Antimicrobial Activities of Tanzania Honey Bees in Relation to Vegetation Types

Honey sampling procedure

A purposive sampling protocol was used to select high honey producing areas and vegetation types. Twelve available areas were selected. Each 3 beekeepers were selected randomly belonging to each area and vegetation type, making a total of 36 beekeepers who were involved in the study. Areas selected and vegetation types are as shown in Table 1.

Table 1.

Selected study sites and vegetation types

Honey was harvested directly from the beehives and bulked according to the area and vegetation type, packaged in sterilized plastic bottles (330ml) then transported and stored at ambient temperature (25±2°C) at laboratory of Micro-Biology at the Nelson Mandela Institute of Science & Technology, Arusha-Tanzania.

Antimicrobial sensitivity test

Antimicrobial sensitivity test of the honey samples was determined by Agar Wells Diffusion method as described by Clinical Laboratory Standard Institute (2009). Where Mueller-Hinton Agar (composed of 2g beef extract, 17.5g acid hydrolysate of casein, 1.5g starch and 17g agar) medium was used for antimicrobial susceptibility test. The medium was prepared following the manufacturer’s instructions. After autoclaving (121°C for 15min) the medium was left to cool to 50°C. Then 25ml per plate (15 ×100mm) was measured and pouring in a way that level pouring surface within the petridish was uniform to a depth of 4 mm and was incubated in an incubator (35±2°C) for 24 h.

The test organisms included bacteria (Escherichia coli (ATCC 25922), Salmonella typhi (ATCC14023) and Staphylococcus aureus (ATCC 25923) and fungus (Candida albicans-isolated from clinical samples), were streaked on to a non-inhibitory agar medium (broth agar) to obtain isolated colonies. After incubation at 35°C overnight, 4 to 5 colonies were picked and inoculated into broth (Mueller- Hinton broth) and incubated at 35°C for 24 h. A sterile cotton swab was dipped into the suspension, pressed firmly against the inside wall of the tube just above the fluid level, then streaked over the entire surface of the medium rotating the plate approximately 60 degree after each application to ensure an even distribution of the inoculums.

Finally cotton were placed all around the edge of the agar surface. Small holes of 5 mm were made on the petri dishes with agar by using sterile loops, and then 100μl of honey sample was placed in the agar hole using sterile micropipette. The plates were inverted and incubated at 37 ±1°C for 24 h for tested microorganisms. After incubation period the diameter (mm) of the zones of complete inhibition (including the diameter of the disk) was measured and recorded. The measurements were made with ruler on the undersurface of the plate without opening the lids.

Honey pH determination

pH of honey samples was determined using a digital portable pH meter (JENWAY, UK 3305P) in accordance with International Honey Commission (2009). In between the readings of different samples, the electrode was washed with distilled water and dried with tissue paper, and inserted into prepared honey samples and recorded. The experiment was done in triplicates.

Honey total sugar and water content

The honey sugar and water content wasdetermined using a digital portable honey refractometer that was held on a flat surface and a drop of honey was inserted on a glass prism that determines refractive index. The measurement key was pressed and results were displayed on the backlit LC-Display. In between the readings of different samples, the surface of the glass prism was washed with distilled water and dried with tissue paper before another honey sample was inserted.

Statistical data analysis

Data obtained from antimicrobial analysis were analyzed using SAS software. Analysis of Variance (ANOVA) was used to determine the influence of main independent variables, the factor influence was considered to be significant when p<0.05. Mean comparison between area and vegetation types was done using Duncan Multiple Range Test (DMRT) and results were presented as Mean±SD.

pH, sugar and water contents of stinging bee honey

Table 2 shows the composition and chemical properties of honey collected from various areas with various vegetation types. The pH of honey samples used in this study ranged from 4.05 to 4.8 which means that all honey samples were acidic.The pH rangeobserved in sampled honeys falls within the range reported by Gidamis et al. (2004) while working with Tanzanian honey collected from Dodoma, Tanga, Morogoro, Same, Arusha and Tabora, reported a pH range of 4.4 - 4.87. Also, complies with the range reported by United Republic of Tanzania - Ministry of Natural Resources and Tourism (2007) who reported a pH range of 3.42 to 6.10 for Tanzanian honey. However, the observed pH range in this study was higher than that reported by Masoud (2014) working with 26 honey producing areas in Tanzania who reported a pH range of 2.6 to 4.4. The difference could be due to difference in soil type and vegetation growing in areas where the samples were collected. Despite the slight differences in pH the honey samples in the present study might have potential medicinal properties as its acidic nature could have resulted from organic acids which remarkably create an acidic micro-environment that threatens pathogenic microbial growth (Aparna and Rajalakshmj, 1999).

Table 2.

pH, sugar and water contents of stinging bee honey collected from different places in and vegetation types in Tanzanian

The total sugar content that ranged from 74~83% (Table 2) was within a range observed by other researchers in Tanzania. Masoud (2014) working on the quality of Tanzanian honey collected from 26 honey potential producing areas reported a sugar content range of 64.2 to 84.8%. Furthermore, it is above the minimum standards of Tanzanian honey of not less than 65% (URT, 2007). The sugar contained in studied honey was sufficient enough to produce high osmotic effect that could dehydrate microbial cells (Molan, 1992 and Bogdanov et al., 1997). Gluconic acids which remarkably create an acidic micro - environment prevents the growth of many microorganisms (Cooper, 2007).

There was an inverse relationship between sugar and water contained in honey. The minimum value observed in this study was 17% with a maximum value of 24%. Some honey in this study had higher water content than the recommended range of 20~22% (URT (2007) for Tanzanian honey. High water content contributes to reduced shelf life of honey that undergoes fermentation and spoil more easily (Cortopassi et al., 2006). However, despite the high water content of some few honey samples, the results of this study suggest that Tanzanian honeys could be among the best honeys that can be used not only as food but also as remedies to various microbial infections.

Effect of area, vegetation and their interaction on microbial growth

Generally, area from which honey was collected, vegetation type on which honey bees foraged, test microorganisms and its interaction influenced significantly (df=47, F=454, P=0.0001) the antimicrobial properties of honey.

It is evident from Table 3 that honey from Gairo where bees foraged on Astripomoea malvaceae and Adansonia digitata was the best honey (df=5, F=101.30, P=0.0001) as it had relatively strongest ability to inhibit microbial growth than other honeys. The Manyoni thicket honey was the second best honey as followed by Morogoro Eastern Arc forest honey that did not differ in inhibiting microbial growth with that from Kisarawe where bees mostly foraged on Euphobia bicompacta. Honey from Inyonga exhibited significantly (df=11, F=316.18, P=0.0001) lowest ability to inhibit microbial growth, generally most of honey from Miombo woodland including Nyakanazi and Tabora had unexpectedly lower ability to inhibit microbial growth (Table 3, Plate 1).

Table 3.

The effect of area and vegetation type on ability to inhibit growth of pathogenic micro-organisms

Plate 1.

Antimicrobial activities of Tanzanian honey collected from various areas.

The difference in ability to resist microbial growth could probably be due to differences in vegetation types between these areas (Kumar et al., 2010). Different vegetation types contain different floral types that vary in pollen and nectar which influence the honey medicinal qualities and composition (Jones, 2001).

The conclusive selection of the best honey can not only be judged by looking at the overall resistance on microbial growth alone. The ability of honey to inhibit multiple microorganisms or to have a broader spectrum antimicrobial ability could be the best property to be looked at.

Antimicrobial spectrum of Tanzania stinging bee honey

Table 4 presents the antimicrobial spectrum of honey in relation to the vegetation types that bees foraged on. It is clearly observed that the Gairo honey obtained from bees foraging on Adansonia digitata and Astripomoea malvaceae, significantly (df=32, F=147.56, P=0.0001) inhibited the growth of both, pathogenic fungus (C. albicans 30.5mm) and bacteria (E. coli 26.5mm, S. aureus and S. typhi 27.5mm) followed by honey originating from Manyoni-thicket (C. albicans 14.5mm, E. coli 22.5mm, S. aureus 31.5mm and S. typhi 25.5mm), Morogoro-Eastern Arc forest (C. albicans 16.5mm, E. coli 22.5mm, S. aureus 30.5mm and S. typhi 28.5mm), Kisarawe-Euphobia bicompacta (C. albicans 12.5mm, E. coli 22.5mm, S. aureus 29.5mm and S. typhi 25.5mm) Itigi-thicket (C. albicans 10.5mm, E. coli 10.5mm, S. aureus 21.5mm and S. typhi 28.5mm) and Dodoma-Miombo woodland (C. albicans 14.5mm, E. coli 7.5mm, S. aureus 23.5mm and S. typhi 25.5mm) and Dodoma-Adansonia digitata (C. albicans 11.5mm, E. coli 18.5mm, S.aureus 23.5mm and S. typhi 30.5mm). Other honeys such as Tabora honey from Miombo woodland could not resist the growth of C. albicans, from Miombo woodland (Nyakanazi, Inyonga and Bukombe) and Dodoma honey from sunflower could not resist the growth of E. coli respectively. Based on the criteria of honey to contain relatively broader medicinal properties and thus be broad spectrum antimicrobial, it can be said that the priory mentioned honeys could be the best as far as this study is concerned.

Table 4.

Variation in ability of Tanzanian honey types to inhibit growth of various pathogenic micro-organisms

The general characteristics of honey to prevent bacterial or fungal growth have been explained by various scientists. Garcial et al. (1986); Wahdan (1998); Molan (1999a) and Khan et al. (2007) reported inhibition of pathogenic microbial growth to be due to presence of hydrogen peroxide resulting from the action of glucose oxidase enzyme produced from hypopharyngeal glands of workers bees on glucose in presence of oxygen that inhibits microbial and fungal growth. Presence of physical-chemical properties such as high sugar content (about 80% w/w) that results into high osmotic effect that dehydrate micro-organism has been reported to inhibit microbial growth (Molan, 1992 and Bogdanov et al., 1997). Aparna and Rajalashmj (1999); White (1978) suggested inhibition of microbial growth to be due to presence of diverse organic acids such as gluconic acids that remarkably creates an acidic micro-environment (pH 3-4.5) that prevents growth of many micro-organisms. Apart from hydrogen peroxide as a factor that inhibits microbial growth, Cabrera et al.(2006) elucidated inhibition of microbial growth to be due to presence of non-peroxidic substances such as polyphenols which possess antimicrobial activity. Furthermore, Molan (1992a) reported that in most of honey antimicrobial growth depends on enzymatic generation of hydrogen peroxide to varying degree, but in some honeys there are additional phytochemical antibacterial factors. Cooper et al. (2002) elucidated that antimicrobial agents have been applied to wounds for 1000 years ago but many ancient remedies have been discontinued because the evidence to support their efficacy was anecdotal. He further, said that failure to identify botanical sources of honey used in many of the studies or to determine their antibacterial potency makes comparison of reported sensitivity unreliable. Jones (2001) also reported that it is remarkable that ancient physicians were selective in the honeys they utilized in their remedies. All these observations reported exhibits that there is variation in medicinal contents depending on area and type of vegetation where honey bees forages. This means that the antimicrobial composition depends on whether the plant foraged is a medicinal plant or not.The broader antimicrobial spectrum observed on Gairo honey where bee forages on Adansonia digitata and Astripomoea malvaceae could be attributed not only by acidity, osmolarity and hydrogen peroxide production but also by phytochemical components of that honey. Baobab fruits pulp and seeds are rich in vitamin C and protein and are apparently effective against dysentery and circulatory disease. On the other hand, Astripomoea malvaceae is a medicinal plant, in Tanzania a root decoction is drunk or the ground roots are taken inorder to treat hookworms. In Malawi a poultice of crushed roots is applied to the swellings and inflammations, especially to treat eye infection, also the sap of leaves and flowers is applied to inflammation of eyeball. In Zimbabwe, a root decoction is drunk to treat coughs, female infertility, dizziness and abdominal pain in babies (Schmetzer and Gurib, 2013). Similar arguments also stand for the Kisarawe honey where honey bees were foraging on Euphobia bicompacta. The plant sap of Euphobia bicompacta is used in Kenya to treat East Coast Fever in cattle whereas a decoction of its leaves and stem bark is given to drink to cattle to control ticks (Schmetzer and Gurib, 2013). Furthermore, in Angola the root fusion of Euphobia is drunk to treat pain in hips and madness. An infusion of Euphobia roots is taken to treat stomach ache, dropsy, stitch, urogenital problems, excessive menstruation, Tuberculosis and cardiac palpitation, the latex is used as fish poison (Schmetzer and Gurib, 2013).

It can be said from findings of this study that honey differs in antimicrobial properties due to differences in constitution and quality of pollen and nectar. Nectar and pollen from medicinal plants is far better to produce honey with broader antimicrobial spectrum. This study supports the study by Tonl et al. (2003) who reported that the Manuka honey produced by bees foraging on Manuka plants in Australia and New Zealand is believed to have superior antimicrobial properties hydrogen peroxide content, phytochemical contents or presence of dihydroacetone in the nectar which is derived from methyglyoxal.

Human activities have been reported to influence so much the honey quality and composition. Schmetzer and Gurib (2013) pointed out that genetic diversity of many plants species in Africa is being eroded sometimes at an alarming rate as a consequence of habitat destruction and over exploitation. The replacement of landraces of cultivated species by modern cultivars by seed companies is another cause of genetic erosion. This was observed in this study that honey from sunflower had relatively low ability in inhibiting microbial growth (C. albicans 8.5mm, E. coli 0.0mm, S. aureus 31.5mm and S. typhi 30.5mm). Surprisingly, honey from Miombo woodland seemed to have lowest medicinal ability (Table 4). This could be attributed by forest encroachment where most of trees are cut down by agro-pastoralists to open up areas for crop farming and creating unfavorable environment for tsetse flies that affects their livestock.