| Antifreeze Protein(AFP) is a kind of specific polypeptides or glycopeptides which produced by creatures, in order to accommodate the extremely cold environments. AFP have been found in a variety of species, including fishes, insects, plants, bacterias and fungus. Though the structures of AFP from the different species often differ greatly, they all depress the non-equilibrium freezing points of water without appreciably changing the melting point of ice. This temperature difference between the melting point and the freezing point is termed thermal hysteresis activity(THA). AFP is characterized by their ability to bind ice, prevent and regulate its growth and recrystallization. Antifreeze protein shows extensive applications in food, medicine, biological and chemical industry.Although classes of macromolecular AFP and their gene sequence have been identified, lower yield and higher cost of AFP restrict their wide application. Due to its peculiar molecule structure, collagen protein riched in livestock may contain highly active AFP. Therefore, collagen of pigskin were extracted by enzyme, THA was taken as indexes to prepare collagen antifreeze peptideis with enzyme, and its functional properties were also studied. The main contents are as follows:THA of collagen was measured with differential scanning calorimetry(DSC) and the influencing of hold temperature, collagen concentrations, and heating and cooling rates on THA were investigated. The results suggested that a maximum THA of 0.61℃ could be obtained with the protein concentration of 20 mg/ml, hold temperature of 0.12℃ and the heating and cooling rate of 10℃/min. The choice of hold temperature was critical for effective measure of THA, while the heating and cooling rates had almost no impact on the THA. According to the study the determination procedure of DSC was finalized as follows: Collagen was dissolved in distilled water, with the final protein content of 20 mg/m L. When DSC was stable, an aliquot of 10 μL of the sample was sealed in the aluminum pan followed by the balancing on the control desk for 5 min; the temperature was droped to-20℃ for 5 min firstly, and then was rose to 10℃ for 5 min, obtaining melting heat and melting point of collagen solution. Again, the temperature was droped to-20℃ for 5 min, then heated up collagen solution to make collagen solution partial melting slowly, namely arrived its hold temperature, keeping for 15 min, then made the temperature reached-20℃ once again. The results showed that the stability, repetition, and accuracy of DSC measurement were high enough for the following studies with lower standard deviation and relative standard deviation of THA, 2.65% and 3.11%.Type-I collagen was prepared from the pigskin using enzyme method, and its purity and amino acid composition were detected. The results suggested that the molecular weight of collagen was about 330 KD with higher purity. The Gly, Ala, and Pro abound in collagen, Gly made up 1/3 of its amino acid constitute, was 30.6%; the content of Ala and Pro were 9.9% and 116%, respectively. Pro was highly hydroxylated with 52.46%. Then taking Type-I collagen as a feedstock, the influence of temperature, amount of enzymes, p H, collagen concentration and DH on the THA of hydrolysates were investigated. The results showed that Alcalase was the most effective enzyme. The p H, collagen concentration and DH had significant impact on THA. And on this basis, response surface experiments were conducted to optimize the preparation of enzymatic hydrolysate of pigskin collagen taking THA as response value, the optimum hydrolysis conditions were estimated as DH of 9%, p H of 7.8, collagen concentration of 20.75 mg/ml. A maximum THA of 5.31℃ could be obtained with enzymatic hydrolysate concentration of 200 mg/ml. The amino acid composition and molecular weight distribution of enzymatic hydrolysate with highly active THA were determined. The relative molecular mass were mainly distributed between 180 to 1000 Da, accounting for about 72.03%。Adding the enzymatic hydrolysate with highly active THA into ice cream, the viscosity, expansion ratio, melting rate, microstructure, the growth of ice crystals recrystallization inhibition activity and glass transition temperature of the ice cream were determined. The results suggested that the viscosity and expansion ratio of ice cream showed increasing trend with the addition of enzymatic hydrolysate; the maximum viscosity and expansion ratio of 34.5m Pa·s and 26.87% were obtained with enzymatic hydrolysate addition of 0.5%. Compared with control group, ice cream with enzymatic hydrolysate laged 8 min behind to begin melting, and the significant improvements of bubble distribution and size were achieved as well; enzymatic hydrolysate could inhibit the growth of ice crystal and recrystallization of ice significantly, and started to show the recrystallization inhibition activity with the additive amount of 0.2%; enzymatic hydrolysate could also increase significantly the glass transition temperature of ice cream, a maximum glass transition temperature of-17.64℃ could be achieved with the additive amount of 0.5%, which make for the vitrification storage of ice cream; In addition, sensory evaluation was carried out to study the influence of enzymatic hydrolysate on the quality of ice cream by h-function method of fuzzy mathematics; enzymatic hydrolysate could obviously improve the quality of ice cream, and got the highest score of organoleptic evaluation with adding enzymatic hydrolysate of 3% into ice cream. |
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