Composite lactic acid bacteria afterbiogen composition as well as preparation method and application of composite lactic acid bacteria afterbiogen composition

Abstract

The invention discloses a compound lactic acid bacteria afterbiogen composition as well as a preparation method and application thereof. According to the compound lactic acid bacteria metagen composition disclosed by the invention, the cell number proportions of all strains are as follows: LRH10 (12%), LPC12 (2%), LF26 (26.3%), ST30 (21.6%) and LH43 (38.1%). The compound lactic acid bacteria metabolite composition prepared by the invention contains various probiotics metabolites, has diversified biological activities, and has a remarkable effect in the aspect of immune health.

Description

Technical Field

[0001] The present invention belongs to the technical field of microorganisms, and in particular relates to a composite lactic acid bacteria postbiotic composition, a preparation method and uses thereof. Background Art

[0002] In recent years, as people's understanding of the relationship between intestinal microecology and health deepens, probiotics and postbiotics, as important means of regulating intestinal microecology, have gradually received widespread attention from the scientific research and industry. Probiotics are a type of active microorganisms that benefit the host by colonizing in the human body and changing the composition of the flora in a certain part of the host. Postbiotics, as a general term for probiotic metabolites and their components after probiotics are processed, including bacteria and metabolites, are believed to have multiple health benefits, especially showing great potential in immune health.

[0003] At present, there are still many deficiencies in the preparation of postbiotics with compound probiotics. First, the activity of probiotics has always been a key factor restricting its application effect. Probiotics that have not been properly treated are easily inactivated during storage and transportation, which greatly reduces the effectiveness of the product. Secondly, there are significant individual differences in probiotics, and the intestinal environment of different people has different acceptance of probiotics, which makes the effect of probiotic products vary from person to person, and it is difficult to ensure stability and consistency.

[0004] Postbiotic components from different strains may have different biological activities, and postbiotics prepared from a single strain may have specific immunomodulatory effects, but this effect is often limited by the specificity of the strain. The immunomodulatory effects of postbiotics from different strains may vary significantly, making it difficult to select the most appropriate strain in practical applications.

[0005] In addition, the specificity of the strain may also lead to inconsistent effects of postbiotics in different populations, thus limiting their widespread application. Postbiotics prepared from a single strain of bacteria have a single function and cannot fully simulate the complex ecosystem of intestinal microorganisms, and cannot provide multiple health benefits, such as immune regulation and metabolic improvement. Summary of the invention

[0006] Purpose of the invention: The purpose of the present invention is to provide a composite lactic acid bacteria postbiotic composition and its preparation method and use in view of the deficiencies of the prior art. The present invention uses composite probiotics to prepare postbiotics. The prepared composite lactic acid bacteria postbiotic composition contains metabolites of multiple probiotics, has diverse biological activities, and has a significant effect on immune health.

[0007] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0008] The invention provides a composite lactic acid bacteria postbiotic composition, comprising Lactobacillus rhamnosus LRH10 with a preservation number of CGMCC NO.24513, Lactobacillus paracasei LPC12 with a preservation number of CGMCC NO.24512, Lactobacillus fermentum LF26 with a preservation number of CGMCC NO.19431, Streptococcus thermophilus ST30 with a preservation number of CGMCC NO.24514; Lactobacillus helveticus LH43 with a preservation number of CGMCC NO.24511;

[0009] The cell number ratio of each strain in the composite lactic acid bacteria postbiotic composition is:

[0010]

[0011] Biomaterial deposit information:

[0012] Lactobacillus rhamnosus LRH10, which was deposited in the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24513;

[0013] Lactobacillus paracasei LPC12, which was deposited in the China General Microbiological Culture Collection Center on March 11, 2022, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO.24512;

[0014] Limosilactobacillus fermentum LF26, which was deposited in the China General Microbiological Culture Collection Center on February 24, 2020, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.19431;

[0015] Streptococcus thermophilus ST30, which was deposited in the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24514;

[0016] Lactobacillus helveticus LH43, this strain was deposited in the China General Microbiological Culture Collection on March 11, 2022. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO.24511.

[0017] The above strains have been recorded in patent application CN116179414A published in the patent gazette.

[0018] The number of cells of the composite lactic acid bacteria postbiotic composition is 10 9 cell / mL.

[0019] The present invention also provides a method for preparing the composite lactic acid bacteria postbiotic composition, comprising the following steps:

[0020] (1) Lactic acid bacteria LRH10, LPC12, LF26, ST30, and LH43 were placed in MRS culture medium and activated once;

[0021] (2) Repeating step (1) with the activated lactic acid bacteria liquid for a second activation;

[0022] (3) sterilizing the lactic acid bacteria liquid activated for the second time;

[0023] (4) centrifuging the sterilized lactic acid bacteria solution;

[0024] (5) The lactic acid bacteria liquid is centrifuged to separate into lactic acid bacteria and supernatant. After removing the supernatant, a sterile phosphate buffer solution is added, and the supernatant is shaken to resuspend the lactic acid bacteria to wash the bacteria.

[0025] (6) Repeat step (5) three times to obtain lactic acid bacteria that have been washed three times;

[0026] (7) Add the washed lactic acid bacteria to a sterilized phosphate buffer solution and adjust the lactic acid bacteria suspension to an OD of 600nm The lower absorbance value was 1.05 ± 0.05;

[0027] (8) The lactic acid bacteria after adjusting the absorbance are mixed evenly according to a certain proportion to obtain the composite lactic acid bacteria postbiotic composition.

[0028] Preferably, in step (1), the activation is performed once by placing the lactic acid bacteria in MRS culture medium and aerobically culturing for 16 hours.

[0029] Preferably, in step (3), the sterilization conditions are: heating at 85°C for 30 minutes.

[0030] Preferably, in step (4), the centrifugation condition is: centrifugation at 3500 rpm for 5 min.

[0031] Preferably, the sterilization condition of the phosphate buffer solution is heating at 85° C. for 30 min.

[0032] Preferably, in step (7), the lactic acid bacteria suspension after adjusting the absorbance is stored at -80°C for later use.

[0033] The present invention also provides use of the composite lactic acid bacteria postbiotic composition in the preparation of immunity-promoting drugs.

[0034] Preferably, the effective bacterial count of the composite lactic acid bacteria postbiotic composition is 10 8 CFU / mL.

[0035] The medicine comprises the composite lactic acid bacteria postbiotic composition and a pharmaceutically acceptable carrier or excipient.

[0036] The auxiliary materials include one or more of preservatives, antioxidants, emulsifiers, wetting agents, diluents, disintegrants or binders.

[0037] The drug of the present invention can be administered in various known ways, such as oral administration, injection, etc. The drug of the present invention can be administered alone or in combination with other drugs. The oral composition can be in any oral acceptable dosage form, including but not limited to tablets, granules, capsules, pills, suspensions and solutions.

[0038] Beneficial effects:

[0039] (1) The composite lactic acid bacteria postbiotic composition prepared by the present invention contains metabolites of various probiotics, has diverse biological activities, and has significant effects on immune health. These metabolites can work synergistically to comprehensively regulate the intestinal microbial community and enhance the function of the immune system. It has diverse functions, strong adaptability, is closer to the composition of the natural intestinal microbial community, and enhances the immune regulation effect.

[0040] (2) The postbiotic composition prepared by the composite probiotics of the present invention can significantly enhance the immunomodulatory effect through the synergistic effect of multiple metabolites. For example, short-chain fatty acids can inhibit the production of inflammatory factors and reduce intestinal inflammation; bacteriocins can inhibit the growth of pathogens and enhance the intestinal barrier function. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The effect of lactic acid bacteria and its composite lactic acid bacteria postbiotic composition on the secretion of IL-10 in mouse peritoneal macrophages;

[0042] Figure 2 The effect of lactic acid bacteria and its composite lactic acid bacteria postbiotic composition on the secretion of TNF-α in mouse peritoneal macrophages;

[0043] Figure 3 The effect of lactic acid bacteria and its composite lactic acid bacteria postbiotic composition on the secretion of IL-6 in mouse peritoneal macrophages; Figure 4 The effect of lactic acid bacteria and its composite lactic acid bacteria postbiotic composition on the secretion of IL-12p40 in mouse peritoneal macrophages. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0045] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0046] The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available products unless otherwise specified.

[0047] The lactic acid bacteria of the present invention, such as LRH10, LRH113, LPC12, LF26, ST30 and LH43, are all from frozen vials in a syngeneic strain library. Before use, the frozen vials are placed in a 37° C. water bath for rapid thawing.

[0048] The centrifuge tube and phosphate buffer used in the present invention are sterilized by heating at 85° C. for 30 minutes before use.

[0049] Example 1 Preparation of MRS culture medium

[0050] The specific components of MRS culture medium are as follows:

[0051]

[0052]

[0053] Preparation method: Add the above ingredients into 1000 mL of distilled water, heat to dissolve, adjust the pH to 6.2±0.2, and sterilize at 121° C. for 15 min to 20 min after packaging to obtain MRS culture medium.

[0054] Example 2 Preparation of composite lactic acid bacteria postbiotic composition

[0055] (1) Place the completely thawed lactic acid bacteria LRH10, LPC12, LF26, ST30, and LH43 in 9 ml of MRS culture medium and culture them aerobically for 16 h, which is the activation step;

[0056] (2) Repeating step (1) with the activated lactic acid bacteria liquid for a second activation;

[0057] (3) Sterilizing the lactic acid bacteria solution after the second activation by heating it at 85° C. for 30 min;

[0058] (4) Transfer the sterilized lactic acid bacteria solution into a sterilized 15 ml centrifuge tube and centrifuge it at 3500 rpm for 5 min.

[0059] (5) The lactic acid bacteria liquid was centrifuged and separated into lactic acid bacteria and supernatant. After removing the supernatant, 9 ml of sterile phosphate buffer solution (pH 6.2) was added and shaken to resuspend the supernatant to wash the bacteria.

[0060] (6) Repeat step (5) three times to obtain lactic acid bacteria that have been washed three times;

[0061] (7) Add the washed lactic acid bacteria to a sterilized phosphate buffer solution and adjust the lactic acid bacteria suspension to an OD of 600nm The absorbance value is 1.05±0.05, and the number of lactic acid bacteria is about 10 9 CFU / mL;

[0062] (8) The lactic acid bacteria suspension after adjusting the absorbance value is stored in a -80°C refrigerator for later use;

[0063] (9) Adjust the OD 600nm Lactic acid bacteria with absorbance values ​​of 1.05±0.05 were evenly mixed according to the ratio of strain cell numbers, LRH10 (12%), LPC12 (2%), LF26 (26.3%), ST30 (21.6%), and LH43 (38.1%), to prepare a composite lactic acid bacteria postbiotic composition.

[0064] Example 3 In vitro cell stimulation test

[0065] 1. Experimental cell types

[0066] Mouse peritoneal macrophage cell line-264.7 (RAW 264.7) was purchased from American Type Culture Collection (ATCC).

[0067] 2. Experimental Drugs

[0068] Fetal bovine serum (FBS): Heat-inactivated fetal bovine serum purchased from Gibco and stored in a -20°C refrigerator.

[0069] Dulbecco's modified Eagle's medium (DMEM): purchased from Gibco, USA, and stored in a 4°C refrigerator.

[0070] Gram-negative bacterial lipopolysaccharide (LPS O55:B5): purchased from Sigma, USA.

[0071] Preparation of Gram-negative bacteria lipopolysaccharide solution for testing:

[0072] (1) Take 1 mg of LPS O55:B5 and mix it evenly with 999 uL of sterile phosphate buffer solution. Use a 0.22 um syringe filter to filter it into a sterile 1.5 mL plastic tube.

[0073] (2) Take 10uL and 100uL of the prepared LPS O55:B5 (1mg / mL) and add 990uL and 900uL of sterile phosphate buffer solution respectively to mix, and the final concentrations are 10ug / mL and 100ug / mL respectively.

[0074] (3) Place LPS O55:B5 (10 ug / mL, 100 ug / mL and 1 mg / mL) in a -80°C refrigerator for later use.

[0075] 0.4% Trypan blue dead cell stain: Trypan blue is often used to count cell survival rate. The principle is that the nucleus of living cells cannot be stained by trypan blue. Therefore, dead cells stained by trypan blue can be easily identified through a microscope. The trypan blue used in the experiment was purchased from Thermo Company.

[0076] 3. Cell culture:

[0077] (1) Preparation of cell culture medium:

[0078] Take the fetal bovine serum out of the -20℃ refrigerator and place it in a 37℃ constant temperature water bath to warm it up. After it is dissolved, dispense it into 50mL sterile centrifuge tubes for later use. Add 50mL FBS to 500mL DMEM to prepare DMEM with 10% FBS, and then add 0.1% double-antibody "penicillin and streptomycin" (Gibco), and then dispense the DMEM containing 10% FBS into sterile 15mL centrifuge tubes and store them in a 4℃ refrigerator.

[0079] (2) Cell thawing and culture:

[0080] After taking out the RAW 264.7 cells from the liquid nitrogen barrel, quickly put them into a 37°C constant temperature water bath for rapid thawing. After they are completely dissolved, take 1 mL of 10% FBSDMEM culture fluid, slowly mix it with the cell solution in the freezing tube, and then take it into a sterilized 15 mL centrifuge tube. Then add three times the volume of about 6 mL DMEM culture fluid to neutralize the toxicity of the antifreeze agent dimethyl sulfoxide. After centrifugation at 1000 rpm for 5 minutes, remove the upper layer of culture fluid in the 15 mL centrifuge tube, add 10 mL DMEM culture fluid, slowly mix it with the bottom cell layer, and slowly put it into a sterilized 10 cm cell culture dish, and place it in a 37°C incubator with 5% CO2. After that, remove the old culture fluid every three days, add 10 mL of fresh 10% FBSDMEM culture fluid, and observe RAW using an inverted microscope. 264.7 Cell growth: On the seventh day of culture, well-growing cells should be close to the bottom of the cell culture dish and appear plump. Subculture or in vitro stimulation test can be performed on about the ninth day of culture.

[0081] IV. In vitro cell stimulation test:

[0082] (1) Use a cell scraper to remove well-growing RAW 264.7 cells from the cell culture dish.

[0083] (2) The scraped RAW 264.7 cells were collected and the number of live and dead cells was counted using 0.4% trypan blue dead cell stain.

[0084] (3) Place the calculated number of viable cells in a 24-well cell plate and adjust the viable cell count to 1.5×10 per ml. 6 cells / well.

[0085] (4) Allow two hours for the cells to settle and adhere to the bottom of the cell plate.

[0086] (5) Use a microscope to observe that the adhesion is good and then stimulate it.

[0087] (6) Take 1uL of LPS O55:B5 (10ug, 100ug and 1000ug / mL) and 100uL of each lactic acid bacteria and its composite lactic acid bacteria postbiotic composition and phosphate buffer solution group and add them into the above-mentioned solution containing 1.5×10 viable cells per ml. 6 The final stimulation concentrations of LPS O55:B5 were 10 ng, 100 ng and 1000 ng / mL in cells / well, and the concentration of lactic acid bacteria group was 10 8 cell / mL, the final concentration of the composite lactic acid bacteria postbiotic composition group was 10 8-7-6-5 The above experiments were repeated three times.

[0088] The composite lactic acid bacteria postbiotic composition prepared in Example 2 was serially diluted by 10, 100, 1000, and 10000 times to obtain 10 8 , 10 7 , 10 6 , 10 5 cell / mL.

[0089] (7) After 24 hours of stimulation, the cell supernatant was collected into a sterile 1.5 mL plastic tube and stored in a -80°C refrigerator for testing of its cytokine content.

[0090] 5. Cytokine determination:

[0091] The cytokine levels secreted by RAW 264.7 cells were measured according to the method in the instruction manual of the enzyme-linked immune attachment kit (Mouse TNF-α, IL-6, IL-10 and IL-12p40 ELISA Ready-SET-Go, purchased from Affymetrix eBioscience, Inc., USA).

[0092] First, the primary antibodies (capture antibodies) of TNF-α, IL-6, IL-10 and IL-12p40 were diluted 250 times with phosphate buffer solution, and 100 μL / well was added to each well of the 96-well plate and stored at 4°C overnight. On the day of the experiment, the samples required for analysis were thawed for testing.

[0093] Wash the primary antibody 3 times with washing solution (1X PBS + 0.05% tween-20), add 200μL / well blocking solution (dilute the 5X ELISA / ELISPOT diluent in the kit 5 times with deionized water), let stand at room temperature for 1 hour, and then wash with washing solution 3 times. Add the prepared standard solution and sample (dissolved in PBS) to the 96-well plate. Let stand at room temperature for 2 hours, wash with washing solution 3 times, add the prepared secondary marker antibody to the 96-well plate (100μL / well), let stand at room temperature in a dark environment for 1 hour, wash with washing solution 3 times, add the prepared avidin-HRP (100μL / well), let stand at room temperature in a dark environment for 30 minutes, add the color reagent (100μL / well), let stand at room temperature in a dark environment for 30 minutes, and add the stop solution (50μL / well) to stop the reaction. The absorbance (OD) was read at a wavelength of 450 nm and the concentrations of TNF-α, IL-6, IL-10 and IL-12p40 were calculated by comparing with the standard curve.

[0094] Statistical analysis:

[0095] Tukey's Honestly Significant Difference Test was used to compare the differences among the groups. The control group was a blank group, the PBS group was a solvent PBS for dissolving the test sample, and the SYN 108 group was a composite lactic acid bacteria postbiotic composition with a bacterial count of 10 8 cell / mL, SYN 107 group is a composite lactic acid bacteria postbiotic composition with a bacterial count of 10 7 cell / mL, SYN 106 group is a composite lactic acid bacteria postbiotic composition with a bacterial count of 10 6 cell / mL, SYN 105 group is a composite lactic acid bacteria postbiotic composition with a bacterial count of 10 5 cell / mL.

[0096] Figure 1 The effect of lactic acid bacteria and its combination on the secretion of IL-10 in mouse peritoneal macrophages. Different letters on the bar graph indicate significant differences (P<0.05) between the groups using Tukey's Honestly Significant Difference Test.

[0097] Depend on Figure 1It can be seen that the lactic acid bacteria composition, LF26 and ST30, and the bacterial counts are all 10 8 CFU / mL, it can significantly increase the IL-10 cytokine secretion compared with other lactic acid bacteria, and there is no significant difference when stimulated with LPS100ng / mL.

[0098] Figure 2 The effect of lactic acid bacteria and its combination on the secretion of TNF-α in mouse peritoneal macrophages. Different letters on the bar graph indicate significant differences (P<0.05) between the groups using Tukey's Honestly Significant Difference Test.

[0099] Depend on Figure 2 It can be seen that the ST30 and LH43 groups were significantly higher than the other lactic acid bacteria groups, the lactic acid bacteria combination group and the LPS group.

[0100] Figure 3 The effect of lactic acid bacteria and its combination on the secretion of IL-6 in mouse peritoneal macrophages. Different letters on the bar graph indicate significant differences (P<0.05) between the groups using Tukey's Honestly Significant Difference Test.

[0101] Depend on Figure 3 It can be seen that LF26 significantly increased the IL-6 cytokine secretion compared with LRH113, LPC12, LRH10, ST30, LH43 and the lactic acid bacteria combination.

[0102] Figure 4 The present invention relates to the effects of lactic acid bacteria and their combination on the secretion of IL-12p40 by mouse peritoneal macrophages.

[0103] Depend on Figure 4 It can be seen that ST30 significantly increased the secretion of cytokines compared with the other stimulation groups.

[0104] In summary, ST30 can significantly increase the content of cytokines (TNF-a and IL-12p40) in the other lactic acid bacteria and lactic acid bacteria combination groups. The reason may be that ST30 is in the form of long chains when cultured in MRS. Its long chains may increase its surface area and thus enhance the stimulation of mouse peritoneal macrophages to secrete cytokines. 8 CFU / mL can significantly increase the secretion of IL-10, and the other cytokines TNF-a, IL-6 and / or IL-12p40 all have the second highest secretion amount, which indicates that the lactic acid bacteria composition of the present invention can combine the immune stimulation advantages of various lactic acid bacteria to achieve the effect of promoting immunity.

[0105] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A composite lactic acid bacteria postbiotic composition, characterized in that: Including Lactobacillus rhamnosus LRH10 with a preservation number of CGMCC NO.24513, Lactobacillus paracasei LPC12 with a preservation number of CGMCC NO.24512, Lactobacillus fermentum LF26 with a preservation number of CGMCC NO.19431, Streptococcus thermophilus ST30 with a preservation number of CGMCC NO.24514; Lactobacillus helveticus LH43 with a preservation number of CGMCC NO.24511; The cell number ratio of each strain in the composite lactic acid bacteria postbiotic composition is:

2. The composite lactic acid bacteria postbiotic composition according to claim 1, characterized in that The number of cells in the composition is 10 9 cell / mL.

3. The method for preparing the composite lactic acid bacteria postbiotic composition according to any one of claims 1 or 2, characterized in that: The following steps are involved: (1) Lactic acid bacteria LRH10, LPC12, LF26, ST30, and LH43 were placed in MRS culture medium and activated once; (2) Repeating step (1) with the activated lactic acid bacteria liquid for a second activation; (3) sterilizing the lactic acid bacteria liquid activated for the second time; (4) centrifuging the sterilized lactic acid bacteria solution; (5) The lactic acid bacteria liquid is centrifuged to separate into lactic acid bacteria and supernatant. After removing the supernatant, a sterile phosphate buffer solution is added, and the supernatant is shaken to resuspend the lactic acid bacteria to wash the bacteria. (6) Repeat step (5) three times to obtain lactic acid bacteria that have been washed three times; (7) Add the washed lactic acid bacteria to a sterilized phosphate buffer solution and adjust the lactic acid bacteria suspension to an OD of 600nm The lower absorbance value was 1.05 ± 0.05; (8) The lactic acid bacteria after adjusting the absorbance are mixed evenly according to a certain proportion to obtain the composite lactic acid bacteria postbiotic composition.

4. The preparation method according to claim 3, characterized in that: In step (1), the activation method is: placing the lactic acid bacteria in MRS culture medium and aerobically culturing for 16 hours.

5. The preparation method according to claim 3, characterized in that: In step (3), the sterilization conditions are: heating at 85°C for 30 minutes.

6. The preparation method according to claim 3, characterized in that: In step (4), the centrifugation condition is: centrifugation at 3500 rpm for 5 min.

7. The preparation method according to claim 3, characterized in that: The sterilization condition of the phosphate buffer solution is heating at 85° C. for 30 min.

8. The preparation method according to claim 3, characterized in that: In step (7), the lactic acid bacteria suspension after adjusting the absorbance is stored at -80°C for later use.

9. Use of the composite lactic acid bacteria postbiotic composition according to claim 1 or 2 in the preparation of immunity-boosting drugs.

10. The use according to claim 9, characterized in that The effective bacterial count of the composition is 10 8 cell / mL.

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