Bifidobacterium longum subsp. Longum and application thereof in improving asthma and high histamine level

Abstract

The invention discloses bifidobacterium longum subsp. Longum and application thereof in improving asthma and high histamine level, and belongs to the technical field of marine microorganisms. The invention discloses a bifidobacterium longum subsp. Longum NHNK-619, which is preserved in the China Center for Type Culture Collection on August 9, 2024, and the preservation number of the bifidobacterium longum subsp. Longum NHNK-619 is CCTCC (China Center for Type Culture Collection) NO: M20241760. The NHNK-619 has the functions of regulating asthma-related flora, agglutinating asthma-related pathogenic bacteria, reducing staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity, reducing airway remodeling and degrading histamine, and a thallus biofilm formed by the NHNK-619 can improve the colonization ability to intestinal epithelial cells. The compound can be used for preparing products for improving asthma and high histamine level.

Description

Technical Field

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

[0002] Bronchial asthma is one of the most common chronic diseases in childhood. Studies have shown that colonization with Streptococcus pneumoniae and Moraxella catarrhalis is associated with the occurrence of asthma and asthma attacks, and that the genera Wesslera and Prevotella are strongly correlated with pathogen scores. Staphylococcus aureus plays an important disease-modifying role in upper and lower airway diseases. In clinical intervention trials, the use of Propionibacterium truncatum by parturients and their infants within six months of cesarean section 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 probability of 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. Histamine receptors mainly include three subtypes, namely H1R, H2R, and H3R.

[0004] Histamine is a key molecule in the human body that is produced by immune cells called "mast cells" that are found in connective tissue and are part of the neuroimmune system. Histamine is also naturally present in many foods. When there is too much histamine in the body to be excreted, the body uses specific enzymes to remove histamine, such as diamine oxidase (DAO) and histamine n-methyltransferase (HNMT), but if there are not enough of these enzymes, histamine will continue to accumulate to a very high level. The clinical symptoms caused by histamine excess from multiple sources are difficult to distinguish from allergic symptoms. Histamine sensitivity is dose-related, and the more you ingest, the more severe the symptoms, which usually appear a few hours after eating. The most common accompanying symptoms are: skin (wheals, urticaria, angioedema, itching, redness), mucous membranes (itching, edema, redness), headaches / migraines, digestive tract dysfunction, etc.

[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, the object of the present invention is to provide a strain of Bifidobacterium longum subspecies longum and its use in improving asthma and high histamine levels.

[0007] The first aspect of the present invention provides a strain of Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp. longum ), the Bifidobacterium longum subspecies long is Bifidobacterium longum subspecies longum NHNK-619, which was deposited in the China Center for Type Culture Collection on August 9, 2024, and its deposit number is CCTCC NO: M 20241760.

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

[0009] The third aspect of the present invention provides the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp. longum ) in the preparation of products for improving asthma and improving high histamine levels.

[0010] Furthermore, the improvement of asthma includes at least one of regulating asthma-related flora, agglutinating asthma-related pathogens, inhibiting Staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity and reducing airway remodeling.

[0011] Furthermore, the regulation of asthma-related flora includes inhibiting the growth of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Prevotella humanus and promoting the growth of Propionibacterium acidipropionici.

[0012] Furthermore, the regulation of asthma-associated flora also includes reducing the formation of biofilms of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Prevotella hominis.

[0013] Furthermore, the reduction of bronchial histamine receptor activation comprises down-regulating 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.

[0014] Further, the improving high histamine levels includes degrading histamine.

[0015] Furthermore, the product also includes auxiliary materials and / or additives.

[0016] The fourth aspect of the present invention provides the Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp. longum ) in the application of bacterial biofilm to increase the colonization ability of intestinal epithelial cells.

[0017] The long bifidobacterium longum subspecies longum NHNK-619 in the present application has a deposit number of CCTCC NO: M 20241760. Experiments have shown that NHNK-619 has the functions of regulating asthma-related flora, agglutinating asthma-related pathogens, inhibiting Staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity, reducing airway remodeling, and degrading histamine.

[0018] Biological Deposit Description Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp. longum ) NHNK-619, 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 20241760. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 1 is a graph showing the results of the separation experiment of Bifidobacterium longum subspecies longum NHNK-619 in Example 1 of the present invention, wherein (a) is a plate colony morphology of Bifidobacterium longum subspecies longum NHNK-619, and (b) is a Gram staining morphology of Bifidobacterium longum subspecies longum NHNK-619; Figure 2 Graphs showing the experimental results of Bifidobacterium longum subspecies longum NHNK-619 agglutinating asthma-related pathogens in Example 6 of the present invention, wherein (a) is a graph showing the bacterial agglutination state of Bifidobacterium longum subspecies longum NHNK-619, (b) is a graph showing the bacterial agglutination state of Moraxella catarrhalis, (c) is a graph showing the bacterial agglutination state of human Prevotella, (d) is a graph showing the bacterial agglutination state of a mixed reaction liquid of Bifidobacterium longum subspecies longum NHNK-619 and Moraxella catarrhalis, and (e) is a graph showing the bacterial agglutination state of a mixed reaction liquid of Bifidobacterium longum subspecies longum NHNK-619 and human Prevotella; Figure 3 This is a graph showing the experimental results of histamine degradation by Bifidobacterium longum subspecies longum NHNK-619 in Example 9 of the present invention; Figure 4This is a graph showing the experimental results of measuring the adhesion ability of Bifidobacterium longum subspecies longum NHNK-619 to human intestinal epithelial cells Caco-2 in Example 11 of the present invention; The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0020] The present invention provides Bifidobacterium longum subspecies longum and its application in improving asthma and high histamine levels. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements 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 realize and apply the technology of the present invention.

[0021] The Bifidobacterium longum subspecies longum NHNK-619 described in the present invention is derived from the intestine of Abalone variegatedis and is identified as Bifidobacterium longum subspecies longum ( Bifidobacterium longum subsp. longum ). This strain is Gram-positive and appears as a long, curved stick, swollen or dumbbell-shaped bacillus under a microscope; it grows on MRS plates and forms shiny, round, neatly edged, and centrally convex circular colonies; it grows evenly turbid in MRS liquid culture medium, and the bacteria form white precipitates after long-term storage. The optimal anaerobic growth temperature is 37°C.

[0022] Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp. longum )NHNK-619, 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: M20241760.

[0023] Furthermore, in the application described in the present invention, the Bifidobacterium longum subspecies longum NHNK-619 provided by the present invention exists in the form of non-sterilized live bacteria or sterilized inactivated bacteria, or in the form of fermentation / secretion products (i.e., supernatant), and the derivative form is preferably selected from: metabolites, metabolic biological products, prebiotics, cell walls and components thereof, extracellular polysaccharides, and compounds containing immunogenic components, preferably selected from: fermentation / secretion products, live bacteria, and inactivated bacteria.

[0024] 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: Example 1 Isolation of NHNK-619 See also Figure 1The 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-619.

[0025] Gram staining microscopy: strain NHNK-619 is a Gram-positive colony, which appears as a long, curved stick, swollen or dumbbell-shaped bacillus under a microscope; it grows on MRS plates and forms shiny, round colonies with neat edges and a raised center; it grows evenly in turbid MRS liquid culture medium, and the bacteria appear as white precipitates after long-term storage.

[0026] Example 2 Nucleic acid identification of NHNK-619 1. 16S rDNA gene sequence analysis 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.

[0027] 2. Results 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-619 strain was Bifidobacterium longum subsp. longum ( Bifidobacterium longum subsp. longum ).

[0028] Example 3 NHNK-619 inhibits the proliferation of asthma-related pathogens 1. Cultivation of asthma-related pathogens 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.

[0029] 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.

[0030] Haemophilus influenzae ( Haemophilus influenzae ) The BNCC359479 frozen tube bacterial liquid was inoculated into a special peptone medium containing 5% defibrinated sheep blood at 2% (v / v), and after shaking culture at 37°C for 18 hours, the OD600 was adjusted to 0.1 with a special peptone medium to obtain a Haemophilus influenzae suspension.

[0031] 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 to obtain Moraxella catarrhalis suspension.

[0032] 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.

[0033] 2. Preparation of NHNK-619 fermentation products A single colony was picked and placed in MRS liquid medium, cultured anaerobically at 37°C for 48 h, and then adjusted to OD 600 =1.0, centrifuge at 5000rpm for 10min, take the supernatant, and filter through a 0.22μm filter membrane to obtain the fermentation product.

[0034] 3. NHNK-619 fermentation products inhibit the proliferation of asthma-related pathogens 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: Table 1 , The results showed that the fermentation products of NHNK-619 could inhibit the growth of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Prevotella spp., with inhibition rates ranging from 14.81% to 50.72%.

[0035] Example 4 NHNK-619 promotes the proliferation of Propionibacterium acidipropionici 1. Propionibacterium acidipropionici ( Propionibacterium acidipropionici ) 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 suspension of Propionibacterium acidipropionici was obtained.

[0036] 2. Preparation of NHNK-619 fermentation products Refer to Example 3.

[0037] 3. NHNK-619 fermentation products promote the proliferation of Propionibacterium acidipropionici 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: Table 2 , The results showed that the growth promotion rate of NHNK-619 fermentation products on Propionibacterium acidipropionici was 38.27%~45.13%.

[0038] Example 5 NHNK-619 inhibits asthma-related pathogenic bacteria biofilm 1. Cultivation of asthma-related pathogens The culture method of Staphylococcus aureus / Streptococcus pneumoniae / Haemophilus influenzae / Mora catarrhalis / Prevotella humains is as described in Example 3; 2. Preparation of NHNK-619 fermentation products Refer to Example 3; 3. NHNK-619 inhibits asthma-related pathogenic bacteria biofilm 100 μL of pathogenic bacteria liquid was added to each well of a 96-well plate, 100 μL of NHNK-619 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.

[0039] 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: Table 3 , The results showed that NHNK-619 fermentation products could inhibit the formation of biofilms of asthma-related pathogens: Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis and Prevotella spp., with an inhibition rate of 60.44% to 84.74%.

[0040] Example 6 NHNK-619 agglutinates asthma-related pathogens 1. Cultivation of asthma-related pathogens 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 60 0=1.0, sterilize at 121℃ for 15min and set aside.

[0041] 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, sterilize at 121℃ for 15min and set aside.

[0042] 2. Preparation of NHNK-619 inactivated bacteria Pick a single colony of NHNK-619 in MRS liquid medium, culture anaerobically at 37°C for 48 h, centrifuge and wash three times with sterile PBS, resuspend with sterile PBS and adjust OD 600 =1.0, sterilize at 121℃ for 15 min and set aside.

[0043] 3. Coagulation The bacterial liquid of each pathogen was mixed with the inactivated bacterial liquid of NHNK-619 at a volume ratio of 1:1, and the flocculation state was observed after standing for 30 minutes.

[0044] 4. Index determination See also Figure 2 , sample the reaction solution of NHNK-619 alone, pathogens, and the mixed reaction solution of NHNK-619 and pathogens at 30 minutes of reaction, the sampling range is the top 50 μL 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.

[0045] Calculation formula: Agglutination rate (%) = [(Ax+Ay)-2Amix] / (Ax+Ay)×100%; Ax represents the OD value of NHNK-619 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-619 and pathogens are mixed. 600 Numeric value.

[0046] The results are shown in Table 4: Table 4 , The results showed that the inactivated bacteria NHNK-619 could agglutinate human Prevotella and Moraxella catarrhalis, with an agglutination rate of 16.87% to 20.27%. That is, the agglutinated asthma-related pathogens included agglutinated Moraxella catarrhalis and human Prevotella.

[0047] Example 7 NHNK-619 inhibits Staphylococcus aureus enterotoxin genes 1. Preparation of NHNK-619 fermentation products Refer to Example 3.

[0048] 2. NHNK-619 down-regulates the expression of Staphylococcus aureus enterotoxin genes (1) Culture and intervention of Staphylococcus aureus 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 of fermentation product to the experimental group and an equal volume of MRS to the control group, and continue to culture at 37°C for 16 hours.

[0049] (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.

[0050] (3) Data processing 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.

[0051] Formula: F=2 -ΔΔCT ,in: △CT 实验 =CT 实验 -CT 内参（实验）; △CT 对照 =CT 对照 -CT 内参（对照）; △△CT=△CT 实验 -△CT 对照。

[0052] The results are shown in Table 5: Table 5 , The results showed that NHNK-619 fermentation products could inhibit the expression of Staphylococcus aureus enterotoxin genes sea The relative expression fold was 0.77~0.88.

[0053] Example 8 NHNK-619 regulates the expression of genes related to Beas-2b histamine receptor, bronchial barrier and airway remodeling in human bronchial epithelial cells 1. Preparation of NHNK-619 inactivated bacteria and fermentation products A single colony of NHNK-619 was selected and placed in MRS liquid medium. After anaerobic culture at 37°C for 48 h, the OD was adjusted to 0. 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.

[0054] 2. NHNK-619 regulates the expression of genes related to histamine receptors, bronchial barrier and airway remodeling in Beas-2b cells (1) Intervention treatment of Beas-2b cells Beas-2b cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the cells were 80% confluent, the cells were collected and inoculated in 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, and cultured at 37°C and 5% CO2 for 16 h.

[0055] (2) RNA extraction and fluorescence quantitative PCR 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 histamine receptor related genes H1R、H2R、H3R、 tight junction protein gene OCLD, E-cadherin gene E-cadherin and airway remodeling-related gene smooth muscle actin gene α-SMA Gene expression.

[0056] (3) Data processing 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.

[0057] The results are shown in Table 6 and Table 7: Table 6 , Table 7 , The results showed that NHNK-619 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 expression.

[0058] Example 9 NHNK-619 Degradation of Histamine 1. NHNK-619 histamine degradation experiment A single colony of NHNK-619 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-619 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.

[0059] 2. Reagent preparation (1) 50 g / L Na2CO3 solution: Dissolve 5 g Na2CO3 in pure water and dilute to 100 mL with pure water.

[0060] (2) 250 g / L NaOH solution: Dissolve 25 g NaOH in pure water and dilute to 100 mL. (3) HCl solution: Add 5 mL of concentrated hydrochloric acid (HCl) to 55 mL of pure water and mix well.

[0061] (4) Liquid A (p-nitroaniline solution): Weigh 0.5g p-nitroaniline, dissolve it in 5mL HCl, dilute to 200mL with pure water, mix well, and prepare before use. Liquid B (NaNO2 solution): Weigh 0.5g NaNO2, dissolve it in pure water and dilute to 100mL, mix well. Azo reagent: Pipette 5mL of Liquid A and 40mL of Liquid B and mix.

[0062] (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.

[0063] 3. Determination of histamine content See also Figure 3 , (1) Pipette 0.00mL, 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 up to 1mL. Add 1mL HCl solution, 3mL 50g / L Na2CO3 solution, and 3mL azo reagent, add water to make up to the scale, and let it stand for 10 minutes before measuring the OD. 480 And draw a standard curve.

[0064] (2) Take 1 mL of the sample to be tested, add 1 mL of HCl solution, 3 mL of 50 g / L Na2CO3 solution, and 3 mL of azo reagent, add water to the scale, and measure the OD after leaving it for 10 minutes. 480 . Degradation rate (%) = (1-absorbance value of experimental group / absorbance value of control group)*100.

[0065] The results are shown in Table 8: Table 8 , The results showed that NHNK-619 could degrade histamine with a degradation rate of 16.72%~19.40%.

[0066] Example 10 NHNK-619 forms biofilm 1. NHNK-619 biofilm formation A single colony of NHNK-619 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.

[0067] 2. Crystal violet staining 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: Table 9 , The results showed that NHNK-619 in OD 600 =0.2 in MRS medium at 37℃ for 24h to form biofilm.

[0068] Example 11 Determination of the Adhesion Ability of NHNK-619 to Human Intestinal Epithelial Cells Caco-2 1. Preparation of NHNK-619 live bacterial suspension Pick a single colony of NHNK-619 in fresh MRS medium, culture anaerobically at 37°C for 24 h, collect the cells by centrifugation at 5000 rpm for 10 min, wash twice with sterile PBS, resuspend the cells in DMEM medium and adjust the OD 600 =0.5 to obtain a live bacterial suspension.

[0069] 2. Preparation of NHNK-619 biofilm bacterial suspension A single colony of NHNK-619 was selected and placed in MRS liquid medium, cultured anaerobically at 37°C for 24 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.

[0070] 3. Determination of the adsorption capacity of NHNK-619 to Caco-2 cells See also Figure 4 Caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37°C and 5% CO2. When the confluence reached 80%, the cells were digested with trypsin and collected, and inoculated into a 6-well plate containing a slide for overnight culture. After the culture was completed, the supernatant was discarded, washed twice with sterile PBS, and 1 mL of DMEM medium without FBS serum and 1 mL of live bacterial suspension or biofilm bacterial suspension were added, and the incubation continued for 2 hours. After the incubation was completed, it was washed twice with PBS to remove non-adherent bacteria. The slide was fixed in 4% paraformaldehyde for 15 minutes, followed by Gram staining and photography.

[0071] The results showed that NHNK-619 could adsorb human intestinal epithelial cells Caco-2 cells, and after NHNK-619 was coated with the biofilm formed by itself, it could increase the adsorption capacity of human intestinal epithelial cells Caco-2 cells.

[0072] 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 longum subsp. longum ( Bifidobacterium longum subsp. longum ), characterized in that, The Bifidobacterium longum subspecies longum is Bifidobacterium longum subspecies longum NHNK-619, which has been deposited in the China Center for Type Culture Collection on August 9, 2024, and its deposit number is CCTCC NO: M 20241760.

2. Based on the Bifidobacterium longum subspecies longum as claimed in claim 1 ( Bifidobacterium longum subsp. longum ) products prepared.

3. The Bifidobacterium longum subspecies longum according to claim 1 ( Bifidobacterium longum subsp. longum ) in the preparation of products for improving asthma and improving high histamine levels.

4. The use according to claim 3, characterized in that: The improvement of asthma includes at least one of regulating asthma-related flora, agglutinating asthma-related pathogens, inhibiting Staphylococcus aureus enterotoxin, reducing bronchial histamine receptor activation, promoting bronchial barrier integrity and reducing airway remodeling.

5. The use according to claim 4, characterized in that: The regulation of asthma-related flora includes inhibiting the growth of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, Prevotella humanus and promoting the growth of Propionibacterium acidi-propionici.

6. The use according to claim 4, characterized in that: The regulation of asthma-associated flora also includes reducing the formation of biofilms of Staphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, Moraxella catarrhalis, and Prevotella hominis.

7. The use according to claim 3, characterized in that: The reduction of bronchial histamine receptor activation comprises down-regulating 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.

8. The use according to claim 3, characterized in that: The improving high histamine levels includes degrading histamine.

9. The use according to claim 3, characterized in that: The product also includes auxiliary materials and / or additives.

10. The Bifidobacterium longum subspecies longum according to claim 1 ( Bifidobacterium longum subsp. longum ) in the application of bacterial biofilm to increase the colonization ability of intestinal epithelial cells.

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