ABSTRACT
Forty honey samples from Apis mellifera were collected in different parts of Algeria during 2007 to 2010 and were analysed for parameters including hydroxymethylfurfural (HMF), invertase and diastase activities. The spectra of pollen of the honeys collected in those areas were also studied. The results show that the amylase activity ranged from 4.40 to 30.15 DN, with only one sample having a low diastase number than 8 DN. The invertase activity ranged from 0 to 138.38 UE. Invertase is generally present in small amounts and is inactivated by heating. The HMF values in our study were low (<40 mg/ kg). 85% of samples (34 samples) had a HMF content less than 15 mg/kg HMF. The results of mellissopalynology show that the NPG/10 g for all samples of honey ranged from 8 × 103 to 2.01 × 106; all samples studied are original flower honey. We also note that the dominant pollen in the majority of samples was: pollen of Eucalyptus, Trifolium sp, Echium sp, Hedysarium corornarium and to a lesser extent, Asteraceae (mostly Carduus), Apiaceae, Ericaceae, Rosaceaeare and Rutaceae. In general, the samples were found to meet the requirements of the international honey standards.
Key words: Honey, hydroxymethylfurfural (HMF), diastase activity, invertase activity, mellissopalynology.
Honey, produced by the honeybee, is a natural supersaturated sugar solution, which is mainly composed of a complex mixture of carbohydrates and contains certain minor constituents like proteins, enzymes (invertase, glucose oxidase, catalase, phosphatases), amino and organic acids (gluconic acid, acetic acid, etc.), lipids, vitamins (ascorbic acid, niacin, pyridoxine etc.), volatile chemicals, phenolic acids, flavonoids, and carotenoid-like substances and minerals (Blasa et al., 2006). The composition of honey depends on the plant species visited by the honeybees and the environmental, processing and storage conditions (Bertoncelj et al., 2007, Guler et al., 2007).
Enzymes are the most important and also the most interesting honey components. They are accountable for the conversion of nectar and honeydew to honey, and serve as a sensitive indicator of the honey treatment. In some countries, the specification of enzymes is a binding legal indicator (Bogdanov et al., 1987; Oddo et al., 1999). Honey naturally contains small amounts of enzymes. The predominant enzymes in honey are diastase (amylase), invertase (a- glucosidase) and glucose oxidase. Others, including catalase and acid phosphatase, can also be present. Honey’s enzyme content can vary widely by floral source and region. The levels of some enzymes such as diastase are relatively easy to measure and have been used for many years to estimate the extent of heating to which a honey has been exposed. Such information has been required by some countries where heating of honey is believed to reduce or destroy potentially health-promoting properties. In fact, because the enzyme content of fresh honeys can vary widely, enzyme levels in packed honey are a poor indicator of processing and storage conditions (White, 1968).
The HMF content depends on the temperature, the duration of storage or heating, the content of various sugars, pH, the mineral content (especially that of iron) and the water content of honey (Gondarski, 1961; Ivanov, 1978; Pichler et al., 1984). According to some authors (Bogdanov, 1999), fresh honey contains practically no HMF but its concentrations increase during storage. Many studies (Ivanov, 1986; Pichler et al., 1984; Von der Oh, 1992; White, 1964) have shown that HMF levels in natural non-heated honey is almost always under 1 mg%. For 1 to 2 years, the HMF content could increase up to 3 to 4 mg% or more depending on the temperature of storage. The high HMF content in honeys is related to its long-term storage or to its overheating for liquidification of crystallized honey (White et al., 1964).
Pollen analysis has been the traditional method to determine the floral origin of the honey (Hermosin et al., 2003; Von der Ohe, 1994). Usually, honey is considered unifloral if the pollen frequency of that plant is >45%. (Terrab et al., 2003c). The identification and quantification of pollen grains in honey sediment (melisopallynological) is still the most important method for determining the botanical species that make up the pollen spectrum of honey samples. The pollen spectrum shows whether the honey samples are from different geographical and floral regions, and also the flowering time of the bee plants and their value as nectar and pollen suppliers .Through quantitative analysis of pollen grains, it is possible to establish the proportion which each plant, as a nectar supplier contributes to the constitution of the honey. However, this proportion depends on the pollen, and on the structure and biology of the different plant species (Anklam, 1998).
The objective of this study was to determine the enzymes activity of α-glucosidase (invertase), ß-amylase (diastase), the contents of hydroxymethylfurfural (HMF = 5-hydroxymethylfuran-2-carbaldehyde) and the contents of pollen spectrum for forty honey samples sorted by their botanical origins.
Honey samples
The forty fresh honey samples (Table 1) were collected directly from professional beekeepers in various regions in Algeria. The vegetation in these areas can be described as being composed of a mixture of various natural plants. Honey samples were filled into glass jars after harvesting and stored at 4°C until analysis. Duplicate analyses were performed for each parameters.
Physico-chemicals analysis
Method of determination of enzymes activities
The diastase enzymes (α- and β-amylase) were analysed with the Phadebas procedure. The determination of the honey diastase activity is a photometric method in which a blue tint insoluble cross-linked type of starch is used as a substrate. This is hydrolyzed by the enzyme, giving soluble blue fragments in water, determined photometrically at 620 nm with a Varian spectrophotometer type UV visible Cary 50. The absorbance of the solution is directly proportional to the diastatic activity of the sample.
Measurement of the invertase enzyme (α- glucosidase) was done following the method of Siegenthaler as harmonised by the International Honey Commission (Bogdanov, 1997). The activity can also be expressed as number invertase. p- nitrophenyl - α -D-glucopyranoside (pNPG) as a substrate is used for the analytical determination of the number of sucrase in honey. pNPG is divided into p -nitrophenol and glucose by α-glucosidase (invertase). By adjusting the pH value to 9.5 the enzyme reaction is stopped, and at the same time is turned into nitrophenol nitrophenolate anion, which corresponds to the amount of substrate converted and which is determined photometrically at 400 nm with a Varian spectrophotometer type UV visible Cary 50.
Determination of hydroxymethylfurfural (HMF)
The HPLC method, HMF is separated on a reversed- phase column, with water and methanol as the mobile phase, and then detected by UV absorbance. HMF was analysed by HPLC method as harmonised by the International Honey Commission (Bogdanov, 1997). One gram of sample was mixed with 9 ml of distilled water. The mixture was then centrifuged at 15,000 rounds for 10 min to remove any fine debris present in the sample. The supernatant was filtered through a 0.45 mm membrane and 20 ml of filtrate was injected to HPLC with autosampler of HPLC Agilent 1200 series, a photo diode array detector (DAD) and Nucleosil 5 C18 column. Mobile phase was a mixture of methanol/ water. The detector was set to 280 nm and duplicate analyses were performed.
Pollen analysis
Pollen analysis was carried out using the methods established by the International Commission of Bee Botany described by Louveaux et al. (1978) and Von der Ohe et al. (2004).
Quantitative melissopalynological
Analysis was performed according to Maurizio’s method (Louveaux et al., 1978) by counting microscopically the number of pollen present in the honey sediment after centrifuging a honey solution. Pollen in 400 fields of view was counted in four fold. The results were based on the average number in the 400 fields of view and expressed as the number of pollen grains in 10 g honey (PG/10 g) in thousands (103) and rounded to the nearest thousand. The honeys were placed into one of the five pollen representativity classes as distinguished by Maurizio (Louveaux et al., 1978; Von der Ohe et al., 2004). Class I includes honeys poor in pollen (PG/10 g < 20 × 103), class II includes honeys with normal pollen representativity (20 × 103 to 100 × 103), class III honeys with over representativity pollen (100 × 103 to 500 × 103), class IV with strongly over-represented pollen (500 × 103 − 106 ) and class V includes pressed honeys (PG/10 g > 106 pollen).
Qualitative melissopalynological analysis
The types of pollen grains with their percentages in the pollen sediment was carried out by dissolving the honey in dilute sulphuric acid, centrifuging the solution and mounting the sediment in Kaiser’s Glycerol Gelatine (glycerine jelly). For each sample, pollen were counted and their relative frequency classes were determined, using the terms predominant (> 45%) secondary pollen (16 to 45%), pollen occurring rarely and sporadically are called important minor pollen (3 to 15%) and minor pollen (< 3%) (Louveaux et al.,1978). For all pollen species in the 40 samples the individual occurrence was calculated and expressed as percentage of the total studied samples in which the determined pollen type was found.
Tables 2, 3 and 4 shows the mean values of the different parameters analysis and the result of pollen spectrum.
Diastase
Diastase is the common name for the enzyme a-amylase. It is found in nectar and is also added by the honeybee during the collection and ripening of nectar. Diastase digests starch to simpler compounds. The amylase activity ranged from 4.40 to 30.15 DN (Table 2); only one sample had a low diastase number than 8 DN. The floral origin of honey also influences its diastase content. For example, citrus and clover honeys tend to contain less diastase. Other factors may affect diastase values: the natural difference in pH among honeys, nectar flow and foraging patterns of the bees. Long storage at moderate temperatures and exposure to high temperatures will inactivate diastase in honey. Diastase levels do not correlate with honey quality. Therefore, specifying the diastase level will not guarantee quality (Babacan et al., 2002). Numerous studies also indicate that honey shows significant variations in amylase content based on composition, pH value and floral source. In fact, research reveals that heating is not the only factor influencing amylase content in honey (White, 1992; Crane, 1980; Low, 1988; Babacan and Rand, 2007).
Invertase
The invertase activity ranged from 0 to 138.38 UE (Table 2). Invertase is the enzyme that hydrolyzes sucrose to fructose and glucose. It is added to the nectar by the bee. The resulting chemical reaction is a key step in the ripening of nectar to honey. Invertase has been considered responsible for most of the chemical changes that take place during the conversion of nectar to honey. Invertase is generally present in small amounts and is inactivated by heating (Babacan et al., 2005).
Hydroxymethylfurfural (HMF)
Since HMF can be formed either by Maillard reaction (heating of reducing sugars in the presence of proteins), or by dehydration under acidic conditions. The HMF values in our study were low (<40 mg/ kg) (Table 3), which is fixed at 40 mg/kg in the honey standards (Codex Alimentarius Commission, 2001; European Commission, 2002). An exception has been made for honeys from tropical regions for which this limit is 80 mg/kg. 85% of samples (34 samples) had a HMF content less than 15 mg/kg. HMF content are widely recognized as parameters indicating the freshness of honey (Mendes et al., 1998; Terrab et al., 2002).The presence of HMF is an indicator for spoiled, adulterated or products that were exposed to heat stress or bad storage conditions. On the other hand, HMF is considered an irritant and is irritating to eyes, upper respiratory tract, skin and mucous membrane. No positive or negative definite reports associating HMF with a cancer risk in humans were identified in available literature (FPA, 2006). However the National Institute of Environmental Health Sciences nominated HMF for testing based on the extensive human exposure, lack of adequate data characterizing its toxicity and carcinogenicity. Miller (1994) in studies based on mice and rats, proposed that sulphonation of HMF may lead to multagenicity and carcinogenicity. Janzowski et al. (2002) studied the DNA damaging potential and reactivity of HMF towards cellular glutathione as an assessment of multagenicity of HMF. It is also reported that HMF damages striated muscles and viscera by combining with protein and thus causing the accumulation of poisons in the body (Pamplona et al., 1995; Chi et al., 1998).
Pollen analysis
The results from quantitative melissopalynological analysis, summarised in Table 3, show that the NPG/10 g for all samples ranged from 8 × 103 to 2.01 × 106. 22 of the 40 samples were poor in pollen (< 20 × 103 in 10 g) and belonged consequently to class I; 14 samples belonged to class II, 3 samples were in class III and one sample in class V; they contained more than 106 pollen in 10 g. The observed that pollen distribution is quite normal for Mediterranean honey (Persano Oddo and Piro, 2004) and also corresponded with the results of published study of Ouchemoukh et al. (2007), who found in their study of 11 Algerian samples, lower PG/10 g values ranging from 20 × 103 till 40 × 103 and a study of Makhloufi et al. (2007). However many factors influence the number of pollen in honey. It is known that the pollen richness depends upon the pollen production of the plant, the weather conditions, the distance of the beehive to the flower field, the filtering by the bee’s proventriculus and consequently the pollen’s diameter, and the mode of honey extraction (Von der Ohe, 1994).The results from qualitative melissopalynological analysis, summarised in Table 4, show that predominant pollen is found in 9 samples. It is generally accepted that a minimum content of 70% Eucalyptus pollen is necessary to classify Eucalyptus honey as unifloral. Eucalyptus honeys are considered among the best honeys and are very valuable from a consumer’s point of view (Terrab et al., 2003a). The results show that all honey samples studied are original flower honey. We also note that the dominant pollen in the majority of samples are: pollen of Eucalyptus, Trifolium sp, Echium sp and Hedysarium corornarium, Brassicaceae, Asteraceae (mostly Carduus) and to a lesser extent, Rubus and Citrus. Our results of pollen analysis are consistent with Louveaux and Abed (1984), Chefrour et al. (2007), Ouchemoukh et al. (2007) and Makhloufi et al. (2007, 2010). The distribution of observed pollen is quite normal for honey of the Mediterranean (Persano Oddo and Piro, 2004). Also, Louveaux and Abed (1984) found that eucalyptus is one of the most important honey plants in Algeria and Terrab et al. (2003c) concluded that the single-flower honeys from Eucalyptus camaldulensis Dehnh are very common in northern Morocco. They mentioned Plantago sp. Thymelaceae as accompanying pollens while Ricciardelli and Vorwohl (1980) found the Acacia and Gleditsia species H. coronarium as pollens companions for Libya. According to the same authors, Tunisian eucalyptus honeys were characterized by Citrus species, Acacia and Erica, Olea europaea, and H. coronarium as pollens carers (Seijo et al., 2003). We also found a pollen of Citrus in our honey in the important minor pollen case. Pollen Citrus is generally under-represented in honey. Unifloral honey citrus were also collected in northwestern Morocco (Terrab et al., 2003b), Spain, Cyprus (Ricciardelli and Vorwohl, 1980) and Tunisia (Louveaux and Abed, 1984). Analysis of pollen honey (melissopalynology) is very important to the quality control of honey. However, the wealth of pollen depends on the production of plant pollen, weather conditions, distance from the hive to flower field, filtering by glandular of the bee and the method of extraction of honey (Von der Ohe, 1994).
On the basis of HMF and enzymes activities of forty Algerian honey samples we can note that the results obtained agreed with requirements of international and European Community Directives. Although the enzymes activities and the HMF content of honey usually bear no direct relationship to the botanical origin of the product of some honey types; like Citrus honey, they are characterized by naturally low enzyme content. In general, these tree parameters are used as quality criteria. The strong heating and too long storage damage the enzyme activity and increase the HMF content. However, honey samples differ in quality on account of various factors such as season, origin of honey, activity of the bee, food of the bee, period and technique of extraction of honey, conditions of storage and the freshness of honey. The analysis of palynological results therefore revealed that the dominant pollen in the majority of samples are: pollen of Eucalyptus, Trifolium sp, Echium sp and H. corornarium, Brassicaceae, Asteraceae (mostly Carduus) and to a lesser extent, Rubus and Citrus, Apiaceae and Ericaceae are most frequently found in the pollen spectrum of Algerian honeys.
The authors have not declared any conflict of interests.
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