Antimicrobial Activities And Physico-chemical Analyses Of Honeys

One of the characteristics that sets honey apart from all other sweetening agents is the presence of enzymes. These conceivably arise from the  bee,  pollen, nectar, or even yeasts or micro-organisms in the honey. Those most prominent are  added by the bee during the conversion of nectar to honey. Enzymes are complex protein materials that under mild conditions bring about chemical changes, which may be very difficult to accomplish in a chemical laboratory without their aid. The changes that enzymes bring about throughout nature are essential to life. Some of the most important honey enzymes are invertase, diastase, and glucose oxidase  (Bogdanov et  al., 2008).

Invertase, also known as sucrase or saccharase splits sucrose into its constitutent simple sugars, dextrose, and levulose. Other more complex sugars have been found recently to form  in small amounts during this  action and in part explain   the complexity of the minor sugars of honey. Although the work of invertase is completed when honey is ripened, the enzyme remains in the honey and retains its activity for some time. Even so, the sucrose content of  honey never  reaches zero.  Since the enzyme also synthesizes sucrose, perhaps the final low value for the sucrose content of honey represents an equilibrium between splitting and forming sucrose (Ortiz-Vázquez et al., 2013).

Diastase (amylase) digests starch to simpler compounds but no  starch  is  found in nectar. What its function is in honey is not clear. Diastase appears to be  present in varying amounts in nearly all honey and it can be measured. It has probably had the greatest attention in the past, because it has been used as a measure of honey quality in several countries (Bogdanov et al., 2008).

Glucose oxidase converts dextrose to a related material, a gulconolactone, which in turn forms gluconic acid, the principal acid in honey. Since this enzyme previously was shown to be in the pharyngeal gland of the honey bee, this is probably the source. Here, as with other enzymes, the amount varies in different honeys. In addition to gluconolactone, glucose oxidase forms hydrogen peroxide during its action on dextrose, which has been shown to be the basis of the heat-sensitive antibacterial activity of honey. Other enzymes are reported to be present in honey,  including  catalase and an acid phosphatase. All the honey enzymes can be destroyed  or  weakened by heat (DebMandal and Mandal, 2011).

The methods for the determination of diastase and invertase activity are described (Bogdanov et al., 1997). Later, another formula was found for the diastase determination with the Phadebas method in honeys with low enzyme content (Persano-Oddo and Pulcini, 2004). For the expression of  invertase  results,  international units (U/kg) were proposed instead of Hadorn numbers (von der Ohe  et al., 1999). These changes were included in the online IHC methods.

Hydroxymethylfurfural Fresh honey does not contain hydroxymethylfurfural (HMF). Thus,  HMF is not a criterion for the botanical classification of honey. However, before determining storage-dependent parameters like enzyme activity and colour, one should ensure that honeys are fresh and unheated. Before testing these parameters, it should be checked that the HMF content is below 15 mg/kg (European Commission, 2002).

Three methods for the determination of HMF are described and validated by the IHC (Bogdanov et al., 1997). Only two of them are recommended for  use: the HPLC and the White method. The Winkler method should not be used because one of the reagents (p-toluidine) is carcinogenic. Since the publication of the IHC methods there is a change in the procedure of the HMF determination by HPLC: a Carrez treatment of the honey solution is necessary in order to prevent HMF break-down (DebMandal and Mandal, 2011).

Phenolic  acids and polyphenols

Phenolic acids and polyphenols are plant derived secondary metabolites.  These compounds have been used as chemotaxonomic markers in plant systematics. They have been suggested as possible markers for the determination  of  botanical  origin of honey. Considerable differences in composition and content of phenolic compounds between different unifloral honeys were found. Dark coloured honeys are reported to contain more phenolic acid derivatives but less flavonoids than light coloured ones (Alvarez-Suarez et al., 2013, 2010, 2009).

Proteins and Amino Acids

The amount of nitrogen in honey is low, 0.04 percent on the average, though   it may range to 0.1 percent. Recent work has shown that only 40 to 65 percent of the total nitrogen in honey is  in protein, and some  nitrogen resides in substances other  than proteins, namely the amino acids. Of the 8 to 11 proteins found  in  various  honeys, 4 are common to all, and appear to originate in the bee, rather than the nectar. Little is known of many proteins in honey, except that the enzymes fall into this class (European Commission, 2002; Alvarez-Suarez et al., 2013).

The presence of proteins causes honey to have a lower surface tension than it would have otherwise, which produces a marked tendency to foam and form scum     and encourages formation of fine air bubbles. Beekeepers familiar with buckwheat honey know how readily it tends to foam and produce surface scum, which is largely due to its relatively high protein content (European Commission, 2002).

The amino acids are simple compounds obtained when proteins are broken down by chemical or digestive processes. They are the “building blocks” of the  proteins. Several of them are essential to life and must be obtained in the diet. The quantity of free amino acids in honey is small and of no nutritional significance. Breakthroughs in the separation and analysis of minute quantities of material (chromatography) have revealed that various honeys contain 11 to 21  free  amino  acids. Proline, glutamic acid, alanine, phenylalanine, tyrosine, leucine, and isoleucine are the most common, with proline predominating (DebMandal and Mandal, 2011).

Amino acids are known to react slowly, or more rapidly by heating,  with sugars to produce yellow or brown materials. Part of the darkening of honey with age   or heating may be due to this. Proline, the main amino acid of  honey,  added to honey by the bee, is a criterion of honey ripeness (von der Ohe et al., 1991). This parameter shows characteristic values in different unifloral honeys (Sabatini and  Grillenzoni, 2002; Persano-Oddo and Piro, 2004), roughly correlated with the enzyme activity (Sabatini and Grillenzoni, 2002).

However, the variation of this parameter in different unifloral honeys is quite high and it is not possible to classify unifloral honey on the basis of proline content  only (Sanchez et al., 2001; Sabatini and Grillenzoni, 2002; Persano-Oddo and Piro, 2004). The proline content is easily determined by photometry (Bogdanov et  al.,  1997).

Sugars

Honey is above all a carbohydrate material according to Codex Alimentarius (2001) and European Commission (2002), with 95 to 99.9 percent of the solids being sugars, and the identity of these sugars has been studied for many years. Sugars are classified according to their size or the complexity of the molecules of which they are made. Dextrose (glucose) and levulose (fructose), the main sugars  in  honey,  are  simple sugars, or monosaccharides, and are the building blocks for the more complex honey sugars. Dextrose and levulose account for about 85 percent of the solids  in honey.

Until the middle of this century, the sugars of honey were thought to be a simple mixture of dextrose, levulose, sucrose (table sugar), and an ill-defined carbohydrate material called “honey dextrin.” With the advent of new methods for separating and analyzing sugars, workers in Europe, the United States, and Japan have identified many sugars in honey after separating them from the complex honey mixture. This task has been accomplished using a variety of physical and chemical methods (European Commission, 2002).

Dextrose and levulose are still by far the major sugars in honey, but 22 others have been found. All of these sugars are more complex than the monosaccharides, dextrose and levulose. Ten disaccharides have been identified: sucrose, maltose, isomaltose, maltulose, nigerose, turanose, kojibiose, laminaribiose, a, B-trehalose, and gentiobiose. Ten trisaccharides are present: melezitose, 3-a-isomaltosylglucose, maltotriose, l-kestose, panose, isomaltotriose, erlose, theanderose, centose, and isopanose. Two more complex sugars, isomaltotetraose and isomaltopentaose,  have been identified. Most of these sugars are present in quite small quantities (Codex Alimentarius, 2001; European Commission, 2002).

Most of these sugars do not occur in nectar, but are formed either as a result of enzymes added by the honeybee during the ripening of honey or by chemical action in the concentrated, somewhat acid sugar mixture we know as honey (Bogdanov et al., 2008).