Simultaneous application of phytase andxylanase to broiler feeds based on wheat: in vitromeasurements of phosphorus and pentoserelease from wheats and wheat-based feeds †

Krzysztof Zyra,1* Dorota Gogol,1 Jerzy Koreleski,2 Sylwester Swiatkiewicz2 andDavid R Ledoux3

1University of Agriculture, Department of Food Biotechnology 29-Listopada Avenue 46, 31–425 Krakow, Poland2Institute of Zootechnics, Research Farm of Balice, Department of Animal Nutrition in Brzezie, 32–080 Zabierzow, Poland3112 Animal Science Research Center, University of Missouri-Columbia, Columbia, MO 65211, USA

Abstract: An in vitro procedure that simulated digestion in growing broilers was tested to predict

phosphorus availability and arabinoxylan hydrolysis in samples of nine wheat varieties and in a wheat-

based diet. Amounts of dialysable phosphorus freed from wheat samples correlated with activities of

endogenous phytase (R =0.913; p<0.0001), whereas amounts of pentoses released were correlated with

viscosities of the digested samples (R =0.899; p<0.0001). Differences in phosphorus release resulting

from graded levels of microbial phytase added to feeds that were either autoclaved or not autoclaved

revealed a decreasing role of endogenous phytase in dephosphorylation as levels of microbial phytase

supplementation grew. Amounts of pentoses released from feeds containing two different xylanase

preparations re¯ected literature data on different in vivo ef®cacies of those preparations. Simul-

taneous addition of phytase and xylanase affected phosphorus release in a manner that depended upon

the form of xylanase preparation used (liquid or powder). There was a positive in¯uence of acid

protease on both phytate and arabinoxylan hydrolysis in feeds supplemented with phytase. Effects

observed by the in vitro procedures corresponded to in vivo phenomena described in the literature.

# 1999 Society of Chemical Industry

Keywords: in vitro procedure; phytase; xylanase; protease; wheat

INTRODUCTIONPhytase and xylanase constitute two successful niches

of microbial enzyme application in poultry nutrition.

Supplementation of diets with phytate phosphorus

content ranging from 1.7 to 3.0g kgÿ1 with microbial

phytase has been shown to increase phosphorus

availability in monogastric animals and reduce phos-

phorus content in animal manure.1±3 Wheat, among

cereals, is known to be high in endogenous phytase

activity that contributes to the availability of phytate

phosphorus.4,5 Larsson et al6 have declared that the

release of free phosphorus during in vitro digestion of

wheats is correlated with the endogenous phytase

activity. Although wheat phosphorus is more available

to monogastrics than phosphorus of many other

feedstuffs, most likely owing to high endogenous

phytase activity,7 a substantial fraction of phytate in

wheat remains undigestible. Kiiskinen et al8 reported

51% phosphorus utilisation in broilers fed on wheat-

and barley-based diets, whereas the inclusion of

microbial phytase in those diets resulted in 60%

phosphorus utilisation. Similar values of phosphorus

retention were reported when microbial phytase was

added to diets containing maize.3 On the other hand,

phosphorus utilisation as high as 80% was observed in

studies with turkeys fed on maize±soybean meal diet

supplemented with a cocktail of phytate-degrading

(phytase, acid phosphatase) and tissue-degrading

(acid protease, pectinase) enzymes.9

Wheat contains substantial amounts of arabinoxy-

lans that, by elevating intestinal viscosity, impair

nutrient bioavailability, decrease metabolisable energy

and consequently lower the performance of birds fed

on wheat-based diets. Addition of a suitable xylanase

of microbial origin to poultry diets alleviates those

problems owing to depolymerysation of the arabi-

noxylans, thereby reducing the impact on intestinal

viscosities.10

Journal of the Science of Food and Agriculture J Sci Food Agric 79:1832±1840 (1999)

* Correspondence to: Krzysztof Zyra, University of Agriculture, Department of Food Biotechnology, 29-Listopada Avenue 46, 31–425 Krakow,Poland† Presented in part at the 11th European Symposium on Poultry Nutrition, Faaborg, Denmark, 24–28 August 1997Contract/grant sponsor: Polish Research Council; contract/grant number: 109/P06/96-6-1185Contract/grant sponsor: United States Department of Agriculture; contract/grant number: RSED-PL25(Received 2 March 1998; revised version received 22 October 1998; accepted 23 April 1999)

# 1999 Society of Chemical Industry. J Sci Food Agric 0022±5142/99/$17.50 1832

Simultaneous application of phytase and xylanase to

broiler feeds based on wheat may be expected to

reduce intestinal viscosity, enhance absorption of

nutrients and possibly enhance absorption of phos-

phorus released from phytate by phytase. It should be

pointed out, however, that Van der Klis et al11 found a

signi®cant positive in¯uence of endoxylanase supple-

mentation to wheat-based diets on the absorption of

calcium, magnesium, sodium and potassium in

broilers, but there was no effect on phosphorus

absorption. First attempts to optimise simultaneous

application of phospholytic and viscosity-reducing

enzymes in poultry feeds have been made by Newkirk

and Classen12 on laying hens. In a study with broilers

performed by Adrizal at al,13 simultaneous application

of phytase and xylanase to broiler diets containing

225g kgÿ1 defatted rice bran did not result in signi-

®cant differences among dietary treatments.

Although methods based on single incubations of

feed samples with phytase2,14 have not been reliable,

there have been successful in vitro approaches used to

predict phosphorus availability from feeds containing

commercial phytase. The in vitro method of Zyla etal15 was designed for studying enzymatic dephos-

phorylation of phytate by phytase in maize±soybean

meal diets for turkey poults. The three-step method,

which simulated digestive conditions of the crop,

gizzard and duodenum of the turkey, allowed the

design of a cocktail of enzymes that caused complete

dephosphorylation of maize±soybean meal feed invitro16 and 80% phosphorus utilisation in growing

turkey.9 The method has subsequently been modi®ed

for studying enzymatic dephosphorylation of phytate

in growing swine17 as well as for studying wheat

digestion in the presence of xylanase and cellulase.18

As the in vitro gastrointestinal model of the turkey

fed on maize±soybean meal diets cannot be directly

applied to broilers receiving diets high in wheats, the

objective of this research was to modify the model

appropriately and examine effects of different enzymes

on phytate dephosphorylation and on arabinoxylan

hydrolysis in a wheat-based feed under simulated

intestinal conditions of growing broilers.

MATERIALS AND METHODSMaterialsDodecasodium phytate, oat spelt xylan and dialysis

tubings (molecular weight cut-off 12000±14000,

diameter 16.0mm) were purchased from Sigma

Chemical Co. All other chemicals used were of

analytical grade. Feed ingredients were obtained from

commercial suppliers.

Wheat samplesRandomly chosen samples of nine wheat varieties

grown in Poland in 1995 were a gift from Dr Jacek

Winiarski (Plant Breeding and Acclimatization Insti-

tute, KrakoÂw). The samples were examined as such

and were not used for preparation of the wheat±

soybean meal diet.

Diet compositionThe detailed composition of the wheat±soybean meal

(WB) diet used in the experiments is given in Table 1.

All the ingredients used for the preparation of the diet

were purchased from a local feed market. Vegetable oil

was chosen to ensure an energy source of constant and

high quality. Levels of calcium and phosphorus were

lowered below the required levels in order to promote

the action of phytase. In some experiments the WB

diet autoclaved at 121°C for 6 or 20min was used.

Enzymes and enzyme assaysA commercial preparation Novo Phytase L (Novo

Nordisk, Denmark) was used. Commercial prepara-

tions of xylanase Bio-Feed Wheat L, Bio-Feed Plus L

as well as Bio-Feed Wheat CT were obtained from the

same producer.

Fungal acid protease (EC 3.4.23.6) type XIII from

Aspergillus saitoi, pepsin and pancreatin (activity

8�United States Pharmacopeia) were purchased

from Sigma Chemical Co, St Louis, MO, USA.

Activities of phytase (EC 3.1.3.8) and acid protease

(EC 3.4.23.6) were determined as described pre-

viously.16 One unit of phytase activity (FTU) was

de®ned as the amount of enzyme required to liberate

1mM of inorganic phosphorus in 1min under the

conditions of the assay, whereas one unit of acid

protease (PRU) was de®ned as the amount of enzyme

which hydrolysed haemoglobin to produce colour

equivalent to 1.0mM tyrosine per minute at pH 2.8

and 40°C. The endogenous phytase activity in wheats

was measured following the procedure of Barrier-

Guillot et al.7 The activity was determined in crude

extracts (125g lÿ1; 0.1M citrate buffer pH 5.5) and

also in extracts subjected to ammonium sulphate

fractionation. Suf®cient quantities of ammonium

sulphate were slowly added to cooled and stirred

crude extracts so as to have 40% saturation. The

resulting precipitate was discarded after centrifuga-

Table 1. Ingredients and chemical composi-tion of experimental diet (WB)

Item gkgÿ1

Ingredients

Wheat 550

Soybean meal 375

Vegetable oil 50

Limestone 12

Lucerne meal 10

Salts, minerals, vitamins 3

Analysed composition

Crude protein 222.5

Ca 6.05

P total 4.35

P phytate 2.73

J Sci Food Agric 79:1832±1840 (1999) 1833

Phytase and xylanase ef®cacies in wheat-based poultry feeds

tion, whereas the solution was fully saturated with

ammonium sulphate. The precipitate was dissolved in

the buffer and ultra®ltrated (membrane cut-off

30kDa) against ®ve volumes of cold buffer.

The activity of endo-1,4-b-xylanase (EC 3.2.1.8)

was assayed by the procedure of Yinbo et al19 using a

1.6% suspension of oat spelt xylan as substrate. One

unit of xylanase activity (FXU) was equivalent to 1mM

of reducing sugars released per minute under the assay

conditions.

Experimental treatmentsExperimental factors, in vitro analyses and statistical

analyses performed in the consecutive experiments are

summarised in Table 2.

Measurement of intestinal pHsMeasurements of intestinal pH in the crop, gizzard

and duodenum of 10 three-week-old broiler chickens

were performed as described previously.15 Chicks

were fed from day 1 to 21 on the WB diet (as speci®ed

in Table 1) supplemented with limestone and calcium

phosphate to meet chickens' requirements for phos-

phorus and Ca.20 The pH values found were 5.82 (SD

0.34), 2.74 (SD 0.49) and 6.11 (SD 0.10) for the crop,

gizzard and duodenum respectively. Thus, in the invitro procedures, feed samples were adjusted to pH

values of 5.80, 2.75 and 6.10 for the three incubation

periods simulating the sections of the intestinal tract.

In vitro digestions and measurementsIn vitro procedure for P

A modi®ed in vitro procedure of Zyla et al15 was used

(Fig 1). Triplicate samples (1�0.001g) of wheat or a

diet, ground through a 1mm screen, were weighed

into a 5ml plastic syringe without Luer-lock tips. The

feed sample was hydrated with doubly distilled water

and HCl solution so that a pH value of 5.80 was

obtained. When enzyme solution was applied, water

was partly (or completely) replaced by the enzyme

solution. The contents of each tube were vortexed,

then the tubes were sealed with para®lm and incu-

bated in a water bath at 40°C for 30min. The ®rst step

of the procedure simulated digestion in the crop. Then

0.5ml of 1.5M HCl (feed) or 0.23ml of 1.5M HCl

(wheat) and 3000 units of pepsin were added to each

tube, mixed well, vortexed, sealed with para®lm and

reincubated for 45min at the same temperature. At the

end of this period, which was designed to simulate

conditions in the gizzard, 0.455ml of 1M NaHCO3

(feed) or 0.5ml of 0.32M NaHCO3 (wheat) contain-

Table 2. Summary of experimental factors, in vitro analyses and statistical analyses in consecutive experiments

In vitro analyses

Consecutive

experiments

Substrate Experimental factors Phosphorus Pentoses Viscosity Statistical analyses

1 Wheats Nine varieties of wheat; endogenous

phytase activities in crude extracts and

after fractionation

Yes Yes Yes Correlation and

regression

2 Wheat-based

feed (WB)

Grinding (yes, no); autoclaving time (0, 6,

20min)

Yes No No 2�3 factorial ANOVA

3 WB Phytase (0, 250, 750, 1000, 1250,

1500FTUkgÿ1)

Yes No No One-way ANOVA

WB autoclaved

for 20min

Phytase (0, 500, 1000, 1500FTUkgÿ1) Yes No No One-way ANOVA

4 WB Xylose (0, 1, 2, 4, 8, 15, 30, 60g kgÿ1) No Yes No Regression

5 WB XylanaseÐpreparations: Bio-Feed

Wheat L, Bio-Feed Plus L; dosages: 0,

0.25, 0.5, 1, 2, 5, 10FXUgÿ1

No Yes No 2�7 factorial ANOVA

6 WB Phytase: 750 or 1000FTUkgÿ1; xylanase:

none, 0.4FXUgÿ1 in solution,

0.4FXUgÿ1 in powder form

Yes No No 2�3 factorial ANOVA

7 WB Phytase: 0, 250, 750, 1000FTUkgÿ1; as

above plus acid protease 42PRUgÿ1;

as above plus xylanase 0.4FXUgÿ1

Yes Yes No One-way ANOVA

Figure 1. Flow chart of in vitro procedures designed for determination ofphosphorus and pentose release from wheats and wheat-based dietsunder simulated intestinal conditions of growing broilers.

1834 J Sci Food Agric 79:1832±1840 (1999)

K ZÇ yta et al

ing 3.7mgmlÿ1 pancreatin was added dropwise with

constant stirring into each tube. The slurry was trans-

ferred quantitatively to segments of dialysis tubing by

means of the syringe piston. Segments were placed in

250ml Erlenmeyer ¯asks containing 100ml of 0.1M

NaCl in 0.05M succinate buffer (pH 6.10) and incu-

bated in a shaking water bath at 41.1°C (temperature

of dialysate was 40°C). In that way the duodenal phase

of digestion was simulated. A ratio of about 25:1 (v/v)

between the dialysing medium and segment contents

was maintained. Samples of the dialysate were with-

drawn after 240min for determining inorganic phos-

phate.21

In vitro procedure for pentoses

Simulation of the digestion in the crop and gizzard of

broilers was performed essentially as described for the

phosphorus procedure, but centrifuge tubes were used

instead of syringes. Digestion in the small intestine was

simulated as described by TervilaÈ-Wilo et al18 for

60min. Amounts of pancreatin and NaHCO3 were the

same as in the phosphorus procedure. After digestions,

2ml of water was added to each tube and solubles were

separated by centrifugation (7500�g). Supernatant

viscosity was analysed at 25°C using a capillary

viscometer. Then supernatants were treated with

potassium ferrocyanide and zinc sulphate solutions

to precipitate protein. The precipitate was removed by

®ltration. Deproteinised samples were hydrolysed in

2M HCl solution (®nal concentration) and analysed

for pentoses as described by Hashimoto et al.22

Other analytical methodsTotal phosphorus in a feed was determined color-

imetrically by the molybdovanadate method23 in

duplicate samples which were digested by a wet-ash

procedure of Hach. The procedure was validated by

including standard reference material 1572 (citrus

leaves) from the National Institute of Standards and

Technology.

Phytate phosphorus in the WB feed was calculated

based on amounts of phytate phosphorus determined

in wheat and soybean meal by the method of FruÈhback

et al.24 Calcium was determined by atomic absorption

spectrophotometry (Varian Techtron 1200 spectro-

photometer).

Statistical analysesExperimental data were collected in six to nine

replicates and analysed by the general linear model

procedure of Statgraphics Plus for Windows.25 Results

of in vitro factorial designs were subjected to analysis of

variance following the mode.26

Xij=m�ai�bj�(ab)ij�eij

where m is the overall mean, ai denotes the ®rst-factor

effect, bj denotes the second-factor effect, (ab)ij

represents the interaction between factors and eij is

the error contribution with average zero and variance

s2, with i =1, ¼, a and j =1, ¼, b.

Mean differences were determined using Fisher's

least signi®cant difference test. Statistical signi®cance

was accepted at p<0.05. Correlation analyses were

performed to determine the relationships between

amounts of xylose added to the diet and amounts

recovered after in vitro digestions, between endogen-

ous phytase activity in wheats and amounts of in vitrodialysable phosphorus, as well as between in vitroviscosity after digestion of samples and their pentose

contents. Linear and quadratic effects on phosphorus

release were tested with different levels of phytase and

feeds subjected or not subjected to autoclaving.

RESULTS AND DISCUSSIONTesting of phosphorus in vitro procedure ondifferent wheatsWhen compared with the data reported by Barrier-

Guillot et al,7 the variability in endogenous phytase

activity among tested wheats was small (512±

686FTUkgÿ1, mean=561, SD=56). Dialysable

phosphorus accounted for 50±68% of total phos-

phorus content in wheats (data not shown). Barrier-

Guillot et al5 reported 45±70% phosphorus retention

when 19 wheat samples were tested in broilers. A

highly signi®cant correlation was found between

endogenous phytase activity in wheats and amounts

of phosphorus released during in vitro digestions

(Table 3). Moreover, the relationship was much

stronger when phytase analysis was performed on

crude extracts that were further puri®ed by ammo-

nium sulphate fractionation and ultra®ltration. Most

probably, puri®cation resulted in removing some acid

phosphatase activity present in crude extracts, and

consequently less acid phosphatase was acting on

sodium phytate during activity determination. On the

other hand, both phytase and acid phosphatase were

active in dephosphorylating plant phytates in wheat

samples subjected to in vitro digestions. Apparently, in

the dephosphorylation processes the contribution of

phytate-degrading enzymes other than phytase present

in wheat is more notable in degrading sodium phytate

than during their action on plant phytates.

Effects of grinding and autoclaving on amounts ofphosphorus liberated from WB feedBoth autoclaving and grinding of feed containing

about 55% wheat might be expected to in¯uence invitro dephosphorylation of wheat phytates. A signi®-

cant in¯uence of both factors studied, as well as a

signi®cant interaction of these factors, on the amounts

of phosphorus released from WB feed was observed

(Table 4). Grinding increased the concentration of

dialysable phosphorus, but only in samples that were

not autoclaved. Autoclaving remarkably decreased

amounts of phosphorus liberated from WB feed. This

leads to the conclusion that a signi®cant part of

phosphorus liberated from wheat-based feed under

simulated conditions of poultry intestine may be

attributed to the action of endogenous wheat enzymes

J Sci Food Agric 79:1832±1840 (1999) 1835

Phytase and xylanase ef®cacies in wheat-based poultry feeds

(phytase and, possibly, acid phosphatase). Mechanical

breakdown of plant tissues improves access of those

enzymes to wheat phytates. This broadens a thesis of

many reports summarised by Fuller27 indicating that

the particle size of a feed sample affects in vitrodigestibility of protein and carbohydrates.

Effect of microbial phytase concentration onphosphorus release determined by in vitro

procedureAs shown in Fig 2, phosphorus release in response to

increasing levels of phytase was quadratic both from

non-autoclaved feed (Y =1.756�0.0011xÿ0.000042x2, p<0.0001; R2=0.879; DF=113) as well

as from feed autoclaved for 20min (Y =0.4165�0.0029xÿ0.0000083x2, p<0.0001; R2=0.986; DF=

27). This is in agreement with our previous study on

dephosphorylation of maize±soybean meal in a turkey

in vitro model,15 with the study of Liu et al,17 who

reported similar ®ndings working with a swine gastro-

intestinal model, as well as with the data from broiler

feeding experiments.3 Analysis of the data presented

here revealed that the increase in phytase supplemen-

tation can be justi®ed up to 750FTUkgÿ1, as higher

levels did not signi®cantly affect amounts of phos-

phorus freed from feed. A similar conclusion may be

drawn from research reported by Kiiskinen et al8

performed on broilers fed on wheat-based diets with

graded levels of commercial phytase. The difference in

phosphorus release between non-autoclaved and

autoclaved feed was highly signi®cant at each phytase

dosage, but most pronounced at low levels of phytase

addition (Fig 2). In non-autoclaved feed the depho-

sphorylation process is attributed both to endogenous

phytase of wheat and to commercial enzyme, but in

autoclaved feed to microbial phytase only. Amounts of

phosphorus freed from non-autoclaved feed that did

Table 3. In vitro analyses of different wheat varieties

Wheat variety Phytase activity in crude

extracts (FTUkgÿ1)

Phytase activity after

puri®cation (FTUkgÿ1)

In vitro dialysable

phosphorus (gkgÿ1)

In vitro viscosity

(mPa s)

In vitro pentoses

(gkgÿ1)

Alba 520 347 2.41 1.74 4.7

Almari 686 447 2.59 1.86 5.2

Emika 652 417 2.51 1.77 4.8

Maltanka 519 322 2.37 2.16 6.1

Olma 575 429 2.56 1.94 5.6

Panda 576 372 2.43 1.70 4.5

Roma 550 368 2.46 1.96 5.2

Sakwa 530 356 2.41 1.80 4.7

Tercja 512 312 2.34 1.93 5.3

N 3 4 9 6 6

SEM 11 9.2 0.014 0.02 0.064

Variable code X1 X2 Y1 Y2 X3

Probability of a variety effect was <0.0001 for each of the factors studied

Signi®cant relationships among factors were the following:

Y1=1.883�0.00103 X1 (p =0.0013; R2=0.367)

Y1=1.773�0.00184 X2 (p<0.0001; R2=0.834)

Y2=0.704�0.232 X3 (p<0.0001; R2=0.809)

Lack of ®t was not signi®cant for each of the above models.

Table 4. Effect of grinding and autoclaving on amounts ofphosphorus (mggÿ1) liberated from WB feed

Grinding

Autoclaving time (min) Yes No Mean

0 1.67a,A 1.20b,A 1.43

6 0.52a,B 0.51a,B 0.52

20 0.40a,C 0.41a,C 0.41

Mean 0.86 0.71 0.79

Probabilities of main effects were: grinding, p<0.01; auto-

claving, p<0.0001; interaction, p<0.0001

a±b Means within rows bearing different superscripts were

signi®cantly different (LSD test, p<0.05).

A±C Means within columns bearing different superscripts

were signi®cantly different (LSD test, p<0.05).

Figure 2. Effect of microbial phytase concentrations in a wheat-based diet(&) and an autoclaved wheat-based diet (&) on phosphorus releasedetermined by in vitro procedure. Broken line (- -* - -) denotes differencesin phosphorus release between non-autoclaved and autoclaved diet.Different superscript letters (a–d) denote differences in phosphorusreleased at specified phytase dosages within a diet.

1836 J Sci Food Agric 79:1832±1840 (1999)

K ZÇ yta et al

not contain microbial phytase were similar to that

released from autoclaved feed supplemented with

900±1000FTUkgÿ1. A difference in phosphorus

release between non-autoclaved and autoclaved feed

may be considered therefore as an indirect measure of

endogenous phytase participation in the dephos-

phorylation process. As the concentration of microbial

phytase in feed is increased, the role played by

endogenous enzyme decreases. Frapin and Nys,28

who studied the relative ef®ciency of microbial and

vegetable phytases in broilers, reported highly coin-

ciding conclusions.

Testing of in vitro procedure for pentoses ondifferent wheatsAs shown in Table 3, amounts of pentoses found after

in vitro digestions of wheats varied from 4.5 to 6.1g

kgÿ1 (mean=5.0, SD=0.474). In a hard red winter

wheat, Hashimoto et al22 reported a soluble pentosan

content of 6.8g kgÿ1, whereas TervilaÈ-Wilo et al18

found 3.0g kgÿ1 xylose and about 3.0g kgÿ1 arabinose

after digestion simulation similar to that used in this

study, followed by analysis of sugars by an HPLC

method. The strong relationship between viscosity and

pentose content in digested wheat samples re¯ects the

well-known function of non-starch polysaccharides in

increasing the viscosity of feeds. Viscosity reduction of

the intestinal contents is thought to be the principal

phenomenon resulting from xylanase addition to

poultry diets based on wheat.10 Viscosity measure-

ment was the basis of an in vitro model of Bedford and

Classen29 used to predict ef®cacies of fungal enzymes

in poultry diets based on rye. In their research the invitro viscosity of feeds was highly correlated with body

gains of broilers. Fuente et al30 found that 90% of the

variation in gut viscosity of broilers fed a barley-based

diet supplemented with enzyme was explained by the

viscosity determined in vitro. There are reports to

indicate that xylanase supplementation of feeds based

on wheat improved the performance of broilers owing

to a concomitant reduction in intestinal viscosity.10,31

It can be expected therefore that the in vitro procedure

used in the study presented here will also produce

meaningful relationships with in vivo data.

Effects of graded levels of xylose or xylanase addedto WB feed determined by in vitro procedureThere was a linear response (p<0.0001; R2=0.998) of

pentose concentrations determined in vitro to increas-

ing levels of xylose added to WB feed over the range

investigated. The regression equation describing

amounts of xylose found as a function of xylose added

was Y =0.8943X (data not shown). Two different

commercial xylanase preparations added to WB feed

produced markedly different responses in amounts of

pentoses found in the dialysates (Fig 3). Analysis of

variance of the entire data set showed that there was no

signi®cant effect of the type of xylanase used

(p =0.372) on pentose concentrations, but there was

a signi®cant interaction between xylanase type and

xylanase dosage (p =0.018). For xylanase concentra-

tions of up to 1000FXUkgÿ1 the addition of Bio-Feed

Wheat xylanase resulted in higher concentrations of

pentoses in dialysates than the application of Bio-Feed

Plus xylanase (p =0.024), whereas at concentrations

higher than 1000FXUkgÿ1 the opposite was true

(p =0.033). As Bio-Feed Plus xylanase is known to be

high in b-glucanase activity, data of this research seem

to support a surprising ®nding of Bedford et al,32 who

claimed that b-glucanase co-presence in a xylanase

preparation is disadvantageous at or around optimal

xylanase level (300±400FXUkgÿ1). Research con-

ducted by Novo Nordisk33 also showed that

monocomponent xylanase enzyme gave higher im-

provements in broiler feed conversion ratio than a

xylanase preparation containing b-glucanase activity.

Although the mechanism behind this phenomenon

remains unknown, it may be possibly related to

xylosidase presence in Bio-Feed Plus that negatively

in¯uences endoxylanase action, whereas at higher

dosages this effect is overcome by b-glucanase co-

operation.

Effects of xylanase addition and of its form on in

vitro phosphorus release from feeds containing twolevels of phytaseAs expected, the increase in concentration of phytase

from 750 to 1000FTUkgÿ1 did not result in sig-

ni®cant changes in amounts of phosphorus freed from

feed, no matter whether xylanase was present or not

(Table 5). Application of xylanase in liquid form

negatively in¯uenced dephosphorylation of feed by

phytase, but the same enzyme applied as a powder

signi®cantly increased the concentration of phos-

phorus in dialysates above the level attained with

phytase as a sole supplemental enzyme. The positive

effect of xylanase on enzymic feed dephosphorylation

had been expected. As has already been mentioned,

the literature concerning simultaneous application of

phytase and xylanase is scarce. Slightly relevant data

come from the work of Newkirk and Classen,12 who

found a positive xylanase and phytase interaction in

egg production in laying hens. In contrast, the negative

Figure 3. Amounts of free pentoses determined in vitro in samples of awheat-based diet supplemented with graded concentrations of either Bio-Feed Wheat (&) or Bio-Feed Plus (*) xylanase.

J Sci Food Agric 79:1832±1840 (1999) 1837

Phytase and xylanase ef®cacies in wheat-based poultry feeds

in¯uence of liquid enzyme on the process is dif®cult to

explain, also because it has not been mentioned in the

literature before. Bedford and Morgan10 showed

impaired diffusion of bradykinin (MW 1000) when

the viscosity of a solution was increased from 1 to

3mPas. It seems therefore that viscosity reduction by

endoxylanase may in¯uence dephosphorylation and

the process may also be followed in vitro.

Effects of simultaneous application of xylanase andfungal protease to WB feed containing graded levelsof phytaseFungal protease has been found to accelerate maize±

soybean feed dephosphorylation by fungal phytase invitro.16 The enzyme produced a positive synergistic

response in growth of young broiler chickens fed on a

wheat/canola meal diet de®cient in available phos-

phorus, supplemented with phytase and a carbo-

hydrase.34 An enzyme feed additive comprising

microbial xylanase and protease has been claimed to

improve ef®ciency of wheat-based feeds in both

poultry and pigs.32 In the work presented here the

WB diet was supplemented with tiered concentrations

of phytase (0±1000FTUkgÿ1), or with phytase and

fungal protease (42PRUgÿ1), or with phytase and

protease and xylanase (400FXUkgÿ1), and analysed

for phosphorus and pentoses by the in vitro proce-

dures. Fungal protease markedly increased amounts of

phosphorus liberated at each phytase level, except

1000FTUkgÿ1 (Fig 4). Similar ®ndings have been

reported already for feeds based on maize.15,16 The

application of Bio-Feed Wheat L xylanase to feeds

supplemented with fungal protease and phytase

increased dephosphorylation at low phytase dosages

(0±250FTUkgÿ1), but at higher phytase concentra-

tions (500±750FTUkgÿ1) lowered dialysable phos-

phorus below the levels attained with phytase and

protease. Over the entire range of phytase concentra-

tions studied there was no signi®cant effect of xylanase

addition (p =0.411) on phosphorus release, whereas

protease application was signi®cant (p =0.0044). Acid

proteinase activity was found in aleurone grains of

plant seeds,35 along with phytates, and thus can be

suspected to play a role in phytate dephosphorylation

during seed germination, probably by accelerating the

breakdown of phytate±protein complexes. Even a low

concentration of phytase (250FTUkgÿ1) caused an

increase in amounts of pentoses released from feeds

(p<0.0001; Fig 5). This effect is surprising as it does

not seem probable that the phytase preparation

contains xylanase side activity. Jayarajah et al36 did

not ®nd any signi®cant changes in non-starch poly-

saccharide contents of wheat bran that underwent

dephytinisation by endogenous or yeast phytase. On

the other hand, dephosphorylation carried out by

microbial phytase may affect the structure of plant

cells and render them more accessible for digestion by

intestinal enzymes. Simultaneous application of phy-

tase (250±750FTUkgÿ1) and fungal protease resulted

in increases in amounts of pentoses freed from feeds to

such an extent that consecutive application of xylanase

did not signi®cantly change pentose concentrations.

As in the case of dialysable P, 1000FTUkgÿ1 phytase

dosage produced a response of free pentoses to

subsequent protease and xylanase application that

differed from the responses observed at lower phytase

levels. Although soluble arabinoxylans of wheat con-

tain a fraction with almost 50g kgÿ1 protein content,37

it is not clear why proteolysis enhances release of

Table 5. Influence of xylanase added either in solution or in powder form onphosphorus release from WB feed containing two levels of phytase

Xylanase added

Phytase dosage (FTUkgÿ1) None In solution

In powder

form

750 2.43 2.30 2.51

1000 2.38 2.26 2.50

Mean 2.406B 2.28A 2.51C

Probabilities of main effects were: phytase, p =0.298; xylanase form,

p<0.0001; interaction, p =0.883

A±C Means bearing different superscripts were signi®cantly different (LSD

test, p<0.05). Figure 4. Influence of graded phytase concentrations (&), phytase andacid protease 42PRUgÿ1 (&), phytase, acid protease and xylanase400FXUkgÿ1 (*) on phosphorus release from a wheat-based feed.

Figure 5. Influence of graded phytase concentrations (&), phytase andacid protease 42PRUgÿ1 (&), phytase, acid protease and xylanase400FXUkgÿ1 (*) on pentose release from a wheat-based feed.

1838 J Sci Food Agric 79:1832±1840 (1999)

K ZÇ yta et al

pentoses from feed. Side activities in Aspergillus saitoiprotease preparation might be the answer. The

possibility is now under study.

The in vitro procedure designed to examine phytate

dephosphorylation and arabinoxylan hydrolysis in

wheat-based feeds under simulated intestinal condi-

tions of growing broilers produced responses related to

nutritional effects known in the literature. The

procedure is postulated therefore to be a valuable tool

in predicting results of simultaneous application of

phytase and xylanase in broiler diets based on wheat,

but it needs additional validation by results coming

from animal experiments.

ACKNOWLEDGEMENTSThis work was supported by the Polish Research

Council (grant 109/P06/96-G1185) as well as by the

United States Department of Agriculture (project

RSED-PL25). The authors wish to express their

gratitude to GrazÇyna Gazda and Dorota Kowalew-

ska-Rapacz for their skilful technical assistance, and

thank Novo Nordisk, Poland for providing enzymes

used throughout the study.

REFERENCES1 Nelson TS, Shieh TR, Wodzinsk RJ and Ware JH, Effect of

supplemental phytase on the utilization of phytate phosphorus

by chicks. J Nutr 101:1289±1294 (1971).

2 Simons PCM, Versteegh HAJ, Jongbloed AW, Kemme PA,

Slump P, Bos KD, Wolters MGE, Beudeker RF and

Verschoor GJ, Improvement of phosphorus availability by

microbial phytase in broilers and pigs. Br J Nutr 64:525±540

(1990).

3 Kornegay ET, Denbow DM, Yi Z and Ravindran V, Response of

broilers to graded levels of microbial phytase added to maize±

soybean-meal-based diets containing three levels of non-

phytate phosphorus. Br J Nutr 75:839±852 (1996).

4 Pointillart A, The importance of cereal phytases. Feed Mix

2(3):12±15 (1994).

5 Barrier-Guillot B, Casado P, Maupetit P, Jondreville C and Gatel

F, Wheat phosphorus availability: 2ÐIn vivo study in broilers

and pigs; relationship with endogenous phytasic activity and

phytic phosphorus content in wheat. J Sci Food Agric 70:69±74

(1996).

6 Larsson M, Minekus M and Havenaar R, Estimation of the

bioavailability of iron and phosphorus in cereals using a

dynamic in vitro gastrointestinal model. J Sci Food Agric 74:99±

106 (1997).

7 Barrier-Guillot B, Casado P, Maupetit P, Jondreville C and Gatel

F, Wheat phosphorus availability: 1ÐIn vitro study; factors

affecting endogenous phytasic activity and phytic phosphorus

content. J Sci Food Agric 70:62±68 (1996).

8 Kiiskinen T, Piironen J and Hakonen T, Effects of supplemental

phytase on performance of broiler chickens. Agric Sci Fin

3:457±466 (1994).

9 ZÇ yta K, Ledoux DR, Kujawski M and Veum TL, The ef®cacy of

an enzymic cocktail and a fungal mycelium in dephosphorylat-

ing corn±soybean meal-based feeds fed to growing turkeys.

Poultry Sci 75:381±387 (1996).

10 Bedford MR and Morgan AJ, The use of enzymes in poultry

diets. World Poultry Sci J 52:61±68 (1996).

11 Van der Klis JD, Kwakernaak C and de Wit W, Effects of

endoxylanase addition to wheat-based diets on physico-

chemical chyme conditions and mineral absorption in broilers.

Anim Feed Sci Technol 51:15±27 (1995).

12 Newkirk RW and Classen HL, The effects of dietary xylanase,

phytase and phosphorus on the performance of laying hens.

Poultry Sci 72(Suppl 1):17 (1993).

13 Adrizal, Palo PE and Sell JL, Utilization of defatted rice bran by

broiler chickens. Poultry Sci 75:1012±1017 (1996).

14 ZÇ yta K and Koreleski J, In-vitro and in-vivo dephosphorylation of

rapeseed meal by means of phytate-degrading enzymes derived

from Aspergillus niger. J Sci Food Agric 61:1±6 (1993).

15 ZÇ yta K, Ledoux DR, Garcia A and Veum TL, An in vitro

procedure for studying enzymic dephosphorylation of phytate

in maize±soybean feeds for turkey poults. Br J Nutr 74:3±17

(1995).

16 ZÇ yta K, Ledoux DR and Veum TL, Complete enzymic depho-

sphorylation of maize±soybean meal feed under simulated

intestinal conditions of the turkey. J Agric Food Chem 43:288±

294 (1995).

17 Liu J, Ledoux DR and Veum TL, In vitro procedure for

predicting the enzymatic dephosphorylation of phytate in

maize±soybean meal diets for growing swine. J Agric Food

Chem 45:2612±2617 (1997).

18 TervilaÈ-Wilo A, Parkkonen T, Morgan A, Hopeakoski-Nurmi-

nen M, Poutanen K, Heikkinen P and Autio K, In vitro

digestion of wheat microstructure with xylanase and cellulase

from Trichoderma reesei. J Cereal Sci 24:215±225 (1996).

19 Yinbo Q, Peiji G, Dong W, Xin Z and Xiao Z, Production,

characterization, and application of cellulase-free xylanase

from Aspergillus niger. Appl Biochem Biotechnol 57/58:375±381

(1996).

20 National Research Council, Nutrient Requirement of Poultry, 9th

edn, National Academy of Science, Washington, DC (1994).

21 Lowry OH and Lopez JA, The determination of inorganic

phosphate in the presence of labile phosphate esters. J Biol

Chem 162:421±428 (1946).

22 Hashimoto S, Shogren MD and Pomeranz Y, Cereal pento-

sans:their estimation and signi®cance. I. Pentosans in wheat

and milled wheat products. Cereal Chem 64:30±34 (1987).

23 Association of Of®cial Analytical Chemists, Of®cial Methods of

Analysis, Association of Of®cial Analytical Chemists, Wa-

shington, DC, p 125 (1970).

24 FruÈhback G, Alonso R, Marzo F and SantidriaÂn S, A modi®ed

method for the indirect quantitative analysis of phytate in

foodstuffs. Anal Biochem 225:206±212 (1995).

25 Statgraphics Plus for Windows, Advanced Regression, Manugis-

tics, Rockville, MA (1996).

26 Wadsworth HM, Handbook of Statistical Methods for Engineers and

Scientists, McGraw-Hill, New York (1990).

27 Fuller MF (ed), In Vitro Digestions for Pigs and Poultry, CAB

International, Wallingford (1991).

28 Frapin D and Nys C, Relative ef®ciency of microbial and vegetable

phytases and additional effect of phosphorus availability in broilers.

Proc 10th Eur Symp on Poultry Nutrition, World Poultry Science

Association Proc, Antalya, pp 352±354 (1995).

29 Bedford MR and Classen HL, An in vitro assay for prediction of

broiler intestinal viscosity and growth when fed on rye-based

diets in the presence of exogenous enzymes. Poultry Sci

72:137±143 (1993).

30 Fuente JM, Perez de Ayala P, Flores A and Villamide MJ, Effect

of storage time and dietary enzyme on the metabolizable

energy and digesta viscosity of barley-based diets for poultry.

Poultry Sci 77:90±97 (1998).

31 Steenfeldt S, MuÈ llerz A and Jensen F, Enzyme supplementation

of wheat-based diets for broilers. 1. Effect on growth per-

formance and intestinal viscosity. Anim Feed Sci Technol 75:27±

43 (1998).

32 Bedford MR, Morgan AJ, Clarkson K and Schultze HK, Enzyme

feed additive and animal feed. US Patent 5612055 (1997).

33 Novo Nordisk, The ef®cacy of Bio-Feed Wheat (CT) compared to

Bio-Feed Plus (CT) in a broiler chicken trial in the UK. Animal

Trial Rep B 924a-GB (1996).

J Sci Food Agric 79:1832±1840 (1999) 1839

Phytase and xylanase ef®cacies in wheat-based poultry feeds

34 Simbaya J, Slominski BA, Guenter W, Morgan A and Campbell

LD, The effects of protease and carbohydrase supplementa-

tion on the nutritive value of canola meal for poultry: in vitro

and in vivo studies. Anim Feed Sci Technol 61:219±234 (1996).

35 Yatsu LY and Jacks TJ, Association of lysosomal activity with

aleurone grains in plant seeds. Arch Biochem Biophys 124:466±

471 (1968).

36 Jayarajah CN, Tang HR, Robertson JA and Selvendran RR,

Dephytinisation of wheat bran and the consequences for ®bre

matrix non-starch polysaccharides. Food Chem 58:5±12

(1996).

37 Shiiba K, Yamada H, Hara H, Okada K and Nagao S,

Puri®cation and characterization of two arabinoxylans from

wheat bran. Cereal Chem 70:209±214 (1993).

1840 J Sci Food Agric 79:1832±1840 (1999)

K ZÇ yta et al