Introduction
Chicken meat and giblets are very important 
sources for animal-derived proteins, essential 
amino acids, and a vast array of vitamins and 
minerals. 
Freezing is  a very useful  method of  meat 
preservation for prolonged times, however, in some 
cases due to bad storage conditions or fluctuation 
of freezing temperatures, mould contamination 
may occur. Mould contamination of frozen chicken 
meat and giblets may lead to their spoilage and 
production of mycotoxins with potential health 
hazards to human due to their carcinogenic effects, 
liver diseases and organ damage3).
The incidence of meat contamination with 
different mould genera was investigated in 
different localities of the world such as Australia, 
Japan, Italy and Spain6,8). Despite of the large-
scale consumption of different chicken meat and 
giblets by Egyptian population, few published 
reports are available about the incidence of mould 
contamination and aflatoxin residues in such 
products. Thus, this study was undertaken to 
estimate the incidence of mould contamination in 
frozen chicken meat-cuts and giblets. Furthermore, 
identification of prevalent mould genera was 
carried out. Aflatoxin residues were measured in 
Mould contamination and aflatoxin residues in 
frozen chicken meat-cuts and giblets
Wageh S. Darwish1,*), Rasha M. El Bayomi1), Amany M. Abd El-Moaty2) and 
Tamer M. Gad3)
1)Food Control Department, Faculty of Veterinary Medicine, Zagazig University, Egypt 
2)Laboratory of Microbiology, Educational Veterinary Hospital, Faculty of Veterinary Medicine, Mansura 
University, Egypt 
3)Educational Veterinary Hospital, Faculty of Veterinary Medicine, Mansura University, Egypt
*Corresponding author Email: wagehdarwish@zu.edu.eg
Abstract
Mould contamination of frozen chicken meat and giblets has a particular public health 
significance in the field of food safety due to its related food spoilage and production of 
mycotoxins. The objectives of this study were firstly, to investigate the incidence of mould 
contamination in frozenchicken meat-cuts and giblets. Secondly, to estimate aflatoxin 
residues in these products. The public health importance of the prevalent mould genera 
and aflatoxin residues was discussed. Frozen gizzards had the highest total mould count 
followed by frozen liver, breast and thigh respectively. The prevalent mould genera were 
Aspergillus, Penicillium, Cladosporium and alternaria. Aspergillus niger, flavus, parasiticus 
and versicolor were the identified Aspergilli. Examined frozen chicken samples had variable 
residual concentrations of total aflatoxins. Thus, strict hygienic precautions during 
processing of frozen chicken products should be adopted to reduce mould contamination and 
mycotoxin production.
Key words: Mould, frozen chicken meat, aflatoxins
Japanese Journal of  Veterinary Research 64(Supplement 2): S167-171,  2016
S168
the examined samples. Public health significance 
of aflatoxins and the existing mould genera were 
discussed.
Materials and Methods  
Food Samples collection:
Eighty random samples of frozen chicken breast, 
thigh, gizzards and livers (twenty of each) were 
collected from different localities in Zagazig 
city, Egypt. Samples were identified, packed 
and transferred to the laboratory in icebox and 
subjected to the mycological examination.
Preparation of samples:  
Twenty five grams from each sample were 
aseptically excised and homogenized in 225 ml 
of sterile buffered peptone water 0.1% at 2500 
rpm for 2 min using a sterile homogenizer (type 
M-P3-302, mechanic, precyzina, Poland). Such 
homogenate represents the dilution of 10-1, and 
then decimal dilutions were done.
Determination of the total mould count: 
The total mould count (TMC) was determined by 
culturing duplicate plates on each of malt extract 
agar media and Czapeck-Dox agar with 5% Nacl 
(Oxoid, Basingstoke, UK) followed by incubation in 
dark at 25 °C for 5-7 days. During the incubation 
time, the plates were examined daily for the star-
shape mould growth, which is picked up under 
aseptic conditions with its surrounding cultivated 
medium and transferred into malt extract slope 
agar (Oxoid) then kept for further examination. 
Estimation of TMC was obtained by counting of 
the cultured agar plates of acidified malt extract 
agar and osmophilic moulds on Czapeck-Dox 
agar1). Mould counts were converted into base 
logarithms of colony forming units per g of meat 
product samples (log cfu/g).
Identification of isolated moulds:
The identification of isolated mould genera were 
carried out based on their micromorphological 
properties10). In brief, the isolates were sub-
cultured on malt extract agar and Czapeck-
Dox agar, incubated at 25 °C for 5-7 days. The 
identification of the colonies was carried out by 
careful observation and measurements of the 
macroscopical and microscopical characteristics of 
the mould colonies, which were recorded in data 
sheet.
Macroscopical examination:
The cultures were examined daily for the rate and 
pattern of growth during the incubation period. 
Observations were made for the consistency of 
the surface growth; the pattern of folding (rugae); 
the distinctness of the colony margin and for 
the presence of pigment either on the surface 
or the reverse of the colony or diffusing into 
the surrounding medium. Both the surface and 
backside of the colony were examined.
Microscopical examination:
From the periphery of 5-7 days old mould colony, 
a triangular piece was transferred to a clean glass 
slide. With two mycological needles, the piece of 
the colony was distributed with one or two drops 
of 70 % alcohol. One drop of lactophenol stain 
was added after evaporation of the alcohol. Then 
covered by a clean cover slide followed by gentle 
pressure to remove the excess of fluid and air 
bubbles as well as to depress the hyphae and other 
structures for facilitating microscopic examination. 
The prepared slides were examined under low 
power and oil immersion lens to characterize the 
measurements and morphological structures of the 
mould growth, concerning the conidial stage, head, 
vesicle, sterigmata, conidiophore and conidia.
Quantitative estimation of total aflatoxins:
The quantitative estimation of total aflatoxins
(B1+B2+G1+G2) by flourometer (VICAM. Series 
4) was done following the method published 
before4) with slight modifications. In brief, 25 g 
of ground samples with 5 g NaCl were extracted 
in 100 mL methanol: water (80:20) three times. 
The extracts were diluted 4 times with DDW 
and filtrated using glass microfibre filter; 4 mL 
filtered diluted extract passed through AflaTest® 
-P affinity column at a rate of about 1-2 drops/
second. Elution of affinity column by passing 1.0 
S169Wageh S. Darwish et al.
mL HPLC grade methanol through column at a 
rate of 1 2 drops/second and collecting all of the 
sample elute (1 mL) in a glass cuvette. Then add 1.0 
mL of AflaTest® Developer to elute in the cuvette. 
Mixing well and place cuvette in a calibrated 
fluorometer. Reading of aflatoxin concentration 
after 60 seconds. The detection limit from 0.1 ppb 
to 300 ppb.
Statistical analysis:
Statistical significance was evaluated using the 
Tukey–Kramer HSD test (JMP statistical package; 
SAS Institute Inc., Cary, NC). In all analyses, P < 
0.05 was taken to indicate statistical significance.
Results and Discussion
Inadequate freezing of chicken meat-cuts and 
giblets may result in mould growth, especially, 
if  the initial microbial load is high. Thus, 
investigating the mould contamination and the 
resultant aflatoxin production is a matter of 
importance for public health. 
In this study, the incidence of mould contamination 
of frozen chicken meat-cuts and giblets distributed 
in Zagazig city, Egypt was done. The obtained 
results  revealed that frozen gizzards had 
significantly higher TMC followed by frozen livers, 
breast and thigh. The recorded mean ± SD (Log 
cfu/g) values in such samples were 3.60 ± 0.14, 
3.47 ± 0.32, 3.17 ± 0.11, 2.69 ± 0.12 respectively 
(Figure 1). 
In line with these results, Iacumin et al.6) recorded 
a mould contamination in 41% of the examined 
sausage samples collected from different cities in 
Italy. However, the level of mould contamination 
recorded in this study was much lower than the 
level recorded by Ismail and Zaky7) who recorded 
100% mould contamination in luncheon samples 
collected from Assiut city markets, Egypt. 
Mould contamination of frozen chicken meat 
samples in this study indicates inadequate 
sanitary measures performed during processing 
of such products leading to increase of the initial 
microbial load. Additionally, inadequate storage 
conditions like fluctuation in freezing temperature 
may lead to high mould contamination of such 
products. The conditions of the environment in 
the manufacturing rooms, stores, refrigerators 
and shops are very suitable for the development of 
moulds inside the products, but more frequently 
on the surface of various sorts of meat and meat 
products9). Serious contamination takes place from 
soil and water, raw meat becomes contaminated 
from meat contact surfaces, equipment, utensils, 
handling by workers and during transportation12).
Four mould genera were identified in this study, 
namely, Aspergillus, Penicillium, Cladosporium 
and Alternaria. Frozen chicken gizzards and 
livers had the highest incidence percentages of 
Aspergillus (90%& 80%), Penicillium (75% & 
60%) and Alternaria (25% & 10%), while breast 
and thigh samples had the highest incidence of 
Figure 1. Mould contamination of different frozen 
chicken meat samples
Total mould count in the examined frozen chicken meat-
cuts and giblets. Values represent means ± SD (Log cfu/
g) of twenty samples for each product and each sample 
was measured in duplicates.  Columns with different 
letters differ significantly at P < 0.05.
S170
Cladosporium (25% each) (Figure 2).  This finding 
goes in agreement with Pitt and Hocking10), who 
reported that, the common isolated mould genera 
from retailed meats are Aspergillus, Penicillium, 
Alternaria, Cladosporium, Mucor and Rhizopus. 
Additionally, Iacumin et al.6), mentioned that 
Penicillium and Aspergillus were the mainly 
isolated moulds from sausage marketed in Italy. 
Furthermore, Saccomori et al.11) observed high 
growth of Penicillium polonicum and Penicillium 
glabrium in frozen chicken nuggets. 
Penicillium and Aspergillus can grow over a 
wide range of pH from 2 to 11; over a water 
activity value ranges from 0.620 to 0.995; over a 
temperature ranges from -10 to around 60°C and 
over a wide range of nutrient limitations10), these 
reasons may explain the frequent isolation of these 
two moulds from frozen chicken meat-cuts in 
Egypt and other countries.
We further identified the different isolated 
Aspergilli. A. niger, A. flavus, A. parasiticus, and A. 
versicolor were the dominant Aspergillus species 
as clear in figure 3. This finding goes partially 
in correspondence with Ismail and Zaky7) and 
Mizakova et al.9) who could isolate these Aspergilli 
from luncheon in Egypt and fermented meat 
products in Slovakia.
Generally, mould growth may introduce a meat 
product to consumers containing aflatoxins, 
ochratoxin and other mould metabolites like 
antibiotics and allergens, which represent a 
potential health hazard to consumers too. Thus, 
total aflatoxin residues were further estimated in 
this study as clear in figure 4.
Parallel to mould contamination level, frozen 
livers had the highest aflatoxin residues followed 
by frozen gizzards, breast and thigh-cuts. The 
estimated concentrations were nearly matching 
with Abd-Elghany and Sallam2). Despite of being 
below the maximum permissible limits (20ppb)5), 
aflatoxins are known as dietary human carcinogens 
of fungal origin and have a well-documented 
genotoxic effects3). Thus, contaminated samples 
should not be consumed and strict precautions 
should be given during freezing of chicken meat 
Figure 3. Incidence (%) of different Aspergilli 
isolated and identified from examined frozen 
chicken meat samples
Figure 4. Total aflatoxin residues in frozen 
chicken meat-cuts and giblets
Total aflatoxin residues in the examined frozen chicken 
meat-cuts and giblets. Values represent means± SD (ppb) 
of twenty samples for each product and each sample was 
measured in duplicates.  Columns with different letters 
differ significantly at P < 0.05. 
Figure 2. Incidence (%) of different mould genera 
isolated from frozen chicken meat samples
S171Wageh S. Darwish et al.
and giblets to avoid initial high microbial load. In 
addition, chicken feed should be investigated for 
their mycotoxin contents.
Conclusions
Mould contamination of meat products marketed 
in Egypt was clearly observed in this study 
indicating unsatisfactory hygienic measures 
adopted during processing and freezing of chicken 
meat-cuts and giblets. The prevalent mould species 
were Aspergillus and Penicillium. The growing 
moulds may represent potential health hazards for 
consumers. Thus, strict hygienic measures should 
be followed during the processing and preservation 
of chicken meat. 
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