Gelatin, the degradation product of the protein collagen, is a heterogeneous mixture of water-soluble proteins of high molecular weight. A progressive aggregation and desolvation occurs when non-solvents are added to gelatin solutions. Since fish gelatin has a different molecular weight profile from that of other types of gelatin, it is expected that the degree of interaction between the molecular weight fractions may be different, and hence, so would be the response of the protein to non-solvents such as ethanol. Modification of the net charge of the protein molecules, by adjusting the solution pH to values ranging about the iso-electric point (IEP) of the protein may influence the same interactions. The objective of this work was to determine the response of poikilothermic fish skin gelatin to the non-solvent ethanol at different pH’s at concentrations ranging from 0 to 80% w/w.
The method used was that of Farrugia and Groves (1999). Unbuffered solutions of gelatin were prepared by stirring aqueous suspensions of gelatin at 25°C, with stirring for 20 minutes. The pH was adjusted to values between pH 4 and pH 10 by adding dilute HCl or dilute NaOH. The gelatin solutions prepared above were incubated at 15°C, 25°C or 39°C for 90 minutes and mixed with ethanol/water mixtures that had been similarly incubated, such that the final solutions contained 0.2% w/w gelatin and increasing ethanol concentrations (0 to 80% w/w). The three component systems were incubated at the same temperature for a further 20 minutes and the turbidity of the solutions measured by percentage transmittance using a Shimadzu 160 UV/Vis spectrophotometer operated at 600nm. The data obtained was subjected to nonlinear regression analysis and the parameter V50 (the ethanol concentration at the % transmittance midway between the initial and final values) was used to monitor the effects of the experimental conditions on the phase behaviour of gelatin in solution, lower V50 values being indicative of a greater sensitivity to desolvation.
The behaviour of fish gelatin solutions was observed to be highly dependent both on the temperature and the pH of the setup. Gelatin solutions adjusted to pH 4 were shown to be insensitive to the desolvating effect of ethanol. This insensitivity was also observed from similar solutions adjusted to pH 5 at 15°C and at 25°C. Solutions adjusted to pH 6, 7, 8 and 9 exhibited increased turbidity with increasing ethanol concentration. The V50 values decreased as the pH was increased from 6 to 9; the lowest values were obtained at pH 9. This trend was observed at all temperatures at which the investigation was carried out. It was also noted that the lowest V50 values were obtained for the lowest temperature, which was 15°C; these values increased as the temperature was increased. Such increase across temperature was more pronounced at pH 8 and 9.
It is known that the stability of a colloidal system is determined by the dominance of the electrical double layer’s repulsive forces over the Van der Waals attractive forces that may occur between colloidal particles. When gelatin solutions were adjusted to pH values at which their molecules exhibited no charge stabilisation then desolvation occurred (pH 8 and 9); when charge stabilisation was present, then the gelatin molecules were insensitive to any desolvating effect. These results contrasted with those obtained in earlier studies concerning other types of gelatin, particularly B-type gelatin. The difference in how various gelatin types are extracted affects the product’s IEP range. Whilst B-type gelatin has an IEP at pH 4.7-5, that of fish skin gelatin has is higher and spans a broader range, as reflected itself in the difference these gelatin types have in their sensitivity towards desolvation. In fact, V50 values for fish skin gelatin solutions increased considerably from solutions at pH 5 to solutions at pH 9. Consequently, fish-skin gelatin solutions experience less charge stabilization at higher pH values compared to B-type gelatin solutions, and are thus more sensitive towards desolvation.