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Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 1984-1992 International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 10 Number 02 (2021) Journal homepage: http://www.ijcmas.com Original Research Article https://doi.org/10.20546/ijcmas.2021.1003.253 Kinetics of Microorganisms in Ready-To-Eat Salads Stored at 4°C Sold in Supermarkets in the City of Abidjan (Côte d’Ivoire) Djédoux Maxime Angaman1*, N’goran Parfait N’zi1,2 and Valérie Carole Gbonon2 1 Department of Biochemistry-Microbiology, Laboratory of Agrovalorization, University Jean Lorougnon Guédé, Daloa, Côte d’Ivoire 2 Department of Bacteriology-Virology, National Reference Center for Antibiotics, Pasteur Institute of Côte d'Ivoire, Abidjan, Côte d'Ivoire *Corresponding author ABSTRACT Keywords Fourth range salads, Mesophilic and psychrophilic bacteria, Fungal flora Article Info Accepted: xx February 2021 Available Online: xx March 2021 Consumption of ready-to-eat salads is increasingly observed in large African cities, in particular Abidjan (Côte d’Ivoire). Thus, monitoring the microbiological quality of these salads packaged in polyethylene terephthalate (PET) plastics is a necessity in order to guarantee their compliance and prevent occurrence of food poisoning. The objective of this work was to assess microbiological quality of fourth-range salads sold in supermarkets in the City of Abidjan during the storage period at 4°C after opening the packages. Our study focused on 4 types of salads, Spinach, Lamb’s lettuce, Lamb’s lettuce and Arugula and Young shoots (lettuce, arugula, spinach, red chard, bull's blood) bought in Abidjan’s supermarkets. We proceeded to an enumeration of mesophilic and psychrophilic aerobic bacteria as well as yeasts and molds by an interval of 3 days. The results revealed the presence of these microorganisms as soon as the packages were opened. Most of ready-to-eat salads were of satisfactory quality from the opening of the packaging but not on the 2nd and 3rd day of analysis. However, composition of salads and shelf life favored the growth of microorganisms. Good practice guides for the purchase, storage and consumption of these foods must be put in place to prevent poisoning. Introduction Fourth range of food products concerns products that are pre-cleaned, mainly packaged and ready to be eaten without preparation or prior cooking (Paudya et al., 2017). Nowadays, the changing lifestyle of consumers and the great attention paid to healthy and nutritious foods have widened the demand for fourth range products (Cofelice et al., 2019). Fourth range therefore plays an important role in the food industry because it meets the demand of consumers, who have little time to cook, but who want to follow a 1984 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 1984-1992 healthy and balanced diet, without having to spend a lot of time in the kitchen. In addition, salads of fresh fruits and vegetables are the most preferred because the WHO, FAO and the World Cancer Research Fund recommend the consumption of 400 to 600g of fruits and vegetables per day (Pollard et al., 2009; Adjrah et al., 2011). However, the production and distribution of these products requires the use of a specific manufacturing process, all phases of which are governed by common health and safety rules (OESAAS, 2018). Despite the many advantages of fourth-range salads, several studies have shown their major involvement in food-borne epidemics (Zhang et al., 2012; MacDonald et al., 2016; Espenhain et al., 2019). In Africa, the prevalence of foodborne pathogens in foods in some African countries is the high prevalence of major pathogens in ready-to-eat foods, almost as high as in raw foods (Paudyal et al., 2017). In Côte d'Ivoire, the trend to move towards a healthy lifestyle is pushing the population to take more and more interest in ready-to-eat salads (fourth range). We are therefore witnessing a flood of several types of salads of various origins in supermarkets in Abidjan. However, consumers sometimes lack knowledge about adequate refrigeration (Marklinder et al., 2004). Anin et al., (2016) assessed the microbiological contamination of certain artisanal fourth range products sold on local markets in Abidjan. Also, Toe & Dadié (2018) determined the prevalence and potential virulence of Escherichia coli in ready-to-eat raw vegetable salads in collective catering in Abidjan. However, scientific data on kinetics of microflora in fourth range salads are not yet available. That’s why we determine devolution of the microflora growth according to conditions of domestic storage over time after packaging opening. Materials and Methods This study took place over a three-month period from July to September 2020. Collection of salads packaged in polyethylene terephthalate (PET) plastics was purchased in supermarkets of Abidjan (Côte d’Ivoire). Indeed, the salads selected are of 4 types (2 from one type of salad, one with 2 types of salads and last one with 5 salads) and have foreign origins (Europe). After collection, samples were stored at -80 ° C. Microbial evolution was determined by time interval (Day 0; Day 3; Day 7). Enumeration of microorganisms focused on mesophilic aerobic flora, psychrotrophic aerobic flora and fungal flora. Techniques used for enumeration refer to AFNOR standards and ISO standards. First, decimal dilutions were carried out in accordance with the AFNOR NF V 08 010 standard of March 1996. A quantity of 25 g of products was diluted in a volume of 225 mL of buffered peptone water, From the suspension obtained (stock suspension), a series of dilutions was carried out in the order of 10-1 to 10-6. Enumeration of mesophilic aerobic flora was carried out on PCA Agar according to UNI EN ISO 4833-1: 2013 standard, incubated at 30 °C for 72 h. Psychrophilic aerobic flora was also counted on the PCA agar according to ISO 17410 (2001) incubated at 6.5 °C for 72 h. As for fungal flora, it was counted on Sabouraud-Chloramphenicol Agar according to ISO/IEC 17025 at 25 °C for 72. Presumptive colonies are counted and then average concentration or bacterial load was reported according to ISO 7218 (2007). The data processing was carried out using two software programs, Excel 2016 and Statistica 7.1. Fisher's LSD Test was performed to distinguish differences between averages. 1985 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 1984-1992 Results and Discussion Microorganisms storage periods growth evolution during Spinach fungi flora was not detected. During storage at 4 °C, mesophilic aerobic flora reproduced significantly from day 3 to day 7.Psychrophilic flora also developed significantly throughout storage period (Table 3). The average loads of microorganisms in spinach sample during storage period are listed in Table 1. On opening the packaging of these samples, the revivable microorganisms were counted. The results show that microbial loads varied between 6.80 102 CFU/g and 2.97 105 CFU/g. During storage period (4°C), mesophilic and psychrophilic aerobic flora developed slowly until the 3rd day (D3).However, after the 3rd day to the 7th day of storage, significant growth was observed in psychrophilic aerobicbacteria while mesophilic remained slow. This salad was also marked by a low load of yeast and mold when opening the package. But this flora underwent significant growth during storage at 4 °C. Mixture of young shoots Lamb’s lettuce Mesophilic aerobic flora Lamb's lettuce when opening the packaging had a very low average load of mesophilic aerobic flora (7.80 102 CFU/g) as did psychrophilic aerobic flora (5.90 102 CFU/g). With regard to the aerobic mesophilic flora, the salad consisting of lamb's lettuce and arugula showed a statistical difference in the average loads during storage time. In this sample the number of mesophilic bacteria which was low when the packaging was opened reproduced slowly on the 3rd day and very rapidly on the 7th day of storage. Psychrophilic bacteria, also with a low initial load, experienced rapid growth from the 3rd day until the 7th day of storage. However, the count did not reveal the presence of mold and yeast in this type of salad (Table 2). It follows that lamb’s lettuce salad showed significant growth on the 7th day. Young shoots (although having high values) and spinach salads did not obtain significant growth over time (Figure 1). Lamb's lettuce and Arugula Microbiological analyzes revealed low average microbial loads varying between 1.20 103 CFU/g and 1.82104 CFU/g. However, The enumeration of this salad gave very high bacterial loads, 1.73 108 CFU/g for the mesophilic flora and 2.81 107 CFU/g for the psychrophilic flora. Yeast and mold had a load of 4.20 102CFU/g. During the storage period, psychrophilic bacteria developed significantly unlike the mesophilic flora and fungal flora which developed slowly (Table 4). Comparison of the evolution of microbial growth in different salads Psychrophilic aerobic flora Psychrophilic bacteria showed significant growth in all of our samples. The increase in average loads was a function of the number of components in the package. This is how the salad made from young people growing with 5 elements (lettuce, arugula, spinach, red chard, bull's blood) had the highest values (figure 2). 1986 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 1984-1992 Table.1 Microbial evolution in spinach during storage time. MAF: Mesophilic Aerobic Flora, PAF: Psychrophilic Aerobic Flora, FF: Fungal Flora Days Flora MAF (CFU/g) PAF (CFU/g) FF (CFU/g) D0 D3 D7 2.97 105a 2.80 105a 6.80 102a 3.29106 a 3.37106 a 1.50103b 5.93107a 4.64107b 2.72103c Values followed by the same letter on the same lines are not significantly different at the 5% level according to Fisher's LSD test. Table.2 Microbial evolution in Lamb’s lettuce during storage time. MAF: Mesophilic Aerobic Flora, PAF: Psychrophilic Aerobic Flora, FF: Fungal Flora Days Flora MAF (UFC/g) PAF (UFC/g) FF (UFC/g) D0 D3 D7 7.80 102a 2.55106a 3.16×107 b 5.90 102a <1 3.04×106 b <1 1.26×107c <1 Values followed by the same letter on the same lines are not significantly different at the 5% level according to Fisher's LSD test. Table.3 Microbial evolution in spinach during storage time. MAF: Mesophilic Aerobic Flora, PAF: Psychrophilic Aerobic Flora, FF: Fungal Flora. Days Flora MAF (UFC/g) PAF (UFC/g) FF (UFC/g) D0 D3 D7 1.20 103 a 1.82104 a <1 2.95106 b 3.25107 b <1 1.68107c 1.60108c <1 Values followed by the same letter on the same lines are not significantly different at the 5% level according to Fisher's LSD test Table.4 Microbial evolution in young shoots during storage time. MAF: Mesophilic Aerobic Flora, PAF: Psychrophilic Aerobic Flora, FF: Fungal Flora Days Flora MAF (UFC/g) PAF (UFC/g) FF (UFC/g) D0 D3 D7 1.73108a 2.81107 a 4.20102 a 2.77108a 1.24108b 7.50102 a 2.96108 a 2.64108c 1.020103a Values followed by the same letter on the same lines are not significantly different at the 5% level according to Fisher's LSD test 1987 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 1984-1992 Fig.1 Evolution of the mesophilic aerobic flora as a function of time in the different types of sample.* p ≤ 0.05 * * Fig.2 Evolution of the psychrophilic aerobic flora as a function of time of the samples from batch 1. * p ≤ 0.05 * * * * 1988 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 1984-1992 Fig.3 Evolution of the fungal flora as a function of the time of the samples. * p ≤ 0.05 * Fungal Flora (FF) Only spinach had significant growth with yeasts and molds (figure 3). In general, the values with yeasts and molds were of the same order. Lamb’s lettuce and Arugula had low values. In this work, we analyzed the microbiological quality of ready-to-eat salads sold in supermarkets in Abidjan and the evolution of microbial growth monitored after opening the packages. Thus, the count of spinach salad showed that this salad was of satisfactory microbiological quality when packaging was opened due to the microbial load which varied between 6.80 102 CFU/g and 2.97 105 CFU/g. Indeed, according to microbiological criteria in accordance with regulation 2073/2005/EC, average acceptable loads in total aerobic flora and fungal flora for fresh little processed foods do not exceed 107 CFU/g and 104 CFU/g respectively (CMADA, 2018). Our results are different to Mritunjay & Kumar(2017) who showed the highest microbial counts in samples of spinach. According to them, this may be linked to poor handling practice during storage and at the point of selling. Valentin-Bon et al., (2008) also demonstrated a strong microbial contamination of this food. The difference observed when opening the packaging of our spinach sample would probably be due to a more controlled situation in the production and distribution chain such as type of washing recommended, stability of the storage temperature, good packaging and shelf life of the product. Indeed, the commercial atmosphere modified by 0.25-3% O2 and 312% CO2 with a balance in N2 (O'Beirne et al., 2015), storage at a maximum temperature of 4 ° C and a shelf life between 7 to 10 days (De Giusti et al., 2010) would be necessary to control spoilage of minimally processed foods. Soendjojo (2012), for its part, showed a low average load of yeast and mold in spinach from grocery stores, which confirms the low average loads of fungal flora obtained in our sample. During the storage period at 4 °C, the growth of the mesophilic aerobicflora was slow while this of aerobic psychrophilic flora was very significant. Garg et al., (1990) 1989 Int.J.Curr.Microbiol.App.Sci (2021) 10(03): 1984-1992 showed in their work that the storage of spinach salads at refrigeration temperature promotes the growth of psychrophilic microorganisms. However, mesophilic bacteria grew slowly at a low temperature which is in line with our results. quality of fresh and minimally processed fruits and vegetables, and sprouts from retail establishments. Regarding the salad composed of lamb's lettuce and arugula, it was also of satisfactory quality when opening the packaging but from the 3rd day of storage it proved to be unsatisfactory due to a significant multiplication of the psychrophilic flora. This result is similar to those of Schuh et al., (2020) on the evaluation of microbiological quality of minimally processed vegetables. The strong growth of the different bacterial flora conferred an unsatisfactory quality on these two salad samples from the 3rd day of storage. Our results are substantially similar to those of Arienzo et al., (2020), who showed that after 2 days of storage at 4 °C, these same types of products analyzed presented an unsatisfactory microbiological quality. This strong contamination could be linked to growing conditions and also to processing operations, such as delays in post-harvest refrigeration or processing after receipt of vegetables in the factory (Caponigro et al., 2010). Which is in agreement with the work of Akoachere et al., (2018) who showed that hygiene and poor storage practices for vegetables can aggravate bacterial contamination. Caleb et al., (2012) also showed that the packaging of fresh, minimally processed products (4th range) must have a specific temperature. However, excessive storage temperature would lead to bacterial growth. Delibato et al., (2018) also showed in their study on the persistence of microbial contamination of products that complete decontamination was not achieved in ready-toeat fresh plant products despite the recommended types of washing. Low temperature storage during our analyzes could also inhibit the growth of the mesophilic aerobicflora as described by Garg et al., (1990). In general, psychrophilic aerobic flora developed more rapidly in the mixed salad composed of young shoots than in the other samples. This result would probably be due to the composition of the salad which could act on the pH. Indeed, an acidic food promotes a significant growth of psychrophilic lactic acid bacteria. Garcia-Gimeno & Zurera-Cosano (1997), showed that the spoilage of mixed salads stored at a temperature of 4 °C was linked to lactic acid bacteria. Foryoung shoots, studies have shown high concentrations of mesophilic aerobic flora (1.73108 CFU/g) and psychrophilic aerobic flora (2.81107 CFU/g) when opening the packaging. Abadias et al., (2008) produced similar results in salads from young shoots during their work on the microbiological The results of the microbiological study showed the presence of mesophilic and psychrophilic aerobic bacteria as well as fungal flora in the salads. When packaging was opened the greatest loads of Mesophilic (1.73 108 CFU/g) and psychrophilic germs (2.81 107 CFU/g) were found in young shoots For lamb's lettuce, microbiological analyzes showed that it was also of satisfactory quality because of the absence of fungal flora and its very low average load in mesophilic aerobic flora (7.80 102 CFU/g) and aerobic psychrophilic flora (5.90 102 CFU/g) which comply with microbiological criteria. However, from the 3rd day of storage, an exponential growth was noticed in psychrophilic aerobic flora. 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How to cite this article: Djédoux Maxime Angaman, N’goran Parfait N’zi and Valérie Carole Gbonon. 2021. Kinetics of Microorganisms in Ready-To-Eat Salads Stored at 4°C Sold in Supermarkets in the City of Abidjan (Côte d’Ivoire). Int.J.Curr.Microbiol.App.Sci. 10(03): 1984-1992. doi: https://doi.org/10.20546/ijcmas.2021.1003.253 1992