Traditional Chinese Medicine Plant-based Medicine and Food Homologous Nanoactive Peptide Preparation and Its Preparation Method

Abstract

Traditional Chinese medicine plant-based medicine and food homology nano-active peptide preparation and its preparation method, belonging to the field of traditional Chinese medicine preparations. The technical key points are to prepare an aqueous solution of micro-crushed plants; add sodium hydroxide to the aqueous solution for alkali treatment; according to the content ratio of different types of sugars in the plants, add glycosidase of the corresponding sugar type and ratio to the alkali-treated solution for sugar hydrolysis; add protease to the sugar-hydrolyzed solution for protein hydrolysis; add sodium tripolyphosphate and disodium EDTA to the protein-hydrolyzed solution and perform ultrasonic treatment to obtain a plant-based nano-active peptide preparation. The effect is that while retaining and improving traditional active ingredients such as the original quality markers of traditional Chinese medicine, the medicine and food homology is prepared into a nano-level active peptide preparation, enabling it to have the unique physical, chemical, and biological properties of nano materials, and can be applied to drugs, foods, or health products.

Description

Technical Field

[0001] The present invention belongs to the field of traditional Chinese medicine preparations, and particularly relates to a plant-based medicine and food homologous nano-active peptide preparation and a preparation method thereof. Background Art

[0002] Medicinal and edible homologous traditional Chinese medicines are a type of traditional Chinese medicine resources gradually discovered by humans in the long-term production and life practice. They can not only satisfy hunger but also regulate the body state and prevent diseases. With the characteristics of both medicine and food, they have received increasing attention worldwide.

[0003] Traditional traditional Chinese medicine extraction and preparation processes include decoction method, maceration method, reflux method, percolation method, fermentation method, etc., which have defects such as long extraction time, more impurities, and easy mildew when water is used as a solvent. On the other hand, modern extraction processes such as ultrasonic-assisted extraction, microwave-assisted extraction, supercritical fluid extraction, ultrafine grinding, and ultra-high pressure extraction also have disadvantages such as easy denaturation and loss of active ingredients, high energy consumption and production costs, and gelatinization of starch, which are not conducive to the retention of active ingredients and large-scale industrial production. In addition, many traditional Chinese medicine components have problems such as poor water solubility, poor stability, low bioavailability, and large adverse reactions, which greatly limit their applications and also pose new problems and challenges to the modern development of traditional Chinese medicine. Introducing nanotechnology into the research and development field of traditional Chinese medicine, processing traditional Chinese medicine at the nanoscale can change the physical and chemical properties and biological activities of traditional Chinese medicine preparations, improve bioavailability and solubility, and provide new ideas for solving such problems.

[0004] At the same time, medicinal and edible homologous traditional Chinese medicines are high-quality natural active peptide libraries. Multiple previous studies have shown that active peptides derived from traditional Chinese medicines have important biological activities such as neuroprotection, liver protection, anti-tumor, anticoagulation, antioxidant, and immune enhancement. For example, neuroactive regulatory peptides extracted from ginseng, angiotensin-converting enzyme (ACE)-short peptides in oysters, etc. However, the basic and applied research on traditional Chinese medicine active peptides is still weak at present, and high-quality formed traditional Chinese medicine active peptide products are yet to be developed. Summary of the Invention

[0005] In order to solve the above problems existing in the extraction and preparation of the existing plant-based traditional Chinese medicines, the present invention provides a traditional Chinese medicine plant-based medicine and food homologous nano-active peptide preparation and a preparation method thereof to simultaneously improve the absorption rate, utilization rate of traditional Chinese medicine and the dissolution rate of functional components.

[0006] In a first aspect, according to the preparation method of the plant-based nano-active peptide preparation in some embodiments of the present application, it includes:

[0007] Preparing an aqueous solution of micronized plants; adding sodium hydroxide to the aqueous solution for alkali treatment;

[0008] According to the content ratio of different types of sugars in the plant, glycosidase corresponding to the type and ratio of the sugar is added to the solution after alkali treatment for sugar hydrolysis; protease is added to the solution after sugar hydrolysis for protein hydrolysis;

[0009] Sodium tripolyphosphate and disodium EDTA are added to the solution after protein hydrolysis, and after ultrasonic treatment, a plant-based nanoactive peptide preparation is obtained.

[0010] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, the median diameter of the micronized plant is 50-100 μm.

[0011] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, sodium hydroxide is added for alkali treatment, the pH is 9.0-10.0, the temperature is 90-100 °C, and the alkali treatment time is 30-60 min.

[0012] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, it further includes inactivating the enzyme after sugar hydrolysis; inactivating the enzyme after protein hydrolysis.

[0013] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, by mass, the glycosidase accounts for about 1%-3% of the total sugar content, wherein the proportion of the content of one type of sugar in the total sugar content is basically the same as the proportion of the dosage of the glycosidase corresponding to the one type of sugar in the total dosage of the glycosidase.

[0014] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, the glycosidase accounts for about 2.0% of the total sugar content.

[0015] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, the sugar includes any one or a combination of cellulose polysaccharides, pectin polysaccharides and starch, and the glycosidase includes any one or a combination of cellulase, pectinase and amylase corresponding to the cellulose polysaccharides, pectin polysaccharides and starch.

[0016] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, the sugar includes cellulose polysaccharides, pectin polysaccharides and starch, and the glycosidase includes cellulase, pectinase and amylase.

[0017] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, by mass, the dosage of the protease accounts for 0.5%-1.5% of the weight of the plant.

[0018] According to the preparation method of the plant-based nanoactive peptide preparation in some embodiments of the present application, the dosage of the protease accounts for 1.0% of the weight of the plant.

[0019] According to the preparation method of the plant-based nano-active peptide preparation in some embodiments of the present application, the protease includes compound protease, and the compound protease is composed of any one or a combination of alkaline protease, neutral protease, flavor protease, trypsin, papain, and bromelain.

[0020] According to the preparation method of the plant-based nano-active peptide preparation in some embodiments of the present application, after protein hydrolysis, centrifugation, filtration, and membrane concentration are carried out to obtain a protein hydrolysate solution with a solid content of 20-25%.

[0021] According to the preparation method of the plant-based nano-active peptide preparation in some embodiments of the present application, the mass ratio of sodium tripolyphosphate to the solid content is (1-2:100); the mass ratio of disodium EDTA to the solid content is (0.5-1):100.

[0022] According to the preparation method of the plant-based nano-active peptide preparation in some embodiments of the present application, the solution added with sodium tripolyphosphate and disodium EDTA is stirred and then subjected to ultrasonic treatment. After ultrasonic treatment, it is frozen and dried to obtain the plant-based nano-active peptide preparation.

[0023] According to the preparation method of the plant-based nano-active peptide preparation in some embodiments of the present application, the ultrasonic treatment time is 3-5 min, and the ultrasonic power is 150-200 W.

[0024] In a second aspect, according to the plant-based nano-active peptide preparation in some embodiments of the present application, it is obtained by any one of the above-mentioned preparation methods.

[0025] Beneficial effects:

[0026] (1) The main components of the primary cell wall of the plant-based traditional Chinese medicine cell wall are cellulose, hemicellulose, and pectin, while the secondary cell wall is cellulose and hemicellulose. The presence of the cell wall poses a great obstacle to the release of active ingredients. At the same time, a large number of starch grains contained in the plant-based traditional Chinese medicine cells are prone to absorb water and expand during the extraction process, resulting in a certain degree of gelatinization, which also greatly affects the subsequent protease hydrolysis effect. The present invention formulates the ratio and addition amount of compound glycosidase by measuring the sugar content such as cellulose polysaccharide, pectin polysaccharide, and starch in different raw materials to achieve good biological enzymatic wall-breaking and starch degradation effects. The present invention adopts a combined technology of mild ultra-fine physical pulverization, alkali treatment chemical wall-breaking, and targeted compound glycosidase enzymatic biological wall-breaking, which has a more thorough wall-breaking effect compared to the traditional ultra-fine physical pulverization process alone, and improves the dissolution rate of active ingredients of traditional Chinese medicine such as ginsenoside and gastrodin as quality markers of traditional Chinese medicine preparations. On the other hand, a large amount of heat energy is generated when the material is usually ultra-finely physically pulverized to less than 10 microns, which may damage the inherent activity of traditional Chinese medicine, while the temperature conditions of the method adopted by the present invention are milder, which is beneficial to retaining the structure of active ingredients of traditional Chinese medicine.

[0027] (2) While retaining and enhancing the active ingredients such as the original quality markers of Chinese medicinal herbs and edible substances used interchangeably, the present invention exploits the protein resources in Chinese medicinal herbs and edible substances used interchangeably, improving the utilization rate of Chinese medicinal herbs and edible substances used interchangeably. At the same time, cell wall fragments that are not completely lysed will affect the enzymatic hydrolysis effect of proteases. Compared with proteins, bioactive peptides have two characteristics: higher absorption efficiency and no antigenicity, and these two characteristics are directly related to the molecular weight of the peptides. A large number of studies have demonstrated that there are two independent transport mechanisms for the absorption of small peptides (mainly dipeptides and tripeptides with a molecular weight of less than 500 Da) and amino acids. There are two peptide carriers in the body, which have the transport activity for di- and tripeptides and use the H+ gradient on the transport membrane as the driving force. Compared with free amino acids, the small peptide transport system with the same amino acid composition has the characteristics of fast transport speed, low energy consumption, and not easy to saturate. Therefore, the proportion of peptides with a molecular weight of <1000 Da and <500 Da is a very important technical index. Benefiting from the combined wall-breaking technology of ultramicro physical pulverization, alkali treatment, and complex glycosidase enzymatic hydrolysis, compared with the complex protease enzymatic hydrolysis after only ultramicro pulverization treatment, the present invention has a better enzymatic hydrolysis effect on the proteins of Chinese medicinal herbs and edible substances used interchangeably, and the proportion of peptide segments with a molecular weight of <1000 Da and 189 - 500 Da is higher.

[0028] (3) On the basis of using ultramicro physical pulverization to make Chinese medicinal herbs and edible substances used interchangeably reach the micron level, the present invention further hydrolyzes the macromolecular carbohydrates and proteins, which are the main components of Chinese medicinal herbs and edible substances used interchangeably, into nanoscale molecules through glycosidase enzymatic hydrolysis and protease enzymatic hydrolysis. However, many Chinese medicinal herbs and edible substances used interchangeably contain a certain amount of elements such as calcium and magnesium, which are likely to form chelates with peptide molecules, leading to molecular aggregation; on the other hand, nanoscale molecular particles have extremely high surface energy and a large contact surface, and will adsorb and easily aggregate through intermolecular forces, hydrogen bonds, and electrostatic interactions; the complex bioactive peptide composition has positive and negative charges due to different amino acid types and forms aggregates through self-assembly. The above reasons result in the actual particle size of the enzymatic hydrolysis products of Chinese medicinal herbs and edible substances used interchangeably in aqueous solution being much larger than their molecular particle size, so that a truly nanoscale Chinese medicine preparation cannot be formed. The present invention first chelates calcium, magnesium and other elements by adding EDTA to reduce the aggregation of peptide molecules caused by chelation, adds sodium tripolyphosphate to increase the absolute value of the surface potential of nanoscale molecules, generates strong double-layer electrostatic repulsion and steric repulsion, and then further disperses the nanoscale molecules by ultrasonic treatment and freeze-drying to increase the adsorption of nanoscale molecules and sodium tripolyphosphate, realizing the dispersion of nanoscale molecules of Chinese medicinal herbs and edible substances used interchangeably after re-dissolution, so as to avoid aggregation and further improve the absorption rate. Detailed implementation mode

[0029] The present invention provides a wall-breaking method combining ultramicrophysical pulverization, alkali treatment and complex glycosidase enzymolysis, and adding chelating competitive inhibition and a dispersant after complex protease enzymolysis to prepare a medicine and food homology active peptide preparation. The technology of the present invention can be applied to various plant-based medicine and food homology traditional Chinese medicines to obtain a nano-level traditional Chinese medicine preparation combining active ingredients such as traditional Chinese medicine quality markers and active peptide components. For the above purpose, the present invention provides a wall-breaking method combining physical, chemical and biological wall-breaking, which can improve the extraction efficiency of traditional Chinese medicine active ingredients such as gastrodin and ginsenosides. On this basis, complex protease enzymolysis is carried out, and a chelating competitive inhibitor and a dispersant are added to prepare a nano-level medicine and food homology active peptide preparation. It can simultaneously improve the absorption rate, utilization rate of traditional Chinese medicine and the dissolution rate of functional ingredients.

[0030] In a first aspect, a preparation method of a plant-based medicine and food homology nano active peptide preparation of the present invention is as follows:

[0031] Step 1. The raw material of the medicine and food homology traditional Chinese medicine is initially crushed by a pulverizer, and then the powder is ultramicrophysically pulverized by an ultramicro pulverizer to a median diameter of 50-100 μm;

[0032] Step 2. Methods such as colorimetry and high performance liquid chromatography are used to measure the content X of cellulose polysaccharides, the content Y of pectin polysaccharides, and the content Z of starch in the raw material of the medicine and food homology traditional Chinese medicine;

[0033] Step 3. The powder obtained in Step 1 is mixed with deionized water, and sodium hydroxide is added to adjust the pH to 9.0-10.0, and alkali treatment is carried out at 90-100 °C for 30-60 min;

[0034] Step 4. According to the content ratio X:Y:Z of cellulose polysaccharides, pectin polysaccharides, and starch obtained in Step 2, a complex glycosidase composed of cellulase, pectinase, and amylase is used to enzymolyze the traditional Chinese medicine solution in Step 2. Among them, the dosage ratio of cellulase, pectinase, and amylase is X:Y:Z, and the total dosage of cellulase, pectinase, and amylase is 1% to 3% of the total content of cellulose polysaccharides, pectin polysaccharides, and starch, preferably 2%. After the enzymolysis is completed, the enzyme is inactivated by heating to obtain a first enzymolysis solution;

[0035] Step 5. A complex protease is added to the first enzymolysis solution obtained in Step 4 for enzymolysis. After the enzymolysis is completed, the enzyme is inactivated by heating, centrifuged and filtered, and then membrane concentrated to obtain a second enzymolysis solution with a solid content of 20-25%;

[0036] In an example of the present invention, the complex protease is composed of two or three of alkaline protease, neutral protease, flavor protease, trypsin, papain, and bromelain;

[0037] Step 6. Sodium tripolyphosphate and disodium EDTA are added to the second enzymatic hydrolysate obtained in Step 5, stirred well, treated by ultrasonic waves, and finally freeze-dried to obtain the medicine and food homology nano-active peptide preparation.

[0038] In one example of the present invention, the mass ratio of sodium tripolyphosphate to the solid content of the enzymatic hydrolysate is 1-2:100;

[0039] In one example of the present invention, the mass ratio of disodium EDTA to the solid content of the enzymatic hydrolysate is 0.5-1:100;

[0040] In one example of the present invention, the ultrasonic treatment time is 3-5 min, and the ultrasonic power is 150-200 W.

[0041] In the present invention, the "medicine and food homology nano-active peptide preparation" refers to a traditional Chinese medicine preparation with a particle size distribution reaching less than 100 nm in an aqueous solution and containing short peptide components of traditional Chinese medicine with medicine and food homology.

[0042] Test description:

[0043] (1-1). Determination of total ginsenosides: The total ginsenoside contents of the main root raw material of ginseng, ginseng nano-active peptide preparations G1, G2, and G3 are determined by the vanillin-ethanol-concentrated sulfuric acid colorimetric method. Weigh 5 mg of the ginsenoside Re standard product, add anhydrous ethanol to prepare a standard solution with a mass concentration of 0.5 mg / mL. Respectively absorb 0.2, 0.4, 0.6, 0.8, and 1.0 mL of the standard solution, evaporate to dryness in a water bath at 80 °C, add 0.5 mL of a 10% vanillin solution by mass fraction, then add 5 mL of a 60% sulfuric acid solution by volume fraction, mix well, keep in a water bath at 60 °C for 15 min, then cool in ice water for 10 min, stand at room temperature for 15 min, and measure the absorbance at a wavelength of 472 nm. Draw a standard curve with the mass concentration as the abscissa and the absorbance as the ordinate.

[0044] (1-2). Determination of the peptide molecular weight of the ginseng nano-active peptide preparation: The peptide molecular weight distribution of the ginseng nano-active peptide preparations G1, G2, and G3 is determined by HPLC-MS / MS high-performance liquid chromatography-mass spectrometry.

[0045] (1-3). Determination of the particle size of the ginseng nano-active peptide preparation: In the present invention, a ZETASIZER NANO nano particle size and potential analyzer is used to measure the average of the aqueous solution of the ginseng nano-active peptide preparation. Weigh the ginseng nano-active peptide preparation powders G1, G2, G3, and G4 respectively, add 1 ml of deionized water to prepare 1% and 0.1% aqueous solutions respectively, inject them into the sample cell, and measure the average particle size.

[0046] (1 - 4). Determination of total gastrodin saponins: The gastrodin contents of gastrodia raw materials, gastrodia nano - active peptide preparations R1, R2, and R3 were determined by ultraviolet spectrophotometry. Accurately weigh 5 mg of gastrodin standard in a 10 - mL volumetric flask, add an appropriate amount of solvent to dissolve, make up the volume, and shake well to obtain a standard stock solution with a concentration of 500 μg / mL. Respectively pipette 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 mL of the standard stock solution into 10 - mL volumetric flasks, dilute to the mark, so that the concentrations are 2.5, 5, 7.5, 10, 12.5, and 15 μg / mL respectively. Measure the absorbance at a wavelength of 221 nm. Take the standard solution concentration (μg / mL) as the abscissa and the absorbance A as the ordinate to draw a standard curve. Take 1.666 - 1.667 g of the sample powder of gastrodia raw materials, gastrodia nano - active peptide preparations R1, R2, and R3, place it in a 50 - ml round - bottom flask. Add 25 ml of 70% ethanol, seal the mouth of the round - bottom flask with a sealing film, and soak for 0.5 h. Heat in a water bath and reflux for 60 min. Cool to room temperature, filter under reduced pressure, and place the filtrate in a 50 - mL volumetric flask. Wash the round - bottom flask 3 times with an appropriate amount of 70% ethanol solution, filter, combine the filtrates, wash the suction flask 3 times with an appropriate amount of 70% ethanol solution, combine and transfer to the 50 - mL volumetric flask, make up the volume, and shake well for standby. Measure the absorbance in the same way and calculate the gastrodin content according to the standard curve.

[0047] (2 - 2). Determination of the peptide molecular weight of gastrodia nano - active peptide preparations: The peptide molecular weight distribution of gastrodia nano - active peptide preparations R1, R2, and R3 was determined by HPLC - MS / MS high - performance liquid chromatography - mass spectrometry.

[0048] (2 - 3). Determination of the particle size of gastrodia nano - active peptide preparations: The average particle size of the aqueous solutions of gastrodia nano - active peptide preparations R1, R2, R3, and R4 was determined by a ZETASIZER NANO nanoparticle size and zeta potential analyzer. Weigh the gastrodia nano - active peptide preparation powder respectively, add 1 ml of deionized water to prepare 1% and 0.1% aqueous solutions respectively, inject into the sample cell, and measure the average particle size.

[0049] The present invention will be described in detail below through examples.

[0050] Example G1. The preparation method of ginseng nano - active peptide preparation G1 is as follows:

[0051] Step 1. Take 1000 g of dried ginseng main root, initially crush it with a pulverizer, and then ultra - micro - crush the powder to a median diameter of 100 μm with an ultra - micro pulverizer to obtain 975 g of ginseng powder;

[0052] Step 2. Determine the total content of cellulose and hemicellulose in the dried main root of ginseng to be 53 g by the high-performance liquid chromatography method described in the standard NY / T 3494-2019 Determination of Cellulose, Hemicellulose and Lignin in Agricultural Biomass Raw Materials; determine the content of pectin in the dried main root of ginseng to be 42 g by the spectrophotometry method described in the standard NY / T 2016-2011 Determination of Pectin Content in Fruits and Their Products; determine the starch content in the dried main root of ginseng to be 340 g by the near-infrared method described in the standard GB / T 25219-2010 Cereals and Oils Inspection Determination of Corn Starch Content;

[0053] Step 3. Mix the ginseng powder obtained in Step 1 with 15 times the volume of deionized water, add sodium hydroxide to adjust the pH to 10.0, and perform alkali treatment at 95 °C for 60 min;

[0054] Step 4. Add 8.7 g of a composite glycosidase, which is 2.0% of the total mass of the three polysaccharides described in Step 2. The composition of this composite glycosidase is cellulase:pectinase:amylase = 53:42:340, and perform enzymatic hydrolysis at 50 °C and pH 5.5 for 2 h. After the enzymatic hydrolysis is completed, raise the temperature to 80 °C to inactivate the enzyme for 10 min to obtain the first enzymatic hydrolysate;

[0055] Step 5. Add 9.75 g of a composite protease, which is 1% of the mass of the dried main root of ginseng, to the first enzymatic hydrolysate obtained in Step 4. The composition of the composite protease is alkaline protease:neutral protease = 1:1, and perform enzymatic hydrolysis at 50 °C and pH 8.0 for 4 h. After the enzymatic hydrolysis is completed, raise the temperature to 80 °C to inactivate the enzyme for 10 min, centrifuge, filter, and perform membrane concentration to obtain a second enzymatic hydrolysate with a solid content of 20%;

[0056] Step 6. Add 4 g of sodium tripolyphosphate and 2 g of disodium EDTA to the second enzymatic hydrolysate obtained in Step 4, stir well, perform ultrasonic treatment, and finally obtain the ginseng nanoactive peptide preparation G1 by freeze-drying.

[0057] Comparative Example G2. The preparation method of the ginseng nanoactive peptide preparation G2 is as follows: Replace the composite glycosidase combination in Example G1 with cellulase:pectinase:amylase = 1:1:1, and perform the remaining steps according to Example G1 to obtain the ginseng nanoactive peptide preparation G2.

[0058] Comparative Example G3. The preparation method of the ginseng nanoactive peptide preparation G3 is as follows:

[0059] Step 1. Take 1000 g of the dried main root of ginseng, perform primary crushing with a pulverizer, and then use an ultrafine pulverizer to ultrafinely pulverize the powder to a median diameter of 10 μm to obtain 966 g of ginseng powder;

[0060] Step 2. Mix the ginseng powder obtained in Step 1 with 15 times the volume of deionized water, add sodium hydroxide to adjust the pH to 10.0, and perform alkali treatment at 95 °C for 60 min;

[0061] Step 3. Add 9.66 g of compound protease, which is 1% of the dry weight of the main root of ginseng. The compound protease is composed of alkaline protease: neutral protease = 1:1. Enzymatically hydrolyze at 50 °C and pH 8.0 for 4 h. After the enzymatic hydrolysis is completed, raise the temperature to 80 °C to inactivate the enzyme for 10 min. Centrifuge and filter, and after membrane concentration, obtain an enzymatic hydrolysate with a solid content of 20%. Freeze-dry to obtain the ginseng nanoactive peptide preparation G3.

[0062] Comparative example G4. The preparation method of the ginseng nanoactive peptide preparation G4 is as follows: After carrying out Steps 1-5 in Example G1, carry out freeze-drying to obtain the ginseng nanoactive peptide preparation G4.

[0063] Experimental example 1. Determine the total ginsenosides of ginseng according to (1-1) in the test instructions: Measure the absorbance of the main root raw material of ginseng, G1, G2, and G3 samples, and calculate the saponin content according to the standard curve. The results are shown in Table 1 below:

[0064] Table 1 Total ginsenoside content

[0065] Group Ginseng main root raw material G1 G2 G3 Saponin content (%) 3.74±0.24 6.11±0.36 4.89±0.22 3.03±0.10

[0066] As can be seen from Table 1, the total ginsenoside content of G1 prepared by ultrafine pulverization, alkali treatment, and targeted glycosidase enzymatic hydrolysis is significantly higher than that of the main root raw material of ginseng, G2 prepared by non-targeted glycosidase enzymatic hydrolysis, and G3 prepared by only ultrafine pulverization and alkali treatment (P < 0.01). This shows that the glycosidase enzymatic hydrolysis step targeting the different polysaccharide content characteristics of the raw materials in the technology of the present invention can effectively improve the release rate and extraction rate of functional components.

[0067] Experimental example 2. Determine the peptide molecular weight and particle size according to (1-2) and (1-3) in the test instructions:

[0068] (1) The results of peptide molecular weight determination are shown in Table 2:

[0069] Table 2 Peptide molecular weight distribution of the ginseng nanoactive peptide preparation

[0070] Group G1 G2 G3 <1000Da (%) 95.84 78.25 67.37 189 - 500Da (%) 51.57 31.04 27.88

[0071] As can be seen from Table 2, the proportions of peptides with a molecular weight of <1000 Da and 189-500 Da in G1 are significantly lower than those in G2 prepared by non-targeted glycosidase enzymatic hydrolysis and G3 without glycosidase enzymatic hydrolysis. It can be seen that the glycosidase enzymatic hydrolysis treatment step significantly improves the subsequent protease hydrolysis effect.

[0072] (2) The results of particle size determination are shown in Table 3:

[0073] Table 3 Average particle size (nm) of the aqueous solution of the ginseng nanoactive peptide preparation

[0074] Group Average particle size (nm) G1(1％) 95.1 G1(0.1％) 69.8 G2(1％) 177.0 G2(0.1％) 130.9 G3(1％) 827.4 G3(0.1％) 703.5 G4(1％) 558.3 G4(0.1％) 480.3

[0075] As can be seen from Table 3, the average particle size of the aqueous solutions of G1 and G2 is significantly reduced compared with that of G3 and G4 (P < 0.01), indicating that the addition of EDTA and sodium tripolyphosphate supplemented by ultrasonic treatment has well improved the molecular aggregation in the aqueous solution of the preparation. The results of G1 compared with G2 and G4 compared with G3 show that the glycosidase enzymatic hydrolysis treatment targeting the characteristics of the raw material polysaccharide types also helps to reduce the average particle size of the ginseng nanoactive peptide preparation. The reduction of the average particle size of the active peptide preparation can indicate an improvement in the absorption effect of the active peptide.

[0076] Example R1. The preparation method of Gastrodia elata nanoactive peptide preparation R1 is as follows:

[0077] Step 1. Take 1000 g of dried Gastrodia elata raw material and perform primary crushing with a pulverizer, and then use an ultrafine pulverizer to ultrafinely pulverize the powder to a median diameter of 100 μm to obtain 984 g of Gastrodia elata powder;

[0078] Step 2. According to the high-performance liquid chromatography method described in the standard NY / T 3494-2019 "Determination of Cellulose, Hemicellulose, and Lignin in Agricultural Biomass Raw Materials", the total content of cellulose and hemicellulose in the dried Gastrodia elata raw material is determined to be 36 g; according to the spectrophotometry method described in the standard NY / T2016-2011 "Determination of Pectin Content in Fruits and Their Products", the pectin content in the dried Gastrodia elata raw material is determined to be 156 g; according to the near-infrared method described in the standard GB / T 25219-2010 "Inspection of Grain and Oil - Determination of Corn Starch Content", the starch content in the dried Gastrodia elata raw material is determined to be 437 g;

[0079] Step 3. Mix the Gastrodia elata powder obtained in Step 1 with 15 times the volume of deionized water, add sodium hydroxide to adjust the pH to 10.0, and perform alkali treatment at 95 °C for 60 min;

[0080] Step 4. Add 12.58 g of a composite glycosidase, which is 2.0% of the total mass of the three polysaccharides described in Step 2. The composition of this composite glycosidase is cellulase: pectinase: amylase = 36:156:437, and perform enzymatic hydrolysis at 50 °C and pH 5.5 for 2 h. After the enzymatic hydrolysis is completed, raise the temperature to 80 °C to inactivate the enzyme for 10 min to obtain the first enzymatic hydrolysate;

[0081] Step 5. Add 9.84 g of a composite protease, which is 1% of the mass of the dried Gastrodia elata raw material, to the first enzymatic hydrolysate obtained in Step 4. The composition of the composite protease is alkaline protease: neutral protease = 1:1, and perform enzymatic hydrolysis at 50 °C and pH 8.0 for 4 h. After the enzymatic hydrolysis is completed, raise the temperature to 80 °C to inactivate the enzyme for 10 min, centrifuge and filter, and after membrane concentration, obtain a second enzymatic hydrolysate with a solid content of 20%;

[0082] Step 6. Add 4 g of sodium tripolyphosphate and 2 g of disodium EDTA to the second enzymatic hydrolysate obtained in Step 4, stir well, perform ultrasonic treatment, and finally obtain Gastrodia elata nanoactive peptide preparation R1 by freeze-drying.

[0083] Example 6. The preparation method of Gastrodia elata nanoactive peptide preparation R2 is as follows: Replace the composite glycosidase combination in Example R1 with cellulase: pectinase = 1:1, and perform the remaining steps according to Example R1 to obtain Gastrodia elata nanoactive peptide preparation R2.

[0084] Example 7. The preparation method of Gastrodia elata nanoactive peptide preparation R2 is as follows:

[0085] Step 1. Take 1000 g of dried Gastrodia elata raw material, perform primary crushing with a pulverizer, and then use an ultrafine pulverizer to ultrafinely pulverize the powder to a median diameter of 10 μm to obtain 973 g of Gastrodia elata powder;

[0086] Step 2. Mix the Gastrodia elata powder obtained in Step 1 with 15 times the volume of deionized water, add sodium hydroxide to adjust the pH to 10.0, and perform alkali treatment at 95 °C for 60 min;

[0087] Step 3. Add 9.73 g of composite protease, which is 1% of the mass of the dried Gastrodia elata raw material. The composite protease composition is alkaline protease: neutral protease = 1:1. Perform enzymatic hydrolysis at 50 °C and pH 8.0 for 4 h. After the enzymatic hydrolysis is completed, raise the temperature to 80 °C to inactivate the enzyme for 10 min, centrifuge and filter, and obtain an enzymatic hydrolysate with a solid content of 20% after membrane concentration, and obtain Gastrodia elata nanoactive peptide preparation R2 by freeze-drying.

[0088] Example 8. The preparation method of Gastrodia elata nanoactive peptide preparation R3 is as follows: Perform Steps 1-5 in Example R1 and then perform freeze-drying to obtain Gastrodia elata nanoactive peptide preparation R3.

[0089] Experimental Example 1. Determine the total saponins of Gastrodia elata according to (2-1) in the test instructions: Measure the absorbance of Gastrodia elata raw material, Gastrodia elata nanoactive peptide preparations R1, R2, and R3, and calculate the content of gastrodin according to the standard curve. The results are shown in Table 4 below:

[0090] Table 4 Content of gastrodin

[0091] Group Gastrodia elata raw material R1 R2 R3 Gastrodin content (%) 0.46±0.08 2.19±0.14 1.43±0.07 0.63±0.11

[0092] As can be seen from Table 4, similar to the results of total ginsenosides, compared with the Gastrodia elata raw material and R2 treated with non-targeted glycosidase enzymatic hydrolysis and R3 not treated with glycosidase enzymatic hydrolysis, the content of gastrodin in R1 prepared through the targeted glycosidase enzymatic hydrolysis step has a significant increase (P < 0.01), indicating that the technology of the present invention has good extraction effects on functional components in different types of plant-based medicine and food homologous traditional Chinese medicines.

[0093] Experimental Example 2. Determination of peptide molecular weight and particle size according to (2-2) and (2-3) in the test instructions:

[0094] (1) The results of peptide molecular weight determination are shown in Table 5:

[0095] Table 5 Peptide molecular weight distribution of Gastrodia elata nanoactive peptide preparation

[0096] Group G1 G2 G3 <1000Da (%) 92.13 70.25 50.42 189 - 500Da (%) 48.40 22.74 10.07

[0097] The proportions of peptides with molecular weight <1000 Da and 189-500 Da in R1 are significantly lower than those in R2 and R3, which also illustrates the promoting effect of glycosidase enzymatic hydrolysis treatment on protease hydrolysis effect.

[0098] (2) The results of particle size determination are shown in Table 3:

[0099] Table 6 Average particle size (nm) of Gastrodia elata nanoactive peptide preparation aqueous solution

[0100]

[0101]

[0102] As can be seen from Table 6, the average particle size of the Gastrodia elata nanoactive peptide preparation G1 prepared by the technology of the present invention is much smaller than that of G2, G3, and G4 in the aqueous solution. The reduction of the average particle size of the active peptide preparation can indicate the improvement of the absorption effect of the active peptide.

[0103] Combined with the effects of the above embodiments, it can be seen that the preparation method of the plant-derived homologous nanoactive peptide preparation of medicine and food provided by the present invention analyzes the content characteristics of cellulose polysaccharides, pectin polysaccharides, and starch in different plant-derived homologous traditional Chinese medicines, and performs targeted glycosidase enzymatic hydrolysis according to this characteristic. Combined with appropriate ultrafine grinding and alkali treatment for sufficient cell wall breaking, and reducing the influence of a large number of starch granules on the further extraction of active ingredients. At the same time, adding EDTA to chelate calcium, magnesium and other elements to reduce the aggregation of peptide molecules caused by chelation, adding sodium tripolyphosphate to change the absolute value of the surface potential of nano-molecules, reducing the adsorption between molecules, and then further dispersing nano-molecules by ultrasonic treatment and freeze-drying to increase the adsorption between nano-molecules and sodium tripolyphosphate, so as to reduce the average particle size of nano-molecules in the aqueous solution of homologous traditional Chinese medicines of medicine and food after re-dissolution, and achieve a better absorption effect. This technology has good application prospects in the fields of food, medicine, etc.

[0104] Thus, the preparation method of a plant-based medicine-food homologous nano-active peptide preparation of the present invention belongs to the field of traditional Chinese medicine preparations. The technical key points are that the plant-based medicine-food homologous raw materials are first ultra-finely ground to obtain traditional Chinese medicine powder with a particle size of micron level, pure water and sodium hydroxide are added in proportion for alkali treatment, the composition of cellulose polysaccharides, pectin polysaccharides and starch in the raw materials is analyzed, and a composite glycosidase composed of cellulase, pectinase and amylase is added in proportion for the first enzymolysis, and physical wall breaking, chemical wall breaking and biological wall breaking are combined for effective wall breaking, and the dissolution rate of active ingredients such as markers is increased. On the basis of the above steps, a composite protease is added for the second enzymolysis, and after the enzymolysis product is separated by an ultrafiltration membrane, sodium tripolyphosphate and disodium EDTA are added, fully stirred and ultrasonically treated, and the medicine-food homologous nano-active peptide preparation is obtained by freeze drying. While retaining and improving the traditional active ingredients such as the original quality markers of traditional Chinese medicine, the medicine-food homologous is prepared into a nano-level active peptide preparation, so that it has the physical, chemical and biological properties unique to nano materials, and can be applied to medicines, foods or health products.

[0105] Finally, it should be noted that the above examples are only some specific embodiments of the present invention, and all derivatives that can be directly derived or associated with the contents disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. A method for preparing a plant-based nano-active peptide preparation, characterized in that: include: preparing an aqueous solution of micro-crushed plants; adding sodium hydroxide to the aqueous solution for alkaline treatment; According to the content ratio of different types of sugars in plants, glycosidase corresponding to the type and ratio of sugars is added to the solution after alkali treatment to hydrolyze sugars; protease is added to the solution after sugar hydrolysis to hydrolyze proteins; Sodium tripolyphosphate and disodium EDTA are added to the protein hydrolyzed solution, and the solution is subjected to ultrasonic treatment to obtain a plant-based nano-active peptide preparation.

2. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: The median diameter of the micro-crushed plants is 50~100μm.

3. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: in, Sodium hydroxide is added for alkali treatment, the pH is 9.0~10.0, the temperature is 90~100℃, and the alkali treatment time is 30~60min.

4. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: It also includes inactivating enzymes after sugar hydrolysis and inactivating enzymes after protein hydrolysis.

5. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: By mass, the glycosidase accounts for 1% to 3% of the total sugar content, wherein the proportion of one type of sugar content to the total sugar content is consistent with the proportion of the amount of glycosidase corresponding to the one type of sugar to the total glycosidase amount.

6. The method for preparing the plant-based nano-active peptide preparation according to claim 5, characterized in that: Glycosidase accounts for 2.0% of the total sugar content.

7. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: The sugar includes any one or a combination of cellulose polysaccharides, pectin polysaccharides and starch, and the glycosidase includes any one or a combination of cellulase, pectinase and amylase corresponding to the cellulose polysaccharide, pectin polysaccharide and starch.

8. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: Sugars include cellulose polysaccharides, pectin polysaccharides and starch, and glycosidases include cellulase, pectinase and amylase.

9. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: In terms of mass, the amount of protease used is 0.5%~1.5% of the plant weight.

10. The method for preparing the plant-based nano-active peptide preparation according to claim 9, characterized in that: The amount of protease used is 1.0% of the plant weight.

11. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: The protease includes a composite protease, which is composed of any one or a combination of alkaline protease, neutral protease, flavor protease, trypsin, papain, and bromelain.

12. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: After protein hydrolysis, the protein is centrifuged, filtered, and concentrated by membrane to obtain a protein hydrolyzed solution with a solid content of 20-25%.

13. The method for preparing the plant-based nano-active peptide preparation according to claim 12, characterized in that: The mass ratio of sodium tripolyphosphate to the solid is (1-2:100); the mass ratio of disodium EDTA to the solid is (0.5-1):

100.

14. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: The solution to which sodium tripolyphosphate and disodium EDTA are added is stirred and then ultrasonically treated, and then frozen and dried to obtain the plant-based nano-active peptide preparation.

15. The method for preparing the plant-based nano-active peptide preparation according to claim 1, characterized in that: The ultrasonic treatment time is 3-5 minutes, and the ultrasonic power is 150-200W.

16. A plant-based nano-active peptide preparation, characterized in that: Prepared by the preparation method described in any one of claims 1 to 15.

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