CN107129946B

CN107129946B - High-planting microbial preparation and application thereof

Info

Publication number: CN107129946B
Application number: CN201710289138.1A
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: CN107129946A

Abstract

The invention discloses a high-colonization microbial preparation, which comprises Enterococcus durans CR-29(Enterococcus durans CR-29) with the CGMCC number of 11870 and Leuconostoc mesenteroides subsp. Also discloses application of the high-colonization microbial preparation in preparation of a preparation for adjusting intestinal microbial flora and preparation of a preparation for preventing and/or treating intestinal pathogenic bacteria.

Description

High-planting microbial preparation and application thereof

Technical Field

The invention belongs to the field of probiotics, and particularly relates to a high-colonization microbial preparation and application thereof.

Background

The intestinal bacteria include two types, namely, domestic bacteria and passerby bacteria, from the source. The former is not taken orally and remains a stable population in the intestinal tract; the latter is taken orally and passed through the gastrointestinal tract, and the native bacteria are factors that make the road bacteria unable to colonize. The indigenous flora of the gastrointestinal tract inhibits colonization or proliferation of other foreign microorganisms in the intestinal tract, which is colonization resistance, also known as competitive exclusion. This colonization resistance arises because the microbes in the body compete with pathogenic bacteria for the adsorption sites of the intestinal epithelium. If these adsorption sites are occupied by more beneficial microorganisms, pathogenic microorganisms are rejected.

Preventing colonization and invasion of pathogenic bacteria, such as Salmonella infection of the host. This interstitial colonization may be due to the presence of specific substances on the surface of the probiotic bacteria that are capable of binding strongly to intestinal mucosal cells, presumably lipoteichoic acid or peptidoglycan, or cell surface layer proteins, or both, or all three. These specific substances are called adhesins, by which lactic acid bacteria bind tightly to the intestinal mucosa, colonize sites on the surface of the intestinal mucosa, and are a major component of the physiological barrier. The barrier can resist invasion of foreign bacteria and maintain microecological balance in intestinal tract. The research on the colonization resistance and ecological occupation of the probiotics is of great importance to the performance of the probiotics on human body.

In recent years, the theory and application of probiotics have been rapidly developed. The development of mixed culture of various microorganisms by utilizing the natural reciprocal symbiosis phenomenon among organisms has wide application prospect, and is also one of the fields of relative shortage of current research. The natural or artificial mixed symbiosis system maintains a stable state by virtue of original synergy, reciprocal symbiosis and biased symbiosis, and antagonism and synergy existing in microbial floras are important self-stabilization mechanisms of the floras. The intestinal flora of human body is a huge microbial symbiosis balance system, which comprises reciprocal symbiosis of human body and intestinal flora, symbiosis of prokaryotes and eukaryotes, symbiosis between bacteria and the like, while natural probiotic symbiont preparation has larger buffering capacity and environmental adaptability in human body micro-ecological environment, wherein the artificial composite microbial symbiont in the fields of medicine and food can be used for more than one hundred years. At present, effective methods and theoretical guidance are lacked for screening and combining strains with a synergistic relationship, and meanwhile, the formula and the proportion relationship of the probiotics are lacked for improving the functions of the probiotics and theoretical basis is lacked.

Disclosure of Invention

The inventor of the present invention surprisingly found a highly colonized probiotic symbiont which produced unexpected effects in terms of intestinal colonization through repeated experiments, and completed the present invention based on the above findings.

The inventor screens a pair of probiotic symbiont comprising an enterococcus faecalis strain SR-19 and enterococcus durans CR-29 from a traditional ethnic dairy product, and researches the reciprocal symbiosis of the probiotic symbionts and the growth promoting capability of the probiotic symbionts on other human intestinal probiotics.

The first aspect of the invention discloses a high-colonization microbial preparation, 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.

Preferably, in the high-colonization microbial preparation, the cfu/g ratio of the enterococcus durans CR-29 to the Leuconostoc mesenteroides SR-19 is 1:10-10: 1.

The second aspect of the invention discloses a culture method for preparing the microbial preparation with high colonization of the first aspect,

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 bacterial powder for preparing a high-colonization microbial preparation, which is characterized by being 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 obtain symbiont probiotic powder; all the operations are carried out under aseptic conditions.

The fourth aspect of the invention discloses a high-colonization microbial preparation which contains the microbial preparation and pharmaceutically acceptable auxiliary materials.

A fifth aspect of the invention is to disclose the use of a high colonizing microbial preparation in the preparation of a formulation for modulating the intestinal microbial flora.

The sixth aspect of the invention discloses application of the high-colonization microbial preparation in preparation of a preparation for regulating intestinal microbial flora, wherein the preparation is resistant to gastric acid, intestinal juice and bile.

The seventh aspect of the invention discloses the application of the microbial preparation with high colonization in the preparation of the medicines for preventing and/or treating the intestinal pathogenic bacteria.

The eighth aspect of the invention discloses application of the high-colonization microbial preparation in preparation of gastric acid resistance, intestinal juice resistance and bile resistance for preventing and/or treating intestinal pathogenic bacteria.

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; eppendorf TGL-168 high speed desktop 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.

Biological material preservation information:

enterococcus durans CR-29, classified and named Enterococcus durans, is preserved in the general microbiological culture Collection center of China Committee for culture Collection of microorganisms 11 months 12 and 11 days 2015, and is preserved in the No. 3 Hospital No.1, North West Lu, the Tokyo area, Beijing, and the preservation number is: CGMCC No. 11870.

The Leuconostoc mesenteroides strain SR-19 is classified and named as Leuconostoc mesenteroides subsp.mesenteroides, is preserved in the general microbiological center of China Committee for culture Collection of microorganisms on 12-11 months in 2015, has the preservation address of No. 3 Hospital No.1 of Xilu of the North Cheng of the sunward area in Beijing, and has the preservation number of: CGMCC No. 11869.

In the present invention, the symbiont and probiotic symbiont are the same as the microbial preparation.

Drawings

FIG. 1 is a photograph of a microscope at magnification 1000, wherein

1) Morphology of CaCo-2 cell adhesion;

2) morphograms of probiotic adhesion to CaCo-2 cells;

3) morphograms of probiotic adhesion to CaCo-2 cells;

4) a morphogram of probiotic symbiont adhesion to CaCo-2 cells;

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 determination of intestinal cell adhesion Capacity of probiotic symbionts

The strength of the ability of probiotics to adhere to intestinal epithelial cells is still often used as an important indicator of the size of their intestinal colonization ability and whether they can exert probiotic functions. The CaCo-2 cell is a human colon adenocarcinoma cell which is easy to culture in vitro, the cell morphology, the adhesion capability and the like of the human colon adenocarcinoma cell are similar to those of human intestinal epithelial cells, and the human colon adenocarcinoma cell is often used as a model cell for in vitro adhesion tests. And (3) determining the adhesion capacity of the symbiont of the single probiotics and the probiotics to CaCo-2 cells. The specific experimental steps are as follows:

1) cell recovery, namely rapidly placing a CaCo-2 cell freezing tube stored in a liquid nitrogen tank into a 37 ℃ water bath kettle to melt the cell, inoculating the cell into an RPMI-1640 complete culture medium, culturing in a 37 ℃ carbon dioxide incubator, and changing the culture solution once every other day;

2) subculturing the cells for 3-4 generations, and counting by using a blood counting chamber after the cells grow to full of a culture dish, washing by using PBS (phosphate buffer solution) and digesting by using pancreatin;

3) cell concentration was adjusted to 5 × 10 using RPMI-1640 basal medium⁵Placing the cell suspension in a 6-hole cell culture plate in which a cover glass is placed in advance, and culturing for 6 hours in a carbon dioxide incubator to allow cells to adhere to the wall;

4) regulating the concentration of single probiotic bacteria and probiotic bacteria symbiont cultured overnight to 10 with RPMI-1640 basic culture medium⁷cfu/mL；

5) Pouring off the cell culture medium, and co-culturing the adherent cells and the bacterial suspension for 4 hours;

6) decanting the bacterial suspension, and washing non-adhered bifidobacteria with PBS buffer solution;

7) fixing with methanol for 5-10min, and dyeing with Swiss-Giemsa dye solution, wherein the dyeing step is carried out according to the Specification of Rapid Giemsa Compound dye solution;

8) washing with PBS buffer solution until the dye solution is colorless, and drying the cell culture plate in an incubator at 37 ℃ or naturally drying;

(9) the number of probiotics adhering to the cells was counted in 50 random fields, observed under an oil lens.

TABLE 1 results of the relationship of individual probiotics and probiotic symbionts to human cell adhesion

Probiotic bacterial strains	Number of CaCo-2 cell adhesion probiotics
		SR-19	645.3±2.0
CR-29	973.4±6.3
		Symbiont of SR-19 and CR-29	2124.9±6.7

The results show that the cell adhesion capacity of the symbiont of SR-19 and CR-29 is greatly beyond the direct superposition of the two, and the cell adhesion capacity has a remarkable synergistic effect.

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 normal indoor temperature, 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. 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 2 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 3 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 4 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. A high colonization microbial preparation, 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;

in the high-colonization microbial preparation, the cfu/g ratio of enterococcus durans CR-29 to Leuconostoc mesenteroides SR-19 is 1:10-10: 1.

2. A culture process for the preparation of a high colonizing microbial preparation according to claim 1,

the initial culture temperature is 44 ℃, and the initial culture time is 1.0-1.5 hours; the middle culture temperature is 42 ℃, the middle culture time is 2.0-2.5 hours, and the pH value of the middle fermentation section is 5.9; the final stage culture temperature is 38 ℃, and the final stage culture time is 1.5-2.0 hours;

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

the formula of the liquid 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.

3. A fungal powder for preparing a high colonisation microbial preparation according to any one of claims 1 to 2, produced by the process of:

viable count is not less than 10¹⁰Filtering or centrifuging cfu/mL probiotic symbiont fermentation liquor, and carrying out vacuum freeze drying by using a freeze-drying protective agent, wherein the freeze-drying curve is as follows: the initial temperature is-42 deg.C, pre-freezing for 4 hr, and the temperature is-42 deg.C to-5 deg.C, and is increased per hourWarming to 5 ℃; 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 obtain symbiont probiotic powder; all the operations are carried out under aseptic conditions.

4. A high colonisation microbial preparation comprising a microbial preparation according to any one of claims 1 to 2 and a pharmaceutically acceptable adjuvant.