Composition of Sugars in Honey Produced in the South-South and South-West Regions of Nigeria

Six samples of honey from two states in Nigeria, Cross River and Oyo were investigated. (Igoli in Ogoja, farm bred and wild species from Obudu, Agoi Ibami, Eruwa and Iseyin) were analysed for their sugar components, Hydroxymethyl furfural (HMF), free acidity and electrical conductivity. Honey is composed of various sugars but the dominant sugars are two monosaccharide, fructose and glucose which are also reducing sugars. Honey is used for various purposes including medicinal, cosmetics, in the food industry, bakery and confectionary. The sugar contents were analysed using the HPLC with refractive Index (RI). The European Commission procedures were used to analyse HMF and electrical conductivity, while free acidity was analysed by AOAC methods (1999). The results of the analysis showed that although fructose and glucose were predominant in all the samples, only two samples, Igoli in Ogoja and Obudu farm bred met the Codex Alimentarius standard for fructose and glucose at 60g/100g. Fructose values (g/100g) reported for Igoli in Ogoja, Obudu farm, Agoi Ibami, Obudu wild, Eruwa and Iseyin were 34.92 ± 0.12, 36.45 ± 0.14, 30.50 ± 0.06, 28.45 ± 0.09, 33.40 ± 0.17, and 30.45 ± 0.09 respectively. Sucrose in all the honey samples met the Codex Alimentarius standard of < 5g/100g limit. Fructose/glucose ratio for all the samples was greater than 1 which indicated slower crystallization of all the honey samples. Values of free acidity ranged from 11.57 ± 0.07 to 18.60 ± 0.10meq/kg, which were lower than the 50meq/kg limit set by the Council of the European Union showing the absence of undesirable fermentation. HMF concentration in the samples all met the International optimum value of 40mg/kg reported by European Union and 80mg/kg for honey from the tropics. Electrical conductivity of the samples varied from 0.41 ± 0.01 to 0.63 ± 0.00mS/cm. The results showed that the honey samples sold locally in Nigeria meet the required international standards for honey.


Introduction:
Honey (Apis mellofera) is a sweet, thick, supersaturated sugar solution produced by honey bees from plant nectars, plant secretion and excretions of plant-suckling insects of the living parts of plants (Codex Alimentation, 2001). It is one of the known natural sources of sweetness and energy for man. Honey is composed mainly of disaccharides which contain two monosaccharaides, glucose and fructose, with a percentage of water and other group of substances (Kamal and Kleen;2010). Small quantities of other sugars are also present, in the form of other disaccharides, trisaccharides and oligosaccharides which are formed during the ripening and storage effects of bee enzymes and acids of honey (Ball, 2007). Chemical compositions of honey differ depending on the plant species on which the bees forage, the climatic conditions and other factors (Buba et al., 2013). The very concentrated solution of several sugars produce the characteristic physical properties of honey like high viscosity, high density, graduation tendencies, tendency to absorb water from the atmosphere and immunity from some types of spoilage (White and Doner, 1980). Honey has been used as food and for medicinal purposes since stone age. It's medicinal properties as remedy for burns, scars, wound healing and ulcers have been established. It is also used as a sweetening agent for children's drugs, treatment of sore throat, cough and hay fever (Adenekan et al., 2012;Aloisi, 2010;Fasasi, 2010). The wax from bees is used in food processing industries as additives, making of chewing gums and in cosmetic industries for making lip balm, lip gloss, hand creams, moisturizers and many others (Ezekiel et al., 2013;Dike and Onwuka, 2016). fungi and viruses Human Metabolomic Databases (2009). It has antioxidant, anti-inflammatory and anti-microbial properties (Alvarez-Suarez et al., 2014). Its antioxidant capacity is due to its wide range of compounds which include peptides, organic acids, phenolic enzymes and Maillard reaction products (Eteraf-Oskouei and Najafi, 2013). Honey is consumed for its high nutritive value and effects on human health. Among the sugars in honey include sucrose, turanose, nigerose, maltose, kejibiose, with traces of trehalose, isomaltose and raffinose (Cantarelli et al., 2008). Kejibiose is a reducing sugar which was accidentally discovered by scientists in Germany. Nigerose is an unfermented sugar obtained by partial hydrolysis of nigeran. It is a product of the caramelization of glucose It is a product of the decomposition of fructose and it is found in trace amounts in honey (Buba et al., 2013;Fasasi, 2012;Canterelli et al., 2008). HMF of honey can be increased by various factors including storage temperature, heat, moisture content and acidity (Buba et al., 2013). The Codex Alimentarius Commission recommended the optimal value for HMF in honey as 40mg/kg for the European Union and 80mg/kg for the tropics (Bogdanov, 2009). Acidity of honey is directly related to its floral sources and in combination with its electrical conductivity, can affect its quality and classification (Yadata, 2014). Electrical conductivity is the indication of ionizable acids and compounds in aqueous solution (Chaker, 2016). Bee-keeping have been the way of life of the people of Cross River and Oyo States of Nigeria, although it had not been on a commercial scale until recently when honey became an alternative for table sugar and also a possible foreign exchange commodity.
This study determines the various sugar components, HMF, free acidity and electrical conductivity of honey from the northern and central senatorial districts of Cross River State and central senatorial district of Oyo State in Nigeria.

Materials and Methods
The honey samples were purchased from farmers from Igoli in Ogoja, Obudu (farm bred and wild), Agoi Ibami, Eruwa and Iseyin, stored in a dark cupboard at room temperature until needed for the analysis.

Analysis of sugars.
The method Bogdanon and Baumann (1988) determines fructose, glucose, sucrose, furanose and maltose in honey, for which precision data were required. It can also be used for the quantification of other saccharides such as melezitose, erlose, isomaltose, raffinose and others as described by Bogdanon and Baumann (1988). Sugar content was determined by High Performance Liquid Chromatography (HPLC ) with Refractive Index(RI) detector and analytical steel column in amine modified silica gel of 5-7micrometer particle size and 250x4.6mm diameter. A measured amount (5g) of honey was dissolved in 40ml of water, 25ml methanol was pipetted into a 100ml volumetric flask and the honey solution transferred into the flask. This was then filled to the mark with water. Thereafter, 2.0g of fructose, 1.5g of glucose, 0.25g of sucrose and 0.15g each of turanose, maltose and all other sugars analysed were used as standards. The solution was poured through a membrane filter and collected in sample vials (Bognadov et al., 1997;Bogdanov and Baumann, 1988). All sugars were purchased from Merck (Switzerland) except turanose and erlose (Senn Chemicals, Dielsdorf, Switzerland).

Hydromethyl furfural (HMF) analysis
HMF was determined according to the European Commission method of analysis (Bogdanov et al., 1997). Twenty grams (20g) of the sample was dissolved in 100ml milli-Q water, filtered through a 0.22mL nylon filter into an HPLC vial, capped and injected with 20microlitre loop into the HPLC. HMF concentration in the honey was calculated by comparing the peak area to that of the standards taking the solution into account (Bogdanon et al., 1997). There is a linear relationship between the concentration and the peak area and the results were expressed in mg/kg honey.

Free Acidity and Electrical Conductivity analyses
Free acidity was determined by titrimetric method. The addition of 0.10N sodium hydroxide (NaOH) was stopped at pH 8.3 (AOAC, 1999). While electrical conductivity was measured on 20% (g/g by dry weight) honey solution in carbon dioxide free deionised distilled water at 20 o C Bognadov et al., (1997) and expressed in millisiemens per cm (mS/cm).

Discussion
Honey has been used in a variety of ways for centuries. Nutritionally, honey is recently used as an alternative to sucrose as sugar and it is also widely used for industrial purposes. It also serves as a foreign exchange earning where it is widely produced.  (Amir et al., 2010). Honey crystallization is slower when the ratio is more than 1.0 (Draiaia et al., 2015) and faster when it is less than 1.0 (Amir et al., 2010). The F/G ratios in all the honey samples were more than 1.0, which indicates slower crystallization. Crystallization also depends on storage temperature. Obudu farm has the lowest F/G ratio at 1.24 but there was no significant difference between the ratios in all the samples studied.
HMF is a representation of the freshness of honey (Cantarell et al., 2008

Conclusion
The sum of fructose and glucose in (Obudu wild) honey presumes that it can be used by diabetic patients as the value is below the 60g/100g recommended by Codex Alimentarius standard. Also, the fructose/glucose ratio for all the samples was more than 1 which indicates slower crystallization. There may be a need for Nigeria to set country specific values for honeys produced in the country as the range in the samples studied, varied widely. Honeys from Igoli and Obudu were within the international standards set by both European Council and Codex Alimentation. Sucrose content for all the honey samples were well within the European Council standard of not more than 5g/100g. Total acidity and HMF values showed good indicators of the freshness of the honey samples.