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Enterococcus exopolysaccharide with immunomodulatory effect as well as preparation method and application of enterococcus exopolysaccharide

An extracellular polysaccharide, immune regulation technology, applied in microorganism-based methods, biochemical equipment and methods, bacteria, etc., can solve the problems of rare immune regulation and less research on biological activity, and achieve significant technical advantages and applications. Clear range, proliferative effect

Active Publication Date: 2021-05-28
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that Enterococcus can produce exopolysaccharides. Compared with lactic acid bacteria such as Lactobacillus plantarum and Lactobacillus rhamnosus, there are few studies on the biological activity of probiotic Enterococcus exopolysaccharides, and their role in immune regulation is even rarer.

Method used

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  • Enterococcus exopolysaccharide with immunomodulatory effect as well as preparation method and application of enterococcus exopolysaccharide
  • Enterococcus exopolysaccharide with immunomodulatory effect as well as preparation method and application of enterococcus exopolysaccharide
  • Enterococcus exopolysaccharide with immunomodulatory effect as well as preparation method and application of enterococcus exopolysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation of crude exopolysaccharides from Enterococcus hirai:

[0041] (1) Inoculate the seed solution of Enterococcus hirae WEHI01 in the aseptic brain heart infusion broth medium with an inoculation amount of 1.0% by volume, and culture it anaerobically at 37° C. for 15 hours to obtain a fermentation broth;

[0042] (2) Centrifuge the fermented liquid obtained in step (1) at 4° C. at 9000×g for 5 min, remove the bacterial cells, and obtain the fermentation supernatant;

[0043] (3) Add 3 times the volume of absolute ethanol to the fermentation supernatant obtained in step (2), place it in a refrigerator at 4°C for alcohol precipitation for 48-72 hours, and centrifuge at 10,000×g for 20 minutes at 4°C to collect the precipitate , and the precipitate was dissolved in water, and dialyzed at 4°C for 3 days with a dialysis bag with a molecular weight cut-off of 8,000-14,000 Da. During this period, the water was changed twice a day. thing;

[0044] (4) After redissolvi...

Embodiment 2

[0046] Preparation of crude exopolysaccharides from Enterococcus hirai:

[0047] (1) Inoculate the seed solution of Enterococcus hirae WEHI01 in the aseptic brain heart infusion broth medium with an inoculation amount of 4.0% by volume, and culture it anaerobically at 37° C. for 25 hours to obtain a fermentation broth;

[0048] (2) centrifuge the fermented liquid obtained in step (1) at 4° C., 7000×g for 5 min, remove the bacteria, and obtain the fermentation supernatant;

[0049] (3) Add 4 times the volume of absolute ethanol to the fermentation supernatant obtained in step (2), place it in a 4°C refrigerator for alcohol precipitation for 72h, and centrifuge at 10000×g for 20min at 4°C to collect the precipitate, and The precipitate was dissolved in water, and dialyzed at 4°C for 3 days with a dialysis bag with a molecular weight cut-off of 8,000-14,000 Da. During this period, the water was changed twice a day, and the dialyzed sample was freeze-dried to obtain the crude extr...

Embodiment 3

[0052] Separation and purification of the exopolysaccharide of Enterococcus hirai to obtain EPS-I02, a component of the exopolysaccharide of Enterococcus hiraeii:

[0053] (1) Use 5 mL of HiTrap Q Sepharose High Performance anion-exchange chromatography column to separate and purify the crude enterococcal exopolysaccharide prepared in Example 1, and use 0, 0.5, 0.7, 1mol / L NaCl solution for segmental elution and separation , the elution rate is 1mL / min, and 0.5mol / L NaCl solution is eluted to obtain the component EPS-I02 of the exopolysaccharide of Enterococcus hirae. In a dialysis bag, dialyze in deionized water for 48 hours at 4°C, change the water every 4 hours, and finally vacuum freeze-dry;

[0054] (2) Further purify with 10mm×300mm Superdex G 200 gel filtration chromatography column, and use 0.2mol / L NH 4 HCO 3 Solution elution, the elution rate is 0.5mL / min, the elution products of the component peaks are combined and collected, and water is repeatedly added and evap...

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Abstract

The invention discloses an enterococcus exopolysaccharide with an immunomodulatory effect as well as a preparation method and application of enterococcus exopolysaccharide; enterococcus hiraei WEHI01 is used for fermentation to generate the exopolysaccharide, the exopolysaccharide is separated and purified, and the enterococcus hiraei WEHI01 is preserved in the China Center for Type Culture Collection, and has the preservation number of CCTCC NO:M 2019068. The prepared enterococcus exopolysaccharide comprises four components: EPS-I01, EPS-I02, EPS-I03 and EPS-I04, the average molecular weight of the component EPS-I02 is 2.28*10<4> Da, and the component EPS-I02 is composed of rhamnose, trehalose, arabinose, xylose, mannose, glucose and galactose according to the molar ratio of 1:1.76:5.48:1.76:2.52:6.2:20.92. The enterococcus exopolysaccharide prepared by the preparation method has an immunoregulation effect, can significantly improve the proliferation rate and phagocytic ability of macrophages, and significantly promote macrophages to secrete NO and cell factors, and the enterococcus exopolysaccharide as an additive is applied to the fields of food and medicine, and has a wide application prospect.

Description

technical field [0001] The invention relates to the technical field of polysaccharides in microbiology, in particular to an enterococcal exopolysaccharide with immune regulation function and its preparation method and application. Background technique [0002] Lactic acid bacteria (LAB), generally refers to bacteria that utilize fermentable carbohydrates and produce a large amount of lactic acid, and are recognized as green and safe (GRAS) food-grade microorganisms. In addition to producing lactic acid, acetic acid, formic acid and folic acid, lactic acid bacteria also produce other secondary metabolites, such as bacteriocin and exopolysaccharides. The exopolysaccharide of lactic acid bacteria can not only improve the texture and flavor of fermented dairy products, such as thickening, stabilizing, emulsifying, gelling and water holding, etc.; it also has a variety of physiological functions, including promoting cell adhesion, anti-oxidation, lowering cholesterol, Anti-tumor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/04C12N1/20C08B37/00C12R1/01
CPCC12P19/04C12N1/20C08B37/0003Y02A50/30
Inventor 陶雪莹魏华贾凯颖王玉洁何瑶魏敏陶悦
Owner NANCHANG UNIV
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