A method for preparing sea cucumber enzyme or postbiotics using Cordyceps militaris as fermentation bacteria

Abstract

The present invention discloses a method for preparing sea cucumber enzyme or postbiotics using Cordyceps militaris as fermentation bacteria, which is a method for preparing sea cucumber enzyme or postbiotics by pure bacterial fermentation using edible and medicinal fungi Cordyceps militaris as a sole fermentation substrate. Sea cucumber enzyme solution provides nutrition for Cordyceps militaris as a substrate, and through the biotransformation of Cordyceps militaris, the prepared sea cucumber enzyme and postbiotics not only have the functional components such as sea cucumber polysaccharides, sea cucumber saponins, cordyceps polysaccharides, cordycepin, etc. present in sea cucumber and Cordyceps militaris itself, but also have sea cucumber-based Cordyceps militaris fermentation products, each component is organically combined, synergistically enhanced, and the fiber dissolving and antioxidant activity of sea cucumber enzymes are significantly improved, as well as the skin care effects of sea cucumber postbiotics including anti-inflammatory, repair, firming, and antioxidant aspects. Because other carbon sources and nitrogen sources do not need to be added during the fermentation process, the fermentation liquid can be enriched without separation, and there are the advantages of simple operation, energy saving, and ecological environmental protection.

Description

Technical Field

[0001] The invention belongs to the technical field of comprehensive utilization of aquatic products and processing by-products, and in particular relates to a method for preparing sea cucumber enzymes or postbiotics by using Cordyceps militaris as fermentation bacteria. Background Art

[0002] Enzymes are products containing specific biologically active ingredients that are made from animals, plants or fungi, with or without auxiliary materials, and fermented by microorganisms. Its biologically active ingredients include various nutrients provided by the raw materials and microorganisms, as well as some physiologically active substances generated by fermentation, such as amino acids, peptides, vitamins, polysaccharides, polyphenols, flavonoids, alcohols, esters, enzymes, mineral elements, organic acids and various probiotics. At present, there are reports on the preparation of sea cucumber enzymes using sea cucumbers as raw materials, but there are two fermentation methods: one is natural fermentation without adding any microorganisms; the other is composite fermentation with the addition of multiple lactic acid bacteria. Its fermentation products only contain the nutrients of sea cucumbers and the fermentation products of sea cucumbers.

[0003] Postbiotics are inactivated microorganisms and / or bacterial components with a clear genetic background that are beneficial to the health of the host, including or excluding their metabolites. The "Research Status and Industrial Application of Postbiotics" published by the Probiotics Branch of the Chinese Society of Food Science and Technology in the Journal of Chinese Institute of Food Science and Technology records the main connotations of postbiotics as follows: 1) The first condition for postbiotics is that they are beneficial to the health of the host, and the health effects must be scientifically based; 2) The microorganisms selected for postbiotics should be strains with recognized safety and clear genetic backgrounds and biological characteristics, and the components of health effects such as inactivated microorganisms, bacterial components and metabolites are clear; 3) Postbiotics include inactivated bacteria, bacterial components (including bacterial lysates) and their metabolites that are beneficial to the health of the host; vaccines, viruses (including bacteriophages), purified strain metabolites, and traditional fermented products with uncertain bacterial composition are not included.

[0004] Cordyceps militaris ( Cordyceps militaris (L.) Link) is also known as northern cordyceps, and is the model species of the genus Cordyceps of the family Clavicipitaceae of the subphylum Ascomycota. It is of the same genus as Cordyceps sinensis but of a different species. It is a medicinal and edible fungus with high nutritional value and medicinal efficacy. It not only contains nutrients such as protein and fat, but also contains active ingredients such as cordycepin, adenosine and cordyceps polysaccharides. Cordycepin is a unique nucleoside active substance in Cordyceps, which has anti-cancer, antibacterial and antiviral functions; adenosine is an endogenous nucleoside that is distributed throughout human cells, participates in myocardial energy metabolism, and has physiological effects on the cardiovascular system and many systems and tissues of the body; cordyceps polysaccharides are considered to be one of the very good immunostimulants, which can enhance the body's immune capacity. Since the artificial cultivation of Cordyceps militaris is obtained by isolating and domesticating wild Cordyceps militaris, and wild Cordyceps militaris usually parasitizes on the larvae or pupae of Lepidoptera insects, the culture medium used for artificial cultivation of Cordyceps militaris is a key factor in determining the success or failure of cultivation. The existing artificial cultivation method of Cordyceps militaris is to culture Cordyceps militaris strains in liquid potato culture medium to obtain Cordyceps militaris strain culture solution, and then inoculate the Cordyceps militaris strain culture solution into a solid culture medium for the cultivation of Cordyceps militaris fruiting bodies.

[0005] So far, there is no report on the preparation of sea cucumber enzymes or postbiotics using sea cucumber hydrolysate as the sole fermentation matrix and Cordyceps militaris as the fermentation bacteria. Summary of the invention

[0006] The present invention aims to solve the above-mentioned technical problems existing in the prior art and provides a method for preparing sea cucumber enzyme or postbiotics by using sea cucumber hydrolysate as the only fermentation matrix and Cordyceps militaris as the fermentation bacteria.

[0007] The technical solution of the present invention is: a method for preparing sea cucumber enzyme or postbiotics using Cordyceps militaris as fermentation bacteria, which is carried out in the following steps:

[0008] Step 1. Pretreatment of sea cucumber: repeatedly soaking the body wall and / or viscera of the sea cucumber in deionized water until the conductivity is less than 1500 μs / cm, thereby obtaining a sea cucumber-based raw material;

[0009] Step 2. preparing a sea cucumber enzymatic hydrolysate: adding distilled water to the sea cucumber raw material to crush it, and then adding protease to perform enzymatic hydrolysis to obtain a sea cucumber enzymatic hydrolysate;

[0010] Step 3. Prepare the secondary seed culture solution of Cordyceps militaris: transfer the Cordyceps militaris from the slant mother culture to liquid culture to obtain a cell density of ≥10 9 spores / mL of secondary seed culture solution of Cordyceps militaris;

[0011] Step 4. Prepare sea cucumber fermentation liquid: add the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate, and ferment with aeration and stirring at 20-24° C. for 4-7 days to obtain the sea cucumber fermentation liquid, wherein the mass ratio of the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate is 2-10%;

[0012] Step 5. Prepare sea cucumber enzyme and postbiotics: freeze-dry the sea cucumber fermentation liquid into powder to obtain sea cucumber enzyme; or sterilize the sea cucumber fermentation liquid at high temperature, centrifuge it, take the supernatant, and freeze-dry it into powder to obtain sea cucumber postbiotics.

[0013] The step 1 is to cut the sea cucumber body wall and / or viscera into strips with a width of 2 to 5 cm, soak them in deionized water at a mass ratio of 1:10 for 4 to 8 hours, and repeatedly soak them 3 to 4 times to obtain the sea cucumber-based raw material.

[0014] The step 2 is to add distilled water to homogenize the sea cucumber raw material at a mass ratio of 1:8-10, then add 1-3% of protease at a mass ratio, control the temperature at 45-55°C, perform magnetic stirring for 2-3 hours of enzymolysis, filter and remove impurities, and sterilize at 115°C for 30 minutes to obtain a sea cucumber enzymolysis solution.

[0015] The step 3 is to take the slant surface of Cordyceps militaris covered with hyphae and store the mother culture of the slant, inoculate the slug into a liquid potato culture medium, and culture the slug in a shaking flask at 130-150 r / min for 5-7 days at 18-22° C. to obtain a first-level seed culture solution of Cordyceps militaris; then transfer the first-level seed culture solution of Cordyceps militaris to a fresh liquid potato culture medium at a ratio of 3-5%, and culture until the cell density is ≥10 9 spores / mL, and the secondary seed culture solution of Cordyceps militaris was obtained.

[0016] The step 5 of sterilizing the sea cucumber fermented liquid at high temperature and then centrifuging to obtain the supernatant is to sterilize the sea cucumber fermented liquid at 115° C. for 30 min, centrifuge at 10,000 r / min for 20 min, and obtain the supernatant.

[0017] The present invention is a method for preparing sea cucumber enzyme or postbiotics by pure bacterial fermentation using sea cucumber hydrolysate as the only fermentation matrix and utilizing edible and medicinal fungi-Cordyceps militaris. Sea cucumber hydrolysate provides nutrition for Cordyceps militaris as a matrix, and through the biotransformation of Cordyceps militaris, the prepared sea cucumber enzyme and postbiotics not only have functional components such as sea cucumber polysaccharides, sea cucumber saponins, cordyceps polysaccharides, cordycepin, etc. present in sea cucumber and Cordyceps militaris itself, but also have sea cucumber-based Cordyceps militaris fermentation products, and each component is organically combined and synergistically enhanced, significantly improving the fiber dissolving and antioxidant activity of sea cucumber enzyme and improving the skin care effects of sea cucumber postbiotics including anti-inflammatory, repair, firming, and antioxidant aspects. Because no other carbon source and nitrogen source need to be added during the fermentation process, the fermentation liquid and thalline can be enriched without separation, and the fishy smell of sea cucumber is significantly reduced during the fermentation process, and no other methods such as physical or chemical de-awakening treatment are required, which has the advantages of simple operation, energy saving, and ecological environmental protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1Schematic diagram of the fibrinolytic activity of sea cucumber enzymes in an embodiment of the present invention and a comparative example.

[0019] Figure 2 Schematic diagram of the antioxidant activity of sea cucumber enzymes in embodiments of the present invention and in comparative examples.

[0020] Figure 3 Schematic diagram of the relative contents of inflammatory factors after cells were treated with sea cucumber postbiotics in an embodiment of the present invention and in a comparative example.

[0021] Figure 4 Schematic diagram of the relative areas of the sea cucumber postbiotics after repairing cell scratches in the embodiment of the present invention and the comparative example.

[0022] Figure 5 Schematic diagram of the relative contents of Procollagen-1 and Elastin after cells were treated with sea cucumber postbiotics in an example of the present invention and a comparative example.

[0023] Figure 6 Schematic diagram of the relative contents of SOD, MDA and GSH in cells treated with sea cucumber postbiotics in an embodiment of the present invention and a comparative example. DETAILED DESCRIPTION

[0024] Example 1: Preparation of sea cucumber enzyme

[0025] Step 1. Pretreatment of sea cucumber: cut the body wall and viscera of fresh sea cucumber into strips with a width of 2 to 5 cm, soak them in deionized water at a mass ratio of 1:10 for 4 h, and repeat the soaking 4 times to obtain a sea cucumber-based raw material with an electrical conductivity of less than 1500 μs / cm;

[0026] Step 2. Prepare sea cucumber enzymatic hydrolysate: add distilled water to the sea cucumber raw material at a mass ratio of 1:10 to homogenize, then add 3% protease (activity unit 100000 U / g) at a mass ratio, control the temperature at 45-55°C, perform enzymatic hydrolysis with magnetic stirring for 3 h, filter and remove impurities, and sterilize at 115°C for 30 min to obtain sea cucumber enzymatic hydrolysate;

[0027] Step 3. Prepare the secondary seed culture of Cordyceps militaris: Take the slant-preserved mother culture of Cordyceps militaris (the strain is commercially available) covered with mycelium, inoculate the slant into liquid potato culture medium, and culture at 18-22°C and 150 r / min for 5 days to form a large number of dense mycelium balls to obtain the primary seed culture of Cordyceps militaris; then transfer the primary seed culture of Cordyceps militaris to fresh liquid potato culture medium at a ratio of 5%, and culture until the cell density is ≥10 9 spores / mL, and obtain the secondary seed culture solution of Cordyceps militaris;

[0028] Step 4. Prepare sea cucumber fermentation liquid: add the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate, and ferment for 4 days with aeration and stirring at 20-24° C. to obtain sea cucumber fermentation liquid, wherein the mass ratio of the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate is 10%;

[0029] Step 5. Prepare sea cucumber enzyme: freeze-dry the sea cucumber fermentation liquid into powder to obtain sea cucumber enzyme.

[0030] Example 2: Preparation of sea cucumber postbiotics

[0031] Step 1. Pretreatment of sea cucumber: cut the thawed sea cucumber body wall and viscera into strips with a width of 2 to 5 cm, soak them in deionized water at a mass ratio of 1:10 for 8 h, and repeat the soaking three times to obtain a sea cucumber-based raw material with an electrical conductivity of less than 1500 μs / cm;

[0032] Step 2. Prepare sea cucumber hydrolysate: add distilled water to the sea cucumber raw material at a mass ratio of 1:8 to homogenize, then add 1% protease (activity unit 100000 U / g) at a mass ratio, control the temperature at 45-55°C, perform enzymolysis with magnetic stirring for 2 h, filter and remove impurities, and sterilize at 115°C for 30 min to obtain sea cucumber hydrolysate;

[0033] Step 3. Prepare the secondary seed culture of Cordyceps militaris: Take the slant-preserved mother culture of Cordyceps militaris (the strain is commercially available) covered with mycelium, inoculate the slant into liquid potato culture medium, and culture at 18-22°C and 130 r / min for 7 days to form a large number of dense mycelium balls to obtain the primary seed culture of Cordyceps militaris; then transfer the primary seed culture of Cordyceps militaris to fresh liquid potato culture medium at a ratio of 3% and culture until the cell density is ≥10 9 spores / mL, and obtain the secondary seed culture solution of Cordyceps militaris;

[0034] Step 4. Prepare sea cucumber fermentation liquid: add the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate, and ferment with aeration and stirring at 20-24° C. for 7 days to obtain the sea cucumber fermentation liquid, wherein the mass ratio of the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate is 2%;

[0035] Step 5. Preparation of sea cucumber postbiotics: sterilize the sea cucumber fermentation liquid at 115°C for 30 min, centrifuge at 10,000 r / min for 20 min, and freeze-dry the supernatant into powder to obtain sea cucumber postbiotics.

[0036] experiment:

[0037] 1. Experiment on the performance of sea cucumber enzyme

[0038] The various experiments of the present invention are divided into multiple treatment groups, and the specific groups and corresponding reagents in the experiments are shown in Table 1:

[0039] Table 1

[0040]

[0041] Experiment 1: Analysis of nutritional components in groups S1 and D1~D3

[0042] The nutritional components of S1 and D1~D3 groups were tested, including the determination of protein and polypeptide content by Kjeldahl nitrogen determination method, the determination of crude fat content by acid hydrolysis method, and the determination of total sugar, sea cucumber saponin, and cordycepin content by high performance liquid chromatography. The results are shown in Table 2.

[0043] Table 2

[0044]

[0045] The results are shown in Table 2. The total sugar, crude fat and sea cucumber saponin nutrients of the S1 group were significantly higher than those of the other groups. At the same time, a higher content of cordycepin was detected, indicating that the sea cucumber enzyme prepared in the embodiment of the present invention can effectively convert protein source nutrients and generate a variety of active ingredients compared with other groups, thereby improving the efficacy of the enzyme.

[0046] Experiment 2: Fibrinolytic activity of sea cucumber enzyme

[0047] The agarose-fibrin plate method is a standard method for determining the activity of thrombolytic enzymes such as urokinase and lumbrokinase. It is also one of the earliest methods used to determine the activity of nattokinase thrombolytic enzymes. It is simple and intuitive. This experiment uses the agarose-fibrin plate method to determine the fibrinolytic activity of each group. The specific determination method is as follows:

[0048] 0.2 mol / L pH7.4 phosphate buffer was mixed with agar powder heated to boiling to prepare an agar solution with a mass concentration of 1%, which was placed in a 55℃ constant temperature water bath to cool down; phosphate buffer was used to dissolve fibrin powder to prepare a fibrin stock solution with a mass concentration of 0.3%; 5 μL of thrombin was dissolved in a small amount of physiological saline to prepare a thrombin solution; the three solutions prepared above were quickly mixed and shaken at 55℃, and then poured into sterilized plates respectively, and cooled and solidified into milky white translucent agarose-fibrin plates, which were kept at a constant temperature of 4℃ for use. In the clean bench, 1000 IU / mL urokinase solution (standard), 10 μL of corresponding reagents of different groups of S1, D1~D3 were taken with a microinjector and spotted on a fresh agarose-fibrin plate, and incubated at 37℃ for 18 h to observe and measure the size of the fibrinolytic area.

[0049] The results are as follows Figure 1As shown, the fibrinolytic area of ​​the S1 group is significantly larger than that of the other test groups, indicating that the sea cucumber enzyme prepared in the embodiment of the present invention can effectively improve the fibrinolytic activity compared with other groups.

[0050] Experiment 3: Antioxidant activity of sea cucumber enzymes

[0051] Using the in vitro oxidation model of Caco-2 cells induced by H2O2, SOD, CAT and MDA were detected using the kit method to evaluate the antioxidant effect of each group. The specific detection method is as follows:

[0052] Normal Caco-2 cells were inoculated into 6-well plates and cultured in an incubator for 24 h. DMEM culture medium was added to the negative control group (NC), DMEM culture medium was added to the model group (M), and DMEM culture medium containing 0.4 mg / mL of the corresponding reagent was added to the S1, D1~D3 groups. Except for the negative control group (NC), the original culture medium in the wells of the other treatment groups was discarded, PBS was washed twice, and DMEM culture medium containing H2O2 was added for 4 h, then the cells were collected and broken, and the relative contents of SOD, CAT and MDA were detected by ELISA kit.

[0053] The results are as follows Figure 2 As shown, compared with the model control group (M), the relative contents of SOD and CAT in the S1 group were significantly increased, and the relative content of MDA was significantly decreased ( P <0.05), indicating that the antioxidant effect of the sea cucumber enzyme in Example 1 of the present invention was significantly improved compared with that in other groups.

[0054] 2. Experiments on the performance of sea cucumber postbiotics

[0055] The various experiments of the present invention are divided into multiple treatment groups, and the specific groups and corresponding reagents in the experiments are shown in Table 3:

[0056] Table 3

[0057]

[0058] Experiment 1: In vitro anti-inflammatory

[0059] Using lipopolysaccharide (LPS)-induced human keratinocytes (HaCaT) as an in vitro inflammatory cell model, the inflammatory factors (IL-1α, IL-8, PGE-2) secreted in the cell supernatant were detected by ELISA kit to evaluate the anti-inflammatory efficacy of the sample to be tested. The specific experimental method is as follows:

[0060] HaCaT cells were routinely cultured, inoculated into 12-well plates, and then placed in an incubator for 18-24 hours. The culture plates were removed, the original culture medium in the wells was discarded, DMEM culture medium was added to the negative control group (NC), DMEM culture medium containing LPS was added to the model group (M), DMEM culture medium containing LPS and dexamethasone was added to the positive control group (PC), and DMEM culture medium containing LPS and corresponding reagents at a concentration of 0.01 mg / mL was added to the S2 and D1-D3 treatment groups, respectively. Each group had 3 replicates and was cultured for 24±1 hours. The supernatant was collected, and the relative content of inflammatory factors (IL-1α, IL-8, PGE-2) was detected by ELISA kit.

[0061] The results are as follows Figure 3 As shown, the relative contents of the three inflammatory factors in the sea cucumber postbiotic group (S2) of Example 2 of the present invention were significantly reduced compared with those in the model control group (M). P <0.05), which is better than the control group (D1-D3), indicating that the sea cucumber postbiotics in Example 2 of the present invention have better anti-inflammatory effects.

[0062] Experiment 2: In vitro repair evaluation

[0063] Through the human keratinocyte (HaCaT) scratch migration test, observation and analysis were performed at 12 h and 24 h, and the cell migration and proliferation conditions were recorded to evaluate the repair performance of each group of samples. The specific experimental method is as follows:

[0064] HaCaT cells were routinely cultured, cell suspension was prepared, 70 μL was inoculated into each well of a 24-well culture plate with a 2-well insert, and the cells were returned to the incubator for 18-24 h until the cells were confluent. The 2-well insert was removed with tweezers, and a flat cell-free area was formed between the 2 wells. The cell layer was washed 3 times with PBS to remove the detached cells. 1 mL of culture medium containing the samples of each group was added to each well. The blank control group (NC) was added with DMEM culture medium containing 1% fetal bovine serum, the positive control group (PC) was added with DMEM culture medium containing 1% fetal bovine serum and epidermal growth factor EGF, and the S2, D1-D3 treatment groups were added with DMEM culture medium containing 1% fetal bovine serum and the corresponding reagent at a concentration of 0.01 mg / mL. At the same time, images were collected under the microscope, recorded as 0 h, and the collection position was marked. Every 12 h, images were collected at the marked position, recorded as 12 h and 24 h, and the relative area of ​​the cell scratch was calculated.

[0065] The results are as follows Figure 4 As shown, compared with the blank control group (NC), the relative area of ​​cell scratches in the sea cucumber postbiotic group (S2) of Example 2 of the present invention at 12 h and 24 h was significantly reduced ( P<0.05), which is better than the control group (D1-D3 groups), indicating that the sea cucumber postbiotics in Example 2 of the present invention have a better repair-promoting effect.

[0066] Experiment 3: In vitro firming effect

[0067] The human skin fibroblasts were treated with the test samples, and the Procollagen-1 and Elastin contents were detected by the kit method to evaluate the firming performance of the samples. The specific experimental methods are as follows:

[0068] Normal human skin fibroblasts were seeded into 96-well plates and cultured in an incubator for 18-24 h. The cell layer was washed twice with PBS. The negative control group (NC) was added with DMEM culture medium containing 1% fetal bovine serum, the positive control group (PC) was added with DMEM culture medium containing 1% fetal bovine serum and Vc, and the S2, D1-D3 treatment groups were added with DMEM culture medium containing LPS, 2% fetal bovine serum and the corresponding reagent at a concentration of 0.05 mg / mL, respectively. Each group was exposed for 72 h with 3 replicates. The supernatant was collected and the relative contents of Procollagen-1 and Elastin were detected by ELISA kit.

[0069] The results are as follows Figure 5 As shown, compared with the negative control group (NC), the relative contents of Procollagen-1 and Elastin in the sea cucumber postbiotic group (S2) were significantly increased ( P <0.05), and the sea cucumber postbiotics S2 treatment group was better than the control group (D1-D3), indicating that the sea cucumber postbiotics in Example 2 of the present invention have better firming effect.

[0070] Experiment 4: In vitro antioxidant efficacy

[0071] Using the H2O2-induced in vitro oxidation model of human skin fibroblasts, the kit method was used to detect SOD, MDA and GSH to evaluate the antioxidant effect of the samples.

[0072] Normal human skin fibroblasts were inoculated into 6-well plates and cultured in an incubator for 18-24 h. DMEM culture medium was added to the negative control group (NC), DMEM culture medium was added to the model group (M), DMEM culture medium containing Vc was added to the positive control group (PC), and DMEM culture medium containing 0.05 mg / mL of the corresponding reagent was added to the other groups (S2, D1-D3). Except for the negative control group (NC), the original culture medium in the wells of the other treatment groups was discarded, washed once with PBS, and DMEM culture medium containing H2O2 was added for 2 h, then replaced with the corresponding culture medium of each group, and incubated for 48 h. The cells were collected and broken, and the relative contents of SOD, MDA and GSH were detected by ELISA kit.

[0073] The results are as follows Figure 6 As shown, compared with the model control group (M), the relative contents of SOD and GSH in the sea cucumber postbiotic treatment group (S2) were significantly increased, and the relative content of MDA was significantly decreased ( P <0.05), which is better than the effect of the control group (D1-D3), indicating that the sea cucumber postbiotics in Example 2 of the present invention have better antioxidant effect.

[0074] Note: Figure 2 , Figure 3 , Figure 6 In the table, # indicates that the difference is statistically significant compared with the negative control group (P<0.05); * indicates that the difference is statistically significant compared with the model control group ( P <0.05). Figure 4 , Figure 5 In the table, * indicates that the difference is statistically significant compared with the blank (negative) control group ( P <0.05).

Claims

1. A method for preparing sea cucumber enzyme or postbiotics using Cordyceps militaris as fermentation bacteria, characterized in that Follow the steps below: Step 1. Pretreatment of sea cucumber: repeatedly soaking the body wall and / or internal organs of the sea cucumber in deionized water until the conductivity is less than 1500 μs / cm, thereby obtaining a sea cucumber-based raw material; Step 2. preparing a sea cucumber enzymatic hydrolysate: adding distilled water to the sea cucumber raw material to crush it, and then adding protease to perform enzymatic hydrolysis to obtain a sea cucumber enzymatic hydrolysate; Step 3. Prepare the secondary seed culture solution of Cordyceps militaris: transfer the Cordyceps militaris from the slant mother culture to liquid culture to obtain a cell density of ≥10 9 spores / mL of secondary seed culture solution of Cordyceps militaris; Step 4. Prepare sea cucumber fermentation liquid: add the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate, and ferment with aeration and stirring at 20-24° C. for 4-7 days to obtain the sea cucumber fermentation liquid, wherein the mass ratio of the secondary seed culture liquid of Cordyceps militaris to the sea cucumber enzymatic hydrolysate is 2-10%; Step 5. Prepare sea cucumber enzyme and postbiotics: freeze-dry the sea cucumber fermentation liquid into powder to obtain sea cucumber enzyme; or sterilize the sea cucumber fermentation liquid at high temperature, centrifuge and take the supernatant, freeze-dry it into powder to obtain sea cucumber postbiotics; The step 1 is to cut the sea cucumber body wall and / or viscera into strips with a width of 2 to 5 cm, soak them in deionized water at a mass ratio of 1:10 for 4 to 8 hours, and repeatedly soak them 3 to 4 times to obtain a sea cucumber-based raw material; The step 2 is to add distilled water to homogenize the sea cucumber raw material at a mass ratio of 1:8-10, then add 1-3% of protease by mass, control the temperature at 45-55°C, perform magnetic stirring for 2-3 hours of enzymolysis, filter and remove impurities, and sterilize at 115°C for 30 minutes to obtain a sea cucumber enzymolysis solution; The step 3 is to take the slant surface of Cordyceps militaris covered with hyphae and store the mother culture of the slant, inoculate the slug into a liquid potato culture medium, and culture the slug in a shaking flask at 130-150 r / min for 5-7 days at 18-22° C. to obtain a first-level seed culture solution of Cordyceps militaris; then transfer the first-level seed culture solution of Cordyceps militaris to a fresh liquid potato culture medium at a ratio of 3-5%, and culture until the cell density is ≥10 9 spores / mL, and the secondary seed culture solution of Cordyceps militaris was obtained.

2. The method for preparing sea cucumber enzyme or postbiotics using Cordyceps militaris as fermentation bacteria according to claim 1, characterized in that The step 5 of sterilizing the sea cucumber fermented liquid at high temperature and then centrifuging to obtain the supernatant is to sterilize the sea cucumber fermented liquid at 115° C. for 30 min, centrifuge at 10,000 r / min for 20 min, and obtain the supernatant.

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