Identification of oligosaccharides formed during stachyose hydrolysis by pectinex ultra SP-L

Article abstract of DOI:10.1021/jf900309x

The commercial enzyme preparation Pectinex Ultra SP-L containing fructosyltransferase activity was used to hydrolyze stachyose. During this reaction, besides the formation of mono-, di-, and trisaccharides (DP ₃), the presence of one pentasacch

Full text of DOI:10.1021/jf900309x

J. Agric. Food Chem. 2009, 57, 5007–5013 5007
DOI:10.1021/jf900309x
Identification of Oligosaccharides Formed during Stachyose
Hydrolysis by Pectinex Ultra SP-L
‡
§
ONTILLA,‡ NIEVES
C
ORZO,*,‡ AGUSTIN
O
LANO
,
AND
M
ARIA
L
UISA IMENO
J
´
A
NTONIA
M
‡Instituto de Fermentaciones Industriales and §Centro Quı
´
mica Organica Lora Tamayo, CSIC,
ꢀ
Juan de la Cierva 3, 28006 Madrid, Spain
The commercial enzyme preparation Pectinex Ultra SP-L containing fructosyltransferase activity
was used to hydrolyze stachyose. During this reaction, besides the formation of mono-, di-, and
trisaccharides (DP3), the presence of one pentasaccharide (DP5) and a new oligosaccharide (DP6)
has been detected by gas chromatography. DP5 and DP6 oligosaccharides were isolated and fully
characterized for the first time by an extensive nuclear magnetic resonance (NMR) study. Complete
structure elucidation and full proton and carbon assignments were carried out using 1D (1H, 13C)
and 2D (gCOSY, multiplicity-edited gHSQC, gHSQC-TOCSY, and gHMBC) NMR experiments. The
two oligosaccharides were shown to be stachyose-based structures; the pentasaccharide has a
fructose unit linked to the C-1 of the fructose end of stachyose, and the hexasaccharide has a
fructose unit linked to the C-1 of the fructose end of the pentasaccharide. The fructosyltransferase
activity present in Pectinex Ultra SP-L allows new uses of this commercial enzyme preparation in
the synthesis of oligosaccharides derived from R-galactosides.
KEYWORDS: Galactosides; stachyose; Pectinex-Ultra SP-L; NMR
INTRODUCTION
length, and linkage types. Therefore, the synthesis of new oligo-
saccharides can contribute to the development of prebiotic
compounds with complementary properties. Enzyme-catalyzed
transglycosylation has been widely used to increase the prebiotic
oligosaccharide groups mainly through the synthesis of galactoo-
ligosaccharides (GOS) by transgalactosylation during lactose
hydrolysis using β-galactosidases from different sources
(11-13) and fructooligosaccharides from sucrose by transfructo-
sylation (14-16).
R-Galactosides are oligosaccharides abundant in grain le-
gumes such as soy, lupin, and chickpea. Although their consump-
tion is associated with the production of flatulence, they might be
used as a prebiotic growth substrate for intestinal bacteria
because they pass undigested to the colon due to the absence of
R-galactosidase among human endogenous enzymes, and they
are therefore available for a few bacteria that are known to have
high R-galactosidase activity (1-5). Several studies provide con-
vincing evidence that R-galactosides have beneficial effects on the
survival of different bifidobacteria strains (6-9). In vitro assays
have shown the ability of Lactobacillus reuteri (4 ) and Bifido-
bacterium adolescentis (5 ) to grow when R-galactosides were used
as the sole carbon source.
Prebiotic carbohydrates may be fermented in different parts of
the digestive tract. Rapidly fermented prebiotics stimulate the
production of bifidobacteria in the proximal colon, whereas
slowly fermented prebiotics can maintain fermentation all the
way to the distal end of the large intestine. In addition to the
health benefits in the colon, other parts of the gastrointestinal
tract are considered to be potential prebiotic targets, and new
prebiotics could be designed to promote the health of the oral
cavity and of the small intestine. Also, the urogenital tract can be
considered to be a new potential target for prebiotics as it harbors
a diverse microbiota susceptible to disturbance (10). The rate at
which oligosaccharides are fermented in the gut depends on,
among other factors, the monosaccharide composition, chain
The tetrasaccharide stachyose and the trisaccharide raffinose
are the main R-galactosides in legume seeds. They are derived
from sucrose by successive condensations of R-D-galactose units
attached by R-1,6 linkages to the glucose moiety. Because these
carbohydrates contain a sucrose residue, they may be used as
both donor and acceptor substrates for transfructosylation.
Purified fructosyltransferase from Aspergillus niger has been used
to catalyze the synthesis of new oligosaccharides from raffinose
(17). Glycosidases usually cleave glycosidic bonds, but can also
be used in a reverse mode for the formation of glycosidic bonds;
they are inexpensive compared to transglycosidases and do not
need complex expensive substrates, like sugar nucleotides (18).
The relatively inexpensive commercial enzyme preparation Pec-
tinex Ultra SP-L, produced by Aspergillus aculeatus and com-
monly used in fruit juice processing to reduce viscosity, was
shown to contain high fructosyltransferase activity (14, 19, 20);
therefore, it could be used as a catalyst in the large-scale produc-
tion of prebiotic oligosaccharides. The present study was carried
out to examine the possibility of using the commercial prepara-
tion Pectinex-Ultra SP-L for the synthesis of fructooligosacchar-
ides derived from stachyose.
*Author to whom correspondence should be addressed (telephone:
+ 34 91 258 74 85; fax: + 34 91 564 48 53; e-mail: ncorzo@ifi.csic.es).
© 2009 American Chemical Society
Published on Web 04/27/2009
pubs.acs.org/JAFC

5008 J. Agric. Food Chem., Vol. 57, No. 11, 2009
Montilla et al.
MATERIALS AND METHODS
with CP-SIL 5CB (methyl silicone from Chrompack, Middelburg,
The Netherlands). The carrier gas (nitrogen) flow rate was 1.1 mL/min.
The injector and detector temperatures were 280 and 340 °C, respectively.
The oven temperature was programmed from 150 to 165 °C at 3 °C/min
and then at 5 °C/min to 340 °C, keeping this temperature for 10 min.
Injections were made in the split mode (1:10). Data acquisition and
integration were done using HP ChemStation software (Hewlett-Packard,
Wilmington, DE).
Materials.
D-Galactose, D-glucose, D-fructose, sucrose, melibiose,
raffinose, stachyose, and phenyl-β-glucoside were purchased from Sigma
(St. Louis, MO). The commercial enzyme preparation Pectinex Ultra
SP-L,
A. aculeatus, was
a
soluble preparation of fructosyltransferase produced by
generous gift from Novozymes (Dittingen,
a
Switzerland) and used as received without further purification.
Enzyme Characterization. The fructosidase and fructosyltransferase
activities of Pectinex Ultra SP-L were measured using sucrose as substrate.
The hydrolysis of sucrose (300 g/L) was assayed at 60 °C in sodium acetate
solution (0.1 M, pH 5.5). Samples of 0.1 mL were withdrawn at different
times. The reaction was stopped by adding 10 μL of 0.1 N acetic acid. The
amounts of fructose and glucose formed were determined by GC using the
method described below. The fructosidase activity of Pectinex Ultra SP-L
was expressed as the amount of free glucose, and its fructosyltransferase
activity was expressed as the difference between the amount of released
glucose and fructose; this indicates the amount of fructose involved in the
transfructosylation reaction. Enzyme activity measurements were re-
peated three times, and the experimental error was <5%.
Pectinex Ultra SP-L expressed a fructosidase activity of 450 units,
where 1 unit is definedas the amount of enzyme releasing 1 μmol of glucose
per minute per milliliter under the assayed conditions. The fructosyltrans-
ferase activity was 400 units, where 1 unit is defined as the amount of
enzyme joining 1 μmol of fructose per minute per milliliter at other
molecules under the assayed conditions.
The TMS-oximes were formed following the method of Brobst and
Lott (24). First, a quantity of sample corresponding to approximately
4 mg of sugars was added to 0.4 mL of internal standard (I.S.) solution
(0.5 mg/mL phenyl-β-glucoside). Afterward, the mixture was dried at
::
38-40 °C in a rotary evaporator (Buchi Labortechnik AG, Flawil,
Switzerland). TMS-sugar oxime derivatives were formed by adding
200 μL of hydroxylamine chloride (2.5%) in pyridine and heating the
mixture at 75 °C for 30 min and then silylated with hexamethyldisilazane
(200 μL) and trifluoroacetic acid (20 μL) and kept at 45 °C for 30 min.
Reaction mixtures were centrifuged at 7000g for 5 min at 5 °C (25).
Supernatants were injected in the GC or stored at 4 °C prior to analysis.
Stock standard solutions of each carbohydrate (galactose, glucose,
fructose, sucrose, melibiose, raffinose, and stachyose) were prepared by
dissolution in water/methanol (50:50, v/v) of an appropriate amount of
carbohydrate. Then, several working standard solutions were prepared at
different concentrations by various mixtures of stock standard solutions.
Response factors (RF) were calculated using phenyl-β-glucoside as inter-
nal standard. Analysis of solutions was performed in triplicate.
The protein concentration in the enzyme preparation was determined
using the bicinchoninic acid (BCA) assay (21). Bovine serum albumin
(BSA) was used as standard. Specific activity (units/mg) of the enzyme was
calculated from the relationship of the activity units (units/mL) over
protein concentration (mg/mL). The protein content in the enzyme
preparation was 82 mg/mL. Therefore, the enzyme expressed a fructosi-
dase specific activity of 5.5 units/mg and a fructosyltransferase specific
activity of 4.9 units/mg.
The repeatability of the GC method was calculated using a standard
solution prepared, derivatized, and analyzed five times on the same day.
Reproducibility of the analytical method was calculated using a standard
solution prepared, derivatized, and analyzed one time on five days. The
relative standard deviation (RSD) was <8%.
Structural Characterization. Acid Hydrolysis. To identify the
monosaccharide residues that constitute the isolated oligosaccharides,
1 mg of each sample was dissolved in 1 mL of 1 N HCl and then heated in a
hot-air oven in sealed tubes at 100 °C for 1 h (26 ). The reaction product
was dried at 38-40 °C in a rotary evaporator and then converted to the
TMS derivatives for GC analysis.
Enzymatic Synthesis of r-Galactosides. Enzymatic syntheses of
modified R-galactosides from stachyose (600 g/L) using 34 units/mL of
commercial enzyme preparations Pectinex Ultra-SP-L in 0.1 M sodium
acetate at pH 5.5 and a temperature of 60 °C were carried out following the
method of Ghazi et al. (20). Samples were withdrawn from the reaction
mixture at different times (0.5, 1, 3, 6, and 24 h). Reactions were carried out
in duplicate with individual tubes of 2 mL, incubated in an orbital shaker
at 300 rpm, and stopped by heating at 100 °C for 5 min. The samples were
stored at -18 °C for subsequent analysis.
Mass Spectrometry (MS) Analysis. The molecular mass of the
purified compounds was determined by MS using a quadrupole HP
1100 mass detector in the electrospray positive mode (API-ES). The mass
spectrometer was operating with a 4000 V needle potential, 330 °C gas
temperature drying gas flow of 10 mL/min, and 40 psi nebulizer pressure.
Scan m/z was from 100 to 1500.
The amount of stachyose remaining and the yield of R-galactosides
were expressed as percentage by weight of the total carbohydrate content
in the reaction mixtures.
Nuclear Magnetic Resonance (NMR) Analysis. NMR spec-
tra were recorded at 303 K, using D2O as the solvent, on a Varian System
500 NMR spectrometer equipped with a 5 mm HCN cold probe. 1H
chemical shifts were referenced to the residual D2O signal at δH
4.72 relative to TMS. 13C chemical shifts were referenced using dioxane
as an internal reference (δC 66.6 relative to external TMS in water). 1H
spectra were obtained using presaturation solvent suppression. gCOSY
spectra were carried out with the following parameters: a delay time of
1.3 s, a spectral width of 1822.2 Hz in both dimensions, 4096 complex
points in t2 and 4 transients for each of 256 time increments, and linear
prediction to 512. The data were zero-filled to 4096 ꢀ 4096 real points.
2D [1H-13C] NMR experiments (multiplicity-edited gHSQC, gHSQC-
TOCSY, and gHMBC) used the same 1H spectral window, a 13C spectral
window of 7353 Hz, 1 s of relaxation delay, 1024 data points, and 256 time
increments, with linear prediction to 512. The data were zero-filled to
2048 ꢀ 1024 real points. Typical numbers of transients per increment were
4, 8, and 16, respectively. For gHSQC-TOCSY spectra a mixing time of
80 ms was used. Multiplicity-edited gHSQC and gHMBC spectra were
optimized for coupling constants of 145 and 8 Hz, respectively.
r-Galactosides Purification. Purification was performed following
the method described by Morales et al. (22). In brief, a total of 2.5 mL
of reaction mixture containing 1.5 g of carbohydrates was dissolved in
300 mL of water and stirred for 30 min with 9 g of activated charcoal
Darco G60, 100 mesh (Sigma) to remove mono- and disaccharides. This
mixture was filtered through Whatman no. 1 filter paper under vacuum,
and the activated charcoal was washed with 50 mL of water. The
oligosaccharides adsorbed onto the activated charcoal were then extracted
by stirring the mixture with 300 mL of a 50:50 (v/v) ethanol/water solution.
Activated charcoal was eliminated by filtering, and the solution was
evaporated under vacuum at 30 °C. The solid residue was dissolved in
5 mL of deionized water, filtered through 0.22 μm filters (Millipore Corp.,
Bedford, MA), and purified by semipreparative high-performance liquid
chromatography (HPLC-RI) using a refractive index detector RID-
10 (Shimadzu) and a Kromasil 100 NH2 5 μm 25 ꢀ 1.0 cm, column
(Tecknocroma, Spain). The mobile phase was acetonitrile/water
(70:30, v/v) at a flow rate of 3 mL/min. Fractions corresponding to the
main peaks were pooled and freeze-dried for mass spectrometry (MS) and
NMR analysis.
RESULTS AND DISCUSSION
Figure 1 shows the GC profiles of TMS-oxime derivatives of
carbohydrates formed during stachyose hydrolysis by Pectinex
Ultra SP-L after 3 h of reaction at pH 5.5, 60 °C, 600 mg/mL
of stachyose, and 34 units/mL of enzyme. Besides stachyose
(peak 6), compounds with retention times of fructose (peak 1),
glucose (peak 2), galactose (peak 3), melibiose (peak 4), and
Gas Chromatographic (GC) Analysis. Trimethylsilylated oximes
(TMS-oxime) of mono-, di-, and R-galactosides were analyzed by GC
following the method of Montilla et al. (23). Chromatographic analysis
was performed on a Varian 3800G (Harbor City, CA) gas chromatograph
equipped with a flame ionization detector (FID). Separation was carried
out in a 8 m ꢀ 0.25 mm ꢀ 0.25 μm film fused silica capillary column coated

Article
J. Agric. Food Chem., Vol. 57, No. 11, 2009 5009
Figure 1. Gas chromatographic profile of the TMS oximes of oligosaccharides formed during enzymatic hydrolysis of stachyose (600 mg/mL) with Pectinex
Ultra SP-L(34 units/mL) after3 h at60 °C inacetatebuffer (0.1 M, pH 5.5). Peaks: 1, fructose; 2, glucose; 3, galactose; 4, melibiose; 5, raffinose; DP3 (aand b);
6, stachyose; DP5; DP6; internal standard (I.S., phenyl-β-glucoside).
raffinose (peak 5), indicators of fructosidase and galactosidase
activities, were observed. Oligosaccharides with retention times
corresponding to trisaccharides, DP3 (a and b), and higher than
tetrasaccharides, DP5 and DP6, were also detected. Peak DP3 was
tentatively assigned as 60 galactosyl melibiose (R-
D
-Galp-(1f6)-
R-D
-Galp-(1f6)-R-
D-Glcp) [isomers of the TMS oximes syn
(a) and anti (b)]; peak DP5 was assigned as 1F fructofuranosyl
stachyose, the presence of which has been previously observed in
stachyose hydrolysates (17), and DP6 corresponded to a un-
known peak.
Mass spectrometry analysis of pure oligosaccharides gave two
peaks at m/z 527.5 (M + Na) and 543.3 (M + K) for DP3,
corresponding to trisaccharide, a peak at m/z 853.5 (M + Na),
corresponding to DP5 and a peak at m/z 1015.8 (M + Na)
corresponding to a DP6. Besides, acid hydrolysis of DP3, DP5,
and DP6 yielded glucose and galactose (1:2); fructose, glucose,
and galactose (2:1:2); and fructose, glucose, and galactose (3:1:2),
respectively.
The hydrolysis of stachyose and formation of oligosaccharides
DP3, DP5, and DP6 are shown in Figure 2. Under the assay
conditions used, 80% of initial stachyose was hydrolyzed after
6 h. The formation of DP3 increased with time, reaching a
maximum of 58.68% (based on total carbohydrates) after 24 h.
At this time 90.76% of stachyose was hydrolyzed. With regard to
DP5 the maximum formation (20.05%) was observed after 1 h at
45.18% stachyose hydrolysis. At prolonged reaction times a
decrease of DP5 was observed, probably contributing to the
DP3 formation. DP6 content increased at a slower rate, reaching
a maximum of 3.83% after 3 h and 72.74% of stachyose of
hydrolysis.
Uhm et al. (17) proposed the formation of DP2, DP4, and DP5
and DP3 and DP5 during enzymatic hydrolysis of raffinose and
stachyose, respectively, catalyzed by fructosyltransferase from
A. niger. However, they did not found oligosaccharides with a
polymerization degree of higher than 5, and only a DP4 com-
pound was characterized by NMR. They suggested that the
maximum chain length of five could fill the active sites of the
enzyme. Because Pectinex Ultra SP-L is an enzyme preparation
obtained from A. aculeatus, the observed differences could be due
Figure 2. Remaining stachyose content (a) and DP3, DP5, and DP6 formation
(b) during the time course of the reaction performed at 60 °C, initial stachyose
concentration of 600 mg/mL, 34 units/mL of enzyme, and pH 5.5.

5010 J. Agric. Food Chem., Vol. 57, No. 11, 2009
Table 1. 1H and 13C NMR Chemical Shift Values for Oligosaccharides Stachyose, DP3, DP5, and DP6
Montilla et al.
terminal galactose
δH δC
internal galactose
glucose
δC
fructose
δC
oligosaccharide position
δH
δC
δH
δH
δC
δH
δH
δC
stachyose29
1
4.99 100.82
4.98
101.15
5.42
94.88
3.66
3.66
64.23
2
3
4
5
6
3.82 71.06
3.85 72.29
3.98 72.01
4.01 73.76
3.74 63.91
3.74
3.83
3.90
4.03
4.13
3.73
3.87
71.21
72.15
72.11
71.58
69.27
3.56
3.75
3.52
4.05
3.65
4.03
73.72
75.50
72.27
74.06
68.66
106.58
79.16
76.79
84.12
65.23
4.22
4.06
3.88
3.76
3.81
DP3
1
2
3
4
5
6
4.98 100.82
4.98 100.77
3.82 71.04
4.98
4.98
3.83
100.88
100.81
71.21
71.19
72.19
72.19
72.14
72.11
71.59
71.53
69.35
5.23a
4.66b
3.54
3.25
3.71
3.48
3.48
3.49
4.00
3.64
3.69
4.00
3.74
3.96
94.95
98.84
74.20
76.83
75.78
78.74
72.48
72.31
72.70
76.95
68.62
3.84 72.25
3.97 72.01
3.99 73.75
3.88
3.90
4.02
4.15
4.17
3.72
3.86
3.72
3.86
3.74 63.93
3.74
69.24
68.51
95.16
DP5
1
4.99 100.82
4.98
101.15
5.44
3.70
3.81
63.78
3.67
3.73
63.24
2
3
4
5
6
3.82 71.06
3.84 72.28
3.98 72.01
3.99 73.76
3.74 63.91
3.74
3.83
3.90
4.03
4.13
3.72
3.86
71.22
72.15
72.12
71.58
69.28
3.55
3.74
3.53
4.04
3.66
4.04
73.76
75.53
72.24
74.09
68.57
106.14
79.39
76.65
84.00
65.05
106.49
79.43
77.27
83.90
65.15
4.27
4.05
3.88
3.74
3.76
4.19
4.08
3.86
3.76
3.82
DP6
1
4.98 100.83
4.98
101.16
5.44
95.16
3.72
3.82
63.91
3.71
3.85
63.66
3.67
3.74
63.19
2
3
4
5
6
3.82 71.06
3.84 72.28
3.97 72.01
4.01 73.76
3.73 63.91
3.73
3.83
3.90
4.03
4.13
3.72
3.86
71.23
72.16
72.12
71.59
69.29
73.80
75.53
72.22
74.11
68.56
106.10
79.46
76.66
84.02
65.06
105.83
80.26
77.25
83.85
65.02
106.43
79.54
77.13
83.85
65.01
3.74
3.54
4.04
3.65
4.04
4.27
4.05
3.87
3.75
3.81
4.21
4.06
3.85
3.75
3.83
4.27
4.09
3.85
3.75
3.83
a J(H1,H2) = 3.8 Hz. b J(H1,H2) = 8.1 Hz.
not only to operating conditions but also to the specificity of the
enzymes. Recently, Ghazi et al. (27) purified and characterized a
fructosyltransferase present in Pectinex Ultra SP-L and observed
that stachyose was not a substrate of the enzyme. They indicated
that the specific activity diminished with longer oligosaccharides
and suggested limitations for binding of large molecules.
Structural Characterization. Unequivocal structural elucida-
tion of purified products was carried out by the combined use of
1D and 2D [1H, 1H] and [1H-13C] NMR experiments (gCOSY,
gHSQC-TOCSY, multiplicity-edited gHSQC and gHMBC). 1H
and 13C NMR assignments for stachyose, DP3, DP5, and DP6 are
given in Table 1. A full set of spectra are collected in the
Supporting Information.
(C-2 of the fructose residue at 106.58 ppm), 18 methine carbons,
and 5 methylene carbons (69.27, 68.66, 65.23, 64.23, and
63.91 ppm) were identified. Taking anomeric protons and car-
bons as a starting point, gCOSY and gHSQC-TOCSY experi-
ments allowed us to assign the signals belonging to each sugar
residue. Finally, for sequential assignment of the sugar residues,
a gHMBC experiment was used. Correlations between the
anomeric proton of the terminal galactose (4.99 ppm) and
the C6 carbon of the internal galactose (69.27 ppm), between
the anomeric carbon of the internal galactose (101.15 ppm)
and the methylene protons of glucose (4.03 and 3.65 ppm), and
finally between the C-2 of the fructose residue (106.58 ppm) and
the anomeric proton ofthe glucose unit (5.42 ppm), completed the
full assignment (see Figures 3 and 5B). Carbon and proton
chemical shifts values found were very similar to those reported
by McIntyre and Vogel (28).
1
The H NMR spectrum of tetrasaccharide stachyose showed
three doublets in the anomeric region (see Figure S7 of the
Supporting Information). Besides, in the anomeric region of
the 13C spectrum, four signals at 100.82, 101.15, 94.88, and
106.58 ppm were observed. A multiplicity-edited gHSQC spec-
trum was used to link the carbon signals to the corresponding
proton resonances. From this experiment, a quaternary carbon
For DP3 two sets of resonances were observed in the NMR
spectra, indicating the presence of two compounds. For the major
component, inspection of the anomeric regions of both 1H
and 13C 1D spectra showed the presence of three anomers.

Article
J. Agric. Food Chem., Vol. 57, No. 11, 2009 5011
Figure 3. Structures of compounds stachyose, DP3, DP5, and DP6. Relevant interglycosidic gHMBC NMR correlations are indicated by arrows.
Therefore, taking the anomeric protons as starting point, two
residues of R-galactose and one residue of R-glucose [J(H1,H2) =
3.8 Hz] were identified from gHSQC-TOCSY and gCOSY
spectra. Finally, the observed gHMBC correlations between the
anomeric proton of the terminal galactose (4.98 ppm) and the C6
carbon of the internal galactose (69.35 ppm) and between the
anomeric carbon of the internal galactose (100.88 ppm) and the
methylene protons of glucose (4.00 and 3.69 ppm) completed
the full assignment (see Figures 3 and 5A), so the major com-
This compound was identified as R-
D
-Galp-(1f6)-R-D-Galp-
(1f6)-β- -Glcp.
D
The 1H NMR spectrum of DP5 showed three doublets in the
anomeric region. Besides, in the anomeric region of the 13C
spectrum, five resonances at 106.49, 106.14, 101.15, 100.82, and
94.88 were observed. From multiplicity-edited gHSQC, 2 qua-
ternary (106.49 and 106.14 ppm), 21 methine, and 7 methylene
carbons (69.28, 68.57, 65.15, 65.05, 63.91, 63.78, and 63.24 ppm)
were found. The combined use of gCOSY, gHSQC-TOCSY, and
multiplicity-edited gHSQC and gHMBC spectra revealed the
presence of two galactose rings, a glucose ring, and two fructose
residues. Therefore, all signals belonging to each sugar residue
resonances of pentasaccharide were assigned. Interglycosidic
pound was identified as galactosyl melibiose (R-
D
-Galp-(1f6)-R-
D
-Galp-(1f6)-R- -Glcp). By using the same procedure for
D
the minor component, two residues of R-galactose and one
residue of β-glucose [J(H1,H2) = 8.1 Hz] were determined.

5012 J. Agric. Food Chem., Vol. 57, No. 11, 2009
Montilla et al.
Figure 4. NMR spectra of DP3, stachyose, DP5, and DP6 oligosaccharides: (A) 500 MHz 1H spectra; (B) 125 MHz proton-decoupled 13C spectra.
Figure 5. gHMBC NMR spectra of (A) DP3, (B) stachyose, (C) DP5, and (D) DP6. Boxed cross-peaks are due to relevant interglycosidic correlations.
linkages were established from the gHMBC spectrum and by
comparison with the completely assigned 1H and 13C spectrum of
stachyose. Interresidual correlations were observed between the
anomeric proton of the terminal galactose (4.99 ppm) and the C6
carbon of the internal galactose (69.28 ppm), between the anome-
ric carbon of the internal galactose (101.15 ppm) and the methy-
lene protons of glucose (4.04 and 3.66 ppm), between the C-2 of the
internal fructose (106.14 ppm) and the glucose anomeric proton
(5.44 ppm), and finally between the C-2 of the terminal fructose
residue (106.49 ppm) and methylene C1 protons of the internal
fructose (3.81 and 3.70 ppm) (see Figures 3 and 5C). From these
results and by comparison with stachyose spectra (see Figure 4),
the structure of DP5 was established as (R-
D
-Galp-(1f6)-R-
D-
Galp-(1f6)-R-
D
-Glcp-(1f2)-β- -Fruf-(1f2)-β-
D
D-Fruf ).
The 1H NMR spectrum of DP6 showed only three doublets in
the anomeric region, whereas six resonances at 106.43, 106.10,
105.83, 101.16, 100.83, and 95.16 ppm were observed in the
anomeric region of the corresponding 13C spectrum. From the
multiplicity-edited gHSQC experiment, 3 quaternary (106.43,
106.10, and 105.83 ppm), 24 methine, and 9 methylene carbons

Article
J. Agric. Food Chem., Vol. 57, No. 11, 2009 5013
were identified. 2D gCOSY, gHSQC-TOCSY, multiplicity-edited
gHSQC and gHMBC NMR spectra revealed the existence of two
galactose rings, a glucose ring, and three fructose residues. Finally,
by the observation of relevant gHMBC correlations (see Figures 3
and 5D) and comparison with 1H and 13C NMR spectra of DP5
(see Figures 4 and 5D), the structure of DP6 was established as the
(11) Matsumoto, K.; Kobayashi, Y.; Ueyama, S.; Watanabe, T.; Tanaka,
R.; Kan, T.; Kuroda, A.; Sumihara, Y. Galactooligosaccharides. In
Oligosaccharides. Production, Properties, and Applications; Nakakuki,
T., Ed.; Japanese Technology Reviews; Gordon and Breach: Tokyo,
Japan, 1993; Vol. 3, pp 90-106.
(12) Mahoney, R. R. Galactosyl-oligosaccharide formation during lac-
tose hydrolysis: a review. Food Chem. 1998, 63 (2), 147–154.
(13) Martinez-Villaluenga, C.; Cardelle-Cobas, A.; Corzo, N.; Olano, A.;
Villamiel, M. Optimization of conditions for galactooligosaccharide
synthesis during lactose hydrolysis by a β-galactosidase from Kluy-
veromyces lactis (Lactozym 3000 L HP G). Food Chem. 2008, 107,
258–264.
hexasaccharide
(1f2)-β- -Fruf-(1f2)-β-
In this work, the formation and characterization of a new
(R-
D
-Galp-(1f6)-R-
D
-Galp-(1f6)-R-
D-Glcp-
D
D
-Fruf-(1f2)-β-
D-Fruf).
oligosaccharide (DP6) R-
Glcp-(1f2)-β- -Fruf-(1f2)-β-
for the first time characterization of pentasaccharide (DP5) R-
Galp-(1f6)-R- -Galp-(1f6)-R- -Glcp-(1f2)-β-
-Fruf, found during stachyose hydrolysis byfructosyltransferase
D
-Galp-(1f6)-R-
D
-Galp-(1f6)-R-
D
-
D
D
-Fruf-(1f2)-β-
D
-Fruf as well as
(14) Hang, Y. D.; Woodams, E. E. Optimization of enzyme production of
fructo-oligosaccharides from sucrose. Lebensm. Wiss. Technol. 1996,
29, 578–580.
D-
D
D
D-Fruf-(1f2)-β-
D
(15) Yun, J. W. Fructooligosaccharides;occurrence, preparation, and
application. Enzyme Microb. Technol. 1996, 19, 107–117.
(16) Sangeetha, P. T.; Ramesh, M. N.; Prapulla, S. G. Recent trends in
the microbial production, analysis and application of fructooligo-
saccharides. Trends Food Sci. Technol. 2005, 16, 442–457.
(17) Uhm, T. B.; Baek, N. I.; Jhon, D. Y.; Kim, D. M.; Hwang, K. T.;
Ryu, E. J. Synthesis the new oligosaccharides from raffinose by
Aspergillus niger fructosyltransferase. Biotechnol. Tech. 1999, 13,
169–171.
activity from Pectinex Ultra SP-L, have been described. These
results provide confirmatory evidence of the fructosyltransferase
activity of Pectinex Ultra SP-L toward stachyose and there-
fore show that R-galactosides seems to be a promising raw
material for the synthesis of new oligosaccharides by the use this
commercial enzyme preparation.
ACKNOWLEDGMENT
(18) Smaali, M. I.; Michaud, N.; Marzouki, N.; Legoy, M. D.; Maugard,
T. Comparison of two β-glucosidases for the enzymatic synthesis of
β-(1-6)-β-(1-3)-gluco-oligosaccharides. Biotechnol. Lett. 2004, 26
(8), 675–679.
(19) Tanriseven, A.; Aslan, Y. Immobilization of Pectinex Ultra SP-L to
produce fructooligosaccharides. Enzyme Microb. Technol. 2005, 36,
550–554.
We thank Ramiro Martınez (Novozymes A/S, Spain) for
´
providing us with Pectinex Ultra SP-L.
Supporting Information Available: Additional figures. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(20) Ghazi, I.; Fernandez-Arrojo, L.; Gomez de Segura, A.; Alcalde, M.;
Plou, F. J.; Ballesteros, A. Beet sugar syrup and molasses as low-cost
feedstock for the enzymatic production of fructo-oligosaccharides. J.
Agric. Food Chem. 2006, 54, 2964–2968.
(21) Smith, P. K.; Krohn, R. I.; Hermanson, G. T.; Mallia, A. K.;
Gartner, F. H.; Provenzano, M. D.; Fujimoto, E. K.; Goeke, N.
M.; Olson, B. J.; Klenk, D. C. Measurement of protein using
bicinchoninic acid. Anal. Biochem. 1985, 150, 76–85.
(22) Morales, V.; Sanz, M. L.; Olano, A.; Corzo, N. Rapid separation on
activated charcoal of high oligosaccharides in honey. Chromatogra-
phia 2006, 64 (3-4), 233–238.
(23) Montilla, A.; van de Lagemaat, J.; Olano, A.; del Castillo
M. D. Determination of oligosaccharides by conventional high-
resolution gas chromatography. Chromatographia 2006, 63 (9-10),
453–458.
LITERATURE CITED
(1) Sakai, K.; Takashi, T.; Kumagai, H.; Tochikura, T. Hydrolysis of
R-
D-galactosyl oligosaccharides in soymilk by R-D-galactosidase of
Bifidobacterium breve 203. Agric. Biol. Chem. 1987, 51 (2), 315–322.
(2) Van Laere, K. M. J.; Hartemink, R.; Beldman, G.; Pitson, S.;
Dijkema, C.; Schols, H. A.; Voragen, A. G. J. Transglycosidase
activity of Bifidobacterium adolescentis DSM 20083 R-galactosidase.
Appl. Biochem. Biotechnol. 1999, 52, 681–688.
(3) Rubio, M. C.; Runco, R.; Navarro, A. R. Invertase from a strain of
Rhodotorula glutinis. Phytochemistry 2002, 61 (6), 605–609.
(4) Tzortzis, G.; Jay, A. J.; Baillon, M. L. A.; Gibson, G. R.; Rastall, R.
A. Synthesis of R-galactooligosaccharides with R-galactosidase from
Lactobacillus reuteri of canine origin. Appl. Biochem. Biotechnol.
2003, 63, 286–292.
(24) Brobst, K. M.; Lott, C. E. Determination of some components in
corn syrup by gas-liquid chromatography of trimethylsilyl deriva-
tives. Cereal Chem. 1966, 43, 35–43.
(5) Van den Broek, L. A. M.; Hinz, S. W. A.; Beldman, G.; Doeswijk-
Voragen, C. H. L.; Vincken, J. P.; Voragen, A. G. J. Glycosyl
hydrolases from Bifidobacterium adolescentis DSM20083. An over-
view. Lait 2005, 85 (1-2), 125–133.
~
(25) Banos, J. L. G.; Olano, A.; Corzo, N. Determination of mono and
disaccharide content of enteral formulations by gas chromatogra-
(6) Trojanova, I.; Vlkova, E.; Rada, V.; Marcunek, M. Different
utilization of glucose and raffinose in Bifidobacterium breve and
Bifidobacterium animalis. Folia Microbiol. 2006, 51 (4), 320–324.
(7) Martinez-Villaluenga, C.; Gulewicz, P.; Perez, A.; Frias, J.; Vidal-
Valverde, C. Influence of the addition of raffinose family oligosac-
charides on probiotic survival in fermented milk during refrigerating
storage. Int. Dairy J. 2006, 16 (7), 768–774.
(8) Martinez-Villaluenga, C.; Gomez, R. Characterization of bifidobac-
teria as starters in fermented milk containing raffinose family of
oligosaccharides from lupin as prebiotic. Int. Dairy J. 2007, 17 (12),
116–122.
phy. Chromatographia 2000, 52 (3-4), 221–224.
(26) Garna, H.; Mabon, N.; Wathelet, B.; Paquot, M. New method for a
two-step hydrolysis and chromatographic analysis of pectin neutral
sugar chains. J. Agric. Food Chem. 2004, 52, 4652–4659.
(27) Ghazi, I.; Fernandez-Arrojo, L.; Garcia-Arellano, H.; Ferrer, M.;
Ballesteros, A.; Plou, F. J. Purification and kinetic characterization
of a fructosyltransferase from Aspergillus aculeatus. J. Biotechnol.
2007, 128, 204–211.
(28) McIntyre, D. D.; Vogel, H. J. Complete assignment of the 1H-NMR
spectrum of stachyose by two-dimensional NMR spectroscopy. J.
Nat. Prod. 1989, 52 (5), 1008–1014.
(9) Farnworth, E. R.; Mainville, I.; Desjardins, M. P.; Gardner, N.;
Fliss, I.; Champagne, C. Growth of probiotic bacteria and bifido-
bacteria in a soy yogurt formulation. Int. J. Food Microbiol. 2007,
116, 174–181.
(10) Ouwehand, A. C.; Derrien, M.; de Vos, W.; Tiihonen, K.; Rautonen,
N. Prebiotics and other microbial substrates for gut functionality.
Curr. Opin. Biotechnol. 2005, 16 (2), 212–217.
Received for review January 28, 2009. Revised manuscript received April
3, 2009. Accepted April 03, 2009. This study was funded by the Spanish
Ministry of Science and Innovation, Projects Consolider Ingenio 2010
Fun-C. Food CSD 2007-00063 and CTQ2006-15279-C03-03, and Project
ALIBIRD S-0505/AGR/000153 from the Comunidad de Madrid.