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Qualitative characterization of Romanian robinia honey according to the European directive and IHC approaches.


The development and the promotion of quality products represent one of the major challenges of European Union. The honey production is differently organized from one European Member State to another. Nowadays, the beekeeping products have to satisfy numerous quality and certification criteria before commercialization, especially in western countries, where there is an increasing need to have food products of high quality with well-defined characteristics. The purpose of the present study was to find suitable physical and chemical parameters to facilitate the qualitative characterization of Romanian robinia honey.

The quality of the honey depends on the floral source, season and honeybee ecotypes. In Romania beekeepers breed the Apis mellifera carpatica, a subspecies of honeybee that is native in Romania. Because of the great variability of landscape, our country is covered by forests consisting in various melliferous species. The most valuable honey sources are black locust tree (Robiniapseudoacacia L.) and limetree (Tilia cordata L.). Currently, black locust trees represent the most important melliferous source, providing important annual production. There are two kinds of forests: pure black locust forests: Valea lui Mihai, Lovrin, Alios, Bujor, Snagov, Baragan, Pogoanele or mixt black locust forests, which are all over the hills and submontaneous areas. This enables the flowering of black locust tree clumps to take place at different periods of time, in connection with the altitude and soil type, providing the opportunity to benefit great harvests in locust trees over the same apiarian year.

On the other hand the quality of honey is mainly determined by its sensorial, chemical, physical and microbiological characteristics. Honey physicochemical quality criteria are well specified by the EC Directive 2001/110 (EU, 2001). The major criteria of interest are moisture content, electrical conductivity, reducing and non-reducing sugars, free acidity and hydroxymethylfurfural (HMF) content. In fact, numerous studies have been reported on the physicochemical parameters of honeys from all over the world [2,8,10,12,18] and several studies have been published on the physicochemical parameters of monofloral honeys, from Romanian production areas especially from Transylvania [3,14].

The aim of this study was to use these markers of quality to facilitate the Romanian markers of quality for acacia honey and to create a physicochemical profile for acacia honey. The physicochemical parameters were particularly investigated as possible certification criteria for a future denomination of origin.


Honey samples

Robinia pseudoacacia honey (n=16) were collected directly from beekeepers in different natural-geographical regions of Romania and were stored in at 4[degrees]C temperature prior to analyses. The type of honey was determined in all samples by means of melissopalynology [13]. A botanical classification was considered to be achieved when the pollen spectra contained even less than 20% in black locust honey. Honey samples were obtained from one production year (table 1) and from different regions of Romania (figure 1).


Statistical testing was made by using Kolmogorov-Smirnov normality test, results being presented in table 2 and 3. Selective physicochemical parameters were determined according to the Romanian standard (STAS 784/1 ... 3-89) and Harmonized Methods of International Honey Commission. Water content was determined refractometrically (Abbe digital refractometer WYA-S Selecta Spain). Then the content was expressed in mg/100g; electrical conductivity was measured at 20[degrees]C in a 20% (w/v) honey solution in water with a KIT consort conductometer (CONSORT nv, Belgium) and expressed as |iS/cm, free acidity, lactone acidity, total acidity was determined by automatic titration (TitroLine Easy Schott, Germany).

Hydroxymethylfurfural (HMF)

Hydroxymethylfurfural was determined using the standard method White. Five grams Of honey were dissolved in 25 mL of distilled water, treated with a clarifying agent (0.5 mL of Carrez I and 0.5 mL of Carrez II solutions) and volume made up to 50 mL. The solution was filtered, and the first 10 mL discarged. The absorbance of the filtered solution was measured at 284 and 336 nm against an aliquot of the filtered solution treated with NaHSO3. HMF was determined as:

HMF/100 g of honey = ((Abs284 -Abs336) x 149.7 x 5)/g of sample

Sugar content

Sugar profile was determined with the help of HPLC method on a Shimadzu system. The system is equipped with a LC-10AD pump, DGU-14A degasser, SIL-10AV VP auto sampler, RID-10A refractive index detector, thermostated at 30[degrees]C with CTO-10AS VP temperature controller of separation column (Altima Amino 100 A 5 |im, 250 mm x 4.6 mm) with a mixture of acetonitrile/water as mobile phase with 1.3 ml/min flow rate. For the quantification of main sugars, a calibration curve in the range 40-0.5 g/100g, with regression coefficient of R2=0.9982 for a mixture of standards (glucose, fructose and saccharose) was obtained. The results were expressed in g/100g honey.


The present study determined the main physicochemical quality parameters of acacia honey, by methods proposed by International Honey Commission [4,5].

Results obtained for the physicochemical quality parameters in the sixteen Romanian robinia honey samples are listed in the table 2.

With the HPLC method used for determining the profile of sugar s from honey we Could identify and quantify seven sugars. Sugars are the main constituents of robinia honey, especially fructose and glucose [3,7,17]. Kolmogorov-Smirnov normality test applied to all parameters indicate that data are from a normally distributed population and confirm that the sample is a good choice.

Furthermore, the experimental values of the sample are compared with quality Parameters fixed by the European Directive concerning honey (2001/110/CE).

All samples were found to meet honeys quality European Legislation (EC Directive2001/110) in all parameters except for water content and the content of reducing sugars.

Honey moisture content depends on the environmental conditions and the manipulation from beekeepers at the harvest period, and it can vary from year to year [1]. The values of moisture content of honey samples ranged between 15.5 and 17.6 g/100g, which are well below to the imposed limit of <20% [9] only sample S15 showed level of moisture higher than the allowed limits of 20 g/100g.

Electrical conductivity and free acidity values are also within the limits (lower than 0.8 mS/cm and 50 mEq/kg, respectively).

None of the analyzed honey samples showed electrical conductivity values superior to 0.8 mS/cm (variation between 0.08 and 0.15 mS/cm), suggesting that all samples are from nectar honey, which is corroborated by the content of total ashes inferior to 0.6% [9].

Honey color depends on various factors, being their minerals content an important one.

The HMF content is widely recognized as a parameter of honey samples freshness, because it is absent in fresh honeys and tends to increase during processing and/or aging of the product. Several factors influence the levels of HMF, such as temperature and time of heating, storage conditions, pH and floral source, thus it provides an indication of overheating and storage in poor conditions [11]. None of the samples showed levels of HMF higher than the allowed limits of 80 mg/kg.

Reducing sugars, mainly fructose and glucose, represented the largest portion of honey composition. The reducing sugars were above 60% in almost all samples, except sample S6, S7, S12 such level complied with the requirements of the European Directive. These samples do not only meet the standards but also correspond to the levels observed in other studies [12, 16].

A physicochemical profile to confirm the quality of robinia honey was developed. This profiling approach has recently been described in more detail by Persano Oddo and Piro [15].

Therefore, we considered it of interest to apply this model for results obtained in our research and the usefulness of this to find quality markers of Romanian robinia honeys. The limits obtained by the IHC research work are compared with the values obtained in this study and these are shown in table 5 and figure 2.


The majority of Romanian robinia honeys presented values below the maximum allowed by International Honey Commission. The samples of Romanian acacia honey showed law values of lactone and free acidity and, consequently, they had law total acidity and high values of glucose, that could be attributed to the fact that samples are obtained from different regions of the different soil characteristics or group may be related to their different floral origins (polen composition) because robinia honey has a relative frequencies of the pollen types of nectariferous species and only 30% of pollen has to be robinia pollen .


In this preliminary research, a set of complementary techniques is proposed that does not require chemical reactants or expensive equipment, to improve quality control of honeys from regions of Romania with different soil characteristics. The analytical results of honey samples have been compared with profiles describing data ranges of robinia honeys. When all the values fit into the respective ranges described for robinia honey, it is assigned to this corresponding honey type. Further studies have to be made on other constituents of honey, of soil and plant, in order to correlate them with origin of honey for a future denomination of origin. From the economical standpoint, the discrimination and assessment of origin may increase the added value of honey.


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[2.] Al-Khalifa, A.S., I.A. Al-Arify, (1999). "Physicochemical characteristics and pollenspectrum of some Saudi honeys," Food Chemistry 67, 21-25.

[3.] Bobis O., L.Marghitas, V. Bonta, D. Dezmirean, O. Cocan, (2006). "Determination of sugars in Romania honeys of different botanical origin using high performance liquid chromatography," Buletin USAMV-CN, vol.63, p. 143-148.

[4.] Bogdanov S., S., P. Martin, C. Lullman, (1997). "Harmonized Methods of the European Honey Commission," Apidologie, extra issue, 1-59.

[5.] Bogdanov, S., C. Lullman, C., P.Martin, W. Von der Ohe, H. Russmann, G.Vorwohl, (1999). "Honey quality methods of analysis and international regulatory standards: Review of the work of the International honey Commission," Mitteilungen Gerbeite Lebensmittel, 108-125.

[6.] Codex Alimentarius Commission Standards, 1981/revised 1987/revised 2001. Codex Standards for honey, 1-7, FAO- Rome.

[7.] Doner L.M., (1977). "The sugars of honey--a review," Journal Science and Food Agriculture 28, 443-456.

[8.] Downey, G.K., Hussey, J.D. Kelly, T.F. Walshe, P.G. Martin, (2005). "Preliminary contribution to the characterization of artisanal honey produced on the island of Ireland by palynological and physico-chemical data," Food Chemistry 91, 347-354.

[9.] European Commission, (2002). Council Directive 2001/110/EC of 20 December 2001 relating to honey, Official Journal of the European Communities L10, 47-52.

[10.] Finola, M.S., M.C. Lasagno, J.M. Marioli, (2007). "Microbiological and chemical characterisation of honey from central Argentina," Food Chemistry 100, 1649-1653.

[11.] Fallico, B., E. Arena, A. Verzera, M. Zappala, (2006). "The European Food Legislation and its impact on honey sector," Accreditation and Quality Assurance 11, 49-54.

[12.] Kucuk, M., Kolayli, S., Karaoglu, S., Ulusoy, E., Baltaci, C., Candan, F., (2007). "Biological activities and chemical composition of three honeys of different types from Anatolia," Food Chemistry 100, 526-534.

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[14.] MarghitaD L., D. Dezmirean, A. Moise, O. Bobis, L. Laslo, S. Bogdanov, (2009). "Physicochemical and bioactive properties of different floral origin honeys from Romania," Food Chemistry 112, 863-867.

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MARIANA NICULINA MADAC, LIVIU ALEXANDRU MARGHITAC, DANIEL SEVERUS DEZMIREAN, CRISTINA BIANCA POCOL, VICTORITA BONTA University of Agricultural Sciences and Veterinary Medicine, Faculty of Animal Science and Biotehnologies, 3-5 ManaDtur Street, Cluj-Napoca, Romania
Table 1. Romanian acacia honey used in this study on the basis
of place of production and date of harvest

                                                        Date of
No.   Sample code           Production place            harvest

1        MS1        Valea lui Mihai, Satu-Mare county    2009
2        MS2              Glogovet, Alba county          2009
3        MS3                Blaj, Alba county            2009
4        MS4              Calafat, Dolj county           2009
5        MS5                  Bacau county               2009
6        MS6                  Valcea county              2009
7        MS7             Vanatori, Galati county         2009
8        MS8              Darlos, Sibiu county           2009
9        MS9                  Timis county               2009
10       MS10               Aiud, Cluj county            2009
11       MS11                Suceava county              2009
12       MS12                Harghita county             2009
13       MS13            Bistrita-Nasaud county          2009
14       MS14                 Brasov county              2009
15       MS15                 Timis county               2009
16       MS16              Turda, Cluj county            2009

Table 2. Physicochemical quality parameters of Romanian acacia honey

         Color      Conductivity    Free Acidity
Sample   mmPfund       mS/cm           mEq/kg

S1           4          0.11            7.44
S2           5          0.13           11.17
S3          20          0.12            9.96
S4          18          0.13            9.67
S5           4          0.10            8.40
S6           8          0.12            8.33
S7           9          0.12            8.33
S8           5          0.11            7.51
S9           4          0.12           10.08
S10          8          0.13            8.27
S11         20          0.11            8.36
S12         23          0.11            7.91
S13         19          0.10            6.63
S14         24          0.15            9.69
S15          5          0.08            8.17
S16          7          0.09            7.69
Mean        18          0.11            8.6
SD         1.2          0.02            1.19

         Lactone    Total
         Acidity   Acidity   Moisture    Hmf
Sample   mEq/kg    mEq/kg     g/100g    mg/kg

S1        0.36       7.80       16.8    13.17
S2        0.75      11.90       15.5     2.69
S3        0.88      10.84       16.9    15.72
S4        0.71      10.38       16.1     1.50
S5        0.16       8.56       17.3     1.05
S6        0.64       8.97       17.4     4.04
S7        1.36       9.69       15.8     0.60
S8        0.64       8.15       16.0    17.96
S9        0.79      10.86       16.8     1.05
S10       0.53       8.79       15.4     1.65
S11       0.16       8.52       16.4     1.35
S12       0.15       7.76       15.7     1.65
S13       0.27       6.90       17.6     4.94
S14       0.31       9.99       17.3     2.84
S15       0.46       8.63       21.3     0.75
S16       0.29       7.98       15.7     1.05
Mean      0.38       9.13      16.75      4.5
SD         0.5       1.35       1.42     5.71

Table 3. Determined sugars in Romanian robinia honey samples
(g/100g of honey)

Sample   Fructose   Glucose   Sucrose   Turanose   Maltose

S1        45.55      30.77     0.17       2.77      4.016
S2        45.77      29.46     0.39       2.58       3.88
S3        44.38      30.26     1.22       2.36       3.75
S4        44.27      30.15     1.60       2.23       3.61
S5        45.54      31.74     1.35       1.70       2.99
S6        39.67      27.98     0.46       1.73       2.24
S7        37.97      40.14     1.63       0.98       1.11
S8        45.54      30.44     1.69       2.43       3.61
S9        45.62      32.43     0.30       2.31       3.63
S10       45.33      31.61     1.25       2.41       3.88
S11       45.66      31.31     1.83       2.77       3.90
S12       40.30      26.06     2.37       2.58       3.56
S13       43.76      26.96     1.13       1.81       2.51
S14       44.52      32.63     0.08       2.49       3.50
S15       44.67      28.46     4.65       2.43       3.36
S16       44.97      30.55     0.89       2.60       3.87
Mean      43.97      30.68     1.31       2.26       3.34
SD         2.42       3.14     1.11       0.48       0.78

Sample   Iso    Erlose    F+G    F/G    G/Water

S1       0.56    1.20    62.35   1.48    1.83
S2       0.55    1.78    61.27   1.55    1.90
S3       0.43    1.90    61.28   1.47    1.79
S4       0.34    2.33    60.37   1.47    1.87
S5       0.29    2.21    62.84   1.43    1.83
S6       0.36    1.66    57.07   1.42    1.61
S7       0.13    0.09    53.77   0.95    2.54
S8       0.53    2.53    61.54   1.50    1.90
S9       0.47    1.82    62.42   1.41    1.93
S10      0.43    2.08    60.73   1.43    2.05
S11      0.55    2.58    62.06   1.46    1.91
S12      0.41    2.50    56.00   1.55    1.66
S13      0.66    1.89    61.36   1.62    1.53
S14      0.60    1.71    61.82   1.36    1.89
S15      0.39    3.19    65.97   1.57    1.34
S16      0.59    2.47    60.68   1.47    1.95
Mean     0.45    1.99    60.72   1.45    1.85
SD       0.14    0.69     2.9    0.15    0.26

Table 4. Quality parameters fixed by the European Directive concerning
honey and experimental values of the samples analyzed in this study

                              EU Directive   Experimental values
Parameter                     limits         of robinia honey

[H.sub.2]O(g/100g)            <20            Max = 21.3
Fructose+Glucose (g/100g)     >60            Max = 78.11
Sucrose (g/100g)              <5             Max = 4.64
Electrical conductivity       <0.8           Max = 0.14
Free acidity (mEq/kg)         <50            Max = 11.17
HMF (mg/kg)                   <40            Max = 17.96

Table 5. Quality parameters obtained by IHC

                                      Experimental values
                                      from IHC databank
parameters                 Unit       min.       mean       max.

Color                      mm Pfund   5.0        12.9       23.9
Electrical conductivity    mS/cm      0.09       0.16       0.23
Free acidity               mEq/kg     4.5        11.2       17.9
Lactones                   mEq/kg     0.0        2.8        7.1
Total Acidity              mEq/kg     7.5        13.4       19.4
Water                      g/100g     14.7       17.1       19.6
Fructose                   g/100g     38.1       42.7       47.3
Glucose                    g/100g     23.1       26.5       29.9
Sucrose                    g/100g     0.0        2.1        6.1
Fructose+Glucose           g/100g     62.7       69.2       75.7
Fructose/Glucose                      1.39       1.61       1.83
Glucose/water                         1.32       1.57       1.82

                           Experimental values
                           of the study
parameters                 min.       mean       max.

Color                      4.0        12.0       24
Electrical conductivity    0.09       0.11       0.15
Free acidity               6.63       8.6        11.17
Lactones                   0.15       0.36       1.36
Total Acidity              6.9        9.13       11.9
Water                      15.4       16.75      21.3
Fructose                   37.97      43.97      45.66
Glucose                    26.06      30.68      40.14
Sucrose                    0.17       1.31       4.65
Fructose+Glucose           53.77      60.72      65.97
Fructose/Glucose           0.95       0.95       1.62
Glucose/water              1.34       1.85       2.54
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Author:Madas, Mariana Niculina; Marghitas, Liviu Alexandru; Dezmirean, Daniel Severus; Pocol, Cristina Bian
Publication:Economics, Management, and Financial Markets
Article Type:Report
Date:Mar 1, 2011
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