CN108191961A - The albuminised method and antioxidant that there is high scavenging capacity to superoxide anion are prepared from coconut cake - Google Patents
The albuminised method and antioxidant that there is high scavenging capacity to superoxide anion are prepared from coconut cake Download PDFInfo
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- CN108191961A CN108191961A CN201810060879.7A CN201810060879A CN108191961A CN 108191961 A CN108191961 A CN 108191961A CN 201810060879 A CN201810060879 A CN 201810060879A CN 108191961 A CN108191961 A CN 108191961A
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
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Abstract
The invention discloses being prepared from coconut cake to albuminised method and antioxidant of the superoxide anion with high scavenging capacity, belong to agricultural product and adopt post-processing field；Using degreasing coconut cake as raw material, coconut cake albumin is fully extracted using dilute salting liquid, secondly for albumin and the difference of globulin solubility, the globulin impurity in the partition method and secondary high speed centrifugation removal coconut cake albumin of being dialysed using film；Again, using the clearance rate to ultra-oxygen anion free radical as evaluation index, further purification and isolation is carried out to coconut cake albumin using chromatographic separation technology；Present invention sieve effectively removes that interference in coconut cake albumin is strong and the globulin of difficult removal, and obtained coconut cake albumin has a higher SOD activity, during a concentration of 100 μ g/mL, to O2 ‑Clearance rate up to 84.14% ~ 87.92%, can be widely applied in cosmetics, food industry, meanwhile, conducive to the intensive processing and level of comprehensive utilization for improving coconut cake.
Description
Technical field
The present invention relates to agricultural product to adopt post-processing field, and in particular to one kind is prepared from coconut cake has superoxide anion
The high albuminised method of scavenging capacity.
Background technology
Modern medicine study show human senility and many diseases generation and interior free yl it is unbalance closely related；
And in the food industry, the degeneration of food quality and it is putrid and deteriorated also with the generation of excessive free radicals in food and oxidationreduction
React unbalance related.Free radical is the as lively as a cricket atom of chemical property self-existent, with unpaired electron or atomic group.
The generation of free radical and disappear that there is dynamic balances in normal body.However, when being induced by certain factors（It is such as excessive
Psychological pressure, smoking, environmental pollution, microbial infection etc.）When, the oxidation-reduction reaction disequilibrium in body generated
The oxygen radical of amount.Excessive oxygen radical can further induced oxidation reaction, generate more free radicals, induction body kernel
Acid, lipid, protein and other react, and in turn result in cell oxidative damage, eventually lead to a series of diseases
Occur.In oxygen radical, ultra-oxygen anion free radical（O2 -·）Property it is very active and toxicity is very big.O2 -It is a variety of
The inducing agent of oxidation reaction, it can react, and reaction speed is exceedingly fast with molecule any in living cells, can generate more
Free radical；These free radicals can cause again etc. more than oxidation reaction, make to generate oxidation chain reaction into the cell；Meanwhile
O2 -Toxicity be also the main reason for oxygen poisoning occurs for body, nucleic acid chain rupture, polysaccharide solution are made by impaired performance caused by it
It is poly-, unrighted acid peroxidating, Mitochondria and protein carbonylation etc..Therefore, timely and effectively removing machine
Excessive O in vivo2 -, it is the effective way safeguarded body health, keep food quality.The O being widely used at present2 -It removes
Agent is mainly artificial synthesized class antioxidant, including BHT（Dibutyl hydroxy toluene）、BHA（Butylated hydroxy anisole）、TBHQ
（Tert-butyl hydroquinone）Deng.These antioxidants are to O2 -Scavenging activity it is all relatively strong, production cost is relatively low.It is but near
Over year the study found that BHT, BHA, TBHQ can cause animal's liver to expand, increase the activity of microsomal enzyme, there is certain poison
Side effect.After particularly Yi Dong of nineteen eighty-three Japan et al. reports the carcinogenicity of BHA, countries in the world are artificial to BHT, BHA etc.
The use of synthetized oxidation preventive agent in the food industry is strictly limited.At the same time, the development and utilization of natural
By increasingly extensive concern.Research shows that some natural components such as polyphenol, polysaccharide and reactive protein etc. in animals and plants,
Both there is higher O2 -Scavenging activity, and having no toxic side effect, in addition abundance, of low cost, therefore have exploitation for day
Right O2 -The potentiality of scavenger.Wherein, superoxide dismutase（Superoxide dismutase, SOD）It is to be widely present in
In animals and plants, it can effectively remove and remove O in vivo2 -Substance.
The most important effects of SOD are catalysis O2 -Occur disproportionated reaction, block intracellular oxidation chain reaction, avoid or
The oxidative damage of cell is reduced, so as to improve immunity of organisms.SOD belongs to metalloprotein, according to the difference of contained metal prothetic group,
It is divided into four kinds：Cu/Zn-SOD, Fe-SOD, Mn-SOD and Ni-SOD, wherein Cu/Zn-SOD, Mn-SOD are present in eukaryon life
In object, Fe-SOD is present in prokaryotes and only a few plant, and Ni-SOD is existed only in streptomyces and cyanobacteria.
SOD has been applied to as important antioxidant in medical product, cosmetics and health food.SOD is mostly from animal blood at present
Middle preparation, but there is it is of high cost, product purity is low, containing pyrexin and the defects of fishy smell.The content of SOD is very in plant
It is low, and extraction process is complicated, equipment requirement is high, and production cost is higher.Recent studies indicate that some protein are from structure
For and be not belonging to SOD, but they have SOD activity, effectively can remove or quench internal O2 -, confrontation is with blocking because of oxygen certainly
It is damaged, and repair damaged cell in time, restored caused by free radical to cell damage, this kind of egg caused by cell as base
White matter is known as SOD activated proteins or has the activated protein of Scavenging activity to superoxipe ion free radical.This proteinoid be compared with
Good natural, can as SOD substitute or effectively supplement and be added in cosmetics or functional food, application
Prospect is good, great market potential.
Coconut（Cocos nuciferaL.）Be the most important woody oleiferous plants crop in torrid areas and food energy crop it
One.Oil content about 60% in coconut meat, coconut oil are important industry and food oil.After coconut flesh squeezes the juice or extracts coconut oil
Main By product be coconut cake（coconut cake）.The yield of coconut cake is also very big, and 2016 are only Filipine yield just up to 300
Ten thousand tons.Coconut cake contains 16%~24% protein, research shows that coconut cake protein contains 18 kinds of amino acid, it is necessary to amino acid ligand ratio
Rationally, nutritive value is higher, while coconut protein also has reducing blood lipid isoreactivity, is high-quality and abundance vegetable protein
Matter resource.However, coconut cake is only used as the auxiliary material of the food such as animal feed or cake at present, protein isoreactivity ingredient does not have
It is exploited, causes the significant wastage of resource.Thus, how effectively protein isoreactivity ingredient in coconut cake to be developed
It utilizes, improves added value of product, be coconut secondary industry urgent problem to be solved.
The content of albumin and globulin is respectively 21% and 40.1% in coconut cake protein, is main protein.This subject
Group early period research shows that, coconut cake albuminised essential amino acid composition is reasonable, and external digestion absorptivity is 92.50%, biology effect
Valency is 58.63, and has the characteristics that low antigen, and human body is easy to digest and absorb, and nutritive value is very high.Meanwhile coconut cake is albuminised
Inoxidizability is preferable, has SOD activity, during a concentration of 100 μ g/mL, to O2 -Clearance rate for 47.82%, be significantly higher than same
The coconut cake globulin of concentration（11.99%）, coconut cake glutelin（7.65%）And BHT（24.13%）.Coconut cake albumin is mainly by 4 ~ 6
Small molecular protein（Subunit）Composition, molecular weight ranges are 18.9 ~ 51.9 kDa, by further detaching and after purification, can sieve
Select the higher albumin of SOD activity.Since the yield of coconut cake is huge, abundance, cheap, coconut cake has exploitation
Potentiality for high SOD reactive proteins.The present invention prepares tool using a set of relatively simple, efficient, economic method from coconut cake
The albumin for having high definition to remove superoxipe ion activity, not only improves comprehensive utilization and intensive processing of the realization to oil palm, and can be with
Turn waste into wealth, the strong development for promoting problem.
Invention content
The purpose of the present invention is be directed to the higher O of coconut cake albumin2 -Scavenging capacity, provide and prepared from coconut cake to super
Oxygen anion free radical has the albuminised method of high scavenging capacity.There is prepared coconut cake albumin higher SOD to live
Property, it can effectively remove internal O2 -, it is excellent natural, can be widely applied in cosmetics, food industry, profit
In the intensive processing and level of comprehensive utilization that improve coconut cake.
In order to achieve the above object, the present invention is achieved through the following technical solutions.
A kind of method to ultra-oxygen anion free radical from coconut cake with high scavenging capacity, using degreasing coconut cake as original
Material, coconut cake albumin is fully extracted using dilute salting liquid, saturating using film secondly for albumin and the difference of globulin solubility
Analyse the globulin impurity in partition method and secondary high speed centrifugation removal coconut cake albumin；Again, with to ultra-oxygen anion free radical
Clearance rate for evaluation index, further purification and isolation is carried out to coconut cake albumin using chromatographic separation technology, is filtered out pair
Ultra-oxygen anion free radical has the albumin of scavenging capacity, as natural.The film dialysis partition method uses
Molecular cut off is the dialysis membrane of 3.5 kDa, and dialyse 24-48 h.
Preferably, the dilute salting liquid is the chlorination of the sodium chloride solution or 0.2-0.4 mol/L of 0.2-0.6 mol/L
Calcium aqueous solution.
Preferably, the chromatographic separation technology is gel chromatography separation or cation chromatography isolation technics.
More preferably, coconut cake albumin sephadex g-100 chromatographic column or SP sephadex C-25 sun from
Further purifying is carried out on sub- exchange chromatography column with detaching.
Preferably, secondary high speed centrifugation is that dialyzate is centrifuged 20-40 min under 8000-12000 × g.
Coconut cake protein is mainly albumin and globulin, and the content of the two is respectively 21% and 40.1%.Wherein, albumin
It is a kind of water soluble protein, can be dissolved in water, dilute salt, diluted acid and dilute alkaline soln, relatively be easily soluble in 0.2 ~ 0.4 mol/L's
In weak brine solution；And coconut cake globulin is relatively easily soluble in the weak brine solution of 0.2 ~ 0.4 mol/L, but molten insoluble in pure water
Liquid.When extracting albumin matter using pure water solution, recovery rate is very low；And utilize dilute salting liquid extraction albumin when, recovery rate compared with
Height, but easily it is mixed with the foreign proteins such as globulin and glutelin.Therefore, one of key points and difficulties of this technology are clear in extraction coconut cake
During albumen, albuminised recovery rate should be improved, also to remove globulin as much as possible, ensures the purity of product.Meanwhile coconut palm
Bran albumin contains multiple component proteins（Subunit）Composition, the molecular size of each component albumen, electrification property and physicochemical property are each
It is different, to O2 -Scavenging activity it is also different.Therefore, the difficult point of this technology second is that ensure protein invariance condition
Under, degreasing coconut cake albumin is purified and detached using effective means, is filtered out to O2 -Scavenging activity it is highest
Component.The present invention fully extracts coconut cake albumin using dilute salting liquid, then for albumin and ball using degreasing coconut cake as raw material
The difference of albumen solubility, it is miscellaneous using the globulin in film dialysis partition method and secondary high-speed centrifugation technology removal coconut cake albumin
Matter.Then with to O2 -Clearance rate for evaluation index, using gel chromatography separation or cation chromatography isolation technics to coconut cake
Albumin carries out further purification and isolation, filters out to the highest albumin of superoxide anion scavenging capacity, as natural anti-
Oxidant.
The present invention has following row advantageous effect compared with prior art.
Partition method that the present invention is just carried by dilute salting liquid, film is dialysed and secondary high-speed centrifugation technology are removed in coconut cake albumin
Globulin impurity.Then with to O2Clearance rate for evaluation index, detached using gel chromatography separation or cation chromatography
Technology carries out further purification and isolation to coconut cake albumin, filters out to the clear egg of the highest coconut cake of superoxide anion scavenging capacity
In vain, effectively remove that interference in coconut cake albumin is strong and the globulin of difficult removal.
The coconut cake albumin that the present invention obtains is white powder, and quality is soft.SDS-PAGE experiments show the clear egg of the coconut cake
In vain rich in arginine and leucine, there is higher SOD activity, during a concentration of 100 μ g/mL, to O2 -Clearance rate reach
84.14% ~ 87.92%, it is significantly higher than common antioxidant BHT, present invention process flow is short, easy to operate, and nothing in the present invention
The use of organic solvent avoids albumen and aggregation or denaturation occurs, and activity is strong, while can reduce the pollution to environment and produce
Insecurity in journey can be widely applied in cosmetics, food industry, meanwhile, conducive to improve coconut cake intensive processing and
Level of comprehensive utilization.
Description of the drawings
Fig. 1 is separating spectrum of the coconut cake albumin in Sephadex G-100 gel chromatographic columns prepared by embodiment 1.
Fig. 2 is the coconut cake albumin molecule amount analytical electrophoresis figure prepared by embodiment 1.
Fig. 3 is separation figure of the coconut cake albumin in SP sephadex C-25 chromatographic columns prepared by embodiment 2
Spectrum.
Specific embodiment
In order to which technical problems, technical solutions and advantages to be solved are more clearly understood, tie below
Embodiment and attached drawing are closed, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used
To explain the present invention, it is not intended to limit the present invention.With reference to the embodiment technical solution that the present invention will be described in detail, but protect
Range is not limited by this.
Embodiment 1
1）Raw material pre-treatment：Dry 48 h, crushing, by mass volume ratio under 45 °C by coconut cake（m/v）The ratio of 1 ﹕ 10 adds in
Petroleum ether, stirs 2h, and filter residue is collected in filtering；Repeatedly after degreasing 3 times, by filter residue in 45 °C of lower 8 h of air heat, separating twice,
80 mesh sieve is crossed, obtains degreasing coconut cake powder.
2）Extract coconut cake albumin：1 ﹕ 15 is pressed in degreasing coconut cake powder（m/v）Ratio add in 0.2 mol/L chlorination
Sodium solution（pH 7.0）, under 4 °C stirring extraction 2h, filtering collect filtrate and filter residue respectively.0.2 mol/L again in filter residue
Sodium chloride solution（pH 7.0）, extract 3 times repeatedly, filtering, merging filtrate, 10000×g30 min of lower centrifugation, in collection
Clear liquid.
3）Film dialysis separation albumin and globulin：It is the saturating of 3.5 kDa that the supernatant of collection is packed into molecular cut off
It analyses in film, dialyse 48 h in 4 °C of distilled water, and first water is changed every 2 h, after stopping dialysis, dialyzate in collection membrane.
4）Secondary globulin and the albumin of being centrifuged at a high speed：By the dialyzate of above-mentioned collection 12000×gLower centrifugation 20
Min collects supernatant, vacuum freezedrying, you can obtain the higher coconut cake albumin powder of purity.
5）The purifying of gel chromatography is with detaching
Make above-mentioned gained coconut cake albumin in sephadex g-100 chromatographic column（Ф16 mm×100 cm）It is upper to carry out further
Purifying, separation and preparation.
Sephadex G-100 are soaked in 65 °C of distilled water first, 48h is swollen, first water is changed every 2h.So
The Sephadex G-100 being swollen are packed into chromatographic column afterwards（Ф16 mm×100 cm）In, using distilled water as mobile phase, balance
After 48h.Above-mentioned gained coconut cake albumin is redissolved in distilled water（A concentration of 1.0 mg/mL）, loading, progress gel chromatography point
From, using distilled water as mobile phase, monitored under flow velocity 0.8 mL/min, 280 nm, automatic collector collect, every 5 min collect 1
Pipe draws chromatography separating effect figure.The result shows that on this condition, coconut cake albumin can be separated into 4 main components,
Wherein the 4th component is to O2 -Clearance rate highest (87.92% ± 2.27%), collect the 4th component（F4）.
6）Freeze-drying：By obtained above to O2 -Albuminised 4th component of the highest coconut cake of clearance rate（F4）'s
Solution is collected, is merged, vacuum freeze drying, you can is obtained to O2 -The highest coconut cake albumin of scavenging capacity.
As shown in Figure 1, according to the degreasing coconut cake albumin prepared by embodiment 1 in Sephadex G-100 gel chromatographic columns
In separating effect F1, F2, F3 and F4 represent isolate first to the 4th component respectively.It is according to implementation shown in Fig. 2
The analysis of degreasing coconut cake albumin molecule amount prepared by example 1, wherein, Mw- molecular weight marker proteins, the clear egg of swimming lane 1- coconut cakes
In vain.
It is white experiments have shown that the highest coconut cake albumin to superoxipe ion scavenging capacity can be obtained using above-mentioned technique
Color powder, quality are soft.SDS-PAGE experiments show the albuminised molecular weight of the coconut cake as 18.9 kDa, rich in arginine and
Leucine, has a higher SOD activity, during a concentration of 100 μ g/mL, to O2 -Clearance rate for 87.92%, be significantly higher than often
Use antioxidant BHT.
Embodiment 2
1）Raw material pre-treatment：Dry 24 h, crushing, in the ratio of 1 ﹕ 15 under 55 °C by coconut cake（Mass volume ratio, m/v）It adds in
Petroleum ether, stirs 6 h, and filter residue is collected in filtering；Repeatedly after degreasing 2 times, by filter residue in 50 °C of lower 4 h of air heat, remove remaining
Organic solvent, separating twice, cross 80 mesh sieve, obtain degreasing coconut cake powder.
2）Extract coconut cake albumin：1 ﹕ 10 is pressed in degreasing coconut cake powder（m/v）Ratio add in 0.3 mol/L chlorination
Sodium solution（pH 7.0）, under 4 °C 4 h of stirring extraction, filtering collect filtrate and filter residue respectively.Add in 0.3 again in filter residue
The sodium chloride solution of mol/L（pH 7.0）, so extract repeatedly 3 times, collect filtrate and filter residue respectively.Merging filtrate, 12000×g20 min of lower centrifugation collect supernatant.
3）Film dialysis separation albumin and globulin：It is the saturating of 3.5 kDa that the supernatant of collection is packed into molecular cut off
It analyses in film, dialyse 36 h in 4 °C of distilled water, and first water is changed every 2 h, after stopping dialysis, dialyzate in collection membrane.
4）Secondary globulin and the albumin of being centrifuged at a high speed：By above-mentioned dialyzate 10000×g30 min of lower centrifugation,
Collect supernatant, vacuum freezedrying, you can obtain the higher coconut cake albumin powder of purity.
5）The purifying of cation-exchange chromatography is with detaching
Make above-mentioned gained coconut cake albumin in SP sephadex C-25 cation-exchange chromatography columns（Ф 26 mm×60
The cm of 35 mm of cm~Ф × 80）On be further purified with being detached.
First by SP sephadexs C-25（SP Sephadex C-25）It is soaked in 60 °C of distilled water, is swollen
48 h change first water every 4 h.Then the SP Sephadex C-25 being swollen are soaked in the acetic acid of 0.1 mol/L
Sodium buffer solution（PH5.2, containing 1 mol/L sodium chloride）In, after pre-equilibrating 48 h, it is packed into chromatographic column（Ф 26 mm×60 cm）
In.Above-mentioned gained coconut cake albumin is redissolved in 1 mol/L sodium chloride (a concentration of 1.0 mg/mL), loading, carry out cation
Exchange chromatography detaches, the elution of binary concentration gradient mobile phase.Wherein, mobile phase A is acetic acid-acetate buffer of 0.1 mol/L
Solution（pH5.2）, Mobile phase B is 1 mol/L sodium chloride solutions, and the concentration of Mobile phase B increases with the extension of elution time, is increased
Speed is 0.01 mol/L per minute.The flow velocity of eluent is 0.6 mL/min, is monitored under 280 nm, and automatic collector is collected, often
1 pipe is collected every 5 min, draws chromatography separating effect figure.The result shows that on this condition, coconut cake albumin can be separated into 5
Main component, wherein, the 4th component is to O2 -Scavenging capacity highest (84.14% ± 1.99%), collect the 4th group
Point.
6）Freeze-drying：By obtained above to O2 -The highest coconut cake Albumin Fraction of clearance rate（4th component）'s
Solution is collected, is merged, vacuum freeze drying, you can is obtained to O2 -The highest coconut cake albumin of scavenging capacity.
As shown in figure 3, it is in SP sephadex C-25 chromatographies according to the coconut cake albumin prepared by embodiment 2
Separating effect in column；F1, F2, F3, F4 and F5 represent isolate first to the 5th component respectively.
Experiments have shown that it can be obtained to O using above-mentioned technique2 -The highest coconut cake albumin of scavenging capacity, molecular weight are
18.9 kDa rich in arginine and leucine, have a higher SOD activity, during a concentration of 100 μ g/mL, to O2 -Removing
Rate is up to 84.14%, is significantly higher than common antioxidant BHT.
The above content is combine specific preferred embodiment to the further description of the invention done, it is impossible to assert
The specific embodiment of the present invention is only limitted to this, for those of ordinary skill in the art to which the present invention belongs, is not taking off
Under the premise of from the present invention, several simple deduction or replace can also be made, should all be considered as belonging to the present invention by being submitted
Claims determine scope of patent protection.
Claims (6)
1. the albuminised method that there is high scavenging capacity to superoxide anion is prepared from coconut cake, it is characterised in that：With degreasing
Coconut cake is raw material, and coconut cake albumin is fully extracted using dilute salting liquid, is secondly directed to the difference of albumin and globulin solubility,
Globulin impurity in the partition method and secondary high speed centrifugation removal coconut cake albumin of being dialysed using film；Again, with to super oxygen the moon from
The clearance rate of sub- free radical is evaluation index, and further purification and isolation is carried out to coconut cake albumin using chromatographic separation technology,
The albumin that there is scavenging capacity to ultra-oxygen anion free radical is filtered out, as natural；
The film dialysis partition method uses dialysis membrane of the molecular cut off for 3.5 kDa, dialysis 24-48 h.
2. according to claim 1 prepare the albuminised side for having high scavenging capacity to superoxide anion from coconut cake
Method, which is characterized in that the dilute salting liquid is the chlorination of the sodium chloride solution or 0.2-0.4 mol/L of 0.2-0.6 mol/L
Calcium aqueous solution.
3. according to claim 1 prepare the albuminised side for having high scavenging capacity to superoxide anion from coconut cake
Method, which is characterized in that the chromatographic separation technology is gel chromatography separation or cation chromatography isolation technics.
4. according to claim 3 prepare the albuminised side for having high scavenging capacity to superoxide anion from coconut cake
Method, which is characterized in that coconut cake albumin is in sephadex g-100 chromatographic column or SP sephadexs C-25 cations
Further purifying is carried out on exchange chromatography column with detaching.
5. according to claim 1 prepare the albuminised side for having high scavenging capacity to superoxide anion from coconut cake
Method, which is characterized in that secondary high speed centrifugation is that dialyzate is centrifuged 20-40 min under 8000-12000 × g.
6. the albuminised method system that there is high scavenging capacity to superoxide anion is prepared from coconut cake as described in claim 1
A kind of standby antioxidant.
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| CN108902437A (en) * | 2018-06-29 | 2018-11-30 | 山西师范大学 | The method and food emulsifying agent of the glutelin -1 of high emulsibility are extracted from the oil palm dregs of rice |
| EP3623012A1 (en) * | 2018-09-14 | 2020-03-18 | Societe Industrielle Limousine d'Application Biologique | Cosmetic active ingredient obtained from the albumen of the fruit of cocos nucifera and cosmetic uses |
| FR3085847A1 (en) * | 2018-09-14 | 2020-03-20 | Societe Industrielle Limousine D'application Biologique | COSMETIC ACTIVE INGREDIENT OBTAINED FROM CUCOS NUCIFERA FRUIT ALBUMEN AND COSMETIC USES |
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