Microbiological analysis of fresh vegetables & fruits and effect of anti-microbial agents on microbial load

Pradnya A. Joshi*, Sonali P. Patel. Department of Microbiology, Birla College of Arts, Science and Commerce, Kalyan 421 304, India Email*: pradnyajoi@rediffmail.com, cool.sonaliap@gmail.com Tel.No:0251-2231196

Abstract : Fresh vegetables & fruits are exposed to potential microbial contamination. Consumer demand is to use fresh vegetables & fruits which are bacteriologically safe. Bacteriological survey of 45 samples of fresh vegetables & fruits, (15 collected from 3 different sources) was done. Samples were analyzed to study the density of microorganisms by standard plate count (SPC), yeast & mould count (YMC), coliform counts (CC). The high contamination & high number of pathogens on the surface of fresh vegetables & fruits in case of local market samples (log 9-10 cfu/ml) as compared to super market samples (log 5-6 cfu/ml). Various pathogens were identified from the surface of these samples. Escherichia coli, Staphylococcus aureus, Pseudomonas were found to be the pre-dominant species in most of the samples. 5 different anti- microbial agents Hydrogen Peroxide, Calcium Chloride, Benzoic Acid, Citric Acid, Ethanol were studied to decrease the microbial load of these samples. Samples rinsed with these solutions for 5mins. & washed with sterile distilled water; showed 40-80 % reduction in total number of organisms. Hydrogen Peroxide was found to be the most effective, where 80 % samples showed more reduction in total number of organisms. Results indicated that the microbial load in local market samples was very high as compared to super market & Hydrogen Peroxide can be used as rinsing agent to improve the quality of fresh vegetables & fruits. Key words : Contaminants, Pathogens, Standard plate count (SPC), Yeast & mould count (YMC), Coliform counts (CC), Anti- microbial agents.

Increasing health awareness has led to consumption of minimally processed foods in recent years (Wells and Butterfield 1997). Minimally processed foods or other raw vegetables have become popular since it suits the present day necessity as need of elaborate preparations are not required (Tournas 2005). Fruits and vegetables carry microbial flora while passing from the farm to the table. The produce is exposed to potential microbial contamination at every step including cultivation, harvesting, transporting, packaging, storage and selling to the final consumers. Microbial spoilage and contaminating pathogens pose a serious problem in food safety. (FDA 2000). The Centers for Disease Control and Prevention (CDC) estimate that there are 76 million cases of food borne illness each year. Outbreaks with identified etiology were predominantly of bacterial origin so that the study about microbial ecosystems on the surface of raw fruits and vegetables is necessary. As consumers, we need to recognize that food safety is important for fresh fruits and vegetables Food from sources like super markets (Spinach, Food land fresh) may be protected from contamination and spoilage (Ibenyassine 2007) during subsequent handling, packaging, storage and while in transit . In cities like Mumbai, with the growing population eating fresh fruits and vegetables has become very popular (Viswanathan and Kaur 2001). Thus comparative study of fresh foods from local market as well as from super market should be done. The lack of an effective antimicrobial treatment at any step from planting to consumption means that pathogens introduced at any point may be present on the final food product, fresh vegetables & fruits must be washed or treated specifically to minimize microbial load.(United States Patent 5869122).

The present work was carried out to compare & determine the total bacterial load of the fresh vegetables & fruits from local market & super market and use of anti- microbial agents to reduce the microbial population in samples. Materials and Methods : Sample collection & Preparation : Fifteen samples of fresh vegetables (10- Tomato, Spinach, Coriander, Potato, Cabbage, Beet, Capsicum, Cauli- flower, Brinjal, Peas) & fruits (5- Lemon, Sweet Lime, Banana, Chickoo, Apple ); each collected from local market & from 2 different super markets (totally 45). The samples were rinsed thoroughly with distilled water. And these were serially 10 fold diluted. Dilution was made depending on cell density. The highest three dilutions were taken for analyzing the total microbial count (Jarvis & Shapton 1986). Enumeration of microbes: Standard Plate Count (SPC) & Yeast & Mold Count (YC) were done on St. GYEA & St. Malt Extract Agar respectively by Spread Plate Technique & Coliform Count (CC) was studied on St. VRBA by Pour Plate Technique. (Koburger & Norden 1975). This count was carried out in triplicates. Isolation of Bacteria: The pathogens from the surface of samples were enriched & isolated on selective media & identification was done according to Bergey`s Manual of Determinative Bacteriology. Effect of anti-microbial agents : Samples were treated with 5 different anti- microbial agents- Hydrogen per-oxide, ethanol, Calcium chloride, Citrate, Benzoate with various concentrations ranging from

0.5-3 % (with 0.5 % interval). Samples were washed with these solutions for 5mins & again washed with Sterile Distilled water. 0.1 ml of these was taken (Test) & inoculated in 3 ml sterile nutrient broth. Control samples were washed only with Sterile Distilled water & then further treated as above. They were incubated at room temperature for 24 hrs. Density was checked colorimetrically at 540nm & effects of these agents were compared with control. Effect was studied on the basis of % decrease in microbial density, which was calculated by, Actual cell density decreased = (Cell density of control) - (Cell density at highest concentration) & then % decrease was calculated (Yueming Jiang 2004). Results and discussion : Standard Plate Count, Yeast Mold Count, coliform count was in the range of 9-10 log cfu / ml for local market sample. For Super market it was in the range of 5-6 log cfu / ml. The high log cfu / ml of viable counts of Local Markets could be because of unhygienic condition of local market & exposure during transport facility, improper storage condition etc. may add organisms. Generally, there is no pre-treatment given to fresh produce before transporting to retailer & hence number of contaminants is so large. Similar findings were observed by Richard L. Thunberg et al (2004). They have reported total viable count as 8.7, 8.6, 7.5, 7.4, and 6.3 log10 CFU/g for various samples collected from various retail markets. Low occurrence of contaminants on Super Market sample could be because the Fresh produce of Super Markets was treated with chlorinated water before it is transported to retailer (Information given by owner of Super Markets).

Escherichia coli, Staphylococcus aureus, were the common pathogens found on the surface of all the samples collected from all the sources. Salmonella spp & Klebsiella spp were found in 60 % of the local Market samples. On the other hand only 20 % of samples of Super Markets showed presence of these organisms (Table-1). Viswanathan and Kaur (2001) also showed presence of Salmonella, Serratia, Enterobacter, Staphylococcus aureus, faecal E. coli and P. aeruginosa in vegetables & fruits. These results indicate that as a consumer, one should buy fresh vegetables & fruits from Super Markets not from Local Market as microbiological quality of these are better in Super market compared to Local Market. All the anti- microbial agents showed maximum reduction in average microbial density at 3 % concentration as compared to microbial density of control (Table-2, fig-1). Hydrogen Peroxide (3 %) decreased the cell density from 0.51 (Control) to o.17 (Test), while other 4 anti- microbial agents decreased the cell density ranging of 0.22-0.27. The anti- microbial agents showed 40-80 % reduction in microbial load (Table-3). Effect of Hydrogen Peroxide (3 %) showed maximum decrease of microbial density in almost 80 % of the cases. Ethanol (3 %) being second effective compound for decreasing microbial density i.e. effective in 47 % of the cases. Benzoate Citrate & Calcium Chloride not showed much decrease in cell density as compared to other 2 agents. The effect of Hydrogen Peroxide (3 %) showed that, it can be used in combination with chlorinated water or alone while washing the fresh produce. This reduction in number of micro-organisms may be due to anti- microbial property of Hydrogen Peroxide. Similar results were reported by Dike O. Ukuku (2004) , revealed that Hydrogen peroxide (2.5%

and 5%) treatments of fruits for 5 min caused a 3 log10 CFU/cm2 reduction of the indigenous surface micro flora. The study sugge sts that fresh vegetables & fruits harbour high number of contaminants & pathogen in case of local market compared to super market samples, hence are more prone to spoilage making it necessary to process them before consumption. Thus use of hydrogen peroxide (As rinsing agent) in fresh vegetables & fruits for reduction of microorganisms can play an important role in food processing. Conclusion : Fresh vegetables & fruits of Local Market are harboring many microbial contaminants & pathogens as compared to of Super Markets, indicating that these are protected from contamination while subsequent handling, packaging, storage & transit. Thus, one should buy the Super Market fresh vegetables & fruits, as they are microbiologically safe. In India majority of the people preferred to buy the Local Markets fresh vegetables & fruits even though the quality of Super Markets fresh produce is better. However, the quality of fresh produce of the Local Market must be maintained in hygienic condition & proper handling, transport, storage must be controlled so that risk of contaminants decreases & chances of food borne outbreaks can be minimized. These can be done by pre-treatment of fresh produce by various anti- microbial agents to decrease the density of microbial contaminant from the surface of the fresh produce.

References : Dike O. Ukuku 2004 Effect of hydrogen peroxide treatment on microbial quality and appearance of whole and fresh-cut melons contaminated with Salmonella spp. International Journal of Food Microbiology .95 (2):137-146 Food Safety Article by World Health Organization 2006.

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NACMC 1999 Microbiological safety evaluation and recommendation on sprouted seeds. National Advisory Committee on Microbiological Criteria for Food. Int J Food Microbiol. 52:123-153.

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Treatment

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5869122

1996;

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Table-1 Samples showing presence of various pathogens (in %)

Organisms

Local Market

Food Land Fresh

Spinach

(%) Escherichia coli Klebsiella aerogene Staphylococcus aurues Proteus mirabilis Proteus vulgaris Salmonella typhi Salmonella paratyphi A Salmonella paratyphi B Shigella dysenteriae Vibrio cholerae Pseudomonas aeruginosa 100 60 100 13 27 53 40 53 47 80 100

(%) 100 33 100 33 47 6 20 20 20 33 60

(%) 100 33 100 33 13 20 20 20 20 26 33

Total Number of Samples-15

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Table-2 Effect of anti-microbial agents on microbial load (Average cell density for 15 samples)

CONC.

H2 02

Ethanol

Benzoate

Citrate

CaCl2

(%) 0.5 1.0 1.5 2.0 2.5 3.0 Control 0.45 0.40 0.33 0.25 0.23 0.17 0.51 0.48 0.42 0.35 0.27 0.24 0.22 0.51 0.51 0.47 0.43 0.37 0.31 0.27 0.51 0.50 0.45 0.39 0.30 0.25 0.24 0.51 0.51 0.48 0.41 0.34 0.27 0.26 0.51

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Table-3 Effectiveness of disinfectants on microbial density (in %)

Compounds

Range of % effectiveness

(Concentration- 3 %) Hydrogen peroxide Ethanol Calcium chloride Citrate Benzoate

(%) 58-80 46-65 45-69 49-66 40-63

Total Number of Samples-15

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Fig-1 Effect of anti-microbial agents on microbial load (Average cell density for 15 samples)

Effect Of Anti-microbial agents on Microbial load

0.6 0.5
Cell Density At 540nm

0.4 0.3 0.2 0.1 0 0.5 1 1.5 2 2.5 3

Concentration of anti-microbial agents H2O2 Ethanol Benzoate Citrate CaCl2 Control

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