Abstract Cagaita (Eugenia dysenterica DC) is a Brazilian cerrado fruit with great economic potential. It can be consumed in natura or as processed products like juices and pulps. The search for products with lower nutritional and sensorial changes led to the development of non-thermal techniques, where membrane processes stand out. The use of immobilized pectinolytic enzymes to reduce juices and pulps turbidity and viscosity has several advantages over the free enzymes use, such as the enzyme reduction costs and their reuse. The objective was the cagaita pulp hydrolysis optimization step using encapsulated commercial pectinase, the evaluation of its reuse in several cycles and its application in the microfiltration process. The activity of free commercial enzymes and encapsulated in calcium alginate in the pulp was evaluated, mainly the viscosity and turbidity reduction. The optimum conditions for hydrolysis with encapsulated enzymes were: temperature (30 °C), without stirring and enzymatic concentration (570 μL/L), considering the clarity increment and viscosity reduction. After 8 cycles, the encapsulated enzymes maintained 30% of its activity in reducing viscosity, resulting in the reuse possibility. The mean flow rate during the microfiltration (MF) of the hydrolyzed cagaita pulp with encapsulated enzymes was 13.4% higher than the non hydrolyzed pulp, indicating that enzymatic treatment was also efficient in process time reduction. Enzymes encapsulated can be applied by the juices and fruit pulps industries as a pre-process step for MF increasing the permeate flows, reducing operational and input costs.

Abstract Cagaita (Eugenia dysenterica DC) is a Brazilian cerrado fruit with great economic potential and can be consumed in natura or as processed products like juices and pulps. The search for products with lower nutritional and sensorial changes led to the non-thermal techniques development where membrane processes stand out. The use of immobilized pectinolytic enzymes to reduce juices and pulps turbidity and viscosity has advantages over free enzymes such as enzyme reduction costs and reuse. The objective was the hydrolysis optimization of cagaita pulp with encapsulated pectinase, evaluation of reuse in cycles and application in microfiltration (MF). The free commercial enzymes and encapsulated activity in calcium alginate in pulp was evaluated, viscosity and turbidity reduction. The optimum hydrolysis with encapsulated enzymes conditions were temperature (30 °C), without stirring, enzymatic concentration (570 μL/L), considering clarity increment and viscosity reduction. After 8 cycles, encapsulated enzymes maintained 30% of its activity in reducing viscosity and reuse possibility. Microfiltration flow rate of hydrolyzed pulp with encapsulated enzymes was 13.4% higher than the nonhydrolyzed, indicating that enzymatic hydrolysis was efficient in time reduction. Encapsulated enzymes can be applied in juices and pulps as a pre-process for increasing the permeate flows, reducing operational and input costs.

Abstract Despite the high catalytic properties of pectinases, the utilization of free enzymes always presents some hindrances such as low stability, a difficulty for product recovery and the impossibility of continuous use. Enzyme encapsulation is one of the methods used to overcome these limitations; however, some kind of effect is expected to occur on its catalytic activity. The objective of this work was to study the parameters involved in the process of encapsulation of a commercial pectinase product in calcium alginate and the effect of this encapsulation on its catalytic activity. The effect of the parameters of sodium alginate, calcium chloride, enzyme concentration and reaction time on enzymatic activity and encapsulation yield were also evaluated. The effect of pH and temperature on the activity of the free and encapsulated enzyme was studied. The highest yield of immobilization was obtained with a concentration of enzyme solution of 4%. Free and encapsulated enzymes showed similar behavior regarding the catalytic activity. The encapsulated enzyme had a narrower pH range (pH 4.0) than the free enzyme (pH 3.0 to 5.0). Besides, the encapsulated enzyme showed an increase in the stability in the pH range between 7 and 8 and above 10 to 12.

O ácido ascórbico é uma vitamina hidrossolúvel de importância nutricional há muito estabelecida, por sua atuação como cofator em diversos processos fisiológicos e como antioxidante. O ser humano depende da ingestão diária desse micronutriente, cujas principais fontes são as frutas e hortaliças. Por ser um nutriente menos estável, o ácido ascórbico sofre perdas no processamento e no armazenamento, influenciadas por diversos fatores, como pH, temperatura, presença de íons, etc. A literatura apresenta escasso material sobre a estabilidade de ácido ascórbico em armazenamento doméstico, além de haver poucas informações sobre essa vitamina em sucos frescos. A metodologia padrão de análise dessa vitamina em sucos é o Método Titulométrico de Tillmans, que pode apresentar ponto de viragem de difícil visualização. Neste trabalho, foram avaliadas a concentração e a estabilidade do ácido ascórbico de sucos comerciais recém-preparados de laranja, abacaxi com hortelã e melancia, armazenados sob refrigeração e em temperatura ambiente, por dois métodos titulométricos distintos. O método alternativo de análise (NBS) superestimou a concentração de ácido ascórbico das amostras. Não houve diferença significativa na estabilidade da vitamina em sucos armazenados em temperaturas entre 6 e 30ºC, no período testado. O ácido ascórbico foi estável por 24h no suco de laranja, porém apresentou decréscimo significativo após 8h de armazenamento nos sucos de abacaxi e melancia, possivelmente em virtude das diferenças de acidez inicial desses sucos. Em comparação com a literatura disponível, verificou-se que sucos frescos apresentaram menor estabilidade de ácido ascórbico do que sucos industrializados.

Ascorbic acid is a water soluble vitamin of long-established nutritional importance for its role as a cofactor in many physiological processes and as an antioxidant. Human beings depend on daily intake of this micronutrient, whose main sources are fruits and vegetables. As the least stable nutrient, ascorbic acid suffers losses during processing and storage, influenced by several factors (pH, temperature, presence of ions, etc.). The literature provides little material on the stability of ascorbic acid in home storage and there is little information about this vitamin in fresh juices. The standard methodology for analysis of this vitamin in juices is the titrimetric method of Tillmans, which can present difficulties in visualizing the turning point. In this study, the concentration and stability of ascorbic acid in freshly prepared commercial juices of orange, pineapple with mint and watermelon, stored under refrigeration or at room temperature, were evaluated with two different titrimetric methods. The alternative method of analysis (NBS) overestimated the ascorbic acid concentration of the samples. There was no significant difference in the stability of this vitamin in juices stored in temperatures between 6 and 30ºC during the testing period. Ascorbic acid was stable for 24 h in orange juice, but showed significant decrease after 8 h storage in pineapple and watermelon juices, possibly due to differences in initial acidity of these juices. In comparison to the available literature it was found that fresh juices had lower ascorbic acid stability than industrialized juices.

Carotenoids have antioxidant activity, but few are converted by the body into retinol, the active form of vitamin A. Among the 600 carotenoids with pro-vitamin A activity, the most common are α- and β-carotene. These carotenoids are susceptible to degradation (e.g., isomerization and oxidation) during cooking. The aim of this study was to assess the total carotenoid, α- and β-carotene, and 9 and 13-Z- β-carotene isomer contents in C. moschata after different cooking processes. The raw pumpkin samples contained 236.10, 172.20, 39.95, 3.64 and 0.8610 µg.g- 1 of total carotenoids, β-carotene, α-carotene, 13-cis-β-carotene, and 9-Z-β-carotene, respectively. The samples cooked in boiling water contained 258.50, 184.80, 43.97, 6.80, and 0.77 µg.g- 1 of total carotenoids, β-carotene, α-carotene, 13-Z-β-carotene, and 9-Z-β-carotene, respectively. The steamed samples contained 280.77, 202.00, 47.09, 8.23, and 1.247 µg.g- 1 of total carotenoids, β-carotene, α-carotene,13-Z-β-carotene, and 9-Z-β-carotene, respectively. The samples cooked with added sugar contained 259.90, 168.80, 45.68, 8.31, and 2.03 µg.g- 1 of total carotenoid, β-carotene, α-carotene, 13-Z- β-carotene, and 9-Z- β-carotene, respectively. These results are promising considering that E- β-carotene has 100% pro-vitamin A activity. The total carotenoid and carotenoid isomers increased after the cooking methods, most likely as a result of a higher availability induced by the cooking processes.

Enzimas pectinolíticas de origem microbiana são conhecidas por desempenhar um papel importante comercialmente em uma série de processos industriais. Dois tipos de levedura podem ser distinguidos para a produção dessas enzimas. Um grupo inclui aqueles que têm capacidade de produzi-las na ausência de um indutor e outro grupo compreende as leveduras que as produzem na presença de um indutor. O objetivo deste estudo foi investigar a influência de substâncias pécticas, de glicose, do pH e da temperatura na atividade da poligalacturonase de Kluyveromyces marxianus CCMB 322. O cultivo foi em caldo de fermentação contendo diferentes concentrações de glicose e substâncias pécticas. A atividade da poligalacturonase foi determinada pelo método do DNS, e pH e temperatura otimizados pelo delineamento experimental central composto de experimentos. A poligalacturonase secretada por K. marxianus CCMB 322 foi parcialmente constitutiva, apresentando pH e temperatura ótima de 7,36 e 70 °C, respectivamente e reteve 93% de sua atividade original após 50 minutos a 50 °C. A termoestabilidade da enzima poligalacturonase foi estudada em várias temperaturas (50, 60, 70 e 80 °C) em diferentes tempos de incubação (0, 10, 20, 30, 40 e 50 minutos). Este estudo mostrou que a glicose tem influência na regulação da síntese da poligalacturonase.

Microbial pectinolytic enzymes are known to play a commercially important role in a number of industrial processes. Two kinds of yeast can be discerned regarding the production of enzymes. One group includes those which can produce enzymes in the absence of an inducer, and the other group comprises the yeasts that produce enzymes in the presence of an inducer. The objective of this study was to investigate the influence of pectic substances, glucose, pH, and temperature on the polygalacturonase activity by Kluyveromyces marxianus CCMB 322. The yeast was grown in a fermentation broth containing different concentrations of glucose and pectic substances. The polygalacturonase activity was determined by the DNS method, and the pH and temperature were optimized using a central composite experimental design. The polygalacturonase secreted by K. marxianus CCMB 322 was partially constitutive showing optimum pH and temperature of 7.36 and 70 °C, respectively, and maintained approximately 93% of its original activity for 50 minutes at 50 °C. Thermal stability of the polygalacturonase enzyme was studied at different temperatures (50, 60, 70, and 80 °C) and different incubation times (0, 10, 20, 30, 40, and 50 minutes). This study showed that glucose can influence the regulation of the synthesis of polygalacturonase.

A mamoneira (Ricinus communis L.) é uma oleaginosa de alto valor econômico pelo fato de apresentar um mercado bem definido para o óleo extraído de suas sementes. A torta, que é um resíduo desta extração, se destaca pelo alto teor em proteínas. Dentre as proteínas encontradas na torta destaca-se a ricina, uma citotoxina, que inviabiliza sua utilização como fonte protéica alternativa para alimentação animal. O presente trabalho tem como objetivo identificar um melhor tratamento experimental para a extração de ricina da torta de mamona, visando futuros estudos de perda de integridade da ricina, o que garantiria a inocuidade do produto. Para tanto, buscou-se identificar a solução de maior capacidade de extração de proteínas, empregando a metodologia de superfície de resposta. Um delineamento composto central rotacional foi elaborado a fim de verificar o melhor pH e concentração de NaCl para a extração. Dos cinco diferentes valores de pH (4,0; 4,6; 6,0; 7,4; 8,0) e concentração de NaCl (0,0M; 0,3M; 1,0M; 1,7M; 2,0M) utilizados, o tratamento associando fosfato de potássio 0,2M/NaCl 1,7M pH 7,4 foi escolhido como o melhor. A concentração de proteína extraída neste tratamento chegou a valores quatro vezes maiores que o encontrado no de mínima extração de proteína. Pela evidenciação do gel de eletroforese não houve extração preferencial de ricina nos tratamentos testados, entretanto etapas de purificação usando diálise e precipitação com sulfato de amônio, permitiram uma evidenciação melhor das duas cadeias polipeptídicas de ricina.

Castor bean (Ricinus communis L.) is an oilseed crop of high economic value due to the fact of presenting a clearly defined market for the oil extracted from its seeds. Castor cake, which is a residue of oil extraction, is at the moment receiving special attention because of its high protein content. However, among the proteins found in this cake it is observed the presence of ricin, a cytotoxin, which turns this seed dangerous to be used as an alternative protein source for animal feed. The present research has the objective of identifying the best experimental treatment for extraction of ricin from castor cake, with the aim of future studies of loss of ricin integrity, which would ensure the safety of the product. With this aim, it was initiated the search for a buffer of higher capacity of proteins extraction, using the response surface methodology. A central composite design was developed in order to determine the best pH and NaCl concentration for extraction. Of the five different pH values (4.0, 4.6, 6.0, 7.4, 8.0) and NaCl (0.0M, 0.3M, 1.0M, 1.7M, 2.0M) used, the treatment containing 0.2M potassium phosphate / 1.7M NaCl pH 7.4 was chosen as the best. The amount of protein extracted in this treatment reached values four times larger than the minimum found in other treatment studied. The electrophoresis analysis showed that there was no preferential extraction of ricin in the treatments; however purification steps using dialysis and precipitation with ammonium sulfate led to a better resolution of the two polypeptide chains presents in ricin.

O extrato bruto de umbu-cajá foi avaliado quanto à influência do pH e da temperatura na atividade e na estabilidade de peroxidases e polifenol-oxidases nativas. Para avaliação das condições de inativação das enzimas por tratamento térmico e por adição de agente redutor (ácido ascórbico), foi realizado um experimento fatorial (n = 3) com aplicação do software Statistica (6.0) para elaboração da planilha de ensaios e das superfícies de resposta. Para as peroxidases, foram encontrados os seguintes parâmetros ótimos: pH = 5,0 e temperatura = 40 ºC. E para polifenol-oxidases: pH = 7,0 e temperatura = 40 ºC. Ambas as enzimas foram estáveis em toda a faixa de pH testada (3,0 - 10,0), peroxidases e polifenol-oxidases mantiveram acima de 60% de sua atividade em temperaturas acima de 30 e 40 ºC, respectivamente. Para inativação total das enzimas em teste, duas alternativas foram sugeridas: 200 mg.L-1 de ácido ascórbico, 92 ºC/3,15 minutos ou 100 mg.L-1 de ácido ascórbico, 96 ºC/2,80 minutos.

A crude extract of Spondias spp. was evaluated for the influence of pH and temperature on the activity and stability of its peroxidases and polyphenol-oxidases. In order to evaluate the conditions for the inactivation of the enzymes by heat treatment and by addition of a reducing agent, a factorial experimental design (n = 3) was employed using the Statistica (6.0) software package for data analysis. The optimal conditions found for peroxidases were: pH = 5.0 and temperature = 40 ºC, and for polyphenol-oxidases they were pH = 7.0 and temperature = 40 ºC. The peroxidases and polyphenol-oxidases were stable at all pH values tested (3.0 - 10.0) and maintained more than 60% of their activity at temperatures above 30 and 40 ºC, respectively. To achieve the total inactivation of these enzymes, two alternatives can be suggested: incubation at 92 ºC for 3.15 minutes with 200 mg.L-1 of ascorbic acid or incubation at 96 ºC for 2.80 minutes with 100 mg.L-1 of ascorbic acid.

O presente trabalho teve por objetivo purificar e caracterizar a fração lipolítica secretada por uma linhagem de Rhizopus sp. Apenas 3 etapas de purificação foram necessárias para alcançar homogeneidade em eletroforese de proteína desnaturada. Uma única banda com massa molecular de 37,5 KDa e atividade específica de 1446U/mg foi obtida. A fração purificada apresentou 2 isoformas de lipase, ambas com temperatura ótima de atividade igual a 50ºC, mantiveram-se estáveis entre valores de pH entre 6,5 e 7,5 e a temperaturas inferiores a 50ºC e mantiveram sua atividade na presença de hexano. A fração lipolítica foi inativada por Hg+ e por n-bromosuccinimida e ativada por Na+.

The present study had as a goal to purify and characterize the lipolytic fraction secreted by a strain of Rhizopus sp. Only 3 steps of purification were necessary to achieve SDS-PAGE homogeneity. One band with 37.5 KDa molecular mass and with 1446 U/mg specific activity was obtained. The purified fraction presented 2 lipase isoforms; both showed optimum activity at 50ºC, and were stable between 6.5 and 7.5 pH values and at temperatures below 50ºC and also kept their activity in hexane. The lipase was inactivated by Hg+2 and by n-bromosuccinimide and activated by Na+.

Lipases, especialmente as de origem microbiana, são largamente utilizadas em processos e na obtenção de produtos para as indústrias química, cosmética, farmacêutica e alimentícia. A produção de enzimas de elevada pureza é importante, principalmente, do ponto de vista do controle dos processos (ausência de interferentes), porém as etapas necessárias à purificação, em geral, provocam perdas na atividade das enzimas e aumentam seu custo final. O objetivo deste trabalho foi propor a melhor metodologia de purificação para a lipase de Rhizopus sp. através do teste de dois diferentes métodos cromatográficos (troca iônica e interação hidrofóbica) e, ainda verificar o melhor planejamento estatístico para caracterização bioquímica da mesma. Foi possível purificar parcialmente a lipase de Rhizopus sp. com o uso de coluna de DEAE Sepharose (troca aniônica) e de FENIL Sepharose (interação hidrofóbica). A primeira, embora mais seletiva para a enzima em questão, parece provocar redução de sua atividade. A presença de maiores concentrações de íons Na+1 na fração purificada por FENIL Sepharose parece contribuir para o aumento de atividade da lipase. Embora os resultados obtidos por análise multivariável para determinação das características bioquímicas da lipase sejam compatíveis com a análise univariável, aquele planejamento não foi considerado indicado no presente caso.

Lipases, especially of microbial origin, are widely applied in processes and in the production of insumesfor the chemical, cosmetic, pharmaceutical and food industries. To produce highly pure enzyme is important to process control (absence of interferentes). However the necessary stages to the purification, in general, cause losses of activity and increases enzyme final cost. The aim of this work was to consider the best methodology of purification for the Rhizopus sp. lipase through the test of two different chromatographic methods (ion exchange and hydrophobic interaction) and also to verify the best statistical design for the biochemical characterization of the enzyme. It was possible to partially purify the Rhizopus sp lipase applying a DEAE Sepharose (anionic exchange) column and a PHENYL Sepharose (hydrophobic interaction) column. The first, although more selective for the enzyme in study, seems to cause reduction of its activity. The presence of higher concentrations of Sodium ions in the PHENYL Sepharose fraction seems to contribute for an increased activity of the lipase. Although the results obtained through the factorial design for the biochemical characteristics of the lipase were compatible with the single variable analysis, that statistical approach should not be indicated in the present case.