Development and validation of an analytical method by HPLC-DAD for determination of caffeine in products based on guarana extracts (Paullinia cupana)

Guarana (Paullinia cupana) is a native plant from the Amazon whose seeds contain a high concentration of caffeine. Aqueous extract of guarana is widely used in the world. In this study, the objective was to develop and validate a High-Performance Liquid Chromatography method for the determination of caffeine in extracts and commercial beverages based on guarana. A sensitive, simple, and viable high performance liquid chromatographic method without the need of an analyte extraction procedure was developed and validated according to Brazilian and international requirements. The method presented high performance, fulfilling Brazilian and international requirements, in addition to allowing product compliance tests. Results confirmed high selectivity and linearity (>0.999) between 5 to 135 ug/mL, with no significant matrix effect. Detection and quantification limits were 0.02 µg/mL and 2 µg/mL, respectively. Precision was less than 4 %, and accuracy varied from 99.9-120 %. Applicability of the method was demonstrated by conducting a limited evaluation in products containing caffeine. Commercial extracts showed quite different caffeine levels, while carbonated drinks follow Brazilian and American recommendations. Our results indicate that the developed method can be used to evaluate the quality of the guarana extract and of products containing caffeine

The importance of heat against antinutritional factors from Chenopodium quinoa seeds

Chenopodium quinoa seeds have high protein content. The nutritional value of quinoa is superior compared with traditional cereals. Its essential amino acid composition is considered next to the ideal, and its quality matches that of milk proteins. In this study, the seed storage proteins from Chenopodium quinoa were extracted, fractionated, partially purified, and characterized. The structural characterization was performed by Tricine-SDS-PAGE and two-dimensional electrophoresis, and it confirmed the presence of proteins of molecular weight of 30 and 7kDa, probably corresponding to lectins and trypsin inhibitors, respectively. The functional characterization of these proteins evidenced their activity as antinutritional factors due to their in vitro digestibility. Quinoa proteins have an excellent amino acid composition with many essential amino acids. In vitro digestibility evaluation indicated that heat-treated samples showed a more complete digestion than the native state samples. Quinoa seeds can be an important cereal in human diet after adequate heat treatment

Production of pectin lyase and polygalacturonase by recombinant strain Penicillium griseoroseum T20

Fungos filamentosos são reconhecidos como excelentes produtores de enzimas extracelulares e as linhagens geneticamente modificadas têm tornado possível a produção de pectinases com maior especificidade e pureza, melhor utilização de matéria-prima e menor produção de resíduos. O processo de produção de pectina liase (PL) e poligalacturonase (PG) pela linhagem geneticamente modificada Penicillium griseoroseum T20 foi estudado. As condições ótimas de cultivo para a produção de PL e PG foram determinadaspor meio da Metodologia de Superfície de Resposta (RSM). A maior produção de PL em frascos Erlenmeyer com 200 mL de meio de cultivo foi obtida após 87,7 h em meio contendo sacarose em concentração inicial de 15,7 g/L, sendo a maior atividade de PL estimada de 2.428 U/mL. A maior produção de PG em frascos Erlenmeyer com 200 mL de meio de cultivo foi obtida após 83,8 h, sendo a maior atividade de PG estimada de 9.465 U/mL. A concentração de sacarose não mostrou influência significativa sobre a produção dessa enzima. Após otimização dos fatores tempo de cultivo e concentração de sacarose em Erlenmeyers, foi feito o escalonamento para biorreator com 10 L de trabalho. A condição de aeração que proporcionou a maior atividade de PL e PG foi de 1,0 L de ar por minuto. Nessa condição, a linhagem apresentou baixa atividade de protease no período final de cultivo e não foi detectada atividade de β-glicosidase. O perfil protéico da linhagem recombinante T20 mostrou a presença de duas bandas de proteínas distintas com aproximadamente 38 e 36 kDa, correspondentes à PG e à PL, respectivamente. O crescimento micelial da linhagem P. griseoroseum T20 foi estudado por meio da RSM, e a massa micelial seca máxima estimada foi de 8,63 g/L, na condição de 30 g/L de sacarose após 120 horas de cultivo. A avaliação da morfologia micelial sugeriu a existência de uma relação entre a ocorrência de hifas livres e dispersas e a produção de PL e PG. Os parâmetros cinéticos da fermentação foram determinados e comparados entre as escalas de produção de PL e PG. A proteína total máxima observada foi de 9,1 e 9,5 mg/L nos cultivos de 200 mL e 10 L, respectivamente. As atividades específicas de PL (PspePLp) e PG (PspePGp) em relação à proteína total foram de 353 e 613 U/μg no cultivo em 200 mL e de 305 e 1.106 U/μg no cultivo em 10 L, respectivamente. As produtividades enzimáticas máximas de PL (PdPL) e PG (PdPG) observadas foram de 33,4 e 73,3 U/mL.h em 200 mL e de 24,1 e 289 U/mL.h em 10 L. Os parâmetros rendimento de PL (RPL/S) e de PG (RPG/S) calculados foram de 214 e 352 no cultivo em 200 mL e de 87,4 e 1.049 no cultivo em 10 L, respectivamente. A produção de PL e PG entre as linhagens P. griseoroseum T20 e P. griseoroseum selvagem foi comparada e aumentos de mais 400 vezes na produção de PL e de pelo menos 14 vezes na produção de PG foram observados. Os resultados sugerem o grande potencial de aplicação industrial dessa linhagem para a produção de PL e PG.Filamentous fungi are recognized as excellent producers of extracellular enzymes and the genetically modified strains have made possible the production of pectinases with greater specificity and purity, better use of raw materials and lower production of waste. The production of pectin lyase (PL) and polygalacturonase (PG) by genetically modified strain Penicillium griseoroseum T20 has been studied. The response surface methodology (RSM) was used to optimize PL and PG production. The parameters sucrose concentration and cultivation time were evaluated. The highest PL production in Erlenmeyer flasks with 200 mL of culture medium was achieved after 87.7 h in sucrose 15.7 g/L and the highest PL estimated activity was 2428 U/ml. The higher PG production in Erlenmeyer flasks with 200 mL of culture medium was achieved after 83.8 hours and the highest PG estimated activity was 9465 U/ml. The sucrose concentration showed no significant influence on the production of this enzyme. The optimization in Erlenmeyer flasks was followed by the scaleup to 10 L bioreactor. The aeration conditions were evaluated and the highest PL and PG activity was observed in 1.0 L of air per minute. Under this condition, the strain showed low protease activity, just in the final period of cultivation, and β-glucosidase activity was not detected. The protein profile of the recombinant strain T20 showed the presence of two distinct bands of with approximately 38 and 36 kDa. These bands corresponded to PL and PG, respectively. The mycelial growth of the strain P. griseoroseum T20 has been studied by RSM, and the maximum mycelial dry weight observed was 8.63 g/L, on 30 g/L sucrose and 120 hours of cultivation. The mycelial morphology suggested a link between the occurrence of free and dispersed hyphae and the production of PL and PG. The kinetic parameters of fermentation were determined and compared between the scales of PL and PG production. The maximum total protein was 9.1 and 9.5 mg/L in cultures of 200 ml and 10 L, respectively. The specific PL (PspePLp) and PG (PspePGp) activities in relation to total protein were 353 and 613 U/g at 200 mL cultivation and 305 and 1106 U/g at 10 L cultivation, respectively. The maximum yield of PL (PdPL) and PG (PdPG) observed were 33.4 and 73.3 U/mL.h at 200 mL cultivation and 24.1 and 289 U/L.mL.h at 10 L. The performance parameters of PL (RPL/S) and PG (RPG/S) were 214 and 352 at 200 ml cultivation and 87.4 and 1049 at 10 L, respectively. The PL and PG production between P. griseoroseum T20 and P. griseoroseum wide type strain was compared. Increases of more than 400 times in the PL production and at least 14 times in the PG production were observed. The results suggest the great potential for industrial application of this strain for the production of PL and PG.Conselho Nacional de Desenvolvimento Científico e Tecnológic

Anti-nutritional factors and digestibility of protein in Cayocar brasiliense seeds

<div><p>Abstract The Brazilian Cerrado presents a wide variety of natural products, including the Pequi (Caryocar brasiliense). The important factor that determines the dietary and nutritional ideality of protein is degestitibility. This work aims to evaluate the protein digestibility of Pequi seeds and the presence of anti-nutritional factors. The protein Pequi almonds were extracted, toasted and untoasted. Evaluation digestibility was structurally characterized by SDS-PAGE 15%, which can be used to analyze simulated gastric fluid (SFG) and digestion in intestinal fluid (SFI) in the presence of pepsin and trypsin/chymotrypsin. The extract from Pequi almonds showed inhibitory activity and was not detected hemagglutination. The intensity of the bands according to 2S albumins, after heat treatment, did not decrease in comparison to the condition native, significantly. In the crisp crude extract, which was incubated under SGF and SIF, the intensity of the corresponding bands at 8 and 3 kDa was resistant to SGF and indigestible after 4 hours under SIF. The characterization of Pequi almonds demonstrated that the product of the crude extract has anti-nutritional factors, which were confirmed by evaluating digestibility.</p></div

Overproduction of polygalacturonase by Penicillium griseoroseum recombinant strains and functional analysis by targeted disruption of the pgg2 Gene

Inactivation of the pgg2 gene, a polygalacturonase-encoding gene from Penicillium griseoroseum, reduced the total activity of polygalacturonase (PG) by 90 % in wild-type P. griseoroseum, which indicates that the pgg2 gene is the major gene responsible for PG production in this species. To increase PG production, the coding region of the pgg2 gene was cloned under the control of the glyceraldehyde 3-phosphate dehydrogenase (gpd) promoter and the terminator region of the tryptophan synthase (trpC) gene from Aspergillus nidulans (pAN52pgg2 vector). This vector was then used to transform P. griseoroseum. The transformed strains were characterized according to PG production using glucose, sucrose, or sugar cane juice as the carbon sources. The recombinant P. griseoroseum T146 strain contained an additional copy of the pgg2 gene, which resulted in a 12-fold increase in PG activity when compared with that detected in the supernatant of the control PG63 strain. The proteins secreted by the recombinant strain T146 showed a strong band at 38 kDa, which corresponds to the molecular weight of PG of the P. griseoroseum. The results demonstrate the significant biotechnological potential of recombinant P. griseoroseum T146 for use in PG production

Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum

Protoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and pectin lyase production (1.2-fold)