Proline content of different honey types

Abstract

In this study we examined the proline content of 143 honey samples with different flower origin. Some of the samples originated from commercial trade, the others came directly from beekeepers. We measured the proline content of beekeepers' honeys and the received results were compared to the results of commercial honeys. The effects of added sugar products and heating on the quality parameters were also studied. Honey is defined by the EU (2002) as "The natural, sweet product produced by Apis Mellifera bees from the nectar of plants or from secretions of living plants, which bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in honeycombs to ripen and mature." Quality of honey depend on several parameters such as sugar-, moisture-, elements-, hidroxy-methyl-furfurol (HMF)-, enzyme-, organic acids-, vitamins-, etc. contents and ratio of glucose and fructose, pH, acidity, electrical conductivity, etc. (Zsidei, 1993; ACquaroNe et al., 2007). The main components of honey do not contain protein (Croft et al., 1986). This material comes from animal or vegetal (e.g. pollen) sources (Lee et al., 1985). The protein content of floral honey is about 1.0–1.5%, while in honeydew honey this quantity is about 3.0%. The amount of amino acids is 1.0%, and proline is the major component with 50–85% of the total amino acids (Pawlowska & ArMstroNg, 1994; ANklaM, 1998). The amino acid profile could give an indication of botanical origin of honey samples (ANklaM, 1998). Besides proline, the honey contains 26 amino acids and their amount depends on the origin of honey (nectar or honeydew). Proline content of honey constantly decreases during storage therefore the proline might be indicator of honey ripeness (VoN der Ohe et al., 1991). The minimum proline content was determined by EU (2002). For proline the 180 mg/kg minimum value is internationally accepted (HerMosíN et al., 2003). In sugar adulterated honeys several parameters such as proline and HMF content, electrical conductivity and enzyme activities are lowered. The proline was suggested as quality criterion for honey with respect to sugar adulteration (BogdaNov & MartiN, 2002). Honey is our essential food since ancient times. In Hungary about 20 000 tons of honey is produced each year and most of this product (about 80%) is exported to different countries in the world. Due to the quantity, Hungary plays an important part in honey production of the world. According to FAO Statistics, our country was the eighteenth higher producer in the world in 2008 and Hungary came in second among the European Union countries (Table 1).

Full text

0239–3006/$ 20.00 © 2011 Akadémiai Kiadó, Budapest
Acta Alimentaria, Vol. 41 (1), pp. 26–32 (2012)
DOI: 10.1556/AAlim.2011.0002
First published online 4 August 2011
PROLINE CONTENT OF DIFFERENT HONEY TYPES
N. Czipa*, M. BorBély and Z. Győri
Institute of Food Science, Quality Assurance and Microbiology
Faculty of Agricultural and Food Sciences and Environmental Management
Centre for Agricultural and Applied Economic Sciences
University of Debrecen, H-4032 Debrecen, Böszörményi út 138. Hungary
(Received: 4 June 2010; accepted: 7 February 2011)
In this study we examined the proline content of 143 honey samples with different ower origin. Some of the
samples originated from commercial trade, the others came directly from beekeepers. We measured the proline
content of beekeepers’ honeys and the received results were compared to the results of commercial honeys. The
effects of added sugar products and heating on the quality parameters were also studied.
Keywords: honey, proline, invert sugar, glucose syrup, saccharose syrup, heating
Honey is dened by the EU (2002) as “The natural, sweet product produced by Apis Mellifera
bees from the nectar of plants or from secretions of living plants, which bees collect, transform
by combining with specic substances of their own, deposit, dehydrate, store and leave in
honeycombs to ripen and mature.” Quality of honey depend on several parameters such as
sugar-, moisture-, elements-, hidroxy-methyl-furfurol (HMF)-, enzyme-, organic acids-,
vitamins-, etc. contents and ratio of glucose and fructose, pH, acidity, electrical conductivity,
etc. (Zsidei, 1993; ACquaroNe et al., 2007).
The main components of honey do not contain protein (Croft et al., 1986). This material
comes from animal or vegetal (e.g. pollen) sources (Lee et al., 1985). The protein content of
oral honey is about 1.0–1.5%, while in honeydew honey this quantity is about 3.0%. The
amount of amino acids is 1.0%, and proline is the major component with 50–85% of the total
amino acids (Pawlowska & ArMstroNg, 1994; ANklaM, 1998). The amino acid prole could
give an indication of botanical origin of honey samples (ANklaM, 1998). Besides proline, the
honey contains 26 amino acids and their amount depends on the origin of honey (nectar or
honeydew). Proline content of honey constantly decreases during storage therefore the
proline might be indicator of honey ripeness (VoN der Ohe et al., 1991). The minimum proline
content was determined by EU (2002). For proline the 180 mg/kg minimum value is
internationally accepted (HerMosíN et al., 2003).
In sugar adulterated honeys several parameters such as proline and HMF content,
electrical conductivity and enzyme activities are lowered. The proline was suggested as
quality criterion for honey with respect to sugar adulteration (BogdaNov & MartiN, 2002).
Honey is our essential food since ancient times. In Hungary about 20 000 tons of honey
is produced each year and most of this product (about 80%) is exported to different countries
in the world. Due to the quantity, Hungary plays an important part in honey production of the
world. According to FAO Statistics, our country was the eighteenth higher producer in the
world in 2008 and Hungary came in second among the European Union countries (Table 1).
* To whom correspondence should be adressed.
Phone: +36-30-366-2383; fax: +36-52-417-572; e-mail: czipa@agr.unideb.hu

27CZIPA et al.: PROLINE CONTENT OF HONEY
Acta Alimentaria 41, 2012
Table 1. Honey production in the European Union
Country Production (t) Country Production (t)
Spain 31250 Slovakia 4200
Hungary 22394 Sweden 3400
Romania 19833 Belgium 2150*
Greece 17960* Lithuania 1907
France 16000* Denmark 1500
Germany 15727 Finland 1500
Poland 14007 Slovenia 1480
Italy 12000* Latvia 688
Bulgaria 11377 Cyprus 630
United Kingdom 7200* Estonia 501
Portugal 6100 Ireland 200*
Czech Republic 6079 Luxembourg 112
Austria 5000* Netherlands No data
* FAO estimation
FAOSTAT / FAO Statistics Division (2010)
1. Materials and methods
1.1. Honey samples
More then 100 honey samples were examined. Most of these samples came from Hungarian
beekeepers (91 samples). Botanical origin of these samples are the following: acacia (19),
linden (13), rape (7), oral (23), fruit (5), sunower (5), milkweed, (5), chestnut (5), coriander
(3), wild garlic (other names: ramsons, bear’s garlic, etc.) (3), lavender (3) and honeydew (4).
Some of the honey samples were purchased in stores. Among the commercial samples 16
honeys were produced in Hungary and 32 samples came from different countries of the
world, e.g. Tasmania, New-Zealand, Malaysia, Thailand, South-Africa, Finland, etc.
1.2. Sample preparation
To examine the effect of added sugar products (invert sugar, glucose syrup and saccharose
syrup) on proline content of acacia and ower honeys, different sugar products were mixed
to honey samples in percentage of 10, 20 and 30% on the basis of total weight.
During the heating experiment the heating temperatures were 40, 50, 60, 80 and 100 °C,
the heating times were 2, 5, 10 and 20 min.
1.3. Analytical methods
The amino acid content of honey samples was determined according to the method of
A.O.A.C. 979.20 and Ough (Meda et al., 2005). The honey was diluted in 50 ml distilled water
and formic acid and ninhidrin solution was added to the mixture. After heating, aqueous iso-
propanol was added to honey solution and mixed. The absorbance was determined at 520 nm

28 CZIPA et al.: PROLINE CONTENT OF HONEY
Acta Alimentaria 41, 2012
against blank of distilled water with spectrophotometer (Spectronic® GenesysTM, Spectronic
Instruments, USA). The proline content was obtained by means of calibration curve.
1.4. Statistical methods
All analyses were carried out in triplicate. SPSS 13.0 statistical programme was used for
statistical analysis.
2. Results and discussion
The proline content of Hungarian honey samples is presented in Table 2. The lowest proline
concentration was measured in acacia honeys (252±38 mg/kg) and the highest amount in
coriander honey (2283±128 mg/kg) and honeydew honey (1089±137 mg/kg). The rape, wild
garlic, and asclepias honeys with value of 377±60 mg/kg, 476±27 mg/kg and 485±114 mg/
kg proline were following the acacia honeys. In the other honey types the proline content is
higher then 500 mg/kg. In ower honeys the proline content changed on a wide range because
in these honeys the nectar and pollen ratios are very different.
Table 2. Proline content of Hungarian honey types
Honey types N Mean
(mg/kg)
Standard
deviation
Minimum
(mg/kg)
Maximum
(mg/kg)
Acacia 19 252 38 186 312
Linden 13 697 248 406 1155
Rape 7 377 60 318 464
Flower 23 542 143 314 890
Fruit 5 694 128 524 855
Sunower 5 809 60 774 899
Asclepias 5 485 114 316 567
Chestnut 5 644 155 466 780
Coriander 3 2283 128 2148 2404
Lavender 3 537 108 445 692
Wild garlic 3 476 27 446 497
Honeydew 4 1089 137 932 1192
As there is no limit value for proline content of honey in Hungary, excluding the special
honey when this value is at least 200 mg/kg (Codex AliMeNtarius huNgariCus 2009) we used
the international value, that is minimum 180 mg/kg, for evaluation (HerMosíN et al., 2003).
In one rape, one sunower and one asclepias honey the measured concentration was below
limit so we omitted these values from the statistic assessment.
The measured proline concentrations of the commercial samples are shown in Table 3.
The proline content of commercial acacia honeys was higher than of the beekeepers’ acacia
honeys, except one honey sample where this value was 207 mg/kg. In ve samples the
measured value was over 400 mg/kg. The proline concentrations of the two linden honeys

29CZIPA et al.: PROLINE CONTENT OF HONEY
Acta Alimentaria 41, 2012
were very different. In case of Hungarian linden honey this value was 313 mg/kg that is too
low according to beekeepers’ honeys. Proline quantity of honeydew honeys were about 1000
mg/kg in beekeepers’ honeys, with the exception of one commercial sample (492 mg/kg).
The evaluation regarding commercial ower honeys was not possible because of the mixed
nectar origin.
Table 3. Proline content of commercial honeys
Honey type Collecting area Proline content
(mg/kg)
Honey type Collecting area Proline content
(mg/kg)
Acacia Hungary 375±7 Floral Hungary 501±14
Acacia Hungary 343±12 Floral Not EU 971±19
Acacia Hungary 429±8 Floral EU and not EU 601±9
Acacia Hungary 395±7 Floral EU and not EU 755±16
Acacia Hungary 397±16 Floral EU and not EU 778±15
Acacia Hungary 447±15 Floral Croatia 601±11
Acacia Hungary 207±8 Floral France 429±20
Acacia Not EU 416±7 Floral Greece 529±10
Acacia EU 457±11 Floral Turkey 453±13
Acacia Croatia 422±10 Floral EU and not EU 689±16
Linden Hungary 313±8 Floral EU and not EU 734±9
Linden EU 739±22 Honeydew Argentina 492±11
Floral Hungary 595±19 Honeydew Not EU 1841±23
Floral Hungary 705±23 Honeydew Greece 2513±28
One part of special honey samples originated from Hungary. In these samples high
proline concentrations were measured except for viper’s bugloss honey. The highest
concentration was measured in lemon tea tree honey but high values were received in
rosemary, vipers bugloss (from New-Zealand), and manuka honeys as well (Table 4).
Table 4. Proline content of special honey types
Honey type Collecting area Mean (mg/kg) Honey type Collecting area Mean (mg/kg)
Blue gum Australia 650±25 Alpine rose Deutschland 513±14
Lemon tea tree Australia 1131±58 Rosemary Spanish 896±20
Eucalyptus Australia 661±12 Sage Croatia 357±5
Latherwood Tasmania 487±11 Clover Finland 479±19
Manuka New-Zealand 809±36 Vipers bugloss Hungary 347±9
Tawari New-Zealand 717±9 Mustard Hungary 628±11
Vipers bugloss New-Zealand 848±22 Oleaster Hungary 601±16
Tropical jungle Malaysia 19±0 Sweet clover Hungary 569±12
Forest Thailand 470±9 Goldenrod Hungary 821±16
Lavender South-Africa 309±4 Golden gaioa Australia 512±40

30 CZIPA et al.: PROLINE CONTENT OF HONEY
Acta Alimentaria 41, 2012
Regarding Hungarian and New-Zealand’s vipers bugloss honeys, the proline content
was twice times higher in the Hungarian than in the New-Zealand’s sample. The same
situation was found in case of Hungarian and South-African lavender honeys, where the
Hungarian samples had a much higher proline content.
The inuence of different added sugar products (invert sugar, glucose syrup and
saccharose syrup) and the heating on the proline content was also investigated. Sugar products
were mixed to acacia honey in 10, 20 and 30% ratio. The results are shown in Fig. 1. The
original acacia honey contained 303.9 mg/kg proline. The proline content of glucose syrup
was high (213 mg/kg) that is similar to the mean proline concentration of acacia honeys.
Therefore the added glucose syrup had no effect on the proline content of acacia honeys. The
reduction was 6% (19 mg/kg) in case of 30% glucose syrup. The invert sugar and saccharose
syrup contained proline in lower quantities (47 and 28 mg/kg). Due to the lower values the
decreasing of proline content in their mixtures were notable, 21.4% (64 mg/kg) in case of
30% invert sugar and 29% (88 mg/kg) in case of 30% saccharose syrup.
304
281
258
239
285
291
298
272
216
235
150.0
170.0
190.0
210.0
230.0
250.0
270.0
290.0
310.0
330.0
0102
03
0
Concentration, %
Proline content, mg/kg
Fig. 1.
Fig. 1. Proline content of honey mixed with different sugar products ▲: Invert sugar; ●: glucose syrup;
■: saccharose syrup
These values still correspond to the standard, but when these sugar products are added
to, e.g. linden honey, the decrease of proline content is more obvious. When we added sugar
products to linden honey the decrease was 157 mg/kg, 206 mg/kg and 218 mg/kg for 30%
glucose syrup, 30% invert sugar and 30% saccharose syrup, respectively.
In honey samples with very low proline content (221 mg/kg) the proline concentration
became lower then 180 mg/kg when invert sugar and saccharose syrup was added.

31CZIPA et al.: PROLINE CONTENT OF HONEY
Acta Alimentaria 41, 2012
300
400
500
600
700
800
900
0 5 10 15 20
Time, min
Proline content, mg/kg
Fig. 2.
Fig. 2. Effect of heating on proline content of honey : 40 °C; : 50 °C; ▲: 60 °C; : 80 °C; : 100°C
For the heating experiment ower honey sample was kept at different temperatures and
the proline content was checked at different times. The results are shown in Fig. 2. The
proline content of the control sample was 832 mg/kg. The decreasing in proline content was
proportional with the temperature and time. The diminution of proline content was 30% at 40
°C, 38% at 50 °C, 42% at 60 °C, 44% at 80 °C and 50% at 100 °C. The highest change was
416 mg/kg in proline. In case of acacia honey with 217 mg/kg original proline content, the
decrease went to 93 mg/kg, which is far below the minimum value given by the regulation
(results are not presented here).
3. Conclusion
Among the examined beekeepers’ honey types, the acacia honey had the lowest proline
content and the value was highest in coriander and honeydew honeys. Acacia, rape, sunower
and coriander honeys can be clearly distinguished from other honey types by their proline
content. In case of other honeys we could not determine an evident limit because the values
of standard deviation are high. Much of the commercial acacia honeys contained more proline
than the beekeeper’s acacia honeys. The measured proline content was very low in one linden
and one honeydew honey. The proline concentration in case of these samples gives grounds
for distrust. The results are not absolute values because the colour of honey samples could
inuence the value of proline content. According to the literature others were facing the same
difculties.
We examined the moisture content and diastase activity of honey types. The moisture
results show that all samples correspond to regulation. The results of diastase activity proved
that the samples were not heated and these honeys were fresh.

32 CZIPA et al.: PROLINE CONTENT OF HONEY
Acta Alimentaria 41, 2012
The addition of different sugar products has variant effect on proline content of honeys.
The proline content decreased gradually with the increasing amount of added sugar products
in all examined honey types.
The proline content decreased also when heat treated. This change is directly proportional
to the increasing temperature and heating time. The highest decrease was measured at 100 °C
and in 20 min. The smallest change was obtained at 40 °C.
In several cases very high proline concentration was measured in honeys. There is a
suspicion of manipulation in these cases, as very high proline content never occurs naturally,
and was never detected in those samples that originated directly from beekeepers.
Unfortunately, there is no method to distinguish the added proline, yet.
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