A method for producing an active peptide with enhanced uric acid-lowering activity

Abstract

The present invention relates to the field of food biotechnology, and particularly relates to a method for producing active peptides with enhanced uric acid-lowering activity. The method comprises the following steps: using tuna meat or tuna processing waste as raw materials, pretreating the raw materials, adding water and stirring evenly, then adding a compound protease for enzymatic hydrolysis, subjecting to enzyme inactivation and cooling treatment, and then performing separation, purification and drying to obtain the product; the compound protease is composed of alkaline protease, flavor protease, trypsin, aminopeptidase and weakly alkaline protease. The active peptides prepared by the method of the present invention have a higher XOD inhibitory activity and enhanced uric acid-lowering activity at the same protein concentration.

Description

Technical Field

[0001] The present invention relates to the field of food biotechnology, and particularly to a method for producing active peptides with enhanced uric acid-lowering activity. Background Art

[0002] Tuna contains partial carnosine and anserine, and carnosine and anserine have significant in vitro and in vivo antioxidant activities. Among them, anserine has been found to have uric acid-lowering effect.

[0003] It is also reported in the market that Hailongyuan extracts tuna peptides from deep-sea tuna bones through internationally advanced bioenzymatic hydrolysis and multiple membrane separation technologies. The provided tuna peptides contain anserine (4 - 10%), and 90% of the molecular weight is between 600 and 900 daltons, which is extremely easy to be absorbed and utilized by the human body and can be applied to anti-fatigue products and uric acid-lowering products.

[0004] The XOD inhibition rate of existing active peptide products still needs to be further improved. Summary of the Invention

[0005] The main object of the present invention is to provide a method for producing active peptides with enhanced uric acid-lowering activity. The method of the present invention uses tuna meat or tuna processing scraps as raw materials, and is enzymatically hydrolyzed with alkaline protease, flavor protease, trypsin, aminopeptidase and weakly alkaline protease. The prepared active peptides have higher XOD inhibitory activity and enhanced uric acid-lowering activity at the same protein concentration.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a method for producing active peptides with enhanced uric acid-lowering activity, the method comprising the following steps: using tuna meat or tuna processing scraps as raw materials, pre-treating the raw materials, adding water and stirring evenly, then adding a compound protease for enzymatic hydrolysis, and after enzyme inactivation and cooling treatment, performing separation, purification and drying to obtain the product; the compound protease is composed of alkaline protease, flavor protease, trypsin, aminopeptidase and weakly alkaline protease.

[0008] Further, based on the dry matter of the raw materials, the addition amounts of each protease in the compound protease are: 50 - 1000 U / g of alkaline protease, 2 - 40 U / g of flavor protease, 2 - 40 U / g of trypsin, 2 - 40 U / g of aminopeptidase and 2 - 40 U / g of weakly alkaline protease.

[0009] Further, based on the dry matter of the raw materials, the mass ratio of the raw materials to the added water is 1:3 - 8.

[0010] Further, enzymatic hydrolysis is carried out at 40 - 55 °C for 1.5 h - 3 h.

[0011] Further, inactivate enzymes at 90 - 100 °C for 10 - 20 min.

[0012] Further, after enzyme inactivation and cooling, centrifuge and separate and purify through a membrane integration system, collect the filtrate with a molecular weight ≤ 3000 Da, and concentrate, spray-dry or freeze-dry the obtained filtrate to obtain the product.

[0013] The present invention also provides the bioactive peptides prepared by the above-mentioned method.

[0014] Compared with the prior art, the present invention has the following technical advantages:

[0015] Through a large number of experiments, the present invention found that when weakly alkaline protease is combined with alkaline protease, flavor protease, trypsin, and aminopeptidase for enzymolysis of tuna meat or tuna processing by-products, it can significantly enhance the inhibition rate of the obtained bioactive peptides on XOD and improve the uric acid-lowering activity. Moreover, in the composite protease, only a small amount of weakly alkaline protease needs to be added to achieve a relatively significant effect. The method steps of the present invention are simple, and the composite protease can catalyze the hydrolysis of raw materials at natural pH without adjusting the pH value.

[0016] Compared with commercially available tuna peptide products, the bioactive peptides prepared by the method of the present invention have an XOD inhibition rate increased by at least 33% or more at the same protein concentration. The bioactive peptides obtained by the present invention can be preferably used in the fields of food processing, additive production, development of uric acid-lowering health products, etc. Specific Embodiments

[0017] It should be noted that the following detailed description is exemplary and is intended to provide further illustration of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0018] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0019] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in combination with specific embodiments.

[0020] The alkaline protease, flavor protease, trypsin, aminopeptidase, and weakly alkaline protease used in the embodiments of the present invention are all produced by Shandong Guoli Biotechnology Co., Ltd. The alkaline protease has an enzyme activity of 300,000 U / ml; the flavor protease has an enzyme activity of 100,000 U / g; the trypsin has an enzyme activity of 4,000 U / g; the aminopeptidase has an enzyme activity of 5,000,000 U / g; the weakly alkaline protease has an enzyme activity of 480,000 U / g.

[0021] Example 1

[0022] A method for producing an active peptide with improved uric acid-lowering activity, the method comprising the following steps: using tuna meat as a raw material, adding water at a ratio of 1:5 based on the dry matter of the raw material, stirring and crushing it into a paste, maintaining the temperature at 50 °C, and adding the following amounts of proteases based on the dry matter of the raw material: 1000 U / g of alkaline protease, 20 U / g of flavor protease, 10 U / g of trypsin, 15 U / g of aminopeptidase, and 20 U / g of weakly alkaline protease, enzymatically hydrolyzing for 2.5 h, raising the temperature to 100 °C to inactivate the enzyme for 15 min, cooling and centrifuging, filtering the supernatant through a membrane integration system, collecting the filtrate with a molecular weight ≤ 3000 Da, concentrating, and spray-drying to obtain a tuna active peptide product.

[0023] Example 2

[0024] A method for producing an active peptide with improved uric acid-lowering activity, the method comprising the following steps: using tuna meat as a raw material, adding water at a ratio of 1:3 based on the dry matter of the raw material, stirring and crushing it into a paste, maintaining the temperature at 55 °C, and adding the following amounts of proteases based on the dry matter of the raw material: 50 U / g of alkaline protease, 40 U / g of flavor protease, 2 U / g of trypsin, 40 U / g of aminopeptidase, and 2 U / g of weakly alkaline protease, enzymatically hydrolyzing for 3 h, raising the temperature to 90 °C to inactivate the enzyme for 20 min, cooling and centrifuging, filtering the supernatant through a membrane integration system, collecting the filtrate with a molecular weight ≤ 3000 Da, and freeze-drying to obtain a tuna active peptide product.

[0025] Example 3

[0026] A method for producing an active peptide with improved uric acid-lowering activity, the method comprising the following steps: using tuna processing by-products (such as fish skin, fish bones, etc.) as a raw material, adding water at a ratio of 1:8 based on the dry matter of the raw material, stirring and crushing it into a paste, maintaining the temperature at 40 °C, and adding the following amounts of proteases based on the dry matter of the raw material: 1000 U / g of alkaline protease, 8 U / g of flavor protease, 2 U / g of trypsin, 2 U / g of aminopeptidase, and 40 U / g of weakly alkaline protease, enzymatically hydrolyzing for 1.5 h, raising the temperature to 100 °C to inactivate the enzyme for 10 min, cooling and centrifuging, filtering the supernatant through a membrane integration system, collecting the filtrate with a molecular weight ≤ 3000 Da, and freeze-drying to obtain a tuna active peptide product.

[0027] Example 4

[0028] A method for producing an active peptide with improved uric acid-lowering activity, the method comprising the following steps: using tuna processing by-products (such as fish skin, fish bones, etc.) as raw materials, adding water at a ratio of 1:6 based on the dry matter of the raw materials, stirring and crushing into a paste, maintaining the temperature at 50 °C, and adding the following amounts of proteases based on the dry matter of the raw materials: alkaline protease 600 U / g, flavor protease 2 U / g, trypsin 4 U / g, aminopeptidase 10 U / g, and weakly alkaline protease 10 U / g, enzymatically hydrolyzing for 2.0 h, heating to 100 °C to inactivate the enzymes for 10 min, cooling and centrifuging, subjecting the supernatant to membrane integrated filtration, collecting the filtrate with a molecular weight ≤ 3000 Da, and spray drying to obtain a tuna peptide product.

[0029] Comparative Example 1

[0030] The difference from Example 1 is that the following amounts of proteases are added: alkaline protease 1000 U / g, flavor protease 20 U / g, trypsin 10 U / g, aminopeptidase 35 U / g, and the others are the same as in Example 1.

[0031] Comparative Example 2

[0032] The difference from Example 1 is that the following amounts of proteases are added: alkaline protease 1000 U / g, flavor protease 20 U / g, trypsin 10 U / g, papain 15 U / g, and weakly alkaline protease 20 U / g, and the others are the same as in Example 1.

[0033] Comparative Example 3

[0034] The difference from Example 1 is that only alkaline protease is used, and the amount of the alkaline protease is 1065 U / g, and the others are the same as in Example 1.

[0035] Comparative Example 4

[0036] The difference from Example 1 is that only neutral protease is used, and the amount of the neutral protease is 1065 U / g, and the others are the same as in Example 1.

[0037] Test Example

[0038] The active peptides prepared by the methods described in Examples 1 to 4, the active peptides prepared by the methods described in Comparative Examples 1 to 4, as well as commercially available tuna peptide product 1 and commercially available tuna peptide product 2 are respectively formulated into solutions with the same protein concentration, and the XOD inhibition rate is detected. The obtained results are shown in Table 1 below.

[0039] Table 1 XOD inhibition rate of different active peptide products

[0040] Active peptide product XOD inhibition rate (%) Active peptide product XOD inhibition rate (%) Example 1 89.37 Comparative example 2 72.39 Example 2 87.05 Comparative example 3 28.94 Example 3 77.85 Comparative example 4 40.02 Example 4 84.58 Commercially available product 1 53.08 Comparative example 1 59.24 Commercially available product 2 58.46

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent substitution methods and are all included in the protection scope of the present invention.

Claims

1. A method for producing an active peptide with improved uric acid-lowering activity, characterized in that: The following steps are involved: Tuna meat or tuna processing waste is used as raw material, the raw material is pre-treated, water is added and stirred evenly, and then composite protease is added for enzymolysis, and after enzyme inactivation and cooling treatment, separation, purification and drying are performed to obtain the product; The composite protease is composed of alkaline protease, flavor protease, trypsin, aminopeptidase and weak alkaline protease; the added amount of each protease in the composite protease is: alkaline protease 50-1000U / g, flavor protease 2-40U / g, trypsin 2-40U / g, aminopeptidase 2-40U / g and weak alkaline protease 2-40U / g; After inactivating the enzyme and cooling, centrifuging and separating and purifying through a membrane integrated system, collecting a filtrate with a molecular weight of ≤3000Da, and concentrating, spray-drying or freeze-drying the obtained filtrate; The mass ratio of the raw materials, calculated on a dry basis, to the added water is 1:3 to 8; Enzymatic hydrolysis at 40-55℃ for 1.5h-3h; Inactivate the enzyme at 90-100℃ for 10-20 min.

2. The method according to claim 1, characterized in that: The added amount of each protease in the composite protease is: 50U / g alkaline protease, 40U / g flavor protease, 2U / g trypsin, 40U / g aminopeptidase and 2U / g weak alkaline protease.