CN110117315B

CN110117315B - Post-acidification related gene of lactobacillus bulgaricus and application of post-acidification related gene in yoghourt

Info

Publication number: CN110117315B
Application number: CN201910507244.1A
Authority: CN
Inventors: 李晨; 田洪涛; 孙永胜; 谷新晰; 卢海强
Original assignee: Hebei Agricultural University
Current assignee: Hebei Agricultural University
Priority date: 2019-06-12
Filing date: 2019-06-12
Publication date: 2021-02-12
Anticipated expiration: 2039-06-12
Also published as: CN110117315A

Abstract

The invention discloses a post-acidification gene of lactobacillus bulgaricus participating in yoghourt and application thereof. The invention designs a primer LDB _ RS05285-F/R (Ldb1239-F/R), connects with an expression vector after PCR amplification, constructs a recombinant plasmid, converts the recombinant plasmid into lactic acid bacteria, constructs a recombinant bacterium, determines the survival rate of the recombinant bacterium and a control bacterium under acid and acid cold treatment, simultaneously inoculates LDB _ RS05285 gene overexpression strain and the control strain into inactivated yoghurt with the same bacterium amount, determines the viable bacterium number and the pH value of the yoghurt in the storage period, and establishes a method for cloning, expressing and function verification of the gene. The overexpression of the gene can effectively weaken the post acidification problem in the storage process of the yoghourt, improve the storage stability of the yoghourt and prolong the quality guarantee period of the yoghourt. Provides a new idea for solving the problem of post acidification of the yoghourt.

Description

Post-acidification related gene of lactobacillus bulgaricus and application of post-acidification related gene in yoghourt

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to cloning of a post-acidification related gene LDB _ RS05285 of lactobacillus bulgaricus and application thereof in yoghourt.

Background

The yoghourt is a dairy product which is prepared by taking fresh milk as a raw material, adding a leavening agent and other ingredients into the fresh milk, fermenting, cooling and filling. The milk is fermented by lactic acid bacteria, and the milk has the unprecedented effects, such as relieving lactose intolerance. The probiotics in the yoghourt also plays an important health-care role in improving the gastrointestinal function of a human body, resisting tumors, delaying aging, promoting the absorption of the human body and the like. Research shows that the pH value of the fermented milk with high product quality is 4.2-4.3 when the fermented milk is eaten, however, the pH value of the fermented milk can be continuously reduced in the storage process of the fermented milk, so that the fermented milk is too sour, a small amount of whey is separated out on the fermented milk, the sensory quality of the fermented milk is reduced, and the like, and the fermented milk is acidified after being fermented. Post-acidification of fermented milk refers to the phenomenon that after the fermentation of yogurt, the cells still grow and propagate during the process of storage, transportation, sale and eating, so that the fermented milk is unacceptable to consumers due to too high acidity and reduced sensory quality, and the phenomenon is called post-acidification of yogurt.

Lactobacillus delbrueckii subsp. bulgaricus (also called Lactobacillus delbrueckii subsp. bulgaricus) is widely applied to the fermented dairy product industry due to excellent fermentation performance, can ferment large-molecular nutrient substances into small-molecular nutrient components and endow products with special flavor, for example, lactose can be fermented to generate glucose and galactose which are easy to be absorbed by human bodies, and lactose intolerance can be relieved to a certain extent. In addition, l.bulgaricus also has effects of regulating gastrointestinal tract, inhibiting growth of harmful bacteria, enhancing immunity, etc. However, l.bulgaricus has a key problem in the fermentation industry production that restricts the development of the industry, namely post-acidification. In recent years, researches show that the control of the growth of lactobacillus bulgaricus is the key of acidification after the solution in the fermentation of the yoghourt, and scholars control the growth and acid production of the lactobacillus bulgaricus by methods such as pasteurization, strain mutagenesis, antibacterial substance supplement and the like. All the methods can relieve the process of post acidification of yogurt, but the effect is not absolutely inhibited, the environmental stress condition of the lactobacillus bulgaricus ATCC 11842 in the post acidification process of the yogurt is simulated in the early stage, the change of the gene expression amount in the process is measured through transcriptomics, and the analysis result shows that the lactobacillus bulgaricus ATCC 11842 can respond to the stress action of a low pH environment through a series of adaptability regulation, and mainly comprises the following steps: increasing the expression level of enzymes related to the glycolytic pathway and providing ATP for cells; the outward direction of the pyruvate metabolites is adjusted, so that the pyruvate metabolism is more shifted to the biosynthesis of fatty acid, however, the metabolic mechanism exits the acid response mechanism under the action of cold stress; degrading misfolded proteins while increasing the accuracy of protein translation; repair of DNA damage, etc. In the approaches, a plurality of genes involved in the post-acidification process of the yogurt are discovered, and the alleviation of the post-acidification degree of the yogurt by regulating and controlling the post-acidification key genes is a new idea and has no related report.

Disclosure of Invention

The present invention aims to provide a post-acidification related gene derived from Lactobacillus bulgaricus ATCC 11842.

The invention also aims to provide the application of the post-acidification related gene in inhibiting the post-acidification process of the lactobacillus bulgaricus yoghurt.

A post-acidification protein of Lactobacillus bulgaricus, which is a protein having one of the following amino acid residue sequences:

1) SEQ ID NO: 1;

2) the sequence of SEQ ID NO: 1 by substitution and/or deletion and/or addition of one or more amino acid residues, and has post-acidification inhibiting activity.

The gene sequence of the post-acidification protein of the lactobacillus bulgaricus is shown in a sequence table SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

A vector containing the post-acidification gene of lactobacillus bulgaricus.

Engineering bacteria containing the gene vector.

And (3) a primer for amplifying any fragment of the post-acidification gene of the lactobacillus bulgaricus.

The post-acidification protein of lactobacillus bulgaricus is applied to the process of delaying post-acidification of yoghourt.

The key gene LDB _ RS05285 of post-acidification of lactobacillus bulgaricus is obtained by the following method:

(1) inoculating lactobacillus bulgaricus ATCC 11842 and MRS culture medium, placing in a 42 ℃ incubator, culturing to the end of logarithm, and extracting bacterial genome;

(2) designing an LDB _ RS05285 specific primer to amplify a target gene, carrying out tailing reaction, connecting with a pMD19-T vector, screening positive clones, extracting plasmids, carrying out enzyme digestion verification and then sequencing;

(3) the three-dimensional structure of the protein coded by the gene is predicted;

(4) detecting the expression quantity of a target gene in the post-acidification process of the yoghourt by using an RT-qPCR technology;

(5) constructing a gene over-expression vector, introducing a recombinant vector into host bacteria by an electrotransformation method, and screening positive clones;

(6) detecting the expression quantity of the target gene in the recombinant bacteria by using an RT-qPCR technology;

(7) the application of the gene in the post acidification process of the yoghourt is verified.

The invention has the beneficial effects that: the LDB _ RS05285 gene cloned by the invention can weaken the post-acidification problem in the storage process of the yoghourt by excessively expressing the synthetic copper ion transporter in the lactobacillus, improve the storage stability of the yoghourt, prolong the quality guarantee period of the yoghourt and provide good guarantee for the research and development of weak post-acidification strains.

Drawings

FIG. 1 shows the results of extraction of the genome of Lactobacillus bulgaricus ATCC 11842 and amplification of a target gene.

FIG. 2 shows the predicted three-dimensional structure of the protein based on the amino acid sequence.

FIG. 3 is a graph showing the analysis of the expression level of a target gene in skim milk at different stages of fermentation.

FIG. 4 shows the PCR verification result of the recombinant vector transformed lactococcus lactis ML23 bacterial liquid.

FIG. 5 shows the measurement of the expression level of a target gene in a recombinant bacterium.

FIG. 6 is a comparison of viable count of recombinant bacteria and control bacteria in post-acidification process of yogurt.

FIG. 7 is a comparison of pH changes of post-acidification process of yogurt by recombinant bacteria and control bacteria.

Detailed Description

The invention is further described with reference to the following figures and specific embodiments.

EXAMPLE 1 cloning of target Gene and prediction of three-dimensional Structure of protein

Based on the gene sequence of LDB _ RS05285 in the genome of Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. bulbgaricus) ATCC 11842 published on NCBI, DNAMAN 6.0 software is adopted to design the upstream and downstream primers of the target gene:

LDB_RS05285-F：5-GACGAGCTCGCATGGAAAATAAAGAGGACC-3；

LDB_RS05285-R：5-AAAACTGCAGCATTGGATTACACGTTTTTAAT-3。

Ldb1239-F：5-GACGAGCTCGCATGGCAAATAAAGAGGACC-3；

Ldb1239-R：5-AAAACTGCAGCATTGGATTACACGTTTTTAAT-3。

PCR amplification is respectively carried out from the extracted lactobacillus genome (the left half picture in figure 1) by using high fidelity enzyme, pre-denaturation is carried out for 5min at 95 ℃, denaturation is carried out for 30s at 95 ℃, annealing is carried out for 30s at 52 ℃, extension is carried out for 2.5min at 72 ℃, 35 cycles are carried out, and extension is carried out for 10min at 72 ℃ after the cycles are finished (the PCR amplification result is shown in the right half picture in figure 1). After amplification is finished, ordinary Taq enzyme is used for carrying out 'A' reaction (72 ℃, 10min), after the reaction is finished, purification is carried out, a cloning vector pMD19-T is connected, plasmid pMD19-LDB _ RS05285 is constructed, escherichia coli DH5a competent cells are transformed, positive cloning strains are screened, and sequencing is carried out.

The amino acid sequence encoded by this gene was subjected to tertiary structure prediction of the protein using the Swiss-Model tool.

Example 2 analysis of expression level of LDB _ RS05285 Gene during fermentation in skim milk

The Lactobacillus bulgaricus ATCC 11842 is inoculated into a skim milk culture medium, and fermented skim milk with the initial fermentation stage, the middle and later stages of the fermentation logarithm, the fermentation end point and 6 different time nodes is selected and refrigerated in a refrigerator at 4 ℃ for 2h, 7d and 14d after the fermentation is finished according to the growth curve and the pH value change of the strain in the skim milk culture medium. Extracting total RNA of 6 periods, measuring the relative expression quantity of the target gene, and analyzing the change of the expression quantity of the gene in the process of storing the yoghourt to show that the gene participates in the post-acidification process of the yoghourt.

EXAMPLE 3 construction of recombinant expression strains

After the sequencing result is determined to be accurate, extracting plasmids pMD19-LDB _ RS05285 and pMG36e, simultaneously carrying out double enzyme digestion on the two plasmids by using PstI and SacI respectively, recovering the enzyme digestion product, connecting by using T4 DNA Ligase, transferring into escherichia coli DH5a competent cells again, screening positive clones, and carrying out sequencing comparison. After the sequencing result is correct, extracting the plasmid pMG36e-LDB _ RS05285 by shaking the bacteria, transferring the recombinant plasmid into lactococcus lactis ML23 competent cells by using an electric transfer instrument, culturing at 30 ℃, selecting a transformant for PCR verification (figure 2), and detecting the correct fragment size as the recombinant bacteria ML23-36e-LDB _ RS05285 by electrophoresis.

Example 4 qRT-PCR detection of Gene LDB _ RS05285 expression in lactococcus lactis

In the test, qRT-PCR technology is adopted to analyze the expression quantity of the target gene LDB _ RS05285 in the recombinant strain and the control strain, and the expression of the target gene is also detected in the control strain because the L.lactis ML23 strain also has a sequence which has the same function with the target gene and has higher homology with the target gene. As shown in FIGS. 3 to 5, the target gene was expressed in both the recombinant strain (ML23-36e-LDB _ RS05285) and the control strain (ML23-36e) and the expression level of the recombinant strain was significantly higher than that of the control strain (P < 0.05).

Example 5 Effect of recombinant strains on post acidification Process of yogurt

Inoculating activated Lactobacillus bulgaricus ATCC 11842 into skim milk culture medium, culturing at 37 deg.C for 11h until skim milk coagulates, pasteurizing, inactivating lactobacillus in yogurt, cooling to room temperature, inoculating the LDB _ RS05285 gene overexpression strain and control strain cultured in advance into inactivated yogurt with the same bacterial amount, and refrigerating at 4 deg.C for 0d, 1d, 2d, 3d, 4d, 5d, 6d, and 7 d. Sampling according to time to determine viable count and pH value. The results show that: with the prolonging of the storage time, the number of viable bacteria is gradually reduced, the decline of 0-3d is obvious, and the decline of 4-7d tends to be gentle (figure 6). In the later period of storage, the total number of colonies of the strain in which the target gene was overexpressed was 92.3% of the control group (P > 0.05). The pH of the control strain was significantly lower than that of the gene of interest overexpressing strain (P <0.05) during the storage period of the yogurt (fig. 7). The result shows that the overexpression of the LDB _ RS05285 gene weakens the post-acidification problem in the storage process of the yoghourt to a certain extent, improves the storage stability of the yoghourt and prolongs the quality guarantee period of the yoghourt.

Sequence listing

<110> university of agriculture in Hebei

<120> post-acidification gene of lactobacillus bulgaricus and application thereof

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<213> Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. bulgaricus)

<400> 1

Met Glu Asn Lys Glu Asp His Met Asn Met Lys Asn Met Ser Ala Lys

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Asn Met Glu Asn Asn Glu Ser Asn Met Cys His Met Asp His Asp Met

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Thr Glu Thr Asp His Asn Gln Met Asn Met Asp His Asp Met Ala Glu

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Thr Asp His Asn Gln Met Asn Met Asp His Asp Met Ala Glu Met Asp

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His Ser Gln Met Asn Met Asn His Gly Asp Met Asp Met Ala Gly Thr

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Asp Met Met Met His Gly Gly Thr Met Met His Met Gly Asn Leu Lys

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Val Lys Phe Trp Val Ser Val Val Leu Ala Ile Pro Val Leu Leu Leu

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Ala Pro Ile Met Gly Leu Asn Val Ser Ile Leu Ser Phe Ser Ser Pro

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Leu Ile Val Gly Ile Ile Ile Val Leu Phe Asp Thr Ala Leu Tyr Phe

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Tyr Gly Gly Met Pro Phe Leu Lys Gly Ala Lys Ala Glu Ile Gln Asn

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Lys Ser Pro Glu Met Met Thr Leu Val Thr Leu Gly Ile Ser Val Ser

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Tyr Phe Tyr Ser Leu Tyr Ala Phe Ile Ala Asn Asn Phe Leu Asn Pro

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Ala Asn His Val Met Asp Phe Ser Phe Glu Leu Ala Thr Leu Ile Leu

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Ile Met Leu Leu Gly His Trp Ile Glu Met Asn Ala Leu Met Gly Ala

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Gly Asp Ala Leu Gln Lys Met Ala Ala Leu Leu Pro Lys Thr Ala His

225 230 235 240

Leu Val Thr Asp Asn Gly Glu Thr Lys Glu Val Pro Val Ser Asp Leu

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Lys Val Gly Gln Ala Phe Gln Val Arg Ser Gly Glu Ser Ile Pro Ala

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Asp Gly Val Ile Thr Ala Gly Glu Ser Thr Val Asn Glu Ala Leu Val

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Thr Gly Glu Ser Ala Ala Val Pro Lys Lys Val Gly Asp Lys Val Ile

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Gly Gly Ala Thr Asn Asn Asn Gly Thr Leu Thr Val Lys Ile Ser Gly

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Thr Gly Asp Ser Gly Tyr Leu Ser Gln Val Met Lys Met Val Lys Asn

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Ala Gln Gln Ala Lys Ser Lys Ala Glu Asp Lys Ala Asp Leu Val Ala

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Lys Tyr Leu Phe Tyr Ala Ala Phe Ser Val Gly Ile Ile Ala Phe Phe

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Ala Trp Leu Pro Gln Gly Leu Ala Thr Ala Met Thr Ile Met Val Thr

370 375 380

Val Phe Val Ile Ala Cys Pro His Ala Leu Gly Leu Ala Ile Pro Leu

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Val Val Ser Arg Ser Thr Thr Ile Gly Ala Gln Asn Gly Leu Leu Val

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Arg Asn Arg Gln Ala Ile Glu Ala Ser Gln His Val Ser His Val Leu

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Leu Asp Lys Thr Gly Thr Leu Thr Glu Gly Lys Phe Thr Val Asn Ala

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Leu Ile Pro Asn Asp Gly Ile Asp Glu Thr Thr Leu Leu Ser Arg Leu

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Ala Ala Leu Glu Ser Asn Ser Thr His Pro Leu Ala Gln Ala Ile Ile

465 470 475 480

Thr Glu Ala Gln Ala Lys Gly Ile Glu Val Val Ala Ala Glu Lys Ser

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Gln Asn Ile Pro Gly Val Gly Ile Ser Gly Asn Val Asp Gly Thr Asp

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Tyr Met Ile Val Asn Gly Asn Tyr Leu Lys Lys Gln Gly Ile Lys Phe

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Asp Glu Ala Ala Ala Asp Lys Trp Ala Ala Lys Gly Asn Ser Val Ser

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Phe Leu Leu Gln Gly Thr Gln Val Gln Gly Met Val Ala Glu Gly Asp

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Thr Ile Lys Ala Gly Ala Lys Glu Leu Ile Ser Gly Leu Gln Lys Arg

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Gly Ile Thr Pro Val Met Leu Thr Gly Asp Asn Pro Lys Ala Ala Glu

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His Val Ala Asn Leu Leu Gly Leu Thr Glu Phe His Ala Gly Leu Leu

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Pro Asp Asp Lys Gln Lys Ile Val Ala Asp Tyr Gln Ala Lys Gly Asn

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His Val Ile Met Val Gly Asp Gly Val Asn Asp Ala Pro Ser Leu Ala

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Ala Ala Asp Ile Gly Ile Ala Ile Gly Ala Gly Thr Asp Val Ala Ile

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His Phe Leu Asp Leu Ala Lys Ile Thr Asn Arg Lys Met Val Gln Asn

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<213> Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. bulgaricus)

<400> 2

atggaaaata aagaggacca tatgaatatg aaaaacatga gtgctaagaa tatggaaaat 60

aatgaatcaa atatgtgtca tatggatcac gatatgaccg aaacggatca taatcaaatg 120

aacatggacc acgatatggc cgaaacggat cataatcaaa tgaacatgga ccacgatatg 180

gccgaaatgg atcacagcca aatgaacatg aatcacggtg atatggatat ggctgggacc 240

gacatgatga tgcacggtgg cacaatgatg catatgggga atttgaaagt taaattttgg 300

gtctccgttg tcttagcgat tccagtttta ctgttagcac caatcatggg tttaaacgtt 360

tccatcctta gtttcagttc accgctaatt gttggcatta tcatcgtttt gtttgataca 420

gcactttact tttatggcgg aatgccattt ttaaaggggg ctaaagcgga aattcagaat 480

aaatctcctg aaatgatgac gctagtaacc cttggaattt ccgtttcgta tttctacagt 540

ttgtacgcat ttattgccaa caacttcttg aacccggcaa atcatgtaat ggatttttcg 600

ttcgaacttg caaccctgat tttaattatg cttctaggac actggattga aatgaatgca 660

ttgatggggg ctggggatgc cttacaaaag atggcggccc tgttgcctaa gacggcccat 720

ctagttacag ataatggtga aacaaaagaa gtgccagtat ctgatttaaa agttggtcaa 780

gctttccaag tgcgttcagg tgagagtatt ccagccgatg gtgttattac ggctggggag 840

tcgaccgtga atgaagcact ggtaaccggt gaatctgctg ccgttcccaa gaaagttggc 900

gataaggtca ttggtggtgc aaccaacaat aacgggacgc taacggttaa aattagtggt 960

actggtgact ccggctatct ttctcaagta atgaaaatgg ttaaaaatgc tcagcaagct 1020

aaatctaaag cagaagataa agctgattta gttgccaagt atctatttta cgcggcattt 1080

agtgttggga ttattgcttt ctttgcctgg ttaccccagg gattggcgac tgcaatgacg 1140

atcatggtga ccgtcttcgt gattgcttgc ccgcatgcat taggattagc gattccatta 1200

gtggtttctc gttctactac gattggcgct caaaatgggc tattagttcg aaatcgccaa 1260

gccattgaag caagtcaaca tgttagccac gttctcttgg ataaaactgg cacgttaaca 1320

gaaggtaaat ttacggtgaa tgcattgatt ccaaatgatg ggattgacga aacaacgtta 1380

ttaagccgac tggccgccct tgaaagtaat tcgactcatc cgctggccca agcaatcatt 1440

actgaagccc aagcgaaggg cattgaagtc gttgcggctg aaaagtctca aaatattccg 1500

ggcgttggta tttccggtaa tgttgatggc actgactata tgattgttaa tggtaactat 1560

ttaaagaagc aagggatcaa gtttgacgag gccgctgctg ataaatgggc tgctaagggt 1620

aattccgtca gcttcctatt gcagggcacc caagttcaag gaatggttgc tgaaggcgac 1680

accatcaaag cgggtgctaa ggaattaatt agtggtcttc agaagcgagg aattacccct 1740

gtaatgctca ctggcgataa tccaaaagcc gcggaacacg ttgctaactt actaggattg 1800

actgaattcc atgcaggcct attaccagat gataagcaaa agattgttgc tgattatcaa 1860

gcaaagggca atcacgtcat catggttggt gacggcgtaa atgacgcacc aagtcttgcc 1920

gcggccgata ttggaattgc aattggtgcc ggaaccgatg ttgccattga ttccgctgat 1980

gttgtgttag ttaaatcaga acccagcgat attttacatt ttcttgattt ggctaaaatc 2040

acaaatcgga aaatggttca aaatctctgg tggggagcag gctacaatat tgtcgcaatt 2100

ccacttgctg ccggtgtgtt gtcatttatt ggaatcattc tagacccagc cgttggtgct 2160

gtggtcatgg ctatgtcgac aattatcgtg gcaattaatg cgatgggatt gactgctgaa 2220

aagattaaaa acgtgtaa 2238

<210> 3

<211> 2289

<212> DNA

<213> Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. bulgaricus)

<400> 3

atggcaaata aagaggacca tatgaacatg aaaaacatga gtgctaagaa tatggaaaat 60

aaagaggacc atatgaatat gaaaaacatg agtgctaaga atatggaaaa taatgaatca 120

aatatgtgtc atatggatca cgatatgacc gaaacggatc ataatcaaat gaacatggac 180

cacgatatgg ccgaaacgga tcataatcaa atgaacatgg accacgatat ggccgaaatg 240

gatcacagcc aaatgaacat gaatcacggt gatatggata tggctgggac cgacatgatg 300

atgcacggtg gcacaatgat gcatatgggg aatttgaaag ttaaattttg ggtctccgtt 360

gtcttagcga ttccagtttt actgttagca ccaatcatgg gtttaaacgt ttccatcctt 420

agtttcagtt caccgctaat tgttggcatt atcatcgttt tgtttgatac agcactttac 480

ttttatggcg gaatgccatt tttaaagggg gctaaagcgg aaattcagaa taaatctcct 540

gaaatgatga cgctagtaac ccttggaatt tccgtttcgt atttctacag tttgtacgca 600

tttattgcca acaacttctt gaacccggca aatcatgtaa tggatttttc gttcgaactt 660

gcaaccctga ttttaattat gcttctagga cactggattg aaatgaatgc attgatgggg 720

gctggggatg ccttacaaaa gatggcggcc ctgttgccta agacggccca tctagttaca 780

gataatggtg aaacaaaaga agtgccagta tctgatttaa aagttggtca agctttccaa 840

gtgcgttcag gtgagagtat tccagccgat ggtgttatta cggctgggga gtcgaccgtg 900

aatgaagcac tggtaaccgg tgaatctgct gccgttccca agaaagttgg cgataaggtc 960

attggtggtg caaccaacaa taacgggacg ctaacggtta aaattagtgg tactggtgac 1020

tccggctatc tttctcaagt aatgaaaatg gttaaaaatg ctcagcaagc taaatctaaa 1080

gcagaagata aagctgattt agttgccaag tatctatttt acgcggcatt tagtgttggg 1140

attattgctt tctttgcctg gttaccccag ggattggcga ctgcaatgac gatcatggtg 1200

accgtcttcg tgattgcttg cccgcatgca ttaggattag cgattccatt agtggtttct 1260

cgttctacta cgattggcgc tcaaaatggg ctattagttc gaaatcgcca agccattgaa 1320

gcaagtcaac atgttagcca cgttctcttg gataaaactg gcacgttaac agaaggtaaa 1380

tttacggtga atgcattgat tccaaatgat gggattgacg aaacaacgtt attaagccga 1440

ctggccgccc ttgaaagtaa ttcgactcat ccgctggccc aagcaatcat tactgaagcc 1500

caagcgaagg gcattgaagt cgttgcggct gaaaagtctc aaaatattcc gggcgttggt 1560

atttccggta atgttgatgg cactgactat atgattgtta atggtaacta tttaaagaag 1620

caagggatca agtttgacga ggccgctgct gataaatggg ctgctaaggg taattccgtc 1680

agcttcctat tgcagggcac ccaagttcaa ggaatggttg ctgaaggcga caccatcaaa 1740

gcgggtgcta aggaattaat tagtggtctt cagaagcgag gaattacccc tgtaatgctc 1800

actggcgata atccaaaagc cgcggaacac gttgctaact tactaggatt gactgaattc 1860

catgcaggcc tattaccaga tgataagcaa aagattgttg ctgattatca agcaaagggc 1920

aatcacgtca tcatggttgg tgacggcgta aatgacgcac caagtcttgc cgcggccgat 1980

attggaattg caattggtgc cggaaccgat gttgccattg attccgctga tgttgtgtta 2040

gttaaatcag aacccagcga tattttacat tttcttgatt tggctaaaat cacaaatcgg 2100

aaaatggttc aaaatctctg gtggggagca ggctacaata ttgtcgcaat tccacttgct 2160

gccggtgtgt tgtcatttat tggaatcatt ctagacccag ccgttggtgc tgtggtcatg 2220

gctatgtcga caattatcgt ggcaattaat gcgatgggat tgactgctga aaagattaaa 2280

aacgtgtaa 2289

Claims

1. The application of the post-acidification protein of lactobacillus bulgaricus in the process of delaying the post-acidification of yoghourt is characterized in that the gene sequence of the post-acidification protein of lactobacillus bulgaricus is shown as a sequence table SEQ ID NO: 2 and SEQ ID NO: 3, respectively.