Optimization of enzymatic hydrolysis of shrimp waste for recovery of antioxidant activity rich protein isolate.

Shrimp waste is an important source of astaxanthin, which occur as a complex with proteins, and protein isolates as well as carotenoids are known to possess antioxidant activity. Investigations were carried out to optimize hydrolysis of shrimp waste using a bacterial protease to obtain antioxidant activity rich protein isolate. The effect of three process variables namely enzyme concentration to waste, incubation temperature and time on carotenoid recovery, protein content, trichloro acetic acid (TCA) soluble peptide content and DiPhenyl Picryl Hydrazylchloride (DPPH) scavenging activity was evaluated using a fractionally factorial design. A high correlation coefficient (>0.90) between the observed and the predicted values indicated the appropriateness of the design employed. Maximum carotenoid recovery was obtained by hydrolysing the shrimp waste with 0.3 % enzyme for 4 h. DPPH radical scavenging activity of carotenoprotein isolate was markedly affected by enzyme concentration, temperature and time of hydrolysis. The study indicated that in order to obtain the carotenoprotein from shrimp waste with higher carotenoid content hydrolysing with an enzyme concentration of 0.2-0.4 %, at lower temperature of 25-30° upto 4 h is ideal. However, in order to obtain the protein isolate with increased antioxidant activity hydrolysing at higher temperature of 50 °C, with higher enzyme concentration of 0.5 % for shorter duration is more ideal.

An autolytic process for recovery of antioxidant activity rich carotenoprotein from shrimp heads.

Studies were carried out to utilize in situ proteases of shrimp heads to recover carotenoproteins possessing antioxidant activity. Highest protease activity of the buffer extract was found at pH 8.0 (9.85 ± 0.61 units). The protease activity increased with temperature up to 50°C and reduced thereafter with highest activity being 19.32 ± 2.0 units. Thus, the autolysis of shrimp heads for recovery of carotenoprotein was carried out at pH 8.0 and at 50°C. Waste to buffer ratio had a significant (p < 0.05) effect on recovery of carotenoids in carotenoprotein filtrate with a maximum of 58.5 ± 6.4% recovery with a waste to buffer ratio of 1:2.5 (w:v). The carotenoid recovery increased significantly to 63.4% ± 3.6% at the end of a 4-h autolysis. The studies on combined effect of waste to buffer ratio and autolysis time indicated increase in protein recovery with increase in waste to buffer ratio but not with autolysis time. DPPH scavenging activity of the carotenoprotein isolate increased with autolysis time up to 100 min, and thereafter, reduced above 160 min of autolysis time. With increase in waste to buffer ratio, the scavenging activity increased, reaching more than 12.5 mg TBHQ equivalent/mg protein at waste to buffer ratio of 1:5. The optimum autolysis condition for obtaining antioxidant activity rich carotenoprotein from shrimp heads was found to be waste to buffer (pH 8.0) ratio of 1:5 and an autolysis time of 2 h at 50°C. The isolated carotenoprotein was found to have antioxidant activity with respect to singlet oxygen quenching, reducing power and metal chelating activity.

Purification of alkaline protease from chicken intestine by aqueous two phase system of polyethylene glycol and sodium citrate.

An aqueous two phase system of polyethylene glycol (PEG) and salts was evaluated for separation and purification of alkaline proteases from chicken intestine. Among the different salts evaluated potassium phosphate and sodium citrate gave higher enzyme yield (73.5% and 69.7% respectively) and enzyme purification (5.3 and 7.4 fold) in PEG rich upper phase. Increase in concentration of sodium citrate in the system resulted in reduction in enzyme yield and enzyme purification factor, with 15% salt showing highest enzyme yield (59.8%) and purification (6.7 fold). Initial protein concentration in the system did not show any specific trend on the partition behavior of the enzyme. The temperature at which the system is incubated did not show any significant (p ≥ 0.05) effect on enzyme partition and purification. Increasing the PEG concentration in the system from 15 to 25% resulted in reduction in enzyme yield from 53.7 to 21.9% and enzyme purification from 5 fold to 1.4 fold in PEG rich upper phase. pH also had a significant (p < 0.05) effect on the partition of the enzyme to the upper phase with highest purification (3.4 fold) at pH 9.0 and higher enzyme yield (46.2%) at pH 10.