Witzia coagulans, afterbiogen and application of Witzia coagulans and afterbiogen

Abstract

The invention relates to the technical field of microorganisms, in particular to Witzia ciliaris, a penicillium and application of the Witzia ciliaris and the penicillium. According to the present invention, the Weizmania coagulans is Weizmania coagulans Naqu.VC-1201, the Weizmania coagulans is preserved in the China Center for Type Culture Collection, and the preservation number of the Weizmania coagulans is CCTCC (China Center for Type Culture Collection): M 20231577; according to the present invention, the bacillus coagulans Naqu.VC-1201 has characteristics of strong antioxidant activity, strong antibacterial ability and artificial digestive juice tolerance; according to the present invention, with the addition of the Witzia ciliaris metagen in the walnut sauce product, the oxidation rancidity of the oil can be effectively inhibited, the shelf life of the walnut sauce can be prolonged, the nutritional value of the walnut sauce can be improved, the probiotic effect can be enhanced, and the human health can be easily achieved. And the problem of poor smearing property caused by addition of an emulsion stabilizer in the prior art can be solved, and the use is convenient.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to Weizmannella coagulans, postbiotics thereof and applications thereof. Background Art

[0002] Walnut, also known as walnut, is one of the main woody oil tree species in my country. Due to its rich nutritional value, it is often called "brain-boosting fruit", "beauty fruit" and "longevity fruit". Walnut paste made from walnut kernels through processes such as baking and grinding can provide high heat energy and rich vitamins and minerals, with a special flavor and good taste, and is a delicious side dish or seasoning food. However, since walnut kernels contain a lot of oil, during the processing of walnut paste, its fat cells are destroyed and oil is precipitated. Long-term storage can easily cause oil to float, solids to aggregate and settle, making the product extremely easy to oxidize and rancid, and the shelf life is sharply shortened, reducing the value of the product.

[0003] In current industrial production, in order to ensure the stability of walnut paste, the shelf life is usually extended by adding emulsifiers such as hydrogenated oil, palm oil, and vegetable protein powder. However, hydrogenated vegetable oil, due to its hard texture, will cause the spreadability of walnut paste to deteriorate, and hydrogenated oil contains trans fatty acids, which can easily cause cardiovascular and cerebrovascular diseases.

[0004] Chinese patent CN102018241A discloses a soy protein walnut paste with health care function and its preparation method, the raw materials of which include by weight: 75-80 parts of walnut kernels, 5-12 parts of soy protein, 4-6 parts of white sugar, 0.5 parts of sodium carboxymethyl cellulose (CMC), 0.3 parts of salt, and the balance is water. The invention maintains the original natural flavor of walnuts, increases the content of protein, vitamins, and mineral elements, increases the functional ingredients of oligosaccharides, saponins, and isoflavones, and improves the nutritional and health care effects. However, the patent adds plant protein powder of soy protein, which will aggravate the solidification of the sauce, especially under low temperature conditions, and seriously affects the spreadability of the walnut paste.

[0005] Therefore, the current market urgently needs to improve the stability and shelf life of walnut paste products, while improving the performance of walnut paste products. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a strain of Weizmannella coagulans and its postbiotics, improve the stability of walnut paste and extend the shelf life of the product, improve the spreadability of the walnut paste product, facilitate the use of the product, and avoid the side effects of existing emulsifiers and antioxidants.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] In a first aspect, the present application provides a Weizmannia coagulans strain, wherein the strain is Weizmannia coagulans Naqu.VC-1201, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20231577.

[0009] In some embodiments of the present application, the 16s rRNA sequence of Weizmannella coagulans comprises the sequence shown in SEQ ID NO: 1.

[0010] In a second aspect, the present application provides a bacterial preparation comprising the above-mentioned Weizmannella coagulans.

[0011] In a third aspect, the present application provides a postbiotic composition, which at least comprises the postbiotics formed by the above-mentioned Weizmannella coagulans.

[0012] In some embodiments of the present application, the postbiotic composition further comprises postbiotics formed by Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ.

[0013] In some embodiments of the present application, the content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition is 30-50wt%; the content of postbiotics formed by Lactobacillus delbrueckiisubsp.lactis Dangxiong LBⅧ is 50-70wt%;

[0014] Preferably, the content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition is 30-45wt%; the content of postbiotics formed by Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ is 55-70wt%;

[0015] More preferably, the content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition is 30-40wt%; the content of postbiotics formed by Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ is 60-70wt%.

[0016] In some embodiments of the present application, the postbiotic composition is prepared by inactivating the strain after fermentation.

[0017] Preferably, the inactivation comprises placing the fermentation liquid of the strain in a supercritical carbon dioxide environment for inactivation; preferably, the inactivation pressure is 10-20 MPa, and more preferably, the inactivation temperature is 40-60°C.

[0018] In some embodiments of the present application, the postbiotic composition is prepared by mixing the fermentation broth of Lactobacillus delbrueckii Dangxiong LBⅧ and the fermentation broth of Weizmannella coagulans Naqu.VC-1201 and then inactivating them. Preferably, the mass ratio of the fermentation broth of Weizmannella coagulans Naqu.VC-1201 and the fermentation broth of Lactobacillus delbrueckii Dangxiong LBⅧ is 0.5-1:1, preferably 0.5-0.8:1, and more preferably 0.5-0.7:1.

[0019] In a fourth aspect, the present application provides a method for preparing the above-mentioned postbiotic composition, the preparation method comprising inactivating the strain after fermentation and culturing,

[0020] Preferably, the inactivation comprises placing the fermentation liquid of the strain in a supercritical carbon dioxide environment for inactivation; preferably, the inactivation pressure is 10-20 MPa, and more preferably, the inactivation temperature is 40-60°C.

[0021] In some embodiments of the present application, the preparation method further comprises the step of freeze-drying after inactivation;

[0022] Preferably, the freeze-drying temperature is -65 to -85°C, and the freeze-drying time is preferably 24 to 48 hours.

[0023] In some embodiments of the present application, the preparation method comprises the following steps:

[0024] (1) inoculating Weizmannella coagulans into MRS medium for fermentation;

[0025] (2) inoculating Lactobacillus delbrueckii into MRS medium for fermentation;

[0026] (3) mixing the fermentation broths of steps (1) and (2) and then inactivating the mixture;

[0027] (4) Drying the inactivated fermentation liquid.

[0028] In some embodiments of the application, the inoculum amount of coagulating Weizmannella in step (1) is 5-10%, preferably 5-8 v / v%, more preferably 5-6.5% v / v%; preferably, the fermentation culture temperature is 30-40°C, preferably 30-35°C; more preferably, the fermentation culture time is 24-48h, preferably 24-36h; and / or,

[0029] In step (2), the inoculum amount of Lactobacillus delbrueckii is 5-10%, preferably 5-8 v / v%, more preferably 5-6.5% v / v%; preferably, the fermentation culture temperature is 30-40° C., preferably 36-38° C.; more preferably, the fermentation culture time is 24-48 h, preferably 24-36 h.

[0030] In some embodiments of the application, in step (3), the mass ratio of the coagulated Weizmannella fermentation broth to the Lactobacillus delbrueckii fermentation broth is 0.5-1:1, preferably 0.5-0.8:1, and more preferably 0.5-0.7:1.

[0031] In a fifth aspect, the present application also provides the use of the above-mentioned postbiotic composition or the postbiotic composition prepared by the above-mentioned preparation method in food anti-oxidation, preferably in walnut paste anti-oxidation.

[0032] In a sixth aspect, the present application provides a walnut paste, which is prepared from the following raw materials: walnut kernels and the above-mentioned postbiotic composition, wherein the mass percentage of the postbiotic composition in the walnut kernels is 0.01-1%, preferably 0.01-0.1%, and more preferably 0.01-0.05%.

[0033] In some embodiments of the application, the walnut kernel is a blanched walnut kernel, and preferably the walnut kernel is a roasted walnut kernel.

[0034] In a seventh aspect, the present application provides a method for preparing the above-mentioned walnut paste, the method comprising the following steps:

[0035] (1) Peeling of walnut kernels;

[0036] (2) roasting the peeled walnut kernels;

[0037] (3) coarse grinding after baking;

[0038] (4) adding the above-mentioned postbiotic composition for fine grinding.

[0039] In some embodiments of the application, the baking temperature in step (2) is 80-140°C, preferably 100-140°C, and more preferably 100-125°C;

[0040] Preferably, the baking time is 20-80 min, preferably 40-70 min.

[0041] In some embodiments of the application, the coarse grinding in step (3) is performed by a pulverizer, preferably to a particle size of 60-100 mesh.

[0042] In some embodiments of the application, the step (4) fine grinding uses a colloid mill to grind the coarsely ground walnut paste, preferably the grinding time is 10-30 minutes, and more preferably the particle size is 325-460 mesh.

[0043] In some embodiments of the application, the method further comprises step (5) sterilization; preferably, the sterilization temperature is 40-70°C, and more preferably, the sterilization time is 20-30 min.

[0044] The present application also provides walnut paste prepared by the above preparation method.

[0045] Beneficial effects of the present invention:

[0046] The coagulated Weizmannella Naqu.VC-1201 of the present invention has strong antioxidant activity and antibacterial ability and can tolerate artificial digestive fluid. The coagulated Weizmannella postbiotic has strong antioxidant property. Adding the postbiotic to the walnut paste product can effectively inhibit the oxidation and rancidity of oil and extend the shelf life of the walnut paste, and can improve the nutritional value of the walnut paste, enhance the prebiotic effect, and be beneficial to human health. In addition, the problem of poor spreadability caused by adding an emulsifier stabilizer in the prior art can be overcome, and the product is easy to use.

[0047] Strain deposit information

[0048] The strain Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ used in the present invention was deposited in the China Center for Type Culture Collection on March 23, 2023, with a deposit number of CCTCC NO: M 2023396, and a deposit address: Wuhan University, Wuhan, China, postal code: 430072; telephone: 02768754052. The strain has been recorded in the patent application with Chinese application number 202310802241.7 (publication number CN116948884A).

[0049] The Weizmannia coagulans Naqu.VC-1201 used in the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on August 31, 2023, with a deposit number of CCTCC NO: M 20231577, and a deposit address of: Wuhan University, Wuhan, China, postal code: 430072; telephone: (027)-68754052. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and technical effect of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention is described clearly and completely. The embodiments described below are part of the embodiments of the present invention, not all of them. In combination with the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0051] Walnut paste products in the prior art have poor stability, short shelf life, and the use of existing emulsifiers and antioxidants may bring health risks and usage problems, such as difficulty in spreading. Therefore, the current market is in urgent need of a walnut paste product that is pure and natural, has good stability, and has a long shelf life.

[0052] In this application, postbiotics refer to preparations of inactivated microorganisms and / or their metabolites for food that are beneficial to the health of the host, excluding purified metabolites. Postbiotics are microbial preparations that are beneficial to human health. Compared with live bacteria, postbiotics have the advantages of long shelf life, convenience and ease of use, and the ability to exert more stable probiotic effects.

[0053] The present application screened out a strain of Weizmannia coagulans Naqu.VC-1201 and made it into a postbiotic, which was applied to walnut paste products. On the one hand, it can effectively inhibit the oxidation and rancidity of oils and extend the shelf life of walnut paste. On the other hand, it can improve the nutritional value of walnut paste, enhance the probiotic effect, and benefit human health.

[0054] In a first aspect, in a specific embodiment of the present application, the present application provides a Weizmannia coagulans, wherein the strain is Weizmannia coagulans Naqu.VC-1201, which is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC No: M 20231577.

[0055] In some embodiments of the present application, the 16s rRNA sequence of the strain comprises the sequence composition shown in SEQ ID NO: 1 in Table 9.

[0056] The Weizmannia coagulans Naqu.VC-1201 of the present application has strong antibacterial properties and the effect of promoting intestinal digestion and absorption, and has high antioxidant activity.

[0057] The present application also provides a bacterial preparation containing the above-mentioned strain.

[0058] The present application also provides a postbiotic composition, which at least comprises the postbiotics formed by the above-mentioned Weizmannia coagulans Naqu.VC-1201.

[0059] Furthermore, the postbiotic composition also includes postbiotics formed by Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ.

[0060] In some embodiments of the present application, the content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition is 30-50wt%, and the content of postbiotics formed by Lactobacillus delbrueckiisubsp.lactis Dangxiong LBⅧ is 50-70wt%.

[0061] In some embodiments of the present application, the content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition may be 30wt%, 35wt%, 40wt%, 45wt% or 50wt% or a range formed by any two of the above values ​​as endpoints. It should be understood that in a specific embodiment, any of the above ranges may be combined with any other ranges.

[0062] In some embodiments of the present application, the content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition may be 50wt%, 55wt%, 60wt%, 65wt% or 70wt% or a range formed by any two of the above values ​​as endpoints. It should be understood that in a specific embodiment, any of the above ranges may be combined with any other ranges.

[0063] In some embodiments of the present application, the postbiotic composition is prepared by mixing and inactivating the fermentation broth of Lactobacillus delbrueckii Dangxiong LBⅧ and the fermentation broth of Weizmannella coagulans Naqu.VC-1201, wherein the mass ratio of the fermentation broth of Lactobacillus delbrueckii Dangxiong LBⅧ and the fermentation broth of Weizmannella coagulans Naqu.VC-1201 is 1:0.5-1, preferably 1:0.5-0.8, and more preferably 1:0.5-0.7.

[0064] In some embodiments of the present application, the fermentation broth of Lactobacillus delbrueckii Dangxiong LBⅧ is prepared by inoculating Lactobacillus delbrueckii into MRS medium for fermentation;

[0065] Preferably, the inoculation amount of Lactobacillus delbrueckii is 5-10 v / v%, preferably 5-8 v / v%, more preferably 5-6.5% v / v%;

[0066] More preferably, the culture temperature is 30-40°C, preferably 30-35°C;

[0067] Further preferably, the culture time is 24-48 h, preferably 24-36 h.

[0068] In some embodiments of the present application, the fermentation broth of Weizmannella coagulans Naqu.VC-1201 is obtained by inoculating Weizmannella coagulans Naqu.VC-1201 into MRS medium for fermentation;

[0069] Preferably, the inoculum amount of the coagulant Weizmannella is 5-10 v / v%, preferably 5-8 v / v%, more preferably 5-6.5% v / v%;

[0070] More preferably, the culture temperature is 30-40°C, preferably 36-38°C;

[0071] Further preferably, the culture time is 24-48 h, preferably 24-36 h.

[0072] This application uses postbiotics prepared from Lactobacillus delbrueckii Dangxiong LBⅧ and Weizmannella coagulans Naqu.VC-1201. These two strains have high antioxidant activity, which can more effectively inhibit the oxidation and rancidity of oils and extend the shelf life of walnut paste. At the same time, postbiotics also have the advantages of regulating intestinal flora, enhancing intestinal barrier function, and improving body immunity.

[0073] Furthermore, the above-mentioned postbiotic composition comprises inactivated bacteria of Weizmannia coagulans Naqu.VC-1201 and fermentation products thereof.

[0074] In some embodiments of the present application, the postbiotic composition is prepared by inactivating the strain after fermentation, wherein the inactivation includes placing the fermentation liquid of the strain in a supercritical carbon dioxide environment for inactivation; preferably, the inactivation pressure is 10-20MPa, and more preferably the inactivation temperature is 40-60°C.

[0075] The present invention adopts supercritical carbon dioxide sterilization technology to prepare postbiotics at low temperature, which can better protect their functional activity.

[0076] In some embodiments of the present application, the postbiotic composition has a DPPH radical scavenging activity of 70-90%, and a hydroxyl radical scavenging activity of 50-75%; preferably, the DPPH radical scavenging activity is 80-90%, and the hydroxyl radical scavenging activity is 60-75%.

[0077] The present application also provides a method for preparing the above-mentioned postbiotic composition, comprising the following steps:

[0078] (1) Inoculating Weizmannia coagulans Naqu.VC-1201 into MRS medium for fermentation;

[0079] (2) inoculating Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ into MRS medium for fermentation;

[0080] (3) mixing the fermentation broths of steps (1) and (2) and then inactivating the mixture;

[0081] (4) freeze-drying the inactivated fermentation broth.

[0082] In some embodiments of the present application, in step (3), the mass ratio of the coagulated Weizmannella fermentation broth to the Lactobacillus delbrueckii fermentation broth is 0.5-1:1, preferably 0.5-0.8:1, and more preferably 0.5-0.7:1.

[0083] In some embodiments of the present application, the inoculum amount of coagulated Weizmannella in step (1) is 5-10%, preferably 5-8 v / v%, and more preferably 5-6.5% v / v%.

[0084] In some embodiments of the present application, the fermentation culture temperature in step (1) is 30-40°C, preferably 30-35°C.

[0085] In some embodiments of the present application, the fermentation culture time in step (1) is 24-48 hours, preferably 24-36 hours.

[0086] In some embodiments of the present application, the inoculation amount of Lactobacillus delbrueckii in step (2) is 5-10%, preferably 5-8 v / v%, and more preferably 5-6.5% v / v%.

[0087] In some embodiments of the present application, the culture temperature in step (2) is 30-40°C, preferably 36-38°C.

[0088] In some embodiments of the present application, the culturing time in step (2) is 24-48 hours, preferably 24-36 hours.

[0089] In some embodiments of the present application, the MRS culture medium includes the following weight component raw materials: 5-15 parts of casein digest, 5-15 parts of beef extract powder, 3-5 parts of yeast extract powder, 1-3 parts of triammonium citrate, 4-6 parts of sodium acetate, 0.1-0.3 parts of magnesium sulfate, 0.04-0.06 parts of manganese sulfate, 1-3 parts of potassium dihydrogen phosphate, 15-25 parts of glucose, and 1.0-1.1 parts of Tween-80. Preferably, the MRS culture medium is prepared by adding the above components to water, wherein the weight of water is 800-1000 parts.

[0090] In some embodiments of the present application, the inactivation comprises placing the fermentation broth of the strain in a supercritical carbon dioxide environment for inactivation; preferably, the inactivation pressure is 10-20 MPa, and more preferably, the inactivation temperature is 40-60°C.

[0091] In some embodiments of the present application, the freeze-drying temperature is -65 to -85°C, and the preferred freeze-drying time is 24 to 48 hours.

[0092] The present application also provides the use of the above composition or the composition prepared by the above preparation method in food anti-oxidation, preferably in walnut paste anti-oxidation.

[0093] The present application also provides a walnut paste, comprising walnut kernels and the above-mentioned postbiotic composition or the postbiotic composition prepared by the above-mentioned preparation method, wherein the mass percentage of the postbiotic composition in the walnut kernels is 0.01-1%, preferably 0.01-0.1%, preferably 0.01-0.05, and more preferably 0.01-0.03%.

[0094] In some embodiments of the present application, the walnut paste does not contain an emulsifier and / or an antioxidant. The emulsifier described herein refers to hydrogenated oil, palm oil, monoglyceride, sucrose ester, soybean lecithin and its derivatives, sodium octenyl succinate starch, wheat fiber, modified cellulose, etc., and the antioxidant refers to vitamin E, ascorbic acid, β-carotene, tea polyphenols, etc.

[0095] The walnut paste of the present invention has a clean formula and does not contain food additives (such as emulsifiers, antioxidants, etc.). The postbiotic composition is prepared by supercritical carbon dioxide sterilization technology and vacuum freeze-drying technology, which can avoid high temperature and ensure the activity of the substance to the greatest extent, thereby producing a walnut paste with a clean formula, a long shelf life, and natural health.

[0096] The present application also provides a method for preparing the above walnut paste, wherein the method comprises the following steps:

[0097] (1) Peeling of walnut kernels;

[0098] (2) roasting the peeled walnut kernels;

[0099] (3) coarse grinding after baking;

[0100] (4) adding the above-mentioned postbiotic composition for fine grinding.

[0101] In some embodiments of the present application, the walnut kernel peeling in step (1) adopts a pure physical peeling process, and the physical peeling comprises the following steps:

[0102] First, soak the walnut kernels in water.

[0103] Secondly, use high-pressure running water to wash the walnut kernel to naturally fall off the inner seed coat;

[0104] The principle is to use the impact force of high-pressure running water to remove the walnut inner seed coat, and the water used can be normal temperature water, which not only has high peeling efficiency, high peeling rate and energy saving. The commonly used physical peeling is to put the walnut kernel into boiling water for a period of time. The principle is to use thermal expansion and contraction to remove the walnut inner seed coat, and the two methods are essentially different. In addition, boiling water will destroy the taste of the walnut kernel, reduce its brittleness, and appear soft. Therefore, the physical peeling method of the present invention is more advantageous.

[0105] Again, rinse the peeled walnut kernels in purified water to remove pigments, floating skin residues, etc., then remove the walnut kernels and drain the water.

[0106] Purified water is water that has been treated with a variety of technologies to remove impurities, microorganisms, heavy metals and other substances in the water to achieve high purity. It is usually used in laboratories, medical, electronics, pharmaceuticals and other fields. It can also be used for drinking water, cooking, etc. Compared with tap water, purified water is cleaner, healthier and more conducive to food processing.

[0107] In some embodiments of the present application, the drained walnut kernels are spread on a baking tray and placed in an oven for baking. The walnut kernels are turned over 1 to 2 times during the baking process to make the walnut kernels evenly baked. The baked walnut kernels have a golden surface and emit a unique walnut aroma. The baking temperature is 80-140°C, preferably 100-140°C, and more preferably 100-125°C; preferably, the baking time is 20-80 minutes, and preferably 40-70 minutes.

[0108] In some embodiments of the present application, in step (3), a pulverizer is used to grind the roasted walnut pieces into a mud cake-like shape, and preferably, the particle size of the particles is 60-100 mesh.

[0109] In some embodiments of the present application, the step (4) fine grinding uses a colloid mill to grind the coarsely ground walnut paste, preferably the grinding time is 10-30 minutes, and more preferably the particle size is ground to 325-460 mesh.

[0110] In some embodiments of the present application, the above preparation method further includes step (5) sterilization; preferably, the sterilization is pasteurization, the sterilization temperature is 40-70°C, preferably 55-70°C, and more preferably, the sterilization time is 20-30min.

[0111] The beneficial effects of the present invention are further illustrated by specific examples below.

[0112] The raw materials and reagents used in the present invention are all purchased from mainstream manufacturers in the market. Those without manufacturer or concentration are all analytically pure raw materials and reagents that can be routinely obtained. As long as they can play the expected role, there is no special limitation.

[0113] If no specific technology or conditions are specified in this embodiment, the process shall be carried out according to the technology or conditions described in the literature in the art or according to the product manual.

[0114] Hereinafter, the present invention will be described in more detail using examples and comparative examples, but the technical scope of the present invention is not limited to these examples. It should be noted that, unless otherwise specified, all percentages, parts, and ratios used in the present invention are based on mass.

[0115] The sources of the reagents and instruments used in the following examples are shown in Table 1.

[0116] Table 1: Raw materials and instrument information used in the examples and comparative examples

[0117] raw material Model / Purity Manufacturers for sale Universal Crusher GH-20 Jiangyin Gaohong Machinery Manufacturing Co., Ltd. Colloid mill JM280QF Wenzhou Colloid Mill Factory Hydrogenated Oil Food Grade Jinan Yuncheng Biotechnology Co., Ltd. Weizmannella coagulans CICC25229 China Industrial Microbiological Culture Collection Management Center NB medium HB8681-1 Haibo Biotechnology Co., Ltd. Escherichia coli CICC10899 China Industrial Microbiological Culture Collection Management Center Staphylococcus aureus CICC10384 China Industrial Microbiological Culture Collection Management Center

[0118] The culture medium used in the examples was prepared as follows:

[0119] MRS medium: Add 10 g of casein digest, 10 g of beef extract powder, 4 g of yeast extract powder, 2 g of triammonium citrate, 5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 2 g of potassium dihydrogen phosphate, 20 g of glucose, and 1.08 g of Tween-80 to distilled water in sequence, and make up to 1000 mL with distilled water. Adjust the pH to 6.3, stir and heat, sterilize at 121°C and 0.1 MPa for 30 min, and set aside.

[0120] Water agar medium: weigh 20 g of agar and add it to 1000 mL of distilled water, heat and boil until completely dissolved, divide into portions, and sterilize under high pressure at 121°C for 15 min. After sterilization, shake well to prevent the agar from settling at the bottom of the vessel and solidifying, and set aside.

[0121] NB medium: Weigh 4.237 g of NB medium, dissolve in 1000 ml of distilled water, divide into portions, sterilize under high pressure at 116°C for 30 min, and set aside.

[0122] Example 1 Isolation, screening and identification of strains

[0123] 1) Take 25g of dried yak meat from Oma Village, Gulu Town, Seni District, Nagqu City, Tibet, and mix it with 225mL of sterile saline to obtain a uniform sample solution. Dilute the sample solution in a gradient manner and take 10 -3 , 10 -4 , 10 -5 , 10 -6 The dilution of the concentration was spread on MRS agar plates for culture. After culture at 36°C for 48 hours, colonies grew on the MRS agar plates.

[0124] 2) Initial screening of bacterial strains

[0125] Pick a single colony and place it in MRS agar medium for separation, purification and continued cultivation at 36°C. Separate and purify continuously for no less than three times to obtain purified colonies.

[0126] 3) Acid production experiment

[0127] The single colony obtained from the initial screening of the strain was incubated in a 0.2% CaCO 3 The colonies were cultured on MRS agar plates at 36°C for 48 hours, and the formation of transparent circles around the colonies was observed. Single colonies with strong acid production ability and large transparent circles were selected for further separation and purification. The colonies grew as round or irregular white colonies in MRS agar medium, showing a dry surface, thick colonies, and neat edges.

[0128] 4) Gram staining

[0129] The strain obtained by repeated screening was subjected to Gram staining, and the Gram staining was typical positive, and the strain obtained was the target strain. Under a microscope, the cells were straight rod-shaped, with a diameter of about 0.5-1.5 μm, without flagella, producing spores, and being motile.

[0130] 5) Identification of strains

[0131] The isolated and purified strain was Gram-positive. 2 O 2The catalase-positive, acid-producing, non-gas-producing strain was sequenced for 16S rDNA gene sequence, and the obtained results were compared with the homology analysis in the NCBI GenBank database. The results showed that the strain was Weizmannia coagulans. The strain was named Weizmannia coagulans Naqu.VC-1201, and its 16S rRNA sequence was shown in SEQ ID NO: 1:

[0132] The strain was deposited in the China Center for Type Culture Collection (CCTCC) on August 25, 2023, with the deposit number CCTCC MNO.20231577. The deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072; Telephone: (027)-68754052.

[0133] Example 2 Evaluation of Physiological and Biochemical Characteristics of Strains

[0134] 1. Antibacterial experiment

[0135] The antimicrobial activity of the strains was determined using the double-layer agar diffusion method. Two foodborne pathogens, including Escherichia coli ATCC 8739 and Staphylococcus aureus ATCC 6538, were used as indicator bacteria. 10 mL of 2 g / 100 mL water agar medium was added to the sterilized plate and placed in a sterilized Oxford cup after the water agar solidified. 10 mL of NB agar medium was added. 6 ~10 7 CFU / mL indicator bacteria, mix thoroughly and pour into the plate. After the culture medium solidifies, take out the Oxford cup, add 200μL of the test bacterial suspension into each well, culture at 37℃ for 24h, and observe and measure the diameter of the inhibition zone (mm).

[0136] Table 2 Antibacterial ability of coagulated Weizmannella

[0137]

[0138]

[0139] 2. Gastrointestinal fluid resistance test

[0140] Preparation of artificial gastric juice: adjust the pH of 0.2% NaCl solution to 2.0 with 1 mol / L HCl, add 0.05 g pepsin (specific activity 1:10000), and filter through a 0.22 μm sterile filter membrane for sterilization.

[0141] Preparation of artificial intestinal fluid: NaH 2 PO 41.2g, trypsin 0.1g, bile salt 0.45g, distilled water 100mL, adjust the pH to 8.0 with sodium hydroxide, and filter sterilize with a 0.22μm sterile filter membrane.

[0142] Survival rate determination: A single colony of Weizmannella coagulans Naqu.VC-1201 was picked and cultured in MRS liquid culture medium overnight, and 1 mL of the culture solution was inoculated into 9 mL of artificial gastric juice or artificial intestinal juice, respectively, and cultured at 37°C for 2 hours. The number of viable bacteria was determined, and the survival rate was calculated; using Weizmannella coagulans CICC 25229 control bacteria as a positive control, Weizmannella coagulans CICC 2522 was inoculated in 9 mL of MRS liquid culture medium and cultured overnight, and 1 mL of the culture solution was inoculated into 9 mL of artificial gastric juice or artificial intestinal juice, respectively, and cultured at 37°C for 2 hours. The number of viable bacteria was determined, and the survival rate was calculated; wherein, the survival rate was calculated according to the following formula, and the results are shown in Table 3.

[0143] Survival rate (%) = number of viable bacteria of the strain after 2 hours of treatment with artificial digestive fluid / initial number of viable bacteria of the strain before treatment with artificial digestive fluid × 100%

[0144] Table 3 Survival rate of coagulant Weizmannella after treatment with artificial gastric juice and artificial intestinal juice

[0145]

[0146] 3. Antioxidant activity test

[0147] 1) DPPH free radical scavenging activity assay

[0148] Mix 2mL of the test bacterial suspension with 1mL of 0.2mmol / L DPPH-anhydrous ethanol solution, react at 37℃ in the dark for 30min, centrifuge at 6000r / min for 10min, take the supernatant, and measure the absorbance A1 at a wavelength of 517nm; the blank group uses an equal volume of anhydrous ethanol instead of DPPH-anhydrous ethanol solution, and the control group uses an equal volume of distilled water instead of the sample solution. An equal volume of distilled water-anhydrous ethanol mixture is used for blank zero adjustment. The DPPH free radical scavenging rate is calculated according to formula (1):

[0149]

[0150] Where: A0 is the absorbance of the blank group; A1 is the absorbance of the sample group; A2 is the absorbance of the control group.

[0151] 2) Hydroxyl free radical scavenging activity assay

[0152] Take 1 mL each of 5 mmol / L ferrous sulfate solution, 5 mmol / L salicylic acid ethanol solution, and 3 mmol / L hydrogen peroxide solution and add them to a 10 mL stoppered test tube, add 2 mL of the bacterial suspension to be tested, fill up to the mark with distilled water, react in a 37°C water bath for 15 min, centrifuge at 6000 r / min for 10 min, take the supernatant, and measure the absorbance at a wavelength of 510 nm. Distilled water is used as a blank control. The hydroxyl radical scavenging rate is calculated according to formula (2):

[0153]

[0154] Where: A0 is the absorbance of the blank group; A1 is the absorbance of the sample group.

[0155] The antioxidant activities of Weizmannella coagulans Naqu.VC-1201 and Weizmannella coagulans CICC 25229 are shown in Table 4.

[0156] Table 4 Antioxidant activity of coagulated Weizmannella

[0157]

[0158] Example 3

[0159] The steps for making walnut sauce are as follows:

[0160] (1) Preparation of postbiotics: ① Inoculate Weizmannella coagulans Naqu.VC-1201 in an MRS medium at a 5% inoculum amount, and culture at 33°C for 24 hours to obtain a fermentation broth; ② Inactivate the fermentation broth by centrifugation, mix the bacteria and supercritical carbon dioxide, and then freeze-dry to obtain a postbiotic powder; wherein the centrifugation conditions are 7000 r / min, centrifugation for 12 minutes; the inactivation treatment pressure is 10 MPa, the treatment temperature is 40°C, and the treatment time is 40 minutes; the freezing temperature is -65°C, and the drying time is 48 hours;

[0161] (2) 2 kg of peeled walnut kernels were used as raw materials, and they were roasted and coarsely ground to obtain 1800 g of material, wherein the roasting temperature was 100 ° C and the roasting time was 65 min.

[0162] (3) Coarse grinding: Grind the peach kernels into a slurry using a universal grinder;

[0163] (4) adding 0.18 g of the postbiotic powder prepared in step (1) (the addition amount is 0.01 wt%) and finely grinding the walnut paste with a colloid mill for 30 min. After grinding, the walnut paste can pass through a 325-mesh sieve;

[0164] (4) The finely ground material is pasteurized and canned to obtain walnut paste, wherein the sterilization temperature is 65° C. and the sterilization time is 20 min.

[0165] Example 4

[0166] The steps for making walnut sauce are as follows:

[0167] (1) Preparation of postbiotics: ① Inoculate Lactobacillus delbrueckii Dangxiong LBⅧ in MRS medium at a 5% inoculation rate and culture at 30°C for 24 hours; ② Inoculate Weizmannella coagulans Naqu.VC-1201 in MRS medium at a 5% inoculation rate and culture at 36°C for 24 hours; ③ Mix Lactobacillus delbrueckii Dangxiong LBⅧ and Weizmannella coagulans Naqu.VC-1201 fermentation liquid at a ratio of 1:0.5 to obtain a fermentation mixture, then obtain the bacterial cells by centrifugation, mix the bacterial cells with supercritical carbon dioxide for inactivation, and then freeze-dry to obtain postbiotic powder. The centrifugation conditions are 7000r / min and centrifugation for 12 minutes; the inactivation treatment pressure is 10MPa, the treatment temperature is 40°C, and the treatment time is 40 minutes; the freezing temperature is -65°C and the drying time is 48 hours;

[0168] (2) 2 kg of peeled walnut kernels were used as raw materials, and the raw materials were roasted and coarsely ground to obtain 1800 g of material, wherein the roasting temperature was 100 ° C and the roasting time was 65 min;

[0169] (3) Coarse grinding: Grind the peach kernels into a slurry using a universal grinder;

[0170] (4) After adding 0.18 g of postbiotic powder (the addition amount is 0.01 wt%), the walnut paste is finely ground with a colloid mill for 30 min. After grinding, the walnut paste can pass through a 460-mesh sieve;

[0171] (4) The finely ground material is pasteurized and canned to obtain walnut paste, wherein the sterilization temperature is 55° C. and the sterilization time is 30 min.

[0172] Example 5

[0173] The steps for making walnut sauce are as follows:

[0174] (1) Preparation of postbiotics: ① Inoculate Lactobacillus delbrueckii Dangxiong LBⅧ in MRS medium at an inoculum size of 6.5% and culture at 35°C for 36 hours; ② Inoculate Weizmannella coagulans Naqu.VC-1201 in MRS medium at an inoculum size of 6.5% and culture at 38°C for 36 hours; ③ Mix Lactobacillus delbrueckii Dangxiong LBⅧ and Weizmannella coagulans Naqu.VC-1201 fermentation broth at a ratio of 1:0.7, inactivate the bacteria and supercritical carbon dioxide by centrifugation, and then freeze-dry to obtain postbiotic powder. The centrifugation conditions are 8000r / min and centrifugation for 10 minutes; the inactivation treatment pressure is 15MPa, the treatment temperature is 50°C, and the treatment time is 35 minutes; the freezing temperature is -75°C and the drying time is 36 hours;

[0175] (2) 3 kg of peeled walnut kernels were used as raw materials, and the raw materials were roasted and coarsely ground to obtain 2640 g of material, wherein the roasting temperature was 125 ° C and the roasting time was 45 min;

[0176] (3) Coarse grinding: Grind the peach kernels into a slurry using a universal grinder;

[0177] (4) After adding 0.792 g of postbiotic powder (addition amount is 0.03 wt%), the walnut paste is finely ground with a colloid mill for 30 min. After grinding, the walnut paste can pass through a 400-mesh sieve;

[0178] (5) The finely ground material is pasteurized and canned to obtain walnut paste, wherein the sterilization temperature is 60° C. and the sterilization time is 20 min.

[0179] Example 6

[0180] The steps for making walnut sauce are as follows:

[0181] (1) Preparation of postbiotics: ① Inoculate Lactobacillus delbrueckii Dangxiong LBⅧ in MRS medium at an inoculum rate of 8% and culture at 40°C for 48 hours; ② Inoculate Weizmannella coagulans Naqu.VC-1201 in MRS medium at an inoculum rate of 8% and culture at 40°C for 48 hours; ③ Mix Lactobacillus delbrueckii Dangxiong LBⅧ and Weizmannella coagulans Naqu.VC-1201 fermentation broth at a ratio of 1:0.8, inactivate by centrifugation, mix the bacteria with supercritical carbon dioxide, and then freeze-dry to obtain postbiotic powder. The centrifugation conditions are 9000r / min and 8min; the inactivation treatment pressure is 20MPa, the treatment temperature is 60°C, and the treatment time is 30min; the freezing temperature is -85°C and the drying time is 24h;

[0182] (2) 4 kg of peeled walnut kernels were used as raw materials, and were roasted and coarsely ground to obtain 3400 g of material, wherein the roasting temperature was 140° C. and the roasting time was 20 min;

[0183] (3) Coarse grinding: Grind the peach kernels into a slurry using a universal grinder;

[0184] (4) After adding 1.7 g of postbiotic powder (the addition amount is 0.05 wt%), the walnut paste is finely ground with a colloid mill for 30 min. After grinding, the walnut paste can pass through a 460-mesh sieve;

[0185] (5) The finely ground material is pasteurized and canned to obtain walnut paste, wherein the sterilization temperature is 70° C. and the sterilization time is 10 min.

[0186] Example 7

[0187] Compared with Example 4, the inactivation method in step (1) is changed to heat treatment, that is, the fermentation mixture is heat treated at a temperature of 110° C. for 20 min, and then centrifuged and freeze-dried to obtain the postbiotic powder. Other proportions and preparation methods are the same as the preparation method of Example 4.

[0188] Example 8

[0189] Compared with Example 4, the freeze drying in the preparation of postbiotics in step (1) is changed to spray drying, that is, the inactivated bacteria are spray dried to obtain postbiotic powder, wherein the inlet air temperature is 150°C and the outlet air temperature is 80°C. Other proportions and preparation methods are the same as the preparation method of Example 4.

[0190] Example 9

[0191] Compared with Example 4, the difference is that the addition amount of the postbiotic powder in step (3) is 1wt% (18g), and the other proportions and preparation methods are the same as the preparation method of Example 4.

[0192] Comparative Example 1

[0193] (1) 2 kg of peeled walnut kernels were used as raw materials, and were roasted and coarsely ground to obtain 1800 g of material, wherein the roasting temperature was 100° C. and the roasting time was 65 min. The coarse grinding was performed using a universal grinder to grind the walnut kernels into a slurry;

[0194] (2) finely grinding the walnut paste with a colloid mill for 30 min;

[0195] (3) The finely ground material is pasteurized and canned to obtain walnut paste, wherein the sterilization temperature is 40° C. and the sterilization time is 20 min.

[0196] Comparative Example 2

[0197] The steps for making walnut sauce are as follows:

[0198] (1) Preparation of postbiotics: ① Inoculate Lactobacillus delbrueckii LBⅧ at a 5% inoculation rate into MRS medium and culture at 30°C for 24 hours; ② Centrifuge the fermentation broth, mix the bacteria with supercritical carbon dioxide to inactivate, and then freeze-dry to obtain postbiotic powder. The centrifugation conditions are 7000r / min, centrifugation for 12 minutes; the inactivation treatment pressure is 10MPa, the treatment temperature is 40°C; the freezing temperature is -65°C, and the drying time is 48 hours;

[0199] (2) 2 kg of peeled walnut kernels were used as raw materials, and the raw materials were roasted and coarsely ground to obtain 1800 g of material, wherein the roasting temperature was 100 ° C and the roasting time was 65 min;

[0200] (3) Coarse grinding: Grind the peach kernels into a slurry using a universal grinder;

[0201] (4) After adding 0.18 g of postbiotic powder (addition amount is 0.01%), the walnut paste is finely ground with a colloid mill for 30 min. After grinding, the walnut paste can pass through a 325-mesh sieve;

[0202] (5) The finely ground material is pasteurized and canned to obtain walnut paste, wherein the sterilization temperature is 55° C. and the sterilization time is 30 min.

[0203] Comparative Example 3

[0204] Compared with Comparative Example 1, the difference is that 0.18 g of hydrogenated oil is added before fine grinding in step (2), and other proportions and preparation methods are the same as the preparation method of Comparative Example 1.

[0205] Experimental Example 1 Antioxidant Activity Experiment

[0206] The postbiotic powders prepared in Examples 3-8 and Comparative Example 2 were formulated into solutions with a concentration of 1 wt%, and the DPPH radical scavenging activity and hydroxyl radical scavenging activity were measured according to the DPPH radical scavenging activity measurement method and the hydroxyl radical scavenging activity measurement method in Example 2, respectively, to compare the activities of the postbiotics prepared by different preparation methods.

[0207] The effects of the postbiotics prepared in Examples 3-8 and Comparative Example 2 on the DPPH free radical scavenging activity and the hydroxyl free radical scavenging activity are shown in Table 5.

[0208] Table 5 Antioxidant activity of postbiotic powder

[0209]

[0210]

[0211] As can be seen from Table 5, the postbiotic powders of the present application examples all have strong DPPH free radical scavenging activity and hydroxyl free radical scavenging ability. The postbiotic powders prepared in Examples 4-8 have a DPPH free radical scavenging activity of 72.16-89.73%, and a hydroxyl free radical scavenging activity of 54.49-70.17%. Among them, the postbiotic powders prepared in Examples 4-6 using supercritical dioxide inactivation have a DPPH free radical scavenging activity of 86.85-89.73%, and a hydroxyl free radical scavenging activity of 66.83-70.17%. Compared with Example 3 and Comparative Example 2, the postbiotic powder prepared by the combination of Lactobacillus delbrueckii Dangxiong LBⅧ and Weizmannella coagulans Naqu.VC-1201 has a DPPH free radical scavenging ability and a hydroxyl free radical scavenging ability that are stronger than the postbiotic powders prepared by the two strains alone, indicating that the combination of the two has a certain synergistic effect. Compared with the heat treatment inactivation in Example 7, the postbiotic agent prepared by supercritical inactivation has stronger DPPH radical scavenging ability and hydroxyl radical scavenging ability; compared with the spray drying in Example 8, the postbiotic powder prepared by freeze drying has stronger DPPH radical scavenging ability and hydroxyl radical scavenging ability.

[0212] Experimental Example 2

[0213] 1) Shelf life stability test

[0214] A 90-day shelf life stability test was conducted on the walnut pastes prepared in Examples 3-9 and Comparative Examples 1-3 at a storage temperature of 4° C., and the sensory properties, acid value, and peroxide value of the walnut pastes were measured.

[0215] ① Sensory evaluation

[0216] A 10-member evaluation team was formed to evaluate the flavor of the product, and then the average of their scores was taken. The scoring criteria were: 8-10 points for a strong flavor of the walnut sauce; 6-8 points for a normal flavor of the walnut sauce; 4-6 points for a weak flavor of the walnut sauce; 2-4 points for a slightly rancid flavor of the walnut sauce; 0-2 points for a very rancid flavor of the walnut sauce. The results are shown in Table 6.

[0217] Table 6 Sensory evaluation results after different storage time

[0218]

[0219]

[0220] ② Determination of acid value

[0221] According to the first method in the national standard GB 5009.229-2016 "National Food Safety Standard Determination of Acid Value in Food", the oil in walnut paste was determined, and the results are shown in Table 7.

[0222] Table 7 Acid value results after different storage time

[0223] serial number Day 0 15 days 30 days 45 days 60 days 75 days 90 days Example 3 0.15 0.19 0.21 0.27 0.33 0.39 0.43 Example 4 0.12 0.15 0.16 0.2 0.32 0.36 0.42 Example 5 0.14 0.17 0.21 0.23 0.35 0.41 0.44 Example 6 0.12 0.14 0.18 0.22 0.29 0.35 0.41 Example 7 0.14 0.26 0.43 0.86 1.28 1.83 2.06 Example 8 0.16 0.34 0.53 0.83 1.26 1.78 2.16 Example 9 0.19 0.36 0.58 0.87 1.32 1.93 2.08 Comparative Example 1 0.15 0.53 0.92 1.38 1.83 2.31 2.42 Comparative Example 2 0.17 0.28 0.32 0.54 1.53 1.97 2.17 Comparative Example 3 0.13 0.17 0.21 0.26 0.29 0.35 0.4

[0224] ③Determination of peroxide value

[0225] The oil in walnut paste was determined by referring to the first method in the national standard GB 5009.227-2016 "Determination of peroxide value in food, national food safety standard", and the results are shown in Table 8.

[0226] Table 8 Peroxide value results after different storage time

[0227]

[0228]

[0229] It can be seen from Tables 6-8 that after 90 days of storage, the flavor of the walnut paste prepared in Examples 3-6 becomes lighter, but there is no rancid taste, the acid value is 0.41-0.44, the peroxide value is 0.0263-0.0337, and the results of the sensory score, acid value and peroxide value are better than those of Comparative Example 3 using hydrogenated oil as a stabilizer; Compared with Example 3 and Comparative Example 2, the sensory score of Example 4 after 90 days of storage is higher than that of Example 3 and Comparative Example 2, and the acid value and peroxide value are lower than those of Example 3 and Comparative Example 2, and the walnut paste prepared using only the postbiotic powder of Lactobacillus delbrueckii Dangxiong LBⅧ has a lower sensory score, higher acid value and peroxide value, and poorer antioxidant effect.

[0230] Table 9 Sequence Listing

[0231]

[0232]

[0233]

[0234] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A coagulant Weizmannella, characterized in that The Weizmannia coagulans is Weizmannia coagulans Naqu.VC-1201, which is deposited in China Center for Type Culture Collection with a deposit number of CCTCC NO: M 20231577.

2. The coagulant Weizmannella according to claim 1, characterized in that The 16srRNA sequence of Weizmannella coagulans comprises the sequence shown in SEQ ID NO:

1.

3. A bacterial preparation comprising the Weizmannella coagulans according to claim 1 or 2.

4. A postbiotic composition, characterized in that It contains at least the postbiotics formed by Weizmannella coagulans according to claim 1 or 2.

5. The postbiotic composition according to claim 4, characterized in that The postbiotic composition also includes Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ formed by epigenetics.

6. The postbiotic composition according to claim 4 or 5, characterized in that The content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition is 30-50wt%; the content of postbiotics formed by Lactobacillus delbrueckiisubsp.lactis Dangxiong LBⅧ is 50-70wt%; Preferably, the postbiotic composition contains Weizmannia coagulans The content of postbiotics formed by Naqu.VC-1201 is 30-45wt%; the content of postbiotics formed by Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ is 55-70wt%; More preferably, the content of postbiotics formed by Weizmannia coagulans Naqu.VC-1201 in the postbiotic composition is 30-40wt%; the content of postbiotics formed by Lactobacillus delbrueckii subsp. lactis Dangxiong LBⅧ is 60-70wt%.

7. The postbiotic composition according to any one of claims 4 to 6, characterized in that The postbiotic composition is prepared by fermenting and culturing the strain and then inactivating it; Preferably, the inactivation comprises placing the fermentation liquid of the strain in a supercritical carbon dioxide environment for inactivation; preferably, the inactivation pressure is 10-20 MPa, and more preferably, the inactivation temperature is 40-60°C.

8. The postbiotic composition according to claim 7, characterized in that The postbiotic composition is prepared by mixing the fermentation broth of Lactobacillus delbrueckii Dangxiong LBⅧ and the fermentation broth of Weizmannella coagulans Naqu.VC-1201 and then inactivating them. Preferably, the mass ratio of the fermentation broth of Weizmannella coagulans Naqu.VC-1201 and the fermentation broth of Lactobacillus delbrueckii Dangxiong LBⅧ is 0.5-1:1, preferably 0.5-0.8:1, and more preferably 0.5-0.7:

1.

9. The method for preparing the postbiotic composition according to any one of claims 4 to 8, characterized in that: The preparation method comprises the steps of fermenting and culturing the strain and then inactivating the strain to obtain the product. Preferably, the inactivation comprises placing the fermentation liquid of the strain in a supercritical carbon dioxide environment for inactivation; preferably, the inactivation pressure is 10-20 MPa, and more preferably, the inactivation temperature is 40-60°C.

10. The preparation method according to claim 9, characterized in that: The preparation method further comprises the step of freeze-drying after inactivation; Preferably, the freeze-drying temperature is -65 to -85°C, and the freeze-drying time is preferably 24 to 48 hours.

11. The preparation method according to claim 9 or 10, characterized in that: The steps include: (1) inoculating Weizmannella coagulans into MRS medium for fermentation; (2) inoculating Lactobacillus delbrueckii into MRS medium for fermentation; (3) mixing the fermentation broths of steps (1) and (2) and then inactivating the mixture; (4) Drying the inactivated fermentation liquid.

12. The preparation method according to claim 11, characterized in that: The inoculum amount of the coagulation Weizmannella in step (1) is 5-10%, preferably 5-8% v / v%, more preferably 5-6.5% v / v%; preferably, the fermentation culture temperature is 30-40° C., preferably 30-35° C.; more preferably, the fermentation culture time is 24-48 hours, preferably 24-36 hours; and / or, In step (2), the inoculum amount of Lactobacillus delbrueckii is 5-10%, preferably 5-8 v / v%, more preferably 5-6.5% v / v%; preferably, the fermentation culture temperature is 30-40° C., preferably 36-38° C.; more preferably, the fermentation culture time is 24-48 h, preferably 24-36 h.

13. The preparation method according to claim 11 or 12, characterized in that: In step (3), the mass ratio of the coagulated Weizmannella fermentation broth to the Lactobacillus delbrueckii fermentation broth is 0.5-1:1, preferably 0.5-0.8:1, and more preferably 0.5-0.7:

1.

14. Use of the postbiotic composition according to any one of claims 4 to 8 or the postbiotic composition prepared by the preparation method according to any one of claims 9 to 13 in food anti-oxidation, preferably in walnut paste anti-oxidation.

15. A walnut paste, characterized in that: The invention is prepared from the following raw materials: walnut kernels and a composition according to any one of claims 4 to 8 or a postbiotic composition prepared by the preparation method according to any one of claims 9 to 13, wherein the mass percentage of the postbiotic composition in the walnut kernels is 0.01-1%, preferably 0.01-0.1%, and more preferably 0.01-0.05%.

16. The walnut paste according to claim 15, characterized in that The walnut kernels are blanched walnut kernels, and preferably the walnut kernels are roasted walnut kernels.

17. The method for preparing walnut paste according to claim 15 or 16, characterized in that: The method comprises the following steps: (1) Peeling of walnut kernels; (2) roasting the peeled walnut kernels; (3) coarse grinding after baking; (4) Fine grinding after coarse grinding: during fine grinding, the composition according to any one of claims 4 to 8 or the postbiotic composition prepared by the preparation method according to any one of claims 9 to 13 is added.

18. The preparation method according to claim 17, characterized in that: The baking temperature in step (2) is 80-140° C., preferably 100-140° C., more preferably 100-125° C.; Preferably, the baking time is 20-80 min, preferably 40-70 min.

19. The preparation method according to claim 17 or 18, characterized in that: The coarse grinding in step (3) is performed by a pulverizer, preferably to a particle size of 60-100 mesh.

20. The preparation method according to any one of claims 17 to 19, characterized in that: Step (4) fine grinding: the walnut paste after coarse grinding is ground by a colloid mill, preferably the grinding time is 10-30 minutes, and more preferably the particle size is ground to 325-460 mesh.

21. The preparation method according to any one of claims 17 to 20, characterized in that: The method further comprises step (5) sterilization; preferably, the sterilization temperature is 40-70° C., and more preferably, the sterilization time is 20-30 min.

22. Walnut paste prepared by the preparation method according to any one of claims 17 to 21.

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