in high effective liquid chromatography for measuring zhenjiang vinegar, specific method is same as Example 1.Analysis
The results show that compared with the control group, lactic acid content is improved to 1.76g/100mL in processing group finished product vinegar, 27.5% is increased；
The ratio of fixed acid is improved from 26.2% to 29.7% in total acid, increases 13.0%, so that the mouthfeel of vinegar is softer, it is whole
Body quality also increases.
The physicochemical characteristics of the addition 12934 front and back solid-state fermented vinegar of bacterial strain CGMCC of table 3
SEQUENCE LISTING
<110> Jiangnan University
<120>one plants of acidproof lactobacillus fermentis and its applications
<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
gagagtttgt aacacccaaa gtcggtgggg taacctttta ggagccagcc gcctaaggtg 1500
ggacagatga ttagggtgaa gtcgtaacaa ggtagccgta g 1541
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.
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| WO2023029569A1 (en) * | 2021-08-30 | 2023-03-09 | 江苏恒顺醋业股份有限公司 | Strain hscy 2073, and isolation and screening therefor and use thereof in improving flavor and quality of vinegar |
| CN115044479A (en) * | 2022-04-13 | 2022-09-13 | 茅台学院 | Geotrichum candidum strain with high lactic acid tolerance and application thereof |
| CN115044479B (en) * | 2022-04-13 | 2024-03-29 | 茅台学院 | Geotrichum candidum strain with high lactic acid tolerance and application thereof |
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