Phoebe X. Qi

Secondary structural studies of bovine caseins: temperature dependence of β-casein structure as analyzed by circular dichroism and FTIR spectroscopy and correlation with micellization

Abstract To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between β-casein and the other caseins, three-dimensional models have been presented. Secondary structural prediction algorithms and molecular modeling techniques were used to predict β-casein structure. The secondary structure of bovine β-casein was re-examined using Fourier transform infrared and circular dichroism spectroscopies to test these predictions. Both methods predict a range of secondary structures for β-casein (28–32% turns, 32–34% extended) at 25°C. These elements were highly stable from 5 to 70°C as viewed by circular dichroism. More flexible conformational elements, tentatively identified as loops, helix and short segments of polyproline II, were influenced by temperature, increasing with elevated temperatures. Another view is that as temperature decreases, these elements are lost (cold denaturation). Several distinct transitions were observed by circular dichroism at 10, 33 and 41°C, and another transition, extrapolated to occur at 78°C. Calculations from analytical ultracentrifugation indicate that the 10, 33 and 41°C transitions occur primarily in the monomeric form of the protein. As β-casein polymers are formed, and increase in size, the transitions at higher temperature may reflect changes in the more flexible conformational elements as they adjust to changes in surface charge during polymer formation. The transition at 10°C may represent an actual general conformational change or cold denaturation. Over the range of temperatures studied, the sheet and turn areas remain relatively constant, perhaps forming a supporting hydrophobic core for the monomers within the micelle-like polymer. This interpretation is in accord with the known properties of β-casein, and those predicted from molecular modeling.

Structure and Function of Protein-Based Edible Films and Coatings

Research and development on films and coatings made from various agricultural proteins has been conducted over the past 20 years, but is of heightened interest, due to the demand for environmentally-friendly, renewable replacements for petroleum-based polymeric materials and plastics. To address this demand, films and coatings have been made from renewable resources, such as casein, whey, soy, corn zein, collagen, wheat gluten, keratin and egg albumen. Those made from agricultural proteins create new outlets for agricultural products, byproducts and waste streams, all of which can positively impact the economics of food processes.

Physical properties, molecular structures, and protein quality of texturized whey protein isolate: Effect of extrusion moisture content1

To explore the complex relationship between processing conditions and functional and nutritional properties of food products containing whey protein isolate (WPI), we investigated the effect of extrusion texturization at various temperatures (50, 75, and 100 °C) and varying moisture levels of the feed (20, 30, 40, and 50%) on changes in the composition, molecular structure, and protein quality of the extrudates. Bradford assay methods were used to determine protein solubility of the extruded WPI as a function of changing level of moisture. Protein compositional changes as a function of extrusion conditions were quantitatively characterized and analyzed by sodium dodecyl sulfate-PAGE and reversed-phase-HPLC techniques. We showed that at a given temperature, increasing the extrusion moisture content resulted in a slight increase in the overall protein water solubility (at 50 and 75 °C), averaging approximately 5% per 10% increase in moisture content. A reduction in β-lactoglobulin content was observed at 50 °C with increasing moisture content, indicative of the sensitive nature of β-lactoglobulin to extrusion treatment, whereas the amount of α-lactalbumin remained unchanged at all moisture contents used at a set temperature. The protein quality of the extruded WPI, determined chemically by available sulfhydryl and primary and secondary amines, remained relatively unchanged as a function of moisture level. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic studies revealed considerable structural changes, both at the secondary structural level and the tertiary contacts as a function of increasing temperature, and higher moisture levels can slightly preserve secondary structures but not the tertiary contacts of the protein molecules. Atomic force microscopy provided direct visualization of the fine difference of the protein particles caused by changing extrusion moisture contents, which is in close agreement with the results obtained using other techniques in this work.

Structural determinants of the rate of protein folding

To understand the mechanism of protein folding and to assist rational design of fast-folding, non-aggregating and stable artificial enzymes, it is essential to determine the structural parameters which govern the rate constants of folding, kf. It has been found that -logkf is a linear function of the so-called chain topology parameter (CTP) within the range of 10(-1)s(-1)< or = kf < or =10(8)s(-1). The correlation between -logkf and CTP is much improved than using previously published contact order (CO) method. It has been further suggested that short sequence separations may be preferred for the establishment of stable interactions for the design of novel artificial enzymes and the modification of slow-folding proteins with aggregating intermediates.

Physical Properties, Molecular Structures, and Protein Quality of Texturized Whey Protein Isolate: Effect of Extrusion Temperature

Although extrusion technology has contributed much to increasing the effective utilization of whey, the effect of extrusion conditions on the functional properties of the proteins is not well understood. In this work, the impact of extrusion temperature on the physical and chemical properties, molecular structures, and protein quality of texturized whey protein isolate (WPI) was investigated at a constant moisture content and compared with WPI treated with simple heat only. The Bradford assay methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and reversed-phase high-performance liquid chromatography techniques were used to determine protein solubility and to analyze compositional changes in the two major whey proteins, α-lactalbumin and β-lactoglobulin. Circular dichroism and intrinsic tryptophan fluorescence spectroscopic techniques were applied to study the secondary and tertiary structures of the proteins. This study demonstrated that extrusion temperature is a critical but not the sole determining factor in affecting the functional properties of extruded WPI.

Investigation of molecular interactions between β-lactoglobulin and sugar beet pectin by multi-detection HPSEC.

Molecular interactions between β-lactoglobulin (β-LG) and sugar beet pectin (SBP) were studied using online multi-detection high performance size exclusion chromatography (HPSEC) at neutral pH and 50mM ionic strength. The hydrodynamic properties of various interacting polymer fractions were characterized in detail and compared with those of β-LG and SBP. Results showed that ∼6.5% (w/w) of native dimeric β-LG molecules formed complexes with over 35% SBP molecules of varying sizes, 800, 110 and 75 kDa. Although the β-LG molecules bind to SBP molecules of all sizes and shapes, they tend to favor the intermediate (110 kDa) and small sized (75 kDa) SBP molecules. All resulting complexes possess altered shapes and hydrodynamic properties when compared to unbound SBP and β-LG. About half of the interacting β-LG (∼3.5%) molecules were thought to bind to a small amount of non-covalently bound feruloyl groups, possibly present in SBP. When pre-heat treated β-LG and SBP were combined, more than 16% of β-LG formed complexes with at least 45% of SBP molecules of varying sizes, Mw∼750-800, 110, and 55-80 kDa. The complexes formed between β-LG aggregates and/or oligomers and the large SBP molecules (750-800 kDa) adopt the shape of β-LG aggregates, random coil. Both groups of complexes formed between β-LG intermediate (110 kDa) and small sized (55-80 kDa) SBP take on the shape of rigid rod. It was speculated that half of the interacting heat-treated β-LG molecules (∼8%) are complexed with non-covalently bound feruloyl groups in SBP.

Enrichment and Purification of Casein Glycomacropeptide from Whey Protein Isolate Using Supercritical Carbon Dioxide Processing and Membrane Ultrafiltration

Whey protein concentrates (WPC) and isolates (WPI), comprised mainly of β-lactoglobulin (β-LG), α-lactalbumin (α-LA) and casein glycomacropeptide (GMP), are added to foods to boost nutritional and functional properties. Supercritical carbon dioxide (SCO2) has been shown to effectively fractionate WPC and WPI to obtain enriched fractions of α-LA and β-LG, thus creating new whey ingredients that exploit the properties of the individual component proteins. In this study, we used SCO2 to further fractionate WPI via acid precipitation of α-LA, β-LG and the minor whey proteins to obtain GMP-enriched solutions. The process was optimized and α-LA precipitation maximized at low pH and a temperature (T) ≥65 °C, where β-LG with 84% purity and GMP with 58% purity were obtained, after ultrafiltration and diafiltration to separate β-LG from the GMP solution. At 70 °C, β-LG also precipitated with α-LA, leaving a GMP-rich solution with up to 94% purity after ultrafiltration. The different protein fractions produced with the SCO2 process will permit the design of new foods and beverages to target specific nutritional needs.

Structural and thermal stability of β-lactoglobulin as a result of interacting with sugar beet pectin.

Changes in the structural and thermal stability of β-lactoglobulin (β-LG) induced by interacting with sugar beet pectin (SBP) have been studied by circular dichroism (CD), Fourier transform infrared, and steady-state as well as time-resolved fluorescence spectroscopic techniques. It has been demonstrated that SBP not only is capable of binding to native β-LG but also causes a significant loss in antiparallel β-sheet, ∼10%, accompanied by an increase in either random coil or turn structures. In addition, the interaction also disrupted the environments of all aromatic residues including Trp, Phe, and Tyr of β-LG as evidenced by near-UV CD and fluorescence. When preheated β-LG was combined with SBP, the secondary structure of β-LG was partially recovered, ∼4% gain in antiparallel β-sheet, and Trp19 fluorescence was recovered slightly. Although forming complexes with SBP did not significantly impact the thermal stability of individual secondary structural elements of β-LG, the environment of Trp19 was protected considerably.

Reactions between β-lactoglobulin and genipin: kinetics and characterization of the products.

In this paper, we present the first detailed study of the reaction kinetics and the characterization of the products from the endothermic reactions between β-lactoglobulin and genipin. The effects of the concentration, temperature, and pH were investigated. In the temperature range studied, the reaction was approximately a pseudo-first-order with respect to genipin and 0.22-order and -0.24-order with respect to β-lactoglobulin for pH 6.75 and 10.5 with corresponding activation energy (E(a)) estimated to be 66.2 ± 3.8 and 9.40 ± 0.36 kJ/mol, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis studies, validated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, showed the presence of oligomeric, i.e., di-, tri-, quadri-, and pentameric, forms of cross-linked β-lactoglobulin by genipin at neutral but not alkaline pH; however, an extensive cross-linked network was not observed, consistent with the atomic force microscopy images. It was demonstrated that the reaction temperature and the concentration of genipin but not that of β-lactoglobulin positively affected the extent of the cross-linking reactions.

Extrusion Texturized Dairy Proteins: Processing and Application

The primary proteins in milk, casein and the whey proteins α-lactalbumin and β-lactoglobulin, have a number of health benefits and desirable functional properties. In a twin-screw extruder, mechanical shear forces, heat, and pressure cause considerable changes in the molecular structures of the dairy proteins, a process known as texturization. These changes further impart unique functional properties to dairy proteins, resulting in new protein-based food ingredients. The new functional behavior depends on the extent of texturization and the degree of structural change imparted and is controlled by adjusting parameters such as extrusion temperature and moisture level. Such texturized proteins can be used to produce puffed high-protein snacks. Softer gels and expanded structures can be made using supercritical fluid extrusion and cold extrusion, techniques that avoid elevated temperatures, minimizing possible damage to the nutritive components and functionality of the texturized dairy proteins. The uses of the texturized dairy ingredient in food products with improved functionality and enhanced nutritive profiles are presented.