Novel bifidobacterium longum as well as microbial preparation, product and application thereof

Abstract

The invention provides a bifidobacterium longum strain aFMT-101, which is classified and named as Bifidobacterium longum subsp.longum, is preserved in Guangdong Microbial Culture Collection Center, and has the preservation number of GDMCC No: 65179. The invention also provides a preparation method of the bifidobacterium longum strain aFMT-101. The invention also provides a microbial preparation containing the strain aFMT-101 and application of the strain aFMT-101 and the microbial preparation in preparation of products for improving intestinal health and regulating immune functions. Meanwhile, the invention also provides a product containing the strain aFMT-101 or a corresponding microbial preparation. The strain aFMT-101 disclosed by the invention can keep a relatively high survival rate in gastric acid and cholate environments, can be rapidly colonized in intestinal tracts, promotes reproduction of beneficial bacteria and inhibits growth of harmful bacteria, so that intestinal health is improved, immunity is enhanced, and the strain aFMT-101 has no adverse effect on hosts and has good biological safety.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a novel Bifidobacterium longum and a microbial preparation, product and application thereof. Background Art

[0002] In recent years, intestinal health has received extensive attention, and the imbalance of intestinal microecology has been shown to be closely related to the occurrence of a variety of diseases. There are many probiotic preparations on the market for regulating intestinal flora, among which Bifidobacterium is widely used in various foods and medicines due to its excellent colonization ability and prebiotic function.

[0003] However, traditional Bifidobacterium longum strains have certain limitations, such as low survival rate under adverse conditions such as gastric acid and bile salts, low colonization efficiency after entering the intestine, and inability to fully exert their prebiotic effects.

[0004] Therefore, there is an urgent need to obtain a new type of Bifidobacterium longum with good acid resistance, bile salt resistance, and strong intestinal colonization ability. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a new type of Bifidobacterium longum and its microbial preparation, product and application. The new type of Bifidobacterium longum is isolated from the human intestine, has good acid resistance, bile salt resistance, and strong intestinal colonization ability, and has a significant effect on maintaining intestinal microecological balance, improving immunity, etc.

[0006] The present invention adopts the following technical solutions to solve the above technical problems:

[0007] A new type of Bifidobacterium longum aFMT-101, classified and named Bifidobacterium longumsubsp.longum, was deposited in the Guangdong Provincial Microbiological Culture Collection Center on September 23, 2024 and showed survival, with the deposit number GDMCC No: 65179, and the deposit address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] A microbial preparation containing the novel Bifidobacterium longum.

[0009] As one of the preferred embodiments of the present invention, the microbial preparation is a bacterial suspension or a bacterial agent, and the number of viable bacteria of Bifidobacterium longum aFMT-101 is not less than 1×10 9 CFU / g.

[0010] A use of the novel Bifidobacterium longum or the microbial preparation in preparing a product for improving intestinal health and regulating immune function.

[0011] A product of the above novel Bifidobacterium longum or microbial preparation.

[0012] As one of the preferred embodiments of the present invention, the product comprises probiotics.

[0013] As one of the preferred embodiments of the present invention, the dosage form of the probiotics includes capsules, powders, tablets or granules.

[0014] As one of the preferred embodiments of the present invention, the product also includes food and medicine.

[0015] The advantages of the present invention compared to the prior art are:

[0016] (1) The present invention isolates and screens a new type of Bifidobacterium longum subspecies strain aFMT-101 (Bifidobacterium longum subsp. longum) from the human intestine. The strain can maintain a high survival rate in the environment of gastric acid and bile salts, and can quickly colonize in the intestine, promote the reproduction of beneficial bacteria, and inhibit the growth of harmful bacteria, thereby improving intestinal health and enhancing immunity;

[0017] (2) The strain of the present invention can further enhance the host's resistance to pathogens by increasing the level of intestinal-related immunoglobulins;

[0018] (3) The strain of the present invention has no adverse effects on the host during long-term use and has good biosafety;

[0019] (4) The strain of the present invention can be widely used in the fields of food, medicine, etc., especially in preparations for promoting intestinal health and improving immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a morphological characteristic diagram of strain aFMT-101 in Example 2;

[0021] Figure 2 is the intestinal colonization ability test result in Example 4 (in the figure, the horizontal axis is the sampling time; the vertical axis is the strain colonization rate, %);

[0022] Figure 3 is the test result of improving immune function in Example 5 (in the figure, the horizontal axis is the sampling time; the vertical axis is the concentration level of IgA and IgG μg / mL, or the increase in the number of CD4+T cells %);

[0023] Figure 4 These are the results of changes in body weight and blood biochemical indexes in the biosafety test in Example 6 (in the figure, the horizontal axis is the detection time point; the vertical axis is body weight in g, or the concentration level of ALT and AST in U / L, or the concentration level of BUN and serum creatinine in mg / dL). DETAILED DESCRIPTION

[0024] The following is a detailed description of the embodiments of the present invention. The embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation methods and specific operation processes are provided, but the protection scope of the present invention is not limited to the following embodiments. Meanwhile, unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0025] The culture medium formula involved in the following examples is as follows:

[0026] MRS medium: Peptone 10 g / L, Beef Extract 10 g / L, Yeast Extract 5 g / L, Glucose 20 g / L, Sodium Acetate Trihydrate 5 g / L, Ammonium Citrate 2 g / L, Dipotassium Phosphate (K2HPO4) 2 g / L, Magnesium Sulfate Heptahydrate (MgSO4·7H2O) 0.2 g / L, Manganese Sulfate Monohydrate (MnSO4·H2O) 0.05 g / L, Tween 80 mL / L.

[0027] Modified MRS medium: MRS medium + 0.05% L-cysteine.

[0028] MRS agar medium: Peptone 10 g / L, Beef Extract 10 g / L, Yeast Extract 5 g / L, Glucose 20 g / L, Sodium Acetate Trihydrate 5 g / L, Ammonium Citrate 2 g / L, Dipotassium Phosphate (K2HPO4) 2 g / L, Magnesium Sulfate Heptahydrate (MgSO4·7H2O) 0.2 g / L, Manganese Sulfate Monohydrate (MnSO4·H2O) 0.05 g / L, Tween 80 mL / L, Agar 15 g / L.

[0029] Modified MRS agar medium: MRS agar medium + 0.05% L-cysteine.

[0030] Example 1: Screening and Isolation of Strain aFMT-101

[0031] A subspecies strain of Bifidobacterium longum was isolated from the feces of healthy people and was confirmed to be Bifidobacterium longum subsp. Longum after preliminary identification and 16S rRNA gene sequencing.

[0032] Based on the identification results, the strain was named Bifidobacterium longum subsp. longum aFMT-101, and was preserved in the Guangdong Provincial Microbiological Culture Collection Center on September 23, 2024 and showed survival, with the preservation number GDMCC No: 65179, and the preservation address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0033] Example 2: Colony morphology of strain aFMT-101

[0034] The strain aFMT-101 was inoculated on a modified MRS agar medium (MRS agar medium + 0.05% L-cysteine) plate, incubated at 37° C. for 1 day, and the colony morphology was observed.

[0035] The colonies of strain aFMT-101 on the plate are white, round, smooth, opaque, and convex in the middle. Figure 1 shown.

[0036] Example 3: Acid and bile resistance test of strain aFMT-101

[0037] 1. Experimental Methods

[0038] (1) Strain culture

[0039] The strain aFMT-101 was inoculated into a modified MRS medium (MRS+0.05% L-cysteine), and cultured at 37°C under anaerobic conditions (90% nitrogen, 5% carbon dioxide, 5% hydrogen) for 17 h until the logarithmic growth phase, and the bacterial liquid was collected for later use.

[0040] (2) Acid resistance test

[0041] ① Preparation of simulated gastric fluid (pH 2.0): Use hydrochloric acid to adjust the pH of modified MRS medium (MRS+0.05% L-cysteine) to 2.0 (accurately measure and calibrate using a pH meter), sterilize, cool to 37° C., and set aside.

[0042] ② Preparation of bacterial suspension: The aFMT-101 bacterial suspension cultured to the logarithmic growth phase was washed twice with sterile saline (5000 rpm, centrifugation for 10 min), and then resuspended in simulated gastric fluid.

[0043] ③ Acid-resistant treatment: transfer the bacterial suspension into simulated gastric fluid (pH 2.0) and incubate at 37°C for 2 h. Maintain a constant temperature during the treatment and mix gently to ensure that the acid evenly contacts the bacteria.

[0044] ④ Survival detection: After the treatment, take a part of the bacterial solution for ten-fold gradient dilution, spread it on a modified MRS agar medium (MRS agar medium + 0.05% L-cysteine) plate, and culture it anaerobically at 37°C for 48h. Count the number of surviving colonies to determine the survival rate.

[0045] (3) Bile salt resistance test

[0046] ① Preparation of bile salt medium: Add 0.3% bile salt to the modified MRS medium (MRS medium + 0.05% L-cysteine ​​+ 0.3% bile salt).

[0047] ② Preparation of bacterial suspension: The bacterial suspension cultured to the logarithmic growth phase was washed twice with sterile physiological saline (5000 rpm, centrifugation for 10 min), and then resuspended in a modified MRS medium containing 0.3% bile salt (MRS medium + 0.05% L-cysteine ​​+ 0.3% bile salt).

[0048] ③ Bile salt resistance treatment: transfer the bacterial suspension into bile salt culture medium and incubate at 37°C for 2 hours. Maintain a constant temperature and mix gently to ensure that the culture medium is in even contact with the bacteria.

[0049] ④ Survival detection: After the treatment, dilution and plate spreading are also performed to detect the number of surviving colonies.

[0050] 2. Experimental Results

[0051] The strain aFMT-101 of the present invention was cultured in simulated gastric fluid (pH 2.0) for 2 hours, and the survival rate of the strain was 92%; and was cultured in a culture medium containing 0.3% bile salt for 4 hours, and the survival rate of the strain was 88%.

[0052] Example 4: Intestinal colonization ability test of strain aFMT-101

[0053] 1. Experimental Methods

[0054] The experimental mice were randomly divided into three groups, 10 mice in each group, namely the control group, low-dose group, and high-dose group. The control group: gavage 1×10^8CFU control strain (commercial standard bifidobacterium strain: Bifidobacterium longum ATCC15707) every day; the low-dose group: gavage 1×10^8CFU of the strain aFMT-101 of the present invention every day; the high-dose group: gavage 1×10^9CFU of the strain aFMT-101 of the present invention every day. Mouse fecal samples were collected on the 3rd, 7th and 14th days, and the number of strains in the intestine was detected by fluorescent quantitative PCR, and the fixed value rate was calculated to evaluate its colonization ability in the mouse intestine.

[0055] 2. Experimental Results

[0056] The results are as follows Figure 2 shown.

[0057] Depend on Figure 2 It can be seen that the strain aFMT-101 of the present invention can effectively colonize in the mouse intestine, and the colonization effect of the high-dose group is significantly better than that of the control group and the low-dose group. In particular, on the 14th day, the colonization rate of the high-dose group reached 70%, showing a strong colonization ability.

[0058] Example 5: Immunity function improvement test of strain aFMT-101

[0059] 1. Experimental Methods

[0060] The mice were randomly divided into three groups, with 10 mice in each group, namely the control group, low-dose group, and high-dose group. The control group: the mice were given an equal amount of normal saline every day without strain treatment. The low-dose group: the mice were gavaged with 1×10^8 CFU of strain aFMT-101 every day. The high-dose group: the mice were gavaged with 1×10^9 CFU of strain aFMT-101 every day. Blood samples were collected from the mice on days 0, 14, and 28 of the experiment to detect their serum immunoglobulins and intestinal immune-related cells.

[0061] Immunoglobulin detection method: Use ELISA (enzyme-linked immunosorbent assay) to detect the IgA and IgG levels in serum to evaluate the enhancement of humoral immunity.

[0062] Intestinal immune-related cell detection method: Collect intestinal contents or mucosa, label immune-related cells, and use flow cytometry to detect CD4+T cells.

[0063] 2. Experimental Results

[0064] The results are shown in Table 1. Figure 3 shown.

[0065] Table 1 Immunoglobulin and intestinal immune-related cell detection data

[0066]

[0067] From Table 1 and Figure 3 The results show that oral administration of the strain aFMT-101 of the present invention can significantly increase the immunoglobulin level of mice and enhance the intestinal-related immune function, especially in the high-dose group, showing a significant immune enhancement effect.

[0068] Example 6: Biosafety Test of Strain aFMT-101

[0069] 1. Experimental Methods

[0070] The mice were randomly divided into two groups, a control group and an experimental group, with 10 mice in each group. The control group was given an equal amount of normal saline (sham treatment group) every day without adding any strains; the experimental group was gavaged with 1×10^9 CFU of the strain aFMT-101 of the present invention every day. The behavior and health status of the mice were observed, and the weight, blood biochemical indicators and major organs of the mice in each group were tested regularly.

[0071] Specific methods:

[0072] (1) Observation of behavior and health status

[0073] Observe and record the behavior, activity level, food intake, and any abnormal manifestations (such as mental state, hair condition, etc.) of the mice daily.

[0074] (2) Weight monitoring

[0075] The body weight of mice was measured at week 0, 2 weeks, and 4 weeks to evaluate the effect of strain on growth.

[0076] (3) Blood biochemical index detection

[0077] At week 0, 2, and 4, blood samples were collected from the tail vein to detect liver function indicators (alanine aminotransferase ALT and aspartate aminotransferase AST) and renal function indicators (blood urea nitrogen BUN and serum creatinine Cr).

[0078] (4) Histopathological analysis of major organs

[0079] At the end of the experiment (week 4), the main organs of the mice (liver, kidney, spleen, intestine) were taken, fixed in 4% paraformaldehyde solution, embedded in paraffin, sliced, and stained with HE (hematoxylin-eosin). Subsequently, the histological changes of the organs were observed under a microscope to evaluate whether there was tissue damage, inflammatory response, or abnormal lesions.

[0080] 2. Experimental Results

[0081] (1) Behavior and health status results

[0082] Both the control group and the experimental group mice showed normal activity levels and eating status, and did not show any adverse symptoms such as lethargy or deterioration of hair condition.

[0083] (2) Body weight and blood biochemical index results

[0084] As shown in Table 2, Figure 4 shown.

[0085] Table 2 Results of body weight and blood biochemical indexes

[0086]

[0087] From Table 2, Figure 4 It can be seen that: the weight gain of mice in the experimental group was normal during the experiment, with no significant difference compared with the control group; there was no significant change in ALT and AST levels between the experimental group and the control group, indicating that the strain had no toxic effect on the liver; there was no significant difference in BUN and serum creatinine levels between the experimental group and the control group, indicating that the strain had no toxic effect on the kidneys.

[0088] (3) Histopathological results

[0089] The liver tissue sections of mice in the control group and the experimental group showed normal liver lobule structure, without cell necrosis or inflammatory reaction; there was no glomerular lesion in both the control group and the experimental group, the renal tubule structure was intact, and there was no inflammatory cell infiltration; the white pulp and red pulp structure of the spleen in the control group and the experimental group were normal, without abnormal proliferation or damage; there was no significant inflammatory reaction in the intestinal sections of the experimental group and the control group, the intestinal villus structure was intact, and no pathological changes were observed. The above results show that the intragastric administration of the strain has no adverse effects on organ tissues.

[0090] Based on this, long-term intragastric administration of the strain aFMT-101 of the present invention has no adverse effects on the body weight, blood biochemical indicators and major organs of mice, and has good biosafety.

[0091] In summary, the present invention has isolated and screened a new type of Bifidobacterium longum subsp. longum strain aFMT-101 (Bifidobacterium longum subsp. longum) from the human intestine. The strain can maintain a high survival rate in a gastric acid and bile salt environment, and can quickly colonize in the intestine, promote the reproduction of beneficial bacteria, and inhibit the growth of harmful bacteria, thereby improving intestinal health and enhancing immunity, and has no adverse effects on the host and has good biosafety.

[0092] The above description is only a preferred embodiment of the present invention and is 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 protection scope of the present invention.

Claims

1. A novel Bifidobacterium longum, characterized in that: The strain is aFMT-101, classified and named Bifidobacterium longum subsp. longum, and deposited in Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 65179.

2. A microbial preparation containing the novel Bifidobacterium longum according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The microbial preparation is a bacterial suspension or a bacterial agent, and the number of live bacteria of Bifidobacterium longum aFMT-101 is not less than 1×10 9 CFU / g.

4. Use of the novel Bifidobacterium longum as claimed in claim 1 or the microbial preparation as claimed in any one of claims 2 to 3 in the preparation of products for improving intestinal health and regulating immune function.

5. A product containing the novel Bifidobacterium longum according to claim 1 or the microbial preparation according to any one of claims 2 to 3.

6. The product according to claim 5, characterized in that The product includes probiotics.

7. The product according to claim 6, characterized in that The dosage forms of the probiotics include capsules, powders, tablets or granules.

8. The product according to claim 5, characterized in that The products also include food and medicine.

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