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Method for purifying lactobacillus rhamnosus extracellular polysaccharides

A technology of Lactobacillus rhamnosus and extracellular polysaccharide, applied in the field of extracellular polysaccharide, can solve problems such as complicated steps and the like

Inactive Publication Date: 2016-10-12
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the main method at present is to optimize the fermentation process of active lactic acid bacteria milk beverage, and the steps are cumbersome

Method used

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  • Method for purifying lactobacillus rhamnosus extracellular polysaccharides
  • Method for purifying lactobacillus rhamnosus extracellular polysaccharides
  • Method for purifying lactobacillus rhamnosus extracellular polysaccharides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1: CaCl 2 Promotes the growth of Lactobacillus rhamnosus

[0044] During the fermentation process, there was a linear correlation between the number of live lactic acid bacteria and the absorbance in the fermented liquid with skim milk as the medium. Therefore, the number of viable lactic acid bacteria in fermented dairy products can be assessed by measuring the absorbance of the fermented broth. Simultaneously detect the pH value of the fermentation broth.

[0045] 1) Medium:

[0046] 1. MRS liquid medium (g / L): glucose 20, beef extract 10, Peptone 10, yeast powder 5, ammonium citrate 1, sodium acetate 5, KH 2 PO 4 2, MgSO 4 ·7H 2 O 0.05, MnSO 4 ·H 2 O 0.05, pH 6-6.5.

[0047] 2. MRS solid medium (g / L): glucose 20, beef extract 10, Peptone 10, yeast powder 5, ammonium citrate 1, sodium acetate 5, KH 2 PO 4 2, MgSO 4 ·7H 2 O 0.05, MnSO 4 ·H 2 O 0.05, agar powder 15, pH 6-6.5.

[0048] 2) Sterile CaCl 2 Preparation of solution

[0049] CaC...

Embodiment 2

[0066] Example 2: CaCl 2 Increase the number of live bacteria of Lactobacillus rhamnosus

[0067] The colony counting method is used to calculate the total number of bacteria, which does not include the number of dead bacteria, so it can accurately reflect the number of viable bacteria in the fermentation broth.

[0068] 1) Cultivate bacteria according to the culture method of Example 1. After culturing for 12h or 24h, take a 1ml fermentation broth sample and dilute it step by step to 10 with sterile water. -7 times. Take 10 of the fermentation broth samples under the two conditions respectively -6 and 10 -7 Double the two dilutions, draw 50 μl of the diluted bacterial solution and evenly spread it on the MRS solid medium plate, and incubate at 37°C for 48h. Take out the petri dish and plate, and pay attention to the distinction of tiny colonies, small insoluble particles or sediments on the plate during the counting process, carefully investigate and deal with suspicious ...

Embodiment 3

[0072] Embodiment 3: application example

[0073] Escherichia coli BL21 (E.coli BL21), Staphylococcus aureus (B.cereus), and Bacillus cereus (S.aureus) were selected for antibacterial experiments. The specific operation is as follows:

[0074] 1) Escherichia coli BL21 and Bacillus cereus were inoculated into LB liquid medium and cultured for 12 hours at a rotation speed of 200 rpm, and set aside. Staphylococcus aureus was inoculated into TSB liquid medium and cultured for 12 hours at a rotation speed of 150 rpm, and then set aside.

[0075] 2) Punch the filter paper with a puncher with an inner diameter of 5 mm to obtain several circular filter paper pieces with uniform inner diameter, and then sterilize.

[0076] 3) Prepare a certain amount of LB and TSB liquid and agar medium, and sterilize.

[0077]4) Put Lactobacillus rhamnosus in MRS liquid medium and add 10mM CaCl 2 The MRS was cultured (37°C, static) for 12 hours, and set aside.

[0078] 5) After heating and meltin...

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Abstract

The invention discloses a method for purifying lactobacillus rhamnosus extracellular polysaccharides and relates to the extracellular polysaccharides. The method includes: inoculating a single colony on the MRS solid culture medium of seed conservation to the activated fermentation liquid cultured in the MRS liquid culture medium; adding CaCl2 with the final concentration being 10mM into the MRS liquid culture medium, using the activated fermentation liquid for inoculation to obtain inoculated fermentation liquid, and measuring the OD600 and pH values of 1ml of inoculated liquid which is cultured for 0, 2, 4, 6, 12 and 24 hours; removing the cells and protein of the inoculated fermentation liquid, adding glacial acetic acid, placing into a refrigerator for one night, centrifuging, removing supernatant, performing freeze-drying on sediment, taking out, dialyzing, performing freeze-drying to obtain crude extracellular polysaccharides, dissolving into pure water, dissolving the extracellular polysaccharides sample after dialyzing and freeze-drying into distilled water to prepare a 20mg / mL solution, centrifuging, and performing ion column chromatography on supernatant to obtain the lactobacillus rhamnosus extracellular polysaccharides.

Description

technical field [0001] The invention relates to exopolysaccharides, in particular to a method for purifying the exopolysaccharides of Lactobacillus rhamnosus. Background technique [0002] The use of probiotics has a long history. A large number of studies have shown that lactic acid bacteria can regulate the balance of normal gastrointestinal flora in the body, lower serum cholesterol, control endotoxins, inhibit the production of spoilage bacteria and spoilage products in the intestine, and produce nutrients, thereby affecting the body's nutritional status, physiological functions, immune response [1] . Lactic acid is the main fermentation product of lactic acid bacteria. Lactic acid not only has a unique flavor, but also has a strong bactericidal power, which is unique among organic acids. Its bactericidal ability is several times greater than that of citric acid, tartaric acid, and succinic acid [2] . Lactic acid is also an attractive precursor for the chemical indu...

Claims

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Application Information

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IPC IPC(8): C12P19/04C08B37/00C12R1/225
CPCC12P19/04C08B37/0003
Inventor 卢英华薛成凤吴意珣凌雪萍敬科举姚传义
Owner XIAMEN UNIV
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