CN114717127B - Lactobacillus reuteri for preventing and relieving allergic asthma symptoms and application thereof - Google Patents
Lactobacillus reuteri for preventing and relieving allergic asthma symptoms and application thereof Download PDFInfo
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- CN114717127B CN114717127B CN202110352326.0A CN202110352326A CN114717127B CN 114717127 B CN114717127 B CN 114717127B CN 202110352326 A CN202110352326 A CN 202110352326A CN 114717127 B CN114717127 B CN 114717127B
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Classifications
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
- A61P11/06—Antiasthmatics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/173—Reuteri
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- General Chemical & Material Sciences (AREA)
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- Molecular Biology (AREA)
- Epidemiology (AREA)
- Nutrition Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention provides a novel lactobacillus reuteri (Lactobacillus reuteri), which is a lactobacillus reuteri VHProbi M07 strain and is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China in 10 months 08 of 2019: m2019779. The lactobacillus reuteri provided by the invention is used for preparing products for preventing and treating allergic asthma. The lactobacillus reuteri VHProbi M07 provided by the invention can be used as a food raw material source, and has no side effect and excessive risk after long-term administration. The lactobacillus reuteri provided by the invention can effectively prevent and relieve allergic asthma symptoms, and the strain can be independently used, has the effect of preventing and relieving allergic asthma without being compounded with prebiotics and/or other probiotics, and has important application value.
Description
Technical Field
The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus reuteri with the function of preventing and relieving allergic asthma symptoms and application of lactobacillus reuteri.
Background
Allergic asthma is a clinical syndrome with heterogeneity that involves the lower respiratory tract. According to epidemiological investigation and research results, the prevalence of asthma patients in China is increasing continuously, about 2000 thousands of people. Asthma is clinically manifested as persistent or intermittent allergic symptoms such as wheezing, cough, dyspnea, and mucus increase after re-exposure to allergen, and pathology is manifested by eosinophilia and cytokine increase associated with Th2 type immune response. In clinical treatment, glucocorticoids and beta 2 receptor agonists are main medicines for treating asthma, but the problems of poor medicine toxicity, poor compliance and the like always plague patients. Thus, finding new therapeutic approaches to asthma is a major issue.
Research of the intestinal flora by the scientific community has shown explosive growth in the last two decades. The research shows that probiotics can help digestion and absorption, promote intestinal flora balance and maintain human health. There is increasing evidence that the health effects of probiotics on humans are not limited to the intestinal tract but also have a broader range of action, such as endocrine balance regulation, immune balance regulation, nervous system regulation, respiratory system regulation, etc. Therefore, the prevention and alleviation of immune-mediated multi-cause diseases such as allergic asthma by administering probiotics is a new idea for treating asthma.
The Chinese patent publication No. CN 109628359A discloses Lactobacillus reuteri for relieving allergic asthma and application thereof, and animal experiments show that the strain can relieve asthma symptoms of mice after 6 weeks of gastric lavage. The Chinese patent publication No. CN 110643542A discloses Lactobacillus reuteri for relieving allergic asthma and application thereof, and animal experiments show that the strain can relieve asthma symptoms of mice after 6 weeks of gastric lavage. However, the existing probiotics for preventing and relieving allergic symptoms have the problems of unknown action effect mechanism, slow effect, weak action effect and the like. Therefore, a new probiotic strain with definite action mechanism and remarkable action effect and capable of rapidly preventing and relieving allergic asthma, relieving lung inflammation and recovering Th1/Th2 type immune reaction balance is needed to be found.
Disclosure of Invention
The invention aims to provide a novel lactobacillus reuteri (Lactobacillus reuteri) and application thereof; the provided lactobacillus reuteri is separated from breast milk of healthy lactating women, can regulate immunity, improve the immune function of human bodies, and effectively prevent and relieve allergic asthma symptoms.
The lactobacillus reuteri provided by the invention is a lactobacillus reuteri (Lactobacillus reuteri) VHProbi M07 strain, and is preserved in China center for type culture collection (CCTCC NO) of university of Wuhan in China in 10 months 08 of 2019: m2019779.
The RAPD fingerprint of the M07 strain is shown in FIG. 2, and the rep-PCR fingerprint is shown in FIG. 3.
The 16s rDNA sequence of the lactobacillus reuteri VHProbi M07 strain provided by the invention is SEQ ID NO. 1.
The lactobacillus reuteri provided by the invention is applied to the preparation of medicines for preventing and treating allergic asthma.
The lactobacillus reuteri VHProbi M07 provided by the invention has no side effect and excessive risk after long-term administration. The lactobacillus reuteri VHProbi M07 provided by the invention can effectively prevent and relieve allergic asthma symptoms, and the strain can be independently used, has the effect of preventing and relieving allergic asthma without being compounded with prebiotics and/or other probiotics, and has important application value.
Drawings
FIG. 1 is a chart of the Riboprinter fingerprint of the M07 strain;
FIG. 2 is a RAPD fingerprint of strain M07;
FIG. 3 shows rep-PCR fingerprint of M07 strain;
FIG. 4 is a graph showing the results of differential leukocyte counts for each group of mice;
FIG. 5 is a graph showing cytokine measurement results of the fine alveolar lavage fluid of each group of mice;
FIG. 6 is a graph showing the results of pathological staining of lung tissue of mice of each group.
Detailed Description
The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.
The present invention will be described in detail with reference to specific examples.
Example 1 isolation screening of Lactobacillus reuteri VHProbi M07
1. Primary screen
Preparing MRS (Man Rogosa Sharpe) agar medium: 1000mL of purified water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of citric acid diamine, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ for 15min.
According to 2019 edition of human genetic resource coulomb regulation, after signing project commitment and informed consent with a sample provider, taking 1mL of fresh breast milk of a parturient in lactation period, which does not eat probiotic preparations within half a year, diluting the fresh breast milk with sterile normal saline, putting the diluted breast milk into a sterile sample bag, and beating and uniformly mixing the diluted breast milk with a homogenizer; and (3) taking 100 mu L of mixed solution for gradient dilution, coating the mixed solution on an MRS agar medium, and performing anaerobic culture at 37 ℃ for 48 hours, and performing microscopic examination on a single colony after the plate grows.
According to the microscopic examination result, the applicant screens out 7 potential lactobacillus strains, which are respectively named as M01, M02, … … M06 and M07.
2. Double screen
Preparing 1L of MRS liquid culture medium, sterilizing at 121deg.C for 15min, cooling, adding 3.2g of pig mucosa pepsin, shaking for dissolving, and placing in a 37 deg.C water bath shaking table for 1 hr to obtain acid-resistant culture medium.
7 strains of lactobacillus M01, M02, … … M06 and M07 obtained by screening are respectively inoculated into the acid-resistant culture medium according to the inoculum size of 6 percent, and are subjected to anaerobic static culture for 48 hours at 37 ℃, and fermentation liquor is taken for bacterial count.
The results show that the logarithmic values of the viable bacteria amounts in the 7 lactobacillus fermentation liquid are 7.23, 6.94, 6.76, 7.56, 6.33, 5.39 and 8.78Log CFU/mL respectively, wherein the M07 strain has the largest viable bacteria amount after being re-screened by the acid-resistant culture medium, and the logarithmic value is as high as 8.78Log CFU/mL. Thus, the strain M07 has the highest acid resistance.
Example 2 identification of strains
1. Colony morphology identification
After the M07 strain is inoculated on an MRS agar culture medium and subjected to anaerobic culture for 24 hours at 37 ℃, the single colony of the M07 strain is milky white, the diameter of the colony is about 1.5-3mm, the surface is moist, and the tail end of the colony is circular campylobacter under a microscope.
2. Identification of physiological and biochemical characteristics
The inoculum was prepared as follows: under the aseptic condition, a proper amount of fresh M07 bacterial liquid is taken, centrifuged for 5min at 5000rpm/min, washed for 2 times by PBS buffer, and then the bacterial cells are diluted by 50 times after the same volume of PBS buffer is used as an inoculation liquid.
2.1 salinity tolerance test
Under aseptic conditions, 190. Mu.L of BSM liquid medium with salt concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7% and 8% was added to the 96-well plate, respectively, 3 replicates of each salt concentration, and then 10. Mu.L of inoculum was added thereto, and the wells without inoculation were used as controls. 50. Mu.L of autoclaved paraffin oil was added to each well to prevent evaporation of water during the culture. Culturing at 37deg.C, and observing whether the culture medium becomes turbid.
The results showed that the M07 strain had a maximum tolerated salt concentration of 1%.
2.2 catalase experiments
The fresh bacterial liquid was taken and dropped onto a clean glass slide, and then a drop of 3% hydrogen peroxide solution was dropped thereon, and no bubbles were observed to be generated by the M07 strain, which was a negative reaction.
2.3 carbon Source metabolism test
The basal medium formulation used in this example is as follows:
1.5g of peptone; 0.6g of yeast extract; tween 80.1 g; 0.5mL of saline solution; 18mg of phenol red; distilled water 100mL; pH 7.4.+ -. 0.2. Salt solution components: mgSO (MgSO) 4 ·7H 2 O 11.5g，MnSO 4 ·4H 2 O2.8 g, distilled water 100mL.
A10 g/100mL solution of sugar, alcohol and glycoside carbohydrate was prepared and filtered with a 0.22 μm sterile filter. Under aseptic conditions, 20. Mu.L of sterilized carbohydrate solution, 4 per carbohydrate, was added to the 96-well plate, then 170. Mu.L of sterilized phenol red-containing basal medium was added, and 10. Mu.L of inoculum was added, without inoculating the reaction well as a control. 50. Mu.L of liquid paraffin was added to each well to prevent evaporation of water during the culture. Anaerobic culture at 37 deg.c with phenol red as indicator to observe the color change of the culture medium; the specific results are shown in Table 1.
Table 1: carbon source metabolism results table of M07 strain
| Cellobiose | Melibiose | Raffinose | Mannitol (mannitol) | Amygdalin | Sucrose | Galactose |
| - | + | + | - | - | + | + |
| Lactose and lactose | Maltose | Mannose | Salicin | Trehalose | Arabinose (Arabic sugar) | Gluconic acid sodium salt |
| + | + | - | - | - | + | + |
| Melezitose | Ribose | Sorbitol | Xylose | Rhamnose (rhamnose) | / | / |
| - | + | - | - | - | / | / |
Note that: a "+" positive response; "-" negative reaction.
3 molecular biological identification
3.1 16s rDNA Gene sequence analysis
3.1.1 genomic DNA extraction
Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).
3.1.2, 16s rDNA Gene amplification
1) Primer sequence:
27F：AGAGTTTGATCCTGGCTCA；
1492R：GGTTACCTTGTTACGACTT。
2) Reaction system (50. Mu.L)
Table 2:16s rDNA PCR amplification System Table
3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.
4) Sequencing of PCR products
The 16s rDNA sequence SEQ ID NO. 1 of the M07 strain was obtained by sequencing, and the sequences were aligned in NCBI database to preliminarily determine that the M07 strain was Lactobacillus reuteri.
3.2 Riboprinter fingerprint
The purified single colony is dipped from an agar culture medium plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then a sample frame is placed into a heater for inactivation and then placed into a Riboprinter system, and a bacterial identification result is obtained after DNA preparation, film transfer, imaging detection and data processing are carried out on the sample. The identification result shows that the M07 strain is lactobacillus reuteri, and the result of the Riboprinter fingerprint is shown in figure 1.
3.3RAPD and rep-PCR fingerprinting identification
3.3.1 RAPD finger print identification
1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');
2) RAPD reaction system
Table 3: RAPD reaction System Table
3) Electrophoresis
1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and finally the electropherograms were detected using a gel imaging system. RAPD finger-prints of M07 strain are shown in FIG. 2. 3.3.2 rep-PCR finger print
1) rep-PCR primer
CTACGGCAAGGCGACGCTGACG。
2) reaction system of rep-PCR
Table 4: table of the reaction System of rep-PCR
3) Electrophoresis
DL2000 DNA Marker served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of the M07 strain is shown in FIG. 3.
To sum up, the colony morphology and physiological and biochemical characteristic results of the M07 strain were uploaded to the website http:// www.tgw1916.net/bacteria_log_desktop. HtmL, and aligned in combination with the results published in De Clerck E, et al systems and applied microbiology,2004,27 (1) 50. From the results of the molecular biology identification, it can be concluded that the M07 strain is a novel strain of Lactobacillus reuteri, which is named Lactobacillus reuteri VHProbi M07.
EXAMPLE 3 preparation of Lactobacillus reuteri VHProbi M07 strain for tolerance test 1 to artificial gastric juice and artificial intestinal juice
5g of peptone, 2.5g of yeast extract, 1g of glucose and 2g of NaCl are weighed respectively, 1000mL of distilled water is added, pH is adjusted to 3.0 by dilute hydrochloric acid, and then sterilization is carried out for 20min at 115 ℃. Then 3.2g of pig mucosa pepsin is added before use, the pig mucosa pepsin is uniformly shaken and dissolved, and the mixture is placed in a water bath shaker at 37 ℃ for warm water bath for 1 hour so as to simulate the temperature of a human body.
2 preparation of artificial intestinal juice
Respectively weighing peptone 5g, yeast extract 2.5g, glucose 1g, KH 2 PO 4 6.8g and 3.0g of ox gall salt, 77mL of 0.2mol/L NaOH solution is added, the volume is fixed to 1000mL, the pH is regulated to 6.8+/-0.1 by dilute hydrochloric acid or sodium hydroxide solution, and the mixture is sterilized for 20min at 115 ℃. Then adding 1g of pancreatin before use, shaking to dissolve, and placing in a water bath shaker at 37 ℃ for warm water bath for 1h to simulate the temperature of human body.
3 test method
2mL of fresh bacterial liquid is taken, the bacterial liquid is collected by centrifugation at 5000rpm/min for 5min, the bacterial liquid is washed 3 times by physiological saline, and then 2mL of physiological saline is used as inoculation liquid for resuspension. 1mL of the inoculation liquid is taken and added into 24mL of artificial intestinal juice, and the mixture is placed on a water bath shaking table (200 rpm/min) at 37 ℃ for 3 hours, 1mL of sample is taken, and the viable bacteria amount is detected.
The viable bacteria counting method is used for measuring the bacterial amount according to national standard GB 4789.35-2016-lactobacillus test for food microorganism test, and the viable bacteria amount (Log CFU/mL) of the strain after artificial intestinal juice digestion is shown in Table 5.
Table 5: viable bacteria scale after digestion of artificial gastrointestinal fluids
As shown in Table 5, the survival rate of the lactobacillus reuteri VHProbi M07 screened by the method can still reach 86.2% after being digested by artificial gastric juice and artificial intestinal juice. Therefore, the strain has strong tolerance to artificial gastric juice and artificial intestinal juice.
EXAMPLE 4 haemolytic and antibiotic resistance experiments with Lactobacillus reuteri VHProbi M07
1. Hemolysis test
(1) Preparing an inoculation liquid: the frozen and preserved lactobacillus reuteri VHProbi M07 strain is streaked and inoculated in an MRS agar culture medium, cultured for 24-48 hours at the temperature of 37 ℃, subcultured for 1 time by the MRS liquid culture medium, and then the lactobacillus reuteri VHProbi M07 is inoculated in a fresh MRS liquid culture medium for 24-48 hours at the temperature of 37 ℃ with the inoculum size of 5 percent, so that fresh bacterial liquid is obtained and is used as the inoculum.
(2) Preparation of blood cell culture medium: weighing the various components of TBS basic culture medium, dissolving, autoclaving at 121deg.C for 15min, cooling to 50deg.C, adding 5% sterilized defibrinated sheep blood, mixing, and plating.
(3) And (3) streaking culture: and streaking the test strain, inoculating the streaked strain to a prepared blood cell plate, culturing the strain in a 37 ℃ incubator, and observing whether the test strain has a hemolysis phenomenon or not in 24-48 hours.
The results show that: the lactobacillus reuteri VHProbi M07 was unable to grow and the blood cell plate was unchanged, indicating that lactobacillus reuteri VHProbi M07 did not produce hemolysin and was unable to lyse blood cells.
2. Antibiotic resistance test
(1) Preparing antibiotics: ampicillin, clindamycin, erythromycin, gentamicin, streptomycin, tetracycline and vancomycin are prepared into stock solution of 2048 mug/mL, and the stock solution is preserved at-20 ℃ for standby. When in use, the stock solution is serially diluted by 2 times by using BSM liquid culture medium to form use solution, and the gradient dilution concentration is 1-1024 mu g/mL and total 11 gradients.
(2) Preparing an inoculation liquid: taking a proper amount of fresh bacterial liquid (24 h,37 ℃ for culture), centrifuging at 5000rpm for 5min, washing once with sterile physiological saline, and diluting 50 times after re-suspending bacterial cells with the same volume of physiological saline to obtain an inoculation liquid.
(3) Determination of minimum inhibitory concentration MIC value of antibiotic for Lactobacillus reuteri VHProbi M07 by micro broth dilution method
an MRS liquid culture medium without antibiotics is added to the 1 st column of the 96-well plate as a negative control, 190 mu L of MRS liquid culture medium with antibiotics with different concentrations is sequentially added to the 2 nd to 12 th columns, 10 mu L of the inoculation liquid is inoculated respectively, 3 parallel wells are made, and 1 well of the non-added bacteria liquid is used as a blank.
b. 50. Mu.L of paraffin oil was added to cover the water and prevent evaporation.
c. The 96-well plate was incubated at 37℃for 24 hours, then removed, and OD was measured 600 Values, MIC values of antibiotics against strains were counted with 24h results, and specific results are shown in table 6.
Table 6: antibiotic MIC value of Lactobacillus reuteri VHProbi M07 μg /mL)
From the results shown in Table 6, the Lactobacillus reuteri VHProbi M07 provided by the invention is sensitive to common antibiotics such as erythromycin and ampicillin, and has good biological safety.
Example 5 hydrophobic cell surface test of Lactobacillus reuteri VHProbi M07
1. Preparing a bacterial liquid to be tested: the purified lactobacillus reuteri VHProbi M07 colony is selected and inoculated in a newly prepared MRS liquid culture medium, and is cultured for 24 to 48 hours at 37 ℃. Inoculating 1% (V/V) of the strain into MRS liquid culture medium, continuously culturing at 37deg.C for 24-48 hr, centrifuging at 6000 Xg for 10min, collecting thallus, washing with sterile physiological saline for 2 times, and sterilizing with 0.1M KNO 3 The bacterial cells were resuspended in 1mL of the solution and used as the bacterial liquid to be tested.
2. Surface hydrophobicity determination: mu.L of the above bacterial suspension was pipetted into 2450. Mu.L of 0.1M KNO 3 And record OD600 as A 0 1.5mL of the bacterial suspension was mixed with 500. Mu.L of xylene, and the mixture was allowed to stand at room temperature for 10 minutes (a two-phase system was formed). Vortex oscillating the two-phase system for 2min, standing for 20min, and reforming into water phase and organic phase. The aqueous phase (not to the organic phase) was carefully aspirated and absorbance A was measured at 600nm 1 . Cell Hydrophobicity = (a) according to the formula hydropathicity = (a) 0 -A 1 )/A 1 X% calculation, measurement of the average of three experiments.
The results show that: the hydrophobicity of the surface of the lactobacillus reuteri VHProbi M07 cell provided by the invention is 64.48%, and the standard deviation is 2.16%.
Example 6 use of Lactobacillus reuteri VHProbi M07 for alleviating allergic asthma in mice
1.1 Experimental materials
1.1.1 laboratory animals
BALB/c mice SPF grade, 24 females, 8-9 weeks, body weight 19-25 g. License number SCXK (robust) 20140007 was produced by the experimental animal breeding limited company, punyue, atanan.
Environmental conditions for experimental animal feeding management: the room temperature is 20-26 ℃, the daily temperature difference is less than or equal to 4 ℃, the relative humidity is 40-70%,
the light and shade alternation time is 12/12h. Animals were kept in standard mouse cages, 6 per cage.
Animal feed, drinking water: can be ingested and drunk freely. The feed is SPF-class mice growth breeding feed, which is provided by Jinan Pengyue laboratory animal breeding Limited (lot number: 20190905). The drinking water is city tap water sterilized at high temperature.
1.1.2 reagent consumable
Ovalbumin (OVA) (lot number: S12016): shanghai Yuan Ye Biotech Co., ltd;
aluminum adjuvant (lot number: UL 292268): simer Feier technology (China);
IL-5 (lot number: E20200605-20187B) kit: shanghai enzyme-linked biotechnology limited company;
IL-10 (lot number: E20200605-20188B) kit: shanghai enzyme-linked biotechnology limited company;
IL-13 (lot number: E20200601-201662B) kit: shanghai enzyme-linked biotechnology limited company;
MCP-1 (lot number: E20200602-20140B) kit: shanghai enzyme-linked biotechnology limited company;
TNF-alpha cytokine (lot number: E20200607-20852B) kit: shanghai enzyme-linked biotechnology limited company;
IFN-gamma (lot number: E20200803-20140B) kit: shanghai enzyme-linked biotechnology limited company;
kit for eotaxin (lot number: E20200803-20622B): shanghai enzyme-linked biotechnology limited company;
eosin (lot number: 20181204): beijing Soy Bao technology Co., ltd;
hematoxylin (lot number: 20181204): beijing Soy Bao technology Co., ltd.
1.2 Experimental methods
1.2.1 preparation of bacterial liquid
Anaerobic culturing single colony on MRS plate at 37 deg.C for 24-48 hr, picking single colony, amplifying culturing in MRS broth culture medium for 16 hr, collecting bacterial liquid and regulating its concentration to 10 9 CFU/mL bacterial suspension.
1.2.2 grouping
Mice were randomly divided into a blank control group, an OVA allergy model group, a probiotic pretreatment group, and a probiotic post-treatment group after 7 days of adaptive rearing, 6 mice per group, and probiotic pretreatment group and post-treatment group were given probiotic bacterial liquid according to a gastric lavage of 0.2mL/10 g.
1.2.3 modeling and probiotic intervention scheme
The probiotic pretreatment group mice were given probiotic bacteria by gastric lavage in advance, for 10 consecutive days, before molding, and the other groups were untreated. In addition to the blank control group, the OVA allergy model group, the probiotic pretreatment group, the probiotic post-treatment group mice were intraperitoneally injected with 200 μl of allergen solution on days 0,6, 50 μg OVA per mouse, 800 μg aluminum hydroxide, and the blank control group was injected with PBS; the OVA allergy model group, the probiotics pretreatment group, the probiotics post-treatment group and the blank control group are replaced by normal saline, the mice are killed after 24 hours of the last atomization excitation, alveolar lavage is carried out, alveolar lavage liquid is collected, centrifugation, smear and Rayleigh staining are carried out, and white blood cell classification counting is carried out, wherein the supernatant is used for cytokine concentration detection; lung tissue was taken, fixed, HE stained and observed for pathological changes.
1.3 index observations
1.3.1 differential counts of mouse alveolar lavage fluid and measurement of cytokine concentration of IL-5, IL-10, IL-13, MCP-1, TNF- α, INF- γ, eotaxin
Each mouse was anesthetized by intraperitoneal injection of 2% sodium pentobarbital solution (0.045 ml/g), the neck skin of the mouse was incised, and neck muscle and connective tissue were blunt-separated, exposing the trachea. The trachea cannula is connected with a 1ml syringe by a self-made puncture needle, the lung alveoli are irrigated with PBS precooled at 4 ℃ and each time is 0.8ml, at least 0.6ml is fully recovered, and the operation is repeated for 3 times. The collected BALF was centrifuged at 1200r/min at 4℃for 10min. Collecting supernatant, and storing at-20deg.C. The sediment is used for preparing cell smears, and is subjected to Rayleigh staining and differential counting of white blood cells; the collected supernatants were assayed for IL-5, IL-10, IL-13, MCP-1, TNF- α, INF- γ, eotaxin cytokine concentrations by the Elisa method.
1.3.2 histopathological examination
After 24h of nebulization excitation, lung tissue was taken, 4% paraformaldehyde fixed, material was taken, dehydrated, paraffin embedded, sectioned, HE stained for lung histopathological changes.
1.3.3 statistical data processing method
All experimental data are expressed as mean ± standard deviation, data statistics and plots are performed using Microsoft EXCEL, and comparisons between two sets of data are determined to be significantly different by P < 0.05.
1.4 experimental results
1.4.1 differential counts of mouse alveolar lavage fluid and measurement of cytokine concentration of IL-5, IL-10, IL-13, MCP-1, TNF- α, INF- γ, eotaxin
Compared with a blank control group after the last excitation, eosinophils and neutrophils in the OVA allergy model group are obviously increased (P is less than 0.05), and the model construction is proved to be successful. Compared with the OVA model group, the probiotics pretreatment group has reduced eosinophils and neutrophils, and has significant difference (P is less than 0.05); the probiotic post-treatment group had a significant difference (P < 0.05) in neutropenia, eosinophilia. The differential leukocyte counts of the alveolar lavage fluid of each group of mice are shown in Table 7 below, and the comparison results are shown in FIG. 4.
Table 7: white blood cell classification counting result table in mouse alveolar lavage fluid
PBS, blank control group, OVA allergy model group, pre, probiotic pretreatment group, pos, probiotic post-treatment group. P <0.05 compared to the blank; compared with OVA allergy model group #, P is less than 0.05
The concentration of IL-5, IL-13, MCP-1, TNF-alpha, eotaxin cytokines in the alveolar lavage fluid of mice in the OVA allergy model group was increased with significant differences (P < 0.05), and the concentration of IL-10, INF-gamma was decreased with significant differences (P < 0.05), compared with the blank control group, indicating that the construction of the OVA-induced mouse allergic asthma model was successful. Compared with OVA allergy model group, the concentration of IL-5, IL-13, MCP-1, TNF-alpha and eotaxin in the alveolar lavage fluid of mice in the probiotic post-treatment group is reduced and significantly different (P < 0.05), and the concentration of IL-10 and INF-gamma is increased and significantly different (P < 0.05); the concentration of IL-5, IL-13, MCP-1, TNF-alpha, eotaxin in the probiotic pretreatment group alveolar lavage fluid is reduced and significantly different (P < 0.05), and the concentration of IL-10, INF-gamma is increased and significantly different (P < 0.05). The cytokine concentration data for IL-5, IL-10, IL-13, MCP-1, TNF- α, INF- γ, eotaxin in alveolar lavage fluid of each group is shown in Table 8 below, and is compared to that shown in FIG. 5.
Table 8: comparison table of cytokine concentration detection results in alveolar lavage fluid of each group of mice
PBS: blank control group; OVA: OVA allergy model group; pre: a probiotic pretreatment group; pos: probiotic post-treatment group. Comparison to the blank control group: * P (P)<0.05，**P<0.01; comparison to OVA model group: # P<0.05， ## P<0.01
1.5.2 histopathological examination
As can be seen under an optical microscope, each level of branch of bronchi in a lung of a blank control group is covered with normal airway epithelium, an alveolus is surrounded by type I and type II alveolus cells, and a small amount of interstitial space between each level of bronchi and the alveolus and around blood vessels of the same are seen, so that inflammatory cell infiltration is avoided; the lung of the OVA model group shows the inflammatory hyperplasia of the oversleeve around the terminal bronchiole, the terminal bronchiole spreads to the periphery of the larger bronchiole, and alveolar macrophage is obviously increased; the main pathological changes of the lung of the probiotic pretreatment group are shown by no obvious inflammatory reaction except alveolar macrophage increase; the main pathological changes of the lung of the probiotic post-treatment group are manifested by inflammatory reactions such as pulmonary vasodilation, inflammatory cell infiltration, alveolar macrophage increase and the like; typical pathological lesions are shown in FIG. 6.
From the above results, it was found that the probiotic pretreatment group reduced airway inflammation by 77.0% and 37.1% compared with the OVA allergy model group, respectively. Th1/Th2 immune response tended to equilibrate, IL-5 decreased by 26.8%, IL-13 decreased by 29.1%, MCP-1 decreased by 14.7%, TNF- α decreased by 43.0%, eotaxin concentration decreased by 27.5%, IL-10 increased by 17.2%, INF-gamma concentration increased by 10.8%; compared with the OVA allergy model group, the airway inflammation of the mice in the probiotic post-treatment group is reduced, and eosinophils and neutrophils are respectively reduced by 72.3 percent and 36.2 percent. Th1/Th2 immune response tended to equilibrate, IL-5 decreased by 20.7%, IL-13 decreased by 38.0%, MCP-1 decreased by 13.2%, TNF-alpha decreased by 59.4%, eotaxin concentration decreased by 31.9%, IL-10 increased by 34.5%, INF-gamma concentration increased by 11.7%; the pathological section results show that no obvious inflammatory reaction is seen in the mice in the probiotic pretreatment group, and inflammatory cell infiltration phenomenon of the mice in the probiotic post-treatment group is reduced.
The lactobacillus reuteri VHProbi M07 provided by the invention has strong tolerance to artificial intestinal juice, and the survival rate in the artificial intestinal juice reaches 86.2%; the strain is sensitive to common antibiotics such as erythromycin and ampicillin, does not produce hemolysin and can not dissolve blood cells. The biological safety is good; the maximum tolerated salt concentration was 1% and the catalase reaction was negative.
Lactobacillus reuteri VHProbi M07 has a good effect of preventing and treating allergic asthma. The lactobacillus reuteri VHProbi M07 provided by the invention is used for perfusing stomach in advance or is used for perfusing stomach allergic asthma mice in modeling, the asthma symptoms of the mice are obviously reduced compared with the mice in an OVA allergy model group, and the white blood cell count result and the cytokine level in alveolar lavage fluid are close to those of the mice in a blank control group, so that the strain can effectively prevent and relieve allergic asthma.
The use of lactobacillus reuteri VHProbi M07 brings the mouse immune response towards an equilibrium of Th1/Th2 type immune responses. Compared with the OVA allergy model group mice, in the probiotic pretreatment group allergic asthma mice, eosinophils were reduced by 77.0%, and neutrophils were reduced by 37.1%. Cytokine IL-5 in alveolar lavage fluid was reduced by 26.8%, IL-13 by 29.1%, MCP-1 by 14.7%, TNF- α by 43.0%, eotaxin concentration by 27.5%, IL-10 by 17.2%, INF-gamma concentration by 10.8%; compared with the OVA allergy model group mice, eosinophils were reduced by 72.3% and neutrophils were reduced by 36.2% in the probiotic post-treatment group allergic asthma mice. Cytokine IL-5 in alveolar lavage fluid was reduced by 20.7%, IL-13 by 38.0%, MCP-1 by 13.2%, TNF- α by 59.4%, eotaxin concentration by 31.9%, IL-10 by 34.5%, INF-gamma concentration by 11.7%; from the results of HE staining of the lung tissue of mice, the probiotic pretreatment group allergic asthma mice have no obvious inflammatory response except alveolar macrophage increase; the main pathological changes of the lung of the mice with allergic asthma in the group after probiotics post-treatment are inflammatory reactions such as pulmonary vasodilation, inflammatory cell infiltration, alveolar macrophage increase and the like.
In conclusion, the lactobacillus reuteri VHProbi M07 provided by the invention has strong tolerance to simulated artificial intestinal gastric juice, which lays a foundation for the probiotic strains to successfully pass through the gastrointestinal tract to perform the probiotic function by colonic colonisation. The antibiotic resistance test proves that the lactobacillus reuteri VHProbi M07 is sensitive to common antibiotics, does not produce hemolysin and has good biological safety. Animal experiments prove that the lactobacillus reuteri VHProbi M07 can effectively prevent and relieve inflammatory response of allergic asthma model mice, can inhibit Th2 type immune response while enhancing Th1 type cell immune response, reduce inflammatory state of organisms and enhance immunity, and has potential application value in preventing and treating allergic asthma symptoms.
Sequence listing
<110> Qingdao blue biological Co., ltd
QINGDAO VLAND BIOTECH GROUP Co.,Ltd.
<120> Lactobacillus reuteri for preventing and alleviating allergic asthma symptoms and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1315
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 1
cgactttggg cgttacaaac tcccatggtg tgacgggcgg tgtgtacaag gcccgggaac 60
gtattcaccg cggcatgctg atccgcgatt actagcgatt ccgacttcgt gtaggcgagt 120
tgcagcctac agtccgaact gagaacggct ttaagagatt agcttactct cgcgagtttg 180
cgactcgttg taccgtccat tgtagcacgt gtgtagccca ggtcataagg ggcatgatga 240
tctgacgtcg tccccacctt cctccggttt gtcaccggca gtctcactag agtgcccaac 300
tcaatgctgg caactagtaa caagggttgc gctcgttgcg ggacttaacc caacatctca 360
cgacacgagc tgacgacgac catgcaccac ctgtcattgc gtccccgaag ggaacgcctt 420
atctctaagg ttagcgcaag atgtcaagac ctggtaaggt tcttcgcgta gcttcgaatt 480
aaaccacatg ctccaccgct tgtgcgggcc cccgtcaatt cctttgagtt tcaaccttgc 540
ggtcgtactc cccaggcgga gtgcttaatg cgttagctcc ggcactgaag ggcggaaacc 600
ctccaacacc tagcactcat cgtttacggc atggactacc agggtatcta atcctgttcg 660
ctacccatgc tttcgagcct cagcgtcagt tgcagaccag acagccgcct tcgccactgg 720
tgttcttcca tatatctacg cattccaccg ctacacatgg agttccactg tcctcttctg 780
cactcaagtc gcccggtttc cgatgcactt cttcggttaa gccgaaggct ttcacatcag 840
acctaagcaa ccgcctgcgc tcgctttacg cccaataaat ccggataacg cttgccacct 900
acgtattacc gcggctgctg gcacgtagtt agccgtgact ttctggttgg ataccgtcac 960
tgcgtgaaca gttactctca cgcacgttct tctccaacaa cagagcttta cgagccgaaa 1020
cccttcttca ctcacgcggt gttgctccat caggcttgcg cccattgtgg aagattccct 1080
actgctgcct cccgtaggag tatggaccgt gtctcagttc cattgtggcc gatcagtctc 1140
tcaactcggc tatgcatcat cgccttggta agccgttacc ttaccaacta gctaatgcac 1200
cgcaggtcca tcccagagtg atagccaaag ccatctttca aacaaaagcc atgcggcttt 1260
tgttgttatg cggtattagc atctgtttcc aaatgttatc ccccgctccg gggca 1315
Claims (4)
1. The lactobacillus reuteri is characterized in that the preservation number of the lactobacillus reuteri is CCTCC NO: m2019779.
2. Lactobacillus reuteri according to claim 1, wherein the RAPD fingerprint of lactobacillus reuteri is shown in figure 2.
3. Lactobacillus reuteri according to claim 1, characterized in that the rep-PCR fingerprint of lactobacillus reuteri is shown in fig. 3.
4. The lactobacillus reuteri of claim 1, wherein the 16s rDNA sequence of the lactobacillus reuteri is SEQ ID NO. 1.
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| CN110643542A (en) * | 2019-10-25 | 2020-01-03 | 江南大学 | Lactobacillus reuteri capable of relieving Th2 reaction of allergic asthma and application thereof |
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| 口服6 种益生菌的混合制剂对哮喘小鼠气道炎症的影响及其机制;马婧一等;山西医科大学学报;第49卷(第6期);596-601 * |
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