Preparation method and application of culture medium suitable for lactobacillus rhamnosus to produce exopolysaccharide

A technology of Lactobacillus rhamnosus and exopolysaccharides, applied in the direction of microorganism-based methods, biochemical equipment and methods, applications, etc., can solve the problem of difficult removal, low yield of exopolysaccharides of Lactobacillus rhamnosus, and incomplete Eliminate interference and other issues

Pending Publication Date: 2022-05-27
ZHEJIANG GONGSHANG UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, due to the influence of medium components and culture conditions, the production of Lactobacillus rhamnosus exopolysaccharide (EPS) has not been high, and the MRS liquid medium commonly used for fermentation and cultivation of Lactobacillus rhamnosus, due to its composition Yeast extract, beef extract, and peptone contain more polysaccharide-like components but cannot be completely consumed by bacterial metabolism, and will be mixed with the exopolysaccharide secreted by the strain, and it is difficult to remove. For the quantification of exopolysaccharide and qualitative interference
Some studies have used some improved media, but these media components more or less contain one or more of yeast extract, beef extract, and peptone, and interference cannot be completely ruled out
In addition, whey medium and semi-defined medium are also used to avoid interference from components, but due to reasons such as poor growth of the strain, the yield of exopolysaccharides will be low

Method used

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  • Preparation method and application of culture medium suitable for lactobacillus rhamnosus to produce exopolysaccharide
  • Preparation method and application of culture medium suitable for lactobacillus rhamnosus to produce exopolysaccharide
  • Preparation method and application of culture medium suitable for lactobacillus rhamnosus to produce exopolysaccharide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1. Acquisition of Lactobacillus rhamnosus strain

[0061] Take 1 mL of fresh milk (fresh milk of New Hope Shuangfeng Milk Cow Breeding Base (Hangzhou)) into a sterile test tube, follow 10 －1 Perform serial dilutions, pipette 10 －2 , 10 －3 , 10 －4 and 10 －5 1ml of the dilution solution was evenly spread on the MRS plate, and placed in a 37°C incubator for 48h. Pick a single colony on the plate, inoculate it on the MRS plate and continue to cultivate for 3 days, inoculate the single colony with signs of acid production into the lactic acid bacteria medium, observe the colony morphology and microscopic morphological characteristics, study the physiological and biochemical changes, and carry out molecular analysis of the bacteria. Biological identification, 40% glycerol was stored at -80 ℃.

[0062] The resulting Lactobacillus rhamnosus ZFM216 activated strain was identified by 16S rDNA sequencing. For strains stored at -80°C, single colonies were selected for...

Embodiment 2

[0065] Embodiment 2, the polysaccharide production situation of Lactobacillus rhamnosus in conventional MRS medium

[0066] 1), the preparation of MRS medium:

[0067] MRS liquid medium: peptone 10g, yeast extract 5g, beef extract 10g, glucose 20g, sodium acetate 5g, diamine hydrogen citrate 2g, Tween-80 1mL, dipotassium hydrogen phosphate 2g, magnesium sulfate heptahydrate 0.2g, Manganese sulfate heptahydrate 0.05g, add ultrapure water to make up to 1L. High temperature sterilization (1.1 atmospheric pressure, sterilization at 115°C for 20min).

[0068] MRS solid medium: 15g / L agar was added to the above-mentioned MRS liquid medium, and the rest were the same.

[0069] 2), the preparation of Lactobacillus rhamnosus MRS fermentation broth:

[0070] Activated strains: Ultraviolet sterilization for 30min, take out the Lactobacillus rhamnosusZFM216 cryopreservation tube stored at -80°C, and thaw at room temperature for use. Take a one-time sterilized inoculation loop and dip ...

Embodiment 3

[0080] Example 3. Preparation of Lactobacillus rhamnosus-producing exopolysaccharide medium

[0081] 1), yeast extract, beef extract and peptone are subjected to polysaccharide removal as follows:

[0082] The yeast extract / beef extract / peptone is fully dissolved in ultrapure water according to the set mass / volume ratio of 10% (yeast extract is 5g / 50mL, beef extract and peptone are 10g / 100mL), and then 4 Double the volume of 4°C pre-cooled absolute ethanol, and place it at 4°C for precipitation for 12 hours; the supernatant was collected by centrifugation at 10,000 rpm for 15 min, and then the ethanol in the supernatant was removed by a rotary evaporator (about 60°C). Corresponding to the yeast extract / beef extract / peptone after depolysaccharide respectively; spare.

[0083] Other conditions are prepared according to MRS liquid medium;

[0084] 2), yeast extract, beef extract and peptone, on the basis of the above polysaccharide removal treatment, the culture time (0-32h), c...

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Abstract

The preparation method comprises the following steps: dissolving yeast extract / beef extract / peptone in ultrapure water, carrying out depolysaccharide treatment by adding an absolute ethyl alcohol precipitation method, and removing ethyl alcohol from supernate; correspondingly obtaining a depolysaccharide yeast extract / depolysaccharide beef extract / depolysaccharide peptone; the depolysaccharide yeast extract / depolysaccharide beef extract / depolysaccharide peptone is used for preparing a culture medium. The invention further discloses a preparation method of the lactobacillus rhamnosus extracellular polysaccharide, and the preservation number of the lactobacillus rhamnosus ZFM216 is CCTCC (China Center for Type Culture Collection) NO: M2020325. The lactobacillus rhamnosus extracellular polysaccharide can be used for preparing the lactobacillus rhamnosus extracellular polysaccharide. The lactobacillus rhamnosus exopolysaccharide disclosed by the invention is used for preparing functional foods.

Description

technical field [0001] The invention relates to the technical field of polysaccharides in microbiology, in particular to the development of an extracellular polysaccharide-producing medium of Lactobacillus rhamnosus, the preparation of extracellular polysaccharides and the biological activity of the polysaccharides. Background technique [0002] Lactobacillus rhamnosus mostly exists in the intestinal tract of humans and animals, and has the functions of improving gastrointestinal function, enhancing immunity, enhancing intestinal barrier function, promoting the growth of other probiotics, and assisting the body to regulate the intestinal micro-ecological environment. It also has important applications in improving the rheological properties of food and improving the flavor properties. Studies have shown that these excellent properties of Lactobacillus rhamnosus are closely related to the extracellular polysaccharides it secretes. Extracellular polysaccharides are macromolec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P19/04A23L33/125C12R1/225
CPCC12N1/20C12P19/04A23L33/125A23V2002/00A23V2200/30A23V2250/51
Inventor 顾青陈亮周涛
Owner ZHEJIANG GONGSHANG UNIVERSITY
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