TWI707689B - Immunomodulatory probiotic bacteria and uses thereof - Google Patents

Abstract

The present invention provides a novel Lactobacillus gasseri strain and use thereof for manufacture of immunomodulatory composition. This probiotic bacterial strain and metabolites thereof enhance the expression of genes regulating innate immunity, and inhibit the gene expression of proinflammatory cytokines when the inflammatory response is induced, thereby improving the innate immunity of individuals and avoiding excessive inflammation and damage to tissue. The invention also provides an immunomodulatory composition containing the probiotic bacterial strain.

Description

Probiotic strains for immune regulation and their uses

The present invention relates to a novel probiotic strain and its use, and particularly relates to a Lactobacillus gasseri and its use for preparing an immunomodulatory composition.

The human immune system can be roughly divided into two parts: "innate immunity" and "acquired immunity". The "innate immunity" system is composed of non-specific immune cells and protective proteins. It is activated when pathogens (such as bacteria, viruses, molds, parasites and other microorganisms that can cause diseases) enter the human body through the skin or mucous membranes. At this time, white blood cells with the ability to phagocytose microorganisms, such as neutrophils, macrophages differentiated from monocytes, etc., will gather at the pathogen invasion to destroy the pathogen. Certain cells, including white blood cells, also secrete pro-inflammatory cytokines, recruiting more white blood cells to the pathogen. At the same time, natural killer cells will also participate in the encirclement and suppression, responsible for poisoning cells infected by pathogens. In addition, complement proteins are activated in body fluids to attack pathogens.

When an individual's immunity becomes weak, diseases are prone to occur, such as frequent cold symptoms, and even a higher risk of cancer. However, there is a price for the immune system to protect the body from pathogens. When the body is chronically inflamed due to a malfunction of the immune system, normal cells or tissues in the body will be damaged. In severe cases, the average life span may be shortened. Therefore, keeping the body's immune system in a balanced state is one of the important principles for maintaining health.

Known methods to maintain the normal function of the immune system include paying attention to nutritional balance in daily diet, avoiding obesity, maintaining a regular schedule, exercising moderately, and maintaining a happy mood. However, for modern people with heavy academic work or busy work, it is difficult to adhere to a healthy lifestyle. Therefore, it is necessary to develop a novel composition that is convenient to use and helps maintain immune balance to achieve the goal of reducing disease occurrence and longevity.

For this reason, one purpose of the present invention is to provide an immunomodulatory probiotic strain, which is a novel strain of Lactobacillus gasseri that can be used for food. The strain is deposited in the Bioresource Collection and Research Center (BCRC) of the Food Industry Development Institute, and its deposit label is BCRC 910886.

Another object of the present invention is to provide a use of the aforementioned Lactobacillus gasseri or its metabolites for preparing an immunomodulatory composition.

In an embodiment of the present invention, the Lactobacillus gasseri or its metabolites improve innate immunity. This effect can be attributed to, but not limited to, the Lactobacillus gasseri or its metabolites enhance interleukin-1β (interleukin 1 beta, IL-1β), tumor necrosis factor alpha (tumor necrosis factor alpha, TNF-α), mediate Gene expression of interleukin-8 (IL-8), interleukin-18 (IL-18), or any combination thereof.

In an embodiment of the present invention, the Lactobacillus gasseri or its metabolite inhibits an inflammatory response. The inflammatory response can be triggered by a pathogen or a substance produced by it, such as lipopolysaccharide (LPS). This effect can be attributed to, but not limited to, that the Lactobacillus gasseri or its metabolites inhibit the gene expression of interleukin-18, tumor necrosis factor-α, or a combination thereof during the inflammation reaction.

Another object of the present invention is to provide an immunomodulatory composition comprising an effective amount of Lactobacillus gasseri or its metabolites, wherein the deposit number of Lactobacillus gasseri is BCRC 910886.

The Lactobacillus gasseri and its metabolites disclosed in the present invention can enhance the expression of innate immunity regulation genes, and therefore help to enhance the innate immunity of individuals to resist the invasion of foreign pathogens. In addition, the strain and its metabolites inhibit the gene expression of pro-inflammatory cytokines when the inflammatory response is induced, so that tissues or organs can be prevented from being damaged by excessive inflammation. Therefore, Lactobacillus gasseri BCRC 910886 can be used to prepare immunomodulatory compositions. The composition can have a powder, granule, solution, or gel dosage form, and can be made into food, drink, nutritional supplement, or medicine, and administered to a body by oral administration.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the inventive features and applications of the present invention, rather than limiting the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the appended patent scope.

Figure 1 shows the relative expression level of the IL-1β gene of the human monocyte line THP-1 after being treated with Lactobacillus medium (MRSD) or a variety of Lactobacillus gasseri culture supernatants.

Figure 2 shows the relative expression level of the TNF-α gene of the human monocyte line THP-1 after being treated with MRSD medium or the supernatant of the designated Lactobacillus gasseri culture.

Figure 3 shows the relative expression level of IL-8 gene of the human monocyte line THP-1 treated with MRSD medium or the supernatant of the designated Lactobacillus gasseri culture.

Figure 4 shows the relative expression level of IL-18 gene of the human monocyte line THP-1 after being treated with MRSD medium or the supernatant of the designated Lactobacillus gasseri culture.

Figure 5 shows the relative expression level of IL-18 gene of human monocyte line THP-1 after being treated with MRSD medium or designated Lactobacillus gasseri culture supernatant and co-cultured with lipopolysaccharide (LPS).

Figure 6 shows the relative expression level of the TNF-α gene of the human monocyte line THP-1 after being treated with MRSD medium or a variety of Lactobacillus gasseri culture supernatants and then co-cultured with lipopolysaccharide (LPS).

Fig. 7 shows the change of the number of bacteria in the artificial gastric juice simulation test of Lactobacillus gasseri of the present invention.

Fig. 8 shows the change of the number of bacteria in the artificial intestinal juice simulation test of Lactobacillus gasseri of the present invention.

The present invention provides a Lactobacillus gasseri with immunomodulatory effect, which is isolated from breast milk, confirmed as a novel strain after culture and strain identification, and has been deposited in the Bioresource Conservation and Research Center of the Food Industry Development Institute ( BCRC), the registration number is BCRC 910886. The following examples show that the strain and its metabolites can enhance the expression of innate immunity regulating genes, and can inhibit the gene expression of pro-inflammatory cytokines when an inflammatory response is induced.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

The "effective amount" used herein refers to the amount of an active substance required to induce a specific effect in an individual. As recognized by those skilled in the art, the effective dose will vary depending on the route of administration, the use of excipients, and the possibility of sharing with other substances.

Materials and Methods Bacterial culture

The following examples use Lactobacillus gasseri isolated from human breast milk and determined by 16S rRNA gene sequence alignment, including the BCRC 910886 strain deposited at the Bioresource Conservation and Research Center of the Food Industry Development Institute, and its 16S rRNA gene has SEQ ID NO:1 nucleotide sequence; and LH009 strain and LH028 strain. Among them, the LH009 strain was selected from breast milk, and the LH028 strain was selected from the human intestine. After thawing and activation of each of the bacteria, 1% of the inoculum was statically cultured in Lactobacillus MRSD medium (55g Lactobacilli MRS Broth (BD Difco Lactobacilli MRS Broth; Thermo Fischer Scientific) at 37°C, dissolved in 1L Ionized water, pH value is about 6.8) 18 hours. After the obtained bacterial culture was centrifuged at 5000 rpm for 20 minutes, the supernatant was collected for further analysis. The supernatant contains bacterial metabolites.

Cell culture

The following examples use human monocytic cell line THP-1 (ATCC TIB-202) purchased from American Type Culture Collection (ATCC) and human colorectal cancer epithelial cell line (human colon adenocarcinoma cell line) CACO2 (ATCC HTB-37). The THP-1 cell line was cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco RPMI medium 1640; Thermo Fisher Scientific), hereinafter referred to as RPMI cell culture medium. The CACO2 cell line was cultured in DMEM medium (Dulbecco's modified Eagle's medium; Thermo Fischer Scientific) supplemented with 10% FBS and 1% penicillin/streptomycin under the conditions of 37°C and 5% carbon dioxide, referred to as DMEM cell medium hereinafter.

Gene expression analysis

Based on the quantitative polymerase chain reaction (qPCR) to determine the gene expression level of cytokines that regulate innate immunity in THP-1 cells, the steps are briefly described as follows. According to the manufacturer’s instructions, use RNA Extraction Kit (Geneaid) to isolate ribonucleic acid (RNA) from cells, and reverse transcribe 2000ng RNA into cDNA at 37°C with SuperScript® III Reverse Transcriptase (Invitrogen) . Afterwards, use the qPCR kit (KAPA CYBR FAST qPCR Kit (2X); KAPA Biosystems) and the primer pair of the target gene and the Glyceraldehyde 3-phosphate dehydrogenase gene GAPDH as an internal control (Table 1) Perform qPCR on the aforementioned cDNA in a PCR reaction machine (Step One Plus Real-Time PCR system; Applied Biosystems), and analyze the melting curve of the PCR product.

Finally, the 2- ^△△CT method was used to determine the relative expression of the target gene. The method is the threshold cycle (C _T) GAPDH gene as an internal control of the reference cycle threshold gene, calculated by the following formula relative fold change: C gene of △ C _T = the experimental group or the control group _T - internal control C _T

△△ C _T = test group △ C _T - △ C _T of the control group

Multiple change=2 ^{-△△Ct average}

In the anti-inflammatory effect test, the gene expression level is determined according to the aforementioned method, but the TATA-box binding protein gene TBP is used as an internal control, and the nucleotide sequences of the forward primer and reverse primer are respectively They are 5'-GCCAGCTTCGGAGAGTTCTGGGATT-3' (SEQ ID NO: 12) and 5'-CGGGCACGAAGTGCAATGGTCTTTA-3' (SEQ ID NO: 13).

The statistical analysis department uses the STDEV function in Excel software to calculate the standard deviation of the relative expression of each gene, and the one-tailed student t test (TTEST) is used to determine statistical significance.

Example 1 Enhancement effect of Lactobacillus gasseri on the expression of innate immunity regulating genes

In order to evaluate the immune enhancement effect of Lactobacillus gasseri of the present invention, qPCR was used to determine the changes in the expression of the innate immunity regulating genes of the human monocyte cell line THP-1 after being treated with the supernatant of the designated Lactobacillus gasseri culture. In short, THP-1 cells were seeded in a 6-well plate at 1×10 ⁶ cells/well, and each well contained 2 mL of RPMI cell culture medium. After culturing the cells at 37°C for 3 to 6 hours, the medium was removed, and the cells in each well were treated in the following manner: (a) 2mL RPMI cell culture medium (control group); (b) 2mL containing 0.5mg/mL RPMI cell culture medium of MRSD medium (MRSD group); (c) 2 mL of RPMI cell culture medium containing 0.5 mg/mL LH009 strain culture supernatant (LH009 group); (d) 2 mL of 0.5 mg/mL LH028 RPMI cell culture medium of strain culture supernatant (LH028 group); or (e) 2 mL of RPMI cell culture medium (BCRC 910886 group) containing 0.5 mg/mL BCRC 910886 strain culture supernatant. The aforementioned groups of cells were cultured at 37°C for 6 hours and then used for qPCR analysis.

Figure 1 shows the relative expression levels of the IL-1β gene of the aforementioned cells in the control group; Figure 2 shows the relative expression levels of the TNF-α gene of the aforementioned cells in the control group; Figure 3 shows the relative expression of the TNF-α gene in the aforementioned groups of cells The relative expression level of IL-8 gene of cells relative to the control group; Figure 4 shows the relative expression level of IL-18 gene of the aforementioned cells in the control group; ** and *** in the figure indicate the relative expression levels respectively The control group was p<0.01 and p<0.001. According to Figures 1, 2, 3 and 4, administering the culture supernatant of LH009, LH028, or BCRC 910886 strains to cells significantly improved the expression of IL-1β , TNF-α , IL-8 , and IL-18 genes, Especially the BCRC 910886 strain has the most significant effect. In view of the fact that IL-1β and TNF-α can integrally strengthen the innate immunity of individuals to pathogens, IL-8 can promote the aggregation and phagocytosis of neutrophils, and IL-18 can increase the activity of natural killer cells. This result indicates that Lactobacillus gasseri BCRC 910886 and its metabolites help to enhance the individual's innate immunity.

Example 2 Inhibitory effect of Lactobacillus gasseri on the expression of pro-inflammatory genes stimulated by lipopolysaccharide

In order to evaluate the anti-inflammatory effect of Lactobacillus gasseri of the present invention, the human monocyte line THP-1 was measured by qPCR after co-cultivation with the designated Lactobacillus gasseri culture supernatant and lipopolysaccharide (LPS) for inducing inflammation. Changes in the expression of hormone genes that promote inflammation. In short, THP-1 cells were seeded in a 6-well plate at 1×10 ⁶ cells/well, and each well contained 2 mL of RPMI cell culture medium. After culturing the cells at 37°C for 3 to 6 hours, the medium was removed, and the cells in each well were treated in the following manner: (a) applied 2 mL of RPMI cell culture medium (control group); (b) applied 2 mL with 100 ng/mL lipid Polysaccharide RPMI cell culture medium (LPS group); (c) Apply 2 mL of RPMI cell culture medium containing 0.5 mg/mL MRSD medium for 1 hour, then apply 100 ng/mL lipopolysaccharide (MRSD+LPS group); (d) Apply 2 mL of RPMI cell culture medium containing 0.5 mg/mL LH009 strain culture supernatant for 1 hour, and then 100 ng/mL lipopolysaccharide (LH009+LPS group); (e) 2 mL of 0.5 mg/mL LH028 strain culture Supernatant RPMI cell culture medium for 1 hour, and then 100ng/mL lipopolysaccharide (LH028+LPS group); or (f) 2mL RPMI cell culture medium containing 0.5mg/mL BCRC 910886 strain culture supernatant 1 After hours, 100ng/mL lipopolysaccharide (BCRC 910886+LPS group) was administered. The aforementioned groups of cells were cultured at 37°C for 3 hours and then used for qPCR analysis.

Figure 5 shows the relative expression levels of the IL-18 gene of the aforementioned cells in the control group; Figure 6 shows the relative expression levels of the TNF-α gene of the aforementioned cells in the control group; *** in the figure indicates Compared with the LPS group, it was p<0.001. According to Figure 5 and Figure 6, the treatment of lipopolysaccharide alone leads to an increase in the gene expression of pro-inflammatory cytokines such as TNF-α and IL-18. However, simultaneously administering the culture supernatant of the BCRC 910886 strain or the LH0028 strain to the cells significantly inhibited the expression of TNF-α and IL-18 genes. In contrast, the culture supernatant of strain LH009 has no such inhibitory effect. This result shows that Lactobacillus gasseri BCRC 910886 and its metabolites can inhibit the generation of inflammatory signals under inflammatory stimuli, and therefore can reduce tissue or organ damage due to excessive inflammation. Moreover, this anti-inflammatory effect is not available in any variety of Lactobacillus gasseri.

Example 3 The acid and bile resistance properties of Lactobacillus gasseri

In order to test whether the Lactobacillus gasseri of the present invention can resist the acidic environment of the stomach and the bile salts in the intestine, the overnight culture broth of this strain (the bacteria amount is about 5x10 ⁹ CFU/mL) is used for artificial gastric juice or intestinal juice simulation test. In the artificial gastric juice simulation test, the bacterial solution was added to the artificial gastric juice (0.2% sodium chloride aqueous solution, pH 3) according to the addition amount of 1%, and cultured with shaking at 37° C. and 50 rpm for 3 hours. In the artificial intestinal juice simulation test, the bacterial solution was added to the artificial intestinal juice (0.68% potassium dihydrogen phosphate and or 0.3% bile salt (oxgall) aqueous solution, pH 6.8) according to the addition amount of 1%, and the temperature was 37°C, 50rpm Incubate with shaking for 3 hours. As a comparison, another control group was set up, and the bacterial solution of Lactobacillus gasseri of the present invention was cultured in 0.2% sodium chloride or 0.68% potassium dihydrogen phosphate aqueous solution with pH 7 and no bile salts. After the aforementioned test, 100 μL of the bacterial solution obtained was spread on Lactobacillus MRS agar medium, and cultured at 37° C. overnight to calculate the bacterial count.

According to Fig. 7, the number of bacteria of the Lactobacillus gasseri of the present invention cultured in artificial gastric juice (pH 3) for 3 hours is equivalent to the number of bacteria cultured at pH 7, indicating that it has gastric acid resistance. According to Fig. 8, the number of bacteria of the Lactobacillus gasseri of the present invention cultured in artificial intestinal juice (0.3% bile salt) for 3 hours and cultured in a bile salt-free environment The number of bacteria is equivalent, showing its tolerance to bile salts. These results indicate that Lactobacillus gasseri BCRC 910886 can survive in the digestive tract after being taken orally into the human body, so it can play the role of immune regulation.

Example 4 Intestinal colonization test of Lactobacillus gasseri

In order to test the ability of Lactobacillus gasseri of the present invention to fix the intestinal tract, a human colon cancer epithelial cell line CACO2 was used to perform an intestinal colonization test. In short, CACO2 cells were seeded on a 24-well plate at 1×10 ⁴ cells/well, and each well contained 1 mL of DMEM cell culture medium. After culturing the cells at 37°C for several days to form a cell monolayer, replace the original medium with antibiotic-free DMEM medium, and then collect the bacterial cells from the septum culture broth of Lactobacillus gasseri BCRC 910886 with phosphate buffer A salt solution (phosphate buffered saline, PBS; Thermo Fischer Scientific) was prepared as a bacterial suspension for infecting CACO2 cells (0.5 mL/well) for about 30 minutes. Thereafter, the CACO2 cells were lysed with a PBS solution containing 0.05% Triton X-100, and the resulting cell lysate was spread on the Lactobacillus MRS agar medium along with the Lactobacillus gasseri attached to the cell surface or entered the cell, and cultured at 37°C 48 hours to calculate the number of bacteria. According to the results of intestinal colonization test, about 33 Lactobacillus gasseri cells are attached to each intestinal cell (the colonization rate is about 33), which shows that Lactobacillus gasseri BCRC 910886 can stably attach to the intestines after being taken orally into the human body Tao.

In summary, the Lactobacillus gasseri disclosed in the present invention can enhance the innate immunity of individuals and have the effect of inhibiting inflammation, so it can be used to prepare an immunomodulatory composition. The composition can have a powder, granule, solution, or gel dosage form, and can be made into food, drink, medicine, or nutritional supplement, and administered to a body by oral administration.

【Biological Material Deposit】

Domestic deposit information [please note in order of deposit institution, date and number]

Food Industry Development Research Institute, April 15, 2019, BCRC 910886

Foreign hosting information [please note in the order of hosting country, institution, date and number]

<110> Dajiang Biomedical Co., Ltd.

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Claims (9)

An immunomodulatory probiotic strain that improves innate immunity is Lactobacillus gasseri with the deposit number BCRC 910886. A use of Lactobacillus gasseri or its metabolites for preparing an immunomodulatory composition for improving innate immunity, wherein the registration number of Lactobacillus gasseri is BCRC 910886. The use described in item 2 of the scope of patent application, wherein the Lactobacillus gasseri or its metabolite enhances interleukin-1β, tumor necrosis factor-α, interleukin-8, interleukin-18, or any of these The combination of genetic performance. The use described in item 2 of the scope of patent application, wherein the Lactobacillus gasseri or its metabolites inhibit an inflammatory reaction. The use described in item 4 of the scope of patent application, wherein the inflammatory reaction is caused by a lipopolysaccharide. The use according to item 4 of the scope of patent application, wherein the Lactobacillus gasseri or its metabolite inhibits gene expression of interleukin-18, tumor necrosis factor-α, or a combination thereof during the inflammation reaction. An immunomodulatory composition for improving innate immunity, comprising an effective amount of Lactobacillus gasseri or its metabolites, wherein the deposit number of Lactobacillus gasseri is BCRC 910886. The composition described in item 7 of the scope of the patent application is in the form of powder, granules, solution, or colloid. The composition described in item 7 of the scope of patent application is a food, a drink, a nutritional supplement, or a medicine.

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