ABSTRACT
Breadfruit is an exotic tree in Brazil, very well acclimatized. Despite of being a laticifer plant, the knowledge about its latex is scarce. However, it is known that proteolytic enzymes represents over 50% of the latex composition in laticifers plants. The aim of this study was to evaluate the potential of breadfruit (Artocarpus altilis var. Apyrena) latex as a source of milk clotting proteases and partially characterize it. The proteolytic activity of the fraction of crude extract was assessed using azocasein and quantitation of total protein, the bicinchoninic acid (BCA). Milk-clotting activity was analyzed using skim milk at 12%. The enzyme activity was analyzed under different temperature conditions (35 to 80°C) and pH (5.8 to 10.7) presenting optimum activity in alkaline pH (8.5) and 50°C; being stable to the two variables at 120 minutes during the test. The clotting activity was directly proportional to the temperature at the better concentration of CaCl2 (10mmol L-1). The results indicate that enzyme is a possible replacement for calf rennet.
A fruta-pão é uma árvore exótica no Brasil, onde se aclimatou muito bem. Embora seja uma planta lactífera, o conhecimento sobre seu látex é escasso. No entanto, sabe-se que enzimas proteolíticas correspondem a mais de 50% da composição do látex em plantas lactíferas. O objetivo deste estudo foi avaliar o potencial do látex da fruta-pão (Artocarpus altilis var. Apyrena) como fonte de protease coagulante do leite e caracterizar parcialmente a enzima. A atividade proteolítica da fração de extrato bruto foi avaliada utilizando azocaseína e, para a quantificação das proteínas totais, o ácido bicinconínico (BCA). A atividade de coagulação do leite foi testada utilizando leite desnatado a 12%. A protease foi testada em diferentes condições de temperatura (35 a 80°C) e pH (5,8 a 10,7) e apresentou atividade ótima a 50°C e pH alcalino (8,5) sendo estável a estas variáveis durante 120 minutos. A atividade coagulante no leite foi diretamente proporcional à temperatura na melhor concentração de CaCl2 a 10µmol L-1. Os resultados indicam que a enzima analisada é uma possível alternativa à quimosina.
ABSTRACT
Objective To evaluate the efficacy and safety of composite milk clotting enzyme capsules in children with functional dyspepsia.Methods A multicenter,randomized,open-label clinical trial was carried out in 4Guangzhou hospitals during Feb.2012 to Mar.2013.Children with functional dyspepsia on basis of Rome Ⅲ criteria were enrolled and treated with composite milk clotting enzyme capsules.Total symptom score and drug-related adverse reactions were evaluated after one-week and two-week therapy.The treatment outcome were divided into very effective,fairly effective and ineffective according to the symptom score changes.Superiority test was done for effectiveness of the treatment based on efficacy difference by 10％ regarded as superiority.Results Two hundred and one children were enrolled.One hundred and ninety-six children completed the study as planned.After two-week therapy,better effective rate and total effective rate were 68.88％ and 87.76％,higher than those of one-week therapy (27.04％,76.02％) (U =2.935,P ＜ 0.05).The lower limits of 95 ％ confidence interval of difference in symptoms improve rate,better efficacy rate and symptom score decrease rate between two-week and one-week therapy were more than 10％,and two-week therapy was superior to one-week therapy.No significant drug-related adverse reaction was found during the study.Conclusions Composite milk clotting enzyme capsules can improve functional dyspepsia symptoms significantly.The effectiveness of two-week of treatment is better than that of one-week treatment.No obvious drug-related adverse reaction is observed.
ABSTRACT
The partial characterization and purification of milk clotting enzyme obtained from the (root latex) of Jacaratia corumbensis O. kuntze was studied, by fractional precipitation with ammonium sulphate and ion exchange chromatography. The ammonium sulphate precipitate showed five fractions (AS1- 0-20 percent; AS2 - 20-40 percent; AS3 - 40-60 percent; AS4 - 60-80 percent; AS5 - 80-100 percent) and among the fractions obtained, the 40-60 percent fraction (AS3) showed the highest milk clotting activity with a purification factor of 1.2 fold in relation to the crude extract. This fraction when applied on Mono Q column yielded two protein peaks (p1 and p2), but p1 pool showed the best milk-clotting activity. The optimal pH for the crude and partially purified extract was 6.5 and 7.0, respectively. The maximum milk-clotting activity was at 55ºC for the both crude and partially purified extracts. The enzyme was inhibited by iodoacetic acid which suggested that this enzyme was a cysteine protease, with molecular weight of 33 kDa.
A enzima coagulante de leite obtida de látex de raiz de Jacaratia corumbensis O. kuntze foi caracterizada parcialmente e purificada, por precipitação fracionária com sulfato de amônio e cromatografia de troca de íon. Foram utilizadas cinco frações de sulfato de amônio (AS1 - 0-20 por cento; AS2 - 20-40 por cento; AS3 - 40-60 por cento; AS4 - 60-80 por cento; AS5 - 80-100 por cento), a fração 40-60 por cento (AS3) mostrou alta atividade coagulante com um fator de purificação de 1,2 vezes em relação ao extrato bruto. Esta fração foi aplicada em coluna Mono Q obtendo dois picos de proteína (p1 e p2), o p1 mostrou melhor atividade coagulante. O pH ótimo para o extrato bruto e parcialmente purificado foi 6,5 e 7,0, respectivamente. A atividade coagulante foi atingida a 55ºC para ambos os extratos, bruto e parcialmente purificado. A enzima foi inibida por ácido iodoacético que sugere que esta enzima é uma cisteína protease, com peso molecular de 33 kDa.