Method for improving blood sugar reducing and antioxidant activity of mulberry polysaccharide extract

Abstract

The invention relates to the technical field of functional polysaccharide preparation, and discloses a method for improving blood sugar reducing and antioxidant activity of mulberry polysaccharide extract.The method comprises the following steps that an enzymolysis system containing mulberry pomace freeze-dried powder, pectinase and cellulase is prepared, after enzymolysis is conducted, ultrasonic-assisted extraction is conducted, a product is separated and purified, and the mulberry polysaccharide extract is obtained. The mulberry polysaccharide extract with high blood sugar reducing and antioxidant activity is obtained. According to the invention, pectinase and cellulase are adopted for pretreatment, and ultrasonic-assisted extraction is combined, so that the blood sugar reducing and antioxidant activity of the mulberry polysaccharide extract can be improved at the same time, and the mulberry polysaccharide extract has better blood sugar reducing and antioxidant effects under the same concentration condition.

Description

Technical Field

[0001] The invention relates to the technical field of functional polysaccharide preparation, and in particular to a method for improving the blood sugar lowering and antioxidant activities of mulberry polysaccharide extracts. Background Art

[0002] Mulberry pomace contains a large amount of water, sugars, organic acids and other substances. If it is not handled in time or is randomly dumped, it is easy to rot and produce bad odors, attracting mosquitoes. A large number of beneficial ingredients such as polyphenols and pectin polysaccharides have not been effectively utilized, thus causing serious environmental pollution and waste of resources. Studies have shown that polysaccharides play an important role in physiological activities such as anti-tumor, anti-inflammatory, antiviral, hypoglycemic, anti-aging, anticoagulation, and immune promotion. Therefore, studying the extraction method and physiological activity of mulberry polysaccharides will help to achieve the effective utilization of active ingredients in mulberry pomace.

[0003] The physiological activity of polysaccharides is closely related to their structural properties (including glycosyl composition, arrangement order, number, distribution and length of branches, spatial structure and other factors), and different structures are affected by the extraction method. Therefore, there is no positive correlation between the physiological activity of polysaccharides and the extraction rate. Improving the extraction rate does not mean that the physiological activity of the obtained polysaccharides can be improved. For example, by comparing the extraction rate and property differences of mulberry polysaccharides extracted by hot water extraction (MFPH), ultrasonic extraction (MFPU), acid (MFPA) and alkali (MFPB), it was found that MFPH had the lowest yield, but the strongest antioxidant and anti-diabetic activities. In addition, different physiological activities have different requirements for the structural properties of polysaccharides. The extraction method that can improve a certain physiological activity of polysaccharides may not necessarily improve other physiological activities of this polysaccharide. For example, the monosaccharide compositions of mulberry polysaccharides (MFP-30 and MFP-90) extracted at different extraction temperatures (30°C and 90°C) were the same, but the molar ratios were different. Higher extraction temperatures would cause degradation of polysaccharide chains, leading to a decrease in molecular weight and polysaccharide fragmentation. In terms of physiological activity, MFP-30 had better antioxidant activity, and MFP-90 had a high binding capacity for bile acids.

[0004] Exploring the effects of different extraction methods on the physiological activity of mulberry polysaccharides can provide a scientific basis for the in-depth development and utilization of mulberry pomace. However, the research on the extraction process of mulberry polysaccharides, their structural characteristics and physiological activity is still in its infancy. There are few studies on the relationship between the structural characteristics of mulberry polysaccharides and their activity. Moreover, as mentioned above, there is no positive correlation between the physiological activity of polysaccharides and the extraction rate, and different physiological activities have different requirements for the structural properties of polysaccharides, which brings great difficulty to the research. At present, no extraction method has been reported that can simultaneously improve the hypoglycemic and antioxidant activities of mulberry polysaccharide extracts. Summary of the invention

[0005] In order to solve the above technical problem, that is, there is no reported extraction method that can simultaneously improve the hypoglycemic and antioxidant activities of mulberry polysaccharide extracts, the present invention provides a method for improving the hypoglycemic and antioxidant activities of mulberry polysaccharide extracts. By adopting this method, the hypoglycemic and antioxidant activities of the extracted mulberry polysaccharide extracts can be improved.

[0006] The specific technical scheme of the present invention is: A method for improving the hypoglycemic and antioxidant activities of a mulberry polysaccharide extract comprises the following steps: preparing an enzymatic hydrolysis system comprising freeze-dried mulberry pomace powder, pectinase and cellulase, performing enzymatic hydrolysis, then performing ultrasonic-assisted extraction, separating and purifying the product, and obtaining a mulberry polysaccharide extract with high hypoglycemic and antioxidant activities.

[0007] The present invention adopts specific enzymes (pectinase + cellulase) for enzymatic pretreatment, combined with ultrasonic-assisted extraction, which can improve the hypoglycemic activity and antioxidant activity of the obtained mulberry polysaccharide extract. The mulberry polysaccharide extract obtained by the method of the present invention has higher antioxidant activity and hypoglycemic activity than the mulberry polysaccharide extract obtained by hot water extraction, enzyme pretreatment + hot water extraction and ultrasonic-assisted extraction under the same concentration conditions.

[0008] Preferably, the preparation process of the enzymatic hydrolysis system comprises: mixing mulberry pomace freeze-dried powder, pectinase solution and cellulase solution to obtain the enzymatic hydrolysis system.

[0009] Furthermore, the mass volume ratio of the mulberry pomace freeze-dried powder to water is 1g:40-100mL.

[0010] Furthermore, in the enzymatic hydrolysis system, the sum of the volumes of the cellulase solution and the pectinase solution is 0.2-4% of the water volume, the mass ratio of cellulase to pectinase is 1:1-1.5; and the concentrations of the cellulase solution and the pectinase solution are 10-15 mg / mL.

[0011] Furthermore, in the enzymatic hydrolysis system, the sum of the volumes of the cellulase solution and the pectinase solution is 1-1.5% of the water volume, the mass ratio of cellulase to pectinase is 1:1-1.5; and the concentrations of the cellulase solution and the pectinase solution are 10-30 mg / mL.

[0012] When the sum of the volumes of the cellulase solution and the pectinase solution is 1-1.5% of the volume of water, the hypoglycemic and antioxidant activities of the mulberry polysaccharide extract can be further improved.

[0013] Furthermore, the pectinase solution is a mixture of pectinase and 0.05-0.5 mol / L citric acid-sodium citrate buffer in a mass volume ratio of 0.1-0.3 g:10 mL; the cellulase solution is a mixture of cellulase and 0.05-0.5 mol / L citric acid-sodium citrate buffer in a mass volume ratio of 0.1-0.3 g:10 mL.

[0014] The concentration of the above citric acid-sodium citrate buffer refers to the total concentration of citric acid and sodium citrate in the citric acid-sodium citrate buffer.

[0015] Preferably, during the enzymatic hydrolysis process, the temperature is 40-60° C. and the time is 50-150 r / min.

[0016] Preferably, during the ultrasonic-assisted extraction process, the ultrasonic power is 300-400 W, the temperature is 50-60° C., and the time is 40-60 min.

[0017] Preferably, after the enzymatic hydrolysis is completed, the enzyme is inactivated and then the ultrasonic-assisted extraction is performed.

[0018] Preferably, the freeze-dried mulberry pomace powder is first subjected to alcohol elution and decolorization treatment before being prepared into the enzymatic hydrolysis system.

[0019] Preferably, the preparation process of the freeze-dried mulberry pomace powder comprises: squeezing mulberry juice to obtain pomace, freeze-drying the obtained pomace, crushing it, and sieving it to obtain the freeze-dried mulberry pomace powder.

[0020] Preferably, the process of separating and purifying the product comprises: after centrifugation, adding ethanol to the supernatant for precipitation, centrifugation, deproteinizing, dialysis, decolorizing, concentrating and freeze-drying the obtained precipitate.

[0021] Furthermore, the deproteinization method is the Sevage method, and the Sevage reagent used is a mixture of n-butanol and chloroform in a volume ratio of 1:3-5.

[0022] Furthermore, the decolorization method is AB-8 macroporous adsorption resin decolorization, and the addition amount of AB-8 macroporous adsorption resin is 1 / 2 to 1 / 5 of the volume of the solution.

[0023] Furthermore, the concentration temperature is 40-55°C.

[0024] Furthermore, during the dialysis process, the dialysis membrane used has a molecular weight cutoff of 10 to 20 kDa, and the dialysis time is 36 to 72 hours.

[0025] Compared with the prior art, the present invention has the following advantages: The present invention adopts pectinase and cellulase pretreatment, combined with an ultrasonic-assisted extraction method, which can simultaneously improve the hypoglycemic and antioxidant activities of the obtained mulberry polysaccharide extract, so that it has better hypoglycemic and antioxidant effects under the same concentration conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of the α-glucosidase inhibition activity test of mulberry polysaccharide extracts obtained by different extraction methods are shown in Figure 2. Figure 1 (A) is the α-glucosidase inhibition rate of mulberry polysaccharide extracts at different concentrations; Figure 1 (B) is the IC of mulberry polysaccharide extract on α-glucosidase inhibition 50 value.

[0027] Figure 2 The results of the α-amylase inhibition activity test of mulberry polysaccharide extracts obtained by different extraction methods are shown in Figure 2. Figure 2 (A) is the α-amylase inhibition rate of mulberry polysaccharide extracts at different concentrations; Figure 2 (B) is the IC of mulberry polysaccharide extract on α-amylase inhibitory activity 50 value.

[0028] Figure 3 The results of the DPPH free radical scavenging activity test of mulberry polysaccharide extracts obtained by different extraction methods are shown in Figure 2. Figure 3 (A) is the DPPH free radical scavenging rate of mulberry polysaccharide extracts at different concentrations; Figure 3 (B) is the IC of DPPH free radical scavenging activity of mulberry polysaccharide extract 50 value.

[0029] Figure 4 The results of the ABTS free radical scavenging activity test of mulberry polysaccharide extracts obtained by different extraction methods are shown in Figure 2. Figure 4 (A) is the ABTS free radical scavenging rate of mulberry polysaccharide extracts with different concentrations; Figure 4 (B) is the IC of mulberry polysaccharide extract on ABTS free radical scavenging activity 50 value.

[0030] Figure 5 is the extraction rate of mulberry polysaccharides under different extraction methods.

[0031] Figure 6 is the extraction rate of mulberry polysaccharides when using different enzymes.

[0032] Figure 7 The results of hypoglycemic and antioxidant activity tests of mulberry polysaccharide extracts obtained by using different enzymes are shown in Figure 2. Figure 7 (A) is the ABTS free radical scavenging rate of mulberry polysaccharide extracts with different concentrations; Figure 7 (B) is the DPPH free radical scavenging rate of mulberry polysaccharide extracts at different concentrations; Figure 7 (C) is the α-amylase inhibition rate of mulberry polysaccharide extracts at different concentrations; Figure 7 (D) is the α-glucosidase inhibition rate of mulberry polysaccharide extracts at different concentrations.

[0033] Figure 8 is the extraction rate of mulberry polysaccharides under different solid-liquid ratios.

[0034] Fig. 9 This is the extraction rate of mulberry polysaccharides under different enzyme addition amounts.

[0035] Fig.10 The results of hypoglycemic and antioxidant activity tests of mulberry polysaccharide extracts obtained with different enzyme addition amounts are shown in Table 1. Fig.10 (A) is the ABTS free radical scavenging rate of mulberry polysaccharide extracts with different concentrations; Fig.10 (B) is the DPPH free radical scavenging rate of mulberry polysaccharide extracts at different concentrations; Fig.10 (C) is the α-amylase inhibition rate of mulberry polysaccharide extracts at different concentrations; Fig.10 (D) is the α-glucosidase inhibition rate of mulberry polysaccharide extracts at different concentrations. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with the embodiments.

[0037] Overall embodiment A method for improving the hypoglycemic and antioxidant activities of a mulberry polysaccharide extract comprises the following steps: preparing an enzymatic hydrolysis system comprising freeze-dried mulberry pomace powder, pectinase and cellulase, performing enzymatic hydrolysis, then performing ultrasonic-assisted extraction, separating and purifying the product, and obtaining a mulberry polysaccharide extract with high hypoglycemic and antioxidant activities.

[0038] As a specific implementation manner, the freeze-dried mulberry pomace powder is first subjected to alcohol elution and decolorization treatment before being prepared into the enzymatic hydrolysis system.

[0039] As a specific implementation, the preparation process of the mulberry pomace freeze-dried powder includes: squeezing mulberry juice to obtain pomace, freeze-drying the obtained pomace, crushing it, and sieving it to obtain the mulberry pomace freeze-dried powder.

[0040] As a specific embodiment, the preparation process of the enzymatic hydrolysis system includes: mixing mulberry pomace freeze-dried powder, pectinase solution and cellulase solution to obtain an enzymatic hydrolysis system. The mass volume ratio of the mulberry pomace freeze-dried powder to water is 1g:40-100mL. In the enzymatic hydrolysis system, the sum of the volumes of the cellulase solution and the pectinase solution is 0.2-4% of the volume of water, and the mass ratio of cellulase to pectinase is 1:1-1.5. The pectinase solution is a mixture of pectinase and 0.05-0.5mol / L citric acid-sodium citrate buffer at a mass volume ratio of 0.1-0.3g:10mL; the cellulase solution is a mixture of cellulase and 0.05-0.5mol / L citric acid-sodium citrate buffer at a mass volume ratio of 0.1-0.3g:10mL.

[0041] As a specific implementation manner, during the enzymatic hydrolysis process, the temperature is 40-60° C. and the time is 50-150 r / min.

[0042] As a specific implementation manner, after the enzymatic hydrolysis is completed, the enzyme is inactivated, and then the ultrasonic-assisted extraction is performed.

[0043] As a specific implementation, during the ultrasonic-assisted extraction process, the ultrasonic power is 300-400W, the temperature is 50-60°C, and the time is 40-60min.

[0044] As a specific embodiment, the process of separating and purifying the product includes: after centrifugation, adding ethanol to the supernatant for precipitation, centrifugation, deproteinization, dialysis, decolorization, concentration and freeze-drying of the obtained precipitate. The deproteinization method is the Sevage method, and the Sevage reagent used is a mixture of n-butanol and chloroform in a volume ratio of 1:3 to 5. The decolorization method is AB-8 macroporous adsorption resin decolorization, and the addition amount of AB-8 macroporous adsorption resin is 1 / 2 to 1 / 5 of the solution volume. The concentration temperature is 40 to 55°C. During the dialysis process, the dialysis membrane used has a molecular weight cutoff of 10 to 20 kDa, and the dialysis time is 36 to 72 hours.

[0045] The present invention is described below by specific examples. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention, and the attached claims and any equivalents thereof are the protection scope of the present invention.

[0046] In the following specific embodiments, the equipment used is as follows: SPARK microplate reader (effective value 0-1.5), Deloitte Austria Co., Ltd.; ultra-low temperature refrigerator, Thermo Fisher Scientific (Suzhou) Co., Ltd.; SCIENTZ-10N freeze dryer, Ningbo Xinzhi Biotechnology Co., Ltd.; RE-52AA rotary evaporator, Shanghai Yarong Biochemical Instrument Factory; SHZ-D (III) circulating water vacuum pump, Hangzhou Mingyuan Instrument Co., Ltd.; HJ-4A multi-head magnetic stirrer, Jintan District Xinrui Instrument Factory; FE28 pH meter, Mettler-Toledo Instrument (Shanghai) Co., Ltd.; LXJ-IIB low-speed centrifuge, Shanghai Anting Scientific Instrument Factory.

[0047] Example 1 The mulberry polysaccharide extract (EUP) was prepared by the following steps: (1) Preparation of mulberry pomace freeze-dried powder: The pomace remaining after squeezing the mulberry juice is freeze-dried and then crushed, passed through a 40-mesh sieve to obtain freeze-dried mulberry pomace powder, and stored in a -20°C refrigerator for later use.

[0048] (2) Preparation of enzyme solution: Weigh 13.89 g of citric acid and 7.15 g of trisodium citrate, dissolve in deionized water and adjust the volume to 1000 mL to obtain 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5). Weigh 100 mg of pectinase and dissolve in 10 mL of 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5) to obtain 10 mg / mL pectinase solution.

[0049] Weigh 3.58 g of citric acid and 8.10 g of trisodium citrate, dissolve in deionized water and dilute to 1000 mL to obtain 0.05 mol / L citric acid-sodium citrate buffer (pH 4.8). Weigh 100 mg of cellulase and dissolve in 10 mL of 0.05 mol / L citric acid buffer (pH 4.8) to obtain 10 mg / mL cellulase solution.

[0050] (3) Preparation of mulberry crude polysaccharide extract: Take 20g of mulberry pomace freeze-dried powder, add 400mL of 95% ethanol solution, stir thoroughly for 12h under magnetic stirring, centrifuge at 3000rpm for 10min; add 1600mL of distilled water to the precipitate, add 10mg / mL cellulase solution and 10mg / mL pectinase solution 16mL each, water bath at 50℃, magnetic stirring for 120min, inactivate at 100℃ for 10min. Then treat with ultrasound at 400W power for 50min at 60℃. Centrifuge at 3000rpm for 10min, add 5 times volume of 95% ethanol to the supernatant for precipitation overnight, centrifuge at 3000rpm for 15min to collect the precipitate, and obtain mulberry crude polysaccharide extract.

[0051] (4) Purification: Add 400 mL of distilled water to the crude mulberry polysaccharide extract obtained in step (3) and stir until it is completely dissolved, add 100 mL of Sevage reagent and stir magnetically at room temperature for 30 min, centrifuge at 4000 rpm for 10 min, retain the supernatant, and repeat 4 times, wherein the Sevage reagent is a mixture of n-butanol and chloroform in a volume ratio of 1:4. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for 72 hours, during which the water is changed 8 times. After the dialysis is completed, 1 / 3 volume of the treated AB-8 macroporous adsorption resin is added according to the volume of the contents of the dialysis bag, and the shaker is used for decolorization for 4 hours, and the resin particles are removed by suction filtration with a Buchner funnel. The filtrate is concentrated to 1 / 4 of the original volume by a rotary evaporator at 45°C, and freeze-dried to obtain a white mulberry polysaccharide extract, which is recorded as EUP.

[0052] Comparative Example 1 The difference between this comparative example and Example 1 is that during the extraction process, the amount of pectinase and cellulase added is 0, and ultrasound-assisted extraction is not performed. Specifically, this comparative example prepares mulberry polysaccharide extract (HWP) by the following steps: (1) Preparation of mulberry pomace freeze-dried powder: The pomace remaining after squeezing the mulberry juice is freeze-dried and then crushed, passed through a 40-mesh sieve to obtain freeze-dried mulberry pomace powder, and stored in a -20°C refrigerator for later use.

[0053] (2) Preparation of citric acid-sodium citrate buffer: Weigh 13.89 g of citric acid and 7.15 g of trisodium citrate, add deionized water to dissolve and then make up to 1000 mL to obtain 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5).

[0054] Weigh 3.58 g of citric acid and 8.10 g of trisodium citrate, add deionized water to dissolve and then make up to 1000 mL to obtain 0.05 mol / L citric acid-sodium citrate buffer (pH 4.8).

[0055] (3) Preparation of mulberry crude polysaccharide extract: Take 20g of mulberry pomace freeze-dried powder, add 400mL of 95% ethanol solution, stir thoroughly for 12h under magnetic stirring, centrifuge at 3000rpm for 10min; add 1600mL of distilled water to the precipitate, add 16mL of citric acid-sodium citrate buffer (pH3.5) and citric acid-sodium citrate buffer (pH 4.8), water bath at 50℃, magnetic stirring for 120min, inactivate at 100℃ for 10min. Then treat at 60℃ for 50min. Centrifuge at 3000rpm for 10min, add 5 times volume of 95% ethanol to the supernatant for precipitation overnight, centrifuge at 3000rpm for 15min to collect the precipitate, and obtain the mulberry crude polysaccharide extract.

[0056] (4) Purification: Add 400 mL of distilled water to the crude mulberry polysaccharide extract obtained in step (3) and stir until it is completely dissolved, add 100 mL of Sevage reagent and stir magnetically at room temperature for 30 min, centrifuge at 4000 rpm for 10 min, retain the supernatant, and repeat 4 times, wherein the Sevage reagent is a mixture of n-butanol and chloroform in a volume ratio of 1:4. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for 72 hours, during which the water is changed 8 times. After the dialysis is completed, 1 / 3 volume of the treated AB-8 macroporous adsorption resin is added according to the volume of the contents of the dialysis bag, and the shaker is used for decolorization for 4 hours. The resin particles are removed by suction filtration with a Buchner funnel, and the filtrate is concentrated to 1 / 4 of the original volume by a rotary evaporator at 45°C, and freeze-dried to obtain a white mulberry polysaccharide extract, which is recorded as HWP.

[0057] Comparative Example 2 The difference between this comparative example and Example 1 is that during the extraction process, the amount of pectinase and cellulase added is 0, and only ultrasound-assisted extraction is used. Specifically, this comparative example prepares mulberry polysaccharide extract (UWP) by the following steps: (1) Preparation of mulberry pomace freeze-dried powder: The pomace remaining after squeezing the mulberry juice is freeze-dried and then crushed, passed through a 40-mesh sieve to obtain freeze-dried mulberry pomace powder, and stored in a -20°C refrigerator for later use.

[0058] (2) Preparation of citric acid-sodium citrate buffer: Weigh 13.89 g of citric acid and 7.15 g of trisodium citrate, add deionized water to dissolve and then make up to 1000 mL to obtain 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5).

[0059] Weigh 3.58 g of citric acid and 8.10 g of trisodium citrate, add deionized water to dissolve and then make up to 1000 mL to obtain 0.05 mol / L citric acid-sodium citrate buffer (pH 4.8).

[0060] (3) Preparation of mulberry crude polysaccharide extract: Take 20g of mulberry pomace freeze-dried powder, add 400mL of 95% ethanol solution, stir for 12h under magnetic stirring, centrifuge at 3000rpm for 10min; add 1600mL of distilled water to the precipitate, add 16mL of citric acid-sodium citrate buffer (pH3.5) and citric acid-sodium citrate buffer (pH 4.8), water bath at 50℃, magnetic stirring for 120min, inactivate at 100℃ for 10min. Then treat with ultrasound at 400W power for 50min at 60℃. Centrifuge at 3000rpm for 10min, add 5 times volume of 95% ethanol to the supernatant for precipitation overnight, centrifuge at 3000rpm for 15min to collect the precipitate, and obtain the mulberry crude polysaccharide extract.

[0061] (4) Purification: Add 400 mL of distilled water to the crude mulberry polysaccharide extract obtained in step (3) and stir until it is completely dissolved, add 100 mL of Sevage reagent and stir magnetically at room temperature for 30 min, centrifuge at 4000 rpm for 10 min, retain the supernatant, and repeat 4 times, wherein the Sevage reagent is a mixture of n-butanol and chloroform in a volume ratio of 1:4. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for 72 hours, during which the water is changed 8 times. After the dialysis is completed, 1 / 3 volume of the treated AB-8 macroporous adsorption resin is added according to the volume of the contents of the dialysis bag, and the shaker is used for decolorization for 4 hours. The resin particles are removed by suction filtration with a Buchner funnel, and the filtrate is concentrated to 1 / 4 of the original volume by a rotary evaporator at 45°C, and freeze-dried to obtain a white mulberry polysaccharide extract, which is recorded as UWP.

[0062] Comparative Example 3 The difference between this comparative example and Example 1 is that during the extraction process, only cellulase and pectinase are added, and ultrasound-assisted extraction is not performed. Specifically, this comparative example prepares mulberry polysaccharide extract (EWP) by the following steps: (1) Preparation of mulberry pomace freeze-dried powder: The pomace remaining after squeezing the mulberry juice is freeze-dried and then crushed, passed through a 40-mesh sieve to obtain freeze-dried mulberry pomace powder, and stored in a -20°C refrigerator for later use.

[0063] (2) Preparation of enzyme solution: Weigh 13.89 g of citric acid and 7.15 g of trisodium citrate, dissolve in deionized water and adjust the volume to 1000 mL to obtain 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5). Weigh 100 mg of pectinase and dissolve in 10 mL of 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5) to obtain 10 mg / mL pectinase solution.

[0064] Weigh 3.58 g of citric acid and 8.10 g of trisodium citrate, dissolve in deionized water and dilute to 1000 mL to obtain 0.05 mol / L citric acid-sodium citrate buffer (pH 4.8). Weigh 100 mg of cellulase and dissolve in 10 mL of 0.05 mol / L citric acid buffer (pH 4.8) to obtain 10 mg / mL cellulase solution.

[0065] (3) Preparation of mulberry crude polysaccharide extract: Take 20g of mulberry pomace freeze-dried powder, add 400mL of 95% ethanol solution, stir thoroughly for 12h under magnetic stirring, centrifuge at 3000rpm for 10min; add 1600mL of distilled water to the precipitate, add 16mL of 10mg / mL cellulase solution and 10mg / mL pectinase solution, water bath at 50℃, stir magnetically for 120min, inactivate at 100℃ for 10min. Then treat at 60℃ for 50min. Centrifuge at 3000rpm for 10min, add 5 times volume of 95% ethanol to the supernatant for precipitation overnight, centrifuge at 3000rpm for 15min to collect the precipitate, and obtain the mulberry crude polysaccharide extract.

[0066] (4) Purification: Add 400 mL of distilled water to the crude polysaccharide extract of mulberry obtained in step (3) and stir until it is completely dissolved, add 100 mL of Sevage reagent and stir magnetically at room temperature for 30 min, centrifuge at 4000 rpm for 10 min, retain the supernatant, and repeat 4 times, wherein the Sevage reagent is a mixture of n-butanol and chloroform in a volume ratio of 1:4. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for 72 hours, during which the water is changed 8 times. After the dialysis is completed, 1 / 3 volume of the treated AB-8 macroporous adsorption resin is added according to the volume of the contents of the dialysis bag, and the shaker is used for decolorization for 4 hours, and the resin particles are removed by suction filtration with a Buchner funnel. The filtrate is concentrated to 1 / 4 of the original volume by a rotary evaporator at 45°C, and freeze-dried to obtain a white mulberry polysaccharide extract, which is recorded as EWP.

[0067] Test Example 1: Effect of extraction method on hypoglycemic activity, antioxidant activity and extraction rate 1.1 Hypoglycemic activity 1.1.1 α-Glucosidase inhibitory activity The mulberry polysaccharide extracts prepared according to the methods of Example 1 and Comparative Examples 1 to 3 were used to detect their inhibitory activity against α-glucosidase, as follows: ① Preparation of mulberry polysaccharide sample solutions with different concentrations: Weigh 10 mg of mulberry polysaccharide extract, add a small amount of ultrapure water to dissolve, transfer to a 10 mL volumetric flask, dilute to volume with ultrapure water, shake well, and obtain 1 mg / mL mulberry polysaccharide sample mother solution. Dilute the mulberry polysaccharide sample mother solution to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL with ultrapure water to obtain mulberry polysaccharide sample solutions of different concentrations, and store at low temperature for later use.

[0068] ② Preparation of acarbose sample solutions with different concentrations: Weigh 10 mg of acarbose, add a small amount of ultrapure water to dissolve, transfer to a 10 mL volumetric flask, dilute to volume with ultrapure water, shake well, and obtain 1 mg / mL acarbose sample mother solution. Dilute the acarbose sample mother solution to 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL with ultrapure water to obtain acarbose sample solutions of different concentrations, and store at low temperature for later use.

[0069] ③ Preparation of α-glucosidase solution: Weigh 3 mg of α-glucosidase, add a small amount of PBS buffer solution (pH = 6.9) to dissolve, transfer to a 10 mL volumetric flask, make up to volume with PBS, and shake well. Take 1 mL of the solution to a 10 mL volumetric flask, make up to volume with PBS buffer solution, shake well, and obtain a 0.03 mg / mL α-glucosidase solution, which is stored at low temperature for later use.

[0070] ④ Preparation of p-nitro-α-D-glucoside (PNPG) solution: Weigh 4.52 mg of p-nitro-α-D-glucoside, add a small amount of PBS buffer solution (pH = 6.9) to dissolve, transfer to a 10 mL volumetric flask, make up to volume with PBS, shake well to obtain a 1.5 mmol / LPNPG solution, and store at low temperature for later use.

[0071] ⑤ Preparation of 1 mol / L sodium carbonate solution: Weigh 10.6 g of sodium carbonate, add a small amount of ultrapure water to dissolve it, transfer it to a 100 mL volumetric flask, make up to volume, and shake well to obtain a 1 mol / L sodium carbonate solution, which should be stored for later use.

[0072] ⑥ Determination of inhibition of α-glucosidase activity: Use a pipette to draw 50 μL of mulberry polysaccharide sample solution of different concentrations and 100 μL of α-glucosidase solution, mix them, vortex and incubate at 37°C for 20 minutes. Then add 100 μL PNPG and continue to incubate at 37°C for 10 minutes. Finally, add 1 mL of Na2CO3 solution (1 mol / L) to terminate the reaction. Use an enzyme reader to measure its absorbance at a wavelength of 400 nm, with acarbose as a positive control. The experiment was repeated three times, and the α-glucosidase inhibition rate was calculated as follows: Wherein, As is the absorbance of the mixed solution of the sample solution, PNPG solution and α-glucosidase solution, Ac is the absorbance of the mixed solution of PBS buffer solution instead of α-glucosidase solution, and Ab is the absorbance of the mixed solution of PBS buffer solution instead of the sample solution.

[0073] ⑦IC 50 Calculation: According to the α-glucosidase inhibition rate test results of mulberry polysaccharide sample solutions with different concentrations, the IC value of mulberry polysaccharide on α-glucosidase inhibition activity was calculated using Graphpad Prism9.1 software 50 value.

[0074] The results of the α-glucosidase inhibition activity test of the mulberry polysaccharide extracts obtained by different extraction methods (Example 1 and Comparative Examples 1 to 3) are shown in Figure 1 .

[0075] 1.1.2 α-Amylase Inhibitory Activity The mulberry polysaccharide extracts prepared according to the methods of Example 1 and Comparative Examples 1 to 3 were used to detect their inhibitory activity against α-amylase, as follows: ① Preparation of mulberry polysaccharide sample solutions of different concentrations and acarbose positive control solutions: Weigh 50 mg of mulberry polysaccharide extract, add a small amount of ultrapure water to dissolve, transfer to a 5 mL volumetric flask, dilute to volume with ultrapure water, shake well, and obtain a 10 mg / mL mulberry polysaccharide sample mother solution. Dilute the mulberry polysaccharide sample mother solution to 1, 2, 3, 4, and 5 mg / mL with ultrapure water to obtain mulberry polysaccharide sample solutions of different concentrations, and store at low temperature for later use.

[0076] ② Preparation of acarbose sample solutions with different concentrations: Weigh 50 mg of acarbose, add a small amount of ultrapure water to dissolve, transfer to a 5 mL volumetric flask, dilute to volume with ultrapure water, shake well, and obtain a 10 mg / mL acarbose sample solution. Dilute the acarbose sample solution to 1, 2, 3, 4, and 5 mg / mL with ultrapure water to obtain acarbose sample solutions of different concentrations, and store at low temperature for later use.

[0077] ③ Preparation of DNS reagent: Weigh 18.2g of potassium sodium tartrate into a beaker, add 50mL of ultrapure water, stir thoroughly, and heat at 50°C to dissolve it. At this temperature, add 0.6300g of 3,5-dinitrosalicylic acid, 2.1000g of sodium hydroxide, and 0.5000g of phenol in sequence, stirring rapidly while adding until they are completely dissolved. After cooling, dilute to 100mL with ultrapure water to obtain DNS reagent, which is stored in a brown volumetric flask.

[0078] ④ Preparation of α-amylase solution: Weigh 5 mg of α-amylase, add a small amount of PBS buffer solution (pH=6.9), dissolve it and transfer it to a 50 mL volumetric flask, make up to volume with PBS, and shake well to obtain a 0.1 mg / mL α-amylase solution, which can be stored at low temperature.

[0079] ⑤ Preparation of 1% (w / v) soluble starch solution: Weigh 0.1g of soluble starch into a beaker, quickly add 50mL of boiling water and stir to dissolve it. Then slowly add 50mL of boiling water and heat in a boiling water bath for 2-3min to obtain a 1% (w / v) soluble starch solution, which is then placed at room temperature for later use.

[0080] ⑥ Determination of α-amylase activity inhibition: Use a pipette to take 500 μL of mulberry polysaccharide solution of different concentrations and 500 μL of α-amylase solution, mix thoroughly, and vortex. Incubate at 37°C for 10 minutes. Then add 500 μL of 1% (w / v) soluble starch solution and continue incubating at 37°C for 10 minutes. At the end of the incubation, add 1 mL of DNS reagent and boil for 5 minutes to terminate the reaction. After cooling to room temperature, the solution was diluted to 10 mL with ultrapure water, and its absorbance at a wavelength of 520 nm was measured with an enzyme reader, with acarbose as the positive control. The experiment was repeated three times, and the α-amylase inhibition rate formula is as follows: Where As is the absorbance of the mixed solution containing α-amylase solution, starch solution and sample solution, Ac is the absorbance of the mixed solution containing PBS buffer solution instead of α-amylase solution, and Ab is the absorbance of the mixed solution containing PBS buffer solution instead of sample solution.

[0081] ⑦IC 50 Calculation: According to the α-amylase inhibition rate test results of mulberry polysaccharide sample solutions with different concentrations, the IC value of mulberry polysaccharide on α-amylase inhibition activity was calculated using GraphpadPrism 9.1 software. 50 value.

[0082] The results of the α-amylase inhibition activity test of the mulberry polysaccharide extracts obtained by different extraction methods (Example 1 and Comparative Examples 1 to 3) are shown in Figure 2 .

[0083] from Figure 1 and Figure 2It can be seen that compared with hot water extraction (HWP), hot water extraction after enzyme treatment (EWP) and ultrasonic extraction (UWP), the mulberry polysaccharide extract obtained by ultrasonic-assisted extraction (EUP) after cellulase and pectinase pretreatment has higher α-glucosidase inhibition activity and α-amylase inhibition activity, indicating that it has a better hypoglycemic effect. At the same concentration, the hypoglycemic activity of mulberry polysaccharide extracts extracted by different methods is EUP>EWP>HWP>UWP.

[0084] 1.2 Antioxidant activity 1.2.1 DPPH free radical scavenging activity The mulberry polysaccharide extracts prepared according to the methods of Example 1 and Comparative Examples 1 to 3 were used to detect their DPPH free radical scavenging activity as follows: ① Preparation of mulberry polysaccharide sample solutions with different concentrations: Weigh 50 mg of mulberry polysaccharide extract, add a small amount of ultrapure water to dissolve, transfer to a 5 mL volumetric flask, dilute to volume with ultrapure water, shake well, and obtain a 10 mg / mL mulberry polysaccharide sample solution. Dilute the mulberry polysaccharide solution to 1, 2, 3, 4, and 5 mg / mL by adding ultrapure water, and store at low temperature for later use.

[0085] ② Preparation of positive control (ascorbic acid) sample solutions of different concentrations: Weigh 50 mg of ascorbic acid, add a small amount of ultrapure water to dissolve, transfer to a 5 mL volumetric flask, dilute to volume with ultrapure water, and shake well to obtain a 10 mg / mL ascorbic acid sample solution. Dilute the ascorbic acid solution to 1, 2, 3, 4, and 5 mg / mL by adding ultrapure water, and store at low temperature for later use.

[0086] ③ Preparation of 0.2mmol / L DPPH free radical solution: Weigh 7.8 mg DPPH, add a small amount of anhydrous ethanol to dissolve, transfer to a 100 mL brown volumetric flask, make up to volume with anhydrous ethanol, and shake well to obtain a 0.2 mmol / L DPPH solution, which should be stored away from light.

[0087] ④DPPH free radical scavenging ability determination: Use a pipette to draw 1 mL of mulberry polysaccharide solution of different concentrations and 1 mL of DPPH solution, mix thoroughly, vortex and shake, and let it stand at room temperature in the dark for 30 minutes. Finally, use an ELISA reader to measure its absorbance at 517 nm, with ascorbic acid as a positive control. The experiment was repeated three times, and the DPPH free radical scavenging activity was calculated as follows: Where As is the absorbance of the mixed solution of the sample solution and the DPPH solution, Ac is the absorbance of the mixed solution of anhydrous ethanol instead of the DPPH solution, and Ab is the absorbance of the mixed solution of anhydrous ethanol instead of the sample solution.

[0088] The results of the DPPH free radical scavenging activity test of the mulberry polysaccharide extracts obtained by different extraction methods (Example 1 and Comparative Examples 1 to 3) are shown in Figure 3 .

[0089] 1.2.2 ABTS free radical scavenging activity The mulberry polysaccharide extracts prepared according to the methods of Example 1 and Comparative Examples 1 to 3 were used to detect their ABTS free radical scavenging activity as follows: ① Preparation of mulberry polysaccharide sample solutions and positive control solutions of different concentrations: Weigh 50 mg of mulberry polysaccharide extract, add a small amount of ultrapure water to dissolve, transfer to a 5 mL volumetric flask, dilute to volume with ultrapure water, shake well, and obtain a 10 mg / mL mulberry polysaccharide sample solution. Dilute the mulberry polysaccharide solution to 1, 2, 3, 4, and 5 mg / mL by adding ultrapure water, and store at low temperature for later use.

[0090] Weigh 50 mg of ascorbic acid, add a small amount of ultrapure water to dissolve, transfer to a 5 mL volumetric flask, dilute to volume with ultrapure water, and shake well to obtain a 10 mg / mL ascorbic acid sample solution. Dilute the ascorbic acid solution to 1, 2, 3, 4, and 5 mg / mL by adding ultrapure water, and store at low temperature for later use.

[0091] ② Preparation of 7.00mmol / L ABTS solution: Weigh 192 mg of ABTS, add a small amount of ultrapure water to dissolve, transfer to a 50 mL brown volumetric flask, make up to volume with ultrapure water, and shake well to obtain a 7.00 mmol / L ABTS solution, which should be stored away from light.

[0092] ③ Preparation of 2.45mmol / L potassium persulfate solution: Weigh 33 mg of potassium persulfate, add a small amount of ultrapure water to dissolve, transfer to a 50 mL volumetric flask, make up to volume with ultrapure water, and shake well to obtain a 2.45 mmol / L potassium persulfate solution, which is stored for later use.

[0093] ④ Preparation of ABTS free radical solution: Equal volumes of ABTS solution and potassium persulfate solution were fully mixed and shaken, and reacted at room temperature in the dark for 12 h, and then the solution was diluted with ultrapure water until its absorbance at 734 nm was 0.7 (±0.2).

[0094] ⑤ABTS free radical scavenging ability determination: Use a pipette to draw 3mL of ABTS free radical solution and 0.4mL of mulberry polysaccharide sample solution of different concentrations, mix, vortex, and react at room temperature in the dark for 30 minutes. Finally, use an ELISA reader to measure the absorbance value As at 732nm. Ascorbic acid is used as a positive control. The experiment is repeated three times, and the calculation formula of ABTS free radical scavenging activity is as follows: Where As is the absorbance of the mixed solution of the sample solution and the ABTS free radical solution, Ac is the absorbance of the mixed solution of ultrapure water instead of the ABTS free radical solution, and Ab is the absorbance of the mixed solution of ultrapure water instead of the sample solution.

[0095] The results of the ABTS free radical scavenging activity test of the mulberry polysaccharide extracts obtained by different extraction methods (Example 1 and Comparative Examples 1 to 3) are shown in Figure 4 .

[0096] from Figure 3 and Figure 4 It can be seen that the antioxidant activity of mulberry polysaccharides obtained by different extraction methods is EUP>EWP>HWP>UWP. 50 The IC values ​​of ABTS free radical scavenging rate were 38.5%, 46.5% and 63.2% lower than those of EWP, HWP and UWP, respectively. 50 It is 28.5%, 39.4% and 46.9% lower than EWP, HWP and UWP respectively.

[0097] 1.3 Extraction rate Mulberry polysaccharide extracts were prepared according to the methods in Example 1 and Comparative Examples 1 to 3, and the extraction rates were calculated as follows: the mass of the freeze-dried powder of mulberry pomace used was recorded as m0, and the supernatant was extracted according to the method in step (3) of each embodiment and comparative example, and then the supernatant was precipitated with alcohol, freeze-dried, and protein was removed, and then the supernatant was re-dissolved and precipitated with alcohol. The precipitate was freeze-dried at -80°C to obtain crude polysaccharide, and the mass of the crude polysaccharide was weighed and recorded as m1. The extraction rate of mulberry polysaccharide was the mass of the crude polysaccharide divided by the mass of the freeze-dried powder of mulberry, that is, m1 / m0×100%.

[0098] The extraction rates of mulberry polysaccharides under different extraction methods (Example 1 and Comparative Examples 1 to 3) are shown in Table 1. Figure 5 .from Figure 5 It can be seen that the extraction rate of mulberry pomace polysaccharides by ultrasound-assisted extraction (EUP) after cellulase and pectinase pretreatment was 4.81%, which was 18.8%, 16.8% and 20.9% higher than those of hot water extraction (HWP), hot water extraction after enzyme treatment (EWP) and ultrasonic extraction (UWP), respectively.

[0099] Comparative Example 4 The difference between this comparative example and Example 1 is that only cellulase is used in the extraction process. Specifically, this comparative example prepares mulberry polysaccharide extract by the following steps: (1) Preparation of mulberry pomace freeze-dried powder: The pomace remaining after squeezing the mulberry juice is freeze-dried and then crushed, passed through a 40-mesh sieve to obtain freeze-dried mulberry pomace powder, and stored in a -20°C refrigerator for later use.

[0100] (2) Preparation of enzyme solution: Weigh 3.58 g of citric acid and 8.10 g of trisodium citrate, dissolve in deionized water and dilute to 1000 mL to obtain 0.05 mol / L citric acid-sodium citrate buffer (pH 4.8). Weigh 100 mg of cellulase and dissolve in 10 mL of 0.05 mol / L citric acid buffer (pH 4.8) to obtain 10 mg / mL cellulase solution.

[0101] (3) Preparation of mulberry crude polysaccharide extract: Take 20g of mulberry pomace freeze-dried powder, add 400mL of 95% ethanol solution, stir for 12h under magnetic stirring, centrifuge at 3000rpm for 10min; add 1600mL of distilled water to the precipitate, add 32mL of 10mg / mL cellulase solution, water bath at 50℃, magnetic stirring for 120min, inactivate at 100℃ for 10min. Then treat with ultrasound at 400W power for 50min at 60℃. Centrifuge at 3000rpm for 10min, add 5 times volume of 95% ethanol to the supernatant for precipitation overnight, centrifuge at 3000rpm for 15min to collect the precipitate, and obtain mulberry crude polysaccharide extract.

[0102] (4) Purification: Add 400 mL of distilled water to the crude mulberry polysaccharide extract obtained in step (3) and stir until it is completely dissolved, add 100 mL of Sevage reagent and stir magnetically at room temperature for 30 min, centrifuge at 4000 rpm for 10 min, retain the supernatant, and repeat 4 times, wherein the Sevage reagent is a mixture of n-butanol and chloroform in a volume ratio of 1:4. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for 72 hours, during which the water is changed 8 times. After the dialysis is completed, add 1 / 3 volume of the treated AB-8 macroporous adsorption resin according to the volume of the dialysis bag content, decolorize on a shaking table for 4 hours, filter with a Buchner funnel to remove the resin particles, and concentrate the filtrate to 1 / 4 of the original volume at 45°C by a rotary evaporator. After freeze-drying, a white mulberry polysaccharide extract is obtained.

[0103] Comparative Example 5 The difference between this comparative example and Example 1 is that only pectinase is used in the extraction process. Specifically, this comparative example prepares mulberry polysaccharide extract by the following steps: (1) Preparation of mulberry pomace freeze-dried powder: The pomace remaining after squeezing the mulberry juice is freeze-dried and then crushed, passed through a 40-mesh sieve to obtain freeze-dried mulberry pomace powder, and stored in a -20°C refrigerator for later use.

[0104] (2) Preparation of enzyme solution: Weigh 13.89 g of citric acid and 7.15 g of trisodium citrate, dissolve in deionized water and adjust the volume to 1000 mL to obtain 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5). Weigh 100 mg of pectinase and dissolve in 10 mL of 0.1 mol / L citric acid-sodium citrate buffer (pH 3.5) to obtain 10 mg / mL pectinase solution.

[0105] (3) Preparation of mulberry crude polysaccharide extract: Take 20g of mulberry pomace freeze-dried powder, add 400mL of 95% ethanol solution, stir thoroughly for 12h under magnetic stirring, centrifuge at 3000rpm for 10min; add 1600mL of distilled water to the precipitate, add 32mL of 10mg / mL pectinase solution, water bath at 50℃, magnetic stirring for 120min, inactivate at 100℃ for 10min. Then treat with ultrasound at 400W power for 50min at 60℃. Centrifuge at 3000rpm for 10min, add 5 times volume of 95% ethanol to the supernatant for precipitation overnight, centrifuge at 3000rpm for 15min to collect the precipitate, and obtain the mulberry crude polysaccharide extract.

[0106] (4) Purification: Add 400 mL of distilled water to the crude mulberry polysaccharide extract obtained in step (3) and stir until it is completely dissolved, add 100 mL of Sevage reagent and stir magnetically at room temperature for 30 min, centrifuge at 4000 rpm for 10 min, retain the supernatant, and repeat 4 times, wherein the Sevage reagent is a mixture of n-butanol and chloroform in a volume ratio of 1:4. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for 72 hours, during which the water is changed 8 times. After the dialysis is completed, add 1 / 3 volume of the treated AB-8 macroporous adsorption resin according to the volume of the dialysis bag content, decolorize on a shaking table for 4 hours, filter with a Buchner funnel to remove the resin particles, and concentrate the filtrate to 1 / 4 of the original volume at 45°C by a rotary evaporator. After freeze-drying, a white mulberry polysaccharide extract is obtained.

[0107] Comparative Example 6 The difference between this comparative example and Example 1 is that during the extraction process, cellulase and pectinase are replaced with papain. Specifically, this comparative example prepares mulberry polysaccharide extract by the following steps: (1) Preparation of mulberry pomace freeze-dried powder: The pomace remaining after squeezing the mulberry juice is freeze-dried and then crushed, passed through a 40-mesh sieve to obtain freeze-dried mulberry pomace powder, and stored in a -20°C refrigerator for later use.

[0108] (2) Preparation of enzyme solution: Weigh 100 mg of papain and dissolve it in 10 mL of phosphate buffer (pH 7.0) to obtain a 10 mg / mL papain solution.

[0109] (3) Preparation of mulberry crude polysaccharide extract: Take 20g of mulberry pomace freeze-dried powder, add 400mL of 95% ethanol solution, stir for 12h under magnetic stirring, centrifuge at 3000rpm for 10min; add 1600mL of distilled water to the precipitate, add 32mL of 10mg / mL papain solution, water bath at 50℃, magnetic stirring for 120min, inactivate at 100℃ for 10min. Then treat with ultrasound at 400W power for 50min at 60℃. Centrifuge at 3000rpm for 10min, add 5 times volume of 95% ethanol to the supernatant for precipitation overnight, centrifuge at 3000rpm for 15min to collect the precipitate, and obtain the mulberry crude polysaccharide extract.

[0110] (4) Purification: Add 400 mL of distilled water to the crude mulberry polysaccharide extract obtained in step (3) and stir until it is completely dissolved, add 100 mL of Sevage reagent and stir magnetically at room temperature for 30 min, centrifuge at 4000 rpm for 10 min, retain the supernatant, and repeat 4 times, wherein the Sevage reagent is a mixture of n-butanol and chloroform in a volume ratio of 1:4. The supernatant is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed for 72 hours, during which the water is changed 8 times. After the dialysis is completed, add 1 / 3 volume of the treated AB-8 macroporous adsorption resin according to the volume of the dialysis bag content, decolorize on a shaking table for 4 hours, filter with a Buchner funnel to remove the resin particles, and concentrate the filtrate to 1 / 4 of the original volume at 45°C by a rotary evaporator. After freeze-drying, a white mulberry polysaccharide extract is obtained.

[0111] Test Example 2: Effect of enzyme types on hypoglycemic activity, antioxidant activity and extraction rate Mulberry polysaccharide extracts were prepared according to the methods in Example 1 and Comparative Examples 4 to 6. The hypoglycemic activity, antioxidant activity and extraction rate were measured using the method in Test Example 1. The results are shown in Table 2. Figure 6 and Figure 7 .from Figure 6 and Figure 7It can be seen that: although the extraction rate of mulberry polysaccharide when papain is used alone is higher than that when cellulase + pectinase is used in combination, the antioxidant activity and hypoglycemic activity of the obtained mulberry polysaccharide extract are significantly lower than that when cellulase + pectinase is used in combination. This shows that there is no positive correlation between the hypoglycemic and antioxidant activities of the mulberry polysaccharide extract and the polysaccharide extraction rate. Improving the polysaccharide extraction rate does not mean that the hypoglycemic and antioxidant activities of the product can be improved. Moreover, the method of extracting with cellulase + pectinase in the present invention can make the obtained mulberry polysaccharide extract have higher hypoglycemic and antioxidant activities.

[0112] Embodiments 2 to 5 In Examples 2 to 5, based on Example 1, only the material-liquid ratio (the mass ratio of the freeze-dried mulberry pomace powder to distilled water in step (3)) was changed. The material-liquid ratio was changed from 1:80 in Example 1 to 1:20, 1:40, 1:60, and 1:100, respectively. That is, in step (3), the amount of distilled water added to the precipitate was changed from 1600 mL in Example 1 to 400 mL, 80 mL, 1200 mL, and 2000 mL, respectively; the remaining operations were the same as in Example 1.

[0113] Test Example 3: Effect of the solid-liquid ratio on the extraction rate Mulberry polysaccharide extracts were prepared according to the methods in Examples 1 and 2 to 5, and the extraction rate was calculated using the method in Test Example 1. The results are shown in Table 1. Figure 8 .from Figure 8 It can be seen that: when the extraction solvent is too little (the solid-liquid ratio is too low), the polysaccharide components in the mulberry pomace are not easy to dissolve, resulting in a low extraction rate; when the extraction solvent is too much (the solid-liquid ratio is too large), the mulberry polysaccharides are more degraded, which will also result in a low extraction rate; the optimal solid-liquid ratio for mulberry polysaccharide extraction is 1:80, and the extraction rate is the highest at this solid-liquid ratio.

[0114] Embodiments 6 to 8 In Examples 6 to 8, based on Example 1, only the amount of enzyme added was changed (enzyme addition amount = enzyme solution volume in step (3) / distilled water volume × 100%). The enzyme addition amount was changed from 1% in Example 1 to 0.5%, 2%, and 4%, respectively. That is, in step (3), the amount of cellulase solution and pectinase solution added was changed from 16 mL in Example 1 to 8 mL, 32 mL, and 64 mL, respectively. The rest of the operations were the same as in Example 1.

[0115] Test Example 4: Effect of enzyme addition on extraction rate Mulberry polysaccharide extracts were prepared according to the methods in Example 1 and Examples 6 to 8, and the hypoglycemic activity, antioxidant activity and extraction rate were measured using the method in Test Example 1. The results are shown in Table 1. Fig. 9 and Fig.10 .from Fig. 9 and Fig.10 It can be seen that when the addition amount of pectinase and cellulase is 2%, the extraction rate is similar to that when the enzyme addition amount is 1%; but when the enzyme addition amount is 1%, the mulberry polysaccharide extract obtained has higher antioxidant and hypoglycemic activities, and the ABTS clearance rate, α-amylase inhibition rate and α-glucosidase inhibition rate are the highest, and the DPPH clearance rate is equivalent to that when the enzyme addition amount is 2%.

[0116] Unless otherwise defined, all technical terms and scientific terms used in the present invention have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the raw materials and equipment used in the present invention are conventional raw materials and equipment in the field and can be obtained from conventional commercial channels; unless otherwise specified, the methods used in the present invention are conventional methods in the field.

[0117] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A method for improving the hypoglycemic and antioxidant activities of mulberry polysaccharide extracts, characterized in that: The following steps are involved: An enzymatic hydrolysis system comprising freeze-dried mulberry pomace powder, pectinase and cellulase is prepared, and after enzymatic hydrolysis, ultrasonic-assisted extraction is performed to separate and purify the product to obtain a mulberry polysaccharide extract with high hypoglycemic and antioxidant activities.

2. The method according to claim 1, characterized in that The preparation process of the enzymatic hydrolysis system comprises: mixing mulberry pomace freeze-dried powder, pectinase solution, cellulase solution and water to obtain the enzymatic hydrolysis system.

3. The method according to claim 2, characterized in that The mass volume ratio of the mulberry pomace freeze-dried powder to water is 1 g: 40-100 mL.

4. The method according to claim 2, characterized in that: In the enzymatic hydrolysis system, the sum of the volumes of the cellulase solution and the pectinase solution is 0.2-4% of the volume of water, the mass ratio of the cellulase to the pectinase is 1:1-1.5, and the concentrations of the cellulase solution and the pectinase solution are 10-30 mg / mL.

5. The method according to claim 1 or 2, characterized in that: During the enzymatic hydrolysis process, the temperature is 40-60° C. and the time is 50-150 r / min.

6. The method according to claim 2, characterized in that The pectinase solution is a mixture of pectinase and 0.05-0.5 mol / L citric acid-sodium citrate buffer in a mass volume ratio of 0.1-0.3 g:10 mL; the cellulase solution is a mixture of cellulase and 0.05-0.5 mol / L citric acid-sodium citrate buffer in a mass volume ratio of 0.1-0.3 g:10 mL.

7. The method according to claim 1, characterized in that During the ultrasonic-assisted extraction process, the ultrasonic power is 300-400 W, the temperature is 50-60° C., and the time is 40-60 min.

8. The method according to claim 1, characterized in that The mulberry pomace freeze-dried powder is first subjected to alcohol washing and decolorization treatment before being prepared into the enzymatic hydrolysis system.

9. The method according to claim 1, characterized in that: The preparation process of the mulberry pomace freeze-dried powder comprises: squeezing mulberry juice to obtain pomace, freeze-drying the obtained pomace, crushing it, and sieving it to obtain the mulberry pomace freeze-dried powder.

10. The method according to claim 1, characterized in that The process of separating and purifying the product comprises: adding ethanol to the supernatant for precipitation after centrifugal separation, centrifugal separation, deproteinizing, dialysis, decolorizing, concentrating and freeze-drying the obtained precipitate.

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