Equine x-amylase : does it limit starch digestion in the small intestine of the horse?

Abstract

191 Equine _amylase: does it limit starch digestion in the small intestine of the horse? N. Richards1, M. Choct1, G.N. Hinch1 and J.B. Rowe 2 1 2 School of Rural Science and Agriculture, Animal Science, University of New England, Armidale NSW 2351 Australian Sheep Industry CRC, Locked Bag 1, Armidale NSW 2350 ghinch@metz.une.edu.au Summary The amylolytic enzyme amylase is essential for the digestion of starch in the small intestine of the horse. While it had been previously documented that the concentration of this amylolytic enzyme in the equine small intestine is low, the ability of equine amylase to degrade cereal grain starch is poorly understood. In an experiment designed to compare the in vitro starch digesting capacity (activity) of equine amylase with that of bacterial amylase it was observed that the equine amylase digested an average of 20% less starch during the in vitro incubations. This result suggests that the apparently limited capacity of equine amylase to digest starch along with the low concentrations of amylase reported to be present in the equine small intestine may limit precaecal starch digestion. A second experiment investigated if the addition of exogenous glycanase enzymes to cereal grain diets fed to horses would improve small intestinal starch digestion. The results showed that the addition of amylase and amyloglucosidase (AMG) to a digestible starch source significantly elevated postprandial glycaemic responses suggesting an improvement in pre caecal starch digestion. Thus it appears that the concentration and activity of equine amylase may be limiting small intestinal starch digestion in the horse. Keywords: horse, amylase, amyloglucosidase, starch digestion, grain increased energy demands placed on them during work. Ideally the starch from these cereal grains will be digested by amylolytic enzymes in the small intestine and absorbed as glucose. The digestion of starch in the small intestine of the horse occurs via a threestep process, beginning with the degradation of starch by the enzyme amylase. This enzyme does not act on 16 or any 14 links adjacent to reducing ends, but efficiently breaks down 14 links in the two principle forms of starch, amylose and amylopectin, into disaccharide (maltose), trisaccharide (maltotriose) and dextrin units. The second phase of starch digestion involves the hydrolysis of maltose, maltotriose and dextrin units by small intestinal brush border glycanases, primarily AMG which successively releases glucose units from the non reducing ends, and dextrinase which acts on 16, to form free glucose units (Gray 1992). Na+dependant active transport and Na +independent facilitated diffusion then transport glucose from the lumen of the small intestine, through intestinal epithelial cells and into the bloodstream of the animal (Bird et al. 1996; Huntington 1997; Thorens 1993). Amylase thus plays an essential role in the digestion of starch in the small intestine of the horse. It has been recognised that amylases from various plant and animal sources vary in their activity, their capacity to degrade cereal grain starch, a function possibly related to the number of subsites (an area within an active site capable of interacting with one glucose molecule) at each of the enzymes active sites and the affinity of each of these subsites for a glucose molecule (Anindyawati et al . 1998; MacGregor 1988; MacGregor 1993; MacGregor et al. 2001). While it has been documented that horses perhaps have low concentrations of amylase in their small intestine (Comline et al. 1969; Kienzle et al. 1994; Roberts 1974) the capacity of equine amylase to degrade cereal grain starch is not clear. This paper describes two experiments; the first investigates the ability of equine amylase to degrade starch from various cereal grain sources in vitro and the second examines the use of exogenous amylolytic Introduction Although horses evolved primarily consuming grass and grasslike plant species (Budiansky 1998), cereal grains now form a common component of performance horse diets. A recent survey of 72 thoroughbred trainers in rural, provincial and metropolitan areas of New South Wales, Australia showed that thoroughbred horses in training are being fed an average of around 7 kg of grain concentrate/d (Richards 2003). Cereal grains are fed to these high performance animals to provide starch as a relatively cheap energy source to help to meet the Recent Advances in Animal Nutrition in Australia, Volume 14 (2003) 192 Richards et al. enzymes to improve the digestion of starch in the equine small intestine. Experiment 1: assessing the activity of equine _amylase Jejunal supernatant collected from a horse following slaughter was used during this study as a source of equine amylase in an in vitro assay that enabled assessment of how much starch was degraded by this enzyme during 15 and 60minute incubations at physiological temperature. The in vitro starch digestion assay of Bird et al. (1999) was used to provide an estimation of the ability of amylase from a bacterial source to degrade starch. It was hypothesised that the equine and bacterial amylases would have the same activity and, due to the more extensive range of digestive enzymes assumed to be present in the equine jejunal supernatant, incubation in the supernatant would result in a more extensive degradation of cereal grain starch in vitro than during the standard enzyme digestion assay of Bird et al. (1999) which employed only the amylolytic enzymes amylase and amyloglucosidase (AMG). The small intestine of each horse was cut into the duodenal, jejunal and ileal sections. Each was weighed individually when full of digesta; the contents were then emptied from each section into labelled plastic containers, the mucosa was lightly scraped to sample the brush border, and each section was reweighed empty. All small intestinal digesta was immediately placed on ice. On return to the laboratory all digesta samples were centrifuged at 8000 g and the supernatant and pellet were stored separately at 18�C prior to analysis. Measurement and analytical procedure Amylase concentration in the digesta supernatant was measured using the Megazyme Ceralpha Method (Megazyme International Ireland Ltd, Ireland) using the Amylase HR reagent (specific for amylase) and non reducingend blocked pnitrophenyl maltoheptaoside (BPNPG7) as substrate. AMG concentration in the small intestinal digesta supernatant was determined using the Megazyme assay for amyloglucosidase using pnitrophenylmaltoside (4 mM) and glucosidase (25 U/ml) as substrate. The method of Bird et al. (1999), which utilises 300 U of amylase, derived from Bacillus licheniformis and 20 U of AMG derived from Aspergillus niger, per grain sample, with both 15 and 60 minute incubation periods was used to assess the starch digesting capacity of bacterial amylase. Two in vitro starch digestion assays, using a modification of the method of Bird et al. (1999) which involved using the jejunal supernatant from horse A to supply amylase, were then carried out. These assays involved the incubation of 100 mg of cereal grain in 5.3 ml of equine jejunal fluid (300 U amylase and 5.6 U AMG) and 14.4 U of AMG, derived from Aspergillus niger (Megazyme). Incubation periods of 15 and 60 minutes were used. Tubes with no grain sample were run as blanks for both assays. Twelve grain feeds commonly included in equine diets were examined during this study. These feeds were two varieties of corn, one variety of each of rice, barley, triticale and oats in unprocessed form, and five of these grains in processed forms. The grains processed were Sample collection Two horses (one thoroughbred gelding aged 9 years and one mare, breeding unknown, aged >20) that were being sent to slaughter by their owners who gave permission to collect gastrointestinal tract samples from the horses at the abattoir. The horses had been fed a cereal grain diet for 7 days prior to slaughter, with each horse consuming 2.8 kg of oats and 2 kg of extruded rice in two meals/d. Lucerne hay was fed ad libitum as the only roughage component of the diet and was the only feed available to the animals for the 18 h prior to slaughter. On the morning of slaughter, horses were transported to a commercial pet food abattoir and were slaughtered using a captive bolt gun and the gastrointestinal tract was immediately removed. Cotton ties were promptly placed at the junctions of the stomach with duodenum, duodenum with jejunum, jejunum with ileum, and the ileum with caecum to segregate these sections of the gut. Table 1 The _amylase concentration (U/mL) and the total units of _amylase in the digesta supernatant collected from the duodenum, jejunum and ileum of horses A and B. Total units were calculated as U/mL multiplied by the total supernatant volume for each segment of the small intestine. a_Amylase Horse A U/mL Duodenum Jejunum Ileum 166 57 80 Total units (U) 1238 47870 1400 concentration Horse B U/mL 8 28 22 Total units (U) 1450 24630 1210 Equine _amylase and starch digestion in the horse 193 corn 1 extruded, corn 2 micronised, white rice extruded, barley expanded, triticale expanded, and triticale steam rolled; there was no processed oats. All grains were finely ground in a mill prior to analysis. The percent of starch digested during incubations in jejunal fluid with equine amylase was typically lower than that digested during the standard in vitro assay for both the 15 and 60 minute incubation times (Table 3). Results Table 1 shows the concentrations of amylase in the three segments of the equine small intestine of both horses. Horse A had higher concentrations of amylase/mL in all three segments of the small intestine than horse B, but owing to a greater digesta pool size in the gastrointestinal tract of horse B the total number of amylase units in the duodenum and ileum did not differ between horses. AMG concentrations in the small intestine of horses A and B are shown in Table 2 and again horse A tended to have higher concentrations of AMG/mL in all sections of the small intestine. However, horse B had a greater number of total units of AMG than horse A in the duodenum and ileum due to a greater digesta pool size. Discussion The amylase concentrations measured in these two horses, if calculated on a units/g wet mucosa basis, are comparable to those reported by Kienzle et al. (1994). Also in support of earlier observations (Kienzle et al. 1994; Roberts 1974), the concentration of enzymes appears to be variable between horses, Horse A having a higher concentration than B of amylase on a units/mL basis in all three segments of the small intestine. Despite previous reports from Kienzle et al. (1993) and Roberts et al. (1974) indicating that small intestinal brush border glycanase concentrations in the equine small intestine are high and comparable to o ther monogastrics, the AMG concentration measured in the Table 2 The amyloglucosidase (AMG) concentration (U/mL) and the total units of AMG in the digesta supernatant collected from the duodenum, jejunum and ileum of horses A and B. Total units were calculated as U/mL multiplied by the total supernatant volume for each segment of the small intestine. AMG concentration Horse A U/mL Duodenum Jejunum Ileum 1.68 1.00 1.71 Total units (U) 13 840 30 U/mL 0.94 0.60 0.70 Horse B Total units (U) 170 528 39 Table 3 The % starch digested from the specified grains during the standard assay (Bird et al. 1999), employing bacterial derived enzymes with 15 and 60 minute incubation periods and the % starch digested during the jejunal fluid assays employing equine _amylase with 15 and 60 minute incubation periods. In vitro starch digestion (% starch digested in specified time) Standard assay (15 min) Unprocessed corn 2 Unprocessed corn 1 Unprocessed white rice Cracked barley Micronised corn Expanded barley Oats Cracked triticale Expanded triticale Steam rolled triticale Extruded corn Extruded rice 12.3 15.8 18.7 25.7 34.0 39.5 40.6 41.9 50.1 60.4 73.9 80.2 Jejunal fluid (15 min) 5.8 8.2 10.8 12.7 13.2 19.2 27.2 16.7 21.3 25.1 40.4 44.9 Standard assay (60 min) 24.4 30.6 32.8 50.3 49.4 62.1 69.5 73.8 80.7 82.3 84.0 86.2 Jejunal fluid (60 min) 15.2 21.6 29.5 30.7 28.8 39.5 61.6 37.1 45.0 52.5 63.4 58.0 194 Richards et al. small intestine of these horses appears to be low. However, brush border glycanases, such as AMG, are attached to the intestinal brush border by a short terminal hydrophobic section of their protein chain (Gray 1992) and thus, even though the epithelium was scraped, it is likely that a majority of AMG remained attached to the small intestinal brush border. The pattern of distribution of AMG throughout the small intestine was, however, consistent with the pattern of distribution of glucosidases reported by Roberts et al. (1974). It was hypothesised that the bacterial and equine amylases would have the same activity and so, due to the greater array of protease and glycanase enzymes assumed to be present in equine jejunal fluid, the in vitro starch digestion within jejunal fluid would be higher than that observed in the standard in vitro starch digestion assay employing only Bacillus licheniformis amylase and Aspergillus niger AMG. However, an average of around 20% less starch was digested in jejunal fluid in comparison to that digested during the standard starch digestion assay (Table 3), suggesting that perhaps the activity of the equine amylase is inferior to that of the B. licheniformis amylase. Such variation between amylases in ability to degrade starch has been previously reported by Anindyawati et al. (1998) who found that of the three forms of amylase produced by the fungi Aspergillus awamori KT11 only two had the ability to degrade raw corn starch, and that of these two one was more effective than the other. Equine amylase appeared to have greater substrate specificity for oat starch (Table 3), with almost the same percentage of starch digested by both amylases during the 60minute incubation period, which may possibly explain the superior precaecal digestibility and thus safety of oats for horses. that the addition of exogenous amylolytic enzymes to cereal grain diets may be used to improve starch digestion in the small intestine of horses and this was the focus of experiment 2. Experiment 2: the use of exogenous amylolytic enzymes to improve starch digestion The hypothesis for experiment 2 was that the addition of amylase or a combination of amylase and AMG to diets containing digestible starch would improve the digestion of starch in the equine small intestine. It was also hypothesised that amylase is the primary limiting factor to starch digestion in the equine small intestine and that the addition of AMG alone would have a non significant effect on precaecal starch digestion. The design of the experiment was based on the assumption that an improvement in small intestinal starch digestion would be reflected in glycaemic responses that are elevated above those observed for the control diet. Methods Three treatment diets and a control diet were used in the experiment. Steamrolled triticale containing per kg dry matter 662 g starch, 120 g nonstarch polysaccharide, 120 g crude protein, 32 g crude fat, 25 g crude fibre, and 17.0 MJ gross energ y, was used as the control grain to which exogenous enzymes were added. Steamrolled triticale had a high in vitro starch digestibility (82% of starch digested in 1 h) when incubated at 39 � C i n the presence o f excess thermostable amylase and AMG using the method of Bird et al. (1999). The control diet consisted of 1.12 kg of steamrolled triticale meal which corresponded to 670 g of starch; meal sizes were not varied according to body weight. Treatment 1 was 1.12 kg steamrolled triticale and 3 mL of heat stable amylase derived from Bacillus licheniformis (450 Kilo Novo amylase units, Termamyl� Classic, Novozym es A/S. DK2880, Bagsvaerd); treatment 2 was 1.12 kg steamrolled triticale and 1 mL AMG, derived from Aspergillus niger (300 amyloglucosidase units, AMG 300L, Novozymes A/S. DK2880, Bagsvaerd); treatment 3 was 1.12 kg steamrolled triticale, 1 mL AMG (AMG 300L) and 3 mL of heat stable amylase (Termamyl� Classic). All enzymes were diluted to 25 mL with distilled water and sprayed over the steamrolled triticale meal using a hand held spray nozzle to thoroughly cover the grain. Enzymes were added to the grain meal 10 minutes prior to feeding and horses were fed twice daily at 0700 h and 1730 h. Twelve horses (eight standardbred horses, 4 geldings and 4 mares, and four thoroughbred horse geldings) aged 4 to 9 years and weighing 432512 kg were used in the study. The horses were Implications With a seemingly limited ability of equine amylase to degrade cereal grain starch in vitro and previous observations that horses have suboptimal concentrations of amylase in their small intestine (Comline 1969; Kienzle 1994; Roberts 1974), it is likely that starch digestion will be limited by the activity and concentration of amylase in the equine small intestine. Starch that escapes digestion in the small intestine will be fermented in the horses enlarged hindgut by amylolytic bacteria, in a less energy efficient process (Black 1971). This hindgut fermentation of starch commonly causes an accumulation of volatile fatty acids and lactic acid in the caecum and colon of equines. Such acid accumulation can lead to hindgut acidosis (Garner et al. 1977), a metabolic condition commonly associated with reduced fibre fermentation (de Fombelle et al. 1999), behavioural changes (Johnson et al. 1998; Willard et al. 1977) and the crippling and potentially fatal disease laminitis (Pollitt 2001). Thus it is desirable from an animal health and feed use e fficiency perspective that cereal grain starch is completely digested in the small intestine. It is possible Equine _amylase and starch digestion in the horse 195 divided into four groups of three; group A was allocated to the control diet, group B to the amylase diet, group C to the AMG diet, and group D to the amylase + AMG diet. Horses were allowed one and a half days to acclimatize to the stables and daily routine and from the evening feed o f the second day were fed the experimental diets. Blood sampling to measure the glycaemic response took place on the morning of the fifth and eighth days; samples via jugular catheter were taken before the start of eating, every quarter hour for one hour following commencement of eating, and then every half hour for the next four hours. Plasma glucose concentrations were determined using the Dimension� clinical chemistry system on a DADE XL clinical auto analyser (Dade Behring Inc, Newark, DE 19714, USA). The GLU Flex reagent cartridge (Cat No DF39A) was used as the in vitro reagent. that a deficiency of amylase is the major limiting factor to starch digestion in the equine small intestine and is in agreement with Kienzle et al. (1993) and Roberts et al. (1974) who suggest that horses possess naturally high levels of brush border glycanases. However, the addition of AMG to a diet also being supplemented with exogenous amylase increased the glycaemic response above that observed for the diet being supplemented with exogenous amylase only. This result indicates that endogenous AMG may not be sufficient to cope with the excess maltose, maltotriose and dextrin units produced through the addition of amylase to the diet, suggesting that a combination of amylase and AMG may be necessary to effectively enhance small intestinal starch digestion. Results and discussion The addition of a combination of amylase and AMG to the steamrolled triticale diet significantly (P0.008) elevated peak, average and peak minus basal plasma glucose concentrations above those initiated by the control diet, while the addition of amylase alone significantly (P0.008) increased average glucose concentrations above those observed for horses consuming the control diet (Table 4). Thus, in support of the hypothesis, these findings indicate that dietary supplementation with amylolytic enzymes improves small intestinal starch digestion and are in accordance with Meyer et al. (1993) who observed a 12% improvement in the precaecal digestion of cracked corn following the addition of powdered amylase to the diet. The addition of AMG to the control diet appeared to make no improvement to the digestion of starch in the small intestine, with similar glycaemic responses observed for horses given the control and AMG diets (Table 4). Thus in support of the hypothesis it appears that endogenous concentrations of AMG present in the equine small intestine are adequate to break down the products of endogenous amylase digestion to glucose. This observation further supports the theory Conclusions It appears that low concentrations of amylase in the equine small intestine and a limited ability of this equine amylase to degrade cereal grain starch may limit starch digestion in the equine small intestine. Dietary supplementation with amylolytic enzymes may be used to overcome these limitations and improve starch digestion in the equine small intestine. Four major benefits may be expected following the dietary supplementation of cereal grain diets with amylolytic enzymes. These are: 1 the feeding efficiency of cereal grains to horses will be improved as starch will be digested and absorbed as glucose in the small intestine in a more energy efficient process than the fermentation of starch in the equine hindgut; 2 the incidence of hindgut starch fermentation and hindgut acidosis will be reduced; 3 the occurrence of diseases associated with hindgut acidosis, such as laminitis, will be reduced; 4 the incidence of adverse behaviours, often associated with acid accumulation in the equine hindgut, may be reduced. Table 4 Mean peak glucose concentration, average glucose concentration, peak minus basal glucose concentration, time to peak glucose and slope to peak glucose for the control, _amylase, amyloglucosidase (AMG) and _amylase + AMG diets. Control Mean Peak glucose (mmol/L) Average glucose (mmol/L) Peak_basal glucose (mmol/L) Time to peak glucose (hours) Slope to peak glucose (mmol/L/h) 8.8 6.9 3.3 a a b a_Amylase SE 0.19 0.08 0.24 0.20 0.16 Mean 10.0 7.9 4.6 ab b ab AMG Mean 8.8 7.0 3.6 a a ab AMG+ a_Amylase SE 0.30 0.19 0.31 0.15 0.12 Mean 10.8 8.4 5.5 b b a SE 0.56 0.41 0.54 0.20 0.33 SE 0.22 0.11 0.23 0.11 0.16 1.6 2.2 1.9 2.5 1.6 2.4 2.2 2.8 Values in same row with different superscripts are significantly different (P0.008) 196 Richards et al. References Anindyawati, T., Melliawati, R., Ito, K., Iizuka, M. and MinamUra, N. (1998). Three different types of amylases from Aspergillus awamori KT11: their purifications, properties and specificities. Bioscience, Biotechnology and Biochemistry 62, 13511357. Bird, A.R., Croom Jr, W.J., Fan, Y.K., Black, B.L., McBride, B.W. and Taylor, I.L. (1996). Peptide regulation of intestinal glucose absorption. Journal of Animal Science 74, 25232540. 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