PaPer Ital. J. Food Sci., vol. 27 - 2015 351 - Keywords: cooking, egg, florfenicol, florfenicol amine, residue, storage - THE EFFECT OF COOKING AND STORAGE ON FLORFENICOL AND FLORFENICOL AMINE RESIDUES IN EGGS AYHAN FILAZI1*, UFUK TANSEL SIRELI2, BEGUM YURDAKOK DIKMEN1, FARAH GONUL AYDIN1 and ASLI GUL KUCUKOSMANOGLU1 1Department of Pharmacology and Toxicology, Faculty of Veterinary Medicine, Ankara University, 06110 Diskapi, Ankara, Turkey 2Department of Food Hygiene and Technology, Faculty of Veterinary Medicine, Ankara University, 06110 Diskapi, Ankara, Turkey *Corresponding author: filazi@veterinary.ankara.edu.tr AbstrAct the aim of this study was to evaluate the effects of storage conditions (room temperature, re- frigerator) and cooking methods (frying, boiling) on florfenicol (FF) and florfenicol amine (FFA) resi- due levels in eggs. Without any significant difference between storage conditions at 20˚c and +4˚c, residue levels decreased within days, but were still present on day 28. Frying and boiling for 1 and 5 min yielded similar results to the storage conditions just described; there was a significant decrease in residue levels, but still not enough for decomposing. these findings indicate that FF and FFA residues are heat-labile. mailto:filazi%40veterinary.ankara.edu.tr?subject= 352 Ital. J. Food Sci., vol. 27 - 2015 IntroductIon When veterinary drugs are administered to farm animals, either therapeutically or to pro- mote growth, residues remain in their meat, milk or eggs if proper precautions are not fol- lowed (botsoGLou and FLEtourIs, 2001). An- tibiotics play an important role among such drugs. In addition to their positive effects, they can also cause health problems, including drug hypersensitivity (PAIGE et al., 1999; donoGHuE, 2003). Antibiotics not only threaten food safe- ty, but also cause the development of some re- sistant bacterial strains from among sensitive bacteria even when used at moderate doses for long periods of time (PAIGE et al., 1999; FILAZI et al., 2005). testing for drug residues are ordinarily per- formed on raw products. Almost no edible ani- mal products or byproducts are consumed raw, but require some type of processing or cooking, such as frying, boiling, or roasting, before con- sumption. these processes can cause denatura- tion of proteins, elevation of temperature, loss of water and fat, and pH variations that can even- tually result in alteration to the concentration, chemical nature, chemical reactions, and solu- bility of drug residues in a particular food item. Many drugs are chemically unstable to varying degrees, and therefore may undergo degradation during storage, cooking or processing in consum- able foods (botsoGLou and FLEtourIs, 2001). In general, the temperatures achieved during cooking are assumed to degrade antibiotic res- idues in food; however, ordinary cooking proce- dures are unreliable for degrading or inactivat- ing several commonly used veterinary drugs. Ear- lier studies have indicated that sulfamethazine (rosE et al., 1995; PAPAPAnAGIotou et al., 2005) chloramphenicol (botsoGLou and FLEtourIs, 2001), streptomycin (InGLIs and KAtZ, 1978; o’brIEn et al., 1980), neomycin (KAtZ and LEV- In, 1978), gentamicin (sIrELI et al., 2006), fluo- roquinolones (bAYdAn et al., 2000a, b; bAYdAn et al., 2002), penicillin G (nouWs and ZIV, 1976; boIson et al., 1992), nitrofurantoin (cooPEr and KEnnEdY, 2007), oxacillin, clindamycin, novo- biocin, trimethoprim, vancomycin, and azlocil- lin are heat-stable (trAub and LEonHArd, 1995), whereas oxytetracycline (KItts et al., 1992) and amphenicols (FrAnJE et al., 2010) heat-labile. on the other hand, several β-lactams including am- picillin and amoxicillin are partially heat-labile (trAub and LEonHArd, 1995). Antibiotics of the same class were reported to vary in heat stabil- ity according to the type of matrix and heating treatment involved (KItts et al., 1992; rosE et al., 1996; FrAnJE et al., 2010). As such, the ef- fect of different matrices on the stability of every veterinary drug should be investigated. Although most edible animal products are consumed after cooking or some type of pro- cessing, for the licensing of veterinary drugs research concerning the effects of storage and cooking of the drugs on different matrices are lacking. Most data on drug residues in edible an- imal products and government regulation con- cern raw products. It is therefore essential to de- termine the effect of processing on all veterinary drugs when assessing human exposure to drug residues in animal food products (IbrAHIM and MoAts, 1994; MoAts, 1988; MoAts 1999; bot- soGLou and FLEtourIs, 2001). Florfenicol (FF) is a wide-spectrum, synthetic antibacterial that is structurally related to d(−) threo-chloramphenicol; however, FF differs from chloramphenicol in that FF contains a p-methyl sulfonyl group instead of a p-nitro group and it contains a fluorine atom instead of a hydrox- yl group in the terminal primary alcohol group (EMEA, 1999). FF has not been approved for use in laying hens; however it is used in cattle, swine, poultry, and fish (EMEA, 2000). FF is metabolized into florfenicol amine (FFA), florfenicol oxamic acid, florfenicol alcohol, and mono-chloroflorfenicol in animals. FFA is the longest-lived metabolite in the bovine liver; therefore, FFA can be used as a marker for the calculation of withdrawal time (AnAdon et al., 2008; XIE et al., 2011). In light of the apparent advantages over chlo- ramfenicol and its availability as an additive, the potential for off label use of FF is high. due to its broad spectrum antibacterial activity, ready avail- ability and low cost, it remains a possibility that FF residues will continue to be found in such ani- mal food products as eggs. For example, Xie et al. (2011) analyzed 50 egg samples obtained from a local supermarket in china and reported 19 ppb of FF and 36 ppb of FFA in only 1 egg. FILAZI et al. (2014) reported that the concentration of FF and FFA in eggs were 0.1%, 0.08% respectively regardless of the route of administration. data on the heat stability of FF is essential for food safety; however, the literature contains few data regarding its heat-stability during cooking. under environmental conditions FF is stable at 25 °c, yet photodegradation occurs at varying rates in water under various lighting conditions (GE et al, 2009). FF was shown to rapidly de- grade to FFA in the deep sediment of marine en- vironments via biodegradation (HEKtoEn et al, 1995). A few studies on the residue of FF and FFA in eggs have been published (XIE et al. 2011; FILAZI et al., 2014); but the data are insufficient. FrAnJE et al. (2010) reported that amphenicols exhibit differential behavior in terms of heat- induced degradation in solutions and protein matrices. Although the level of amphenicol deg- radation in soybean sauce and meat was high, heating may generate product with antimicro- bial activity; therefore, heating amphenicol res- idues in food cannot always be considered safe. FF is the most commonly used veterinary anti- microbial agent in turkey, particularly so due to its illegally use in laying hens. nonetheless, Ital. J. Food Sci., vol. 27 - 2015 353 few studies have examined FF residue levels in eggs. An earlier study reported that FF and FFA were detected in the eggs of hens administered with FF (FILAZI et al., 2014) chicken eggs are widely used in the prepara- tion of many types of food, including many baked goods. some of the most common preparation methods include fried in oil, hard-boiled, soft- boiled and omelets. data regarding FF and its main metabolites in cooked and stored eggs are lacking. As such, the aim of the present study was to determine the effects of different stor- age conditions (room temperature and refriger- ation) on FF residue levels in eggs stored up to 28 days and to determine the effect the differ- ent cooking methods (frying and boiling) on FF and FFA residue levels. MAtErIALs And MEtHods Animals the study protocol was approved by the Anka- ra university Ethics committee (2007-15-45). the study included 50 IsA brown laying hens aged 48 weeks and weighing 1.9-2.4 kg. the hens were housed individually in fiber cages (30x35x45 cm), in a ventilated room maintained at 20°c under 14 h day light condition. the hens received standard commercial layer mash (120 g/d) and water ad libitum. the hens were fed for 1 week and their eggs were collected for preliminary analysis to de- termine if they were analyte-free. Trials A veterinary drug containing 300 mg of FF in 1 mL was used (Mediflor 30% oral solution, Medicavet company, turkey) for the clinical tri- als. FF was administered at a dose of 20 mg/kg/ day via gavage for 3 days to the 50 laying hens, and then their eggs were collected daily thereaf- ter. the effect of storage procedures on the res- idues was determined on the first day using 44 eggs. the effects of cooking procedures were de- termined on the second day using 32 eggs. In all, 20 of the eggs collected on the first day were kept at 4°c in a refrigerator, and 20 were kept at 15-20°c (room temperature). In addition, 4 eggs were analyzed on day 4, 7, 14, 21, 28 of storage to determine FF and FFA residue levels. Lastly, 4 uncooked eggs collected on day 1 were ana- lyzed as a control group; of the eggs collected on day 2, 8 uncooked, 8 fried in oil, 8 undercooked (1 min in boiling water) and 8 overcooked (5 min in boiling water) were then analyzed. Sample preparation and analysis FF and FFA were extracted from homogenized eggs via phosphate buffer (pH:7) and ethyl ace- tate. Following purification, the samples under- went high-performance liquid chromatography (HPLc) using a photodiode array detector (PdA) and c18 column; the method was validated ac- cording to IcH guidelines, as described elsewhere (FILAZI et al., 2014). According this method, limits of detection and of quantitation values were 1.94 and 6.45 ppb for FF, respectively, and 0.48 and 1.58 ppb for FFA, respectively. relative standard deviation values of intra-day and inter-day var- iation below 11% also confirmed the usefulness of the method for analysing FF and FFA in eggs. stAtIstIcAL AnALYsIs Variance analysis was performed with all data and a multiple range test was used to determine the differences between groups. All analyses were performed using sPss v. 17.0 for Windows. rEsuLts And concLusIon the effects of different storage temperatures and durations on FF and FFA residue levels in table 1 - Mean±sd* concentration (in ppb) of florfenicol and florfenicol amine residues in eggs stored at room temperature (15-20°c) and in a refrigerator (+4°c). Days Florfenicol Florfenicol amine (n=4) Room temperature Refrigerator Room temperature Refrigerator (15-20°C) (+4°C) (15-20°C) (+4°C) 0 290.65±11.02a 290.65±11.02a 91.79±6.77a 91.79±6.77a 4 151.24±10.69b 167.43±8.18b 58.26±5.98b 58.61±5.85b 7 79.65±9.43cx 105.10±4.25cy 28.95±5.03c 35.40±2.33c 14 68.23±8.74dx 87.84±5.01dy 20.52±3.92d 24.37±1.20d 21 29.43±4.91ex 61.82±2.11ey 10.42±1.54e 8.54±1.04e 28 18.57±3.48f 22.14±0.03f 6.74±0.79f 7.06±1.21f *SD: Standard Deviation. abcdef: Differences between values with different letters in the same columns are significant (P<0.05). xy: Differences between values with different letters in the same rows are significant (P<0.05). 354 Ital. J. Food Sci., vol. 27 - 2015 eggs are shown in table 1. both FF and FFA amine residue levels in eggs were observed on day 28, though their levels had decreased signif- icantly (P<0.05). HEKtoEn et al. (1995) report- ed that FF rapidly depurated in the sediment of marine environments and that its metabolite (FFA) was isolated from the sediment. this find- ing suggests that FF is degraded to FFA in the sediment via metabolization or leaching; howev- er, the present study FF residues in eggs follow- ing storage for 28 day at room temperature and in a refrigerator were observed. FF residue lev- els in eggs were higher than FFA residue levels in the present study, which indicates that the in vitro degradation of FF might occur at a very low level or that it differs from its biological deg- radation. Further research would be required to understand the effect of storing on the FF and FFA residues in the eggs. FrAnJE et al. (2010) studied the heat stabili- ty of amphenicols in chicken meat and reported that 5-min heating of amphenicols in water in a microwave oven generated a comparable per- centage of degradation as did boiling in a water bath for 30 min 1 h; FF produced thiampheni- col (tAP) as a product of its breakdown, but not FFA. It was reported that although a higher lev- el of degradation of amphenicols was observed in soybean sauce, heating treatment might still generate product with antimicrobial activity (FF to tAP) and as such, heating amphenicol resi- dues in food cannot always be safe. FF was reported to be hydrolytically stable and to have a hydrolysis half-life > 1 year at 25°c in natural waters (HAYEs et al., 2003; PouLIQuEn et al., 2007; GE et al., 2009). GE et al. (2009) per- formed photodegradation experiments on tAP and FF in aqueous solutions under irradiation from different light sources. they reported that under uV-Vis irradiation (λ>200 nm) photodeg- radation in seawater was fastest, followed by pure water and freshwater, whereas under so- lar or simulated sunlight (λ > 290 nm), photo- degradation occurred only in freshwater. under uV-Vis irradiation, cl- (dominant sea water con- stituent) was observed to promote singlet oxy- gen formation and accelerated the photodegra- dation of phenicols, whereas phenicols did not photolyze under simulated solar irradiation, ir- respective of the presence of cl-. In contrast, HAYEs et al. (2003) reported that FF was stable under a range of simulated field conditions, including various pipe materials and conditions of hard and soft and chlorinated or non-chlorinated water at low or high pH; there- fore not only cl- but also some other minerals might effect the stability of FF. the effects of different cooking procedures on FF and FFA residues in eggs observed in the present study are shown in table 2. Even though, none of the cooking methods complete- ly destroyed FF or FFA residues in eggs, there was a significant decrease in the level of detect- able FF and FFA residues (P<0.05). concentra- tions of both analytes were reduced by 78%- 97% via frying and boiling. these findings sug- gest that FF and FFA heat labile in eggs, which indicates that both do not bind to proteins in eggs with high affinity. FrAnJE et al. (2010) re- ported that amphenicol degradation was appar- ent following as little as 30 min of heating and that it was correlated with the length of heat- ing, implying that as cooking time increased the degree of residual drug present in samples de- creased; as such, it could be assumed that there was a strong correlation between the decrease in FF and FFA concentrations in observed eggs during different cooking methods and the dura- tion of cooking (P<0.05, table 2). sHAKILA et al. (2006) studied the stability of chloramphenicol (cHP) residues in white shrimp (Penaeus indicus) subjected to cooking (100 °c) for 10, 20 and 30 min as well as retorting (121°c) for 10 and 15 min, based on a microbial assay method using Photobacterium leiognathi as the test organism. they reported that the loss of cHP increased as temperature and duration of heating increased, where the drug could be completely destroyed. on the other hand botsoGLu and FLEutorIs (2010) reported that cHP was quite stable un- der heating conditions when added to water or milk; after 2 h of boiling, it was decreased by <8%. these findings indicate that the heat sta- bility of amphenicols is matrix dependent, where results from different matrices could not be at- tributed to eggs when interpreting. Even though, FF is not approved for use in laying hens, its off label use for severe indica- tions can result in antibiotic residues in eggs that both farmers and consumers should be in- formed about. As such, drug withdrawal periods should be extended prior to poultry slaughter table 2 - Mean±sd*, quantity of florfenicol and florfenicol amine residues (in ppb) after different cooking methods. Residues (n=8) Raw Fried Undercooked Overcooked (1 minute) (5 minutes) Florfenicol 265.45±13.67a 56.51±9.68b 35.67±4.57c 5.68±1.17d Florfenicol amine 110.31±12.73a 19.77±4.71b 10.20±1.72c 4.57±0.92d *SD: Standard Deviation. abcd: Differences between values with different letters in the same row are significant (P<0.05). Ital. J. Food Sci., vol. 27 - 2015 355 or egg distribution to avoid antimicrobial resis- tance. thermal treatments may reduce the con- centration of veterinary drug residues in foods and thereby might reduce the pharmacological and/or toxic effects of these compounds. (HsIEH et al., 2011). In the current study, FF and FFA were observed to be heat labile in chicken eggs, the level of which depended on cooking meth- od and duration. the findings show that FF and FFA residue levels in eggs from treated laying hens were not completely eliminated via cooking or of up to 28 d; however, cooking did significantly decreased the level of the drug in eggs. 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