Application of Bifidobacterium lactis BLa80 in improving and alleviating allergic reactions

Abstract

The present invention belongs to the field of microbial technology, and relates to the application of Bifidobacterium lactis BLa80 in improving and alleviating allergic reactions. The present invention first provides the application of Bifidobacterium lactis BLa80 in the preparation of a medicine for improving and alleviating allergic reactions. Experiments of the present invention have shown that BLa80 can significantly reduce the scratching behavior and back skin lesion inflammation scores of mice, and significantly reduce the behavioral observation scores and the degree of ear swelling. Bifidobacterium lactis BLa80 significantly reduces the IgE level in the serum of the model group, significantly increases the IFN‑γ level, significantly reduces the IL‑4 level, and Bifidobacterium lactis BLa80 can significantly increase the IFN‑γ / IL‑4 ratio, indicating that Bifidobacterium lactis BLa80 can regulate the balance of Th1 / Th2. The present invention also provides the application of Bifidobacterium lactis BLa80 in the preparation of health foods and cosmetics for improving and allergic relief.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to application of Bifidobacterium lactis BLa80 in improving and relieving allergic reactions. Background Art

[0002] Allergy, also known as atopic reaction or allergic reaction, refers to the specific abnormal tendency to harmless and ubiquitous environmental allergens. It may occur in the intestines, skin and respiratory tract, and the main clinical manifestations include abdominal pain and diarrhea, atopic dermatitis, allergic rhinitis, asthma, etc. Among them, atopic dermatitis (AD), also known as "atopic dermatitis", "genetic allergic dermatitis", "atopic eczema", "atopic eczema dermatitis syndrome (AEDS)", etc., is a chronic, recurrent, inflammatory skin disease related to genetic allergic constitution. In recent years, AD is considered to be a biphasic inflammatory skin disease. The interactive relationship between Th1 and Th2 cytokines is closely related to the changes in AD disease. The immune inflammatory response is manifested as a dynamic imbalance of Th1 / Th2 cell subsets. Th2 cells secrete excessive interleukin-4 (IL-4) and other cytokines, stimulating B cells to produce excessive IgE antibodies. It is generally believed that IgE levels or content play multiple important roles in the onset of AD. Due to the high reactivity of Th2 cells in AD patients, the body's immune cells are stimulated to secrete a large amount of IgE, which recognizes and binds to certain external allergens, thereby triggering subsequent allergic reactions and causing a series of systemic immune regulation abnormalities.

[0003] The skin barrier is the first line of defense of the human body. When the skin barrier function is damaged, external irritants can more easily enter through the epidermis, causing the body's antigen-presenting cells to combine with allergens, stimulating immune cells to release related mediators such as histamine and substance P, causing severe itching in patients. The itching in turn induces scratching in patients, further destroying the integrity of the skin barrier, thus forming a vicious cycle of "damage-scratch-damage".

[0004] The Bifidobacterium lactis BLa80 of the present invention can effectively alleviate and improve the allergic reaction of atopic dermatitis and improve the quality of life of patients with atopic dermatitis. Summary of the invention

[0005] Based on the above-mentioned needs in the art, the present invention provides the use of Bifidobacterium lactis BLa80 strain in improving and allergic reactions.

[0006] The present invention first provides the use of Bifidobacterium lactis BLa80 in preparing a drug for improving and relieving allergic reactions, wherein the drug includes but is not limited to microecological preparations and external-use drugs.

[0007] The present invention relates to Bifidobacterium lactis BLa80, which is classified and named Bifidobacterium animalis subsp. lactis. The strain was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration Committee on May 17, 2021, with an address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and a deposit number of CGMCC No. 22547. The strain was first disclosed in Chinese patent application CN 113564078 A "A strain of cholesterol-lowering Bifidobacterium lactis BLa80 and its application", which was published on October 29, 2021, and applied on July 29, 2021.

[0008] The in vitro experiments of the present invention prove that the Bifidobacterium lactis BLa80 of the present invention has good gastrointestinal tolerance and has a strong inhibitory effect on Escherichia coli, Salmonella and Staphylococcus aureus.

[0009] The Bifidobacterium lactis BLa80 can significantly reduce the scratching behavior and back skin lesion inflammation scores of mice, and significantly reduce the behavioral observation scores and ear swelling degree.

[0010] Bifidobacterium lactis BLa80 significantly reduced the IgE level in the serum of the model group, significantly increased the IFN-γ level, and significantly reduced the IL-4 level. Bifidobacterium lactis BLa80 significantly increased the IFN-γ / IL-4 ratio, indicating that Bifidobacterium lactis BLa80 can regulate the balance of Th1 / Th2. Bifidobacterium lactis BLa80 can effectively improve and alleviate the allergic reactions of mice with atopic dermatitis.

[0011] Bifidobacterium lactis BLa80 can effectively improve and relieve skin conditions and allergic reactions in patients with atopic dermatitis. Bifidobacterium lactis BLa80 can improve and enhance the quality of life of patients with atopic dermatitis.

[0012] In addition, the present invention also provides the use of Bifidobacterium lactis BLa80 in the preparation of cosmetics. During the use of cosmetics, it is inevitable to contact and irritate the skin, and BLa80 can play a role in improving and relieving allergies in cosmetics.

[0013] The cosmetics include lotions, emulsions, creams and sprays.

[0014] The lactobacillus BLa80 used in the present invention is derived from breast milk, is safe and non-pathogenic, and is on the list of common foods recognized by the state. It has been proven to have the effect of improving and alleviating allergies through animal experiments and human experiments. It is widely used in the field of food and daily necessities, increases the possibility of consumer consumption, and can achieve the purpose of improving and alleviating allergies through daily intake. Of course, the lactobacillus BLa80 used can also be used in the preparation of medicines for treatment and allergy relief. DETAILED DESCRIPTION

[0015] The following specific experimental examples are used to illustrate the implementation of the present invention. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The following experimental examples are used to illustrate the present invention, but are not used to limit the scope of the present invention.

[0016] Example 1 Evaluation of the environment of Bifidobacterium lactis BLa80 passing through the gastrointestinal tract

[0017] Simulate artificial gastric juice: prepare 0.5% NaCl solution, add 0.3% pepsin, adjust the pH value to 2.5 with 1 mol / L HCL, then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving.

[0018] Simulated artificial intestinal fluid: prepare 0.5% NaCl solution, add 0.1% trypsin, adjust the pH value to 8.0 with 0.1 mol / L NaOH, then filter and sterilize with a 0.22 μm microporous filter membrane after fully dissolving.

[0019] Bifidobacterium lactis BLa80 was activated and cultured for 2 generations under anaerobic conditions. The activated bacterial suspension of Bifidobacterium lactis BLa80 was centrifuged and the cells were collected. 0.4 mL of the bacterial suspension was added to 1.6 mL of the prepared simulated artificial gastric juice of pH 2.5 and the simulated artificial intestinal juice of pH 8.0, and mixed. The mixture was digested at 37°C. The number of live bacteria was detected by taking the digestion fluid at 0 h and 3 h, and the survival rate was calculated. The results are shown in the table below. Among them, the survival rate of the strain (%) = N t / N0×100%, where N0 represents the number of viable bacteria of the strain at 0h (CFU / mL), N t It indicates the number of viable bacteria of the strain at 3h (CFU / mL).

[0020] Table 1 Experimental data of Bifidobacterium lactis BLa80 in simulated digestive tract environment

[0021]

[0022] The survival rate of Bifidobacterium lactis Bla80 in artificial gastric juice of pH 2.5 for 3 hours was 96.7%, and the survival rate in artificial intestinal juice of pH 8.0 for 3 hours was 93.6%. The experiment showed that Bifidobacterium lactis BLa80 had a strong ability to tolerate the gastrointestinal environment (see Table 1).

[0023] Experimental Example 2: Evaluation of the antibacterial activity of Bifidobacterium lactis BLa80

[0024] Take the antagonistic strain and inoculate it into an anaerobic glass tube containing 0.1% L-cysteine ​​hydrochloride MRS liquid medium at 2% (V / V), and culture it at 37°C for 12 hours. Inoculate the pathogenic strains of Escherichia coli, Salmonella, and Staphylococcus aureus into liquid beef extract peptone medium, culture it at 37°C, 250 rpm, and a constant temperature shaker overnight, and then prepare the pathogen suspension. Cool the MRS solid medium to about 55°C, mix it with the pathogen suspension in a certain proportion, and make the number of viable pathogens in the system reach 10 6 CFU / mL order of magnitude, and then quickly pour it into a plate with an Oxford cup placed in advance. After the culture medium cools and solidifies, take out the Oxford cup, inject 200μL of antagonistic strain solution into each well, cover the plate lightly and place it upright in a 37℃ constant temperature incubator, observe after the appropriate incubation time, and measure the diameter of the inhibition zone with a vernier caliper.

[0025] Table 2 Inhibitory effect of Bifidobacterium lactis BLa80 on pathogenic bacteria

[0026]

[0027] As shown in Table 2, the antagonistic diameters of Bifidobacterium lactis BLa80 against Escherichia coli, Salmonella typhi and Staphylococcus aureus were 42 mm, 41 mm and 27 mm respectively, showing a strong inhibitory effect on intestinal pathogens, especially against Escherichia coli and Salmonella.

[0028] Experimental Example 3: Adhesion ability of Bifidobacterium lactis BLa80 to HT-29 cells

[0029] Activation of strains: Bifidobacterium lactis BLa80 was inoculated at 2% in an anaerobic glass tube containing 0.1% L-cysteine ​​hydrochloride MRS liquid medium, cultured at 37°C for 14 h, the fermentation broth was centrifuged, the bacteria were collected, washed 3 times with PBS, and then resuspended in DMEM culture medium without double antibodies. The concentration of the bacterial suspension was adjusted to 10 8 CFU / mL.

[0030] HT-29 cell culture: Add 5 mL of DMEM complete culture medium (containing 10% fetal bovine serum, 1% penicillin and streptomycin solution) to a cell culture flask and incubate at 37°C in a 5% CO2 constant temperature incubator. Change the culture medium once a day. When the cells are in good condition (70% to 80% fusion), use 0.2% digestion solution (trypsin-EDTA) for digestion and passage.

[0031] Adhesion assay: Digested HT-29 cells were adjusted to a concentration of 10 5 cell / mL, inoculated into a 12-well cell culture plate, 1 mL per well, incubated in an incubator with a 5% CO2 concentration until the cells grow to a monolayer, washed twice with sterile PBS, one well was digested with trypsin, and the cells were counted using a hemocytometer; 1 mL of BLa80 bacterial suspension and control bacteria Lactobacillus rhamnosus LGG bacterial suspension were added to 2 wells respectively, incubated at 37°C in a 5% CO2 incubator for 2 hours, washed the cells 5 times with sterile PBS, removed the non-adherent bacterial suspension, added 0.2 mL of trypsin-EDTA buffer to each well to digest the cells for 5 minutes, added 0.8 mL of PBS after digestion, and evenly blown, and the bacterial solution was taken for dilution and viable bacterial count. The above cell adhesion experiment used Lactobacillus rhamnosus LGG as a comparison strain, and each experiment was performed in 3 parallels. Lactobacillus rhamnosus LGG is a commercial strain recognized in the industry for good adhesion. The adhesion of the strain to HT-29 cells was tested by adhesion experiment, and the results are shown in Table 3.

[0032] Table 3 Test results of adhesion characteristics of Bifidobacterium lactis BLa80

[0033]

[0034] From the results in Table 3, it can be seen that the adhesion performance of the Bifidobacterium lactis BLa80 strain of the present invention has obvious advantages.

[0035] Experimental Example 4: Bifidobacterium lactis BLa80 relieves and improves atopic dermatitis in mice

[0036] Experimental animals and groups: 40 C57BL / 6 female mice, SPF grade, 6-7 weeks old, maintained a circadian rhythm of 12 hours each. Free access to food and water, the breeding environment temperature is 18-24 ° C, the relative humidity is 50%-70%. They were randomly divided into 4 groups, namely blank group (Blank), model group (Model), Bifidobacterium lactis BB12 group, and Bifidobacterium lactis BLa80 group. After the mice adapted for one week, gavage began in the second week until the end of the experiment. The Bifidobacterium lactis BB12 group and the Bifidobacterium lactis BLa80 group were gavaged with the corresponding strains (1×10 9The blank group and the model group were gavaged with the same amount of PBS solution. The model was established from the third week. The hair on the back of the mice was removed one day before the model was established. The area was about 2.5 cm × 1.5 cm. On the day of the experiment, except for the blank group, 25 μL of 0.5% DNFB (2,4-dinitrofluorobenzene) solution was applied to the back of the other three groups of mice for sensitization. On the 5th, 8th and 14th days, 20 μL of 0.2% DNFB solution was applied to the back of the right ear and back of the mice to induce dermatitis. 20 μL of acetone / olive oil matrix solution was applied to the left ear as a control.

[0037] Evaluation of mouse scratching behavior: Before and after the experiment, place the mice separately and observe the number of times the mice scratched their ears and head with their front paws, scratched their trunk and back with their hind paws, and gnawed various parts of the body with their mouths within 10 minutes. Continuous scratching was counted as one time.

[0038] Observation and evaluation of mouse behavior: Before and after the experiment, the mouse behavior was evaluated according to the following scoring criteria.

[0039] Table 4 Evaluation criteria for mouse behavior observation

[0040]

[0041] Evaluation of skin lesions on the back of mice: The inflammation of the back skin lesions was evaluated according to the clinical manifestation standards; erythema, edema / papule, epidermal exfoliation / scratches, and scales (dry) were divided into four levels: none, mild, moderate, and severe, and were scored as 0, 1, 2, and 3 points respectively. Among them, no symptoms were scored as 0 points; erythema: vaguely visible, pink (1 point); clearly visible, dark red (2 points); dark red or fiery red (3 points). Edema / papule: almost imperceptible separation from the skin surface (1 point); clearly visible above the skin surface but not significantly (2 points); significantly separated from the skin surface (3 points). Epidermal exfoliation / scratches: a few shallow epidermal exfoliations (1 point); many superficial and / or some deeper epidermal exfoliations (2 points); diffuse superficial and / or many deep epidermal exfoliations (3 points). Scales (dry): bran-like exfoliation (1 point); thin coverage of flaky scales (2 points); thick coverage (3 points)

[0042] Evaluation of mouse ear swelling: On day 0 of modeling and after modeling, the thickness of the middle part of both ears of the mouse was measured with a vernier caliper.

[0043] Serum cytokine detection: Serum was collected before mice were killed, and the ELISA method was used to detect the levels of IgE, IL-4, IFN-γ, and IL-10 in the serum.

[0044] After modeling, the behavioral observation scores of mice were significantly lower than those of the control group mice. The scratching behavior scores, back skin lesion inflammation scores, and ear swelling were all significantly different from those of the control group, indicating that the DNFB-induced atopic dermatitis mouse model was successfully established.

[0045] Bifidobacterium lactis BLa80 can reduce the number of scratching times in mice, and the effect is extremely significant, and is significantly better than the BB12 group.

[0046] Bifidobacterium lactis BLa80 significantly improved the behavioral observation scores of atopic dermatitis mice and basically restored them to the scores of the control group, and was better than the BB12 group.

[0047] Bifidobacterium lactis BLa80 improved the inflammation of skin lesions on the back of mice, and there was a very significant difference compared with the model group, and the effect was better than that of the BB12 group.

[0048] Bifidobacterium lactis BLa80 significantly improved ear swelling in mice, which was significantly different from the model group, and the improvement effect was better than that of the BB12 group.

[0049] Table 5 Bifidobacterium lactis relieves and improves allergic reactions in mice with atopic dermatitis

[0050]

[0051] Note: Compared with the control group: #p<0.05, ##p<0.01; compared with the model group: *p<0.05, **p<0.01.

[0052] As shown in Table 5, Bifidobacterium lactis BLa80 can significantly reduce the scratching behavior and back skin lesion inflammation scores of mice, significantly reduce the behavioral observation scores and ear swelling, and the control strain Bifidobacterium lactis BB12 can significantly reduce the scratching behavior, behavioral observation scores and back skin lesion inflammation scores of mice. Bifidobacterium lactis BLa80 is better than Bifidobacterium lactis BB12 in alleviating and improving the allergic reactions of mice with atopic dermatitis.

[0053] Table 6 Effect of Bifidobacterium lactis BLa80 on cytokine expression levels in atopic dermatitis mice

[0054]

[0055] Note: Compared with the control group: #p<0.05, ##p<0.01; compared with the model group: *p<0.05, **p<0.01.

[0056] As shown in Table 6, Bifidobacterium lactis BLa80 significantly reduced the IgE level of the model group, significantly increased the IFN-γ level, and significantly reduced the IL-4 level. Bifidobacterium lactis BLa80 can significantly increase the IFN-γ / IL-4 ratio. IFN-γ and IL-4 are characteristic cytokines secreted by Th1 and Th2 cells, respectively. The balance between the two, that is, the ratio, indirectly reflects the balance between pro-inflammatory and anti-inflammatory. The IFN-γ / IL-4 ratio of the BLa80 group was increased to a level with no significant difference from the control group. It can be seen that Bifidobacterium lactis BLa80 can effectively improve and alleviate the allergic reactions of mice with atopic dermatitis.

[0057] Experimental Example 5: Preparation of Bifidobacterium lactis BLa80 powder

[0058] 1) Bifidobacterium lactis BLa80 powder fermentation process:

[0059] The Bifidobacterium lactis strain stored in a glycerol tube was inoculated into 10 mL of L-MRS medium, and anaerobically cultured at 37°C for 18 hours to obtain the first-level seeds; the first-level seed bacterial liquid was inoculated into the L-MRS medium at an inoculation amount of 2% (V / V), and cultured at 37°C under anaerobic conditions for 12 hours to obtain the second-level seeds, which were then inoculated into the L-MRS medium at an inoculation amount of 2%, and cultured at 37°C under anaerobic conditions for 12 hours to obtain the third-level seeds.

[0060] The fermentation medium formula is: lactose 20 / L, beef extract powder 10 / L, yeast extract powder 10 / L, bovine bone peptone 10g / L, dipotassium hydrogen phosphate 3g / L, ammonium hydrogen citrate 3g / L, sodium acetate 5g / L, magnesium sulfate 0.5g / L, manganese sulfate 0.1g / L, Tween-801mL / L, L-cysteine ​​hydrochloride 1g / L.

[0061] Fermentation: 10L fermentation medium was added to a 15L anaerobic fermentation tank, sterilized at 121°C for 20 minutes, and the pH of the fermentation medium was adjusted to 7 with NaOH after cooling. 200mL of the third-grade seed solution of Bifidobacterium lactis was accessed into the fermentation medium and fermented at a temperature of 37°C and a speed of 100rpm. During the fermentation process, 25% ammonia water was automatically added to maintain the pH value of the fermentation liquid at 5.5-6.0, and nitrogen was introduced throughout the fermentation process. When the OD600 of the fermentation liquid no longer increased after 16h of fermentation, the fermentation was terminated.

[0062] 2) The preparation process of the Bifidobacterium lactis BLa80 powder:

[0063] (1) Centrifuge the fermentation liquid obtained by the above-mentioned high-density fermentation of Bifidobacterium lactis, remove the supernatant, and collect the bacterial sludge. The centrifugation method is preferably performed at 4°C and 8000 rpm for 10 minutes.

[0064] (2) The bacterial sludge collected by centrifugation was mixed evenly with the freeze-drying protective agent in a mass ratio of 1:1, placed in a freeze dryer, pre-frozen at -40°C for 2 h, then heated to -10°C for 4 h under 10 Pa conditions and freeze-dried for 15 h, and then heated to 25°C for 2 h under 20 Pa conditions and freeze-dried for 10 h to obtain freeze-dried Bifidobacterium lactis powder.

[0065] The freeze-dried protective agent comprises the following components in percentage by mass: 6% trehalose, 8% maltodextrin, 2% sucrose, 0.02% vitamin C, 3% glycerol, 0.5-0.8% L-arginine, and the balance is water.

[0066] Example 6: Human food trial of Bifidobacterium lactis BLa80 in improving and alleviating atopic dermatitis

[0067] A total of 124 people were recruited for this human food trial and randomly divided into two groups: the treatment group (70 people) took one probiotic Bifidobacterium lactis BLa80 product (containing 1×10 9 CFU / strip), and the placebo group (46 people) took the same amount of maltodextrin. Dosage: It can be taken directly or with warm water (not exceeding 37°C). The intervention period is 4 weeks. After the end of the experiment, the patients with atopic dermatitis will continue to be followed up for 2 weeks to observe whether there are any adverse reactions.

[0068] Inclusion criteria: ① Itching; ② Typical morphology and location (flexural dermatitis) or atypical morphology and location accompanied by xeroderma; ③ Chronic or chronic recurrent course. AD can be diagnosed if all three of the above are present. Typical morphology and location (flexural dermatitis) include facial and extremity involvement. Atypical morphology and location include: ① Typical eczematous rash occurring in non-flexural areas (scalp dermatitis, eyelid eczema, nipple eczema, vulvar eczema, nummular eczema, fingertip eczema, nonspecific hand or foot dermatitis / atopic winter foot, nail or periungual eczema, and eczematous rash in other parts of the body); ② Atypical eczematous rash, simple pityriasis, cheilitis, subauricular and postauricular / subnasal fissures, prurigo, sweat herpes, papular lichenoid variant.

[0069] Exclusion criteria: ① Patients with serious diseases of important organs such as heart, kidney, brain, etc. and patients who have used antihistamines within 7 days before enrollment. ② Patients who have consumed yogurt or probiotic drinks for a long time.

[0070] The volunteers were informed of the nature and content of the trial and signed an informed consent form. After 4 weeks of trial, the original questionnaire data were counted, and 8 people were lost to follow-up. Before and after the trial, the standardized severity index of atopic dermatitis (SCORAD index) and the dermatology life quality index (DLQI) scale were used to investigate and record the volunteers' conditions.

[0071] Table 7 SCORAD scores of the two groups before and after the trial

[0072]

[0073]

[0074] Note: Comparison between the two groups after the trial: #p<0.05, ##p<0.01; Comparison between before and after the trial: *p<0.05, **p<0.01.

[0075] Before the trial, there was no significant difference in SCORAD scores between the BLa80 group and the placebo group. After taking Bifidobacterium lactis BLa80, the SCORAD scores were significantly reduced compared with those before the trial. At the same time, after the trial, the SCORAD scores of the BLa80 group and the placebo group were significantly different. This shows that Bifidobacterium lactis BLa80 can effectively improve and relieve allergic reactions such as skin conditions in patients with atopic dermatitis.

[0076] Table 8 DLQI scores of the two groups before and after the trial

[0077]

[0078] Note: Comparison between the two groups after the trial: #p<0.05, ##p<0.01; Comparison between before and after the trial: *p<0.05, **p<0.01.

[0079] Before the trial, there was no significant difference in DLQI scores between the BLa80 group and the placebo group. After taking Bifidobacterium lactis BLa80, the DLQI score was significantly lower than that before the trial. At the same time, after the trial, the DLQI score of the BLa80 group was significantly different from that of the placebo group. This shows that Bifidobacterium lactis BLa80 can improve and enhance the quality of life of patients with atopic dermatitis.

Claims

1. The use of Bifidobacterium lactis BLa80 in the preparation of drugs for improving and relieving atopic dermatitis. The Bifidobacterium lactis BLa80 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on May 17, 2021, with the deposit number CGMCC No.22547.

2. The use according to claim 1, characterized in that: The drugs include but are not limited to microecological preparations and external-use drugs.

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