ABSTRACT
A ß-Lactoglobulin fraction (r-ßLg) was isolated from milk whey hydrolysates produced with cardosins from Cynara cardunculus. The impact of the technological process on the r-ßLg structure and how in turn this determined its heat-induced gelation was investigated. Results were analysed taking pure ß-Lg (p-ßLg) as control sample. The process induced changes in the r-ßLg native conformation causing exposure of hydrophobic groups, lower thermal stability and also, shorter thermal treatments needed to give rise to non-native and aggregated species. At pH 3.2, r-ßLg and p-ßLg solutions exhibited two gelation steps, with the advantage that r-ßLg protein may form stable gels at lower temperature than p-ßLg. At pH 7.2, a specific thermo-viscoelastic stability to 73 °C was found, which corresponded to the gel point in both protein solutions. The difference was that while for p-ßLg solution in sol state δ<45° (solid-like), however for r-ßLg solution δ>45° (fluid-like).
Subject(s)
Lactoglobulins/chemistry , Whey Proteins/chemistry , Whey/chemistry , Animals , Cattle , GelsABSTRACT
Three mathematical models, two logistic models (previously published in previous works) and one mechanistic, developed in this work and based on Michaelis-Menten kinetics, were compared to select the most adequate model in describing the angiotensin-converting enzyme (ACE)-inhibitory activity of bioactive peptide mixtures obtained from cheese whey protein. The significance of both the model and its parameters as well as the value of the regression coefficient was used as criteria to select the most adequate model for obtaining the IC(50) values corresponding to each bioactive peptides mixture. The best results were obtained with the Michaelis-Menten-based model because it provided the best fits and in addition the values for its parameters were always significant. As parameters of this model have a physical meaning, it could be used for inhibition-testing experiments in the development of novel bioactive peptides. The results obtained indicated that the peptide mixture derived from the neutrase hydrolysis exhibited strong ACE inhibition activity. The main active peptides were short, with molecular masses below 1 kDa (IC(50) = 40.37 ± 2.66 µg/mL) and represent 38% of the initial protein content in the hydrolysate.
Subject(s)
Angiotensin-Converting Enzyme Inhibitors/chemistry , Cheese/analysis , Milk Proteins/chemistry , Peptides/chemistry , Protein Hydrolysates/chemistry , Animals , Cattle , Hydrolysis , Kinetics , Models, Theoretical , Peptidyl-Dipeptidase A/chemistry , Rabbits , Whey ProteinsABSTRACT
Two N-terminally truncated variants of the esterase E34Tt from Thermus thermophilus HB27 (YP_004875.1) were expressed in Kluyveromyces lactis. Production and biochemical properties of both recombinant proteins were investigated. The esterase activity was greatly increased compared to the wild-type strain. In particular, the extracellular production of the ΔN16 variant (KLEST-3S) was 50-fold higher than that obtained with T. thermophilus HB27. Response surface methodology was applied to describe the pH and temperature dependence of both activity and stability. When compared with the wild type esterase, the optimal temperature of reaction decreased 35 and 15 °C for ΔN16 and ΔN26, respectively. KLEST-3S showed a maximum of activity at pH 7.5 and 47.5 °C, and maximal stability at pH 8.1 and 65 °C. KLEST-5A (ΔN26) did not show an absolute maximum of activity. However, best results were obtained at 40 °C and pH 8.5. KLEST-5A showed also a lower stability. In the presence of a surfactant, both proteins showed lower stability at 85 °C (t(½)< 5 min) than the wild-type enzyme (t(½)=135 min). However, in the absence of detergent, the stability of KLEST-3S was higher (t(½)=230 min, at 85 °C) than that of the mutant KLEST-5A (12 min) or the wild type enzyme (19 min). Minor differences were observed in the substrate specificity. Our results suggest that the N-terminal segment is critical for maintaining the hyperthermophilic function and stability.