CN101921316A - Frost-resisting polypeptide prepared by using pigskin collagen - Google Patents
Frost-resisting polypeptide prepared by using pigskin collagen Download PDFInfo
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Abstract
The invention provides a frost-resisting polypeptide prepared by carrying out enzymolysis on pigskin collagen with alkali protease. The purified specific frost-resisting polypeptide is obtained by the steps of carrying out enzymolysis with the alkali protease and then separating and purifying, wherein the amino acid complete sequence of the specific frost-resisting polypeptide is Ser-Gly-Arg-Gly-Glu-Arg-Gly-Phe-Hyp-Gly-Glu-Arg-Gly. The invention breaks through the idea and the method of researching the frost-resisting protein (polypeptide) existing at home and abroad, overcomes the limit of the quantity of the purified frost-resisting protein in the natural organism, obtains highly-efficient frost-resisting polypeptide based on a food source, and lays the theoretical foundation of developing the frost-resisting polypeptide based on the food source and exploring wide application thereof to the medicine and the food.
Description
Technical field
The present invention relates to a kind of antifreeze peptide, related more specifically to a kind of antifreeze peptide that utilizes the pigskin collagen protein Preparation, belong to biological technical field.
Background technology
The food and medicine product in low-temperature storage and transportation since the fluctuation of envrionment temperature change and suffer the problem of ice-crystal growth and recrystallization more and more to be subjected to people's attention repeatedly.The lifting repeatedly of temperature makes constantly growth of ice crystal, freeze thawing and recrystallization, havoc cell and weave construction and lose the due quality of product.Worldwide scientist is faced with serious challenge: how controlling ice-crystal growth and recrystallization, realize the ice-crystal growth control on the low temperature cold chain, is the key point of the numerous food and medicine product quality of restriction.
Antifreeze protein (antifreeze proteins, AFPs) or claim " ice structural protein " (ice structuring proteins), be that a class suppresses the growth of ice crystal and the active protein of recrystallization attached to ice crystal (ice crystals) surface.Antifreeze protein is owing to it has the control ice-crystal growth, reduces cell injury and keeps the original weave construction of product, quality and quality characteristics and outstanding meaning to become the hot research theme, very active to freeze proof proteic research both at home and abroad in recent years, " Nature " and " Science " follow up on closely latest Progress of antifreeze protein aspect.Present research mainly concentrates on from organisms such as polar region fish, land insect, plant and bacterium and is separated to multiple antifreeze protein, and seeks them in food, medical science and other industrial application.Be accompanied by the discovery of multiple antifreeze protein and going deep into of research, the research of its restriction organism antifreeze protein and two big key issues of application also show especially day by day: 1. the resulting quantity pettiness of natural separation and purification, very limited quantity limitation it in food and big rule application prospect medically; 2. be devoted to transgenic technology when enlarging the antifreeze protein output in organism source as scientist, the safety concerns of transgenosis antifreeze protein in food applications becomes the focal issue that consumers in general, European Union's tissue and international FDA tissue institute worry jointly again.
Same research also shows, the freeze proof active part of antifreeze protein only be present in partial specific polypeptides chain structure territory and be not whole protein in action, even sublimed antifreeze protein, freeze proof activity are often not high, still need to probe into its active territory and further improve freeze proof efficient.So, how to obtain the high reactivity antifreeze peptide of the compact construction in food source, just become the urgent research direction of antifreeze protein.
Summary of the invention
In order to address the above problem, the invention provides a kind of antifreeze peptide that utilizes the pigskin collagen protein Preparation, freeze proof activity is realized efficiently.
A kind of antifreeze peptide of the present invention is by 14 polypeptide that amino acid constituted.The amino acid total order of described polypeptide is classified as: Ser-Gly-Ala-Arg-Gly-Glu-Arg-Gly-Phe-Hyp-Gly-Glu-Arg-Gly.
The preparation method of described antifreeze peptide is as follows:
With pigskin collagen albumen is raw material, adopts Sumizyme MP that it is carried out enzymolysis, and separation and purification, lyophilize obtain antifreeze peptide; Enzymatic hydrolysis condition is: enzymolysis pH is 9.0,45 ℃ of temperature, enzymolysis time be that 30 minutes, enzyme-substrate proportioning are 1:15(w/w); Described enzyme is a Sumizyme MP; The concrete steps of described separation and purification are: enzymolysis product at first utilizes Sephadex G-50 gel chromatography to separate, and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm; Collection has best freeze proof active peak, separates with Sulfopropyl-Sepadex C-25 cation-exchange chromatography again, and elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-1.0M for the NaCl concentration gradient, and flow velocity is 0.5mL/min; Collection has best freeze proof active peak, utilize the RP-HPLC RPLC further to separate again, the separation condition of reversed-phase HPLC is as the elutriant gradient elution with the 10%-90% acetonitrile solution, flow velocity is 1mL/min, collect the elution peak that 30% acetonitrile and 70% water (v/v) are located, obtain described antifreeze peptide.
In order to realize such scheme, the concrete steps that the present invention takes are as follows:
(1) optimization of gelatin enzymatic hydrolysis condition
Enzyme that present technique adopted and pigskin collagen albumen are available from Sigma biological reagent company (Chinese Shanghai).
Adopt experiment of single factor, respectively three enzymolysis factors are investigated, be respectively hydrolysis temperature (37 ℃, 45 ℃ and 50 ℃), enzymolysis time (10,20,30,40 and 60 minutes) and enzyme-substrate proportioning (1:100,1:50,1:20,1:15 and 1:10 w/w).Take by weighing 1.65 gram pigskin collagen protein dissolutions in 6ml Mili-Q water, use 2mol/L NaoH then its pH regulator to 9.0.Earlier this solution water-bath being heated to needs temperature, then adds the enzyme of respective amount again by different enzyme-substrate proportionings, begins to react according to the predetermined reaction times.Then in boiling water bath, go out again enzyme 10 minutes, centrifugal 10 minutes of 1000rpm again after the cooling.Supernatant liquor is measured its freeze proof activity respectively after collecting, to determine optimum enzymolysis condition.The enzymatic hydrolysis condition that obtains having maximum freeze proof active enzymolysis solution is: enzymolysis pH is that pH7-10, temperature 30-55 ℃, enzymolysis time are that 20-60 minute, enzyme-substrate proportioning are 1:15-1:20(w/w).
(2) separation of enzymolysis enzymolysis product, purifying
Carry out earlier enzymolysis under optimum enzymolysis condition, the enzymolysis product that obtains separates through Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm) earlier, and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm.Collection has best freeze proof active peak, use Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm again, diameter 2.0cm) separate, elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-1.0M for the NaCl concentration gradient, and flow velocity is 0.5mL/min.Collection has best freeze proof active peak, utilize the RP-HPLC-C18 RPLC further to separate again, the separation condition of reversed-phase HPLC is to use the 10-90% acetonitrile solution as elutriant, and flow velocity is 1mL/min, obtains highly purified specificity antifreeze peptide.
(3) freeze proof active test
Utilize ice-creams matrix (Ice Cream Base Mix) to be antifreeze peptide research carrier.Get the ice-creams of 5uL, utilize the cold-heat-stage system, make its with the speed fast cooling of 40 ℃/min to-40 ℃, under this temperature, keep 5min and take pictures.Speed with 1 ℃/min is warming up to-14 ℃ more then, and then with every 3min frequency once reciprocation cycle 7 times between-14 ℃ and-12 ℃, with the variation of temperature of simulation ice-creams in two months storage transportations, and observes ice crystal and changes.
(4) determined amino acid sequence
Utilize protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) the amino acid complete sequence of mensuration specificity antifreeze peptide.
The present invention sees through the quantity limitation of present organism antifreeze protein purified research and the edible safety misgivings of transgenosis antifreeze protein, the freeze proof active part that is based on antifreeze protein only is present in special peptide chain structure territory (domain) rather than whole protein theoretical basis in action, with the collagen protein that comes from pigskin is starting point, be conceived to cutting condition control by Sumizyme MP, be cut into active polypeptide, and freeze proof activity is realized efficiently with specific peptide chain length and structural domain composition.
The present invention has broken through the research ideas and methods of domestic and international existing antifreeze protein (polypeptide), overcome from the natural biological body quantity limitation of antifreeze protein purified and international FDA tissue to the safety concerns of transgenosis antifreeze protein in food applications, acquisition is based on the efficient antifreeze peptide in food source, for exploitation based on the antifreeze peptide in food source and explore its widespread use based theoretical in food, medicine.
Description of drawings
Fig. 1 is a collagen protein enzymolysis thing Sephadex G-50 elution profile;
Fig. 2 is the Sulfopropyl-Sepadex C-25 elution profile of Fraction2;
Fig. 3 is the CLC-HPL-C18C elution profile of SP;
Fig. 4 is the effect of the specificity antifreeze peptide of purifying to Ice Crystal in Ice Cream growth and inhibition ice-crystal growth; Wherein, A, B represent blank ice-creams respectively, add 0.5%(w/w) the ice-crystal growth situation map of specificity antifreeze peptide sample.
Embodiment
Antifreeze peptide of the present invention is by 14 polypeptide that amino acid constituted.The amino acid total order of described polypeptide is classified as: Ser-Gly-Ala-Arg-Gly-Glu-Arg-Gly-Phe-Hyp-Gly-Glu-Arg-Gly.
The preparation method is as follows:
With pigskin collagen albumen is raw material, use Sumizyme MP that it is carried out enzymolysis, and according to the active standard that detects of international antifreeze protein, set up freeze proof activity monitor system, optimize its enzymatic hydrolysis condition, the enzymatic hydrolysis condition that obtains having maximum freeze proof active enzymolysis solution is: enzymolysis pH is 9.0, temperature is that 45 ℃, enzymolysis time are that 30 minutes, enzyme-substrate proportioning are 1:15; Carry out enzymolysis under optimum enzymolysis condition, the enzymolysis product that obtains separates through Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm) earlier, and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm.Collection has best freeze proof active peak, use Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm again, diameter 2.0cm) separate, elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-0.5M for the NaCl concentration gradient, and flow velocity is 0.5mL/min.Collection has best freeze proof active peak, utilize RP-HPLC RPLC (long 15cm, diameter 0.8cm) further separates again, the separation condition of reversed-phase HPLC is as elutriant with the 10-90% acetonitrile solution, the mixed solution of self-contained 10% acetonitrile of elutriant and 90% water (v/v) begins, mixed solution to 90% acetonitrile and 10% water (v/v) finishes, carry out gradient elution, flow velocity is 1mL/min, collect the elution peak that 30% acetonitrile and 70% water (v/v) are located, obtain highly purified specificity antifreeze peptide.
Lyophilize obtains antifreeze peptide of the present invention, is by 14 polypeptide that amino acid constituted.The amino acid total order of described polypeptide is classified as: Ser-Gly-Ala-Arg-Gly-Glu-Arg-Gly-Phe-Hyp-Gly-Glu-Arg-Gly.
Instrument, detection means that the present invention adopts are as follows:
The freeze proof activity monitor system of preparation antifreeze peptide of the present invention adopts in conjunction with cold cycling (cold-heat-stage) system, many power microscopes and automatic photography system, the freeze proof active inhibition ice-crystal growth detection system platform of group structure.Keep the low temperature environment of operating process with liquid nitrogen.The temperature fluctuation of analog equipment in the low-temperature storage process.By the system program setting, the temperature fluctuation that sample is set changes and 7～25 circulations repeatedly continuously, connects many power microscopes and photographic recording system in real time, and the record sample is in the cold-hot real-time ice-crystal growth situation in the working cycle repeatedly.
Use multiple separation and purification means such as Sephadex G-50 molecular sieve, Sepharose SP C-25 ion-exchange chromatography, RP-HPLC RPLC, dialysis, realize the high efficiency separation purifying of the special antifreeze peptide of remarkable activity.
Utilize protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) the amino acid complete sequence of mensuration specificity antifreeze peptide.
In order further to understand content of the present invention, characteristics and effect, exemplify following examples now:
Take by weighing 1.65 gram pigskin collagen protein dissolutions in 6ml Mili-Q water, use 2mol/L NaOH then its pH regulator to 9.0.Earlier this solution water-bath being heated to 45 ℃, is the enzyme of the ratio adding respective amount of 1:15 according to the enzyme-substrate proportioning more then, and enzymolysis time is 30 minutes.Then in boiling water bath, go out enzyme 10 minutes, centrifugal 10 minutes of 14000rpm again after the cooling, it is standby to collect supernatant liquor.
Supernatant liquor is separated with Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm), and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm, sees Fig. 1.Collect each peak and measure freeze proof activity.Freeze proof active testing shows, Fraction2 has best freeze proof activity, and statistical analysis shows that the ice crystal mean diameter is 5.67 μ m, be significantly less than Fraction1(18.83 μ m) and Fraction3(9.02 μ m), significant difference (P ﹤ 0.05) had.
Separate Fraction2 and carry out next step separation again, with Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm, diameter 2.0cm) separates, elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-0.5M for the NaCl concentration gradient, flow velocity is 0.5mL/min, sees Fig. 2.Collect each peak sample and measure freeze proof activity, the result shows that SP has best freeze proof activity, collects the sample at SP peak and does further separation and purification.
Utilize the RP-HPLC-C18 RPLC further to separate again to the sample of separating the SP part, the mixed solution of self-contained 10% acetonitrile of elutriant and 90% water (v/v) begins, mixed solution to 90% acetonitrile and 10% water (v/v) finishes, carry out gradient elution, flow velocity is 1mL/min, collect the elution peak that 30% acetonitrile and 70% water (v/v) are located, obtain highly purified specificity antifreeze peptide (Fig. 3).The HP2 peak is the chromatographic peak of this specificity antifreeze peptide.
The specificity antifreeze peptide of purifying has the ability of very strong inhibition ice-crystal growth, and as seen from Figure 4, (Fig. 4 A) compares with blank, has added the specificity antifreeze peptide of 0.5% (w/w), ice crystal grow hardly (Fig. 4 B).
Specificity antifreeze peptide to purifying utilizes protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) aminoacid sequence of mensuration specificity antifreeze peptide.The amino acid total order of described polypeptide is classified as: Ser-Gly-Ala-Arg-Gly-Glu-Arg-Gly-Phe-Hyp-Gly-Glu-Arg-Gly.
Claims (2)
1. antifreeze peptide, it is characterized in that: described amino acid sequence of polypeptide is: Ser-Gly-Ala-Arg-Gly-Glu-Arg-Gly-Phe-Hyp-Gly-Glu-Arg-Gly.
2. the preparation method of an antifreeze peptide as claimed in claim 1, it is characterized in that: with pigskin collagen albumen is raw material, adopts Sumizyme MP that it is carried out enzymolysis, separation and purification, lyophilize obtain antifreeze peptide; Enzymatic hydrolysis condition is: enzymolysis pH is 9.0,45 ℃ of temperature, enzymolysis time be that 30 minutes, enzyme-substrate proportioning are 1:15(w/w); Described enzyme is a Sumizyme MP; The concrete steps of described separation and purification are: enzymolysis product at first utilizes Sephadex G-50 gel chromatography to separate, and elutriant is a deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm; Collection has best freeze proof active peak, separates with Sulfopropyl-Sepadex C-25 cation-exchange chromatography again, and elutriant is the phosphoric acid buffer of the 0.01mol/L pH7.0 of 0-0.5 M for the NaCl concentration gradient, and flow velocity is 0.5mL/min; Collection has best freeze proof active peak, utilize the RP-HPLC RPLC further to separate again, the separation condition of reversed-phase HPLC is to use 10%-90%(v/v) acetonitrile solution is as the elutriant gradient elution, flow velocity is 1mL/min, collect the elution peak that 30% acetonitrile and 70% water (v/v) are located, obtain described antifreeze peptide.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104798868A (en) * | 2015-05-20 | 2015-07-29 | 福州大学 | Compound antifreeze agent and application thereof |
| CN106544385A (en) * | 2015-09-16 | 2017-03-29 | 上海理工大学 | The separation method of collagen antifreeze peptide |
| CN114404605A (en) * | 2022-01-29 | 2022-04-29 | 陕西未来多肽生物科技有限公司 | Vitamin C gold-collagen peptide nanocomposite and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7442769B2 (en) * | 2002-03-15 | 2008-10-28 | National Institute Of Advanced Industrial Science And Technology | Antifreeze proteins from basidiomy cetes |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7442769B2 (en) * | 2002-03-15 | 2008-10-28 | National Institute Of Advanced Industrial Science And Technology | Antifreeze proteins from basidiomy cetes |
Non-Patent Citations (4)
| Title |
|---|
| 《食品科技》 20071031 李燕 胶原蛋白的分离纯化及氨基酸组成分析 , 第10期 * |
| 《食品科技》 20071031 李燕 胶原蛋白的分离纯化及氨基酸组成分析 , 第10期 2 * |
| <中国食品科学技术学会第六届年会暨第五届东西方食品业高层论坛论文摘要集> 20091231 汪少芸 天然来源的抗冻－冰结构多肽的研究 , * |
| <中国食品科学技术学会第六届年会暨第五届东西方食品业高层论坛论文摘要集> 20091231 汪少芸 天然来源的抗冻－冰结构多肽的研究 , 2 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104798868A (en) * | 2015-05-20 | 2015-07-29 | 福州大学 | Compound antifreeze agent and application thereof |
| CN106544385A (en) * | 2015-09-16 | 2017-03-29 | 上海理工大学 | The separation method of collagen antifreeze peptide |
| CN106544385B (en) * | 2015-09-16 | 2020-11-27 | 上海理工大学 | Method for separating collagen antifreeze peptide |
| CN114404605A (en) * | 2022-01-29 | 2022-04-29 | 陕西未来多肽生物科技有限公司 | Vitamin C gold-collagen peptide nanocomposite and application thereof |
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