Purification and characterisation of an extracellular phytase from Aspergillus niger 11T53A9

An extracellular phytase from Aspergillus niger 11T53A9 was purified about 51-fold to apparent homogeneity with a recovery of 20.3% referred to the phytase activity in the crude extract. Purification was achieved by ammonium sulphate precipitation, ion chromataography and gel filtration. The purified enzyme behaved as a monomeric protein with a molecular mass of about 85 kDa and exhibited maximal phytate-degrading activity at pH 5.0. Optimum temperature for the degradation of phytate was 55°C. The kinetic parameters for the hydrolysis of sodium phytate were determined to be K M = 54 µmol l-1 and k cat = 190 sec-1 at pH 5.0 and 37°C. The purified enzyme was rather specific for phytate dephosphorylation. It was shown that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 to finally Ins(2)P.

Evaluation of viability of Aspergillus flavus and aflatoxins degradation in irradiated samples of maize

One of the currently most important fungi in stored grains is Aspergillus flavus, which produce aflatoxins. This fungus can grow on diverse substrates and represents a serious public health and animal nutritional problem. Therefore, the study of techniques that can be applied to the control of aflatoxins is of great importance. The objective of the present study was to determine the effects of gamma radiation on the growth of Aspergillus flavus Link and on degradation of aflatoxin B1 and B2 (AFB1 and AFB2) at a relative humidity of 97 99% and a water activity (Aw) of 0.88-0.94. Samples of corn grains were irradiated using a cobalt 60 source emitting gamma rays at doses of 2, 5 and 10 kGy. Irradiation was found to be effective in reducing the number colony-forming units of A. flavus, per gram, in the corn samples analyzed. In addition, the fluorescent viability test (fluorescein diacetate and ethidium bromide) revealed a decrease in the number of viable cells with increasing irradiation doses and three different fluorescence patterns. Furthermore, irradiation induced a partial reduction in AFB1 and AFB2 levels at the doses of 2 and 5 kGy, whereas complete degradation of aflatoxins was observed in the assay employing 10 kGy.

Um dos fungos mais importantes atualmente em grãos armazenados é o Aspergillus flavus, o qual produz aflatoxinas. Este fungo pode crescer em diversos substratos e representa uma séria preocupação em saúde pública e nutrição animal. Portanto, o estudo de técnicas que possam ser aplicadas no controle das aflatoxinas é de grande importância. Assim sendo, o objetivo do presente trabalho foi estudar os efeitos da radiação gama no crescimento de Aspergillus flavus Link e na degradação das aflatoxinas B1 e B2, (AFB1 e AFB2) em umidade relativa (UR) de 97-99% e atividade de água (Aa) de 0,88-0,94. Amostras de grãos de milho foram irradiadas, utilizando-se uma fonte de Cobalto 60, emissora de raios gama, com as doses de 2; 5 e 10 kGy. A irradiação foi efetiva na redução do número de Unidades Formadoras de Colônias de A. flavus, por grama, nas amostras de milho analisadas. Adicionalmente, o teste de viabilidade fluorescente (solução de diacetato de fluoresceína e brometo de etídio) revelou diminuição no número de células viáveis com o aumento das doses de irradiação e três diferentes padrões de fluorescência. Além disso, a irradiação induziu a uma parcial redução dos níveis de AFB1 e AFB2, nas doses de 2 e 5 kGy, ao passo que uma completa degradação das aflatoxinas foi observada no ensaio empregado com 10 kGy.

Bacterial phytase: potential application, in vivo function and regulation of its synthesis

The stepwise release of phosphate from phytate, the major storage form of phosphate in plant seeds and pollen, is initiated by a class of enzymes that have been collectively called phytases. The classification is solely due to the in vitro capability of these enzymes to accept phytate as a substrate. Phytases have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. They have a wide distribution in plants, microorganisms, and in some animal tissues. Due to several biological characteristics, such as substrate specificity, resistance to proteolysis and catalytic efficiency, bacterial phytases have considerable potential in commercial applications. In bacteria, phytase is an inducible enzyme and its expression is subjected to a complex regulation, but phytase formation is not controlled uniformly among different bacteria. It was suggested that phytase is not required for balanced growth of bacterial cells, but may be synthesised in response to a nutrient or energy limitation.

Bacterial phytase: potential application, in vivo function and regulation of its synthesis

The stepwise release of phosphate from phytate, the major storage form of phosphate in plant seeds and pollen, is initiated by a class of enzymes that have been collectively called phytases. The classification is solely due to the in vitro capability of these enzymes to accept phytate as a substrate. Phytases have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. They have a wide distribution in plants, microorganisms, and in some animal tissues. Due to several biological characteristics, such as substrate specificity, resistance to proteolysis and catalytic efficiency, bacterial phytases have considerable potential in commercial applications. In bacteria, phytase is an inducible enzyme and its expression is subjected to a complex regulation, but phytase formation is not controlled uniformly among different bacteria. It was suggested that phytase is not required for balanced growth of bacterial cells, but may be synthesised in response to a nutrient or energy limitation.

Bacterial phytase: potential application, in vivo function and regulation of its synthesis

The stepwise release of phosphate from phytate, the major storage form of phosphate in plant seeds and pollen, is initiated by a class of enzymes that have been collectively called phytases. The classification is solely due to the in vitro capability of these enzymes to accept phytate as a substrate. Phytases have been studied intensively in recent years because of the great interest in such enzymes for reducing phytate content in animal feed and food for human consumption. They have a wide distribution in plants, microorganisms, and in some animal tissues. Due to several biological characteristics, such as substrate specificity, resistance to proteolysis and catalytic efficiency, bacterial phytases have considerable potential in commercial applications. In bacteria, phytase is an inducible enzyme and its expression is subjected to a complex regulation, but phytase formation is not controlled uniformly among different bacteria. It was suggested that phytase is not required for balanced growth of bacterial cells, but may be synthesised in response to a nutrient or energy limitation.

The application of PCR in the detection of mycotoxigenic fungi in foods

It is estimated that 25 to 50% of the crops harvested worldwide are contaminated with mycotoxins. Because of the toxic and carcinogenic potential of mycotoxins, there is an urgent need to develop detection methods that are rapid and highly specific. The highly advanced physico-chemical methods for the analysis of mycotoxins in use, have the disadvantage that highly sophisticated clean-up and/or derivatization procedures must be applied. An alternative could be the detection of the mycotoxigenic moulds themselves, especially as molecular techniques have been introduced recently as powerful tools for detecting and identifying fungi. PCR methods for the detection of aflatoxigenic Aspergilli, patulin-producing Penicillum and trichothecene- as well as fumonisin-producing Fusaria strains have been described. The usefulness of the PCR methods developed so far to monitor quality and safety in the food an feed industry was already demonstrated. Thus, PCR may be applied to the screening of agricultural commodities for the absence of mycotoxin producers prior to or even after processing. Negative results in this assay indicate that a sample should be virtually free of mycotoxins. Only the positive samples left must be analyzed for the presence of mycotoxins using physico-chemical standard methods. This review does not only summarize the so far developed qualitative and quantitative PCR assays for the detection of mycotoxigenic fungi in agricultural commodities, foods and animal feeds, but describes also strategies to develop new specific PCR assays for such a detection.