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Assessment of bacterial quality of honey produced in Tamale metropolis (Ghana)

The bacterial quality of honey from different production sites within Tamale metropolis, Ghana, was estimated using standard microbiological methods. Honey samples were bought from six different production sites within Tamale metropolis and labeled. Samples that were taken from location B recorded the least mean bacterial count of 6.0 × 104 colony forming units/mL with samples taken from location D showing the highest, 1.1 × 105 colony forming units/mL. However, samples from production sites E and F recorded no bacteria growth. Bacteria isolated included Escherichia coli, Staphylococcus spp., Shigella spp., Streptococcus spp., and Bacillus spp. The pH values of honey samples from the various locations were found to be directly correlated to the average bacteria load. The variation in bacteria load and species at the various production sites and the absence of bacteria growth in two production sites is an indication of the differences in production practices, as well as hygienic conditions at these sites. The presence of these isolates is a cause for concern as pathogenic strains of these bacteria can cause serious health related problems.


Introduction
According to Codex Alimentarius commission [1], honey is defined as a natural sweet substance produced by honey bees from the nectar blossoms or the secretion of the living part of plants, which honey bees collect, transform, combine with specific substances of their own, store, and leave in the honeycomb to ripen and mature.
The composition of carbohydrate (82.3%) in honey is more than any other animal product [2]. Honey is composed primarily of the sugars glucose and fructose (monosaccharides). It also contains numerous other types of sugars, disaccharides, like maltose, sucrose, kojibiose, turanose, isomaltose, and maltulose, which make up over 7% of its composition. In addition, honey also contains carbohydrates known as oligosaccharides [3,4]. It also subsumes other ranges of elements such as minerals, proteins, carbohydrates, vitamins, enzymes, free amino acids, and numerous volatile compounds [5,6]. The composition of honey has been shown to depend largely on its floral source, and also varies greatly according to its geographical origin [7,8].
Honey can be used as a natural "sweetening agent" without further processing [9]. Honey is considered as one of the sweetest natural foods in Ghana in terms of its nourishment and therapeutic properties [10]. It can be used as food, for religious ceremonies, and as medicine for both humans and animals [11,12]. It also serves to feed animals and for sweetening drugs for children [10].
The benefits obtained from the consumption of honey can be overshadowed by adulteration. Adulteration of honey occurs by the addition of different materials. Addition of foreign substances such as molasses, starch solution, glucose, sucrose, water, and inverted sugar to honey has been reported [11]. The addition of some of foreign substances can microbiologically contaminate honey [12]. Microorganisms in the honey may arise from the nectar and parts of plant flower, as well as from the processing area.
Good quality honey must lack pathogenic microorganisms that cause enteric illnesses [12]. The present study was therefore carried out to evaluate bacterial quality of honey from production sites, and also to determine the type of bacteria implicated in honey contamination within Tamale metropolis.

Study location
The study was carried out in the Tamale metropolis. Tamale is the capital town of the Northern region of Ghana.

Sampling
Honey samples were aseptically collected in sterile bottles from different production sites A, B, C, D, E, and F within Tamale metropolis, Ghana. The samples were then transported in an ice chest containing ice to the Spanish Laboratory of University for Development Studies, Nyanpkala campus for immediate analysis.

pH analysis of honey samples
The pH of the honey samples was determined using a pH meter (Crison, Barcelona, Spain). Ten mL of each honey sample was measured into a clean beaker. The pH electrodes were first immersed in standard solution to calibrate the pH meter before putting in the honey sample. The pH value was then recorded.

Media preparation
All media were prepared as indicated by the manufacturers. The media used include MacConkey agar (Oxoid Ltd., Basingstoke, Hampshire, England), nutrient agar (Techno Pharmchem, Vardhman, India), and Salmonella Shigella agar (Techno Pharmchem, Vardhman, India). All of the media were autoclaved at 121 C for 15 minutes. Then, they were cooled to about 45 C and poured into sterile Petri dishes to solidify.

Preparation of sample
With the aid of the laminar flow hood, serial dilution of the honey samples was carried out with 10 mL of each honey sample in 90 mL of sterile 0.1% peptone water. This was stirred very well using a sterile glass rod.

Inoculation and incubation
One mL each of 10 À4 and 10 À5 dilutions was taken aseptically under the lamina flow hood and inoculated on a solidified nutrient agar for total plate count. The inoculated plates were inverted and incubated at 37 C for 24 hours. After 24 hours of incubation, plates with countable colonies [30e300 colony forming units (cfu)] were removed and counted using the colony counter (J.P. Selecta, Barcelona, Spain). The number of colonies was recorded as cfu/mL. The number of cfu/mL of the sample was calculated as follows:

Bacteria isolation, identification, and confirmation
Sixteen colonies were randomly selected from sampled nutrient agar plates and streaked on fresh nutrient agar plates. These plates were then incubated at 37 C for 24 hours. This was carried out to obtain pure cultures for identification purposes. Morphological characteristics, gram staining, and other biochemical tests were also executed to identify the isolates.
One mL each of 10 À4 and 10 À5 dilutions was also inoculated on the solidified McConkey agar and incubated at 37 C for 48 hours. Unique colonies were selected and streaked on fresh McConkey agar plates to obtain pure cultures. Morphological characteristics of the pure cultures as well as other biochemical tests were then used to confirm the species.
One mL each of 10 À4 and 10 À5 dilutions was again inoculated on SS agar and incubated at 37 C for 48 hours. Again, distinct colonies were selected and streaked on fresh SS agar plates to obtain pure culture. Pure cultures were again identified and confirmed using morphological features as well as other biochemical tests.

Gram staining and biochemical tests
Biochemical tests carried out included, catalase, modified oxidase, oxidative-fermentative, furazolidone and bacitracin susceptibility, oxidase, sugar fermentation, indole, citrate utilization, urease, and motility tests. Gram staining and all biochemical tests were carried out according to [13].

Results
The microorganisms counts ranged from 6.0 Â 10 4 cfu/mL (Location B) to 1.1 Â 10 5 cfu/mL (Location D). Samples taken from locations E and F showed no growth. Different genera of bacteria were isolated from honey samples at different production sites. E. coli and Shigella spp. were isolated from all the samples except samples from locations E and F.
It can be seen from Figure 1 that lower pH values correspond to less bacteria load while higher pH values also correspond to high bacteria load. The last two locations showed no growth because the pH values were the lowest.

Bacteria isolated from honey samples from different production sites
The presence of the isolated bacteria (Table 1) may be attributed to the extraction, unhygienic handling, and processing of the honey. At some of the production sites, people were seen carrying honey in unhygienic plastic containers on their head, motorbikes, and sometimes bicycles. Some honey was also kept in unhygienic environments and at times not covered well to prevent flies and other insects from settling on it. At some of the production sites, people were seen conversing during extraction of honey, handling, and processing without realizing that they may be contaminating the honey by introducing saliva in to it. According to a previous work [14], contamination from the skin, mouth, and nose of food handlers can be introduced directly into food during processing. The primary source of honey contamination includes the pollen, the digestive tracts of honeybees, dust, air, and nectar which are difficult to control. Others like honey handlers, cross contamination, equipment, and buildings are secondary sources which can be controlled by good manufacturing practices [15]. A previous work [12] reported that fungi and bacteria contamination is an indication of inadequate hygienic conditions during collecting, manipulating, processing, and storing. Microbiological quality of honey may serve as an indicator of the hygienic conditions under which the product was processed, handled, and stored [16].

Variation in bacteria load and species of honey samples
Variations in bacteria load and species in honey samples at different production sites (Table 1) may be attributed to differences in the processing and handling of honey samples at these locations. The absence of bacteria growth in samples    Table 1 Escherichia coli indicates presence of bacteria, (À) indicates absence of bacteria.
j o u r n a l o f f o o d a n d d r u g a n a l y s i s 2 5 ( 2 0 1 7 ) 3 6 9 e3 7 3 from two locations (E and F) also supports this fact. It was found that producers at these two locations had special training in apiaries and hence carried out the extraction and processing of honey under good hygienic conditions. Honeys from these locations are well preserved to prevent contaminations of all forms. Honeys that are well preserved provide unfavorable conditions for bacteria to survive [19]. According to [20], microbiological contamination during or after processing of honey was demonstrated by the absence of the microorganisms in the samples collected from primary sources (producers) and by the presence of bacterium (Bacillus spp.) and various types of fungi in the collected samples from local markets. Previous work [9] also reported that honey found in Akwa-Ibom, Ondo, and Ogun had no coliforms and total viable counts, while honey samples from Shaki, Yola, and Ibadan had some total viable counts.

Effect of bacteria presence in honey samples
E. coli is a Gram-negative, facultative anaerobic, rod-shaped bacterium that can be found in the intestine of warmblooded organisms [21]. Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in their host. Some strains of E. coli (0157:H7) can cause serious anemia or kidney failure, which can lead to death. Shigella spp. is also a Gram-negative bacterium that can infest the digestive tract and cause infection called shigellosis with wide range of symptoms from diarrhea, cramping, vomiting, and nausea to more serious complications and illness. However, antibiotics can shorten the illness [22].
Staphylococcus spp. can be part of the normal flora on the skin of humans and can be transmitted from person to product by unhygienic practices [23]. According to previous work [24], infections caused by Staphylococcus spp. includes arthritis, boil, bumble foot, pneumonia, endocarditis, meningitis, black pox, bronchitis, scaled skin, cystitis, carbuncle, and osteomyelitis.
Some Streptococcus sp. might cause sore throat, scarlet fever and in the most virulent form, necrotizing fasciitis [25].
Bacillus is a genus of Gram-positive, rod-shaped bacteria and a member of the phylum Firmicutes. Bacillus spp. can be obligate aerobes or facultative anaerobes [26].
The bacteria can produce oval endospores that are not true spores during stressful conditions, but can reduce themselves and remain dormant for very long period [27]. Other species of Bacillus are important pathogens; Bacillus anthracis causes anthrax and Bacillus cereus causes food poisoning [28].

4.4.
Effect of pH on bacterial load of honey samples The pH of honey is very important, as it has a major influence on microbial growth in honey. Locations where samples presented low pH had minimum bacteria load when compared to those with higher pH. According to a previous work [29] honey is characteristically quite acidic with pH ranges between 3.2 and 4.5. The pH of honey was low enough to inhibit the growth of many species of bacteria. According to [30], the low pH of honey is inhibitory to many animal pathogens. Under experimental conditions, especially with heavily diluted honeys, the growth medium tends to neutralize the acidity of the honey so that it does not cause inhibition of growth.
The pH of the honey samples used in this study (Figure 1) fell within the range specified by [31] and the [1] this contributed to the inhibitory properties of the honey to microbial growth.

Conclusion
In the present study, incidence of honey contamination was observed and may be attributed to several factors. Among these factors are unhygienic handling of the products and bad storage conditions. The genera of bacteria isolated include E. coli, Shigella spp., Staphylococcus spp., Bacillus spp., and Streptococcus spp. These findings testify to an urgent need to monitor microbial status of honey produced in different production sites in the Northern region of Ghana.

Conflicts of interest
The authors declare that there are no conflicts of interest.