Antifreeze polypeptide prepared by utilizing acid protease to carry out hydrolysis on fish-derived collagens
A technology of acid protease and collagen, applied in the preparation method of peptides, peptides, peptide sources, etc., can solve the problems of losing quality, destroying cells and tissue structures, etc.
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[0031] The antifreeze activity detection system for preparing antifreeze polypeptides of the present invention uses low-temperature freeze-thawed bacteria to detect the growth protection effect of adding antifreeze polypeptides on bacteria after low-temperature freeze-thaw. Inoculate the activated Lactobacillus bulgaricus into the liquid medium at 37°C, and cultivate overnight on a shaker at 130r / min as the seed liquid, inoculate the seed liquid into a new liquid medium at a ratio of 1:100, at 37°C, 130r / min Shaker culture to OD 600 =1.0 or so. Take a number of 1.5mL sterilized centrifuge tubes, add 900μL of the sample to be tested at a concentration of 250μg / mL that has been sterilized by filtration, and dilute the bacterial solution by 10 4 times, pipette 100 μL and add it to the sample to be tested, mix evenly, spread, incubate upside down in an incubator at 37°C for 18 hours, and count the number of colonies. Put the remaining sample-bacteria solution mixture at -20°C fo...
Embodiment 1
[0036] Weigh 1.65 g of fish skin collagen and dissolve it in 6 ml of Mili-Q water, then adjust its pH to 5.4 with 2 mol / L HCL. First heat the solution to 37°C in a water bath, then add the corresponding amount of acid protease according to the enzyme-substrate ratio of 1:15 (w / w), and the enzymatic hydrolysis time is 30 minutes. Then inactivate the enzyme in a boiling water bath for 10 minutes, then centrifuge at 14000 rpm for 10 minutes after cooling, and collect the supernatant for later use.
[0037] The supernatant was separated by Sephadex G-50 gel chromatography (length 100cm, diameter 2.6cm), the eluent was deionized water, the flow rate was 2mL / min, and the elution peak was measured at 225nm. Elution peak of antifreeze activity.
[0038] The elution peak with the best antifreeze activity separated by Sephadex G-50 gel chromatography is separated in the next step, separated by Sulfopropyl-Sepadex C-25 cation exchange chromatography (length 55cm, diameter 2.0cm), and el...
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