Chinese yam active peptide as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biology, in particular to Chinese yam active peptide and a preparation method and application thereof.The Chinese yam active peptide comprises active peptide and free amino acid, the mass content of the active peptide with the molecular weight of 180-1000 Da is larger than or equal to 65%, and the mass content of hydrophobic amino acid in the free amino acid is larger than or equal to 30%. The preparation method of the Chinese yam active peptide is easy and convenient to operate, through the synergistic effect of the neutral protease and the alkaline protease, on the premise that the hydrolysis degree of the Chinese yam peptide is improved, it can be prevented that a large number of amino acids are generated through excessive hydrolysis, glycoprotein in Chinese yam is subjected to sufficient enzymolysis, and the yield of the Chinese yam active peptide is increased. The Chinese yam peptide prepared by the method can protect the vitality of Vero cells and MRC5 cells, has the characteristics of high activity, small molecular weight and easiness in absorption, and can be widely applied to the fields of medicines, foods, health care products, cell culture additives and the like.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a yam bioactive peptide, its preparation method, and its application. Background Technology

[0002] Yam, also known as Huai yam, is a perennial herb used in both medicine and food. It is rich in polysaccharides, proteins, mucilage, digestive enzymes, and various amino acids. Traditional medicine believes it has the effects of strengthening the spleen and stomach, invigorating qi, and calming the mind. Modern research shows that the special glycoprotein complexes in yam have potential biological activities such as anti-aging, anti-fatigue, immune regulation, and promoting energy metabolism. However, the native proteins and polysaccharides in yam have large molecular weights, resulting in low bioavailability, difficulty in direct absorption by the human body, and even the potential to trigger allergic reactions. This greatly limits the development and application of its high-value-added products.

[0003] Peptides are small molecular fragments generated from proteins through enzymatic hydrolysis. Compared with traditional proteins, they have advantages such as small molecular weight, good solubility, low viscosity, easy absorption, and diverse biological activities, exhibiting significant functions in antioxidation, blood pressure reduction, immune regulation, and anti-fatigue. Therefore, the preparation of bioactive peptides by enzymatic hydrolysis has become a research hotspot in the fields of functional foods and biomedicine. Currently, some studies have attempted to prepare bioactive peptides from yam using enzymatic hydrolysis technology, but several key technical bottlenecks remain: First, the enzymatic hydrolysis process is extensive, often using single enzymes, resulting in limited hydrolysis sites, low efficiency, and difficulty in achieving sufficient targeted protein hydrolysis; second, the proportion of target bioactive peptides (molecular weight <1000 Da) in the product is low, the molecular weight distribution is wide, and over-hydrolysis easily occurs, generating a large number of free amino acids, leading to insufficient content of active ingredients and unstable function; third, existing technologies lack in-depth understanding and precise control of the structure-activity relationship between product structure and activity, especially research on the key correlation between hydrophobic amino acid content and functions such as immune regulation and enhanced cell viability is still limited, and further development and utilization of these peptides in the research of pharmaceuticals, foods, health products, and cell culture additives are needed.

[0004] Therefore, this invention is proposed to solve the above-mentioned technical problems. Summary of the Invention

[0005] In order to overcome the technical problems existing in the prior art, the purpose of this invention is to provide a yam active peptide, its preparation method and application. The experiment of this invention proves that the yam active peptide can promote cell growth and maintain cell activity.

[0006] The technical solution of the present invention is as follows: The present invention first provides a yam active peptide, comprising active peptide and free amino acids, wherein the active peptide with a molecular weight of 180~1000 Da has a mass content of ≥65%, and the hydrophobic amino acids account for ≥30% of the mass content of the free amino acids.

[0007] The yam active peptide of the present invention includes 17 free amino acids, including 8 essential amino acids, namely threonine, valine, methionine, isoleucine, leucine, phenylalanine, lysine, and histidine, and 9 non-essential amino acids, namely aspartic acid, serine, glutamic acid, glycine, alanine, cysteine, tyrosine, arginine, and proline. The proportion of hydrophobic amino acids is ≥30%, and the hydrophobic amino acids refer to valine, isoleucine, leucine, proline, methionine, alanine, and glycine.

[0008] This invention also provides a method for preparing the yam active peptides described in the above technical solution, the preparation method comprising the following steps: (1) After washing and removing the skin of fresh yam, add water and blend into a paste to obtain yam paste; (2) After adjusting the pH of the yam homogenate, add neutral protease for enzymatic hydrolysis to obtain the first hydrolysate; (3) After adjusting the pH of the first hydrolysate, alkaline protease was added for enzymatic hydrolysis to obtain the second hydrolysate; (4) After heating and inactivating the second enzyme hydrolysate, vacuum filter to remove impurities. The pore size selected for vacuum filtration is suitable for yam peptides with a diameter of <1000 Da.

[0009] Preferably, the mass ratio of fresh yam to water in step (1) is 1:10.

[0010] Preferably, in step (2), the pH is 6.50~8.50, the enzymatic hydrolysis temperature is 40~60℃, the amount of neutral protease added is 1%~2%, and the enzymatic hydrolysis time is 1~2h.

[0011] Preferably, in step (3), the pH is 7.50~9.50, the enzymatic hydrolysis temperature is 40~60℃, the amount of alkaline protease added is 1%~2%, and the enzymatic hydrolysis time is 2~4h.

[0012] Preferably, the heating temperature in step (4) is 60°C and the heating time is 10 min.

[0013] This invention provides the application of the yam active peptide described in the above technical solution in the preparation of products that enhance cell vitality.

[0014] Preferably, the cells are VERO cells or MRC5 cells.

[0015] Preferably, the product is a pharmaceutical, food, health product, or cell culture additive.

[0016] VERO cells are epithelial cells derived from the kidneys of African green monkeys, commonly used in biomedical research fields such as virus culture and vaccine preparation, exhibiting good proliferative capacity and biological stability. MRC5 cells are human embryonic lung fibroblasts derived from normal fetal lung tissue, frequently used to study physiological and pathological processes such as cellular senescence and pulmonary fibrosis. They can secrete various extracellular matrix components, participating in the structural maintenance and repair of lung tissue. The yam active peptides provided in this invention can enhance the activity of VERO cells and MRC5 cells, demonstrating their role in kidney and lung protection. Therefore, yam active peptides can be used as active ingredients in related products.

[0017] The beneficial effects of this invention are: This invention employs a stepwise enzymatic hydrolysis method. It utilizes the primary hydrolysis achieved by neutral proteases specifically recognizing adjacent peptide bonds of hydrophobic amino acids, followed by secondary hydrolysis using alkaline proteases. These alkaline proteases not only further hydrolyze peptide bonds but also selectively cleave amide and ester bonds. Through the sequential synergistic effect of these two enzyme systems, the degree of hydrolysis of yam glycoproteins is significantly enhanced, while the reaction process is precisely controlled, preventing excessive hydrolysis and the generation of excessive free amino acids. This process successfully converts large glycoprotein molecules into small, bioactive peptides, improving both the filtration efficiency and bioavailability of the product.

[0018] This invention provides the application of yam bioactive peptides in the preparation of products that enhance cell viability. The yam bioactive peptides used in this invention include yam bioactive peptides and free amino acids; wherein, the bioactive peptide content with a molecular weight of 180-1000 Da is ≥65%, and the hydrophobic amino acid content is ≥30%. Experiments of this invention have confirmed that, compared with the control group without the addition of the yam bioactive peptides of this invention, the experimental group with added yam bioactive peptides can enhance the activity of VERO cells and MRC5 cells. Therefore, yam bioactive peptides can be used to prepare pharmaceuticals, foods, health products, or cell culture additives with corresponding effects. Attached Figure Description

[0019] Figure 1 The molecular weight distribution diagrams of the yam active peptides prepared in Examples 1-3 and Comparative Examples 1-2 are shown below. Figure 2 The images show the results of culturing VERO cells with the yam active peptides prepared in Examples 1-3 and Comparative Examples 1-2 under a 100x optical microscope. Figure 3 The images show the results of culturing MRC5 cells with the yam active peptides prepared in Examples 1-3 and Comparative Examples 1-2 under a 100x optical microscope. Detailed Implementation

[0020] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] Example 1 Step 1: Wash and peel the fresh yam, weigh 100g and add it to a blender. Add 1000mL of purified water and blend until homogeneous to obtain yam homogenate. Step 2: After adjusting the pH of the yam homogenate to 6.5, add neutral protease and react on a shaker to obtain the first enzymatic hydrolysate. The amount of neutral protease added is 2.0% of the fresh yam, the enzymatic hydrolysis time is 2.0 h, and the enzymatic hydrolysis temperature is 40℃. Step 3: After adjusting the pH of the first enzymatic hydrolysate to 9.5, add alkaline protease and react on a shaker to obtain the second enzymatic hydrolysate. The amount of alkaline protease added is 2.0% of the fresh yam, the enzymatic hydrolysis time is 4.0 h, and the enzymatic hydrolysis temperature is 60℃. Step 4: After heating the second enzyme hydrolysate at 60°C for 10 minutes to inactivate it, vacuum filter it to remove impurities. The pore size selected for vacuum filtration is suitable for yam peptides with a diameter of <1000 Da.

[0022] Example 2 Step 1: Wash and peel the fresh yam, weigh 100g and add it to a blender. Add 1000mL of purified water and blend until homogeneous to obtain yam homogenate. Step 2: After adjusting the pH of the yam homogenate to 8.5, add neutral protease and react in a shaker to obtain the first enzymatic hydrolysate. The amount of neutral protease added is 1.00% of the fresh yam, the enzymatic hydrolysis time is 1.0 h, and the enzymatic hydrolysis temperature is 60℃. Step 3: After adjusting the pH of the first enzymatic hydrolysate to 7.5, add alkaline protease and react in a shaker to obtain the second enzymatic hydrolysate. The amount of alkaline protease added is 1.00% of the fresh yam, the enzymatic hydrolysis time is 2.0 h, and the enzymatic hydrolysis temperature is 40℃. Step 4: After heating the second enzyme hydrolysate at 60°C for 10 minutes to inactivate it, vacuum filter it to remove impurities. The pore size selected for vacuum filtration is suitable for yam peptides with a diameter of <1000 Da.

[0023] Example 3 Step 1: Wash and peel the fresh yam, weigh 100g and add it to a blender. Add 1000mL of purified water and blend until homogeneous to obtain yam homogenate. Step 2: After adjusting the pH of the yam homogenate to 7.6, add neutral protease and react on a shaker to obtain the first enzymatic hydrolysate. The amount of neutral protease added is 1.60% of the fresh yam, the enzymatic hydrolysis time is 1.5 h, and the enzymatic hydrolysis temperature is 51.2℃. Step 3: After adjusting the pH of the first enzymatic hydrolysate to 8.6, add alkaline protease and react on a shaker to obtain the second enzymatic hydrolysate. The amount of alkaline protease added is 1.50% of the fresh yam, the enzymatic hydrolysis time is 3.0 h, and the enzymatic hydrolysis temperature is 49.0 ℃. Step 4: After heating the second enzyme hydrolysate at 60°C for 10 minutes to inactivate it, vacuum filter it to remove impurities. The pore size selected for vacuum filtration is suitable for yam peptides with a diameter of <1000 Da.

[0024] Comparative Example 1 Step 1: Wash and peel the fresh yam, weigh 100g and add it to a blender. Add 1000mL of purified water and blend until homogeneous to obtain yam homogenate. Step 2: After adjusting the pH of the yam homogenate to 7.6, add neutral protease and react on a shaker to obtain the first enzymatic hydrolysate. The amount of neutral protease added is 1.60% of the fresh yam, the enzymatic hydrolysis time is 1.5 h, and the enzymatic hydrolysis temperature is 51.2℃. Step 3: Heat the first enzymatic hydrolysate at 60°C for 10 minutes to inactivate it, then remove impurities by vacuum filtration. The pore size selected for vacuum filtration is suitable for yam peptides with a diameter of <1000 Da.

[0025] Comparative Example 2 Step 1: Wash and peel the fresh yam, weigh 100g and add it to a blender. Add 1000mL of purified water and blend until homogeneous to obtain yam homogenate. Step 2: After adjusting the pH of the yam homogenate to 8.6, add alkaline protease and react on a shaker to obtain the first enzymatic hydrolysate. The amount of alkaline protease added is 1.50% of the fresh yam, the enzymatic hydrolysis time is 3.0 h, and the enzymatic hydrolysis temperature is 49.0 ℃. Step 3: Heat the first enzymatic hydrolysate at 60°C for 10 minutes to inactivate it, then remove impurities by vacuum filtration. The pore size selected for vacuum filtration is suitable for yam peptides with a diameter of <1000 Da.

[0026] The molecular weight distribution of the yam active peptides in Examples 1-3 and Comparative Examples 1-2 was determined by gel chromatography. Gel chromatography is based on the molecular sieving effect. High molecular weight proteins cannot enter the interior of the gel particles but flow through the gaps between them, resulting in a shorter path, faster flow, and shorter elution time. Conversely, low molecular weight proteins need to pass through the interior of the gel particles, resulting in a longer path, slower flow, and longer elution time. Chromatographic column: TSK gel G2000SWXL 300mm × 7.8mm gel chromatography column; mobile phase: acetonitrile:water:trifluoroacetic acid = 45:55:0.1 (v / v); detection wavelength: UV 220nm; flow rate: 0.5mL / min; column temperature: 30℃; injection volume: 10μL.

[0027] The specific results are shown in Table 1 and... Figure 1 : Table 1. Molecular weight distribution of yam peptides obtained in Examples 1-3 and Comparative Examples 1-2 As can be seen from Table 1, the yam peptides extracted by Examples 1-3 of the preparation method provided by the present invention have small molecular weights and narrow distributions. The content of peptides with molecular weights of 180-1000 Da reaches more than 65%, and the content of peptides with molecular weights of 1000-10000 Da is more than 17%. In contrast, the yam peptides prepared by Comparative Examples 1-2 have large molecular weights and wide distribution ranges.

[0028] from Figure 1 It can be seen that the molecular weights of Examples 1-3 are mainly concentrated in the range of 180-1000 Da, indicating that the enzymatic hydrolysis of Examples 1-3 is effective and thorough; while the molecular weights of yam peptides in the enzymatic hydrolysates obtained in Comparative Examples 1-2 are concentrated above 10000 Da, indicating that the enzymatic hydrolysis is incomplete.

[0029] The free amino acids in the active peptides of yam were detected using an L-8800 automatic amino acid analyzer. The amino acid content is shown in Table 2 below.

[0030] Table 2. Amino acid content of yam peptides in Examples 1-3 Assessing VERO cell viability VERO cells were fed at a rate of 1×10⁻⁶. 4 One well per well is inoculated into a 96-well plate; The experiment was divided into an experimental group and a blank control group; The experimental group was treated with the yam active peptides (2 mg / mL) provided in Examples 1-3 and Comparative Examples 1-2, while the blank control group was not treated with yam active peptides. Cells were cultured in 2% FBS + DMEM medium for 24 hours. Cell morphology was observed under a microscope and photographed. The results are shown below. Figure 2 .

[0031] from Figure 2 It can be seen that VERO cells in Examples 1-3 all showed good growth, and their growth was better than that of the control group. Among them, the growth of VERO cells in Example 3 was the best. The effect of yam peptides prepared in Comparative Examples 1-2 on VERO cells was not significantly different from that of the control group, indicating that the yam active peptides of the present invention can promote the activity of VERO cells.

[0032] Measurement of MRC5 cell viability MRC5 cells were fed at a rate of 1×10 4 One well per well is inoculated into a 96-well plate; The experiment was divided into an experimental group and a blank control group; The experimental group was treated with the yam active peptides (2 mg / mL) provided in Examples 1-3 and Comparative Examples 1-2, while the blank control group was not treated with yam active peptides. Cells were cultured in 2% FBS + DMEM medium for 24 hours. Cell morphology was observed under a microscope and photographed. The results are shown below. Figure 3 .

[0033] from Figure 3 It can be seen that MRC5 cells in Examples 1-3 all showed good growth, and the growth was better than that of the control group. Among them, the growth was the best in Example 3. The effect of the yam peptides prepared in Comparative Examples 1-2 on the culture of MRC5 cells was not significantly different from that of the control group, indicating that the yam active peptides of the present invention can promote the activity of MRC5 cells.

[0034] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A yam active peptide, characterized in that, It includes bioactive peptides and free amino acids, wherein the bioactive peptides with a molecular weight of 180~1000 Da account for ≥65% of the total mass, and the hydrophobic amino acids account for ≥30% of the total mass of the free amino acids.

2. A method for preparing yam active peptides according to claim 1, characterized in that, Includes the following steps: Step (1) After washing and removing the skin of the fresh yam, add water and blend into a paste to obtain a yam paste; Step (2) After adjusting the pH of the yam homogenate, add neutral protease for enzymatic hydrolysis to obtain the first hydrolysate; Step (3) After adjusting the pH of the first hydrolysate, add alkaline protease for enzymatic hydrolysis to obtain the second hydrolysate; Step (4) After heating and inactivating the second enzymatic hydrolysate, vacuum filter is used to remove impurities. The pore size selected for vacuum filtration is suitable for yam peptides with a diameter of <1000 Da.

3. The method for preparing yam active peptides according to claim 2, characterized in that, In step (1), the mass ratio of fresh yam to water is 1:

10.

4. The method for preparing yam active peptides according to claim 2, characterized in that, In step (2), the pH is 6.50~8.50, the enzymatic hydrolysis temperature is 40~60℃, the amount of neutral protease added is 1%~2%, and the enzymatic hydrolysis time is 1~2h.

5. The method for preparing yam active peptides according to claim 2, characterized in that, In step (3), the pH is 7.50~9.50, the enzymatic hydrolysis temperature is 40~60℃, the amount of alkaline protease added is 1%~2%, and the enzymatic hydrolysis time is 2~4h.

6. The method for preparing yam active peptides according to claim 2, characterized in that, In step (4), the heating temperature is 60°C and the heating time is 10 min.

7. The use of the yam active peptide of claim 1 in the preparation of products that enhance cell vitality.

8. The application according to claim 7, characterized in that, The cells are either VERO cells or MRC5 cells.

9. The application according to claim 7 or 8, characterized in that, The product is a pharmaceutical, food, health product, or cell culture additive.

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