Comparative Studies of the Physicochemical Properties and Mineral Elements of Honey Produced in the Guinea Savannah Zones of Nigeria

Honey is a viscous natural sweet substance produced by
honeybees from the nectars of plant flowers or from secretions...

The quality standard and freshness of honey is represented by its diastase value which is within the range of three to eight [13].
Similarly, hydroxymethylfurfural (HMF) content in bee honey gives an indication of the degree of freshness, overheating at extraction or adulteration with commercial invert syrup (Gomes, et al., 2010;Leticia, et al., 2013). Codex Alimentarius (Alinorm 01/25 2000) established that the HMF, content of honey after processing or blending must not be higher than 80mg/kg, while the European Union (EU Directive 110/2001), recommends a lower limit of 40mg/kg with the exceptions that 80mg/kg is allowed for honey that originates from countries or regions with Tropical temperature, while a lower limit at only 15mg/kg is allowed for honey with low enzymatic levels. Honey is produced in a commercial quantity in the Guinea Savannah zones of Nigeria; but there have not been a scientific document that compares the quality with those prescribed in Codex Alimentarius and European standards. This study, therefore, is aimed at evaluating the physicochemical properties and mineral elements of honeys produced in the Guinea Savannah zones of Nigeria, with a view of comparing the quality with the Codex Alimentarius and European Standards honeys produced by honeybees.

Honey Samples Collection
A total of sixteen (16) honey samples that were traditionally processed were randomly collected from four (4) different markets in Guinea Savannah zones namely: Benue (Naka), Kaduna (Zangokataf), Niger (Gbako) and Taraba (Sardauna). Two liters (2L) of four (4) honey samples were collected per commercial market (n = 4). Particles of wax, dead bees, combs and other dirt in the honey samples were removed by straining through sieve. The honey samples were stored, preserved, protected from heat and light to avoid deterioration of enzymes, Hydroxymethylfurfural (HMF), viscosity and moisture content [3]. These samples were used for the laboratory analyses.

Duration of the Study
The study was carried out between March 2017 and September 2018 with first nursing of beehives for one year. Thereafter, the maturation of the bee honey and honeycombs were harvested and further processed.

Moisture Content
The water content of honey sample was determined by hot airdried oven method (A.OA. C, 1990). Determination was in triplicate for each sample. Clean and dried crucibles were weighed empty (Wo g) using aeAdam, analytical weighing balance, model N17250.
Two grams (2g) of honey sample was pipetted into the empty porcelain crucible and were weighed (W 1 g). Thereafter, the set up were put in the oven, Genlab, hot air oven, model MINO/50, and the temperature of the oven was set at 100 o C and was switched on.
Crucible and content were heated for two (2) hours. Afterwards, crucibles and content were transferred to a desiccator containing desiccant (Silica gel), where heated honey samples and crucibles cool and maintained dryness for forty-five (45) minutes. Thereafter, crucibles and contents were weighed. Repeated heating and weighing of crucible and content was performed till a constant weight was obtained (W 2 g). The Moisture Content of honey samples were obtained from the relation of loss in weight of sample (g), upon weight of sample (g), multiplied by one hundred (%).

Ash Content of Honey
The ash content of honey was determined according to the Furnace ash method (A.O.A. C, 1990). Determination was in triplicate of each sample. Gallenkamp electric Muffle Furnace, model 3, was used for the ash content. Analytical weighing balance "aeAdam", model N17250; was used for weighing. Clean and dried crucibles were weighed empty (W o g). Ten grams (10g) of honey samples were pipetted into the empty porcelain crucibles and were weighed (W 1 g). The crucible and content were preheated on a gas flame to darkness/charred, to avoid foaming. Thereafter, the preheated crucible and content were ashed (oxidize organic matter) in an electric muffle furnace at a temperature of 550 o C, for five (5) hours. The obtained ash was allowed to cool in desiccators for forty five(45) minutes, after which the weight of crucible and ash content were determined (W 2 g).The ash content of honey was calculated from the ratio of weight of ash to sample weight multiplied by one hundred.

Total Solids of Honey
This was the difference of moisture content of honey sample from 100. Total solids of honey sample were calculated from the expression: % Total Solids = 100 % moisture Content.

Fat Content of Honey
The lipid content of honey samples was determined according in the hot air oven at 60 o C, for one hour; afterwards, were allowed to cool in the desiccators. Then, the dried flask and content were weighed (W 1 g).Fat content of honey was calculated from the ratio of the difference between W 1 -W 0 to sample weight multiply by one hundred.

Total Protein Content of Honey
Protein content of honey was determined by the method of Kjeldahl (A.O.A.C, 1990). Parameters were determined in triplicates of each sample. Ten (10) grams of honey was weighed using analytical balance, aeAdam, model N17250, and the honey was introduced into micro-Kjedahl digestion flask, followed with the addition of twenty milliliter (20ml) concentrated sulphuric acid (H 2 SO 4 ), which transformed organic nitrogen to mineral nitrogen (NH 4 ) 2 SO 4 , in presence of ten gram added mixed catalyst (mixture of 9grams of potassium sulphate plus 15grams of copper sulphate).
The setup was subjected to two processes i.e. digestion and distillation. The resulting solution was distilled after adding 30ml of 40% sodium hydroxide solution, and the distillate was collected in a flask with 40% boric acid and mixed indicator. The mixture was titrated with a standard solution of 0.01MHCL. The crude protein content was calculated using the conversion factor of 6.25 (i.e. N X 6.25).

Total Carbohydrate Content of Honey
Total carbohydrate of honey sample was obtained by difference from 100 of the sum of % Ash, Protein and Lipid, according to [14].

Acidity (Free, Lactone and Total) of Honey Samples
Acidity of honey sample was determined according to potentiometric titration method (A.OA.C, 1990). Parameters were assayed in triplicates of each sample. Free, Lactones and Total acidity were measured by titration method using a solution containing 10grams (weighed with aeAdam analytical balance model N17250), of honey sample dissolved in 75ml of carbon dioxide free distilled water (prepared by boiling the distilled water for 20minutes and allowed it to cool at room temperature),in a 250ml glass beaker. The honey solution was titrated with 0.05M NaOH solution from 50ml glass burette at the rate of 5.0ml minute -1 . Immediately the pH meter read 8.50 (Free acidity); the addition of 0.05M NaOH solution was stopped and the volume of consumed 0.05M NaOH solution recorded. Excess 10ml of 0.05M NaOH solution was immediately added and without delay was back titrated with 0.05M HCL solution from ten milliliter (10ml) glass burette to a pH of 8.30(Lactones acid) and the volume of consumed 0.05M HCL solution recorded. The blank sample was also treated as the sample. Total acidity of the honey sample was determined by adding Free acidity and Lactones acidity. The result was expressed in mEq/kg.

Optical Density (O.D.) of Honey Samples
Honey colour intensity otherwise known as Optical Density (O.D) was measured using the UV-Visible Spectrophotometer method. Measurement was in triplicates of each sample. Ten gram (10g) of honey sample was weighed, into a 250ml beaker, using aeAdam analytical balance model N17250. The honey was diluted to 100ml with distilled water. The resulting honey solution was transferred into plastic centrifuge tube and the set up was put in centrifuge bucket (Centurion Scientific Refrigerated Centrifuge, K3   Series, and Model K24IR), and spun for 10 minutes at 3000rpm.   Thereafter, the absorbance and transmission of the supernatant   filtrate was measured at 530 nm, against distilled water as   blank using UV-Visible Spectrophotometer (T60 UV-Visible Spectrophotometer, PG Instrument, model T60U) [15].

Density/Specific Gravity of Honey Samples
The Specific Gravity (S.G.), of honey samples was determined as the ratio of the weight of sample to that of equal volume of water.

Analysis of Mineral Element of Honey Samples
Honey samples were analyzed for mineral elements according to A.O.A. C (1990). Analysis was carried out in triplicates of each sample. Ten grams (10 g) of honey sample was weighed into a clean and dried empty porcelain crucible, using aeAdam analytical balance model N17250. Then, crucible and content were preheated on a gas flame to darkness/charred, to avoid foaming. Thereafter, the preheated crucible and content were ashed (oxidize organic matter) in Gallenkamp electric muffle furnace, model 3, at a temperature of 525 o C, for five (5) hours. Thereafter, the crucible and ash were allowed to cool. Afterwards, the calcined ash of honey was

Statistical Analysis
Each parameter analyzed was done in triplicates. The data were subjected to statistical analyses to verify and evaluate the differences between chemical compositions of honey samples investigated.
The data were expressed as mean and standard deviation (n=16) where "n" represents the number of samples. Comparison of means was analyzed using one-way analysis of variances (ANOVA) on a statistical programmed (Statistical Package for Social Sciences SPSS) version 16.0. The difference was considered significant at P ˂ 0.05.

Result
The results were as shown in Tables 1-5        a) **. Correlation is significant at the 0.01 level (2-tailed).

Physicochemical Analysis
The mean values of physicochemical characteristics of the studied honey samples were presented in Table 1

Proximate Analysis
In
There were statistical significance differences (P <0.05) in the ob-

Variables of Honey Samples
There is an indication of a high positive correlation at P < 0.01 and P < 0.05 between the chemical qualities of the investigated honey samples (Table 5). This was established at P<0.01 between fc and mc(r = 0.428); nc and mc(r = 0.426); mc and te (r = 0.471), fc and te (r = 0.760) tcho and ts (r = 0.43. P < 0.01) while significant negative correlations at P<0.01occured between mc and ts (r =-0.996); mc and tcho (r = -0. 440); ts and fc (r = -0.398), ts and nc (r =-0.419), ts and te (r = -0.458); ac and te (r = -0.425); nc and tcho (r = -0.998). In addition, positive and negative significant correlations were established at P<0.05 between nc and te (r = 0.305) as well as tcho and te (r = -0. 319) respectively. Generally, honey is acidic in nature disregarding its geographical origin. The average pH of honey was 3.9 with a typical range of 3.4 to 6.1 [1]. Low pH in the acidic range is an indication of good shelf life and excellent stability of honey against microorganisms and natural flavour. The pH means range and the overall mean for the studied honey indicated that all the studied honey samples were acidic and were within the standard limit (3.4 to 6.1), and insures freshness of honey samples. The investigated honey pHmean range were much higher than that of Manuka honey of 3.99±0.02 and may suggest Nigeria. The honey samples analyzed by Khalil, et al. [9], and Selene, et al. [11], could have been fermented. This is so because, the content of gluconic acid, produced from glucose by the action of the glucose oxidase enzyme increases during the storage of honey since the enzyme remains in activity after processing, and this increases the acidity of honey during storage with the resultant effect in the decrease of the pH [17].

Discussion
Also, the mean range and the overall mean values of Lactone acid (Lactones Acid represents the combined acidity which is not directly titratable), were found to be from 31. with over 20% water content will ferment [20]. Also, honey with carbohydrate content greater than 83%, moisture content less than 17.1% and storage temperature less than 11 oC, will not ferment [1]. Regarding the studied honey samples, the moisture content mean values for all the honey samples were less than the maximum recommended standard limit 20% for good quality honey and 14% ascribed as valuable (Ojeleye, 1999). The investigated honeys were Ash content is a reflection of inorganic mineral content and it is a quality criterion for botanical and geographical origin of honey.
The values of the ash content of the studied honey range from 0.67% to 0.68%. The obtained ash value of the study was higher than the, and the European honey Commission specified value of 0.6%. This could be that the analyzed honeys contained higher quantities of essential inorganic minerals or be attributed to the soil where the honeybee plants grew [20]. However, reported that the mineral content of blossom honeys is within the range of 0.1 to 0.3% and that of honeydew honeys is up to 1.0%.
The obtained ash value was higher than the values reported by Ndife, et al. [10] who reported mean range value of 0.26±0.05% to 0.38±0.04%, in comparison study with an imported U.S.A, honey; Olugbemi, et al. [16] reported range value of 0.33% to 0. Food of a higher fat content stands the risk of rancid spoilage during storage [14]. The fat content recorded in this study that ranged from 0.31% to 0.35% is higher than the previous records by Ndife et al. [10] who reported a range of fat value of 0.12±0.01% to 0.20±0.03%, in comparison study with an imported U.S.A, honey, which were lower than the obtained value while, reported value (0.01% to 0.50%) and lower compared to Leticia et al. [8] reported value of between 0.37% and 0.39%. Accordingly, these literatures The main constituents of honey are the carbohydrates which constitute about 95% of honey dry weight. The main sugars found in honey are the fructose and glucose [21], reported that honey is a high energy carbohydrate food and that the sugar content in honey is digestible similar to the sugars found in fruits. Also, honey with carbohydrate content greater than 83%, moisture content less than 17.1% and storage temperature less than 11 o C, will not ferment [1].  Dietary Allowance (RDA) of Sodium for men ranged between 400mg to 420mg and for women varied from 310mg to 320mg [1989]. The average amount of Sodium per 100g honey is 2.85mg and per one tablespoon serving 21g is 0.6mg [25]. Minerals in honey vary according to the botanical origin and soil composition.  (Pamplona, 2006).
The average amount of Potassium per 100g honey is 50.0mg and per one tablespoon serving 21g is 11.0mg [25].
The Calcium content in the analyzed honey samples from the four various locations of savanna zones of Nigeria, varied from 6.57mg/l to 11.23mg/l with an overall mean value of 9.16±1.79mg/l. The Calcium content obtained in this study agreed with results recorded by [24], who documented mean range from 2.8mg/100g to 16.6mg/100g, on the study of Blossom honey and  Iron is 0.05mg [25].
Phosphorus content of honey in this investigation varied from 8.43mg/l to 31.58mg/l, with an overall mean of 24.85±9.67mg/l.
In this study, the obtained Phosphorus value is in agreement with the reported mean range 3.0 to 31.5mg/100g, with a mean value of 8.8±5.5mg/100g, by [24], on the study of Blossom and Honeydew honeys from Northwest Spain. In an average amount of 100g honey, the quantity of Phosphorus is 5.0mg and an average amount of 21g honey of one tablespoon, the quantity of Phosphorus is 1.0mg [25]. Differences in soils composition, locations and floral origin are ascribed to variations in values of obtained Phosphorus.
In this investigation, the mean contents of Nickel in the studied honey samples varied from 0.05mg/l to 1.01mg/l, with an overall mean of 0.30±0.41mg/l. These variations in the obtained values of Nickel could be attributed to differences in soils composition and floral origin [22]. Also, the mean value of Copper in the study ranged between 0.00mg/l to 0.16mg/l, with an overall mean value of 0.04±0.07mg/l. This indicated the relationship between plant nutrient uptake and soils. The Copper results of the analyzed honey samples of this study are lower than the mean range values (0.00 to 3.7mg/100g, with a mean value 0.4±0.8mg/100g) obtained by [24], on the study of Blossom and Honeydew honeys from Northwest Spain; the mean values (0.8mg/100g) obtained by [26] on the analysis of honeys from Marche region in Italy; the mean value(243µg/kg) reported by [12] on the analysis of honeys from Ardabil, in Iran and the mean ranged value(0.057 to 0.168mg/l) obtained by (2011), on the study of honeys from stations in South South Nigeria, in comparison with the Manuka honey. In an average amount of 100g honey, the quantity of Copper is 0.05mg and an average amount of 21g honey of one tablespoon, the quantity of Phosphorus is 0.01mg [25]. Differences in geographical locations, floral origin and soils composition could be attributed to the variations in the values of copper contents of honeys [22,23].
The analyzed honey samples in this study recorded mean ranged values of Cadmium from 0.00mg/l to 2.96mg/l, with an overall mean of 0.78±2.14mg/l. The differences in soils composition and floral origin could account for the variations in the Cadmium values [22]. The reported Cadmium values in this study agreed with the result (27.62µg/kg, and the ranged value from 1.36 to 125.88µg/ kg) obtained by Hassan, et al. [12] on the analysis of honeys from Ardabil, in Iran while the mean Lead values in this investigation ranged between 0.02mg/l and 0.05mg/l, with an overall mean of 0.03±0.02mg/l. The variations in soils composition and floral origin could also be ascribed to the differences in lead obtained values. although the order differs from the study of the investigated honey samples, the main minerals were the same [28][29][30][31].

Conclusion
This study evaluated the physicochemical properties and mineral elements of honeys produced in Guinea Savannah Zone of Nigeria, via: Benue (Naka), Kaduna (Zangokataf), Niger (Gbako) and Taraba (Sardauna); with a view of comparing the quality with those described in Codex Alimentarius and European regulatory commissions for bee honeys. The values of quality parameters of all the investigated honeys were within the acceptable limits of international standards and showed that the studied honeys met the physicochemical and mineral quality requirements established in international market. The moisture content of all the studied honeys is less than 17% and this confers on the samples stability, against osmophilic bacterial activities, long period preservation.
Honeys from the four locations of savanna zones of Nigeria, are good for food security, industrial value as raw materials and for export.