Preparation method of phosphorylated peptidoglycan

A technology for phosphorylating peptidoglycan and peptidoglycan, which can be applied in biochemical equipment and methods, microorganism-based methods, microorganisms, etc. , the effect of mild reaction conditions

Active Publication Date: 2015-07-08
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, the phosphorylation modification is mainly concentrated on starch, chitosan and cellulose, etc., and the research on the phosphorylation modification of peptidoglycan has not been reported so far.

Method used

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  • Preparation method of phosphorylated peptidoglycan
  • Preparation method of phosphorylated peptidoglycan
  • Preparation method of phosphorylated peptidoglycan

Examples

Experimental program
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Effect test

Embodiment 1

[0036] The invention uses common commercially available Lactobacillus acidophilus bacteria powder as a raw material to extract the peptidoglycan. First, inoculate 2 mg of bacterial powder into 200 mL of MRS liquid medium, and culture it statically at 37°C for 24 hours to activate the strain. A method for preparing phosphorylated peptidoglycan of the present invention specifically includes the following steps:

[0037] (1) Preparation of peptidoglycan

[0038] a. Bacteria collection: Inoculate the activated Lactobacillus acidophilus into the MRS liquid medium with an inoculum volume of 4% by volume, culture it in a constant temperature incubator at 38.5°C for 20 hours, centrifuge at 6500r / min for 15min to collect the bacteria, and use distilled water repeatedly Wash until the cells are milky white; the preparation method of the MRS liquid medium is as follows: 10g of peptone, 10g of beef extract, 5g of yeast extract, 10g of glucose, 2g of triamine citrate, 2g of dipotassi...

Embodiment 2

[0047] With embodiment 1, its difference is:

[0048] (1) Preparation of peptidoglycan

[0049] Inoculate the activated Lactobacillus acidophilus into the MRS liquid medium with an inoculation amount of 3% by volume, and culture it in a constant temperature incubator at 37°C for 16 hours, then centrifuge at 5000r / min for 20 minutes to collect the bacteria, and wash them repeatedly with distilled water until the bacteria are milky white ;Suspend the bacteria in 40% hydrofluoric acid solution at a solid-to-liquid ratio of 1g:9ml, place overnight at 4°C, centrifuge at 7000 r / min for 20min to collect cell wall precipitates without teichoic acid, and wash the precipitates with distilled water To neutrality; dissolve the precipitate in chymotrypsin phosphate buffer according to the solid-liquid ratio of 2g:5ml, shake at 110r / min on a shaker at 37°C for 17h, inactivate in a boiling water bath for 4min, and centrifuge at a speed of 1500r / min for 6min. Discard the undissolved pr...

Embodiment 3

[0055] With embodiment 1, its difference is:

[0056] (1) Preparation of peptidoglycan

[0057] Inoculate the activated Lactobacillus acidophilus into the MRS liquid medium with an inoculation amount of 5% by volume, and culture it in a constant temperature incubator at 40°C for 24 hours, then centrifuge at 8000r / min for 10 minutes to collect the bacteria, and wash them repeatedly with distilled water until the bacteria are milky white ;Suspend the bacteria in a 50% hydrofluoric acid solution at a solid-to-liquid ratio of 1g:9ml, place at 4°C overnight, centrifuge at 9000 r / min for 10min to collect the cell wall precipitate, and wash the precipitate with distilled water until neutral; Dissolve in chymotrypsin phosphate buffer at a solid-to-liquid ratio of 2g:5ml, shake on a shaker at 40°C at 130r / min for 15h, inactivate in a boiling water bath for 6min, centrifuge at a speed of 2500r / min for 4min, and put the supernatant at 12000r Centrifuge at 9000r / min for 10min, and ...

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Abstract

The invention discloses a preparation method of phosphorylated peptidoglycan. The method is characterized by comprising the following steps of: inoculating Lactobacillus acidophilus on a culture medium for cultivation, centrifuging to collect thallus, repeatedly washing the thallus, suspending the thallus in hydrofluoric acid solution for one night at 4 DEG C, centrifugally collecting cell wall sediment, and washing the sediment to become neutral; dissolving the sediment into Trypsin chymotrypsin phosphate buffer solution, inactivating in boiling water, centrifuging the liquid supernatant, washing the sediment; dissolving the sediment in diethyl ether, stirring and centrifuging to collect sediment, and washing the sediment with water until completely removing the smell of diethyl ether, then obtaining the peptidoglycan; dissolving the peptidoglycan in Lysostaphin phosphate buffer solution for performing enzymolysis and centrifuging, taking the liquid supernatant, centrifuging the liquid supernatant, and centrifugally washing the sediment with water to get micromolecule peptidoglycan; and finally, dissolving the micromolecule peptidoglycan in mixed phosphate solution, precipitating with alcohol after reaction, treating the alcohol precipitated peptidoglycan by freeze drying and redissolving, and adding the redissolved solution in a dialysis bag, performing dialysis concentration, and then, performing freeze drying to get the phosphorylated peptidoglycan. The method has the advantages of increasing the solubility remarkably and improving the immunological effect to a certain extent.

Description

technical field [0001] The invention relates to a method for preparing peptidoglycan, in particular to a method for preparing phosphorylated peptidoglycan. Background technique [0002] As an important intestinal probiotic, lactic acid bacteria play an important role in regulating the intestinal microbial flora, enhancing the body's immunity and preventing diseases such as tumors. In recent years, in addition to paying attention to the long-term research of Bifidobacterium and Bacillus subtilis at home and abroad, they have begun to focus on another important member-Lactobacillus acidophilus. These bacteria use their unique peptidoglycan components to induce the release of various cytokines to regulate the immune function of the host. [0003] Peptidoglycan is one of the main components of bacterial cell walls. Its monomer is composed of a tetrapeptide tail and a disaccharide unit. This microbial polysaccharide has different immune effects due to the particularity of its ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/14C12P19/04C12R1/23
Inventor 潘道东王琳珺曾小群孙扬赢曹锦轩
Owner NINGBO UNIV
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