**4. Adulteration**

Adulteration means addition of external chemical compounds to a food product containing naturally similar substances. With more than 200 major and minor components, and a constantly increasing market value, honey ranks high in the category of merchandises subjected to forgery. Honey adulteration can be carried out directly, by deliberately adding certain substances into it, or indirectly, by feeding the honeybees with the adulterating compound. Although most adulterating agents do not represent health hazards, any change in the composition or physico-chemical parameters values outside the standardized intervals may be classified as a fraud attempt and are to be sanctioned accordingly in the trading activities.

Mehryar and Esmaiili [49] have reviewed the normal values of principal physico-chemical honey parameters, drawing attention to adulteration possibilities and means of investigation. There are several possibilities to determine and report these parameters; they mainly refer to sugar content (total sugar, total reducing sugar, inverted sugar, fructose, glucose, fructose/glucose ratio), acidity (pH, free acidity, lactonic acidity, and total acidity), nitrogenous compounds (protein content, nitrogen content, proline content, diastase index, invertase index) phenolic compounds (total polyphenols, total flavonoids), HMF, minerals, and other trace elements, water content and water activity, viscosity, glass transition temperature, and colour. Authors point out that honey is adulterated directly by addition of inverted sugar or syrup (corn syrup, high fructose corn syrup, high fructose inulin syrup, and inverted syrup), intruders being difficult to detect by sugar analysis, as they have properties similar to those of natural honey. Many of the techniques involved in adulteration detection require specialized personnel and equipment, being prone to exceptional rather than routine analysis.

Plants, sources of substances used for indirect adulteration, are either C3 of C4 plants, a classification based on the carbon metabolism. The C3 plants are able to fix atmospheric carbon dioxide using the Calvin cycle, while the C4 plants use the Hatch-Slack cycle. C3 plants are characterized by a lower <sup>13</sup>C/12C ratio than the C4 plants. Beet, rice, and wheat are C3 plants, whilst maize and sugarcane are C4 plants. Zabrodska and Vorlova [50] have discussed adulterant detection methods employed over the time, indirect adulteration of honey included, and botanical and geographical authentication issues. According to the national legislation [11] and European legislation, Council Regulation (EC) no. 797/2004 and Commission Regulation (EC) no. 917/2004 [26, 27] honey is defined as the product of the *Apis mellifera* honeybee species. Still there are other bee species, which also produce 'honey'; yet according to the regulations in force, this cannot be considered true honey. Therefore, entomological origin is another issue that needs addressing and asks for some sort of regulations, especially in South American countries where *Melipona* and *Melipona seminigra merrillae* bees produce 'honey' with extremely high antioxidant and antimicrobial activity, but higher moisture, free fatty acids, and pollen content.

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

Adulteration means addition of external chemical compounds to a food product containing naturally similar substances. With more than 200 major and minor components, and a constantly increasing market value, honey ranks high in the category of merchandises subjected to forgery. Honey adulteration can be carried out directly, by deliberately adding certain substances into it, or indirectly, by feeding the honeybees with the adulterating compound. Although most adulterating agents do not represent health hazards, any change in the composition or physico-chemical parameters values outside the standardized intervals may be classified as a fraud attempt and are to be sanctioned accordingly in the trading activities.

Mehryar and Esmaiili [49] have reviewed the normal values of principal physico-chemical honey parameters, drawing attention to adulteration possibilities and means of investigation. There are several possibilities to determine and report these parameters; they mainly refer to sugar content (total sugar, total reducing sugar, inverted sugar, fructose, glucose, fructose/glucose ratio), acidity (pH, free acidity, lactonic acidity, and total acidity), nitrogenous compounds (protein content, nitrogen content, proline content, diastase index, invertase index) phenolic compounds (total polyphenols, total flavonoids), HMF, minerals, and other trace elements, water content and water activity, viscosity, glass transition temperature, and colour. Authors point out that honey is adulterated directly by addition of inverted sugar or syrup (corn syrup, high fructose corn syrup, high fructose inulin syrup, and inverted syrup), intruders being difficult to detect by sugar analysis, as they have properties similar to those of natural honey. Many of the techniques involved in adulteration detection require specialized

personnel and equipment, being prone to exceptional rather than routine analysis.

Plants, sources of substances used for indirect adulteration, are either C3 of C4 plants, a classification based on the carbon metabolism. The C3 plants are able to fix atmospheric carbon dioxide using the Calvin cycle, while the C4 plants use the Hatch-Slack cycle. C3 plants are characterized by a lower <sup>13</sup>C/12C ratio than the C4 plants. Beet, rice, and wheat are C3 plants, whilst maize and sugarcane are C4 plants. Zabrodska and Vorlova [50] have discussed adulterant detection methods employed over the time, indirect adulteration of honey included, and botanical and geographical authentication issues. According to the national legislation [11] and European legislation, Council Regulation (EC) no. 797/2004 and Commission Regulation (EC) no. 917/2004 [26, 27] honey is defined as the product of the *Apis mellifera* honeybee species. Still there are other bee species, which also produce 'honey'; yet according to the regulations in force, this cannot be considered true honey. Therefore, entomological origin is another issue

and a high F/G ratio, are very unlikely to granulate.

**4. Adulteration**

42 Honey Analysis

Using a set of 10 acacia honey samples from Valea lui Mihai, Bihor County, Marghitas et al. [51] have concentrated on clarifying their biochemical profile in relation to adulteration. The discussion basis comprises selected physico-chemical parameters (moisture, electrical conductivity, *p*H, pollen, total and free acidity, fructose, glucose, along with their sum and ratio, maltose, sucrose), phenolic and flavonoids data (total phenolic and flavonoids content, punctual levels of three phenolic acids and five free flavonoids) and elemental content (sodium, potassium, calcium, magnesium, copper, zinc, iron, and manganese). The natural variation of *R. pseudoacacia* pollen grains falls in the 21–36% range, in line with the national regulations. Phenolic acids rise to 12.11 mg/kg, ferulic acid representing 29% of the total amount; levels of *p*-coumaric and vanillic acid have been also determined, but appearance is random. Acacetine, pinobanksine, pinocembrine, and chrysin are present in all samples (0.38–2.28 mg/kg), quantified levels being characteristic to the Romanian acacia honey, lower than the European acacia studied by Tomas-Barberan et al. [30], but higher than the Croatian values reported by Kenjeric et al. [52]. Apart from offering a valuable instrument to confirm the compositional formula and lack of adulteration, the authors recommend the polyphenolics profile as starting point for geographic authentication.

Indirect adulteration has gained momentum in the 1970, when high fructose corn syrup became available at low costs. With an oligosaccharides profile very similar to that of natural honey, these syrups have been used as bees fed with little restriction; direct sugar analysis could not make any difference between honey produced by honeybees fed on natural honey and those produced by honeybees fed on solutions of industrial sugars. Within less than a decade, a sensitive and precise technique based on analysis of <sup>13</sup>C/12C stable isotopes ratio has been released [53], and proved to be effective for C3 and C4 sugars adulteration. The <sup>13</sup>C/12C isotopic ratio (or δ<sup>13</sup>C, ‰) varies with the photosynthetic paths, so that the C4 plants, present δ<sup>13</sup>C values ranging from –8 to –12‰, while for C3 plants it varies between –22 and –30‰. If honey has not been pampered with by syrup honeybee feeding, δ<sup>13</sup>C of its protein extract is very close to the value of honey itself. Dordai et al. [54] have used Eq. (1) in calculating the adulteration degree, drawing the attention on the fact that C4 syrups affect only the honey isotopic ratio, with little effect on its protein composition:

$$\stackrel{\circ}{\text{isotopic ratio, with little effect on its protein composition:}} \quad \text{(\text{\textquotedblleft}b\text{\textquotedblright})}$$

$$\text{Adulteration, \text{\textquotedblleft}b = \frac{\delta^{13}\text{C}\_{\text{reaction}} - \delta^{13}\text{C}\_{\text{heavy}}}{\delta^{14}\text{C}\_{\text{reaction}} - \delta^{13}\text{C}\_{\text{HCS}}} \times 100\tag{1}$$

They have used an elemental analyser coupled with an isotope ratio mass spectrometer to gain access to experimentally determined δ<sup>13</sup>C values for 12 samples of Romanian acacia, linden, sunflower, and polyfloral honeys, and their corresponding protein extracts. Some δ<sup>13</sup>Cprotein–δ<sup>13</sup>Choney differences are positive, indicating no adulteration. Others present negative values (–0.06 to –0.98‰), thus leading to an apparent adulteration of 0.38 and 6.39%. Since –1‰ value (7% adulteration) is internationally accepted as critical threshold, only one of the 12 samples should be reported as adulterated up to 10.8% with high fructose corn syrup. The study gives access to an average δ13C value of –25.35‰ for Romanian honey, in line with values reported for other samples harvested in temperate climate areas of Europe. The authors point out that δ<sup>13</sup>C values vary with time, location, pollen content, but there is a levelling effect characteristic to the system itself. Honey is collected from more than one colony, over a period of several weeks. As the season starts, honeybees are fed with syrups, so there is high chance that the honey produced reflects the syrup isotopic ratio. Since hive population is renewed every 3–4 weeks, newer generations feed on the previously collected honey, so the adulterating effect of the syrup on the protein δ<sup>13</sup>C value will quickly decrease.

The stable isotopic ratio methods for adulteration with C4 sugars is expensive in terms of time, consumables, personnel, and equipment, so the efforts of Puscas et al. [55] in developing a simple and reproducible high-performance thin-layer chromatographic method are welcome. It has been tested on some Romanian honey samples, being based on the F/G ratio and sucrose content evaluation. Using a suitable composition of ethyl acetate : pyridine : water : acetic acid, 6:3:1:0.5 volume ratios, high-performance thin-layer chromatographic aluminium silica gel sheets, a chromatographic twin through chamber, a dipping acetone solution of diphenylamine and aniline hydrochloride, and a visible light TLC visualization device, the authors have managed to validate the proposed procedure for the determination of the glucose, fructose, and sucrose levels. The newly validated method has given trustworthy results during the analysis of 15 Romanian acacia, linden, and polyfloral honey samples harvested by five individual producers. Almost half of the investigated samples have been declared adulterated with fructose from other sources than the natural ones. As F/G is 0.88, a polyfloral sample is declared adulterated with industrial glucose. When determined sucrose levels run above the admitted limit, there is an indication of adulteration by honeybees feeding with sucrose syrup. The acacia honey samples present a higher fructose/glucose ratio than the admitted value, effect of some producers' initiative to improve sensory properties by fructose addition (acacia honey being not too sweet).

EC regulation 470/2009 [21] states that honey should be free from antibiotics residues, serious health hazard agents. Antibiotics are generally used for the treatment of bacterial brood diseases produced by *Paenibacillus larvae*, known as American foulbrood (AFB). Even if they are effective only against the hives infestation with AFB, many beekeepers, the Romanians included, practice preventive antibiotics usage. Streptomycin, often used in veterinary medicine, opens up the human organism to deafness and kidney failure at higher concentrations, causing allergies, destroying intestinal flora, and inducing resistance of certain microorganisms at lower concentrations. So there is a multitude of antibiotics screening tests and confirmatory methods. High-performance liquid chromatography with post-column derivatization and fluorescence detection (HPLC-FD) is one of the most versatile and reliable methods in antibiotics residues analysis. Equally effective are the immunochemical assay kits based on antigen-antibody interactions to detect a large variety of antibiotics. The lower rate of false-negative samples, short analysis time, simple operating procedures, good selectivity, low costs are counterbalanced by the possibility to identify and quantify a single target analyte. Cara et al. [56] have used an enzyme-linked immunosorbent assay (ELISA) test kit for streptomycin to determine the antibiotic loadings in acacia, linden, and polyfloral honey samples collected from the Romanian market and get more information on the kinetic law governing the contaminant degradation on storage in the dark and different temperatures. The method has been validated (in terms of repeatability, recovery, precision, specificity, and variation coefficient), and cross-validated by high-performance liquid chromatography with post-column derivatization and fluorescence detection. Running a *F*-distribution test on the experimental results dispersions obtained by the two methods demonstrates that both sets of analysis are equally reproducible, no matter the method. No residue has been detected in the samples tested. Experiments on spiked (20 and 200 μg/kg streptomycin) honey samples in the 4–70°C temperature range, for 20 weeks revealed that degradation fits a second-order multiple linear regression model for all three types of honey.
