Comparison of the microbial quality of pork and poultry meat with or without grill marinade available in Polish retail markets

During April and September 2012, a study on pork and poultry meat was undertaken to assess the differences in microbial quality of pork and poultry between the marinated and unmarinated products available in local trade network. Examination of the meats revealed that the vast majority of samples (98.5%) were of satisfactory microbiological quality according to Commission Regulation (EC) No 2073/2005, as amended. Unsatisfactory quality was due to the presence of Salmonella spp. in 5 pork necks with grill marinade. Campylobacter spp. was detected in 81% of poultry meat samples, and Listeria monocytogenes in 43% of all types of analysed meat. The total bacterial count was from 1.3 x 10 5 CFU/g for neck without marinade to 7.9 x 10 7 CFU/g for neck with marinade 1. The number of Enterobacteriaceae ranged between < 10 CFU/g (for neck without marinade) and 1.4 x 10 7 CFU/g (for chicken shashlik). The one-way ANOVA showed the significant influence of marinade on the number of Enterobacteriaceae and total bacterial count in all tested meat samples. No significant differences were observed taking into account the mean number of lactic acid bacteria and yeast (p>0.05). The relatively high contamination of marinated and unmarinated pork and poultry meat with food spoilage microorganisms and pathogens decreases the quality and constitutes a public health hazard but it is comparable with meat quality of developed countries.


INTRODUCTION
Grilling is a form of cooking dishes without fat, on grill placed over heat source.Nowadays, it is a very fashionable way of preparing food.In Poland, grilling tradition started in the first half of the 1990s.Initially, consumers usually bought grilling meat products, such as thin sausage, black pudding and bacon.Currently, the most popular are: neck-ends, knuckle ribs and small pieces of chicken: wings, legs, breast fillets.The demand for this kind of products is growing by up to 30% in the peak season between April and September.Simul-taneously, a decrease in sale (by 20-25%) of traditional meat for polish dinner, the loin and ham can be observed.Consumers are more willing to get new products, especially products previously seasoned or marinated.Industrial marinades include: oil, water, spices, organic acids and glucose or sucrose.
With the marinade, time of meal preparation is reduced significantly, and the dishes have a wonderful aroma, a deep and attractive colour, enhanced flavour and juiciness fragility (Björkroth, 2005).During the heat treatment marinated meat evenly grills and does not burn up.In the available commercial offer, ready seasoned and marinated products constitute over a quarter of all of meat on the grill.
Despite the large variety of grill equipment facilities, the most popular used during the spring and summer in Poland are charcoal grills.These are the oldest types of grills, with temperatures of 260°C, suggesting a safe food preparation.However, the minimum time required for a trained fire up the grill is about 20-25 min.
As people are leading a very busy life style and not finding sufficient time for grilling, there is a high risk of food poisoning and when the meat is uncooked.Therefore, one of the most important issues, in addition to sensory value, is to ensure the microbiological safety of meat, spices and marinades.Marinating has been considered to increase safety of meat products.However, it may not necessarily have an effect on the survival of some enteric pathogens, like Campylobacter spp.(Björkroth, 2005).Moreover, the critical components, such as spices can decrease the microbial quality of marinades and have influence on their safety.In a study conducted by Dababneh (2013), 87.5% of the spices and herbs from local market were unsatisfactory because of the large population of fungi (87.5%) and coliforms (37.5%).Although plant materials used for producing commercially available herbs and spices contain natural antimicrobial substances, their microbiological quality is very diverse (Nokwanda and Ijabadeniyi, 2013).Microorganisms isolated most often from spices are: non sporing bacteria, spore-forming bacteria, pathogenic bacteria, yeast and moulds.Particularly high microbial load was detected in spices like: black pepper, mustard, fenugreek, chilli,marjoram, cumin, red pepper, basil (Dababneh, 2013;Keller et al., 2013;Nokwanda and Ijabadeniyi, 2013;Van Doren et al., 2013b), commonly used in grill marinades.
Taking into account this fact, the Laboratory of Microbiology, Department of Refrigeration and Food Quality, Institute of Agricultural and Food Biotechnology has undertaken research aimed at determining the differences in microbial quality of pork and poultry between the marinated and unmarinated products available in local trade network.

Sampling
A total of 340 samples of 10 types of commercially available meat were analyzed for their microbiological quality.The samples of meat were delivered to the Laboratory of Microbiology in isothermal containers, maintaining the temperature at 0 -2°C, in the period from April to September 2012 and processed within 24 h.The material included 160 samples of pork and 180 samples of poultry meat.The pork consisted of 8 samples of neck without marinade and 24 samples of neck in three different types of marinades (herbs and garlic (1), paprika (2) and Bavarian (3)).Poultry meat consisted of 6 samples of chicken schnitzels, 6 samples of chicken schnitzels in paprika marinade, 6 samples of chicken wings, 6 samples of chicken wings in herbal paprika marinade, 6 samples of chicken stew and 6 samples of chicken shashlik.Microbiological analyses were carried out in 5 replicates.

Microbiological and statistical analyses
The meat was evaluated in terms of food safety and hygiene of the process.All microbiological parameters were sampled and analysed following Polish Standards (PN-EN ISO 4833: 2003;PN-EN ISO 6579: 2003+ A1:2007;PN-EN ISO 6888-1: 2001;PN-ISO 7954: 1999;PN-EN ISO 11290-2:2000+ A1:2005+ Ap1:2006;PN-ISO 10272-1:2007;PN-ISO 21528-2: 2005) and Commission Regulation (EC) No 2073/2005 of 15.11.05 on the microbial criteria for foodstuffs, as amended.The pathogens investigated included the presence of: Salmonella spp., Listeria monocytogenes, and poultry Campylobacter spp.Additionally, total bacterial count (TBC), the number of Enterobacteriaceae (E), lactic acid bacteria (LAB), coagulase-positive Staphylococcus (CPS), yeast and moulds were enumerated.API-tests (Biomerieux), Microgen Listeria ID (MicrogenBioproducts), and Singelpath (Merck) were used for biochemical and serological determination.All microbiological tests were carried out in 5 replicates, and the results expressed as log10 CFU/g.Data analysis was performed using STATISTICA software package (StatSoft, 2012).A one-way ANOVA analysis of variance was used for determination of the effect of marinades and spices on microbial quality of pork and poultry available in local trade network.The significance differences were indicated by ANOVA when p= 0.05 or less and F>1.

RESULTS
The frequency of detection of the pathogens in the tested meat samples is shown in Table 1.Examinations revealed that the vast majority of samples (98.5%) were of satisfactory microbiological quality according to Commission Regulation (EC) No 2073/2005 as amended.Unsatisfactory quality was caused by the presence of Salmonella spp. in 5 samples of necks in marinade 2. The spiced chicken shashlik had the highest prevalence of L. monocytogenes and Campylobacter spp.
The changes in counts of bacteria, yeast and moulds (CFU/g) for different types of meats are shown in Table 2. TBC displayed loads from 1.3 x 10 5 CFU/g (neck without marinade) to 7.9 x 10 7 CFU/g (neck with marinade 1).CPS were below the detection limit, except for three cases: neck with marinade 2, chicken wings in marinade and chicken shashlik, whose counts increased to 5.0 x 10 1 CFU/g, 8.0 x 10 1 CFU/g,1.0x 10 2 CFU/g, respectively.The number of Enterobacteriaceae ranged between < 10 CFU/g (for neck without marinade) and 1.4 x 10 7 CFU/g (for spiced chicken shashlik), while LAB grew up to 1.9 x 10 7 CFU/g (neck with marinade 3).Moulds generally reached values between 10 1 -10 2 CFU/g with the highest values of 4.2 x 10 3 CFU/g for spiced chicken shashlik.In contrast, the viable counts of yeast were higher and grew over 10 6 CFU/g (neck with marinade 1).The mean count of analysed microorganisms of marinated and unmarinated meat samples is depicted in Figure 1.In the present study, the average number of total bacterial count was within the range of 5.87 (chicken   4 -4.9 x 10 5 stew) -7.47 log10 CFU/g (chicken shashlik).The average count of Enterobacteriaceae ranged from 2.52 log10 CFU/g (neck unmarinated) to 5.87 log10 CFU/g (chicken shashlik).The population of LAB was 3.22 log10 CFU/g for chicken wings and 5.48 log10 CFU/g for chicken shashlik.

Product
Irrespective of the analysed group of microorganisms, the mean microbial load of meat (neck and chicken) with marinade and spices was higher compared to that without marinade and spices.The mean TBC for samples of neck with marinade 1, 2, 3 was 7.3, 7.19, 7.38, respectively, and for neck unmarinated was 6.28 log10 CFU/g.The mean TBC of marinade chicken schnitzels was 6.66; in contrast the mean TBC of chicken schnitzels without marinade was 6.07 log10 CFU/g.The mean number of Enterobacteriaceae in chicken shashlik was 5.87 as compared to 4.64 log10 CFU/g in chicken stew.
The one-way ANOVA showed the significance influence of marinade on the number of Enterobacteriaceae and total bacterial counts in all tested meat samples.No significant differences were observed taking into account mean number of lactic acid bacteria and yeast for meat with or without marinade (p>0.05).

DISCUSSION
According to the Regulative EU (Commission Regulation 2073/2005, as amended), the only parameters that can disqualify the tested meat is: the presence of Salmonella spp.(in pork) and the presence of Salmonella Typhimurium and Enteritidis (in poultry).These two serovars remain the most important in foodborne salmonellosis worldwide.International research indicates wide variation in the rates of meat samples contaminated by Salmonella spp.The presence of this pathogen in meat is dependent on a number of factors such as: the quality of meat, spices used or hygienic condition during production.The occurrence of Salmonella spp. in spices has been reported in many countries (Keller et al., 2013;Moreira et al., 2009;Van Doren et al., 2013a;Zweifel and Roger, 2012).Results obtained by the present study provided evidence that 4.4% of analysed samples were contaminated with Salmonella spp.None of the bacteria isolates belonged to Salmonella Enteritidis or Typhimurium.The survey performed by Greeson et al. (2013), Madden et al. (2011) and Zdragas et al. (2012) found 5.1, 13.7 and 39.5% samples of poultry meat to be positive for Salmonella, respectively.On the other hand, the results obtained by Chagas et al. (2013) indicated that 94.0% of the chicken carcass samples contained Salmonella; among these isolates, 32% were genotyped by multiplex polymerase chain reactions (mPCR) as Salmonella Enteritidis while none were identified as Salmonella Typhimurium.Furthermore, the prevalence of Salmonella in ground beef and pork was 0.4% (Schwaiger et al., 2012;Vipham et al., 2012).
L. monocytogenes in the current study was detected in 42.6% of analysed samples of meat that is comparable with the results obtained by other researchers.In the work conducted by Aarnisalo et al. (2008) the prevalence of L. monocytogenes in marinated broiler legs varied between 36 and 57% for the samples stored at 6 and 10°C, respectively.Survey performed by Goh et al. (2012) showed that the occurrence of L. monocytogenes in raw chicken samples was 20%.
There are no safety criteria for pork and poultry meat, in terms of total bacterial count, Enterobacteriaceae, lactic acid bacteria, molds, yeast and the presence of Campylobacter spp. in the Polish standard regulations.
The average number of total bacterial count in the current study was within the range of 5.87-7.47log10 CFU/g.This level is in accordance with Cohen et al. (2007) and Andritsos et al. (2012), who found the mean value of 5.9 log10 CFU/g (poultry) and 6.8 log10 CFU/g (minced pork), respectively.As expected, in all marinated pork and poultry meat tested, the higher total bacterial counts were observed.The reason for this fact is probably due to the use of spices, which could be a cause of increased microbial contamination.From the report of Steinhauserová et al. (2012), it is known that the quality and age of the raw meat play an important role.Some producers often try to "salvage" raw meat before its expiratory date.The salt content and low pH of the marinade slow down the multiplication of microorganisms and thus extend their shelf life.
The average count of Enterobacteriaceae in the present research ranged from 2.52 log10 CFU/g (neck unmarinated) to 5.87 log10 CFU/g (chicken shashlik).These values are close to the counts reported by some other researchers.Andritsos et al., (2012) indicated that samples of minced pork marketed in Greece contained Enterobacteriaceae at the level of 3.6 ± 1.2 log10 CFU/g.In another work (Kilonzo-Nthenge et al., 2013), Enterobacteriaceae numbers were between 3.26 (chicken wings) and 4.75 log10 CFU/g (chicken breast).Our results have shown different effect of marinades on the proportion of Enterobacteriaceae in meat microflora, from that observed by Finnish scientists.Finnish research showed that marination may extend sensory shelf-life of broiler meat by delaying the growth of Enterobacteriaceae that is associated with spoilage of poultry (Björkroth, 2005;Nieminen et al., 2012a).To our knowledge, this is the first report comparing the effect of grill marinade and spices on the microbial quality of pork and poultry meat available in Polish retail markets.More research in this field should be performed in order to determine the differences between the proportion of Enterobacteriaceae in marinated and unmarinated meat.Despite this fact, high level of total bacterial count and Enterobacteriaceae found in current research, independent of marinades and spices used, is an indication of fecal contamination and poor hygiene during processing.On the other hand, in the present study, we observed the low level of contamination with Staphylococcus aureus (2.0 log10 CFU/g).This level suggests strict personal hygiene of the workers (Cohen et al., 2007).
Lactic acid bacteria are considered to be the Specific Spoilage Organisms that contribute to the meat spoilage stored under packaging conditions in which the concentration of carbon dioxide is increased (Doulgeraki et al., 2010).Lactobacillus, Leuconostoc, and Carnobacterium are the most frequently encountered genera on marinated and unmarinated meat and play a significant role in the spoilage of refrigerated raw meat (Doulgeraki et al., 2010, Nieminen et al., 2012b).In a study conducted by Nieminen et al. (2012b), the LAB colony counts measured from marinated broiler fillets strips ranged from 8.2 to 9.2 log10 CFU/g.In the present research, the average count of LAB for poultry products with grill marinade ranged from 4.14 (chicken wings in marinade) to 5.48 log10 CFU/g (chicken shashlik).
Molds and yeasts constituted a minor portion of the microflora on meat.However, it is practically impossible to eliminate them completely from meat.Especially, spices added to meat can considerably contribute to total molds and yeast contamination.In the present study, the marinades and spices used have little influence on the numbers of molds and yeasts detected.Analysed samples were contaminated with molds and yeasts in values varying up to 2.78 and 5.13 log10 CFU/g, respectively.Similar results have been noted by Ismail et al. (2000).According to them, mycoflora has not been considered as playing a major role in spoilage, as it is seldom present in high numbers.The mean populations of yeast and molds observed by these researchers ranged from 1.65 to 5.12 log10 CFU/g.The growth of the mycoflora was not inhibited by marination.
Our analysis revealed that Campylobacter spp. was detected in poultry product with the prevalence of 38.9% and 61.1% for non marinated and marinated products, respectively.The data presenting contamination of chicken with Campylobacter spp. in industrialized nations are very diverse.In Alabama (USA), during the seven years of the study, the prevalence of Campylobacter spp. in retail broiler meat did not change and was at the level of 41.0% (Williams and Oyarzabal, 2012).Madden et al. (2011) reported, that in the Republic of Ireland, 84.3% of chicken samples were positive for Campylobacter spp.This result is in agreement with that presented by Moran et al. (2009).A year-long survey of fresh poultry products in Northern Ireland showed that 91.0% of chicken samples were contaminated by this pathogen.Luber and Bartelt (2007) obtained similar results.They analysed the prevalence of Campylobacter spp. in 100 German fresh broilers chicken breast, and found that 87% of the filets were Campylobacter-positive on the surface and 20.0% in the deep tissue.The research carried out in Finland by Perko-Mäkelä et al. (2000) and Björkroth (2005) showed that marinating may not have inhibiting effect on the survival of Campylobacter spp.This may be due to the buffering capability of meat quickly neutralizing the pH of the acidic marinade and resulting in dissociation of the lipophilic acids, making their antimicrobial effect invisible.The high occurrence of Campylobacter spp. in retail poultry meat suggests the high prevalence of this microorganism in broilers in farms.However, more research is needed to clarify the cause of the high numbers of this pathogen located in different parts of chicken meat.This kind of data may be useful for future study of risk assessment.

Conclusion
The relatively high contamination of marinated and unmarinated pork and poultry meat with food spoilage microorganisms and pathogens decreases their quality and constitutes a public health hazard, but it is comparable with the meat quality of developed countries.

Table 1 .
Frequency of isolated pathogens from tested meats.

Table 2 .
Microbiological quality of tested meat.