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CN-122005616-A - Preparation method and application of roxburgh rose polysaccharide-mucin-philic Ackermans mixture

CN122005616ACN 122005616 ACN122005616 ACN 122005616ACN-122005616-A

Abstract

The invention discloses a preparation method of a Rosa roxburghii polysaccharide-mucin-philic Ackermans mixture and application of the Rosa roxburghii polysaccharide-mucin-philic Ackermans mixture in regulating intestinal microecology and improving lipid metabolism, and belongs to the technical field of probiotic products. According to the invention, the in vitro anaerobic culture of the mucin-philin Ackermans is carried out, so that the Rosa roxburghii polysaccharide has a promoting effect on the growth and proliferation of the mucin-philin Ackermans and has a remarkable effect. The invention can obviously improve the disorder of intestinal flora, improve lipid metabolism disorder caused by high-fat diet, improve intestinal barrier function, reduce inflammatory reaction, activate the expression of key proteins such as PPARgamma, ABCG1 and the like, promote reverse transportation of cholesterol and inhibit lipogenesis through the mucin Achroman cultured by the roxburgh rose polysaccharide, thereby improving lipid metabolism steady state. The roxburgh rose polysaccharide-mucin-philic ackermannin mixture provides scientific basis for the synergic regulation of lipid metabolism by regulating and controlling the effect of intestinal microecology to improve lipid metabolism disorder and the mechanism thereof.

Inventors

  • CAI KUN
  • PENG JINGYI
  • GAO XIAOJIAO
  • TIAN WEIYI

Assignees

  • 贵州中医药大学

Dates

Publication Date
20260512
Application Date
20260107

Claims (10)

  1. 1. A preparation method of a Rosa roxburghii polysaccharide-mucin-philic Ackermans mixture is characterized by comprising the following steps: (1) Preparing fructus Rosae Normalis polysaccharide mother liquor, namely preparing mother liquor with concentration of 0-120mg/mL by using standard fructus Rosae Normalis polysaccharide, and filtering with 0.1-0.3 μm sterile filter membrane to obtain fructus Rosae Normalis polysaccharide mother liquor; (2) Preparing a thioglycolate liquid culture medium, namely mixing and proportioning all the components of the thioglycolate liquid culture medium, sterilizing for 10-20min under high-pressure steam at the temperature of 100-140 ℃, and cooling to the pH value of 5-9 for later use, wherein the components of the thioglycolate liquid culture medium consist of 15.0 parts of casein peptone, 5.0 parts of yeast extract powder, 5.0 parts of glucose, 0.5 parts of sodium thioglycolate, 0.5 part of L-cystine, 2.5 parts of sodium chloride, 0.001 part of resazurin and 0.75 part of agar according to the mass ratio; (3) Anaerobic treatment of the culture medium, namely, putting the thioglycolate liquid culture medium in the step (2) in an anaerobic working station for deoxidizing for 20-30 hours, wherein the temperature of the anaerobic working station is 30-50 ℃, and the anaerobic working station consists of 70-90% of N 2 、5-15%H 2 and 1-10% of CO 2 for standby; (4) Absorbing 0.5mL of the anaerobic thioglycolate liquid culture medium in the step (3), adding the anaerobic thioglycolate liquid culture medium into 1X 10 6 of the Achroman mucilaginosus freeze-dried bacteria for full dissolution, culturing in an anaerobic workstation for 2-6 days to obtain the 1 st-generation bacteria, inoculating the 1 st-generation bacteria to the anaerobic thioglycolate liquid culture medium according to the 5% volume inoculum size for culturing to obtain the 2 nd-generation bacteria, and inoculating the 2 nd-generation bacteria to the anaerobic thioglycolate liquid culture medium according to the 5% volume inoculum size for culturing to obtain the 3 rd-generation bacteria for later use; (5) And (3) calibrating the 3 rd generation bacteria in the logarithmic growth phase to 1X 10 9 CFU/mL by using a Mahalanobis turbidimetry method, re-suspending the bacteria with normal saline, and mixing the re-suspended bacteria with 0.2mL of Rosa roxburghii polysaccharide mother liquor to prepare the Rosa roxburghii polysaccharide-mucin-philic Ackermans mixture.
  2. 2. The method for preparing the roxburgh rose polysaccharide-mucin-philic ackermannin mixture according to claim 1, wherein in the step (1), the roxburgh rose polysaccharide mother liquor is prepared by taking a standard substance of roxburgh rose polysaccharide, preparing the mother liquor with the concentration of 20-80mg/mL by using ultrapure water, and filtering the mother liquor by using a sterile filter membrane with the concentration of 0.15-0.25 mu m.
  3. 3. The method for preparing the roxburgh rose polysaccharide-mucin-philic ackerman mixture according to claim 2, wherein in the step (1), the roxburgh rose polysaccharide mother liquor is prepared by taking a standard substance of roxburgh rose polysaccharide, preparing mother liquor with the concentration of 40mg/mL by ultrapure water, and filtering the mother liquor by a 0.22 mu m sterile filter membrane.
  4. 4. The preparation method of the roxburgh rose polysaccharide-mucin-philic ackermannin mixture according to claim 1, wherein in the step (2), the preparation of a thioglycolate liquid culture medium is carried out by taking the components of the thioglycolate liquid culture medium, mixing and proportioning, sterilizing for 13-17min under high-pressure steam at the temperature of 110-130 ℃, and cooling to obtain the pH value of 6-8 for later use, wherein the components of the thioglycolate liquid culture medium consist of 15.0 parts of casein peptone, 5.0 parts of yeast extract powder, 5.0 parts of glucose, 0.5 parts of sodium thioglycolate, 0.5 parts of L-cystine, 2.5 parts of sodium chloride, 0.001 parts of resazurin and 0.75 parts of agar according to the mass ratio.
  5. 5. The preparation method of the roxburgh rose polysaccharide-mucin-philic ackerman bacteria mixture according to claim 4, wherein in the step (2), the preparation of a thioglycolate liquid culture medium is carried out by taking all components of the thioglycolate liquid culture medium, mixing and proportioning, sterilizing for 15min under high-pressure steam at the temperature of 121 ℃, and cooling to obtain the pH value of 7.2 for later use, wherein the thioglycolate liquid culture medium component consists of 15.0 parts of casein peptone, 5.0 parts of yeast extract powder, 5.0 parts of glucose, 0.5 parts of sodium thioglycolate, 0.5 parts of L-cystine, 2.5 parts of sodium chloride, 0.001 parts of resazurin and 0.75 parts of agar according to the mass ratio.
  6. 6. The method for preparing the roxburgh rose polysaccharide-mucin-philic ackerman mixture according to claim 1, wherein in the step (3), the culture medium is subjected to anaerobic treatment, namely the thioglycolate liquid culture medium in the step (2) is placed in an anaerobic working station for deoxidizing for 22-27 hours, the temperature of the anaerobic working station is 35-40 ℃, and the anaerobic working station consists of 75-85% N 2 、7-13H 2 and 3-7% CO 2 .
  7. 7. The method for preparing the roxburgh rose polysaccharide-mucin-philic ackermannin mixture according to claim 6, wherein in the step (3), the culture medium is subjected to anaerobic treatment, namely the thioglycolate liquid culture medium in the step (2) is placed in an anaerobic working station for deoxidizing for 24 hours, the temperature of the anaerobic working station is 37 ℃, and the anaerobic working station consists of 85% N 2 、10H 2 and 5% CO 2 .
  8. 8. The preparation method of the roxburgh rose polysaccharide-mucin-philic ackermannin mixture according to claim 1, wherein in the step (4), 0.5mL of the anaerobic thioglycolate liquid culture medium in the step (3) is absorbed, and after the anaerobic thioglycolate liquid culture medium is fully dissolved in 1×10 6 of mucin-philic ackermannin freeze-dried bacteria, the culture is carried out in an anaerobic workstation for 3-5 days to obtain the 1 st-generation bacteria, the 1 st-generation bacteria are inoculated to the anaerobic postthioglycolate liquid culture medium according to the inoculum size of 5% by volume for culture to obtain the 2 nd-generation bacteria, and then the 2 nd-generation bacteria are inoculated to the anaerobic postthioglycolate liquid culture medium according to the inoculum size of 5% by volume for culture to obtain the 3 rd-generation bacteria.
  9. 9. The preparation method of the roxburgh rose polysaccharide-mucin-philic ackermannin mixture according to claim 8, which is characterized in that in the step (4), 0.5mL of the anaerobic thioglycolate liquid culture medium in the step (3) is absorbed, fully dissolved in 1X 10 6 of mucin-philic ackermannin freeze-dried bacteria, the mixture is cultured in an anaerobic workstation for 4 days to obtain the 1 st-generation bacteria, the 1 st-generation bacteria are inoculated to the anaerobic thioglycolate liquid culture medium according to the inoculum size of 5% by volume for culturing to obtain the 2 nd-generation bacteria, and the 2 nd-generation bacteria are inoculated to the anaerobic thioglycolate liquid culture medium according to the inoculum size of 5% by volume for culturing to obtain the 3 rd-generation bacteria.
  10. 10. Application of Rosa roxburghii polysaccharide-mucin-philin Ackermans mixture in preparing intestinal microecology regulating and lipid metabolism improving product is provided.

Description

Preparation method and application of roxburgh rose polysaccharide-mucin-philic Ackermans mixture Technical Field The invention relates to the technical field of probiotic products, in particular to a preparation method and application of a Rosa roxburghii polysaccharide-mucin-philic Ackermans mixture. Background The roxburgh rose serving as a traditional plant with homology of medicine and food has rich nutrition and unique biological activity, and polysaccharide (Polysaccharide from Rosa roxburghii Tratt, RTFP) in the roxburgh rose is a typical non-digestible polysaccharide, and the non-digestible polysaccharide is a macromolecular saccharide compound polymerized by various sugar molecules in vegetable food. Achroman muciniphila (AKKERMANSIA MUCINIPHILA, akk) is a gram-negative, spore-free, anaerobic intestinal symbiotic bacterium with special mucous degradation function. It mainly colonizes the intestinal mucus layer, maintaining its growth by degrading mucins secreted by intestinal epithelial cells. It is often used in metabolic diseases such as obesity, type 2 diabetes, etc. Currently, the research of the rosa roxburghii polysaccharide (Polysaccharide from Rosa roxburghii Tratt, RTFP) and the mucin-philic ackermanni (AKKERMANSIA MUCINIPHILA, akk) is still in an independent exploration stage, and the development of the rosa roxburghii polysaccharide as a synergistic mixture faces a remarkable bottleneck. The primary difficulty is that the mucin-philic Achroman is used as strict anaerobe, the abundance of mucin-philic Achroman is obviously reduced in metabolic diseases such as obesity, type 2 diabetes and the like, and the molecular structure, active ingredients and functional stability of the Rosa roxburghii polysaccharide are difficult to uniformly control due to various extraction processes, so that the preparation and quality consistency of the Rosa roxburghii polysaccharide-mucin-philic Achroman mixture are severely tested. At present, preparation products of the roxburgh rose polysaccharide and the mucin-philin Ackermans probiotics are not explored, and the specific ways of regulating and controlling the integrity of intestinal barriers, the metabolism of intestinal lipids and systemic inflammation are disclosed by researching the roxburgh rose polysaccharide and the mucin-philin Ackermans probiotics, so that the preparation method of the roxburgh rose polysaccharide-mucin-philin Ackermans probiotics and the application of the roxburgh rose polysaccharide-mucin-philin Ackermans probiotics in regulating intestinal micro-ecology to improve lipid metabolism are key to promoting the mixture to practical application. Disclosure of Invention Aiming at the defects in the technology, the invention provides a preparation method and application of a Rosa roxburghii polysaccharide-mucin-philic Ackermans mixture, and solves the problem of lipid metabolism disorder and intestinal microecology incapacity of regulation. The technical scheme of the invention is as follows: a preparation method of a Rosa roxburghii polysaccharide-mucin-philic Ackermans mixture comprises the following steps: (1) Preparing fructus Rosae Normalis polysaccharide mother liquor, namely preparing mother liquor with concentration of 0-120mg/mL by using standard fructus Rosae Normalis polysaccharide, and filtering with 0.1-0.3 μm sterile filter membrane to obtain fructus Rosae Normalis polysaccharide mother liquor; (2) Preparing a thioglycolate liquid culture medium, namely mixing and proportioning all the components of the thioglycolate liquid culture medium, sterilizing for 10-20min under high-pressure steam at the temperature of 100-140 ℃, and cooling to the pH value of 5-9 for later use, wherein the components of the thioglycolate liquid culture medium consist of 15.0 parts of casein peptone, 5.0 parts of yeast extract powder, 5.0 parts of glucose, 0.5 parts of sodium thioglycolate, 0.5 part of L-cystine, 2.5 parts of sodium chloride, 0.001 part of resazurin and 0.75 part of agar according to the mass ratio; (3) Anaerobic treatment of the culture medium, namely, putting the thioglycolate liquid culture medium in the step (2) in an anaerobic working station for deoxidizing for 20-30 hours, wherein the temperature of the anaerobic working station is 30-50 ℃, and the anaerobic working station consists of 70-90% of N 2、5-15%H2 and 1-10% of CO 2 for standby; (4) Absorbing 0.5mL of the anaerobic thioglycolate liquid culture medium in the step (3), adding the anaerobic thioglycolate liquid culture medium into 1X 10 6 of the Achroman mucilaginosus freeze-dried bacteria for full dissolution, culturing in an anaerobic workstation for 2-6 days to obtain the 1 st-generation bacteria, inoculating the 1 st-generation bacteria to the anaerobic thioglycolate liquid culture medium according to the 5% volume inoculum size for culturing to obtain the 2 nd-generation bacteria, and inoculating the 2 nd-generation bacteria to the anaerobic thioglycolate liqu