Lactobacillus rhamnosus strain with antiallergic activity and preparation method thereof

Abstract

The invention discloses a lactobacillus rhamnosus strain with antiallergic activity and a preparation method thereof, the strain is lactobacillus rhamnosus Lrn-PG-06, the classification name of the strain is Lactobacillus rhamnosus, the preservation number is CGMCC (China General Microbiological Culture Collection Center) No.3077, the preservation time is March 21, 2024, the preservation unit is China General Microbiological Culture Collection Center, the preservation number is CGMCC No.3077, and the preservation number is CGMCC No.3077. The address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The lactobacillus rhamnosus Lrn-PG-06 strain is high in acid resistance, alkali resistance and cholate resistance, high in simulated gastrointestinal fluid resistance and high in oxidation resistance, has the advantage of resisting allergy, and solves the problems that at present, allergic diseases are mainly treated by drugs or are prevented from being in contact with allergens in the environment and food, but due to the fact that the drugs are large in toxic and side effects, the allergens cannot be in contact with the allergens in the environment and food. And allergens causing anaphylaxis are difficult to determine, so that allergic diseases are difficult to treat or prevent.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a Lactobacillus rhamnosus strain with anti-allergic activity and a preparation method thereof. Background Art

[0002] Allergic diseases are common and multi-morbid conditions. Allergies are caused by foreign substances entering the human body. The body's immune system identifies these substances as foreign bodies, causing immune system disorders and leading to allergies and inflammation. The vast majority of allergic diseases are type I allergic reactions related to hyaluronidase activity in the body. Hyaluronidase is a hydrolase that degrades hyaluronic acid and is a major component of the extracellular matrix of the body's connective tissue. It is associated with most type I allergic reactions mediated by IgE and type IV allergic reactions mediated by T cells. Therefore, the hyaluronidase in vitro inhibition test is an effective method for rapid in vitro screening of anti-allergic drugs, and is also a method for screening probiotics with anti-allergic activity.

[0003] Currently, allergic diseases are mainly treated with drugs or avoiding contact with allergens in the environment and food. However, due to the large toxic side effects of drugs, the allergens that cause allergic reactions are difficult to identify, making the treatment or prevention of allergic diseases more difficult. Some probiotics that are beneficial to human health and have no side effects can effectively slow down allergic reactions. Therefore, rhamnosus lactis strains with anti-allergic activity are needed as probiotics to prevent allergic diseases. Summary of the Invention

[0004] The purpose of the present invention is to provide a Lactobacillus rhamnosus strain with anti-allergic activity and a preparation method thereof. The strain has strong acid resistance, alkali resistance and bile salt resistance, strong resistance to simulated gastrointestinal fluid, strong antioxidant capacity, and has the advantages of anti-allergy. It solves the problem that allergic diseases are currently mainly treated with drugs or avoidance of contact with allergens in the environment and food, but due to the large toxic side effects of drugs, the allergens of allergic reactions are difficult to determine, thereby making the treatment or prevention of allergic diseases difficult.

[0005] To achieve the above-mentioned object, the present invention provides the following technical solution: a Lactobacillus rhamnosus strain having anti-allergic activity, the strain being Lactobacillus rhamnosus Lrn-PG-06, classified as Lactobacillus rhamnosus, with a deposit number of CGMCC No. 3077 and a deposit date of March 21, 2024, deposited at the General Microbiology Center of the China Culture Collection Administration, located at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0006] A method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity comprises the following steps:

[0007] S1. Dilute the stool sample with physiological saline, spread it on MRS agar solid medium, and incubate it at 37°C for 48 hours. Pick single colonies of different morphology and size on the plate, streak them multiple times on MRS agar medium for purification until they are confirmed to be single colonies. Pick single colonies for microscopic examination, physiological and biochemical analysis, and 16S rDNA homology comparison, and store the isolated strains in glycerol freezer.

[0008] S2, fully activating and expanding the Lactobacillus rhamnosus Lrn-PG-06 stored in glycerol freeze-stored;

[0009] S3. The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial solution was inoculated into MRS medium with different pH values, cultured at 37°C for 24 hours, and then the acid and alkali resistance test of the strain was performed;

[0010] S4. Inoculate the fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial suspension into MRS medium with different bile salt concentrations, incubate at 37°C for 24 hours, and then perform a bile salt tolerance test on the strain;

[0011] S5. Centrifuging the fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial suspension, collecting the bacterial sludge, and preparing a bacterial suspension with physiological saline for a naked bacteria simulated gastrointestinal tract test;

[0012] S6. Activate the indicator bacteria and inoculate them into LB liquid medium. Activate Lactobacillus rhamnosus Lrn-PG-06 and inoculate them into MRS liquid medium. After culturing them at 37° C. for 24 h, perform an antibacterial test on Lactobacillus rhamnosus Lrn-PG-06.

[0013] S7. Streak the activated Lactobacillus rhamnosus Lrn-PG-06 bacterial solution onto Columbia blood agar plates, invert the plates and incubate them in an incubator for 48 hours to perform a hemolysis test.

[0014] S8, centrifuging the fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial solution, taking the supernatant, and determining the hyaluronidase inhibition rate;

[0015] S9. The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 was subjected to in vitro antioxidant capacity determination.

[0016] As a preferred method for preparing a Lactobacillus rhamnosus strain with anti-allergic activity of the present invention, when activating and expanding the Lactobacillus rhamnosus Lrn-PG-06 in S2, the Lactobacillus rhamnosus Lrn-PG-06 strain preserved in -20°C glycerol is first melted, and then inoculated into a liquid MRS culture medium at an inoculum rate of 2% (V / V), and then cultured at 37°C for 24 hours. The revived bacterial liquid is then inoculated into an MRS liquid culture medium, and cultured at 37°C for another 24 hours to fully activate and expand the strain.

[0017] As a preferred method for preparing a Lactobacillus rhamnosus strain with anti-allergic activity of the present invention, when the Lactobacillus rhamnosus Lrn-PG-06 in S3 is subjected to acid and alkali resistance tests, the pH values ​​of multiple MRS liquid culture media with a pH of 6.0 are adjusted to 5.0, 4.0, 3.0, 2.0 and 1.0, respectively, with HCl at a concentration of 4.0 mol / L, and the pH values ​​of multiple MRS liquid cultures with a pH of 6.0 are adjusted to 11.0, 10.0, 9.0, 8.0 and 7.0, respectively, with NaOH, the strain is inoculated into culture media with different pH values, and after culturing at 37°C for 24 hours, the absorbance of the bacterial suspension is measured at a wavelength of 600 nm, and the survival rate is calculated to test the acid and alkali tolerance of Lactobacillus rhamnosus Lrn-PG-06.

[0018] As a preferred method for preparing a Lactobacillus rhamnosus strain with anti-allergic activity of the present invention, when the Lactobacillus rhamnosus Lrn-PG-06 in S4 is subjected to a bile salt tolerance test, the strain is inoculated into MRS culture medium containing bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively, at a 3% inoculation rate. After culturing at 37°C for 24 hours, the absorbance of the bacterial suspension is measured at a wavelength of 600nm. The absorbance of the strain at a wavelength of 600nm in MRS culture medium without adding bile salts is used as a control. The survival rate is calculated to evaluate the effect of bile salts on the activity of Lactobacillus rhamnosus Lrn-PG-06.

[0019] As a preferred method for preparing a strain of Lactobacillus rhamnosus with anti-allergic activity of the present invention, when the Lactobacillus rhamnosus Lrn-PG-06 in S5 is subjected to a simulated gastrointestinal tract assay, the strain is activated and passaged in MRS medium at 37°C for two generations, 1.0 g of bacterial mud is taken out by centrifugation and added to 5.0 mL of artificial gastric juice, and treated at 37°C for 3 hours. Under sterile conditions, the precipitate is collected, and sterile physiological saline is added according to the ratio of 1.0 g of bacterial mud: 5.0 mL of physiological saline, mixed, diluted and coated, and the colony counted, and 1.0 g of artificial gastric juice is taken out. The bacterial sludge treated with gastric juice was added to 5.0 mL of artificial bile and treated at 37°C for 20 min. The mixture was centrifuged at 8000 r / min for 10 min, and the colony count was performed on the bacterial sludge after centrifugation. 1.0 g of the bacterial sludge treated with artificial bile was added to 5.0 mL of artificial intestinal fluid and treated at 37°C for 4 h and 8 h respectively. The mixture was centrifuged at 8000 r / min for 10 min, and the colony count was performed on the bacterial sludge after centrifugation. The survival rate was calculated using the colony count in MRS culture medium that had not been treated with simulated gastrointestinal tract as a control.

[0020] As a preferred method for preparing a Lactobacillus rhamnosus strain with anti-allergic activity of the present invention, when the Lactobacillus rhamnosus Lrn-PG-06 in S5 is subjected to an antibacterial test, the Oxford cup agar diffusion method is adopted, and Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes are used as indicator bacteria for plate coating, and physiological saline is used as a blank control. Four Oxford cups are evenly placed on each plate, 200 μL of Lactobacillus rhamnosus Lrn-PG-06 bacterial suspension is added to three Oxford cups, and physiological saline is added to one Oxford cup. After culturing at 37° C. for 24 hours, the size of the inhibition zone is measured with a vernier caliper.

[0021] As a preferred method for preparing a Lactobacillus rhamnosus strain with anti-allergic activity of the present invention, when the Lactobacillus rhamnosus Lrn-PG-06 in S6 is subjected to a hemolytic test, the strain is streaked on a blood agar plate, cultured at 37°C for 48 hours, and then taken out for observation. The hemolytic effect is manifested as the presence of a transparent zone, a green zone, or an absence of a zone around the colony. The transparent zone represents β hemolysis, the green zone represents α hemolysis, and the absence of a zone represents γ hemolysis.

[0022] As a preferred method for preparing a strain of Lactobacillus rhamnosus with anti-allergic activity of the present invention, when the Lactobacillus rhamnosus Lrn-PG-06 in S7 is subjected to an in vitro hyaluronidase inhibition test, the strain is activated and cultured in MRS medium at 37°C for two generations, and then the supernatant is taken by centrifugation. 0.1 mL of 2.5 mmol / LCaCl2 and 0.5 mL of 600 U / mL hyaluronidase are added to the test tube, and the mixture is treated at 37°C for 20 min. Then, 0.5 mL of bacterial suspension is added, and the mixture is treated at 37°C for 20 min. 0.5 mL of 0.5 mg / mL sodium hyaluronate solution is added, and the mixture is treated at 37°C for 20 min. Treat for 30 minutes, let it stand at room temperature for 5 minutes, add 0.5mL of acetylacetone solution and 0.1mL of 0.4mol / LNaOH solution, the acetylacetone solution is 1.4mL of acetylacetone dissolved in 20mL of 1.0mol / LNa2CO3 solution, boil in water bath for 15 minutes, immediately ice bath for 5 minutes, add 1mL of Ehrlich reagent, the Ehrlich reagent is 0.8g of p-dimethylaminobenzaldehyde dissolved in 15mL of concentrated hydrochloric acid and 15mL of anhydrous ethanol, color at room temperature for 20 minutes, measure its absorbance at a wavelength of 530nm, and use the absorbance value of LGG bacterial solution treated under the same conditions as the control.

[0023] As a preferred method for preparing a rhamnosus Lactobacillus strain with anti-allergic activity of the present invention, when the rhamnosus Lactobacillus Lrn-PG-06 in S8 is subjected to an in vitro antioxidant capacity test, the strain is activated and cultured in MRS medium for two generations at 37°C, and then the DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, ABTS+ free radical scavenging capacity, and superoxide anion free radical scavenging capacity are measured, and the antioxidant capacity of VC and the LGG bacterial liquid treated in the same manner are used as controls.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The rhamnosus Lactobacillus strain of the present invention is isolated and purified from infant feces. The strain has high in vitro hyaluronidase inhibition activity, with an in vitro hyaluronidase inhibition rate of 92.91±4.41%, and has strong antibacterial ability. In addition, the strain has strong acid resistance, alkali resistance, bile salt resistance, tolerance to artificial simulated gastrointestinal fluid and antioxidant capacity, and has the characteristics of a probiotic.

[0026] 2. The rhamnosus Lactobacillus strain of the present invention can be used as a probiotic. When used as a probiotic, it can maintain the body's immune system by regulating intestinal flora, improving intestinal lymphoid tissue function, and regulating the body's cellular immunity, humoral immunity, and nonspecific immunity, thereby achieving a probiotic effect of enhancing immune function. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 2 is the plate colony morphology and Gram staining result of the strain of the present invention;

[0028] Figure 2 Graph showing the acid resistance test results of the strain of the present invention;

[0029] Figure 3 This is a graph showing the alkali resistance test results of the strain of the present invention;

[0030] Figure 4 This is a graph showing the bile salt tolerance test results of the strain of the present invention;

[0031] Figure 5 This is a graph showing the results of a test on the strain's ability to tolerate artificial simulated gastrointestinal fluid;

[0032] Figure 6 This is a graph showing the results of a pathogenic bacteria inhibition test using the fermentation supernatant of the strain of the present invention;

[0033] Figure 7 This is a graph showing the results of a hemolytic test of the strain of the present invention;

[0034] Figure 8 DPPH free radical scavenging ability test results of the strain of the present invention;

[0035] Figure 9 This is a graph showing the test results of the strain of the present invention's ability to scavenge hydroxyl radicals;

[0036] Figure 10 This is a graph showing the test results of the strain of the present invention's ability to scavenge ABTS+ free radicals;

[0037] Figure 11 This is a graph showing the results of a test on the superoxide anion free radical scavenging ability of the strain of the present invention. DETAILED DESCRIPTION

[0038] Example 1

[0039] A Lactobacillus rhamnosus strain with anti-allergic activity, the strain is Lactobacillus rhamnosus Lrn-PG-06, its classification name is Lactobacillus rhamnosus, the preservation number is CGMCC No.3077, the preservation date is March 21, 2024, and the preservation unit is the General Microbiology Center of the China Culture Collection Administration, the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[0040] Furthermore, the rhamnosus Lactobacillus Lrn-PG-06 strain was isolated and purified from infant feces. The strain has high in vitro hyaluronidase inhibition activity, with an in vitro hyaluronidase inhibition rate of 92.91±4.41%, and has strong antibacterial ability. In addition, the strain has strong acid resistance, alkali resistance, bile salt resistance, tolerance to artificial simulated gastrointestinal fluid and antioxidant capacity, and has the characteristics of a probiotic.

[0041] Furthermore, the rhamnosus Lactobacillus strain can be used as a probiotic. When used as a probiotic, it can maintain the body's immune system by regulating intestinal flora, improving intestinal lymphoid tissue function, and regulating the body's cellular immunity, humoral immunity, and nonspecific immunity, thereby achieving a probiotic effect of enhancing immune function.

[0042] Example 2

[0043] A method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity comprises the following steps:

[0044] First, 1.0 g of infant fecal sample was diluted with normal saline, and 100 μL of dilution gradient was taken, namely 10 -2 ~10 -7 The fermentation broth was evenly spread on MRS agar solid culture medium and cultured at a constant temperature of 37°C for 48 hours. Single colonies with different morphologies and sizes on the plate were picked and streaked multiple times on the MRS agar culture medium for purification until they were confirmed to be single colonies. Single colonies were picked for microscopic examination, physiological and biochemical characteristics, and 16SrDNA homology comparison, and the isolated strains were frozen and preserved with glycerol.

[0045] When performing microscopic examination, pick a single colony and inoculate it into MRS broth medium. After culturing for 24 hours, perform Gram staining. Smear the test bacterial solution. After Gram staining, observe the cell morphology and arrangement under an optical microscope. Record and photograph the cells. Finally, Gram-positive bacteria are identified as suspected lactic acid bacteria. Pipette 1.0 mL of pure culture into a preservation tube containing 50% glycerol.

[0046] refer to Figure 1 When conducting physiological and biochemical experiments, physiological and biochemical tests such as hydrogen peroxide test, indole test and carbohydrate utilization test were used to identify and screen bacterial strains. The isolated and purified strain was milky white, with a convex middle, a smooth and moist surface, a diameter of 1.14 mm, and Gram-positive. The cells were rod-shaped, solitary or arranged in pairs. Its physical and chemical characteristics were negative for hydrogen peroxide test and positive for indole reaction; the hydrolysis test of esculin was positive, and it could ferment cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin and lactose.

[0047] Furthermore, when performing 16sRNA homology comparison, the strain was expanded and cultured, the bacterial solution was aspirated and centrifuged to obtain the bacteria, and sequencing was performed by Nanjing Paisonno Gene Technology Co., Ltd. The 16S rDNA sequence of the strain Lrn-PG-06 submitted after sequencing was as follows:

[0048] (CTTTGTGACCTTAGACGGCTCGCTCCCTAAAAGGGTTACGCCACCGGCTTCGGGTGTTACAAAC TCTCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCGTGTAGGCGAGTTGCAGCCTACAGTCCGAACTGAGAATGGCTTTAAGAGATTAGCTTGACCTCGCGGTCTCGCAACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTTACTAGAGTGCCCAACTAAATGCTGGCAACTAGTCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCATTTTGCCCCCGAAGGGGAAACCTGATCTCTCAGGTGATCAAAAGATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAATGCTTAATGCGTTAGCTGCGGCACTGAAGGGCGGAAACCCTCCAACACCTAGCATTCATCGTTTACGGCATGGACTACCAGGGTATCTAATCCTGTTCGCTACCCATGCTTTCGAGCCTCAGCGTCAGTTACAGACCAGACAGCCGCCTTCGCCACTGGTGTTCTTCCATATATCTACGCATTTCACCGCTACACATGGAGTTCCACTGTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCGATGCACTTCCTCGGTTAAGCCGAGGGCTTTCACATCAGACTTAAAAAACCGCCTGGGCTCGCTTTACGCCCAATAAATCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGGTGGGCCCGAAGT) was compared in the NCBI BLAST database,The results showed that the gene sequence of strain Lrn-PG-06 was 99.97% identical to that of Lactobacillus rhamnosus. Combined with Gram staining results and physiological and biochemical characteristics, strain Lrn-PG-06 was identified as Lacticaseibacillus rhamnosus.

[0049] Example 3

[0050] The process of fully activating and expanding the Lactobacillus rhamnosus Lrn-PG-06 frozen and preserved in glycerol comprises the following steps:

[0051] First, the -20℃ glycerol-stored Lactobacillus rhamnosus Lrn-PG-06 strain was thawed and inoculated into liquid MRS culture medium at a 2% (V / V) inoculation rate. Then, the culture was incubated at 37℃ for 24 hours. The revived bacterial liquid was then inoculated into MRS liquid culture medium and cultured at 37℃ for another 24 hours to fully activate the strain.

[0052] Example 4

[0053] The acid and alkali resistance test of a Lactobacillus rhamnosus strain having antiallergic activity comprises the following steps:

[0054] The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial liquid was inoculated into MRS culture medium with different pH values, cultured at a constant temperature of 37°C for 24 hours, and then the acid and alkali resistance test of the strain was carried out.

[0055] Furthermore, when the acid and alkali resistance test of Lactobacillus rhamnosus Lrn-PG-06 was carried out, the pH values ​​of multiple MRS liquid culture media with a pH of 6.0 were adjusted to 5.0, 4.0, 3.0, 2.0 and 1.0, respectively, using HCl at a concentration of 4.0 mol / L, and the pH values ​​of multiple MRS liquid cultures with a pH of 6.0 were adjusted to 11.0, 10.0, 9.0, 8.0 and 7.0, respectively, using NaOH. The strain was inoculated into culture media with different pH values. After culturing at 37°C for 24 hours, the absorbance of the bacterial suspension was measured at a wavelength of 600 nm, and its survival rate was calculated to test the acid and alkali tolerance of Lactobacillus rhamnosus Lrn-PG-06.

[0056] Furthermore, the survival rate was calculated, survival rate = Nt / N0

[0057] Where: Nt is the absorbance value of MRS culture medium with pH values ​​of 1.0-11.0;

[0058] N0 is the absorbance value of MRS medium at pH 6.0.

[0059] The pH value of gastric juice of a normal person is 0.9-1.8 when fasting. After taking food, the pH value of gastric juice will change to about 1.8-5.0. Therefore, this experiment selected pH values ​​of 1-5 to test the strain's tolerance to acid. The results are as follows Figure 2 As shown by Figure 2 As can be seen, Lactobacillus rhamnosus Lrn-PG-06 had poor tolerance at pH 1.0, with a survival rate of only 6.51% after 24 hours of treatment. However, at pH 4.0, it showed strong tolerance, with a survival rate of 46.98% after 24 hours of treatment, indicating that the bacterium has a strong ability to tolerate acidic conditions.

[0060] The pH value of 7-11 was selected to test the strain's tolerance to alkali. Figure 3 As shown by Figure 3 It can be seen that the survival rate of Lactobacillus rhamnosus Lrn-PG-06 gradually increased as the pH value tended to neutral, but after culturing at a pH of 11.0 for 24 hours, its survival rate was still 64.55%, indicating that the bacteria has a strong ability to tolerate alkaline conditions.

[0061] Example 5

[0062] The bile salt tolerance test of a Lactobacillus rhamnosus strain having antiallergic activity comprises the following steps:

[0063] The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial liquid was inoculated into MRS culture medium with different bile salt concentrations, cultured at a constant temperature of 37°C for 24 hours, and then a bile salt tolerance test of the strain was performed.

[0064] Furthermore, when Lactobacillus rhamnosus Lrn-PG-06 was subjected to a bile salt tolerance test, the strain was inoculated into MRS culture medium containing bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively, at a 3% inoculum size. After culturing at 37°C for 24 hours, the absorbance of the bacterial suspension was measured at a wavelength of 600nm. The absorbance value of the strain at a wavelength of 600nm in MRS culture medium without bile salts was used as a control. The survival rate was calculated to evaluate the effect of bile salts on the activity of Lactobacillus rhamnosus Lrn-PG-06.

[0065] Furthermore, survival rate = Nt / N0;

[0066] Where: Nt is the number of viable bacteria in MRS medium containing different concentrations of bile salts, and N0 is the number of viable bacteria in MRS medium without bile salts.

[0067] The bile salt concentration in the small intestine of most human bodies is about 0.3% (w / v). Probiotics need to tolerate bile salts in the intestine to play a probiotic role in the small intestine. By treating Lactobacillus rhamnosus Lrn-PG-06 strain with 5 different mass fractions of bile salts (0.1%, 0.3%, 0.5%, 0.7%, 0.9%) for 24 hours (the strain cultured without bile salts was used as the control), the results are as follows: Figure 4 As shown, from Figure 4 It can be seen that with the increase of bile salt concentration, the survival rate of the strain gradually decreased. After 24 hours of treatment with 0.3% bile salt group, the survival rate was still 45.19%, which shows that Lactobacillus rhamnosus Lrn-PG-06 has good bile salt tolerance.

[0068] Example 6

[0069] When a strain of Lactobacillus rhamnosus with anti-allergic activity is subjected to a naked bacteria simulated gastrointestinal tract test, the following steps are included:

[0070] The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial solution was centrifuged, and the bacterial mud was collected and prepared into a bacterial suspension with physiological saline for a naked bacteria simulated gastrointestinal tract test.

[0071] Furthermore, when Lactobacillus rhamnosus Lrn-PG-06 was subjected to a simulated gastrointestinal tract assay, the strain was activated and passaged for two generations in MRS medium at 37°C, 1.0 g of bacterial sludge was centrifuged and added to 5.0 mL of artificial gastric fluid. After treatment at 37°C for 3 h, the precipitate was collected under sterile conditions, and sterile saline was added according to the ratio of 1.0 g bacterial sludge: 5.0 mL of normal saline to mix, dilute and spread, and count the colonies. Then 1.0 g of bacterial sludge treated with artificial gastric fluid was taken and added to 5.0 mL of human The cells were treated in artificial bile at 37°C for 20 minutes and then centrifuged at 8000 r / min for 10 minutes. The bacterial sludge after centrifugation was counted for colony count. Then 1.0 g of the bacterial sludge treated with artificial bile was added to 5.0 mL of artificial intestinal fluid. The cells were treated at 37°C for 4 hours and 8 hours respectively, and then centrifuged at 8000 r / min for 10 minutes. The bacterial sludge after centrifugation was counted for colony count. The survival rate was calculated using the colony count in MRS culture medium that had not been treated with simulated gastrointestinal tract as a control.

[0072] Furthermore, the survival rate of artificial gastrointestinal fluid and bile = Nt / N0

[0073] Where: Nt is the number of viable bacteria after treatment with artificial simulated gastric juice, bile, and intestinal juice; N0 is the number of viable bacteria before treatment.

[0074] Gastric juice contains mucus, gastric acid, pepsin, etc. The premise for probiotics to play a probiotic role is that they can survive in the stomach, that is, they need to have the ability to tolerate the gastric acid environment and resist pepsin. Usually food stays in the stomach for 3 hours, so simulated gastric juice was selected to treat rhamnosus casei Lrn-PG-06 bacterial liquid for 3 hours. After gastric digestion, food enters the intestine. The small intestine environment is weakly alkaline and contains trypsin and bile. The number of live probiotics in the intestine must reach 106 cfu / mL or more to play a probiotic function. Therefore, probiotics need to tolerate the weak alkaline environment in the intestine and have the ability to resist trypsin and bile. Food generally stays in the small intestine for 3-8 hours, so simulated intestinal fluid was selected to treat rhamnosus casei Lrn-PG-06 bacterial liquid for 4 hours. The results are as follows Figure 5 As shown by Figure 5 It can be seen that after 3 hours of gastric juice treatment, the survival rate of Lactobacillus rhamnosus Lrn-PG-06 was 80.20%, and the number of viable bacteria reached 14.79±0.01lg (cfu / g); after 20 minutes of bile treatment, the survival rate was 75.57%, and the number of viable bacteria reached 13.94±0.03lg (cfu / g); after 4 hours of intestinal juice treatment, the survival rate was still 71.11%, and the number of viable bacteria reached 13.12±0.02lg (cfu / g). It can be seen that Lactobacillus rhamnosus Lrn-PG-06 has a strong ability to tolerate artificial simulated gastrointestinal juice and bile.

[0075] Example 7

[0076] The antibacterial test of a Lactobacillus rhamnosus strain having antiallergic activity comprises the following steps:

[0077] The indicator bacteria were activated and inoculated into LB liquid culture medium, and the Lactobacillus rhamnosus Lrn-PG-06 was activated and inoculated into MRS liquid culture medium. After culturing at 37°C for 24 hours, the Lactobacillus rhamnosus Lrn-PG-06 antibacterial test was performed.

[0078] Furthermore, when conducting an antibacterial test on Lactobacillus rhamnosus Lrn-PG-06, the Oxford cup agar diffusion method was used, and plates were spread with Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes as indicator bacteria, and physiological saline was used as a blank control. Four Oxford cups were evenly placed on each plate, 200 μL of Lactobacillus rhamnosus Lrn-PG-06 bacterial suspension was added to three Oxford cups, and physiological saline was added to one Oxford cup. After incubation at 37°C for 24 hours, the size of the inhibition zone was measured with a vernier caliper.

[0079] From Table 1 and Figure 6It can be seen that Table 1 is the experimental data table of the antibacterial ability of Lactobacillus rhamnosus Lrn-PG-06. Inhibition zones appeared in the culture dishes, and the diameters of the inhibition zones all reached 14.0 mm or more, indicating that Lactobacillus rhamnosus Lrn-PG-06 had an inhibitory effect on the growth of Escherichia coli, Staphylococcus aureus, and Listeria monocytogenes. The diameters of the inhibition zones were 15.06±0.36 mm, 16.43±0.23 mm, and 14.63±0.50 mm, respectively, indicating that Lactobacillus rhamnosus Lrn-PG-06 had a significant antibacterial effect and strong antibacterial ability.

[0080] indicator bacteria Diameter of inhibition zone (mm) Escherichia coli 15.06±0.36 Listeria monocytogenes 16.43±0.23 Staphylococcus aureus 14.63±0.50

[0081] Table 1

[0082] Example 8

[0083] A strain hemolysis test of a Lactobacillus rhamnosus strain having antiallergic activity comprises the following steps:

[0084] The activated Lactobacillus rhamnosus Lrn-PG-06 bacterial liquid was streaked onto Columbia blood agar plate culture medium, placed upside down in an incubator and cultured for 48 hours, and a hemolysis test of the strain was performed.

[0085] Furthermore, when Lactobacillus rhamnosus Lrn-PG-06 was subjected to a hemolytic test, the strain was streaked on a blood agar plate, cultured at 37°C for 48 hours, and then taken out for observation. The hemolytic effect was manifested as the presence of a transparent zone, a green zone, or an absence of a zone around the colony. The transparent zone represented β hemolysis, the green zone represented α hemolysis, and the absence of a zone represented γ hemolysis.

[0086] The test results of Lactobacillus rhamnosus Lrn-PG-06 are as follows Figure 7 As shown, Staphylococcus aureus was used as the control strain (left), and Lactobacillus rhamnosus Lrn-PG-06 (right) was non-hemolytic and was a safe strain.

[0087] Example 9

[0088] The determination of the hyaluronidase inhibition rate of a Lactobacillus rhamnosus strain having anti-allergic activity comprises the following steps:

[0089] The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial solution was centrifuged, and the supernatant was collected to determine the hyaluronidase inhibition rate.

[0090] In the in vitro hyaluronidase inhibition test of Lactobacillus rhamnosus Lrn-PG-06, the strain was activated and passaged twice in MRS medium at 37°C, and the supernatant was collected by centrifugation. 0.1 mL of 2.5 mmol / LCaCl2 and 0.5 mL of 600 U / mL hyaluronidase were added to the test tube and treated at 37°C for 20 min. Then 0.5 mL of bacterial suspension was added and treated at 37°C for 20 min. 0.5 mL of 0.5 mg / mL sodium hyaluronate solution was added and treated at 37°C for 30 min. The mixture was allowed to stand at room temperature for 5 min, and 0. 5mL of acetylacetone solution and 0.1mL of 0.4mol / LNaOH solution. The acetylacetone solution is 1.4mL of acetylacetone dissolved in 20mL of 1.0mol / LNa2CO3 solution. After boiling in a water bath for 15 minutes, immediately ice bath for 5 minutes, add 1mL of Ehrlich reagent. The Ehrlich reagent is 0.8g of p-dimethylaminobenzaldehyde dissolved in 15mL of concentrated hydrochloric acid and 15mL of anhydrous ethanol. Color develop at room temperature for 20 minutes, and measure its absorbance at a wavelength of 530nm. The absorbance value of LGG bacterial solution treated under the same conditions is used as a control.

[0091] The inhibition rate was calculated as follows:

[0092] Where: A is the absorbance of the control group (acetate buffer replaces the bacterial suspension); B is the absorbance of the blank group (acetate buffer replaces the enzyme solution and bacterial suspension); C is the absorbance of the experimental group; D is the absorbance of the sample blank group (acetate buffer replaces the enzyme solution).

[0093] Refer to Table 2, which is a table of experimental data on the hyaluronidase inhibition ability of strains;

[0094] Strain name Inhibition rate (%) LGG 64.26±4.16 Lrn-PG-06 92.91±4.41

[0095] Table 2

[0096] Hyaluronidase is strongly correlated with inflammation and allergies. Many anti-allergic drugs have a strong inhibitory effect on hyaluronidase activity. A hyaluronidase inhibition rate of 70% or above indicates a strong anti-allergic effect, between 50% and 70% indicates a moderate anti-allergic effect, and an inhibition rate of less than 30% indicates no anti-allergic effect. As shown in Table 2, the in vitro hyaluronidase inhibition rate of Lactobacillus rhamnosus Lrn-PG-06 in this experiment was 92.91±4.41%, which was significantly higher than LGG (64.26±4.16%). Therefore, Lactobacillus rhamnosus Lrn-PG-06 has a strong anti-allergic effect.

[0097] Table 3 shows the comparison of hyaluronidase inhibition ability in vitro with other probiotic strains;

[0098]

[0099] Table 3

[0100] Example 10

[0101] The in vitro antioxidant capacity assay of a Lactobacillus rhamnosus strain having anti-allergic activity comprises the following steps:

[0102] The in vitro antioxidant capacity of Lactobacillus rhamnosus Lrn-PG-06 after full activation and expansion was determined.

[0103] Furthermore, when Lactobacillus rhamnosus Lrn-PG-06 was tested for its in vitro antioxidant capacity, the strain was activated and cultured in MRS medium at 37°C for two generations, and then its DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, ABTS+ free radical scavenging capacity, and superoxide anion free radical scavenging capacity were measured, with the antioxidant capacity of VC and the same treated LGG bacterial solution used as a control.

[0104] Furthermore, to determine the DPPH radical scavenging ability, a 0.1 mmol / L DPPH solution was prepared with anhydrous ethanol and stored in the dark. Before use, the absorbance at 517 nm was adjusted to 1.25 ± 0.05 with anhydrous ethanol. 2.0 mL of the bacterial solution was placed in a 10 mL test tube, and 2.0 mL of the DPPH solution was added. The tube was incubated at room temperature in the dark for 20 minutes. The absorbance at 517 nm was measured and recorded as A1. The absorbance with ethanol replacing DPPH was recorded as A2, and the absorbance with ethanol replacing the sample was recorded as A0. The DPPH radical scavenging rate was calculated according to the formula.

[0105] The formula is as follows:

[0106]

[0107] like Figure 8 As shown in the results, the DPPH radical scavenging rate of Lactobacillus rhamnosus Lrn-PG-06 was 96.45±0.32%, which was significantly higher than that of LGG (79.34±7.04%).

[0108] Furthermore, when determining the hydroxyl radical scavenging ability, 1 mL of bacterial liquid was placed in a 10 mL test tube, and then 1 mL of 9 mmol / L ferrous sulfate solution, 1 mL of 9 mmol / L salicylic acid ethanol solution and 1 mL of 8.8 mmol / L H2O2 solution were added in sequence, and shaken evenly. The test tube was warmed at 37°C for 30 minutes, and cooled to room temperature. The absorbance value was measured at a wavelength of 510 nm as A1, the absorbance value of H2O replacing H2O2 as A2, and the absorbance value of H2O replacing the sample as A0. The hydroxyl radical scavenging rate was calculated according to the formula.

[0109] The formula is as follows:

[0110]

[0111] like Figure 9 The hydroxyl radical scavenging rate of Lactobacillus rhamnosus Lrn-PG-06 was 99.02±0.44%, which was close to VC and LGG (96.83±1.09%).

[0112] Furthermore, to determine the ABTS+ free radical scavenging ability, a 7 mmol / L ABTS solution and a 2.45 mmol / L K2S2O8 solution were first prepared and mixed in equal proportions. The mixture was allowed to react in the dark at 4°C for 12-16 hours before use. The mixture was diluted with anhydrous ethanol to an absorbance of 0.7±0.02 at 734 nm before use. 0.5 mL of the bacterial solution was placed in a test tube, and 4.5 mL of the diluted ABTS stock solution was added. The mixture was reacted at 30°C for 10 minutes. The absorbance at 734 nm was measured as A1. The absorbance at 734 nm of H2O replacing ABTS was A2, and the absorbance at 734 nm of H2O replacing the sample was A0. The ABTS+ free radical scavenging rate was calculated according to the formula.

[0113] The formula is as follows:

[0114]

[0115] like Figure 10 The ABTS+ free radical scavenging rate of Lactobacillus rhamnosus Lrn-PG-06 was 95.72±0.75%, which was significantly higher than that of LGG (81.86±8.69%).

[0116] Furthermore, when determining the superoxide anion radical scavenging ability, 1 mL of bacterial solution was placed in a test tube, and 1 mL of 10 mmol / L pyrogallol solution and 5 mL of 50 mmol / L Tris-HCl (pH 8.2) were added. After reacting at 25°C for 4 minutes, 1 mL of 8 mol / L HCl was added to terminate the reaction. The absorbance at a wavelength of 320 nm was measured as A1, the absorbance at a wavelength of 320 nm when H2O replaced pyrogallol as A2, and the absorbance at a wavelength of 320 nm when H2O replaced the sample as A0. The superoxide anion radical scavenging rate was calculated according to the formula.

[0117] The formula is as follows:

[0118]

[0119] like Figure 11 The superoxide anion radical scavenging rate of Lactobacillus rhamnosus Lrn-PG-06 was significantly lower than that of LGG but higher than 50%, and the superoxide anion radical scavenging rate of Lactobacillus rhamnosus Lrn-PG-06 was 63.04±1.57%.

[0120] In summary, the above results show that Lactobacillus rhamnosus Lrn-PG-06 has a high antioxidant capacity, and studies have shown that strains with antioxidant properties may play a positive role in alleviating allergic reactions.

[0121] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A Lactobacillus rhamnosus strain having anti-allergic activity, characterized in that: The strain is Lactobacillus rhamnosus Lrn-PG-06, its classification name is Lactobacillus rhamnosus, the preservation number is CGMCC No.3077, the preservation time is March 21, 2024, and the preservation unit is the General Microbiology Center of China Culture Collection Administration, the address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

2. A method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity, applicable to the Lactobacillus rhamnosus strain having anti-allergic activity according to claim 1, characterized in that: The following steps are involved: S1. Dilute the stool sample with physiological saline, spread it on MRS agar solid medium, and incubate it at 37°C for 48 hours. Pick single colonies of different morphology and size on the plate, streak them multiple times on MRS agar medium for purification until they are confirmed to be single colonies. Pick single colonies for microscopic examination, physiological and biochemical analysis, and 16S rDNA homology comparison, and store the isolated strains in glycerol freezer. S2, fully activating and expanding the Lactobacillus rhamnosus Lrn-PG-06 stored in glycerol freeze-stored; S3. The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial solution was inoculated into MRS medium with different pH values, cultured at 37°C for 24 hours, and then the acid and alkali resistance test of the strain was performed; S4. Inoculate the fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial suspension into MRS medium with different bile salt concentrations, incubate at 37°C for 24 hours, and then perform a bile salt tolerance test on the strain; S5. Centrifuging the fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial solution, collecting the bacterial sludge, and preparing a bacterial suspension with physiological saline for a naked bacteria simulated gastrointestinal tract test; S6. Activate the indicator bacteria and inoculate them into LB liquid medium. Activate Lactobacillus rhamnosus Lrn-PG-06 and inoculate them into MRS liquid medium. After culturing them at 37° C. for 24 h, perform an antibacterial test on Lactobacillus rhamnosus Lrn-PG-06. S7. Streak the activated Lactobacillus rhamnosus Lrn-PG-06 bacterial solution onto Columbia blood agar plates, invert the plates and incubate them in an incubator for 48 hours to perform a hemolysis test. S8, centrifuging the fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 bacterial solution, taking the supernatant, and determining the hyaluronidase inhibition rate; S9. The fully activated and expanded Lactobacillus rhamnosus Lrn-PG-06 was subjected to in vitro antioxidant capacity determination.

3. The method for preparing a Lactobacillus rhamnosus strain having antiallergic activity according to claim 2, wherein: When activating and expanding the Lactobacillus rhamnosus Lrn-PG-06 in S2, the Lactobacillus rhamnosus Lrn-PG-06 strain stored in -20°C glycerol was first thawed and inoculated into liquid MRS culture medium at a 2% (V / V) inoculation rate, and then cultured at 37°C for 24 hours. The revived bacterial liquid was then inoculated into MRS liquid culture medium and cultured at 37°C for another 24 hours to fully activate and expand the strain.

4. The method for preparing a Lactobacillus rhamnosus strain having antiallergic activity according to claim 3, wherein: When the acid and alkali resistance test of Lactobacillus rhamnosus Lrn-PG-06 in S3 was performed, the pH values ​​of multiple MRS liquid culture media with a pH of 6.0 were adjusted to 5.0, 4.0, 3.0, 2.0 and 1.0, respectively, with HCl at a concentration of 4.0 mol / L, and the pH values ​​of multiple MRS liquid cultures with a pH of 6.0 were adjusted to 11.0, 10.0, 9.0, 8.0 and 7.0, respectively, with NaOH. The strain was inoculated into culture media with different pH values. After culturing at 37° C. for 24 hours, the absorbance of the bacterial suspension was measured at a wavelength of 600 nm, and the survival rate was calculated to test the acid and alkali tolerance of Lactobacillus rhamnosus Lrn-PG-06.

5. The method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity according to claim 4, characterized in that: When the bile salt tolerance test was performed on the Lactobacillus rhamnosus Lrn-PG-06 in the S4, the strain was inoculated into MRS culture medium containing bile salt concentrations of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%, respectively, at a 3% inoculum size. After culturing at 37° C. for 24 hours, the absorbance of the bacterial suspension was measured at a wavelength of 600 nm. The absorbance of the strain at a wavelength of 600 nm in MRS culture medium without bile salt was used as a control. The survival rate was calculated to evaluate the effect of bile salts on the activity of Lactobacillus rhamnosus Lrn-PG-06.

6. The method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity according to claim 5, characterized in that: When the Lactobacillus rhamnosus Lrn-PG-06 in S5 was subjected to a simulated gastrointestinal tract assay, the strain was activated and passaged in MRS medium at 37°C for two generations, 1.0 g of bacterial mud was centrifuged and added to 5.0 mL of artificial gastric juice. After treatment at 37°C for 3 h, the precipitate was collected under sterile conditions, and sterile physiological saline was added according to the ratio of 1.0 g bacterial mud: 5.0 mL of normal saline to mix, diluted and spread, and the colony count was performed. Then 1.0 g of bacterial mud treated with artificial gastric juice was taken and added to 5.0 mL In artificial bile, after treatment at 37°C for 20 minutes, the culture was centrifuged at 8000 r / min for 10 minutes. The bacterial sludge after centrifugation was taken for colony counting. Then 1.0 g of the bacterial sludge treated with artificial bile was taken and added to 5.0 mL of artificial intestinal fluid. After treatment at 37°C for 4 hours and 8 hours respectively, the culture was centrifuged at 8000 r / min for 10 minutes. The colony count of the bacterial sludge after centrifugation was taken, and the survival rate was calculated using the colony count in MRS culture medium that had not been treated with simulated gastrointestinal tract as a control.

7. The method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity according to claim 6, characterized in that: When the rhamnosus casei Lrn-PG-06 in the S5 was subjected to an antibacterial test, the Oxford cup agar diffusion method was used, and Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes were used as indicator bacteria for plate coating. Physiological saline was used as a blank control. Four Oxford cups were evenly placed on each plate, 200 μL of the rhamnosus casei Lrn-PG-06 bacterial suspension was added to three Oxford cups, and physiological saline was added to one Oxford cup. After incubation at 37° C. for 24 hours, the size of the inhibition zone was measured with a vernier caliper.

8. The method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity according to claim 7, characterized in that: When the hemolytic test of Lactobacillus rhamnosus Lrn-PG-06 in S6 was performed, the strain was streaked on a blood agar plate, cultured at 37°C for 48 hours, and then taken out for observation. The hemolytic effect was manifested by the presence of a transparent zone, a green zone, or an absence of a zone around the colony. The transparent zone represents β hemolysis, the green zone represents α hemolysis, and the absence of a zone represents γ hemolysis.

9. The method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity according to claim 8, characterized in that: When the Lactobacillus rhamnosus Lrn-PG-06 in S7 was subjected to an in vitro hyaluronidase inhibition test, the strain was activated and cultured in MRS medium at 37° C. for two generations, and then the supernatant was taken by centrifugation. 0.1 mL of 2.5 mmol / LCaCl2 and 0.5 mL of 600 U / mL hyaluronidase were added to the test tube and treated at 37° C. for 20 min. Then, 0.5 mL of the bacterial suspension was added and treated at 37° C. for 20 min. 0.5 mL of 0.5 mg / mL sodium hyaluronate solution was added and treated at 37° C. for 30 min. The mixture was allowed to stand at room temperature for 5 min, and 0.5 mL of acetylacetone solution and 0.1 mL of 0.4 mol / L NaOH solution were added. The acetylacetone solution was 1.4 mL of acetylacetone dissolved in 20 mL of 1.0 mol / L Na2 CO3 solution, after boiling in water bath for 15 minutes, immediately ice bath for 5 minutes, add 1 mL of Ehrlich reagent, Ehrlich reagent is 0.8 g of p-dimethylaminobenzaldehyde dissolved in 15 mL of concentrated hydrochloric acid and 15 mL of anhydrous ethanol, color development at room temperature for 20 minutes, and measure its absorbance at a wavelength of 530 nm. The absorbance value of LGG bacterial solution treated under the same conditions is used as a control.

10. The method for preparing a Lactobacillus rhamnosus strain having anti-allergic activity according to claim 9, characterized in that: When the in vitro antioxidant capacity test of Lactobacillus rhamnosus Lrn-PG-06 in S8 was performed, the strain was activated and cultured in MRS medium for two generations at 37° C., and then the DPPH free radical scavenging capacity, hydroxyl free radical scavenging capacity, ABTS+ free radical scavenging capacity, and superoxide anion free radical scavenging capacity were measured, and the antioxidant capacity of VC and the LGG bacterial solution treated in the same manner was used as a control.

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