1437

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

Biochemical- and bioprotection

* For correspondence.

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 impor-tant role in heat regulation, in insulation against drying and prevents microorgan-isms 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

1438

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 perform-ance 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 refrig-erator used is an electrolux eNB43691S. The carcasses weight varied between

1439

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 suit-able 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 re-search 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 Sigma-Aldrich companies. installations and equipment used for biogenic amine determi-nation: 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 sys-tem 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 DE-TeCTOr, 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

1440

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

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 psy-chotrophic 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 dur-ing considered storage time. in the 21st day of refrigeration the total viable count (10.4 log CFU/cm2) was doubled compared to initial contamination.

456789

1011

0 2 4 6 8 10 12 14 16 18 20 22storage (days)

tota

l via

ble

coun

t (l

g C

FU/c

m2 )

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 experi-mental 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.

1441

3

5

7

9

11

0 2 4 6 8 10 12 14 16 18 20 22

storage (days)

psyc

hrot

roph

ic c

ount

(lg

CFU

/cm

2 )

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 psy-chrotrophic 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.

1442

3456789

1011

1 3 5 7 14 21storage (days)

lg C

FU/c

m2

psychrotrophic total viable count

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

1443

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 decreas-ing 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.

0

5

10

15

20

25

30

35

40

45

50

0 2 4 6 8 10 12 14 16 18 20 22

storage (days)

bioa

ctiv

e am

ines

(mg/

kg d

.m.)

TRP FEA PUT CAD HISTSER TIR SPMD SPM

Fig. 5. Bioactive amines variation in refrigerated chicken skin during three weeks of storageTrP 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

1444

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.

reFereNCeS1. C. J. THOMAS, T. A. McMeeKiN: Spoilage of Chicken Skin at 2C: electron Microscopy

Study. Applied and environmental Microbiology, 2, 495 (1981).2. B. M. luNd, T. C. BAird-PArKer, G. W. GOuld: The Microbiological Safety and Quality

of Food. Springer-Verlag, New York, llC, 1, 451 (2000).3. M. l. MOrGAN, F. BAuer, A. WHiTe: Biogenically Active Amines in Food. Cost Action

917, 7, 270 (2005).4. Y. H. Hui: Handbook of Food Science, Technology and engineering. Taylor and Francis CrC,

1, 13.1 (2005).5. G. ViNCi, M. ANTONelli: Biogenic Amines: Quality index of Freshness in red and White

Meat. elsevier, Oxford, royaume uni, Food control, 13 (8), 520 (2002).6. P. APOSTOlOS, i. CHOuliArA, ek. PAleOlOGOS et al.: relation of Biogenic Amines

to Microbial and Sensory Changes of Precooked Chicken Meat Stored Aerobically and under Modified Atmosphere Packaging at 4C. European Food Res. and Tech., 5, 685 (2006).

7. C. C. BAlAMATSiA, ek. PAleOlOGOS, M. G. KONTOMiNAS, i. N. SAVVAidiS: Cor-relation between Microbial Flora, Sensory Changes and Biogenic Amines Formation in Fresh Chicken Meat Stored Aerobically or under Modified Atmosphere Packaging at 4oC: Possible

1445

role of Biogenic Amines as Spoilage indicators. Antonie van leeuwenhoek, Springerlink , 89, 13 (2006).

8. C. ruiz-CAPillAS, F. JiMeNez-COlMeNerO: Biogenic Amine Content in Spanish retail Market Meat Products Treated with Protective Atmosphere and High Pressure. eur Food res. Technol., 218, 237 (2004).

9. A. l. STrOiA, G. MeNCiNiCOPSCHi, S. MuSu et al.: Active Biogenic Amines Contained in Foods and Agro-foods raw Materials of romania. Cost Action 917, Biogenically Active Amines in Food, 7, 194 (2005).

10. M. M. ABdel-MONeM, K. OHNO: Separation of the dansyl derivatives of Polyamines and related Compounds by Thin-layer and High-pressure liquid Chromatography. J. of Chroma-tography, (1975).

11. A. r. SHAlABY: Multidetection, Semiquantitative Method for determining Biogenic Amines in Foods. Food Chemistry, 52, 367 (1995).

12. A. M. SPiNelli, l. lAKriTz, A. e. WASSerMAN: effects of Processing on the Amine Content of Pork Bellies. J. of Agr. and Food Chem., 22, 1027 (1974).

13. C. GAGeT, e. WOlF, B. HeiNTzelMANN, J. WAGNer: Separations of the enantiomers of Substituted Putrescine and Cadaverine Analogues by Gas Chromatography on Chiral Stationary Phases. J. of Chromatography, 395, 597 (1987).

14. W. F. STAruSzKieWiCz, J. F. BONd: Gas Chromatographic determination of Cadaverine, Putrescine and Histamine in Foods. J. of the Assoc. of Analytical Chemists, 64, 584 (1981).

15. S. YAMAMOTO, H. iTANO, H. KATAOKA, M. MAKiTA: Gas-liquid Chromatographic Method for Analysis of di- and Polyamines in Foods. J. of Agr. and Food Chem., 30, 435 (1982).

16. M. A. deSideriO, P. dAVAlli, A. PeriN: Simultaneous determination of Aminobutyric Acid and Polyamines by High-performance liquid Chromatography. J. of Chrom., 419, 285 (1987).

17. J. l. MieTz, e. KArMAS: Chemical Quality index of Canned Tuna as determined by High-pressure liquid Chromatography. J. of Food Sci., 42, 155 (1977).

18. S. SuzuKi, K. KOBAYASHi, J. NOdA et al.: Simultaneous determination of Biogenic Amines by reverse-phase High-performance liquid Chromatography. J. of Cromatography, 508, 225 (1990).

19. G. ruGGieri, F. BOTre, M. C. dAleSSANdrO et al.: Meat Freshness evaluation by Biogenic Amines determination: Comparison between HPlC and Bioelectrical Techniques. in: The 2nd Congresso Nazionale di Chimica degli Alimenti, Giardini Naxos, 2427 Maggio, 1095, 1995.

20. S. eerOlA, r. HiNKKANeN, e. liNdOrFS, T. HirVi: liquid Chromatographic determina-tion of Biogenic Amines in dry Sausages. J. AOAC international, 76 (3), 575 (1993).

Received 7 July 2009 Revised 11 September 2009