Carbohydrase inclusion in a corn-soybean diet improves broiler growth performance

An experiment was conducted to evaluate the effect of diets with reduced energy level content, supplemented with carbohydrase, on broiler performance and the coefficient of metabolizability of nutrients. A total of 720 one-day-old male Cobb-500 chicks were distributed in a completely randomized design, with six treatments, eight repetitions of 15 birds each. The treatments were: (1) a positive control, basal diet to meet the requirements of broiler chickens (PC); (2) a negative control, basal diet with a reduction of 80 kcal kg-1 (NC); (3) NC + alphagalactosydase; (4) NC + xylanase; (5) NC + xylanase and alphagalactosydase, and (6) NC + enzymatic blend (alphagalactosydase, xylanase, pectinase and amylase). The nutrient digestibility was not improved by use of enzymes. At 7 days of age, the broilers which were fed diets supplemented with enzymes showed a lower feed intake (FI) and better feed conversion ratio (FCR) than the broilers fed on PC. Both the NC and enzymatic blend resulted in a worse performance of the broilers at 21, 35 and 42 days old. The use of alphagalactosydase and xylanase, isolated or in combination, in a corn-soybean meal-based diet is effective in improving the growth performance of broilers fed energy-reduced diets.(AU)

Polysaccharide storage myopathy in two Percheron horses / Miopatia por acúmulo de polissacarídeo em dois equinos da raça Percheron

Polysaccharide storage myopathy (PSSM) is a genetic disorder or by undetermined cause characterized by the abnormal accumulation of glycogen and polysaccharides in skeletal muscle fibers. The present report describes two Percheron horses, from different properties in the municipality of Pouso Redondo -SC, with locomotor clinical signs that started after work. The clinical signs observed were rigid gait, mainly of the pelvic limbs, muscle weakness, and recumbency. In the first case, the animal was diagnosed with severe multifocal rhabdomyonecrosis, moderate multifocal myoglobin nephrosisand severe diffuse transmural fibrinonecrotic gastritis. PSSM was confirmed by the presence of polysaccharide inclusions in myofiber sarcolemma visualized by periodic acid-Schiff (PAS) staining. In the second case, the horse presented two episodes ofmuscle disorder after work. The therapeutic protocol was based on penicillin, flunixin meglumine and fluid therapy with ringer lactate. An increase in creatine kinase (669.0 UI/L) and aspartate aminotransferase (669.0 UI/L) was observed in the serum biochemical evaluation. Research for mutation in the GYS1 gene was performed, with a positive heterozygote result. After rest and gradual recovery, the horse was only submitted to light work, with no return of the clinical condition since then. Thus, this report describes two cases of PSSM with distinct clinical evolution and diagnostic methods.(AU)

A miopatia por acúmulo de polissacarídeos (PSSM) é uma desordem genética ou de causa indeterminada caracterizada pelo acúmulo anormal de glicogênio e polissacarídeos nas fibras musculares esqueléticas. O presente relato descreve dois cavalos Percheron, oriundos de diferentes propriedades do município de Pouso Redondo ­SC, com sinais clínicos locomotores que iniciaram após o trabalho. Os sinais clínicos observados foram marcha rígida, principalmente dos membros pélvicos, fraqueza muscular, e decúbito. No primeiro caso, o animal foi diagnosticado com rabdomionecrose multifocal acentuada, nefrose mioglobínica multifocal moderada e gastrite fibrinonecrótica transmural difusa acentuada. A PSSM foi confirmada pela presença de inclusões polissacarídicas no sarcolema de miofibras visualizadas pela coloração com ácido periódico de Schiff (PAS). No segundo caso, o equino apresentou dois episódios de disfunção muscular após o trabalho. O protocolo terapêutico foi baseado em penicilina, flunixin megluminee fluidoterapia com ringer lactato. Um aumento de creatina quinase (669,0 UI/L) e aspartato aminotransferase (669,0 UI/L) foi observado na avaliação bioquímica sérica. Foi realizada pesquisa de mutação nogene GYS1, com resultado heterozigoto positivo. Após repouso e recuperação gradual, o cavalo foi submetido apenas a trabalhos leves, sem retorno do quadro clínico desde então. Assim, este relato descreve dois casos com PSSM com evolução clínica e métodos diagnósticos distintos.(AU)

Suppresses of Astragalus Polysaccharide on E. coli-Induced Injured Intestinal Microvascular through TLR4-NF-B Signal Pathways in Chickens

To investigate the hypothesis that APS can attenuate E. coli-induced microvascular dysfunction in chicken intestine, 60 0-day old male chickens were divided into three groups with 5 replications of 4 chicks. Chicken in the APS group were treated with 15 mg APS daily while the others were given 0 mg APS for 6 days. Then all 7-day old chicken were injected intraperitoneally by E. coli, except for the chicken in the control group. After 4 h, all chickens intestinal samples were collected to detect gene expressions involved in inflammatory factors and adhesion molecules. Results showed that APS attenuated the signs of edema and hemorrhage in 7-day old chicken intestinal mucosa which were induced by E. coli injection. Consistently, APS significantly reduced the increasing mRNA levels of inflammatory factors such as Tumor necrosis factor-a (TNF-a), interleukin (IL) -1 and inducible nitric oxide synthase (iNOS) (p 0.05), the same results were observed in vascular adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Moreover, we observed that APS supplementation in water suppressed significantly (p 0.05) the decline of reparative factors such as epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) in E. coli injected group. Furthermore, supplementation with APS substantially blocked (p 0.05) the increase in Toll-like receptor-4 (TLR4) and Nuclear factor (NF)-B mRNA abundance (p=0.087) induced by E. coli infection. This study indicated that microvascular injured chicken intestine induced by E. coli would be attenuated with feeding APS, and the mechanism of repairing were probably mediated through TLR4-NF-B signal pathways.(AU)

Suppresses of Astragalus Polysaccharide on E. coli-Induced Injured Intestinal Microvascular through TLR4-NF-B Signal Pathways in Chickens

To investigate the hypothesis that APS can attenuate E. coli-induced microvascular dysfunction in chicken intestine, 60 0-day old male chickens were divided into three groups with 5 replications of 4 chicks. Chicken in the APS group were treated with 15 mg APS daily while the others were given 0 mg APS for 6 days. Then all 7-day old chicken were injected intraperitoneally by E. coli, except for the chicken in the control group. After 4 h, all chickens intestinal samples were collected to detect gene expressions involved in inflammatory factors and adhesion molecules. Results showed that APS attenuated the signs of edema and hemorrhage in 7-day old chicken intestinal mucosa which were induced by E. coli injection. Consistently, APS significantly reduced the increasing mRNA levels of inflammatory factors such as Tumor necrosis factor-a (TNF-a), interleukin (IL) -1 and inducible nitric oxide synthase (iNOS) (p 0.05), the same results were observed in vascular adhesion molecules such as E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). Moreover, we observed that APS supplementation in water suppressed significantly (p 0.05) the decline of reparative factors such as epithelial growth factor (EGF) and basic fibroblast growth factor (bFGF) in E. coli injected group. Furthermore, supplementation with APS substantially blocked (p 0.05) the increase in Toll-like receptor-4 (TLR4) and Nuclear factor (NF)-B mRNA abundance (p=0.087) induced by E. coli infection. This study indicated that microvascular injured chicken intestine induced by E. coli would be attenuated with feeding APS, and the mechanism of repairing were probably mediated through TLR4-NF-B signal pathways.

Optimized acid hydrolysis of the polysaccharides from the seaweed Solieria filiformis (Kützing) P.W. Gabrielson for bioethanol production / Hidrólise ácida otimizada dos polissacarídeos da macroalga Solieria filiformis (Kützing) P.W.Gabrielson para produção de bioetanol

The seaweeds are bio-resource rich in sulfated and neutral polysaccharides. The tropical seaweed species used in this study (Solieria filiformis), after dried, shows 65.8% (w/w) carbohydrate, 9.6% (w/w) protein, 1.7% (w/w) lipid, 7.0% (w/w) moisture and 15.9% (w/w) ash. The dried seaweed was easily hydrolyzed under mild conditions (0.5 M sulfuric acid, 20 min.), generating fermentable monosaccharides with a maximum hydrolysis efficiency of 63.21%. Galactose and glucose present in the hydrolyzed were simultaneously fermented by Saccharomyces cerevisiae when the yeast was acclimated to galactose and cultivated in broth containing only galactose. The kinetic parameters of the fermentation of the seaweed hydrolyzed were Y(PS) = 0.48 ± 0.02 g.g1, PP = 0.27 ± 0.04 g.L1.h1, h = 94.1%, representing a 41% increase in bioethanol productivity. Therefore, S. filiformis was a promising renewable resource of polysaccharides easily hydrolyzed, generating a broth rich in fermentable monosaccharides for ethanol production.(AU)

As algas marinhas são recursos naturais ricos em polissacarídeos sulfatados e neutros. A espéciede macroalga tropical utilizada neste estudo (Solieria filiformis) apresentou teores de carboidratos de 65,8%(m/m), proteínas de 9,6% (m/m), lipídios de 1,7% (m/m), umidade de 7,0% (m/m) e 15,9 % (m/m) decinzas. A macroalga seca foi facilmente hidrolisada em condições brandas, na presença de ácido sulfúrico0,5 M, por 20 min, produzindo monossacarídeos fermentáveis com uma eficiência de hidrólise máxima de63,21%. A galactose e a glicose presentes no hidrolisado foram fermentadas simultaneamente porSaccharomyces cerevisiae, após aclimatação da levedura cultivada em meio contendo apenas galactose comofonte de carbono. Os parâmetros cinéticos da fermentação do hidrolisado algáceo pela levedura aclimatada agalactose foram Y(P/S) = 0,48 ± 0,02 g.g-1, PP = 0,27 ± 0,04 g.L-1.h-1, η = 94,1%. Portanto, a macroalga S.filiformis se mostrou um recurso renovável promissor como fonte de polissacarídeos facilmente hidrolisados,gerando um meio nutritivo rico em glucose e galactose para a produção de etanol.(AU)

Efficient production of pullulan by Aureobasidium pullulans grown on mixtures of potato starch hydrolysate and sucrose

Pullulan is a natural exopolysaccharide with many useful characteristics. However, pullulan is more costly than other exopolysaccharides, which limits its effective application. The purpose of this study was to adopt a novel mixed-sugar strategy for maximizing pullulan production, mainly using potato starch hydrolysate as a low-cost substrate for liquid-state fermentation by Aureobasidium pullulans. Based on fermentation kinetics evaluation of pullulan production by A. pullulans 201253, the pullulan production rate of A. pullulans with mixtures of potato starch hydrolysate and sucrose (potato starch hydrolysate:sucrose = 80:20) was 0.212 h-¹, which was significantly higher than those of potato starch hydrolysate alone (0.146 h-¹) and mixtures of potato starch hydrolysate, glucose, and fructose (potato starch hydrolysate:glucose:fructose = 80:10:10, 0.166 h-¹) with 100 g L-¹ total carbon source. The results suggest that mixtures of potato starch hydrolysate and sucrose could promote pullulan synthesis and possibly that a small amount of sucrose stimulated the enzyme responsible for pullulan synthesis and promoted effective potato starch hydrolysate conversion effectively. Thus, mixed sugars in potato starch hydrolysate and sucrose fermentation might be a promising alternative for the economical production of pullulan.(AU)

High-quality total RNA isolation from melon (Cucumis melo L. ) fruits rich in polysaccharides / Isolamento de RNA total de alta qualidade a partir de frutos de melão (Cucumis melo L. ) contendo altos níveis de polissacarídeos

Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazils main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/?-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/?-mercaptoethanol; T5) SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.(AU)

O melão pertencente à família Cucurbitaceae é o quarto fruto mais importante no mercado mundial e a fruta mais exportada pelo Brasil. Muitas técnicas moleculares utilizadas para compreender a maturação destes frutos requerem o uso de RNA altamente purificado e em alta concentração. Entretanto, o elevado nível de compostos polifenólicos e polissacarídeos nos frutos tornam a extração de RNA um desafio. Este trabalho teve por objetivo determinar o método mais adequado para extração de RNA total em frutos de melão. Seis diferentes tampões de extração foram testados: T1) tiocianato de guanidina/fenol/clorofórmio; T2) azida de sódio/?-mercaptoetanol; T3) fenol/tiocianato de guanidina, T4) CTAB/PVP/?-mercaptoetanol, T5) SDS/perclorato de sódio/PVP/?-mercaptoetanol e T6) sarcosil/PVP/tiocianato de guanidina associado com AxyPrep TM Multisource Total RNA Miniprep Kit. O melhor método para extração do RNA tanto de fruto verde quanto maduro foi o baseado no tampão SDS/PVP/?-mercaptoetanol, por gerar material íntegro e de qualidade em grande quantidade, associado à rapidez de execução. Em geral, maiores quantidades de RNA foram obtidas a partir de frutos verdes, provavelmente devido à baixa concentração de polissacarídeos e água. O material purificado poderá ser utilizado como molde em técnicas de estudos moleculares como microarrays, RT-PCR e bibliotecas de cDNA e RNAseq, pelo qual foi testado.(AU)

Bran palm giant with exogenous enzymes addition in pig nutrition on growth / Farelo de palma gigante (Opuntia fícus-indica) na alimentação de suínos em crescimento

The objective of this study was to evaluate the use of giant palm meal (FPG) in diets with or without enzymatic supplementation for growing pigs. For the evaluation of the digestibility, ten castrated male pigs with a mean weight of 34.3±6.7 kg, housed in metabolic cages for 15 days (nine of adaptation and six of total collection of feces and urine) were used. The animals were fed twice a day (eight and 15 hours) according to the metabolic weight, five of which received the control diet and five received a test diet with a 30% inclusion of the FPG. The samples were taken daily, homogenized and a 20% aliquot was taken and stored in a freezer. Subsequently, the samples were analyzed in the laboratory of animal nutrition. In the performance test, twenty animals with 28.62±3.66kg were used, distributed in randomized blocks, with five treatments and four replicates. The treatments consisted of inclusion levels of the giant palm meal, associated or not to the enzymatic complex (EC) (0; 5; 10; 5 + EC and 10% + EC). The FPG digestible energy value for pigs in the growing phase was 1113.83 kcal/kg. It was observed that the weight gain and the absolute weight of the organs of the pigs were not influenced by the diets. Feed conversion, feed intake, economic viability of the diets and the relative weight of the organs presented differences between the treatments. It is recommended to include 10% FPG associated with the complex.

Objetivou-se avaliar a utilização do farelo de palma gigante (FPG) em dietas com ou sem suplementação enzimática para suínos em crescimento. Para avaliação da digestibilidade foram utilizados dez suínos machos castrados com peso médio de 34,3 ± 6,7kg, alojados em gaiolas metabólicas por quinze dias (nove de adaptação e seis de coleta total de fezes e urina). Os animais foram alimentados duas vezes ao dia (oito e 15hs) de acordo com o peso metabólico, sendo que cinco receberam a ração controle e cinco receberam dieta teste com 30% de inclusão do FPG. As coletas foram realizadas diariamente, homogeneizadas e retirada uma alíquota de 20% e armazenada em freezer. Posteriormente, as amostras foram analisadas no laboratório de nutrição animal. No ensaio de desempenho foram utilizados vinte animais com 28,62 ± 3,66kg, distribuídos em blocos ao acaso, com cinco tratamentos e quatro repetições. Os tratamentos consistiram em níveis de inclusão do farelo de palma gigante, associado ou não ao complexo enzimático (CE) (0; 5; 10; 5 + CE e 10% + CE). O valor da energia digestível do FPG para suínos na fase de crescimento foi de 1113,83 kcal/kg. Observou-se que o ganho de peso e o peso absoluto dos órgãos dos suínos não foram influenciados pelas dietas. A conversão alimentar, o consumo de ração, a viabilidade econômica das dietas e o peso relativo dos órgãos apresentaram diferenças entre os tratamentos. Recomenda-se a inclusão de 10% de FPG associado ao complexo.

Polysaccharide extract of Mimosa tenuiflora stem barks stimulates acute inflammatory response via nitric oxide / Extrato polissacarídico oriundo das cascas do caule de Mimosa tenuiflora induz resposta inflamatória aguda via óxido nítrico

Mimosa tenuiflora (Mimosaceae) or jurema-preta is well distributed in the northeast Brazil, being popularly used to treat skin lesions, burns and inflammation. The healing effect of the alcoholic extract prepared with its barks corroborates the popular use. This study aimed to evaluate the inflammatory response of polysaccharides extracted from M. tenuiflora barks (EP-Mt) by methanol/NaOH and ethanol precipitation. Inflammatory activity was assessed in rat models of acute inflammation (paw edema and peritonitis), by the following parameters: edema, vascular permeability, leukocyte migration, myeloperoxidase activity and pharmacological modulation of nitric oxide and prostaglandins. EP-Mt presented 3.8% yield, 41% carbohydrate and 0.34% protein. EP-Mt (0.01, 0.1, 1.0 mg kg-1) injected by subcutaneous route elicited paw edema that lasted from 30-420 min, with maximal effect at 1 mg kg-1 (40x vs. saline), and was inhibited by L-NAME (52%) and dexamethasone (26%). EP-Mt (1 mg kg-1, via intraperitoneal) stimulated leukocytes migration (2.2x), mainly neutrophils (6.5x) and MPO activity (96%). The leukocyte migration elicited by EP-Mt was inhibited by dexamethasone (39%) and L-NAME (38%). EP-Mt containing high carbohydrate content induces acute inflammation via nitric oxide, which open perspectives of application in pathological conditions of immunosuppression.(AU)

Mimosa tenuiflora (Mimosaceae) ou "jurema-preta", amplamente distribuída no nordeste brasileiro, é utilizada popularmente no tratamento de lesões de pele, queimaduras e inflamação. O efeito cicatrizante do extrato alcoólico de suas cascas corrobora o uso popular. Avaliou-se o efeito inflamatório dos polissacarídeos da casca de M. tenuiflora (EP-Mt), obtidos por extração com metanol/NaOH e precipitação com etanol. O efeito inflamatório foi avaliado em modelos agudos em ratos (edema de pata, peritonite) por meio dos seguintes parâmetros: edema, permeabilidade vascular, migração leucocitária, atividade da mieloperoxidase e modulação farmacológica de prostaglandinas e óxido nítrico. EP-Mt apresentou 3,8% de rendimento, 41% de carboidratos totais e 0,34% de proteína. EP-Mt (0,01, 0,1, 1,0 mg kg-1), administrado por via subcutânea, induziu edema de pata com duração de 30 a 420 min e efeito máximo na dose de 1 mg kg-1 (40x vs. salina), o qual foi inibido por L-NAME (52%) e dexametasona (26%). EP-Mt (1 mg kg-1, via intraperitoneal) estimulou a migração de leucócitos (2,2x vs. salina), principalmente de neutrófilos (6,5x), com aumento da atividade da mieloperoxidase (96%). A migração de leucócitos foi inibida por dexametasona (39%) e L-NAME (38%). EP-Mt contendo elevado teor de carboidratos induz inflamação aguda via óxido nítrico com perspectivas de aplicação em condições patológicas(AU)

Bran palm giant with exogenous enzymes addition in pig nutrition on growth / Farelo de palma gigante (Opuntia fícus-indica) na alimentação de suínos em crescimento

The objective of this study was to evaluate the use of giant palm meal (FPG) in diets with or without enzymatic supplementation for growing pigs. For the evaluation of the digestibility, ten castrated male pigs with a mean weight of 34.3±6.7 kg, housed in metabolic cages for 15 days (nine of adaptation and six of total collection of feces and urine) were used. The animals were fed twice a day (eight and 15 hours) according to the metabolic weight, five of which received the control diet and five received a test diet with a 30% inclusion of the FPG. The samples were taken daily, homogenized and a 20% aliquot was taken and stored in a freezer. Subsequently, the samples were analyzed in the laboratory of animal nutrition. In the performance test, twenty animals with 28.62±3.66kg were used, distributed in randomized blocks, with five treatments and four replicates. The treatments consisted of inclusion levels of the giant palm meal, associated or not to the enzymatic complex (EC) (0; 5; 10; 5 + EC and 10% + EC). The FPG digestible energy value for pigs in the growing phase was 1113.83 kcal/kg. It was observed that the weight gain and the absolute weight of the organs of the pigs were not influenced by the diets. Feed conversion, feed intake, economic viability of the diets and the relative weight of the organs presented differences between the treatments. It is recommended to include 10% FPG associated with the complex.(AU)

Objetivou-se avaliar a utilização do farelo de palma gigante (FPG) em dietas com ou sem suplementação enzimática para suínos em crescimento. Para avaliação da digestibilidade foram utilizados dez suínos machos castrados com peso médio de 34,3 ± 6,7kg, alojados em gaiolas metabólicas por quinze dias (nove de adaptação e seis de coleta total de fezes e urina). Os animais foram alimentados duas vezes ao dia (oito e 15hs) de acordo com o peso metabólico, sendo que cinco receberam a ração controle e cinco receberam dieta teste com 30% de inclusão do FPG. As coletas foram realizadas diariamente, homogeneizadas e retirada uma alíquota de 20% e armazenada em freezer. Posteriormente, as amostras foram analisadas no laboratório de nutrição animal. No ensaio de desempenho foram utilizados vinte animais com 28,62 ± 3,66kg, distribuídos em blocos ao acaso, com cinco tratamentos e quatro repetições. Os tratamentos consistiram em níveis de inclusão do farelo de palma gigante, associado ou não ao complexo enzimático (CE) (0; 5; 10; 5 + CE e 10% + CE). O valor da energia digestível do FPG para suínos na fase de crescimento foi de 1113,83 kcal/kg. Observou-se que o ganho de peso e o peso absoluto dos órgãos dos suínos não foram influenciados pelas dietas. A conversão alimentar, o consumo de ração, a viabilidade econômica das dietas e o peso relativo dos órgãos apresentaram diferenças entre os tratamentos. Recomenda-se a inclusão de 10% de FPG associado ao complexo.(AU)

Calorimetric techniques applied to the thermodynamic study of interactions between proteins and polysaccharides / Técnicas calorimétricas aplicadas ao estudo termodinâmico das iterações entre proteínas e polissacarídeos

The interactions between biological macromolecules have been important for biotechnology, but further understanding is needed to maximize the utility of these interactions. Calorimetric techniques provide information regarding these interactions through the thermal energy that is produced or consumed during interactions. Notable techniques include differential scanning calorimetry, which generates a thermodynamic profile from temperature scanning, and isothermal titration calorimetry that provide the thermodynamic parameters directly related to the interaction. This review described how calorimetric techniques can be used to study interactions between proteins and polysaccharides, and provided valuable insight into the thermodynamics of their interaction.(AU)

As interações entre macromoléculas biológicas têm tido importante aplicação na biotecnologia, mas, para sua devida utilização, estudos mais detalhados são necessários. As técnicas calorimétricas permitem estudá-las ao serem capazes de fornecer informações referentes a essas interações através da energia térmica que é gerada ou absorvida durante o processo de interação. Dentre as técnicas que mais se destacam estão a Calorimetria Exploratória Diferencial, que é capaz de fornecer um perfil termodinâmico a partir de uma varredura de temperatura, e a Calorimetria de Titulação Isotérmica, que fornece parâmetros termodinâmicos diretamente relacionados ao processo de interação. Nesta revisão, descrevemos como essas técnicas calorimétricas podem ser efetivamente aplicadas no estudo das interações entre proteínas e polissacarídeos, com o propósito de obter informações valiosas sobre a termodinâmica da interação.(AU)

High-quality total RNA isolation from melon (Cucumis melo L. ) fruits rich in polysaccharides / Isolamento de RNA total de alta qualidade a partir de frutos de melão (Cucumis melo L. ) contendo altos níveis de polissacarídeos

Melon, a member of the family Cucurbitaceae, is the fourth most important fruit in the world market and, on a volume basis, is Brazils main fresh fruit export. Many molecular techniques used to understand the maturation of these fruits require high concentrations of highly purified RNA. However, melons are rich in polyphenolic compounds and polysaccharides, which interfere with RNA extraction. This study aimed to determine the most appropriate method for total RNA extraction from melon fruits. Six extraction buffers were tested: T1) guanidine thiocyanate/phenol/chloroform; T2) sodium azide/?-mercaptoethanol; T3) phenol/guanidine thiocyanate; T4) CTAB/PVP/?-mercaptoethanol; T5) SDS/sodium perchlorate/PVP/?-mercaptoethanol, and T6) sarkosyl/PVP/guanidine thiocyanate, using the AxyPrepTM Multisource Total RNA Miniprep Kit. The best method for extracting RNA from both mature and green fruit was based on the SDS/PVP/?-mercaptoethanol buffer, because it rapidly generated a high quality and quantity of material. In general, higher amounts of RNA were obtained from green than mature fruits, probably due to the lower concentration of polysaccharides and water. The purified material can be used as a template in molecular techniques, such as microarrays, RT-PCR, and in the construction of cDNA and RNA-seq data.

O melão pertencente à família Cucurbitaceae é o quarto fruto mais importante no mercado mundial e a fruta mais exportada pelo Brasil. Muitas técnicas moleculares utilizadas para compreender a maturação destes frutos requerem o uso de RNA altamente purificado e em alta concentração. Entretanto, o elevado nível de compostos polifenólicos e polissacarídeos nos frutos tornam a extração de RNA um desafio. Este trabalho teve por objetivo determinar o método mais adequado para extração de RNA total em frutos de melão. Seis diferentes tampões de extração foram testados: T1) tiocianato de guanidina/fenol/clorofórmio; T2) azida de sódio/?-mercaptoetanol; T3) fenol/tiocianato de guanidina, T4) CTAB/PVP/?-mercaptoetanol, T5) SDS/perclorato de sódio/PVP/?-mercaptoetanol e T6) sarcosil/PVP/tiocianato de guanidina associado com AxyPrep TM Multisource Total RNA Miniprep Kit. O melhor método para extração do RNA tanto de fruto verde quanto maduro foi o baseado no tampão SDS/PVP/?-mercaptoetanol, por gerar material íntegro e de qualidade em grande quantidade, associado à rapidez de execução. Em geral, maiores quantidades de RNA foram obtidas a partir de frutos verdes, provavelmente devido à baixa concentração de polissacarídeos e água. O material purificado poderá ser utilizado como molde em técnicas de estudos moleculares como microarrays, RT-PCR e bibliotecas de cDNA e RNAseq, pelo qual foi testado.