CN112852670B - Bifidobacterium infantis DH231 and fermentation product, application and microecological preparation thereof - Google Patents
Bifidobacterium infantis DH231 and fermentation product, application and microecological preparation thereof Download PDFInfo
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- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
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Abstract
The invention relates to the field of biological medicines, and particularly relates to bifidobacterium infantis DH231, and a fermentation product, application and a microecological preparation thereof. The preservation number of the bifidobacterium infantis is CGMCC No. 18372. The bifidobacterium infantis DH231 microecological preparation has obvious curative effects of preventing and treating autism, resisting allergy, improving immunity and the like, and has quick response, good effect and no adverse reaction or side effect compared with other treatment modes. Can be used as medicine, veterinary medicine, feed additive, etc., and has great social significance.
Description
Technical Field
The invention relates to the field of biological medicines, and particularly relates to bifidobacterium infantis DH231, and a fermentation product, application and a microecological preparation thereof.
Background
Currently, the incidence of diseases such as autism and allergy is increasing year by year with the change of living environment.
Autism
Autism is a neurodevelopmental disorder, and Autism Spectrum Disorder (ASD) is a generic term for such disorders. The incidence rate of the ASD increases year by year, and statistical data issued by the us disease prevention and control center in 2014 show that the prevalence rate of the ASD in the us child in 2010 reaches 14.7 per thousand, which is equivalent to 1 ASD in 68 children. Currently, there is no effective method for treating autism core disorder. However, doctors, researchers, and parents of children patients have tried various treatment methods and accumulated much experience. The common treatment methods include behavioral intervention, special education, medication, biomedical intervention, psychological intervention, and the like. However, most of these methods lack a complete and effective evaluation of safety and efficacy, and still require more systematic studies and evaluations.
Statistically, about 50% to 70% of patients with autism will use biologically relevant therapies, but most lack a complete and effective assessment of safety and efficacy, and still require more systematic studies and assessments. With the development of research on the relationship between human microorganisms and autism, the corresponding intervention or treatment methods, such as food intervention, probiotic and fecal bacteria transplantation, gradually show unique safety and effectiveness, and are biological intervention methods with wide application prospects.
In 1998, it was reported that secretin (secretin) was used to intervene in autism and achieved a certain effect, but since only three samples were available, the subsequent studies found that secretin was not effective and there was a possible placebo effect, and thus, it was not promoted. At present, no specific medicine for autism exists. Among them, risperidone (risperidone) and aripiprazole (aripiprazole) are drugs for autism which are only approved by the Food and Drug Administration (FDA) and only used for children aged 5 to 16 years, and other selective 5-HT reuptake inhibitors citalopram (citalopram), escitalopram (escitalopram) and fluoxetine (fluoxetine) and the like can be used for treating serious behavior problems such as self-injury behavior and repetitive behavior. Although doctors prescribe patients with autism, the medication does not provide substantial treatment of the core deficits of autism, i.e., social and communication disorders. These drugs are also less effective than other diseases, have certain side effects, still lack sufficient clinical experience in terms of children's medication, and therefore, it is necessary to limit the large-scale and long-term medication of patients with autism.
After some of the intestinal symptoms of autism have been discovered, attempts have been made to treat the intestinal symptoms with antibiotics, which also help with the symptoms of autism. Some autistic patients have some therapeutic effect after oral administration of two vancomycin (vancomy) and metronidazole (metronidazole or flagyl) which are widely used for anaerobic infections. Among them, vancomycin may act mainly by affecting gram-positive anaerobes, and high concentrations of vancomycin also eliminate clostridium difficile and most gram-negative anaerobes. However, it was found that after vancomycin withdrawal, there was a gradual regression of intestinal and behavioral symptoms in all subjects. Vancomycin is effective for autism therapy for a short period of time, with repetition once discontinued, and with each interruption during each course of treatment, with the possibility that certain microorganisms in the gut contribute to autism symptoms. Oral vancomycin cannot enter a blood system and is hardly absorbed by a human body, but mainly plays a role by influencing intestinal microorganisms, spores possibly generated by the intestinal microorganisms are repeated after the medicine is stopped, when the vancomycin is stopped, the spores are activated into bacteria to continuously influence the intestinal tract and the nervous system, and clostridium capable of generating the spores is most doubtful. In addition, vancomycin and gentamicin (gentamycin), which are commonly used in serious diseases when other antibiotics are ineffective, are considered to be the last line of defense against drug-resistant bacteria, which are likely to cause bacterial resistance upon misuse. Therefore, such antibiotics should be carefully selected for use in the treatment of autism.
Many environmental factors affect the gut-brain axis, especially the daily food intake of an individual. Studies have shown that food can affect the composition of the intestinal flora. For example, in the gut flora of breast-fed infants, where clostridia and escherichia coli are significantly lower than in formula-fed infants, dietary emulsifiers may also alter the localization, composition and pro-inflammatory potential of the gut microbiota. Changes in the microbial flora caused by diet may affect serum metabolism and modulate brain activity in an individual. Thus, certain foods may restore the microbial-gut-brain axis balance and have therapeutic effects on the defects associated with autism spectrum disorders. Although there have been a number of related studies showing that GF/CF food intervention has a better effect on the symptoms of autism, the specific mechanism of action is not yet well understood. The theory of opioid-overdose suggests that ingestion of gluten and casein by children may have adverse effects on their behavior and development, or that food may cause gastrointestinal discomfort when it enters the intestine, due to allergy to food or the intrinsic abdominal disease, in autistic children, and that it is likely that the child exhibits inappropriate behavior that is a reaction to gastrointestinal discomfort. This therapy is very popular in the world, but its mechanism of action is still unclear, and it is not effective in all autistic children, and may cause side effects such as malnutrition, and thus is questioned.
Second, allergic diseases
By 2015, 40-50% of people worldwide have had allergies, and WHO has classified allergic diseases as one of three major diseases that need intensive research and control in the 21 st century. According to statistics, the incidence rate of allergic diseases in China reaches 37.3%, and the main influence group is infants. The search for effective allergy prevention measures is urgent. The current more common approach to the treatment of allergic diseases is the use of drug therapy. Such as histamine antagonists, which inhibit histamine responses by blocking histamine receptors, and steroids and drugs which inhibit the release of inflammatory substances from mast cells, bronchodilator drugs, etc. However, these drugs only provide temporary relief of symptoms, do not really change the allergic immune response, and treat the symptoms and not the root cause.
Allergen-specific desensitization therapy is the only cause therapy for allergic diseases recognized by the world health organization, and the simple principle is that substances allergic to patients are injected into the patients intermittently and subcutaneously according to a certain rule, the dosage is increased from small to large, so that the patients can tolerate the allergens gradually, and no allergy phenomenon or allergy alleviation is generated any more when the allergens are contacted again. Desensitization treatment is a long-term treatment scheme, and has the advantages of slow response, long treatment course and good curative effect, and patients need to be subjected to heart-tolerant combination treatment. Generally, the treatment lasts for 3-4 months, and a maintenance course of treatment lasts for 2-5 years.
A more common approach to the treatment of allergic diseases is the use of drug therapy. Such as histamine antagonists, which inhibit histamine responses by blocking histamine receptors, and steroids and drugs which inhibit the release of inflammatory substances from mast cells, bronchodilator drugs, etc. However, these drugs only provide temporary relief of symptoms, do not really change the allergic immune response, and treat the symptoms and not the root cause.
At present, measures for improving immunity include healthy eating habits, proper exercise, pleasant mood maintenance and traditional Chinese medicine treatment; the organic acids, polysaccharides, common substances, alkaloids, volatile oils and other substances contained in various traditional Chinese medicines can fully exert and comprehensively adjust the self adaptability and immune function of the organism so as to achieve the purpose of preventing diseases. The polysaccharide plays a certain role in regulating the immunity of an organism, so that immune cells can be activated, and the important influence on a complement system can be exerted to induce the generation of cytokines. However, these therapies are slow and long lasting and only last for a period of time, and are therefore not a good way for most people.
Recently, China has taken the national effort to combat the new type of coronavirus pneumonia, and human immunity plays a very important role in the battle. In the case of fighting against novel coronavirus pneumonia, the most important point of personal protection is to enhance physique and improve immunity.
The probiotics is a good immune activator, can promote specific and nonspecific immunity at the same time, can regulate an immune system to perform an immune function under the condition of not causing inflammatory reaction, and is generated by activating macrophages, improving the expression level of anti-inflammatory cytokines and enhancing the activities of natural killer cells and immunoglobulin. In addition, probiotics are planted in the intestinal tract to inhibit pathogenic bacteria from adhering to the intestinal mucosa, so that the number of the pathogenic bacteria can be effectively reduced, and the balance of intestinal flora is maintained, thereby indirectly influencing the immune system.
Disclosure of Invention
In view of the above, the present invention provides bifidobacterium infantis DH231, and a fermentation product, applications and a microecological preparation thereof. The Bifidobacterium infantis and the microecological preparation have effects of preventing or treating organism anaphylaxis, relieving autism, and enhancing immunity.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides bifidobacterium infantis, the preservation number of which is CGMCC No. 18372.
The invention also provides application of the bifidobacterium infantis in preparing a microecological preparation with the effect of relieving autism.
The invention also provides application of the bifidobacterium infantis in preparing a microecological preparation with the effect of relieving allergy.
The invention also provides application of the bifidobacterium infantis in preparing a microecological preparation with the effect of improving immunity.
The invention also provides a fermentation product prepared from the bifidobacterium infantis.
The invention also provides a microecological preparation which comprises the bifidobacterium infantis or the fermentation product and pharmaceutically acceptable auxiliary materials.
Preferably, the dosage form of the microecological preparation is one of powder, pulvis, tablets, capsules, aqueous solution, gels, paste, dripping pills, pills and granules.
Preferably, the probiotic is a solid or liquid formulation.
The total viable count contained in the solid preparation prepared from the microecological preparation is not less than 1 multiplied by 10 6 CFU/g, typically 1X 10 9 CFU/g is more than 1 multiplied by 10 at most 11 CFU/g or 1X 10 11 CFU/g is above; the total viable count of the prepared liquid preparation is not less than 1 × 10 6 CFU/mL, typically at 1X 10 9 CFU/mL or more, up to 1 × 10 11 CFU/mL or 1X 10 11 CFU/mL or more.
The microecological preparation can be prepared into a pharmaceutical composition, a veterinary drug or a feed additive and the like.
The bifidobacterium infantis refers to a living organism.
The invention provides bifidobacterium infantis DH231, a fermentation product, application and a microecological preparation thereof. The preservation number of the bifidobacterium infantis is CGMCC No. 18372. The invention has the following technical effects:
the bifidobacterium infantis DH231 microecological preparation has obvious curative effects of preventing and treating autism, resisting allergy, improving immunity and the like, and has quick response, good effect and no adverse reaction or side effect compared with other treatment modes. Can be used as medicine, veterinary medicine, feed additive, etc., and has great social significance.
Biological preservation Instructions
Biomaterial DH231, taxonomic nomenclature: bifidobacterium infantis (Bifidobacterium infantis) is preserved in China general microbiological culture Collection center (CGMCC) in 2019 at 06.08, wherein the preservation center addresses No. 3 of Xilu 1 of Beijing Korean district and No.18372 of China academy of sciences microbiological research institute.
Detailed Description
The invention discloses bifidobacterium infantis DH231 and a fermentation product, application and a microecological preparation thereof, and can be realized by appropriately improving process parameters by taking the contents as reference by the technical personnel in the field. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Description of the preparation examples: the preparation method of other bifidobacterium infantis bacterial preparations is easily mastered by the skilled person in the art through the embodiment, and the preparation method of other dosage forms is easily mastered by the skilled person in the art through the implementation, and the description is not repeated here. The preparation method is not limited to the examples of the invention, and any known method capable of achieving the preparation purpose can be used, and the preparation description of the examples is only illustrative of the invention and is not limiting to the protection scope of the invention.
The bifidobacterium infantis DH231 and the fermentation product and the application thereof as well as the reagents, instruments or auxiliary materials used in the microecological preparation can be purchased from the market.
The invention is further illustrated by the following examples:
EXAMPLE 1 preparation of fungal powder and identification of bacterial species
5-10g of naturally discharged fresh excrement is collected by a sterile container, quickly put into an anaerobic tank and taken back to a laboratory for strain separation. Taking 1.5g of a feces sample, suspending and uniformly mixing the feces sample by using 15mL of PBS buffer solution, centrifuging the feces sample for 5min by 400g, collecting supernatant, adding 8000g of PBS buffer solution with a proper amount, centrifuging the feces sample for 10min, and collecting the supernatant to obtain a test sample.
Sucking 1mL of the supernatant, adding the supernatant into a test tube filled with 9mL of PBS diluent (containing 5 per mill L-cysteine), and mixing uniformly in a vortex manner to obtain 10 -2 Diluting the solution to 10 deg.C by the same method -3 Respectively taking out the stock solution of excrement and 10 -2 Diluent, 10 -3 Spreading 0.1mL each of the diluted solutions on BBL plate, culturing in 37 deg.C constant temperature incubator for 24 hr, selecting bacterial colony with round shape, smooth, neat edge, raised shape, milky semitransparent and diameter of 0.5-2mm, inoculating in BBL liquid culture medium, culturing at 37 deg.C for 24 hr, centrifuging the obtained culture solution (12000rpm), separating out thallus, freeze vacuum drying to obtain dry powder with viable count of 1 × 10 9 CFU/g is higher than the standard.
And meanwhile, physiological and biochemical characteristics of the strain are identified. And respectively carrying out a catalase test and a curd test on the separated strains to remove strains with positive catalase and without curd phenomenon, removing non-target strains, continuously carrying out physiological and biochemical characteristic identification on the rest strains through BIOLOG and API 50CH, and finally extracting DNA for sequencing to further define the classification status of the strains.
Confirming the taxonomic status of the strain according to the colony and thallus morphological characteristics of the strain, 16SrDNA sequence analysis and analysis results of an API bacteria identification system, and finding that the strain with the number DH231 is bifidobacterium infantis; the strain is characterized by the following morphological and general properties:
1. cell and colony morphology
The shape of the thallus: the thallus is in various branched, V-shaped, Y-shaped, single, paired and other irregular shapes under a microscope, the length is 1.5-3 microns, the width is 0.6-0.8 micron, the thallus does not move, spores do not exist, and gram stain is positive.
Colony morphology: the colony morphology on the solid culture medium is round, smooth, neat in edge, raised, milky and translucent. The colony diameter is generally 0.5-2 mm.
2. Physiological and biochemical characteristics
The optimum growth temperature is 35-37 deg.C, anaerobic, and the optimum growth pH value of the strain is 5.5-6.2. Has no pathogenicity and produces no toxin.
In the sugar fermentation, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, inositol, maltose, lactose, melibiose, sucrose, raffinose, L-fucose are fermented; non-fermented glycerol, erythritol, D-arabinose, L-xylose, arabitol, beta-methyl-D-xyloside, L-sorbose, L-rhamnose, dulcitol, mannitol, sorbitol, alpha-methyl-D-mannoside, alpha-methyl-D-glucoside, N-acetyl-glucosamine, amygdalin, arbutin, esculin, salicin, cellobiose, trehalose, inulin, melezitose, starch, glycogen, xylitol, gentiobiose, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, gluconate, 2-keto-gluconate, and catalase negative.
Example 2 toxicity test
1. Animals and groups
20 SPF-level mice are taken, aged 6-8 weeks and weighed 18-22 g, and are randomly divided into each bifidobacterium infantis group and each group of 10 mice.
2. Preparation of bacterial liquid
Respectively concocting the above Bifidobacterium infantis powder with purified water to obtain powder with bacteria content of 1 × 10 7 CFU/mL of bacterial liquid.
3. Method of producing a composite material
The same basic feed is fed to the bifidobacterium infantis group and the same basic feed is fed to the non-dosed group, the feeding conditions are consistent, the bifidobacterium infantis group is irrigated with 0.5mL of bifidobacterium infantis bacterial liquid every day, the non-dosed group is irrigated with 0.5mL of purified water every day, and the feeding is carried out for 6 months to observe toxic reaction.
4. Results
All mice of each group have no abnormal conditions, such as tremor, spasm, dyskinesia, abnormal posture, no eyeball protrusion, normal urination, normal skin and respiration and no death, and the bifidobacterium infantis has no toxicity.
Example 3 preparation of Bifidobacterium infantis powder
After the separation and identification according to the steps and the method, the bifidobacterium infantis strain can be prepared into the strain powder by experimental verification of no toxicity, and then related auxiliary materials are added according to the requirements to prepare various dosage forms, preferably, the powder is prepared according to the viable count of the bifidobacterium infantis strain powder, and the main raw materials are as follows: bifidobacterium infantis and other auxiliary materials are as follows: stachyose, maltodextrin, xylitol, crystalline fructose, non-dairy creamer, food additives including microcrystalline cellulose and stevioside, nutrition enhancers including vitamin B, vitamin E powder and folic acid, and the preparation method comprises the following main processes of crushing or not crushing, blending, uniformly mixing, packaging and the like. The viable count is not less than 1 × 10 7 CFU/g, and then bagging.
Application effects description of the examples: the invention takes the preservation number of the bifidobacterium infantis DH231 CGMCC13872 as a representative to illustrate the application effect of the bifidobacterium infantis, but the invention is not limited. Application effects example 1: study of Bifidobacterium infantis autism-relieving test
1. Materials and methods
1.1 materials
1.1.1 test products
Bifidobacterium infantis viable bacteria DH231 powder with viable bacteria count of 1 × 10 7 CFU/g is higher than the standard.
1.1.2 test animals
SPF clean grade C57BL/6 inbred mice, 5 females, 10 males, 20 + -2 g body mass, and 8 weeks of fertile age. The experimental mice are bred in a laboratory with the temperature of 18-22 ℃ and the humidity of 55-60% and the illumination of 12 hours a day, and are fed with common mouse feed, and general observation and quarantine are carried out before the experiment is started.
1.2 methods
Closing the cages at 5 nights according to the male-female ratio of 2:1, observing the vaginal embolus in 8 nights, performing vaginal smear if the vaginal embolus cannot be observed, recording as 1 day of pregnancy when sperm is observed, breeding female mice in cages after conception, and randomly dividing the female mice into a model group and a control group; the injection of sodium Valproate (VPA)600mg/kg is given to the abdominal cavity on the 12.5 th day after the pregnancy of the model group, the injection of physiological saline with the same amount (the physiological saline is prepared into 250mg/mL solution) is given to the pregnant mice of the control group at the same time, and the weaning of the young mice is carried out 21 days after birth.
1.3 establishing an autism animal model
The weaned young mice were randomly divided into a negative control group and an experimental group, and 6 mice were used in each group.
TABLE 1 grouping and administration methods
1.4 Observation index
1.4.1 open field experiments
(1) An adaptation stage: gently placing the mouse into the device (made of acrylic glass, 40cm × 40cm × 30cm), and allowing the mouse to move freely for 10 min.
(2) And (3) a testing stage: the number of crossing times of the mice tested was recorded within the next 10 min. The device is divided into 16 lattices, the middle 4 lattices are central lattices, and the peripheral 12 lattices are peripheral lattices. The cross-grid criteria are: the 4 limbs all cross the lattice border. After the test was completed, the test mouse was gently taken out and returned to the cage, and then the device was wiped with a 75% alcohol solution, and after it was dried, the next mouse was tested.
1.4.2 three-Chamber social experiment
(1) An adaptation stage: the mouse to be tested is placed in a device (material: acrylic glass, 120cm multiplied by 20cm multiplied by 22cm), and is allowed to freely move in 3 communicated chambers for 10min so as to adapt to the environment.
(2) And (3) a testing stage: the well-adapted test mice were taken out, and the cages with strange mice (mice of the same strain raised in the same cage and of the same sex and age as the test mice) were placed in the left and right chambers (left and right randomly). The mice which are well adapted before are gently placed into the middle chamber to be subjected to internal activity for 10min, and the mice are gently taken out after the operation is finished. Another strange mouse was placed in the empty cage, and the test mouse was again gently placed in the middle chamber. The time that the test mouse stayed in the middle chamber during the next 10min was recorded. And after the test is finished, sequentially taking out the tested mouse and the strange mice in the empty cages at two sides, putting the tested mouse and the strange mice in different cages, washing the experimental device by using a 75% ethanol solution, and testing the next tested mouse after the experimental device is dried. And the Any-maze software and a matched camera system automatically record the motion trail and the time of the Any-maze software in three boxes.
1.4.3 Water maze test
The MWM water maze system (SLY-WMS2.0) required by the experiment was installed and any-maze software was set up.
Clean water was injected into a black circular water tank (diameter 130cm, depth 45cm) until the liquid level reached 1cm above the platform. The shading cloth is used for surrounding the periphery of the water tank, and the shading cloth and the periphery of the tank wall are respectively pasted with marks in different shapes, so that the experimental rat can conveniently recognize and memorize the direction. After the experimental rat is placed in water with the surface facing the pool wall, the experimenter leaves the periphery of the water tank quickly and pulls the shading cloth, and the any-maze software automatically records the time from the time when the experimental rat is placed in the water to the time when the experimental rat finds the platform and the motion track of the experimental rat in the water.
The whole experimental process lasts for 7 days and is divided into three stages of testing and training. Day 1, acclimation: the platform is removed, the experimental rat is placed in water from the first quadrant, and the rat is taken out after 1 min. And (3) positioning navigation training on days 2-6: fixing the platform at the center of the quadrant III and 1cm below the water surface, adding ink into the water until the underwater platform cannot be seen by naked eyes, putting the experimental rat into the water from N (normal), E (east), S (south) and W (west)4 different initial points, and recording the swimming track of the experimental rat in the maze by software. If the rat finds and climbs the platform within 1min, the software automatically stops recording, and the time for finding the platform is counted as the escape latency time. If the platform is not found within 1min, the experimental rat is guided to the platform to enhance memory for 10s, and the latency period is recorded as 60 s. Day 7, platform exploration test: and (5) dismantling the platform again, and recording the total times of each experimental rat to swim past the position of the original platform within 1 min.
1.4.4 repetitive engraving action detection
Two groups of postnatal 35d rats are respectively taken, and each rat is independently placed in a clean observation box for 10min to keep the environment quiet and dark light. The cumulative time of repeated stereotypy of the rats was observed and recorded. The repeated carving action here means that the rat turns round, jumps and trims the fur of all parts of the body.
1.5 data statistics
SPSS19.0 statistical software is adopted for analysis, the measured data is represented by means of +/-standard deviation, t test is adopted for comparison between two groups, single-factor analysis of variance test is adopted for comparison between multiple groups, and SNK-q test is adopted for comparison between every two groups. P <0.05 differences were statistically significant.
2. Test results and analysis
2.1 test results
Table 2 effect of bifidobacterium infantis DH231 strain on autism behaviour in mice (n ═ 6)
2.2 analysis
Bifidobacterium infantis DH231 was able to significantly alleviate autism behavior with statistical differences (P < 0.05). Therefore, the probiotic has good function of relieving autism.
Application effects example 2: study on Bifidobacterium infantis allergy relief test
1. Materials and methods
1.1 materials
1.1.1 test products
Bifidobacterium infantis viable bacteria DH231 powder with viable bacteria count of 1 × 10 7 CFU/g is higher than the standard.
1.1.2 test animals
14 mice of the weaning male BALB/c inbred line at 3 weeks of clean-grade age.
1.2 methods
14 mice were randomly divided into 2 groups of 7 mice each. Dissolving probiotics of each group in normal saline, and mixing to obtain turbid solution with final concentration of 1 × 10 8 cfu/mL. The administration amount is 20mL/kg, and the daily dose is divided into two times. Negative control group: the freeze-drying protective agent is dissolved in normal saline to prepare turbid liquid, the mice are subjected to intragastric administration of 20mL/kg every day, and the intragastric administration is carried out twice in daily dose.
1.3 establishing delayed type hypersensitivity animal model
The abdomen of the mice was shaved in the range of 3cm × 3cm on the second day of the first gavage to fully expose the sensitized site. 20 μ L of 0.5% DNFB solution (dissolved in 4:1 acetone: olive oil) was uniformly applied for sensitization. The mice of each group were continuously gavaged for 14 days according to the above method, the last administration was carried out for 1 hour, and 10 μ L of 0.25% DNFB solution was evenly spread on both sides of the left ear of the mouse for challenge to induce the delayed hypersensitivity model of the mouse. After 60min, the ears are cut off and weighed.
1.4 Observation index
Ear weight: and taking the middle ear tissue block by using a puncher, weighing by using an analytical balance, and calculating the weight difference of the left and right ear tissue blocks. The values of each group were compared and statistically processed by t-test for significance of sample mean difference.
1.5 calculation formula
Swelling degree (mg) is equal to weight of right ear piece-weight of left ear piece
The inhibition ratio (%) was (average degree of swelling in placebo-average degree of swelling in each administration group)/average degree of swelling in placebo × 100%
2 test results and analysis
2.1 test results
TABLE 3 Effect of Bifidobacterium infantis DH231 Strain on delayed hypersensitivity in mice (n ═ 7)
| Group number | Swelling degree mg | Inhibition ratio of swelling% |
| Bifidobacterium infantis DH231 | 1.06±0.31 | 69.09％ |
| Negative control group | 3.43±1.44 | / |
2.2 analysis
Bifidobacterium infantis DH231 was found to significantly inhibit the ear swelling in mice delayed-type hypersensitivity induced by DNFB at a rate of 69.09% with statistical differences (P < 0.05). Therefore, the probiotics has good function of inhibiting the anaphylactic reaction of organisms.
Application effects example 3: study on bifidobacterium infantis immunity improvement test
1. Materials and methods
1.1 materials
1.1.1 test products
Bifidobacterium infantis viable bacteria DH231 powder with viable bacteria count of 1 × 10 7 CFU/g is higher than the standard.
1.1.2 test animals
SPF-grade BALB/c mice 24, male, 18-21 g.
1.2 Experimental methods
1.2.1 animal grouping and administration
Mice were acclimatized for 1 week, randomized into 2 groups: negative control group and Bifidobacterium infantis intervention group, each group contains 12 mice, the control group is administered with distilled water by intragastric administration, the Bifidobacterium infantis intervention group is administered with lyophilized powder of water for soaking 0.1g/kg, and the intragastric volume of each group of mice is 20mL/kg (viable count is 1.5 × 10) 7 CFU/g), 1 time per day, and gavage for 4 weeks. Animals were weighed weekly and gavage doses were adjusted according to body weight.
1.2.2 organ coefficient measurement
And (3) continuously performing intragastric administration for 28 days, weighing, removing cervical vertebrae, killing the mice, taking and weighing thymus and spleen, and calculating the ratio of the weight of the viscera to the weight to be used as the thymus coefficient and the spleen coefficient respectively.
1.2.3 cellular immune function assay (ConA-induced mouse lymphocyte transformation experiment)
Gavage to 28d, sacrifice mice by dislocation of cervical vertebrae, aseptically taking out spleen, preparing spleen cell suspension, washing with Hank's solution for 2 times (1000r/min, 10min), suspending cells with complete culture medium, adjusting cell concentration to 3 × 10 6 cell/mL, added in two wells (1 mL/well) to a 24-well plate, test wells added 75. mu.L of ConA solution (0.1mg/mL), control wells untreated. Is placed in CO 2 Incubator 5% CO 2 After culturing at 37 ℃ for 68 hours, 0.7mL of the supernatant was aspirated from each well, and 0.7mL of RPMI1640 culture medium and 50. mu.L of MTT (5mg/mL) were added thereto, followed by further culturing for 4 hours. And adding 1mL of acidic isopropanol into each well after the culture is finished, blowing and beating until the purple crystals are completely dissolved, subpackaging the mixture into 96-well plates, and measuring the absorbance at 570nm to represent the proliferation capacity of the lymphocytes by the difference value of the absorbance of the test wells and the control wells.
1.2.4 humoral immune function assay
1.2.4.1 antibody-producing cell assays
The mice were immunized by intragastric gavage to 28d with 2% Sheep Red Blood Cells (SRBC), 0.2 mL/mouse. After 5 days, the spleen was aseptically harvested to prepare a spleen cell suspension, and the cell concentration was adjusted to 5X 10 6 cell/mL. The bottom layer medium was prepared by adding 0.5% agarose in physiological saline to a six-well plate. Hank's solution containing 0.5% agarose was added at 0.5 mL/tube to a 50 ℃ thermostatic tube for use. Sequentially adding 50 μ L of 20% SRBC and 200 μ L of spleen cell suspension into a constant temperature test tube, immediately mixing, pouring into a six-well plate, laying flat, and adding CO 2 Incubating for 1h in incubator, adding 500 μ L complement diluted 11 times with complete culture medium into each well, culturing for 2h, removing upper liquid, taking picture, counting number of hemolytic plaques in each well with ImageJ software, and counting result as number of plaques/10 6 Splenocytes are shown.
1.2.4.2 determination of the half-maximal hemolysis value HC50
Gavage to 28d, mice were immunized i.p. with 2% SRBC, 0.2 mL/mouse. After 4 days, blood was collected from the orbit to prepare serum, and 1mL of serum diluted 200 times with SA buffer was placed in a test tube, and 0.5mL of 10% SRBC was added, followed by 1mL of SA solution to dilute 9 times of complement. The control tube replaces serum with SA solution. The reaction is stopped by constant temperature water bath for 20min at 37 ℃. Centrifuging at 2000r/min for 10min, collecting 1mL of supernatant, adding 3mL of Du's reagent, simultaneously collecting 0.25mL of 10% SRBC and 3.75mL of Du's reagent as half of hemolysis control tube, mixing well, standing for 10min, blank with the control tube, and measuring absorbance of each tube at 540nm wavelength.
Sample HC50 ═ absorbance of sample/absorbance at half-maximal hemolysis of SRBC × 200
1.2.5 monocyte-macrophage function assay
1.2.5.1 carbon Clearance test
Gavage to 30d, and injecting Indian ink diluted 4 times with physiological saline solution 0.1mL/10g body weight from mouse tail vein. 20 μ L of blood was collected from the canthus 2min (t1) and 10min (t2) after the injection of the ink, and then added to 2mL of 0.1% Na 2 CO 3
And (4) mixing the solution evenly. With 0.1% Na 2 CO 3 The solution was blanked and the optical density values (OD1, OD2) were measured at 600nm wavelength with a spectrophotometer, respectively. Mice were sacrificed by dislocation of cervical vertebrae, livers and spleens were taken and weighed, and the phagocytic index was used to express the mouse carbon clearance ability.
1.2.5.2 Abdominal macrophage phagocytosis fluorescent microsphere experiment
When the stomach is irrigated to 24 days, 0.2mL of 2% SRBC is injected into the abdominal cavity of each mouse to activate the macrophages of the mouse, the mouse is killed after 4 days of cervical dislocation, the abdominal skin is cut to expose the peritoneum, 3mL of Hank's solution containing 5% calf serum is injected into the abdominal cavity, the abdomen is gently kneaded for 20 times, a small opening is cut into the peritoneum, 2mL of abdominal cavity solution is sucked and filtered into a test tube through a 200-mesh sieve, the number of the macrophages is adjusted to be 4 multiplied by 10 5 -6×10 5 cell/mL. The diluted cell suspension was added to a 6-well plate at 1 mL/well, and 1% BSA pre-conditioned fluorescent microspheres (1X 10) 7 Plate) in CO 2 Incubating in cell incubator in dark for 90min, washing with precooled PBS buffer solution gently for 2 times (1000r/min, 5min), adding PBS buffer solution 0.3mL, scraping with cell scraperAnd (3) attaching cells to the wall, blowing and beating uniformly, filtering by using a 200-mesh sieve, detecting by using a flow cytometer, analyzing and counting the proportion of the non-phagocytosed fluorescent microspheres and the macrophages engulfed with different numbers of fluorescent microspheres by using CytExpert2.0 software, and calculating the phagocytosis percentage and the phagocytosis index of the fluorescent microspheres.
Percent (%) phagocytosis ═ number of macrophages engulfed fluorescent microspheres/number of macrophages counted × 100%
Phagocytosis index-total number of fluorescent microspheres phagocytosed/number of macrophages counted
1.2.6 NK cell Activity assay
Gavage to 28 days, remove cervical vertebrae and sacrifice mice, aseptically take spleen and prepare single cell suspension. Filtering with 200 mesh sieve, washing with Hank's solution for 2 times (1000r/min, 10min), discarding supernatant, lysing erythrocytes, adding 8mL Hank's solution, centrifuging at 1000r/min for 10min, discarding supernatant, adjusting cell concentration to 2 × 10 with complete culture medium 7 cell/mL. Adding 100 mu L of target cells (YAC-1 cells, 24h passage before experiment) and effector cells (splenocytes) into a U-shaped 96-hole culture plate according to an effect-target ratio of 50:1, wherein 100 mu L of target cells and 100 mu L of complete culture solution are added into a target cell natural release hole and a target cell maximum release hole respectively; to the background blank wells 200. mu.L of complete medium was added. All the above-mentioned materials are equipped with three compound holes, and at 37 deg.C CO 2 After 3h of culture in the incubator, 20 microliter of LDH release reagent is added into the maximum release hole of the target cell, the mixture is evenly blown and beaten, and the culture is continued for 1 h. The culture plate is kept still for 10min, 100 mu L of each well is absorbed and added into a flat-bottom 96-well plate, 50 mu L of LDH detection working solution is added at the same time, the reaction is carried out for 30min in a dark place, and the absorbance (OD) is measured at the 490nm wavelength of an enzyme labeling instrument. NK cell activity was calculated as follows:
NK cell activity (%) - (reaction well OD value-natural release well OD value)/(maximum release well OD value-natural release well OD value) × 100%
1.3 data processing
The experimental data are expressed as mean ± standard deviation, the number n of each group of experimental animals is 12, GraphPadPrism7 statistical software is used, two-factor analysis of variance is used for body weight, one-factor analysis of variance is used for other indexes, Tukey post-hoc tests (multiple comparisons) are all performed, and the difference is statistically significant when P is less than 0.05.
2. Test results and analysis
2.1 test results
Table 4 effect of bifidobacterium infantis DH231 strain on immunity in mice (n ═ 12)
| Item | Negative control group | Bifidobacterium infantis intervention group |
| Coefficient of thymus (mg/g) | 1.82±0.33 | 2.02±0.28 |
| Spleen coefficient (mg/g) | 4.8±0.4 | 5.4±0.5 |
| Proliferative capacity of lymphocytes | 0.28±0.05 | 0.36±0.06 |
| Antibody cell-producing ability | 980±90 | 1150±173 |
| HC50 | 127±5 | 136±4 |
| Carbon clearing ability | 3.5±0.5 | 4.4±0.6 |
| Percent% fluorescent microspheres | 48.00±7.09 | 59.01±5.49 |
| Phagocytic index | 0.78±0.11 | 1.01±0.09 |
| NK cell Activity (%) | 30.47±7.07 | 45.21±0.62 |
2.2 analysis
The bifidobacterium infantis DH231 can obviously improve the immunity of various organisms of mice, and the difference has statistical significance (P is less than 0.05). Therefore, the probiotic has good function of improving immunity.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. Bifidobacterium infantis is characterized in that the preservation number is CGMCC No. 18372.
2. Use of bifidobacterium infantis as claimed in claim 1 in the preparation of a probiotic with efficacy in alleviating autism.
3. Use of bifidobacterium infantis as claimed in claim 1 in the preparation of a probiotic with an allergy-relieving effect.
4. Use of bifidobacterium infantis as claimed in claim 1 in the preparation of a probiotic with an immune enhancing effect.
5. A probiotic formulation comprising Bifidobacterium infantis as claimed in claim 1 together with pharmaceutically acceptable adjuvants.
6. The microecological formulation according to claim 5, wherein the microecological formulation is in the form of one of a powder, a tablet, a capsule, a water aqua, a gel, a paste, a pill, and a granule.
7. The microecological formulation according to claim 6, wherein the microecological formulation is in the form of a drop pill.
8. The microecological formulation according to claim 5, wherein the microecological formulation is a solid formulation or a liquid formulation.
9. The microecological preparation according to claim 8, wherein the total viable count of the solid preparation is not less than 1 x 10 6 CFU/g。
10. The microecological formulation according to claim 8, wherein the total viable count of the liquid formulation is not less than 1 x 10 6 CFU/mL。
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