CN109207544A - A kind of preparation method of chlorella antioxidation polypeptide - Google Patents

A kind of preparation method of chlorella antioxidation polypeptide Download PDF

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CN109207544A
CN109207544A CN201811308304.9A CN201811308304A CN109207544A CN 109207544 A CN109207544 A CN 109207544A CN 201811308304 A CN201811308304 A CN 201811308304A CN 109207544 A CN109207544 A CN 109207544A
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chlorella
enzymatic hydrolysis
vacuum
polypeptide
liquid
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CN109207544B (en
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林陈胜
张彦定
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Fujian Chenrun Biotech Co ltd
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Fujian Normal University
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    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/06Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K1/00General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14Extraction; Separation; Purification
    • C07K1/34Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
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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

A kind of preparation method of chlorella antioxidation polypeptide
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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CN112266944A (en) * 2020-10-31 2021-01-26 润科生物工程(福建)有限公司 Industrial production method for obtaining chlorella protein peptide by acid-enzyme method
CN112451651A (en) * 2020-11-30 2021-03-09 湖北瑞邦生物科技有限公司 Application of chlorella pyrenoidosa peptide
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CN112876548B (en) * 2021-02-05 2022-08-09 东莞理工学院 Chlorella pyrenoidosa antioxidant peptide and preparation method thereof
CN113278049A (en) * 2021-05-19 2021-08-20 国民康健(北京)自然科学研究有限公司 Selenium-rich flavor peptide separated from selenium-rich seaweed, preparation method and application
CN113278049B (en) * 2021-05-19 2022-04-29 国民康健(北京)自然科学研究有限公司 Selenium-rich flavor peptide separated from selenium-rich seaweed, preparation method and application

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