CN116083323A - Bifidobacterium lactis HC2786 capable of relieving anaphylactic reaction, and product and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, in particular to bifidobacterium lactis HC2786 capable of relieving anaphylactic reaction and a production method thereofAnd products and applications. Bifidobacterium lactis @Bifidobacterium animalis subsp.lactis) HC2786 was preserved in China center for type culture Collection (CCTCC NO) at the 12-month 23-year 2022 with the preservation address of China, the university of Wuhan and Wuhan: m20222008. The bifidobacterium lactis HC2786 provided by the invention can promote the secretion of the levels of interleukin-4 (IL-4) and gamma interferon (IFN-gamma) by cells, has stronger survival rate in the gastrointestinal tract, has stronger colonisation ability in the intestinal tract, and can relieve allergic symptoms by improving the autoimmunity of a host.

Description

A strain of Bifidobacterium lactis HC2786 capable of alleviating allergic reactions and its products and applications

technical field

The invention relates to the technical field of microbes, in particular to a bifidobacterium lactis HC2786 capable of relieving allergic reactions and its products and applications.

Background technique

Allergy, also known as hypersensitivity, is a pathological immune response. As the global prevalence increases year by year, it has become a common disease that plagues human health. Allergy is a pathological immune response in which the body is continuously stimulated by certain allergens or re-stimulated by the same allergen, resulting in physiological dysfunction and/or tissue cell damage. There are four types (type I, type II, type III and Type IV). Common type I hypersensitivity diseases include eczema, asthma, atopic rhinitis, contact dermatitis, and food allergy.

Traditional Chinese and Western medicine generally believe that allergies are a problem of low human immunity, but many allergic patients actively exercise and take supplements on time, but their allergies have not improved. Clinical medicine can alleviate the clinical symptoms of allergic diseases, but it can bring dry mouth, drowsiness, insomnia and other adverse reactions, which affect the quality of life of patients. In recent years, the research on flora has provided a new basis for the use of intestinal flora to treat diseases, and also made the treatment more targeted.

Probiotics are an important class of immune regulators. They regulate the stability of the intestinal environment and the balance of the immune system through a series of metabolic activities in the human intestine, enhance the body's gastrointestinal digestion and absorption capacity, and at the same time protect the body's intestinal immune system. Prevents the occurrence of allergic reactions against harmful external sources. Studies have shown that probiotics can specifically participate in the body's immune response, relieve clinical symptoms of allergic diseases, and improve the quality of life of patients with diseases. The therapeutic/preventive effects of probiotics in allergic diseases mainly have four mechanisms: regulating the balance of Th1/Th2; inducing the production of Treg; promoting the production of antigen-specific IgG/IgA and inhibiting the production of antigen-specific IgE; antagonizing the pathogenic Bacteria, maintain the strong membrane barrier, promote the production of antimicrobial peptides, etc. The imbalance of Th1/Th2 dominated by Th2 is an important mechanism leading to allergies: Th2 cells are important cells that mediate the body's humoral immunity. The increase in the number of cells and the enhancement of their functions eventually lead to the secretion of high levels of IgE by B cells, causing The occurrence of allergic reactions; Th1 cells can secrete a large amount of cytokines such as IL-2, IL-12, and IFN-γ, which can inhibit the activity of Th2 cells, thereby blocking the synthesis of IgE, and finally alleviating allergic symptoms. Animal experiments and a large number of clinical trials have shown that probiotics can regulate the balance of Th1/Th2 immune responses in patients with allergic diseases, induce the production of regulatory T cells, increase the diversity of intestinal flora, antagonize allergic inflammation, and possibly correct The patient's "allergic constitution".

Based on this, it is necessary to provide a probiotic and its product with the effect of alleviating allergic reactions.

Contents of the invention

Aiming at the technical problem that clinical treatment can alleviate the clinical symptoms of allergic diseases, but it can cause adverse reactions such as dry mouth, drowsiness, and insomnia, and affect the quality of life of patients, the invention provides a strain of Bifidobacterium lactis that can relieve allergic reactions HC2786 and its products and applications, Bifidobacterium lactis HC2786 can increase the level of interleukin-4 (IL-4) and interferon-γ (IFN-γ) secreted by cells, and has a strong survival rate in the gastrointestinal tract, and It also has a strong colonization ability in the intestinal tract, which can relieve allergic symptoms by enhancing the host's own immunity.

In the first aspect, the present invention provides a strain of Bifidobacterium animalis subsp. lactis HC2786 that can alleviate allergic reactions. The strain has been deposited in the China Center for Type Culture Collection on December 23, 2022, and the preservation address is China , Wuhan, Wuhan University, the deposit number is CCTCC NO: M 20222008.

In the second aspect, the present invention provides a product containing Bifidobacterium lactis HC2786.

Further, the specification of Bifidobacterium lactis HC2786 in the product can be ≥10 billion CFU/g, ≥20 billion CFU/g, ≥50 billion CFU/g, ≥100 billion CFU/g or ≥200 billion CFU/g g.

Further, the product is prepared according to the following preparation method:

(1) Expansion of strains

The Bifidobacterium lactis HC2786 is subjected to primary culture and secondary culture sequentially under anaerobic conditions, and then the seed liquid is transferred to a fermenter for anaerobic fermentation to obtain a fermented liquid;

(2) Preparation of bacteria powder

Centrifuge the fermented liquid to obtain the bacteria slime, vacuum freeze-dry after coating the bacteria sludge, crush the freeze-dried block to obtain the original powder of Bifidobacterium lactis, mix the original powder of Bifidobacterium lactis with fructo-oligosaccharide and isomalto-oligosaccharide have to.

Further, in step (1), the medium components used for primary culture include peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, citric acid Triammonium 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, cysteine hydrochloride 0.5g/L and Tween 80 1.0g/L;

The medium components used in secondary culture include peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, cysteine hydrochloride 0.5g/L and edible oil 1.0g/L.

Further, the medium components used for fermentation include whey powder 22g/L, glucose 35g/L, yeast powder 32g/L, carrot juice 5g/L, manganese sulfate 0.05g/L, magnesium sulfate 0.1g/L, phosphoric acid Dipotassium hydrogen 1.0g/L, triammonium citrate 1.0g/L, sodium acetate 2.0/L, cysteine hydrochloride 0.3g/L and edible oil 1.0g/L.

In the third aspect, the present invention also provides an application of Bifidobacterium lactis HC2786 in the preparation of a product for alleviating allergic symptoms.

Further, the product for relieving allergic symptoms is a product for relieving allergic rhinitis, allergic dermatitis, allergic conjunctivitis and/or allergic asthma.

The beneficial effects of the present invention are:

The Bifidobacterium lactis HC2786 provided by the present invention has excellent colonization and adhesion capabilities, and can improve its microbiota or its metabolites by remaining in the host's gastrointestinal tract for a long time, thereby enhancing the host's own immunity. Moreover, the bifidobacterium lactis can affect the concentration of cytokines in cells that are closely related to the alleviation or treatment mechanism of allergic diseases, such as IL-4 and IFN-γ. It has been verified by clinical trials that the Bifidobacterium lactis HC2786 has the function of alleviating allergic reactions.

Description of drawings

In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art. Obviously, for those of ordinary skill in the art, In other words, other drawings can also be obtained from these drawings on the premise of not paying creative work.

FIG. 1 is a histogram comparing the secretion levels of interleukin-4 in the cell culture supernatant in Example 1. FIG.

FIG. 2 is a histogram comparing the secretion levels of interferon-γ in the cell culture supernatant in Example 1. FIG.

Fig. 3 is a line graph of the colonization survival rate of each strain in the simulated gastric juice in Example 1 as a function of time.

Fig. 4 is a line graph of the colonization survival rate of each strain in the simulated intestinal fluid in Example 1 as a function of time.

Fig. 5 is a histogram comparing colonization survival rates of each strain in Example 1 after gastric juice-intestinal juice two-step simulated digestion.

Fig. 6 is a histogram comparing the adhesion rate of each strain to mucin and the adhesion rate of Caco-2 cells in Example 1.

7 is a histogram comparing the allergic remission rates of patients in each test group in Example 3.

Detailed ways

In order to enable those skilled in the art to better understand the technical solutions in the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described The embodiments are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

Example 1 Screening of Bifidobacterium lactis HC2786

1. Isolation of Probiotic Strains

(1) Sampling: In June 2020, in Weifang City, Shandong Province, breast milk samples were taken from healthy and normal women who were breastfeeding, stored in refrigerators, and numbered and recorded.

(2) Bacteria enrichment: Selective enrichment of the sampled samples in liquid MRS medium.

(3) Separation: Operate under sterile conditions, perform ten-fold gradient dilution on the enriched sample, apply 100 μL of each gradient to MRS solid medium, and culture under aerobic and anaerobic conditions respectively. Incubate at 37°C for 24h.

Eight lactic acid bacteria strains were initially screened, temporarily numbered as RG-1, RG-2, YRG-3, RG-4, RQ-5, RQ-6, YRG-7, and YRG-8. At the same time, the existing lactic acid bacteria strains WT-003, ZW-001, and LYS-006 in the laboratory were used for comparison, that is, a total of 11 strains were used for subsequent experiments.

2. Screening of lactic acid bacteria strains with anti-allergic function

(1) Bacterial suspension preparation: 11 strains of lactic acid bacteria were respectively transferred to liquid MRS medium, cultivated at 37°C until the stationary phase, and centrifuged at 6000r/min for 8min to collect the bacteria. Use sterile PBS buffer to resuspend, measure the number of bacteria by plate coating, and adjust the concentration of the bacteria suspension to about 10 ⁹ CFU/mL for later use.

(2) Isolation and culture of spleen cells: Disinfect the surface of the mice sacrificed by decapitation with 75% alcohol, take the spleens of the mice under aseptic conditions, and put them into a plate containing RPMI-1640 culture medium (containing 10% fetal Bovine serum and 1% penicillin and streptomycin), the spleen was washed repeatedly with a 1mL syringe until the dark red color changed to light red during the operation, then the spleen cells were collected, centrifuged to remove the supernatant, added to erythrocyte lysate for lysis for 1 min, and cultured in RPMI-1640 The splenocytes were obtained by washing with liquid three times, and the cell concentration was adjusted to 5×10 ⁶ mL ^-1 for later use.

(3) Determination of cytokine secretion level: Splenocytes were co-cultured with 11 lactic acid bacteria strains and sterile medium blank at a ratio of 1:1, and the stimulator ovalbumin was added to adjust the final mass concentration to 1 mg/mL. After 72 hours of culture, the cell supernatant was collected by centrifugation, and the secretion levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in the cell culture supernatant were detected by enzyme-linked immunosorbent assay (ELISA).

(4) Conclusion: IL-4 is mainly produced by activated T cells and belongs to Th2 cytokines, which can promote the proliferation of activated B cells, the production of molecules and Ig, and the class conversion into IgE and IgG1. When the body has an allergic reaction, IL-4 will Inhibits the differentiation of Th cells into Th2. IFN-γ is mainly produced by activated T cells and NK cells, and belongs to the Th1 type cytokine. When the body has an allergic reaction, IFN-γ promotes the differentiation of Th cells into Th1. Th1 cells have the effect of inhibiting the activity of Th2 cells, and by inhibiting their activity, they can block the synthesis of IgE and finally relieve allergic symptoms.

Compared with the blank group CK, the secretion levels of IL-4 in the experimental groups WT-003, RG-2, YRG-3, RQ-5, RQ-6 and YRG-8 decreased by 25.80%, 25.47%, 38.63%, respectively. 35.49%, 26.22% and 30.62%, compared with the other 5 experimental groups, the reduction effect is the most significant.

Compared with the blank group CK, the IFN-γ secretion levels of the experimental groups WT-003, RG-2, YRG-3, RG-4, RQ-5, RQ-6 and YRG-8 were all significantly increased, respectively 42.89%, 42.84%, 52.30%, 39.86%, 40.16%, 42.64%, and 44.20%.

Based on the above results, six strains of WT-003, RG-2, YRG-3, RQ-5, RQ-6 and YRG-8 were selected for subsequent experiments.

3. Determination of Strain Colonization Ability

(1) Solution preparation:

①Gastric buffer: accurately weigh 0.5144g KCl, 0.1225g KH ₂ PO ₄ , 2.7585g NaCl, 2.1002g NaHCO ₃ , 0.0203g MgCl ₂ 6H ₂ O, 0.0480g (NH ₄ ) ₂ CO ₃ , 0.0083g CaCl ₂ , and set the volume to 1000mL.

②Simulated gastric juice: Dissolve 3 g of pepsin in 1000 mL of gastric juice buffer, adjust the pH to 3.0 with 1 mol/L HCl, filter and sterilize with a 0.22 μm filter membrane, and make up immediately.

③Intestinal fluid buffer: Accurately weigh 0.5069g KCl, 0.1089g KH ₂ PO ₄ , 2.2442g NaCl, 7.1408g NaHCO ₃ , 0.0067g MgCl ₂ ·6H ₂ O, 0.0333g CaCl ₂ and make up to 1000mL.

④Simulated intestinal fluid: Dissolve 1g of trypsin and 3g of bile salt in 1000mL of intestinal fluid buffer, adjust the pH to 8.0 with 1mol/L NaOH, filter and sterilize with a 0.22μm filter membrane, and prepare and use immediately.

⑤ Bacteria enrichment: 6 strains of lactic acid bacteria were cultured in MRS liquid medium overnight, centrifuged at 6000r/min for 10min, and the bacteria sludge was washed 3 times with sterile PBS buffer, and then set aside.

(2) Simulated digestion with gastric juice: take 1 mL of enriched bacterial cells, add 9 mL of simulated gastric juice, set up 3 parallel sets of bacteria for each strain, in a constant temperature water bath at 37°C, rotate at 100 r/min, shake for 0h, 1h, 2h, 3h, 4h, respectively The number of live bacteria was calculated by the diluted coating plate method, and each sample was coated with 3 replicates. Calculate the colonization survival rate of lactic acid bacteria according to the formula: colonization survival rate = the number of lactic acid bacteria that survived colonization/the initial number of lactic acid bacteria × 100%.

(3) Intestinal juice simulated digestion: take 1 mL of enriched bacteria, add 9 mL of simulated intestinal juice, set 3 parallels for each strain of bacteria, 37 ° C constant temperature water bath, speed 100 r/min, shake 0h, 1h, 2h, 3h, 4h, respectively The number of live bacteria was calculated by the diluted coating plate method, and each sample was coated with 3 replicates. Calculate the colonization survival rate of lactic acid bacteria according to the formula: colonization survival rate = the number of lactic acid bacteria that survived colonization/the initial number of lactic acid bacteria × 100%.

(4) Gastric juice-intestinal juice two-step simulated digestion: take 1 mL of enriched bacteria, add 9 mL of simulated gastric juice, set up 3 parallels for each strain of bacteria, 37 ° C constant temperature water bath, speed 100 r/min, shake for 3 hours, take out and centrifuge quickly, Add 9mL of simulated intestinal fluid to it, shake and culture under the same conditions for 2h, and finally count by dilution coating plate method, and each sample is coated with 3 replicates. Calculate the colonization survival rate of lactic acid bacteria according to the formula: colonization survival rate = the number of lactic acid bacteria that survived colonization/the initial number of lactic acid bacteria × 100%.

(5) Conclusion: The colonization and adhesion of lactic acid bacteria in the gastrointestinal tract of the host can prolong their residence time in the body, and then affect the health of the host by improving the local microbiota or its metabolites.

The results of the survival rate of strains at different times of simulated gastric juice digestion are shown in Figure 3. After 0h, 1h, 2h, 3h, and 4h of simulated gastric juice digestion, the survival rate of the strains decreased to varying degrees with time. See, in the simulated gastric juice with time, the colonization ability of YRG-3 strain is the strongest, followed by RQ-6, followed by WT-003, and then RG-2, RQ-5 and YRG-8 respectively.

The survival rate of the strains at different times of simulated intestinal juice digestion is shown in Figure 4. After 0h, 1h, 2h, 3h, and 4h of simulated intestinal juice digestion, the survival rate of the strains decreased to varying degrees with the prolongation of time. The curves of the six strains were combined. , in the simulated intestinal fluid over time, the order of colonization ability from strong to weak was YRG-3>WT-003>RQ-6>YRG-8>RG-2>RQ-5.

The survival rate of the strains after the two-step digestion of simulated gastric juice-intestinal juice is shown in Figure 5. During the experiment of the two-step simulated gastric juice-intestinal juice method, the survival rates of the six bacterial strains were different after 3 hours of gastric juice digestion and 2 hours of intestinal juice digestion. Among them, the colonization ability The order from strong to weak is YRG-3>WT-003>RQ-6>YRG-8>RG-2>RQ-5. The strains with a survival rate of over 70% were YRG-3, WT-003 and RQ-6, so these three strains were selected for subsequent experiments.

4. Determination of Strain Adhesion Ability

(1) Determination of the strain's ability to adhere to mucin:

① Mucin model establishment: Weigh 10 mg of mucin and prepare a mucin solution with a concentration of 1 mg/mL in sterile PBS buffer, and store at -20°C. Take 500 µL of mucin solution and fix it on a 24-well cell culture plate for 1 h, culture at 4°C overnight, then add an equal volume of mucin and continue to culture at 37°C for 2 h to make up for blank spots, and then wash twice with sterile PBS buffer.

②Measurement of adhesion ability: The 3 bacterial strains cultivated overnight were collected and resuspended in sterile PBS buffer respectively, the number of bacteria was adjusted to 10 ⁸ CFU/mL, counted by spreading and recorded. Add 500 µL of bacterial suspension to the mucin model, incubate at 37°C for 1 hour, wash 5 times with sterile citrate buffer to remove unbound bacteria, add 1 mL of 0.5% (v/v) Tween 80 to collect adherent bacteria . Finally, counting was performed by the dilution coating plate method, and each sample was coated with 3 replicates. Calculate the adhesion rate of lactic acid bacteria according to the formula: adhesion rate = number of lactic acid bacteria adhered/initial number of lactic acid bacteria × 100%.

(2) Determination of the adhesion ability of strains to Caco-2 cells: inoculate 0.5 mL of Caco-2 cells into a 24-well cell culture plate, in which the Caco-2 cells were adjusted to 2×10 ⁵ cell/mL, and cultured to a monolayer, After washing twice with PBS, add 500 µL of lactic acid bacteria suspension (10 ⁸ CFU/mL), incubate at 37°C for 2 hours, wash with PBS three times to remove non-adhered lactic acid bacteria, add 150 µL of trypsin cell digestion solution, and add 350 µL of MEM for complete culture after the cells are completely detached The solution was used to stop the digestion, and the final count was performed by the dilution coating plate method, and each sample was coated with 3 replicates. Calculate the adhesion rate of lactic acid bacteria according to the formula: adhesion rate = number of lactic acid bacteria adhered/initial number of lactic acid bacteria × 100%.

(3) Conclusion: The colonization and adhesion of lactic acid bacteria in the gastrointestinal tract of the host can prolong their residence time in the body, and then affect the health of the host by improving the local microbiota or its metabolites.

The determination results of mucin adhesion rate and Caco-2 cell adhesion rate are shown in Fig. 6 . According to the test results of the adhesion ability of strains to mucin, it can be seen that among the three strains of WT-003, YRG-3 and RQ-6, the strain with the highest adhesion rate to mucin is YRG-3; the adhesion ability to Caco-2 cells The measurement results show that the adhesion rate from high to low is YRG-3, RQ-6, WT-003. Combining the two results, YRG-3 had the highest adhesion rate to mucin and Caco-2 cells, so this strain was selected for subsequent strain identification and clinical trials.

5. Strain identification

The 16S rDNA identification of the strain YRG-3 with the best effect was carried out. After identification, the strain was Bifidobacterium animalis subsp. lactis , named HC2786. The sequencing results are as follows:

CctgcggggatgctgcgcgcgcgcgcgccAcAacggggggccccccgcgcgCTGGGGGGGGGGGGGGGGGGGGCGCCCCTGCCTGCTGCACCC GGAATAGCTCTCTCTCTGGGGGGGTGTGTGTAATAATACCGGGCCCCTCCCATCATCATCATGGGGGGGGGGGGCTTTGCGGGGGGGGGGGGGGCGCGCGGGGGGGGGGGGGGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCT GTTGACGGTAGCGCCCCTGAGGGGGGACCCCCCACACATGGGGGGCCCCCCCTCTCTCTCTCTCTCGGGGGGGGGCAATGCCGCCGCCGCCCCCGCGCGCGTGTG CGGGGGGGGCCTCGGGGGGGTGTGTGCGCGCGCAAGGCAGGCAGCAGCACACGGGGCCCCCCGGGGGGGGGAATCCCCCCCCCCCGCCGCGCGCGCGCGGCGGGGGGGGGGGGGCGGCGCGCGCGCGCGCGCCGCCGCCGCCCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCACCGCCCCCCCCCACCGCCCCCCCCCGCCCCCCCCCCCCC GTGCGCGCGTTTTCGGGATTTTTGGGGGCGTAAAGGGCTCGTCGCGCGTCGTCGTCGGTGTGTCCCCGCGCGGGCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCGTCGTCGTCGTCGTCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCC ActggaattCCCCCCGGTGGGGGGGGGTGTGTGTGTAGGGGGGGGGGGGGAACCAATGCGCGCGCGTCTGGGGGGGGCGCGCGCGAgCGGGGGGGGAgGAgGAGAGAGAGAGAGACCCCTG GTAGTCCCCGCCGCGGGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCGCCGCCAAAAAAGAAAAGAAAAGAAAAAAAAAATTGAC GGGGGCCCCACAAGCGGGGGGGCATGCGGGGAATGCGCGCGCGAACGACTTACTTGGGGGGGGGGCCCCGCGCGGCGGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCGCAACGCGCAAT AtggtcGTCGTCGTCGTCGTGTGTGTGAGAGAGTGTGTGTCCCCCCGCGCGCGCGCGCCGCCCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGAga GTGGGGGACGTCACATCATCATGCCCCCCCCCCCCCAGGCATCTAAACGGCGCGCGCGCGCGCGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCGCAG TCTGCACTCGACGACGCGCGTGCGGCGGGGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCGCCCCCGCCCCCGCCCCCCCG GTGGCCCCCCCTTGGGGGGAG.

The Bifidobacterium animalis subsp. lactis HC2786 was sent to the China Center for Type Culture Collection for preservation, the preservation address is China, Wuhan, Wuhan University, the preservation number is CCTCC NO: M20222008, and the preservation date is December 23, 2022 day.

Example 2 Preparation of products containing Bifidobacterium lactis HC2786

(1) Strain expansion:

①First-level cultivation: Warm the refrigerated Bifidobacterium lactis HC2786 at room temperature for 1 hour, inoculate it in 3.5L liquid medium-1 and culture it anaerobically for 20 hours to make first-level seeds;

The components of liquid medium 1 include peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g /L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, cysteine hydrochloride 0.5g/L and Tween 80 1.0g/L.

②Secondary cultivation: transfer the primary seeds to a 100L seed tank, and culture in liquid medium 2 with nitrogen for 12 hours to obtain secondary seeds;

The components of liquid medium two include peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g /L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, cysteine hydrochloride 0.5g/L and edible oil 1.0g/L.

③Fermentation: Subsequently, with 10% inoculum amount, the secondary seeds were replanted into a 1000L fermenter, and fermented anaerobically with nitrogen in the liquid medium 3 to obtain a fermented liquid;

The components of liquid medium three include whey powder 22g/L, glucose 35g/L, yeast powder 32g/L, carrot juice 5g/L, manganese sulfate 0.05g/L, magnesium sulfate 0.1g/L, dipotassium hydrogen phosphate 1.0g/L, triammonium citrate 1.0g/L, sodium acetate 2.0/L, cysteine hydrochloride 0.3g/L and edible oil 1.0g/L.

(2) Bacteria powder preparation: Centrifuge the fermentation liquid to obtain the bacteria slime, after coating the bacteria slime, vacuum freeze-dry to get the freeze-dried block, crush the freeze-dried block to get the original powder of Bifidobacterium lactis, mix the original powder of Bifidobacterium lactis with oligomerization Mix fructose and isomaltooligosaccharide to obtain a product containing Bifidobacterium lactis HC2786.

By adjusting the amount of Bifidobacterium lactis raw powder added, products with different specifications of viable bacteria can be obtained, such as the number of viable bacteria ≥ 10 billion CFU/g, ≥ 20 billion CFU/g, ≥ 50 billion CFU/g, ≥ 100 billion CFU/g or ≥200 billion CFU/g, etc.

Example 3 Clinical trial verification of the anti-allergic alleviating ability of Bifidobacterium lactis HC2786

(1) Test method: According to the method of Example 2, a probiotic product containing Bifidobacterium lactis HC2786 with a viable count of 10 billion CFU/g and 2 g/bar was prepared. Recruit 120 subjects with allergic rhinitis, 108 subjects with allergic dermatitis, 102 subjects with allergic conjunctivitis and 94 subjects with allergic asthma, each divided into normal medication (ZCFY) group and normal medication plus bacterial powder (ZCFY+RSQ) The clinical trial was carried out in the group, and the trial period was 24 weeks, during which the subjects were not allowed to consume other types of probiotics.

During the whole clinical trial process, the allergic symptom score sheet, quality of life questionnaire and medication situation of the patients were collected every week, and the remission rate was 0 without any intervention as the baseline, and the symptom remission was counted according to the symptom score.

(2) Test groups:

ZCFY group: The ZCFY group only needs to take medication during the allergic period, and live a normal life in the rest of the period, without using other probiotics during the period. Among them, patients with allergic rhinitis, allergic dermatitis, and allergic conjunctivitis all take levocetirizine hydrochloride normally during the allergic period, once a day, one tablet each time; allergic asthma patients need to take it according to the doctor's prescription during the allergic period. inhaled drugs.

ZCFY+RSQ group: On the basis of ZCFY group, patients with allergic rhinitis, allergic dermatitis, allergic conjunctivitis and allergic asthma need to take milk-containing formula with 10 billion CFU/g and 2g/bar per day. Two probiotic products of Bifidobacterium HC2786 are taken directly in the mouth or dissolved in 100mL of warm water (water temperature <40°C).

(3) Conclusion: The clinical test results of HC2786's anti-allergic alleviating ability are shown in Figure 7. Compared with the baseline, after 24 weeks, the relief rate of allergic rhinitis, allergic asthma, allergic dermatitis, and allergic conjunctivitis was 69.35%, 63.63%, and 66.85% in the ZCFY+RSQ group; The relief rate of allergic rhinitis was 83.68%, that of allergic asthma was 77.36%, that of allergic dermatitis was 85.32%, and that of allergic conjunctivitis was 89.48%. It can be seen that after the intervention of Bifidobacterium lactis HC2786, the remission rates of allergic rhinitis, allergic asthma, allergic dermatitis, and allergic conjunctivitis were 1.17, 1.22, 1.23, and 1.34 times those before the intervention, respectively.

Although the present invention has been described in detail in conjunction with preferred embodiments with reference to the accompanying drawings, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, those skilled in the art can make various equivalent modifications or replacements to the embodiments of the present invention, and these modifications or replacements should be within the scope of the present invention/any Those skilled in the art can easily think of changes or substitutions within the technical scope disclosed in the present invention, and all should be covered within the protection scope of the present invention.

Claims (8)

1. A strain of Bifidobacterium animalis subsp. lactis HC2786 that can alleviate allergic reactions is characterized in that it has been preserved in the China Center for Type Culture Collection on December 23, 2022, and the preservation address is Wuhan, China. Wuhan University, the deposit number is CCTCC NO: M 20222008. 2. A product containing Bifidobacterium lactis HC2786 as claimed in claim 1. 3. The product according to claim 2, characterized in that the specification of Bifidobacterium lactis HC2786 in the product is that the number of viable bacteria is ≥10 billion CFU/g, ≥20 billion CFU/g, ≥50 billion CFU/g, ≥ 100 billion CFU/g or ≥200 billion CFU/g. 4. product as claimed in claim 2, is characterized in that, product is made according to following preparation method: (1) Expansion of strains The Bifidobacterium lactis HC2786 is subjected to primary culture and secondary culture sequentially under anaerobic conditions, and then the seed liquid is transferred to a fermenter for anaerobic fermentation to obtain a fermented liquid; (2) Preparation of bacteria powder Centrifuge the fermented liquid to obtain the bacteria slime, vacuum freeze-dry after coating the bacteria sludge, crush the freeze-dried block to obtain the original powder of Bifidobacterium lactis, mix the original powder of Bifidobacterium lactis with fructo-oligosaccharide and isomalto-oligosaccharide have to. 5. The product according to claim 4, characterized in that, in step (1), the medium components used in the primary culture include peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L , dipotassium hydrogen phosphate 2.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, cysteine hydrochloride 0.5g/L L and Tween 80 1.0g/L; The medium components used in secondary culture include peptone 10.0g/L, yeast extract powder 5.0g/L, glucose 20.0g/L, dipotassium hydrogen phosphate 2.0g/L, triammonium citrate 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.1g/L, manganese sulfate 0.05g/L, cysteine hydrochloride 0.5g/L and edible oil 1.0g/L. 6. The product according to claim 4, wherein the medium components used for fermentation include whey powder 22g/L, glucose 35g/L, yeast powder 32g/L, carrot juice 5g/L, manganese sulfate 0.05 g/L, magnesium sulfate 0.1g/L, dipotassium hydrogen phosphate 1.0g/L, triammonium citrate 1.0g/L, sodium acetate 2.0/L, cysteine hydrochloride 0.3g/L and edible oil 1.0 g/L. 7. An application of Bifidobacterium lactis HC2786 as claimed in claim 1 in the preparation of a product for alleviating allergic symptoms. 8. The application according to claim 7, wherein the product for relieving allergic symptoms is a product for relieving allergic rhinitis, allergic dermatitis, allergic conjunctivitis and/or allergic asthma.

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