Application of phytobacterium plantarum BT1-1, strain composition and compound preparation

Abstract

The invention relates to the technical field of microbial engineering, in particular to application of phytobacterium plantarum BT1-1, a strain composition and a compound preparation. The lactobacillus plantarum BT1-1 is applied to any one of the following items (1) to (5): (1) preparing a product capable of improving the immunity, (2) preparing a product capable of improving the immunity, (3) preparing a product capable of improving the immunity, (4) preparing a product capable of improving the immunity, and (5) preparing a product capable of 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 is prepared from the plant lactobacillus BT1-1.

Description

Technical Field

[0001] The invention relates to the technical field of microbial engineering, and in particular to the application of Lactobacillus plantarum BT1-1 and a strain composition and a compound 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 new probiotic strains that can relieve and inhibit inflammation. At the same time, the types of inflammatory factors that can be regulated by existing plant lactobacilli are limited, but in inflammation caused by different reasons, the types of inflammatory factors that change are different, and the effects of different inflammatory factors on the body are different. Summary of the invention

[0007] The purpose of the present invention is to solve the shortcomings of the prior art and to propose the application of Lactobacillus plantarum BT1-1 and the strain composition and compound preparation.

[0008] Lactobacillus plantarum ( Lactiplantibacillus plantarum) Application of BT1-1 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 plantarum BT1-1 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration on September 20, 2024, with the deposit number CGMCC NO.32011.

[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 the Lactobacillus plantarum BT1-1 used in the above application.

[0014] Preferably, it also includes: fermentation of mucus lactobacillus ( Limosilactobacillus fermentum )FM-12; the ratio of the number of viable bacteria of plant lactobacillus BT1-1 and fermented mucus lactobacillus FM-12 is 0.8-1.2:0.8-1.2.

[0015] A composite preparation, the raw materials of which include, by mass, 5-10 parts of the above-mentioned strain composition, 1-2 parts of amino-terminated polyamidoamine, 1-5 parts of glutathione, 0.01-0.1 parts of transglutaminase, 1-2 parts of sodium alginate, 1-2 parts of surfactant, and 0.03-0.15 parts of calcium chloride; the total live bacteria concentration of the strain composition is 1.5-3.5×10 9 cfu / g.

[0016] Preferably, the amino-terminated polyamidoamine has a generation number of 1.0-3.0.

[0017] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add amino-terminated polyamidoamine to water and stir evenly, add glutathione thereto and stir for 10-20 min, add transglutaminase thereto, stir at 50-60° C. for 1-2 h, heat to inactivate the enzyme, cool, add the above strain composition and sodium alginate and stir evenly to obtain phase A; S2. Stir the surfactant and vegetable oil evenly, add phase A dropwise thereto under stirring, stir for 10-30 minutes after the addition is complete, add 3-5% calcium chloride solution by mass and continue stirring for 5-15 minutes, let stand for 10-15 hours, filter, wash, and freeze-dry.

[0018] Preferably, the vegetable oil is at least one of soybean oil, rapeseed oil, peanut oil and olive oil.

[0019] Preferably, the surfactant is anhydrous sorbitan fatty acid ester or / and polyoxyethylene anhydrous sorbitan fatty acid ester. Beneficial effect: The plant lactobacillus BT1-1 of the present invention can effectively reduce the content of pro-inflammatory factors and increase the content of anti-inflammatory factors, has a significant anti-inflammatory effect, effectively enhances immunity, and has a good bile salt tolerance effect. The present invention has been found through experiments that the anti-inflammatory effect of compounding plant lactobacillus BT1-1 with fermented mucus lactobacillus FM-12 is excellent, which is much higher than using plant lactobacillus BT1-1 alone, and significantly enhances the body's immunity.

[0020] The present invention combines the amino-terminated polyamidoamine with glutathione, which can not only significantly enhance the stability of glutathione, but also effectively load the strain composition, significantly enhance the stability of the preparation, have good bile salt tolerance, and have a high survival rate of the strain in artificial gastric juice and intestinal juice, and can stably exert the activity of the strain composition for a long time, and at the same time, have excellent adhesion performance, good growth and colonization effects, significantly enhance the antioxidant activity of probiotics, can be widely used in microbial preparations and medicines, improve the body's antioxidant capacity and immunity, and is suitable for large-scale promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a photo of the colony morphology of Lactobacillus plantarum BT1-1.

[0022] Figure 2 This is a microscopic photo of Lactobacillus plantarum BT1-1.

[0023] Figure 3 This is the curve of the tolerance level of Lactobacillus plantarum BT1-1 changing with pH value.

[0024] Figure 4 This is the curve of bile salt tolerance of Lactobacillus plantarum BT1-1 changing with bile salt concentration.

[0025] Figure 5 This is a photo of the colony morphology of fermented Lactobacillus mucilaginosus FM-12.

[0026] Figure 6 This is a microscopic photo of fermented mucus Lactobacillus FM-12.

[0027] Figure 7This 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, BT1-1 bacterial solution group, FM-12 bacterial solution group and bacterial solution combination group.

[0028] Figure 8 This is the tolerance level curve of Lactobacillus plantarum BT1-1, Lactobacillus fermentative mucus FM-12 and the combined strain of Lactobacillus plantarum BT1-1 + Lactobacillus fermentative mucus FM-12 as a function of pH value.

[0029] Fig. 9 This is the bile salt tolerance curve of Lactobacillus plantarum BT1-1, Lactobacillus fermentative mucus FM-12 and the combined strain of Lactobacillus plantarum BT1-1 + Lactobacillus fermentative mucus FM-12 as the bile salt concentration changes.

[0030] 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, BT1-1 group, FM-12 group, and BF group.

[0031] Fig.11 This is a comparison chart of the spleen index of mice in the normal control group, model group, BT1-1 group, FM-12 group, and BF group.

[0032] Fig.12 This is a graph showing the changes in intestinal microbial community genus level in the normal control group, model group, and BF group of mice.

[0033] Fig.13 It is a comparison chart of the embedding efficiency of the composite preparations obtained in Example 12 and Comparative Examples 1-2.

[0034] Fig.14 This 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.

[0035] 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.

[0036] 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, BF group, SS12 group, DB1 group, and DB2 group. DETAILED DESCRIPTION

[0037] The present invention will be further explained below in conjunction with specific embodiments.

[0038] The MRS culture medium used below was purchased from Qingdao Hibo Biotechnology.

[0039] 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.

[0040] 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.

[0041] The isolated strains were identified by morphology (colony morphology such as Figure 1 As shown, the microscopic morphology Figure 2 as shown).

[0042] 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'.

[0043]

[0044] The sequence was compared and analyzed using the BLAST program in NCBI. The results showed that it was Lactobacillus plantarum, and its homology with the known Lactobacillus plantarum in the GeneBank database reached 99.93%. It was named Lactobacillus plantarum ( Lactiplantibacillus plantarum )BT1-1.

[0045] Lactobacillus plantarum BT1-1 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.32011.

[0046] Example 2 Effect of Lactobacillus plantarum BT1-1 on anti-inflammatory factors IL-10 and IL-12 (1) Preparation of strain supernatant Lactobacillus plantarum BT1-1 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 put it at 4°C for use.

[0047] (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.

[0048] (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 (BT1-1): LPS with a final mass concentration of 2 μg / mL and the above-mentioned Lactobacillus plantarum BT1-1 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.

[0049] 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 plantarum BT1-1 was 54.620 pg / mL.

[0050] Example 3 Effect of different pH on the survival rate of Lactobacillus plantarum BT1-1 Lactobacillus plantarum BT1-1 was inoculated at an inoculation rate of 2% 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.

[0051] like Figure 3 As shown, as the pH value increased, the OD value of Lactobacillus plantarum BT1-1 increased and then remained stable.

[0052] Example 4 Effect of different bile salt concentrations on bile salt tolerance of Lactobacillus plantarum BT1-1 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.

[0053] The bacterial suspension of Lactobacillus plantarum BT1-1 was adjusted 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.

[0054] 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.

[0055] like Figure 4 As shown, with the increase of bile salt concentration, the bile salt tolerance of Lactobacillus plantarum BT1-1 showed an overall downward trend.

[0056] Example 5 Detection of anti-inflammatory factors IL-10 and IL-12 (1) Preparation of FM-12 supernatant Lactobacillus fermentans FM-12 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 put it at 4°C for use.

[0057] Among them, the colony morphology of fermented mucus Lactobacillus FM-12 is as follows Figure 5 Its microscopic morphology is shown in Figure 6

[0058] Limosilactobacillus fermentum FM-12 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.32012.

[0059] (2) Preparation of supernatant of composite strains Plant lactobacillus BT1-1 and fermentative mucus lactobacillus FM-12 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.

[0060] (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.

[0061] (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 Bacteria solution treatment group (FM-12): LPS with a final mass concentration of 2 μg / mL and the above-mentioned fermented Lactobacillus mucosa FM-12 supernatant were added to the cell culture wells; ⑤ Bacterial solution complex group (BT1-1 + FM-12): 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.

[0062] 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.

[0063] The content of anti-inflammatory factors in the BT1-1 group was slightly higher than that in the PC group, that is, the supernatant of Lactobacillus plantarum BT1-1 showed an anti-inflammatory effect similar to that of quercetin; and the content of anti-inflammatory factors in the BT1-1+FM-12 group was the highest, more than 1.4 times that of the PC group, showing an excellent anti-inflammatory effect.

[0064] Example 6 Effect of different pH on the survival rate of strains Fermented mucus Lactobacillus FM-12 was inoculated into MRS culture medium with initial pH = 3.0, 4.0, 5.0, 6.0, and 7.0 at an inoculation rate 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.

[0065] Lactobacillus plantarum BT1-1 and Lactobacillus fermentans FM-12 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 carried out 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.

[0066] 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 the single strain at pH=3.0.

[0067] Example 7 Effect of different bile salt concentrations on the bile salt tolerance of strains The bacterial suspension of fermented Lactobacillus mucilaginosus FM-12 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.

[0068] Lactobacillus plantarum BT1-1 and Lactobacillus fermentans FM-12 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.

[0069] 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.

[0070] like Fig. 9 As shown, as the bile salt concentration increased, the bile salt tolerance of the composite strain decreased; when the bile salt concentration reached 0.3% (human intestinal bile salt concentration), the composite strain still had a certain bile salt tolerance.

[0071] 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%, light exposure of 12 h per day, free access to food and water, and regular replacement of bedding.

[0072] The mice were randomly divided into 5 groups, with 8 mice in each group: normal control group (CK), model group (MC), BT1-1 group (BT1-1), FM-12 group (FM-12) and BF group.

[0073] 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.

[0074] The intervention treatments for each group are shown in Table 1 Table 1 Intervention treatments in each group

[0075] 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℃.

[0076] 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.

[0077] 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 BT1-1 group, FM-12 group, and BF 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 BF group was significantly lower than that of the BT1-1 group and the FM-12 group, while the serum IL-10 content was significantly higher than that of the BT1-1 group and the FM-12 group.

[0078] This shows that oral administration of Lactobacillus plantarum BT1-1 or fermented Lactobacillus mucin FM-12 alone can effectively inhibit the inflammatory response in mice, while the combined oral administration of Lactobacillus plantarum BT1-1 and fermented Lactobacillus mucin FM-12 can produce a synergistic effect and further inhibit the inflammatory response in mice.

[0079] like Fig.11 As shown in the figure, compared with the CK group, the spleen index of the mice in the MC group was significantly smaller, indicating that the immune function of the mice in the MC group was weakened; compared with the MC group, the spleen index of the mice in the BT1-1 group, FM-12 group, and BF group was significantly increased, indicating that the immune function of the mice in the BT1-1 group, FM-12 group, and BF group was enhanced. The spleen index of the mice in the BF group was significantly higher than that in the BT1-1 group and FM-12 group, confirming that the combined intragastric administration of Lactobacillus plantarum BT1-1 and Lactobacillus fermentum FM-12 can produce a synergistic effect to enhance the immune function of mice.

[0080] through Fig.10 and Fig.11 It can be seen that the combined intragastric administration of Lactobacillus plantarum BT1-1 and Lactobacillus fermentans FM-12 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, MC group, and BF group for 16S rRNA gene high-throughput sequencing to analyze the intestinal flora structure of mice.

[0081] like Fig.12 As shown, compared with the normal control group, the harmful bacteria Helicobacter genus ( Helicobacter The relative abundance of Ruminococcaceae increased significantly (P < 0.05). Related studies have shown that Ruminococcaceae is negatively correlated with immune response.

[0082] Beneficial bacteria such as host-associated Lactobacillus spp. Ligilactobacillus ), Bacteroides ( Bacteroides ) abundance was significantly decreased (P<0.05). Bacteroides is a resident flora in the human intestine. Related studies have shown that Bacteroides can regulate the immune system and enhance the host's immune function by producing capsular polysaccharide A, and can also produce short-chain fatty acids to maintain intestinal homeostasis.

[0083] Compared with the normal control group, the number of harmful bacteria Helicobacter spp. in the intestine of mice in the BF group was higher than that in the control group. Helicobacter) decreased in abundance from 4.95% to 1.54%, while the abundance of beneficial bacteria, Muribaculaceae, increased. Muribaculaceae is a type of intestinal symbiotic bacteria commonly found in the intestines of animals and is essential for the health of the host. They maintain intestinal homeostasis through multiple pathways such as competition, production of antibacterial substances, and regulation of the immune system. In addition, intestinal probiotics, Lactobacillus spp. ( Lactobacillus ), host-associated Lactobacillus spp. Ligilactobacillus ) and Lactobacillus mucilaginosus ( Limosilactobacillus ) increased significantly, from 2.01% to 10.83%, from 2.61% to 5.19%, and from 1.46% to 3.46%, respectively. The abundance of Rikenellaceae increased from 0.73% to 1.77%. Related studies have shown that this bacterium is an anti-inflammatory probiotic. Increasing the abundance of Rikenellaceae in the intestinal flora can help improve the body's anti-inflammatory ability, improve intestinal health, and enhance the body's immunity. This shows that oral administration of composite bacterial liquid can improve the intestinal flora of immunosuppressed mice, positively regulate the relative abundance of immune-related flora, and enhance the body's immunity.

[0084] Example 9 A strain composition comprising: Lactobacillus plantarum BT1-1 and Lactobacillus mucilaginosus fermentation FM-12. The live bacterial concentration of Lactobacillus plantarum BT1-1 is 2×10 9 cfu / g, and the live bacterial concentration of fermented Lactobacillus mucilaginosus FM-12 was 2×10 9 cfu / g.

[0085] Example 10 A composite preparation, whose raw materials include: 5 g of the strain composition obtained in Example 9, 1 g of 1.0 generation amino-terminated polyamidoamine, 1 g of glutathione, 0.01 g of transglutaminase, 1 g of sodium alginate, 1 g of Span 80, and 0.03 g of calcium chloride.

[0086] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add amino-terminated polyamidoamine to 20 g of water and stir evenly, add glutathione thereto, stir at 100 r / min for 10 min, add transglutaminase thereto, stir at 50° C. for 1 h, heat to inactivate the enzyme, cool, add the strain composition obtained in Example 9 and sodium alginate and stir evenly to obtain phase A; S2. Stir Span 80 and rapeseed oil evenly, add phase A dropwise thereinto while stirring, stir at 1000 r / min for 10 min after complete addition, add 3% calcium chloride solution, continue stirring for 5 min, let stand for 10 h, filter, wash, and freeze-dry.

[0087] Example 11 A composite preparation, whose raw materials include: 10g of the strain composition obtained in Example 9, 2g of 3.0-generation amino-terminated polyamidoamine, 5g of glutathione, 0.1g of transglutaminase, 2g of sodium alginate, 2g of Tween 60, and 0.15g of calcium chloride.

[0088] The preparation method of the above-mentioned composite preparation comprises the following steps: S1, adding amino-terminated polyamidoamine to 40 g of water and stirring evenly, adding glutathione thereto, stirring at a speed of 500 r / min for 20 min, adding transglutaminase thereto, stirring at a temperature of 60° C. for 2 h, heating to inactivate the enzyme, cooling, adding the strain composition obtained in Example 9 and sodium alginate and stirring evenly to obtain phase A; S2. Stir Tween 60 and peanut oil evenly, add phase A dropwise thereinto while stirring, stir at 2000 r / min for 30 min after complete addition, add 5% calcium chloride solution, continue stirring for 15 min, let stand for 15 h, filter, wash, and freeze-dry.

[0089] Example 12 A composite preparation, whose raw materials include: 8g of the strain composition obtained in Example 9, 1.5g of 2.0-generation amino-terminated polyamidoamine, 3g of glutathione, 0.05g of transglutaminase, 1.5g of sodium alginate, 1.5g of Tween 80, and 0.09g of calcium chloride.

[0090] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add amino-terminated polyamidoamine to 30 g of water and stir evenly, add glutathione thereto, stir at 300 r / min for 15 min, add transglutaminase thereto, stir at 55° C. for 1.5 h, heat to inactivate the enzyme, cool, add the strain composition obtained in Example 9 and sodium alginate and stir evenly to obtain phase A; S2. Stir Tween 80 and soybean oil evenly, add phase A dropwise thereinto while stirring, stir at 1500 r / min for 15 min after complete addition, add 4% calcium chloride solution, continue stirring for 10 min, let stand for 12 h, filter, wash, and freeze-dry.

[0091] Comparative Example 1 A strain composition comprising: Lactobacillus plantarum Mle-3 (a strain owned by the applicant, with a deposit number of CGMCC NO.30556) and Lactobacillus mucilaginosus FM-12. The live bacterial concentration of Lactobacillus plantarum Mle-3 is 2×10 9 cfu / g, and the live bacterial concentration of fermented Lactobacillus mucilaginosus FM-12 was 2×10 9 cfu / g.

[0092] A composite preparation, whose raw materials include: 8g of the strain composition obtained in this comparative example, 1.5g of 2.0-generation amino-terminated polyamidoamine, 3g of glutathione, 0.05g of transglutaminase, 1.5g of sodium alginate, 1.5g of Tween 80, and 0.09g of calcium chloride.

[0093] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add amino-terminated polyamidoamine to 30 g of water and stir evenly, add glutathione thereto, stir at 300 r / min for 15 min, add transglutaminase thereto, stir at 55° C. for 1.5 h, heat to inactivate the enzyme, cool, add the strain composition obtained in this comparative example and sodium alginate and stir evenly to obtain phase A; S2. Stir Tween 80 and soybean oil evenly, add phase A dropwise thereinto while stirring, stir at 1500 r / min for 15 min after complete addition, add 4% calcium chloride solution, continue stirring for 10 min, let stand for 12 h, filter, wash, and freeze-dry.

[0094] Comparative Example 2: A composite preparation, whose raw materials include: 8 g of the strain composition obtained in Example 9, 1.5 g of 2.0-generation amino-terminated polyamidoamine, 3 g of glutathione, 1.5 g of sodium alginate, 1.5 g of Tween 80, and 0.09 g of calcium chloride.

[0095] The preparation method of the above-mentioned composite preparation comprises the following steps: S1. Add amino-terminated polyamidoamine to 30 g of water and stir evenly, add glutathione thereto, stir at 300 r / min for 15 min, add the strain composition obtained in Example 9 and sodium alginate and stir evenly to obtain phase A; S2. Stir Tween 80 and soybean oil evenly, add phase A dropwise thereinto while stirring, stir at 1500 r / min for 15 min after complete addition, add 4% calcium chloride solution, continue stirring for 10 min, let stand for 12 h, filter, wash, and freeze-dry.

[0096] 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 compound preparation was added to an appropriate amount of 0.06 mol / L sodium citrate solution and mixed. The mixture was shaken at 180 r / min in a constant temperature shaker at 37°C for 60 min. The live Lactobacillus plantarum (BT1-1 or Mle-3) in each group was counted and the encapsulation rate was calculated.

[0097] Wherein, N is the number of live Lactobacillus plantarum released from the co-formulation; N0 is the number of free Lactobacillus plantarum in the mixture during the production of the co-formulation.

[0098] like Fig.13 As shown, the embedding rates of the composite preparations obtained in Example 12 and Comparative Example 1 are similar, with no significant difference (P>0.05), but the embedding rates of both are higher than those of Comparative Example 2 (P<0.05).

[0099] The applicant believes that this is because comparative example 2 does not use transglutaminase, which cannot effectively promote the effective combination of amino-terminated polyamidoamine and glutathione, which not only leads to a decrease in its embedding rate, but also fails to effectively load the strain.

[0100] 2. Bile salt tolerance test Each group of compound 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, and the live Lactobacillus plantarum (BT1-1 or Mle-3) in each group was counted.

[0101] 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 plantarum in Comparative Example 2 decreased significantly after bile salt treatment.

[0102] The applicant believes that this is because comparative example 2 does not use transglutaminase, which cannot effectively promote the effective combination of amino-terminated polyamidoamine and glutathione, resulting in the embedding wall material being not tight enough.

[0103] 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.

[0104] 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.

[0105] 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.

[0106] 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.

[0107] Among them, A1 is the number of Lactobacillus plantarum (BT1-1 or Mle-3) cells during the test, cfu / g; A0 is the number of Lactobacillus plantarum (BT1-1 or Mle-3) cells at the beginning of digestion, cfu / g.

[0108] 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 plantarum in Comparative Example 2 decreased significantly after being treated with simulated digestive fluid.

[0109] The applicant believes that this is because comparative example 2 does not use transglutaminase, which cannot effectively promote the effective combination of terminal amino polyamidoamine and glutathione, reducing the stability of glutathione, resulting in insufficient stability of the composite preparation and a significant decrease in the number of live Lactobacillus plantarum.

[0110] 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.

[0111] 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).

[0112] 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: 0.5 g / d / mouse).

[0113] 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.

[0114] 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.

[0115] The normal control group, model group, and BF group continued to use the data in Example 8 above.

[0116] like Fig.16 As shown in the figure, compared with the MC group and the BF group, the serum IL-10 content of the 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 the 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.

[0117] At the same time, 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.

[0118] This shows that the composite preparation obtained in Example 12 can effectively inhibit inflammatory response and enhance the body's immunity. That is, the raw materials and methods of the composite preparation of the present invention can further produce a synergistic effect with Lactobacillus plantarum BT1-1 and Lactobacillus mucilaginosus FM-12, further inhibiting the inflammatory response in mice.

[0119] 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 plantarum Lactiplantibacillus plantarum ) Application of BT1-1 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 plantarum BT1-1 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 20, 2024, with the deposit number CGMCC NO.32011.

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: Including the use of Lactobacillus plantarum BT1-1 as claimed in claim 1.

6. The strain composition according to claim 5, characterized in that: Also includes: Lactobacillus mucilaginosus fermentation Limosilactobacillus fermentum )FM-12; the ratio of the number of viable bacteria of plant lactobacillus BT1-1 and fermented mucus lactobacillus FM-12 is 0.8-1.2:0.8-1.

2.

7. A composite preparation, characterized in that: The raw materials include, by mass: 5-10 parts of the strain composition as claimed in claim 5 or 6, 1-2 parts of amino-terminated polyamidoamine, 1-5 parts of glutathione, 0.01-0.1 parts of transglutaminase, 1-2 parts of sodium alginate, 1-2 parts of surfactant, and 0.03-0.15 parts of calcium chloride; The total viable bacterial concentration of the strain composition according to claim 5 or 6 is 1.5-3.5×10 9 cfu / g.

8. The composite formulation according to claim 7, characterized in that: The amino-terminated polyamidoamine has a generation number of 1.0-3.

0.

9. A method for preparing the composite formulation according to claim 7 or 8, characterized in that: The steps include: S1. Add amino-terminated polyamidoamine to water and stir evenly, add glutathione thereto and stir for 10-20 min, add transglutaminase thereto, stir at 50-60° C. for 1-2 h, heat to inactivate the enzyme, cool, add the strain composition as claimed in claim 5 or 6 and sodium alginate and stir evenly to obtain phase A; S2. Stir the surfactant and vegetable oil evenly, add phase A dropwise thereto under stirring, stir for 10-30 minutes after the addition is complete, add 3-5% calcium chloride solution by mass and continue stirring for 5-15 minutes, let stand for 10-15 hours, filter, wash, and freeze-dry.

10. The method for preparing the composite formulation according to claim 9, characterized in that: The vegetable oil is at least one of soybean oil, rapeseed oil, peanut oil and olive oil; the surfactant is sorbitan fatty acid ester and / or polyoxyethylene sorbitan fatty acid ester.

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