High-purity scallop polysaccharide as well as extraction method and application thereof

Abstract

The invention discloses high-purity scallop polysaccharide as well as an extraction method and application thereof, and belongs to the technical field of extraction of natural products from marine animals. In order to solve the technical problems that in the prior art, a scallop polysaccharide extraction and purification method is difficult to consider the aspects of efficiency, purity and operability, and the like, scallop columns are used as raw materials, and firstly, a scallop enzymolysis extracting solution is obtained by adopting hot bath extraction and protease enzymolysis technologies; then adding ammonium sulfate and tert-butyl alcohol to carry out ultrasonic-assisted synergistic three-phase extraction on the enzymatic hydrolysate, and carrying out dialysis, concentration and freeze drying to obtain crude polysaccharide; and purifying the crude polysaccharide by adopting an ammonium sulfate fractional precipitation method and gel column chromatography to obtain the scallop polysaccharide with high purity and relatively high antioxidant activity. The scallop polysaccharide prepared by the extraction method provided by the invention can be applied to the development of products with antioxidant effects.

Description

Technical Field

[0001] This invention belongs to the field of extraction technology of natural products from marine animals, specifically relating to a high-purity scallop polysaccharide, its extraction method, and its application. Background Technology

[0002] Scallops are a valuable aquatic product, rich in various nutrients such as polysaccharides, peptides, taurine, and trace elements, making them an important marine fishery resource. Scallop polysaccharides are a class of natural high-molecular-weight compounds extracted from scallops. Studies have shown that they possess various physiological activities, including anti-tumor, immunomodulatory, lipid-lowering, and antioxidant effects, and have broad application prospects in the development of functional foods, health products, and pharmaceuticals.

[0003] Currently, the extraction of scallop polysaccharides mainly relies on traditional methods. Water extraction followed by alcohol precipitation is the most common method, using water as a solvent. The polysaccharide is extracted by heating, concentrated, and then precipitated with ethanol. While this method is simple, mild, and safe, it generally suffers from long extraction times, high energy consumption, and low extraction rates. Furthermore, the crude polysaccharide obtained often contains large amounts of protein, pigments, and small molecule impurities, resulting in low purity. In addition, enzymatic hydrolysis (such as using proteases) promotes polysaccharide dissolution through biological enzymatic hydrolysis, which can improve extraction efficiency and reduce protein impurities to some extent. However, it also faces the problem of complex extract composition and a heavy burden on subsequent purification.

[0004] In terms of purification, existing technologies primarily rely on column chromatography to obtain high-purity scallop polysaccharides. A common procedure is the combined use of ion-exchange column chromatography and gel filtration column chromatography. However, this method has significant limitations: firstly, the process is lengthy, with two or more column chromatography cycles taking tens of hours to several days; secondly, sample loss is significant, with adsorption residues and dilution losses of the target polysaccharide occurring during loading, elution, and collection; thirdly, it is costly, requiring expensive chromatographic media and equipment. Most importantly, studies have shown that scallop polysaccharides are mainly neutral polysaccharides composed of glucose, lacking strong ionization groups on their molecular surface and exhibiting weak binding to ion-exchange media, resulting in poor purification effects and limited applicability of ion-exchange column chromatography. Therefore, purification strategies relying on traditional column chromatography are inefficient and uneconomical.

[0005] Ultrasonic extraction, as a physical enhancement technique, has been widely used in the extraction of natural products. Through the cavitation, mechanical vibration, and thermal effects generated by ultrasound, it can effectively disrupt cells, accelerate solvent penetration and component diffusion, thereby significantly shortening extraction time and increasing the yield of the target substance. This technology has also been explored for polysaccharide extraction and for enhancing phase separation and mass transfer in systems such as aqueous two-phase extraction. However, existing research on ultrasound-assisted extraction largely focuses on single solvent systems (such as water or low-concentration alcohols). An effective solution remains lacking for integrating it with efficient fractionation purification steps to achieve simultaneous highly selective extraction and primary purification from complex matrices.

[0006] In summary, existing methods for extracting and purifying scallop polysaccharides suffer from a trade-off between efficiency, purity, and operability: traditional extraction methods are time-consuming, labor-intensive, and yield low purity; while column chromatography-based purification methods are complex, prone to losses, and lack selectivity for neutral polysaccharides. Therefore, there is an urgent need in this field to develop a novel integrated extraction and purification method for scallop polysaccharides that is simple to operate, time-efficient, highly effective, and can directly yield high-purity scallop polysaccharides. Summary of the Invention

[0007] To address the technical challenges of balancing efficiency, purity, and operability in existing scallop polysaccharide extraction and purification methods, this invention uses scallop adductor muscles as raw material. First, a hot water bath extraction and protease hydrolysis technique are employed to obtain a scallop enzymatic extract. Then, ammonium sulfate and tert-butanol are added to the hydrolysate for ultrasonic-assisted synergistic three-phase extraction. Following dialysis, concentration, and freeze-drying, crude polysaccharide is obtained. Finally, ammonium sulfate fractionation precipitation and gel column chromatography are used to purify the crude polysaccharide, resulting in high-purity scallop polysaccharide with high antioxidant activity.

[0008] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution: The first objective of this invention is to provide a method for extracting high-purity scallop polysaccharides, the extraction method comprising the following steps: (1) The adductor muscles of scallops are freeze-dried and then pulverized to obtain scallop powder; (2) Mix the scallop powder obtained in step (1) with water, extract it in a water bath to obtain an aqueous extract, cool it to room temperature, add protease to the aqueous extract for enzymatic hydrolysis, and collect the supernatant after enzyme inactivation, cooling and centrifugation to obtain the hydrolysate. (3) Add ammonium sulfate and tert-butanol to the enzymatic hydrolysate obtained in step (2) in sequence, and treat with ultrasound to form a three-phase extraction system. Take the lower clear liquid. The amount of ammonium sulfate added is 10%-30% of the mass of the enzymatic hydrolysate, and the volume ratio of tert-butanol to the enzymatic hydrolysate is 0.5:1-2:1. (4) Take the lower layer of clear liquid obtained in step (3), dialyze it through a dialysis bag with a molecular weight of 6 kDa, concentrate it, and freeze-dry it to obtain scallop crude polysaccharide; (5) Take the crude scallop polysaccharide obtained in step (4), add water to redissolve it to obtain a crude polysaccharide solution, add ammonium sulfate with a mass fraction of 40% to the crude polysaccharide solution, stir evenly, let it stand to precipitate, centrifuge, and take the supernatant; continue to add ammonium sulfate to the supernatant, stir evenly to obtain a 50% saturated ammonium sulfate solution, let it stand to precipitate, centrifuge, and take the supernatant; continue to add ammonium sulfate to the supernatant, stir evenly to obtain a 60% saturated ammonium sulfate solution, let it stand to precipitate, centrifuge, take the precipitate, and obtain the polysaccharide component by dissolving, dialysis, and freeze-drying. (6) The polysaccharide component obtained in step (5) is reconstituted and purified by column chromatography to obtain high-purity scallop polysaccharide.

[0009] In one embodiment of the present invention, the ratio of scallop powder to water in the water bath extraction process in step (2) is 1 g: 30 mL, the extraction temperature is 90℃, and the extraction time is 4 h.

[0010] In one embodiment of the present invention, the enzymatic hydrolysis process in step (2) uses papain, the amount added is 2% of the mass of the water extract, the enzymatic hydrolysis temperature is 55℃, and the enzymatic hydrolysis time is 5 h.

[0011] The beneficial effects of the above scheme are that by using hot water method combined with protease hydrolysis as a pretreatment method, the release of polysaccharides in scallop raw material tissue can be effectively promoted, thereby improving the polysaccharide extraction rate.

[0012] In one embodiment of the present invention, the amount of ammonium sulfate added in step (3) is 30% of the mass of the enzymatic hydrolysate.

[0013] In one embodiment of the present invention, the volume ratio of tert-butanol to enzymatic hydrolysate in step (3) is 1:1-1.5:1.

[0014] Preferably, the volume ratio of tert-butanol to enzymatic hydrolysate is 1.5:1.

[0015] The beneficial effects of adopting the above-mentioned further scheme are as follows: the present invention can simultaneously complete the separation of polysaccharides, removal of proteins, and defatting steps through three-phase extraction technology, and the obtained scallop polysaccharides have high purity and certain changes in polysaccharide structure, which may have certain antioxidant capacity.

[0016] In one embodiment of the present invention, the temperature of the ultrasound in step (3) is 35-50°C and the duration of the ultrasound is 6-12 min.

[0017] Preferably, the temperature of the ultrasound is 35°C and the duration of the ultrasound is 10 min.

[0018] The beneficial effects of adopting the above-mentioned further scheme are as follows: adding ultrasonic assistance to the three-phase extraction technology can enhance mass transfer and yield, significantly shorten the extraction time, and improve the extraction rate.

[0019] The purpose of dialysis in step (4) is to remove the influence of inorganic salts and solvents used in step (3).

[0020] In one embodiment of the present invention, the static precipitation in step (5) is performed by standing at 4°C for 24 h.

[0021] The advantage of ammonium sulfate fractionation in step (5) is that it is simple to operate and can further improve the purity of the obtained polysaccharide.

[0022] In one embodiment of the present invention, the column chromatography in step (6) is Sephacryls-400 gel chromatography column chromatography.

[0023] The advantage of using gel column chromatography is that it eliminates the need for ion exchange column chromatography, allowing for the separation of sugars of different molecular weights, further removal of impurities, and rapid and efficient acquisition of high-purity scallop polysaccharides.

[0024] A second objective of this invention is to provide scallop polysaccharides obtained by the above extraction method.

[0025] A third objective of this invention is to provide the application of the above-mentioned scallop polysaccharide in the preparation of functional foods, health products, or pharmaceuticals with antioxidant effects.

[0026] To be further defined, the products include, but are not limited to, functional foods, health products, and pharmaceuticals.

[0027] The beneficial effects of this invention are: This invention provides a method for extracting high-purity scallop polysaccharides. The method first employs water extraction followed by enzymatic pretreatment to promote the release of polysaccharides from scallop tissue and initially induce the degradation of scallop proteins. Then, an ultrasound-assisted three-phase extraction method is used to reduce impurity interference, rapidly separating the polysaccharide layer from the protein impurity layer, reducing the difficulty of subsequent purification and ensuring polysaccharide purity. This is further combined with a fractionated ammonium sulfate precipitation method, which is mild, simple to operate, and time-efficient. Finally, gel column chromatography is used for further purification, yielding high-purity scallop polysaccharides with certain antioxidant activity.

[0028] This invention employs an extraction method combining enzymatic hydrolysis-ultrasound-assisted three-phase extraction with ammonium sulfate fractionation precipitation and gel column chromatography. This method solves the technical problems of low efficiency and purity caused by traditional extraction methods for preparing scallop polysaccharides. It offers advantages such as simple operation, high efficiency, environmental friendliness, economy, and high safety. The scallop polysaccharides prepared by this invention not only have high purity (up to 96.77%) but also possess high antioxidant activity, exhibiting a DPPH free radical scavenging rate of 45.63% at 5 mg / mL, making it more suitable for the development of products with antioxidant properties. Attached Figure Description

[0029] Figure 1 The above are the HPGPC spectra of SPS-E, SPS-T, and SPS-U extracted in this invention; wherein, Figure 1 In the image, A represents the HPGPC map of SPS-E. Figure 1 In the image, B represents the HPGPC map of SPS-T. Figure 1 C in the figure represents the HPGPC spectrum of SPS-U; Figure 2 The graph shows the detection results of the DPPH free radical scavenging ability of SPS-E, SPS-T and SPS-U extracted in this invention; Figure 3 The above are the HPGPC spectra of SPSU-40, SPSU-50, and SPSU-60 extracted in this invention; wherein, Figure 3 In the image, A represents the HPGPC spectrum of SPSU-40. Figure 3 In the image, B represents the HPGPC spectrum of SPSU-50. Figure 3 C in the figure represents the HPGPC spectrum of SPSU-60; Figure 4 The graph shows the detection results of the DPPH free radical scavenging ability of SPSU-40, SPSU-50 and SPSU-60 extracted in this invention. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the objectives of the invention. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, any embodiments obtained by other skilled personnel without creative effort are protected by this invention.

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials, reagents and instruments used are conventional materials, reagents and instruments in the art, which can be obtained by those skilled in the art through commercial channels.

[0032] The detection method involved in this invention is as follows: 1. Detection of polysaccharide content Sugar content was determined using the phenol-sulfuric acid method, with glucose as the standard. 0.0, 0.1, 0.3, 0.4, 0.5, 0.7, 0.9, and 1.0 mL of 0.2 mg / mL glucose solution were placed in test tubes, and distilled water was added to a final volume of 1.0 mL. 0.5 mL of 6% phenol and 2.5 mL of concentrated sulfuric acid were added to each tube, respectively. The tubes were gently shaken and allowed to stand at room temperature for 10 min. The absorbance of tubes 2-8 was measured at 490 nm using tube 1 (blank control) as the zero point. A standard curve was plotted with glucose concentration on the x-axis and absorbance on the y-axis.

[0033] Take 1 mL of sample solution, follow the above procedure, measure the absorbance value, substitute the measurement result into the standard curve, and calculate the total sugar content of the sample.

[0034] 2. Calculation of scallop polysaccharide extraction rate The formula for calculating the extraction rate of scallop polysaccharides is as follows: .

[0035] M a Indicates the quality of the scallop raw material; V a Indicates the volume of the lower layer solution; C a This indicates the concentration of polysaccharides in the lower layer.

[0036] 3. Calculation of the purity of crude polysaccharide from scallops The formula for calculating the purity of crude polysaccharides from scallops is as follows: .

[0037] 4. Determination of the molecular weight of polysaccharides The relative molecular mass of the samples was determined by high performance gel size exclusion chromatography (HPLC). The HPLC conditions were: 0.2 M NaCl solution, flow rate of 0.5 mL / min, injection volume of 20 μL, and column temperature of 25℃. A standard curve was plotted using Dextran Blue 2000, Dextran T-10, Dextran T-20, and Dextran T-500 as standards, with retention time on the x-axis and -lgMw (where Mw is the relative molecular mass) on the y-axis.

[0038] 5. Determination of DPPH antioxidant index Prepare crude polysaccharide samples and vitamin C solutions (2, 4, 6, 8, and 10 mg / mL as positive controls) at concentrations of 2, 4, 6, 8, and 10 mg / mL. Accurately weigh 4.0 mg of DPPH and prepare a 0.1 mmol / L DPPH ethanol solution, using freshly prepared solutions. Take 1 mL of each concentration of crude polysaccharide sample or vitamin C control solution as the sample solution, add 1 mL of 0.1 mmol / L DPPH solution to each, mix well, and let stand for 30 min. Measure the absorbance value (A1) at 517 nm. Use deionized water instead of the sample solution and measure the absorbance value (A2) under the same conditions. Use anhydrous ethanol instead of the DPPH solution and measure the absorbance value (A0) under the same conditions. Perform three replicates for each sample. The formula for calculating the free radical scavenging capacity of DPPH is as follows: .

[0039] Example 1: A method for extracting high-purity scallop polysaccharides includes the following steps: Scallop adductor muscles were collected, freeze-dried, and pulverized to obtain scallop powder. Water was added to the scallop powder at a material-to-liquid ratio of 1 g:30 mL, and pre-extracted in a 90℃ water bath for 4 h to obtain an aqueous extract. The extract was then cooled to room temperature. 2% (w / w) papain was added to the aqueous extract, and enzymatic hydrolysis was performed in a 55℃ constant temperature water bath for 5 h. After hydrolysis, the enzyme was inactivated for 10 min. The extract was cooled to room temperature, centrifuged at 9000 rpm for 10 min, and the supernatant was collected as the hydrolysate. A certain amount of the hydrolysate was taken, and 10% (w / w) ammonium sulfate was added. Tert-butanol was then added to achieve a volume ratio of tert-butanol to hydrolysate of 1.5:1. The mixture was sonicated at 35℃ for 10 min to form a three-phase mixture. The lower supernatant was collected to determine the polysaccharide content, and the extraction rate of scallop polysaccharides was calculated to be 11.77%.

[0040] Example 2: Compared to Example 1, the only difference was the addition of 20% ammonium sulfate by mass to the enzymatic hydrolysate. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 10.15%.

[0041] Example 3: Compared to Example 1, the only difference was the addition of 30% ammonium sulfate to the enzymatic hydrolysate. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 15.26%.

[0042] Example 4: Compared to Example 1, the only difference was the addition of 40% ammonium sulfate to the enzymatic hydrolysate. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 2.16%.

[0043] Therefore, as shown by the extraction rates in Examples 1-4, the extraction rate of scallop crude polysaccharide is highest when 30% ammonium sulfate is added to the enzymatic hydrolysate.

[0044] Example 5: Compared to Example 3, the only difference is that the volume ratio of tert-butanol to enzymatic hydrolysate is 0.5:1. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 13.53%.

[0045] Example 6: Compared to Example 3, the only difference is that the volume ratio of tert-butanol to enzymatic hydrolysate is 1.0:1. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 14.91%.

[0046] Example 7: Compared to Example 3, the only difference is that the volume ratio of tert-butanol to enzymatic hydrolysate is 2.0:1. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 9.35%.

[0047] Therefore, as shown by the extraction rates in Examples 3 and 5-7, the extraction rate of scallop crude polysaccharide is relatively high when the volume ratio of tert-butanol to enzymatic hydrolysate is 1:1-1.5:1; and the extraction rate of scallop crude polysaccharide is highest when the volume ratio of tert-butanol to enzymatic hydrolysate is 1.5:1.

[0048] Example 8: Compared to Example 3, the only difference is the ultrasonic time, which is 6 minutes. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 13.04%.

[0049] Example 9: Compared to Example 3, the only difference is that the ultrasonic time was 8 minutes. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 12.97%.

[0050] Example 10: Compared to Example 3, the only difference is that the ultrasonic time was 12 min. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 12.72%.

[0051] Therefore, as shown by the extraction rates in Examples 3 and 8-10, the extraction rate of crude polysaccharide from scallops is highest when the ultrasonic time is 10 min.

[0052] Example 11: Compared to Example 3, the only difference is that the ultrasonic temperature was 40°C. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 12.41%.

[0053] Example 12: Compared to Example 3, the only difference is that the ultrasonic temperature was 45°C. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 14.00%.

[0054] Example 13: Compared to Example 3, the only difference is that the ultrasonic temperature was 50°C. Based on the determination of polysaccharide content, the extraction rate of crude scallop polysaccharides obtained in this example was calculated to be 10.01%.

[0055] Therefore, as shown by the extraction rates in Examples 3 and 11-13, the extraction rate of crude polysaccharide from scallops is highest when the ultrasonic temperature is 35°C.

[0056] Example 14: A method for extracting high-purity scallop polysaccharides includes the following steps: Scallop adductor muscles were collected, freeze-dried, and pulverized to obtain scallop powder. Water was added to the scallop powder at a material-to-liquid ratio of 1 g:30 mL, and pre-extracted in a 90℃ water bath for 4 h to obtain an aqueous extract. The extract was then cooled to room temperature. 2% (w / w) papain was added to the aqueous extract, and enzymatic hydrolysis was performed in a 55℃ constant temperature water bath for 5 h. After hydrolysis, the enzyme was inactivated for 10 min. The extract was cooled to room temperature, centrifuged at 9000 rpm for 10 min, and the supernatant was collected as the hydrolysate. A certain amount of the hydrolysate was taken, and 30% (w / w) ammonium sulfate was added. Tert-butanol was then added to maintain a volume ratio of tert-butanol to hydrolysate of 1.5:1. The mixture was sonicated at 35℃ for 10 min to form a three-phase mixture, and the lower clear layer was collected. The clear layer was dialyzed through a 6 kDa dialysis bag, concentrated, and freeze-dried to obtain crude scallop polysaccharide SPS-U. The HPGPC spectrum is shown below. Figure 1 As shown in C, the polysaccharide purity is 89.57% and the molecular weight is 7211.024 kDa.

[0057] Comparative Example 1: Compared with Example 14, this comparative example did not use ultrasound-assisted three-phase extraction technology, but instead used alcohol precipitation technology. The specific method is as follows: Scallop adductor muscles were taken, freeze-dried, and pulverized to obtain scallop powder. Water was added to the scallop powder at a material-to-liquid ratio of 1 g: 30 mL, and pre-extracted in a 90℃ water bath for 4 h to obtain an aqueous extract. The extract was then cooled to room temperature. 2% (mass percentage) papain was added to the aqueous extract, and enzymatic hydrolysis was performed in a 55℃ constant temperature water bath for 5 h. After enzymatic hydrolysis, the enzyme was inactivated for 10 min. The extract was cooled to room temperature, centrifuged at 9000 rpm for 10 min, and the supernatant was collected as the hydrolysate. 95% ethanol (3 times the volume of the hydrolysate) was added to the hydrolysate, and the mixture was allowed to stand overnight. After centrifugation (9000 rpm, 10 min), the precipitate was collected, dissolved in a small amount of deionized water, and the ethanol was evaporated in a 60℃ water bath. The precipitate was then freeze-dried to obtain crude scallop polysaccharide, named SPS-E. The HPGPC spectrum is shown below. Figure 1 As shown in A in the diagram.

[0058] Comparative Example 2: Compared with Example 14, no ultrasonic-assisted treatment was performed during the extraction process, and the three-phase extraction time was extended to 30 min. The remaining steps were the same, and the specific method is as follows: Scallop adductor muscles were taken, freeze-dried, and pulverized to obtain scallop powder. Water was added to the scallop powder at a material-to-liquid ratio of 1 g: 30 mL, and pre-extracted in a 90℃ water bath for 4 h to obtain an aqueous extract. The extract was then cooled to room temperature. 2% (mass percentage) papain was added to the aqueous extract, and enzymatic hydrolysis was performed in a 55℃ constant temperature water bath for 5 h. After enzymatic hydrolysis, the enzyme was inactivated for 10 min. The extract was cooled to room temperature, centrifuged at 9000 rpm for 10 min, and the supernatant was collected as the enzymatic hydrolysate. A certain amount of the enzymatic hydrolysate was taken, and 30% (mass fraction) ammonium sulfate was added. Tert-butanol was then added to make the volume ratio of tert-butanol to the enzymatic hydrolysate 1.5:1. The extract was then extracted at 35℃ for 30 min to form a three-phase mixture, and the lower supernatant was collected. The supernatant was dialyzed through a dialysis bag with a molecular weight of 6 kDa, concentrated, and freeze-dried to obtain scallop crude polysaccharide SPS-T. The HPGPC spectrum is shown below. Figure 1 As shown in B in the diagram.

[0059] The purity and molecular weight of the polysaccharides obtained in Example 14 and the two comparative examples are shown in Table 1. The results of the DPPH free radical scavenging ability test are as follows: Figure 2 As shown in the figure, a comparison of the three extraction methods shows that the polysaccharide extracted by the enzyme pretreatment and ultrasound-assisted three-phase extraction method in Example 14 of this invention has higher purity and better antioxidant activity.

[0060] Table 1. Results of purity and molecular weight analysis of crude scallop polysaccharides obtained in Example 14 and two comparative examples.

[0061] Example 15: After extracting crude scallop polysaccharides according to Example 14, the scallop polysaccharides were further separated using ammonium sulfate fractionation precipitation. 3.0 g of SPS-U powder was weighed and added to 3000 mL of distilled water. After stirring to dissolve, 40% ammonium sulfate was added to the solution and stirred until completely dissolved. The solution was then incubated in a chromatography cabinet at 4°C for 24 h, centrifuged at 9000 rpm for 10 min, and the precipitate was dissolved, dialyzed, and freeze-dried to obtain the polysaccharide fraction named SPSU-40 (HPGPC chromatogram as shown). Figure 3 (As shown in A in the diagram). Add ammonium sulfate powder to the supernatant to obtain a 50% saturated ammonium sulfate solution. Following the above procedure, the polysaccharide component was named SPSU-50 (HPGPC spectrum shown in the diagram). Figure 3 (As shown in B in the diagram). Continue adding ammonium sulfate powder to the obtained supernatant to obtain a 60% saturated ammonium sulfate solution. Following the above procedure, the polysaccharide component, named SPSU-60, was obtained (HPGPC spectrum as shown in the diagram). Figure 3 (as shown in C). The purity and molecular weight of the three polysaccharides are shown in Table 2. The results of the test on the DPPH free radical scavenging ability are as follows. Figure 4 As shown.

[0062] Table 2. Results of purity and molecular weight tests for SPSU-40, SPSU-50, and SPSU-60.

[0063] The results showed that after ammonium sulfate fractionation precipitation, the purity of scallop polysaccharide SPSU-60 obtained in Example 15 was further improved, and it also had good antioxidant activity.

[0064] Example 16: After extracting crude polysaccharides from scallops as described in Example 15, SPSU-60 was further purified using a Sephacryls-400 gel chromatography column. 10 mg of sample was accurately weighed, dissolved in 1 mL of deionized water, and filtered through a 0.45 μm aqueous membrane to obtain the loading solution. The solution was isocratically eluted with deionized water at a flow rate of 0.3 mL / min, with 3 mL of sample solution collected from each tube. The total sugar content was measured in each tube, and an elution curve was plotted with the number of tubes on the x-axis and absorbance on the y-axis. The sample solutions from the tubes corresponding to the elution peaks were collected, lyophilized, and the fraction SPSU-60-S was obtained, with a purity of 96.77% and a molecular weight of 2.11 × 10⁻⁶. 6Da, at 5 mg / mL, showed a DPPH radical scavenging rate of 45.63%.

[0065] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be defined by the claims.

Claims

1. A method for extracting high-purity scallop polysaccharides, characterized in that, Includes the following steps: (1) The adductor muscles of scallops are freeze-dried and then pulverized to obtain scallop powder; (2) Mix the scallop powder obtained in step (1) with water, extract it in a water bath to obtain an aqueous extract, cool it to room temperature, add protease to the aqueous extract for enzymatic hydrolysis, and collect the supernatant after enzyme inactivation, cooling and centrifugation to obtain the hydrolysate. (3) Add ammonium sulfate and tert-butanol to the enzymatic hydrolysate obtained in step (2) in sequence, and treat with ultrasound to form a three-phase extraction system. Take the lower clear liquid. The amount of ammonium sulfate added is 10%-30% of the mass of the enzymatic hydrolysate, and the volume ratio of tert-butanol to the enzymatic hydrolysate is 0.5:1-2:

1. (4) Take the lower layer of clear liquid obtained in step (3), dialyze it through a dialysis bag with a molecular weight of 6 kDa, concentrate it, and freeze-dry it to obtain scallop crude polysaccharide; (5) Take the crude scallop polysaccharide obtained in step (4), add water to redissolve it to obtain a crude polysaccharide solution, add ammonium sulfate with a mass fraction of 40% to the crude polysaccharide solution, stir evenly, let it stand to precipitate, centrifuge, and take the supernatant; continue to add ammonium sulfate to the supernatant, stir evenly to obtain a 50% saturated ammonium sulfate solution, let it stand to precipitate, centrifuge, and take the supernatant; continue to add ammonium sulfate to the supernatant, stir evenly to obtain a 60% saturated ammonium sulfate solution, let it stand to precipitate, centrifuge, take the precipitate, and obtain the polysaccharide component by dissolving, dialysis, and freeze-drying. (6) The polysaccharide component obtained in step (5) is reconstituted and purified by column chromatography to obtain high-purity scallop polysaccharide.

2. The extraction method according to claim 1, characterized in that, In step (2), the ratio of scallop powder to water in the water bath extraction process is 1 g: 30 mL, the extraction temperature is 90℃, and the extraction time is 4 h.

3. The extraction method according to claim 1, characterized in that, In step (2), papain was used for the enzymatic hydrolysis process. The amount added was 2% of the mass of the water extract. The hydrolysis temperature was 55℃ and the hydrolysis time was 5 h.

4. The extraction method according to claim 1, characterized in that, The amount of ammonium sulfate added in step (3) is 30% of the mass of the enzymatic hydrolysate.

5. The extraction method according to claim 1, characterized in that, The volume ratio of tert-butanol to enzymatic hydrolysate in step (3) is 1:1 to 1.5:

1.

6. The extraction method according to claim 1, characterized in that, The temperature of the ultrasound in step (3) is 35-50℃ and the duration of the ultrasound is 6-12 min.

7. The extraction method according to claim 1, characterized in that, The static precipitation in step (5) is to stand at 4°C for 24 hours.

8. The extraction method according to claim 1, characterized in that, The column chromatography in step (6) is Sephacryls-400 gel chromatography column chromatography.

9. Scallop polysaccharide obtained by any of the extraction methods described in claims 1-8.

10. The use of the scallop polysaccharide according to claim 9 in the preparation of products with antioxidant properties.

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