PROCESSING, PRODUCTS, AND FOOD SAFETY Effects of packaging, mineral oil coating, and storage time on biogenic amine levels and internal quality of eggs T. C. Figueiredo,*1 D. C. S. Assis,* L. D. M. Menezes,† D. D. Oliveira,‡ A. L. Lima,* M. R. Souza,* L. G. D. Heneine,§ and S. V. Cançado* *Escola de Veterinária da Universidade Federal de Minas Gerais, Avenida Antônio Carlos, 6627, Belo Horizonte, 30123-970, Brazil; †Instituto Mineiro de Agropecuária, Rodovia Américo Gianetti, 4001, Belo Horizonte, CEP: 31.630-901, Brazil; ‡Aviário Santo Antônio, and §Fundação Ezequiel Dias, Rua Conde Pereira Carneiro, 80, Belo Horizonte, 30510-010, Brazil amines in the egg yolk and albumen. The application of mineral oil to the eggshell resulted in higher Haugh unit values throughout storage, and the use of plastic packages altered the internal quality. The application of mineral oil and the use of packaging had no effects on the microbiological and biogenic amine results. Microbiological analyses showed the absence of Salmonella spp., Staphylococcus aureus, thermal-tolerant coliforms, and fungi. However, the highest counts of mesophilic (1.1 × 107 cfu/g) and psychrotrophic (6.7 × 107 cfu/g) microorganisms were recorded. The highest values of biogenic amines detected and quantified were putrescine (2.38 mg/kg) and cadaverine (7.27 mg/kg) in the egg yolk and putrescine (1.95 mg/kg), cadaverine (2.83 mg/kg), and phenylethylamine (2.57 mg/kg) in the albumen. Despite these results, the biogenic amine levels recorded were considered low and would not be harmful to consumer health.
Key words: egg quality, mineral oil treatment, packaging, biogenic amine, microbiological quality 2014 Poultry Science 93:1–8 http://dx.doi.org/10.3382/ps.2014-04268
INTRODUCTION
them from becoming a source of toxo-infection and to ensure that they reach consumers with a high standard of quality. The internal quality of eggs can be evaluated based on their physical, chemical, biological, and functional characteristics. Factors such as the temperature, air relative humidity, storage conditions, and time influence the quality of eggs. Among the chemical reactions that take place inside eggs during storage is the transformation of dense albumen into thin albumen. This change may involve carbonic acid (H2CO3), one of the components of the buffering system in albumen, which is dissociated into water and carbon dioxide gas (CO2), which are lost via the eggshell pores (Brake et al., 1997; Figueiredo et al., 2011). The preservation of the internal quality of eggs can be achieved through the use of packages for storage
Eggs are a great and inexpensive source of high-quality protein. Eggs provide a unique and well-balanced source of nutrients, especially iron, various vitamins, and phosphorus, for people of all ages. Their availability, modest cost, ease of preparation, taste, and low caloric value give eggs a primary advantage for human nutritional needs. Eggs are used in the food processing industry for various applications, such as emulsifying, foaming, gelling, and whipping. Because of the high nutrient content of eggs, care must be taken to prevent ©2014 Poultry Science Association Inc. Received June 19, 2014. Accepted September 5, 2014. 1 Corresponding author: [email protected]
1
Downloaded from http://ps.oxfordjournals.org/ at University of Idaho on November 3, 2014
ABSTRACT This study was carried out with the aim of evaluating the effects of mineral oil application on eggshells and the use of plastic packages with lids on the physical-chemical and microbiological quality and biogenic amine contents of eggs stored under refrigeration for up to 125 d. A total of 1,920 eggs from 46-wk-old Hyline W36 laying hens were randomly distributed into 4 groups soon after classification: (i) 480 eggs were stored in pulp carton tray packages; (ii) 480 eggs were stored in plastic packages with lids; (iii) 480 eggs were stored in carton packages after the application of mineral oil; and (iv) 480 eggs were stored in plastic packages with lids after the application of mineral oil. The internal quality was measured by Haugh units, by the counts of mesophilic and psychrotrophic microorganisms, by the most probable number of total and thermal-tolerant coliforms, by the counts of molds and yeasts, by the analysis of Salmonella spp. and Staphylococcus spp., and by the levels of biogenic
2
Figueiredo et al.
MATERIALS AND METHODS Samples and Experimental Conditions A total of 1,920 eggs, weighing from 55 to 59 g, were produced by 46-wk-old hens of the same strain (Hyline W36). They were housed in the same laying facility and were fed a commercial laying hen diet and water ad libitum. The eggs were collected at the same time. Shortly thereafter, the unwashed eggs were selected and classified and then were randomly distributed into 2 treatments: a group in which the eggshell was coated with mineral oil (Chemistry Anastacio, São Paulo, Brazil) using a spray machine (Yamasa, Rinópolis, São Paulo, Brazil) and another group without eggshell coat-
ing. After this procedure, the eggs were again divided into 2 treatments: in one group the eggs were packed in plastic cases with a lid, with 12 eggs per package (Crystal Form, Caxias do Sul, RS, Brazil), and in the other group the eggs were packed in conventional pulp carton trays with a capacity of 30 units. The mean refrigeration temperature during the experiment was 5.0 ± 1°C, and the average relative air humidity at the storage location was 65%. For the physical-chemical and microbiological analyses, the experiment was designed in a 2 × 2 × 12 factorial arrangement, consisting of 2 types of packaging (plastic cases with a lid or pulp carton trays), 2 types of eggshell covering (with or without the application of mineral oil), and 12 periods of storage under refrigeration (1, 6, 13, 20, 27, 41, 55, 69, 83, 97, 111, and 125 d), totaling 48 treatments with 4 repetitions of 5 eggs each for physical-chemical analyses (n = 960) and 4 repetitions with a pool of 5 eggs each for microbiological analyses (n = 192). For biogenic amine determination, the treatments were designed in a 2 × 2 × 8 factorial arrangement consisting of 8 periods of storage under refrigeration (1, 6, 13, 20, 27, 55, 83, and 125 d), totaling 32 treatments with 3 repetitions of a pool of 5 eggs each (n = 96) using the same eggs used in the physicalchemical analyses.
Methods of Analysis Determination of HU. After the eggs were weighed, the HU values were determined using an Egg Quality Micrometer, model S-8400 (AMES, Melrose, MA). The HU values were calculated from the egg weight (W) and albumen height (H) using the following formula: HU = 100 log (H + 7.57 – 1.7 W37), as described by Brant et al. (1951) and Silversides and Budgell (2004). The USDA Egg Quality Standards (USDA, 2000) define the conditions that should be present from production to consumption. Under these standards, excellent quality (AA) eggs must have HU values higher than 72, eggs of high quality (A) must have HU values of 60 to 72, and eggs of inferior quality (B) must have values below 60. Determination of Microbiological Quality. Before procedures of microbiological analyses, the eggs were broken aseptically, placed in a sterile sample bag, and then mixed by a sample homogenizer for 1 min. Salmonella spp. analyses were carried out by a VIDAS device (bioMerieux, Hazelwood, MO) based on an enzyme-linked fluorescent immunoassay (AOAC International, 2005). Briefly, 25 mL of mixed egg contents was preenriched in 225 mL of 1% sterile peptone saline solution and incubated for 24 h at 35°C. Afterward, 0.1 mL was transferred to Salmonella Xpress broth (bioMerieux) and incubated for 24 h at 41°C. Next, 0.5 mL of the solution was transferred to a test well of reagent strip (bioMerieux) and heated to 100°C for 15 min. Readings were performed after 4 h.
Downloaded from http://ps.oxfordjournals.org/ at University of Idaho on November 3, 2014
and by applying mineral oil to the shell. Both methods may slow down the loss of water and CO2, causing obstruction of the eggshell pores, which hinders the penetration of microorganisms into the eggs (FAO, 2003; Xavier et al., 2008). The Haugh unit (HU) value has been adopted to estimate the internal quality of eggs. It measures the albumen height corrected to the egg weight (Brant et al., 1951). Its estimation is used worldwide because of its ease of application and high correlation to the appearance of an egg after breaking it on a plain surface (Williams, 1992). According to Silversides et al. (1993), the HU value has also been used by the egg industry, and its interpretation provides a reliable indication of egg shelf life as well of the storage conditions to which they were submitted. Various bacterial genera are associated with egg deterioration, such as Pseudomonas, Actinobacter, Proteus, Aeromonas, Alcaligenes, Escherichia, Micrococcus, Serratia, Enterobacter, Flavobacterium, Salmonella, and Staphylococcus. Moreover, the storage conditions and temperature are fundamental to the preservation of the microbiological quality of eggs (Keller et al., 1995; Cardoso et al., 2001; Radkowski, 2001; Jones et al., 2004; Adesiyun et al., 2006; Rêgo et al., 2014). The content of biogenic amines has been used as an indicator of quality for many foods, such as fish, meat, eggs, cheese, fruit, vegetables, beer, and wine (Shalaby, 1996; Lima and Gloria, 1999; Kalac and Krausová, 2005; Figueiredo et al., 2013). The quantity and the type of amines in a food depend on the nature of the food and its microbiota. Some microorganisms, namely enterobacteria, lactobacilli, pediococci, and enterococci, are particularly active in the production of biogenic amines. However, little information is available on the amine content in eggs, and some of the published data are based on small numbers of egg samples (Okamoto et al., 1997; Lima et al., 2006; Nishimura et al., 2006; Cipolla et al., 2007). Thus, the objective of this study was to verify the influence of the packaging, mineral oil coating, and storage time on the microbiological characteristics and levels of biogenic amines in eggs.
BIOGENIC AMINE LEVELS AND INTERNAL QUALITY OF EGGS
filtered through a PTFE membrane with 0.45-µm pores and a 13 to 15 mm diameter (Millipore Corp., Milford, MA) before injection and chromatographic analysis. The HPLC determinations were performed with an ÄKTAmicro (GE HealthCare, Buckinghamshire, UK) using 2 type P-900 pumps and a type INV-907 manual injector with a 100-μL loop. The detector operated in the UV-visible (UV-900), and Unicorn 5.11 Software (GE HealthCare, Buckinghamshire, UK) was also used. Detection was performed by measuring the absorbance at 254 nm. The column was a reversed-phase Kromasil C18 column (5 µm, 100 Angstron, 25 cm × 4.6 mm; AkzonNobel, Amsterdam, the Netherlands). The eluents were water (A) and acetonitrile (B). The elution program consisted of a gradient system with a 1.0 mL/ min flow rate. The gradient applied was as follows: 60 to 72% (vol/vol) B in A within 4.1 min, maintained at 72% (vol/vol) B in A for 0.58 min, from 72 to 75% (vol/ vol) B in A within 12 min, maintained at 75% (vol/vol) B in A for 2.28 min, from 75 to 95% (vol/vol) B in A within 6.1 min, and maintained at 95% (vol/vol) B in A for 6.6 min. The used amine standards were spermine tetrahydrochloride (SPM), spermidine trihydrochloride (SPD), putrescine dihydrochloride (PUT), cadaverine dihydrochloride (CAD), histamine dihydrochloride (HIS), β-phenylethylamine hydrochloride (PHE), and tyramine (TYR). Amine standards were purchased from Sigma-Aldrich Co. (St. Louis, MO). The limit of quantification for the yolk matrix was 0.7 mg/kg for PUT, HIS, and SPD; 0.8 mg/kg for TYR; and 1.0 mg/kg for PHE, CAD, and SPM. The limit of quantification for the albumen was 0.7 mg/kg for PUT and HIS, 0.8 mg/kg for CAD and TYR, 0.9 mg/kg for ESD, 1.0 mg/kg for SPM, and 1.1 mg/kg for PHE. Statistical Analysis. The differences between treatments in terms of HU values were compared by the Student-Newman-Keuls test at a 5% significance level, and the evaluation periods were estimated by regression models. The results of the microbiological analyses were submitted to nonparametric analyses using the Kruskal-Wallis test at a 5% significance level. The results of the biogenic amine analyses were submitted to nonparametric analyses using the Kruskal-Wallis test at a 5% significance level. Correlation analyses between variables were submitted to Pearson and Spearman correlations.
RESULTS AND DISCUSSION Physical-Chemical and Microbiological Characteristics and Levels of Biogenic Amines in Fresh Eggs The results (Table 1) represent the quality of the eggs 1 d after they were laid. In regard to the internal quality, the HU average was 98.55. The values were much higher than 72, which are considered by the
Downloaded from http://ps.oxfordjournals.org/ at University of Idaho on November 3, 2014
The total aerobic mesophilic microorganism counts were performed using the Petrifilms Aerobic Count Plate method (3M, St. Paul, MN; Curiale et al., 1990). The incubation was carried out at 36 ± 1°C for 48 h. The psychrotrophic microorganism count was performed by the same technique; however, the plates were incubated at 7 ± 1°C for 7 d. Mold and yeast counts and total and thermal-tolerant coliform counts were performed by the most probable number (MPN) method using a Simplate (Biocontrol Systems Inc., Bellevue, WA; Feldsine et al., 2003, 2005). The samples were processed following the instructions from the manufacturer. The plates were incubated at 35°C for both analyses and read at 24 and 48 h. Wells showing any color change from the initial blue were considered positive. Wells were counted positive for Escherichia coli on the basis of their color change and fluorescence under UV light. The total coliform, E. coli, mold, and yeast counts were determined on the basis of the number of positive wells correlated with the SimPlate conversion table, which generated a most probable number (MPN or cfu) per gram of sample. For Staphylococcus aureus analysis, 1 mL of the diluted samples was spread over the surface of Baird-Parker agar (Oxoid Ltd., Basingstoke, UK), and the plates were incubated at 35°C for 48 h. After incubation, the isolated bacteria were characterized by a coagulase test, by Gram staining, and by the production of catalase and thermostable nuclease test (Lancette and Tatini, 2001). Determination of Biogenic Amines. The biogenic amines were separated by ion-pairing HPLC in a reverse-phase column and quantified by UV detection after column derivatization with dansyl chloride. The analyses were carried out in duplicate. Amines were extracted from 3 g of sample using 20 mL of 50 g/L of trichloroacetic acid in 3 successive extractions (7, 7, and 6 mL). After agitation for 10 min in a vortex mixer, the slurry was centrifuged at 12,100 × g for 21 min at 4°C, and the supernatants were collected, combined, and filtered through a Whatman qualitative filter paper, grade 1 (Sigma-Aldrich Co., St. Louis, MO). The derivation protocol and chromatographic conditions were carried out according to Malle et al. (1996) and Duflos et al. (1999). A 200-μL volume of supernatant was added to 400 μL of saturated Na2CO3 and 800 μL of dansyl chloride solution (75 mg/L acetone) in a tube that was hermetically sealed. The tube was shaken and left in a water bath at 60°C for 5 min. Next, 200 mL of proline solution was added (0.1 mg/L of distilled water), and the mixture was shaken and allowed to stand at room temperature for 30 min in the dark. A 1-mL volume of toluene was then added, and the mixture was shaken and centrifuged at 4,350 × g for 10 min at 4°C to separate the phases. The organic phase (supernatant) was recovered and evaporated under a stream of nitrogen. The pellet was suspended in 600 μL of acetonitrile (Merck, Darmstadt, Germany) and
3
4
Figueiredo et al. Table 1. Physical-chemical and microbiological characteristics and biogenic amine contents of fresh eggs Value Part of the egg/parameters units1
Maximum
Mean
97.90 1.8 × 102 0.05) of the package on the HU values was observed throughout the storage period (Table 2). The regression equation for the HU values of the eggs as a function of the days of storage under refrigeration is shown in Figure 1. The albumen height decreased with increasing storage time, and consequently the HU values decreased. Despite the long period of storage, according to the Quality Control criteria (USDA, 2000), all eggs, irrespective of the use or not of packages and the application or not of mineral oil on the shell, are classified as
excellent quality (AA) when they display HU values higher than 72.
Microbiological Analyses The results of the microbiological analyses of the internal qualities of the eggs were similar (P > 0.05) throughout the storage time regardless of the application of mineral oil to the shell and of the type of packaging. The presence of Salmonella spp., Staphylococcus aureus, and thermal-tolerant coliforms was not detected in any of the samples. This may be determined by the adequate sanitary conditions of the birds and the hygienic conditions of their housing. A similar result was observed by Radkowski (2001) and Figueiredo et al. (2013). No mold or yeast were present in the internal contents of eggs from any sample. Of the 192 samples analyzed in the experiment, 13 (6.8%) were positive for Staphylococcus spp. The count of this bacterium was low in all samples, reaching 2.2 × 101 cfu/g at the maximum. However, Staphylococcus Table 2. Mean and SD values of Haugh units in eggs with and without mineral oil application on the shell and packed or not in plastic containers under refrigeration for 125 d Application of mineral oil Packaging
Noncoated
Oil-coated
Mean
Not packed Packed Mean
84.28 ± 7.3 83.76 ± 7.2 84.02b
87.57 ± 7.3 88.22 ± 6.5 87.89a
85.92x 85.99x
a,bMeans followed by different letters in the same row are different (P < 0.05; by Student-Newman-Keuls test). xMeans followed by different letters in the same column are different (P < 0.05; by Student-Newman-Keuls test). n = 960; CV = 2.21.
Downloaded from http://ps.oxfordjournals.org/ at University of Idaho on November 3, 2014
Haugh Microbiological analysis2 (cfu/g) Total coliforms Mesophilic Psychrotrophic Biogenic amine3 (mg/kg) Albumen Putrescine Cadaverine Phenylethylamine Yolk Putrescine Cadaverine Spermidine Spermine
Minimum
5
BIOGENIC AMINE LEVELS AND INTERNAL QUALITY OF EGGS
Figure 1. Regression curve of the Haugh unit values of the eggs according to the days of storage under refrigeration.
Biogenic Amines Putrescine and cadaverine were detected in the egg yolks, and putrescine, cadaverine, and phenylethylamine were detected in the albumen at levels higher than the quantification threshold of the test (Tables 5 and 6). Spermine and spermidine showed values in egg yolks mostly near the detection limits of the ana-
lytical method used. Some results of these amines were quantified in low levels, reaching at maximum 8.6 mg/ kg of SPM and 4.3 mg/kg of SPD. The application of mineral oil, the packaging, and their association did not influence (P > 0.05) the amine concentrations throughout the storage period. The requirements for polyamines are higher during phases of intense growth, which may explain the presence of these substances in eggs, which are also rich in nutrients. The presence of putrescine, cadaverine, and phenylethylamine may indicate initial deterioration. However, these amines were detected in fresh eggs from the beginning of the experiment, and their concentrations were not altered (P > 0.05) during storage. According to Glória (2005), the presence of cadaverine in spoiled samples could indicate contamination by enterobacteria because they can participate in lysine decarboxylation. Cipolla et al. (2007) recorded 3.84 mg/kg of putrescine in the egg yolk and 88 μg/kg in the albumen of commercial eggs. Nishimura et al. (2006) found cadaverine in egg yolks, but in higher values (26.6 mg/kg) than those found in the present study. A lower concentration
Table 3. Total coliforms (cfu/g) in the internal contents of the eggs with and without mineral oil application and packed or not in plastic containers under refrigeration for 125 d Treatment1 Storage time (d)
NC + NP
1 6 13 20 27 41 55 69 83 97 111 125 Mean
1.8 5.2 1.8
Key words: egg quality, mineral oil treatment, packaging, biogenic amine, microbiological quality 2014 Poultry Science 93:1–8 http://dx.doi.org/10.3382/ps.2014-04268
INTRODUCTION
them from becoming a source of toxo-infection and to ensure that they reach consumers with a high standard of quality. The internal quality of eggs can be evaluated based on their physical, chemical, biological, and functional characteristics. Factors such as the temperature, air relative humidity, storage conditions, and time influence the quality of eggs. Among the chemical reactions that take place inside eggs during storage is the transformation of dense albumen into thin albumen. This change may involve carbonic acid (H2CO3), one of the components of the buffering system in albumen, which is dissociated into water and carbon dioxide gas (CO2), which are lost via the eggshell pores (Brake et al., 1997; Figueiredo et al., 2011). The preservation of the internal quality of eggs can be achieved through the use of packages for storage
Eggs are a great and inexpensive source of high-quality protein. Eggs provide a unique and well-balanced source of nutrients, especially iron, various vitamins, and phosphorus, for people of all ages. Their availability, modest cost, ease of preparation, taste, and low caloric value give eggs a primary advantage for human nutritional needs. Eggs are used in the food processing industry for various applications, such as emulsifying, foaming, gelling, and whipping. Because of the high nutrient content of eggs, care must be taken to prevent ©2014 Poultry Science Association Inc. Received June 19, 2014. Accepted September 5, 2014. 1 Corresponding author: [email protected]
1
Downloaded from http://ps.oxfordjournals.org/ at University of Idaho on November 3, 2014
ABSTRACT This study was carried out with the aim of evaluating the effects of mineral oil application on eggshells and the use of plastic packages with lids on the physical-chemical and microbiological quality and biogenic amine contents of eggs stored under refrigeration for up to 125 d. A total of 1,920 eggs from 46-wk-old Hyline W36 laying hens were randomly distributed into 4 groups soon after classification: (i) 480 eggs were stored in pulp carton tray packages; (ii) 480 eggs were stored in plastic packages with lids; (iii) 480 eggs were stored in carton packages after the application of mineral oil; and (iv) 480 eggs were stored in plastic packages with lids after the application of mineral oil. The internal quality was measured by Haugh units, by the counts of mesophilic and psychrotrophic microorganisms, by the most probable number of total and thermal-tolerant coliforms, by the counts of molds and yeasts, by the analysis of Salmonella spp. and Staphylococcus spp., and by the levels of biogenic
2
Figueiredo et al.
MATERIALS AND METHODS Samples and Experimental Conditions A total of 1,920 eggs, weighing from 55 to 59 g, were produced by 46-wk-old hens of the same strain (Hyline W36). They were housed in the same laying facility and were fed a commercial laying hen diet and water ad libitum. The eggs were collected at the same time. Shortly thereafter, the unwashed eggs were selected and classified and then were randomly distributed into 2 treatments: a group in which the eggshell was coated with mineral oil (Chemistry Anastacio, São Paulo, Brazil) using a spray machine (Yamasa, Rinópolis, São Paulo, Brazil) and another group without eggshell coat-
ing. After this procedure, the eggs were again divided into 2 treatments: in one group the eggs were packed in plastic cases with a lid, with 12 eggs per package (Crystal Form, Caxias do Sul, RS, Brazil), and in the other group the eggs were packed in conventional pulp carton trays with a capacity of 30 units. The mean refrigeration temperature during the experiment was 5.0 ± 1°C, and the average relative air humidity at the storage location was 65%. For the physical-chemical and microbiological analyses, the experiment was designed in a 2 × 2 × 12 factorial arrangement, consisting of 2 types of packaging (plastic cases with a lid or pulp carton trays), 2 types of eggshell covering (with or without the application of mineral oil), and 12 periods of storage under refrigeration (1, 6, 13, 20, 27, 41, 55, 69, 83, 97, 111, and 125 d), totaling 48 treatments with 4 repetitions of 5 eggs each for physical-chemical analyses (n = 960) and 4 repetitions with a pool of 5 eggs each for microbiological analyses (n = 192). For biogenic amine determination, the treatments were designed in a 2 × 2 × 8 factorial arrangement consisting of 8 periods of storage under refrigeration (1, 6, 13, 20, 27, 55, 83, and 125 d), totaling 32 treatments with 3 repetitions of a pool of 5 eggs each (n = 96) using the same eggs used in the physicalchemical analyses.
Methods of Analysis Determination of HU. After the eggs were weighed, the HU values were determined using an Egg Quality Micrometer, model S-8400 (AMES, Melrose, MA). The HU values were calculated from the egg weight (W) and albumen height (H) using the following formula: HU = 100 log (H + 7.57 – 1.7 W37), as described by Brant et al. (1951) and Silversides and Budgell (2004). The USDA Egg Quality Standards (USDA, 2000) define the conditions that should be present from production to consumption. Under these standards, excellent quality (AA) eggs must have HU values higher than 72, eggs of high quality (A) must have HU values of 60 to 72, and eggs of inferior quality (B) must have values below 60. Determination of Microbiological Quality. Before procedures of microbiological analyses, the eggs were broken aseptically, placed in a sterile sample bag, and then mixed by a sample homogenizer for 1 min. Salmonella spp. analyses were carried out by a VIDAS device (bioMerieux, Hazelwood, MO) based on an enzyme-linked fluorescent immunoassay (AOAC International, 2005). Briefly, 25 mL of mixed egg contents was preenriched in 225 mL of 1% sterile peptone saline solution and incubated for 24 h at 35°C. Afterward, 0.1 mL was transferred to Salmonella Xpress broth (bioMerieux) and incubated for 24 h at 41°C. Next, 0.5 mL of the solution was transferred to a test well of reagent strip (bioMerieux) and heated to 100°C for 15 min. Readings were performed after 4 h.
Downloaded from http://ps.oxfordjournals.org/ at University of Idaho on November 3, 2014
and by applying mineral oil to the shell. Both methods may slow down the loss of water and CO2, causing obstruction of the eggshell pores, which hinders the penetration of microorganisms into the eggs (FAO, 2003; Xavier et al., 2008). The Haugh unit (HU) value has been adopted to estimate the internal quality of eggs. It measures the albumen height corrected to the egg weight (Brant et al., 1951). Its estimation is used worldwide because of its ease of application and high correlation to the appearance of an egg after breaking it on a plain surface (Williams, 1992). According to Silversides et al. (1993), the HU value has also been used by the egg industry, and its interpretation provides a reliable indication of egg shelf life as well of the storage conditions to which they were submitted. Various bacterial genera are associated with egg deterioration, such as Pseudomonas, Actinobacter, Proteus, Aeromonas, Alcaligenes, Escherichia, Micrococcus, Serratia, Enterobacter, Flavobacterium, Salmonella, and Staphylococcus. Moreover, the storage conditions and temperature are fundamental to the preservation of the microbiological quality of eggs (Keller et al., 1995; Cardoso et al., 2001; Radkowski, 2001; Jones et al., 2004; Adesiyun et al., 2006; Rêgo et al., 2014). The content of biogenic amines has been used as an indicator of quality for many foods, such as fish, meat, eggs, cheese, fruit, vegetables, beer, and wine (Shalaby, 1996; Lima and Gloria, 1999; Kalac and Krausová, 2005; Figueiredo et al., 2013). The quantity and the type of amines in a food depend on the nature of the food and its microbiota. Some microorganisms, namely enterobacteria, lactobacilli, pediococci, and enterococci, are particularly active in the production of biogenic amines. However, little information is available on the amine content in eggs, and some of the published data are based on small numbers of egg samples (Okamoto et al., 1997; Lima et al., 2006; Nishimura et al., 2006; Cipolla et al., 2007). Thus, the objective of this study was to verify the influence of the packaging, mineral oil coating, and storage time on the microbiological characteristics and levels of biogenic amines in eggs.
BIOGENIC AMINE LEVELS AND INTERNAL QUALITY OF EGGS
filtered through a PTFE membrane with 0.45-µm pores and a 13 to 15 mm diameter (Millipore Corp., Milford, MA) before injection and chromatographic analysis. The HPLC determinations were performed with an ÄKTAmicro (GE HealthCare, Buckinghamshire, UK) using 2 type P-900 pumps and a type INV-907 manual injector with a 100-μL loop. The detector operated in the UV-visible (UV-900), and Unicorn 5.11 Software (GE HealthCare, Buckinghamshire, UK) was also used. Detection was performed by measuring the absorbance at 254 nm. The column was a reversed-phase Kromasil C18 column (5 µm, 100 Angstron, 25 cm × 4.6 mm; AkzonNobel, Amsterdam, the Netherlands). The eluents were water (A) and acetonitrile (B). The elution program consisted of a gradient system with a 1.0 mL/ min flow rate. The gradient applied was as follows: 60 to 72% (vol/vol) B in A within 4.1 min, maintained at 72% (vol/vol) B in A for 0.58 min, from 72 to 75% (vol/ vol) B in A within 12 min, maintained at 75% (vol/vol) B in A for 2.28 min, from 75 to 95% (vol/vol) B in A within 6.1 min, and maintained at 95% (vol/vol) B in A for 6.6 min. The used amine standards were spermine tetrahydrochloride (SPM), spermidine trihydrochloride (SPD), putrescine dihydrochloride (PUT), cadaverine dihydrochloride (CAD), histamine dihydrochloride (HIS), β-phenylethylamine hydrochloride (PHE), and tyramine (TYR). Amine standards were purchased from Sigma-Aldrich Co. (St. Louis, MO). The limit of quantification for the yolk matrix was 0.7 mg/kg for PUT, HIS, and SPD; 0.8 mg/kg for TYR; and 1.0 mg/kg for PHE, CAD, and SPM. The limit of quantification for the albumen was 0.7 mg/kg for PUT and HIS, 0.8 mg/kg for CAD and TYR, 0.9 mg/kg for ESD, 1.0 mg/kg for SPM, and 1.1 mg/kg for PHE. Statistical Analysis. The differences between treatments in terms of HU values were compared by the Student-Newman-Keuls test at a 5% significance level, and the evaluation periods were estimated by regression models. The results of the microbiological analyses were submitted to nonparametric analyses using the Kruskal-Wallis test at a 5% significance level. The results of the biogenic amine analyses were submitted to nonparametric analyses using the Kruskal-Wallis test at a 5% significance level. Correlation analyses between variables were submitted to Pearson and Spearman correlations.
RESULTS AND DISCUSSION Physical-Chemical and Microbiological Characteristics and Levels of Biogenic Amines in Fresh Eggs The results (Table 1) represent the quality of the eggs 1 d after they were laid. In regard to the internal quality, the HU average was 98.55. The values were much higher than 72, which are considered by the
Downloaded from http://ps.oxfordjournals.org/ at University of Idaho on November 3, 2014
The total aerobic mesophilic microorganism counts were performed using the Petrifilms Aerobic Count Plate method (3M, St. Paul, MN; Curiale et al., 1990). The incubation was carried out at 36 ± 1°C for 48 h. The psychrotrophic microorganism count was performed by the same technique; however, the plates were incubated at 7 ± 1°C for 7 d. Mold and yeast counts and total and thermal-tolerant coliform counts were performed by the most probable number (MPN) method using a Simplate (Biocontrol Systems Inc., Bellevue, WA; Feldsine et al., 2003, 2005). The samples were processed following the instructions from the manufacturer. The plates were incubated at 35°C for both analyses and read at 24 and 48 h. Wells showing any color change from the initial blue were considered positive. Wells were counted positive for Escherichia coli on the basis of their color change and fluorescence under UV light. The total coliform, E. coli, mold, and yeast counts were determined on the basis of the number of positive wells correlated with the SimPlate conversion table, which generated a most probable number (MPN or cfu) per gram of sample. For Staphylococcus aureus analysis, 1 mL of the diluted samples was spread over the surface of Baird-Parker agar (Oxoid Ltd., Basingstoke, UK), and the plates were incubated at 35°C for 48 h. After incubation, the isolated bacteria were characterized by a coagulase test, by Gram staining, and by the production of catalase and thermostable nuclease test (Lancette and Tatini, 2001). Determination of Biogenic Amines. The biogenic amines were separated by ion-pairing HPLC in a reverse-phase column and quantified by UV detection after column derivatization with dansyl chloride. The analyses were carried out in duplicate. Amines were extracted from 3 g of sample using 20 mL of 50 g/L of trichloroacetic acid in 3 successive extractions (7, 7, and 6 mL). After agitation for 10 min in a vortex mixer, the slurry was centrifuged at 12,100 × g for 21 min at 4°C, and the supernatants were collected, combined, and filtered through a Whatman qualitative filter paper, grade 1 (Sigma-Aldrich Co., St. Louis, MO). The derivation protocol and chromatographic conditions were carried out according to Malle et al. (1996) and Duflos et al. (1999). A 200-μL volume of supernatant was added to 400 μL of saturated Na2CO3 and 800 μL of dansyl chloride solution (75 mg/L acetone) in a tube that was hermetically sealed. The tube was shaken and left in a water bath at 60°C for 5 min. Next, 200 mL of proline solution was added (0.1 mg/L of distilled water), and the mixture was shaken and allowed to stand at room temperature for 30 min in the dark. A 1-mL volume of toluene was then added, and the mixture was shaken and centrifuged at 4,350 × g for 10 min at 4°C to separate the phases. The organic phase (supernatant) was recovered and evaporated under a stream of nitrogen. The pellet was suspended in 600 μL of acetonitrile (Merck, Darmstadt, Germany) and
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Figueiredo et al. Table 1. Physical-chemical and microbiological characteristics and biogenic amine contents of fresh eggs Value Part of the egg/parameters units1
Maximum
Mean
97.90 1.8 × 102 0.05) of the package on the HU values was observed throughout the storage period (Table 2). The regression equation for the HU values of the eggs as a function of the days of storage under refrigeration is shown in Figure 1. The albumen height decreased with increasing storage time, and consequently the HU values decreased. Despite the long period of storage, according to the Quality Control criteria (USDA, 2000), all eggs, irrespective of the use or not of packages and the application or not of mineral oil on the shell, are classified as
excellent quality (AA) when they display HU values higher than 72.
Microbiological Analyses The results of the microbiological analyses of the internal qualities of the eggs were similar (P > 0.05) throughout the storage time regardless of the application of mineral oil to the shell and of the type of packaging. The presence of Salmonella spp., Staphylococcus aureus, and thermal-tolerant coliforms was not detected in any of the samples. This may be determined by the adequate sanitary conditions of the birds and the hygienic conditions of their housing. A similar result was observed by Radkowski (2001) and Figueiredo et al. (2013). No mold or yeast were present in the internal contents of eggs from any sample. Of the 192 samples analyzed in the experiment, 13 (6.8%) were positive for Staphylococcus spp. The count of this bacterium was low in all samples, reaching 2.2 × 101 cfu/g at the maximum. However, Staphylococcus Table 2. Mean and SD values of Haugh units in eggs with and without mineral oil application on the shell and packed or not in plastic containers under refrigeration for 125 d Application of mineral oil Packaging
Noncoated
Oil-coated
Mean
Not packed Packed Mean
84.28 ± 7.3 83.76 ± 7.2 84.02b
87.57 ± 7.3 88.22 ± 6.5 87.89a
85.92x 85.99x
a,bMeans followed by different letters in the same row are different (P < 0.05; by Student-Newman-Keuls test). xMeans followed by different letters in the same column are different (P < 0.05; by Student-Newman-Keuls test). n = 960; CV = 2.21.
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Haugh Microbiological analysis2 (cfu/g) Total coliforms Mesophilic Psychrotrophic Biogenic amine3 (mg/kg) Albumen Putrescine Cadaverine Phenylethylamine Yolk Putrescine Cadaverine Spermidine Spermine
Minimum
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BIOGENIC AMINE LEVELS AND INTERNAL QUALITY OF EGGS
Figure 1. Regression curve of the Haugh unit values of the eggs according to the days of storage under refrigeration.
Biogenic Amines Putrescine and cadaverine were detected in the egg yolks, and putrescine, cadaverine, and phenylethylamine were detected in the albumen at levels higher than the quantification threshold of the test (Tables 5 and 6). Spermine and spermidine showed values in egg yolks mostly near the detection limits of the ana-
lytical method used. Some results of these amines were quantified in low levels, reaching at maximum 8.6 mg/ kg of SPM and 4.3 mg/kg of SPD. The application of mineral oil, the packaging, and their association did not influence (P > 0.05) the amine concentrations throughout the storage period. The requirements for polyamines are higher during phases of intense growth, which may explain the presence of these substances in eggs, which are also rich in nutrients. The presence of putrescine, cadaverine, and phenylethylamine may indicate initial deterioration. However, these amines were detected in fresh eggs from the beginning of the experiment, and their concentrations were not altered (P > 0.05) during storage. According to Glória (2005), the presence of cadaverine in spoiled samples could indicate contamination by enterobacteria because they can participate in lysine decarboxylation. Cipolla et al. (2007) recorded 3.84 mg/kg of putrescine in the egg yolk and 88 μg/kg in the albumen of commercial eggs. Nishimura et al. (2006) found cadaverine in egg yolks, but in higher values (26.6 mg/kg) than those found in the present study. A lower concentration
Table 3. Total coliforms (cfu/g) in the internal contents of the eggs with and without mineral oil application and packed or not in plastic containers under refrigeration for 125 d Treatment1 Storage time (d)
NC + NP
1 6 13 20 27 41 55 69 83 97 111 125 Mean
1.8 5.2 1.8
