A strain of Bifidobacterium longum VB234 capable of inhibiting carbohydrate absorption and its application

Abstract

The present invention belongs to the technical field of probiotics and their application, and specifically relates to a probiotic Bifidobacterium longum ( Bifidobacterium longum )VB234. The longum Bifidobacterium VB234 is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with the deposit number CGMCC NO.25841. The strain can inhibit the activity of α-glucosidase and a variety of intestinal pathogens, and has good tolerance to gastric acid and choline. It can be used to prepare products for controlling blood sugar and improving intestinal flora imbalance.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics and their application, and in particular to Bifidobacterium longum and its application in blood sugar lowering and antibacterial products. Background Art

[0002] Bifidobacterium ( Bifidobacterium ) is a Gram-positive, non-motile, rod-shaped, strictly anaerobic bacterium that is sometimes forked at one end. Bifidobacterium is a beneficial microorganism in the intestine that plays an important role in maintaining the health of the body. Bifidobacterium longum ( Bifidobacterium longum ) is the most common Bifidobacterium species found in the gut microbiota of infants and adults.

[0003] As a unique type of probiotic, Bifidobacterium longum has been widely used in food production, including fermented yogurt, fermented juice, synbiotic preparations, microecological preparations, etc. In addition, Bifidobacterium longum also plays an important role in medical care, with the effects of preventing and treating constipation, regulating intestinal balance, lowering cholesterol, promoting digestion and absorption of nutrients, delaying aging and enhancing the body's immune activity. The diversity of sources of Bifidobacterium longum leads to its genetic diversity and functional diversity. However, there are still few studies on the isolation and identification of Bifidobacterium longum and its probiotic properties, which to a certain extent also affects the development and utilization of Bifidobacterium longum. Currently, there are very few types of Bifidobacterium longum put into commercial production.

[0004] The incidence of diabetes has increased dramatically around the world. Most T2DM patients in China have elevated postprandial blood glucose (PPG). Elevated PPG is directly related to the development of diabetic complications. Controlling PPG is an important strategy to control the occurrence and development of diabetic complications. α-glucosidase inhibitors inhibit α-glucosidase on the intestinal mucosa, slow down the rate at which starch breaks down into glucose, and reduce the absorption of glucose in the small intestine, which has a very obvious control effect on PPG. Currently, the commonly used α-glucosidase inhibitors in clinical practice are mainly acarbose and voglibose. This type of drug often causes gastrointestinal adverse reactions, such as abdominal discomfort, gastrointestinal flatulence, increased flatulence, and diarrhea.

[0005] Gastrointestinal dysfunction is one of the most common complications of diabetes. Relevant survey data show that 75% of diabetic patients have obvious gastrointestinal symptoms, mainly vomiting, constipation, diarrhea, and fecal incontinence, among which diarrhea is the most common. Diabetic diarrhea not only has a serious impact on the quality of life of patients, but also has a significant impact on the control of diabetes. At present, the pathogenesis of diabetic diarrhea has not been fully clarified, but many studies have shown that intestinal flora imbalance plays an important role in the onset of diabetic diarrhea.

[0006] The present invention provides a Bifidobacterium longum which can effectively inhibit the activity of alpha-glucosidase and gastrointestinal pathogens. The Bifidobacterium longum can inhibit the activity of alpha-glucosidase and the colonization and reproduction of intestinal pathogens by utilizing the characteristics of intestinal colonization and strict anaerobicity. The Bifidobacterium longum has good tolerance to gastric acid and choline and can be used to prepare products for controlling blood sugar and improving diarrhea, and is particularly suitable for preparing diabetic diarrhea drugs. Summary of the invention

[0007] The present invention provides a Bifidobacterium longum ( 双歧杆菌属 长双歧杆菌 ) VB234, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 28, 2022, with the deposit number CGMCC NO.25841. The strain obtained by isolating from adult feces has higher safety and is more suitable for the production of health products.

[0008] Furthermore, the 16S rDNA sequence of the Bifidobacterium longum VB234 is shown in SEQ ID NO:1.

[0009] The present invention also provides the use of Bifidobacterium longum VB234 in the preparation of alpha-glucosidase inhibitors.

[0010] Studies have shown that Bifidobacterium longum VB234 can effectively inhibit the activity of α-glucosidase, which is related to type 2 diabetes. Inhibiting α-glucosidase is one of the methods to control postprandial hyperglycemia, suggesting that Bifidobacterium longum VB234 is expected to be used in areas such as lowering blood sugar and inhibiting obesity.

[0011] The present invention also provides the use of Bifidobacterium longum VB234 in preparing an intestinal pathogen antibacterial agent.

[0012] Studies have found that Bifidobacterium longum VB234 can effectively inhibit intestinal pathogens, especially Staphylococcus aureus, Escherichia coli and Clostridium perfringens, suggesting that Bifidobacterium longum VB234 is expected to be used for symptoms such as diarrhea, bloating, and indigestion caused by intestinal flora imbalance.

[0013] The present invention also provides application of Bifidobacterium longum VB234 in preparing a medicine for diabetic diarrhea.

[0014] Considering the inhibitory activity of α-glucosidase and the inhibitory activity of intestinal pathogens, Bifidobacterium longum VB234 is particularly suitable for the preparation of diabetic diarrhea drugs.

[0015] At the same time, Bifidobacterium longum VB234 has good tolerance to gastric acid and choline.

[0016] The present invention provides a bacterial agent with probiotic function, wherein the bacterial agent comprises the Bifidobacterium longum VB234 described above.

[0017] Furthermore, the bacterial agent is a fermentation product of Bifidobacterium longum VB234 described above.

[0018] Beneficial effects:

[0019] The Bifidobacterium longum VB234 provided by the present invention has a strong inhibitory effect on α-glucosidase and can inhibit a variety of intestinal

[0020] It has good tolerance to gastric acid and choline and can be used to prepare products for controlling blood sugar and improving intestinal flora imbalance, especially for preparing drugs for diabetic diarrhea. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The results of the inhibition test of α-glucosidase activity by acarbose, blank culture medium, Bifidobacterium infantis and Bifidobacterium longum VB234 provided by the present invention are shown. DETAILED DESCRIPTION

[0022] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0023] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0024] The MAGEN bacterial genomic DNA extraction kit used was purchased from Shanghai Maigen Biotechnology Co., Ltd. Phusion high-fidelity DNA polymerase was purchased from Thermo Scientific.

[0025] The present invention will be further described below by way of examples, which are not intended to further limit the present invention. It should be understood by those skilled in the art that equivalent replacements or corresponding improvements made to the present invention still fall within the scope of protection of the present invention.

[0026] In the culture medium used in the following examples, the percentage (%) used in the material ratio is the mass percentage unless otherwise specified.

[0027] Example 1 Source of bacteria

[0028] Bifidobacterium longum ( Bifidobacterium longumVB234 was isolated from adult feces. Take fresh adult feces and add sterile PBS buffer in a sterile container. After sufficient shaking, dilute and spread on MRS solid culture medium plates. After 48 hours of culture, pick single colonies of different sizes and shapes and streak on fresh plates. Then pick single colonies to the slant. After the slant is cultured, the colony morphology is examined under a microscope. After no other bacteria are found under the microscope, the identification is carried out. Using adult feces as the isolation source, the obtained strain has higher safety and is more suitable for the production of health products.

[0029] Example 2: Bacterial species identification

[0030] The experiment was conducted according to the relevant contents in the Molecular Cloning Experiment Guide. After the purified strains were cultured in MRS medium for 1 day, 1 mL of bacterial solution was centrifuged at 10,000 rpm for 1 min, the supernatant was discarded, the bacteria were collected, and DNA was extracted according to the operation steps of the MAGEN Bacterial Genomic DNA Extraction Kit. The extracted DNA was used as a template, and 16S rDNA was amplified by PCR (polymerase chain reaction) using universal primers 337F (SEQ ID NO: 2, 5′-GACTCCTACGGGAGGCWGCAG-3′) and 1492R (SEQ ID NO: 3, 5′-TACGGCTACCTTGTTACGACTT-3′). The PCR reaction system was 50 μL, and the reaction system and program were configured according to the instructions of Phusion high-fidelity DNA polymerase, and the annealing temperature was 55°C. The PCR product was sent to Shanghai Bioengineering for sequencing.

[0031] The 16S rDNA sequence of the strain was collated and then compared with the sequences of related species and genera in the GenBank database by BLAST to determine the taxonomic status of the strain. 双歧杆菌属 长双歧杆菌猪亚种 strain 2909 has a homology of 99.82% with Bifidobacterium longum 猪亚种 strain2705 had a homology of up to 99.91%, so the strain was identified as Bifidobacterium longum ( Bifidobacterium longum ) strain. The strain is internally numbered VB234 and has been deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration on September 28, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.25841. The 16S rDNA sequence of Bifidobacterium longum VB234 is shown in SEQ ID NO: 1.

[0032] Example 3: Bacteria fermentation

[0033] 1. Bacteria activation

[0034] Take the VB234 original strain glycerol tube, thaw it at room temperature, draw 0.1ml of bacterial suspension and inoculate it onto the MRS solid plate, spread it evenly, put the plate into an anaerobic bag, and place it in a 37℃ incubator for 48h to obtain a well-activated single colony. 2. Seed preparation Take the activated single colony, scrape a ring of colonies with an inoculation loop, and then inoculate it into a 500ml triangular flask containing 100ml MRS liquid culture medium, wrap it and put it into an anaerobic bag, shake and culture on a shaker at 37℃ and 70rpm for 48h, and control the OD value of the seed liquid to be ≥2.0. Before disinfecting the MRS liquid culture medium, adjust the pH to 7.0±0.2, and the disinfection conditions are 121~123℃ and 30min. 3. Fermentation: Prepare the cultured seed liquid and inoculate it into a 500ml Erlenmeyer flask containing 100ml MRS liquid medium at a ratio of 3-5% (V / W). Wrap it and put it into an anaerobic bag. Incubate it on a shaker at 37℃ and 70rpm for 24-48h. Control the OD value of the fermentation liquid to be ≥3.0. Adjust the pH of the MRS liquid medium to 7.0±0.2 before disinfection. The disinfection conditions are 121~123℃ and 30min.

[0035] MRS liquid culture medium: yeast extract 0.5%, peptone 1.0%, beef extract 1.0%, glucose 2.0%, dipotassium hydrogen phosphate 0.2%, diammonium hydrogen citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.02%, manganese sulfate 0.005% (all in mass percentage), Tween 80 0.1% (volume / mass ratio).

[0036] MRS solid medium: Add 1.5% agar powder to MRS liquid medium.

[0037] Example 4: Detection of the inhibitory ability of α-glucosidase activity

[0038] The activated VB234 and Bifidobacterium longum subsp. infantis (purchased from the General Microbiology Center of the China Microbiological Culture Collection Administration, with the culture collection number of CGMCC NO.1.2202) were inoculated into MRS liquid medium and cultured at 37°C for 48h. The culture medium was centrifuged to obtain the fermentation supernatant. The reaction system for the α-glucosidase activity experiment was 100 μL. First, 25 μL of the substrate 4-nitrophenol-α-D-pyranoglucoside (PNPG, 10mmol / L, Solebao) and 25 μL of the sample were mixed and incubated at 37°C for 10min. Then 50 μL of α-glucosidase (40U / L, Sigma) was added and mixed and reacted at 37°C for 30min. Subsequently, 100 μL of Na2 CO3 (0.1mol / L) was quickly added to terminate the reaction, and the absorbance at 405 nm was measured with an enzyme reader. Three parallel experiments were set up for each group of experiments. The absorbance of the sample was calibrated with the absorbance of the sample blank control (50 μL PBS was used to replace α-glucosidase in the system). The negative control (no sample that inhibits α-glucosidase in the system) was replaced with 25 μL PBS, and the negative blank control (no α-glucosidase activity in the system) was replaced with 25 μL PBS and 50 μL PBS was used to replace α-glucosidase.

[0039] The calculation formula of α-glucosidase inhibition rate is:

[0040] α-glucosidase inhibition rate = (1-(AB) / (CD)) × 100%

[0041] Wherein: A is the absorbance of the sample at 405 nm; B is the absorbance of the sample blank control at 405 nm; C is the absorbance of the negative control at 405 nm; D is the absorbance of the negative blank control at 405 nm.

[0042] See attached for the results Figure 1 The inhibition rates of 10 µM acarbose on α-glucosidase were 82.7% (MRS), 59.0% (artificial intestinal fluid), and 88.3% (artificial intestinal fluid + starch); the inhibition rates of blank medium were 0.7% (MRS), 2.0% (artificial intestinal fluid), and 1.0% (artificial intestinal fluid + starch); the inhibition rates of Bifidobacterium longum subsp. infantis were 41.3% (MRS), 7.9% (artificial intestinal fluid), and 19.0% (artificial intestinal fluid + starch); the inhibition rates of Bifidobacterium longum VB234 were 64.2% (MRS), 74.3% (artificial intestinal fluid), and 56.2% (artificial intestinal fluid + starch). The results showed that under the conditions of simulated artificial intestinal fluid, including with and without the addition of starch, the fermentation supernatant of Bifidobacterium longum VB234 had a strong inhibitory effect on α-glucosidase, and was significantly higher than that of Bifidobacterium longum subsp. infantis.

[0043] Example 5 Antagonistic Pathogen Ability Test

[0044] The antibacterial ability of the strains was evaluated by double-layer plate culture method.

[0045] Preparation of the lower layer probiotics: After thawing the activated frozen tubes of different single bacteria, spot them on the MRS solid plate, put them in an anaerobic bag, and culture them anaerobically at 37°C for 48 hours. The inoculation amount of the bacteria is 2ul / spot.

[0046] Preparation of the upper pathogenic bacteria layer: The high temperature sterilized upper culture medium is cooled to 40-50°C, and different pathogens are added as required (the final concentration of pathogens in the culture medium is about 10 6 / ml) and mix well. Add 7ml / dish of the medium containing pathogenic bacteria to the above-mentioned probiotic single bacterial plates that have grown. After solidification, culture them according to the growth conditions of different pathogenic bacteria (Table 1). After the upper plate is grown, observe whether there is an antibacterial circle around the probiotics to determine whether it has antibacterial ability. All pathogenic bacteria strains were purchased from Qingdao Haibo Biological.

[0047] Table 1 Types of pathogenic strains and culture conditions

[0048]

[0049] As shown in Table 2, Bifidobacterium longum VB234 can inhibit all 7 common pathogens included in Table 2 and has the ability to antagonize pathogens.

[0050] Table 2 Antibacterial effect of Bifidobacterium longum VB234

[0051]

[0052] Example 6 Acid and bile salt resistance

[0053] Take 1 mL of 24h bacterial solution, centrifuge and remove the supernatant, add artificial gastric juice (Coolaber, pH3.0) or 0.1% ox bile salt (Coolaber, PBS) to resuspend, and treat at 37℃ for 2h. Gradient dilution and colony count on the plate to calculate the survival rate of the tested bacteria. The results showed that the survival rate of Bifidobacterium longum VB234 in artificial gastric juice with pH3.0 was 98.4% for 2h and 95.2% for 4h; the survival rate of Bifidobacterium longum VB234 in 0.1% ox bile salt was 97.7% for 2h and 50.6% for 4h, which showed strong acid and bile salt resistance.

[0054] Table 3 Acid and bile resistance of Bifidobacterium longum VB234

[0055]

[0056] 16S sequencing sequence

[0057]

Claims

1. A Bifidobacterium longum ( Bifidobacterium longum )VB234, characterized in that The Bifidobacterium longum VB234 is deposited in the General Microbiology Center of the China Microorganism Culture Collection Administration, with a deposit number of CGMCCNO.25841 and a deposit date of September 28, 2022. The 16S rDNA sequence of the Bifidobacterium longum VB234 is shown in SEQ ID NO:

1.

2. Use of the Bifidobacterium longum VB234 according to claim 1 in the preparation of α-glucosidase inhibitors.

3. Use of the Bifidobacterium longum VB234 according to claim 1 in the preparation of an intestinal pathogen antibacterial agent, wherein the intestinal pathogen is Staphylococcus aureus, Escherichia coli or Clostridium perfringens.

4. Use of the Bifidobacterium longum VB234 described in claim 1 in the preparation of a drug for treating diabetic diarrhea.

5. A bacterial agent with probiotic function, characterized in that: The bacterial agent includes the Bifidobacterium longum VB234 described in claim 1.

6. The bacterial agent according to claim 5, characterized in that The bacterial agent comprises the fermentation supernatant of Bifidobacterium longum VB234 described in claim 1.

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