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Evaluation of minimally processed salads commercialized in Portugal
Food Control 23 (2012) 275e281
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Short communication
Evaluation of minimally processed salads commercialized in Portugal M.I. Santos a, b, c, A. Cavaco d, J. Gouveia e, M.R. Novais a, P.J. Nogueira a, L. Pedroso b, c, M.A.S.S. Ferreira d, * a
Instituto Nacional da Saúde Doutor Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal Núcleo de Investigação e Formação em Segurança Alimentar, Instituto Superior de Ciências da Saúde Egas Moniz, Campus Universitário, Quinta da Granja, Monte da Caparica, 2829-511 Caparica, Portugal c Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal d Centro de Botânica Aplicada Agricultura, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal e Laboratório de Estudos Técnicos, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2011 Received in revised form 10 June 2011 Accepted 21 June 2011
During the last decades food borne outbreaks associated with consumption of raw vegetables have been increasing and green leafy vegetables seem to be the most frequently implicated products. In order to determine the microbial quality and the incidence of the major food borne pathogens in Minimally Processed (MP) salads commercialized in Portugal, a survey was conducted in Lisbon Retail Markets, from July 2007 to August 2008, to determine microbial contamination loads and identify potential pathogenic bacteria. A total of 151 samples were purchased from 2 supermarkets: 38 romaine lettuce, 12 various spinach and 101 mixed salads with three or four different ingredients. The samples were tested for aerobic psychotropic micro-organisms (APM), Enterobacteriaceae, Escherichia coli, Listeria spp., presumptive Bacillus cereus, Aeromonas hydrophila and Clostridium perfringens counts as well as for presence of Salmonella spp., Listeria monocytogenes and E. coli O157. Samples were also evaluated for taste quality. The results showed that APM counts of romaine lettuce and mixed salads had a similar median, respectively, 6.2 and 6.5 log cfu/g and mixed spinach had the highest one (7.6 log cfu/g). The median value found for Enterobacteriaceae was 5.44 log cfu/g. Only four samples showed positive result for E. coli (2.65%) but just one (0.8%) had a slightly higher load of contamination. Although the percentage and levels of contamination loads were low, these mustn’t be ignored, for the reason that the strains in three of the samples belong to VTEC group (1.99%). E. coli O157, Salmonella spp. and C. perfringens weren’t detected in any sample. For the enumeration of Listeria spp., two samples (1.32%) had presence of Listeria innocua and L. monocytogenes had an incidence of 0.66%. A. hydrophila was identified in 11 samples (7.28%), and in 8 of them with considerable counts (>105). Contamination with B. cereus was found in 22.7% of samples analyzed, though in small numbers, which doesn’t represent a major concern to food safety. All isolated strains were assessed for its potential toxin production and it was found that 40% of these strains had this ability. Results from sensory panel showed organoleptic differences in salads during its shelf-life period. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Minimally processed salads Bacterial pathogens Sensory analyses Quality evaluation
1. Introduction The dietary habits in the Western world are quite different from those of the first half of the twentieth century. The changes that have occurred in society, related to the phenomenon of globalization have deeply changed the eating habits. Previously the foods
* Corresponding author. Present address: Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749024 Lisboa, Portugal. Tel.: þ351 21 365 3240/3435; fax: þ351 21 365 3238. E-mail address: [email protected] (M.A.S.S. Ferreira). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.06.022
consumed were locally produced, served at the moment, had a low risk of contamination and were healthier (Santos & Campos Cunha, 2007). Nowadays meals aren’t always carried out at home, the consumers can find a wide range of alternatives and very often this kind of food is high in calories. On the other hand, the influence of diet on health is unquestionable and is now universally accepted that fruit and vegetables are essential components of a healthy diet. The insufficient intake of fruits and vegetables is recognized as one of the biggest factors contributing to the increase in chronic noncommunicable diseases worldwide, causing 2.7 million deaths annually. These facts have led the World Health Organization (WHO) and many health authorities in various countries, to
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stimulate the intake of 400 g of vegetables and fruits, equivalent to 5 portions by day (Agudo, 2005; Anonymous, 2005a). The growing consumption trends of raw vegetables for nutritional reasons, associated with the shortest time available, due to the increasingly role of women in the labor market, led to the emergence of a growing industry of MP ready-to-eat vegetables. Such products help to make life easier and the preparation of healthy pleasant and varied meals simpler and permit time gain, reduced waste, since the consumer takes home only the edible part of the product. The consumption of this type of product presents an increasing trend in most countries and Portugal is no exception, since the market of produce reached 16 million Euros, constituting 15% of total sales of fresh products in 2008 (Iberian Salad, personal communication). Produce with high quality and available to consumers throughout the year, comes as a result of improvements in agricultural practices, processing, preservation, distribution and sale but at the same time, these practices also expanded the geographic distribution of food borne outbreaks associated with its consumption (FDA, 1998). According to Beuchat (1998), recognition of fruits and vegetables as vehicles of pathogens is not new. In 1899 a typhoid infection was associated with celery and in 1903 another outbreak of this disease was attributed to the consumption of watercress, grown in soil fertilized with sewage. However, only with the most recent outbreaks associated with consumption of fruits and vegetables it was considered to be an important source of food borne diseases. In recent decades have occurred increasing interests by scientists and health authorities to investigate aspects of food safety related to microbial contamination of fresh produce (Ayers, 2006; Mukherjee, Spee, Jones, Buesing, & Dez-Gonzalez, 2006; Sivapalasingam, Friedman, Cohen, & Tauxe, 2004). This is due to several factors that include changes in agricultural practices, increased consumption of raw or MP vegetables, increasing numbers of immunocompromised consumers and changes in distribution and trade of such products (Beuchat, 2002; Brandl, 2006; Fonseca & Ravishankar, 2007; Tauxe, 1997). It should be noted that these products don’t undergo any heat treatment during production,
processing or preparation, which intensifies the difficulty to eliminate the risk associated with it consumption (FDA, 2001; Tauxe et al., 1997). So it can be pointed out that the great majority of pathogenic organisms have been found in produce (Beuchat, 1996; Nguyen-the & Carlin, 2000). The Food and Agriculture Organization (FAO, 2008) refer outbreaks in several countries of Europe and other regions of the world and we can observe that the green leafy vegetables are the product most frequently implicated in outbreaksand Salmonella and Escherichia coli O157 the most common bacteria in these situations. The aim of this study was to determine the microbial quality and the incidence of the main food borne bacterial pathogens in MP salads commercialized in Portugal.
2. Material and methods 2.1. Samples Individual packs of 100 g MP leafy salads (151 samples) from different producers and three different brands, were purchase in two different supermarkets in Lisbon, Portugal, from July 2007 to August 2008. The samples analyzed include 38 romaine lettuce, 12 various spinach and 101 mixed salads, with three or four different ingredients such as endive, radicchio, canonigo, green lettuce, purple lettuce, arugula, carrot, corn, cabbage, chicory and nuts. The samples were collected on the day of the supermarket stock reposition, from the refrigerator exhibitor displayer. After that were transported in a maximum period of 30 min after acquisition in a cooler box, at temperature between 1 C and 8 C, monitored regularly with a Tomprobe (Ref. MD30100, LaboratoiresÔ AES, Rennes, France). They were analyzed in the same day or the day after always within the sell-by-date period presented on the packaged, in the Microbiology Laboratory of Departamento de Alimentação e Nutrição (DAN) of Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), accredited by NP EN ISO/IEC 17025 (NP, 2005).
Table 1 Methodologies used to determine microbial parameters. Parameter
Method
Description
Psychrotrophic Micro-organisms Enterobacteriaceae
ISO 17410: 2001
Escherichia colia
ISO 16649-2: 2001
Listeria spp.
ISO 11290-2: 1998; Amd 1: 2004
Presumptive Bacillus cereusb Aeromonas hydrophila Clostridium perfringens Salmonella spp.
ISO 7932: 2004
Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Enumeration of Psychrotrophic Micro-organisms Microbiology of Food and Animal Feeding Stuffs e Horizontal Methods for the Detection and Enumeration of Enterobacteriaceae - Part 2: Colony-count Method Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Enumeration of b-glucuronidase-positive Escherichia coli e Part 2: Colony-count Technique at 44 C using 5-bromo-4-chloro-3-indolyl b-D-glucuronide Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Detection and Enumeration of Listeria monocytogenes e Part 2: Enumeration Method. AMENDMENT 1 (2004) Modification of the Isolation Media and the Haemolysis Test, and Inclusion of Precision Data Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Enumeration of Presumptive Bacillus cereus e Colony-count Technique at 30 C Method described in Practical Food Microbiology, pp. 136e138, Blackwell Publishing, Oxford, UK. Microbiology of Food and Animal Feeding Suffs e Horizontal Method for the Enumeration of Clostridium perfringens e Colony-count Technique VIDAS SLM e bioMérieuxÒ SA, Marcy l’Étoile, France (performed according to the manufacter’s instructions) VIDAS LMO2 e bioMérieuxÒ SA, Marcy l’Étoile, France (performed according to the manufacter’s instructions) VIDAS E. coli O157 (ECO) - bioMérieuxÒ SA, Marcy l’Étoile, France (performed according to the manufacter’s instructions)
Listeria monocytogenesc E. coli O157 a
ISO 21528-2: 2004
HPA method ISO 7937: 2004 ELFA ELFA ELFA
E. coli stains from the positive samples were sent to Unidade de Enterobactérias, Instituto Nacional de Saúde for pathogenicity factors study. All B. cereus stains found were tested for toxin production with the kit B. cereus Enterotoxin Test - Reversed Passive Latex Agglutination (BCET-RPLA) (OxoidÔ, Basingstone, Hampshire, UK), according to the manufacter’s instrutions. c To study the genetic profile of L. monocytogenes was performed by Amplified Fragment Length Polymorphism (AFLP), based on selective amplification of DNA fragments previously obtained from the digestion of the whole bacterial genome. b
M.I. Santos et al. / Food Control 23 (2012) 275e281
277
Table 3 Statistics results of sensory analysis of paired samples.
a {1} b {2} c {3} d {4}
{1}
{2}
{3}
2375
4.5
3
4.5
0.104712
0.625733 0.246513
0.104712 1 0.246513
0.104712 0.625733 0.104712
0.246513 1
{4}
0.246513
Tripticase Soya Broth (Becton, Dickinson and Company, Le Pont de Claix, France) supplemented with novobiocin (Sigma, St Louis, Missouri, USA), 20 mg/L, (mTSB þ N) for E. coli O157 detection. 2.3. Sensory analysis
Fig. 1. Results for aerobic psychrotrophic micro-organisms cell counts.
2.2. Microbial analysis On reaching the laboratory, the samples were identified and stored into original package in a refrigerator with continuously monotirized temperature at 3 C 2 C with a Tomprobe, until beginning of analysis. Reception and identification were made by assigning a coded number to samples, to ensure follow-up till the exit of the final result. The following information was registered: product type, purchase date, brand, variety, batch number, expiry date and place of purchase. Microbial parameters were determined using the standard methodologies described in Table 1. All portions were collected, respecting the aseptic rules listed in ISO 7218 (ISO, 2007). Original containers were disinfected with cotton soaked in 70% alcohol and cut with a sterile scissors. After homogenization, 25 g of each sample were diluted 1/10 with Buffered Peptone Water (BPW) (bioMérieuxÒ SA, Marcy l’Étoile, France) using the “Pinch Diluter” (Ref 7577 e bioMérieuxÒ SA, Marcy l’Étoile, France) into sterile stomacher bags (Seward Limited, London, UK). Samples were then homogenized in a StomacherÒ (Model 400 Circulator, Seward Limited, London, UK), during 1 min at normal speed. Serial dilutions of the initial suspension were made in Tryptone Salt (TS) (bioMerieuxÒ SA, Marcy L’Étoile, France) and analyzed for aerobic psychrotrophic micro-organisms (APM), Enterobacteriaceae, Escherichia coli, Listeria spp., Aeromonas hydrophila, Bacillus cereus and Clostridium perfringens. Remaining suspension was incubated at 37 C 1 C, for Salmonella detection. Another 25 g test portion was diluted in 225 ml of Half-Fraser broth (bioMérieuxÒ SA, Marcy l’Étoile, France) for Listeria monocytogenes detection. Finally another 25 g test portion was mixed with Modified
Evaluation of the sensory quality of samples was carried out by a seven member trained sensory panel in Laboratório de Estudos Técnicos (LET), Instituto Superior de Agronomia (ISA), that were asked to evaluate differences in taste of fresh samples and the same type of salad in the last day of shell-by-date, performing a test of paired comparison. Samples were scored by a 10-0 scale were 0 means no differences and 10 means extremely different (Louro and Nunes, 1988). 3. Results and discussion As far as we know this is the first study of this nature performed in our country, so we believed that it allows having a general idea on the microbiological quality of fresh-cut salads commercialized in Portugal. As it can be seen on Fig. 1, APM counts ranged from 4.65 to 8.48 cfu/g, romaine lettuce and mixed salads showed a similar median count (6.2 and 6.5 log cfu/g, respectively), without significant differences (p > 0.05), while mixed spinach had the highest one (7.6 log cfu/g) with a significant difference (p > 0.05). Since the samples were all treated in the same way, these results may be explained by a greater soil contamination. In this study, APM counts at >106 were found in 108 of salads (71.47%) and most samples (41.06%) were between 106 and 107. About 75% of mixed salads and 55% of lettuce samples revealed APM counts >106 cfu/g, while 100% of spinach samples had counts of this order (Table 2). Unlike the APM, the Enterobacteriaceae median from all types of salad doesn’t show significant differences between one another (Fig. 2), in 79
Table 2 Populations of aerobic psychrotrophic microorganism, Enterobacteriaceae, E. coli and Listeria spp. Population (ufc/g) <10 10e102 102103 103104 104105 105106 106107 107108 >108
N (%) of samples APM
ENT
E. coli
Listeria spp.
0 0 0 0
0 0
148 (98.02) 2 (1.32) 1 (0.66) 0 0 0 0 0 0
149 (98.68) 2 (1.32) 0 0 0 0 0 0 0
2 41 62 41 5
(1.32) (27.15) (41.06) (27.15) (3.32)
1 7 29 79 30 5 0
(0.66) (4.64) (19.21) (52.32) (19.87) (3.31)
Fig. 2. Results for Enterobacteriaceae cell counts.
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samples (52.32%), the population was between 105 and 106 cfu/g and 23.18% were above 106. Microbial loads found, namely APM and Enterobacteriaceae tended to be high. Nevertheless, other countries have studies with similar results (Table 4). It should be noted, however, that somehow the comparison of results is only indicative since the methods used between works varied for the same parameter; there was also differences in the type of samples and point of the food chain where the samples were collected. Furthermore, the results may also reflect the great diversity of conditions prevailing during cultivation and post harvest phase. Vegetables are raw products from agricultural sources, therefore are expected to contain micro-organisms, including pathogens. In fact, the contamination of vegetables reflects the micro flora environment in which they are grown (Sagoo, Little, Ward, Gillespie, & Mitchell, 2003a), so the levels of micro-organisms found on vegetables “in natura”, the raw material for MP vegetables, are high. According to Nguyen-the and Carlin (2000), the level of aerobic mesophilic micro-organisms (AMM) in raw vegetables, varies between 102 and 109 which supports Ângelo, Santos, and Cabral (2007) load detected between 105 and 107 on lettuce “in natura”. Studies indicate the presence of Enterobacteriaceae in loads not less than 103 cfu/g, with some samples showing levels of 105 to 106 cfu/g (Litle, Roberts, Youngs, & Louvois, 1999), but Ângelo et al. (2007), found values greater than 104 cfu/g in 60% of their samples. The difficulty is that vegetables are raw agricultural issues, composed by living tissues, and ready-to-eat products (Litle et al., 1999). Additionally, the processing stages of fresh-cut products such as washing, cutting, shredding and slicing are potential sources of contamination (Abadias, Usall, Anguera, Solsona, & Viñas, 2008). As a result, the incidence of micro-organisms in processed vegetables is related with: the hygiene quality of processing steps and microbiological quality of raw products upon arrival at the processing plant (Jay, Loessner, & Golden, 2005). The washing and decontamination process decreased only 1 or 2 log cycles the microbiological population (Beuchat, 1998; Sapers, 2003). It’s important to remember the fact that the microbial populations that survive disinfectants, grow faster than the corresponding populations in non disinfected products and during marketing, consequently, those micro-organisms can grow and reach higher levels than the initial ones (Abadias et al., 2008; Nguyen-the & Carlin, 2000). Nevertheless, high levels of micro-organisms are usual in this kind of products and aren’t indicative of fecal contamination but they can compromise the sensorial and nutritive quality. The results of sensory analysis of our samples (Table 3) allowed concluding, through a test of Least Significance Differences (LSD), that all pairs of salads were considered different, taking into account the average ratings of different tasters. Values obtained for all combinations of pairs, were significant for p > 0.05. Thus, it appears that changes occur in this type of ready-to-eat salads during shelf life. Ragaert, Devlieghere, and Debevere (2007), in a review article, reported that counts exceeding 7e8 log cfu/g, not always produce changes in sensory quality of vegetables MP, but both microbiological and physiological activity play a role in spoilage. In our work, 30.47% of samples showed a APM >107 and some showed signs of flawless texture, color and alterations in taste were recorded by the tasting panel. It is worth mentioning that the International Commission on Microbiological Specifications for Foods (ICMSF) states that foods with the presence of aerobic micro-organisms, above 106 cfu/g, do shows apparent decomposition by smell, taste and appearance (ICMSF, 1988). Regarding the indicator E. coli, is considered the best indicator of fecal contamination, which could explain their inclusion in the process hygiene criteria for these products by European legislation
(Anonymous, 2005b, 2007). And once the fecal matter may contain pathogenic micro-organisms of enteric origin, is extremely important that the contamination with E. coli be kept to a minimum. Generally, if we consider the scientific literature, it has been found in low percentages and when present in samples is at low levels (Table 4). The present work confirmed this trend, since only four samples were found positive (2.65%) and only three showed a level above the detection limit of the method (<10 cfu/g). Two of these mixed salads, showed a level of contamination very low (1.0 101 and 2.0 101), which according to ISO 7218 (ISO, 2007), is considered only as presence. But the 3rd sample of mixed spinach, showed a slightly higher level (2.2 102), exceeding the hygienic criteria established by European legislation (Anonymous, 2005b, 2007) and a fourth sample, was positive in the enrichment step for the detection of E. coli O157:H7. The percentage and levels of contamination found, although low shouldn’t be overlooked because the strains present in three samples (1 of mixed salads and 2 of mixed spinach), belonged to the VTEC group (1, 99%). Abadias et al. (2008), found a percentage of positive samples much higher than ours but the discrepancy may be explained by the fact that these researchers have used the most probable number (MPN) method, which implies an enrichment step, allowing detection of lower levels. Ours previous study in Portugal, performed with 50 (25 before sanitation and 25 ready-to-eat) samples of salad, revealed 5 positive samples, collected from a mass catering, (Ângelo et al., 2007). E. coli O157, Salmonella spp. and C. perfringens weren’t detected in any of our samples. In what concerns E. coli O157:H7, none of the samples showed positive results and this is consistent with many other published (Table 4). Similarly, in a surveillance study carrying out with 1028 samples of fruits and vegetables, none of them tested positive for this organism (FDA, 2003). However, this bacterium has been the source of numerous food borne outbreaks associated produce (Ayers, 2006; DeWaal & Bhuiya, 2007; EFSA, 2006; Hallman, Cuite, Nucci, Pleasant, & Chess, 2008; Itoh et al., 1998; Kasuga, 1999; Meng, Doyle, Zhaot, & Zhaot, 2007; Tauxe, 2008). Likewise, none of samples revealed the presence of Salmonella spp. and if we compare with others, can be observed that many have reached the same conclusion (Table 4). A surveillance work carried out in 2003, found a positive sample of lettuce in 142 tested (0.70%) (FDA, 2003). But Al-Mohizea (1996) found higher values, which may reflect the increase in Good Agricultural Practices (GAP) and Good Hygiene Practices (GHP) occurred since that time. For the enumeration of Listeria spp., indicator of possible presence of L. monocytogenes, only two samples (1.32%), one of mixed salads and another of mixed spinach, revealed the presence of Listeria innocua (1.0 102 and 2.0 102 respectively). Since the detection limit of the method used was 102, it is possible that other samples have proved to be positive with a lower limit of detection. L. monocytogenes was found in only 1 of our 151 samples, which was a sample of mixed spinach, in a level of <102, representing an incidence of 0.66% and is in compliance with the European legislation (Anonymous, 2007, 2005b; Luber, 2010). This isolate was confirmed by AFLP and as for the aforementioned pathogens in this case the results are in agreement with those found by other authors (Table 4). Liao and Fett (2001) have shown that some species of microorganisms isolated from plants, which includes P. fluorescens, Bacillus mojavensis, Bacillus megaterium and a yeast (unidentified species) are antagonists to pathogenic micro-organisms such as L. monocytogenes and Salmonella. As Pseudomonas spp. and Bacillus spp. were some of the genus of bacteria found in this study, we can infer that its presence may contribute to the low prevalence of L. monocytogenes and Salmonella spp. we found. Another pathogenic microorganism that we assessed was A. hydrophila. In 11 positive samples this strain was identified by
Table 4 Percentage of species and microbial groups found in produce of other countries. Authors
Arthur, Jones, Fabri, and Odumeru (2007) Sagoo, Little, Ward et al. (2003a) Sagoo, Little, and Mitchell (2003b) Sagoo, Little, and Mitchell (2001) Guerra, McLauchlin, and Bernardo (2001) 37 samples Ângelo et al. (2007) Bohaychuk et al. (2009) Al-Mohizea, 1996
APM/AMM
Enterobacteriaceae NP e not performed
E. coli
106
>105<106
106
MPN
<10
>10<103
>103<105
spp.
monocytogenes
MP Saladas (133e181) MP cut vegetables (236) Vegetables in natura (106) Lettuce in natura (90)
51.1 89.9 e e
42.1 33.9 NP NP
16.5 26.3 e e
NP 11.4 e e
NP NP e e
e e 19 3.3e20
e e 81 e
NP NP NP NP
0.6 0.7 NP NP
Bagged and loose saladas (297) Whole lettuce (357)
59.26
16.5
29.13
1.96
Listeria
7.74
e
0
e
100
e
90.24
6.73
3.03
Salmonella
E. coli O157
3.0 1.3 0 NP
NP NP NP N^P
0.98
0.68
0
NP
e
0
0
0
NP 0
NP
NP
e
e
100
e
e
NP
0.5
0
91.5
NP
e
e
NP
e
e
2.7
0.9
0.67
NP
NP
e
e
e
e
NP
Bagged salad (3852)
NP
13.68
2.77
e
98.42
1.22
0.13
4.47
Open, prepared ready-to-eat salads (2950) Ready-to-eat organic vegetables (3200) Ready-to-eat vegetables (23)
NP
11.32
3.59
e
93.1
5.76
0.94
NP
NP
e
e
1.2
0.3
NP
NP
e
e
NP
22.0 NP
12.0 NP
8.0 e
e 8.2
NP
NP
e
e
Lettuce (200) and pre-cut salads (100) Fresh produce (445) (including 159 MP salads) Lettuce (530)
Salads in natura (50) Fruiys and vagetables (644e673) Lettuce (68)
5.7
NP
0.17
0
2.34
0.13
0
4.3
3.0
0
0
e
0.2
0
0
0
e
e
4
0
NP
NP
90.0 e
10.0 e
0 e
ND ND
2.0 ND
0 0
ND 0
NP
e
e
e
12.0
6.0
e
M.I. Santos et al. / Food Control 23 (2012) 275e281
Froder et al. (2007) Abadias et al. (2008) Erkan and Vural (2008) Aycicek, Oguz, and Karci (2006) Williamson, Allen, and Bolton (2006) Williamson, Allen, and Bolton (2003) Johannessen, Loncareviv, and Kruse (2002) Badosa et al. (2008)
Products (n of samples)
279
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M.I. Santos et al. / Food Control 23 (2012) 275e281
biochemical tests (API 20E bioMérieuxÒ SA, Marcy l’Étoile, France) as A. hydrophila/caviae/sobria (Good ID% of ID > 98%) which corresponds to 7.3%. However, because of the culture medium used, although reported in the literature as suitable for this enumeration, hasn’t proved to be very inhibitory to the background flora and most plates were full of colonies morphologically very similar, therefore it was very difficult to distinguish suspect colonies. It is possible that this organism was present in many other samples but it was impossible to be identified, although the reading was always done by comparison to a control strain, incubated under the same conditions. This microorganism counts ranged between 3.15 and >5.18 log cfu/g and in 8 samples the level found was >105. Since this is a pathogenic microorganism and some strains can produce Shiga-like toxins, these scores are considered very high. Xanthopoulos, Tzanetakis, and Litopoulou-Tzanetaki (2009), tested in duplicate 26 samples of vegetables MP, in a much smaller assay, but using an enrichment method, refers 46.1% of positivity, higher than the one found in this work and proved the idea that it wasn’t possible to isolate this organism in many samples. A work with 12 samples of leafy vegetables “in natura”, done in duplicate, by Calister and Agger (1987), verified that all samples, except one replicate of chicory, proved to be positive for Aeromonas spp. The samples we analyzed for B. cereus, 15 out of 66 (22.70%) showed to be contaminated. Curiously the great majority was from the same brand (6 mixed spinach and 8 mixed salads) and just 1 sample of mixed salads was from a different brand. It was observed, by analyzing the results, that this organism was present in small numbers, between <2.0 and 3.20 log cfu/g, showing no meaning in terms of food safety. Nevertheless all the strains isolated, were assessed for their potential Hbl enterotoxin production and it was found that 6 strains (40%) had this ability. 4. Conclusion As a conclusion we may say that Enterobacteriacea are considered normal flora in this type of products, and therefore have proven not to be an indicator of fecal and pathogenic contamination of salads MP, as pointed the results of E. coli and pathogens reveled in this work. The high level we found, shows a very decayed hygienic conditions and is probably responsible for the deterioration of sensory quality and the flawless aspect presented by this salads. Nevertheless, it might have occurred an antagonist effect against the pathogenic micro-organisms that might support the very low contamination level detected. We can say after all, that the incidence of pathogenic microorganisms found was very low. Only the incidence of A. hydrophila was high, however the association of this microorganism to food borne outbreaks remains unclear. It is important to note that international data also point to a low prevalence of pathogenic micro-organisms, however, despite this, there have been food borne outbreaks associated to produce. The existing information shows that the pathogens are opportunistic contaminants that can occur sporadically in fruits and vegetables and consequently, these products may pose a risk to the consumers’ health. A proper assessment of risk is limited due to the infrequent nature of this occurrence (Monaghan, Thomas, Goodburn, & Hutchison, 2009). Most reported outbreaks found that the source of infection occurred in the field, during cultivation, thus making it very important to a strict observance of Good Agricultural Practices (GAP). Moreover, during transport and processing in plant, it is important to adhere to Good Manufacturing Practices (GMP) and GHP to avoid cross-contamination. These products are only subject to a disinfection step that doesn’t ensure the elimination of pathogens. Keeping the product under refrigeration by distributors, retailers and consumers, is another important factor. In addition,
researchers should continue to develop new methods of disinfection to remove micro-organisms and to assay new preservatives effective in reduce the microbial growth along storage.
Acknowledgments We would like to acknowledge the two supermarkets for providing some salad samples used in this work. This research is part of M.I. Santos Ms.C. theses and was funded by Centro de Botanica Aplicada a Agricultura and Instituto Nacional de Saúde Dr. Ricardo Jorge.
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Evaluation of minimally processed salads commercialized in Portugal M.I. Santos a, b, c, A. Cavaco d, J. Gouveia e, M.R. Novais a, P.J. Nogueira a, L. Pedroso b, c, M.A.S.S. Ferreira d, * a
Instituto Nacional da Saúde Doutor Ricardo Jorge, Av. Padre Cruz, 1649-016 Lisboa, Portugal Núcleo de Investigação e Formação em Segurança Alimentar, Instituto Superior de Ciências da Saúde Egas Moniz, Campus Universitário, Quinta da Granja, Monte da Caparica, 2829-511 Caparica, Portugal c Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749-024 Lisboa, Portugal d Centro de Botânica Aplicada Agricultura, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal e Laboratório de Estudos Técnicos, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, Tapada da Ajuda, 1349-017 Lisboa, Portugal b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 January 2011 Received in revised form 10 June 2011 Accepted 21 June 2011
During the last decades food borne outbreaks associated with consumption of raw vegetables have been increasing and green leafy vegetables seem to be the most frequently implicated products. In order to determine the microbial quality and the incidence of the major food borne pathogens in Minimally Processed (MP) salads commercialized in Portugal, a survey was conducted in Lisbon Retail Markets, from July 2007 to August 2008, to determine microbial contamination loads and identify potential pathogenic bacteria. A total of 151 samples were purchased from 2 supermarkets: 38 romaine lettuce, 12 various spinach and 101 mixed salads with three or four different ingredients. The samples were tested for aerobic psychotropic micro-organisms (APM), Enterobacteriaceae, Escherichia coli, Listeria spp., presumptive Bacillus cereus, Aeromonas hydrophila and Clostridium perfringens counts as well as for presence of Salmonella spp., Listeria monocytogenes and E. coli O157. Samples were also evaluated for taste quality. The results showed that APM counts of romaine lettuce and mixed salads had a similar median, respectively, 6.2 and 6.5 log cfu/g and mixed spinach had the highest one (7.6 log cfu/g). The median value found for Enterobacteriaceae was 5.44 log cfu/g. Only four samples showed positive result for E. coli (2.65%) but just one (0.8%) had a slightly higher load of contamination. Although the percentage and levels of contamination loads were low, these mustn’t be ignored, for the reason that the strains in three of the samples belong to VTEC group (1.99%). E. coli O157, Salmonella spp. and C. perfringens weren’t detected in any sample. For the enumeration of Listeria spp., two samples (1.32%) had presence of Listeria innocua and L. monocytogenes had an incidence of 0.66%. A. hydrophila was identified in 11 samples (7.28%), and in 8 of them with considerable counts (>105). Contamination with B. cereus was found in 22.7% of samples analyzed, though in small numbers, which doesn’t represent a major concern to food safety. All isolated strains were assessed for its potential toxin production and it was found that 40% of these strains had this ability. Results from sensory panel showed organoleptic differences in salads during its shelf-life period. Ó 2011 Elsevier Ltd. All rights reserved.
Keywords: Minimally processed salads Bacterial pathogens Sensory analyses Quality evaluation
1. Introduction The dietary habits in the Western world are quite different from those of the first half of the twentieth century. The changes that have occurred in society, related to the phenomenon of globalization have deeply changed the eating habits. Previously the foods
* Corresponding author. Present address: Faculdade de Medicina Veterinária, Universidade Lusófona de Humanidades e Tecnologias, Campo Grande 376, 1749024 Lisboa, Portugal. Tel.: þ351 21 365 3240/3435; fax: þ351 21 365 3238. E-mail address: [email protected] (M.A.S.S. Ferreira). 0956-7135/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2011.06.022
consumed were locally produced, served at the moment, had a low risk of contamination and were healthier (Santos & Campos Cunha, 2007). Nowadays meals aren’t always carried out at home, the consumers can find a wide range of alternatives and very often this kind of food is high in calories. On the other hand, the influence of diet on health is unquestionable and is now universally accepted that fruit and vegetables are essential components of a healthy diet. The insufficient intake of fruits and vegetables is recognized as one of the biggest factors contributing to the increase in chronic noncommunicable diseases worldwide, causing 2.7 million deaths annually. These facts have led the World Health Organization (WHO) and many health authorities in various countries, to
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stimulate the intake of 400 g of vegetables and fruits, equivalent to 5 portions by day (Agudo, 2005; Anonymous, 2005a). The growing consumption trends of raw vegetables for nutritional reasons, associated with the shortest time available, due to the increasingly role of women in the labor market, led to the emergence of a growing industry of MP ready-to-eat vegetables. Such products help to make life easier and the preparation of healthy pleasant and varied meals simpler and permit time gain, reduced waste, since the consumer takes home only the edible part of the product. The consumption of this type of product presents an increasing trend in most countries and Portugal is no exception, since the market of produce reached 16 million Euros, constituting 15% of total sales of fresh products in 2008 (Iberian Salad, personal communication). Produce with high quality and available to consumers throughout the year, comes as a result of improvements in agricultural practices, processing, preservation, distribution and sale but at the same time, these practices also expanded the geographic distribution of food borne outbreaks associated with its consumption (FDA, 1998). According to Beuchat (1998), recognition of fruits and vegetables as vehicles of pathogens is not new. In 1899 a typhoid infection was associated with celery and in 1903 another outbreak of this disease was attributed to the consumption of watercress, grown in soil fertilized with sewage. However, only with the most recent outbreaks associated with consumption of fruits and vegetables it was considered to be an important source of food borne diseases. In recent decades have occurred increasing interests by scientists and health authorities to investigate aspects of food safety related to microbial contamination of fresh produce (Ayers, 2006; Mukherjee, Spee, Jones, Buesing, & Dez-Gonzalez, 2006; Sivapalasingam, Friedman, Cohen, & Tauxe, 2004). This is due to several factors that include changes in agricultural practices, increased consumption of raw or MP vegetables, increasing numbers of immunocompromised consumers and changes in distribution and trade of such products (Beuchat, 2002; Brandl, 2006; Fonseca & Ravishankar, 2007; Tauxe, 1997). It should be noted that these products don’t undergo any heat treatment during production,
processing or preparation, which intensifies the difficulty to eliminate the risk associated with it consumption (FDA, 2001; Tauxe et al., 1997). So it can be pointed out that the great majority of pathogenic organisms have been found in produce (Beuchat, 1996; Nguyen-the & Carlin, 2000). The Food and Agriculture Organization (FAO, 2008) refer outbreaks in several countries of Europe and other regions of the world and we can observe that the green leafy vegetables are the product most frequently implicated in outbreaksand Salmonella and Escherichia coli O157 the most common bacteria in these situations. The aim of this study was to determine the microbial quality and the incidence of the main food borne bacterial pathogens in MP salads commercialized in Portugal.
2. Material and methods 2.1. Samples Individual packs of 100 g MP leafy salads (151 samples) from different producers and three different brands, were purchase in two different supermarkets in Lisbon, Portugal, from July 2007 to August 2008. The samples analyzed include 38 romaine lettuce, 12 various spinach and 101 mixed salads, with three or four different ingredients such as endive, radicchio, canonigo, green lettuce, purple lettuce, arugula, carrot, corn, cabbage, chicory and nuts. The samples were collected on the day of the supermarket stock reposition, from the refrigerator exhibitor displayer. After that were transported in a maximum period of 30 min after acquisition in a cooler box, at temperature between 1 C and 8 C, monitored regularly with a Tomprobe (Ref. MD30100, LaboratoiresÔ AES, Rennes, France). They were analyzed in the same day or the day after always within the sell-by-date period presented on the packaged, in the Microbiology Laboratory of Departamento de Alimentação e Nutrição (DAN) of Instituto Nacional de Saúde Doutor Ricardo Jorge (INSA), accredited by NP EN ISO/IEC 17025 (NP, 2005).
Table 1 Methodologies used to determine microbial parameters. Parameter
Method
Description
Psychrotrophic Micro-organisms Enterobacteriaceae
ISO 17410: 2001
Escherichia colia
ISO 16649-2: 2001
Listeria spp.
ISO 11290-2: 1998; Amd 1: 2004
Presumptive Bacillus cereusb Aeromonas hydrophila Clostridium perfringens Salmonella spp.
ISO 7932: 2004
Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Enumeration of Psychrotrophic Micro-organisms Microbiology of Food and Animal Feeding Stuffs e Horizontal Methods for the Detection and Enumeration of Enterobacteriaceae - Part 2: Colony-count Method Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Enumeration of b-glucuronidase-positive Escherichia coli e Part 2: Colony-count Technique at 44 C using 5-bromo-4-chloro-3-indolyl b-D-glucuronide Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Detection and Enumeration of Listeria monocytogenes e Part 2: Enumeration Method. AMENDMENT 1 (2004) Modification of the Isolation Media and the Haemolysis Test, and Inclusion of Precision Data Microbiology of Food and Animal Feeding Stuffs e Horizontal Method for the Enumeration of Presumptive Bacillus cereus e Colony-count Technique at 30 C Method described in Practical Food Microbiology, pp. 136e138, Blackwell Publishing, Oxford, UK. Microbiology of Food and Animal Feeding Suffs e Horizontal Method for the Enumeration of Clostridium perfringens e Colony-count Technique VIDAS SLM e bioMérieuxÒ SA, Marcy l’Étoile, France (performed according to the manufacter’s instructions) VIDAS LMO2 e bioMérieuxÒ SA, Marcy l’Étoile, France (performed according to the manufacter’s instructions) VIDAS E. coli O157 (ECO) - bioMérieuxÒ SA, Marcy l’Étoile, France (performed according to the manufacter’s instructions)
Listeria monocytogenesc E. coli O157 a
ISO 21528-2: 2004
HPA method ISO 7937: 2004 ELFA ELFA ELFA
E. coli stains from the positive samples were sent to Unidade de Enterobactérias, Instituto Nacional de Saúde for pathogenicity factors study. All B. cereus stains found were tested for toxin production with the kit B. cereus Enterotoxin Test - Reversed Passive Latex Agglutination (BCET-RPLA) (OxoidÔ, Basingstone, Hampshire, UK), according to the manufacter’s instrutions. c To study the genetic profile of L. monocytogenes was performed by Amplified Fragment Length Polymorphism (AFLP), based on selective amplification of DNA fragments previously obtained from the digestion of the whole bacterial genome. b
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277
Table 3 Statistics results of sensory analysis of paired samples.
a {1} b {2} c {3} d {4}
{1}
{2}
{3}
2375
4.5
3
4.5
0.104712
0.625733 0.246513
0.104712 1 0.246513
0.104712 0.625733 0.104712
0.246513 1
{4}
0.246513
Tripticase Soya Broth (Becton, Dickinson and Company, Le Pont de Claix, France) supplemented with novobiocin (Sigma, St Louis, Missouri, USA), 20 mg/L, (mTSB þ N) for E. coli O157 detection. 2.3. Sensory analysis
Fig. 1. Results for aerobic psychrotrophic micro-organisms cell counts.
2.2. Microbial analysis On reaching the laboratory, the samples were identified and stored into original package in a refrigerator with continuously monotirized temperature at 3 C 2 C with a Tomprobe, until beginning of analysis. Reception and identification were made by assigning a coded number to samples, to ensure follow-up till the exit of the final result. The following information was registered: product type, purchase date, brand, variety, batch number, expiry date and place of purchase. Microbial parameters were determined using the standard methodologies described in Table 1. All portions were collected, respecting the aseptic rules listed in ISO 7218 (ISO, 2007). Original containers were disinfected with cotton soaked in 70% alcohol and cut with a sterile scissors. After homogenization, 25 g of each sample were diluted 1/10 with Buffered Peptone Water (BPW) (bioMérieuxÒ SA, Marcy l’Étoile, France) using the “Pinch Diluter” (Ref 7577 e bioMérieuxÒ SA, Marcy l’Étoile, France) into sterile stomacher bags (Seward Limited, London, UK). Samples were then homogenized in a StomacherÒ (Model 400 Circulator, Seward Limited, London, UK), during 1 min at normal speed. Serial dilutions of the initial suspension were made in Tryptone Salt (TS) (bioMerieuxÒ SA, Marcy L’Étoile, France) and analyzed for aerobic psychrotrophic micro-organisms (APM), Enterobacteriaceae, Escherichia coli, Listeria spp., Aeromonas hydrophila, Bacillus cereus and Clostridium perfringens. Remaining suspension was incubated at 37 C 1 C, for Salmonella detection. Another 25 g test portion was diluted in 225 ml of Half-Fraser broth (bioMérieuxÒ SA, Marcy l’Étoile, France) for Listeria monocytogenes detection. Finally another 25 g test portion was mixed with Modified
Evaluation of the sensory quality of samples was carried out by a seven member trained sensory panel in Laboratório de Estudos Técnicos (LET), Instituto Superior de Agronomia (ISA), that were asked to evaluate differences in taste of fresh samples and the same type of salad in the last day of shell-by-date, performing a test of paired comparison. Samples were scored by a 10-0 scale were 0 means no differences and 10 means extremely different (Louro and Nunes, 1988). 3. Results and discussion As far as we know this is the first study of this nature performed in our country, so we believed that it allows having a general idea on the microbiological quality of fresh-cut salads commercialized in Portugal. As it can be seen on Fig. 1, APM counts ranged from 4.65 to 8.48 cfu/g, romaine lettuce and mixed salads showed a similar median count (6.2 and 6.5 log cfu/g, respectively), without significant differences (p > 0.05), while mixed spinach had the highest one (7.6 log cfu/g) with a significant difference (p > 0.05). Since the samples were all treated in the same way, these results may be explained by a greater soil contamination. In this study, APM counts at >106 were found in 108 of salads (71.47%) and most samples (41.06%) were between 106 and 107. About 75% of mixed salads and 55% of lettuce samples revealed APM counts >106 cfu/g, while 100% of spinach samples had counts of this order (Table 2). Unlike the APM, the Enterobacteriaceae median from all types of salad doesn’t show significant differences between one another (Fig. 2), in 79
Table 2 Populations of aerobic psychrotrophic microorganism, Enterobacteriaceae, E. coli and Listeria spp. Population (ufc/g) <10 10e102 102103 103104 104105 105106 106107 107108 >108
N (%) of samples APM
ENT
E. coli
Listeria spp.
0 0 0 0
0 0
148 (98.02) 2 (1.32) 1 (0.66) 0 0 0 0 0 0
149 (98.68) 2 (1.32) 0 0 0 0 0 0 0
2 41 62 41 5
(1.32) (27.15) (41.06) (27.15) (3.32)
1 7 29 79 30 5 0
(0.66) (4.64) (19.21) (52.32) (19.87) (3.31)
Fig. 2. Results for Enterobacteriaceae cell counts.
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samples (52.32%), the population was between 105 and 106 cfu/g and 23.18% were above 106. Microbial loads found, namely APM and Enterobacteriaceae tended to be high. Nevertheless, other countries have studies with similar results (Table 4). It should be noted, however, that somehow the comparison of results is only indicative since the methods used between works varied for the same parameter; there was also differences in the type of samples and point of the food chain where the samples were collected. Furthermore, the results may also reflect the great diversity of conditions prevailing during cultivation and post harvest phase. Vegetables are raw products from agricultural sources, therefore are expected to contain micro-organisms, including pathogens. In fact, the contamination of vegetables reflects the micro flora environment in which they are grown (Sagoo, Little, Ward, Gillespie, & Mitchell, 2003a), so the levels of micro-organisms found on vegetables “in natura”, the raw material for MP vegetables, are high. According to Nguyen-the and Carlin (2000), the level of aerobic mesophilic micro-organisms (AMM) in raw vegetables, varies between 102 and 109 which supports Ângelo, Santos, and Cabral (2007) load detected between 105 and 107 on lettuce “in natura”. Studies indicate the presence of Enterobacteriaceae in loads not less than 103 cfu/g, with some samples showing levels of 105 to 106 cfu/g (Litle, Roberts, Youngs, & Louvois, 1999), but Ângelo et al. (2007), found values greater than 104 cfu/g in 60% of their samples. The difficulty is that vegetables are raw agricultural issues, composed by living tissues, and ready-to-eat products (Litle et al., 1999). Additionally, the processing stages of fresh-cut products such as washing, cutting, shredding and slicing are potential sources of contamination (Abadias, Usall, Anguera, Solsona, & Viñas, 2008). As a result, the incidence of micro-organisms in processed vegetables is related with: the hygiene quality of processing steps and microbiological quality of raw products upon arrival at the processing plant (Jay, Loessner, & Golden, 2005). The washing and decontamination process decreased only 1 or 2 log cycles the microbiological population (Beuchat, 1998; Sapers, 2003). It’s important to remember the fact that the microbial populations that survive disinfectants, grow faster than the corresponding populations in non disinfected products and during marketing, consequently, those micro-organisms can grow and reach higher levels than the initial ones (Abadias et al., 2008; Nguyen-the & Carlin, 2000). Nevertheless, high levels of micro-organisms are usual in this kind of products and aren’t indicative of fecal contamination but they can compromise the sensorial and nutritive quality. The results of sensory analysis of our samples (Table 3) allowed concluding, through a test of Least Significance Differences (LSD), that all pairs of salads were considered different, taking into account the average ratings of different tasters. Values obtained for all combinations of pairs, were significant for p > 0.05. Thus, it appears that changes occur in this type of ready-to-eat salads during shelf life. Ragaert, Devlieghere, and Debevere (2007), in a review article, reported that counts exceeding 7e8 log cfu/g, not always produce changes in sensory quality of vegetables MP, but both microbiological and physiological activity play a role in spoilage. In our work, 30.47% of samples showed a APM >107 and some showed signs of flawless texture, color and alterations in taste were recorded by the tasting panel. It is worth mentioning that the International Commission on Microbiological Specifications for Foods (ICMSF) states that foods with the presence of aerobic micro-organisms, above 106 cfu/g, do shows apparent decomposition by smell, taste and appearance (ICMSF, 1988). Regarding the indicator E. coli, is considered the best indicator of fecal contamination, which could explain their inclusion in the process hygiene criteria for these products by European legislation
(Anonymous, 2005b, 2007). And once the fecal matter may contain pathogenic micro-organisms of enteric origin, is extremely important that the contamination with E. coli be kept to a minimum. Generally, if we consider the scientific literature, it has been found in low percentages and when present in samples is at low levels (Table 4). The present work confirmed this trend, since only four samples were found positive (2.65%) and only three showed a level above the detection limit of the method (<10 cfu/g). Two of these mixed salads, showed a level of contamination very low (1.0 101 and 2.0 101), which according to ISO 7218 (ISO, 2007), is considered only as presence. But the 3rd sample of mixed spinach, showed a slightly higher level (2.2 102), exceeding the hygienic criteria established by European legislation (Anonymous, 2005b, 2007) and a fourth sample, was positive in the enrichment step for the detection of E. coli O157:H7. The percentage and levels of contamination found, although low shouldn’t be overlooked because the strains present in three samples (1 of mixed salads and 2 of mixed spinach), belonged to the VTEC group (1, 99%). Abadias et al. (2008), found a percentage of positive samples much higher than ours but the discrepancy may be explained by the fact that these researchers have used the most probable number (MPN) method, which implies an enrichment step, allowing detection of lower levels. Ours previous study in Portugal, performed with 50 (25 before sanitation and 25 ready-to-eat) samples of salad, revealed 5 positive samples, collected from a mass catering, (Ângelo et al., 2007). E. coli O157, Salmonella spp. and C. perfringens weren’t detected in any of our samples. In what concerns E. coli O157:H7, none of the samples showed positive results and this is consistent with many other published (Table 4). Similarly, in a surveillance study carrying out with 1028 samples of fruits and vegetables, none of them tested positive for this organism (FDA, 2003). However, this bacterium has been the source of numerous food borne outbreaks associated produce (Ayers, 2006; DeWaal & Bhuiya, 2007; EFSA, 2006; Hallman, Cuite, Nucci, Pleasant, & Chess, 2008; Itoh et al., 1998; Kasuga, 1999; Meng, Doyle, Zhaot, & Zhaot, 2007; Tauxe, 2008). Likewise, none of samples revealed the presence of Salmonella spp. and if we compare with others, can be observed that many have reached the same conclusion (Table 4). A surveillance work carried out in 2003, found a positive sample of lettuce in 142 tested (0.70%) (FDA, 2003). But Al-Mohizea (1996) found higher values, which may reflect the increase in Good Agricultural Practices (GAP) and Good Hygiene Practices (GHP) occurred since that time. For the enumeration of Listeria spp., indicator of possible presence of L. monocytogenes, only two samples (1.32%), one of mixed salads and another of mixed spinach, revealed the presence of Listeria innocua (1.0 102 and 2.0 102 respectively). Since the detection limit of the method used was 102, it is possible that other samples have proved to be positive with a lower limit of detection. L. monocytogenes was found in only 1 of our 151 samples, which was a sample of mixed spinach, in a level of <102, representing an incidence of 0.66% and is in compliance with the European legislation (Anonymous, 2007, 2005b; Luber, 2010). This isolate was confirmed by AFLP and as for the aforementioned pathogens in this case the results are in agreement with those found by other authors (Table 4). Liao and Fett (2001) have shown that some species of microorganisms isolated from plants, which includes P. fluorescens, Bacillus mojavensis, Bacillus megaterium and a yeast (unidentified species) are antagonists to pathogenic micro-organisms such as L. monocytogenes and Salmonella. As Pseudomonas spp. and Bacillus spp. were some of the genus of bacteria found in this study, we can infer that its presence may contribute to the low prevalence of L. monocytogenes and Salmonella spp. we found. Another pathogenic microorganism that we assessed was A. hydrophila. In 11 positive samples this strain was identified by
Table 4 Percentage of species and microbial groups found in produce of other countries. Authors
Arthur, Jones, Fabri, and Odumeru (2007) Sagoo, Little, Ward et al. (2003a) Sagoo, Little, and Mitchell (2003b) Sagoo, Little, and Mitchell (2001) Guerra, McLauchlin, and Bernardo (2001) 37 samples Ângelo et al. (2007) Bohaychuk et al. (2009) Al-Mohizea, 1996
APM/AMM
Enterobacteriaceae NP e not performed
E. coli
106
>105<106
106
MPN
<10
>10<103
>103<105
spp.
monocytogenes
MP Saladas (133e181) MP cut vegetables (236) Vegetables in natura (106) Lettuce in natura (90)
51.1 89.9 e e
42.1 33.9 NP NP
16.5 26.3 e e
NP 11.4 e e
NP NP e e
e e 19 3.3e20
e e 81 e
NP NP NP NP
0.6 0.7 NP NP
Bagged and loose saladas (297) Whole lettuce (357)
59.26
16.5
29.13
1.96
Listeria
7.74
e
0
e
100
e
90.24
6.73
3.03
Salmonella
E. coli O157
3.0 1.3 0 NP
NP NP NP N^P
0.98
0.68
0
NP
e
0
0
0
NP 0
NP
NP
e
e
100
e
e
NP
0.5
0
91.5
NP
e
e
NP
e
e
2.7
0.9
0.67
NP
NP
e
e
e
e
NP
Bagged salad (3852)
NP
13.68
2.77
e
98.42
1.22
0.13
4.47
Open, prepared ready-to-eat salads (2950) Ready-to-eat organic vegetables (3200) Ready-to-eat vegetables (23)
NP
11.32
3.59
e
93.1
5.76
0.94
NP
NP
e
e
1.2
0.3
NP
NP
e
e
NP
22.0 NP
12.0 NP
8.0 e
e 8.2
NP
NP
e
e
Lettuce (200) and pre-cut salads (100) Fresh produce (445) (including 159 MP salads) Lettuce (530)
Salads in natura (50) Fruiys and vagetables (644e673) Lettuce (68)
5.7
NP
0.17
0
2.34
0.13
0
4.3
3.0
0
0
e
0.2
0
0
0
e
e
4
0
NP
NP
90.0 e
10.0 e
0 e
ND ND
2.0 ND
0 0
ND 0
NP
e
e
e
12.0
6.0
e
M.I. Santos et al. / Food Control 23 (2012) 275e281
Froder et al. (2007) Abadias et al. (2008) Erkan and Vural (2008) Aycicek, Oguz, and Karci (2006) Williamson, Allen, and Bolton (2006) Williamson, Allen, and Bolton (2003) Johannessen, Loncareviv, and Kruse (2002) Badosa et al. (2008)
Products (n of samples)
279
280
M.I. Santos et al. / Food Control 23 (2012) 275e281
biochemical tests (API 20E bioMérieuxÒ SA, Marcy l’Étoile, France) as A. hydrophila/caviae/sobria (Good ID% of ID > 98%) which corresponds to 7.3%. However, because of the culture medium used, although reported in the literature as suitable for this enumeration, hasn’t proved to be very inhibitory to the background flora and most plates were full of colonies morphologically very similar, therefore it was very difficult to distinguish suspect colonies. It is possible that this organism was present in many other samples but it was impossible to be identified, although the reading was always done by comparison to a control strain, incubated under the same conditions. This microorganism counts ranged between 3.15 and >5.18 log cfu/g and in 8 samples the level found was >105. Since this is a pathogenic microorganism and some strains can produce Shiga-like toxins, these scores are considered very high. Xanthopoulos, Tzanetakis, and Litopoulou-Tzanetaki (2009), tested in duplicate 26 samples of vegetables MP, in a much smaller assay, but using an enrichment method, refers 46.1% of positivity, higher than the one found in this work and proved the idea that it wasn’t possible to isolate this organism in many samples. A work with 12 samples of leafy vegetables “in natura”, done in duplicate, by Calister and Agger (1987), verified that all samples, except one replicate of chicory, proved to be positive for Aeromonas spp. The samples we analyzed for B. cereus, 15 out of 66 (22.70%) showed to be contaminated. Curiously the great majority was from the same brand (6 mixed spinach and 8 mixed salads) and just 1 sample of mixed salads was from a different brand. It was observed, by analyzing the results, that this organism was present in small numbers, between <2.0 and 3.20 log cfu/g, showing no meaning in terms of food safety. Nevertheless all the strains isolated, were assessed for their potential Hbl enterotoxin production and it was found that 6 strains (40%) had this ability. 4. Conclusion As a conclusion we may say that Enterobacteriacea are considered normal flora in this type of products, and therefore have proven not to be an indicator of fecal and pathogenic contamination of salads MP, as pointed the results of E. coli and pathogens reveled in this work. The high level we found, shows a very decayed hygienic conditions and is probably responsible for the deterioration of sensory quality and the flawless aspect presented by this salads. Nevertheless, it might have occurred an antagonist effect against the pathogenic micro-organisms that might support the very low contamination level detected. We can say after all, that the incidence of pathogenic microorganisms found was very low. Only the incidence of A. hydrophila was high, however the association of this microorganism to food borne outbreaks remains unclear. It is important to note that international data also point to a low prevalence of pathogenic micro-organisms, however, despite this, there have been food borne outbreaks associated to produce. The existing information shows that the pathogens are opportunistic contaminants that can occur sporadically in fruits and vegetables and consequently, these products may pose a risk to the consumers’ health. A proper assessment of risk is limited due to the infrequent nature of this occurrence (Monaghan, Thomas, Goodburn, & Hutchison, 2009). Most reported outbreaks found that the source of infection occurred in the field, during cultivation, thus making it very important to a strict observance of Good Agricultural Practices (GAP). Moreover, during transport and processing in plant, it is important to adhere to Good Manufacturing Practices (GMP) and GHP to avoid cross-contamination. These products are only subject to a disinfection step that doesn’t ensure the elimination of pathogens. Keeping the product under refrigeration by distributors, retailers and consumers, is another important factor. In addition,
researchers should continue to develop new methods of disinfection to remove micro-organisms and to assay new preservatives effective in reduce the microbial growth along storage.
Acknowledgments We would like to acknowledge the two supermarkets for providing some salad samples used in this work. This research is part of M.I. Santos Ms.C. theses and was funded by Centro de Botanica Aplicada a Agricultura and Instituto Nacional de Saúde Dr. Ricardo Jorge.
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