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Research concerning the content of amylase and hydroxymethylfurfuraldehyde of Romanian honeys.


Honey is defined as "the natural sweet substance produced by honey bees from the nectar of blossoms or from secretion of living parts of plants." It must contain at least 60% reducing sugars and no more than 21% moisture. Honey composition is highly influenced by the types of flowers used by the bees as well as regional and climatic conditions [1]. Fresh honey is usually heated in order to facilitate processing and to maintain good quality. However, excessive heat treatment leads to the formation of 5- hydroxymethylfurfuraldehyde (HMF) and reduced honey quality. HMF value is virtually absent or very low in fresh honey and is high in honey that has been heated, stored in non-adequate conditions, or adulterated with invert syrup [2,5,7]. Chemical properties of honey such as pH, mineral content and total acidity also affect HMF content. The presence of organic acids and low water activity also favors the production of HMF [2,4,9]. The Codex Alimentarius (2001) set the maximum concentration of HMF to 40 mg/kg for honey from non-tropical regions and 80 mg/kg for honey from tropical regions. Extremely high (>500 mg/kg) HMF values demonstrate adulteration with invert syrup [10]. Codex Alimentarius (2001) proposed two quality indicators for honey, namely, 5-hydroxymethylfurfuraldehyde and amylase (diastase) activity to measure the freshness of honey. Many countries have set the national limit for HMF content in honey to 40 mg/kg.

Adulteration of pure honey with synthetic honey has become much more prevalent in recent years and more difficult to identify, due to the availability of cheap fructose corn syrup. Consequently, this project was developed to thoroughly examine the physiochemical properties of Romanian honey and to identify a standard for HMF content in comparison to the Codex Alimentarius standard.


Experimental design

Fresh unprocessed honey samples were obtained from local production units, in the metropolitan area of Bucharest. Each fresh unprocessed honey sample weighed 500 g, and was from season 2009. The commercially-processed Romanian honey samples (500 g each) were purchased from a local supermarket in Bucharest in July 2009, and included acacia and linden honey. The samples were kept in the original containers and stored in a dark room at room temperature throughout the analysis. Each honey sample was divided into three sub-samples, to represent three replicates. All analyses were performed at least three times.

Heat treatments of honey samples

Honey samples, 5 g in tubes were submerged in a water bath at 70oC for 5 minutes and cooled down in an ice water bath for another 5 min. Heat-treated samples were subjected to HMF, and amylase activity analyses.


Analytical standard-grade HMF was obtained and sulfuric acid was used as analytical-reagent grade. Methanol of HPLC grade was obtained and water was purified by using a Milli-Q water system from Millipore. All other chemicals were purchased from the local Chemical companies in the country.

Determination of moisture, pH, free acidity, lactones and total acidity contents in honey

Moisture content was determined using a standard direct drying method. In this method, 5 g of each honey sample were placed in an aluminium dish, mixed with 2 g of analytical-grade acid-washed sand, and dried in the oven at 100[degrees]C, following the standard method. The pH was measured using a solution containing 10 g honey in 75 ml carbon dioxide-free water in a 250 ml beaker. The free acidity lactones and total acidity were determined by a titrimetric method and the following equations:

Free acidity = (ml 0.05 N NaOH from burette--ml blank) x 50/g sample

Lactone = (10:00 - ml 0.05 N HCl from burette) x 50/g sample

Total acidity = free acidity + lactone

Measurement of color

Color was measured using a color meter, with a specific presentation of honey samples to avoid variations. The honey sample (15 g) was poured into a small disposable petri dish (55 mm) covered with the lid and left at room temperature for 20 min before taking the color measurement. Readings were taken at three different points with the lid on and the average values were calculated.

Evaluation of amylase activity

Heat-treated honey (10 g) was dissolved in 25 ml Milli-Q water and 10 ml acetate Buffer solution (pH 5.5), transferred to a 100-ml volumetric flask containing 10 ml sodium chloride solution and diluted to volume. Using a volumetric pipette, 25 ml of honey solution were transferred into a 100-ml flask and placed in a 50oC water bath along with a second flask containing 25 ml of 2% starch solution. After 30 min, 5 ml starch solution were added to the honey solution, mixed and timed. At periodic intervals, for the first time after 5 min, 0.5 ml aliquots of the mixture were mixed with 5 ml diluted iodine solution and 22 ml Milli-Q water, vortexed and immediately measured at 660 nm against a water blank. A plot of Absorbance against time was used to determine the time, at which the specified absorbance of 0.235 was reached. The diastase number was calculated following the standard method.

Determination of disaccharide, glucose and fructose contents

The sugar composition was determined by a HPLC. A honey sample (1 g) was dissolved in 19 ml Milli-Q water, filtered through a 0.22 |im nylon filter into an HPLC vial, capped and injected (20 into the HPLC. The mobile phase was 0.005 M sulfuric acid at a flow rate of 0.50 ml/min. External calibration curves constructed from standard solutions were used to quantify the amount of sugars in the sample. Results were expressed as gram sugar per 100 g of honey.

Quantitation of HMF

A honey sample (10 g) was dissolved in approximately 30 ml Milli-Q water and transferred quantitatively to a 100-ml volumetric flask. To clarify the honey samples and to stop HMF breakdown, 1.5 ml of a 15% (w/v) Carrez I (potassium hexacyanoferrate) solution and 1.5 ml of a 30% (w/v) Carrez II (zinc acetate dehydrate) solution were added and made up to 50 ml with Milli-Q water. The solution was filtered through Whatman filter paper, and the first 10 ml of the filtrate was discarded. The filtrate was passed through a 0.25 |im membrane filter before injection on the HPLC for HMF analysis. The mobile phase was water: methanol (90:10, v/v), and the flow rate was 0.50 ml/min with an injection volume of 20 Serial standard solutions of HMF (1-50 mg/l) were made in Milli-Q water, to generate a calibration curve at 285 nm.


pH of honeys

The pH of the honey samples varied from 4.01 to 4.06. Linden honey was found to have the highest pH value of 4.06 (table 1).

The pH values of honey are of great importance during extraction and storage, since acidity can influence the texture, stability, and shelf life of honey [15].

Total acidity of honeys

Total acidity of the honey ranged from 42.9 to 46.7 mEq acid/kg in linden honey and (table 1). The acidity of honey is due to the presence of organic acids, particularly gluconic acid, in equilibrium with their lactones or esters, and inorganic ions, such as phosphate and chloride.

Free acidity of honey

Free acidity of all seven samples fell within the permitted range proposed by Codex Alimentarius (2001) of no more than 50 mEq acid/kg. The free acidity of honey samples in this study ranged from 17.8 to 20.1 mEq acid/kg in acacia honey and rape honey, respectively (table 1). High free acidity values may indicate the fermentation of honey sugar by yeasts. It is well known that during fermentation, glucose and fructose are converted into carbon dioxide and alcohol. Alcohol is further hydrolyzed in the presence of oxygen and converted to acetic acid, which contributes to the level of free acidity in honey.

Lactones contents

Lactone contents in all honey samples except linden honey (29.7 mEq/kg) showed an average lactone content of >30 mEq/kg.

Moisture content

All the moisture values were under the allowed limit of 21% moisture content. The moisture content of the studied honey samples ranged from 12.5% to 15.1% (table 1). High moisture (>21%) honey indicates a premature extraction or extraction under high humidity conditions.

Sugar content

Honey samples had total sugar amounts ranging from 83.4 g/100 g to 88.5 g/100g, with the highest value registered for rape honey and the lowest for acacia honey (table 2).

The calculated ratio of fructose: glucose ranged from 0.92 in linden honey to 1.15 in acacia honey (table 2). Honey with high fructose: glucose ratio would remain liquid for Longer periods because of the modification of the saturated level of glucose by the presence of the larger amount of fructose [12]. The actual proportion of fructose to glucose in any particular honey depends largely on the source of the nectar [10]. The fructose: glucose ratio may also have an impact on honey flavor since fructose is much sweeter than glucose. Unlike to the amounts of monosaccharides recorded in various processed and unprocessed honey samples, the disaccharides contents were much smaller, and ranged from 11.2% for acacia to 16.8% in sunflower honey.

Analysis of amylase activity

The amylase activity is usually expressed as diastase number, symbol DN, and also known as Gothe units. A Gothe unit is defined as ml of 1% starch solution hydrolyzed at 40oC for one hour by the enzyme present in 1 g of honey [3]. The initial amylase activity was 10.21 and 11,05 in the tested commercial honeys and 10.92 and 10,57 in unprocessed honeys (table 3).

Linden honey, a commercial type of honey, presented higher value of DN in comparison to the unprocessed ones. Also, acacia honey had a DN value of only 10.21, lower than the ones of unprocessed honey. Such low DN values in some commercial honey samples may indicate severe heat treatments that caused a significant decline in amylase contents.

Heating honey samples in a water bath for 5 minutes at 50, 65 and 70oC revealed a positive correlation between temperature and level of amylase destruction. Heating at 70oC caused the largest decline in amylase activities in all honey samples (table 3). Nevertheless, the initial DN values did not affect the level of amylase inactivation.

HMF contents in commercial and unprocessed honey samples

Acacia and linden honey revealed HMF contents of 51.5 and 73.2 mg/kg, respectively, which did not fall within the international limit of 40 mg/kg (table 4). Consequently, it was concluded that the commercial Romanian honeys were not treated under appropriate temperature and storage conditions.

On the contrary, sunflower and rape honeys contained normal initial HMF levels of 20.1 and 34.2 mg/kg, respectively, which were under the maximum limit of 40 mg/kg.

Effect of heating on HMF contents

All honey samples registered increments in HMF contents when subjected to heating. These findings may suggest that these honey samples had been heated to the extent that most amylase activities and the substrate for Maillard reaction had been exhausted.

Considering the positive correlation between heat treatment and the increment in HMF contents in all fresh (unprocessed) honeys and two commercial samples it can be Suggested that HMF could be used as an indicator to judge honey quality.


Diastase number (amylase activity) varied between honey samples, had no uniform Starting point, and was not as sensitive to applied heating as we would expect.

HMF content was normal in unprocessed honeys, and seriously high considering the commercial types of honey subjected to analysis during this study.

Also, it is obvious that heating is not the only factor influencing HMF formation in honey but also honey composition, pH value and floral source can contribute.

This experiment found 40 mg/kg standard to be too strict on some honeys and too permissive for others.


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Faculty of Veterinary Medicine, University of Agronomic Sciences and Veterinary Medicine,

Splaiul Independentei Street 150, Bucharest, Romania
Table 1. Physiochemical parameters of various processed and unprocessed
honey samples. Results represent the average of three measurements

                    Total acidity   Free acidity
Honey type    pH      (mEq/kg)        (mEq/kg)

Acacia       4.05       45.8            17.8
Linden       4.06       46.7            19.6
Sunflower    4.01       42.9            18.5
Rape         4.02       45.3            20.1

             Lactone     Moisture
Honey type   (mEq/kg)   content (%)

Acacia         31.2        12.5
Linden         29.7        14.6
Sunflower      31.5        15.1
Rape           33.4        13.8

Table 2. The concentration of glucose, fructose and disaccharides
in the honey samples (g/100 g) and fructose/glucose ratio

                    Sugar (g/100g)

Honey type   Glucose   Fructose   Disaccharides

Acacia        33.5       38.7         11.2
Linden        37.8       34.9         15.7
Sunflower     32.6       35.9         16.8
Rape          34.5       39.1         14.9

                    Sugar (g/100g)

Honey type   Total sugar   Fructose/glucose

Acacia          83.4             1.15
Linden          88.4             0.92
Sunflower       85.3             1.11
Rape            88.5             1.13

Table 3. Analysis of amylase activity, expressed as diastase number
(Gothe unit), in various honey samples heated at different
temperatures for 5 min

                                     Gothe units

Honey type        Initial   50[degrees]C   65[degrees]C   70[degrees]C

Acacia honey       10.21       10.10           9.88           8.94
Linden honey       11.05       10.94          10.02           9.95
Sunflower honey    10.92       10.54          10.13          10.01
Rape honey         10.57       10.45          10.31          10.24

Table 4. HMF (mg/kg) in commercial and fresh honeys heated (5 min)
at 50, 65 and 70[degrees]C determined by HPLC method


Honey type        Initial   50[degrees]C   65[degrees]C   70[degrees]C

Acacia honey       51.5         52.2           53.4           55.6
Linden honey       73.2         73.9           74.5           75.1
Sunflower honey    20.1         20.9           21.2           21.5
Rape honey         34.2         34.8           35.1           35.3
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Author:Tudor, Laurentiu; Mitranescu, Elena; Galis, Anca Maria; Ceausi, Constantin
Publication:Economics, Management, and Financial Markets
Article Type:Report
Date:Mar 1, 2011
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