Binding effect of proline-rich-proteins (PRPs) on in vitro antimicrobial activity of the flavonoids

The interaction of the cyanidin, pelargonidin, catechin, myrecetin and kaempferol with casein and gelatin, as proline rich proteins (PRPs), was studied. The binding constants calculated for both flavonoid-casein and flavonoid-gelatin were fairly large (105–107 M−1) indicating strong interaction. Due to higher proline content in gelatin, the binding constants of flavonoid-gelatin (2.5 × 105–6.2 × 107 M−1) were found to be higher than flavonoid-casein (1.2 × 105–5.0 × 107 M−1). All the flavonoids showed significant antibacterial activity against the tested strains. Significant loss in activity was observed due to the complexation with PRPs confirming that binding effectively reduced the concentration of the free flavonoids to be available for antibacterial activity. The decline in activity was corresponding to the values of the binding constants. Though the activities of free catechin and myrecetin were higher compared to pelargonidin, cyanidin and kaempferol yet the decline in activity of catechin and myrecetin due to complexation with casein and gelatin was more pronounced.

Seleção de basidiomicetos proteolíticos / Selección de basidiomicetos proteolíticos / Selection of proteolytic basidiomycetes

Basidiomicetos têm sido amplamente utilizados como produtores de enzimas, no entanto são pouco explorados quanto a sua capacidade de produção de proteases. Estes fungos são reconhecidos pelas suas propriedades antitumorais, hipocolesterolêmicas, antimutagênicas, antioxidantes entre outras. Assim, a associação destas propriedades aos derivados do leite pode potencializar estes produtos como alimentos funcionais. Neste sentido, o objetivo deste trabalho foi selecionar basidiomicetos produtores de proteases, com potencial uso no processo de fabricação de derivados do leite. Foram utilizadas 27 linhagens de fungos crescidas em meio mínimo adicionado de 0,2% de caseína. A atividade proteolítica foi verificada pela formação de halo pela adição de uma solução saturada de (NH4)2SO4. Concluiu-se que a produção de proteases não apresenta relação com o crescimento micelial. O melhor produtor de proteases é a linhagem Lentinula edodes U8/1, seguida por Pleurotus sp (U2/9, U6/10 e U2/11). Os basidiomicetos Agaricus blazei (U4/3), Agaricus sp (U5/1), Flamulina sp (U5/4), Lycoperdon sp (U8/8), Agaricus blazei (U2/7), Agaricus blazei (U7/2), Agaricus blazei (U7/4) e Agaricus blazei (U7/5) não produzem proteases suficientes para serem medidas pela metodologia. Desta forma, estes resultados embasam o uso de Lentinula edodes e Pleurotus sp para o desenvolvimento de potenciais aplicações na hidrólise de proteínas em alimentos.

Basidiomycetes have been widely used as enzyme producers, but are poorly explored about their ability to produce protease. These fungi are known as antitumor, cholesterol-lowering, antimutagenic, antioxidant among other biological activities. Thus, the combination of basidiomycete properties to dairy products can improve them as functional foods. Therefore, the objective of this work was to screen basidiomycete protease producers to prospect the use of these fungi on dairy products. 27 basidiomycete strains grown on minimal medium supplemented with 0.2% casein were used. The proteolytic activity was verified by halo formation after a (NH4)2SO4 saturated solution addition on the culture medium. The production of proteases is not associated with mycelial growth. The best producers of proteases is Lentinula edodes U8/1 and after Pleurotus sp (U2/9, U6/10 e U2/11). The basidiomycetes of Agaricus blazei (U4/3), Agaricus sp (U5/1), Flamulina sp (U5/4), Lycoperdon sp (U8/8), Agaricus blazei (U2/7), Agaricus blazei (U7/2), Agaricus blazei (U7/4) and Agaricus blazei (U7/5) do not produce enough proteases to be measured by the methodology. Thus, these results support the use of Lentinula edodes and Pleurotus sp as potencial basidiomycetes for protein hydrolysis on food.

Basidiomicetos han sido ampliamente utilizados como productores de enzimas, pero poco exploradas en su capacidad de producción de proteasa. Estos hongos son reconocidos por sus propiedades antitumorales, reductor de colesterol, antimutagénicos, antioxidantes entre otras. Así, la asociación de estas propiedades a los derivados de la leche puede potencializar estos productos como alimentos funcionales. En este sentido, el objetivo de este trabajo fue seleccionar basidiomicetos productores de proteasas, con potencial uso en el proceso de fabricación de productos lácteos. Se utilizó 27 cepas de hongos crecidos en medio mínimo adicionado de 0,2% de caseína. La actividad proteolítica fue verificada por formación de halo por la adición de solución saturada de (NH4)2SO4. Se concluyó que la producción de proteasas no presenta relación con el crecimiento del micelio. El mejor productor de proteasas es la cepa de Lentinula edodes U8/1, seguida por Pleurotus sp (U2/9, U6/10 y U2/11). Los basidiomicetos Agaricus blazei (U4/3), Agaricus sp (U5/1), Flamulina sp (U5/4), Lycoperdon sp (U8/8), Agaricus blazei (U2/7), Agaricus blazei (U7/2), Agaricus blazei (U7/4) y Agaricus blazei (U7/5) no producen proteasas suficientes para que sean medidos por la metodología. Por lo tanto, estos resultados apoyan el uso de Lentinula edodes y Pleurotus sp para el desarrollo de potenciales aplicaciones en hidrólisis de proteínas en alimentos.

Production of puerarin and isoflavones in cell suspension cultures of Pueraria lobata (Willd.): effects of medium supplementation with casein hydrolysate and coconut milk.

Callus induced from leaf explants of Pueraria lobata seedlings were suspended in Gamborg B5 medium supplemented with 1 mg l(-1) 2,4-dichlorophenoxyacetic acid, 1 mg l(-1) naphthalene acetic acid, 0.5 mg l(-1) kinetin and 30 g l(-1) sucrose. The effects of coconut milk and casein hydrolysate (CH) on cell growth and yields of puerarin and isoflavones in cells suspension were studied. The contents of total isoflavones and puerarin in suspension cultures were determined by spectrophotometry and HPLC. Coconut milk (10%, filter sterilized) decreased the growth of cell cultures and the accumulation of total isoflavones, while 0.2% CH promoted the growth of cell cultures and the accumulation and release of puerarin and total isoflavones. The total yield of puerarin and isoflavones were 34% and 40.8% higher than in the control, respectively. The optimum medium for cell cultures of leaves of P. lobata seedlings was B5 liquid medium supplemented with 2% sucrose, 1.0 mg l(-1) 2,4-D, 1.0 mg l(-1) NAA, 0.5 mg l(-1) kinetin and 20 mg l(-1) CH. The procedure use is a potentially useful for the production of isoflavones.

Behavior of Araujiain, a new cysteine phytoprotease, in organic media with low water content

In this paper we studied the effect of different organic solvents (1-octanol, trichloroethylene, ethanol, ethyl acetate, tetrahydrofuran, cyclohexane, propanone, acetonitrile, dichloromethane, chlorobenzene, N,N-dimethylformamide, acetophenone, diethyl ether, methanol, ethylene glycol and toluene) with low and constant water content on substrate preferences, thermostability and stability (caseinolytic activity retention after 4 h) of proteases of Araujia hortorum Fourn. (Asclepiadaceae). The stability of araujiain was high in N,N-dimethylformamide and ethanol at 40 ºC, but decreased at higher temperature. Araujiain substrates preferences in buffer Tris-HCl (pH 8), ethylene glycol and N,N-dimethylformamide exhibited different patterns, but the enzyme showed a high preference by glutamine derivative in all cases. According to FTIR spectroscopy studies, araujiain changed its secondary structure and as a consequence, it also changed its substrate preferences. This enzyme showed lower beta-helical character and greater beta-sheet folding in buffer than in organic media. A larger amount of antiparallel beta-sheet residues indicates the formation of tighter intermolecular hydrogen bonds and enzymatic aggregates. These facts could explain the higher esterolytic activities, the greater stability and good hydrolytic potential of araujiain in some organic media such as N,N-dimethylformamide.

Characterization of HC58cDNA, a putative cysteine protease from the parasite Haemonchus contortus

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.

Obtenção e caracterização química e nutricional de diferentes concentrados de caseína / Obtention and chemical and nutritional characterization of different bovine casein concentrates

OBJETIVO: O objetivo deste trabalho foi avaliar a composição centesimal, o perfil de aminoácidos e as características nutricionais de três concentrados de caseína, obtidos do leite bovino por diferentes processos. MÉTODOS: Os concentrados de caseína foram analisados pelos seguintes processos: uma caseína comercial, obtida por precipitação ácida seguida de neutralização; caseína obtida pela coagulação enzimática; caseína micelar obtida, respectivamente, pelos processos de microfiltração e diafiltração em membrana. A composição centesimal foi determinada por meio de procedimentos descritos no manual Official Methods of Analysis. O perfil de aminoácidos foi determinado após hidrólise ácida da proteína (HCl 6N, 105ºC, 22h) em auto-analisador de aminoácidos, dotado de coluna de troca catiônica e reação pós-coluna com ninidrina. Os perfis de aminoácidos essenciais dos diferentes concentrados de caseína foram comparados e estão de acordo com o padrão Food and Agriculture Organization/World Health Organization, para crianças de 2 a 5 anos de idade. O valor nutritivo foi determinado em ratos da linhagem Wistar, recém desmamados, por meio dos índices, digestibilidade aparente da proteína, quociente de eficiência líquida da proteína e quociente de eficiência protéica operacional. RESULTADOS: A caseína comercial apresentou maior concentração de proteína (92,0 por cento), que a caseína micelar (86,0 por cento) e o coágulo de caseína (72,0 por cento). Os animais nas dietas com as diferentes fontes de proteína, não apresentaram diferencas significativas quanto ao ganho de peso e ingestão de dieta. Maior digestibilidade (93,8 por cento) foi verificada na caseína comercial, comparada à dos outros dois concentrados (91,0 por cento). CONCLUSÃO: Os concentrados de caseína apresentaram diferenças quanto à composicão centesimal, sendo a caseína comercial superior na concentração protéica. O coágulo de caseína apresentou resultados inferiores aos demais concentrados, quanto aos índices quociente de eficiência líquida da proteína e quociente de eficiência protéica operacional.

Contact of Entamoeba histolytica with baby hamster kidney-21 (BHK-21) cell line on cysteine proteinase activity.

BACKGROUND & OBJECTIVES: Entamoeba histolytica, the causative agent of amoebiasis and amoebic liver abscess, lyses host cells by direct contact using surface lectins and releases cysteine proteinase (CP). Virulence of E. histolytica is directly related to activity of its CP. The relationship of CP activity and cytotoxicity has not been established. The present study was carried out to explore the events following contact of E. histolytica with target cells. METHODS: Protease activity of E. histolytica was measured by azocaseine and haemoglobin assays, and cysteine proteinase activity was assessed by substrate gel electrophoresis. Target cell lysis was measured by chromium release assay. RESULTS: Protease activity of E. histolytica was increased 2.5-fold following contact with BHK-21 cell line. CP activity of trophozoites alone was visualized at position 56, 35 and 29 kDa in substrate gel electrophoresis. Contact of trophozoites with target cells augmented the cytotoxic activity of amoebic CP. The increase in CP activity seen by substrate gel electrophoresis and cytotoxicity assay was blocked by pretreatment with E 64, a specific CP inhibitor and GalNAc, a contact inhibitor. INTERPRETATION & CONCLUSION: The present data showed the involvement of amoebic CP in cytotoxicity and that the CP activity was enhanced on lectin-mediated contact of E. histolytica to the target cells. Further studies need to be done to understand the mechanism at the molecular level.

Nicotine induced alterations on reproductive organs of female rats under protein deficient condition.

Nicotine causes decrement in body weight, reduction in ovarian and uterine weight, irregularity in estrous cycle and histological damage in ovary and uterus in rats maintained on normal (18% casein) and protein restricted diet (5% casein). The degree of nicotine toxicity increases in protein inadequacy.

Adições crescentes de ácido fítico à dieta não interferiram na digestibilidade da caseína e no ganho de peso em ratos / Increasing quantities of phytic acid in the diet did not affect casein digestibility and weight gain in rats

O crescente consumo de alimentos de origem vegetal, sejam como fontes protéicas com baixo teor de gordura ou como fontes de fibras, tem acrescido à dieta humana o ácido fítico. Devido à sua carga altamente negativa, o ácido fítico tem sido visto como componente de ação antinutricional capaz de quelar minerais bivalentes, proteínas e amido, podendo comprometer a biodisponibilidade destes nutrientes. No presente estudo investigou-se a influência da adição de ácido fítico à dieta de caseína, em concentrações iguais ou até oito vezes superiores àquelas encontradas no feijão-comum Phaseolus vulgaris, cultivar IAC-Carioca (14,7mg de ácido fítico/g feijão cru), durante período experimental de dez dias, sobre os índices nutricionais Ganho de Peso, Quociente de Eficiência da Dieta, Quociente de Eficiência Protéica Líquida, Digestibilidade Aparente e Digestibilidade Verdadeira. Trinta e seis ratos machos SPF da linhagem Wistar, recém-desmamados, divididos em grupos experimentais com seis ratos cada, foram alimentados com dieta purificada AIN-93G isenta de ácido fítico (Controle) e dietas teste AIN-93G acrescidas de 218, 436, 872 e 1744mg de ácido fítico/kg de dieta (Tratamentos). Os ganhos de peso (g) e os índices de qualidade dietética e protéica não apresentaram diferença estatística (p>0,05), e os valores médios entre os grupos foram: Ganho de Peso: 59,5 ± 5,0g; Quociente de Eficiência da Dieta: 0,39 ± 0,01; Quociente de Eficiência Protéica Líquida: 3,64 ± 0,12; Digestibilidade Aparente: 92,7 ± 1,1 por cento e Digestibilidade Verdadeira: 94,4 ± 0,9 por cento. Os resultados demonstraram que nas condições experimentais utilizadas, o ácido fítico não foi capaz de alterar o valor nutritivo da caseína.

Influência das isoflavonas extraídas da soja na hipercolesterolemia e na aterosclerose experimental induzida pela caseína / Isoflavones influences extracted of soy in the experimental hypercholesterolemia and atherosclerosis by casein-fed

A oxidação da lipoproteína da baixa densidade (LDL) é um evento crucial na aterogênese. Os antioxidantes, que protegem a LDL contra a oxidação, potencialmente, podem exercer efeitos benéficos quanto ao desenvolvimento e progressão da aterosclerose. O objetivo principal deste trabalho foi avaliar o efeito das isoflavonas da soja, que apresentam atividade antioxidante, sobre a formação da fração da LDL oxidada in vivo, denominada LDL- e anticorpos anti-LDL-, a colesterolemia e a formação de lesões ateroscleróticas, em coelhos com hipercolesterolemia induzida por uma dieta contendo caseína. Coelhos machos da raça Nova Zelândia (n=15) foram divididos randomicamente em três grupos, a saber: grupo X, que consumiu...

Efecto de la caseína en la absorción de d-xilosa medida mediante la prueba de H2 espirado / Effect of casein on d-xylose absorption measured by expired H2 test

Different experimental studies suggest that the presence of food in the alimentary tract, promote small intestinal absorption. The mechanism involved are not completely understood and might be related to motility changes or to humoral factors. Since studies have shown a decrease of small bowel motility after casein administration, the aim was to analyze the effect of this protein on small intestinal absorption and orocecal transit time. The hydrogen breath test was used to estimate d-xylose absorption. H2 production is dependent on the amount of this carbohydrate reaching the colon and therefore inversely proportional to d-xylose absorption. Six normal volunteers ingested 25 g d-xylose and 25 g de-xyllose + 30 g casein, and alveolar breath samples were obtained at 15 min intervals. Results: by adding casein to d-xylose solution a statistically significant decrease of maximal H2 concentration was observed from a mean of 40ñ11 ppm to a mean of 26ñ8 ppm. Similarly the area under the curve (which reflects the amount of xylose that was not absorbed) was also significantly decreased from a mean of 3281ñ1399 ppm to mean of 1394ñ700 ppm of H2. The orocecal transit time was significantly prolonged from 85.5ñ40 min to 112ñ38 min. Our results suggest that casein increased d-xylose absorption in normal subjects and that this effect might be related to a slower transit time

Phosphorylation of casein, fibrinogen and calmodulin by a glycoprotein protein kinase from monkey cerebellum: a casein kinase II-like enzyme.

A glycoprotein protein kinase was isolated from monkey cerebellum by polylysine-Sepharose chromatography and affinity chromatography on Sepharose 4B coupled to the lectin, Concanavalin A. The protein kinase phosphorylated casein on serine and threonine residues and was stimulated by polylysine, polyarginine, spermine, histone, protamine and sphingosine, but was inhibited by heparin, poly (Glu, Ala, Tyr) and poly (Glu, Tyr). These characteristics were typical of casein kinase II. The protein kinase also phosphorylated fibrinogen and calmodulin and exhibited similar characteristics of stimulation by polylysine or polyarginine. The phosphorylation of fibrinogen (a glycoprotein), but not casein or calmodulin (non-glycoproteins), was significantly inhibited by Concanavalin A. Unlike casein kinase II, the enzyme did not undergo autophosphorylation. The collective results suggested that the enzyme from monkey cerebellum was a casein kinase II-like protein kinase and that phosphorylation of a glycoprotein substrate (fibrinogen) by the kinase could be influenced by a carbohydrate binding lectin.

Nylon-6 cylinders and a sponge-like derivative as supports for immobilizing trypsin

1. Two types of nylon-6 supports (small cylinders and a sponge-like derivative) were prepared for immobilizing enzymes. Nylon-6 beads were solubilized by immersion in 80 formic acid and then reprecipitated using two different types of non-solvent solutions (distilled water or a 1:1 acetone:water solution) giving rise to a sponge-like derivative and to a colloidal suspension, respectively. The latter was molded into a thin thread which was cut into small cylinders. 2. Trypsin (EC 3.4.21.4) was covalently bound to glutaraldehyde-activated nylon-6 cylinders as well as to the sponge-like derivative. The maximum (100) apparent initial enzymatic activity was found for the trypsin bound to small cylinders, while the initial activity of trypsin bound to the sponge-like material was 61 in comparison with that of trypsin-small cylinders, under the same conditions of enzyme immobilization reaction (1 g of nylon support and 5 ml of 1.3 mg/ml trypsin in 0.1 M sodium phosphate buffer, pH 8.5, at 10 degrees C for 18 h) and of enzymatic reaction (1 g of trypsin-nylon in a batch reactor, 2 ml of 0.7 w/v azocasein solution in 50 mM borate buffer, pH 8.5, at 37 degrees C, with shaking, for 1 h). However, the decrease of activity after enzyme immobilization was more conspicuous for the trypsin-small cylinders than for the trypsin-sponge. The former retained approximately 25 of its initial activity, while the latter retained approximately 67 of its initial activity, after seven cycles of utilization for 1 h, pH 8.5, at 37 degrees C and 8 days of storage, pH 8.5, at 4 degrees C in the presence of azocasein. 3. Scanning electron microscopy was performed to visualize the surface of the support after each step of the immobilization process. The electron micrographs show that the two types of nylon supports had a rough surface, which became rougher and full of craters after treatment with 5 N HCl. On the other hand, the partially hydrolyzed nylon surface acquired the appearance of Swiss cheese after treatment with 2.5 glutaraldehyde. After reaction with the enzyme molecules the surface became rougher again.

Hidrolisado de colageno: utilizacao biologica em misturas proteicas e seu efeito no tecido cutaneo / Hidrolised collagen: biological utilization in proteics mix and its effect in cutaneous tissue

Estudou-se a adicao de hidrolisado de colageno a racoes com caseina nas proporcoes de 0 por cento, 25 por cento, 50 por cento, 65 por cento, 75 por cento e 100 por cento. As racoes com 25 por cento e 65 por cento de hidrolisado proteico foram acrescidas com prolina e aminoacidos essenciais (tirosina, triptofano, metionina e leucina). As reacoes continham 10 por cento e 20 por cento de proteina. Determinou-se o coeficiente de eficacia proteica, coeficiente de digestibilidade, composicao centesimal, aminograma e avaliou-se histologicamente o figado e rim e a pele. Doseou-se o teor de hidroxiprolina da pele dos animais testados. Verificou-se que: a adicao de 25 por cento de hidrolisado de colageno a caseina nao promoveu modificacao significativa no valor biologico da racao. A adicao de prolina em racao com 25 por cento de colageno para ratos em crescimento nao demonstrou alteracao do peso dos animais. Entretanto com adicao de 65 por cento de colageno houve queda no peso dos animais em relacao ao controle. O figado dos animais estudados (quando utilizados racoes com 10 por cento de proteina), nao demonstrou alteracao significativa em relacao as diferentes adicoes de colageno. Quando a racao foi acrescida de 50 porcento e 65 por cento de hidrolisado de colageno, o doseamento de hidroxiprolina na pele aumentou e os cortes histologicos de tecido cutaneo apresentaram ausencia de hipoderme

Influência da fonte lipídica da dieta na utilizaçäo de caseína e proteína de soja por ratos Wistar / Influence of dietary lipid on utilization of casein and soybean protein by Wistar rats

Estudou-se a influência das fontes lipídicas, óleo de soja e banha de porco, nas propriedades nutricionais de caseína e isolado protéico de soja, sob forma de dieta balanceada, com ratos Wistar, em termos de ganho de peso, quociente de eficiência alimentar e proteíca e índices aparentes de digestibilidade, valor biológico e utilizaçäo líquida da proteína. Verificou-se que, para uma mesma fonte protéica, a fonte lipídica näo influiu nesses índices de qualidade, mas ocorreu influência a comparar fontes protéicas diferentes. Assim, inferiu-se que a combinaçäo caseína e óleo de soja forneceu os melhores resultados e a combinaçäo isolado protéico de soja e óleo de soja, o pior desempenho. Entretanto, ao combinar-se o isolado protéico de soja com a banha de porco, o valor nutritivo da proteína de soja näo diferiu daquele da caseína

Utilizaçäo biológica de cálcio em ratos alimentados com dieta contendo caseina como única fonte de cálcio / Biological utilization of calcium in rats feed a diet with casein as the only source of calcium

Wealing Wistar rats fed a diet with casein as the only source calcium showed less weight gain as compared to controls receiving calcium carbonate besides casein in the diet. Food intake was also diminished in that group, although the animals ingested sufficient calcium for normal growth. Apparent absorption of the mineral was twice that of the controls but this was not enough for normal mineralization, as showed by bone rarefaction in the experimental group. Bone calcium was also lower in theese animals. The data indicate that when using casein as a protein source in experiments concerning calcium bioavailability one must take into consideration that part of the requirements of the mineral will be supplied by asein itself. Key words: calcium; biovailability; casein