Bifidobacterium longum subsp. Longum aFMT-102 strain as well as microbial preparation, product and application thereof

Abstract

The invention provides a bifidobacterium longum subsp. Longum aFMT-102 strain, which is classified and named as Bifidobacterium longum subsp. Longum, is preserved in Guangdong Microbial Culture Collection Center, and has a preservation number of GDMCC No: 65180. The invention also provides a preparation method of the bifidobacterium longum subsp. Longum aFMT-102 strain. The invention further provides a microbial preparation containing the aFMT-102 strain and application of the aFMT-102 strain and the microbial preparation in preparation of products for relieving intestinal tracts and relieving stress. Meanwhile, the invention also provides a product containing the strain aFMT-102 or a corresponding microbial preparation. The strain aFMT-102 is separated from excrement of healthy adults, has good acid resistance and bile salt resistance and relatively strong intestinal colonization ability, and has a remarkable effect in the aspects of relieving intestinal tracts and relieving pressure.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and in particular to a Bifidobacterium longum subspecies longum aFMT-102 strain and a microbial preparation, product and application thereof. Background Art

[0002] In recent years, the bidirectional regulatory effect of intestinal health and mental health has received increasing attention. Many studies have shown that intestinal microbiota plays an important role in regulating emotions, relieving stress, and maintaining mental health. With the accelerated pace of life, stress-related health problems (such as irritable bowel syndrome, anxiety, etc.) in modern society are gradually increasing, and the demand for probiotics that can soothe the intestines and relieve stress is increasing.

[0003] Bifidobacterium longum ssp. Longum is a strain that has been shown to have potential benefits for intestinal health and psychological state. However, traditional Bifidobacterium strains have limited survival in the intestines, especially after passing through the harsh environment of gastric acid and bile salts, and the colonization effect is not ideal, which affects its effect of soothing the intestines and relieving stress.

[0004] Therefore, there is an urgent need to obtain a new strain of Bifidobacterium longum subspecies 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 Bifidobacterium longum subspecies long aFMT-102 strain and its microbial preparation, product and application. The aFMT-102 strain is isolated from the feces of healthy adults, has good acid resistance, bile salt resistance, and strong intestinal colonization ability, and has a significant effect on soothing the intestines and relieving stress.

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

[0007] A Bifidobacterium longum subsp. longum strain aFMT-102 was isolated from the feces of healthy adults and classified as Bifidobacterium longum subsp. longum. It was deposited in the Guangdong Provincial Microbiological Culture Collection on September 23, 2024 and showed survival. The deposit number is GDMCC No: 65180. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0008] As one of the preferred embodiments of the present invention, the full-length sequence of the 16S rDNA gene of the aFMT-102 strain is shown as SEQ ID NO.1, and the target sequence for subsequent amplification is shown as SEQ ID NO.2.

[0009] A microbial preparation containing the above-mentioned Bifidobacterium longum subspecies longum aFMT-102 strain.

[0010] As one of the preferred embodiments of the present invention, the microbial preparation is a bacterial suspension or a bacterial preparation of the aFMT-102 strain.

[0011] As one of the preferred embodiments of the present invention, the number of viable bacteria of the aFMT-102 strain contained in the microbial preparation is not less than 1×10 9 CFU / g.

[0012] An application of the above-mentioned Bifidobacterium longum subspecies longum aFMT-102 strain or microbial preparation in the preparation of products for soothing the intestines and relieving stress.

[0013] A product containing the above-mentioned Bifidobacterium longum subspecies longum aFMT-102 strain or microbial preparation.

[0014] As one of the preferred embodiments of the present invention, the product comprises probiotics.

[0015] As one of the preferred embodiments of the present invention, probiotics are prepared by combining the aFMT-102 strain or the corresponding microbial preparation with other prebiotics (such as oligofructose).

[0016] As one of the preferred embodiments of the present invention, the dosage form of the probiotics includes capsules, powders, tablets or granules to ensure their effective release in the intestine and promote the balanced growth of intestinal flora.

[0017] As one of the preferred embodiments of the present invention, the product also includes food and medicine.

[0018] The advantages of the present invention compared to the prior art are:

[0019] (1) Significantly improved acid resistance and bile salt resistance: The aFMT-102 strain of the present invention can maintain a high survival rate in a gastric acid and bile salt environment, ensuring that it can reach the intestine smoothly;

[0020] (2) High intestinal colonization efficiency: The aFMT-102 strain of the present invention can quickly colonize in the intestine, effectively promote the reproduction of beneficial bacteria and inhibit the growth of harmful bacteria;

[0021] (3) Soothing the intestines and relieving stress: The aFMT-102 strain of the present invention significantly relieves stress-related behavioral manifestations and improves mental health by improving the balance of intestinal flora;

[0022] (4) Good biosafety: The aFMT-102 strain of the present invention has no adverse effects on the host during long-term use and has good biosafety;

[0023] (5) Wide application: The aFMT-102 strain of the present invention can be widely used in the fields of food, medicine, etc., especially in preparations for promoting intestinal health and relieving stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the MOLDI-TOF chromatogram of strain aFMT-102 in Example 1;

[0025] Figure 2 is a morphological characteristic diagram of strain aFMT-102 in Example 2;

[0026] Figure 3 is a graph showing the survival status of strain aFMT-102 in simulated gastric fluid (pH 2.0) in Example 3;

[0027] Figure 4 is a graph showing the survival status of strain aFMT-102 in Example 3 in 0.3% bile salt;

[0028] Figure 5 is the open field test result in Example 5;

[0029] Figure 6 is the forced swimming test and physiological stress test results in Example 5;

[0030] Figure 7 is the detection result of inflammatory cytokines (TNF-α and IL-6) in Example 6;

[0031] Figure 8 is the detection result of short-chain fatty acids (acetic acid, propionic acid and butyric acid) in Example 6;

[0032] Fig. 9 This is the detection result of neurotransmitters (5-hydroxytryptamine and norepinephrine) in Example 6. DETAILED DESCRIPTION

[0033] 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.

[0034] The culture medium formula involved in the following examples is as follows:

[0035] 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.

[0036] Modified MRS medium: MRS medium + 0.05% L-cysteine.

[0037] 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.

[0038] Modified MRS agar medium: MRS agar medium + 0.05% L-cysteine.

[0039] Example 1: Screening and Isolation of aFMT-102 Strain

[0040] A new strain isolated from the feces of a healthy adult was initially identified and sequenced with 16S rDNA gene (SEQ ID NO.1), MOLDI-TOF chromatographic information ( Figure 1), and confirmed it to be Bifidobacterium longum ssp. Longum.

[0041] Based on the identification results, the strain was named Bifidobacterium longum subspecies longum aFMT-102 strain, and was preserved in the Guangdong Provincial Microbiological Culture Collection Center on September 23, 2024 and showed survival, with the preservation number GDMCC No: 65180, and the preservation address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0042] Example 2: Colony morphology of strain aFMT-102

[0043] The strain aFMT-102 was inoculated on a modified MRS agar medium (MRS agar medium + 0.05% L-cysteine) plate, cultured inverted at 37°C for 2 days, and its colony morphology was observed.

[0044] The colonies of strain aFMT-102 on the plate are white, round, smooth, opaque, and convex in the middle. Figure 2 shown.

[0045] Example 3: Acid and bile resistance test of strain aFMT-102

[0046] 1. Experimental Methods

[0047] (1) Strain culture

[0048] The strain aFMT-102 was inoculated into a modified MRS medium (MRS+0.05% L-cysteine), cultured at 37°C under anaerobic conditions (90% nitrogen, 5% carbon dioxide, 5% hydrogen) until the logarithmic growth phase, and set aside.

[0049] (2) Acid resistance test

[0050] 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.

[0051] ② Preparation of bacterial suspension: The aFMT-102 strain 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 to obtain a bacterial suspension.

[0052] ③ Acid-resistant treatment: transfer the bacterial suspension into simulated gastric fluid (pH 2.0) and incubate at 37°C for 2 h.

[0053] 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, culture it anaerobically at 37°C for 48h, and count the colony forming units (CFU) to determine the survival rate.

[0054] (3) Bile salt resistance test

[0055] ① Preparation of bile salt medium: Add 0.3% bile salts to the modified MRS medium (MRS medium + 0.05% L-cysteine ​​+ 0.3% bile salts).

[0056] ② Preparation of bacterial suspension: The strain cultured to the logarithmic growth phase was washed twice with sterile saline (5000 rpm, centrifugation for 10 min), and then resuspended in a modified MRS medium containing 0.3% bile salts (MRS medium + 0.05% L-cysteine ​​+ 0.3% bile salts) to obtain a bacterial suspension.

[0057] ③ Bile salt resistance treatment: transfer the bacterial suspension into bile salt culture medium and incubate at 37°C for 4 hours.

[0058] ④ Survival test: After the treatment, dilution and plate spreading were performed in the same way, and the colony forming units (CFU) were counted to determine the survival rate.

[0059] 2. Experimental Results

[0060] The strain aFMT-102 of the present invention was cultured in simulated gastric fluid (pH 2.0) for 2 h, and the survival status was as follows: Figure 3 As shown, the survival rate was 93%; after culturing in a medium containing 0.3% bile salt for 4 hours, the survival status was as follows Figure 4 As shown, the survival rate was 90%.

[0061] The above results show that the strain aFMT-102 of the present invention has good acid and bile salt resistance and can maintain a high survival rate under the harsh conditions of gastric acid and bile salt.

[0062] Example 4: Intestinal colonization ability test of strain aFMT-102

[0063] 1. Experimental Methods

[0064] 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-102 of the present invention every day; the high-dose group: gavage 1×10^9CFU of the strain aFMT-102 of the present invention every day. Mouse fecal samples were collected on the 3rd, 7th, 14th and 28th days, diluted and homogenized with sterile saline, and appropriate samples were taken for DNA extraction. The number of strains in the mouse intestine was detected by fluorescent quantitative PCR to evaluate its colonization ability.

[0065] Specific steps:

[0066] (1) Oral gavage of bacterial strains: Use an oral gavage needle to administer the corresponding dose of bacterial strain suspension every day to ensure that each mouse consumes a sufficient amount of probiotics.

[0067] (2) Sample collection: Fecal samples were collected at designated time points, immediately placed in sterile centrifuge tubes and stored at −20°C until further analysis.

[0068] (3) DNA extraction: Use a stool DNA extraction kit (MolPure Stool DNA Kit (Stool DNA Extraction Kit, Yisheng Bio) extracts total DNA from stool samples to ensure that the probiotic DNA in the sample can be successfully extracted.

[0069] (4) Fluorescence quantitative PCR: A specific primer pair (SEQ ID NO.3, SEQ ID NO.4) was used to amplify the V1-V3 fragment (SEQ ID NO.2) of the 16S rDNA gene of the target strain to evaluate the colonization level of the strain in the mouse intestine.

[0070] 2. Experimental Results

[0071] The statistical results are shown in Table 1.

[0072] Table 1 Results of intestinal colonization ability test

[0073] Colonization rate Control group Low dose group High dose group Day 3 20％ 40％ 60％ Day 7 35％ 50％ 65％ Day 14 40％ 55％ 75％ Day 28 45％ 60％ 80％

[0074] From the results in Table 1, it can be seen that the strain aFMT-102 of the present invention can effectively colonize in the mouse intestine, and the colonization effect of the strain in the high-dose group in the mouse intestine is significantly better than that in the control group and the low-dose group, especially on the 28th day, the colonization rate reached 80%.

[0075] Example 5: Test of stress relief effect of strain aFMT-102

[0076] 1. Experimental Methods

[0077] 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 was given an equal amount of normal saline every day without strain treatment; the low-dose group was gavaged with 1×10^8 CFU of strain aFMT-102 every day; the high-dose group was gavaged with 1×10^9 CFU of strain aFMT-102 every day. The experiment lasted for 4 weeks, and the anxiety and depression behaviors of the mice were evaluated by open field test (OFT), forced swimming test (FST) and physiological stress test.

[0078] (1) Open Field Test (OFT)

[0079] Once a week, the anxiety level of mice was assessed and the time they spent in the central area of ​​the open field and their total distance traveled were recorded.

[0080] (2) Forced swim test (FST)

[0081] A 6-min test was performed on day 28 to record the immobility time of the mice to assess depressive-like behavior.

[0082] (3) Physiological stress detection

[0083] During the experiment, the following stressor types were set:

[0084] Restraint stress: Mice were confined in a narrow plastic tube for 1 hour.

[0085] Wet-bed stress: The mouse cage bedding was soaked with water for 24 hours.

[0086] Empty cage starvation: Place mice in an empty cage and remove all food for 12 h.

[0087] Noise stress: Random high-frequency noise is played continuously for 6 hours.

[0088] Circadian reversal: prolonging or shortening the light hours to disrupt the circadian rhythm.

[0089] Cold water foot immersion: Immerse the mouse's front feet in 4°C cold water for 30 minutes.

[0090] At the end of the experiment, serum samples were collected from mice and the levels of corticosterone (CORT) were measured using enzyme-linked immunosorbent assay (ELISA) as a quantitative indicator of stress response.

[0091] 2. Experimental Results

[0092] (1) Open Field Test (OFT) results are as follows Figure 5As shown: In the first week, the average stay time of mice in the control group in the central area was 25 seconds, 30 seconds in the low-dose group, and 45 seconds in the high-dose group; in the second week, the stay time of mice in the high-dose group in the central area increased significantly to 60 seconds, while that in the low-dose group was 40 seconds; in the fourth week, the control group remained at 30 seconds, the low-dose group increased to 45 seconds, and the high-dose group increased to 80 seconds, indicating that the anxiety level of the high-dose group was significantly reduced.

[0093] (2) Results of the forced swim test (FST) and physiological stress test Figure 6 As shown: ① Regarding the immobility time, the average of the control group was 180 seconds, which was reduced to 150 seconds in the low-dose group and significantly reduced to 120 seconds in the high-dose group, showing an obvious antidepressant effect; ② Regarding the serum corticosterone level, the corticosterone level of the control group was 350 ng / mL; the low-dose group was reduced to 280 ng / mL; and the high-dose group was significantly reduced to 200 ng / mL, indicating that the stress response of mice in the high-dose group was significantly inhibited.

[0094] Example 6: Intestinal stress relief test of strain aFMT-102

[0095] 1. Experimental Methods

[0096] 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 same amount of normal saline was given every day without strain treatment; the low-dose group: 1×10^8CFU of strain aFMT-102 was gavaged every day; the high-dose group: 1×10^9CFU of strain aFMT-102 was gavaged every day. The experiment lasted for 4 weeks. At the end of the experiment, serum, feces, and brain tissue samples were collected from the mice to analyze physiological markers related to the gut-brain axis.

[0097] (1) Collection and detection of serum samples: Blood was collected from the eye sockets of mice to obtain serum for the detection of inflammatory cytokines. Then, the concentrations of inflammatory cytokines (TNF-α and IL-6) were detected using enzyme-linked immunosorbent assay (ELISA).

[0098] (2) Collection and testing of fecal samples: Feces were collected from mice, and the concentrations of acetic acid, propionic acid, and butyric acid in the feces were measured using a short-chain fatty acid (SCFA) analysis kit.

[0099] (3) Collection and detection of brain tissue samples: Mouse brain tissue was obtained and the content of neurotransmitters (such as 5-hydroxytryptamine and norepinephrine) was analyzed by high performance liquid chromatography (HPLC).

[0100] 2. Experimental Results

[0101] (1) Inflammatory cytokines (TNF-α and IL-6) Figure 7As shown: the TNF-α concentration in the control group was 150pg / mL, and the IL-6 concentration was 120pg / mL; in the low-dose group, TNF-α was reduced to 120pg / mL, and IL-6 was reduced to 100pg / mL; in the high-dose group, TNF-α was significantly reduced to 90pg / mL, and IL-6 was reduced to 80pg / mL, indicating that the level of inflammation was significantly reduced.

[0102] (2) Short-chain fatty acids (SCFAs) Figure 8 As shown: ① Acetic acid, the control group was 3.5mmol / g, the low-dose group increased to 4.0mmol / g, and the high-dose group significantly increased to 5.5mmol / g; ② Propionic acid, the control group was 1.2mmol / g, the low-dose group was 1.5mmol / g, and the high-dose group was 2.0mmol / g; ③ Butyric acid, the control group was 0.8mmol / g, the low-dose group increased to 1.0mmol / g, and the high-dose group increased to 1.5mmol / g. The SCFA level in the high-dose group increased significantly, indicating that probiotics have a positive regulatory effect on intestinal metabolic activity.

[0103] (3) Neurotransmitters (5-hydroxytryptamine and norepinephrine) Fig. 9 As shown: ① 5-hydroxytryptamine (5-HT): the content of the control group was 250ng / g brain tissue, the low-dose group increased to 300ng / g, and the high-dose group significantly increased to 400ng / g, indicating that probiotics can increase 5-HT levels and improve depressive symptoms; ② Norepinephrine (NE): the content of the control group was 100ng / g brain tissue, the low-dose group increased to 120ng / g, and the high-dose group significantly increased to 150ng / g, indicating that probiotics can improve stress-related neurotransmitter changes.

[0104] In summary, the present invention is a novel long subspecies of Bifidobacterium longum strain aFMT-102 (Bifidobacterium longum ssp. Longum) isolated from healthy adult feces; the strain can maintain a high survival rate in a gastric acid and bile salt environment, and can quickly colonize in the intestine, improve the balance of intestinal flora, relieve stress, improve mental health, and have no adverse effects on the host, and has good biosafety. The strain aFMT-102 of the present invention can be prepared into a probiotic (a combination of strain and oligofructose) or food or medicine for application.

[0105] 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 strain of Bifidobacterium longum subspecies longum aFMT-102, characterized in that The strain was classified and named Bifidobacterium longum subsp. longum and deposited in Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 65180.

2. The Bifidobacterium longum subspecies longum aFMT-102 strain according to claim 1, characterized in that The full-length sequence of the 16S rDNA gene of the aFMT-102 strain is shown in SEQ ID NO.

1.

3. A microbial preparation containing the Bifidobacterium longum subspecies longum aFMT-102 strain according to claim 1 or 2.

4. The microbial preparation according to claim 3, characterized in that The microbial preparation is a bacterial suspension or a bacterial agent of the aFMT-102 strain.

5. The microbial preparation according to claim 4, characterized in that The number of viable bacteria of the aFMT-102 strain contained in the microbial preparation is not less than 1×10 9 CFU / g.

6. Use of the Bifidobacterium longum subspecies longum aFMT-102 strain as claimed in any one of claims 1 to 2 or the microbial preparation as claimed in any one of claims 3 to 5 in the preparation of products for soothing the intestines and relieving stress.

7. A product containing the Bifidobacterium longum subspecies longum aFMT-102 strain according to any one of claims 1 to 2 or the microbial preparation according to any one of claims 3 to 5.

8. The product according to claim 7, characterized in that The product includes probiotics.

9. The product according to claim 8, characterized in that The probiotics are prepared by combining the aFMT-102 strain or the corresponding microbial preparation with oligofructose.

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

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