Lactobacillus mucilaginosus for high-yield production of protease and application of lactobacillus mucilaginosus in preparation of sea cucumber peptide

Abstract

The invention provides fermented lactobacillus mucus with high protease yield and application of the fermented lactobacillus mucus in preparation of sea cucumber peptides, the fermented lactobacillus mucus is numbered as CL-6 and preserved in China Center for Type Culture Collection on November 15, 2024, called CCTCC for short, and the address is Wuhan University, Wuchang District, Wuhan City, Hubei Province; the postal code is 430072, the preservation number is CCTCC No: M 20242563, and the Latin literature name is Limosilicobacterium fermentum CL-6, the preservation number is CCTCC No: M 20242563, the preservation number is CCTCC No: M 20242563, and the Latin literature name is CCTCC No: M 20242563. The sea cucumber peptide with excellent functions can be obtained by applying extracellular protease or mixed crude enzyme of intracellular protease and extracellular protease of the strain CL-6 to enzymolysis of Illicium verum; the preparation process of the sea cucumber peptide has the advantages of simplicity in operation, greenness and no pollution, and has relatively high popularization and application values.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a fermented mucus lactobacillus strain with high protease production and application thereof in preparing sea cucumber peptides. Background Art

[0002] Sea cucumber is a material that can be used as both medicine and food. It is rich in active substances such as protein, polysaccharides, saponins, etc., which make it have the effects of lowering blood pressure, lowering blood lipids, lowering blood sugar, preventing cardiovascular and cerebrovascular diseases, improving immunity, anti-tumor, and delaying aging. Sea cucumber peptides are peptide products with different molecular weights obtained after processing sea cucumber proteins. Compared with sea cucumber proteins, they are more conducive to human absorption and utilization, so they have broad application prospects in the fields of functional foods and functional cosmetics.

[0003] There are many types of sea cucumbers, and the protein content, amino acid content, etc. in various sea cucumbers also vary greatly. For example, the protein content of Icelandic red sea cucumber is 80-83% (dry weight, w / w), the protein content of rough sea cucumber is 75-78% (dry weight, w / w), the protein content of eggplant sea cucumber is 75-79% (dry weight, w / w), and the protein content of sea cucumber is 75-78% (dry weight, w / w); the essential amino acid content in Icelandic red sea cucumber is 23-25% (dry weight, w / w), the essential amino acid content in rough sea cucumber is 16-17% (dry weight, w / w), the essential amino acid content in eggplant sea cucumber is 14-15% (dry weight, w / w), and the essential amino acid content in sea cucumber is 18-19% (dry weight, w / w); comparison shows that Icelandic red sea cucumber is rich in nutrients such as protein and amino acids, and is of great development value.

[0004] In the application document with application number CN202410475512.7 and subject name "A method for preparing sea cucumber peptide", sea cucumber is first fermented with Lactobacillus casei and Bifidobacterium, and then the fermentation product is ground and mixed with a composite enzymatic hydrolysis solution of papain and bromelain for enzymolysis to obtain sea cucumber peptide; in this method, the Bifidobacterium used is a strict anaerobic bacteria, and the Lactobacillus casei is a facultative anaerobic bacteria, and co-cultivation is difficult, which makes the method difficult to promote and apply; in addition, in this method, acetaldehyde with a concentration of 1% is used as an enzyme inactivator, and its residue is likely to cause product quality and safety problems.

[0005] In the patent document with application number CN201810644150.4 and subject name "Sea Cucumber Peptide and Enzymatic Extraction Process thereof", sea cucumber peptide is prepared by homogenization, washing, alkali washing, enzymatic hydrolysis, decolorization and deodorization, ultrafiltration and freeze-drying process steps. In this method, resin is used for decolorization and deodorization. The optimal method has a nitrogen recovery rate of 93.49%, a decolorization rate of 76.32% and a deodorization rate of 67.16%.

[0006] It can be seen that there is currently a relatively mature process for the preparation of sea cucumber peptides. However, if the protein is over-hydrolyzed during the conversion into peptides, bitterness and other phenomena may occur, and the molecular weight range of the obtained product is difficult to control; for example, in the application document with application number CN202010595805.0 and subject name "A Method for Preparing Sea Cucumber Peptides", it is disclosed that the use of exo-aminopeptidase at an amount of 0.3-0.5% of the mass of fresh sea cucumber can effectively remove bitterness, and when the amount is less than 0.3%, bitterness still remains. When the amount is greater than 0.5%, excessive hydrolysis will occur, which will increase the free amino acids in the product, which will not only affect the taste of the product, but also weaken the biological activity of the product.

[0007] It can be seen that in the process of hydrolyzing sea cucumber protein into sea cucumber peptides, not only the enzymatic efficiency but also the degree of protein hydrolysis should be considered; currently, composite proteases are usually used to hydrolyze proteins, and the composition of the composite enzyme, hydrolysis conditions, and application conditions of each enzyme will affect the enzymatic efficiency and degree of hydrolysis, resulting in the protein hydrolysis process being difficult to control, which in turn is not conducive to the production of sea cucumber peptide products with stable quality. Summary of the invention

[0008] In view of this, the present invention provides a fermented mucus lactobacillus with high protease production and its application in preparing sea cucumber peptides; the fermented mucus lactobacillus, numbered CL-6, was deposited in the China Center for Type Culture Collection, referred to as CCTCC, on November 15, 2024, address: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072, the deposit number is CCTCC No: M 20242563, the Latin name is Limosilactobacillus fermentum CL-6; applying the extracellular protease of the strain CL-6 or a mixed crude enzyme solution of intracellular and extracellular proteases in the enzymatic hydrolysis of Icelandic red sea cucumber can obtain sea cucumber peptides with excellent functions; the preparation process of the sea cucumber peptides has the advantages of simple operation, green and pollution-free, and has strong promotion and application value.

[0009] The technical solution of the present invention is as follows: A strain of Lactobacillus mucilaginosus with high protease production, numbered CL-6, was deposited in the China Center for Type Culture Collection, referred to as CCTCC, on November 15, 2024, with an address of: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072, with a deposit number of CCTCC No: M 20242563, and a Latin name of Limosilactobacillus leaven CL-6; the enzyme activity of the fermentation broth of the strain CL-6 after fermentation is as high as 100.1 U / ml; after the fermentation broth is crushed, the enzyme activity of the crude protease solution obtained is as high as 128.3 U / ml; after concentration by membrane ultrafiltration, the enzyme activity of the protease concentrate is as high as 2587.6 U / ml.

[0010] Preferably, the strain CL-6 is screened from Icelandic red sea cucumber, which is imported from Iceland (fishing vessel name number: Klettur IS-808), and the seller is Yantai Yedao Ocean Food Co., Ltd.

[0011] The application of the fermented mucus lactobacillus CL-6 in the preparation of sea cucumber peptides.

[0012] Preferably, in the above application, the fermentation broth, crude protease solution or protease concentrate prepared by strain CL-6 is used in the preparation of sea cucumber peptides; the fermentation broth, crude protease solution or protease concentrate is used to enzymolyze sea cucumber protein, which can prevent excessive hydrolysis of protein while ensuring high enzymolysis efficiency, improve the controllability of the degree of enzymolysis of sea cucumber protein, and the obtained sea cucumber peptide has high physiological activity.

[0013] Preferably, the fermentation broth of strain CL-6 is prepared as follows: S1, preparation of seed solution Take the strain CL-6 stored on the plate, inoculate it into the liquid seed culture medium, and culture it statically at 35±0.5℃ for 48-52 hours to obtain the seed liquid; S2, fermentation The seed liquid is inoculated into a liquid fermentation medium at an inoculation rate of 3-7% (v / v), and cultured at 35±0.5°C for 24-60 h with a stirring speed of 20-50 rpm to obtain a fermentation broth containing a highly active protease.

[0014] Preferably, in step S1, the formula of the seed culture medium is: 10 g peptone, 5 g beef extract powder, 5 g yeast extract powder, 2 g dipotassium hydrogen phosphate, 2 g ammonium citrate, 5 g sodium acetate, 20 g glucose, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, 1 g Tween 80, 1000 ml water, sterilized at 121°C for 30 min and then cooled to room temperature for use.

[0015] Preferably, in step S2, the formula of the fermentation medium is 15 kg of peptone, 2 kg of dipotassium hydrogen phosphate, 2 kg of ammonium citrate, 5 kg of sodium acetate, 20 kg of glucose, 0.5 kg of magnesium sulfate, 0.25 kg of manganese sulfate, and 1 kg of Tween 80. Water is added to 1000 L, sterilized at 121°C for 30 min, and then cooled to room temperature for use.

[0016] Preferably, the crude protease solution of strain CL-6 is prepared by adjusting the pH of the fermentation broth to 6-8, adding lysozyme with an enzyme activity of 2 million U / g to a final concentration of lysozyme of 1 g / L, controlling the temperature to 35-45°C, and processing for 0.5-5 h to obtain the crude protease solution.

[0017] Preferably, the strain CL-6 protease concentrate is prepared by centrifuging the crude protease solution, collecting the filtrate, and concentrating the filtrate by membrane ultrafiltration to obtain the protease concentrate.

[0018] Preferably, in the membrane ultrafiltration concentration, an ultrafiltration membrane with a molecular weight cutoff of 1000-5000 Da is used.

[0019] Preferably, the protease concentrate is used to enzymolyze the sea cucumber protein, and the process is as follows: SS1, Preparation of sea cucumber homogenate Preparation of sea cucumber homogenate: remove the internal organs of fresh Icelandic red sea cucumbers, clean them, and grind them in a blender to obtain sea cucumber homogenate; SS2, preparation of sea cucumber peptides Mixing sea cucumber homogenate with water in a mass ratio of 1:3-5 to obtain a slurry; Adding protease concentrate to the slurry, controlling the enzymatic hydrolysis temperature to 40-50°C and the enzymatic hydrolysis time to 2-8 h, to obtain an enzymatic hydrolyzate; The enzymatic hydrolysis solution was heated to 100°C and treated for 15-20 min to obtain the sea cucumber peptide enzymatic hydrolysis product.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1. The present invention provides a strain of fermented mucus lactobacillus, numbered CL-6, which was deposited in the China Center for Type Culture Collection, referred to as CCTCC, on November 15, 2024, with an address of: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072, with a deposit number of CCTCC No: M 20242563, and a Latin name of Limosilactobacillus leaven CL-6; This strain can produce protease, and the enzyme activity of the fermentation broth after fermentation is as high as 100.1 U / ml; after the fermentation broth is crushed, the enzyme activity of the obtained crude protease liquid is as high as 128.3 U / ml; after membrane ultrafiltration and concentration, the activity of the protease concentrate can reach 2587.6U / ml.

[0021] 2. When the fermentation liquid, crude enzyme liquid or concentrated liquid containing protease obtained by fermenting the strain CL-6 provided by the present invention is used in the enzymatic hydrolysis of Icelandic red sea cucumber, among the obtained sea cucumber peptides, the sea cucumber peptides with a molecular weight of 200~1000Da account for the highest proportion of 85.3%; when the method is used for enzymatic hydrolysis of sea cucumber protein, the process is easy to control, and while ensuring the enzymatic hydrolysis efficiency, the molecular weight of the sea cucumber peptide obtained by hydrolysis can be relatively stably controlled between 200~1000Da, so that the sea cucumber peptide has higher physiological activity and the quality of the obtained sea cucumber peptide product is guaranteed to be stable.

[0022] 3. The enzymatic hydrolysis of Icelandic red sea cucumber using the strain CL-6 protease prepared by the present invention has mild conditions and low cost, and the enzymatic hydrolysis product is rich in active ingredients such as probiotic metabolites such as polysaccharides, short-chain fatty acids, rare sea cucumber saponins, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, for ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0024] Figure 1 The colony morphology of strain CL-6. DETAILED DESCRIPTION

[0025] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0026] Example 1 Screening and identification of strains 1. Strain screening (1) Preparation of screening medium Screening medium: 2 g potassium hydrogen phosphate, 2 g ammonium citrate, 5 g sodium acetate, 20 g glucose, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, 1 g Tween 80, 20 g agar, 10 g calcium carbonate, x-gal (5-bromo-4-chloro-3-indole-β-D-galactoside, 60 mg / ml), 1000 ml water, mix well, sterilize at 121°C for 30 min, and then cool to room temperature before use; (2) Cultivation Sea cucumber homogenate: remove the internal organs of fresh Icelandic red sea cucumbers, clean them, and grind them in a blender to obtain sea cucumber homogenate; Icelandic red ginseng is produced in the deep sea waters of the Republic of Iceland near the Arctic Circle, where the water is pure and cold. It is rich in resources and has a protein content of 80.5% (dry weight, w / w). Icelandic red ginseng is imported from Iceland (fishing vessel name number: KletturIS-808) and the seller is Yantai Yedao Ocean Food Co., Ltd. 100 g of sea cucumber homogenate was spread on the screening medium and cultured at 35°C for 24 h; Select a single colony and continue to streak culture on the screening medium to purify it to obtain a single strain. Figure 1 ; The strain was numbered CL-6 and then stored in glycerol tubes.

[0027] 2. Identification The strain CL-6 preserved in the glycerol tube of Example 1 was selected and identified. The 16S rDNA nucleotide sequence of the strain CL-6 is shown in the sequence table SEQ ID No. 1, as follows: SEQ ID No.1 The 16S rDNA nucleotides of strain CL-6 were compared by BLAST tool through sequence homology analysis of National Center for Biotechnology Information (NCBI). The comparison results showed that strain CL-6 belonged to the genus Lactobacillus. Figure 1 Morphological analysis of strain CL-6 showed that the single colony of the strain was light blue, smooth, round, and surrounded by a white transparent circle; The physiological and biochemical characteristics of the strain CL-6 were tested, and the results are shown in Table 1; Table 1 Physiological and biochemical characteristics of strain CL-6

[0028] Combination Figure 1 As shown in Table 1, the strain CL-6 is a fermentative Lactobacillus mucosa in the genus Lactobacillus mucosa; the strain CL-6 was deposited in the China Center for Type Culture Collection, referred to as CCTCC, on November 15, 2024, with an address of: Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University; Postal Code: 430072, the deposit number is CCTCC No: M 20242563, and the Latin name is Limosilactobacillus fermentum CL-6.

[0029] In the present invention, the protease activity detection is carried out with reference to the method specified in GB / T 23527.1-2023 (Enzyme Preparation Quality Requirements Part 1: Protease Preparation Appendix A Folin Method).

[0030] Example 2 Preparation of crude protease enzyme solution using strain CL-6 fermentation The crude protease solution was prepared using the strain CL-6 preserved in Example 1, and the process was as follows: S1, preparation of seed solution The formula of seed culture medium is: peptone 10 g, beef extract powder 5 g, yeast extract powder 5 g, dipotassium hydrogen phosphate 2 g, ammonium citrate 2 g, sodium acetate 5 g, glucose 20 g, magnesium sulfate 0.5 g, manganese sulfate 0.25 g, Tween 80 1 g, water 1000 ml, sterilized at 121 ° C for 30 min and cooled to room temperature before use; Take strain CL-6, inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of liquid seed culture medium, and culture it at 35°C for 48 h to obtain seed liquid; The seed solution was expanded at a ratio of 1:10 to prepare 50 L of seed solution for fermentation, which was set aside; S2, fermentation to prepare fermentation broth The formula of the fermentation medium is 15 kg of peptone, 2 kg of dipotassium hydrogen phosphate, 2 kg of ammonium citrate, 5 kg of sodium acetate, 20 kg of glucose, 0.5 kg of magnesium sulfate, 0.25 kg of manganese sulfate, 1 kg of Tween 80, add water to 1000 L, sterilize at 121°C for 30 min, and then cool to room temperature for use; According to the inoculation amount of 5% (v / v), 50 L of seed liquid was inoculated into 1000 L of liquid fermentation medium, and cultured at 35 °C for 48 h with a stirring speed of 40 rpm to obtain a fermentation broth with an enzyme activity of 100.1 U / ml. S3, preparation of crude protease solution The pH of the fermentation liquid was adjusted to 7.0, and lysozyme with an enzyme activity of 2 million u / g was added to a final concentration of 1 g / L of lysozyme. The temperature was controlled at 45°C and the treatment time was 4.5 h to obtain a crude protease solution. The activity of the protease in the crude enzyme solution was 128.3 U / ml. S4. Preparation of protease concentrate After the crude protease solution is centrifuged, the filtrate is collected and concentrated by membrane ultrafiltration to obtain a protease concentrate; in the membrane ultrafiltration concentration, an ultrafiltration membrane with a molecular weight cutoff of 3000 Da is used; the enzyme activity of the protease concentrate is 2587.6 U / ml.

[0031] The activity and optimal catalytic conditions of the protease in the fermentation broth of Example 2 were tested, and the results are shown in Tables 2-3, as follows: Table 2 pH stability of strain CL-6 protease

[0032] Table 3 Temperature stability of strain CL-6 protease

[0033] Combining Tables 2 and 3, it can be seen that the protease of strain CL-6 has high stability in the range of pH 4-8 and temperature 30-45°C, and the highest enzyme activity is 100.1 U / ml.

[0034] Detection 1. In vitro antioxidant capacity test (1) O 2-· Clearance rate: Accurately pipette 4.5 ml of 50 mmol / L pH 8.2 Tris-HCl buffer solution into a test tube, keep it in a 25℃ water bath for 20 min, add 0.4 ml of sample solution and 0.08 ml of 25 mmol / L pyrogallol (prepared with 10 mmol / L HCl), seal and mix well; then measure the absorbance at a wavelength of 325 nm, record the absorbance every 0.5 min, and record for a total of 4 min; 10 mmol / L HCl replaces the pyrogallol solution, and distilled water replaces the sample solution as a self-oxidation tube (control group); O 2- The oxidation rate is calculated according to formula 1, as follows: Formula 1, Oxidation rate / ( ΔA / min)=( D 1- D 2) / T O 2- Oxidation rate / %=[( V 1- V 2) / V 1]×100 In the formula, D 1 is the absorbance value recorded for 4 min of the sample; D 2 is the absorbance value recorded at 0 min of the sample; T The absorbance value was recorded every 0.5 min for a total of 4 min; V 1 is the oxidation rate of the self-oxidation tube, ΔA / min; V 2 is the oxidation rate of the sample tube, ΔA / min; (2) OH scavenging rate: Accurately pipette 1 ml of the sample solution after different drying treatments, add 0.5 ml of 20 mmol / L hydrogen peroxide, 1.0 ml of 2.5 mmol / L o-phenanthroline, and 1.0 ml of 2.5 mmol / L ferrous sulfate in sequence, adjust the pH to 7.0 with phosphate buffer, let stand at room temperature (25°C) for 1 h, measure the absorbance at 536 nm, and calculate the OH scavenging rate according to the following formula: Formula 2: OH removal rate / %=[( A 2- A 0) / ( A 1- A 0)]×100 In the formula, A 2 is the absorbance value of the sample group; A 1 is the absorbance value of distilled water instead of hydrogen peroxide solution (control group); A 0 is the absorbance value when distilled water replaces the test solution (control group); (3) Antioxidant enzyme detection: Catalase (CAT) and superoxide dismutase (SOD) detection kits from Nanjing Jiancheng Bioengineering Institute were used according to the instructions.

[0035] 2. Immune function test Experimental animals: BALB / c adult mice, weighing 18-22 g, 10 mice per group; The experimental group used the sea cucumber peptide prepared in the example, and in addition to daily basic feed and water, was gavaged once a day according to the weight of the mice according to the dosage design in Table 4 for a total of 28 days; The control group was gavaged with normal saline in addition to the basic feed and water every day for 28 days; Control group 1, in addition to daily basic feed and water, was gavaged once a day according to the weight of mice according to the dosage design in Table 4 for a total of 28 days; Control group 2, in addition to daily basic feed and water, was gavaged once a day according to the weight of mice according to the dosage design in Table 4 for a total of 28 days; Table 4 Group design of the test for enhancing immunity activity

[0036] After 28 days, the mice were killed, and the splenocyte proliferation and the expression of interleukin IL-4 and TNF-α were detected as follows: 1) Determination of spleen cell proliferation ability: The spleen was removed aseptically and placed in a small dish containing sterile Hank's solution to prepare a cell suspension, which was filtered through a 200-mesh sieve; the cells were washed twice with Hank's solution, each time centrifuged at 1000 r / min for 10 min, and the cells were collected; The cells were suspended in 1 ml of complete culture medium, the number of live cells was counted, and the cell concentration was adjusted to 3 × 10 6 / ml; then the cell suspension was added to two wells of a 24-well culture plate, 1 ml per well, 75 µl ConA solution (equivalent to 7.5 µg / ml) was added to one well, and the other well was left untreated as a control, and the culture plate was incubated at 5% CO2 and 37°C for 72 h; 4 h before the end of the culture (i.e., 68 h), 0.7 ml of the supernatant was gently aspirated from each well, RPMI 1640 culture medium without calf serum was added, and 50 µl / well of MTT (5 mg / ml) was added, and the culture was continued for 4 h; after the end of the culture, 1 ml of acidic isopropanol was added to each well, and the solution was evenly blown to completely dissolve the purple crystals; Then dispense into 96-well culture plates, with 3 parallel wells in each well, and use an ELISA reader to measure the optical density at a wavelength of 570 nm; calculate the proliferation capacity of spleen cells according to the following formula: Formula three, Proliferation capacity of spleen cells = OD Con A -OD 无 In the formula, OD Con A is the OD value of the sample with ConA solution added; OD 无 is the OD value of the sample without adding ConA solution.

[0037] 2) Detection of interleukin IL-4 expression ability: The interleukin 4 (IL-4) and tumor necrosis factor α (TNF-α) ELISA detection kits of Jiangsu Jingmei Biotechnology Co., Ltd. were used according to the instructions.

[0038] 3. Fatigue relief function test Experimental animals: BALB / c adult mice, weighing 18-22 g, 10 mice per group; The experimental group used the sea cucumber peptide prepared in the example; in addition to daily administration of basic feed and water, the mice were gavaged once a day according to the weight of the mice according to the dosage design in Table 5 for a total of 28 days; The control group was gavaged with normal saline in addition to the basic feed and water every day for 28 days; Control group 1, in addition to daily basic feed and water, was gavaged once a day according to the weight of mice according to the dosage design in Table 5 for a total of 28 days; Control group 2, in addition to daily basic feed and water, was gavaged once a day according to the weight of mice according to the dosage design in Table 5 for a total of 28 days; Table 5 Group design of the activity experiment for relieving physical fatigue

[0039] experiment: 1) Weighted swimming test 30 min after the last gavage, mice with a lead sheet of 5% of their body weight at the base of their tails were placed in a swimming bucket for swimming; the water depth was not less than 30 cm and the water temperature was 25±1.0℃. The time from the start of swimming to the death of the mice was recorded, i.e., the weighted swimming time of the mice.

[0040] 2) Liver glycogen determination 30 min after the last gavage, the animals were killed and fresh liver samples were taken. The liver samples were rinsed with physiological saline, dried with filter paper and weighed. The liver samples were fully homogenized with a handheld homogenizer and centrifuged to obtain the supernatant, which was then assayed using the liver glycogen assay kit (Nanjing Jiancheng Bioengineering Institute).

[0041] Example 3 Preparation of Sea Cucumber Peptide 1 The protease concentrate prepared in Example 2 (with an activity of 2587.6 U / ml) was used to enzymolyze the red ginseng, and the process was as follows: SS1, Preparation of sea cucumber homogenate Sea cucumber homogenate, prepared in the same manner as in Example 1, using Icelandic red sea cucumber having a protein content of 80.5% (dry weight, w / w); SS2, preparation of sea cucumber peptides Taking 100 kg of sea cucumber homogenate, mixing the sea cucumber homogenate with water in a mass ratio of 1:3 to obtain a slurry; Add 100 L of the protease concentrate of Example 2 to the slurry, mix well, control the enzymolysis temperature to 48° C., and the enzymolysis time to 6 h to obtain an enzymolysis solution; The enzymatic hydrolysate was heated to 100°C and treated for 15 min to obtain the enzymatic hydrolysate of sea cucumber peptide; The sea cucumber peptide hydrolysate was centrifuged and the solid was spray dried to obtain sea cucumber peptide powder with a yield of 33.9% (by dry weight), and sea cucumber peptides with a molecular weight of 200-1000Da accounted for 85.3%; the sea cucumber peptide powder was white and odorless, and the aqueous solution was clear and transparent. The functional test results are shown in Table 6, as follows: Table 6 Results of sea cucumber peptide function test

[0042] Example 4 Preparation of Sea Cucumber Peptide 2 The protease concentrate was prepared by fermentation using the strain CL-6 preserved in Example 1, and the process was as follows: S1, preparation of seed solution Same as Example 2; S2, fermentation The formula of the fermentation medium is the same as that of Example 2; According to the inoculation amount of 5% (v / v), 50 L of seed liquid was inoculated into 1000 L of liquid fermentation medium, and cultured at 35°C for 24 h with a stirring speed of 20 rpm to obtain a protease-containing fermentation broth; the enzyme activity of the fermentation broth was 20.6 U / ml; S3, preparation of crude protease solution The pH of the fermentation liquid was adjusted to 6.0, and lysozyme with an enzyme activity of 2 million u / g was added to a final concentration of 1 g / L of lysozyme. The temperature was controlled at 35°C and the treatment time was 0.5 h to obtain a crude protease solution with an enzyme activity of 25.8 U / ml. S4. Preparation of protease concentrate After the crude protease solution is centrifuged, the filtrate is collected, and the filtrate is concentrated by membrane ultrafiltration to obtain a protease concentrate; in the membrane ultrafiltration concentration, an ultrafiltration membrane with a molecular weight cutoff of 3000 Da is used; the enzyme activity of the protease concentrate is 589.3 U / ml; The above-mentioned protease concentrate was used to enzymatically hydrolyze the red ginseng, and the process was as follows: SS1, preparing sea cucumber homogenate, same as in Example 3; SS2, preparation of sea cucumber peptides Taking 100 kg of sea cucumber homogenate, mixing the sea cucumber homogenate with water in a mass ratio of 1:3 to obtain a slurry; Add 100 L of protease concentrate to the slurry, mix well, control the enzymatic hydrolysis temperature to 40°C, and the enzymatic hydrolysis time to 2 h to obtain an enzymatic hydrolyzate; The enzymatic hydrolysate was heated to 100°C and treated for 20 min to obtain the enzymatic hydrolysate of sea cucumber peptide; The sea cucumber peptide hydrolysate was centrifuged and the solid was spray dried to obtain sea cucumber peptide powder with a yield of 6.8% (by dry weight), and sea cucumber peptides with a molecular weight of 200-1000 Da accounted for 55.6%; the sea cucumber peptide powder was slightly yellow in color and had no fishy smell, and the aqueous solution was clear and transparent. The functional test results are shown in Table 7, as follows: Table 7 Results of sea cucumber peptide function test

[0043] Example 5 Preparation of Sea Cucumber Peptide 3 The protease concentrate was prepared by fermentation using the strain CL-6 preserved in Example 1, and the process was as follows: S1, preparation of seed solution Same as Example 2; S2, fermentation The formula of the fermentation medium is the same as that of Example 2; According to the inoculation amount of 5% (v / v), 50 L of seed liquid was inoculated into 1000 L of liquid fermentation medium, and cultured at 35°C for 36 h with a stirring speed of 50 rpm to obtain a protease-containing fermentation broth; the enzyme activity of the fermentation broth was 98.1 U / ml; S3, preparation of crude protease solution The pH of the fermentation liquid was adjusted to 6.0, and lysozyme with an enzyme activity of 2 million u / g was added to a final concentration of 1 g / L of lysozyme. The temperature was controlled at 40°C and the treatment time was 2 h to obtain a crude protease solution with an enzyme activity of 108.6 U / ml. S4. Preparation of protease concentrate After the crude protease solution is centrifuged, the filtrate is collected, and the filtrate is concentrated by membrane ultrafiltration to obtain a protease concentrate; in the membrane ultrafiltration concentration, an ultrafiltration membrane with a molecular weight cutoff of 3000 Da is used; the enzyme activity of the protease concentrate is 1679.1U / ml; The above-mentioned protease concentrate was used to enzymatically hydrolyze the red ginseng, and the process was as follows: SS1, preparing sea cucumber homogenate, same as in Example 3; SS2, preparation of sea cucumber peptides Taking 100 kg of sea cucumber homogenate, mixing the sea cucumber homogenate with water in a mass ratio of 1:3 to obtain a slurry; Add 100 L of protease concentrate to the slurry, mix well, control the enzymatic hydrolysis temperature to 50°C, and the enzymatic hydrolysis time to 8 h to obtain an enzymatic hydrolyzate; The enzymatic hydrolysate was heated to 100°C and treated for 15 min to obtain the enzymatic hydrolysate of sea cucumber peptide; The sea cucumber peptide hydrolysate was centrifuged and the solid was spray dried to obtain sea cucumber peptide powder with a yield of 25.8% (by dry weight). The sea cucumber peptide with a molecular weight of 200-1000Da accounted for the highest proportion of 75.5%; the sea cucumber peptide powder was white and odorless, and the aqueous solution was clear and transparent. The functional test results are shown in Table 8, as follows: Table 8 Sea cucumber peptide function test results

[0044] Example 6 Preparation of Sea Cucumber Peptide 4 The protease concentrate was prepared by fermentation using the strain CL-6 preserved in Example 1, and the process was as follows: S1, preparation of seed solution Same as Example 2; S2, fermentation The formula of the fermentation medium is the same as that of Example 2; According to the inoculation amount of 5% (v / v), 50 L of seed liquid was inoculated into 1000 L of liquid fermentation medium, and cultured at 35°C for 60 h with a stirring speed of 35 rpm to obtain a protease-containing fermentation liquid; the enzyme activity of the protease was 99.6 U / ml; S3, Preparation of Protease Concentrate The pH of the fermentation liquid was adjusted to 8.0, and lysozyme with an enzyme activity of 2 million u / g was added to a final concentration of 1 g / L of lysozyme. The temperature was controlled at 40°C and the treatment time was 3 h to obtain a crude protease solution with an enzyme activity of 117.7 U / ml. S4. Preparation of protease concentrate After the crude protease solution is centrifuged, the filtrate is collected and concentrated by membrane ultrafiltration to obtain a protease concentrate. In the membrane ultrafiltration concentration, an ultrafiltration membrane with a molecular weight cutoff of 3000 Da is used; the enzyme activity of the protease concentrate is 1903.3U / ml; The above-mentioned protease concentrate was used to enzymatically hydrolyze the red ginseng, and the process was as follows: SS1, preparing sea cucumber homogenate, same as in Example 3; SS2, preparation of sea cucumber peptides 100 kg of sea cucumber homogenate was taken, and the sea cucumber homogenate and water were mixed in a mass ratio of 1:3 to obtain a slurry; 100 L of protease concentrate was added to the slurry, mixed well, and the enzymolysis temperature was controlled to be 45°C and the enzymolysis time was 4 h to obtain an enzymolysis solution; The enzymatic hydrolysate was heated to 100°C and treated for 20 min to obtain the enzymatic hydrolysate of sea cucumber peptide; The enzymatic hydrolysis product of sea cucumber peptide was filtered and spray-dried to obtain sea cucumber peptide powder with a yield of 29.5% (by dry weight), and sea cucumber peptides with a molecular weight of 200-1000Da accounted for 80.4%; the sea cucumber peptide powder was white and odorless, and the aqueous solution was clear and transparent. The functional test results are shown in Table 9, as follows: Table 9 Results of sea cucumber peptide function test

[0045] Comparative Example 1 The red ginseng was enzymatically hydrolyzed using flavor protease, and the process was as follows: SS1, preparing sea cucumber homogenate, same as in Example 3; SS2, preparation of sea cucumber peptides 100 kg of sea cucumber homogenate was taken, and the sea cucumber homogenate was mixed with water in a weight ratio of 1:3 to obtain a slurry, the pH of the slurry was adjusted to 7.2, 4000 U / g protein of flavor protease was added, the enzymolysis temperature was 50°C, and the enzymolysis time was 8 h; After the enzymatic hydrolysis was completed, the termination conditions were a temperature of 100°C and a processing time of 15 min, and an enzymatic hydrolysis product containing sea cucumber peptides was obtained.

[0046] The sea cucumber peptide hydrolysate was filtered and spray-dried to obtain sea cucumber peptide powder. The yield was 21.7% (by dry weight), and the sea cucumber peptide with a molecular weight of 200-1000Da accounted for 85.0%; the sea cucumber peptide powder was white and odorless, and the aqueous solution was clear and transparent. The functional test results are shown in Table 10, as follows: Table 10 Results of sea cucumber peptide function test

[0047] Combined with Table 10, it can be seen that compared with the control group, the catalase (CAT) activity of mice in comparative example 1 increased by 5.0%, and the superoxide dismutase (SOD) inhibition rate increased by 9.4%; The sea cucumber peptide obtained in Example 3 increased the catalase (CAT) activity in mice by 10.0% compared with the control group, and increased the superoxide dismutase (SOD) inhibition rate by 18.9% compared with the control group; That is to say, the performance of the sea cucumber peptide in Example 3 is significantly improved compared with the sea cucumber peptide in Comparative Example 1.

[0048] Comparative Example 2 The difference from Example 3 is that the Icelandic red sea cucumber is replaced by sea cucumber, and the rest is the same as Example 3; The obtained sea cucumber peptide hydrolysate has a sea cucumber peptide content of 28.0% (by dry weight), and sea cucumber peptides with a molecular weight of 200-1000 Da account for 80.1%; The enzymatic hydrolysis product of the sea cucumber peptide was filtered and spray-dried, and the obtained sea cucumber peptide powder was white and odorless, and the aqueous solution was clear and transparent. The functional test results are shown in Table 11, as follows: Table 11 Results of sea cucumber peptide function test

[0049] Combined with Table 11, it can be seen that compared with the control group, the catalase (CAT) activity of mice in comparative example 2 increased by 3.0%, and the superoxide dismutase (SOD) inhibition rate increased by 8.9%; The sea cucumber peptide obtained in Example 3 increased the catalase (CAT) activity in mice by 10.0% compared with the control group, and increased the superoxide dismutase (SOD) inhibition rate by 18.9% compared with the control group; That is to say, the performance of the sea cucumber peptide in Example 3 is significantly improved compared with the sea cucumber peptide in Comparative Example 2.

[0050] Although the present invention has been described in detail by reference to the preferred embodiments, the present invention is not limited thereto. Without departing from the spirit and essence of the present invention, a person of ordinary skill in the art may make various equivalent modifications or substitutions to the embodiments of the present invention, and these modifications or substitutions shall be within the scope of the present invention. Any person of ordinary skill in the art may easily think of changes or substitutions within the technical scope disclosed by the present invention, and these shall be within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the claims.

Claims

1. A strain of Lactobacillus mucilaginosus that produces high levels of protease, characterized in that: The strain is numbered CL-6 and was deposited in the China Center for Type Culture Collection, abbreviated as CCTCC, on November 15, 2024. The address is: Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province; Postal Code: 430072, the deposit number is CCTCC No: M 20242563, and the Latin name is Limosilactobacillus fermentum CL-6; the enzyme activity of the fermentation broth of the strain CL-6 after fermentation is as high as 100.1 U / ml; after the fermentation broth is crushed, the enzyme activity of the obtained crude protease liquid is as high as 128.3 U / ml; after concentration by membrane ultrafiltration, the enzyme activity of the protease concentrate is as high as 2587.6 U / ml.

2. The fermented Lactobacillus mucilaginosus with high protease production according to claim 1, characterized in that: The strain CL-6 was screened from Icelandic red sea cucumber, which was imported from Iceland. The fishing vessel name and number was Klettur IS-808, and the seller was Yantai Yedao Ocean Food Co., Ltd.

3. Application of a strain of fermented mucus Lactobacillus with high protease production in the preparation of sea cucumber peptides.

4. The use according to claim 3, characterized in that Application of fermentation broth, crude protease solution or protease concentrate prepared by strain CL-6 in the preparation of sea cucumber peptides.

5. The use according to claim 4, characterized in that The fermentation broth of strain CL-6 was prepared as follows: S1, preparation of seed solution Take the strain CL-6 stored on the plate, inoculate it into the liquid seed culture medium, and culture it statically at 35±0.5℃ for 48-52h to obtain the seed liquid; S2, fermentation The seed liquid is inoculated into a liquid fermentation medium at an inoculation rate of 3-7% (v / v), and cultured at 35±0.5°C for 24-60 h with a stirring speed of 20-50 rpm to obtain a fermentation broth containing a highly active protease.

6. The use according to claim 5, characterized in that In step S1, the formula of the seed culture medium is: 10 g peptone, 5 g beef extract powder, 5 g yeast extract powder, 2 g dipotassium hydrogen phosphate, 2 g ammonium citrate, 5 g sodium acetate, 20 g glucose, 0.5 g magnesium sulfate, 0.25 g manganese sulfate, 1 g Tween 80, 1000 ml water, sterilized at 121°C for 30 min and then cooled to room temperature for use.

7. The use according to claim 4, characterized in that In step S2, the formula of the fermentation medium is 15 kg of peptone, 2 kg of dipotassium hydrogen phosphate, 2 kg of ammonium citrate, 5 kg of sodium acetate, 20 kg of glucose, 0.5 kg of magnesium sulfate, 0.25 kg of manganese sulfate, and 1 kg of Tween 80. Water is added to 1000 L, sterilized at 121°C for 30 min, and then cooled to room temperature for use.

8. The use according to claim 5, characterized in that The preparation process of the crude protease solution of strain CL-6 is as follows: adjusting the pH of the fermentation liquid to 6-8, adding lysozyme with an enzyme activity of 2 million U / g to a final concentration of lysozyme of 1 g / L, controlling the temperature to 35-45°C, and processing time to 0.5-5 h to obtain the crude protease solution.

9. The use according to claim 8, characterized in that The protease concentrate of strain CL-6 is obtained by centrifuging the crude protease solution, collecting the filtrate, and concentrating the filtrate by membrane ultrafiltration to obtain the protease concentrate; In membrane ultrafiltration concentration, an ultrafiltration membrane with a molecular weight cutoff of 1000-5000 Da is used.

10. The use according to claim 9, characterized in that The protease concentrate is used to enzymolyze the sea cucumber protein, and the process is as follows: SS1, Preparation of sea cucumber homogenate Preparation of sea cucumber homogenate: remove the internal organs of fresh Icelandic red sea cucumbers, clean them, and grind them in a blender to obtain sea cucumber homogenate; SS2, preparation of sea cucumber peptides Mixing sea cucumber homogenate with water in a mass ratio of 1:3-5 to obtain a slurry; Adding protease concentrate to the slurry, controlling the enzymatic hydrolysis temperature to 40-50°C and the enzymatic hydrolysis time to 2-8 h, to obtain an enzymatic hydrolyzate; The enzymatic hydrolysis solution was heated to 100°C and treated for 15-20 min to obtain the sea cucumber peptide enzymatic hydrolysis product.

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