CN103467586A - Antifreeze polypeptide for protecting bacteria from low temperature freeze-thawing damage and preparation method thereof - Google Patents
Antifreeze polypeptide for protecting bacteria from low temperature freeze-thawing damage and preparation method thereof Download PDFInfo
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Abstract
The invention discloses an antifreeze polypeptide for protecting bacteria from low temperature freeze-thawing damage and a preparation method thereof. The preparation method is characterized by using fish-derived collagens as raw materials, adopting alkaline protease to carry out enzymolysis on the fish-derived collagens and carrying out separation and purification and freeze drying, thus obtaining the antifreeze polypeptide for protecting bacteria from low temperature freeze-thawing damage. The amino acid complete sequence of the antifreeze polypeptide is Gly-Gln-Arg-Gly-Gly-Arg-Gly-Leu-Pro-Gly-Glu-Arg-Gly-Arg-Val-Gly-Pro-Ser-Gly-Pro-Ala-Gly-Ala-Arg-Gly-Ala-Asp-Gly. By adopting the antifreeze polypeptide and the preparation method, the research ideas and methods of antifreeze proteins (polypeptides) existing at home and abroad are broken through, limitation of the quantity of the antifreeze proteins purified form natural organisms and considerations of international FDA (food and drug administration) for security of transgenic antifreeze proteins in food application are overcome, an efficient antifreeze polypeptide based on food sources is obtained, and a theoretical foundation is laid for developing the antifreeze polypeptide based on food sources and exploring extensive application of the antifreeze polypeptide to food and medicines.
Description
Technical field
The present invention relates to a kind of antifreeze peptide, related more specifically to a kind of antifreeze peptide that utilizes Sumizyme MP enzymolysis source of fish collagen protein to prepare, belong to biological technical field.
Background technology
The food and medicine product suffers repeatedly the problem of ice-crystal growth and recrystallization more and more to receive people's concern in low-temperature storage and transportation due to the fluctuation variation of envrionment temperature.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 that the ice-crystal growth on the low temperature cold chain is controlled, is the key point of the numerous food and medicine product quality of restriction.
Antifreeze protein, also claim " ice structural protein ", is that a class is attached to the ice crystal surface and suppresses the growth of ice crystal and the active protein of recrystallization.Antifreeze protein is controlled ice-crystal growth, reduces cell injury and is kept characteristics and the outstanding meaning of the original weave construction of product, quality and quality to become the hot research theme because it has.Same research also shows, the Activity of Antifreeze segment of antifreeze protein only be present in local specific polypeptides chain structure territory and be not whole protein in action, even sublimed antifreeze protein, Activity of Antifreeze is often not high, still needs to probe into its active territory and further improves freeze proof efficiency.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
The object of the present invention is to provide a kind of antifreeze peptide of protecting bacterium low temperature freezing-thawing damage and preparation method thereof; the research ideas and methods of domestic and international existing antifreeze protein (polypeptide) have been broken through; the limitation and the international FDA that overcome antifreeze protein purified quantity from the natural biological body organize to the transgenosis antifreeze protein safety concerns in food applications; the efficient antifreeze peptide of acquisition based on the food source, for developing the antifreeze peptide based on the food source and exploring its widespread use based theoretical in food, medicine.
For achieving the above object, the present invention adopts following technical scheme:
A kind of aminoacid sequence of the antifreeze peptide of bacterium low temperature freezing-thawing damage of protecting is: Gly-Gln-Arg-Gly-Gly-Arg-Gly-Leu-Pro-Gly-Glu-Arg-Gly-Arg-Val-Gly-Pro-Ser-Gly-Pro-Ala – Gly-Ala-Arg-Gly-Ala-Asp-Gly.
Prepare the method for the antifreeze peptide of protection bacterium low temperature freezing-thawing damage as above, take source of fish collagen protein as raw material, adopt Sumizyme MP to carry out enzymolysis to it, separation and purification, lyophilize obtain the antifreeze peptide of described protection bacterium low temperature freezing-thawing damage.
Described source of fish collagen protein comprises the source of fish collagen protein from fish-skin, fish scale.
Enzymatic hydrolysis condition is: the pH value is 9.0, and temperature is 45 ℃, and the time is 30 minutes, and the mass ratio of enzyme-to-substrate is 1:20.
The concrete steps of described separation and purification are: at first enzymolysis product utilizes Sephadex G-50 gel chromatography to be separated, and elutriant is deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm; Collection has the peak of best Activity of Antifreeze, then is separated with Sulfopropyl-Sepadex C-25 cation-exchange chromatography, the phosphoric acid buffer that elutriant is the NaCl concentration gradient 0.01mol/L pH7.0 that is 0-0.5 M, and flow velocity is 0.5mL/min; Collection has the peak of best Activity of Antifreeze, utilize the RP-HPLC RPLC further to separate again, the acetonitrile solution that the separation condition of reversed-phase HPLC is is 10%-90% by volume fraction is as the elutriant gradient elution, flow velocity is 1mL/min, the elution peak that the collected volume mark is 35% acetonitrile solution place.
Remarkable advantage of the present invention is: the Activity of Antifreeze segment that the present invention is based on antifreeze protein only is present in special peptide chain structure territory rather than whole protein theoretical basis in action, take and come from source of fish collagen protein as starting material, be conceived to control by the cutting condition of Sumizyme MP, be cut into the active polypeptide with specific peptide chain length and structural domain composition, and Activity of Antifreeze is realized efficiently.The present invention is for developing the antifreeze peptide based on the food source and exploring its widespread use based theoretical in food, medicine.
The accompanying drawing explanation
Fig. 1 is the CLC-HPLC-C18 color atlas of purifying collagen of fish skin antifreeze peptide.
Fig. 2 is the low-temperature protection effect of the specificity antifreeze peptide of purifying to bulgaricus ccm; Wherein, the A in figure, B mean respectively blank bulgaricus ccm, add 0.5%(w/w) the bulgaricus growth situation map of specificity antifreeze peptide sample.
Embodiment
The optimization of gelatin enzymatic hydrolysis condition
The enzyme that present technique adopts and fish (fish-skin, fish scale) collagen protein is purchased 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 1.65 gram source of fish collagen proteins and be dissolved in 6ml Mili-Q water, then use 2mol/L NaOH by its pH regulator to 9.0.First this solution water-bath is heated to and needs temperature, then add again the enzyme of respective amount by different enzyme-substrate proportionings, start to react according to the predetermined reaction times.Then go out in boiling water bath again enzyme 10 minutes, cooling after centrifugal 10 minutes of 1000rpm again.Supernatant liquor is measured its Activity of Antifreeze respectively, to determine optimum enzymolysis condition after collecting.The enzymatic hydrolysis condition that obtains having the enzymolysis solution of maximum Activity of Antifreeze 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).
The separation of enzymolysis product, purifying
First carry out enzymolysis under optimum enzymolysis condition, the enzymolysis product obtained first passes through Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm) to be separated, and elutriant is deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm.Collection has the peak of best Activity of Antifreeze, use again Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm, diameter 2.0cm) separated, the phosphoric acid buffer that elutriant is the NaCl concentration gradient 0.01mol/L pH7.0 that is 0-0.5 M, flow velocity is 0.5mL/min.Collection has the peak of best Activity of Antifreeze, utilize the RP-HPLC 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.
The test of Activity of Antifreeze
The present invention, according to the active standard detected of international antifreeze protein, sets up the Activity of Antifreeze detection system.Activity of Antifreeze adopts the detection antifreeze peptide to test the provide protection of low temperature freeze thawing bacterium.
The bulgaricus ccm of activation is inoculated in liquid nutrient medium to 37 ℃, and the 130r/min overnight incubation is as seed liquor, and the ratio by seed liquor with 1:100 is inoculated in new liquid nutrient medium, and 37 ℃, the 130r/min shaking table is cultured to OD
600=1.0 left and right.Get the sterilized centrifuge tube of some 1.5mL, the concentration that adds degerming after filtration is 250 μ g/mL testing sample 900 μ L, by bacterium liquid dilution 10
4doubly, draw 100 μ L and be added in testing sample, mix, coating, be inverted for 37 ℃ and cultivate 18h, the meter colony number.Remaining sample-bacterium liquid mixed solution is placed in to-20 ℃ of subzero treatment 24h, takes out and to melt and be coated with, be inverted cultivation 18h for 37 ℃, count colony number, every group do two parallel.Then calculate the survival rate of bulgaricus ccm according to following formula:
Determined amino acid sequence
Utilize protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) to measure the amino acid complete sequence of specificity antifreeze peptide.
In order further to understand content of the present invention, Characteristic, hereby exemplify following examples:
embodiment 1
Take 1.65 gram collagen of fish skin and be dissolved in 6ml Mili-Q water, then use 2mol/L NaOH by its pH regulator to 9.0.First this solution water-bath is heated to 45 ℃, the ratio that is then 1:20 according to the enzyme-substrate proportioning again adds the enzyme of respective amount, and enzymolysis time is 30 minutes.Then go out in boiling water bath enzyme 10 minutes, cooling after centrifugal 10 minutes of 14000rpm again, collect supernatant liquor standby.
Supernatant liquor is separated with Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm), and elutriant is deionized water, and flow velocity is 2mL/min, and elution peak is measured under 225nm, collects the elution peak with best Activity of Antifreeze.
The elution peak with best Activity of Antifreeze to Sephadex G-50 gel chromatography separation carries out next step separation again, with Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm, diameter 2.0cm) separated, the phosphoric acid buffer that elutriant is the NaCl concentration gradient 0.01mol/L pH7.0 that is 0-0.5M, flow velocity is 0.5mL/min.Collect each peak sample and measure Activity of Antifreeze, the sample that obtains best Activity of Antifreeze utilizes the RP-HPLC-C18 RPLC to carry out further separation and purification again.The mixed solution of self-contained 10% acetonitrile of elutriant and 90% water (v/v) starts, mixed solution to 90% acetonitrile and 10% water (v/v) finishes, and carries out gradient elution, collects the elution peak that 35% acetonitrile and 65 % water (v/v) are located, obtain highly purified specificity antifreeze peptide of the present invention, as shown in Figure 1.The chromatographic peak that the YP3 peak is this specificity antifreeze peptide.
The specificity antifreeze peptide of purifying has the ability of very strong bacterium low-temperature protection, as seen from Figure 2, with blank (Fig. 2 A), compares, and has added the specificity antifreeze peptide of 0.5% (w/w), (Fig. 2 B).The survival rate of calculating bulgaricus ccm is 92.6%.
Specificity antifreeze peptide to purifying utilizes protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) to measure the aminoacid sequence of specificity antifreeze peptide.The amino acid total order of described polypeptide is classified as: Gly-Gln-Arg-Gly-Gly-Arg-Gly-Leu-Pro-Gly-Glu-Arg-Gly-Arg-Val-Gly-Pro-Ser-Gly-Pro-Ala – Gly-Ala-Arg-Gly-Ala-Asp-Gly.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110 > University of Fuzhou
<120 > a kind of antifreeze peptide of protecting bacterium low temperature freezing-thawing damage and preparation method thereof
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 28
<212> PRT
<213 > artificial sequence
<400> 1
Gly Gln Arg Gly Gly Arg Gly Leu Pro Gly Glu Arg Gly Arg Val Gly
1 5 10 15
Pro Ser Gly Pro Ala Gly Ala Arg Gly Ala Asp Gly
20 25
Claims (5)
1. an antifreeze peptide of protecting bacterium low temperature freezing-thawing damage, it is characterized in that: the aminoacid sequence of described antifreeze peptide is: Gly-Gln-Arg-Gly-Gly-Arg-Gly-Leu-Pro-Gly-Glu-Arg-Gly-Arg-Val-Gly-Pro-Ser-Gly-Pro-Ala – Gly-Ala-Arg-Gly-Ala-Asp-Gly.
2. one kind prepares the method for protecting the antifreeze peptide of bacterium low temperature freezing-thawing damage as claimed in claim 1; it is characterized in that: take source of fish collagen protein as raw material; adopt Sumizyme MP to carry out enzymolysis to it, separation and purification, lyophilize obtain the antifreeze peptide of described protection bacterium low temperature freezing-thawing damage.
3. the preparation method of the antifreeze peptide of protection according to claim 2 bacterium low temperature freezing-thawing damage, it is characterized in that: described source of fish collagen protein comprises the source of fish collagen protein from fish-skin, fish scale.
4. the preparation method of the antifreeze peptide of protection according to claim 2 bacterium low temperature freezing-thawing damage, it is characterized in that: enzymatic hydrolysis condition is: the pH value is 9.0, and temperature is 45 ℃, and the time is 30 minutes, and the mass ratio of enzyme-to-substrate is 1:20.
5. the preparation method of the antifreeze peptide of protection according to claim 2 bacterium low temperature freezing-thawing damage, it is characterized in that: the concrete steps of described separation and purification are: at first enzymolysis product utilizes Sephadex G-50 gel chromatography to be separated, elutriant is deionized water, flow velocity is 2mL/min, and elution peak is measured under 225nm; Collection has the peak of best Activity of Antifreeze, then is separated with Sulfopropyl-Sepadex C-25 cation-exchange chromatography, the phosphoric acid buffer that elutriant is the NaCl concentration gradient 0.01mol/L pH7.0 that is 0-0.5 M, and flow velocity is 0.5mL/min; Collection has the peak of best Activity of Antifreeze, utilize the RP-HPLC RPLC further to separate again, the acetonitrile solution that the separation condition of reversed-phase HPLC is is 10%-90% by volume fraction is as the elutriant gradient elution, flow velocity is 1mL/min, the elution peak that the collected volume mark is 35% acetonitrile solution place.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103804471A (en) * | 2014-03-06 | 2014-05-21 | 福州大学 | Method for preparing metal chelated peptide by enzyme method |
| CN104945469A (en) * | 2015-06-30 | 2015-09-30 | 石狮海星食品有限公司 | ACE (angiotensin converting enzyme) inhibitory tripeptide |
| CN105394029A (en) * | 2016-01-05 | 2016-03-16 | 潘时辉 | Cell cryopreservation liquid used for treatment of leukemia |
| CN105432599A (en) * | 2016-01-04 | 2016-03-30 | 潘时辉 | Cell freezing medium for treating leukemia |
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| US8211487B2 (en) * | 2008-11-26 | 2012-07-03 | Srinivasan Damodaran | Inhibition of ice crystal growth |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103804471A (en) * | 2014-03-06 | 2014-05-21 | 福州大学 | Method for preparing metal chelated peptide by enzyme method |
| CN103804471B (en) * | 2014-03-06 | 2015-10-14 | 福州大学 | A kind of enzyme process prepares the method for metal chelating peptide |
| CN104945469A (en) * | 2015-06-30 | 2015-09-30 | 石狮海星食品有限公司 | ACE (angiotensin converting enzyme) inhibitory tripeptide |
| CN104945469B (en) * | 2015-06-30 | 2018-09-28 | 石狮海星食品有限公司 | ACE inhibitory tripeptides |
| CN105432599A (en) * | 2016-01-04 | 2016-03-30 | 潘时辉 | Cell freezing medium for treating leukemia |
| CN106973889A (en) * | 2016-01-04 | 2017-07-25 | 潘时辉 | A kind of cells frozen storing liquid of leukemia treating |
| CN106973889B (en) * | 2016-01-04 | 2018-08-24 | 南京三生生物技术股份有限公司 | A kind of cells frozen storing liquid of leukemia treating |
| CN105394029A (en) * | 2016-01-05 | 2016-03-16 | 潘时辉 | Cell cryopreservation liquid used for treatment of leukemia |
| CN107027742A (en) * | 2016-01-05 | 2017-08-11 | 潘时辉 | A kind of cells frozen storing liquid for leukemia treating |
| CN107027742B (en) * | 2016-01-05 | 2019-01-18 | 河北生命原点生物科技有限公司 | A kind of cells frozen storing liquid for leukemia treating |
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