Method for preparing metagen by using fermented supernate and application of metagen

Abstract

The invention belongs to the technical field of food industry, and particularly relates to a method for preparing metabiotics from fermented supernate and application. The invention provides a preparation method of metabiotics, which comprises the following steps: mixing lactobacillus fermentation supernate with a carbon source to obtain first feed liquid; and inoculating lactic acid bacteria into the first feed liquid for culturing to obtain the metabiotics. According to the method disclosed by the invention, the waste of the lactobacillus fermentation supernatant is used as a main raw material, so that the reutilization of the lactobacillus fermentation supernatant is realized, and residual proteins, saccharides, vitamins and other nutrients in the supernatant are used for carrying out secondary fermentation culture to prepare the metabiotics; meanwhile, in consideration of relative lack of nutritional ingredients in the supernate, a carbon source is added to enrich nutrition in the supernate, and the prepared metagen is high in thallus content and has the effects of relieving constipation and diarrhea, improving immunity, resisting oxidation (delaying senescence), relieving pressure, promoting sleep, inhibiting bacteria and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of microecological product production, and specifically relates to a method for preparing postbiotics by utilizing fermentation supernatant and its application. Background Art

[0002] Postbiotics are a general term for the metabolites of probiotics after probiotics have been processed, including bacteria and metabolites. In recent years, studies have found that inactivated probiotics and their lysates, extracts or separated parts have similar effects to probiotics, and have higher gastric acid tolerance and are easier to preserve than probiotics.

[0003] During the production process, probiotic production enterprises centrifuge or filter the fermentation liquid of probiotic production to collect the bacteria, and obtain the fermentation supernatant. The fermentation supernatant is generally the waste of probiotic production enterprises, and is usually discharged into the sewage treatment system after being treated by acid-base adjustment, activated sludge method, membrane bioreactor, etc., which increases the treatment cost on the one hand, and on the other hand, the fermentation supernatant is not effectively utilized, wasting resources. Therefore, it is urgent to develop the fermentation supernatant, realize waste utilization, and improve the economic value of the enterprise. However, there is no report on the preparation of postbiotics using fermentation supernatant waste. Summary of the invention

[0004] The purpose of the present invention is to provide a method and application of preparing postbiotics using fermentation supernatant. The present invention prepares postbiotics using fermentation supernatant waste, thereby realizing the recycling of fermentation supernatant waste.

[0005] In order to solve the above technical problems, the present invention proposes the following technical solutions:

[0006] The present invention provides a method for preparing postbiotics, comprising the following steps:

[0007] Mixing the lactic acid bacteria fermentation supernatant with a carbon source to obtain a first feed liquid;

[0008] Lactic acid bacteria are inoculated into the first liquid for culturing to obtain liquid postbiotics.

[0009] Preferably, the components of the lactic acid bacteria fermentation supernatant include: a total solid content of 4.96% to 7.04%, a total sugar content of 0.11% to 0.60%, a total protein content of 1.67% to 2.45% and a total acid content of 2.41 to 7.12 g / L; the total acid content is calculated as lactic acid content.

[0010] Preferably, after obtaining the liquid postbiotics, the method further comprises drying the liquid postbiotics to obtain solid postbiotics.

[0011] Preferably, the carbon source comprises glucose, or the carbon source comprises glucose, yeast extract and soy peptone.

[0012] Preferably, when the carbon source includes glucose, the mass volume ratio of the glucose to the lactic acid bacteria fermentation supernatant is (2-4) g:100 mL; or,

[0013] When the carbon source includes glucose, yeast extract powder and soy peptone, the mass volume ratio of the glucose to the lactic acid bacteria fermentation supernatant is (2-4) g:100 mL, the mass volume ratio of the yeast extract powder to the lactic acid bacteria fermentation supernatant is (1-1.5) g:100 mL, and the mass volume ratio of the soy peptone to the lactic acid bacteria fermentation supernatant is (1-1.5) g:100 mL.

[0014] Preferably, the culture temperature is 36-38° C., and the culture time is 24-72 hours.

[0015] Preferably, the inoculation form of lactic acid bacteria includes lactic acid bacteria seed liquid.

[0016] The present invention provides a postbiotic, characterized in that it is prepared by adopting the preparation method of the postbiotic described in the above technical scheme.

[0017] The present invention provides the use of the postbiotics described in the above technical solution in the preparation of one or more of the following products 1) to 4);

[0018] 1) Food;

[0019] 2) Antibacterial products;

[0020] 3) Medicines;

[0021] 4) Cosmetics.

[0022] Preferably, the dosage form of the product includes one or more of capsules, tablets and powders.

[0023] Beneficial effects of the present invention: The present invention provides a method for preparing postbiotics, comprising the following steps:

[0024] The lactic acid bacteria fermentation supernatant is mixed with a carbon source to obtain a first feed liquid; lactic acid bacteria are inoculated into the first feed liquid for aerobic culture to obtain a culture solution; the inactivated culture solution is freeze-dried to obtain postbiotics.

[0025] The present invention uses lactic acid bacteria fermentation supernatant waste as the main raw material, realizes the reuse of lactic acid bacteria fermentation supernatant, and uses the residual protein, sugar, vitamins and other nutrients in the supernatant for secondary fermentation culture to prepare postbiotics; at the same time, considering the relative lack of nutrients in the supernatant, a carbon source is added to enrich the nutrition in the supernatant, and the prepared postbiotics have a high bacterial content, and the postbiotics have the effects of relieving constipation and diarrhea, improving immunity, anti-oxidation (delaying aging), relieving stress, promoting sleep, and inhibiting bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.

[0027] Figure 1 The figure is a graph showing the change in the number of viable bacteria in the secondary culture solution obtained in Examples 1 to 2 and Comparative Examples 1 to 3;

[0028] Figure 2 This is a graph showing the effect of postbiotic samples on alleviating the frequency of bowel movements due to constipation;

[0029] Figure 3 This is a graph showing the effect of postbiotic samples on alleviating stool characteristics in constipation;

[0030] Figure 4 This is a graph showing the effect of postbiotic samples on alleviating the frequency of diarrheal stools;

[0031] Figure 5 This is a graph showing the effect of postbiotic samples on alleviating diarrhea stool traits;

[0032] Figure 6 This is the inhibition zone diagram of postbiotics on Escherichia coli solution diluted 100 times;

[0033] Figure 7 This is the antibacterial zone diagram of postbiotics on Escherichia coli solution diluted 1000 times;

[0034] Figure 8 This is the antibacterial zone diagram of postbiotics on Escherichia coli solution diluted 10,000 times;

[0035] Fig. 9 This is a picture of liquid postbiotic samples prepared using fermentation supernatant;

[0036] Fig.10 Figure 2 shows a solid postbiotic sample prepared using fermentation supernatant;

[0037] Fig.11 It is the stool characteristics scale, namely the Bristol stool classification system. DETAILED DESCRIPTION

[0038] The present invention provides a method for preparing postbiotics, comprising the following steps:

[0039] Mixing the lactic acid bacteria fermentation supernatant with a carbon source to obtain a first feed liquid;

[0040] Lactic acid bacteria are inoculated into the first liquid for culturing to obtain liquid postbiotics.

[0041] In the present invention, the method for preparing the lactic acid bacteria fermentation supernatant preferably includes: obtaining the lactic acid bacteria fermentation supernatant after centrifuging the lactic acid bacteria fermentation broth. The present invention does not specifically limit the species of the lactic acid bacteria, and conventional lactic acid bacteria can be used. In an embodiment of the present invention, postbiotics are prepared using the fermentation supernatant of Lactobacillus casei LC001. The present invention does not specifically limit the source of the lactic acid bacteria fermentation broth, and the waste of lactic acid bacteria fermentation broth obtained by conventional lactic acid bacteria production enterprises can be used. The lactic acid bacteria fermentation supernatant of the present invention is preferably prepared using the lactic acid bacteria fermentation broth after constant pH culture, and the constant pH range is preferably 6.0 to 6.5, and the selection of this pH range is conducive to the growth of lactic acid bacteria. If the pH of the lactic acid bacteria fermentation broth is not within the range of 6.0 to 6.5, it can be adjusted using an acidic regulator or an alkaline regulator. As an embodiment, the composition of the lactic acid bacteria fermentation supernatant of the present invention includes: a total solid content of 4.96% to 7.04%, a total sugar content of 0.11% to 0.60%, a total protein content of 1.67% to 2.45%, and a total acid content of 2.41 to 7.12 g / L, wherein the total acid content is calculated as lactic acid content. The reason why the composition of the lactic acid bacteria fermentation supernatant is limited is that the lactic acid bacteria fermentation supernatant is required to contain a certain amount of nutrients. If the nutrients of the lactic acid bacteria fermentation supernatant are too low, the effect of inoculating lactic acid bacteria for cultivation and reproduction will be poor, which is not conducive to the growth of lactic acid bacteria to prepare postbiotics. If lactic acid bacteria are fermented using a conventional fermentation medium formula, the supernatant obtained after fermentation will have a relatively low nutrient content, and nutrients such as carbon sources or nitrogen sources must be appropriately added before it can be used to prepare postbiotics.

[0042] Obtain lactic acid bacteria fermentation supernatant, the present invention mixes lactic acid bacteria fermentation supernatant with a carbon source to obtain a first feed liquid. The carbon source of the present invention preferably includes glucose, or the carbon source preferably includes yeast extract powder and soy peptone; more preferably glucose. When the carbon source preferably includes glucose, the mass volume ratio of glucose to lactic acid bacteria fermentation supernatant is preferably (2-4) g:100 mL, more preferably 3 g:100 mL. When the carbon source preferably includes glucose, yeast extract powder and soy peptone; the mass volume ratio of glucose to lactic acid bacteria fermentation supernatant is preferably (2-4) g:100 mL, more preferably 3 g:100 mL; the mass volume ratio of yeast extract powder to lactic acid bacteria fermentation supernatant is preferably (1-1.5) g:100 mL, more preferably 1 g:100 mL; the mass volume ratio of soy peptone to lactic acid bacteria fermentation supernatant is (1-1.5) g:100 mL. The present invention adds single glucose as a carbon source, and uses the lactic acid bacteria fermentation supernatant after adding glucose to culture lactic acid bacteria, and the number of live lactic acid bacteria reaches 10 9 CFU / mL or above, which can not only meet the live bacteria count requirements of postbiotics but also reduce the production cost of postbiotics.

[0043] The present invention mixes the lactic acid bacteria fermentation supernatant with a carbon source. The present invention has no particular limitation on the mixing parameters, and the mixing can be uniformly stirred. The container for the mixing application of the present invention preferably includes a culture tank, a batching tank or a fermentation tank, and more preferably a batching tank.

[0044] The lactic acid bacteria fermentation supernatant in the prior art is waste, which is discharged after being treated, increasing the processing cost of the enterprise and causing a certain waste of resources. The present invention realizes the reuse of the lactic acid bacteria fermentation supernatant. Considering that the lactic acid bacteria fermentation supernatant is relatively lacking in nutrients, relatively cheap glucose is added as a carbon source to increase the nutrition in the supernatant, and the residual protein, sugar, vitamins and other nutrients in the supernatant and the additionally added carbon source are used for secondary fermentation and culture to prepare postbiotics, thereby reducing the production cost of postbiotics.

[0045] A first liquid is obtained, and the present invention inoculates lactic acid bacteria in the first liquid for fermentation culture to obtain a liquid postbiotic. The inoculation form of lactic acid bacteria of the present invention preferably includes lactic acid bacteria seed liquid. The preparation method of the lactic acid bacteria seed liquid of the present invention preferably includes: the freeze-dried strain of Lactobacillus casei LC001 is activated and cultured twice, and the obtained culture liquid is the seed liquid. The temperature of the activation culture is preferably 36-38°C, more preferably 37°C; the time of the activation culture is preferably 20-24h, more preferably 22-23h. The inoculation amount of the second activation culture is preferably 4% of the volume of the culture medium.

[0046] The temperature of the fermentation culture of the present invention is preferably 36-38°C, more preferably 37°C; the time of the fermentation culture is preferably 24-72h, more preferably 48-72h. During the fermentation culture, the inoculation amount of the seed liquid is preferably 2%-5% of the volume of the culture medium, more preferably 4%. The fermentation culture of the present invention can be a closed culture in a culture container without the need to fill it with inert gas. In an embodiment of the present invention, the culture container used in the present invention is preferably a fermentation tank, and the fermentation culture is preferably a closed culture in a fermentation tank. The selection of the fermentation culture temperature and fermentation culture time of the present invention is to promote the growth of lactic acid bacteria and the production of their metabolites.

[0047] After obtaining the liquid postbiotic, the present invention also preferably sterilizes the liquid postbiotic, mixes with the auxiliary material after sterilization and dries to obtain the solid postbiotic. The auxiliary material preferably includes skim milk powder. The mass volume ratio of the skim milk powder and the postbiotic of the present invention is preferably (10-20) g: 100 mL, more preferably 15 g: 100 mL. The drying of the present invention preferably includes freeze drying and / or spray drying. As an embodiment, the freeze drying of the present invention includes first stage freeze drying, second stage freeze drying, third stage freeze drying, fourth stage freeze drying, fifth stage freeze drying and sixth stage freeze drying. The temperature of the first stage freeze drying of the present invention is preferably -47 to -43 ° C, more preferably -45 ° C, and the time of the first stage freeze drying is preferably 4 to 5 h, more preferably 5 h. The temperature of the second stage freeze drying of the present invention is preferably -27 to -23 ° C, more preferably -25 ° C, and the time of the second stage freeze drying is preferably 8 to 10 h, more preferably 9 to 10 h, more preferably 10 h. The temperature of the third stage freeze-drying is preferably -8°C; the drying time of the third stage is preferably 5 to 6 hours, more preferably 6 hours. The temperature of the fourth stage freeze-drying is preferably 8°C; the drying time of the fourth stage is preferably 4 to 5 hours, more preferably 5 hours. The temperature of the fifth stage freeze-drying is preferably 20°C; the drying time of the fifth stage is preferably 4 to 5 hours, more preferably 5 hours. The temperature of the sixth stage freeze-drying is preferably 32°C; the drying time of the sixth stage is preferably 6 to 8 hours, more preferably 8 hours. The time of the first stage freeze-drying, the second stage freeze-drying, the third stage freeze-drying, the fourth stage freeze-drying, the fifth stage freeze-drying and the sixth stage freeze-drying is preferably 30 to 38 hours, more preferably 38 hours. In the present invention, staged freeze-drying is carried out under low temperature conditions. This drying method is suitable for heat-sensitive substances, can fully retain the nutrients and bioactive components in the postbiotic products, so that these nutrients and bioactive components are not destroyed, and some volatile components in the postbiotic products are lost less; in addition, the postbiotic products of staged freeze-drying have strong rehydration properties, and the products dissolve quickly and completely after adding water, and can immediately restore their original properties. The step-by-step freeze-drying operation of the present invention is to directly start the freeze-drying machine by inputting the corresponding freeze-drying program. The postbiotics of the present invention contain bacteria and various metabolites and beneficial components of the bacteria. The inactivated bacteria and beneficial components are retained by freeze-drying or spray drying. The number of inactivated dead bacteria in the postbiotics can reach 5×10 10 CFU / g or more. The prepared postbiotics increase the content of lactic acid, acetic acid, polysaccharides, polypeptides, proteins, γ-aminobutyric acid and fatty acids. The method for determining the number of inactivated dead bacteria preferably uses a flow cytometer or a cell counter.

[0048] After obtaining the solid postbiotics, the present invention also preferably includes crushing and sieving the solid postbiotics to obtain postbiotic dry powder. The present invention does not specifically limit the parameters of the crushing, and a conventional method can be used. The sieving of the present invention preferably includes a 40-mesh sieve, and after sieving, the sieve-down material, i.e., postbiotic dry powder, is preferably obtained.

[0049] The present invention provides a postbiotic, which is prepared by the preparation method of the postbiotic described in the above technical solution. The postbiotic dry powder prepared by the present invention contains bacteria and various metabolites and beneficial components of the bacteria. The lactic acid bacteria of the postbiotic dry powder reaches 10 10 CFU / g, the composition of the postbiotics preferably includes lactic acid, acetic acid, polysaccharides, polypeptides, proteins and fatty acids.

[0050] The present invention provides the use of the postbiotics described in the above technical solution in the preparation of one or more of the following products 1) to 4);

[0051] 1) Food;

[0052] 2) Antibacterial products;

[0053] 3) Medicines;

[0054] 4) Cosmetics.

[0055] In the present invention, the raw materials of the product include postbiotics and oligosaccharides; the added form of the postbiotics is more preferably postbiotic dry powder. The mass content of postbiotic dry powder in the product of the present invention is preferably ≥30%, more preferably 30% to 50%. The mass content of oligosaccharides in the product is preferably 8% to 12%, more preferably 10%; the oligosaccharides are added as a functional raw material, which has the function of regulating gastrointestinal function and improving human immunity. The preparation method of the product of the present invention preferably includes mixing the postbiotic dry powder, oligosaccharides and auxiliary materials. The present invention does not specifically limit the parameters of the mixing, and conventional methods can be used to ensure uniform mixing. The mixing of the present invention is preferably completed in a mixer, and the mixing time is preferably 15 to 30 minutes.

[0056] After adding auxiliary materials to the postbiotics, the present invention prepares a microecological preparation product containing inactivated bacteria and metabolites. The product is easy to store, has good stability, is convenient to use and has good effects.

[0057] In the present invention, when the product is a food, the food preferably includes ordinary food and / or health food. The health food of the present invention is preferably prepared in the form of a beverage or a solid preparation. The present invention has no special restrictions on the auxiliary materials used in the preparation of ordinary food and / or health food, and conventional auxiliary materials can be used.

[0058] In the present invention, when the product is an antibacterial product and / or cosmetic, there is no special limitation on the dosage form of the product, and conventional dosage forms can be used. There is no special limitation on the auxiliary materials used in the preparation of antibacterial products and / or cosmetics, and conventional auxiliary materials can be used.

[0059] In the present invention, when the product is a medicine, the dosage form of the medicine preferably includes one or more of capsules, tablets and powders.

[0060] When the dosage form is preferably a capsule, the excipients for preparing the capsule preferably include one or more of a diluent, a lubricant, a glidant, a disintegrant and a wetting agent; the lubricant preferably includes magnesium stearate. The diluent preferably includes maltodextrin.

[0061] When the dosage form is preferably a tablet, the auxiliary materials for preparing the tablet preferably include one or more of a diluent, a wetting agent, a binder and a disintegrant; the lubricant is preferably magnesium stearate. The diluent preferably includes maltodextrin.

[0062] When the dosage form is preferably a powder, the excipients for preparing the powder preferably include one or more of a filler, a rheology regulator, a lubricant and a sweetener; the lubricant is preferably magnesium stearate. The filler preferably includes maltodextrin.

[0063] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below in combination with embodiments and drawings, but they should not be understood as limiting the protection scope of the present invention.

[0064] Example 1

[0065] The preparation method of postbiotics is as follows:

[0066] 1. Collection of fermentation centrifugal supernatant: centrifuge the Lactobacillus casei LC001 fermentation liquid to obtain a supernatant stock solution; collect the supernatant stock solution in a suitable batching tank; the fermentation liquid is the fermentation culture liquid of Lactobacillus casei LC001 after constant pH culture, and the constant pH range is 6.0-6.5.

[0067] 2. Ingredients: Add glucose to the batching tank in step 1, the mass volume ratio of glucose to supernatant stock solution is 3g:100mL, then stir and dissolve evenly to obtain a feed liquid.

[0068] 3. Sterilization: sterilize the liquid in step 2 by high pressure steam in a culture tank or fermentation tank at a temperature of 121°C for 7 minutes.

[0069] 4. Inoculation of Secondary Culture

[0070] The preparation method of lactic acid bacteria seed liquid is as follows: freeze-dried strains of Lactobacillus casei LC001 are activated and cultured twice, and the obtained culture liquid is the seed liquid. The temperature of the activation culture is 36-38° C., and the time of the activation culture is 24 hours. The inoculum amount of the second activation culture is 4% of the volume of the culture medium.

[0071] The temperature of the sterilized liquid in step 3 is reduced to 36-38° C., and the seed liquid of Lactobacillus casei LC001 is inoculated into the sterilized liquid at a volume ratio of 4% for aerobic culture at a temperature of 36-38° C. for 72 hours to obtain a secondary culture liquid;

[0072] 5. The secondary culture liquid obtained in step 4 is heated and inactivated; the inactivation temperature is 95° C., the inactivation time is 30 min, and the stirring in the tank is turned on during the inactivation process, and the speed is controlled to be 60 rpm; after inactivation, the lactic acid bacteria inactivated secondary culture liquid, i.e., liquid postbiotics, is obtained; Fig. 9 Figure 2 shows liquid postbiotic samples prepared using fermentation supernatant.

[0073] 6. Add skim milk powder to the liquid postbiotics obtained in step 5, dissolve and mix, then freeze-dry, and obtain postbiotic dry powder after crushing and sieving; the mass volume ratio of skim milk powder to liquid postbiotics is 15g:100mL. Fig.10 A picture of solid postbiotic samples prepared using fermentation supernatant.

[0074] The freeze-drying process parameters are: temperature of -45℃, freeze-drying time of 5h; temperature of -25℃, freeze-drying time of 5h; temperature of -8℃, freeze-drying time of 10h; temperature of 8℃, freeze-drying time of 5h; temperature of 20℃, freeze-drying time of 5h; temperature of 32℃, freeze-drying time of 8h; the total freeze-drying time is 38h.

[0075] Example 2 Preparation of product

[0076] The postbiotic dry powder, maltodextrin, oligosaccharides, magnesium stearate and other auxiliary materials prepared in Example 1 are mixed in a mixer for 20 minutes to obtain a mixture. The mass proportion of postbiotic dry powder in the mixture is 50%, the mass proportion of maltodextrin in the mixture is 35%, the mass proportion of oligosaccharides in the mixture is 10%, the mass proportion of magnesium stearate in the mixture is 0.6%, and the mass proportion of other auxiliary materials in the mixed material is 4.4%. Other auxiliary materials are citric acid and microcrystalline cellulose, the mass proportion of citric acid is 0.4%, and the mass proportion of microcrystalline cellulose is 4%.

[0077] The mixture is made into capsules to obtain postbiotic capsules; the mixture is made into tablets to obtain postbiotic tablets; the mixture is made into powder to obtain postbiotic powder.

[0078] Example 3

[0079] The same as Example 1, except that the carbon sources are yeast extract powder, soy peptone and glucose; the volume ratio of the added mass of yeast extract powder to the supernatant stock solution is 1 g:100 mL, the volume ratio of the added mass of soy peptone to the supernatant stock solution is 1 g:100 mL; the volume ratio of the added mass of glucose to the supernatant stock solution is 3 g:100 mL.

[0080] Comparative Example 1

[0081] Same as Example 1, the only difference is that the supernatant obtained by centrifugation of Lactobacillus casei LC001 fermentation broth is replaced with pure water.

[0082] Comparative Example 2

[0083] Same as Example 1, except that glucose was not added. The culture medium was only the supernatant obtained by centrifugation of the fermentation liquid of Lactobacillus casei LC001.

[0084] Comparative Example 3

[0085] The same as Example 1, the only difference is that glucose is replaced by yeast extract powder and soy peptone; yeast extract powder and soy peptone are added to the supernatant obtained by centrifugation of Lactobacillus casei LC001 fermentation liquid, the addition amount of yeast extract powder is 1% of the mass of the supernatant stock solution, and the addition amount of soy peptone is 1% of the mass of the supernatant stock solution.

[0086] The viable bacterial counts in the secondary culture fluids obtained in Example 1, Example 3 and Comparative Examples 1 to 3 were tested, and the results are shown in Tables 1 and Figure 1 . Figure 1 Formula 3 represents Example 1; Formula 5 represents Example 3; Formula 1 represents Comparative Example 1; Formula 2 represents Comparative Example 2; Formula 4 represents Comparative Example 3.

[0087] Table 1 The number of viable bacteria in the secondary culture fluid obtained in Example 1, Example 3 and Comparative Examples 1 to 3 (×10 8 CFU / mL)

[0088] Secondary culture time Example 1 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 24h 9.2 17.1 0.35 1.01 1.68 48h 8.9 19.3 0.27 1.14 3.9 72h 12.8 25 0.21 1.76 6.2

[0089] According to Table 1 and Figure 1 It can be seen that although the viable count of Lactobacillus casei LC001 in Example 3 is the highest, considering the cost of the culture medium, the technical solution of adding only glucose in Example 1 is adopted, and relatively cheap glucose is added to the supernatant stock obtained by centrifugation of the Lactobacillus casei LC001 fermentation liquid for re-cultivation, and the final viable count can reach 10 9 CFU / mL, which can meet actual production needs.

[0090] Comparative Example 4 Postbiotic Dry Powder

[0091] Postbiotic dry powder prepared by fermentation in conventional medium

[0092] 1. Ingredients: Prepare an appropriate amount of liquid culture medium according to the conventional culture medium composition, and then stir and dissolve evenly to obtain a feed liquid; the conventional fermentation medium refers to the composition of the culture medium for conventionally culturing Lactobacillus casei, which contains carbon sources, nitrogen sources, inorganic salts and other components. The composition of the conventional fermentation medium is shown in Table 2.

[0093] Table 2 The culture medium composition of conventional culture of Lactobacillus casei

[0094] name unit quantity lactose g 8 Beef extract g 5 Casein Peptone g 8 Soybean Peptone g 5 Yeast extract powder g 5 Disodium hydrogen phosphate g 2.0 Potassium dihydrogen phosphate g 2.0 *Magnesium sulfate g 0.1 *L-Cysteine ​​hydrochloride g 0.5 Twain-80 g 0.25 Purified water L 1

[0095] Note: Substances marked with "*" are dissolved separately and the pH is adjusted to 7.0-7.2 with 25% NaOH solution. Sterilization of the culture medium is carried out at 121℃ for 15min.

[0096] 2. Sterilization: sterilize the liquid in step 1 by high pressure steam in a culture tank or fermentation tank at a temperature of 121°C for 15 minutes;

[0097] 3. Inoculation of secondary culture:

[0098] The preparation method of lactic acid bacteria seed liquid is as follows: freeze-dried strains of Lactobacillus casei LC001 are activated and cultured twice, and the obtained culture liquid is the seed liquid. The temperature of the activation culture is 36-38° C., and the time of the activation culture is 24 hours. The inoculum amount of the second activation culture is 4% of the volume of the culture medium.

[0099] The temperature of the sterilized liquid in step 3 is reduced to 36-38° C., and the activated seed solution of Lactobacillus casei LC001 is inoculated into the sterilized liquid at a volume ratio of 4% for aerobic culture at a temperature of 36-38° C. for 72 h to obtain a culture solution;

[0100] 4. The culture solution obtained in step 3 is heated and inactivated; the inactivation temperature is 95° C., the inactivation time is 30 min, and the stirring in the tank is turned on during the inactivation process, and the speed is controlled to be 60 rpm; after inactivation, a lactic acid bacteria inactivated culture solution is obtained, i.e., a conventional liquid postbiotic;

[0101] 5. Add skim milk powder to the liquid postbiotics obtained in step 4, dissolve and mix, then freeze-dry, and obtain postbiotic dry powder after crushing and sieving; the mass volume ratio of skim milk powder to liquid postbiotics is 15g:100mL.

[0102] The freeze-drying process parameters are: temperature of -45℃, freeze-drying time of 5h; temperature of -25℃, freeze-drying time of 5h; temperature of -8℃, freeze-drying time of 10h; temperature of 8℃, freeze-drying time of 5h; temperature of 20℃, freeze-drying time of 5h; temperature of 32℃, freeze-drying time of 8h; the total freeze-drying time is 38h.

[0103] The components of the postbiotic dry powder prepared when the fermentation culture time was 72 h in Example 1 and Comparative Example 4 were tested, and the testing unit was Stand Science Pharmaceutical Technology (Qingdao) Co., Ltd. The results are shown in Tables 3 to 5. According to Tables 3 to 5, the postbiotics prepared by the present invention increase the content of lactic acid, acetic acid, polysaccharides, polypeptides and proteins.

[0104] Table 3 Postbiotic sample composition test results

[0105] Serial number Test items and units Test Data Conventional postbiotics obtained in Comparative Example 4 1 Acetic acid, mg / kg 17648.483 14562.34 2 Lactic acid, mg / kg 162358.382 29693.40 3 Polysaccharide, g / 100g 0.90 0.75 4 Peptide content, g / 100g 18.9 15.7 5 26 fatty acids See Table 5 6 Glucose, g / 100g 1.758 —— 7 Protein, g / 100g 35.4 32.6 8 GABA, mg / kg 726.48 254.17 9 15 kinds of free amino acids See Table 4

[0106] Table 4 Determination results of 15 free amino acids in postbiotics (unit: mg / 100g)

[0107] composition content Aspartic acid (Asp) 5.0 Threonine (Thr) 12 Serine (Ser) 14 Glutamate (Glu) 49 Glycine (Gly) 320 Alanine (Ala) 83 Cystine (Cys) 4.7 Valine (Val) 20 Isoleucine (Ile) 130 Leucine (Leu) 32 Phenylalanine (Phe) 80 Lysine (Lys) 1.4 Histidine (His) 9.3 Arginine (Arg) 51 Proline (Pro) 36

[0108] Table 5 Determination results of 26 fatty acids in postbiotics (unit: mg / 100g)

[0109]

[0110]

[0111] Example 3 Postbiotics for Relief of Constipation and Diarrhea

[0112] 1. Relieve Constipation

[0113] (1) Experiment on the effect of postbiotic samples on the frequency of constipation bowel movements: The experiment was set up in two groups, the experimental group (i.e., the sample group) and the control group (i.e., the placebo group). Each group selected 20 volunteers with constipation symptoms, half of whom were male and half were female. The experimental group took postbiotic tablets as the experimental material and took 4 tablets per day; the control group took placebo and took 4 tablets per day. The preparation method of the placebo was the same as that of the postbiotic tablets, the only difference being that no postbiotic dry powder was added. The raw material for the preparation of the placebo was a mixture obtained by mixing maltodextrin, resistant dextrin, oligosaccharides, magnesium stearate and other excipients. The mass proportion of maltodextrin in the mixture was 65%, the mass proportion of resistant dextrin was 20%, the mass proportion of oligosaccharides was 10%, the mass proportion of magnesium stearate was 0.6%, and the mass proportion of other excipients was 4.4%. The other excipients were citric acid and microcrystalline cellulose, with the mass proportion of citric acid being 0.4% and the mass proportion of microcrystalline cellulose being 4%. The number of spontaneous bowel movements of the 20 people in each group was recorded and the average value was calculated.

[0114] Results Figure 2 ,according to Figure 2 It can be seen that compared with the control group, the proportion of people in the experimental group with an average of ≥3 complete spontaneous bowel movements (CSBMs) per week reached a maximum of 65% on the 14th day, while the control group only reached a maximum of 45% on the 21st day, indicating that postbiotics can effectively increase the frequency of bowel movements in people with constipation.

[0115] (2) Experiment on the effect of postbiotic samples on alleviating constipation stool characteristics: The experiment set up two groups, the experimental group (i.e., the sample group) and the control group (i.e., the placebo group). Each group selected 10 volunteers with constipation symptoms, half of whom were male and half were female. The experimental group took the postbiotic tablets prepared in Example 2, 4 tablets per day; the control group took a placebo. The preparation method of the placebo was the same as 1 in Example 3 to relieve constipation (1). The two groups took it for 7 days each, with the 0th day being the first day of taking it, and the 0th to 6th days being a total of 7 days. The stool characteristics of the two groups of volunteers were scored, and the stool characteristics scale, i.e., the Bristol stool classification system, was used. Fig.11 , Fig.11 The different numbers from 1 to 7 in the table represent their respective scores. For example, if the stool characteristic is 1, the score is 1. Fig.11 Among them, 1 represents separate nut-shaped particles; 2 represents sausage-shaped strips with uneven surfaces; 3 represents sausage-shaped strips with cracks on the surface; 4 represents smooth and soft sausage and snake shapes; 5 represents soft blocks with smooth cross-section; 6 represents loose, mushy feces with broken edges; 7 represents no solids and completely liquid; the same below.

[0116] Both groups of volunteers recorded the stool characteristics scale scores, and the average values ​​of each group were calculated.

[0117] Results Figure 3 , Figure 3 The vertical axis represents the score of the rating, in points. Figure 3 It can be seen that compared with the control group, the stool characteristics scale score of the experimental group reached a maximum of 3.8 points on the 5th day, and the control group reached a maximum of 3.2 points on the 5th day, indicating that postbiotics can effectively improve the degree of dry stool in constipated patients. It can be seen that the postbiotics prepared by the present invention have the effect of relieving constipation.

[0118] 2. Relieve diarrhea

[0119] (1) Experiment on the effect of postbiotic samples on the frequency of diarrhea and bowel movements:

[0120] Experimental conditions: The experiment was set up with two groups, namely the experimental group (i.e., the sample group) and the control group (i.e., the placebo group). Ten volunteers with diarrhea symptoms were selected from each group, half of whom were male and half were female. The experimental group took the postbiotic tablets prepared in Example 2, 4 tablets per day, for a total of 7 days; the control group took the placebo for a total of 7 days. The raw materials for the preparation of the placebo were the same as those for the preparation of the constipation relief (1), and the preparation method of the placebo was the same as that of Example 3, 1, for the constipation relief (1). The average number of diarrhea times for each volunteer in the two groups was recorded.

[0121] Results Figure 4 , Figure 4 The vertical axis represents the number of bowel movements (unit: times). Figure 4 It can be seen that the average number of diarrhea episodes in the experimental group on the third day dropped from 5 times a day at the beginning to 2 times a day, while the average number of diarrhea episodes in the placebo group on the third day was 3 times, indicating that postbiotics can effectively reduce the number of diarrhea episodes in patients with diarrhea.

[0122] (2) Experiment on the effect of postbiotic samples on the characteristics of diarrhea stool

[0123] Experimental conditions: The experiment was set up with two groups, the experimental group (i.e., the sample group) and the control group (i.e., the placebo group). Ten volunteers with diarrhea symptoms were selected from each group, half of whom were male and half were female. The experimental group took the postbiotic tablets prepared in Example 2, 4 tablets per day, and the control group took a placebo. The preparation method of the placebo was the same as that of Example 3, 1 to relieve constipation (1). The two groups took the medicine for 7 days each, with the 0th day being the first day of taking the medicine, and the 0th to 6th days being the total 7 days of taking the medicine. The stool characteristics of the two groups of volunteers were scored, and the stool characteristics scale, i.e., the Bristol stool classification system, was used. Fig.11 .

[0124] according to Figure 5 It can be seen that Figure 5 The vertical axis represents the score obtained by the scoring. The stool characteristics score of the experimental group on the third day decreased from 6.2 to 4.2, and the stool characteristics score of the control group on the third day decreased from 6.3 to 5.2, indicating that postbiotics can effectively improve the watery stool state of patients with diarrhea.

[0125] It can be seen that the postbiotics prepared by the present invention have the effect of alleviating diarrhea.

[0126] Example 4 Antibacterial Effect

[0127] Bacterial culture: Escherichia coli strains were inoculated and streaked onto a tryptic soy agar medium plate (2 cm in height). Culture conditions: the plate was cultured at 37°C for 1 day to obtain a cultured Escherichia coli plate.

[0128] Antibacterial test:

[0129] Take 2 well-cultured E. coli plates, add 1 mL of saline solution to each plate, scrape the bacterial mud with a coating rod, suck it into an empty triangular flask, add 100 mL of saline solution to dilute, and obtain the original E. coli solution. Take 1 mL of the original solution for gradient dilution, dilute the original solution 100, 1000, and 10000 times, and perform antibacterial experiments respectively. The antibacterial experiment is as follows: 0.5 mL of E. coli solution of the three concentrations is drawn into the blank culture medium plate, the coating rod is evenly coated, and an Oxford cup is placed. Each concentration of bacterial solution is coated on 3 plates, one is the postbiotic sample experimental plate, and the other two are negative and positive control plates. Put 3 Oxford cups on each plate, add 0.1 mL of postbiotic sample liquid to the 3 Oxford cups of the sample experimental plate, add 0.1 mL of saline to the 3 Oxford cups of the negative control plate, and add levofloxacin solution to the 3 Oxford cups of the positive control plate, and observe after cultivation. The negative control used normal saline; the positive control used levofloxacin solution (Handan Kangye Pharmaceutical Co., Ltd.), and the levofloxacin concentration was 5 mg / mL (the stock solution was diluted 10 times with normal saline). The concentration of the postbiotic sample solution was 500 mg / mL, and the preparation method was as follows: the postbiotic powder prepared in Example 1 was dissolved in normal saline to prepare a sample solution, and the concentration of the sample solution was 500 mg / mL.

[0130] Results Figure 6 to Figure 8 , Figure 6 This is the inhibition zone diagram of postbiotics on 100-fold diluted Escherichia coli solution; Figure 7 This is the antibacterial zone diagram of postbiotics on Escherichia coli solution diluted 1000 times; Figure 8 This is the inhibition zone diagram of postbiotics on Escherichia coli solution diluted 10,000 times.

[0131] according to Figure 6 to Figure 8 It can be seen that the diameters of the inhibition zones of Escherichia coli liquid diluted 100 times are 14mm, 12mm, and 10mm respectively; the diameters of the inhibition zones of the positive control of levofloxacin are 20mm, 20mm, and 20mm respectively; the diameters of the inhibition zones of the Escherichia coli liquid diluted 1000 times are 13mm, 11mm, and 10mm respectively; the diameters of the inhibition zones of the positive control of levofloxacin are 20mm, 20mm, and 20mm respectively; the diameters of the inhibition zones of the Escherichia coli liquid diluted 10000 times are 14mm, 13mm, and 15mm respectively; the inhibition zones of the antibiotic positive control are 20, 20, and 20mm respectively. The postbiotics prepared by the present invention have an inhibitory effect on Escherichia coli.

[0132] In summary, the culture solution after inactivation in the present invention contains bacteria and various metabolites and beneficial components of the bacteria. The inactivated bacteria and beneficial components are retained by freeze-drying or spray-drying, and the number of inactivated dead bacteria can reach 5×10 10CFU / g or above, after adding auxiliary materials, it is made into a microecological preparation containing inactivated bacteria and metabolites. The product is easy to store, has good stability, is convenient to use, and has good effects. The prepared postbiotics can relieve constipation and diarrhea and have antibacterial effects.

[0133] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing postbiotics, characterized in that: The steps include: Mixing the lactic acid bacteria fermentation supernatant with a carbon source to obtain a first feed liquid; Lactic acid bacteria are inoculated into the first liquid for culturing to obtain liquid postbiotics.

2. The preparation method according to claim 1, characterized in that: The components of the lactic acid bacteria fermentation supernatant include: a total solid content of 4.96% to 7.04%, a total sugar content of 0.11% to 0.60%, a total protein content of 1.67% to 2.45% and a total acid content of 2.41 to 7.12 g / L; the total acid content is calculated as lactic acid content.

3. The preparation method according to claim 1, characterized in that: After obtaining the liquid postbiotics, the method further includes drying the liquid postbiotics to obtain solid postbiotics.

4. The preparation method according to claim 1, characterized in that: The carbon source includes glucose, or the carbon source includes glucose, yeast extract and soy peptone.

5. The preparation method according to claim 4, characterized in that: When the carbon source includes glucose, the mass volume ratio of the glucose to the lactic acid bacteria fermentation supernatant is (2-4) g:100 mL; or, When the carbon source includes glucose, yeast extract powder and soy peptone, the mass volume ratio of the glucose to the lactic acid bacteria fermentation supernatant is (2-4) g:100 mL; the mass volume ratio of the yeast extract powder to the lactic acid bacteria fermentation supernatant is (1-1.5) g:100 mL, and the mass volume ratio of the soy peptone to the lactic acid bacteria fermentation supernatant is (1-1.5) g:100 mL.

6. The preparation method according to claim 1, characterized in that: The culture temperature is 36-38° C., and the culture time is 24-72 hours.

7. The preparation method according to claim 1, characterized in that: The inoculation form of lactic acid bacteria includes lactic acid bacteria seed liquid.

8. A postbiotic, characterized in that: The postbiotics are prepared by the preparation method of claims 1 to 7.

9. Use of the postbiotics according to claim 8 in the preparation of one or more of the products in 1) to 4) below; 1) Food; 2) Antibacterial products; 3) Medicines; 4) Cosmetics.

10. The use according to claim 9, characterized in that: The dosage form of the product includes one or more of capsules, tablets and powders.

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