Abstract The goal of this study was to evaluate the α-amylases production by basidiomycetes in submerged cultivation (SmC), using wheat milling by-products as substrate. Enzymatic activity was evaluated to select the best strain, the ideal concentration, the by-product and the influence of supplementation in the culture medium. The best producer was chosen among three strains, Ganoderma applanatum (MR-56), Schizophyllum commune (MR-01), and Ganoderma stipitatum (MR-72). All of them were cultivated under the same conditions. MR-56 was selected for an initial screening activity of 5.10±0.31 U/mL in 192h. The Ganoderma applanatum strain was cultured in a medium with various starch concentrations to determine which concentration resulted in the highest α-amylase activity. After 120h of cultivation, activity in the medium containing 4.5% (w/v) had increased from 0.40±0.03 U/mL to 20.70±0.9 U/mL. Subsequently, the wheat milling by-products [Glue Flour (GF), Clean-Out Flour (CF4) Flour, and Low-grade Flour (LGF)] were evaluated in aqueous suspension with and without nutritional supplementation. It was shown that GF and LGF are potential starch sources which do not require nutritional supplementation, exhibiting increases in α-amylase activity of 31.79 U/mL and 30.98 U/mL, respectively. This is the first report involving the application of Ganoderma applanatum in SmC for α-amylase production using synthetic and wheat milling by-products. The results also demonstrated that wheat milling by-products may also be used without supplementation for cell growth and enzyme production, contributing to the development of more sustainable forms of enzyme production through the innovative use of edible organisms with medicinal properties.

RESUMO: A farinha de trigo especial (FT), farinha de ervilha (FE) e farinha de trigo integral (FTI) foram misturadas em diferentes proporções, mediante a aplicação de um delineamento de misturas simplex-centroide, com a finalidade de avaliar o impacto dessas combinações nas propriedades físicas e sensoriais dos muffins. A interação entre FT e FTI produziu muffins com maior luminosidade na crosta e os muffins preparados com maior proporção de FE foram mais duros. O teste de ordenação foi realizado com objetivo de os julgadores ordenarem os experimentos de muffins mais preferidos de acordo com o atributo sabor. Os experimentos com as menores somas de ordem foram selecionados e submetidos ao teste de aceitação. No teste de aceitação foram avaliados os atributos de cor, sabor, textura e aparência global, em que os muffins obtiveram notas maiores que sete (“gostei moderadamente”), indicando uma boa aceitação de todos os experimentos. O experimento (a) (80% de FT, 10% de FE e 10% de FTI) foi escolhido por apresentar o maior conjunto de notas.

ABSTRACT: Wheat flour (WF), pea flour (PF) and whole wheat flour (WWF) were mixed in different ratios by applying a simplex-centroid mixture design, in order to evaluate the impact of these combinations on the physical and sensorial properties of muffins. The interaction between WF and WWF produced muffins with brighter crusts and muffins prepared with higher ration of PF were harder. The ranking test was performed with the objective of identifying the most preferred muffin experiments according to the flavor attribute. The experiments with the lowest ranking scores were selected and submitted to the acceptance test. In the acceptance test, the attributes of color, taste, texture and overall acceptance were evaluated, where muffins obtained scores higher than 7 (“moderately liked”), indicating good acceptance of all experiments. The experiment (a) (80% of WF, 10% of PF and 10% of WWF) was chosen for presenting the highest set of scores.

Resumo A extração de bioativos de plantas assistida por enzimas tem sido estudada como uma tecnologia verde alternativa. As carboidrases foram candidatas para melhorar o processo de extração de numerosos compostos bioativos de plantas. Os polifenóis são metabólitos secundários de plantas geralmente envolvidos em defesa contra diferentes tipos de estresse. A erva-mate (Ilex paraguariensis A. St.-Hil., Aquifoliaceae) é uma fonte natural desses compostos antioxidantes. O objetivo deste trabalho foi avaliar a extração de polifenóis auxiliada por enzimas para determinar as melhores condições para o uso, empregando metodologia de superfície de resposta (RSM). As variáveis independentes foram: temperatura (33,2 to 66,8°C), concentração enzimática (0 to 336 FGB / 100 g), tempo de reação (19 to 221 minutos) e pH (2,82 to 6,18). O uso de carboidrase aumentou a extração de polifenóis de 38,67% para 52,08%. Os resultados mostraram que todas as variáveis independentes foram significativas a nível linear e a temperatura e o pH não se mostraram significativos nos níveis quadráticos. As interações de temperatura e pH, tempo de reação e concentração da enzima e pH e concentração da enzima foram significativas. O modelo de regressão apresentou coeficiente de determinação (R2) próximo de 0,85 e ajustado perto de 0,45. Considerando-se os resultados deste estudo e a viabilidade industrial, as melhores condições para a obtenção de polifenóis de erva-mate são temperatura de 50,0°C, concentração de enzima 168 FGBU / g de folhas secas, 120 minutos de tempo de reação e pH 4,5. Este estudo é o primeiro relato de otimização da extração de polifenóis de erva-mate verde assistida por enzimas baseada em RSM.

Abstract The enzyme-assisted extraction of bioactive compounds from plants has been studied as an alternative green technology and the carbohydrases have been candidates to improve the extraction process of numerous such compounds from plants. Polyphenols are secondary plant metabolites, generally involved in the defense against different types of stress and yerba mate (Ilex paraguariensis A. St.-Hil., Aquifoliaceae) is a natural source of these antioxidant compounds. The aim of this work was to evaluate the enzyme-assisted extraction of polyphenols from green yerba mate employing response surface methodology (RSM), in order to determine the best extraction conditions. The independent variables were temperature (33.2 to 66.8 °C), enzyme concentration (0 to 336 FGBU/100g), reaction time (19 to 221 minutes) and pH (2.82 to 6.18). The use of carbohydrases increased the extraction of polyphenols from about 38.67% to 52.08%. The present results showed that all the independent variables were significant at the linear level and that temperature and pH were not significant at the quadratic level. The interactions of temperature and pH; enzyme and reaction time; and enzyme and pH were significant. The regression model presented a determination coefficient (R2) close to 0.85 and a fitted value close to 0.45. Considering the results of this study and their industrial viability, the best conditions for the extraction of polyphenols from green yerba mate are a temperature of 50.0 °C, enzyme concentration of 168 FGB/100 g, reaction time of 120 minutes and pH value of 4.50. This study was the first RSM-based report of the optimization of the enzyme-assisted extraction of total phenolic compounds from green yerba mate.

The aim of this study was to evaluate the probiotic properties of Pediococcus acidilactici B14 and to study its resistance in the gastrointestinal system when combined with Lactobacillus acidophilus ATCC 4356 and used in a potentially symbiotic aerated soy based dessert. P. acidilactici B14 showed some important probiotic characteristics such as survival rate of 45.9% at pH 2.5; 72.4% in 0.3% bile salts and 95.8% after gastrointestinal transit at pH 4.0. Tolerance against the antibiotics cephalexin, neomycin, vancomycin, cefotaxime and penicillin G was also observed. The strain inhibited antagonism against the following cultures: Escherichia coli ATCC 25922, Bacillus cereus ATCC 33018, Staphylococcus aureus ATCC 6538P and Salmonella sp. The mixed culture of P. acidilactici B14 with L. acidophilus ATCC 4356 showed a survival rate of 92.4% after the passage through the gastrointestinal system at pH 4.0. Furthermore, in the presence of the food matrix, an average increase in cell viability, after being subjected to the gastrointestinal system of 9.9% at pH 2.0 and 6.1% at pH 4.0, was observed. This characterized the adequacy of the associated culture as probiotic in the development of a functional food such as soy based aerated symbiotic dessert.

Enzymes obtained by fermentation processes offer a number of advantages and have been widely researched and used throughout the world. This study aimed to partially characterise an inulinase produced from palm and cassava peel. The enzyme was produced via the solid-state fermentation of Aspergillus japonicus URM5633. The optimal temperatures were 50ºC and 55ºC, and the optimal pH values were 5.2 and 3.4 for inulinase fermentatively produced from palm and cassava peel, respectively. The thermostability measurements for inulinase produced in palm showed that the relative activity remained below 100% until 30 minutes of stability for all temperatures, but reached 106.8% at a temperature of 50ºC after 60 minutes. Inulinase from the crude extract of cassava peel was pH stable and only decreased to 55% of the maximal activity over the course of the assay, suggesting that this enzyme can be used in inulinase production and can be utilized in food industries.

A new formulated product containing high yield of phytase from Schizophyllum sp., an important mushroom used for medicinal studies, was developed for application in feed industries and for future use in food processing. The enzyme presented a high activity yield 55.5 U/mL and 6240 U/gds in liquid and solid formulated product, respectively. It showed a good shelf-life in concentrated product, retaining 67.8% of its activity after 60 days of storage at room temperature and 90% of the activity was maintained in the liquid formulation after the same period. Powder bioformulated product maintained 77% of its activity after two months of storage, without the addition of chemical additives, which was named as a new bioformulated product containing high quantities of phytase. After separation and concentration steps, enzyme stability was monitored in two forms: liquid and solid. The liquid product was stable with the presence of manitol and polyethylene glycol at 1% (w/v), while solid product was the most stable product without the presence of chemical additives.

The aim of this study was to investigate the potential of lactic acid bacteria, bacilli and yeasts isolates to produce antimicrobial substances, especially against Gram-negative bacteria isolated from the animal faeces, searching for a new alternative to control the enteritis diseases in animal health. Evaluations were performed by microdilution in broth using crude extract obtained from the cultivation of 272 strains against Escherichia coli, S. Typhimurium, S. Enteritidis and Pseudomonas aeruginosa. Thirty-five lactic acid bacteria presented some antimicrobial action. One Lactobacillus isolate, named FLPB-1, was selected to studies the kinetics of the production. An important increase in the production was obtained when the producer strain had cell-to-cell contact with a Gram-positive inducer culture. Maximal production of antimicrobial compounds was obtained in the beginning of stationary phase and the addition of urea in the medium increased the inhibition rate.

Um modelo simplificado para descrever o crescimento fúngico durante a fermentação em polpa citric para a produção da fitase foi descrita pela primeira vez. Dados experimentais para a formação de biomassa foram ajustados a modelos clássicos de crescimento microbiano (Monod e Logístico). O modelo Monod previsto mostrou boa correlação aos resultados experimentais para a concentração de biomassa até 96 horas de fermentação. Parâmetros como rendimento de biomassa a partir de oxigênio (Y X/O), coeficiente de manutenção (m) e taxa específica de crescimento (µ) foram comparados mostrando uma boa correlação entre os dados e o modelo. Um método alternativo para a determinação de biomassa neste processo foi desenvolvido a partir de uma excelente correlação encontrado entre o crescimento microbiano e a formação da enzima.

A simplified model to describe fungal growth during citric pulp fermentation for phytase production was described for the first time. Experimental data for biomass growth were adjusted to classical mathematical growth models (Monod and Logistic). The Monod model predictions showed good agreement with the experimental results for biomass concentration during 96 hours of fermentation. Parameters such as yield of biomass from oxygen (Y X/O), maintenance coefficient (m) and specific growth rate (µ) were compared showing a good correlation between the data and the model. An alternative method for biomass determination in this process was developed since a great correlation was found between biomass growth and enzyme formation.

A busca por novas técnicas de cultivo in vitro vem sendo amplamente estudadas, visando otimizar e baixar o custo de produção das mudas que tenham interesse agronômico. O objetivo deste trabalho foi comparar diferentes sistemas de cultivo in vitro de Ananas comosus L. Merril. Para tanto, o crescimento in vitro de plântulas foi promovido em sistemas de biorreatores (B.I.B.® e R.I.T.A.®) com ciclo de imersão a cada 2 horas por 15 minutos e o sistema tradicional em frascos de 200 mL. Em todos os sistemas de cultivo, foram utilizadas solução nutritiva líquida MS, suplementado com 1 mg L-1 de BAP, 0,25 mg L-1 de ANA, 30 g L-1 de sacarose e 0,5 µL de Tween® 20, pH ajustado para 5,8 e autoclavagem a 120°C por 15 minutos. As culturas foram mantidas em sala de crescimento durante 30 dias, temperatura controlada de 25±2°C, sob luz branca fria (46,8 µmol.m-2.s-1), com 16 horas de fotoperíodo. O delineamento foi inteiramente casualizado, com três tratamentos, três repetições e dez plantas por estágios. Para os sistemas avaliados, o biorreator de imersão por bolhas apresentou os melhores resultados dentre as variáveis analisadas, com destaque ao número total de brotações, sendo 2,3 e 3,1 vezes superiores quando comparado com o sistema R.I.T.A.® e sistema tradicional respectivamente. Portanto, o sistema de imersão por bolhas torna-se um equipamento eficaz na produção de mudas de abacaxizeiro em larga escala.

The research for new techniques of in vitro cultivation is being object of many studies around the world, in order to optimize and decrease production costs of seedlings with agronomical interest. The main goal of this work was to compare different systems of in vitro cultivations using Ananas comosus L. Merril. So, the in vitro growth of the plantlets was promoted in two different bioreactors: Bioreactor of Immersion by Bubbles (B.I.B.®) and the Reactor of Temporary Immersion (R.I.T.A.®) with immersion cycle every 2 hours for 15 minutes and the traditional system in flasks with 200 mL. All cultivation systems used the MS liquid nutritive solution, supplemented with BAP (1 mgL-1), ANA (0.25 mgL-1), sucrose (30 gL-1) and Tween 20® (0.5 µL). The pH was adjusted to 5.8 and sterilized at 120°C for 15 minutes. The cultures were kept into a growth room during 30 days, with controlled temperature of 25±2°C, under white cold light (46.8 µmol.m-2.s-1), with photoperiod of 16 hours. The experimental design used was randomized, with three treatments, three repetitions and ten plants each stage. Among the evaluated systems, the BIB® presented the best results for the tested variables, mainly the total number of shoots, being 2.3 e 3.1 times superior when compared with the system R.I.T.A.® and the traditional consecutively. So the system of immersion by bubbles turns into an effective equipment to produce seedlings of pineapple in large scale.