Microbiological status of some commonly available food items and the effects of microwave oven heat on the existence micro flora

F U L L P A P E R Microbiological status of some commonly available food items and the effects of microwave oven heat on the existence microflora Islam, M.F., Nur, I.T., Islam, T., Sultana, R., Rezanujjaman, Md. and Acharjee, M. Department of Microbiology, Stamford University Bangladesh, 51 Siddeswari Road, Dhaka-1217, Bangladesh Department of Molecular and Developmental Biology, Graduate School of Science and Technology, Shizuoka University, Oya 836, Suruga-ku, Shizuoka, 422-8529, Japan


Introduction
For the maintenance of healthy life, feasting good and safe food has no alternative in our daily lifestyle Noor et al., 2013;Acharjee, Fatema, Jahan et al., 2013;Acharjee, Jahan, Rahman et al., 2013;Jane et al., 2018). As foods are the major source of energy and nutrition for all the living organisms including human, animal and even plants, therefore, measuring the food safety and security is the vital concern nowadays Acharjee, Fatema, Jahan et al., 2013). There are several reasons such as physical, chemical and biological matters behind the vigorous deterioration of the quality of different food and food products (Balbani and Montovani, 2012). Among these three factors of food spoilage, biological matters are so serious as because of the direct involvement of pathogenic microorganisms which are mainly responsible for food borne intoxication and infection (CDC, 2014). As explained in many previous studies that the common food borne pathogens are Campylobacter spp., E. coli, Listeria monocytogenes, Salmonella spp. and Staphylococcus spp. (FSANZ 2001a;FSANZ 2001b;FAO 2004). However, several methods are available in modern decades to eradicate the dissemination of food born microorganisms such as irradiation, cooking, drying, pasteurization, microwave heating and even adding of preservatives during the food preparation (Hossain et al., 2019 Many studies previously reported that the gamma irradiation with different doses (3, 4, 6 and 8 Kilo Gray) have considered a very useful method to inhibit the excessive growth of bacteria in raw fish and meat sample (Acharjee, Fatema, Jahan et al., 2013;Acharjee, Israt and Noor, 2019;Acharjee, Sultana and Noor, 2019). Another study focused on the role of drying and cooking process on the bacterial proliferation during food preparation (Nur et al., 2019). The most common source of radiation is microwave-operating devices including radars, electric oven and diathermy (Balbani and Montovani, 2012). Meanwhile, short time microwave oven heating (60-second) process is now very common not only in the megacity of Bangladesh but also very popular in all over the world especially for the ready to eat food preparation (Hossain et al., 2019). Earlier studies revealed that the few second heat treatments are much quicker and easier methods to reduce the growth of food born microorganisms than the other conventional heating process (Cross and Fung, 1982;Rosenberg and Bogl, 1987;Kakita et al., 1995;Canumir et al., 2002). Thus, one of the common factors is still making controversy among the scientists about the thermal effects and the non-thermal effects of radiation (Dreyfuss and Chipley, 1980;Welt et al., 1994;Wayland et al., 1997;Kothari et al., 2011;Trivedi et al., 2011). Microorganisms can be killed using the thermal effect of microwave radiation while non-thermal effect has been suggested to inactivate microbial propagation (Jeng et al., 1987;Barnabas et al., 2011;Woo et al., 2000). Dissemination of drug-resistant bacteria through spoilage food is now very common issue in many developing countries, which may create massive obstacle in disease medication (Dutta et al., 2013).
Along these lines, the present study attempted to (1) introduce the microbiological profiling of some common ready to eat food, (2) the drug-resistant attributes of the isolates and (3) the effects of 60 seconds microwave oven heat treatment on food microflora.

Study area, sampling, sample processing and microbiological analysis
A total of five samples of each food type (chicken sandwich, French pizza, chicken pie, pasta and hot dog) (n = 25) was randomly collected following the standard protocol of APHA (1998). All the samples were transported to the laboratory as soon as possible for microbiological assay. Samples were divided equally into two portions. The first portion was assessed without microwave oven heat treatment while the other portion was assessed after microwave oven heat treatment.
A total of 10 g of each sample portion was homogenized with 90 mL of buffered peptone water (pH 7.2±0.2) in 9: 1 ratio and serially diluted up to 10 -3 . From the 10 -2 dilution, 0.1 mL was introduced on to the nutrient agar and Sabouraud dextrose agar for the isolation of total viable bacteria and fungi, respectively. Subsequently, MacConkey agar, Mannitol Salt agar Salmonella Shigella agar and Cetrimide agar were used as selective media for the quantification of coliforms, Staphylococcus spp., Pseudomonas spp. consecutively (Cappuccino and Sherman, 1996;Acharjee et al., 2014). All the inoculated plates were incubated at 37°C for 24 hours except SDA plates, which were incubated at 25°C for 48 hours.

Biochemical identification of the isolates
The biochemical properties of identified isolates were confirmed through standard biochemical methods (Cappuccino and Sherman, 1996).

Antibiotic susceptibility test of the identified bacteria
The pathogenic isolates were examined for the detection of antibiotic susceptibility traits (either drugresistant or sensitive) by disc diffusion assay on Mueller-Hinton agar (Difco, Detroit, MI) against commonly used antibiotics by following the standard protocol (Bauer et al., 1966;Ferraro et al., 2011). Lawns of bacterial suspensions including Escherichia coli, Pseudomonas spp., and Staphylococcus spp. (turbidity compared with the McFarland standard OD 600 -0.5) were prepared and introduced on to Muller Hinton agar. Some common antibiotics such as Kanamycin (30 µg), Streptomycin (10 µg), Gentamycin (10 µg), Azythromycin (15 µg), Tetracycline (30 µg) were introduced against the targeted bacteria. All the plates were incubated at 37 °C for 12-18 hrs and examined for formation of the zone of inhibitions (mm).

Results and discussion
Propagation of different pathogenic strains including coliform and fecal coliform in food samples due to the poor sanitation and hygienic condition are the main causative agent of several food borne diseases like diarrhea and dysentery (Mead et al., 1999;Munshi et al., 2012;Tamang et al., 2016). Such diseases are very common in developing countries like Bangladesh due to the lack of proper knowledge on personnel hygiene as well as poor maintenance quality of raw materials (Tamag et al., 2011, Acharjee, Jahan, Rahman et al., 2013. All the street food items (chicken sandwich, French pizza, chicken pie, pasta, hot dog) were highly contaminated with various pathogenic microorganisms such as E. coli, Staphylococcus spp, and Pseudomonas spp. before and after heat treatment (Figure 1). However, the presence of E. coli was noticed only in chicken sandwich before the heat treatment (10 2 CFU/g) and the growth was totally eradicated after post heat treatment. Due to heat treatment, the total viable bacteria both in chicken sandwich and french pizza was observed to reduce up to 5 log. Whereas 6 log reductions of total viable bacteria were found in chicken pie but 2 log reduction was notified in pasta after heat treatment. On the contrary, the fungal growth was deducted about 1 log, 2 log, 3 log and 4 log in the post-heat samples of chicken pie, french pizza, pasta and chicken sandwich respectively (Figure 1). Though Staphylococcus spp. and Pseudomonas spp. were found in pre-heat sample of chicken pie, pasta and french pizza, the heat treatment was able to completely eliminate them but only in chicken sandwich 2 log reduction of Staphylococcus spp. was notified. Couples of previous studies investigated that the existence of contaminating microflora in food samples may occur during the processing of raw material, mixing of ingredients and packaging of the end products (Anon., 2000;FSANZ, 2001a;FSANZ, 2001b;Anon., 2003;Anon., 2004;Anon., 2006). According to the International Commission on Microbiological Specifications for Foods (ICMSF) 1986 the presence of a specific pathogen in food should not be exceed 10 2 CFU/ g. In this study, maximum post-heated food harbored excessive amount of pathogens which exited the marginal limit provided by International Commission on Microbiological Specifications for Foods (ICMSF) 1986. However, the presence of fecal coliform, colifrom and other pathogens in different food samples are the principle reason of food borne diseases (Acharjee, Fatema, Jahan et al., 2013). So, proper methodology including microbial load reduction method should be used to eliminate the contamination of this microflora.

Biochemistry of the isolates
A total of five types of isolates showed their physiological properties through several biochemicals tests and confirmed the pathogens (Table 1) To evaluate the efficacy of commonly available antibiotics as well as the clinical significance of the bacterial isolates, the present study introduced antibiotic susceptibility test. Identified bacterial isolates were experimented to determine the antibiotic susceptibility against the common antibiotics. All the isolates from the samples were found to be 100% sensitive against streptomycin (10 µg), azithromycin (15 µg) and gentamycin (10 µg) while all the strains showed resistance against more than one antibiotics such as penicillin G (10 µg), erythromycin (15 µg), amoxicillin (30 µg), ceftriaxone (30 µg), ciprofloxacin (5 µg), ampicillin (10 µg), tetracycline (30 µg), chloramphenicol (30 µg) and cefixime (5 µg) ( Table 2). A number of research findings unveiled, the resistance gene can be transferred through contaminated food into the human body, which may hinder the disease medication (Bennett and Klich, 2008;Canteón, 2009;Acharjee, Fatema, Jahan et al., 2013).

Conclusion
Overall, the present investigation discussed the compact microbiological profile of some popular food items as well as their drug-resistant properties. Previously huge information has been gathered on the propagation of bacterial strain in different foods but unfortunately, very few studies described the methodology by which the existence pathogens might be reduced. This study showed the 60 seconds of heat treatment by microwave oven was so effective to reduce the substantial amount of bacteria. Moreover, the presence of resistant strain in food is very much alarming which indicates the poor management set up and lack of proper legislation in the food sector. In this perspective, the given information on the microbiological quality in accordance with the recommended microbiological criteria of different food items would impart a practical outcome in knowledge dissemination on food safety in Bangladesh.
(2014). Validation of γ-irradiation in controlling FULL PAPER  The experiments were conducted three times independently, and the results were found to be reproducible. One representative data has been shown. TSI: Triple Sugar Iron Test, Y: Yellow (Acid), R: Red (