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Recombinant food-grade lactobacillus containing catalase genes

A technology of catalase and recombinant lactic acid bacteria, which is applied in the field of genes and food preparations, and can solve problems such as resistance to environmental stress

Inactive Publication Date: 2011-01-12
ZHAOQING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In terms of innovation, at present, in the biological research of anti-oxidative stress, the research reports on SOD are more common, while there are few reports on the anti-stress research on CAT. The research on the recombination of CAT in lactic acid bacteria and its resistance to environmental stress research, not yet reported

Method used

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  • Recombinant food-grade lactobacillus containing catalase genes
  • Recombinant food-grade lactobacillus containing catalase genes
  • Recombinant food-grade lactobacillus containing catalase genes

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Example 1: Construction of food-grade recombinant lactic acid bacteria containing catalase gene

[0031] ①Cultivate Escherichia coli, extract chromosomal DNA, use restriction enzyme Sau3AI for partial digestion, and separate and purify DNA fragments above 2.5kb.

[0032] ② After the E. coli plasmid pUC18 / 19 was digested with BamHI, it was ligated with T4 DNA ligase with different fragments that were partially digested with Sau3AI, and transformed into E. coli TG1 by electric shock to construct an E. coli genome library of different size fragments.

[0033] ③Brain heart infusion (BHI) containing 1% tannic acid and 150ug / ml ampicillin was selected as the culture medium. The culture medium was anaerobic at 37℃ for 2 days, and then aerobic culture at 37℃ for 24 hours. Catalase activity. As in aerobic culture, catalase can cause tannic acid degradation. Therefore, a clone that can cause tannin degradation on the culture plate is selected as a catalase-positive bacterial clone.

[...

Embodiment 2

[0056] Example 2: For the present invention, the expression of Escherichia coli CAT protein in Lactococcus lactis

[0057] ①Cultivate recombinant Lactococcus lactis in MG17 medium at 30℃ anaerobic, and measure the OD value (600nm0.5). After about 3.5 hours, use nisin to induce its expression, and collect the culture supernatant and bacteria. After the bacterial cells are ultrasonically broken, do SDS-PAGE together with the culture supernatant to observe the expression of cat gene, purchase CAT antibody, and do Western-blot detection. See the result image 3 with Figure 4 .

[0058] ②When the CAT protein appears in the recombinant lactic acid bacteria, the next step of environmental stress resistance experiment is carried out. Artificially create different physiological environments, first cultivate in an anaerobic environment for 3.5h, and then place the lactic acid bacteria in cold (4℃-0℃--20℃), hot (30℃-80℃), salt NaCl (0-500mmol) , Oxygen (add H2O20.2mmol / L) or shaking cultur...

Embodiment 3

[0061] Example 3: Detection of Catalase Activity

[0062] (a) Crush the acid bacteria and centrifuge to get the supernatant;

[0063] (b) Prepare a 10% Tween 80 solution with a buffer of phosphate (K2 HPO4+KH2PO4, 0.1 mol / L) and NaCl (2 mol / L), and mix 5 ml with 5 ml of 30% hydrogen peroxide. Separate control tube and cat tube;

[0064] (c) Add different volumes of debacterial supernatant (20, 40, 80, 160 μl) into the cat tube;

[0065] (d) Add the supernatant of lactic acid bacteria (20, 40, 80, 160, 320 μl) containing only plasmid pRV300 to the control tube in the hydrogen peroxide reaction system;

[0066] (e) Comparing the bubble generation situation, it is found that each tube added with the supernatant of the strain containing the pRV-cat expression plasmid has bubble generation, and they are all more obvious than the control tube;

[0067] (f) It is suggested that the recombinant catalase (cat) expression product has catalase activity.

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Abstract

The invention relates to recombinant food-grade lactobacillus containing catalase genes. The lactobacillus is the food-grade lactobacillus which contains the catalase genes of a nucleotide sequence shown in SEQ ID NO.1 and is constructed through recombining gene engineering by taking lactococcus lactis NZ9000 as a strain. In the invention, colon bacillus gene group libraries with different sizes of segments are constructed by using a shotgun method, clones containing complete catalase genes are screened through heart brain dipping culture medium containing gallotannic acid, and the catalase genes are inserted into a lactococcus lactis carrier pGK12 or pRV300 to convert the lactococcus lactis NZ9000 to construct the recombinant food-grade engineering recombinant lactobacillus containing the catalase genes. The recombinant lactococcus lactis has certain anti-oxidization capability and capability for resisting environmental stress, thereby providing novel food-grade engineering recombinant lactococcus lactis.

Description

Technical field [0001] The invention belongs to the field of genes and food preparations, and relates to the construction of a new catalase gene and a new food engineering bacteria and related preparation processes. Background technique [0002] Lactic acid bacteria are heterologous bacteria and are found in various environments of animals, humans and plants. In sewage, fermentation production (such as silage, fruit wine, beer, kimchi, soy sauce, yogurt, cheese) culture, animal digestive tract, etc., the content of lactic acid bacteria is higher. This kind of bacteria has been used for the production of various fermented foods for a long time and has important utilization value in the food industry. In addition, lactic acid bacteria have a very close relationship with human health. Intestinal lactic acid bacteria can prevent the growth of spoilage bacteria and play a role in extending life. In medicine, lactic acid bacteria can be used for acute diarrhea caused by rotavirus and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/74C12R1/01
Inventor 李充璧李妍李赛男孙帆梁盛年李广宏李芸瑛黄丽华陈庆华赵建芬
Owner ZHAOQING UNIV
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