Beeswax： A minireview of its antimicrobial activity and its application in medicine

Filippo Fratini， Giovanni Cilia， Barbara Turchi， Antonio Felicioli，2

1Department of Veterinary Sciences， Viale delle Piagge 2， University of Pisa， Italy

2Interdepartmental Research Center， Nutraceuticals and Food for Health， Via del Borghetto 80， University of Pisa， Italy

Beeswax： A minireview of its antimicrobial activity and its application in medicine

Filippo Fratini1，2✉， Giovanni Cilia1， Barbara Turchi1， Antonio Felicioli1，2

1Department of Veterinary Sciences， Viale delle Piagge 2， University of Pisa， Italy

2Interdepartmental Research Center， Nutraceuticals and Food for Health， Via del Borghetto 80， University of Pisa， Italy

Beeswax

Article history:

Received 15 May 2016

Received in revised form 16 June 2016

Accepted 1 July 2016

Available online 20 September 2016

Beeswax is the substance that forms the structure of a honeycomb； the bees secrete wax to build the honeycombs where to store honey. Thanks to its rich hydrophobic protective properties，the beeswax is in fact present within cosmetics and body products. Also， beeswax is used in the food industry: as a fi lm to wrap cheese for maturing or as a food additive （E901） to give shine to the products. Exactly as the honey which it contains， beeswax is also characterized by several therapeutic properties of great interest to us； it is thought to be particularly ef f ective in healing bruises， inf l ammation and burns. Recently， the interest of researchers has moved even on antimicrobial properties of beeswax although there are still few studies in the literature focused only on the action of beeswax. The few studies showed an anitimicrobic ef f ectiveness of beeswax against overall Staphylococcus aureus， Salmonella enterica， Candida albicans and Aspergillus niger； these inhibitory ef f ects are enhanced synergistically with other natural products such as honey or olive oil. This minireview aims to be a collection of major scientif i c works that have considered the antimicrobial activity of beeswax alone or in combination with other natural products in recent years.

1. Introducttiioonn

In nature， there are some insects that produce wax， but some Apoidea ［1］， especially bees， produce wax more appreciated and used by man. The most used wax， beeswax， is produced by species

Apis mellifera and Apis cerana， which are the most bred by humans and， therefore， it provides easier access to this bee product that has a wide spectrum of uses.

Beeswax is a complex product secreted in liquid form by special wax glands in the abdomen of younger worker bees （aged between 12 and 18 days， that is to say at the end of the period in which the bees act as nurses） ［2］. In contact with the air， it solidif i es in scales（that the bees model with jaws to build the honeycombs， adding pollen and propolis） ［3，4］.

When secreted by the bee， the pure beeswax is almost white； only after contact with honey and pollen it assumes a variably intense yellowish color and turns brown after about four years， because itcontains the cocoon［5］. It resists the action of acids and gastric juices of honeybees and is insoluble in water and cold alcohol； it dissolves partially in boiling alcohol， and completely in chloroform， in carbon disulf i de， and in the essence of hot turpentine ［4，6］. When the wax is treated with boiling alcohol the part that melts is formed by cerotic acid， free or mixed with small amounts of melisic acid， while the one that does not dissolve is formed by ether-melisil palmitic mixed with small amounts of ethers compounds of palmitic and stearic acid. Its density at 15 ℃°is about 0.960 kg/m3to 0.970 kg/m3and it melts at temperatures between 63.5 ℃ and 64.5 ℃ ［3，5，6］. If subjected to a dry distillation it turns into a buttery mass which is called wax oil［7］.

1.1. Composition of beeswax

Beeswax is a complex mixture （more then 300 components） of hydrocarbons， free fatty acids， esters of fatty acids and fatty alcohol，diesters and exogenous substances ［8，9］.

The beeswax composition is: hydrocarbons （12%-16%） with a predominant chain length of C27-C33， mainly heptacosane，nonacosane， hentriacontane， pentacosane and tricosane ［10］； freefatty acids （12%-14%）， with a chain length of C24-C32 ［11］； free fatty alcohols （ca. 1%） of C28-C35 ［5］； linear wax monoesters and hydroxymonoesters （35%-45%） with chain lengths generally of C40-C48， derived fundamentally from palmitic，15-hydroxypalmitic and oleic acids ［11］； complex wax esters （15%-27%） containing 15-hydroxypalmitic acid or diols， which through their hydroxyl group，are linked to another fatty-acid molecule ［7］； exogenous substances that are mainly residues of propolis， pollen， small pieces of fl oral component factors and pollution ［5］. The composition of the beeswax may vary between and among the dif f erent families and dif f erent breeds of bees， because it is probable that wax production is closely related to bee genetics and diet ［12］.

1.2. Pharmaceutical and traditional use of beeswax

"Pharmaceutical" use of beeswax dates back to ancient Egypt: as reported by the Ebers Papyrus （1550 B.C.）， beeswax was the main ingredient in many recipes for the preparation of ointments and creams used to help pull plugs， to treat burns and wounds and to soothe joint pain. The "father of medicine"， Hippocrates，recommended the use of beeswax in case of purulent tonsillitis. In ancient Rome， many doctors of the time used to apply a cream known as "cold cream"， which contained olive oil， beeswax and rose water for the treatment of burns， wounds， cuts， bruises and fractures［2］. Beeswax was one of the components of the fi rst cosmetic cream， which was created by Galen， the great Greek physician， in 150 B.C.， composed of beeswax and olive oil， with emulsion of water （or rose water） ［13］. Beeswax plays an important role also in Ayurvedic medicine， the ancient and traditional Indian medicine，with the name of Madhuchishtha ［14］. In Western countries， the search for natural products to be used together with drugs or， even，to replace them has lead to a "rediscovery" of Ayurvedic medicine. The Madhuchishtha （beeswax） is used for the care of wounds from abrasion or even from burns with topical application ［15］； it has been shown to be particularly ef f ective in the treatment of heel cracking［16］. Some reports highlight the use of Madhuchishtha in combination with other natural products or mixtures， like Madhu（Honey） or Guda （Jaggery） or Taila （Oil） ［17］. Today beeswax is widely studied and used for human medicine.

2. Antimicrobial activity of crude beeswax

During recent years， the antimicrobial activity of natural products and especially products of the hive is gaining importance and unlike other bee products， beeswax has been only recently studied. Crude beeswax showed antibacterial activity against several bacterial strains and against the Candida albicans （C. albicans） yeast ［18］. The sample of beeswax was ef f ective against both Gram-positive bacteria， in particular S. aureus ATCC25923 （S. aureus ATCC25923） （7 mm），Streptococcus epidermidis ATCC12228 （6.5 mm） and Streptococcus pyogenes ATCC19615 （6.5 mm）， and against Gram-negative bacteria，in particular Bacillus subtilis ATCC27853 （B. subtilis ATCC27853）（7 mm）， Pseudomonas aeruginosa ATCC27853 （4 mm）， Escherichia coli ATCC25922 （E. coli ATCC25922）， and a particular inhibitory ef f ect was found against C. albicans NCTC2708 （20 mm）； no ef f ect was found instead against Salmonella typhimurium ATCC14028 and Proteus mirabilis ATCC14153 ［18］.

Considerable interest was aroused by beeswax methanol and ethanol extracts ［19］. Beeswax was extracted with four different solvents: methanol aqueous medium at concentrations of 99.9% and 70%， respectively denominated Meh and Mel； ethanol aqueous medium at concentrations of 96% and 70%， respectively denominated Eh and El.

The results， expressed in millimeters by measuring the zone of inhibition obtained from the ef f ect of the extracts on the tested microrganisms （Table 1） were very successful. As shown in Table 1， the most sensitive strains with WMeh were S. enterica CCM4420 and Candida tropicalis （C. tropicalis） ， while， for WMel the most sensitive strains were E. coli CCM3988， C. albicans， C. tropicalis and Aspergillus niger （A. niger） . With Weh the strains with a higher zone of inhibition were A. niger， C. albicans and Candida glabrata （C. glabrata） ， and with WEl they were found to be Listeria monocytogenes CCM4699， E. coli CCM3988 and C. glabrata［19］.

Table 1Diameters in mm of beeswax inhibition zones against pathogenic bacteria，moulds and yeasts［19］.

The reasons for the different antimicrobial actions of beeswax extracted with methanol and beeswax extracted with ethanol are still unknown， but it is assumed that they were derived from the dif f erent extraction method that inhibits or blocks certain molecules ［19］. At present， this is the only available research in literature that have considered the antimicrobial activity of beeswax alone.

3. Antimicrobial activity of beeswax in synergy with other natural products

Instead， a little more has been studied and tested regarding the antimicrobial action of beeswax in synergy with the other products of the hive and other natural products. Honey， beeswax and olive oil mixture （1:1:1， v/v） are useful to inhibit the growth of S. aureus and C. albicans， isolated by human patients ［20］. After being incubated at 37 ℃， the bacteria （for 24 hours） and and the yeast （for 48 hours）， a zone of inhibition of 4 mm and 3.5 mm was measured for S. aureus and C. albicans， respectively ［20］. After the fi rst incubation， the two microorganisms were incubated separately in different cultured media to test the effi ciency of the various components. As shown in Table 2， S. aureus was not able to grow in media added with honeyand agar， with the mixture and with agar and mix （both at 66% versus 50%）； a slight growth was also shown in the media with agar and olive oil， while moderate and heavy growth was seen in other media. With regard to C. albicans and S. aureus （Table 2）， growth was inhibited in media with agar - honey， with the mixture of honey，beeswax and olive oil （1:1:1） and with agar - mixture 66%， while in other media was noted a growth ranging from moderate to heavy ［20］.

Table 2Growth of C. albicans and S. aureus on dif f erent media ［20］.

The antifungal activity of the mixture of honey， beeswax and olive oil was also tested in vivo in patients with pityriasis versicolor， tinea cruris， tinea corporis and tinea faciei ［21］. Pityriasis versicolor is a superf i cial skin infection caused by four of the eleven species of Malassezia （Malassezia furfur， Malassezia sympodialis， Malassezia globular and Malassezia obtusa）， characterized by changes in skin pigmentation， with irregular and well demarcated patches and may be related to pregnancy， disease like diabetes， state of chronic malnutrition or ineffi cient personal hygiene； tinea cruris is a fungal infection of the groin area caused by Trichophyton rubrum and Epidermophyton floccosum that affects mainly males； tinea corporis is a form of superf i cial mycosis by Trichophyton rubrum，Microsporum canis and Trichophyton mentagrophytes that primarily af f ects the face and arms and spreads by contact with pets ［22］； tinea faciei is a very rare infection that af f ects the face whose causative agents are Trichophyton rubrum and Trichophyton tonsurans ［22.23］.

The mixture was applied topically in 37 patients （14 suf f ering from pityriasis versicolor， 8 from tinea corporis， 14 from tinea cruris， and 1 from tinea faciei） every eight hours for 4 weeks ［21］. For patients with pityriasis versicolor at the beginning of the therapy the average score ± s.d. was （7.10±1.35） while at the end of the treatment（fourth week） was （1.00±1.20）， showing clinical sign in 12 out of 14 patients. The average score for patients with tinea corporis from（8.70±2.40） is decreased to （1.30±1.50） with clinical sign in 6/8 of patients， while the average score for patients with tinea cruris from（8.50±2.40） is decreased to （1.00±1.30） with clinical sign in 11/14 of 14 patients ［21］.

The antimicrobial effi cacy of the mixture of honey， beeswax and olive oil was also tested on strains of C. albicans isolated from four infants with diaper dermatitis. Patients who had candidiasis， showed severe erythema with pustules and/or ulceration. Diaper dermatitis associated with C. albicans was treated topically with the mixture of honey， beeswax and olive oil for 7 days. After the treatmnet period， clinical improvments were observed. At the end of treatment 50% of infants with C. albicans completely or almost completely reduced the level of injury， while the remaining 50% still showed yeast infection and the level of lesions slightly decreased with the disappearance of ulcers and pustules ［21］.

Recently， the antimicrobial activity of propolis and beeswax in synergy （1:1， v/v） has also been investigated in order to inhibit the growth of S. aureus ATCC25923， Staphylococcus epidermidis ATCC12228， B. subtilis ATCC27853 and C. albicans NCTC270 ［25］. Propolis is a resinous product that bees collect from the buds of trees and which is subsequently treated with the addition of beeswax，pollen and enzymes. For its insulating properties it is used by bees as a glue to seal the cell walls. For its antimicrobial， antiviral， antifungal and anti-inf l ammatory properties， its use in medicine dates back to ancient times ［26，27］.

S. aureus and C. albicans were the most sensitive microrganisms to the mixture with a zone of inhibition， respectively， 20 and 22 mm［25］.

Staphylococcus epidermidis and B. subtilis have proved to be slightly more resistant to the mixture （13.5 mm and 10.5 mm）， but still have conf i rmed the antibacterial action of propolis and wax together ［28］.

4. Other properties of beeswax

The use of mixture of honey， beeswax and olive oil has proved to be very ef f ective for the treatment of atopic dermatitis， psoriasis and diaper dermatitis ［20， 28-30］. All patients subjected to therapy showed signif i cant improvements after a month of local application to three times a day. Therapy with the mixture is found to be more ef f ective than treatment with the conventional medicines used for the treatment of the diseases specif i ed before. Signif i cant ef f ects regarding the application of the mixture of honey， beeswax and olive oil have also been demonstrated in patients with hemorrhoids and anal fi ssure ［29］. A new mixture of honey， olive oil， propolis extract and beeswax （called HOPE by authors ［29］） was used in treatment of chemotherapy-induced mucositis proving to be a very fast method for healing the patients with high degrees of mucositis ［31］. C. albicans proliferation is often associated with diaper dermatitis， and the application of mixture of honey， olive oil and beeswax in some cases has the same ef f ect of nistatine cream； thus the synergy of nistatine cream with the mixture could represent an ef f ective therapeutic tool ［32］. Moreover， the same mixture showed high ef f ectiveness in treatment of canine deep second-degree burns， so it is possible an use in veterinary medicine［33］. A component of beeswax of major interest in medicine is D-002. D-002， a natural alcoholic mixture of high molecular weight which has been isolated and purif i ed from the beeswax of Apis mellifera ［34，35］. The D-002， in addition to having among its components a high amount of triacontanol （fatty alcohol which stimulates the growth of many plants）， has anti-ulcer activity［34，36］， anti-inflammatory activity ［37，38］， protective action for the gastrointestinal mucosa ［39，40］， ef f ect in patients with non-alcoholic fatty liver disease ［41］ and osteoarthritis symptoms ［42］， and， in studies carried out on murine animal models， was found to be particularlyef f ective on gastroesophageal ref l ux ［43］ and intestinal problems of acidity ［38，44-47］.

Beeswax is also often used in the food packaging to protect the cheese during the seasoning as a polishing agent E901 or as edible coverage mixed with the polylactic-acid PLA［48-52］.

5. Conclusions

In conclusion it would be appropriate to carry out further studies on the antimicrobial activity of beeswax alone and in combination with other natural products and in particular with beehive products. It would also be interesting to investigate the antimicrobial activity of the individual substances that characterize the beeswax produced under dif f erent environmental and geographical conditions.

Moreover， although the information provided by the studies collected in this review are important and show the ef f ectiveness of beeswax， it will be crucial in future investigations to develop the quantitative tests incorporating the use of the MIC （Minimum Inhibitory Concentration） and the FIC （Fractional Inhibitory Concentration） to determine quantitatively the real contribution of this product to the antimicrobial activity.

Furthermore， antibiotics， pesticides， herbicides， fungicides and acaricides are widely used in beekeeping. The residues of these substances， especially antibiotics， are a relevant risk for the quality of bee products and for human health and the accumulation of pesticides overall in beeswax is well documented as a result from environmental pollution［54-59］.

No reference and/or information on this issue have been reported in the studies analyzed， but it is possible that the data can be skewed by the presence of antibiotic residues that continue to have a certain residual antibacterial and antifungal activity even against bacterial strains used in the laboratory.

For this reason in future investigations it will be necessary to employ pure and residue free beeswax with guaranteed chemical composition.

Conflict of interest statement

We declare that we have no conf l ict of interest.

Acknowledgments

We wish to thank Salvatore Dolores for linguistic assistance provided and Angelo Sommaruga for the essential technical information.

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10.1016/j.apjtm.2016.07.003

✉First and corresponding author: Filippo Fratini， Department of Veterinary Sciences， Viale delle Piagge 2， University of Pisa， Italy.

Tel: +390502216959， +390502216941

E-mail: fi lippo.fratini@unipi.it

ARTICLE INFO

Antimicrobial activity

Honeybees

Natural product