Available online at www.sciencedirect.

com

International Journal of Food Microbiology 123 (2008) 121 129 www.elsevier.com/locate/ijfoodmicro

Microbiological quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail establishments
M. Abadias a,, J. Usall a , M. Anguera a , C. Solsona a , I. Vias b
b

IRTA, Centre UdL-IRTA, XaRTA-Postharvest, 191 Rovira Roure, 25198-Lleida, Catalonia, Spain University of Lleida, Centre UdL-IRTA, XaRTA-Postharvest, 191 Rovira Roure, 25198-Lleida, Catalonia, Spain Received 7 March 2007; received in revised form 10 December 2007; accepted 18 December 2007

Abstract A survey of fresh and minimally-processed fruit and vegetables, and sprouts was conducted in several retail establishments in the Lleida area (Catalonia, Spain) during 20052006 to determine whether microbial contamination, and in particular potentially pathogenic bacteria, was present under these commodities. A total of 300 samplesincluding 21 ready-to-eat fruits, 28 whole fresh vegetables, 15 sprout samples and 237 readyto-eat salads containing from one to six vegetableswere purchased from 4 supermarkets. They were tested for mesophilic and psychrotrophic aerobic counts, yeasts and moulds, lactic acid bacteria, Enterobacteriaceae, presumptive E. coli and Listeria monocytogenes counts as well as for the presence of Salmonella, E. coli O157:H7, Yersinia enterocolitica and thermotolerant Campylobacter. Results for the fresh-cut vegetables that we analyzed showed that, in general, the highest microorganism counts were associated with grated carrot, arugula and spinach (7.8, 7.5 and 7.4 log cfu g 1 of aerobic mesophilic microorganisms; 6.1, 5.8 and 5.2 log cfu g 1 of yeast and moulds; 5.9, 4.0 and 5.1 log cfu g 1 lactic acid bacteria and 6.2, 5.3 and 6.0 log cfu g 1 of Enterobacteriaceae). The lowest counts were generally associated with fresh-cut endive and lettuce (6.2 and 6.3 log cfu g 1 of aerobic mesophilic microorganisms; 4.4 and 4.6 log cfu g 1 of yeast and moulds; 2.7 and 3.8 log cfu g 1 lactic acid bacteria and 4.8 and 4.4 log cfu g 1 of Enterobacteriaceae). Counts of psychrotrophic microorganisms were as high as those of mesophilic microorganisms. Microbiological counts for fresh-cut fruit were very low. Sprouts were highly contaminated with mesophilic (7.9 log cfu g 1), psychrotrophic microorganisms (7.3 log cfu g 1) and Enterobacteriaceae (7.2 log cfu g 1) and showed a high incidence of E. coli (40% of samples). Of the samples analyzed, four (1.3%) were Salmonella positive and two (0.7%) harboured L. monocytogenes. None of the samples was positive for E. coli O157:H7, pathogenic Y. enterocolitica or thermotolerant Campylobacter. 2008 Elsevier B.V. All rights reserved.
Keywords: Incidence; Foodborne pathogens; Fresh-cut; Ready-to-eat; Salmonella; E. coli; L. monocytogenes; Campylobacter; Y. enterocolitica

1. Introduction Fresh fruit and vegetables are essential components of the human diet and there is considerable evidence of the health and nutritional benefits associated with the consumption of fresh fruit and vegetables. In the USA, Canada, New Zealand and several European Union states, public health institutions have run campaigns recommending the daily consumption of at least five daily servings of fruit and vegetables. As well as a significant rise in the consumption of fresh produce for health benefits, there have also been significant changes in lifestyles and major shifts in
Corresponding author. Tel.: +34 973 003 430; fax: +34 973 238 301. E-mail address: isabel.abadias@irta.cat (M. Abadias). 0168-1605/$ - see front matter 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2007.12.013

consumption trends. These changes have produced a demand for a wider range of products, and have led people to spend less time cooking at home and to eat out more often. Such trends have been reflected in an increase in the popularity of salad bars and have prompted the appearance of minimally-processed convenience foods that are ready-to-eat. Among them, the consumption of freshcut or minimally-processed fruit and vegetables has undergone a sharp increase. Consumption of fresh-cut fruits and vegetables in Spain is still low (11.5 kg per person per year) compared with the rest of Europe (UK, 12 kg; France, 6 kg; Italy, 4 kg and Germany, Belgium and Netherlands with more than 3 kg) and USA (30 kg per person). However, in Spain this market is showing an annual increase of sales of about 20%, with 53,465 t sold in 2006 (Anonymous, 2007).

122

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129

Fresh produce can be a vehicle for the transmission of bacterial, parasitic and viral pathogens capable of causing human illness and a number of reports refer to raw vegetables harbouring potential foodborne pathogens (Nguyen-the and Carlin, 1994; Beuchat, 1996). Listeria monocytogenes (Schlech et al., 1983), Salmonella (Doyle, 1990), and Escherichia coli (Nguyen-the and Carlin, 1994) have been isolated from raw vegetables, which can become contaminated while growing or during harvesting, postharvest handling, or distribution. The incidence of foodborne outbreaks caused by contaminated fresh fruit and vegetables has increased in recent years (Mukherjee et al., 2006). The pathogens most frequently linked to produce-related outbreaks include bacteria (Salmonella, E. coli), viruses (Norwalk-like, hepatitis A), and parasites (Cryptosporidium, Cyclospora) (Tauxe et al., 1997), with Salmonella and E. coli O157:H7 being the leading causes of produce-related outbreaks in the USA (Olsen et al., 2000). Fresh produce and sprouts have been implicated in a number of documented outbreaks of illness in countries such as Japan (Nat'l. Inst. Inf. Dis., 1997; Gutierrez, 1997), the USA (De Roever, 1998) and EU (Emberland et al., 2007; Nygrd et al., 2004; Pezzoli et al., 2007; PHLS, 2000; Sderstrm et al., 2005). In September 2006, an E. coli O157:H7 outbreak affected 26 US States which involved about 200 cases of illness, including some of Hemolytic Uremic Syndrome (HUS) and resulted in three deaths (FDA, 2006). Data indicated that fresh spinach grown in three Californian counties was the source of the bacterium. Fresh fruit and vegetables may therefore pose a food safety risk because they are consumed raw and are susceptible to be contaminated by fecal material and soil on the farm (Mukherjee et al., 2004). Several national microbiological guidelines have been published for ready-to-eat food in countries such as: the UK (PHLS,
Table 1 List of methodologies used to determine microbial quality Determination Aerobic mesophilic count Psychrotrophic microorganisms Yeasts and moulds Lactic acid bacteria Enterobacteriaceae Presumptive E.colia Salmonellab L. monocytogenes Y. enterocoliticac Campylobacter
a

2000), Spain (Real Decreto 3484/2000); France (Nguyen-the and Carlin, 1994), Germany (Lund, 1993) and Japan (FEHD, 2002). The European Commission has also recently published a new regulation (n 2073/2005Official Journal of the European Union, 2005, L 338) that establishes a common food safety and process hygiene criteria for food in EU countries, and which covers pre-cut fruit and vegetables and sprouts. Although there have been a number of reports about microbiological contamination involving whole fresh produce (GarcaVillanova et al., 1987a,b; Johannessen et al., 2002; McMahon and Wilson, 2001; Salleh et al., 2003; Johnston et al., 2005; Mukherjee et al., 2004, 2006; FDA, 2001, 2003), there is still little information about microbial contamination of fresh-cut fruit and vegetables (FEHD, 2002; Sagoo et al., 2003, Tournas, 2005, Tournas et al., 2006) and sprouts (Robertson et al., 2002) and even less relating to the EU. A report by the EU Scientific Committee on Food (2002) stated that the prevalence of foodborne pathogens on fruit and vegetables and their involvement in outbreaks are not well documented from a European perspective. The aim of this study was therefore to investigate the microbiological quality of fresh and minimally-processed fruit and vegetables, and sprouts commercialized in Spain and the incidence of the main bacterial foodborne pathogens in this area. 2. Materials and methods 2.1. Samples A total of 300 samples of whole vegetables, minimallyprocessed (fresh-cut) fruit and vegetables and sprouts were analyzed during the period 20052006. The samples analyzed

Methodology ISO 4833:2003 ISO 17410:2001 ISO 7954:1987 ISO 15214:1998 ISO 21528-2:2004 ISO 7251:2005 ISO 6579:2002 ISO 11290-2:1998 ISO 10273:2003 ISO 10272:1995

Description Microbiology of food and animal feeding stuffsHorizontal methods for the enumeration of microorganisms. Colony-count technique at 30 C. Microbiology of food and animal feeding stuffsHorizontal methods for the enumeration of psychrotrophic microorganisms. MicrobiologyGeneral guidance for enumeration of yeasts and mouldsColony count technique at 25 C Microbiology of food and animal feeding stuffsHorizontal methods for the enumeration of mesophilic lactic acid bacteriaColony-count technique at 30 C. Microbiology of food and animal feeding stuffsHorizontal methods for the detection and enumeration of EnterobacteriaceaePart 2: Colony-count method Microbiology of food and animal feeding stuffsHorizontal methods for the detection and enumeration of presumptive Escherichia coliMost probable number technique Microbiology of food and animal feeding stuffsHorizontal methods for the detection of Salmonella spp. Microbiology of food and animal feeding stuffsHorizontal methods for the detection and enumeration of Listeria monocytogenes. Part 2: Enumeration method. Microbiology of food and animal feeding stuffsHorizontal methods for the detection of presumptive pathogenic Yersinia enterocolitica Microbiology of food and animal feeding stuffsHorizontal methods for the detection of thermotolerant Campylobacter

E. coli strains isolated were plated in Tergitol BCIG agar and Sorbitol MacConkey Agar and incubated at 44 1 C for the detection of -glucuronidase and sorbitol positive strains, respectively. b Presumptive Salmonella were sent to the National Reference Laboratory (Instituto de Salud Carlos III, Mahadahonda, Spain) for confirmation. c Strains identified as Y. enterocolitica by biochemical tests were sent to the Laboratorio de Microbiologa, Universidad de Navarra in Spain to determine their pathogenicity.

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129

123

included: 104 single-ingredient salad samples (5 of arugula, 18 of grated carrot, 21 of corn salad, 21 of endive, 29 of lettuce (Iceberg, Batavia and Romaine) and 10 of spinach); 132 readyto-eat salads, containing from three to six ingredients such as lettuce (different varieties), endive, carrots, corn salad, spinach, red beet and soybean sprouts; 21 single-ingredient ready-to-eat fruits (apple, pineapple, orange, mango and peach); 15 sprout samples (soybean and alfalfa) and 28 samples of whole fresh vegetables (Iceberg, Oakleaf, Romaine and Trocadero lettuces, endive and lettuce hearts). Samples of all of the main brands of fresh-cut vegetables and sprouts sold in Spain were randomly purchased from different supermarkets in Lleida (Catalonia, Spain), in their original, individual packages. Whole vegetables were drawn from bulk trays and fresh-cut fruit samples were obtained from vending machines. 2.2. Microbial analysis All samples were purchased before their best-before date, transported to the laboratory and immediately analyzed. Several product-related data were recorded, including: manufacturer, type of salad, composition, batch number, expiry date and type of packaging (modified or non-modified atmosphere). Microbial determinations were carried out using the standard methodologies described in Table 1. Twenty-five grams of each sample of ready-to-use fruit, vegetables or sprouts were diluted in 225 ml of peptone saline solution (PS, 1 g peptone and 8.5 g NaCl per litre) and homogenized for 2 min at normal speed in a Stomacher (Model 400 Circulator, Seward, Norfolk, England). Serial dilutions of the suspension were made in PS and analyzed for aerobic mesophilic (AM), psychrotrophic microorganisms, yeasts and moulds (YM), Enterobacteriaceae, mesophilic lactic acid bacteria (LAB) and presumptive E. coli. Another 25 g were diluted in 225 ml of buffered peptone water (Oxoid, CM1049) for
Table 2 Results of aerobic mesophilic count (AM) in the samples analyzed

the enumeration of L. monocytogenes and detection of Salmonella. Another test sample was inoculated into two enrichment media (a peptone, sorbitol and bile salts broth and an irgasan, ticarcillin and potassium chlorate broth) for the detection of presumptive pathogenic Y. enterocolitica. Finally, another test portion was enriched with Preston broth for the detection of thermotolerant Campylobacter. For whole products, the 34 outer leaves were discarded and the inner portion was cut up in the laboratory under aseptic conditions. We then proceeded as previously described. 3. Results In general, among the fresh-cut vegetables and sprouts that we analyzed, the samples with the highest microorganism counts were sprouts, grated carrot, arugula and spinach, while fresh-cut endive and lettuce were those with the lowest. Freshcut fruit had the lowest microorganism counts. There were no significant differences between commercial brands. All samples were collected before their respective best-before dates and even those with high counts were not spoiled based on their visual appearance (data not shown). The aerobic mesophilic count (AM) ranged from 4.3 to 8.9 log10 cfu g 1 in fresh-cut vegetables, from 2.0 to 7.1 in fresh-cut fruit, from 7.1 to 9.2 in sprouts and from 2.7 to 8.0 log10 cfu g 1 in whole vegetables (Table 2). Sprouts, grated carrot, arugula and spinach had the highest mean AM counts (7.9, 7.8, 7.5 and 7.4 log10 cfu g 1, respectively), and fresh-cut fruit had the lowest (3.8 log10 cfu g 1). About 90% of the spinach and grated carrot and 100% of the sprout samples had AM counts of N107 cfu g 1. On the contrary, the number of AM in fresh-cut fruit was b105 cfu g 1 in 90.4% of the samples. AM counts in whole vegetables were, in general, lower that in minimally-processed salads, with Iceberg lettuce and lettuce

Percentage of samples in the indicated interval n Fresh-cut vegetables Arugula Carrot Corn salad Endive Lettuce Spinach Mixed salads Fresh-cut fruit Sprouts Whole vegetables Iceberg Lettuce hearts Oakleaf Trocadero Romaine Endive
a

b105 1.7 0 0 0 9.5 6.9 0 0 90.4 0 14.3 60.0 33.3 0 0 0 0

105106 10.2 0 0 0 19.0 37.9 0 6.8 4.8 0 32.1 40.0 66.6 20.0 40.0 40.0 0

106107 34.3 40.0 11.1 47.6 66.7 27.6 10.0 33.3 0 0 35.7 0 0 60.0 40.0 60.0 40.0

107108 47.9 60.0 55.6 52.4 4.8 27.6 80.0 54.5 4.8 60.0 17.9 0 0 20.0 20.0 0 60.0

N108 5.9 0 33.3 0 0 0 10.0 5.3 0 40.0 0 0 0 0 0 0 0

Rangeb 4.38.9 6.78.0 6.58.9 6.47.8 4.37.2 4.97.6 6.28.0 5.48.5 2.07.1 7.19.2 2.78.0 3.35.9 2.75.3 5.88.0 5.17.3 5.46.6 6.77.2

Meanb 7.0 7.5 7.8 7.1 6.2 6.3 7.4 7.1 3.8 7.9 5.9 4.6 4.4 6.7 6.2 6.0 7.0

236 5 18 21 21 29 10 132 21 15 28 5 3 5 5 5 5

n: Number of samples. :Range in cfu g 1 of product. b :Counts are given in terms of log10 cfu g 1 of product.

124

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129

Table 3 Results of phsychrotrophic microorganisms (6.5 C) in the samples analyzed Percentage of samples in the indicated interval n Fresh-cut vegetables Arugula Carrot Corn salad Endive Lettuce Spinach Mixed salads Fresh-cut fruit Sprouts Whole vegetables Iceberg Lettuce hearts Oakleaf Trocadero Romaine Endive
a

b105 1.3 0 0 0 9.5 3.4 0 0 85.7 0 14.3 60.0 33.3 0 0 0 0

105106 8.9 20.0 0 4.8 19.0 25.6 0 5.3 9.5 0 35.7 40.0 66.6 20.0 40.0 60.0 0

106107 35.2 20.0 5.6 47.6 61.9 44.8 20.0 32.6 0 46.6 35.7 0 0 60.0 60.0 40.0 40.0

107108 46.2 40.0 55.6 47.6 9.5 24.1 70.0 53.8 4.8 26.7 14.3 0 0 20.0 0 0 60.0

N108 8.5 20.0 38.9 0 0 0 10.0 8.3 0 26.7 0 0 0 0 0 0 0

Rangeb 4.38.9 5.78.2 6.68.9 5.97.8 4.37.1 4.97.8 6.18.1 5.28.5 1.77.1 6.38.9 3.07.8 3.25.9 3.05.2 5.87.8 5.26.7 5.36.5 6.27.2

Meanb 7.0 7.3 7.9 7.0 6.1 6.4 7.4 7.1 3.6 7.3 5.8 4.6 4.4 6.6 6.0 5.9 6.8

236 5 18 21 21 29 10 132 21 15 28 5 3 5 5 5 5

n: Number of samples. :Range in cfu g 1 of product. b :Counts are given in terms of log10 cfu g 1 of product.

hearts being the items with the lowest mean AM counts (4.6 and 4.4 log10 cfu g 1, respectively). Psychrotrophic microorganism counts were very similar to those of mesophilic microorganisms (Table 3), with carrot, spinach, arugula and sprouts being the vegetables with the highest mean counts (7.9, 7.4, 7.3 and 7.3 log10 cfu g 1). Yeasts and moulds (YM) were present in smaller numbers than bacteria, but results varied greatly (Table 4). The ranges for YM in fresh-cut vegetables, fresh-cut fruit, sprouts and whole vegetables were 2.07.8, 1.74.9, 2.87.6 and 2.26.1 log10 cfu g 1, respectively. Grated carrot, arugula, corn salad and mixed salads were the items with the highest mean counts for moulds and yeasts
Table 4 Results of yeasts and moulds (YM) in the samples analyzed

(6.1, 5.8, 5.5 and 5.4 log10 cfu g 1, respectively). Whole vegetables generally contained relatively small numbers of YM, with 64.3% of samples having counts of b105 cfu g 1. Lactic acid bacteria (LAB) (Table 5) were also present in large numbers in carrots, sprouts and spinach (5.9, 5.5 and 5.1 log10 cfu g 1). Whole vegetables, corn salad, fresh-cut endive and fresh-cut fruit had the smallest numbers of LAB, with all samples containing b 105 cfu g 1. Sprouts were highly contaminated with Enterobacteriaceae (Table 6), with a mean level of 7.2 log10 cfu g 1, followed by grated carrot, spinach and mixed salads (6.2, 6.0 and 5.5 log10 cfu g 1 ). The range for Enterobacteriaceae in fresh-cut

Percentage of samples in the indicated interval n Fresh-cut vegetables Arugula Carrot Corn salad Endive Lettuce Spinach Mixed salads Fresh-cut fruit Sprouts Whole vegetables Iceberg Lettuce hearts Oakleaf Trocadero Romaine Endive
a

b105 36.9 0 5.6 14.3 71.4 75.9 50.0 31.1 95.2 46.7 64.3 100 100 40.0 60.0 80.0 20.0

105106 45.3 80.0 33.3 61.9 28.6 17.2 40.0 52.3 4.8 26.7 32.1 0 0 60.0 40.0 20.0 60.0

106107 14.8 20.0 50.0 19.0 0 6.9 10.0 13.6 0 20.0 3.6 0 0 0 0 0 20.0

107108 3.0 0 11.1 4.8 0 0 0 3.0 0 6.7 0 0 0 0 0 0 0

Rangeb 2.07.8 5.76.1 4.77.2 2.07.5 2.05.5 2.96.4 4.06.0 3.87.8 1.74.9 2.87.6 2.26.1 2.24.5 2.64.6 4.65.7 3.95.3 3.75.2 4.36.1

Meanb 5.2 5.8 6.1 5.5 4.4 4.6 5.2 5.4 3.6 5.2 4.5 3.4 3.6 5.1 4.6 4.6 5.4

236 5 18 21 21 29 10 132 21 15 28 5 3 5 5 5 5

n: Number of samples. :Range in cfu g 1 of product. b :Counts are given in terms of log10 cfu g 1 of product.

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129 Table 5 Results of Lactic Acid Bacteria (LAB) counts in the samples analyzed Percentage of samples in the indicated interval n Fresh-cut vegetables Arugula Carrot Corn salad Endive Lettuce Spinach Mixed salads Fresh-cut fruit Sprouts Whole vegetables Iceberg Lettuce hearts Oakleaf Trocadero Romaine Endive
a

125

b105 a 66.9 100 22.2 100 100 89.7 50.0 57.6 100 40.0 100 100 100 100 100 100 100

105106 14.0 0 27.8 0 0 6.9 30.0 17.4 0 20.0 0 0 0 0 0 0 0

106107 12.3 0 33.3 0 0 3.4 20.0 22.7 0 13.3 0 0 0 0 0 0 0

107108 2.5 0 16.7 0 0 0 0 2.3 0 26.7 0 0 0 0 0 0 0

Rangeb b1.08.5 3.05.9 4.37.6 b1.04.0 b1.04.6 1.76.3 3.76.9 b1.08.5 1.74.8 3.47. 5 b1.03.3 b1.01.2 b1.02.0 b1.02.8 b1.03.3 b1.01.9 1.72.7

Meanb 4.2 4.0 5.9 2.2 2.7 3.8 5.1 4.5 3.0 5.5 1.6 1.0 1.3 1.7 1.9 1.2 2.2

236 5 18 21 21 29 10 132 21 15 28 5 3 5 5 5 5

n: Number of samples. :Range in cfu g 1 of product. b :Counts are given in terms of log10 cfu g 1 of product.

vegetables was very broad, from b 1.0 to 8.0 log10 cfu g 1. The incidence of Enterobacteriaceae in whole vegetables, corn salad and fresh-cut fruit was very rare with 78.6, 100 and 100% of samples, respectively, containing less than 105 cfu g 1. E. coli was not detected in fresh-cut fruit, but was present in 2 of 28 whole vegetable samples (7.1%) and in 27 of 236 fresh-cut vegetable samples (11.4%) (Table 7). Nevertheless, only two fresh-cut vegetable samples (0.8%) had E. coli counts of N100 MPN g 1 (data not shown). The incidence of E. coli in sprouts was higher (40%) and 4 samples (26.6%) had E. coli counts of between 1100 and 4600 MPN g 1 (data not shown). None of the
Table 6 Results of Enterobacteriaceae in the samples analyzed

isolates was identified as E. coli O157:H7. Salmonella was found in 4 out of 300 samples (1.3%) from 3 different brands: one mixed salad, one lettuce, one spinach and one corn salad. Of the 300 samples analyzed, two from the same brand (one bag of fresh-cut lettuce and one of mixed salad) tested positive for L. monocytogenes, but only one of them (fresh-cut lettuce) had more than 100 cfu g 1 (130 cfu g 1). Salmonella and L. monocytogenes strains were subsequently confirmed by Spanish reference laboratories. In the case of fresh-cut vegetables, 5 out of 236 samples (two mixed salads, one grated carrot, one fresh-cut lettuce and one fresh-cut spinach) contained non-pathogenic Y.

Percentage of samples in the indicated interval n Fresh-cut vegetables Arugula Carrot Corn salad Endive Lettuce Spinach Mixed salads Fresh-cut fruit Sprouts Whole vegetables Iceberg Lettuce hearts Oakleaf Trocadero Romaine Endive
a

b105a 39.8 40.0 16.7 100 57.1 62.1 20.0 27.3 100 0 78.6 100 100 60.0 80.0 80.0 60.0

105106 33.9 60.0 22.2 0 28.6 20.7 40.0 43.2 0 0 14.3 0 0 20.0 20.0 20.0 20.0

106107 21.2 0 38.9 0 14.3 13.8 30.0 25.0 0 33.3 7.1 0 0 20.0 0 0 20.0

N107 5.1 0 22.2 0 0 3.4 10.0 4.5 0 66.7 0 0 0 0 0 0 0

Rangeb b1.08.0 4.35.9 4.57.2 b1.03.3 3.26.2 b1.07.1 4.78.0 2.77.9 1.74.8 6.38.1 b1.06.0 b1.03.3 1.83.6 2.26.0 b1.05.6 b1.05.0 3.96.0

Meanb 3.5 5.3 6.2 2.1 4.8 4.4 6.0 5.5 3.0 7.2 3.5 2.0 2.5 3.9 3.4 3.7 4.9

236 5 18 21 21 29 10 132 21 15 28 5 3 5 5 5 5

n: Number of samples. :Range in cfu g 1 of product. b :Counts are given in terms of log10 cfu g 1 of product.

126

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129

Table 7 Results of incidence of foodborne pathogens in the samples analyzed Percentage of positive samples n Fresh-cut vegetables Arugula Carrot Corn salad Endive Lettuce Spinach Mixed salads Fresh-cut fruit Sprouts Whole vegetables Iceberg Lettuce hearts Oakleaf Trocadero Romaine Endive Total
a

E. coli 11.4 40.0 ND ND ND 3.4 20.0 16.7 ND 40.0 7.1 ND ND ND 20.0 ND 20.0 14.8

Salmonella 1.7 ND ND 4.8 ND 3.4 10 0.8 ND ND ND ND ND ND ND ND ND 1.3

L. monocytogenes NDa ND ND ND ND 3.4 ND 0.8b ND ND ND ND ND ND ND ND ND 0.7

236 5 18 21 21 29 10 132 21 1 28 5 3 5 5 5 5 300

n: Number of samples. ND: not detectable in 25 g. b N100 cfu g 1 of L. monocytogenes.

enterocolitica strains: they were not Ca-dependant and did not form the Mucoid Yersinia factor, Myf, which is associated with enteropathogenicity (data not shown). Neither pathogenic Y. enterocolitica nor thermotolerant Campylobacter were found in any of the analyzed samples. 4. Discussion This is the first report on the microbiological quality of freshcut fruit and vegetables and sprouts conducted in Spain. Although samples were purchased locally, the brands selected were the ones with the greatest national sales and are sold throughout the country. This study is therefore quite representative. Microbial loads tended to be high for leafy vegetables, carrots and sprouts, which contained large numbers of mesophilic and psychrotrophic microorganisms. However, Nguyen-the and Carlin (1994) also reported similar results for minimally-processed fresh vegetables. Despite the high counts, it is important to underline that products did not show any visible signs of spoilage. Microbial loads on the edible parts (without wrapper leaves) of unprocessed (whole) vegetables were, in general, lower than those associated with fresh-cut vegetables. For example, 82% of whole vegetables had AM counts of b107 cfu g 1 while the corresponding percentage for fresh-cut vegetables was only 46%. Similar tendencies were observed with yeasts and moulds, LAB and Enterobacteriaceae. In fact, vegetable crops produced in a natural environment cannot be expected to remain free from microbial agents (Sagoo et al., 2003) and the processing stages of fresh-cut products, such as handling, cutting, shredding, slicing and grating, are all potential sources of contamination, which could further increase the microbial load. Effective washing and decontamination of fresh-cut vegetables is difficult to achieve and the reduction of microbial counts on fruits and vegetables are typically 100-fold at

best (Beuchat, 1998; Seymour, 1999). Moreover, some of these microorganisms could grow during marketing on fresh-cut fruit and vegetables, especially those that are phsychrotrophic and/or when packaged products are not stored at the temperature recommended by the provider (usually between 1 and 5 C). In general, the commodities with the highest microbial counts were sprouts, grated carrot, arugula and spinach, with lettuce (different cultivars) and endive being the items with the lowest counts. Carrots are subterranean crops and are directly in contact with soil and water and therefore also with manure, fertilizers and irrigation water. Arugula and spinach are topsoil crops, but have open leaves that could be in contact with soil and irrigation water. Sprouts have been identified as a special problem because of the potential for pathogen growth during the sprouting process and because there is no specific step in the general production process to reduce or eliminate pathogens present on raw sprouts (NACMCF, 1999). Similarly, Paterson and Woodburn (1980) reported aerobic plate counts of 108109 cfu g 1 in alfalfa and bean sprouts. Even so, the NACMCF (1999) pointed out that high microbial levels per se are not necessarily of public health concern in sprouts; it is the presence and growth of pathogenic microbes that cause concern. In the case of the sprouts that we analyzed, the manufacturer recommended washing before eating. Over 50% of the fresh-cut vegetables and 100% of sprout samples had AM counts N 107 cfu g 1), while in fruit, 90.4% of samples had counts of b105 cfu g 1. The number of mesophilic aerobic microflora present was very similar to that of psychrotrophic microflora and therefore most of the microorganisms were able to grow at storage temperatures. Garg et al. (1990) pointed out that mesophilic and psychrotrophic counts may be of similar magnitude at the time of processing. Storage at refrigeration temperatures generally selects in favour of the growth of psychrotrophic microorganisms, including the pectinolitic pseudomonads (Nguyen-the and Carlin, 1994). Nevertheless, mesophilic microorganisms may continue to grow at low temperature, albeit at reduced growth rates (Vescovo et al., 1996; Carlin et al., 1990). In a recent review by Ragaert et al. (2007), it was concluded that microbial counts that produce changes in sensory quality factors of minimally-processed vegetables resulting in rejection of the product are in most cases N 78 log cfu g 1. However, exceeding this microbiological limit does not always result in occurrence of visual defects as both microbiological and physiological activity play a role in spoilage of these products (Ragaert et al., 2007). Observed numbers of yeasts and moulds were lower than bacteria, with grated carrot and arugula as the items with the highest counts. Tournas (2005) obtained similar results with samples of fresh and minimally-processed vegetables, and sprouts. The role of yeasts in the spoilage of fresh vegetables is not well studied, although they have been implicated in the spoilage of fermented vegetable products and the development of soft rot (Fleet, 1992). Ragaert et al. (2006) pointed out that some volatiles linked to spoilage yeasts were detected in a simulation medium of shredded mixed-lettuce when Pichia fermentans populations were 5.0 log cfu cm 2. Later, Ragaert et al. (2007) mentioned that these counts do not necessarily cause off-odours and neither do they necessarily result in visual defects in minimally-processed fruits and vegetables. Spoilage

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129

127

as a result of mould growth does not appear to be a major problem in ready-to-eat salads, although the problem is not well documented in the literature (Heard, 1999). However, some authors (Tournas, 2005; Tournas and Katsoudas, 2005) have pointed out the possible health problems associated with the presence of moulds in fruit and vegetables, as some may produce mycotoxins and others are known to cause allergies when they are able to produce large numbers of conidia. In contrast, the fresh-cut fruit analyzed (apple, peach, orange, mango and pineapple) harboured small microbial populations and these mainly consisted of yeasts and moulds. None Enterobacteriaceae were detected. Fruits are more acidic than other types of fresh produce and the combination of low pH and low temperature during storage also tends to inhibit growth. The acid environment is more inhibitory at refrigeration (5 C) than at higher temperatures (10 C) (Heard, 1999). Moreover, the peel is a good barrier against microorganisms and there is no contact with either soil or irrigation water. Under these conditions, numbers of indigenous microbiota would possibly be smaller and they would tend not proliferate on cut fruit due to the low temperature and low pH of these fruits. In contrast, Tournas et al. (2006) found higher yeasts and moulds counts in other types of fruit salad (cantaloupe, honeydew melon, citrus fruit, pineapple, strawberry, watermelon and mixtures of these fruits) with a range between b2.0 and 9.72 log10 cfu g 1. Large numbers of lactic acid bacteria were found, particularly in grated carrot, sprouts and spinach. LAB have been reported in a wide variety of salads including shredded carrots and shredded mixed vegetable salads, with amounts ranging from 102 cfu g 1 in shredded chicory to 108 cfu g 1 in mixed salad vegetables (Heard, 1999). Both pseudomonads and LAB are normal flora of vegetables, whereas coliforms, yeasts and moulds may arise from the raw material or from contamination during processing (Nguyen-the and Carlin, 1994). LAB are known as spoilage organisms in grated, sliced or shredded carrots. Leuconostoc mesenteroides was the main LAB associated with spoilage, causing exudation, acidity (sourness) and loss of texture in shredded carrots stored at 10 C for 10 days (Carlin et al., 1989). The number of Enterobacteriaceae was very high in sprouts and carrots, while fresh-cut fruit and corn salad had the lowest concentrations. In fact, it should be noted that Enterobacteriaceae are not used to indicate the microbiological quality of fresh vegetables; high levels of Enterobacteriaceae are common in raw vegetables and such vegetables often have high levels of these organisms as part of their normal flora (Little et al., 1999). Therefore, levels of faecal organisms, such as E. coli are a better indicator of contamination (Nguyen-the and Carlin, 1994) and this could explain why this organism has been included as a hygienic criterion in the new EU regulation (n2073/2005). Other Enterobacteriaceae such as Erwinia herbicola, Rahnella aquatilis, Enterobacter agglomerans and Serratia spp. are often present in high numbers in minimally-processed vegetables (Nguyen-the and Carlin, 1994; Ragaert et al., 2007). In the present work, E. coli was present in 7.1% of whole vegetables, 11.4% of fresh-cut samples and 40% of sprouts, but none of the samples contained E. coli O157:H7. Only 0.8% of fresh-cut vegetables exceeded the hygienic criteria established

by the 2073/2005 guidelines, while this limit was exceeded in 26.7% of sprout samples. While the presence of Salmonella is a food safety criterion at the market place, that of E. coli is a hygienic criterion that should be applied during the manufacturing process. E. coli was isolated in 2% of sprout samples in Norway (Robertson et al., 2002). In two separate studies carried out in 1987, Garca-Villanova et al. (1987a,b) found that 86.1% and 75% of whole vegetables harboured and E. coli. E. coli O157:H7 was not detected in recent surveys of whole and freshcut fruit and vegetables conducted in Hong Kong (FEHD, 2002), USA (FDA, 2003; Mukherjee et al., 2006), UK (Sagoo et al., 2003), Ireland (McMahon and Wilson, 2001) and Norway (Johannessen et al., 2002). However, as previously indicated, some recent outbreaks linked to the consumption of fresh fruit, vegetables or juices have been attributed to this pathogenic bacterium (FDA, 2006; Sderstrm et al., 2005). Salmonella sp. was isolated in 4 out of 300 samples (1.3%, one bag of corn salad, 1 of spinach, 1 of lettuce and 1 of mixed salad). However, no cases of salmonellosis linked to the consumption of fresh-cut vegetables were reported in Spain in the study period. In two further studies conducted in Spain in 1987, Garca-Villanova et al. (1987a,b) found that 7.5% and 3.3% of whole vegetables harboured Salmonella. Other studies of fresh, unprocessed produce conducted in Minnesota and Wisconsin (Mukherjee et al., 2006), the UK (Sagoo et al., 2003), the southern USA (Johnston et al., 2005), the USA (with imported fresh produce, FDA, 2001) and Malaysia showed widely varying incidences of Salmonella: 0, 0.2%, 3.3%, 3.5% and 35%, respectively. In a survey conducted in Hong Kong, 0.7% of ready-to-eat salad samples contained Salmonella (FEHD, 2002). In a previous study, Abadias et al. (2006) analyzed the incidence of E. coli, E. coli O157:H7 and Salmonella in whole fresh apples in the Lleida area. They found that 7% of samples contained E. coli but the enterohaemorragic strain and Salmonella were not found. The incidence of L. monocytogenes in the samples analyzed was 0.7% (2 out of 300) but only one (0.4%, 130 cfu g 1) did not accomplish the EU microbiological regulation. L. monocytogenes was not detected in a survey conducted in the southern USA (Johnston et al., 2005) nor in another carried out in Northern Ireland (McMahon and Wilson, 2001). On the contrary, three cabbage samples (1%) were positive for L. monocytogenes in a survey of US and Mexican fresh produce (Johnston et al., 2006) and 0.3% of fresh produce samples in Norway (Johannessen et al., 2002). In a microbiological study of ready-to-eat salad vegetables, Sagoo et al. (2003) found L. monocytogenes in 2.3% of samples (90 of 3849), but only one sample contained levels of N 102 cfu g 1. Similarly, the FEHD (2002) isolated L. monocytogenes in 6 of 573 samples (1.0%) of ready-to-eat salads. Pathogenic Y. enterocolitica and thermotolerant Campylobacter were not detected in any of the samples examined. McMahon and Wilson (2001) did not found Campylobacter in any of 86 samples of commercial whole organic vegetables in Northern Ireland. Y. enterocolitica was found by PCR analysis in 3.0% of produce samples in Norway but was not found by culturing methods (Johannessen et al., 2002). Although the number of samples studied was small for some items due to sampling limitations, we believe this study

128

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129 EU Scientific Committee on Food, 2002. Risk profile on the microbiological contamination of fruits and vegetables eaten raw. URL: http://europa.eu.int/ comm/food/fs/sc/scf/out125_en.pdfs. FDA (Food and Drug Administration, USA), 2001. FDA Survey of imported fresh produce. URL: http://www.cfsan.fda.gov/~dms/prodsur6.html. FDA (Food and Drug Administration, USA), 2003. FDA Survey of domestic fresh produce. URL: http://www.cfsan.fda.gov/~dms/prodsu10.html. FDA (Food and Drug Administration, USA), 2006. Spinach and E. coli outbreak. URL: http://www.fda.gov/oc/opacom/hottopics/spinach.html. FEHD (Food and Environmental Hygiene Department, HKSAR), 2002. Microbiological risk assessment on salads in Hong Kong. Risk Assessment Studies, Report n9. URL: http://fehd.gov.hk/safefood/report/salad/report.pdf. Fleet, G.H., 1992. Spoilage yeasts. Critical Reviews in Biotechnology 12, 144. Garca-Villanova, B., Cueto-Espinar, A., Garca-Villanova, R., 1987a. Hygienic quality of vegetables cultivated and consumed in the area of Granada. L'Igiene Moderna 87, 117125. Garca-Villanova, B., Galvez-Vargas, R., Garca-Villanova, R., 1987b. Contamination on fresh vegetables during cultivation and marketing. International Journal of Food Microbiology 4, 285291. Garg, N., Churey, J.J., Splittsotesser, D.F., 1990. Effect of processing conditions on the microflora of fresh-cut vegetables. Journal of Food Protection 53, 701703. Gutierrez, E., 1997. Japan prepares as O157 strikes again. Lancet 349, 1156. Heard, G., 1999. Microbial safety of ready-to-eat salads and minimally processed vegetables and fruits. Food Australia 51, 414420. Johannessen, G.S., Lancarevic, S., Kruse, H., 2002. Bacteriological analysis of fresh produce in Norway. International Journal of Food Microbiology 77, 199204. Johnston, L.M., Jaykus, L., Moll, D., Martinez, M.C., Anciso, J., Mora, B., Moe, C.L., 2005. A field study on the microbiological quality of fresh produce. Journal of Food Protection 68, 18401847. Johnston, L.M., Jaykus, L., Moll, D., Anciso, J., Mora, B., Moe, C.L., 2006. A field study of the microbiological quality of fresh produce of domestic and Mexican origin. International Journal of Food Microbiology 112, 8395. Little, C.L., Roberts, D., Youngs, E., de Louvouis, J., 1999. Microbiological quality of retail imported unprepared whole lettuce. A PHLS food working group study. Journal of Food Protection 62, 325328. Lund, B.M., 1993. The microbiological safety of prepared salad vegetables. In: Turner, A. (Ed.), Food Technology International Europe. Sterling Publications International Ltd, London, pp. 196200. McMahon, M.A.S., Wilson, I.G., 2001. The occurrence of enteric pathogens and Aeromonas species in organic vegetables. International Journal of Food Microbiology 70, 155162. Mukherjee, A., Speh, D., Dyck, E., Diez-Gonzalez, F., 2004. Preharvest evaluation of coliforms, Escherichia coli, Salmonella and Escherichia coli O157:H7 in organic and conventional produce grown by Minnesota farmers. Journal of Food Protection 67, 894900. Mukherjee, A., Speh, D., Jones, A.T., Buesing, K.M., Diez-Gonzalez, F., 2006. Longitudinal microbiological survey of fresh produce grown by farmers in the upper Midwest. Journal of Food Protection 69, 19281936. NACMFC (National Advisory Committee on Microbiological Criteria for Food), 1999. Microbiological safety evaluations and recommendation on sprouted seeds. URL: http://vm.cfsan.fda.gov/~mow/sprouts2.html. Nat'l. Inst. Inf. Dis. (National Institute of Infectious Diseases and Infectious Diseases Control Division, Ministry of Health and Welfare of Japan), 1997. Verocytotoxinproducing Escherichia coli (enterohemorrhagic E. coli) infection, Japan, 1996 1997. Infectious Agents Surveillance Reports 18, 53154. Nguyen-the, C., Carlin, F., 1994. The microbiology of minimally processed fresh fruits and vegetables. Critical Reviews in Food Science and Nutrition 34, 371401. Nygrd, K., Lassen, J., Vold, L., Aavitsland, P., 2004. E-alert: outbreak of Salmonella Thompson infections caused by contaminated ruccola (rocket) salad. URL: http://www.eurosurveillance.org/ew/2004/041125.asp. Olsen, S.J., MacKinon, L.C., Goulding, J.S., Slutsker, L., 2000. Surveillance for foodborne disease outbreaksUnited States, 19931997. Morbidity and Mortality Weekly Report 49, 151. Paterson, J.E., Woodburn, M.J., 1980. Klebsiella and other bacteria on alfalfa and bean sprouts at the retail level. Journal of Food Science 45, 492495.

provides a general overview of the microbiological quality of fresh-cut fruit and vegetables, and sprouts commercialized in Spain. It also shows that the incidence of pathogenic bacteria in the samples analyzed was quite low: E. coli O157:H7, pathogenic Y. enterocolitica, and thermotolerant Campylobacter were not found, while the respective incidences of L. monocytogenes and Salmonella were 1.3 and 0.7%. No foodborne pathogens were found in fresh-cut fruit, whole vegetables or sprouts. Nevertheless, the results indicated that fresh-cut packaged vegetables may contain pathogenic bacteria and therefore could represent a risk to the consumers. Minimally-processed salads are exposed to a variety of different conditions during growth, harvest, preparation, packaging and distribution that could increase natural contamination. Such products only suffer from disinfection that would not ensure the complete elimination of pathogens. Good hygiene practices must be implemented by producers and processors in order to prevent contamination and bacterial growth. Distributors, retailers and consumers must also guarantee that refrigerated conditions are always maintained. Finally, researchers should develop new techniques to improve microbial disinfection and reduce microbial growth during storage. Acknowledgements The authors are grateful to the Spanish Government [Ministerio de Educacin y Ciencia, MEC, Research Project AGL-2004-06027 and INIA research contract CTE/3597/2003 (BOE 23/12/2003)] and FEDER for their financial support. References
Abadias, M., Caams, T.P., Asensio, A., Anguera, M., Vias, I., 2006. Microbial quality of commercial Golden Delicious apples throughout production and shelf-life in Lleida. International Journal of Food Microbiology 108, 404409. Anonymous, 2007. Fresh-cut. The sector takes off in a big way but there is still a long road ahead. URL: http://www.fruittoday.com/articulos.php?id=118416 1180215227&idioma=E. Beuchat, L.R., 1996. Pathogenic microorganisms associated with fresh produce. Journal of Food Protection 59, 204216. Beuchat, L.R., 1998. Surface decontamination of fruits and vegetables eaten raw: a review. WHO/FSF/FOS/98.2. Food Safety Unit. World Health Organisation. URL: http://www.who.int/foodsafety/publications/fs_management/en/ surface_decon.pdf. Carlin, F., Nguyen-the, C., Cudennec, P., Reich, M., 1989. Microbiological spoilage of fresh ready-to-use grated carrots. Science des Aliments 9, 371386. Carlin, F., Nguyen-the, C., Chambroy, Y., Reich, M., 1990. Effects of controlled atmospheres on microbial spoilage, electrolyte leakage and sugar content of fresh ready to use grated carrots. International Journal of Food Science and Technology 25, 110119. De Roever, C., 1998. Microbiological safety evaluations and recommendations on fresh produce. Food Control 9, 321347. Doyle, M.P., 1990. Fruit and vegetable safety-microbiological considerations. Hortscience 25, 14781481. Emberland, K.E., Ethelberg, S., Kuusi, M., Vold, L., Jensvoll, L., Lindstedt, B.A., Nygrd, K., Kjels, C., Torpdahl, M., Srensen, G., Jensen, T., Lukinmaa, S., Niskanen, T., Kapperud, G., 2007. Outbreak of Salmonella Weltevreden infections in Norway, Denmark and Finland associated with alfalfa sprouts, JulyOctober 2007. URL: http://www.eurosulveillance.org/ew/2007/ 071129.asp.

M. Abadias et al. / International Journal of Food Microbiology 123 (2008) 121129 Pezzoli, L., Elson, R., Little, C., Fisher, I., Yip, H., Peters, T., Hampton, M., De Pinna, E., Coia, J.E., Mather, H.A., Brown, D.J., Mller-Nielsen, E., Ethelberg, S., Heck, M., de Jager, C., Threlfall, J., 2007. International outbreak of Salmonella Senftenberg in 2007. URL: http://www.eurosurveillance.org/ew/2007/070614.asp. PHLS (Public Health Laboratory Service), 2000. Guidelines for the microbiological quality of some ready-to-eat foods sampled at the point of sale. Communicable Disease and Public Health (CDPH) 3, 163167. Ragaert, P., Devlieghere, F., Devuyst, E., Dewulf, J., Van Langenhove, H., Debevere, J., 2006. Volatile metabolite production of spoilage micro-organisms on a mixed-lettuce agar during storage at 7 C in air and low oxygen atmosphere. International Journal of Food Microbiology 112, 162170. Ragaert, P., Devlieghere, F., Debevere, J., 2007. Role of microbiological and physiological spoilage mechanisms during storage of minimally processed vegetables. Postharvest Biology and Technology 44, 185194. Real Decreto 3484/2000, de 29 de diciembre, por el que se establecen las normas de higiene para la elaboracin, distribucin y comercio de comidas preparadas (BOE n.11 del 12 de enero de 2001, 14351441). Robertson, L.J., Johannessen, G.S., Gjerde, B.K., Loncarevic, S., 2002. Microbiological analysis of seed sprouts in Norway. International Journal of Food Microbiology 75, 119126. Sagoo, S.K., Little, C.L., Ward, L., Gillespie, I.A., Mitchell, R.T., 2003. Microbiological study of ready-to-eat salad vegetables from retail establishments uncovers a national outbreak of salmonellosis. Journal of Food Protection 66, 403409.

129

Salleh, N.A., Rusul, G., Hassan, Z., Reezal, A., Isa, S.H., Nishibuchi, M., Radu, S., 2003. Incidence of Salmonella spp. in raw vegetables in Selangor, Malaysia. Food Control 14, 475479. Schlech, W.F., Lavigne, P.M., Bortolussi, R.A., Allen, A.C., Haldane, E.V., Wort, A.J., Hightower, A.W., 1983. Epidemic listeriosisevidence for transmission by food. New England Journal of Medicine 308, 203206. Seymour, I.J., 1999. Review of Current Industry Practice on Fruit and Vegetable Decontamination. CCFRA Review no. 14. Chipping Campden, UK, Campden & Chorley Food Research Association Group. Sderstrm, A., Lindberg, A., Andersson, Y., 2005. EHEC O157 outbreak in Sweden from locally produced lettuce, AugustSeptember 2005. URL: http://www.eurosurveillance.org/ew/2005/050922.asp. Tauxe, R., Kruse, H., Hedberg, C., Potter, M., Madden, J., Wachsmuth, K., 1997. Microbial hazards and emerging issues associated with produce. A preliminary report to the national advisory committee on microbiological criteria for foods. Journal of Food Protection 60, 14001408. Tournas, V.H., 2005. Moulds and yeasts in fresh and minimally processed vegetables, and sprouts. International Journal of Food Microbiology 99, 7177. Tournas, V.H., Katsoudas, E., 2005. Mould and yeast flora in fresh berries, grapes and citrus fruits. International Journal of Food Microbiology 105, 1117. Tournas, V.H., Heeres, J., Burgess, L., 2006. Moulds and yeast in fruit salads and fruit juices. Food Microbiology 23, 684688. Vescovo, M., Torriano, S., Orsi, C., Macchiarolo, F., Scolari, G., 1996. Application of antimicrobial-producing lactic acid bacteria to control pathogens in ready-touse vegetables. Journal of Applied Bacteriology 81, 113119.