Use of whey protein beads as a new carrier system for recombinant yeasts in human digestive tract.

A new immobilizing protocol using whey protein isolates was developed to entrap recombinant Saccharomyces cerevisiae. The model yeast strain expresses the heterologous P45073A1 that converts trans-cinnamic acid into p-coumaric acid. Beads resulted from a cold-induced gelation of a whey protein solution (10%) containing yeasts (7.5 x 10(7)cells ml(-1)) into 0.1M CaCl(2). The viability and growth capability of yeasts were not altered by our entrapment process. The release and activity of immobilized yeasts were studied in simulated human gastric conditions. During the first 60 min of digestion, 2.2+/-0.9% (n=3) of initial entrapped yeasts were recovered in the gastric medium suggesting that beads should cross the gastric barrier in human. The P45073A1 activity of entrapped yeasts remained significantly higher (p<0.05) than that of free ones throughout digestion (trans-cinnamic acid conversion rate of 63.4+/-1.6% versus 51.5+/-1.8% (n=3) at 120 min). The protein matrix seemed to create a microenvironment favoring the activity of yeasts in the stringent gastric conditions. These results open up new opportunities for the development of drug delivery system using recombinant yeasts entrapped in whey protein beads. The main potential medical applications include biodetoxication or the correction of digestive enzyme deficiencies.

Composition and structure of whey protein/gum arabic coacervates.

Complex coacervation in whey protein/gum arabic (WP/GA) mixtures was studied as a function of three main key parameters: pH, initial protein to polysaccharide mixing ratio (Pr:Ps)(ini), and ionic strength. Previous studies had already revealed under which conditions a coacervate phase was obtained. This study is aimed at understanding how these parameters influence the phase separation kinetics, the coacervate composition, and the internal coacervate structure. At a defined (Pr:Ps)(ini), an optimum pH of complex coacervation was found (pH(opt)), at which the strength of electrostatic interaction was maximum. For (Pr:Ps)(ini) = 2:1, the phase separation occurred the fastest and the final coacervate volume was the largest at pH(opt) = 4.0. The composition of the coacervate phase was determined after 48 h of phase separation and revealed that, at pH(opt), the coacervate phase was the most concentrated. Varying the (Pr:Ps)(ini) shifted the pH(opt) to higher values when (Pr:Ps)(ini) was increased and to lower values when (Pr:Ps)(ini) was decreased. This phenomenon was due to the level of charge compensation of the WP/GA complexes. Finally, the structure of the coacervate phase was studied with small-angle X-ray scattering (SAXS). SAXS data confirmed that at pH(opt) the coacervate phase was dense and structured. Model calculations revealed that the structure factor of WP induced a peak at Q = 0.7 nm(-1), illustrating that the coacervate phase was more structured, inducing the stronger correlation length of WP molecules. When the pH was changed to more acidic values, the correlation peak faded away, due to a more open structure of the coacervate. A shoulder in the scattering pattern of the coacervates was visible at small Q. This peak was attributed to the presence of residual charges on the GA. The peak intensity was reduced when the strength of interaction was increased, highlighting a greater charge compensation of the polyelectrolyte. Finally, increasing the ionic strength led to a less concentrated, a more heterogeneous, and a less structured coacervate phase, induced by the screening of the electrostatic interactions.

Iron derivatives of modified milk protein.

A product containing a high amount of iron and maintaining all the necessary characteristics of stability and solubility for a drug can be prepared by succinylating the proteins, in the specific case the milk proteins, before the reaction with the iron salt. For these derivatives a particular advantage can be achieved, the iron succinyl protein precipitates at the pH of the stomach further keeping the iron bonded and it re-solubilizes at pH higher than 7, the intestine pH level. Electrophoresis indicates that the iron succinyl protein is homogeneous and gel filtration shows an apparent high molecular weight that contributes to the stability of the complexed iron. The preliminary structure determined by analytical and physical-chemical methods indicates that iron, in the form of oligomeric complexes, is tightly bonded to the protein not in chelated structures with basic residues but involving several sites of the protein chain. The solubility of the iron complex is assured by the increased availability of carboxyl groups that follows the succinylation reaction.