The Chemical Analysis of Brazilian Pepper, Clover and Citrus Honeys Produced by The Honey Bee Workers, Apis mellifera L. (Hymenoptera: Apidae)

INTRODUCTION

Honey is defined as "the natural sweet substance", which is produced by honey bees from the nectar of plants, secretions of living parts of plants and excretions of plant sucking insects (honey dew). Bees collect nectar, transform it by combining with specific substances of their own, deposit, dehydrate, store and leave the honey in the combs to be ripened and mature according to the international standards of honey products (European Union, 2002 and Codex Alimentarius, 2010).

It is well known that there are three seasons of yielding honey in many parts of Egypt according to blooming of plant species that can be available for bees to collect their food (nectar and pollen). For example, Citrus honey is produced by honey bee workers from the nectar of flowers of different Citrus trees at the beginning of spring until the end of flowering period of Citrus species ( 1^st season of yield). Citrus honey has a light yellowish or golden color with light consistency, light acidic taste and aroma of citrus flowers.

Clover honey is being produced by honey bee workers in the period of May-June during the flowering of clover (2^nd season). Clover honey has a light brownish or amber yellow color, with thick consistency and good sweet taste.

Cotton honey is also produced at the mid of August (3^rd season). The period between November and February (or March) is defined as "the critical dearth period" due to the lack of food sources. Therefore, the artificial feeding is very important and necessary during that period.

Brazilian pepper honey can be also produced in Egypt due to the occurrence of some cultivated pepper trees. Brazilian pepper trees (Schinus terebinthifolius L.) are often encountered as shrub or small trees that can grow to a height of 3-7 meters. Brazilian pepper tree is native to Brazil, Argentina, and Paraguay and it was introduced into United States (Florida) as an ornamental plant (Mytinger and Williamson, 1987).  The tree can grow to be substantially taller to as much as 13 meter (Ferriter, 1997; Langland and Baker, 1994). Some individual trees can live as long as 35 years (Hall et al., 2006). The main flowering season of that tree occurs during the period of September to October or November (the beginning of dearth period) (Mohanna, 1989), although in Florida some trees can usually be seen flowering at any time of the year (Ewel et al., l982). Mohamed (2005) reported that the highest peak of egg laying by Apis mellifera queen occurred during the flowering period of Brazilian pepper trees in Egypt. At the apiary of El-Sabahia Research Station, Alexandria, all the hives were found to have considerable amounts of honey and pollen grains. Earlier, Mohanna (1989) estimated the honey yield by 6.85 kg/colony in November when the hives were replaced near by the Brazilian pepper trees.

Therefore, the objective of the present study is directed to determine the chemical components of Brazilian pepper honey and to be compared with citrus and clover honey. The honey yield/colony for the experimented honeys (Brazilian pepper, clover and Citrus) during the period of 2012-2014 is also considered.

MATERIALS AND METHODS

Brazilian pepper honey was collected from the apiary of Agric. Research Station, El-Sabahia in Alexandria which contains about 15 colonies. Clover honey was collected from Abis the 2^nd village in Alexandria and Citrus honey was collected from Abo-Elyouser village, Beheira Governorate.  All honey samples (0.5 kg, each) were collected in glass bottles and stored in dark at 4°C until the analytical procedure is ready.

A. Chemical analysis of the collected honey samples

1- Ash, HMF and pH

The ash content was determined following the method of AOAC (1995).   Hydroxy methyl furfural (HMF) content in honey samples was determined according to the Winkier method (AOAC, 1995).

The pH of the different honey samples was determined for 10% (w/v) of sample prepared in distilled water using the pH meter (Accument AB15, Fisher Scientific) according to AOAC (1995).

2- Determination of minerals content

Minerals content (elements) measurement was done at the City for Scientific Research and Technological Applications, Arid Lands Cultivation Research Institute (ALCRI) (Borg El-Arab, Alexandria Governorate). The mineral contents were determined by the atomic absorption spectrophotometer according to the method described by Hernandez et al. (2005).

3- Determination of sugars

Samples were prepared according to the method described by Ouchemoukh et al. (2010) and detection was done according to Zappala et al. (2005).  Sugar analysis was done by HPLC (Shimadza model) where HMF is separated on a reversed phase column, with water and methanol as the mobile phase and then detected by UV absorbance.

B. Honey yield

The honey production was taken into consideration during the period of November 2012 till February 2014. The production of the three types of honey, i.e. Brazilian pepper, clover and Citrus were estimated by weighing the honey after extraction (kg/15colonies) and the mean weight as kg/colony was calculated.

Results and Discussion

Chemical analysis of the collected honey samples

1- Ash, HMF and pH

Data presented in Table (1) represented the ash content (%), HMF and pH values of the samples of  Brazilian pepper, clover and Citrus honeys. It was noticed that ash content is widely different among the experimented samples and ranged from 0.06 for Citrus honey to 0.46% for Brazilian pepper. The ash mass fraction is a quality criterion for determining the botanical origin of honey (Felsner et al., 2004).

Hydroxy methyl furfural (HMF), or 5-hydroxymethyl- 2-furaldehyde, is a water-soluble heterocyclic organic compound derived from sugars. HMF is a decomposition product of fructose (dehydration of fructose), which is naturally found in a trace amount and its concentration increases with storage and prolonged heating of honey. The HMF concentration is increased as honey undergoes heat treatment even for short term at higher temperature to reduce viscosity and prevent crystallization to facilitate filling (Subramanian et al., 2007). HMF is high in honeys that have been heat treated, stored in non-adequate conditions or adulterated with invert syrup (Ajlouni and Sujirapinyokul, 2010).  Therefore, it is the most used index to evaluate thermal damages or ageing in food products. It is also an indicator of excessive heat-treatment, spoilage, and of possible adulteration with other sugars or syrups. As a result, many countries impose restrictions on maximum levels of HMF in food and beverages (Codex Alimentarius, 2000). Many countries impose maximum levels for HMF of 40 mg/kg as the maximum permitted level in the EU for table honey (Codex Alimentarius, 1981). Data indicated that clover honey contained a high level of HMF (77.13 mg/kg), while Citrus honey had the lowest one of 10.73 mg/kg. Brazilian pepper honey was found to contain an amount of 25.0 mg/kg of HMF. The fluctuation of HMF in honeys could depend on the equilibrium between the accumulation and the degradation processes.

Both samples of Citrus and Brazilian pepper honey are suitable and meet the international upper limit. In this respect, the results of   Windsor et al. (2013) showed that among the studied honeys stored at 4°C, all six L. polygalifolium and one L. liversidgei honeys exceeded the HMF upper the limit (40mg/kg) of the International Honey Commission (IHC) (Codex Alimentarius, 2000). Clover honey might be exposed to a heat treatment and therefore it showed a high value of HMF content (77.13) (Table, 1).

The pH values of the three experimented honeys are also presented in Table (1). The pH values ranged from 3.55 (Citrus honey) to 4.50 (Brazilian pepper honey). According to the National Honey Board Food Technology (2006), the average pH of honey is 3.9 with a typical range of 3.4 to 6.1and therefore the determined pH values are consistent.

The pH value is of a great importance during the extraction and storage of honey as it influences the texture stability and shelf life. In general, honey is acidic in nature irrespective of its variable geographical region. The pH values of Algerian, Brazilian, Spanish and Turkish honey types also have been found to vary between 3.49 to 4.53, 3.10 to 4.05, 3.63 to 5.01 and 3.67 to 4.54, in respect (Azeredo et al., 2003; Ouchemoukh et al., 2007; Kayacier and Karaman, 2008).     

Table (1). Ash, hydroxyl methyl furfural (HMF) and pH of samples collected from different types of honey

2- Mineral contents (Elements)

Data presented in Table (2) referred to the elements content (mg /100g) of different honeys collected from different flora. The minerals and trace elements in honey samples could give an indication of environmental pollution and herewith also an indication of the geographical origin of honey. Generally, data pointed that the minerals content of Brazilian pepper honey samples were higher than that of both clover and citrus honeys; the latter had the lowest minerals content. Citrus honey seems to be a bright one and in this respect, Felsner et al. (2004) reported that the brighter honeys usually containing fewer elements than those darker ones. Meanwhile, Brazilian pepper honey seems to be dark assuring that the concentrations of its elements were generally high.

Potassium (K) was also shown to be the most abundant element in both honey samples of clover and Citrus (306.600 and 115.300 mg/100g, successively) as compared with the other detected elements. On the other hand, magnesium (Mg) was the most abundant element that was found in a high concentration in Brazilian pepper honey (422.80 mg/100g). Fernandez- Torres et al., (2005) noticed that potassium (K) and sodium ions were the most fully sufficient minerals in honey collected from Spain. The minerals content of honey is depending on the flower elemental composition gathered by the bees (Rashed and Sultan, 2004). It was also noticed that among these honey types, great differences existed, in both HMF (Table,1)and the mineral contents of Ca as well as Na, Mg and zinc (Table, 2).

Table (2). Minerals content of different the honeys collected from of experimented honey

3- Sugars

The results listed in Table (3) showed clearly the total sugars (sucrose, glucose and fructose), glucose/fructose (Gl/Fr) ratio and the reducing sugars of the different types of honey. Sugars are considered to be the main component of honey. Reducing sugars, mainly fructose and glucose had been found to be the major constituents of honey (Mendes et al., 1998). The presented data displayed sucrose (a disaccharide) percentage in honey that ranged from 3.26 (Brazilian pepper honey) to 4.25 (citrus honey). The determined percentages of sucrose in all the tested types of honey are still consistent with the standards.  A high sucrose content in honey most of the time means an early harvest of honey because sucrose had not been fully transformed to glucose and fructose by the action of invertase (Azeredo et al., 2003).

Monosaccharides (glucose and fructose) are the main sugars in any honey sample. Glucose content was found to be 24.96, 28.26 and 36.79% for the experimented honeys (clover, Citrus and Brazilian pepper, respectively). Fructose is also the most important sugar, quantitatively. It represented the highest percentage as compared with glucose. Brazilian pepper honey was found to contain the highest percentage of fructose (41.50%). Fructose and glucose values of the honey depend on the source of nectar. Meanwhile, the sum of glucose and fructose contents was also the highest in Brazilian pepper honey (78.29 % of the total sugar). Brazilian pepper honey was found to have a lower glucose /fructose ratio (1:1.13), which expressed the slow speed of honey granulation. On the other hand, the high ratio of citrus (1:1.38) might affect honey flavor, since fructose is sweeter than glucose. Some consumers of Brazilian pepper honey placed complaints about the better taste and that might be due to the higher content of minerals, low glucose /fructose ratio and the high acidic content which represented a high value of pH (4.5).

The determined percentages of the total reduced sugars content in honey were presented in Table (3). It could be shown that Brazilian pepper honey contained the highest percentage of reduced sugars (78.29%) as compared with the other samples of clover and Citrus honeys.

Table (3). Total sugars (sucrose, glucose and fructose) and reducing sugars collected from different types of experimented honey

It is well known that the majority of soluble sugars in honey are reducing sugars and therefore, it could be concluded that Brazilian pepper honey contained more soluble sugars than the other types of honey.     

Honey yield

Honey yield of the three experimented types was determined for three successive seasons during the period of 2012-2014 (Table, 4). Data revealed that the yield of Brazilian pepper was the highest all over the experimental period with a general mean of 7.21 kg/colony. The production of Brazilian pepper honey was superior as compared with the other honeys. Citrus and clover showed more or less than the same productivity with a general mean of 4.50 kg/colony. The presented results are in agreement with those reported by Mohhana (1989) who found that the general mean of Brazilian pepper honey production was about 6.85 kg/colony. Brazilian pepper honey was found to be dark greenish yellow, thick and has strong characteristic aroma with peculiar flavor. So, it would be recommended to grow Brazilian pepper trees in Egypt around the local apiaries to act as a good source for nectar plus pollen and to keep the bees foraging during the autumn season getting a valuable and nutritionally rich honey. In this regard, the beekeeper will not be obligated to feed the bees with artificial food during the period of Brazilian pepper trees flowering during winter season (October – February), the critical dearth period. Also, it could be concluded that honey composition depends basically on the nectar composition of each producing plant species.

Table (4). Total honey yield (kg/colony) of different types of the tested honey throughout the period 0f 2012-2014