The microbial distribution and antimicrobial effect of AgZnO and AgTiO nanoparticles against microorganisms isolated from floury snacks sold in Federal University of Technology, Akure (FUTA) was investigated. Three common floury snacks viz; meat pie, eggroll and puff-puff were collected from these vendors and examined for microbial load and types using standard methods. The microbial load recorded ranged from 1.2x10² to 1.8x10⁴ cfu/g. The microbial load of puff-puff was the lowest (1.2x10²–1.9x10³ cfu/g). The microorganisms isolated from these products include, Staphylococcus aureus, Staphylococcus epidermidis, Bacillus spp, Serratia marcescens, Aspergillus niger and Aspergillus fumigatus. The microbial isolates showed varying degree of resistance to commercial antibiotics while the AgZnO and AgTiO nanoparticles were able to inhibit all the isolates with zones of inhibition ranging from 9mm to 20mm at 100mg/mL. The results from this study revealed that AgZnO and AgTiO nanoparticles (NPs) has significant antimicrobial activity on microorganisms isolated from floury snacks, however, the issue of safety is still very crucial.

Keywords: antimicrobial, nanoparticles (NP), floury, snacks, microorganisms

Street foods are edibles prepared by vendors for immediate consumption at the point of sale. These foods could be raw or cooked, hot or chilled and can be consumed without further processing.¹ Floury snacks such as meat pie, puff-puff, sausage, and burger form major part of these readymade street foods. Street foods are common in Nigeria and many developing countries. They are prepared and sold in public places such as schools, markets, pupil school’s gates, campuses, interstate highways and stalls at corner of the streets to attract the consumers. Fast food enterprise is one of the major businesses that contribute to the socio-economic development in many countries.²

These street foods and fast foods had been the choice of people in the urban areas because the food is ready-to-eat, cheap, convenient, sweeten with a different flavor and accessible as immediate want.^3‒5 However, the preparation of some of street foods do not meet the sanitary qualities and specification of food safety bodies.⁶ Street foods have partially alleviated the problem of food insecurity and hunger by its steady availability but; the major concerns is its hygienic status during production and marketing system.⁷ The incidence of foodborne diseases is increasing rapidly due to unhygienic methods of processing food.⁸ The following microorganisms Staphylococcus aureus, Escherichia coli, Salmonella typhi, Shigella dysenteriae, Klebsiella pneumoniae, Proteus vulgaris, Enterobacter aerogenes, Streptococcus lactis, Pseudomonas aeruginosa, Bacillus spp, Vibrio parahaemolyticus, Saccharomyces cerevisiae, Aspergillus spp, Penicillium spp, Rhizopus stolonifer, Mucor mucedo and Candida albicans were isolated from street foods sold in Akure metropolis.⁷ The authors also reported that the isolates showed varying degree of resistance to commercially available antibiotics.

In the last three decades, the issue of antibiotic resistance has taken the centre stage. This is because most microbial etiologic agents of foodborne diseases have developed resistance to commonly used antibiotics.⁹ It has been reported that the transmission of antibiotic resistance genes to another pathogenic organism has contributed to food illnesses, higher morbidity and mortality rates.¹⁰ There is a continuous search for alternative antimicrobial agent that will be more effective than commonly available antimicrobial agent. Nanomolecules are considered as an alternative to conventional antimicrobial agents. Nanomolecules can be synthesized from polymers, lipids and metals.¹¹ The present study was therefore designed to investigate the microbial quality and susceptibility pattern of isolates from floury snacks to AgZnO and AgTiO nanoparticles (NPs).

Sample collection

Ready to eat floury food samples such as egg roll, meat pie, puff-puff were collected from South gates, Oba-Kekere and Oba-Nla sales point at the Campus of The Federal University of Technology, Akure. Collection was done in the morning and at evening. Food samples were aseptically collected from October 2017 to March 2018. The food samples in ice bag were transferred to the laboratory for immediate analysis.

Isolation of microorganisms

Floury food samples collected were homogenized, and one gram of each homogenate was weighed out into 10 ml of sterile water as a stock solution. Serial dilution was carried out to obtain appropriate aliquot. Serially diluted sample (1.0 ml) was transferred into Petri dish containing Plate Count Agar (PCA, Lab M) for bacteria and Potato Dextrose Agar (PDA, Lab M) for fungi. The plates were incubated at 37°C for 24 h and 48 h for bacteria and fungi, respectively. At the end of the incubation period, colonies were counted using the colony counter (TT-20, Techmel and Techmel, USA). The counts for each plate were expressed as colony forming unit per gram (CFU g-1) for bacteria and spore forming unit per gram (SFU g-1) for fungi. Microbial growth from the plate was subcultured to obtain pure cultures and these were kept at 4^oC for further study.

Identification of microorganisms

For the preliminary identification was carried out using Gram stain technique and biochemical tests^12,13 and the results obtained were interpreted according to Bergey’s Manual of Systematic Bacteriology.¹⁴ Identification of fungal isolates was carried out by staining with lactophenol blue and examined under a microscope. The feature characteristics of fungi were used in their identification.¹⁵

Antibiotic sensitivity test

Antibiotic susceptibility test was carried out using the agar disc diffusion method following the recommendation of the Clinical and Laboratory Standards Institute.¹⁶ The inoculum of the isolates obtained from the floury foods was prepared from 18 h old broth culture of each isolate and their absorbance was adjusted to 0.5 McFarland equivalent. Inoculum size (0.1mL) was spread on Mueller-Hinton agar and the antibiotic discs were placed at the equidistance of the plate. The antibiotics used include amoxicillin (30µg), ofloxacin (10 µg), streptomycin (10 µg), chloramphenicol (30 µg), gentamycin (10 µg), pefloxacin (10 µg), cotrimoxazole (30 µg), ciprofloxacin (10 µg), erythromycin (10 µg) and tetracycline (25 µg) obtained from Abtek Biological Ltd, Liverpool, L9 7AR, UK. The zones of inhibition were measured and interpreted according to CLSI (2012). The fungal isolates were tested against antifungal drugs, namely ketoconazole (15 μg), fluconazole (25 μg) and nystatin (1μg). This was done according to the standard methods described by CLSI (2009). Already prepared green synthesized AgZnO and AgTiO nanoparticles (NPs) were obtained from Prof. A.E. Okoronkwo of Department of Chemistry, Federal University of Technology, Akure, Nigeria. Antimicrobial activity of nanoparticles was checked against microbial isolates by agar well diffusion assay.¹⁷

Most food vendors that are involved in the sale of street foods lack knowledge of basic food hygiene.¹⁸ Hence, foods served by them are suspect in terms of microbial quality. It has also been observed that food-handlers’ hygienic status is a major factor contributing to the poor microbiological quality and safety of street foods.¹⁹ Hence, the issue of the microbial quality and antimicrobial sensitivity pattern of isolates from floury snacks patronized by most students of The Federal university of Technology, Akure is germane. Results from the microbial load of the floury snacks revealed that meat pie has the highest load (8.0x10² to 1.5x10⁴ cfu/g). The bacterial load recorded for floury snacks collected in the evening was slightly higher than in the morning (Table 1).

The following microorganisms were isolated from the floury snacks viz; Staphylococcus aureus, Staphylococcus epidermidis, Bacillus sp, Serratia marcescens, Aspergillus niger and Aspergillus fumigatus. Staphylococcus aureus has highest percentage of occurrence being present in all the samples collected in the morning and evening (Table 3).

Commercial antibiotics used displayed varying inhibitory zones ranging from 18mm to 22mm against the microbial isolates from floury snack (Table 4).

CN, Chloramphenicol; Ery, Erythromycin; Sep, Septrin; Amo, Amocicillin; Cipro, Ciprofloxacin; Co, Cotrimoxazole; Pef, Perfloxacin; Amp, Ampiclox; Ery, Erythromycin; Tetr, Tetracycline

The green synthesized nanoparticles, AgZnO and AgTiO nanoparticles (NPs) inhibited all the microbial isolates from floury snacks. NPs had been reported to possess broad-spectrum antibacterial properties against both Gram-positive and Gram-negative bacteria.²⁸ ZnO NPs were found to inhibit Staphylococcus aureus, and Ag NPs exhibit concentration-dependent antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa.²⁹ In a recent study,^30,31 respectively reported inhibition of some microorganisms by AgTiO and AgZnO NP. Three mechanisms of action had been proposed for the antimicrobial action of NPs. These are oxidative stress induction;³²  metal ion release³³ and non-oxidative mechanisms.³⁴ These three types of mechanisms can occur simultaneously.²⁸

Conclusively, the results from this study revealed that AgZnO and AgTiO nanoparticles (NPs) significantly inhibit bacterial and fungal isolates obtained from floury snacks sold around and within the campus of The Federal University of Technology. This NPs may be exploited in the preservation of these snacks after the appropriate in-situ safety assays might have been carried out.

None.

Authors declare that there is no conflict of interest.

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