Phytase supplementation of broiler diets is a routine practice and is used primarily to liberate the P component (282 g/kg) of the phytate molecule (myo-inositol hexaphosphate; IP6). Phytase also generates ‘extra-phosphoric’ responses because phytate interacts with protein, starch, fat, calcium and trace minerals but these responses are less well understood and accepted (Selle and Ravindran, 2007). In particular, interactions between phytate and starch have not been clarified and the effects of phytase on starch digestion are elusive. Rickard and Thompson (1997) nominated several mechanisms whereby phytate may influence starch digestion, which included inhibition of amylase activity either directly or via chelation of Ca, which is a requisite co-factor for amylase and it was also suggested that phytate may complex starch directly or indirectly by binding proteins that are closely associated with starch. The present study was designed to evaluate the inclusion of exogenous phytase in broiler diets based on wheat and maize and to identify whether phytase has an effect on starch digestive dynamics.
Data from two separate but similar 40-day feeding studies were compiled and analysed as a 2 x 2 factorial array of treatments consisting of wheat- and maize-based diets, without or with bacterial phytases at 500 FTU/kg. Protein contents of wheat and maize were 110 and 81 g/kg, respectively, and the wheat-based diet was formulated to be 12.97 MJ and 217 g/kg protein while the maizebased diet was formulated to be 12.92 MJ and 178g/kg protein. The two cereals were mediumlyground (3.2 mm hammer-mill screen) prior to incorporation into the diets that were steampelleted at 84°C. CeliteTM (World Minerals, Lompoc, CA, USA) was included in diets at 20 g/kg as an inert marker to determine apparent digestibility coefficients of starch in four small intestinal sites. The small intestines were removed from birds euthanized by intravenous injection of sodium pentobarbital and digesta in their entirety were gently expressed from the proximal jejunum (PJ), distal jejunum (DJ), proximal ileum (PI) and distal ileum (DI) and pooled for each cage. Proximal jejunal samples were taken from the end of the duodenal loop to the mid-point with Meckel’s diverticulum and distal jejunal samples from the mid-point to the diverticulum. Proximal ileal samples were taken from Meckel’s diverticulum to the mid-point with the ileocaecal junction and distal ileal samples were taken from below this mid-point. The digesta samples were freeze-dried and acid insoluble ash (AIA) concentrations were determined by the method of Siriwan et al. (1993).
Starch concentrations in diets and digesta were determined by a procedure based on dimethyl sulfoxide, α-amylase and amyloglucosidase, as described by Mahasukhonthachat et al. (2010). The apparent digestibility coefficients for starch at four small intestinal segments were calculated from the following equation:
Starch disappearance rates (g/bird/day) were calculated from the following equation from the defined regions of the small intestine:
Disappearance rate = feed intake (g/bird/day) × dietary starch (g/kg) × starch (digestibility coefficient)
Experimental data was analysed as a 2x2 factorial array of dietary treatments using the IBM® SPSS® Statistics 20 program (IBM Corporation. Somers, NY USA). A probability level of less than 5% was considered to be statistically significant.
The effects of dietary treatments on apparent starch digestibility coefficients and starch disappearance rates are shown in Table 1. Interactions between grain-type and phytase were not observed for any of the parameters determined. There were significantly different starch digestibility coefficients between wheat and maize in the PJ and DI. Starch from wheat-based diets was 34.8% more digestible in the proximal jejunum (0.822 versus 0.610; P < 0.001) but, in the distal ileum, starch digestibility coefficients were 4.53% higher for maize-based diets (0.947 versus 0.906; P < 0.02). Maize had significantly slower starch disappearance rates compared to wheat in the PJ (50.7 vs 67.5 g/bird/day; P < 0.001), however in the DI, this was reversed where wheat had lower starch disappearance rates than maize (74.2 vs. 78.6 g/bird/day; P< 0.04). Phytase increased proximal jejunal starch digestibility coefficients by 17.6% (0.774 versus 0.658; P < 0.005). Phytase significantly (P < 0.01) increased starch disappearance rates in all segments of the small intestine. Starch digestibility rates with phytase supplementation were increased by 23.7% (65.3 vs. 52.8 g/bird/day) in the PJ, 10.1% (73.8 vs. 67.0 g/bird/day) in the DJ, 9.91% (78.7 vs. 71.6 g/bird/day) in the PI and 7.89% (79.3 vs. 73.5 g/bird/day) in the DI.
This study presents evidence that suggests that phytate interacts with starch and this is declared by the accelerated starch disappearance rates generated by phytase. One possibility is that phytate impedes starch digestion and, as mentioned, several mechanisms have been proposed. However, there is little evidence to support the existence of direct starch-phytate complexes and it seems more likely that phytate may indirectly bind starch via starch granule-associated proteins (SGAP) as either binary or ternary protein–phytate complexes involving starch and cereal proteins. In theory, phytate has the capacity to complex proteins that are located in and on starch granules (Baldwin, 2001). Interactions between starch granules with soy and wheat proteins have been investigated (Mohamed and Rayas-Duarte, 2003) where SGAP removal from the surface of starch granules decreased protein-starch interactions.
Table 1 - The effects of 500 FTU/kg phytase on apparent digestibility coefficients and disappearance rates (g/bird/day) of starch in the proximal jejunum (PJ), distal jejunum (DJ), proximal ileum (PI) and distal ileum (DI) in broilers offered wheat- or maize-based diets.
The first study to investigate the effect of phytate on sodium was by Cowieson et al. (2004), where broilers were fed atypical diets of glucose and phytate displayed increased total tract sodium excretion but with phytase addition total tract sodium excretion was reduced by 44%. These findings were subsequently confirmed where phytase increased Na digestibility at the ileal level (Ravindran et al., 2006; 2008; Selle et al., 2009). There is evidence that phytate depresses sodium pump activity in rats which was associated with reduced blood glucose levels (Dilworth et al., 2005). In addition, Liu et al. (2008) found that phytase increased sodium pump and glucose concentrations in duodenal and jejunal enterocytes in chickens. For example in the jejunum, 1000 FTU/kg phytase significantly increased concentrations of the sodium pump by 18.4% (13.59 versus 11.48 mmol/mg) and of glucose by 46.8% (13.73 versus 9.35 mmol/mg) in birds offered maize-based diets containing 2.2 g/kg phytate-P. The implication is that phytate was depressing sodium pump activity and, in turn, intestinal uptakes of glucose.
It has been proposed that phytate may impede absorption of glucose by depressing the functionality of the so-called ‘sodium pump’ (Na+ -K+ -ATPase) (Truong et al., 2014). In essence, glucose absorption from the gut lumen into the systemic circulation is driven by the sodium pump, which is located in the baso-lateral membrane of enterocytes in extreme numbers (Glynn, 1993). The sodium pump maintains an electrochemical gradient across the enterocyte by the active exchange of three Na+ ions exiting for two K+ ions entering the enterocyte. The Nadependent absorption of glucose from the gut lumen into enterocytes via SGLT-1 co-transporters is driven by the activity of the sodium pump. Concentrations of Na within enterocytes are pivotal for sodium pump function (Therein and Blostein, 2003). Phytate may deplete Na concentrations in enterocytes, thereby depressing sodium pump activity. By limiting P bioavailability phytate could interfere with the rephosphorylation of the sodium pump but the diets in the present study were P-adequate. However, the depletion of Na may be a consequence of sodium bicarbonate hypersecretion into the duodenum especially from the pancreas. It has been proposed that this is a compensatory mechanism because phytate increases secretions of HCl and pepsin as phytatebound protein is refractory to pepsin digestion in the stomach (Selle et al., 2012).
The differences in starch digestive dynamics of wheat and maize are noteworthy as the disappearance rate of wheat starch from the proximal jejunum was 33% more rapid than maize. As a consequence, 91% of wheat starch digestion took place in the PJ as opposed to 64% in maize-based diets. Slowly digestible starch is that which is digested in the three posterior segments of the small intestine and therefore 36% of maize starch was slowly digestible as opposed to only 9% for wheat. Giuberti et al. (2012) reported that the digestion rate of wheat starch was double that of maize under in vitro conditions. Differences in rates and sites of starch digestion along the small intestine can influence broiler performance, where several studies (Weurding et al. 2001, 2003) have indicated that the provision of slowly digestible starch improves feed conversion ratios in broilers. This suggests that the rate at which wheat starch is digested may be excessively rapid.
There are few studies which have demonstrated starch effects from phytase addition to diets in broilers, however the current study provides compelling evidence that phytase influences starch digestion dynamics where responses in starch digestion and starch disappearance rate where observed in phytase addition to wheat- and maize-based diets.
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