Antimicrobial Activity of Donkey Milk against Dermatomycotic Fungi and Foodborne Bacteria

The antimicrobial activity of donkey milk was examined against 3 bacterial and 3 fungal strains selected on the basis of their relevance as human pathogens. All samples of donkey milk exhibited antimicrobial activity against dermatomycotic fungi and foodborne pathogen bacteria. The highest antimicrobial activity was recorded against Trichophyton mentagrophytes and T. rubrum with minimal lethal concentration of 32 mg/ml. In respect to foodborne pathogenic bacteria, Grampositive bacteria (Bacillus cereus and Staphylococcus aureus) were more sensitive to donkey milk than Gram-negative bacteria (E. coli) with minimal lethal concentrations of 32, 64 and 128 mg/ml respectively. Donkey milk kept about 60 to 62% of its antimicrobial activity against B. cereus and S. aurous respectively, after digestion with pepsin (2 mg/ml), suggesting that the fatty acids of donkey milk has the highest antimicrobial effect. While the antimicrobial activity against dermatomycotic fungi and Gram-negative bacteria not affected after digestion with pepsin. To explain the antifungal capability of donkey milk against dermatomycotic fungi, fatty acids were analyzed by gas chromatography. Fatty acids analysis indicated that the major constituents in donkey milk lipid are oleic (25.4%), palmitic (23.75%), linolenic (20.04%), arachidic (3.58%) and stearic (3.26%), which have antimicrobial activity. Finally, on the basis of results obtained in the current study, the antimicrobial activity of donkey milk against Staphylococcus aureus and dermatomycotic fungi, specially T. mentagrophytes and T. rubrum which frequently cause acute or chronic inflammatory tinea corporis; may be considered as a valuable natural product with novel functional protection properties in cosmetics and pharmaceutical industries.


Introduction
Milk is a complex medium containing a variety of nutrients, minerals, vitamins as well as other molecules of functional or bioactive properties [1]. Donkey milk is considered to be helpful in the treatment of human immunerelated diseases; discovery of ass' milk properties find its roots in antiquity, when doctors recommended it to treat several afflictions, due to its healing and cosmetic virtues; Cleopatra, Queen of Ancient Egypt, took baths in donkey milk to preserve the beauty and youth of her skin and used in the manufacture of soaps and moisturizers. Also it is been found that Hippocrates (460-370 BC) prescribed donkey milk for several purposes, such as liver troubles, infectious diseases, fevers, edema, nose bleeds, poisonings and wounds. Trichophyton mentagrophytes and T. rubrum are the common causal agents of tinea, [2] while, Candida species were frequently isolated as causative agents of catheter-related bloodstream infections. In the last years, great interest has been developed toward donkey milk, because of its high likeness to human milk and unique functional properties with low casein content and high percentage of essential amino acids. Because of that, recently it is considered to be a good alternative to infant formulas, since its nutrient composition, particularly the proteins, are very similar to those of human milk [1,3]. Donkey milk contains a number of antimicrobial factors, especially milk proteins, which play a major role in milk protection. Enzymes, especially lysozyme, in donkey milk possess some unique characteristics such as bactericidal property which makes it different from other mammal milks [3,4]. The antimicrobial activity of donkey milk was reported previously against Salmonella choleraesuis and Shigella dysenteriae [5].
One of the defense factors in milk is free fatty acids which protect milk from different microbial infections [6]. Antimicrobial activity of fatty acids was stated to be dependent on chain length and unsaturation degree [7]. Longchain unsaturated fatty acids exhibit inhibitory activity against many bacteria even including methicillin-resistant S. aureus [8]. For instance, oleic and linoleic acids were reported as potent antibacterial [9]. However, milk and dairy products are great sources of saturated fatty acids; donkey milk appears to contain lower saturated fatty acids and higher amounts of essential fatty acids than cow milk. The role and the importance of donkey milk fatty acids are related to nutritional and extra-nutritional properties [10]. This study aims to (1) assess the antimicrobial activity of some Egyptian samples of donkey milks against the dermatomycotic fungi and some foodborne pathogenic bacteria, (2) to determine the minimal lethal concentration of donkey milks against tested pathogenic microorganisms, (3) to detect the major component corresponding to the antimicrobial activity of donkey milk.

Milk Samples Collection
Individual milk samples from different milk animals were collected from El-Minia and Fayoum governorates in Egypt. These milk types were; Buffalo, Donkey, Camel, Cow, Goat, and Sheep. Milk samples were collected immediately after milking in sterilized bottles and chilled in ice box to 4°C, then stored at -80ºC until using.

Gas Chromatographic Analysis of Donkey Milk
To evaluate fatty acids content of donkey milk, three analytical methods were used; DB-Wax, DB-23 and HP-88 methods [13]. Freeze dried milk samples (100 mg) were dissolved in 10 ml hexane, and then 100 µl of potassium hydroxide (2.0 N) was added. After centrifugation, clear supernatant was transferred into a 2.0 ml autosampler vial. The analyses were performed in an Agilent 6890 GC (University of Basilicata-Italy).

Target Microorganisms
Three fungal strains (Candida albicans DSM 2361, Trichophyton mentagrophytes AUMC 5505, T. rubrum AUMC 5488) and 3 bacterial strains (Bacillus cereus ATCC 14579, Staphylococcus aureus ATCC 8095 and Escherichia coli ATCC 25922) were used to investigate the antimicrobial activity of donkey milk samples. Bacteria and Candida strains were obtained from the Department of Agricultural Microbiology, Faculty of Agriculture, Fayoum University, Fayoum, Egypt, while, the other fungal strains were obtained from Assiut University Mycological Centre (AUMC), Assiut, Egypt. Stock cultures of bacteria were maintained on slants of nutrient agar. While, fungal cultures were maintained on slants of potato dextrose agar at 4°C.

Enzymatic Hydrolysis of Lysozyme in Donkey Milk
To polish the antimicrobial activity of lysozyme fraction, hydrolysis of donkey milk was carried out in two steps [14]. Initially, donkey milk was acidified with HCl to a pH 3.5, and then incubated with 2 mg/ml pepsin (Sigma) at 37°C for 2h. Then the enzymatic reaction was stopped by adjusting the pH to 8.0 with 0.5M sodium carbonate (NaHCO3) solution, followed by heating at 75°C for 5 min. The samples were cooled then kept at 4°C till used to determine the antimicrobial activity by agar disk diffusion method. As a control experiment, the lysozyme solution (2 mg/ mL) was subjected to the process described above without the addition of pepsin.

Antimicrobial Activity Test
Antimicrobial activity of different milk types were assessed against fungal and bacterial strains by agar well diffusion method [15], [16]. For each bacterial or fungal strain, sterilized Mueller Hinton and potato dextrose agar medium were poured into sterilized Petri dishes, left to solidify at room temperature (25°C), and then swabbed from fresh bacterial or fungal culture strain. Wells in the centre of agar plate were created using a sterile cork borer (8 mm) and loaded by 250 µl of milk samples (concentrated to 50 folds by lyophilization and suspended in sterile water then pasteurized before tested). Plates with pathogenic bacteria or Candida albicans were incubated at 37°C and 30°C for 24h respectively. Other pathogenic fungi were incubated at 30°C for 48-72 h. The antimicrobial activity was determined by measuring the clear zones diameter (CZD) around each well in mm. Distilled water without test compounds was used as a control. Antibiotics such as Tetracycline (30 µg), Chloramphenicol (30 µg), Flucoral (fluconazole, 100 µg/mL) and Mycosat (nystatin BP, 100 µg/mL) were used as references for comparison in antibacterial and antifungal tests, respectively. All experimental procedures were performed in triplicates.

Determination of Minimum Lethal Concentrations (MLC)
The MLC of donkey milk was determined according to the dilution method described [17], [18]. For pathogenic bacteria or Candida albicans, serial of two-fold concentrations of donkey milk (8,16,32,64,128,256 and 512 mg/ml) were pipetted into tubes containing 4 mL of LB or potato dextrose broth medium, respectively. Each tube was inoculated with 0.4 mL (0.5 McFarland medium) of a standardized suspension of bacterial test species containing 1x10 6 cell/ml. For fungi, liquid media was inoculated with a fungus strain and incubated for approximately 48 h at 30°C. Subsequently, the culture was filtered through a thin layer of sterile sintered Glass G2 to remove mycelia fragments. The titer of spores of each fungus was determined microscopically using a hemocytometer. A suspension containing the spores was used for inoculation on PDA medium. Serial of two-fold concentrations of donkey milk were pipetted into tubes containing 4 mL of PD broth. Each tube was inoculated with 1x10 6 spores/ml.
All inoculated tubes were incubated at appropriate temperature and time for each strain of tested microorganisms. After the incubation period, 0.1 mL from each tube was subcultured on LB agar or PDA plates and incubated at appropriate temperature and time for each microorganism. The least concentration of tested donkey milk which gave a viable count less than 0.1% of the original inoculum (1x10 6 cell/ml) was assumed as the MLC.

Chemical Composition and Antimicrobial Activity of Milk Samples
Data in Table 1 shows the gross composition of donkey milk compared to different milk types used in this experiment. In this study, the antimicrobial activity of donkey milk was examined against 3 bacterial and 3 fungal strains selected on the basis of their relevance as human pathogens. The examination of antimicrobial activity of the different milk samples by the agar diffusion method revealed that only donkey milk inhibited growth of most tested microorganisms. Different samples of donkey milk exhibited various degrees of antimicrobial activity against fungi and bacteria (Table 2 and Figure 1). Dermatomycotic fungi including Trichophyton rubrum are anthropophilic fungi which frequently cause acute or chronic inflammatory tinea corporis which is a superficial fungal infection (Dermatomycosis) of the arms and legs, especially on glabrous skin. The data in Table 2 and Figure 1 showed that different donkey milk samples had a significant antifungal potential against the tested dermatomycotic fungi. The highest antimicrobial activity of donkey milk was recorded against the dermatomycotic fungi including T. mentagrophytes (ranged from 17-25 mm) and T. rubrum (ranged from 15-20 mm) with minimal lethal concentration 32 mg/ml. On the other hand, foodborne pathogen bacteria differed in their sensitivity to donkey milk samples tested, whereas, the Gram-positive bacteria were more sensitive than Gram-negative. The antibacterial activity of donkey milk against B. cereus and S. aureus ranged from 15-23, and 17-20 mm with minimal lethal concentration of 32 and 64 mg/ml respectively, while, E. coli recorded the least inhibition zone values (13-15 mm) with minimal lethal concentration 128 mg/ml ( Table 2). It is worthy to mention that the antimicrobial activity of donkey milk significantly decreased (38-40%) after digestion with pepsin (2 mg/ml) and kept about 60 to 62 % of its antimicrobial activity against B. cereus and S. aurous, respectively, suggesting that the fatty acids of donkey milk has the majority of the antimicrobial effect ( Figure 2). Similar results were reported that monoglycerides of fatty acids possessed the most potent antimicrobial activity against S. aureus [19]. It has been indicated that, donkey's native and digested milk have different effects against two pathogenic bacterial strains; Escherichia coli and Listeria monocytogenes [20]. Lysozyme concentration in donkey milk reaches to 4000 mg/l, whereas only traces were found in bovine milk [21]. Lysozyme is well known as an antibacterial protein. It catalyzes the hydrolysis of bacterial cell wall by cleavage of β-1, 4 linkages between N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) of peptidoglycan in the bacterial cell wall and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens [22]. The in vitro antimicrobial activity of lysozyme is directed against certain Gram-positive bacteria such as Staphylococcus aureus, Micrococcus luteus, Bacillus stearothermophilus, Clostridium tyrobutyricum [23], and to a lesser degree against Gram-negative bacteria [24]. Cow (10 mg
The chemical composition of donkey milk revealed that the lipid fraction was characterized by high levels of linoleic (average 8.15 g·100 g-1 of total fatty acids) and linolenic acid with an average 6.32 g·100 g-1 of total fatty acids. The essential fatty acids (linoleic and linolenic acids) were the most represented components of the poly-unsaturated fatty acids class in donkey's milk [27] showing higher values than human milk and that of the other animal species. So, donkey's milk can be considered a functional food for human nutrition and its potential utilization for infant nutrition as well as adult diets, particular for the elderly [26], [36].
Fatty acids are widely occurring in natural fats and dietary oils and they are known to have antibacterial and antifungal properties. Fatty acid methyl ester extracts of Excoecaria agallocha have been reported to possess antibacterial and antifungal activities against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Klebsiella pneumonia, Candida albicans, C. krusei, C. tropicalis. This extract was rich in palmitic, luric acids and also contains myristic, stearic, pentadecanoic, heptadecanoic acids and lower amount of arachidic acid [28].
The antibacterial effect of fatty acids has been studied against 12 strains, which cause oral pathogens and they found that ω-3 PUSFAs and ω-6 PUSFAs showed an antibacterial effect against 11 of them [29]. Donkey milk fat contains 9.5% free fatty acids, while cow, sheep, goat and human milk fat have low levels of free fatty acids (0.7-1.5%) [30], [31]. Compared to ruminants, donkey milk fat contains a higher percentage of polyunsaturated fatty acids (PUFA) and a lower percentage of saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA). In absolute values, donkey milk contains on average 1.69 g/l PUFA, 5.46 g/l SFA and 1.96 g/l MUFA compared to respectively 5.78, 15.2 and 16.9 g/l in human milk and 1.31, 25.8 and 9.2 g/l in bovine milk [25]. The fatty acids of horse and donkey milk are mainly unsaturated or short-chained, which is interesting from a nutritional point of view. Horse and donkey milks have additionally higher levels of linoleic acid (n 6 C18: 2) and a-linolenic acid (n-3 C18: 3) than bovine milk (respectively 5 and 224 times more) [32]. The antifungal activities exhibited by linolenic, linoleic and oleic acids may be useful in controlling important plant pathogens [33].
It was indicated that C12 (lauric acid) is the most inhibitory saturated fatty acid against gram-positive organisms [34].
Although lysozyme is designated as the main antimicrobial agent in donkey milk owing to its high concentration some fatty acids which have already been determined in donkey milk could be important for overall antibacterial [35]. Longchain polyunsaturated fatty acids are attracting attention as potential new topical treatments for Gram-positive infections due to their antimicrobial potency and anti-inflammatory properties. It has been found that S. aureus cells were killed within 15-30 min during exposure to the Long-chain polyunsaturated fatty acids [36], [37]. In addition it was demonstrated that fatty acid and monoglycerides with 8-12 carbons were more strongly antiviral and antibacterial when added to milk and infant formula than long chain monoglycerides [38]. Moreover it has been found that linoleic, lauric and oleic acid among the detected fatty acids in donkey milk have antibacterial activity against Listeria monocytogenes and Staphylococcus aureus [39].

Conclusion
Finally, on the basis of results obtained in this study, the antimicrobial activity of donkey milk against Staphylococcus aureus and the dermatomycotic fungus Trichophyton rubrum which frequently cause acute or chronic inflammatory tinea corporis; may be considered as a valuable natural product with novel functional properties in cosmetics and pharmaceutical industries.