Lactobacillus rhamnosus RH22 with anti-inflammatory and anti-allergic effects and application thereof

Abstract

The present invention relates to a rhamnosus lactobacillus RH22 with anti-inflammatory and anti-allergic effects and an application thereof. The rhamnosus lactobacillus RH22 is classified and named as rhamnosus lactobacillus Lacticaseibacillus rhamnosus , the deposit number is CGMCC No.31054, and the deposit date is June 24, 2024. The present invention separates and preserves a new strain of Lactobacillus rhamnosus RH22 with anti-inflammatory and anti-allergic effects from the Tibetan snow lotus fungus sample. The bacterium has excellent anti-inflammatory and anti-allergic abilities, high activity, acid and bile resistance, and excellent antioxidant properties.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and relates to Lactobacillus rhamnosus RH22 with anti-inflammatory and anti-allergic effects and application thereof. Background Art

[0002] Skin sensitivity is becoming more and more common among the population. Harmful substances in the environment, such as dust, chemicals and heavy metals, constantly attack people's skin, causing damage to the skin barrier. At the same time, bad living habits such as staying up late, unhealthy diet and lack of exercise also aggravate the occurrence of skin sensitivity problems. Skin sensitivity symptoms are often more troublesome. Slight stimulation or touch may cause skin discomfort, such as tingling, redness, dryness and itching. These symptoms are not only related to people's appearance, but also have a significant impact on their daily life and mood. In order to cope with this problem, the raw materials for sensitive skin care products on the market are also being continuously researched and developed to help sensitive skin return to a healthy state.

[0003] An imbalance in the intestinal microbiome is considered one of the key factors causing allergies. The intestine is the largest immune organ in the human body, and intestinal microorganisms are closely related to the host's health status by affecting the intestinal barrier function and regulating the host's immune response. Probiotics, as an important component of the intestinal microbiome, can maintain the balance of intestinal microorganisms, enhance the intestinal barrier function, and regulate the immune system, thereby alleviating allergic symptoms.

[0004] Lactobacillus rhamnosus, also known as Lactobacillus rhamnosus, belongs to the genus Lactobacillus. It is an anaerobic, non-spore-forming Gram-positive probiotic that is mostly found in the intestines of humans and animals. It has biological characteristics such as acid resistance, bile salt resistance, and resistance to multiple antibiotics. Lactobacillus rhamnosus can ferment a variety of monosaccharides (glucose, arabinose, maltose, etc.), but cannot utilize lactose. Most Lactobacillus rhamnosus can produce a small amount of soluble ammonia, but do not produce indole and hydrogen sulfide. Lactobacillus rhamnosus is widely present in the intestines of humans and animals and is excreted with feces. As part of the normal human flora, Lactobacillus rhamnosus is an important component of the normal intestinal microbial system and accompanies the host throughout life, which is of great significance for maintaining the balance of intestinal microecology. Lactobacillus rhamnosus is a probiotic lactobacillus with great potential. As a probiotic in the intestinal flora, it is also an edible lactic acid bacterium. It has become a hot topic of research in recent years and is being continuously used to make probiotic preparations suitable for humans and animals.

[0005] Different strains of Lactobacillus rhamnosus have been reported to have different probiotic functions, such as regulating intestinal flora, regulating the structure and function of the microecological community in the host's intestine and achieving a balanced state, thereby improving the function of the host's digestive system; enhancing the body's immunity; preventing and treating diarrhea, etc. At present, there are few reports on the efficacy of Lactobacillus rhamnosus in anti-inflammatory and anti-allergic aspects. Therefore, it is very meaningful to develop more Lactobacillus rhamnosus with other effects. Summary of the invention

[0006] In view of the deficiencies in the prior art, the object of the present invention is to provide a Lactobacillus rhamnosus RH22 with anti-inflammatory and anti-allergic effects and its application.

[0007] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0008] In a first aspect, the present invention provides a rhamnosus Lactobacillus RH22 with anti-inflammatory and anti-allergic effects. The rhamnosus Lactobacillus RH22 is classified and named as Lacticaseibacillus rhamnosus, with a preservation number of CGMCC No.31054 and a preservation date of June 24, 2024.

[0009] The present invention separates and preserves a new strain of Lactobacillus rhamnosus RH22 with anti-inflammatory and anti-allergic effects from a sample of Tibetan snow lotus fungus. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is Lactobacillus rhamnosus. The bacterium has excellent fermentation activity, acid and choline salt resistance, and excellent in vitro antioxidant performance, which is specifically manifested in excellent scavenging ability and iron reducing ability (FRAP) for DPPH free radicals and ABTS+ free radicals; it also has excellent anti-inflammatory and anti-allergic ability, which is specifically manifested in regulating the content of cellular inflammatory factors, reducing the aggregation of neutrophils in zebrafish models and the level of tryptase. It has a promising prospect for using it to prepare anti-inflammatory, anti-allergic, antioxidant and anti-aging products.

[0010] In a second aspect, the present invention provides a use of the Lactobacillus rhamnosus RH22 described in the first aspect in the preparation of a product with anti-inflammatory and anti-allergic effects.

[0011] Preferably, the product includes medicine, food, health care product, daily chemical product or cosmetics.

[0012] Preferably, the bacterial count of Lactobacillus rhamnosus RH22 in the product is not less than 1×10 8 / mL or 1×10 8 Pieces / g, for example 1×108 pieces / g (pieces / mL), 1×10 9 pieces / g (pieces / mL), 1×10 10 pieces / g (pieces / mL), 5×10 10 pieces / g (pieces / mL), 1×10 11 pieces / g (pieces / mL), 3×10 11 pieces / g (pieces / mL), 5×10 11 pieces / g (pieces / mL), 1×10 12 Pieces / g (pieces / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0013] In the above products, Lactobacillus rhamnosus RH22 can exist in either a live or inactivated state. It is more preferred to use it in an inactivated state (postbiotics), because postbiotics are safer and more stable than live bacteria, giving them greater advantages in processing, transportation and storage. Compared with live probiotics, the longer shelf life ensures that postbiotic products can enter many areas with inconvenient transportation, making it easier to promote and expand their scope of use.

[0014] In a third aspect, the present invention provides a microbial preparation having anti-inflammatory and anti-allergic effects, wherein the strain in the microbial preparation includes the Lactobacillus rhamnosus RH22 described in the first aspect.

[0015] Preferably, the strain in the microbial preparation also includes Lactobacillus plantarum MH95; the classification name of Lactobacillus plantarum MH95 is Lactiplantibacillus plantarum, the preservation number is CGMCC No.31053, and the preservation date is June 24, 2024.

[0016] The present invention also creatively discovered that the above-mentioned Lactobacillus rhamnosus RH22 strain can be used in combination with Lactobacillus plantarum MH95 strain for anti-inflammatory and anti-allergic effects, and also has excellent effects. Compared with a single bacterial agent, the composite bacterial agent has more significant anti-inflammatory and anti-allergic effects, indicating that the RH22 strain and the MH95 strain have potential synergistic effects in the above-mentioned effects.

[0017] In the above-mentioned microbial preparations, Lactobacillus rhamnosus RH22 and Lactobacillus plantarum MH95 can exist in a live bacterial state or in an inactivated state.

[0018] Preferably, the ratio of the bacterial counts of Lactobacillus rhamnosus RH22 to Lactobacillus plantarum MH95 is (2-10):1, for example 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, etc. Other specific point values ​​within this numerical range can be selected and will not be described one by one here.

[0019] Based on the potential interaction between the two strains in anti-inflammatory and anti-allergic aspects, the effect will be better when the two meet the above-mentioned specific ratio relationship.

[0020] Preferably, the total bacterial count in the microbial preparation is not less than 1×10 8 / mL or 1×10 8 Pieces / g, for example 1×10 8 pieces / g (pieces / mL), 1×10 9 pieces / g (pieces / mL), 1×10 10 pieces / g (pieces / mL), 5×10 10 pieces / g (pieces / mL), 1×10 11 pieces / g (pieces / mL), 3×10 11 pieces / g (pieces / mL), 5×10 11 pieces / g (pieces / mL), 1×10 12 Pieces / g (pieces / mL), etc. Other specific point values ​​within this numerical range can be selected and will not be described here one by one.

[0021] Preferably, the microbial preparation is in the form of a solution or a powder.

[0022] The dosage form of the microbial preparation involved in the present invention is not limited, including the most commonly used solutions, powders, or further prepared capsules, tablets or granules.

[0023] A fourth aspect is the use of the microbial preparation according to the third aspect in the preparation of a product with anti-inflammatory and / or anti-allergic effects.

[0024] A fifth aspect is the use of the microbial preparation according to the third aspect in the preparation of products with antioxidant and / or anti-aging effects.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention separates and preserves a new strain of Lactobacillus rhamnosus RH22 with anti-inflammatory and anti-allergic effects from a sample of Tibetan snow lotus fungus. The strain is sequenced and analyzed, and its 16S rDNA sequence is shown in SEQ ID No: 1. The sequence obtained by sequencing is compared with the nucleic acid sequence in GeneBank, and the result shows that the strain is Lactobacillus rhamnosus. The bacterium has excellent fermentation activity, acid and choline salt resistance, and excellent in vitro antioxidant performance, which is specifically manifested in excellent scavenging ability and iron reducing ability (FRAP) for DPPH free radicals and ABTS+ free radicals; it also has excellent anti-inflammatory and anti-allergic ability, which is specifically manifested in regulating the content of cellular inflammatory factors, reducing the aggregation of neutrophils in zebrafish models and the level of tryptase. It has a promising prospect for using it to prepare anti-inflammatory, anti-allergic, antioxidant and anti-aging products.

[0027] The RH22 strain involved in the present invention is classified and named as Lactobacillus rhamnosus, and the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration. The preservation time is June 24, 2024, the preservation number is CGMCC No.31054, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0028] The MH95 strain involved in the present invention is classified and named as Lactiplantibacillus plantarum, and the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Administration. The preservation time is June 24, 2024, the preservation number is CGMCC No.31053, and the address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a microscopic image of the Lactobacillus rhamnosus strain RH22.

[0030] Figure 2 This is the appearance of the colony of Lactobacillus rhamnosus RH22 strain. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0032] All zebrafish used in the experiments described below were purchased from the National Zebrafish Resource Center.

[0033] The culture medium formula involved in the following content is as follows:

[0034] MRS liquid culture medium: glucose 20 g / L, yeast peptone 10 g / L, beef extract powder 10 g / L, yeast extract powder 5 g / L, anhydrous sodium acetate 5 g / L, Tween-80 1 g / L, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.58 g / L, manganese sulfate monohydrate 0.19 g / L, water 1000 mL, pH 6.8.

[0035] The RH22 strain involved below is classified and named Lacticaseibacillus rhamnosus, the preservation date is June 24, 2024, and the preservation number is CGMCC No.31054.

[0036] The MH95 strain involved below is classified and named Lactiplantibacillus plantarum, the preservation date is June 24, 2024, and the preservation number is CGMCC No.31053.

[0037] The inactivated bacterial suspension involved below is obtained as follows: the seed liquid of the desired strain is inoculated into the MRS medium at an inoculation amount of 3% of the total mass of the culture medium, and cultured at 37°C to obtain a bacterial suspension; the bacterial suspension is transferred to a sterile centrifuge tube, the precipitate is collected by centrifugation, and resuspended with a sterile PBS buffer solution, and the centrifugation and resuspension are repeated twice to remove the residue in the culture medium; the bacterial mud and the protective agent (30% trehalose solution) are mixed and resuspended at a mass ratio of 1:2, and then heat-inactivated at 70°C for 30 min.

[0038] Example 1

[0039] Isolation and screening of Lactobacillus rhamnosus RH22:

[0040] (1) Screening of Tibetan snow lotus fungus: a certain amount of snow lotus fungus was homogenized in physiological saline with a mass concentration of 0.85%. 100 μL of the homogenate was spread on an MRS agar plate with cycloheximide (2%) added, and cultured anaerobically at 37°C for 48 h. The colonies with milky white, smooth surface and 1-3 mm in diameter were selected and cultured in 10 mL of sterile MRS broth. After cultured at 37°C for 24 h, the colonies were streaked on a sterile MRS agar plate and cultured anaerobically at 37°C for 48 h. Finally, single colonies on the four zones were selected and cultured in MRS liquid culture medium at 37°C for 24 h to obtain pure strains.

[0041] (2) The fermentation broth of the isolated different strains was screened out by catalase test to identify negative strains, and then screened out positive strains by Gram staining, and these strains were preliminarily determined to be lactic acid bacteria based on colony morphology. The above bacterial broth was mixed with 50% glycerol in a 1:1 volume ratio in a cryopreservation tube and stored in a -80℃ refrigerator.

[0042] (3) The above-mentioned preserved lactic acid bacteria are screened for the following functional live bacteria: fermentation activity, acid and bile salt resistance, free radical scavenging antioxidant capacity (including the determination of water-soluble vitamin E equivalent antioxidant capacity, DPPH free radical scavenging capacity, ABTS+ free radical scavenging capacity, and iron reduction antioxidant capacity). The specific operation process is as follows:

[0043] Use an inoculation loop to pick up the lactic acid bacteria stored in the glycerol tube and inoculate it into MRS broth to culture for 24 hours to obtain the first-level seed solution, and then reactivate it with an inoculation amount of 2% to obtain the second-level seed solution for the following experiments:

[0044] (3.1) Fermentation activity determination:

[0045] The secondary seed solution was diluted to a certain gradient and counted on MRS agar medium. The more viable bacteria in the secondary seed solution, the better the fermentation activity. The 20 strains with the best activity were selected for the next screening. The activity was between 5×10 9 CFU / mL-2×10 11 CFU / mL.

[0046] (3.2) Acid and bile salt resistance performance test:

[0047] Acid resistance test group: inoculate the first-level seed liquid into MRS liquid medium for acid resistance test at a rate of 2%, culture at 37℃ for 4 h, and count the viable bacteria.

[0048] Bile salt tolerance test group: inoculate the first-level seed solution at a 2% inoculation rate into MRS liquid medium for bile salt tolerance test, culture at 37℃ for 4 h, and count the viable bacteria.

[0049] Control group: Inoculate the first-level seed liquid into MRS liquid medium at a 2% inoculation rate, culture at 37℃ for 4 h, and count the viable bacteria. Take the logarithm of each viable bacteria count to get the viable bacteria logarithm value, and the difference between the viable bacteria logarithm value of the control is the viable bacteria logarithm value, which is used to evaluate the acid and bile salt resistance. 10 dominant strains were selected for the following test.

[0050] (3.3) Determination of DPPH free radical scavenging ability:

[0051] Weigh DPPH, dissolve it with an appropriate amount of anhydrous ethanol, and dissolve it completely under ultrasound in the dark. Then use anhydrous ethanol to prepare a DPPH working solution with a final concentration of 0.05 mg / mL. Take an appropriately diluted bacterial solution and mix it with the DPPH working solution at a ratio of 1:1 (v:v) (tube A); take anhydrous ethanol and DPPH working solution at a ratio of 1:1 (v:v) (tube B); take anhydrous ethanol and an appropriately diluted bacterial solution and mix them at a ratio of 1:1 (v:v) (tube C). After reacting at 25℃ for 30 min, measure the absorbance values ​​of tubes A, B, and C at 517 nm. Calculate the DPPH free radical scavenging rate using the following formula:

[0052] DPPH free radical scavenging rate (%) = (A B +A C -A A ) / A B ×100.

[0053] (3.4) Determination of ABTS+ free radical scavenging ability:

[0054] Mix 7 mM ABTS solution and 2.45 mM potassium persulfate solution in equal volumes, react at 25°C in the dark for 12 h to make ABTS working solution, dilute with pH 7.4 phosphate buffer until the absorbance at 734 nm is 0.700±0.02; then take 0.2 mL of appropriately diluted bacterial solution and mix with 3.8 mL ABTS working solution, react at 25°C in the dark for 6 min, and measure the absorbance at 734 nm. Use distilled water as the blank control, and calculate the ability of the sample to scavenge ABTS free radicals based on its absorbance value. The final result is calculated as the ABTS+ free radical scavenging rate, and the formula is as follows:

[0055] ABTS+ free radical scavenging rate (%) = (A1-A2) / A1×100.

[0056] Where: A1 is the absorbance value of the blank group, and A2 is the absorbance value of the sample.

[0057] (3.5) Determination of iron ion reducing ability:

[0058] 10 mM TPTZ solution, 20 mM FeCl3 solution and 0.3 M acetic acid buffer (pH 3.6) were mixed in a ratio of 1:1:10 (v:v:v) and placed in a 37°C dark water bath for 1 h to prepare a working solution. 0.1 mL of appropriately diluted bacterial solution was added to 0.3 mL of distilled water and 3 mL of working solution and mixed evenly. The mixture was placed in a 37°C dark water bath for 6 min. The absorbance was measured at 593 nm using distilled water as a blank control. A standard curve was drawn using FeSO4 as the standard. The iron ion reducing ability of the sample was calculated by substituting the absorbance value of the sample into the standard curve. The results were expressed as mmol Fe 2+ / L indicates.

[0059] The results of each group of strains are shown in Table 1. Combined with the results of antioxidant activity, No. 7 was selected as the dominant strain through comprehensive judgment and named RH22.

[0060] Table 1

[0061]

[0062] Example 2

[0063] Identification of Lactobacillus rhamnosus RH22:

[0064] (1) Observe the selected RH22 strain under a microscope. Figure 1 As shown, the strains are short rod-shaped, arranged singly or in pairs, without flagella and non-motile, and Gram-positive.

[0065] (2) The appearance of the RH22 colonies on the plate is as follows: Figure 2 As shown, the colonies are round, with a convex surface, neat edges, milky white and opaque.

[0066] (3) 16S rDNA molecular biological identification:

[0067] The screened RH22 strain was inoculated into a centrifuge tube containing 20 mL MRS liquid medium at a ratio of 2%, and centrifuged at 37°C for 18 hours, centrifuged at 5000rpm for 10 minutes, and the supernatant was removed to collect the bacteria. The genome of the strain was extracted, and bacterial universal primers were added for PCR amplification, and the amplified product was sequenced and identified. The strain was sequenced and analyzed, and its 16S rDNA sequence was shown in SEQ ID No: 1. The sequence obtained by sequencing was compared with the nucleic acid sequence in GeneBank, and the result showed that the strain was Lactobacillus rhamnosus.

[0068] Example 3

[0069] Anti-inflammatory evaluation at the cellular level:

[0070] (1) Cell safety evaluation:

[0071] Raw264.7 mouse macrophages in the logarithmic phase were collected, 100 μL of culture medium was added, and then cultured in a 96-well plate. The concentration of the cell suspension was adjusted to 10 cells per well. 4cells; after observing that the cells are completely attached to the wall, add different concentrations of inactivated Lactobacillus rhamnosus RH22 suspension or inactivated Lactobacillus plantarum MH95 suspension as the sample group, add culture medium as the sample control group, and transfer the 96-well plate to 37°C for 24 hours; centrifuge to remove the culture medium, add 20 μL of 0.5% MTT solution to each well and culture for 1 day; after removing the supernatant, add 150 μL of DMSO solution and shake, observe that the blue-purple formazan crystals are completely dissolved, and measure the absorbance with an enzyme marker at a wavelength of 490 nm. Set three parallel groups for each concentration, and use the wells without bacterial suspension and culture medium as the blank group. The cell survival rate formula is as follows:

[0072] Cell survival rate = (Ai-A0) / (Aj-A0) × 100%.

[0073] Where: A0 is the absorbance of the blank group; Ai is the absorbance of the sample group; Aj is the absorbance of the sample control group.

[0074] The results showed that the concentration of Lactobacillus rhamnosus RH22 suspension or Lactobacillus plantarum MH95 suspension was less than 1×10 13 When the concentration of 50 μg / mL was 400 μg / mL, the cell survival rate was above 92%, and it had no cytotoxicity to Raw264.7 mouse macrophages.

[0075] (2) Experimental groups:

[0076] The experiment was divided into a blank control group (not treated with probiotic solution), a model group, a RH22 group, a MH95 group, a RH22+MH95 group 1 (the ratio of the number of bacteria was 2:1), a RH22+MH95 group 2 (the ratio of the number of bacteria was 10:1), and a commercially available Lactobacillus rhamnosus + MH95 group (the ratio of the number of bacteria was 2:1, and the commercially available Lactobacillus rhamnosus was ATCC7469).

[0077] (3) Cellular intervention methods:

[0078] Raw264.7 mouse macrophages were collected in the logarithmic phase and inoculated into 6-well plates, with 1×10 5 The cells were treated after reaching 80-90% confluence. The control group used DMEM medium, and the other groups used DMEM medium supplemented with 1 μg / mL LPS to culture the cells. After 1 h, the strain intervention groups continued to add DMEM medium with different bacterial suspensions (the final bacterial concentration in the culture system was 1×10 11 The control group and the model group were added with the same amount of DMEM medium.

[0079] (4) Index detection methods and results:

[0080] The cell culture supernatant of each group was collected, and the concentrations of TNF-α, IL-6, IL-10, and NO inflammatory factors were detected according to the instructions of the ELISA kit.

[0081] The results are shown in Table 2:

[0082] Table 2

[0083]

[0084] It can be seen from the data results in Table 2 that the Lactobacillus rhamnosus RH22 and the microbial agent involved in the present invention have excellent anti-inflammatory effects, and the combined use of the RH22 strain and the Lactobacillus plantarum MH95 strain has a more excellent effect on the above-mentioned effects.

[0085] Example 4

[0086] Anti-inflammatory and anti-allergic evaluation of zebrafish:

[0087] (1) Experimental groups:

[0088] The experiment was divided into model control group (not treated with probiotic solution), RH22 group, MH95 group, RH22+MH95 group 1 (bacterial number ratio was 2:1), RH22+MH95 group 2 (bacterial number ratio was 10:1), commercially available Lactobacillus rhamnosus + MH95 group (bacterial number ratio was 2:1, the commercially available Lactobacillus rhamnosus was ATCC7469), positive control group (indomethacin or sodium cromolyn), and normal control group.

[0089] (2) Intervention methods:

[0090] (2.1) Neutrophil aggregation inhibition test:

[0091] Maximum tolerance concentration test: Zebrafish 3 days after fertilization were randomly selected and placed in a 6-well plate, with 24 zebrafish treated in each well. The volume of each well was 3 mL. Copper sulfate pentahydrate was given in water and treated at 28°C for 1 day. The maximum tolerance concentration of copper sulfate pentahydrate for model zebrafish was determined to guide subsequent experiments.

[0092] Healthy zebrafish 3 days after fertilization were selected. Among them, 24 fish embryos were randomly selected from the model control group and placed in a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 0.16 mg / L copper sulfate pentahydrate was added; among them, 24 fish embryos were randomly selected from the positive control group and placed in a 3 cm culture dish, and 5 mL of fish embryo culture medium containing 0.1 mg / L copper sulfate pentahydrate and 0.0036 mg / L indomethacin was added; among them, 24 fish embryos were randomly selected from the probiotic intervention group and placed in a 3 cm culture dish, and 5 mL of 0.16 mg / L copper sulfate pentahydrate and sterilized probiotic solution of each group were added (the final bacterial concentration in the culture system was 1×108 In the blank control group, 24 healthy fish embryos were randomly selected and placed in a 3 cm culture dish, and 5 mL of fish embryo culture medium was added.

[0093] Each group was placed in a 28℃ incubator for 40 min, and then each group of fish embryos was fixed in paraformaldehyde for 1 h, treated with PBST 3 times, 5 min each time, and then treated with 50% ethanol for 3 min. After the fish embryos were stained with Sudan black staining solution for 1 h, they were washed with 70% ethanol 4 times, 5 min each time, and then treated with PBST 2 times, 5 min each time. The fish embryos were treated with bleaching solution for 10 min, then treated with 70% ethanol solution for 5 min, PBST for 1 min, treated with clear solution 1 for 15 min, clear solution 2 for 10 min, and then treated with PBST for 3 min.

[0094] The fish embryos were placed sideways, and then placed under a stereo microscope to observe the tail of the fish embryos. The number of neutrophils in the three-quarters tail region of each fish embryo from the anus was counted, and the neutrophil aggregation inhibition rate was calculated. The inhibition rate (%) = [(AB) / A] × 100%, where B represents the average number of neutrophils in the fish embryos of the test substance treatment group, in units of granules per tail (granules / tail); A represents the average number of neutrophils in the fish embryos of the model control group, in units of granules per tail (granules / tail); the average values ​​of each test group were calculated and expressed, and the results are shown in Table 3.

[0095] Table 3

[0096]

[0097] It can be seen from the data results in Table 3 that the rhamnosus Lactobacillus RH22 and the microbial agent involved in the present invention have excellent efficacy in inhibiting neutrophil aggregation in the zebrafish model, that is, have excellent anti-inflammatory efficacy, and the combined use of the RH22 strain and the MH95 strain has a more excellent effect on the above efficacy.

[0098] (2.2) Allergic reaction marker (tryptase) content evaluation test:

[0099] Maximum tolerance concentration test: Zebrafish 3 days after fertilization were randomly selected and placed in a 6-well plate, with 24 zebrafish treated in each well. The volume of each well was 3 mL. C48 / 80 (polymer produced by condensation of N-methyl-p-methoxyphenylethylamine and formaldehyde) was administered in water. After treatment at 28°C for 1 day, the maximum tolerance concentration of C48 / 80 for model zebrafish was determined to guide subsequent experiments.

[0100] Healthy zebrafish 3 days after fertilization were selected for the experiment. A 96-well cell culture plate was used as the test carrier. Three parallel groups were set up in each group. Ten zebrafish were added to each well. 100 μL of the test bacterial solution was added to the test sample group, 100 μg / mL sodium cromoglycate solution was added to the positive control group, and 100 μL of culture medium was added to the model group and the blank control group. The plates were placed in a constant temperature incubator at 28°C and incubated for 2 h.

[0101] After incubation, 100 μL of 10 μg / mL C48 / 80 was added to each group except the blank control group, and 100 μL of culture medium was added to the blank control group, and the cells were incubated in a constant temperature incubator at 28°C for 1.5 h.

[0102] After incubation, 100 μL of the supernatant from each group was taken to a new 96-well cell culture plate, and 100 μL of 1 mg / mL N-benzoyl-DL-arginine p-nitrobenzamide hydrochloride (BAPNA) was added. In addition, a zero control group was set up (200 μL of 1 mg / mL N-benzoyl-DL-arginine p-nitrobenzamide hydrochloride was added to each well) and the plates were incubated at 37°C for 24 h.

[0103] After the incubation was completed, the OD405 value of each group was detected by a multifunctional microplate reader, and the trypsin inhibition rate was calculated. Inhibition rate (%) = [(AB) / A] × 100%, where B represents the average expression level of trypsin in the fish embryos of the test substance treatment group (OD405 value); A represents the average expression level of trypsin in the fish embryos of the model group (OD405 value); the average values ​​of each test group were calculated, and the results are shown in Table 4.

[0104] Table 4

[0105]

[0106] It can be seen from the data results in Table 4 that the rhamnosus Lactobacillus RH22 and the microbial agent involved in the present invention have excellent efficacy in inhibiting the level of trypsin in the zebrafish model, that is, they have excellent anti-allergic efficacy, and the combined use of the RH22 strain and the MH95 strain has a more excellent effect on the above efficacy.

[0107] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0108] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0109] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A microbial preparation with anti-inflammatory and anti-allergic effects, characterized in that: The strains in the microbial preparation are composed of Lactobacillus rhamnosus RH22 and Lactobacillus plantarum MH95; The classification of the Lactobacillus rhamnosus RH22 is named Lactobacillus rhamnosus Lacticaseibacillus rhamnosus , the deposit number is CGMCC No.31054, and the deposit date is June 24, 2024; The classification of the plant lactobacillus MH95 is named plant lactobacillus Lactiplantibacillus plantarum , the deposit number is CGMCC No.31053, and the deposit date is June 24, 2024; The ratio of the bacterial counts of Lactobacillus rhamnosus RH22 to Lactobacillus plantarum MH95 is (2-10):

1.

2. The microbial preparation according to claim 1, characterized in that The total bacterial count in the microbial preparation is not less than 1×10 8 / mL or 1×10 8 Pieces / g.

3. The microbial preparation according to claim 1, characterized in that The dosage form of the microbial preparation includes solution or powder.

4. Use of the microbial preparation according to any one of claims 1 to 3 in the preparation of medicines or daily chemical products with anti-inflammatory and / or anti-allergic effects.

5. Use of the microbial preparation according to any one of claims 1 to 3 in the preparation of medicines or daily chemical products with antioxidant and / or anti-aging effects.

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