Antistaphylococcal activity of Omani honey in combination with bovine milk

British Journal of Biomedical Science, 2005 by Al-Jabri, A A, Hosni, S A Al, Nzeako, B, Nsanze, H

Honey is well known for its antibacterial properties,1-4 which act against a range of organisms including those that are highly resistant to antibiotics.5,6 Honey has been used as an adjuvant to accelerate wound healing since ancient times and has been used sporadically in the treatment of burns.2 The mild laxative, bactericidal, sedative and antiseptic characteristics of honey make it an important medicine.7

Bovine and human milk is reported to possess antimicrobial activities8 and contains an array of bioactive substances including lysozyme, lactoferrin, immunoglobulins, growth factors and hormones, all of which are secreted in their active form by the mammary gland.8,9 In combination with milk, honey provides excellent nutritional value and is recommended as the main source of nutrition in children.10,11

The antimicrobial activity of various Omani honeys has been investigated, and some possess excellent antibacterial activity against a wide spectrum of bacterial strains including Staphylococcus aureus.12 In the present study, the extent to which Omani bovine milk inhibits the growth of S. aureus is assessed and whether or not a combination of honey and milk enhances antistaphylococcal activity.

An Omani honey sample (OH26), which previously demonstrated excellent activity against S. aureus (NCTC 6571), was compared to 29 other Omani honey samples after storage in sterile universal containers at room temperature (22-26�C) for no more than two months. Four different bovine milk samples (Al Marai, Al Rawabi, A'Safwa and Sohar) were obtained from farms immediately after pasteurisation and used directly. Honey and milk samples were diluted (1 in 2 to 1 in 16) using sterile distilled water.

A large S. aureus colony was emulsified in 0.1% peptone water (4 mL), yielding approximately 1 x 10^sup 6^ organisms, and used to swab diagnostic sensitivity test (DST, Oxoid, England) agar plates. Four wells (each 6 mm diameter) were cut in the DST agar plate and dilutions of honey or milk (50 �L) were added to each. Honey and milk samples were tested in triplicate. Plates were allowed to stand at room temperature for 10 min and then were incubated for 24 h at 37�C. Inhibition zone diameters were measured in millimetres and the average recorded.

Dilutions of honey and milk were made separately and in combination, using 2-mL volumes. A typical test included honey (diluted 1 in 2), milk (diluted 1 in 2) and a honey and milk combination (diluted 1 in 2). Tests were performed in triplicate and so a total of nine samples were prepared. To each sample was added 20 �L S. aureus (1 x 10^sup 6^ colony forming units [cfu]/mL, standardised with McFarlane turbidity tube 0.5) and mixed thoroughly. From these dilutions, a 10-�L volume was plated on blood agar and incubated for 24 h at 37�C. Subculture of each sample was repeated every two hours up to 24 h. The tests were run five times on five successive days. All colonial growth on the blood agar plates was counted and averaged for each set.

Honey sample OH26, from the Al Batinah region (Al Kaborah), showed the highest level of S. aureus inhibition, and activity was evident up to a 1 in 8 dilution (12.5%). Table 1 shows the sizes of the zones of inhibition obtained with various dilutions of this honey sample and the four milk samples tested. Table 2 shows the percentage growth inhibition of S. aureus by honey, milk and a combination thereof at various time points.

Over the past decade, research into bioactive or biogenic substances derived from foods has been undertaken. Bioactive substances of food origin are considered to be dietary components that exert a regulatory activity beyond basic nutrition.

Although many studies of the antimicrobial activity of honey have been undertaken, it is believed that the present study is the first to address the antimicrobial effects of Omani honey in combination with bovine milk. The antistaphylococcal activity of Omani honey varies considerably,12 and in this study it has been shown that not all milk samples possess antibacterial activity against S. aureus (Table 1). Two of the samples (Al-Rawabi and A'Safwa) not only failed to demonstrate antistaphylococcal activity but actually allowed significant growth of the organism. Combinations of honey and milk showed greater antistaphylococcal activity, enhancing the effect of honey alone by approximately 20%.

Honey is known to contain, phenol, fatty acids, lipids, amylases, ascorbic acid, peroxidases and fructose, and has a high osmolarity and low pH. These elements, acting alone or synergistically, may contribute significantly to the antibacterial activity of honey.13-15 Although honey is known to have an antibacterial effect on different microorganisms, including those that are resistant to antibiotics,5,6 its exact mode of action remains unclear.

Milk is generally accepted to have antimicrobial activities. Many bioactivities attributed to milk are encrypted in the primary structure of milk proteins and they require proteolysis for their effect to be realised. Proteolysis may release these biogenic peptides during gastrointestinal transit or during food processing.8-9 These molecules include: opioid agonist and antagonist peptides; hypotensive peptides that inhibit angiotensin-I-converting enzyme (ACE); and mineral binding, immunomodulatory, antibacterial, and antithrombotic peptides. Other antimicrobial fragments from bovine milk proteins are isracidin, casocidin-I and lactoferricin, which are released by pepsin digestion and have an effect against Gram-positive and Gram-negative bacteria.8,9,16