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Rapid screening method for Lactobacillus helveticus rich in ace-inhibiting active peptides, combined sequence and construction method for realizing the method

A construction method and screening method technology, which is applied in the field of joint sequence, rapid screening of Lactobacillus helveticus rich in ACE-inhibiting active peptides, can solve the problems of Lactobacillus helveticus lacking ACE active peptides, and achieve low screening cost, simple operation, High throughput effect

Active Publication Date: 2019-02-26
INNER MONGOLIA MENGNIU DAIRY IND (GRP) CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] It can be seen that although there have been studies on the housekeeping genes of Lactobacillus helveticus, there is no clear conclusion that the Lactobacillus helveticus that inhibits the ACE active peptide depends on which genes, and there is also a lack of rapid, rapid and effective genetic information through bioengineering methods. Method for accurately screening Lactobacillus helveticus rich in ACE-inhibiting active peptides

Method used

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  • Rapid screening method for Lactobacillus helveticus rich in ace-inhibiting active peptides, combined sequence and construction method for realizing the method
  • Rapid screening method for Lactobacillus helveticus rich in ace-inhibiting active peptides, combined sequence and construction method for realizing the method
  • Rapid screening method for Lactobacillus helveticus rich in ace-inhibiting active peptides, combined sequence and construction method for realizing the method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Activation, cultivation and identification of embodiment 1 bacterial strain

[0065] The samples of Lactobacillus helveticus (Lactobacillus helveticus) that have been analyzed for the characteristics of ACE-inhibiting active peptides in the lactic acid bacteria strain resource bank of Inner Mongolia Agricultural University were used as the analysis objects, and a total of 12 strains were collected. The strain information is as follows:

[0066] Table 2 Isolation sources and ACE inhibitory peptide characteristics of Lactobacillus helveticus strains

[0067]

[0068] Do the following for each sample:

[0069] Activation of bacterial strains: Put the freeze-dried bacterial powder stored in the ampoule tube into skim milk for activation and rejuvenation (37°C, 24h), microscopically examine the bacterial liquid smear, and the microscopic examination results are Gram-positive pure cultures Continue to subculture to the third generation.

[0070] Use liquid nitrogen free...

Embodiment 2

[0071] The construction of embodiment 2 joint sequence

[0072] 2.1 Design primers

[0073] Using Lactobacillus helveticus H9 (genbank number CP002427) as a template, the key genes of pepX, pepN and pepO involved in the proteolysis system were selected as targets. Primer Premier 5.0 software was used to design specific amplification primers, and each primer was artificially synthesized as shown in Table 1 above.

[0074] 2.2

[0075] The primers in Table 1 were used to amplify the template DNA by PCR, respectively, to obtain three gene fragments of each strain, and the template DNA came from the 12 isolates in Table 2.

[0076] Amplification system: template DNA 50ng, 10×PCR buffer 5μL, dNTPs (2.5mmol / μL) 4μL, upstream and downstream primers 10μM each 1.5μL, Taq DNA polymerase (5U / μL) 0.5μL, ddH 2 O to make up to 50 μL.

[0077] Amplification conditions:

[0078] Pre-denaturation at 95°C for 5min; denaturation at 95°C for 1min, annealing at 50°C for 45s, extension at 72°C...

Embodiment 3

[0083] Example 3 Construction of developmental tree

[0084] Using the disclosed Lactobacillus helveticus H9 genomic DNA as a template, amplified in the same steps as in Example 2, the resulting gene fragments were concatenated in the order of pepX-pepN-pepO to obtain the combined gene sequence of SEQ No.1, Used as a standard for screening methods.

[0085] The sequence of each strain of Example 2 was concatenated in the order of pepX-pepN-pepO to obtain the combined gene sequences of 12 strains to be screened respectively, and the nucleic acid sequences thereof are shown in SEQ No.2-13.

[0086] Adopt MEGA6.0 software package to analyze joint sequence, through Clustal W alignment, the joint gene sequence of 12 bacterial strains that embodiment 2 isolates obtains and the joint sequence of SEQ No.1 carries out pairwise comparison, calculates the distance between the sequences, Construct a distance matrix to reflect the genetic relationship between sequences.

[0087] Then con...

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Abstract

The invention provides application of a union sequence, containing Lactobacillus helveticus genomes pepX and pepN and a gene pepO, in screening high-yielding ACE (angiotensin converting enzyme) bioactive peptide restraining Lactobacillus helveticus. The invention further provides a rapid screening method for the high-yielding ACE bioactive peptide restraining Lactobacillus helveticus. The rapid screening method includes: (1) extracting genome DNA (deoxyribonucleic acid) of Lactobacillus helveticus bacterial strains; (2) constructing union gene sequences of the to-be-screened bacterial strains; comparing the union gene sequences of the to-be-screened bacterial strains with a union gene nucleotide sequence as shown in the SEQ No.1, constructing a phylogenetic tree through a neighbor-joining method, and acquiring classifications of the Lactobacillus helveticus through the phylogenetic tree; (4) identifying the bacterial strains, shown in the same branch of Lactobacillus helveticus H9, in the phylogenetic tree as the high-yielding ACE bioactive peptide restraining Lactobacillus helveticus. The rapid screening method is capable of screening the high-yielding ACE bioactive peptide restraining Lactobacillus helveticus effectively and has the advantages of low screening cost and simplicity in operation.

Description

【Technical field】 [0001] The invention relates to the field of genetic engineering, in particular to a method for rapidly screening Lactobacillus helveticus rich in ACE-inhibiting active peptides, and a combination sequence for realizing the method. 【Background technique】 [0002] Lactobacillus helveticus (Lactobacillus helveticus) is a recognized probiotic lactic acid bacteria, widely used in fermented milk, cheese and other dairy industries. According to literature reports, Lactobacillus helveticus has a strong proteolytic enzyme system, and the fermented milk produced by it can metabolize a large amount of biologically active tripeptides VPP and IPP, which has the effect of lowering blood pressure. Whether fermented milk products have the effect of lowering blood pressure and the strength of the function of lowering blood pressure are closely related to the type of starter used and the content and type of antihypertensive peptides in fermented milk. VPP and IPP are the mo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/689C12Q1/04C12N15/57C12N15/10C12R1/225
CPCC12N9/485C12N15/10C12Q1/689
Inventor 陈永福张和平孙志宏
Owner INNER MONGOLIA MENGNIU DAIRY IND (GRP) CO LTD
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