Characterisation of physicochemical parameters and antibacterial properties of New Caledonian honeys.

Honey is an attractive natural product with various health benefits. A few honey-based commercial products have successfully been adopted in clinics to improve wound healing. However, screening of other potential sources of medical-grade honey, in particular, honeys from territories with high floral species diversity and high endemicity, is highly needed. The goal of this study was to characterise the physicochemical and antibacterial properties of New Caledonian honey samples (n = 33) and to elucidate the major mechanism of their antibacterial action. Inhibitory antibacterial activity of honeys against Staphylococcus aureus and Pseudomonas aeruginosa was determined with a minimum inhibitory concentration (MIC) assay. Enzymatic activity of glucose oxidase and the content of hydrogen peroxide (H2O2) in honey samples were analysed. Furthermore, total protein content of honeys together with their electrophoretic protein profiles were also determined in the study. The antibacterial efficacy of 24% of the tested honey samples was slightly superior to that of manuka honey with unique manuka factor 15+. The antibacterial activity of catalase-treated honey sample solutions was significantly reduced, suggesting that H2O2 is a key antibacterial compound of diluted honeys. However, the kinetic profiles of H2O2 production in most potent honeys at a MIC value of 6% was not uniform. Under the experimental conditions, we found that a H2O2 concentration of 150 µM in diluted honeys is a critical concentration for inhibiting the growth of S. aureus. In contrast, 150 µM H2O2 in artificial honey solution was not able to inhibit bacterial growth, suggesting a role of phytochemicals in the antibacterial activity of natural honey. In addition, the continuous generation of H2O2 in diluted honey demonstrated an ability to counteract additional bacteria in re-inoculation experiments. In conclusion, the tested New Caledonian honey samples showed strong antibacterial activity, primarily based on H2O2 action, and therefore represent a suitable source for medical-grade honey.

Physicochemical Characterization and Biological Properties of Pine Honey Produced across Greece.

Pine honey is a honeydew honey produced in the East Mediterranean region (Greece and Turkey) from the secretions of the plant sucking insect Marchalina hellenica (Gennadius) (Coccoidea: Marchalini-dae) feeding on living parts of Pinus species. Nowadays, honeydew honey has attracted great attention due to its biological activities. The aim of this study was to study unifloral pine honey samples produced in Greece regarding their physicochemical parameters and antioxidant and antibacterial activity against five nosocomial and foodborne pathogens. These honeys showed physicochemical and microscopic characteristics within the legal limits, except for diastase activity, a parameter known to be highly variable, depending on various factors. Substantially higher levels of H2O2 were estimated compared to other types of honeydew honey, whereas protein content was similar. The total phenolic content was 451.38 ± 120.38 mg GAE/kg and antiradical activity ranged from 42.43 to 79.33%, while FRAP values (1.87 to 9.43 mmol Fe+2/kg) were in general higher than those reported in the literature. Various correlations could be identified among these parameters. This is the first attempt to investigate in depth the antibacterial activity of pine honey from Greece and correlate it with honey quality parameters. All tested honeys exerted variable but significant antibacterial activity, expressed as MIC and MBC values, comparable or even superior to manuka honey for some tested samples. Although honey antibacterial activity is mainly attributed to hydrogen peroxide and proteins in some cases (demonstrated by elevated MICs after catalase and Proteinase K treatment, respectively), no strong correlation between the antibacterial activity and hydrogen peroxide concentration or total protein content was demonstrated in this study. However, there was a statistically significant correlation of moisture, antioxidant and antibacterial activity against Klebsiella pneuomoniae, as well as antioxidant and antibacterial activity against Salmonella ser. Typhimurium. Interestingly, a statistically significant negative correlation has been observed between diastase activity and Staphylococcus aureus antibacterial activity. Overall, our data indicate multiple mechanisms of antibacterial activity exerted by pine honey.

Effects of the Carbohydrate Sources Nectar, Sucrose and Invert Sugar on Antibacterial Activity of Honey and Bee-Processed Syrups.

Honey is a functional food with health-promoting properties. Some types of honey are used in wound care for the treatment of acute and chronic infected wounds. Increased interest in using honey as a functional food and as a base for wound care products causes limited availability of raw honey. Numerous studies suggest that the protein content of honey is mainly comprised of bee-derived proteins and peptides, with a pronounced antibacterial effect. Therefore, the aim of our study was to characterize for the first time the antibacterial activity of raw honeys and bee-processed syrups which were made by processing sucrose solution or invert sugar syrup in bee colonies under field conditions. Furthermore, we compared the contents of glucose oxidase (GOX) and the levels of hydrogen peroxide (H2O2) in honey samples and bee-processed syrups. These parameters were also compared between the processed sucrose solution and the processed invert sugar syrup. Our results clearly show that natural honey samples possess significantly higher antibacterial activity compared to bee-processed syrups. However, no differences in GOX contents and accumulated levels of H2O2 were found between honeys and bee-processed syrups. Comparison of the same parameters between bee-processed feeds based on the two artificial carbohydrate sources revealed no differences in all measured parameters, except for the content of GOX. The amount of GOX was significantly higher in bee-processed sucrose solutions, suggesting that processor bees can secrete a higher portion of carbohydrate metabolism enzymes. Determination of honey color intensity showed that in bee colonies, bee-processed syrups were partially mixed with natural honey. Further research is needed to identify the key botanical compounds in honey responsible for the increased antibacterial potential of honey.

Natural Products and Skin Diseases.

The skin is the largest multifunctional organ in the human body, serving as an excellent barrier against chemical and biological hazards [...].

Demanding New Honey Qualitative Standard Based on Antibacterial Activity.

Honey is a functional food with health-beneficial properties and it is already used as a medical device in wound care management. Whether ingested orally or applied topically, honey must fulfill the requirements of international standards based on physicochemical characteristics. However, there is an urgent need for some additional standards reflecting biological properties. The aim of the study was to evaluate the antibacterial activity of 36 commercial honey samples purchased from supermarkets and local food shops and compare their efficacy to that of three honey samples from local beekeepers and three types of medical-grade honey. Furthermore, the hydrogen peroxide (H2O2) content and protein profile were assessed in all honey samples. Analysis of the antibacterial activity of commercial honeys revealed that 44% of tested samples exhibited low antibacterial activity, identical to the activity of artificial honey (sugars only). There was a significant correlation between the overall antibacterial activity and H2O2 content of honey samples. However, in some cases, honey samples exhibited high antibacterial activity while generating low levels of H2O2 and vice versa. Honey samples from local beekeepers showed superior antibacterial activity compared to medical-grade honeys. The antibacterial activity of honey can be easily altered by adulteration, thermal treatment or prolonged storage, and therefore it fulfils strict criteria to be suitable new additional quality standard.

Vitamin C Enhances the Antibacterial Activity of Honey against Planktonic and Biofilm-Embedded Bacteria.

Multifactorial antibacterial action is an important feature of honey; however, its bactericidal efficacy against biofilm-embedded bacteria is limited. The aim of this study was to investigate the impact of vitamin C (Vit C) on the antibacterial activity of natural honeys against planktonic as well as biofilm-embedded bacterial pathogens. The antibacterial activity of four honey samples supplemented with Vit C was expressed as the minimum inhibitory concentration (MIC). At sub-MICs, Vit C significantly increased the antibacterial activity of the tested honeys against Pseudomonas aeruginosa in planktonic cultures. However, after supplementation, honeydew honey, the most active honey, was ineffective against Staphylococcus aureus. On the other hand, when 100% honeydew honey was supplemented with Vit C (100 mg/g of honey) in a multispecies wound biofilm model, complete eradication of almost all bacterial isolates, including S. aureus, was observed. Furthermore, a mixture of honey and Vit C was partially effective against Enterococcus faecalis, whereas honey alone exhibited no antibacterial activity against this bacterium. Vit C counteracted hydrogen peroxide in honey solution and, thus, eliminated the major antibacterial compound present in honey. It is likely that a combination of honey with Vit C may trigger the intracellular production of reactive oxygen species in bacterial cells, but the exact cellular mechanisms warrant further investigations.

Antibacterial Activity of Different Blossom Honeys: New Findings.

Antibacterial activity is the most investigated biological property of honey. The goal of this study was to evaluate the antibacterial activity of 57 Slovak blossom honeys against Staphylococcus aureus and Pseudomonas aeruginosa and investigate the role of several bioactive substances in antibacterial action of honeys. Inhibitory and bactericidal activities of honeys were studied to determine the minimum inhibitory and bactericidal concentrations. The contents of glucose oxidase (GOX) enzyme, hydrogen peroxide (H2O2), and total polyphenols (TP) were determined in honeys. We found that honey samples showed different antibacterial efficacy against the tested bacteria as follows: wildflower honeys > acacia honeys > rapeseed honeys. Overall antibacterial activity of the honeys was statistically-significantly correlated with the contents of H2O2 and TP in honeys. A strong correlation was found between the H2O2 and TP content. On the other hand, no correlation was found between the content of GOX and level of H2O2. Antibacterial activity of 12 selected honeys was markedly reduced by treatment with catalase, but it remained relatively stable after inactivation of GOX with proteinase-K digestion. Obtained results suggest that the antibacterial activity of blossom honeys is mainly mediated by H2O2 levels present in honeys which are affected mainly by polyphenolic substances and not directly by GOX content.

Honey: An Effective Regenerative Medicine Product in Wound Management.

Honey has successfully been used in the treatment of a broad spectrum of injuries including burns and non-healing wounds. It acts as an antibacterial and anti-biofilm agent with anti/pro-inflammatory properties. However, besides these traditional properties, recent evidence suggests that honey is also an immunomodulator in wound healing and contains several bee and plant-derived components that may speed up wound healing and tissue regeneration process. Identifying their exact mechanism of action allows better understanding of honey healing properties and promotes its wider translation into clinical practice. This review will discuss the physiological basis for the use of honey in wound management, its current clinical uses, as well as the potential role of honey bioactive compounds in dermal regenerative medicine and tissue re-modeling.

Effect of thermal liquefying of crystallised honeys on their antibacterial activities.

Thermal liquefying of crystallised honey is the most convenient option for beekeepers to fully liquefy honey. A controlled mild thermal treatment is widely used for effective and safe liquefying of crystallised honey. In this study, we demonstrated that thermal liquefying at different temperatures of 45, 55 and 65 °C does not affect the overall honey antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus, and, in some cases, it may even increase the antibacterial potential of honey. A mild thermal treatment of crystallised honey was able to significantly increase its glucose oxidase (GOX) enzyme activity. On the other hand, the increased GOX activity in treated honey samples did not affect levels of accumulated H2O2, suggesting that some phytochemicals may contribute to or modulate antibacterial activity. Taken together, a mild thermal liquefaction of crystallised honey may increase the potential bioactivity of this matrix, which is also affected by the botanical origin.

Phytochemicals-mediated production of hydrogen peroxide is crucial for high antibacterial activity of honeydew honey.

Honeydew honey is increasingly valued due to its pronounced antibacterial potential; however, the underlying mechanism and compounds responsible for the strong antibacterial activity of honeydew honey are still unknown. The aim of this study was to investigate the inhibition of bacterial growth of 23 honeydew honey samples. Activity of bee-derived glucose oxidase (GOX) enzyme, the content of defensin-1 (Def-1) and hydrogen peroxide (H2O2), and total polyphenol content were determined in the 23 honey samples. Our results demonstrated that antibacterial activity of honeydew honey was equivalent to medical-grade manuka and kanuka honey and was abolished by catalase. Although H2O2 is an important factor in the inhibition of bacterial growth, polyphenolic compounds and their interaction with H2O2 are the key factors responsible for high antibacterial activity of honeydew honey. In addition, our results indicated that the antibacterial activity of honeydew honey is not dependent on GOX-mediated production of H2O2 or the presence of Def-1.

Microwave processing of honey negatively affects honey antibacterial activity by inactivation of bee-derived glucose oxidase and defensin-1.

Microwave (MW) thermal heating has been proposed as an efficient method for honey liquefaction, while maintaining honey quality criteria. However, little is known about the effects of MW thermal heating on honey antibacterial activity. In this study, we aimed to determine the effects of MW heating on the antibacterial activity of raw rapeseed honeys against Pseudomonas aeruginosa and Staphylococcus aureus, with a particular focus on two major bee-derived antibacterial components, defensin-1 and hydrogen peroxide (H2O2). Our results demonstrated that MW thermal heating completely abolished honey antibacterial activity whereas conventional thermal treatment at 45 and 55°C did not affect the antibacterial activity of honey samples. A significant decrease in both glucose oxidase activity and H2O2 production as well as defensin-1 amount was observed in MW-treated samples. Given that defensin-1 and H2O2 are regular antibacterial components of all honeys, MW heating may have similar negative effects on every type of crystallized/liquid honey.

Bee-derived antibacterial peptide, defensin-1, promotes wound re-epithelialisation in vitro and in vivo.

Royal jelly (RJ) has successfully been used as a remedy in wound healing. RJ has multiple effects, including antibacterial, anti-inflammatory and immunomodulatory activities, in various cell types. However, no component(s) (other than antibacterial) have been identified in RJ-accelerated wound healing. In this study, we demonstrate that keratinocytes are responsible for the elevated production of matrix metalloproteinase-9 (MMP-9) after incubation with a water extract of RJ. Furthermore, the keratinocyte migration and wound closure rates were significantly increased in the presence of RJ extract. MMP-9 production was reduced significantly following proteinase K treatment but remained stable after heat treatment, indicating that active component(s) have a proteinous character. To identify the component responsible for inducing MMP-9 production, RJ extract was fractionated using C18 RP-HPLC. In fractions exhibiting stimulatory activity, we immunochemically detected the bee-derived antibacterial peptide, defensin-1. Defensin-1 was cloned, and recombinant peptide was produced in a baculoviral expression system. Defensin-1 stimulated MMP-9 secretion from keratinocytes and increased keratinocyte migration and wound closure in vitro. In addition, defensin-1 promoted re-epithelisation and wound closure in uninfected excision wounds. These data indisputably demonstrate that defensin-1, a regular but concentration variable factor found in honey and RJ, contributes to cutaneous wound closure by enhancing keratinocyte migration and MMP-9 secretion.

Antibiofilm efficacy of honey and bee-derived defensin-1 on multispecies wound biofilm.

Many clinically relevant biofilms are polymicrobial. Examining the effect of antimicrobials in a multispecies biofilm consortium is of great clinical importance. The goal of this study was to investigate the effect of different honey types against bacterial wound pathogens grown in multispecies biofilm and to test the antibiofilm activity of honey defensin-1 (Def-1) in its recombinant form. A modified Lubbock chronic wound biofilm formed by four bacterial species (Staphylococcus aureus, Streptococcus agalactiae, Pseudomonas aeruginosa and Enterococcus faecalis) was used for evaluation of honey and recombinant bee-derived Def-1 antibiofilm efficacy. Recombinant Def-1 was prepared by heterologous expression in Escherichia coli. We showed that different types of honey (manuka and honeydew) were able to significantly reduce the cell viability of wound pathogens (Staphylococcus aureus, Streptococcus agalactiae and Pseudomonas aeruginosa) in mature polymicrobial biofilm. None of the tested honeys showed the ability to eradicate Enterococcus faecalis in biofilm. In addition, recombinant Def-1 successfully reduced the viability of Staphylococcus aureus and Pseudomonas aeruginosa cells within established polymicrobial biofilm after 24 and 48 h of treatment. Interestingly, recombinant Def-1 did not affect the viability of Streptococcus agalactiae cells within the biofilm, whereas both natural honeys significantly reduced the viable bacteria. Although Enterococcus faecalis was highly resistant to Def-1, Def-1 significantly affected the biofilm formation of Enterococcus faecalis and Streptococcus agalactiae after 24 h of treatment, most likely by inhibiting its extracellular polymeric substances production. In conclusion, our study revealed that honey and Def-1 are effective against established multispecies biofilm; however, Enterococcus faecalis grown in multispecies biofilm was resistant to both antimicrobials.

Honeybee glucose oxidase--its expression in honeybee workers and comparative analyses of its content and H2O2-mediated antibacterial activity in natural honeys.

Antibacterial properties of honey largely depend on the accumulation of hydrogen peroxide (H2O2), which is generated by glucose oxidase (GOX)-mediated conversion of glucose in diluted honey. However, honeys exhibit considerable variation in their antibacterial activity. Therefore, the aim of the study was to identify the mechanism behind the variation in this activity and in the H2O2 content in honeys associated with the role of GOX in this process. Immunoblots and in situ hybridization analyses demonstrated that gox is solely expressed in the hypopharyngeal glands of worker bees performing various tasks and not in other glands or tissues. Real-time PCR with reference genes selected for worker heads shows that the gox expression progressively increases with ageing of the youngest bees and nurses and reached the highest values in processor bees. Immunoblot analysis of honey samples revealed that GOX is a regular honey component but its content significantly varied among honeys. Neither botanical source nor geographical origin of honeys affected the level of GOX suggesting that some other factors such as honeybee nutrition and/or genetic/epigenetic factors may take part in the observed variation. A strong correlation was found between the content of GOX and the level of generated H2O2 in honeys except honeydew honeys. Total antibacterial activity of most honey samples against Pseudomonas aeruginosa isolate significantly correlated with the H2O2 content. These results demonstrate that the level of GOX can significantly affect the total antibacterial activity of honey. They also support an idea that breeding of novel honeybee lines expressing higher amounts of GOX could help to increase the antibacterial efficacy of the hypopharyngeal gland secretion that could have positive influence on a resistance of colonies against bacterial pathogens.