3. FOOD ChEMISTRy & bIOTEChNOLOGy 3.1. Lectures

Chem. Listy, 102, s265–s1311 (2008) Food Chemistry & Biotechnology
P66 ANALySIS OF ACTIVE SubSTANCES IN
hONEy – A CONTRIbuTION TO hONEy
AuThENTICITy
IVAnA MáROVá a , ZUZAnA JELénKOVá a ,
KATEřInA DUROňOVá a , RADKA KOČí a , PETRA
ŽDánSKá b and VEROnIKA EHREnBERGEROVá b
a Institute of Food Science and Biotechnology, Brno University
of Technology, Purkyňova 118, 612 00 Brno, Czech
Republic,
b The Secondary Technical School of Chemisty, Vranovská 65,
614 00 Brno, Czech Republic,
marova@fch.vutbr.cz
Introduction
Honey belongs to the oldest delicacies of natural origin.
Moreover, honey contains many biologically active substances
with positive effect on human health. This work was
focused on study of antioxidant content, enzyme activities,
protein and saccharide composition in 26 sorts of honey,
which differed in geographical and botanical origins. Several
qualitative as well as quantitative parameters were determined
with the aim to contribute to identification of these compounds
which could be considered as markers of the floral
origin of honey.
Material and Methods
26 samples of honey, one sample of propolis and one
sample of royal jelly were analyzed. Honey were harvested in
year 2006 and 2007 and bought in retail chain, special shops
and directly from bee-keeper. The study was focused on analysis
of antioxidants. Followed groups of antioxidants were
determined: flavonoids, catechins, carotenoids, vitamins E,
C, A. Authenticity of quality were determined by hydroxymethylfurfural
analysis.
A c t i v e S u b s t a n c e s A n a l y s i s
Total phenolics were analyzed colometrically with Folin
Ciocalteu reagent (750 nm). Total flavonoid content was analyzed
colometrically with nanO 2 + AlCl 3 (510 nm).Total
antioxidant capacity was measured by Randox kit.
Individual flavonoids were analyzed by RP- HPLC/
UV-VIS method. Using extrenal standards concentration
of ((-)catechin, catechin gallate, chlorogenic acid, epicatechin,
morin, quercetin, rutin was done. Samples (20 μl) were
injected into the RP-18 column (Biospher PSI 200 C18, 7 μm,
150 mm × 4.6 mm). Mobile phases were methanol/water
(55 : 45) for catechins and methanol/acetonitrile/water + 1%
phosporic acid (20 : 30 : 50) for flavonoid analysis. The flow
rate was maintained at 0.75 ml min –1 , analysis was performed
at 30 °C. Carotenoids (beta-carotene, lycopene, luteine) were
analyzed by RP-HPLC with spectrophotometric detection
using Hypersil C18, 5 μm, 250 mm × 4.6 mm column, isocratic
elution by methanol at the flow rate 1.1 ml min –1 and at
45 °C.
s720
Identifiaction of individual flavonoids and catechins was
performed by on-line LC/MS/ESI analysis (Mass spectrometer
LCQ Advantage Max). Optimalization of mass spectrometry
analysis in negative mode was done using chlorogenic
acid. Samples of beer were mixed with 5 times higher amount
of 2% HCl and extracted by SPE (Amid-2 column). Isocratic
elution was performed by mobile phase acetonitrol:1% acetic
acid 50 : 50 at flow rate 0.4 ml min –1 at 30 °C. Individual
components were detected in full scan module.
Ascorbic acid was determined usin RP-HPLC on Hypersil
APS-2, nH2, 5 μm, 150 mm × 4.6 mm column. Samples
were stabilized by 2% HPO 3 , 20 μl was injected. Mobile
phase was natrium acetate/acetonitrile (95 : 5). Analysis was
performed at flow rate 0.6 ml min –1 and 30 °C. Microtitration
metod with 2,6-dichlorindofenol was used as comparative
mthod too. The end point of titration was determined by pink
colour.
5 - h y d r o x y m e t h y l f u r f u r a l A s s a y
Concentration of 5-HMF was analyzed by RP- HPLC/
UV-VIS method using external calibration. Honey extracts
(20 μl) were injected into the RP-18 column (Kromasil C18,
7 μm, 150 × 4.6 mm). Isocratic elution was performed by
mobile acetonitrile/water + 1% acetin acid (3:97). The flow
rate was maintained at 1 ml min –1 , analysis was performed at
30 °C, UV detection was done at 284 nm.
S e n s o r y A n a l y s i s
A group of 21 respondents were enrolled into orientation
sensory study. They tested several fruit tea and evaluated
basic sensory parameters. The droup of respondents was divi-
ded into two age-different groups:
•
•
seniors: total 13, age 68.5 ± 7.16; 10 F/3 M,
juniors: total 8, age 27.13 ± 3.63; 6 F/2 M.
Additionally, consumer questionnaire was completed
by 35 student respondents. Preferences and consumption of
dried fruit and cereal bars were evaluated.
Results
Average values of total antioxidant capacity ranged
(12.75–137.49) mmol 100 g –1 . Average values of total phenolic
ranged (8.51–61.34) mg 100 g –1 and average values
of total flavonoids ranged (0.75–6.04) mg 100 g –1 . Honey
samples contained (41.83–585.10) µg 100 g –1 of rutin, (9.30–
313.40) µg 100 g –1 of myricetin, (6.5–171.90) µg 100 g –1
of luteolin, (3.19–436.37) µg 100 g –1 of quercetin, (2.10–
242.66) µg 100 g –1 of apigenin, (0.15–105.12) µg 100 g –1
of caempferol and (0.07–17.52) mg 100 g –1 of naringenin.
From group of catechins there were measured (5.98–
310) µg 100 g –1 of catechin, (17.77–486.29) µg 100 g –1 of
epicatechin, (0.18–64.90) µg 100 g –1 of catechin gallate
and (0.59–140.56) µg 100 g –1 of epicatechin gallate. From
lipophilic compounds the most abundant in honey samples
was tocopherol, its value ranged (29.20–8531.17) µg 100 g –1 .