Probiotic strain with antibacterial activity and application thereof

Abstract

The invention belongs to the technical field of probiotics, and particularly relates to a probiotic strain with antibacterial activity and application thereof.The probiotic strain is bifidobacterium animalis subsp. Lactis HN1000 and preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC NO.30862, the preservation number is CGMCC NO.30862, the preservation number is CGMCC NO.30862, and the preservation number is CGMCC NO.30862. According to the application of the probiotic strain in the oral antibacterial agent, the use form is HN1000 freeze-dried powder prepared after culture of an induction culture medium, prebiotics such as fructo-oligosaccharide, inulin, resistant starch and the like and functional synergistic factors such as tea polyphenol, lactoferrin and the like in the oral antibacterial agent further improve the antibacterial activity and gastrointestinal targeting of the probiotic strain; and a safe and efficient microbial solution is provided for infection prevention and treatment of pathogenic bacteria.

Description

Technical Field

[0001] The present invention belongs to the technical field of probiotics, and in particular relates to a probiotic strain with antibacterial activity and an application thereof. Background Art

[0002] Probiotic therapy has gradually become an important research direction to replace antibiotics for its biosafety and potential for microecological regulation. However, in actual applications, the inherent antibacterial activity of most strains is limited, making it difficult to effectively inhibit the colonization and proliferation of multidrug-resistant pathogens in the intestine. In addition, the erosion of gastric acid and bile salts during oral delivery leads to a large number of live bacteria being inactivated, significantly weakening their functional expression after reaching the intestine. In addition, traditional probiotic preparations generally lack the ability to respond to the intestinal infection microenvironment and cannot form an antibacterial effect in areas where pathogens are enriched. Especially for strains with basic antibacterial ability, existing technologies have not yet established an effective activity enhancement pathway, and although physical encapsulation methods can partially improve gastric acid tolerance, it is difficult to coordinate the temporal release of live bacteria in the gastrointestinal area. These limitations make it impossible for existing probiotic products to achieve the expected antibacterial effect when dealing with refractory or drug-resistant pathogen infections.

[0003] Therefore, there is an urgent need to break through the technical difficulties in the antibacterial application of probiotics through strain function remodeling and gastrointestinal delivery design, and provide new biological solutions for the prevention and treatment of bacterial infections. Summary of the Invention

[0004] In response to the above situation, the present invention provides a probiotic strain with antibacterial activity and its application. By functionalizing and inducing a new strain that has been discovered and isolated, it is endowed with stronger antibacterial properties, and at the same time provides the application of probiotics in the field of preparing oral antibacterial agents.

[0005] To achieve the above object, the technical solution adopted by the present invention is as follows: The present invention provides a probiotic strain with antibacterial activity. The probiotic strain is obtained by culturing animal Bifidobacterium lactis subsp. lactis HN1000 through an induction medium. The animal Bifidobacterium lactis subsp. lactis HN1000 is isolated from dairy products and is deposited at the General Microbiology Center of the China Culture Collection of Microorganisms at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing on June 5, 2024. The probiotic strain is classified and named as animal Bifidobacterium lactis subsp. lactis. The viability test result shows that the probiotic strain is alive, and the deposit number is CGMCC NO.30862.

[0006] Further, the Bifidobacterium animalis lactis HN1000 has the following characteristics: gram-positive staining, bacillus form, no spore formation, round at both ends, spherical and milky white in colony on MRS agar medium, upward protrusion, moist, and smooth colony edge.

[0007] Further, the Bifidobacterium animalis lactis HN1000 is sequenced by 16S, and the base sequence of the primer used for sequencing is as follows: 27F: AGAGTTTGATCMTGGCTCAG, 1492R: GGTTACCTTGTTACGACTT, and the base sequence of the 16S sequencing result is shown in SEQ ID NO. 1.

[0008] Further, the Bifidobacterium animalis lactis HN1000 is used in the preparation of an antibacterial product, and the antibacterial product is an oral antibacterial agent.

[0009] Further, in the application of the Bifidobacterium animalis lactis HN1000 in the preparation of an oral antibacterial agent, the antibacterial activity induction culture is used, and the induction culture medium includes the following raw materials: MRS basic medium, ferric citrate, low-fat pectin, bovine bile salt, furanone, and distilled water.

[0010] Further, the mass ratio of the MRS basic medium, ferric citrate, low-fat pectin, bovine bile salt, furanone, and distilled water is 66:0.1:3:2:0.04:1000.

[0011] Further, in the application of the Bifidobacterium animalis lactis HN1000 in the preparation of an oral antibacterial agent, the HN1000 is in the form of a freeze-dried powder, and the HN1000 freeze-dried powder contains 8% skimmed milk and 0.5% sodium glutamate by mass fraction.

[0012] Further, the preparation method of the HN1000 freeze-dried powder is as follows: S1: weigh the MRS basic medium and dissolve it in distilled water to obtain a basic broth, weigh the MRS basic medium, ferric citrate, low-fat pectin, and bovine bile salt, and dissolve them in distilled water, and then add furanone after sterilization to obtain an induction culture medium; S2: activate the Bifidobacterium animalis lactis HN1000 in the basic broth twice to obtain a seed liquid; S3: inoculate the seed liquid into the induction culture medium and program the culture for three times to obtain an induced seed liquid; S4: ferment and centrifuge the induced seed liquid, collect the bacterial slurry precipitate, mix skimmed milk and sodium glutamate, and then freeze-dry to obtain HN1000 freeze-dried powder.

[0013] The application provides application of a probiotic strain with antibacterial activity in preparation of an oral antibacterial agent, and the oral antibacterial agent comprises the following raw materials in parts by weight: HN1000 freeze-dried powder 30-50 parts, Lactobacillus rhamnosus freeze-dried powder 7-12 parts, fructooligosaccharide 45-55 parts, inulin 12-15 parts, trehalose 6-8 parts, resistant starch 2 parts, tea polyphenol 1.5 parts, lactoferrin 1 part, blueberry powder 0.5 part and steviol glycoside 0.05 part.

[0014] The preparation method of the oral antibacterial agent is as follows: Step 1: 1.5 parts of tea polyphenol, 1 part of lactoferrin and 2 parts of resistant starch are weighed and mixed to prevent agglomeration to obtain a mixed powder; 45-55 parts of fructooligosaccharide and 12-15 parts of inulin are placed in a mixer and mixed for 15 min; then the mixed powder and 6-8 parts of trehalose are added successively and mixed for 10 min and 5 min respectively to obtain a base powder; Step 2: 30-50 parts of HN1000 freeze-dried powder and 7-12 parts of Lactobacillus rhamnosus freeze-dried powder are added to the base powder, and low-speed mixing is performed for 5 min to avoid mechanical shear damage to the bacterial body to obtain a probiotic powder; Step 3: 0.5 part of blueberry powder and 0.05 part of steviol glycoside are sieved through a 120-mesh screen and mixed with the probiotic powder for 3 min to obtain the oral antibacterial agent.

[0015] The application has the following beneficial effects: The probiotic strain animal Bifidobacterium lactis HN1000 provided by the application has good acid and alkali resistance and the function of inhibiting pathogenic bacteria such as Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Helicobacter pylori; on this basis, the antibacterial activity of the strain is further improved through an antibacterial induction medium; the iron-limited environment constructed by the ferric citrate in the antibacterial induction medium triggers the bacteriocin synthesis pathway; the low-ester pectin simulates the structure of the pathogenic bacterial biofilm to promote the competitive adhesion ability of the animal Bifidobacterium lactis HN1000; the bovine bile salt exposure further strengthens the intestinal tolerance of the strain; and the furanone as a quorum sensing inducer synergistically regulates the efficient expression of antibacterial substances, so that the basal metabolic activity of the animal Bifidobacterium lactis HN1000 in the antibacterial aspect is enhanced.

[0016] The application of animal Bifidobacterium lactis subsp. HN1000 in preparing oral antibacterial agent, the induced and strengthened probiotic strain and Lactobacillus rhamnosus form a functional complementary antibacterial double core, fructo-oligosaccharides and inulin can activate the metabolic activity of Bifidobacterium and Lactobacillus, promote the proliferation of probiotics to produce long-acting antibacterial activity, resistant starch is used for supplementing dietary fiber on the one hand, and used as a colon targeting carrier and a slow-release matrix on the other hand, and the anti-enzymatic property thereof can help the efficient enrichment of probiotics in the site of intestinal pathogenic bacteria to improve the antibacterial efficiency; tea polyphenols and lactoferrin synergistically enhance the penetration of bacteriocin and the chelation of iron ions to form a synergistic barrier to inhibit the biofilm of pathogenic bacteria in the intestinal microenvironment, and natural antioxidants in trehalose and blueberry powder constitute a cell activity protection network, which improves the palatability while avoiding interference with the balance of flora. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The strain phylogenetic tree of animal Bifidobacterium lactis subsp. HN1000; Figure 2 The antibacterial property investigation results of the oral antibacterial agents prepared from animal Bifidobacterium lactis subsp. HN1000 and Example 2 and Comparative Examples 1-2; Figure 3 The colony and bacterial morphology characterization results of animal Bifidobacterium lactis subsp. HN1000; Figure 4 The acid and alkali resistance investigation results of the oral antibacterial agents prepared from Example 1-3 and Comparative Example 1. DETAILED DESCRIPTION

[0018] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments of the present application; based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of the present application.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art. In addition, any method and material similar or equivalent to those described herein can be used in the present application. The preferred implementation methods and materials described herein are only used for demonstration, but cannot limit the content of the present application.

[0020] In the following examples, unless otherwise specified, all are conventional methods; in the following examples, unless otherwise specified, the raw materials used are new materials purchased on the market, and the parts are all weight parts, wherein the phylogenetic tree of animal Bifidobacterium lactis subsp. HN1000 is shown in Figure 1 The live bacteria number of the used HN1000 freeze-dried powder is about 3.0×10 11CFU / g, moisture ≤3%; the viable cell count of the Lactobacillus rhamnosus freeze-dried powder used is about 1.0 x 10 11 CFU / g; the degree of polymerization of the fructooligosaccharide used is 2-8, and the ash content is ≤0.3%; the inulin used is derived from chicory roots and is of long-chain type; the degree of esterification of the low-fat pectin used is 26-30%, and the galacturonic acid content is ≥85%; the trehalose used is dihydrate, and the purity is ≥99.5%; the resistant starch used has an anti-enzymatic hydrolysis rate of ≥85%, and the average particle size is 25 μm; the tea polyphenol used is derived from green tea extraction; the lactoferrin used has an iron saturation degree of ≤15%, and the purity is ≥95%; the steviol glycoside used is steviol glycoside Reb M; the ferric citrate used is in the form of hydrate, and the iron ion content is 16-18%; the sodium cholate in the bovine bile salt used has a content of ≥80%, and the combined bile acid is ≤0.5%; the furanone used is 4-hydroxy-2,5-dimethyl-3(2H)-furanone in full name, and the colority is ≤50.

[0021] In the following examples and comparative examples, the preparation method of the HN1000 freeze-dried powder is as follows: S1: 71 parts of MRS basic medium were weighed and dissolved in 1000 parts of 80°C distilled water, and high-pressure steam sterilization was performed to obtain a basic broth; 66 parts of MRS basic medium, 0.1 part of ferric citrate, 3 parts of low-fat pectin, and 2 parts of bovine bile salt were weighed and dissolved in 1000 parts of 60°C distilled water, and high-pressure steam sterilization was performed, and then 0.04 parts of furanone filtered through a 0.22 μm filter was added to obtain an induction medium; S2: the preserved animal Bifidobacterium lactis HN1000 strain was taken into the basic broth, and cultured at 37°C under facultative anaerobic conditions for 24 h to obtain a first-stage seed liquid; and then the first-stage seed liquid was inoculated into the basic broth at an inoculation amount of 5% (v / v) for secondary activation culture for 24 h to obtain a second-stage seed liquid; S3: the second-stage seed liquid was inoculated into the induction medium at an inoculation amount of 10%, and after anaerobic culture at 37°C for 12 h, micro-aerobic culture at 30°C for 4 h, and normal culture at 30°C for 3 h, a first-stage induction seed liquid was obtained; and then the above induction culture process was repeated twice with an inoculation amount of 5% to obtain a third-stage induction seed liquid; S4: the third-stage induction seed liquid was inoculated into a fermentation tank medium at an inoculation amount of 10%, and fermentation was performed at 37°C, 60 rpm, a ventilation amount of 0.5 L / min, and pH 5.0 for 8 h to obtain a fermentation liquid; the fermentation liquid was centrifuged at 10000 rpm for 15 min, and the collected bacterial sludge precipitate was mixed with skimmed milk and sodium glutamate and then freeze-dried to obtain the HN1000 freeze-dried powder, and the contents of the skimmed milk and the sodium glutamate in the HN1000 freeze-dried powder were 8% and 0.5%, respectively.

[0022] Example 1: This example provides a probiotic strain with antibacterial activity, wherein the probiotic strain is Bifidobacterium animalis subsp. lactis HN1000, which has the activity of inhibiting Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Helicobacter pylori pathogens. The Bifidobacterium animalis subsp. lactis HN1000 is isolated from dairy products and is currently deposited in the China General Microorganism Culture Collection Center with the deposit number: CGMCC No. 30862.

[0023] This embodiment also provides a use of a probiotic strain with antibacterial activity in preparing an oral antibacterial agent, wherein the oral antibacterial agent comprises the following raw materials in parts by weight: 30 parts of HN1000 freeze-dried powder, 12 parts of Lactobacillus rhamnosus freeze-dried powder, 55 parts of oligofructose, 15 parts of inulin, 8 parts of trehalose, 2 parts of resistant starch, 1.5 parts of tea polyphenols, 1 part of lactoferrin, 0.5 parts of blueberry powder, and 0.05 parts of steviol glycoside. The specific preparation method is as follows: Step 1: Weigh 1.5 parts of tea polyphenols, 1 part of lactoferrin, and 2 parts of resistant starch and premix them in a three-dimensional motion mixer at 15 rpm for 10 minutes to prevent agglomeration to obtain a mixed powder with no visible lumps. Place 55 parts of oligofructose and 15 parts of inulin in a V-type mixer at 40 rpm and mix for 15 minutes. Add the mixed powder and mix for 10 minutes. Then add 8 parts of trehalose and mix for 5 minutes to obtain a matrix powder. Step 2: Add 30 parts of HN1000 freeze-dried powder and 12 parts of Lactobacillus rhamnosus freeze-dried powder to the matrix powder, and mix at a low speed of 25 rpm for 5 minutes to avoid mechanical shear damage to the bacteria, to obtain probiotic powder; Step 3: After passing 0.5 parts of blueberry powder and 0.05 parts of stevioside through a 120-mesh sieve, mix them with probiotic powder for 3 minutes to obtain an oral antibacterial agent.

[0024] Example 2: This example provides a probiotic strain with antibacterial activity, wherein the probiotic strain is Bifidobacterium animalis subsp. lactis HN1000, which has the activity of inhibiting Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Helicobacter pylori pathogens. The Bifidobacterium animalis subsp. lactis HN1000 is isolated from dairy products and is currently deposited in the China General Microorganism Culture Collection Center with the deposit number: CGMCC No. 30862.

[0025] This embodiment also provides a use of a probiotic strain with antibacterial activity in preparing an oral antibacterial agent, wherein the oral antibacterial agent comprises the following raw materials in parts by weight: 40 parts of HN1000 freeze-dried powder, 10 parts of Lactobacillus rhamnosus freeze-dried powder, 50 parts of oligofructose, 13 parts of inulin, 7 parts of trehalose, 2 parts of resistant starch, 1.5 parts of tea polyphenols, 1 part of lactoferrin, 0.5 parts of blueberry powder, and 0.05 parts of steviol glycoside. The specific preparation method is as follows: Step 1: Take 1.5 parts of tea polyphenol, 1 part of lactoferrin and 2 parts of resistant starch in a three-dimensional motion mixer at 15 rpm and mix for 10 min to prevent agglomeration, to obtain a mixed powder, and the mixed powder has no visible clumps. Put 50 parts of fructooligosaccharide and 13 parts of inulin into a V-type mixer at 40 rpm and mix for 15 min. Add the mixed powder to the V-type mixer and mix for 10 min. Then add 7 parts of trehalose and mix for 5 min to obtain a base powder; Step 2: Add 40 parts of HN1000 freeze-dried powder and 10 parts of Lactobacillus rhamnosus freeze-dried powder to the base powder, and mix at 25 rpm for 5 min to avoid mechanical shear damage to the bacterial body, to obtain a probiotic powder; Step 3: Screen 0.5 parts of blueberry powder and 0.05 parts of stevioside through a 120-mesh sieve, and mix with the probiotic powder for 3 min to obtain an oral antibacterial agent.

[0026] In this embodiment, a probiotic strain with antibacterial activity is provided, which is animal Bifidobacterium lactis HN1000 with inhibitory activity on Escherichia coli, Salmonella typhi, Staphylococcus aureus and Helicobacter pylori pathogenic bacteria. The animal Bifidobacterium lactis HN1000 is isolated from dairy products and is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 30862.

[0027] In this embodiment, a probiotic strain with antibacterial activity is provided, which is animal Bifidobacterium lactis HN1000 with inhibitory activity on Escherichia coli, Salmonella typhi, Staphylococcus aureus and Helicobacter pylori pathogenic bacteria. The animal Bifidobacterium lactis HN1000 is isolated from dairy products and is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC No. 30862. Step 1: Take 1.5 parts of tea polyphenol, 1 part of lactoferrin and 2 parts of resistant starch in a three-dimensional motion mixer at 15 rpm and mix for 10 min to prevent agglomeration, to obtain a mixed powder, and the mixed powder has no visible clumps. Put 50 parts of fructooligosaccharide and 13 parts of inulin into a V-type mixer at 40 rpm and mix for 15 min. Add the mixed powder to the V-type mixer and mix for 10 min. Then add 7 parts of trehalose and mix for 5 min to obtain a base powder; Step 2: Add 40 parts of HN1000 freeze-dried powder and 10 parts of Lactobacillus rhamnosus freeze-dried powder to the base powder, and mix at 25 rpm for 5 min to avoid mechanical shear damage to the bacterial body, to obtain a probiotic powder; Step 3: Screen 0.5 parts of blueberry powder and 0.05 parts of stevioside through a 120-mesh sieve, and mix with the probiotic powder for 3 min to obtain an oral antibacterial agent.

[0028] Embodiment 4: The embodiment provides a probiotic strain with antibacterial activity, the probiotic strain being animal Bifidobacterium lactis subsp. HN1000 with inhibitory activity on Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Helicobacter pylori pathogenic bacteria, the animal Bifidobacterium lactis subsp. HN1000 being isolated from dairy products and being preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.30862.

[0029] The embodiment also provides application of the probiotic strain with antibacterial activity in preparation of an oral antibacterial agent, the oral antibacterial agent comprising the following raw materials in parts by weight: 30 parts of HN1000 freeze-dried powder, 7 parts of Lactobacillus rhamnosus freeze-dried powder, 55 parts of fructooligosaccharide, 15 parts of inulin, 8 parts of trehalose, 2 parts of resistant starch, 1.5 parts of tea polyphenol, 1 part of lactoferrin, 0.5 part of blueberry powder and 0.05 part of steviol glycoside, and the specific preparation method is as follows: Step 1: 1.5 parts of tea polyphenol, 1 part of lactoferrin and 2 parts of resistant starch are weighed in a three-dimensional motion mixer and pre-mixed at 15 rpm for 10 min to prevent agglomeration, to obtain a mixed powder, and the mixed powder has no visible agglomeration; 55 parts of fructooligosaccharide and 15 parts of inulin are placed in a V-type mixer and mixed at 40 rpm for 15 min, 8 parts of trehalose is added and mixed for 5 min, to obtain a base powder; Step 2: 30 parts of HN1000 freeze-dried powder and 7 parts of Lactobacillus rhamnosus freeze-dried powder are added to the base powder, and low-speed mixing is performed at 25 rpm for 5 min to avoid mechanical shear damage to the bacterial bodies, to obtain probiotic powder; Step 3: 0.5 part of blueberry powder and 0.05 part of steviol glycoside are sieved through a 120-mesh sieve and mixed with the probiotic powder for 3 min, to obtain the oral antibacterial agent.

[0030] Embodiment 5: The embodiment provides a probiotic strain with antibacterial activity, the probiotic strain being animal Bifidobacterium lactis subsp. HN1000 with inhibitory activity on Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Helicobacter pylori pathogenic bacteria, the animal Bifidobacterium lactis subsp. HN1000 being isolated from dairy products and being preserved in the China General Microbiological Culture Collection Center with a preservation number of CGMCC No.30862.

[0031] The embodiment also provides application of the probiotic strain with antibacterial activity in preparation of an oral antibacterial agent, the oral antibacterial agent comprising the following raw materials in parts by weight: 30 parts of HN1000 freeze-dried powder, 7 parts of Lactobacillus rhamnosus freeze-dried powder, 55 parts of fructooligosaccharide, 15 parts of inulin, 8 parts of trehalose, 2 parts of resistant starch, 1.5 parts of tea polyphenol, 1 part of lactoferrin, 0.5 part of blueberry powder and 0.05 part of steviol glycoside, and the specific preparation method is as follows: Step 1: Take tea polyphenols 1.5 parts, lactoferrin 1 part and resistant starch 2 parts into a three-dimensional motion mixer and premix at 15 rpm for 10 min to prevent agglomeration, to obtain a mixed powder. The mixed powder has no visible agglomerates. Place fructooligosaccharide 45 parts and inulin 12 parts into a V-type mixer and mix at 40 rpm for 15 min. Add the mixed powder into the V-type mixer and mix for 10 min. Add trehalose 6 parts and mix for 5 min to obtain a base powder; Step 2: Add HN1000 freeze-dried powder 50 parts and Lactobacillus rhamnosus freeze-dried powder 12 parts into the base powder, and mix at 25 rpm for 5 min to avoid mechanical shear damage to the bacterial bodies, to obtain a probiotic powder; Step 3: Pass blueberry powder 0.5 parts and steviol glycoside 0.05 parts through a 120-mesh sieve, and mix with the probiotic powder for 3 min to obtain an oral antibacterial agent.

[0032] Comparative Example 1 differs from Example 2 in that the animal bifidobacterium lactis HN1000 strain is not treated with an induced culture medium, and the rest is the same as Example 2. Comparative Example 2 differs from Example 2 in that an equal weight of edible starch is used instead of HN1000 freeze-dried powder, and the rest is the same as Example 2.

[0033] In vitro antibacterial experiment Preparation of samples: Take the oral antibacterial agents prepared from the animal bifidobacterium lactis HN1000 not treated with an induced culture medium, Example 2, Comparative Example 1 and Comparative Example 2, respectively, into 10 m sterile normal saline to obtain bacterial suspensions. The concentration of each group of bacterial suspensions is 1×10 7 CFU / mL, calculated based on the animal bifidobacterium lactis HN1000. Preparation of detection plates: Take Escherichia coli (ATCC 25922), Salmonella typhimurium (ATCC 14028), Staphylococcus aureus (ATCC 6538) and Helicobacter pylori (ATCC 43504) after culture, and prepare them into bacterial solutions with a concentration of 1×10 7 CFU / mL and an OD 600 of 0.5. Take 800 μL of each and spread on LB plates. The Helicobacter pylori is spread on a Brucella agar culture medium containing 7% defibrinated horse blood. After the surface is dry, place a filter paper disc with a diameter of 6 mm. Add 200 μL of each bacterial suspension. Incubate at 37℃ for 12 h. Detect the diameters of the antibacterial rings. The antibacterial rings of Example 2, the animal bifidobacterium lactis HN1000 not treated with an induced culture medium, Comparative Example 1 and Comparative Example 2 are represented by A, B, C and D, respectively. The results are shown in Table 1. Figure 2

[0034] Strain morphology investigation ​The morphological characteristics of the animal Bifidobacterium lactis HN1000 were observed by optical microscopy, and the results are shown in Figure 3 .

[0035] Gastrointestinal tolerance The artificial gastric juice with pH 3.0 and containing 0.3% pepsin, the artificial simulated intestinal fluid with pH 6.8 and containing 0.3% trypsin, and the artificial simulated bile with pH 7.0 and containing 0.3% bile salt were respectively prepared, and the plate counting method was used to investigate the change in the number of colonies of the strains of Examples 1-3 and Comparative Example 1 before and after the treatment with the artificial gastric juice, the artificial simulated intestinal fluid, and the artificial simulated bile, and the survival rate was calculated, and the results are shown in Figure 4 .

[0036] Investigation of bacteriostatic substances To determine the properties of the bacteriostatic substances of the animal Bifidobacterium lactis HN1000, the following tests were performed, and each group was tested in parallel for 6 times: Acid-base effect verification: the fermentation broth obtained in step S4 during the preparation of the HN1000 freeze-dried powder was centrifuged, and the supernatant was taken, the pH of the supernatant was detected, and the pH was adjusted to be consistent with that before fermentation by using 3 M NaOH or 3 M HCl, and the bacteriostatic activity of the supernatant before and after the pH adjustment was determined, and the results are shown in Table 1; High-temperature treatment: the bacteriostatic activity of the supernatant before and after the high-temperature treatment at 115°C for 30 min was determined, and the results are shown in Table 1; Protease treatment: protease K was added to the supernatant with pH 7.0, and pepsin was added to the supernatant with pH 3.0, and the mixture was incubated at 37°C for 4 h, and then the pH was adjusted back to the original pH, and the bacteriostatic activity was detected, and the results are shown in Table 1; Salt-out treatment: ammonium sulfate was added to the supernatant to a saturation degree of 65% for salt-out, and the precipitate was dissolved with PBS (phosphate) buffer with pH 7 to 10 mL, and the bacteriostatic activity of the supernatant after the PBS dissolution and desalination in a 2 kDa dialysis bag was determined, and the results are shown in Table 1; Dialysis treatment: 10 mL of the supernatant was treated in an 8 kDa dialysis bag, the liquid in and outside the dialysis bag was concentrated to 1 mL, and then diluted with PBS buffer to 10 mL, and the bacteriostatic activity was determined, and the results are shown in Table 1.

[0037] Table 1 Investigation of the properties of bacteriostatic substances

[0038] Figure 1 The results of the phylogenetic tree show that the animal Bifidobacterium lactis HN1000 of the present application is similar to the MT585567.1 colony genetic sequence, and has high homology.

[0039] Figure 2The results of the antibacterial detection show that the oral antibacterial agent prepared in Example 2 (A) has stronger antibacterial effect than the oral antibacterial agent prepared in Comparative Example 1 (C) without antibacterial induction, and the oral antibacterial agent has significantly enhanced inhibitory effect on Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Helicobacter pylori than the single strain (B).

[0040] Figure 3 The results show that the animal Bifidobacterium lactis HN1000 has medium colony size, is spherical, and the single probiotic is rod-shaped under microscopic staining, and grows well.

[0041] Figure 4 The results of the tolerance show that under the component composition of Example 1-3 and Comparative Example 1, the oral antibacterial agent has good acid and alkali resistance, and can maintain good enzyme activity in a strong acid and enzyme environment.

[0042] The results of Table 1 show that the antibacterial activity of the animal Bifidobacterium lactis HN1000 on Staphylococcus aureus is enhanced in an alkaline environment, excluding the antibacterial mechanism dominated by organic acids, and the antibacterial activity significantly decreases after high-temperature treatment, it is speculated that the secondary or tertiary structure of polypeptide proteins and other substances may be heat-deformed, the antibacterial activity decreases after two enzyme treatments, and the antibacterial activity of the dialysis liquid is higher, which further indicates that the antibacterial mechanism is related to the expression of polypeptide proteins.

[0043] Although the embodiments of the present application have been shown and described, it can be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

[0044] The above describes the present application and its embodiments, which are not restrictive, and the embodiments shown in the drawings are only one of the embodiments of the present application, and the actual application is not limited thereto. In summary, if those skilled in the art are inspired by it, without departing from the purpose of the present application, without creative design, similar ways and embodiments of the technical solution should belong to the protection scope of the present application.

Claims

1. A probiotic strain with antibacterial activity, characterized in that: The probiotic strain is Bifidobacterium animalis subspecies lactis HN1000, and the preservation number of Bifidobacterium animalis subspecies lactis HN1000 is CGMCC NO.30862; The animal Bifidobacterium lactis subspecies HN1000 is cultured in an induction medium; The induction culture medium comprises the following raw materials: MRS basal culture medium, ferric citrate, low-fat pectin, ox bile salt, furanone and distilled water.

2. A probiotic strain with antibacterial activity according to claim 1, characterized in that The animal Bifidobacterium lactis subsp. lactis HN1000 was sequenced by 16S sequencing. The base sequences of the primers used in the 16S sequencing were as follows: 27F: AGAGTTTGATCMTGGCTCAG, 1492R: GGTTACCTTGTTACGACTT. The base sequence of the 16S sequencing result was shown as SEQ ID NO.

1.

3. The probiotic strain with antibacterial activity according to claim 1, characterized in that The mass ratio of the MRS basal medium, ferric citrate, low-fat pectin, ox bile salt, furanone and distilled water is 66: 0.1：3：2：0.04：1000。 4. A use of a probiotic strain with antibacterial activity according to any one of claims 1 to 3, characterized in that: The animal Bifidobacterium lactis subspecies HN1000 is used to prepare an oral antibacterial agent; The animal Bifidobacterium lactis subspecies HN1000 is in the form of HN1000 freeze-dried powder in the application of preparing an oral antibacterial agent; The oral antibacterial agent comprises the following raw materials in parts by weight: 30-50 parts of HN1000 freeze-dried powder, 7-12 parts of Lactobacillus rhamnosus freeze-dried powder, 45-55 parts of oligofructose, 12-15 parts of inulin, 6-8 parts of trehalose, 2 parts of resistant starch, 1.5 parts of tea polyphenols, 1 part of lactoferrin, 0.5 parts of blueberry powder and 0.05 parts of steviol glycoside.

5. The use of a probiotic strain with antibacterial activity according to claim 4, characterized in that: The specific preparation method of the oral antibacterial agent is as follows: Step 1: Weigh tea polyphenols, lactoferrin and resistant starch to obtain a premixed powder, mix oligofructose and inulin, and then add the premixed powder and trehalose to obtain a matrix powder; Step 2: Mixing the HN1000 freeze-dried powder and the Lactobacillus rhamnosus freeze-dried powder with the matrix powder to obtain probiotic powder; Step 3: After sieving the blueberry powder and stevioside, mix them with the probiotic powder to obtain an oral antibacterial agent.

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