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CN114717168B - Screening method of functional probiotics based on inhibition of lipopolysaccharide production - Google Patents

Screening method of functional probiotics based on inhibition of lipopolysaccharide production Download PDF

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CN114717168B
CN114717168B CN202210639204.4A CN202210639204A CN114717168B CN 114717168 B CN114717168 B CN 114717168B CN 202210639204 A CN202210639204 A CN 202210639204A CN 114717168 B CN114717168 B CN 114717168B
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probiotics
lipopolysaccharide
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culture solution
screening
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CN114717168A (en
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方曙光
盖忠辉
董瑶
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Luohe Weikang Bio Tech Co ltd
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WeCare Probiotics Co Ltd
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Abstract

The invention relates to a screening method of functional probiotics based on lipopolysaccharide production inhibition, which comprises the following steps of testing the lipopolysaccharide production inhibition capacity of candidate probiotics, and specifically comprises the following steps: co-culturing candidate probiotics and a bacterial strain producing lipopolysaccharide to obtain a culture solution, detecting the concentration of lipopolysaccharide in the culture solution, and selecting corresponding candidate probiotics with low concentration of lipopolysaccharide as screened functional probiotics; the bacterial strain for producing lipopolysaccharide comprises any one or the combination of at least two of Escherichia coli, salmonella enteritidis, enterobacter cloacae, Shigella flexneri, helicobacter pylori, Klebsiella pneumoniae, Acinetobacter baumannii or pseudomonas aeruginosa. The screening method is simple to operate, low in reagent cost, suitable for high-throughput experiments, high in efficiency and success rate, and has important application value.

Description

Screening method of functional probiotics based on inhibition of lipopolysaccharide production
Technical Field
The invention belongs to the technical field of microorganisms, and relates to a method for screening functional probiotics based on lipopolysaccharide production inhibition.
Background
Since probiotic preparations have been widely used in the prevention or treatment of various diseases, particularly diseases related to gastrointestinal function and metabolism, due to their advantages of good safety and no side effects, researchers have been working on the identification of probiotic strains capable of producing reproducible beneficial effects on human health for the prevention or treatment of the corresponding diseases in recent years.
However, the identification of functional probiotics is a complex and time-consuming and laborious process, and in fact, the discovery of functional probiotics is now largely by an empirical, top-down approach, i.e. studying the distribution of microorganisms in individuals in different health states, those microorganisms which are usually enriched in individuals in a health state being considered beneficial, which are then tested and validated. This empirical approach led to the discovery of a range of probiotic candidates over the past century, including some strains of bifidobacteria and lactobacilli, followed by e.coli Nissle 1917, and more recently Akkermansia muciniphila (Akkermansia muciniphila). Although this empirical top-down approach provides a powerful lead for probiotic development, it inherently requires multiple cycles of trial and error to determine the beneficial health of the strain in the absence of prior mechanistic information, which results in sometimes conflicting studies (evidence of their efficacy is sometimes inconsistent) between different documents, complicating the formulation of probiotic usage rules following clinical guidelines.
In summary, the problems of the prior art are as follows: the existing screening method for functional probiotics is complex to operate and low in efficiency, and screening results may have contradictions. Therefore, how to provide a simple and efficient method for screening functional probiotics to improve the success rate of screening functional probiotics becomes a problem to be solved urgently by those skilled in the art.
Disclosure of Invention
In view of the shortcomings of the prior art, the present invention aims to provide a method for screening functional probiotics based on inhibition of lipopolysaccharide production.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a screening method for functional probiotics based on inhibition of lipopolysaccharide production, the screening method comprising testing the ability of candidate probiotics to inhibit lipopolysaccharide production, specifically comprising: co-culturing candidate probiotics and a bacterial strain producing lipopolysaccharide to obtain a culture solution, detecting the concentration of lipopolysaccharide in the culture solution, and selecting corresponding candidate probiotics with low concentration of lipopolysaccharide as screened functional probiotics;
the bacterial strain for producing lipopolysaccharide comprises any one or the combination of at least two of Escherichia coli, salmonella enteritidis, enterobacter cloacae, Shigella flexneri, helicobacter pylori, Klebsiella pneumoniae, Acinetobacter baumannii or pseudomonas aeruginosa.
Combinations of the at least two species are, for example, a combination of Escherichia coli and Salmonella enteritidis, a combination of Enterobacter cloacae and Shigella flexneri, a combination of helicobacter pylori and Klebsiella pneumoniae, and the like, in any other combinations.
Preferably, the temperature of the co-culture is 35-40 ℃ and the time is 12-24 h.
Specific values of the above-mentioned temperature range of 35-40 ℃ are, for example, 35 ℃, 35.5 ℃, 36 ℃, 36.5 ℃, 37 ℃, 37.5 ℃, 38 ℃, 38.5 ℃, 39 ℃, 39.5 ℃ and 40 ℃.
Specific numerical values of the above-mentioned 12 to 24 h are, for example, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h and the like.
Preferably, the culture medium used in the co-culture includes any one or a combination of at least two of tryptone, yeast powder, sodium chloride, beef extract, dipotassium phosphate, glucose, bovine heart extract powder or fetal bovine serum, for example, a combination of tryptone and yeast powder, dipotassium phosphate and glucose, glucose and bovine heart extract powder, and the like, and any other combination may be used.
Preferably, the components in the culture medium comprise 8-12 g/L of tryptone, 3-7 g/L of yeast powder, 8-12 g/L of sodium chloride, 1-3 g/L of beef extract, 1-3 g/L of dipotassium hydrogen phosphate, 1-3 g/L of glucose, 8-12 g/L of bovine heart extract powder and 1-5% (mass percentage content) of fetal bovine serum.
Specific values among the above 8 to 12 g/L are, for example, 8 g/L, 8.5 g/L, 9 g/L, 9.5 g/L, 10 g/L, 10.5 g/L, 11 g/L, 11.5 g/L, 12 g/L and the like.
Specific values among the above 3 to 7 g/L are, for example, 3 g/L, 3.5 g/L, 4 g/L, 4.5 g/L, 5 g/L, 5.5 g/L, 6 g/L, 6.5 g/L, 7 g/L and the like.
Specific values among the above 1 to 3 g/L are, for example, 1 g/L, 1.5 g/L, 2 g/L, 2.5 g/L, 3 g/L, etc.
Specific values of the above-mentioned 1% to 5% are, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, etc.
Preferably, the ratio of the inoculum size of the candidate probiotic bacteria to the lipopolysaccharide producing strain in the co-culture is (1-2): (1-2).
Specific numerical values in the above (1-2) include, for example, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2 and the like.
Preferably, the detection refers to detection using lipopolysaccharide ELISA (enzyme linked immunosorbent assay) detection kit.
Preferably, the detection comprises centrifuging the culture solution, collecting the supernatant, filtering, collecting the supernatant, and detecting the concentration of lipopolysaccharide in the supernatant using a lipopolysaccharide ELISA detection kit.
Preferably, the rotation speed of the centrifugation is 200-600 g, and the centrifugation time is 5-15 min.
The specific values in 200-600 g are, for example, 200 g, 300 g, 400 g, 500 g, 600 g, etc.
The specific value of 5-15 min is 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, etc.
Preferably, the filtration comprises first filtering through a 0.45 μm filter and then filtering the supernatant through a 0.22 μm filter.
Preferably, the screening method further comprises subjecting the candidate probiotic to a gastrointestinal fluid resistance test and/or a bile salt resistance test.
Preferably, the gastrointestinal fluid resistance test comprises: mixing the candidate probiotics with the artificial simulated gastric juice, incubating for 2-4 h at 36-38 ℃ to obtain first mixed liquid, mixing the first mixed liquid with the artificial simulated intestinal juice, incubating for 6-10 h at 36-38 ℃ to obtain second mixed liquid, detecting the number of viable bacteria in the second mixed liquid, calculating the survival rate of the candidate probiotics, wherein the higher the survival rate is, the stronger the gastrointestinal fluid resistance of the candidate probiotics is.
Specific values in the above 36-38 ℃ range include, for example, 36 ℃, 36.1 ℃, 36.2 ℃, 36.3 ℃, 36.4 ℃, 36.5 ℃, 36.6 ℃, 36.7 ℃, 36.8 ℃, 36.9 ℃, 37 ℃, 37.1 ℃, 37.2 ℃, 37.3 ℃, 37.4 ℃, 37.5 ℃, 37.6 ℃, 37.7 ℃, 37.8 ℃, 37.9 ℃ and 38 ℃.
Specific values in the above 2 to 4 hours are, for example, 2 hours, 2.2 hours, 2.4 hours, 2.6 hours, 2.8 hours, 3 hours, 3.2 hours, 3.4 hours, 3.6 hours, 3.8 hours, 4 hours and the like.
Specific values in the above 6 to 10 h are, for example, 6 h, 6.5 h, 7 h, 7.5 h, 8 h, 8.5 h, 9 h, 9.5 h, 10 h and the like.
Preferably, the pH of the simulated gastric fluid is 1.5-3.5, such as 1.5, 1.7, 2, 2.2, 2.5, 2.7, 3, 3.2, 3.5, and the like.
Preferably, the simulated gastric fluid contains 2-3 g/L pepsin, such as 2 g/L, 2.1 g/L, 2.2 g/L, 2.3 g/L, 2.4 g/L, 2.5 g/L, 2.6 g/L, 2.7 g/L, 2.8 g/L, 2.9 g/L, 3 g/L, etc.
Preferably, the artificial simulated intestinal fluid contains 0.6-1 g/L trypsin, such as 0.6 g/L, 0.65 g/L, 0.7 g/L, 0.75 g/L, 0.8 g/L, 0.85 g/L, 0.9 g/L, 0.95 g/L, 1 g/L, etc.
Preferably, the pH of the artificial simulated intestinal fluid is 6.5-8.5, such as 6.5, 6.7, 7, 7.2, 7.5, 7.7, 8, 8.2, 8.5, and the like.
Preferably, the bile salt resistance test comprises: inoculating the candidate probiotics into a culture medium containing 0.2-0.4% of bile salt by mass percent, incubating for 3-5 h at 36-38 ℃ to obtain a culture solution, and detecting the absorbance of the culture solution at 600 nm, wherein the higher the absorbance is, the stronger the bile salt resistance of the candidate probiotics is.
Specific values of the above-mentioned 0.2% to 0.4% include, for example, 0.2%, 0.22%, 0.24%, 0.26%, 0.28%, 0.3%, 0.32%, 0.34%, 0.36%, 0.38%, 0.4%, etc.
Specific values in the above 36-38 ℃ range include, for example, 36 ℃, 36.1 ℃, 36.2 ℃, 36.3 ℃, 36.4 ℃, 36.5 ℃, 36.6 ℃, 36.7 ℃, 36.8 ℃, 36.9 ℃, 37 ℃, 37.1 ℃, 37.2 ℃, 37.3 ℃, 37.4 ℃, 37.5 ℃, 37.6 ℃, 37.7 ℃, 37.8 ℃, 37.9 ℃ and 38 ℃.
Specific values in the above-mentioned 3-5 h are, for example, 3 h, 3.2 h, 3.5 h, 3.7 h, 4 h, 4.2 h, 4.5 h, 4.7 h, 5 h, etc.
Preferably, the culture medium in the bile salt resistance test comprises an MRS culture medium containing 0.2-0.4% by mass of bile salt.
Preferably, the screening method comprises the following steps:
(1) mixing candidate probiotics with artificial simulated gastric juice, incubating for 2-4 h at 36-38 ℃ to obtain a first mixed solution, mixing the first mixed solution with artificial simulated intestinal juice, incubating for 6-10 h at 36-38 ℃ to obtain a second mixed solution, detecting the number of viable bacteria in the second mixed solution, calculating the survival rate of the candidate probiotics, and selecting the candidate probiotics with high survival rate for next testing;
(2) inoculating the candidate probiotics obtained by screening in the step (1) into a culture medium containing bile salt with the mass percentage content of 0.2-0.4%, incubating for 3-5 h at 36-38 ℃ to obtain a culture solution, detecting the absorbance of the culture solution at 600 nm, and selecting the candidate probiotics with high absorbance for next test;
(3) co-culturing the candidate probiotics obtained by screening in the step (2) and the bacterial strain producing lipopolysaccharide at 35-40 ℃ for 16-20 h to obtain a culture solution, centrifuging the culture solution, collecting the supernatant, filtering, collecting the supernatant, detecting the lipopolysaccharide concentration in the supernatant by using a lipopolysaccharide ELISA detection kit, and selecting the corresponding candidate probiotics with low lipopolysaccharide concentration as the functional probiotics obtained by screening.
Preferably, the functional probiotic has anti-helicobacter pylori efficacy.
Preferably, the functional probiotic has the efficacy of ameliorating or treating chronic non-infectious diseases including gastritis, colitis or obesity.
Preferably, the colitis comprises ulcerative colitis.
The recitation of numerical ranges herein includes not only the above-recited values, but also any values between any of the above-recited numerical ranges not recited, and for brevity and clarity, is not intended to be exhaustive of the specific values encompassed within the range.
Compared with the prior art, the invention has the following beneficial effects:
the screening method is simple to operate, low in reagent cost, suitable for high-throughput experiments, high in efficiency and high in success rate, and the screened probiotics with strong lipopolysaccharide production inhibition capacity have excellent performance in resisting helicobacter pylori infection and treating chronic non-infectious diseases such as gastritis, colitis, obesity and the like. Therefore, the screening method can be used for screening strains resisting helicobacter pylori infection and improving or treating chronic non-infectious diseases such as gastritis, colitis, obesity and the like, and in practical application, a person skilled in the art only needs to screen out candidate strains by using the method and then carries out animal experiments or clinical test verification, so that the cost is greatly saved, the screening efficiency is improved, and the method has important application value.
Drawings
FIG. 1 is a graph showing the change in body weight of each group of mice in example 6.
FIG. 2 is a graph of statistical Disease Activity Index (DAI) scores for groups of mice in example 6.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
In the following examples, unless otherwise specified, reagents and consumables were purchased from conventional reagent manufacturers in the field; unless otherwise indicated, all experimental methods and technical means used are those conventional in the art.
The material and the method are as follows:
the probiotics used in the examples below, such as lactobacilli or bifidobacteria, were obtained from the strain stock of microbiota probiotic (suzhou) gmbh;
fetal bovine serum, PBS and trypsin were purchased from Thermo;
columbia medium was purchased from OXOID, UK;
general medium: 10 g/L tryptone, 5 g yeast powder, 10 g sodium chloride, 2 g beef extract and K 2 HPO 4 ·3H 2 1 g of O, 2 g of glucose, 10 g of bovine heart extract powder and 30 mL of fetal bovine serum, adding water to a constant volume of 1L, sterilizing and cooling for later use;
MRS solid medium: weighing 10 g of peptone, 10 g of beef extract, 20 g of glucose, 2 g of sodium acetate, 5 g of yeast powder, 2 g of diammonium hydrogen citrate and K 2 HPO 4 ·3H 2 O 2.6 g、MgSO 4 ·7H 2 O 0.1 g、MnSO 4 Dissolving 0.05 g of agar, 20 g of agar and 0.5 g of cysteine hydrochloride by using deionized water, adding 1mL of Tween 80, diluting to a constant volume of 1L, sterilizing, cooling, and pouring into a sterilized culture dish for later use;
MRS liquid medium: weighing 10 g of peptone, 10 g of beef extract, 20 g of glucose, 2 g of sodium acetate, 5 g of yeast powder, 2 g of diammonium hydrogen citrate and K 2 PO 4 ·3H 2 O 2.6 g、MgSO 4 ·7H 2 O 0.1 g、MnSO 4 0.05 g and 0.5 g of cysteine hydrochloride are dissolved by deionized water, 1mL of Tween 80 is added, the volume is adjusted to 1L, and the mixture is sterilized and cooled for standby.
Example 1: activation and culture of candidate probiotics
The probiotic candidate strain used in the present example is from the strain resource library of micro-health probiotic (suzhou) gmbh, and 100 strains (numbered P1-P100) of lactobacillus or bifidobacterium were randomly selected in this experiment, and the activation and culture steps were as follows:
(1) and respectively inoculating candidate probiotics frozen in glycerin pipes at the temperature of-80 ℃ into a liquid MRS culture medium, culturing at the constant temperature of 37 ℃ for 18 h for activation, and using the activated probiotics for subsequent tests.
(2) Preparing a candidate probiotic bacteria liquid: centrifuging the candidate probiotic culture solution obtained in step (1) (8000 rpm, 10 min), collecting bacterial sludge, washing with PBS for 3 times to obtain bacterial suspension (concentration adjusted to 1 × 10) 9 CFU/mL）。
Example 2: screening of gastrointestinal fluid resistant probiotics
The screening steps are as follows:
(1) preparation of simulated gastric fluid: pepsin was dissolved in PBS at pH 3.0 to a final concentration of 2.5 g/L and filtered through a 0.22 μm filter to prepare a simulated gastric fluid.
(2) Preparation of simulated intestinal fluid: trypsin was dissolved in PBS at pH 8.0 to a final concentration of 0.8 g/L, and filtered through a 0.22 μm filter to prepare a simulated intestinal fluid.
(3) Determination of the survival rate of candidate probiotics in simulated gastric fluid-simulated intestinal fluid: resuspending candidate probiotic bacteria with 0.85% physiological saline, mixing with simulated gastric juice (pH 3.0), and adjusting the density of the bacteria liquid to 1 × 10 8 CFU/mL, mixing uniformly, and incubating at 37 ℃ for 3 h; ② adding 1mL of the culture solution obtained by incubation into 9 mL of simulated intestinal fluid (pH 8.0), uniformly mixing, culturing at 37 ℃ for 8 h, and detecting the viable count. The survival rate of the candidate probiotics is calculated by the following formula: survival = lg N1/lg N0; n = viable count of candidate probiotic after simulated gastric fluid-simulated intestinal fluid treatment, N0= viable count of candidate probiotic before untreated. The results are shown in Table 1.
TABLE 1 screening results for gastrointestinal fluid resistant probiotic
Figure 640849DEST_PATH_IMAGE001
TABLE 1 continuation
Figure 777432DEST_PATH_IMAGE002
As shown in Table 1, the survival rate of each candidate probiotic in simulated gastric fluid-simulated intestinal fluid is different, and candidate strains (46 strains) with the survival rate of more than 0.5 are selected for further screening.
Example 3: screening of bile salt resistant probiotics
Candidate strains (46 strains) with the survival rate of more than 0.5 in example 2 were selected for the bile salt resistance test.
Growth assay of candidate probiotics in bile salts: 1mL of the solution was added at a concentration of 1X 10 9 The bacterial suspension of the CFU/mL candidate probiotics is inoculated in an MRS-THIO (MRS contains 0.2% sodium thioglycollate) culture medium containing 0.3% of bile salt, the culture solution is obtained by culturing at 37 ℃ for 4 h, the absorbance value is measured at 600 nm, and the larger the OD600 value is, the stronger the bile salt tolerance of the strain is. The results are shown in Table 2.
TABLE 2 screening results for bile salt resistant probiotic
Figure 118415DEST_PATH_IMAGE003
As shown in Table 2, the tolerance of each candidate probiotic to bile salt is different, and the candidate strain (23 strains) with OD600 of more than 0.30 is selected for further screening.
Example 4: construction of lipopolysaccharide-producing flora
Construction of lipopolysaccharide-producing flora Escherichia coli ATCC25922, Salmonella enteritidis ATCC14028, Enterobacter cloacae ATCC13047, Shigella flexneri CMCC51572, helicobacter pylori ATCC43504, Klebsiella pneumoniae ATCC700603, Acinetobacter baumannii ATCC19606 and Pseudomonas aeruginosa ATCC27853 were all purchased from North Nor Biotech, Shanghai, Inc.
The lipopolysaccharide producing bacteria activation and culture steps are as follows:
(1) inoculating the lipopolysaccharide producing bacteria frozen in a glycerol tube at-80 deg.C into a general culture medium, and culturing at constant temperature of 37 deg.C for 18 h for activation;
(2) preparing a lipopolysaccharide producing bacteria liquid: centrifuging the culture solution of lipopolysaccharide producing bacteria obtained in step (1) (3000 rpm, 10 min), washing with PBS for 3 times to obtain bacterial suspension, and adjusting concentration to 1 × 10 8 CFU/mL；
(3) And (3) uniformly mixing 1mL of each bacterial suspension of the 8 lipopolysaccharide-producing bacteria to obtain a lipopolysaccharide-producing bacteria group mixed solution.
Example 5: screening of functional probiotics for inhibiting lipopolysaccharide production
Candidate strains (23 strains) having strong bile salt tolerance (OD 600 > 0.3) in example 3 were selected and tested for inhibition of lipopolysaccharide production.
1mL of a mixture of lipopolysaccharide-producing bacterial colonies (1X 10) 8 CFU) and 1mL of bacterial suspension of the candidate strain (1X 10) 8 CFU) was added to 8 mL of the general medium and incubated at 37 ℃ for 18 h. And setting a comparison group: 1mL of a mixture of lipopolysaccharide-producing bacterial colonies (1X 10) 8 CFU) and 1mL of PBS solution were added to 8 mL of the universal medium and incubated at 37 ℃ for 18 h. After 18 h, the culture broth of each group was centrifuged at 400 Xg for 10 min, and the supernatant was filtered through a 0.45 μm filter and then refiltered through a 0.22 μm filter and collected. The Lipopolysaccharide (LPS) concentration in the supernatant was then determined in. mu.g/mL using a lipopolysaccharide ELISA detection kit (purchased from Shanghai enzyme-linked biosciences, Inc.). The results are shown in Table 3.
TABLE 3 screening results for lipopolysaccharide production inhibiting probiotics
Figure 377358DEST_PATH_IMAGE004
As shown in table 3, different probiotic candidates differed in their ability to inhibit lipopolysaccharide production, with P29 and P89 being the strongest, followed by P2 and P83. These probiotics are therefore potential strains of functional probiotics.
Example 6: functional verification for improving ulcerative colitis
In this example, functional strains P29 and P89 with the strongest ability to inhibit the production of lipopolysaccharide and P6 and P36 with little inhibition were selected as comparison and tested to study the effects of these four strains on the body weight and Disease Activity Index (DAI) of mice with ulcerative colitis. Query for micro-health probiotics (Su)Oncorhyncho GmbH Strain Bank archive, P29 is Lactobacillus rhamnosus (C.)Lacticaseibacillus rhamnosus) P89 is Lactobacillus reuteri: (A)Limosilactobacillus reuteri) P6 is Lactobacillus plantarum (B)Lactiplantibacillus plantarum) P36 is Bifidobacterium adolescentis (Bifidobacterium adolescentis）。
The experimental procedure was as follows:
(1) preparation of P29, P89, P6 and P36 lyophilized powders:
inoculating P29 into MRS culture medium according to the inoculation amount accounting for 3% of the total mass of the culture medium, and culturing at 37 ℃ for 18 h to obtain a culture solution; centrifuging the culture solution to obtain thalli; resuspending the thallus with a freeze-drying protective agent (containing trehalose 100 g/L, skimmed milk powder 100 g/L, and water as solvent) (the mass ratio of the freeze-drying protective agent to the thallus is 2: 1) to obtain a resuspension solution; and (3) freeze-drying the heavy suspension by adopting a vacuum freezing method to obtain P29 freeze-dried powder, and detecting that the viable count is 2000 hundred million CFU/g. The lyophilized powders P89, P6 and P36 were obtained by the same method, and the viable count was 3000 hundred million CFU/g, 2500 hundred million CFU/g and 3000 hundred million CFU/g, respectively.
(2) Animals were grouped and the Ulcerative Colitis (UC) mouse model was established:
c57BL/6 mice were randomly divided into 6 groups of 10 mice each, normal control group (CTL group), UC model group (UC group), P29 group of caseobacter rhamnosus (P29 group), P89 group of lactobacillus reuteri (P89 group), P6 group of lactobacillus plantarum (P6 group) and P36 group of bifidobacterium adolescentis (P36 group), respectively.
Experimental colitis was induced by oral administration of 3% dextran sodium sulfate DSS (w/v) in drinking water for 7 days. The CTL group was given normal drinking water daily for one week, and the other group was given 3% dextran sodium sulfate (w/v) 1 time daily for one week. Meanwhile, mice in groups P29, P89, P6 and P36 were orally administered the corresponding probiotic lyophilized powder solution prepared in step (1) (dissolved in physiological saline, each mouse was gavaged with 1X 10 per day) 9 CFU), whereas the CTL group and UC group were given physiological saline (0.1 mL/10 g).
(3) All mice were recorded daily for weight, stool consistency, rectal bleeding, food intake, and observed for clinical signs such as mental, activity, food intake, diarrhea, and stool traits. Disease Activity Index (DAI) score is an essential clinical performance evaluation index of UC and an important index for evaluating colon damage, and the DAI score criteria are shown in table 4:
TABLE 4 disease Activity index Scoring Table
Figure 803791DEST_PATH_IMAGE005
The results were: normal control group (CTL group) mice were active, fed normally, and had no diarrhea and hematochezia; the UC model group (UC group) mice have less activity, and mucus, pus and bloody stool can be seen by naked eyes; the P29 and P89 mice had significantly less diarrhea and hematochezia compared to the UC group.
According to the change of the body mass of each group of mice, a body mass change curve is drawn, as shown in fig. 1, as can be seen from fig. 1, the body mass of the mice in the CTL group is gradually increased, the body mass of the mice in the UC group is obviously reduced, the body mass loss of the mice in the P29 group and the P89 group is obviously improved compared with the body mass loss of the mice in the UC group, and the improvement effect of the mice in the P6 group and the P36 group is poorer.
DAI score results As shown in FIG. 2, the mouse DAI scores of the P29 and P89 groups were significantly reduced (P < 0.001) compared to the UC group, while the mouse DAI scores of the P6 and P36 groups were still higher.
The results fully prove that the functional probiotics P29 and P89 obtained by screening have the effect of remarkably improving or treating ulcerative colitis, and meanwhile, the screening method of the probiotics based on the inhibition of the generation of lipopolysaccharide is very efficient and has high success rate.
Example 7: functional verification of anti-helicobacter pylori
This example explores the growth inhibitory effect of P29, P89, P6 and P36 on helicobacter pylori:
(1) taking out helicobacter pylori ATCCA43504 from-80 deg.C, spreading on Columbia blood agar medium containing 5% sheep blood (v/v), activating, culturing at 37 deg.C for 48 hr in microaerophilic environment;
(2) respectively inoculating P29, P89, P6 and P36 in MRS liquid culture medium, culturing at 37 deg.C for 18 h for activation, and continuously activating for 2 times to obtain activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at 37 ℃ for 18 h to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000 g for 10 min, and resuspending the thallus by PBS to obtain probiotic bacterial suspension (the concentration of each probiotic is consistent);
(3) bacteriostatic zone experiment:
100 μ L of helicobacter pylori ATCCA43504 (density 10) 9 CFU/mL) was applied to the surface of a 5% sheep blood-containing columbia blood agar medium (medium volume 20 mL) containing no antibiotic and perforated, and either of the following liquids was added, respectively, to form different experimental groups: 100 mu L of probiotic bacteria suspension to be detected; positive control (metronidazole solution with mass concentration of 0.025%) 100 μ L; negative control (PBS buffer) 100. mu.L; blank control (MRS liquid medium) 100. mu.L. The effect of inhibiting the growth of H.pylori at 48 h and 72 h was determined by the Oxford cup method and the results are shown in Table 5.
TABLE 5 results of zone of inhibition
Figure 71961DEST_PATH_IMAGE006
As can be seen from Table 5, the MRS liquid culture medium has no inhibition effect on helicobacter pylori, and different probiotic bacterial suspensions have inhibition effects on the growth of the helicobacter pylori, wherein the inhibition effect of the P29 bacterial suspension is most obvious, the diameter of an inhibition zone can reach more than 13.2 +/-1.7 mm when the culture is carried out for 48 hours, and the diameter of the inhibition zone can reach more than 18.2 +/-2.5 mm when the culture is carried out for 72 hours; and the two bacterial suspensions are P89 bacterial suspensions, and the bacteriostatic effect of the two bacterial suspensions is obviously better than that of the bacterial suspensions P6 and P3, even better than that of a positive control.
The results fully prove that the functional probiotics P29 and P89 obtained by screening have obvious anti-helicobacter pylori effect, and meanwhile, the screening method of the probiotics based on the inhibition of the generation of lipopolysaccharide is very efficient and has high success rate.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above examples to be carried out. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

Claims (8)

1. Use of a screening method based on functional probiotics that inhibit the production of lipopolysaccharides for screening strains that are resistant to helicobacter pylori infection, wherein the screening method comprises testing candidate probiotics for their ability to inhibit the production of lipopolysaccharides, and specifically comprises: co-culturing candidate probiotics and a bacterial strain producing lipopolysaccharide to obtain a culture solution, detecting the concentration of lipopolysaccharide in the culture solution, and selecting corresponding candidate probiotics with low concentration of lipopolysaccharide as screened functional probiotics;
the strain for producing lipopolysaccharide is helicobacter pylori.
2. The use according to claim 1, wherein the co-cultivation is carried out at a temperature of 35-40 ℃ for a period of 12-24 h.
3. The use according to claim 1, wherein the medium used in the co-cultivation comprises any one or a combination of at least two of tryptone, yeast powder, sodium chloride, beef extract, dipotassium hydrogen phosphate, glucose, bovine heart extract powder or fetal bovine serum.
4. The use of claim 1, wherein the detection is performed using a lipopolysaccharide ELISA test kit.
5. The use of claim 1, wherein the screening method further comprises subjecting the candidate probiotic to a gastrointestinal fluid resistance test and/or a bile salt resistance test.
6. The use of claim 5, wherein the gastrointestinal fluid resistance test comprises: mixing the candidate probiotics with the artificial simulated gastric juice, incubating for 2-4 h at 36-38 ℃ to obtain first mixed liquid, mixing the first mixed liquid with the artificial simulated intestinal juice, incubating for 6-10 h at 36-38 ℃ to obtain second mixed liquid, detecting the number of viable bacteria in the second mixed liquid, calculating the survival rate of the candidate probiotics, wherein the higher the survival rate is, the stronger the gastrointestinal fluid resistance of the candidate probiotics is.
7. The use of claim 5, wherein said bile salt resistance test comprises: inoculating the candidate probiotics into a culture medium containing 0.2-0.4% of bile salt by mass percent, incubating for 3-5 h at 36-38 ℃ to obtain a culture solution, and detecting the absorbance of the culture solution at 600 nm, wherein the higher the absorbance is, the stronger the bile salt resistance of the candidate probiotics is.
8. Use according to claim 1, wherein the screening method comprises the steps of:
(1) mixing candidate probiotics with artificial simulated gastric juice, incubating for 2-4 h at 36-38 ℃ to obtain a first mixed solution, mixing the first mixed solution with artificial simulated intestinal juice, incubating for 6-10 h at 36-38 ℃ to obtain a second mixed solution, detecting the number of viable bacteria in the second mixed solution, calculating the survival rate of the candidate probiotics, and selecting the candidate probiotics with high survival rate for next testing;
(2) inoculating the candidate probiotics obtained by screening in the step (1) into a culture medium containing bile salt with the mass percentage content of 0.2-0.4%, incubating for 3-5 h at 36-38 ℃ to obtain a culture solution, detecting the absorbance of the culture solution at 600 nm, and selecting the candidate probiotics with high absorbance for next test;
(3) co-culturing the candidate probiotics obtained by screening in the step (2) and the bacterial strain producing lipopolysaccharide at 35-40 ℃ for 16-20 h to obtain a culture solution, centrifuging the culture solution, collecting the supernatant, filtering, collecting the supernatant, detecting the lipopolysaccharide concentration in the supernatant by using a lipopolysaccharide ELISA detection kit, and selecting the corresponding candidate probiotics with low lipopolysaccharide concentration as the functional probiotics obtained by screening.
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