<p> Lambda carrageenan when injected subcutaneously causes the formation of a connective tissue granuloma. Initially there is a proliferation of connective tissue elements up to about fourteen days followed by regression so that by six weeks most collagenous tissue has disappeared and been replaced by adipose tissue. Lambda carrageenan has been identified in the granuloma by staining reactions with toluidine blue and other stains for acid polysaccharides. The present study was undertaken to localize the carrageenan by means of fluorescent antibody and autoradiography. For this purpose labelling of carrageenan by photoassimilation of 14CO2 into carrageenan was done. Different parameters affecting the incorporation of 14C into the carrageenan fractions were studied.</p> / Thesis / Master of Science (MSc)

O presente estudo investigou o efeito do veneno de Crotalus durissus terrificus (VCdt) e da sua principal fração, crotoxina (ctx) sobre a resposta inflamatória induzida pela carragenina (cg). Foram avaliados o efeito do pré e do pós-tratamento com o VCdt sobre o desenvolvimento do edema de pata, a migração celular para a cavidade peritoneal e a interação leucócito-endotélio na microcirculação do músculo cremaster de camundongos induzidos por carragenina. O pré (14, 7 dias ou 1 h) e pós-tratamento (48, 4 ou 1 h) com o VCdt (1,5 &#956;g/50 &#956;L s.c.) inibiu o desenvolvimento do edema de pata induzido pela injeção intraplantar de carragenina em todos os tempos analisados (300 &#956;g/50 &#956;L). O mesmo efeito inibitório foi observado para o pré (21, 14, 7 dias ou 1 h) e pós-tratamento (1 h) com o VCdt (1,5 &#956;g/50 &#956;L s.c.) sobre a migração celular para a cavidade peritoneal induzida pela carragenina (300 &#956;g/50 &#956;L). A administração do VCdt por via oral (8,8 &#956;g/200 &#956;L) antes (14, 7 dias ou 1 h) ou após (1 h) a injeção de carragenina (300 &#956;g/200 &#956;L) também inibiu o influxo celular para a mesma cavidade. Em relação à interação leucócito-endotélio, o tratamento com o VCdt 1 h antes ou após a injeção de carragenina no subcutâneo da bolsa escrotal (300 &#956;g/100 &#956;L), aumentou o número de células em estado de rolling. Por outro lado, reduziu a adesão das células ao endotélio e a migração celular para o tecido extravascular. Da mesma forma que o VCdt, a ctx (0,89 &#956;g/50 &#956;L s.c.) administrada 1 h antes ou após a carragenina inibiu todos os eventos acima citados. Os efeitos inibitórios do VCdt sobre o edema e sobre a migração celular foi comparado ao de clássicas drogas antiinflamatórias: a dexametasona, a indometacina, o L-NAME e o nimesulide. Para o edema, foi observado que somente o VCdt e o nimesulide impediram a formação do segundo pico de edema de carragenina. Para a migração celular, não foi observada diferença entre o tratamento com o VCdt e as drogas antiinflamatórias. A associação do VCdt com as drogas antiinflamatórias acima citadas não potencializou as suas ações inibitórias sobre a migração celular. Os dados demonstram que o VCdt apresenta um efeito antiinflamatório prolongado, e ainda, que a ctx pode contribuir para o efeito antinflamatório do VCdt. / In the present study, we investigated the effects of Crotalus durissus terrificus venom (CdtV) and crotoxin (ctx) on vascular and cellular events of inflammation induced by carrageenan (cg) in mice. It was evaluated the effect of the pre or post-treatment with CdtV on development of paw edema, cell migration and leukocyte-endothelium interaction at the cremaster muscle microcirculation induced by cg in mice. The pre (14, 7 days or 1h) and posttreatment (48, 4 or 1h) with CdtV (1,5 &#956;g/50 &#956;L s.c.), inhibited the paw edema induced by carrageenan (300 &#956;g/50 &#956;L) in all periods of time evaluated. The same inhibitory effect of CdtV (1,5 &#956;g/50 &#956;L s.c.) was observed to the pre (21, 14, 7 days or 1 h) and post-treatment (1h) on cell migration to the peritoneal cavity induced by cg (300 &#956;g/200 &#956;L). The oral administration of CdtV (8,8 &#956;g/200 &#956;L) before (14, 7 days or 1h) or after (1h) intraperitoneal injection of cg (300 &#956;g/200 &#956;L), also inhibited the cell influx to the same cavity. To evaluate leukocyte-endothelium interaction, the treatment with CdtV 1h before or after the cg injection (300 &#956;g/100 &#956;L) into the scrotal bag of the animals induced an increase in leukocyte rolling, but reduced the cellular adhesion to the endothelium surface and cellular migration to the extravascular tissue. The administration of ctx (0,89 &#956;g/50 &#956;L s.c.) 1 h before or after cg injection, could inhibit all inflammatory events mentioned above. The inhibitory effect of CdtV on paw edema and cellular migration were also compared with that observed for classical anti-inflammatory drugs, such as: dexamethasone, indomethacin, LNAME and nimesulide. Only CdtV and nimesulide blocked the second peak of edema formation induced by cg. For cell migration, no differences were observed among the treatment with CdtV and anti-inflamatory drugs. The association of CdtV with drugs did not potentialize their actions on cell migration. These results demonstrate that CdtV exhibits a long-lasting antiinflammatory effect and that ctx could contribute for the anti-inflammatory effect of CdtV.

Disfunções temporomandibulares são secundárias à inflamação e dor. Estudos mostram a participação da ET na nocicepção. Investigamos o papel da ET1 na sinovite induzida pela injeção de CGN na ATM de ratos. Injeções intra-articulares de CGN, ET1 ou do agonista do receptor ETB BQ3020 foram realizadas na ATM. Após 4h, a hiperalgesia foi avaliada usando um analgesímetro digital e as cavidades articulares foram lavadas para contagem total e diferencial de leucócitos. Antagonistas dos receptores ETA e ETB foram co-injetados com CGN e ET1, e o antagonista de TRPV1 capsazepina com a ET1. A ET1, o BQ3020 e a CGN induziram hiperalgesia que foi inibida pela injeção simultânea (mas não individual) dos antagonistas ETA e ETB. A capsazepina não afetou o efeito nociceptivo da ET1. Nem a ET1, o BQ3020 nem os antagonistas dos receptores da ET afetaram a infiltração de leucócitos. Com base nestes resultados concluímos que a ET1 induz hiperalgesia e está envolvida na nocicepção induzida por CGN através dos receptores ETA e ETB, sem qualquer efeito sobre a migração de leucócitos. / Temporomandibular disorders are secondary to inflammation and pain. Studies show the involvement of ET in nociception. Weve investigated the role of ET1 in synovitis induced by CGN injection on the rats TMJ. Intra-articular injections of CGN, ET1 or ETB receptor agonist BQ3020 were performed at the TMJ. After 4h, hyperalgesia was assessed using a digital analgesimeter and the joint cavities were washed for total and differential leukocyte counting. ETA and ETB receptors antagonists were co-injected with CGN and ET1, and the TRPV1 antagonist capsazepine with ET1. ET1, BQ3020 and CGN induced hyperalgesia, which was inhibited by simultaneous (but not single) antagonists ETA and ETB injection. The capsazepine did not affect the ET1 nociceptive effect. Neither ET1, BQ3020 nor antagonists of ET receptors affect leukocyte infiltration. Based on these results we conclude that the ET1 induces hyperalgesia and its involved in CGN induced nociception through ETA and ETB receptors, without any effect on the leukocytes migration.

A literatura relata que o veneno de Crotalus durissus terrificus (VCdt) ou suas toxinas isoladas modulam a resposta inflamatória. A crotoxina (CTX) é a principal toxina do VCdt, representando aproximadamente 65% do conteúdo do veneno bruto. Dando continuidade aos estudos que evidenciam o efeito modulador do VCdt sobre a inflamação, foi demonstrado que o VCdt apresenta um efeito anti-inflamatório prolongado sobre a resposta inflamatória induzida pela carragenina (Cg), em camundongos. Esse estudo mostrou que uma única dose de VCdt, administrada pela via subcutânea, 7 ou 21 dias antes da injeção de Cg inibe, respectivamente, o desenvolvimento do edema de pata e a migração celular para a cavidade peritoneal, induzidos por este agente inflamatório. Este efeito anti-inflamatório também foi observado após a instalação da resposta inflamatória (Nunes et al., 2007). Além disso, foi demonstrado também que a CTX, é a toxina responsável por este efeito anti-inflamatório prolongado. Ainda, dados recentes mostram que os receptores para peptídeo formil, tais como lipoxina/anexina, mediadores com potente ação anti-inflamatória, estão envolvidos no efeito da CTX. Em continuidade a essa linha de investigação, este trabalho teve por objetivo caracterizar o efeito da CTX sobre a expressão de mediadores pró-inflamatórios e de moléculas de adesão envolvidas na resposta inflamatória induzida pela Cg. em camundongos. Além de avaliar também o efeito desta toxina sobre a translocação da subunidade p65 do NF-&kappa;B para o núcleo celular. Para tanto, foi, investigado o efeito de uma única dose de CTX (44 &mu;g/kg) sobre: a expressão de P-selectina, ICAM-1, PECAM-1 e Mac-1; sobre a secreção de TNF-&alpha;, IL-1&beta;, IL-6, PGE2, e LTB4 e sobre a expressão de iNOS e p65. Cabe destacar ainda que, um inibidor da síntese de glicocorticoides (Mifepristone), bem como um antagonista de receptor para glicocorticoides (Metirapona) foram administrados antes do tratamento da CTX, para avaliar também a participação de glicocorticoides endógenos no efeito anti-inflamatório da CTX. A administração subcutânea de uma única dose de CTX produziu: 1- diminuição da secreção de TNF-&alpha;, IL-1&beta;, IL-6; 2- diminuição da expressão de P-selectina e ICAM-1 e 3- diminuição da expressão de p65. Por outro lado, a CTX não alterou os níveis de PGE2, e LTB4, como também não alterou a expressão de iNOS e Mac-1. Além disso, nossos resultados sugerem que os glicocorticóides endógenos não interferem no efeito anti-inflamatório da CTX, uma vez que o pré-tratamento com Mifepristone ou Metirapona não alteraram o efeito inibitório desta toxina sobre a migração celular. Em conjunto, os resultados caracterizam o efeito anti-inflamatório da CTX sobre a migração celular induzida pela Cg e sugerem que esta toxina pode inibir a expressão de importantes substâncias pró-inflamatórias envolvidas na resposta inflamatória pela Cg ao inibir a ativação do fator de transcrição, NF-&kappa;B, uma vez que este fator favorece a transcrição de vários genes, cujas proteínas são importantes no desenvolvimento da resposta inflamatória. Esses resultados contribuem para a elucidação dos mecanismos envolvidos na ação modulatória da CTX sobre a resposta inflamatória / The literature shows that Crotalus durissus terrificus snake venom (CdtV) or their toxins isolated modulate the inflammatory response. The crotoxin (CTX) is the main toxin of CdtV, representing approximately 65% of the content of the crude venom. It was demonstrated that CdtV presents a long-lasting anti-inflammatory effect induced by carrageenan (Cg) in mice. This study showed that a single dose of CdtV inhibits respectively, the development of paw edema and cell migration to the peritoneal cavity induced by this inflammatory agent. This anti-inflammatory effect was also observed after installation of the inflammatory response (Nunes et al., 2007). Furthermore, it was also demonstrated that CTX is responsible for this long-lasting anti-inflammatory effect. Still, recent data show that the formil peptide receptors, such as lipoxin/anexin, mediators with potent anti-inflammatory action, are involved in the effect of CTX. The aim of this study is characterize the effect of CTX on the expression of proinflammatory mediators and adhesion molecules involved in the inflammatory response induced by Cg in mice and also evaluate the effect of the toxin on translocation of the p65 subunit of NF-&kappa;B to the nucleus. Therefore, it was investigated the effect of a single dose of CTX (44 &mu;g/kg) on: P-selectin, ICAM-1, PECAM-1 and Mac-1 expression; TNF-&alpha;, IL-1&beta;, IL-6, PGE2 and LTB4 secretion and, on iNOS and p65 expression. It should be noted that a synthesis of glucocorticoids inhibitor (Mifepristone) and a glucocorticoid antagonist receptor (Metyrapone) were administrated before CTX treatment to evaluate the involvement of endogenous glucocorticoids in the anti-inflammatory effect of CTX. Our results show that a single dose of CTX produced: reduction of TNF-&alpha;, IL-1&beta; and IL-6 secretion; reduction of P-selectin and ICAM-1 expression and reduction of p65 expression. Moreover, CTX did not alter levels of PGE2 and LTB4 secretion and did not alter iNOS and Mac-1 expression. Furthermore, our results suggest that endogenous glucocorticoids do not interfere with anti-inflammatory effect of CTX, since that pre-treatment with Mifepristone and Metyrapone did not alter the inhibitory effect of this toxin on cell migration induced by Cg and suggest that this toxin can inhibit the expression of important proinflammatory substances involved in the inflammatory response induced by Cg to inhibit the NF-&kappa;B activation, since this factor promotes the transcription of several genes whose proteins are important in the development inflammatory response. These results contribute to the elucidation of the mechanisms involved in the modulatory action of CTX on the inflammatory response

Nossa hipótese é de que os efeitos pró-inflamatórios iniciais da ativação do receptor ativado por protease 2 (PAR2) na articulação temporomandibular (ATM) sejam mediados por mecanismos neurogênicos. A análise por imunofluorescência revelou um alto grau de imunorreatividade ao PAR2 em aferentes primários trigeminais da ATM. Além do mais, a imunorreatividade ao PAR2 também foi observada na camada íntima da sinóvia, além de co-localizar com o marcador neuronal PGP9.5 e o neuropeptídeo substância P. A injeção intra-articular de agonistas PAR2 na ATM induziu um aumento dependente da dose no extravasamento plasmático, influxo de neutrófilos e indução de alodinia mecânica. O bloqueio farmacológico de receptors NK1 inibiu o aumento no extravasamento plasmático, influxo de neutrófilos e alodinia induzido pela ativação do PAR2. Em conclusão, a ativação do PAR2 é pró-inflamatório na ATM, via mecanismos neurogênicos envolvendo receptores NK1, sugerindo que o PAR2 é um importante componente da resposta imunológica inata na ATM. / We hypothesised that the early pro-inflammatory effects of proteinase-activated receptor 2 (PAR2) activation in the temporomandibular joint (TMJ) are mediated by neurogenic mechanisms. Immunofluorescence analysis revealed a high degree of neurons expressing PAR2 in retrogradely labelled trigeminal ganglion neurons. Furthermore, PAR2 immunoreactivity was observed in the lining layer of the TMJ, co-localizing with the neuronal marker PGP9.5 and substance P-containing peripheral sensory nerve fibres. The intra-articular injection of PAR2 agonists into the TMJ triggered a dose-dependent increase in plasma extravasation, neutrophil influx and induction of mechanical allodynia. The pharmacological blockade of NK1 receptors abolished PAR2-induced plasma extravasation and inhibited neutrophil influx and mechanical allodynia. We conclude that PAR2 activation is pro-inflammatory in the TMJ, through a neurogenic mechanism involving NK1 receptors. This suggests that PAR2 is an important component of innate neuro-immune response in the TMJ.

O presente estudo investigou o efeito do veneno de Crotalus durissus terrificus (VCdt) e da sua principal fração, crotoxina (ctx) sobre a resposta inflamatória induzida pela carragenina (cg). Foram avaliados o efeito do pré e do pós-tratamento com o VCdt sobre o desenvolvimento do edema de pata, a migração celular para a cavidade peritoneal e a interação leucócito-endotélio na microcirculação do músculo cremaster de camundongos induzidos por carragenina. O pré (14, 7 dias ou 1 h) e pós-tratamento (48, 4 ou 1 h) com o VCdt (1,5 &#956;g/50 &#956;L s.c.) inibiu o desenvolvimento do edema de pata induzido pela injeção intraplantar de carragenina em todos os tempos analisados (300 &#956;g/50 &#956;L). O mesmo efeito inibitório foi observado para o pré (21, 14, 7 dias ou 1 h) e pós-tratamento (1 h) com o VCdt (1,5 &#956;g/50 &#956;L s.c.) sobre a migração celular para a cavidade peritoneal induzida pela carragenina (300 &#956;g/50 &#956;L). A administração do VCdt por via oral (8,8 &#956;g/200 &#956;L) antes (14, 7 dias ou 1 h) ou após (1 h) a injeção de carragenina (300 &#956;g/200 &#956;L) também inibiu o influxo celular para a mesma cavidade. Em relação à interação leucócito-endotélio, o tratamento com o VCdt 1 h antes ou após a injeção de carragenina no subcutâneo da bolsa escrotal (300 &#956;g/100 &#956;L), aumentou o número de células em estado de rolling. Por outro lado, reduziu a adesão das células ao endotélio e a migração celular para o tecido extravascular. Da mesma forma que o VCdt, a ctx (0,89 &#956;g/50 &#956;L s.c.) administrada 1 h antes ou após a carragenina inibiu todos os eventos acima citados. Os efeitos inibitórios do VCdt sobre o edema e sobre a migração celular foi comparado ao de clássicas drogas antiinflamatórias: a dexametasona, a indometacina, o L-NAME e o nimesulide. Para o edema, foi observado que somente o VCdt e o nimesulide impediram a formação do segundo pico de edema de carragenina. Para a migração celular, não foi observada diferença entre o tratamento com o VCdt e as drogas antiinflamatórias. A associação do VCdt com as drogas antiinflamatórias acima citadas não potencializou as suas ações inibitórias sobre a migração celular. Os dados demonstram que o VCdt apresenta um efeito antiinflamatório prolongado, e ainda, que a ctx pode contribuir para o efeito antinflamatório do VCdt. / In the present study, we investigated the effects of Crotalus durissus terrificus venom (CdtV) and crotoxin (ctx) on vascular and cellular events of inflammation induced by carrageenan (cg) in mice. It was evaluated the effect of the pre or post-treatment with CdtV on development of paw edema, cell migration and leukocyte-endothelium interaction at the cremaster muscle microcirculation induced by cg in mice. The pre (14, 7 days or 1h) and posttreatment (48, 4 or 1h) with CdtV (1,5 &#956;g/50 &#956;L s.c.), inhibited the paw edema induced by carrageenan (300 &#956;g/50 &#956;L) in all periods of time evaluated. The same inhibitory effect of CdtV (1,5 &#956;g/50 &#956;L s.c.) was observed to the pre (21, 14, 7 days or 1 h) and post-treatment (1h) on cell migration to the peritoneal cavity induced by cg (300 &#956;g/200 &#956;L). The oral administration of CdtV (8,8 &#956;g/200 &#956;L) before (14, 7 days or 1h) or after (1h) intraperitoneal injection of cg (300 &#956;g/200 &#956;L), also inhibited the cell influx to the same cavity. To evaluate leukocyte-endothelium interaction, the treatment with CdtV 1h before or after the cg injection (300 &#956;g/100 &#956;L) into the scrotal bag of the animals induced an increase in leukocyte rolling, but reduced the cellular adhesion to the endothelium surface and cellular migration to the extravascular tissue. The administration of ctx (0,89 &#956;g/50 &#956;L s.c.) 1 h before or after cg injection, could inhibit all inflammatory events mentioned above. The inhibitory effect of CdtV on paw edema and cellular migration were also compared with that observed for classical anti-inflammatory drugs, such as: dexamethasone, indomethacin, LNAME and nimesulide. Only CdtV and nimesulide blocked the second peak of edema formation induced by cg. For cell migration, no differences were observed among the treatment with CdtV and anti-inflamatory drugs. The association of CdtV with drugs did not potentialize their actions on cell migration. These results demonstrate that CdtV exhibits a long-lasting antiinflammatory effect and that ctx could contribute for the anti-inflammatory effect of CdtV.

In recent years, studies have increasingly recognized that many natural products with biological activity have been isolated from marine organisms, while the chemical structures are very different from those of land-based organisms. Therefore, the ocean is a natural drug source. Regarding drug screening, anti-inflammatory activity has become a key point, and many studies confirm that inflammation plays an important role in many human diseases. Many different compounds are now in the clinical evaluation stage. However, the inflammation-related diseases being closely linked, there is an urgent need to study the anti-inflammatory effects as well as screen the therapeutic drugs for research and development. In this study, we isolated and purified compounds from Formosan gorgonian (Briareum excavatum) and Formosan soft coral (Lobophytum sarcophytoides) and investigated biological activities. We confirmed that the natural compound Brei from B. excavatum and the compounds Sac-1 and Sac-2 from L. sarcophytoides produced significant inhibition of the proinflammatory proteins inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in the lipopolysaccharide (LPS)-induced murine macrophages (RAW 264.7) cell model. We examined in vivo whether the B. excavatum Brei has anti-inflammatory and antinociceptive effects by using the carrageenan-induced inflammation model. Using the paw-edema assay, we performed several important investigations such as the plantar analgesia test, mechanical hyperalgesia test (allodynia), and weight-bearing analysis of animal behavior to evaluate the degree of pain and inflammation. Our results demonstrate that the natural product Brei can reduce paw-pad swelling, thermal hyperalgesia, threshold latency, and improve the affected limb in the carrageenan-induced inflammatory model. In the histopathology analysis, we showed that Brei significantly inhibited the aggregation and infiltration of inflammation-related blood cells and improved the inflammatory status of the tissues. Therefore, the marine natural compound Brei has anti-inflammatory activity and it can be used as a therapeutic compound for acute inflammation in the near future.

The skin, by weight, is the largest organ of the body. Human skin serves to provide several important functions that may be classified. in a general context, as protective, maintaining homeostasis and sensing. The outermost layer of the skin, the stratum corneum, has an essential role as a barrier against the transport of water and of chemical and biological agents. In this study acyclovir (ACV), an antiviral used for treating the varicella zoster virus, was used. It is sensible to say that a hydrophilic drug like acyclovir needs a delivery vehicle or penetration enhancer to permeate the skin with more ease. In an attempt to enhance the permeation of acyclovir, it was formulated in a delivery vehicle with the same formulation as for a micro-emulsion. Increasing percentages of the surfactant, Brij 97, were incorporated in the formulation to determine which of the four formulations is indeed a micro-emulsion. A gelating agent. carrageenan, was used to make the emulsion transdermally more applicable; the influence of this component on the transdermal delivery of acyclovir was also determined. Therefore the aim of this study was to determine: -The effect of a drug delivery vehicle on the transdermal delivery of acyclovir; -The specific formulation of a micro-emulsion and -The influence of a gelating agent on the transdermal delivery of acyclovir. Diffusion studies were performed in vertically mounted glass Franz diffusion cells. The epidermis of female abdominal skin, obtained after abdomeoplasty, was heat separated from the dermis. One millilitre of emulsion (0.1%: 1mg/ml ACV) was added to the skin sample in the donor side of the diffusion cell. The control solution had an equivalent amount of active in water and was added to the donor compartment in a separate experiment. The receptor phase was PBS (phosphate buffered solution). The entire receptor phase of the cells was removed every second hour and was replaced with fresh receptor phase at 37°C. The amount of acyclovir in the receptor phase was determined by HPLC analysis. The cumulative amounts of the active that permeated the skin over the 24 hour period were plotted with the slope of the graphs representing the flux in ng/cm²/h. The average flux values of the experimental cells and control cells were compared. Results of the diffusion studies without carrageenan showed that increasing the concentration of the surfactant increased the diffusion of acyclovir. Permeation studies with carrageenan had a totally different outcome. The enhancement ratio of the experimental cells was much lower than that of the control cells. However the experimental cells showed a small increase as the concentration of the surfactant increased. From VanKel dissolution studies it could be seen that release of acyclovir from the emulsion was not a problem and that the active was available for absorption. Confocal studies were done to determine whether there were any vesicles in the emulsions. Vesicles were expected in the 25% Brij 97 emulsion because it was the same formulation as a micro-emulsion, but vesicles could only be found in the 4% and 8% Brij 97 emulsion. A previous study with acyclovir and three different delivery vehicles gave enhancement ratios between 0.32 to 2.92. Values obtained in this study of the 4% and 8% Brij 97 emulsion without carrageenan were more or less the same but the 15% and 25% Brij 97 emulsion had a much higher enhancement ratio. For the emulsions with carrageenan not one exceeded an enhancement ratio of 0.57. More studies still have to be done on micro-emulsions to determine which specific concentration of surfactant forms a micro-emulsion. The active itself and its physicochemical properties also play an important role in the diffusion studies with the specific delivery vehicle and further research has to be done with different model drugs. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

In this thesis, the macroscopic phase separation of milk proteins and high molecular weight oat beta-glucan was investigated. A better knowledge of this model system will improve our ability to control structure in dairy gels containing nutritionally significant concentrations of dietary fiber. A phase behaviour diagram was obtained experimentally, and the results were then modelled using theoretical models based on thermodynamic incompatibility between casein micelles and beta-glucan and demonstrated that casein micelles are the main contributors to the instability of these mixtures. Water in water emulsion systems formed at high concentrations of protein and beta-glucan upon mixing, and were visualized using confocal scanning laser microscopy. For the first time, the dynamics of phase separation of these mixtures were followed using diffusing wave and ultrasonic spectroscopy, as well as with rheological methods. The work explored the formation of different bi-continuous networks by controlling the gelation of the protein phase using chymosin. This enzymatic reaction specifically destabilizes the casein micelles, allowing for a kinetic control of protein gelation within or between phase separated domains. The addition of -carrageenan and the effect of shear on the mixtures were evaluated as possible strategies for controlling the growth of the phase separated domains in dairy gels containing concentrations of beta-glucan high enough to be nutritionally significant. Results indicated that different structures could be obtained depending on the processing conditions, for example, the mode of addition of the polysaccharides or the pre-shearing conditions. This work represents a novel approach for incorporating nutritionally significant concentrations of beta-glucan in dairy foods, and serves as proof of concept for further development of an important application area linked to the development of reduced fat dairy products with additional health benefits. / Canadian Dairy Commission (CDC) and Natural Sciences and Engineering Research Council (NSERC)

In this thesis, mechanisms for enhancing the stability of whey protein emulsions using two approaches were investigated. First, the physicochemical and emulsifying properties of whey protein isolate (WPI), and its two main proteins, alpha-lactalbumin (ALA) and beta-lactoglobulin (β-LG), were investigated in response to changes in pH and temperature pre-treatments. Solvent conditions which inhibit protein aggregation, such as pHs away from the isoelectric point, were found to form stable emulsions. In contrast, thermal treatments were found to negatively affect emulsion stability, where the most stable emulsions for WPI, ALA and β-LG were formed at room temperature (i.e. 25°C) at pH 7.0. It was also determined that emulsions formed using WPI, ALA and β-LG were stabilized by electrostatically repulsive forces which prevent flocculation and creaming. Secondly, the use of tailored protein-polysaccharide interactions involving WPI and carrageenan (CG) were explored as a means of enhancing emulsion stability. Carrageenan (CG) partakes in electrostatic attraction with WPI when acidified, leading to the formation of coupled gel networks. CG was selected for its anionic properties and for its well-characterized structure in that kappa-, iota- and lambda-type CG contain 1-, 2- and 3-sulfated groups per disaccharide repeating unit respectively. WPI-CG mixtures formed gel networks once acidified, where WPI-kappa-CG and WPI-iota-CG mixtures formed stiff networks, whereas WPI-lambda-CG formed a weak fluid network. WPI-CG complexes were found to be surface active, causing changes to the interfacial tension and interfacial rheology at pHs corresponding to where electrostatic attraction occurs upon acidification. Electrostatically coupled gel networks were formed in an emulsion, where oil droplets became entrapped within the biopolymer matrix. WPI-CG mixtures were sensitive to WPI-CG mixing ratio as stiffer gels were formed at higher CG content. Furthermore, WPI-iota-CG gels were stiffer than those made with WPI-kappa-CG gels presumably due to the higher number of sulfated groups lending greater opportunities for iota-CG to form bonds with neighboring polymers compared to kappa-CG.