Sea cucumber flower enzyme with in-vitro uric acid reducing and blood sugar reducing activity and preparation method of sea cucumber flower enzyme

Abstract

The invention belongs to the technical field of biology, and discloses sea cucumber flower enzyme as well as a preparation method and application thereof. According to the preparation method of the sea cucumber and ova enzyme, the protease is used for carrying out enzymolysis on the sea cucumber and ova raw material so as to realize full release of sea cucumber cell contents; then, high-pressure homogenization treatment is adopted for fully and evenly mixing the enzymolysis product with milk and saccharides, so that emulsification embedding of the milk on fishy smell substances is achieved, and the uptake rate of microorganisms such as lactobacillus bulgaricus and streptococcus thermophilus is effectively increased while the fishy smell is reduced; and finally, the added saccharides are used for optimizing the microbial metabolism condition and better synergic cooperation, so that fishy smell substances are metabolized and converted into fishy smell-free substances, and related biological activity precursor substances are fully decomposed and converted, so that the finally obtained sea cucumber flower enzyme has no obvious fishy smell or even no fishy smell, has potential uric acid and blood sugar reducing activity, and has a good application prospect. Therefore, the method has good market development and application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a sea cucumber flower enzyme having both in vitro uric acid-lowering and blood sugar-lowering activities and a preparation method thereof. Background Art

[0002] Sea cucumber gonads (commonly known as sea cucumber flowers) contain a variety of bioactive substances, such as precious sea urchin purpurone, beta-carotene, astaxanthin, canthaxanthin, zeaxanthin and other gonadal pigments, as well as rich arginine, saponin, sea cucumber lipids, vanadium, selenium, iron, zinc and other nutrients. These bioactive substances have multiple biological activity functions such as anti-oxidation, immune enhancement, hypoglycemia, and uric acid reduction. However, sea cucumber flowers are often directly discarded as scraps or sold as feed during sea cucumber processing, causing serious waste of resources. In addition, sea cucumber flowers are also rich in protein, and biologically active polypeptides can be prepared through certain enzymolysis or biological treatment.

[0003] However, sea cucumber flowers are rich in substances that produce fishy smell precursors, such as basic amino acids and unsaturated fatty acids. During the extraction process of the above-mentioned bioactive substances, the substances that produce fishy smell precursors will also be decomposed, making the resulting product have a heavier fishy smell, which limits its application.

[0004] Therefore, it is of great significance to improve the processing technology of sea cucumber flowers, improve their flavor characteristics, make them acceptable to consumers, and increase the active ingredients in the resulting products, in order to lay a solid foundation for the high-value utilization of sea cucumber by-products and product development. Summary of the invention

[0005] The first purpose of the present invention is to provide a method for preparing sea cucumber flower enzyme. The sea cucumber flower enzyme prepared by the preparation method exhibits obvious xanthine oxidase and α-glucosidase activity inhibition, has potential uric acid and blood sugar lowering activity; and the obtained sea cucumber flower enzyme has no obvious fishy smell, so it has good market development and application prospects.

[0006] The second purpose of the present invention is to provide sea cucumber flower enzyme prepared by the above method.

[0007] The third purpose of the present invention is to use the above-mentioned sea cucumber flower enzyme in the preparation of products for improving uric acid levels and / or blood sugar levels.

[0008] Specifically, the preparation method of sea cucumber flower enzyme provided by the present invention includes: S1, taking sea cucumber flower raw materials and mixing them with protease, and then performing enzymolysis treatment to obtain an enzymolysis product; S2, taking milk, sugar and the enzymolysis product, mixing them, and then successively performing high-pressure homogenization treatment and sterilization treatment to obtain a fermentation raw material liquid; S3, taking fermentation bacteria powder and inoculating it into the fermentation raw material liquid for fermentation treatment to obtain the sea cucumber flower enzyme; wherein the fermentation bacteria powder includes a basic fermentation agent, and the basic fermentation agent includes Lactobacillus bulgaricus and Streptococcus thermophilus.

[0009] Furthermore, in step S1, the protease is selected from one or more of papain, bromelain and trypsin.

[0010] Furthermore, in step S1, the added mass ratio of solid matter and protease in the sea cucumber flower raw material is (1-20):1.

[0011] Furthermore, in step S1, the temperature of the enzymatic hydrolysis treatment is 40-65° C., the pH value is 5.5-6.5, and the time is 1-11 hours.

[0012] Furthermore, in step S2, the milk is selected from one or more of pure cow's milk, buffalo milk, goat's milk and camel's milk.

[0013] Furthermore, in step S2, the sugar is selected from one or more of sucrose, maltose, trehalose and lactose.

[0014] Furthermore, in step S2, the added mass ratio of the milk, sugar and solid matter in the enzymatic hydrolysis product is 100:(1-7):(1-4).

[0015] Furthermore, in step S2, the pressure of the high-pressure homogenization treatment is 80-150 MPa, and the time is 1-10 min.

[0016] Furthermore, in step S2, the sterilization treatment comprises: taking the mixture of milk, sugar and enzymatic hydrolysis product treated by the high-pressure homogenization treatment and treating it at 85-95° C. for 5-10 minutes.

[0017] Furthermore, in step S3, the mass ratio of Lactobacillus bulgaricus and Streptococcus thermophilus added to the basic starter is 1:(1-4).

[0018] Furthermore, in step S3, based on the weight of the fermentation raw material liquid, the inoculation amount of the basic starter is 0.1-0.4%.

[0019] Furthermore, in step S3, the fermented bacterial powder further comprises an auxiliary fermentation agent, and the auxiliary fermentation agent comprises one or more of yeast, bifidobacterium, Pediococcus pentosaceus and Lactobacillus paracasei.

[0020] Furthermore, in step S3, based on the weight of the fermentation raw material liquid, the inoculation amount of the auxiliary fermentation agent is 0.1-0.4%.

[0021] Furthermore, in step S3, the fermentation treatment method includes: taking the fermentation raw material liquid for a first fermentation treatment at 35-40°C for 5-10 hours, and then performing a second fermentation treatment at 0-5°C for 8-16 hours to obtain the sea cucumber flower enzyme.

[0022] The sea cucumber flower enzyme provided by the present invention is prepared by the above-mentioned sea cucumber flower enzyme preparation method.

[0023] The present invention also provides the use of the sea cucumber flower enzyme in the preparation of products for improving uric acid levels and / or blood sugar levels.

[0024] Furthermore, the product for improving uric acid level and / or blood sugar level is a medicine and / or a health food.

[0025] Beneficial effects:

[0026] In the preparation method of sea cucumber flower enzyme provided by the present invention, the sea cucumber flower raw material is firstly enzymatically hydrolyzed by protease to fully catalyze the cleavage of peptide bonds in polypeptides or protein molecules, thereby decomposing them into smaller peptides or amino acids; then the enzymatic hydrolysis product is fully mixed with milk and sugar by a high-pressure homogenization treatment method, and the protein and lipid rich in milk are used to achieve emulsification and embedding of fishy substances in the enzymatic hydrolysis product, which can reduce the fishy smell and effectively improve the uptake rate of fishy substances by microorganisms such as Lactobacillus bulgaricus and Streptococcus thermophilus in subsequent treatment; finally, a fermentation bacteria containing Lactobacillus bulgaricus and Streptococcus thermophilus is used to prepare the sea cucumber flower enzyme. The powder is fermented and the added sugars are used to optimize the metabolism in the cells of each microorganism, so that microorganisms such as Lactobacillus bulgaricus and Streptococcus thermophilus can cooperate with each other to work together to convert the fishy substances in the enzymatic hydrolysis products into odorless substances, and achieve sufficient decomposition and conversion of related biologically active precursor substances in the enzymatic hydrolysis products, and finally obtain a sea cucumber flower enzyme with no obvious fishy smell or even no fishy smell, and the sea cucumber flower enzyme shows obvious inhibitory effect on the activity of α-glucosidase and xanthine oxidase, which is a functional food raw material with great potential for lowering blood sugar and uric acid, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is one of the experimental result graphs of the fishy smell sensory evaluation score provided in Test Example 1 of the present invention;

[0028] Figure 2 This is the second experimental result diagram of the fishy smell sensory evaluation score provided in Test Example 1 of the present invention. DETAILED DESCRIPTION

[0029] Based on the problem of heavy fishy smell in existing sea cucumber flower enzymatic hydrolysis products, the inventor of the present invention found after in-depth research that the addition of pure milk, pure goat milk and other milk and sugars such as sucrose, maltose, trehalose and lactose to the sea cucumber flower enzymatic hydrolysis products for high-pressure homogenization treatment can make the subsequent introduction of microorganisms such as Lactobacillus bulgaricus and Streptococcus thermophilus be able to make good use of metabolism to convert fishy substances into odorless substances, greatly reducing the fishy smell of the final sea cucumber flower enzyme; at the same time, the relevant metabolic activities of the microorganisms can hydrolyze and convert the relevant biologically active precursor substances in the enzymatic hydrolysis products into small molecule biologically active substances with more excellent activity, and the final sea cucumber flower enzyme has a high content of effective ingredients and has significant uric acid and blood sugar lowering effects. Based on this, the technical solution of the present invention is obtained.

[0030] In the present invention, the preparation method of the sea cucumber flower enzyme specifically includes: S1, taking sea cucumber flower raw materials and mixing them with protease, and then performing enzymolysis treatment to obtain an enzymolysis product; S2, taking milk, sugar and the enzymolysis product and mixing them, and then performing high-pressure homogenization treatment and sterilization treatment in sequence to obtain a fermentation raw material liquid; S3, taking fermentation bacteria powder and inoculating it into the fermentation raw material liquid for fermentation treatment to obtain the sea cucumber flower enzyme; wherein, the fermentation bacteria powder includes a basic fermentation agent, and the basic fermentation agent includes Lactobacillus bulgaricus and Streptococcus thermophilus.

[0031] In the present invention, in step S1, the protease refers to a substance that catalyzes the breaking of peptide bonds in polypeptides or protein molecules, thereby decomposing them into smaller peptides or amino acids. It is a type of substance commonly used in the prior art, and is limited to achieving sufficient digestion of sea cucumber flower cells and sufficient release of cell contents. The present invention does not impose any special limitation on it.

[0032] In some specific embodiments, specific examples of the protease include but are not limited to: one or more of papain, bromelain and trypsin.

[0033] In the present invention, in step S1, the preparation of the sea cucumber flower raw material preferably includes: freeze-drying and grinding the sea cucumber flower to obtain freeze-dried sea cucumber flower powder; mixing the freeze-dried sea cucumber flower powder with water to obtain the sea cucumber flower raw material. At this time, the sea cucumber flower is pre-treated by freeze-drying combined with grinding, which can achieve preliminary crushing of the sea cucumber flower, increase the contact area between the sea cucumber flower raw material and the protease, and achieve a better hydrolysis effect.

[0034] In the present invention, the freeze-drying and grinding are technical means conventionally used in the prior art, which are limited to achieving sufficient drying and crushing of the sea cucumber flowers, and the present invention does not impose any special limitation thereto.

[0035] In some specific embodiments, the added mass ratio of the freeze-dried sea cucumber flower powder to water is preferably 1:(5-20), such as 1:5, 1:8, 1:10, 1:15, 1:20 or any value therebetween.

[0036] In the present invention, in step S1, the added mass ratio of the solid matter and the protease in the sea cucumber flower raw material is preferably (1-20):1, such as 1:1, 3:1, 5:1, 10:1, 12.5:1, 15:1, 20:1 or any value therebetween. At this time, the protease can fully hydrolyze the sea cucumber flower raw material, which is conducive to the full release of the fishy smell substances and related bioactive precursor substances.

[0037] In the present invention, in step S1, the enzymatic hydrolysis treatment refers to the process of using the above-mentioned protease to decompose the sea cucumber flower raw material to obtain an enzymatic hydrolysis product.

[0038] In some specific embodiments, the conditions of the enzymatic treatment specifically include a temperature of preferably 40 to 65°C, such as 40°C, 45°C, 50°C, 55°C, 60°C, 65°C or any value therebetween; a pH value of preferably 5.5 to 6.5, such as 5.5, 5.8, 5.9, 6.0, 6.2, 6.5 or any value therebetween; and a time of preferably 1 to 11 hours, such as 1 hour, 3 hours, 5 hours, 7 hours, 9 hours, 11 hours or any value therebetween. At this point, the protease can more efficiently catalyze protein hydrolysis to achieve a better release effect of fishy smell substances and related bioactive precursor substances.

[0039] In the present invention, step S2 preferably further comprises: taking the enzymatic hydrolysis product for freeze drying and grinding. At this time, the enzymatic hydrolysis product is treated again by freeze drying combined with grinding, which can effectively achieve the concentration of fishy substances and related biologically active precursor substances in the enzymatic hydrolysis product, and increase its contact with milk in the subsequent high-pressure homogenization process, so as to achieve a more ideal treatment effect.

[0040] In the present invention, in step S2, the milk refers to the milk of mammals, which is rich in carbohydrates, proteins and lipids, can achieve effective emulsification and embedding of fishy substances in high-pressure homogenization treatment, and can affect the metabolism of microorganisms such as Lactobacillus bulgaricus and Streptococcus thermophilus in subsequent fermentation treatment, so as to achieve multiple effects such as converting fishy substances into odorless substances and hydrolyzing and converting related bioactive precursor substances into small molecule bioactive substances with better activity.

[0041] In some specific embodiments, specific examples of the milk include but are not limited to: one or more of pure cow's milk, buffalo milk, goat's milk and camel's milk.

[0042] In the present invention, in step S2, the carbohydrate refers to a type of carbohydrate that provides energy for microorganisms such as Lactobacillus bulgaricus and Streptococcus thermophilus. Its addition can optimize the metabolism of the above-mentioned microorganisms to promote related metabolic pathways in the cells, thereby achieving better conversion of fishy substances into odorless substances and hydrolysis of related bioactive precursor substances into small molecule bioactive substances with better activity.

[0043] In some specific embodiments, specific examples of the sugar include but are not limited to: one or more of sucrose, maltose, trehalose and lactose.

[0044] In the present invention, in step S2, the added mass ratio of the milk, sugars and solid matter in the enzymatic hydrolysis product is preferably 100:(1-7):(1-4), such as 100:1:1, 100:3:1, 100:5:1, 100:7:1, 100:7:3, 100:7:4 or any value therebetween. At this time, the milk can fully achieve the emulsification and embedding of the fishy substances and / or related bioactive precursor substances, and is conducive to the subsequent fermentation process to obtain a sea cucumber flower enzyme with more obvious functionality.

[0045] In the present invention, in step S2, the high-pressure homogenization treatment refers to the technology of using high pressure to pass liquid substances through extremely small pores or gaps to achieve fine dispersion and uniform mixing. This technology can fully dissolve the solid matter in the enzymatic hydrolysis product and achieve sufficient emulsification and embedding of fishy substances and / or related bioactive precursor substances in milk.

[0046] In some specific embodiments, the conditions of the high-pressure homogenization treatment specifically include a pressure of preferably 80 to 150 MPa, such as 80 MPa, 85 MPa, 90 MPa, 100 MPa, 120 MPa, 140 MPa, 150 MPa or any value therebetween; and a time of preferably 1 to 10 min, such as 1 min, 2 min, 2.5 min, 3 min, 4 min, 8 min, 10 min or any value therebetween.

[0047] In the present invention, the sterilization treatment refers to the process of eliminating or inhibiting the growth and reproduction of harmful microorganisms such as bacteria, viruses, and fungi by physical or chemical methods. Through this process, the fermentation raw material liquid is free of other bacteria, thereby ensuring the biological safety of the finally obtained sea cucumber flower enzyme.

[0048] In some specific embodiments, the sterilization treatment method specifically includes: taking the mixture of milk, sugars and enzymatic hydrolysis products that have undergone the high-pressure homogenization treatment and treating it at 85-95° C. for 5-10 minutes.

[0049] In the present invention, in step S3, the addition mass ratio of Lactobacillus bulgaricus and Streptococcus thermophilus in the basic leavening agent is preferably 1:(1-4), such as 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:4 or any value therebetween. At this time, the Lactobacillus bulgaricus and Streptococcus thermophilus have a better synergistic effect to achieve a more ideal treatment effect.

[0050] In the present invention, in step S3, based on the weight of the fermentation raw material liquid, the inoculation amount of the basic starter is preferably 0.1-0.4%, such as 0.1%, 0.15%, 0.2%, 0.3%, 0.35%, 0.4% or any value therebetween.

[0051] In the present invention, in step S3, the fermented bacteria powder preferably also includes an auxiliary fermentation agent, and the auxiliary fermentation agent specifically includes one or more of yeast, bifidobacterium, Pediococcus pentosaceus and Lactobacillus paracasei. At this time, the introduction of the auxiliary fermentation agent can further synergistically enhance the treatment effect of the basic fermentation agent to give the finally obtained sea cucumber flower enzyme more significant biological activity.

[0052] In some specific embodiments, based on the weight of the fermentation raw material liquid, the inoculation amount of the auxiliary fermentation agent is preferably 0.1-0.4%, such as 0.1%, 0.13%, 0.18%, 0.2%, 0.25%, 0.3%, 0.4% or any value therebetween.

[0053] In the present invention, in step S3, the fermentation treatment refers to the process of decomposing or transforming the relevant organic matter in the fermentation raw material liquid by utilizing the action of microorganisms in the fermentation bacteria powder, through which the fishy substances in the enzymatic hydrolysis products are transformed into odorless substances, and the relevant biologically active precursor substances in the enzymatic hydrolysis products are fully decomposed and transformed.

[0054] In the present invention, in step S3, the fermentation treatment method preferably includes: taking the fermentation raw material liquid for a first fermentation treatment and a second fermentation treatment to obtain the sea cucumber flower enzyme. Wherein, the conditions of the first fermentation treatment include a temperature of 35 to 40°C, such as 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or any value therebetween; the time is 5 to 10h, such as 5h, 5.5h, 6h, 7h, 8h, 9h, 10h or any value therebetween. The conditions of the second fermentation treatment include a temperature of 0 to 5°C, such as 0°C, 1°C, 2°C, 3°C, 4°C, 5°C or any value therebetween; the time is 8 to 16h, such as 8h, 9h, 12h, 14h, 16h or any value therebetween. At this time, compared with the conventional fermentation treatment method used in the prior art, the present invention adopts a multi-temperature fermentation method, and utilizes temperature control to optimize the metabolism of each microorganism in the fermentation powder, so as to better achieve the effect of converting fishy substances into odorless substances and hydrolyzing and converting related bioactive precursor substances into small molecule bioactive substances with better activity.

[0055] The present invention also provides a sea cucumber flower enzyme, which is prepared by the above-mentioned sea cucumber flower enzyme preparation method. The sea cucumber flower enzyme not only has no obvious fishy smell or even no fishy smell, but also exhibits obvious xanthine oxidase and α-glucosidase activity inhibition, and has potential uric acid and blood sugar lowering activities.

[0056] Based on the remarkable performance of the above-mentioned sea cucumber flower enzyme, the present invention also provides the use of the above-mentioned sea cucumber flower enzyme in the preparation of products for improving uric acid and / or blood sugar.

[0057] In the present invention, the product for improving uric acid and / or blood sugar is specifically a medicine and / or health food. Wherein, the medicine refers to a substance used to prevent, diagnose, and treat diseases related to abnormal uric acid and / or abnormal blood sugar. The health food refers to a food containing a certain amount of nutrients or having a specific health function that helps maintain a healthy level of uric acid and / or helps maintain a healthy level of blood sugar.

[0058] Embodiments of the present invention are described in detail below, and the examples of the embodiments are intended to be used to explain the present invention, but should not be construed as limiting the present invention. In the embodiments, if specific techniques or conditions are not specified, the techniques or conditions described in the literature in this area or the product specifications are used. If the manufacturer is not specified for reagents or instruments used, they are all conventional products that can be obtained commercially.

[0059] Preparation Example

[0060] This preparation example is used to illustrate the preparation of freeze-dried sea cucumber flower powder, which specifically includes: freeze-drying the frozen sea cucumber flowers and grinding them into fine powder, passing them through a 100-mesh sieve, and the sieved material is the freeze-dried sea cucumber flower powder.

[0061] Example 1

[0062] This example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0063] S1. (1) mixing the freeze-dried sea cucumber flower powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0064] (2) Papain (Nanning Pangbo Bioengineering Co., Ltd., product number PBD-01-1) was added to the sea cucumber flower raw material solution in an amount of 2 wt %, and the solution pH was adjusted to 6 with sodium hydroxide. The solution was enzymatically hydrolyzed at 40° C. for 3 h to obtain an enzymatic solution. The enzymatic solution was freeze-dried and ground into a fine powder to obtain an enzymatic product.

[0065] S2. Pure milk, sucrose and enzymatic hydrolysis product were mixed evenly in a mass ratio of 100:7:1, and high-pressure homogenization was performed at 100 MPa for 3 min, followed by sterilization in a 90° C. water bath for 5 min to obtain a fermentation raw material liquid.

[0066] S3. Inoculate Lactobacillus bulgaricus (Xi'an Mixianer Biotechnology Co., Ltd., product number LB42), Streptococcus thermophilus (Xi'an Mixianer Biotechnology Co., Ltd., product number ST81) and Lactobacillus paracasei (Xi'an Mixianer Biotechnology Co., Ltd., product number LC86) in inoculation amounts of 0.1%, 0.1% and 0.2% respectively into the fermentation raw material liquid, and carry out the first fermentation treatment at 37°C for 7 hours; then carry out the second fermentation treatment at 4°C for 12 hours to obtain the fermentation liquid, and freeze-dry the fermentation liquid to obtain sea cucumber flower enzyme.

[0067] Example 2

[0068] This example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0069] S1. (1) mixing the freeze-dried sea cucumber powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0070] (2) Add papain in an amount of 2 wt % to the sea cucumber flower raw material solution, and use sodium hydroxide to adjust the solution pH to 6, perform enzymatic hydrolysis at 40° C. for 3 h to obtain an enzymatic hydrolyzate, and freeze-dry the enzymatic hydrolyzate and grind it into fine powder to obtain an enzymatic hydrolyzate product.

[0071] S2. Pure milk, sucrose and enzymatic hydrolysis product were mixed evenly in a mass ratio of 100:7:1, and high-pressure homogenization was performed at 100 MPa for 3 min, followed by sterilization in a 90° C. water bath for 5 min to obtain a fermentation raw material liquid.

[0072] S3. Inoculate Lactobacillus bulgaricus and Streptococcus thermophilus in the fermentation raw material liquid at inoculation amounts of 0.1% and 0.1% respectively, and perform the first fermentation treatment at 37°C for 7 hours; then perform the second fermentation treatment at 4°C for 12 hours to obtain fermentation liquid, and freeze-dry the fermentation liquid to obtain sea cucumber flower enzyme.

[0073] Example 3

[0074] This embodiment adopts the method provided in Example 1 to prepare sea cucumber flower enzyme, except that in step S2, sucrose is replaced by maltose of equal mass, and other conditions are the same to obtain sea cucumber flower enzyme.

[0075] Example 4

[0076] This embodiment adopts the method provided in Example 1 to prepare sea cucumber flower enzyme, the difference being that in step S2, sucrose is replaced by trehalose of equal mass, and other conditions are the same to obtain sea cucumber flower enzyme.

[0077] Example 5

[0078] This embodiment adopts the method provided in Example 1 to prepare sea cucumber flower enzyme, except that in step S2, an equal mass of lactose is used instead of sucrose, and other conditions are the same to obtain sea cucumber flower enzyme.

[0079] Example 6

[0080] This embodiment adopts the method provided in Example 1 to prepare sea cucumber flower enzyme, except that, in step S3, the inoculation amount of Lactobacillus bulgaricus is 0.05%, the inoculation amount of Streptococcus thermophilus is 0.05%, and other conditions are the same to obtain sea cucumber flower enzyme.

[0081] Example 7

[0082] This embodiment adopts the method provided in Example 1 to prepare sea cucumber flower enzyme, except that, in step S3, the inoculation amount of Lactobacillus bulgaricus is 0.2%, the inoculation amount of Streptococcus thermophilus is 0.2%, and other conditions are the same to obtain sea cucumber flower enzyme.

[0083] Example 8

[0084] This example adopts the method provided in Example 1 to prepare sea cucumber flower enzyme, except that in step S3, an equal inoculation amount of Pediococcus pentosaceus (Xi'an Mixianer Biotechnology Co., Ltd., product number S698) is used instead of Lactobacillus paracasei, and other conditions are the same to obtain sea cucumber flower enzyme.

[0085] Comparative Example 1

[0086] This comparative example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0087] (1) mixing the freeze-dried sea cucumber flower powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0088] (2) adding papain in an amount of 2 wt % to the raw material solution of sea cucumber flower, adjusting the pH of the solution to 6 with sodium hydroxide, performing enzymolysis at 40° C. for 3 h to obtain an enzymolysis solution, and freeze-drying the enzymolysis solution and grinding it into a fine powder to obtain an enzymolysis product;

[0089] (3) Pure milk and the enzymatic hydrolysis product are mixed evenly in a mass ratio of 100:1 and freeze-dried to obtain sea cucumber flower enzyme.

[0090] Comparative Example 2

[0091] This comparative example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0092] S1. (1) mixing the freeze-dried sea cucumber flower powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0093] (2) Add papain in an amount of 2 wt % to the sea cucumber flower raw material solution, and use sodium hydroxide to adjust the solution pH to 6, perform enzymatic hydrolysis at 40° C. for 3 h to obtain an enzymatic hydrolyzate, and freeze-dry the enzymatic hydrolyzate and grind it into fine powder to obtain an enzymatic hydrolyzate product.

[0094] S2. Pure milk and enzymatic hydrolysis product are mixed evenly in a mass ratio of 100:1, and high-pressure homogenization is performed at 100 MPa for 3 minutes, followed by freeze-drying to obtain sea cucumber flower enzyme.

[0095] Comparative Example 3

[0096] This comparative example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0097] S1. (1) mixing the freeze-dried sea cucumber flower powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0098] (2) Add papain in an amount of 2 wt % to the sea cucumber flower raw material solution, and use sodium hydroxide to adjust the solution pH to 6, perform enzymatic hydrolysis at 40° C. for 3 h to obtain an enzymatic hydrolyzate, and freeze-dry the enzymatic hydrolyzate and grind it into fine powder to obtain an enzymatic hydrolyzate product.

[0099] S2, pure milk and enzymatic hydrolysate were mixed evenly in a mass ratio of 100:1, and high-pressure homogenization was performed at 100 MPa for 3 min, followed by sterilization in a 90° C. water bath for 5 min to obtain a fermentation raw material liquid;

[0100] S3. Inoculate Lactobacillus bulgaricus and Streptococcus thermophilus in the fermentation raw material liquid at inoculation amounts of 0.1% and 0.1% respectively, and perform the first fermentation treatment at 37°C for 7 hours; then perform the second fermentation treatment at 4°C to obtain fermentation liquid, and freeze-dry the fermentation liquid to obtain sea cucumber flower enzyme.

[0101] Comparative Example 4

[0102] This comparative example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0103] S1. (1) mixing the freeze-dried sea cucumber flower powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0104] (2) Add papain in an amount of 2 wt % to the sea cucumber flower raw material solution, and use sodium hydroxide to adjust the solution pH to 6, perform enzymatic hydrolysis at 40° C. for 3 h to obtain an enzymatic hydrolyzate, and freeze-dry the enzymatic hydrolyzate and grind it into fine powder to obtain an enzymatic hydrolyzate product.

[0105] S2. Pure milk, sucrose and enzymatic hydrolysis product were mixed evenly in a mass ratio of 100:1, and high-pressure homogenization was performed at 100 MPa for 3 min, followed by sterilization in a 90° C. water bath for 5 min to obtain a fermentation raw material liquid.

[0106] S3. Inoculate Lactobacillus paracasei into the fermentation raw material liquid at an inoculation rate of 0.2%, and perform a first fermentation treatment at 37° C. for 7 hours; then perform a second fermentation treatment at 4° C. to obtain a fermentation liquid, and freeze-dry the fermentation liquid to obtain sea cucumber flower enzyme.

[0107] Comparative Example 5

[0108] This comparative example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0109] S1. (1) mixing the freeze-dried sea cucumber powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0110] (2) Add papain in an amount of 2 wt % to the sea cucumber flower raw material solution, and use sodium hydroxide to adjust the solution pH to 6, perform enzymatic hydrolysis at 40° C. for 3 h to obtain an enzymatic hydrolyzate, and freeze-dry the enzymatic hydrolyzate and grind it into fine powder to obtain an enzymatic hydrolyzate product.

[0111] S2. Pure milk, sucrose and enzymatic hydrolysis product are mixed evenly in a mass ratio of 100:7:1, and high-pressure homogenization is performed at 100 MPa for 3 minutes, followed by freeze-drying to obtain sea cucumber flower enzyme.

[0112] Comparative Example 6

[0113] This comparative example is used to illustrate a method for preparing sea cucumber flower enzyme, and the preparation specifically includes:

[0114] S1. (1) mixing the freeze-dried sea cucumber powder provided in the preparation example with water at an addition mass ratio of 1:10 (g:mL) to obtain a sea cucumber flower raw material liquid;

[0115] (2) Add papain in an amount of 2 wt % to the sea cucumber flower raw material solution, and use sodium hydroxide to adjust the solution pH to 6, perform enzymatic hydrolysis at 40° C. for 3 h to obtain an enzymatic hydrolyzate, and freeze-dry the enzymatic hydrolyzate and grind it into fine powder to obtain an enzymatic hydrolyzate product.

[0116] S2, taking water, sucrose and enzymatic hydrolysis products according to a mass ratio of 100:7:1 and mixing them uniformly, performing high-pressure homogenization treatment at 100 MPa for 3 minutes, and then sterilizing them in a water bath at 90° C. for 5 minutes to obtain a fermentation raw material liquid;

[0117] S3. Inoculate Lactobacillus bulgaricus and Streptococcus thermophilus in the fermentation raw material liquid at inoculation amounts of 0.1% and 0.1% respectively, and perform the first fermentation treatment at 37°C for 7 hours; then perform the second fermentation treatment at 4°C to obtain fermentation liquid, and freeze-dry the fermentation liquid to obtain sea cucumber flower enzyme.

[0118] Test Example 1

[0119] This test example is used to illustrate the sensory evaluation of the fishy smell of sea cucumber flower enzymes provided in the above embodiments and comparative examples. The test specifically includes: 20 graduate students were selected from the School of Marine Biology and Food Engineering of Jimei University for sensory evaluation training. The fishy smell sensory evaluation adopts a 15-point system, and the specific reference standard is: sea cucumber flower hydrolysate is 0 points, and sea cucumber flower hydrolysate that has not been deodorized is 15 points; the evaluators conduct sensory evaluation according to the scoring rules shown in Table 1, and take the average score as the final score for each group for the sensory evaluation of the fishy smell of sea cucumber flower enzymes. The test results are as follows: Figure 1 and 2 shown.

[0120] Table 1.

[0121] score Fishy smell Acceptance 10-15 Strong fishy smell, not easy to accept Totally acceptable 5-10 Slight fishy smell, acceptable Basically acceptable 0-5 Basically no fishy smell, easy to accept Hard to accept

[0122] Depend on Figure 1 and 2From the test results shown, it can be seen that by adding pure milk, pure goat milk and other milk products as well as sugars such as sucrose, maltose, trehalose and lactose to the sea cucumber flower enzymatic hydrolysate and subjecting it to high-pressure homogenization treatment, the subsequently introduced microorganisms such as Lactobacillus bulgaricus and Streptococcus thermophilus can make good use of metabolism to convert fishy substances into odorless substances, thereby greatly reducing the fishy smell of the final sea cucumber flower enzyme.

[0123] Test Example 2

[0124] This test example is used to illustrate the biological activity of the sea cucumber flower enzymes provided in the above embodiments and comparative examples. The test specifically includes:

[0125] 1. Hypoglycemic activity

[0126] (1) 1 mg of α-glucosidase was dissolved in 49 mL of PBS solution (0.2 M, pH = 7) to obtain an α-glucosidase solution with a concentration of 1 U / mL;

[0127] (2) 0.075 g of p-nitrophenol-α-D-pyranoglucoside (PNPG) was dissolved in 10 mL of PBS solution (0.2 M, pH = 7) to obtain a 2.5 mM PNPG solution;

[0128] (3) taking sea cucumber flower enzyme and PBS solution (0.2 M, pH = 7) at a final concentration of 25 mg / mL to obtain a sample solution;

[0129] (4) Take 50 μL of sample solution and 50 μL of α-glucosidase solution, mix them evenly, and react them in a 37°C water bath for 15 minutes. Then add 50 μL of PNPG solution and mix them evenly. Mix them in a 37°C water bath for 10 minutes. After the reaction, measure the absorbance of the solution at 405 nm, and calculate the α-glucosidase inhibition rate according to the following formula. The test results are shown in Table 1.

[0130] α-glucosidase inhibition rate (%) = [1-(A1-A3) / (A2-A0)] × 100%

[0131] Among them, A0 is the absorbance value of the experimental group; A1 is the absorbance value measured by the sample solution; A2 is the absorbance value measured by mixing only 50 μL of α-glucosidase solution and 50 μL of PNPG solution, and mixing and reacting in a 37°C water bath for 10 minutes; A3 is the absorbance value measured by mixing 50 μL of sample solution and 50 μL of PNPG solution, and mixing and reacting in a 37°C water bath for 10 minutes.

[0132] 2. Uric acid lowering activity

[0133] (1) 6.946 g of disodium hydrogen phosphate, 0.956 g of sodium dihydrogen phosphate and 0.0186 g of ethylenediaminetetraacetic acid (EDTA) were mixed, dissolved in distilled water and diluted to 500 mL to obtain a PBS solution with a concentration of 0.2 M and a pH of 7.5;

[0134] (2) 0.1 mg of xanthine oxidase was dissolved in 250 mL of PBS solution (0.2 M, pH = 7.5) to obtain a xanthine oxidase solution with a concentration of 0.02 U / mL;

[0135] (3) 0.0073 g of xanthine was dissolved in 400 μL of 1 mol / L sodium hydroxide solution, and the volume was fixed to 100 mL with PBS solution (0.2 M, pH = 7.5) to obtain a xanthine solution (protected from light);

[0136] (4) taking sea cucumber flower enzyme and PBS solution (0.2 M, pH = 7) at a final concentration of 25 mg / mL to obtain a sample solution;

[0137] (5) Take 50 μL of sample solution and 50 μL of xanthine oxidase solution and mix them evenly. Mix and react at room temperature for 5 minutes. Then add 150 μL of xanthine solution and mix them evenly. Mix and react at room temperature for 25 minutes. After the reaction, measure the absorbance of the solution at 290 nm, and calculate the xanthine oxidase inhibition rate according to the following formula. The test results are shown in Table 1.

[0138] Xanthine oxidase inhibition rate (%) = [1-(A1-A2) / (A3-A4)] × 100%

[0139] Among them, A1 is the absorbance value measured by the experimental group; A2 is the absorbance value measured by mixing only 50 μL of sample solution and 150 μL of xanthine solution and reacting at room temperature for 25 minutes; A3 is the absorbance value measured by mixing 50 μL of xanthine oxidase solution and 150 μL of xanthine solution and reacting at room temperature for 25 minutes; A4 is the absorbance value of xanthine solution.

[0140] Table 1.

[0141]

[0142] It can be seen from the test results shown in Table 1 that, compared with comparative examples 1 to 6, the sea cucumber flower enzyme prepared by the method provided by embodiments 1 to 8 of the present invention can effectively decompose and transform the relevant biologically active precursor substances in the freeze-dried sea cucumber flower powder, so that the finally prepared sea cucumber flower enzyme exhibits more significant xanthine oxidase activity and α-glucosidase activity inhibitory effects, and is a highly potential active ingredient for lowering blood sugar and uric acid, and has good application prospects.

[0143] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention without departing from the principles and intent of the present invention.

Claims

1. A method for preparing sea cucumber flower enzyme, characterized in that: The preparation method comprises: S1, taking the sea cucumber flower raw material and mixing it with protease, and then performing enzymolysis treatment to obtain an enzymolysis product; S2, mixing milk, sugar and the enzymatic hydrolysis product, and sequentially subjecting the mixture to high pressure homogenization and sterilization to obtain a fermentation raw material liquid; S3. Take fermentation bacteria powder and inoculate it into the fermentation raw material liquid for fermentation treatment to obtain the sea cucumber flower enzyme; wherein the fermentation bacteria powder includes a basic fermentation agent, and the basic fermentation agent includes Lactobacillus bulgaricus and Streptococcus thermophilus.

2. The method for preparing sea cucumber flower enzyme according to claim 1, characterized in that: In step S1, the protease is selected from one or more of papain, bromelain and trypsin; Optionally, the added mass ratio of solid matter and protease in the sea cucumber flower raw material is (1-20):

1.

3. The method for preparing sea cucumber flower enzyme according to claim 1, characterized in that: In step S1, the temperature of the enzymatic hydrolysis treatment is 40-65°C, the pH is 5.5-6.5, and the time is 1-11 hours.

4. The method for preparing sea cucumber flower enzyme according to claim 1, characterized in that: In step S2, the milk is selected from one or more of pure cow's milk, buffalo milk, goat's milk and camel's milk; Optionally, the sugar is selected from one or more of sucrose, maltose, trehalose and lactose; Optionally, the added mass ratio of the milk, sugar and solid matter in the enzymatic hydrolysis product is 100:(1-7):(1-4).

5. The method for preparing sea cucumber flower enzyme according to claim 1, characterized in that: In step S2, the pressure of the high-pressure homogenization treatment is 80-150 MPa, and the time is 1-10 min; Optionally, the sterilization treatment comprises: treating the mixture of milk, sugar and enzymatic hydrolysis product after the high-pressure homogenization treatment at 85-95° C. for 5-10 minutes.

6. The method for preparing sea cucumber flower enzyme according to claim 1, characterized in that: In step S3, the mass ratio of Lactobacillus bulgaricus and Streptococcus thermophilus added to the basic starter is 1:(1-4); Optionally, based on the weight of the fermentation raw material liquid, the inoculation amount of the basic starter is 0.1-0.4%; Optionally, the fermented bacteria powder further comprises an auxiliary fermentation agent, wherein the auxiliary fermentation agent comprises one or more of yeast, bifidobacterium, Pediococcus pentosaceus and Lactobacillus paracasei; Optionally, based on the weight of the fermentation raw material liquid, the inoculation amount of the auxiliary fermentation agent is 0.1-0.4%.

7. The method for preparing sea cucumber flower enzyme according to claim 1, characterized in that: In step S3, the fermentation treatment method includes: taking the fermentation raw material liquid for a first fermentation treatment at 35-40°C for 5-10 hours, and then performing a second fermentation treatment at 0-5°C for 8-16 hours to obtain the sea cucumber flower enzyme.

8. A sea cucumber flower enzyme, characterized in that: The sea cucumber flower enzyme is prepared by the sea cucumber flower enzyme preparation method described in any one of claims 1 to 7.

9. Use of the sea cucumber flower enzyme described in claim 8 in the preparation of a product for improving uric acid levels and / or blood sugar levels.

10. The use of sea cucumber flower enzyme according to claim 9 in the preparation of a product for improving uric acid level and / or blood sugar level, characterized in that: The product for improving uric acid level and / or blood sugar level is a medicine and / or a health food.

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