Os compostos antimicrobianos naturais são um tema de grande interesse devido ao aumento da demanda por alimentos seguros e de alta qualidade. A utilização de lipossomas é uma alternativa interessante para proteger antimicrobianos nos alimentos, além de fornecer compostos naturais de liberação controlada. Os lipossomas revestidos com polissacarídeos apresentam melhor estabilidade, representando uma alternativa aos lipossomas convencionais. Inicialmente, os nanolipossomas que encapsulam a nisina foram preparados com fosfatidilcolina de soja (PC) e pectina ou ácido poligalacturônico. Os lipossomas desenvolvidos apresentaram alta eficiência de encapsulação, baixo índice de polidispersão e foram estáveis durante 21 dias a 7 °C e 25 °C. A atividade antimicrobiana foi observada contra cinco cepas diferentes de Listeria em placas de ágar de leite, com uma melhor eficiência contra L. innocua 6a. Em um segundo momento, as características estruturais dos lipossomas foram estudadas por dispersão de raios-X de pequeno ângulo (SAXS) e as amostras foram submetidas a ciclos de temperatura (20-60 °C). Para isso, os lipossomas foram desenvolvidos contendo pectina ou ácido poligalacturônico pelos métodos de hidratação de filme e evaporação em fase reversa, para encapsular nisina. A análise de SAXS confirmou a presença de estruturas lamelares em todas as amostras. Além disso, parte da estrutura multilamelar tornou-se cúbica, provavelmente devido à presença de nisina nos lipossomas. A adição de polissacarídeos mostrou diferenças entre as fases cúbicas formadas. Em última análise, a mistura de lisozima e nisina foi encapsulada em lipossomas contendo polissacarídeos. O diâmetro médio dos lipossomas foi de 85,6 e variou para 77,3 e 79,9 nm com a incorporação de pectina ou ácido poligalacturônico, respectivamente. O potencial zeta dos lipossomas com polissacarídeos foi de cerca de -30 mV, mostrando alta eficiência de encapsulação. A atividade antimicrobiana foi avaliada a 37 °C, mostrando que a PC-pectina reduziu a população de L. monocytogenes em 2 log UFC/mL e 5 log UFC/mL em leite integral e desnatado, respectivamente. Em refrigeração, a PC-pectina reduziu a população de L. monocytogenes para quase zero por até 25 dias em leite desnatado. Portanto, pode dizer-se que os lipossomas que contêm polissacarídeos podem ser uma tecnologia promissora para o encapsulamento da lisozima e nisina. Além disso, a existência de estrutura cúbica nos lipossomas pode proporcionar liberação controlada de antimicrobianos. / Natural antimicrobial compounds are a topic of utmost interest due to the increased demand for safe and high-quality foods. The use of liposomes is an interesting alternative to protect antimicrobials in food, also providing controlled release natural compounds. Polysaccharides coated liposomes present better stability, representing an alternative to conventional liposomes. Initially, nanoliposomes encapsulating nisin were prepared with soy phosphatidylcholine (PC) and pectin or polygalacturonic acid. The liposomes developed presented high encapsulation efficiency, low polydispersity index, and were stable for 21 days at 7°C and 25°C. The antimicrobial activity was observed against five different strains of Listeria in milk-agar plates, with a better efficiency against L. innocua 6a. In a second moment, structural characteristics of liposomes were studied by small angle X-ray scattering (SAXS) and the samples were submitted to temperature cycles (20-60°C). For this, liposomes were developed containing pectin or polygalacturonic acid by the thin-film hydration method and reverse phase evaporation method for nisin encapsulation. The analysis of SAXS confirmed the presence of lamellar structures in all the samples. In addition, part of the multilamellar structure became cubic, probably due to the presence of nisin in the liposomes. The addition of polysaccharides showed differences between the cubic phases formed. Ultimately, the mixture of lysozyme and nisin were encapsulated in liposomes containing polysaccharides. The mean diameter of the liposomes was 85.6 and varied to 77.3 and 79.9 nm with the incorporation of pectin or polygalacturonic acid, respectively. The zeta potential of liposomes with polysaccharides were around -30 mV, showing high encapsulation efficiency. The antimicrobial activity was assessed at 37 °C, showing that PC-pectin reduced the population of L. monocytogenes to 2 log CFU/mL and 5 log CFU/mL in whole and skim milk, respectively. At under refrigeration, PC-pectin reduced the population of L. monocytogenes to almost zero for up to 25 days in skim milk. Therefore, it can say that the liposomes containing polysaccharides can be a promising technology for the encapsulation of lysozyme and nisin. In addition, the existence of cubic structure in the liposomes can provide controlled release of antimicrobials.

This diploma thesis was focused on basic physicochemical properties of caffeine and investigation of the interaction between the polysaccharide sodium hyaluronan (HA), pectin (PEC) or lignohumate (HUM) and the alkaloid caffeine (CAF). The reason why this thesis was focused on study of aqueous solutions of hyaluronan or pectin with caffeine is that the presence of polysaccharide- caffeine complex could be beneficial for slowing the absorption of caffeine. In the theoretical part of the diploma thesis there is described a group of alkaloids, which caffeine belongs to and also polysaccharides hyaluronic acid and pectin. The most attention has been paid to caffeine, its properties and effects on the human organism. This diploma thesis also deals with non-alcoholic beverages with caffeine, 4-methylimidazole, and phosphoric acid. The last chapter of the theoretical part is dedicated to fluorescence spectroscopy. In the experimental part of this work I deal with the basic properties of caffeine and interaction between the caffeine and selected polysaccharides or lignohumate. At first, the chemical and physical properties of caffeine were determined. Based on these results the interaction of caffeine and hyaluronan or pectin was studied by using the fluorescence and absorbance. The HA of molecular weight of 1,7 MDa and pectin from citrus fruits were chosen for the study of the interaction with the caffeine. These two substances were mixed with the caffeine of concentration contained in Coca-Cola. The results of interaction that would lead to the influence of the emission or absorption properties of caffeine, were not proved. As a part of the work there was determined how pectin behaves in strongly acidic solution and then there was monitored the pH, conductivity and solubility of caffeine in water by using thermogravimetry and visual experiments. The obtained results of the interaction of hyaluronan-caffeine, pectin caffeine or caffeine-lignohumate can be used for description of the behavior of caffeine in the presence of selected polysaccharides and natural organic substance.

No description available.

No description available.

Mixed solutions of β-lactoglobulin and anionic polysaccharides, specifically pectin, were formed into associative complexes through pH reduction from neutral conditions. Thermal treatment of these associative complexes was investigated as a function of biopolymer composition, heating conditions, pH, and ionic strength. Thermal treatment of β-lactoglobulin-pectin complexes at pH 4.5 – 5.0 was found to create protein-based particulates of consistent and narrow size distribution (diameter ~ 150 – 400 nm). These particulates were relatively stable to further pH adjustment and to high levels of salt (200 NaCl). Particle characteristics were maintained after re-suspending them in aqueous solutions after they have been either frozen or lyophilized. Thermal analysis of β- lactoglobulin-pectin complexes using calorimetry (DSC) and turbidity-temperature scanning indicated that the denaturation of β-lactoglobulin was unaffected by pectin, but protein aggregation was limited by the presence of pectin. Biopolymer particles formed using two different methods were compared: Type 1 – forming β-lactoglobulin nanoparticles by heating, then coating them with pectin; Type 2 – forming particles by heating β-lactoglobulin and pectin together. Type 2 particles had smaller diameters and had better pH and salt stability than Type 1 particles. It was proposed that Type 2 particles had a pectin-saturated surface that limited their aggregation, whereas Type 1 particles had “gaps” in the pectin surface coverage that led to greater aggregation. Finally, the possibility of controlling the size and concentration of biopolymer particles formed by heating β-lactoglobulin-pectin complexes by controlling preparation conditions was studied. Biopolymer particle size and concentration increased with increasing holding time (0 to 30 minutes), decreasing holding temperature (90 to 70 ºC), increasing protein concentration (0 to 2 wt%), increasing pH (4.5 to 5.0), and increasing salt concentration (0 to 50 mol/kg). The influence of these factors on biopolymer particle size was attributed to their impact on protein-polysaccharide interactions, protein denaturation, and protein aggregation kinetics. The knowledge gained from this study will facilitate the rational design of biopolymer particles with specific physicochemical and functional attributes that can be used in the food and other industries, e.g., for encapsulation, texture modification, optical properties modification.

No description available.

No description available.

The purpose of the present study is to determine the ascorbic acid values of orange marmalades made with and without commercial pectin; and to compare these values with those obtained by testing random samples of home and commercial prepared orange marmalades.

Made available in DSpace on 2017-07-21T18:53:11Z (GMT). No. of bitstreams: 1 dissertacao parcial.pdf: 389132 bytes, checksum: 1072407aabab8f91b6ea13999acc01d4 (MD5) Previous issue date: 2009-02-06 / Pectinases, a group of enzymes that degrade pectic substances and break glycosidic linkages, are produced by fungi, yeasts and bacteria, but are also in plants in general and fruit in particular.In the juice industry the pectinolytic enzymes are added to increase the efficiency of the process, decrease the viscosity and reduce the time of filtration. The pectin methylesterase, PME, hydrolyzes the methyl ester groups, forming carboxyl groups in pectin chain, releasing methanol end H3O+. Therefore, its knowledge is vital in order to control the effectiveness of the treatment. The purpose of this study was to determine the optimum conditions of the activity of the pectin methylesterase in industry preparations, proposing a potentiometric procedure for determining the PME activity and compare the data with those obtained by traditional potentiometry and Uv-Vis, evaluate the efficacy of this method in determining the residual activity exogenous of PME in mango juice. The activity of PME in the three commercial samples, Pectinex 100L Plus, Panzym Univers and Panzym Clears, was determined by potentiometry, Uv-Vis spectroscopy, with the bromophenol blue indicator, and the action of alcohol oxidase with acetyl acetone. The reaction consisted of 5.00 mg.mL-1 apple pectin, 0.100 mol.L-1 sodium chloride and 50 μL commercial pectinolytic enzyme for a volume of 30 mL. In all experiments the enzyme deesterification showed first-order kinetics, with increased activity at pH 4.0 to 4.5 and 45 ºC, whereas the complete inactivation occurred at 75 ºC for 10 minutes, in the three industrial preparations. The thermal inactivation of the PME of Pectinex 100L Plus and Panzym Clears preparations occurred under the same conditions, when the activity was measured by the procedures of ΔVNaOH / Δttime or of ΔpH/ Δttime. The activity of PME in industrial preparations at 25 °C and pH 4.5, determined by UV-Vis spectroscopy with bromophenol blue indicator, showed good correlation with the activity determined by the procedures by potentiometry. The stability of the indicator in the pectin solution allows its use to determine the PME activity in samples in which the optimum pH is located in acid band. The release of methanol as measured by alcohol oxidase, followed by the reaction with acetyl acetone to determine the formaldehyde, showed good agreement with the results of the enzyme activity measuring procedures used in this research. The inactivation of residual PME in mango juice occurred at 75 ºC for 20 minutes of exposure in the procedure ΔVNaOH / Δttime and 10 minutes of exposure during the procedure ΔpH/ Δttime. The residual activity of PME in 70 °C for 10, 20 and 30 minutes of exposure in the presence of juice was higher than in the control, indicating its protective effect. The procedure of ΔpH/ Δttime shows good correlation with other methods, with the advantage of precise and direct measures of [H+], excusing a series of reagents and high costs materials. / As pectinases, um grupo de enzimas que degradam substâncias pécticas e rompem ligações glicosídicas, são produzidas por fungos filamentosos, leveduras e bactérias, mas encontram-se também em plantas em geral e em frutas, em particular. Na indústria de sucos as enzimas pectinolíticas são adicionadas para aumentar o rendimento do processo, diminuir a viscosidade e reduzir o tempo de filtração. A pectina metilesterase, PME, hidrolisa os grupos metil éster, formando grupos carboxilícos na cadeia da pectina, produzindo metanol e H3O+.Portanto, é fundamental o seu conhecimento, a fim de controlar a eficiência do tratamento. O objetivo deste trabalho foi determinar as condições ótimas da atividade da PME presente em preparações industriais, propor um procedimento potenciométrico para determinação da atividade da enzima e comparar os dados com os obtidos por potenciometria tradicional e Uv- Vis, avaliar a eficiência do método proposto na determinação da atividade residual da PME exógena no suco de manga. A atividade da PME nas três amostras comerciais, Pectinex 100L Plus, Panzym Univers e Panzym Clears, foi determinada por potenciometria, espectroscopia Uv-Vis, com o indicador azul de bromofenol, e pela ação do álcool oxidase com acetil acetona. A reação consistiu de 5,00 mg.mL-1 de pectina de maçã, 0,100 mol.L-1 de cloreto de sódio e 50 μL de enzima pectinolítica comercial para um volume de 30 mL. Em todos os experimentos a desmetoxilação enzimática mostrou uma cinética de primeira ordem, com maior atividade em pH 4,0 a 4,5 e 45 ºC, sendo que a inativação completa ocorreu a 75 ºC por 10 min, nas três preparações industriais. A inativação térmica da PME das preparações Pectinex 100L Plus e da Panzym Clears ocorreu sob mesmas condições, quando a atividade foi medida pelos procedimenos de ΔVNaOH / Δttempo ou de ΔpH/ Δttempo. A atividade da PME nas preparações industriais a 25 ºC e pH 4,5, determinada por espectroscopia Uv-Vis com o indicador azul de bromofenol, apresentou boa correlação com a atividade determinada pelos procedimentos por potenciometria. A estabilidade do indicador em solução com a pectina permite a sua utilização para determinar a atividade da PME em amostras nas quais o pH ótimo localiza-se na faixa ácida. A liberação do metanol medida pela álcool oxidase, seguida da reação com a acetil acetona para determinar o formaldeído, mostrou boa concordância com os resultados dos procedimentos de medida de atividade enzimática utilizados neste trabalho. A inativação da PME residual em suco de manga ocorreu na temperatura de 75 ºC por 20 min de exposição no procedimento ΔVNaOH / Δttempo e durante 10 min de exposição pelo procedimento ΔpH/ Δttempo. A atividade residual da PME a 70 ºC por 10, 20 e 30 min de exposição em presença do suco foi maior do que no controle, indicando o seu efeito protetor. O procedimento da ΔpH/ Δttempo apresenta boa correlação com os demais métodos, com a vantagem de medidas precisas e diretas da [H+], dispensando uma série de reagentes e materiais de custos elevados.

La pectine, un composant de la matrice de la paroi primaire, joue un rôle dans le contrôle de la porosité de la paroi, l’élongation et l’adhésion cellulaire et est un facteur important dans le développement de la plante. Homogalacturonane (HG), le polymère pectique le plus abondant, est sécrété sous forme méthylestérifiée et ne porte donc peu ou pas de charges négatives. La déméthylestérification d’HG par des pectines méthylestérases (PMEs) expose ensuite des charges négatives et a des conséquences importantes sur les propriétés mécaniques de la paroi, affectant des processus physiologiques et développementaux comme l’ouverture des stomates, l’initiation d’organes et la croissance cellulaire anisotropique. De multiples PME existent (chez Arabidopsis thaliana) qui peuvent être inhibées par des « PME inhibitors » (PMEIs) endogènes. L’HG déméthylestérifiée peut former des liaisons Ca²⁺-pectate qui peuvent rigidifier la paroi, mais des études récentes montrent que la déméthylestérification de HG peut également promouvoir le relâchement de la paroi et l’expansion cellulaire à travers un mécanisme inconnu. Dans ma thèse j’ai adressé ce paradoxe en étudiant le lien entre le métabolisme des pectines, le pH et l’extensibilité de la paroi. À cette fin j’ai développé et utilisé des outils génétiques et pharmacologiques pour la manipulation in vivo de l’activité PME. J’ai généré des lignées permettant la surexpression inductible de deux PMEIs différents, mais qui malheureusement s’avéraient non fonctionnelles. J’ai produit PMEI3 dans une levure ce qui m’a permis de montrer que la protéine a une activité inhibitrice pH-dépendante sur un large éventail de PMEs. Par ailleurs, j’ai utilisé et développé des senseurs ratiométriques pour la mesure du pH à la surface de la cellule. En dehors des senseurs existants, j’ai aussi essayé d’adresser les mêmes biosenseurs à la paroi. À l’aide de ces outils, j’ai ensuite étudié l’impact de la modification de l’activité PME sur le pH et la croissance cellulaire. J’ai pu observer qu’un inhibiteur chimique de la PME, l’(-)-epigallocatechin gallate (EGCG) entraînait une augmentation du pH apoplastique (pHApo) dans la racine, et ceci indépendamment de la H⁺-ATPase ; une inhibition de la croissance et une perte de l’anisotropie des cellules. Par ailleurs, un traitement avec PMEI3 ralentissait la croissance et un apport de PME entraînait une réduction du pHApo et une augmentation de la croissance. Enfin, une induction de 24h de PMEI5 causait une réduction du pHApo, ce qui est en accord avec l’activation compensatoire d’autres PMEs suite à une signalisation liée eux brassinostéroide accrue décrit précédemment. En conclusion, nos résultats suggèrent que la démethylesterification des HG crée domaines anioniques qui peuvent séquestrer des protons ce qui pourrait activer localement des protéines qui stimulent le relâchement de la paroi avec un pH optimum acide entraînant une augmentation de la vitesse de croissance cellulaire. / Pectin, a matrix component in the primary cell wall, plays a role in controlling cell wall porosity, cell elongation and cell adhesion and constitutes an important factor in plant development. The demethylesterification of homogalacturonan (HG), the most abundant pectic polymer, has vast consequences on the mechanical properties of the cell wall, and affects developmental processes such as stomata opening, organ initiation and anisotropic cell growth. HG is selectively demethylesterified in muro by pectin methylesterases (PME), which in turn can be inhibited by endogenous PME inhibitor proteins (PMEIs). Demethylesterified HG is thought to form Ca²⁺-pectate complexes, which contribute to wall stiffening, but recent evidence suggest that it can also promote cell wall loosening and expansion, through a so far unknown mechanism. In this study I addressed this paradox by investigating the link between pectin metabolism, cell wall pH and extensibility. To this end I developed and used genetic and pharmacological tools for the in vivo manipulation of PME activity. I generated inducible overexpression lines for two distinct PMEIs, which unfortunately were not functional. I also produced PMEI3 from Arabidopsis in a yeast and showed that the protein displayed an inhibiting activity on a broad range of PMEs. In addition, I developed and used tools to monitor the cell surface pH. In addition to using existing genetically-encoded ratiometric apoplastic pH sensors, I also tried to generate similar sensors targeted to the cell wall. Using these tools I then studied the impact of changes in pectin methylesterification on the cell wall pH and cell expansion. I discovered that a chemical inhibitor of PME, (-)-epigallocatechin gallate (EGCG), promoted an increase in apoplastic pH (pHApo) in root cells, independently from the inhibition of the H⁺-ATPase, and triggered root growth inhibition and abnormal cell shape. Exogenous PMEI3 application also inhibited root growth. In addition, PME application caused a decrease in pHApo and enhanced root growth. Interestingly, long-term induction of PMEI5 could reduce pHApo, consistent with the previously described activation of brassinosteroid signaling causing a compensatory increase in PME activity. Together, my study provides evidence that HG demethylesterification leads to a decrease in pHApo and an increase in cell growth in the Arabidopsis root. Our results support the view that the negatively charged pectate can sequester protons and thus may contribute to the activation of cell wall loosening proteins and cell growth.