Application of lactobacillus casei BT5-B6, strain composition and compound preparation

Abstract

The invention relates to the technical field of microbial engineering, in particular to application of casei casei BT5-B6, a strain composition and a compound preparation. The lactobacillus casei BT5-B6 is applied to any one of the following items (1) to (5): (1) preparing a product for improving the immunity, (2) preparing a product for improving the immunity, (3) preparing a product for improving the immunity, (4) preparing a product for improving the immunity, and (5) preparing a product for improving the immunity; (2) preparing a product for reducing the content of proinflammatory factors; (3) preparing a product for increasing the content of anti-inflammatory factors; (4) preparing a product for inhibiting the growth of helicobacter in intestinal tracts; and (5) preparing a product for promoting growth of murine bacilliaceae or / and physicochemical bacteria in intestinal tracts. The bacterial strain composition comprises the casei casei BT5-B6. The lactobacillus casei BT5-B6 can effectively reduce the content of proinflammatory factors and improve the content of anti-inflammatory factors, has a remarkable anti-inflammatory effect, and effectively enhances the immunity.

Description

Technical Field

[0001] The invention relates to the technical field of microbial engineering, and in particular to the application of Lactobacillus casei BT5-B6 and a strain composition and a composite preparation. Background Art

[0002] Generally speaking, the inflammatory response is the following defensive response process of the organism: when cells or tissues are invaded by any organic changes, they will try to repair and regenerate the damaged area. Therefore, this series of reactions includes local blood vessels, various tissue cells of body fluids, immune-related cells, etc.

[0003] In recent years, with the development of molecular biology, people are trying to explain inflammatory diseases at the molecular level of cytokines. With the occurrence of inflammation, MCP-1 is produced, which promotes macrophage infiltration and subsequent secretion of inflammatory factors (such as IL-6, TNF-α and NO, etc.). Once macrophages are activated, the activated macrophages release inflammatory cytokines, which trigger the infiltration of other immune cells (such as neutrophils, CD8 and CD4 T cells, etc.).

[0004] Some clinical studies have shown that probiotics have beneficial effects in preventing and improving inflammation and metabolic disorders. They can increase the abundance of beneficial bacteria such as lactic acid bacteria and bifidobacteria, reduce the abundance of Gram-negative bacteria, and improve the intestinal microecological environment.

[0005] Bifidobacterium animalis subsp. lactis can significantly reduce serum lipopolysaccharide levels and liver inflammation, as well as the adhesion of Escherichia coli in the intestine, thereby improving the inflammatory state. Live and inactivated strains of Lactobacillus reuteri GMNL-263 can significantly restore the reduction of beneficial bacteria in intestinal microorganisms, improve the intestinal barrier, downregulate the expression of pro-inflammatory genes in fat and liver tissues, effectively inhibit the infiltration of macrophages in adipose tissue, and improve metabolic-related chronic inflammation.

[0006] However, these strains are dose- and strain-specific, and individual intestinal flora and physiological states vary significantly, resulting in differences in the effects of different strains. Therefore, there is still a need to develop other new strains of probiotics that can relieve and inhibit inflammation. At the same time, the types of inflammatory factors that can be regulated by existing strains are limited, but in inflammation caused by different reasons, the types of inflammatory factors that change are different, and different inflammatory factors have different effects on the body. Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings in the prior art and to propose an application of Lactobacillus casei BT5-B6, a strain composition and a composite preparation.

[0008] Lactobacillus casei Lacticaseibacilus casei) BT5-B6 is used in any of the following (1)-(5): (1) Preparation of products for improving immunity; (2) preparing products that reduce the content of pro-inflammatory factors; (3) preparing products with increased content of anti-inflammatory factors; (4) Preparation of products for inhibiting the growth of Helicobacter in the intestine; (5) Preparing products that promote the growth of Bacillus family and / or Bacillus family in the intestine.

[0009] Preferably, Lactobacillus casei BT5-B6 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 20, 2024, with a deposit number of CGMCC NO.32013 and a classification name of Lacticaseibacilus casei.

[0010] Preferably, the product is a microbial preparation or a pharmaceutical product.

[0011] Preferably, the pro-inflammatory factor is interleukin-6.

[0012] Preferably, the anti-inflammatory factor is interleukin 10 and / or interleukin 12.

[0013] A strain composition comprises Lactobacillus casei BT5-B6 used in the above application.

[0014] Preferably, it also includes: Lactobacillus plantarum ( Lactiplantibacillus plantarum )BM2-LA7; the ratio of the number of viable bacteria of Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 was 0.8-1.2:0.8-1.2.

[0015] A composite preparation, the raw materials of which include, by mass, 5-9 parts of the above-mentioned strain composition, 1-2 parts of gelatin, 1-2 parts of guar gum, 1-2 parts of surfactant, 1-3 parts of transglutaminase solution with a concentration of 600-1000U / mL, 0.1-0.24 parts of calcium chloride, 1-2 parts of oxidized polysaccharide, and 0.1-1 parts of amino-terminated polyamidoamine; the total live bacteria concentration of the above-mentioned strain composition is 1-3×10 9 cfu / g.

[0016] Preferably, the oxidized polysaccharide is oxidized dextran, which is obtained by oxidizing dextran with sodium periodate.

[0017] Oxidized dextran was prepared by the following steps: 4 g of dextran and 3.4 g of sodium periodate were dissolved in 50 mL of deionized water, stirred and reacted for 24 h in the dark, and the solution was bright yellow; 1 g of ethylene glycol was added to terminate the reaction. The solution was transferred to a dialysis bag and dialyzed with deionized water for 72 h in the dark until the solution was clear and colorless; after freeze drying, it was placed in a 4°C refrigerator for later use.

[0018] Preferably, the surfactant is at least one of polyoxyethylene polyoxypropylene pentaerythritol ether, polyglycerol fatty acid ester, polyglycerol ricinoleate, and calcium stearoyl lactylate.

[0019] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add the above strain composition, gelatin, guar gum and surfactant to water and stir evenly, then add dropwise to the stirred vegetable oil, stir for 20-30 min after complete addition, add transglutaminase solution and stir for 5-15 min, and let stand at 40-50° C. for 1-2 h to obtain a prefabricated material; S2, adding a 10-12% by mass calcium chloride aqueous solution to the prefabricated material, stirring for 5-15 minutes, standing for 5-10 hours, filtering, washing, and vacuum drying to obtain pellets; S3. Add oxidized polysaccharide and amino-terminated polyamidoamine to a phosphate buffer solution with a pH of 7.1-7.4, stir at 70-80°C for 5-15 minutes, cool to room temperature, spray the solution on the surface of the pellets for encapsulation, and vacuum dry.

[0020] Preferably, the vegetable oil is at least one of soybean oil, rapeseed oil, peanut oil and olive oil. Beneficial effect: The present invention finds that Lactobacillus casei BT5-B6 can effectively reduce the content of pro-inflammatory factors and increase the content of anti-inflammatory factors, has significant anti-inflammatory effect, effectively enhances immunity, and has good bile salt tolerance. The present invention has found through experiments that the anti-inflammatory effect of compounding Lactobacillus casei BT5-B6 with Lactobacillus plantarum BM2-La7 is excellent, which is much higher than using Lactobacillus casei BT5-B6 alone, and significantly enhances the body's immunity.

[0021] The present invention compounds the strain composition with gelatin and cross-links them through transglutaminase catalysis, and then intersperses them with guar gum polysaccharide macromolecules after cross-linking, which has extremely high microbial coating stability and enhanced mechanical strength. The granules are further embedded, which not only has good biocompatibility, but also can further improve the stability of the microorganisms and effectively exert their anti-inflammatory activity. At the same time, it can promote the colonization of the strain composition in the intestine and exert a strong anti-inflammatory effect in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the colony morphology of Lactobacillus casei BT5-B6.

[0023] Figure 2 This is a microscopic photo of Lactobacillus casei BT5-B6.

[0024] Figure 3 This is the curve of the tolerance level of Lactobacillus casei BT5-B6 changing with pH value.

[0025] Figure 4 This is the bile salt tolerance curve of Lactobacillus casei BT5-B6 changing with bile salt concentration.

[0026] Figure 5 This is a photo of the colony morphology of Lactobacillus plantarum BM2-La7.

[0027] Figure 6 This is a microscopic photo of Lactobacillus plantarum BM2-La7.

[0028] Figure 7 This is a comparison chart of the sum of IL-10 and IL-12 levels released by THP-1 macrophages in the normal group, model group, experimental group, BT5-B6 bacterial solution group, BM2-LA7 bacterial solution group and bacterial solution combination group.

[0029] Figure 8 This is the tolerance level curve of Lactobacillus casei BT5-B6, Lactobacillus plantarum BM2-La7 and the combined strain of Lactobacillus casei BT5-B6 + Lactobacillus plantarum BM2-La7 changing with pH value.

[0030] Fig. 9 This is the bile salt tolerance curve of Lactobacillus casei BT5-B6, Lactobacillus plantarum BM2-La7 and the combined strain of Lactobacillus casei BT5-B6 + Lactobacillus plantarum BM2-La7 as the bile salt concentration changes.

[0031] Fig.10 This is a comparison chart of the serum IL-6 and IL-10 levels of mice in the normal control group, model group, BT5-B6 group, BM2-LA7 group, and BB group.

[0032] Fig.11 This is a comparison chart of the spleen index of mice in the normal control group, model group, BT5-B6 group, BM2-LA7 group, and BB group.

[0033] Fig.12 This is a graph showing the changes in intestinal microbial community genus level in the normal control group, model group, and BB group of mice.

[0034] Fig.13 It is a comparison chart of the embedding efficiency of the composite preparations obtained in Example 12 and Comparative Examples 1-2.

[0035] Fig.14This is a comparison chart of the index of the number of viable bacteria after the composite preparations obtained in Example 12 and Comparative Examples 1-2 were treated with bile salts.

[0036] Fig.15 This is a comparison chart of the survival rates of the composite preparations obtained in Example 12 and Comparative Examples 1-2 after being treated with simulated digestive fluid in vitro.

[0037] Fig.16 This is a comparison chart of the serum IL-6 and IL-10 levels of mice in the normal control group, model group, BB group, SS12 group, DB1 group, and DB2 group. DETAILED DESCRIPTION

[0038] The present invention will be further explained below in conjunction with specific embodiments.

[0039] The MRS culture medium used below was purchased from Qingdao Hibo Biotechnology.

[0040] Example 1: The strain was isolated and collected by Huang Dasheng from a homemade fermentation broth sample in Shangcheng District, Hangzhou City, Zhejiang Province, China in May 2024.

[0041] Isolation and purification of strains: From the homemade fermentation broth sample, 100 μL of the sample was taken into 900 μL of sterile water, and then the sample was diluted to 10 -4 , 10 -5 concentration, apply 100 μL to the MRS plate, place it in a 37℃ incubator for 48 hours, and observe the bacterial growth on the plate. After the colonies grow, pick a typical single colony and streak it on the MRS medium for strain isolation and purification. Inoculate a single colony into 5 mL of sterilized MRS broth medium, incubate at 37℃ for 48 hours, and then store it in a -80℃ refrigerator with 50% glycerol for later use.

[0042] The isolated strains were identified by morphology (colony morphology such as Figure 1 As shown, the microscopic morphology Figure 2 as shown).

[0043] The isolated and purified single colonies were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing identification. The primers are as follows: 27F: 5'-AGAGTTTGATCMTGGCTCAG-3'; 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0044]

[0045] The sequence was compared and analyzed using the BLAST program in NCBI. The results showed that it was Lactobacillus casei, and its homology with the known Lactobacillus casei in the GeneBank database reached 99%. It was named Lactobacillus casei ( Lacticaseibacilus casei )BT5-B6.

[0046] Lactobacillus casei BT5-B6 was deposited in the General Microbiology Center of China Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​September 20, 2024, with the deposit number CGMCC NO.32013 and the classification name: Lacticaseibacilus casei.

[0047] Example 2 Effect of Lactobacillus casei BT5-B6 on anti-inflammatory factors IL-10 and IL-12 (1) Preparation of strain supernatant Lactobacillus casei BT5-B6 strain OD 600nm After adjusting to 1.0, inoculate it into MRS liquid culture medium at a 2% inoculation rate. After culturing at 37°C overnight for 24 hours, centrifuge at 5000r / min for 5 minutes, take the supernatant and filter it through a bacterial filter membrane, and place it at 4°C for use.

[0048] The supernatant of Lactobacillus plantarum BM2-La7 was obtained according to the above operation.

[0049] (2) Preparation of THP-1 macrophages The THP-1 macrophage suspension and RPMI-1640 medium were placed in a T25 flask at a ratio of 1:1 and cultured in a 37°C 5% CO2 cell culture incubator. When the cell density grew to 2×10 5 cells / mL, keep aside.

[0050] (3) Anti-inflammatory experiment of strain supernatant The above THP-1 macrophages were inoculated into a 24-well cell culture plate (1 mL / well). After the cells adhered overnight, the following were added: ① Normal group (CK): only the same volume of complete culture medium was added to the cell culture wells; ②Model group (MC): LPS with a final mass concentration of 2 μg / mL was added to the cell culture wells; ③ Drug treatment group (PC): LPS and quercetin with a final mass concentration of 2 μg / mL were added to the cell culture wells; ④ Bacteria solution treatment group (BT5-B6): LPS with a final mass concentration of 2 μg / mL and the above-mentioned Lactobacillus casei BT5-B6 supernatant were added to the cell culture wells; Then, after culturing in a 37°C 5% CO2 cell culture incubator for 24 hours, the cell supernatant was collected and the levels of inflammatory factors IL-10 and IL-12 were detected using an ELISA kit.

[0051] Through the above experimental method, the sum of the contents of macrophage inflammatory factors IL-10 and IL-12 detected in the supernatant of Lactobacillus casei BT5-B6 was 55.725 pg / mL.

[0052] Example 3 Effect of different pH on the survival rate of Lactobacillus casei BT5-B6 Lactobacillus casei BT5-B6 was inoculated into MRS culture medium with initial pH=3.0, 4.0, 5.0, 6.0, and 7.0 at an inoculum size of 2%, and cultured anaerobically at 37°C for 24 h. The OD value of the strain in the culture medium with different initial pH was measured at a wavelength of 600 nm to analyze its tolerance level.

[0053] like Figure 3 As shown, as the pH value increased, the OD value of Lactobacillus casei BT5-B6 continued to increase.

[0054] Example 4 Effect of different bile salt concentrations on bile salt tolerance of Lactobacillus casei BT5-B6 Add 0.2% ( w / v ) sodium thioglycolate, and then continue to add 0%, 0.03%, 0.1%, 0.2%, 0.3% ( w / v ) bile salts were added to adjust the pH to 7.2, and MRS liquid culture medium without bile salts was used as a control.

[0055] The bacterial suspension of Lactobacillus casei BT5-B6 was diluted to 2% ( v / v ) were added into MRS liquid culture medium containing different bile salt concentrations, and three parallel replicate groups were set up and cultured anaerobically at 37°C for 24 h.

[0056] Using MRS liquid culture medium as blank control, the OD value of the strain in culture medium with different bile salt concentrations was measured at a wavelength of 600nm to analyze its tolerance level.

[0057] like Figure 4 As shown, with the increase of bile salt concentration, the bile salt tolerance of Lactobacillus casei BT5-B6 showed an overall downward trend.

[0058] Example 5 Detection of anti-inflammatory factors IL-10 and IL-12 (1) Preparation of BM2-LA7 supernatant OD of Lactobacillus plantarum BM2-La7 strain 600nmAfter adjusting to 1.0, inoculate it into MRS liquid culture medium at a 2% inoculation rate. After culturing at 37°C overnight for 24 hours, centrifuge at 5000r / min for 5 minutes, take the supernatant and filter it through a bacterial filter membrane, and put it at 4°C for use.

[0059] Among them, the colony morphology of Lactobacillus plantarum BM2-La7 is as follows Figure 5 Its microscopic morphology is shown in Figure 6

[0060] Lactobacillus plantarum BM2-La7 was deposited in the General Microbiology Center of China Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​September 20, 2024, with the deposit number CGMCC NO.32014 and the classification name: Lactiplantibacillus plantarum.

[0061] (2) Preparation of supernatant of composite strains Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 were inoculated into MRS liquid culture medium at the same concentration and volume, cultured overnight at 37°C for 24 hours, centrifuged at 5000r / min for 5 minutes, and the supernatant was filtered through a bacterial filter and placed at 4°C for later use.

[0062] (3) Preparation of THP-1 macrophages The THP-1 macrophage suspension and RPMI-1640 medium were placed in a T25 flask at a ratio of 1:1 and cultured in a 37°C 5% CO2 cell culture incubator. When the cell density grew to 2×10 5 cells / mL, keep aside.

[0063] (4) Conducting anti-inflammatory experiments on bacterial supernatants The above THP-1 macrophages were inoculated into a 24-well cell culture plate (1 mL / well). After the cells adhered overnight, the following were added: ① Normal group (CK): only the same volume of complete culture medium was added to the cell culture wells; ②Model group (MC): LPS with a final mass concentration of 2 μg / mL was added to the cell culture wells; ③ Drug treatment group (PC): LPS and quercetin with a final mass concentration of 2 μg / mL were added to the cell culture wells; ④ Bacterial solution treatment group (BM2-LA7): LPS with a final mass concentration of 2 μg / mL and the above-mentioned Lactobacillus plantarum BM2-La7 supernatant were added to the cell culture wells; ⑤ Bacterial solution complex group (BT5-B6 + BM2-LA7): LPS with a final mass concentration of 2 μg / mL and the supernatant of the above-mentioned complex strains were added to the cell culture wells; Then, after culturing in a 37°C 5% CO2 cell culture incubator for 24 hours, the cell supernatant was collected and the content of anti-inflammatory factors IL-10 and IL-12 was detected using an ELISA kit.

[0064] like Figure 7As shown, the MC group used lipopolysaccharide to stimulate macrophages, resulting in the lowest content of anti-inflammatory factors; while quercetin, as an anti-inflammatory drug, can promote the secretion of anti-inflammatory factors by macrophages, making the content of anti-inflammatory factors in the PC group higher than that in the MC group, but still lower than that in the CK group.

[0065] The content of anti-inflammatory factors in the BT5-B6 group was higher than that in the PC group, that is, the supernatant of Lactobacillus casei BT5-B6 showed better anti-inflammatory effect than quercetin; and the content of anti-inflammatory factors in the BT5-B6+BM2-LA7 group was the highest, which was 1.33 times that of the PC group, showing excellent anti-inflammatory effect.

[0066] Example 6 Effect of different pH on strain survival rate Lactobacillus plantarum BM2-La7 was inoculated at a 2% inoculation rate into MRS culture medium with initial pH = 3.0, 4.0, 5.0, 6.0, and 7.0, respectively, and cultured anaerobically at 37°C for 24 h. The OD value of the strain in the culture medium with different initial pH was measured at a wavelength of 600 nm to analyze its tolerance level.

[0067] Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 were mixed at the same concentration and volume, and then inoculated into MRS culture medium with initial pH = 3.0, 4.0, 5.0, 6.0, and 7.0 at an inoculum size of 2%, respectively. The culture was anaerobically cultured at 37°C for 24 h, and the OD values ​​of the strains in the culture medium with different initial pH values ​​were measured at a wavelength of 600 nm to analyze their tolerance levels.

[0068] The results are as follows Figure 8 As shown, with the increase of pH value, the tolerance level of each group increased and then remained stable, while the tolerance level of the composite strain was better than that of single Lactobacillus casei BT5-B6 at pH 4.0-6.0.

[0069] Example 7 Effect of different bile salt concentrations on the bile salt tolerance of strains The bacterial suspension of Lactobacillus plantarum BM2-La7 was diluted to 2% ( v / v ) were added into MRS liquid culture medium containing different bile salt concentrations, and three parallel replicate groups were set up and cultured anaerobically at 37°C for 24 h.

[0070] Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 were mixed at the same concentration and volume, and then 2% ( v / v ) were added into MRS liquid culture medium containing different bile salt concentrations, and three parallel replicate groups were set up and cultured anaerobically at 37°C for 24 h.

[0071] Using MRS liquid culture medium as blank control, the OD value of the strain in culture medium with different bile salt concentrations was measured at a wavelength of 600nm to analyze its tolerance level.

[0072] like Fig. 9 As shown, with the increase of bile salt concentration, the bile salt tolerance of the composite strain also showed a downward trend.

[0073] Example 8: The experimental animals were 4 / cage (5-week-old male BALB / c mice), with room temperature of 23-25°C, humidity of 40-45%, 12 hours of light per day, free access to food and water, and regular replacement of bedding.

[0074] The mice were randomly divided into five groups, with eight mice in each group: normal control group (CK), model group (MC), BT5-B6 group (BT5-B6), BM2-LA7 group (BM2-LA7) and BB group.

[0075] The mice were adaptively fed for 1 week and then intervened for 4 weeks. During the intervention period, they were allowed to eat and drink freely, and were fed with conventional feed (25% bran, 20% flour, 20% corn, 20% bean feed, 10% rice flour, 2.5% fish meal, and 2.5% bone meal). The mice were given 12 hours of light per day and were gavaged once a day at a fixed time (gavage volume: 0.2 mL / d / mouse). Except for the normal control group, the other groups were intraperitoneally injected with cyclophosphamide (80 mg / kg) for 3 consecutive days starting from the 22nd day of intervention.

[0076] The intervention treatments of each group are shown in Table 1: Table 1 Intervention treatments of each group

[0077] One day before the end of the experiment, collect 3-5 pieces of mouse feces and fast the mice for less than 12 hours. Then store the collected feces at -80℃.

[0078] After the experiment, the blood of mice was collected to measure the levels of serum IL-6 and IL-10 inflammatory factors; at the same time, the spleen was collected, accurately weighed, and its immune organ index was calculated.

[0079] like Fig.10 As shown in the figure, compared with the CK group, the serum IL-6 content of the mice in the MC group was significantly increased, and the serum IL-10 content was significantly decreased; compared with the MC group, the serum IL-10 content of the BT5-B6 group, the BM2-LA7 group, and the BB group was significantly increased, and the serum IL-6 content was significantly decreased, but none of them reached the level of the CK group. The serum IL-6 content of the BB group was significantly lower than that of the BT5-B6 group and the BM2-LA7 group, while the serum IL-10 content was significantly higher than that of the BT5-B6 group and the BM2-LA7 group.

[0080] This indicates that oral administration of Lactobacillus casei BT5-B6 or Lactobacillus plantarum BM2-La7 alone can effectively inhibit the inflammatory response in mice, while the combined oral administration of Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 can produce a synergistic effect and further inhibit the inflammatory response in mice.

[0081] like Fig.11 As shown in the figure, compared with the CK group, the spleen index of mice in the MC group was significantly reduced, indicating that the immune function of mice in the MC group was weakened; compared with the MC group, the spleen index of mice in the BT5-B6 group, BM2-LA7 group, and BB group was significantly increased, indicating that the immune function of mice in the BT5-B6 group, BM2-LA7 group, and BB group was enhanced. The spleen index of mice in the BB group was significantly higher than that in the BT5-B6 group, confirming that the combined intragastric administration of Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 can synergistically enhance the immune function of mice.

[0082] through Fig.10 and Fig.11 It can be seen that the combined intragastric administration of Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 can produce a synergistic effect, enhance the immunity of mice, and inhibit the inflammatory response in mice. Therefore, the applicant selected the feces of mice in the CK group and the BB group for 16S rRNA gene high-throughput sequencing to analyze the intestinal flora structure of mice.

[0083] The intestinal microbiota is mainly composed of Bacteroidetes and Firmicutes, which are essential for metabolism and immunity. Unclassified Bacteroidetes is a bacterial family in the order Bacteroidetes. It produces short-chain fatty acids through endogenous (mucin polysaccharides) and exogenous polysaccharides (dietary fiber), providing part of the energy required by the human body, regulating electrolyte balance, and protecting the intestinal mucosal barrier. At the same time, this family has a cross-feeding relationship with probiotics such as Bifidobacterium and Lactobacillus, which can promote the growth of probiotics in the intestine. And related studies have found that the alleviating effect of a plant-based diet on inflammatory bowel disease, obesity, and type 2 diabetes is related to the increase in its relative abundance.

[0084] Depend on Fig.12 It can be seen that the relative abundance of unclassified Murine Bacillus family in the BB group was 24.74%, while the relative abundance of unclassified Murine Bacillus family in the CK group was 6.58%, indicating that the combined gavage of Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 to inflammatory mice increased the relative abundance of unclassified Murine Bacillus family by about 18.16% compared with the CK group, indicating that the combined gavage of BT5-B6 and BM2-La7 can promote the significant increase in the relative abundance of unclassified Murine Bacillus family in the intestines of inflammatory mice, thereby achieving the purpose of regulating the balance of intestinal flora in inflammatory mice, reducing the content of inflammatory factors in mice, and promoting the production of anti-inflammatory factors.

[0085] Example 9: A bacterial strain composition, comprising: Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7. The live bacterial concentration of Lactobacillus casei BT5-B6 is 1×10 9 cfu / g, and the live bacterial concentration of Lactobacillus plantarum BM2-La7 was 1×10 9 cfu / g.

[0086] Example 10: A composite preparation, whose raw materials include: 5 g of the strain composition obtained in Example 9, 1 g of gelatin, 1 g of guar gum, 1 g of calcium stearoyl lactylate, 1 g of transglutaminase solution with a concentration of 6000 U / mL, 0.1 g of calcium chloride, 1 g of oxidized dextran, and 0.1 g of 2.0-generation amino-terminated polyamidoamine.

[0087] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add the above strain composition, gelatin, guar gum, and calcium stearoyl lactylate to 20 g of water and stir evenly, then add dropwise to 50 g of rapeseed oil in a stirring state, stir at a speed of 2000 r / min for 20 min after complete addition, add transglutaminase solution thereto, stir for 5 min, and stand at 40 ° C for 1 h to obtain a prefabricated material; S2, adding 10% by mass calcium chloride aqueous solution to the prefabricated material, stirring for 5 minutes, standing for 5 hours, filtering, washing, and vacuum drying to obtain pellets; S3. Add oxidized dextran and amino-terminated polyamidoamine to 5 g of phosphate buffer solution with a pH of 7.1-7.4, stir at 70°C for 5 min, cool to room temperature, spray on the surface of the pellets for embedding, and vacuum dry.

[0088] Example 11: A composite preparation, whose raw materials include: 9 g of the strain composition obtained in Example 9, 2 g of gelatin, 2 g of guar gum, 2 g of polyglycerol ricinoleate, 3 g of transglutaminase solution with a concentration of 1000 U / mL, 0.24 g of calcium chloride, 2 g of oxidized dextran, and 1 g of 2.0-generation amino-terminated polyamidoamine.

[0089] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add the above strain composition, gelatin, guar gum and polyglycerol ricinoleate to 30 g of water and stir evenly, then add dropwise to 100 g of peanut oil in a stirring state, stir at a speed of 5000 r / min for 30 min after complete addition, add transglutaminase solution thereto and stir for 15 min, and let stand at 50° C. for 2 h to obtain a prefabricated material; S2, adding a 12% by mass calcium chloride aqueous solution to the prefabricated material, stirring for 15 minutes, standing for 10 hours, filtering, washing, and vacuum drying to obtain pellets; S3. Add oxidized dextran and amino-terminated polyamidoamine to 12 g of phosphate buffer solution with a pH of 7.1-7.4, stir at 80°C for 15 min, cool to room temperature, spray on the surface of the pellets for embedding, and vacuum dry.

[0090] Example 12: A composite preparation, whose raw materials include: 7 g of the strain composition obtained in Example 9, 1.5 g of gelatin, 1.5 g of guar gum, 1.5 g of polyglycerol ricinoleate, 1.5 g of transglutaminase solution with a concentration of 800 U / mL, 0.2 g of calcium chloride, 1.5 g of oxidized dextran, and 0.8 g of 2.0-generation amino-terminated polyamidoamine.

[0091] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add the above strain composition, gelatin, guar gum and polyglycerol ricinoleate to 25 g of water and stir evenly, then add dropwise to 80 g of soybean oil in a stirring state, stir at a speed of 3500 r / min for 25 min after complete addition, add transglutaminase solution thereto and stir for 10 min, and let stand at 45° C. for 1.5 h to obtain a prefabricated material; S2, adding a 10% by mass calcium chloride aqueous solution to the prefabricated material, stirring for 10 minutes, standing for 8 hours, filtering, washing, and vacuum drying to obtain pellets; S3. Add oxidized dextran and amino-terminated polyamidoamine to 8 g of phosphate buffer solution with a pH of 7.1-7.4, stir at 75°C for 10 min, cool to room temperature, spray on the surface of the pellets for embedding, and vacuum dry.

[0092] Comparative Example 1: A strain composition, comprising: Lactobacillus casei BT5-B6 and Lactobacillus plantarum Mle-3 (a strain owned by the applicant, with a deposit number of CGMCC NO.30556). The live bacterial concentration of Lactobacillus casei BT5-B6 is 1×10 9 cfu / g, and the live bacterial concentration of Lactobacillus plantarum Mle-3 was 1×10 9 cfu / g.

[0093] A composite preparation, whose raw materials include: 7g of the strain composition obtained in this comparative example, 1.5g of gelatin, 1.5g of guar gum, 1.5g of polyglycerol ricinoleate, 1.5g of transglutaminase solution with a concentration of 800U / mL, 0.2g of calcium chloride, 1.5g of oxidized dextran, and 0.8g of 2.0-generation amino-terminated polyamidoamine.

[0094] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add the above strain composition, gelatin, guar gum and polyglycerol ricinoleate to 25 g of water and stir evenly, then add dropwise to 80 g of soybean oil in a stirring state, stir at a speed of 3500 r / min for 25 min after complete addition, add transglutaminase solution thereto and stir for 10 min, and let stand at 45° C. for 1.5 h to obtain a prefabricated material; S2, adding a 10% by mass calcium chloride aqueous solution to the prefabricated material, stirring for 10 minutes, standing for 8 hours, filtering, washing, and vacuum drying to obtain pellets; S3. Add oxidized dextran and amino-terminated polyamidoamine to 8 g of phosphate buffer solution with a pH of 7.1-7.4, stir at 75°C for 10 min, cool to room temperature, spray on the surface of the pellets for embedding, and vacuum dry.

[0095] Comparative Example 2: A composite preparation, whose raw materials include: 7 g of the strain composition obtained in Example 9, 1.5 g of gelatin, 1.5 g of guar gum, 1.5 g of polyglycerol ricinoleate, 1.5 g of transglutaminase solution with a concentration of 800 U / mL, 0.2 g of calcium chloride, 1.5 g of oxidized dextran, and 0.8 g of 2.0-generation amino-terminated polyamidoamine.

[0096] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add the above strain composition, gelatin, guar gum and polyglycerol ricinoleate to 25 g of water and stir evenly, then add dropwise to 80 g of soybean oil in a stirring state, stir at a speed of 3500 r / min for 25 min after complete addition, add transglutaminase solution thereto and stir for 10 min, and let stand at 45° C. for 1.5 h to obtain a prefabricated material; S2. Add 10% by mass calcium chloride aqueous solution to the prefabricated material and stir for 10 minutes, let stand for 8 hours, filter, wash and vacuum dry.

[0097] A comparative experiment was conducted using the composite preparations obtained in Example 12 and Comparative Examples 1-2, as follows: 1. Determination of embedding rate 1 g of each composite preparation was added to an appropriate amount of 0.06 mol / L sodium citrate solution, and the mixture was shaken at 180 r / min in a constant temperature shaker at 37°C for 60 min. The live Lactobacillus casei in each group was counted and the embedding rate was calculated.

[0098] Wherein, N is the number of live Lactobacillus casei released from the composite formulation; N0 is the number of free Lactobacillus casei in the mixture during the production of the composite formulation.

[0099] like Fig.13As shown, the embedding rates of the composite preparations obtained in Example 12 and Comparative Example 1 were similar, with no significant difference (P>0.05), but the embedding rates of both were lower than those of Comparative Example 2 (P<0.05).

[0100] The applicant believes that this is because both Example 12 and Comparative Example 1 adopt double-layer embedding, which leads to a decrease in the embedding rate of both.

[0101] 2. Bile salt tolerance test Each group of composite preparations was inoculated into MRS liquid culture medium with a bile salt concentration of 0.3% (w / v) at a volume fraction of 10%, and then placed in a 37°C incubator for 4 hours. Then, an appropriate amount of 0.06 mol / L sodium citrate solution was added and mixed, and the mixture was shaken at a speed of 180 r / min for 60 minutes in a 37°C constant temperature shaker to count the live Lactobacillus casei in each group.

[0102] like Fig.14 As shown, the composite preparations obtained in Example 12 and Comparative Example 1 still maintained high activity after bile salt treatment, but there was no significant difference between the two (P>0.05), while the number of live Lactobacillus casei in Comparative Example 2 decreased significantly after bile salt treatment.

[0103] The applicant believes that this is because Comparative Example 2 only uses a single-layer embedding, and its embedding wall material is not tight enough, resulting in a significant decrease in the number of Lactobacillus casei.

[0104] 3. In vitro simulated digestive fluid tolerance test The simulated gastric fluid used was as follows: 0.5% NaCl solution was adjusted to pH 2.0 with 1 mol / L hydrochloric acid, pepsin was added to make the final concentration of the enzyme in the solution 3.0 g / L, and then sterilized by filtration with a 0.22 μm aqueous phase filter membrane.

[0105] The simulated intestinal fluid used was as follows: Trypsin, bile salts and phenol were added to 0.5% NaCl solution to make them evenly dispersed, and the final concentrations were 1.0 g / L, 5.0 g / L and 4.0 g / L, respectively. The pH value of the system was adjusted to 8.0 with 1 mol / L NaOH solution, and then filtered and sterilized with a 0.22 μm aqueous phase filter membrane.

[0106] Take 1 g of each composite preparation, add 9 mL of simulated gastric fluid, shake and mix the microcapsules and artificial gastric fluid evenly, then shake at 160 r / min in a constant temperature shaker at 37±1°C, centrifuge after 180 minutes to discard the simulated gastric fluid, add 9 mL of simulated intestinal fluid, shake and mix the remaining composite preparation and artificial intestinal fluid evenly, then shake at 160 r / min in a constant temperature shaker at 37±1°C, and count after 420 minutes.

[0107] For counting, the remaining compound preparation was washed with saline, dissolved in 9 mL of 0.06 mol / L sodium citrate solution, and after gradient dilution, live bacteria were counted using the spread plate method.

[0108] Among them, A1 is the number of Lactobacillus casei cells during the test, cfu / g; A0 is the number of Lactobacillus casei cells at the beginning of digestion, cfu / g.

[0109] like Fig.15 As shown, the composite preparations obtained in Example 12 and Comparative Example 1 still maintained high activity after being treated with simulated digestive fluid, but there was no significant difference between the two (P>0.05). However, the number of live Lactobacillus casei in Comparative Example 2 decreased significantly after being treated with simulated digestive fluid.

[0110] The applicant believes that this is because Comparative Example 2 only uses a single layer of embedding, and its embedding wall material is not tight enough, resulting in insufficient stability of the composite preparation, causing a significant decrease in the number of live Lactobacillus casei.

[0111] 4. In vivo experiments in mice The experimental animals were 4 / cage (5-week-old male BALB / c mice), with room temperature of 23-25°C, humidity of 40-45%, light for 12 h per day, free access to food and water, and regular replacement of bedding.

[0112] The mice were randomly divided into 3 groups, with 8 mice in each group: Example 12 group (SS12), Comparative Example 1 group (DB1) and Comparative Example 2 group (DB2).

[0113] The mice were adaptively fed for 1 week and then intervened for 4 weeks. During the intervention period, they were allowed to eat and drink freely, and were fed with conventional feed (25% bran, 20% flour, 20% corn, 20% soybean feed, 10% rice flour, 2.5% fish meal, and 2.5% bone meal), with 12 hours of light per day, and were gavaged once a day at a fixed time (gavage volume: 0.75 g / d / mouse).

[0114] Among them, Example 12 group was gavaged with the composite preparation obtained in Example 12, Comparative Example 1 group was gavaged with the composite preparation obtained in Comparative Example 1, and Comparative Example 2 group was gavaged with the composite preparation obtained in Comparative Example 2.

[0115] Each group received continuous intraperitoneal injection of cyclophosphamide (80 mg / kg) for 3 days starting from the 22nd day of intervention. After the experiment, the blood of mice was collected to measure the levels of serum IL-6 and IL-10 inflammatory factors.

[0116] The normal control group, model group, and BB group continued to use the data in Example 8 above.

[0117] like Fig.16As shown in the figure, compared with the MC group and the BB group, the serum IL-10 content of mice in the SS12 group and the DB2 group was significantly increased, and the serum IL-6 content was significantly decreased. The serum IL-10 content of mice in the SS12 group was significantly higher than that in the DB2 group, while the serum IL-6 content was significantly lower than that in the DB2 group.

[0118] Meanwhile, the serum IL-10 content of mice in the SS12 group was significantly higher than that in the CK group, while the serum IL-6 content was significantly lower than that in the CK group, indicating that the composite preparation obtained in Example 12 can effectively inhibit inflammatory response and enhance immunity.

[0119] The applicant believes that this is because the strain composition is compounded with gelatin in the present invention, cross-linked by transglutaminase, and then interspersed with guar gum polysaccharide macromolecules after cross-linking, which has extremely high microbial coating stability and enhanced mechanical strength. The granules are further embedded, which not only has good biocompatibility, but also can further improve the stability of the microorganisms and effectively exert their anti-inflammatory activity. At the same time, it can promote the colonization of the strain composition in the intestine and exert a strong anti-inflammatory effect in the body.

[0120] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Lactobacillus casei ( Lacticaseibacilus casei ) BT5-B6 is used in any of the following (1)-(5): (1) Preparation of products for improving immunity; (2) preparing products that reduce the content of pro-inflammatory factors; (3) preparing products with increased content of anti-inflammatory factors; (4) Preparation of products for inhibiting the growth of Helicobacter in the intestine; (5) Preparing products that promote the growth of Bacillus family and / or Bacillus family in the intestine.

2. The application according to claim 1, characterized in that: Lactobacillus casei BT5-B6 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 20, 2024, with the deposit number CGMCC NO.32013.

3. The application according to claim 1, characterized in that: The product is a microbial preparation or medicine.

4. The use according to claim 1, characterized in that: The pro-inflammatory factor is interleukin-6; the anti-inflammatory factor is interleukin-10 and / or interleukin-12.

5. A strain composition, characterized in that: The invention comprises the Lactobacillus casei BT5-B6 used in the application as claimed in claim 1.

6. The strain composition according to claim 5, characterized in that: Also includes: Lactobacillus plantarum ( Lactiplantibacillus plantarum )BM2-LA7; the ratio of the number of viable bacteria of Lactobacillus casei BT5-B6 and Lactobacillus plantarum BM2-La7 was 0.8-1.2:0.8-1.

2.

7. A composite preparation, characterized in that: The raw materials include, by mass: 5-9 parts of the strain composition as claimed in claim 5 or 6, 1-2 parts of gelatin, 1-2 parts of guar gum, 1-2 parts of surfactant, 1-3 parts of transglutaminase solution with a concentration of 600-1000 U / mL, 0.1-0.24 parts of calcium chloride, 1-2 parts of oxidized polysaccharide, and 0.1-1 parts of amino-terminated polyamidoamine; The total viable bacterial concentration of the strain composition according to claim 5 or 6 is 1-3×10 9 cfu / g.

8. The composite formulation according to claim 7, characterized in that: The oxidized polysaccharide is oxidized dextran, which is obtained by oxidizing dextran with sodium periodate.

9. The composite formulation according to claim 7, characterized in that: The surfactant is at least one of polyoxyethylene polyoxypropylene pentaerythritol ether, polyglycerol fatty acid ester, polyglycerol ricinoleate, and calcium stearoyl lactylate.

10. A method for preparing the composite formulation according to any one of claims 7 to 9, characterized in that: The steps include: S1. Add the strain composition as claimed in claim 5 or 6, gelatin, guar gum, and surfactant into water and stir evenly, add dropwise into the stirred vegetable oil, stir for 20-30 min after complete addition, add transglutaminase solution thereto and stir for 5-15 min, and stand at 40-50° C. for 1-2 h to obtain a prefabricated material; S2, adding a 10-12% by mass calcium chloride aqueous solution to the prefabricated material, stirring for 5-15 minutes, standing for 5-10 hours, filtering, washing, and vacuum drying to obtain pellets; S3. Add oxidized polysaccharide and amino-terminated polyamidoamine to a phosphate buffer solution with a pH of 7.1-7.4, stir at 70-80°C for 5-15 minutes, cool to room temperature, spray the solution on the surface of the pellets for encapsulation, and vacuum dry.

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