The effect of diet, freezing method and time on gastric concentrations of phytate and its hydrolysis products (InsP2 through InsP5 and MYO) is presented in Table 5.1. In
line with the hydrolysis data, gastric InsP6 concentration was influenced by time
(P<0.001) and a significant diet by freezing temperature interaction (P<0.05). The concentration of InsP6 in the gastric digesta decreased by 13.4% from 0 to 15 min at a
constant rate of approximately 32 nmol /mg TiO2/min (P<0.001). As expected, the
diet by freezing temperature interaction was the same as it was for the InsP6 hydrolysis
data, with less InsP6 being measured in samples frozen at -26 °C than those frozen at -
78.5 °C, and the effect being greater in the PC and NC than in the STD and SD
0 20 40 60 80 100 120 0 5 10 15 D egr adati on (% ) Min D*** T† FT*** D x FT*
treatments. The concentration of lower inositol phosphates and MYO in the gastric digesta was influenced by a freezing temperature x diet interaction (P<0.01), but not by time (P>0.05). The data show that freezing temperature had no influence on the total concentration of lower phytate esters and MYO in the PC and NC diets; however, in diets with supplementary phytase, freezing at -78.5 °C produced greater gastric InsP2-5 and MYO concentrations than freezing at -26 °C.
The effect of time on the concentrations of phytate hydrolysis products in the gastric digesta is presented in Figure A.3 (appendix). Leaving the gastric digesta to sit at room temperature for up to 15 min following sampling had no effect on InsP5, InsP4,
InsP2 or MYO concentrations (P>0.05). InsP3 concentration increased in a linear
manner (P<0.05) over time in the PC, NC and STD diets, but remained fairly constant in the SD diet, resulting in a tendency for a time x diet interaction (P = 0.06).
The effects of freezing temperature and diet on the concentration of the individual phytate hydrolysis products in the gastric digesta, including MYO, are presented in Figure 5.2. Within freezing temperature, the gastric inositol phosphate and MYO composition of pigs fed the PC and NC diets was almost identical. Adding phytase to the NC at either 500 or 2,000 FTU/kg significantly reduced InsP5 content (P<0.001),
though there were no differences between the two phytase doses (P>0.05). Digesta frozen at -26 °C measured 30.5% less InsP5 than that frozen at -78.5 °C (402 vs. 280
nmol/mg TiO2; P<0.001). InsP4, InsP3 and InsP2 concentration were all influenced by
a significant freezing temperature x diet interaction (P<0.001, P<0.001 and P<0.01 respectively). In the PC and NC diets, digesta frozen at -26 °C tended to have higher levels of InsP4 than that frozen at -78.5 °C (P<0.1), whereas in the STD diet, freezing
the digesta at -26 °C had the reverse effect and resulted in lower levels of measured InsP4(P<0.05). The freezing temperature x diet interaction for gastric InsP3 was
similar to that described for InsP4. Within freezing temperature, increasing the phytase
dose from a standard dose (500 FTU/kg) to a super-dose (2,000 FTU/kg) reduced the amount of InsP4 and InsP3 present in the stomach digesta (P<0.01). Levels of
measured InsP2 were similar irrespective of freezing temperature for the PC, NC and
STD diets; however, in the SD diet, InsP2 concentration was higher when frozen at -
78.5 °C than at -26 °C. The concentration of gastric MYO was not influenced by treatment (P>0.05).
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Table 5.1. Effect of diet, freezing temperature and time on gastric InsP6and ∑InsP2-5 + MYO(nmol/mg TiO2) concentration following samplinga
FT -26 °C -78.5 °C
Diet PC NC STD SD PC NC STD SD Average Significanceb SEM
InsP6 D ***, FT***, T†, D x FT* 125 0 min 835 1098 110 166 1338 1489 327 356 715 5 min 846 1091 47 185 1280 1425 274 329 685 10 min 801 1085 50 135 1259 1380 223 280 651 15 min 766 985 72 142 1222 1331 183 250 619 Average 812 1064 70 157 1275 1406 252 304 FT average 526 809 ∑InsP2-5+ MYO D *, FT**, D x FT** 192 0 min 1942 1822 1360 940 1857 1856 1859 1386 1426 5 min 1927 1755 1370 996 1876 1750 1844 1355 1415 10 min 2019 1837 1368 806 1948 1786 1887 1328 1425 15 min 1958 1905 1422 1013 2002 1910 1904 1327 1471 Average 1962 1830 1380 939 1921 1826 1874 1349 FT average 1528 1742
a Values represent the mean of 10 observations.
b Significance level: *P<0.05,** P<0.01,*** P<0.001, † linear effect P<0.001. D = diet, FT = freezing temperature, T = time, and D x FT = diet by freezing temperature interaction. No D x T, FT x T or 3 way interactions were observed (P>0.05).
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Figure 5.2 Interactive effects of freezing temperature and diet on gastric inositol pentakisphosphate (InsP5), inositol tetrakisphosphate (InsP4), inositol trisphosphate (InsP3), inositol bisphosphate (InsP2) and myo-inositol (MYO) concentration (nmol/mg TiO2).
5.5 Discussion
The present study demonstrates that the inositol phosphate composition of pig gastric digesta is influenced by both freezing temperature and time taken to freeze the sample following collection. It should be noted that although gastric phytate hydrolysis was measured in the present study, these data do not provide an accurate measure of post- prandial gastric phytate hydrolysis, as time between feed intake and sampling was not controlled. Nevertheless, gastric phytate hydrolysis provides a suitable means of assessing the influence of time and freezing temperature on phytate degradation and has thus been included in this thesis.
Phytase induced phytate hydrolysis is a time-dependent process which in the pig is often limited by the retention time of the digesta in the stomach (Blaajberg et al., 2011). Thus, it was unsurprising to learn that this enzyme catalysed reaction continues in the gastric digesta after sample collection from pigs fed diets with added phytase. Perhaps a more surprising discovery was that phytate continues to be hydrolysed in the gastric digesta after sampling from pigs fed diets with no added phytase. This is contrary to the results of Kemme et al. (2006), who found almost no phytate was degraded in the stomach of pigs when fed a low phytase diet. Both wheat and barley are known to possess high levels of phytase activity (Eeckhout and de Paepe, 1994; Viveros et al., 2000); although, this is generally lost during the pelleting process. In this study, dietary phytase analysis revealed that the inactivation of the intrinsic phytase activity during the pelleting process was more effective in the NC than in the PC (<50 vs 85 FTU/kg) treatment. It is possible that intrinsic phytase activity was responsible for the phytate hydrolysis occurring in the gastric digesta of pigs fed the non-phytase supplemented diets. However, this seems unlikely, particularly in the NC diet for which the analysed phytase activity was below the limit of detection (< 50 FTU/kg). The possibility of an additional source of microbial phytase cannot be excluded. Several studies have shown that some species of lactic acid bacteria reside within the stomach of pigs (Cranwell et al., 1976; Tannock, 1992; Hojberg et al., 2003; Chow and Lee, 2006); however, the ability of these to produce extracellular phytase is a contentious issue (Reale et al., 2007). Some have found that certain species of lactic acid bacteria are capable of producing extracellular phytase
al., 2005). Nevertheless, if indeed microbes residing in the stomach of the pig are capable of producing phytase, their quantitative importance for in vivo phytate hydrolysis is untested and would be worthy of further study.
Another key finding of the current study is that freezing temperature has a significant influence on gastric phytate hydrolysis; samples frozen at -26 °C recovered less analysed phytate than those frozen at -78.5 °C. This suggests that phytate hydrolysis continues during the freezing process, and digesta must be frozen rapidly in order to terminate the enzyme catalysed reaction and prevent possible erroneous calculation of in vivo phytate hydrolysis. There was a significant diet by freezing temperature
interaction on gastric phytate hydrolysis, as freezing at -26 °C recovered proportionally less phytate than that frozen at -78.5 °C in diets devoid of added phytase than in diets with added phytase. This interaction may reflect the fact that digesta samples used in this study likely reflect digesta at different stages of digestion. As a result, some of the pigs receiving a diet with added phytase, most likely at the later stages of gastric digestion, had completely degraded (i.e. no phytate detected) or were close to completely degrading the ingested phytate. Logically, the choice of freezing
temperature will have little to no effect on phytate hydrolysis in these samples. Gastric phytate hydrolysis was much lower in pigs fed diets devoid of added phytase, and thus the potential scope for continued phytate hydrolysis during processing in such samples is much greater. It would be interesting to see if this interaction would stand if time from feeding to sampling had been controlled and all pigs were at an early stage in gastric digestion.
The gastric lower inositol phosphate and MYO profiles in diets without added phytase were almost identical. This suggests that the phytate in these diets is likely being degraded by the same mechanism, through similar phytases with similar specificities and reaction kinetics. Supplementing the NC diet with a standard dose of phytase changed the inositol phosphate composition of the digesta, with reductions in InsP5
and concurrent increases in InsP4 and InsP3 content. Adding a super-dose of phytase to
the diet effectively diminished the build of InsP4 and InsP3, resulting in more complete
phytate hydrolysis. These findings are consistent with the ileal inositol phosphate data from the previous chapter and confirms that InsP6 and InsP5 are the primary substrates
for standard doses of this E. coli phytase, whereas InsP4 and InsP3 phytate esters are
Interestingly, the more complete phytate hydrolysis associated with the standard phytase dose frozen at -26 °C and the super-dosing diets was not met with clear changes in InsP2 or MYO concentration. Furthermore, the total sum of measured
phytate hydrolysis products in the digesta of these pigs was lower than that of the controls, which likely indicates InsP1 formation. Unfortunately, it was not possible to
measure InsP1 using the inositol phosphate quantitation methodology used in the
present study. InsP1 is considered to be a transient compound that is present in trace
amounts in ileal digesta, as it is rapidly dephosphorylated by endogenous phosphatases or possibly absorbed directly by the small intestine (Adeola and Cowieson, 2011). Thus, it is frequently dismissed as having minor quantitative importance in ileal digesta; however, this study would suggest that this is not the case in gastric digesta. Moreover, these results indicate that this particular E. coli phytase is unable to completely dephosphorylate phytate to MYO, which is in agreement with the current view that most microbial phytases are not capable of completely dephosphorylating phytate. This is thought to be due to the axial arrangement of the phosphate group positioned at C2 on the inositol nucleus (Wyss et al., 1999). However, the MYO data presented in the previous chapter clearly demonstrate that supplementing this phytase increased MYO levels in the ileal digesta of the pigs. Thus, it seems probable that the phytase degraded ingested phytate to lower, more soluble, inositol phosphate esters (InsP2 and InsP1) in the gastric phase, which were then available to endogenous
luminal and mucosal phosphatases in the intestine for further dephosphorylation to MYO.
It can be concluded that significant phytate hydrolysis occurs in the gastric digesta during collection and processing. In the current experiment, delaying the freezing of gastric digesta by just 5 min resulted in significant InsP6 hydrolysis. Therefore,
digesta should be processed as quickly as possible and frozen on dry ice in order to minimise post-collection phytate degradation and changes in the gastric inositol phosphate profile. At present, many papers fail to provide an adequate level of detail in their methodology with regards to the sampling and processing of digesta prior to inositol phosphate quantitation, making it difficult for one to draw firm conclusions and draw cross-study comparisons. Clearly, differences in sample processing
methodologies can have a large impact on the digesta inositol phosphate composition and it seems highly likely that such differences have contributed to the variable
outcomes surrounding the effect of phytase in the literature. This highlights the necessity for the implementation of standardised methodology in phytate degradation studies.