A method for preparing bioactive peptides by enzymatic hydrolysis of enzyme residues and its application

Abstract

The present invention belongs to the field of bioengineering technology, and specifically provides a method for preparing bioactive peptides by enzymatically hydrolyzing enzyme residues, comprising the following steps: (1) preparing ginger homogenate containing ginger protease; (2) taking the enzyme residue for pretreatment, and then adding the ginger homogenate for enzymatic hydrolysis to obtain an enzymatic solution; (3) heating and inactivating the enzymatic solution, filtering and centrifuging, and the supernatant is the bioactive peptide product. The method for preparing bioactive peptides by enzymatically hydrolyzing enzyme residues provided by the present invention directly uses ginger to enzymatically hydrolyze the residual crude protein in the enzyme residue, replacing the complicated ginger protease extraction process, and reusing the enzyme residue substrate, greatly reducing the environmental pressure of waste residue treatment in the enzyme production process, and the reaction raw materials are all pure natural and edible raw materials, which are green and environmentally friendly.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a method for preparing bioactive peptides by enzymolysis of enzyme residues and application thereof. Background Art

[0002] Bioactive peptide (BAP) is a general term for 20 kinds of peptides in different compositions and arrangements in proteins, ranging from dipeptides to complex linear and ring structures. It is a multifunctional compound derived from protein. Bioactive peptides have a variety of human metabolism and physiological regulation functions, are easy to digest and absorb, and have the effects of promoting immunity, hormone regulation, antibacterial, antiviral, lowering blood pressure, and lowering blood lipids.

[0003] Enzyme residue is a by-product of the enzyme production process. It is the residual solid that is insoluble in the enzyme liquid after fermentation. It contains organic matter, nitrogen and phosphorus nutrients and enzyme bacteria. It is mostly used to make organic fertilizers or repair contaminated soil. Ginger protease is a plant protease found in ginger. It belongs to the papain class and together with papain, bromelain and kiwi protease, it constitutes 4 important plant proteases. Ginger protease has good enzymatic properties and still has high enzyme activity at around 50°C. It can effectively degrade crude protein in the reaction system to form small molecule peptides and amino acids.

[0004] Currently, raw materials for preparing bioactive peptides are mostly soybeans, peanuts, fish meal, etc. The present invention utilizes ginger protease to degrade crude protein in enzyme residues, thereby providing a new method for preparing bioactive peptides. Summary of the invention

[0005] The purpose of the present invention is to provide a novel method for preparing bioactive peptides.

[0006] To this end, the present invention provides a method for preparing bioactive peptides by enzymatic hydrolysis of enzyme residues, comprising the following steps:

[0007] (1) preparing ginger homogenate containing ginger protease;

[0008] (2) taking enzyme residue for pretreatment, and then adding ginger homogenate for enzymolysis to obtain enzymolysis solution;

[0009] (3) The enzymatic hydrolysate is heated and inactivated, filtered and centrifuged, and the supernatant is the bioactive peptide product.

[0010] Specifically, the above step (1) is as follows: cutting ginger rhizomes into pieces, squeezing out juice, and homogenizing, adding vitamin C during the homogenization process to obtain ginger homogenate containing ginger protease.

[0011] Specifically, based on the weight of ginger, the added amount of the vitamin C is 0.1%-5%.

[0012] Specifically, the enzyme residue is red date enzyme residue or wolfberry enzyme residue.

[0013] Specifically, the pretreatment in the above step (2) is as follows: adding water or buffer solution to the enzyme residue and boiling it.

[0014] Specifically, the buffer is PBS buffer or pH 6.0 phosphate buffer.

[0015] Specifically, in the above step (2), the amount of ginger homogenate added is more than twice the mass of the enzyme residue.

[0016] Specifically, the enzymatic hydrolysis condition in the above step (2) is a constant temperature water bath at 50° C. for 4-16 hours.

[0017] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0018] The method for preparing bioactive peptides from enzyme residues provided by the present invention directly uses ginger to hydrolyze the residual crude protein in the enzyme residues, replacing the complicated ginger protease extraction process, and reusing the enzyme residue substrate, greatly reducing the environmental pressure of waste residue treatment in the enzyme production process, and the reaction raw materials are all pure natural, edible raw materials, green and environmentally friendly. In addition, the present invention adds ginger to the enzyme residues. On the one hand, natural plant protein is used to continue to hydrolyze crude protein. On the other hand, the active substances in ginger are mixed with peptide chains and amino acids formed by the decomposition of crude protein, which can increase the final antioxidant activity of the product. Compared with simply using ginger protease to hydrolyze protein, it makes full use of the various active substances contained in ginger itself, simplifies the production process, and greatly improves the utilization rate of raw materials. The molecular weight of the bioactive peptides in the bioactive peptide product prepared by the method of the present invention is mainly distributed between 10kDa-240KDa, and the antioxidant activity (DPPH clearance rate) IC 50 The value is between 20mg / mL and 120mg / mL. The source of bioactive peptides is clear, green and natural. They have the functions of promoting digestion and absorption, enhancing immunity and lowering blood lipids.

[0019] The present invention will be further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is the SDS-PAGE electrophoresis diagram in Example 1 of the present invention.

[0021] Figure 2 It is the SDS-PAGE electrophoresis diagram in Example 2 of the present invention.

[0022] Figure 3 It is the SDS-PAGE electrophoresis diagram in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0023] The technical scheme in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Although the representative embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that various modifications and changes can be made to the present invention without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the embodiments, but should be limited by the attached claims and their equivalents.

[0024] The present invention provides a method for preparing bioactive peptides by enzymatic hydrolysis of enzyme residues, comprising the following steps:

[0025] (1) preparing ginger homogenate containing ginger protease;

[0026] The ginger rhizome is cut into pieces, juice is squeezed, and homogenized. Vitamin C is added during the homogenization process to obtain a ginger homogenate containing ginger protease. The amount of vitamin C added is preferably 0.1%-5% of the weight of the ginger. Vitamin C can maintain the activity of ginger protease and maintain the physiological activity of effective substances in ginger.

[0027] (2) Take the enzyme residue, add water or buffer, preferably PBS buffer or pH 6.0 phosphate buffer, boil for about 15 minutes, add ginger slurry after cooling, and place in a constant temperature water bath at 50° C. for 4-16 hours to obtain an enzymatic hydrolyzate.

[0028] The amount of ginger homogenate added is more than twice the mass of the enzyme residue

[0029] (3) The enzymatic hydrolysate is heated and boiled for 15 minutes to inactivate the enzyme, filtered and centrifuged, and the supernatant is filled and sterilized to obtain the bioactive peptide product.

[0030] The following is a study of the method for preparing bioactive peptides from enzyme residues by enzymolysis and the effects of its application through specific examples.

[0031] Embodiment 1:

[0032] This embodiment provides a method for preparing bioactive peptides by enzymatic hydrolysis of enzyme residues, comprising the following steps:

[0033] (1) Cut ginger rhizomes into pieces, squeeze out juice and homogenize. During the homogenization process, 5% vitamin C is added to obtain ginger homogenate containing ginger protease.

[0034] (2) Take 0.5 kg of red date enzyme residue, add 1.5 kg of water, boil for 15 min, cool, add 2 kg of ginger slurry, and perform enzymolysis in a constant temperature water bath at 50°C for 16 h to obtain enzymolysis solution.

[0035] (3) Heat and boil the enzymatic hydrolysate for 15 minutes to inactivate it, then filter and centrifuge it to detect the DPPH clearance rate IC of the supernatant. 50 The value was 50 mg / mL, and the enzymatic solution was subjected to SDS-PAGE protein separation gel. The results were as follows Figure 1 As shown, its molecular weight distribution was determined to be around 15-25 KDa.

[0036] The supernatant is filled and sterilized to obtain the bioactive peptide product.

[0037] Embodiment 2:

[0038] This embodiment provides a method for preparing bioactive peptides by enzymatic hydrolysis of enzyme residues, comprising the following steps:

[0039] (1) Cut ginger rhizomes into pieces, squeeze out juice and homogenize. During the homogenization process, 1% vitamin C is added to obtain ginger homogenate containing ginger protease.

[0040] (2) Take 50 g of red date enzyme residue, add 150 g of water, boil for 15 min, cool, add 200 g of ginger slurry, and perform enzymolysis in a constant temperature water bath at 50°C for 16 h to obtain enzymolysis solution.

[0041] (3) Heat and boil the enzymatic hydrolysate for 15 minutes to inactivate it, then filter and centrifuge it to detect the DPPH clearance rate IC of the supernatant. 50 The value was 86 mg / mL, and the enzymatic solution was subjected to SDS-PAGE protein separation gel. The results were as follows Figure 2 As shown, its molecular weight distribution was determined to be around 10-15 KDa.

[0042] The supernatant is filled and sterilized to obtain the bioactive peptide product.

[0043] Comparative Example 1:

[0044] This comparative example provides a method for preparing bioactive peptides by enzymatic hydrolysis of enzyme residues, comprising the following steps:

[0045] Take 0.5 kg of red date enzyme residue, add 1.5 kg of water, boil for 15 minutes, cool, add 2 kg of ginger protease with an enzyme activity of 300 U / g, and perform enzymolysis in a constant temperature water bath at 50°C for 16 hours to obtain an enzymolysis solution.

[0046] (3) Heat and boil the enzymatic hydrolysate for 15 minutes to inactivate it, then filter and centrifuge it to detect the DPPH clearance rate IC of the supernatant. 50 The value was 20 mg / mL, which was less than the DPPH clearance rate in Example 2. The enzymatic solution was subjected to SDS-PAGE protein separation gel. The results were as follows Figure 3 As shown, its molecular weight distribution was determined to be around 10-15 KDa.

[0047] The above examples are merely illustrative of the present invention and do not constitute a limitation on the protection scope of the present invention. All designs that are the same or similar to the present invention fall within the protection scope of the present invention.

Claims

1. A method for preparing bioactive peptides by enzymatic hydrolysis of enzyme residues, characterized in that: The following steps are involved: (1) Preparing ginger homogenate containing ginger protease; (2) Add water or buffer to the red date enzyme residue, boil it, then add ginger homogenate for enzymolysis, and perform enzymolysis in a constant temperature water bath at 50°C for 16 hours to obtain an enzymolysis solution; the amount of ginger homogenate added should be more than twice the mass of the enzyme residue; (3) After the enzymatic hydrolysate is heated and inactivated, it is filtered and centrifuged, and the supernatant is the bioactive peptide product.

2. The method for preparing bioactive peptides from enzyme residues according to claim 1, characterized in that: The step (1) specifically comprises: cutting ginger rhizomes into pieces, squeezing out juice and homogenizing, and adding vitamin C during the homogenization process to obtain ginger homogenate containing ginger protease.

3. The method for preparing bioactive peptides from enzymatic enzyme residues as claimed in claim 2, characterized in that: Calculated based on the weight of ginger, the added amount of vitamin C is 0.1%-5%.

4. The method for preparing bioactive peptides from enzyme residues according to claim 1, characterized in that: The buffer is PBS buffer or pH 6.0 phosphate buffer.

5. A bioactive peptide, characterized in that: The bioactive peptide is prepared by the method according to any one of claims 1 to 4, and its molecular weight distribution is 10-15 KDa.

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