A breakthrough in our understanding of phytase enzyme mode of action and the associated matrix values in feed formulation has been provided in a recent paper published by Tran et al. in the January 2011 edition of Analytical Biochemistry entitled ‘A simple and fast kinetic assay for phytases using phytic acid–protein complex as substrate’.
While at first glance the relevance of the title to animal nutritionists may not be immediately obvious, this collaborative research by scientists from Lund University (Sweden) and Genencor studied interactions of phytate with dietary proteins as measured by the formation of insoluble complexes and the suitability of using the phytate-protein complex as a substrate for phytase assays. The authors demonstrated that, as phytate-protein complexes occurred in feed and the digestive tract of animals, this may be a more relevant substrate for the evaluation of phytases than sodium phytate, a laboratory chemical routinely used in traditional phytase assays. Of commercial interest was that the study included an independent evaluation of four different commercial phytases in their effectiveness to hydrolyze either synthetic sodium phytate, or naturally occurring protein-phytate complexes in feed at a pH of 3.0. Results shown in Table 1 firstly confirm that when sodium phytate (IP6-Na) is used as a substrate at low pH, the degree of Na-phytate hydrolysis is similar between the two E. coli phytases, but is significantly lower when fungal phytases (P. lycii and A. niger) are applied. These differences between E. coli and fungal phytase sources are not new and have been shown to be a function of the lower pH optimum of the E. coli phytases versus their fungal phytase counterparts (Wyss et al., 1999). However, of greater relevance to nutritionists are the large differences shown between commercial phytases in their ability to degrade protein-phytate complexes, being not only significantly different between E. coli and fungal phytases, but also between different E. coli phytase sources. As the main anti-nutritional effect of phytate is caused by the formation of insoluble protein-phytate complexes in the acid stomach this data provides the first strong evidence that the nutritional benefit from phytase enzymes can no longer be viewed as being constant within classes of phytase such as E. coli. Consequently, in practice, different matrix values should be applied in feed formulation to account for these differences.
To fully understand the research results shown in Table 1 it is important to know that while sodium phytate is used as the substrate to determine phytase activity in-vitro, in nature and also in the acid part of the digestive tract of poultry (proventriculus and gizzard) and swine (stomach), phytic acid does not occur in its free acid or sodium salt form, but rather in association with proteins. The reason for the association of phytate and protein in the gut is that most proteins of plant origin, such as those derived from maize, soybean, sunflower and rapeseed (canola) meal, have their iso-electric points (pI values) in the acidic range (pH 4.5). Hence, when the pH in the digestive tract drops below the iso-electric point of the protein, the protein is now positively charged, which allows the negatively charged phytic acid molecule to bind to it, thereby altering the proteins iso-electric point and rendering the protein insoluble (Reddy et al., 1989; Konietzny and Greiner, 2003). This formation of insoluble protein-phytate complexes in the acid part of the digestive tract can have important nutritional consequences due to a decreased accessibility to pepsin proteases resulting in increased pepsin secretion, greater endogenous losses and inefficient protein digestion, as indicated by a reduced ileal amino acid digestibility from phytate. (Vaintraub and Bulmaga, 1991, Konietzny and Greiner, 2003, Kies et al., 2006, Cowieson et al., 2008).
Of further importance are new data that have shown the formation of insoluble protein-phytate complexes to be proportional to the amount of phytate present. This is shown in Figure 1 where the presence of insoluble protein-phytate complexes from either soy protein or casein, measured by absorbance at 600 nm, increased almost linearly as the phytate concentration increased. Stated another way, the anti-nutritional effect of phytate in the acid stomach of the animal will be proportional to the concentration of undigested phytate (IP6) present in the acid stomach and is further supported by previous work by Cowieson et al. (2009), who demonstrated a step-wise increase in endogenous amino acid losses at the terminal ileum as dietary phytate levels increased. Converse to the negative effects of phytate on energy and amino acid digestibility, nutritional advantages of phytase enzymes will depend primarily on the ability of the source of phytase to rapidly hydrolyze phytate-protein complexes at a low pH (gizzard and provetriculus region), thereby reducing their anti-nutritional effects and resulting in net improvements in energy and protein