Antimicrobial Activities And Physico-chemical Analyses Of Honeys

1.5.2. Honeys as Modern Medicine

In the last 30 years interest in the use of honey as a treatment agent  has  increased. Most research that has been undertaken has focused on the employment of honey in wound treatment (Ahmed et al., 2003; Berguman et al., 1983; Dumronglert, 1983; Emarah, 1982; Wadi et al., 1987; Ingle et al., 2006).

Efem (1988) reported the first large clinical cohort study involving 59 patients who had a  variety of wounds such as Fournier’s gangrene,  burns and ulcers. The use  of honey on these patients resulted in successful wound healing and the clearance of infection. In addition, Subrahmanyam (1993,  1994, 1996,  1998) and Subrahmanyam  et al., 2001, 2003, 2007) reported several clinical trials on burns patients with honey compared to various different treatments.

Clinically, many researchers have studied the uses of honey in wound management and it has been reported to clear wound pathogens rapidly (Al-Waili and Saloom, 1999; Lusby et al., 2002), to stimulate immune response and to reduce inflammation (Molan and Betts, 2004; Tonks et al., 2003) and to support the debridement of wounds by autolysis (Stephen-Haynes, 2004). In addition, honey has been reported to have a deodorising property on wounds, due to the oxidation  of glucose by bacteria resulting in production of lactic acid rather than malodorous compounds such as ammonia, sulphur compounds and amines produced by the breakdown of amino acids (Cooper, 2005; Molan, 2002; Stephen-Haynes, 2004). Moreover, honey has been used effectively on skin grafts (Schumacher, 2004),

diabetic foot ulcers (Eddy and Gideonsen, 2005), malignant ulcers (Simon  et  al.,  2005) and abscesses (Okeniyi et al., 2005). Some researchers have  observed  that  honey promotes tissue regeneration through the stimulation of angiogenesis and the growth of fibroblasts and epithelial cells (Efem, 1988, 1993; Stephen-Haynes, 2004; Subrahmanyam, 1994, 1998). Fast healing can therefore minimise the need for skin grafts (Subrahmanyam, 1998).

Recently, (Gethin et al., 2008) observed that the use of manuka honey as a wound dressing reduced wound pH which in turn decreased  protease  activity,  increased fibroblast activity and released more oxygen from haemoglobin to promote rapid wound healing. Furthermore, after honey is applied to the wound, it forms a film of liquid between the wound and  the  dressing that prevents the dressing from sticking to the wound, reducing pain and not damaging the newly formed cells. As honey has   no adverse effects on tissue, it can be used on wounds safely and introduced into  cavities and sinuses to clear infection (Molan, 2000).

1.5.3. Some Physical and Chemical Composition of Natural Honey

Honey according to Codex Alimentarius Commission (1989) has several important qualities in addition to composition and taste. Freshly extracted honey is a viscous liquid. Its viscosity depends on large variety of  substances  and  therefore  varies with its composition and particularly with its water content. Hygroscopicity is another property of honey and describes the ability of honey to absorb and hold  moisture from environment. Normal honey with water content of 18.8% or less will absorb moisture from air of a relative humidity of above 60%. The surface tension of honey varies with the origin of the honey and is probably due to colloidal substances. Together with high viscosity, it is responsible for the foaming characteristics of honey (Olaitan et al., 2007).

Natural honey contains about 200 substances, including  amino  acids, vitamins, minerals and enzymes, but it primarily contains sugar and water. Sugar accounts for 95–99% of honey dry matter. The principal carbohydrate constituents of honey are fructose (32.56 to 38.2%) and glucose (28.54 to 31.3 %), which represents 85–95% of total sugars that are readily absorbed in the gastrointestinal tract (Ezz El- Arab et al., 2006; Moundoi et al., 2001).

Other sugars include disaccharides such as maltose, sucrose, isomaltose turanose, nigerose, meli-biose, panose, maltotriose, melezitose.  A  few  oligosaccharides are also present. Honey contains 4 to 5% fructo-oligosaccharides, which serve as probiotic agents (Chow, 2002; Ezz El-Arab et al., 2006). Water is the second most important component of honey. Organic acids constitute 0.57% of honey and include gluconic acid which is a by-product of  enzymatic  digestion of  glucose. The organic acids are responsible for the acidity of honey and contribute largely to its characteristic taste (Olaitan et al., 2007). The concentration of mineral compounds ranges from 0.1% to 1.0 %. Potassium is the major metal, followed by calcium, magnesium, sodium, sulphur and phosphorus. Trace elements include  iron,  copper, zinc and manganese (Sampath et al., 2010; Lachman et al., 2007; Rashed and Soltan, 2004).

Nitrogenous compounds, vitamins C, B1 (thiamine)  and  B2  complex vitamins like riboflavin, nicotinic acid, B6 and panthothenic acid are also found  (Jagdish and Joseph, 2004). Honey contains proteins only in minutequantities, 0.1–0.5 percent (Lee et al., 1998). According to a recent report, specific  protein quantities  differ according to the honeybee origin (Won et al., 2009).

A variety of enzymes such as oxidase, invertase, amylase, catalase, etc. are present in honey. However, the main enzymes in honey are invertase (saccharase), diastase (amylase) and glucose oxidase. They have  an important role in the formation  of honey (Olaitan et al., 2007). The enzyme glucose oxidase produces hydrogen peroxide (which provides antimicrobial properties) along with gluconic acid from glucose which helps in calcium absorption. Invertase converts sucrose to fructose and glucose. Dextrin and maltose are produced from long starch chains by the activity of amylase enzyme. Catalase helps in producing oxygen and water from hydrogen  peroxide (Bansal et al., 2005).

Colour Characteristics

The colour in liquid honey varies from clear and colourless (like  water)  to dark amber or black. The various honey colours are basically all shades of yellow and amber. Colour varies with botanical origin, age, and storage conditions,  but transparency or clarity depends on the amount of suspended particles such as pollen (Olaitan et al., 2007). Less common honey colours are bright yellow (sunflower), reddish undertones (chest nut), grayish (eucalyptus) and greenish (honeydew). Once crystallized, honey turns lighter in colour because the glucose crystals are  white.  Honey crystallization results from the formation of monohydrate glucose crystals,  which vary in number, shape, dimension, and quality with the honey composition and storage conditions. The lower the water and the higher the glucose content of  honey,  the faster the crystallization (Olaitan et al., 2007).

In all over the world, dark honeys are especially appreciated. The most commonly used methods are based on optical comparison,  using  simple  colour grading after Pfund (Fell, 1978) or Lovibond (Aubert  and Gonnet, 1983). The  values  of these comparators give a measure of colour intensity, but only along the normal amber tone of honey. The Lovibond comparators are easier to handle than the Pfund graders, but honey is generally marketed according to the Pfund colour scale. That is why at present Lovibond graders with a Pfund scale are marketed.

Electrical conductivity

The measurement of electrical conductivity (EC) was introduced a long time ago (Vorwohl, 1964). At present it is the most useful quality parameter for the classification of unifloral honeys, which can be determined by relatively inexpensive instrumentation. This has been confirmed by the data, published in this issue (Persano-Oddo and Piro, 2004). On the basis of an extensive survey of EC values on honeys originating from different parts of the world (Bogdanov et al., 2002), this parameter was included recently in the new international standards for honey (Codex Alimentarius, 2001; European Commission, 2002), replacing the determination of ash content. Indeed, EC correlates well with the mineral content of honey, (Accorti et al., 1987). In these standards maximal EC values for blossom honeys (except chestnut honey) are introduced for differentiation between honeydew and blossom honeys.

The method for the determination of electrical conductivity is described in Bogdanov et al. (2002). Honey EC values are expressed in milli Siemens/cm at 20 °C, while nowadays the international reference measurements should be carried out at 25

°C. This contradiction needs to be resolved.

pH and acidity

All honeys are acidic with a pH-value generally lying between 3.5 and 5.5,  due to the presence of organic acids that contribute to honey flavour and stability  against microbial spoilage. In honey the main acid is gluconic acid, which is found together with the respective glucono-lactone in a variable equilibrium (White et al., 1958). Free acidity, total acidity and pH have some classification power for the discrimination between unifloral honeys, while lactones, due to  their  strong  variability, do not provide useful information (Persano-Oddo and Piro, 2004). The methods for the determination of free acidity by titration to pH 8.3 or to an

equivalence point have a poor reproducibility (Bogdanov et al., 2002), due to lactone hydrolysis during titration. The reproducibility of the measurement of total activity  (free acidity +lactones) is slightly better (Conte et al., 2002).

Acids

According to Codex Alimentarius (2001) and European Commission (2002), the flavour of honey results from the blending of many “notes,” not the least being a slight tartness or acidity. The acids of honey account for less than 0.5 percent of the solids, but this level contributes not only to the flavour,  but is in part responsible for  the excellent stability of honey against microorganisms. Several  acids  have  been  found in honey, gluconic acid being the major one. It arises from dextrose through the action of an enzyme called glucose oxidase. Other acids in honey are formic, acetic, butyric, lactic, oxalic, succinic, tartaric, maleic, pyruvic, pyroglutamic, a-ketoglutaric, glycollic, citric, malic, 2- or 3-phosphoglyceric acid, a- or B-glycerophosphate, and glucose 6-phosphate.

Water content

The water content is a quality parameter, important above all for honey shelf life. It has a minor importance for the characterisation of unifloral honeys. However, depending on the production season and the climate, unifloral honeys show  some typical differences in water content, which affect the physical properties of honey (viscosity, crystallisation) and also influence the value of the glucose/water ratio (Persano-Oddo and Piro, 2004). However, water content can be artificially altered during honey processing.

Moisture is routinely determined by refractometry by an Abbé analogue refractometer. Digital refractometers can also be used for the determination of water content, as the results achieved are not significantly different from those obtained with the analogue ones. The values, determined by refractometry are somewhat lower than the true water content, which can be measured only by Karl Fischer  titration (Bogdanov, 1999).

Enzymes: diastase and invertase.