Thesis (Ph. D.)--University of Missouri-Columbia, 2000. / Typescript. Vita. Includes bibliographical references. Also available on the Internet.

In Arabidopsis, the mucilage secretory cells (MSC) of the seed coat produce a pectinaceous mucilage. Very little is known about which genes are involved in the synthesis of pectins. A reverse genetic approach was used to identify genes involved in mucilage synthesis. A publicly available microarray database was screened with expression visualization tools, and was complemented by in-lab microarray experiments between wild type and known MSC mutants to identify candidate cell wall genes highly expressed at the time of mucilage synthesis. Several cell wall genes were also chosen based on their putative functions which would implicate them in mucilage synthesis. Phenotyping of mutant lines obtained for the cell wall candidate genes revealed no abnormal mucilage phentoypes in single or select double mutant lines. These results indicate that significant genetic redundancy exists in cell wall genes and/or the genes studied do not play significant roles in mucilage synthesis.

Digestive proteases were isolated from the pancreas of the stomachless cunner fish (Tautogolabrus adspersus) and characterized in terms of their physicochemical properties, their ability to hydrolyze native pectin methylesterase (PME) from orange and polyphenol oxidases (PPO) from mushroom and the ability of the cunner enzyme(s) to maintain the stability of orange juice cloud. / The cunner trypsin fraction exhibited exceptional capacity to hydrolyze native proteins versus the bovine trypsin. Incubation of native PME with cunner or bovine trypsin resulted in a loss of 75% or 35% in PME activity respectively. Similarly, a 75% or 55% loss in PPO activity was observed after treatment with cunner and bovine trypsin respectively. Bovine trypsin, however, hydrolyzed the heat-denatured PME and PPO better than the cunner trypsin. Also, there was no reactivation of both PME and PPO activity after treatment with either the cunner or bovine enzyme during storage at 4$ sp circ$C for 3 weeks. However, PPO retained up to 20% or 50% of the initial activity after treatment with cunner or bovine trypsin, respectively. / A 3 x 3 factorial design involving the factors of temperature, enzyme concentration and incubation time carried out gave an r$ sp2$ of 0.92 and 0.95 for cunner and bovine trypsin treated PME respectively. On the other hand, an r$ sp2$ of 0.91 and 0.94 was obtained for the combined effects using cunner and bovine trypsin for PPO inactivation. Validation of the model of PME inactivation measured as the % cloud remaining revealed that the cunner trypsin fraction upheld the cloud stability of cloud juice better than bovine trypsin, with cunner trypsin retaining more than 90% of the cloud whereas the juice treated with bovine trypsin only resulted in a 70% retention of the juice cloud. (Abstract shortened by UMI.)

The FRIABLE1 (FRB1) gene is identified to be a novel glycosyltransferase involved in cell adhesion, based on reverse genetics and immunocytochemistry studies. A total of 31 FRB1 paralogues were found in Arabidopsis thaliana using a bioinformatics approach. The following expression analysis has revealed 6 FRB1 paralogues to be pollen-specific. One pollen-specific FRB1 paralogue, At1g14970, exhibits longer silique lengths when exposed to higher than normal temperature at 28oC in its T-DNA insertional knockout when compared to Columbia wildtype plants. This may be due to the loss of temperature sensing and the continuous stimulated pollen tube cell wall growth or the up-regulation of genes that encode other glycosyltransferases. Thus, the identification of FRB1 paralogues and homologues in both rice and poplar may have tremendous potential to increase their yield in global warming for agricultural and industrial benefits.

Accumulated genetic and epigenetic abnormalities contribute to the development of colon cancer. We have shown that a combination of fish oil (containing decosahexaenoic acid, DHA, 22:6 n-3) and pectin (fermented to butyrate by colonic microflora) is protective against colon carcinogenesis in part by regulating the expression of genes involved in apoptosis, leading to apoptosis induction. To determine how FO/P enhances apoptosis, we measured the expression of genes involved in apoptosis. We performed a pathway analysis on differentially expressed genes identified at three times during colon tumorigenesis: initiation, aberrant crypt foci (ACF) formation, and tumor stage, and compared these results with phenotypic observations at those times. At initiation, FO/P down-regulated the expression of genes involved with cell adhesion and enhanced apoptosis compared with corn oil/cellulose (CO/C). At the ACF stage, expression of genes involved in cell cycle regulation was modulated by FO/P and proliferation was reduced in FO/P rats compared with CO/C rats. FO/P increased apoptosis and the expression of genes that promote apoptosis at the tumor endpoint compared with CO/C. We next determined if changes in expression of genes involved in apoptosis by FO/P are associated with changes in promoter methylation of a key apoptosis regulator, Bcl-2. Genomic DNA was isolated from carcinogen-induced colon tumors and non-involved tissues. FO/P increased Bcl-2 promoter methylation in tumor tissues and colonocyte apoptosis relative to those observed with CO/C. A negative correlation between Bcl-2 DNA methylation and Bcl-2 mRNA levels was observed in the tumors. Additionally, we examined gene specific promoter methylation of 24 apoptosis-related genes using human colon cancer cells. Cells were treated with DHA or linoleic acid (18:2 n-6), and select cultures were also treated with butyrate. The combination of DHA and butyrate led to a significant reduction in the methylation of pro-apoptotic genes and an increase in apoptosis. These data suggest that part of the mechanisms involved in the induction of apoptosis by FO/P may be through epigenetic regulation of genes involved in apoptosis throughout colon carcinogenesis.

Starch residue samples from two Australian sweet potato varieties (Beauregard and Northern Star) and two Indonesian sweet potato varieties (Bis192 and Bis183), and commercial sample of sweet potato starch residue, were studied for their pectins. Pectins were extracted using 0.1M HCl, 0.05M NaOH, 0.1M HCl/0.75%SHMP, and 0.05M NaOH/0.75% SHMP. Hydrolysis of residual starch in the cell wall of sweet potato using heat stable α-amylase and amyloglucosidase was employed prior to pectin extraction to eliminate starch contamination. Pectins were characterised for yield, moisture, ash, galacturonic acid content (GA), degree of esterification (DE), neutral sugars content and molecular weight (MW). Selected pectins were also characterised for their dispersion and gel properties. Pectin with the highest yield was compared to commercial low methoxyl pectin (GRINSTED??YF 450) for its application in milk pudding. The pectin characteristics are dependent on variety and extraction process; however, the extraction methods were dominant. Yields were between 7 and 30 % of the cell wall. Ash varied from 2.9 to 6.8%. NaOH/SHMP extraction gave higher yields and ash contents. Moisture was between 5.5 and 6.7. GA varied from 27 to 80% with the highest found in Bis192 extracted using NaOH/SHMP. DE varied between traceable and 57%. HCl extraction gave higher DE, while NaOH/SHMP caused demethylation. Neutral sugars varied from 10 to 25% where galactose was the predominant sugar, followed by arabinose, rhamnose, glucose and xylose. Pectin MW was very heterogeneous. HCl extraction gave higher MW than the others. Dispersion of sweet potato pectins displayed shear-thinning. Consistency coefficient (K) increased, while flow behaviour index (n) decreased with increasing pectin concentration. HCl-extracted pectins formed stiff sugar-gels at concentrations higher than 2%. The optimum Ca2+ concentration for NaOH/SHMP-extracted pectin gels was 20 mg/g pectin. Pectin with the highest yield and stiffness (Norther Star extracted using NaOH containing SHMP) was used for milk pudding preparation. This pudding had comparable texture to pudding prepared from commercial low methoxyl citrus pectin (GRINSTED??YF 450). Overall, this study demonstrated that pectin from sweet potato starch residue is mainly low methoxyl pectin with possible chemical structure of rhamnogalacturonan I (RGI) that has potential for food applications.

In strongly acidic soils (pH <4.5) aluminium (Al) becomes soluble in quantities that can lead to Al phytotoxicity. It is estimated that approximately 30 % of the worlds’ potentially arable lands are acidic, with Al toxicity the most limiting factor for plant growth on acid soils. With increasing use of marginal land in cropping systems, this area could reach 70 %. Cell wall pectin provides up to 70 % of the root cation exchange capacity. Pectin is suggested to control a number of physiological properties of the plant cell wall such as porosity, charge density, microfibril spacing and pH. The ability of pectin to bind cations is not only important for the uptake of nutrients but is implicated in metal toxicity, in particular Al. Despite over a century of research, the mechanisms of Al toxicity are yet to be fully elucidated or agreed upon. Gluconacetobacter xylinus is a gram-negative, soil dwelling bacterium which produces extracellular cellulose. It is an established archetype for the study of cellulose biogenesis. In the presence of pectin in the growth medium, the bacterium can form cellulose-pectin composites. Recently, the bacterium has been used to form composites as model cell walls to understand plant cell wall deposition. Additionally, bacterial cellulose composites in their natural hydrated state mimic the hydration state of primary plant cell walls. The aim of this project was to attempt to incorporate this novel cell wall analog into laboratory investigations into metal interactions with plant cell walls. Preliminary work was undertaken to optimise the bacterial culture medium, growth conditions, analysis of the composites and developing an overall general methodology. The medium buffering system was altered, growth under non-optimal pH conditions was evaluated and Al was successfully incorporated into the composites. Appropriate sample preparation for scanning electron microscopy (SEM) of the composites was determined. This work resulted in the successful production of bacterial cellulose-pectin composites with 30 % w/w pectin incorporation. The effect of Al on the tensile properties of the composites was examined. Aluminium had no effect on the stress/strain profiles, confirming the hypothesis that pectin is not the main load bearing component of the cell wall. The composites were used to investigate the effects of Al and other trace metals (copper (Cu), gadolinium (Gd), lanthanum (La), ruthenium (Ru) and scandium (Sc)) on the hydraulic conductivity of the composites. Hydraulic conductivity was reduced to ≈ 30 % of the initial flow rate by 39 μM Al and 0.6 Cu μM, ≈ 40 % by 4.6 μM La, 3 μM Sc and 4.4 μM Ru, and ≈ 55 % by 3.4 μM Gd. These metal concentrations were selected based on the concentrations causing a 50 % reduction in root elongation in cowpea (Vigna unguiculata L.). This study demonstrated that all the trace metals caused a similar decrease of hydraulic conductivity, despite the different concentrations of the metals used. Scanning electron microscopy showed changes in pectin porosity with metal binding which may account for the decreases in hydraulic conductivity observed. As the composites could not be used as a model material in all investigations, pectin-only systems were employed in a rheological study to investigate the effect of increasing concentrations of Al, Ca, Cu or La at pH 4 on pectin (degree of esterification 30 %, 1 % w/v) gel physical strength. Comparing similar saturation levels, La formed the weakest gel, followed by Ca, which was similar to Al, while the strength of Cu gels was almost an order of magnitude stronger than the other cations. This study was the first to investigate Al and La pectate gel strength. The swelling of the gels also varied, with Ca gels being the most swollen. Pectin was also used to determine the exchange selectivity of Al, Cu, Gd, La, Ru and Sc toward Ca pectate. The order of selectivity was found to be Sc>Gd>La>Cu>Ru>Al. There were some parallels between this sequence and the rhizotoxicity data of the metals, suggesting that the strength with which metals bind to pectin is an indication of their rhizotoxicity. Through the use of synthetic pectate gel systems new information was discovered about the strength of pectin gels and the selectivity of trace metals towards pectin. These findings were in keeping with those of a number of related studies, as well as with studies of plant root tissue. Overall, the novel bacterial cellulose-pectin cell wall analog was successfully integrated into research into Al and other metal toxicity in plants, and offers a useful system that can overcome some of the difficulties encountered when using plant cell wall tissue. Further research may be warranted on manipulating the growth system to produce composites in the presence of the metal (ie. metal added to the growth medium), as opposed to post-formation treatments. Moreover, the production of a three way composite of cellulose, hemicellulose and pectin would likely be another useful analog for plant cell wall material.

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.

Submitted by Geise Jesus (geise.crj@gmail.com) on 2018-04-17T00:23:24Z No. of bitstreams: 2 Dissertação-Geise Cristina de Jesus.pdf: 1627240 bytes, checksum: cd3bd55063ba96f48854e00923ac8b0e (MD5) Carta comprovante.pdf: 353788 bytes, checksum: 3b2d5be6eb455c3a33e7fb4ae9adcc11 (MD5) / Approved for entry into archive by Alini Demarchi (ri.bar@ufscar.br) on 2018-07-10T12:57:32Z (GMT) No. of bitstreams: 2 Dissertação-Geise Cristina de Jesus.pdf: 1627240 bytes, checksum: cd3bd55063ba96f48854e00923ac8b0e (MD5) Carta comprovante.pdf: 353788 bytes, checksum: 3b2d5be6eb455c3a33e7fb4ae9adcc11 (MD5) / Approved for entry into archive by Alini Demarchi (ri.bar@ufscar.br) on 2018-07-10T12:58:21Z (GMT) No. of bitstreams: 2 Dissertação-Geise Cristina de Jesus.pdf: 1627240 bytes, checksum: cd3bd55063ba96f48854e00923ac8b0e (MD5) Carta comprovante.pdf: 353788 bytes, checksum: 3b2d5be6eb455c3a33e7fb4ae9adcc11 (MD5) / Made available in DSpace on 2018-09-17T18:54:56Z (GMT). No. of bitstreams: 2 Dissertação-Geise Cristina de Jesus.pdf: 1627240 bytes, checksum: cd3bd55063ba96f48854e00923ac8b0e (MD5) Carta comprovante.pdf: 353788 bytes, checksum: 3b2d5be6eb455c3a33e7fb4ae9adcc11 (MD5) Previous issue date: 2018-02-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Among the main industrial wastewaters, sugarcane vinasse figures as an actual environmental concern, due to its polluting potential and large volumes available, about 10 liters per liter of ethanol. Considering the alternatives to vinasse disposal, fertirrigation is the most commonly used. However, it is currently being questioned due to its effects on the soil and on groundwaters, caused by nutrient lixiviation such as potassium. The application of immobilized microalgae for wastewater treatment with emphasis on the removal of nutrients has increased over the last years. The aim of this study was to develop uniform alginate and pectin beads for immobilization of Desmodesmus subspicatus and evaluate its growth and ability to carbon, nitrogen and potassium removal in vinasse. The process parameters of bead production, type and concentration of biopolymer (alginate 1, 2, and 3% w/v and pectin 5, 7 and 10% w/v) and crosslinking agent concentration (calcium chloride 2, 5 and 10% w/v), were varied in order to evaluate their influence on bead characteristics. Results indicated that stable alginate and pectin beads were produced and according to the preliminary particle characterization, concentrations of 2% alginate and 7% pectin were chosen for immobilization of D. subspicatus and growth in vinasse. Immobilized D. subspicatus showed cellular growth in vinasse, with maximum specific rates of 0.009 h-1 and 0.002 h-1 in alginate and pectin beads, respectively. In the tests performed with 2% alginate, the immobilized microalgae reached 42, 49 and 48% carbon; 34, 35 and 34% nitrogen and 22, 23 and 32% potassium removal; and for pectin 7%, the removals were 32, 39 and 41% for carbon; 11, 24 and 34% for nitrogen and 39, 36 and 35% for potassium, for 2, 5 and 10% of calcium chloride, respectively. The microalgae were able to grow and remove appreciable amounts of nutrients from the vinasse. Compared with the free microalgae cultivation, immobilized microalgae indicate good prospects for the use of nutrient removal from vinasse. / A vinhaça é considerada a principal água residuária do setor sucroalcooleiro, sendo obtida pela destilação alcoólica do vinho para a obtenção do etanol. Considerando as alternativas para sua disposição, a fertirrigação na cultura da cana-de-açúcar é a mais utilizada. No entanto, o seu uso deve ser cauteloso, uma vez que em excesso pode culminar na contaminação dos lençóis freáticos, acarretando problemas ambientais. A aplicação de microalgas imobilizadas no tratamento de águas residuárias com ênfase principalmente na remoção de nutrientes tem aumentado nos últimos anos. Quanto aos métodos de imobilização celular, o sistema de encapsulamento em matrizes de macromoléculas como alginato e pectina vem despertando interesse devido às suas características de biodegradabilidade, biocompatibilidade e não toxicidade. Neste contexto, o objetivo do trabalho foi o desenvolvimento de esferas de alginato e de pectina para a imobilização da microalga Desmodesmus subspicatus, assim como a avaliação do seu crescimento e habilidade na remoção de carbono, nitrogênio e potássio da vinhaça. Os parâmetros do processo de produção das esferas, tais como tipo e concentração de biopolímero (alginato 1, 2, e 3% m/v e pectina 5, 7 e 10% m/v) e concentração de reticulante (cloreto de cálcio 2, 5 e 10% m/v) foram estudados quanto a sua influência nas características das esferas. Os resultados indicaram a obtenção de esferas estáveis de alginato e de pectina e, de acordo com os testes, optou-se por utilizar alginato 2% e pectina 7% para imobilização da D. subspicatus e seu cultivo na vinhaça. A microalga D. subspicatus imobilizada apresentou crescimento celular em vinhaça, com velocidades específicas máximas de 0,009 h-1, e 0,002 h-1 em esferas de alginato e de pectina, respectivamente. Nos ensaios realizados com alginato 2%, a microalga imobilizada atingiu remoções de 42, 49 e 48% de carbono; 34, 35 e 34% de nitrogênio e 22, 23,2 e 31,6% de potássio; e para a pectina 7%, as remoções foram de 32, 39 e 41% para carbono; 11, 24 e 34% para nitrogênio e 39,2, 35,8 e 35,2% para potássio para 2, 5 e 10% de cloreto de cálcio, respectivamente. Os resultados demonstraram a viabilidade do cultivo desta microalga, assim como a capacidade de remoção de compostos da vinhaça.

Misturas aquosas de polissacarídeos e proteínas são normalmente instáveis e separam-se em fases devido às interações repulsivas ou atrativas existentes entre os polímeros. O efeito da temperatura, do pH e da concentração polimérica no comportamento de misturas de pectina/caseína foi estudado nesse trabalho. Um diagrama de fases construído em pH 7 revelou que a mistura é estável apenas em baixas concentrações. Concentrações mais elevadas levam à incompatibilidade termodinâmica, governada por forças puramente entrópicas, e ao aparecimento de duas fases: uma rica em caseína (inferior) e outra rica em pectina (superior). A decomposição espinodal pôde ser visualizada nos estágios iniciais da separação de fases e, nos estágios intermediários, observou-se a formação de emulsões água/água. Quando o pH dessas emulsões é reduzido para abaixo de 6, a pectina é atraída para a fase de caseína, resultando na formação de partículas de complexo pectina/caseína que não coalescem e são resistentes à adição de sal (NaCl 100 mM), apresentando um diâmetro médio aproximado de 4 m. As micropartículas de pectina/caseína produzidas por este método demonstraram ser capazes de encapsular com alta eficiência tanto substâncias hidrofóbicas quanto hidrofílicas, possibilitando sua aplicação na encapsulação de compostos variados para fins diversos. Neste trabalho, as micropartículas foram utilizadas para encapsular cristais de metronidazol e sua utilização na obtenção de filmes de aplicação intra-bolsa periodontal foi avaliada in vitro. As dispersões de pectina/caseína contendo as micropartículas carregadas foram submetidas à secagem para a obtenção dos filmes. Estes, reticulados ou não com cálcio, sustentaram a liberação in vitro do fármaco por pelo menos 7 dias in vitro. A reticulação foi importante para reduzir a desintegração dos filmes, contribuindo para aumentar o tempo de permanência deles no local de aplicação e para melhorar suas propriedades mecânicas, facilitando seu manuseio e inserção na bolsa periodontal. Com esses resultados, conclui-se que os filmes de micropartículas de complexo pectina/caseína contendo metronidazol desenvolvidos neste trabalho são excelentes candidatos a sistemas de liberação local para o tratamento da doença periodontal. / Aqueous mixtures of polysaccharides and proteins are usually unstable and phase-separate either because of repulsive or attractive interactions. The effect of temperature, pH, and biopolymer concentration on the phase behavior of pectin/casein mixtures was investigated. A phase diagram built at pH 7 revealed that the mixture is stable at low polymer concentrations. Higher concentrations lead to thermodynamic incompatibility, driven purely by entropic forces, and to the appearance of two phases: one enriched with casein (lower) and the other, with pectin (upper). Spinodal decomposition was visualized in the early stages of phase separation. In the intermediate stages, water-in-water emulsions were observed. When the pH of these emulsions was lowered below 6, pectin was attracted by casein-rich phase, resulting in the formation of particles (diameter ~ 4 m) of pectin/casein complex, which do not coalesce and are insensitive to salt addition (100 mM NaCl). Pectin/casein microparticles obtained by this method were able to encapsulate with high efficiency either hydrophobic or hydrophilic substances and, due to that, could be applied in the encapsulation of a great variety of compounds for different purposes. In this work, the pectin/casein microparticles were used to encapsulate metronidazole crystals and the preparation of intra-periodontal pocket films with them was evaluated. Therefore, dispersions of loaded pectin/casein microparticles were dried in an oven. Films cross-linked or not with calcium sustained the in vitro drug release at least for 7 days. The cross-linking with calcium was important to reduce film disintegration, accounting for its permanence in the applied region, and to improve the mechanical properties, which facilitates manipulation and insertion into the periodontal pocket. With these results we conclude that the films formed by microparticles of pectin/casein complex loaded with metronidazole are excellent candidates for local drug delivery systems for the treatment of periodontal disease.