I have question about the protein hydrolysis, we doing some research about the antimicrobial activities of peptides and their zinc complexes extracted from fish. The problem is after hydrolysis; we used ultrafiltration for Fractionation of peptides and then use the lyophilizing to get the powder but every time after lyophilizing we got nothing and we have repeated this for three times the results always same, our experiment steps are as following: 1- Ground fish muscle mixed with deionized water at a ratio of 1:10 (W/V). 2- The pH of the mixture was adjusted to 8.0 with 1 mol/L NaOH. 3- Two different types of enzymes (alkaline protease and papain) 4- The alkaline protease was added to the mixture at a ratio of 22000u/g the hydrolysis was performed at 45°C for 9 h and the pH kept at 8.0 during the hydrolysis time. 5- After 9 h of alkaline protease hydrolysis, the mixture pH was adjusted to 9.0 with 1 mol/L NaOH and then the papain was added to the same mixture at a ratio of 22000u/g the hydrolysis was performed at 45°C for 8 h. 6- The hydrolysis process followed by heating at 90 °C for 30 min to inactivate the enzymes. 7- Filter by centrifugation at 15000 rpm for 10 min 4°C to remove the fat. 8- Chelating the peptides with zinc: the pH of the hydrolysates was readjusted to 7.0, and ZnCl2 2% (50 ml ZnCl2 with final concentration of 1 mol/l was prepared and HCL was used to adjusted the pH, then ZnCl2 was added to hydrolysates (for 250 ml hydrolysate add 2.5 ml ZnCl2). The chelating was performed at 30°C for 30min in water bath. 9- Then absolute ethanol was added to the mixture till ethanol concentration up to 80%, and left for 30 min to precipitate the PZCs (peptides zinc complex). The mixture was then centrifuged at 5000 rpm at 4 °C for 15 min. The precipitation was washed several times with absolute ethanol till free Zn+2 was not detected 10- The precipitation was re-dissolved in dd water and fractionated using ultrafiltration with 3.5 and 1 kDa molecular weight (MW) cut off, then two peptide fractions ( 1-3.5 k and < 1 k) were collected and stored at −80 °C one night then freeze dried 11- After freeze dried we got nothing I do not know what the wrong with this method or is there anything missing in the method? Thanks
Did you see your peptides before drying the sample? To our experience peptides often won't migrate through ultrafiltration membranes. Our 2 kDa peptides did not pass even through membrane with a 16 kDa cut-off. So did you test the sample prep method with a known peptide chelate (reference substance) without any matrix?
What method are you using for the estimation of freeze dried protein. It is next to impossible that you get nothing after lyophilisation. Try ninhydrin reaction to find whether the peptides that you purified by ultrafiltration have further degraded to individual aminoacids or not...
I suggest to analyse the soluble protein (Lowry method) after each treatment and you can see which is the amount of protein after each stage. You know the protein in the initial extract (total protein extract) and in the final product (in solution). The Lowry method is quite simple. Or, another method (UV). You will be cleared about this problem.
Spray drying is another alternative method to get powdered form but then the yield will be lesser than freeze dried product...But, definitely you will get powdered form after spray drying of sample..
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