CN103467586B

CN103467586B - Antifreeze polypeptide for protecting bacteria from low temperature freeze-thawing damage and preparation method thereof

Info

Publication number: CN103467586B
Application number: CN201310441148.4A
Authority: CN
Inventors: 汪少芸; 邵彪; 赵立娜; 饶平凡
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2013-09-25
Filing date: 2013-09-25
Publication date: 2015-04-15
Anticipated expiration: 2033-09-25
Also published as: CN103467586A

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

A kind ofly protect antifreeze peptide of bacterium low temperature freezing-thawing damage and preparation method thereof

Technical field

The present invention relates to a kind of antifreeze peptide, related more specifically to a kind of antifreeze peptide utilizing Sumizyme MP enzymolysis fish source collagen albumen to prepare, belonged to biological technical field.

Background technology

Food and medicine product suffers the problem of ice-crystal growth and recrystallization more and more to receive the concern of people repeatedly due to the fluctuation change of envrionment temperature in low-temperature storage and transportation.The lifting repeatedly of temperature makes ice crystal constantly grow, freeze thawing and recrystallization, havoc biological cells and tissues structure and lose the due quality of product.Worldwide scientist is faced with serious challenge: how to control ice-crystal growth and recrystallization, and the ice-crystal growth realized on low temperature cold chain controls, and is the key point restricting numerous food and medicine product quality.

Antifreeze protein, also claims " ice structural protein ", is that a class is attached to ice crystal surface and suppresses the growth of ice crystal and the active protein of recrystallization.Antifreeze protein controls ice-crystal growth because it has, reduce cell injury and keep the feature of product original weave construction, quality and quality and outstanding meaning and become hot research theme.Same research also shows, the Activity of Antifreeze segment of antifreeze protein be only present in local specific polypeptides hinge domain and be not overall protein in action, even sublimed antifreeze protein, Activity of Antifreeze is often not high, still needs to probe into its active territory and improves freeze proof efficiency further.So, how to obtain the high reactivity antifreeze peptide of the compact construction of food source, just become the research direction that antifreeze protein is urgent.

Summary of the invention

The object of the present invention is to provide and a kind ofly protect antifreeze peptide of bacterium low temperature freezing-thawing damage and preparation method thereof; breach the research ideas and methods of existing antifreeze protein (polypeptide) both at home and abroad; the limitation and the international FDA that overcome antifreeze protein purified quantity from native organism organize the safety concerns of transgenosis antifreeze protein in food applications; obtain based on the efficient antifreeze peptide of food source, for exploitation is based on the antifreeze peptide of food source and explore 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.

The method of the antifreeze peptide of preparation protection as above bacterium low temperature freezing-thawing damage, with fish source collagen albumen for 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 fish source collagen albumen comprises the fish source collagen albumen from fish-skin, fish scale.

Enzymatic hydrolysis condition is: pH value is 9.0, and temperature is 45 DEG C, 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: 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; Collect and have the peak of best Activity of Antifreeze, then be separated with Sulfopropyl-Sepadex C-25 cation-exchange chromatography, the phosphoric acid buffer of elutriant to be NaCl concentration gradient the be 0.01mol/L pH7.0 of 0-0.5 M, flow velocity is 0.5mL/min; Collect the peak with best Activity of Antifreeze, RP-HPLC RPLC is utilized further to be separated again, the separation condition of reversed-phase HPLC is that the acetonitrile solution of 10%-90% is as elutriant gradient elution by volume fraction, flow velocity is 1mL/min, and collected volume mark is the elution peak at 35% acetonitrile solution place.

Remarkable advantage of the present invention is: the present invention is only present in special peptide chain structure territory instead of overall protein theoretical basis in action based on the Activity of Antifreeze segment of antifreeze protein, to come from fish source collagen albumen for starting material, be conceived to be controlled 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 that exploitation is based on the antifreeze peptide of food source and explore its widespread use based theoretical in food, medicine.

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 specificity antifreeze peptide to bulgaricus ccm of purifying; Wherein, A, B in figure represent blank bulgaricus ccm respectively, 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 this technology adopts and fish (fish-skin, fish scale) collagen protein are purchased from Sigma biological reagent company (Chinese Shanghai).Adopt experiment of single factor, respectively three enzymolysis factors are investigated, be respectively hydrolysis temperature (37 DEG C, 45 DEG C and 50 DEG C), 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 grams of fish source collagen protein dissolutions 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 the enzyme of respective amount again by different enzyme-substrate proportionings, start reaction according to the predetermined reaction times.Then go out enzyme 10 minutes again in boiling water bath, centrifugal 10 minutes of 1000rpm again after cooling.After supernatant collection, respectively its Activity of Antifreeze is measured, to determine optimum enzymolysis condition.The enzymatic hydrolysis condition obtaining the enzymolysis solution with maximum Activity of Antifreeze is: enzymolysis pH is pH7-10, temperature 30-55 DEG C, enzymolysis time is 20-60 minute, enzyme-substrate proportioning is 1:15-1:20(w/w).

The separation of enzymolysis product, purifying

First under optimum enzymolysis condition, carry out enzymolysis, the enzymolysis product obtained first is separated through 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.Collect the peak with best Activity of Antifreeze, use Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm again, diameter 2.0cm) be separated, the phosphoric acid buffer of elutriant to be NaCl concentration gradient the be 0.01mol/L pH7.0 of 0-0.5 M, flow velocity is 0.5mL/min.Collect the peak with best Activity of Antifreeze, RP-HPLC RPLC is utilized further to be separated again, the separation condition of reversed-phase HPLC is that flow velocity is 1mL/min, obtains highly purified specificity antifreeze peptide with 10-90% acetonitrile solution as elutriant.

The test of Activity of Antifreeze

The present invention, according to the standard of international antifreeze protein Activity determination, sets up Activity of Antifreeze detection system.Activity of Antifreeze adopts the provide protection of detection antifreeze peptide to low temperature freeze thawing bacterium to test.

The bulgaricus ccm of activation to be inoculated in liquid nutrient medium 37 DEG C, seed liquor, as seed liquor, is inoculated in new liquid nutrient medium with the ratio of 1:100 by 130r/min overnight incubation, and 37 DEG C, 130r/min shaking table is cultured to OD ₆₀₀about=1.0.Get the sterilized centrifuge tube of some 1.5mL, adding concentration degerming is after filtration 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, mixing, coating, be inverted for 37 DEG C and cultivate 18h, meter colony number.Remaining sample-bacterium liquid mixed solution is placed in-20 DEG C of subzero treatment 24h, take out melt and be coated with, 37 DEG C be inverted cultivate 18h, count colony number, often group do two parallel.Then according to the survival rate of following formulae discovery bulgaricus ccm:

Determined amino acid sequence

Protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) is utilized to measure the overall amino acid sequence of specificity antifreeze peptide.

In order to understand content of the present invention, Characteristic further, hereby exemplify following examples:

embodiment 1

Take 1.65 grams of collagen of fish skin to 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 DEG C, the ratio being then 1:20 according to enzyme-substrate proportioning again adds the enzyme of respective amount, and enzymolysis time is 30 minutes.Then go out enzyme 10 minutes in boiling water bath, and centrifugal 10 minutes of 14000rpm again after cooling, collects supernatant liquor for subsequent use.

Be separated by supernatant liquor Sephadex G-50 gel chromatography (long 100cm, diameter 2.6cm), 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 of Sephadex G-50 gel chromatography separation is carried out again to next step separation, with Sulfopropyl-Sepadex C-25 cation-exchange chromatography (long 55cm, diameter 2.0cm) be separated, the phosphoric acid buffer of elutriant to be NaCl concentration gradient the be 0.01mol/L pH7.0 of 0-0.5M, flow velocity is 0.5mL/min.Collect each peak sample and measure Activity of Antifreeze, the sample obtaining best Activity of Antifreeze utilizes 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) terminates, and carries out gradient elution, collects the elution peak at 35% acetonitrile and 65 % water (v/v) places, obtain highly purified specificity antifreeze peptide of the present invention, as shown in Figure 1.YP3 peak is the chromatographic peak of 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, compared with blank (Fig. 2 A), with the addition of the specificity antifreeze peptide of 0.5% (w/w), (Fig. 2 B).The survival rate calculating bulgaricus ccm is 92.6%.

Protein sequencer (Applied Biosystems Model 476A, Perkin Elmer Co. MA, U.S.A) is utilized to measure the aminoacid sequence of specificity antifreeze peptide to the specificity antifreeze peptide of purifying.The overall amino acid sequence of described 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.

The foregoing is only preferred embodiment of the present invention, all equalizations done 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> mono-kind protects antifreeze peptide of 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

1. protect an antifreeze peptide for 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.