Compound composition based on enzymolysis technology as well as preparation method and application of compound composition

Abstract

The invention discloses a compound composition based on an enzymolysis technology as well as a preparation method and application thereof, and belongs to the technical field of health food. The invention discloses a compound composition based on an enzymolysis technology. The compound composition is prepared from the following raw materials in parts by weight: 30-45 parts of radix polygonati officinalis powder, 20-35 parts of fructus lycii powder, 10-20 parts of sea cucumber enzymatic hydrolysate, 10-25 parts of colla corii asini enzymatic hydrolysate, 3-5 parts of collagen peptide, 5-8 parts of jujube powder and 4-8 parts of hawthorn fruit powder. The compound composition based on the enzymolysis technology has the effects of increasing the number of red blood cells and hemoglobin, promoting hematopoiesis, improving anemia of zebrafish and improving immunity.

Description

Technical Field

[0001] The present invention relates to the technical field of health food, and more particularly to a compound composition based on enzymatic hydrolysis technology, a preparation method and application thereof. Background Art

[0002] Anemia is common among women today, primarily due to physiological characteristics, nutritional intake, and lifestyle factors. Currently, the main blood-enriching products on the market are iron supplements, donkey-hide gelatin, and medicinal and edible products. Traditional donkey-hide gelatin preparations have a low absorption rate due to their large molecular structure. Iron supplements are easily affected by dietary ingredients, limiting absorption and can cause gastrointestinal irritation, leading to constipation and other problems. For example, ferrous fumarate and ferrous gluconate are easily affected by phytic acid, tannic acid, carbonates, cellulose, and oxidizing substances in food, resulting in limited absorption.

[0003] At present, there are endless types of blood-enriching products on the market. For example, the Chinese invention patent with application number CN202411764591.X is a Huangqi and Huangjing oral liquid and its preparation method, which is prepared with 13 kinds of traditional Chinese medicines and has the effects of invigorating qi and blood, and resisting fatigue; the Chinese invention patent with application number CN202510517638.0 is a traditional Chinese medicine composition with the effects of invigorating qi and nourishing blood and strengthening the spleen and kidneys and its preparation method, which is prepared with 16 kinds of traditional Chinese medicines. The combination of these medicines can achieve multiple effects such as invigorating qi and nourishing blood, strengthening the spleen and kidneys, regulating immunity, and improving metabolism. It has a comprehensive conditioning effect on various symptoms such as qi and blood deficiency, spleen and stomach weakness, and kidney deficiency. However, the ingredients of traditional Chinese medicine are too complex, which may increase the risk of adverse reactions between the ingredients. At the same time, it lacks clear specificity and it is difficult to highlight the advantages of the product in specific effects. In addition, blood-enriching products are mainly iron preparations, donkey-hide gelatin, etc. The products are single, and the ingredients are mainly traditional Chinese medicinal materials. There is a lack of modern bioactive ingredients, and the traditional donkey-hide gelatin preparations have a low absorption rate due to their macromolecular structure; iron preparations are easily affected by ingredients in food, and absorption is limited. They are also prone to gastrointestinal irritation, leading to problems such as constipation. For example, patent CN118947913A provides an iron-rich composition with the function of replenishing qi and blood and a preparation method thereof. The composition can improve iron absorption, promote the formation of red blood cells and hemoglobin, and synergistically improve the blood-enriching effect. However, ferrous fumarate and ferrous gluconate are easily affected by phytic acid, tannic acid, carbonates, cellulose and oxidizing substances in food, resulting in limited absorption.

[0004] Therefore, providing a compound composition based on enzymatic hydrolysis technology and its preparation method and application is a problem that needs to be solved urgently by those skilled in the art. Summary of the Invention

[0005] In light of this, the present invention provides a compound composition based on enzymatic hydrolysis technology, its preparation method, and application. This compound composition has the efficacy of increasing the number of red blood cells and hemoglobin, promoting hematopoiesis, improving anemia in zebrafish, and enhancing immunity. The formula is simple, easily absorbed, and has rapid effects.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] A compound composition based on enzymatic hydrolysis technology is composed of the following raw materials in parts by weight: 30-45 parts of polygonatum powder, 20-35 parts of wolfberry powder, 10-20 parts of sea cucumber enzymatic hydrolyzate, 10-25 parts of donkey-hide gelatin enzymatic hydrolyzate, 3-5 parts of collagen peptide, 5-8 parts of jujube powder, and 4-8 parts of hawthorn powder.

[0008] The effects and synergistic effects of the raw materials used in the present invention are briefly described as follows:

[0009] As the saying goes, "There are ginseng on land, and sea cucumbers in water." Sea cucumbers have high nutritional and therapeutic value. Traditional Chinese Medicine believes that their salty flavor and warm nature have the benefits of nourishing the kidneys and essence, nourishing the blood and moistening dryness, and beautifying the complexion. Modern medical research shows that sea cucumbers contain a variety of active substances, including minerals, saponins, polysaccharides, and a large amount of protein, which have anti-fatigue effects. They also nourish yin and blood, replenish yang and moisten dryness, greatly enhancing their qi and blood-tonifying functions.

[0010] Collagen peptide is an extract from animal connective tissue. It is rich in amino acids and can be quickly absorbed by the human body. Regular consumption can repair the body's skin cells, restore skin elasticity, restore skin luster, delay aging, and moisturize hair.

[0011] Jujubes have the benefits of tonifying the middle and replenishing qi, nourishing the blood, and calming the mind. They are often used in combination with donkey-hide gelatin. Jujubes nourish the blood and harmonize the Ying and Wei, while donkey-hide gelatin nourishes yin, replenishes blood, and stops bleeding. Together, these two herbs nourish the blood, stop bleeding, and harmonize the Ying and Wei.

[0012] Polygonatum odoratum has the benefits of nourishing yin, moistening dryness, promoting body fluid and quenching thirst. Modern medical research shows that the polysaccharides and saponins contained in Polygonatum odoratum can promote erythropoietic tree (EPO) secretion by regulating the hematopoietic microenvironment and improving iron absorption efficiency. When combined with donkey-hide gelatin, this can enhance these yin-nourishing and moistening effects. Furthermore, Polygonatum odoratum can significantly increase whole-blood levels of superoxide dismutase (SOD) and glutathione peroxide, significantly inhibit the formation of lipid peroxides (LPO), scavenge free radicals generated by metabolism, and delay aging.

[0013] Donkey-hide gelatin has the benefits of nourishing blood, tonifying yin, moistening the lungs, and stopping bleeding. "Medical Compendium: Medicinal Properties" states: "It nourishes the heart and harmonizes blood, dissipates heat and nourishes yin." In the Compendium of Materia Medica, it is hailed as a "blood-tonifying holy medicine," emphasizing that its primary function is to "tonify blood and fluid." It can fundamentally address blood deficiency and effectively replenish qi and blood. Long-term use can improve qi and blood sufficiency.

[0014] Goji berries nourish the liver and kidneys, improve vision, and enhance vision. Traditional Chinese Medicine believes that "the liver stores blood, the kidneys store essence, and that essence and blood share the same origin." Goji berries nourish the liver and kidneys, regulating overall body function and helping to alleviate symptoms of blood deficiency caused by liver and kidney deficiency, such as pale complexion, dizziness, and fatigue. Modern medical research shows that goji berries are rich in various nutrients, including vitamins, minerals, and amino acids. Their beta-carotene and polysaccharides, in particular, promote blood circulation and enhance hematopoiesis. Goji berries also have anti-aging, anti-tumor, lipid-lowering, liver-protecting, anti-fatty liver, and blood sugar- and blood pressure-lowering properties.

[0015] Hawthorn has the effects of promoting digestion and strengthening the stomach, promoting qi and dispersing blood stasis, clearing turbidity and reducing lipids, and can help promote the smooth flow of qi and blood. Modern medical research shows that hawthorn is rich in organic acids (such as citric acid and malic acid), which can promote the conversion of trivalent iron into divalent iron and relieve iron deficiency anemia.

[0016] Donkey-hide gelatin is sweet and mild, entering the liver and kidneys. Sea cucumber is salty and warm, entering the kidneys, and the mutual transformation of essence and blood forms the core of "dual tonification of essence and blood." Polygonatum is sweet and mild, moistening the lungs. Metal generates water to support kidney essence. Collagen peptides "replenish form with form," creating a "qi-form" synergy. Goji berries are sweet and mild, entering the liver and kidneys, nourishing them, improving essence and improving eyesight. Jujubes are sweet and warm, entering the spleen, tonifying the spleen and harmonizing the vital energy, harmonizing the various herbs. Hawthorn is sour and sweet, transforming yin, invigorating the spleen and promoting digestion, forming a "tonification and elimination balance." The monarch herbs directly replenish the visible blood, the minister herbs improve the hematopoietic microenvironment, and the adjuvant herbs ensure tonification without stagnation, in line with the therapeutic principle of the "Inner Canon of Medicine" that "form deficiency should be warmed with qi, and essence deficiency should be replenished with flavor."

[0017] Further, the preparation method of the sea cucumber enzymatic hydrolysate is as follows:

[0018] (1) grinding the sea cucumber after removing the internal organs and cleaning it to obtain a slurry for standby use;

[0019] (2) adding water to the slurry at a ratio of 1:2.5-5, adjusting the pH to 6-8, adding a composite protease for enzymolysis to obtain an enzymolysis solution; the enzymolysis temperature is 45°C-65°C, and the enzymolysis time is 2-5 hours; the amount of the composite protease is 3000-6800 U / g (based on the slurry after pH adjustment); the composite protease is composed of flavor protease, papain, trypsin and neutral protease, and the mass ratio thereof is 3-5:2-5:4-6:4-8; the enzymatic activity of the flavor protease is 150,000 U / g; the enzymatic activity of the papain is 1,000,000 U / g; the enzymatic activity of the trypsin is 150,000 U / g; and the enzymatic activity of the neutral protease is 200,000 U / g;

[0020] (3) sterilizing the obtained enzymatic solution at a temperature of 88°C to 95°C for 20 to 30 minutes;

[0021] (4) After sterilization and enzyme inactivation, the mixture is filtered to obtain the sea cucumber enzymatic hydrolysate.

[0022] Furthermore, the preparation method of the donkey-hide gelatin enzymatic hydrolysate is as follows:

[0023] (1) placing donkey-hide gelatin in a high-speed centrifuge for pulverization to obtain donkey-hide gelatin powder;

[0024] (2) adding water to the donkey-hide gelatin powder to prepare a donkey-hide gelatin powder solution with a mass concentration of 15-20%; adding dilute hydrochloric acid to adjust the pH to 1.5-3, adding 0.5-1% (based on the donkey-hide gelatin powder solution after pH adjustment) of pepsin, and isothermally enzymolyzing at 37-42°C for 1-2 hours; then adding sodium hydroxide solution to adjust the pH to 7.5-8.5, adding 0.5-1% (based on the solution after pH adjustment with sodium hydroxide solution) of trypsin, and isothermally enzymolyzing at 37-45°C for 2.5-3.5 hours to obtain an enzymatic solution; the enzymatic activity of the pepsin is 200,000 U / g; the enzymatic activity of the trypsin is 150,000 U / g;

[0025] (3) sterilizing the obtained enzymatic solution at a temperature of 80°C to 90°C for 20 to 40 minutes;

[0026] (4) After sterilization and enzyme inactivation, the mixture is filtered to obtain the enzymatic hydrolyzate of donkey-hide gelatin.

[0027] Furthermore, the preparation method of the compound composition based on enzymatic hydrolysis technology comprises the following specific steps: mixing sea cucumber enzymatic hydrolysate, donkey-hide gelatin enzymatic hydrolysate, polygonatum powder, wolfberry powder, collagen peptide, jujube powder and hawthorn powder in proportion to obtain the compound composition.

[0028] Furthermore, a compound composition preparation based on enzymatic hydrolysis technology comprises the compound composition and pharmaceutically acceptable excipients as well as common food excipients and food additives; the dosage form of the compound composition preparation is powder, decoction, granules, pills, capsules, tablets or oral liquid.

[0029] Furthermore, the dosage form of the compound composition preparation is an oral liquid.

[0030] Furthermore, the compound composition or the compound composition preparation is used in the preparation of products for increasing the number of red blood cells and hemoglobin, promoting hematopoiesis, and improving anemia.

[0031] Furthermore, the compound composition or the compound composition preparation is used in the preparation of a rapid blood-enriching product.

[0032] Furthermore, the compound composition or the compound composition preparation is used in the preparation of a product for improving immunity.

[0033] It can be seen from the above technical solution that compared with the existing technology, the present invention discloses a compound composition based on enzymatic hydrolysis technology, a preparation method and application thereof, which integrates traditional Chinese medicine theory and modern biotechnology. While inheriting the essence of traditional Chinese medicine theory, it combines the development of modern biotechnology and combines "marine active substances-traditional tonic medicinal materials-modern biotechnology". According to the formula of the combination of medicinal and edible ingredients that has been verified by multiple times, it has the characteristics of simple and effective formulation, easy absorption and fast effect, which can effectively increase the number of red blood cells and hemoglobin, improve anemia and enhance immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0035] Figure 1 is the staining intensity of zebrafish heart red blood cells in the normal control group;

[0036] Figure 2 is the staining intensity of zebrafish cardiac erythrocytes in the model control group;

[0037] Figure 3 is the staining intensity of zebrafish heart red blood cells in the positive control group;

[0038] Figure 4 The zebrafish heart erythrocyte staining intensity in Example 6;

[0039] Figure 5 The change rate of zebrafish heart red blood cell staining intensity in Example 6 and Comparative Example 2;

[0040] Figure 6 is the fluorescence intensity of macrophages in the tail vein of zebrafish in the normal control group;

[0041] Figure 7 is the fluorescence intensity of zebrafish tail vein macrophages in the model control group;

[0042] Figure 8 is the fluorescence intensity of zebrafish tail vein macrophages in the positive control group;

[0043] Figure 9 This is the fluorescence intensity of zebrafish tail vein macrophages in Example 6. DETAILED DESCRIPTION

[0044] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the 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.

[0045] Polygonatum odoratum powder was purchased from Henan Jianjiu Industrial Co., Ltd., wolfberry powder, jujube powder and hawthorn powder were purchased from Xi'an Tianyi Biotechnology Co., Ltd.; collagen peptide was purchased from Hubei Jianpeptide Biotechnology Co., Ltd.

[0046] Example 1

[0047] A compound composition based on enzymatic hydrolysis technology contains the following raw materials: 30g of polygonatum powder, 20g of wolfberry powder, 10g of sea cucumber enzymatic hydrolyzate, 10g of donkey-hide gelatin enzymatic hydrolyzate, 3g of collagen peptide, 5g of jujube powder, and 4g of hawthorn powder.

[0048] The preparation method of the compound composition oral liquid preparation comprises the following steps: mixing the above raw materials to obtain the compound composition; adding 8.2g of isomalt and 6.6g of xylitol, and adding water to make the volume to 1000ml to obtain the compound composition oral liquid preparation.

[0049] Wherein, the preparation method of sea cucumber enzymatic hydrolysate is as follows:

[0050] (1) grinding the sea cucumber after removing the internal organs and cleaning it to obtain a slurry for standby use;

[0051] (2) Add water to the slurry at a ratio of 1:2.5, adjust the pH to 6.0, and add a composite protease for enzymolysis to obtain an enzymatic solution; the enzymatic hydrolysis temperature is 45°C, the enzymatic hydrolysis time is 2 h, the amount of the composite protease is 3000 U / g, the composite protease is composed of flavor protease, papain, trypsin and neutral protease, and the mass ratio is 3:2:4:4; the enzymatic activity of flavor protease is 150,000 U / g; the enzymatic activity of papain is 1,000,000 U / g; the enzymatic activity of trypsin is 150,000 U / g; and the enzymatic activity of neutral protease is 200,000 U / g;

[0052] (3) The obtained enzymatic hydrolysate is sterilized at a temperature of 88°C for 20 minutes.

[0053] (4) After sterilization and enzyme inactivation, the solution was filtered on a cardboard with a pore size of 0.22 μm to obtain the sea cucumber enzymatic hydrolysate.

[0054] The preparation method of donkey-hide gelatin enzymatic hydrolysate is as follows:

[0055] (1) placing donkey-hide gelatin in a high-speed centrifuge for pulverization to obtain donkey-hide gelatin powder;

[0056] (2) Add water to the donkey-hide gelatin powder to prepare a gelatin powder solution with a mass concentration of 15%; add dilute hydrochloric acid to adjust the pH to 1.5, add 0.5% pepsin, and incubate at 37°C for 1 hour; then add sodium hydroxide solution to adjust the pH to 7.5, add 0.5% trypsin, and incubate at 37°C for 2.5 hours to obtain an enzymatic solution; the enzymatic activity of pepsin is 200,000 U / g; the enzymatic activity of trypsin is 150,000 U / g;

[0057] (3) sterilizing the obtained enzymatic solution at a temperature of 80° C. for 20 min;

[0058] (4) After sterilization, the enzyme was inactivated and the resulting solution was filtered through a cardboard with a pore size of 0.45 μm to obtain the enzymatic hydrolyzate of donkey-hide gelatin.

[0059] Example 2

[0060] A compound composition based on enzymatic hydrolysis technology contains the following raw materials: 35g of polygonatum powder, 25g of wolfberry powder, 15g of sea cucumber enzymatic hydrolyzate, 15g of donkey-hide gelatin enzymatic hydrolyzate, 4g of collagen peptide, 6g of jujube powder, and 5g of hawthorn powder.

[0061] The preparation methods of the sea cucumber hydrolyzate and the donkey-hide gelatin hydrolyzate in this example are the same as those in Example 1.

[0062] The preparation method of the compound composition oral liquid preparation comprises the following steps: mixing the above raw materials to obtain the compound composition; adding 10.5g of isomalt and 7g of xylitol, and adding water to make the volume to 1000ml to obtain the compound composition oral liquid preparation.

[0063] Example 3

[0064] A compound composition based on enzymatic hydrolysis technology contains the following raw materials: 40g of polygonatum powder, 30g of wolfberry powder, 20g of sea cucumber enzymatic hydrolyzate, 20g of donkey-hide gelatin enzymatic hydrolyzate, 4g of collagen peptide, 6g of jujube powder, and 5g of hawthorn powder.

[0065] The preparation methods of the sea cucumber hydrolyzate and the donkey-hide gelatin hydrolyzate in this example are the same as those in Example 1.

[0066] The preparation method of the compound composition oral liquid preparation comprises the following steps: mixing the above raw materials to obtain the compound composition; adding 10.5g of isomalt and 8g of xylitol, and adding water to make the volume to 1000ml to obtain the compound composition oral liquid preparation.

[0067] Example 4

[0068] A compound composition based on enzymatic hydrolysis technology contains the following raw materials: 45g of polygonatum powder, 35g of wolfberry powder, 20g of sea cucumber enzymatic hydrolyzate, 25g of donkey-hide gelatin enzymatic hydrolyzate, 5g of collagen peptide, 8g of jujube powder, and 8g of hawthorn powder.

[0069] The preparation methods of the sea cucumber hydrolyzate and the donkey-hide gelatin hydrolyzate in this example are the same as those in Example 1.

[0070] The preparation method of the compound composition oral liquid preparation comprises the following steps: mixing the above raw materials to obtain the compound composition; adding 10.8g of isomalt and 8.2g of xylitol, and adding water to make the volume to 1000ml to obtain the compound composition oral liquid preparation.

[0071] Example 5

[0072] A compound composition based on enzymatic hydrolysis technology contains the following raw materials: 45g of polygonatum powder, 35g of wolfberry powder, 20g of sea cucumber enzymatic hydrolyzate, 25g of donkey-hide gelatin enzymatic hydrolyzate, 4g of collagen peptide, 7g of jujube powder, and 6g of hawthorn powder.

[0073] The preparation methods of the sea cucumber hydrolyzate and the donkey-hide gelatin hydrolyzate in this example are the same as those in Example 1.

[0074] The preparation method of the compound composition oral liquid preparation comprises the following steps: mixing the above raw materials to obtain the compound composition; adding 11g of isomalt and 8g of xylitol, and adding water to make the volume to 1000ml to obtain the compound composition oral liquid preparation.

[0075] Example 6

[0076] A compound composition based on enzymatic hydrolysis technology, the original ingredient ratio is the same as that of Example 4.

[0077] The preparation method of the oral liquid preparation of the compound composition is the same as that of Example 4.

[0078] Wherein, the preparation method of sea cucumber enzymatic hydrolysate is as follows:

[0079] (1) grinding the sea cucumber after removing the internal organs and cleaning it to obtain a slurry for standby use;

[0080] (2) Add water to the slurry at a ratio of 1:3.5, adjust the pH to 7.0, and add compound protease for enzymolysis to obtain an enzymatic solution; the enzymatic hydrolysis temperature is 55°C, the enzymatic hydrolysis time is 3.5 hours, the amount of compound protease is 4800 U / g, the compound protease is composed of flavor protease, papain, trypsin and neutral protease, and the mass ratio is 4:3.5:5:6; the enzymatic activity of flavor protease is 150,000 U / g; the enzymatic activity of papain is 1,000,000 U / g; the enzymatic activity of trypsin is 150,000 U / g; the enzymatic activity of neutral protease is 200,000 U / g;

[0081] (3) The obtained enzymatic hydrolysate is sterilized at a temperature of 90°C for 25 minutes.

[0082] (4) After sterilization and enzyme inactivation, the solution was filtered on a cardboard with a pore size of 0.22 μm to obtain the sea cucumber enzymatic hydrolysate.

[0083] The preparation method of donkey-hide gelatin enzymatic hydrolysate is as follows:

[0084] (1) placing donkey-hide gelatin in a high-speed centrifuge for pulverization to obtain donkey-hide gelatin powder;

[0085] (2) Add water to the donkey-hide gelatin powder to prepare a donkey-hide gelatin powder solution with a mass concentration of 18%; add dilute hydrochloric acid to adjust the pH to 2.0, add 0.8% pepsin, and perform enzymolysis at a constant temperature of 40°C for 1.5 hours; then add sodium hydroxide solution to adjust the pH to 8.0, add 0.8% trypsin, and perform enzymolysis at a constant temperature of 40°C for 3 hours to obtain an enzymatic solution; the enzymatic activity of pepsin is 200,000 U / g; the enzymatic activity of trypsin is 150,000 U / g;

[0086] (3) sterilizing the obtained enzymatic solution at a temperature of 85° C. for 30 min;

[0087] (4) After sterilization, the enzyme was inactivated and the resulting solution was filtered through a cardboard with a pore size of 0.45 μm to obtain the enzymatic hydrolyzate of donkey-hide gelatin.

[0088] Example 7

[0089] A compound composition based on enzymatic hydrolysis technology, the original ingredient ratio is the same as that of Example 4.

[0090] The preparation method of the oral liquid preparation of the compound composition is the same as that of Example 4.

[0091] Wherein, the preparation method of sea cucumber enzymatic hydrolysate is as follows:

[0092] (1) grinding the sea cucumber after removing the internal organs and cleaning it to obtain a slurry for standby use;

[0093] (2) The slurry was added with water at a ratio of 1:5, the pH was adjusted to 8.0, and a composite protease was added for enzymolysis to obtain an enzymatic solution; the enzymatic hydrolysis temperature was 65°C, the enzymatic hydrolysis time was 5 h, the amount of the composite protease was 6800 U / g, the composite protease consisted of flavor protease, papain, trypsin, and neutral protease in a mass ratio of 5:5:6:8; the enzymatic activity of flavor protease was 150,000 U / g; the enzymatic activity of papain was 1,000,000 U / g; the enzymatic activity of trypsin was 150,000 U / g; and the enzymatic activity of neutral protease was 200,000 U / g;

[0094] (3) The obtained enzymatic hydrolysate is sterilized at a temperature of 95°C for 30 minutes.

[0095] (4) After sterilization and enzyme inactivation, the solution was filtered on a cardboard with a pore size of 0.22 μm to obtain the sea cucumber enzymatic hydrolysate.

[0096] The preparation method of donkey-hide gelatin enzymatic hydrolysate is as follows:

[0097] (1) placing donkey-hide gelatin in a high-speed centrifuge for pulverization to obtain donkey-hide gelatin powder;

[0098] (2) Add water to the donkey-hide gelatin powder to prepare a donkey-hide gelatin powder solution with a mass concentration of 20%; add dilute hydrochloric acid to adjust the pH to 3.0, add 1% pepsin, and perform enzymolysis at a constant temperature of 42°C for 2 hours; then add sodium hydroxide solution to adjust the pH to 8.5, add 1% trypsin, and perform enzymolysis at a constant temperature of 45°C for 3.5 hours to obtain an enzymatic solution; the enzymatic activity of pepsin is 200,000 U / g; the enzymatic activity of trypsin is 150,000 U / g;

[0099] (3) sterilizing the obtained enzymatic solution at a temperature of 90° C. for 40 min;

[0100] (4) After sterilization, the enzyme was inactivated and the resulting solution was filtered through a cardboard with a pore size of 0.45 μm to obtain the enzymatic hydrolyzate of donkey-hide gelatin.

[0101] Comparative Example 1

[0102] Different from Example 6, the donkey-hide gelatin was not subjected to enzymatic hydrolysis, but only sterilized and filtered. The specific steps are as follows:

[0103] The donkey-hide gelatin was crushed in a high-speed centrifuge, and the obtained donkey-hide gelatin powder was prepared into a donkey-hide gelatin powder solution with a mass concentration of 18%, sterilized at 85°C for 30 minutes, and filtered on a cardboard with a pore size of 0.45 μm. 25 g of the sterilized and filtered donkey-hide gelatin powder solution was taken and mixed with other materials.

[0104] Comparative Example 2

[0105] Different from Example 6, neither donkey-hide gelatin nor sea cucumber was subjected to enzymatic hydrolysis, but only sterilized and filtered. The specific steps are as follows:

[0106] The sea cucumber was cleaned after evisceration and then ground. The resulting slurry was added with water at a ratio of 1:3.5 and sterilized at 90°C for 25 minutes. The slurry was filtered on a cardboard with a pore size of 0.22 μm. 20 g of the sterilized and filtered sea cucumber solution was mixed with other materials.

[0107] The donkey-hide gelatin was crushed in a high-speed centrifuge, and the obtained donkey-hide gelatin powder was prepared into a donkey-hide gelatin powder solution with a mass concentration of 18%, sterilized at 85°C for 30 minutes, and filtered on a cardboard with a pore size of 0.45 μm. 25 g of the sterilized and filtered donkey-hide gelatin powder solution was taken and mixed with other materials.

[0108] Comparative Example 3

[0109] The difference from Example 6 is that no donkey-hide gelatin enzymatic hydrolysate is added.

[0110] Comparative Example 4

[0111] The difference from Example 6 is that no sea cucumber enzymatic hydrolysate is added.

[0112] Comparative Example 5

[0113] The difference from Example 6 is that no sea cucumber hydrolyzate and donkey-hide gelatin hydrolyzate were added.

[0114] In order to further verify the superior effect of the oral liquid preparation of the compound composition of the present invention compared with the prior art, the inventors also studied the effect of the oral liquid preparation of the compound composition of the present invention on the staining intensity of zebrafish red blood cells and the fluorescence intensity of macrophages to verify that the oral liquid preparation of the compound composition of the present invention has the effect of replenishing blood and improving immunity. The test method and results are as follows:

[0115] 1. Evaluation of the blood-tonifying efficacy of the compound oral liquid preparation

[0116] Red blood cells are the most numerous cells in the blood and are key cells for transporting oxygen and carbon dioxide. The hemoglobin contained in them is responsible for transporting oxygen from the lungs to tissues throughout the body and transporting carbon dioxide produced by metabolism back to the lungs for excretion. In clinical medicine, red blood cell counts can be used to assess the degree of anemia. When the number of red blood cells is too small or the hemoglobin content in red blood cells is too low, anemia can result. In anemia model experiments, the zebrafish red blood cell staining intensity is directly related to the number of red blood cells in the blood, which can be used to evaluate the effect of anemia improvement.

[0117] 1.1 Testing materials

[0118] 1.1.1 Sample preparation information

[0119] The solvent of the compound oral liquid preparation is standard dilution water.

[0120] Positive control: Shengxuening tablets, hereinafter referred to as Shengxuening, are brown-red tablets, batch number 20211003, produced by Wuhan United Pharmaceutical Co., Ltd., and the solvent is standard dilution water.

[0121] 1.1.2 Experimental animals

[0122] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3). They were bred and provided by our company's fish farming center. The experimental animal use license number is: SYXK(Zhejiang)2022-0004. The breeding and management of zebrafish complies with the requirements of the international AAALAC accreditation (certification number: 001458) and the IACUC ethics review number is: IACUC-2025-12624-01.

[0123] 1.1.3 Instruments, consumables, and reagents

[0124] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tusen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, America); 6-well plate (Zhejiang Bellanbo Biotechnology Co., Ltd., China).

[0125] Phenylhydrazine (Batch No. YH0170509, Shanghai Yihe Biotechnology Co., Ltd., China); o-dianisidine (Batch No. MKBX3619V, Sigma, USA); anhydrous sodium acetate (Batch No. F20090306, Sinopharm Chemical Reagent Co., Ltd., China); anhydrous ethanol (Batch No. 20220414, Shanghai Aladdin Biochemical Technology Co., Ltd., China); dimethyl sulfoxide (DMSO, Batch No. BCCD8942, Sigma, Switzerland).

[0126] 1.2 Detection method

[0127] Wild-type AB strain zebrafish at 4 dpf (days post fertilization, meaning the developmental stage of zebrafish larvae), were randomly selected and plated in 24-well plates, with 30 zebrafish treated per well (experimental group). Samples (concentrations shown in Table 1) were administered in water, along with a positive control, Shengxuening, at a concentration of 500 μg / mL. A normal control group and a model control group were also established, with the volume per well being 3 mL. After 6 hours of treatment at 28°C, all experimental groups except the normal control group were administered phenylhydrazine in water to establish an anemia model in zebrafish. The concentration of phenylhydrazine in each well was 0.5 μg / mL. After treatment at 28°C for 18 hours, o-dianisidine staining was performed. Ten zebrafish were randomly selected from each experimental group and photographed under a dissecting microscope. Data were acquired using NIS-Elements D 3.20 advanced image processing software, and the staining intensity of zebrafish cardiac erythrocytes was analyzed. Statistical analysis of this indicator was used to evaluate the blood-tonifying efficacy of the samples. Statistical results are expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p < 0.05 indicated that the difference was statistically significant.

[0128] 1.2.1 Effect on the staining intensity of zebrafish cardiac erythrocytes

[0129] As shown in Table 1, the zebrafish heart red blood cell staining intensity in the normal control group was significantly enhanced compared with the model control group (P < 0.001), indicating that the zebrafish anemia model was successfully established. Among them, the zebrafish heart red blood cell staining intensity in Examples 1-7 was significantly enhanced compared with the model control group (P < 0.001), and the enhancement amplitude of Example 6 was the largest, which was 12292; the zebrafish heart red blood cell staining intensity in Comparative Examples 1-5 was somewhat enhanced compared with the model control group (P < 0.05), but there was a large difference compared with Examples 1-7 (P < 0.01), indicating that the compound composition oral liquid preparation of the present invention has the effect of enriching blood. It may be that the small molecule components after enzymatic hydrolysis are more easily absorbed by zebrafish, promoting erythropoiesis; or the other effective ingredients contained in the compound composition oral liquid preparation (lycium barbarum polysaccharides, sea cucumber saponins, polysaccharides, etc.) can reduce the destruction of red blood cells and indirectly achieve the effect of enriching blood.

[0130] Table 1 Sample blood-enriching efficacy evaluation test results (n=10)

[0131]

[0132]

[0133] Note: Compared with the model control group, ***p<0.001; *p<0.05; compared with the embodiment group, ## p<0.01.

[0134] Figure 1 is the staining intensity of zebrafish heart red blood cells in the normal control group; Figure 2 is the staining intensity of zebrafish cardiac erythrocytes in the model control group; Figure 3 is the staining intensity of zebrafish heart red blood cells in the positive control group; Figure 4 This is the zebrafish heart red blood cell staining intensity in Example 6.

[0135] 2. Evaluation of the blood-enriching efficiency of the oral liquid preparation of the compound composition

[0136] In order to verify the superior difference of the oral liquid preparation of the compound composition of the present invention in terms of blood-enriching efficiency, the changes in the staining intensity of zebrafish heart red blood cells in Example 6 and Comparative Example 2 at different time periods were also detected during the zebrafish blood-enriching efficacy experiment.

[0137] Change rate of red blood cell chromosome intensity (%) = (experimental group - model group) / model group * 100

[0138] like Figure 5As shown, after 6, 12, and 18 hours of staining, the change rate of chromosome intensity in cardiac erythrocytes in Example 6 was significantly higher than that in Comparative Example 2 (P < 0.05), indicating that the oral liquid formulation of the compound composition of the present invention has a rapid effect in replenishing blood. This may be because after enzymatic hydrolysis of donkey-hide gelatin and sea cucumber, macromolecules such as collagen are broken down into small molecular peptides or amino acids, which are more easily absorbed and improve blood replenishment efficiency.

[0139] 3. Evaluation of the Immunity Efficacy of the Oral Liquid Preparation of the Compound Composition

[0140] Macrophages are important immune cells that initiate both innate and adaptive immune responses and possess a wide range of biological activities. They exert their immune functions by phagocytizing pathogens, presenting antigens, and secreting various cytokines. This study examined the effects of the oral solution of the compound composition on the fluorescence intensity of macrophages in the tail vein of zebrafish, establishing an immunosuppressive zebrafish model.

[0141] 3.1 Test materials

[0142] 3.1.1 Sample preparation information

[0143] The solvent of the compound oral liquid preparation is standard dilution water.

[0144] Positive control: Bailing capsules, hereinafter referred to as Bailing capsules, are brown powders, batch number 2307029D, manufactured by Hangzhou Sino-US Huadong Pharmaceutical Co., Ltd. The solvent is standard dilution water.

[0145] 3.1.2 Experimental animals

[0146] Zebrafish were raised in fish farming water at 28°C (water quality: 200 mg of instant sea salt was added to 1 L of reverse osmosis water, conductivity was 450-550 μS / cm; pH was 6.5-8.5; hardness was 50-100 mg / LCaCO3). They were bred and provided by our company's fish farming center. The experimental animal use license number is: SYXK(Zhejiang)2022-0004. The breeding and management of zebrafish complies with the requirements of the international AAALAC accreditation (certification number: 001458) and the IACUC ethics review number is: IACUC-2025-12225-01.

[0147] 3.1.3 Instruments, consumables and reagents

[0148] Dissecting microscope (SZX7, OLYMPUS, Japan); CCD camera (VertA1, Shanghai Tushen Vision Technology Co., Ltd., China); precision electronic balance (CP214, OHAUS, USA); 6-well plate (Zhejiang Belambra Biotechnology Co., Ltd., China); microinjector (IM300, Smith-Root, USA); puller (PC-10, Narishige, Japan); motorized continuous variable fluorescence microscope (AZ100, Nikon, Japan).

[0149] 0.9% Sodium chloride injection (Batch No. S21091609, Hunan Kelun Pharmaceutical Co., Ltd., China); Methyl cellulose (Batch No. C2004046, Shanghai Aladdin Bio-Chem Technology Co., Ltd., China); Vinorelbine tartrate injection (Batch No. 600211003, Jiangsu Haosen Pharmaceutical Co., Ltd., China).

[0150] 3.2 Detection method

[0151] Randomly selected 3 dpf (zebrafish embryos develop to the third day of the stage) transgenic macrophage green fluorescent zebrafish in 24-well plates, each hole (experimental group) was treated with 30 zebrafish. Respectively, water soluble administration of samples (concentration see Table 2), positive control bailing capsule 15.0 μg / mL concentration, while setting up normal control group and model control group, each hole volume was 3 mL. Except for the normal control group, the rest of the experimental groups were intravenously administered with 200 μg / ml vinorelbine tartrate injection 0.5 nL to establish the zebrafish immunocompromised model. After 2 days of treatment at 28℃, 10 zebrafish were randomly selected from each experimental group and placed under a fluorescence microscope for photography and image saving. NIS-Elements D 3.20 advanced image processing software was used for analysis and data collection, and the fluorescence intensity of zebrafish tail vein macrophages was analyzed. The statistical analysis results of the index were used to evaluate the immune function of the sample. The statistical processing results were expressed as mean ± SE. Statistical analysis was performed using SPSS software, and p<0.05 indicated that the difference was statistically significant.

[0152] 3.2.1 Effect on fluorescence intensity of zebrafish tail vein macrophages

[0153] As shown in Table 2, the fluorescence intensity of the tail vein macrophages of the normal control group zebrafish was significantly enhanced compared with the model control group (P<0.001), indicating that the zebrafish immunocompromised model was successfully modeled. Among them, the fluorescence intensity of the tail vein macrophages of the zebrafish in Example 1-7 was significantly enhanced compared with the model control group (P<0.001), and the enhancement amplitude of Example 6 was the largest, which was 557829; the fluorescence intensity of the tail vein macrophages of the zebrafish in Comparative Example 1-5 was certain enhancement compared with the model control group (P<0.05), but there was a large difference compared with Example 1-7 (P<0.01), indicating that the compound composition oral liquid preparation of the application has the effect of improving immunity. It may be because the compound composition oral liquid preparation of the application has the effect of tonifying blood, promoting blood circulation, improving the oxygen supply capacity of tissues, and helping to maintain the normal metabolism and function of macrophages. In addition, it may also be that more immune-regulating components (holothurian polysaccharides, saponins, small molecule peptides, etc.) are produced after enzymolysis to activate macrophages.

[0154] Table 2 Sample immune-enhancing efficacy experiment results (n=10)

[0155]

[0156] Note: compared with the model control group, ***p<0.001, *p<0.05; compared with the example group, ## P<0.01.

[0157] Figure 6 The fluorescence intensity of the tail vein macrophages of the normal control group zebrafish is; Figure 7 The fluorescence intensity of the tail vein macrophages of the model control group zebrafish is; Figure 8 The fluorescence intensity of the tail vein macrophages of the positive control group zebrafish is; Figure 9 The fluorescence intensity of the tail vein macrophages of Example 6 zebrafish is.

[0158] The above description of the disclosed examples enables one skilled in the art to make or use the application. Numerous modifications to these examples will be apparent to those skilled in the art, and the general principles defined herein can be applied to other examples without departing from the spirit or scope of the application. Therefore, the present application is not to be limited to the examples shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A compound composition based on enzymatic hydrolysis technology, characterized in that: The invention is composed of the following raw materials in parts by weight: 30-45 parts of polygonatum powder, 20-35 parts of wolfberry powder, 10-20 parts of sea cucumber enzymatic hydrolysate, 10-25 parts of donkey-hide gelatin enzymatic hydrolysate, 3-5 parts of collagen peptide, 5-8 parts of jujube powder and 4-8 parts of hawthorn powder.

2. The compound composition based on enzymatic hydrolysis technology according to claim 1, characterized in that: The preparation method of the sea cucumber enzymatic hydrolyzate is as follows: (1) grinding the sea cucumber after removing the internal organs and cleaning it to obtain a slurry for standby use; (2) adding water to the slurry at a ratio of 1:2.5-5, adjusting the pH to 6-8, adding a composite protease for enzymolysis to obtain an enzymolysis solution; the enzymolysis temperature is 45°C-65°C, and the enzymolysis time is 2-5 hours; the amount of the composite protease is 3000-6800 U / g; the composite protease is composed of flavor protease, papain, trypsin and neutral protease, and the mass ratio thereof is 3-5:2-5:4-6:4-8; the enzymatic activity of the flavor protease is 150,000 U / g; the enzymatic activity of the papain is 1,000,000 U / g; the enzymatic activity of the trypsin is 150,000 U / g; and the enzymatic activity of the neutral protease is 200,000 U / g; (3) sterilizing the obtained enzymatic solution at a temperature of 88°C to 95°C for 20 to 30 minutes; (4) After inactivating the enzyme and sterilizing, filter to obtain the sea cucumber enzymatic hydrolysate.

3. The compound composition based on enzymatic hydrolysis technology according to claim 1, characterized in that: The preparation method of the donkey-hide gelatin enzymatic hydrolyzate is as follows: (1) placing donkey-hide gelatin in a high-speed centrifuge for pulverization to obtain donkey-hide gelatin powder; (2) adding water to the donkey-hide gelatin powder to prepare a donkey-hide gelatin powder solution with a mass concentration of 15-20%; adding dilute hydrochloric acid to adjust the pH to 1.5-3, adding 0.5-1% pepsin, and performing enzymolysis at a constant temperature of 37-42°C for 1-2 hours; then adding sodium hydroxide solution to adjust the pH to 7.5-8.5, adding 0.5-1% trypsin, and performing enzymolysis at a constant temperature of 37-45°C for 2.5-3.5 hours to obtain an enzymatic solution; the enzymatic activity of the pepsin is 200,000 U / g; the enzymatic activity of the trypsin is 150,000 U / g; (3) sterilizing the obtained enzymatic solution at a temperature of 80°C to 90°C for 20 to 40 minutes; (4) After sterilization and enzyme inactivation, the mixture is filtered to obtain the enzymatic hydrolyzate of donkey-hide gelatin.

4. The method for preparing a compound composition based on enzymatic hydrolysis technology according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: sea cucumber enzymatic hydrolysate, donkey-hide gelatin enzymatic hydrolysate, polygonatum powder, wolfberry powder, collagen peptide, jujube powder and hawthorn powder are evenly mixed in proportion to obtain a compound composition.

5. A compound composition preparation based on enzymatic hydrolysis technology, characterized in that: The compound composition comprises the compound composition according to any one of claims 1 to 3 and pharmaceutically acceptable excipients as well as common food excipients and food additives; the compound composition preparation is in the form of powder, decoction, granules, pills, capsules, tablets or oral liquid.

6. The compound composition preparation based on enzymatic hydrolysis technology according to claim 5, characterized in that: The dosage form of the compound composition preparation is oral liquid.

7. Use of the compound composition according to any one of claims 1 to 3 or the compound composition preparation according to any one of claims 5 to 6 in the preparation of products for increasing the number of red blood cells and hemoglobin, promoting hematopoiesis, and improving anemia.

8. Use of the compound composition according to any one of claims 1 to 3 or the compound composition preparation according to any one of claims 5 to 6 in the preparation of a rapid blood-enriching product.

9. Use of the compound composition according to any one of claims 1 to 3 or the compound composition preparation according to any one of claims 5 to 6 in the preparation of a product for improving immunity.

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