**3.1. Legal basis of honey trade**

The European Union has established food hygiene and safety regulations stricter than those in force in other regions of the world. Moreover, European buyers often apply even stricter requirements of their own, depending on the market. These can vary from composition specifications to colour and taste preferences and organic/fair trade certifications.

As honey is generally used as food, the European Union legislation on food applies to all honey present on the European Union market, locally processed and imported. The basis for food legislation is laid down in the EU General Food Law, Regulation (EC) 178/2002 [8], defining responsibilities and requirements for food business operators supplying food to the European Union. Directive (EC) 110/2001 [9] sets European requirements concerning honey quality standards and labelling. It has been amended by Directive (EC) 63/2014 [10], stating that pollen is not considered an ingredient anymore and labelling of honey originating in more than one member state or third country is compulsory. It also defines the right of the commission to set methods of analysis in order to verify the compliance with provisions of the current directive and the procedures of issuing and applying new decisions.

Requirements regarding honey composition and quality standards on the Romanian market are stated in this SR 784, parts 1 and 2 [11, 12]. Part 3 of the standard establishes the analysis methods for the sensory evaluation and quantification of the mandatory physical and chemical parameters (moisture, ash, acidity, reducing and easily hydrolysable sugars, total water insoluble matter, diastase and invertase, hydroxymethyl furfural content, colour index, electrical conductivity, and palynological evaluation) [13]. It also states the methods for determining adulteration with industrial glucose, starch, gelatine, glues, and aniline pigments. In addition to these requirements, all honey must comply with the general food and safety regulations mentioned above. The Romanian standard requires evaluation of routine physico-chemical parameters and identification of handful of adulterants. The recommended methods for evaluation of hydroxymethyl furfural (HMF) content are based on its reaction with resorcinol in acidic conditions or with barbituric acid in the presence of the carcinogenic *p*-toluidine [13]. Commercial contracts, even within the European Union, may contain a larger number of quality requirements than the national standard, and any importer should comply. Limited compliance with specific regulations may restrict access to certain categories of buyers.

Tesalios [7] have reported that 11% of the adult population does not consume honey, while 35% of the population consumes less than 750 g/year. An average consumption between 750 and 2000 g/year is acknowledged by 20%, and only 20% consume more than 2000 g/year. A correlation between age and consumption has been identified, stating that subjects in the 46–60 years category consume average and large amounts; this age range is negligible in the non-consumers category. Median age subjects (32–45) reported a normal consumption, while people below 30 consume reduced amounts of honey. These signal that status and economic determinants play an important part in honey consumption in Romania. Unfortunately, no linear dependency could be found between the amount of honey purchased and consumed and the economic and status variables, higher consumption being associated with mediumhigh status and income. As for cultural, demographic, and environmental variables, only age, cultural area, and nationality discriminate between categories. The authors conclude that honey in Romania is not part of the general dietary habits, being associated with a medium

The European Union has established food hygiene and safety regulations stricter than those in force in other regions of the world. Moreover, European buyers often apply even stricter requirements of their own, depending on the market. These can vary from composition speci-

As honey is generally used as food, the European Union legislation on food applies to all honey present on the European Union market, locally processed and imported. The basis for food legislation is laid down in the EU General Food Law, Regulation (EC) 178/2002 [8], defining responsibilities and requirements for food business operators supplying food to the European Union. Directive (EC) 110/2001 [9] sets European requirements concerning honey quality standards and labelling. It has been amended by Directive (EC) 63/2014 [10], stating that pollen is not considered an ingredient anymore and labelling of honey originating in more than one member state or third country is compulsory. It also defines the right of the commission to set methods of analysis in order to verify the compliance with provisions of the

Requirements regarding honey composition and quality standards on the Romanian market are stated in this SR 784, parts 1 and 2 [11, 12]. Part 3 of the standard establishes the analysis methods for the sensory evaluation and quantification of the mandatory physical and chemical parameters (moisture, ash, acidity, reducing and easily hydrolysable sugars, total water insoluble matter, diastase and invertase, hydroxymethyl furfural content, colour index, electrical conductivity, and palynological evaluation) [13]. It also states the methods for determining adulteration with industrial glucose, starch, gelatine, glues, and aniline pigments. In addition to these requirements, all honey must comply with the general food and safety regulations mentioned above. The Romanian standard requires evaluation of routine

fications to colour and taste preferences and organic/fair trade certifications.

current directive and the procedures of issuing and applying new decisions.

to high welfare.

34 Honey Analysis

**3. Quality assurance**

**3.1. Legal basis of honey trade**

As botanical and geographical authentication has become a marked feature of the national and international honey trade, conformity evaluation laboratories and different research groups in Romania have taken steps to evaluate a larger portfolio of parameters to be used for the classification of honey samples, including geographical origin traceability [14–20].

As regards contaminants, the national Romanian regulations for beekeeping and honey do not give details, but on the European Union territory, the Regulation (EC) 470/2009 [21], in conjunction with the annexes of Regulation (EC) 2377/90 [22], is in function and establishes the maximum residue levels (MRLs) for use of authorized veterinary drugs (mainly antibiotics) applied to honeybees. The use of veterinary drugs containing pharmacological substances not listed in the annexes of the mentioned document is prohibited.

The systematic use of pesticides in the European agriculture has led to worrying declines in bee colonies, phenomenon known as colony collapse disorder (CCD). Following the negative trend and the extensive research by the European Food Safety Authority (ESFA) [23], the European Union has decided to ban the clothianidin, imidacloprid, and thiametoxam pesticides. The European proposal targets pesticides used in the treatment of cereals and plants attractive for bees and other pollinators.

In the European Union, there are strict guidelines concerning genetically modified organisms (GMO) used as food. The ruling issued by the European Court of Justice in September 2011 stipulated that honey with traces of pollen from genetically modified crops needed special authorization and labelling before it could be commercialized in Europe. Then European Parliament authorized the shift of pollen from the 'constituent' to the 'ingredient' category, in effect from July 2014 [10]. Therefore, honey containing genetically modified pollen should no longer be labelled as containing GMOs.

An important segment of the European market is the organic honey. Regulations have become stricter in time and European honey importers will increasingly require proof of organic certification of honey before entering this market. If honey is to be marketed as 'organic', it has to comply with the Council Regulation (EC) 834/2007 [24]. The specified requirements for organic beekeeping are


Honey laundering is an increasingly worrying issue and refers to the re-labelling of honey from one origin to allege that it comes from another region, perceived by honey buyers as offering better quality. There is a constant race to discover affordable markers and techniques for authenticating geographical origin, with authorities and researchers on one side and international traders on the other side. The 2011 dossier on the Chinese honey shipped to India and Thailand and re-labelled before entering the European Union and the USA has prompted for concerted measures over the world. European buyers have established a working group in the International Federation of Beekeepers' Associations (Apimondia) with the aim to set up a consequent framework to prevent and fight unfair trading [25].

Generating more than €400 million per annum, European beekeeping sector is a significant economic player. Therefore, it is assisted by the European Union through subsidies, as laid down in Council Regulation 917/2004 [26, 27]. These subsidies are mostly directed to national apiculture programmes, which support research in the field of beekeeping and physical and chemical analysis of honey, technical assistance for trade, etc. Unfortunately, current production levels within the union are falling. This trend is characteristic mainly to Western European countries such as Belgium, France, Germany, Switzerland, the United Kingdom, and the Netherlands, but it was also spotted in the South in Italy, Greece, and Cyprus.

#### **3.2. Physico-chemical characterization**

Apart from the mandatory characteristics imposed by Standardization Association of Romania [28], different research groups have been engaged in the last 25 years in studying honey effects on the human body, setting up new analytical procedures, optimizing and validating those destined to routine operation, and building up an image as detailed as possible of its chemical and biochemical profile. Starting with 2005, a significant national financial support has contributed to the creation of a solid infrastructure for research and conformity compliance purposes. Some contributions are further presented, shedding light on the achievements obtained so far in exhaustively characterizing Romanian honey.

While the major sugars present in honey are readily accessible titrimetrically or spectrophotometrically, minor carbohydrates in Transylvanian acacia honey have been determined by liquid chromatography, along with individual phenolics [29]. An elaborate extraction procedure has been used prior to the identification and quantification by refractive index, UV, and mass spectrometry (MS) detection. Fructose and glucose, amounting to 42.4 and 31.9%, respectively, have been accompanied by 2.94% maltose, 2.16% sucrose, and 0.91% trehalose. Out of the 13 phenolic acids and flavonoids identified in the black locust honey, ferulic acid, abscisic acid, pinobanksine, pinocembrine, chrysin, and acacetine have been found in all studied samples, *p*-hydroxybenzoic acid, *t*-cinnamic acid, kaempherol, and apigenine have appeared in 50% of the samples, while vanillic acid, *p*-coumaric acid, and vanilline have been detected only in a quarter of the lot. This phenolic profile has been reported previously [30]. Abscisic acid with an average 16.2 mg/kg level (the highest concentration in the 13 phenolics detected) plays a major role in mediating plant adaptation to stress. Since ferulic acid and acacetine are found only in acacia honey samples, when comparison to the rest of honey samples produced in the area is carried out, they might be a candidate for the role of markers in botanical origin discrimination.

• artificial honeybee fodder should also be certified as organic;

• honeybees should not be stupefied while harvesting honey.

up a consequent framework to prevent and fight unfair trading [25].

obtained so far in exhaustively characterizing Romanian honey.

substances;

36 Honey Analysis

Cyprus.

**3.2. Physico-chemical characterization**

• diseases should not be treated with veterinary medicines, only with approved organic

Honey laundering is an increasingly worrying issue and refers to the re-labelling of honey from one origin to allege that it comes from another region, perceived by honey buyers as offering better quality. There is a constant race to discover affordable markers and techniques for authenticating geographical origin, with authorities and researchers on one side and international traders on the other side. The 2011 dossier on the Chinese honey shipped to India and Thailand and re-labelled before entering the European Union and the USA has prompted for concerted measures over the world. European buyers have established a working group in the International Federation of Beekeepers' Associations (Apimondia) with the aim to set

Generating more than €400 million per annum, European beekeeping sector is a significant economic player. Therefore, it is assisted by the European Union through subsidies, as laid down in Council Regulation 917/2004 [26, 27]. These subsidies are mostly directed to national apiculture programmes, which support research in the field of beekeeping and physical and chemical analysis of honey, technical assistance for trade, etc. Unfortunately, current production levels within the union are falling. This trend is characteristic mainly to Western European countries such as Belgium, France, Germany, Switzerland, the United Kingdom, and the Netherlands, but it was also spotted in the South in Italy, Greece, and

Apart from the mandatory characteristics imposed by Standardization Association of Romania [28], different research groups have been engaged in the last 25 years in studying honey effects on the human body, setting up new analytical procedures, optimizing and validating those destined to routine operation, and building up an image as detailed as possible of its chemical and biochemical profile. Starting with 2005, a significant national financial support has contributed to the creation of a solid infrastructure for research and conformity compliance purposes. Some contributions are further presented, shedding light on the achievements

While the major sugars present in honey are readily accessible titrimetrically or spectrophotometrically, minor carbohydrates in Transylvanian acacia honey have been determined by liquid chromatography, along with individual phenolics [29]. An elaborate extraction procedure has been used prior to the identification and quantification by refractive index, UV, and mass spectrometry (MS) detection. Fructose and glucose, amounting to 42.4 and 31.9%, respectively, have been accompanied by 2.94% maltose, 2.16% sucrose, and 0.91% trehalose. Out of the 13 phenolic acids and flavonoids identified in the black locust honey, ferulic acid, abscisic acid, pinobanksine, pinocembrine, chrysin, and acacetine have been found in all studied samMarghitas et al. [18] were among the first to contribute to Romanian honey characterization in terms of antioxidant properties. Knowledge about phenols and flavonoids levels, as well as the radical scavenging activity completes the Romanian honey profile and helps understand and predict part of its dietary and health effects. Using a lot of 24 nectar and honeydew honey collected from beekeepers in 2005–2006, they determined the sugars profiles by high-performance liquid chromatography (HPLC), water, colour, and ash content according to the International Honey Commission recommendations [31]. The total phenolic content was accessible by a modification of Folin-Ciocalteu method, using gallic acids equivalents to report results, while the flavonoids were evaluated as quercitin equivalents in basic solution. All studied samples passed the Romanian quality requirements. The honeydew honey has higher ash content than the nectar honey samples evaluated. Melezitose is present only in the honeydew samples, being a good candidate as discriminant for honeydew. As for the fructose/glucose ratio, all samples with values below 1 were crystallized, while the rest were fluid at the moment of investigations. In the nectar honey category, sunflower samples contain the largest levels of phenols, as high as 45 mg gallic acid/100 g sample; this maximum is easily exceed by honeydew honey samples, whose content is 23–125 mg gallic acid/100 g sample. While the honeydew phenols content resembles that of other European studied samples [32], the Romanian nectar honey samples contain fewer phenols than the values reported by other groups [33]. A significant correlation between phenols and radical scavenging activity was found, which was better than the correlation between flavonoids and radical scavenging activity (0.94 as compared to 0.83). The honeydew honey presents the highest flavonoids content, the highest percent of inhibition towards free radicals, being followed by sunflower, lime, and acacia honey.

The special situation of honeydew honey has been further addressed by Chis et al. [34], when they compared the total phenolic compounds, flavonoids, and vitamin C levels in 10 samples from Bihor, Romania, and Podcarpackie, Poland, collected from beekeepers in 2012–2013. Two Polish samples were labelled organic. Apart from the attempt to standardize the evaluation procedure for radical scavenging activity using 2,2-di(phenyl-1-hydrazyl-hydrate) by using the percentage concentration of honey inducing a 50% inhibition of the free radical, IC50%, and the inhibition degree induced by a 1% honey solution, AA1%, the authors reported higher homogeneity of the evaluated parameters for the Romanian samples, compared to the Polish samples. Even if the entire Polish lot was labelled as honeydew honey, samples were different in appearance: 'usual' samples were dark brown, highly viscous, opaque, and completely liquid, while the 'organic' samples were light brown, opaque, and crystallized. The hypothesis of floral honey addition has been rejected based on the lower levels of phenolic compounds in Polish colza and sunflower honey, the possible candidates for adulteration. Ascorbic acid, flavonoids, and polyphenols are present in significant amounts, Polish samples being richer in all three compounds. The good correlation between the polyphenols levels and the radical scavenging activity points out that polyphenols are the main contributors for the antioxidant properties of honey.

Information on the polycyclic aromatic hydrocarbons is mainly required when exporting Romanian honey on European and American markets. Nectar honey samples and other byproducts (propolis, royal jelly, bee venom, bee wax) are prone to contamination by products resulted from the partial combustion of organic matter during different industrial processes, polycyclic aromatic hydrocarbons. Since many of these hydrocarbons have been proved to have mutagenic and/or carcinogenic effect [35], there has been an increasing concern about the levels of polycyclic aromatic hydrocarbons in foodstuff, not only in water, air, and soil. Investigations of Dobrinas et al. [19] lead to a successful procedure for extraction of polycyclic aromatic hydrocarbons from honey and propolis originating from 15 Romanian regions using hexane, followed by separation on aluminium oxide and silica gel chromatographic column and gas spectrography-mass spectrophotometry (GS-MS) dosage. Fourteen different aromatic hydrocarbons were determined, acenaphthene, and fluorine being the most abundant, at levels ranging from 2.0 to 55.0 ng/g. According to Environment Protection Agency, benzo[α]anthracene, benzo[*k*]fluoranthene, chrysene, benzo[α]pyrene, dibenzo[α,*h*]anthracene, and indenol[1, 2, 3,-*cd*]pyrene are potential carcinogens. Chrysene, benzo[α]anthracene, and dibenzo[α,*h*]anthracene were below the limit of quantification in all samples. Benzo[*k*] fluoranthene, and benzo[α]pyrene varied in the 1–155 ng/g, while indenol[1, 2, 3,-*cd*]pyrene appeared at levels below 23 ng/g, being absent in the samples from Deva rural area and Pecineaga. The highest level was obtained for samples from Bucharest urban area. The lowest levels were recorded in samples collected from Pecineaga and Dragasani rural areas. Samples originating from urban areas are characterized by much higher levels of the six carcinogenic polycyclic aromatic hydrocarbons. Whenever a forest has surrounded the beehives, levels of contamination have been much lower. The same has been found for propolis, so the authors have concluded that polycyclic aromatic hydrocarbons contamination of samples originating from the rural and mountain areas is significantly lower than for samples collected from urban areas. Contamination comes from atmospheric sources or from the soil on which plants grow. The levels of polycyclic aromatic hydrocarbons measured in honey and propolis are comparable with values found in grains, milk, and lettuce, lower than those found in olives. Luckily, the detected polycyclic aromatic hydrocarbons levels do not raise any concern for the human health.

How does organic honey perform from the quality parameters point of view had been reported by Badescu et al. [36] after measuring moisture, HMF, colour, and antibiotics residues of acacia, linden, and polyfloral honey samples collected in 2012–2015 from beekeepers members of the Romanian Beekeepers Association, in Bacau and Deva. Three samples were taken from each type of honey, for each year, amounting to 54 samples. Water content varied in the 17–19.5% range stating all samples as superior quality honeys. Only one acacia sample collected from Bacau region in 2014 out of 54 in the studied lot had 1.23 mg HMF/100 g samples. As for the antibiotics residues, they were not put in evidence, thus meeting the national requirements for antibiotics residues in food stuff. It is thus gratifying that the organic honey originating from Bacau and Deva regions observe the quality standards for honey, as well as the European provision for organic honey.

in Polish colza and sunflower honey, the possible candidates for adulteration. Ascorbic acid, flavonoids, and polyphenols are present in significant amounts, Polish samples being richer in all three compounds. The good correlation between the polyphenols levels and the radical scavenging activity points out that polyphenols are the main contributors for the antioxidant

Information on the polycyclic aromatic hydrocarbons is mainly required when exporting Romanian honey on European and American markets. Nectar honey samples and other byproducts (propolis, royal jelly, bee venom, bee wax) are prone to contamination by products resulted from the partial combustion of organic matter during different industrial processes, polycyclic aromatic hydrocarbons. Since many of these hydrocarbons have been proved to have mutagenic and/or carcinogenic effect [35], there has been an increasing concern about the levels of polycyclic aromatic hydrocarbons in foodstuff, not only in water, air, and soil. Investigations of Dobrinas et al. [19] lead to a successful procedure for extraction of polycyclic aromatic hydrocarbons from honey and propolis originating from 15 Romanian regions using hexane, followed by separation on aluminium oxide and silica gel chromatographic column and gas spectrography-mass spectrophotometry (GS-MS) dosage. Fourteen different aromatic hydrocarbons were determined, acenaphthene, and fluorine being the most abundant, at levels ranging from 2.0 to 55.0 ng/g. According to Environment Protection Agency, benzo[α]anthracene, benzo[*k*]fluoranthene, chrysene, benzo[α]pyrene, dibenzo[α,*h*]anthracene, and indenol[1, 2, 3,-*cd*]pyrene are potential carcinogens. Chrysene, benzo[α]anthracene, and dibenzo[α,*h*]anthracene were below the limit of quantification in all samples. Benzo[*k*] fluoranthene, and benzo[α]pyrene varied in the 1–155 ng/g, while indenol[1, 2, 3,-*cd*]pyrene appeared at levels below 23 ng/g, being absent in the samples from Deva rural area and Pecineaga. The highest level was obtained for samples from Bucharest urban area. The lowest levels were recorded in samples collected from Pecineaga and Dragasani rural areas. Samples originating from urban areas are characterized by much higher levels of the six carcinogenic polycyclic aromatic hydrocarbons. Whenever a forest has surrounded the beehives, levels of contamination have been much lower. The same has been found for propolis, so the authors have concluded that polycyclic aromatic hydrocarbons contamination of samples originating from the rural and mountain areas is significantly lower than for samples collected from urban areas. Contamination comes from atmospheric sources or from the soil on which plants grow. The levels of polycyclic aromatic hydrocarbons measured in honey and propolis are comparable with values found in grains, milk, and lettuce, lower than those found in olives. Luckily, the detected polycyclic aromatic hydrocarbons levels do not raise any concern for

How does organic honey perform from the quality parameters point of view had been reported by Badescu et al. [36] after measuring moisture, HMF, colour, and antibiotics residues of acacia, linden, and polyfloral honey samples collected in 2012–2015 from beekeepers members of the Romanian Beekeepers Association, in Bacau and Deva. Three samples were taken from each type of honey, for each year, amounting to 54 samples. Water content varied in the 17–19.5% range stating all samples as superior quality honeys. Only one acacia sample collected from Bacau region in 2014 out of 54 in the studied lot had 1.23 mg HMF/100 g samples. As for the antibiotics residues, they were not put in evidence, thus meeting the national

properties of honey.

38 Honey Analysis

the human health.

Next to the routine physico-chemical parameters, Stihi et al. [37] investigated the presence of a series of metals by energy dispersive X-ray fluorescence (Ca, K) and atomic absorption spectrometry (Fe, Cu, Zn, and Pb) in 18 unifloral honey samples (acacia, lime tree, colza, and sunflower) from different sites of Romania. The quality requirements according to the national and European requirements have been fulfilled by most of the lot, with the exception of four samples, some adulteration suspicions and the likelihood of fermentation being signalled. Using an yttrium internal standard, the authors have found an average potassium level of 269.8 mg/kg in 2012 and a 271.9 mg/kg in 2013 and almost five times less calcium. Iron and copper levels have been as high as 6.46 and 3.1 mg/kg, respectively. Only six honey samples contained copper up to 2.2 mg/kg, while lead exceed the limit imposed for drinking water and foodstuff of 1 mg/kg. Results evaluation by two-tailored *t* test and principal component analysis demonstrate that K, Ca, and Cu levels are connected to the honeybee activity and nectar plants visited by the honeybees, while Fe, Zn, and Pb appear as a result of air and soil pollution.

Volatile organic compounds are present in honey in very different amounts and their profile has been expected to vary with the botanical origin of the flowers supplying the nectar for honey production. Sample workup is crucial to the investigation success, so a variety of approaches has been used, such as solid phase microextraction [38], liquid-liquid extraction, static head space [39], or purge and trap [40]. Several Romanian acacia and linden honey samples, along with other samples originating from Slovakia, Serbia, Poland, Georgia, Germany, Ukraine, Czech Republic, Italy, France, Greece, and Moldavia have been subjected to twodimensional GC-MS, the volatiles being first separated using a non-chiral stationary phase and further fed to a chromatographic system containing a chiral stationary phase [38]. Over 270 compounds have been detected: alkanes, alcohols, aldehydes, ketones, carboxylic acids, and their methyl and/or ethyl esters. Hotrienol, linalool, and linalool oxides have been present at the highest concentration levels, while α-terpineol, 4-terpineol, and isomers of lilac aldehydes have been reported at significantly lower amounts. All these compounds have been found in all investigated samples. Enantiomer ratios of these compounds have been determined by multidimensional GC, results demonstrating that distribution varies with the botanical origin. Although present at significant levels in all samples, (2R,5S)-*cis*-linalool oxide exceeds 80% with respect to its (2S,5R) enantiomer only in linden honey. Rapeseed, orange, acacia, and linden honey contain almost racemic mixtures of *trans*-linalool oxide. A slight predomination of (2R,5R)-*trans*-linalool oxide over its second enantiomer is observed in sunflower honey. As Italian chestnut honey present a predomination of the (2S,5S)-enantiomer of *trans*-linalool oxide, it results that the enantiomer ratio of *trans*-linalool oxide is a potential marker for sunflower and chestnut honey. The list of good candidates continues with (S)-4 terpineol marker for sunflower honey origin, (2S,2'S,5'S)-lilac aldehydes A, B, or C for orange and acacia honey. The authors recommend that a larger pool of chiral volatile organic compounds should be evaluated when botanical origin is under scrutiny. Since all enantiomeric ratios have been observed in samples regardless their country of origin, this information cannot be exploited for geographical authentication.

#### **3.3. Pollen spectrum**

Given the characteristics of the vegetation zones in the country, 77 pollen types from 35 families were found in the 54 unifloral and polyfloral honey samples studied by Dobre et al. [41]. The international melissopalynological nomenclature recommends four different terms to be used when reporting a pollen spectrum: *dominant pollen* is present as at least 45% of the grains counted, the *accompanying pollen* should be found between 15 and 45%, the *important minor pollen* varies in the 3–15% range and the pollen present at less than 1% is just *minor pollen*. The average number of pollen forms per sample varied in the 12–44 range, with an average of 37, spread in the four categories mentioned. Current botanical classification occurs solely on the pollen count, *R. pseudoacacia* being the dominant pollen for acacia honey (present as 5–58% from the total count), *Tilia* pollen for linden honey (28.3–88.3%), *Brassica* for colza honey (52– 93%), *H. annuus* for sunflower (57.7–65.5%). The rest falls in the category of polyfloral and honeydew honey. Accompanying pollens found are *Prunus, Quercus, Castanea sativa, Echium, Trifolium repens, Filipendula*, and *Vitis vinifera*.

The total pollen content was also investigated; it varied from 525 to 19,525 grains per gram of honey, thus placing the studied lot in the low and very low level categories. The differences in the pollen content is attributed to the climatic conditions, pollen production of the parent plant, distance between beehive and flower field, diameter of pollen grains, and even the procedure used for extraction of honey. A principal component analysis of the pollen spectrum demonstrated that 77.89% of the entire variability of the pollen spectrum is explained by the first four principal components. The main contribution in the new components comes from *B. napus, Tilia*, and *H. annuus* types of grains.

#### **3.4. Rheological behaviour**

The complex chemical composition has a large impact on the honey viscosity, as moisture, variable sugars ratios, acids, proteins, phenolics, minerals, and pigments contribute to yield a mixture with changing molecular structure. This issue has enjoyed special attention over the time, due to the part played in processing and storage operations. Crystallization is a serious issue, causing problems during the extraction, filtration, mixing, and packaging stages. As crystallization decreases with the temperature, it looks that heating may overcome some of the processing troubles, but at the same time induces hydroxymethyl furfural formation, a strictly regulated quality parameter [11, 12].

Studies have identified a temperature-dependent Newtonian behaviour for acacia, heather, sunflower, lime, and rape honey, as well as non-Newtonian behaviour for certain crystallized samples [42, 43]. Several anomalies in terms of yield point, shear thinning, and rheodynamic behaviour of the crystallized honey in the temperature range investigated have been detected. It has been concluded that crystallization is significantly affected by the botanic origin, temperature profile, and storage time. Modelling of the viscoelastic properties and their relation to moisture, palynological spectrum, and sugars have been addressed by several groups, using either domestic or European honey for study [44–48]. The declared objectives were correct prediction of the rheological behaviour and identification of further correlation with the botanical origin.

Using a set of 52 artisanal honey samples collected directly from Romanian beekeepers during the 2009–2010 flowering season, Dobre et al. [46] have verified the pollen spectrum, moisture, carbohydrate composition, and rheological parameters. Six specific carbohydrates (fructose, glucose, sucrose, maltose, melezitose, and trehalose) and rheological parameters (loss modulus and shear stress) were used as predictors in the viscosity function. It was confirmed that granulation is favoured by a glucose/fructose ratio (F/G) larger than 1.3, as it is the case with sunflower and rape, while honeys with higher fructose content present a very low crystallization rate, maintaining the liquid appearance for years (typical for black locust honey). F/G ratio favours rapid solid phase formation: crystallization is slow or absent for a ratio lower than 1.7, but becomes complete if it exceeds two. Some correlations between pollen content and each type of carbohydrate were noticed for at least 45% pollen. On the other hand, significant amounts of crystallized glucose lead to lower deformation stress values, as the molecular network is already destroyed when the shear is applied. Colza and honeydew honeys present non-Newtonian shear thinning behaviour, as viscosity decreases with increasing shear rate. This is not a surprise, as honeydew honey contains large amounts of proteins (of high molecular mass), and sunflower honey presents the highest content of carbohydrates, in line with the findings of other groups for colza [42] and heather [43] honey.

**3.3. Pollen spectrum**

40 Honey Analysis

*Trifolium repens, Filipendula*, and *Vitis vinifera*.

*napus, Tilia*, and *H. annuus* types of grains.

strictly regulated quality parameter [11, 12].

**3.4. Rheological behaviour**

the botanical origin.

Given the characteristics of the vegetation zones in the country, 77 pollen types from 35 families were found in the 54 unifloral and polyfloral honey samples studied by Dobre et al. [41]. The international melissopalynological nomenclature recommends four different terms to be used when reporting a pollen spectrum: *dominant pollen* is present as at least 45% of the grains counted, the *accompanying pollen* should be found between 15 and 45%, the *important minor pollen* varies in the 3–15% range and the pollen present at less than 1% is just *minor pollen*. The average number of pollen forms per sample varied in the 12–44 range, with an average of 37, spread in the four categories mentioned. Current botanical classification occurs solely on the pollen count, *R. pseudoacacia* being the dominant pollen for acacia honey (present as 5–58% from the total count), *Tilia* pollen for linden honey (28.3–88.3%), *Brassica* for colza honey (52– 93%), *H. annuus* for sunflower (57.7–65.5%). The rest falls in the category of polyfloral and honeydew honey. Accompanying pollens found are *Prunus, Quercus, Castanea sativa, Echium,* 

The total pollen content was also investigated; it varied from 525 to 19,525 grains per gram of honey, thus placing the studied lot in the low and very low level categories. The differences in the pollen content is attributed to the climatic conditions, pollen production of the parent plant, distance between beehive and flower field, diameter of pollen grains, and even the procedure used for extraction of honey. A principal component analysis of the pollen spectrum demonstrated that 77.89% of the entire variability of the pollen spectrum is explained by the first four principal components. The main contribution in the new components comes from *B.* 

The complex chemical composition has a large impact on the honey viscosity, as moisture, variable sugars ratios, acids, proteins, phenolics, minerals, and pigments contribute to yield a mixture with changing molecular structure. This issue has enjoyed special attention over the time, due to the part played in processing and storage operations. Crystallization is a serious issue, causing problems during the extraction, filtration, mixing, and packaging stages. As crystallization decreases with the temperature, it looks that heating may overcome some of the processing troubles, but at the same time induces hydroxymethyl furfural formation, a

Studies have identified a temperature-dependent Newtonian behaviour for acacia, heather, sunflower, lime, and rape honey, as well as non-Newtonian behaviour for certain crystallized samples [42, 43]. Several anomalies in terms of yield point, shear thinning, and rheodynamic behaviour of the crystallized honey in the temperature range investigated have been detected. It has been concluded that crystallization is significantly affected by the botanic origin, temperature profile, and storage time. Modelling of the viscoelastic properties and their relation to moisture, palynological spectrum, and sugars have been addressed by several groups, using either domestic or European honey for study [44–48]. The declared objectives were correct prediction of the rheological behaviour and identification of further correlation with A deeper insight in the rheological behaviour of Romanian honey has been offered by Stoica-Guzun et al. [48]. They studied acacia, lime, coriander, peppermint, colza, sunflower, and polyfloral honey before and after heating at 50°C, looking for the compatibility degree with the Newtonian law of viscosity. Viscosity, Arrhenius constant at 20°C, and activation energies were measured for all unheated and heated samples. The qualitative analysis of the flow curves signalled the presence of a thixotropic behaviour for peppermint and colza honey, which diminished and even disappeared at higher temperatures. Using thixotropic relative areas (ratio of the thixotropic area to the area limited by the upper flow curves) at 30, 40, and 45°C, the authors attempted to classify honey samples using cluster analysis. Regardless the presences or absence of preheating, two clusters were formed, with cluster composition depended on the thermal regime. Thixotropy appears more often for unheated samples, but regresses with heating. The authors have pointed out that honey likely to crystallize (having higher glucose contents) are those prone to thixotropic behaviour.

The general model proposed by Oroian et al. [44] to describe the viscoelastic properties of honey is a fourth-order polynomial equation, applicable to all honey types (unifloral, polyfloral, or honeydew), for a 5–40°C temperature range. Validation on a set of Spanish honey samples having 32–42% fructose, 24–35% glucose, 79–83% reducing sugars, 16–19% water, and 3.4% sucrose demonstrated a Newtonian behaviour of all samples [45]. The loss modulus, G″, and viscosity show increase with moisture content, and decrease with temperature. The fourth-order polynomial equation described the combined effect of fructose, glucose, other sugars content, and moisture. A series of exponential and power models were analysed, to fit the experimental data.

A Spanish-Romanian research group [47] extended the crystallization tendency study on 136 unifloral honey samples (bramble, chestnut, eucalyptus, heather, acacia, colza, honeydew, lime, and sunflower) originating from Romania and north-west of Spain, by adding a new descriptor to the customary pollen spectrum, sugars profile, and moisture: the ratio between the major carbohydrates. It has been found a close relation between the fructose/glucose, glucose/water, sum of the first two sugars and main pollen types in honey, namely *B. napus, H. annuus, C. sativa, Rubus*, and *Eucalyptus*. This demonstrates that the botanical source influences not only the sugar ratios, but also the crystallization process. Such descriptors bring in close proximity colza and sunflower samples, discriminating them from acacia, bramble, chestnut, eucalyptus, honeydew, and heather. The last two, containing less than 30% glucose and a high F/G ratio, are very unlikely to granulate.
