CN106906302B - A kind of method of lactobacillus acetotolerans in quick screening, identification and quantitative fermented food - Google Patents
A kind of method of lactobacillus acetotolerans in quick screening, identification and quantitative fermented food Download PDFInfo
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Abstract
The invention discloses a kind of methods of lactobacillus acetotolerans in quickly screening, identification and quantitative fermented food, belong to biotechnology and fermented food field.The present invention is directed to the specificity of lactobacillus acetotolerans, design the specific primer of lactobacillus acetotolerans, by the lactic acid bacteria in modified MRS selective medium separation and fermentation food, purpose bacterial strain can be rapidly and accurately identified using specific primer and bacterium colony polymerase chain reaction (PCR).The screening of primer pair lactobacillus acetotolerans suitable for traditional fermented food is identified and is quantified.
Description
Technical field
The present invention relates to a kind of methods of lactobacillus acetotolerans in quickly screening, identification and quantitative fermented food, belong to biology
Technology and fermented food field.
Background technique
Lactobacillus acetotolerans are widely present in various fermented food brewing systems, including white wine, yellow rice wine, vinegar, pickles, sauce
Oil, Yoghourt, cheese, fermented glutinous rice, fermented soya bean, fermented bean curd, yellow rice wine, beer, grape wine etc., and be the advantage in fermented food brewing system
Microorganism.By high-flux sequence, the result shows that, in white wine fermented grain fermentation middle and later periods bacterium having 90%, the above are the bars of resistance to yogurt
Bacterium, the advantage stage occupies the time of entire fermentation stage 2/3, this is because lactobacillus acetotolerans are resistant to or are adapted to peracid
What the characteristic of the unfavorable conditions such as degree, high ethano concentration, low oxygen content was determined.Studies have found that the liquor fermentation middle and later periods is second
The stage that the important flavor substance such as acid, lactic acid, ethyl acetate, ethyl lactate, ethyl caprilate is formed, and lactobacillus acetotolerans are considered
It is to generate the critical strain of these flavor substances, therefore the bacterium has huge contribution to the flavor of white wine.So for acidproof
The separation identification of lactobacillus is extremely important.But lactobacillus acetotolerans are amphimicrobe, nutritional condition is very harsh, and resistance to
Difference is not significant between most lactic acid bacterias on colonial morphology and physicochemical property for Lactobacillus lactis, this to lactobacillus acetotolerans screening,
The main reason for identification and paced work bring great difficulty, and less for lactobacillus acetotolerans research at present.
Unique growth factor needed for lacking the growth of lactobacillus acetotolerans bacterium due to conventional medium, lactobacillus acetotolerans are in routine
It can hardly be grown on the culture mediums such as MRS, tomato juice agar (TJA)；In addition, at present for the screening and identification of lactobacillus acetotolerans,
The method of main physicochemical property or 16srDNA by bacterial strain.This identification method is long, costly there are the period, success rate is low
The problems such as.Therefore, in terms of lactobacillus acetotolerans screening and identification there are many urgent problems to be solved, establish a kind of quickly screening,
The method of lactobacillus acetotolerans is very necessary and is of great significance in identification and quantitative fermented food.
Summary of the invention
To solve the above problems, the deficiency of existing lactic acid bacteria screening, identification and quantitative technique is made up, firstly, the present invention mentions
A kind of specific primer for effectively improving lactic acid bacteria screening, identification and quantitative efficacy is supplied.Secondly, the present invention improve it is traditional
MRS culture, adds the survival rate that a variety of trophic factors improve lactic acid bacteria, and addition Nysfungin inhibits the growth of miscellaneous bacteria, adds bromine
Cresols is green quickly to identify the sour lactic acid bacteria of production.Finally, can quickly and accurately be sieved from the sour lactic acid bacteria of production using specific primer
Select lactobacillus acetotolerans.
There is provided a kind of screenings of lactobacillus acetotolerans in fermented food, identification and its special for first content of the invention
Property quantitative approach, the method is using the nucleotide sequence of the encoding gene of glycosyl hydrolase in above-mentioned lactobacillus acetotolerans as mould
Plate design primer pair.
In one embodiment of the invention, the nucleotides sequence of the glycosyl hydrolase enzyme coding gene is classified as
SEQIDNO.1。
There is provided a pair of of specific primers for second content of the invention, to improve screening, the identification of lactobacillus acetotolerans
And quantitative efficacy, the specific primer are LaF1:5 '-AACATCCCCAGAGCGTCAAG-3 ' to sequence；LaR1:5′-
GGCACTACCCCAAGCATCTT-3′。
Third content of the invention is the specific primer to the application in terms of lactobacillus acetotolerans screening and identification.
In one embodiment of the invention, the sample be any source contain the thallus of lactobacillus acetotolerans, bacterium colony,
Bacterium solution and genomic samples.
In one embodiment of the invention, the screening is addition on the basis of every liter of MRS culture medium main solution
1~5mL pair solution, which mixes, to be used；The composition of secondary solution: indicator 0.01~0.02g/mL of bromocresol green, trophic factors D- asparagus fern
Propylhomoserin diformazan ester hydrochloride, hiochic acid lactones and vitamin B11, wherein D-Asp diformazan ester hydrochloride 0.01~
0.02g/mL, 0.005~0.015g/mL of hiochic acid lactones, 0.01~0.015g/mL of vitamin B11, Nysfungin 0.04~
0.06g/mL, solvent are dimethyl sulfoxide, use sterilised membrane filter filtration sterilization.
In one embodiment of the invention, the component of the MRS culture medium includes: 8.0~15.0g/ of tryptone
L, 5.0~10.0g/L of beef extract, 2.0~6.0g/L of yeast extract, 18.0~25.0g/L of glucose, anhydrous sorbierite oil
0.5~1.5mL/L of acid esters, K2HPO41.0~5.0g/L, 1.0~10.0g/L of sodium acetate trihydrate, Triammonium citrate 1.0~
5.0g/L、MgSO4·7H2O0.1~0.5g/L, MnSO4·4H2O0.01~0.1g/L, 15.0~25.0g/L of agar (liquid training
Feeding base does not add) adjust pH be 6.8 ± 0.3, addition 1000mL distilled water be configured to main solution, 121 DEG C of sterilizing 15min；Bromine first
Green 0.01~the 0.02g/mL of phenol, 0.01~0.02g/mL of D-Asp diformazan ester hydrochloride, hiochic acid lactones 0.005~
0.04~0.06g/mL of 0.015g/mL, 0.01~0.015g/mL of vitamin B11 and Nysfungin, addition dimethyl sulfoxide are configured to
Secondary solution uses 0.2 μm of sterilised membrane filter filtration sterilization；When main solution is cooled to 40~60 DEG C, addition 1~5mL pair solution is mixed
It uses.
In an embodiment of the present invention, the purpose bacterium colony selected is single colonie surrounding media on plate by blue
Become the bacterium colony of yellow.
In one embodiment of the invention, the specific primer PCR, refers to using specific primer to progress
PCR shows the segment having between 100~250bp, then represents the bacterium colony as lactobacillus acetotolerans if PCR product detects.Such as
Fruit PCR product is not between 100~250bp or do not have band, then represents the bacterium colony without lactobacillus acetotolerans.
In one embodiment of the invention, the PCR reaction system are as follows: 25~50 μ L:Prime of total system
The μ L of STAR12.5~25, each 0.5~1.0 μ L of upstream and downstream primer, 1~2 μ L of bacterium solution, 11~22 μ L of ultrapure water.
In one embodiment of the invention, the PCR reaction condition are as follows: 90~98 DEG C of 2~10min of initial denaturation become
Property 95~98 DEG C of 5~15s, anneal 50~60 DEG C of 0.5~1.5min, extend 68~75 DEG C of 1~5min, totally 20~45 circulation,
68~75 DEG C of 8~15min.
4th content of the invention is the primer pair right using fluorescent quantitative poly chain reaction (RTFQ PCR)
Application in terms of lactobacillus acetotolerans specific quantification
In one embodiment of the invention, the sample be any source contain the thallus of lactobacillus acetotolerans, bacterium colony,
Bacterium solution and genomic samples.
In one embodiment of the invention, the RTFQ PCR reaction system is, 10~40 μ L:2 of total system ×
FastqPCR Master Mix (with loading dye) 5~20 μ L, each 0.2~0.8 μ L of upstream and downstream primer, genome 0.5
~1.5 μ L, 4.1~16.4 μ L of ultrapure water.
In one embodiment of the invention, the RTFQ PCR reaction condition are as follows: 92~98 DEG C 3 of initial denaturation~
8min is denaturalized 90~98 DEG C of 5~15s, and anneal 40~60 DEG C of 15~40s, extends 65~75 DEG C of 0.5~1.5min, and totally 30~50
A circulation, 65~75 DEG C of 8~12min.
Beneficial effects of the present invention:
The MRS culture medium that the present invention is improved can be improved the survival rate of lactobacillus acetotolerans in fermented food, inhibit miscellaneous bacteria
Growth, convenient for identification produce sour lactic acid bacteria；
Primer pair designed by the present invention can be sieved rapidly and accurately from fermented food by specific primer round pcr
Choosing identifies lactobacillus acetotolerans；
Primer pair designed by the present invention can be used in the fast of lactobacillus acetotolerans in fermented food by RTFQ round pcr
Speed, accurate quantitative analysis；
Primer pair designed by the present invention, for lactobacillus acetotolerans it is qualitative, quantitative when, have that speed is fast, accuracy is high
With feature applied widely.
Detailed description of the invention
Fig. 1: the pcr amplification product electrophoresis verifying of specific primer pair；
Fig. 2: the specificity screening pcr amplification product electrophoretic analysis of purpose bacterial strain；
Fig. 3: purpose bacterial strain 16sr DNA pcr amplification product electrophoretic analysis；
Fig. 4: cycle threshold and the dense standard curve of lactobacillus acetotolerans bacterium；
Fig. 5: the reaction result of lactobacillus acetotolerans quantitative fluorescent PCR.
Specific embodiment
Embodiment 1: the design of lactobacillus acetotolerans (Lactobacillus acetotolerans) specific primer pair
The design method of specific primer pair:
(1) in Fenyang wine fermentation process main lactic acid bacteria kind be lactobacillus acetotolerans (L.acetotolerans), short newborn bar
Bacterium (L.brevis), Lactobacillus casei (L.casei), dry ferment lactobacillus (L.diolivorans), Lactobacillus farciminis
(L.farciminis) and Lactobacillus saki (L.sakei) (existence rate > 1% of the lactic acid bacteria in fermented grain)；
(2) by capital of a country gene and genomic encyclopedia metabolic pathway (KEGG Pathway) find lactobacillus acetotolerans with
Enzyme-specific-glycosyl hydrolase (glycosyl hydrolase) between remaining 6 strains of lactic acid bacteria, obtains the enzyme-specific
The nucleotide sequence of 2328bp, sequence such as SEQ ID NO.1；
(3) nucleotide sequence is examined by US National Biotechnology Information center (NCBI) using local alignment
Rope basic tool (BLAST) is compared, and genome sequence of the comparison result only with lactobacillus acetotolerans (NBRC13120) has
100% similarity shows that 2328bp amino acid sequence corresponding to glycosyl hydrolase is the height spy of L.acetotolerans
Anisotropic sequence；
(4) NCBI Primer-BLAST tool is utilized, is set primer parameter (Primer Parameters): polymerase chain
Formula reacts (PCR) product length (PCR product size) 80~300；Set primer pair specificity verification parameter (Primer
Pair Specificity Checking Parameters): database (Database) nr；Remaining parameter is set according to default
It is fixed；
(5) from obtained in primer pair, being selected using DNAMAN software can not itself cyclization (self-
Complementarity) upstream primer the LaF1:5 '-AACATCCCCAGAGCGTCAAG-3 ' of Serial No. SEQ ID NO.2
Draw with Serial No. SEQ ID NO.3 downstream primer LaR1:5 '-GGCACTACCCCAAGCATCTT-3 ' as possibility purpose
Object pair, PCR product length 151bp, Tm are 60 DEG C, GC% 55%；
(6) BLAST, comparison result and resistance to yogurt are carried out to for upstream and downstream primer in NCBI with possibility purpose primer
Bacillus has 100% similarity, this shows selected LaF1/LaR1 primer pair possibility purpose primer to for suitable under this environment
Specific primer pair.
Embodiment 2: the verifying of lactobacillus acetotolerans (Lactobacillus acetotolerans) specific primer pair
The verifying of specific primer pair:
(1) two plants of purpose lactobacillus acetotolerans (LA1, LA2) are selected to carry out bacterium colony PCR, negative control is the fermentation of white wine fermented grain
Superior microorganism in the process: Lactobacillus brevis (LB), Lactobacillus casei (LC), Lactobacillus pentosus (LP), Lactobacillus
diolivorans(LD)、Lactobacillus farciminis(LF)、Lactobacillus sakei(LS)、
Pediococcus parvulus(PP)、Pseudomonas hunanensis(PH)、Pichia kudriavzevii(PK)、
Geotrichum candidum (GC), Naumovozyma castellii (NC) and redistilled water (dd H2O)；
(3) PCR reaction system is 25 μ L:Prime STAR12.5 μ L, upstream and downstream primer each 0.5 μ L, bacterium solution 1mL；
(4) response procedures of PCR: 94 DEG C of 4min of initial denaturation are denaturalized 98 DEG C of 10s, and anneal 55 DEG C of 1min, extend 72 DEG C
2min, totally 30 recycle, 72 DEG C of 10min；
(5) agarose gel electrophoresis: 2% Ago-Gel, 2 μ L dye liquors, 4 μ L PCR products, Mark DL2000,
160V, 20min；
(6) specificity of primer is observed by gel imager, LA1, LA2 have apparent purpose item near 151bp
Band, and remaining negative control does not have band (as shown in Figure 1) then shows designed specific primer to having pair
The specificity of L.acetotolerans.
Embodiment 3: the screening of lactobacillus acetotolerans Lactobacillus acetotolerans
In lactobacillus acetotolerans incubation, a certain proportion of 3 kinds of trophic factors-D- are added by repeatedly attempting discovery
Aspartic acid diformazan ester hydrochloride, hiochic acid lactones and vitamin B11 can greatly improve the survival rate of lactobacillus acetotolerans.
In addition, according to the production of the lactobacillus acetotolerans sour time earlier than other lactic acid bacterias, and acid producing ability is better than other lactic acid bacterias, for cream
It is extremely important that the characteristic of lactic acid production of sour bacterium designs effective indicator medium.
The preparation of culture medium: tryptone 10.0g/L, beef extract 8.0g/L, yeast extract 4.0g/L, glucose
18.0g/L, anhydrous sorbitan oleate 0.8mL/L, K2HPO42.5g/L, sodium acetate trihydrate 6.0g/L, Triammonium citrate
2.0g/L、MgSO4·7H2O0.3g/L、MnSO4·4H2It is 6.8 ± 0.3 that O0.08g/L, agar 20.0g/L, which adjust pH, addition
1000mL distilled water is configured to main solution, 121 DEG C of sterilizing 15min；Bromocresol green 0.015g/mL, D-Asp dimethyl ester salt
Hydrochlorate 0.01g/mL, hiochic acid lactones 0.01g/mL, vitamin B11 0.01g/mL and Nysfungin 0.05g/mL add diformazan
Base sulfoxide is configured to secondary solution, uses 0.2 μm of sterilised membrane filter filtration sterilization；In use, main solution is cooled to 50 DEG C, 2mL is added
Secondary solution, which mixes, to be used.
Sample pre-treatments: weighing 10g fermented food, equipped with 100mL sterile PBS buffer, (pH7.4 contains in having sterilized
0.5g/LD- aspartic acid diformazan ester hydrochloride) and the 250mL triangular flask of 3g bead in, be subsequently placed in 37 DEG C, 200r/min
Shaking table oscillation mixes 30min, stands 5min and obtains bacteria suspension.
Microculture: 10 are taken-3、10-4With 10-53 sample bacteria suspension dilutions are coated on above-mentioned MRS culture medium flat plate
In cultivate 84h in 37 DEG C of anaerobic box, selecting surrounding media on picking plate is become the single colonie and number of yellow from blue,
Bacterium colony PCR is carried out using specific primer.PCR reaction condition and agarose gel electrophoresis are the same as " specific primer verifying ".
Experimental result specificity screening pcr amplification product electrophoretogram is as shown in Figure 2.Bacterium colony number D6, F1 and F6 have mesh
Band.
Embodiment 4: lactobacillus acetotolerans Lactobacillus acetotolerans screening verification
By gel imager, all single colonies being numbered, use bacterial universal primers pair in selection example 3
1492R/F27 carries out 16srDNA bacterium colony PCR (as shown in Figure 3), and positive control (CK+) is purpose bacterial strain, negative control (CK-)
For redistilled water.PCR product is subjected to nucleotide sequencing, nucleotide sequence is subjected to BLAST comparison, comparison result knot using NCBI
Fruit shows the corresponding 16srDNA bacterium colony PCR result and lactobacillus acetotolerans of bacterium colony number D6, F1 and F6 with purpose band
(LC202658.1) there is 100% similarity, to quickly learn that the corresponding bacterium colony numbered of D6, F1 and F6 is purpose lactic acid
Bacterium.
Embodiment 5: the screening of lactobacillus acetotolerans Lactobacillus acetotolerans
The preparation of culture medium: tryptone 15.0g/L, beef extract 10.0g/L, yeast extract 6.0g/L, glucose
25.0g/L, anhydrous sorbitan oleate 1.5mL/L, K2HPO45.0g/L, sodium acetate trihydrate 10.0g/L, Triammonium citrate
5.0g/L、MgSO4·7H2O0.5g/L、MnSO4·4H2It is 6.8 ± 0.3 that O0.1g/L, agar 25.0g/L, which adjust pH, addition
1000mL distilled water is configured to main solution, 121 DEG C of sterilizing 15min；Bromocresol green 0.02g/mL, D-Asp dimethyl ester salt
Hydrochlorate 0.02g/mL, hiochic acid lactones 0.015g/mL, vitamin B11 0.015g/mL and Nysfungin 0.06g/mL, addition two
Methyl sulfoxide is configured to secondary solution, uses 0.2 μm of sterilised membrane filter filtration sterilization；In use, main solution is cooled to 60 DEG C, addition
5mL pair solution, which mixes, to be used.
Sample pre-treatments: weighing 10g fermented food, equipped with 100mL sterile PBS buffer, (pH7.4 contains in having sterilized
0.5g/LD- aspartic acid diformazan ester hydrochloride) and the 250mL triangular flask of 3g bead in, be subsequently placed in 37 DEG C, 200r/min
Shaking table oscillation mixes 30min, stands 5min and obtains bacteria suspension.
Microculture: 10 are taken-3、10-4With 10-53 sample bacteria suspension dilutions are coated on above-mentioned MRS culture medium flat plate
In cultivate 84h in 37 DEG C of anaerobic box, selecting surrounding media on picking plate is become the single colonie and number of yellow from blue,
Bacterium colony PCR is carried out using specific primer.
PCR reaction condition and agarose gel electrophoresis are the same as " specific primer verifying ".
Experimental result has band with embodiment 3, the PCR product of three plants of bacterium colonies between 100~250bp.
Embodiment 6: the screening of lactobacillus acetotolerans Lactobacillus acetotolerans
The preparation of culture medium: tryptone 8.0g/L, beef extract 5.0g/L, yeast extract 2.0g/L, glucose
18.0g/L, anhydrous sorbitan oleate 0.5mL/L, K2HPO41.0g/L, sodium acetate trihydrate 1.0g/L, Triammonium citrate
1.0g/L、MgSO4·7H2O0.1g/L、MnSO4·4H2It is 6.8 ± 0.3 that O0.01g/L, agar 15.0g/L, which adjust pH, addition
1000mL distilled water is configured to main solution, 121 DEG C of sterilizing 15min；Bromocresol green 0.01g/mL, D-Asp diformazan ester hydrochloride
Salt 0.01g/mL, hiochic acid lactones 0.005g/mL, vitamin B11 0.01g/mL and Nysfungin 0.04g/mL add dimethyl
Sulfoxide is configured to secondary solution, uses 0.2 μm of sterilised membrane filter filtration sterilization；In use, main solution is cooled to 40 DEG C, 1mL pair is added
Solution, which mixes, to be used.
Sample pre-treatments: weighing 10g fermented food, equipped with 100mL sterile PBS buffer, (pH7.4 contains in having sterilized
0.5g/LD- aspartic acid diformazan ester hydrochloride) and the 250mL triangular flask of 3g bead in, be subsequently placed in 37 DEG C, 200r/min
Shaking table oscillation mixes 30min, stands 5min and obtains bacteria suspension.
Microculture: 10 are taken-3、10-4With 10-53 sample bacteria suspension dilutions are coated on above-mentioned MRS culture medium flat plate
In cultivate 84h in 37 DEG C of anaerobic box, selecting surrounding media on picking plate is become the single colonie and number of yellow from blue,
Bacterium colony PCR is carried out using specific primer.PCR reaction condition and agarose gel electrophoresis are the same as " specific primer verifying ".
Experimental result has band with embodiment 3, the PCR product of three plants of bacterium colonies between 100~250bp.
Embodiment 7: fermentation fermented grain in lactobacillus acetotolerans Lactobacillus acetotolerans) RTFQ PCR
Establish CT value and the dense standard curve of lactobacillus acetotolerans bacterium (as shown in Figure 4).
Total genome in fermented grain sample is extracted, genome concentration is 10ng/ μ L.
RTFQ PCR is carried out to the genome more extracted using LaF1/LaR1 primer pair
RTFQ PCR reaction system are as follows: 20 μ L:2 × Fast qPCR Master Mix (with loading of total system
Dye) 10 μ L, each 0.4 μ L of upstream and downstream primer, 1 μ L of genome, 8.2 μ L of ultrapure water.
The RTFQ PCR reaction condition are as follows: 95 DEG C of 5min of initial denaturation are denaturalized 95 DEG C of 10s, and anneal 50 DEG C of 0.5min, extend
70 DEG C of 1min, totally 40 recycle, 70 DEG C of 10min.
According to the CT value (as shown in Figure 5) that reaction terminates, lactobacillus acetotolerans are calculated in sample by the standard curve established
Concentration in product is C=8.2 × 108CFU/mL。
Reference examples 1:
The preparation of culture medium: tryptone 10.0g/L, beef extract 8.0g/L, yeast extract 4.0g/L, glucose
18.0g/L, anhydrous sorbitan oleate 0.8mL/L, K2HPO42.5g/L, sodium acetate trihydrate 6.0g/L, Triammonium citrate
2.0g/L、MgSO4·7H2O0.3g/L、MnSO4·4H2It is 6.8 ± 0.3 that O0.08g/L, agar 20.0g/L, which adjust pH, addition
1000mL distilled water is configured to main solution, 121 DEG C of sterilizing 15min；Bromocresol green 0.015g/mL, D-Asp dimethyl ester salt
Hydrochlorate 0.01g/mL and Nysfungin 0.05g/mL, addition dimethyl sulfoxide are configured to secondary solution, are filtered using 0.2 μm of sterilised membrane filter
Sterilizing；In use, main solution is cooled to 50 DEG C, addition 2mL pair solution, which mixes, to be used.
Sample pre-treatments: weighing 10g fermented food, equipped with 100mL sterile PBS buffer, (pH7.4 contains in having sterilized
0.5g/L D-Asp diformazan ester hydrochloride) and the 250mL triangular flask of 3g bead in, be subsequently placed in 37 DEG C, 200r/min
Shaking table oscillation mixes 30min, stands 5min and obtains bacteria suspension.
Microculture: 10 are taken-3、10-4With 10-53 sample bacteria suspension dilutions are coated on above-mentioned MRS culture medium flat plate
In cultivate 84h in 37 DEG C of anaerobic box, selecting surrounding media on picking plate is become the single colonie and number of yellow from blue,
Bacterium colony PCR is carried out using specific primer.PCR reaction condition and agarose gel electrophoresis are the same as " specific primer verifying ".
Experimental result shows that PCR product is not between 100~250bp or do not have band, illustrates lactobacillus acetotolerans upper
It states and is not grown in MRS culture medium.
Reference examples 2:
The preparation of culture medium: tryptone 10.0g/L, beef extract 8.0g/L, yeast extract 4.0g/L, glucose
18.0g/L, anhydrous sorbitan oleate 0.8mL/L, K2HPO42.5g/L, sodium acetate trihydrate 6.0g/L, Triammonium citrate
2.0g/L、MgSO4·7H2O0.3g/L、MnSO4·4H2It is 6.8 ± 0.3 that O0.08g/L, agar 20.0g/L, which adjust pH, addition
1000mL distilled water is configured to main solution, 121 DEG C of sterilizing 15min；Bromocresol green 0.015g/mL, hiochic acid lactones 0.01g/
ML and Nysfungin 0.05g/mL, addition dimethyl sulfoxide are configured to secondary solution, use 0.2 μm of sterilised membrane filter filtration sterilization；It uses
When, main solution is cooled to 50 DEG C, and addition 2mL pair solution, which mixes, to be used.
Sample pre-treatments: weighing 10g fermented food, equipped with 100mL sterile PBS buffer, (pH7.4 contains in having sterilized
0.5g/LD- aspartic acid diformazan ester hydrochloride) and the 250mL triangular flask of 3g bead in, be subsequently placed in 37 DEG C, 200r/min
Shaking table oscillation mixes 30min, stands 5min and obtains bacteria suspension.
Microculture: 10 are taken-3、10-4With 10-53 sample bacteria suspension dilutions are coated on above-mentioned MRS culture medium flat plate
In cultivate 84h in 37 DEG C of anaerobic box, selecting surrounding media on picking plate is become the single colonie and number of yellow from blue,
Bacterium colony PCR is carried out using specific primer.PCR reaction condition and agarose gel electrophoresis are the same as " specific primer verifying ".
The experimental results showed that PCR product is not between 100~250bp or do not have band, illustrate lactobacillus acetotolerans upper
It states and is not grown in MRS culture medium.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with time skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Sequence table
<110>Southern Yangtze University
<120>in a kind of quickly screening, identification and quantitative fermented food lactobacillus acetotolerans method
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 2328
<212> DNA
<213>lactobacillus acetotolerans (Lactobacillus acetotolerans)
<400> 1
atggcaagtg taaataaaca tgttaattat aaattgatta cttcaattgc tgctgcgaca 60
ttattaggat taattgaatt acaaagtcaa acggtaaaag cagaggtaaa tgctacaccc 120
cttataatac aacaacgggt taaccaagat gataataatg atccgttgtt taacggtgct 180
gatgtttcaa atgggtatac tgataaaggc tatacctatg tagaaccaac tttaacatcc 240
ccagagcgtc aagactcata tcatttaacg actaatcaag gttggtcgaa tgatttgcaa 300
actattaact ataatcaaaa agataataat tataatttat attatcttca tacagagggt 360
ggagctaatg gcaatgcaac cggtggtcaa aactggcagc gtgtaacgac caaagacttt 420
actcacttta gcaaacccaa tactgctatt caggataaag gaaatattaa agatgcttgg 480
ggtagtgcct ggacaggatc aattattact aataaaggta atattaccgg tgtacctaaa 540
ggtgcacaag tagcttattt ttcagggtta agtattaagg ataaacaaca gaatatttgg 600
gcagcctggt cggataatga tggtcaaagt tttgatcata tcctttataa tggctttcca 660
gtaattgatc attattggga ttggacttct aaaaataaat ctgatgaaag agatccttcg 720
gttttttatt ggcataataa actaattatg tatgcggctg aaggtaatga tttaggtgtt 780
tatcaatccg cagatggtct ccattggtca aaagctaatc ccgaaaatga aagtaaggtt 840
tataacgatc agtattttaa agggctaaag tttgaagcac ctgtagaatg tcctgttgtt 900
cgaagcatga aaattgcaaa tggaactgta aaacaagttt tattctttgg tgctaaatcg 960
ccgcaagatg gtcaaactac tggtacctat tatatagttg gtcatttaga taacaatggg 1020
atctttttcc cagaaaacga tgttaagagg ttagatcagg ggacagatta ttacggtgcc 1080
aattttagtg gtagcgatga cctgtcaaaa gcagatgatt cgctgattag catggcttgg 1140
gtaggaaatt ggaattatat taattctggt ataaaaacta atcaagaaga tatgcccgta 1200
acttccaaga gattgggtgc ttatacttta gcaaggaaat tagttctaaa taatgataat 1260
actatttcaa gtacaccgat tactactaat cttgaagaaa aaagtggtaa tgaagttaat 1320
ggcaatgtta gttctaaacg agttgatgaa aatggtaatc acgagttagt taatttgaaa 1380
aagcaaccgg ctaatagtaa atatgttctc catttctcta caaaaaataa tgagaattat 1440
aatggtgctg ttcagataaa atttactcaa ggcaaagata ataattcaat tatttttaat 1500
ccggctaatg gacaatatcg tgtttatgga aatagcagtg aattaacagg tgcagctgca 1560
gattattata aaaatggatt atatagcggg ttaggttatc gtaatgattc tggattgaaa 1620
gataaacaag attttacttt aaccatttat actgataagg attcaattga attatttttc 1680
ccaaatgggg aaacttatac aatggcccgt ttttgtgtaa ataatattca agatgtatct 1740
attttaagtc aggatccgaa taagaataat aaatttaata ttagttttaa tcaggtagga 1800
cctgatttag ttggatatgt ggctaaacct aatgattcaa aggcaagttc tggaaatact 1860
gataacaata atattgactt taacagtaat aatattgtta cacctgataa ttatgtaaag 1920
ccagaaaagg aaccgaataa tatggctgaa attaaaggaa ctgctagtaa tagtcagttg 1980
atgcataatg catatattta tgatcaaaat ggtaagaaga taagtaatat cattttgcag 2040
gcttattcga ttttgaaaac ttacggtact aaaatgatta acggtaagaa gtattttgtt 2100
ttaagcaatg atcattatat tgctgcgggt aatatttctg gtagcaaacg taaacttagc 2160
cataatgctt atatttataa taaatgtggt aaacgtagtg ggaagcgtgt attaaagaaa 2220
aataaaagag ttaatactta tggtagtgct aaaaaaatta atggtaagaa atattatgct 2280
attggcaaaa ataaatttat aaaaaaagct aattttcaaa tcaaataa 2328
<210> 2
<211> 20
<212> DNA
<213>artificial synthesized
<400> 2
aacatcccca gagcgtcaag 20
<210> 3
<211> 20
<212> DNA
<213>artificial synthesized
<400> 3
ggcactaccc caagcatctt 20
Claims (9)
1. a kind of screening of lactobacillus acetotolerans, identification or quantitative approach, which is characterized in that the method is to be with nucleotides sequence column
The Cellulase gene of SEQ ID NO.1 is as template design primer to progress PCR；The nucleotides sequence of the primer pair is classified as
LaF1:5 '-AACATCCCCAGAGCGTCAAG-3 '；LaR1:5 '-GGCACTACCCCAAGCATCTT-3 '.
2. the method according to claim 1, wherein the band that the primer pair carries out the product that PCR is obtained exists
Between 100~250bp.
3. a kind of for lactobacillus acetotolerans screening, identification or quantitative primer pair, which is characterized in that the nucleotide of the primer pair
Sequence is LaF1:5 '-AACATCCCCAGAGCGTCAAG-3 '；LaR1:5 '-GGCACTACCCCAAGCATCTT-3 '.
4. application of the primer pair described in claim 3 in terms of lactobacillus acetotolerans screening and identification, which is characterized in that the application is
Bacterium colony PCR is carried out using primer pair as claimed in claim 3.
5. application according to claim 4, which is characterized in that the screening is the base in every liter of main solution of MRS culture medium
On plinth, addition 1~5mL pair solution, which mixes, to be used；The composition of secondary solution: indicator 0.01~0.02g/mL of bromocresol green, nutrition
The factor, 0.04~0.06g/mL of Nysfungin, solvent are dimethyl sulfoxide, wherein the trophic factors is D-Asp diformazan
0.01~0.015g/ of 0.01~0.02g/mL of ester hydrochloride, 0.005~0.015g/mL of hiochic acid lactones and vitamin B11
mL。
6. application according to claim 4, which is characterized in that the application field of the application is fermented food field.
7. primer pair described in claim 3 is in the application of the quantitative aspect of lactobacillus acetotolerans, which is characterized in that the application is to utilize
Primer pair described in claim 3 carries out fluorescent quantitative poly chain reaction.
8. application according to claim 7, which is characterized in that the application field of the application is fermented food field.
9. application according to claim 7, which is characterized in that cycle threshold x and lactobacillus acetotolerans bacterium solution in the application
The standard curve of log concentration y is y=-0.2237x+8.9658, R2=0.999.
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| CN107699610A (en) * | 2017-10-13 | 2018-02-16 | 哈尔滨工业大学（威海） | For detecting two pairs of specific primers of acidproof Bacillus acidi lactici content and its application during liquor fermentation |
| CN109371100B (en) * | 2018-11-20 | 2023-09-22 | 四川农业大学 | Culture medium for detecting vinegar gas-producing bacteria and method thereof |
| CN109680082A (en) * | 2019-01-07 | 2019-04-26 | 江南大学 | A kind of lactobacillus specific data library and its application |
| CN113913351B (en) * | 2021-11-29 | 2023-07-25 | 山西杏花村汾酒厂股份有限公司 | Screening method of lactic acid bacteria in fermented grains of fen-flavor liquor |
| CN114292929B (en) * | 2021-11-30 | 2024-01-02 | 绍兴文理学院 | Molecular marker for quantifying lactobacillus-resistant bacteria and method for absolutely quantifying bacterial colony composition in yellow wine fermentation process |
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