**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.


**Table 2.**

 *Summary of studies on effects of storage system on the quality of selected root vegetables.* sugar content is linked to the development of harsh, and oily flavor in carrots during prolonged storage [96].

Significant differences have been observed in the content of main beetroot betcyanins (betanin and isobetanin) during cold storage of red beetroot at 5°C for 196 days [71]. The content of betanin in red beetroot peel decreased in the first 140 days of cold storage and then slightly increased (**Table 2**). In terms of isobetanin, until 98 days, an increasing trend and afterward, up to 140 days of storage, a light decrease were noticed [71]. In [72] it was shown that after 1 month of cold storage, there was an increase in beetroot water loss that resulted into increased total dry matter content. The total phenolic content of beetroot in cold storage increased after 4 months of storage. In another study, the nitrate content in beetroot increased significantly after 1 month of cold storage [73], and no further significant increase was observed after a cold storage period of 4 months. In [61], there was no significant change in total sugar content of 11 beetroot genotypes stored under optimal cold storage conditions at 1 ± 1°C and 90–95% RH for 7 months. Additionally, beetroots retained their levels of total soluble sugar contents after 1 month of cold storage, and the levels decreased after 4 months of cold storage. Therefore, it is beneficial to use these beetroot genotypes freshly or within the first month of storage, when a high sugar content is desired.

Freshly harvested cassava roots transpire and loose moisture, which reduces their quality during storage. Cassava undergoes postharvest physiological deterioration (PPD) once the roots are separated from the main plant. As a result of this mechanical damage (wounding), the roots respond with a healing mechanism that initiates about 15 min after damage, and fails to switch off in harvested roots [97]. This is observed as a blue/black or brown discoloration of the vascular parenchyma (vascular streaking) within 24–72 h of harvest [20] rendering it unpalatable. As described by Beeching [98, 99], this mechanism involves an oxidative burst of the superoxide radical (O2−), which is followed by the further production of reactive oxygen species, altered gene expression, and the accumulation of secondary metabolites. This physical and biochemical change is often followed by microbial deterioration and root tissue softening.

Traditional methods have been shown to extend cassava shelf life for few days during which, the cyanide, moisture, and starch content in the root decreases while the ash, sugar, crude fiber as well as the acidic content increases with the length of storage [49, 58, 59]. Generally, temperatures of about 20–30°C and low RH between 65 and 80% encourage deterioration [100]. Therefore, storage of cassava under high relative humidity and limited oxygen conditions, for example, in polyethylene bags, storage boxes, and coating with paraffin wax, can reduce water loss and oxidative stress [101]. Besides, Rickard [102] reported that at 80–90% RH, cassava roots showed a typical wound-healing response with periderm formation occurring in 7–9 days at 35°C and 10–14 days at 25°C.

Prolonged in-ground storage has been shown to increase the size of the root. However, the roots become more woody and fibrous, and with decreased palatability and amount of extractable starch, besides becoming more susceptible to attack by pathogenic microorganisms (**Table 2**) [49, 58]. Covering cassava roots with paraffin wax by dipping the root in paraffin wax (at a temperature of 55–65°C for a few seconds) after treatment with fungicide has been reported to prolong shelf-life of cassava roots up to 2 months (**Table 1**) [49, 68].

Cassava roots can also be stored for 2 weeks between 0 and 4°C without any internal deterioration, and after 6.5 months of storage, the part of the root without decay usually is in excellent condition for human consumption [49]. At temperatures

#### *Phytochemical Changes in Root Vegetables during Postharvest Storage DOI: http://dx.doi.org/10.5772/intechopen.106554*

above 4°C, roots develop the PPD symptoms more rapidly and have to be discarded after 2 weeks of storage [49]. A study on the effect of the total carotenoids content in cassava root on the reduction and delay of postharvest deterioration showed that cassava roots kept at 10°C and 80% RH can remain fresh till after 2 weeks [100]. Thus, higher carotenoid can reduce or delay the onsets of PPD and extend the shelf life of the root. Wijesinghe and Sarananda [103] reported that freezing of fresh cassava for up to 8 weeks resulted into loss in water and increase in dry matter, that was driven by the high vapor pressure deficit created between the product and the low RH in the refrigerator environment. Consequently, the water stress which remained of acceptable eating quality although none remained as good as the freshly harvested ones.

Physicochemical parameters that determine quality of radish are well maintained in lower storage temperature of 0°C hence it is recommended for extended storage period of radish. Studies by Chandra et al. [90] showed that weight losses of radish roots were remarkably lower (<3%) in radish packed with micro perforated HDPE film in plastic crate and that cured then packed in micro perforated HDPE film in PC while the cured sample maintained its total soluble solids, and flesh and skin firmness. Both samples also recorded lower scores of black spot, surface shrinkage, and fungal infection incidence (**Table 2**). About 1.3% weight loss of unpacked topped radish root was noted after 9 days of storage at either 5°C or 10°C [104]. However, severe weight loss of radishes (about 52%) was noted just after 3 days of storage when radishes were stored at room temperature (20 ± 2°C) without leaves [91]. In a related study, a weight loss of about 2.5% was noted when whole radishes were stored at 1°C or 5°C for 10 days, whereas this loss reached nearly double when they were stored at 10°C [92]. These results indicate that storage temperature greatly affects the fresh weight retention ability of radish. In another study, the concentration of isothiocyanate sulforaphene and myrosinase activity were measured in two radish cultivars, namely "Chungwoon plus" (CP) and "Taebaek" (TB), during storage at 0°C for 4 months. After the storage period, the sulforaphene concentrations in the CP and TB radish cultivars decreased by 81% and 40%, respectively [89]. Also, the myrosinase activity decreased in both cultivars which subsequently decreased the formation of sulforaphene [89]. Glucosinolates, lipid-soluble vitamins E and K contents, and primary metabolites were measured from topped radish root stored at 1°C for 90 days. The results indicated that the tested storage conditions had no effect on the concentration of aliphatic glucosinolates present in radish [88].

Use of low temperatures (i.e., 2–4°C) and potato sprout inhibitors are the widely used storage treatments on freshly harvested potatoes. Curing potatoes allows the formation of a protective layer (wound periderm) over areas of potato that could have been damaged during harvesting. Curing has been reported to limit weight loss and to prevent the penetration of pathogenic microorganisms. In a study in which the effects of various curing and storage conditions (i.e., duration, temperature, and RH) on the quality of two potato cultivars, "Moonlight" and "Nadine" were investigated, high curing RH (93%) led to significantly lower skin browning, shriveling, and weight loss in both cultivars, and significantly lower incidence of rot in "Nadine" than low curing RH (62%) [105]. A 7 days curing at >90% RH and at least 15°C was recommended.

Sprout inhibitors (e.g., ethylene) and treatments to inhibit microbial establishment on harvested potatoes are reported to be effective but with varied secondary effects on potatoes. Previous studies have reported that ethylene application can either shorten or delay potato dormancy period depending on both treatment duration and concentration [106]. Furthermore, a continuous application of ethylene [69] and/or early application (applied after appearance of first sprouting) [70] is reported

to prevent potato sprouting. However, this can also increase the content of reducing sugars (primarily glucose and fructose) in potato, thus limiting its use for processing potatoes. High levels of reducing sugars in processing potatoes causes cold induced sweetness [107] that is responsible for the dark brown color on processed potato products that gives a bitter taste [108]. In a separate study, a single treatment with HPP or CIPC resulted, after 6 months of storage at 10 ± 1°C, in sprouting rates of 61% and 58%, respectively, vs. 87% in the untreated control [54]. From preliminary experiments in [48], potatoes exposed to UV-C light at five different intensities (0.0, 3.4, 7.1, 10.5, and 13.6 kJ m−2) and stored in the dark at 20°C and 80% RH for 40 days showed reduction in sprout length and development up to 20 d. However, this effect diminished during storage. Also in [47], potatoes exposed to gamma Irradiation (0, 50, 100, and 150 Gy levels) on different dates (10, 30, and 50 days after harvest) were studied during prolonged storage at 8 and 16°C. Results indicated that indicated that early and higher irradiation levels significantly decreased sprouting, percent weight loss and specific gravity of potato. However, the loss of ascorbic acid, and reducing and non-reducing sugars significantly increased by delay in irradiation whereas the sugars and ascorbic acid content was decreased by irradiation. Higher storage temperature (16°C) caused greater loss of ascorbic acid. A delay in irradiation and storage at high temperature was not recommended [47]. A study in [83] showed that potatoes buried at deeper depths (overground/pit storage) accumulated a lot of respiratory heat. This heat has been reported to promote potato sprouts [84]. Sprouting results in remobilization of storage compounds mainly starch and proteins as sprout tissue is built from the potato reserves. This increases the rate of respiration as well as evaporation [109], and consequently weight loss. Also, vitamin C is adversely affected by sprouting [47]. Sprouting and sprout growth contributes to formation of toxic glycoalkaloids (TGA). This involves the buildup of chlorophyll beneath the peel, a process known as "greening" [110]. Greening in potatoes is associated with the TGA solanine accumulation. In a study where six potato cultivars were analyzed for TGA content after 6 and 14 weeks of storage at either 10 or 4°C, results indicated that the exposure of some cultivars, to low temperatures within 2 weeks of harvest resulted in a relatively rapid accumulation of TGA to levels close to or exceeding the recommended safe maximum level of 200 mg of TGA per kilogram of fresh weight [86].

Nevertheless, storage of potatoes at low temperatures has been shown to significantly decreases the content of vitamin C [87, 111, 112], although in [82] it was noted that even with the decrease in vitamin C, a significant amount was still retained. The decrease in vitamin C content is attributed to its use, in the potato, as an antioxidant compound in response to oxidative stress caused by low temperature storage. In [87], an evaluation for antioxidant parameters of potatoes stored at room temperature, 15°C and 4°C for 90 days showed that all phenolic acid content increased with storage time, except para-coumaric acid which decreased at 4°C. Similarly, a separate study showed an increase in chlorogenic acid, caffeic acid, and sinapic acid content during storage of potatoes for 90 days at 3°C [81].

High temperatures can also influence respiration rate, development of decay causing organisms, greening, and shrinkage in stored potatoes [113]. A study on the effect of three store types (ambient charcoal-cooled, traditional grass thatched hut and diffused light store [DLS]) on the quality of potatoes stored for 56 days at temperatures 9.7–19.4°C and RH 65–93%, results showed increased weight loss, greening ,and shrinkage in potatoes stored in DLS (temperature 16.15–19.35°C; RH 65–89%) [85]. Additionally, there was a significant increase in the reducing sugar content. The starch content in potato samples from the three stores decreased with increasing storage time.


**5. Conclusion**

 Vitamin A is one of the fat-soluble vitamins that are vital for various biological roles in the human body, as it is essential for embryogenesis up to adulthood. It can be sourced from both animal-based (preformed vitamin A) and plant-based (provitamin A) foods. The evaluation of whether a population is vitamin A deficient or excess is determined by status monitoring. Biological and biochemical indicators are the most widely applied parameters in assessment of vitamin A status. Vitamin A deficiency or toxicity state arises under conditions where the dietary intake does not comply with recommended levels. It is crucial to note that both conditions could lead to various health complications with VAD leading to mainly xerophthalmia, increased infection risk and anaemia, while toxicity could result in chronic hypervitaminosis and hypercarotenemia. In line with this, prevention efforts that could improve vitamin A status are widely explored. Dietary diversification, fortification and supplementation are the three main approaches that are widely applied for this purpose. These continuous efforts are believed to be able in improving vitamin A status among the vitamin A-deficient populations.

## **Acknowledgements**

Utmost appreciation is conveyed to the Director General of Health Malaysia and the Director of Institute for Medical Research (IMR) for giving permission for this manuscript to be published as a book chapter. Special thanks to all staff of the Nutrition Unit, IMR, for their continuous support.