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THE DEVELOPMENTAL EVALUATION OF ASPERGILLUS
TUBINGENSIS CTM 507 GLUCOSE OXIDASE TOXICITY IN
WISTAR RATS
MOUNA KRIAA1, KAIS MNAFGUI2, SAHLA BELHADJ2, ABDELFATTAH EL FEKI2and
RADHOUANE KAMMOUN1,3,4
1Laboratory of Microorganisms and Biomolecules, Center of Biotechnology of Sfax, 2Laboratory of Animal Ecophysiology, Faculty of Sciences of
Sfax, University of Sfax, Road Sidi Mansour Km 6, B.P. 1177, Sfax 3018, Tunisia
3Biotechnology High School of Sfax (ISBS), Sfax 3038, Tunisia
4Corresponding author.
TEL: +216-74-870-451;
FAX: +216-74-870-451;
EMAIL: radhouan.kammoun@cbs.rnrt.tn
Received for Publication April 8, 2014
Accepted for Publication August 14, 2014
doi: 10.1111/jfs.12154
ABSTRACT
The glucose oxidase (GOD) produced by the fungus Aspergillus tubingensis will be
used to improve the quality of bread. The GOD preparation was assessed in a set
of toxicological tests to document its safety in use. The oral administration of the
GOD preparation to rats at a dose level of 0.4 mg/kg body weight/day did not
cause any overt toxic effect on the survival, food intake and body weight gain
throughout the 4-week treatment period. The statistically significant changes in
serum biochemical indices in treated groups like the increase in the blood glucose
levels and the enzymatic activities of alpha amylase, aspartate aminotransferase
and alkaline phosphatase were not concomitant with pertinent histopathological
changes and were hence toxicologically inconsequential. No microscopic lesions
attributable to the treatment were manifested in the experimental groups. The
tested dose level has no-observed-adverse-effect level for developmental toxicity in
rats. This GOD can be considered as a candidate for future application especially
in the food industry.
PRACTICAL APPLICATIONS
The GOD has gained considerable importance in biotechnological applications. In
fact, this enzyme is a potential candidate for both biomedical and agricultural field
including the biological control of crop pest as well as food processing and formu-
lation. It also has great benefits to biopharmaceutical field. Few studies have so far
dealt with the toxicity of GOD. Indeed, studying the toxicity of GOD from
A. tubingensis, a new isolate fungus, was necessary to assess its potential applica-
tion in many other fields. Results showed that GOD preparation was not toxic and
could be potentially used for large-scale application. This study is of great interest
as it gives us the chance to use the GOD preparation as a food additive and in
bio-pharmacy.
INTRODUCTION
A considerable interest has developed in recent years for the
production of glucose oxidase (GOD) since it is well docu-
mented that GOD has several commercial and biotechno-
logical applications in food, beverage and pharmaceutical
industries. GOD has been used largely to remove glucose
and oxygen from various sources such as dried egg, fruit
juices, wine and beer in order to prolong their shelf life
(Wong et al. 2008; Bankar et al. 2009). In fact, the removing
of dissolved oxygen not only prevents but also stabilizes
the color, texture and aroma of different food products
(soft drinks, beer, tinned foods and fish). It importantly
inhibits food spoilage microbes (Cichello 2014). Oxygen is
an important factor in food spoilage. Therefore, it is
important to remove oxygen in the package (Gea et al.
bs_bs_banner
Journal of Food Safety ISSN 1745-4565
263Journal of Food Safety 35 (2015) 263–269 © 2014 Wiley Periodicals, Inc.
2012). Furthermore, GOD was found to have an antagonis-
tic potential effect against different foodborne pathogens
like Staphylococcus aureus,Salmonella infantis,Clostridium
perfringens,Bacillus cereus and Listeria monocytogenes (Tiina
and Sandholm 1989; Bankar et al. 2009). Likewise, GOD
was successively used in baking industries. Its addition to
flour, it improves bread quality. GOD increases dough con-
sistency and ameliorates rheological properties. During
bread making, addition of GOD produces changes in
crumbs texture and increases the bread volume (Rasiah
et al. 2005; Steffolani et al. 2010). In medicine, it is used as a
diagnostic reagent for the measurement of glucose level in
body fluids such as blood and urine (Khurshid et al. 2011).
Filamentous fungi have demonstrated a great ability to
secrete GOD. The genera Penicillium (Eryomin et al. 2004;
Sukhacheva et al. 2004) and black Aspergillus such as
A. niger (Zia et al. 2010; Khurshid et al. 2011) and A. terreus
(Anas et al. 2012) are the most extensively studied and
reviewed among the GOD producing fungi.
Numerous investigations on the safety evaluation of
GOD preparations from A. niger have been well established.
However, because the use of preparations derived from
other fungal species is of interest, an evaluation of their
safety is so necessary (Konishi et al. 2013). To the best of
our knowledge, no reports described toxicological data on
the Aspergillus tubingensis GOD preparation. In the present
study, we report a number of toxicological studies that were
carried out to evaluate the safety of this new GOD prepara-
tion as a food additive. The enzyme preparation was sub-
jected to a subchronic oral toxicity study in rats.
MATERIALS AND METHODS
Biological Material
A. tubingensis strain was originally isolated from the con-
taminated cereal sample based on its capacity to produce
GOD. The strain was deposited at the CBS Bank database
under the accession number CTM 507.
GOD Production and Purification
GOD production was carried out by inoculating the liquid
medium that contains (in g/L): 20 sucrose; 4 yeast extract; 2
casein peptone; 2 corn steep liqueur; 30 CaCO3; 5 KCl; 5
MgSO4; and 1.5 KH2PO4; and (in μg/L): 5 Mncl24H2O, 35
ZnSO47H2O and 25 CuSO45H2O with 1.5% (v/v) of the
inoculum preparation of A. tubingensis fungus (unpub-
lished data). The pH was adjusted to 6.5 using 0.1 N NaOH.
The flasks were then incubated at 30C and 150 rpm in an
orbital shaker. Following 32 h of cultivation, the extracellu-
lar enzyme was separated from the culture medium by fil-
tration and centrifugation. The enzyme was then partially
purified by heat treatment (10 min at 50C) followed by 55%
ammonium sulfate fractionation steps. The precipitate
obtained after centrifugation at 9,000 g for 30 min was sus-
pended in sodium citrate buffer and dialyzed overnight
against repeated changes of the same buffer. The dialyzate
was then used as the partially purified enzyme.
The commercial GOD was obtained from Sigma-Aldrich,
France.
Enzyme Activity
The GOD activity was assayed in a reaction mixture (3.1 mL)
containing 100 μL of enzyme, 1 mL of benzoquinone solution
(0.1%) and 2 mL of glucose solution (1 M) prepared in a
sodium citrate buffer (0.1 M, pH 5.0). The reaction mixture
was incubated for 10 min at 35C. The hydroquinone liberated
in the reaction mixture was measured according to the fast
spectrometric method in 290 nm (Liu et al. 2001).
Animal
To implement this study, Wistar strain male rats weighing
150 ±5 g were chosen. The animals were obtained from the
local central pharmacy, Tunisia. Each group was housed in
polypropylene cages under hygienic and standard environ-
mental conditions (20 ±2C, humidity 60 ±5%, 12 h light/
dark cycle). They were allowed a standard diet and a free
access to tap water.
The diet contained 56.2% carbohydrate (nitrogen free),
22% crude protein, 7.5% ash, 4.2% crude oil, 3% crude
fiber, 2.5% glucose, 1.8% vitamin, 1.4% silica, 0.8% calcium
and 0.6% phosphorus, and provided metabolizable energy
of 3,600 kcal (Ganesan et al. 2011). The experiment was
carried out following the guidelines of an animal laboratory
issued by the University of Sfax, Tunisia, and approved by
the Committee of Animals Ethics.
Experimental Protocol
The experimental animals were divided into three groups of
eight rats each. The control group rats received only the
standard diet. The treated groups received the standard diet
as that of the normal rats every day and were orally admin-
istered with 1 mL of commercial GOD solution or enzyme
preparation via a gastric gavage by intragastric intubation
foraperiodof30days.
The GOD preparation was used for bread making. To be
in the same bread conditions, both GOD solutions
(commercial and synthetic GOD) were inactivated by heat
treatment in 100C for 10 min before use.
The GOD dose was calculated based on preliminary esti-
mates of the human daily intake of bread which was equiva-
lent approximately to 0.4 mg/kg body weight/day. After 4
ASPERGILLUS TUBINGENSIS GLUCOSE OXIDASE TOXICITY M. KRIAA ET AL.
264 Journal of Food Safety 35 (2015) 263–269 © 2014 Wiley Periodicals, Inc.
weeks of the experiment, animals were killed by decapita-
tion, and blood was collected in a heparinized tube for the
separation of plasma by centrifugation (1,500 ×g, 15 min,
4C) and stored at −80C until biochemical analysis.
The kidney and liver tissues were excised immediately
and were stored at −80C until used. For histological studies,
pieces of liver were fixed in a Bouin solution for 24 h and
were then embedded in paraffin. Sections of 5 μm thickness
were stained with hematoxylin eosin. The slides were photo-
graphed with an Olympus U-TU1X-2 camera linked to an
Olympus CX41 microscope (Tokyo, Japan).
Biochemical Analysis
The activities of alpha amylase, alanine transaminase (ALT)
and aspartate transaminase (AST), lactate dehydrogenase
and alkaline phosphatase (ALP), as well as the levels of cre-
atinine, uric acid and urea rates, and the levels of total cho-
lesterol, triglyceride and high-density lipoprotein (HDL)
cholesterol in the serum were measured using the corre-
sponding commercial kits from Biolabo (Tunis, Tunisia).
Statistical Analysis
Data are expressed as means ±standard deviation. Statistical
significance was assessed by the Student’s t-test. P<0.05
was considered statistically significant.
RESULTS
Clinical Signs
During the experimental period, all the animals survived
and in their cages appeared normal with no allergic reac-
tions or overt clinical signs of toxicity. No incidence of con-
stipation, diarrhea or other gastrointestinal disorders was
observed. General conditions and behavior were not
adversely affected by the enzyme preparation in any of the
treatment groups.
Effect of the GOD on Food Intake and
Body Weight
The comparative study of body weight and food intake
between untreated and treated rats by GOD was performed.
Figure 1 depicts the evolution of the body weight of the
control and that of the experimental groups (rats treated by
commercial and synthetic GOD). The treated rats showed a
marked increase in the body weight throughout the entire
experimental period but this increase was limited when
compared with the control rats. Body weights of the treated
animals with GOD were significantly (P<0.05) less
than those of the vehicle controls. However, there are no
significant changes in body weight between the animals
treated by commercial and synthetic GOD, indicating that
there are no differences between commercial and synthetic
GOD effects. The difference of the body weight between the
treated and untreated rats can be explained by the lack of
the power supply, thus the treated rats consume less than
the control group rats. Interestingly, the food consumption
of treated rats was significantly less than that of the control
rats during the 4 weeks; it is approximately ranging between
15.48 ±0.25, 14.34 ±0.4 and 14.67 ±0.41 for the control
and the treated rats with commercial and synthetic GOD,
respectively (Fig. 2).
Effect of the GOD on α-Amylase Activity on
Plasma and Blood Glucose Level of the
Experimental Rats
To evaluate the GOD effect on experimental rats,
the α-amylase activity in serum was determined (Table 1).
The finding showed that compared to the control group
(35.41 ±0.99 U/mL), the treated rats both with commercial
and produced GOD underwent significant increases in
FIG. 1. BODY WEIGHT CHANGES OF RATS ADMINISTERED GOD
PREPARATION AND COMMERCIAL GOD ORALLY FOR 30 DAYS
FIG. 2. FOOD CONSUMPTION OF RATS ADMINISTERED GOD PREPA-
RATION AND COMMERCIAL GOD ORALLY FOR 30 DAYS
M. KRIAA ET AL.ASPERGILLUS TUBINGENSIS GLUCOSE OXIDASE TOXICITY
265Journal of Food Safety 35 (2015) 263–269 © 2014 Wiley Periodicals, Inc.
terms of plasma α-amylase activities reaching
47.4 ±0.99 U/mL (P<0.05) and 46.26 ±1.71 U/mL
(P<0.05), respectively. This stimulated effect of GOD on
α-amylase activity was followed by an increase in the rate of
blood glucose of the treated rats (Fig. 3). There are no sig-
nificant variations between the levels of the blood glucose of
the treated rats by the synthetic and commercial enzyme.
However, a significant increase in the rate of the blood
glucose reaching 1.09 ±0.013 (P<0.05) was observed when
compared to untreated rats (control).
Effect of the GOD Preparation on
Liver-Kidney Functions in the Treated Rats
The effect of the GOD on liver and kidney functions in the
experimental rats was studied. The finding indicated that the
administration of GOD preparation had no significant effect
on the indices related to kidneys but caused a slight variation
in the activity of some liver enzymes. Thus, urea, uric acid and
creatinine rates in the serum of the groups treated with com-
mercial and synthetic enzymes showed normal levels which
were similar to the control rats (Table 2). Table 3 depicts a
significant (P<0.05) increase in the terms of AST and
alkaline phosphatase (ALP) of the two experimental groups
treated when compared to the control animals. In fact,
treated rats with commercial GOD, compared to treated
rats with GOD preparation, showed approximately an equal
PAL level (309.28 ±5.51 UI/L) while the AST activity was
significantly important (134.44 ±2.02 UI/L). Despite these
significant variations, the levels of these liver markers (AST
and PAL in serum) remain in the standards. The ALT
activity of the three groups was also measured. The treated
groups with the synthetic and commercial GOD showed
the same ALT in serum when compared with the control
group reaching 62.56 ±0.71 UI/L, 61.4 ±1.23 UI/L and
60.41 ±2.64, respectively.
Effect of GOD on Lipids Profile in Serum
Biochemical analysis of the lipids profile in experimental rat
serum (Table 4) indicated that the administration of the
GOD had no significant effect on the lipids profile in serum.
The levels of the total cholesterol, triglyceride and HDL
cholesterol in the serum of the experimental rats treated by
commercial and synthetic GOD were equivalent to the
control rats which were 1.64 ±0.12, 0.96 ±0.08 and
1.01 ±0.12, respectively.
Histopathological Examination
In order to study the effect of GOD on rat organs and
confirm the biochemical analysis results in serum, histologi-
cal sections of the liver and kidney were released. The
microscopic analysis revealed no histological abnormalities
in liver and kidneys between control and treated groups
TABLE 1. Α-AMYLASE ACTIVITIES IN THE SERUM OF CONTROL AND
TREATED RATS
Groups Control Commercial GOD GOD preparation
α-Amylase (UI/L) 35.41 ±0.99 47.4 ±0.99 46.26 ±1.71
FIG. 3. GLUCOSE RATE IN THE SERUM OF CONTROL AND TREATED
RATS
TABLE 2. KIDNEY PARAMETERS (CREATININE,
UREA AND URIC ACID) OF CONTROL AND
EXPERIMENTAL GROUPS OF RATS
Groups Control Commercial GOD GOD preparation
Creatinine (μmol/L) 44.57 ±0.4 49.1 ±1.59* 50.7 ±0.55*
Uric acid (μmol/L) 62.86 ±0.85 63.12 ±1.27 64.67 ±1.26
Urea (mmol/L) 6.54 ±0.2 6.43 ±0.1 6.23 ±0.19
Note: Values are given as mean SD for groups of eight animals each.
* Significant differences (P<0.05) via control group.
TABLE 3. LIVER PROFILE INDICES (AST, ALT, PAL) IN SERUM OF
CONTROL AND EXPERIMENTAL GROUPS OF RATS
Groups (UI/L) Control Commercial GOD GOD preparation
ALT 60.41 ±2.64 61.4 ±1.23 62.56 ±0.71
AST 108.54 ±4.61 123.67 ±2.57 134.44 ±2.02*†
PAL 286.125 ±7.03 309.28 ±5.51 309.28 ±2.72*
Note: Values are given as mean SD for groups of eight animals each.
* Significant difference (P<0.05) via control group.
† Significant difference (P<0.05) via treated group with the commer-
cial GOD.
ASPERGILLUS TUBINGENSIS GLUCOSE OXIDASE TOXICITY M. KRIAA ET AL.
266 Journal of Food Safety 35 (2015) 263–269 © 2014 Wiley Periodicals, Inc.
with prepared and commercial GOD. There are no adverse
effects on cellular structure of these organs after treatment
by GOD (Fig. 4).
DISCUSSION
The GOD has considerable industrial importance. It is used
in different food products (dried egg, bread, beverages and
vegetables) as preservative and in pharmaceutical industries
as a component of biosensors in the medical diagnostics
field (Wong et al. 2008; Bankar et al. 2009; Ostafe et al.
2014).
The purpose of this study was to evaluate the toxicity of
A. tubingensis GOD preparation. A 28-day study is consid-
ered as a subacute toxicity testing, which is well accepted for
eliciting any toxicity on long-term feeding. It gives valuable
information on the cumulative toxicity of a substance,
forget organs, physiological organ and metabolic effects of a
compound on prolonged exposure at low dose (Sathya and
Kokilavani 2012; Gandhare et al. 2013; Ghode and
Rajkapoor 2013). The feeding of GOD preparation to male
Wistar rats for 4 weeks was not associated with any abnor-
malities in growth, food intake and some clinical chemistry
biomarkers. There is no death case in all experimental
groups and the treated rats as well as the control rats
appeared active. Body weight gain and food consumption
were slightly low for treated rats (with commercial GOD
and GOD preparation) in comparison with the control
ones, but the food conversion efficiency was unaffected by
the treatment, indicating that the lower body weight gain
was partially due to the food intake reduction and was
therefore considered to be of no toxicological significance.
The reduction of the food intake was probably due to their
inability to feed. It is very likely that they were stressed after
treatment via gavage method. Stress was defined as psycho-
logical and physical modifications that disrupt the homeo-
stasis and the balance of organisms.
Regarding serum biochemistry, a significant increase in
the α-amylase activity was revealed in the rats treated with
GOD. This increase of α-amylase activity may be caused by
the presence of calcium, a necessary cofactor for the stability
and optimal functioning of amylase, on the enzyme prepa-
ration. Further, gluconic acid, a natural reaction product of
GOD, may either impair amylase activity and stability or
regulate the digestion process. Thus, the stimulated effects
of α-amylase seem to have excited the process of carbohy-
drate hydrolysis and absorption in the intestine, which led
to an increase in serum glucose levels (Hamden et al. 2012).
α-Amylase hydrolyzes the glycosidic linkages of starch to
generate glucose, maltose and various α-limit dextrin con-
taining α-(1–6) bonds (De Castro and Sato 2013). After
α-amylase action, the completion of starch digestion is cata-
lyzed to glucose by saccharidases attached to the brush
border of the small intestine (Liu et al. 2014). Accordingly,
we can therefore conclude that GOD preparation can inter-
fere with sugar digestion and blood glucose in rats and can
also impair digestive competence.
Additionally, the present study showed an important
fluctuation of glucose rate in the serum of all experimental
groups under the treatment period. This fluctuation could
be attributed to the fluctuation of diet consumption along
the experimental period. The increase in consumption diet
TABLE 4. TOTAL CHOLESTEROL (T-CH),
TRIGLYCERIDES (TGS) AND HDL
CHOLESTEROL (HDL-C) IN SERUM OF
TREATED RATS WITH GOD
Groups (mmol/L) Control Control +commercial GOD Control +GOD preparation
T-Ch 1.64 ±0.12 1.76 ±0.1 1.72 ±0.15
TGs 0.96 ±0.08 0.92 ±0.02 0.94 ±0.01
HDL-C 1.01 ±0.12 1.16 ±0.14 1.13 ±0.12
FIG. 4. MICROSCOPIC EXAMINATION OF A
HISTOLOGICAL SECTION OF NORMAL KIDNEY
(A), KIDNEY FROM TREATED RAT WITH GOD
PREPARATION (B), KIDNEY FROM TREATED
RAT WITH COMMERCIAL GOD (C), NORMAL
LIVER (D), LIVER FROM TREATED RAT WITH
GOD PREPARATION (E), AND LIVER FROM
TREATED RAT WITH COMMERCIAL GOD (F)
M. KRIAA ET AL.ASPERGILLUS TUBINGENSIS GLUCOSE OXIDASE TOXICITY
267Journal of Food Safety 35 (2015) 263–269 © 2014 Wiley Periodicals, Inc.
which contains chow could increase the activity of
α-amylase, a key digestive enzyme related to the digestion
and absorption of glucose. However, the glucose rate is still
in the normal range in all animals and did not show a
pathological increase exceeding 1.2 g/L.
The current work also showed notable changes in liver
function parameters of the rats treated with GOD such as
the significant increase in serum aspartate aminotransferase
(AST). This change is not considered of toxicological sig-
nificance in hepatic tissue regarding the histopathological
examination of liver which revealed no signs of toxicity
such as necrosis or leucocyte infiltration. Moreover, the lack
of a corresponding change in alanine aminotransferase
(ALT), a more reliable marker of liver integrity in rats, indi-
cated a lack of toxicological relevance for the increase in
AST (Ojiako and Nwanjo 2006; Adenuga et al. 2014). Thus,
Ojiako and Nwanjo (2006) demonstrated that the elevation
in the aspartate aminotransferase activity alone may there-
fore be of extrahepatic origin specifically in skeletal muscle.
On the other hand, the body weight of treated rats that
remained lower than that of the control rats may be evi-
denced by a less developed skeletal muscle which could
explain the slight increase in serum AST activity. Accord-
ingly, a significant increase in serum ALP (PAL) was
observed. This increase was not considered of toxicological
significance as the values of these enzymes were well within
habitual control range for rats and, thus, represented
normal variation. Indeed, Adenuga et al. (2014) noted that
without hepatocellular degeneration, the increase of
PAL activity caused by the rats treatment with 1,3,5-
trimethylbenzene did not indicate a potential adverse
hepatic effect. These authors interpreted the increase of PAL
activity as an adaptive rather than an adverse response to
chemical exposure.
The finding showed that no toxicological adverse in
growth, clinical chemistry and histopathology related to
the oral administration of GOD preparation from
A. tubingensis CTM 507 in Wistar rats. Similarly, Konishi
et al. (2013) demonstrated that GOD preparation was not
associated with any toxic effects in treated rats and it was
demonstrated to be nongenotoxic in a series of in vitro and
in vivo genotoxicity tests.
This study provides assurance that GOD produced by
A. tubingensis CTM 507 may be safely used in the food and
pharmaceutical fields.
CONCLUSION
The results of the present study clearly showed that the
GOD preparation did not induce subchronic or acute toxic
effects in rats. No treatment related to histopathological
changes was observed between the control and the treated
rats at a dose of 0.4 mg/kg body weight/day for 4 weeks.
Further, the GOD preparation was well tolerated as evi-
denced by the absence of any adverse effects on growth,
body weight gain and serum enzyme levels.
CONFLICT OF INTEREST AND ETHICAL
STANDARDS
The authors state explicitly that no potential conflicts of
interest exist. The experimental protocols were conducted in
accordance with the Guide for the Care and Use of Labora-
tory Animals issued by the University of Sfax, Tunisia, and
approved by the Tunisia Committee of Animal Ethics.
ACKNOWLEDGMENTS
This work was funded by the Tunisian Ministry of
Higher Education and Scientific Research and Technology
(contract program LMB-CBS, Grant No. RL02CBS01).
The authors would like to express their sincere gratitude to
Mrs. Salma Karray for her constructive proofreading and
valuable language polishing services.
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