Protein structure affects the bioactivity and functionality of whey protein ingredients in food systems. Bioactivity of whey proteins and their derivatives are highly dependent upon primary, secondary and tertiary structure. The degree of denaturation of whey proteins is an important factor for determining how whey protein ingredients will perform in a food system. Several analytical methods have been developed to quantify protein denaturation of whey proteins. The goal of this project was to use a variety of analytical methods to quantify whey protein denaturation and to evaluate the correlation of denaturation to the functionality of whey protein powders. The objective of the first series of experiments was to compare three different analytical methods to measure denaturation of whey proteins in liquid whey obtained by various methods of separation and with varying degrees of heat treatment. A split plot experimental design was used. Raw bovine milk was skimmed and liquid whey was separated from the skim milk at natural pH. Three separation methods: 1) centrifugation, 2) membrane filtration and 3) enzyme coagulation, made up the first split plot. Each sub-plot of liquid whey was then divided into three split plots to receive heat treatment. Heat treatments were no heat, 76°C for fifteen seconds and 85°C for three minutes. Each of the resulting nine treatment combinations was analyzed by 1) polyacrylamide gel electrophoresis, 2) bicinchoninic acid-soluble protein assay and 3) fluorescence spectroscopy to determine the amount of denatured protein in the liquid whey. Fluorescence spectroscopy was found to be the most sensitive and reliable method for detecting differences in structure due to denaturation, while native polyacrylamide gel electrophoresis was found to be the least sensitive method. The sample which received the centrifugal treatment of isolation with no heat was found to be the most undenatured in structure while the sample which received the enzyme treatment of isolation with high heat was found to be the most denatured in structure. The objective of the second series of experiments was to evaluate the effect of denaturation on whey protein solubility in dried whey protein powders. Solubility is one of the most important functional properties to consider when selecting a whey protein ingredient, especially for beverage systems. Processing parameters are often manipulated in efforts to improve solubility. The protein structures of whey are considered to have an effect on solubility. Specifically, the degree of denaturation of whey proteins is thought to play a role in solubility. In this experimental design, raw bovine milk was skimmed and pasteurized then enzyme-coagulated at natural pH to separate the whey. Liquid whey was then split into three aliquots and each received one of the following treatments: 1) mild heat/ freeze dry, 2) mild heat/spray dry and 3) high heat/spray dry. Heat treatment was applied to liquid whey prior to concentration. Heat treated whey was then concentrated and dried. Powders were reconstituted and analyzed for denaturation using 1) bicinchoninic acid assay for soluble protein and 2) fluorescence spectroscopy and for solubility using an insolubility index. pH 4.6 solubility and fluorescence spectroscopy for quantifying denaturation correlated well to one another. Both found that the low heat treated samples were less denatured in structure than the sample which received the high heat treatment, regardless of drying method. However, the drying method of the protein powders was correlated to solubility rather than heat treatment. A correlation of denaturation measured in whey protein powders and solubility was apparent for the low heat, freeze dried sample and the high heat, spray dried sample. Several conclusions were made in this research. 1) Centrifugal force causes less denaturation than membrane filtration and enzyme coagulation, thus unheated liquid whey obtained by centrifugal force can be used as a control in research on denaturation. 1) Fluorescence spectroscopy is a better method for quantifying denaturation in liquid and powdered whey compared to native PAGE and pH 4.6 solubility measured by BCA. 3) Functional solubility is dependent on denaturation and can be correlated to analytical methods of measuring denaturation.

Yes / It is important for the formulators of biopharmaceuticals to predict the folding–unfolding transition of proteins. This enables them to process proteins under predetermined conditions, without denaturation. Depending on the apparent denaturation temperature (Tm) of lysozyme, we have derived an equation describing its folding–unfolding transition diagram. According to the water content and temperature, this diagram was divided into three different areas, namely, the area of the water-folded lysozyme phase, the area of the water-folded lysozyme phase and the bulk water phase, and the area of the denatured lysozyme phase. The water content controlled the appearance and intensity of the Raman band at ∼1787 cm–1 when lysozyme powders were thermally denatured at temperatures higher than Tm. / MAM gratefully acknowledges CARA (Stephen Wordsworth and Ryan Mundy) and University of Bradford for providing an academic fellowship.

Trypsin and alpha-chymotrypsin were immobilized on nylon pellets or porous glass by covalent methods to change molecular properties and functional characteristics of soybean proteins. The amount of trypsin immobilized on nylon pellets using the glutaraldehyde method was high when the pellets were treated with methanolic solution and 6 - 8% glutaraldehyde as well as high concentrations of soluble trypsin. Immobilized trypsin and chymotrypsin had uniform pKm and were stable at high temperatures. The optimum pH for activity of immobilized enzymes could be changed by using different supports and different methods of immobilization. A multi-enzyme system with immobilized trypsin and chymotrypsin was designed to produce an efficient hydrolysis and various desirable products of hydrolysis. Controlled hydrolysis of soybean proteins by immobilized enzyme(s) increased water holding capacity, oil holding capacity, and relative viscosity, and improved emulsifying and foaming characteristics. Hydrolysis by immobilized protease(.s) increased solubility, relative viscosity and foaming ability of partially purified fractions. Succinylated soybean proteins had high oil holding capacity, viscosity, emulsifying ability, emulsion stability, and foaming ability. The order in which succinylation and hydrolysis by immobilized enzymes were done, conferred on soybean proteins various functional properties. Evaluation of the molecular size of modified soybean proteins with sodium dodecyl sulfate (SDS) indicated that immobilized trypsin and chymotrypsin preferentially hydrolyzed specific protein components, and that succinylation enhanced hydrolysis, expanded protein molecules, and dissociated subunits. Measurement of molecular size and charge of the modified soybean proteins without SDS showed that immobilized trypsin hydrolyzed the intermediate subunits relatively fast, and succinylation separated the intermediate subunits. Succinylation increased the average molecular charge of soybean proteins, while hydrolysis decreased their average molecular size and their average molecular charge. The ratio of the average molecular weight to the average molecular charge could explain various functional properties. When the ratio was less than 5.0 x 10⁵, the modified soybean proteins had high soluble amino groups, high foaming ability, low water holding capacity and low foam stability. When the ratio was 9.0 x 10⁵, oil holding capacity, emulsifying ability, and emulsion stability were maximum. Relative viscosity was high at a constant value of 2.5 x 10⁵. / Ph. D.

As terapias térmicas vêm sendo utilizadas com muita freqüência em áreas da saúde, como ortopedia, dermatologia e oftalmologia entre outras. Em média a literatura revela que na prática de tratamentos com calor os tecidos são submetidos a temperaturas que não ultrapassam os 45 ºC (igual aproximadamente 8,5 acima da média da temperatura corpórea normal). Este trabalho procurou verificar de fato a possível influência das variações térmicas sobre o colágeno, quanto à possibilidade de desnaturação do mesmo. Utilizou-se 48 ratos (Rattus Novergicus Albinus) da raça Wistar machos, divididos em oito grupos: grupo I Controle (sem alteração térmica), grupo II (35 ºC), III (40 ºC), IV (42 ºC), V (45 ºC), VI (48 ºC), VII (50 ºC), VIII (55 ºC) (cujos membros inferiores foram submersos por 10 minutos em água aquecida com temperatura variável). Em seguida os animais do grupo controle e experimental foram sacrificados, retirando por dissecação e tenotomia os tendões calcâneos que conferidos às lâminas histológicas passaram por análise microscópica de medidas de birrefringência, análise histológica por microscopia de luz e análises por calorimetria exploratória diferencial (DSC). Os resultados mostram que existe uma diferença significativa, dos grupos I a IV, em relação aos grupos V a VII, obtidos pela média de valores de retardos ópticos (RO), após as análises de medidas de birrefringência, confirmados pela análise visual histológica e pela calorimetria exploratória diferencial, sugerindo que em temperaturas elevadas ocorre uma desnaturação da proteína colágeno. / Thermal therapies are being used in orthopedic, dermatology and ophthalmology, among other medical specialties. Literature reveals in the clinical practice of thermal therapies, 45 ºC is the temperature of choice (= 8.5 below human body temperature). The purpose of this study was to quantify changes in the tendon of calcaneous after a range of thermal exposure, and correlate these results with tissue denaturation. It was used 48 Wistar rats (Rattus Novergicus Albinus), male. They grouped as: I (Control) II (35 ºC), III (40 ºC), IV (42 ºC), V (45 ºC), VI(48 ºC), VII (50 ºC), VIII (55 ºC), and had their pad submerged during 10 minutes in hot water (varying from 35 to 55 ºC. Animals were sacrificed and tendon of calcaneous were analyzed by birefringence microscopy, histology (H & E and Trichromic of Masson) and differential scanning calorimetry (DSC). Results showed that there is a significant difference between groups I-IV and V-VIII. Groups I-IV not shows signals of denaturation by heat treatment. Heatings above 45 ºC resulted in thermal denaturation, decreasing o birefringence and changes in histological aspect.

Although the native conformation of a protein is thermodynamically its most stable form, this stability is only marginal. As a consequence, globular proteins have a certain amount of flexibility in their backbones which allows for conformational changes in the course of their biological function. In the course of this thesis, we study protein models at the edge of stability in different contexts: (1) First, we use molecular dynamics to determine the force needed to rupture a chain molecule (an unfolded protein) being stretched at constant loading rate and temperature. When all energy bonds of the molecule are identical, we find that the force F depends on the pulling rate r and temperature T according to F ~ const -- T 1/3|ln(r/T)|1/3 When a single weak bond is introduced, this result is modified to F ~ const -- T2/3|ln(r/ T)|2/3 This scaling, which is model independent, can be used with force-spectroscopy experiment to quantitatively extract relevant microscopic parameters of biomolecules. (2) Second, we study the structural stability of models of proteins for which the selected folds are unusually stable to mutation, that is, designable. A two-dimensional hydrophobic-polar lattice model is used to determine designable folds and these folds were investigated under shear through Langevin dynamics. We find that the phase diagram of these proteins depends on their designability. In particular, highly designable folds are found to be weaker, i.e. easier to unfold, than low designable ones. This is argued to be related to protein flexibility. (3) Third, we study the mechanism of cold denaturation through constant-pressure simulations for a model of hydrophobic molecules in an explicit solvent. We find that the temperature dependence of the hydrophobic effect is the driving force for cold denaturation. The physical mechanism underlying this phenomenon is identified as the destabilization of hydrophobic contact in favor of solvent separated configurations, the same mechanism seen in pressure induced denaturation. A phenomenological explanation proposed for the mechanism is suggested as being responsible for cold denaturation in real proteins.

No description available.

Photocopy of typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1979. / Bibliography: leaves 174-182. / Microfiche. / xvi, 182 leaves ill. 29 cm

Accurate and reliable measurement of meat quality is essential for the Australian beef industry to remain competitive in both the domestic and export markets. Recent developments of the resonator technique have lead to the commercial availability of the High Resolution Ultrasound Spectroscope (HR-US). This research project was designed to assess the potential of HR-US for the analysis of post-mortem bovine muscle. This was accomplished by; 1) establishing a suitable measurement protocol that considered sources of variability, 2) the effects of post-mortem aging on HR-US parameters, 3) analysis of thermal related changes that occur in muscle, and extracted connective tissue during heating, and 4) the use of HR-US for the measurement of the intramuscular fat. A procedure for the measurement of bovine muscle with HR-US was established. Briefly, an external semisolid cell was used as the measurement cell. The frequency range of 2000 KHz to 3000 KHz was selected as the most suitable for whole muscle analysis and all five resonance peaks within this range were analysed and used to obtain velocity and attenuation values of the meat sample. Water was used as the reference media, and measurements were conducted at 250C. Changes were made to this method during experimental work depending on the sample being run and the objectives of the study. The measurement protocol was shown to be repeatable. Factors likely to cause variation in measurements of the samples, such as water loss and freeze-thaw, were also considered when developing the operational parameters of the study. High resolution ultrasound spectroscopy was applied to measure the post-mortem changes that occur in bovine muscle. Using two muscle types, Semitendinosus and Psoas major, significant changes were observed in HR-US parameters with ageing. Significant increases in the acoustic impedance of bovine muscle with increased ageing time were attributed to degradation of the muscle structure. This was confirmed in transmission electron microscopy images where clear disruption the myofibillar structure was apparent in the muscle at 21 days post-mortem.In localised regions the Z bands and the adjoining actin fibres were totally degraded. Water loss from the muscle system had a significant influence on HR-US measurements. Thermal related changes that occur in whole bovine muscle and in isolated intramuscular connective tissue were observed with HR-US. Heat induced changes were identified in whole muscles and included the melting of the fat within samples at 48oC, coagulation of sarcoplasmic proteins between 450C and 55oC, and the shrinkage of collagen fibres at 630C. An 80% reduction in the attenuating properties of extracted connective ageing in buffer solution was observed within the first 5 days. This is attributed to the degradation of proteoglycans and the resulting disassociation of collagen fibrils. Structural changes occurring in extracted connective tissue were observed with TEM. HR-US measurements proved to be highly sensitive to identifying temperatures at which transitions occurred. Unfolding of the triple helix structure of collagen was identified in velocity transitions between 59°C and 63oC. HR-US results suggested a greater sensitivity to thermal related changes in extracted intramuscular connective tissue when compared with differential scanning calorimeter results. An increase in temperature was observed for thermal denaturation of collagen with ageing, however a reduction was also observed in the temperature range at which the denaturation process occurred. Temperature ramps conducted on extracted intramuscular bovine showed a reduction in velocity from 1613.1m/s at 250C to 1343.1 m/s at 900C equalling an overall reduction in velocity of 270m/s. A transition in the velocity trend seen at 46°C indicates the majority of the triglycerides are melted (or in liquid state) above this temperature. Results are confirmed with differential scanning calorimeter thermogram. HR-US measurements showed high sensitivity to increasing concentration of bovine fat in prepared emulsions with an adjusted R2 99.46% for velocity measurements taken at 5100 kHz. Attenuation values at 8100 kHz also showed a strong linear response to increasing fat concentration in the emulsion (R2 98.77). The use of HR-US for the measurement of intramuscular bovine fat demonstrated a high sensitivity to extracted bovine fat in prepared emulsions. An increase in the intramuscular fat content of whole bovine muscles resulted in a reduction in the velocity measurements and an increase in the attenuation of the ultrasonic signal. This provides the basis for potential method for the prediction intramuscular fat in bovine muscle. The present studies have highlighted the complexities of investigations relating to meat quality and have demonstrated the diversity of data required to assess quality. Only when comprehensive data are available, can we hope to accurately determine meat quality and predict how it will vary with changes in animal production and meat processing.

As terapias térmicas vêm sendo utilizadas com muita freqüência em áreas da saúde, como ortopedia, dermatologia e oftalmologia entre outras. Em média a literatura revela que na prática de tratamentos com calor os tecidos são submetidos a temperaturas que não ultrapassam os 45 ºC (igual aproximadamente 8,5 acima da média da temperatura corpórea normal). Este trabalho procurou verificar de fato a possível influência das variações térmicas sobre o colágeno, quanto à possibilidade de desnaturação do mesmo. Utilizou-se 48 ratos (Rattus Novergicus Albinus) da raça Wistar machos, divididos em oito grupos: grupo I Controle (sem alteração térmica), grupo II (35 ºC), III (40 ºC), IV (42 ºC), V (45 ºC), VI (48 ºC), VII (50 ºC), VIII (55 ºC) (cujos membros inferiores foram submersos por 10 minutos em água aquecida com temperatura variável). Em seguida os animais do grupo controle e experimental foram sacrificados, retirando por dissecação e tenotomia os tendões calcâneos que conferidos às lâminas histológicas passaram por análise microscópica de medidas de birrefringência, análise histológica por microscopia de luz e análises por calorimetria exploratória diferencial (DSC). Os resultados mostram que existe uma diferença significativa, dos grupos I a IV, em relação aos grupos V a VII, obtidos pela média de valores de retardos ópticos (RO), após as análises de medidas de birrefringência, confirmados pela análise visual histológica e pela calorimetria exploratória diferencial, sugerindo que em temperaturas elevadas ocorre uma desnaturação da proteína colágeno. / Thermal therapies are being used in orthopedic, dermatology and ophthalmology, among other medical specialties. Literature reveals in the clinical practice of thermal therapies, 45 ºC is the temperature of choice (= 8.5 below human body temperature). The purpose of this study was to quantify changes in the tendon of calcaneous after a range of thermal exposure, and correlate these results with tissue denaturation. It was used 48 Wistar rats (Rattus Novergicus Albinus), male. They grouped as: I (Control) II (35 ºC), III (40 ºC), IV (42 ºC), V (45 ºC), VI(48 ºC), VII (50 ºC), VIII (55 ºC), and had their pad submerged during 10 minutes in hot water (varying from 35 to 55 ºC. Animals were sacrificed and tendon of calcaneous were analyzed by birefringence microscopy, histology (H & E and Trichromic of Masson) and differential scanning calorimetry (DSC). Results showed that there is a significant difference between groups I-IV and V-VIII. Groups I-IV not shows signals of denaturation by heat treatment. Heatings above 45 ºC resulted in thermal denaturation, decreasing o birefringence and changes in histological aspect.

Orientador: Elias Basile c / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Quimica / Made available in DSpace on 2018-08-09T16:09:35Z (GMT). No. of bitstreams: 1 Godoi_PatriciaHelenade_M.pdf: 865544 bytes, checksum: 0a09a500bcd26f04fe7be64851f3522a (MD5) Previous issue date: 2007 / Resumo: Bromelina é uma enzima de origem vegetal obtida de diversas espécies da família Bromeliaceae, presente na casca, no talo e no fruto do abacaxi. O Brasil encontra-se entre um dos maiores produtores mundiais de abacaxi, ocupando o terceiro lugar no ranking mundial. O desenvolvimento de novas técnicas de extração e purificação da bromelina vem sendo bem explorado, entretanto, tornouse necessário um estudo de estabilidade desta protease, proporção pela qual a bromelina conserva sua conformação estrutural ou sua atividade quando sujeita à estocagem, isolamento e purificação ou várias outras manipulações físicas ou químicas, incluindo autodigestão, outras enzimas proteolíticas e aquecimento. Tornou-se de fundamental importância saber em que condições a bromelina se mantém estável, ou seja, ativa e por qual período de tempo, já que a meta mais significante da enzimologia aplicada é obter compostos úteis para biocatálise. Este trabalho apresenta um estudo das condições de pH e temperatura nas quais a Bromelina P.A em solução aquosa em concentração próxima ao do suco extraído da polpa da fruta, mantém-se ativa, ou seja, não desnaturada. A atividade enzimática da bromelina em solução foi medida através da hidrólise da caseína e a condição de pH e temperatura mais próxima da ideal foi determinada / Abstract: Bromelain is a vegetable enzyme found in many species of Bromeliaceae family, its present in pineapple skins, stem and fruit. Brazil is one of the world¿s largest producers of pineapples, its production being the third one in the world. The development of new extraction and purification processes of bromelain have been studied, however, its necessary a enzyme stabilization investigation, state that bromelain remains its structure or biological activity when stored, isolated, purified or any other manipulation, included autodigestion, proteolytic enzymes and heating. It became very important to know the conditions and the time which bromelain remain stabilized, active. In applied enzymology, the most significant goal is to achieve useful compounds by biocatalysis. This work presents a study about pH and temperature conditions which a bromelain aqueous solution, in the same concentration of a pineapple fruit extract, remains with biological activity. The bromelain aqueous solution enzyme activity was tested across the casein hydrolysis and the ideal pH and temperature was determinated. / Mestrado / Sistemas de Processos Quimicos e Informatica / Mestre em Engenharia Química