Evaluation of the consumption and contamination level of Vegetables and Fruits in Ethiopia

Background; Vegetables and Fruits have got major acceptance in the modern scientic world nowadays and advisable to use more per daily food consumption. Although these food products have got more acceptances, their preparation and sanitation before consumption at the household level have to get proper attention. Objective: The objective was to assess the microbial load of vegetables and fruits which had been submitted at the Public health microbiology laboratory of Ethiopian Public Health Institute, from the year 2008- 2017, ( 10 years Retrospective data). Methods: Samples were tested for the presence of Mold, Yeast, Mesophilic aerobic bacteria, Total coliform, Thermo-tolerant coliform and the Indicator E.coli, to determine the contamination level based on the NMKL protocol. For viable bacteria count, APHA protocol was applied. Result: One hundred ninety-ve 5.9% (195/ 3279) raw and processed products had been received per ten years. Of these, 15% (29/195) of the samples revealed an intolerable microbial quality of the mesophilic aerobic plate count, followed by total coliforms 7.7% (15/195), thermo-tolerant coliforms 10.8 % (21/ 195), E.coli 3 % (6/ 195), mold count 1.5% (3/195) and yeast count 1.5% (3/195) (ICMSF protocol). Discussion and Conclusion: Although vegetables and fruits are currently proved to be the best healthy foods worldwide and are available with the low cost relatively, in the developing countries like Ethiopia, their consumption rate is overwhelmed by cereals and animal products based on the live status of the community. Therefore, encouraging use heart on sanitation procedures like immediate disinfection the community from environmental


Introduction
Fruit and vegetable cocktails are unfermented raw fruit or vegetable products prepared by cutting, mixing and/ or diluting (in case of fruits) of the product after removing the unwanted portion. Based on the global trend observed, unlike in the developing countries, fresh fruit and vegetable consumption rate increased by 25.8 and 32.6%, respectively in the United States (US) from the year 1970-2004. Thus it was predicted that it can be exceeded processed fruit and vegetable consumption if it continues with a similar tendency. It was very important to acknowledge such a positive shift in nutritional preference and diet selection, for every country. However, it was also described that, in the year 1995, 18.9 billion pounds of fresh fruits and vegetables were lost annually due to spoilage, which accounts for 19.6% of all US economic losses of edible foods for that particular year (European commission 2002).
Vegetables and fruits are easily invaded by many microbes since they grow friendly with the environment. Moreover, their tissue is composed of the polysaccharides cellulose, hemicellulose, pectin and starch which is their storage polymer. Therefore, they can be easily spoiled by bacteria and fungi species which have the particular extracellular lytic enzymes like pectinases and hemicellulase (European commission 2002, Asha 'et al.' 2014). The direct consumption by the consumers without heat treatment is the primary concern. The cleanness of all utensils used for squeezing/ mixing, types of the water samples used and their microbial content and loads, storage conditions until serving at the household level are also another concern. These steps may not be e cient to eliminate contamination of ready to eat (RTE) vegetables and fruits from parasites and viruses. Besides, the storage under refrigeration may favour the growth of psychotropic pathogenic and spoilage microorganisms.
Globally, signi cant food safety concern (Mortality and Morbidity) has been linked with the microbiological hazards. Illnesses associated by microbial degradation of such food samples by the microbial proliferation and their toxins lead to major outbreaks. Such contamination of fresh fruits and vegetables by various bacterial pathogens (Salmonella spp., E. coli O157: H7 and Shigella spp.) was seen (Rajvan Shi A 2010, Victor 'et al.' 2017, Mirtunjay 'et al.' 2015. Food-borne mold can produce mycotoxins, and some yeasts and mold are responsible for human and animal infections (Jeddi 'et al.' 2014). Microbes, mainly the coliforms group has been used extensively as an indicator of the main indicators of microbiological quality of water and food. Their presence indicates improper treatment or post-disinfection contamination (Badasa 'et al. ' , Sewan 'et al.' 2012.
Despite the overall prevalence of contamination seen, a notable seasonal trend was observed in the leafy vegetable groups and higher bacterial contamination rates were recognized in the summer. (Denis 'et al.' 2016, Back 'et al.' 2003. Although such outbreaks were infrequently reported in the country, the main objective of this evaluative study focused to assess the microbial quality of such vegetables and fruits by testing the common indicator and pathogenic organisms. Speci cally, to look into their distribution and to estimate the level of consumption rate in the country.

Materials And Methods
a. Study Design and sites of the study A retrospective study had been undertaken using a routine, previously analyzed and reported data. The study included all fresh vegetable and fruit samples which had been o cially submitted at Food microbiology laboratory of EPHI for routine testing within the indicated period. Samples from manufacturers or service providers OR from shops or different markets were collected using sterile plastic bags, by the health professionals. The samples were kept in appropriate storage condition during transportation time and until it was processed. Subsequently, it was submitted for different laboratories of EPHI, which one of them is Food microbiology laboratory. Then, each sample was tested by homogenizing of its contents and a portion from every ve units was taken into a sterile Petri dish (Composite). For detection of pathogens and indicators, 25 g of the composite representative sample was weighed and blended in 225 ml of sterile Buffer peptone and in 1% saline peptone solution under sterile conditions respectively.
All samples had been analyzed and checked for the presence of Mold/yeast count, Aerobic plate count, Total coliform count, thermotolerant coliform count and E.coli, based on the Nordic Committee for National Reference laboratory (NMKL) protocol. For viable bacteria count, the American Public Health Association (APHA) protocol was applied; using a pour plate enumeration culture technique. Pathogen detection (Salmonella and Shigella species) were processed following the instructions of NMKL protocol on XLD media after inoculating on the primary (Buffer peptone broth) and secondary (Selenite cysteine broth) enrichment broth.

Method used
Plate count agar (Oxoid CM463) was used for detection of the total aerobic bacterial count after incubation at 30 °C for 48 h. Both total coliforms and E.coli were determined using Violet Red Bile Agar (Oxoid, CM463) followed by incubation at 37 °C and 44 °C respectively. Typical colonies on the plates were enumerated and colony counts per 1 g sample were determined. Only typical red colonies were included for the calculations after con rming the formation of gas production in Brilliant Green broth (BGB) and EC broth respectively. To detect the presence of mould and yeast, Baird Parker selective agar with chloramphenicol supplement per 0.1 ml of sample suspension was used by streak plate method and after preparing a 1:10 dilution. The nal count then reported by multiplying with a total dilution factor. Staphylococcus and Staph aureus were also counted similarly using Mannitol salt agar after 1:10 dilution prepared with 1% Saline peptone. To isolate Salmonella and Shigella species 25 g of each vegetable and fruit samples were transferred to sterile plastic bags and homogenized with 225 ml of 1% (w/v) buffered peptone water (BPW) (Oxoid) and kept at room temperature for 30 min followed by incubation at 37 0 C. The analyses of pathogenic bacteria like Salmonella, Shigella were performed using primary and secondary enrichment broth and con rmed by isolation, puri cation and identi cation on XLD selective media and Nutrient Agar. Results were obtained as presence or absence of Salmonella species in 25 g of vegetables and fruit samples.

Data quality and Statistical analysis
Suspected colonies from each category were picked and further checked using different biochemical media for con rmation and the report had been made per nal volume. For determining the testing e ciency of each culture media/ broths, S. aureus and E.coli standard strains were used as a positive control for Gram-positive and Gramnegative organisms' detection respectively. SPSS version 20.00 had been used for statistical analysis and to test the values of the signi cance of the results respectively. Unacceptable food types over the total were calculated to nd the single proportion value (P) followed by calculating the standard error of the proportion using the following formula. SE = square root of (P x (1-P)/ N). The P values < 0.05 was taken as statistical signi cance.
This research was ethically cleared by EPHI Scienti c and Research Ethical Clearance Committee (SERC).

Results
A total number of one hundred ninety-ve freshly eaten vegetables and fruits were received per ten years, which is around 5.9% (195/ 3279), of all food types which had been submitted during the indicated period. Twenty-four solely served or combined commonly used vegetables and fruits were tested for determining their microbiological contamination level (Table 1). Around 90% of the samples were collected from Addis Ababa city, six samples from SNNPR and one from Harar town.

Conclusions And Recommendation
Consumption of fruits and vegetables provide more antioxidants and vitamins for the body and support in preventing health risk diseases associated with consuming more heavy and fatty foods (animal products). These make them to be selected to use more recently. Most people become vegetarian or Lacto vegetarian to avoid such general risks especially when they become around adulthood ages.
Although, such food products have got more acceptances on a daily basis, their preparation and sanitation before consumption at household level has to get proper attention. Consideration has to be given to the dangerous habit of the Ethiopian custom of consuming raw food without treating with insu cient heat or detergents as nationwide. This is because vegetables and fruits which are sold in the market can bring other side health risks since it mostly acts as a reservoir for many microorganisms. In most of the study, it was reported that higher microbial loads as the result of using different water sources, from the rivers, ponds or other similar sources which have easily in contact with animal fecal matters. Their contact with dust and soil has also played a major role as a contamination source since soil harbours millions of parasites and bacteria which are capable of surviving these environments, like roundworms and several bacterial pathogens.
On the other hand, the existing regulatory system of food products in Ethiopia has to be actively engaged in all regions like that of Addis Ababa. Capacitating the laboratory facilities and skills of the personnel in regional Food microbiology laboratories throughout the country has to get series devotion.
In summary, joint efforts have to be exerted by different branches of MOH for encouraging the community to use vegetables and fruits as its main food source for better health and to reduce nasty illnesses like diabetes, obesity, heart diseases and so on. However, while doing so, series health education has to be provided on sanitation This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors. Figure 1 Unacceptable number of bacteriological and mycological contaminants of fruits and vegetable samples (ICMSF reference protocol was used per this food type category) Figure 2 Trends of Veg & Fruit samples submitted in comparison with other food samples per ten years