Biogenic Amines and Their Role as Index of Freshness in Chicken Meat

Aims: To evaluate the biogenic amines formation in chicken breast and thigh meat during chilled storage and their potential use as index of freshness. February 2014 May 2014. Methodology: The biogenic amines (BA) concentrations in chicken breast and thigh meat samples were determined by HPLC analysis and were monitored during storage at two temperature regimes (5.0±1.0ºC and 1.0±1.0ºC). The changes in biogenic amines content, microbiological and sensory quality of meat were studied. Results: It was found that during the storage period, the content of putrescine, cadaverine and tyramine increased in all meat samples. The biogenic amine histamine was not detected. The first signs of chicken meat sensory deterioration were found when BAI values reached 20 mg/kg. Poultry samples with total viable count (TVC) higher than 10 7 cfu/g always had BAI>20 mg/kg. Conclusion: The fallowing limits of biogenic amine index (BAI) (putrescine + cadaverine + histamine + tyramine) for chicken breast and thigh meat quality were proposed: BAI < 10 mg/kg for good quality fresh meat, 10 mg/kg < BAI < 20 mg/kg for acceptable meat and BAI > 20 mg/kg for unacceptable meat with initial spoilage signs.


INTRODUCTION
Chilled meat is perishable food stuff and its quality deteriorates progressively throughout storage. The spoilage during storage of refrigerated chicken meat is due to the microbial activity and the biochemical transformations inside the product. Meat freshness evaluation is becoming increasingly important because of the consumer awareness about food quality and safety [1]. Chicken meat quality could be evaluated by determination of its microbiological and sensorial characteristics. Preliminary information about meat freshness could be obtained by sensory evaluation, but taken alone this analysis is not sufficient for estimation of meat quality and safety. Standard microbiological analyses are time consuming and they could be replaced by the analysis of the chemical changes which are caused by the meat microflora. It also could provide important information about the degree of the meat spoilage caused by the microbial decarboxylases. Under appropriate conditions they can convert some amino acids to their corresponding toxic amine [2]. For identifying incipient spoilage some chemical indices were proposed: meat pH values, total volatile basic nitrogen (TVBN), biogenic amines content etc [3]. Undesired accumulation of biogenic amines in to food requires both the availability of precursors (i.e. amino acids) and the presence of microorganisms possessing amino acid decarboxylases [4,5,6,7].
Among chemical indicators, biogenic amines, particularly putrescine and cadaverine, have been proposed for determining meat quality. According to Balamatsia et al. [8] the biogenic amine determination is important not only because of their toxicity but also for their application as potential freshness indicators. The fresh meat contains very low quantities of these compounds, during the storage period these concentrations increase due to bacterial growth [9]. Galgano et al. [10] and Hernández-Jover et al. [11] reported that tyramine, putrescine and cadaverine can be produced during storage of the meat. According to Ruiz-Capillas and Jiménez-Colmenero [12] the most prevalent biogenic amines in meat and meat products are tyramine, cadaverine, putrescine and histamine.
The toxic effects of biogenic amines and their potential effects on human health are not fully clarified. According to the scientific opinion on risk based control of biogenic amine formation in fermented foods of EFSA [13] no adverse health effects were observed after exposure to 50 mg histamine (per person per meal) and 600 mg tyramine for healthy individuals. The information in that respect for putrescine and cadaverine is insufficient.
Different studies on the biogenic amines formation in refrigerated chicken meat showed that the tyramine, cadaverine, putrescine and histamine concentrations are increasing in time, while spermine and spermidine concentrations are decreasing during storage [1,8,14,15,16]. Identification and quantification of biogenic amines is less time consuming in comparison with the microbiological analysis and they could be used as a complex indicator for chicken meat freshness evaluation. However, the practical implementation of this parameter requires more studies.
The aim of the present study was to evaluate the biogenic amines formation in chicken breast and thigh meat during chilled storage at two temperature regimes (5.0±1.0ºC and 1.0±1.0ºC) and their potential use as index of freshness.

Meat Samples and Refrigeration Treatment
The refrigerated chicken breast and thigh meat samples were delivered from local processor

Microbiological Analysis
Determination of the total viable count and psychrotrophic bacteria counts were performed according to the standard procedures [17,18]. Мeat samples (10 g) were dispersed in 90 mL of saline solution (0.85% NaCl) by using of a Stomacher Lab Blender. Decimals dilutions were prepared and plated on Plate Count Agar (PCA). Inoculated plates were incubated in aerobic condition at 30ºC for 3 days for total viable count and at 7ºC for 10 days for psychrotrophic bacteria determination. After incubation period viable colonies were counted.

Analysis of Biogenic Amines by HPLC
Sample treatments were the same for each kind of meat (chicken breast and thigh meat). The meat cuts were minced. 20 ml of 0.4 M HClO 4 were added to an amount of 5 g minced meat. The samples were homogenised and then centrifuged for 5 min at 2500 rpm. The supernatant was pooled and diluted to 50 ml with 0.4 M HClO 4 . The centrifuged acid extract was derivatised according to the following procedure: 200 µl of 2N NaOH were added to 1 ml portions of the diluted supernatant, then buffered by adding 300 µl of saturated NaHCO 3 solution and then 2 ml of dansylchloride solution (10 mg/ml in acetone) were added. The dansylation reaction proceeds at room temperature [19]. 100 µl of NH 4 OH were added after 15 min to stop the reaction and to remove residual dansylchloride. The final volume was adjusted to 5 ml by adding acetonitrile. The obtained dansylated solution was filtered and injected into the Liquid chromatograph (Agilent Technologies, Model 1260 Infinity) equipped with UV detector and Spherisorb ODS2 (C18) (4.6 x 150 mm, 5 µm, Waters, Milford, USA) column. The mobile phase was a mixture of acetonitrile and water (80:20). Chromatographic conditions: injected volume 20 µl, flow rate 0.5 ml/min; detection wavelength (λ) = 254 nm were used. After each run the column was conditioned for 10 min. Each HPLC run took about 18 min and afterwards the column must be conditioned again for 10 min.
The presence and abundance of biogenic amines (putrescine, cadaverine, histamine, tyramine -Sigma-Aldrich) were determinate by comparing sample peak retention time to standards. Additionally 1.4-diaminoheptane was used as internal standard. One milliliter of the standard solutions was derivatised as previously described for the acid extracts.

Sensory Evaluation
Sensory quality of all studied samples was evaluated by five member expert panel. The color, appearance, texture and aroma acceptability of chicken breast and thigh samples were evaluated. A five point's hedonistic scale was used: 5 points -good quality (without any off -odors or off -flavours); 4 points -acceptable quality (slight changes in meat color and texture); 3 points -medium quality (significant changes in meat color and texture); 2 points -low quality (slight off-odors and/or off-flavors); 1 -very low quality (spoiling stage). Total sensory evaluation score (the average of sensory quality attributes) for each sample was calculated and submitted at this article. The samples with total sensory evaluation score lower than 3 were evaluated as unacceptable for consumption.

Statistical Analysis
Statistical analyses were carried out on the averages of the triplicate results. Data were analyzed by the analysis of variance (one-way ANOVA) method with a significant level of P= .05 [20]. The Duncan's multiple comparison test (SPSS) with a significant difference set at P= .05 was used to compare sample means. Significant differences between means less than 0.05 were considered statistically significant [21]. All statistical procedures were computed using the Microsoft Excel 5.0 software.

Microbiological Analysis
Total viable count (TVC) represents the total bacterial load in the studied meat sample. The TVC tests could reflect the general hygiene condition of the poultry. The psychrotrophic bacteria count (PBC) represents that part of microbial population which is able to growth at low temperatures. The results for TVC and PBC of studied poultry samples are shown on Fig. 1. The initial microbial loads of chicken breast and thigh samples were 3.0 x 10 4 cfu/g and 2.4 x 10 5 cfu/g, respectively (Fig. 1). Balamatsia et al. [1] established similar initial contamination of modified atmosphere-packaged chicken fillets. Higher microbial load of thigh samples is probably due to the presence of skin which is more contaminated during the processing. The psychrotrophic bacteria count of breast and thigh meat at the beginning of cold storage was 3.2 x 10 4 cfu/g and 1.6 x 10 5 cfu/g, respectively. The initial values of TVC and PBC were in the same range. Moreover, PBC of the studied samples had a similar variation trend as TVC (Fig. 1). According to Baston et al. [3] during the cold storage of meat the majority of the microorganisms incline to become mostly psychrotrophic-type due to the adaptation at the new environment conditions. This could explain the similarity in the variation trend of TVC and PBC found in the present study. Different limits for total microbial load were proposed to classify meat quality. Values lower than 10 6 cfu/g would indicate good quality meat [22]. TVC values between 10 6 cfu/g and 10 7 cfu/g -acceptable quality and counts higher than 10 7 cfu/gunacceptable [23]. In the present study, an arbitrary value for total microbial load of 10 7 cfu/g was taken for the upper acceptability limit of fresh chicken meat.
For chicken thigh and breast samples stored at 5.0±1.0ºС such values were exceeded on day 5 and day 8, respectively (Fig. 1A). The TVC and PBC of chicken samples stored at 1.0±1.0ºС remained below the values of 10 7 cfu/g up to the 10-th day of the storage (Fig. 1B). These results demonstrate the significant impact of storage temperature on the microbial growth in chicken meat. Delayed microbial growth in chicken samples stored at 1.0±1.0ºС is a main reason for the extended shelf-life and sensory acceptability of these samples in comparison with the samples stored at 5.0±1.0ºС.

Sensory Evaluation
Sensory evaluation is one of the most popular means for assessing meat freshness. At the present study, greatest sensory score received by chicken breast and thigh samples at the first stages of cold storage (Fig. 2).
With the increasing of storage time some alterations in meat texture and flavour were observed. The appearance of off flavours and off odours as well as some great textural changes at the last stages of the storage indicated for meat deterioration processes started. Usually, such changes are recognized as first signs of deterioration and makes meat sensory quality unacceptable (less than 3 points). It was found ( Fig. 2A), that the chicken thigh and breast samples stored at 5.0±1.0ºС preserved acceptable sensory quality for 5 and 8 days, respectively. These results are in agreement with the findings of other authors [8,24,25] who established that the shelf-life of chicken breast meat stored at 4.0ºС is between 7 and 9 days. The longer shelf-life of chicken breast meat in comparison with thigh meat could be due to the lower microbial load of this product (Fig. 1). The results (Fig. 2B) showed that the chicken thigh and breast samples stored at 1.0±1.0ºС preserved their acceptable sensory quality up to the 10-th day of storage. The decreasing of storage temperature with 4.0ºС (from 5.0±1.0ºС to 1.0±1.0ºС) delayed significantly deterioration processes in chicken meat which results in approximately double increase of its shelf-life.

Biogenic Amines Analysis
The biogenic amines formation in meat is associated with amino acid decarboxylase activity of microorganisms during storage and can be used as a quality indicator for poultry products. The changes in biogenic amines content of studied chicken breast and thigh samples during the storage at 5.0±1.0ºC are shown on Fig. 3. At the beginning of the storage period concentrations of putrescine, cadaverine and tyramine were in the range from 1.2 mg/kg to 3.4 mg/kg. The biogenic amine histamine was not detected in studied poultry samples during the whole storage period. During the first 5 days of storage at 5.0±1.0ºC biogenic amines content of poultry samples did not changed significantly (P= .05). After this period, gradual increase of biogenic amines concentrations was observed. The tyramine content of chicken breast samples increased significantly (P= .05) and at the 10-th day of storage reached up to 25.5 mg/kg. At the same storage period the putrescine and cadaverine concentrations increased from 4.5 mg/kg to 17.0 mg/kg and from 7.2 mg/kg to 15.8 mg/kg, respectively. Similar trends in biogenic amines accumulation were found during storage of chicken thigh samples. Significant increase in biogenic amines concentration of these samples was observed after 8 -th day of storage. At the 10 -th day the content of putrescine, cadaverine, and tyramine in chicken thigh meat was 15.2 mg/kg, 12.8 mg/kg and 18.7 mg/kg, respectively. The results (Fig. 3 and Fig. 4) showed delayed formation of biogenic amines in samples stored at lower temperatures. Probably, this is due to the greater inhibitory effect of lower temperatures on the microbial growth and lower decarboxylase activity of the bacteria responsible for the production of biogenic amines in the muscle tissue. Our results are in agreement with the study of Hutařová et al. [26] who found dependence of biogenic amines formation with temperature and length of storage of eviscerated pheasants. The generation of biogenic amines is a complex biochemical process, but mainly it is due to the activity of the meat microflora. Therefore, the biogenic amines formation in poultry (Figs. 3 and 4) is related to variations of the microbial population (Fig. 1). Amino acid decarboxylases are enzymes present in many microorganisms such as species of the genera Bacillus, Clostridium, Pseudomonas, Photobacterium, as well as in genera of the family Enterobacteriaceae, such as Citrobacter, Klebsiella, Escherichia, Proteus, Salmonella and Shigella, and Micrococcaceae such as Staphylococcus, Micrococcus and Kocuria. [6,10]. According to Dainty [9] the biogenic amines putrescin, cadaverine, histamine and tyramine are found in very low levels in fresh meat, and their formation is associated with bacterial spoilage.
In this respect the so-called "biogenic amine index" (BAI) could be used for evaluation of the poultry products freshness. Wortberg and Woller [27] and Hernández-Jover et al. [11] defined BAI as a sum of the putrescine + cadaverine + histamine + tyramine concentrations in meat. In the present study, the first signs of chicken meat sensory deterioration were found when BAI values reached 20 mg/kg. Moreover, the samples with total viable count (TVC) higher than 10 7 cfu/g always had BAI>20 mg/kg. Based on the results from sensory and microbiological analysis of studied poultry samples and the TVC -thigh meat TVC -breast meat PBC -thigh meat PBC -breast meat А В acceptability limits of 10 7 cfu/g for TVC and 3 point for sensory evaluation score, the fallowing limits of BAI for chicken breast and thigh meat quality were proposed: BAI < 10 mg/kg for good quality fresh meat, 10 mg/kg < BAI < 20 mg/kg for acceptable meat and BAI > 20 mg/kg for unacceptable meat with initial spoilage signs. For the practical implementation of these BAI limits further investigations on the effect of breed and feeding conditions, as well as microbial contamination on the biogenic amines content of cold stored poultry are needed.

CONCLUSION
The shelf-life of the poultry samples stored at 5.0±1.0ºC and 1.0±1.0ºC was evaluated on the basis of the microbiological and sensory parameters. Based on the obtained results the BAI (putrescine + cadaverine + histamine + tyramine) values higher than 20 mg/kg of muscle are proposed as the upper limits for initiation of spoilage in fresh chicken meat stored aerobically. The results obtained demonstrate the applicability of biogenic amine index (BAI) as indicator for poultry quality and freshness evaluation.