CN111393518B - Ruditapes philippinarum antifreeze protein and extraction method and application thereof - Google Patents
Ruditapes philippinarum antifreeze protein and extraction method and application thereof Download PDFInfo
- Publication number
- CN111393518B CN111393518B CN202010280662.4A CN202010280662A CN111393518B CN 111393518 B CN111393518 B CN 111393518B CN 202010280662 A CN202010280662 A CN 202010280662A CN 111393518 B CN111393518 B CN 111393518B
- Authority
- CN
- China
- Prior art keywords
- ice
- ruditapes philippinarum
- round
- temperature
- bath
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 108010053481 Antifreeze Proteins Proteins 0.000 title claims abstract description 62
- 241000620877 Ruditapes philippinarum Species 0.000 title claims abstract description 47
- 238000000605 extraction Methods 0.000 title claims abstract description 16
- 238000000746 purification Methods 0.000 claims abstract description 39
- 238000000034 method Methods 0.000 claims abstract description 19
- 239000000287 crude extract Substances 0.000 claims abstract description 13
- 235000013372 meat Nutrition 0.000 claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 117
- 239000007788 liquid Substances 0.000 claims description 45
- 239000008367 deionised water Substances 0.000 claims description 20
- 229910021641 deionized water Inorganic materials 0.000 claims description 20
- 239000006228 supernatant Substances 0.000 claims description 13
- 239000000047 product Substances 0.000 claims description 12
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000007853 buffer solution Substances 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 238000000502 dialysis Methods 0.000 claims description 9
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 8
- 238000002844 melting Methods 0.000 claims description 8
- 230000008018 melting Effects 0.000 claims description 8
- FULZLIGZKMKICU-UHFFFAOYSA-N N-phenylthiourea Chemical compound NC(=S)NC1=CC=CC=C1 FULZLIGZKMKICU-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 6
- 239000012535 impurity Substances 0.000 claims description 6
- 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 claims description 5
- 238000005119 centrifugation Methods 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 4
- 239000007798 antifreeze agent Substances 0.000 claims description 3
- 239000002826 coolant Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 235000013373 food additive Nutrition 0.000 claims description 2
- 239000002778 food additive Substances 0.000 claims description 2
- 229910017053 inorganic salt Inorganic materials 0.000 claims description 2
- 238000002156 mixing Methods 0.000 claims description 2
- OAHKWDDSKCRNFE-UHFFFAOYSA-N phenylmethanesulfonyl chloride Chemical compound ClS(=O)(=O)CC1=CC=CC=C1 OAHKWDDSKCRNFE-UHFFFAOYSA-N 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims description 2
- 150000003839 salts Chemical class 0.000 claims description 2
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 12
- 230000000694 effects Effects 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 230000002528 anti-freeze Effects 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 235000020639 clam Nutrition 0.000 description 5
- 238000007710 freezing Methods 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 230000008014 freezing Effects 0.000 description 4
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000001179 sorption measurement Methods 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 241000237519 Bivalvia Species 0.000 description 3
- 241001052560 Thallis Species 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- WKVZMKDXJFCMMD-UVWUDEKDSA-L (5ar,8ar,9r)-5-[[(2r,4ar,6r,7r,8r,8as)-7,8-dihydroxy-2-methyl-4,4a,6,7,8,8a-hexahydropyrano[3,2-d][1,3]dioxin-6-yl]oxy]-9-(4-hydroxy-3,5-dimethoxyphenyl)-5a,6,8a,9-tetrahydro-5h-[2]benzofuro[6,5-f][1,3]benzodioxol-8-one;azanide;n,3-bis(2-chloroethyl)-2-ox Chemical compound [NH2-].[NH2-].Cl[Pt+2]Cl.ClCCNP1(=O)OCCCN1CCCl.COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3C(O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 WKVZMKDXJFCMMD-UVWUDEKDSA-L 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000013078 crystal Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 239000003223 protective agent Substances 0.000 description 2
- 235000015170 shellfish Nutrition 0.000 description 2
- 238000010257 thawing Methods 0.000 description 2
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 241000251468 Actinopterygii Species 0.000 description 1
- 235000019750 Crude protein Nutrition 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 235000013960 Lactobacillus bulgaricus Nutrition 0.000 description 1
- 241000186672 Lactobacillus delbrueckii subsp. bulgaricus Species 0.000 description 1
- 241000237852 Mollusca Species 0.000 description 1
- 241000194020 Streptococcus thermophilus Species 0.000 description 1
- 241000237551 Veneridae Species 0.000 description 1
- 238000003450 affinity purification method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000013375 chromatographic separation Methods 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000000136 cloud-point extraction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011491 glass wool Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 239000011810 insulating material Substances 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- 229940004208 lactobacillus bulgaricus Drugs 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000012474 protein marker Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000001953 recrystallisation Methods 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
-
- 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
- A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
- A23L29/03—Organic compounds
-
- 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
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Nutrition Science (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses Ruditapes philippinarum antifreeze protein and an extraction method and application thereof, belonging to the technical field of biology. The extraction method comprises the following steps: (1) culturing Ruditapes philippinarum at 4 deg.C, and collecting meat; (2) extracting crude extract of anti-freeze protein of Ruditapes philippinarum (3) preparing ice shell (4), purifying anti-freeze protein of Ruditapes philippinarum (5) and collecting anti-freeze protein. The method has the advantages of easy operation, simple purification process, short time consumption, high purity of the obtained product, contribution to actual production, capacity increase and cost reduction.
Description
Technical Field
The invention relates to Ruditapes philippinarum antifreeze protein and an extraction method and application thereof, belonging to the technical field of biology.
Background
Ruditapes philippinarum is an active stress process against low temperature stress. Antifreeze proteins (AFPs) are proteins that inhibit the growth and recrystallization of ice crystals, and they reduce the freezing point of aqueous solutions in a non-linear fashion with little effect on their melting point, and the difference between the melting point and the freezing point that results is known as thermal hysteresis activity, and thus are also known as thermal hysteresis proteins. Antifreeze proteins are distributed in plants, marine fishes, insects and microorganisms, and no report of extracting antifreeze proteins from Ruditapes philippinarum is found at present.
Ruditapes philippinarum (Ruditapes philippinarum) belongs to the phylum mollusca, class Bivalvia, order Veneridae. Is the buried shellfish which widely lives in the coastal areas of south and north China and has important economic value. Currently, about 90% of the total Ruditapes philippinarum production in about 200 million tons of the world is
From breeding and mainly from breeding in continents of China (Goulletquer, 1997). In the north of China, the clams are subjected to low temperature in winter, even frozen, the living environment is extremely poor, but the ruditapes philippinarum still normally overwinter, so that the clams embody strong cold resistance and are excellent economic shellfish which can adapt to the low temperature in the sea area in the north of China.
At present, the separation and purification of antifreeze protein mainly comprises the traditional chromatographic separation method, SDS-PAGE electrophoresis and cloud point extraction method. The traditional method has the defects of long separation period, complex process, low yield, unstable separation process, high cost, poor repeatability and the like.
Disclosure of Invention
According to the invention, the anti-freeze protein of Ruditapes philippinarum is extracted by adopting an ice specific adsorption separation method, the ice specific adsorption separation method utilizes the specific adsorbability of ice to the anti-freeze protein, the purification multiple of the anti-freeze protein can be obviously improved by one-step separation, the method is quicker and more efficient than the traditional chromatographic method, and the loss of anti-freeze activity can be avoided.
A method for extracting Ruditapes philippinarum antifreeze protein comprises the following steps:
(1) culturing Ruditapes philippinarum at 0-4 deg.C for at least one month, collecting Ruditapes philippinarum meat, washing with water, and storing;
(2) extracting the crude extract of anti-freeze protein of Ruditapes philippinarum: mixing the Ruditapes philippinarum meat with a buffer solution according to the proportion of 1: 2.8-3.2 (m/v), homogenizing, centrifuging the obtained homogenate, retaining a supernatant, adding water into the supernatant until the volume of the supernatant is the same as that of the homogenate before centrifugation, and storing for later use;
(3) preparing an ice shell: adding deionized water into a round-bottom flask, and rotating the round-bottom flask in a liquid nitrogen-absolute ethyl alcohol bath or an inorganic salt-water-ice mixed bath at a constant speed for 60-80 seconds to enable the deionized water to form ice shells with uniform thickness on the inner wall of the round-bottom flask, wherein the volume of the deionized water for forming the ice shells is 1/3-1/5 of the volume of the initial deionized water; pouring out the rest deionized water, putting the round-bottom flask into a liquid nitrogen-absolute ethyl alcohol bath again, and rotating at a constant speed for 20-40 s to completely freeze the ice shell in the round-bottom flask and form a crack;
(4) purifying the anti-freeze protein of Ruditapes philippinarum:
first purification: adding the crude extract obtained in the step (2) into the ice shell prepared in the step (3), rotating the ice shell in a low-temperature bath at the temperature of between-1 and-1.5 ℃ for 1 to 2 hours at the speed of 50 to 70rpm/min, pouring out the residual liquid when the volume of the residual liquid in the round-bottomed flask is half of the initial volume, flushing the surface of the ice shell with a buffer solution, and removing impurities on the surface of the ice shell;
and (3) second purification: melting the ice shell after the first purification at room temperature, adding a buffer solution and deionized water in a volume ratio of 1: 4-5, pouring the mixture into another ice shell, rotating the ice shell in a low-temperature bath at-0.5 to-2 ℃ at a speed of 50 to 70rpm/min for 1 to 2 hours, pouring out the residual liquid when the volume of the residual liquid in the round-bottomed flask is half of the initial volume, flushing the surface of the ice shell with the buffer solution, and removing impurities on the surface of the ice shell; (ii) a
And (3) third purification: repeating the step of the second purification once;
(5) collecting antifreeze protein: and (4) melting the product obtained after the third purification in the step (4) at room temperature, concentrating the product by using a dialysis bag until the volume of the product is 1/10 before dialysis, centrifuging the concentrated solution, and freeze-drying the supernatant to obtain white powder, namely the antifreeze protein.
Further, in the technical scheme, the Ruditapes philippinarum in the step (1) is selected to be consistent in specification, healthy and complete; the preservation temperature is-80-4 ℃.
Further, in the technical scheme, the temperature of the buffer solution is 0-4 ℃; the buffers consisted of 50mM Tris-HCl pH 8.0, 100mM NaCl, 1mM phenylthiourea, 1mM EDTA and 0.1mM phenylmethylsulfonyl chloride.
Further, in the technical scheme, the time for homogenizing in the step (2) is 30-60 s; centrifuging at 20000g at 0-4 ℃ for 30-40 min in the centrifuging condition in the step (2); and (3) preserving at the temperature of 0-4 ℃.
Further, in the technical scheme, the temperature of the deionized water in the step (3) is 0-4 ℃; the liquid nitrogen-anhydrous ethanol bath in the step (3) is as follows: putting a coverless container filled with absolute ethyl alcohol into a coverless container filled with liquid nitrogen, and uniformly rotating a round-bottom flask in the coverless container filled with absolute ethyl alcohol to form an ice shell; the coolant in the inorganic salt-water-ice mixed bath in the step (3) is as follows: inorganic salts: water: the ice ratio is 10:2:5(m/v/m), and the inorganic salt comprises calcium chloride.
Further, in the above technical scheme, the method for adding the crude extract obtained in step (2) into the ice shell prepared in step (3) in the first purification in step (4) comprises: inserting long-neck funnel into the center of round-bottom flask, pouring crude extractive solution or injecting the crude extractive solution with injector, and pouring or injecting the crude extractive solution into ice shell for 1-2 min.
Further, in the above technical scheme, a bath liquid is added to the low temperature bath in the step (4), and the bath liquid comprises absolute ethyl alcohol; the temperature of the deionized water in the step (4) is 0-4 ℃.
Further, in the technical scheme, the dialysis temperature in the step (5) is 0-4 ℃; the molecular weight cut-off of the dialysis bag in the step (5) is 14000 MwCO; the centrifugation in the step (5) is carried out for 0.8-1 h at 12000-15000 g at 0-4 ℃.
The invention also provides the Ruditapes philippinarum antifreeze protein extracted by the extraction method.
The invention also provides application of the Ruditapes philippinarum antifreeze protein in preparation of food additives and antifreeze agents.
Advantageous effects of the invention
The invention provides a method for extracting Ruditapes philippinarum antifreeze protein, which adopts a rotating round-bottom flask to prepare an ice shell, so that the ice shell grows in a large area from outside to inside (the outside refers to the side close to the bottle wall of the round-bottom flask, and the inside refers to the side close to the center of the round-bottom flask), and the rapid purification of the Ruditapes philippinarum antifreeze protein is realized. In the prior art, at least 1 day is needed for one-time purification of the antifreeze protein, the purification can be completed for 1-2 hours, the steps are simple, experimental material equipment is simple and easy to obtain, the method is more beneficial to actual production, the capacity is increased, and the cost is reduced. The method can obviously improve the concentration of the antifreeze protein through one-step purification step, is quicker and more efficient than the traditional method, and can also avoid the loss of antifreeze activity. According to the invention, the clam meat which is simple and easy to obtain, low in price and high in quality is used as the raw material for preparing the antifreeze protein, and a preparation method for simply and efficiently extracting the antifreeze protein from the clam meat is developed through a large number of experiments, so that the industrial production is easy to realize, and the clam meat antifreeze protein has a huge application prospect and market value. The purity of the Ruditapes philippinarum antifreeze protein extracted by the method is 81.92%.
Drawings
FIG. 1 is an ice shell prepared according to example 1 of the present invention.
FIG. 2 shows the liquid from the round bottom flask without ice crust formed during the second purification.
FIG. 3 shows the product of example 1 after the ice shell has been melted after the third purification.
FIG. 4 is a Coomassie blue stained SDS-PAGE image of Ruditapes philippinarum antifreeze protein prepared in example 1 of the present invention; wherein lane 1 is marker, lane 2 is crude extract of philippine antifreeze protein, lane 3 is liquid 1, lane 4 is ice 1, lane 5 is liquid 2, lane 6 is ice 2, lane 7 is liquid 3, and lane 8 is ice 3.
Detailed Description
The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.
Example 1
According to the traditional ice specific adsorption, a metal rod is used as an attachment base, ice crystals wrap the metal rod from inside to outside, and the attachment area is small. The method optimizes the ice specific adsorption attachment group, the traditional ice affinity purification method needs one day for adsorbing and purifying the antifreeze protein, and only 1-2 hours are needed for completing one-time purification at present. Traditionally, ice shells grow from inside to outside, and the invention enables ice shells to grow from outside to inside in a rotating round-bottom flask. The materials used in the method can be scaled up in equal proportion for practical production.
A method for extracting Ruditapes philippinarum antifreeze protein comprises the following steps:
(1) sample preparation: selecting Ruditapes philippinarum with consistent specification, and culturing in 4 deg.C seawater for one month to increase anti-freeze Protein (AFP) content. Taking the Ruditapes philippinarum meat, washing with water, and storing at-80-4 deg.C.
(2) Extracting the rough extract of Ruditapes philippinarum AFPs: 50g of Ruditapes philippinarum meat was homogenized with 150mL of buffer in a homogenizer for 30s in advance in a 4 ℃ precooling buffer (50mM Tris-HCl (pH 8.0), 100mM NaCl, 1mM phenylthiourea (phenylthiocarbamide), 1mM ethylenediaminetetraacetic acid (EDTA), 0.1mM phenylmethylsulfonyl fluoride (PMSF)). Centrifuging the homogenate at 20000 × g for 30min at 4 deg.C, skimming off lipids on the surface of the centrifuge tube, filtering with glass wool, transferring the supernatant from the centrifuge tube to a flask pre-cooled to 0 deg.C, and removing lipids remaining in the supernatant. The supernatant volume was then made up to 200mL with deionized water.
(3) Preparing an ice shell: ice crust preparation was done in a 1L round bottom flask and ice crust was prepared in a liquid nitrogen-absolute ethanol bath at-80 deg.C (placed in a non-lidded container with liquid nitrogen into which was placed a non-lidded container with absolute ethanol and the round bottom flask rotated in a non-lidded container with absolute ethanol to form an ice crust). When the ice shell is manufactured, the absolute ethyl alcohol can be placed into a refrigerator at the temperature of minus 80 ℃ for precooling so as to reduce the use amount of liquid nitrogen. If there is no liquid nitrogen, 100g of CaCl is contained2A frozen mixture of 20mL of water and 50g of ice will produce a water bath at about-55 c, in which case the ice shell may take longer to form).
200mL of low temperature deionized water (0-4 ℃) is added into a 1L round bottom flask, the flask is rotated in a bath with liquid nitrogen-anhydrous ethanol for 60-80 s, and the excess water is poured into a measuring cylinder for measurement to ensure that the volume of water forming the ice shell is between 30-50mL (for larger or smaller flasks, the time can be properly adjusted to control the volume of water forming the ice shell). After removing the water, the round bottom flask was put into a liquid nitrogen-anhydrous ethanol bath for 30-50s to completely freeze the ice shell in the flask and form a crack. Excess water was added to the round bottom flask because: if less water is initially added, the ice shells will be formed with a non-uniform thickness due to the lower temperature, and therefore an excess of water is required to form the ice shells with a uniform thickness.
(4) Purification of Ruditapes philippinarum antifreeze proteins (AFPs):
immersing half of a round-bottomed flask precooled to 0-4 ℃ in a low-temperature bath at the temperature of-1 ℃ to-1.5 ℃, wherein the bottle mouth of the round-bottomed flask is higher than the height of liquid in the low-temperature bath. The low-temperature bath is added with bath liquid which can be any nonvolatile fluid with the temperature of-1.5 ℃, and absolute ethyl alcohol is commonly used. The coolant does not need reagent grade materials, and is food grade or industrial grade, so that the cost can be saved.
First purification: precooling the crude extract obtained in the step (2) to 0-4 ℃, and adding 200mL of the crude extract obtained in the step (2) into the ice shell prepared in the step (3), wherein the ice shell is prevented from melting in the adding process. A long-neck funnel can be used to insert into the center of the flask and slowly pour the sample; a syringe connected to a tube sufficient to reach the bottom of the flask may also be used. The crude extract was introduced for a time of 1 to 2 minutes, thus reducing the risk of melting the ice crust, since the heat transfer from the flask is very limited. The flask was rotated at 60rpm/min for 1.5h in a cryogenic bath at-1.5 c, and the bath of the cryogenic bath and the round bottom flask were covered with an insulating material (e.g., bubble wrap) as the flask rotated to reduce temperature changes due to convection. Or the experimental setup was placed in a cold room (-2-1 ℃) to reduce the formation of condensation and significant temperature differences. The bath temperature and the extraction time can be adjusted according to the actual situation so as to achieve the purpose of remaining half of the liquid components. The liquid component was poured into a graduated cylinder and the volume of supernatant liquid entering the ice shell was calculated. After removing water, the ice shells were slowly washed with 0-4 ℃ buffer (0.5M Tris-HCl (pH 8.0), 1M NaCl, 10mM phenylthiocarbamide (10 mM ethylenediaminetetraacetic acid (EDTA)) to remove impurities from the surfaces of the ice shells.
And (3) second purification: after thawing the ice shells after the first purification, 10mL of buffer (0.5M Tris-HCl (pH 8.0), 1M NaCl, 10mM phenylthiocarbamide (10 mM ethylenediaminetetraacetic acid (EDTA)) and low temperature deionized water were added to make the volume 200 mL. Then poured into another ice shell for extraction, rotated in a circulating bath at-1.6 ℃ for 1h to reduce the liquid content in the flask to 100mL and then rinsed with low temperature deionized water.
And (3) third purification: the second purification step was repeated.
(5) Collecting antifreeze protein: after thawing the product obtained in step (4), 200mL of the liquid was concentrated to 20mL with a dialysis bag (3,000MWCO) at 4 ℃ and then the concentrate was centrifuged at 15000 × g for 1h at 4 ℃ to remove insoluble impurities. And (4) centrifuging to obtain supernatant, and freeze-drying to obtain white powder which is the antifreeze protein. The antifreeze protein can be freeze-dried and stored at-80 deg.C.
Example 2
SDS-PAGE electrophoresis experiment is carried out on the crude extract of the anti-freezing Philippine protein obtained in example 1 and the ice shell obtained by three times of purification.
Each lane was loaded with 10 μ L, the first lane was protein marker; the second lane is crude protein extract; the third lane (liquid 1) is a liquid in which no ice shell is formed in the round-bottomed flask during the first purification, the fourth lane (ice 1) is a liquid in which the ice shell after the first purification is melted, the fifth lane (liquid 2) is a liquid in which the ice shell is not formed in the round-bottomed flask during the second purification, the sixth lane (ice 2) is a liquid in which the ice shell after the second purification is melted, the seventh lane (liquid 3) is a liquid in which the ice shell is not formed in the round-bottomed flask during the third purification, and the eighth lane (ice 3) is a liquid in which the ice shell after the third purification is melted.
The experimental result is shown in figure 4, a large amount of impure protein is removed in the first purification, and the relatively pure Ruditapes philippinarum antifreeze protein can be obtained in the third purification. The molecular weight of the Ruditapes philippinarum antifreeze protein is 44.32 kDa. After 50g of Ruditapes philippinarum is purified, 0.625g of crude extract protein is obtained, wherein the antifreeze protein is 0.512g, the purity of the antifreeze protein is 81.92%, and the yield of the antifreeze protein is 1.02%.
Example 3
Respectively adding different antifreeze agents (namely antifreeze protein of a first extract, antifreeze protein of a second extract and antifreeze protein of a third extract, antifreeze product of sucrose and glycerol with the weight percentage of 5 percent respectively, and combination of the third extract, the sucrose and the glycerol with the weight percentage of 3-5 percent in the invention) into the centrifuged and concentrated lactobacillus thallus (Lactobacillus bulgaricus and Streptococcus thermophilus), placing the thallus in a freeze dryer at-40 ℃, freezing and drying the thallus in a vacuum degree of 0.01mbar for 48 hours after freezing bacterial liquid, and then rehydrating under proper conditions to determine the survival rate of the thallus.
TABLE 1 Effect of antifreeze experiment results
From the experimental results it can be seen that: the survival rate of thalli can be greatly improved by using the Ruditapes philippinarum antifreeze protein of the invention, and can be improved by 12 percent compared with the freeze-drying protective agent using sucrose and glycerol, about 40 percent compared with the freeze-drying protective agent without adding the antifreeze, and more than 20 percent compared with the first extract (which shows that if the product is not purified, the antifreeze effect of the product is obviously inferior to that of the product subjected to purification treatment); the survival rate of the thalli can reach more than 50 percent; if the Ruditapes philippinarum antifreeze protein, sucrose and glycerol are used together, the survival rate of thalli can reach more than 80%.
Claims (10)
1. A method for extracting Ruditapes philippinarum antifreeze protein is characterized by comprising the following steps:
(1) culturing Ruditapes philippinarum at 0-4 deg.C for at least one month, collecting Ruditapes philippinarum meat, washing with water, and storing;
(2) extracting the crude extract of anti-freeze protein of Ruditapes philippinarum: mixing the Ruditapes philippinarum meat with a buffer solution according to the proportion of 1: 2.8-3.2 (m/v), homogenizing, centrifuging the obtained homogenate, retaining a supernatant, adding water into the supernatant until the volume of the supernatant is the same as that of the homogenate before centrifugation, and storing for later use;
(3) preparing an ice shell: adding deionized water into a round-bottom flask, and rotating the round-bottom flask in a liquid nitrogen-absolute ethyl alcohol bath or an inorganic salt-water-ice mixed bath at a constant speed for 60-80 seconds to enable the deionized water to form ice shells with uniform thickness on the inner wall of the round-bottom flask, wherein the volume of the deionized water for forming the ice shells is 1/3-1/5 of the volume of the initial deionized water; pouring out the rest deionized water, putting the round-bottomed flask into a liquid nitrogen-absolute ethyl alcohol bath again, and rotating at a constant speed for 20-40 s to completely freeze ice shells in the round-bottomed flask and form cracks;
(4) purifying the anti-freeze protein of Ruditapes philippinarum:
first purification: adding the crude extract obtained in the step (2) into the ice shell prepared in the step (3), rotating the ice shell in a low-temperature bath at the temperature of between-1 and-1.5 ℃ for 1 to 2 hours at the speed of 50 to 70rpm/min, pouring out the residual liquid when the volume of the residual liquid in the round-bottomed flask is half of the initial volume, flushing the surface of the ice shell with a buffer solution, and removing impurities on the surface of the ice shell;
and (3) second purification: melting the ice shell after the first purification at room temperature, adding a buffer solution and deionized water in a volume ratio of 1: 4-5, pouring the mixture into another ice shell, rotating the ice shell in a low-temperature bath at-0.5 to-2 ℃ at a speed of 50 to 70rpm/min for 1 to 2 hours, pouring out the residual liquid when the volume of the residual liquid in the round-bottomed flask is half of the initial volume, flushing the surface of the ice shell with the buffer solution, and removing impurities on the surface of the ice shell;
and (3) third purification: repeating the step of purifying for the second time;
(5) collecting antifreeze protein: and (4) melting the product obtained after the third purification in the step (4) at room temperature, concentrating the product by using a dialysis bag until the volume of the product is 1/10 before dialysis, centrifuging the concentrated solution, and freeze-drying the supernatant to obtain the antifreeze protein.
2. The extraction method according to claim 1, wherein the Ruditapes philippinarum of step (1) is selected to be healthy and complete with consistent specification; the preservation temperature is-80-4 ℃.
3. The extraction method according to claim 1, wherein the buffer solution is at a temperature of 0-4 ℃; the buffers consisted of 50mM Tris-HCl pH 8.0, 100mM NaCl, 1mM phenylthiourea, 1mM EDTA and 0.1mM phenylmethylsulfonyl chloride.
4. The extraction method according to claim 1, wherein the time for homogenizing in step (2) is 30-60 s; centrifuging at 20000g at 0-4 ℃ for 30-40 min in the centrifuging condition in the step (2); and (3) preserving at the temperature of 0-4 ℃.
5. The extraction method according to claim 1, wherein the temperature of the deionized water in the step (3) is 0-4 ℃; the liquid nitrogen-anhydrous ethanol bath in the step (3) is as follows: putting a coverless container filled with absolute ethyl alcohol into a coverless container filled with liquid nitrogen, and uniformly rotating a round-bottom flask in the coverless container filled with absolute ethyl alcohol to form an ice shell; the coolant in the inorganic salt-water-ice mixed bath in the step (3) is as follows: inorganic salts: water: the ice ratio is 10:2:5(m/v/m), and the inorganic salt comprises calcium chloride.
6. The extraction method according to claim 1, wherein the step (4) of adding the crude extract obtained in the step (2) to the ice shells prepared in the step (3) in the first purification comprises: inserting long-neck funnel into the center of round-bottom flask, pouring crude extractive solution or injecting the crude extractive solution with injector, and pouring or injecting the crude extractive solution into ice shell for 1-2 min.
7. The extraction method according to claim 1, wherein a bath solution is added to the low temperature bath in step (4), and the bath solution comprises anhydrous ethanol; and (4) controlling the temperature of the deionized water in the step (4) to be 0-4 ℃.
8. The extraction method according to claim 1, wherein the dialysis temperature in the step (5) is 0-4 ℃; the molecular weight cut-off of the dialysis bag in the step (5) is 14000 MwCO; the centrifugation condition in the step (5) is 12000-15000 g centrifugation at 0-4 ℃ for 0.8-1 h.
9. Ruditapes philippinarum antifreeze protein extracted by the extraction method of any one of claims 1-8.
10. Use of Ruditapes philippinarum antifreeze protein according to claim 9 for the preparation of food additives and antifreeze agents.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010280662.4A CN111393518B (en) | 2020-04-10 | 2020-04-10 | Ruditapes philippinarum antifreeze protein and extraction method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010280662.4A CN111393518B (en) | 2020-04-10 | 2020-04-10 | Ruditapes philippinarum antifreeze protein and extraction method and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111393518A CN111393518A (en) | 2020-07-10 |
| CN111393518B true CN111393518B (en) | 2022-07-08 |
Family
ID=71429496
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010280662.4A Expired - Fee Related CN111393518B (en) | 2020-04-10 | 2020-04-10 | Ruditapes philippinarum antifreeze protein and extraction method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111393518B (en) |
-
2020
- 2020-04-10 CN CN202010280662.4A patent/CN111393518B/en not_active Expired - Fee Related
Non-Patent Citations (5)
| Title |
|---|
| Antifreeze proteins;Irena Roterman等;《Bioinformation》;20171231;第13卷(第12期);400-401 * |
| Ice-Shell Purification of Ice-Binding Proteins;Craig J Marshall 等;《Cryobiology》;20160326;第72卷(第3期);258-263 * |
| Purification of Antifreeze Protein from Wheat Bran (Triticum aestivum L.) Based on Its Hydrophilicity and Ice-binding Capacity;Chao Zhang等;《J. Agric. Food Chem.》;20070823;第55卷(第19期);7654-7658 * |
| The antifreeze protein type I (AFP I) increases seabream (Sparus aurata) embryos tolerance to low temperatures;V. Robles 等;《Theriogenology》;20070607;第68卷(第2期);284-289 * |
| 越冬期前后菲律宾蛤仔体成分变化研究;杨东敏等;《大连海洋大学学报》;20171231;第32卷(第06期);694-699 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111393518A (en) | 2020-07-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111393518B (en) | Ruditapes philippinarum antifreeze protein and extraction method and application thereof | |
| HRP20210525T1 (en) | Cryopreservation of juvenile stages of barnacles | |
| CN112843003A (en) | Lyophilized powder of latamoxef sodium for injection and its lyophilization process | |
| CN112841175A (en) | Human umbilical cord mesenchymal stem cell injection frozen stock solution with high proliferation capacity | |
| CN104255901B (en) | A kind of method for protecting swimming crab shell | |
| JPS6143356B2 (en) | ||
| CN102524510A (en) | Preparation method of low-fluorine Euphasia superb protein base stock | |
| CN104255900A (en) | Method for preventing shell crack during freezing process of portunid | |
| CN105875800B (en) | Frozen red shrimp quality improver and preparation method and application thereof | |
| CN106615024A (en) | Method for freeze drying of aquatic products and antifreezing agent | |
| CN110499276B (en) | Preparation method of Escherichia coli DH5 alpha high-efficiency competent cell | |
| CN108660045A (en) | A kind of coconut alcoholic drink mixed with fruit juice industrialized preparing process and gained coconut alcoholic drink mixed with fruit juice | |
| CN107811027A (en) | A kind of compound antifreeze of Quick-freezing meat balls containing ice structural protein | |
| CN106857790A (en) | A kind of method of the fluidisation ice preservation butterfish of bamboo vinegar liquid antistaling agent | |
| CN107502567B (en) | Photobacterium freeze-dried powder and preparation method thereof | |
| CN105918738A (en) | Method for raising nucleation temperatures of citrus juice freeze-concentrated crystals by utilizing synergistic effects of ice nucleation protein-ultrasonic waves | |
| CN105532641A (en) | Placenta preserving fluid and use method thereof | |
| CN107177572A (en) | A kind of lyophilized technique of Proteinase K | |
| CN108070022B (en) | Seaweed antifreeze polypeptide concentrated solution and preparation method thereof | |
| CN111713488B (en) | Cryopreservation agent, preparation method thereof and application thereof in coccidian oocysts | |
| KR102292656B1 (en) | Composition for cryopreservating semen containing Amerokana egg yolk extract as an active ingredient | |
| CN101473881A (en) | Technique for making chrysanthemum plum tea | |
| CN106332949A (en) | Frozen surimi antifreeze agent, and preparation method and application thereof | |
| CN113925082A (en) | Egg white storage method | |
| CN102268063A (en) | Method for separating active materials from marine animals by freezing and concentrating |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220708 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |