HONCEA Adina1, BURLOI Elena2, SCHRÖDER Verginica3*, MITITELU Magdalena4, DUMITRESCU Denisa Elena3
1Railway Hospital, Bulevardul 1 Mai 3-5, 900123, Constanța, ROMANIA
2Sensiblu Pharmacy, Constanta Street, Bloc C3, 905700, Navodari, ROMANIA
3Faculty of Pharmacy, Ovidius University, Mamaia Blvd. 124, 900527, Constanta, ROMANIA
4Clinical Laboratory and Food Hygiene Department, Faculty of Pharmacy, ”Carol Davila” University of Medicine and Pharmacy, 6, Traian Vuia Street, 020956, Bucharest, ROMANIA
A variety of natural products are approved and used in the prevention or treatment of infectious pathologies, due to their content in active principles with remarkable antimicrobial action. Concerns about the study of these natural sources meet the biomedical research whose interest in the study is antibiotic resistance.
Honey bee falls into the category of such a product, both food and with significant potential in the manufacture of products with multiple medical applications.
The present study makes a comparative analysis of several types of honey of Romanian production, by evaluating the antimicrobial activity on some bacterial and fungal strains involved in major infectious pathology. In this regard, 5 types of honey were tested on the reference bacterial strains Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 23235), Streptococcus pyogenes (ATCC 19615), Pseudomonas aeruginosa (ATCC 9027) and Candida parapsilosis (ATCC 22019) using the Kirby–Bauer diffusimetric method.
The results confirm the antimicrobial effect of honey and highlight the inhibitory potential on these tested bacterial strains, sensitive or resistant to antibiotics. We noticed, compared to antibiotics, the superior inhibitory effect on some strains with major implications in infectious pathology (E. coli, P. pseudomonas, S. aureus, Streptococcus pyogenes) as well as on the resistant strain of E. coli, used in the test.
Our results showed that there is an obvious antimicrobial and antifungal activity and they support the importance of using natural products in the prevention of microbial infections. The tested Romanian products can be considered valuable in terms of the anticipated therapeutic effect.
Keywords: Honey, food, antimicrobial and antifungal activity, COVID-19
In the context of the pandemic caused by coronavirus, the consumption of immunostimulatory foods is beneficial for the body. The category of foods with a beneficial effect on respiratory infections also includes bee products: honey, propolis, pollen, royal jelly.
Honey bee (Apis melifera) is a miraculous food that people have been consuming since ancient times, a natural sweetener, flavoring and preservative  used worldwide, which contains over 200 chemical compounds: saccharides (80-85 %) of which monosaccharides (glucose, fructose), disaccharides (sucrose, maltose, isomaltose, furanose), oligosaccharides (maltotriose, panose); water (15-17%); powders (0.2%) ; vitamin C (ascorbic acid), B1 (thiamine), B3 (niacin), B5 (pantothenic acid), B6 (pyridoxine), B9 (folic acid), B12 (cyanocobalamine), H (biotin), A (retinol), E (tocopherol), K (phytomenadione) , , , , , , ; amino acids and proteins (0.1-0.4%) (arginine, cysteine, glutamic acid, aspartic acid, proline, reduced glutathione) , ; enzymes (CAT catalase, invertase, superoxide dismutase, amylase, peroxidase, acid phosphorylase, glucose oxidase) , , , , ; flavonoids, divided into three classes with similar structure – flavonols, flavones and flavonones – (apigenin, chrysin, galangin, kaempeferol, hesperetin, pinocembrin, quercetin), with antioxidant properties; minerals (sodium, potassium, calcium, magnesium, phosphorus, sulfur, iron, zinc, copper, manganese, selenium) , , ; carotenoids , ; wax, pollen, pigments , ; fatty acids, ethyl ester ; phenolic compounds – among the most important constituents, classified as benzoic acid derivatives, cinnamic acid derivatives (caffeic acid, ferulic acid, p-coumaric acid), polyphenols (ellagic acid); antioxidants .
Each constituent has unique nutritional and medicinal properties, it acts synergistically, which makes honey a food with multiple biomedical applications .
The chemical composition and physical properties of honeys (pH, color, enzymatic activity, electrical conductivity, taste) vary depending on several factors , .
Honey has a medium acidity (pH between 3.2 and 4.5) , responsible for the inhibitory action on many pathogenic bacteria, and a high osmolarity which, in combination with the enzyme assembly and hydrogen peroxide, exerts an antimicrobial action . The pH of honey indicates the purity or rawness of honey and depends on geographical areas .
The color of honey varies from light yellow to dark red and even black, depending on the source of the plant. The dark color is mainly due to temperature changes.
Humidity is the most important characteristic for determining the solidity of honey. The tendency to form granules is a characteristic of honey that distinguishes it from other sweeteners .
Due to the complex chemical composition, the nutritional quality of honey is very high and is combined with its use as an alternative drug in therapy , .
There are numerous studies that confirm the biological properties of honey: antioxidant, anti-inflammatory, antibacterial, antiviral, antiulcer, anti-hyperlipidemic, antidiabetic and anti-cancer (breast, cervical, prostate cancer), liver and heart protector .
Due to its remarkable antimicrobial, anti-inflammatory and antioxidant activity honey has a stimulating effect on the immune system , .
In terms of antimicrobial activity, honey has been shown to act on various types of viruses, fungi and bacteria . Honey was found to have a pronounced inhibitory effect (85.7%) on Gram-negative bacteria (Pseudomonas aeruginosa, Enterobacter spp., Klebsiella, Escherichia coli, Acinetobacter, Stenotrophomonas, Shighella spp., Helicobacter pylori, Salmonella) and inhibitory effect on 100% on methicillin-resistant Gram-positive bacteria (Staphylococcus aureus) , .
Honey has been shown to be effective against methicillin-resistant bacteria (Staphylococcus aureus), MRSA, Vancomycin-resistant Enterococcus, due to its osmolarity and acidity, through which bacteria are dehydrated and their proliferation is prevented , , , as well as hydrogen peroxide generated by the enzyme glucose oxidase, phenolic compounds , .
The antibacterial action of honey is very useful in healing wounds and intestinal diseases.
In addition to antibacterial activity, honey reduces inflammation, thus avoiding surgical debridement, neutralizes the bad smell of the wound and accelerates the wound healing process . It has been found that antimicrobial activity depends on the type of honey , .
We can say that honey is not just a food, but a combination of chemicals with medical importance. Therefore, honey is used in the manufacture of drugs, the combined effects of honey and plant extracts paving the way for the production of strong natural drugs against contagious diseases such as tuberculosis, tetanus, influenza, hepatitis, AIDS .
Materials and methods
We analyzed 5 samples including 5 different types of marketed honey: sunflower honey (1 Sf code), polyfloral honey (2 Po code), rapeseed honey (3 Ra code), linden honey (4 Ti code) and raspberry honey (5 Zm code). Organoleptic control was performed according to FR-X, before testing, to determine the quality of the samples. This analysis is performed in order to verify the appearance, smell or taste of a substance, compared to the provisions of the reference rules. If the organoleptic control does not comply with the provisions of FR X, it means that the test substance contains impurities.
The reference bacterial strains were used, respectively – Escherichia coli – ATCC 25922, Staphilococcus aureus – ATCC 23235, Streptococcus pyogenes – ATCC 19615, Pseudomonas aeruginosa – ATCC 9027 and Candida parapsilosis – ATCC 22019.
Culture media used: Glucose broth, Columbia blood agar, Muller-Hinton + 5% blood, Sabouraud.
The Kirby-Bauer diffusimetric method was used, a standardized technique for testing the antibacterial sensitivity of antibiotics. It is a very simple and fast method in order to calculate the therapeutic doses of antibiotics, which allows the simultaneous determination of the sensitivity spectrum of the microorganism and the value of the minimum inhibitory concentration. The method has several variants. In practice it is commonly used the technique of discs impregnated with antibiotics, standardized, recommended by NCCLS (National Committee for Clinical Laboratory Standardization).
On the surface of an agarized medium sown “in cloth” with a standardized inoculum, obtained from the test strain, were placed at equal distances discs impregnated with antibiotic solutions of a certain concentration that diffuse in the medium were placed at equal distances, achieving a concentration gradient inversely proportional to the diameter of the diffusion zone, and therefore also to the distance from the disc.
First the reference bacterial strains Escherichia coli – ATCC 25922, Staphilococcus aureus – ATCC 23235, Streptococcus pyogenes – ATCC 19615, Pseudomonas aeruginosa – ATCC 9027 and Candida parapsilosis – ATCC 22019 were reconstituted. Thus, on the Columbia Agar medium with 5% ram blood, the cryobiles were placed (Corning) for each of the respective strains. The plates were thermostated at 37°C, 24-48 hours, under conditions of aerophilia. Thus pure microbial culture was obtained for each of the respective strains.
This inoculation was performed sterile, using sterile loops, in the bacteriological hood. After 24 hours, specific colonies could be observed on each Petri dish. For the Candida parapsilosis strain, it took 48 hours for the characteristic colonies to appear.
Next, tests were performed to observe the inhibition of bacterial growth for each selected type of honey. For each bacterial strain was used a rigorously nephelometric standardized inoculum (density 0.5 MacFarland units).
Honey samples diluted w / v (1/5 and 1/10) and undiluted were used for testing. For each type of test 3 repetitions were performed.
The bacterial inoculum was distributed on the Muller Hinton medium for each strain. The distribution was made with sterile swab in 3 different directions, for a more uniform coverage of the environment.
In order to be able to observe by comparison the bacterial inhibition, in box 6 micro-tablets of antibiotic were deposited. Antibiotics with known susceptibility to that strain were chosen. Thus, ciprofloxacin was used for Escherichia coli ATCC 25922, for Staphylococcus aureus ATCC 23235 – erythromycin, for Streptococcus pyogenes ATCC 19615 – penicillin, for Pseudomonas aeruginosa ATCC 9027 – piperacillin.
In the case of Candida parapsilosis strain – ATCC 22019, we did not use the antifungal micro-tablet because we do not have a reference range available in EUKAS or CLSI.
The evaluation of the antibacterial activity was performed after 24 hours, during which time the samples were kept at 37° C, under thermostatic conditions.
After 24 hours, susceptibility to the antibiotic was assessed for each strain, according to EUKAST and CLSI standards.
For the interpretation, the diameters of the zones of inhibition (DI) were measured and analyzed in comparison with the reference table regarding the interpretation of the results of quantitative or diffusimetric sensitivity tests .
Results and discussions
Table 1. Organoleptic characteristics of the analyzed honey samples, according to FR-X
|Type of honey||Quality / Color||Smell and taste||Consistency|
yellow-reddish to yellow-brown
|pleasant, sweet, specific||homogeneous, fluid, viscous|
|pleasant, sweet, specific||homogeneous, fluid|
|Ra||light yellow with olive tint||pleasant, sweet, specific||vâscoasă usor cristalizată|
|Ti||orange-yellow to dark brown||sweet, with a pronounced linden aroma||homogeneous, fluid|
|Zm||greenish yellow to reddish||pleasant, sweet, with specific raspberry flavor||viscous, slightly crystallized|
The observations made regarding the antimicrobial activity revealed a reduced response, even non-existent to the use of diluted samples.
Undiluted samples are highlighted by differences in the manifestation of antimicrobial activity (Table 2).
Table 2. DI antimicrobial activity (diameter of inhibition) identified in the analyzed honey samples.
|S. aureus||E. coli||C. parapsilosis||P.aeruginosa||S.pyogenes|
|Polyfloral||22.67 ± 2.05||20.00 ± 0.2||11.33 ± 0.94||27.33 ± 0.94||25.33 ± 0.47|
|Sunflower||25.67 ± 0.94||20.17 ± 0.62||25.33 ± 0.47||25.33 ± 0.47||25.33 ± 0.47|
|Rape honey||18.54 ± 2.36||16.15 ± 0.47||14.61 ± 0.94||22.14 ± 1.25||20.48 ± 1.25|
|Lime honey||25.67 ± 3.30||23.00 ± 1.63||10.33 ± 0.47||33.00 ± 2.16||23.00 ± 2.16|
|Raspberry||30.33 ± 0.47||19.67 ± 0.47||10.33 ± 0.47||26.33 ± 0.94||27.67 ± 0.47|
|Antibiotic*||27.00 ± 0.82||11.50 ± 0.50||–||19.67 ± 0.47-||30.00 ± 1.41-|
Mean values and ± standard deviation, * ciprofloxacin for E. coli, erythromycin for S. aureus, penicillin for S. pyogenes, piperacillin for P. aeruginosa
The measurements performed on the sensitivity of the strains, according to the standards , reveal that the strains used are sensitive to the antibiotics used, with one exception, E. coli which falls into the category of resistant strains.
Thus, in the case of E. coli and P. aeruginosa strains, all tested honey samples showed relevant inhibition quantified by DI, the average values being significantly higher compared to those of DI measured for the antibiotic used for comparison, ciprofloxacin for E. coli and piperacillin for P. aeruginosa.
These results are valuable in that both species have important pathological implications. P aeruginosa ranks first among pseudomonads in terms of the frequency of isolations in the clinical laboratory, the variety, morbidity and mortality of certain infections .
Undiluted samples of sunflower honey (SF) and lime honey (Ti) show an inhibition close to that induced by erythromycin on the Staphylococcus aureus strain (Table 2). In the same strain, the Zm sample is distinguished by higher DI than the same antibiotic. Raspberry honey is less known to consumers and these observations may open up new study opportunities for protection against S. aureus contamination.
The bacterium Staphylococcus is a Gram-positive bacterium. The genus Staphylococcus is composed of 33 species, most of which are represented by the normal skin flora and mucous membranes. Certain coagulase-negative strains, infection-producing agents in immuno-compromised individuals, have developed resistance to antibiotics. These bacteria colonize devices implanted in the human body, such as nails, industrial devices and joints used in bones, heart valves and catheters of various types, as well as in peritoneal dialysis.
An increase in the prevalence and incidence of methicillin-resistant Staphylococcus aureus has been observed, making it more difficult to treat such infections. Coagulase-negative staphylococci are considered among the most common microorganisms involved in hospital infections, so honey has been used to inhibit these bacteria, as well as to treat skin and other infections. The natural ingredients of honey show activities against various microorganisms, this having a more pronounced inhibitory effect on Gram-negative bacteria compared to frequently used antimicrobial agents.
Similar studies have shown 100% inhibition in methicillin-resistant Staphylococcus aureus in parallel with the use of antibiotics .
Our studies as well as those in the literature indicate that the effectiveness of honey against the bacterium Staphylococcus aureus depends on its type and the concentration at which it is administered.
In the case of Candida parapsilosis we observe a fairly obvious area of inhibition in sunflower honey (SF) and rapeseed honey (Ra) and lower in the other samples, lime honey (Ti), polyfloral honey (Po) and raspberry honey (Zm) (Table 2).
This study makes interesting contributions to existing knowledge highlighting the in vitro antibacterial and antifungal effect of honey, using the Kirby Bauer diffusimetric method on specific medium for antibiograms and antifungi grams, Muller Hinton with blood and Sabouraud medium, respectively.
Our results are promising in terms of the purpose of this study to identify some sources of products with antibacterial activity, given the observations that undiluted samples show inhibitory activity on strains with major implications in infectious pathology (E. coli, P. pseudomonas, S. aureus, Streptoccus pyogenes).
The study also highlights the inhibitory effect on a resistant strain of E. coli used in testing superior to the antibiotic ciprofloxacin.
The antifungal effects on the Candida parapsilosis strain were also highlighted.
The results showed that there is an obvious significant antimicrobial and antifungal activity and supports the importance of using natural products in the prevention of microbial infections.
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