Abstract C-phycocyanin is a potential nutraceutical/pharmaceutical candidate with functionalities that are better documented than those of health foods in general. Studies have demonstrated that C-phycocyanin has antioxidant and antitumor properties and potential activities against other diseases. However, its large-scale purification remains problematic and expensive. The aim of this work was to establish the best process for obtaining C-phycocyanin of different purities for different applications. The first step of this study was the maximization of the ultrafiltration process. Under the best ultrafiltration conditions, we evaluated the application of ultrafiltration, precipitation, and ion exchange chromatography (expanded and fixed beds) in different sequences and their effects on C-phycocyanin recovery and purification. It was possible to obtain C-phycocyanin that could be used as food dye with purity of 0.95 employing only diafiltration/ultrafiltration. With different process configurations, we obtained extracts that could be used as cosmetic dye with purity of 2.1 and biomarkers with purity of 3.0, and for therapeutic and biomedicine applications with an analytical grade (purity > 4.0).
The interest in carotenoid production from natural sources has increased based in their possible use as dyes and their powerful biological antioxidant capacity. This study evaluated the use of rice parboiling wastewater (RPW) as an alternative substrate for the bioproduction of carotenoids using the yeast Phaffia rhodozyma and found it to be promising as the only source of nutrients, reaching a concentration of 0.6 μg mL-1 (259.1 μg g-1) in 48 h. To increase the potential use of this industrial effluent, a study of supplementation was carried out to enhance the production of carotenoids using the methodology of experimental design (a 2IV 6-2 fractional factorial design sequenced by a 24 central composite design). The conditions for maximizing the production of carotenoids were (g L-1) malt extract (16.25), peptone (8.75), sucrose (15) and rice parboiling wastewater (87.5), with a pH of 5 at 25 °C and 150 rpm for 144 h, which produced a concentration 5.3 μg mL-1 (628.8 μg g-1).
O interesse pela produção de carotenoides por fontes naturais é crescente, em decorrência da possibilidade de atuar como corante e sua capacidade antioxidante biológica potente. Neste estudo, avaliou-se o uso da água de parboilização do arroz, como substrato alternativo para a bioprodução de carotenoides, usando a levedura Phaffia rhodozyma que, como única fonte de nutrientes, foi promissora, alcançando 0,6 μg mL-1 (259,1 μg g-1) em 48 h. Para aumentar o potencial de aproveitamento desse efluente industrial, um estudo da suplementação do meio de produção foi realizado para incrementar a obtenção dos carotenoides, utilizando a metodologia do planejamento fatorial (um planejamento fatorial 2IV 6-2 sequenciado por um planejamento fatorial completo 24). As condições maximizadas de produção de carotenoides foram (g L-1): extrato de malte, 16,25; peptona, 15 e água de parboilização de arroz, 87,5, com pH 5, a 25 ºC, 150 rpm por 144 h, alcançando 5,3 μg mL-1 (628,8 μg g-1).
C-phycocyanin from Spirulina platensis was purified in aqueous two-phase systems (ATPS) of polyethylene glycol (PEG)/potassium phosphate, varying the molar mass of the PEG. Results using a full factorial design showed that an increase in the concentration of salt and decrease in the concentration of PEG caused an increment in the purification factor for all the ATPS studied. Optimization of the conditions of the purification was studied using a central composite rotatable design for each molar mass of PEG. The ATPS composed of 7% (w/w) PEG 1500 or 4% (w/w) PEG 8000 (g/gmol) and 23 or 22.5% (w/w) of phosphate resulted a purification factor of 1.6-fold for C-phycocyanin, with total and 57% recovery, respectively. Process conditions were optimized for the purification factor for the system with PEG 1500. The ATPS with 4% (w/w) PEG 4000 or 4% (w/w) PEG 6000 and 21% (w/w) phosphate resulted purification factors of 2.1 and 2.2-fold, recovering 100% and 73.5%, respectively of C-phycocyanin in the top phase.
The demand for functional foods, including milk fermented with probiotic microbial cultures, has increased the interest in research to maintain these microorganisms viable in the product. Thus the objective of this work was to formulate strawberry flavoured fermented milk with added potentially beneficial prebiotics and probiotics, and with suitable sensory characteristics. A 2³ experimental design was used to optimize the product flavour, evaluating the addition of sucrose (50, 85 and 120 g.L-1), the natural dye cochineal carmine (0.5, 1.0 and 1.5 g.L-1) and a natural strawberry aroma (0.10, 0.25 and 0.40 g.L-1). Different concentrations of Bio Rich culture (L. acidophilus, Bifidobacterium and S. thermophilus) were tested (1.00, 0.50 and 0.33 g.L-1) ranking the products in according to sensory preference. Two fructo-oligosaccharide syrup concentrations (50 and 100 g.L-1) were evaluated according to their sensory acceptability and microbiological analyses. A strawberry flavoured fermented dairy beverage was developed using (g.L-1) 0.33 culture, 120 sucrose, 1.5 dye, 0.4 aroma and 100 fructo-oligosaccharide syrup. The increase in concentration of the prebiotics positively influenced product preference, with acceptance rates of 88% and increased microbiological counts of L. acidophilus and Bifidobacterium at 1.7 × 10(7) and 4.8 × 10(6) CFU.mL-1 for 100 g.L-1 of prebiotics.
A procura por alimentos funcionais, como leites fermentados com culturas microbianas probióticas, aumentou o interesse em pesquisas para manter estes micro-organismos viáveis no produto. Neste sentido, o objetivo deste trabalho foi formular um leite fermentado sabor morango agregado de benefícios potencialmente prebióticos e probióticos, apresentando características sensoriais adequadas. Um planejamento fatorial 2³ com ponto central foi realizado para otimizar a saborização do produto, avaliando a adição de sacarose (50, 85 e 120 g.L-1), corante natural carmim cochonilha (0,5; 1,0 e 1,5 g.L-1) e aroma natural de morango (0,10; 0,25 e 0,40 g.L-1). Diferentes concentrações da cultura Bio Rich (L. acidophilus, Bifidobacterium e S. thermophilus) foram testadas (1,00; 0,50 e 0,33 g.L-1), ordenando-se sensorialmente conforme a preferência. Concentrações do xarope de fruto-oligossacarídeos (50 e 100 g.L-1) foram avaliadas por meio de aceitação sensorial e análises microbiológicas. Um leite fermentado sabor morango utilizando, em g.L-1, 0,33 de cultura, 120 de sacarose, 1,5 de corante, 0,4 de aroma e 100 de xarope de fruto-oligossacarídeos, foi desenvolvido. O incremento da concentração de prebiótico influenciou positivamente na preferência do produto, apresentando índice de aceitação de 88%; microbiologicamente, aumentou a contagem de L. acidophilus e Bifidobacterium, sendo 1,7 × 10(7) e 4,8 × 10(6) UFC.mL-1, respectivamente, com 100 g.L-1 de prebiótico.
The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).
β-galactosidase is an important enzyme that acts on lactose hydrolysis and can be used to obtain food for consumers intolerant to this disaccharide. The yeast Kluyveromyces marxianus presents a good growth yield, is a safe microorganism for industrial applications and has been used for enzyme production using the submerged process. The β-galactosidase obtained was fractionated with ammonium sulphate and characterized with respect to its optimum temperature and pH, thermal stability and its D and z values, as well as its kinetic and thermodynamic parameters. The optimum temperature and pH were 45-50 °C and 7.0, respectively. The activation energy and the deactivation reaction of the enzymatic reaction were, respectively, 9.8 kcal.mol-1 and 64.2 kcal.mol-1. The Km and Vmax values obtained were 3.7 mM and 99.0 U.mL-1, respectively. The Gibbs free energy decreased with increasing temperature and the enzyme was more stable at 30 °C (∆G* = 106.8 kJ.mol-1). The enthalpy was 313.04 kJ.mol-1 and entropy 0.68 kJ.mol.k-1. The D value confirmed that the enzyme was more stable at temperatures around 30 °C (D = 11,513.0 min) and the z value was 5.8 °C.
A β-galactosidase é uma enzima importante que atua na hidrólise da lactose, podendo ser empregada na obtenção de alimentos destinados a consumidores intolerantes a esse dissacarídeo. A levedura Kluyveromyces marxianus apresenta bom rendimento de crescimento, além de ser um micro-organismo seguro em aplicações industriais, e tem sido utilizada para produção da enzima por cultivo submerso. A β-galactosidase obtida foi fracionada com sulfato de amônio e caracterizada quanto a temperatura e pH ótimos, estabilidade térmica, valores D e z, e parâmetros cinéticos e termodinâmicos. A temperatura e pH ótimos foram de 45-50 °C e 7,0, respectivamente. A energia de ativação da reação enzimática foi de 9,8 kcal.mol-1 e da reação de desativação, 64,2 kcal.mol-1. Os valores de Km e Vmax obtidos foram de 3,7 mM e 99,0 U.mL-1, respectivamente. A energia livre de Gibbs reduziu com o aumento de temperatura, sendo a enzima mais estável a 30 °C (∆G* = 106,8 kJ.mol-1). A entalpia foi de 313,04 kJ.mol-1 e a entropia 0,68 kJ.mol-1.k-1. O valor D confirmou que a enzima foi mais estável em temperaturas próximas de 30 °C (D = 11.513,0 min) e o valor z foi de 5,8 °C.
The main goal of this work was to study the biodegradation of phenol in batch mode by a filamentous fungus isolated from a contaminated site in Southern Brazil. A better performance was obtained by previous adaptation of the microorganism to the toxic chemical. A 2³ experimental design was proposed and it could be observed total phenol degradation in 72 h using 500 mg L-1 glucose, inoculum of 20% and agitation of 200 rpm, resulting a biodegradation rate of 3.76 mg L-1 h-1. In relation to phenol tolerance, Aspergillus sp. LEBM2 was able to consume up to 989 ± 15 mg L-1.
Isoamyl butyrate production was investigated using free and immobilized lipases by esterification of butyric acid with isoamyl alcohol in a solvent-free system and in an organic media. Among the enzymes studied, Lipozyme TL IM was found to be the most active catalyst in n-hexane as a solvent. The effects of different solvents and the amount of water added on conversion rates were studied. A maximum conversion yield of 80% in n-hexano at 48 h was obtained under the following conditions: 3 g L-1 of Lipozyme TL IM, 30 ºC, 180 rpm of agitation, isoamyl alcohol to butyric acid molar ratio of 1:1 and acid substrate concentration of 0.06 M.
Amyloglucosidase enzyme was produced by Aspergillus niger NRRL 3122 from solid-state fermentation, using deffated rice bran as substrate. The effects of process parameters (pH, temperature) in the equilibrium partition coefficient for the system amyloglucosidase - resin DEAE-cellulose were investigated, aiming at obtaining the optimum conditions for a subsequent purification process. The highest partition coefficients were obtained using 0.025M Tris-HCl buffer, pH 8.0 and 25ºC. The conditions that supplied the highest partition coefficient were specified, the isotherm that better described the amyloglucosidase process of adsorption obtained. It was observed that the adsorption could be well described by Langmuir equation and the values of Qm and Kd estimated at 133.0 U mL-1 and 15.4 U mL-1, respectively. From the adjustment of the kinetic curves using the fourth-order Runge-Kutta algorithm, the adsorption (k1) and desorption (k2) constants were obtained through optimization by the least square procedure, and the values calculated were 2.4x10-3 mL U-1 min-1 for k1 and 0.037 min-1 for k2 .
A enzima amiloglicosidase foi produzida por Aspergillus niger NRRL 3122 através de fermentação em estado sólido, tendo como substrato farelo de arroz desengordurado. Os efeitos dos parâmetros de processo (pH e temperatura) no coeficiente de partição no equilíbrio, para o sistema amiloglicosidase - resina DEAE-celulose foram investigados, com o objetivo de se obter as melhores condições para um posterior processo de purificação. Os maiores coeficientes de partição foram obtidos usando tampão Tris-HCl 0,025M pH 8,0 e 25°C. Determinadas as condições que forneceram o maior coeficiente de partição obteve-se a isoterma que melhor descrevia o processo de adsorção de amiloglicosidase. Foi verificado que adsorção pode ser bem descrita pela equação de Langmuir e os valores de Qm e Kd foram estimados em 133,0 U mL-1 e 15,4 U mL-1 respectivamente. A partir do ajuste das curvas cinéticas utilizando o método de Runge-Kutta de quarta ordem, obteve-se as constantes de adsorção (k1) e dessorção (k2) através da otimização pelo método dos mínimos quadrados, os valores encontrados foram 2,4x10-3 mL U-1 min-1 para k1 e 0,037 min-1 para k2.
Recently lipases have been increasing in prominence due to its wide industrial application. The lipase production can be influenced by different variables such as the producing microorganism, carbon sources, aeration and agitation conditions, inductor type and the geometry of the reactor. Biosurfactants are composites of surface active produced by microbial cells which reduce superficial and interfacial tensions. The objective of this study was to verify the influence of different process variables in the lipase production during a fermentative process. The results showed that the concomitant production of lipases and biosurfactant was possible in different cultivation conditions.
The optimization of a traditional technique of cellular disruption by abrasion was carried out and a process using ultrasonic waves associated with glass pearls to extract beta-galactosidase from Kluyveromyces marxianus proposed. In the first case, the effects of the diameter and weight of the pearls in relation to the volume of cellular suspension and amount of time for cellular disruption were evaluated. The efficiency of the new process of cellular disruption was evaluated by varying the length of time of sonification and comparing with the method of abrasion under the same conditions. The proposed method can be efficiently applied to obtain beta-galactosidase at laboratory scale.