A method for preparing sea cucumber polysaccharides using sea cucumber by-products

Abstract

The present 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 using sea cucumber by-products. The present invention obtains sea cucumber crude polysaccharides by subjecting sea cucumber by-products to steps such as protease hydrolysis and alcohol precipitation, and then prepares sea cucumber polysaccharides through steps such as hydrogen peroxide and ascorbic acid degradation. The present invention not only solves the resource problem 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 improves its hypoglycemic activity, and provides technical support for the development of functional foods.

Description

Technical Field

[0001] The present 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 by-products. Background Art

[0002] Sea cucumber by-products (such as viscera and blanching liquid) are often considered waste during the processing of sea cucumbers. Discarding them in large quantities not only pollutes the environment but also results in the waste of highly nutritious ingredients. These by-products 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 high molecular weight sea cucumber polysaccharides have limited absorption and utilization in the human body, limiting their 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, allowing it to stand, and centrifuging it 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 polysaccharide;

[0010] 4) preparing the crude sea cucumber polysaccharide obtained in step 3) into a crude sea cucumber polysaccharide solution, mixing the solution 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 to 70° C. and time of 0.5 to 2.5 h;

[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 polysaccharide.

[0017] Preferably, the concentration of hydrogen peroxide in the mixture in 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 in 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 hour.

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

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

[0024] The concentration of EDTA in the buffer solution 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 solution is 6.0;

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

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

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

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

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

[0030] Preferably, the freeze-drying conditions in 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 at 4°C overnight;

[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, measuring the absorbance using a phenol-sulfuric acid method, drawing an elution curve and collecting the eluted product, and performing freeze-drying after dialysis to obtain sea cucumber polysaccharide.

[0037] The core innovation of this invention is the use of enzymatic hydrolysis combined with the green and efficient non-metallic Fenton reaction technology. By enzymatically removing the protein component, hydrogen peroxide and ascorbic acid generate hydroxyl radicals, which selectively break the glycosidic bonds of the polysaccharide chains while retaining 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 damages the activity of polysaccharides.

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

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

[0041] Through this innovative process, not only the resource utilization 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 their blood sugar-lowering activity and provides technical support for the development of functional foods.

[0042] Furthermore, compared with conventional methods, the metal-free Fenton reaction system significantly improves product purity and bioactivity without destroying the active structural groups and functionality of sea cucumber polysaccharides. The development of this highly efficient preparation method provides a theoretical and technical foundation for promoting the transformation and upgrading of the sea cucumber deep processing industry and the high-value utilization of low-value byproducts.

[0043] Improved hypoglycemic activity: After Fenton degradation, the inhibition rate of sea cucumber polysaccharide 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 polysaccharide.

[0044] High degradation efficiency: The degraded polysaccharides are more active and show concentration dependence.

[0045] Purity and structure optimization: The high degree of sulfation 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, 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 for use 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, allowing it to stand, and centrifuging it 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 polysaccharide;

[0058] 4) preparing the crude sea cucumber polysaccharide obtained in step 3) into a crude sea cucumber polysaccharide solution, mixing the solution 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 to 70° C. and time of 0.5 to 2.5 h;

[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 polysaccharide.

[0065] In the present invention, sea cucumber byproducts are subjected to protease hydrolysis, enzyme inactivation, and centrifugation to obtain a supernatant. In the present invention, the sea cucumber byproducts are preferably a powder obtained by concentrating and drying sea cucumber viscera and sea cucumber blanching liquid. In the present invention, the protease preferably comprises papain. In the present invention, the mass percentage of the protease in the buffer is preferably 10%. In the present invention, the enzymatic hydrolysis conditions preferably include a temperature of 60°C and a time of 24 hours. In the present invention, the sea cucumber byproducts are mixed with the buffer and protease before enzymatic hydrolysis, and the volume ratio of the sea cucumber byproducts to the buffer is 1g:20mL. In the present invention, the concentration of EDTA in the buffer is preferably 5mmol / L, the concentration of cysteine ​​is preferably 5mmol / L, and the concentration of sodium acetate is preferably 0.1mol / L. The pH of the buffer is preferably 6.0. In the present invention, the enzyme inactivation conditions preferably include a temperature of 100°C and a time of 15 minutes. In the present invention, the centrifugation conditions preferably include a speed of 6000 rpm and a time of 10 minutes.

[0066] In the present invention, the obtained supernatant is subjected to alcohol precipitation, allowed to stand, and centrifuged to obtain a precipitate. In the present invention, the alcohol precipitation conditions preferably include: mixing the supernatant with anhydrous ethanol and then subjecting the supernatant to alcohol precipitation, wherein the volume ratio of the supernatant to anhydrous ethanol is 1:4. In the present invention, the standing conditions preferably include: standing at 4°C overnight. In the present invention, the centrifugation conditions preferably include: centrifugation at 4000 rpm for 10 minutes.

[0067] The present invention dialyzes the obtained precipitate using a dialysis bag with a molecular weight of 8 to 14 kDa, and freeze-dries it to obtain crude sea cucumber polysaccharide. 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, which is then mixed with hydrogen peroxide and ascorbic acid to obtain a mixture, and then 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; and the concentration of ascorbic acid in the mixture is 10-30 mmol / L. The degradation conditions include: a temperature of 30-70°C and a time of 0.5-2.5 hours. 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 include: a temperature of 50°C and a time of 1 hour. In the present invention, the degradation conditions include: a temperature of 51°C and a time of 0.95 hours.

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

[0070] The present invention dialyzes the obtained precipitate using a dialysis bag with a molecular weight of 500Da, freeze-dries it, and obtains sea cucumber polysaccharide. In the present invention, the freeze-drying conditions preferably include: freeze-drying at -80°C for 12 hours. The present invention preferably further purifies 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, 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 polysaccharide.

[0071] In order to further illustrate the present invention, the present invention is described in detail below with reference to the 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 solution.

[0073] Sodium chloride, glacial acetic acid, sodium hydroxide, phenol, anhydrous ethanol, hydrochloric acid, concentrated sulfuric acid, dextran standards, gel filtration media, 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 of analytical grade. α-Amylase was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; papain and alkaline protease were purchased from Tianjin Taijin Technology Co., Ltd. Dextran standards and gel filtration media were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0074] Instruments and equipment: High performance liquid chromatograph (1260infinity), Agilent Technologies, Inc.; 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.; multifunctional microplate reader (Epoch2), ThermoFisher Scientific, USA; Fourier transform infrared spectrometer (IS50), ThermoFisher Scientific, USA.

[0075] Example 1

[0076] Extraction of crude sea cucumber polysaccharides:

[0077] Take 1g of sea cucumber powder and dissolve it in 20mL of 0.1M sodium acetate solution (pH 6.0) containing 5mM EDTA and 5mM cysteine, and use 10% papain by mass to enzymatically hydrolyze 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, and dialyze the resulting precipitate with an 8kDa-14kDa dialysis bag. After dialysis, the polysaccharide is freeze-dried at -80℃ for 12h to obtain crude sea cucumber polysaccharide.

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

[0079] A 5 mg / mL solution of crude sea cucumber polysaccharide was prepared by adding 20 mM hydrogen peroxide and ascorbic acid, each in a 1:1 molar ratio. After degradation at 50°C for 1 hour, the solution was adjusted to neutral pH with 0.01 M sodium hydroxide solution. Four volumes of anhydrous ethanol were added and the solution was incubated at 4°C overnight. The solution was centrifuged at 4000 rpm for 10 minutes. The resulting precipitate was dialyzed using a 500 Da dialysis bag and freeze-dried at -80°C for 12 hours to obtain the degraded sea cucumber polysaccharide.

[0080] Purification of sea cucumber polysaccharide degradation products

[0081] An ultrafiltration membrane system was used to fractionate the sea cucumber polysaccharides before and after degradation according to molecular weight. After fractionation, they were separated using a dextran gel column, and the fraction with a single symmetrical peak in the liquid chromatogram was purified to obtain degraded sea cucumber polysaccharides. Detailed steps: The sea cucumber polysaccharides were fractionated using a 100 kDa ultrafiltration membrane. After ultrafiltration, a 24 × 500 mm chromatography column was selected with an elution flow rate of 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 fractions was measured using the phenol-sulfuric acid method. The elution curve was plotted and the fractions were collected. The fractions were dialyzed using a 500 Da dialysis bag and freeze-dried at -80°C for 12 hours 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] Example 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 polysaccharide and degraded polysaccharide samples were prepared into different concentrations (2 mg / mL, 4 mg / mL, 6 mg / mL, 8 mg / mL, 10 mg / mL), 0.3 mL was added to 1 mL phosphate buffer and 1 mL α-amylase solution, and the reaction was carried out in a water bath at 37°C for 10 minutes. 1 mL of 1% starch solution was added and the reaction was continued in a water bath for 20 minutes. 5 mL of 3,5-dinitrosalicylic acid (DNS) solution was added to terminate the reaction and the mixture was rapidly cooled. The absorbance value A1 was measured at a wavelength of 540 nm. The enzyme solution was replaced with distilled water to obtain A2. The sample solution was replaced with distilled water to obtain A3. Both the sample solution and the enzyme solution were replaced with distilled water as a blank control A4. The inhibition rate calculation formula is:

[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 ]]> Example 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 ]]> Comparative Example 1 <![CDATA[1.24±0.30 g ]]> <![CDATA[0.95±0.04 h <!-- 6 -->]]> 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 concentrations

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

[0117] (2) Influence of degradation time

[0118] When the degradation time in Example 1 was 1 hour, the degradation rate reached a maximum of 60.26%, and the α-amylase inhibition rate reached a maximum of 56.07%. Comparison with other examples shows that with the extension of reaction time, the hydrolysis rate of sea cucumber polysaccharides and the inhibition rate of α-amylase first increase and then decrease. After 1 hour of degradation, due to the presence of residual hydroxyl radicals in the oxidative degradation reaction system of hydrogen peroxide and ascorbic acid solution, they react with the hydrolysis products, resulting in a decrease in the polysaccharide hydrolysis rate and no longer decrease in polysaccharide molecular weight or a slowing of the molecular weight reduction trend. Therefore, the degradation time of Example 1 was selected as the optimal reaction time.

[0119] (3) Influence of degradation temperature

[0120] When the degradation temperature in Example 1 reached 50°C, the polysaccharide degradation rate reached a maximum of 60.26%, and the α-amylase inhibition rate reached 56.70%. A comparison of Examples 10-13 shows that the degradation rate increases rapidly with increasing temperature. The increase in temperature leads 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 hydrogen peroxide partially decomposes, resulting in a decrease in the hydroxyl radical content 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 beneficial to the degradation effect and activity of polysaccharides.

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

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

[0123] Chemical composition Sea Cucumber Polysaccharide Example 1 Degradation of sea cucumber polysaccharides Total sugar content (%) <![CDATA[89.42±0.93 c ]]> <![CDATA[89.72±1.68 c ]]> Uronic acid content (%) <![CDATA[11.97±2.80 e ]]> <![CDATA[7.85±0.88 e ]]> Protein content (%) <![CDATA[7.45±2.49 f ]]> <![CDATA[6.98±3.27 e ]]> Sulfate content (%) <![CDATA[17.81±1.58 d ]]> <![CDATA[16.33±2.16 d ]]> Molecular weight (kDa) <![CDATA[1862.20±42.04 a ]]> <![CDATA[439.66±13.16 a ]]> Particle size (nm) <![CDATA[863.53±135.81 b ]]> <![CDATA[186.83±40.33 b ]]>

[0124] The process conditions of Example 1 were used to degrade sea cucumber polysaccharides, and further separation and purification were performed to determine the main indicators. It was found that 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, while the protein content and uronic acid content that caused the polysaccharide to have a high molecular weight and carry a negative charge were reduced. The molecular weight of crude sea cucumber polysaccharides was 1862.20 kDa, and the degraded polysaccharides were reduced to 439.66 kDa. At the same time, as the molecular weight decreased, the particle size of the sea cucumber polysaccharides after degradation was significantly reduced to 186.83 nm, and the smaller particle size verified the reduction in the molecular weight of the polysaccharides. The sea cucumber polysaccharides were subjected to ultrafiltration and fractionation treatment ( Figure 4 ), and the obtained liquid chromatogram of the purified degraded sea cucumber polysaccharide was ( Figure 5 ), this component is a single symmetrical peak, indicating good purification results and successful degradation of sea cucumber polysaccharides. The results show that hydroxyl radical degradation targets uronic acid and hardly attacks the sugar chain branches and sulfate esters necessary for its biological activity. At the same time, the molecular weight of the polysaccharide is also effectively reduced. The above analysis shows that the degradation results of Example 1 are reliable.

[0125] Although the above embodiment provides a detailed description of the present invention, 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 scope of protection 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, allowing it to stand, and centrifuging it 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 polysaccharide; 4) preparing the crude sea cucumber polysaccharide obtained in step 3) into a crude sea cucumber polysaccharide solution, mixing the solution 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 to 70° C. and time of 0.5 to 2.5 h; 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 polysaccharide.

2. The method according to claim 1, characterized in that 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.

3. The method according to claim 1, characterized in that 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.

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

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

6. The method according to claim 1, wherein The protease in step 1) comprises papain; The enzymatic hydrolysis conditions include: temperature of 60°C and time of 24h; The sea cucumber by-product is mixed with a buffer solution and a protease and then enzymatically hydrolyzed, wherein the mass ratio of the sea cucumber by-product to the buffer solution is 1 g:20 mL; the mass percentage of the protease in the buffer solution is 10%; The concentration of EDTA in the buffer solution 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 solution is 6.0; The enzyme inactivation conditions include: temperature of 100°C and time of 15 minutes; The centrifugal conditions include: a rotation speed of 6000 rpm and a time of 10 min.

7. The method according to claim 1, wherein 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 anhydrous ethanol is 1:4; The standing conditions include: standing at 4°C overnight; 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 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 at 4°C overnight; 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, measuring the absorbance using a phenol-sulfuric acid method, drawing an elution curve and collecting the eluted product, and performing freeze-drying after dialysis to obtain sea cucumber polysaccharide.

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