Methodology for Determination of Degree of Hydrolysis
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AbstractThe utilization of rest raw materials is becoming increasingly important due to the increasing demand for marine food sources. One way to do this is by enzymatic hydrolysis of the rest raw material from fish. Important rest raw materials produced from herring, salmon and cod result in protein hydrolysates with high amounts of proteins with nutritional value, and can therefore help meet the increased demand for marine food sources. The aim of this master thesis is to compare and determine the degree of hydrolysis (DH) of three different types of hydrolysates (herring, salmon and cod) analysed with three different methods (ninhydrin, TNBS and formoltitration). The properties and composition of the hydrolysate were analysed by molecular weight distribution, amount of total and free amino acids and amount of acid soluble peptides to evaluate effects of process parameters on DH. In addition analysis of the amount of hydroxyproline and the lipid content (%) were performed. The present work shows that the DH varies with the method used to analyse the fish protein hydrolysates. The formoltitration method gave a DH, for the herring hydrolysates: 15,3±0,003 mmol/g to 29,7±0,3 mmol/g. The salmon hydrolysates had values: 32,3±0,280 mmol/g to 48,3±0,451 mmol/g. The cod hydrolysates had values of: 11,4±0,009 mmol/g to 15,9±0,006 mmol/g. Utilization of the standard L-Leucine in the ninhydrin method, gave a DH for the herring hydrolysates between 0,8±0,08 to 1,5±0,06mmol/g. Salmon hydrolysates gave a value of 2,3±0,07 to 3,6±0,1 mmol/g and the cod hydrolysates a value of 0,4±0,01 to 1,2±0,1 mmol/g. Analysis of the ninhydrin method with the L-Proline standard gave a DH for herring hydrolysates of 0,4±0,01 mmol/g to 0,7 ±0,01 mmol/g, salmon hydrolysates 0,9±0,05 mmol/g to 1,8±0,1 mmol/g and for the cod hydrolysates 0,1±0,004, mmol/g 0,3±0,01 mmol/g and 0,5±0,03 mmol/g. The TNBS method gave a DH for herring hydrolysates between 89,9±2,7 mmol/g to 161,6±1,2 mmol/g, for the salmon hydrolysates a DH of 37,5±4,8 mmol/g to 79,6±2,2 mmol/g and for the cod hydrolysates a DH in the range 16,0±1,6 mmol/g to 22,4±1,9 mmol/g. Based on a comparison of the different methods, the TNBS method gave the highest DH. The DH found in the TNBS method were opposite for salmon and cod hydrolysates when they were compared to the formoltitration and the ninhydrin method. This could be caused by the inaccuracy of the method, leading to a higher DH. These findings needs to be analysed further to find the cause to this variability. The findings from the formoltitration method gave the next highest values for the DH, with salmon hydrolysates dominating, then the herring hydrolysates and last the cod hydrolysates. This is probably due to the high activity of the endogenous enzymes present in viscera during heating in enzymatic hydrolysis. This is probably also related to the next highest value of herring, containing amongst others viscera. Hydrolysates from cod head, most likely contain a lower amount of endogenous enzymes. These findings might also be correlated to the low content of hydrophobic amino groups that shields the protein from enzymatic hydrolysis, because the amount of these groups increase from the salmon hydrolysates, to the herring hydrolysates and finally to the cod hydrolysates. Heat denaturation and lipid protein interaction are also probable causes of the differences between the three hydrolysates. It is an easy method to apply and it does not need much instruments. It might give some overestimation of the DH and it does mean working with formaldehyde, a health hazardous compound. In this master thesis the DH values from the ninhydrin method were lowest for all the fish hydrolysates compared to both the TNBS and the formoltitration. This is probably due to the method, which has previously been shown in the literature to give low results. Additional evidence to support this assumption it that the overall results from the ninhydrin reaction with both the standards L-Leucine and L-Proline can be explained by the same findings as in the formoltitration method. Further investigation of the ninhydrin is necessary because of the small sample selection. In this study, an inverse relationship was found between the amount of acid soluble peptides and the degree of hydrolysis. A positive correlation was expected. This phenomenon should therefore be studied further. Further analysis of the ninhydrin method with L-Proline, total amino acid composition of the remaining herring, salmon and cod hydrolysates should be performed. Future prospects might be to analyse the hydrolysates with the OPA and pH stat might be valuable as well as multivariate statistical analysis of the results in this master thesis.