Preparation method of multi-strain symbiotic fermentation precisely-controlled metabiotics

Abstract

The invention discloses a preparation method of metabiotics capable of precisely controlling symbiotic fermentation of multiple strains. Preparing a base from the following components in percentage by mass: 20-25% of skim milk powder, 0.1-0.5% of yeast powder, 1-5% of white granulated sugar and the balance of water; the substrate is sterilized at the temperature of 90-95 DEG C for 10-15 min; cooling to the inoculation temperature of 40-44 DEG C; inoculating lactic acid bacteria according to the cell number proportion of each strain; the viable count of the substrate after inoculation is 1.0 * 10 < 5 > CFU / ml; fermenting at 40-44 DEG C for 5-8 hours; adjusting the test pH to 4.8-5.0, and adding 0.5-1% of yeast extract and 0-1% of isomaltooligosacharide in terms of a substrate before inoculation; fermenting at 37-39 DEG C for 10-16 hours; adding 15 to 20 percent of maltodextrin; and carrying out spray drying to obtain the metagen. According to the invention, the tedious steps of separate fermentation and mixing of each lactic acid bacterium in the traditional process are avoided, the equipment investment is reduced, and the production cycle is shortened.

Description

Technical Field

[0001] The present invention belongs to the field of microbial technology, and specifically relates to a method for preparing postbiotics with precise control of multi-strain symbiotic fermentation. Background Art

[0002] In the rapid development of intestinal microbial research, the research perspective is gradually expanding from a single bacterial species to a comprehensive and systematic understanding of the smallest functional group, which is crucial to deeply revealing the role of intestinal microorganisms in health maintenance and disease development, especially type 2 diabetes. One of the research focuses is to explore the core ecological network - the key microbial interaction network that maintains the balance of intestinal ecology, and its identification has opened up new avenues for the development of disease-specific intervention strategies.

[0003] In this context, targeted intervention strategies, especially keystone species targeted supplementation strategies, have emerged to restore the intestinal ecological balance by supplementing key keystone species. Scientists are actively identifying and testing the role of putative important species in different environments. Single-species deletion experiments have preliminarily revealed significant differences in the distribution of bacterial strains in the intestinal region and in the adaptability of culture media. These differences involve key functional mechanisms that depend on the external environment, such as polysaccharide utilization and bacteriocin production.

[0004] Notably, keystone taxa, as a highly interconnected group, have a significant impact on the structure and function of the microbiome, regardless of their spatiotemporal abundance. Symbiotic relationships between species, such as growth promotion through secretion of autoinducing molecules, and cross-feeding phenomena, where one species relies on nutrients produced by other species, are common interaction patterns in microbial communities. These interactions often involve the degradation of complex molecules, such as carbohydrates being broken down by specific species to release simple sugars for utilization by other species.

[0005] In the study of lactic acid bacteria, especially in the process of compound lactic acid fermentation, a series of challenges are faced. Lactic acid bacteria can absorb and utilize a variety of sugars, but their growth characteristics, morphology and physiological characteristics vary depending on the sugar substrate, resulting in different resistance to stress treatments. In addition, slight changes in fermentation conditions, whether normal or abnormal, may significantly affect the fermentation characteristics of lactic acid bacteria, resulting in changes in the composition of metabolites. For example, when homofermentative bacteria grow on sugars other than glucose, in addition to lactic acid, other compounds such as mannitol are formed, and the accumulation of these metabolites depends on the carbon source in the growth medium.

[0006] The phenomenon of cross-feeding between species also exists in natural ecosystems. For example, the bacterial community of kefir (a fermented milk) is an example of dynamic change. Kefir from different regions also shows the concept of core minimum functional groups.

[0007] Postbiotics, as metabolites of probiotics, also show potential probiotic effects (i.e., they partially have prebiotic-like functions and have some value-added functions for probiotics), but their preparation methods and applications still need further research. The existing preparation methods of postbiotics are inefficient and it is difficult to ensure the stability of postbiotics. Directly adding probiotics also faces problems such as strain stability, low intestinal colonization rate, and individual differences:

[0008] Low survival rate of probiotics: Probiotics are easily affected by environmental factors and become inactivated during production, storage and transportation, resulting in reduced efficacy of the final product.

[0009] Low intestinal colonization rate: Not all ingested probiotics can effectively colonize and exert their effects in the intestines, especially for the elderly, whose intestinal environment is complex and colonization is more difficult.

[0010] Strain homogeneity: Many existing probiotic preparations contain only a single strain, such as only one type of Bifidobacterium, while ignoring the synergistic effects between different strains, and their efficacy is limited.

[0011] Lack of synergy: The lack of in-depth research on the synergistic effects of probiotic combinations (such as the combination of multiple bifidobacteria with other probiotics) and their metabolites (postbiotics) makes it difficult to develop efficient and stable probiotic preparations.

[0012] In summary, the difficulty of compound lactic acid bacteria fermentation lies in understanding and controlling the interaction between different lactic acid bacteria and their sensitivity to environmental conditions, as well as how to precisely regulate the fermentation process to obtain a stable metabolite composition. It is of great significance to develop efficient and stable postbiotic products through in-depth research and technological innovation. Summary of the invention

[0013] Purpose of the invention: The purpose of the present invention is to provide a method for preparing postbiotics with precise control of multi-species symbiotic fermentation. The present invention uses genomics and other technologies to isolate and identify 6 core bacterial species from natural kefir. By precisely controlling the fermentation process, these core bacterial species can be effectively utilized to obtain a stable number of lactic acid bacteria when preparing postbiotics, thereby avoiding the cumbersome steps of fermenting each lactic acid bacteria separately and then mixing them in the traditional process, reducing equipment investment and shortening the production cycle.

[0014] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0015] The present invention provides a method for preparing postbiotics with precise control of multi-strain symbiotic fermentation, comprising the following steps:

[0016] (1) Prepare a base according to the ratio of 20-25% skim milk powder, 0.1-0.5% yeast powder, 1-5% white sugar, and the rest water by mass percentage, and stir to dissolve;

[0017] (2) sterilizing the substrate of step (1): 90-95° C., 10-15 min;

[0018] (3) Cooling: Cool to the inoculation temperature of 40-44°C;

[0019] (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain; the base number of live bacteria after inoculation is 1.0×10 5 CFU / ml;

[0020] Strain No. Strain Cell Number %

[0021] LRH10 40%

[0022]

[0023] (5) First fermentation: 40-44°C for 5-8 hours;

[0024] (6) Adjust the test pH to 4.8-5.0, add 0.5-1% yeast extract and 0-1% isomaltooligosaccharide based on the base before inoculation;

[0025] (7) Secondary fermentation: fermentation at 37-39°C for 10-16 hours;

[0026] (8) Preparation: adding 15-20% maltodextrin to the fermentation product obtained in step (7);

[0027] (9) Spray drying: air inlet temperature 180°C, air outlet temperature 110°C;

[0028] After spray drying, the postbiotics with precisely controlled multi-strain symbiotic fermentation are obtained.

[0029] Biomaterial deposit information:

[0030] 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;

[0031] 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;

[0032] 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;

[0033] 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;

[0034] 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.

[0035] The above strains have been recorded in patent application CN116179414A published in the patent gazette.

[0036] After spray drying, due to the instant inactivation of cells, the structure of the lactic acid bacteria strains was basically the same as before spraying, and a postbiotic with precisely controlled multi-species symbiotic fermentation was obtained, in which the cell number proportion of each fermentation strain was: LRH10 (12±0.5%), LPC12 (2±0.5%), LF26 (26.3±0.5%), ST30 (21.6±0.5%), and LH43 (38.1±0.5%).

[0037] The number of cells in the postbiotics precisely controlled by the multi-strain symbiotic fermentation is ≥ 1.0×10 9 cell / g.

[0038] The present invention also provides a specific and preferred preparation method, comprising the following steps:

[0039] (1) Prepare a base in the following proportions by mass: 20% skim milk powder, 0.5% yeast powder, 5% white sugar, and the remainder water, and stir to dissolve;

[0040] (2) sterilizing the substrate of step (1): 95°C, 10 min;

[0041] (3) Cooling: Cool to the inoculation temperature of 42°C;

[0042] (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain; the base number of live bacteria after inoculation is 1.0×10 5CFU / ml;

[0043] (5) First fermentation: 42°C for 8 h;

[0044] (6) Adjust the test pH to 4.8-5.0, add 1% yeast extract and 1% isomaltooligosaccharide based on the base before inoculation;

[0045] (7) Secondary fermentation: fermentation at 37°C for 16 h;

[0046] (8) Preparation: adding 15% maltodextrin to the fermentation product obtained in step (7);

[0047] (9) Spray drying: air inlet temperature 180°C, air outlet temperature 110°C;

[0048] After spray drying, the postbiotics with precisely controlled multi-strain symbiotic fermentation are obtained.

[0049] The cell number proportion of each fermentation strain in the postbiotics precisely controlled by the multi-strain symbiotic fermentation is: LRH10 12.1%, LPC12 2%, LF26 26.1%, ST30 21.6%, LH43 38.2%.

[0050] The postbiotics prepared by the present invention with precise control of multi-species symbiotic fermentation have stable bacterial numbers of various lactic acid bacteria, and can combine the immune stimulation advantages of various lactic acid bacteria, thereby achieving the effect of promoting immunity.

[0051] Beneficial effects:

[0052] (1) The preparation method of the present invention can accurately supplement nutrients with targeted carbon and nitrogen sources at a fixed time and location during fermentation.

[0053] (2) The preparation method of the present invention avoids the cumbersome steps of fermenting each lactic acid bacteria separately and then mixing them in the traditional process, reduces equipment investment, and shortens the production cycle.

[0054] (3) The number of various lactic acid bacteria in the postbiotics prepared by the present invention with precise control of multi-species symbiotic fermentation is stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] 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;

[0056] 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;

[0057] 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;

[0058] 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

[0059] 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.

[0060] 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.

[0061] 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.

[0062] The lactic acid bacteria of the present invention, such as LRH10, 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.

[0063] The method for detecting the number of LRH10, LPC12, LF26, ST30, and LH43 fermented lactic acid bacteria cells in the postbiotics accurately controlled by multi-strain symbiotic fermentation prepared in an embodiment of the present invention is:

[0064] 1. Preparation of MRS medium

[0065] Peptone 10.0g; beef powder 5.0g; yeast powder 4.0g; glucose 20.0g; Tween 80 1.0mL; triammonium citrate 2.0g; sodium acetate 5.0g; magnesium sulfate 0.2g; manganese sulfate 0.05g; dipotassium hydrogen phosphate 2.0g; agar powder 15.0g.

[0066] 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 20 min after packaging.

[0067] 2. Preparation of (MRS+Ciprofloxacin) medium

[0068] (1) Preparation of Ciprofloxacin (Sigma) Solution: Weigh 40 mg of ciprofloxacin and dissolve it in 10 mL of 0.1 M HCl. Mix well using an oscillator and filter the mixture using a 0.22 μm filter into a sterile 15 mL centrifuge tube for later use.

[0069] (2) Add the ingredients obtained in step (1) to 1000 mL of distilled water, heat to dissolve, adjust the pH to 6.4, and sterilize by autoclave at 121° C. for 20 min after packaging. Heat to melt agar before use, cool to 48° C. in a water bath, transfer 5 mL of ciprofloxacin solution to 1000 mL of MRS medium using a syringe with a 0.22 μm microporous filter membrane, mix well, pour 12 mL of the solution into a culture dish, and wait for solidification before use.

[0070] 3. Sample preparation and dilution

[0071] Accurately weigh 10.00g of the sample to be tested, put it into a 250mL Erlenmeyer flask filled with 90mL sterile water and small glass beads, and shake it thoroughly on a shaker to make a 1:10 dilution; then use a 1mL sterile pipette to draw 1mL of the 1:10 dilution, transfer it into a test tube filled with 9mL sterile water, and mix it thoroughly to make a 1:100 dilution. Take another 1mL sterile pipette and make 10-fold incremental dilutions according to the above operation sequence to make 10 according to the bacterial content of the sample. -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 A series of dilutions of bacterial solution were taken, 10 -6 , 10 -7 , 10 -8 Three dilutions for inoculation of plates.

[0072] 4. Coating:

[0073] According to the estimation of the total number of viable bacteria in the sample to be tested, use a 0.1mL sterile pipette to take 0.1mL of three different dilutions respectively, repeat 3 times for each dilution, add them to the plates containing culture medium respectively, and use a sterile glass spreading rod to evenly spread the dilutions on the surface of the culture medium.

[0074] 5. Cultivation

[0075] Place the coated culture medium dish upside down in a constant temperature incubator for culture.

[0076] MRS medium was cultured anaerobically at 37°C for 48 hours.

[0077] (MRS+ciprofloxacin) medium was anaerobically cultured at 37°C for 48 hours.

[0078] 6. Colony morphology identification and counting

[0079] (1)LPC12

[0080] In the (MRS+Ciprofloxacin) culture medium, yellow colonies can be seen at the bottom of the plate. Obvious larger colonies are milky white, round, raised, with neat edges and a diameter of 1.8 mm±0.2 mm, which are LPC12.

[0081] (2)LRH10

[0082] In the (MRS+Ciprofloxacin) culture medium, the bottom of the plate was yellow and obviously smaller colonies were visible. They were milky white, round, raised, with neat edges and a diameter of 0.7 mm ± 0.3 mm, which was LRH10.

[0083] (3)LH43

[0084] In the (MRS+Ciprofloxacin) culture medium, yellow colonies can be seen at the bottom of the plate. Obvious smaller colonies are milky white, round, with raised colonies and neat edges. The diameter is 2.5 mm ± 0.2 mm, which is LH43.

[0085] (4) LF26

[0086] Lactobacillus fermentum LF26 does not grow in (MRS+ciprofloxacin) medium. The total number of bacteria on MRS medium minus the total number of bacteria on (MRS+ciprofloxacin) medium is the number of Lactobacillus fermentum colonies.

[0087] The calculation formula of the number of each fermented lactic acid bacteria is:

[0088]

[0089] The principle of this method is based on the fact that each bacteria has a different inhibitory effect on antibiotics, and the strains can be distinguished based on their colony size.

[0090] Example 1 Preparation method of postbiotics with precise control of multi-strain symbiotic fermentation

[0091] (1) Prepare a base in the following proportions by mass: 20% skim milk powder, 0.5% yeast powder, 5% white sugar, and the remainder water, and stir to dissolve;

[0092] (2) sterilizing the substrate of step (1): 95°C, 10 min;

[0093] (3) Cooling: Cool to the inoculation temperature of 42°C;

[0094] (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain in Table 1; the base viable bacterial count after inoculation is 1.0×10 5 CFU / ml;

[0095] Table 1 Proportion of inoculated cells of each strain

[0096] Lactobacillus rhamnosus LRH10 40％ Lactobacillus mucilaginosus fermentation LF26 17％ Lactobacillus paracasei LPC12 17％ Lactobacillus helveticus LH43 17％ Streptococcus salivarius subsp. thermophilus ST30 9％

[0097] The cell number of each lactic acid bacteria after inoculation was detected, and the actual cell number ratio of each strain, the bacterial number of each strain, and the total bacterial number were calculated. The results are shown in Table 2.

[0098] (5) First fermentation: 42°C for 8 h;

[0099] (6) Adjust the test pH to 4.8-5.0, add 1% yeast extract (based on the base before inoculation) and 1% isomaltooligosaccharide (based on the base before inoculation);

[0100] The cell count of each lactic acid bacteria in the intermediate stage was detected, and the actual cell count ratio of each strain, the bacterial count of each strain, and the total bacterial count were calculated. The results are shown in Table 2.

[0101] (7) Secondary fermentation: fermentation at 37°C for 16 h;

[0102] (8) Preparation: adding 15% maltodextrin to the fermentation product obtained in step (7);

[0103] (9) Spray drying: air inlet temperature 180°C, air outlet temperature 110°C.

[0104] After spray drying, due to the instant inactivation of cells, the structure of the lactic acid bacteria strains was basically the same as before spraying, and a postbiotic with precise control of multi-species symbiotic fermentation was obtained. The cell number of each lactic acid bacteria was detected, and the actual cell number ratio of each strain, the bacterial number of each strain, and the total bacterial number were calculated. The results are shown in Table 2.

[0105] Among them, the cell number percentage of each fermentation strain is: LRH10 (12.1%), LPC12 (2%), LF26 (26.1%), ST30 (21.6%), and LH43 (38.2%).

[0106] Table 2 Results of precise control scheme

[0107]

[0108] It can be seen from Table 2 that the fermentation of the present invention accurately supplements nutrients with targeted carbon sources and nitrogen sources at a fixed time and place; the number of various lactic acid bacteria in the postbiotics accurately controlled by the multi-species symbiotic fermentation prepared by the present invention is stable.

[0109] Comparative Example 1 Conventional preparation method of postbiotic fermentation powder

[0110] (1) Prepare a base in the following proportions by mass: 20% skim milk powder, 0.5% yeast powder, 5% white sugar, and the remainder water, and stir to dissolve;

[0111] (2) sterilizing the substrate of step (1): 95°C, 10 min;

[0112] (3) Cooling: Cool to the inoculation temperature of 42°C;

[0113] (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain in Table 3; the base number of viable bacteria after inoculation is 1.0×10 5 CFU / ml;

[0114] Table 3 Proportion of inoculated cells of each strain

[0115] Lactobacillus rhamnosus LRH10 40％ Lactobacillus mucilaginosus fermentation LF26 17％ Lactobacillus paracasei LPC12 17％ Lactobacillus helveticus LH43 17％ Streptococcus salivarius subsp. thermophilus ST30 9％

[0116] (5) Fermentation: 42° C. for 24 h;

[0117] (6) Preparation: adding 15% maltodextrin to the fermentation product obtained in step (5);

[0118] (7) Spray drying: inlet air temperature 180°C, outlet air temperature 110°C.

[0119] After spray drying, due to the instant inactivation of cells, the structure of the lactic acid bacteria strains was basically the same as before spraying, and a postbiotic fermentation powder was obtained. The number of cells of each lactic acid bacteria was detected, and the actual cell number ratio of each strain, the number of bacteria of each strain, and the total number of bacteria were calculated. The results are shown in Table 4.

[0120] Among them, the cell number percentage of each fermentation strain is: LRH10 (20%), LPC12 (16%), LF26 (16%), ST30 (33%), and LH43 (16%).

[0121] Table 4 Results of conventional regimen

[0122]

[0123] As can be seen from Table 4, the conventional preparation method cannot accurately control the cell number of each lactic acid bacteria strain in the postbiotics.

[0124] In previous studies, we found that a composite lactic acid bacteria postbiotic composition with a specific strain cell number ratio can combine the immune stimulation advantages of each lactic acid bacteria, thereby achieving the effect of promoting immunity. However, the conventional preparation scheme cannot accurately control the cell number of each lactic acid bacteria strain in the postbiotic. Therefore, the present invention provides a method for preparing a postbiotic with precise control of multi-species symbiotic fermentation, and the number of various lactic acid bacteria in the postbiotic with precise control of multi-species symbiotic fermentation prepared by the preparation method of the present invention is stable.

[0125] The following are the inventor's preliminary research results:

[0126] 1. Preparation of MRS culture medium

[0127] The specific components of MRS culture medium are as follows:

[0128]

[0129] 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.

[0130] 2. Preparation of composite lactic acid bacteria postbiotic composition

[0131] (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;

[0132] (2) Repeating step (1) with the activated lactic acid bacteria liquid for a second activation;

[0133] (3) heating the lactic acid bacteria solution activated for the second time at 85° C. for 30 min to sterilize;

[0134] (4) Transfer the sterilized lactic acid bacteria solution into a sterilized 15 ml centrifuge tube and centrifuge it at 3500 rpm for 5 min.

[0135] (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.

[0136] (6) Repeat step (5) three times to obtain lactic acid bacteria that have been washed three times;

[0137] (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;

[0138] (8) storing the lactic acid bacteria suspension after adjusting the absorbance in a -80°C refrigerator for later use;

[0139] (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.

[0140] 3. In vitro cell stimulation test

[0141] 1. Test cell types

[0142] Mouse peritoneal macrophage cell line-264.7 (RAW 264.7) was purchased from American Type Culture Collection (ATCC).

[0143] 2. Experimental drugs

[0144] Fetal bovine serum (FBS): Heat-inactivated fetal bovine serum purchased from Gibco and stored in a -20°C refrigerator.

[0145] Dulbecco's modified Eagle's medium (DMEM): purchased from Gibco, USA, and stored in a 4°C refrigerator.

[0146] Gram-negative bacterial lipopolysaccharide (LPS O55:B5): purchased from Sigma, USA.

[0147] Preparation of Gram-negative bacteria lipopolysaccharide solution for testing:

[0148] (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.

[0149] (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.

[0150] (3) Place LPS O55:B5 (10 ug / mL, 100 ug / mL and 1 mg / mL) in a -80°C refrigerator for later use.

[0151] 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.

[0152] 3. Cell culture:

[0153] (1) Preparation of cell culture medium:

[0154] 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.

[0155] (2) Cell thawing and culture:

[0156] 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.

[0157] 4. In vitro cell stimulation test:

[0158] (1) Use a cell scraper to remove well-growing RAW 264.7 cells from the cell culture dish.

[0159] (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.

[0160] (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.

[0161] (4) Allow two hours for the cells to settle and adhere to the bottom of the cell plate.

[0162] (5) Use a microscope to observe that the adhesion is good and then stimulate it.

[0163] (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.

[0164] The prepared composite lactic acid bacteria postbiotic composition was serially diluted by 10, 100, 1000, and 10000 times to obtain 10 8 , 10 7 , 10 6 , 10 5 cell / mL.

[0165] (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.

[0166] 5. Cytokine determination:

[0167] The cytokine levels secreted by RAW 264.7 cells were measured according to the method in the instruction manual of the enzyme-linked immunosorbent assay kit (Mouse TNF-α, IL-6, IL-10 and IL-12p40 ELISA Ready-SET-Go, purchased from Affymetrix eBioscience, Inc., USA).

[0168] 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.

[0169] 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.

[0170] Statistical analysis:

[0171] 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.

[0172] 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. Figure 1 It can be seen that the lactic acid bacteria composition, LF26 and ST30, and the bacterial counts are all 10 8CFU / 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.

[0173] Figure 2 The effect of lactic acid bacteria and its combination on the secretion of TNF-α in mouse peritoneal macrophages. The different letters on the bar graph indicate that there is a significant difference between the groups using Tukey's Honestly Significant Difference Test (P<0.05). 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.

[0174] Figure 3 The effect of lactic acid bacteria and its combination on the secretion of IL-6 in mouse peritoneal macrophages. The different letters on the bar graph indicate that there is a significant difference between the groups using Tukey's Honestly Significant Difference Test (P<0.05). 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.

[0175] Figure 4 The present invention relates to the effects of lactic acid bacteria and their compositions on the secretion of IL-12p40 in mouse peritoneal macrophages. Figure 4 It can be seen that ST30 significantly increased the secretion of cytokines compared with the other stimulation groups.

[0176] In summary, the lactic acid bacteria composition utilizes 10 8 CFU / mL can significantly increase the secretion of IL-10, while the other cytokines TNF-a, IL-6 and / or IL-12p40 all have the second highest secretion amount. This indicates that the composite lactic acid bacteria postbiotic composition containing a specific strain cell number ratio can combine the immune stimulating advantages of each lactic acid bacteria, thereby achieving the effect of promoting immunity.

[0177] 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 method for preparing postbiotics with precise control of multi-strain symbiotic fermentation, characterized in that: The following steps are involved: (1) Prepare a base according to the ratio of 20-25% skim milk powder, 0.1-0.5% yeast powder, 1-5% white sugar, and the rest water by mass percentage, and stir to dissolve; (2) sterilizing the substrate of step (1): 90-95° C., 10-15 min; (3) Cooling: Cool to the inoculation temperature of 40-44°C; (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain; the base number of live bacteria after inoculation is 1.0×10 5 CFU / ml; (5) First fermentation: 40-44°C for 5-8 hours; (6) Adjust the test pH to 4.8-5.0, add 0.5-1% yeast extract and 0-1% isomaltooligosaccharide based on the base before inoculation; (7) Secondary fermentation: fermentation at 37-39°C for 10-16 hours; (8) Preparation: adding 15-20% maltodextrin to the fermentation product obtained in step (7); (9) Spray drying: air inlet temperature 180°C, air outlet temperature 110°C; After spray drying, the postbiotics with precisely controlled multi-strain symbiotic fermentation are obtained.

2. The preparation method according to claim 1, characterized in that: The cell number proportion of each fermentation strain in the postbiotics precisely controlled by the multi-strain symbiotic fermentation is: LRH10 12±0.5%, LPC12 2±0.5%, LF26 26.3±0.5%, ST30 21.6±0.5%, LH43 38.1±0.5%.

3. The preparation method according to claim 1, characterized in that: The number of cells in the postbiotics precisely controlled by the multi-strain symbiotic fermentation is ≥ 1.0×10 9 cell / g.

4. The preparation method according to claim 1, characterized in that: The following steps are involved: (1) Prepare a base in the following proportions by mass: 20% skim milk powder, 0.5% yeast powder, 5% white sugar, and the remainder water, and stir to dissolve; (2) sterilizing the substrate of step (1): 95°C, 10 min; (3) Cooling: Cool to the inoculation temperature of 42°C; (4) Inoculation: Inoculate lactic acid bacteria according to the cell number ratio of each strain; the base number of live bacteria after inoculation is 1.0×10 5 CFU / ml; (5) First fermentation: 42°C for 8 h; (6) Adjust the test pH to 4.8-5.0, add 1% yeast extract and 1% isomaltooligosaccharide based on the base before inoculation; (7) Secondary fermentation: fermentation at 37°C for 16 h; (8) Preparation: adding 15% maltodextrin to the fermentation product obtained in step (7); (9) Spray drying: air inlet temperature 180°C, air outlet temperature 110°C; After spray drying, the postbiotics with precisely controlled multi-strain symbiotic fermentation are obtained.

5. The preparation method according to claim 4, characterized in that: The cell number proportion of each fermentation strain in the postbiotics precisely controlled by the multi-strain symbiotic fermentation is: LRH10 12.1%, LPC12 2%, LF26 26.1%, ST3021.6%, LH43 38.2%.

Images