Screening the susceptibility of bacteria to honeys and determination of minimal inhibitory concentration
In the preliminary screening, all the honeys samples were tested against C. perfringens type A strain. The concentrations of the honey solutions were 50 % (w/v) (5 mg). The activity of the tested honeys was expressed by zone of inhibition (Table 1). From all the tested honeys, Finnish organic honeys B, C, E, and F showed higher antimicrobial activity than water control (diameter of zone of inhibition >5.2 mm ± 0.5) and artificial honey sugar solution (diameter of zone of inhibition 6.1 mm ± 1.5). The broadest zone of inhibition against C. perfringens was induced by honey F (diameter of zone of inhibition 14.3 mm ± 0.6) followed by honey E (diameter of zone of inhibition 11 mm ± 2) and honey B with the zone of inhibition diameter of 8.3 mm ± 2 and honey C with the zone of inhibition diameter of 7.5 mm ± 0.7; honeys A and D did not show any antibacterial activity against C perfringens. Negative water control did not show any activity, and positive antibiotic control was constantly high (diameter of zone of inhibition of 30.8 mm ± 1.4).
After screening the organic honeys against C. perfringens, honey F that showed the highest antibacterial activity was tested for the minimum inhibitory concentration (MIC), the lowest concentration of honey inhibiting visible growth of the bacteria. MIC was determined by measuring the zone of inhibition (Table 2). Twenty microliters of the honey concentrations 50, 25, 20, and 15 % were pipetted on the discs containing 10, 5, 4, and 3 mg of the honey, respectively. Honey concentrations of 50, 25, and 20 % induced better antimicrobial activity than artificial honey, sugar solution SS (Table 2). Down to the dilutions of 25 and 20 %, the sugar effect increased in the control, but was lower than induced by the corresponding honey dilution. At 15 % concentrations, the results were not clear and the zones of inhibition could not be distinguished. Honey F showed higher antimicrobial activity against C. perfringens down to the concentration of 20 % than the control sugar solution. For honey F, the MIC value was thus 20 % (4 mg).
There are many factors in honey that effect on the growth of C. perfringens. C. perfringens is not tolerant of low water activity (aw), reported values for tolerance being between 0.93 and 0.97 (Labbe 1989). Honey is a supersaturated sugar solution with 0.56–0.62 aw (Molan 1992), which partly explains the inhibitory activity against C. perfringens. In our study, a sugar control, artificial honey, was used to eliminate the hyperosmotic effect of honey in the results. Antimicrobial activity was recorded when the zone of inhibition was higher than induced by control. Thus, in honeys B, C, E, and F, the antimicrobial activity results from additional factors to sugar.
Like most microorganisms, C. perfringens initiate growth most readily at neutral pH, although excellent growth occurs between pH 6 and 7. Smith (1972) reported that growth of C. perfringens is severely limited at pH ≤5.0 and pH ≥8.3. The pH of honey is between 3.2 and 4.5. The most active honey F showed activity in dilutions up to 20 % with raised pH. In honey F, at least, this suggests that there may be also other factors than pH that act as antimicrobials.
The variation on antibacterial activity of the honeys could be attributed to the floral source. In honey F, the main floral source was willow herb. In our previous study with Finnish monofloral honeys (Huttunen et al. 2013), we found that the best antimicrobial activities were received with willow herb (E. angustifolium), heather (Calluna vulgaris), and buckwheat (Fagopyrum esculentum) honeys against the studied human pathogenic streptococcal and staphylococcal strains. In the present study, honey E had the second best activity after honey F. In honey E, the major nectar source was clover. Clover honey has been reported to possess antimicrobial activity against Pseudomonas aeruginosa (Lu et al. 2013). In honeys B and C, the antimicrobial activity was quite equal. The major floral sources in honeys B and C were wild raspberry, willow herb, lingonberry, and bilberry. In honey D, which was negative, the major sources for the nectar were wild raspberry and lingonberry. The floral source of honey A was not reported.
The geographic region where the honey is produced may influence on antimicrobial activity of honey. In the present study, four of the five Finnish organic honeys had antimicrobial activity. The main difference compared to the active honeys was that the non-active honey D had been treated by heating shortly at 50 °C to melt crystals. One may speculate that heating have destroyed the active components. All the other Finnish honeys had crystals and were untreated. Honey A from Argentina and Hungary was reported to be untreated and contained crystals, but it did not show inhibitory activity. The flower source was not reported by the manufacturer. The reason for the inactivity remains unsolved. In the present work, the effect of hydrogen peroxide cannot be excluded because neither the heat treatment nor catalase addition was included in the study.
Kokubo et al. (1984) found spores of Bacillus and Clostridium in honeys from processing plants and retailers. Of the studied 71 samples, 6 contained C. perfringens. In connection to this, it was reported that the growth of Bacillus cereus strains was not inhibited by honeys they investigated (Taormina et al. 2001). Higher tolerance of C. perfringens against honey was not seen in our study. Native organic honeys have not been investigated before as regards their antimicrobial activity, and they may even have unknown antimicrobial factors. In Finland, organic honey production is regulated by the European Commission and controlled by the Finnish Food Safety Authority, Evira.
Quantification of methylglyoxal in the honeys
In addition to the main antimicrobial factors of honey, namely high osmolarity, low pH, and hydrogen peroxide, in Manuka honey, the main active component is methylglyoxal (MGO) (Mavric et al. 2008). In Manuka honey, the MGO concentrations are high ranging from 38 to 761 mg/kg, up to 100-fold higher compared to non-Manuka honeys (Mavric et al. 2008). In the present study, we showed that the amounts of MGO were quite equal in all the studied organic honeys including inactive honeys A and D. MGO contents varied from 22 to 27 mg/kg (Table 3), which is tenfold higher than in conventionally produced non-Manuka honeys. There was no correlation between MGO and antimicrobial activity of the honeys. In the studied organic honeys, the antimicrobial activity against C. perfringens is thus due to other factors than MGO, and those factors remain here unknown.
Because of increasing drug resistance also against C. perfringens strains, new antimicrobials are needed. Antibacterial activity of several conventional honeys has been investigated for their potential action against foodborne pathogens (Taormina et al. 2001). Our data show that from the six organic honeys tested, four had antibacterial action, and two of the honeys had no activity against C. perfringens strain. There are no previous studies on the antimicrobial activity of honeys against C. perfringens. The effect of organic regime of the bees on antimicrobial activity of the organic honeys remains open. The importance of the present work is especially in protection against food spoilage bacteria, here shown for C. perfringens.