WO2012121172A1 - 植物種子由来の氷結晶化阻害物質 - Google Patents
植物種子由来の氷結晶化阻害物質 Download PDFInfo
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- WO2012121172A1 WO2012121172A1 PCT/JP2012/055460 JP2012055460W WO2012121172A1 WO 2012121172 A1 WO2012121172 A1 WO 2012121172A1 JP 2012055460 W JP2012055460 W JP 2012055460W WO 2012121172 A1 WO2012121172 A1 WO 2012121172A1
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- ice crystallization
- protein
- ice
- crystallization inhibitor
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G9/00—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor
- A23G9/32—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds
- A23G9/38—Frozen sweets, e.g. ice confectionery, ice-cream; Mixtures therefor characterised by the composition containing organic or inorganic compounds containing peptides or proteins
-
- 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
- A23L33/185—Vegetable proteins
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- 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
- A61K8/645—Proteins of vegetable origin; Derivatives or degradation products thereof
-
- 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
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/16—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from plants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
Definitions
- the present invention relates to an ice crystallization inhibitory substance derived from plant seeds, an antibody that specifically binds to the ice crystallization inhibitory substance, a composition containing the ice crystallization inhibitory substance, a food, a biological sample protective agent, and a cosmetic. And a peptide serving as an index of a protein having an ice crystallization inhibitory action.
- AFP ice crystallization inhibitory proteins
- AFPs derived from fish, insects, and microorganisms that have been found so far include those derived from sika deer fishes, those derived from insects such as the larvae of the beetle, and microorganisms such as the genus Flavobacterium These have high ice crystallization inhibitory activity (Patent Documents 1 to 3).
- plant-derived AFP for example, those derived from winter rye and carrot are known (Non-Patent Documents 1 and 2).
- AFP derived from fungi those derived from basidiomycetes such as Ishikarigamanotake and Antarctic enokitake (Flamulina velutipes KUAF-1) are known (Patent Documents 4 to 5).
- Patent Documents 6 to 7 Recently, attempts have been made to use the above properties industrially and use AFP to maintain the quality of frozen confectionery products such as ice cream and frozen foods.
- JP 2004-83546 A Special table 2002-507889 gazette JP 2004-161761 A JP 2004-24237 A JP 2004-275008 A International Publication No. 92/22581 Pamphlet International Publication No. 94/03617 Pamphlet
- AFP derived from fish it is difficult to completely remove odor from AFP derived from fish.
- insects and microorganisms are difficult to use as food materials, AFP derived from insects and microorganisms is not suitable for use in foods.
- the problem to be solved by the present invention is industrially useful, a novel ice crystal having excellent functions and properties, which can be produced easily, efficiently and stably by a safe method suitable for food production. It is to provide a chemical inhibitor.
- the present invention also provides an antibody that specifically binds to the ice crystallization inhibitor, a composition containing the ice crystallization inhibitor, a food, a biological sample protective agent and a cosmetic, and an ice crystallization inhibitory action.
- Another object of the present invention is to provide a peptide that serves as an index of the protein possessed.
- the present inventors have intensively studied to solve the above problems. As a result, it has been found that an ice crystallization inhibitor having excellent characteristics can be obtained from the seeds of the cowpea plant among the legumes, and the present invention has been completed.
- the ice crystallization inhibitor according to the present invention is characterized in that it comprises leguminous cowpea plants, related varieties of legume cowpea plants, or seed proteins derived from these improved varieties.
- red beans or mung beans are suitable. According to the experimental findings of the present inventors, some seed proteins obtained from the seeds of these plants have very excellent ice crystallization inhibitory activity.
- seed protein As seed protein, ripening protein, LEA protein (Late embryogenesis abundance protein), and each amino acid sequence thereof is SEQ ID NO: 1-2 (SEQ ID NO: 1-2), or 1 or Those having a sequence in which a plurality of amino acids are deleted, substituted or added and having a sequence serving as an index of a protein having an inhibitory effect on ice crystallization are preferred.
- the excellent ice crystallization inhibitory activity of these seed proteins has been demonstrated by the present inventors.
- the seed protein contained in the ice crystallization inhibitor according to the present invention may form a complex with other denatured proteins.
- Some seed proteins that have been found by the present inventors and exhibit an ice crystallization inhibitory action have structural characteristics that they form a complex with other denatured proteins.
- the antibody according to the present invention is characterized in that it specifically reacts with the ice crystallization inhibitor.
- composition, food, cryoprotectant and cosmetic according to the present invention are characterized by including the ice crystallization inhibitor according to the present invention.
- the peptide according to the present invention is a protein having an ice crystallization inhibitory action, which is the sequence of SEQ ID NO: 2 (SEQ ID NO: 2) or a sequence in which one or more amino acids of the sequence are deleted, substituted or added It contains the arrangement
- the ice crystallization inhibitory substance according to the present invention can be easily and efficiently produced from leguminous cowpea plants, related varieties of leguminous cowpea plants, or seeds of these improved varieties. Can be supplied automatically. In addition, since it is derived from plants without problems such as odors, it can be used with confidence in foods. Therefore, the ice crystallization inhibitor of the present invention can be used for maintaining the quality of frozen foods, for example. It can also be used effectively for protective agents in cosmetics such as organs, cells, blood itself and platelets for cryopreservation.
- FIG. 1 is an enlarged photograph of an ice crystal whose form is controlled by an ice crystallization inhibitor according to the present invention.
- FIG. 2 is an enlarged photograph of ice crystals whose form is controlled by the ice crystallization inhibitor according to the present invention.
- the ice crystallization inhibitor means a substance having a function of binding to the crystal plane of ice crystals and inhibiting the growth thereof. The bond also inhibits ice recrystallization by preventing further binding of free water to the ice crystal. That is, the ice crystallization inhibitor according to the present invention has an ice crystallization inhibitory action defined by any known method such as measurement of ice recrystallization inhibitory activity, measurement of thermal hysteresis, or observation of ice crystal structure. Means a substance.
- the ice crystallization inhibitory activity can be measured, for example, as follows. An ice crystallization inhibitor solution containing 30 w / v sucrose is cooled to ⁇ 40 ° C., then the temperature is raised to ⁇ 6 ° C., and the average area of ice crystals observed after 30 minutes is measured. Separately, as a control, a 30 w / v% aqueous solution of sucrose is subjected to the same treatment, and the average area of ice crystals is measured.
- the average area of the ice crystals can be analyzed, for example, by using a commercially available image analysis software such as an image factory made by Imsoft, Image Factory made by Imsoft, Adobe PHOTOSHOP ELEMENT8 made by Adobe, or Scion Image made by Scion. It can be measured by dividing the total area of ice crystals in the image by the number of ice crystals in the image.
- the ice crystallization inhibitory activity can be quantitatively evaluated using the numerical value obtained by dividing the measured average area of ice crystals by the average area of the control as an index. That is, the stronger the ice crystallization inhibitory activity of the ice crystallization inhibitor, the smaller the average area of the ice crystals that are formed. If inhibited, the above numerical value becomes smaller than 1, and it can be determined that the compound has ice crystallization inhibitory activity.
- Thermal hysteresis is the difference between the melting temperature of ice and the freezing temperature of water caused by the presence of an ice crystallization inhibitor, and the higher this value, the higher the activity of the ice crystallization inhibitor.
- the measurement of thermal hysteresis can be performed as follows, for example.
- phase contrast microscope capable of temperature control at a low temperature, holding a glass petri dish at ⁇ 20 ° C., placing 1 mL of the sample thereon, reducing the temperature to ⁇ 40 ° C. at a rate of 100 ° C./min, and ice Crystals are formed.
- the formed ice crystals are heated to ⁇ 5 ° C. at a rate of 100 ° C./minute, and then heated at a rate of 5 ° C./minute to form a single crystal by dissolving the ice crystals, and then 1 ° C./minute.
- the temperature is decreased at a rate of, and the time for the ice crystals made into single crystals to start growing is measured, and the thermal hysteresis can be calculated from the following equation.
- Thermal hysteresis [60-1] (° C / sec) x measurement time (sec) If the obtained value is 0.02 ° C. or higher, it can be determined that the compound has ice crystallization inhibitory activity.
- thermal hysteresis 0.025 degreeC or more is more preferable, and 0.03 degreeC or more is further more preferable.
- whether or not the substance is an ice crystallization inhibitor can also be determined by observing an ice crystal structure generated from water or an aqueous solution to which a test substance is added. That is, ice crystals obtained by cooling ordinary water have a flat disk shape, but the form of ice crystals formed upon addition of an ice crystallization inhibitor is, for example, hexagonal (flat hexagonal columnar) or Since it takes various forms depending on the crystal plane of the ice to which the substance binds, such as a bipyramid shape, when adding ice crystals to a form other than a flat disk shape, It can be judged that there is.
- the ice crystallization inhibitor according to the present invention comprises leguminous cowpea (Vigna) plants, related varieties of leguminous cowpea plants, or seed proteins derived from these improved varieties.
- Vigna leguminous cowpea
- red beans such as Sahoroshozu; white red beans such as Kita-hotaru and Hockaishiroshozu; mung beans (jaenari); cowpea and the like.
- red beans are preferable, and large-sized red beans are more preferable because they have abundant food experience, are easily available, and have excellent ice crystallization inhibitory activity and thermal hysteresis activity of the extract obtained per unit weight. preferable.
- a kind of leguminous cowpea genus plant refers to, for example, a plant that belongs to the same legume family and that does not belong to the cowpea genus, and that is close to the cowpea genus in the scientific classification.
- the specific varieties of the leguminous cowpea genus plants are varieties that are close in scientific classification to the plant.
- the “improved varieties of leguminous cowpea genus plants” refer to leguminous cowpea genus plants improved by artificial selection, crossing, mutation, genetic recombination and the like.
- the seed protein is not particularly limited as long as it is a seed protein extracted from the plant, and may be a fraction containing the protein.
- various ripening proteins generally defined as proteins that are synthesized and accumulated in the process of “ripening”, which means that plant seeds mature and enlarge, such as albumin, globulin, glutelin, prolamin And various seed storage proteins; stress proteins such as heat shock proteins; LEA protein (Late embryogenesis abundance protein) and the like.
- the LEA protein is particularly preferable.
- the ice crystallization inhibitor according to the present invention may contain only one seed protein or may contain two or more seed proteins.
- the seed protein may form a complex with other proteins in the ice crystallization inhibitor according to the present invention.
- LEA protein is known to interact with other denatured proteins.
- the denatured protein that forms a complex with the seed protein according to the present invention may be derived from a seed containing the seed protein, or may be derived from the seed.
- the said denatured protein may be only 1 type, and 2 or more types may be sufficient as it.
- LEA protein is a group of highly hydrophilic proteins that are specifically induced when water stress occurs in cells, and is synthesized and accumulated during the ripening process when plant seeds mature and enlarge.
- LEA protein may be accumulated not only in seeds but also in leaves, roots, pollen and the like.
- a protein existing in addition to the seed is also included in the scope of the invention.
- the molecular weight of the ice crystallization inhibitor according to the present invention is not particularly limited, for example, the average molecular weight measured by gel filtration chromatography is preferably 1 kDa or more and 200 kDa or less. If the molecular weight is 1 kDa or more, ice crystallization inhibitory activity is sufficiently exhibited. On the other hand, if the molecular weight is too large, the viscosity of the solution may become excessively high, and therefore the molecular weight is preferably 200 kDa or less. The molecular weight is more preferably 180 kDa or less, and further preferably 160 kDa or less.
- the ice crystallization inhibitor may be a complex, and the average molecular weight includes the average molecular weight of the monomer.
- various decomposition products of the ice crystallization inhibitor having the above molecular weight are also included in the scope of the present invention as long as the ice crystallization inhibitory activity and the like are exhibited.
- the amino acid sequence thereof is SEQ ID NO: 1 to 3 (SEQ ID NO: 1 to 3), or one or more amino acids of each sequence is deleted or substituted.
- SEQ ID NO: 1 to 3 amino acids of each sequence is deleted or substituted.
- those having an added sequence and a sequence serving as an index of a protein having an ice crystallization inhibitory action are preferable.
- the number of amino acids to be deleted, substituted or added is preferably 1 or more, 5 or less, more preferably 1 or more and 3 or less, and even more preferably 1 or 2.
- ice crystallization inhibitory substance “becomes an index of a protein having an ice crystallization inhibitory activity” means that a protein having a sequence having the above-described deletion or the like measures ice recrystallization inhibitory activity or thermal hysteresis. It means that it can be said to have an ice crystallization inhibitory action defined by any known method such as observation of ice crystal structure.
- Examples of the method for obtaining the ice crystallization inhibiting substance of the present invention include a method of extracting from seeds of the leguminous cowpea plant and the like. A detailed manufacturing method will be described below.
- the seeds of the leguminous cowpea genus plant subjected to extraction can further induce an ice crystallization inhibitor by acclimation at a low temperature in advance.
- seeds may be stored at 20 ° C. or lower for 3 days or longer.
- a sufficient amount of ice crystallization inhibitor can be obtained from the legume seeds without the low temperature acclimation treatment.
- a pulverized product As the form of the leguminous cowpea genus plant seed subjected to the extraction, a pulverized product, a crushed product, a ground product, or the like can be used. These pulverized materials may be obtained by pulverizing raw seeds, or by pulverizing dried seeds.
- Extraction efficiency can be increased by grinding seeds.
- Various general means can be used as a means for pulverizing plant seeds.
- physical crushing methods such as pressure fracture, mechanical grinding, ultrasonic treatment, and homogenizer can be used.
- Typical examples of pulverizing means include homogenizers such as Potter-Elvegem homogenizers, blenders such as Waring blenders, pulverizers such as dyno mills, French presses, mortars and pestles, rabies, freezing and crushing with liquid nitrogen, ultrasonic Means such as processing may be mentioned.
- fine pulverization it may be further finely ground or crushed with a mortar and pestle, a ball mill or a hammer mill.
- the seed pulverized material is suspended in the extraction medium, and the contents are extracted.
- the extraction medium In addition, when using a dry seed, in order to make extraction easy, it is preferable to immerse in an extraction medium previously.
- the extraction medium is preferably one having little modification of the target substance and low toxicity.
- Preferred extraction media include water; saline; buffers such as sodium acetate buffer, phosphate buffer, Tris-HCl buffer; hydrophilic organic solvents such as methanol, ethanol, and acetone; other organic solvents such as ethyl acetate; And a mixed solution thereof.
- a preferable extraction medium is water, saline, sodium acetate aqueous solution, or a mixture thereof appropriately.
- the composition and pH of the extraction medium can be selected as appropriate. In general, the pH of the extraction medium is preferably in the vicinity of neutrality of about 5 or more and 9 or less.
- the amount of the extraction medium relative to the seed can be selected as appropriate, and is generally set so that the entire seed is immersed in the extraction medium. For example, what is necessary is just to make the quantity of an extraction medium into 1 mL / g or more and about 5 mL / g or less with respect to the seed to be used.
- the seeds may be pulverized in the extraction medium using a mixer or the like after mixing the raw state or dried seed with the extraction medium.
- a mixer or the like after mixing the raw state or dried seed with the extraction medium.
- only immersion may be performed, vacuum extraction may be performed, or stirring may be performed during extraction.
- the extraction temperature may be adjusted as appropriate.
- the extraction temperature can be 0 ° C. or higher and 100 ° C. or lower, more preferably 2 ° C. or higher and 10 ° C. or lower.
- the temperature is 10 ° C. or lower, the activity of protease contained in seeds can be reduced, and protein degradation can be more reliably suppressed.
- the mixture containing seeds may be extracted while adding the extraction medium while adjusting the temperature to a predetermined temperature, or the temperature may be maintained by adding the extraction medium adjusted to a predetermined temperature in advance. It may be extracted. For example, after the seed is immersed in water or the like and held at a temperature of 2 ° C. or higher and 10 ° C. or lower for a sufficient time, the seed can be extracted while being replaced with an extraction medium and pulverized.
- the extraction time may be adjusted as appropriate, but it can usually be 5 minutes or more and 100 hours or less.
- the obtained extract can be separated by filtration, centrifugation, etc., and used as an ice crystallization inhibitor. Furthermore, the same extraction process may be repeated for the extraction residue, and the resulting extract may be collected and used as an ice crystallization inhibitor.
- An alkali treatment step of adding a base to the obtained extract may be performed.
- the alkali treatment step can increase the ice crystallization inhibitory activity of the protein.
- the conditions for the alkali treatment are not particularly limited and may be adjusted as appropriate.
- alkali metal hydroxides such as sodium hydroxide and potassium hydroxide can be used.
- the pH is preferably 10.0 or higher, more preferably 10.5 or higher.
- the upper limit of the pH is not particularly limited, but is preferably 13.0 or less, and more preferably 12.0 or less.
- the pH may be neutralized to about 6.0 or more and 8.0 or less using hydrochloric acid or the like.
- the ice crystallization inhibitor obtained as described above may be further purified as necessary.
- decantation, filtration, centrifugation, etc. are suitably combined to remove solids, and then, for example, salting out or precipitation with an organic solvent, affinity chromatography, ion exchange column chromatography, gel filtration, low-speed cooling device is used.
- Purification by binding to ice or the like, and concentration by dialysis or ultrafiltration may be suitably combined.
- the form of the ice crystallization inhibitor of the present invention varies depending on its use, and may be a solid, a solution, a concentrated liquid, a suspension, or the like. Furthermore, you may process into arbitrary forms, such as a powder form, a granular form, and a tablet, as needed.
- the processing method is not particularly limited.
- the above-described extract is pulverized according to a conventional method such as spray drying or freeze-drying, or the extract is adsorbed and supported on an excipient and solidified in a powder or granular form. And the like. These operations are known to those skilled in the art, and can be appropriately selected and used according to the application.
- the ice crystallization inhibitor according to the present invention can be used for the purpose of suppressing this failure in various fields in which failure occurs due to ice crystallization of water.
- it can be used in the food field, machine field, civil engineering field, cosmetics field, medical field using biomaterials, and the like.
- the form of the ice crystallization inhibitor of the present invention varies depending on its use, and may be a solution, a concentrated solution, a suspension, a lyophilized product, a powder, a granule, a tablet, or the like as it is. Moreover, it can also be set as the composition mixed with the excipient
- the antibody according to the present invention specifically reacts with the ice crystallization inhibitor, and tests for the presence or absence of the ice crystallization inhibitor in basidiomycetes or a culture solution thereof, or from a basidiomycete culture solution or the like. It can be used to identify polysaccharides having ice crystallization inhibitory activity.
- the antibody according to the present invention may be prepared according to a conventional method. For example, mice and rats are immunized with the ice crystallization inhibitor, and hybridomas are obtained by fusing antibody-producing cells, spleen cells and myeloma cells. The hybridoma is cloned, and a clone producing an antibody that specifically reacts with the ice crystallization inhibitor is screened. This clone may be cultured and the secreted monoclonal antibody may be purified.
- the taste of the food can be prevented from deteriorating.
- the quality of frozen foods and the like can be reduced by causing water in foods to crystallize into ice, physically pressing proteins and fat components, etc., and suppressing deterioration in taste and quality due to changes in the structure Can be improved.
- the cosmetics field it can be used as an additive to prevent deterioration of cosmetic quality.
- a cosmetic containing an oil / fat component when frozen, water contained in the cosmetic may crystallize in ice, and the oil / fat component may be physically pressed to break the structure, thereby deteriorating quality and feeling of use.
- the ice crystallization inhibitor according to the present invention is used, the structure of the oil and fat component is maintained by preventing ice crystallization of water, so that deterioration of quality and the like can be suppressed.
- the medical field it can be used as a protective agent when cryopreserving a biological sample.
- biological samples such as cells, blood, organs, biologically active proteins, biologically active peptides, biologically-derived low molecular weight compounds
- the water in the storage solution freezes and ice crystals
- the biological sample may be damaged by such ice crystals.
- the ice crystallization inhibitor according to the present invention is added, the generation and growth of ice crystals can be suppressed, so that the biological sample can be protected from damage due to ice crystals.
- the protein concentration in the following examples was measured by a bicinchoninic acid (BCA) method using a commercially available measurement kit (Thermo SCIENTIFIC, BCA Protein Assay).
- BCA bovine serum albumin
- Example 1 To a 1,000 mL beaker, commercially available large peanut red beans (100 g) were added, distilled water was added to the extent that the red beans were hidden, and the mixture was immersed at 4 ° C. for 48 hours. After removing distilled water, 5 mM Tris-HCl buffer (pH 8.0, 500 mL) was added, and the mixture was crushed at 4 ° C. using a mixer, and then the crushed solution was filtered with gauze and filtered at 4 ° C., 8,000. Centrifuged for 30 minutes at xg. The supernatant after centrifugation (460 mL) was recovered as a crude extract. The protein concentration of the obtained crude extract was measured by the BCA method and found to be 42 mg / mL.
- Tris-HCl buffer pH 8.0, 500 mL
- Example 2 To the crude extract (460 mL) obtained in Example 1, acetone cooled to ⁇ 30 ° C. is added little by little, and when acetone is added to 25 v / v%, insoluble matter (insoluble fraction 1) and acetone solution are added. Separated. For the insoluble fraction 1, ice crystallization inhibitory activity was not confirmed. Thereafter, acetone was added to the acetone solution until it reached 75 v / v%, and then the insoluble content (insoluble fraction 2) and the 75 v / v% acetone solution were separated. The obtained insoluble fraction 2 was dissolved in 5 mM Tris-HCl buffer (pH 8.0, 100 mL).
- the resulting solution was dialyzed with a dialysis membrane (fraction molecular weight: 14,000) and then replaced with the same buffer to obtain a solution (230 mL).
- the solution thus obtained was recovered as an acetone fraction, and its protein concentration was measured by the BCA method and found to be 8.3 mg / mL.
- Example 3 The solution (230 mL) obtained in Example 2 was charged on a DEAE column (manufactured by Tosoh Corporation, product name “DEAE TOYOPARRL 650M”) equilibrated with the same buffer, and a 0-0.5M NaCl gradient was applied. And eluted at a flow rate of 2.0 mL / min, and the non-adsorbed fraction was recovered as the active fraction.
- the obtained fraction was dialyzed with a dialysis membrane (fraction molecular weight: 14,000), and the solvent was replaced with 20 mM sodium acetate buffer (pH 5.0) to obtain a solution (485 mL).
- the protein concentration of the obtained DEAE column chromatography active fraction was measured by BCA method and found to be 1.8 mg / mL.
- Example 4 The solution (485 mL) obtained in Example 3 was charged to a cation exchange column (product name “CM TOYOPEARL 650M” manufactured by Tosoh Corporation) equilibrated with the same buffer, and a NaCl gradient of 0 to 0.5 M was applied. And eluted at a flow rate of 2.0 mL / min, and the adsorbed fraction was recovered as the active fraction.
- the obtained fraction was dialyzed and desalted with a dialysis membrane (fraction molecular weight: 14,000), and the solvent was replaced with 20 mM phosphate buffer (pH 6.0) to obtain a solution (132 mL).
- the protein concentration of the obtained active fraction (CM1 fraction) was measured by the BCA method and found to be 0.73 mg / mL.
- Example 5 The solution (132 mL) obtained in Example 4 was charged to a cation exchange column (product name “CM TOYOPEAL 650M” manufactured by Tosoh Corporation) equilibrated with the same buffer, and a NaCl gradient of 0 to 0.5 M was applied. The column was eluted at a flow rate of 2.0 mL / min, and the column non-adsorbed fraction (CM2 non-adsorbed fraction) and the column adsorbed fraction (CM2 adsorbed fraction) were collected. The column adsorption fraction was dialyzed and desalted with a dialysis membrane (fraction molecular weight: 14,000), and the solvent was replaced with 20 mM phosphate buffer (pH 6.0).
- CM TOYOPEAL 650M manufactured by Tosoh Corporation
- CM2 non-adsorbed fraction 126 mL
- CM2 adsorbed fraction 64 mL
- Example 6 The CM2 non-adsorbed fraction and CM2 adsorbed fraction obtained in Example 5 were each dissolved in 20 mM phosphate buffer.
- the protein concentration of each obtained aqueous solution 1000 ⁇ L was 5000 ⁇ g / mL and 5000 ⁇ g / mL, respectively.
- Each aqueous solution was charged as a sample into a gel filtration column (manufactured by GE Healthcare, product name “Sephacl S300 HR”), and 5 mM Tris-HCl buffer solution (pH 8.0, 0.15 M NaCl) at a temperature of 4 ° C.
- Test Example 1 Measurement of ice crystallization inhibitory activity The ice crystallization inhibitory activity of each of the solutions of Examples 1 to 6 was measured. Specifically, after diluting each solution obtained in Examples 1 to 6 to adjust to the protein concentration shown in Table 1, an equal amount of these solutions and a 60 w / v% sucrose aqueous solution were mixed to prepare a sample. A 30 w / v% sucrose solution was prepared. As an evaluation of the ice crystallization inhibitory activity, under a microscope having a stage with a cooling control function, the 30 w / v% sucrose solution of the sample was cooled to ⁇ 40 ° C. and then the temperature was raised to ⁇ 6 ° C.
- the average area of ice crystals observed when observed at -6 ° C for 30 minutes was measured.
- the average area of the ice crystal is analyzed using a commercially available image analysis processing software (Image Factory, Image Factory), and the total area of the ice crystal in the image is determined by analyzing the ice crystal microscopic image. It was measured by dividing by the number. As a control, the same measurement was performed on a 30 w / v% sucrose aqueous solution, and the average area of ice crystals was calculated.
- the numerical value (RI value) obtained by dividing this average area by the average ice crystal area of the 30 w / v% sucrose aqueous solution as a control is As an index, ice crystallization inhibitory activity was quantitatively evaluated. Furthermore, the protein concentration of the sample was measured by the BCA method, the specific activity per protein was determined by dividing the reciprocal of the RI value by the protein concentration, and the relative value with respect to the crude extract of Example 1 was calculated. The results are shown in Table 1.
- Test Example 2 Measurement of Thermal Hysteresis Activity
- CM2 non-adsorbed S300 fraction and CM2 adsorbed S300 fraction obtained in Example 6 was dissolved in water so that the protein concentration was 0.25 mg / mL, and thermal hysteresis activity was obtained.
- thermal hysteresis activity was obtained.
- a phase contrast microscope capable of temperature control at low temperature manufactured by Olympus, product name “L600A”
- a glass petri dish is kept at ⁇ 20 ° C.
- a 1 mL sample is placed on the glass petri dish
- the temperature was decreased to ⁇ 40 ° C. at a rate of minutes to form ice crystals.
- the generated ice crystals were heated to ⁇ 5 ° C.
- FIG. 1 shows an enlarged photograph of ice crystals whose form is controlled by the CM2 non-adsorbed S300 fraction
- FIG. 2 shows an enlarged photograph of ice crystals whose form is controlled by the CM2 adsorbed S300 fraction.
- Example 6 As shown in Table 2, it is clear that the purified protein obtained in Example 6 has excellent thermal hysteresis as an ice crystallization inhibitor.
- the ice crystals obtained by adding the purified protein obtained in Example 6 are all loose hexagons, and are flattened from normal water or aqueous solution. It is different from disk-shaped ice crystals. Such a morphological change indicates that the obtained purified protein is bound to a specific crystal plane of the ice crystal.
- the experimental results provide evidence that these purified proteins are ice crystallization inhibitors.
- the photograph in FIG. 1 is a hexagonal crystal viewed from the side.
- Test Example 3 Determination of Amino Acid Sequence of Purified Protein CM2 adsorbed S300 fraction (4.0 ⁇ g) obtained in Example 6 was dissolved in distilled water (5 ⁇ L) and subjected to protease treatment according to a conventional method. Subsequently, the sample buffer (product name “EzApply”, manufactured by ATTO, Inc.) (5 ⁇ L) was mixed at a solution ratio of 1: 1, and then heat treated at 99 ° C. for 3 minutes. The heat-treated sample was applied to a 10-20% polyacrylamide gel (manufactured by ATTO, product name “e-Pagel”), and subjected to SDS-PAGE at 20 mA for 85 minutes.
- sample buffer product name “EzApply”, manufactured by ATTO, Inc.
- the SDS-PAGE gel was transferred to a PVDF membrane (manufactured by Millipore, product name “Immobilon PSQ”) by a semi-dry method, and then CBB staining was performed.
- a plurality of bands mainly consisting of 52 kDa digested with protease were cut out, and amino acid sequences were determined by Edman method using a protein sequencer (manufactured by Shimadzu Corporation, product name “PPSQ-33A”). The obtained sequences are as shown in SEQ ID NOs: 1 to 3 (SEQ ID NO: 1 to 3).
- Example 7 In the same manner as in Example 1, a crude extract was prepared from commercially available mung beans, white azuki beans, and Tokachi red beans belonging to the leguminous cowpea genus. The protein concentration was adjusted to 2.0 mg / mL, and the ice crystallization inhibitory activity was measured in the same manner as in Test Example 1. The results are shown in Table 3.
- Example 8 The crude mung bean extract obtained in Example 7 was frozen at ⁇ 20 ° C. to remove the precipitate, centrifuged after thawing, and the supernatant was collected.
- the protein concentration of the obtained crude extract was measured by the BCA method, it was 15.5 mg / mL.
- Sodium hydroxide was added to the crude extract (200 mL) to adjust the pH to 11.0. Further, the pH was neutralized to 7.0 by adding hydrochloric acid, and then dialyzed with a dialysis membrane (fraction molecular weight: 14,000), and the solvent was replaced with water to obtain a solution (230 mL). .
- the protein concentration of the obtained solution was measured by the BCA method, it was 10.0 mg / mL.
- Example 9 The alkali-treated crude extract (100 mL) obtained in Example 8 was subjected to the ice-binding substance concentration operation (cold finger (CF) method) shown below. That is, a cylindrical cooling rod was inserted into the crude extract of Example 8 containing an ice crystallization inhibitor, and the surrounding solution was cooled at a low speed (from -0.5 ° C to -1.5 ° C in 16 hours). did. After 16 hours, ice adsorbed by the ice crystallization inhibitor formed around the cooling rod was collected. This operation was repeated twice to obtain a solution (50 mL) after CF treatment. When the protein concentration of the obtained solution was measured by the BCA method, it was 4.4 ⁇ g / mL. As a result of SDS-PAGE using this solution, a single band was confirmed at a position of about 48 kDa.
- CF cold finger
- Example 10 To the crude extract (230 mL) after alkali treatment obtained in Example 8, acetone cooled to ⁇ 30 ° C. was dropped little by little, and acetone was added to 25 v / v%. When insoluble (insoluble fraction 3) was added. ) And the acetone solution. Thereafter, acetone was added to the acetone solution until 50 v / v%, and then the insoluble matter (insoluble fraction 4) and the acetone solution were separated. Further, acetone was added to the acetone solution until 75 v / v%, and then the insoluble matter (insoluble fraction 5) and the acetone solution were separated.
- Insoluble fractions 3 to 5 were dissolved in 5 mM Tris-HCl buffer (pH 8.0), dialyzed with a dialysis membrane (fraction molecular weight: 14,000), and the solution was replaced with the same buffer. Insoluble fraction 3 and insoluble fraction 5 were not observed to have ice crystallization inhibitory activity, and only insoluble fraction 4 (30 mL) had strong ice crystallization inhibitory activity. Therefore, the insoluble fraction 4 was recovered as an acetone fraction, and the protein concentration measured by the BCA method was 3.0 mg / mL.
- Example 11 The acetone fraction (3.0 mg) obtained in Example 10 was charged onto a DEAE column (product name “DEAE TOYOPARRL 650M” manufactured by Tosoh Corporation) equilibrated with 5 mM Tris-HCl buffer (pH 8.0). And a gradient of 0 to 0.5 M NaCl was used to elute under the condition of a flow rate of 2.0 mL / min. The fraction of the adsorbed fraction whose peak was detected at an absorption wavelength of 215 nm was collected as the active fraction. The obtained fraction was desalted by dialysis with a dialysis membrane (fraction molecular weight: 14,000), and the solvent was replaced with deionized water. When the protein concentration of the obtained fraction was measured by the BCA method, it was 12 ⁇ g / mL (yield: 30 mL).
- Test Example 4 Measurement of Ice Crystallization Inhibitory Activity With respect to each solution of Examples 7 to 11 obtained by extraction from mung beans, ice crystallization inhibitory activity was measured in the same manner as in Test Example 1. From the protein concentration of each solution measured by the BCA method and the RI value that is an index of the ice crystallization inhibitory activity, the specific activity per protein was determined by the same formula as in Test Example 1, and the crude extract of Example 7 The relative value with respect to was calculated. Moreover, the total protein amount extracted from 100 g of mung beans was calculated from the solution amount and the protein concentration. The results are shown in Tables 4 and 5.
- the mung bean ice crystallization inhibitor As shown in Table 4, by the purification treatment of Examples 7 to 9, the mung bean ice crystallization inhibitor was purified to a specific activity of 1.3 times and a protein concentration of 1/3100. Further, as shown in Table 5, in the purification treatment of Examples 10 to 11, the mung bean ice crystallization inhibitor was purified to a specific activity of 5.9 times and a protein concentration of 11/3100.
- Example 8 increased the ice crystallization inhibitory action 1.3 times. This result indicates that the antifreezing activity is enhanced depending on the conditions under which denaturation occurs in the protein such as alkali treatment. Since this result is related to the characteristics of LEA protein that is known to suppress aggregation of denatured protein, Test Example 5 and Test Example 6 were performed below.
- Example 5 Protein Aggregation Inhibitory Effect Using Purified Fraction
- Commercially available lactate dehydrogenase (LDH) 15 ⁇ g was dissolved in water 1.0 mL. 9 ⁇ g of the mung bean purified protein after CF treatment obtained in Example 9 was dissolved in the solution.
- LDH lactate dehydrogenase
- each solution was frozen with liquid nitrogen and thawed at room temperature. The thawed solution was measured for absorbance at 340 nm. The results are shown in Table 6.
- the turbidity of the solution increased due to the denaturation and aggregation of the LDH protein by freezing and thawing.
- the increase in turbidity was remarkably suppressed in the solution to which the mung bean purified protein after CF treatment was added.
- This result shows that the mung bean purified protein obtained in Example 9 has a characteristic function of the LEA protein that suppresses protein aggregation, and this mung bean purified protein is free from protein denaturation caused by freeze-thawing. This indicates that it has a protective effect.
- Test Example 6 Increase in Ice Crystallization Inhibitory Activity of Mung Bean Crude Extract Associated with Aggregation Suppression
- the protein concentration of the mung bean crude extract obtained in Example 7 was adjusted to 60 mg / mL.
- 240 ⁇ L of this solution was dissolved 30 ⁇ g of the mung bean purified protein after CF treatment obtained in Example 9.
- each solution was frozen with liquid nitrogen and thawed at room temperature. After the thawed solution was centrifuged, the protein concentration was adjusted to 0.95 mg / mL.
- the ice crystallization inhibitory activity of the solution was measured in the same manner as in Test Example 1. The results are shown in Table 7.
- Example 9 By adding a very small amount of the CF-treated mung bean purified protein obtained in Example 9 and freeze-thawing, the ice crystallization inhibitory activity of the mung bean crude extract was remarkably increased. This increase in ice crystallization inhibitory activity does not occur only by adding CF-treated mung bean purified protein. Therefore, when freeze-thaw stress is applied, mung bean purified protein interacts with proteins in the crude extract. It is considered that the ice crystallization inhibitory activity was increased.
- Example 12 Using 4.5 mg of the DEAE fraction obtained in Example 11 as a sample, this was charged in a gel filtration column (manufactured by GE Healthcare, product name “Sephacryl S300 HR”), and 5 mM under a temperature condition of 4 ° C. A Tris-HCl buffer solution (pH 8.0, containing 0.15 M NaCl) was allowed to flow at a flow rate of 0.5 mL / min to elute the non-adsorbed fraction and detected at an absorption wavelength of 215 nm. Standard proteins with different molecular weights were eluted under the same conditions. By the above column chromatography, an absorption peak was confirmed at the same molecular weight (160,000 and 130,000) as that of Dainamon red beans purified in Example 6.
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Abstract
Description
得られた値が0.02℃以上であれば、氷結晶化阻害活性を有すると判断することができる。熱ヒステリシスとしては、0.025℃以上がより好ましく、0.03℃以上がさらに好ましい。
1,000mL容のビーカーに市販の大納言小豆(100g)を加え、小豆が隠れる程度の蒸留水を加え、4℃で48時間浸漬した。蒸留水を除いた後、5mM Tris-HCl緩衝液(pH8.0,500mL)を加え、ミキサーを用いて4℃で破砕処理した後、破砕液をガーゼで濾過して、4℃、8,000×gで30分間遠心分離した。遠心分離後の上清(460mL)を粗抽出液として回収した。得られた粗抽出液のタンパク質濃度をBCA法にて測定したところ、42mg/mLであった。
実施例1で得られた粗抽出液(460mL)に、-30℃に冷やしたアセトンを少量ずつ滴下し、25v/v%までアセトンを加えたところで、不溶分(不溶画分1)とアセトン溶液を分離した。不溶画分1については、氷結晶化阻害活性は確認されなかった。その後、上記アセトン溶液に75v/v%になるまでアセトンを加えた後、不溶分(不溶画分2)と75v/v%アセトン溶液とを分離した。得られた不溶画分2を5mM Tris-HCl緩衝液(pH8.0,100mL)に溶解した。得られた溶液を透析膜(分画分子量:14,000)で透析した後、同緩衝液で置換して溶液(230mL)を得た。このようにして得られた溶液をアセトン画分として回収し、そのタンパク質濃度をBCA法にて測定したところ、8.3mg/mLであった。
実施例2で得られた溶液(230mL)を、同緩衝液で平衡化したDEAEカラム(東ソー社製,製品名「DEAE TOYOPEARL 650M」)にチャージし、0~0.5MのNaCl勾配をつけて、流速2.0mL/minの条件で溶出し、非吸着画分を活性画分として回収した。得られた画分を透析膜(分画分子量:14,000)で透析し、溶媒を20mM 酢酸ナトリウム緩衝液(pH5.0)に置換して溶液(485mL)を得た。得られたDEAEカラムクロマトグラフィ活性画分のタンパク質濃度をBCA法にて測定したところ、1.8mg/mLであった。
実施例3で得られた溶液(485mL)を、同緩衝液で平衡化した陽イオン交換カラム(東ソー社製,製品名「CM TOYOPEARL 650M」)にチャージし、0~0.5MのNaCl勾配をつけて、流速2.0mL/minの条件で溶出し、吸着画分を活性画分として回収した。得られた画分を透析膜(分画分子量:14,000)で透析・脱塩し、溶媒を20mM リン酸緩衝液(pH6.0)に置換して溶液(132mL)を得た。得られた活性画分(CM1画分)のタンパク質濃度をBCA法にて測定したところ、0.73mg/mLであった。
実施例4で得られた溶液(132mL)を、同緩衝液で平衡化した陽イオン交換カラム(東ソー社製,製品名「CM TOYOPEAL 650M」)にチャージし、0~0.5MのNaCl勾配をつけて、流速2.0mL/minの条件で溶出し、カラム非吸着画分(CM2非吸着画分)とカラム吸着画分(CM2吸着画分)をそれぞれ回収した。カラム吸着画分は透析膜(分画分子量:14,000)で透析・脱塩し、溶媒を20mM リン酸緩衝液(pH6.0)に置換した。得られたCM2非吸着画分(126mL)とCM2吸着画分(64mL)のタンパク質濃度をBCA法にて測定したところ、それぞれ0.22mg/mL、0.29mg/mLであった。
実施例5で得られたCM2非吸着画分とCM2吸着画分をそれぞれ20mM リン酸緩衝液に溶解した。得られた各水溶液(1000μL)のタンパク質濃度は、それぞれ5000μg/mLと5000μg/mLであった。各水溶液を試料としてゲル濾過カラム(GEヘルスケア社製,製品名「Sephacrl S300 HR」)にチャージし、4℃の温度条件下で、5mM Tris-HCl緩衝液(pH8.0,0.15M NaClを含む)を流速0.5mL/minの条件で流し、非吸着画分を溶出させ、吸収波長215nmにて検出した。また分子量の異なる標準タンパク質を同条件にて溶出させた。
実施例1~6の各溶液について、氷結晶化阻害活性を測定した。具体的には、実施例1~6で得られた各溶液を希釈して表1に記載のタンパク質濃度に調整した後、これら溶液と60w/v%ショ糖水溶液を等量混合して、試料の30w/v%ショ糖溶液を調製した。氷結晶化阻害活性の評価として、冷却調節機能が付いたステージを有する顕微鏡下、試料の30w/v%ショ糖溶液を-40℃に冷却した後に-6℃まで温度を上げて氷結晶を溶かし、-6℃を保った状態で30分間観察したときに認められる氷結晶の平均面積を測定した。なお、氷結晶の平均面積は、氷結晶の顕微鏡画像を市販の画像解析処理ソフト(Imsoft社製,Image Factory)を用いて解析し、画像中の氷結晶の総面積を画像中の氷結晶の個数で除することにより測定した。また、対照として、30w/v%ショ糖水溶液について同様に測定し、氷結晶の平均面積を算出した。
実施例6で得られたCM2非吸着S300画分とCM2吸着S300画分それぞれを、タンパク質濃度が0.25mg/mLとなるように水に溶解し、熱ヒステリシス活性を次のようにして測定した。即ち、低温での温度制御が可能な位相差顕微鏡(Olympus社製,製品名「L600A」)を使用し、ガラスシャーレを-20℃に保持し、その上に1mLの試料をおき、100℃/分の速度で-40℃まで温度を低下させて氷結晶を形成させた。生成した氷結晶を100℃/分の速度で-5℃まで加温し、そこから5℃/分の速度で加温して氷結晶を溶解させ、単結晶にした。次に、1℃/分の速度で温度を低下させ、単結晶にした氷結晶が成長し始める時間を測定し、以下の式から熱ヒステリシスを算出した。
この数値が高い程、氷結晶化阻害物質の活性が高いことを意味している。結果を表2に示す。また、CM2非吸着S300画分により形態が制御された氷結晶の拡大写真を図1に、CM2吸着S300画分により形態が制御された氷結晶の拡大写真を図2に示す。
実施例6で得られたCM2吸着S300画分(4.0μg)を蒸留水(5μL)に溶解し、常法に従ってプロテアーゼ処理を行った。次いで、サンプルバッファー(ATTO社製,製品名「EzApply」)(5μL)と溶液比1:1で混合した後、99℃で3分間熱処理した。熱処理後の試料を10~20%ポリアクリルアミドゲル(ATTO社製,製品名「e‐Pagel」)にアプライし、20mAで85分間SDS-PAGEを行った。セミドライ法にてSDS-PAGE後のゲルをPVDF膜(ミリポア社製,製品名「イモビロンPSQ」)に転写した後、CBB染色を行った。プロテアーゼにより消化された52kDaをメインとする複数のバンドを切り出し、プロテインシーケンサー(島津製作所社製,製品名「PPSQ-33A」)を用いて、エドマン法によりアミノ酸配列を決定した。得られた配列は、配列番号1~3(SEQ ID NO:1~3)に示すとおりである。
実施例1と同様にして、マメ科ササゲ属に属する市販の緑豆、白小豆および十勝産小豆より粗抽出液を調製した。タンパク質濃度を2.0mg/mLとなるように濃度調整し、試験例1の方法と同様にして氷結晶化阻害活性を測定した。その結果を表3に示す。
実施例7で得られた緑豆の粗抽出液を、沈殿を除去するため-20℃で凍結し、解凍後に遠心分離し、上清を回収した。得られた粗抽出液のタンパク質濃度をBCA法にて測定したところ、15.5mg/mLであった。当該粗抽出液(200mL)に水酸化ナトリウムを加え、そのpHを11.0に調整した。さらに、塩酸を加えることによりそのpHを7.0に中和した後、透析膜(分画分子量:14,000)にて透析し、溶媒を水に置換して、溶液(230mL)を得た。得られた溶液のタンパク質濃度をBCA法にて測定したところ、10.0mg/mLであった。
実施例8で得られたアルカリ処理後の粗抽出液(100mL)を、以下に示す氷結合物質濃縮操作(コールドフィンガー(CF)法))に供した。即ち、氷結晶化阻害物質を含んだ実施例8の粗抽出液にシリンダー状の冷却棒を挿入し、周囲の溶液を低速(16時間で-0.5℃から-1.5℃)で冷却した。16時間後、冷却棒の周囲に生成した氷結晶化阻害物質が吸着した氷を回収した。この操作を2回繰り返し、CF処理後の溶液(50mL)を得た。得られた溶液のタンパク質濃度をBCA法にて測定したところ、4.4μg/mLであった。この溶液を用いてSDS-PAGEを行った結果、約48kDaの位置に単一のバンドが確認された。
実施例8で得られたアルカリ処理後の粗抽出液(230mL)に、-30℃に冷やしたアセトンを少量ずつ滴下し、25v/v%までアセトンを加えたところで、不溶分(不溶画分3)とアセトン溶液を分離した。その後、上記アセトン溶液に50v/v%になるまでアセトンを加えた後、不溶分(不溶画分4)とアセトン溶液を分離した。さらに上記アセトン溶液に75v/v%になるまでアセトンを加えた後、不溶分(不溶画分5)とアセトン溶液を分離した。不溶画分3~5を5mM Tris-HCl緩衝液(pH8.0)に溶解し、透析膜(分画分子量:14,000)で透析した後、溶液を同緩衝液で置換した。不溶画分3と不溶画分5については氷結晶化阻害活性が認められず、不溶画分4(30mL)にのみ、強い氷結晶化阻害活性を認めた。よって、不溶画分4をアセトン画分として回収し、そのタンパク質濃度をBCA法にて測定したところ、3.0mg/mLであった。
実施例10で得られたアセトン画分(3.0mg)を、5mM Tris-HCl緩衝液(pH8.0)で平衡化したDEAEカラム(東ソー社製,製品名「DEAE TOYOPEARL 650M」)にチャージし、0~0.5MのNaCl勾配をつけて、流速2.0mL/minの条件で溶出した。吸収波長215nmでピークを検出した吸着画分のフラクションを活性画分として回収した。得られた画分を透析膜(分画分子量:14,000)で透析することにより脱塩し、溶媒を脱イオン水に置換した。得られた画分のタンパク質濃度をBCA法にて測定したところ、12μg/mL(収量:30mL)であった。
緑豆より抽出して得られた実施例7~11の各溶液について、試験例1と同様の方法で氷結晶化阻害活性を測定した。BCA法により測定した各溶液のタンパク質濃度と、氷結晶化阻害活性の指標であるRI値より、試験例1と同様の計算式にてタンパク質当たりの比活性を求め、実施例7の粗抽出液に対する相対値を算出した。また緑豆100g当たりから抽出された総タンパク質量を溶液量とタンパク質濃度から算出した。その結果を表4と表5に示す。
市販の乳酸脱水素酵素(Lactate Dehydrogenase:LDH)15μgを水1.0mLに溶解した。当該溶液へ、実施例9で得られたCF処理後の緑豆精製タンパク質9μgを溶解した。CF処理後の精製タンパク質を添加していないLDH水溶液を対照として、それぞれの溶液を液体窒素で凍結させ、これを室温にて解凍した。解凍後の溶液について、340nmの吸光度を測定した。その結果を表6に示す。
実施例7で得られた緑豆粗抽出液のタンパク質濃度を60mg/mLに調整した。この溶液240μLに実施例9で得られたCF処理後の緑豆精製タンパク質30μgを溶解した。CF処理後の精製タンパク質を添加していない緑豆粗抽出液を対照として、それぞれの溶液を液体窒素で凍結させ、これを室温にて解凍した。解凍後の溶液を遠心分離した後、タンパク質濃度を0.95mg/mLに調整した。溶液の氷結晶化阻害活性を試験例1と同様の方法にて測定した。その結果を表7に示す。
実施例11で得られたDEAE画分4.5mgを試料として、これをゲル濾過カラム(GEヘルスケア社製,製品名「Sephacryl S300 HR」)にチャージし、4℃の温度条件下で、5mM Tris-HCl緩衝液(pH8.0,0.15M NaClを含む)を流速0.5mL/minの条件で流し、非吸着画分を溶出させ、吸収波長215nmで検出した。また分子量の異なる標準タンパク質を同条件で溶出させた。上記カラムクロマトグラフィにより、実施例6で精製した大納言小豆と同じ分子量(16万と13万)に吸収ピークが確認された。
Claims (13)
- マメ科ササゲ属植物、もしくはマメ科ササゲ属植物の類縁品種、またはこれらの改良品種に由来する種子タンパク質からなることを特徴とする氷結晶化阻害物質。
- マメ科ササゲ属植物が小豆または緑豆である請求項1に記載の氷結晶化阻害物質。
- 種子タンパク質が登熟タンパク質である請求項1または2に記載の氷結晶化阻害物質。
- 種子タンパク質がLEAタンパク質(Late embryogenesis abundant protein)である請求項1~3のいずれかに記載の氷結晶化阻害物質。
- 種子タンパク質のアミノ酸配列が、配列番号1の配列、または当該配列の1もしくは複数のアミノ酸が欠失、置換または付加された配列であり且つ氷結晶化阻害作用を有するタンパク質の指標となる配列を含むものである請求項4に記載の氷結晶化阻害物質。
- 種子タンパク質のアミノ酸配列が、配列番号2の配列、または当該配列の1もしくは複数のアミノ酸が欠失、置換または付加された配列であり且つ氷結晶化阻害作用を有するタンパク質の指標となる配列を含むものである請求項4に記載の氷結晶化阻害物質。
- 種子タンパク質が他のタンパク質と複合体を形成しているものである請求項1~6のいずれかに記載の氷結晶化阻害物質。
- 請求項1~7のいずれかに記載の氷結晶化阻害物質と特異的に反応することを特徴とする抗体。
- 請求項1~7のいずれかに記載の氷結晶化阻害物質を含むことを特徴とする組成物。
- 請求項1~7のいずれかに記載の氷結晶化阻害物質を含むことを特徴とする食品。
- 請求項1~7のいずれかに記載の氷結晶化阻害物質を含むことを特徴とする生体試料保護剤。
- 請求項1~7のいずれかに記載の氷結晶化阻害物質を含むことを特徴とする化粧品。
- 配列番号2の配列、または当該配列の1もしくは複数のアミノ酸が欠失、置換または付加された配列であり且つ氷結晶化阻害作用を有するタンパク質の指標となる配列を含むものであることを特徴とするペプチド。
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EP12754617.4A EP2682402A4 (en) | 2011-03-04 | 2012-03-02 | PLANT SENSORY RECOVERED ICE CRYSTALLIZATION INHIBITOR |
CN2012800117014A CN103415533A (zh) | 2011-03-04 | 2012-03-02 | 源自植物种子的冰结晶化抑制物质 |
US14/002,849 US20140213663A1 (en) | 2011-03-04 | 2012-03-02 | Ice crystallization inhibitor derived from plant seed |
JP2013503520A JPWO2012121172A1 (ja) | 2011-03-04 | 2012-03-02 | 植物種子由来の氷結晶化阻害物質 |
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JP (1) | JPWO2012121172A1 (ja) |
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WO2016178426A1 (ja) * | 2015-05-07 | 2016-11-10 | 学校法人 関西大学 | 抗氷核活性剤 |
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CN105969788B (zh) * | 2016-06-21 | 2019-09-17 | 深圳大学 | 一种大豆抗冻蛋白的制备方法、应用 |
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JP2010285359A (ja) * | 2009-06-09 | 2010-12-24 | Kaneka Corp | 植物由来氷結晶化阻害剤 |
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- 2012-03-02 EP EP12754617.4A patent/EP2682402A4/en not_active Withdrawn
- 2012-03-02 WO PCT/JP2012/055460 patent/WO2012121172A1/ja active Application Filing
- 2012-03-02 CN CN2012800117014A patent/CN103415533A/zh active Pending
- 2012-03-02 JP JP2013503520A patent/JPWO2012121172A1/ja active Pending
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Cited By (2)
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WO2016178426A1 (ja) * | 2015-05-07 | 2016-11-10 | 学校法人 関西大学 | 抗氷核活性剤 |
JPWO2016178426A1 (ja) * | 2015-05-07 | 2018-03-01 | 学校法人 関西大学 | 抗氷核活性剤 |
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EP2682402A1 (en) | 2014-01-08 |
EP2682402A4 (en) | 2014-09-03 |
JPWO2012121172A1 (ja) | 2014-07-17 |
CN103415533A (zh) | 2013-11-27 |
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