Polygonatum sibiricum peptide preparation method based on synergistic enzymolysis of supramolecular solvent

Abstract

The invention discloses a polygonatum sibiricum peptide preparation method based on supramolecular solvent synergistic enzymolysis, and belongs to the technical field of natural active ingredient extraction. The method comprises the following steps: S1, crushing rhizoma polygonati, adding purified water and supramolecular NaDES (a natural deep eutectic solvent formed by betaine and carboxylic acid compounds), decocting, cooling, and adding a compound enzyme containing at least two of cellulase, amylase, pectinase and saccharifying enzyme for first enzymolysis; s2, carrying out enzyme deactivation and centrifugation, and then carrying out membrane separation to remove small molecular impurities; s3, adding alkaline protease into the product which is not permeable to the membrane to carry out secondary enzymolysis; s4, after secondary enzyme deactivation and centrifugation, filtering through a ceramic membrane, concentrating and drying to obtain the supramolecular polygonatum sibiricum polypeptide. Through the synergistic effect of two times of enzymolysis and supramolecular NaDES, the extraction rate (more than or equal to 80%) and the peptide content (more than or equal to 70%) of polygonatum sibiricum peptide are remarkably improved, and the product is good in water solubility and free of bitter taste and can be widely applied to the fields of food, health care products and medicines.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural active ingredient extraction technology, specifically to a method for preparing polygonatum peptide based on supramolecular solvent (NaDES)-assisted enzymatic hydrolysis technology. This method efficiently extracts high-purity, highly water-soluble supramolecular polygonatum peptide from polygonatum through a double enzymatic hydrolysis process combined with membrane separation technology. The method is suitable for large-scale production of functional ingredients in the food, health care, and pharmaceutical industries. Background Art

[0002] Polygonatum spp. is a traditional medicinal and edible plant. Its rhizomes are rich in active ingredients such as polysaccharides, saponins, and peptides, which have the effects of regulating immunity, lowering blood sugar, and anti-oxidation (Pharmacopoeia of the People's Republic of China 2020 edition). Currently, the industrial extraction of Polygonatum peptide mainly relies on the following technologies:

[0003] Traditional water extraction method: extracting crude extract of Polygonatum sibiricum by high-temperature decoction, but there are problems such as low extraction rate (≤60%), many impurities (such as starch, cellulose), and easy denaturation and inactivation of peptide components.

[0004] Single enzymatic hydrolysis method: Using a single protease (such as alkaline protease) to hydrolyze Polygonatum protein can increase the yield of peptides, but the enzymatic hydrolysis efficiency is limited due to the dense cell wall structure, and residual polysaccharide impurities in the product result in a low peptide content (about 50-65%) and a bitter taste.

[0005] Organic solvent extraction method: Using solvents such as ethanol and methanol to assist extraction can partially remove fat-soluble impurities, but solvent residues affect product safety and cannot solve the problem of poor water solubility of peptides.

[0006] In recent years, supramolecular solvents (Natural Deep Eutectic Solvents, NaDES) have begun to be applied in the field of natural product extraction due to their environmental friendliness and the ability to design solvent properties. For example, Chinese patent CN202410106142.X discloses a method and application for extracting characteristic polyphenolic components from Polygonatum odoratum using ultrasound-assisted natural deep eutectic solvents. The method uses a natural deep eutectic solvent combined with ultrasonic extraction to prepare a polyphenolic extract from Polygonatum odoratum, establishing a method for efficiently and greenly extracting polyphenolic compounds from Polygonatum odoratum using an environmentally friendly natural deep eutectic solvent as an extraction solvent. However, the method does not involve the coordinated application of enzymatic hydrolysis processes, and the extraction efficiency is still insufficient when extracting raw materials with complex cell wall structures, such as Polygonatum odoratum.

[0007] Summary of existing technology defects:

[0008] 1. Single enzymatic hydrolysis or solvent extraction is difficult to effectively break the cell wall of Polygonatum sibiricum, resulting in a low dissolution rate of peptides;

[0009] 2. Impurities (such as polysaccharides and fibers) are not completely separated during the extraction process, affecting the purity and taste of the product;

[0010] 3. The products of traditional methods have poor water solubility, which limits their direct application in food and medicine.

[0011] Therefore, an innovative process combining NaDES and multi-enzyme synergistic enzymatic hydrolysis was developed, aiming to efficiently break the wall and deeply extract Polygonatum sibiricum peptide, and purify it through membrane separation technology to ensure high water solubility and high purity of the product, and broaden its application prospects in functional food and medicine. Summary of the Invention

[0012] In response to the existing technical problems, the present invention provides a method for preparing Polygonatum sibiricum peptide based on supramolecular solvent-assisted enzymatic hydrolysis. Through two enzymatic hydrolysis processes combined with membrane separation technology, high-purity, highly water-soluble supramolecular Polygonatum sibiricum polypeptide is efficiently extracted from Polygonatum sibiricum.

[0013] In order to achieve the above object, the technical solution of the present invention is as follows:

[0014] A method for preparing polygonatum peptide based on supramolecular solvent-assisted enzymatic hydrolysis comprises the following steps:

[0015] S1: After crushing Polygonatum sibiricum, adding purified water and supramolecular NaDES, stirring evenly and then boiling, cooling to 40-50°C, adding complex enzyme for the first enzymatic hydrolysis;

[0016] S2: heating the enzymatic hydrolysate from step S1 to inactivate the enzyme, collecting the centrifuge after centrifugation, and separating the product that does not permeate the membrane by membrane separation;

[0017] S3: adding purified water and alkaline protease to the non-permeabilized product of step S2 to perform a second enzymatic hydrolysis;

[0018] S4: heating the enzymatic hydrolyzate from step S3 to inactivate the enzyme, collecting the centrifuge after centrifugation, filtering through a ceramic membrane, concentrating, and drying to obtain the supramolecular Polygonatum sibiricum polypeptide;

[0019] The complex enzyme comprises at least two enzymes selected from the group consisting of cellulase, amylase, pectinase and saccharifying enzyme; and the supramolecular NaDES is a natural deep eutectic solvent formed by betaine and carboxylic acid compounds.

[0020] Preferably, in step S1, the mass ratio of cellulase, amylase, pectinase and saccharifying enzyme in the complex enzyme is 1:(0.2-1):(2-3):(4-5).

[0021] Preferably, in step S1, the carboxylic acid compound is selected from at least one of citric acid, lactic acid, malic acid or tartaric acid.

[0022] Preferably, in step S1, the amount of purified water added is 5 to 20 times the weight of polygonatum, and the amount of supramolecular NaDES added is 0.3 to 6.0% of the weight of polygonatum.

[0023] Preferably, in step S1, the decoction temperature is 90-100°C, and the decoction time is 1-4 hours.

[0024] Preferably, in step S1, the first enzymatic hydrolysis time is 1 to 8 hours, and the added amount of the complex enzyme is 0.3 to 3.0% of the weight of the polygonatum.

[0025] Preferably, in step S3, the amount of purified water added is 2 to 6 times the weight of the non-membrane-permeable product, and the amount of alkaline protease added is 0.3 to 3.0% of the weight of the non-membrane-permeable product.

[0026] Preferably, in step S4, the pore size of the ceramic membrane is 50 to 300 nm.

[0027] Preferably, in step S4, the drying method is spray drying or freeze drying.

[0028] Due to the adoption of the above solution, the beneficial effects of the present invention are as follows:

[0029] 1. Extraction efficiency and purity are significantly improved:

[0030] Through the synergistic effect of two enzymatic hydrolysis and supramolecular NaDES, the extraction rate of Polygonatum sibiricum peptide can reach more than 80%, and the peptide content can reach more than 70%, which is 20% to 40% higher than the traditional single enzyme method.

[0031] 2. Impurities are completely removed and the product has excellent water solubility:

[0032] The composite enzyme degrades polysaccharide impurities (cellulose, starch, etc.), and is separated twice by nanofiltration membrane and ceramic membrane. The residual impurities in the product are reduced to less than 5%. The obtained supramolecular polyanthocyanidin is completely soluble in water (solubility ≥ 99%) without precipitation or turbidity, and can be directly used in liquid food or medicine.

[0033] 3. Taste and safety optimization:

[0034] Supramolecular NaDES replaces traditional organic solvents (such as methanol and ethanol) to avoid toxic residues; the enzymatic hydrolysis process degrades bitter peptides, and the product has no bitterness or odor (sensory evaluation score ≥8.5 / 10), meeting food-grade raw material standards.

[0035] 4. The process is green and efficient, suitable for industrialization:

[0036] The total time of the two enzymatic hydrolysis steps is ≤12 hours, which is more than 50% shorter than the traditional acid / alkali extraction method (24 to 48 hours); the supramolecular NaDES can be recycled (recovery rate ≥90%), reducing production costs by 30% to 40%, and there is no wastewater discharge, meeting the requirements of green chemistry.

[0037] 5. Wide range of applications:

[0038] The product has both high water solubility and physiological activity, and can be added to solid beverages (addition amount 1% to 5%), health care capsules (drug loading ≥ 80%) or functional foods (such as oral liquids, dairy products) to meet diverse market needs. DETAILED DESCRIPTION

[0039] The technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0040] Example 1

[0041] A method for preparing Polygonatum peptide based on supramolecular solvent-assisted enzymatic hydrolysis, comprising the following steps:

[0042] S1: 100 g of Polygonatum rhizome was crushed, 1000 mL of purified water and 2 g of betaine citrate NaDES were added, stirred evenly, and then decocted at 95°C for 2 hours. The temperature was then lowered to 45°C, and 1.5 g of complex enzyme (cellulase: amylase: pectinase: saccharifying enzyme = 1:0.5:2.5:4.5) was added, and the mixture was stirred and enzymolyzed for 3 hours.

[0043] S2: The enzymatic hydrolysate from step S1 was heated to 100°C for 10 minutes to inactivate the enzyme, and the centrifuge was collected (4000 rpm, 15 minutes). The centrifuge was filtered through a nanofiltration membrane (molecular weight cut-off 500 Da) to retain the product that did not pass through the membrane.

[0044] S3: Add 300 mL of purified water and 1.0 g of alkaline protease to the non-permeable product of step S2, and stir and enzymolyze for 4 hours;

[0045] S4: The enzymatic hydrolyzate of step S3 is heated to 100° C. for 10 minutes to inactivate the enzyme, centrifuged, collected the centrifuge, filtered through a 200 nm ceramic membrane, concentrated to a solid content of 20%, and spray-dried to obtain the supramolecular Polygonatum sibiricum polypeptide.

[0046] The polygonatum sibiricum polypeptide prepared by the above method has an extraction rate of 89.42%, a peptide content of 79.26%, a water solubility of ≥99%, and no bitter taste (see Table 1).

[0047] Example 2

[0048] A method for preparing Polygonatum peptide based on supramolecular solvent-assisted enzymatic hydrolysis, comprising the following steps:

[0049] S1: 100 g of Polygonatum sibiricum rhizome was crushed, added with 1500 mL of purified water and 0.5 g of betaine citrate NaDES, stirred evenly, and decocted at 95°C for 3 hours. The temperature was then lowered to 50°C, and 2 g of complex enzyme (cellulase: amylase: pectinase: saccharifying enzyme = 1:0.2:3:5) was added, and the mixture was stirred and enzymolyzed for 6 hours.

[0050] S2: The enzymatic hydrolysate from step S1 was heated to 100°C for 10 minutes to inactivate the enzyme, and the centrifuge was collected (4000 rpm, 15 minutes). The centrifuge was filtered through a nanofiltration membrane (molecular weight cut-off 500 Da) to retain the product that did not pass through the membrane.

[0051] S3: Add 500 mL of purified water and 0.5 g of alkaline protease to the non-permeable product of step S2, and stir and enzymolyze for 6 hours;

[0052] S4: The enzymatic hydrolyzate of step S3 is heated to 100° C. for 10 minutes to inactivate the enzyme, centrifuged, collected the centrifuge, filtered through a 100 nm ceramic membrane, concentrated to a solid content of 20%, and spray-dried to obtain the supramolecular Polygonatum sibiricum polypeptide.

[0053] The polygonatum sibiricum polypeptide prepared by the above method has an extraction rate of 81.36%, a peptide content of 78.95%, a water solubility of ≥99%, and no bitterness (see Table 1).

[0054] Example 3

[0055] 5% betaine lactate NaDES was used instead of betaine citric acid, and other steps were the same as in Example 1.

[0056] The Polygonatum sibiricum polypeptide prepared in this example had an extraction rate of 83.14%, a peptide content of 72.15%, a water solubility of ≥99%, and no bitterness (see Table 1).

[0057] Example 4

[0058] The complex enzyme only contains cellulase and pectinase (mass ratio 1:3), and the other steps are the same as those in Example 1.

[0059] The Polygonatum sibiricum polypeptide prepared in this example had an extraction rate of 86.94%, a peptide content of 77.33%, a water solubility of ≥99%, and no bitterness (see Table 1).

[0060] Example 5

[0061] The addition amount of betaine citrate NaDES is 0.3% (weight of polygonatum), and the other steps are the same as those in Example 1.

[0062] The Polygonatum sibiricum polypeptide prepared in this example had an extraction rate of 80.12%, a peptide content of 71.56%, a water solubility of ≥99%, and no bitterness (see Table 1).

[0063] Comparative Example 1

[0064] No betaine citrate NaDES was added, and other steps were the same as in Example 1.

[0065] The Polygonatum sibiricum polypeptide prepared in this comparative example had an extraction rate of 68.73%, a peptide content of 65.15%, a water solubility of ≤80%, and a slightly bitter taste (see Table 1).

[0066] Comparative Example 2

[0067] After the enzymatic hydrolysis in step S1, centrifugation was performed directly, and the second enzymatic hydrolysis was omitted. Other aspects were the same as those in Example 1.

[0068] The Polygonatum sibiricum polypeptide prepared in this comparative example had an extraction rate of 70.68%, a peptide content of 53.43%, a water solubility of ≤85%, and a slightly bitter taste (see Table 1).

[0069] Comparative Example 3

[0070] The complex enzyme only contains cellulase (1.5%), and the other ingredients are the same as those in Example 1.

[0071] The Polygonatum sibiricum polypeptide prepared in this comparative example had an extraction rate of 62.4%, a peptide content of 58.2%, residual impurities of ≥15%, and a slightly bitter taste (see Table 1).

[0072] Comparative Example 4

[0073] 70% ethanol was used instead of supramolecular NaDES, and the rest was the same as in Example 1.

[0074] The extraction rate of the Polygonatum sibiricum polypeptide prepared in this comparative example was 58.9%, the peptide content was 49.7%, and the solvent residue was 0.1% (see Table 1).

[0075] Table 1

[0076] Group Extraction rate (%) Peptide content (%) Water-soluble Bitterness score (1-0) Example 1 89.42 79.26 ≥99％ 9.0 Example 2 81.36 78.95 ≥98％ 8.5 Example 3 83.14 72.15 ≥97％ 8.7 Example 4 86.94 77.33 ≥97％ 8.5 Example 5 80.12 71.56 ≥97％ 8.5 Comparative Example 1 68.73 65.15 ≤80％ 6.5 Comparative Example 2 70.68 53.43 ≤85％ 5.0 Comparative Example 3 62.42 58.21 ≤80％ / Comparative Example 4 58.93 49.72 ≤80％ /

[0077] In summary, the supramolecular NaDES of the present invention and the enzyme mutually promote each other to improve the enzymatic hydrolysis effect, which is beneficial to the removal of impurities. Alkaline protease is then used to enzymatically hydrolyze the polysaccharin, and combined with centrifugation and ceramic membranes, the prepared polysaccharin peptide product has a higher extraction rate and a higher peptide content, and has a short production cycle, low production cost, and does not produce any toxic or harmful substances during the preparation process. The product obtained by the present invention is completely soluble in water, has a good taste, is odorless, and has no bitterness. It can be widely used in the fields of food, health products, medicines, etc., and has broad application prospects.

Claims

1. A method for preparing Polygonatum peptide based on supramolecular solvent-assisted enzymatic hydrolysis, characterized in that: The following steps are involved: S1: After crushing Polygonatum sibiricum, add purified water and supramolecular Na DES, stir evenly and boil, cool to 40-50°C, add complex enzyme for the first enzymatic hydrolysis; S2: heating the enzymatic hydrolysate from step S1 to inactivate the enzyme, collecting the centrifuge after centrifugation, and separating the product that does not permeate the membrane by membrane separation; S3: adding purified water and alkaline protease to the non-permeabilized product of step S2 to perform a second enzymatic hydrolysis; S4: heating the enzymatic hydrolyzate from step S3 to inactivate the enzyme, collecting the centrifuge after centrifugation, filtering through a ceramic membrane, concentrating, and drying to obtain the supramolecular Polygonatum sibiricum polypeptide; The complex enzyme comprises at least two enzymes selected from the group consisting of cellulase, amylase, pectinase and saccharifying enzyme; and the supramolecular NaDES is a natural deep eutectic solvent formed by betaine and carboxylic acid compounds.

2. The method for preparing Polygonatum peptide according to claim 1, wherein: In step S1, the mass ratio of cellulase, amylase, pectinase and saccharifying enzyme in the complex enzyme is 1:(0.2-1):(2-3):(4-5).

3. The method for preparing Polygonatum peptide according to claim 1, wherein: The carboxylic acid compound is selected from at least one of citric acid, lactic acid, malic acid or tartaric acid.

4. The method for preparing Polygonatum peptide according to claim 1, wherein: In step S1, the amount of purified water added is 5 to 20 times the weight of polygonatum, and the amount of supramolecular NaDES added is 0.3 to 6.0% of the weight of polygonatum.

5. The method for preparing Polygonatum peptide according to claim 1, wherein: In step S1, the decoction temperature is 90-100° C., and the decoction time is 1-4 hours.

6. The method for preparing Polygonatum peptide according to claim 1, wherein: In step S1, the first enzymatic hydrolysis time is 1 to 8 hours, and the added amount of the complex enzyme is 0.3 to 3.0% of the weight of the polygonatum.

7. The method for preparing Polygonatum peptide according to claim 1, wherein: In step S3, the amount of purified water added is 2 to 6 times the weight of the non-membrane-permeated product, and the amount of alkaline protease added is 0.3 to 3.0% of the weight of the non-membrane-permeated product.

8. The method for preparing Huangjing peptide according to claim 1, wherein: In step S4, the pore size of the ceramic membrane is 50 to 300 nm.

9. The method for preparing Polygonatum peptide according to claim 1, wherein: In step S4, the drying method is spray drying or freeze drying.