CN112898410B - Tachypleus tridentatus hemocyanin and preparation method and application thereof - Google Patents
Tachypleus tridentatus hemocyanin and preparation method and application thereof Download PDFInfo
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- 108060003552 hemocyanin Proteins 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 241000239224 Tachypleus tridentatus Species 0.000 title abstract description 13
- 241001529572 Chaceon affinis Species 0.000 claims abstract description 43
- 239000000243 solution Substances 0.000 claims abstract description 40
- 230000003078 antioxidant effect Effects 0.000 claims abstract description 25
- 238000005119 centrifugation Methods 0.000 claims abstract description 19
- 239000006228 supernatant Substances 0.000 claims abstract description 14
- 241000239218 Limulus Species 0.000 claims abstract description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 11
- 238000000502 dialysis Methods 0.000 claims abstract description 9
- 239000000287 crude extract Substances 0.000 claims abstract description 8
- 239000002244 precipitate Substances 0.000 claims abstract description 8
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000011259 mixed solution Substances 0.000 claims abstract description 3
- 239000013049 sediment Substances 0.000 claims abstract 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 14
- 239000002953 phosphate buffered saline Substances 0.000 claims description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 11
- 230000002000 scavenging effect Effects 0.000 claims description 9
- 239000003963 antioxidant agent Substances 0.000 claims description 7
- 230000003859 lipid peroxidation Effects 0.000 claims description 4
- 230000014759 maintenance of location Effects 0.000 claims 1
- 239000012460 protein solution Substances 0.000 claims 1
- 238000000034 method Methods 0.000 abstract description 13
- 239000002537 cosmetic Substances 0.000 abstract description 10
- 235000013305 food Nutrition 0.000 abstract description 8
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- 238000007254 oxidation reaction Methods 0.000 abstract description 4
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- 238000011068 loading method Methods 0.000 abstract description 2
- 238000002835 absorbance Methods 0.000 description 20
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- 108090000765 processed proteins & peptides Proteins 0.000 description 9
- -1 superoxide radicals Chemical class 0.000 description 8
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 8
- 239000000843 powder Substances 0.000 description 7
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 108090000623 proteins and genes Proteins 0.000 description 6
- WQGWDDDVZFFDIG-UHFFFAOYSA-N pyrogallol Chemical compound OC1=CC=CC(O)=C1O WQGWDDDVZFFDIG-UHFFFAOYSA-N 0.000 description 6
- 241000238421 Arthropoda Species 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 239000000413 hydrolysate Substances 0.000 description 5
- 150000003254 radicals Chemical class 0.000 description 5
- 102000009027 Albumins Human genes 0.000 description 4
- 108010088751 Albumins Proteins 0.000 description 4
- 241000209094 Oryza Species 0.000 description 4
- 235000007164 Oryza sativa Nutrition 0.000 description 4
- 239000000284 extract Substances 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 238000004321 preservation Methods 0.000 description 4
- 235000009566 rice Nutrition 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000000385 dialysis solution Substances 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
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- 229940079877 pyrogallol Drugs 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
- GLDQAMYCGOIJDV-UHFFFAOYSA-N 2,3-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC=CC(O)=C1O GLDQAMYCGOIJDV-UHFFFAOYSA-N 0.000 description 2
- WXTMDXOMEHJXQO-UHFFFAOYSA-N 2,5-dihydroxybenzoic acid Chemical compound OC(=O)C1=CC(O)=CC=C1O WXTMDXOMEHJXQO-UHFFFAOYSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 239000004365 Protease Substances 0.000 description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 238000010266 Sephadex chromatography Methods 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- ABBQHOQBGMUPJH-UHFFFAOYSA-M Sodium salicylate Chemical compound [Na+].OC1=CC=CC=C1C([O-])=O ABBQHOQBGMUPJH-UHFFFAOYSA-M 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000032683 aging Effects 0.000 description 2
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 2
- 235000011130 ammonium sulphate Nutrition 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000001641 gel filtration chromatography Methods 0.000 description 2
- 239000013067 intermediate product Substances 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 229940067606 lecithin Drugs 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 150000002978 peroxides Chemical class 0.000 description 2
- 230000002797 proteolythic effect Effects 0.000 description 2
- 229960004025 sodium salicylate Drugs 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 229940082044 2,3-dihydroxybenzoic acid Drugs 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 101800000068 Antioxidant peptide Proteins 0.000 description 1
- 108091005658 Basic proteases Proteins 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- 206010018612 Gonorrhoea Diseases 0.000 description 1
- 102000003820 Lipoxygenases Human genes 0.000 description 1
- 108090000128 Lipoxygenases Proteins 0.000 description 1
- 241000361919 Metaphire sieboldi Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 230000002292 Radical scavenging effect Effects 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000003064 anti-oxidating effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000004146 energy storage Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 229960002089 ferrous chloride Drugs 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000005452 food preservative Substances 0.000 description 1
- 235000019249 food preservative Nutrition 0.000 description 1
- 230000007760 free radical scavenging Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 208000001786 gonorrhea Diseases 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 210000000087 hemolymph Anatomy 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- NMCUIPGRVMDVDB-UHFFFAOYSA-L iron dichloride Chemical compound Cl[Fe]Cl NMCUIPGRVMDVDB-UHFFFAOYSA-L 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
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- 230000000241 respiratory effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 150000003873 salicylate salts Chemical class 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/795—Porphyrin- or corrin-ring-containing peptides
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/17—Amino acids, peptides or proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- Y—GENERAL 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
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Polymers & Plastics (AREA)
- Food Science & Technology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nutrition Science (AREA)
- Biochemistry (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Birds (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Gerontology & Geriatric Medicine (AREA)
- Dermatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Cosmetics (AREA)
- Compounds Of Unknown Constitution (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the field of biotechnology, in particular to horseshoe crab hemocyanin with antioxidant activity and a preparation method thereof. The preparation method comprises the following steps: (1) Discarding the sediment and remaining supernatant after low-temperature low-speed primary centrifugation of the horseshoe crab plasma; (2) Mixing the supernatant with saturated ammonium sulfate solution and standing for 10-24 h; (3) Collecting precipitate after low-temperature low-speed secondary centrifugation of the mixed solution, and re-suspending in PBS solution with pH of 7.0-7.6 to obtain limulus hemocyanin crude extract; (4) And (3) loading the crude extract into a dialysis bag, and dialyzing at 0-4 ℃ for 12-48 h. The method can obtain Tachypleus tridentatus hemocyanin with antioxidant activity. The method uses the residual horseshoe crab waste plasma of the horseshoe crab reagent as raw material, reduces the cost, has simple operation and saves time, and the obtained horseshoe crab hemocyanin has stronger oxidation resistance, can be used for medicines, cosmetics, foods, food storage and the like, and has good market prospect and industrial value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of horseshoe crab hemocyanin with antioxidant activity and freeze-dried powder thereof.
Background
Hemocyanin is a protein that is widely found in arthropod and mollusc blood gonorrhea and is known as the three major respiratory proteins in nature with hemoglobin, earthworm hemoglobin. However, with the intensive research, it has been found that hemocyanin functions are far from this, and in addition to oxygen transport, hemocyanin is involved in energy storage, animal molting, and immune function regulation in arthropods. The horseshoe crab is also called horseshoe crab, which is an arthropod living on the sea floor. As a member of arthropods, the blood of Tachypleus tridentatus contains a large amount of hemocyanin. The research proves that the hemocyanin has various catalytic effects, and under specific conditions, the hemocyanin can denature and express activities of phenol oxidase, catalase, lipoxygenase and the like.
Cosmetics are highly popular commodity, and the consumption of the cosmetics moves along with the trend of society, so that the cosmetics have vivid times features. With the improvement of life quality due to economic development, how to delay the aging process is the most popular topic. Nowadays, the age characteristics of population aging and the special industry background of cosmetics provide a wide space for the application of natural extracts with physiological activities such as free radical resistance, oxidation resistance and the like. With intensive research into free radical theory, natural extracts having antioxidant activity are increasingly being used in the pharmaceutical industry and in food storage.
CN104788541a discloses a preparation method of small peptide with antioxidant activity, wheat germ albumin is hydrolyzed by trypsin to obtain albumin hydrolysate i; separating the component with molecular weight less than 10kDa from the hydrolysate I by gel filtration chromatography, and taking the component with antioxidant activity as albumin extract II; carrying out enzymolysis on the extract II by using alkaline protease to obtain albumin hydrolysate III; separating the hydrolysate III by gel filtration chromatography and reversed phase high performance liquid chromatography in sequence to obtain small peptide with antioxidant activity.
CN102080118A discloses a preparation method of rice bran antioxidant active protein peptide, fresh rice bran is subjected to low-temperature degreasing treatment, rice bran protein is prepared by an alkaline method, and composite protease is used for preparing the rice bran antioxidant peptide.
The existing methods have complex preparation methods, and the finished products are not easy to store and transport.
The horseshoe crab is one of arthropods, and its haemolymph contains a large amount of hemocyanin. Because of the need for the preparation of a limulus reagent, tachypleus tridentatus is often taken and blood cells are used to prepare the limulus reagent, and the remaining plasma is discarded in its entirety. In fact, these discarded plasma still contain significant amounts of hemocyanin. These hemocyanins present in horseshoe crab plasma are all available.
It has been found that a hydrolyzed peptide having antioxidant activity is prepared from these residual plasma, CN106148465a discloses a horseshoe crab plasma proteolytic peptide having antioxidant activity, a preparation method and application thereof, heat-treating and denaturing horseshoe crab plasma in a constant temperature water bath at 70-100 ℃, and then suction-filtering and washing the horseshoe crab plasma with water; hydrolyzing the obtained horseshoe crab plasma with protease, and centrifuging the horseshoe crab plasma hydrolysate to obtain supernatant; the obtained supernatant is passed through Sephadex G-50 Sephadex gel column, and the component with maximum absorbance at wavelength of 230nm is collected to obtain the limulus plasma proteolytic peptide.
The method is characterized in that the raw material of the method is horseshoe crab plasma, and aims to obtain hydrolyzed peptide with antioxidant activity, and the steps of suction filtration, enzymolysis and the like are needed, and because the method of sephadex chromatography is adopted, the sephadex chromatography is complex in operation, long in time consumption in experiments, and extremely time-consuming in the steps of column loading, pH adjustment of a column bed and repeated balancing of a chromatographic column by using buffer solution are needed before the experiments; chromatographic columns are also prone to clogging during the experiment, take more time to process once clogged, and have narrow uses, mainly in foods and medicines. And the problems of inconvenient product preservation and transportation and the like exist.
Therefore, the prior art has the disadvantages of complex operation, long time consumption, narrow application of the prepared hydrolyzed peptide, and difficult preservation of the prepared hydrolyzed peptide.
Based on the invention, the hemocyanin and the freeze-dried powder with the antioxidant activity are developed, and the purpose of the invention is to efficiently and simply obtain the hemocyanin with the antioxidant activity so as to meet the market demand.
Disclosure of Invention
The invention aims to provide a horseshoe crab hemocyanin with antioxidant activity, freeze-dried powder and a preparation method thereof.
A preparation method of Tachypleus tridentatus hemocyanin comprises the following steps:
(1) Removing precipitate from Limulus plasma after low-temperature low-speed primary centrifugation to obtain supernatant;
(2) Mixing the supernatant obtained by primary centrifugation in the step (1) with a saturated ammonium sulfate solution according to the following ratio of 1: mixing in the volume ratio of 1.5-3 and setting for 10-24 hr;
(3) Collecting the precipitate after the mixed solution is placed in the step (2) and subjected to low-temperature low-speed secondary centrifugation, and re-suspending the precipitate in PBS (phosphate buffered saline) solution with the pH of 7.0-7.6 to obtain a limulus hemocyanin crude extract;
(4) Adding the limulus hemocyanin crude extract obtained in the step (3) into a dialysis bag, dialyzing for 12-48 h at 0-4 ℃ by using PBS solution with pH of 7.0-7.6, and taking a part with molecular weight of more than or equal to 3500 Da.
The primary centrifugation in the step (1) and the secondary centrifugation in the step (3) are carried out at the temperature of 0-4 ℃, the centrifugation speed of 6000-8000 rpm and the time of 0.5-2 h. More preferably, the temperature of the primary or secondary centrifugation is 4℃and the centrifugation speed is 8000rpm for 1 hour.
Preferably, the saturated ammonium sulfate solution of step (2) has ph=7.
Preferably, in the step (2), the volume ratio of the supernatant to the saturated ammonium sulfate solution is 1:2.
the preparation method of the saturated ammonium sulfate solution in the step (2) comprises the following steps: ammonium sulfate was dissolved in water at 55-65 ℃, cooled to room temperature, filtered and the filtrate pH was adjusted to 7.0 with ammonia.
In step (2), a saturated ammonium sulfate solution is slowly added dropwise to the supernatant.
In steps (3) and (4), the pH of the PBS solution is preferably 7.0.
In the steps (3) and (4), the concentration of the PBS solution is 0.005 to 0.1mol/L, more preferably 0.005 to 0.05mol/L. In a preferred embodiment of the invention, the PBS solution has a pH of 7.0 and a concentration of 0.01mol/L.
In the step (4), the dialysis time is 24 hours, and the dialysis solution is changed at the 2 nd, 8 th and 22 th of the dialysis.
Preferably, the horseshoe crab plasma in step (1) is horseshoe crab waste plasma from which horseshoe crab reagent residues are prepared.
And (3) freeze-drying the hemocyanin solution obtained in the step (4), and performing vacuum freeze-drying or spray-drying to obtain the limulus hemocyanin freeze-dried powder.
Preferably, the horseshoe crab hemocyanin solution obtained in the step (4) is freeze-dried by a vacuum freeze dryer and ground into powder by a low temperature grinder. More preferably, the freeze-drying temperature is-70 to-80 ℃ and the time is 48 to 96 hours. More preferably, the temperature of freeze-drying is-80℃for 72 hours.
A horseshoe crab hemocyanin is prepared by the above method.
The above Limulus blood blue protein has good antioxidant activity, and can be used for preparing antioxidant. The horseshoe crab hemocyanin can be used for preparing cosmetics, foods, food preservative or medicines, and is applied to the fields of functional foods, food storage and preservation, pharmacy and cosmetics.
A pharmaceutical or cosmetic with antioxidant activity contains the above Tachypleus tridentatus hemocyanin. Preferably, the above-mentioned Tachypleus tridentatus hemocyanin is used as an active ingredient.
The obtained Tachypleus tridentatus hemocyanin has strong oxidation resistance, and can effectively remove various free radicals and peroxides and inhibit the generation of free radicals and peroxides, such as superoxide scavenging or inhibiting superoxide generation of superoxide radicals, hydroxyl radicals, hydrogen peroxide and lipid.
The invention has the beneficial effects that:
(1) The horseshoe crab waste plasma remained in the preparation of horseshoe crab reagent is used as a raw material to obtain horseshoe crab hemocyanin with antioxidant activity, byproducts are fully utilized, and the cost is reduced;
(2) The extraction and preparation method is simple, and the time is saved; the method of ammonium sulfate fractional precipitation and dialysis is adopted, the whole operation is simple, convenient and quick, and the obtained horseshoe crab hemocyanin has good antioxidation effect;
the purified horseshoe crab hemocyanin is subjected to freeze drying and grinding into powder, so that the horseshoe crab hemocyanin is convenient to store and transport, and the quality problems such as protein property change and the like of the horseshoe crab hemocyanin in the subsequent use process are avoided;
(3) The obtained horseshoe crab hemocyanin product has strong antioxidant activity and wide application, can be widely used as an antioxidant in the antioxidant market, such as the fields of medicine, food storage and preservation, cosmetic preparation and the like, and has good market prospect and industrial value.
Detailed Description
The technical scheme of the invention is described below with reference to specific examples.
Example 1
A preparation method of Tachypleus tridentatus hemocyanin with antioxidant activity comprises the following steps:
(1) Taking out the residual horseshoe crab plasma from-80deg.C refrigerator, and thawing at-20deg.C and 4deg.C;
(2) After the plasma is completely thawed, 5mL of the plasma is taken out in a centrifuge tube, and the low-temperature and low-speed centrifugation is carried out under the conditions that the temperature is 4 ℃ and the rotating speed is 8000rpm/min, and the centrifugation time is 1h. After centrifugation, the pellet was discarded and the supernatant was retained for further use.
(3) Taking supernatant and saturated ammonium sulfate solution with pH of 7.0, and using a pipette to mix the saturated ammonium sulfate solution according to the following weight ratio of 2: slowly adding the solution into the supernatant in the step (2) dropwise in a proportion of 1; the obtained suspension was placed in a refrigerator at 4 ℃ overnight.
(4) Taking the suspension, and centrifuging at low temperature and low speed under the conditions of the temperature of 4 ℃ and the rotating speed of 8000rpm/min, wherein the centrifuging time is 1h. After centrifugation, the blue precipitate of the bottom part of the horseshoe crab hemocyanin can be seen, the supernatant is discarded, and the precipitate is resuspended in PBS solution with pH of 7.0, thus obtaining the horseshoe crab hemocyanin crude extract.
(5) The crude extract of horseshoe crab hemocyanin was added to the dialysis bag of MD3500, and the bag was placed in a refrigerator at 4℃for 24 hours, and the dialysis solution was changed at 2 hours, 8 hours, and 22 hours after dialysis, respectively. The dialysis solution was a PBS solution with pH=7.0, 0.01mol/L. Taking the part with molecular weight more than or equal to 3500 Da.
(6) Freezing and crystallizing the dialyzed limulus hemocyanin solution at-80deg.C for 72 hr, pulverizing into powder with a low temperature grinder, and preserving at 4deg.C.
EXAMPLE 2 measurement of antioxidant Capacity of Tachypleus tridentatus hemocyanin
(1) Determination of superoxide radical scavenging Capacity
Superoxide radicals are one of the most abundant oxygen radicals produced in living organisms. The pyrogallol undergoes oxidative decomposition in alkaline (pH 8.2) buffer solvent to produce a superoxide anion and a yellow intermediate product having a maximum absorption peak at a wavelength of 320 nm. When an antioxidant substance is added into the reaction system, the oxidation reaction of the pyrogallol is inhibited, the intermediate product is reduced, and the absorbance of the sample solution at 320nm is reduced. The specific steps for measuring the scavenging ability of the superoxide radical are as follows:
0.5mL of the Tachypleus tridentatus hemocyanin solution is taken in a sample tube, 2.3mL of 50mmol/L Tris-HCl buffer solution with pH of 8.2 is added, when 20 mu L of 10mmol/L pyrogallol is added, the solution is rapidly and uniformly mixed for reaction, the absorbance is measured at 320nm after 1min, and the absorbance value is recorded every 30s until the reaction is carried out for 6min. According to the formula [ A ] Blank space -(A Sample of -A Control )]/A Blank space X 100 calculated clearance, where a Blank space That is, absorbance of the group treated without hemocyanin, A Sample of That is, the absorbance of the treated group to which hemocyanin is added, A Control I.e. only the absorbance of hemocyanin is added.
The results show that the IC for eliminating superoxide radical by the horseshoe crab hemocyanin 50 The value was 0.42mg/mL.
(2) Determination of the Hydrogen peroxide scavenging Capacity
The ability to scavenge hydrogen peroxide was determined by the method referenced Gurcin, which comprises the following steps: 3.4mL of hemocyanin solution was taken in a sample tube, and 0.6mL of 40mmol/L H was added 2 O 2 A solution. Standing for 200min, zeroing with distilled water, and measuring the absorbance at 230 nm. According to the formula [ A ] Blank space -(A Sample of -A Control )]/A Blank space X 100 calculated clearance, where a Blank space That is, absorbance of the group treated without hemocyanin, A Sample of That is, the absorbance of the treated group to which hemocyanin is added, A Control I.e. only the absorbance of hemocyanin is added.
IC for removing hydrogen peroxide from horseshoe crab hemocyanin 50 The value was 0.89mg/mL.
(3) Determination of lipid peroxidation resistance
With reference to Zain, 200g of lecithin was dissolved in 200mL of 0.1mol/L phosphate buffer pH7.4 with shaking in an ice bath. 4mL of hemocyanin solution is taken in a sample tube, and 3.6mL of lecithin solution, 0.4mL of 10mmol/L ferrous dichloride solution and 0.4mL of 10mmol/L VC solution are added. The mixture was subjected to a constant temperature water bath at 37℃in the absence of light for 60min. Cooled to room temperature, 1mL of 0.8% TBA solution, 1mL of 20% TCA solution were added, respectively, and the mixture was boiled in water for 15min, cooled to room temperature and centrifuged for 10min, and absorbance was measured at 535 nm.
According to the formula [ A ] Blank space -(A Sample of -A Control )]/A Blank space X 100 calculated clearance, where a Blank space That is, absorbance of the group treated without hemocyanin, A Sample of That is, the absorbance of the treated group to which hemocyanin is added, A Control I.e. only the absorbance of hemocyanin is added.
IC of horseshoe crab hemocyanin with lipid peroxidation resisting capability 50 The value was 0.05mg/mL.
(4) Determination of the scavenging ability of hydroxyl radicals
Sodium salicylate free-forms salicylates in hydrogen peroxide solution which react with hydroxyl radicals to form the colored materials 2, 3-dihydroxybenzoic acid and 2, 5-dihydroxybenzoic acid. The coloured substance has a maximum absorption peak at 510 nm. The addition of a substance having a function of scavenging hydroxyl radicals to the reaction can reduce the absorbance of the reaction. The specific steps for measuring the free radical scavenging ability of the hydroxyl group are as follows:
0.2mL of hemocyanin solution is taken in a sample tube, 1mL of 2mmol/L FeSO is added 4 Solution, 0.4mL of 2mmol/L sodium salicylate solution and 1mL of 0.1% H 2 O 2 A solution. The absorbance was measured at 510nm after cooling in a constant temperature water bath at 37℃for 1h. According to the formula [ A ] Blank space -(A Sample of -A Control )]/A Blank space X 100 calculated clearance, where a Blank space That is, absorbance of the group treated without hemocyanin, A Sample of That is, the absorbance of the treated group to which hemocyanin is added, A Control I.e. only the absorbance of hemocyanin is added.
IC for scavenging hydroxy free radical of Tachypleus tridentatus hemocyanin 50 The value was 1.63mg/mL.
The horseshoe crab hemocyanin prepared by the method has good capability of scavenging free radicals and resisting lipid peroxidation, and can be used in the fields of medicine preparation, food storage, cosmetic preparation and the like.
Claims (4)
1. The application of the horseshoe crab hemocyanin in preparing the antioxidant with the functions of scavenging superoxide radicals, scavenging hydrogen peroxide and resisting lipid peroxidation is characterized in that the preparation method of the horseshoe crab hemocyanin comprises the following steps:
(1) Preparing horseshoe crab reagent residual horseshoe crab plasma, and discarding sediment retention supernatant after low-temperature low-speed primary centrifugation;
(2) Mixing the supernatant obtained by primary centrifugation in the step (1) with a saturated ammonium sulfate solution according to the following ratio of 1: mixing in the volume ratio of 1.5-3 and setting for 10-24 hr;
(3) Collecting the precipitate after the mixed solution is placed in the step (2) and subjected to low-temperature low-speed secondary centrifugation, and re-suspending the precipitate in PBS (phosphate buffered saline) solution with the pH of 7.0-7.6 to obtain a limulus hemocyanin crude extract; the concentration of the PBS solution is 0.005-0.1mol/L;
(4) Adding the limulus hemocyanin crude extract obtained in the step (3) into a dialysis bag, dialyzing for 12-48 h at 0-4 ℃ by using PBS solution with pH of 7.0-7.6, and taking a part with molecular weight more than or equal to 3500 Da; the concentration of the PBS solution is 0.005-0.1mol/L.
2. The use according to claim 1, wherein the primary centrifugation in step (1) and the secondary centrifugation in step (3) are carried out at a temperature of 0 to 4 ℃, at a centrifugation speed of 6000 to 8000rpm, for a period of 0.5 to 2 hours.
3. The use according to claim 1, wherein the concentration of the PBS solution is 0.01mol/L.
4. The use according to claim 1, wherein the protein solution after dialysis in step (4) is freeze-dried or spray-dried.
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