Assessment of the microbiological quality and safety of marinated chicken products from Greek retail outlets

Journal Pre-proof Assessment of the microbiological quality and safety of marinated chicken products from Greek retail outlets

Anastasia E. Lytou, Chrissanthi T. Renieri, Agapi I. Doulgeraki, George-John E. Nychas, Efstathios Z. Panagou PII:

S0168-1605(19)30437-4

DOI:

https://doi.org/10.1016/j.ijfoodmicro.2019.108506

Reference:

FOOD 108506

To appear in:

International Journal of Food Microbiology

Received date:

9 August 2019

Revised date:

30 December 2019

Accepted date:

31 December 2019

Please cite this article as: A.E. Lytou, C.T. Renieri, A.I. Doulgeraki, et al., Assessment of the microbiological quality and safety of marinated chicken products from Greek retail outlets, International Journal of Food Microbiology (2018), https://doi.org/10.1016/ j.ijfoodmicro.2019.108506

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© 2018 Published by Elsevier.

Journal Pre-proof Assessment of the microbiological quality and safety of marinated chicken products from Greek retail outlets Anastasia E. Lytoua, Chrissanthi T. Renieria, Agapi I. Doulgerakib, George-John E. Nychasa*, Efstathios Z. Panagoua

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Laboratory of Microbiology and Biotechnology of Foods, Department of Food

Science and Human Nutrition, School of Food and Nutritional Sciences, Agricultural

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Hellenic Agricultural Organization – DEMETER, Institute of Technology of

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Agricultural Products, Sof. Venizelou 1, Lycovrissi 14123, Athens, Greece

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*Corresponding Author: [email protected]

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University of Athens, Iera Odos 75, Athens 11855, Greece

Journal Pre-proof Abstract The prevalence of three pathogens in marinated chicken products and the evaluation of their quality by microbiological and sensory analysis were assessed. Eighty (80) samples obtained from several meat retail markets in Greece were analyzed for the presence of Campylobacter spp., Salmonella and Listeria monocytogenes. Concerning Campylobacter, rep-PCR and species specific PCR were applied for the differentiation and identification of isolates, respectively. The samples

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were subsequently stored aerobically at 4 oC for 5 days. Microbiological analysis,

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sensory assessment and HPLC analysis were carried out for the evaluation of spoilage

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microorganisms, sensory quality and the presence of preservatives (potassium sorbate

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and sodium benzoate). Τhe prevalence of Campylobacter spp., Salmonella, and Listeria monocytogenes was 50%, 11% and 44%, respectively. In the case of

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Campylobacter, from a total of 40 isolates, 27 were identified as Campylobacter coli,

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4 as Campylobacter jejuni, whereas the remaining 9 belonged to unidentified Campylobacter species. Pseudomonas spp. was the dominant spoilage microbial

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genus in 43% of the samples, while in 31% of them a co-dominance of Pseudomonas spp. and Brochothrix thermosphacta was observed. Total aerobic counts increased to 7.0 log CFU/g at the 1st, 2nd or 3rd day of storage in 71% of the samples, while sensory analysis showed that 80% of the samples were characterized as spoiled after 3, 4 or 5 days. The presence of preservatives was confirmed in 31% of the samples and slightly affected the microbiological profile. In conclusion, the obtained data demonstrated the occurrence of foodborne pathogens and allowed the acquisition of an overall view about the microbiological quality of marinated chicken products. Keywords: poultry meat, marination, Campylobacter, Salmonella, Listeria, spoilage microorganisms

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Journal Pre-proof 1. Introduction Marinated products have been a significant segment of the meat market as the consumer interest for ready-to-cook, convenience products is steadily increasing (Jackson et al., 2018). The use of marinades to flavor and tenderize meat is not a new concept; however it is widely applied nowadays by the meat industry. In recent years there is a wide variety of choices for meat marination, by using either homemade or

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commercial marinades. Commercial marinades usually contain a combination of

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flavorful and functional ingredients such as phosphate, salt, modified starch, gums, organic acids, colorants, stabilizers, powders of herbs and spices together with

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vegetable oils that are basic constituents of many marinades (Yusop et al., 2010).

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Marinades target to (a) the enhancement of sensory quality of meat products, (b) the

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introduction of new products in the market with different sensory attributes from the raw ones and, occasionally, (c) the extension of products’ shelf life (Pathania et al.,

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2010; Van Haute et al., 2016). Marination has always been a matter of interest for

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both the research and the retail market, regarding the microbiological quality and safety of meat, either by delaying spoilage and reducing pathogens due to the antimicrobial constituents of marinades (Birk and Knøchel, 2009; Thanissery and Smith, 2014) or by increasing the microbial load of meat due to contamination during marination (Jung et al., 2018). Poultry meat is associated with foodborne pathogens such as Salmonella and Campylobacter. As far as Campylobacter is concerned, poultry meat is well known as the most common foodborne source of human campylobacteriosis (up to 80% of foodborne infections). From 2011 to 2018 over 200,000 confirmed cases of campylobacteriosis mainly from poultry flocks and meat were reported per year (EFSA, 2018). Contaminated poultry meat is the cause of 20%-30% of all 3

Journal Pre-proof campylobacteriosis cases, with 50%-80% of them attributed to the chicken reservoir as a whole (Bahrndorff et al., 2013; EFSA, 2011). Salmonellosis was the second most commonly reported gastrointestinal infection in humans in the EU (91 857 cases reported in 2018), while Salmonella was the most common cause of foodborne outbreaks. Moreover, it should be underlined that the declining trend for salmonellosis in humans has leveled off since 2014 (EFSA, 2019). The aim of this study was to evaluate the safety and quality of marinated chicken

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products from Greek retail outlets. Regarding safety, the presence of Salmonella,

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Campylobacter spp. and Listeria monocytogenes was investigated. More specifically,

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the identification of C. jejuni and C. coli species among the positive Campylobacter

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isolates, as well as their discrimination at strain level were attempted. Moreover, this work aimed to the assessment of the microbiological quality of marinated products

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both at the time of purchase and throughout refrigerated storage.

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2. Materials and Methods

2.1 Sample collection and storage Eighty (80) marinated chicken products were purchased from forty (40) different meat retail markets from several areas of Attica. The obtained samples originated from all parts of the broiler, including breast, thighs, drumsticks and wings in several cuts. The marinades employed in this study were of unknown origin (i.e., their ingredients were unknown), and further the time from slaughter to sale was unknown. The samples were transferred immediately to the laboratory (in less than 1 hour at refrigerated temperature), placed into polystyrene trays, covered with transparent plastic membrane and stored at 4 oC for 5 days under aerobic conditions, simulating 4

Journal Pre-proof consumer practice. Samples (8 batches) were purchased and analyzed microbiologically and organoleptically in duplicate in different time periods within 3 months (February to April).

2.2 Microbiological analysis On arrival, the collected samples were analyzed for the presence of Salmonella, Campylobacter spp. and Listeria monocytogenes, according to the procedures

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suggested by ISO standards (Table 1). Moreover, samples were analyzed at regular

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time intervals throughout storage (0, 1, 3, 4, and 5 days) for monitoring spoilage

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microorganisms. Fillets were weighed aseptically, added to sterile 1/4 strength

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Ringer's solution (LAB0023, LAB M) and homogenized in a stomacher (Lab Blender 400, SewardMedical, London, UK) for 60 s at room temperature. Serial decimal

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dilutions were prepared in 1/4 strength Ringer's solution and duplicate 1 or 0.1 mL

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aliquots of appropriate dilutions were plated on the appropriate media: (i) Plate Count Agar (402145, Biolife) for enumeration of total aerobic count (TAC), incubated at 30 o

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C for 72 h (ISO 4833-2:2013); (ii) MRS Agar (401728, Biolife) for enumeration of

lactic acid bacteria (LAB), overlaid with the same medium and incubated at 30 °C for 96 h (ISO 15214:1998); (iii) Pseudomonas agar (LAB 108, LAB M), supplemented with CFC (cetrimide, fucidin and cephaloridine) selective supplement (X108, LAB M), for the enumeration of Pseudomonas spp., incubated at 25 °C for 48 h (ISO 13720:2010); (iv) Violet Red Bile Dextrose Agar (402188, Biolife) for the enumeration of Enterobacteriaceae, overlaid with the same medium and incubated at 37 °C for 24 h (ISO 21528-2:2017); (v) STA Agar Base (402079, supplemented with selective supplement 4240052, Biolife) for Brochothrix thermosphacta, incubated at 25 oC for 72 h (ISO 13722:1996). The pH of the samples was recorded after the end

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Journal Pre-proof of microbiological analysis by a pH meter (Metrohm 691 pH meter, Ion Analysis, Switzerland) with immersion of the glass electrode in the homogenate of chicken fillet (1:10).

2.3 Sensory analysis Sensory evaluation was performed by 15 experienced assessors. Their ages ranged from 22 to 45 years old, while the percentage of gender was 67% females and

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33% males. . The samples that were raw and marinated with different marinades, were

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tested for their overall acceptance, taking into consideration the attributes of odor and

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appearance (i.e., visual perception of meat quality). They were served at room

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temperature in individual dishes, in groups of 6 samples in each testing booth. The sensory assessment was conducted using a 5 point hedonic scale (5- very bad; 4- bad;

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3- accepted; 2- good; 1- very good) and the samples were presented in random order.

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A score of 3 was the threshold for the sample acceptance . It needs to be mentioned that assessors were asked to evaluate the samples based on spoilage level and not on

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their individual preferences for marinades.

2.4 Determination of preservatives The samples were examined for the presence of potassium sorbate and sodium benzoate which are commonly used preservatives in food processing. The purpose of this analysis was to investigate the presence of these preservatives in marinades as antimicrobial agents and use it as a tool for the interpretation of microbiological and sensory results. For the extraction of the two preservatives, 5 g of homogenized meat sample was thoroughly mixed with 10 mL of water (HPLC-grade) and blended for 2 min. The sample homogenate was then centrifuged at 4500 g for 10 min at 4 °C and

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Journal Pre-proof 1.0 mL of the supernatant was diluted 1:10 with water. After dilution, all samples were filtered through 0.22 μm filter. The HPLC system consisted of a JASCO LC-Net II/ADC system controller, a JASCO AS-2055 Plus Autosampler with a Model PU980 Intelligent pump, a Model LG-980-02 ternary gradient unit pump and a MD-910 multiwavelength detector. The chromatography column was a Waters spherisorb C18 analytical column, 5 μm ODS2 (4.6 × 250 mm) (Resteck Co., Pinnacle II, Bellefonte, USA). The mobile phase consisted of 81% water, 17% acetonitrile and 2% 0.005 M

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ammonium acetate buffered at pH 4.2 with glacial acetic acid. The flow rate was 1.0

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mL/min and the injection volume 20 μL. The chromatogram was recorded for 15 min

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while the detection of benzoic and sorbic acids was carried out at the wavelengths of

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maximum absorption of the compounds, namely 228 and 260 nm, respectively. The peaks of benzoic and sorbic acids in the samples were identified by their UV spectrum

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(200–400 nm) and by comparing the retention time with that of the standard solution.

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The software used for spectra collection and processing was Jasco Chrompass Chromatography Data system v1.7.403.1. Seven (7) different concentrations of

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potassium sorbate and sodium benzoate standard solutions (100, 50, 10, 5.0, 1.0, 0.5, 0.05 mg/L) were used to prepare the calibration curves for the quantification of the two preservetives in meat samples. In order to verify the accuracy and precision of the analytical procedure, recovery studies were carried out by spiking selected samples of chicken meat matrix with standards. The spiked samples as well as the unspiked controls were analysed in duplicate. Recoveries were calculated from the differences in total amount of either benzoic or sorbic acid between the spiked and unspiked samples.

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Journal Pre-proof 2.5 Molecular identification of Campylobacter jejuni and Campylobacter coli

After confirmation of Campylobacter spp. with the ISO protocol, DNA extraction and identification of C. coli and C. jejuni were conducted according to the protocol suggested by DTU National Food Institute (2013). In brief, multiplex PCR amplification with mapA and ceuE (for C. jejuni and C. coli, respectively) derived primers was performed to Campylobacter isolates to identify and differentiate C.

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jejuni and C. coli, giving PCR products of about 590 and 460 bp, respectively.

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Additionally, a 16S primer set was included as quality assurance of the DNA-

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preparation and analysis (internal control) (800bp). PCR amplifications were

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conducted in a final volume of 25 μL containing 1.25 U of thermostable (Taq) DNA polymerase (Kapa Biosystems, Boston, MA, USA), 2.5 mL Taq buffer, 0.2 mM each

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dNTP’s, 0.34 mM of each primer, 1.75 mM MgCl2 and 0.5 μL of DNA template.

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PCR reaction consisted of an initial denaturation step at 94 οC for 10 min, followed by 30 cycles (denaturation at 94 οC for 30 s, primer annealing at 50 οC for 90 s, primer

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extension at 72 οC for 60 s) and a final extension step at 72 οC for 10 min. Aliquots (10 μL) of PCR products were checked on 1.5% agarose gels at 100V for 45 min by electrophoresis. Reference strains included in this study were C. jejuni ATCC 29428 and C. jejuni ATCC 33560 (type strain). Repetitive element polymerase chain reaction (rep-PCR) was performed using the (GTG)5-primer for the discrimination of the obtained isolates in different strains. One hundred nanograms of the DNA extracted from isolates was subjected to rep-PCR analysis using primer (GTG)5 (5′-GTGGTGGTGGTGGTG-3′) according to Papadopoulou et al. (2012) with slight modifications. In brief, reactions were carried out in a final volume of 25 μL containing 2 mM MgCl2, 0.8 mM mix dNTPs, 10 μM

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Journal Pre-proof primer (GTG)5 and 1.25U Taq-polymerase. PCR reactions consisted of an initial denaturation step at 95 °C for 5 min, followed by 30 cycles of denaturation at 95 °C for 30 s, primer annealing at 40 °C for 1 min and primer extension at 72 °C for 8 min, and completed by a final extension step at 72 °C for 16 min. Amplicons were separated in a 2% (w/v) agarose gel in TBE 1× at 120 V for 2 h. After the run, gels were stained with ethidium bromide 0.5 μL mL−1 for 30 min. Normalization and further analysis were performed using the Dice coefficient and UPGMA cluster

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analysis with Bionumerics software, version 6.1 (Applied Maths, Sint-Martens-

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Latem, Belgium).

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2.6 Data analysis

For the identification of any grouping between the samples, Discriminant

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Analysis (DA) was carried out using XLSTAT software 2019 (Addinsoft, France).

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The initial and final pH values, the initial TAC, the presence/absence of potassium sorbate and sodium benzoate as well as the dominant spoilage microorganisms

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(Pseudomonas spp., B. thermosphacta, their co-dominance and lactic acid bacteria) were used as explanatory variables. The dependent (qualitative) variable was the storage time needed for TAC and sensory score values to exceed 7.0 log CFU/g (microbiological threshold of spoilage) and 3, respectively.

3. Results 3.1 Safety assessment of marinated chicken products The results as far as the presence of the three pathogens is concerned are presented in Table 2. Campylobacter spp. was detected in 50% of the samples, Salmonella was present in 11% of the samples, and L. monocytogenes was found in

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Journal Pre-proof 44% of the samples, whereas no presence of the above mentioned pathogens was observed in 16% of total samples (Table 2A). As presented in Table 2B, Campylobacter spp. and L. monocytogenes were concurrent in 11% of the samples, whereas 2.5% of the samples were positive in the concurrent presence of Salmonella and L. monocytogenes, or in Campylobacter spp. and Salmonella, respectively. Finally, a percentage of 3.75% of the samples was positive in all pathogens assayed. In the particular case of Campylobacter spp., a more thorough examination was

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conducted to identify the isolates that belonged to C. jejuni or C. coli species and

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discriminate them at strain level. From the 40 Campylobacter isolates, 27 were

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identified as C. coli, 4 as C. jejuni and 9 belonged to neither species (Table 2, Fig. 1).

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Subsequently, C. coli and C. jejuni isolates were subjected to rep-PCR for strain differentiation. Analysis of genotypic patterns assorted the C. coli and C. jejuni strains

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into 6 groups with a coefficient of similarity of 70% (Fig. 2). It needs to be noted that

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8 out of 12 strains of Group C had a coefficient of similarity higher than 80%, while 3 out of 8 strains, isolated from different meat markets and different sampling days,

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presented 100% similarity. Moreover, the linkage distance of 3 out of 4 C. jejuni strains assorted in group F was lower than 10%. It needs to be mentioned that although the two species were well distinguished into different groups, a single C. coli strain was assorted with C. jejuni strains into Group F.

3.2 Quality assessment of marinated chicken products Several parameters associated with the quality of marinated chicken products were assessed (Table 3, Fig. 3, Supplementary File). In 46.3% of the samples, the initial pH value was higher than 6.1. In a similar proportion (45%) of samples, the pH ranged within 5.6-6.0, while in 8.8% of the samples the pH was in the range of 5.0-

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Journal Pre-proof 5.5. Regarding the microbiological quality, the initial population of total aerobes was in the range of 6.1-6.5 log CFU/g in 42.5% of the samples, whereas the initial population in 20% and 38.8% of the samples was higher than 6.6 log CFU/g and lower than 5.5 log CFU/g, respectively. Enterobacteriaceae exceeded 5.1 log CFU/g in 33.4% of the samples, whereas lower counts (< 4.0 log CFU/g) were obtained in 26.3% of the samples (Fig. 3). Concerning the spoilage microorganisms, it was found that 73% of the samples were spoiled by Pseudomonas spp. or a combination of

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Pseudomonas spp. and B. thermosphacta, since these microbial groups were first to

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reach levels of 7.0 log CFU/g. The number of samples spoiled by LAB or B.

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thermosphacta was significantly lower (ca. 13% of the samples for each microbial

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group) (Table 3A). As regards the presence of preservatives, sodium benzoate or/and potassium sorbate were detected in ca. 31% of the samples, with the latter being the

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most frequently occurring preservative (in 23.8% of the samples) (Table 3D).

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However, it should be noted that in the majority of the samples that were positive in the presence of preservatives, the concentration was lower than 100 mg/kg of meat

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(Supplementary File). Considering as microbiological shelf life of marinated chicken products the time needed for TAC to reach 7.0 log CFU/g (microbiological threshold for spoilage), it was found that in 71.3% of the samples this level was reached within 3 days of storage (Table 3B). It must be noted that 63.2% of these samples reached the threshold of 7.0 log CFU/g on days 2 or 3, while the respective percentage for the shelf life as derived by sensory analysis (i.e., sensory score > 3) was 48% within the first 3 days of storage, from which 43.8% on days 2 or 3. According to the sensory panel, 52.5% of the samples were characterized as spoiled after 4-5 days of storage, whereas the respective percentage for the microbiological shelf life was 28.7% (Table 3C) within the same time period. A further multivariate analysis on the obtained data

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Journal Pre-proof revealed certain correlations (Fig. 4). As it can be observed in the discriminant analysis map for the results of the microbiological analysis (Fig. 4A), the time needed to reach 7.0 log CFU/g is clearly differentiated by Discriminant Factor 1 (F1), explaining almost 82% of the variability in the dataset, with gradual transition of storage time from the right (1-2 days) to the left (4-5 days) side of the discriminant map. The discrimination was less clear for the sensory analysis data as significant overlapping was observed among the five classes of storage time. As in the previous

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case, Discriminant Factor 1 accounted for the storage time needed to exceed the

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sensory threshold score of 3 and presented gradual transition from low (left side) to

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high (right side) values of storage time. Taking into consideration the plots of

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variables and scores for the microbiological analysis (Fig. 4A), the samples in which a lower level of spoilage was recorded (less than 7.0 log CFU/g on days 4 and 5) were

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associated with sensory score > 3 on day 4 or 5, while these groups of samples were

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correlated with the dominance of B. thermosphacta or LAB and – to a lesser extent – with the presence of sorbate or benzoate. On the other hand, early spoilage of the

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samples (1st or 2nd day) was associated with higher pH values and higher initial microbial population, while Pseudomonas spp. and mainly the co-dominance of Pseudomonas spp. and B. thermosphacta characterized these samples. A similar pattern was observed in the variables that discriminate storage time in which spoilage occurred based on sensory analysis (Fig. 4B).

4. Discussion The prevalence of Campylobacter in marinated samples from the Greek retail market (50.0%) was within the range (20-80%) recorded by previous studies for poultry meat (Skarp et al., 2016). Moreover, it was quite close to the occurrence in

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Journal Pre-proof fresh meat from broilers (37.4%) reported by the European Union summary report on zoonoses, zoonotic agents and foodborne outbreaks (EFSA, 2018). According to this report, C. jejuni and C. coli are the two most frequently isolated species representing 81% and 8.4% of total isolates, respectively. However, the results from this study showed high C. coli presence in marinated samples. Only in a few studies C. coli has been reported as the most prevalent species (Kovaļenko et al., 2014; Torralbo et al., 2015) since in the majority of relevant studies C. jejuni was most frequently isolated

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(Guyard-Nicodème et al., 2015; Wei et al., 2016; Wieczorek et al., 2013). However, it

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has been reported that C. coli was most frequently isolated during the whole

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processing line at different slaughterhouses (Spain, Italy and Bulgaria) and in some

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cases it has even doubled its occurrence in the final product compared to C. jejuni. This could be attributed to higher resistance of C. coli to environmental conditions

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such as temperature and exposure to oxygen or antimicrobials (Damjanova et al.,

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2011; Oyarzabal et al., 2010; Stella et al., 2017; Torralbo et al., 2015). Additionally, the effect of latitude on the relative frequency of the two species was also reported,

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with a marked predominance of C. jejuni in Northern Europe (up to 100% on carcasses in the Baltic countries) and a gradual increase of C. coli prevalence in Southern countries (EFSA, 2010a). Regarding the identification methods of Campylobacter spp., it should be underlined that the accurate and rapid detection of Campylobacter spp. is of critical importance. In this framework, traditional culture-based techniques present certain limitations as they are time consuming, they have relatively high limit of detection and present consistency and reliability issues. Culture-independent methods could play a significant role in Campylobacter detection and identification and contribute to

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Journal Pre-proof the improvement of speed, sensitivity and specificity of the analysis (Buss et al, 2019; Couturier, 2016; Ricke et al., 2019). The occurrence of Listeria monocytogenes in meat products has been also widely investigated. Poultry has been reported as a vehicle for L. monocytogenes transmission and thus, the main concern with raw chicken meat is the potential for cross-contamination to other foods (Tod and Notermans, 2010). A prevalence of L. monocytogenes similar to that reported in the present work was observed in poultry

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in previous studies (Alonso-Hernando et al., 2012; Filiousis et al., 2009; Pesavento et

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al., 2010; Vitas et al., 2004;). The high incidence of L. monocytogenes in carcasses is

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likely to arise from cross-contamination during processing (Moura et al., 2016). At

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the end of the process, storage of meat at low temperatures is a typical step to inhibit the growth of pathogens. However, it was observed that in chilled poultry meats, the

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prevalence of L. monocytogenes was still high, indicating that refrigeration is not

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enough to inhibit the proliferation of this cold-resistant pathogen (Gonçalves-Tenório et al., 2018). Salmonella is a pathogen of major concern for food safety authorities,

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meat industries and consumers. There was a declining trend for many years concerning Salmonella foodborne outbreaks, which has been stabilized and slightly increased in recent years (EFSA, 2018). Salmonella enterica is among the most tracked human pathogens, with the serovar Enteritidis associated mainly with poultry meat and major outbreaks (Jackson et. al, 2013). The prevalence of Salmonella in this study was higher compared to the mean estimate for Salmonella in chicken meat reported by a Meta-Analysis of European Published Surveys (3.2%) (GonçalvesTenório et al., 2018) as well as by EFSA (6.4%) (EFSA, 2017). However, taking into consideration that marinated chicken meat requires further treatment and processing, a

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Journal Pre-proof scenario of contamination from additional ingredients and post-processing could not be excluded. As far as the microbiological quality of marinated chicken meat is concerned, there are two contradictory scenarios. The first is that meat marination enhances its quality due to the presence of antimicrobial ingredients that marinades may contain (Karam et al., 2019; Moon et al., 2017; Sengun et al., 2019) and the second is that meat retailers choose to marinate the meat that is close to the end of shelf life in order to make it

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more attractive for consumers. It is widely known that marinades contain several

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flavor enhancers that could mask the undesirable flavors of spoiled chicken meat. In

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this study, the high percentage of samples with increased initial population of spoilage

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microorganisms (i.e., Pseudomonas spp. and/or B. thermosphacta) indicates that the second scenario may be true for the majority of the samples that belonged to this

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group. The outcome of the sensory analysis showed that marinades contributed to the

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moderation of the off-flavors produced by the spoilage microorganisms and consequently the taste panel, in many cases, failed to perceive the actual level of

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spoilage. Moreover, the increased level of enterobacteria that are considered as a hygiene indicator (EFSA BIOHAZ Panel, 2017), showed that the compliance with hygiene requirements regarding chicken meat was probably inadequate. However, there were few samples, for which the low microbial population combined with the low pH value of the meat could indicate that the used marinade contained probably an antimicrobial agent (e.g., organic acid, preservative, etc.). Nevertheless, it should be noted that, in this study, the concentrations of these substances on meat were at low levels, probably due to the fact that their role was to act as preservative agents for the marinades solutions only. It has been reported that for bacteria, the minimum inhibitory concentration (MIC) of benzoic and sorbic acid at pH 5.0-6.6 is 1,500 –

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Journal Pre-proof 10,000 mg/L and 700-10,000 mg/L, respectively, while these concentrations are significantly lower at more acidic environments (pH lower than 4.5) (Lund and Eklund, 2000). Concerning the microorganisms that induce spoilage, LAB are usually reported as the main microbial group associated with spoilage of marinated meat (Nieminen et al., 2012; Rouger et al., 2017). However, it should be taken into account that in the aforementioned studies, meat marination was combined with modified atmosphere

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packaging (MAP) with reduced oxygen concentration, a condition that favors the

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growth of LAB. In this study, Pseudomonas spp. clearly dominated in most samples

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and also correlated with reduced shelf life, since it was enumerated in high

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populations even by the 1st or 2nd day of storage. Apart from the high populations, Pseudomonas spp., correlated with short shelf life by panelists probably due to the

2015).

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5. Conclusion

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offensive flavors of metabolites produced by this microbial group (Casaburi et al.,

The results obtained in this study demonstrated the presence of pathogens in marinated chicken meat products from the Greek retail market, while bacteria of Campylobacter genus were isolated with high frequency, with C. coli being the most prevalent. Regarding the microbiological quality, the high initial microbial populations and the resulting short shelf life in the majority of the samples, indicated that their microbiological quality was not satisfactory at the day of purchase. Moreover, the outcome of the sensory analysis showed that the assessors encountered a difficulty in evaluating the actual level of products’ microbiological quality, especially in the samples close to the acceptance threshold.

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Journal Pre-proof References Alonso-Hernando, A., Prieto, M., García-Fernández, C., Alonso-Calleja, C., Capita, R., 2012. Increase over time in the prevalence of multiple antibiotic resistance among isolates of Listeria monocytogenes from poultry in Spain. Food Contr. 23, 37–41. Bahrndorff, S., Rangstrup-Christensen, L., Nordentoft, S., Hald B., 2013. Foodborne disease prevention and broiler chickens with reduced Campylobacter infection.

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Birk, T. & Knøchel, S., 2009. Fate of food-associated bacteria in pork as affected by

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marinade, temperature, and ultrasound. J. Food Prot. 72, 549–555.

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Buss, J.E., Cresse, M., Doyle, S., Buchan, B.W., Craft, D.W., Young, S., 2019. Campylobacter culture fails to correctly detect Campylobacter in 30% of

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positive patient stool specimens compared to non-cultural methods. Eur. J. Clin.

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Microbiol. Infect. Dis. 38, 1087-1093. Casaburi, A., Piombino, P., Nychas, G.-J.E., Villani, F., Ercolini, D., 2015. Bacterial

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populations and the volatilome associated to meat spoilage. Food Microbiol. 45, 83–102.

Couturier, M.R., 2016. Revisiting the roles of culture and culture independent detection tests for Campylobacter. J. Clin. Microbiol. 54, 1186 –1188. Damjanova, I., Jakab, M., Farkas, T., Meszaros, J., Galantai, Zs, Turcsanyi, I., Bistyak, A., Juhasz, A., Paszti, J., Kiss, I., Kardos, G., 2011. Form farm to fork follow-up of thermotolerant campylobacters throughout the broiler production chain and in human cases in a Hungarian county during a ten-months period. Int. J. Food Microbiol. 150, 95-102.

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Journal Pre-proof Karam, L., Roustom, R., Abiad, M. G., El-Obeid, T., & Savvaidis, I. N., 2019. Combined effect of thymol, carvacrol and packaging on the shelf-life of marinated chicken. Int. J. Food Microbiol. 291, 42-47. Kovaļenko, K., Roasto, M., Šantare, S., Bērziņš¸ A., Hörman, A., 2014. Campylobacter species and their antimicrobial resistance in Latvian broiler chicken production. Food Contr. 46, 86-90. Lund, B.M., Eklund, T., 2000. Control of pH and use of organic acids. In: Lund,

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chicken meat. J. Food Nutr. Res. 4, 436–441. Nieminen, T.T., Koskinen, K., Laine, P., Hultman, J., Sade, E., Paulin, L., Paloranta, A., Johansson, P., Björkroth, J., Auvinen, P., 2012. Comparison of microbial communities in marinated and unmarinated broiler meat by metagenomics. Int. J. Food Microbiol. 157, 142–149. Oyarzabal, O.A., Oscar, T.P., Speegle, L. and Nyati, H., 2010. Survival of Campylobacter jejuni and Campylobacter coli on retail broiler meat stored at 20, 4, or 12oC and development of Weibull models for survival. J. Food Prot. 73, 1438–1446.

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Journal Pre-proof Papadopoulou, O.S., Doulgeraki, A.I., Botta, C., Cocolin, L., Nychas, G.-J.E., 2012. Genotypic characterization of Brochothrix thermosphacta isolated during storage of minced pork under aerobic or modified atmosphere packaging conditions. Meat Sci. 92, 735-738. Pathania, A., McKee, S.R., Bilgili, S.F., Singh, M., 2010. Antimicrobial activity of commercial marinades against multiple strains of Salmonella spp. Int. J. Food Microbiol. 139, 214–217.

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Journal Pre-proof Thanissery, R., & Smith, D. P., 2014. Marinade with thyme and orange oils reduces Salmonella Enteritidis and Campylobacter coli on inoculated broiler breast llets and whole wings. Poultry Sci. 93, 1258-1262. Todd, E. C. D., & Notermans, S., 2010. Surveillance of listeriosis and the causative pathogen, Listeria monocytogenes. Food Control 22(9), 1484-1490. Torralbo, A., Borge, C., García-Bocanegra, I., Méric, G., Perea, A., Carbonero, A., 2015. Higher resistance of Campylobacter coli compared to Campylobacter

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monocytogenes in fresh and processed foods in Navarra (Spain). Int. J. Food Microbiol. 90(3), 349-356.

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Wei, B., Cha, S.Y., Yoon, R.H., Kang, M., Roh, J.H., Seo, H.S., Lee, J.A., 2014. Antimicrobial susceptibility profiles and molecular typing of Campylobacter jejuni and Campylobacter coli isolates from ducks in South Korea. Appl. Environ. Microbiol. 80(24), 7604-10. Wieczorek, K., Kania, I., Osek, J. J., 2013. Prevalence and antimicrobial resistance of Campylobacter spp. isolated from poultry carcasses in Poland. J. Food Prot. 76, 1451-1455. Yusop, S.M., O'Sullivan, M.G., Kerry, J.F., Kerry, J.P., 2010. Effect of marinating time and low pH on marinade performance and sensory acceptability of poultry meat. Meat Sci. 85, 657–663.

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Figure 1. Representative patterns of multiplex PCR targeting mapA (590bp), ceuE

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(460bp) and 16S rRNA (800bp) genes of Campylobacter jejuni (8D,3A) and

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Campylobacter coli (3E, 2A, 2E, 6G, 6D, 6E, 6Z, 6H) isolates from marinated

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jejuni ATCC 33560.

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chicken meat products. L: 100bp DNA ladder; C1: C. jejuni ATCC 29428; C2: C.

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Figure 2. Cluster analysis based on rep-PCR genotypic patterns of Campylobacter coli (co) and Campylobacter jejuni (je) calculated by the unweighted average pair

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grouping method. The distance between the pattern of each strain is indicated by the

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mean Dice correlation coefficient (r%). Groups (A, B, C, D, E and F) include strains with coefficient similarity of 70%

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Figure 3. Distribution (%) of tested samples among several ranges of initial pH (A),

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final pH (B), initial TAC (C) and Enterobacteriaceae population (D).

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Figure 4. Discriminant analysis map (a) and plot of variables (b) determined by Discriminant Factors 1 (F1) and 2 (F2) for the storage time (days) needed for TAC (A) and sensory score (B) values to exceed 7.0 log CFU/g and 3, respectively. (PS: Pseudomonas spp., LAB: Lactic Acid Bacteria, BR: B. thermosphacta, PS + BR: Pseudomonas spp. + B. thermosphacta, TAC (TVC): Total Aerobic Counts). (days)

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Table 1. Enrichment and isolation media, incubation conditions and confirmation tests of the methods followed for the detection of Salmonella, Campylobacter spp. and Listeria monocytogenes. Target microorganism Salmonella

Method

ISO 6579:2002

Campylobacter ISO spp. 10272-1: 2006

Enrichment Media 1. Preenrichment : Buffered peptone water 2. Selective enrichment a. RVS broth b. MKTTn Broth

Incubation 1. 18 h ± 2 h at 37 °C ± 1°C 2a. 24+24 h ± 3 h at 43°C 2b. 24 h ± 3 at 37°C

Media Streaking on XLD Agar after each enrichment step

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Incubation 24 h ± 3 h at 37 °C

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Selective enrichment :Bolton Broth+supple ment

Confirmation tests

Isolation

4−6 h at 37°C and then for 44 h ± 4 at 41.5°C in a microaerobic atmosphere

1. mCCD Agar 2. Skirrow

44 h ± 4 at 41.5°C in a microaerobic atmosphere

Columbia Blood Agar: 1. Incubation for 48 h at 41.5°C in aerobic conditions (negative) 2. Incubation for 48 h at 25 oC in a microaerobic atmosphere (negative) 3. Oxidase test (positive)

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Listeria ISO monocytogenes 112901:1996

1. Primary enrichment : Half Fraser Broth 2.Secondary enrichment : 0.1 mL of primary enrichment culture (after 24h of incubation) to 10 mL of Fraser Broth

1. 24 + 24 h ± 2 at 30°C

48 h at 37°C

1. ALOA 2.Palcam

2. 48 h ± 2 at 37°C

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4. Microscopic observation Tryptone Soya Yeast Extract 1. Catalase Test 2. Haemolysis Test (Sheep Blood Agar ) 3. Carbohydrate Utilization 4. CAMP Test

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Table 2. Prevalence* (%) of the three pathogenic bacteria (A) and percentage (%) of their co-occurrence (B) in marinated chicken products.

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Frequencies A Campylobacter spp. 40 C. jejuni 4 C.coli 27 Unidentified 9 Listeria monocytogenes 35 Salmonella 9 N/D 13 Frequencies B Campylobacter spp. + Listeria monocytogenes 9 Campylobacter spp. + Salmonella 2 Salmonella + Listeria monocytogenes 2 Campylobacter spp. + Listeria monocytogenes + + Salmonella 3  Total of 80 chicken meat samples

% 50.00

43.75 11.25 16.25 % 11.25 2.50 2.50 3.75

Journal Pre-proof Table 3. Parameters associated with quality of marinated chicken products (% of the samples) % of the samples

Pseudomonas spp.

34

42.50

Pseudomonas spp. + B. thermosphacta

25

31.25

B. thermosphacta

11

13.75

Lactic Acid Bacteria

10

12.50

A. Spoilage microorganism

B. Microbiological shelf life*

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Frequencies

2

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7

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(days) 1

% of the samples 8.75 33.75

23

28.75

10

12.50

13

16.25

Frequencies

% of the samples

3

3.75

13

16.25

22

27.50

4

23

28.75

5 ***

19

23.75

Frequencies

% of the samples

Sodium benzoate

3

3.75

Potassium sorbate

19

23.75

Sorbate + benzoate

3

3.75

4 5 ***

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C. Sensorial spoilage time**

2 3

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(days) 1

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Frequencies

D. Preservatives

*TAC > 7 log CFU/g

**Sensory score > 3

***5 days or higher

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Journal Pre-proof Highlights

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  

Foodborne pathogens were detected in marinated chicken products of the Greek market Campylobacter coli was the most prevalent pathogenic microorganism High initial microbial load and short shelf life were observed in most products The flavor of marinades could mask the spoilage off-odors

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

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