CN104878061A

CN104878061A - Double-enzyme preparation method of oyster meat nutrient solution with antioxidation function

Info

Publication number: CN104878061A
Application number: CN201510245154.1A
Authority: CN
Inventors: 段振华; 刘艳; 李园
Original assignee: Hainan University
Current assignee: Hainan University
Priority date: 2015-05-15
Filing date: 2015-05-15
Publication date: 2015-09-02

Abstract

The invention discloses a double-enzyme preparation method of an oyster meat nutrient solution with antioxidation function. The method mainly comprises the following steps: 1) pretreatment: collecting meat of fresh oysters, removing shells, cleaning, draining and pulping to obtain a homogenate solution, bagging, and carrying out freezing storage at -20 DEG C for later use; 2) preparation of enzymolysis substrate: taking out the homogenate solution, defreezing in flowing tap water, regulating the material/water ratio at 1:2 (m/V), and stirring uniformly to obtain an enzymolysis substrate; 3) enzymolysis: heating the enzymolysis substrate to required temperature, and adding enzymes to carry out double-enzyme enzymolysis under the following technological parameters: the mass ratio of ficin to flavor proteinase is 5:1-5:1, the total enzyme amount is 4-12 wt% (relative to the enzymolysis substrate), the initial pH value for enzymolysis is 5.5-7.5, the enzymolysis temperature is 45-55 DEG C, and the enzymolysis time is 2-6 hours; 4) enzyme inactivation: after the enzymolysis finishes, carrying out enzyme inactivation on the enzymolysis solution at the constant temperature of 100 DEG C; 5) cooling: cooling the enzymolysis solution subjected to enzyme inactivation in a water bath; and 6) centrifuging: centrifuging the cooled enzymolysis solution at the rotation speed of 6000 r/min, and collecting the supernate.

Description

A kind of two enzyme preparation methods of Oyster anti-oxidant function nutritive medium

Technical field

The present invention relates to a kind of preparation method of anti-oxidant function nutritive medium, particularly the method for two enzyme enzymolysis.

Background technology

Free radical is one of mesostate of multiple biochemical reaction in human life activity.In human body, the accumulation of oxyradical is caused by the degraded due to biomacromolecule in human body, and in human body cell, free radical produces with accumulating constantly is the major cause causing various disease, produce constantly as hydroxy radical qiao and accumulate, directly can change the structure of Nucleotide, protein, attacking unsaturated fatty acids, is the major cause causing the diseases such as cancer, hepatitis, parkinson's syndrome.Free-radical scavengers can be used as the free radical that people's cylinder accumulation removed to some extent by polyphenoils, and experimental study shows, the disease that antioxidant causes the accumulation because of free hydroxyl, free oxygen base all has treatment and prophylactic effect.Synthetic antioxidant is because of the impact of side effect, and its use is subject to strict restriction; And natural antioxidants side effect is less.Therefore, the natural anti-reflecting oxide research and preparation of safety non-toxic is enjoyed to the concern of scientists.

Generate many small-molecular peptides and amino acid after proteolysis, be not only conducive to absorption of human body, also improve the utilization ratio of protein, the small-molecular peptides that research produces after finding proteolysis has multiple biological activity.At present, the preparation of protein enzyme solution, adopts single enzymolysis more.But the specificity of enzyme determines the peptide bond that certain proteolytic enzyme only may act on some peptide bond or be formed with the amino acid of certain group, single enzymolysis mainly utilizes peptide ending enzyme or endopeptidase enzymolysis protein, and deficiency is that the consumption of zymin is large, and degree of hydrolysis is lower.And adopt prozyme, can select targetedly according to the restriction enzyme site difference of different enzyme to peptide bond, thus increase restriction enzyme site, efficient enzymolysis Oyster prepares polyphenoils.

In order to the anti-oxidant nutritive medium of the mixed polypeptide obtaining high degree of hydrolysis and high anti-oxidation activity, the present invention adopts ficin and flavor protease Combined Processing Oyster first, proposes the method for two anti-oxidant nutritive medium of enzyme enzyme-squash techniqued.Ficin, is a kind of sulfydryl endopeptidase, has the characteristic of thiol proteinase, and its reactive site is containing cysteine residues, and selective hydrolysis tyrosine and phenylalanine residue, have stronger proteolytic activity.Flavor protease is a kind of compound proteolytic enzyme based on peptide ending enzyme, and when protein hydrolysate, the main effect playing peptide ending enzyme, from the terminal hydrolysis polypeptide of polypeptide chain.The difference of these two kinds of protease cleavage site, mutually promotes, and a kind of proteolytic enzyme for another proteolytic enzyme provides more restriction enzyme site, improves the hydrolysis rate of substrate, can obtain the nutritive medium compared with high anti-oxidation activity after cutting off peptide chain.

Ficin and flavor protease be enzymolysis material protein simultaneously, and due to the specificity of enzyme, two kinds of proteolytic enzyme interact, and promote mutually, thus protein hydrolysate efficiently, obtain more active fragments.Compared with single enzyme enzymolysis, ficin and flavor protease enzymolysis protein matter simultaneously, enzymolysis time is short, energy consumption is low, thus greatly reduces production cost, and enzymolysis process is simple and convenient, easy handling, and the nutritive medium anti-oxidant activity of preparation is high.

The while of current also not useful ficin and flavor protease, the method for enzymolysis prepares the report of polyphenoils.

Summary of the invention

The object of this invention is to provide a kind of method utilizing two enzyme to prepare Oyster anti-oxidant function nutritive medium, make easy and simple to handle, anti-oxidant activity is high, energy consumption is low, cost is low, the cycle is short, and can obtain the anti-oxidant nutritive medium of greater activity.

Technical scheme of the present invention is: a kind of method utilizing pair enzyme to prepare Oyster anti-oxidant function nutritive medium, specifically comprises the following steps:

1) pre-treatment: by fresh oyster through adopting meat, shell, clean, drain, pull an oar after, obtain equal slurries, it is for subsequent use that pack is placed on-20 DEG C of cold storage;

2) enzymolysis substrate is prepared: take out the equal slurries of cold storage and thaw in flowing tap water, regulate material-water ratio 1:2(m/V), stir, obtain enzymolysis substrate;

3) enzymolysis: enzymolysis substrate is warmed up to required temperature, enzyme-addedly carry out two enzyme enzymolysis, enzymolysis process parameter is: the mass ratio 5:1 ~ 5:1 of ficin and flavor protease, total enzyme amount (enzyme accounts for equal slurries mass percent) 4 % ~ 12 %, the initial pH of enzymolysis is 5.5 ~ 7.5, hydrolysis temperature 45 DEG C ~ 55 DEG C, enzymolysis time 2 ~ 6 h;

4) go out enzyme: enzymolysis completes, and go out enzymolysis solution at constant temperature 100 DEG C ferment treatment;

5) cool: enzymolysis solution complete for the enzyme that goes out is carried out water-bath cooling;

6) centrifugal: cooled enzymolysis solution is carried out centrifugal under rotating speed 6000 r/min, collect supernatant liquor.

The invention has the beneficial effects as follows: compared with Oyster enzyme solution before, ficin and flavor protease combine enzymolysis Oyster by the present invention first, make full use of the difference of ficin and flavor protease restriction enzyme site, two kinds of proteolytic enzyme interact, mutual promotion, after a kind of proteolytic enzyme cuts off peptide chain, for another proteolytic enzyme provides more restriction enzyme site, thus protein hydrolysate efficiently, both shortened extraction time, the nutritive medium compared with high anti-oxidation activity can be obtained again.Two enzyme preparation methods of anti-oxidant function nutritive medium provided by the invention are applicable to the processing of other shellfish too, be the marine products of an effective exploitation China's abundant, freshwater shellfish resource, improve the practicable technological method of China's marine products, freshwater shellfish resources economy value.

Embodiment

With specific embodiment, technical scheme of the present invention is described below.

Embodiment 1

Fresh oyster is adopted meat shell, clean, drain, carries out homogenate with tissue mashing machine, equal slurries pack and at being placed in-20 DEG C cold storage for subsequent use; With flowing tap water, 15min is frozen to freezing homogenate lyolysis, take the equal slurries of 10.0 g and be placed in 100 mL Erlenmeyer flasks, by material-water ratio 1: 2(m/V) add 20.0 mL tap water, stir, regulate pH to 6.5 with 0.1mol/L NaOH and 0.1mol/L HCl, ficin and flavor protease are according to the ratio of 1:3, and total enzyme amount adds according to 10 % (in the equal slurries quality of Oyster) simultaneously.Enzymolysis substrate is put into water-bath 45 DEG C insulation enzymolysis, and stir 1 time every 20 min, after enzymolysis 2 h, go out under 100 DEG C of boiling water baths enzyme 10 min, enzymolysis solution complete for the enzyme that goes out is carried out water-bath and cools 10 min, again with centrifugal 15 min of 6000 r/min after cooling, get supernatant liquor and namely obtain anti-oxidant nutritive medium, the refrigeration of 4 DEG C, refrigerator is for subsequent use.The DPPH clearance rate of anti-oxidant nutritive medium is 72.50% after measured, and degree of hydrolysis is 23.18%.

Embodiment 2

Fresh oyster is adopted meat, cleans, drains, carries out homogenate with tissue mashing machine, equal slurries pack and at being placed in-20 DEG C cold storage for subsequent use; With flowing tap water, freezing equal slurries are thawed 20min, take the equal slurries of 10.0 g oyster and be placed in 100 mL Erlenmeyer flasks, by material-water ratio 1: 2(m/V) add 20.0 mL tap water, regulate pH to 7.0 with NaOH and HCl of 0.1mol/L, ficin and flavor protease are according to the ratio of 5:1, and total enzyme amount adds according to 10 % (in the equal slurries quality of Oyster) simultaneously.Enzymolysis substrate is put into water-bath 50 DEG C insulation enzymolysis, and stir 1 time every 20 min, after enzymolysis 4 h, go out under 100 DEG C of boiling water baths enzyme 13 min, enzymolysis solution complete for the enzyme that goes out is carried out water-bath and cools 15 min, again with centrifugal 15 min of 6000 r/min after cooling, get supernatant liquor and namely obtain anti-oxidant nutritive medium, the refrigeration of 4 DEG C, refrigerator is for subsequent use.The DPPH clearance rate of anti-oxidant nutritive medium is 79.40% after measured, and degree of hydrolysis is 24.71%.

Embodiment 3

Fresh oyster is adopted meat, cleans, drains, carries out homogenate with tissue mashing machine, equal slurries pack and at being placed in-20 DEG C cold storage for subsequent use, with flowing tap water, freezing equal slurries are thawed 18min, take the equal slurries of 10.0 g oyster and be placed in 100 mL Erlenmeyer flasks, by material-water ratio 1: 2(m/V) add 20.0 mL tap water, regulate pH to 7.0 with NaOH and HCl of 0.1mol/L, ficin and flavor protease are according to the ratio of 1:1, total enzyme concentration 6% (with the equal slurries Mass Calculation of Oyster), first add flavor protease, enzymolysis substrate is put into water-bath 50 DEG C insulation, and stir 1 time every 20 min, cool after enzymolysis 3 h, enzymolysis substrate pH is regulated to be 6.0, add ficin again and continue 55 DEG C of insulation enzymolysis, again after enzymolysis 3 h, go out under 100 DEG C of boiling water baths enzyme 10 min, enzymolysis solution complete for the enzyme that goes out is carried out water-bath and cools 10 min, again with centrifugal 25 min of 6000 r/min after cooling, get supernatant liquor and namely obtain anti-oxidant nutritive medium, the refrigeration of 4 DEG C, refrigerator is for subsequent use.The DPPH clearance rate of anti-oxidant nutritive medium is 77.40% after measured, and degree of hydrolysis is 26.35%.

Claims

1. utilize two enzyme to prepare a method for Oyster anti-oxidant function nutritive medium, it is characterized in that, specifically comprise the following steps:

Pre-treatment: after being shelled through adopting meat by fresh oyster, cleaning, drain, pulling an oar, obtain equal slurries, it is for subsequent use that pack is placed on-20 DEG C of cold storage;

Prepare enzymolysis substrate: take out the equal slurries of cold storage and thaw in flowing tap water, regulate material-water ratio 1:2(m/V), stir, obtain enzymolysis substrate;

Enzymolysis: enzymolysis substrate is warmed up to required temperature, enzyme-addedly carries out two enzyme enzymolysis,

Enzymolysis process parameter is: the mass ratio 5:1 ~ 1:5 of ficin and flavor protease, total enzyme amount (enzyme accounts for equal slurries mass percent) 4 % ~ 12 %, and the initial pH of enzymolysis is 5.5 ~ 7.5, hydrolysis temperature 45 DEG C ~ 55 DEG C, enzymolysis time 2 ~ 6 h;

Go out enzyme: enzymolysis completes, and go out enzymolysis solution at constant temperature 100 DEG C ferment treatment;

Cooling: enzymolysis solution complete for the enzyme that goes out is carried out water-bath cooling;

Centrifugal: cooled enzymolysis solution is carried out centrifugal under rotating speed 6000 r/min, collect supernatant liquor.

2. the two enzyme of utilization according to claim 1 prepares the method for Oyster anti-oxidant function nutritive medium, it is characterized in that: described cold storage homogenate liquid thawing time is 15 ~ 20min, go out enzyme time 10 ~ 13 min, and water-bath min cooling time 10 ~ 15, centrifugation time is 15 ~ 25min.