Enzymatic Quantification of Phytate in Animal Manure

Abstract

Phytate (inositol hexaphosphate) has been identified as a major organic phosphorus (P) form in soil, animal manure, and other environmental samples. Although a number of methods are available for quantitative isolation and determination of phytate, they are time-consuming and not amenable to routine analysis. We developed a simple, rapid method for enzymatic determination of phytate in animal manure. Animal manure was extracted by H 2 O, 1 M hydrochloric acid (HCl), 0.1 M sodium acetate (NaOAc, pH 5.0) with or without 0.05 M ethylenediaminetetraacetate (EDTA), and 0.25 M or 0.5 M sodium hydroxide (NaOH)-0.05 M EDTA. Extracts were diluted (1/10-1/150) and adjusted to pH 5.0 in sodium acetate buffer. The diluted extracts were then incubated at 37 uC for 1 h in the absence and presence of fungal 3-phytase (PHY) and potato acid phosphatase (PAP). Enzymatic hydrolyzable organic P was calculated as the difference in inorganic P (P i ) between the mixtures with and without enzymes. Our data indicated that enzymatic incubation of properly diluted and pH-adjusted HCl or NaOH/EDTA extracts released phytate P. The Address correspondence to Zhongqi He, U.S. Department of AgricultureAgricultural Research Service, New England Plant, Soil, and Water Laboratory, Orono, ME 04469. E-mail: Zhongqi.He@ars.usda.gov Trade or manufacturers' names mentioned in the article are for information only and do not constitute endorsement, recommendation, or exclusion by the USDA-ARS. 566This article is a U.S. government work, and is not subject to copyright in the United States.complementary substrate specificity of the two enzymes is considered to enhance the effectiveness of enzymatic hydrolysis. Consequently, we recommend this method of combining PAP and PHY for quantifying phytate P. Additional research is being conducted to verify the effectiveness of this method for general use across a wider range of soils and manures.

Investigation of the Inorganic and Organic Phosphorus Forms in Animal Manure

“…2). These results agree with the literature (He and Honeycutt, 2001; He et al, 2004a, 2007, 2009, 2010).…”

“…Turner and Leytem (2004) reported that the P recovery from the sequential fractionation was 94, 79, and 92% for broiler litter, cattle manure, and swine manure, respectively, values that are similar to those reported in this study (98, 77, and 92% for chicken, dairy, and swine manures, respectively) ( Table 2). He et al (2009) also reported incomplete recovery of P t during the fractionation scheme.…”

Phosphorus Flow and Characterization in Dry‐Grind Corn Ethanol Plants

“…These two P pools were selected primarily for their predominance and impact on coproduct P bioavailability. Due to procedural simplicity and its widespread use for soil and manure extracts (Toor et al, 2003; He et al, 2009), we used the enzymatic hydrolysis method to quantify the phytate‐P fraction.…”

Changes in mineral concentrations and phosphorus profile during dry-grind processing of corn into ethanol

“…Inorganic P (also known as phosphate P or free P) has higher bioavailability than phytate P. The rest of P represents the sum of all P-containing compounds in a sample other than phytate P and inorganic P. It includes, for example, P found in DNA, RNA, proteins, phospholipids and starch. Since phytate P is not well utilized by monogastric animals, it contributes to increased P discharge into the environment (He et al, 2009). In addition, phytate has been shown to interact directly and indirectly with various dietary components, and in particular certain minerals such as Ca and Zn, and thus reduces their availability to humans and animals (Bohn et al, 2008).…”