METABOLISM AND NUTRITION
Effect of Enzyme Addition on the Digestibilities of Cell Wall
Polysaccharides and Oligosaccharides from Whole, Dehulled,
and Ethanol-Extracted White Lupins in Chickens
A. Brenes,*,1 B. A. Slominski,† R. R. Marquardt,† W. Guenter,† and A. Viveros‡
*Instituto del Frı́o, Departamento Metabolismo y Nutrición, José Antonio Novais, 10, 28040 Madrid, Spain;
†Department of Animal Science, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2;
and ‡Departamento de Producción Animal, Facultad de Veterinaria, Ciudad Universitaria, 28040 Madrid, Spain
ABSTRACT Three experiments were conducted to examine the effects of a commercial enzyme preparation
on chicks performance and digestibilities of nonstarch
polysaccharides (NSP), raffinose (R), stachyose (S), and
total oligosaccharides (O) in diets containing whole and
dehulled lupin and ethanol-extracted dehulled lupin
meal. Ethanol extract was also used to produce a rich
oligosaccharide fraction. In the first experiment, the dehulling treatment and the addition of enzyme to the diet
improved (P < 0.05) weight gain (24 and 15%), feed-togain ratio (13 and 9%), dry matter retention (32 and 8%),
apparent protein digestibility (6 and 3%) and ileal digestibilities of raffinose (19 and 119%), stachyose (85 and
204%), and total oligosaccharides (68 and 178%), respectively. In addition, enzyme treatment improved (P < 0.05)
excreta digestibility values for NSP (from −1.7 to 5.5%),
R (from 64.6 to 92.6%), S (from 48.8 to 82.4%), and O
(from 55.8 to 83.5%). In the second experiment, extraction
of the ethanol soluble components from dehulled lupin
decreased (P < 0.0001) weight gain (51%), and increased
(P < 0.0001) feed consumption (34%), feed-to-gain ratio
(32%), relative gizzard weight (14%), and the relative ceca
length (20%). The addition of the isolated fraction of oligosacharides to the corn-extracted lupin diet (7.5 and 15%)
yielded performance values similar to those obtained
with the unextracted dehulled lupin. The addition of enzymes to the diets significantly improved the weight gain
(11%) and feed to gain ratio (6%), and decreased relative
gizzard weight (12%) and relative ceca length (7%). Ileal
digestibilities of R, S, and O were considerably lower than
corresponding excreta digestibilities. The excreta digestibility of NSP and excreta and ileal digestibilities of R, S,
and O were lower (P < 0.05) in chicks fed the lupin diet
containing the high concentration of extract compared
with those fed the same diet containing the lower concentration of extract. Enzymes, when added to the diets,
increased (P < 0.05) the digestibilities of R, S, and O with
the relative effects being much greater for ileal than excreta samples. In the third experiment, the ethanol extract
(7.5 and 15%) added to a corn-soybean diet improved
weight gain, feed consumption, and feed-to-gain ratio by
19, 13, and 6%, respectively. Ileal digestibilities of R, S,
and O were low (<45%), especially in chicks fed the diets
containing the high amount of added oligosaccharide
fraction. There was no effect of enzyme addition on the
performance parameters. However, supplemental dietary
enzymes improved (P < 0.05) ileal and excreta digestibilities of oligosaccharides from 15.6 and 68.6% to 52.3 and
84.8%, respectively. In summary, dehulling greatly improved the nutritional value of lupin, while removal of
the ethanol soluble materials, including raffinose and stachyose considerably decreased its value. The oligosaccharide fraction does not seem to have an antinutritive effect
and the addition of enzymes increased the digestibilities
of NSP, raffinose, and stachyose but did not significantly
improve chick performance.
(Key words: chickens, enzymes, lupins, NSP, oligosaccharides)
2003 Poultry Science 82:1716–1725
INTRODUCTION
During the past few years interest has been expressed
on the possible use of low-alkaloid lupin seeds as a protein
source in poultry diets (Olver, 1987; Watkins et al., 1988;
Cheeke and Kelly, 1989). Up to 25% of lupin can be in2003 Poultry Science Association, Inc.
Received for publication January 24, 2003.
Accepted for publication May 9, 2003.
1
To whom correspondence should be addressed: [email protected].
cluded in the diets, without detrimental effects on growth
or other production measurements when compared with
commercial diets containing protein supplements, such as
soybean meal (Bekric et al., 1990; Centeno et al., 1990;
Brenes et al., 1993; Roth-Maier and Kirchgessner, 1994).
A limitation of legume seeds, in general, and lupins, in
Abbreviation Key: NSP = nonstarch polysaccharides; O = oligosaccharides; R = raffinose; S = stachyose.
1716
DIGESTIBILITIES OF CELL WALL COMPONENTS
particular, is the relatively high content of a mixture of
complex nonstarch polysaccharides (NSP) and oligosaccharides (α-galactosides). For poultry the fibrous hull of
lupin seed may contribute to observed growth depression.
Cerning and Filiatre (1980) determined that the 24% hull
from sweet yellow lupins (L. luteus) consisted mainly of
cellulose, hemicellulose, and lignin. These components are
poorly digested by poultry, and the growth depression in
turkeys fed white lupins was partially due to its high content of crude fiber (Halvorson et al., 1988). Lupin kernels
contain pectic-like substances with the major polysaccharides being β-(1-4)-galactan consisting of a mixture of Dgalactose, L-arabinose, L-rhamnose, and galacturonic acid
(Carre et al., 1985; Van Barneveld, 1997; Bach-Knudsen,
1997).
The cotyledon part of lupin seeds, in contrast to the
hulls, also contains significant levels (5 to 8%) of oligosaccharides of the raffinose family (Saini, 1989; Brenes et al.,
1989; Van Kempen and Jansman, 1994). It has been suggested that the α-galactose-containing oligosaccharides
may have a detrimental osmotic effect and promote the
growth of microorganisms (Fleming, 1981; Rubio et al.,
1998). These oligosaccharides have been reported to be
only partly digested in the intestinal tract of the singlestomached animal due to a lack of a significant activity of
α-galactosidase enzyme (Chibowska et al., 1997). Although
some studies on the nutritive effects of galactose-containing
oligosaccharides have been reported, the results are ambiguous (Coon et al., 1990; Treviño et al., 1990; Slominski et
al., 1994).
The use of supplementary enzymes in lupin diets for
poultry has met with varying degrees of success in improving animal performance and nutrient utilization. The response appears to be dependent on the type and quantity
of the lupins as well as the enzymes used (Brenes et al.,
1993, 2002; Annison et al., 1996; Ferraz de Oliveira, 1998;
Naveed et al., 1999). The role of lupin NSP in poultry
nutrition is by no means clear, and detailed knowledge
of the substrates and their breakdown in the intestine of
chickens are required. For this reason, the objective of the
current study was to evaluate the effect of whole lupins,
dehulled lupins, oligosaccharide-free dehulled lupin meal
and its oligosaccharide fraction on the performance of
chicks, and the excreta and ileal digestibilities of nonstarch
polysaccharides and oligosaccharides. Exogenous enzymes
and ethanol extraction were employed to produce dietary
treatments with and without enzymes or the ethanol-soluble lupin components.
MATERIAL AND METHODS
Birds and Diets
One-day old male broiler (experiment 1) and Leghorn
cockerels (experiments 2 and 3) were purchased from a
2
Petersime Incubator Co., Gettysburg, OH.
Novozymes A/S, Denmark.
3
1717
commercial hatchery and raised in a Jamesway battery
brooder for 7 d. They were fed commercial chick starter
crumbles containing 21% (N × 6.25) protein for 7 (Leghorn)
or 14 d (broilers). At 7 d of age the birds were randomly
distributed in Petersime battery brooders2. The experimental diets were formulated to meet the minimum National
Research Council (1994) requirements only in the case of
Leghorn chicks. All diets were given in mash form with
birds having free access to water and feed throughout the
entire experiment. The crude enzyme preparation used in
these studies was Energex (multienzyme complex produced from a selected strain of Aspergillus niger that hydrolyzes a broad range of carbohydrates), Bio-Feed Pro (proteolytic enzyme produced from a selected strain of Bacillus
licheniformis), and Novozyme SP-230 (alpha-galactosidase
preparation).3 The enzymes activities per gram crude product as determined by the manufacturers were as follows:
Energex, 75 fungal β-glucanase units, 150,000 hemicellulase
units, 10,000 pectinase units, and 400 endoglucanase units;
Bio-Feed Pro, 150,000 protease units; Novozyme SP 230,
500 α-galactosidase units. The composition of the diets is
given in Tables 2, 3 and 4.
The white lupins (cv. Amiga) were grown in Saskatchewan (Canada). The whole seeds were dehulled in a commercial pea splitter containing a plate-type grinder, followed by mechanical removal of the hulls. The whole seeds
and the cotyledon preparation (dehulled lupin seeds) were
milled to pass through a 0.7-mm screen.
Extraction of Oligosaccharides
Alcohol-extracted dehulled lupin meal was obtained by
extracting the meal with 50% (vol/vol) ethanol. The process
involved extraction of 1 kg of meal with 10 L of ethanol
(50%) for 11 h with continuous mixing. Following filtration
through cheesecloth, the residue was placed in trays and
dried at room temperature. The ethanol extracts were combined, concentrated under vacuum, and the water phase
was frozen and freeze-dried. The dried product was subjected to chemical analyses and used in the feeding trials.
The concentration of raffinose, stachyose, and total oligosaccharides in whole lupin, dehulled lupin, ethanol-extracted dehulled lupin, and ethanol extract are given in
Table 1.
Experiment 1
This experiment was designed to determine the effect
of the addition of enzymes to whole and dehulled lupin
diets on the bird performance and the digestibilities of
nonstarch polysaccharides and oligosaccharides (raffinose,
stachyose, and total oligosaccharides) in broiler chickens.
Each dietary treatment (Table 2) consisted of six replicates
of two birds per replicate. The balance trial used in this
experiment involved a 4-d pretrial (adaptation) and 1-d
collection period (14 to 18 d of age) in which excreta were
collected, frozen, and freeze-dried. All the birds were sacrificed by cervical dislocation, and the ileum was removed
1718
BRENES ET AL.
TABLE 1. Oligosaccharide composition of different dietary ingredients
Whole lupin
Dehulled lupin
Ethanol-extracted dehulled lupin
Ethanol extract
Soybean protein concentrate
Raffinose1
(mg/g)
Stachyose
(mg/g)
Total
oligosaccharides
(mg/g)
10.1
11.6
1.1
66.5
ND2
48.7
51.8
5.9
288.0
0.2
58.8
63.4
7.0
354.5
0.2
1
As pentahydrate.
Not detected.
2
and its contents collected. The digesta was frozen and
freeze-dried for further analysis.
tracts were removed. Ileum content was collected, frozen,
and freeze-dried for further analysis.
Experiments 2 and 3
Analytical Methods
The objectives of these experiments were to investigate
the effect of the oligosaccharide extracted fractions obtained from dehulled lupin seed on the performance of
Leghorn chicks and the digestibilities of nonstarch polysaccharides and oligosaccharides. Two different kinds of diets
were used for each experiment. In experiment 2, the residue
of the meal following extraction of the oligosaccharide fraction was the major component in the diet (50%, Table 3).
In experiment 3, a diet containing 82% corn-soy-fish meal
was supplemented with different amounts of the ethanol
extract of lupin (0, 7.5 and 15%, Table 4). In both experiments, each dietary treatment consisted of five replicates
of three birds per replicate. After the 12-d growth studies,
the excreta were collected, frozen, and freeze-dried. All
birds were killed by cervical dislocation, and the digestive
Dry matter (7.003) and crude protein (N x 6.25; 7.015)
were determined by methods according to the Association
of Official Agricultural Chemists (AOAC, 1984). Chromic
oxide was determined spectrophotometrically by the
method of Williams et al. (1963). Feed and excreta were
analyzed for nonstarch polysaccharides (NSP). The NSP
were determined by gas-liquid-chromatography (component neutral sugars) using the procedure described by Englyst and Cummings (1984) with some modifications
(Slominski and Campbell, 1990). Uronic acids were determined by the method of Scott (1979) as modified by Englyst
and Cummings (1984). The oligosaccharides were extracted (50 mg of sample) with 3 mL of 80% ethanol for
5 h. Following centrifugation (3,000 rpm), 1 mL of clear
supernatant was transferred into a 4-mL sililation vial, and
TABLE 2. Composition of experimental diets used in experiment 1
Item
Ingredient
Corn
Starch
Lupin meal
Dehulled lupin
Sunflower oil
Dicalcium phosphate
Calcium carbonate
DL-Methionine
Vitamins1
Minerals2
Chromic oxide
Enzyme mix3
Calculated analysis
Crude protein (N × 6.25)
ME, kcal/kg
Lysine, %
Methionine + cystine, %
Calcium, %
Available phosphorus, %
Whole lupin
(%)
Dehulled lupin
(%)
5.00
12.85
70.00
—
7.00
1.50
1.61
0.39
1.00
0.35
0.30
−/+
5.00
24.05
—
58.80
7.00
1.50
1.61
0.39
1.00
0.35
0.30
−/+
22.47
2,926
1.13
0.83
1.00
0.45
22.65
3,086
1.13
0.83
1.00
0.45
1
Vitamins supplied the following per kilogram of diet: vitamin A, 8,250 IU; cholecalciferol, 1,000 IU; vitamin
E, 11 IU; vitamin K, 1.1 mg; vitamin B12, 12.5 mcg; riboflavin, 5.5 mg; Ca panthotenate, 11 mg; niacin, 53.3 mg;
choline chloride, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; delaquin, 125 mg; DL-methionine, 500 mg; amprol,
1 g.
2
Mineral mix supplied the following per kilogram of diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se,
0.1 mg; I, 0.18 mg; NaCl, 2,500 mg.
3
Enzyme added to the diets were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
1719
DIGESTIBILITIES OF CELL WALL COMPONENTS
TABLE 3. Composition of experimental diets used in experiment 2
Corn/
dehulled
lupin
(%)
Item
Ingredient
Corn
Starch
Dehulled lupin meal
Extracted dehulled lupin meal
Ethanol extract
Lupin hulls
Sunflower oil
Dicalcium phosphate
Calcium carbonate
DL-Methionine
Vitamins1
Minerals2
Chromic oxide
Enzyme mix3
Calculated analysis
Protein (N × 6.25)
ME (kcal/kg)
Lysine, %
Methionine + cystine
Calcium, %
Available phosphorus, %
Corn/ethanolextracted
dehulled lupin
(%)
Corn/ethanolextracted
dehulled
lupin+ethanol
extract
(%)
Corn/ethanolextracted
dehulled
lupin+ethanol
extract
(%)
8.51
10.10
58.80
—
—
11.20
7.00
1.17
1.42
0.15
1.00
0.35
0.30
−/+
8.51
18.80
—
50.00
—
11.20
7.00
1.27
1.42
0.15
1.00
0.35
0.30
−/+
8.51
11.30
—
50.00
7.50
11.20
7.00
1.27
1.42
0.15
1.00
0.35
0.30
−/+
8.51
3.80
—
50.00
15.00
11.20
15.00
1.27
1.42
0.15
1.00
0.35
0.35
−/+
23.14
2,940
1.09
0.65
0.86
0.40
23.86
3,080
1.13
0.66
0.85
0.40
23.86
3,080
1.13
0.66
0.85
0.40
23.86
3,080
1.13
0.66
0.85
0.40
1
Vitamin supplied the following per kilogram of diet: vitamin A, 8,250 IU; cholecalciferol, 1,000 IU; vitamin
E, 11 IU; vitamin K, 1.1 mg; vitamin B12, 12.5 mcg; riboflavin, 5.5 mg; Ca panthotenate, 11 mg; niacin, 53.3 mg;
choline chloride, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; delaquin, 125 mg; DL-methionine, 500 mg; amprol,
1 g.
2
Mineral mix supplied the following per kilogram of diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se,
0.1 mg; I, 0.18 mg; NaCl, 2,500 mg.
3
Enzyme added to the diets were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
TABLE 4. Composition of experimental diets used in experiment 3
Control
(%)
Ingredient
Corn
Starch
Ethanol extract
Soybean concentrate
Fish meal
Tallow
Calcium carbonate
Vitamins1
Minerals2
Chromic oxide
Enzyme mix3
Calculated analysis
Crude protein, (N × 6.25)
ME, kcal/kg
Lysine, %
Methionine + cystine, %
Calcium, %
Available phosphorus, %
Control + ethanol
extract
(%)
Control + ethanol
extract
(%)
61.99
15.00
—
10.00
10.00
1.00
0.36
1.00
0.35
0.30
−/+
61.99
7.50
7.50
10.00
10.00
1.00
0.36
1.00
0.35
0.30
−/+
61.99
—
15.00
10.00
10.00
1.00
0.36
1.00
0.35
0.30
−/+
19.98
3,326
1.23
0.64
0.80
0.44
19.98
3,326
1.23
0.64
0.80
0.44
19.98
3,326
1.23
0.64
0.80
0.44
1
Vitamins supplied the following per kilogram of diet: vitamin A, 8,250 IU; cholecalciferol, 1,000 IU; vitamin
E, 11 IU; vitamin K, 1.1 mg; vitamin B12, 12.5 mcg; riboflavin, 5.5 mg; Ca panthotenate, 11 mg; niacin, 53.3 mg;
choline chloride, 1,020 mg; folic acid, 0.75 mg; biotin, 0.25 mg; delaquin, 125 mg; DL-methionine, 500 mg; amprol,
1 g.
2
Mineral mix supplied the following per kilogram of diet: Mn, 55 mg; Zn, 50 mg; Fe, 80 mg; Cu, 5 mg; Se,
0.1 mg; I, 0.18 mg; NaCl, 2,500 mg.
3
Enzyme added to the diets were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
1720
BRENES ET AL.
TABLE 5. Growth performance, dry matter retention (DMR), and apparent protein digestibility (APD)
of broiler chicks (2 to 3 wk) fed enzyme supplemented diets containing whole
and dehulled lupins (experiment 1)
Weight gain
(g)
Item
Feed consumption
(g)
Feed-to-gain
ratio
(g/g)
DMR
(%)
APD
(%)
Diet
CL1
CDL2
225b
280a
389
425
1.75a
1.52b
47.3b
62.5a
80.3b
85.2a
Enzyme3
No enzyme
Enzyme
235b
271a
397
417
1.71a
1.56b
52.9b
56.9a
81.7b
83.8a
4.47
1.95
0.0001
0.0417
0.7426
0.0001
0.0195
0.0528
Pooled SEM
Source of variation
Diet
Enzyme
Diet × enzyme
29.07
0.0001
0.0069
0.0735
42.61
0.11
Probability
0.0512
0.2581
0.1398
0.0001
0.0042
0.8375
Means in each column not showing common letter are significantly different (P < 0.05).
CL = corn-lupin. This diet contained 70% lupins.
2
CDL = corn-dehulled lupin. This diet contained 58.8% dehulled lupins.
3
E = enzyme added to the diet were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
a,b
1
the solvent was evaporated under a stream of air at 40°C.
The dry residue was derivatized at room temperature by
adding 200 µL of a mixture of anhydrous acetone:BSA
[N,O-bis(trimethylsilyl)acetamide]: TMCS(trimethylchlorosilane):1-methylimidazole (2:1:0.1:0.05 vol/vol). The oligosaccharides were then analyzed by gas-liquid chromatography using the procedure described by Slominski et
al. (1994).
Statistical Analysis
The data were subjected to analysis of variance using
the general linear models procedure of SAS software (SAS
Institute, 1986). Experiments 1, 2, and 3 were analyzed by
ANOVA in 2 (lupin fractions) × 2 (enzyme concentration),
4 × 2, and 3 × 2 factorial arrangements of treatments, respectively, and single df contrast were used to separate treatment means in the factorial analysis.
RESULTS
Oligosaccharide Content
The oligosaccharide content of whole and dehulled
lupin, ethanol-extracted lupin, and the lupin extract is
given in Table 1. The concentration of R and S in the
extracted lupin were greatly decreased, while the concentration of R and S in the ethanol extract was relatively
high with total oligosaccharides (R + S) accounting for
approximately 35% of the dry matter content.
Experiment 1
Analysis of variance of the data demonstrated that there
were main effects (P < 0.05) for both type of lupin fed
(whole vs. dehulled) and enzyme treatment (Tables 5 and
6). A significant diet × enzyme interaction effect was only
observed on digestibility of NSP (P < 0.05) and a marginal
interaction effect on weight gain (P < 0.1). It is not discussed
as it accounted for only 21% of the total treatment variation,
whereas the enzyme main effect accounted for 30% of this
variation. In regards to the main effects, both dehulling
of lupin and enzyme addition to the diet, respectively,
improved (P < 0.05) weight gains (24 and 15%), the feed
to gain ratio (13 and 9%), dry matter retention (32 and
8%), apparent protein digestibility (6 and 3%), and ileal
digestibilities of raffinose (19 and 119%), stachyose (85 and
204%), and total oligosaccharides (68 and 178%). In addition, enzyme treatment improved (P < 0.05) excreta digestibility values for NSP (from −1.7 to 5.5%), R (from 64.6 to
92.6%), S (from 48.8 to 82.4%), and O (from 55.8 to 83.5%).
Experiment 2
The content of R, S, and O was greatly reduced in ethanol
extracted lupin and correspondingly increased in the ethanol extract obtained from dehulled lupin (Table 1). Extraction of the ethanol soluble components from dehulled lupin
(diet corn dehulled lupin vs. corn-extracted dehulled lupins, Table 7) decreased (P < 0.05) weight gain (51%), feed
consumption (34%), and increased feed to gain ratio (32%),
relative gizzard weight (14%), and relative ceca length
(20%). In general, the addition of the extract to the extracted
dehulled lupin yielded performance values similar to those
obtained with the unextracted dehulled lupin. Enzyme addition to the lupin-containing diets improved (P < 0.05)
weight gain (11%) and feed-to-gain ratio (6%) and decreased relative gizzard weight (12%) and relative ceca
length (7%).
Ileal digestibilities of R, S, and O for chicks fed the extracted lupin with added ethanol extract were considerably
lower (P < 0.05) than the corresponding excreta digestibili-
1721
DIGESTIBILITIES OF CELL WALL COMPONENTS
TABLE 6. Excreta and ileal digestibilities of nonstarch polysaccharides (NSP), raffinose (R), stachyose (S),
and total oligosaccharides (O) in broiler chicks (2 to 3 wk) fed enzyme supplemented
diets containing whole and dehulled lupins (experiment 1)
Excreta digestibility (%)
Item
NSP
Diet
CL1
CDL2
Enzyme3
No enzyme (NE)
Enzyme (E)
Pooled SEM
Ileal digestibility (%)
R
S
O
R
S
O
0.3
3.5
77.4
79.7
61.8
69.4
67.1
72.2
53.6b
63.9a
23.8b
44.1a
28.1b
47.1a
−1.7b
5.5a
4.69
64.6b
92.6a
8.61
48.8b
82.4a
11.12
36.9b
80.7a
8.09
16.8b
51.0a
8.27
19.9b
55.4a
7.98
Source of variation
Diet
Enzyme
Diet × enzyme4
0.1154
0.0013
0.0055
0.5189
0.0001
0.1617
55.8b
83.5a
10.46
Probability
0.1096
0.0001
0.1178
0.2406
0.0010
0.0758
0.0491
0.0001
0.9393
0.0027
0.0001
0.1507
0.0034
0.0010
0.1858
Means in each column not showing common letter are significantly different (P < 0.05).
CL = corn=lupin. This diet contained 70% lupins.
2
CDL = corn-dehulled lupin. This diet contained 58.8% dehulled lupins.
3
E = enzyme added to the diet were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
4
The percentages of total treatment sum of square for the E and D × E interaction were 30 and 21% for NSP
digestibility. Individual values for significant D × E interaction for NSP digestibilities were CL + NE, −0.3; Cl
+ E, 0.9; CDL + NE, −3.1, and CDL + E, 10.0.
a,b
1
ties of R, S, and O (Table 8). Also, both ileal and excreta
digestibilities of R, S, and O were lower (P < 0.05) in chicks
fed the lupin diet containing the high concentration of
extract (15%) compared with those fed the same diet containing the lower concentration of extract (7.5%). Improvements in the excreta digestibility of NSP (230%) and excreta
and ileal digestibilities of R (60 and 249%), S (28 and 238%),
and O (36 and 271%) were also observed by the addition
of enzymes.
The diet × enzyme interactions (P < 0.05) for ileal digestibilities of R, S, and O demonstrated that the relative effectiveness of enzymes were lower when the amount of extract
or R, S, and O in the diet were increased.
Experiment 3
The extract at a concentration of 7.5 or 15% of total
dietary ingredients improved (P < 0.05) weight gain (19%),
TABLE 7. Growth performance, relative gizzard weight (RGW), and relative ceca length (RCL) of Leghorn
chicks (5 to 12 d) fed enzyme supplemented diets containing dehulled lupins, ethanol-extracted
dehulled lupins, and ethanol extract of oligosaccharides (experiment 2)
Item
Diet
CDL1
CEDL2
CEDL + ethanol extract3
CEDL + ethanol extract4
Enzyme5
No enzyme
Enzyme
Pooled SEM
Source of variation
Diet
Enzyme
Diet × enzyme
Weight gain
(g)
Feed
consumption
(g)
67b
33d
60c
72a
122b
80d
115c
134a
55b
61a
4.03
111
115
6.96
0.0001
0.0002
0.7375
0.0001
0.6230
0.8464
Feed-to-gain
ratio
(g/g)
RGW
RCL
1.83b
2.42a
1.92b
1.86b
3.7c
4.2a
4.0ab
3.9b
6.4c
7.7a
7.0b
6.6c
2.02a
1.89b
0.14
Probability
4.2a
3.7b
0.39
7.2a
6.7b
0.70
0.0001
0.0001
0.6749
0.0001
0.0001
0.0964
0.0001
0.0230
0.7698
Means in each column not showing common letter are significantly different (P < 0.05).
CDL = corn-dehulled lupin. This diet contained 58.8% dehulled lupins.
2
CEDL = corn-extracted dehulled lupins. This diet contained 50% extracted dehulled lupin and 11.2% lupin
hulls.
3
Oligosaccharide containing fraction at 7.5%.
4
Oligosaccharide containing fraction at 15%.
5
E = enzyme added to the diet were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
a–d
1
1722
BRENES ET AL.
TABLE 8. Excreta and ileal digestibilities of nonstarch polysaccharides (NSP), raffinose (R), stachyose (S),
and total oligosaccharides (O) in Leghorn chicks (5 to 12 d) fed enzyme supplemented diets containing
dehulled lupins, ethanol-extracted dehulled lupins, and ethanol extract of oligosaccharides (experiment 2)
Excreta digestibility (%)
Item
NSP
Ileal digestibility (%)
R
S
O
R
S
O
5.4a
9.7a
5.5a
−0.7b
52.6b
—
69.3a
55.4b
43.6c
—
68.7a
52.3b
45.8c
—
67.5a
52.5b
47.9a
—
35.0b
20.8c
22.9a
—
25.7a
11.3b
28.1a
—
26.2a
12.3b
Enzyme5
No enzyme (NE)
Enzyme (E)
2.3b
7.6a
45.5b
72.7a
48.1b
61.6a
46.7b
63.7a
15.4b
53.7a
9.1b
30.8a
9.4b
34.9a
Pooled SEM
6.15
7.46
7.39
7.56
6.54
7.60
Source of variation
Diet
Enzyme
Diet × enzyme6
0.0069
0.0110
0.4107
0.0001
0.0001
0.4298
0.0001
0.0001
0.0085
0.0002
0.0001
0.0002
0.0057
0.0001
0.0007
0.0069
0.0001
0.0020
Diet
CDL1
CEDL2
CEDL + ethanol extract3
CEDL + ethanol extract4
7.05
Probability
0.0001
0.0001
0.0087
Means in each column not showing common letter are significantly different (P < 0.05).
CDL = corn-dehulled lupin. This diet contained 58.8% dehulled lupins.
2
CEDL = corn-extracted dehulled lupins. This diet contained 50% extracted dehulled lupins and 11.2% lupin
hulls.
3
Oligosaccharide containing fraction at 7.5%.
4
Oligosaccharide containing fraction at 15%.
5
E = enzyme added to the diet were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
6
Respective mean square values for diet × enzyme interaction for excreta digestibilities of S and O represented
only 10 and 9% total mean square variation and therefore are not given (Little, 1981). However, corresponding
values for R, S, and O ileal digestibilities were 18, 27, and 22%, respectively. The individual, R, S, and O ileal
digestibilities (%) values for the diet × enzyme interactions were 37.9, 18.3, and 21.2 for diets CDL + NE; 57.9,
27.5, and 35.0 for diets CDL + E; 0.5, 3.1, and 1.6 for diets CEDL + OF1 + NE; 69.4, 48.2, and 50.7 for diets CEDL
+ OF1 + E; 7.9, 5.8, and 5.5 for diets CEDL + OF2 + NE and 33.7, 16.7, and 19.0 for CEDL + OF2 + E. The
respective individual R, S, and O excreta and ileal digestibilities (%) values for diets CEDL + NE and CEDL +
E were not determined.
a–c
1
feed consumption (13%), and feed to gain ratio (6%). Enzyme addition did not affect performance values (P > 0.05)
(Table 9). However, dietary enzymes improved excreta and
ileal digestibilities of R (44 and 374%), S (27 and 207%),
and O (24 and 235%), respectively (Table 10). In addition,
the excreta digestibilities of R, S, and O decreased (18, 16,
and 14%, respectively) when the dietary amounts of the
extract were increased from 7.5 to 15%. The interaction for
TABLE 9. Growth performance of Leghorn chicks (5 to 12 d) fed enzyme supplemented diets
containing ethanol extract of oligosaccharides (experiment 3)
Item
Diet
C1
C + ethanol extract2
C + ethanol extract3
Enzyme4
No enzyme
Enzyme
Pooled SEM
Weight gain
(g)
Feed
consumption
(g)
Feed-to-gain
ratio
(g/g)
63b
75a
75a
127b
141a
144a
2.01a
1.89b
1.93b
70
72
4.65
138
137
7.59
1.96
1.92
0.08
Probability
Source of variation
Diet
Enzyme
Diet × enzyme
0.0001
0.4675
0.8962
0.0001
0.8678
0.6706
Means in each column not showing common letter are significantly different (P < 0.05).
C = control diet contained 62% corn, 10% soybean concentrate, and 10% fish meal.
2
Oligosaccharide containing fraction at 7.5%.
3
Oligosaccharide containing fraction at 15%.
4
E = enzyme added to the diet were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
a,b
1
0.0064
0.1592
0.9428
1723
DIGESTIBILITIES OF CELL WALL COMPONENTS
TABLE 10. Excreta and ileal digestibilities of nonstarch polysaccharides (NSP), raffinose (R), stachyose
(S), and total oligosaccharides (O) in Leghorn chicks (5 to 12 d) fed enzyme supplemented diets
containing ethanol extract of oligosaccharides (experiment 3)
Excreta digestibility (%)
Item
NSP
Ileal digestibility (%)
R
S
O
R
S
O
Diet
C1
C + ethanol extract2
C + ethanol extract3
17.1
19.2
14.4
—
78.1a
64.0b
—
75.9a
63.4b
—
76.1a
65.2b
—
44.9
33.7
—
37.2
28.9
—
38.4
29.4
Enzyme4
No enzyme (NE)
Enzyme (E)
15.8
17.9
58.2b
83.9a
61.4b
77.9a
68.6b
84.8a
13.7b
64.9a
16.2b
49.8a
15.6b
52.3a
9.16
10.11
9.95
Pooled SEM
5.27
7.67
8.13
8.35
Probability
Source of variation
Diet
Enzyme
Diet × enzyme5
0.1530
0.2857
0.0351
0.0008
0.0001
0.8319
0.0033
0.0003
0.9094
0.0001
0.0001
0.3008
0.0663
0.0001
0.7745
0.1945
0.0004
0.9912
0.1556
0.0002
0.9776
Means in each column not showing common letter are significantly different (P < 0.05).
C = control diet contained 62% corn, 10% soybean concentrate, and 10% fish meal.
2
Oligosaccharide containing fraction at 7.5%.
3
Oligosaccharide containing fraction at 15%.
4
E = enzyme added to the diet were 0.1% each of Energex + Bio-Feed Pro + Novozyme.
5
The individual values for the diet × enzyme interaction for excreta digestibilities of NSP (%) of chicks fed
the different diets were C + NE, 19.6; C + E, 14.5; C + OF1 + NE, 17.4; C + OF1 + E, 21; C + OF2 + NE, 10.5;
and C + OF2 + E, 18.3. The respective individual R, S, and O excreta and ileal digestibilities (%) values for diets
C + NE and C + E were not determined. The respective individual NSP excreta and R ileal digestibilities (%)
values for diets C + NE and C + E were not determined. Over 21% of the total treatment variation was associated
with the interaction and only 3% with the enzyme effect. The enzyme was therefore not discussed.
a,b
1
excreta digestibilities of NSP indicated that enzyme was
most effective when the enzymes were added to the higher
oligosaccharide extracted fraction.
DISCUSSION
The results of the current study demonstrated that dehulling dramatically improved broiler performance but affected ileal digestibilities of the raffinose (R) and stachyose
(S) to a much lesser degree. The improved performance of
chicks fed the dehulled lupins is attributed to changes in
the proportions of nutrients. The increase (20%) in the
crude protein content and the decrease (70%) in crude fiber
content (Brenes et al., 1993, 2002; Smulikowska et al., 1995;
Flis et al., 1997; Gdala, 1998) could explain the higher digestibility of energy (18%) and that of protein (7%) obtained
by Brenes et al. (1993) when comparing dehulled to whole
lupin chickens diets. The positive effects of feeding dehulled lupins on ileal digestibility of dry matter by poultry
have also been demonstrated by Jansman and Mieczkowska (1998). Presumably low amounts of indigestible fibrous
material in the diets containing dehulled lupins, when compared with that of a nondehulled lupin diet, facilitated dry
matter retention, apparent protein digestibility and ileal
digestibility of R, S and total oligosaccharides. The improved excreta digestibilities of R, S, and O when compared
with ileal samples can be attributed to prolonged action
of the added enzymes and the presence of microorganisms
in the caeca and colon as compared with conditions in
the ileum.
Carre and Leclercq (1985) reported that the ability of
cockerels to digest plant cell walls is very low. Lupin NSP,
observed to be digested extensively in rats, remained practically undigested by the cockerels and ducks (Carre et al.,
1990). These results are in agreement with the digestibilities
of NSP of the birds fed corn-lupin diet in experiments
1 and 2. The digestibility values tended to be negative,
indicating that nondietary polysaccharides were probably
excreted in addition to the lupin polysaccharides. Negative
values have been noted by others (Longstaff and McNab,
1989; Petersson and Aman, 1989) and can also arise from
sampling problems when the transit time of the marker is
different from that of the NSP (Annison et al., 1996). That
cecal bacteria of domestic birds are devoid of cellulolytic
activity (McNab, 1973), the short transit time for cockerels
(Sibbald, 1980), and the separation mechanism located in
the caecum-colon region, which prevent large cell-wall particles from entering the caeca (Björnhag and Sperher, 1977)
probably explain the low ability of cockerels to digest plant
cell walls.
Enzymes dramatically improved ileal digestibilities of
O and had a less pronounced effect on excreta digestibilities
of O, dry matter retention, apparent protein digestibility
and chick performance. The low digestibility of R, S, and
O in the ileum of chicks fed the lupin diets without added
enzyme is attributed to the lack of both intestinal and
microbial α-galactosidases. These deficiences were partially overcome by the addition of the exogenous enzyme
(Table 6). In contrast, the corresponding excreta digestibilities were much higher. This, in part, is attributed to a much
1724
BRENES ET AL.
higher concentration of microorganisms, which have the
ability to ferment R and S. This effect was further enhanced
by the addition of exogenous enzymes. Kocher et al. (2000)
also showed that the addition of a commercial enzyme
preparation to a lupin based-diet resulted in a significant
increase in the ileal digestibility of NSP related mainly to
the digestibility of glucose, xylose, arabinose, and polymers
present predominantly in the hull fraction. The depolymerization pattern of the NSP was also investigated by Hughes
et al. (2000), demonstrating that the inclusion of lupin kernel isolate with enzymes in a sorghum-casein diet, improved dietary apparent metabolizable energy. The results
of previous experiments reported by Brenes et al. (1993,
2002) demonstrated that the addition of enzymes in lupin
diets produced an improvement in the nutritive value of
low alkaloid lupin diet. These results are also in agreement
with those of other researchers (Annison et al., 1996; Ferraz
de Oliveira, 1998; Naveed et al., 1999).
The results of the second experiment, contrary to what
may have been expected, demonstrated that removal of
the ethanol soluble fraction from dehulled lupins resulted
in a dramatic decrease in chick performance. Chick performance was restored when this fraction was added to the
diet. The content of R and S in the extract was moderately
high. These results, therefore, suggest that the ethanol soluble fraction of lupins contains nutrients (i.e., sucrose) that
greatly improve chick performance. However, the specific
contribution of R and S cannot be established from this
study as they comprised only 35% of the total extract dry
matter content (354 mg/g). Nevertheless, the results from
the digestibility studies would suggest that the oligosaccharides do not greatly affect chick performance and that in the
presence or absence of enzymes they also do not contribute
significantly to its nutritional value since only 9 to 15 and
31 to 54% were digested in the ileum, respectively, in the
absence and presence of added enzymes.
In poultry the role of dietary oligosaccharides is not
clear. Some studies (Coon et al., 1990; Leske et al., 1991,
1993; Leske and Coon, 1999) have demonstrated that removal of the α-galactose-containing oligosaccharides (raffinose and stachyose) from soybean meal by ethanol extraction increases its nitrogen-corrected true metabolizable energy value and protein content. Interestingly, however,
addition of oligosaccharide extract to pea-based diets had
no effect on bird performance (Treviño et al., 1990). Similar
experiments in which the α-galactosides of canola meal
were extracted and then added back also failed to indicate
any detrimental nutritive effects associated with these components (Slominski et al., 1994). In another study, the removal of oligosaccharides by ethanol extraction or pretreatment with α-galactosidase enzyme had no beneficial effect
on the nutritive value of soybean meal for chickens (Irish
et al., 1995). Our results also demonstrated that the addition
of the ethanol-extracted fraction obtained from dehulled
lupin to the diet improved the performance of the birds
and confirmed that oligosaccharides in lupin should not be
considered antinutritional. Moreover, the effects of dietary
additions of ethanol-extracted lupin resulted in a significant reduction in the performance of the birds. These re-
sults are similar to those reported by Hughes et al. (1998)
using raw seeds or ethanol-extracted seeds to remove oligosaccharides, showing a significantly reduced apparent metabolizable energy content and chicken performance.
In conclusion, dehulling and enzyme addition to diets
containing lupins greatly improved its nutritional value,
while removal of the ethanol soluble materials, including
R and S considerably decreased its value. Raffinose and S
appear to be poorly digested in the ileum or upper section
of the gastrointestinal tract. These digestibilities, however,
can be dramatically improved by the addition of an appropriate dietary enzyme supplement. This may not be economical as enzyme addition to a corn-based diet containing
an ethanol extract of lupins that was also high in R and S
did not seem to affect chick performance. These results,
therefore, suggest that R and S do not greatly affect chick
performance, and the use of enzymes to enhance their
digestibility does not significantly improve chick performance. Enzymes probably affect other constituents in lupins, thereby producing a beneficial effect in chick performance. These constituents were not identified.
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