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African Journal of Food Agriculture Nutrition and Development, Vol. 4, No. 1, 2004 MICROBIOLOGICAL QUALITIES OF GREEN CHUTNEYS SOLD IN PATIALA CITY, INDIA La qualité microbiologique des condiments verts vendus dans la ville de Patiala (Inde) Bansal S1, Ghosh M1 and A Ganguli*1 *Corresponding Author Email: aganguli@mail.tiet.ac.in Code Number: nd04008 ABSTRACT The purpose of this study was to determine the microbiological quality of green chutney, a popular traditional Indian food sold by the street vendors in Patiala city. Street vendors serving at least 100 customers, comprising of upper, middle and low-income groups per day were identified in five major areas of the city (which represented almost the whole of the city). A total of 150 samples of green chutney were collected in sterile containers, transported to the laboratory on ice and analyzed within two hours using standard microbiological methods- Total Aerobic Plate Counts (TAC), Total Staphylococcal Counts (TSC), Total Faecal Coliforms Counts (TFCC) and Total Faecal Streptococcal Counts (TFSC) as well as for specific bacterial pathogens: Salmonella, Shigella and enterotoxigenic S.aureus. For detecting Salmonella, portions were pre-enriched in Universal pre-enrichment broth followed by enrichment on Rappaport Vassilidis medium and selection on Xylose-Lysine Deoxycholate agar, presumptive colonies were serotyped; for Shigella loopful of enrichment broth were streaked onto Salmonella, Shigella and Xylose –Lysine Deoxycholate agar and presumptive colonies were subjected to requisite biochemical tests. S.aureus was selected on Baird –Parker agar, characterized for production of coagulase, Thermostable DNAse activity, catalase and hemolysine production and mannitol fermentation. Colonies of coagulase positive S.aureus were further tested for the production of enterotoxins A, B, C and D by reverse passive latex agglutination kits (SET –RPLA). The mean TAC and TSC values obtained were 5.4 (±0.4) log cfu/g and 5.9 (±0.2) log cfu/g; whereas the mean TFCC and TFCC values were 4.8 (±0.4) log cfu/g 4.9 (±0.3) log cfu/g, respectively. Coagulase positive S.aureus was detected in 66 (44%) samples, of which 27 produced enterotoxins A and B. None of the samples showed the presence of Shigella, however, Salmonella enteridis was found in 3 (2%) samples. The results of this study demonstrate the poor microbiological status of a popular, traditional Indian street vended food which is consumed raw, and suggest government intervention to ensure that the standards of safety are attained for this food. Key words: Green chutneys, Street foods, Salmonella, S.aureus, E.coli RESUME La présente étude avait pour objectif la détermination de la qualité microbiologique du condiment vert, un aliment traditionnel indien largement vendu par les marchands ambulants dans la ville de Patiala. Les vendeurs ambulants ayant au moins 100 clients par jour appartenant aux groupes à hauts revenus, à moyens et à faibles revenus ont été identifiés dans les cinq principaux quartiers de la ville (représentant presque toute la ville). Au total 150 échantillons de chutneys verts ont été collectés dans des récipients stériles, transportés en laboratoire sur la glace et analysés en l'espace de deux heures en utilisants des méthodes classiques d'analyse microbiologique : dénombrement des bactéries aérobies totales sur plaque (TAC), dénombrement de staphylocoques totaux (TSC), dénombrement de coliformes fécaux totaux (TFCC) et le dénombrement de streptocoques fécaux totaux (TFSC). Ont été également soumis à cette analyse les bactéries pathogènes suivantes : S almonella, Shigella et le S.aureus producteur d'entérotoxines. Pour le dépistage des Salmonella , des portions ont été enrichies d'avance dans du bouillon de pré-enrichissement universel suivi par l'enrichissement sur milieu de Rappaport Vassilidis ; et pour l'isolement sur gélose Xylose-lysine Desoxycholate, les colonies présumées ont été sérotypées. Pour l'identification des Shigella le bouillon d'enrichissement a été ensemencé sur une gélose Salmonella-Shigella et Xylose-lysine Desoxycholate et les colonies présumées ont été soumises aux tests biochimiques requis. S.aureus a été isolé sur une gélose Baird-Parker et a été caractérisé pour déceler la production de coagulase, l'activité d'une ADNase thermostable, la production de catalase et d'hémolysine et la fermentation du mannitol. Les colonies de S.aureus à coagulase positive ont été en outre analysées pour détecter la production des entérotoxines A, B, C et D par des kits d'agglutination passive inverse au latex (TROUSSE - APIL). Les valeurs moyennes en TAC et TSC obtenues étaient de 5.4 (± 0.4) log cfu/g et 5.9 (± 0.2) log cfu/g tandis que les valeurs moyennes en TFCC et TFCC étaient de 4.8(± 0.4) log cfu/g 4.9(± 0.3) log cfu/g. S. aureus à coagulase positive a été isolé dans 66 (44%) échantillons et la production des entérotoxines A et B a été décelée dans 27 échantillons. Aucun échantillon n'a révélé la présence de Shigella; cependant, Salmonella enteridis a été détecté dans trois (2%) échantillons. Les résultats de l'étude révèlent une mauvaise qualité microbiologique d'un aliment populaire traditionnel indien vendu dans les rues et consommé cru. Les résultats préconisent l'intervention du gouvernement pour que des normes de sécurité soient atteintes pour cet aliment.Mots-clés : Condiments verts, aliments de rue, Salmonella, S.aureus, E.coli INTRODUCTION Being ubiquitous in distribution, microorganisms may contaminate and grow in many food products. The entry of microorganisms into the food chain can occur from various sources during different stages of processing, storage and serving. Additionally, by providing a suitable nutritional and physical environment for growth and multiplication of microorganisms, the foods possess an inherent capacity to sustain them in large numbers. The possible routes of contamination of foods include environmental sources such as soil, dust and air, water, through food handlers or unhygienic practices, raw materials and materials used in preparing the food and also utensils or other articles used during serving of foods[1,2,3]. In developing countries like India, with the changing life styles, a huge section of people, including those in the metropolis have started eating ready-to-eat foods, especially those sold on the streets; most of which are accompanied with green chutneys. Green chutneys are prepared by blending leaves of coriander, mint and green chilies, approximately in a ratio of 3:1:1 for about 5 minutes followed by addition of water, just sufficient to obtain a paste; requisite taste is obtained by further addition of table salt and tamarind extract. Green chutneys have a very high consumer preference in the Northern; Western and Eastern parts of India and like all street foods, are exposed to the abundant sources of contamination. Surprisingly, little work has been done on the microbiological qualities of green chutneys in India. In the present study, the authors selected the city of Patiala (Punjab) where green chutneys are greatly appreciated and consumed with traditional ready -to-eat foods by people of all age groups. MATERIALS AND METHODS Sample collection A total of 150 samples were collected from 5 major areas, which represented almost whole of the city and comprised of shops which prepared ready-to-eat foods and served at least 100 customers (comprising of upper, middle and low-income groups) per day. All samples were collected from the vendors in pre-sterilized 250 ml bottles and transported to the laboratory on ice. The samples were analyzed within 1-2 hours of procurement. Portions of chutneys weighing 25 g were diluted with 250 ml of Sterile Butterfields phosphate buffer, blended for 2 minutes and subsequently diluted tenfold with the same. Appropriate dilutions were then enumerated for total aerobic plate counts using Tryptone Soya agar, total faecal coliforms using Violet Red Bile agar, total faecal Streptococcus using Bile Esculin Azide agar and Staphylococcus aureus using Baird –Parker agar. Inoculated plates were incubated at requisite time-temperature combinations [4, 5, 6]. For detecting the presence of Salmonella, 10g portions of chutneys were pre-enriched with 100 ml Universal preenrichment broth for 24 hour at 37 oC, 0.1ml of the preenriched culture was inoculated in semisolid Rappaport Vassilidis agar and incubated at 42 oC for 24 hour, loopful portions of the latter were streaked on Xylose –Lysine Deoxycholate agar; presumptive isolates of Salmonella were sent to the National Centre for Escherichia coli and Salmonella, Central Research Institute, Kasauli, India for serotyping and further confirmation. Presence of Shigella was assessed by streaking a loopful of enrichment broth onto plates of Salmonella-Shigella agar and XLD agar- colonies exhibiting typical characteristics of Shigella were isolated and confirmed by biochemical tests. Isolates of S.aureus obtained on Baird –Parker agar were characterized for their production of coagulase, Thermostable DNAse activity, catalase, hemolysine production and mannitol fermentation [4,5]. The production of enterotoxins, A, B, C and D by all isolates of coagulase positive S.aureus strains were confirmed using a reversed passive latex agglutination based kit (SET RPLA, Oxoid). RESULTS Quantitative analysis revealed that the trends for total aerobic counts (TAC) were around 5 log cfu/g, 6 log cfu/g of TSC, 5.2 log cfu/g of TFCC and 4.97 log cfu/g of TFSC (Table: 1).Total Staphylococcal counts of all chutney samples were around 6 log cfu/g; 44% of the total Staphylococcus were identified to be coagulase positive S.aureus, of these 27 produced enterotoxins A and B. The total faecal coliforms counts were found to be around 5 log cfu/g. Qualitative analysis showed the presence of Salmonella enteridis in three samples. DISCUSSION Since green chutneys are prepared as pastes of coriander, mint, and green chillies and undergo no processing; the presence of S.aureus might be due to the contaminated raw ingredients, poor handling and improper storage. The presence of high numbers of coagulase positive S.aureus in green chutneys is of concern as S.aureus is an important food-borne pathogen and staphylococcal food poisoning ranks as one of the most prevalent causes of gastroenteritis worldwide [7]. The presence of faecal coliforms and Salmonella enteridis in the samples further confirms the poor microbiological qualities of these products. Hazard Analysis and Critical Control Points- Total Quality Management lay down the microbial qualities of raw food, according to which, food containing < 4 log10 cfu/ml of organisms is rated “good” and those containing >approximately 8 log10 cfu/ml is rated as “spoiled food”, also, the suggested level of E.coli for the purchaser is > 10cfu/g. Although not spoiled, in comparison to the average total aerobic counts obtained for Indian street vended salad vegetables, green chutneys had lower total aerobic counts [9]. However the presence of Salmonella, the high coliforms counts (5 log cfu/g) prevalence of both E.coli and Streptococci and the incidence of enterotoxigenic S.aureus, indicate the possible potential of green chutneys to cause food-borne illness. This study revealed the potential hazard of street-vended green chutneys- the prevalence and growth of bacterial pathogens on street vended salad vegetables, fruits and sprouts in India is not uncommon [8,9]. Since, green chutneys are eaten raw and undergo no treatment either before eating or during preparation and usually consumed with almost all ready-to-eat foods sold on the street, they can serve as important vehicles for food-borne pathogens. Additionally, people of almost all ages and income groups have a high preference for this product – possibility of food related outbreaks of diseases cannot be ruled out. Like other Asian, African and Latin American developing countries the street vended foods form a significant part of urban and semi-urban food supplies in India [10, 11, 12].Therefore if the consumer is to get the full benefit of street vended foods (like green chutneys) with minimal risks of diseases, government intervention is required to ensure that the standard of safety of such foods is attainable. ACKNOWLEDGEMENTS The authors wish to thank the Head, Departmentof Biotechnology & Environmental Sciences, Thapar Institute of Engineering and Technology, for providing the requisite financial and infrastructural support. REFERENCES
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