Preparation and application of bifidobacterium adolescentis B-201 and metagen of active substances thereof

Abstract

The invention provides bifidobacterium adolescentis B-201, a metagen containing the bifidobacterium adolescentis B-201 and rich in active substances and application, and belongs to the technical field of probiotics. The bifidobacterium adolescentis B-201 is separated from healthy breast milk, and is preserved in the China General Microbiological Culture Collection Center (CGMCC), and the preservation number is CGMCC No.31756. The bifidobacterium adolescentis B-201 metabolite is rich in functional active substances such as amino acid and metabolite thereof, benzene and derivative thereof, organic acid alcohol, flavone, hormone and hormone related substances, alkaloid, organic acid and derivative thereof, heterocyclic compound and fatty acyl thereof, has broad-spectrum antibacterial activity, and can effectively inhibit pathogenic bacteria and food-borne putrefying bacteria. The invention provides a brand new thought for deep research and utilization of the probiotic postbiotics with functional active substances, and has great research and application values.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics, and particularly relates to Bifidobacterium adolescentis B-201 and a postbiotic rich in active substances containing the same and an application thereof. Background Art

[0002] Postbiotics are a general term for the metabolites of probiotics after probiotics have been processed, including bacteria and metabolites. The International Scientific Association of Probiotics and Prebiotics (ISAPP) defines postbiotics as "preparations of inanimate microorganisms and / or their components that are beneficial to the health of the host." Killed probiotics are a type of postbiotics and have gradually attracted attention in research and application in recent years. Unlike traditional probiotics, although killed probiotics have lost their ability to reproduce, they retain their cell wall components, proteins, metabolites and other functional substances, and can still provide a variety of health benefits, such as immune regulation, anti-inflammatory effects, and improved intestinal barrier function. They have a significant improvement effect on irritable bowel syndrome, allergic diseases and periodontitis, and at the same time have higher safety, and have broad development and application prospects in the food and pharmaceutical fields.

[0003] As an important probiotic in the human intestine, Bifidobacterium has many beneficial effects, such as maintaining the balance of intestinal flora, enhancing the intestinal barrier and immune system function, promoting nutrient absorption and metabolism, and improving digestive function. Therefore, the preparation, evaluation and development of active metabolites of Bifidobacterium have positive significance for regulating human immunity and improving intestinal function. Summary of the invention

[0004] In view of this, the purpose of the present invention is to improve a strain of Bifidobacterium adolescentis and its application. Based on the research on the preparation of Bifidobacterium adolescentis B-201 and its postbiotic bioactive substances, the present invention develops Bifidobacterium adolescentis B-201 postbiotics rich in active ingredients and its application in improving intestinal diseases.

[0005] The present invention provides a bacterial agent of Bifidobacterium adolescentis B-201 or an inactivated product of Bifidobacterium adolescentis B-201 or a fermented product containing Bifidobacterium adolescentis B-201 and / or the inactivated product of Bifidobacterium adolescentis B-201.

[0006] Furthermore, a use of the above-mentioned bacterial agent in producing functional active substances and / or preparing intestinal regulating preparations is provided.

[0007] The Bifidobacterium adolescentis B-201 is deposited in the General Microbiological Culture Collection Center of China Microorganism Culture Collection Administration, and the deposit number is: CGMCC No.31756.

[0008] The functionally active substances include one or more of amino acids and their metabolites, benzene and its derivatives, organic acid alcohols, flavonoids, hormones and hormone-related substances, alkaloids, organic acids and their derivatives, heterocyclic compounds and their fatty acyl compounds.

[0009] Preferably, the fermentation product of Bifidobacterium adolescentis B-201 is obtained by fermentation culture of Bifidobacterium adolescentis B-201.

[0010] Preferably, the fermentation product of Bifidobacterium adolescentis B-201 includes intracellular and extracellular products of Bifidobacterium adolescentis B-201.

[0011] Preferably, the antibacterial product includes one or more of a broad-spectrum antibacterial agent, a disinfectant, a food preservative and a preservative.

[0012] Preferably, the bacteriostatic objects of the bacteriostatic or bacteriostatic products include pathogenic bacteria and / or food-borne spoilage bacteria.

[0013] Preferably, the Bifidobacterium adolescentis B-201 postbiotic has the effect of alleviating DSS-induced IBD disease symptoms and inflammatory response in rats.

[0014] The present invention provides the application of Bifidobacterium adolescentis B-201 or fermentation product of Bifidobacterium adolescentis B-201 or functional active ingredients produced by Bifidobacterium adolescentis B-201 or postbiotics of Bifidobacterium adolescentis B-201 in the field of improving intestinal diseases; the functional active substances include one or more of amino acids and metabolites thereof, benzene and its derivatives, organic acid alcohols, flavonoids, hormones and hormone-related substances, alkaloids, organic acids and their derivatives, heterocyclic compounds and their fatty acyl groups; the Bifidobacterium adolescentis B-201 of the present invention is preserved in the General Microbiological Culture Collection Center of the China Microbiological Culture Collection Administration Committee, and the preservation number is: CGMCC No.31756. Postbiotics of Bifidobacterium adolescentis B-201 have broad-spectrum antibacterial activity and can effectively inhibit pathogenic bacteria and food-borne spoilage bacteria. The present invention provides a new idea for the in-depth research and utilization of probiotics with functional active substances, and has great research and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. Among them:

[0016] Figure 1 This is the genome circle map of Bifidobacterium adolescentis B-201;

[0017] Figure 2 The effect of Bifidobacterium adolescentis B-201 postbiotics on body weight in IBD rats;

[0018] Figure 3 This is a schematic diagram of the results of the effect of Bifidobacterium adolescentis B-201 postbiotics on the disease index (DAI) of IBD rats;

[0019] Figure 4 This is a schematic diagram of the results of the effect of Bifidobacterium adolescentis B-201 postbiotics on serum IL-6 levels in IBD rats;

[0020] Figure 5 This is a schematic diagram of the results of the effect of Bifidobacterium adolescentis B-201 postbiotics on serum TNF-α levels in IBD rats;

[0021] Figure 6 This is a schematic diagram of the effects of Bifidobacterium adolescentis B-201 postbiotics on the pathological changes in colon tissue of IBD rats.

[0022] Biological Deposit Description

[0023] Bifidobacterium adolescentis B-201 was deposited in the General Microbiological Culture Collection Center of the China Microbiological Culture Collection Administration on August 23, 2024. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is: CGMCC No.31756. DETAILED DESCRIPTION

[0024] The present invention provides the use of Bifidobacterium adolescentis B-201 or a fermentation product of Bifidobacterium adolescentis B-201 or a fermentation product containing Bifidobacterium adolescentis B-201 and / or Bifidobacterium adolescentis B-201 in the production of functional active ingredients and Bifidobacterium adolescentis B-201 postbiotics for improving intestinal diseases; the functional active substances include one or more of amino acids and metabolites thereof, benzene and derivatives thereof, organic acid alcohols, flavonoids, hormones and hormone-related substances, alkaloids, organic acids and derivatives thereof, heterocyclic compounds and fatty acyl compounds thereof, and the like; the Bifidobacterium adolescentis B-201 of the present invention is preserved in the General Microbiological Culture Collection Center of China Microorganism Culture Collection Administration, and the preservation number is: CGMCC No.31756.

[0025] The Bifidobacterium adolescentis B-201 of the present invention is isolated from healthy breast milk.

[0026] The Bifidobacterium adolescentis B-201 of the present invention is isolated by the following method: the breast milk sample is appropriately diluted and spread on a modified MRS solid medium, and anaerobically cultured at 37°C for 72 hours. After selecting typical colonies for Gram staining microscopy, they are streaked on a modified MRS solid medium for 2 to 3 times to obtain pure colonies. The strain is identified as a Bifidobacterium adolescentis lactis subspecies through physiological and biochemical identification combined with 16S rRNA molecular identification, and is named Bifidobacterium adolescentis B-201.

[0027] The adolescent bifidobacterium B-201 of the present invention has the following biological characteristics: Gram-positive bacteria, no flagella, no movement, no spore formation, anaerobic; the bacteria are rod-shaped and have various shapes, mostly V-shaped, Y-shaped or baseball-shaped, and the colonies are smooth and have complete edges and are milky white. The optimal growth temperature is 36-38°C; the optimal pH value is 6.0-7.0.

[0028] In the present invention, the 16S rRNA sequence of the Bifidobacterium adolescentis B-201 is shown in SEQ ID No. 1, specifically:

[0029] Figure 1 .

[0030] In the present invention, the antibacterial product preferably includes one or more of a broad-spectrum antibacterial agent, a disinfectant, a food preservative and a fresh-keeping agent; the antibacterial product is obtained by adding the intracellular and extracellular products of the Bifidobacterium adolescentis B-201 to a substrate.

[0031] In the present invention, the bacteriostatic objects of the bacteriostatic agent or bacteriostatic product preferably include pathogenic bacteria and / or food-borne spoilage bacteria.

[0032] In the present invention, when the Bifidobacterium adolescentis B-201 postbiotics are used for antibacterial and / or preparation of antibacterial products, the fermentation product and postbiotics of Bifidobacterium adolescentis B-201 preferably include intracellular and extracellular products of Bifidobacterium adolescentis B-201. In the present invention, the Bifidobacterium adolescentis B-201 postbiotics include broad-spectrum antibacterial products. Bifidobacterium adolescentis B-201 postbiotics have broad-spectrum antibacterial activity and can effectively inhibit pathogenic bacteria and foodborne spoilage bacteria.

[0033] The present invention also provides the use of Bifidobacterium adolescentis B-201 or a fermentation product of Bifidobacterium adolescentis B-201 or a fermentation product containing Bifidobacterium adolescentis B-201 and / or Bifidobacterium adolescentis B-201 in the production of functional active ingredients and Bifidobacterium adolescentis B-201 postbiotics to improve intestinal diseases; the functional active substances include one or more of amino acids and their metabolites, benzene and its derivatives, organic acid alcohols, flavonoids, hormones and hormone-related substances, alkaloids, organic acids and their derivatives, heterocyclic compounds and their fatty acyl groups, etc.; the improvement of intestinal diseases includes improving disease activity index, body weight, immune factors and colon tissue pathology. The Bifidobacterium adolescentis B-201 described in the present invention is deposited in the General Microbiological Culture Collection Center of the China Microbiological Culture Collection Administration, and the deposit number is: CGMCC No.31756.

[0034] Exemplary embodiments will be described in detail herein, examples of which are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The embodiments described in the following exemplary embodiments do not represent all embodiments consistent with the present invention. Instead, they are merely examples of methods consistent with some aspects of the present invention as detailed in the appended claims.

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0036] The methods and culture media used in the embodiments of the present invention are as follows:

[0037] (I) Methods for counting viable bacteria

[0038] The pour plate counting method was adopted, and the specific operation steps were as follows: detection was carried out according to GB 4789.35-2016 method.

[0039] (II) Culture medium for determination of viable probiotic counts

[0040] The culture medium used for determining the number of viable probiotic bacteria in the experimental examples of this application is a modified MRS culture medium. The modified MRS synthetic culture medium was purchased from Guangdong Huankai Microbiological Technology Co., Ltd. for microbiological detection.

[0041] Example 1 Isolation, Screening and Identification of Bifidobacterium adolescentis B-201

[0042] 1 Materials and methods

[0043] 1.1 Source, isolation and identification of Bifidobacterium adolescentis B-201 strain

[0044] Take a healthy breast milk sample, weigh 0.5mL of the sample, dilute it with 4.5mL of sterile PBS, take 0.1mL and spread it on a modified MRS solid medium, and culture it anaerobically at 37℃ for 72h. Pick a typical colony for Gram staining microscopy, select a Gram-positive strain, and then streak it on a modified MRS solid medium to obtain a pure colony. This strain is a Gram-like bacterium observed under a microscope. The bacteria are rod-shaped and have various shapes, mostly V-shaped, Y-shaped or baseball-shaped; on the modified MRS solid medium, the colony diameter and colony edge are neat, creamy white and opaque.

[0045] The isolated strain was subjected to 16S rRNA molecular identification. The DNA sample of the strain was extracted and the sample was subjected to 16S rRNA molecular identification by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The molecular sequence was compared with blastn in the NCBI database, and it was confirmed that B-201 was Bifidobacterium adolescentis, and its 16S rRNA sequence was shown in SEQ ID No.1.

[0046] Example 2 Preparation and Analysis of Bifidobacterium adolescentis B-201 Postbiotics

[0047] 2.1 Preparation method of Bifidobacterium adolescentis B-201 postbiotics

[0048] 2.1.1 Activation of strains: The test strains stored at -80°C were inoculated into the modified TPY liquid medium and activated at 37°C under anaerobic conditions. After activation, the test strains were inoculated into the seed medium at a 2% inoculation rate using a pipette, and then continuously cultured at 37°C under anaerobic conditions for 10 to 14 hours. To ensure the purity of the test strains, the microbial morphology was observed during the passage.

[0049] 2.1.2 Expanded culture: The above seed solution is expanded with an inoculation volume of 10% (V / V). After the inoculation, add ammonia water or sodium hydroxide to adjust the initial pH of the fermentation liquid to 7.0±0.02, sterile nitrogen pressure is maintained at 0.02-0.03MPa, the stirring speed is set at 80rpm / min, and fermentation begins. The culture temperature is 37±0.2℃, and ammonia water is used to maintain a constant pH (5.9-6.2)±0.02.

[0050] 2.1.3 Preparation of postbiotics of Bifidobacterium adolescentis B-201: The fermentation broth at the end point of fermentation was collected, concentrated and spray-dried to prepare the powder, which was reconstituted by diluting 10 times (equivalent to about 10% dry matter), and the pH value of the reconstituted solution was adjusted to 7.0. The antibacterial ability against Pseudomonas fluorescens, Pseudomonas aeruginosa, Staphylococcus aureus and Escherichia coli was determined by measuring the diameter of the inhibition zone.

[0051] 2.2 Results and Analysis

[0052] 2.2.1 Bifidobacterium adolescentis B-201 postbiotics Through metabolomics detection technology, a total of functional metabolites were identified, including amino acids and their metabolites, benzene and its derivatives, organic acid alcohols, flavonoids, hormones and hormone-related substances, alkaloids, organic acids and their derivatives, heterocyclic compounds and their fatty acyl compounds and other functional active substances, as shown in Table 1.

[0053]

[0054]

[0055]

[0056] 2.2.2 Bifidobacterium adolescentis B-201 postbiotics had inhibitory effects on 4 types of bacteria, and the results are shown in Table 2.

[0057] Table 2 Antibacterial test of Bifidobacterium adolescentis B-201 postbiotics

[0058]

[0059] Staphylococcus aureus and Escherichia coli are common foodborne pathogens that can contaminate food through various channels and methods, becoming one of the important pathogens of bacterial food poisoning. They are also conditional pathogens of zoonotic diseases, threatening human and animal health. The postbiotics of Bifidobacterium adolescentis B-201 have active substances that have good antibacterial effects on Staphylococcus aureus and Escherichia coli.

[0060] Pseudomonas fluorescens is one of the main bacteria that causes spoilage of milk and eggs when stored at low temperatures. The postbiotics of Bifidobacterium adolescentis B-201 have a significant antibacterial effect on Pseudomonas fluorescens.

[0061] Pseudomonas aeruginosa is a common conditional pathogen and one of the main pathogens of hospital-acquired infections. The postbiotics of Bifidobacterium adolescentis B-201 have an inhibitory effect on pathogens.

[0062] According to the antibacterial test results of this example, it can be seen that the Bifidobacterium adolescentis B-201 postbiotic has an inhibitory effect on foodborne pathogens and spoilage bacteria, and has potential application value as a food preservative and antistaling agent.

[0063] Example 3 Bifidobacterium adolescentis B-201 postbiotics improve IBD symptoms and inflammatory response in rats

[0064] 3.1 Experimental design and methods

[0065] The Wistar wild-type rats involved in the embodiment were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. 24 7-week-old male Wistar rats without specific pathogens were raised in an SPF animal room at a temperature of 22±2°C, a relative humidity of 50±10%, a 12-hour light / dark cycle, and free access to food and water for 1 week, and then randomly divided into a control group (Control, intragastric administration of normal saline), a model group (Model, fed with 3% DSS and intragastric administration of normal saline) and a postbiotic group (B-201 postbiotics, intragastric administration of 3% DSS and B-201 postbiotics, a dose of 4×10 9 CFU / animal / day). The experiment lasted for 28 days.

[0066] The IBD rat model was established by giving drinking water containing 3% DSS for 7 consecutive days starting from day 0. After day 8, all groups were provided with normal drinking water. The postbiotic group was gavaged with B-201 postbiotics, and the other groups were gavaged with the same volume of normal saline. All rats were killed on day 28, and blood and colon tissue were collected. After the blood was allowed to stand for 2 hours, it was centrifuged at 3000g for 15 minutes, and the supernatant was collected and frozen at -80°C. Fresh colon tissue was quickly frozen with liquid nitrogen and stored at -80°C.

[0067] Disease activity index (DAI) is a comprehensive score of physical signs based on the DAI score table. The weight loss rate, stool characteristics, and bloody stools are scored. The higher the score, the more serious the disease state. That is, DAI = weight change rate score + stool characteristics score + fecal occult blood score.

[0068] Serum cytokines were detected by enzyme-linked immunosorbent assay using rat serum TNF-α and IL-6 kits. After rewarming, the positions of the standard wells and sample wells were set. 50 μL of standard wells with different concentrations were added, and 50 μL of serum samples to be tested were added to the sample wells. According to the requirements of the kit, 100 μL of the corresponding detection antibodies were added to the standard wells and sample wells respectively; incubated at 37°C in the dark for 60 min; washed the plate 5 times and patted the reaction plate dry. 20 μL of solution A and solution B were added to all wells; after incubation at 37°C in the dark for 15 min, 50 μL of stop solution was added to all wells, and the absorbance (OD value) of each well was read using an enzyme reader at a wavelength of 450 nm. A standard curve was drawn using the standard concentration and OD value, and the concentration value was calculated using the OD value of the sample well.

[0069] 3.2 Results and Analysis

[0070] The results are as follows Figure 2-Figure 6 As shown. Body weight and disease activity index (DAI) are key indicators for evaluating the effect of IBD modeling. The results of animal experiments showed that rats fed with 3% DSS had symptoms of loose stools and bloody diarrhea, indicating that the model was successfully established. The IBD disease activity index can be significantly reduced by taking Bifidobacterium adolescentis B-201 postbiotics (P<0.05). IBD disease causes weight loss in rats, and the weight of rats in the model group is significantly lower than that in the normal group (P<0.05), while the intervention of Bifidobacterium adolescentis B-201 postbiotics slows down the weight loss of IBD rats.

[0071] Among serum cytokines, TNF-α and IL-6 are important indicators of disease inflammation in the IBD model. The experimental results showed that the serum TNF-α and IL-6 levels of rats in the model group were higher than those in the normal group. Compared with the model group, the intake of Bifidobacterium adolescentis B-201 postbiotics can significantly reduce the serum TNF-α and IL-6 levels of rats with inflammatory bowel disease (P<0.05). This indicates that Bifidobacterium adolescentis B-201 postbiotics can regulate the release levels of cytokines TNF-α and IL-6, change the state of excessive intestinal immune response, and thus improve the related symptoms of IBD.

[0072] Colon histopathology results showed that the colon tissue structure of rats in the normal group was intact, arranged regularly, and without obvious inflammation, while the structure of the intestinal mucosal lamina propria of rats in the model group was severely damaged, with a large number of epithelial cells falling off and obvious inflammatory cell infiltration. The intake of Bifidobacterium adolescentis B-201 postbiotics effectively alleviated intestinal damage.

[0073] According to the results of this example, the Bifidobacterium adolescentis B-201 postbiotic has the effect of alleviating the disease symptoms and inflammatory response of DSS-induced IBD rats.

Claims

1. A strain of Bifidobacterium adolescentis B-201, characterized in that: The Bifidobacterium adolescentis B-201 has been deposited in the China General Microbiological Culture Collection Center, with the deposit number: CGMCC No. 31756.

2. The Bifidobacterium adolescentis B-201 according to claim 1, characterized in that The sequence of 16S rRNA of the Bifidobacterium adolescentis B-201 is shown in SEQ ID NO.

1.

3. A preparation containing the Bifidobacterium adolescentis B-201 and / or the inactivated product according to claim 1 or 2.

4. A bacterial agent comprising the Bifidobacterium adolescentis B-201 and / or an inactivated product thereof according to claim 1 or 2.

5. Use of the Bifidobacterium adolescentis B-201 according to claim 1 or 2 in the preparation of products for improving intestinal diseases or inhibiting bacteria.

6. Use of Bifidobacterium adolescentis B-201 according to claim 1 or 2 in producing functional active substances.

7. The use according to claim 7, characterized in that: The active substances include one or more of amino acids and their metabolites, benzene and its derivatives, organic acid alcohols, flavonoids, hormones and hormone-related substances, alkaloids, organic acids and their derivatives, heterocyclic compounds and their fatty acyl compounds.

8. The Bifidobacterium adolescentis B-201 postbiotic according to claim 1 or 2.

9. The method for preparing the Bifidobacterium adolescentis B-201 postbiotic according to claim 8, characterized in that: The bifidobacterium adolescentis B-201 is obtained by fermentation, culture and inactivation.

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