Microbiological Quality of Fresh Vegetables and Fruits Collected from Supermarkets in Istanbul, Turkey
Serkan Kemal Buyukunal1, Ghassan Issa2, Filiz Aksu3, Aydin Vural4, *
1Istanbul Arel University, School of Health Sciences, Department of Nutrition and Dietetics, Istanbul, Turkey
2Avrupa Vocational School, Culinary Programme, Istanbul, Turkey
3Istanbul University, Vocational School of Veterinary Medicine, Istanbul, Turkey
4Dicle University, Faculty of Veterinary Medicine, Department of Food Hygiene and Technology, Diyarbakir, Turkey
To cite this article:
Serkan Kemal Buyukunal, Ghassan Issa, Filiz Aksu, Aydin Vural. Microbiological Quality of Fresh Vegetables and Fruits Collected from Supermarkets in Istanbul, Turkey. Journal of Food and Nutrition Sciences. Vol. 3, No. 4, 2015, pp. 152-159. doi: 10.11648/j.jfns.20150304.13
Abstract: Two hundred sixty one samples, collected from supermarkets in Istanbul, Turkey. All samples were analysed for aerobic mesophilic bacterial counts (AMC), aerobic psychrotrophic bacterial counts (APC), enumeration of yeasts and moulds (YM), coliforms, Escherichia coli and detection of Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes, thermotolerant Campylobacter spp. AMC ranged from 2.95 to 3.75 log10 CFU/g. APC ranged from 0 to 3.55 log10 CFU/g. The highest counts of coliforms were found in carrot, spinach, green leaf lettuce, cos lettuce and iceberg lettuce. The highest counts of YM were found in tomato, spinach, green leaf lettuce, cos lettuce and iceberg lettuce. Green leaf lettuces, cos lettuces, iceberg lettuces, spinach and carrot were highly contaminated with aerobic mesophilic (3.6 log10 CFU/g), psychrotrophic microorganisms (3.4 log10 CFU/g), and showed a high incidence of E.coli (41.77% of samples). Of the samples analysed, 10 (3.83%) were Salmonella spp. positive and 17 (6.51%) were thermotolerant Campylobacter spp. positive. None of the samples was positive for Escherichia coli O157:H7 and Listeria monocytogenes. Fresh vegetables and fruits, sold in Istanbul, Turkey, are needed to control foodborne pathogens especially Salmonellosis and Campylobacteriosis.
Keywords: Food Safety, Fresh Fruit, Microbiological Quality, Raw Vegetable
Hygienic fresh vegetables and fruits are important components of the human diet and there is strong relationship between fresh vegetable and fruit consumption and health . Raw vegetables and fruits provide proteins, carbonhydrates and many vitamins, minerals which are essential in healthy human life. These commodities are convenient meal for contemporary lifestyles. Various types of fruits and raw eaten vegetables and salads are also very popular due to their attributes. In many countries, including Turkey, consumption rates of raw fruits and vegetables is increasing day by day. To prevent cancer, diabetes, heart disease, obesity and micronutrient deficiencies, joint FAO/WHO Expert Consultation Panel recommends anadequate daily intake of 400–500 g of fruit and vegetables .
Fresh produce may be contaminated with a different kind of microorganisms [3-5]. Microbial contamination can occur during any steps of the farm-to-table and this contamination can arise from environmental, animal, human sources and technological applications. Microorganisms could spread not only by direct contact, but also through air and water [6-9]. Raw eaten vegetables and fruits are consumed without enough heating process, and therefore the possibility of food poisoning and food-borne infections always exists . The number of reported food-borne outbreaks associated with raw fruits and vegetables has increased in the late years. Identified outbreaks etiology has primarily bacteriological origin . However, most of farmers don’t still have enough information about hygienic production and good agricultural practices. This is a highly potential risk for public health. Increasing consumption of fresh produce has associated outbreaks of foodborne illnesses [5,12,13]. Fresh produce can be a vehicle for the contamination of enterotoxigenic and enterohemorrhagic Escherichia coli [1,10,14,16], Salmonella spp. [1,14,17], Listeria monocytogenes [1,17,18,19], thermotolerant Campylobacter spp. [20,21], parasitic and viral pathogens capable of causing human illness and a number of reports refer to raw vegetables and fruits harbouring potential foodborne pathogens [3,13,16,22,25].
The Centers for Disease Control and Prevention has reported an increase of fresh produce related foodborne disease outbreaks between 1995 and 2005 around the world .
Istanbul which has a geographic and geopolitics importance due to representing a transit corridor between Europa and Asia is a rapidly developing market cause of high population growth rate and continuous migration from various regions of Turkey and the neighboring countries. This high density of the population is regarded as a comprehensive supply of foods. Especially, the rapid growth of supermarkets chains in Turkey over the past ten years and also the subsequent development of quality requirements like volume, regularity, quality homogeneity, range of varieties, packaging have given rise to new market opportunities for local agriculture .
The present survey was intended to supply some assessment on the microbiological quality of marketed fresh vegetables and fruits in Istanbul, Turkey .
2. Materials and Methods
2.1. Collection of Samples
Two hundred sixty one fresh vegetables and fruits samples were analyzed between the period May 2012 and January 2013. The samples analyzed included: 161 samples of whole fresh vegetables, 100 samples of fresh fruits. Samples of all of the fresh produce sold in Istanbul were randomly purchased from different major supermarkets and local chain markets, in their original, individual packages. Production data of the samples are shown in table 1.
2.2. Preparation for Microbial Analysis
All samples were analysed as soon as possible after purchase. Before samples were taken out of their original packaging, the possible contact surfaces were carefully sterilised using polyurethane sponges to prevent cross-contamination. Damaged samples were discarded before analysis. For lettuce, spinach and purslane approximately 25 g of each sample were placed in a sterile stomacher bag and homogenized using a stomacher (Interscience - BagMixer 400 P, France) with 225 ml of sterile 0.1% buffered peptone water (BPW) (Oxoid, Cambridge, UK) for 2 min. For all other commodities, i.e. cucumber, tomato, green bean, squash, carrot, plum and peach each sample (whole) was aseptically transferred into a stomacher bag filled with equal weight of BPW. Each whole sample was then agitated and rubbed by hand in the stomacher bag for 2 min to suspend surface microbes [23,29]. Appropriate 1:10 dilutions of the resultant homogenate or the rinse fluid were prepared using BPW.
2.3. Aerobic Mesophilic Plate Count and Aerobic Psychrotrophic Plate Count
Laboratory analyses to enumarate of aerobic mesophilic plate count and psychrotrophic plate count were performed in accordance with the ISO 4833-1:2003 standard  and ISO 17410:2001 , respectively.
2.4. Enumeration of Yeasts and Moulds
Laboratory analyses to enumarate of yeasts and moulds were performed in accordance with the ISO 7954:1987 standard .
2.5. Enumeration of Coliforms and Escherichia coli
Samples were prepared as described above. Homogenate or the rinse fluid was prepared using BPW. For each selected dilution, 0.1 ml of sample was spread-plated onto brilliance E. coli/coliform agar (Oxoid, Cambridge, UK). The plates were incubated at 37 °C for 24 h, following which, the number of pink (coliform) and purple (presumptive E. coli) colonies was counted . Identification of E. coli was carried out with IMVIC tests .
2.6. Isolation of E. coli O157:H7
Samples were prepared as described above. Homogenate or the rinse fluid was prepared using BPW. Laboratory analyses to detect of E. coli O157:H7 were performed in accordance with the ISO 16654:2001 standard [35)].
2.7. Isolation of Salmonella Spp
Samples were prepared as described above. Laboratory analyses to detect of Salmonella spp. were performed in accordance with the ISO 6579:2002 standard .
2.8. Isolation of Listeria monocytogenes
Samples were prepared as described above. Laboratory analyses to detect of L. monocytogenes were performed in accordance with the ISO 11290 method .
2.9. Isolation and Confirmation of Thermotolerant Campylobacter spp
Laboratory analyses to detect thermotolerant Campylobacter spp. were performed in accordance with the ISO 10272-1:2006 standard (qualitative analysis) . To confirm suspect isolates polymerase chain reaction (PCR) methods  and a commercially available real-time PCR kit (Taq Man Campylobacter spp. Kit, AB Applied Biosystems) were applied. For quality control, the C. jejuni ATCC 33560 reference strain was used.
2.10. Confirmation of Presumptive Colonies
API®(bioMerieux, Marcy-l’Etoile, France) was used for the confirmation of presumptive colonies. The biochemical tests were carried out according to the manufacturer’s instructions.
2.11. Statistical Analysis
Colony counts were converted into log10 CFU/g. The mean values obtained from the microbiological evaluation of fruits and vegetables were analysed by independent samples t-test and to determine any statistically significant difference (P<0.05) among the all commodities means by one-way analysis of variance (ANOVA) followed by post hoc Tukey's test using SPSS 17.0 software (SPSS Inc. Chicago, IL, USA).
|samples||production region||production area||source of water||method of irrigation|
|Green leaf lettucea||Antalyab||greenhouse||ground water||drip|
|Iceberg lettucea||Antalyab||greenhouse||ground water||drip|
|Cos lettucea||Antalyab||greenhouse||ground water||drip|
|Green bean||Antalya-Serikb||greenhouse||ground water||drip|
|Kidney bean||Antalya-Serikb||greenhouse||ground water||drip|
aUntreated bovine or ovine originated manure are applied during pre-harvest on the greenhouse
bMediterranean climatic condition (mildly during spring, autumn and winter seeasons, hot and dry during summer season)
cColdly during spring, autumn and winter seasons, hot and dry during summer season)
AMC, APC and YM counts are shown in table 2. Coliform and Escherichia coli counts and percentages are shown in table 3. Results of incidence of Escherichia coli O157:H7, Salmonella spp., Listeria monocytogenes and thermotolerant Campylobacter spp.in the samples analysed are shown in table 4.
AMC and APC were up to 3.75 and 3.55 log10 CFU/g. Coliform and Escherichia coli counts were up to 3.45 and 2.65 log10 CFU/g. Escherichia coli contamination was highest in green leaf lettuce (80%). The highest YM counts were detected in tomatoes (3.15 log10 CFU/g). Statistically, whole fresh vegetable samples had significantly higher microbial loads than fresh fruit samples (p<0.05) and green leaf lettuce, iceberg lettuce, cos lettuce and spinach had significantly higher microbial loads than other commodities (p<0.05). No pathogenic bacteria could be isolated from fresh fruit samples, purslane, cucumber, tomato, green bean, kidney bean, squash, eggplant, and pepper. Salmonella spp. was isolated from 10 whole fresh vegetable samples. Thermotolerant Campylobacter spp. was isolated from 17 whole fresh vegetable samples.
|Type of samples||n||AMC/g||APC/g||YM/g|
|Green leaf lettuce||15||3.6-3.75||3.7||3.4-3.45||3.45||2.95-3.05||3.0|
|Type of samples||n||Coliform count (log10 CFU/g)||E.coli count E.coli presence (log10 CFU/g)|
|range||median||range||median||number of positive samples(%)|
|Green leaf lettuce||15||3.25-3.45||3.3||0-2.65||2.5||12 (80)|
|Iceberg lettuce||15||3.25-3.45||3.35||0-2.55||2.25||7 (46.67)|
|Cos lettuce||16||3.2-.3.4||3.3||0-2.6||2.35||9 (56.25)|
|Number and percentage (%) of positive samples|
|Type of samples||n||E.coli O157:H7||Salmonella spp.||Listeria monocytogenes||Campylobacter spp.|
|Green leaf lettuce||15||nd||4 (26.67)||nd||5 (33.33)|
|Iceberg lettuce||15||nd||1 (6.67)||nd||4 (26.67)|
|Cos lettuce||16||nd||3 (18.75)||nd||4 (25)|
|Spinach||19||nd||1 (5.26)||nd||3 (15.79)|
|Carrot||14||nd||1 (7.14)||nd||1 (7.14)|
|Total||261||nd||10 (3.83)||nd||17 (6.51)|
Abbrevations: n, number of samples; nd: not detected
Fresh vegetables and fruits can be contaminated with pathogenic bacteria in any steps of all the process from cultivation to consuming. These bacteria cause major public health concern worldwide in terms of human ilnesses. In addition, food-borne diseases have a large economic loss. Different studies on this subject are available in the world. But in our country, number and content of the study is not satisfactory. Our study was planned for this reason. The AMC for the whole vegetables and fresh fruits examined was around 3.35 log10 CFU/g, with a range of 2.95-3.75 log10 CFU/g and 3.30 log10 CFU/g, with a range of 2.95-3.50 log10 CFU/g, respectively. Pianetti et al.  reported that aerobic colony count does not relate to food poisoning and infections, generally. However, it acts as an indicator for food quality and shelf life.
According to the HACCP-TQM technical guidelines, raw foods containing <104 CFU/g (<4 log10 CFU/g), 104-5x106 CFU/g (4-6.7 log10 CFU/g), 5x106-5x107CFU/g (6.7-7.7 log10 CFU/g) and >5x107 CFU/g (>7.7 log10 CFU/g) (number of spoilage microorganisms aerobic plate count at 70°F (21.1°C) are rated as "good", "average", "poor" and "spoiled food", respectively (41). In our study, the quality of 261 samples was regarded as "good".
Aycicek et al.  reported some raw eaten vegetables (lettuce, cos lettuce, iceberg lettuce, parsley, dill, carrot) total aerobic counts between 0 and 7.4 log10 CFU/g. Abadias et al.  found the AMC ranged from 4.3 to 8.9 log10 CFU/g in fresh-cut vegetables, from 2.0 to 7.1 log10 CFU/g in fresh-cut fruits, from 7.1 to 9.2 log10 CFU/g in sprouts and from 2.7 to 8 log10 CFU/g in whole vegetables. APC counts were similar to those of mesophilic microorganism, with carrot (7.9 log10 CFU/g) and spinach (7.4 log10 CFU/g) being the vegetables with the highest mean counts. Badosa et al.  reported fruits had AMC ranging from 1 to 8 log10 CFU/g, most of them ranging between 3 and 4 log10 CFU/g. Oliveira et al.  noted thatthe AMC ranged from <3 to 7 log10 CFU/g in conventional and from 5 to >7 log10 CFU/g in organic lettuce. Psychrotrophic microorganism is very similar to those of mesophilic microorganisms, with ranges between 3 to >7 log10 CFU/g in organic lettuce and between <3 to 7 log10 CFU/g in conventional lettuce. Seow et al. (23) reported that AMC of fresh vegetables and fruits counts ranged from 1.6 to 9.1 log10 CFU/g with the lowest counts recorded for orange and highest counts recorded for bean sprouts. Fresh-cut salads had the highest mean APC of 4.9 log10 CFU/g. In another study done by Viswanathan and Kaur , it was reported that AMC for raw salad vegetables, fruits and sprouts were in the range between 5-10 log10 CFU/g, 6-8 log10 CFU/g and 9-12 log10 CFU/g, respectively. These findings were higher than our study. These differences may be originated from regions, geographical and climatical conditions, irrigation techniques and post-harvest processes.
Regarding the YM range of fresh produces, our findings is similar to other author’s studies. Generally, lettuces, tomatoes and carrots contamination is higher than the other commodities for YM. Abadias et al.  found that the ranges for YM in fresh-cut vegetables, fresh-cut fruit, sprouts and whole vegetables were 2.0-7.8, 1.7-4.9, 2.8-7.6 and 2.2- 6.1 log10 CFU/g, respectively. Grated carrot was the highest mean counts for YM (6.1 log10 CFU/g). Oliveira et al.  reported that 51.4% of the organic lettuce, 52.8% conventional lettuce was found in the range between 4 and 5 log10 CFU/g, respectively. The YM mean was 4.7 and 4.2 log10 CFU/g. Tournas (4) noted that YM counts ranged between 3.1-5.95 log10 CFU/g in lettuce, <2-5.3 log10 CFU/g in carrots, 2-3.8 log10 CFU/g in cucumbers, 4-4.1 log10 CFU/g in iceberg lettuce, 3.4-3.6 log10 CFU/g in spinach, 2-6.2 log10 CFU/g in different kind of tomatoes (cherry, grape, Roma). Tournas  and Tournas and Katsoudas  declared the some health problems related with the presence of yeasts and moulds in vegetables and fruits. Some of these may produce mycotoxins and others are known to cause allergic reactions.
Aycicek et al.  found coliform counts up to 6.9 log10 CFU/g, E.coli counts up 3.8 log10 CFU/g. Microbial loads of outer leaves of lettuce, cos lettuce and icerberg lettuce, parsley and dill samples detected the same levels. E.coli positive sample percentages of raw eaten vegetable was among 10% (iceberg lettuce and carrot)-70% (parsley). Seow et al.  noted that highest level of coliforms was found in bean sprouts and fresh-cut salads. Viswanathan and Kaur  found coliform counts ranged between 6-9 log10 CFU/g in raw salad vegetables, 4-7 log10 CFU/g in fruits and 8-11 log10 CFU/g in sprouts, respectively. Also, it was reported that prevalances of E.coli 50% of lettuces and 10% of carrots in their study. Abadias et al.  noted that E.coli was not detected in fresh-cut fruit, but was present in 7.1% whole vegetable samples and 11.4% fresh-cut vegetable samples. Our findings about E.coli prevalences showed similarity with these results. Especially, lettuces varieties, spinach, purslane, carrot etc. are risky products. Applying agricultural techniques should be reviewed for hygienic conditions.
HACCP-TQM technical guidelines give threshold and quality levels for food-borne illness hazards according to the volunteer feeding test of healthy people groups. For, E.coli, the estimated illness dose is 6-10 log10 CFU/g and suggested level of E.coli for purchasing 1 log10 CFU/g (41). In our study, purchasing limit for E.coli was exceeded 19.76% of vegetable samples (33 of the 167). On the other hand, none of the fruit sampleswere exceeded this level.
A lot of studies were undertaken about food-borne disease related to consumption of fresh fruit and vegetable showed that the number of the outbreaks has increasing day by day. Each year millions of cases occur that most of this E.coli 0157:H7, Salmonella spp., Listeria monocytogenes, Campylobacter spp. etc. Infections cause mild ilness, severe infections and serious complications-including death . Abadias et al.  noted that none of the fresh, minimally-processed fruit and vegetables, and sprouts samples was positive for E.coli O157:H7 and thermotolerant Campylobacter. Of the samples analyzed 1.3% was Salmonella spp. and 0.7% were L.monocytogenes positive. Seow et al.  reported that E.coli O157:H7 and Salmonella spp. was not detected in the analyzed of total 125 fresh fruits and vegetables samples. Al-Hindawi and Rished  reported that 7% of 43 vegetables samples were detected positive for Salmonella spp. Ercolani  noted that 68.3% rate of Salmonella in lettuces. Garcia-Villanova Ruiz et al.  reported that analyzed of 80 lettuce samples, 6.3% were positive and 5.2% of analyzed 28 spinach samples were positive for Salmonella spp. Garcia-Villanova Ruiz et al.  at the another study reported that analyzed 46 of 849 vegetables samples were detected positive Salmonella spp. Jerngklinchan and Saitanu  reported 8.7% rate of Salmonella in bean sprouts. Viswanathan and Kaur  tested of 72 vegetables and 33.3% of the samples were Salmonella spp. positive; tested of 24 fruits and 37.5% of the samples were Salmonella spp. detected positive.
In the present study E.coli O157:H7 was not detected. E.coli O157:H7 presents sporadically at very low levels together with very high levels of competitor organisms that’s why it is very difficult to detect. Salmonella spp. contamination in lettuces, spinach and carrots may cause by the use of contaminated irrigation water and untreated bovine or ovine originated manure.
Arumugaswamy et al.  reported of the analyzed 22 lettuce, 5 cucumber, 7 bean sprouts sample, 22.7%, 80%, 85% were positive for L.monocytogenes, respectively. Breer and Baumgartner  noted 2.3% of 263 salad vegetable samples were detective L.monocytogenes positive. De Simon et al.  reported the L.monocytogenes prevalence rate of 7.8% of vegetables samples in Spain. Gunasena et al.  reported 10 of 20 lettuce sample were positive for L.monocytogenes. Harvey and Gilmour  reported 25% L. monocytogenes prevalence rate in salad vegetables in Northern Ireland. In another study was undertaken Hesick et al. , L.monocytogenes contamination weredetected in 2 of 92 eggplant samples. MacGowan et al.  noted that 6.2% L.monocytogenes prevalence rate in vegetables in UK. Wong et al.  reported that 6 of 49 vegetable samples were detective L.monocytogenes positive. Our findings in this study showed contrary with these studies results.
Chai et al.  reported that 153 of 309 vegetables samples were detected Campylobacter spp. by polimerase chain reaction (PCR) techniques. Kumar et al.  reported that out of 56 vegetables samples examined, two (3.57%) revealed the presence of Campylobacter spp. Park and Sanders  reported that 1 of 40 leafy vegetables samples, 2 of 67 lettuce samples, 1 of 63 pepper and 2 of 60 spinach samples were positive for Campylobacter spp., respectively. Verhoeff –Bakkenes et al.  noted that 10 of 4691 (0.21%) vegetables samples, 2 of 790 (0.25%) fruits samples were positive for Campylobacter spp. Whyte et al.  reported that 2 of 279 (0.72%) vegetables samples were positive for Campylobacter spp.
The most important source of the Campylobacter species is birds, rodents, sheep and cattle intestinal tracts. Agricultural areas may attack by birds during the cultivation and/or storage; use of untreated manure may be source of this contamination .
In conclusion, considering the public health, fresh fruits and vegetables are common sources for various microorganisms and also pathogenic bacteria. Therefore, it is essential to ensure applying good agricultural practices GAP(s) and good manufacturing practices GMP(s) during production. Farmers should be informed about the sources of microbial contamination and should be trained in hygienic production. It is emphasized that whole fresh vegetables and fresh fruits should be protected from contamination by human, animal and other wastes which may constitute a hazard to health of the consumer through fresh produces. The use of poor microbiological quality of irrigation water should be avoided. On the other hand, post-harvest washing of fresh vegetables and fruits with organic antimicrobial agent and use of edible coatings containing antimicrobial is an important method for pathogen reduction.Transport vehicles and warehouse should be designed specifically for the transport and storage of fruits and vegetables. Also, strict temperature control from harvesting to consumption by farmer, handler, transporter, warehouseman etc. and consumers is highly important.