CN114317310B - Antiallergic bifidobacterium infantis preparation and preparation method thereof - Google Patents
Antiallergic bifidobacterium infantis preparation and preparation method thereof Download PDFInfo
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Abstract
The invention relates to an antiallergic bifidobacterium infantis preparation and a preparation method thereof, and the bifidobacterium infantis (Bifidobacterium infantis) is a bifidobacterium infantis Miuyo-21 strain with a preservation number of CGMCC 20868. The bifidobacterium infantis preparation has large number of viable bacteria and high activity, contains metabolic products with antiallergic effect generated by probiotics, and has excellent antiallergic effect in human gastrointestinal tract.
Description
Technical Field
The invention belongs to the technical field of probiotic production, and particularly relates to an antiallergic bifidobacterium infantis preparation and a preparation method thereof.
Background
Allergy is a phenomenon in which an organism's susceptibility to some external stimulus (antigen) is abnormally increased, i.e. an abnormal, too high immune response, simply being oversensitive to some substance (antigen). The occurrence and development of allergic diseases are related to the immune system of the organism, wherein the type 2 immune response mechanism plays an important role in the allergic diseases, and can generate specific IgE antibodies aiming at allergens so as to lead the organism to be in a sensitized state. When the sensitized organism contacts the same allergen again, igE antibody adsorbed on the surfaces of the mast cells and the eosinophils is combined with the corresponding antigen to cause degranulation of the mast cells and release allergic mediums such as histamine and the like, so that allergic reaction is caused, the organism is subjected to tissue injury or dysfunction, common symptoms are abdominal pain, diarrhea and vomiting, or skin itching and difficult decoction are caused, and shock can be caused for more serious people.
For people with allergic constitution, the method of avoiding contact with allergen is adopted to reduce the occurrence of allergy, but the effect is very weak, and the allergen is avoided, so that the allergic symptoms can be temporarily reduced, but the allergic symptoms can not be radically cured. Thus, methods of inducing immune tolerance have emerged, which, although somewhat viable, are difficult to fully desensitize under current hygienic conditions.
A common immune organ in humans is a lymphoid organ, and T cells expressing CD4 in the gonorrhea are called helper T cells (Th). T helper cells can be subdivided into two major parts, depending on their function, or on their response to different cytokines and their ability to secrete cytokines, i.e. T helper cells can be divided into two types (Th 1 and Th 2). In general, th1 is beneficial for enhancing cellular immunity, while Th2 is beneficial for enhancing humoral immunity, with a dynamic balance between Th1 and Th2 normally present. And the two can mutually balance: th1 immune response is vigorous, th2 activity and function is inhibited and greatly reduced, and vice versa. The main effect of Th1 is to resist the stimulation of various microbes outside, and the more the external infection is, the more the Th1 is stimulated by microbes, and the more the Th1 is activated, the more the Th2 immune response is inhibited. Or the following: stimulation of a Th1 type immune response reduces the Th2 type immune response that can lead to allergy. Studies have shown that allergic diseases are associated with T helper cell imbalance and Th2 is too strong to cause allergy. Thus, stimulating a Th1 type immune response and thus reducing a Th2 type immune response will reduce the incidence of allergic reactions.
Allergic diseases have a significant trend to rise in the 80 s of the 20 th century, especially in western or mostly modern countries. Students find that long urban children in very clean and sanitary environments suffer from a much higher proportion of allergic diseases than long rural children in dirty environments, and thus find the relationship between the living environment of the person and the allergic diseases, thereby proposing a "sanitary hypothesis". "hygienic hypothesis" considers: too little exposure to pathogens such as bacteria and viruses during childhood results in insufficient stimulation of Th1 cells and thus failure to equilibrate with Th2 cells, ultimately leading to an allergic predisposition. From the "hygienic hypothesis" point of view, moderate microbial stimulation is considered necessary for the formation of normal immune functions and oral tolerance of the body, which is advantageous for inducing differentiation of Th0 cells into Th1 cells while suppressing Th2 cell responses, thereby playing an immune balance role. The more microorganisms that children are exposed to early, the lower the chance of developing allergic diseases later on. However, the presence of pathogenic microorganisms in the environment can cause serious epidemic diseases, and thus, there is a substantial risk of exposing children directly to the environmental microorganisms.
Then if certain non-pathogenic microorganisms can be utilized without the risk of truly infecting the disease, but can stimulate the relevant mechanisms of the hygiene hypothesis, thereby preventing the occurrence of allergic disease?
Clearly, probiotic therapy is based on this view. Probiotics are non-pathogenic microorganisms, but can stimulate the immune system of intestinal tracts, change the balance of Th1/Th2, enable immune response to be carried out towards the direction of Th1 cells, inhibit the reaction of Th2 cells and play a role in regulating immunity, thereby reducing the occurrence of allergic diseases. In addition, probiotics and their metabolites also have the effect of enhancing intestinal barrier function, reducing the penetration of food antigens through the intestinal mucosa, avoiding their exposure to the immune system. Most probiotics are nonpathogenic gram-positive bacteria, and the cell walls of the probiotics are mainly composed of Peptidoglycan (PG), bacterial Polysaccharide (PS) and lipoteichoic acid (LTA), and all have immunostimulating properties.
The invention patent application number 201610280825.2 'application of bifidobacterium infantis in food or medicine for preventing and treating food allergy' discloses bifidobacterium infantis capable of preventing and treating allergy and application of bifidobacterium infantis in food or medicine; liu Meng et al, 8 months of 2015, in the paper "study of the effect of bifidobacteria infantis on airway inflammation in asthmatic mice" published in journal of China microecology: the oral administration of the bifidobacterium infantis can relieve allergic symptoms, reduce inflammatory infiltration degree of lung tissues and inhibit Th2 immune response. These patents and papers provide theoretical basis for preventing and treating allergic diseases by using bifidobacterium infantis.
A large number of microorganisms are planted in human intestinal tracts, and the intestinal microorganisms have a very close relationship with human health and diseases. The bifidobacterium infantis exists in large quantity in the intestinal tract of breast-fed infants, has a plurality of benefits for infants, such as nutrition, immunity and anti-infection effects, and also has the effects of resisting allergy, resisting tumor, adjusting intestinal functions, improving nutrition and the like. Bifidobacterium infantis is a gram-positive bacterium, a non-motile, bifurcated rod-like, strictly anaerobic bacterium. The bifidobacterium infantis is used as a physiologically beneficial bacterium, and has various important physiological functions of biological barrier, nutrition, anti-tumor effect, immunity enhancement, gastrointestinal tract function improvement, aging resistance and the like for human health. Clinically, bifidobacteria have the effect of regulating intestinal dysfunction, preventing diarrhea and reducing constipation, namely bidirectional regulation, and the regulation can play a role in preventing and treating various intestinal diseases. However, since bifidobacterium infantis belongs to the obligate anaerobe, the requirements on nutrition conditions are very high, and the bifidobacterium infantis is acid-proof and has no motility, which brings great difficulty to industrial production.
At present, in the production industry of probiotics, liquid fermentation is almost adopted in the process of producing various probiotics bacterial powder (including bifidobacterium infantis bacterial powder), moisture is removed by centrifugal separation or other solid-liquid separation methods after the liquid fermentation is completed to obtain wet thalli, and then a drying protective agent or a carrier is added for uniform mixing, freeze drying, crushing, packaging and the like. The production method removes the metabolite produced by the probiotics in the liquid fermentation process, and the effect of the bifidobacterium infantis metabolite cannot be reflected in the product, so that the probiotic product produced by the method only utilizes the effect of the probiotics and cannot utilize the effect of the metabolite; in addition, the strain death rate of the thallus is high during drying, and the quantity of the thallus actually obtained after the liquid fermentation product is dried is reduced due to the addition of a proper amount of a drying protective agent or a carrier into the wet thallus.
Therefore, the existing probiotics production process has the defects of low strain content, poor fermentation effect, high bacterial death rate during drying, low strain activity in finished products and the like.
Disclosure of Invention
A first object of the present invention is to provide a bifidobacterium infantis.
A second object of the present invention is to provide the use of bifidobacteria infantis as described above, in particular in the manufacture of a medicament for the treatment of allergy.
A third object of the present invention is to provide a preparation comprising the above bifidobacterium infantis.
The fourth object of the present invention is to provide a method for preparing the preparation of bifidobacterium infantis, wherein the preparation of bifidobacterium infantis prepared by the method has a large number of viable bacteria and high activity, and contains metabolites produced by probiotics, so that the preparation has a better antiallergic effect in the gastrointestinal tract of human body.
In order to achieve the above object of the present invention, the present invention adopts the following technical scheme:
the invention provides a bifidobacterium infantis (Bifidobacterium infantis) strain which is a bifidobacterium infantis Miuyo-21 strain and is stored in the national institute of microbiology and microbiological culture Collection center of China general microbiological culture Collection center, with the preservation number of CGMCC 20868, of the North west road No. 1, no. 3, the Korean region of Beijing, 10 months and 12 days in 2020.
The invention also provides application of the bifidobacterium infantis (Bifidobacterium infantis) strain, in particular application in preparing antiallergic drugs.
The invention also provides an antiallergic preparation comprising the aforementioned bifidobacterium infantis (Bifidobacterium infantis) strain.
The preparation method of the antiallergic preparation comprises the following steps:
(1) Activating the bifidobacterium infantis Miuyo-21 strain, and then performing gradual expansion culture in a liquid culture medium to obtain a liquid culture product;
the expansion culture method can be carried out according to a conventional probiotics liquid culture method, and as an implementation scheme, bifidobacterium infantis is inoculated into a triangular flask liquid culture medium after being subjected to slant culture or flat culture activation, and is cultured for 24 hours under anaerobic conditions at 37 ℃ to obtain a triangular flask liquid strain; inoculating the triangular bottle liquid strain into fermentation tank liquidCulturing in a body culture medium at 37 ℃ under anaerobic condition for 24 hours to obtain a liquid culture product; the culture medium for slant culture or plate culture comprises the following raw material components in concentration: glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, agar 15g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 g/L, mgS0 4 .7H 2 00.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L; the triangular flask liquid culture medium and the fermentation tank liquid culture medium both comprise the following raw material components in concentration: glucose powder 20g/L, peptone powder 10g/L, beef extract 10g/L, yeast extract 5g/L, tomato sauce 10g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 1g/L, mgS0 4 .7H 2 0 0.5g/L、MnS0 4 .4H 2 00.05g/L, L-cysteine hydrochloride 0.5g/L.
(2) Carrying out solid-liquid separation on the liquid culture product and removing a liquid part to obtain bifidobacterium infantis wet thalli;
the specific mode of solid-liquid separation can be carried out according to a conventional liquid culture method of probiotics, for example, a tube centrifuge or a disc centrifuge can be adopted under the aseptic condition, when the two centrifuges are adopted for solid-liquid separation, the centrifugal speed can be controlled to 8000-12000r/min, the centrifugal time is controlled to 15-25min, and the centrifugal speed of 10000r/min is preferably adopted for 20min.
(3) Inoculating the bifidobacterium infantis wet thalli into a solid fermentation culture medium for culture, preferably at a culture temperature of 37 ℃, and drying, crushing, granulating and coating at a low temperature to obtain the antiallergic preparation.
The aim of inoculating the bifidobacterium infantis wet thalli into a solid fermentation culture medium for culture is that (1) the quantity and the vitality of thalli are greatly improved, and probiotics with high vitality are easier to colonize in intestinal tracts; (2) Obtaining the bifidobacterium infantis metabolite with antiallergic effect, including Peptidoglycan (PG), bacterial Polysaccharide (PS) and lipoteichoic acid (LTA). Compared with the bifidobacterium infantis preparation prepared by the traditional method, the bifidobacterium infantis preparation containing the metabolite of the invention has better antiallergic effect in human gastrointestinal tract.
As a preferred embodiment, it further comprises adjusting the water content of the inoculated solid fermentation medium to 45-55%. The water content is far lower than that of wet thallus obtained from liquid culture, so that the damage and death rate of dry solid culture to thallus are far lower than those of dry liquid culture, and the viable count of the final product can reach 10 11 cfu/g or more. Specifically, the solid culture medium after inoculation can be adjusted to reach the water content range by utilizing wet thalli, sterile water or water-containing materials such as liquid parts obtained by separation in the step (2).
As an embodiment, the solid fermentation medium comprises the following raw material components in parts by weight, based on 100 parts by weight of wet cells: glucose powder 5 weight portions, oligosaccharide powder 15 weight portions, peptone powder 10 weight portions, yeast extract powder 10 weight portions, tomato powder 10 weight portions, K 2 HP0 4 2 parts by weight, L-cysteine hydrochloride 0.5 parts by weight and food grade calcium carbonate 2 parts by weight. Further, the present inventors have found through a number of experiments that adding whey powder to a solid fermentation medium or replacing part of glucose with an appropriate amount of an oligosaccharide powder, such as isomaltose or fructooligosaccharide, has a certain benefit for obtaining a larger amount of bifidobacterium infantis with higher activity, and therefore, as a preferred embodiment, the solid fermentation medium comprises the following raw material components in parts by weight: glucose powder 5 weight portions, oligosaccharide powder 15 weight portions, peptone powder 10 weight portions, yeast extract powder 10 weight portions, whey powder 10 weight portions, tomato powder 10 weight portions, K 2 HP0 4 2 parts by weight, L-cysteine hydrochloride 0.5 parts by weight and food grade calcium carbonate 2 parts by weight.
All the raw materials of the solid fermentation culture medium are preferably food-grade or medical-grade powder raw materials, and are subjected to high Wen Ganre sterilization by powder high-temperature instant sterilization equipment (sterilization at 150 ℃ for 10 s) or sterilization equipment such as a high-temperature dry hot air sterilization box (high-temperature oven at 130 ℃ for 1 h) before use, and are subjected to aseptic cooling to 38 ℃ or lower for reuse. The low-temperature drying refers to freeze-drying at-40 ℃ or vacuum drying at 30-40 ℃, and preferably freeze-drying at-40 ℃. The steps of crushing, granulating and coating can be carried out by referring to the conventional granule preparation process.
The invention has the beneficial effects that:
1. the bifidobacterium infantis Miuyo-21 strain obtained by separation has the following characteristics:
(1) The culture and the growth are good under the environment with the pH value of 6.5-7.5, and the optimal growth pH value is pH7.0;
(2) The growth capacity is good under the culture temperature environment of 34-40 ℃, and the optimal growth temperature is 37 ℃;
(3) Has antiallergic effect.
2. The invention comprises a process preparation method combining liquid culture, solid-liquid separation and solid culture, fully utilizes the high liquid culture speed, the high number of wet strain thalli after the solid-liquid separation, and the solid culture can ensure that the strain activity and the number of bifidobacterium infantis are recovered and increased greatly, and can also produce metabolic products with antiallergic effect such as Peptidoglycan (PG), bacterial Polysaccharide (PS), lipoteichoic acid (LTA) and the like in the solid culture process. In addition, the solid culture medium added in the solid culture and the culture product thereof also have the function of a drying protective agent, and the strain produced by the solid culture has small stress during drying, low death rate during the drying of the strain, and the finally obtained product has high strain activity and contains a proper amount of metabolites with antiallergic effects, such as Peptidoglycan (PG), bacterial Polysaccharide (PS), lipoteichoic acid (LTA) and the like, so that the product has better antiallergic capability in the actual use process.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, based on the examples herein, which are within the scope of the invention as defined by the claims, will be within the scope of the invention as defined by the claims.
Example 1 isolation and characterization of Strain
1.1 sample collection
Healthy infants, which have not recently used antibacterial drugs 3 months to 1 year after birth, are selected, and samples are taken from their faeces, i.e. the strain collected is from the intestinal tract of the healthy infant.
1.2 isolation of strains
Taking infant feces sample 5-10g, placing into a sterile 50ml centrifuge tube, adding 20ml sterile physiological saline, shaking, mixing, placing into an anaerobic incubator at 37deg.C, standing for culturing overnight, sucking 1ml sample solution, sequentially diluting with 10 times of sterile physiological saline to 10 times -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Each gradient is respectively coated with 100 mu l of bacterial suspension on an improved MRS flat plate, placed in an anaerobic incubator at 37 ℃ for inversion culture, colonies are selected from coated culture flat plates with different gradients for transfer culture after 24 hours, on the basis, colonies conforming to the typical characteristics of bifidobacterium infantis are selected, repeated streak culture separation is carried out on the improved MRS flat plate, and single colonies conforming to the growth form of the bifidobacterium colonies are selected and inoculated into an improved MRS liquid culture medium for culture in the anaerobic incubator at 37 ℃. Finally, a strain with good growth performance is obtained.
1.3 identification of strains
(1) Colony morphology and physiological biochemical experimental analysis
The bacterial colony of the strain is milky white, round or star-shaped, small in size, smooth in surface, fine and branched in shape. No movement, negative H2O2 enzyme activity test and negative hydrogen sulfide test; gram positive staining; the growth is good at pH6.5-7.5, and the most suitable pH is 7.0; the optimal growth temperature is 34-40 ℃ and 37 ℃; can ferment glucose, sucrose, maltose, fructose, raffinose, lactose, galactose, xylose, melibiose, sorbitol, mannitol, etc.; acid and no gas are produced when glucose is fermented.
(2) 16S rRNA partial sequence analysis
Genomic DNA of the selected strain was amplified by PCR and detected by gel electrophoresis using 1% agarose. The PCR amplification was performed using bacterial universal primer 27F (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R (5'-GGTTACCTTGTTACGACTT-3'), the PCR product was submitted to the gene technology service company for purification and sequencing, and the obtained PCR product sequence was subjected to homology comparison in GenBank by BLAST, and strain identification was performed.
The strain was identified as Bifidobacterium infantis by combining the results of colony morphology analysis, physiological biochemical experimental analysis and 16S rRNA partial sequence analysis, and was named "Bifidobacterium infantis Miuyo-21".
1.4 characteristics of bifidobacterium infantis Miuyo-21
The bifidobacterium infantis has the following characteristics:
(1) The growth is good under the environment with pH of 6.5-7.5, and the pH is optimally 7.0.
(2) The growth is good under the temperature environment of 34-40 ℃, and the optimal growth temperature is 37 ℃;
(3) The in vivo experiment has antiallergic effect.
These features are described in detail below:
1. is suitable for growth under neutral pH environment
The bifidobacterium infantis Miuyo-21 grows well in culture in the environment of pH6.5-7.5, and the optimal growth pH is pH7.0.
The experimental method is as follows:
inoculating activated bifidobacterium infantis Miuyo-21 strain into an improved MRS liquid culture medium (namely adding 0.5 g/L-cysteine hydrochloride on the basis of the MRS liquid culture medium), culturing to obtain strain seed liquid, inoculating 1ml of bifidobacterium infantis Miuyo-21 seed liquid into 19ml of improved MRS liquid culture medium with pH of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0 respectively, anaerobically culturing for 24 hours at 37 ℃, and determining OD600 values (namely absorbance values at 600nm wavelength) after the initial and the final steps, wherein the OD600 values can be used for comparing cell densities or cell growth in the culture liquid.
The initial and final OD600 values were measured separately, and the cell growth in the different pH media was calculated as follows, using the OD600 value at pH7.0 as a control (i.e., the number of cells cultured at pH7.0 was 100%):
cell growth (%) = (OD of other pH culture)/OD of pH7.0 culture×100%
The results are shown in Table 1:
TABLE 1 growth of Bifidobacterium infantis Miuyo-21 in different pH Medium
| pH | Blank control (without culture) | 5.0 | 5.5 | 6.0 | 6.5 | 7.0 | 7.5 | 8.0 |
| Growth of cells (%) | 0.1 | 30.3 | 80.5 | 85.6 | 95.5 | 100 | 95.3 | 83.2 |
The results in table 1 show that: the experiment was performed with the OD of the bacterial liquid grown at the initial pH7.0 as a control (i.e., 100%) and the bacterial liquid grown in the medium at other pH's, although lower than the OD of the bacterial liquid grown in the medium at pH7.0, still had a higher growth rate than the control without the culture.
2. Is suitable for growth at 37 DEG C
The optimal growth temperature is 37 ℃, and the growth is good under the temperature environment of 34-40 ℃.
The experimental method is as follows:
inoculating activated bifidobacterium infantis Miuyo-21 strain into an improved MRS liquid culture medium (adding L-cysteine hydrochloride 0.5 g/L), culturing to obtain strain seed liquid, inoculating 1ml of bifidobacterium infantis Miuyo-21 seed liquid into 19ml of improved MRS liquid culture medium with pH of 7.0, respectively, anaerobically culturing at 31 ℃, 34 ℃,37 ℃, 40 ℃ and 43 ℃ for 24 hours, and measuring OD600 values (namely absorbance values at 600nm wavelength, which can be used for comparing cell density or cell growth in the culture liquid) after initial and final culture.
The initial and final OD600 values were determined separately, and the control was performed at 37℃OD600 (i.e., at 37 DEG C
The number of cultured cells was 100%), the growth of cells in the different pH media was calculated according to the following formula:
cell growth (%) = (number of cells cultured at other temperature)/number of cells cultured at 37℃multiplied by 100%
The results are shown in Table 2:
TABLE 2 growth of Bifidobacterium infantis Miuyo-21 at different temperatures
| Temperature (. Degree. C.) | Control (not culture) | 31 | 34 | 37 | 40 | 43 |
| Growth of cells (%) | 0 | 70.3 | 89.5 | 100 | 91.3 | 75.2 |
The results in table 2 show that: the bifidobacterium infantis Miuyo-21 has good growth capacity in a culture temperature environment of 34-40 ℃, and the optimal growth temperature is 37 ℃.
Example 2 preparation of Bifidobacterium infantis preparation (liquid method control)
2.1 Medium
(1) Improved MRS solid medium (for slant culture and plate culture, used in isolation and preservation of strain):
glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, agar 15g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 g/L, mgS0 4 .7H 2 00.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L. The pH was adjusted to 7.0 with 1mol/L NaOH at 121℃highSterilizing for 20min, and cooling to 40deg.C, and chamfering or plating.
(2) Improved MRS liquid medium (for delta flask culture and fermenter culture):
glucose powder 20g/L, peptone powder 10g/L, beef extract 10g/L, yeast extract 5g/L, tomato sauce 10g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 g/L, mgS0 4 .7H 2 0 0.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L. Adjusting pH to 7.0 with 1mol/L NaOH, and sterilizing at 121deg.C for 20min.
2.2 preparation method of the preparation
The preparation method of the bifidobacterium infantis preparation of the embodiment comprises the following steps:
(1) Activating strains:
the modified MRS solid medium is used for slant culture or plate culture of strain.
(2) Liquid culture:
inoculating the strain obtained by slant or plate culture into a triangle bottle modified MRS liquid culture medium, and performing liquid anaerobic culture at 37 ℃ for 24 hours to obtain a triangle bottle liquid strain; inoculating the triangular flask liquid strain into an improved MRS liquid culture medium of a fermentation tank, and performing liquid anaerobic culture at 37 ℃ for 24 hours to obtain a liquid culture product;
(3) Solid-liquid separation:
performing solid-liquid separation on the liquid culture product by using a tube centrifuge or a disc centrifuge, wherein the rotation speed of the centrifuge is 10000r/min, the centrifugation time is 20min, and wet thalli with the moisture content of 80% are obtained;
(4) And (3) drying:
and (3) placing the wet thalli in a freeze dryer, and freeze-drying for 20 hours at the temperature of minus 40 ℃ to obtain the bifidobacterium infantis raw powder prepared by a liquid method. The measurement is as follows: the total bacterial count of the bifidobacterium infantis raw powder product prepared by the example is 2.23 multiplied by 10 11 CFU/g。
(5) Granulating, coating and packaging.
Adding proper amount of auxiliary materials (such as trehalose, skimmed milk powder, etc.) into the preparation obtained in the step (4), and granulating to obtain the Bifidobacterium infantis preparation prepared by a liquid method.
The measurement is as follows: the bifidobacterium infantis preparation product prepared in this example has a total bacterial count of 1.35X10 11 CFU/g。
Example 3 preparation of Bifidobacterium infantis preparation (solid state method 1)
3.1 Medium
(1) Improved MRS solid medium (for slant culture and plate culture, used in isolation and preservation of strain):
glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, agar 15g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 g/L, mgS0 4 .7H 2 00.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L. Regulating pH to 7.0 with 1mol/L NaOH, sterilizing at 121deg.C for 20min, and cooling to about 40deg.C.
(2) Improved MRS liquid medium (for delta flask culture and fermenter culture):
glucose powder 20g/L, peptone powder 10g/L, beef extract 10g/L, yeast extract 5g/L, tomato sauce 10g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 1g/L, mgS0 4 .7H 2 0 0.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L. Adjusting pH to 7.0 with 1mol/L NaOH, and sterilizing at 121deg.C for 20min.
(3) Culture medium for solid culture (for solid culture):
the solid-state culture materials were prepared according to the following formulation ratio:
100g of wet thalli, 20g of glucose powder, 10g of peptone powder, 10g of yeast extract powder, 10g of tomato powder and K 2 HP0 4 2g, L-cysteine hydrochloride 0.5g and food grade calcium carbonate 2g.
Except for wet bacteria, the other raw materials are sterilized respectively (sterilized respectively by high-temperature instantaneous sterilization equipment at 150 ℃ for 10 s), and are mixed uniformly after cooling, and are used as raw materials of a solid culture medium for standby.
3.2 preparation method of preparation
The preparation method of the bifidobacterium infantis preparation of the embodiment comprises the following steps:
(1) Activating strains:
the modified MRS solid medium is used for slant culture or plate culture of strain.
(2) Liquid culture:
inoculating the strain obtained by slant or plate culture into a triangle bottle modified MRS liquid culture medium, and performing liquid anaerobic culture at 37 ℃ for 24 hours to obtain a triangle bottle liquid strain; inoculating the triangular flask liquid strain into an improved MRS liquid culture medium of a fermentation tank, and performing liquid anaerobic culture at 37 ℃ for 24 hours to obtain a liquid culture product;
(3) Solid-liquid separation:
performing solid-liquid separation on the liquid culture product by using a tubular centrifuge or a disc centrifuge, wherein the rotation speed of the centrifuge is 10000r/min, the centrifugation time is 20min, and wet thalli with the moisture content of 80% are obtained;
(4) Solid state culture:
the wet cells were mixed with a sterile solid medium in a sterile room, and the water content of the resulting mixture was adjusted to 50%. Then placing the mixture in an anaerobic solid state fermentation tank, and sealing the anaerobic solid state fermentation tank at 37 ℃ for 72 hours.
(5) And (3) drying:
vacuum drying the solid culture product obtained in the step (4) at 30 ℃ for 10 hours to obtain the bifidobacterium infantis raw powder prepared by the solid method (1); the measurement is as follows: the total bacterial count of the bifidobacterium infantis raw powder product prepared by the example is 2.87 multiplied by 10 11 CFU/g。
(6) Granulating, coating and packaging.
Granulating and coating the bifidobacterium infantis raw powder obtained in the step (5) to obtain the bifidobacterium infantis preparation.
The measurement is as follows: the bifidobacterium infantis preparation product prepared in the example has a total bacterial count of 2.35 multiplied by 10 11 CFU/g。
Example 4 preparation of Bifidobacterium infantis preparation (solid state method 2)
The process operation of example 4 was essentially the same as that of example 3, with only a portion of the operating parameters being modified to demonstrate the effect of producing a solid state cultured probiotic product under different operating conditions.
4.1 Medium
The only difference from example 3 is that: the medium composition of example 4 was varied to increase the whey powder by 10g.
4.2 preparation method
The solid state incubation times were different, the solid state incubation time of example 4 was 96 hours; the drying was carried out in a different manner, and example 4 was carried out by freeze-drying at-40℃for 20 hours.
Other steps are the same as in example 3, and a raw powder product and a preparation product are respectively prepared.
The measurement is as follows: the total bacterial count of the bifidobacterium infantis raw powder product prepared in the embodiment is 3.45 multiplied by 10 11 CFU/g; the total bacterial count of the preparation product obtained by preparing the microbial inoculum into the preparation is 2.91 multiplied by 10 11 CFU/g。
Example 5 preparation of Bifidobacterium infantis preparation (solid state method 3)
The process operation of example 5 was essentially the same as example 3, with only a portion of the operating parameters being modified to demonstrate the effect of producing a solid state cultured probiotic product under different operating conditions.
5.1 Medium
The only difference from example 3 is that the solid medium of example 5 replaces part of the glucose (15 g) with isomaltooligosaccharide (15 g); namely, the carbon source of example 3 was 20g of glucose, and the carbon source of example 5 was 15g of glucose 5 g+isomaltooligosaccharide.
5.2 preparation method
The difference from example 3 was only that the culture temperature was different, and the culture temperature was 35℃for both the liquid culture and the solid culture in example 5, and the solid culture time was 96 hours.
Other steps are the same as in example 3, and a raw powder product and a preparation product are respectively prepared.
The measurement is as follows: the total bacterial count of the bifidobacterium infantis raw powder product prepared in the embodiment is 3.51X10 11 CFU/g; the total bacterial count of the preparation product obtained by preparing the microbial inoculum into the preparation is 2.93 multiplied by 10 11 CFU/g. Example 6 validation of antiallergic Effect of Bifidobacterium infantis preparation
6.1 experimental materials:
test animals: SPF-class Balb/c female mice were selected for 5-6 weeks of age (body weight 18-20 g).
Crude peanut proteins (crude peanut extract protein, CPE) were self-produced in the literature (Liu Zhigang et al, establishment of Balb/c mouse peanut allergy model and pathogenesis, university of Shenzhen journal, published, 2012 (2)).
Phosphate Buffered Saline (PBS), pH 7.2-7.4.
6.2 antiallergic effects of probiotic products at the same weight concentration
6.2.1 doses and groupings:
each group of mice is sensitized by using a blank control group, a model group or a positive control group and the other groups as treatment groups respectively, and is treated by using the crude peanut protein with the same content, and the bifidobacterium infantis preparation prepared by the method is used for lavage, and the specific grouping method is as follows:
blank control group: the PBS buffer solution is adopted for injection, so that the bifidobacterium infantis is not sensitized and is not treated by the bifidobacterium infantis preparation;
model group or positive control group: the peanut crude protein is sensitized, but the bifidobacterium infantis preparation is not used for treatment, and the mice have obvious sensitization symptoms;
example 2 (conventional liquid method control) group: sensitization with crude peanut protein, but treatment with bifidobacterium infantis formulation (0.1 g dose/dose) produced in example 2 (traditional liquid method);
example 3 (solid state method 1) group: priming with crude peanut protein, but with bifidobacterium infantis formulation produced in example 3 (solid state method 1) (0.1 g dose/time) for intragastric treatment;
example 4 (solid state method 2) group: intragastric treatment with crude peanut protein but with bifidobacterium infantis formulation produced in example 4 (solid state method 2) (0.1 g dose/time);
example 5 (solid state method 3) group: intragastric treatment with crude peanut protein but with bifidobacterium infantis formulation produced in example 5 (solid state method 3) (0.1 g dose/time);
6.2.2 sensitization and challenge in mice:
balb/c mice were purchased 10% -20% more than actually needed. Firstly, 10 mice are separated out to be used as a blank control group, and the blank control group is injected by PBS buffer solution and is not sensitized; all other mice were sensitized by injection with crude peanut protein solution.
Sensitization pattern in Balb/c mice of each group:
the model group was sensitized twice by subcutaneous injection, 3 weeks apart, with 100 μg of crude peanut protein and 1mg of aluminum hydroxide adjuvant per injection; after 1 week of immunization 2, 200 μg of crude peanut protein was injected subcutaneously for priming;
allergic mice were then selected and randomly grouped into 10 mice per group, with excess mice discarded. Allergic mice were subjected to intragastric treatment with probiotic solutions daily according to the samples and doses of table 3, and continuously intragastric administration was performed for 10 days, 2 times daily, and the blank control group and the positive control group were administered with sterile purified water.
The blank group replaced crude peanut protein with PBS solution of the same volume of solution, and the other steps were the same.
Detection of total IgE antibodies in 6.2.3 serum:
after the bifidobacterium infantis preparation feeding experiment is finished, blood is taken from each group of mice, the mice are kept stand at room temperature for 2 hours, and the mice are centrifuged at 4000r/min for 10 minutes, and the upper serum is carefully taken out. The content of total IgE antibodies in serum is detected by ELISA reagent, and the detection is carried out according to the specification of the product.
6.2.4 experimental results:
the experimental results of the effect of bifidobacterium infantis formulation on the prevention and treatment of allergy in mice are shown in table 3:
TABLE 3 control Effect of Bifidobacterium infantis formulations produced by different technologies on allergy of mice
Note that: the symptoms of the allergy of the mice are observed by naked eyes, and the symptoms of the allergy of the mice are obviously reduced or healed by naked eyes, so the mice are classified as symptoms are reduced or obvious.
IgE-mediated allergic reactions are the main cause of allergy, and IgE antibodies in serum are key contributing factors to the development of allergic reactions.
From the experimental results in table 3, it can be seen that: the total IgE antibody content in the serum of mice in the blank control group and the model group corresponds to the IgE content of mice, and the allergic symptoms are that: the mice in the blank group with no allergic symptoms had a lower serum IgE antibody content, while the mice in the model group with allergic symptoms had a higher serum IgE antibody content. After the same weight of probiotics is used for lavage treatment, the content of IgE antibodies in each probiotic treatment group is increased; the method has the advantages that the allergic symptoms are correspondingly improved or relieved, and a good therapeutic allergic effect is shown, wherein the liquid-state-method-produced probiotic product only has 6 allergic symptoms improved or relieved in 10 mice, namely only has 60% of the allergic symptoms improved, and compared with the liquid-state-method-produced probiotic product, the liquid-state-method-produced probiotic product has a better antiallergic effect.
6.3 antiallergic Effect of the preparation product at the same bacterial count
The same weight of probiotics was used in each example in table 3, but since the total number of probiotics was different in each example, the number of bacteria and the metabolite content were different, and although the antiallergic effect of each example was seen, the effect of the metabolite in each example could not be distinguished. For this purpose, the following examples were carried out by adjusting the number of bacteria, and using samples of preparations having the same number of probiotics to conduct experiments and determine the antiallergic effect. By comparing the antiallergic differences between the examples, the antiallergic effects of the metabolites of the probiotics can be compared since the number of probiotics is the same.
6.3.1 dose and grouping:
preparation of probiotic experimental samples: respectively weighing 1g of probiotic preparation, adding appropriate amount of sterile water, measuring bacterial count, and adding sterile water to adjust total probiotic concentration to 1×10 10 CFU/ml, ready for use.
6.3.2 doses and groupings:
each group of mice is sensitized by using a blank control group, a model group or a positive control group and the other groups as treatment groups respectively, and the mice are sensitized by using the crude peanut proteins with the same content, and the bifidobacterium infantis preparation prepared by the invention is used for gastric lavage treatment after the bacterial count is regulated, and the specific grouping method comprises the following steps:
blank control group: the PBS buffer solution is adopted for injection, so that the bifidobacterium infantis is not sensitized and is not treated by the bifidobacterium infantis preparation;
model group or positive control group: the peanut crude protein is sensitized, but the bifidobacterium infantis preparation is not used for treatment, and the mice have obvious sensitization symptoms;
example 2 (conventional liquid method control) group: bifidobacterium infantis preparation (1×10) sensitized with crude peanut protein but produced by example 2 (conventional liquid method) 10 CFU/time) gastric lavage treatment;
example 3 (solid state method 1) group: bifidobacterium infantis preparation (1×10) sensitized with crude peanut protein but produced by example 3 (solid state method 1) 10 CFU/time) gastric lavage treatment;
example 4 (solid state method 2) group: bifidobacterium infantis preparation (1×10) sensitized with crude peanut protein but produced by example 4 (solid state method 2) 10 CFU/time) gastric lavage treatment;
example 5 (solid state method 3) group: bifidobacterium infantis preparation (1×10) sensitized with crude peanut protein but produced by example 5 (solid state method 3) 10 CFU/time) gastric lavage treatment;
mice were sensitized and challenged as in example 6.2.1.
Allergic mice were then selected and randomly grouped into 10 mice per group, with excess mice discarded. Allergic mice were subjected to intragastric treatment with the same bacterial count of probiotic solution daily according to the samples and doses of table 4, and were continuously intragastric for 10 days, 2 times daily, and the blank control group and the positive control group were perfused with sterile purified water.
The blank group replaced crude peanut protein with PBS solution of the same volume of solution, and the other steps were the same.
6.3.3 detection of total IgE antibodies in serum:
after the bifidobacterium infantis preparation feeding experiment is finished, blood is taken from each group of mice, the mice are kept stand at room temperature for 2 hours, and the mice are centrifuged at 4000r/min for 10 minutes, and the upper serum is carefully taken out. The content of total IgE antibodies in serum is detected by ELISA reagent, and the detection is carried out according to the specification of the product.
6.3.4 experimental results:
the experimental results of the effect of bifidobacterium infantis formulation on the prevention and treatment of allergy in mice are shown in table 4:
TABLE 4 control Effect of Bifidobacterium infantis formulations produced by different technologies on allergy of mice
Note that: the symptoms of the allergy of the mice are observed by naked eyes, and the symptoms of the allergy of the mice are obviously reduced or healed by naked eyes, so the mice are classified as symptoms are reduced or obvious.
From the experimental results in table 4, it can be seen that: after the probiotics with the same bacterial count are adopted for treatment, the probiotic sample prepared by the solid state method has better antiallergic effect than the probiotic sample prepared by the liquid state method. The reason for this difference may be due to the same number of probiotics used: the probiotic sample produced by the liquid state method is directly added with auxiliary materials for drying after centrifugation, and the solid state rule is that the solid state culture is carried out for the second time after the solid state culture medium is added, so that the activity of the probiotics is enhanced in the solid state culture process, and metabolic products with antiallergic effect are produced in the growth process, thereby increasing the antiallergic effect of the product.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any person skilled in the art will readily recognize that variations or substitutions are within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (9)
1. The bifidobacterium infantis (Bifidobacterium infantis) strain is characterized by being a bifidobacterium infantis Miuyo-21 strain with a preservation number of CGMCC 20868;
the bifidobacterium infantis strain is obtained by culturing and separating the bifidobacterium infantis strain in the following steps:
taking infant feces sample 5-10g, placing into a sterile 50ml centrifuge tube, adding 20ml sterile physiological saline, shaking, mixing, placing into an anaerobic incubator at 37deg.C, standing for culturing overnight, sucking 1ml sample solution, sequentially diluting with 10 times of sterile physiological saline to 10 times -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 Each gradient is respectively coated with 100 mu l of bacterial suspension on an improved MRS flat plate, the improved MRS flat plate is placed in an anaerobic incubator at 37 ℃ for inversion culture, colonies are selected from coated culture flat plates with different gradients for transfer culture after 24 hours, on the basis, colonies conforming to the typical characteristics of bifidobacterium infantis are selected, repeated streak culture separation is carried out on the improved MRS flat plate, single colonies conforming to the growth form of the bifidobacterium colonies are selected and are connected into an improved MRS liquid culture medium, and the strain with good growth performance is finally obtained after the culture is cultured in the anaerobic incubator at 37 ℃;
the improved MRS liquid culture medium comprises the following components: glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, agar 15g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 g/L, mgS0 4 .7H 2 0 0.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L.
2. Use of a bifidobacterium infantis (Bifidobacterium infantis) strain as claimed in claim 1 in the manufacture of an antiallergic medicament.
3. An antiallergic preparation, characterized in that it comprises the bifidobacterium infantis (Bifidobacterium infantis) strain of claim 1.
4. A method of preparing an antiallergic agent according to claim 3, comprising the steps of:
(1) Activating the bifidobacterium infantis Miuyo-21 strain, and then performing gradual expansion culture in a liquid culture medium to obtain a liquid culture product;
(2) Carrying out solid-liquid separation on the liquid culture product and removing a liquid part to obtain bifidobacterium infantis wet thalli;
(3) Inoculating the bifidobacterium infantis wet thalli into a solid fermentation culture medium for culture, and drying at low temperature, crushing, granulating and coating to obtain the antiallergic preparation.
5. The method for preparing an antiallergic agent according to claim 4, wherein in the step (1), bifidobacterium infantis is inoculated into a liquid culture medium of a triangular flask after being activated by slant culture or plate culture, and is cultured for 24 hours under anaerobic conditions at 37 ℃ to obtain a liquid strain of the triangular flask; inoculating the triangular flask liquid strain into a liquid culture medium of a fermentation tank for culturing, and culturing for 24 hours at 37 ℃ under anaerobic conditions to obtain a liquid culture product;
the culture medium for slant culture or plate culture comprises the following raw material components in concentration: glucose 20g/L, peptone 10g/L, beef extract 10g/L, yeast extract 5g/L, agar 15g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 g/L, mgS0 4 .7H 2 0 0.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L;
the triangular flask liquid culture medium and the fermentation tank liquid culture medium both comprise the following raw material components in concentration: glucose powder 20g/L, peptone powder 10g/L, beef extract 10g/L, yeast extract 5g/L, tomato sauce 10g/L, K 2 HP0 4 2g/L, diammonium hydrogen citrate 2g/L, sodium acetate 5g/L, tween-80 1g/L, mgS0 4 .7H 2 0 0.5g/L、MnS0 4 .4H 2 0.05g/L, L-cysteine hydrochloride 0.5g/L.
6. The method for producing an antiallergic agent according to claim 4, wherein in the step (3), the solid fermentation medium comprises, based on 100 parts by weight of wet cells, the following parts by weightIs prepared from the following raw materials: glucose powder 5 weight portions, oligosaccharide powder 15 weight portions, peptone powder 10 weight portions, yeast extract powder 10 weight portions, tomato powder 10 weight portions, K 2 HP0 4 2 parts by weight, L-cysteine hydrochloride 0.5 parts by weight and food grade calcium carbonate 2 parts by weight.
7. The method for producing an antiallergic agent according to claim 4, wherein in the step (3), the solid fermentation medium comprises the following raw material components in parts by weight, based on 100 parts by weight of wet cells: glucose powder 5 weight portions, oligosaccharide powder 15 weight portions, peptone powder 10 weight portions, yeast extract powder 10 weight portions, whey powder 10 weight portions, tomato powder 10 weight portions, K 2 HP0 4 2 parts by weight, L-cysteine hydrochloride 0.5 parts by weight and food grade calcium carbonate 2 parts by weight.
8. The method for producing an antiallergic agent according to claim 4, further comprising adjusting the water content of the inoculated solid fermentation medium to 45-55% in step (3).
9. The method of preparing an antiallergic agent according to claim 4, wherein in both step (1) and step (3), the temperature of the culture is 37 ℃.
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| CN105018379A (en) * | 2015-07-16 | 2015-11-04 | 山东凤凰生物有限公司 | Lactobacillus plantarum with high antioxidant activity and application of lactobacillus plantarum |
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