Studies towards the stabilisation of a mushroom phytase produced by submerged cultivation.

A novel phytase from Ganoderma australe G24 was produced by submerged cultivation and recovery. Liquid and solid forms of phytase were developed; both types of product were formulated using different additives. Ganoderma australe G24 phytase was very stable in liquid form with NaCl and sodium acetate buffer. Solid form products were obtained by spray-drying using different polymers to encapsulate the phytase and the capsules obtained were analyzed by electron microscopy. Micrographs confirmed micro and nanoparticles formed with maltodextrin (300 nm to 7-8 µm) without the presence of agglomerates. The use of maltodextrin for solid formulation of G. australe G24 phytase is recommended, and resulted in good stability after the drying process and during storage (shelf life). Kinetic models of phytase inactivation in the microencapsulated powders over time were proposed for the different stabilizing additives. Inactivation rate constants, half-lives and D values (decimal reduction time) were obtained. Phytase encapsulated with maltodextrin remained stable after 90 days, with k 0.0019 day(-1) and a half-life (t1/2) of 367.91 days(-1).

Production of partially phosphorylated myo-inositol phosphates using phytases immobilised on magnetic nanoparticles.

Phytases of different origin were covalently bound onto Fe3O4 magnetic nanoparticles (12 nm). Binding efficiencies of all three phytases were well above 70% relative to the number of aldehyde groups available on the surface of the magnetic nanoparticles. Temperature stability for all three phytases was enhanced as a consequence of immobilisation, whereas pH dependence of enzyme activity was not affected. Maximum catalytic activity of the immobilised phytases was found at 60°C (rye), 65°C (Aspergillus niger) and 70°C (Escherichia albertii). The immobilised enzymes exhibited the same excellent substrate specificities and unique myo-inositol phosphate phosphatase activities as their soluble counterparts. However, the catalytic turnover number dropped drastically for the immobilised phytases. The amount of the desired partially phosphorylated myo-inositol phosphate isomer could be easily controlled by the contact time of substrate solution and immobilised enzymes. The immobilised phytases showed a high operational stability by retaining almost full activity even after fifty uses.

myo-inositol phosphate isomers generated by the action of a phytase from a malaysian waste-water bacterium.

Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P(5), D-I(2,3,4,5)P(4), D-I(2,3,4)P(3), D-I(2,3)P(2) to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P(5). Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P(5), D/L-I(1,2,4,5)P(4), D/L-I(1,2,4)P(3), D/L-I(2,4)P(2) to finally I(2)P.

The importance of lactic acid bacteria for phytate degradation during cereal dough fermentation.

Lactic acid fermentation of cereal flours resulted in a 100 (rye), 95-100 (wheat), and 39-47% (oat) reduction in phytate content within 24 h. The extent of phytate degradation was shown to be independent from the lactic acid bacteria strain used for fermentation. However, phytate degradation during cereal dough fermentation was positively correlated with endogenous plant phytase activity (rye, 6750 mU g(-1); wheat, 2930 mU g(-1); and oat, 23 mU g(-1)), and heat inactivation of the endogenous cereal phytases prior to lactic acid fermentation resulted in a complete loss of phytate degradation. Phytate degradation was restored after addition of a purified phytase to the liquid dough. Incubation of the cereal flours in buffered solutions resulted in a pH-dependent phytate degradation. The optimum of phytate degradation was shown to be around pH 5.5. Studies on phytase production of 50 lactic acid bacteria strains, previously isolated from sourdoughs, did not result in a significant production of intra- as well as extracellular phytase activity. Therefore, lactic acid bacteria do not participate directly in phytate degradation but provide favorable conditions for the endogenous cereal phytase activity by lowering the pH value.

Functional properties of phytate / Propriedades funcionais do fitato

Phytate [myo-inositol (1,2,3,4,5,6) hexakisphosphate], a naturally compound formed during maturation of plant seeds and grains, is a common constituent of plant-derived foods. The major concern about the presence of phytate in thediet is its negative effect on mineral uptake. Minerals of concern in this regardin clude Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, and Cu2+. Especially zinc and iron deficiencies were reported as a consequence of high phytate intakes. In addition, a negative effect on the nutritional value of protein by dietary phytate is discussed. Consumption of phytate, however, seems not to have only negative effects on human health. Dietary phytate was reported to prevent kidney stone formation, protect against diabetes mellitus, caries, atherosclerosis and coronary heart disease as well as against a variety of cancers. Furthermore, individual myo-inositol phosphate esters have been proposed to be metabolicall y active. D-myo-inositol(1,2,6)trisphosphate, for example, has been studied in respect to prevention of diabetes complications and treatment of chronic inflammations as well as cardiovascular diseases and due to its antiangiogenic and antitumour effects myo-inositol(1,3,4,5,6) pentakisphosphate was suggested as a promising compound for anticancer therapeutic strategies

El fitato, (1,2,3,4,5,6) hexafosfato de mio-inositol, compuesto que se forma naturalmente durante la maduración de las semillas ygranos, es un constituyente común de los alimentos vegetales. La mayor preocupación con la presencia de fitato es su efecto negativo en la absorción de minerales, particularmente Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, e Cu2+.Las deficiencias de zinc y hierro, fueron relacionadas con altas ingestas de fitato. Es discutido también su efecto negativo en el valor biológico de las proteínas. Sin embargo, el consumo de fitato parece no tener solamente efectos negativos para la salud humana. Fue descripto, por ejemplo, un efecto protector del fitato contra la formación de cálculos renales, contra la diabetes mellitus, formación de caries, ateriosclerosis y enfermedades coronarias, como también contra una gran variedad de tipos de tumores malignos. Además, ha sido propuesto que, individualmente, algunos ésteres de fosfato de mio-inositol sean metabólicamente activos. Hay estudios relacionando el (1,2,6) trisfosfato de D-mio-inositol con la prevención de complicaciones de la diabetes y con el tratamiento de inflamaciones crónicas y de enfermedades cardiovasculares; el (1,3,4,5,6)pentafosfato de mio-inositol, debido a sus efectosanti-angiogénicos y anti-tumorales, fue sugerido como un compuesto promisor en las estratégias terapéuticas contra el cáncer

O fitato, (1,2,3,4,5,6) hexafosfato de mio-inositol], composto que ocorre naturalmente e é formado durante a maturação de sementes e grãos, é um constituinte comum de alimentos vegetais. A maior preocupação com a presença de fitato na dieta é seu efeito negativo na absorção de minerais, particularmente Zn2+, Fe2+/3+, Ca2+, Mg2+, Mn2+, e Cu2+. As deficiências de zinco e de ferro, em especial, foram relacionadas com altas ingestões de fitato. É discutido, também, o efeito negativo do fitato no valor biológico de proteínas. Por outro lado, o consumo de fitato parece não ter somente efeitos negativos para a saúde humana. Foi descrito, por exemplo, um efeito protetor do fitato contra a formação de cálculos renais, contra diabetes Mellitus, formação de cáries, aterosclerose e doença coronariana, bem como contra uma grande variedade de tipos de tumores malignos. Além disso, foi proposto que, individualmente, alguns ésteres de fosfato de mio-inositol sejam metabolicamente ativos. Assim, há estudos relacionando o (1,2,6) trisfosfato de D-mio-inositol com a prevenção das complicações do diabetes e com o tratamento de inflamações crônicas e de doenças cardiovasculares; o(1,3,4,5,6) pentafosfato de mio-inositol, devido a seus efeitos anti-angiogênicos e anti-tumorais, foi sugerido como um composto promissor nas estratégias terapêuticas contra o câncer

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 percent 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.