A W Jongbloed, Z Mroz and P A Kemme

acid in different sections of the alimentary tract.

concentration and apparent digestibility of dry matter, total phosphorus, and phytic
The effect of supplementary Aspergillus niger phytase in diets for pigs on
1992, 70:1159-1168. J ANIM SCI
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The Effect of Supplementary Aspergillus niger
Phytase in Diets for Pigs on Concentration
and Apparent Digestibility of Dry Matter,
Total Phosphorus, and Phytic Acid
in Different Sections of the Alimentary Tract'
A. W. Jongbloed, Z. Mroz, and P. A. Kemme
Research Institute for Livestock Feeding and Nutrition,
Lelystad, The Netherlands
ABSTRACT, Six barrows of approximately 37 kg
BW, fitted with two simple T-cannulas in the
duodenum (25 cm posterior to the pylorus) and
terminal ileum (12 to 15 cm anterior to the
ileocecal junction), were fed two diets containing
2.1 g of P/kg in the form of phytic acid and a low
intrinsic phytase activity (comsoybean meal
based diet [Diet AI or a typical Dutch diet [Diet BI)
without or with supplementary microbial phytase
from Aspergillus niger (var. ficuuml equal to 1,500
phytase units per kilogram of diet, in a crossover
design. The apparent duodenal, ileal, and total
tract (overall) digestibilities of DM, total P, and
phytate P (phytic acid x .282) were calculated using
both Cr-NDR (neutral detergent residue mor-
danted with Cr) and Co-EDTA as dual-phase
markers. Concentration of total P in the ileal
digesta (P e .01) and feces (P c .0011 of pigs fed
microbial phytase was lower than without this
enzyme, irrespective of the diet. Ileal digestibility
of total P was 18.5 and 29.8 percentage units
higher (which was a 1.7- to 2.9-fold increase) due to
added Aspergillus niger phytase (P e .05). Also,
total tract (overall) digestibility increased by 27.0
to 29.7 percentage units (P e .011. Phytic acid
concentration in the duodenal and ileal digesta of
pigs receiving microbial phytase was lower (P e
.01 or .0011, resulting in its higher ileal digestibility
(dephosphorylation rate) by 50.1 percentage units
for Diet A and by 75.4 percentage units for Diet B.
Irrespective of the treatment, no phytase activity
could be detected in the ileal digesta of pigs.
Key Words: Pigs, Cannulation, Phytase, Phosphorus
Introduction
Supplementary microbial phytase from Asper-
gilli in diets for pigs is used to enhance digestibil-
ity of myo-inositol phosphates, insoluble com-
plexes of phytic acid, and minerals from feedstuffs
of plant origin (Han, 1989; Zyla et al., 1989; Simons
et al., 1990). A degree of phytate degradation
(dephosphorylation, hydrolysis) may also be
related to the presence of intrinsic plant phytases,
'The authors wish to thank the Gist-brocades (The Nether-
lands) for financial support, all the coworkers at the IVVO
CLelystad), CIVO (Zeist), Gist-brocades (Delftl, and the Phospho-
rus Working Group from the Commodity Board for Feedstuffs
(The Netherlands) for their excellent advice.
Received J anuary 25, 1991.
Accepted October 31, 1991.
J . Anim. Sci. 1992. 703159-1168
phytases from the bacterial flora in the gut, and
intestinal mucosal phytases Williams and Taylor,
19851, although Pointillart (1988) concluded that
intestinal phytase activity in pigs is negligible.
According to Cosgrove (19801, Aspergilli produce
3-phytase (EC 3.1.3.81, a nonspecific phos-
phomonoesterase, catalyzing the following reac-
tion: myo-inositol hexakisphosphate +H20 + D-
myo-inositol - 1,2,4,5,6-pentakisphosphate + or-
thophosphate. This reaction proceeds in a step-
wise manner, producing five classes of intemedi-
ate products (myo-inositol pentakis-, tetrakis-, tris-,
bis-, and monophosphates) of variable
stereochemistry (Maga, 1982; Frnrlich et al., 1986).
However, the effect of this enzyme on phytate
degradation in the gastrointestinal tract of pigs is
still not well elucidated and in vitro studies of Han
and Wilfred (19881, Han (19891, and Bos (19881 do
1159
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1160 J ONGBLOED ET AL.
not necessarily relate quantitatively to the in vivo
conditions, in which factors such as gastric empty-
ing, retention time, variation of pH and digesta
composition, and interactions between minerals
and other nutrients (dietary and endogenous) may
affect the dephosphorylation reactions. Besides,
myo-inositol phosphate is capable of forming
insoluble complexes containing Ca and other
cations, and the extent to which such complexes
are formed in vivo in animals fed practical diets is
dependent on the concomitant presence of high
concentrations of each constituent in the intestinal
chyme (Oberleas and Moody, 1982; Suttle, 1983).
Therefore, the present experiment was conducted
to study the effect of Aspergillus niger bar. ficuuml
phytase in diets for pigs on the concentration and
apparent digestibility of DM, total P, and phytic
acid in different sections of the alimentary tract.
Materials and Methods
AnimaZs. Six barrows from a three-breed rota-
tional cross (Yorkshire, Finnish Landrace, and
Dutch Landrace) of 37-kg average initial BW, fitted
with two simple T-cannulas under inhalation
anaesthesia (one located in the duodenum approx-
imately 25 cm posterior to the pylorus and another
in the terminal ileum, approximately 12 to 15 cm
anterior to the ileocecal junction), were used in
this experiment. After a 14-d recovery period, the
pigs were housed individually in pens of 2.00 m x
1.45 m, at ambient temperature (18OC1, and as-
signed to four experimental treatments (providing
six replications) according to a crossover design as
follows: 1) corn-soybean meal diet (Diet A) without
microbial phytase, 21 corn-soybean meal diet (Diet
A) with microbial phytase, 3) typical Dutch diet
(Diet B) without microbial phytase, and 41typical
Dutch diet (Diet B) with microbial phytase. Final
BW of the animals was approximately 75 to 80 kg.
Diets. The major components of Diet A were
ground corn and extracted soybean meal, whereas
Diet B contained tapioca, extracted soybean meal,
hominy feed, extracted sunflower meal, and soy-
bean oil (Table 1). These components are known to
have a low intrinsic phytase activity ( e 50 phytase
units per kilogram of feed). Both diets were
calculated to have similar protein (approximately
11.5% digestible protein) and energy levels (approx-
imately 12.5 MJ of ME/kg or 3,000 kcal of ME/kg).
Treatments 2 and 4 were supplemented with 4 g/
kg of crude microbial phytase preparation from
Aspergillus niger (var. ficuum) (equal to approxi-
mately 1,500 phytase units/kg of diet). This en-
zyme was obtained from A. niger (var. ficuurn)
strain NRRL 3135 according to the procedure
described by Simons et al. (1990). The activity of
the crude microbial phytase showed pH optima at
pH 5.5 and 2.5. The enzyme was able to hydrolyze
phytate complexes in vitro in soybean meal, corn,
and liquid compound feed for pigs. The thermal
stability of the microbial phytase was good &e.,
95% of phytase remained active when a meal was
steam-heated at 50C, as part of the pelleting
process). Pigs were fed twice daily (0700 and 1500)
in a wet, mash form at a feeding level of 2.3 times
maintenance requirement (maintenance require-
ment = 418 kJ of ME/BW.75). Daily rations were
supplemented with 4 g of neutral detergent resi-
due mordanted with Cr per kilogram of diet as an
indigestible marker for the solid phase and Co-
EDTA (5 g/kg of diet) for the liquid phase and
mixed with water at a ratio of 2.5:l (vol/wt) just
before feeding. The pigs had no access to water
between feedings.
Collection Procedures. Each treatment was tested
for 14 d. After the first 3 d, samples of duodenal
digesta were collected five times at 1- or
1.5-h intervals after the morning meal on the 4th
and 6th d of the test period. Feces were collected
at random on the 9th and 10th d, whereas samples
of ileal digesta were collected quantitatively on
the loth, Wth, and 14th d (seven times in
1- to 2-h intervals, beginning at 0700). The samples
of digesta were collected into sterilized polyethyl-
ene bags attached to the cannula barrel and were
frozen immediately after the pH was measured.
Table 1. Ingredients and nutritive value
of experimental diets
Diet A Diet B
(corn-soybean (Typical
Item meal) Dutchl
Ingredient
g/kg
Corn 859.50 -
Soybean meal 124.55 124.55
Tapioca - 421.00
Hominy feed - 336.00
Sunflower meal - 80.00
Soybean oil - 26.00
Limestone 11.80 8.30
Salt 2.50 2.50
Trace mineral-vitamin premix* 1.40 1.40
Choline chloride .25 .25
GE, MJ k g 15.9 16.2
ME, MJ/kg 12.7 12.4
Calculated nutritive value
Digestible protein, g/kg 114 119
Digestible P, g k g .9 1.2
Digestible lysine, g/kg 5.0 5.0
%ace mineral-vitamin premix contained the following in
mi l l i grams per kilogram of diet: 2.4 (8,000 IU) vitamin A, .04
(1,600 IU) vitamin D3, 8.0 (8.0 IU) vitamin E, 4.0 riboflavin, 20.0
nicotinic acid, 8.0 panthotenic acid, .02 vitamin BIZ, 125.0 antiox-
idant (4 to 5% BHA, 4 to 5% ethoxyquin, 4 to 5% citric acid, 2 to
3% o-phosphoric acid, 2 to 3% E 471 fatty acid, and Si02 as
carrier), 50 MnO, 155 %SO4, 40 CuS04, 2 KI, 430 FeS04, .3 Se,
and 555.5 carrier.
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ASPERGILLUS NIGER PHYTASE IN DIETS FOR PIGS 1161
Thereafter, they were freeze-dried and ground to
pass a 1-mm sieve before analysis.
Analytical Procedures. Dry matter was deter-
mined after freeze drying and nitrogen content
waa determined by the Kjeldahl method [AOAC,
1080). Crude protein was calculated as Kjeldahl N
x 6.25. Content of Ca, Mg, Na, K, Cr, and Co was
assayed by atomic absorption spectrophotometry
after ashing the samples at 46OOC and preparing
mineral solutions in .48 M HC1. Total P was deter-
mined colorimetrically by the vanadomolybdate
procedure (AOAC, 10801. Phytic acid was extracted
and quantitatively assayed by the HPLC method
of Simons et al. (10001, whereas separation of myo-
inositol tetrakis- and pentakisphosphates from
degraded phytates was carried out using an anion-
exchange column (Dionex, AS 3, Dionex, Sun-
nyvale, CAI with .OO M H N 0 3 at a flow rate of 1.0
ml/min IBos, 1088). Measured concentrations of
particular nutrients in the samples of digesta from
the duodenum and the ileum of the experimental
pigs were related to the total volume of digesta
passing these sections within the time of our
sampling by an extrapolation from the pattern of
flow of digesta described by Braude et al. (10761.
Phytase activity in the diets was assayed by
measuring the amount of o-phosphates released
from phytic acid within a period of linear increase
with time (Simons et al. 1990). One unit of phytase
activity is equal to 1 p o l of o-phosphate liberated
from 1 mo l of phytic acid within 1 min at 40C
and pH 5.5.
Statistical Analysis. Each pig was the experi-
mental unit. All data were statistically analyzed
using the analysis of variance according to the
where N = overall mean, A = effect of animal, P
= effect of period, T = effect of treatment, and e
= error contribution with average 0 and variance
02,andi = l . . . a, j = l . . . b, k= l ...n.Therewas
no evidence of a P x T interaction. Treatment
means were compared by the Student t-test at P <
.05, .01, and .001.
following model: Yijk = N +Ai +Pj +TK +eijk,
Results
Corn-soybean meal Diet A contained 3.3 g of
total P/kg, of which 78.8% was as phytate-bound P
(myo-inositol hexakisphosphate [IPS, 63.6%1, myo-
inositol pentakisphosphate DP5, 9.1/01, and myo-
inositol tetrakisphosphate [IP4, 6.1/01) (Table 21. The
typical Dutch Diet B had 4.1 g of total PAg, of
which 70.7% was in the form of phytate P (IPS,
51.2%; IPS, 12.2%; and IP4, 7.3%). The pen-
takisphosphates aP5) were separated aa Denan-
tiomers (D-myo-inositol 1,2,3,4,5-pentakis phos-
phates, IP,,) and Lenantiomers (L-myo-inositol
Table 2. Analyzed chemical composition of experimental diets
Nutrient
Diet A Diet B
- Phytase +Phytase - Phytase +Phytase
DM
Ash
Crude protein
Crude fat
Crude fiber
Calcium
Magnesium
Total phosphorus
Potassium
sodium
Myo-inositol phosphates8
Wl
p 5 b
P4a
-58
PAh
--
P I C
Ip6
Phytate-bound phosphorusb
863
35
136
34
25
5.2
1.5
3.3
6.0
1.1
863
35
136
32
27
5.0
1.5
3.3
5.9
1.8
882
64
158
47
64
5.6
2.5
4.1
10.0
1.5
882
65
156
47
06
5.4
2.3
4.1
10.0
2.2
7.4 7.3 7.6 7.4
.4 .4 .7 .7
.9 1.1 1.4 .6
.4 .4 .6 .0
.2 .2 .4 .4
.2 .2 .4 .4
2.1 2.1 2.1 2.1
.3 .3 .5 .5
.2 .2 .3 .3
Tot& (IPle +I ps +IP4) 2.6 2.6 2.9 2.9
~
aMyo-inositol hexakisphosphate (IPB), myo-inositol pentalcisphosphate (Pd, and myo-inositol
bPhosphorus content in IP6, P5, and P4 is 28.2, 22.2, and 20.0%, respectively.
tetrakisphosphate (IP4).
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1162 JONGBLOED ET AL.
7 -
0 -
6 -
4 -
9 -
2 -
1 -
0 -
CORN-SOYBEAN BASED DIET
(DUODENUM)
g / b FRESH
180
140 I I I I
120
100
80
00
40
20
rl

at hodlng aftor 1 h 2 h 9.6 h 6 h
TIME
WITHOUT PHYTME Wl TH PHYTME 8lMMnD DEVlATlON
Figure 1. Effect of microbial phytase from Aspergillus
niger on changes of dry matter content in the duodenal
digesta of pigs measured at feeding and after feeding.
1,2,3,4,5-pent akisphosphates, IP~b). The tetrakis-
phosphates (IP4) were isolated as L-myo-inositol
1,2,3,44etrakisphosphates (IP4,3, D-myo-inositol
1,2,5,6-tetrakisphosphates CIP4bl, and D-chiro-in-
ositol 1,2,3,6-tetrakisphosphates CIP4,). Ratios of
Ca:total P:Mg were 1.5:1:.5 in Diet A and 1.3:1:.6 in
Diet B. In both diets, the proportion of Ca:phytate-
bound P was similar, (approximately 1.9:ll.
Concentrations. Concentrations of DM, total P,
and phytic acid UP6) in the duodenal and ileal
digesta and in the feces (except IP61 of pigs are
presented in Table 3. One pig was excluded from
this experiment in the third period because of a
sudden health problem, and therefore the mean
values for Diet B are from five animals only.
Irrespective of the diet, an average concentration
of DM in the duodenal and ileal digesta of pigs fed
diets containing A. niger phytase was slightly
lower than that in the digesta of pigs fed diets
without this enzyme. During the time course after
feeding, this difference was more apparent in the
duodenal digesta (Figure 11 than in the ileal
digesta (Figure 21, although not significantly. Pres-
ence of microbial phytase in both diets did not
significantly affect total P concentration in the
duodenal digesta, whereas phytic acid concentra-
tion was substantially reduced from 8.2 to 2.9 g/kg
of DM in Diet A and from 9.4 to .6 g/kg of DM in
Diet B ( P < .OOll. Phytate complexes were hydro-
lyzed in the stomach, but liberated o-phosphates
were not absorbed anterior to the duodenal cannu-
la. This effect of microbial phytase on changes of
phytic acid concentration in the duodenal digesta
measured at feeding and after feeding is illus-
trated in Figure 3. Levels of phytic acid were
consistently lower when phytase was added, and
in the case of Diet A, were more related to the
diurnal flow of digesta, whereas in the case of Diet
CORN-SOYBEAN BASED DIET
(ILEUM)
g/kg FRESH
140
120 I
100
80
80
40
20
n
-
at hodl ng dt or 1 h 2 h 9.6 h 6 h 7 h Oh
TIME
=WITHOUT PHYTME WITH PHYTME 8TANDARD DEVIATION
Figure 2. The effect of microbial phytase from
Aspergillus niger on changes of dry matter content in the
ileal digesta of pigs measured at feeding and after
feeding.
CORN-SOYBEAN BASED DIET
(DUODENUM)
al ko DM
.
4
8t I
at feeding
1 attor 1 h
2 h
TIME
9.6 h 6 h
WITHOUT PHYTME WITH PHYTME h 8 TA NMRD DEVIATION
Figure 3. Effect of microbial phytase from Aspergillus
niger on the variation of myo-inositol hexakisphosphate
( 1 ~ ~ ) concentration in the duodenal digesta of pigs
measured at feeding and after feeding.
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ASPERGILLUS NIGER PHYTASE IN DIETS FOR PIGS 1163
B, no phytic acid was found later than 2 h after
feeding. Content of total P in the ileal digesta of
pigs given the microbial phytase preparation was
30.8 and 28.6% lower (P < .01 or .001) for Diets A
and B, respectively. Similarly, a lower phytic acid
concentration was noted at this section of the
alimentary tract (29.5 vs 12.5 g/kg of DM for Diet A
and 20.8 vs 5.6 g/kg of DM for Diet B). These
differences in the concentration of phytic acid in
the ileal digesta increased during the time course
up to 5 to 6 h and thereafter slightly decreased
again (Figure 41. Concentration of DM in feces of
pigs fed either Diets A or B was not influenced by
added microbial phytase, whereas total P concen-
tration was significantly reduced from 21.0 to 13.6
g/kg of DM in Diet A and from 15.8 to 10.4 g/kg of
DM in Diet B. Concentrations of myo-inositol
pentakis- and tetrakisphosphates in the duodenal
and ileal digesta of pigs are presented in Table 3.
The averages are based on two replicates onl y,
because amounts of digesta collected from the
pigs were limited. Nevertheless, for a preliminary
orientation it seemed worthwhile to provide these
data, particularly because no such information
was available in the literature. The amount of
pentakis- and tetrakisphosphates in DM of digesta
from the duodenum of pigs fed without microbial
phytase was higher than with this enzymatic
preparation, irrespective of the diet. A similar
effect was noted at the end of the small intestine of
pigs fed Diet B, whereas for unknown reasons the
level of pentakisphosphates was higher when Diet
A with microbial phytase was given.
CORN-SOYBEAN BASED DIET
(DUODENUM)
PH
10
Q t
7 t
at kodl ng aftor 1 h 2 h 3.6 h 6 h
TIME
I WITHOUT PHYTME a WITH PHYTME fi OTANMAD DEVIATION
Figure 5. Effect of microbial phytase from Aspergillus
niger on pH changes in the duodenal digesta of pigs
measured at feeding and after feeding.
pH. Changes of p H in the duodenal and ileal
digesta, respectively, of pigs fed diets without or
with microbial phytase are presented in Figures 5
and 6. Irrespective of the treatment, the pH of
duodenal digesta gradually decreased from 5.7 to
6.1 to 3.3 to 4.2. The pH values measured in the
CORN-SOYBEAN BASED DIET
(ILEUM)
DM
30 I I 1
CORN-SOYBEAN BASED DIET
(ILEUM)
-
PH
i n ,
I"
9 1
26
a
7
0
16 6
4
3
2
1
20
10
6
n n
"
at kodlng mfhr 1 h 2 h 3.6 h 6 h 7 h Oh
TIME
WITHOUT PHYTME a WITH PHY- OTANDARD DEVIATION
Figure 4. Effect of microbia1 phytase from Aspergillus
niger on the variation of myo-inositol hexakisphosphate
(IP6) concentration in the ileal digesta of pigs measured
at feeding and after feeding.
Y
at foodlng aftor 1 h 2 h 8.6 h 6 h 7 h Q h
TIME
WITHOUT PHYTAOE a WITH PHYTME OTANMRD DEVIATION
Figure 6. Effect of microbial phytase from Aspergillus
niger on pH changes in the ileal digesta of pigs
measured at feeding and after feeding.
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1164 JONGBLOED ET AL.
Table 3. Concentration of nutrients in digesta and feces of pigs fed diets
without or with microbial phytase from Aspergillus niger
Diet A Diet B
Item - Phytase +Phytase SEDa - Phytase +Phytase SED8
DM g/kg fresh
Duodenal digesta 113.2 108.5 8.3 131.3 125.0 6.0
Ileal digesta 88.3 87.5 2.8 100.0 95.3 2.4
Feces 309.5 296.5 5.0 300.2 30 1.8 3.8
Total phosphorus g/kg DM
Duodenal digesta 3.6 3.8 .2 4.5 4.4 .1
Ileal digesta 9.5 6.6 .6** 7.7 5.5 .2* *
Feces 21.0 13.6 1.3** 15.8 10.4 .4**'
Duodenal digesta 8.2 2.9 .3*** 9.4 .6 2***
Duodenal digesta .8 .6 .OB 1.2 .3 .21
Phytic acid (IPS)
5.6 e***
Ileal digesta 29.5 12.5 2.1.. 20.8
Myo-inositol pentakisphosphate (IF5) SDb
Ileal digesta 1.7 2.4 .15 2.9 2.4 .13
Duodenal digesta .2 .1 .05 .5 .1 .14
Myo-inositol tetrakisphosphate UP4)
Ileal digesta .7 .4 .10 1.5 .7 .20
%andard error of difference.
bStandard deviation because number of replicates was only 2).
**P < .01.
***P < .001.
duodenal digesta of pigs fed the diets with micro-
bial phytase were slightly lower 5 h after feeding.
The pH of the ileal digesta vaned between 6.8 and
7.9 and was not clearly related to the treatments
or to the time course.
DigestibiIiiy. Apparent digestibility coefficients
of the nutrients in different sections of the alimen-
tary tract are expressed as the mean values
obtained by using Cr and Co (as markers for the
solid and liquid phases, respectively). Digestibility
of DM (duodenal, ileal, and total tract) was not
significantly influenced by the treatments ff able
4). Negative values obtained at the duodenal site
can be explained by the exocrine secretion of
gastric, biliary, and pancreatic juices. Similarly,
no statistical differences were noted in the digesti-
Table 4. The effect of supplementary Aspergillus niger phytase in diets for pigs
on apparent digestibility of nutrients in different sections of the alimentary tract
Diet A Diet B
- Phytase +Phytase SED8 - Phytase +Phytase SED8 Item
DM
Duodenal
Ileal
Total tract
(Overalll
Duodenal
Ileal
Total tract
(Overall)
Phytic acidb
Duodenal
Ileal
Total phosphorus
,"
10.3
2.0
-11.2
48.8
-4.8
54.2
6.5
3.3
-4.2
67.9
3.8
70.0
83.9 84.1 1.2 78.2 79.8 .5
8.4
26.4
-4.4
44.9
12.1
4.4'
-7.0
16.0
1.6
45.8
6.7
6.1**
12.9 42.6 4.0"' 27.8 54.8 2.0**
21.5
9.6
69.2
59.7
5.4"*'
6.2**
1.2
-1.4
93.2
74.0
5.5***
6.4***
%andard error of difference.
bPhytic acid was not present in feces.
*P .05.
**P < .01.
***P < ,001.
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ASPERGILLUS NIGER PHYTASE IN DIETS FOR PIGS
Table 5. Phytase activities in diets and digesta of pigs fed
without or with microbial phytase fromAspergillus niger
Diet A Diet B
Item - Phytase +Phytase - Phytase +Phytase
1165
Phytase activity in
-
X
SDb
Phytase activity in
-
X
SD
Phytase activity in
-
X
SD
mo l . mh-' . kg-'
diets
c 50a 1,585
- 150
c 50 1,330
- 125
< 50 <50
duodenal digesta
ileal digesta
- -
diet
< 50 1,550
- 150
< 50 955
- 150
aThe lower detection limit.
bStandard deviation.
bility of total P in the duodenum due to the
treatments. However, the ileal digestibility of total
P was 18.5 to 29.8 percentage units higher (which
was a 1.7- to 2.9-fold increase) due to added A.
niger phytase (P < .05 or .01). Also, its total tract
(overall) digestibility increased by 27.0 to 29.7
percentage units (P < .011. Gastroduodenal degra-
dation (dephosphorylation) rate of myo-inositol
hexakisphosphate (expressed as the apparent di-
gestibility of phytic acid) in Diets A and B
supplemented with microbial phytase was higher
by 47.7 and 92.0 percentage units, respectively (P
< .01 or .001). The relative values of phytic acid
digestibility measured at the terminal ileum of
pigs have also been increased by 50.1 and 75.4
percentage units due to microbial phytase. Nei-
ther the concentration nor the apparent digestibil-
ity of phytic acid in feces was measured, because
ortho-phosphates liberated by enzymes of micro-
organisms present in the large intestine did not
seem to be absorbed. Effective utilization of
liberated ortho-phosphates from myo-inositol hex-
akisphosphate by the animal was assumed to take
place in the small intestine only (Smith et al., 1955;
Gueguen et al., 1968).
Phytase Activity. Although the values of phytase
activity presented in Table 5 represent the activity
of the supplementary microbial phytase from A.
niger, dietary plant phytases, phytases from the
bacterial flora in the gut, and intestinal mucosal
phytases, it should be noted that there was no
detectable phytase activity in either of the unsup-
plemented diets or in the duodenal and ileal
digesta of pigs fed these diets. This suggests that
the other sources of phytase were negligible. Total
phytase activity in Diets A and B was lowered by
235 and 595 phytase units after the gastroduo-
denal degradation, respectively. Irrespective of the
treatment, no phytase activity was detected in the
ileal digesta. A. niger phytase was active anterior
to the ileal cannula only.
Discussion
Some attempts have been made to measure the
practical effects of microbial phytase in diets for
poultry and pigs (Shieh et al., 1969; Nelson et al.,
1971; Shurson et al., 1984; Han, 1989; Zyla et al.,
1989; J ongbloed et al., 1990; Simons et al., 1990).
However, the degree of dephosphorylation of
phytate compounds by this enzyme in different
sections of the gastrointestinal tract of pigs has
not been studied. In all previous in vivo experi-
ments on pigs, only dietary plant phytase was
evaluated (Schulz and Oslage, 1972; Pointillart et
al., 1987; Scheuemann et al., 1988; Kemme and
J ongbloed, 1989, 1990; Lantzsch et al., 1992). In our
study, the effect of A. niger phytase on the
apparent DM and total P digestibility and phytic
acid degradation was measured in three sections
of the gastrointestinal tract: 11 in the stomach and
the first part of the duodenum (approximately 25
cm), 2) in the small intestine (between the duode-
num and terminal ileum), and 3) in the large
intestine (except for phytic acid, which was as-
sumed to be hydrolyzed by bacterial microflora
and liberated o-phosphates excreted in feces).
In general, the relative measurements of the
duodenal, ileal, and total tract (overall) digestibili-
ties of those nutrients indicated a positive influ-
ence of A. niger phytase on dephosphorylation of
the dietary inositol phosphates. It was found that
from 60 to 74% of phytic acid could be hydrolyzed
at the end of the small intestine, whereas only up
to 10% from the unsupplemented diets could be
hydrolyzed. Lantzsch et al. (1992) reported that
due to dietary plant phytase approximately 38% of
phytate P from corn was absorbed in the stomach
and proximal half of the small intestine, whereas
up to approximately 55% was absorbed by the end
of the small intestine. However, these results were
obtained using the postslaughter technique and
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1166 J ONGBLOED ET AL.
the authors reported some experimental difficul-
ties with markers. Also, Gukguen et al. (1968)
observed that plant phytase hydrolyzed phytate P
from activated wheat bran mainly in the stomach
of pigs. From postslaughter studies on the extent
of intestinal hydrolysis of phytates, Schulz and
Oslage (1972) reported that, due to plant and
microbial phytases in the stomach and small
intestine, approximately 33% of ingested phytate P
was hydrolyzed within 3 h, and an average of 42%
of the phytate P was released 9 h after the last
feeding of pigs with cereals, milling byproducts,
and oilseed residues. In our experiment, addition
of the enzymatic preparation increased the appar-
ent ileal digestibility of total P by 18.5 to 29.8
percentage units and the overall apparent digesti-
bility of total P by 27.0 to 29.7 percentage units. It
is somewhat confusing that the degree of phytic
acid degradation in the stomach and anterior to
the duodenal cannula was higher than that mea-
sured at the terminal ileum, irrespective of the
treatments (Table 4). This may be explained by the
fact that the results obtained at the duodenal level
were corrected for the total flow of digesta,
according to the pattern of flow described by
Braude et al. (1976). It is evident that the rate of
passage of digesta is influenced by many factors,
such as dietary composition, feeding level, water
supply, stress, exercise, gastrointestinal anatomy,
and procedures employed to measure it [Low,
1980). The data of Braude et al. (1976) were used as
a base-point for our estimation of phytate phos-
phorus availability in the duodenum and the
ileum, assuming that most of the above-mentioned
factors might have been similar in both trials.
However, such an extrapolation presumably over-
estimated the extent of phytic acid hydrolysis
anterior to the duodenal cannula. Besides, it was
reported that the composition of the spot samples
of the duodenal digesta is more variable than the
composition of the ileal digesta, because the liquid
phase may flow in the duodenum much faster than
the solid phase, a phenomenon associated with
gastric emptying (Low et al., 1978). In this context,
it may be also hypothesized that the lower values
of phytic acid digestibility obtained at the ileal
than at the duodenal level might be partly due to
de novo synthesis of myo-inositol phosphates in
the small intestine of pigs. This hypothesis is
indirectly supported by the fact that in blood of
birds myo-inositol pentakisphosphate (IPS) was
found, which must have been synthesized in the
blood, and not just absorbed in this form (Cos-
grove, 1980). From the results presented in Table 4,
there is evidence of a net disappearance of P
posterior to the ileal cannulas of pigs fed with Diet
B and P influx into the lumen of the large intestine
of pigs fed the corn-soybean meal-based diet. In
studies by Partridge (19781, growing pigs fed diets
based on starch, sucrose, and peanut meal had a
significant net secretion of P in the large intestine.
Also, there was a tendency for a net absorption in
this region when diets consisting of starch, su-
crose, and casein were fed. Drochner (1984) re-
ported that a control diet based on wheat and
soybean meal fed to growing minipigs resulted in a
negative apparent digestibility in the post-ileal
region, whereas the addition of fibrous products to
the diet such as crude wood fiber product, isolated
wood cellulose, or pectin resulted in a positive
apparent digestibility of P that largely compen-
sated the prececal negative effects. Therefore,
although urinary excretion seems to be the main
process responsible for P homeostasis in nomumi-
nants (Irving, 19641, other regulation mechanisms
of the efficiency of absorption of P according to the
P intake may also occur (Challa and Braithwaite,
1989). Further studies of the metabolic fate of P in
the large intestine of pigs should elucidate this
phenomenon. Apparently, in our studies there is a
relatively weak correlation between the extent of
IPS (phytic acid) digestibility and total P digestibil-
ity at the ileal level. Degradation of phytic acid
proceeds in a stepwise manner, producing five
classes of intermediate products that cannot be
absorbed. Only released o-phosphate ions (PO4)
from this dephosphorylation reaction can pass
through the gastrointestinal wall and become
available for the animal. In addition, we were not
able to determine how much endogenous P was
present in the digesta. Smith et al. (1955) con-
cluded that endogenous P seemed to enter the
proximal half of the small intestine and be
reabsorbed to a greater extent than dietary P in
the end of the small intestine. The mechanisms
regulating the flow of endogenous P in the small
intestine of pigs are still not known. To gain more
insight into the differences in overall absorption
percentages of P between feedstuffs, some authors
assume that the amount of endogenous P in feces
ranges from 5 to 10 mg/kg of BW (J ongbloed,
19871, but the same amount should not necessarily
be expected at the ileal level. Lowering of phytase
activity by 235 and 595 phytase units after the
gastroduodenal degradation resulted in an in-
crease of phytic acid degradation of 47.7 and 92.0
percentage units in Diets A and B, respectively.
Therefore, for each reduction of one phytase unit,
phytic acid degradation between the diet and
duodenum increased .2O% (47.7%/235) in Diet A
and by .l6% (92.0%/595) in Diet B. Bearing in mind
that microbial phytase is most active at pH 2.5 and
5.5 (Simons et al., 19901 and that posterior to the
duodenum the pH is above 6.0 (minimizing its
capability to hydrolyze phytic acid), it can be
concluded that a substantial degradation of phytic
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ASPERGILLUS NIGER PHYTASE IN DIETS FOR PIGS 1167
acid takes place in the gastroduodenum, whereas
liberated ortho-phosphates are absorbed in the
small intestine. This conclusion is supported by
the fact that the further lowering of phytase
activity posterior to the duodenal cannula was not
related to an increased rate of phytic acid degra-
dation between the duodenum and the terminal
ileum.
Implications
Supplementary microbial phytase from Aspergil-
lus niger (var. ficuurn) in the amount of 1,500
phytase units per kilogram of feed significantly
reduced the concentration of total P in the ileal
digesta and feces of pigs. This resulted in higher
ileal digestibility of total P from 18.5 to 29.8
percentage units and total tract (overall) digestibil-
ity from 27.0 to 29.7 percentage units. No phytase
activity was detected in the ileal digesta.
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