CN109207544A - A kind of preparation method of chlorella antioxidation polypeptide - Google Patents
A kind of preparation method of chlorella antioxidation polypeptide Download PDFInfo
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Abstract
The present invention provides a kind of preparation methods of chlorella antioxidation polypeptide, it is characterized in that, chlorella albumen is extracted using high pressure homogenization method association fibre element enzyme/pectinase enzymatic hydrolysis, after specific protein enzymatic hydrolysis liquid enzymatic hydrolysis, Ultra filtration membrane obtains the chlorella Peptides in different molecular weight section.The chlorella polypeptide that molecular weight ranges of the present invention after specific protein enzymatic hydrolysis liquid enzymatic hydrolysis are 0-3.5 KDa has preferably removes DPPH and hydroxyl radicals than the chlorella polypeptide between other protease hydrolyzed products and other molecular weight areas in vitro, external to remove liver cancer cells reactive oxygen species ability, more effective animal body endoperoxides product malonaldehyde forms rejection ability and activity of glutathione peroxidase promotes ability.The chlorella active peptide of more preferably anti-oxidation function is made dependent on specific proteases enzymolysis liquid by the present invention, is conducive to the development and utilization of anti-oxidation function health food and medical product.
Description
Technical field
The present invention provides a kind of preparation methods of chlorella antioxidation polypeptide, belong to field of biotechnology.
Background technique
Chlorella (Chlorella) is general natural disposition monoplast green alga, extensive in distributed in nature, can autotrophy, also can be
Growth and breeding is carried out using organic carbon source under the conditions of heterotrophism, biomass is big, is the life that can uniquely increase by 4 times on the earth at 20 hours
Object.Chlorella nutrition is extremely abundant, and wherein crude protein content is up to 50% or more, it is necessary to which amino acid composition is in a basic balance, simultaneously
Also containing unsaturated fatty acid, bioactive polysaccharide, nucleic acid, vitamin, microelement, minerals, chlorophyll etc., nutritive value
It is high, there is prevention and treatment peptic ulcer, antitumor, strengthen immunity, anti-radiation, resisting pathogenic microbes, prevention and treatment anaemia, reducing blood lipid
With a variety of bio-pharmacology activity such as antiatherosclerosis.Therefore, chlorella is other than being widely used in aquaculture, not
Food, beverage and medicines and health protection product etc. are successively developed into few countries and regions.
The production of chlorella health care product at present is using broken wall dry powder as raw material.Through broken wall, it is concentrated and is obtained by extraction bead
Algae dry powder be it is a kind of by nucleoprotein, ribonucleic acid (RNA), DNA (DNA), multivitamin, amino acid, polysaccharide,
The mixture of the material compositions such as complicated aleuroplast, ferment, glycoprotein, various plants hormone.Since chlorella has such as
Its powder mixture is usually also known as chlorella growth factor (Chlorella Growth by this effective healthcare function
Factor, CGF).Meanwhile there are also many researchs to think in its dry powder also containing having unique biological activity and physiological function
" the baffled factor ".But correlative study there is no to prove which kind of group is the main matter for playing high bioactivity in chlorella have at present
Divide and constitute, what respective biological activity and function is different components have.Obviously, the deficiency in these researchs seriously constrains small
The further exploitation of ball algae medical value, the exploitation of especially high-valued chlorella health care product.
Protein is the main undertaker of vital movement, it is closely related with life and various forms of vital movements, raw
The dynamic too busy to get away protein of destiny.It is believed that the objects such as nucleic acid, vitamin, microelement, minerals, the chlorophyll in chlorella
Matter nutriment similar with other sources does not have difference substantially.Protein content is up to dry weight in chlorella
50%, the healthcare function that chlorella has must be related to proteins and peptides substantial connection contained by it, and active function has very
Big a part should be from the protein and polypeptide component contained by it.
Biologically active peptide be it is a kind of have anti-oxidant, antifatigue, aided blood pressure-lowering, adjust immunity, antitumor, antibacterial,
The important active substances of the functions such as hypoglycemic usually contain 3 to 20 amino acid residues.The short peptide chain of these amino acid composition is in parent
It is inactive in the sequence of this protein, but by pipe intestinal digesting, is released after food processing or fermentation.Food proteins
The features such as biologically active peptide obtained by hydrolysis is absorbed with high security and easily.The biology that biologically active peptide has
Activity is closely related with architectural characteristic, amino acid sequence, relative molecular mass, the amino acid side groups of itself peptide chain etc..Together
After different protease hydrolyzeds, since different protease sites are different, different peptides can be obtained in the food proteins in one source
Chain structure, amino acid sequence, molecular size range and side-chain radical polypeptide.Existing very more domestic and international research confirms, same
The biologically active peptide that the albumen in source obtains after different protease hydrolyzeds often has different bioactivity, while different
The albumen in source often bioactivity having the same after same protease hydrolyzed, such as: same milk protein is through different micro-
The not homopolypeptide obtained after the enzymatic hydrolysis such as biological enzyme or trypsase, pepsin is respectively provided with blood pressure lowering, strengthen immunity, antioxygen
The different bioactivity such as change;And the protein in different food (milk, soybean, fish, microalgae etc.) sources is through pepsin enzyme
The polypeptide in the different molecular weight section and amino acid sequence that obtain after solution is often all with the health care function of apparent strengthen immunity
Effect.Therefore, it is necessary to be screened according to the different bioactivity of acquisition required for us most suitable for digesting specific food
The protease species of albumen.
Anti-oxidation peptide is a kind of peptides having compared with the peroxidating of high inhibition large biological molecule and remove interior free yl function.
The mechanism of action of anti-oxidation peptide is to act directly on free radical, or consume the substance for being easy to generate free radical indirectly, is prevented
Further reaction occurs.Currently, many studies have reported the protein hydrolysates of a variety of plant and animal materials to all have anti-oxidant work
Property.Although having correlative study using chlorella albumen as raw material, using antioxidation polypeptide is obtained after different protease hydrolyzeds, such as:
Wang, which is equal to 2017, uses alkalinity extraction chlorella albumen, digests through flavor protease, obtains the enzyme with antioxidant activity
Solve polypeptide;Han et al. has separated a kind of tripeptides from chlorella vulgaris with antioxidant activity in 2015;Wang Shaoyun is equal to
It using chlorella albumen as raw material, is digested by acid protease within 2014, isolates and purifies to obtain different antioxidation polypeptides;
Sheih, which is equal to 2009, digests chlorella using pepsin, and isolates and purifies to obtain a kind of with antioxidant activity
11 peptides.But the antioxidant effect of the antioxidation polypeptide of these research acquisitions is mostly based on external and cellular level detection,
Seldom based on interior animal experiment as a result, being difficult to determine which class enzymolysis product has most between these different enzymolysis polypeptides simultaneously
Good antioxidation polypeptide.
Existing research further demonstrates in microalgae derived Protein, the polypeptide product tool obtained through different protease hydrolyzeds
Standby different degrees of antioxidant activity.Alzahrani, which is equal to 2017, extracts 3 kinds of microalgae albumen by ultrasonic method, further divides
Not after alkali protease, flavor protease and trypsase individually digest, compare the antioxidant effect of these enzymolysis products, ties
Fruit finds that these enzymolysis polypeptides all have certain antioxidant activity, but is only had using the polypeptide after hydrolysis by novo
Standby optimal antioxidant activity.In consideration of it, to chlorella, this high-quality microalgae protein resource carries out height using modern biotechnology
Value exploitation, by selecting most suitable proteinase combination, optimization enzymatic hydrolysis formula of liquid and enzymolysis process, it is expected to it is good to obtain antioxidant effect
Chlorella antioxidation polypeptide, for further developing the relevant food of antioxidation polypeptide, health care and medical product.
Summary of the invention
The present invention provides a kind of preparation method of chlorella antioxidation polypeptide, can obtain in vitro, cell and animal
The good chlorella antioxidation polypeptide of internal antioxidant effect, to more preferably expand chlorella in food, health care and field of medicaments
Using.
The present invention adopts the following technical scheme:
1. the preparation of chlorella cells wall enzymolysis liquid and specific protein enzymatic hydrolysis liquid
1) the chlorella cells wall enzymolysis liquid component proportion described in are as follows:
Chlorella cells wall enzymolysis liquid, which matches to postpone, adjusts pH to 3.0~5.0 with the concentrated hydrochloric acid that mass fraction is 36%;
2) the specific protein enzymatic hydrolysis liquid component proportion described in are as follows:
Specific protein enzymatic hydrolysis liquid, which matches to postpone, adjusts pH to 7.5~9.5 with 1mol/L NaOH solution;
2. chlorella antioxidation polypeptide is prepared by the following steps:
It 1), will for 1:7~10 according to chlorella powder and NaOH dilute alkaline soln mass ratio using chlorella pyrenoidosa powder as raw material
Chlorella powder is added in 0.2~0.3mol/L NaOH dilute alkaline soln, under the conditions of 60 DEG C, impregnates 60min, it is small to obtain pretreatment
Ball algae solution;
2) pretreatment bead algae solution is centrifuged under 5000rpm high-pressure homogeneous 2~4 times under 30~60MPa pressure
20min, the supernatant A of acquisition save under the conditions of being temporarily placed in 4 DEG C, and the sediment of acquisition is added in chlorella cells wall enzymolysis liquid
Digested, enzymatic hydrolysis condition is 40~60 DEG C of hydrolysis temperature, 3~7h of enzymolysis time, speed of agitator 150rpm, after enzymatic hydrolysis in
It is centrifuged 20min under 5000rpm, obtains supernatant B;
3) supernatant A and supernatant B are mixed, carries out single-action energy conservation concentration in atmospheric conditions, until concentration front and back body
Product ratio reaches 3~6:1, and concentrate can obtain chlorella albumen powder through vacuum freeze drying;
4) chlorella albumen powder is added in specific protein enzymatic hydrolysis liquid and is digested, digested under the conditions of 40~70 DEG C, enzymatic hydrolysis 30
After~60min in 90 DEG C of water destroy the enzyme treatment 30min, be rapidly cooled to room temperature, 20min be centrifuged under 4000rpm, obtain supernatant
Liquid C;
5) it is respectively by molecular cut off in the case where 1MPa enters film pressure, 40mL/min dialysis flow condition by supernatant C
The ultrafiltration membrane of 10KDa, 5KDa, 3.5KDa, ultra-filtration and separation obtain molecular size range range be respectively > 10KDa, 5-10kDa,
The chlorella antioxidation polypeptide liquid of 3.5-5kDa and 0-3.5kDa collects different molecular weight chlorella antioxidation polypeptide liquid, respectively
It is the different molecular weight chlorella antioxidation polypeptide of powdery through vacuum freeze drying.Chlorella through 0-3.5kDa is anti-oxidant more
Peptide liquid antioxidant activity is most strong.
The vacuum freeze drying, condition are as follows: freezing control -40 DEG C of set temperature, time 30min, retention time
120min;Primary drying sets the 1st -20 DEG C of stage drying temperature, drying time 30min, retention time 120min, vacuum
0.25;2nd -15 DEG C of stage drying temperature, drying time 30min, retention time 120min, vacuum 0.25;3rd stage dry temperature
Spend -5 DEG C, drying time 30min, retention time 500min, vacuum 0.25;4th 0 DEG C of stage drying temperature, drying time
30min, retention time 1500min, vacuum 0.3;5th 5 DEG C of stage drying temperature, drying time 30min, retention time
120min, vacuum 0.3;6th 10 DEG C of stage drying temperature, drying time 30min, retention time 120min, vacuum 0.3;Parsing
20 DEG C of dryer settings temperature, time 30min, retention time 120min, vacuum 0.3.
Compared with prior art, the present invention has advantage as is evident below and better functional effect: the present invention is with bead
Algae albumen is that starting point is effectively improved small by the cellulase after high-pressure homogeneous combined optimization/pectase cell wall enzymolysis liquid
Ball algae protein extracting ratio;The chlorella albumen of acquisition is had through specific protein enzymatic hydrolysis liquid, enzymolysis process control and ultra-filtration and separation
There is the chlorella polypeptide product in the specified molecular weight section (0~3.5KDa) of best antioxidant activity.The bead that the present invention obtains
Algae antioxidation polypeptide has optimal antioxidant activity: (1) having more preferable than the chlorella enzymolysis product of other protease hydrolyzeds
External total reducing power;(2) there is the polypeptide product than other molecular size ranges after same specific protein enzymatic hydrolysis liquid enzymatic hydrolysis
Preferably external to remove DPPH and hydroxyl radicals, external removing liver cancer cells reactive oxygen species (ROS) ability is more effectively dynamic
Object endoperoxides product malonaldehyde (MDA) forms rejection ability and glutathione peroxidase (GSH-PX) activity promotes energy
Power.Thus, the chlorella antioxidation polypeptide that the present invention obtains is more advantageous to the further relevant food of exploitation antioxidation polypeptide, guarantor
Strong and medical product.
Detailed description of the invention
Fig. 1 be in the embodiment of the present invention 1 the chlorella albumen for preparing through specific proteases enzymolysis liquid enzyme used in the present invention
The external removing DPPH and hydroxyl radicals of different molecular weight polypeptide after solution compare figure.
Fig. 2 is that the chlorella antioxidation polypeptide that the molecular weight that the present invention obtains is 0-3.5KDa reduces activity in liver cancer cells
The effect picture of oxygen.
After Fig. 3 is the chlorella polypeptide for the different molecular weight size that aged mouse stomach-filling is obtained through the present invention, MDA in serum
Content and GSH-PX expression activitiy figure.
Specific embodiment
Specific implementation of the invention is described further with attached drawing with reference to embodiments, it is all special according to the present patent application
The equivalent changes and modifications done in sharp range, all should belong to covering scope of the invention.
Embodiment 1
1. the preparation of chlorella cells wall enzymolysis liquid and specific protein enzymatic hydrolysis liquid
Cellulase, pectase and all kinds of protease used are purchased from Beijing Suo Laibao Science and Technology Ltd.
1) the chlorella cells wall enzymolysis liquid component proportion described in are as follows:
Above-mentioned chlorella cells wall enzymolysis liquid uses the concentrated hydrochloric acid that mass fraction is 36% to adjust pH to 4.0 after preparing.
2) the specific protein enzymatic hydrolysis liquid component proportion described in are as follows:
Above-mentioned specific protein enzymatic hydrolysis liquid uses 1mol/L NaOH solution to adjust pH to 9.5 after preparing;
2. chlorella antioxidation polypeptide is prepared by the following steps:
1) it weighs 500g chlorella powder to be added in 4.5L 0.3mol/L NaOH dilute alkaline soln, under the conditions of 60 DEG C, impregnate
60min obtains pretreatment bead algae solution;
2) the pretreatment bead algae solution of the step 1) acquisition, high-pressure homogeneous 3 times under 45MPa pressure, under 5000rpm from
Heart 20min, the supernatant A of acquisition save under the conditions of being temporarily placed in 4 DEG C, and the sediment of acquisition is added to chlorella cells wall enzymolysis liquid
In, 6h is digested under the conditions of being 150rpm in 45 DEG C, speed of agitator, 20min is centrifuged after enzymatic hydrolysis under 5000rpm, obtains supernatant
B;
3) supernatant A of the step 2) acquisition and supernatant B mixing, carry out single-action energy conservation concentration in atmospheric conditions,
Until concentration front and back volume ratio reaches 5:1, concentrate can obtain chlorella albumen powder through vacuum freeze drying;
4) the chlorella albumen powder that step 3) obtains is added in specific protein enzymatic hydrolysis liquid and is digested, digested under the conditions of 50 DEG C
Time 60min, the destroy the enzyme treatment 30min in 90 DEG C of water, is rapidly cooled to room temperature later, and 20min is centrifuged under 4000rpm, obtains
Supernatant;
5) supernatant described in step 4) is in the case where 1MPa enters film pressure, 40mL/min dialysis flow condition through retention molecule
Amount is respectively the ultrafiltration membrane of 10KDa, 5KDa, 3.5KDa, and it is respectively > 10KDa, 5- that ultra-filtration and separation, which obtains molecular size range range,
The chlorella antioxidation polypeptide liquid of 10kDa, 3.5-5kDa and 0-3.5kDa, the chlorella for collecting different molecular weight section are anti-oxidant
Polypeptide liquid is the chlorella antioxidation polypeptide of powdery through vacuum freeze drying.
Vacuum freeze drying condition in above-mentioned steps are as follows: when -40 DEG C of set temperature of freezing control, time 30min, holding
Between 120min;Primary drying sets the 1st -20 DEG C of phase temperature, time 30min, retention time 120min, vacuum 0.25, the 2nd rank
Duan Wendu -15 DEG C, time 30min, retention time 120min, the 0.25, the 3rd -5 DEG C of phase temperature of vacuum, time 30min, holding
Time 500min, the 0.25, the 4th 0 DEG C of phase temperature of vacuum, time 30min, retention time 1500min, vacuum 0.3, the 5th stage
5 DEG C of temperature, time 30min, retention time 120min, the 0.3, the 6th 10 DEG C of phase temperature of vacuum, time 30min, retention time
120min, vacuum 0.3;20 DEG C of parsing-desiccation set temperature, time 30min, retention time 120min, vacuum 0.3.
3. the measurement of antioxidation activity in vitro
1) measurement of total reducing power
2.5mL chlorella antioxidation polypeptide sample solution manufactured in the present embodiment is taken, the phosphate buffer of 2.5mL is added
The 0.01g/mL K of (0.2mol/L, pH=6.6) and 2.5mL3Fe(CN)6Solution, after 50 DEG C of water-bath 20min, fast quickly cooling
But, 10% solution of trichloroacetic acid of 2.5mL is added, extracts reaction solution the ddH that 5mL is added in 5mL2O and 1mg/mL FeCl3Solution 1mL,
It is uniformly mixed, measures its absorbance value after 10min at 700nm, using water as blank, the bigger expression reducing power of absorbance value is more
By force.
2) measurement of hydroxyl radical free radical Scavenging activity
Chlorella antioxidation polypeptide sample solution 2mL manufactured in the present embodiment is taken, 2mL 9mM FeSO is sequentially added4、2mL
9mM salicylic acid (dehydrated alcohol preparation), 2mL 8.8mM H2O2And 2mL ddH2O, 37 DEG C of reaction 30min, with ddH2O is ginseng
According to the measurement light absorption value at wavelength 510nm calculates Hydroxyl radical-scavenging ability.Using ascorbic acid as positive control, bead is evaluated
The ability of algae enzymolysis polypeptide scavenging hydroxyl.
Scavenging action to hydroxyl free radical (%) ﹦ [1- (A1-A2)/A0] × 100%
In formula, A1 is the light absorption value of sample sets;A2 is that color developing agent H is not added2O2The light absorption value of solution group;A0 is distilled water generation
For the light absorption value of sample blank group.
3) measurement of DPPH free radical scavenging ability
Chlorella antioxidation polypeptide sample solution 2mL manufactured in the present embodiment is taken, the DPPH that 2mL 0.04g/mL is added is molten
Liquid (dehydrated alcohol configuration), it is dark after mixing to stand 30min, light absorption value is measured at wavelength 517nm, and it is clear to calculate DPPH free radical
Removing solid capacity.Positive control is done with ascorbic acid, evaluation chlorella enzymolysis polypeptide removes the ability of DPPH free radical.
DPPH clearance rate (%)=[1- (A1-A2)/A0] × 100%
In formula, A1 is the light absorption value that sample is reacted with DPPH;A2 is replaced the light absorption value of sample by ethyl alcohol;A0 is distilled water generation
For the light absorption value light absorption value of sample.
4. the test of cellular level antioxidant activity
7402 liver cancer cells of Bel that growth conditions are good, in logarithmic growth phase are taken, are inoculated in six orifice plates, to thin
Born of the same parents it is adherent for 24 hours after, be separately added into 100 μ g/mL, 200 μ g/mL, 400 μ g/mL and 800 μ g/mL molecular weight be 0-3.5kDa it is small
Ball algae antioxidation polypeptide detects the ROS in cell horizontal in accordance with the following methods: being matched with serum-free 1640 culture medium after processing for 24 hours
The ROS fluorescence probe DCFH-DA of 10 μM of system, sucks the culture solution in six orifice plates, and the DCFH- that 1mL concentration is 10 μM is added in every hole
DA (except blank control wells), is placed in CO2It is protected from light in constant incubator and is incubated for 20min.It is molten that DCFH-DA is sucked under the conditions of being protected from light
Liquid passes through fluorescence microscope fluorescence intensity after washing cell 3 times using serum-free 1640 culture medium.Fluorescence intensity is stronger
It is higher to represent intracellular ROS level.
5. the measurement of antioxidant activity in animal body
12 monthly age aged mouse adaptable feds are randomly divided into 5 groups, every group 10: control group, that is, model after 7 days, by weight
Group, total polypeptide stomach-filling group, 5-10kDa polypeptide stomach-filling group, 3.5-5kDa polypeptide stomach-filling group, 0-3.5kDa polypeptide stomach-filling group.Daily
The given low of 50mg/kg, only, control group gives equivalent distilled water by stomach-filling volume 0.2mL/, and continuous gavage 5 weeks.During experiment,
Dosage is fed by changes of weight adjustment, all mouse do not limit feed and drinking-water.
After last stomach-filling, all mouse are deprived of food but not water 12h, take eyeball blood into anticoagulant tube under narcosis, room temperature is quiet
30min is set, in 4 DEG C, 10min is centrifuged under conditions of 3000rpm, the supernatant of acquisition is mice serum.It (is purchased from according to kit
Bioengineering Research Institute is built up in Nanjing) specification detects MDA content in animal blood serum, SOD and GSH-PX activity level respectively.
In vitro, cell and the anti-oxidant testing result of internal animal show: molecular weight is that 0-3.5KDa chlorella is anti-oxidant more
Peptide has optimal antioxidant activity, has external total reduction preferably than the chlorella enzymolysis product of other protease hydrolyzeds
Ability, by scheming (1) it can be seen that it has the polypeptide product of other molecular size ranges after digesting than same protein enzymatic hydrolyzate more
Good external removing DPPH and hydroxyl radicals, by scheming (2) it can be seen that it can be effectively reduced the ROS water in liver cancer cells
It is flat, it can more effectively inhibit Peroxidation Product MDA generation in animal body by scheming (3) it can be seen from it, and significantly improve GSH-
PX activity.
Embodiment 2
1. the preparation of chlorella cells wall enzymolysis liquid and specific protein enzymatic hydrolysis liquid
Cellulase, pectase and all kinds of protease used are purchased from Beijing Suo Laibao Science and Technology Ltd.
1) the chlorella cells wall enzymolysis liquid weight part ratio described in are as follows:
PH to 4.0 is adjusted with the concentrated hydrochloric acid that mass fraction is 36%;
2) specific protein described in digests liquid weight part ratio are as follows:
PH to 7.5~9.5 is adjusted with 1mol/L NaOH solution;
2. being prepared by the following steps:
1) it weighs 750g chlorella powder to be added in 4.25L 0.3mol/L NaOH dilute alkaline soln, under the conditions of 60 DEG C, impregnate
60min obtains pretreatment bead algae solution;
2) the pretreatment bead algae solution of the step 1) acquisition, high-pressure homogeneous 2 times under 55MPa pressure, under 5000rpm from
Heart 20min, the supernatant A of acquisition save under the conditions of being temporarily placed in 4 DEG C, and the sediment of acquisition is added to chlorella cells wall enzymolysis liquid
In, 5h is digested under the conditions of being 150rpm in 50 DEG C, speed of agitator, 20min is centrifuged after enzymatic hydrolysis under 5000rpm, obtains supernatant
B;
3) supernatant A of the step 2) acquisition and supernatant B mixing, carry out single-action energy conservation concentration in atmospheric conditions,
Until concentration volume ratio reaches 1:4, concentrate can obtain chlorella albumen powder through vacuum freeze drying;
4) the chlorella albumen powder that step 3) obtains is added in specific protein enzymatic hydrolysis liquid and is digested, digested under the conditions of 45 DEG C
Time 50min, the destroy the enzyme treatment 30min in 90 DEG C of water, is rapidly cooled to room temperature later, and 20min is centrifuged under 4000rpm, obtains
Supernatant;
5) supernatant described in step 4) is in the case where 1MPa enters film pressure, 40mL/min dialysis flow condition through retention molecule
Amount is respectively the ultrafiltration membrane of 10KDa, 5KDa, 3.5KDa, and it is respectively > 10KDa, 5- that ultra-filtration and separation, which obtains molecular size range range,
The chlorella enzymolysis polypeptide liquid of 10kDa, 3.5-5kDa and 0-3.5kDa collect the chlorella enzymolysis polypeptide in different molecular weight section
Liquid is chlorella polypeptide powder through vacuum freeze drying.In vitro, cell and the anti-oxidant detection method of internal animal and result are the same as real
Apply example 1.
Claims (8)
1. a kind of preparation method of chlorella antioxidation polypeptide, it is characterised in that:
1. being 1:7~10 by chlorella powder according to chlorella powder and NaOH solution mass ratio using chlorella pyrenoidosa powder as raw material
It is soaked in NaOH solution, obtains pretreatment bead algae solution;
2. pretreatment bead algae solution is centrifuged 20min high-pressure homogeneous 2~4 times under 30~60MPa pressure under 5000rpm, obtain
Supernatant A and save under the conditions of being temporarily placed in 4 DEG C;The sediment of acquisition is added to stirring bar in chlorella cells wall enzymolysis liquid
Chlorella cells wall enzymolysis liquid enzymatic hydrolysis is carried out under part, is centrifuged 20min under 5000rpm after enzymatic hydrolysis, is obtained supernatant B;
3. supernatant A and supernatant B are mixed, single-action energy conservation concentration is carried out in atmospheric conditions, until concentration front and back volume ratio
Reach 3~6:1, concentrate can obtain chlorella albumen powder through vacuum freeze drying;
It is digested 4. chlorella albumen powder is added in specific protein enzymatic hydrolysis liquid, 20min is centrifuged under 4000rpm, obtains supernatant C;
5. supernatant C is in the case where 1MPa enters film pressure, 40mL/min dialysis flow condition by ultrafiltration membrane, ultra-filtration and separation obtains bead
Algae aoxidizes polypeptide liquid living, obtains chlorella polypeptide powder after further vacuum freeze drying.
2. a kind of preparation method of chlorella antioxidation polypeptide according to claim 1, it is characterised in that the bead
Frustule wall enzymolysis liquid component proportion are as follows:
Chlorella cells wall enzymolysis liquid uses the concentrated hydrochloric acid that mass fraction is 36% to adjust pH to 3.0~5.0 after preparing.
3. a kind of preparation method of chlorella antioxidation polypeptide according to claim 1, it is characterised in that described is specific
Protein enzymatic hydrolyzate component proportion are as follows:
Specific protein enzymatic hydrolysis liquid, which matches to postpone, adjusts pH to 2.0~3.0 with the concentrated hydrochloric acid solution that mass fraction is 37%.
4. a kind of preparation method of chlorella antioxidation polypeptide according to claim 1, it is characterised in that the immersion,
The molar concentration of NaOH solution is that 0.2~0.3mol/L impregnates 60min under the conditions of the temperature of NaOH solution is 60 DEG C.
5. a kind of preparation method of chlorella antioxidation polypeptide according to claim 1, it is characterised in that the bead
Frustule wall enzymolysis liquid enzymatic hydrolysis, condition are as follows: 40~60 DEG C of hydrolysis temperature, 3~7h of enzymolysis time, speed of agitator 150rpm.
6. a kind of preparation method of chlorella antioxidation polypeptide according to claim 1, it is characterised in that described is specific
It is digested in protein enzymatic hydrolyzate, condition are as follows: 40~70 DEG C of hydrolysis temperature, 30~60min of enzymolysis time, in 90 DEG C of water after enzymatic hydrolysis
Middle destroy the enzyme treatment 30min, is rapidly cooled to room temperature.
7. according to a kind of preparation method of chlorella antioxidation polypeptide as described in claim 1, it is characterised in that described is super
Filter membrane, molecular cut off are respectively 10kDa, 5kDa, 3.5kDaa;Ultra-filtration and separation obtains point of chlorella antioxidation polypeptide liquid
Son amount magnitude range is respectively > 10kDa, 5~10kDa, 3.5~5kDa and 0~1kDa.
8. according to a kind of preparation method of chlorella antioxidation polypeptide as described in claim 1, it is characterised in that described is true
Vacuum freecing-dry, condition are as follows: freezing control -40 DEG C of set temperature, time 30min, retention time 120min;Primary drying setting
1st -20 DEG C of stage drying temperature, drying time 30min, retention time 120min, vacuum 0.25;2nd stage drying temperature -15
DEG C, drying time 30min, retention time 120min, vacuum 0.25;3rd -5 DEG C of stage drying temperature, is protected drying time 30min
Hold time 500min, vacuum 0.25;4th 0 DEG C of stage drying temperature, drying time 30min, retention time 1500min, vacuum
0.3;5th 5 DEG C of stage drying temperature, drying time 30min, retention time 120min, vacuum 0.3;6th stage drying temperature 10
DEG C, drying time 30min, retention time 120min, vacuum 0.3;When 20 DEG C of parsing-desiccation set temperature, time 30min, holding
Between 120min, vacuum 0.3.
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