CN106906302A - A kind of quick screening, identification and the method for quantifying lactobacillus acetotolerans in fermented food - Google Patents
A kind of quick screening, identification and the method for quantifying lactobacillus acetotolerans in fermented food Download PDFInfo
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Abstract
The invention discloses a kind of quick screening, identification and the method for quantifying lactobacillus acetotolerans in fermented food, belong to biotechnology and fermented food field.Specificity of the present invention for 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 PCR (PCR).The screening of primer pair lactobacillus acetotolerans suitable for traditional fermented food, identification and quantitative.
Description
Technical field
The present invention relates to a kind of quick screening, identification and the method for quantifying lactobacillus acetotolerans in fermented food, belong to biological
Technology and fermented food field.
Background technology
Lactobacillus acetotolerans is widely present in during various fermented foods brewage system, 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 during fermented food brewages system
Microorganism.Shown by high-flux sequence result, it is the bar of resistance to yogurt to have more than 90% in white wine fermented grain fermentation middle and later periods bacterium
Bacterium, its advantage stage occupies the time of whole fermentation stage 2/3, and this is because lactobacillus acetotolerans is resistant to or is 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 is considered as
It is the critical strain for generating these flavor substances, therefore the bacterium has huge contribution to the local flavor of white wine.So for acidproof
The separation identification of lactobacillus is extremely important.But lactobacillus acetotolerans is amphimicrobe, nutritional condition is very harsh and resistance to
Lactobacillus lactis difference between most lactic acid bacterias on colonial morphology and physicochemical property is not notable, this to lactobacillus acetotolerans screening,
Identification and paced work bring great difficulty, are also to study less main cause for lactobacillus acetotolerans at present.
Unique growth factor needed for lacking lactobacillus acetotolerans bacteria growing due to conventional medium, lactobacillus acetotolerans is in routine
Can hardly be grown on the culture mediums such as MRS, tomato juice agar (TJA)；Additionally, at present for the Screening and Identification of lactobacillus acetotolerans,
The method of main physicochemical property or 16srDNA by bacterial strain.This authentication method has that the cycle is long, costly, success rate is low
The problems such as.Therefore, there are many problem demanding prompt solutions in terms of lactobacillus acetotolerans Screening and Identification, set up a kind of quick screening,
The method of lactobacillus acetotolerans is very necessary and significant in identification and quantitative fermented food.
The content of the invention
To solve the above problems, the deficiency of existing lactic acid bacteria screening, identification and quantitative technique is made up, first, the present invention is carried
A kind of specific primer for effectively improving lactic acid bacteria screening, identification and quantitative efficacy is supplied.Secondly, the present invention improves traditional
MRS is cultivated, and adds the survival rate that various trophic factors improve lactic acid bacteria, and addition Nysfungin suppresses the growth of miscellaneous bacteria, adds bromine
Cresols is green quickly to recognize the sour lactic acid bacteria of product.Finally, quickly and accurately can be sieved from sour lactic acid bacteria is produced using specific primer
Select lactobacillus acetotolerans.
First content of the invention there is provided a kind of screening of lactobacillus acetotolerans in fermented food, identify and its special
Property quantitative approach, methods described is the nucleotide sequence of the encoding gene of glycosyl hydrolase using in above-mentioned lactobacillus acetotolerans as mould
Plate designs primer pair.
In one embodiment of the invention, the nucleotides sequence of the glycosyl hydrolase enzyme coding gene is classified as
SEQIDNO.1。
Second content of the invention there is provided a pair of specific primers, to improve screening, the identification of lactobacillus acetotolerans
And quantitative efficacy, the specific primer is LaF1 to sequence:5′-AACATCCCCAGAGCGTCAAG-3′；LaR1:5′-
GGCACTACCCCAAGCATCTT-3′。
3rd 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 it is any source the thalline containing lactobacillus acetotolerans, bacterium colony,
Bacterium solution and genomic samples.
In one embodiment of the invention, the screening is the addition on the basis of every liter of main solution of MRS culture mediums
1~5mL pair solution is mixed and used；The composition of secondary solution：Indicator 0.01~0.02g/mL of bromocresol green, trophic factors D- asparagus ferns
Propylhomoserin diformazan ester hydrochloride, hiochic acid lactones and VB11, 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 VB11, Nysfungin 0.04~
0.06g/mL, solvent is dimethyl sulfoxide (DMSO), uses sterilised membrane filter filtration sterilization.
In one embodiment of the invention, the component of the MRS culture mediums 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, MnSO4·4H2(liquid is trained for O0.01~0.1g/L, 15.0~25.0g/L of agar
Support base without) regulation pH is 6.8 ± 0.3, addition 1000mL distilled water is 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~
0.015g/mL, 0.04~0.06g/mL of 0.01~0.015g/mL of VB11 and Nysfungin, addition dimethyl sulfoxide (DMSO) 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
Use.
In an embodiment of the present invention, the purpose bacterium colony selected be flat board on single bacterium colony surrounding media by blueness
It is changed into the bacterium colony of yellow.
In one embodiment of the invention, the specific primer PCR, refers to carrying out using specific primer
PCR, if PCR primer is detected, with the fragment between 100~250bp, then it is lactobacillus acetotolerans to represent the bacterium colony for display.Such as
Fruit PCR primer then represents the bacterium colony without lactobacillus acetotolerans not between 100~250bp or without band.
In one embodiment of the invention, the PCR reaction systems are：The μ L of total system 25~50：Prime
STAR12.5~25 μ L, each 0.5~1.0 μ L of upstream and downstream primer, the μ L of bacterium solution 1~2, the μ L of ultra-pure water 11~22.
In one embodiment of the invention, the PCR reaction conditions are：90~98 DEG C of 2~10min of predegeneration, 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 it is any source the thalline containing lactobacillus acetotolerans, bacterium colony,
Bacterium solution and genomic samples.
In one embodiment of the invention, the RTFQ PCR reaction systems are, the μ L of total system 10~40：2×
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, the μ L of ultra-pure water 4.1~16.4.
In one embodiment of the invention, the RTFQ PCR reaction conditions are：92~98 DEG C 3 of predegeneration~
8min, is denatured 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, totally 30~50
Individual circulation, 65~75 DEG C of 8~12min.
Beneficial effects of the present invention：
The MRS culture mediums that the present invention is improved can improve the survival rate of lactobacillus acetotolerans in fermented food, suppress miscellaneous bacteria
Growth, be easy to identification to produce sour lactic acid bacteria；
Primer pair designed by the present invention can be sieved rapidly and accurately by specific primer round pcr from fermented food
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 pcrs
Speed, accurate quantitative analysis；
Primer pair designed by the present invention, for lactobacillus acetotolerans it is qualitative, quantitative when, with speed is fast, the degree of accuracy is high
And it is applied widely the characteristics of.
Brief description of the drawings
Fig. 1：The pcr amplification product electrophoresis checking 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 method for designing of specific primer pair：
(1) lactic acid bacteria kind main in Fenyang wine fermentation process is lactobacillus acetotolerans (L.acetotolerans), short newborn bar
Bacterium (L.brevis), Lactobacillus casei (L.casei), dry ferment lactobacillus (L.diolivorans), Lactobacillus farciminis
And Lactobacillus saki (L.sakei) (existence rate ＞ 1% of the lactic acid bacteria in fermented grain) (L.farciminis)；
(2) by capital of a country gene and genome 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 the 2328bp amino acid sequences corresponding to glycosyl hydrolase are the height spies of L.acetotolerans
Different in nature sequence；
(4) NCBI Primer-BLAST instruments, setting primer parameter (Primer Parameters) are utilized：Polymerase chain
Formula reacts (PCR) product length (PCR product size) 80~300；Setting primer pair specificity verification parameter (Primer
Pair Specificity Checking Parameters)：Database (Database) nr；Remaining parameter sets according to acquiescence
It is fixed；
(5) from being obtained to primer pair, being selected using DNAMAN softwares can not itself cyclization (self-
Complementarity) the sense primer LaF1 of Serial No. SEQ ID NO.2：5’-AACATCCCCAGAGCGTCAAG-3’
With Serial No. SEQ ID NO.3 anti-sense primers LaR1：5 '-GGCACTACCCCAAGCATCTT-3 ' draw as possibility purpose
Thing pair, PCR primer length 151bp, Tm are 60 DEG C, and GC% is 55%；
(6) with possibility purpose primer to carrying out BLAST, comparison result and resistance to yogurt in NCBI for upstream and downstream primer
Bacillus has 100% similarity, and this shows selected LaF1/LaR1 primer pairs possibility purpose primer to be suitable under this environment
Specific primer pair.
Embodiment 2：The checking of lactobacillus acetotolerans (Lactobacillus acetotolerans) specific primer pair
The checking of specific primer pair：
(1) selecting two plants of purpose lactobacillus acetotoleranses (LA1, LA2) carries out bacterium colony PCR, and negative control ferments for white wine fermented grain
During superior microorganism：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 systems are 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 predegeneration, are denatured 98 DEG C of 10s, and anneal 55 DEG C of 1min, extends 72 DEG C
2min, totally 30 circulations, 72 DEG C of 10min；
(5) agarose gel electrophoresis：2% Ago-Gel, 2 μ L dye liquors, 4 μ L PCR primers, Mark is DL2000,
160V, 20min；
(6) specificity of primer is observed by gel imaging instrument, LA1, LA2 have obvious purpose bar near 151bp
Band, and remaining negative control does not have band (as shown in Figure 1), then show designed specific primer to right
The specificity of L.acetotolerans.
Embodiment 3：The screening of lactobacillus acetotolerans Lactobacillus acetotolerans
In lactobacillus acetotolerans incubation, by repeatedly attempting finding a certain proportion of 3 kinds of trophic factors-D- of addition
Aspartic acid diformazan ester hydrochloride, hiochic acid lactones and VB11 can be greatly enhanced the survival rate of lactobacillus acetotolerans.
Additionally, the product acid time according to lactobacillus acetotolerans is earlier than other lactic acid bacterias, and acid producing ability is better than other lactic acid bacterias, for breast
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、MnSO4·4H2O0.08g/L, agar 20.0g/L regulation pH are 6.8 ± 0.3, 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, VB11 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；When using, main solution is cooled to 50 DEG C, adds 2mL
Secondary solution is mixed and used.
Sample pre-treatments：(pH7.4 contains in having sterilized equipped with 100mL sterile PBS buffers to weigh 10g fermented foods
0.5g/LD- aspartic acid diformazans ester hydrochloride) and the 250mL triangular flasks of 3g beades in, be subsequently placed in 37 DEG C, 200r/min
Shaking table vibration mixes 30min, stands 5min and obtains bacteria suspension.
Microculture：Take 10-3、10-4With 10-53 sample bacteria suspension dilution factors coat above-mentioned MRS culture medium flat plates
In cultivate 84h in 37 DEG C of anaerobic box, select surrounding media on picking flat board and be changed into the single bacterium colony of yellow from blueness and numbered,
Bacterium colony PCR is carried out using specific primer.PCR reaction conditions and agarose gel electrophoresis are with " specific primer checking ".
Experimental result specificity screening pcr amplification product electrophoretogram is as shown in Figure 2.Bacterium colony numbering D6, F1 and F6 have mesh
Band.
Embodiment 4：Lactobacillus acetotolerans Lactobacillus acetotolerans screening verifications
By gel imaging instrument, all single bacterium colonies being numbered in selection embodiment 3 use bacterial universal primers pair
1492R/F27 carries out 16srDNA bacterium colonies PCR (as shown in Figure 3), and positive control (CK+) is purpose bacterial strain, negative control (CK-)
It is redistilled water.PCR primer is carried out into nucleotide sequencing, nucleotide sequence is carried out into BLAST comparisons, comparison result knot using NCBI
Bacterium colony numbering D6, F1 and F6 corresponding 16srDNA bacterium colony PCR result and lactobacillus acetotolerans of the fruit display with purpose band
(LC202658.1) there is 100% similarity, so that the bacterium colony for quickly learning the corresponding numbering 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、MnSO4·4H2O0.1g/L, agar 25.0g/L regulation pH are 6.8 ± 0.3, 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, VB11 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；When using, main solution is cooled to 60 DEG C, addition
5mL pair solution is mixed and used.
Sample pre-treatments：(pH7.4 contains in having sterilized equipped with 100mL sterile PBS buffers to weigh 10g fermented foods
0.5g/LD- aspartic acid diformazans ester hydrochloride) and the 250mL triangular flasks of 3g beades in, be subsequently placed in 37 DEG C, 200r/min
Shaking table vibration mixes 30min, stands 5min and obtains bacteria suspension.
Microculture：Take 10-3、10-4With 10-53 sample bacteria suspension dilution factors coat above-mentioned MRS culture medium flat plates
In cultivate 84h in 37 DEG C of anaerobic box, select surrounding media on picking flat board and be changed into the single bacterium colony of yellow from blueness and numbered,
Bacterium colony PCR is carried out using specific primer.
PCR reaction conditions and agarose gel electrophoresis are with " specific primer checking ".
Experimental result has band with embodiment 3, the PCR primer 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、MnSO4·4H2O0.01g/L, agar 15.0g/L regulation pH are 6.8 ± 0.3, 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, VB11 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；When using, main solution is cooled to 40 DEG C, adds 1mL pairs
Solution is mixed and used.
Sample pre-treatments：(pH7.4 contains in having sterilized equipped with 100mL sterile PBS buffers to weigh 10g fermented foods
0.5g/LD- aspartic acid diformazans ester hydrochloride) and the 250mL triangular flasks of 3g beades in, be subsequently placed in 37 DEG C, 200r/min
Shaking table vibration mixes 30min, stands 5min and obtains bacteria suspension.
Microculture：Take 10-3、10-4With 10-53 sample bacteria suspension dilution factors coat above-mentioned MRS culture medium flat plates
In cultivate 84h in 37 DEG C of anaerobic box, select surrounding media on picking flat board and be changed into the single bacterium colony of yellow from blueness and numbered,
Bacterium colony PCR is carried out using specific primer.PCR reaction conditions and agarose gel electrophoresis are with " specific primer checking ".
Experimental result has band with embodiment 3, the PCR primer of three plants of bacterium colonies between 100~250bp.
Embodiment 7：Fermentation fermented grain in lactobacillus acetotolerans Lactobacillus acetotolerans) RTFQ PCR
Set up CT values and the dense standard curve of lactobacillus acetotolerans bacterium (as shown in Figure 4).
The total genome in fermented grain sample is extracted, genome concentration is 10ng/ μ L.
RTFQ PCR are carried out to the genomes for extracting more using LaF1/LaR1 primer pairs
RTFQ PCR reaction systems are：The μ L of total system 20：2×Fast qPCR Master Mix(with loading
Dye) 10 μ L, each 0.4 μ L of upstream and downstream primer, the μ L of genome 1, the μ L of ultra-pure water 8.2.
The RTFQ PCR reaction conditions are：95 DEG C of 5min of predegeneration, are denatured 95 DEG C of 10s, and anneal 50 DEG C of 0.5min, extends
70 DEG C of 1min, totally 40 circulations, 70 DEG C of 10min.
According to the CT values (as shown in Figure 5) that reaction terminates, pass through set up standard curve and calculate lactobacillus acetotolerans in sample
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、MnSO4·4H2O0.08g/L, agar 20.0g/L regulation pH are 6.8 ± 0.3, 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 (DMSO) is configured to secondary solution, is filtered using 0.2 μm of sterilised membrane filter
Sterilizing；When using, main solution is cooled to 50 DEG C, and addition 2mL pair solution is mixed and used.
Sample pre-treatments：(pH7.4 contains in having sterilized equipped with 100mL sterile PBS buffers to weigh 10g fermented foods
0.5g/L D-Asp diformazans ester hydrochloride) and the 250mL triangular flasks of 3g beades in, be subsequently placed in 37 DEG C, 200r/min
Shaking table vibration mixes 30min, stands 5min and obtains bacteria suspension.
Microculture：Take 10-3、10-4With 10-53 sample bacteria suspension dilution factors coat above-mentioned MRS culture medium flat plates
In cultivate 84h in 37 DEG C of anaerobic box, select surrounding media on picking flat board and be changed into the single bacterium colony of yellow from blueness and numbered,
Bacterium colony PCR is carried out using specific primer.PCR reaction conditions and agarose gel electrophoresis are with " specific primer checking ".
Experimental result shows that PCR primer illustrates lactobacillus acetotolerans upper not between 100~250bp or without band
State no growth in MRS culture mediums.
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、MnSO4·4H2O0.08g/L, agar 20.0g/L regulation pH are 6.8 ± 0.3, 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 (DMSO) is configured to secondary solution, uses 0.2 μm of sterilised membrane filter filtration sterilization；Use
When, main solution is cooled to 50 DEG C, and addition 2mL pair solution is mixed and used.
Sample pre-treatments：(pH7.4 contains in having sterilized equipped with 100mL sterile PBS buffers to weigh 10g fermented foods
0.5g/LD- aspartic acid diformazans ester hydrochloride) and the 250mL triangular flasks of 3g beades in, be subsequently placed in 37 DEG C, 200r/min
Shaking table vibration mixes 30min, stands 5min and obtains bacteria suspension.
Microculture：Take 10-3、10-4With 10-53 sample bacteria suspension dilution factors coat above-mentioned MRS culture medium flat plates
In cultivate 84h in 37 DEG C of anaerobic box, select surrounding media on picking flat board and be changed into the single bacterium colony of yellow from blueness and numbered,
Bacterium colony PCR is carried out using specific primer.PCR reaction conditions and agarose gel electrophoresis are with " specific primer checking ".
Test result indicate that, PCR primer illustrates lactobacillus acetotolerans upper not between 100~250bp or without band
State no growth in MRS culture mediums.
Although the present invention is disclosed as above with preferred embodiment, it is not limited to the present invention, any to be familiar with time skill
The people of art, without departing from the spirit and scope of the present invention, can do various changes and modification, therefore protection model of the invention
Enclose being defined of being defined by claims.
Sequence table
<110>Southern Yangtze University
<120>A kind of quick screening, identification and the method for quantifying lactobacillus acetotolerans in fermented food
<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>It is artificial synthesized
<400> 2
aacatcccca gagcgtcaag 20
<210> 3
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 3
ggcactaccc caagcatctt 20
Claims (10)
1. a kind of screening of lactobacillus acetotolerans, identification or quantitative approach, it is characterised in that methods described is to be with nucleotides sequence row
The Cellulase gene of SEQ ID NO.1 is as template design primer to entering performing PCR.
2. method according to claim 1, it is characterised in that the band that the primer pair enters the product that performing PCR is obtained exists
Between 100~250bp.
3. method according to claim 1, it is characterised in that the nucleotides sequence of the primer pair is classified as LaF1：5′-
AACATCCCCAGAGCGTCAAG-3′；LaR1：5′-GGCACTACCCCAAGCATCTT-3′.
4. it is a kind of for lactobacillus acetotolerans screening, identification or quantitative primer pair, it is characterised in that the nucleotides of the primer pair
Sequence is LaF1：5′-AACATCCCCAGAGCGTCAAG-3′；LaR1：5′-GGCACTACCCCAAGCATCTT-3′.
5. application of the primer pair described in claim 4 in terms of lactobacillus acetotolerans Screening and Identification, it is characterised in that the application is
Bacterium colony PCR is carried out using the primer pair described in claim 4.
6. application according to claim 5, it is characterised in that the screening is in every liter of base of the main solution of MRS culture mediums
On plinth, addition 1~5mL pair solution is mixed and 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 is dimethyl sulfoxide (DMSO), wherein, the trophic factors is D-Asp diformazan
0.01~0.02g/mL of ester hydrochloride, 0.01~0.015g/ of 0.005~0.015g/mL of hiochic acid lactones and VB11
mL。
7. application according to claim 5, it is characterised in that the application field of the application is fermented food field.
8. application of the primer pair described in claim 4 at the quantitative aspect of lactobacillus acetotolerans, it is characterised in that the application is to utilize
Primer pair described in claim 4 carries out fluorescent quantitative poly chain reaction.
9. application according to claim 8, it is characterised in that the application field of the application is fermented food field.
10. application according to claim 8, it is characterised in that cycle threshold is dense with lactobacillus acetotolerans bacterium in the application
Standard curve be y=-0.2237x+8.9658, R2=0.999.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109371100A (en) * | 2018-11-20 | 2019-02-22 | 四川农业大学 | A kind of culture medium and its method for the detection of vinegar aerogenic bacteria |
| CN109680082A (en) * | 2019-01-07 | 2019-04-26 | 江南大学 | A kind of lactobacillus specific data library and its application |
| CN113913351A (en) * | 2021-11-29 | 2022-01-11 | 山西杏花村汾酒厂股份有限公司 | Method for screening lactic acid bacteria in fermented grains of fen-flavor liquor |
| CN114292929A (en) * | 2021-11-30 | 2022-04-08 | 绍兴文理学院 | Molecular marker for quantitative lactobacillus acidophilus resistance and method for absolutely quantifying bacterial community composition in yellow wine fermentation process |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680546A (en) * | 2002-10-30 | 2005-10-12 | 食品工业发展研究所 | Acid and choline-resistant separated strain of lactobacillus with ability of reducing and assimilating cholesterol |
| CN101812521A (en) * | 2010-04-08 | 2010-08-25 | 黑龙江省乳品工业技术开发中心 | Method for identifying plant lactobacillus |
| CN103525736A (en) * | 2013-10-21 | 2014-01-22 | 广州南沙珠江啤酒有限公司 | Method for inducing and restoring beer-spoilage lactic acid bacteria into VBNC (Viable But Non Culturable) state |
| CN106010997A (en) * | 2016-04-29 | 2016-10-12 | 周礼红 | Lactobacillus plantarum and culture and separation method, screening method and application thereof |
-
2017
- 2017-04-27 CN CN201710285328.6A patent/CN106906302B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1680546A (en) * | 2002-10-30 | 2005-10-12 | 食品工业发展研究所 | Acid and choline-resistant separated strain of lactobacillus with ability of reducing and assimilating cholesterol |
| CN101812521A (en) * | 2010-04-08 | 2010-08-25 | 黑龙江省乳品工业技术开发中心 | Method for identifying plant lactobacillus |
| CN103525736A (en) * | 2013-10-21 | 2014-01-22 | 广州南沙珠江啤酒有限公司 | Method for inducing and restoring beer-spoilage lactic acid bacteria into VBNC (Viable But Non Culturable) state |
| CN106010997A (en) * | 2016-04-29 | 2016-10-12 | 周礼红 | Lactobacillus plantarum and culture and separation method, screening method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| HIROYUKI NAKAI等: "Substrate Recognition by Two Members of Glycoside Hydrolase Family 32 Involved in Fructo-oligosaccharide Metabolism in Lactobacillus acidophilus NCFM", 《BULL.FACUL.AGRIC.NIIGATA UNIV.》 * |
| 杜晓华等: "蒙古国地区酸乳中乳酸菌的鉴定及耐酸菌株筛选", 《微生物学通报》 * |
| 石晶红等: "内蒙古地区传统酸乳中耐酸乳酸菌的分离鉴定及其耐酸性能的研究", 《食品科技》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN109371100A (en) * | 2018-11-20 | 2019-02-22 | 四川农业大学 | A kind of culture medium and its method for the detection of vinegar aerogenic bacteria |
| 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 |
| CN113913351A (en) * | 2021-11-29 | 2022-01-11 | 山西杏花村汾酒厂股份有限公司 | Method for screening lactic acid bacteria in fermented grains of fen-flavor liquor |
| 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 |
| CN114292929A (en) * | 2021-11-30 | 2022-04-08 | 绍兴文理学院 | Molecular marker for quantitative lactobacillus acidophilus resistance and method for absolutely quantifying bacterial community composition in yellow wine fermentation process |
| 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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