CN119144530B

CN119144530B - Bifidobacterium longum subsp. longum and its application in improving asthma and high histamine level

Info

Publication number: CN119144530B
Application number: CN202411667754.2A
Authority: CN
Inventors: 陈奕兴; 谭磊磊; 宁长春; 赵兰慧; 冯海霞; 崔庆宇; 谢璐璐; 解维俊; 张宗博
Original assignee: Qingdao Nuohe Nuokang Biotechnology Co ltd
Current assignee: Qingdao Nuohe Nuokang Biotechnology Co ltd
Priority date: 2024-11-21
Filing date: 2024-11-21
Publication date: 2025-03-25
Anticipated expiration: 2044-11-21
Also published as: CN119144530A

Abstract

The present invention discloses Bifidobacterium longum subsp. longum and its application in improving asthma and high histamine levels, belonging to the field of marine microbial technology. Bifidobacterium longum subsp . longum NHNK‑619 has been deposited in the China Center for Type Culture Collection on August 9, 2024, and its deposit number is CCTCCNO: M20241760. NHNK‑619 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. The bacterial biofilm formed by it can increase the colonization ability of intestinal epithelial cells. It can be used to prepare products that improve asthma and high histamine levels.

Description

Bifidobacterium longum subspecies longum and use thereof for improving asthma and high histamine levels

Technical Field

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 level.

Background

Bronchial asthma (bronchial asthma) is simply called asthma, and is one of the most common chronic diseases in childhood. Studies have shown that there is a correlation between the colonization of Streptococcus pneumoniae and Moraxella catarrhalis and the occurrence of asthma and asthma attacks, and that the relevance of the Welch and Prevotella and pathogen scores is strong, with Staphylococcus aureus playing an important role in the regulation of diseases of the upper and lower airways. In clinical intervention trials, the use of propionibacterium for parturients and infants within june after delivery via caesarean section resulted in a significant reduction in IgE-associated asthma. Clinical studies have shown that children with higher butyrate and propionate content in the feces at 1 year of age have significantly reduced asthma and less likelihood of developing asthma when they grow to 3-6 years of age.

Airway inflammation, tissue damage and subsequent abnormal repair lead to changes in airway wall structure in asthmatic patients, known as airway remodeling, whose major pathological changes mainly include epithelial cell changes, mucous gland hyperplasia, subepithelial fibrosis, airway smooth muscle cell proliferation and migration, airway wall revascularization, etc. Smooth muscle actin (α -SMA) is an important indicator of airway remodeling, while E-cadherin (E-cadherin) can regulate the barrier function of airway epithelial cells, inhibiting the activity of NK-kappa b inflammatory pathways. The histamine receptors mainly comprise three subtypes, H1R, H R and H3R.

Histamine is a key molecule in the human body and is produced by immune cells called "mast cells" which are present in connective tissue and are part of the nervous immune system. Histamine is also naturally present in many foods. When histamine is too much to be excreted in the body, the human body scavenges histamine by specific enzymes such as diamine oxidase (DAO) and histamine n-methyltransferase (HNMT), but if these enzymes are not enough, histamine is accumulated up to a high level, and clinical symptoms caused by excess histamine due to various sources are hardly distinguished from allergic symptoms. Histamine sensitivity is dose-dependent, with more symptoms at the intake, and symptoms typically appear hours after eating. The most common accompanying symptoms are skin (wheal, urticaria, angioneurotic oedema, itching, redness), mucous membranes (itching, oedema, redness), headache/migraine, digestive tract dysfunction, etc.

Marine microorganisms are one of the most well known sources of marine bioactive substances, including algae, bacteria, fungi, protozoa, and the like. The bioactive substances have important application values in various fields such as medicines, foods, cosmetics and the like. Therefore, the marine industry related product developed by utilizing the microbiological technology is provided, and the marine industry related product has important practical significance.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a bifidobacterium longum subspecies longum and its use for improving asthma and high histamine levels.

The first aspect of the invention provides a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum), which is a bifidobacterium subspecies longum NHNK-619 and is preserved in China Center for Type Culture Collection (CCTCC) No. M20241760 at the year 08 and 09 of 2024.

In a second aspect the invention provides a preparation prepared on the basis of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) as described in the first aspect above.

In a third aspect the present invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) as described in the first aspect above for the manufacture of a product for the amelioration of asthma and for the amelioration of high histamine levels.

Further, the improving asthma includes at least one of modulating asthma-associated flora, aggregating asthma-associated pathogenic bacteria, inhibiting staphylococcus aureus enterotoxin, reducing activation of bronchohistamine receptors, promoting bronchial barrier integrity, and reducing airway remodeling.

Further, the regulation of asthma-related flora includes inhibition of staphylococcus aureus, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, prasuvorexant in humans, and promotion of the growth of propionibacterium propionicum.

Further, the regulation of asthma-related flora also includes reduction of biofilm formation by staphylococcus aureus, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis, prasugrel bacteria in humans.

Further, the reducing bronchohistamine receptor activation includes down-regulating expression of broncho-epithelial histamine receptor genes HR1, HR2, HR3, the promoting broncho-barrier integrity up-regulates expression of broncho-epithelial barrier-related genes OCLD and E-cadherein, and the reducing airway remodeling includes down-regulating expression of broncho-epithelial airway remodeling-related genes α -SMA.

Further, the improving high histamine levels includes degrading histamine.

Further, the product also comprises auxiliary materials and/or auxiliary agents.

In a fourth aspect, the invention provides the use of a bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum) as described in the first aspect above in a bacterial biofilm to increase colonisation capacity for intestinal epithelial cells.

The preservation number of the bifidobacterium longum subspecies NHNK-619 is CCTCC NO: M20241760. Experiments show that NHNK-619 has the functions of regulating asthma-related flora, agglomerating asthma-related pathogenic bacteria, inhibiting staphylococcus aureus enterotoxin, reducing activation of a bronchial histamine receptor, promoting the integrity of a bronchial barrier, reducing airway remodeling and degrading histamine.

Description of biological preservation

Bifidobacterium longum subspecies (Bifidobacterium longum subsp. Longum) NHNK-619 deposited at China center for type culture collection (CCTCC, address: eight ways 299 of Wuchang district of Wuhan, university of Wuhan, post code 430072) at 08-09 of 2024, with a deposit number of CCTCC NO: M20241760.

Drawings

FIG. 1 is a diagram showing the results of an experiment for separating Bifidobacterium longum subspecies NHNK-619 of example 1 of the present invention, wherein (a) is a plate colony morphology map of Bifidobacterium longum subspecies NHNK-619 and (b) is a gram staining morphology map of Bifidobacterium subspecies NHNK-619;

FIG. 2 is a graph showing experimental results of aggregation of pathogenic bacteria related to asthma by Bifidobacterium longum subspecies NHNK-619 of example 6 of the present invention, wherein (a) is a graph showing the aggregation of cells of Bifidobacterium longum subspecies NHNK-619, (b) is a graph showing the aggregation of cells of Moraxella catarrhalis, (c) is a graph showing the aggregation of cells of Prevotella in human being, (d) is a graph showing the aggregation of cells of a mixed reaction solution of Bifidobacterium longum subspecies NHNK-619 and Prevotella catarrhalis, and (e) is a graph showing the aggregation of cells of a mixed reaction solution of Bifidobacterium longum subspecies NHNK-619 and Prevotella in human being;

FIG. 3 is a graph showing the results of experiments on degradation of histamine by Bifidobacterium longum subspecies NHNK-619 of example 9 of the present invention;

FIG. 4 is a graph showing experimental results of measurement of the ability of bifidobacterium longum subspecies NHNK-619 of example 11 to adhere to human intestinal epithelial cells Caco-2;

The invention will be further described in the following detailed description in conjunction with the above-described figures.

Detailed Description

The present invention provides bifidobacterium longum subspecies longum and uses thereof for improving asthma and high histamine levels. Those skilled in the art can, with the benefit of this disclosure, suitably modify the process parameters to achieve this. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.

The bifidobacterium longum subspecies NHNK-619 of the invention are derived from variegated Bao Changdao and are identified as bifidobacterium subspecies longum (Bifidobacterium longum subsp. Longum) by 16S rDNA. The strain is gram positive, and has long and curved rod-like, expanded or dumbbell shape under microscope, and can form glossy, round colony with regular edge and raised center after growing on MRS plate, and has white precipitate after long-term bacteria growth at optimal anaerobic growth temperature of 37 deg.c.

Bifidobacterium longum subspecies (Bifidobacterium longum subsp. Longum) NHNK-619, the preservation unit is China center for type culture Collection, address is No. 299 of Wuhan City Wu Changou, university of Wuhan, preservation date is 09 of 2024, and preservation number is CCTCCNO: M20241760.

Further, the bifidobacterium longum subspecies NHNK-619 provided by the present invention are in the use according to the present invention in the form of non-sterilized live bacteria or sterilized inactivated bacteria, or in the form of fermentation/secretion products (i.e. supernatant), said derivative forms preferably being selected from the group consisting of metabolites, metabolic biological products, probiotics, cell walls and components thereof, extracellular polysaccharides and compounds containing immunogenic components, preferably from the group consisting of fermentation/secretion products, live bacteria, inactivated bacteria.

The reagent consumables adopted in the invention are all common commercial products. The invention will now be further illustrated with reference to examples:

Examples 1NHNK-619 isolation

Referring to FIG. 1, samples were derived from adult abalone plants, 5 cm in shell length and 3 days in empty stomach, were dissected with sterile tools to obtain the gut, washed 3 times with sterile saline, broken up into intestinal tissue by means of homogenization grinding, the homogenate was collected in 1 ml of sterile saline, diluted in gradient, the supernatant was streaked onto MRS solid plates, placed in anaerobic bags at 37℃for 48h, and white colonies were picked up and streaked repeatedly until a single colony with a regular and uniform shape was obtained, designated NHNK-619.

Gram staining microscopic examination shows that strain NHNK-619 is gram positive colony, and has long bent rod-like, expanded or dumbbell shape bacillus, and the bacterial colony with glossy, regular edge, round and raised center may be produced on MRS plate and after long time setting, the bacterial colony may be produced in MRS liquid culture medium in homogeneous turbidity.

Nucleic acid identification of examples 2NHNK-619

1. 16SrDNA Gene sequence analysis

Single colonies were picked up in MRS liquid medium, cultured overnight at 37℃and centrifuged at 8000 rpm for 1min to collect the colonies, which were then subjected to the procedure according to the instructions of the gram-positive DNA extraction kit. The primers were bacterial 16S sequencing universal primers 27F and 1492R, and the PCR amplification system was 20. Mu.L. The PCR amplification procedure was 95℃pre-denatured for 5min,94℃15s,57℃15s,72℃1min,35 cycles, 72℃extension for 10min.

2. Results

The sequencing result of the PCR product is shown as SEQ ID NO.1, and the result of homology comparison (BLASTN) with the published standard sequence in GenBank shows that NHNK-619 strain is bifidobacterium longum subspecies longum (Bifidobacterium longum subsp. Longum).

Examples 3NHNK-619 inhibit the proliferation of asthma-associated pathogenic bacteria

1. Cultivation of asthma-associated pathogenic bacteria

Staphylococcus aureus (Staphylococcus aureus) CGMCC 1.8721 frozen tube bacterial liquid is inoculated into a BHI liquid culture medium by 2% (v/v), and after shaking culture for 24 hours at 37 ℃, the temperature is adjusted to OD ₆₀₀ =0.1 by the BHI culture medium, thus obtaining staphylococcus aureus bacterial suspension.

Streptococcus pneumoniae (Streptococcus pneumoniae) BNCC338425 frozen tube bacterial liquid is inoculated into a TSB liquid culture medium containing 5% defibrinated sheep blood by 2% (v/v), and after shaking culture for 2d at 37 ℃, the culture medium is adjusted to OD ₆₀₀ =0.1 by the TSB, so as to obtain Streptococcus pneumoniae bacterial suspension.

The haemophilus influenzae (Haemophilus influenzae) BNCC359479 frozen tube bacterial liquid is inoculated into a special peptone culture medium containing 5% defibrinated sheep blood by 2% (v/v), and after shaking culture for 18 hours at 37 ℃, the special peptone culture medium is adjusted to OD600 = 0.1, thus obtaining the haemophilus influenzae bacterial suspension.

The Moraxella catarrhalis (Moraxella catarrhalis) BNCC275822 frozen tube bacterial liquid is inoculated into a BHI liquid culture medium by 2% (v/v), and after shaking culture for 2d at 37 ℃, OD ₆₀₀ =0.1 is adjusted by the BHI culture medium, thus obtaining Moraxella catarrhalis suspension.

Inoculating 2% (v/v) of human Prevotella (Prevotella corporis) BNCC361375 frozen tube bacterial liquid into a special peptone culture medium containing 5% defibrinated sheep blood, anaerobic culturing at 37 ℃ for 2 days, and regulating OD ₆₀₀ =0.1 by the special peptone culture medium to obtain human Prevotella bacterial suspension.

2. Preparation of NHNK-619 fermentation products

Selecting single colony in MRS liquid culture medium, anaerobic culturing at 37deg.C for 48 hr, adjusting to OD ₆₀₀ = 1.0 with MRS, centrifuging at 5000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain fermentation product.

3. NHNK-619 fermentation product for inhibiting proliferation of asthma-associated pathogenic bacteria

The pathogen suspension was added 160 μl per well to 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. The absorbance at OD ₆₀₀ nm was determined after 48 hours of incubation at 37 ℃. Inhibition% = (1-experimental absorbance/control absorbance) ×100%. The results are shown in Table 1:

TABLE 1

,

The result shows that NHNK-619 fermentation product can inhibit the growth of staphylococcus aureus, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and human Prevotella, and the inhibition rate is 14.81% -50.72%.

Examples 4NHNK-619 promote the proliferation of Propionibacterium propionicum

1. Culture of Propionibacterium propionicum (Propionibacterium acidipropionici)

The propionibacterium propionicum CGMCC 1.2232 frozen tube bacterial liquid is inoculated to a special peptone culture medium by 2% (v/v), anaerobic culture is carried out for 5-6 d at 37 ℃, and then the special peptone culture medium is adjusted to OD ₆₀₀ =0.5, thus obtaining the propionibacterium propionicum suspension.

2. NHNK-619 fermentation product preparation

Reference is made to example 3.

3. NHNK-619 fermentation product for promoting proliferation of propionibacterium propionicum

Propionibacterium propionicum suspension was added to 96-well plates at 160. Mu.L per well, 40. Mu.L of fermentation product was added to the experimental group, an equivalent amount of MRS medium was added to the control group, and the absorbance at OD ₆₀₀ nm was measured after 48 hours of incubation at 37 ℃. Proliferation rate = (experimental group absorbance-control group absorbance)/control group absorbance 100%. The results are shown in Table 2:

TABLE 2

,

The result shows that the acceleration rate of the NHNK-619 fermentation product to the propionibacterium propionicum is 38.27% -45.13%.

Examples 5NHNK-619 inhibition of asthma-associated pathogenic bacteria biofilm

1. Cultivation of asthma-associated pathogenic bacteria

The method of culturing staphylococcus aureus/streptococcus pneumoniae/haemophilus influenzae/moraxella catarrhalis/prasugrel human being is described in example 3;

2. NHNK-619 fermentation product preparation

Reference is made to example 3;

3. NHNK-619 biological membrane for inhibiting asthma-related pathogenic bacteria

The pathogen solution was added to 96-well plates at 100 μl per well, 100 μl of fermentation product per well was added to the experimental group, an equal volume of MRS medium was added to the control group, 3 replicates per group, and then incubation was continued for 24 hours at 37 ℃.

After the incubation was completed, the supernatant was discarded, and 100. Mu.L of sterile PBS was added to each well and washed twice, followed by adding 100. Mu.L of 4% paraformaldehyde fixing solution to each well, and fixing at room temperature for 30min. The fixative was discarded, 100. Mu.L of crystal violet was added to each well and stained at room temperature for 30min. After the dyeing is finished, the substrate is washed twice with sterile PBS and dried in the air, 100 mu L of absolute ethyl alcohol is added into each hole, and after standing for 1min, the absorbance at 600nm is measured. Inhibition% = (1-experimental/control group) 100%, the results are shown in table 3:

TABLE 3 Table 3

,

The result shows that NHNK-619 fermentation products can inhibit the formation of biological films of asthma-related pathogenic bacteria such as staphylococcus aureus, streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and human Prevotella, and the inhibition rate is 60.44% -84.74%.

Examples 6NHNK-619 agglutination of asthma-associated pathogenic bacteria

1. Cultivation of asthma-associated pathogenic bacteria

The frozen tube bacterial liquid of the Moraxella catarrhalis (Moraxella catarrhalis) BNCC275822 is inoculated into a BHI liquid culture medium at 2% (v/v), after shaking culture for 2d at 37 ℃, the bacterial cells are centrifugally taken and washed 3 times with sterile PBS, resuspended with sterile PBS and sterilized for 15min at the temperature of OD ₆₀ 0= 1.0,121 ℃ for standby.

Human Prevotella (Prevotella corporis) BNCC361375 frozen stock solution is inoculated into special peptone culture medium containing 5% defibrinated sheep blood at 2% (v/v), after anaerobic culture for 2d at 37 ℃, the thalli are centrifugally taken and washed 3 times with sterile PBS, resuspended with sterile PBS and OD ₆₀₀ = 1.0,121 ℃ is adjusted for sterilization 15min for later use.

2. Preparation of NHNK-619 inactivated thallus

Single colonies of NHNK-619 were picked in MRS liquid medium, anaerobically cultured for 48h at 37 ℃, centrifuged to obtain the thalli and washed 3 times with sterile PBS, resuspended with sterile PBS and OD ₆₀₀ = 1.0,121 ℃ sterilized 15 min for later use.

3. Co-coagulation

Mixing the pathogenic bacteria liquid and NHNK-619 inactivated bacteria liquid according to the volume ratio of 1:1, standing for 30min, and observing flocculation.

4. Index measurement

Referring to fig. 2, at the time of reaction for 30min, the reaction solution of NHNK-619 alone, pathogenic bacteria and the mixture of NHNK-619 and pathogenic bacteria was sampled in a range of 50 μl of the uppermost liquid surface, and transferred to a 96-well plate after pipetting, and the absorbance at od=600 nm was measured. Meanwhile, the aggregated precipitate was stained with a gram-type dye and then observed for the state of bacterial aggregation.

The calculation formula is as follows:

aggregation ratio (%) = [ (ax+ay) -2Amix ]/(ax+ay) ×100%;

Where Ax represents the OD ₆₀₀ value measured at the reaction time for individual NHNK-619, ay represents the OD ₆₀₀ value measured at the reaction time for individual pathogens, and Amix represents the OD ₆₀₀ value measured at the reaction time after mixing NHNK-619 with pathogens.

The results are shown in Table 4:

TABLE 4 Table 4

,

The result shows that NHNK-619 inactivated bacteria can agglutinate human Prevotella and Moraxella catarrhalis, and the agglutination rate is 16.87% -20.27%. Namely, the pathogenic bacteria related to the agglutination asthma comprise the agglutination catarrhalis and the human Prevotella.

Examples 7NHNK-619 inhibition of Staphylococcus aureus enterotoxin Gene

1. Preparation of NHNK-619 fermentation products

Reference is made to example 3.

2. NHNK-619 down-regulating staphylococcus aureus enterotoxin gene expression

(1) Staphylococcus aureus culture and intervention

Staphylococcus aureus CGMCC 1.8721 was inoculated at 2% (v/v) into fresh BHI medium, shake-cultured at 37 ℃ for 24h, followed by adjustment of OD ₆₀₀ = 0.5 with BHI medium. The staphylococcus aureus suspension was added to the centrifuge tube with 1mL of additional 2mL of BHI medium per tube. The experiment was added with 1mL of fermentation product, respectively, and the control was added with an equal volume of MRS, at 37℃for further incubation for 16h.

(2) After the culture is finished, extracting staphylococcus aureus RNA by using a gram positive bacterium RNA extraction kit, detecting the concentration and purity of the RNA, performing reverse transcription to obtain cDNA, using 16S rRNA as an internal reference gene, and detecting the expression of the enterostaphylococcus aureus enterotoxin A gene sea by using a qPCR fluorescent quantification technology.

(3) Data processing

The relative expression fold f=1 for the control group genes, and the F value for each sample was calculated using the 2 ^-ΔΔCT method.

Formula f=2 ^-ΔΔCT, wherein:

△CT_Experiment=CT_Experiment-CT_{Internal reference （ Experiment ）;}

△CT_Control=CT_Control-CT_{Internal reference （ Control ）;}

△△CT=△CT_Experiment-△CT_{Control .}

The results are shown in Table 5:

TABLE 5

,

The result shows that NHNK-619 fermentation product can inhibit the expression of staphylococcus aureus enterotoxin gene sea, and the relative expression multiple is 0.77-0.88.

Example 8NHNK-619 modulation of human bronchial epithelial cells Beas-2b histamine receptor, bronchial barrier and airway remodeling related Gene expression

1. NHNK-619 inactivated bacteria and preparation of fermentation products

Picking NHNK-619 single colony in MRS liquid culture medium, anaerobic culturing at 37deg.C for 48: 48 h, adjusting to OD ₆₀₀ =0.5 with DMEM, centrifuging at 5000rpm for 10min, collecting supernatant, and filtering with 0.22 μm filter membrane to obtain fermentation product. Washing the living thalli precipitated by centrifugation with PBS, re-suspending the living thalli by proper amount, sterilizing at a high temperature of 121 ℃ for 15min, removing the supernatant after centrifugation, and re-suspending the living thalli by DMEM until the OD600 = 0.5 to obtain the inactivated bacterial suspension.

2. NHNK-619 modulate Beas-2b cell histamine receptor, bronchial barrier and airway remodeling related gene expression

(1) Intervention treatment of Beas-2b cells

Beas-2b cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37℃under 5% CO ₂. When the cells were fused to 80%, the cells were collected and inoculated into 6-well plates, the supernatant was discarded after overnight culture, 1mL of fermentation product or inactivated bacteria and 1mL 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 ℃ under 5% co ₂ for 16 hours.

(2) RNA extraction and fluorescent quantitative PCR

After the culture is finished, RIPA lysate is added to lyse cells, total RNA is extracted, after the concentration and purity of the RNA are detected, reverse transcription is carried out to obtain cDNA, GAPDH is used as an internal reference gene, and qPCR fluorescent quantitative technology is adopted to detect the expression of histamine receptor related genes H1R, H, R, H R, a tight junction protein gene OCLD, an E-cadherin gene E-cadherin and airway remodeling related genes smooth muscle actin gene alpha-SMA.

(3) Data processing

The results are shown in Table 6 and Table 7:

TABLE 6

,

TABLE 7

,

The results show that NHNK-619 can down regulate the expression of histamine receptor genes H1R, H R and H3R, up regulate the expression of a claudin gene OCLD and an E-cadherin gene E-cadherin, and down regulate the expression of a smooth muscle actin gene alpha-SMA.

Examples 9NHNK-619 degradation of histamine

1. NHNK-619 test for degradation of Histamine

Single colonies of NHNK-619 were picked up in fresh MRS medium, anaerobically cultured at 37 ℃ for 48h, then adjusted to OD ₆₀₀ =1.0 with MRS to obtain NHNK-619 broth, inoculated at 2% (v/v) in MRS medium containing 500 μg/mL histamine, anaerobically cultured at 37 ℃ for 48h, and control was inoculated into an equal volume of MRS medium.

2. Reagent preparation

(1) 50G/L Na ₂CO₃ solution 5g Na ₂CO₃ was dissolved in pure water and diluted to 100mL with pure water.

(2) 250G/L NaOH solution 25g NaOH was dissolved in pure water and diluted to 100mL.

(3) 5ML of concentrated HCl is added into 55mL of pure water, and the mixture is uniformly mixed.

(4) The solution A (paranitroaniline solution) is prepared by weighing 0.5g paranitroaniline, adding 5mLHCl to dissolve, diluting with pure water to 200mL, mixing, and preparing for immediate use. Solution B (NaNO ₂ solution) is prepared by weighing 0.5: 0.5gNaNO ₂, dissolving with pure water, diluting to 100mL, and mixing. And (3) sucking 5mL of first solution and mixing 40mL of second solution.

(5) 1000 Mug.mL ^-1 histamine phosphate standard solution 0.025g histamine dihydrochloride standard is precisely weighed, dissolved in pure water, transferred into a 25mL volumetric flask, and mixed with pure water to a scale. 20 mu g/mL ^-1 standard use solution of histamine phosphate 1mL1000 mu g/mL ^-1 standard use solution of histamine phosphate is sucked into a 50mL volumetric flask, purified water is added to the volume to scale, and the mixture is uniformly mixed.

3. Histamine assay

Referring to figure 3 of the drawings in which,

(1) Respectively sucking 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL and 1.0mL of standard solution of the histamine phosphate of 20 mug/mL into a 10mL colorimetric tube with a plug, adding water to a volume of 1mL, adding 1mL of HCl solution, 3mL of 50g/LNa ₂CO₃ solution and 3mL of azo reagent, adding water to a volume of scale, standing for 10min, measuring OD ₄₈₀ and drawing a standard curve.

(2) 1ML of a sample to be detected is sucked, 1mL of HCl solution, 3mL of 50g/L Na ₂CO₃ solution and 3mL of azo reagent are additionally added, water is added to fix the volume to the scale, and after the sample is placed for 10min, OD ₄₈₀ is measured. Degradation (%) = (1-absorbance of experimental group/absorbance of control group) ×100.

The results are shown in Table 8:

TABLE 8

,

The result shows that NHNK-619 can degrade histamine with a degradation rate of 16.72% -19.40%.

Examples 10NHNK-619 biofilm formation

1. NHNK-619 formation of biofilm

Single colonies of NHNK-619 were picked in MRS liquid medium, anaerobically cultured for 48h at 37 ℃ and adjusted to OD ₆₀₀ = 0.2 with fresh MRS, 100 μl of bacterial liquid was added per well in 96 well plates, 3 replicates per group, and then anaerobically cultured for 24 hours at 37 ℃.

2. Crystal violet staining

After the incubation was completed, the supernatant was discarded, and 100. Mu.L of sterile PBS was added to each well and washed twice, followed by adding 100. Mu.L of 4% paraformaldehyde fixing solution to each well, and fixing at room temperature for 30min. The fixative was discarded, 100. Mu.L of crystal violet was added to each well and stained at room temperature for 30min. After the dyeing was completed, the substrate was washed twice with sterile PBS and dried in the air, 100. Mu.L of absolute ethanol was added to each well, and after standing for 1min, the absorbance at 600nm was measured, and the results are shown in Table 9:

TABLE 9

,

The results showed that NHNK-619 was able to form biofilms at OD ₆₀₀ = 0.2 at 37 ℃ anaerobic resting culture for 24h in MRS medium.

Examples 11NHNK-619 determination of the adhesion Capacity of human intestinal epithelial cells Caco-2

1. Preparation of NHNK-619 viable bacteria suspension

Picking NHNK-619 single colony, anaerobic culturing at 37 ℃ for 24h in fresh MRS culture medium, centrifuging at 5000rpm for 10min, collecting thalli, washing twice with sterile PBS, re-suspending thalli with DMEM culture medium, and adjusting OD ₆₀₀ =0.5 to obtain viable bacteria suspension.

2. Preparation of NHNK-619 biological membrane bacterial suspension

Single colonies of NHNK-619 were picked in MRS liquid medium, anaerobically cultured for 24h at 37 ℃ and added to bacterial dishes with fresh MRS adjusted to OD ₆₀₀ = 0.2, followed by continued anaerobic culture at 37 ℃ for 24 hours. After the culture is finished, the upper culture medium is discarded, the bottom biomembrane is resuspended after the two times of washing by PBS, the thalli is collected after centrifugation for 10min at 5000rpm, the thalli is resuspended by the DMEM culture medium, and the OD ₆₀₀ = 0.5 is adjusted, thus obtaining the biomembrane bacterial suspension.

3. NHNK-619 determination of adsorption Capo-2 cell Capacity

Referring to FIG. 4, caco-2 cells were inoculated in DMEM medium containing 10% FBS serum and cultured at 37℃under 5% CO ₂. Cells were digested with pancreatin and harvested at 80% confluence, inoculated into 6-well plates containing slide plates, and cultured overnight. After the completion of the incubation, the supernatant was discarded, washed 2 times with sterile PBS, and 1mL of DMEM medium free of FBS serum and 1mL of viable bacteria suspension or biofilm bacteria suspension were added to continue the incubation for 2 hours. After the incubation was completed, the cells were washed with PBS 2 times to remove non-adherent cells. The slide was soaked in 4% paraformaldehyde for 15min, then gram stained and photographed.

The result shows that NHNK-619 can adsorb the human intestinal epithelial cells Caco-2 cells, and NHNK-619 can increase the adsorption capacity to the human intestinal epithelial cells Caco-2 cells after coating the self-formed biological film.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A strain of Bifidobacterium longum subsp. longum , characterized in that the Bifidobacterium longum subsp. longum is Bifidobacterium longum subsp. 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. A product prepared based on the Bifidobacterium longum subsp. longum according to claim 1.

3. Use of Bifidobacterium longum subsp. longum as claimed in claim 1 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 humanis and promoting the growth of Propionibacterium acidipropionici.

6. The use according to claim 4, characterized in that the regulation of asthma-related 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 4, characterized in that the reducing of bronchial histamine receptor activation comprises down-regulating the expression of bronchial epithelial cell histamine receptor genes HR1, HR2, and HR3 , the promoting of bronchial barrier integrity comprises up-regulating the expression of bronchial epithelial cell barrier-related genes OCLD and E-cadherin , and the reducing of airway remodeling comprises down-regulating the expression of bronchial epithelial cell airway remodeling-related gene α-SMA .

8. The use according to claim 3, characterized in that the improving high histamine level comprises degrading histamine.

9. The use according to claim 3, characterized in that the product further comprises auxiliary materials and/or additives.