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CN-122012241-A - Separation, culture and freeze-drying method of parabacteroides dieldrin

CN122012241ACN 122012241 ACN122012241 ACN 122012241ACN-122012241-A

Abstract

The invention discloses a method for separating, culturing and freeze-drying Paramycolatopsis Diels. The method for separating the parabacteroides dieldahl comprises the following steps of S1, collecting a fecal sample of a healthy child to prepare a fecal suspension, S2, diluting the fecal suspension, coating the fecal suspension in a culture medium containing antibiotics, culturing under anaerobic conditions, S3, picking single bacterial colonies with consistent forms, obtaining the single bacterial colonies through pure culture, carrying out MALDI-TOF MS identification on the single bacterial colonies, and then carrying out bidirectional Sanger sequencing to confirm that the bacterial strains are parabacteroides dieldahl. The invention provides the whole process technical parameters related to hypoxia exposure control, selective culture, separation and purification, genome identification, amplification culture and freezing or freeze-drying, and can obtain the parabacteroides dirachta strain resources which have stable activity and phenotype after resuscitation and are suitable for culture amplification and preparation preservation. The method can be used for basic research, large-scale culture and related product development of the parabacteroides dirachta.

Inventors

  • DAI DONGLING
  • LIN BAIXIAN
  • CHEN HU
  • Zhuang Zeling
  • MENG SHIYUN
  • QIU PEIJIAN
  • ZHU ZHONGSHENG
  • LUO MINGJING
  • Zhou Haokui
  • Zhang Chuqing
  • Guan Jiaojie
  • Zou Yigui
  • LI WENWEN
  • LI ZIYUAN

Assignees

  • 深圳市儿童医院
  • 深圳先进技术研究院

Dates

Publication Date
20260512
Application Date
20251231

Claims (10)

  1. 1. The method for separating the parabacteroides dieldrin is characterized by comprising the following steps of: S1, collecting a fecal sample of a healthy child to prepare a fecal suspension; S2, diluting the fecal suspension, coating the fecal suspension in a culture medium containing antibiotics, and culturing the fecal suspension under anaerobic conditions; S3, picking single colony with consistent forms, obtaining single colony through pure culture, carrying out MALDI-TOF MS identification on the single colony, and then carrying out bidirectional Sanger sequencing to confirm that the strain is Paralogue parapsilosis; The antibiotic-containing medium comprises adding 0.1-1 g/L cysteine, 0.2-5 mg/L vitamin K1, 1-10 mg/L heme and 0.01-0.1% L-cysteine to the basal medium.
  2. 2. The method of claim 1, wherein the antibiotic-containing medium comprises adding 0.5 g/L cysteine, 1 mg/L vitamin K1, 5 mg/L heme, and 0.05% L-cysteine to a basal medium, wherein the basal medium is mGAM.
  3. 3. The method of isolation according to claim 1 or 2, wherein the antibiotic-containing medium further comprises 1-3. Mu.g/mL vancomycin and 10-100. Mu.g/mL kanamycin.
  4. 4. The separation method according to claim 1, wherein the steps S1 to S3 are all performed under anaerobic conditions, wherein the anaerobic conditions are N 2 :CO 2 :H 2 = 80:10:10.
  5. 5. The method of claim 1, wherein the healthy child is a 6 year old healthy child who has not used antibiotics or other drugs 3 months prior to collection, without any disease.
  6. 6. A parabacteroides dieldrin, characterized in that the parabacteroides dieldrin obtained by the separation method of claims 1-5.
  7. 7. An amplification culture method of Paralopecuroides dirachta is characterized in that a culture medium containing 0.2-5 mg/L vitamin K1 and 1-10 mg/L heme is adopted for culture under anaerobic conditions; The Paralopecuroides dirachta is obtained by adopting the separation method of claims 1-5.
  8. 8. The method according to claim 7, wherein a mGAM medium containing 1 mg/L of vitamin K1 and 5 mg/L of heme is used.
  9. 9. The freeze-preserving method of the parabacteroides dieldschii is characterized in that the freeze-preserving agent adopted in the freeze-preserving method comprises 1-10% of sucrose, 5-20% of skimmed milk powder and 0.01-0.1% of L-cysteine hydrochloride; The Paralopecuroides dirachta is obtained by adopting the separation method of claims 1-5.
  10. 10. The method of claim 9, wherein the lyoprotectant composition used in the method comprises 5% sucrose, 10% nonfat dry milk, 0.05% calcium chloride, 0.05% magnesium chloride, 0.01% L-cysteine hydrochloride.

Description

Separation, culture and freeze-drying method of parabacteroides dieldrin Technical Field The invention relates to the technical field of microorganisms, in particular to a method for separating, culturing and freeze-drying parabacteroides dirachta. Background Paramycolatopsis Diels (P. Distasonis) is an important symbiotic bacterium in human intestinal tract, and has polysaccharide utilization site (PUL) and short chain fatty acid metabolism potential. In recent years, research shows that P.distasonis has potential probiotic functions in the aspects of maintaining intestinal microecological balance, regulating host metabolism, immunity, nerve function and the like. However, the isolation, purification and preservation of p. In the separation stage, p. distasonis is often preempted in the growth space by dominant anaerobic bacteria such as Bacteroides paraqualis (Parabacteroides) of the same genus, bacteroides pseudoqualis (bacterioides) or Prevotella (Prevotella) due to weak competitiveness, resulting in low separation efficiency of the target strain. After pure culture, strains are susceptible to phenotypic degradation or reduced activity over multiple passages. Accordingly, there is a need for improvement and development in the art. Disclosure of Invention In view of the shortcomings of the prior art, the invention aims to provide a method for separating, culturing and freeze-drying parabacteroides dirachta, and aims to solve the problem of low separation efficiency of the parabacteroides dirachta in the prior art. The technical scheme of the invention is as follows: in a first aspect of the present invention, a method for separating parabacteroides dirachta is provided, comprising the steps of: S1, collecting a fecal sample of a healthy child to prepare a fecal suspension; S2, diluting the fecal suspension, coating the fecal suspension in a culture medium containing antibiotics, and culturing the fecal suspension under anaerobic conditions; S3, picking single colony with consistent forms, obtaining single colony through pure culture, carrying out MALDI-TOF MS identification on the single colony, and then carrying out bidirectional Sanger sequencing to confirm that the strain is Paralogue parapsilosis; The antibiotic-containing medium comprises adding 0.1-1 g/L cysteine, 0.2-5 mg/L vitamin K1, 1-10 mg/L heme and 0.01-0.1% L-cysteine to the basal medium. Optionally, the antibiotic-containing medium comprises adding 0.5 g/L cysteine, 1mg/L vitamin K1, 5 mg/L heme, and 0.05% L-cysteine to a basal medium, wherein the basal medium is mGAM. Optionally, the antibiotic-containing medium further comprises 1-3. Mu.g/mL vancomycin and 10-100. Mu.g/mL kanamycin. Optionally, each of the steps S1 to S3 is performed under anaerobic conditions, where the anaerobic conditions are N 2:CO2:H2 =80:10:10. Optionally, the healthy child is a 6 year old healthy child who does not use antibiotics or other drugs 3 months before collection, without any disease. In a second aspect of the invention, a parabacteroides dirachta is provided, and the parabacteroides dirachta is obtained by the separation method. In a third aspect of the invention, an amplification culture method of Paramycolatopsis Diels is provided, a culture medium containing 0.2-5 mg/L vitamin K1 and 1-10 mg/L heme is adopted, and culture is carried out under anaerobic conditions; the parabacteroides dieldrin is obtained by adopting the separation method. Alternatively, mGAM medium containing 1 mg/L vitamin K1 and 5 mg/L heme was used. The invention provides a freezing method of parabacteroides dieldae, which comprises the following steps of adopting freeze-drying protective agent components including 1-10% of sucrose, 5-20% of skim milk powder and 0.01-0.1% of L-cysteine hydrochloride; the parabacteroides dieldrin is obtained by adopting the separation method. Optionally, the freeze-drying protective agent adopted in the freezing and preserving method comprises 5% of sucrose, 10% of skimmed milk powder, 0.05% of calcium chloride, 0.05% of magnesium chloride and 0.01% of L-cysteine hydrochloride. The invention has the beneficial effects that the invention provides a method for separating, culturing and freeze-drying the parabacteroides dieldahl, compared with the prior art, the method has the advantages that under the anaerobic environment, the method combines the culture medium containing cysteine, L-cysteine, vitamin K1 and heme, and adopts antibiotics to form certain selection pressure, thereby providing a more proper growth environment for the parabacteroides dieldahl under the mixed flora condition. On the basis, the parabacteroides dieldahl can be obtained by diluting, coating, picking out colonies, purifying and passaging, and carrying out identification by combining MALDI-TOF and 16S rRNA sequencing. In addition, by genome analysis of the isolated parabacteroides dieldae, it was found that it carries a plurality of genes related to amino acid metabolism, synth