Frederico José Vieira Passos

Xylose metabolism in Debaryomyces hansenii UFV-170. Effect of the specific oxygen uptake rate.

The new yeast Debaryomyces hansenii UFV‐170 was tested in this work in batch experiments under variable oxygenation conditions. To get additional information on its fermentative metabolism, a stoichiometric network was proposed and checked through a bioenergetic study performed using the experimental data of product and substrate concentrations. The yeast metabolism resulted to be practically inactive under strict oxygen‐limited conditions ( qO2 = 12.0 mmolO2 C‐molDM−1 h−1), as expected by the impossibility of regenerating NADH2+. Significant fractions of the carbon source were addressed to both respiration and biomass growth under excess oxygen levels ( qO2 ≥ 55.0 mmolO2 C‐molDM−1 h−1), thus affecting xylitol yield ( YP/S = 0.41–0.52 g g−1). Semi‐aerobic conditions ( qO2 = 26.8 mmolO2 C‐molDM−1 h−1) were able to ensure the best xylitol production performance ( Pmax = 76.6 g L−1), minimizing the fractions of the carbon source addressed either to respiration or biomass production and increasing YP/S up to 0.73 g g−1. An average P/ O ratio of about 1.0 molATP molO−1 allowed estimation of the main kinetic‐bioenergetic parameters of the biosystem. The overall ATP requirements of biomass were found to be particularly high and dependent on the oxygen availability in the medium as well as on the physiological state of the culture. Under semi‐aerobic and aerobic conditions, they varied in the ranges 13.5–15.4 and 9.74–10.2 molATP C‐molDM−1, respectively, whereas during the best semi‐aerobic bioconversion they progressively increased from 5.68 to 24.7 molATP C‐molDM−1. After a starting phase of adaptation to the medium, the cell achieved a phase of decelerated growth during which its excellent xylose‐to‐xylitol capacity kept almost constant after 112 h up to the end of the run.

A mechanistical mathematical model to predict lactose hydrolysis by β-galactosidase in a permeabilized cell mass of Kluyveromyces lactis: validity and sensitivity analysis

Abstract The kinetics of lactose hydrolysis by intracellular β-galactosidase in a preparation of Kluyveromyces lactis cells permeabilized with ethanol was assessed in the presence of lactose and its hydrolysis reaction products galactose and glucose. Enzyme inhibition by the reaction products was tested using different concentrations of galactose (1, 5, 10, 20, 30 and 40 mM) and glucose (10, 30 and 50 mM) and a combination of both. Galactose was a competitive inhibitor. The competitive or non-competitive nature of the inhibition was defined by the smallest value presented by the residual sum of squares of regression as determined using the Marquardt iterative method of the non-linear regression (NLIN) program of the statistical analysis system (SAS). The kinetic constants V max , K m and K i , with values corresponding to 0.291±0.01 mM min −1 ; 2.536±0.412 mM and 10.88±8.86 mM, respectively, were also estimated by the same program. Enzyme activity was not affected by glucose at the concentrations tested. Glucose affected enzyme activity only when galactose was present. A mechanistic mathematical model was developed to describe lactose hydrolysis, taking into consideration the inhibitory effect of galactose. Sensitivity analysis of the coefficients of the model proposed was performed. Varying estimated K m and K i values by ±20% had no effect on lactose hydrolysis kinetics. However, with a 20% increase in the estimated V max value, the model more closely approximated the experimental data for initial concentrations of 30 mM glucose and 10 mM galactose. Model simulation closely approximated experimental data from lactose hydrolysis in a 5% phosphate-buffered lactose solution as well as in skim milk.

Production of pectin lyase by Penicillium griseoroseum in bioreactors in the absence of inducer

Penicillium griseoroseum was grown in bioreactors on mineral medium supplemented with yeast extract and sucrose. The influence of inoculum and carbon source concentrations, aeration and pH on pectin lyase (PL) production, as well as the capacity of P. griseoroseum to produce PL when grown on sugar cane syrup as carbon source were evaluated. Inoculum concentration did not influence PL production. Production was higher in non-aerated than in aerated medium. The best results were obtained using 60 mM sucrose at pH 6.3-7.2. Production using cane syrup 25% (v/v), without yeast extract supplement, was equal to that obtained under the conditions cited above.

ATP - bioluminescence as a technique to evaluate the microbiological quality of water in food industry

ATP-bioluminescence was used to evaluate the microbiological quality of water samples collected from the water supply, the water treatment system and from a dairy plant, including ammonia-cooling water and industrial water. For industrial water, there was relation between the ATP-bioluminescence technique and microbial count. There were no differences (p>0.05) between water supply and ammonia-cooling water samples for total and free ATP concentrations nor for the microbial counts. Different microbial ATP concentrations were found for these water samples. The results suggested that the physical chemical quality of ammonia cooling water decreased the RLU measurements slightly. It could be concluded that the total ATP concentration was the most effective technique to evaluate the microbiological quality of water used in the food indsutry by ATP-bioluminescence.

Influence of different mixing and aeration regimens on pectin lyase production by Penicillium griseoroseum

Abstract Penicillium griseoroseum was grown in a 20 l fermenter under different degrees of mixing and aeration to evaluate the effect of these variables on fungal macroscopic morphology and growth, as well as on production of pectin lyase (PL). A 2 4−1 fractional factorial design was used. Growth time was divided into two periods: the initial phase, comprised the first 25 h and the final phase, 25 to 60–72 h of growth. Pellet formation was the predominant growth form and no morphological differences were observed between treatments. Increasing the initial mixing rate from 50 to 200 rpm favored increased PL activity (U/g), but increasing the final mixing rate from 200 to 300 rpm did not influence it significantly. Combined analyses of the initial and final aeration rates showed that only the combination of no initial aeration and a final aeration rate of 20 l/min reduced PL activity (U/g).

Pectin lyase production by Penicillium griseoroseum grown in sugar cane juice in repeated batch cultures

Penicillium griseoroseum cultured in the presence of sucrose and yeast extract produces pectin lyase (EC 4.2.2.10) (PL) in the absence of its natural inducer pectin. This fungus was cultured in a fermenter at an aeration rate of 0.5 l/min for the first 25 h and 1.0 l/min for the remainder of the culture period, and at a stirring rate of 200 rev/min for the entire culture period. Fungal spores were inoculated directly into the fermenter at a final concentration of 5 × 104 spores/ml. The fungus was cultured in minimal medium supplemented with powdered dehydrated sugar cane juice, producing PL without added yeast extract. Maximum PL activity (0.067 IU/ml) was obtained after 65 h in batch culture. Pellet morphology of the mycelia made it possible to carry out three cycles of repeated batch culture. The same medium was used for renewal as for the single batch culture. The initial cycle was 53 h, after which approximately 0.103 IU/ml of PL was obtained. After this period, the medium was renewed and fermentation continued for two more cycles, which lasted approximately 20 h. Activity of PL obtained in the second cycle was approximately 0.118 IU/ml and in the third, approximately 0.109 IU/ml.

Cloning and expression of a functional core streptavidin in Pichia pastoris: Strategies to increase yield

Streptavidin is widely used as an analytical tool and affinity tag together with biotinylated surfaces or molecules. We report for the first time a simple strategy that yields high biomass of a Pichia pastoris strain containing a methanol induced core streptavidin (cStp) gene. Three factors were evaluated for biomass production: glycerol concentration, aeration, and feed flow rates in a bioreactor. Recycling of recombinant cells, either free or immobilized, was investigated during induction. Concentration of 2.0 M glycerol, feeding flow rate of 0.11 mL min−1, and aeration by air injection dispersed with a porous stone combined with agitation at 500 rpm were the set of conditions resulting into maximum biomass yield (150 g L−1). These parameters yielded 4.0 g L−1 of cStp, after 96 h of induction. Recombinant biomass was recycled twice before being discarded, which can reduce production costs and simplify the process. Immobilized P. pastoris biomass produced 2.94 and 1.70 g L−1 of cStp in the first and second induction cycle, respectively. Immobilization and recycling of recombinant P. pastoris biomass opens new possibilities as a potential strategy to improve volumetric productivity for heterologous protein expression.

Interference of Some Organic Substances and Microorganisms Adhered to Stainless Steel in ATP- bioluminescence Measurement

Adhesion of organic substances and microorganisms on stainless steel surface was evaluated. The log RLU measurement was affected at a lower or higher degree, by type or concentration of the organic substances (casein, lipid, sucrose and their combinations) adhered to surface. However, if the samples analyzed were from the food processing surfaces, they would have been considered to be under satisfactory hygienic conditions with log RLU 0.05) on ATP-bioluminescence measurements. However, the surfaces adhered with 2.9x103 DMC.cm-2 of B. subtilis spores and organic substances showed an increase in the log RLU measurement compared to surfaces containing only the organic substances.

Isolamento de esporos de equipamentos de abatedouros avícolas e avaliação de sua resistência a sanificantes químicos

Com o objetivo de fornecer subsidios para o controle de microrganismos em abatedouro de aves, avaliou-se a acao dos agentes quimicos sanificantes comprovadamente mais eficientes (hipoclorito de sodio, acido peracetico, e dicloroisocianurato de sodio) em suspensoes de esporos de bacterias aerobias mesofilas e termofilas, isoladas de equipamentos de abatedouro de aves. Pelo teste de Duncan, constatou-se que dentre os esporos isolados o mais resistente obteve 1,5 e 1,2 RD quando em contato com o hipoclorito de sodio e ao acido peracetico com 30 minutos de tempo de contato, que foram os sanificantes mais eficientes (P > 0,05). O dicloroisocianurato de sodio proporcionou 0,1 RD em 30 minutos de tempo de contato com a mesma suspensao de esporos. Os esporos isolados apresentaram diferentes resistencias aos sanificantes avaliados, indicando a necessidade de uma selecao criteriosa de agentes quimicos para o procedimento de sanificacao, sendo importante um rodizio entre os sanificantes mais eficientes testados, que foram o hipoclorito de sodio e o acido peracetico.