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PubMed · 10579503

A novel staining method for detecting phytase activity.

H D Bae·L J Yanke·K J Cheng·L B Selinger
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Abstract

Differential agar media for the detection of microbial phytase activity use the disappearance of precipitated calcium or sodium phytate as an indication of enzyme activity. When this technique was applied to the study of ruminal bacteria, it became apparent that the method was unable to differentiate between phytase activity and acid production. Strong positive reactions (zones of clearing around microbial colonies) observed for acid producing, anaerobic bacteria, such as Streptococcus bovis, were not corroborated by subsequent quantitative assays. Experimentation revealed that acidic solutions generated false positive results on the selected differential medium. Empirical studies undertaken to find a solution to this limitation determined the false positive results could be eliminated through a two step counterstaining treatment (cobalt chloride and ammonium molybdate/ammonium vanadate) which reprecipitates acid solubilized phytate. This report discusses the application of the developed two step counterstaining treatment for the screening of phytase producing ruminal bacteria as well as its use in phytase zymogram assays.

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6-PhytaseAnimalsAspergillusCattleCobaltColoring AgentsCulture MediaElectrophoresis, Polyacrylamide GelMolybdenumPhytic AcidSelenomonasStaining and LabelingStreptococcus bovisVanadatesJournal Article

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H D Bae, L J Yanke, K J Cheng, L B Selinger. 1999. A novel staining method for detecting phytase activity.. https://doi.org/10.1016/s0167-7012(99)00096-2

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PubMed2007

Improved iron bioavailability in an oat-based beverage: the combined effect of citric acid addition, dephytinization and iron supplementation.

BACKGROUND: Iron deficiency in children is a major worldwide nutritional problem. An oat beverage was developed for 1- to 3-year-old children and different treatments were used to improve the iron bioavailability. AIM OF THE STUDY: To investigate the effects of citric acid addition, phytase treatment and supplementation with different iron compounds on non-heme iron absorption in human from a mineral-supplemented oat-based beverage. METHODS: A 240 g portion of a 55Fe-labeled test product (T) or a 59Fe-labeled reference dose (R) was served as breakfast after overnight fasting on four consecutive days in the order of TRRT. On day 18 the retention of 59Fe was measured by a whole-body counter and the erythrocytes uptake of 55Fe and 59Fe by a liquid-scintillation counter. Forty-two healthy subjects (men and women) were randomized into four study groups, members of each being given one of the studied four products (A, B, C, and D) supplemented with Fe (1.3 mg/portion), Zn, Ca, Se and P. Ferric ammonium citrate (FeAC) was added to products A, B, and C and ferric pyrophosphate (FePP) to product D. Citric acid (60 mg/portion) was added to products B, C, and D and phytase treatment applied to products C and D. RESULTS: Citric acid improved iron absorption by 54% from 3.9% in product A to 6.0% in product B (p = 0.051). Phytase treatment increased iron absorption by 78% (from 6.0 to 10.7%, p = 0.003) by reducing the phytate-phosphorus content per portion from 16.3 mg in product B to 2.8 mg in product C. The two compounds gave similar iron absorption rates (p = 0.916). CONCLUSIONS: A combination of citric acid addition, dephytinization and iron supplementation significantly increased the iron absorption in an oat-based beverage. Such a beverage can be useful in the prevention of iron deficiency in 1- to 3-year-old children.

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PubMed2007

Age-related influence of a cocktail of xylanase, amylase, and protease or phytase individually or in combination in broilers.

This 21-d experiment was conducted to determine if the response of chicks to a cocktail of xylanase, amylase, and protease (XAP) or Escherichia coli-derived phytase individually or in combination when fed a nutritionally marginal corn-soybean meal diet is age-dependent. Six hundred 1-d-old chicks were allocated to 5 dietary treatments in a randomized complete block design. The treatments were as follows: 1) positive control with supplemental inorganic P; 2) negative control (NC) marginal in P and ME; 3) NC plus XAP to provide (per kg of diet) 650, 1,650, and 4,000 U of xylanase, amylase, and protease, respectively; 4) NC plus phytase added to provide 1,000 phytase units/kg; and 5) NC plus a combination of XAP and phytase. Low ME and P in the NC diet depressed weight gain and gain:feed (P < 0.001). A cocktail of XAP alone did not improve performance, but phytase supplementation improved (P < 0.001) weight gain. The enzymes were additive in their effects on growth performance. The enzymes had no effect on ileal digestible energy. Ileal N digestibility was higher (P < 0.05) in diet with XAP or phytase individually compared with NC. Both phytase and XAP individually and in combination improved (P < 0.01) ileal P digestibility compared with NC. Total tract nutrient retention and ME increased (P < 0.01) as the birds grew older. There were age x diet interactions (P < 0.001) on total tract retention of P and Ca; improvement in P retention due to phytase use decreased by 50% as the chicks matured. The current study shows that a combination of XAP and phytase improved performance, but the enhancement in performance appears to be mainly from phytase. Both XAP and phytase were effective in improving P digestibility and retention of chicks receiving nutritionally marginal corn-soybean meal. The data also shows that the chicks benefited more from the enzyme addition at a younger age and that the contribution of the enzymes to nutrient retention decreased with age in chickens.

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PubMed2006

Diversity of phytases in the rumen.

Examples of a new class of phytase related to protein tyrosine phosphatases (PTP) were recently isolated from several anaerobic bacteria from the rumen of cattle. In this study, the diversity of PTP-like phytase gene sequences in the rumen was surveyed by using the polymerase chain reaction (PCR). Two sets of degenerate primers were used to amplify sequences from rumen fluid total community DNA and genomic DNA from nine bacterial isolates. Four novel PTP-like phytase sequences were retrieved from rumen fluid, whereas all nine of the anaerobic bacterial isolates investigated in this work contained PTP-like phytase sequences. One isolate, Selenomonas lacticifex, contained two distinct PTP-like phytase sequences, suggesting that multiple phytate hydrolyzing enzymes are present in this bacterium. The degenerate primer and PCR conditions described here, as well as novel sequences obtained in this study, will provide a valuable resource for future studies on this new class of phytase. The observed diversity of microbial phytases in the rumen may account for the ability of ruminants to derive a significant proportion of their phosphorus requirements from phytate.

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