Combined preparation method of abalone viscera polysaccharide and polypeptide, abalone viscera polysaccharide and polypeptide composition and application

Abstract

The invention belongs to the technical field of biology, and discloses a combined preparation method of abalone viscera polysaccharide and polypeptide, an abalone viscera polysaccharide and polypeptide composition and application. According to the combined preparation method of the abalone viscera polysaccharide and polypeptide, a treatment process of combining staged enzymolysis and aspergillus oryzae fermentation is adopted, and all the steps jointly form an organic whole and cooperate with one another to degrade and modify substances contained in the abalone viscera; the abalone viscera polysaccharide polypeptide composition which has an excellent inhibition effect on alpha-glucosidase and DPP-IV is finally obtained, and the abalone viscera polysaccharide polypeptide composition has a good application prospect in development of hypoglycemic drugs and / or hypoglycemic functional foods.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a combined preparation method of abalone viscera polysaccharide and polypeptide, and an abalone viscera polysaccharide polypeptide composition and application. BACKGROUND

[0002] Diabetes refers to a metabolic disorder disease marked by hyperglycemia caused by absolute or relative insulin secretion deficiency and utilization disorder, and is mainly divided into four types of type I diabetes (T1DM), type II diabetes (T2DM), special type diabetes and gestational diabetes. Among them, type II diabetes accounts for 90%, and patients may have symptoms such as diabetic foot, blurred vision, cardiovascular disease, numbness of hands and feet, etc. At present, commonly used drugs for treating hypoglycemia include insulin sensitizers, SGLT-2 inhibitors and insulin secretion promoters, but the above drugs may cause adverse reactions such as gastrointestinal disorders, hypoglycemia, liver and kidney damage, etc.

[0003] Abalone contains nutrients such as protein, calcium, iron, iodine and vitamins, and has low fat content, which helps to enhance immunity and promote metabolism. In the abalone processing process, abalone viscera accounts for about 20% to 30% of the total weight of abalone, and the main by-product viscera mass is usually discarded as waste, and the waste of a large amount of viscera not only causes resource waste, but also may cause environmental pollution problems. Abalone viscera also contains rich bioactive components such as polysaccharides and protein polypeptides, which can be used as raw materials for preparing efficient and safe alternative hypoglycemic drugs.

[0004] Therefore, obtaining a method capable of realizing efficient combined extraction of polysaccharides and polypeptides in abalone viscera has important significance for the development of hypoglycemic functional foods. SUMMARY

[0005] The first object of the present application is to provide a combined preparation method of abalone viscera polysaccharide and polypeptide. The preparation method can realize efficient combined extraction of polysaccharides and polypeptides in abalone viscera, and obtain a polysaccharide polypeptide composition with excellent hypoglycemic biological activity.

[0006] The second object of the present application is to provide an abalone viscera polysaccharide polypeptide composition.

[0007] The third object of the present application is to provide the application of the above-mentioned abalone viscera polysaccharide polypeptide composition in the development of hypoglycemic drugs and / or hypoglycemic functional foods.

[0008] Specifically, the application provides a combined preparation method of abalone viscera polysaccharide and polypeptide, which comprises the following steps: S1, abalone viscera is mixed with water, and then subjected to wall breaking treatment and homogenization treatment to obtain abalone viscera slurry; the abalone viscera slurry is subjected to abalone viscera self-enzyme enzymolysis treatment to obtain abalone viscera self-enzyme enzymolysis liquid; S2, β-mannanase is mixed with the abalone viscera self-enzyme enzymolysis liquid, and then subjected to polysaccharidase enzymolysis treatment to obtain polysaccharidase enzymolysis liquid; S3, alkaline protease is mixed with the polysaccharidase enzymolysis liquid, and then subjected to protease enzymolysis treatment to obtain protease enzymolysis liquid; S4, the protease enzymolysis liquid is subjected to enzyme inactivation treatment and centrifugal treatment to obtain final enzymolysis liquid; and S5, Aspergillus oryzae liquid is mixed with the final enzymolysis liquid, and then subjected to fermentation culture to obtain Aspergillus oryzae fermentation liquid.

[0009] S6, the Aspergillus oryzae fermentation liquid is subjected to ceramic membrane filtration treatment, ultrafiltration membrane concentration treatment and freeze-drying treatment to obtain abalone viscera polysaccharide polypeptide composition.

[0010] Further, in step S1, the mixing mass ratio of the abalone viscera to water is 1:(1-20).

[0011] Further, in step S1, the abalone viscera self-enzyme enzymolysis treatment is carried out at a temperature of 30-60 DEG C, a pH value of 2-9 and for a time of 0.001-7 h.

[0012] Further, in step S2, the adding mass ratio of the β-mannanase to the abalone viscera is (1-6):50.

[0013] Further, in step S2, the polysaccharidase enzymolysis treatment is carried out at a temperature of 35-60 DEG C, a pH value of 4-9 and for a time of 2-6 h.

[0014] Further, in step S3, the adding mass ratio of the alkaline protease to the abalone viscera is (1-5):50.

[0015] Further, in step S3, the protease enzymolysis treatment is carried out at a temperature of 35-60 DEG C, a pH value of 6-11 and for a time of 3-7 h.

[0016] Further, in step S4, the enzyme inactivation treatment is carried out at a temperature of 95-105 DEG C for a time of 5-15 min.

[0017] Further, in step S4, the centrifugal treatment is carried out at a temperature of 20-25 DEG C, a rotation speed of 8000-10000 r / min and for a time of 10-20 min.

[0018] Further, in step S5, the cell density of the Aspergillus oryzae liquid is 1x10 4 CFU / g-1x10 7 CFU / g.

[0019] Further, in step S5, the mass ratio of the Aspergillus oryzae liquid to the final enzymatic hydrolysate is (0.8-1.2):1.

[0020] Further, in step S5, the fermentation culture temperature is 18-30℃, and the time is 24-48h.

[0021] Further, in step S6, the pore size of the ceramic membrane in the ceramic membrane filtration treatment is 40-60nm.

[0022] Further, in step S6, the pore size of the ultrafiltration membrane in the ultrafiltration membrane concentration treatment is 5-10nm.

[0023] Further, in step S6, the temperature of the freeze-drying treatment is -50- -40℃, and the time is 24-50h.

[0024] The abalone viscera polysaccharide polypeptide composition is prepared by the combined preparation method of the abalone viscera polysaccharide and polypeptide.

[0025] Further, the alpha-glucosidase inhibition rate of the abalone viscera polysaccharide polypeptide composition is 65-90%, and the DPP-IV inhibition rate is 30-70%.

[0026] The application provides application of the abalone viscera polysaccharide polypeptide composition in development of hypoglycemic drugs and / or blood sugar-reducing functional foods.

[0027] Beneficial effects:

[0028] In the combined preparation method of the abalone viscera polysaccharide and polypeptide, a treatment process combining phased enzymolysis and Aspergillus oryzae fermentation is adopted, that is, first, the abalone viscera slurry is subjected to step-by-step enzymolysis treatment by using the abalone viscera self-enzyme system, beta-mannanase and alkaline protease, so as to effectively release the cell contents and realize sufficient separation of polysaccharides and proteins, and in the process of phased enzymolysis, the decomposition of polysaccharides and proteins is gradually realized, then the phased enzymolysis products are treated by using Aspergillus oryzae, so as to further realize the degradation of residual macromolecular polysaccharides or proteins, and realize the transformation or modification of the substances contained in the system, such as glycosylation and peptide bond modification, so as to improve the water solubility, stability and biological activity of the polysaccharides and polypeptides, and each treatment step cooperates to form an organic whole, so as to realize the degradation and modification of the active substances contained in the abalone viscera, so as to finally obtain an abalone viscera polysaccharide polypeptide composition which has excellent inhibitory effects on alpha-glucosidase and DPP-IV, and has good application prospects in development of hypoglycemic drugs and / or blood sugar-reducing functional foods. DETAILED DESCRIPTION

[0029] The application provides a combined preparation method of abalone viscera polysaccharide and polypeptide, and specifically comprises the following steps: S1, abalone viscera is mixed with water, and then subjected to wall breaking treatment and homogenate treatment to obtain abalone viscera slurry; the abalone viscera slurry is subjected to abalone viscera self-enzyme enzymolysis treatment to obtain abalone viscera self-enzyme enzymolysis liquid; S2, β-mannanase is mixed with the abalone viscera self-enzyme enzymolysis liquid, and then subjected to polysaccharidase enzymolysis treatment to obtain polysaccharidase enzymolysis liquid; S3, alkaline protease is mixed with the polysaccharidase enzymolysis liquid, and then subjected to protease enzymolysis treatment to obtain protease enzymolysis liquid; S4, the protease enzymolysis liquid is subjected to enzyme inactivation treatment and centrifugal treatment to obtain final enzymolysis liquid; S5, Aspergillus oryzae liquid is mixed with the final enzymolysis liquid, and then subjected to fermentation culture to obtain Aspergillus oryzae fermentation liquid; and S6, the Aspergillus oryzae fermentation liquid is subjected to ceramic membrane filtration treatment, ultrafiltration membrane concentration treatment and freeze-drying treatment to obtain abalone viscera polysaccharide polypeptide composition.

[0030] In the application, in step S1, the mixing mass ratio of the abalone viscera to water is preferably 1:(1-20), and can be 1:1, 1:2, 1:4, 1:8, 1:10, 1:14, 1:16, 1:20 or any value between them.

[0031] In the application, in step S1, the wall breaking treatment refers to a process of destroying the abalone viscera and its cell structure by physical means to release the cell contents, which is a technical means commonly used in the prior art, and the person skilled in the art can make adaptive selection according to the actual needs, and the application does not particularly limit it.

[0032] In the application, in step S1, the homogenate treatment refers to a process of dispersing the broken abalone viscera by physical means to form a suspension or emulsion with uniform and delicate texture, which is a technical means commonly used in the prior art, and the person skilled in the art can make adaptive selection according to the actual needs, and the application does not particularly limit it.

[0033] In the application, in step S1, the abalone viscera self-enzyme enzymolysis treatment refers to a process of realizing the enzymolysis of the abalone viscera slurry by using the enzyme system of the abalone viscera itself. More specifically, the conditions of the abalone viscera self-enzyme enzymolysis treatment include that the temperature is preferably 30-60°C, and can be 30°C, 32°C, 34°C, 38°C, 40°C, 45°C, 50°C, 58°C, 60°C or any value between them; the pH value is preferably 2-9, and can be 2.0, 2.3, 2.5, 2.7, 2.9, 3.0, 3.5, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or any value between them; and the time is preferably 0.001-7h, and can be 0.001h, 0.01h, 0.1h, 0.5h, 1h, 1.5h, 2h, 4h, 6h, 7h or any value between them.

[0034] In the present application, in step S2, the mass ratio of the β-mannanase to the abalone viscera is preferably (1-6):50, and more specifically, 1:50, 1.3:50, 2:50, 3:50, 4.7:50, 5:50, 6:50, or any value therebetween.

[0035] In the present application, in step S2, the polysaccharase enzymolysis treatment refers to the process of using β-mannanase to realize the enzymolysis of the abalone viscera itself. More specifically, the conditions of the polysaccharase enzymolysis treatment include a temperature of preferably 35-60°C, and more specifically, 35°C, 38°C, 39°C, 40°C, 42°C, 45°C, 48°C, 50°C, 55°C, 60°C, or any value therebetween; a pH of preferably 4-9, and more specifically, 4.0, 4.2, 4.3, 4.8, 5.0, 5.2, 5.4, 6.0, 6.5, 7.0, 7.5, 8.0, 9.0, or any value therebetween; and a time of preferably 2-6h, and more specifically, 2h, 2.5h, 2.8h, 3h, 3.5h, 4h, 4.5h, 5h, 6h, or any value therebetween.

[0036] In the present application, in step S3, the mass ratio of the alkaline protease to the abalone viscera is preferably (1-5):50, and more specifically, 1:50, 1.3:50, 1.8:50, 2:50, 2.5:50, 3:50, 4:50, 5:50, or any value therebetween.

[0037] In the present application, in step S3, the protease enzymolysis treatment refers to the process of using alkaline protease to realize the enzymolysis of the polysaccharase enzymolysis liquid. More specifically, the conditions of the protease enzymolysis treatment include a temperature of preferably 35-60°C, and more specifically, 35°C, 38°C, 39°C, 40°C, 43°C, 45°C, 48°C, 50°C, 55°C, 58°C, 60°C, or any value therebetween; a pH of preferably 6-11, and more specifically, 6.0, 6.5, 7.0, 7.8, 9.0, 10.0, 11.0, or any value therebetween; and a time of preferably 3-7h, and more specifically, 3h, 3.5h, 3.8h, 4h, 4.5h, 5h, 6h, 7h, or any value therebetween.

[0038] In the present application, in step S4, the enzyme inactivation treatment refers to a process of inactivating enzyme molecules contained in the protease enzymolysis solution by using high temperature conditions. More specifically, the conditions of the enzyme inactivation treatment include a temperature of preferably 95-105°C, specifically 95°C, 96°C, 97.5°C, 98°C, 99°C, 100°C, 101°C, 105°C, or any value therebetween; and a time of preferably 5-15 min, specifically 5 min, 5.3 min, 5.5 min, 5.8 min, 6 min, 7 min, 8 min, 10 min, 11.5 min, 12 min, 15 min, or any value therebetween.

[0039] In the present application, in step S4, the centrifugal treatment refers to a process of effectively separating polypeptides and polysaccharides and other substances in the protease enzymolysis solution subjected to the enzyme inactivation treatment by using centrifugal force. More specifically, the conditions of the centrifugal treatment include a temperature of preferably 20-25°C, specifically 20°C, 23°C, 24°C, 25°C, or any value therebetween; a rotation speed of preferably 8000-10000 r / min, specifically 8000 r / min, 8500 r / min, 9000 r / min, 9500 r / min, 10000 r / min, or any value therebetween; and a time of preferably 10-20 min, specifically 10 min, 11.5 min, 12 min, 13 min, 15 min, 18 min, 20 min, or any value therebetween.

[0040] In the present application, in step S5, the cell density of the A. oryzae bacterial solution is preferably 1×10 4 CFU / g to 1×10 7 CFU / g, specifically 1×10 4 CFU / g, 4×10 4 CFU / g, 1×10 5 CFU / g, 1×10 6 CFU / g, 1×10 7 CFU / g, or any value therebetween.

[0041] In the present application, in step S5, the addition mass ratio of the A. oryzae bacterial solution to the final enzymolysis solution is preferably (0.8-1.2):1, specifically 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, or any value therebetween.

[0042] In the present application, in step S5, the fermentation culture refers to the process of using Aspergillus oryzae to degrade and transform the substances in the final enzymatic hydrolysate. More specifically, the conditions of the fermentation culture include a temperature of preferably 18-30°C, specifically 18°C, 19°C, 20.5°C, 23°C, 25°C, 28°C, 30°C, or any value therebetween; and a time of preferably 24-48h, specifically 24h, 25h, 28h, 30h, 34h, 36h, 38h, 40h, 42h, 48h, or any value therebetween.

[0043] In the present application, in step S6, the ceramic membrane filtration treatment refers to the process of using the porous structure and pore size selectivity of the ceramic membrane to separate the components in the Aspergillus oryzae fermentation broth. More specifically, the pore size of the ceramic membrane in the ceramic membrane filtration treatment is preferably 40-60nm, specifically 40nm, 40.5nm, 41nm, 42nm, 43nm, 45nm, 48nm, 50nm, 52nm, 55nm, 58nm, 60nm, or any value therebetween.

[0044] In the present application, in step S6, the ultrafiltration membrane concentration treatment refers to the process of using the selective permeability of the ultrafiltration membrane to realize the concentration and purification of polysaccharides and polypeptides in the solution under pressure driving. More specifically, the pore size of the ultrafiltration membrane in the ultrafiltration membrane concentration treatment is preferably 5-10nm, specifically 5nm, 5.5nm, 6nm, 7nm, 8nm, 9nm, 10nm, or any value therebetween.

[0045] In the present application, in step S6, the conditions of the freeze-drying treatment include a temperature of preferably -50- -40°C, specifically -50°C, -48°C, -45°C, -43°C, -41°C, -40°C, or any value therebetween; and a time of 24-50h, specifically 24h, 30h, 42h, 48h, 50h, or any value therebetween.

[0046] The present application also provides a kind of abalone viscera polysaccharide polypeptide composition, which is prepared by the above-mentioned combined preparation method of abalone viscera polysaccharide and polypeptide.

[0047] In the present application, the alpha-glucosidase inhibition rate of the abalone viscera polysaccharide polypeptide composition is preferably 65% to 90%, and specifically can be 65%, 65.87%, 68%, 70%, 72%, 77%, 80%, 80.35%, 86%, 89%, 90% or any value between them; the DPP-IV inhibition rate is preferably 30% to 70%, and specifically can be 30%, 30.5%, 32%, 34%, 38%, 40%, 41%, 43%, 45%, 48%, 50%, 55%, 58%, 60%, 65%, 70% or any value between them.

[0048] The present application also provides the use of the above-mentioned abalone viscera polysaccharide polypeptide composition in the development of hypoglycemic drugs and / or blood sugar-lowering functional foods.

[0049] The embodiments of the present application are described in detail below, and the examples are intended to explain the present application and cannot be understood as a limitation of the present application. If a specific technique or condition is not specified in the embodiments, the technique or condition described in the literature in the art or according to the product manual is used. If the manufacturer of the reagent or instrument is not specified, it is a conventional product that can be obtained by purchase.

[0050] Example 1

[0051] This example is used to illustrate a combined preparation method of abalone viscera polysaccharide and polypeptide, which specifically comprises:

[0052] S1, abalone viscera and water are mixed according to a mass ratio of 1:4, and then subjected to wall breaking treatment (wall breaking machine) and homogenization treatment (homogenizer), and hydrochloric acid (6 mol / L) or sodium hydroxide solution (6 mol / L) is used to adjust the pH of the solution to 5.0 to obtain abalone viscera slurry; the abalone viscera slurry is placed in a water bath at 40°C for 5h to obtain abalone viscera self-enzyme hydrolysis solution.

[0053] S2, according to the mass ratio of abalone viscera to β-mannanase (Shanghai Maikelin Biochemical Technology Co., Ltd., item number G888856) of 10:1, β-mannanase is added to the abalone viscera self-enzyme hydrolysis solution, and hydrochloric acid (6 mol / L) or sodium hydroxide solution (6 mol / L) is used to adjust the pH of the solution to 5.5, and then placed in a water bath at 45°C for 4h to obtain polysaccharase enzyme hydrolysis solution.

[0054] S3, according to the mass ratio of abalone viscera to alkaline protease (Nanning Pangbo Biological Engineering Co., Ltd., item number 2505163) is 25:1, take alkaline protease into polysaccharide enzymolysis enzyme solution, and use hydrochloric acid (6 mol / L) or sodium hydroxide solution (6 mol / L) to adjust the pH of the solution to 8.0, and place it in a water bath at 50℃ for protease enzymolysis treatment for 5h, to obtain a protease enzyme solution.

[0055] S4, take the protease enzyme solution to perform enzyme inactivation treatment at 100℃ for 10min, centrifuge at 9000r / min for 15min, remove the upper layer of oil and the lower layer of precipitate, to obtain the final enzyme solution.

[0056] S5, take the aspergillus oryzae strain (Beinnai Biological, item number BNCC185870) to inoculate in the potato dextrose medium (Shanghai Bomei Biological Technology Co., Ltd., item number BW010), and culture at 28℃ until OD 600 is 1.9, to obtain the aspergillus oryzae liquid; take the final enzyme solution to perform sterilization treatment at 121℃ for 15min and cool to room temperature, take the aspergillus oryzae liquid and the final enzyme solution to mix according to the mass ratio of 1:1, and perform fermentation culture at 25℃ for 36h, to obtain the aspergillus oryzae fermentation liquid.

[0057] S6, take the aspergillus oryzae fermentation liquid to perform ceramic membrane filtration treatment (50nm ceramic membrane), ultrafiltration membrane concentration treatment (8nm ultrafiltration membrane) and freeze-drying treatment (temperature is-45℃, time is 48h) in sequence, to obtain the abalone viscera polysaccharide polypeptide composition.

[0058] Example 2

[0059] The method provided in example 1 is adopted to prepare the abalone viscera polysaccharide polypeptide composition, and the difference lies in that in step S3, the pH value of the solution in the third enzymolysis treatment is 9.0, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0060] Example 3

[0061] The method provided in example 1 is adopted to prepare the abalone viscera polysaccharide polypeptide composition, and the difference lies in that in step S3, the time of the third enzymolysis treatment is 4h, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0062] Example 4

[0063] The method provided in example 1 is adopted to prepare the abalone viscera polysaccharide polypeptide composition, and the difference lies in that in step S3, the mass ratio of abalone viscera to alkaline protease is 50:1, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0064] Example 5

[0065] The embodiment adopts the method provided in Example 1 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the mass ratio of abalone viscera to alkaline protease is 50:3, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0066] Example 6

[0067] The embodiment adopts the method provided in Example 1 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the mass ratio of abalone viscera to alkaline protease is 25:2, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0068] Example 7

[0069] The embodiment adopts the method provided in Example 1 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the mass ratio of abalone viscera to alkaline protease is 10:1, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0070] Example 8

[0071] The embodiment adopts the method provided in Example 2 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the pH value of the solution in the third enzymatic treatment is 6.0, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0072] Example 9

[0073] The embodiment adopts the method provided in Example 2 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the pH value of the solution in the third enzymatic treatment is 7.0, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0074] Example 10

[0075] The embodiment adopts the method provided in Example 2 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the pH value of the solution in the third enzymatic treatment is 10.0, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0076] Example 11

[0077] The embodiment adopts the method provided in Example 3 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the pH value of the solution in the third enzymatic treatment is 11.0, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0078] Example 12

[0079] This example adopts the method provided in Example 3 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the third enzymolysis treatment time is 3h, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0080] Example 13

[0081] This example adopts the method provided in Example 3 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the third enzymolysis treatment time is 6h, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0082] Example 14

[0083] This example adopts the method provided in Example 3 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S3, the third enzymolysis treatment time is 7h, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0084] Comparative Example 1

[0085] This comparative example adopts the method provided in Example 1 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S1, the abalone viscera self-enzyme enzymolysis treatment is not performed, that is, the abalone viscera slurry is directly mixed with β-mannanase for polysaccharide enzyme enzymolysis treatment, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0086] Comparative Example 2

[0087] This comparative example adopts the method provided in Example 1 to prepare the abalone viscera polysaccharide polypeptide composition, except that in step S1, the abalone viscera self-enzyme enzymolysis treatment is replaced by enzyme inactivation treatment, which specifically includes: taking the abalone viscera and water in a mass ratio of 1:4, mixing and performing wall breaking treatment (wall breaking machine) and homogenization treatment (homogenizer), and adjusting the solution pH to 5.0 using hydrochloric acid (6mol / L) or sodium hydroxide solution (6mol / L) to obtain the abalone viscera slurry; taking the abalone viscera slurry, the abalone viscera slurry is subjected to enzyme inactivation treatment at 100℃ for 10min to obtain the enzyme-inactivated abalone viscera slurry, and other conditions remain unchanged, to obtain the abalone viscera polysaccharide polypeptide composition.

[0088] Comparative Example 3

[0089] The comparative example 1 was prepared by the method provided in the example 1, except that the self-enzyme of abalone viscera, polysaccharase and protease were simultaneously subjected to enzymatic hydrolysis treatment, and the specific steps included: S1, abalone viscera and water were mixed at a mass ratio of 1:4, and then subjected to wall breaking treatment (wall breaking machine) and homogenization treatment (homogenizer), to obtain abalone viscera slurry. S2, β-mannanase and alkaline protease were added into the abalone viscera slurry at a mass ratio of 50:1:2, and then subjected to complex enzymatic hydrolysis treatment in a water bath at 45°C for 13h, and then subjected to enzyme inactivation treatment at 100°C for 10min, and then subjected to centrifugation at 9000r / min for 15min, to remove the upper oil and the lower precipitate, to obtain an enzymatic hydrolysate. S3, Aspergillus oryzae was inoculated in a potato glucose medium, and then cultured at 28°C until the OD 600 was 1.9, to obtain Aspergillus oryzae liquid; the enzymatic hydrolysate was sterilized at 121°C for 15min, and then cooled to room temperature, and then mixed with the Aspergillus oryzae liquid at a mass ratio of 1:1, and then subjected to fermentation culture at 25°C for 36h, to obtain Aspergillus oryzae fermentation liquid. S4, the Aspergillus oryzae fermentation liquid was subjected to ceramic membrane filtration treatment (50nm ceramic membrane), ultrafiltration membrane concentration treatment (8nm ultrafiltration membrane) and freeze-drying treatment (temperature of-45°C, time of 48h) in sequence, to obtain abalone viscera polysaccharide polypeptide composition.

[0090] Comparative example 4

[0091] The comparative example 4 was prepared by the method provided in the example 1, except that the fermentation culture was not performed in step S5, that is, the obtained final enzymatic hydrolysate was directly subjected to the operation of step S6, and other conditions were kept the same, to obtain abalone viscera polysaccharide polypeptide composition.

[0092] Test example

[0093] The test example was used to illustrate the hypoglycemic activity of the abalone viscera polysaccharide polypeptide composition provided in the above examples and comparative examples, and the test specifically included:

[0094] 1, Abalone viscera polysaccharide polypeptide composition yield: the abalone viscera polysaccharide polypeptide composition yield (%) was calculated according to the following formula.

[0095] Abalone viscera polysaccharide polypeptide composition yield (%) = m1 / m0x100%

[0096] Wherein, m0 is the added mass of abalone viscera, unit: g; m1 is the mass of the obtained abalone viscera polysaccharide polypeptide composition, unit: g.

[0097] 2. α-glucosidase inhibition rate: Abalone viscera polysaccharide polypeptide composition and PBS solution (0.2M, pH=6.8) were mixed to prepare a test solution with a concentration of 0.25 g / mL. 20 μL of the test solution and 40 μL of PNPG solution (2.5 mM) were mixed evenly and reacted at 37℃ for 15 min. Then, 20 μL of α-glucosidase solution (1 U / mL) was added and mixed evenly. The mixture was reacted at 37℃ for 15 min. After the reaction, the absorbance of the solution was measured at 405 nm, and the α-glucosidase inhibition rate (in %) was calculated according to the following formula.

[0098] α-glucosidase inhibition rate (%) = [1 - (A1 - A3) / (A2 - A4)] × 100%

[0099] Wherein, A1 is the absorbance value measured by the test solution; A2 is the absorbance value measured by replacing 20 μL of the test solution with 20 μL of PBS solution (0.2 M, pH = 6.8) in the reaction; A3 is the absorbance value measured by replacing 20 μL of α-glucosidase solution with 20 μL of PBS solution (0.2 M, pH = 6.8) in the reaction; and A4 is the absorbance value measured by mixing 40 μL of PBS solution (0.2 M, pH = 6.8) and 40 μL of PNPG solution in the reaction.

[0100] 3. DPP-IV Inhibition Rate: Abalone viscera polysaccharide-peptide composition and PBS solution (0.2M, pH=6.8) were mixed to prepare a test solution with a concentration of 0.25 g / mL. 15 μL of the test solution, 50 μL of Tris-HCl buffer (20 mM, pH=8) and 5 μL of DPP-IV were mixed evenly and reacted at 37℃ for 15 min. Then, 30 μL of Gly-Pro-MCA (10 μmol / L) was added and mixed evenly, and reacted at 37℃ for 15 min. After the reaction, the fluorescence intensity was measured using an FP-8200 fluorescence spectrophotometer (excitation wavelength 380 nm, emission wavelength 450 nm), and the DPP-IV inhibition rate (in %) was calculated according to the following formula.

[0101] DPP-IV inhibition rate (%) = [1-(A 实验 -A 空白 ) / (A 对照 -A 空白 )]×100%

[0102] Among them, A 实验 A is the fluorescence intensity measured in the sample solution. 空白 The fluorescence intensity was measured using only 20 μL of Tris-HCl buffer (20 mM, pH = 8) instead of 15 μL of the test solution and 5 μL of DPP-IV; A对照 The fluorescence intensity was measured by using 15 μL of Tris-HCl buffer (20 mM, pH = 8) instead of 15 μL of the test solution. The results are shown in Table 1.

[0103] Table 1.

[0104]

[0105] As can be seen from the results shown in Table 1, compared with Comparative Examples 1-4, the method provided in Examples 1-14 of the present application can achieve efficient preparation of polysaccharides and polypeptides in abalone viscera, and the yield is more than 20%; and the obtained abalone viscera polysaccharide polypeptide composition has excellent inhibitory effect on α-glucosidase and DPP-IV, and has good application prospect in the development of hypoglycemic drugs and / or blood sugar-lowering functional foods.

[0106] Although the embodiments of the present application have been shown and described above, it should be understood that the above embodiments are exemplary and should not be construed as limiting the present application, and those of ordinary skill in the art can make changes, modifications, replacements and variations to the above embodiments without departing from the principles and spirit of the present application within the scope of the present application.

Claims

1. A combined preparation method of abalone viscera polysaccharide and polypeptide, characterized in that, The preparation method comprises the following steps: S1, mixing the abalone viscera with water, performing wall breaking treatment and homogenization treatment to obtain abalone viscera slurry, and performing abalone viscera self-enzyme enzymolysis treatment on the abalone viscera slurry to obtain abalone viscera self-enzyme enzymolysis liquid; S2, mixing β-mannanase and the abalone viscera self-enzyme enzymolysis liquid, and performing polysaccharase enzymolysis treatment to obtain polysaccharase enzymolysis liquid; S3, mixing alkaline protease and the polysaccharase enzymolysis liquid, and performing protease enzymolysis treatment to obtain protease enzymolysis liquid; S4, performing enzyme inactivation treatment and centrifugal treatment on the protease enzymolysis liquid to obtain final enzymolysis liquid; S5, mixing Aspergillus oryzae liquid and the final enzymolysis liquid, and performing fermentation culture to obtain Aspergillus oryzae fermentation liquid; S6, performing ceramic membrane filtration treatment, ultrafiltration membrane concentration treatment and freeze-drying treatment on the Aspergillus oryzae fermentation liquid to obtain abalone viscera polysaccharide and polypeptide composition.

2. The combined preparation method of the abalone viscera polysaccharide and polypeptide according to claim 1, characterized in that, In step S1, the mixing mass ratio of the abalone viscera to water is 1:(1-20); Optionally, the temperature of the abalone viscera self-enzyme enzymolysis treatment is 30-60°C, the pH value is 2-9, and the time is 0.001-7h.

3. The combined preparation method of the abalone viscera polysaccharide and polypeptide according to claim 1, characterized in that, In step S2, the adding mass ratio of the β-mannanase to the abalone viscera is (1-6):50; Optionally, the temperature of the polysaccharase enzymolysis treatment is 35-60°C, the pH value is 4-9, and the time is 2-6h.

4. The combined preparation method of the abalone viscera polysaccharide and polypeptide according to claim 1, characterized in that, In step S3, the adding mass ratio of the alkaline protease to the abalone viscera is (1-5):50; Optionally, the temperature of the protease enzymolysis treatment is 35-60°C, the pH value is 6-11, and the time is 3-7h.

5. The combined preparation method of the abalone viscera polysaccharide and polypeptide according to claim 1, characterized in that, In step S4, the temperature of the enzyme inactivation treatment is 95-105°C, and the time is 5-15min; Optionally, the temperature of the centrifugal treatment is 20-25°C, the rotating speed is 8000-10000r / min, and the time is 10-20min.

6. The combined preparation method of the abalone viscera polysaccharide and polypeptide according to claim 1, characterized in that, In step S5, the cell density of the Aspergillus oryzae bacterial solution is 1 x 10 4 CFU / g ~ 1 x 10 7 CFU / g; Optionally, the adding mass ratio of the Aspergillus oryzae liquid to the final enzymolysis liquid is (0.8-1.2):1; Optionally, the temperature of the fermentation culture is 18-30°C, and the time is 24-48h.

7. The combined preparation method of the abalone viscera polysaccharide and polypeptide according to claim 1, characterized in that, In step S6, the pore size of the ceramic membrane in the ceramic membrane filtration treatment is 40-60nm; Optionally, the pore size of the ultrafiltration membrane in the ultrafiltration membrane concentration treatment is 5-10nm; Optionally, the temperature of the freeze-drying treatment is -50--40°C, and the time is 24-50h.

8. A polypeptide composition of abalone visceral glycan, characterized in that, The abalone viscera polysaccharide and polypeptide composition is prepared by the combined preparation method of the abalone viscera polysaccharide and polypeptide according to any one of claims 1-7.

9. The polypeptide composition of claim 8, wherein the polypeptide composition is a polypeptide composition of a polyprenolsaccharide from the viscera of abalone. The α-glucosidase inhibition rate of the abalone viscera polysaccharide and polypeptide composition is 65-90%, and the DPP-IV inhibition rate is 30-70%.

10. The abalone viscera polysaccharide and polypeptide composition according to claim 8 or 9 is applied to the development of hypoglycemic drugs and / or blood sugar reducing functional foods.

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