CN107034159B

CN107034159B - Pathogenic bacteria antagonist and application thereof

Info

Publication number: CN107034159B
Application number: CN201710289720.8A
Authority: CN
Inventors: 郭聃洋; 王金达; 李永平; 张宇; 孙嘉祺
Original assignee: Changchun China-Russia Science-Tech Park Pig
Current assignee: Changchun China-Russia Science-Tech Park Pig
Priority date: 2017-04-27
Filing date: 2017-04-27
Publication date: 2021-06-08
Anticipated expiration: 2037-04-27
Also published as: CN107034159A

Abstract

The invention discloses a pathogenic bacteria antagonist, which comprises Enterococcus durans CR-29(Enterococcus durans CR-29) with the CGMCC number of 11870 and Leuconostoc mesenteroides SR-19(Leuconostoc mesenteroides subsp. mesenteroides SR-19) with the CGMCC number of 11869. Also discloses the application of the pathogenic bacteria antagonist in preparing the medicament for preventing and/or treating diarrhea.

Description

Pathogenic bacteria antagonist and application thereof

Technical Field

The invention belongs to the field of probiotics, and particularly relates to a pathogenic bacteria antagonist and application thereof.

Background

Salmonella, shigella, staphylococcus aureus, clostridium difficile and listeria monocytogenes are common pathogenic bacteria in intestinal tracts of human bodies and animals, have strong infectivity, can cause intestinal diseases such as diarrhea of human bodies and the like by a small amount of the bacteria, and are difficult to treat after being infected by the bacteria, particularly clostridium difficile. Some strains of E.coli may also cause intestinal disease, and its stable amount is also important for intestinal balance. Part of probiotics have certain inhibiting effect on pathogenic bacteria in intestinal tracts, and the inhibiting mechanism is relatively complex. The in vitro study on the inhibition capability of single thallus on clostridium difficile, staphylococcus aureus and escherichia coli provides reference for the inhibition of the single thallus on intestinal tract conditioned bacteria in vivo.

Disclosure of Invention

Through continuous experiments, the inventor of the invention separates a pair of natural reciprocal symbiotic lactic acid bacteria from a traditional dairy product, wherein the symbiont probiotics SR-19 and CR-29 are gram-positive facultative anaerobes, which have unexpected effects on pathogenic bacteria antagonism and can be used as the pathogenic bacteria antagonist of the invention.

In a first aspect, the invention discloses a pathogenic bacteria antagonist, which comprises Enterococcus durans CR-29(Enterococcus durans CR-29) with CGMCC No. 11870 and Leuconostoc mesenteroides Enterococcus SR-19(Leuconostoc mesenteroides SR-19) with CGMCC No. 11869.

Preferably, in the pathogenic bacteria antagonist, the cfu/g ratio of the enterococcus durans CR-29 to the Leuconostoc mesenteroides SR-19 is 1:10-10: 1.

Preferably, the pathogenic bacteria are Salmonella enterica (Salmonella enterica), Shigella flexneri (Shigella flexneri), Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Clostridium difficile (Clostridium difficile), Escherichia coli (Escherichia coli).

In a second aspect of the invention, a method of culturing an antagonist of a pathogenic bacteria is disclosed,

adopting a liquid culture medium, wherein the inoculum sizes of the leuconostoc mesenteroides mesenterium subspecies SR-19 and the enterococcus durans CR-29 are respectively 5 percent and 7 percent, and the initial pH value is 6.8;

the initial incubation temperature was 44 deg.C (1.0-1.5 hours); the middle culture temperature is 42 deg.C (2.0-2.5 hr), and the middle fermentation stage has pH of 5.9; the final culture temperature is 38 ℃ (1.5-2.0 hours);

culturing under the condition of oscillation frequency of 140r/min, adding supplementary nutrient substances after culturing for 4h, and continuing culturing until the viable count is not less than 1010 cfu/mL;

the cultured viable bacteria can be obtained by low-temperature centrifugal separation;

the formula of the liquid culture medium, namely an industrial culture medium, is as follows: 100g/L of skimmed milk powder, 10g/L of yeast powder, 50g/L of sucrose, 2g/L of sodium citrate, 0.5g/L of sodium acetate and 6.8-7.2 of pH; the formula of the supplementary nutrient substances is as follows: and mixing the beef extract and the carrot juice by 2: 1.

The third aspect of the invention discloses a bacterium powder of a pathogenic bacterium antagonist, which is prepared by the following method:

filtering or centrifuging the probiotic symbiont fermentation liquor with the viable count of not less than 1010cfu/mL, and performing vacuum freeze drying by using a freeze-drying protective agent, wherein the freeze-drying curve is as follows: pre-freezing at-42 deg.c for 4 hr, and heating to-42-5 deg.c and 5 deg.c per hr; heating to 5-11 deg.c at 2 deg.c per hour and heating to 10-40 deg.c at 5 deg.c per hour; raising the temperature from 40 ℃ to 50 ℃ per hour to 2 ℃, and completing freeze-drying preparation within 30 hours to prepare the pathogenic bacteria antagonist probiotic powder; all the operations are carried out under aseptic conditions.

The fourth aspect of the invention discloses a pathogenic bacteria antagonist, which comprises the pathogenic bacteria antagonist of the first, second and third aspects and pharmaceutically acceptable auxiliary materials.

A fifth aspect of the invention discloses the use of a pathogenic bacteria antagonist in the preparation of a pathogenic bacteria antagonist.

The sixth aspect of the invention discloses the application of the pathogenic bacteria antagonist in preparing the pathogenic bacteria antagonist with gastric acid resistance, intestinal juice resistance and bile resistance.

The seventh aspect of the invention discloses the application of the pathogenic bacteria antagonist in preparing a medicament for preventing and/or treating diarrhea.

The eighth aspect of the invention discloses application of the pathogenic bacteria antagonist in preparing medicaments for preventing and/or treating diarrhea, wherein the medicaments are resistant to gastric acid, intestinal juice and bile.

In some embodiments, the symbiont probiotic is produced by: inoculating, rejuvenating, fermenting and culturing, separating, freeze-drying and the like. In particular, the method comprises the following steps of,

the inoculum sizes of the symbiont probiotic leuconostoc mesenteroides subspecies SR-19 and enterococcus durans CR-29 are 5% and 7%, and the culture medium is: milk (goat milk or horse milk), skimmed milk powder, lactalbumin, vitamin K, and mineral elements as raw materials, and initial culture temperature is 44 deg.C (1.0-1.5 hr); the middle culture temperature is 42 ℃ (2.0-2.5 hours); the final culture temperature was 38 deg.C (1.5-2.0 hr). The initial pH value is 6.8, the middle stage of fermentation is pH5.9, the culture is carried out under the condition of oscillation frequency of 140r/min, nutrient substances (mixed solution of beef extract and carrot juice 2: 1) are supplemented at the feed supplement speed of 6.3m/h after the culture is carried out for 4h, and the culture is continued until the harvest, so that the viable count of the probiotics is not less than 1010 cfu/mL. The cultured viable bacteria can be obtained by low temperature centrifugation (6000r/min), and vacuum freeze drying is carried out by using sodium alginate, xylitol and colostrum protein (2:1:1) as freeze-drying protective agent, wherein the freeze-drying curve is as follows: pre-freezing at-42 deg.c for 4 hr, and heating to-42-5 deg.c and 5 deg.c per hr; heating to 5-11 deg.c at 2 deg.c per hour and heating to 10-40 deg.c at 5 deg.c per hour; raising the temperature from 40 ℃ to 50 ℃ per hour by 2 ℃, and completing the freeze-drying preparation within 30 hours to prepare the symbiont probiotic powder.

The formula of the culture medium and the freeze-drying protective agent is as follows:

the detailed formula of the culture medium of the probiotic symbiont comprises components and contents;

laboratory media: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 0.5g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar, and adding distilled water to 1000mL of the mixture until the pH value is 6.4.

The industrial culture medium comprises 100g/L of skimmed milk powder, 10g/L of yeast powder, 50g/L of sucrose, 2g/L of sodium citrate, 0.5g/L of sodium acetate and pH of 6.8-7.2. And (3) supplement of nutrient substances: supplementing the mixed solution of Beef Extract and carrot raw juice 2:1 by 0.8% of the total fermentation amount;

the culture medium is sterilized by moist heat at 121 ℃ for standby.

Freeze-drying protective agent: 50% of sodium alginate, 25% of xylitol and 25% of colostrum protein (2:1:1) as a freeze-drying protective agent are mixed with dry matters of the fermentation liquor according to the proportion of 1: 10.

The invention relates to main instruments and equipment:

4 ℃ refrigerator SC-276, Haier group; refrigerator DW-FW 3-51, Mike Mimex Low temperature science and technology, Inc. at-86 deg.C; electronic balance JA2003, shanghai precision scientific instruments ltd; digital display acidometer PHS-2_5, Shanghai apparatus, electroscience instruments, Inc.; a sterile superclean workbench SW-CJ-1F, Sujing AIRTECH; autoclave LDZX-30KB, Shanghai Sanshen medical instruments, Inc.; a constant temperature incubator JB202, shanghai brocade screen instruments ltd; sterile filtration apparatus 250mL, Nalgene USA; digital display constant temperature water bath HH-2, Fuhua instruments Limited of gold Tan city; HITACHI U-2000 ultraviolet-visible spectrophotometer; EppendorfTGL-168 high speed bench centrifuge; model olympussbx 50 optical microscope; an OLYMPUS BX50 type imaging system; a type 722 spectrophotometer; anaerobic jars (BBL Gas park); OLYMPUS PM-20 type camera system; 250m1 polypropylene centrifuge tube, (Gilson), and the bacteria coating plate are made in China; pasteur pipette, microsyringe 8-channel discharge gun, Eppendorf Co.

The main experimental materials: enterococcus durans CR-29 and Leuconostoc mesenteroides SR-19 are preserved in China general microbiological culture Collection center (CGMCC), and the numbers are CGMCC No. 11870 and CGMCC No. 11869 respectively.

Lactobacillus bulgaricus, Lactobacillus acidophilus are from Russian bioengineering and technology union research and development center in Russian science and technology park of Changchun; bifidobacterium adolescentis was from university of Moscow Russia; enterococcus faecalis F-3 was provided by the Russian St.Peterburg national university of Foundation medicine and medical technology. Salmonella enterica (Salmonella enterica), Shigella flexneri (Shigella flexneri), Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Clostridium difficile (Clostridium difficile), Escherichia coli (Escherichia coli) are available from the institutes of public health of the university of Guillain.

The pathogen antagonists and the probiotics in the invention have the same meaning.

Description of the drawings:

FIG. 1 is an electron microscope photograph of SR-19 and CR-29 probiotic symbiont fermentation supernatant co-cultured with Shigella flexneri (5ml,37 ℃);

1) for comparison, 2) is the phenomenon that pathogenic bacteria cells swell and perforate cell membranes after the supernatant is acted for 30min, and 3) the condition that the cells are cracked after 60 mi.

FIG. 2 shows that the fermentation supernatant of the probiotic symbiont SR-19 and CR-29 is cultured together with Staphylococcus aureus (5ml,37 ℃, 4):60 min; 5) 90 min; 6) 30min) electron microscope picture;

1) and 2), 3) is a control group; 4) 5), 6) are experimental groups. 4) And 5) the condition of the disruption of the staphylococcus aureus thalli under the transmission electron microscope; 6) scanning electron microscope image, showing the surface of tested thallus has rugged phenomenon, suspected by probiotic bacteriocin effect cracking result.

FIG. 3 shows the variation of the number of live bacteria preserved for different probiotics at 4 ℃;

FIG. 4 shows the variation of the number of preserved viable bacteria of different probiotics at 20 ℃;

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 in vitro inhibition assay of representative enteropathogenic bacteria by probiotic symbionts

The in-vitro inhibition degree of the single probiotic thallus and the probiotic symbiont to the representative pathogenic bacteria is measured by measuring the diameter of the inhibition zone. In vitro experiments: the inhibition effect of single probiotic thallus and probiotic symbiotic bacteria on Salmonella enterica (Salmonella enterica), Shigella flexneri (Shigella flexneri), Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Clostridium difficile (Clostridium difficile) and Escherichia coli (Escherichia coli) is as follows:

1) respectively inoculating activated single probiotic thallus and probiotic symbiont on an MRS culture medium plate, carrying out anaerobic culture for 24 hours, inoculating activated Salmonella enterica (Salmonella enterica) with 1% inoculum size to a liquid BS agar culture medium at about 50 ℃, quickly and uniformly pouring the culture medium on the plate on which the probiotic is cultured, and carrying out inverted culture for 24 hours at 37 ℃.

2) Respectively inoculating activated single probiotic thallus and probiotic symbiont to an MRS culture medium plate, carrying out anaerobic culture for 24 hours, then inoculating activated Shigella flexneri (Shigella flexneri) with 1% inoculum size to LB culture medium liquid at about 50 ℃, respectively pouring the culture medium onto the plate on which the probiotic is cultured quickly and uniformly, and carrying out inverted culture for 24 hours at 37 ℃.

3) Respectively inoculating activated single probiotic bacteria and probiotic bacteria on an MRS culture medium plate, carrying out anaerobic culture for 24 hours, then inoculating 1% of activated Listeria monocytogenes (Listeria monocytogenes) to an LB culture medium liquid at about 50 ℃, respectively pouring the culture medium onto the plate cultured with the probiotic bacteria rapidly and uniformly, and carrying out inverted culture at 37 ℃ for 24-36 hours.

4) Respectively inoculating activated single probiotic thallus and probiotic symbiont on an MRS culture medium plate, carrying out anaerobic culture for 24 hours, inoculating 1% of activated Staphylococcus aureus (Staphylococcus aureus) to a TSP agar culture medium liquid at about 50 ℃, quickly and uniformly pouring the culture medium on the plate in which the probiotic bacteria are cultured, and carrying out inverted culture for 24-30 hours at 37 ℃.

5) Respectively inoculating activated single probiotic thallus and probiotic symbionts to an MRS culture medium plate, carrying out anaerobic culture for 24 hours, inoculating 1% of activated clostridium difficile with a liquid BHI agar culture medium at about 50 ℃, quickly and uniformly pouring the culture medium onto the plate in which the probiotics are cultured, and carrying out inverted anaerobic culture at 37 ℃ for 24-48 hours.

6) Respectively inoculating activated single probiotic thallus and probiotic symbiont on an MRS culture medium plate, carrying out anaerobic culture for 24 hours, inoculating 1% of activated escherichia coli to a liquid-state mauve cholate agar culture medium at about 50 ℃, quickly and uniformly pouring the culture medium on the plate cultured with the lactobacillus, and carrying out inverted culture at 37 ℃ for 18-24 hours.

The bacteriostatic ability of the single probiotic thallus and the probiotic symbiont is compared by measuring the diameter of the bacteriostatic zone of the probiotics spotted on each flat plate. The results are shown in Table 1

TABLE 1 zone of inhibition diameter of probiotics

In the POINT AGAR test, a single probiotic strain shows a certain inhibition effect on salmonella enterica, Shigella flexneri, Listeria monocytogenes, staphylococcus aureus, Clostridium difficile and Escherichia coli, the antibacterial ability of the probiotic symbiont is obviously higher than that of the single strain, the antibacterial diameter on Clostridium difficile is 11mm, the antibacterial diameter on Escherichia coli is 17mm, and the two probiotic strains show extremely strong antibacterial synergistic effect, and the synergistic mechanism of the two probiotic strains needs to be further researched. This may be a nutritional competition, producing H ₂0₂And bacteriocin and the like.

Preliminary study on bacteriostatic mechanism of probiotic symbiont on pathogenic bacteria

And (3) researching the bacteriostasis mechanism of the probiotic symbiont on the pathogenic bacteria by utilizing a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM). Thereby further confirming the bacteriostatic effectiveness of the probiotic symbiont on the pathogenic bacteria; the antibacterial mechanism of metabolic products of the probiotics for antagonizing pathogenic bacteria is studied from the aspect of cell morphology through electron microscope photos. The specific method comprises the following steps:

1) the preparation of the probiotic supernatant comprises respectively taking bacterial liquid of single probiotic SR-19, CR-29 and symbiont after culturing for 24h, and centrifuging under aseptic condition (8000r/min,40C,20 min). The centrifugation supernatant was collected in a sterile 4.0mLEP tube and stored in a 40C freezer for further use. The pellet was eluted with 0.5mL of physiological saline, collected in a sterile 1.5mLEP tube and stored in a 40C freezer for use.

2) The experimental method comprises the following steps: respectively culturing Salmonella enterica (Salmonella enterica), Shigella flexneri (Shigella flexneri), Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Clostridium difficile (Clostridium difficile) and Escherichia coli (Escherichia coli) in a corresponding culture medium under growth conditions, picking up the obtained bacterial colonies in 1ml of sterile PBS in a sterile box, fully vibrating the obtained bacterial colonies into turbid liquid, respectively adding 4ml of the above probiotic supernatant, performing mixed culture under aerobic or anaerobic conditions corresponding to pathogenic bacteria (370C, 30min,60min,90min and 120min), preparing heavy metal and slice specimens according to the requirements of a Scanning Electron Microscope (SEM) and a Transmission Electron Microscope (TEM), and observing by an electron microscope.

TABLE 2 proportion of lysis (or apoptosis) of pathogenic bacteria cells under the action of probiotic supernatant

Note: the values are the percentage of pathogenic cell lysis (or apoptosis) in all cells per electron microscope (P <0.05) field, 50 pictures per group.

As proved by the research of bacteriostatic cell morphology through an electron microscope, the SR-19 and CR-29 probiotic symbiont supernatant has stronger bacteriostatic activity compared with the fermentation supernatant of single probiotic, which is consistent with the experimental result of the prior POINT AGAR method. The research on bacteriostatic electron microscope photos (> 400) of a large number of different pathogenic bacteria shows that the tested pathogenic bacteria cells have obvious phenomena of cell membrane perforation and cell sap overflow, and the phenomena are the effect result of bacteriocin in the supernatant of the probiotics to a great extent, but the research and the verification need to be further carried out by different methods.

Example 2

The mixed microorganism can maintain the activity in gastric acid, alkali environment and intestinal enzyme of human body;

single strain and double symbiont strain external tolerance detection method and result

The probiotics can play the probiotic function only when the living bodies enter and are fixedly planted in the intestinal tract, the external temperature and the storage time have direct influence on the activity of the probiotics, and the probiotics enter the intestinal tract and must pass through the oral cavity to reach the intestinal tract after passing through the stomach, wherein gastric acid and pepsin in the stomach and bile salt and trypsin in the small intestine have antibacterial effect and can influence the number of live bacteria, thereby influencing the probiotics to play the effect. Therefore, considering the influence of external factors on the activity of bacteria and the fact that the bacteria pass through the digestive tract and survive and colonize the intestinal tract are one of the important criteria for screening probiotics.

1. Comparison of preservation characteristics of symbiont probiotics to artificially mixed probiotics at different temperatures

Respectively inoculating the activated four probiotics into 5mL of sterilized MRS broth with the inoculation amount of 10%, respectively placing the Leuconostoc mesenteroides SR-19, the enterococcus durans CR-29 and the mixed inoculum into a 42 ℃ water jacket constant temperature incubator to be cultured to the end of logarithmic growth, respectively placing the Lactobacillus bulgaricus, the Lactobacillus acidophilus and the mixed inoculum thereof into a 38 ℃ water jacket constant temperature incubator to be cultured to the end of logarithmic growth, respectively placing into a refrigerator at 4 ℃ and a constant temperature incubator at 20 ℃ for storage, repeating three parallels in each group, and respectively carrying out viable bacteria plate counting on the 0 th, 2 nd, 4 th, 7 th, 10 th, 14 th and 21.28 th days.

Due to different probiotic genetic factors, the number of live bacteria is different even if different products in the same environment are stored for the same time. The temperature of 4 ℃ is the temperature of the preserved food which is commonly used, the temperature of 20 ℃ is the ordinary indoor temperature, and as can be seen from the graph 1 and the graph 2, the viable count of the three strains is reduced to a certain extent during the preservation at the temperature of 4 ℃ and the preservation at the temperature of 20 ℃, which is probably caused by the inhibition effect of metabolites contained in the culture medium, the acid damage effect under the acidic environment and the like during the preservation. However, the survival rate of the four strains at 4 ℃ is still high in the single strain or mixed strain after 28 days, and the viable count is still as high as 10⁸About CFU/ml, wherein the viable bacteria of the mixed liquid of the symbiont of the Leuconostoc mesenteroides SR-19 and enterococcus durans CR-29 is reduced to the minimum, and the survival of the bacteria is still 10 days after 28 days⁹CFU/ml above, and 10 at 20 deg.C for 28 days⁷CFU/ml viable bacteria amount shows that the probiotic symbiont has extremely strong stability.

2. Determination of gastric acid resistance

For the lactobacillus for regulating the intestinal flora, the lactobacillus must enter the human intestinal tract to exert the curative effect, however, the lactobacillus must pass through the stomach of the human body in the process from the oral cavity to the intestinal tract, so that the research on the tolerance of the lactobacillus in gastric juice is necessary. In the fasting state, the pH value of the gastric juice is 1.5-1.8, in the eating process, the pH value of the gastric juice generally fluctuates between 1.8-5.0, usually about 3.0, due to the differences of food components, food intake and individuals, the pH value of the gastric juice can reach 1.5 when the gastric juice is eaten, and can reach 4-5 when the gastric juice is eaten; in addition, different food morphologies also affect their digestion time in the stomach, with fluid foods typically having an average digestion time of 1.5-2 hours in the stomach.

In the experiment, the viable count of a single bacterial strain SR-19 and a single bacterial strain CR-29 and a probiotic symbiont consisting of the single bacterial strain SR-19 and the probiotic symbiont are measured in artificial gastric juice with different pH values at different times.

(1) And (4) preparing artificial gastric juice. Diluting with water to obtain lmol/L diluted hydrochloric acid, adjusting pH to 1.5,2.0,2.5, and 3.5, respectively, adding 1g pepsin (enzyme activity 453U/mg) into each 100mL liquid, mixing, and filtering with 0.20 μm sterile filter.

(2) The experimental method comprises the steps of respectively inoculating the activated three probiotic liquids into the artificial gastric juice with different pH values according to the inoculation amount of 1%, culturing in a constant-temperature water bath at 37 ℃, and counting viable bacteria plates within 0,0.5,1,1.5,2 and 3 hours. Each group was repeated three replicates and algebraically averaged.

The results of the survival conditions of the individual probiotics in the artificial gastric juice at different pH with the probiotic symbiont of their composition are shown in the following table:

TABLE 3 results of artificial gastric juice experiments with single cell and symbiont at different pH values (lgCFU/mL)

The results show that the tested single strain and the symbiont formed by the two strains can not survive at the pH value of 1.5; at a pH of 2.0, three strains of lactobacillus can survive, but the number of surviving bacteria detected in a shorter time is obviously higher than that of the surviving bacteria detected at a longer time point; at pH values of 2.5 and above, the artificial gastric juice had some effect on a single strain, but had little effect on the probiotic symbiont composed of SR-19 and CR-29 strains, indicating good acid stability of the symbiont. At pH values above 2.5 even growth of some bacteria occurred, probably because: 1) the artificial gastric juice has almost no influence on the bacteria under the pH value, and 2)1 percent of the inoculation amount brings certain nutrient substances; 3) the strain is a bacterium with vigorous growth in logarithmic growth phase.

The results show that the acid resistance of the symbiont is stronger than that of a single strain, and the symbiont strains have synergistic action.

3. Determination of intestinal fluid resistance

In the small intestine, a large number of microorganisms exist, not only in large numbers, but also in a large variety, and are sites where various oral probiotics act, and in any case, it is a prerequisite to maintain a certain number of viable bacteria. The action of intestinal fluids, the peristalsis of the small intestine, etc. are factors that influence the number of probiotics. The pH of the intestinal fluid was about 7.6 and the average time for food to pass through the small intestine was about 1.5 h.

The experiment adopts three strains of bacteria to culture in the artificial intestinal juice with different pH values in a constant temperature water bath at 37 ℃ and measures the viable count of the three strains at different time.

(1) The preparation of the artificial intestinal juice comprises the steps of taking 0.8g of monopotassium phosphate, adding 500mL of water for dissolution, adjusting the pH value to 6.8 by using a sodium hydroxide solution with the mass fraction of 0.4%, adding water for dilution to 1000mL, adding 1g of trypsin (with the enzyme activity of 11800U/mg) into each 100mL of liquid, uniformly mixing, and filtering by using a sterile filter with the particle size of 0.20 mu m for later use.

(2) The experimental method comprises the steps of respectively inoculating the activated three probiotic liquids into the artificial intestinal juice with different pH values according to the inoculation amount of 1%, culturing in a constant-temperature water bath at 37 ℃, and counting viable bacteria plates within 0,0.5,1,1.5,2 and 3 hours. Each group was repeated three replicates and algebraically averaged.

The results obtained according to the experimental conditions are shown in the following table:

TABLE 4 results of tolerance test (lg (CFU/mL))

The results show that: the single thallus has higher survival number in the artificial intestinal juice, and the artificial intestinal juice has almost no influence on the number of the viable bacteria in the time change process for the symbiont consisting of the single thallus. Therefore, the SR-19 and CR-29 strains form a probiotic symbiont with excellent tolerance performance in a simulated human intestinal fluid environment.

The results show that the intestinal juice resistance of the symbiont is stronger than that of a single strain, and the symbiont strains have synergistic action.

4. Determination of the bile salt resistance

Bile tolerance is one of the important characteristics of lactobacillus, and the lactobacillus must have certain tolerance to bile salt in order to reach and colonize human intestinal tracts, and the high or low tolerance means the survival capability of the strain in the intestinal tracts. The content of bile juice salt in the small intestine of the human body fluctuates within the range of 0.03-0.3%.

Adding the porcine cholate into an MRS broth to ensure that the mass fractions of the porcine cholate are respectively 0.15%, 0.2%, 0.25%, 0.3%, 0.35% and 0.4%, subpackaging the porcine cholate into centrifugal test tubes, wherein each tube has 3mL and 5 tubes, and the mass fraction of the porcine cholate is 0.4% of bromopotassium phenol purple which is used as an indicator. Sterilizing at 121 deg.C for 15 min. Inoculating the activated three probiotics into the treated culture medium in an inoculation amount of 1%, culturing in a constant-temperature incubator at 37 ℃ for 24h, repeating three times for each group, and observing the color change of each group.

TABLE 5 growth of single and symbiotic bacteria in different concentrations of bile salts

Note "-" does not grow; growth of "+"; "+ +" grew well; "+ + + +" grew very well

The experimental result shows the growth conditions of the single and symbiotic thalli in the ox bile salt with different concentrations, and it can be seen that the single thalli has excellent bile acid resistance, while the symbiotic thalli has extremely strong bile salt resistance, can grow vigorously at 0.2 percent and still can grow at 0.4 percent, and the highest bile salt concentrations of SR-19 and CR-29 which can grow are 0.25 percent and 0.3 percent.

The results show that the bile resistance of the symbiont is stronger than that of a single strain, and the symbiont strains have synergistic action.

Although specific embodiments of the invention have been described in detail, those skilled in the art will appreciate. Various modifications and substitutions of those details may be made in light of the overall teachings of the disclosure, and such changes are intended to be within the scope of the present invention. The full scope of the invention is given by the appended claims and any equivalents thereof.

Claims

1. Use of a pathogenic antagonist in the manufacture of a medicament for the prevention and/or treatment of diarrhoea, wherein the pathogenic antagonist comprises Enterococcus durans CR-29(Enterococcus durans CR-29) having CGMCC number 11870 and Leuconostoc mesenteroides Enterococcus SR-19(Leuconostoc mesenteroides bessbsp. mesenteroides SR-19) having CGMCC number 11869; in the pathogenic bacteria antagonist, the cfu/g ratio of enterococcus durans CR-29 to Leuconostoc mesenteroides subsp.

2. The use according to claim 1, wherein the pathogenic bacteria are Salmonella enterica (Salmonella enterica), Shigella flexneri (Shigella flexneri), Listeria monocytogenes (Listeria monocytogenes), Staphylococcus aureus (Staphylococcus aureus), Clostridium difficile (Clostridium difficile), Escherichia coli (Escherichia coli).

3. Use of a pathogenic bacteria antagonist according to any of claims 1-2 for the manufacture of a medicament for the prevention and/or treatment of gastric acid, intestinal juice and bile tolerance.

Use of Enterococcus durans CR-29(Enterococcus durans CR-29) having CGMCC No. 11870 and Leuconostoc mesenteroides Enterococcus SR-19(Leuconostoc mesenteroides SR-19) having CGMCC No. 11869 for preparing pathogenic bacteria antagonist.

5, the application of Enterococcus durans CR-29(Enterococcus durans CR-29) with CGMCC No. 11870 and Leuconostoc mesenteroides Enterococcus SR-19(Leuconostoc mesenteroides SR-19) with CGMCC No. 11869 in the preparation of pathogenic bacteria antagonist for resisting gastric acid, intestinal juice and bile.