Journal of Agricultural and Food Chemistry
ARTICLE
Figure 1. Structure of diglycoconjugated aroma precursors found in Vitis vinifera (common grape wine). R: The aglycon part is formed by terpenols,
terpenic polyols, norisoprenoids, volatile phenols, or phenolic acids.
main.php?menu_id=2).
The purified products were analyzed by ultraviolet matrix-assisted
laser desorptionÀionization mass spectrometry (UV-MALDI-MS) and
by ultraviolet laser desorptionÀionization mass spectrometry (UV-LDI-MS)
performed on the Bruker Ultraflex Daltonics TOF/TOF mass
spectrometer in positive and negative ion modes. For UV-MALDI-
MS, matrix solutions were prepared by dissolving nor-harmane and 2,5-
dihydrobenzoic acid (DHB) (2 mg/mL) in acetonitrile/water (1:1 [v/v])
solution. Analyte solutions were prepared with methanol (approximately
0.7 mg/mL). For UV-MALDI-MS experiments, the dry droplet sample
preparation or sandwich method was used according to Nonami et al.,15
loading successively 0.5 μL of matrix solution, analyte solution, and
matrix solution after drying each layer at normal atmosphere and room
temperature. For UV-LDI-MS experiments, two portions of analyte
solution (0.5 μL Â 2) were loaded on the probe, as two dry layers,
desorption/ionization was obtained by using a 355 nm solid laser. The
accelerating potential was 20 kV. External mass calibration was made
using β-cyclodextrin (MW 1134) with nor-harmane as the matrix in
positive and negative ion mode. The matrix mass was used as an
additional standard for calibration. Spectra were obtained and analyzed
with the programs FlexControl and FlexAnalysis, respectively.
α-Rhamnosyl-β-glucosidase Activity. For quantification of
α-rhamnosyl-β-glucosidase activity, each reaction (1 mL) contained
495 μL of substrate (0.11% w/v hesperidin in 50 mM sodium citrate
buffer, pH 5.0) and 5 μL of suitably diluted enzyme solution. The reaction
was performed for 60 min at 30 °C and stopped by adding 500 μL of 3,5-
dinitrosalicylic acid.13 The samples were incubated at 100 °C for 10 min,
and the concentration of reducing sugars was measured at 540 nm using
rutinose as standard. One enzyme unit was defined as the amount of
enzyme that released 1 μmol of rutinoside per minute.
Transglycosylation Reactions. The transglycosylation reactions
(500 μL) were performed using 1.8 mM hesperidin as the disaccharide
donor and 2% (v/v) aroma compounds as acceptors (linalool, geraniol,
nerol, and 2-phenylethanol). For the optimization of the acceptor
concentration, 0À60% (v/v) of 2-phenylethanol was used.
Hydrolysis of the Rutinoside Compounds. It was carried out
in presence of 12% (v/v) ethanol as the OH acceptor. Each reaction
(100 μL) contained 0.23 mM substrate products: neryl-, geranyl-, or
2-phenylethyl-rutinoside and 0.4 U/mL α-rhamnosyl-β-glucosidase in
50 mM sodium citrate buffer (pH 5.0) for 2 h (otherwise indicated in
text) in an orbital shaker (250 rev/min) at 30 °C.
’ RESULTS AND DISCUSSION
Analytical Assays. The products of enzymatic reaction were analyzed
by thin-layer chromatography (TLC). The reaction products (5 μL) were
loaded onto silica gel TLC 0.2 mm layer thickness with medium pore
diameter (60 Å) (Fluka Chemika GmbH, Switzerland). The mobile phase
was ethyl-acetate/2-propanol/water (3:2:2, [v/v]). The chromatograms
were stained using anthrone reagent according to the procedure described
for Witham et al.14 The blue complex produced for each glycosidic com-
pound was quantified as integrated optical density units using rutinose as the
standard. The TLC images were analyzed using the software ImageJ 1.38x
(National Institutes of Health, United States; http://rsb.info.nih.gov/ij/).
The 32-bit color images were split into red, green, and blue (RGB) com-
ponents. Images corresponding to the red component were chosen, due to
the highest signal-to-noise ratio, and integrated optical density units were
used for quantification of rutinose and rutinosylated compounds.
Synthesis of Aroma Precursors (Scale-up). Bench-scale synthe-
ses of rutinosides were performed in an agitated reactor (10 mL) contain-
ing 0.4 U/mL α-rhamnosyl-β-glucosidase, 1.8 mM hesperidin, and 10%
(v/v) aromatic compound (geraniol, nerol, or 2-phenylethanol) as the
OH acceptor in 50 mM sodium citrate buffer (pH 5.0) for 16 h at 30 °C.
The reaction was stopped by placing the samples in a water bath for
10 min at 100 °C.
Purification of Rutinosylated Compounds and UV-MALDI-
TOF/TOF MS and UV-LDI-TOF/TOF MS Analysis. The rutino-
sides were purified by solid-phase extraction using a C18 Reversed-
Phase cartridge (Varian Bond Elut LAC C18). Reaction mixtures
were loaded on the cartridge and washed with water (3 volumes).
Elution was performed with 60% (v/v) ethanol (3 volumes). The
alcohol content in samples containing the purified rutinosides was
reduced by evaporation in vacuum. After that, the samples were freeze-
dried and stored at 4 °C.
Rutinosides Syntheses. The enzyme α-rhamnosyl-β-gluco-
sidase was used to transfer the rutinose moiety from hesperidin
(sugar donor) to various alcoholic aroma acceptors in aqueous
medium (30 °C, 2À22 h). Linalool, nerol, geraniol, and 2-phe-
nylethanol were chosen on the basis of their abundance in grape.
The reaction products were analyzed by TLC and a new sugar
spot (Rf values ∼ 0.82À0.84) corresponding to the transglyco-
sylation product was detected for each acceptor except for the
tertiary alcohol, linalool (Figure 2a). These results are in agree-
ment with those reviewed by van Rantwijk et al.,6 who describe
absolute selectivity of glycosidase with regard to the stereochem-
istry at the anomeric center and show a high degree of chemos-
electivity for different hydroxyl groups in the order of reactivity:
primary > secondary alcohols > phenols, with tertiary alcohols
being unreactive. The transglycosylation reaction was higher than
the hydrolysis when 2-phenylethanol was utilized as the sugar
acceptor, with the transglycosylation/hydrolysis ratio being 6/1,
respectively. Figure 2B shows an scheme of the transglycosylation
reaction using 2-phenylethanol as the acceptor and the hydrolysis
of hesperidin by Acremonium sp. α-rhamnosyl-β-glucosidase.
This acceptor was selected to establish the reaction conditions
to acheive transglycosylation products up to a millimolar scale.
Acceptor Concentration and Time Course. The effect of
2-phenylethanol concentration (0À60% [v/v]) on transglyco-
sylation yield was studied using a fixed hesperidin concentration
(1.8 mM) (2 h, 30 °C). 2-Phenylethanol solubility in water is
around 2% (v/v), and as a consequence, the reaction mixture was
a monophasic system in the range of 0À1% (v/v) acceptor and a
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dx.doi.org/10.1021/jf202412e |J. Agric. Food Chem. 2011, 59, 11238–11243