CN114317789B - Marker reflecting acidification degree after yoghurt and application thereof - Google Patents
Marker reflecting acidification degree after yoghurt and application thereof Download PDFInfo
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Abstract
The invention discloses a marker for reflecting the acidification degree after yoghurt and application thereof. The marker is a transcription or post-transcription translation product of the LDB_RS05555 gene. Compared with the traditional method for evaluating the post-acidification degree by measuring the acidity value change in the storage period of the yoghurt, the LDB_RS05555 gene marker disclosed by the invention can be used for rapidly and accurately predicting the post-acidification performance of the strain, is time-saving and labor-saving, and has the predictive advantage which is not possessed by the traditional method.
Description
Technical Field
The invention belongs to the technical field of post-yoghurt acidification inhibition, and particularly relates to a marker for reflecting the post-yoghurt acidification degree and application thereof.
Background
The post-acidification of the yoghurt refers to the phenomenon that after the fermentation of the yoghurt is finished, particularly in the storage process, lactobacillus bulgaricus with stronger acid resistance continuously produces acid to cause the deterioration of the mouthfeel of the yoghurt. In response to this problem, many scholars have proposed different measures to suppress the occurrence of post-acidification. For example, by changing the ratio of cocci to bacilli in the starter culture (Torriani et al,1996,International Dairy Journal 6,625-636); glucose oxidase (Cruz et al,2013,Food Research International 51,723-728) and peroxidase and the like (Nakada et al,1996,International Dairy Journal 6,33-42) were added. Evaluating the extent of post-acidification inhibition by these measures often requires measuring the pH or acidity change throughout storage after the yoghurt fermentation is completed. It is therefore necessary to find biomarkers that can quickly and accurately reflect the degree of acidification after yoghurt. The inventors simulate the acid-post-acidification generation process in vitro in the early stage, and reveal candidate genes involved in post-acidification through a transcriptome sequencing combined with fluorescent quantitative PCR technology. These genes are quite closely related to post-acidification, and the inventors have found a post-acidification specific gene among these genes. The method for screening the weak post-acidification strain and evaluating post-acidification inhibition measures by using the biomarker is particularly important.
The current evaluation of post-acidification performance of the strain and the effect of post-acidification inhibition measures is mainly based on determining the acidity or pH change of the yoghurt during the whole storage process. The traditional method has the advantages that on one hand, the workload is large, the time is tedious, and the evaluation of the acidification degree after the yoghurt cannot be realized in a short period; on the other hand, the traditional method can not predict the change trend of the acidification degree after the yoghurt. At present, from the gene perspective, a molecular biological means is utilized to screen genes which can rapidly and accurately reflect the acidification degree after yoghurt as biomarkers, and no report is yet made.
Disclosure of Invention
The invention aims to provide a marker reflecting the acidification degree after yoghurt and application thereof.
A marker reflecting the degree of post-yoghurt acidification, said marker being the transcriptional or post-transcriptional translation product of the ldb_rs05555 gene.
The nucleotide sequence of the LDB_RS05555 gene is shown in a sequence table SEQ ID NO. 1, and the amino acid sequence of the encoded protein is shown in a sequence table SEQ ID NO. 2.
The coded protein of the LDB_RS05555 gene also comprises an amino acid sequence with the same function and formed by replacing, deleting or adding one or more amino acids in the amino acid sequence shown in a sequence table SEQ ID NO. 2, and a nucleotide sequence for coding the amino acid sequences.
The LDB-RS 05555 gene has 855 bases and codes for 284 amino acids. The protein has a molecular formula of C 1445 H 2289 N 387 O 417 S 6 The theoretical isoelectric point is 9.04, and consists of 20 common amino acids, wherein the highest amino acid content is threonine (Leu) 11.3%, the second alanine (Ala) 8.1%, glycine (Gly) 7.7%, and the lowest content is cysteine (Cys) 0.7%. The instability index is 38.37, indicating that the protein of interest is a stable protein. The average hydrophobicity of the LDB_RS05555 gene is-0.168, which indicates that the protein is a hydrophilic protein. NCBI describes this gene as a LysR family transcriptional regulator.
The fermented bacteria after acidosis are lactobacillus bulgaricus.
Use of the expression level of ldb_rs05555 gene in predicting post-yoghurt acidification process.
The invention utilizes five lactobacillus bulgaricus (named as Lb-S1, LD, DR,11842, S-1) with different post acidification properties to respectively ferment yoghourt, and after the yoghourt is cured, the yoghourt is stored at a low temperature of 4 ℃ for 2 hours, and then is respectively sampled for RNA extraction and cDNA synthesis. The rest yoghurt is kept under the condition of 4 ℃ for storage, the titrating acidity of the yoghurt is measured every 3.5 days, and the acidity change of the yoghurt in the 21-day low-temperature storage process is measured. Using cDNA as a template and rpob as an internal reference gene, the inventors selected 55 genes from 69 post-acidification candidate genes for relative expression amount measurement. By using correlation analysis, the relation between the expression level of LDB_RS055555 and the difference between the acidity values on day 3.5 is explored and a corresponding regression formula is obtained. The screened genes showing significant correlation were verified using a strain of lactobacillus bulgaricus (R2-6) isolated from xinjiang yoghurt. R2-6 was inoculated into skim milk, fermented with yogurt, and stored at 4℃for 2 hours after curd, sampled, and RNA was extracted for reverse transcription to synthesize cDNA, in the same manner as described above. The expression level of the target gene was measured by the same method as described above using cDNA as a template. And carrying the measured expression quantity into a corresponding regression formula to calculate a theoretical acidity value difference value. The remaining yoghurt also continued to be stored at 4 ℃ and the titrated acidity of the yoghurt over a 21-day shelf life was measured every 3.5 days. And performing significance difference analysis on the theoretical acidity value and the actual measured acidity value by using T test. For the genes with insignificant differences, the acid production performance of the strain in the fermentation process can be predicted according to the expression quantity of the genes, and the genes are just post-acidification biomarker genes to be screened.
The invention has the beneficial effects that: compared with the traditional method for evaluating the post-acidification degree by measuring the acidity value change in the storage period of the yoghurt, the LDB_RS05555 gene marker disclosed by the invention can be used for rapidly and accurately predicting the post-acidification performance of the strain, is time-saving and labor-saving, and has the predictive advantage which is not possessed by the traditional method. In addition, more potential post-acidification inhibition measures can be screened by using the biomarker genes.
Drawings
FIG. 1 is a correlation analysis of the difference between the relative expression level of a target gene and the acidity value.
FIG. 2 shows the suitability analysis of the biomarker gene LDB-RS 05555 in mutants.
Detailed Description
The present invention will be described more fully hereinafter in order to facilitate an understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
Example 1 acidity determination during fermentation and storage of yogurt
Five different Lactobacillus bulgaricus strains (Lb-S1, LD, DR,11842, S-1) cultured to mid-log stage were inoculated into 10mL of skim milk medium at an inoculum size of 1%, and were subjected to stationary culture at 42℃to perform subculture in the skim milk medium at the inoculum size until the curd time was stable (about 5 passages). The acidity change of the yoghurt during fermentation is measured by referring to the method of GB5009.239-2016, and the acidity value of the fermentation end point is recorded. After curd, the yoghurt is stored in a refrigerator at 4 ℃. After 2 hours of storage, each group of samples was individually aspirated 2mL of yoghurt in an RNase-free EP tube and stored at-80℃for subsequent RNA extraction. The remaining yoghurt samples were kept at 4 ℃ for storage and the titrated acidity of the yoghurt was measured on days 3.5, 7, 10.5, 14, 17.5 and 21 after the curd and the difference in acidity values was calculated on these days, respectively.
Difference in acidity values = acidity value measured on the day-fermentation end point acidity value.
Example 2 RNA extraction, cDNA Synthesis and determination of relative Gene expression levels
And taking out the yogurt samples stored at low temperature, and respectively placing the yogurt samples in a mortar precooled by liquid nitrogen. Liquid nitrogen was added and the samples were powdered and transferred to RNase-free EP tubes. Total cellular RNA was extracted using Total RNA Extraction Kit from Beijing Soy Corp. RNA quality was assessed using the A280/A260 value of the Thermo Scientific NanoDrop ND-1000 assay sample RNA. Further, beijing full-type gold organism is adopted for RNA meeting the quality requirement
One-Step gDNA Removal and cDNA Synthesis SuperMix cDNA was synthesized. Using cDNA as template, beijing full gold organism +.>
Green qPCR SuperMix the relative expression level of 14 target genes selected from the post-acidification candidate genes screened in the earlier stage was determined. The reaction was performed on a Light Cycler 96 instrument manufactured by Roche company, using rpob as an internal reference gene. Based on the genomic sequence of Lactobacillus bulgaricus ATCC11842 published on NCBI, the upstream and downstream primers of the target gene and the reference gene were designed using DNAMAN 6.0 software (Table1). The reaction system is as follows: />
Green qPCR SuperMix 10. Mu.L, 1. Mu.L each for upstream and downstream primers, 2. Mu. L, ddH for template 2 O6. Mu.L. The PCR reaction procedure was as follows: 94℃pre-denatured 300S,94℃denatured 5S,51℃annealed 15S,72℃extended 10S for 45 cycles. After the reaction was completed, 2 was used -ΔΔCt The relative expression level of the target gene is calculated by the method.
TABLE 1 primers upstream and downstream of the target and reference genes for correlation analysis based on transcriptome sequencing screening
TABLE 2LDB_RS05555 Gene expression level predicted acidity value and actual measured acidity value difference significance analysis
Note that: * Representing the difference in significance level 0.05, representing the difference in significance level 0.01
Example 3 screening and validation of post-acidification biomarker genes
Correlation analysis was performed on the difference between the relative expression level of the target gene and the acidity value by using GraphPad Prism 8.0.1 software, and as a result, it was found that there was a significant correlation between the ldb_rs05555 gene at 3.5 days (fig. 1). Further, the reliability of these several genes was verified by fermenting yogurt with a strain of Lactobacillus bulgaricus (R2-6) isolated from Xinjiang yogurt. Likewise, the titrated acidity of the yogurt was measured on days 3.5, 7, 10.5, 14, 17.5 and 21 after curd and the difference in acidity values at these days was calculated, respectively. RNA extraction, cDNA synthesis and qPCR reactions were performed according to the methods and procedures described above. And substituting the measured gene expression quantities into corresponding regression equations to obtain the predicted acidity value difference value. And adding the acidity value difference value and the fermentation end point acidity value to obtain a predicted acidity value. The SPSS 19.0 software is used for carrying out independent sample T test on the predicted acidity value and the actual acidity value, and the result shows that the predicted result and the actual result of the LDB_RS05555 gene are not obvious, so that the LDB_RS05555 gene can be used as a biomarker for predicting the acidification degree after yoghurt.
Example 4 determination of acid production Performance of post-chemically induced weakened acidification strains by LDB_RS05555 Gene as post-yoghurt acidification biomarker
The weak post-acidification strain rp-1 stored in a laboratory fungus library is taken as a starting strain (Lb-S1 is wild type, thioneomycin is adopted for mutation breeding), and the applicability of LDB_RS05555 serving as a weak post-acidification biomarker in mutants is explored. The overnight activated Lactobacillus bulgaricus Lb-S1 and rp-1 are inoculated into skimmed milk according to the inoculation amount of 1%, and the passages are repeated until the curd time is stable, and the acidity value at the fermentation end point is recorded respectively. And (3) storing the curd yoghurt at 4 ℃, sampling and extracting RNA respectively after 2 hours, and measuring the relative expression quantity of the LDB_RS05555 genes. The remaining yoghurt was kept at 4 ℃ for 21 days and the titrated acidity of the yoghurt was measured every 3.5 days. As a result, as shown in FIG. 2, the acidity value increased by about 9.62℃T and 7.16℃T when the yogurt fermented with Lb-S1 and rp-1 was stored at 4℃until day 3.5. However, strain rp-1 was significantly higher than Lb-S1 in terms of the relative gene expression level. This shows that in the mutant, the expression level of the LDB_RS05555 gene is still in negative correlation with the acid production energy of the strain, and that LDB_RS05555 is still applicable in the mutant as a weak post-acidification biomarker.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Sequence listing
<110> university of agriculture in Hebei
<120> a marker reflecting the degree of acidification after yoghurt and its use
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 855
<212> DNA
<213> Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus)
<400> 1
atgaatttca agcagctcaa ttactttctg gtcgtagccg aggaagggca gattacttca 60
gccgccaagc ggctctttgt cggccagcca gccctcagct accaattaaa gcaattagaa 120
gaggagctag gggctaagct ctttatccgt cagccttatg gaattgaact gaccgaggct 180
ggaaaaaagc tgcgggatta tgctggtcag attttgacat tagccaaaaa cgtccaaact 240
gaaattacgg agctggaccg gggcgggtgg ggaagggtta aattaggttc cgtctcttct 300
tcaatcggcg agcttccttc cgccaaattg acggcttttg cccgccagca tcccggcgtc 360
tcctttgaca tctacgaaga caacactttc ggcatcctgg acaagctgca gggcaacctg 420
ctggacctgg ccatcgtccg caccccctac aaccgggtca acttgaactt tgtcgacatc 480
agcgatgagc cgatgaccgc ggttacctgc caggactttt tccctggcaa gggaaaagta 540
gggctgcagg acctggccgg ccagcccttg gtcatctacc ggcgctttga ggacatgttc 600
aaggaagcct tcgcggaaaa aggtctcacc ccaaattttg tggttaagtg cgatgattcc 660
agaaccgcta tccgctggag cgactctggc ctggggattg ccctggtgcc ggaatcaatc 720
gccaaaactt atgccaaagg ccacatctac ccaatcgact cggctaagtg gcggacccgc 780
ctgcagctag tctggcgcaa agaccgggtg gttaccccct taatgcggca aattattgcc 840
ctatatgcaa aataa 855
<210> 2
<211> 122
<212> PRT
<213> Lactobacillus bulgaricus (Lactobacillus delbrueckii subsp. Bulgaricus)
<400> 2
Met Ser Lys Phe Gly Ser Phe Leu Leu Gly Ala Val Phe Gly Ala Ala
1 5 10 15
Ala Gly Val Ala Ala Ala Ser Leu Ile Ser Asp Asp Lys Leu Lys Glu
20 25 30
Val Lys Asp Lys Ile Lys Ser Asn Pro Asn Val Glu Asp Leu Lys Glu
35 40 45
Lys Tyr Asp Asn Gly Thr Glu Ile Leu Lys Asn Gln Leu Ala Ser Phe
50 55 60
Pro Lys Asn Ile Glu Asp Asp Ser Glu Leu Lys Asp Phe Asp Asp Ile
65 70 75 80
Val Ile Asp Asp Ser Ala Asn Gly Ala Gly Glu Gln Asp Ala Ser Asp
85 90 95
Leu Asp Asn Ala Glu Lys Ser Glu Glu Gly Glu Glu Lys Pro Glu Glu
100 105 110
Pro Ser Thr Pro Thr Ser Pro Asp Gln Glu
115 120
Claims (1)
1.LDB_RS05555Use of a gene for predicting the post-yoghurt acidification degree, characterized in that the following is saidLDB_RS05555The nucleotide sequence of the gene is shown in a sequence table SEQ ID NO. 1; the fermented bacteria after acidosis are lactobacillus bulgaricus.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110117315A (en) * | 2019-06-12 | 2019-08-13 | 河北农业大学 | Post-acidification related gene of lactobacillus bulgaricus and application of post-acidification related gene in yoghourt |
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| CN110117315A (en) * | 2019-06-12 | 2019-08-13 | 河北农业大学 | Post-acidification related gene of lactobacillus bulgaricus and application of post-acidification related gene in yoghourt |
Non-Patent Citations (7)
| Title |
|---|
| Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 complete genome;GenBank;GenBank;CR954253 * |
| Niacin inhibits post-acidification of yogurt based on the mining of LDB_RS00370 biomarker gene;Xin Zhang等;Food ResearchInternational;全文 * |
| 保加利亚乳杆菌ATCC11842中LDB_RS05285基因在酸奶后酸化中的功能研究;孙永胜;万方数据库;全文 * |
| 利用转录组学技术对保加利亚乳杆菌ATCC11842后酸化分子机制的研究;许沙;万方数据库;全文 * |
| 弱后酸化保加利亚乳杆菌KLDS1.1011的筛选及其全基因组注释研究;王成凤等;食品工业科技;全文 * |
| 酸奶后酸化中保加利亚乳杆菌关键基因表达分析;李晨;张国文;赵云;卢海强;田洪涛;罗云波;;中国食品学报(第07期);全文 * |
| 高效阴离子交换色谱-脉冲安培检测法测定脱脂酸奶后酸化生物标志物;薛香菊;刘宁;;食品与发酵工业(第08期);全文 * |
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