A method for preparing postbiotics with complete cells, uniform dispersion and strong stability

Abstract

The present invention relates to a method for preparing a postbiotic with complete cells, uniform dispersion and strong stability. The preparation method comprises: taking a fermentation broth of probiotics, centrifuging it, collecting the precipitate, mixing the precipitate with an inactivation protective agent, inactivating it at 55-95°C to obtain an inactivated emulsion, mixing the inactivated emulsion with a drying protective agent, atomizing and drying it to obtain the postbiotic; the inactivation protective agent contains mannitol; the drying protective agent contains gelatin, chitosan, skim milk and β-cyclodextrin. The present invention designs a completely new method for preparing postbiotics, including designing a specific inactivation protective agent, an inactivation treatment process and a drying protective agent, which cooperate with each other to maintain cell integrity, improve bacterial dispersibility, and have high quality stability of postbiotics.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and relates to a method for preparing postbiotics with complete cells, uniform dispersion and strong stability. Background Art

[0002] Postbiotics refer to preparations of non-living microorganisms and / or their components that are beneficial to the health of the host. They are artificially inactivated microbial cells. Metabolites or cellular components that have been proven to be beneficial to health may or may not be added.

[0003] In the actual application scenarios of probiotic products, China's cosmetics regulations clearly stipulate that "live bacteria" are not allowed to be added to any cosmetics, and the rapid reproduction of live probiotics, coupled with the strict requirement that the total number of bacteria in cosmetics must be controlled at <1000CFU / mL(g), makes the use of live probiotics impossible. Therefore, postbiotics have become a more practical and feasible option in the field of daily chemical products.

[0004] Since postbiotics have lost their activity, they will not proliferate in the body, are not likely to cause infection or other adverse reactions, and can tolerate a wider range of temperatures and storage conditions. They do not require refrigeration and are safer and more stable than live probiotics. Many studies have compared the differences between live bacteria and heat-killed bacteria, proving that inactivated probiotics are as capable of adhering to and binding to macromolecules as live probiotics, and have higher binding stability. A prerequisite for inactivated bacteria to function is to adhere to and occupy a position in the environment of action, and then exert their effects by affecting the microecology in the environment. Postbiotics also have the functions of adsorbing toxins, adhering to antagonistic pathogens, and enhancing immunity. Therefore, postbiotics not only meet regulatory requirements, but also provide many advantages of live probiotics, so that they can be more widely used in various daily chemical products and have broad application prospects.

[0005] At present, the main preparation methods of postbiotics include heat inactivation, etc. For example, CN116042469A discloses a lactic acid bacteria postbiotic complex with antibacterial function and its preparation method and application, wherein the lactobacillus seed liquid obtained by two amplification cultures is inoculated into the fermentation medium for fermentation culture to obtain the first fermentation liquid; the metal antibacterial ion solution is continuously dripped into the first fermentation liquid, stirred while adding, and the chelation reaction is carried out at 40-50 ° C for 2-3 hours to obtain the second fermentation liquid; the second fermentation liquid is centrifuged to obtain a bacterial mud complex; the bacterial mud complex is inactivated by high temperature heat to obtain an inactivated lactic acid bacteria cell complex; the inactivated cell complex is dried and crushed to obtain a lactic acid bacteria postbiotic complex. However, killing the cell activity by high temperature cannot keep the cell structure intact, which affects the qualitative detection; the poor solubility of the bacterial powder and the high energy consumption of the drying method affect the application of the raw materials in the terminal product; and the inactivated bacteria product finally obtained cannot maintain good stability in terms of quality.

[0006] In summary, the development of new methods for preparing postbiotics can effectively maintain cell integrity and uniform dispersion, which is of great significance for the application field of postbiotics. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for preparing postbiotics with intact cells, uniform dispersion and strong stability, so as to achieve the preparation of postbiotics that maintain cell integrity, strong bacterial dispersion and support qualitative detection.

[0008] To achieve this object, the present invention adopts the following technical solutions:

[0009] In a first aspect, the present invention provides a method for preparing postbiotics with complete cells, uniform dispersion and strong stability, the preparation method comprising:

[0010] The fermentation liquid of probiotics is taken, centrifuged, and the precipitate is collected, the precipitate is mixed with an inactivation protective agent, and inactivated at 55 to 95° C. (for example, 56, 60, 65, 70, 75, 80, 85, 90, or 95° C.) to obtain an inactivated emulsion, the inactivated emulsion is mixed with a drying protective agent, and atomized and dried to obtain the postbiotic; the inactivation protective agent contains mannitol; the drying protective agent contains gelatin, chitosan, skim milk and β-cyclodextrin.

[0011] In the present invention, a completely new method for preparing postbiotics is designed, including designing a specific inactivation protective agent, an inactivation treatment process, and a drying protective agent, which work together to maintain cell integrity, improve bacterial dispersibility, support qualitative detection, and have high quality stability of postbiotics.

[0012] In the present invention, the gelatin refers to a protein obtained by partial hydrolysis of collagen, and the skim milk refers to a product obtained by removing fat from fresh milk and drying it.

[0013] Preferably, the temperature of the inactivation treatment is 60-90°C.

[0014] In the present invention, the temperature of the inactivation treatment is further controlled, so that efficient inactivation can be maintained while effectively maintaining the integrity of the cell structure.

[0015] Preferably, the inactivation treatment time is 5 to 30 minutes, including but not limited to 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 18, 20, 22, 25, 26, 28 or 29 minutes.

[0016] Preferably, the inactivation protective agent contains 6% to 20% mannitol (for example, 7%, 8%, 9%, 10%, 11%, 15%, 16%, 17%, 18% or 19%, etc.) based on 100% by mass, and the remainder is water.

[0017] In the present invention, a specific inactivation protective agent is designed to further improve the integrity of the cell structure.

[0018] Preferably, the mass ratio of the precipitate to the inactivation protective agent is 1:(0.6-1.4), including but not limited to 1:0.7, 1:0.8, 1:0.9, 1:1.1, 1:1.2 or 1:1.3, etc.

[0019] Preferably, the drying protective agent contains 2% to 8% gelatin (e.g., 3%, 4%, 5%, 6% or 7%), 2% to 4% chitosan, 8% to 14% skim milk (e.g., 9%, 10%, 11%, 12% or 13%) and 2% to 5% β-cyclodextrin based on 100% by mass, and the remainder is water.

[0020] Preferably, the mass ratio of the inactivated emulsion to the drying protective agent is 1:(1-5), including but not limited to 1:2, 1:3 or 1:4.

[0021] In the present invention, a specific drying protective agent and a mixing ratio are designed to further improve the dispersibility of the postbiotic bacterial powder and the integrity of the cell structure.

[0022] Preferably, the probiotics include any one or a combination of at least two of Lactobacillus probiotics, Bifidobacterium probiotics, Streptococcus probiotics, Enterococcus probiotics, Lactococcus probiotics, Pediococcus probiotics, Staphylococcus probiotics or Leuconostoc probiotics.

[0023] Preferably, the Lactobacillus probiotics include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, Lactobacillus sakei, Lactobacillus crispatus, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, Lactobacillus sakei, Lactobacillus crispatus, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, Lactobacillus salivarius ... crispatus or Propionibacterium freudenreichii, or a combination of at least two thereof.

[0024] Preferably, the Bifidobacterium probiotic includes any one or a combination of at least two of Bifidobacterium animalis, Bifidobacterium longum subsp. longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis subsp. infanttis or Bifidobacterium bifidum.

[0025] Preferably, the Streptococcus probiotic includes Streptococcus salivarius subsp. thermophilus.

[0026] Preferably, the Enterococcus probiotics include Enterococcus faecalis and / or Enterococcus faecium.

[0027] Preferably, the Lactococcus probiotic includes Lactococcus lactis.

[0028] Preferably, the Pediococcus probiotics include Pediococcus acidilactici and / or Pediococcus pentosaceus.

[0029] Preferably, the Staphylococcus probiotics include any one of Mammaliicoccus vitulinus, Staphylococcus xylosus or Staphylococcus carnosus, or a combination of at least two thereof.

[0030] Preferably, the Leuconostoc probiotic enteric bacteria includes Leuconostoc mesenteroides.

[0031] Preferably, the preparation method further comprises the step of adjusting the temperature and pH of the fermentation liquid of the probiotics, specifically comprising adjusting the temperature of the fermentation liquid of the probiotics to 10-20° C. and the pH to 6.2-7.2.

[0032] Preferably, the centrifugal treatment has a rotation speed of 3000-10000 rpm, including but not limited to 3500, 4000, 4500, 5000, 6000, 7000, 8000, 9000 or 9500 rpm, and a time of 5-20 min, including but not limited to 6, 7, 8, 9, 10, 15, 16, 17, 18 or 19 min, etc.

[0033] Preferably, the temperature of the atomization treatment is 130-180°C, including but not limited to 131, 132, 135, 140, 150, 160, 170, 175, 176, 177, 178 or 179°C, etc.

[0034] Preferably, the drying method comprises vacuum freeze drying.

[0035] As a preferred technical solution, the method for preparing postbiotics with complete cells, uniform dispersion and strong stability includes:

[0036] The fermentation liquid of probiotics is taken, the temperature of the fermentation liquid of the probiotics is adjusted to 10-20° C. and the pH value is adjusted to 6.2-7.2, centrifuged, and the precipitate is collected, the precipitate is mixed with an inactivation protective agent at a mass ratio of 1:(0.6-1.4), and inactivation treatment is performed at 55-95° C. for 5-15 minutes to obtain an inactivated emulsion, the inactivated emulsion is mixed with a drying protective agent at a mass ratio of 1:(1-5), and atomized and dried to obtain the postbiotic; the inactivation protective agent contains 6%-20% mannitol by mass percentage, and the balance is water; the drying protective agent contains 2%-8% gelatin, 2%-4% chitosan, 8%-14% skim milk and 2%-5% β-cyclodextrin by mass percentage, and the balance is water.

[0037] Compared with the prior art, the present invention has the following beneficial effects:

[0038] The present invention designs a completely new method for preparing postbiotics, including designing a specific inactivation protective agent, an inactivation treatment process and a drying protective agent, which cooperate with each other to maintain cell integrity while efficiently inactivating the cells, so that the postbiotics have strong bacterial dispersion and high quality stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a diagram of the cell structure morphology of Example 1;

[0040] Figure 2 This is a diagram of the cell structure morphology of Example 2;

[0041] Figure 3 This is a diagram of the cell structure morphology of Example 3;

[0042] Figure 4 This is the cell structure morphology diagram of Comparative Example 2;

[0043] Figure 5 This is a cell structure morphology diagram of Comparative Example 3;

[0044] Figure 6 This is the cell structure morphology diagram of Comparative Example 4;

[0045] Figure 7 This is a cell structure morphology diagram of Comparative Example 5;

[0046] Figure 8 This is a cell structure morphology diagram of Comparative Example 6;

[0047] Fig. 9 This is a cell structure morphology diagram of Comparative Example 7;

[0048] Fig.10 This is a cell structure morphology diagram of Comparative Example 8;

[0049] Fig.11This is a cell structure morphology diagram of Comparative Example 9;

[0050] Fig.12 This is a cell structure morphology diagram of Comparative Example 10;

[0051] Fig.13 It is a diagram of the structure and morphology of living cells;

[0052] Fig.14 This is a complete morphological diagram of the cell structure of Comparative Example 11;

[0053] Fig.15 This is a cell structure morphology diagram of Comparative Example 12;

[0054] Fig.16 This is a cell structure morphology diagram of Comparative Example 13;

[0055] Fig.17 This is the result diagram of cell structure integrity and dispersion in Example 1;

[0056] Fig.18 This is the result diagram of cell structure integrity and dispersion of Comparative Example 11;

[0057] Fig.19 This is the result diagram of cell structure integrity and dispersion of Comparative Example 12;

[0058] Fig. 20 This is the result diagram of cell structure integrity and dispersion of Comparative Example 13. DETAILED DESCRIPTION

[0059] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[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] At present, the thermal inactivation technology for the preparation of postbiotics simply kills the cell activity through high temperature, and cannot keep the cell structure intact, which affects the qualitative detection; the poor solubility of the bacterial powder and the high energy consumption of the drying method affect the application of the raw materials in the terminal product; and the final inactivated bacteria product cannot maintain good stability in quality and has poor dispersibility. Based on the above problems, the present invention designs a new method for preparing postbiotics, designs specific inactivation protective agents, inactivation processes, and drying protective agents, etc., which can maintain cell integrity, improve bacterial dispersibility, support qualitative detection, and the quality stability of postbiotics is high.

[0062] In the specific embodiment of the present invention, Lactobacillus salivarius LS97 is taken as an example to verify the technical scheme of the present invention. Mannitol is purchased from Zhejiang Yinuo Biotechnology Co., Ltd., gelatin is purchased from Liaoning Zhongfa Biotechnology Co., Ltd., chitosan is purchased from Weifang Haizhiyuan Biological Products Co., Ltd., skim milk is purchased from New Zealand Fonterra Group, and β-cyclodextrin is purchased from Shanghai Yuanye Biological Co., Ltd.

[0063] Example 1

[0064] This example prepares postbiotics.

[0065] 1. Pretreatment of Lactobacillus salivarius LS97 before inactivation: ferment and culture Lactobacillus salivarius LS97 using a general method, collect the fermentation liquid, and obtain the highly active Lactobacillus salivarius LS97 fermentation liquid (the viable count level is required to be not less than 1×10 9 CFU / mL) was immediately cooled down, and after cooling to 15°C, the pH of the fermentation broth was adjusted to 7.0 with NaOH, and the broth was centrifuged at 8000rpm for 5min, the supernatant was discarded, and the centrifugal precipitate (bacterial mud) was collected. The inactivation protective agent (bacterial mud: 10% mannitol 1:1 (W / W)) was added to the bacterial mud in proportion, and the broth was stirred continuously at 900rpm for 2.5h at 15°C;

[0066] 2. Inactivation of Lactobacillus salivarius LS97: preheat the mixture obtained in step 1 to 55° C., and then inactivate the mixture at 55° C. and 200 rpm for 5 min to obtain an inactivated emulsion.

[0067] 3. Pre-granulation treatment: The inactivated Lactobacillus salivarius LS97 emulsion obtained in step 2 was mixed with a drying protective agent (gelatin 5%, chitosan 2%, skim milk 11%, β-cyclodextrin 3%, and the balance was water) in a mass ratio of 1:3, stirred at 800 rpm for 30 min, mixed evenly, and the current material temperature was precooled to -40°C.

[0068] 4. Atomization: The mixed liquid obtained in step 3 is atomized into fine droplets by using an atomizing device in a spray tower. During the spraying process, a cryogenic liquid pump is used to add the cryogenic liquid in the storage container to the collection container. The atomization temperature is 150°C.

[0069] 5. Freeze

[0070] The above droplets are rapidly cooled and frozen under the action of low-temperature fluid (low-temperature nitrogen above liquid nitrogen) to form frozen particles. A magnetic stirrer is installed at the bottom of the collection container to continuously stir the sample, so that the sample is frozen evenly.

[0071] 6. Drying

[0072] The frozen particles obtained in step 5 are collected and vacuum condensed to obtain a dry powder, which is the final product (i.e., postbiotics).

[0073] The number of cells in the final product is not less than 1×10 12 cells / g, moisture content not higher than 3.0%, water activity not higher than 0.1aw; the product is milky white to light yellow microparticles with a particle size of 1 to 20μm, which can be completely dissolved in water within 5 to 10 seconds without suspension or agglomeration.

[0074] Example 2

[0075] This example prepares postbiotics.

[0076] 1. Pretreatment of Lactobacillus salivarius LS97 before inactivation: ferment and culture Lactobacillus salivarius LS97 using a general method, collect the fermentation liquid, and obtain the highly active Lactobacillus salivarius LS97 fermentation liquid (the viable count level is required to be not less than 1×10 9 CFU / mL) was immediately cooled down, and after cooling to 10°C, the pH of the fermentation broth was adjusted to 7.2 with NaOH, and the broth was centrifuged at 3000 rpm for 20 min, the supernatant was discarded, and the centrifugal precipitate (bacterial mud) was collected. The inactivation protective agent (bacterial mud: 6% mannitol 1:1.4 (W / W)) was added to the bacterial mud in proportion, and the broth was stirred continuously at 900 rpm for 2.5 h at 15°C;

[0077] 2. Inactivation of Lactobacillus salivarius LS97: preheat the mixture obtained in step 1 to 70° C., then inactivate at 70° C. and 200 rpm for 15 min to obtain an inactivated emulsion.

[0078] 3. Pre-granulation treatment: The inactivated Lactobacillus salivarius LS97 emulsion obtained in step 2 was mixed with a drying protective agent (glycerol 2%, chitosan 4%, skim milk 14%, β-cyclodextrin 2%, and the balance was water) in a mass ratio of 1:1, stirred at 800 rpm for 30 min, mixed evenly, and the current material temperature was precooled to -40°C.

[0079] 4. Atomization: The mixed liquid obtained in step 3 is atomized into fine droplets by using an atomizing device in a spray tower. During the spraying process, a cryogenic liquid pump is used to add the cryogenic liquid in the storage container to the collection container. The atomization temperature is 130°C.

[0080] 5. Freeze

[0081] The above droplets are rapidly cooled and frozen under the action of low-temperature fluid (low-temperature nitrogen above liquid nitrogen) to form frozen particles. A magnetic stirrer is installed at the bottom of the collection container to continuously stir the sample, so that the sample is frozen evenly.

[0082] 6. Drying

[0083] The frozen particles obtained in step 5 are collected and vacuum condensed to obtain a dry powder, which is the final product (i.e., postbiotics).

[0084] The number of cells in the final product is not less than 1×10 12 cells / g, water content not higher than 3.0%, water activity not higher than 0.1aw; product properties are milky white to light yellow microparticles, particle size 1-20μm, which can be completely dissolved in water within 5-10 seconds without suspension or agglomeration

[0085] Example 3

[0086] This example prepares postbiotics.

[0087] 1. Pretreatment of Lactobacillus salivarius LS97 before inactivation: ferment and culture Lactobacillus salivarius LS97 using a general method, collect the fermentation liquid, and obtain the highly active Lactobacillus salivarius LS97 fermentation liquid (the viable count level is required to be not less than 1×10 9 CFU / mL) was immediately cooled down, and after cooling to 20°C, the pH of the fermentation liquid was adjusted to 6.2 with NaOH, and centrifuged at 10000rpm for 5min, the supernatant was discarded, and the centrifugal sediment (bacterial mud) was collected. The inactivation protective agent (bacterial mud: 20% mannitol 1:0.6 (W / W)) was added to the bacterial mud in proportion, and the mixture was stirred at 900rpm for 2.5h at 15°C;

[0088] 2. Inactivation of Lactobacillus salivarius LS97: preheat the mixture obtained in step 1 to 95° C., then inactivate at 95° C. and 200 rpm for 5 min to obtain an inactivated emulsion.

[0089] 3. Pre-granulation treatment: The inactivated Lactobacillus salivarius LS97 emulsion obtained in step 2 was mixed with a drying protective agent (gelatin 8%, chitosan 2%, skim milk 8%, β-cyclodextrin 5%, and the balance was water) in a mass ratio of 1:5, stirred at 800 rpm for 30 min, mixed evenly, and the current material temperature was precooled to -40°C.

[0090] 4. Atomization: The mixed liquid obtained in step 3 is atomized into fine droplets by using an atomizing device in a spray tower. During the spraying process, a cryogenic liquid pump is used to add the cryogenic liquid in the storage container to the collection container. The atomization temperature is 180°C.

[0091] 5. Freeze

[0092] The above droplets are rapidly cooled and frozen under the action of low-temperature fluid (low-temperature nitrogen above liquid nitrogen) to form frozen particles. A magnetic stirrer is installed at the bottom of the collection container to continuously stir the sample, so that the sample is frozen evenly.

[0093] 6. Drying

[0094] The frozen particles obtained in step 5 are collected and vacuum condensed to obtain a dry powder, which is the final product (i.e., postbiotics).

[0095] The number of cells in the final product is not less than 1×10 12 cells / g, water content not higher than 3.0%, water activity not higher than 0.1aw; product properties are milky white to light yellow microparticles, particle size 1-20μm, which can be completely dissolved in water within 5-10 seconds without suspension or agglomeration

[0096] Comparative Example 1

[0097] This comparative example prepares postbiotics.

[0098] Compared with Example 1, the only difference is that the preheating and inactivation temperatures in step 2 are both adjusted to 53°C.

[0099] Comparative Example 2

[0100] This comparative example prepares postbiotics.

[0101] Compared with Example 1, the only difference is that the preheating and inactivation temperatures in step 2 are both adjusted to 97°C.

[0102] Comparative Example 3

[0103] This comparative example prepares postbiotics.

[0104] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of 10% trehalose.

[0105] Comparative Example 4

[0106] This comparative example prepares postbiotics.

[0107] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of 10% sucrose.

[0108] Comparative Example 5

[0109] This comparative example prepares postbiotics.

[0110] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of 10% glucose.

[0111] Comparative Example 6

[0112] This comparative example prepares postbiotics.

[0113] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of 10% mannitol+trehalose (the mass ratio of mannitol to trehalose is 1:1).

[0114] Comparative Example 7

[0115] This comparative example prepares postbiotics.

[0116] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of 10% glucose+mannitol (the mass ratio of glucose to mannitol is 1:1).

[0117] Comparative Example 8

[0118] This comparative example prepares postbiotics.

[0119] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of 10% trehalose+glucose (the mass ratio of trehalose to glucose is 1:1).

[0120] Comparative Example 9

[0121] This comparative example prepares postbiotics.

[0122] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of 10% mannitol+trehalose+glucose (the mass ratio of mannitol, trehalose and glucose is 1:1:1).

[0123] Comparative Example 10

[0124] This comparative example prepares postbiotics.

[0125] Compared with Example 1, the only difference is that the inactivation protective agent is replaced by an equal amount of water.

[0126] Comparative Example 11

[0127] This comparative example prepares postbiotics.

[0128] Compared with Example 1, the only difference is that the drying protective agent is replaced by an equal amount of β-cyclodextrin (β-cyclodextrin replaces gelatin, chitosan and skim milk in the original components in equal amounts).

[0129] Comparative Example 12

[0130] This comparative example prepares postbiotics.

[0131] Compared with Example 1, the only difference is that the drying protective agent is replaced by an equal amount of glycerol+sucrose+skim milk+maltodextrin (an equal amount of glycerol replaces gelatin, an equal amount of sucrose replaces chitosan, and an equal amount of maltodextrin replaces β-cyclodextrin).

[0132] Comparative Example 13

[0133] This comparative example prepares postbiotics.

[0134] Compared with Example 1, the only difference is that the drying protective agent is replaced by an equal amount of dextran + skim milk + sodium starch octenyl succinate (dextran replaces gelatin and chitosan in equal amounts, and sodium starch octenyl succinate replaces β-cyclodextrin in equal amounts).

[0135] Test Case

[0136] This test example tests the preparation process of each embodiment and comparative example, including inactivation rate, cell integrity and dispersibility, etc.

[0137] Inactivation rate test method:

[0138] MRS plate count, accurately weigh 1.0g of the emulsion before and after inactivation, and use the dilution plate count method to count live bacteria. Select 1mL of the bacterial solution from 3 dilution gradients and put it into the plate, pour it into the modified MRS agar medium, mix it well, and invert it at 37℃ for 48h. Record the number of colonies (CFU) on the culture dish, and the total number of colonies is expressed as CFU / g. The calculation formula for the bacterial inactivation rate is as follows:

[0139] Inactivation rate = (1-N / N 0 )×100%;

[0140] Where N 0 ——The number of viable bacteria per unit volume of the sample before inactivation (CFU / g);

[0141] N——The number of viable bacteria per unit volume of the sample after inactivation (CFU / g).

[0142] Cell Integrity Test Methods:

[0143] Scanning electron microscopy

[0144] After fresh sampling, fix the sample with 2.5% glutaraldehyde fixative, place it at 4℃ overnight, pour off the 2.5% glutaraldehyde fixative, rinse the sample with 0.1M, pH7.0 phosphate buffer three times, 15min each time; fix the sample with 1% osmium acid solution for 1-2h; carefully remove the osmium acid waste solution, rinse the sample with 0.1M, pH 7.0 phosphate buffer three times, 15min each time; dehydrate the sample with gradient concentration (including 30%, 50%, 70%, 80%, 90% and 95% five concentrations) of ethanol solution, each concentration treatment for 15min, and then treat it with 100% ethanol twice, 20min each time. Treat the sample with a mixture of ethanol and isoamyl acetate (V / V=1 / 1) for 30min, and then treat the sample with pure isoamyl acetate for 1h or place it overnight. Critical point drying / freeze drying treatment. Coating and observation. The treated sample is observed under a scanning electron microscope.

[0145] Dispersion test method:

[0146] Blood cell count and electron microscope observation

[0147] Under sterile conditions, weigh 1.0g of dry sample and dilute it 10 times to a certain gradient to obtain an isogradient dilution solution. Observe the counting chamber under a microscope. According to the local control principle of random block design, divide the counting chamber into the upper left, lower left, upper right, lower right and central areas. Take 5 fields of view in each area, that is, 5×5 fields of view for a total of 25 fields of view. Count the 25 fields of view and calculate the average value. Then, the total number of bacteria contained in each gram of sample can be calculated according to the formula.

[0148] Stability test method:

[0149] Accelerated test 6 months (42℃, 75%)

[0150] An accelerated test was carried out at a constant temperature of 42°C for 6 months to examine the sensory indicators, physicochemical indicators, microbiological indicators and bacterial count indicators of postbiotics, and to preliminarily predict the stability and shelf life of postbiotics.

[0151] Comparing the inactivation rates and cell structural integrity of Examples 1-3 and Comparative Examples 1 and 2, the inactivation rate of Examples 1-3 is 100%, and the cell morphology is shown in the figure below. Figure 1-3 As shown (the cell membrane surface is smooth and the cells are round); the inactivation rate of comparative example 1 is only 99.9375%, which cannot reach 100%, and the total colony count of the product exceeds the standard and does not meet the product requirements; the inactivation rate of comparative example 2 is 100%, and the cell morphology is as shown Figure 4 As shown, the cell structure integrity is poor (the cell membrane is severely damaged, accompanied by cell breakage, deformation, collapse, and cytoplasm leakage); it can be seen that the present invention controls a specific inactivation temperature, which can effectively maintain cell integrity and achieve efficient inactivation.

[0152] Comparing the cell structure integrity of Example 1 and Comparative Examples 3-10, the cell morphology diagrams of Comparative Examples 3-10 are as follows: Figure 5-Figure 12 As shown, living cells were used as control ( Fig.13 ), it can be seen that the cell structure integrity of Comparative Examples 3-10 was damaged to a certain extent, while Example 1 maintained good cell structure integrity, indicating that the specific inactivation protective agent designed by the present invention can cooperate with the overall process to effectively maintain the cell structure integrity.

[0153] Comparing the cell structure integrity and dispersion of Example 1 and Comparative Examples 11-13, the cell morphology diagrams of Comparative Examples 11-13 are as follows: Figure 14-16 As shown, the dispersion results of Example 1 are as follows Fig.17 As shown, the dispersibility results of Comparative Examples 11-13 are as follows Figure 18-Figure 20As shown, it can be seen that only Example 1 can achieve good dispersibility and cell structure stability, indicating that the specific drying protectant designed in the present invention can cooperate with the overall process to effectively maintain the integrity and dispersibility of the cell structure and prepare high-quality postbiotics.

[0154] The product prepared in Example 1 was tested for stability. The results are shown in Table 1. After the final postbiotic product was packaged, it was placed at a temperature of 40°C ± 2°C and a relative humidity of 75% ± 5%. The product's appearance, water activity, pH, microbial indicators and functional indicators were observed for 6 months. None of the indicators changed significantly, and the intrinsic indicators and hygiene indicators of the product met the product enterprise standards, proving that this postbiotic product is stable.

[0155] Table 1

[0156]

[0157] In summary, the present invention aims to solve the problems that the current postbiotic preparation cannot well keep the cell structure intact and has poor dispersibility, and designs a new postbiotic preparation method, designs specific inactivation protective agents, inactivation processes and drying protective agents, etc. The overall process is coordinated to maintain cell integrity, improve bacterial dispersibility and support qualitative detection, and the quality stability of postbiotics is high.

[0158] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing postbiotics with complete cells, uniform dispersion and strong stability, characterized in that: The preparation method comprises: The fermentation liquid of the probiotics is centrifuged to collect the precipitate, the precipitate is mixed with an inactivation protective agent at a mass ratio of 1:(0.6-1.4), and the inactivation treatment is performed at 55-95° C. for 5-15 min to obtain an inactivated emulsion, the inactivated emulsion is mixed with a drying protective agent at a mass ratio of 1:(1-5), and the mixture is atomized and dried to obtain the postbiotic; The probiotic is Lactobacillus salivarius LS97; The inactivation protective agent contains 6% to 20% mannitol based on 100% by mass, and the remainder is water; The desiccant protective agent contains 2% to 8% gelatin, 2% to 4% chitosan, 8% to 14% skim milk and 2% to 5% beta-cyclodextrin in terms of mass percentage, and the balance is water.

2. The method for preparing postbiotics with complete cells, uniform dispersion and strong stability according to claim 1, characterized in that: The preparation method further comprises the step of adjusting the temperature and pH of the fermentation liquid of the probiotics, specifically comprising adjusting the temperature of the fermentation liquid of the probiotics to 10-20° C. and the pH to 6.2-7.

2.

3. The method for preparing postbiotics with intact cells, uniform dispersion and strong stability according to claim 1, characterized in that: The rotation speed of the centrifugal treatment is 3000-10000 rpm, and the time is 5-20 min.

4. The method for preparing postbiotics with complete cells, uniform dispersion and strong stability according to claim 1, characterized in that: The temperature of the atomization treatment is 130-180°C.

5. The method for preparing postbiotics with complete cells, uniform dispersion and strong stability according to claim 1, characterized in that: The drying method includes vacuum freeze drying.

6. The method for preparing postbiotics with complete cells, uniform dispersion and strong stability according to claim 1, characterized in that: The preparation method comprises: Taking a fermentation broth of probiotics, adjusting the temperature of the fermentation broth of the probiotics to 10-20° C. and the pH to 6.2-7.2, performing a centrifugal treatment, collecting a precipitate, mixing the precipitate with an inactivation protective agent at a mass ratio of 1:(0.6-1.4), performing an inactivation treatment at 55-95° C. for 5-15 min to obtain an inactivated emulsion, mixing the inactivated emulsion with a drying protective agent at a mass ratio of 1:(1-5), performing an atomization treatment and a drying treatment to obtain the postbiotic; The inactivation protective agent contains 6% to 20% mannitol based on 100% by mass, and the remainder is water; The desiccant protective agent contains 2% to 8% gelatin, 2% to 4% chitosan, 8% to 14% skim milk and 2% to 5% beta-cyclodextrin in terms of mass percentage, and the balance is water.

Images