Food and Chemical Toxicology 48 (2010) 1250–1254

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Food and Chemical Toxicology

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Effects of Aspergillus niger-fermented Terminalia catappa seed meal-based diet

on selected enzymes of some tissues of broiler chicks
N.O. Muhammad *, O.B. Oloyede
Dept. of Biochemistry, University of Ilorin, Ilorin, Nigeria

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

Article history: Effects of Aspergillus niger-fermented Terminalia catappa seed meal-based diet on the activities of alkaline
Received 31 October 2009 phosphatase (ALP), alanine transaminase (ALT), aspartate transaminase (AST) and gamma-glutamate
Accepted 12 February 2010 transferase (c-GT) in the crop, small intestine, gizzard, heart, liver and serum of broiler chicks were inves-
tigated. Milled T. catappa seed was inoculated with spores of A. niger (2.21  104 spores per ml) for
3 weeks. Forty-five day-old broiler chicks weighing between 27.62 and 36.21 g, were divided into three
Keywords: groups. The first group was fed soybean-based (control) diet; the second on raw T. catappa seed meal-
T. catappa seed meal
based diet; and the third on A. niger-fermented T. catappa seed meal-based diet for 7 weeks. The results
A. niger
Fermentation
revealed a significantly increased (p < 0.05) activity of ALP in the tissues. Contrarily, there were significant
Broiler chicks reductions (p < 0.05) in the activities of ALP, ALT, AST and c-GT in the liver and heart of the broilers fed
Enzymes the raw T. catappa seed meal-based diet while there were significant increase (p < 0.05) in the activities of
these enzymes in the serum of the broilers in this group. The data obtained showed that A. niger-fer-
mented T. catappa seed meal reduced the toxic effects of the raw seed meal on the tissues of broiler
chicks.
Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction 2001; Oloyede and Muhammad, 2001; Muhammad et al., 2006).

Consumption of certain ingredients in foods and feedstuffs
could be deleterious to the cells of the body (Muhammad et al.,
2004; Muhammad and Oloyede, 2004). This is because foods and
feedstuffs contain in addition to the useful nutrients, some antinu-
trients (Osagie, 1998). Antinutrients are known to elicit their dele-
terious effects on the tissues and cells of the body (Butler, 1989,
1992; Osagie, 1998). Tannins had been implicated as impairing
the intestinal absorption of iron, reduce the digestibility of starch
and proteins (Reed, 1995; Giner-Chavez, 1996). In fact, the pres-
ence of tannins in food sources for monogastric animals, is gener-
ally viewed adversely. Even in ruminants, levels of tannins
exceeding 6% of the diet resulted in negatively affecting the growth
rates and milk yield of lactating animals (Reed, 1995; Giner-Cha-
vez, 1996). Tannin–protein complexes can cause inactivation of
digestive enzymes and reduce protein digestibility by interaction
of protein substrate with ionizable iron (Salunkhe et al., 1990). It
has also been reported that phytate chelates, virtually all divalent
ions, reducing their bioavailability in the living system (Reddy
et al., 1989). Some of these ions are known to be important cofac-
tors for the activity of some enzymes (Muhammad and Oloyede,
* Corresponding author. Tel.: +234 8033931900.
E-mail addresses: alphamno2@yahoo.com, muno@unilorin.edu.ng (N.O. Mu-
hammad).
Terminalia catappa seed has been reported to contain certain
antinutrients like phytate, oxalate, tannins and hydrocyanic acid
(Jeremiah, 1992; Muhammad and Oloyede, 2004; Muhammad,
2007). These antinutrients are known to affect the digestibility
and bioavailability of starch, protein and minerals (Reed, 1995;
Giner-Chavez, 1996), and consequently the growth of animals
(Muhammad et al., 2004; Muhammad and Oloyede, 2004, 2010).
That raw T. catappa seed meal could damage the tissues of animals
was first hinted by Muhammad and Oloyede (2004) and Muham-
mad et al. (2006).
The effects of antinutrients on the proper synthesis and func-
tioning of enzymes cannot be overemphasized. Enzymes are
known to give the signal of damage to the cells long before histo-
logical studies could reveal such (Malomo, 2000). Antinutritional
factors have been suggested to decrease protein digestibility by
partly complexing with trypsin and pepsin (Reddy and Pierson,
1994). Trypsin inhibitors have been known to interfere with the
physiological process of digestion through interference with the
normal functioning of the pancreatic proteolytic enzymes in non-
ruminants, leading to severe growth depression (White et al.,
1989).
However, processing methods like cooking, autoclaving, heat-
ing, germination and microbial fermentation have been used, suc-
cessfully, to either eliminate or reduce these antinutrients in foods
(Aderibigbe et al., 1997; Bedford, 2000; Owoyele et al., 2003;

0278-6915/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.fct.2010.02.018
 N.O. Muhammad, O.B. Oloyede / Food and Chemical Toxicology 48 (2010) 1250–1254 1251

Muhammad, 2007; Muhammad and Oloyede, 2010). Microbial fer- clot after which it was centrifuged at 1000g for 10 min. After centrifugation, the so-
lid blood settled and the supernatant which was the serum was obtained using a
mentation using Aspergillus. niger, for example, has been reported
Pasteur pipette. The sera obtained were appropriately labeled and stored in the
to reduce the phytate, tannins and hydrocyanic acid of T. catappa freezer, at 5 °C, and used for analysis within 24 h (Ogbu and Okechukwu, 2001).
seed meal (Muhammad, 2007). The present work was therefore
aimed at evaluating the effect of A. niger-fermented T. catappa seed 2.4. Preparation of tissue homogenates
meal-based diet on the activities of some enzymes in selected tis-
sues of broiler chicks. The animals were quickly dissected, the tissues excised and immersed in ice-
cold 0.25 M sucrose solution (to maintain the integrity of the tissues). Homogenates
were prepared for the liver, heart, crop, gizzard and small intestine. This was done
2. Materials and methods
by cutting a known weight of the tissue finely with a clean scissors. The tissues
were thereafter homogenized in ice-cold, 0.25 M sucrose solution (1:5 w/v) using
2.1. Sources of materials
pestle and mortar. Triton x-100 was added to a final concentration of 1% (Ngaha
et al., 1989; Muhammad et al., 2006). All operations were carried out at between
Ripe fruits of T. catappa (authenticated at FRIN, Ibadan, Nigeria, with a voucher
0 and 4 °C. The homogenates were stored in the freezer (each in a labeled specimen
number of FHI 107767) were picked from the premises of the main campus of the
bottle) and used for analysis within 24 h (Ogbu and Okechukwu, 2001).
University of Ilorin, Ilorin, Nigeria. The fruits were cracked using a 125 mm Bench
vice, FUKUNG Brand and milled using the magic blender SHB-515 model. Stock of
Aspergillus niger was obtained from the Plant Health Management Department of 2.5. Determination of enzyme activities in the tissues studied
International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria. The broiler
chicks were obtained from Rofat Feed Limited, Ilorin, Nigeria. The paranitrophenyl Alkaline phosphatase (ALP) was assayed using the method described by Bassey
phosphate used was a product of Sigma–Aldrich Chemie GmbH, Germany; the AST et al. (1946) and modified by Wright et al. (1972), which employs the use of p-nitro-
and ALT kits used were products of Dialab GmbH, Austria; while the GGT kit was phenyl phosphate as substrate. In this method the amount of phosphate ester that
produced by Cypress Diagnostics, Belgium. is split within a given period of time is a measure of the phosphatase enzyme activ-
ity. q-Nitrophenyl phosphate (qNPP) is hydrolysed to q-nitrophenol and phospho-
ric acid at a pH 10.1. The q-nitrophenol confers a yellowish colour on the reaction
2.2. Methods
mixture and its intensity, measured spectrophotometrically at 400 nm gives the
measure of the enzyme activity.
The ripe T. catappa fruits were oven-dried at 60 °C and cracked to remove the
The activity of aspartate transaminase (AST) in the serum and tissue homoge-
seeds, using 125 mm Bench vice and the seeds were milled using magic blender.
nates of the broilers was determined following the method reported by Reitman
The milled sample of T. catappa seed was inoculated with spores of A. niger
and Frankel (1957) as modified by Schmidt and Schmidt (1963). AST catalyses
(2.21  104 spores per ml) and fermented for 3 weeks as described by Muhammad,
the formation of oxaloacetate from L-aspartate and a-ketoglutarate. The oxaloace-
(2007). The fermented substrate was oven-dried at 60 °C for 48 h to terminate the
tate generated is unstable and it spontaneously decarboxylated to form pyruvate.
growth of A. niger and the dried substrate was used as a source of protein in the for-
The absorbance of the red coloured complex formed from the reaction of pyruvate
mulating diet as shown in Table 1. Proximate analysis of the formulated feed was
with p-nitrophenyl hydrazine was then read on a spectrophotometer at 546 nm.
carried out as described in AOAC (1990). The metabolisable energy was obtained
The activity of glutamate–pyruvate transaminase (ALT) in the serum and tissue
as described by Pike and Brown (1975).
homogenates of the broilers was determined following the method reported by
Forty-five-day-old broiler chicks of both sexes, weights ranging from 27.62 to
Reitman and Frankel (1957) as modified by Schmidt and Schmidt (1963). ALT catal-
36.21 g, were randomly assigned into three dietary treatment groups. Each treat-
yses the reaction between L-alanine and a-ketoglutarate to produce L-glutamate
ment had three replicates with five birds per replicate. The broilers were kept in
and pyruvate. The method measures spectrophotometrically the absorbance of
an environment that was warm (40–45 °C) and disinfected. The animals were al-
the red coloured complex formed from the reaction between pyruvate and 2,4-
lowed to acclimatize to the laboratory environment, on commercial feed, for 1 week
dinitrophenylhydrazine.
and weighed prior to the commencement of the feeding experiment and, thereafter
The method of Szasz (1969) was used to assay for gamma-glutamyl transferase.
on weekly basis for 7 weeks. Group feeding was carried out and the animals were
Gamma-glutamyl transferase catalyses the transfer of the glutamyl from a peptide
supplied their different feeds and water ad libitum. Appropriate medications and
to an acceptor molecule, glycylglycine. The change in absorbance at 405 nm due to
vaccinations were administered at the 1st, 2nd and 3rd weeks of the feeding trial.
the p-nitroaniline formed in the reaction measured spectrophotometrically gives
the activity of the enzyme.
2.3. Collection of blood for serum preparation

Blood was collected from the broilers by simply incising the neck and evacuat- 2.6. Statistical analysis
ing the blood into sample bottles without anticoagulant for serum separation. Blood
samples for serum were allowed to stand at room temperature for 30 min to form The data obtained were subjected to analysis of variance and the means were
compared using the Duncan Multiple range test (Steel and Torrie, 1980).

Table 1 3. Results
Percentage composition of the diets (g/100 g).

Ingredients A B C The proximate compositions of the diets are shown in Table 2.

Maize 47.00 32.00 49.00 There were no significant differences (p > 0.05) in the quantity of
Soybean meal 35.00 – – nutrients of the formulated diets. That is, the diets were isocaloric
Raw T. catappa seed meal – 50.00 – and isonitrogenous. The alkaline phosphatase activity in the tissues
Fermented T. catappa seed meal – – 33.00
of the broiler chicks fed the control, raw T. catappa seed meal-
Maize bran 6.00 6.00 6.00
Wheat offal 8.00 8.00 8.00
based diet and A. niger treated T. catappa seed meal-based diet over
Bone meal 2.54 2.54 2.54 a period of 7 weeks is shown in Table 3. The ALP activity in the liver
Oyster shell 1.00 1.00 1.00 and heart of the broilers fed the raw T. catappa seed meal-based
NaCl 0.20 0.20 0.20 diet was significantly low (p < 0.05) compared with those on the
Vit/Min premixa 0.25 0.25 0.25
control and A. niger-fermented T. catappa seed meal-based diets.
Lysine 0.01 0.01 0.01
Methionine 0.01 0.01 0.01 Whereas, the activity of the ALP in the crop, gizzard and small
Total 100 100 100 intestine of the raw T. catappa seed meal-based diet was signifi-
cantly higher (p < 0.05) than those of the animals on the control
A = Soybean meal-based diet (Control).
B = Raw Terminalia catappa seed meal-based diet. and A. niger-fermented T. catappa seed meal-based diets. Further-
C = Aspergillus niger treated Terminalia catappa seed meal-based diet. more, the serum ALP activity of the broilers on raw T. catappa seed
a
Vit. A, 4,000,000 IU; Vit. D3, 800,000 IU; tocopherols, 4000 IU; Vit. K3, 800 mg; meal-based diet was significantly higher (p < 0.05) than those on
folacin, 200 mg; thiamine, 600 mg; riboflavin, 1800 mg; niacin, 6000 mg; calcium the control or A. niger-fermented T. catappa seed meal-based diet.
panthothenate, 2000 mg; pyridoxine, 600 mg; cyanocobalamin, 4 mg; biotin, 8 mg;
manganese, 30,000 mg; zinc, 20,000 mg; iron, 8000 mg; choline chloride,
The AST activity of the tissues of the broilers fed the control,
80,000 mg; copper, 2000 mg; iodine, 480 mg; cobalt, 80 mg; selenium, 40 mg; BHT, raw T. catappa seed meal-based and A. niger-fermented T. catappa
25,000; anticaking agent, 6000 mg. seed meal-based diets for 7 weeks are shown in Table 4. The AST
1252 N.O. Muhammad, O.B. Oloyede / Food and Chemical Toxicology 48 (2010) 1250–1254

activity was significantly low (p < 0.05) in the liver and heart of the
broilers fed the raw T. catappa seed meal-based diets while it was
significantly high in the serum when compared with those on the
control and A. niger-fermented T. catappa seed meal-based diets.
Table 5 shows the ALT activity of the selected tissues of broilers
placed on the control, raw T. catappa seed meal-based and A. niger-
fermented T. catappa seed meal-based diets over a period of
7 weeks. The ALT activity in the liver of the broilers fed raw T. cat-
appa seed meal-based diet was significantly reduced (p < 0.05)
when compared with those on the control and A. niger-fermented
T. catappa seed meal-based diets. It was however, significantly
higher (p < 0.05) in the heart and serum of the animals on the
raw T. catappa seed meal-based diet than the corresponding tissues
of the broilers on the control or A. niger-fermented T. catappa seed
meal-based diets.
The results of the gamma-glutamate transferase (c-GT) in the li-
ver and serum of the broilers fed the control, raw T. catappa seed
meal-based and A. niger-fermented T. catappa seed meal-based
diets for 7 weeks are shown in Table 6. The c-GT activity of the li-
ver of the broilers placed on the raw T. catappa seed meal-based

Table 2
Percentage proximate composition of the formulated diet.

Parameters Control Raw T. catappa A. niger treated T. catappa

Dry matter (%) 96.35 ± 2.64 97.02 ± 2.38 96.65 ± 2.87
Crude protein (%) 21.25 ± 0.35 20.89 ± 0.40 21.00 ± 0.32
Ether extract (%) 5.82 ± 0.34 6.25 ± 0.15 6.21 ± 0.17
Crude fibre (%) 7.10 ± 0.12 6.93 ± 0.09 6.89 ± 0.15
Ash content (%) 6.75 ± 0.05 6.70 ± 0.06 6.72 ± 0.03
N.F.E (CHO) (%) 59.09 ± 0.07 59.23 ± 0.05 59.17 ± 0.05
Calorific value (kcal/100 g) 373.74 ± 4.86 376.73 ± 3.49 376.57 ± 3.65

Values are means of four determinations ± SD.

Table 3
Alkaline phosphatase activities* of selected tissues of broilers fed Aspergillus niger-fermented T. catappa seed meal-based diet for 7 weeks.

Tissue Control Raw T. catappa seed meal A. niger-fermented T. catappa seed meal
a b
Liver 65.35 ± 1.10 24.01 ± 0.90 31.25 ± 1.10c
Heart 28.26 ± 0.35a 7.53 ± 0.30b 16.89 ± 0.27c
Crop 115.07 ± 12.62a 281.47 ± 11.06b 182.70 ± 9.47c
Gizzard 50.40 ± 5.26a 207.10 ± 12.68b 140.04 ± 10.64c
Small Intestine 96.39 ± 8.13a 2583.87 ± 48.78b 463.48 ± 18.26c
Serum 15.52 ± 0.58a 52.50 ± 0.69b 29.93 ± 0.21c

Values are means of four determinations ± S.D.

Row values with different superscripts are significantly (p < 0.05) different.
*
Specific activity (nmol/min/mg protein).

Table 4
AST activity (U/L) of selected tissues of broiler chicks fed A. niger-fermented T. catappa seed meal-based diet for 7 weeks.

Tissue Control Raw T. catappa seed meal A. niger-fermented T. catappa seed meal
Liver 1485.00 ± 16.09a 907.00 ± 10.62b 1057.00 ± 20.61c
Heart 607.00 ± 17.68a 255.00 ± 12.00b 727.00 ± 10.23c
Serum 45.00 ± 2.36a 145.00 ± 4.51b 100.00 ± 2.54c

Values are means of four determinations ± S.D.

Row values with different superscripts are significantly (p < 0.05) different.

Table 5
ALT activity (U/L) of selected tissues of broiler chicks fed A. niger-fermented T. catappa seed meal-based diet for 7 weeks.

Tissues Control Raw T. catappa seed meal A. niger-fermented T. catappa seed meal
a b
Liver 24.00 ± 0.41 8.90 ± 0.29 27.00 ± 0.57c
Heart 93.75 ± 6.29a 490.00 ± 5.77b 427.00 ± 10.90c
Serum 89.38 ± 0.42a 115.50 ± 0.70b 56.25 ± 1.98c

Values are means of four determinations ± S.D.

Row values with different superscripts are significantly (p < 0.05) different.

Table 6
c-GT activity (U/L) of the serum and liver of broiler chicks fed A. niger-fermented T. catappa seed-based diet for 7 weeks.
Tissues Control Raw T. catappa seed meal A. niger-fermented T. catappa seed meal
Liver 166.67 ± 1.20a 99.99 ± 1.52b 133.32 ± 1.43c
Serum 5.56 ± 0.30a 16.67 ± 0.41b 11.11 ± 0.25c

Values are means of four determinations ± S.D.

Row values with different superscripts are significantly (p < 0.05) different.
 N.O. Muhammad, O.B. Oloyede / Food and Chemical Toxicology 48 (2010) 1250–1254
diet was significantly lower while its activity in the serum was sig-
nificantly higher (p < 0.05) when compared to those on the control
or A. niger-fermented T. catappa seed meal-based diets.
4. Discussion
Alkaline phosphatase (ALP) is a ‘marker’ enzyme for the plasma
membrane and endoplasmic reticulum (Wright and Plummer,
1974), it is therefore an ectoenzyme of the plasma membrane
(Shahjahan et al., 2004). It is often used to assess the integrity of
the plasma membrane (Akanji et al., 1993), such that any alteration
in the activity of the enzyme in the tissue and serum would indi-
cate likely damages to the external boundary of cells (plasma
membrane). Of all the tissues studied, alkaline phosphatase activ-
ity was highest in the small intestine, crop, gizzard and least in the
heart. This was in agreement with previous reports that the mam-
malian organs, which have very high ALP activities, are those in-
volved in active transport (Bonting et al., 1960).
The significantly higher serum alkaline phosphatase (ALP) activ-
ity of the broiler chicks fed the raw T. catappa seed meal-based diet
(Table 2) is an indication of plasma membrane damage from the tis-
sues in the body and the subsequent leakage of this enzyme from
the tissues into the serum. High level of serum ALP activity is usu-
ally encountered during liver (especially obstructive jaundice) and
bone diseases (especially osteomalacia, rickets among others) (Dic-
kes et al., 1957). Similarly, increase in serum ALP activities is no-
ticed in liver damage, cancer and heart infections (Anderson, 1968).
The reduction in alkaline phosphatase activities in the liver and
heart of the broilers placed on the raw T. catappa seed meal-based
diet (Table 3) might be adduced to either loss of membrane com-
ponents (including ALP) into the extracellular fluid, the serum
(Malbica and Hart, 1971), inactivation of the enzyme molecule in
situ (Umezawa and Hooper, 1982), or inhibition of the enzyme
activity at the cellular/molecular level. Such reduction in ALP activ-
ity from the tissues could be attributable to disruption of the or-
dered lipid-bilayer of the membrane structure leading to escape
of detectable quantity of ALP out of the cell into the extracellular
fluid. The corresponding increase in broilers’ serum ALP activity
confirmed that damage has been inflicted on the plasma mem-
brane, which might lead to the compromise of the integrity (Akanji
et al., 1993) of the tissues of these animals. The consequence of this
is hindered adequate transportation of required ions or molecules
across the cell membrane and this may lead to starvation of the
cells (Akanji et al., 1993). Reduction in ALP activities as observed
in this study might also adversely affect other metabolic processes
where the enzyme is involved such as the synthesis of nuclear pro-
teins, nucleic acids and phospholipids as well as in the cleavage of
phosphate esters.
On the contrary, the significant increase in the ALP activity of
the small intestine, crop and gizzard of the broilers fed on raw T.
catappa seed meal-based diet (Table 3) may be attributed to induc-
tion in the enzyme synthesis probably by de novo or the response
of the tissues to assaults (Umezawa and Hooper, 1982) by the
antinutrients in the feed. The antinutritional factors in the raw T.
catappa seed meal-based diet may be toxic to the cell, thus causing
lysis of the cell membrane. Tannins are known to irritate the gut
due to their astringency (Reed, 1995); damage the mucosal lining
of the gastrointestinal tract; alter excretion of cations; and increase
excretion of proteins and amino acids (Reddy and Pierson, 1994).
Phytate is equally known to reduce the bioavailability of minerals,
and the solubility, functionality and digestibility of proteins and
carbohydrates (Reddy et al., 1989). These toxic effects elicited in
the tissues of the broilers fed the raw T. catappa seed meal-based
diet are, however, alleviated in those animals fed the A. niger-fer-
mented T. catappa seed meal-based diet.
1253
The significantly high activities of aspartate transaminase (AST),
alanine transaminase (ALT) and gamma-glutamate transferase (c-
GT) in the serum and their corresponding significant reductions
in the liver and heart of the broiler chicks fed the raw T. catappa
seed meal-based diet (Tables 4–6, respectively) may be an indica-
tion of a damage to the liver and heart, thus the consequent leak-
age of these enzymes into the serum. This is what is usually
observed in obstructive jaundice or hepatobiliary diseases and
myocardial infarction (Mayne, 1998). Serum AST and ALT activities
are sensitive indicators of parenchymal liver damage. Increase in
the activities of both enzymes in the serum is commonly found
in liver disease (Tietz, 1995).
c-GT is more sensitive than alkaline phosphatase, aminopepti-
dase and the transaminases in detecting obstructive jaundice. It
was reported to be the most sensitive enzymatic indicator of hepa-
tobiliary disease (Mayne, 1998). It is a membrane-localized en-
zyme that plays a major role in glutathione metabolism and
resorption of amino acids from the glomerular filtrate and intesti-
nal lumen (Kaplan and Pesce, 1996). Thus, the significant decrease
in the activities of c-GT in the liver of the broilers fed raw T. catap-
pa seed meal-based diet might be due to leakage from the organs
to the extracellular fluid since there was corresponding significant
increase in the c-GT activity in the serum of these animals. Such
elevations in the serum c-GT apart from indicating damage to
the plasma membrane of the liver also indicated damage to the li-
ver (Kaplan and Pesce, 1996). These effects were however, allevi-
ated in the liver of the animals fed A. niger-fermented T. catappa
seed meal-based diet.
Overall, it is considered that fermentation of T. catappa with A.
niger greatly alleviates the negative effects of the raw seed on
the tissues of the broiler chicks. Thus, the A. niger-fermented T. cat-
appa seed meal could be used as a source of protein in animal feed.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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