Extraction method and application of cassava peel crude polysaccharide

Abstract

The invention relates to the technical field of cassava peel crude polysaccharide extraction, and discloses a cassava peel crude polysaccharide extraction method and application, the cassava peel crude polysaccharide extraction method comprises the following steps: S1, cassava peel pretreatment: cleaning cassava peel, chopping, placing in a grinding bowl, placing on ice, and grinding and crushing to 80-120 meshes to prepare cassava peel pulp; s2, ultrasonic-assisted enzymolysis: adding deionized water according to a material-liquid ratio of 1: 2-1: 8, and adding an enzyme preparation according to 1-5KU / g; s3, performing centrifugal separation, and sub-packaging the enzymatic hydrolysate into centrifugal tubes; s4, performing deproteinization treatment; s5, ethanol precipitation and purification; s6, dialysis refining and product obtaining. Low-temperature alpha-amylase, medium-temperature alpha-amylase, pullulanase or hemicellulase are combined with ultrasonic parameters and enzyme species adaptation design, and the ultrasonic cavitation effect enhances the efficiency of enzyme molecule permeation and substrate combination, reduces the enzyme dosage, avoids thermal degradation, and improves the yield and functional activity.

Description

Technical Field

[0001] The invention relates to the technical field of cassava peel crude polysaccharide extraction, in particular to an extraction method and application of cassava peel crude polysaccharide. Background Art

[0002] Cassava (Manihot esculenta Crantz) is a food crop widely grown in tropical regions around the world, with an annual output of more than 300 million tons. It is a food crop that more than 1 billion people in tropical regions of the world rely on for survival. Cassava is also an important economic crop. During the processing, a large amount of by-products such as cassava residue, cassava leaves and cassava peels will be produced. Cassava peels are thin skins and leathery parenchyma tissues attached to the outside of cassava roots, accounting for about 20% of the total mass of cassava, and are rich in crude polysaccharides. Polysaccharides are a class of biological macromolecules formed by monosaccharides connected together by glycosidic bonds. They have attracted much attention due to their unique biological activities, such as antioxidant, immunomodulatory, anti-tumor, and hypoglycemic effects, and have wide application value in medicine, food, cosmetics and other fields. Studies have shown that cassava tubers are an important material basis for the preparation of active polysaccharides, while traditional extraction methods such as hot water extraction and acid-base extraction,

[0003] In current technology, although hot water extraction and acid-base extraction are simple to operate, they can easily cause the breakage of heat-sensitive groups of polysaccharides, cause deacetylation or oxidation reactions of polysaccharides, and lead to the reduction of polysaccharide molecular weight and loss of antioxidant activity. Summary of the Invention

[0004] In view of the shortcomings of the existing technology, the present invention provides a method for extracting crude cassava peel polysaccharides and its application, which solves the problem that polysaccharides are easily broken by heat-sensitive groups, deacetylated and oxidized, resulting in reduced molecular weight of polysaccharides and loss of antioxidant activity.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions: A method for extracting crude polysaccharides from cassava peels, comprising the following steps:

[0006] S1, pre-treatment of cassava peels: washing and chopping the cassava peels, placing them in a grinding bowl on ice and grinding them to 80-120 mesh, to obtain cassava peel pulp;

[0007] S2, ultrasonic-assisted enzymatic hydrolysis, adding deionized water at a material-liquid ratio of 1:2-1:8, adding enzyme preparation at 1-5 KU / g, and then treating at an ultrasonic temperature of 45-70°C and an ultrasonic power of 300W for 60-360 minutes;

[0008] S3. Centrifugal separation: the enzymatic hydrolysate was divided into centrifuge tubes and centrifuged at 8000 r / min and 25°C for 10 min. The precipitate was removed and the supernatant was retained.

[0009] S4, deproteinization treatment, adding 4% trichloroacetic acid to the supernatant, mixing evenly, and removing the precipitate by centrifugation;

[0010] S5, ethanol precipitation and purification: add 4 times the volume of anhydrous ethanol, let it stand for 2 hours, then centrifuge to remove the supernatant, and wash the precipitate with 95% ethanol and centrifuge, repeat 3 times;

[0011] S6. Dialysis purification and product acquisition: the precipitate was redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed in deionized water for 72 h, with the liquid changed 4 times a day. The liquid in the dialysis bag was then collected and freeze-dried to obtain crude cassava peel polysaccharide.

[0012] Preferably, the step S1 further comprises transferring the cassava peel pulp to a microwave reactor, adjusting the pH to 6.0-7.5, applying microwave-low-frequency ultrasound synergistic treatment, gradually increasing the temperature to 60-80° C., and treating for 15-25 minutes.

[0013] Preferably, the enzyme preparation in S2 includes low-temperature α-amylase, medium-temperature α-amylase, pullulanase or hemicellulase.

[0014] Preferably, the enzyme preparation is a low-temperature α-amylase, and the S2 parameters include:

[0015] The ultrasonic temperature was 45°C, the enzyme dosage was 1 KU / g, the material-liquid ratio was 1:2, and the ultrasonic time was 360 min.

[0016] Preferably, the enzyme preparation is a mesophilic α-amylase, and the S2 parameters include:

[0017] The ultrasonic temperature was 70°C, the enzyme dosage was 5 KU / g, the solid-liquid ratio was 1:2.5, and the ultrasonic time was 240 min.

[0018] Preferably, the enzyme preparation is pullulanase, and the S2 parameters include:

[0019] The ultrasonic temperature was 55°C, the enzyme dosage was 4 KU / g, the solid-liquid ratio was 1:2, and the ultrasonic time was 60 min.

[0020] Preferably, the enzyme preparation is cellulase, and the S2 parameters include:

[0021] The ultrasonic temperature was 50°C, the enzyme dosage was 2.4 KU / g, the material-liquid ratio was 1:8, and the ultrasonic time was 120 min.

[0022] A use of crude cassava peel polysaccharide, wherein the crude polysaccharide is used as a natural antioxidant for preparing functional foods, anti-aging cosmetics, or pharmaceutical preparations for assisting in the treatment of oxidative stress-related diseases, wherein:

[0023] The functional food is a solid beverage or dietary supplement, the addition amount is 1% to 10% by weight, and it is compounded with vitamin C to increase the DPPH free radical scavenging activity by ≥20%;

[0024] The anti-aging cosmetic is a sunscreen or repair essence, and the added amount is 0.5% to 5% by mass

[0025] , synergistically inhibits lipid peroxidation with β-carotene;

[0026] The pharmaceutical preparation is an oral capsule or nanoliposome with a dosage of 50-200 mg / day, and is targeted at clearing free radicals in the intestine or skin.

[0027] The present invention provides a method for extracting crude cassava peel polysaccharides and its application, which has the following beneficial effects:

[0028] 1. The present invention combines low-temperature α-amylase, medium-temperature α-amylase, pullulanase or hemicellulase with ultrasonic parameters and enzyme adaptability design. The ultrasonic cavitation effect enhances the efficiency of enzyme molecule penetration and substrate binding, reduces enzyme dosage and avoids thermal degradation, and improves yield and functional activity.

[0029] 2. The present invention combines ice grinding with microwave and ultrasonic synergistic treatment to efficiently destroy the lignin and cellulose composite structure of the cassava peel cell wall at low temperature, inhibiting oxidase activity and the release of toxic substances. Microwave uniform heating and ultrasonic cavitation synergistically expand cell pores, accelerate solvent penetration and polysaccharide dissolution, and gradient heating avoids high temperature damage to the polysaccharide chain, ultimately obtaining a highly active, low-toxic homogeneous slurry, significantly improving the yield and purity.

[0030] 3. The present invention uses trichloroacetic acid deproteinization, ethanol precipitation and dialysis for three-stage purification to selectively remove proteins, salts and small molecule impurities, retain the natural structure of polysaccharides, and accurately control the molecular weight by combining dialysis with periodic liquid exchange. Freeze-drying maintains the porous structure and activity. The final product has few impurities, high purity, and stable physical and chemical properties, which is suitable for the needs of the food, medicine and cosmetics fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a flow chart of a method for extracting crude polysaccharides from cassava peels of the present invention;

[0032] Figure 2 Schematic diagram of the effects of different cassava crude polysaccharides on DPPH free radical scavenging rate of the present invention;

[0033] Figure 3 Schematic diagram of the effects of different cassava crude polysaccharides on hydroxyl radical scavenging rates of the present invention;

[0034] Figure 4Schematic diagram of the effect of different cassava crude polysaccharides on the superoxide anion radical scavenging rate of the present invention.

[0035] Among them, 1#. Low-temperature α-amylase enzymatic hydrolysis of cassava peel crude polysaccharide; 4#. Medium-temperature α-amylase enzymatic hydrolysis of cassava peel crude polysaccharide; 7#. Pullulanase enzymatic hydrolysis of cassava peel crude polysaccharide; 10#. Hemicellulase enzymatic hydrolysis of cassava peel crude polysaccharide. DETAILED DESCRIPTION

[0036] The following will clearly and completely describe the technical solution of the present invention in conjunction with the accompanying drawings. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0037] Please see the attached Figure 1 The embodiment of the present invention provides a method for extracting crude polysaccharides from cassava peel, comprising the following steps:

[0038] S1, pre-treatment of cassava peels: washing and chopping the cassava peels, placing them in a grinding bowl on ice and grinding them to 80-120 mesh, to obtain cassava peel pulp;

[0039] S2, ultrasonic-assisted enzymatic hydrolysis, adding deionized water at a material-liquid ratio of 1:2-1:8, adding enzyme preparation at 1-5 KU / g, and then treating at an ultrasonic temperature of 45-70°C and an ultrasonic power of 300W for 60-360 minutes;

[0040] S3. Centrifugal separation: the enzymatic hydrolysate was divided into centrifuge tubes and centrifuged at 8000 r / min and 25°C for 10 min. The precipitate was removed and the supernatant was retained.

[0041] S4, deproteinization treatment, adding 4% trichloroacetic acid to the supernatant, mixing evenly, and removing the precipitate by centrifugation;

[0042] S5, ethanol precipitation and purification: add 4 times the volume of anhydrous ethanol, let it stand for 2 hours, then centrifuge to remove the supernatant, and wash the precipitate with 95% ethanol and centrifuge, repeat 3 times;

[0043] S6. Dialysis purification and product acquisition: the precipitate was redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed in deionized water for 72 h, with the liquid changed 4 times a day. The liquid in the dialysis bag was then collected and freeze-dried to obtain crude cassava peel polysaccharide.

[0044] Specifically, in S1, the cassava peel is pretreated by shredding and then grinding on ice to destroy the cell wall structure of the cassava peel and release bound polysaccharides. At the same time, the low temperature environment inhibits the activity of oxidases and reduces the release of toxic substances, forming a uniform and fine slurry to optimize the contact efficiency of the subsequent enzymatic hydrolysis reaction, laying the foundation for the extraction of high-purity and high-activity crude cassava peel polysaccharides, ensuring stable and efficient extraction and controllable product safety.

[0045] S2 uses the synergistic effect of ultrasonic cavitation and enzymatic hydrolysis to efficiently destroy the cell walls and parenchyma structure of cassava peels under mild temperature conditions, prompting enzyme preparations to act directional on substrates such as starch, cellulose and hemicellulose, accelerating the dissolution and release of polysaccharides. The mechanical vibration and microfluidics generated by ultrasound enhance the contact efficiency between enzymes and substrates, shorten the enzymatic hydrolysis reaction time and reduce the enzyme dosage, while avoiding the damage of high temperature to the molecular structure of polysaccharides and ensuring the integrity of the polysaccharide biological activity. By regulating the adaptability of ultrasonic parameters and enzyme types and dosages, selective extraction of different polysaccharide components is achieved, improving product purity and functionality, and providing a high-activity, low-impurity reaction system for subsequent separation and purification.

[0046] S3 effectively separates the solid-liquid mixture produced after the enzymatic hydrolysis reaction by dispensing the enzymatic hydrolysis solution into centrifuge tubes and centrifuging them under specific centrifugal force and temperature conditions. This removes incompletely degraded plant fiber fragments, cell residues, and macromolecular impurities, while retaining the polysaccharide-rich supernatant. By optimizing the centrifugation parameters, the polysaccharide molecules maintain their structural integrity under mild conditions, avoiding activity loss caused by excessive mechanical shearing or temperature fluctuations. At the same time, the clarity of the supernatant is significantly improved, providing a high-purity intermediate for subsequent deproteinization and purification steps, ensuring that the final extracted crude polysaccharide has a low impurity content and stable biological activity.

[0047] S4 adds trichloroacetic acid to the polysaccharide-rich supernatant, using its acidic conditions to denature the protein in the solution and form an insoluble precipitate. The precipitate is then removed by centrifugation, effectively reducing the protein impurity content in the polysaccharide extract, thereby maximizing the separation efficiency of protein and polysaccharide, retaining the biological activity and functional properties of the polysaccharide, and significantly improving the product purity. It provides a low-protein, high-polysaccharide content intermediate for subsequent ethanol precipitation and dialysis purification, ensuring that the final crude polysaccharide meets the strict requirements for impurity limits in the food, pharmaceutical and other fields;

[0048] S5 adds anhydrous ethanol to the deproteinized supernatant, utilizing the property of ethanol reducing the polarity of the solution after being miscible with water, thereby promoting the selective precipitation of crude polysaccharides in the dissolved state due to a sharp drop in solubility, forming a flocculent precipitate. The static standing process ensures that the polysaccharide molecules are fully aggregated and form a stable precipitate. Subsequently, the ethanol, residual small molecule impurities and unbound organic solvents in the supernatant are removed by centrifugation, retaining the polysaccharide precipitate, and further washing the precipitate multiple times with high-concentration ethanol to thoroughly remove impurities such as salts, pigments and trace proteins adsorbed on the surface of the precipitate, significantly improving the purity of the polysaccharide, thereby achieving effective separation of polysaccharides from impurities, and providing a high-purity, low-impurity intermediate product for subsequent dialysis and freeze-drying, thereby ensuring the stability and biological activity of the final crude polysaccharide product;

[0049] S6 dissolves the polysaccharide purified by ethanol precipitation in deionized water and places it into a dialysis bag with a specific molecular weight cutoff. It uses the selective permeability of the dialysis membrane to continuously remove small molecule impurities (such as salts, unbound organic solvents and low molecular weight pigments) remaining in the solution, while retaining the target polysaccharide components. The concentration gradient is maintained by periodically replacing the dialysis external fluid to ensure that small molecules are fully diffused into the external solution, and finally a highly purified polysaccharide solution is obtained. The water is then removed by freeze-drying, and the natural structure and biological activity of the polysaccharide are completely retained, forming a loose, porous, and easy-to-preserve solid crude polysaccharide product, thereby effectively avoiding the destruction of the molecular structure by chemical or thermal degradation, and ensuring that the crude polysaccharide product has a uniform molecular weight distribution, low impurity residues and stable physical and chemical properties.

[0050] S1 further comprises transferring the cassava peel pulp to a microwave reactor, adjusting the pH to 6.0-7.5, applying microwave-low frequency ultrasound synergistic treatment, gradually increasing the temperature to 60-80° C., and treating for 15-25 minutes.

[0051] Specifically, the microwave-low-frequency ultrasound synergistic treatment added in S1 further destroys the lignin-cellulose composite structure of the cassava peel cell wall in a weakly alkaline to neutral environment (pH 6.0-7.5) through the synergistic effect of the electromagnetic heating effect of microwaves and the mechanical cavitation effect of low-frequency ultrasound, thereby enhancing the dissolution of cell contents. The rapid and uniform heating generated by microwaves promotes the expansion of cell wall pores, accelerates the penetration of solvents and the diffusion of polysaccharides, while the cavitation microjets of low-frequency ultrasound break up the residual dense fibers of the cell wall through local high-pressure impact, releasing bound polysaccharides. The gradient heating (60-80°C) reduces the resistance of the cell wall through a staged thermal stress relaxation mechanism, avoids the breakage of polysaccharide molecular chains caused by instantaneous high temperature, improves the cell disruption efficiency and polysaccharide dissolution rate, reduces the substrate resistance in the subsequent enzymatic hydrolysis stage, and at the same time inactivates some endogenous oxidases and microorganisms to ensure the stability of the extraction system, ultimately obtaining a polysaccharide raw material with low impurity content and high molecular integrity.

[0052] The enzyme preparation in S2 includes low-temperature α-amylase, medium-temperature α-amylase, pullulanase or hemicellulase.

[0053] Specifically, the enzyme preparations used in step S2 (low-temperature α-amylase, medium-temperature α-amylase, pullulanase and hemicellulase) target the degradation of biomacromolecules with different structures in cassava peel through specific catalysis. Among them, low-temperature α-amylase selectively hydrolyzes amylose and part of amylopectin at mild temperature, destroying the wrapping effect of starch matrix on polysaccharides. Medium-temperature α-amylase efficiently decomposes complex starch networks at higher temperatures and releases bound polysaccharides. Pullulanase deconstructs branched polysaccharides and glycogen by cutting α-1,6 glycosidic bonds, thereby improving the dissolution efficiency of soluble polysaccharides. Hemicellulase directionally degrades the hemicellulose components in the cell wall, weakens the mechanical strength of the fiber-lignin composite structure, and promotes the full release of intracellular polysaccharides.

[0054] The enzyme preparation is low-temperature α-amylase, and the S2 parameters include:

[0055] The ultrasonic temperature was 45°C, the enzyme dosage was 1 KU / g, the material-liquid ratio was 1:2, and the ultrasonic time was 360 min.

[0056] Specifically, when low-temperature α-amylase is used in step S2, by adapting to a mild ultrasonic temperature environment, the enzyme activity is directed to act on the straight-chain and amylopectin in the cassava peel under non-thermal denaturation conditions, cutting the α-1,4 glycosidic bonds to decompose the physical wrapping effect of the starch network on the intracellular polysaccharides. Combined with the microjet impact generated by the ultrasonic cavitation effect, the cell wall rupture and the diffusion of the enzymatic hydrolysis products are accelerated, thereby promoting the complete hydrolysis of the starch matrix and releasing the bound polysaccharides.

[0057] The enzyme preparation is a mesophilic α-amylase, and the S2 parameters include:

[0058] The ultrasonic temperature was 70°C, the enzyme dosage was 5 KU / g, the solid-liquid ratio was 1:2.5, and the ultrasonic time was 240 min.

[0059] Specifically, when medium-temperature α-amylase is used in S2, by adapting to a higher ultrasonic temperature environment, the enzyme activity is fully activated to efficiently decompose the complex starch network and branched polysaccharide structure in cassava peel, cutting α-1,4 and α-1,6 glycosidic bonds to disintegrate the physical wrapping of the starch matrix on the intracellular polysaccharides.

[0060] The enzyme preparation is pullulanase, and the S2 parameters include:

[0061] The ultrasonic temperature was 55°C, the enzyme dosage was 4 KU / g, the solid-liquid ratio was 1:2, and the ultrasonic time was 60 min.

[0062] Specifically, when S2 uses pullulanase, it targets and cuts the α-1,6 glycosidic bonds in the cassava peel branched polysaccharides through the synergistic effect of adapted ultrasonic temperature and enzyme activity, destroying the complex network structure of amylopectin and glycogen, and releasing the encapsulated intracellular polysaccharides.

[0063] The enzyme preparation is cellulase, and the S2 parameters include:

[0064] The ultrasonic temperature was 50°C, the enzyme dosage was 2.4 KU / g, the material-liquid ratio was 1:8, and the ultrasonic time was 120 min.

[0065] Specifically, when cellulase is used in S2, the cellulose components in the cassava peel cell walls are targeted and degraded through adapted ultrasonic temperature and enzymatic hydrolysis conditions, thereby destroying the cellulose-hemicellulose composite structure and disintegrating its physical constraints on intracellular polysaccharides.

[0066] Please see the attached Figure 2 -Attached Figure 4 , an application of crude cassava peel polysaccharide, the crude polysaccharide is used as a natural antioxidant for preparing functional foods, anti-aging cosmetics or pharmaceutical preparations for assisting in the treatment of oxidative stress-related diseases, wherein:

[0067] Functional foods are solid beverages or dietary supplements, with an added amount of 1% to 10% by mass, and are compounded with vitamin C to increase DPPH free radical scavenging activity by ≥20%;

[0068] Anti-aging cosmetics are sunscreen lotions or repair essences, with an addition amount of 0.5% to 5% by mass, which synergizes with β-carotene to inhibit lipid peroxidation;

[0069] The pharmaceutical preparation is an oral capsule or nanoliposome, with a dosage of 50-200 mg / day, which targets the removal of free radicals in the intestine or skin.

[0070] Specifically, cassava peel crude polysaccharides act as natural antioxidants, which enhance the oxidative stability of food or cosmetic systems by scavenging various free radicals (such as DPPH, hydroxyl radicals and superoxide anions); in functional foods, they synergize with vitamin C to greatly improve the efficiency of free radical scavenging and delay the oxidative degradation of nutrients; in anti-aging cosmetics, they are combined with β-carotene to form a photoprotective barrier to inhibit ultraviolet-induced lipid peroxidation damage, while promoting skin barrier repair; in pharmaceutical preparations, they are targeted through targeted delivery systems (such as nanoliposomes) to bind to reactive oxygen species in the intestine or skin tissue, blocking the oxidative stress reaction chain and assisting in the relief of chronic inflammation or tissue aging, thereby achieving cross-domain and multi-scenario antioxidant and health maintenance functions.

[0071] Among them, 1. Determination of DPPH free radical scavenging ability:

[0072] The electron-withdrawing -N oxygen and the large π bond of the benzene ring in the DPPH molecule stabilize the nitrogen free radical. DPPH-ethanol solutions are dark purple and exhibit strong absorption at 517nm. The stronger the antioxidant's ability to scavenge DPPH, the lighter the color of the reaction solution and the lower the absorbance at 517nm.

[0073] Depend on Figure 2 It can be seen that all cassava crude polysaccharides have a certain scavenging effect on DPPH free radicals, and are concentration-dependent. Sample 4# is a medium-temperature α-enzymatic hydrolyzed cassava peel crude polysaccharide. As the sample concentration increases, the DPPH free radical scavenging effect is positively correlated. The scavenging ability of 6 mg / mL is 81.83%, which is the highest DPPH scavenging ability among all samples. As the sample concentration increases, the DPPH free radical scavenging effect of samples 1# and 10# is negatively correlated, indicating that the antioxidant activity of cassava crude polysaccharides at lower concentrations may be stronger. As the sample concentration increases, the DPPH free radical scavenging effect of sample 7# is not significant;

[0074] 2. Determination of hydroxyl radical scavenging ability

[0075] Hydroxyl radicals can react with functionally important biomolecules within cells, such as carbohydrates, proteins, lipids, and DNA, leading to tissue damage and cell death. In this study, Fe2+ reacted with hydrogen peroxide to generate hydroxyl radicals, which then reacted with salicylic acid to produce colored 2,3-dihydroxybenzoic acid or 2,5-dihydroxybenzoic acid. Substances with antioxidant properties can bind to hydroxyl radicals, thereby terminating the reaction.

[0076] Depend on Figure 3 All crude cassava polysaccharides demonstrated hydroxyl radical scavenging activity. The scavenging rate reached 100% at 500 μg / mL of vitamin C, and 66.25% at 250 μg / mL. Increasing sample concentration positively correlated with hydroxyl radical scavenging activity in all samples, suggesting that increasing crude polysaccharide concentration may enhance hydroxyl radical scavenging activity. The hydroxyl radical scavenging capacity of polysaccharide #7 (64.43%) was greater than that of polysaccharide #4 (53.135%), polysaccharide #10 (31.14%), and polysaccharide #1 (30.77%).

[0077] 3. Determination of superoxide anion radical scavenging ability

[0078] Excessive oxygen-ions can damage cellular DNA and easily lead to lipid peroxidation, which can be pathogenic. Therefore, the study of substances with oxygen-ion scavenging properties is of great significance. In this study, pyrogallol rapidly auto-oxidized under alkaline conditions, releasing oxygen-ions and forming a colored intermediate. The reaction initially turned yellow-green, then yellow after a few minutes, with a linear time of 3-4 minutes. Addition of substances with antioxidant properties inhibited the auto-oxidation rate of pyrogallol by combining with oxygen-ions. The stronger the substance's oxygen-ion scavenging ability, the lighter the color of the reaction solution and the lower the absorbance at 420 nm.

[0079] Depend on Figure 4 It can be seen that all samples have a certain ability to scavenge superoxide anion free radicals and are concentration-dependent. The scavenging ability is within 40%, and the vitamin C concentration is positively correlated with the ability to scavenge hydroxyl free radicals.

[0080] Example 1

[0081] S101, washing and chopping the cassava peels, placing them in a grinding bowl on ice, and grinding them into 100 mesh sizes to obtain cassava peel pulp;

[0082] S102, adding deionized water at a material-liquid ratio of 1:2, adding low-temperature α-amylase (1 KU / g), and ultrasonically treating at 45°C and 300W for 360 min;

[0083] S103. Dispense the enzymatic hydrolysate into centrifuge tubes, centrifuge at 8000 r / min and 25°C for 10 min, remove the precipitate, and retain the supernatant.

[0084] S104, adding 4% trichloroacetic acid to the supernatant, mixing uniformly, and removing the resulting precipitate by centrifugation;

[0085] S105, add 4 times the volume of anhydrous ethanol, let it stand for 2 hours, then centrifuge to remove the supernatant, and wash the precipitate with 95% ethanol and centrifuge, repeating 3 times;

[0086] S106. Redissolve the precipitate in deionized water, put it into a 3.5 kDa dialysis bag, and dialyze it in deionized water for 72 hours, changing the liquid four times a day. Then collect the liquid in the dialysis bag and freeze-dry it to obtain crude cassava peel polysaccharide.

[0087] Example 2

[0088] The difference from the first embodiment is that:

[0089] S202: Use medium-temperature α-amylase (5 KU / g), solid-liquid ratio 1:2.5, and ultrasonic treatment at 70°C for 240 min.

[0090] Example 3

[0091] The difference from the first embodiment is that:

[0092] S302: Pullulanase (4 KU / g) was used at a solid-liquid ratio of 1:2 and ultrasonic treatment was performed at 55°C for 60 min.

[0093] Example 4

[0094] The difference from the first embodiment is that:

[0095] S402, using hemicellulase (2.4 KU / g), solid-liquid ratio 1:8, ultrasonic treatment at 50°C for 120 min.

[0096] Table 1 shows the extraction of cassava peel polysaccharides by different enzymatic hydrolysis processes

[0097] Example 1 Example 2 Example 3 Example 4 enzyme preparations Low-temperature α-amylase Mesophilic α-amylase Pullulanase Hemicellulase Crude polysaccharide yield (%) 3.99±0.12 7.45±0.33 8.48±0.68 1.92±0.19

[0098] Among them, the four enzymes have an effect on the yield of crude polysaccharides from cassava peel in the following order: pullulanase > medium-temperature α-amylase > low-temperature α-amylase > hemicellulase. That is, the yield of crude polysaccharides under enzyme-added conditions is significantly higher than that under no enzyme-added conditions. Ultrasonic-assisted enzymatic hydrolysis can significantly improve the extraction efficiency of crude polysaccharides from cassava peel.

[0099] While embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the invention, and that the scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A method for extracting crude polysaccharides from cassava peel, characterized in that: The following steps are involved: S1, pre-treatment of cassava peels: washing and chopping the cassava peels, placing them in a grinding bowl on ice and grinding them to 80-120 mesh, to obtain cassava peel pulp; S2, ultrasonic-assisted enzymatic hydrolysis, adding deionized water at a material-liquid ratio of 1:2-1:8, adding enzyme preparation at 1-5 KU / g, and then treating at an ultrasonic temperature of 45-70°C and an ultrasonic power of 300W for 60-360 minutes; S3. Centrifugal separation: the enzymatic hydrolysate was divided into centrifuge tubes and centrifuged at 8000 r / min and 25°C for 10 min. The precipitate was removed and the supernatant was retained. S4, deproteinization treatment, adding 4% trichloroacetic acid to the supernatant, mixing evenly, and removing the precipitate by centrifugation; S5, ethanol precipitation and purification: add 4 times the volume of anhydrous ethanol, let it stand for 2 hours, then centrifuge to remove the supernatant, and wash the precipitate with 95% ethanol and centrifuge, repeat 3 times; S6. Dialysis purification and product acquisition: the precipitate was redissolved in deionized water, placed in a 3.5 kDa dialysis bag, and dialyzed in deionized water for 72 h, with the liquid changed 4 times a day. The liquid in the dialysis bag was then collected and freeze-dried to obtain crude cassava peel polysaccharide.

2. The method for extracting crude cassava peel polysaccharides according to claim 1, wherein The S1 further comprises transferring the cassava peel pulp to a microwave reactor, adjusting the pH to 6.0-7.5, applying microwave-low frequency ultrasound synergistic treatment, gradually increasing the temperature to 60-80° C., and treating for 15-25 minutes.

3. A method for extracting cassava peel crude polysaccharides according to claim 2, characterized in that, The enzyme preparation in S2 includes low-temperature α-amylase, medium-temperature α-amylase, pullulanase or hemicellulase.

4. The method for extracting crude cassava peel polysaccharides according to claim 3, wherein The enzyme preparation is a low-temperature α-amylase, and the S2 parameters include: The ultrasonic temperature was 45°C, the enzyme dosage was 1 KU / g, the material-liquid ratio was 1:2, and the ultrasonic time was 360 min.

5. The method for extracting crude cassava peel polysaccharides according to claim 1, wherein The enzyme preparation is a mesophilic α-amylase, and the S2 parameters include: The ultrasonic temperature was 70°C, the enzyme dosage was 5 KU / g, the solid-liquid ratio was 1:2.5, and the ultrasonic time was 240 min.

6. The method for extracting crude cassava peel polysaccharides according to claim 1, wherein The enzyme preparation is pullulanase, and the S2 parameters include: The ultrasonic temperature was 55°C, the enzyme dosage was 4 KU / g, the solid-liquid ratio was 1:2, and the ultrasonic time was 60 min.

7. The method for extracting crude cassava peel polysaccharides according to claim 1, wherein The enzyme preparation is cellulase, and the S2 parameters include: The ultrasonic temperature was 50°C, the enzyme dosage was 2.4 KU / g, the material-liquid ratio was 1:8, and the ultrasonic time was 120 min.

8. An application of cassava peel crude polysaccharide, characterized in that: A method for extracting crude cassava peel polysaccharides according to any one of claims 1 to 9, wherein the crude polysaccharides are used as natural antioxidants for preparing functional foods, anti-aging cosmetics, or pharmaceutical preparations for assisting in the treatment of oxidative stress-related diseases, wherein: The functional food is a solid beverage or dietary supplement, the addition amount is 1% to 10% by weight, and it is compounded with vitamin C to increase the DPPH free radical scavenging activity by ≥20%; The anti-aging cosmetic is a sunscreen lotion or a repair essence, the addition amount of which is 0.5% to 5% by mass, and synergistically inhibits lipid peroxidation with β-carotene; The pharmaceutical preparation is an oral capsule or nanoliposome with a dosage of 50-200 mg / day, and is targeted at clearing free radicals in the intestine or skin.

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