CN101851593B - Lactobacillus plantarum strain LP28 and its use for alleviating allergic reactions - Google Patents

Abstract

The present invention relates to a plant lactobacillus strain LP28 (Lactobacillus plantarum), which is deposited in the China General Microorganism Collection Center with a deposit number of CGMCC No. 3346. This lactobacillus strain or its metabolites have excellent ability to alleviate allergic reactions and can be widely used in the preparation of food compositions or pharmaceutical compositions.

Description

Lactobacillus plantarum strain LP28 and its use for alleviating allergic reactions

technical field

The present invention relates to a lactobacillus strain capable of alleviating allergic reactions. the

Background technique

Allergic reaction refers to the strong reaction of the immune system to some non-invasive foreign substances. In recent decades, the number of patients with allergic diseases such as bronchial asthma, allergic rhinitis and atopic dermatitis has increased dramatically. Human immune center cells T lymphocytes can be divided into two types according to the different cytokines secreted. The first type T lymphocytes (Th1) mainly secrete interferon γ (Interferon-γ, IFN-γ), and the second type T lymphocytes (Th2) mainly secrete interleukin-4 (Interleukin-4, IL-4), interleukin-5 (Interleukin-5, IL-5) and other cytokines; however, type 1 and type 2 T lymphocytes There is a dynamic balance between them. If the effect of type 1 T lymphocytes is weak, it will easily cause allergic reactions in the human body. At present, most of the treatment of allergic diseases is to treat the symptoms with drugs. However, the treatment of allergic diseases with drugs is prone to side effects, which makes many research institutions in many countries eager to find safer and more effective treatments for allergic diseases. the

Researchers have found that the ratio of harmful bacteria to probiotics in the gastrointestinal tract has a great correlation with the occurrence of allergic diseases. If there are more probiotics in the gastrointestinal tract, the probiotics will protect the mucous membrane of the digestive tract, promote the proliferation of immunoglobulins, and produce natural Antibodies and immune cells can easily infiltrate the gastrointestinal mucosa; while there are many harmful bacteria in the gastrointestinal tract, harmful bacteria can easily damage the gastrointestinal mucosa, resulting in decreased lymphocytes and insufficient immunoglobulin types. However, the destruction of the gastrointestinal mucosa will lead to antigenicity increase, and then cause sensitization and allergic diseases; therefore, the types of bacteria that humans are exposed to are enough to affect the development of the immune system. Many researchers hope that by adjusting the number and types of probiotics in the gastrointestinal tract, further control allergic reactions happened. the

Lactic acid bacteria are the most common probiotics in the gastrointestinal tract, and they are extremely safe microorganisms. Lactic acid bacteria have the ability to inhibit the growth of harmful bacteria, regulate immune response, remove peroxides and lower cholesterol. In addition, lactic acid bacteria contain pleasant aroma substances and can produce antibacterial substances such as lactic acid or bacteriocin, so they are widely used to make fermented milk and other foods. the

However, in order to use lactic acid bacteria to achieve the purpose of treating allergic diseases, the efficacy of the current lactic acid bacteria is not satisfactory. The acidity of human stomach acid is as high as pH 2.5. Lactic acid bacteria are damaged or dead after being eroded by gastric acid and choline, and cannot effectively attach to the intestinal mucosa and are excreted with feces. The intestinal tract is the largest immune organ in the human body. Most of the lactic acid bacteria ingested by the human body cannot survive in the intestinal tract healthily, that is, they cannot exert the function of lactic acid bacteria, and it is difficult to effectively improve the allergic reaction caused by the immune system. Therefore, screening out lactic acid bacteria strains that can improve the action of type I T lymphocytes and resist gastric acid and choline is an urgent task for the treatment of allergic diseases. the

Contents of the invention

The purpose of the present invention is to provide a lactic acid bacteria strain, which can promote the action of the first type T lymphocytes to secrete interferon gamma. the

Another object of the present invention is to provide such a lactobacillus strain, which can effectively resist gastric acid and choline. the

Another object of the present invention is to provide the Lactobacillus strain, which can slow down allergic reactions. the

The present invention mainly provides a plant Lactobacillus strain LP28 (Lactobacillus plantarum), which was deposited in the China General Microorganism Collection and Management Center on October 19, 2009, and the deposit number is CGMCCNo.3346. The lactobacillus strain or its metabolite has excellent ability to relieve allergic reaction, and can be widely used in the preparation of food composition or pharmaceutical composition. the

The plant Lactobacillus strain LP28 of the present invention is determined as a Gram-positive bacterium by Gram staining, has no motility, has a short-rod appearance, and has an optimum growth environment temperature of 37°C. the

Table 1 shows the results of using API KIT CHL 50 (purchased from bioMerieux) to analyze the fermentability of LP28 to different carbohydrates, and it was determined to be Lactobacillus plantarum (please refer to the bioMerieux operation manual for the abbreviations of various carbohydrates in Table 1). The results of 16S rDNA sequencing of LP28 are shown in Table 2. the

Table 1: Fermentation capacity of LP28 to different carbohydrates (API KIT 50CH)

the 24H 48H the 0 the the 0 GLY the the 1 ERY the the 2 DARA the the 3 LARA the the 4 RIB the the 5 DXYL the the 6 LZYL the the 7 ADO the the 8 MDX the the 9 GAL the + 10 GLU the + 11 FRUs the + 12 MNE the + 13 SBE the + 14 RHA the the 15 DUL the the 16 INO the the 17 MAN the + 18 SOR the + 19 MDM the the 20 MDG the the twenty one NAG the + twenty two AMY the + twenty three ARB the + twenty four ESC the + 25

the 24H 48H the SAL the + 26 CEL the + 27 MAL the + 28 LAC the + 29 MEL the the 30 SAC the + 31 TRE the + 32 INU the the 33 MLZ the + 34 RAF the + 35 AMD the the 36 GLYG the the 37 XLT the the 38 GEN the + 39 TUR the the 40 LYX the the 41 TAG the the 42 DARL the the 43 LFUC the the 44 DARL the the 45 LARL the the 46 GNT the + 47 2KG the the 48 5KG the the 49

Table 2: LP28-16S rDNA sequence:

CGGCCGGGGGGGTGTCCTATACTGCAAGTCGAACGCGTTGACCCAATTGATTGATGGTGCTTGCACCTGATTGATTTTGGTCGCCAACGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCAGAAGCGGGGGACAACATTTGGAAACAGATGCTAATACCGCATAACAACGTTGTTCGCATGAACAACGCTTAAAAGATGGCTTCTCGCTATCACTTCTGGATGGACCTGCGGTGCATTAGCTTGTTGGTGGGGTAACGGCCTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGGACTGAGACACGGCCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGA GCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTAAAGAAGAACACGTATGAGAGTAACTGTTCATACGTTGACGGTATTTAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGAGAGTGCAGGCGGTTTTCTAAGTCTGATGTGAAAGCCTTCGGCTTAACCGGAGAAGTGCATCGGAAACTGGATAACTTGAGTGCAGAAGAGGGTAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTACCTGGTCTGCAACTGACGCTGAGACTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGACCTTACCAGTCTTGACATCTT GCGCAACCCTAGAGATAGGGCGTTTCCTCGGGACGCAATGACAGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCACCCTTGTTACTAGTTGCCAGCATTAAGTTGGGCACTCTATTGA

Description of drawings

Figure 1 is a histogram of the number of surviving bacteria of lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45 resistant to artificial gastric acid at pH 2.0 for two periods. the

Figure 2 is a histogram of the number of surviving bacteria of lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45 resistant to artificial gastric acid at pH 3.0 for two periods. the

Fig. 3 is a histogram of the test results of lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45 resistant to 0.3% bile salts. the

Fig. 4 is a histogram of the results of stimulating human peripheral blood mononuclear cells to produce IFN-γ by lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45. the

Fig. 5 is a histogram of the results of stimulation of human peripheral blood mononuclear cells to produce IL-4 by lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45. the

Figure 6 is a histogram of the results of the Interferon-r/IL-4 ratio produced by human peripheral blood mononuclear cells stimulated by lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45. the

Detailed ways

In order to make the above-mentioned and other objects, features and advantages of the present invention more obvious and understandable, the preferred embodiments of the present invention are specifically cited below, and in conjunction with the accompanying drawings, the detailed description is as follows:

After extensive and in-depth experiments, the applicant screened out a strain of Lactobacillus plantarum LP28 (Lactobacillus plantarum; deposit number: CGMCC No. 3346) with excellent allergy-reducing effects. The screening process of the Lactobacillus strain is as follows:

1. Screening of lactic acid bacteria strains from samples

The first step in the screening of lactic acid bacteria in the present invention: take dried tofu in the traditional market as a source sample for screening of lactic acid bacteria. The samples were inoculated in 10wt% skimmed milk powder culture solution, and the colony culture (Enrichment culture) was carried out at 37°C for 16-20 hours, and the strains that produced curdling phenomenon were screened out. . the

The second step of screening lactic acid bacteria strains of the present invention: take the samples after colonization and culture, carry out serial dilution and plate streak culture, and cultivate them under anaerobic conditions at 37°C for 2 to 3 days to select a single colony with different bacterial phases . the

The third step of screening lactic acid bacteria strains of the present invention: carry out Gram staining and catalase reaction tests on the single colony, and screen out several strains that are positive in Gram staining and negative in catalase reaction, which is preliminary The lactic acid bacteria strains screened, and the identification of the strains of the lactic acid bacteria strains that were initially screened out, among the several strains of lactic acid bacteria strains that were initially screened out, included a plant Lactobacillus plantarum (Lactobacillus plantarum), three fermenting Lactobacillus strains ( Lactobacillus fermentum) and a strain of Lactobacillus helveticus (Lactobacillus helveticus), the applicant named the five lactic acid bacteria strains as LP28, LF22, LF44, LF46 and LH45 respectively. the

2. Acid and bile salt resistance test

In the present invention, the five lactic acid bacteria strains (LP28, LF22, LF44, LF46 and LH45) are subjected to acid resistance and choline resistance tests respectively. the

The first step of the acid resistance test: inoculate the five strains of lactic acid bacteria into the MRS (deMan, Rogosa, Sharpe, Difco) culture solution, and cultivate them at 37°C for several hours to produce the MRS culture solution of each of the lactic acid bacteria strains . the

The second step of the acid resistance test: take a certain amount of MRS culture solution of each strain of lactic acid bacteria, centrifuge at 5000rpm for 5 minutes, wash and centrifuge with sterile PBS to obtain bacteria, then dissolve them in sterile PBS, add In the artificial gastric juice, the acidity of the artificial gastric juice is pH2.0 and pH3.0, and cultured at 37°C for two hours, and the number of bacteria is analyzed every hour. the

The results of the acid resistance test pH 2.0 and pH 3.0 of the five lactic acid bacteria strains (LP28, LF22, LF44, LF46 and LH45) are shown in Figure 1 and Figure 2 respectively. .0 two hours later, Lactobacillus strains LP28, LF22 and LF46 had the highest number of surviving bacteria (>106 Log CFU/mL), and the results of artificial gastric acid resistance to pH 3.0 for two hours showed that Lactobacillus strains LP28 and LF22 had the highest The number of surviving bacteria (>109LogCFU/mL), the above results show that lactic acid bacteria LP28 and LF22 have good acid resistance, the results of the number of surviving bacteria of these lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45 resistant to artificial gastric acid pH2.0 As shown in Figure 1; in addition, the number of surviving bacteria of these lactic acid bacteria resistant to artificial gastric acid pH 3.0 in two periods is shown in Figure 2. the

The first step of the bile salt tolerance test: the five strains of lactic acid bacteria (LP28, LF22, LF44, LF46 and LH45) were respectively inoculated in the MRS culture medium, and cultured at 37°C for several hours to produce each of the lactic acid bacteria strains MRS and culture medium. the

The second step of the bile salt tolerance test: take a certain amount of MRS culture fluid of each strain of lactic acid bacteria, add them to the MRS culture fluid containing 0.3% bile salt (0.3% ox gall) respectively, as the experimental group, and Take the culture solution of each strain of lactic acid bacteria and add it to the MRS culture solution without adding 0.3% bile salt as the control group; after the experimental group and the control group were cultured at 37°C for several hours, the number of surviving bacteria was analyzed. The results of the bile salt tolerance test of the five lactic acid bacteria strains (LP28, LF22, LF44, LF46 and LH45) are shown in Figure 3. It has the highest number of surviving bacteria with LF46, and the results of the surviving bacteria of the five lactic acid bacteria strains LP28, LF22, LF44, LF46 and LH45 resistant to bile salts are shown in Figure 3:

According to the results of the acid-resistant and choline-resistant tests of five lactic acid bacteria strains in the present invention, two lactic acid bacteria strains LP28 and LF22 with the highest resistance to artificial gastric juice and bile salts were screened out, and these lactic acid bacteria strains LP28, LF22, and LF44 were selected. , LF46 and LH45 were tested for immune activation. the

3. Immune activation test

The invention uses cell experiments to evaluate the ability of lactic acid bacteria to reduce allergic immune response. It is used to stimulate human peripheral blood mononuclear cells (human peripheral blood mononuclear cells, hPBMC) immune activation test with lactic acid bacteria, and to detect the type 1 T lymphocytes by enzyme immunolinked immunosorbent assay (ELISA) The production of the cytokine IFN-γ secreted by the lymphocytes and the cytokine IL-4 secreted by the second type T lymphocytes. the

The first step of the immune activation test is to prepare human peripheral blood lymphocytes, which are type I T lymphocytes. Take human leukocyte thick liquid and phosphate buffered saline (Phosphate Buffered Saline, PBS) and evenly mix; The mixed liquid is separated by cell gradient centrifugation to separate human peripheral blood mononuclear cells. the

The second step of the immune activation test: After the human peripheral blood mononuclear cells were evenly mixed and washed with Hanks balanced salt solution (HBSS), they were placed in RPMI-1640 medium (purchased from GIBCO, product No. 11875), the number of human peripheral blood mononuclear cells was calculated by Trypan blue staining method, and the number of peripheral blood mononuclear cells was adjusted to 2×106Cells/ml by using RPMI-1640 medium , divided into 24-well plates, and co-cultured with different concentrations of lactic acid bacteria. The negative control group was human peripheral blood mononuclear cells mixed with 10 μg/ml phytohemagglutinin (PHA) and 2×10 ⁶ Cells/ml evenly, and stimulated human peripheral blood mononuclear cells with other lactic acid bacteria. Compare.

The third step of the immune activation test: stimulating cytokine production. The five strains of lactic acid bacteria (LP28, LF22, LF44, LF46 and LH45) were used as the experimental group, and human peripheral blood mononuclear cells (PBMC) without adding any lactic acid bacteria were used as the control group and the production of IFN-γ was induced Phytohemagglutinin (PHA) was used as the control group, and the culture medium and the cell fluid were uniformly mixed and cultured for tens of hours at an appropriate ratio, and the culture supernatants of the experimental group, the control group and the control group were taken for enzyme immunolinkage adsorption analysis. To detect the production of cytokines IFN-γ and IL-4. The results of various lactic acid bacteria strains stimulating human peripheral blood mononuclear cells to produce IFN-γ, IL-4 and the ratio between the two are shown in Figure 4, Figure 5 and Figure 6 respectively, and the experimental group and the control group Group stimulates human peripheral blood mononuclear cells to produce the magnitude of IFN-γ as shown in Figure 4:

It can be seen from Figure 4 that the lactic acid bacteria strain LP28 can stimulate human peripheral blood mononuclear cells to produce more IFN-γ, thereby increasing the role of type 1 T lymphocytes and balancing type 1 and type 2 T lymphocytes The dynamic relationship between them can further slow down the allergic reaction of human beings. the

The amount of IL-4 produced by the human peripheral blood mononuclear cells stimulated by the experimental group and the control group is shown in Figure 5: IL-4 is a cytokine that activates mast cells in the Th2 immune system and can be combined with IgE in the body. Through message transmission, cells are stimulated to release chemical substances such as histamine to cause inflammation. These chemical substances cause allergies in tissues in different parts of the body, so IL-4 will help Th2 cells differentiate and inhibit Th1 cell differentiation. It can be seen from Figure 5 and the table above that the lactic acid bacteria strain LP28 can significantly reduce the concentration of IL-4 produced by human peripheral blood mononuclear cells, and contribute to the balance between Th1 and Th2 immune responses. the

The ratio of IFN-γ to IL-4 produced by human peripheral blood monocytes stimulated by the experimental group and the control group is shown in Figure 6:

As mentioned above, the present invention stimulates the first type T lymphocytes (Th1) to secrete a large amount of interferon gamma (Interferon-γ) by the three lactic acid bacteria strains LP28 of the present invention beneficial to the organism, and reduces the level of the second type T lymphocytes. (Th1) The secretion of IL-4, and then can slow down allergic reaction, also can utilize the metabolite of lactic acid bacteria strain LP28 of the present invention, this metabolite is widely used in the processing of food composition or pharmaceutical composition, to make can slow down allergic reaction Food, pharmaceutical composition or therapeutic agent, wherein the food can be dairy products such as fermented milk. the

Although the present invention has been disclosed by the above-mentioned preferred embodiments, it is not intended to limit the present invention. Any skilled person can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the scope of the appended patent application. the

Claims (4)

1. A strain of Lactobacillus plantarum LP28 (Lactobacillus plantarum), which is deposited in the China General Microbiology Collection and Management Center, and the deposit number is CGMCC No.3346. 2. The application of the plant Lactobacillus strain LP28 and its metabolites according to claim 1 in the preparation of a drug for slowing down the allergic reaction caused by the imbalance of the dynamic relationship between the first type and the second type T lymphocytes. 3. The application of the plant Lactobacillus strain LP28 and its metabolites according to claim 1 in the preparation of foods that slow down the allergic reaction caused by the imbalance of the dynamic relationship between the first type and the second type T lymphocytes. 4. as claimed in claim 3, Lactobacillus plantarum strain LP28 and its metabolites are prepared to slow down the application of the allergic reaction caused by the imbalance of the dynamic relationship between the first type and the second type of T lymphocytes, characterized in The food is fermented milk. the

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