阅读说明：本技术 一种乳酸菌胞外多糖及其衍生物的制备方法 (Preparation method of lactobacillus extracellular polysaccharide and derivatives thereof ) 是由 胡风庆 王博君 于 2020-07-15 设计创作，主要内容包括：本发明涉及一种乳酸菌胞外多糖及其衍生物的制备方法。将活化的乳杆菌菌种按3％的接种量接种至改进的MRS选择性培养基上,培养24h后离心去除沉淀菌体,取上清液；将上清液依次进行除蛋白,减压浓缩,醇沉,离心取沉淀,超纯水溶解,冷冻干燥,得粗乳酸菌胞外多糖；将粗乳酸菌胞外多糖进行纯化,得纯乳酸菌胞外多糖。通过本发明的方法,提高了乳酸菌胞外多糖的纯度和产量。并对乳酸菌胞外多糖进行了磷酸和硫酸化修饰,得到了乳酸菌胞外多糖的磷酸化和硫酸化衍生物,提高了乳酸菌胞外多糖及其衍生物的生物活性,在功能性保健食品领域中具有一定的应用价值。(The invention relates to a preparation method of extracellular polysaccharide of lactic acid bacteria and derivatives thereof. Inoculating the activated lactobacillus strain to an improved MRS selective culture medium according to the inoculation amount of 3%, culturing for 24h, centrifuging to remove precipitated bacteria, and taking supernatant; sequentially removing protein from the supernatant, concentrating under reduced pressure, precipitating with ethanol, centrifuging to obtain precipitate, dissolving with ultrapure water, and freeze drying to obtain crude extracellular polysaccharide of lactobacillus; purifying the crude lactobacillus extracellular polysaccharide to obtain pure lactobacillus extracellular polysaccharide. The method of the invention improves the purity and yield of extracellular polysaccharide of lactobacillus. And the lactobacillus extracellular polysaccharide is subjected to phosphoric acid and sulfation modification to obtain phosphorylated and sulfated derivatives of the lactobacillus extracellular polysaccharide, so that the biological activity of the lactobacillus extracellular polysaccharide and the derivatives thereof is improved, and the lactobacillus extracellular polysaccharide has a certain application value in the field of functional health-care food.)

1. The preparation method of the extracellular polysaccharide of the lactic acid bacteria is characterized by comprising the following steps:

(1) preparing crude lactobacillus exopolysaccharides: inoculating the activated lactobacillus strain to an improved MRS selective culture medium according to the inoculation amount of 3%, culturing for 24h, centrifuging to remove precipitated bacteria, and taking supernatant; sequentially removing protein from the supernatant, concentrating under reduced pressure, precipitating with ethanol, centrifuging to obtain precipitate, dissolving with ultrapure water, and freeze drying to obtain crude extracellular polysaccharide of lactobacillus;

(2) preparing pure lactobacillus exopolysaccharides: dissolving the crude extracellular polysaccharide of the lactobacillus in ultrapure water, slowly adding the ultrapure water and a NaCl solution into a DEAE-52 cellulose column along the column wall, sequentially carrying out gradient elution by using the ultrapure water and the NaCl solution, collecting gradient eluent, carrying out reduced pressure concentration, dialyzing for 24 hours, and carrying out freeze drying to obtain the extracellular polysaccharide of the lactobacillus.

2. The method of claim 1, wherein: the improved MRS selective medium comprises the following components: contains 1 percent of galacto-oligosaccharide, 2 percent of soybean peptone, 0.2 percent of phosphate and the balance of water according to weight percentage; the carbon nitrogen ratio is 1: 2, initial pH 6.5.

3. The method of claim 1, wherein: in the step (1), the protein removal is to add polyamide into the supernatant with the addition of 120g/L and stir for 8 hours by magnetic force.

4. The method of claim 1, wherein: in the step (1), the alcohol precipitation is to add 95% ethanol with 4 times volume of the concentrate after decompression concentration, and the alcohol precipitation time is 12 h.

5. The method of claim 1, wherein: in the step (2), the gradient elution is carried out by using ultrapure water and NaCl solution, and the method for collecting the gradient eluent comprises the following steps: sequentially carrying out gradient elution by using ultrapure water and 0.1, 0.2, 0.3 and 0.4mol/L NaCl solution at the flow rate of 1mL/min, wherein each 15mL is a tube, and collecting gradient eluent; and tracking and detecting the concentration of the polysaccharide in each tube of eluent by using a phenol-sulfuric acid method, taking the tubes of the eluent with the highest concentration of the polysaccharide, and combining the eluents.

6. A preparation method of extracellular polysaccharide derivatives of lactic acid bacteria is characterized in that: the lactobacillus extracellular polysaccharide derivative is a phosphorylated lactobacillus extracellular polysaccharide derivative, and the preparation method comprises the following steps: taking the pure extracellular polysaccharide of lactic acid bacteria and sodium hexametaphosphate according to claim 1, mixing uniformly, adjusting the pH of the mixed solution to 6.0, reacting at 90 ℃ for 4h, cooling to room temperature, precipitating with ethanol, rotary evaporating the obtained precipitate, redissolving in a water bath at 50 ℃, dialyzing, and freeze-drying in vacuum to obtain the phosphorylated extracellular polysaccharide derivative of lactic acid bacteria.

7. The method of claim 6, wherein: in mass ratio, m_{Pure lactobacillus exopolysaccharides}：m_{Sodium hexametaphosphate}＝6:1。

8. The method of claim 6, wherein: the alcohol precipitation is to add 95% ethanol with three times volume into the product cooled to room temperature, and carry out alcohol precipitation for 24 h.

9. A preparation method of extracellular polysaccharide derivatives of lactic acid bacteria is characterized in that: the extracellular polysaccharide derivative of the lactic acid bacteria is a sulfated extracellular polysaccharide derivative of the lactic acid bacteria, and the preparation method comprises the following steps: suspending the pure extracellular polysaccharide of lactic acid bacteria of claim 1 in dimethylformamide, stirring for 20min, adding into an esterification reagent dropwise, reacting at 60 ℃ for 2h, cooling to room temperature, neutralizing with 4mol/L NaOH to pH 7, precipitating with 95% ethanol at 4 ℃ for 12h, centrifuging, collecting precipitate, washing with 95% ethanol, redissolving with deionized water, dialyzing with running water bag for 24h, and lyophilizing to obtain the sulfated extracellular polysaccharide derivative of lactic acid bacteria.

10. The method of claim 9, wherein: the esterification reagent is chlorosulfonic acid (CSA) -pyridine (Pyr), and the preparation method comprises the following steps: in an ice-water bath, chlorosulfonic acid was slowly added to pyridine, slowly stirred, reacted well, and when a large amount of pale yellow solids were observed, the ice-water bath was removed to give chlorosulfonic acid (CSA) -pyridine (Pyr), which was completed within 30 min.

Technical Field

The invention belongs to the field of microorganisms, and particularly relates to a preparation method of extracellular polysaccharide of lactic acid bacteria and derivatives thereof.

Background

Lactic acid bacteria are a kind of production strains with relatively high safety in food industry, and the probiotic function of the lactic acid bacteria is realized by the metabolism of the probiotic bacteria on one hand, and exopolysaccharides on the other hand, and the Exopolysaccharides (EPS) generated by the lactic acid bacteria are secondary metabolites secreted out of cells in the growth and metabolism process of the lactic acid bacteria. It has no toxic side effect on body, has very low cytotoxicity, is a potential excellent antioxidant substance, also has immunological activity, and can inhibit proliferation and metastasis of cancer cell, which is related to the kind and structure of lactic acid bacteria. It can be used as adhesion factor to help lactobacillus adhere and fix in intestinal tract, thereby competitively inhibiting proliferation of harmful bacteria, reducing harmful substance production, and has antitumor activity closely related to adhesion, oxidation resistance and immunoregulation of lactobacillus.

The activity of extracellular polysaccharide of lactic acid bacteria is directly or indirectly related to the molecular structure thereof, and the structure of extracellular polysaccharide molecules is modified by a certain method, so that the obtained derivative can improve the biological activity thereof, even add new functions, and is also receiving more and more attention. The phosphorylation reaction of polysaccharide is mainly a covalent modification, and is a process of substituting hydroxyl on a branched chain by phosphate radical. Among the polysaccharide derivatives, the sulfuric acid derivatives are also important ones, and the substitution of the hydroxyl group of the polysaccharide with a sulfate group not only improves the water solubility of the polysaccharide, but also improves the bioactivity of the polysaccharide by forming a helical structure in a local region of the sugar chain.

The lactobacillus rhamnosus is an important probiotic in human intestinal tracts and has the effects of regulating intestinal flora, efficiently reducing cholesterol, preventing and treating diarrhea, improving the immunity of organisms and the like. Although the extracellular polysaccharide of lactic acid bacteria has wide application prospect, the problems of low yield, low purity and the like exist, so that the application is limited to a certain extent, and the extracellular polysaccharide of lactic acid bacteria is also a main influence factor for restricting industrial large-scale production. Therefore, increasing the yield and activity of exopolysaccharides is the focus of current research.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provides a preparation method of extracellular polysaccharide of lactic acid bacteria and derivatives thereof, which can improve the yield of extracellular polysaccharide of lactic acid bacteria.

The technical scheme adopted by the invention is as follows: a preparation method of exopolysaccharide of lactic acid bacteria comprises the following steps:

(1) preparing crude lactobacillus exopolysaccharides: inoculating the activated lactobacillus strain to an improved MRS selective culture medium according to the inoculation amount of 3%, culturing for 24h, centrifuging to remove precipitated bacteria, and taking supernatant; sequentially removing protein from the supernatant, concentrating under reduced pressure, precipitating with ethanol, centrifuging to obtain precipitate, dissolving with ultrapure water, and freeze drying to obtain crude extracellular polysaccharide of lactobacillus;

(2) preparing pure lactobacillus exopolysaccharides: dissolving the crude extracellular polysaccharide of the lactobacillus in ultrapure water, slowly adding the ultrapure water and a NaCl solution into a DEAE-52 cellulose column along the column wall, sequentially carrying out gradient elution by using the ultrapure water and the NaCl solution, collecting gradient eluent, carrying out reduced pressure concentration, dialyzing for 24 hours, and carrying out freeze drying to obtain the extracellular polysaccharide of the lactobacillus.

Further, in the preparation method, the improved MRS selective medium consists of: contains 1 percent of galacto-oligosaccharide, 2 percent of soybean peptone, 0.2 percent of phosphate and the balance of water according to weight percentage; the carbon nitrogen ratio is 1: 2, initial pH 6.5.

Further, in the preparation method, in the step (1), the deproteinization is that polyamide is added into the supernatant, the adding amount is 120g/L, and the mixture is magnetically stirred for 8 hours.

Further, in the preparation method, in the step (1), the alcohol precipitation is to add 95% ethanol with 4 times volume of the concentrate after the concentration under reduced pressure, and the alcohol precipitation time is 12 hours.

Further, in the above preparation method, in the step (2), the gradient elution is performed by using ultrapure water and NaCl solution, and the gradient eluent is collected by a method comprising: sequentially carrying out gradient elution by using ultrapure water and 0.1, 0.2, 0.3 and 0.4mol/L NaCl solution at the flow rate of 1mL/min, wherein each 15mL is a tube, and collecting gradient eluent; and tracking and detecting the concentration of the polysaccharide in each tube of eluent by using a phenol-sulfuric acid method, taking the tubes of the eluent with the highest concentration of the polysaccharide, and combining the eluents.

A preparation method of extracellular polysaccharide derivative of lactic acid bacteria is characterized in that the extracellular polysaccharide derivative of lactic acid bacteria is phosphorylated extracellular polysaccharide derivative of lactic acid bacteria, and the preparation method comprises the following steps: taking pure extracellular polysaccharide of lactobacillus and sodium hexametaphosphate, mixing uniformly, adjusting the pH of the mixed solution to 6.0, reacting for 4h at 90 ℃, cooling to room temperature, precipitating with ethanol, carrying out rotary evaporation on the obtained precipitate, redissolving in a water bath at 50 ℃, dialyzing, and carrying out vacuum freeze drying to obtain the extracellular polysaccharide derivative of the lactobacillus phosphorylated.

Further, the preparation method comprises the following steps of (by mass ratio) m_{Pure lactobacillus exopolysaccharides}：m_{Sodium hexametaphosphate}＝6:1。

Further, in the above preparation method, the alcohol precipitation is carried out by adding three times volume of 95% ethanol into the product cooled to room temperature, and carrying out alcohol precipitation for 24 h.

A preparation method of extracellular polysaccharide derivative of lactic acid bacteria is characterized in that the extracellular polysaccharide derivative of lactic acid bacteria is sulfated extracellular polysaccharide derivative of lactic acid bacteria, and the preparation method comprises the following steps: suspending pure extracellular polysaccharide of lactic acid bacteria in dimethylformamide, stirring for 20min, dropwise adding the mixture into an esterification reagent, reacting at 60 ℃ for 2h, cooling to room temperature, neutralizing with 4mol/L NaOH until the pH value is 7, precipitating with 95% ethanol at 4 ℃ for 12h, centrifuging, taking the precipitate, washing with 95% ethanol, redissolving with deionized water, dialyzing with a running water bag for 24h, and freeze-drying to obtain the extracellular polysaccharide derivative of sulfated lactic acid bacteria.

Further, in the above method, the esterifying reagent is chlorosulfonic acid (CSA) -pyridine (Pyr), and the method comprises the following steps: in an ice-water bath, chlorosulfonic acid was slowly added to pyridine, slowly stirred, reacted well, and when a large amount of pale yellow solids were observed, the ice-water bath was removed to give chlorosulfonic acid (CSA) -pyridine (Pyr), which was completed within 30 min.

The invention has the beneficial effects that: the method of the invention improves the purity and yield of extracellular polysaccharide of lactobacillus. And the lactobacillus extracellular polysaccharide is subjected to phosphoric acid and sulfation modification to obtain phosphorylated and sulfated derivatives of the lactobacillus extracellular polysaccharide, so that the biological activity of the lactobacillus extracellular polysaccharide and the derivatives thereof is improved, and the lactobacillus extracellular polysaccharide has a certain application value in the field of functional health-care food.

Drawings

FIG. 1 is a standard curve of glucose measurement by phenol-sulfuric acid method.

FIG. 2 is a graph showing the ability of extracellular polysaccharide of lactic acid bacteria and derivatives thereof to scavenge hydroxyl radicals in example 4.

FIG. 3 is a graph of the DPPH radical scavenging activity of exopolysaccharides and derivatives of lactic acid bacteria in example 4.

Detailed Description