Method for preparing sea cucumber polysaccharide by using sea cucumber byproducts

Abstract

The invention relates to the technical field of efficient development and utilization of marine biological resources, in particular to a method for preparing sea cucumber polysaccharide from sea cucumber byproducts. The sea cucumber polysaccharide is prepared by performing protease enzymolysis, alcohol precipitation and the like on sea cucumber byproducts to obtain sea cucumber crude polysaccharide, and performing hydrogen peroxide, ascorbic acid degradation and the like. According to the method, the recycling problem of waste sea cucumber byproducts is solved, the low-molecular-weight sea cucumber polysaccharide with definite alpha-amylase and alpha-glucosidase inhibitory activity is prepared, the hypoglycemic activity of the low-molecular-weight sea cucumber polysaccharide is remarkably improved, and a technical support is provided for development of functional foods.

Description

Technical Field

[0001] The invention relates to the technical field of efficient development and utilization of marine biological resources, and in particular to a method for preparing sea cucumber polysaccharides by utilizing sea cucumber byproducts. Background Art

[0002] Sea cucumber byproducts (such as viscera, blanching liquid, etc.) are often regarded as waste during the processing of sea cucumbers. Large-scale disposal not only pollutes the environment, but also causes a waste of high nutritional ingredients. These byproducts are rich in polysaccharides, proteins and other active substances, but there is a lack of high-purity sea cucumber polysaccharide raw materials on the market, and the absorption and utilization rate of high-molecular-weight sea cucumber polysaccharides in the human body is limited, which limits its development potential in the field of high-value-added functional foods and health products.

[0003] Traditional polysaccharide extraction methods often use traditional processes such as alkaline extraction and hot water extraction, which have the disadvantages of low extraction efficiency, complex process and unstable polysaccharide activity, making it difficult to prepare high-purity and high-activity products. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a method for preparing sea cucumber polysaccharides using sea cucumber by-products, which not only solves the problem of resource utilization of discarded sea cucumber by-products, but also prepares low molecular weight sea cucumber polysaccharides with clear α-amylase and α-glucosidase inhibitory activity, significantly improving its blood sugar lowering activity, and providing technical support for the development of functional foods.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides a method for preparing sea cucumber polysaccharides using sea cucumber by-products, comprising the following steps:

[0007] 1) hydrolyzing the sea cucumber by-products with protease, inactivating the enzyme and centrifuging to obtain a supernatant;

[0008] 2) subjecting the supernatant obtained in step 1) to alcohol precipitation, standing, and centrifuging to obtain a precipitate;

[0009] 3) dialyzing the precipitate obtained in step 2) using a dialysis bag with a molecular weight of 8 to 14 kDa, and freeze-drying to obtain crude sea cucumber polysaccharides;

[0010] 4) preparing the crude sea cucumber polysaccharide obtained in step 3) into a crude sea cucumber polysaccharide solution, mixing it with hydrogen peroxide and ascorbic acid to obtain a mixture, and degrading the mixture to obtain a degraded material;

[0011] The concentration of the sea cucumber crude polysaccharide solution is 5 mg / mL;

[0012] The concentration of hydrogen peroxide in the mixture is 10 to 30 mmol / L;

[0013] The concentration of ascorbic acid in the mixture is 10 to 30 mmol / L;

[0014] The degradation conditions include: temperature of 30-70°C and time of 0.5-2.5h;

[0015] 5) adjusting the pH value of the degradation material in step 4) to neutral, and sequentially performing alcohol precipitation, standing and centrifugation to obtain a precipitate;

[0016] 6) The precipitate obtained in step 5) is dialyzed using a dialysis bag with a molecular weight of 500 Da, and freeze-dried to obtain sea cucumber polysaccharides.

[0017] Preferably, the concentration of hydrogen peroxide in the mixture of step 4) is 20 mmol / L, and the concentration of ascorbic acid is 20 mmol / L.

[0018] Preferably, the concentration of hydrogen peroxide in the mixture of step 4) is 20.52 mmol / L, and the concentration of ascorbic acid is 20.52 mmol / L.

[0019] Preferably, the degradation conditions in step 4) include: temperature of 50° C. and time of 1 h.

[0020] Preferably, the degradation conditions in step 4) include: temperature of 51° C. and time of 0.95 h.

[0021] Preferably, the protease in step 1) comprises papain;

[0022] The conditions of the enzymatic hydrolysis include: temperature of 60°C and time of 24h;

[0023] The sea cucumber by-product is mixed with a buffer and a protease and then enzymolyzed, wherein the mass ratio of the sea cucumber by-product to the buffer is 1 g:20 mL; the mass percentage of the protease in the buffer is 10%;

[0024] The concentration of ethylenediaminetetraacetic acid in the buffer is 5 mmol / L, the concentration of cysteine ​​is 5 mmol / L, the concentration of sodium acetate is 0.1 mol / L, and the pH value of the buffer is 6.0;

[0025] The enzyme inactivation conditions include: temperature of 100°C and time of 15 min;

[0026] The centrifugal conditions include: a rotation speed of 6000 rpm and a time of 10 min.

[0027] Preferably, the conditions of the step 2) of alcohol precipitation include: mixing the supernatant and anhydrous ethanol and then performing alcohol precipitation, wherein the volume ratio of the supernatant to the anhydrous ethanol is 1:4;

[0028] The standing conditions include: standing overnight at 4°C;

[0029] The centrifugal conditions include: centrifugation at 4000 r / min for 10 min.

[0030] Preferably, the freeze-drying conditions of steps 3) and 6) both include freeze-drying at -80°C for 12 hours.

[0031] Preferably, in step 5), a 0.01 mol / L sodium hydroxide solution is used to adjust the pH value of the degradation material;

[0032] The alcohol precipitation conditions include: mixing the degradation material and anhydrous ethanol and then precipitating with alcohol, wherein the volume ratio of the degradation material to the anhydrous ethanol is 1:4;

[0033] The standing conditions include: standing overnight at 4°C;

[0034] The centrifugal conditions include: centrifugation at 4000 r / min for 10 min.

[0035] Preferably, the freeze-drying step 5) is followed by purification to obtain sea cucumber polysaccharides;

[0036] The purification conditions include: using a 100kDa ultrafiltration membrane for classification, selecting a 24×500mm chromatography column, an elution flow rate of 0.5mL / min, separation and purification through a Sephadex G-100 dextran gel column and a Sephadex G-10 dextran gel column, determining the absorbance using a phenol-sulfuric acid method, drawing an elution curve and collecting, and freeze-drying after dialysis to obtain sea cucumber polysaccharides.

[0037] The core innovation of the present invention is to use enzymatic hydrolysis combined with green and efficient non-metallic Fenton reaction technology to remove protein components through enzymatic hydrolysis, generate hydroxyl radicals through hydrogen peroxide and ascorbic acid, selectively break the glycosidic bonds of polysaccharide chains, and retain their key active groups (such as sulfate groups). Compared with traditional physical, chemical and biological degradation methods, this combined method has the following significant advantages:

[0038] Mild reaction conditions: Avoid high temperature or extreme pH environment that may damage the activity of polysaccharides.

[0039] High degradation efficiency: rapid reduction of polysaccharide molecular weight and improvement of absorption and utilization rate in the human intestine.

[0040] Environmentally friendly and pollution-free: Abandoning metal ion catalysis to avoid potential harm to the environment and products.

[0041] Through this innovative process, not only the resource problem of discarded sea cucumber by-products is solved, but also low molecular weight sea cucumber polysaccharides with clear α-amylase and α-glucosidase inhibitory activity are prepared, which significantly improves its blood sugar-lowering activity and provides technical support for the development of functional foods.

[0042] In addition, compared with conventional methods, the non-metallic Fenton reaction system significantly improves the purity and biological activity of the product without destroying the active structural groups and functionality of sea cucumber polysaccharides. The development of this efficient preparation method provides a theoretical basis and technical foundation for promoting the transformation and upgrading of the sea cucumber deep processing industry and the high-value utilization of low-value by-products.

[0043] Improved hypoglycemic activity: After Fenton degradation, the inhibition rate of sea cucumber polysaccharides on α-amylase reached 56.23%, and the inhibition rate on α-glucosidase was as high as 90.22%, which were significantly higher than those of untreated crude polysaccharides.

[0044] High degradation efficiency: The degradation rate of polysaccharides reaches 90%, and the degraded polysaccharides are more active and show concentration dependence.

[0045] Purity and structure optimization: The total sugar content of the purified sea cucumber polysaccharide is as high as 86.39%, the sulfate content is 20.90%, and the degree of sulfation is high. It can significantly promote the expression of key genes in glucose metabolism and stabilize blood sugar levels.

[0046] Industrialization and economic value: Using low-cost sea cucumber by-products as raw materials can significantly reduce production costs.

[0047] The extraction process is green and environmentally friendly, easy to scale up for production, and has significant economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0049] Figure 1 The effects of different degradation conditions on the hypoglycemic ability and degradation rate of sea cucumber polysaccharides in vitro;

[0050] Figure 2 This is the infrared spectrum of sea cucumber polysaccharide;

[0051] Figure 3 is the particle size distribution diagram of sea cucumber polysaccharide;

[0052] Figure 4 This is the gel chromatogram of separation and purification of sea cucumber polysaccharide;

[0053] Figure 5 This is the liquid chromatogram of sea cucumber polysaccharide. DETAILED DESCRIPTION

[0054] The present invention provides a method for preparing sea cucumber polysaccharides using sea cucumber by-products, comprising the following steps:

[0055] 1) hydrolyzing the sea cucumber by-products with protease, inactivating the enzyme and centrifuging to obtain a supernatant;

[0056] 2) subjecting the supernatant obtained in step 1) to alcohol precipitation, standing, and centrifuging to obtain a precipitate;

[0057] 3) dialyzing the precipitate obtained in step 2) using a dialysis bag with a molecular weight of 8 to 14 kDa, and freeze-drying to obtain crude sea cucumber polysaccharides;

[0058] 4) preparing the crude sea cucumber polysaccharide obtained in step 3) into a crude sea cucumber polysaccharide solution, mixing it with hydrogen peroxide and ascorbic acid to obtain a mixture, and degrading the mixture to obtain a degraded material;

[0059] The concentration of the sea cucumber crude polysaccharide solution is 5 mg / mL;

[0060] The concentration of hydrogen peroxide in the mixture is 10 to 30 mmol / L;

[0061] The concentration of ascorbic acid in the mixture is 10 to 30 mmol / L;

[0062] The degradation conditions include: temperature of 30-70°C and time of 0.5-2.5h;

[0063] 5) adjusting the pH value of the degradation material in step 4) to neutral, and sequentially performing alcohol precipitation, standing and centrifugation to obtain a precipitate;

[0064] 6) The precipitate obtained in step 5) is dialyzed using a dialysis bag with a molecular weight of 500 Da, and freeze-dried to obtain sea cucumber polysaccharides.

[0065] The present invention hydrolyzes the sea cucumber by-products with protease, inactivates the enzyme and centrifuges to obtain a supernatant. In the present invention, the sea cucumber by-products are preferably powders of sea cucumber viscera and sea cucumber blanching liquid concentrated and dried. In the present invention, the protease preferably includes papain. In the present invention, the mass percentage of the protease in the buffer is preferably 10%. In the present invention, the conditions for the enzymatic hydrolysis preferably include: a temperature of 60°C and a time of 24h. In the present invention, the sea cucumber by-products are mixed with a buffer and a protease and then enzymolyzed, and the mass ratio of the sea cucumber by-products to the volume of the buffer is 1g:20mL. In the present invention, the concentration of ethylenediaminetetraacetic acid in the buffer is preferably 5mmol / L, the concentration of cysteine ​​is preferably 5mmol / L, the concentration of sodium acetate is preferably 0.1mol / L, and the pH value of the buffer is preferably 6.0. In the present invention, the conditions for inactivating the enzyme preferably include: a temperature of 100°C and a time of 15min. In the present invention, the conditions for centrifugation preferably include: a rotation speed of 6000rpm and a time of 10min.

[0066] The present invention performs alcohol precipitation on the obtained supernatant, allows it to stand, and centrifuges to obtain a precipitate. In the present invention, the alcohol precipitation conditions preferably include: mixing the supernatant and anhydrous ethanol and then performing alcohol precipitation, wherein the volume ratio of the supernatant to the anhydrous ethanol is 1:4. In the present invention, the standing conditions preferably include: standing overnight at 4°C. In the present invention, the centrifugal conditions preferably include: centrifugation at 4000r / min for 10min.

[0067] The present invention dialyzes the obtained precipitate using a dialysis bag with a molecular weight of 8 to 14 kDa, and freeze-dries to obtain crude sea cucumber polysaccharides. In the present invention, the freeze-drying conditions preferably include freeze-drying at -80°C for 12 hours.

[0068] The present invention prepares the obtained sea cucumber crude polysaccharide into a sea cucumber crude polysaccharide solution, and then mixes it with hydrogen peroxide and ascorbic acid to obtain a mixture, and degrades the mixture to obtain a degraded material; the concentration of the sea cucumber crude polysaccharide solution is 5 mg / mL; the concentration of hydrogen peroxide in the mixture is 10-30 mmol / L; the concentration of ascorbic acid in the mixture is 10-30 mmol / L; the degradation conditions include: temperature of 30-70°C, time of 0.5-2.5h. In the present invention, the concentration of hydrogen peroxide in the mixture is preferably 20 mmol / L, and the concentration of ascorbic acid is preferably 20 mmol / L. In the present invention, the concentration of hydrogen peroxide in the mixture is preferably 20.52 mmol / L, and the concentration of ascorbic acid is preferably 20.52 mmol / L. In the present invention, the degradation conditions preferably include: temperature of 50°C, time of 1h. In the present invention, the degradation conditions preferably include: temperature of 51°C, time of 0.95h.

[0069] The present invention adjusts the pH value of the degradation material to neutral, and sequentially performs alcohol precipitation, standing and centrifugation to obtain a precipitate. The present invention preferably uses a sodium hydroxide solution with a concentration of 0.01 mol / L to adjust the pH value of the degradation material. In the present invention, the alcohol precipitation conditions preferably include: mixing the degradation material and anhydrous ethanol and then precipitating with alcohol, and the volume ratio of the degradation material to anhydrous ethanol is preferably 1:4. In the present invention, the standing conditions preferably include: standing overnight at 4°C. In the present invention, the centrifugal conditions preferably include: centrifugation at 4000r / min for 10min.

[0070] The present invention dialyzes the obtained precipitate using a dialysis bag with a molecular weight of 500Da, freeze-dries, and obtains sea cucumber polysaccharides. In the present invention, the freeze-drying conditions preferably include: freeze-drying at -80°C for 12 hours. The present invention preferably also purifies after freeze-drying to obtain sea cucumber polysaccharides; the purification conditions include: using a 100kDa ultrafiltration membrane for classification, selecting a 24×500mm chromatography column, an elution flow rate of 0.5mL / min, separating and purifying through a Sephadex G-100 dextran gel column and a Sephadex G-10 dextran gel column, determining the absorbance using a phenol-sulfuric acid method, drawing an elution curve and collecting, and freeze-drying after dialysis to obtain sea cucumber polysaccharides.

[0071] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0072] In the following examples, the sea cucumber by-products are sea cucumber viscera and powder obtained by concentrating and drying the sea cucumber blanching liquid.

[0073] Sodium chloride, acetic acid, sodium hydroxide, phenol, anhydrous ethanol, hydrochloric acid, concentrated sulfuric acid, dextran standard, gel filtration filler, α-amylase, dinitrosalicylic acid, phosphate buffer, p-nitrophenol glucopyranoside, sodium carbonate, acarbose, ascorbic acid, hydrogen peroxide, 1-phenyl-3-methyl-5-pyrazolone, trifluoroacetic acid, and methanol were all analytically pure; α-amylase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; papain and alkaline protease were purchased from Tianjin Taijin Technology Co., Ltd. Dextran standard and gel filtration filler were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0074] Instruments and equipment: High performance liquid chromatograph (1260infinity), Agilent Technologies Co., Ltd.; scanning electron microscope (SU-1510), Hitachi, Japan; thermogravimetric analyzer (TGA-Q50), TA Instruments, USA; desktop high-speed refrigerated centrifuge (Neofuge 13R), Heal Force Development Ltd; pulverizer (30b), Changzhou Panfeng Drying Equipment Co., Ltd.; multi-function microplate reader (Epoch2), ThermoFisher Scientific, USA; Fourier transform infrared spectrometer (IS50), ThermoFisher Scientific, USA.

[0075] Example 1

[0076] Extraction of crude polysaccharides from sea cucumber:

[0077] Take 1g of sea cucumber powder and dissolve it in 20mL of 0.1M sodium acetate solution (pH6.0) containing 5mM EDTA and 5mM cysteine, and use 10% papain by mass to enzymolyze at 60℃ for 24h. Inactivate the enzyme at 100℃ for 15min, centrifuge at 6000r / min for 10min, and take the supernatant. Add anhydrous ethanol with a volume ratio of 1:4 to the enzymatic supernatant for alcohol precipitation, and let it stand at 4℃ overnight. After standing, centrifuge at 4000r / min for 10min, dialyze the obtained precipitate with 8kDa-14kDa dialysis bag, and freeze-dry the polysaccharide at -80℃ for 12h after dialysis to obtain crude sea cucumber polysaccharide.

[0078] Degradation of sea cucumber crude polysaccharides by non-metallic Fenton reaction:

[0079] Prepare a 5 mg / mL sea cucumber crude polysaccharide solution, add 20 mM hydrogen peroxide and ascorbic acid, and the molar ratio of the two reagents is 1:1. After degradation at 50°C for 1 hour, adjust the solution pH to neutral with 0.01 M sodium hydroxide solution, add 4 times the volume of anhydrous ethanol, and place it at 4°C overnight. Centrifuge at 4000 r / min for 10 minutes, dialyze the obtained precipitate with a 500Da dialysis bag, and freeze-dry at -80°C for 12 hours to obtain the degraded sea cucumber polysaccharide.

[0080] Purification of sea cucumber polysaccharide degradation products

[0081] The sea cucumber polysaccharides before and after degradation were graded according to molecular weight using an ultrafiltration membrane system, and after the graded, they were separated using a dextran gel column, and the components with a single symmetrical peak in the liquid chromatogram were purified to obtain degraded sea cucumber polysaccharides. Detailed steps: The sea cucumber polysaccharides were graded using a 100 kDa ultrafiltration membrane, and after ultrafiltration, a 24×500 mm chromatography column was selected, and the elution flow rate was 0.5 mL / min. The polysaccharides were separated and purified using a Sephadex G-100 dextran gel column and a Sephadex G-10 dextran gel column. The absorbance of the purified separated components was determined using a phenol-sulfuric acid method, and the elution curve was drawn and the separated components were collected, dialyzed using a 500 Da dialysis bag, and freeze-dried at -80°C for 12 hours after dialysis to obtain purified sea cucumber polysaccharides and degraded sea cucumber polysaccharides.

[0082] Example 2

[0083] Example 2 is similar to Example 1, except that hydrogen peroxide and ascorbic acid solutions with a concentration of 10 mM are added.

[0084] Example 3

[0085] Example 3 is similar to Example 1, except that hydrogen peroxide and ascorbic acid solutions with a concentration of 15 mM are added.

[0086] Example 4

[0087] Example 4 is similar to Example 1, except that hydrogen peroxide and ascorbic acid solutions with a concentration of 25 mM are added.

[0088] Example 5

[0089] Example 5 is similar to Example 1, except that hydrogen peroxide and ascorbic acid solutions with concentrations of 30 mM are added.

[0090] Example 6

[0091] Example 6 is similar to Example 1, except that the degradation time is 0.5 h.

[0092] Example 7

[0093] Example 7 is similar to Example 1, except that the degradation time is 1.5 h.

[0094] Example 8

[0095] Example 8 is similar to Example 1, except that the degradation time is 2 h.

[0096] Example 9

[0097] Example 9 is similar to Example 1, except that the degradation time is 2.5 h.

[0098] Example 10

[0099] Example 10 is similar to Example 1, except that it is degraded at 30°C.

[0100] Embodiment 11

[0101] Example 11 is similar to Example 1, except that it is degraded at 40°C.

[0102] Example 12

[0103] Example 12 is similar to Example 1, except that it is degraded at 60°C.

[0104] Example 13

[0105] Example 13 is similar to Example 1, except that it is degraded at 70°C.

[0106] Comparative Example 1

[0107] Comparative Example 1 is similar to Example 1, except that hydrogen peroxide and ascorbic acid are not added.

[0108] Comparative Example 2

[0109] Comparative Example 2 is similar to Example 1, except that dilute sulfuric acid is added, the pH value is adjusted to 2, the degradation temperature is 95° C., and the degradation time is 1 h.

[0110] α-Amylase inhibitory activity assay

[0111] Acarbose was used as a positive control. The purified sea cucumber crude polysaccharides and degraded polysaccharide samples were prepared into different concentrations (2mg / mL, 4mg / mL, 6mg / mL, 8mg / mL, 10mg / mL), 0.3mL was added to 1mL phosphate buffer and 1mL α-amylase solution, and reacted in a water bath at 37°C for 10 minutes. Add 1mL 1% starch solution and continue the water bath reaction for 20 minutes. Add 5mL 3,5-dinitrosalicylic acid (DNS) solution to terminate the reaction and cool rapidly. Measure the absorbance value A1 at a wavelength of 540nm. Replace the enzyme solution with distilled water to measure A2. Replace the sample solution with distilled water to measure A3. Replace both the sample solution and the enzyme solution with distilled water as a blank control A4. Inhibition rate calculation formula:

[0112]

[0113] Table 1 α-amylase inhibition rate and degradation rate of sea cucumber polysaccharides

[0114] sample α-Amylase inhibition rate (%) Degradation rate (%) Example 1 <![CDATA[56.70±0.27 a ]]> <![CDATA[60.26±0.13 a ]]> Example 2 <![CDATA[20.54±0.13 f ]]> <![CDATA[29.74±0.21 g ]]> Example 3 <![CDATA[33.20±0.35 c ]]> <![CDATA[39.75±0.04 f ]]> Example 4 <![CDATA[40.33±0.11 b ]]> <![CDATA[56.09±0.06 b ]]> Example 5 <![CDATA[37.44±0.21 c ]]> <![CDATA[55.03±0.06 b ]]> Example 6 <![CDATA[26.86±0.19 d ]]> <![CDATA[43.97±0.11 d ]]> Example 7 <![CDATA[30.28±0.27 d ]]> <![CDATA[52.40±0.37 b ]]> Example 8 <![CDATA[27.43±0.13 d ]]> <![CDATA[51.92±0.33 b ]]> Example 9 <![CDATA[24.33±0.12 e ]]> <![CDATA[48.77±0.27 c ]]> Example 10 <![CDATA[26.33±0.17 d ]]> <![CDATA[50.08±0.29 c ]]> Embodiment 11 <![CDATA[28.69±0.23 d ]]> <![CDATA[57.34±0.35 b ]]> Example 12 <![CDATA[26.42±0.09 d ]]> <![CDATA[48.76±0.51 c ]]> Example 13 <![CDATA[24.83±0.13 d ]]> <![CDATA[47.13±0.42 c <!-- 6 -->]]> Comparative Example 1 <![CDATA[1.24±0.30 g ]]> <![CDATA[0.95±0.04 h ]]> Comparative Example 2 <![CDATA[21.92±0.13 f ]]> <![CDATA[27.74±0.31 g ]]>

[0115] (1) Effect of hydrogen peroxide and ascorbic acid solution concentration

[0116] As shown in Table 1, when the concentration of hydrogen peroxide and ascorbic acid solution in Example 1 was 20 mmol / L, the degradation rate of sea cucumber polysaccharide reached a maximum value of 60.26%, and the inhibition rate of α-amylase reached a maximum value of 56.07%. Compared with other examples, it was found that the reaction rate was accelerated by appropriately increasing the concentration of the degradation agent. When the concentration exceeded 20 mmol / L in Example 1, the inhibition rate decreased, which may be due to the intensification of the free radical oxidation reaction, resulting in desulfurization and decarboxylation of the polysaccharide.

[0117] (2) Influence of degradation time

[0118] When the degradation time of Example 1 is 1h, the degradation rate reaches a maximum value of 60.26%, and the α-amylase inhibition rate reaches a maximum value of 56.07%. Compared with other examples, it is shown that as the reaction time is prolonged, the hydrolysis rate of sea cucumber polysaccharides and the inhibition rate of α-amylase show a trend of first increasing and then decreasing. After 1h of degradation, due to the presence of residual hydroxyl radicals in the oxidation degradation reaction system of hydrogen peroxide and ascorbic acid solution, they react with the hydrolysis product, resulting in a decrease in the hydrolysis rate of polysaccharides, and the molecular weight of polysaccharides no longer decreases or the molecular weight decrease trend slows down. Therefore, the explanation time of Example 1 is selected as the optimal reaction time.

[0119] (3) Effect of degradation temperature

[0120] When the degradation temperature of Example 1 reaches 50°C, the degradation rate of polysaccharides reaches a maximum value of 64.08%, and the inhibition rate of α-amylase reaches 56.70%. The comparison of Examples 10-13 shows that the degradation rate increases rapidly with the increase of temperature. The increase of temperature will lead to an increase in the reaction rate, and more collisions per unit time form a higher average molecular kinetic energy. When the temperature is higher than 50°C in Example 1, the hydrolysis rate and inhibition rate begin to decrease. The reason is that the temperature is too high, the activity of ascorbic acid decreases and the hydrogen peroxide partially decomposes, resulting in a decrease in the content of hydroxyl radicals in the reaction system, a decrease in the ability to degrade polysaccharides, and a decrease in the hydrolysis rate. Properly increasing the reaction temperature is conducive to the enhancement of the degradation effect and activity of polysaccharides.

[0121] The results showed that Example 1 had the best degradation effect, under which the hydrolysis rate of polysaccharide and the inhibition rate of α-amylase were the highest. The optimal degradation conditions were determined to be 20 mM concentration of hydrogen peroxide and ascorbic acid solution, 50°C degradation temperature, and 1 h degradation time.

[0122] Table 2 Chemical composition and molecular weight particle size of sea cucumber polysaccharides before and after degradation

[0123]

[0124]

[0125] The process conditions of Example 1 were used to degrade sea cucumber polysaccharides, and the main indicators were further separated and purified to determine the results: the total sugar content of sea cucumber polysaccharides after degradation was as high as 89.72%, indicating that the polysaccharide extraction purity was high, the uronic acid content of the polysaccharide was reduced by 4.12%, the protein content was low, and the difference in sulfate content before and after degradation was small, indicating that the sulfate esters of the extracted polysaccharides had a high retention rate during the degradation process and were not destroyed, resulting in a decrease in the protein content and uronic acid content of the polysaccharide with a high molecular weight and carrying a negative charge. The molecular weight of crude sea cucumber polysaccharides is 1862.20 kDa, and the degraded polysaccharides are reduced to 439.66 kDa. At the same time, as the molecular weight decreases, the particle size of the sea cucumber polysaccharides after degradation is significantly reduced to 186.83 nm, and the smaller particle size verifies the reduction in the molecular weight of the polysaccharide. The sea cucumber polysaccharides were subjected to ultrafiltration and classification treatment ( Figure 4 ), and the obtained liquid chromatogram of the purified degraded sea cucumber polysaccharide was ( Figure 5 ), this component is a single peak symmetrical peak, indicating that the purification effect is good and the degraded sea cucumber polysaccharide is successfully obtained. The results show that the target of hydroxyl radical degradation is uronic acid, and it hardly attacks the sugar chain branches and sulfate esters necessary for its biological activity, and the molecular weight of the polysaccharide is also effectively reduced. The above analysis shows that the degradation results of Example 1 are true and reliable.

[0126] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing sea cucumber polysaccharides using sea cucumber by-products, characterized in that: The following steps are involved: 1) hydrolyzing the sea cucumber by-products with protease, inactivating the enzyme and centrifuging to obtain a supernatant; 2) subjecting the supernatant obtained in step 1) to alcohol precipitation, standing, and centrifuging to obtain a precipitate; 3) dialyzing the precipitate obtained in step 2) using a dialysis bag with a molecular weight of 8 to 14 kDa, and freeze-drying to obtain crude sea cucumber polysaccharides; 4) preparing the crude sea cucumber polysaccharide obtained in step 3) into a crude sea cucumber polysaccharide solution, mixing it with hydrogen peroxide and ascorbic acid to obtain a mixture, and degrading the mixture to obtain a degraded material; The concentration of the sea cucumber crude polysaccharide solution is 5 mg / mL; The concentration of hydrogen peroxide in the mixture is 10 to 30 mmol / L; The concentration of ascorbic acid in the mixture is 10 to 30 mmol / L; The degradation conditions include: temperature of 30-70°C and time of 0.5-2.5h; 5) adjusting the pH value of the degradation material in step 4) to neutral, and sequentially performing alcohol precipitation, standing and centrifugation to obtain a precipitate; 6) The precipitate obtained in step 5) is dialyzed using a dialysis bag with a molecular weight of 500 Da, and freeze-dried to obtain sea cucumber polysaccharides.

2. The method according to claim 1, characterized in that: In the step 4), the concentration of hydrogen peroxide in the mixture is 20 mmol / L, and the concentration of ascorbic acid is 20 mmol / L.

3. The method according to claim 1, characterized in that In the step 4), the concentration of hydrogen peroxide in the mixture is 20.52 mmol / L, and the concentration of ascorbic acid is 20.52 mmol / L.

4. The method according to claim 1, characterized in that The conditions for the degradation in step 4) include: a temperature of 50° C. and a time of 1 hour.

5. The method according to claim 1, characterized in that The degradation conditions of step 4) include: temperature of 51° C. and time of 0.95 h.

6. The method according to claim 1, characterized in that The protease in step 1) includes papain; The conditions of the enzymatic hydrolysis include: temperature of 60°C and time of 24h; The sea cucumber by-product is mixed with a buffer and a protease and then enzymolyzed, wherein the mass ratio of the sea cucumber by-product to the buffer is 1 g:20 mL; the mass percentage of the protease in the buffer is 10%; The concentration of ethylenediaminetetraacetic acid in the buffer is 5 mmol / L, the concentration of cysteine ​​is 5 mmol / L, the concentration of sodium acetate is 0.1 mol / L, and the pH value of the buffer is 6.0; The enzyme inactivation conditions include: temperature of 100°C and time of 15 min; The centrifugal conditions include: a rotation speed of 6000 rpm and a time of 10 min.

7. The method according to claim 1, characterized in that The conditions of step 2) alcohol precipitation include: mixing the supernatant and anhydrous ethanol and then performing alcohol precipitation, wherein the volume ratio of the supernatant to the anhydrous ethanol is 1:4; The standing conditions include: standing overnight at 4°C; The centrifugal conditions include: centrifugation at 4000 r / min for 10 min.

8. The method according to claim 1, characterized in that The freeze-drying conditions of steps 3) and 6) both include: freeze-drying at -80°C for 12 hours.

9. The method according to claim 1, characterized in that: In the step 5), a 0.01 mol / L sodium hydroxide solution is used to adjust the pH value of the degradation material; The alcohol precipitation conditions include: mixing the degradation material and anhydrous ethanol and then precipitating with alcohol, wherein the volume ratio of the degradation material to the anhydrous ethanol is 1:4; The standing conditions include: standing overnight at 4°C; The centrifugal conditions include: centrifugation at 4000 r / min for 10 min.

10. The method according to claim 1, characterized in that The step 5) is further subjected to purification after freeze drying to obtain sea cucumber polysaccharide; The purification conditions include: using a 100kDa ultrafiltration membrane for classification, selecting a 24×500mm chromatography column, an elution flow rate of 0.5mL / min, separation and purification through a Sephadex G-100 dextran gel column and a Sephadex G-10 dextran gel column, determining the absorbance using a phenol-sulfuric acid method, drawing an elution curve and collecting, and freeze-drying after dialysis to obtain sea cucumber polysaccharides.

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