Bifidobacterium bifidum and its application in improving asthma and histamine intolerance

Abstract

The present invention discloses Bifidobacterium bifidum and its application in improving asthma and histamine intolerance, belonging to the technical field of marine microorganisms. Bifidobacterium bifidum ) NHNK‑618, which was deposited in the China Center for Type Culture Collection on August 9, 2024, and its deposit number is CCTCCNO: M20241761. NHNK‑618 has the functions of regulating asthma-related flora, agglutinating asthma-related pathogens, reducing Staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity, reducing airway remodeling, and degrading histamine. In addition, NHNK‑618 bacterial biofilm can increase the colonization ability of intestinal epithelial cells. It can be used to prepare products that improve asthma and histamine intolerance.

Description

Technical Field

[0001] The invention belongs to the technical field of marine microorganisms, and particularly relates to Bifidobacterium bifidum and application thereof in improving asthma and histamine intolerance. Background Art

[0002] Bronchial asthma is one of the most common chronic diseases in childhood and is influenced by risk factors during pregnancy and early infancy. Many of these are related to microbial exposure, and prospective cohort studies have shown an association between early life airway flora and the development of asthma. Studies have shown that colonization of the pathogens Streptococcus pneumoniae and Moraxella catarrhalis in the hypopharyngeal region of 1-month-old neonates detected by culture is associated with the occurrence of asthma and asthma exacerbations. In addition, in addition to Streptococcus and Moraxella, Wesleyan and Prevotella genera have the strongest correlation with pathogen scores, especially Prevotella. Staphylococcus aureus plays an important disease-modifying role in upper and lower airway diseases. In cross-sectional studies, sensitization to S. aureus enterotoxins (SEs) has been associated with an increased risk of severe asthma, and sensitization to S. aureus enterotoxins (SEs) has been identified as one of the risk factors for asthma. In clinical intervention trials, the use of Propionibacterium spp. in mothers and their infants within six months of cesarean section delivery can significantly reduce IgE-related asthma. Clinical studies have shown that children with higher levels of butyrate and propionate in their feces at the age of 1 have a significantly lower chance of developing asthma, and are less likely to develop asthma when they grow to 3 to 6 years old.

[0003] Airway inflammation, tissue damage, and subsequent abnormal repair lead to changes in the airway wall structure of asthma patients, which is called airway remodeling. Its main pathological changes include epithelial cell changes, mucous gland hyperplasia, subepithelial fibrosis, airway smooth muscle cell proliferation and migration, and airway wall angiogenesis. Smooth muscle actin (α-SMA) is an important indicator of airway remodeling, while E-cadherin can regulate the barrier function of airway epithelial cells and inhibit the activity of the NK-kappaB inflammatory pathway, thereby playing an important immunomodulatory role. Histamine receptors mainly include three subtypes, namely H1R, H2R, and H3R. Histamine acting on H1R can cause contraction of endothelial cells of small veins after airway microvessels, thereby increasing vascular permeability and mucosal edema; H2R excitement can increase the secretion of mucus glycoproteins in airway mucosal glands; H3R receptor activation is related to respiratory inflammation such as rhinitis.

[0004] Histamine intolerance (HIT) is an adverse reaction caused by the accumulation of histamine in the body due to an imbalance between histamine accumulation and degradation. The incidence rate is about 1%, and about 80% occur in middle-aged people. The level or activity of the patient's histamine metabolic enzymes diamine oxidase (DAO) and histamine-N-methyltransferase (HNMT) is reduced, which impairs the ability to degrade histamine. The accumulation of histamine in the body can induce a series of symptoms similar to allergic reactions. Since the clinical manifestations of HIT are similar to those of food allergies induced by specific foods, intolerance to sulfites or other biogenic amines (such as tyramine), etc., it is often misdiagnosed or missed in clinical practice. The clinical manifestations of HIT are similar to those of type I hypersensitivity reactions and are considered to be a pseudoallergic reaction. Its symptoms involve multiple systems such as the digestive tract, skin, respiratory tract, cardiovascular, and nervous systems. In severe cases, life-threatening reactions such as hypotension, bronchospasm, and shock may occur.

[0005] Marine microorganisms are one of the earliest sources of marine bioactive substances known to people, including algae, bacteria, fungi, protozoa and other microorganisms. These bioactive substances have important application value in various fields such as medicine, food, and cosmetics. Therefore, it is of great practical significance to provide a marine industry-related product developed using microbial technology. Summary of the invention

[0006] In view of this, an object of the present invention is to provide a strain of Bifidobacterium bifidum and its use in improving asthma and histamine intolerance.

[0007] The first aspect of the present invention provides a strain of Bifidobacterium bifidum ( Bifidobacterium bifidum ), the Bifidobacterium bifidum is Bifidobacterium bifidum NHNK-618, which has been deposited in the China Center for Type Culture Collection on August 9, 2024, and its deposit number is CCTCC NO: M 20241761.

[0008] The second aspect of the present invention provides a method based on the Bifidobacterium bifidum ( Bifidobacterium bifidum ) products prepared.

[0009] Furthermore, the product is the Bifidobacterium bifidum ( Bifidobacterium bifidum ) of live bacteria, fermentation products or inactivated bacteria.

[0010] The third aspect of the present invention provides the Bifidobacterium bifidum ( Bifidobacterium bifid ) or the use of the product as described in the second aspect above in the preparation of a product for improving asthma.

[0011] Furthermore, the improvement of asthma includes regulating asthma-related flora, agglutinating asthma-related pathogens, reducing Staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity and reducing airway remodeling.

[0012] The fourth aspect of the present invention provides the Bifidobacterium bifidum ( Bifidobacterium bifid ) or the use of the preparation as described in the second aspect above in the preparation of a product for improving histamine intolerance.

[0013] Furthermore, the improving histamine intolerance includes degrading histamine.

[0014] The fifth aspect of the present invention provides the Bifidobacterium bifidum ( Bifidobacterium bifid ) in the application of bacterial biofilm to increase the colonization ability of intestinal epithelial cells.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: the bifidobacterium bifidum NHNK-618 in the present application has a deposit number of CCTCC NO: M 20241761. NHNK-618 has the functions of regulating asthma-related flora, agglutinating asthma-related pathogens, reducing Staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity, reducing airway remodeling, and degrading histamine. In addition, the NHNK-618 bacterial biofilm can increase the colonization ability of intestinal epithelial cells. It can be used to prepare products that improve asthma and histamine intolerance.

[0016] Biological Deposit Description

[0017] Bifidobacterium bifidum ( Bifidobacterium bifidum ) NHNK-618, was deposited in the China Center for Type Culture Collection (CCTCC for short, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, Postal Code 430072) on August 9, 2024, and its deposit number is CCTCC NO: M 20241761. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a graph showing the detection results of Bifidobacterium bifidum NHNK-618 in Gram staining microscopy in Example 1 of the present invention;

[0019] Figure 2 This is a graph showing the experimental results of Bifidobacterium bifidum NHNK-618 agglutinating asthma-related pathogens in Example 6 of the present invention;

[0020] Figure 3 This is a graph showing the experimental results of histamine degradation by Bifidobacterium bifidum NHNK-618 in Example 9 of the present invention;

[0021] Figure 4This is a graph showing the experimental results of measuring the adhesion ability of Bifidobacterium bifidum NHNK-618 to human intestinal epithelial cells Caco-2 in Example 11 of the present invention;

[0022] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0023] The present invention provides Bifidobacterium bifidum and its application for improving asthma and histamine intolerance. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0024] The Bifidobacterium bifidum NHNK-618 described in the present invention is derived from the intestine of Abalone diversicolor and is identified as Bifidobacterium bifidum ( Bifidobacterium bifidum ). This strain is Gram-positive and has a variable rod shape under a microscope. It grows on MRS plates to form round colonies with smooth, translucent surfaces, white in color, and neat edges. It grows evenly in MRS liquid culture medium and forms white precipitates after long-term storage. The optimal anaerobic growth temperature is 37°C.

[0025] Bifidobacterium bifidum ( Bifidobacterium bifidum )NHNK-618, depository unit: China Center for Type Culture Collection, address: No. 299, Bayi Road, Wuchang District, Wuhan City, Wuhan University, deposit date: August 9, 2024, deposit number: CCTCCNO: M20241761.

[0026] Furthermore, in the application described in the present invention, the Bifidobacterium bifidum NHNK-618 provided by the present invention exists in the form of non-sterilized live bacteria or sterilized inactivated bacteria, or in the form of a fermentation product (i.e., supernatant), and the derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and their components, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation products, live bacteria, and inactivated bacteria.

[0027] The preparation method of the live bacteria is as follows: Bifidobacterium bifidum ) was inoculated into the culture medium, cultured at 37° C. for 48 h, fermentation liquid was obtained, precipitated by centrifugation, and the precipitate was washed to obtain the live bacteria.

[0028] The preparation method of the fermentation product is: the Bifidobacterium bifidum ( Bifidobacterium bifidum) was inoculated into the culture medium, cultured at 37°C for 48 hours, a fermentation liquid was obtained, the supernatant was obtained by centrifugation, and filtered at a precision of 0.22 μm. The obtained filtrate was the fermentation product;

[0029] The preparation method of the inactivated bacteria is: Bifidobacterium bifidum ) was inoculated into the culture medium, cultured at 37°C for 48 hours to obtain the fermentation liquid, centrifuged to obtain the precipitate, washed the precipitate, inactivated at 121°C for 30 minutes to obtain the inactivated bacteria.

[0030] It should be noted that the reagents and consumables used in the present invention are all common commercial products. The present invention is further described in conjunction with the embodiments:

[0031] Example 1 Isolation of NHNK-618

[0032] See also Figure 1 The sample was obtained from an adult individual of Abalone variegata obtained from a farm. The shell length was 5 cm and the patient had fasted for 3 days. The digestive tract was obtained by dissection with sterile tools. After being rinsed with sterile saline for 3 times, the intestinal tissue was broken by homogenization and grinding. The homogenate was collected in 1 ml of sterile saline. After gradient dilution, the supernatant was streaked on an MRS solid plate, placed in an anaerobic bag and cultured at 37°C for 48 hours, and white colonies were picked and repeatedly streaked and screened until a single colony with a regular and uniform shape was obtained, which was named NHNK-618.

[0033] Gram staining microscopy: strain NHNK-618 is a Gram-positive colony, showing variable rod-shaped morphology under a microscope; it grows on MRS plates to form white, smooth, rounded, translucent circular colonies with neat edges; it grows evenly in MRS liquid culture medium in a turbid state, and the bacteria show white precipitation after long-term storage.

[0034] Example 2 Nucleic acid identification of NHNK-618

[0035] 1. 16S rDNA gene sequence analysis

[0036] Pick a single colony in MRS liquid medium, culture at 37℃ overnight, centrifuge at 8000 rpm for 1min to collect the bacteria, and operate according to the instructions of the Gram-positive bacteria DNA extraction kit. The primers used were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20μL. The PCR amplification program was 95℃ pre-denaturation for 5min, 94℃ for 15s, 57℃ for 15s, 72℃ for 1min, 35 cycles, and 72℃ extension for 10min.

[0037] 2. Results

[0038] The sequencing result of the PCR product is shown in SEQ ID NO.1. After homology comparison (BLASTN) with the standard sequence published in GenBank, it was concluded that the NHNK-618 strain was Bifidobacterium bifidum ( Bifidobacterium bifidum ).

[0039] Example 3 NHNK-618 inhibits the proliferation of asthma-related pathogens

[0040] 1. Cultivation of asthma-related pathogens

[0041] Staphylococcus aureus ( Staphylococcus aureus ) CGMCC 1.8721 frozen tube bacterial solution was inoculated into BHI liquid medium at 2% (v / v), cultured at 37℃ with shaking for 24h, and then adjusted to OD 600 = 0.1, and the Staphylococcus aureus suspension was obtained. It should be noted that Staphylococcus aureus ( Staphylococcus aureus ) CGMCC 1.8721 frozen tube bacterial solution was purchased from China General Microbiological Culture Collection Center.

[0042] Streptococcus pneumoniae ( Streptococcus pneumoniae ) The frozen tube culture liquid of BNCC338425 was inoculated into TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v), and cultured at 37℃ for 2 days. After that, the OD value was adjusted to 0. 600 = 0.1, and the pneumococcal suspension is obtained. It should be noted that pneumococcal ( Streptococcus pneumoniae )BNCC338425 frozen tube bacterial solution was purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0043] Haemophilus influenzae ( Haemophilus influenzae ) The frozen tube bacterial solution of BNCC359479 was inoculated into a special peptone medium containing 5% defibrinated sheep blood at 2% (v / v), and then cultured at 37°C for 18 hours and adjusted to OD600 = 0.1 with a special peptone medium to obtain a suspension of Haemophilus influenzae. It should be noted that Haemophilus influenzae ( Haemophilus influenza )BNCC359479 frozen tube bacterial solution was purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0044] Moraxella catarrhalis Moraxella catarrhalis ) The frozen tube culture solution of BNCC275822 was inoculated into BHI liquid medium at 2% (v / v), and cultured at 37℃ for 2 days. After that, the OD was adjusted with BHI medium. 600 = 0.1, then the suspension of Moraxella catarrhalis was obtained. It should be noted that Moraxella catarrhalis ( Moraxella catarrhalis )BNCC275822 frozen tube bacterial solution was purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0045] Prevotella humanis ( Body hair ) The frozen tube bacterial solution of BNCC361375 was inoculated into a special peptone medium containing 5% defibrinated sheep blood at 2% (v / v). After anaerobic culture at 37°C for 2 days, the OD was adjusted with a special peptone medium. 600 = 0.1, and the human Prevotella suspension was obtained. It should be noted that human Prevotella ( Prevotella body )BNCC361375 frozen tube bacterial solution was purchased from Beina Chuanglian Biotechnology Co., Ltd.

[0046] 2. Preparation of NHNK-618 fermentation products

[0047] NHNK-618 glycerol tube bacterial solution was inoculated into fresh MRS medium at 2% (v / v), and then cultured anaerobically at 37°C for 48 h and adjusted to OD with MRS. 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter through a 0.22μm filter membrane to obtain the fermentation product.

[0048] 3. NHNK-618 fermentation products inhibit the proliferation of asthma-related pathogens

[0049] 160 μL of pathogen suspension was added to each well of a 96-well plate. 40 μL of fermentation product was added to the experimental group, and an equal volume of MRS medium was added to the control group. After culturing at 37°C for 48 hours, the OD 600 The absorbance value at nm. Inhibition rate % = (1-absorbance value of experimental group / absorbance value of control group) * 100%. The results are shown in Table 1:

[0050] Table 1

[0051] ,

[0052] The results showed that the fermentation product of NHNK-618 can inhibit the growth of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and human Prevotella, with an inhibition rate of 20.00% to 65.11%. That is, the regulation of asthma-related flora includes inhibiting the growth of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and human Prevotella.

[0053] Example 4 NHNK-618 promotes the proliferation of Propionibacterium acidipropionici

[0054] 1. Propionibacterium acidipropionici ( Propionibacterium acidipropionici )

[0055] The frozen tube culture liquid of Propionibacterium acidipropionici CGMCC 1.2232 was inoculated into a special peptone medium at 2% (v / v), and then cultured anaerobically at 37°C for 5-6 days and then adjusted to OD 0. 600 =0.5, and the bacterial suspension of Propionibacterium acidipropionici was obtained. It should be noted that the frozen tube bacterial suspension of Propionibacterium acidipropionici CGMCC 1.2232 was purchased from China General Microbiological Culture Collection Center.

[0056] 2. Preparation of NHNK-618 fermentation products

[0057] Refer to Example 3.

[0058] 3. NHNK-618 fermentation products promote the proliferation of Propionibacterium acidipropionici

[0059] 160 μL of the suspension of Propionibacterium acidipropionici was added to each well of a 96-well plate. 40 μL of the fermentation product was added to the experimental group, and the same amount of MRS medium was added to the control group. After culturing at 37°C for 48 hours, the OD 600 The absorbance value at nm. Proliferation promotion rate % = (absorbance value of experimental group - absorbance value of control group) / absorbance value of control group * 100%. The results are shown in Table 2:

[0060] Table 2

[0061] ,

[0062] The results showed that the growth promotion rate of NHNK-618 fermentation product on Propionibacterium acidipropionici was 43.01%-56.59%. That is, the regulation of asthma-related flora also includes promoting the proliferation of Propionibacterium acidipropionici.

[0063] Example 5 NHNK-618 inhibits asthma-related pathogenic bacteria biofilm

[0064] 1. Cultivation of asthma-related pathogens

[0065] The culture method of Staphylococcus aureus / Streptococcus pneumoniae / Haemophilus influenzae / Mora catarrhalis / Prevotella humains is as described in Example 3;

[0066] 2. Preparation of NHNK-618 fermentation products

[0067] Refer to Example 3;

[0068] 3. NHNK-618 inhibits asthma-related pathogenic bacteria biofilm

[0069] 100 μL of pathogenic bacteria liquid was added to each well of a 96-well plate, 100 μL of NHNK-618 fermentation product was added to each well of the experimental group, and an equal volume of MRS medium was added to the control group. Each group had 3 parallels, and then the culture was continued at 37°C for 24 hours.

[0070] After the incubation, the supernatant was discarded, 100 μL of sterile PBS was added to each well for washing twice, and then 100 μL of 4% paraformaldehyde fixative was added to each well for fixing at room temperature for 30 minutes. The fixative was discarded, 100 μL of crystal violet was added to each well, and staining was performed at room temperature for 30 minutes. After staining, the cells were washed twice with sterile PBS and dried, and 100 μL of anhydrous ethanol was added to each well. After standing for 1 minute, the absorbance at 600 nm was measured. Inhibition rate % = (1-experimental group / control group) * 100%, and the results are shown in Table 3:

[0071] Table 3

[0072] ,

[0073] The results showed that the fermentation product of NHNK-618 can inhibit the formation of biofilms of asthma-related pathogens Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and human Prevotella, with an inhibition rate of 46.15% to 91.88%. That is, the regulation of asthma-related flora also includes inhibiting the formation of biofilms of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and human Prevotella.

[0074] Example 6 NHNK-618 Agglutination of Asthma-Related Pathogens

[0075] 1. Cultivation of asthma-related pathogens

[0076] Moraxella catarrhalis Moraxella catarrhalis ) The frozen tube bacterial solution of BNCC275822 was inoculated into BHI liquid medium at 2% (v / v), cultured at 37℃ for 2 days, centrifuged and washed 3 times with sterile PBS, resuspended with sterile PBS and adjusted to OD 600 =1.0, and you get the Moraxella catarrhalis suspension.

[0077] Streptococcus pneumoniae ( Streptococcus pneumoniae ) The frozen tube bacterial solution of BNCC338425 was inoculated into TSB liquid medium containing 5% defibrinated sheep blood at 2% (v / v). After shaking culture at 37°C for 2 days, the bacteria were centrifuged and washed three times with sterile PBS. The cells were resuspended in sterile PBS and the OD was adjusted. 600 =1.0, and the Streptococcus pneumoniae suspension is obtained.

[0078] Prevotella humanis ( Body hair) The frozen tube bacterial solution of BNCC361375 was inoculated into a special peptone medium containing 5% defibrinated sheep blood at 2% (v / v). After anaerobic culture at 37°C for 2 days, the bacteria were centrifuged and washed three times with sterile PBS. The cells were resuspended in sterile PBS and the OD was adjusted. 600 =1.0, and the human Prevotella suspension is obtained.

[0079] 2. Preparation of NHNK-618 inactivated bacteria

[0080] The NHNK-618 bacterial suspension was inoculated into fresh MRS medium at 2% (v / v), cultured anaerobically at 37°C for 48 h, centrifuged and washed three times with sterile PBS, resuspended with sterile PBS and adjusted to OD 600 =1.0, sterilize at 121℃ for 15 min and set aside.

[0081] 3. Coagulation

[0082] The NHNK-618 bacterial solution was inoculated into fresh MRS medium at 2% (v / v), cultured anaerobically at 37°C for 48 h, centrifuged and washed three times with sterile PBS, resuspended with sterile PBS and adjusted to OD 600 =1.0, sterilize at 121℃ for 15 min and set aside.

[0083] 4. Index determination

[0084] See also Figure 2 , sample the reaction solution of NHNK-618 alone, pathogens, and the mixed reaction solution of NHNK-618 and pathogens at 30 minutes of reaction, the sampling range is the top 50μL of the liquid surface, transfer it to a 96-well plate, and measure its absorbance at OD=600nm. At the same time, the agglutination precipitate is stained with Gram stain to observe its bacterial agglutination state.

[0085] Calculation formula:

[0086] Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%;

[0087] Ax represents the OD value of NHNK-618 alone at the reaction time. 600 Value; Ay represents the OD value of a single pathogen measured at the reaction time 600 Value; Amix means the OD value measured at the reaction time after NHNK-618 and pathogens are mixed. 600 Numeric value.

[0088] The results are shown in Table 4:

[0089] Table 4

[0090] ,

[0091] The results showed that the inactivated bacteria of NHNK-618 could agglutinate Moraxella catarrhalis, Streptococcus pneumoniae and human Prevotella, with agglutination rates of 19.27% ​​to 33.81%. That is, the agglutinated asthma-related pathogens included agglutinated Moraxella catarrhalis, Streptococcus pneumoniae and human Prevotella.

[0092] Example 7 NHNK-618 inhibits Staphylococcus aureus enterotoxin genes

[0093] 1. Preparation of NHNK-618 inactivated bacteria and fermentation products

[0094] NHNK-618 bacterial suspension was inoculated into fresh MRS medium at 2% (v / v) and cultured anaerobically at 37°C for 48 h. After that, the OD was adjusted to 0. 600 = 0.5, centrifuged at 5000 rpm for 10 min, and the supernatant was filtered through a 0.22 μm filter membrane to obtain the fermentation product. The centrifuged precipitated live cells were washed with MRS and adjusted to OD 600 =0.5, sterilize at 121℃ for 15min to obtain inactivated bacterial suspension.

[0095] 2. NHNK-618 down-regulates the expression of Staphylococcus aureus enterotoxin genes

[0096] (1) Culture and intervention of Staphylococcus aureus

[0097] Staphylococcus aureus CGMCC 1.8721 was inoculated into fresh BHI medium at 2% (v / v) and cultured at 37°C with shaking for 24 h. The OD 600 =0.5. Add Staphylococcus aureus suspension to centrifuge tubes, 1 mL per tube, and add 2 mL BHI medium. Add 1 mL NHNK-618 inactivated bacteria or fermentation products to the experimental group, and add an equal volume of MRS to the control group, and continue to culture at 37°C for 16 hours.

[0098] (2) After the culture, the RNA of Staphylococcus aureus was extracted using a Gram-positive bacteria RNA extraction kit. After testing the RNA concentration and purity, it was reverse transcribed into cDNA. Using 16S rRNA as the internal reference gene, qPCR fluorescence quantitative technology was used to detect the enterotoxin A gene of Staphylococcus aureus. sea expression.

[0099] (3) Data processing

[0100] The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0101] Formula: F=2 -ΔΔCT ,in:

[0102] △CT 实验 =CT 实验 -CT 内参（实验）;

[0103] △CT 对照 =CT 对照 -CT 内参（对照）;

[0104] △△CT=△CT 实验 -△CT 对照。

[0105] The results are shown in Table 5:

[0106] Table 5

[0107] ,

[0108] The results showed that both NHNK-618 fermentation products and inactivated bacteria could downregulate the enterotoxin gene of Staphylococcus aureus sea The relative expression multiple is 0.29-0.89. That is, the reduction of Staphylococcus aureus enterotoxin includes down-regulating the Staphylococcus aureus enterotoxin gene sea expression.

[0109] Example 8 NHNK-618 regulates the expression of genes related to Beas-2b histamine receptor, bronchial barrier and airway remodeling in human bronchial epithelial cells

[0110] 1. Preparation of NHNK-618 inactivated bacteria and fermentation products

[0111] NHNK-618 bacterial suspension was inoculated into fresh MRS medium at 2% (v / v), cultured anaerobically at 37°C for 48 h, and then adjusted to OD 600 =0.5, centrifuge at 5000rpm for 10min, take the supernatant, and filter through a 0.22μm filter membrane to obtain the fermentation product. The centrifuged precipitated live bacteria were washed with PBS and resuspended in an appropriate amount, sterilized at 121℃ for 15min, centrifuged and removed the supernatant, resuspended in DMEM and adjusted to OD600=0.5, to obtain the inactivated bacterial suspension.

[0112] 2. NHNK-618 regulates the expression of genes related to histamine receptors, barrier and airway remodeling in Beas-2b cells

[0113] (1) Intervention treatment of Beas-2b cells

[0114] Beas-2b cells were inoculated in DMEM medium containing 10% FBS and incubated at 37°C with 5% CO 2When the cells reached 80% fusion, the cells were collected and inoculated into 6-well plates. After overnight culture, the supernatant was discarded. 1 mL of fermentation product or inactivated bacteria and 1 mL of serum-free DMEM medium were added to each well of the experimental group, and the control group was replaced with an equal volume of DMEM. The plates were incubated at 37°C and 5% CO 2 The cells were cultured for 16 h under the same conditions.

[0115] (2) RNA extraction and fluorescence quantitative PCR

[0116] After the culture was completed, RIPA lysis buffer was added to lyse the cells, and total RNA was extracted. After testing the RNA concentration and purity, it was reverse transcribed into cDNA. GAPDH As the internal reference gene, qPCR fluorescence quantitative technology was used to detect H1R、H2R、H3R、OCLD、E-cadherin as well as α-SMA Gene expression.

[0117] (3) Data processing

[0118] The relative expression multiple of the gene in the control group is F=1, and 2 -ΔΔCT The F value of each sample was calculated by this method.

[0119] The results are shown in Table 6 and Table 7:

[0120] Table 6

[0121] ,

[0122] Table 7

[0123] ,

[0124] The results showed that NHNK-618 could downregulate histamine receptor genes H1R , H2R and H3R Upregulation of tight junction protein gene expression OCLD and E-cadherin gene E-cadherin downregulates the expression of smooth muscle actin gene α-SMA The reduction of bronchial histamine receptor activation includes down-regulating the bronchial epithelial cell histamine receptor gene HR1, HR2, HR3 The expression of bronchial epithelial cell barrier-related genes is upregulated to promote bronchial barrier integrity OCLD and E-cadherin The reduction of airway remodeling includes down-regulating the expression of airway remodeling-related genes in bronchial epithelial cells α-SMA expression.

[0125] Example 9 NHNK-618 Degradation of Histamine

[0126] 1. NHNK-618 histamine degradation experiment

[0127] A single colony of NHNK-618 was selected and placed in fresh MRS medium. After anaerobic culture at 37°C for 48 h, the OD was adjusted to 0. 600 =1.0, NHNK-618 bacterial suspension was obtained, inoculated into MRS medium containing 500 μg / mL histamine at 2% (v / v), and cultured anaerobically at 37°C for 48 h. The control group was inoculated with an equal volume of MRS medium.

[0128] 2. Reagent preparation

[0129] (1) 50 g / L Na 2 CO 3 Solution: 5g Na 2 CO 3 Dissolve in pure water and dilute to 100 mL with pure water.

[0130] (2) 250 g / L NaOH solution: Dissolve 25 g NaOH in pure water and dilute to 100 mL.

[0131] (3) HCl solution: Add 5 mL of concentrated hydrochloric acid (HCl) to 55 mL of pure water and mix well.

[0132] (4) Solution A (p-nitroaniline solution): Weigh 0.5 g of p-nitroaniline, add 5 mL of HCl to dissolve, then dilute to 200 mL with pure water, mix well, and prepare before use. Solution B (NaNO 2 Solution): weigh 0.5gNaNO 2 , dissolve and dilute to 100mL with pure water, and mix well. Azo reagent: Pipette 5mL of liquid A and 40mL of liquid B and mix.

[0133] (5) 1000 μg mL -1 Histamine phosphate standard solution: accurately weigh 0.025g of histamine dihydrochloride standard, dissolve it in pure water, transfer it into a 25mL volumetric flask, and dilute to the mark with pure water, and mix well. 20μg·mL -1 Histamine phosphate standard solution 1mL 1000μg mL -1 Place the histamine phosphate standard solution in a 50 mL volumetric flask, add pure water to the mark and mix.

[0134] 3. Determination of histamine content

[0135] See also Figure 3 ,

[0136] (1) Pipette 0.0mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, and 1.0mL of 20μg / mL histamine phosphate standard solution into 10mL stoppered colorimetric tubes, which are equivalent to 0μg, 4μg, 8μg, 12μg, 16μg, and 20μg of histamine, respectively. Add water to make the volume to 1mL, and add 1mL HCl solution, 3mL 50g / L Na 2 CO 3 Solution, 3mL azo reagent, add water to the scale, and measure OD after 10min 480 And draw a standard curve.

[0137] (2) Take 1 mL of the sample to be tested, add 1 mL of HCl solution, 3 mL of 50 g / L Na 2 CO 3 Solution, 3mL azo reagent, add water to the scale, and measure OD after 10min 480 . Degradation rate (%) = (1-absorbance value of experimental group / absorbance value of control group)*100.

[0138] The results are shown in Table 8:

[0139] Table 8

[0140] ,

[0141] The results showed that NHNK-618 could degrade histamine with a degradation rate of 23.71%~26.27%.

[0142] Example 10 NHNK-618 forms biofilm

[0143] 1. Formation of NHNK-618 biofilm

[0144] A single colony of NHNK-618 was selected and placed in MRS liquid medium, cultured anaerobically at 37°C for 48 h, and the OD was adjusted to 0. 600 =0.2, 100 μl of bacterial solution was added to each well of a 96-well plate, with 3 parallels per group, and then anaerobic culture was continued at 37°C for 24 h.

[0145] 2. Crystal violet staining

[0146] After the incubation, the supernatant was discarded, 100 μL of sterile PBS was added to each well for washing twice, and then 100 μL of 4% paraformaldehyde fixative was added to each well for fixing at room temperature for 30 minutes. The fixative was discarded, 100 μL of crystal violet was added to each well, and staining was performed at room temperature for 30 minutes. After staining, the cells were washed twice with sterile PBS and dried, and 100 μL of anhydrous ethanol was added to each well. After standing for 1 minute, the absorbance at 600 nm was measured. The results are shown in Table 9:

[0147] Table 9

[0148] ,

[0149] The results showed that NHNK-618 in OD 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0150] Example 11 Determination of the Adhesion Ability of NHNK-618 to Human Intestinal Epithelial Cells Caco-2

[0151] 1. Preparation of NHNK-618 live bacterial suspension

[0152] Pick a single colony of NHNK-618 in fresh MRS medium, culture anaerobically at 37°C for 48 h, collect the bacteria by centrifugation at 5000 rpm for 10 min, wash twice with sterile PBS, resuspend the bacteria in DMEM medium and adjust the OD 600 =0.5 to obtain a live bacterial suspension.

[0153] 2. Preparation of NHNK-618 biofilm bacterial suspension

[0154] A single colony of NHNK-618 was selected and placed in MRS liquid medium, cultured anaerobically at 37°C for 48 h, and the OD was adjusted to 0. 600 =0.2 was added to the bacterial culture dish, and then anaerobic culture was continued at 37°C for 24 hours. After the culture was completed, the upper culture medium was discarded, the bottom biofilm was resuspended after washing twice with PBS, and the bacteria were collected by centrifugation at 5000rpm for 10min. The bacteria were resuspended in DMEM culture medium and the OD was adjusted. 600 =0.5 to obtain biofilm bacterial suspension.

[0155] 3. Determination of the adsorption capacity of NHNK-618 on Caco-2 cells

[0156] See also Figure 4 Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum at 37°C and 5% CO 2 Cultivate under the following conditions. When the confluence reaches 80%, the cells are digested with trypsin and collected, and inoculated into a 6-well plate containing a slide for overnight culture. After the culture is completed, the supernatant is discarded, washed twice with sterile PBS, 1mL of DMEM medium without FBS serum and 1mL of live bacterial suspension or biofilm bacterial suspension are added, and the incubation continues for 2h. After the incubation is completed, wash twice with PBS to remove non-adherent bacteria. The slide is fixed in 4% paraformaldehyde for 15min, followed by Gram staining and photography.

[0157] The results showed that NHNK-618 could adsorb human intestinal epithelial cells Caco-2 cells, and after NHNK-618 coated itself with the biofilm, it could increase its adsorption capacity to human intestinal epithelial cells Caco-2 cells.

[0158] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A strain of Bifidobacterium bifidum ( Bifidobacterium bifidum ), characterized in that, The Bifidobacterium bifidum is Bifidobacterium bifidum NHNK-618, which has been deposited in the China Center for Type Culture Collection on August 9, 2024, and its deposit number is CCTCC NO: M 20241761.

2. Based on the Bifidobacterium bifidum as claimed in claim 1 ( Bifidobacterium bifidum ) products prepared.

3. The product according to claim 2, characterized in that The product is the Bifidobacterium bifidum ( Bifidobacterium bifidum ) of live bacteria, fermentation supernatant or inactivated bacteria.

4. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum ) or the use of the product as claimed in claim 2 in the preparation of a product for improving asthma.

5. The use according to claim 4, characterized in that: The improvement of asthma includes regulating asthma-related flora, agglutinating asthma-related pathogens, reducing Staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity and reducing airway remodeling.

6. The Bifidobacterium bifidum according to claim 1 ( Bifidobacterium bifidum ) or the preparation as claimed in claim 2 in the preparation of a product for improving histamine intolerance.

7. The use according to claim 6, characterized in that: The improving histamine intolerance includes degrading histamine.

Images