Journal of Environmental Protection and Ecology 11, No 4, 14371445 (2010)

Biochemical- and bioprotection

Microbial ecology and bioactive amines content

of skin during chicken Carcasses storage at 4C
O. Baston*, O. Barna, E. Pricop
Faculty of Food Science and Engineering, Dunarea de Jos University of
Galati, 111 Domeasca Street, Galati, Romania
E-mail: octavian.baston@ugal.ro
Abstract. The skin is an epithelial tissue witch prevents microorganisms from entering the chicken
body, acting as a protective layer against microbial contamination. It is well known that most of the
spoilage bacteria are found on the chicken skin. We studied the initial microbial content of the skin of
refrigerated chicken carcasses after slaughter, especially total viable count and psychotrophic count.
In the first day of storage we found a total viable count of 5.11 log CFU/cm2 and psychotrophic count
of 4 log CFU/cm2. Also, we were interested to determine the microbial variation during storage at
4C correlated with skin pH. We found that chicken skin pH was steadily increasing during the 21
days of storage at 4C, and also the microbial content was increasing. Thus, in the first day of storage
total viable count was higher than psychotrophic count with 21.72%, after 14 days of storage the
counts were approximately equal and after 21 days of storage the psychotrophic count was higher
than total viable count with 0.38%. In chicken skin bioactive amines occur mainly due to enzymatic
processes of microbial activity. We studied the presence and variation of following bioactive amines:
tryptamine, -phenylethylamine, putresceine, cadaverine, histamine, serotonin, tyramine, spermidine
and spermine in chicken skin during the refrigeration of carcasses. The obtained results indicated
that during carcasses storage the content of tryptamine, -phenylethylamine, putresceine, cadaverine,
histamine, serotonin, and tyramine in chicken skin increased. Spermidine and spermine content in
chicken skin decreased. Cadaverine was not detected in the first day of storage.
Keywords: chicken skin, psychotrophic count, bioactive amines, HPLC, refrigeration storage.

Aims and background

Chicken skin protects chicken body against mechanical damage, having an important role in heat regulation, in insulation against drying and prevents microorganisms from entering the body. We know that bacteria can be found on skin surface
and in feather follicles1. Some of the microorganisms are attached on chicken
skin surface. The attachment of microorganisms to chicken skin is not a simple
matter because of the nature of the skin surface and the fact that changes occur as
the carcass pass through different stages of processing. A major change in surface
structure is due to defeathering which lives many holes in the skin that can trap
bacteria. Also the skin has a complex surface with irregular topography with many
*

For correspondence.

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microscopic channels. After chicken carcass chilling, the skin can support microbial
growth during storage. Conditions of pH and temperature influence the bacterial
survival and attachment. When birds arrive at the processing plant they carry a
large microbial load on the skin, among the feathers and in alimentary tract. The
process of converting a bird into carcass leads to removal of the high proportion of
microorganisms, but during processing can occur further contamination, especially
during scalding, plucking, evisceration, from aerosols, processing equipment and
the hands of operators2.
Having this information, we were interested to determine the content of total
viable count comparatively with psychotrophic count on chicken skin.
It is well known that certain amines fulfill a number of important metabolic
and physiologic functions in living organisms. Here we refer to them after the
death of the chicken. Some of them exist in living organism and are named natural
amines spermine, spermidine, putrescine and histamine and are formed de novo
during biosynthesis. Biogenic amines are formed by bacterial decarboxylation
of free amino acids. So, histamine can be either natural (stored in mast cells or
basophils) or biogenic. Generically we refer these amines to bioactive amines. In
literature we found references only to levels of bioactive amines in fresh chicken
breast and tight, breast stored at different refrigeration temperatures, chicken
chunks and different chicken products: hot dog, mortadella, sausage, meatball,
hamburger, nuggets, etc.39
Bioactive amines determination in chicken skin can be suitable for detecting
incipient spoilage of chicken meat. We found interesting to correlate the microbiota
content and bioactive amines content in raw chicken skin and its storage at 4C,
because in the scientific literature there is a lack of information in this area. For the
analysis of bioactive amines in foods were developed various methods. We used
HPLC for quantifying the bioactive amines. Different chromatographic methods
for quantitative determination of bioactive amines in foods have been employed:
thin-layer chromatography1012, gas chromatography1315 and high liquid performance chromatography (HPLC)10,1620. In this paper we did a correlation between
microbial content, skin pH, bioactive amine content and producer shelf-life of raw
chicken skin, keeping chicken carcasses at constant temperature of 4C.
Experimental
The chicken carcasses were purchased from the Agricola International Bacau
company slaughterhouse. The meat was analysed after cooling, packaging and
transportation from the plant the first day after slaughter. The carcasses were stored
aerobically for 21 days at a temperature of 41C in the refrigerator. The refrigerator used is an Electrolux ENB43691S. The carcasses weight varied between

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1.21.5 kg. The samples were analysed the first day when the meat was received,
recorded as day 1, then the 3rd, 5th, 7th, 14th, and 21st day.
The dry matter determination was done according to the Romanian STAS
9065/3-73. The pH was measured using a standardised method (according to the
Romanian STAS 9065/8-74) with a WTW Ino Lab pH 730 pH-meter.
Pieces of raw chicken skin (16 cm2 in area) were aseptically excised from
carcass and each piece was homogenised with 100 ml of saline water (0.8% NaCl)
by using a homogeniser model Bagmixer 400. Duplicate 0.1 ml aliquots of suitable dilutions of each skin homogenate were spread on the surface of nutrient agar
plates. Inoculated plates were incubated in an ATICH 9082 incubator in aerobic
condition at 4C for 14 days for psychrotrophic microorganisms and at 30C for
2/3 days for total viable count. After that viable colonies were counted using an
automatic colony counter SC6.
The measurement of biogenic amines content using high performance liquid
chromatography, was performed according to the method proposed by Food Research Institute of Helsinki, Finland20. The method principle is as follows:
bioactive amines are extracted from a homogenised sample with diluted
perchloric acid;
an aliquot of the extract is derivatised with dansyl chloride reagent;
separation and quantification of dansylated amines are performed by reversed
phase liquid chromatography with ultraviolet detection at 254 nm.
All the reagents used were analytic pure, for HPLC use. The water used was
deionised. The necessary reagents were purchased from the Merck and SigmaAldrich companies. Installations and equipment used for biogenic amine determination: Philips 7768 food processor, homogenisation device 7011S, Kern 77060
analytical balance, Silent CrusherM homogenisation device, centrifuge EBA 21,
filter paper for quick filtering with 55 mm diameter, syringe filters with porosity
of 0.45 m and 13 mm diameter, Heidolph REAX control agitator, ultrasonic
water tank Aquawave TM, incubator BMT INCUCELL 55, water deionising system EASY pure RoDi, filtering assembly with vacuum pump. The device for the
HPLC determination was a liquid chromatograph model SURVEYOR produced
by Thermo Electron company, configured with detector model PDA PLUS DETECTOR, auto-sampler model AUTOSAMPLER PLUS, pump model LC PUMP
PLUS and detector UV-vis. Chromatography column is type BDS Hipersyl C18.
The biogenic amines quantification: quantitative measurement was performed
depending on the internal standard using peaks for each biogenic amine. The 254
nm wavelength absorbance was measured and the resulted peaks were integrated
with CromQuest software. The concentration of each biogenic amine was expressed
in mg/kg d.m. (d.m. = dry matter).
The statistical analysis of the obtained data was done using Microsoft Excel
features for 6 samples in each of the storage days. The results obtained are presented
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as the mean standard deviation (SD). The standard deviation is a measure of

the dispersion of outcomes around the mean. The differences among means were
determined using the method of the smallest squares and the significance level
was p< 0.05.
Results and discussion

total viable count

2
(lg CFU/cm )

After the chicken carcasses were refrigerated for three weeks at 4C, we excised
the skin and we determined the variation in time of total viable count and psychotrophic count. In Fig. 1 we show the variation on refrigerated storage of total
viable count. The average initial contaminations of the chicken skin were 5.11 log
CFU/cm2 (CFU colony forming unit), and in the 5th day of storage the total viable
count increased to 7.1 log CFU/cm2. The shelf life of the carcass, as stated by the
food manufacturer, was the fifth day of storage. After two weeks of refrigeration
the average microbial ecology of the skin were 9.44 log CFU/cm2. As can be seen
from Fig. 1, the total viable count from refrigerated skin is steadily increasing during considered storage time. In the 21st day of refrigeration the total viable count
(10.4 log CFU/cm2) was doubled compared to initial contamination.
11
10
9
8
7
6
5
4

8 10 12 14
storage (days)

16

18

20

22

Fig. 1. Total viable count variation for three weeks of storage

As it can be observed from Fig. 2, the psychotrophic count increased steadily

along all the storage period of refrigerated chicken skin. In the first day of experimental study, the average organisms were found in number of 4 log CFU/cm2.
After 5 days refrigeration of chicken carcasses, the excised skin revealed average
psychrotrophic ecology of 5.44 log CFU/cm2. Two weeks of refrigeration lead to
an increased number of microorganisms at 9.33 log CFU/cm2, and a week later at
10.44 log UFC/cm2. So, in the 21st day of refrigerated storage the psychotrophic
count was by 2.6 times grater than in the first day of refrigeration.

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(lg CFU/cm )

psychrotrophic count

11
9
7
5
3

10

12

14

16

18

20

22

storage (days)

Fig. 2. Psychotrophic count variation for three weeks of storage

Thomas and McMeekin1 found an initial load of total viable count on leg and
breast skin of chicken carcasses stored at 2C of 4105, respectively 4.83105. Those
results are slightly higher than those found by our team. The total viable count
data in the following days, as we determined, are increasing from those found by
the Australian authors: in the 4th day of storage we have an approximate 6.7 log
UFC/cm2 and they have 7.85105. Also in the 16th day of storage they found a
value of 1.29109 and 2.57109 for leg, respectively breast and we found a value
of 9.8 log UFC/cm2. Our data concerning the total viable count data of chicken
skin are not very different of those compared.
In Fig. 3 we compare the total viable count and the psychotrophic counts of
chicken skins that were refrigerated for three weeks. In the first day of storage the
psychotrophic microorganisms are found under the total viable count (respectively
4 log CFU/cm2 and 5.11 log CFU/cm2). At the beginning of the storage total viable
counts were with 21.72% greater than psychotrophic count. At superior shelf life
limit of carcasses, the chicken skin had a total viable count with 23.38% larger
than psychotrophic count. In time, after two weeks of storage, the psychrotrophic
microorganisms are increasing in number and have a tendency to equal the number
of total viable count. After three weeks the psychotrophic microorganisms are in
greater number than total viable count. We can say that after refrigeration storage
of the skin, it happened a selection in the microbial ecology in advantage of psychrotrophic organisms. Those microorganisms, after adaptation at environmental
conditions (t = +4C), multiply rapidly becoming predominant.
Making a comparison with the psychotrophic count found by Thomas and
McMeekin1 we can say that the initial load of the Romanian chicken skin is a little
bigger: we found a value of 4 log CFU/cm2 and the Australian authors found 6103.
The trend is keeping for the 4th, 8th, and 16th day. In this case, the differencees
between our values found in 16th day of refrigerated skin storage and Thomas and
McMeekin data were of about two logarithmical cycle.

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lg CFU/cm

psychrotrophic
11
10
9
8
7
6
5
4
3

total viable count

5
7
storage (days)

14

21

Fig. 3. Comparative variation of psychotrophic and total viable counts from chicken skin after three
weeks of storage

During refrigeration storage, the pH of chicken skin is increasing, as we

shown in Fig. 4. In the first day of storage, the pH has an average value of 6.16,
at the end of shelf-life of carcasses it increased at 6.35. Three weeks of storage
raised the skin pH at 7.53. Those increased values of pH are due to biochemical
and microbiological changes that occur in the skin. It seems that an increased pH
help microorganisms to multiply, creating a favourable environment.

Fig. 4. pH variation during refrigerated skin storage

All the bioactive amines studied were found in chicken skin. It is known that
some of them are made by metabolic activities in the living organisms and can be
identified after their death, others are made by decarboxylation of free amino acids
by spoilage microorganisms, especially bacteria. Some of the studied amines had
a small content in the first day of storage: putrescine 1.43 mg/kg d.m, histamine
1.86 mg/kg d.m, cadaverine was not detected. It is normal to found putrescine in
chicken skin into a small quantity because putrescine is an amine that participates
to polyamine interconversion pathway, next to spermine and spermidine. Also,
putrescine is a metabolite of spoilage bacteria, but in the first day of storage, is
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unlikely to occur. Cadaverine was not found because, as putrescine, it is made as

a result of microbial spoilage. Spermine had the highest content in the first day
followed by spermidine and serotonin. As it can be seen from Fig. 5, tryptamine,
-phenylethylamine, putresceine, cadaverine, histamine, serotonin, and tyramine
content is increasing during storage. Spermidine and spermine content is decreasing in refrigerated skin storage, due to enzymatic activity of the microorganisms
that need those polyamines as nitrogen sources and by metabolic activities that
take place in the postmortem skin.
TRP
SER

FEA
TIR

PUT
SPMD

CAD
SPM

HIST

50
45

bioactive amines (mg/kg d.m.)

40
35
30
25
20
15
10
5
0

10 12 14 16 18 20 22

storage (days)

Fig. 5. Bioactive amines variation in refrigerated chicken skin during three weeks of storage
TRP tryptamine, FEA phenylethylamine, PUT putrescine, CAD cadaverine, HIST histamine,
SER serotonin, TIR tyramine, SPMD spermidine, SPM spermine

The biggest increment in the 21st day of storage had cadaverine (46.66 mg/kg
d.m.) followed by putrescine (31.19 mg/kg d.m.) and tyramine (22.41 mg/kg d.m.).
The same sequence was followed in the second week of chicken skin storage.
The major increase of those three biogenic amines begins after the seventh day of
storage. So, putrescine, cadaverine and tyramine are biogenic amines produced
by microbial spoilage activity. Also, putrescine and cadaverine are amines that
can be identified by smell because their unpleasant odour. Very important for the
human health is the content of tyramine, phenylethylamine and histamine, because
those amines are responsible for migraines attacks. Comparing the initial content
of those amines from the amines pool, we can say that they are small to medium
(we refer as medium values between 35 mg/kg d.m. and small under 2 mg/kg
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d.m.). At end of the carcasses shelf-life the contents of those amines in the skin
are not exceeding medium values, which is for health a beneficial thing. Anyway,
1 mg of tyramine can trigger a migraine attack to predisposed individuals. So, it
depends on the health and metabolism of each individual.
Conclusions
During chicken carcasses storage at 4C for three weeks, total viable count and
psychrotrophic count are steadily increasing. The skin pH is increasing and it seems
beneficial for microbial development.
In the first day of storage the contents of microorganisms were the least,
microbial ecology becoming double after three weeks of storage at refrigerated
conditions.
Spoilage microorganisms acted more intense after the seventh day of storage,
because the putrescine, cadaverine and tyramine content were increasing very
much after that day.
At the end of carcasses shelf-life the skin microbial ecology was as follows:
7.1 log CFU/cm2 for total viable count and 5.44 log CFU/cm2 for psychotrophic
count; pH value at the fifth day of storage was 6.35. The refrigeration temperature
of 4C has an inhibitor action on development and enzymatic decarboxylation
activity of spoilage microorganism during chicken skin storage.
Acknowledgement. We like to thank the research team of the Institute for ResearchDevelopment of
the Horticultural Products Marketing and Industrialisation Horting Bucharest, for helping us with
our experiments, and especially to Mrs. Daniela Moise who helped with HPLC method validation.

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Received 7 July 2009
Revised 11 September 2009

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