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.

Production of proline and protease with different organic wastes in bacteria (Production proline and protease with organic wastes)

Abstract 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.

Resumo 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.

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.

Cysteine and Aspartic Proteases Underlie the Digestion of Egg Yolk Proteins during the Development of Columba livia domestica Embryo

Yolk proteins undergo digestion either inside the egg yolk or in the surrounding yolk sac membrane (YSM) before being consumed by the developing avian embryo. However, the mechanisms underlying the digestion of yolk proteins during embryogenesis are largely unexplored in the pigeon Columba livia domestica. To better understand these mechanisms, the present study examined the classes of activated proteases in the egg yolk and the gene expression patterns of cathepsin B (CTSB) and cathepsin D (CTSD), which encode for lysosomal cysteine and aspartic proteases, respectively, in the YSM. We investigated the activated proteases by applying different types of protease inhibitors to yolk samples taken from incubation day 16. Then, we detected the mRNA levels of CTSB and CTSD in the YSM at incubation days 6, 8, 10, and 12-17. Both cysteine and aspartic proteases appeared to be activated in the egg yolk. Moreover, CTSB expression increased progressively and reached the maximum value on day 13; however, it decreased significantly on days 14 and 15 and further reduced toward hatching (day 17). In contrast, CTSD expression was weak and fluctuated insignificantly during development. Our results suggest that the degradation of yolk proteins at late developmental stages largely occurs in the egg yolk itself, probably by the activated cysteine and aspartic proteases. Furthermore, cathepsin B in the YSM seems to have a primary role in protein digestion, but this role decreases toward hatching.(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)

Effects of a Novel Protease from Bacillus Subtilis K-5 in Low Protein Corn Distiller Dried Grains with Solubles (cDDGS) Based Diets on Performance and Nutrients Digestibility in Broiler Chickens

This experiment was conducted to evaluate the supplemental effects of a novel protease produced from Bacillus subtilis in low crude protein (CP) corn distiller dried grain with solubles (cDDGS) based diets on growth performance, carcass attributes, nutrients digestibility, blood chemistry, and intestinal histomorphometry of broiler chickens. One hundred and sixty, one-day-old chicks were randomly allotted to one of 4 dietary treatments. Each dietary treatment had four replicates, with 10 birds in each replicate. Two basal diets were formulated for both starter (1-21d) and finisher (22-35d) phase; (PC) a corn soybean meal based diet as per standard recommendations of Ross 308; (NC) 5% cDGGS with 5% reduction in CP with concomitant reduction in essential amino acids (EAAs) compared with PC. The negative control diet was further subdivided into 3 parts. One part was without enzyme supplementation, while the other two parts were supplemented with a novel protease (PROT1) and a commercial protease (PROT2), respectively. The same procedure was adopted for finisher diets. A digestibility assay (32-35d) was carried out using acid insoluble ash (AIA), an external digestibility marker. At the end of 35d, ileal digesta were collected from four birds per experimental unit for nutrient digestibility measurement. Tissue samples of duodenum, jejunum, and ileum were collected for villus height, villus width, crypt depth, and crypt width. Body weight gain (BWG) and feed:gain were improved (p<0.05) with protease supplementation. No effect was observed on carcass parameters. However, CP digestibility, apparent digestibility coefficient for nitrogen (ADCn), nitrogen retention (Nret ), and apparent ileal amino acid digestibility (AIAAD) were improved (p<0.05). However, there was no effect on apparent metabolizable energy (AME) and apparent metabolizable energy corrected for nitrogen (AMEn), blood glucose, total protein and cholesterol (p>0.05) and intestinal integrity (p>0.05). It was concluded that protease enzyme can improve nitrogen and CP digestibility, resulting in improved amino acids availability in low protein diets.(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.

Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)

Design, synthesis, and evaluation of Bothrops venom serine protease peptidic inhibitors

In Central and South America, snakebite envenomation is mainly caused by Bothrops spp. snakes, whose venoms feature significant biochemical richness, including serine proteases. The available bothropic antivenoms are efficient in avoiding fatalities, but do not completely neutralize venom serine proteases, which are co-responsible for some disorders observed during envenomation. Methods: In order to search for tools to improve the antivenom's, 6-mer peptides were designed based on a specific substrate for Bothrops jararaca venom serine proteases, and then synthesized, with the intention to selectively inhibit these enzymes. Results: Using batroxobin as a snake venom serine protease model, two structurally similar inhibitor peptides were identified. When tested on B. jararaca venom, one of the new inhibitors displayed a good potential to inhibit the activity of the venom serine proteases. These inhibitors do not affect human serine proteases as human factor Xa and thrombin, due to their selectivity. Conclusion: Our study identified two small peptides able to inhibit bothropic serine proteases, but not human ones, can be used as tools to enhance knowledge of the venom composition and function. Moreover, one promising peptide (pepC) was identified that can be explored in the search for improving Bothrops spp. envenomation treatment.(AU)

Use of different combinations of enzyme complexes in broiler diets / Utilização de diferentes combinações de complexos enzimáticos em rações para frangos de corte

Two experiments were carried out to investigate the effect of "on top" addition of different enzyme complexes, the enzyme α-galactosidase and three sources of the enzyme phytase available on the market, in broiler diets. In the first experiment, 1260 one-day-old Cobb 500® chicks were distributed into seven treatments in a completely randomized design (CRD) with six replicates and 30 birds/replicate. Treatments consisted of combinations of different enzyme complexes, namely, complex A (phytase, protease, xylanase, ß-glucanase, cellulase, amylase, pectinase), complex B (protease and cellulase) and complex C (xylanase, amylase and protease); isolated α-galactosidase (GAL); and three sources of phytase (P1, P2 and P3) in the diet. The treatments were formulated as follows: T1 - basal diet (BD); T2 - BD + enzyme complex A + enzyme complex B (BDAB); T3 - BDAB + GAL; T4 - BD + complex A + GAL; T5 - BD + complex C + P1 + GAL (BDCG); T6- BDCG + P2; and T7 - BDCG + P3. The following variables were measured in the experimental period of 42 days: feed intake (FI), weight gain (WG), average final weight (AFW), feed conversion (FC), and carcass yield. Significant differences occurred for AFW, WG and FC in the pre-starter phase. In the second experiment,112 Cobb 500® chicks aged 25 days were distributed into seven treatments in a CRD with four replicates and four birds/replicate. Treatments were the same as in the first experiment. Nutrient digestibility was evaluated in an experimental period of seven days. Differences were found in the metabolism coefficient of ether extract (MCEE). Dietary inclusion of enzyme complexes improves the AFW and WG of chickens from1 to 7 days of age and MCEE in the grower phase.

Foram realizados dois experimentos com o objetivo de avaliar o efeito da adição "on top" de diferentes complexos enzimáticos, enzima α-galactosidade e três fontes de enzima fitase disponíveis no mercado em dietas para frangos de corte. No primeiro experimento 1260 pintos Cobb 500® com um dia de idade foram distribuídos em delineamento inteiramente casualizado (DIC), sete tratamentos, seis repetições e 30 aves/repetição. Tratamentos consistiram na associação de diferentes complexos enzimáticos: complexo A (fitase, protease, xilanase, ß-glucanase, celulase, amilase, pectinase); complexo B (protease e celulase); e complexo C (xilanase, amilase e protease); α-galactosidase isolada (GAL); e três fontes de fitase (F1, F2 e F3) nas rações. Em que: T1 - ração basal (RB); T2 - RB + complexo enzimático A + complexo enzimático B (RBAB); T3 - RBAB + GAL; T4 - RB + complexo A + GAL; T5 - RB + complexo C + F1 + GAL (RBCG); T6 - RBCG+F2; T7 - RBCG + F3. O período experimental foi de 42 dias. Avaliaram-se: consumo de ração (CR), ganho de peso (GP), peso médio final (PMF), conversão alimentar (CA), e rendimento de carcaça. Foram observadas diferenças significativas para PMF, GP e CA na fase pré-inicial. No segundo experimento 112 pintos Cobb 500® com 25 dias de idade foram distribuídos em DIC, sete tratamentos, quatro repetições e quatro aves/repetição. Os tratamentos foram os mesmos do primeiro experimento. O período experimental foi de sete dias, avaliou-se a digestibilidade dos nutrientes. Foram observadas diferenças para coeficiente de metabolizabilidade do extrato etéreo (CMEE). A inclusão de complexos enzimáticos nas rações melhora o PMF e GP de frangos de 1 a 7 dias de idade e o CMEE na fase de crescimento.

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.

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)

Use of different combinations of enzyme complexes in broiler diets / Utilização de diferentes combinações de complexos enzimáticos em rações para frangos de corte

Two experiments were carried out to investigate the effect of "on top" addition of different enzyme complexes, the enzyme α-galactosidase and three sources of the enzyme phytase available on the market, in broiler diets. In the first experiment, 1260 one-day-old Cobb 500® chicks were distributed into seven treatments in a completely randomized design (CRD) with six replicates and 30 birds/replicate. Treatments consisted of combinations of different enzyme complexes, namely, complex A (phytase, protease, xylanase, ß-glucanase, cellulase, amylase, pectinase), complex B (protease and cellulase) and complex C (xylanase, amylase and protease); isolated α-galactosidase (GAL); and three sources of phytase (P1, P2 and P3) in the diet. The treatments were formulated as follows: T1 - basal diet (BD); T2 - BD + enzyme complex A + enzyme complex B (BDAB); T3 - BDAB + GAL; T4 - BD + complex A + GAL; T5 - BD + complex C + P1 + GAL (BDCG); T6- BDCG + P2; and T7 - BDCG + P3. The following variables were measured in the experimental period of 42 days: feed intake (FI), weight gain (WG), average final weight (AFW), feed conversion (FC), and carcass yield. Significant differences occurred for AFW, WG and FC in the pre-starter phase. In the second experiment,112 Cobb 500® chicks aged 25 days were distributed into seven treatments in a CRD with four replicates and four birds/replicate. Treatments were the same as in the first experiment. Nutrient digestibility was evaluated in an experimental period of seven days. Differences were found in the metabolism coefficient of ether extract (MCEE). Dietary inclusion of enzyme complexes improves the AFW and WG of chickens from1 to 7 days of age and MCEE in the grower phase.(AU)

Foram realizados dois experimentos com o objetivo de avaliar o efeito da adição "on top" de diferentes complexos enzimáticos, enzima α-galactosidade e três fontes de enzima fitase disponíveis no mercado em dietas para frangos de corte. No primeiro experimento 1260 pintos Cobb 500® com um dia de idade foram distribuídos em delineamento inteiramente casualizado (DIC), sete tratamentos, seis repetições e 30 aves/repetição. Tratamentos consistiram na associação de diferentes complexos enzimáticos: complexo A (fitase, protease, xilanase, ß-glucanase, celulase, amilase, pectinase); complexo B (protease e celulase); e complexo C (xilanase, amilase e protease); α-galactosidase isolada (GAL); e três fontes de fitase (F1, F2 e F3) nas rações. Em que: T1 - ração basal (RB); T2 - RB + complexo enzimático A + complexo enzimático B (RBAB); T3 - RBAB + GAL; T4 - RB + complexo A + GAL; T5 - RB + complexo C + F1 + GAL (RBCG); T6 - RBCG+F2; T7 - RBCG + F3. O período experimental foi de 42 dias. Avaliaram-se: consumo de ração (CR), ganho de peso (GP), peso médio final (PMF), conversão alimentar (CA), e rendimento de carcaça. Foram observadas diferenças significativas para PMF, GP e CA na fase pré-inicial. No segundo experimento 112 pintos Cobb 500® com 25 dias de idade foram distribuídos em DIC, sete tratamentos, quatro repetições e quatro aves/repetição. Os tratamentos foram os mesmos do primeiro experimento. O período experimental foi de sete dias, avaliou-se a digestibilidade dos nutrientes. Foram observadas diferenças para coeficiente de metabolizabilidade do extrato etéreo (CMEE). A inclusão de complexos enzimáticos nas rações melhora o PMF e GP de frangos de 1 a 7 dias de idade e o CMEE na fase de crescimento.(AU)

The Effects of Sodium Chloride versus Sodium Bicarbonate at the Same Dietary Sodium Concentration on Efficacy of Dietary Phytase and a Carbohydrase-Protease Cocktail in Broilers

A trial was conducted to determine the effect of phytase (PHY) or a carbohydrase/protease cocktail (CPX) on broilers fed diets with two different levels of chloride (0.28% or 0.43%) created by altering dietary salt (NaCl) and sodium bicarbonate (NaHCO3). There were 6 combination dietary treatments (3 enzyme x 2 NaCl treatments) applied to 4 replicate pens. The treatments were as follows: Control diet (CON), CON+PHY and CON+CPX, with 0.5% or 0.25% NaCl. The 0.25% NaCl versions contained 0.35% sodium bicarbonate. The 0.5% salt versions had no sodium bicarbonate. Replicate pen BW, and feed consumption (FC) were measured at 1, 14, and 35 d, and mortality was weighed daily for feed conversion ratio (FCR) calculations. Feed consumption at 14 d tended to be lower (p 0.10) for CON+CPX diets compared to CON and CON+PHY diets. The birds fed CON+CPX diet consumed less feed but exhibited improved FCR in the presence of 0.5% NaCl at 14 d. The birds fed the CON, and CON+PHY diets exhibited higher BW at 14 d (p 0.05) and 35 d (p 0.01) of age than did CON+CPX birds. From 15 d to 35 d, birds fed the CON+CPX diet exhibited poorer BW gain (BWG) in the presence of 0.25% NaCl (p 0.05). In conclusion, Cl, as NaCl versus NaH2CO3, could affect CPX but not PHY feed enzyme function in broilers. Further, it may be suggested that certain feed enzymes may be best utilized at later broiler ages rather than in initial feeds.(AU)

The Effects of Sodium Chloride versus Sodium Bicarbonate at the Same Dietary Sodium Concentration on Efficacy of Dietary Phytase and a Carbohydrase-Protease Cocktail in Broilers

A trial was conducted to determine the effect of phytase (PHY) or a carbohydrase/protease cocktail (CPX) on broilers fed diets with two different levels of chloride (0.28% or 0.43%) created by altering dietary salt (NaCl) and sodium bicarbonate (NaHCO3). There were 6 combination dietary treatments (3 enzyme x 2 NaCl treatments) applied to 4 replicate pens. The treatments were as follows: Control diet (CON), CON+PHY and CON+CPX, with 0.5% or 0.25% NaCl. The 0.25% NaCl versions contained 0.35% sodium bicarbonate. The 0.5% salt versions had no sodium bicarbonate. Replicate pen BW, and feed consumption (FC) were measured at 1, 14, and 35 d, and mortality was weighed daily for feed conversion ratio (FCR) calculations. Feed consumption at 14 d tended to be lower (p 0.10) for CON+CPX diets compared to CON and CON+PHY diets. The birds fed CON+CPX diet consumed less feed but exhibited improved FCR in the presence of 0.5% NaCl at 14 d. The birds fed the CON, and CON+PHY diets exhibited higher BW at 14 d (p 0.05) and 35 d (p 0.01) of age than did CON+CPX birds. From 15 d to 35 d, birds fed the CON+CPX diet exhibited poorer BW gain (BWG) in the presence of 0.25% NaCl (p 0.05). In conclusion, Cl, as NaCl versus NaH2CO3, could affect CPX but not PHY feed enzyme function in broilers. Further, it may be suggested that certain feed enzymes may be best utilized at later broiler ages rather than in initial feeds.

Feasibility of bioethanol production from rice bran / Viabilidade da produção de bioetanol a partir do farelo de arroz

Rice bran is a by-product of rice production with a high carbohydrate and starch content and the potential for bioethanol production by alcoholic fermentation. This article describes bioethanol production by Saccharomyces cerevisiae from hydrolyzed defatted rice bran (DRB) a rice by-product applying ultrasonic treatment and protease addition, as well as a sequential strategy of experimental design (SEED). In the first Central Composite Rotatable Design (CCRD), the temperature (25-30 °C) and inoculum concentration (0.5-50 g L-¹) had positive effects on bioethanol production, while the effect of pH (4.0-6.0) was not significant. In the second CCRD, the temperature (28-35 °C) and inoculum concentration (10-70 g L-¹) had negative and positive effects on bioethanol production (p< 0.05). Protease addition (15 µL g-¹) increased the conversion of substrate into bioethanol by 76%. The optimized conditions for the production of 40.7 g L-¹ bioethanol were a temperature of 31.5 °C and an inoculum concentration of 70 g L-¹. Validation in a benchtop bioreactor produced 40.0 g L-¹ of bioethanol from hydrolyzed DRB, and the SEED was characterized as a useful tool to improve bioethanol production from DRB. Furthermore, the DRB proved to be a by-product with great potential for bioethanol production, derived from alternative sources not commonly used in human food.

O farelo de arroz é um subproduto com alto teor de carboidratos e amido, com potencial para produção de bioetanol por fermentação alcoólica. O presente artigo descreve a produção de bioetanol pela ação da Saccharomyces cerevisiae no farelo de arroz desengordurado hidrolisado (DRB) - um subproduto do arroz - com a aplicação do tratamento ultrassônico e adição de protease, e estratégia sequencial de planejamento experimental (SEED). No primeiro Delineamento Composto Central Rotacional (CCRD), a temperatura (25-30 °C) e a concentração de inóculo (0,5-50 g L-¹) tiveram efeitos positivos na produção de bioetanol, enquanto o pH (4,0-6,0) não foi significativo. No segundo CCRD, a temperatura (28-35 °C) e a concentração do inóculo (10-70 g L-¹) tiveram efeitos negativo e positivo, respectivamente, na produção de bioetanol (p < 0,05). A adição de protease (15 µL g-¹) aumentou a conversão do substrato em bioetanol em 76%. Na temperatura de 31,5 °C e concentração de inóculo de 70 g L-¹ obteve-se a condição otimizada para produção de bioetanol, com a produção de 40,7 g L-¹. Na validação, realizada em um fermentador de bancada, foram produzidos 40,0 g L-¹ de bioetanol a partir de DRB hidrolisado; e o SEED foi caracterizado como uma ferramenta útil para otimizar a produção de bioetanol a partir de DRB. Além disso, o DRB provou ser um subproduto com grande potencial para a produção de bioetanol, derivado de fontes alternativas normalmente não utilizadas na alimentação humana.

Feasibility of bioethanol production from rice bran / Viabilidade da produção de bioetanol a partir do farelo de arroz

Rice bran is a by-product of rice production with a high carbohydrate and starch content and the potential for bioethanol production by alcoholic fermentation. This article describes bioethanol production by Saccharomyces cerevisiae from hydrolyzed defatted rice bran (DRB) a rice by-product applying ultrasonic treatment and protease addition, as well as a sequential strategy of experimental design (SEED). In the first Central Composite Rotatable Design (CCRD), the temperature (25-30 °C) and inoculum concentration (0.5-50 g L-¹) had positive effects on bioethanol production, while the effect of pH (4.0-6.0) was not significant. In the second CCRD, the temperature (28-35 °C) and inoculum concentration (10-70 g L-¹) had negative and positive effects on bioethanol production (p< 0.05). Protease addition (15 µL g-¹) increased the conversion of substrate into bioethanol by 76%. The optimized conditions for the production of 40.7 g L-¹ bioethanol were a temperature of 31.5 °C and an inoculum concentration of 70 g L-¹. Validation in a benchtop bioreactor produced 40.0 g L-¹ of bioethanol from hydrolyzed DRB, and the SEED was characterized as a useful tool to improve bioethanol production from DRB. Furthermore, the DRB proved to be a by-product with great potential for bioethanol production, derived from alternative sources not commonly used in human food.(AU)

O farelo de arroz é um subproduto com alto teor de carboidratos e amido, com potencial para produção de bioetanol por fermentação alcoólica. O presente artigo descreve a produção de bioetanol pela ação da Saccharomyces cerevisiae no farelo de arroz desengordurado hidrolisado (DRB) - um subproduto do arroz - com a aplicação do tratamento ultrassônico e adição de protease, e estratégia sequencial de planejamento experimental (SEED). No primeiro Delineamento Composto Central Rotacional (CCRD), a temperatura (25-30 °C) e a concentração de inóculo (0,5-50 g L-¹) tiveram efeitos positivos na produção de bioetanol, enquanto o pH (4,0-6,0) não foi significativo. No segundo CCRD, a temperatura (28-35 °C) e a concentração do inóculo (10-70 g L-¹) tiveram efeitos negativo e positivo, respectivamente, na produção de bioetanol (p < 0,05). A adição de protease (15 µL g-¹) aumentou a conversão do substrato em bioetanol em 76%. Na temperatura de 31,5 °C e concentração de inóculo de 70 g L-¹ obteve-se a condição otimizada para produção de bioetanol, com a produção de 40,7 g L-¹. Na validação, realizada em um fermentador de bancada, foram produzidos 40,0 g L-¹ de bioetanol a partir de DRB hidrolisado; e o SEED foi caracterizado como uma ferramenta útil para otimizar a produção de bioetanol a partir de DRB. Além disso, o DRB provou ser um subproduto com grande potencial para a produção de bioetanol, derivado de fontes alternativas normalmente não utilizadas na alimentação humana.(AU)

Partial characterization of digestive proteases in juveniles of Microphis brachyurus (short-tailed pipefish) (Syngnathiformes: Syngnathidae)

Short-tailed pipe fish (Microphis brachyurus) is a freshwater organism with high economic potential for the aquarium hobby, so it is necessary to implement methods to promote its culture through studies of digestive physiology. General activities of acid and alkaline proteases were evaluated, as well as the effect of pH, temperature and inhibitors. The optimal pH of stomach proteases was 2, while the optimal pH of intestinal proteases was 10. Optimal temperature for the acidic proteases was 35 ºC, while for alkaline proteases it was 45 ºC. Thermal stability showed high resistance at 35 ºC for both acid and alkaline proteases (above 100% residual activity). Acid proteases are resistant at pH 2 (50% of residual activity), meanwhile alkaline proteases were highly resistant at pH 10 (90% of residual activity). Acid proteases were inhibited by 80% with pepstatin A and alkaline proteases were inhibited with TLCK and TPCK for trypsin (75%) and chymotrypsin (80%), respectively. Finally, metallo-proteases were 75% partially inhibited some serine proteases by 75% with EDTA. In conclusion, M. brachyurus has a good digestive capacity, since they can degrade a wide variety of proteins due to their greater proteolytic activity.(AU)

El pez pipa (Microphis brachyurus) es un organismo dulceacuícola con alto potencial económico para la acuarofilia; sin embargo, es necesario implementar su cultivo a través de estudios de fisiología digestiva. Se evaluó el efecto del pH, temperatura e inhibidores sobre las actividades enzimáticas de proteasas ácidas y alcalinas. El pH óptimo de proteasas estomacales es de 2, mientras que el de proteases intestinales es de 10. La temperatura óptima de proteasas ácidas es de 35 ºC y las alcalinas de 45 ºC. La estabilidad térmica para proteasas ácidas y alcalinas es a los 35 ºC (más de 100% de actividad residual). La estabilidad a los diferentes pH de las proteasas ácidas es en 2 (50 % de la actividad residual), mientras que para las proteasas alcalinas es en 10 (90 % de la actividad residual). Las proteasas ácidas fueron inhibidas en 80% con pepstatina A y las proteasas alcalinas fueron altamente inhibidas con TLCK para tripsina (75%) y TPCK quimitripsina (80%). Finalmente, las metaloproteasas fueron inactivadas con EDTA en 70%. En conclusión, M. brachyurus tiene una buena capacidad digestiva al degradar una amplia variedad de proteinas debido a su alta actividad proteolítica.(AU)

Partial characterization of digestive proteases in juveniles of Microphis brachyurus (short-tailed pipefish) (Syngnathiformes: Syngnathidae)

RESUMEN El pez pipa (Microphis brachyurus) es un organismo dulceacuícola con alto potencial económico para la acuarofilia; sin embargo, es necesario implementar su cultivo a través de estudios de fisiología digestiva. Se evaluó el efecto del pH, temperatura e inhibidores sobre las actividades enzimáticas de proteasas ácidas y alcalinas. El pH óptimo de proteasas estomacales es de 2, mientras que el de proteases intestinales es de 10. La temperatura óptima de proteasas ácidas es de 35 ºC y las alcalinas de 45 ºC. La estabilidad térmica para proteasas ácidas y alcalinas es a los 35 ºC (más de 100% de actividad residual). La estabilidad a los diferentes pH de las proteasas ácidas es en 2 (50 % de la actividad residual), mientras que para las proteasas alcalinas es en 10 (90 % de la actividad residual). Las proteasas ácidas fueron inhibidas en 80% con pepstatina A y las proteasas alcalinas fueron altamente inhibidas con TLCK para tripsina (75%) y TPCK quimitripsina (80%). Finalmente, las metaloproteasas fueron inactivadas con EDTA en 70%. En conclusión, M. brachyurus tiene una buena capacidad digestiva al degradar una amplia variedad de proteinas debido a su alta actividad proteolítica.

ABSTRACT Short-tailed pipe fish (Microphis brachyurus) is a freshwater organism with high economic potential for the aquarium hobby, so it is necessary to implement methods to promote its culture through studies of digestive physiology. General activities of acid and alkaline proteases were evaluated, as well as the effect of pH, temperature and inhibitors. The optimal pH of stomach proteases was 2, while the optimal pH of intestinal proteases was 10. Optimal temperature for the acidic proteases was 35 ºC, while for alkaline proteases it was 45 ºC. Thermal stability showed high resistance at 35 ºC for both acid and alkaline proteases (above 100% residual activity). Acid proteases are resistant at pH 2 (50% of residual activity), meanwhile alkaline proteases were highly resistant at pH 10 (90% of residual activity). Acid proteases were inhibited by 80% with pepstatin A and alkaline proteases were inhibited with TLCK and TPCK for trypsin (75%) and chymotrypsin (80%), respectively. Finally, metallo-proteases were 75% partially inhibited some serine proteases by 75% with EDTA. In conclusion, M. brachyurus has a good digestive capacity, since they can degrade a wide variety of proteins due to their greater proteolytic activity.

Complexos enzimáticos sobre a energia metabolizável e digestibilidade dos nutrientes do milheto para frangos de corte / Enzyme complexes on metabolizable energy and nutrient digestibility of millet for broiler chickens

Objetivou-se avaliar o efeito de complexos enzimáticos sobre a energia metabolizável e o coeficiente de digestibilidade de nutrientes do milheto para frangos de corte. Quinhentos e setenta e seis frangos machos foram distribuídos em 36 gaiolas, com três tratamentos: T1 - composição de milheto sem complexo enzimático; T2 - composição de milheto com complexo enzimático (CES) e T3 - composição de milheto com complexo enzimático (CEV). Os tratamentos foram definidos com base em seis dietas (três dietas referências e três dietas testes). As dietas testes foram obtidas pela substituição de 40% da dieta referência por milheto inteiro, e a adição de enzimas consistiu de dois complexos enzimáticos: CES, constituído pelas enzimas fitase, protease, xilanase, ß-glucanase, celulase, amilase e pectinase; e CEV. constituído pelas enzimas protease, celulase e amilase. Na fase de 11 a 20 dias, a suplementação com o CEV reduziu os valores de EMA, EMAn e CDPB. A suplementação com CES melhorou o CDPB, e não houve efeito significativo para CDMS e CDEB. Na fase de 21 a 30 dias, houve menor aproveitamento da energia e dos nutrientes com as suplementações CES e CEV. Na fase de 31 a 40 dias, as suplementações reduziram os valores de EMA, EMAn, e o complexo CEV foi efetivo em aumentar o valor de CDPB. A inclusão dos complexos enzimáticos CES (fitase, protease, xilanase, ß-glucanase, celulase, amilase e pectinase) e CEV (protease, celulase e amilase) não favoreceu a utilização da energia do milheto, no entanto melhorou o coeficiente de digestibilidade da proteína do milheto nos períodos de 11 a 20 e de 31 a 40 dias de idade.(AU)

The objective of this study was to evaluate the effect of enzymatic complexes on metabolizable energy and nutrient digestibility coefficient of millet for broilers chickens. 576 male chickens, were distributed in 36 cages with three treatments: T1 - millet composition without enzymatic complex; T2 - millet composition with enzymatic complex (ECS); and T3 - millet composition with enzymatic complex (ECV). The treatments were defined from six diets (3 reference diets and 3 test diets). The test diets were obtained from the substitution of 40% for reference diet by whole millet, and the enzyme addition consisted of two enzymatic complex, ECS constituted by phytase, protease, xylanase, ß-glucanase, cellulase, amylase and pectinase enzymes, and ECV constituted by protease, cellulase and amylase enzymes. In the 11 to 20 days phase, a supplementation with the ECV reduced the AME, AMEn and CDPB values, a ECS supplementation improved the CDPB, and there was no significant effect for CDMS and CDEB. In the 21 to 30 days phase, there were less profit of the energy and nutrients with ECS and ECV supplements. In the 31 to 40 days phase as supplements reduced the values of AME, AMEn, and the ECV complex was effective in increasing the value of CDPB. The inclusion of ECS enzymatic complexes, (phytase, protease, xylanase, ß-glucanase, cellulase, amylase and pectinase) and ECV (protease, cellulase and amylase), did not favor millet's energy utilization, however, favored the millet's protein digestibility coefficient on 11 to 20 and 31 to 40 periods.(AU)