Lactobacillus D8 and application thereof

A technology of Lactobacillus and intestinal tract, applied in Lactobacillus D8 and its application field, can solve the problems of no Lactobacillus-intestinal organoid-lamina propria lymphocyte co-culture model, no separation, no immunological effect of Lactobacillus, etc. , to achieve the effect of maintaining intestinal health, improving depth and improving immune level

Active Publication Date: 2017-03-15
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] At present, there is no report on the isolation and identification of Lactobacillus D8 isolated from healthy pigs, nor any report on the establishment of an in vitro Lactobacillus-intestinal organoid-lamina propria lymphocyte co-culture model, let alone about Using this Lactobacillus D8 to explore the immunological effect of Lactobacillus and its effect on intestinal mucosa

Method used

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  • Lactobacillus D8 and application thereof
  • Lactobacillus D8 and application thereof
  • Lactobacillus D8 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Isolation and Identification of Lactobacillus D8

[0045] 1.1 Source of samples and preliminary screening of strains

[0046] The sample came from a 30-day-old Duroc piglet, which was purchased from Jiangsu Academy of Agricultural Sciences. After the piglet was slaughtered, the abdominal cavity was opened, and the two ends of the duodenum were tied with sterilized cotton rope. Send it to the laboratory within 30 minutes for strain separation. The separation operation is carried out in an ultra-clean workbench. Cut the intestinal segment longitudinally with sterile scissors, then rinse the contents of the intestinal surface with sterile PBS buffer, and then use a razor blade Gently scrape about 0.5 g of the intestinal wall mucosa, place it in a conical flask filled with glass beads and oscillate fully, let it stand at room temperature for 2-3 minutes, take 100 μl of the suspension and coat the surface of MRS agar medium (pH 5.8), 37 Cultivate in a candle jar f...

Embodiment 2

[0052] Example 2 Establishment of a co-culture model of Lactobacillus-intestinal organoid-lamina propria lymphocytes in vitro

[0053] 2.1 Isolation and culture of intestinal organoids

[0054] 2.1.1 Mice were purchased from the Experimental Animal Center of Yangzhou University. Four-week-old C57 mice were taken. The small intestine was about 15 cm long. Wash several times with sterile PBS buffer solution;

[0055] 2.1.2 Cut the washed small intestine into 3-5mm fragments, transfer it to a 50ml sterile centrifuge tube, add 10mL of 20mM pre-cooled EDTA, digest on ice for 20min, suck up the liquid with a pipette gun and discard it, and keep the small intestine tissue ;

[0056] 2.1.3 Add pre-cooled PBS to the small intestine tissue, pipette gently and continuously with a pipette gun, and pass through an optical microscope in time to discard the supernatant containing a large number of villi until a large number of small intestinal crypts appear in the field of vision, and begi...

Embodiment 3

[0068] Example 3 Establishment of a co-culture model of Lactobacillus-intestinal organoid-lamina propria lymphocytes in vitro

[0069] Basically consistent with Example 2, the difference is that the lamina proprialymphocytes (lamina proprialymphocytes) and intestinal organoids (intestinal organoids) are mixed according to the volume ratio of 3:1 (the specific amount is 3 μl lamina proprialymphocytes and 1 μl intestinal organoid), add (50 μl per well) Matrigel resuspended, spread in 24-well plate, add 50 μl Matrigel to each well, add 500 μl DMEM / F-12 medium (Gibco, USA) (containing 25ng of recombinant mouse EGF, 50ng of recombinant mouse Noggin and 125ng of recombinant human R-spondin), placed in a cell incubator for cultivation. Lactobacillus treatment group needs to add 1×10 per well 3 CFU of Lactobacillus D8, an in vitro Lactobacillus-intestinal organoid-lamina propria lymphocyte co-culture model was constructed.

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Abstract

The invention discloses lactobacillus D8 and application thereof. The lactobacillus D8 disclosed by the invention is separated from healthy pigs, the classification name is Lactobacillus sp., and the preservation number is CGMCC No.13112. Construction of an in-vitro lactobacillus-intestinal tract organ-lamina propria lymphocyte co-culture model shows that proliferation and differentiation of intestinal stem cells can be effectively promoted by using the lactobacillus D8, and furthermore completeness of an intestinal mucosa barrier can be maintained. Further animal tests show that the length of intestinal villi and depth of small intestinal crypts can be remarkably improved with orally taken lactobacillus D8, and enteritis symptoms can be alleviated. Therefore, the strain is expected to be developed as probiotics for preventing and / or treating enteritis.

Description

technical field [0001] The invention relates to biological control technology, in particular to Lactobacillus D8 and its application. Lactobacillus D8 can effectively promote the proliferation and differentiation of intestinal stem cells and repair damage induced by TNF-α; oral administration of animal Lactobacillus D8 can significantly improve enteritis symptoms. Background technique [0002] 1. Current status of research on Lactobacillus and its immunological effects [0003] Lactobacillus belongs to Firmicutes, Bacilli, Lactobacillales, and Lactobacillaceae in taxonomy. It is a Gram-positive bacillus that does not form Bud buds break down sugar to produce lactic acid. Lactobacillus is a normal bacterium in the intestinal tract of humans and animals, and is closely related to the absorption of host nutrients and the development of the intestinal mucosal immune system. Studies have shown that Lactobacillus, as a probiotic, plays an important role in inhibiting the coloniz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12N5/0781C12N5/0783C12N5/071A61K35/747A61P1/00C12R1/225
CPCA61K35/747C12N5/0635C12N5/0636C12N5/0679C12N1/205C12R2001/225
Inventor 庾庆华侯起航杨倩
Owner NANJING AGRICULTURAL UNIVERSITY
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