CN-121991828-A - Helicobacter pylori culture medium, culturing method and application thereof

CN121991828ACN 121991828 ACN121991828 ACN 121991828ACN-121991828-A

Abstract

The invention provides a helicobacter pylori culture medium, a culture method and application thereof, and relates to the technical field of microbial culture, wherein the helicobacter pylori culture medium comprises a basic culture medium and antibiotics, and when the basic culture medium is a brain heart infusion agar culture medium, the basic culture medium is a solid culture medium which is used for activation by adding defibrinated sheep blood or goat serum; when the basic culture medium is a Brucella broth culture medium, the basic culture medium is a liquid culture medium, the essential amino acid is provided by adding the L-cysteine component, and the basic culture medium is used as a reducing agent, so that the basic culture medium is beneficial to enrichment culture of anaerobic bacteria. Meanwhile, the ferric pyrophosphate, the L-cysteine and the antibiotics are used together to promote the rapid growth of helicobacter pylori, effectively reduce the pollution of mixed bacteria and provide a more stable growth environment for the helicobacter pylori, and the data show that the culture medium is used for culturing the helicobacter pylori, compared with the traditional solid culture method, the yield of the collected wet thalli is improved by nearly 3 times, and the high survival rate can be maintained while the helicobacter pylori is subjected to high-speed amplification culture.

Inventors

WANG DIAO
HE BIN
LIU WEI
LUO HAIFENG
XI JIANHONG
GUO YAN
WU XIAOCHENG
WANG WENFENG
Hou Sulin
YUAN HUIMEI

Assignees

杭州华葵金配生物科技有限公司

Dates

Publication Date: 20260508
Application Date: 20260326

Claims (10)

1. A helicobacter pylori culture medium, characterized by comprising a basal medium and an antibiotic; The basic culture medium comprises brain heart infusion agar culture medium or Brucella broth culture medium; said antibiotics comprises vancomycin trimethoprim, a amphotericin B and polymyxin B; when the basic culture medium is brain heart infusion agar culture medium, the helicobacter pylori culture medium also comprises defibrinated sheep blood or goat serum; When the basal medium is a Brookfield broth medium, L-cysteine, ferric pyrophosphate and fetal bovine serum are also included.
2. The helicobacter pylori medium according to claim 1, characterized in that the vancomycin content is 2.5-10 mg/L, trimethoprim content is 2.5-10 mg/L, amphotericin B content is 2.5-10 mg/L, and polymyxin B content is 0.25-1 mg/L, based on the total volume of the helicobacter pylori medium.
3. The helicobacter pylori culture according to claim 1 or 2, characterized in that the defibrinated sheep blood and the goat serum are added in an amount of 80 to 120 ml, preferably 100 ml, per 1000 ml helicobacter pylori culture, independently of each other.
4. The helicobacter pylori culture medium according to claim 1 or 2, characterized in that the contents of L-cysteine and ferric pyrophosphate are each independently 30 to 50 g/L based on the total volume of the helicobacter pylori culture medium; Preferably, the addition amount of the fetal bovine serum is 80-120 ml, preferably 100 ml ml, of the fetal bovine serum per 1000ml of the helicobacter pylori culture medium.
5. A method for culturing helicobacter pylori, characterized by comprising activating helicobacter pylori using the helicobacter pylori culture medium of which the basal medium is brain heart infusion agar medium according to any one of claims 1 to 4, and amplifying and culturing the activated helicobacter pylori using the helicobacter pylori culture medium of which the basal medium is Brucella broth medium according to any one of claims 1 to 4.
6. A culture method according to claim 5, wherein the activating helicobacter pylori comprises applying frozen helicobacter pylori to a culture medium of helicobacter pylori having a basal medium of brain heart infusion agar medium and containing defibrinated sheep blood, subjecting the culture medium to preliminary activation culture, and inoculating the colony of the preliminary activation culture to a culture medium of helicobacter pylori having a basal medium of brain heart infusion agar medium and containing goat serum again for secondary activation culture.
7. The method according to claim 5, wherein the amplification culture comprises inoculating helicobacter pylori obtained by the activation culture to a helicobacter pylori medium whose basal medium is a broths broth, and performing the amplification culture; Preferably, the inoculation amount is 1.5X10. 10 6 cfu/ml of bacterial liquid with concentration of 1.1 ml is inoculated in each 100 ml liquid culture medium; Preferably, the rotation speed of the amplification culture is 100-140 r/min, preferably 120 r/min.
8. The culture method according to claim 6 or 7, wherein the conditions of the activation culture and the amplification culture each independently comprise 35-38 ℃, 8-12% co 2 ,3~7%O 2 , preferably 37 ℃,10% co 2 ,5%O 2 ; preferably, the humidity of the activation culture and the amplification culture is 87-93%, preferably 90%, respectively and independently; Preferably, the time of the activation culture and the amplification culture is 46 to 50 hours, respectively.
9. Use of the helicobacter pylori culture medium of any one of claims 1 to 4 or the culture method of any one of claims 5 to 8 for the preparation of helicobacter pylori antigens.
10. A method for producing a helicobacter pylori antigen, characterized by comprising culturing helicobacter pylori by using the helicobacter pylori medium according to any one of claims 1 to 4 or the culturing method according to claim 5 to 8, centrifuging and collecting wet cells for ultrasonic breakage to obtain the helicobacter pylori antigen; Preferably, the rotational speed of the centrifugation is 8000-12000 r/min, preferably 10000 r/min; Preferably, the centrifugation time is 13-17 min, preferably 15 min.

Description

Helicobacter pylori culture medium, culturing method and application thereof Technical Field The invention relates to the technical field of microorganism culture, in particular to a helicobacter pylori culture medium, a helicobacter pylori culture method and application thereof. Background Helicobacter pylori (Helicobacter pylori, abbreviated as Hp) is the only microorganism species known to survive in the human stomach and is listed as a class of carcinogens by the world health organization international cancer research institute as the main cause of chronic gastritis, peptic gastric ulcer, and the like. Helicobacter pylori is a gram-negative, microaerophilic bacterium, and requires 5-8% of ambient oxygen, and cannot grow in atmospheric or absolute anaerobic environments. Diagnostic assays for H.pylori are currently an important research direction in the art, and the culture medium for H.pylori includes both non-selective and selective media. Common non-selective media are based on brain heart infusion agar, columbia agar, tryptone soy agar, and Wilkins-Chalgren agar. The culture medium is selected by adding certain antibacterial agents such as vancomycin, boscalid, amphotericin B, polymyxin B, trimethoprim (TMP), etc. In order to improve the culture efficiency, the prior art is generally added with necessary nutritional ingredients for the growth and development of helicobacter pylori on the basis of a traditional culture medium, the culture and enrichment speed is improved by 2 times compared with that of the traditional culture, and the form of helicobacter pylori can be maintained unchanged for 96 hours. However, the culture reagent has complicated operation steps, and the survival rate of the culture reagent cannot be ensured when the growth speed is increased. In view of this, the present invention has been made. Disclosure of Invention The invention aims to provide a helicobacter pylori culture medium, which solves the technical problem that the survival rate cannot be ensured when the helicobacter pylori cultured by the culture medium grows at a high speed in the prior art. The second object of the present invention is to provide a method for culturing helicobacter pylori. The third object of the present invention is to provide the use of the above helicobacter pylori culture medium or the above culture method for preparing helicobacter pylori antigen. The fourth object of the present invention is to provide a method for producing helicobacter pylori antigen. In order to achieve the above object of the present invention, the following technical solutions are specifically adopted: In a first aspect, the present invention provides a helicobacter pylori culture medium comprising a basal medium and an antibiotic; The basic culture medium comprises brain heart infusion agar culture medium or Brucella broth culture medium; said antibiotics comprises vancomycin trimethoprim, a amphotericin B and polymyxin B; when the basic culture medium is brain heart infusion agar culture medium, the helicobacter pylori culture medium also comprises defibrinated sheep blood or goat serum; When the basal medium is a Brookfield broth medium, L-cysteine, ferric pyrophosphate and fetal bovine serum are also included. Further, based on the total volume of the helicobacter pylori culture medium, the content of vancomycin is 2.5-10 mg/L, the content of trimethoprim is 2.5-10 mg/L, the content of amphotericin B is 2.5-10 mg/L, and the content of polymyxin B is 0.25-1 mg/L. Further, the addition amount of the defibrinated sheep blood and the goat serum is 80-120 ml, preferably 100 ml, of each 1000ml helicobacter pylori culture medium independently. Further, the content of L-cysteine and ferric pyrophosphate is respectively and independently 30-50 g/L based on the total volume of the helicobacter pylori culture medium; Preferably, the addition amount of the fetal bovine serum is 80-120 ml, preferably 100 ml ml, of the fetal bovine serum per 1000ml of the helicobacter pylori culture medium. In a second aspect, the present invention provides a method for culturing helicobacter pylori, comprising activating helicobacter pylori using the above-mentioned helicobacter pylori medium whose basal medium is brain heart infusion agar medium, and amplifying and culturing the activated helicobacter pylori using the above-mentioned helicobacter pylori medium whose basal medium is broths medium. Further, the method for activating the helicobacter pylori comprises the steps of coating frozen helicobacter pylori on a helicobacter pylori culture medium which is a brain heart infusion agar culture medium and contains defibrinated sheep blood as a basic culture medium, performing primary activation culture, inoculating a colony subjected to primary activation culture on the helicobacter pylori culture medium which is a brain heart infusion serum agar culture medium and contains goat serum as a basic culture medium, and performing secondary activati