Production of mycelial biomass, proteases and protease inhibitors by Ganoderma lucidum under different submerged fermentation conditions / Produção de biomassa micelial, proteases e inibidores de proteases por Ganoderma lucidum cultivado sob diferentes condições de fermentação submersa

Ganoderma lucidum is a medicinal mushroom widely recognized as a source of biomolecules with pharmacological properties, however, little is known about the factors that influence the synthesis of bioactive proteins by this fungus when cultivated under submerged fermentation. The objective of this work was to evaluate the production of mycelial biomass and intracellular proteases and protease inhibitors by G. lucidum cultivated under different submerged fermentation conditions. The cultivation was carried out in a medium composed of glucose (10 or 20 g.L-1), soy peptone (2.5 or 5 g.L-1) and yeast extract (5 g.L-1), with incubation under agitation (120 rpm) and non-agitation, totaling 8 experimental conditions. Biomass production was determined from the dry weight, while glucose consumption was estimated by quantification of reducing sugars. The proteins were extracted in NaCl (0.15 M), and the protein extracts were submitted to protein quantification by the Bradford method, total proteolytic activity using azocasein, caseinolytic and fibrinolytic activity in Petri dishes, activity of serine (trypsin and chymotrypsin) and cysteine (papain) protease inhibitors. Cultivation in agitated condition showed higher biomass production with a maximum value of 7 g.L-1, in addition to higher activities of trypsin, chymotrypsin and papain inhibitors, with 154 IU.mg-1, 153 IU.mg-1 e 343 IU.mg-1 of protein, respectively. The non-agitated condition showed a greater potential for obtaining proteins, total proteases, caseinolytic and fibrinolytic enzymes, with maximum values of 433 mg.g-1 of extract, 71 U.mL-1 of extract, 63.62 mm2 and 50.27 mm2, respectively. Thus, a medium composed of soy peptone, yest extract and glucose in a 1:2:4 proportion is recommended, under agitation to produce protease inhibitors, and the non-agitated condition when the target is, mainly caseinolytic and fibrinolytic enzymes.

Ganoderma lucidum é um cogumelo medicinal amplamente reconhecido como fonte de biomoléculas com propriedades farmacológicas, entretanto, pouco se conhece acerca dos fatores que influenciam a síntese de proteínas bioativas por esse fungo, quando cultivado sob fermentação submersa. O objetivo deste trabalho foi avaliar a produção de biomassa micelial e de proteases e inibidores de proteases intracelulares por G. lucidum cultivado em diferentes condições de fermentação submersa. O cultivo foi realizado em meio contendo glicose (10 ou 20 g.L-1), peptona de soja (2,5 ou 5 g.L-1) e extrato de levedura (5 g.L-1), com incubação sob agitação (120 rpm) ou não-agitado, totalizando 8 condições experimentais. A produção de biomassa foi determinada a partir do peso seco e o consumo de glicose a partir da quantificação de açúcares redutores. As proteínas foram extraídas em NaCl (0,15 M) e os extratos proteicos foram submetidos à quantificação de proteínas pelo método de Bradford, atividade proteolítica total usando azocaseína, atividade caseinolítica e fibrinolítica em placa de Petri, atividade de inibidores de serino-proteases (tripsina e quimotripsina) e cisteíno-protease (papaína). O cultivo em condição agitada apresentou maior produção de biomassa com valor máximo de 7g.L-1, além de maiores atividades de inibidores de tripsina, quimotripsina e papaína, com 154 UI.mg-1, 153 UI.mg-1 e 343 UI.mg-1 de proteína, respectivamente. A condição não-agitada demonstrou maior potencial para a obtenção de proteínas, proteases totais, enzimas caseinolíticas e fibrinolíticas, com valores máximos de 433 mg.g-1 de extrato, 71 U.mL-1 de extrato, 63,62 mm2 e 50,27 mm2, respectivamente. Assim, recomenda-se o meio composto de peptona de soja, extrato de levedura e glicose na proporção 1:2:4, em condição agitada para a produção de inibidores de proteases, e a condição não-agitada para a síntese de proteases, principalmente enzimas caseinolíticas e fibrinolíticas.

Body composition, growth performance and enzyme activities of Labeo rohita fed different commercial fish feeds / Composição corporal, desempenho de crescimento e atividades enzimáticas de diferentes alimentos para peixes comerciais alimentados por Labeo rohita

We have evaluated the effects of different fish feeds on the body composition, growth, and enzyme activities of Labeo rohita (Rohu). In total, 240 fishes between the average weights of 24.77±2.15g were studied. The treatments were applied in a completely randomized design, with 4 treatments of 60 fishes each. Treatments consisted of four different fish feeds [Oryza (T1), AMG (T2), Aqua (T3), and Supreme (T4)]. Body composition, growth performance, and enzyme activities were evaluated. There was a significant variation in performance of fishes fed with different type of feed; as fishes having Oryza feed showed the highest weight gain, specific growth rate (SGR), and best feed conversion ratio (FCR) as compared to other groups that were considered to be significant (P ≤ 0.05). High net weight gain was obtained in T4 when compared with T2 and T3. FCR value of T4 was less than T1 but higher than T2, T3 and T2, which showed the lowest values. The specific growth rate was recorded as average in T4, but T2 led a high SGR than T3. Similarly, crude protein level and digestive enzymes activity was recorded significantly highest in fed with Oryza (T1) as compared to AMG (T2), Aqua (T3), and Supreme (T4). Water quality parameters were recorded significant in all treatments except pH and DO of treatment (T1), significantly different from other treatments. It was concluded that Rohu (Labeo rohita) could show a promising growth rate and protease enzyme activity when fed with the Oryza feed of 25% protein.

Avaliamos os efeitos de diferentes alimentos para peixes em relação à composição corporal, crescimento e atividades enzimáticas de Labeo rohita (Rohu). No total, foram estudados 240 peixes com pesos médios de 24,77 ± 2,15 g. Os tratamentos foram aplicados em delineamento inteiramente casualizado, com quatro tratamentos de 60 peixes cada. Os tratamentos consistiram em quatro alimentos diferentes para peixes: Oryza (T1), AMG (T2), Aqua (T3) e Supreme (T4). Foram avaliados a composição corporal, o desempenho de crescimento e as atividades enzimáticas. Houve uma variação significativa no desempenho dos peixes alimentados com diferentes tipos de ração. Peixes com alimentação Oryza apresentaram maior ganho de peso, taxa de crescimento específico (SGR) e melhor taxa de conversão alimentar (FCR) em comparação com outros grupos que foram considerados significativos (P ≤ 0,05). Elevado ganho de peso líquido foi obtido em T4 quando comparado com T2 e T3. O valor da FCR de T4 foi menor que T1, mas maior que T2 e T3, que apresentaram os menores valores. A taxa de crescimento específico foi registrada como média em T4, mas T2 teve uma SGR alta do que T3. Da mesma forma, o nível de proteína bruta e a atividade das enzimas digestivas foram registrados significativamente mais altos nos peixes alimentados com Oryza (T1) em comparação com AMG (T2), Aqua (T3) e Supreme (T4). Os parâmetros de qualidade da água foram registrados como significativos em todos os tratamentos, exceto pH e OD do tratamento (T1), significativamente diferente dos demais tratamentos. Concluiu-se que Rohu (Labeo rohita) pode apresentar uma taxa de crescimento promissora e atividade da enzima protease quando alimentado com Oryza de 25% de proteína.

Body composition, growth performance and enzyme activities of Labeo rohita fed different commercial fish feeds / Composição corporal, desempenho de crescimento e atividades enzimáticas de diferentes alimentos para peixes comerciais alimentados por Labeo rohita

We have evaluated the effects of different fish feeds on the body composition, growth, and enzyme activities of Labeo rohita (Rohu). In total, 240 fishes between the average weights of 24.77±2.15g were studied. The treatments were applied in a completely randomized design, with 4 treatments of 60 fishes each. Treatments consisted of four different fish feeds [Oryza (T1), AMG (T2), Aqua (T3), and Supreme (T4)]. Body composition, growth performance, and enzyme activities were evaluated. There was a significant variation in performance of fishes fed with different type of feed; as fishes having Oryza feed showed the highest weight gain, specific growth rate (SGR), and best feed conversion ratio (FCR) as compared to other groups that were considered to be significant (P ≤ 0.05). High net weight gain was obtained in T4 when compared with T2 and T3. FCR value of T4 was less than T1 but higher than T2, T3 and T2, which showed the lowest values. The specific growth rate was recorded as average in T4, but T2 led a high SGR than T3. Similarly, crude protein level and digestive enzymes activity was recorded significantly highest in fed with Oryza (T1) as compared to AMG (T2), Aqua (T3), and Supreme (T4). Water quality parameters were recorded significant in all treatments except pH and DO of treatment (T1), significantly different from other treatments. It was concluded that Rohu (Labeo rohita) could show a promising growth rate and protease enzyme activity when fed with the Oryza feed of 25% protein.(AU)

Avaliamos os efeitos de diferentes alimentos para peixes em relação à composição corporal, crescimento e atividades enzimáticas de Labeo rohita (Rohu). No total, foram estudados 240 peixes com pesos médios de 24,77 ± 2,15 g. Os tratamentos foram aplicados em delineamento inteiramente casualizado, com quatro tratamentos de 60 peixes cada. Os tratamentos consistiram em quatro alimentos diferentes para peixes: Oryza (T1), AMG (T2), Aqua (T3) e Supreme (T4). Foram avaliados a composição corporal, o desempenho de crescimento e as atividades enzimáticas. Houve uma variação significativa no desempenho dos peixes alimentados com diferentes tipos de ração. Peixes com alimentação Oryza apresentaram maior ganho de peso, taxa de crescimento específico (SGR) e melhor taxa de conversão alimentar (FCR) em comparação com outros grupos que foram considerados significativos (P ≤ 0,05). Elevado ganho de peso líquido foi obtido em T4 quando comparado com T2 e T3. O valor da FCR de T4 foi menor que T1, mas maior que T2 e T3, que apresentaram os menores valores. A taxa de crescimento específico foi registrada como média em T4, mas T2 teve uma SGR alta do que T3. Da mesma forma, o nível de proteína bruta e a atividade das enzimas digestivas foram registrados significativamente mais altos nos peixes alimentados com Oryza (T1) em comparação com AMG (T2), Aqua (T3) e Supreme (T4). Os parâmetros de qualidade da água foram registrados como significativos em todos os tratamentos, exceto pH e OD do tratamento (T1), significativamente diferente dos demais tratamentos. Concluiu-se que Rohu (Labeo rohita) pode apresentar uma taxa de crescimento promissora e atividade da enzima protease quando alimentado com Oryza de 25% de proteína.(AU)

Effects of a monocomponent protease from Bacillus licheniformis on broiler performance, digestibility, and carcass yield

Two experiments were carried out to evaluate the effectsof protease addition to the diet of broilers at a higher level (1× or 2×) thanthe nutritional value proposed for the enzyme. The first experiment, 1280 day-old chicks (Cobb500®) were randomly allocated (randomized block design, 2×2+1 factorial arrangement),five treatments, eight replicates containing32 birds/replicate. Treatmentsconsisted: control diet without protease (CD); CD + 1× nutritional value of the enzyme (CDM1); CD + 2× nutritional value of the enzyme (CDM2); CDM1 + protease; and CDM2 + protease. The experimental period was 42 days. The mean weight (AFW), feed intake (FI), weight gain (WG), feed conversion, and carcass yield were evaluated. Significant differences were observed for AFW, WG, FI, abdominal fat yield, and feet percentage in the carcass. In the second experiment, 120 Cobb500®chicks at 14 days of age were allotted in a completely randomized design, 2×2+1 factorial arrangement, five treatments, six replicates withfour birds/replicate. The treatments were consistent with the first experiment. Significant improvements in the nitrogen balance wereobserved for the broilersthat received protease.The use of the enzyme tested is recommended with the recommended nutritional matrix, improving the zootechnical indices of broilers.(AU)

Saccharomyces cerevisiae OS303 expression of an alkaline protease from a newly isolated Bacillus subtilis D9 / Expressão de Saccharomyces cerevisiae OS303 de uma protease alcalina de um Bacillus subtilis D9 recém-isolado

The aim of this study was to produce high yield of a local bacterial alkaline protease in the yeast system because the scientific involvement of microorganisms in enzyme production is still not given enough attention in Saudi Arabia. Soil samples were collected from the rhizosphere of some desert plants in Saudi Arabia. Ninety-three alkaline protease producing bacterial isolates were recovered on skimmed-milk agar at pH 9.4 and 45°C for 48 hr. Isolate D9 obtained from the rhizosphere of Heliotropium digynum at Dhahran City was the most potent isolate in respect to enzyme productivity (184.6 U/ml). The full gene of alkaline protease was amplified and showed the expected size (1300 bp). Restriction enzymes analysis also verified the integrity of the PCR product. The sequence of the protease gene revealed an open reading frame of 1329 nt correspond to the full length of the protease gene of isolate D9 encoding a 443 aa protein. After ligation of the amplified gene by the TA cloning method, digestion with appropriate restriction enzymes confirmed the integrity of the cloned gene. The insert was prepared by two PCRs that were conducted with a pair of primers specifically designed for this purpose. The digested and purified cloning vector pRS426/GAL1p-207-Glu-MS was ligated with the insert then transformed into various strains of Saccharomyces cerevisiae via the electroporation method. Maximum protease expression was done by recombinant OS303 in galactose containing media (145.5 U/ml) with an approximately 2-fold increase when compared with the wild OS303 strain., this may be due to ability to activate gal operon.

O objetivo deste estudo foi produzir alto rendimento de uma protease alcalina bacteriana local no sistema de leveduras, já que o envolvimento científico de microrganismos na produção de enzimas ainda não recebe atenção suficiente na Arábia Saudita. Amostras de solo foram coletadas da rizosfera de algumas plantas do deserto na Arábia Saudita. Noventa e três isolados bacterianos produtores de protease alcalina foram recuperados em ágar de leite desnatado a pH 9,4 e 45°C por 48 horas. O isolado D9 obtido da rizosfera de Heliotropium digynum na cidade de Dhahran foi o mais potente em relação à produtividade da enzima (184,6 U/ml). O gene completo da protease alcalina foi amplificado e apresentou o tamanho esperado (1300 pb). A análise de enzimas de restrição também verificou a integridade do produto de PCR. A sequência do gene da protease revelou uma fase de leitura aberta de 1329 nt, correspondendo ao comprimento total do gene da protease do isolado D9 que codifica uma proteína 443 aa. Após a ligação do gene amplificado pelo método de clonagem TA, a digestão com enzimas de restrição apropriadas confirmou a integridade do gene clonado. A inserção foi preparada por dois PCRs que foram conduzidos com um par de primers projetados especificamente para esta finalidade. O vetor de clonagem digerido e purificado pRS426/GAL1p-207-Glu-MS foi ligado com a inserção e então transformado em várias cepas de Saccharomyces cerevisiae por meio do método de eletroporação. A expressão máxima da protease foi feita por OS303 recombinante em meio contendo galactose (145,5 U/ml) com um aumento de aproximadamente duas vezes quando comparado com a cepa OS303 selvagem, e isso pode ser por causa da capacidade de ativar o operon gal.

Enzimas exógenas na dieta de leitões desmamados / Exogenous enzymes in the diet of weaned piglets

O desmame é caracterizado como o período mais crítico da produção de suínos devido à imaturidade digestiva e à baixa concentração de enzimas digestivas, como carboidrases, proteases e fitases, nas primeiras semanas do pós-desmame, o que ocasiona o baixo desempenho dos leitões. Neste caso, há a necessidade de suplementação exógena de enzimas que atuam na degradação de frações do alimento, aumentando o aproveitamento da dieta. A nutrição enzimática exógena tem sido considerada uma alternativa eficaz frente à baixa concentração de enzimas endógenas nas primeiras semanas após o desmame por contribuir para o aumento na digestibilidade das dietas sólidas e, deste modo, para um maior aproveitamento dos ingredientes. Na nutrição de suínos, as enzimas exógenas mais utilizadas são as carboidrases, as proteases e as fitases. Ademais, a utilização de blends enzimáticos também é considerada uma ótima alternativa, pois contribui para a inibição de fatores antinutricionais, os quais são responsáveis por indisponibilizar nutrientes necessários para o desenvolvimento e desempenho dos animais. Diante do exposto, o objetivo deste estudo foi apresentar as principais enzimas exógenas utilizadas na nutrição de leitões desmamados, bem como seus efeitos no desempenho animal.

Weaning is characterized as the most critical period in swine production due to digestive immaturity and low concentration of digestive enzymes, such as carbohydrases, proteases, and phytases, in the first weeks after weaning, which causes poor performance of piglets. In this case, there is the need for exogenous supplementation of enzymes that act on the degradation of food fractions, increasing diet utilization. Exogenous enzyme nutrition has been considered an effective alternative to the low concentration of endogenous enzymes in the first weeks after weaning, as it contributes to an increase in the digestibility of solid diets and, consequently, greater use of the ingredients. In swine nutrition, the most used exogenous enzymes are carbohydrases, proteases, and phytases. Furthermore, the use of enzymatic blends is also considered a great alternative, as they contribute to the inhibition of anti-nutritional factors, which are responsible for making nutrients necessary for the development and performance of animals unavailable. Therefore, this study aimed to present the main exogenous enzymes used in the nutrition of weaned piglets, as well as their effects on animal performance.

Obtainment and characterization of digestive aspartic proteases from the fish Caranx hippos (Linnaeus, 1766) / Obtenção e caracterização de proteases aspárticas digestórias do peixe Caranx hippos (Linnaeus, 1766)

This work aimed to obtain aspartic proteases of industrial and biotechnological interest from the stomach of the crevalle jack fish (Caranx hippos). In order to do so, a crude extract (CE) of the stomach was obtained and subjected to a partial purification by salting-out, which resulted in the enzyme extract (EE) obtainment. EE proteases were characterized physicochemically and by means of zymogram. In addition, the effect of chemical agents on their activity was also assessed. By means of salting-out it was possible to obtain a purification of 1.6 times with a yield of 49.4%. Two acid proteases present in the EE were observed in zymogram. The optimum temperature and thermal stability for EE acidic proteases were 55 ºC and 45 °C, respectively. The optimum pH and pH stability found for these enzymes were pH 1.5 and 7.0, respectively. Total inhibition of EE acid proteolytic activity was observed in the presence of pepstatin A. dithiothreitol (DTT) and Ca2+ did not promote a significant effect on enzyme activity. In the presence of heavy metals, such as Al3+, Cd2+ and Hg2+, EE acidic proteases showed more than 70% of their enzymatic activity. The results show that it is possible to obtain, from the stomach of C. hippos, aspartic proteases with high proteolytic activity and characteristics that demonstrate potential for industrial and biotechnological applications.

Este trabalho objetivou obter proteases aspárticas de interesse industrial e biotecnológico a partir do estômago do peixe xaréu (Caranx hippos). Para isso, foi obtido um extrato bruto do estômago, o qual foi submetido a uma purificação parcial por salting-out onde se obteve o extrato enzimático (EE). As proteases do EE foram caracterizadas físico-quimicamente e através de zimograma. Além disso, o efeito de agentes químicos sobre sua atividade também foi avaliado. Através de salting-out foi possível obter uma purificação de 1,6 vezes com rendimento de 49,4%. Foram observadas duas proteases ácidas presentes no EE através de zimograma. A temperatura ótima e a estabilidade térmica para as proteases ácidas do EE foram de 55 ºC e 45 °C, respectivamente. O pH ótimo e a estabilidade ao pH encontrados para estas enzimas foram o pH 1,5 e 7,0, respectivamente. Observou-se a inibição total da atividade proteolítica ácida do EE na presença de pepstatina A. O ditiotreitol (DTT) e o Ca2+ não promoveram efeito significativo na atividade enzimática. Na presença de metais pesados, como Al3+, Cd2+ e Hg2+, o EE manteve mais de 70% de atividade enzimática do EE. Os resultados mostram que é possível obter, a partir do estômago de C. hippos, proteases aspárticas com alta atividade proteolítica e características que demonstram potencial para aplicações industriais e biotecnológicas.

Carangoides bartholomaei (Cuvier, 1833) stomach: a source of aspartic proteases for industrial and biotechnological applications / Estômago de Carangoides bartholomaei (Cuvier, 1833): uma fonte de proteases aspárticas para aplicações industriais e biotecnológicas

The viscera and other residues from fish processing are commonly discarded by the fishing industry. These byproducts can be a source of digestive enzymes with industrial and biotechnological potential. In this study, we aimed at the extraction, characterization, and application of acidic proteases from the stomach of Carangoides bartholomaei (Cuvier, 1833). A crude extract from the stomachs was obtained and submitted to a partial purification process by salting-out, which obtained a Purified Extract (PE) with a specific proteolytic activity of 54.0 U·mg-1. A purification of 1.9 fold and a yield of 41% were obtained. The PE presents two isoforms of acidic proteases and a maximum proteolytic activity at 45 °C and pH 2.0. The PE acidic proteolytic activity was stable in the pH range of 1.5 to 7.0 and temperature from 25 °C to 50 °C. Purified Extract kept 35% of its proteolytic activity at the presence of NaCl 15% (m/v) but was totally inhibited by pepstatin A. Purified Extract aspartic proteases presented high activity in the presence of heavy metals such as Cd2+, Hg2+, Pb2+, Al3+, and Cu2+. The utilization of PE as an enzymatic addictive in the collagen extraction from Nile tilapia scales has doubled the process yield. The results indicate the potential of these aspartic proteases for industrial and biotechnological applications.

As vísceras e outros resíduos do processamento de peixes são geralmente descartados pela indústria pesqueira. Esses resíduos podem ser uma fonte de enzimas digestivas com potencial industrial e biotecnológico. Neste estudo, objetivamos a extração, caracterização e aplicação de proteases aspárticas do estômago de Carangoides bartholomaei (Cuvier, 1833). Um extrato bruto do estômago foi obtido e submetido a um processo de purificação parcial, que obteve um Extrato Purificado (EP) com uma atividade proteolítica específica de 54,0 U·mg-1. Foi obtida uma purificação de 1,9 vezes e um rendimento de 41%. O EP apresenta duas isoformas de proteases ácidas e atividade proteolítica máxima a 45 °C e pH 2,0. A atividade proteolítica do EP foi estável na faixa de pH de 1,5 a 7,0 e temperatura de 25 °C a 50 °C. O EP manteve 35% de sua atividade proteolítica na presença de NaCl a 15% (m/v), mas foi totalmente inibida pela pepstatina A. As proteases ácidas do EP apresentaram alta atividade na presença de metais pesados como o Cd2+, Hg2+, Pb2+, Al3+ e Cu2+. A utilização de EP como aditivo enzimático na extração de colágeno a partir de escamas de tilápia do Nilo dobrou o rendimento do processo. Os resultados indicam um potencial dessas proteases para aplicações industriais e biotecnológicas.

Production of proline and protease with different organic wastes in bacteria (Production proline and protease with organic wastes) / Produção de prolina e protease com diferentes resíduos orgânicos em bactérias (produção de prolina e protease com resíduos orgânicos)

In this study, we investigated the proline and protease production of different bacteria in several organic waste materials. Our aim was to produce proline and protease economically in waste that is abundantly available while reducing its environmental impact. 5 ml of different organic waste materials (OWW: Olive waste water; N.B: Nutrient Broth; EW: Eggshell; PBS: PBS buffer; PLW: Peach leaf wastes; TCW: Turkish coffee wastes; TWW: Tea waste water; WCW: Waste cheese whey; WFO: Waste frying oil) were placed in 10 ml grow tubes, inoculated and incubated for 24 h. Phosphate-buffered saline and 10% solutions of different organic wastes were added. These cultures were subsequently incubated at 37°C for 24 h. Cells were harvested at 24 h for L-proline assay. 1 ml of culture was transferred by pipette into an Eppendorf tube and centrifuged at 14,000 rpm for 20 min at room temperature. Cellular debris was removed by centrifuge and the supernatant was used for proline activity assays. Protease activity was determined using a modified method with casein as the substrate. We found that proline and protease can easily be produced economically using Turkish coffee wastes (TCW), Waste cheese whey (WCW) and Olive waste water (OWW) organic waste. We believe that this study will result in similar research leading to the economical use of these waste materials thus reducing their impact on the environment.

Neste estudo, investigamos a produção de prolina e protease de diferentes bactérias em diversos resíduos orgânicos. Nosso objetivo era produzir prolina e protease economicamente em resíduos que estão disponíveis em abundância, reduzindo seu impacto ambiental. Cinco ml de diferentes materiais de resíduos orgânicos (OWW: resíduos de azeitona; NB: caldo nutriente; EW: casca de ovo; PBS: tampão PBS; PLW: resíduos de folhas de pêssego; TCW: resíduos de café turco; TWW: resíduos de chá; WCW: resíduos de queijo soro de leite; WFO: óleo de fritura residual) foram colocados em tubos de cultivo de 10 ml, inoculados e incubados por 24 horas. Adicionaram-se solução salina tamponada com fosfato e soluções a 10% de diferentes resíduos orgânicos. Essas culturas foram subsequentemente incubadas a 37° C durante 24 h. As células foram colhidas às 24 h para o ensaio de L-prolina. Um ml de cultura foi transferido por pipeta para um tubo Eppendorf e centrifugado a 14.000 rpm, por 20 min, em temperatura ambiente. Os detritos celulares foram removidos por centrifugação e o sobrenadante foi usado para ensaios de atividade de prolina. A atividade da protease foi determinada usando um método modificado com caseína como substrato. Descobrimos que a prolina e a protease podem ser facilmente produzidas economicamente, usando resíduos de café turco (TCW), resíduos de soro de queijo (WCW) e resíduos orgânicos de água de oliva (OWW). Acreditamos que este estudo resultará em pesquisas semelhantes, levando ao uso econômico desses materiais residuais, reduzindo, assim, seu impacto no meio ambiente.

Protease Supplementation in the Diet of Light Laying Hens

This work was developed to evaluate the impact of the addition of proteases on the performance characteristics, egg quality, relative weight of digestive organs, and intestinal morphometry of laying hens. 390 Hy Line W36® hens were allocated into five treatments and six replicates with 13 animals. The treatments were: 1) Control (standard formulation), 2) Negative control A - NCA (nutritional reduction according to protease A matrix), 3) Negative control B - NCB (nutritional reduction according to protease B matrix), 4) NCA+protease A (0.250 g/kg of feed) and 5) NCB+protease B (0.125 g/kg of feed). Hens fed the NCA, NCB, and NCA+protease A diets showed reductions in feed intake and egg mass. The addition of protease B provided better results for egg production in both percentage and per dozen as compared to the group fed with the NCA+protease A diets. The hens subjected to diets NCA and NCB showed eggs with a reduced eggshell and thickness percentage. However, supplementation with proteases A and B improved these parameters to values similar to the controls. There was no significant effect of the treatments on the relative weight of the liver, proventricle, gizzard, pancreas, and small intestine. However, the addition of protease A resulted in a decreased value for the relative weight of the large intestine. The jejunum and ileum crypt depths were, respectively, smaller in hens fed the control diet in relation to the NCB diet and the NCA and NCB diets. As it can be concluded, Protease B supplementation provided the best performance results.(AU)

Production of proline and protease with different organic wastes in bacteria (Production proline and protease with organic wastes) / Produção de prolina e protease com diferentes resíduos orgânicos em bactérias (produção de prolina e protease com resíduos orgânicos)

In this study, we investigated the proline and protease production of different bacteria in several organic waste materials. Our aim was to produce proline and protease economically in waste that is abundantly available while reducing its environmental impact. 5 ml of different organic waste materials (OWW: Olive waste water; N.B: Nutrient Broth; EW: Eggshell; PBS: PBS buffer; PLW: Peach leaf wastes; TCW: Turkish coffee wastes; TWW: Tea waste water; WCW: Waste cheese whey; WFO: Waste frying oil) were placed in 10 ml grow tubes, inoculated and incubated for 24 h. Phosphate-buffered saline and 10% solutions of different organic wastes were added. These cultures were subsequently incubated at 37°C for 24 h. Cells were harvested at 24 h for L-proline assay. 1 ml of culture was transferred by pipette into an Eppendorf tube and centrifuged at 14,000 rpm for 20 min at room temperature. Cellular debris was removed by centrifuge and the supernatant was used for proline activity assays. Protease activity was determined using a modified method with casein as the substrate. We found that proline and protease can easily be produced economically using Turkish coffee wastes (TCW), Waste cheese whey (WCW) and Olive waste water (OWW) organic waste. We believe that this study will result in similar research leading to the economical use of these waste materials thus reducing their impact on the environment.

Neste estudo, investigamos a produção de prolina e protease de diferentes bactérias em diversos resíduos orgânicos. Nosso objetivo era produzir prolina e protease economicamente em resíduos que estão disponíveis em abundância, reduzindo seu impacto ambiental. Cinco ml de diferentes materiais de resíduos orgânicos (OWW: resíduos de azeitona; NB: caldo nutriente; EW: casca de ovo; PBS: tampão PBS; PLW: resíduos de folhas de pêssego; TCW: resíduos de café turco; TWW: resíduos de chá; WCW: resíduos de queijo soro de leite; WFO: óleo de fritura residual) foram colocados em tubos de cultivo de 10 ml, inoculados e incubados por 24 horas. Adicionaram-se solução salina tamponada com fosfato e soluções a 10% de diferentes resíduos orgânicos. Essas culturas foram subsequentemente incubadas a 37° C durante 24 h. As células foram colhidas às 24 h para o ensaio de L-prolina. Um ml de cultura foi transferido por pipeta para um tubo Eppendorf e centrifugado a 14.000 rpm, por 20 min, em temperatura ambiente. Os detritos celulares foram removidos por centrifugação e o sobrenadante foi usado para ensaios de atividade de prolina. A atividade da protease foi determinada usando um método modificado com caseína como substrato. Descobrimos que a prolina e a protease podem ser facilmente produzidas economicamente, usando resíduos de café turco (TCW), resíduos de soro de queijo (WCW) e resíduos orgânicos de água de oliva (OWW). Acreditamos que este estudo resultará em pesquisas semelhantes, levando ao uso econômico desses materiais residuais, reduzindo, assim, seu impacto no meio ambiente.

Effect of phytase and protease combination on performance, metabolizable energy, and amino acid digestibility of broilers fed nutrient-restricted diets

Three experiments were carried out to evaluate the effect of dietary combination of different enzymes (phytase + protease) on performance, metabolizable energy, and amino acid digestibility of broiler chickens fed diets with nutritional reduction. A total of 1,400, 336, and 384 male chickens were distributed in a completely randomized design, in the experiments of performance, metabolism, and digestibility, respectively. Treatments were divided as follows: positive control (PC), negative control - NC1 (PC minus 0.16% Ca, 0.15% available P (aP), and 0.5% crude protein (CP)), NC2 (PC minus 0.16% Ca, 0.15% aP, and 1% CP), NC1 added with phytase deriving from citrobacter and protease deriving from Bacillus licheniformis (CBE), NC1 added with phytase deriving from E. coli and protease deriving from microbial fermentation (SE), NC2 added with CBE, and NC2 added with SE. A protein-free diet was included in the digestibility experiment. The nutritional restriction did not affect feed intake of birds in the first experiment; however, the restriction inhibited body weight gain and feed conversion ratio in all phases. In experiment 2, the nutritional restriction decreased AME and AMEn values, although the addition of phytase and protease in diets improved both parameters, mainly in NC2. Treatment NC2 impaired the standardized digestibility of total essential amino acids of animals subjected to experiment 3, although the addition of enzymes helped to recover the digestibility to levels similar to PC. Supplementation of phytase in association with protease is effective to improve performance, energy metabolism, and standardized amino acid digestibility of broilers fed diets with nutrient restriction.(AU)

Impact of the Supplementation of Exogenous Protease and Carbohydrase on the Metabolizable Energy and Standardized Ileal Amino Acid Digestibility of Soybean Meals in Two Brazilian Regions

This study aimed to evaluate the effects of different exogenous protease and carbohydrase in broiler diets on the nitrogen-corrected apparent metabolizable energy (AMEn) and standardized ileal amino acid digestibility (SIAAD) of soybean meals (SBM) in two Brazilian regions (Minas Gerais-MG and Rio Grande do Sul-RS). The total excreta collection of 528 14-d-old chicks was used to determine AMEn in a completely randomized design in a 2 (SBM MG and RS) x 5 (enzyme A, B, C, D and basal diet) + 1 (reference diet, RD) factorial arrangement, totaling 11 treatments, 8 repetitions, and 6 birds per experimental unit. Two experimental treatments (T1 and T6) without enzyme supplementation formulated with SBM MG and RS were used as negative control (NC). The RD without the inclusion of SBM MG and RS was used to correct the nitrogen balance. To determine the SIAAD, ileal content was collected from of broilers and the same experimental design and treatments of the previous trial were used except for the RD, which was replaced with a nitrogen-free diet (NFD) to quantify the excretion of endogenous amino acids. Soybean meal from MG showed the highest levels (p<0.05) of AME and AMEn (3,188 kcal/kg and 2,700 kcal/kg, respectively) in comparison to SBM RS (3,121 kcal/kg and 2,549 kcal/kg, respectively) and, when supplemented with the exogenous enzyme C, also improved the SIAAD (p<0.05), as compared to other enzymes.(AU)

Production of proline and protease with different organic wastes in bacteria (Production proline and protease with organic wastes) / Produção de prolina e protease com diferentes resíduos orgânicos em bactérias (produção de prolina e protease com resíduos orgânicos)

In this study, we investigated the proline and protease production of different bacteria in several organic waste materials. Our aim was to produce proline and protease economically in waste that is abundantly available while reducing its environmental impact. 5 ml of different organic waste materials (OWW: Olive waste water; N.B: Nutrient Broth; EW: Eggshell; PBS: PBS buffer; PLW: Peach leaf wastes; TCW: Turkish coffee wastes; TWW: Tea waste water; WCW: Waste cheese whey; WFO: Waste frying oil) were placed in 10 ml grow tubes, inoculated and incubated for 24 h. Phosphate-buffered saline and 10% solutions of different organic wastes were added. These cultures were subsequently incubated at 37°C for 24 h. Cells were harvested at 24 h for L-proline assay. 1 ml of culture was transferred by pipette into an Eppendorf tube and centrifuged at 14,000 rpm for 20 min at room temperature. Cellular debris was removed by centrifuge and the supernatant was used for proline activity assays. Protease activity was determined using a modified method with casein as the substrate. We found that proline and protease can easily be produced economically using Turkish coffee wastes (TCW), Waste cheese whey (WCW) and Olive waste water (OWW) organic waste. We believe that this study will result in similar research leading to the economical use of these waste materials thus reducing their impact on the environment.(AU)

Neste estudo, investigamos a produção de prolina e protease de diferentes bactérias em diversos resíduos orgânicos. Nosso objetivo era produzir prolina e protease economicamente em resíduos que estão disponíveis em abundância, reduzindo seu impacto ambiental. Cinco ml de diferentes materiais de resíduos orgânicos (OWW: resíduos de azeitona; NB: caldo nutriente; EW: casca de ovo; PBS: tampão PBS; PLW: resíduos de folhas de pêssego; TCW: resíduos de café turco; TWW: resíduos de chá; WCW: resíduos de queijo soro de leite; WFO: óleo de fritura residual) foram colocados em tubos de cultivo de 10 ml, inoculados e incubados por 24 horas. Adicionaram-se solução salina tamponada com fosfato e soluções a 10% de diferentes resíduos orgânicos. Essas culturas foram subsequentemente incubadas a 37° C durante 24 h. As células foram colhidas às 24 h para o ensaio de L-prolina. Um ml de cultura foi transferido por pipeta para um tubo Eppendorf e centrifugado a 14.000 rpm, por 20 min, em temperatura ambiente. Os detritos celulares foram removidos por centrifugação e o sobrenadante foi usado para ensaios de atividade de prolina. A atividade da protease foi determinada usando um método modificado com caseína como substrato. Descobrimos que a prolina e a protease podem ser facilmente produzidas economicamente, usando resíduos de café turco (TCW), resíduos de soro de queijo (WCW) e resíduos orgânicos de água de oliva (OWW). Acreditamos que este estudo resultará em pesquisas semelhantes, levando ao uso econômico desses materiais residuais, reduzindo, assim, seu impacto no meio ambiente.(AU)

Addition of a blend of exogenous enzymes to broiler chickens diets: impacts on performance and production costs / Adição de uma mistura de enzimas exógenas a dietas de frangos de corte: impactos no desempenho e custos de produção

Broiler diets are based on corn, soybean and wheat production; however, some protein ingredients have many antinutritional factors and low digestibility. The objective of this study was to add a blend of exogenous enzymes to the feed with low nutritional value for broilers to reduce production costs and improve digestibility while maintaining good zootechnical performance. The experimental design was completely randomized, including three treatments with five replications (n=15) each: a) positive control (PC), diet calculated for males with average performance; b) negative control + exogenous enzymes (NC+EE), minimum nutrient diet according to the requirements of the production phase, and the enzymatic blend was added; and c) negative control (NC), minimum levels of nutrients for each phase. At 21 days, the PC group showed greater weight gain and lower feed conversion than the NC (P<0.05). At 42 days, PC had lower feed intake than NC (P=0.040), while lower feed conversion was observed in groups PC and NC+EE than NC (P=0.001). The production efficiency index was higher in the PC treatment, but the NC+EE treatment was higher than the NC (P=0.001). Considering production costs and body weight, we found that NC+EE birds had greater profitability. Therefore, we conclude that the blend of exogenous enzymes added to a diet with minimal nutritional levels has practical application in the broiler production system.(AU)

As dietas de frangos de corte são baseadas na produção de milho, soja e trigo; no entanto, alguns ingredientes proteicos possuem muitos fatores antinutricionais e baixa digestibilidade. O objetivo deste estudo foi adicionar à ração uma mistura de enzimas exógenas com baixo valor nutricional para frangos de corte para reduzir os custos de produção e melhorar a digestibilidade, mantendo o bom desempenho zootécnico. O delineamento experimental foi inteiramente casualizado, incluindo três tratamentos com cinco repetições (n=15) cada: a) controle positivo (PC), dieta calculada para machos com desempenho médio; b) controle negativo + enzimas exógenas (CN+EE), dieta mínima de nutrientes de acordo com as exigências da fase de produção, e foi adicionada a mistura enzimática; c) controle negativo (CN), teores mínimos de nutrientes para cada fase. Aos 21 dias, o grupo PC apresentou maior ganho de peso e menor conversão alimentar que o NC (P<0,05). Aos 42 dias, PC apresentou menor consumo de ração que NC (P=0,040), enquanto foi observada menor conversão alimentar nos grupos PC e NC+EE que NC (P=0,001). O índice de eficiência de produção foi maior no tratamento PC, mas o tratamento NC+EE foi maior que o NC (P=0,001). Considerando os custos de produção e peso corporal, verificamos que as aves NC+EE tiveram maior rentabilidade. Portanto, concluímos que a mistura de enzimas exógenas adicionada a uma dieta com níveis nutricionais mínimos tem aplicação prática no sistema de produção de frangos de corte.(AU)

Bacillus licheniformis SMIA-2 protease used as an additive in detergent formulations

Microbial proteases, especially from Bacillus spp., have been widely exploited for a broad variety of applications, such as the improvement of the cleaning efficiency of conventional detergents. In this work, the statistical design of the experiment was used to optimize the concentrations of a three-component mixture: Bacillus licheniformis SMIA-2 protease, Linear Alkylbenzene Sulphate and hydrogen peroxide, in an attempt to prepare an environmentally correct cleaning formulation. The results demonstrated that the combination of 1% (w/v) protease with 1.5% (w/v) LAS and 1% (w/v) H2O2 was effective in removing blood from cloth pieces and that a protease concentration decrease from 1.0% to 0.5% (w/v) would not have a signi&64257;cant impact on percent blood removal if LAS concentrations between 1.5-2.0% (w/v) in combination with lower (<0.5%, w/v) concentrations of H2O2 were used. Thus, the protease from Bacillus licheniformis SMIA-2 can be effectively incorporated into cleaning formulations together with LAS and hydrogen peroxide to formulate more sustainable detergents.

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.

Brazilian Theraphosidae: a toxicological point of view

The Theraphosidae family includes the largest number of species of the Mygalomorphae infraorder, with hundreds of species currently catalogued. However, there is a huge lack on physiologic and even ecologic information available, especially in Brazil, which is the most biodiverse country in the world. Over the years, spiders have been presented as a source of multiple biologically active compounds with basic roles, such as primary defense against pathogenic microorganisms or modulation of metabolic pathways and as specialized hunters. Spider venoms also evolved in order to enable the capture of prey by interaction with a diversity of molecular targets of interest, raising their pharmaceutical potential for the development of new drugs. Among the activities found in compounds isolated from venoms and hemocytes of Brazilian Theraphosidae there are antimicrobial, antifungal, antiparasitic and antitumoral, as well as properties related to proteinase action and neuromuscular blockage modulated by ionic voltage-gated channel interaction. These characteristics are present in different species from multiple genera, which is strong evidence of the important role in spider survival. The present review aims to compile the main results of studies from the last decades on Brazilian Theraphosidae with special focus on results obtained with the crude venom or compounds isolated from both venom and hemocytes, and their physiological and chemical characterization.(AU)

Milk-clotting proteases from Pleurotus albidus: an innovative alternative for the production of Minas frescal cheese

Pleurotus albidus, a naturally growing species in the Amazon region, has been considered a promising source of milk-clotting proteases. The production of such enzymes using lignocellulosic residues is a sustainable alternative to replace mammalian rennet. The application of P. albidus milk-clotting proteases in cheese making has not yet been reported in the scientific literature. The aim of this study was to characterize the milk-clotting proteases of P. albidus and use these enzymes in the production of Minas frescal cheese. For the production of coagulating proteases, the mushroom was grown in açaí seeds supplemented with rice bran (10%, w/w). The parameters affecting the production of coagulant, such as inoculum size, fermentation time, initial pH of cultivation medium and age of the inoculum were evaluated. The coagulant extract obtained under optimal production conditions was evaluated for optimal pH and temperature, pH and temperature stability, effect of ions and inhibitors. Significant production of coagulating proteases was obtained under the following conditions: inoculum size (2.5%), fermentation time (10 days), initial pH of the cultivation medium (6), and inoculum age (10 days). The coagulant exhibited significant catalytic activity in pH 5.0 at 55°C, with stability at 45°C and was completely inhibited by iodoacetic acid. The milk-clotting proteases of P. albidus were efficient for making Minas frescal cheese that presented 55.0% of moisture, 20.0% of lipids and 17.20% of protein. Pleurotus albidus is a potential source of milk-clotting proteases that can be applied in dairy industry for production of fresh Minas frescal cheese.(AU)

Bacillus licheniformis SMIA-2 protease used as an additive in detergent formulations

Microbial proteases, especially from Bacillus spp., have been widely exploited for a broad variety of applications, such as the improvement of the cleaning efficiency of conventional detergents. In this work, the statistical design of the experiment was used to optimize the concentrations of a three-component mixture: Bacillus licheniformis SMIA-2 protease, Linear Alkylbenzene Sulphate and hydrogen peroxide, in an attempt to prepare an environmentally correct cleaning formulation. The results demonstrated that the combination of 1% (w/v) protease with 1.5% (w/v) LAS and 1% (w/v) H2O2 was effective in removing blood from cloth pieces and that a protease concentration decrease from 1.0% to 0.5% (w/v) would not have a signi&64257;cant impact on percent blood removal if LAS concentrations between 1.5-2.0% (w/v) in combination with lower (<0.5%, w/v) concentrations of H2O2 were used. Thus, the protease from Bacillus licheniformis SMIA-2 can be effectively incorporated into cleaning formulations together with LAS and hydrogen peroxide to formulate more sustainable detergents.(AU)