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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.

Citric pulp is an agro-industrial residue from the citrus processing industry with low inorganic phosphorus content applied in animal feed. A new bioprocess was developed to produce and purify a new phytase generated on citric pulp fermentation by Aspergillus niger FS3. The phytase was purified by cationic-exchange, anionic-exchange chromatography and chromatofocusing steps. From SDS–PAGE analysis, the molecular weight of the purified phytase was calculated to be 108 kDa. The phytase had an optimum pH of 5.0–5.5 and an optimum temperature of 60°C. The phytase displayed high affinity for phytate, and the Km was 0.52 mM. The purified phytase was sufficiently able to withstand pelleting temperatures, retaining sufficiently high phytate-degrading activity.

The aim of this study was to verify the effects of gamma radiation process on the fungal DNA and the application of PCR in the detection of Aspergillus flavus in irradiated maize grains. The samples were inoculated with a toxigenic strain and incubated under controlled conditions of relative humidity, water activity, and temperature for 15 days. After incubation, the samples were treated with gamma radiation with doses of 5 and 10 kGy and individually analyzed. The use of PCR technique showed the presence of DNA bands of Aspergillus flavus in all irradiated samples that showed no fungal growth in agar medium.

Qualitative and quantitative polymerase chain reaction-based methods to detect genetically modified soy (RoundupReady™ soy) and maize (Bt176 Maximizer™ maize, Bt11 maize, MON810 YieldGard™ corn, T25 LibertyLink™ maize) were applied to processed foods sold commercially in Brazil. From 2000 to 2005, 100 food products containing maize and 100 food products containing soy were analysed every single year. The presence of genetically modified soy increased steadily from 13% in 2000 to 78% in 2005. The number of food products containing genetically modified soy in a proportion above 1.0% on the ingredient level, the threshold for labelling according to Brazilian legislation, increased from 11% in 2000 to 36% in 2005. No clear trend was found within maize containing food products. Eight to eleven percent were shown to consist of material derived from genetically modified maize and 4–6% were found to contain more than 1% of genetically modified maize.

Qualitative and quantitative polymerase-chain-reaction-based methods to detect genetically modified (gm) soy (RoundupReadyTM soy) and maize (Bt176 Maximizer maize; Bt11 maize, MON810 Yield Gard corn, T25 LibertyR Link maize) were applied to processed foods sold commercially in Brazil. In total 100 foods containing maize and 100 foods containing soy were analysed in 2000 and again in 2001. In 2000, 13% of the soy containing products and 8% of those containing maize were shown to consist of material derived from the corresponding genetically modified organisms. In five samples of the products containing gm soy 4% of the soy derived material was identified as coming from gm soybeans, whereas in the case of products containing gm maize five samples of the maize derived material were found to contain 4% of gm maize. In 2001, gm soy was found in 21% of the soy containing foods, and gm maize was found in 9% of the maize containing products. Eight samples of the products containing gm soy were shown to contain 4% of gm soy, the corresponding values for gm maize were five samples 4%.

Three soybean varieties were analyzed to evaluate the irradiation influence on the detection of genetic modification. Samples were treated in a 60Co facility at dose levels of 0, 500, 800, and 1000 Gy. The seeds were at first analyzed by Comet Assay as a rapid screening irradiation detection method. Secondly, germination test was performed to detect the viability of irradiated soybeans. Finally, because of its high sensitivity, its specificity and rapidity the polimerase chain reaction was the method applied for genetic modified organism detection. The analysis of DNA by the single technique of microgel electrophoresis of single cells (DNA Comet Assay) showed that DNA damage increased with increasing radiation doses. No negative influence of irradiation on the genetic modification detection was found.

Summary Phytate-degrading enzymes catalyse the step-wise release of phosphate from phytate, the principle storage form of phosphorus in plant seeds and pollen.They are widespread in nature, occurring in plants and micro-organisms, as well as in some animal tissues. Phytate-degrading enzymes 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.Phytate-degrading enzymes are also of interest for producing defined breakdown products of phytate for kinetic and physiological studies.Certain myo-inositol phosphates have been proposed to have novel metabolic effects and therefore, the physiological role of different myo-inositol phosphates is presently undergoing extensive research.Generally, phytase behaves like a monomeric enzyme with molecular masses between 40 and 70 kDa.Up to now, two main types of phytate-degrading enzymes have been identified; acid phytate-degrading enzymes with an pH optimum around pH 5 and alkaline phytate-degrading enzymes with an pH optimum around pH 8.Most of the so far described phytate-degrading enzymes belong to the acidic type, and their optimal pH ranges from 4.5 to 6.0. This review summarises the molecular features as well as catalytic properties of phytate-degrading enzymes and also discusses enzymatic phytate degradation.

Activation of endogenous or addition of exogenous phytases during food processing gives a chance to reduce the phytate content in the final product to a nutritionally acceptable level. Optimal conditions for the endogenous phytases of black beans, this is 60C and pH 6. 0, resulted in a 55% reduction in IP 6 after soaking and cooking. The sum of IP 6 and IP 5 was reduced by 54%. IP 6 reduction was most extensive when black beans were soaked at 50C while adding exogenous phytases during the last 2 h of soaking. After soaking and cooking IP 6 was degraded by 85% and the sum of IP 6 and IP 5 was reduced by 82% compared to the values in raw beans when adding Escherichia coli phytase. Using rye phytase the values were estimated to be 73% and 70%, thereby a clear accumulation of IP 4 occured. Thus, a significant improvement in reduction of myo-inositol phosphates with adverse effects on mineral bioavailability has been achieved in comparison to the usual household procedure.

The three dry nonprocessed bean varieties contain high amounts of phytate ranging from 9.3 to 15.9 μmol g -1 dry basis, representing 85 to 89% of total myo-inositol phosphates, while the other myo-inositol phosphates were found only in small or trace amounts. Myo-inositol phosphate concentrations were not affected by soaking in water for 15h at 25C, whereas cooking resulted in a significant reduction in phytate content (16 to 24%) with a concomitant increase in the concentrations of the lower myo-inositol phosphates. The sum of phytate and myo-inositol pentakisphosphate after soaking and cooking represents about 93% of the amount in raw beans. Therefore, preparing of beans has only a limited effect on the content of myo-inositol phosphates with inhibitory effects on mineral absorption. Phytate-degrading enzymes (phytases) were identified as responsible for phytate removal during cooking, since a good correlation between phytase activity and phytate hydrolysis was observed.

A cytoplasmatic phytase was purified about 410-fold to apparent homogeneity with a recovery of 28%. The enzyme is induceable under carbon limitation in the presence of phytate. It behaves as a monomeric protein of a molecular mass of about 40 kDa. The phytase is rather specific for phytate and exhibits optimal conditions for phytate degradation at pH 5.0 and 58 degrees C. Kinetic parameters for the hydrolysis of Na phytate are KM 300 microM and kcat 180 s-1 at 35 degrees C and pH 5.0. Phytate is hydrolyzed in a stepwise manner; the penta- and tetrakisphosphate were identified as I(1,2,4,5,6)P5 and I(1,2,5,6)P4. Consequently, this enzyme is a 3-phytase (EC 3.1.3.8).

Model systems for investigating the influence of the food matrix and depth of processing on detection limits for foods deriving from genetic engineering were established using the phytase-encoding gene ofEscherichia coliATCC 33965. For detection the polymerase chain reaction was applied directly to DNA extracted from foods based on cereals.E. colicells andE. coliDNA were added to musli, rolled oats, and rye flour in concentrations of 1010to 102CFU and 10 mg to 10 pg per gram, respectively. After soaking of the food samples in water or yoghurt, detection limits were determined to be 103to 104CFU and 10 to 100 pg by specific DNA hybridization. After sourdough fermentation of rye flour a hybridization signal was observed only with rather highE. coliconcentrations (>1010CFU), whereas detection was impossible in ready-baked breads.

Four soluble phytases were identified in germinating spelt. Although numerous purification strategies were applied, none of the four phytases could be purified to homogeneity. The purest phytase preparation, called D21, contained a phytase (major component) and an acid phosphatase (APH) (minor component). The phytase behaves like a monomeric protein of a molecular mass of about 68 kDa and shows a broad substrate specificity. Optimal pH for degradation of phytate was 6.0 and the optimal temperature 45C. Kinetic parameters for the hydrolysis of Na-phytate were K M 400 μmol l -1 and k cat 368 s -1 at pH 6.0. The spelt phytase D21 degrades phytate stepwise

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.

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.

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.

In this study, the behavior of enzyme activity as a function of pH and temperature is modeled on the basis of fundamental considerations. A formulation is developed that includes the activation of enzymes with increasing temperatures and the deactivation of enzymes at higher temperature, together with the effect of protonation and hydroxylation on activity at various constant pH levels. The model is calibrated and validated against an extensive set of experimental data on phytases from seven different origins. The percentage variance accounted for (R(2)(adj)), obtained by statistical nonlinear regression analysis on all data sets, was shown to range from 97.6% to 99.5%. The equilibrium constant of protonation and hydroxylation proved to be independent of temperature.

To elucidate if there is any possibility to identify highly processed foods as produced through genetic engineering, beer, soya bean oil, processed tomato (ketch-up, paste, pizza tomatoes, peeled tomatoes, soup) and potato (french fries, crisps, mashed potatoes, flour, starch, fried potatoes) products as well as an enzyme preparation (Natuphos) were investigated by PCR. In pizza tomatoes, peeled tomatoes, french fries, fried potatoes, potato flour and potato crisps DNA suitable for PCR was found. Therefore, it is possible to identify these products as produced through genetic engineering. Such an identification is impossible in certain beers (pilsener, export, Nutfield lyte), soya bean oil, tomato soup, potato starch, mashed potatoes and Natuphos since PCR-analysis gave no indication of the presence of DNA in these products. As it was shown by adding Escherichia coli DNA the used method is, in principle, capable of detecting specifically small amounts of DNA in such products.

Die Entdeckung der Restriktionsendonukleasen zu Beginn der 70er Jahre wird als Geburtsstunde der Gentechnik angesehen. Damit wurde es moglich, mehr oder weniger gezielt in das Erbgut von Mikroorganismen, Pflanzen und Tieren einzugreifen, indem einzelne Gene isoliert und auch uber Art grenzen hinweg in das Erbgut einer Empfangerzelle eingeschleust werden. Vor allem in den Bereichen Medizin, Landwirtschaft und Nahrungsmittelproduktion werden vielfaltige Anwendungsmoglichkeiten fur die Gentechnik gesehen. Mit Hilfe der Gentechnik produzierte Medikamente, Impfstoffe und Diagnostika sind inzwischen auf dem Markt/und werden von der uberwiegenden Mehrheit der Bevolkerung auch akzeptiert. Die Gentechnik wird in der Landwirtschaft und der Lebensmittelproduktion in Zukunft wahrscheinlich in sehr grosem Ausmas Eingang finden. Die Anwendungsbereiche der Gentechnik bei der Nahrungsmittelproduktion erstrecken sich vom Einsatz gentechnisch veranderter Organismen zur fermentativen Gewinnung von Einzelsubstanzen uber den direkten Einsatz von gentechnisch veranderten Organismen bei der Herstellung von Lebensmitteln bis zur Zuchtung von transgenen Pflanzen und Tieren. Der Einsatz der Gentechnik in der Lebensmittelherstellung und -Verarbeitung hat bereits fur einige Produkte zur Markteinfuhrung gefuhrt und in naher Zukunft kann mit weiteren Produkten gerechnet werden. Fur den deutschen Markt sind bis heute keine Lebensmittel mit gentechnisch veranderten Organismen zugelassen.

Two periplasmatic phytases, called P1 and P2, were purified about 16,500-fold to an apparent homogeneity with a recovery of 7 and 18%, respectively. The enzymes behave as monomeric proteins with molecular masses of about 42 kDa. Because of the limited amounts recovered, the amino terminal sequence of only one of the phytases was determined. Both enzymes are very specific for phytate and have little or no activity on other phosphate esters tested. The kinetic parameters for the hydrolysis of Na-phytate and p-nitrophenyl phosphate are kcat/KM 478 x 10(5) s-1 M-1 and 0.6 x 10(5) s-1 M-1 at pH 4.5. The hydrolysis pathway for phytate was elucidated for P2; consequently, this enzyme is a 6-phytase. The chemical and kinetic properties of the purified phytase P2 points to an identity with an enzyme described by Dassa et al. (1982, J. Biol. Chem. 257, 6669-6676) as a pH 2.5 acid phosphatase. Because of the kinetic parameters it would be better to denote this enzyme as a phytase.