Antihypertensive recombinant lactic acid bacteria and culture method thereof

A lactic acid bacteria and blood pressure lowering technology, applied in the biological field, can solve the problems of high reaction conditions, many side reactants and residual compounds, difficult purification of products, etc., achieve simple preparation method and production process, enhance mucosal immunity, and have significant antihypertensive effect Effect

Inactive Publication Date: 2012-05-23
王春凤
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0011] In the process of enzymatic hydrolysis, the utilization rate of raw protein is limited, resulting in a certain degree of waste, and the product is difficult to be purified; ACE inhibitory peptides are obtained by artificial synthesis, so The required reaction conditions are high, the cost is high, and there are many side reactants and residual compounds. It is not suitable for large-scale industrial production.

Method used

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  • Antihypertensive recombinant lactic acid bacteria and culture method thereof
  • Antihypertensive recombinant lactic acid bacteria and culture method thereof
  • Antihypertensive recombinant lactic acid bacteria and culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Obtaining of the blood pressure-lowering peptide gene sequence ACEIPn

[0040] Design two short peptides with high blood pressure lowering activity, which are connected in series by arginine (Arg) and have two repeats. The amino acid sequence is as shown in SEQ ID No.1; according to the preferred codons of lactic acid bacteria, Sac I and Kpn I enzyme cutting site, designed ACEIPn gene sequence, respectively named pep1, pep2, pep3, pep4, and its gene sequence is shown in SEQ ID No.1, 2, 3, 4, synthesized by Beijing Huada Gene Company, The synthetic form is pUC57-ACEIPn; wherein pep4 is composed of two ACE inhibitory peptides connected in series by arginine (Arg) and has two repeats, and its amino acid sequence is shown in SEQ ID No.5.

Embodiment 2

[0041] Example 2 Construction of recombinant expression vector pW425et-pep4

[0042] 1) Preparation of plasmids pUC57-pep4 and pW425et

[0043] Escherichia coli liquids containing pUC57-pep4 and pW425et were prepared respectively, and the plasmid was extracted according to the instructions of the plasmid DNA extraction kit, and 2 μL was taken for 0.8% agarose gel electrophoresis detection. As a result, the plasmid pUC57-pep4 was successfully obtained, such as figure 1 shown; plasmid pW425et, as figure 2 shown.

[0044] 2) Recovery, connection and transformation of the target gene

[0045] Recovery of the target gene:

[0046] Sac I and Kpn I restriction endonucleases were used to double-digest pUC57-pep4 and pW425et respectively to obtain pep4 gene and pW425et large fragments; the double-digestion system is as follows:

[0047] 10 × L Buffer 2.0μL

[0048] Recombinant plasmid (or vector) 2.0μL

[0049] Sac I (12U / μL) 1.0μL

[0050] Kpn I (12U / μL) 1.0μL

[0051] wxya...

Embodiment 3

[0073] Example 3 Preparation of recombinant Lactobacillus Lb.planturm Wang with hypotensive function

[0074] 1) Preparation and transformation of Lactobacillus competent cells

[0075] Incubate Lactobacillus plantarum, Lactobacillus bulgaricus, Lactobacillus helveticus, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus reuteri or Lactobacillus fermentum frozen at -70°C until thawed and spread on MRS plate (containing thymidylic acid 100 μg / mL), anaerobic static culture at 37°C overnight. The next day, pick colonies and inoculate them in fresh 10mL MRS liquid medium (containing 1% glycine), and anaerobic static culture at 37°C until the cells OD 600 The value is 0.6-0.8. Take the bacterial cell culture solution, inoculate it in fresh MRS liquid medium (containing 1% glycine) according to the dose of 1%-2%, and culture it anaerobically at 37°C until the cell OD 600 When the value is 0.2-0.3, collect and reserve;

[0076] Place the bacterial culture collected abo...

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Abstract

The invention discloses antihypertensive recombinant lactic acid bacteria and a culture method thereof. According to preferred codons of lactic acid bacteria, a plurality of ACE inhibitory peptide gene sequences are connected in series by arginine, restriction enzyme cutting sites Sac I and Kpn I are inserted into the upper and lower streams, ACEIPn is synthesized manually, an Escherichia coli-lactic acid bacteria shuttling expression recombinant plasmid pW42535-ACEIPn is constructed, the recombinant plasmid is transferred into the lactic acid bacilli and is planted in intestinal tracts by oral administration, antihypertensive peptides can be expressed continuously and stably, and patients do not have to take medicaments every day. The antihypertensive peptides, which are expressed by the recombinant lactic acid bacilli, have no antihypertensive on people with normal blood pressure, and cannot be consumed by protease in the intestinal tracts, the absorption efficiency of the antihypertensive peptides is high, the antihypertensive peptides have the advantages of obvious antihypertensive effect, high safety, no toxic nor side effects and the like and have many nutritive and health-care functions, a new policy for preventing and curing hypertension is provided, and the preparation method and production process of the antihypertensive peptides are relatively simple and convenient and are suitable for large-scale production.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a recombinant lactic acid bacterium with the function of lowering blood pressure and a preparation method thereof. Background technique [0002] Hypertension is one of the main diseases that endanger human health. It is the most common chronic cardiovascular and cerebrovascular disease. It has a high incidence rate and can induce coronary heart disease, cerebral hemorrhage and other diseases. A series of injuries and serious lesions, even functional failure, of important organs such as the kidney, as well as glucose and lipid metabolism disorders, pose a great threat to people's health. According to statistics, the prevalence of hypertension in the world population is as high as 10%-20%, the number of people suffering from hypertension has reached 1 billion, and more than 12 million people die of various diseases caused by hypertension every year. The prevalence of hypertension in our...

Claims

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Application Information

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Patent Type & AuthorityPatents(China)
IPC IPC(8): C12N15/12C12N15/74C12N1/21C12R1/225C12R1/25
Inventor王春凤杨桂连杜芳
Owner王春凤