CN115590893A - Compound probiotic composition capable of inhibiting helicobacter pylori and application thereof - Google Patents
Compound probiotic composition capable of inhibiting helicobacter pylori and application thereof Download PDFInfo
- Publication number
- CN115590893A CN115590893A CN202211394768.2A CN202211394768A CN115590893A CN 115590893 A CN115590893 A CN 115590893A CN 202211394768 A CN202211394768 A CN 202211394768A CN 115590893 A CN115590893 A CN 115590893A
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- China
- Prior art keywords
- helicobacter pylori
- lactobacillus casei
- bifidobacterium lactis
- composite
- probiotic composition
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Abstract
The invention belongs to the technical field of probiotic preparations, relates to a composite probiotic composition capable of inhibiting helicobacter pylori, and further discloses application of the composite probiotic preparation in preparation of a product for treating or preventing helicobacter pylori infection. The composite probiotics provided by the invention take Lactobacillus casei Zhang and Bifidobacterium lactis Probio-M8 as active ingredients, and experiments prove that the composite probiotics provided by the invention have the effect of inhibiting helicobacter pylori, can effectively prevent or treat related diseases caused by helicobacter pylori infection, can effectively inhibit the helicobacter pylori by taking the Lactobacillus casei Zhang and the Bifidobacterium lactis Probio-M8, and has a great application prospect in preparation of products for preventing or treating the helicobacter pylori infection.
Description
Technical Field
The invention belongs to the technical field of probiotic preparations, relates to a composite probiotic composition capable of inhibiting helicobacter pylori, and further discloses application of the composite probiotic preparation in preparation of a product for treating or preventing helicobacter pylori infection.
Background
Helicobacter Pylori (HP) is a microaerophilic gram-negative bacillus which can be adhered and colonized on the surface of gastric mucosa, is one of pathogenic factors of gastritis and peptic ulcer, and is well known to play a causative role in gastric ulcer, duodenal ulcer, gastric cancer and the like. In addition, gastritis caused by infection with helicobacter pylori is also considered as a risk factor for peptic ulcer and its associated complications. Therefore, the elimination of the helicobacter pylori not only can obviously reduce the risk of gastric cancer, but also can prevent peptic ulcer, directly or indirectly improve the eradication rate of the helicobacter pylori, and has important significance for preventing and treating the occurrence of the helicobacter pylori and related diseases thereof.
At present, the conventional treatment scheme for eradicating HP is mainly a four-combination scheme containing bismuth, and the new treatment scheme such as sequential therapy, levofloxacin therapy and the like is mainly to eliminate helicobacter pylori in a patient through triple or four-combination antibiotic combination therapy so as to recover gastrointestinal symptoms caused by helicobacter pylori infection. However, as a plurality of antibiotics are required to be applied in a combined manner in the treatment process, the treatment course is long, side effects such as drug resistance, flora imbalance and compliance reduction are often caused, the probability of adverse reaction of patients is high, and the treatment efficiency is reduced.
A large number of clinical practices prove that the oral administration of intestinal probiotics can play a certain positive auxiliary effect when the helicobacter pylori is clinically treated. This is mainly because probiotics enhance mucosal barrier, reduce the incidence of inflammatory reactions, help patients to improve clinical symptoms, and improve treatment compliance. Therefore, the development of probiotic preparations which can assist in improving the eradication rate of helicobacter pylori has positive significance for preventing and treating helicobacter pylori infection and related diseases.
Disclosure of Invention
To this end, a first object of the present invention is to provide a complex probiotic composition capable of inhibiting helicobacter pylori, which is effective in eradicating helicobacter pylori;
the second purpose of the invention is to provide the application of the composite probiotic preparation in preparing products for preventing or treating helicobacter pylori infection;
the third purpose of the invention is to provide the application of the lactobacillus casei Zhang and/or the bifidobacterium lactis Probio-M8 in preparing a composite probiotic preparation capable of inhibiting helicobacter pylori.
In order to solve the technical problems, the invention discloses a composite probiotic composition capable of inhibiting helicobacter pylori, wherein the active ingredients of the composite probiotic composition comprise lactobacillus casei Zhang and bifidobacterium lactis Probio-M8;
the Lactobacillus casei Zhang is classified and named as Lactobacillus casei, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of China institute of sciences, and has the preservation address of No. 3 of No.1 Xilu of Beijing Korean district, and the preservation number of CGMCC No.5469;
the Bifidobacterium lactis Probio-M8 is classified and named as Bifidobacterium lactis and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of China academy of sciences, the preservation address is No. 3 of the Xilu No.1 of the North Chen of the Inward area of Beijing, and the preservation number is CGMCC No.18610.
Preferably, the composite probiotic composition capable of inhibiting helicobacter pylori has the total viable count of the lactobacillus casei Zhang and the bifidobacterium lactis Probio-M8 not less than 1 x 10 6 CFU/mL or 1X 10 6 CFU/g。
Preferably, the composite probiotic composition capable of inhibiting helicobacter pylori has the viable count ratio of (1-10) of lactobacillus casei Zhang to bifidobacterium lactis Probio-M8: (10-1).
The invention also discloses a probiotic preparation which is prepared from the composite probiotic composition and/or an inactivated substance and a metabolite thereof and can inhibit helicobacter pylori.
Specifically, the probiotic preparation comprises a liquid preparation or a solid preparation.
The invention also discloses a method for preparing the probiotic preparation, which comprises the steps of culturing the thalli of the composite probiotic composition and processing selected dosage forms.
The invention also discloses application of the composite probiotic composition and an inactivated product and a metabolite thereof in preparing a product for preventing or treating helicobacter pylori infection.
In particular, the product comprises a pharmaceutical formulation;
preferably, the pharmaceutical preparation comprises a powder, tablet or capsule.
Specifically, the product comprises functional food;
preferably, the functional food comprises a dairy product, a soy product or a beverage.
The invention also discloses application of the lactobacillus casei Zhang and/or the bifidobacterium lactis Probio-M8 in preparing a probiotic preparation capable of inhibiting helicobacter pylori.
The composite probiotics provided by the invention take Lactobacillus casei Zhang and Bifidobacterium lactis Probio-M8 as active ingredients, and experiments prove that the composite probiotics provided by the invention have the effect of inhibiting helicobacter pylori, can effectively prevent or treat related diseases caused by helicobacter pylori infection, can effectively inhibit the helicobacter pylori by taking the Lactobacillus casei Zhang and the Bifidobacterium lactis Probio-M8, and has a great application prospect in preparation of products for preventing or treating the helicobacter pylori infection.
Drawings
In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,
FIGS. 1 to 3 show the effect of the complex probiotics according to the present invention on the ultrastructure of H.pylori (observed by scanning electron microscopy), wherein FIG. 1 shows the morphology of H.pylori, FIG. 2 shows the morphology of the complex strain and H.pylori before treatment, and FIG. 3 shows the morphology of the complex strain and H.pylori after treatment;
FIGS. 4-6 show the effect of the complex probiotics of the present invention on the ultrastructure of H.pylori (observed by transmission electron microscopy), wherein FIG. 4 shows the morphology of H.pylori, FIG. 5 shows the morphology of the complex strain and H.pylori before treatment, and FIG. 6 shows the morphology of the complex strain and H.pylori after treatment.
Detailed Description
In the following embodiment of the invention, the strains involved include Lactobacillus casei Zhang and Bifidobacterium lactis Probio-M8.
The Lactobacillus casei Zhang (Lactobacillus casei Zhang) is classified and named as Lactobacillus casei, the strain is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of China institute of academy of sciences, the preservation address is No. 3 of Xilu No.1 of Beijing Kogyo of Chaoyang, the preservation date is 11 months and 18 days in 2011, and the preservation number is CGMCC No.5469; the lactobacillus casei Zhang grows on the MRS culture medium to form a milk white colony which is opaque, round, smooth in surface, convex in the center and about 1.2-1.5mm in diameter.
The Bifidobacterium lactis Probio-M8 (Bifidobacterium lactis Probio-M8) is classified and named as Bifidobacterium lactis, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of institute of China academy of sciences, has the preservation address of No. 3 Xilu of No.1 of the national institute of microbiology, 9-20 months in 2019 and the preservation number of CGMCC No.18610; the bifidobacterium lactis Probio-M8 is gram-positive bacteria, the agar plate generates a convex milky colony with the diameter of 1-2mm, the edge is complete, the surface is smooth, the cell shape is dumbbell-shaped bacillus, the two ends are regular, a plurality of obvious nodes are arranged, and the bifidobacterium lactis Probio-M8 is heterotypic fermentation.
The lactobacillus casei Zhang and the bifidobacterium lactis Probio-M8 are two probiotic strains with good gastrointestinal fluid and bile salt tolerance.
In the following examples of the invention the effect of the complex probiotic was compared to a pylopas DSM17648, known in the art to inhibit helicobacter pylori.
EXAMPLE 1 determination of the Redox Capacity of the Strain
Preparation of cell suspensions and cell-free suspensions
Preparation of cell suspension: culturing the two stored microbial inoculum at 37 deg.C for 24h, activating anaerobically to three generations, centrifuging at 4 deg.C and 8000 Xg for 10min, and collecting fermentation supernatant. Respectively washing the thalli for three times by using sterilized PBS, then re-suspending and adjusting the concentration of the bacteria liquid to 10 8 CFU/mL(OD 595nm Value 1.0), 1:1 mixing to prepare cell suspension.
Preparation of cell-free suspension: the two cell suspensions were separately subjected to ultrasonic ice-bath for 10min (10 s work; interval 10s 280w; Φ 2), centrifuged at 10000 × g for 10min at 4 ℃, examined under a microscope for the absence of intact cells, and the supernatant was collected in a sterile centrifuge tube, 1:1 mixing to obtain cell-free suspension.
1. Determination of DPPH free radical scavenging capability of strain
1mL of the above fermentation supernatants (cell suspension, cell-free suspension) were each added to 1mL of DPPH absolute ethanol solution (0.2 mmol/L), shaken, left at room temperature in the dark for 30min, centrifuged at 6000 Xg at 4 ℃ for 10min, and the supernatant was each measured for absorbance Ai at 517nm and recorded. The blank group Aj is formed by replacing DPPH absolute ethyl alcohol solution with equal volume of absolute ethyl alcohol; the control group A0 was prepared by substituting equal volume of distilled water for the sample solution; blank zeroing was performed using a mixture of equal volumes of distilled water and absolute ethanol. The calculation formula is as follows:
in the formula: a0-absorbance of control group;
ai-sample set absorbance;
aj-blank absorbance.
The test results are shown in table 1 below.
TABLE 1 ability of complex strains to scavenge DPPH free radicals
| Strain name | Fermentation supernatant (%) | Cell-free suspension (%) | Cell suspension (%) |
| Composite strain | 78.28±0.14 | 45.11±0.09 | 40.63±0.09 |
| Pylopass DSM17648 | 74.15±0.21 | 46.23±0.01 | 38.18±0.03 |
Therefore, each component of the lactobacillus casei Zhang and bifidobacterium lactis Probio-M8 composite strain has certain DPPH removing capacity, but the DPPH removing capacity of the strain fermentation supernatant is better than that of other two groups and is higher than 75.0%.
2. Determination of the ability of a Strain to scavenge hydroxyl radicals
Absorbance was measured and calculated at 550nm, according to the protocol of hydroxyl radical assay kit (Fenton colorimetry).
The test results are shown in table 2 below.
TABLE 2 Complex strains ability to scavenge hydroxyl radicals
| Name of Strain | Fermentation supernatant (%) | Cell-free suspension (%) | Cell suspension (%) |
| Composite strain | 46.10±0.38 | 32.22±0.46 | 29.11±0.75 |
| Pylopass DSM17648 | 41.35±0.25 | 30.56±0.07 | 32.17±0.43 |
Therefore, each component of the composite strain has certain scavenging capacity for hydroxyl radicals, and the hydroxyl radical scavenging capacity of the strain fermentation supernatant is higher and reaches more than 40.0%.
3. Determination of the reducing ability of the Strain
0.5mL of each of the above fermentation supernatants (cell suspension and cell-free suspension) was taken, added with 1% (w/v) potassium ferricyanide solution and 0.5mL of PBS (0.2 mol/L; pH 6.6) and mixed in a 10mL centrifuge tube, quenched after being subjected to 50 ℃ water bath for 20min, added with 0.5mL of 10% trichloroacetic acid and centrifuged at 4000 Xg for 10min, 1mL of the supernatant was taken, added with 0.1% ferric trichloride solution and 1mL of distilled water and mixed and left for 10min, and the absorbance As was measured at 700nm and recorded. Blank Ab was replaced with an equal volume of PBS. The formula is as follows:
in the formula: as-sample set absorbance;
ab-blank absorbance.
The test results are shown in table 3 below.
TABLE 3 composite strain reduction Capacity determination
Therefore, each component of the composite strain has certain reducing capability, and the reducing capability of the strain fermentation supernatant is obviously higher than that of cell-free suspension and cell suspension, OD 700nm The value reached 1.55.
EXAMPLE 2 determination of the ability of the strains to inhibit helicobacter pylori
Activation and culture of helicobacter pylori
Taking out the cryopreserved tube of helicobacter pylori from-80 deg.C, thawing in 37 deg.C water bath for 2 min, mixing, sucking helicobacter pylori suspension on Columbia blood plate, covering the surface of culture medium with the suspension uniformly with coating rod, and placing in a three-gas incubator (85% N) 2 、10％ CO 2 、5％ O 2 ) And (5) standing and culturing for 72h. After two generations of activation, the colony on the surface of the Columbia blood plate is eluted by normal saline to prepare bacterial suspension for later use.
Activation and culture of strains
Taking out the strain cryopreservation tube from-80 ℃, then placing the tube to unfreeze at room temperature, carrying out aerobic culture in an MRS liquid culture medium at 37 ℃, and preparing a strain suspension for later use after the strain is activated for two generations.
Different proportions of composite strains and helicobacter pylori
In this example, the different ratios of the complex strains and the ratio of H.pylori are shown in Table 4 below.
TABLE 4 details of the ratio of the complex strains and H.pylori in different ratios
1. Determination of capability of strain to inhibit urease activity of helicobacter pylori
The urease is arranged on the surface of the helicobacter pylori thallus, and can decompose urea in a host body to generate ammonia, neutralize gastric acid and protect the thallus from being damaged. Urease is capable of breaking down urea to raise the solution pH, discoloring the indicator solution, and its activity may be reflected by the absorbance value of the assay solution at a wavelength of 550 nm.
Adjusting the viable count of the composite strain bacterial suspension and the helicobacter pylori bacterial suspension according to the proportion of the composite strain to the helicobacter pylori 1: 1. 5:1 and 10:1 in a 96-well plate, respectively absorbing 40 mu L of helicobacter pylori suspension and 10 mu L of composite strain suspension, uniformly mixing, putting into a three-gas culture box for co-culture for 48h, adding 150 mu L of urease test solution, and measuring the light absorption value at 550nm by using an enzyme-labeling instrument after shaking. The result of the measurement of the urease test solution is used as a blank group, and the BHI liquid culture medium is used for replacing the result of the measurement of the bacterial suspension of the compound strain to be used as a control group.
The results of the capability of the composite strain of the invention to inhibit the urease activity of helicobacter pylori are shown in the following table 5.
TABLE 5 determination of the ability of the Complex strains to inhibit the urease activity of helicobacter pylori
It can be seen that in the case of the complex strain, the ratio of H.pylori 1: 1. 5:1 and 10:1 ratio of OD 550nm The values are all lower than the control group, which shows that the compound strains with different concentrations can effectively inhibit the urease activity of the helicobacter pylori.
2. Determination of the ability of a Strain to inhibit the growth of helicobacter pylori
Pouring sterilized vegetarian agar into a disposable flat plate, standing for 30 minutes for waiting for solidification, placing a sterilized Oxford cup on a just poured culture medium according to a proper interval, inoculating helicobacter pylori into 15mL Columba culture medium (containing 7% sterile sheep blood) according to the inoculation amount of 1%, pouring the mixture into the flat plate after fully and uniformly mixing to ensure that the surface of the flat plate is uniformly covered by the culture medium without bubbles, taking out the Oxford cup by using tweezers after waiting for solidification, and mixing the agar and the Pylori cup according to the ratio of a composite strain to helicobacter pylori 1: 1. 5:1 and 10:1, respectively sucking 100 mu L of composite bacterial suspension with different viable counts into the round holes, placing the flat plate in a three-gas culture box, standing and culturing for 48h at 37 ℃, and measuring the size of a bacteriostatic circle (mm).
An oxford cup method is used for carrying out bacteriostatic experiments, the diameter of a bacteriostatic zone is measured, the inhibitory action of the composite strains on helicobacter pylori under different proportions is preliminarily researched, and the test results are shown in the following table 6.
TABLE 6 determination of the ability of the Complex strains to inhibit the growth of helicobacter pylori
It can be seen that the diameter of the inhibition zone is gradually increased along with the increase of the concentration of the compound strain. The passing ratio is 5:1 and 10:1, the complex strain can effectively inhibit the helicobacter pylori.
EXAMPLE 3 determination of the self-agglutination Capacity of the strains
1. Self-agglutination ability of compound strain
Preparing bacterial suspension by using the composite bacterial strain, and adjusting OD by using an enzyme labeling instrument 600nm After =0.5 ± 0.05, 1mL of bacterial suspension is taken and placed in a sterilized 1.5mL centrifuge tube, and is statically cultured at room temperature, and after being respectively statically placed for 1h, 2h, 3h, 4h, 5h, 6h, 24h and 48h, upper liquid is absorbed, and the absorbance at 600nm is tested, and the self-aggregation rate (%) is calculated as follows:
in the formula: a0-initial absorbance at 600nm before self-agglutination of the strain;
at-absorbance At 600nm after standing for t hours.
The results of the self-aggregation ability test of the complex strain are shown in Table 7 below.
Results of self-aggregation ability of the composite strains described in Table 7
As can be seen, the self-aggregation capability of the surface of the strain is enhanced along with the prolonging of the time; after standing for 24 hours, the composite strain shows more obvious self-agglutination capacity which reaches more than 90.0%.
2. Capability of mutual agglutination of composite strain and helicobacter pylori
Preparing bacterial suspension from the composite strain and helicobacter pylori according to the method, and adjusting OD with enzyme labeling instrument 600nm After =0.5 +/-0.05, taking the same amount of experimental strain and helicobacter pylori bacterial suspensionMixing the solution in a sterilized 1.5mL centrifuge tube, thoroughly oscillating for 5min, standing and culturing at room temperature, respectively standing for 1h, 2h, 3h, 4h, 5h, 6h, 24h and 48h, absorbing the upper layer liquid, testing the light absorption value at 600nm, and calculating the interaction agglutination rate (%), wherein the formula is as follows:
in the formula: absorbance of the composite strain at 600nm prior to Ax-cross agglutination;
ay-absorbance at 600nm of H.pylori before intercross agglutination;
amin-standing for t hours, and then mixing the light absorption values of the bacteria liquid.
The results of the determination of the mutual agglutination ability of the complex strain with helicobacter pylori are shown in Table 8 below.
TABLE 8 determination of the reciprocal agglutination ability of the Complex Strain with helicobacter pylori
It can be seen that the mutual cohesion of the lactobacillus casei Zhang and bifidobacterium lactis Probio-M8 complex strain with helicobacter pylori is also enhanced with the extension of the standing time. After standing for 24 hours, the interactive agglutination rate reaches over 90.0 percent.
In conclusion, in the embodiment, the composite strain of lactobacillus casei Zhang and bifidobacterium lactis Probio-M8 can be found to have relatively stable ability of antagonizing helicobacter pylori through the indexes, and has a certain effect of inhibiting helicobacter pylori.
EXAMPLE 4 Effect of the strains on the ultrastructure of helicobacter pylori
1. Observation by scanning electron microscope
Double fixation
The samples were fixed in 2.5% glutaraldehyde solution at 4 ℃ overnight; pouring off the stationary liquid, and rinsing the sample with 0.1M phosphate buffer solution with pH of 7.0 for three times, each time for 15min; samples were fixed with 1% osmic acid solution for 1-2h, the osmic acid waste solution was carefully removed, and the samples were rinsed three times for 15min each with 0.1M, pH7.0 phosphate buffer.
Dewatering
Dehydrating the sample with ethanol solution with gradient concentration (including six concentrations of 30%, 50%, 70%, 80%, 90% and 95%), each concentration for 15min; and treating once again with 100% ethanol for 20min, and finally, placing the sample in new 100% ethanol, and finishing the pretreatment.
Drying and Observation
And (4) drying at a critical point, coating, and observing, wherein the results are shown in the attached figures 1-3.
As is clear from the results shown in FIGS. 1 to 3, H.pylori which had not been co-cultured with the suspension of the complex strain was well-shaped and in a curved rod shape; and after the helicobacter pylori is treated by the composite strain suspension for 4 hours, the helicobacter pylori has obvious morphological structure changes such as pits, cracks and the like.
2. Transmission Electron Microscopy (TEM) Observation
Double fixation
The samples were fixed overnight at 4 ℃ in 2.5% glutaraldehyde solution, the fixative was decanted, the samples were rinsed three times with 0.1M phosphoric acid buffer at pH7.0 for 15min each, the samples were fixed with 1% osmic acid solution for 1-2h, the osmic acid waste was carefully removed, and the samples were rinsed three times with 0.1M phosphoric acid buffer at pH7.0 for 15min each.
Dewatering
The sample is dehydrated by ethanol solution with gradient concentration (including four concentrations of 30%, 50%, 70% and 80%), each concentration is treated for 15min, and then the sample is transferred to 90% and 95% acetone solutions for 15min. Finally, the mixture is treated twice with pure acetone for 20min each time.
Penetration of
The sample was treated with a mixture of sprirr embedding medium and acetone (V/V = 1/1) for 1h; the sample was treated with a mixture of Spurr embedding medium and acetone (V/V = 3/1) for 3h; samples were treated with neat embedding medium overnight at room temperature.
Embedding, sectioning and staining observation
Embedding the sample subjected to the permeation treatment, and heating the sample at 70 ℃ overnight to obtain an embedded sample; slicing the sample in LEICA EM UC7 ultrathin microtome to obtain 70-90nm slices; staining the slices with lead citrate solution and 50% ethanol saturated solution of uranyl acetate for 5-10min; the results of observation in a Hitachi H-7650 type transmission electron microscope are shown in the attached figures 4-6.
As can be seen from the results shown in FIGS. 4 to 6, the cells of H.pylori that had not been co-cultured with the complex strain suspension remained good; after the helicobacter pylori is treated by the composite strain suspension for 4 hours, the phenomena of thallus rupture, content loss and the like of the helicobacter pylori occur.
Example 5 application of Complex strains
In this example, a powder sample can be prepared based on the lactobacillus casei Zhang and bifidobacterium lactis Probio-M8 composite strain.
Streaking the preserved strain on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing for two continuous activation generations again at the temperature of 37 ℃ to obtain an activation solution; inoculating the activated solution into a culture medium according to the inoculation amount of 2% (v/v), and continuously culturing at 37 ℃ for 20h to obtain a bacterial solution; centrifuging the bacterial liquid at 12000g for 10min to obtain bacterial sludge; cleaning bacterial sludge with normal saline, and then putting the bacterial sludge into a protective agent to obtain bacterial suspension; finally freeze-drying the bacterial suspension to obtain bacterial powder with viable count of 1 × 10 11 CFU/g。
Respectively mixing the bacterial powder of the lactobacillus casei Zhang and the bifidobacterium lactis Probio-M8 bacterial strains according to the proportion of 1: mixing at a ratio of 1, adding maltodextrin, and diluting until viable count is 5 × 10 10 CFU/g, adding 1-20g, and weighing other adjuvants 1-50g, wherein the adjuvants can be galactooligosaccharide, inulin, stachyose or erythritol, maltitol and resistant dextrin. Weighing the freeze-dried powder of the composite strain according to the formula, uniformly mixing, adding the auxiliary materials, and uniformly mixing. Mixing the above materials to obtain Lactobacillus casei Zhang and Bifidobacterium lactisBacillus M8 composite powder sample.
Example 6 use of Complex strains
In this example, the lactobacillus casei Zhang and bifidobacterium lactis Probio-M8 composite strain can be used for preparing fermented milk.
Streaking the strain on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing for two continuous activation generations again at the temperature of 37 ℃ to obtain an activation solution; inoculating the activated solution into a culture medium according to the inoculation amount of 2% (v/v), and continuously culturing at 37 ℃ for 20h to obtain a bacterial solution; centrifuging the bacterial liquid at 12000g for 10min to obtain bacterial sludge; cleaning bacterial sludge with normal saline, and then putting the bacterial sludge into a protective agent to obtain bacterial suspension; finally freeze-drying the bacterial suspension to obtain bacterial powder with viable count of 1 × 10 11 CFU/g。
Mixing Lactobacillus casei Zhang and Bifidobacterium lactis Probio-M8 with basic starter at a certain ratio, making fermented milk, adding sucrose at a certain ratio into pure milk, heating to 65 deg.C, homogenizing under 18MPa-20MPa, sterilizing at 95 deg.C for 5min, and cooling; inoculating the starter at an inoculation amount of 0.03% (v/v) when the temperature of the sample is reduced to 37 ℃, and fermenting at 37 ℃; after the fermentation is finished, the mixture is refrigerated at 4 ℃ for 24 hours for after-ripening to obtain a fermented milk sample.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications derived therefrom are intended to be within the scope of the invention.
Claims (10)
1. A composite probiotic composition capable of inhibiting helicobacter pylori is characterized in that active ingredients of the composite probiotic composition comprise lactobacillus casei Zhang and bifidobacterium lactis Probio-M8;
the Lactobacillus casei Zhang is classified as Lactobacillus casei, and is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of China institute of academy of sciences, with the preservation number of CGMCC No.5469;
the Bifidobacterium lactis Probio-M8 is Bifidobacterium lactis with the classification name of CGMCC (China general microbiological culture Collection center) with the collection number of CGMCC No.18610.
2. The complex probiotic composition for inhibiting helicobacter pylori according to claim 1, wherein the total viable count of said lactobacillus casei Zhang and said bifidobacterium lactis Probio-M8 is not less than 1 x 10 6 CFU/mL or 1X 10 6 CFU/g。
3. The composite probiotic composition for inhibiting helicobacter pylori according to claim 1 or 2, wherein the ratio of viable count of lactobacillus casei Zhang to bifidobacterium lactis Probio-M8 is (1-10): (10-1).
4. A helicobacter pylori inhibiting probiotic preparation prepared from the complex probiotic composition of any one of claims 1 to 3 and/or an inactivated substance or metabolite thereof.
5. The probiotic formulation according to claim 4, characterized in that it comprises a liquid or solid formulation.
6. A method for preparing the probiotic preparation according to claim 4 or 5, characterized in that it comprises the steps of culturing the composite probiotic composition according to any one of claims 1 to 3, and processing the probiotic composition into a selected dosage form.
7. Use of a complex probiotic composition according to any one of claims 1 to 3, and the inactivates and metabolites thereof, for the preparation of a product for the prevention or treatment of helicobacter pylori infection.
8. Use according to claim 7, wherein the product comprises a pharmaceutical formulation;
the pharmaceutical preparation comprises powder, tablets or capsules.
9. Use according to claim 7, wherein the product comprises a functional food;
the functional food comprises dairy products, bean products or beverages.
10. The use of said lactobacillus casei Zhang and/or said bifidobacterium lactis Probio-M8 for the preparation of a probiotic preparation capable of inhibiting helicobacter pylori;
the Lactobacillus casei Zhang is classified as Lactobacillus casei, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms of China institute of academy of sciences, and has the preservation number of CGMCC No.5469;
the Bifidobacterium lactis Probio-M8 is Bifidobacterium lactis with the classification name of CGMCC (China general microbiological culture Collection center) with the collection number of CGMCC No.18610.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116751732A (en) * | 2023-07-11 | 2023-09-15 | 内蒙古农业大学 | Culture method for increasing number of living cells of cheese bacillus |
| CN116751732B (en) * | 2023-07-11 | 2024-05-24 | 内蒙古农业大学 | Culture method for increasing number of living cells of cheese bacillus |
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