Nutritional management of PKU with glycomacropeptide from cheese whey.

Individuals with phenylketonuria (PKU) must follow a lifelong low-phenylalanine (Phe) diet to prevent neurological impairment. Compliance with the low-Phe diet is often poor owing to restriction in natural foods and the requirement for consumption of a Phe-free amino acid formula or medical food. Glycomacropeptide (GMP), a natural protein produced during cheese-making, is uniquely suited to a low-Phe diet because when isolated from cheese whey it contains minimal Phe (2.5-5 mg Phe/g protein). This paper reviews progress in evaluating the safety, acceptability and efficacy of GMP in the nutritional management of PKU. A variety of foods and beverages can be made with GMP to improve the taste, variety and convenience of the PKU diet. Sensory studies in individuals with PKU demonstrate that GMP foods are acceptable alternatives to amino acid medical foods. Studies in the PKU mouse model demonstrate that GMP supplemented with limiting indispensable amino acids provides a nutritionally adequate source of protein and improves the metabolic phenotype by reducing concentrations of Phe in plasma and brain. A case report in an adult with classical PKU who followed the GMP diet for 10 weeks at home indicates safety, acceptability of GMP food products, a 13-14% reduction in blood Phe levels (p<0.05) and improved distribution of dietary protein throughout the day compared with the amino acid diet. In summary, food products made with GMP that is supplemented with limiting indispensable amino acids provide a palatable alternative source of protein that may improve dietary compliance and metabolic control of PKU.

Solvent type affects the number, distribution, and relative quantities of volatile compounds found in sweet whey powder.

This study compares the performance of diethyl ether, methylene chloride, methyl formate, and pentane in the analysis of volatile flavor components in sweet whey powder. Extracts were prepared from sweet whey powder using each solvent. Volatile components were isolated by solvent extraction followed by solvent-assisted flavor evaporation. Gas chromatography-mass spectroscopy, coelution with known standards, and retention indices were used to identify the volatile compounds. Sixty total compounds were either positively or tentatively identified across all 4 solvents, but the number, distribution between the molecular classes, and relative quantities detected depended on solvent type. The highest number, widest distribution, and greatest relative quantities were found using methylene chloride and methyl formate, whereas diethyl ether and especially pentane were noticeably less effective. Results are characterized using molecular-based characteristics of solvents and solutes including dipole moment, dielectric constant, Log P (octanol-water partition coefficient), polarizability, water solubility, and Lewis acidity/basicity. Polarity and acidity/basicity were the primary factors that determined solvent performance. This work establishes a molecular-level basis for the selection of solvents in the analysis of sweet whey powder flavors.

Equilibrium adsorption of LDL and gold immunoconjugates to affinity membranes containing PEG spacers.

The objective of this study was to provide insight into the effects of spacer chemistry on immunoaffinity separations for the capture of large macromolecules and biological complexes. Immunoaffinity membranes were prepared by immobilization of immunoglobulin G (IgG) to flat sheet microporous membranes. Two different systems were examined: immobilized IgG for the immunoadsorption of human low-density lipoprotein (LDL) and immobilized IgG for the immunoadsorption of gold particle immunoconjugate. The IgG was immobilized either directly to the membrane or via a polyethylene glycol (PEG) spacer. Adsorption of LDL was significantly greater for anti-LDL IgG immobilized via PEG than for IgG immobilized directly to the membrane. With the PEG spacer, the adsorption capacity for LDL matched the theoretical density of a monolayer of LDL particles on the membrane surface. The gold particle immunoconjugate, similar in size to LDL, was examined as a generalized model of restrictions to immunoaffinity adsorption of large (>20 nm) biological complexes. Adsorption of gold particles was greater for IgG immobilized via PEG than for IgG immobilized directly to the membrane. It is postulated that the PEG spacer allows lateral movement of the immobilized IgG and dense monolayer packing of adsorbed particles on the membrane surface. These results are pertinent to the removal of LDL from human plasma and the purification of gene therapy delivery vectors, viral vaccines, and other large biological complexes.

Mass transfer limitations in protein separations using ion-exchange membranes.

Sorption of bovine serum albumin to commercial 150-micron pore size membranes was measured in batch and flow experiments. For residence times of 2-40 min, early and broad breakthrough curves and broad asymmetric elution peaks were observed that depended strongly on flow-rate. System dispersion could not explain the flow-rate dependence. Breakthrough and elution curves were analyzed using new models that included Langmuir sorption, convection and diffusion. From the analysis, film mass transfer resistance was found to be the rate-limiting factor. The maximum allowable pore size that eliminates this limitation was calculated for different molecular weight solutes.

Sorption kinetics and breakthrough curves for pepsin and chymosin using pepstatin A affinity membranes.

Isotherms and kinetic parameters for pepsin and chymosin sorption to immobilized pepstatin A were measured in batch experiments. The measured single-solute parameters were used in an affinity-membrane model which included competitive sorption kinetics, axial diffusion and dead volume mixing. The predictions made using the affinity-membrane model matched the experimental breakthrough curves, whereas predictions made using local-equilibrium theory were a distinct mismatch. The performance of affinity-membrane separations was dominated by slow sorption kinetics.

Competitive adsorption of alpha-lactalbumin and bovine serum albumin to a sulfopropyl ion-exchange membrane.

Breakthrough curves were measured for pure solutions of alpha-lactalbumin (ALA) and bovine serum albumin (BSA) individually, and for a binary mixture of the proteins, using a sulfopropyl ion-exchange membrane. The breakthrough curves were qualitatively consistent with local-equilibrium theory predictions. Competitive adsorption caused displacement of bound BSA monomer by the more strongly binding BSA dimer, illustrating that even apparently single-protein systems may display multicomponent competitive behavior. In the two-protein experiment, ALA was competitively displaced by the more strongly binding BSA monomer and dimer, indicating that the binding strength was in the order: BSA dimer > BSA monomer > ALA.

Conversion of sugars to 1,2-propanediol by Thermoanaerobacterium thermosaccharolyticum HG-8.

The purpose of this study was to explore a fermentation route for the production of 1,2-propanediol (1,2-PD) from renewable sugars: lactose found in cheese whey, and D-glucose, D-galactose, L-arabinose, and D-xylose found in corn and wood byproducts. Thermoanaerobacterium thermosaccharolyticum, a naturally occurring organism, was found to ferment a wider range of sugars to 1,2-PD than previously reported. The specific sugar had a significant effect on the selectivity for 1,2-PD vs other fermentation products such as ethanol, D- and L-lactate, and acetate. T. thermosaccharolyticum potentially provides an environmentally friendly route to a major commodity chemical now made from petrochemicals.

Charged ultrafiltration membranes increase the selectivity of whey protein separations.

Ultrafiltration is widely used to concentrate proteins, but fractionation of one protein from another is much less common. This study examined the use of positively charged membranes to increase the selectivity of ultrafiltration and allow the fractionation of proteins from cheese whey. By adding a positive charge to ultrafiltration membranes, and adjusting the solution pH, it was possible to permeate proteins having little or no charge, such as glycomacropeptide, and retain proteins having a positive charge. Placing a charge on the membrane increased the selectivity by over 600% compared to using an uncharged membrane. The data were fit using the stagnant film model that relates the observed sieving coefficient to membrane parameters such as the flux, mass transfer coefficient, and membrane Peclet number. The model was a useful tool for data analysis and for the scale up of membrane separations for whey protein fractionation.

In vitro characterization of a membrane-based low-density lipoprotein affinity adsorption device.

The objective of this study was to explore the use of microporous membranes as an alternative substrate to porous beads in affinity adsorption of low-density lipoprotein (LDL) for therapeutic purposes. Flat sheet immunoaffinity membranes containing a polyclonal antibody preparation were utilized as the affinity substrate. The antibody was covalently immobilized to the surface through a poly(ethylene glycol) (PEG) spacer. Equilibrium adsorption of LDL from plasma was measured. Adsorption from plasma and elution of bound LDL using citrate buffer were studied as a function of flow rate. Specific capacity was as great as 2 mg apolipoprotein B per milliliter membrane volume. The superior transport properties of the membrane allowed rapid adsorption and regeneration, which translated to a large number of adsorptive cycles that can be performed within a given treatment time. On the basis of in vitro performance characteristics, it is estimated that an immunoaffinity membrane device can provide a reduction in patient plasma LDL concentration comparable to that provided by packed columns, but with almost an 80% reduction in the device volume.