CN103131785A - Method for detecting acetic bacteria and lactic acid bacteria in vinegar culture by using fluorescent quantitative PCR (Polymerase Chain Reaction) - Google Patents

Method for detecting acetic bacteria and lactic acid bacteria in vinegar culture by using fluorescent quantitative PCR (Polymerase Chain Reaction) Download PDF

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CN103131785A
CN103131785A CN2013100754663A CN201310075466A CN103131785A CN 103131785 A CN103131785 A CN 103131785A CN 2013100754663 A CN2013100754663 A CN 2013100754663A CN 201310075466 A CN201310075466 A CN 201310075466A CN 103131785 A CN103131785 A CN 103131785A
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bacteria
dna
vinegar
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acid bacteria
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许正宏
史劲松
陆震鸣
王宗敏
钱建瑛
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Jiangnan University
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Jiangnan University
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Abstract

The invention provides a method for conducting quantitative analysis on acetic bacteria and lactic acid bacteria in vinegar culture microorganism communities. The method has higher specificity and sensitivity and can rapidly and accurately quantify microorganisms of specific species in complex communities.

Description

The method of acetic bacteria and milk-acid bacteria in fluorescence quantitative PCR detection vinegar unstrained spirits
Technical field
The invention belongs to technical field of bioengineering, be specifically related to the quantitative examination that fluorescent quantitative PCR technique is applied to specific kind of microorganism belonging to genus in solid-state vinegar generation microflora.
Background technology
The production history of China's vinegar is long, but the fermenting process of numerous vinegar manufacturer is all take traditional experience as main, and process controllability is relatively poor, and there is batch difference in product quality, and especially local flavor differs greatly.This is mainly because microorganism complexity in brewing process is various, and is unclear to the microbial process mechanism in vinegar unstrained spirits fermenting process.The making method of China's vinegar is multiple bacteria compound fermentation technique, by the vinegar that metabolism generation local flavor is full, tart flavour is soft of multiple-microorganism.Two main fermentation stages are arranged: zymamsis and acetic fermentation in the production of vinegar.In the zymamsis stage, starchy material such as glutinous rice, Chinese sorghum etc. finally generate alcohol through a series of metabolism of yeast and mold; And it is main to be in the acetic fermentation stage that numerous bacteriums of comprising acetic bacteria, milk-acid bacteria play a part, and this stage is the committed step that determines vinegar flavor and quality.Acetic acid Pseudomonas and genus lactubacillus are the superior microorganisms of acetic fermentation process, both the abundance sum accounts for more than 90% in whole bacterial flora, and the dynamic change of illustrating acetic acid Pseudomonas and genus lactubacillus biomass in fermenting process has important production meaning for the local flavor of controlling the Vinegar Fermentation process and improving product.In view of this, explore and introduce new technological method and means understanding all sidedly the microbe species in the traditional fermentation system, and then illustrate the mechanism of action of microorganism in brewing process, become the active demand of re-recognizing and rebuilding traditional industry.
Fluorescent quantitative PCR technique refers to add fluorophor in the PCR reaction system, utilizes the whole PCR process of fluorescent signal accumulation Real-Time Monitoring, by typical curve, unknown template is carried out at last the method for quantitative analysis.This technology not only realized DNA profiling quantitatively, and have high specificity, highly sensitive, high-throughput, totally-enclosed reaction, quantitatively accurately, speed reaches the level of automation high soon.The present invention has successfully used fluorescent quantitative PCR technique that the microorganism of the superior microorganism in the vinegar unstrained spirits-acetic acid Pseudomonas and genus lactubacillus is carried out quantitatively, and obtains the dynamic change of biomass in the Vinegar Fermentation process of two kinds of microorganisms.
Summary of the invention
The object of the invention is to solve the deficiency of above-mentioned existing vinegar solid brewing analytical technology, the dynamic change that provides a kind of molecular ecology technology to be used for detection by quantitative solid brewing Vinegar Fermentation process acetic acid Pseudomonas, genus lactubacillus is with Guiding Practice.
The present invention is achieved by the following scheme, and the acetic acid Pseudomonas, the genus lactubacillus that the present invention is directed in vinegar unstrained spirits group design respectively Auele Specific Primer, the glucose vinegar acidfast bacilli that extraction separation and purification obtains (Gluco nacetobacter sp.) and the pentose milk-acid bacteria ( Lactobacillus pentosus) genome, then utilize respectively the Auele Specific Primer that designs to carry out PCR and obtain specificity purpose fragment, again the purpose fragment is connected with pMD19-T Vector, obtain recombinant plasmid, with recombinant plasmid transformed JM109 competent escherichia coli cell, the transformant that picking successfully transforms carries out enlarged culturing again, obtains the high density plasmid, so this high density plasmid is done 10 doubling dilutions, as the standard substance of quantitative fluorescent PCR.Utilize the method for liquid nitrogen grinding, enzyme process and high salt binding to extract total DNA of different time points vinegar unstrained spirits in fermenting process group as testing sample, standard model and testing sample are carried out quantitative fluorescent PCR simultaneously, standard model forms typical curve, then calculates respectively the content of acetic bacteria and milk-acid bacteria in testing sample according to typical curve.
For the fluorescence quantifying PCR method of acetic acid Pseudomonas, detect copy number 6.94 * 10 4-6.94 * 10 11During copies/ μ L scope, the amplification curve of acetic bacteria Real-time PCR is one group of typically curve of falling S, and the amplification curve baseline is smooth, and the exponential region is obvious and steepness is large, and platform area can be compiled in together, and linearity range is wider.The concentration of plasmid standard is higher, and cycle threshold CT is lower.The linear equation of this typical curve is y=-0.353x+13.897, and linearly dependent coefficient is 0.9975, has linear relationship preferably, according to formula E=10 -k-1 (slope of k-typical curve) calculated to such an extent that the amplification efficiency of Real-time PCR is 1.18, and between 0.8-1.2, amplification efficiency is desirable.Acetic bacteria Real-time PCR melting curve, single melting peak occurs at about 84 ℃, without primer dimer and non-specific product in addition, curve is steady, and the peak is sharp and narrow, and the melting temperature (Tm) homogeneous of each concentration plasmid is described, the amplified production specificity is good, is quantitatively reliable based on this.
For the fluorescence quantifying PCR method of genus lactubacillus, detect copy number 1.94 * 10 5-1.94 * 10 12During copies/ μ L scope, the amplification curve of milk-acid bacteria Real-time PCR is one group of typically curve of falling S, and this group amplification curve baseline is smooth, and the exponential region is obvious and steepness is large, and platform area can be compiled in together substantially, and linearity range is also very wide.The concentration of plasmid standard is higher, and cycle threshold CT is lower.The linear equation of this typical curve is y=-0.3534x+14.261, and linearly dependent coefficient is 0.996, has linear relationship preferably, according to formula E=10 -k-1 (slope of k-typical curve) calculated to such an extent that the amplification efficiency of Real-time PCR is 1.19, and between 0.8-1.2, amplification efficiency is desirable.Milk-acid bacteria Real-time PCR melting curve in addition, single melting peak appears at about 84.5 ℃, without primer dimer and non-specific product, curve is steady, peak point and narrow, the melting temperature (Tm) homogeneous of each concentration plasmid is described, the amplified production specificity is good, is quantitatively reliable based on this.
The method of acetic bacteria and milk-acid bacteria in fluorescent quantitative PCR technique detection vinegar unstrained spirits microflora, concrete steps are as follows:
1, design, the synthetic Auele Specific Primer that is directed to acetic acid Pseudomonas and genus lactubacillus.
2, the glucose vinegar acidfast bacilli that separation and purification is obtained (Gluco nacetobacter sp.) and the pentose milk-acid bacteria ( Lactobacillus pentosus) carry out shake-flask culture, utilize bacterium to extract test kit and respectively both nutrient solution is carried out DNA extraction.
3, respectively with the glucose vinegar acidfast bacilli (Gluco nacetobacter sp.) and the pentose milk-acid bacteria ( Lactobacillus pentosus) DNA be template, carry out pcr amplification take Ace-F/AceR and Lac-F/Lac-R as primer, obtain the purpose fragment, and the rubber tapping of PCR product reclaimed.
4, construction recombination plasmid, the purpose fragment is connected with pMD19-T Vector, transform the JM109 competent escherichia coli cell after connecting, picking transformant enlarged culturing, extract plasmid, and whether inserting pMD19-T Vector with PCR checking purpose fragment, the plasmid that is proved to be successful is as the standard model of quantitative fluorescent PCR.
5, the method for utilizing liquid nitrogen grinding, enzymic digestion and high salt to extract combination is extracted total DNA of different time points vinegar unstrained spirits in fermenting process group as testing sample.
6, the establishment of quantitative fluorescent PCR reaction system and reaction conditions.By repetition test, determine peak optimization reaction system and cycling condition.
,The Criterion curve, 8 gradients of recombinant plasmid doubling dilution, carry out simultaneously quantitative fluorescent PCR with testing sample, then the copy number of according to standard sample and CT value Criterion curve carry out quantitative analysis by acetic bacteria and milk-acid bacteria in the CT value Dichlorodiphenyl Acetate fermenting process different time vinegar unstrained spirits group of typical curve and testing sample.
Described primer refers to that its sequence is:
Acetic bacteria Genus-specific primers sequence (5 ' → 3 '):
Ace-F：CGCAAGGGACCTCTAACACA
Ace-R：ACCTGATGGCAACTAAAGATAGGG
Milk-acid bacteria specific primer sequence (5 ' → 3 '):
Lac-F：AGAACACCAGTGGCGAAGG
Lac-R：CAGGCGGAGTGCTTAATGC
Described quantitative fluorescent PCR, its reaction system is as follows:
SsoFast EvaGreen premixed liquid 10 mL
Forward primer 1 mL
Reverse primer 1 mL
Template DNA
10 ng
ddH2O To 20 mL
Described quantitative fluorescent PCR, the reaction conditions of its reaction system is:
95℃ Denaturation 30 s
95℃ Sex change 5 s
56℃ 10 s anneal
56 ℃ are read the fluorescent signal data, and cyclic amplification is 40 times altogether.
Described sample, the method of extracting combination with liquid nitrogen grinding, enzymic digestion and high salt is extracted total DNA of the group of different time points vinegar unstrained spirits in fermenting process, its concrete steps are as follows: take 2 g vinegar unstrained spirits, add liquid nitrogen fully to grind in mortar, be transferred to 50 mL centrifuge tubes.Add 6 mL DNA extraction buffers in centrifuge tube, then add 100 μ L N,O-Diacetylmuramidases (50 mg/mL), 37 ℃ are shaken 30 min on 225 rpm shaking tables.Add 1.5 mL 10% SDS in centrifuge tube, 65 ℃ of water-bath 2 h, every 15-20 min put upside down gently several under, centrifugal 10 min of room temperature 6000 * g.Collect supernatant.With supernatant liquor with isopyknic chloroform-primary isoamyl alcohol (24:1 volume ratio), extracting once, Virahol precipitation at room temperature 1 h with 0.6 times of volume, centrifugal 20 min of 16000 g, collection nucleic acid precipitation, with 70% washing with alcohol precipitation, be dissolved in 100 μ L TE or ddH2O after DNA precipitation drying, adding final concentration is 0.5 ug/mL RNaseA, and at 37 ℃ of lower water-bath digestion 2 h, to remove RNA, effect with 1.5% agarose gel electrophoresis validating DNA extraction, if band occurs about 10kb, DNA extraction success is placed in-20 ℃ of Refrigerator stores standby.
In the present invention, " standard substance " refer to the recombinant plasmid dna of known copy number, measure its OD value with Nanodrop2000 nucleic acid determination instrument, when the OD value during in 0.3 left and right, the result that records is comparatively credible, and every pipe is measured 3 times, averages, calculate the copy number of recombinant plasmid, specific formula for calculation is as follows:
C Mark=(OD 260nm* A * N * 6.02 * 10 23* 10 -6)/(660 * base logarithm)
Wherein: C Mark– DNA profiling concentration (copies/mL)
A – 0.05, reduction factor, i.e. 1 OD 260nm=0.05 mg/ (mL double-stranded DNA)
N – extension rate
6.02 * 10 23The – Avogadro constant number
Adopt in the present invention outer object of reference to make quantitative PCR, take the goal gene of concentration known as template, according to certain doubling dilution, then make canonical plotting, unknown sample is found out the copy number of correspondence according to typical curve.
In the present invention, fluorescence threshold usually with the fluorescent value of 10-15 circulation as threshold value, the cycle index of process when the CT value refers to that the fluorescent signal of amplified production reaches the threshold value of setting.
Description of drawings
The copy number dynamic changing curve during the fermentation of acetic bacteria microorganism belonging to genus in Fig. 1 unstrained spirits sample.
The copy number dynamic changing curve during the fermentation of genus lactubacillus microorganism in Fig. 2 unstrained spirits sample.
Embodiment
The feasibility of this invention is described below in conjunction with specific embodiment, below the test method of unreceipted actual conditions in embodiment, usually carry out extraction as bacterial genomes DNA according to the test kit explanation of normal condition or manufacturer.
Embodiment one: the preparation of standard model
1, the synthetic Auele Specific Primer that is directed to acetic acid Pseudomonas, milk-acid bacteria of design.
Primer sequence is:
Acetic bacteria Genus-specific primers sequence (5 ' → 3 '):
Ace-F：CGCAAGGGACCTCTAACACA
Ace-R：ACCTGATGGCAACTAAAGATAGGG
Milk-acid bacteria specific primer sequence (5 ' → 3 '):
Lac-F：AGAACACCAGTGGCGAAGG
Lac-R：CAGGCGGAGTGCTTAATGC
2, the extraction of pure bacterium DNA.
The glucose vinegar acidfast bacilli that separation and purification is obtained (Gluco nacetobacter sp.) and the pentose milk-acid bacteria ( Lactobacillus pentosus) carry out shake-flask culture, utilize bacterium to extract test kit and respectively both nutrient solution is carried out DNA extraction.
3, the clone of purpose fragment.
Respectively take the DNA of Pasteur's acetic bacteria and lactobacterium helveticus as template, carry out pcr amplification take Ace-F/AceR and Lac-F/Lac-R as primer, amplified production detects with 2% agarose gel electrophoresis, the purpose fragment of acetic acid Pseudomonas is approximately 110 bp, genus lactubacillus purpose clip size is 170 bp, and the rubber tapping of PCR product is reclaimed.
4, the preparation of escherichia coli jm109 competent cell.
On picking JM109 flat board in single bacterium colony to 5 mL LB substratum, 37 ℃, 220 rpm overnight incubation; The seed liquor of incubated overnight is transferred in 15 mL LB substratum with 2% inoculum size, and 37 ℃, 220 rpm are cultivated 1.5 h left and right to OD 600Between 0.3-0.5; In packing 1 mL bacterium liquid to 1.5 mL centrifuge tube, centrifugal rapidly after ice bath 20 min, 4 ℃, centrifugal 10 min of 5000 rpm; Thorough supernatant discarded adds the CaCl of 400 μ L 0.1 mol/L precoolings 2The resuspended thalline of solution, then ice bath 30 min; 4 ℃, centrifugal 10 min of 5000 rpm, abandoning supernatant is inverted 1 min; CaCl with 80 μ L 0.1 mol/L precoolings 2The resuspended thalline of solution is placed 30 min on ice.
5, the structure of recombinant plasmid.
1:3 ratio that pMD19-T Vector is connected with the purpose fragment connects, 16 ℃ of connections are spent the night, then will connect product and transform JM109 competence Bacillus coli cells, the competence Bacillus coli cells that then will transform is coated on the LB flat board that contains acillin (100 ug/mL), cultivate 12-16 h for 37 ℃, the positive bacterium colony of picking, concussion is cultivated in the liquid LB substratum that contains penbritin (100 ug/mL), 37 ℃, 8-10 h.Extract plasmid with the bacterium liquid of cultivating, utilize the primer of design to carry out the plasmid amplification checking at this, if sepharose has visible purpose band about 110 bp and 170 bp, can determine that the purpose fragment successfully inserts pMD19-T Vector, the plasmid of its extraction is done 10 doubling dilutions, measure the OD value, the calculating copy number of averaging, specific formula for calculation is as follows:
C Mark=(OD 260nm* A * N * 6.02 * 10 23* 10 -6)/(660 * base logarithm)
Wherein: C Mark– DNA profiling concentration (copies/mL)
A – 0.05, reduction factor, i.e. 1 OD 260nm=0.05 mg/ (mL double-stranded DNA)
N – extension rate
6.02 * 10 23The – Avogadro constant number
Embodiment two: the quantitative analysis of acetic acid Pseudomonas and genus lactubacillus in vinegar unstrained spirits microflora.
1, vinegar unstrained spirits sample collecting.
Take the fermentation of zhenjiang vinegar as research object, gather the vinegar unstrained spirits sample of different time points in the Vinegar Fermentation process, after sample collecting as can not in time extract DNA and should carry out immediately freezing treatment.
2, microorganism total DNA extracting method in the vinegar unstrained spirits.
Take 2 g vinegar unstrained spirits, add liquid nitrogen fully to grind in mortar, be transferred to 50 mL centrifuge tubes.Add 6 mL DNA extraction buffers in centrifuge tube, then add 100 μ L N,O-Diacetylmuramidases (50 mg/mL), 37 ℃ are shaken 30 min on 225 rpm shaking tables.Add 1.5 mL 10% SDS in centrifuge tube, 65 ℃ of water-bath 2 h, every 15～20 min put upside down gently several under, centrifugal 10 min of room temperature 6000 * g.Collect supernatant.With supernatant liquor with isopyknic chloroform-primary isoamyl alcohol (24:1 volume ratio), extracting once, Virahol precipitation at room temperature 1 h with 0.6 times of volume, centrifugal 20 min of 16000 g, collection nucleic acid precipitation, with 70% washing with alcohol precipitation, be dissolved in 100 μ L TE or ddH2O after DNA precipitation drying, adding final concentration is 0.5 ug/mL RNaseA, and at 37 ℃ of lower water-bath digestion 2 h, to remove RNA, with the effect of 1.5% agarose gel electrophoresis validating DNA extraction, if band occurs about 10 kb, DNA extraction success.
3, the optimization of quantitative fluorescent PCR system.
SsoFast EvaGreen premixed liquid 10 mL
Forward primer 1 mL
Reverse primer 1 mL
Template DNA
10 ng
ddH2O To 20 mL
4, the reaction conditions of quantitative fluorescent PCR the best.
95℃ Denaturation 30 s
95℃ Sex change 5 s
56℃ 10 s anneal
56 ℃ are read the fluorescent signal data, and cyclic amplification is 40 times altogether.
Melting curve is drawn, and is warming up to 95 ℃ from 65 ℃, reads plate one time every 0.5 ℃, keeps 0.05 s.
5, testing sample is quantitative.
In vinegar unstrained spirits sample, the acetic acid Pseudomonas is quantitative: according to the linear equation y=-0.353x+13.897 (R that is obtained by acetic acid Pseudomonas standard substance quantitative fluorescent PCR 2=0.9975) calculate the copy number of acetic bacteria microorganism belonging to genus in different time points vinegar unstrained spirits sample, thereby can access the variation tendency (showing as Fig. 1) of acetic acid Pseudomonas biomass in the Vinegar Fermentation process.
In vinegar unstrained spirits sample, genus lactubacillus is quantitative: according to the linear equation y=-0.3534x+14.261 (R that is obtained by genus lactubacillus standard substance quantitative fluorescent PCR 2=0.996) calculate the copy number of genus lactubacillus microorganism in different time points vinegar unstrained spirits sample, thereby can access the variation tendency (showing as Fig. 2) of genus lactubacillus biomass in the Vinegar Fermentation process.

Claims (4)

1. the method for acetic bacteria and milk-acid bacteria in a fluorescence quantitative PCR detection vinegar unstrained spirits, is characterized in that, concrete steps are as follows:
1. design, synthesize the Auele Specific Primer that is directed to acetic acid Pseudomonas and genus lactubacillus;
2. the glucose vinegar acidfast bacilli that separation and purification is obtained (Gluco nacetobacter sp.) and pentose milk-acid bacteria (Lactobacillus pentosus) carry out shake-flask culture, utilize bacterium to extract test kit and respectively both nutrient solution are carried out DNA extraction;
3. respectively take the DNA of glucose vinegar acidfast bacilli (Gluco nacetobacter sp.) and pentose milk-acid bacteria (Lactobacillus pentosus) as template, carry out pcr amplification take Ace-F/AceR and Lac-F/Lac-R as primer, obtain the purpose fragment, and the rubber tapping of PCR product is reclaimed;
4. construction recombination plasmid, the purpose fragment is connected with pMD19-T Vector, transform the JM109 competent escherichia coli cell after connecting, picking transformant enlarged culturing, extract plasmid, and whether inserting pMD19-TVector with PCR checking purpose fragment, the plasmid that is proved to be successful is as the standard model of quantitative fluorescent PCR;
5. the method for utilizing liquid nitrogen grinding, enzymic digestion and high salt to extract combination is extracted total DNA of different time points vinegar unstrained spirits in fermenting process group as testing sample;
6. establish quantitative fluorescent PCR reaction system and reaction conditions;
7. 8 gradients of recombinant plasmid doubling dilution, carry out simultaneously quantitative fluorescent PCR with testing sample, then the copy number of according to standard sample and CT value Criterion curve carry out quantitative analysis by acetic bacteria and milk-acid bacteria in the CT value Dichlorodiphenyl Acetate fermenting process different time vinegar unstrained spirits group of typical curve and testing sample.
2. the method for claim 1, is characterized by, and step Auele Specific Primer 1. is: acetic bacteria Genus-specific primers sequence (5 ' → 3 '):
Ace-F：CGCAAGGGACCTCTAACACA
Ace-R：ACCTGATGGCAACTAAAGATAGGG；
Milk-acid bacteria specific primer sequence (5 ' → 3 '):
Lac-F：AGAACACCAGTGGCGAAGG
Lac-R：CAGGCGGAGTGCTTAATGC。
3. the method for claim 1, is characterized by, and the step 5. described method of utilizing liquid nitrogen grinding, enzymic digestion and high salt to extract combination total DNA of extracting different time points vinegar unstrained spirits in fermenting process group as the method concrete operations of testing sample is:
take 2g vinegar unstrained spirits, add liquid nitrogen fully to grind in mortar, be transferred to the 50mL centrifuge tube, add the 6mLDNA extraction buffer in centrifuge tube, then add 100 μ L N,O-Diacetylmuramidases (50mg/mL), 37 ℃ are shaken 30min on the 225rpm shaking table, add 1.5mL 10%SDS in centrifuge tube, 65 ℃ of water-bath 2h, every 15～20min put upside down gently several under, the centrifugal 10min of room temperature 6000 * g, collect supernatant, with supernatant liquor with isopyknic chloroform-primary isoamyl alcohol (24: 1 volume ratios), extracting once, Virahol precipitation at room temperature 1h with 0.6 times of volume, the centrifugal 20min of 16000g, collection nucleic acid precipitation, with 70% washing with alcohol precipitation, be dissolved in 100 μ L TE or ddH2O after DNA precipitation drying, adding final concentration is 0.5ug/mlRNaseA, and at 37 ℃ of lower water-bath digestion 2h, to remove RNA, effect with 1.5% agarose gel electrophoresis validating DNA extraction, if band occurs about 10kb, DNA extraction success, the successful DNA solution that extracts is placed in-20 ℃ of Refrigerator stores standby.
4. the method for claim 1, is characterized by, and quantitative fluorescent PCR reaction system and reaction conditions that 6. step establishes are as follows:
Reaction system adopts the 20uL system, SsoFast EvaGreen premixed liquid 10 μ L wherein, forward primer 1 μ L, reverse primer 1 μ L, Template DNA10ng, ddH 2O adds to 20 μ L; Reaction conditions is 95 ℃ of denaturation 30s, 95 ℃ of sex change 5s, and 56 ℃ of annealing 10s, 56 ℃ are read the fluorescent signal data, and cyclic amplification is 40 times altogether.
CN2013100754663A 2013-03-11 2013-03-11 Method for detecting acetic bacteria and lactic acid bacteria in vinegar culture by using fluorescent quantitative PCR (Polymerase Chain Reaction) Pending CN103131785A (en)

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CN107523625A (en) * 2017-09-20 2017-12-29 贵州茅台酒股份有限公司 The quantitative analysis method of producing and ethanol and lactic acid producing key microorganisms in more micro- solid state fermentation
CN112048454A (en) * 2020-09-10 2020-12-08 兰州大学 Microbial composition, kit and method for evaluating quality of slurry
CN112143820A (en) * 2020-09-29 2020-12-29 广东省微生物研究所（广东省微生物分析检测中心） Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus
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CN103834745A (en) * 2014-03-27 2014-06-04 泸州品创科技有限公司 Quantitative analysis method of lactic acid bacteria and bacilli in yeast microbial community
CN103834745B (en) * 2014-03-27 2016-02-24 泸州品创科技有限公司 The quantitative analysis method of milk-acid bacteria and genus bacillus in Daqu microflora
CN103834744B (en) * 2014-03-27 2016-03-23 泸州品创科技有限公司 The quantitative analysis method of clostridium, milk-acid bacteria, genus bacillus, methanobacteria in the mud of cellar for storing things
CN107523625A (en) * 2017-09-20 2017-12-29 贵州茅台酒股份有限公司 The quantitative analysis method of producing and ethanol and lactic acid producing key microorganisms in more micro- solid state fermentation
CN107523625B (en) * 2017-09-20 2020-06-26 贵州茅台酒股份有限公司 Quantitative analysis method for key microorganisms producing ethanol and lactic acid in multi-microorganism solid state fermentation
CN112048454A (en) * 2020-09-10 2020-12-08 兰州大学 Microbial composition, kit and method for evaluating quality of slurry
CN112048454B (en) * 2020-09-10 2023-01-03 兰州大学 Microbial composition, kit and method for evaluating quality of serous fluid
CN112143820A (en) * 2020-09-29 2020-12-29 广东省微生物研究所（广东省微生物分析检测中心） Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus
CN112143820B (en) * 2020-09-29 2022-05-06 广东省微生物研究所（广东省微生物分析检测中心） Molecular marker, detection primer and detection method for identifying lactobacillus plantarum and lactobacillus pentosus
CN112980980A (en) * 2021-04-23 2021-06-18 江南大学 Molecular marker and kit for specific quantification of lactobacillus kunmaku and application
CN112980980B (en) * 2021-04-23 2023-08-15 江南大学 Molecular marker and kit for specific quantification of Jin Shancu lactobacillus and application

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