Experimental Results and Discussion:
Dataset collection
By-products of 120 fresh farmed tilapia (oreochromus niloticus) were collected every month over a year at Kafrelsheikh Governorate, Egypt (One of the most important areas in the production of tilapia in the world). We collected fish byproduct under measured parameters (weight, Sex, length, water quality, ration). Then, they were minced and stored at −30 °C till use. The following steps shows the Enzymatic hydrolysis reaction process for preparing the data samples.
Thawing the stored by product over night in cold place(4 °C).
15% of the samples volume mixed with 50 ml phosphate buffer saline (pH 7.5).
Pre-incubation at 60 °C for 20 minutes.
Adding alcalase enzyme (2.5%)to initiate the enzymatic hydrolysis reaction.
Heating in water bath (90 °C) for 15 minutes.
Cooling in ice.
Centrifuge the cooling mixture for twenty minutes at 10000 rpm then the hydrolysis degree was measured to the supernatant according to
27.
Supernatant extraction then freeze dried and characterized.
Preparation Phase
Analysis of Tilapia fish by-product and its hydrolysates powder
The contents of tilapia fish by-product and its hydrolysates were measured according to AOAC method
28, The protein content was determined using kjeldal method. Moisture percentage was estimated with drying method. In addition, Ash content was measured by muffle furnace. Within our study, the protein hydrolysates were extracted with alcalase enzyme with appropriate PH and temperature
29.
Amino acid sequence analysis
According to
30, stacking and separating gel were prepared using gel buffer with percentage 4% and 16% respectively. Heating the sample mixture with the buffer till 90 °C for 10 min, then loading into specific wells. Protein standards (1.06 kDa to 26.6 kDa) were also performed on the gels. Fixing, staining and destaining solutions were mixed with gel, after electrophoresis then comparing the resulted protein bands with the standard ones
31.
Tricine SDS-PAGE analysis
According to
30, we performed Tricine-SDS-PAGE by preparing gel buffer with 4% and 16% stacking and separating gel respectively, then fixing solution was added to gels. After that staining solution was added before the destaining solution. Comparing the resulted bands with standard protein bands.
Measuring the ACE inhibition Activity:
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Results