Livestock Science 134 (2010) 91–93

Contents lists available at ScienceDirect

Livestock Science
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / l i v s c i

Gastro-intestinal digesta pH, pepsin activity and soluble mineral

concentration responses to supplemental phytic acid and
phytase in piglets☆
T.A. Woyengo a, O. Adeola b, C.C. Udenigwe c, C.M. Nyachoti a,⁎
a
Department of Animal Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada
b
Department of Animal Science, Purdue University, West Lafayette , IN 47907-2054, USA
c
Department of Human Nutritional Sciences, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

a r t i c l e i n f o a b s t r a c t

Keywords: Effect of phytic acid (PA) and phytase on gastro-intestinal digesta pH, soluble mineral
Phytic acid concentration and pepsin activity in piglets was investigated. Twenty four piglets (initial body
Phytase weight = 7.60 ± 0.73 kg; mean ± SD) were randomly assigned to 3 experimental diets to give
Pepsin activity 8 piglets per diet. The diets consisted of a casein-cornstarch-based diet with 0, 2% PA (as
Gastro-intestinal pH and mineral
sodium phytate), or 2% PA plus phytase at 500 FTU/kg. The basal diet was formulated to meet
concentration
Piglets
NRC (1998) amino acids requirements for piglets. After consuming experimental diets for
10 days, the piglets were killed and digesta sampled from the stomach and jejunum for pH,
pepsin activity, and soluble mineral determination. Phytic acid decreased (P b 0.01) jejunal
digesta pH from 7.13 to 6.61. Phytic acid also decreased (P b 0.05) stomach digesta pepsin
activity by 46%; jejunal Ca2+ and Mg2+ concentrations by 60 and 85%, respectively; but
increased (P b 0.0001) jejunal Na+ concentration by 57%. Phytase did not influence (P N 0.05)
any of the response criteria measured in this study. It is concluded that supplemented PA
reduces pepsin activity in the stomach, pH in jejunum, and concentrations of soluble Ca and Mg
in the jejunum; and increases Na secretion in the jejunum of piglets. Thus, it appears that PA
reduces nutrient utilization in pigs partly through reduced stomach pepsin activity and altered
mineral solubility in the small intestine.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction Mg in piglets to negative values (Woyengo et al., 2009),

Phytase supplementation improves mineral utilization in
pigs and poultry due to hydrolysis of its substrate, phytic acid
(PA), which is naturally present in plant feedstuffs (Bedford,
2000). Phytic acid is negatively charged and hence it can
reduce the availability of positively minerals by binding to
them (Bedford, 2000). Phytic acid has also been shown to
increase endogenous mineral losses in poultry (Cowieson et
al., 2004), and to reduce the apparent digestibility of Na and
☆ This paper is part of the special issue entitled “11th International
Symposium on Digestive Physiology of Pigs”.
⁎ Corresponding author. Tel.: +1 204 474 7323; fax: +1 204 474 7628.
E-mail address: martin_nyachoti@umanitoba.ca (C.M. Nyachoti).
indicating increased endogenous losses of these minerals in
pigs due to PA. However, there is no information on the
mechanisms by which PA increases the endogenous mineral
losses. Binding of tannic acid to pepsin in the stomach of rats
was shown to result in increased pepsin and HCl secretions
(de Mitjavila et al., 1973). Thus, PA may bind to positively
charged amino acids on pepsinogen in the stomach, leading
to reduced pepsin activity and hence increased pepsin and
HCl secretions via negative feedback mechanisms. The
resulting acidic digesta may then require neutralization by
mineral-bicarbonates in small intestine, resulting in in-
creased endogenous mineral losses. The objective of this
study was to determine the effect of PA and phytase on
stomach digesta pepsin activity, and pH and total and soluble

1871-1413/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.livsci.2010.06.107
92 T.A. Woyengo et al. / Livestock Science 134 (2010) 91–93

mineral concentrations in stomach and jejunal digesta of (one for determining pepsin activity and another one for
piglets. determining pH and mineral concentration), and together
with jejunal contents, they were frozen in liquid nitrogen and
2. Materials and methods stored at −80 °C for determination of pepsin activity, pH and
total and soluble Ca, Mg, Na and K contents.
All experimental procedures were reviewed and approved Stomach digesta samples for pepsin activity analysis were
by the University of Manitoba Animal Care Protocol Manage- thawed, and an aliquot was taken and centrifuged for 15 min
ment and Review Committee, and pigs were handled in at 14,000 × g at 4 °C. The supernatants were then collected
accordance with guidelines described by the Canadian and the pepsin activity was determined as described by Rick
Council on Animal Care (CCAC, 1993). Twenty-four Genesus and Fritsch (1974). Samples for pH and mineral concentration
(Yorkshire-Landrace♀ X Duroc♂) piglets (balanced for sex) analyses were thawed, pH immediately determined and
with an initial body weight of 7.60 ± 0.73 kg (mean ± SD) sampled for total and soluble mineral concentrations deter-
were obtained immediately after weaning, and group-housed mination. Aliquots for determining total mineral contents
in pens and fed a commercial starter diet. After 3 days, piglets were freeze-dried, finely ground, ashed for 12 h, digested
were housed individually in pens and randomly assigned to 3 according to AOAC (1990) procedures (method 990.08), and
experimental diets to give 8 piglets per diet. The diets were a read on a Varian Inductively Coupled Plasma Mass Spectrom-
casein–cornstarch-based diet that was supplemented with 0, eter (Varian Inc, Palo Alto, CA, USA). Aliquots for determining
2% PA (as sodium phytate), or 2% PA plus an Escherichia coli- soluble mineral contents were centrifuged for 10 min at
derived phytase at 500 FTU/kg. The basal diet was formulated 2,440 × g at 4 °C, and supernatants were obtained and read as
to meet NRC (1998) energy, amino acids (AA), minerals and described for total mineral content determination. Data were
vitamins requirements for piglets (Table 1). After consuming subjected to analysis of variance using the GLM procedure
experimental diets ad-libitum for 10 days, the piglets were (SAS software release 9.1, SAS Inst., Inc., Cary, NC). The effects
killed, and the contents of the stomach and jejunum (from of PA and phytase were determined using specific contrasts.
80 cm below the pylorus to 80 cm above the ileal-cecal
junction) were obtained and thoroughly mixed. The stomach 3. Results and discussion
contents were immediately divided into 2 equal portions
The effects of PA and phytase on pepsin activity, pH and
soluble mineral concentration in digesta are presented in
Table 2. Phytic acid decreased (P b 0.05) stomach digesta
pepsin activity, which could be due to binding of the PA to
Table 1 pepsinogen. Pig pepsinogen, but not pepsin contains basic AA
Composition of basal diet as fed basis.
(Stepanov et al., 1973), which are positively charged at acidic
Ingredient Amount, g/kg pH found in the stomach, and hence they can form
Cornstarch 486.0
electrostatic bonds with PA, which is negatively charged at
Lactose 200.0 the same pH (Prattley et al., 1982). By binding to the basic AA
Casein 200.0 of pepsinogen, PA may interfere with its conversion to pepsin,
Vegetable oil 30.0 leading to reduced activity of the latter.
Other a 69.0
The stomach and jejunal pH values were similar to values
Vitamin/mineral premix b 15.0
Calculated nutrient content reported by Moore and Tyler (1955) in piglets. Phytic acid did
DE, MJ/kg 15.2 not affect (P N 0.05) stomach pH, but decreased (P b 0.01)
CP, g/kg 177.4 jejunal digesta pH. The reduced pepsin activity due to PA was
Digestible amino acids content, g/kg expected to result in increased secretion of the enzyme and
Lysine 14.0
Methionine 5.2
HCl, resulting in reduced pH of the digesta in the stomach and
Threonine 7.0 the upper part of small intestine (duodenum and jejunum). It
Analysed composition c is, thus, not clear why PA did not affect the stomach digesta
Ca, g/kg 10.6 pH. It could have been due to method errors as it was
K, g/kg 2.55
determined in samples that had been frozen and thawed.
Mg, g/kg 0.93
Na, g/kg 5.30 Phytic acid did not affect (P N 0.05) soluble Na, Ca, Mg and
P, g/kg 5.29 K concentrations in the stomach digesta (data not presented),
Phytase, FTU/kg 56 but decreased Ca2+ and Mg2+ concentrations and increased
a
Other includes: cellulose, 30 g/kg; limestone, 13.0 g/kg; monocalcium Na+ concentration in the jejunal digesta (P b 0.05). The PA-
phosphate, 13.0 g/kg; salt, 5.0 g/kg; potassium carbonate, 4.0 g/kg; magne- related reduction in Ca2+ and Mg2+ concentrations in the
sium oxide, 1.0 g/kg; chromic oxide, 3.0 g/kg. jejunal digesta could be due to lower solubility of these
b
Supplied the per kg of finished diet: retinol, 2479 μg; cholecalciferol,
cations that were bound to PA because the total contents of
25 μg; α-tocopherol, 13.4 mg; phylloquinone, 1.1 mg; riboflavin, 5 mg;
nicotinamide, 36.8 mg; cyanocobalamin, 25 mg; pyridoxine, 4.4 mg; biotin, these minerals in jejunal digesta were not reduced by PA
200 mg; Pteroyl(mono)glutamic acid, 1 mg; choline, 781 mg; copper, 6 mg; (data not presented). The increased Na+ content could be due
iodine, 0.28 mg; iron, 100 mg; manganese, 40; selenium, 0.30 mg; and zinc, to its reduced absorption or increased endogenous secretion
100 mg. or both because its total concentration was also increased by
c
The analysed P and Na contents, and phytase activity in the basal diet
plus phytic acid and basal diet plus phytic acid and phytase were: 10.3 and
PA (data not presented). However, the increased Na+ content
9.36 g/kg for P, 8.84 and 8.53 g/kg for Na, and 78 and 554 FTU/kg for phytase in jejunal digesta could mainly have been due to its increased
activity, respectively. endogenous secretion because we have observed negative
 T.A. Woyengo et al. / Livestock Science 134 (2010) 91–93 93

Table 2
Effect of phytic acid and phytase on gut digesta pepsin activity, pH and soluble mineral content.

Item Diet a SEM Contrasts

Control PA Phytase Control vs. PA PA vs. phytase

Pepsin activity b, PU/ml 265.5 142.5 161.9 35.5 0.028 0.713

Stomach digesta pH 4.60 4.84 4.48 0.277 0.554 0.379
Jejunal digesta pH 7.13 6.61 6.91 0.122 0.0089 0.113
Jejunal mineral content, ppm
Ca2+ 270.2 108.5 128.2 36.8 0.0071 0.719
K+ 653.4 691.2 740.7 75.8 0.737 0.661
Mg2+ 468.5 69.1 113.4 98.7 0.012 0.762
Na+ 2670.2 4191.9 3987.8 163.9 b0.0001 0.405
a
PA = control plus phytic acid, phytase = control plus phytic acid plus phytase.
b
Determined in stomach digesta.

apparent ileal digestibility of Na due to PA (Woyengo et al., References

2009), indicating its increased endogenous loss due to the
same (PA). Phytase did not influence (P N 0.05) any of the
response criteria measured in this study, which could
attributed to the fact that the basal diet was formulated to
be adequate in Ca and available P, which can reduce the
efficacy of phytase.
In conclusion, supplemented PA in the form of sodium
phytate reduced pepsin activity in the stomach digesta, pH
and soluble Ca and Mg contents in the jejunal digesta; and
increased soluble Na content in the jejunal digesta of piglets.
The reduced pepsin activity implies reduced protein digest-
ibility, whereas reduced soluble Ca and Mg contents, and
increased soluble Na content implies reduced Ca and Mg
availability for absorption, and increased endogenous secre-
tion of Na, respectively. Thus, it appears that supplemented
PA reduces nutrient utilization in pigs partly through reduced
pepsin activity in the stomach and altered mineral solubility
in the small intestine.
Conflict of Interest
All authors acknowledge that there that are no conflicts of
interest concerning the information that is provided in the
paper.
AOAC, 1990. Official Methods of Analysis, 15th ed. Assoc. Off. Anal. Chem,
Washington, DC.
Bedford, M.R., 2000. Exogenous enzymes in monogastric nutrition — their
current value and future benefits. Anim. Feed Sci. Technol. 86, 1–13.
CCAC, 1993. Guide to the Care and Use of Experimental Animals. Canadian
Council on Animal Care, Ottawa, ON, Canada.
Cowieson, A.J., Acamovic, T., Bedford, M.R., 2004. The effects of phytase and
phytic acid on the loss of endogenous amino acids and minerals from
broiler chickens. Br. Poult. Sci. 45, 101–108.
de Mitjavila, S., Saint Blanquat, G., Derache, R., 1973. Effet de I'acide tannique
sur la sécrétion gastrique chez le rat. Ann. Nutr. Metab. 15, 163–170.
Moore, J.H., Tyler, C., 1955. Studies on the intestinal absorption and excretion
of calcium and phosphorus in the pig. 3. The effect of beryllium
carbonate on the absorption of phosphorus. Br. J. Nutr. 9, 389–397.
NRC, 1998. Nutrient Requirements of Swine, 10th ed. Natl. Acad. Press,
Washington, D.C.
Prattley, C.A., Stanley, D.W., van de Voort, F.R., 1982. Protein-phytate
interactions in soybeans. II. Mechanism of protein-phytate binding as
affected by calcium. J. Food Biochem. 6, 255–271.
Rick, W., Fritsch, W.P., 1974. Chymotrypsin, Trypsin and Pepsin, second ed.
In: Bergmeyer, H.U. (Ed.), Methods of Enzymatic Analysis, vol. 2. Acad.
Press, New York, pp. 1006–1057.
Stepanov, V.M., Baratova, L.A., Pugacheva, I.B., Belyanova, L.P., Revina, L.P.,
Timokhina, E.A., 1973. N-terminal sequence of swine pepsinogen and
pepsin. The site of pepsinogen activation. Biochem. Biophys. Res.
Commun. 54, 1164–1170.
Woyengo, T.A., Cowieson, A.J., Adeola, O., Nyachoti, C.M., 2009. Ileal
digestibility and endogenous losses of nutrients responses to dietary
phytic acid in piglets. Br. J. Nutr. 102, 428–433.