阅读说明：本技术 一种抑制结直肠癌致病菌的益生菌配方及其筛选方法 (Probiotic formula for inhibiting colorectal cancer pathogenic bacteria and screening method thereof ) 是由 陈卫华 江浦滋 吴思成 聂庆庆 刘智 于 2019-09-25 设计创作，主要内容包括：本发明公开了一种抑制结直肠癌致病菌的益生菌配方及其筛选方法,益生菌的筛选方法为：根据正常人和结直肠癌患者的肠道菌群的宏基因组数据及丰度找到结直肠癌致病菌,然后构建肠道菌群互作网络筛选可抑制结直肠癌致病菌的益生菌,调节益生菌配比最后进行验证。利用该方法得到的益生菌配方为：丁酸梭菌1.3×10<Sup>10</Sup>CFU/ml,粪肠球菌1×10<Sup>9</Sup>CFU/ml,短乳杆菌1×10<Sup>9</Sup>CFU/ml,植物乳杆菌1×10<Sup>9</Sup>CFU/ml,鼠李糖乳杆菌1×10<Sup>9</Sup>CFU/ml,清酒乳杆菌1×10<Sup>9</Sup>CFU/ml,肠系膜明串珠菌1×10<Sup>9</Sup>CFU/ml。本发明的优点在于：(1)构建了肠道菌群互作网络,使得筛选得到的益生菌配方能更好定殖于肠道；(2)得到的益生菌配方不会产生耐药性,并且安全、有效,性能优良；(3)构建了人体肠道微生物组在线仓库数据库,使结果更加普适、可靠。(The invention discloses a probiotic formula for inhibiting pathogenic bacteria of colorectal cancer, a screening method thereof and a screening method of probioticsComprises the following steps: finding out the colorectal cancer pathogenic bacteria according to the metagenome data and abundance of the intestinal flora of normal people and colorectal cancer patients, then constructing an intestinal flora interaction network, screening probiotics capable of inhibiting the colorectal cancer pathogenic bacteria, adjusting the ratio of the probiotics and finally verifying. The probiotic formula obtained by the method is as follows: clostridium butyricum 1.3X 10 10 CFU/ml, enterococcus faecalis 1X 10 9 CFU/ml, Lactobacillus brevis 1X 10 9 CFU/ml, Lactobacillus plantarum 1X 10 9 CFU/ml, Lactobacillus rhamnosus 1X 10 9 CFU/ml, Lactobacillus sake 1X 10 9 CFU/ml, Leuconostoc mesenteroides 1X 10 9 CFU/ml. The invention has the advantages that: (1) an intestinal flora interaction network is constructed, so that the screened probiotic formula can better colonize the intestinal tract; (2) the obtained probiotic formula does not generate drug resistance, and is safe, effective and excellent in performance; (3) an online warehouse database of the human intestinal microbiome is constructed, so that the result is more universal and reliable.)

1. A screening method of a probiotic formula for inhibiting colorectal cancer pathogenic bacteria is characterized by comprising the following steps:

s1, constructing a human intestinal microbiome online warehouse database, searching and downloading metagenome data of intestinal flora of normal people and colorectal cancer patients based on the human intestinal microbiome online warehouse database, performing quality control on the metagenome data, and evaluating the metagenome data after quality control to obtain the metagenome data after quality control;

s2, performing metagenome species annotation analysis on the metagenome data after quality control to obtain abundance information of species of intestinal flora of normal people and colorectal cancer patients;

s3, analyzing the difference of intestinal flora between a normal person and a colorectal cancer patient based on the macro-genome data after quality control and the abundance information, further screening to obtain differential bacteria with difference between the normal person and the colorectal cancer patient, inputting the differential bacteria into an online warehouse database of the human intestinal microbiome, checking the abundance of the differential bacteria in the intestinal flora of the normal person, and identifying pathogenic bacteria of the colorectal cancer;

s4, respectively selecting bacteria with the abundance ranks 50 above of the species of the intestinal flora of normal people and colorectal cancer patients according to the abundance information in S2, merging the bacteria, acquiring the growth rates of the merged bacteria under the conditions of independent culture and pairwise co-culture in intestinal tracts according to a genome scale metabolic network model from the literature, and constructing an intestinal flora interaction network based on the growth rates;

s5, screening probiotics for inhibiting the colorectal cancer pathogenic bacteria in the S3 based on the intestinal flora interaction network of S4;

s6, optimizing the probiotics in the S5 by combining the abundance of the probiotics in the intestinal flora of normal people to obtain a probiotic formula for inhibiting colon cancer pathogenic bacteria;

and S7, carrying out in-vivo experimental animal verification on the probiotic formula in S6.

2. The method of screening a probiotic formulation for the inhibition of pathogenic bacteria of colorectal cancer according to claim 1, wherein in S1 the quality control is performed using trimmatic software and the post-quality control evaluation is performed using Fastqc software.

3. The method of screening for a probiotic formulation for the suppression of pathogenic bacteria of colorectal cancer according to claim 1, wherein the metagenomic species annotation analysis was performed in S2 using MetaPhIAn2 software.

4. The method of screening a probiotic formulation for the inhibition of pathogenic bacteria of colorectal cancer according to claim 1, wherein said analysis is performed using LefSe and said screening of said differentiating bacteria is performed using a random forest algorithm in S3.

5. The method for screening a probiotic formula for inhibiting pathogenic bacteria of colorectal cancer according to claim 1, wherein in S4, the growth rate of bacteria cultured in the intestinal tract individually is denoted as S, the growth rate of bacteria cultured in the intestinal tract together in pairs is denoted as P, the weight W between bacteria is calculated as log2(P/S), the bacteria with the top abundance ranking 50 are used as nodes, the weight W between bacteria is used as sides, the intestinal flora interaction network is drawn by open source software Gephi, meanwhile, the modulariarithm function in the Gephi software is used for identifying the subgroup in the intestinal flora metabolic network, and the PageRankalgorithm function in the Gephi software is used for identifying the important nodes in the intestinal flora metabolic network.

6. A probiotic formulation obtained by a method of screening a probiotic formulation for the inhibition of pathogenic bacteria of colorectal cancer according to any one of claims 1 to 5, characterized in that the Clostridium butyricum (Clostridium butyricum) content is 1 x 10⁹～5×10¹⁰CFU/ml, Enterococcus faecalis (Enterococcus faecalis) content of 1 × 10⁸～5×10⁹CFU/ml, Lactobacillus brevis (Lactobacillus brevis) content of 1 × 10⁸～5×10⁹CFU/ml, Lactobacillus plantarum (Lactobacillus) content of 1 × 10⁸～5×10⁹CFU/ml, Lactobacillus rhamnosus (Lactobacillus rhamnosus) content of 1 × 10⁸～5×10⁹CFU/ml, Lactobacillus sake (Lactobacillus sakei) 1X 10⁸～5×10⁹CFU/ml, Leuconostoc mesenteroides content 1X 10⁸～5×10⁹CFU/ml。

7. The probiotic formulation according to claim 6, characterized in that the Clostridium butyricum (Clostridium butyricum) content is 1.3 x 10¹⁰CFU/ml, Enterococcus faecalis (Enterococcus faecalis) content of 1 × 10⁹CFU/ml, Lactobacillus brevis (Lactobacillus brevis) content of 1 × 10⁹CFU/ml, Lactobacillus plantarum (Lactobacillus) content of 1 × 10⁹CFU/ml, Lactobacillus rhamnosus (Lactobacillus rhamnosus) content of 1 × 10⁹CFU/ml, Lactobacillus sake (Lactobacillus sakei) 1X 10⁹CFU/ml, Leuconostoc mesenteroides content 1X 10⁹CFU/ml。

Technical Field

The invention relates to the technical field of biological medicines, and particularly relates to a probiotic formula for inhibiting colorectal cancer pathogenic bacteria and a screening method thereof.

Background

Colorectal cancer (CRC), a common malignancy in humans, is currently listed as the third largest cancer in the world. With the improvement of living standard and the change of dietary structure, the morbidity and mortality of people are on an increasing trend, and the health of people is seriously threatened. Numerous studies have reported that CRC is associated with a variety of factors, such as obesity, diabetes, sedentary lifestyle, high fat diet, smoking, alcohol abuse, age, sex, family history, etc., but the precise pathogenesis remains unclear.

More and more studies in this year suggest that the intestinal flora is a very important factor in relation to the development of CRC. The intestinal flora, as an organoid, has a deep interaction relationship with host intestinal mucosal epithelial cells and immune cells, and its composition, quantity and activity play a crucial role in many physiological processes, especially in inflammation and immune response. The gut flora has been shown to be likely the pivotal point for cancer development and to be involved directly or indirectly in the development of gut tumors. The literature reports that the composition of intestinal flora changes in colon cancer, and the ecological imbalance of the intestinal flora plays an important role in the occurrence and development of colorectal cancer. Some bacterial species, such as enterococcus faecalis, escherichia coli, fusobacterium nucleatum, streptococcus hemolyticus, bacteroides fragilis, etc., have a high proportion in the stool and tumor tissue of CRC patients, while butyrate-producing bacteria are depleted in CRC patients.

The development of a drug for resisting pathogenic bacteria in the intestinal tract of a patient with colorectal cancer and recovering the intestinal bacteria homeostasis of the patient with cancer is likely to become an effective means for treating colorectal cancer. Currently, the abuse of antibiotics causes a series of troublesome problems such as drug resistance, and the like, and people are in urgent need to find an antibacterial therapeutic drug which can replace the antibiotics. The probiotics can compete with pathogenic bacteria for adhesion positions through a mechanism of resisting by colonists, resist the invasion of the pathogenic bacteria, regulate the immune response of a host and protect the immune system of a human body. In addition, the probiotics are more acid-resistant than the pathogenic bacteria, and can generate acidic substances such as organic acid and the like to create an acidic microenvironment, so that the growth of the pathogenic bacteria is inhibited. The use of probiotics can resist the imbalance of intestinal flora of CRC patients, thereby restoring intestinal bacteria ecological imbalance caused by diseases, and reducing inflammation, genetic toxicity, carcinogenic path and the like caused by bacteria.

However, at present, the targeted therapy method of the probiotics has not been studied in depth in CRC, and the use of the probiotics still has certain limitations. The antibacterial activity of the probiotics is small, the colonization ability of the bacterial strain in the intestinal tract is poor, and the difficulty in developing the probiotics into the medicine capable of exerting stable drug effect is high; it is greatly influenced by factors such as environment, diet and the like, and no probiotic product aiming at the physique of Chinese people exists at present. The development of probiotic drugs is of great interest and also faces drastic challenges.

Chinese patent CN107937504A discloses a screening method of feed probiotics for resisting pig escherichia coli infection, which comprises the following steps: screening of piglets suffering from colibacillus infection and piglets with disease resistance and collecting intestinal tract content samples; high-throughput sequencing of intestinal microbial flora of piglets with colibacillus infection diseases and piglets with disease resistance; carrying out intestinal microbial metagenomics comparative analysis on escherichia coli infected sick piglets and disease-resistant piglets; and screening and identification of probiotics for resisting escherichia coli infection, however, few samples of pathogenic bacteria for resisting colorectal cancer can be collected at present, the method cannot be used for screening the probiotics for resisting the pathogenic bacteria for the colorectal cancer, and meanwhile, the method does not screen the probiotics from the ecological integrity of intestinal flora, so that the screened probiotic formula is possibly poor in colonization capability in the intestinal tract.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a safe and effective probiotic formula capable of better colonizing intestinal tracts and inhibiting pathogenic bacteria of colorectal cancer and a screening method thereof

In order to achieve the purpose, the invention adopts the technical scheme that:

a screening method of a probiotic formula for inhibiting colorectal cancer pathogenic bacteria comprises the following steps:

s1, constructing a human intestinal microbiome online warehouse database (GMrepo, http:// GMrepo. humangut. info/home), recording 58,903 human intestinal samples and 253 items, searching and downloading metagenome data of intestinal flora of normal people and colorectal cancer patients based on the human intestinal microbiome online warehouse database, performing quality control on the metagenome data, namely trimming and removing joints and low-quality sequences in original data, including two-end sequencing and single-end sequencing, and then evaluating the metagenome data after quality control to obtain the metagenome data after quality control;

s2, performing metagenome species annotation analysis on the metagenome data after quality control to obtain abundance information of species of intestinal flora of normal people and colorectal cancer patients;

s3, analyzing the difference of intestinal flora between a normal person and a colorectal cancer patient based on the macro-genome data after quality control and the abundance information, further screening to obtain differential bacteria with difference between the normal person and the colorectal cancer patient, inputting the differential bacteria into the online warehouse database of the human intestinal microbiome, and checking the abundance of the differential bacteria in the intestinal flora of the normal person, wherein the bacteria which do not exist in the intestinal tract of the normal person are identified as colorectal cancer pathogenic bacteria;

s4, respectively selecting bacteria with the abundance ranks 50 above of the species of the intestinal flora of normal people and colorectal cancer patients according to the abundance information in S2, merging the bacteria, obtaining the growth rates of the merged bacteria under the conditions of independent culture and pairwise co-culture in intestinal tracts from documents according to a gene scale metabolic network model, and constructing an intestinal flora interaction network based on the growth rates;

s5, based on the intestinal flora interaction network and the bacteria antagonism principle of S4, screening probiotics for inhibiting the colorectal cancer pathogenic bacteria of S3, combining all the probiotics for inhibiting the pathogenic bacteria, and then further screening the probiotics from the following two aspects: firstly, excluding probiotics which can promote the growth of other pathogenic bacteria; secondly, mutual inhibition between probiotics is avoided as much as possible;

s6, optimizing the probiotics in the S5 by combining the abundance of the probiotics in the intestinal flora of normal people, and adjusting the proportion of the probiotics to finally obtain a probiotic formula capable of inhibiting colon cancer pathogenic bacteria;

s7, carrying out in-vivo experimental animal verification on the probiotic formula in S6: establishing a colorectal cancer mouse model; preparing probiotic suspension of the probiotic formula in S6, and randomly dividing colorectal cancer mice into a low-dose group, a high-dose group and a blank control group, wherein the low-dose group mice and the high-dose group mice are respectively subjected to intragastric administration of low-concentration probiotic suspension and high-concentration probiotic suspension with the same volume, the blank control group mice are subjected to intragastric administration of normal saline with the same volume, the intragastric administration is performed once a day, and the intragastric administration is continuously performed for 28 days. And respectively taking the intestinal contents of the mice with the same quality under the aseptic condition at the 0d, 7d, 14d, 21d and 28d after the mice are subjected to intragastric administration, preparing metagenome sequencing, and analyzing the abundance of pathogenic bacteria and probiotics in the excrement of the colorectal cancer mice before and after the intragastric administration, thereby determining the inhibition effect of the probiotic formula on the pathogenic bacteria of the colorectal cancer.

Preferably, in S1, trimmatic software is used for the quality control, and Fastqc software is used for the evaluation after the quality control, both of which are common software in the art.

Preferably, in S2, MetaPhIAn2 software is used to perform annotation analysis of the metagenomic species, which collates 17000 multiple reference genomes including 13500 bacteria and archaea, 3500 viruses and 110 eukaryotes, and after downloading the corresponding database, the software can be used to achieve accurate taxonomic group assignment and accurate calculation of the relative abundance of the species, which can achieve species-level accuracy and strain-level identification and tracking.

Preferably, in S3, LefSe, which is a software commonly used in the art, is used for the analysis, a random forest algorithm is used for screening the differential bacteria, the random forest algorithm utilizes an integrated learning strategy of a plurality of decision trees to train and predict a sample, bacteria with significant differences in colorectal cancer obtained by LefSe analysis and screening are input, the random forest can easily check the relative importance of model input features, identify cancer patients and normal persons, and finally obtain the differential bacteria with differences in normal persons and colorectal cancer patients.

Preferably, in S4, the growth rate of bacteria cultured in the intestinal tract individually is denoted as S, the growth rate of bacteria cultured in the intestinal tract two by two is denoted as P, the weight W between the bacteria is calculated to be log2(P/S), the merged bacteria are taken as nodes, the weight W between the strains is taken as a side, the intestinal flora interaction network is drawn by using open source software Gephi commonly used in the field, meanwhile, the modelling algorithm function in the Gephi software is used for identifying the subgroup in the intestinal flora metabolic network, and the PageRank algorithm function carried by the Gephi software is used for identifying the important nodes in the intestinal flora metabolic network.

A probiotic formula for inhibiting colorectal cancer pathogenic bacteria, which is obtained by the screening method: wherein the content of Clostridium butyricum (Clostridium butyricum) is 1 × 10⁹～5×10¹⁰CFU/ml, enterococcus faecalis (enterococcus faecium) content of 1 × 10⁸～5×10⁹CFU/ml, Lactobacillus brevis (Lactobacillus brevis) content of 1 × 10⁸～5×10⁹CFU/ml, Lactobacillus plantarum (Lactobacillus) content of 1 × 10⁸～5×10⁹CFU/ml, Lactobacillus rhamnosus (Lactobacillus rhamnosus) content of 1 × 10⁸～5×10⁹CFU/ml, and Lactobacillus sake (Lactobacillus sakei) content of 1 × 10⁸～5×10⁹CFU/ml, Leuconostoc mesenteroides (Leuconostoc cmesenteroides) content 1X 10⁸～5×10⁹CFU/ml。

Preferably, the probiotic formulation has a Clostridium butyricum (Clostridium butyricum) content of 1.3X 10¹⁰CFU/ml, Enterococcus faecalis (Enterococcus faecalis) content of 1 × 10⁹CFU/ml, Lactobacillus brevis (Lactobacillus brevis) content of 1 × 10⁹CFU/ml, Lactobacillus plantarum (Lactobacillus) content of 1 × 10⁹CFU/ml, Lactobacillus rhamnosus (Lactobacillus rhamnosus) content of 1 × 10⁹CFU/ml, and Lactobacillus sake (Lactobacillus sakei) content of 1 × 10⁹CFU/ml, Leuconostoc mesenteroides (Leuconostoc cmesenteroides) content 1X 10⁹CFU/ml。

Compared with the prior art, the invention has the beneficial effects that:

(1) the method is based on a gene scale metabolic network model of the intestinal flora and an inter-bacterium antagonism principle, makes full use of multiple sets of mathematical information such as genomes, transcriptomes, proteomes and metabolomes of the intestinal flora, and constructs the intestinal flora interaction network by taking the intestinal flora as an ecological whole. Compared with the traditional probiotic formula, the probiotic formula obtained by screening can better colonize in the intestinal tract and play an antibacterial role to the maximum extent by considering the ecological system of the intestinal flora and the interaction relationship between bacteria and bacteria.

(2) Compared with the traditional antibiotics, the probiotic formula developed by the application cannot generate serious side effects such as drug resistance and the like. And the probiotics is taken from people, is safe and effective, has excellent performance, and can effectively relieve the serious threat to human health and ecological environment caused by biological abuse resistance.

(3) According to the screening method, an online warehouse database of human intestinal microbiome is constructed, 58,903 human intestinal samples and 253 projects are recorded, based on the database, LEfSe analysis and random forest modeling analysis are combined, so that the identified differential pathogenic bacteria are more reliable, meanwhile, the database is used for analyzing the intestinal flora condition of normal people, the probiotic formula is optimized, and the result is more universal and reliable.

Drawings

FIG. 1 is a graph of pathogenic bacteria of colorectal cancer screened by the random forest algorithm in S3 of example 1;

FIG. 2 is a graph of the interaction network of the intestinal flora of example 1, at the level of the bacterial strain of the 62 combined bacteria in S4 and W > 4;

FIG. 3 is a graph showing the effect of 33 probiotics selected in S5 of example 1 on pathogenic bacteria of colorectal cancer;

FIG. 4 is a graph of the interaction between 33 probiotics screened in S5 of example 1;

FIG. 5 is a graph showing the abundance changes of the probiotics and pathogenic bacteria in the intestinal contents of the colorectal cancer mice after the probiotic formula in example 1 is subjected to intragastric administration, wherein A is the abundance change graph of the probiotics and B is the abundance change graph of the pathogenic bacteria;

FIG. 6 is a graph showing the abundance changes of the probiotics and pathogenic bacteria in the intestinal contents of the colorectal cancer mice after the probiotic formula is used for gastric lavage in example 2, wherein A is the abundance change graph of the probiotics and B is the abundance change graph of the pathogenic bacteria;

FIG. 7 is a graph showing the abundance changes of the probiotics and pathogenic bacteria in the intestinal contents of the colorectal cancer mice after the probiotic formula is used for gastric lavage in example 3, wherein A is the abundance change graph of the probiotics and B is the abundance change graph of the pathogenic bacteria;

FIG. 8 is a graph showing the abundance changes of the probiotics and the pathogenic bacteria in intestinal contents of colorectal cancer mice after the probiotic formula in comparative example 1 is subjected to intragastric administration, wherein A is the graph showing the abundance changes of the probiotics and B is the graph showing the abundance changes of the pathogenic bacteria;

FIG. 9 is a graph showing the abundance changes of the probiotics and the pathogenic bacteria in intestinal contents of colorectal cancer mice after the probiotic formula in comparative example 2 is subjected to intragastric administration, wherein A is the graph showing the abundance changes of the probiotics and B is the graph showing the abundance changes of the pathogenic bacteria;

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.