Sütte Plazmin ve Kimozin Aktivitesi Üzerine Bazı Fenolik Bileşiklerin Etkisi ve Süreçlere Uyarlanabilirliği

Abstract

Phenolic compounds have been of great interest to food scientists and technologists due to epidemiologically approved health benefits. There are a number of studies investigating possible use of polyphenols in dairy such as yoğurt, ice cream and cheese for developing final product quality and functionality. Besides, inhibitory properties of several phenolics against diverse enzymes have also been reported. However, the interactions between proteolytic enzymes important for dairy industry and polyphenols have been studied to a lesser extent.

In this thesis, the potential inhibition of plasmin and chymosin via phenolic compounds have been investigated with molecular and kinetic studies and the consequences of changes in enzyme activities during cheesemaking and ripening were evaluated. The findings of this study revealed that, interactions between chymosin and phenolics are not strong enough or/and the possible interactions does not lead a conformational change on active site of chymosin leading to unchanged chymosin activity upon binding of polyphenols. However, ECG, EGCG, QUER, MYR and CURC showed 60, 86, 65, 90, 52 % inhibition against plasmin, respectively. The inhibition ratios, however, diminished to 35, 47, 44, 46 ve 40 % in the presence of milk proteins. Spectroscopic and calorimetric studies showed that, the interactions between inhibitory phenolics and plasmin were dominated by H-bonds and electrostatic attractions which leads to a change in secondary structure elements of plasmin, having more ordered structure upon binding to polyphenols. Inhibition modes of plasmin by EGCG, QUER ve MYR were non-competitive, whereas ECG and CURC showed a mixed-type inhibition against plasmin.

Green tea extract (GTE), as a natural source of phenolics, was preferred for model cheesemaking and ripening studies. During cheesemaking, kinetics of rennet coagulation were affected by GTE addition. CMP (caseinomacropeptide) formation rate was found to be decreased by GTE phenolics during enzymatic phase of renneting. In addition, aggregation of para-casein micelles measured by dynamic light scattering spectroscopy (DLS) also retarded in the presence of GTE.

Plasmin activity did not change in the presence of GTE whereas, residual chymosin activity decreased in GTE-added cheeses, compared to control cheese samples. The results of pH 4.6 soluble nitrogen amount analysis, as an indicator of ripening in cheese, showed that the primary proteolysis in GTE cheeses was hindered to some extent. Besides, urea-PAGE anaylysis revealed that, αs1-casein hydrolysis rate was higher for control cheese samples. However, the difference of β-casein hydrolysis among control and GTE added cheeses was statistially insignificant. RP-HPLC anaylsis was carried out in order to evaluate the peptide profile of cheese samples during ripening period. The results revealed that, green tea phenolics did not cause any change in proteolysis pathway, whereas the formation rate of some peptides were found to be less in the presense of GTE phenolics. These results were in agreement with enzyme activity results during ripening.

The effects of phenolics on plasmin and chymosin activity in model systems were described in molecular level. However, the findings observed during cheesemaking and ripening period were not in agreement with model system studies. Knowing the fact that the structure of cheese matrice during ripening has a dynamic structure due to the changes in many parameters, the phenolic-enzyme interactions were probably affected during this processes. Nevertheless, in the light of the findings of this study, the interactions between phenolics and the proteolytic enzymes and the consequences of these interactions were thought to be critical.