Study of influential factors on oligosaccharide formation by fructosyltransferase activity during stachyose hydrolysis by pectinex ultra SP-L

Article abstract of DOI:10.1021/jf202472p

The influence of reaction conditions for oligosaccharide synthesis from stachyose using a commercial enzymatic preparation from Aspergillus aculeatus (Pectinex Ultra SP-L) was studied. Oligosaccharides were analyzed by gas chromatography with flame ionization detection (GC-FID) and matrix-assisted laser desorption/ionization-time-of-flight-mass spectrometry (MALDI-TOF-MS). Galactosyl-melibiose (DP₃) was synthesized as a result of fructosidase activity, whereas fructosyl-stachyose (DP₅) and difructosyl-stachyose (DP₆) were formed as a consequence of the fructosyltransferase activity of Pectinex Ultra SP-L. The optimal reaction conditions for the synthesis of penta-and hexasaccharides were 60 °C, pH 5.5, 600 mg/mL stachyose, and 34 U/mL enzyme. Reaction time played an important role in oligosaccharide mixture composition constituted by 20% DP₅, 0.7% DP₆, 55% stachyose, 21% galactosyl-melibiose, and 1% monosaccharides after 1 h and 16% DP₅, 4% DP₆, 27% stachyose, 44% galactosyl-melibiose, and 2% monosaccharides after 3 h. In conclusion, stachyose could be used as a substrate for the enzymatic synthesis of new oligosaccharides that may open new opportunities in the development of future prebiotics.

Full text of DOI:10.1021/jf202472p

ARTICLE
pubs.acs.org/JAFC
Study of Influential Factors on Oligosaccharide Formation by
Fructosyltransferase Activity during Stachyose Hydrolysis
by Pectinex Ultra SP-L
Antonia Montilla,† Agustín Olano,† Cristina Martínez-Villaluenga,‡ and Nieves Corzo*,†
†Instituto de Investigaciꢀon en Ciencias de la Alimentaciꢀon CIAL, CSIC-UAM, C/Nicolꢀas Cabrera 9, Campus de la Universidad
Autꢀonoma de Madrid, 28049 Madrid, Spain
‡Instituto de Ciencia y Tecnología de Alimentos y Nutriciꢀon ICTAN, CSIC, C/Juan de la Cierva 3, 28006 Madrid, Spain
ABSTRACT: The influence of reaction conditions for oligosaccharide synthesis from stachyose using a commercial enzymatic
preparation from Aspergillus aculeatus (Pectinex Ultra SP-L) was studied. Oligosaccharides were analyzed by gas chromatography
with flame ionization detection (GC-FID) and matrix-assisted laser desorption/ionizationꢀtime-of-flightꢀmass spectrometry
(MALDI-TOF-MS). Galactosyl-melibiose (DP3) was synthesized as a result of fructosidase activity, whereas fructosyl-stachyose
(DP5) and difructosyl-stachyose (DP6) were formed as a consequence of the fructosyltransferase activity of Pectinex Ultra SP-L.
The optimal reaction conditions for the synthesis of penta- and hexasaccharides were 60 °C, pH 5.5, 600 mg/mL stachyose, and 34
U/mL enzyme. Reaction time played an important role in oligosaccharide mixture composition constituted by 20% DP5, 0.7% DP6,
55% stachyose, 21% galactosyl-melibiose, and 1% monosaccharides after 1 h and 16% DP5, 4% DP6, 27% stachyose, 44% galactosyl-
melibiose, and 2% monosaccharides after 3 h. In conclusion, stachyose could be used as a substrate for the enzymatic synthesis of
new oligosaccharides that may open new opportunities in the development of future prebiotics.
KEYWORDS: α-galactosides, stachyose, Pectinex Ultra SP-L, transfructosylation, oligosaccharide synthesis
’ INTRODUCTION
could be an interesting alternative for their utilization as raw
material in the manufacture of future prebiotics.
The consumption of prebiotic carbohydrates has gained
increased interest due to their recognition as agents inducing
beneficial physiological effects in the colon and in extraintestinal
compartments or reducing the risk of associated intestinal and
systemic pathologies.1 Prebiotic carbohydrates may be fermen-
ted in different parts of the digestive tract. Rapidly fermented
prebiotics stimulate the growth and activity of beneficial bacteria
in the proximal colon, whereas slowly fermented prebiotics may
reach the distal colon, where most chronic diseases (e.g., color-
ectal cancer and ulcerative colitis) take place.2,3 Consequently,
there is greater interest in finding prebiotics that can persist to
more distal regions of the colon.4
Currently, the development of future prebiotics targets the
modulation of microbial fermentation along the gastrointestinal
tract. Future prebiotics could be mixtures of oligosaccharides that
have different effects in different parts of the gastrointestinal
tract. Besides the colon, the oral cavity and the small intestine are
also considered to be potential prebiotic targets.5
α-Galactosides (raffinose, stachyose, verbascose, and ajugose)
are sucrose-based oligosaccharides found more abundantly in
grain legumes.6 These carbohydrates derive from sucrose and
contain 1ꢀ4 units of galactose linked by α-(1,6) linkages to the
glucose moiety. These oligosaccharides are resistant to digestion,
promoting the growth of beneficial bacteria in the colon.7
In vitro assays have shown the ability of Bifidobacterium and
Lactobacillus strains to grow using α-galactosides as carbon
source, due to the production of α-galactosidase.8ꢀ10 Today,
α-galactosides are obtained on an industrial scale as a byproduct
from the production of soy protein isolates and soy protein
concentrates.6 The recovery of α-galactosides from soy byproduct
Transglycosylation reactions catalyzed by enzymes are being
used for the production of oligosaccharide mixtures with a
specific composition. Production of galactooligosaccharides mix-
tures from lactose or lactulose using galactosyltransferases from
different sources has been carried out.11ꢀ15 In addition, fructoo-
ligosaccharide mixtures were produced from sucrose by fructosyl-
transferases from bacteria or molds.16ꢀ19 Because α-galactosides
contain a fructosyl end, fructosyltransferases may transfer the
fructosyl moiety to a suitable acceptor to give rise to new
oligosaccharides. Purified fructosyltransferase from Aspergillus
niger has been used to catalyze the synthesis of new oligosac-
charides from raffinose.20 The relatively inexpensive com-
mercial enzyme preparation Pectinex Ultra SP-L (Pectinex),
produced by A. aculeatus, was shown to contain fructosyltrans-
ferase activity;16,19,21,22 therefore, it could be used as a catalyst
in the large-scale production of new oligosaccharides. The
objective of this work has been to investigate in more detail the
fructosyltransferase activity from A. aculeatus of the commercial
enzymatic preparation Pectinex over stachyose hydrolysis.
’ MATERIALS AND METHODS
Chemicals. D-Galactose, D-glucose, D-fructose, melibiose, raffi-
nose, stachyose, and phenyl-β-glucoside were purchased from Sigma
Received: June 21, 2011
Revised:
August 31, 2011
Accepted: September 1, 2011
Published: September 01, 2011
r
2011 American Chemical Society
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Figure 1. MALDI-TOF-MS profile of the purified fraction of oligosaccharides formed during enzymatic hydrolysis of stachyose with Pectinex Ultra
SP-L (DP, degree of polymerization).
(St. Louis, MO). The commercial enzyme preparation from A. aculeatus,
Pectinex Ultra SP-L, containing fructosyltransferase activity was a
generous gift from Novozymes (Dittingen, Switzerland).
different times and immediately immersed in boiling water for 5 min to
inactivate the enzyme. Samples were stored at ꢀ18 °C for subsequent
analysis. Experiments were carried out in duplicate.
The amount of remaining stachyose and the yield of oligosaccharides
in the reaction mixtures were expressed as weight percentage of total
carbohydrate content.
Determination of Enzymatic Activity. The fructosyltransferase
activity of Pectinex was measured using sucrose as substrate. The activity
was assayed at 60 °C using sucrose at 300 g/L in 0.1 M sodium acetate
buffer (pH 5.5). Aliquots (0.1 mL) were withdrawn at different times.
The reaction was stopped by adding 10 μL of 0.1 N acetic acid. The
amount of fructose and glucose released was determined by gas
chromatography (GC-FID) using the method described below. Fructo-
syltransferase activity was calculated as the difference between the
amount of free glucose and fructose, which indicated the amount of
fructose involved in the transfructosylation reaction. The fructosyltrans-
ferase activity was 400 units (U), where 1 U is defined as the amount of
enzyme transferring 1 μmol of fructose per minute per milliliter under
assayed conditions. Enzyme activity measurements were performed in
triplicate, and the experimental error (RSD relative standard deviation)
was <5%.
The soluble protein concentration in the enzymatic preparation was
determined using the bicinchoninic acid (BCA) assay.23 Bovine serum
albumin (BSA) was used as standard. Specific activity was defined as
units per milligram of protein. The soluble protein concentration in the
enzyme preparation was 82 mg/mL. Therefore, the enzyme expressed a
fructosyltransferase specific activity of 4.9 U/mg soluble protein.
Enzymatic Synthesis of Oligosaccharides. Enzymatic synth-
esis of oligosaccharides from stachyose using Pectinex was carried out
under different reaction conditions such as temperature (50, 60, and
70 °C), pH (3.5, 4.5, 5.5, 6.5, and 7.5), stachyose concentration (100,
300, and 600 g/L), enzyme concentration (17, 34, and 78 U/mL), and
time (0.5, 1, 3, 6, and 24 h). Reactions were performed at a final volume
of 1.5 mL in microtubes incubated in an orbital shaker at 300 rpm.
Aliquots (120 μL) were withdrawn from the reaction mixture at the
Purification and Characterization of Reaction Mixtures.
Purification was performed following the method described by Morales
et al.24 Briefly, a total of 2.5 mL of reaction mixture, containing 1.5 g of
carbohydrates, was dissolved in 300 mL of a 1% aqueous solution of
ethanol and stirred for 30 min with 9 g of activated charcoal Darco G60,
100 mesh (Sigma, St. Louis, MO), to remove mono- and disaccharides.
This mixture was vacuum-filtered through Whatman no. 1 filter paper,
and activated charcoal was washed with 50 mL of water. The oligosac-
charides adsorbed onto the activated charcoal were extracted by stirring
for 30 min with 300 mL of ethanol/water solution (1:1, v/v) and then
vacuum-filtered. The ethanol/water solution was evaporated under
vacuum at 30 °C. The sample was dissolved in 5 mL of deionized water
and filtered through 0.22 μm filters (Millipore Corp., Bedford, MA) for
further characterization by mass spectrometry. This methodology
allowed very good recoveries to be obtained for high molecular weight
oligosaccharides (48% trisaccharides, 91% tetrasaccharides, and 100% of
DP5 and DP6).
The enriched fraction containing the main synthesis products was
characterized by matrix-assisted laser desorption/ionizationꢀtime-of-
flightꢀmass spectrometry (MALDI-TOF-MS) on a Voyager DE-PRO
mass spectrometer (Applied Biosystems, Foster City, CA) equipped
with a pulsed nitrogen laser (λ = 337 nm, 3 ns pulse width, and 3 Hz
frequency) and a delayed extraction ion source. Ions generated by laser
desorption were introduced into a time-of-flight analyzer (1.3 m flight
path) with an acceleration voltage of 20 kV, 74% grid voltage, 0.001% ion
guide wire voltage, and delay time of 300 ns in the reflector positive ion
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Figure 2. Effect of temperature on stachyose hydrolysis and oligosaccharide production during the enzymatic treatment of stachyose (300 mg/mL)
with Pectinex Ultra SP-L (34 U/mL) in 0.1 M sodium acetate buffer (pH 5.5): ([) 50 °C; (9) 60 °C; (2) 70 °C.
mode. Mass spectra were obtained over the m/z range 100ꢀ1500.
External mass calibration was applied using the monoisotopic [M + H]+
values of des-Arg1 bradykinin and angiotensin I of the Calibration
Mixture 1, Sequazyme Peptide Mass Standards Kit, Applied Biosystems.
2,5-Dihydroxybenzoic acid (>98%, Fluka) at 10 mg/mL in water was
used as matrix. Sample was diluted 100 times in water and mixed with the
matrix at a ratio of 1:4 (v/v). One microliter of this solution was spotted
onto a flat stainless steel sample plate and dried in air before analysis.
Carbohydrate Analysis by Gas Chromatography (GC-FID).
Trimethylsilylated oximes (TMS-oxime) of mono-, di-, and oligosac-
charides were analyzed by GC following the method of Montilla et al.25
GC analysis was performed with a gas chromatograph (3800GC, Varian,
Palo Alto, CA) equipped with a flame ionization detector (FID). The
trimethylsilyl oximes were separated using a 8 m ꢁ 0.25 mm ꢁ 0.25 μm
film fused silica capillary column coated with CP-SIL 5CB (methyl
silicone from Chrompack, Middelburg, The Netherlands), giving rise to
two peaks, corresponding to the syn (E) and anti (Z) isomers. The carrier
gas (nitrogen) flow rate was 1.1 mL/min. The injector temperature was
280 °C, the detector temperature was 340 °C, and the oven temperature
was programmed from 150 to 165 °C at 3 °C/min, then at 5 °C/min to
340 °C, and held at this temperature for 10 min. Injections were made in
the split mode(1:10). Data acquisition and integration wasdone using HP
ChemStation software (Hewlett-Packard, Wilmington, DE).
Quantitative analysis was carried out by the internal standard method.
Response factors were calculated using a triplicate analysis of seven
standard solutions (galactose, glucose, fructose, sucrose, melibiose, raffi-
nose, and stachyose) at a concentration ranging from 0.06 to 2 mg/mL
(from 0.06 to 4 mg/mL for stachyose).
’ RESULTS AND DISCUSSION
The fructosidase, galactosidase, and fructosyltransferase activities
of Pectinex resulted in the production of melibiose (α-D-Galp-
(1f6)-α-D-Glcp), raffinose (α-D-Galp-(1f6)-α-D-Glcp-(1f2)-
β-D-Fruf), 60-galactosyl melibiose (α-D-Galp-(1f6)-α-D-Galp-
(1f6)-α-D-Glcp), fructosyl stachyose (DP5) (1F fructofuranosyl
stachyose, α-D-Galp-(1f6)-α-D-Galp-(1f6)-α-D-Glcp-(1f2)-
β-D-Fruf-(1f2)-β-D-Fruf), and difructosyl stachyose (DP6)
(1F (fructofuranosyl)2 stachyose (α-D-Galp-(1f6)-α-D-Galp-
(1f6)-α-D-Glcp-(1f2)-β-D-Fruf-(1f2)-β-D-Fruf-(1f2)-β-D-
Fruf) as has been previously reported.22 Besides, MALTI-TOF-
MS analysis of oligosaccharide-enriched fractions allowed the
detection of oligosaccharides with DP7 (m/z 1175.4 and 1191.3)
and DP8 (m/z 1338.5) (Figure 1), whereas previous studies have
reported only the formation of oligosaccharides DP3 and DP5
from stachyose by fructosyltransferase from A. niger.20
Several papers have demonstrated that glycosidic linkages and
degree of polymerization of oligosaccharides contribute toward the
selectivity of fermentation by beneficial bacteria.2,3,27 In addition, a
wide molecular weight range could give rise to modulation of
microbial fermentation. Higher molecular oligosaccharides may be
slowly fermented, exhibiting higher colonic persistence than low
molecular weight carbohydrates, reaching the most distal regions
where most intestinal disorders are encountered.3 On the basis of
this information, the production of α-galactoside mixtures from
stachyose composed of carbohydrates with DP from 3 to 8 may
represent an opportunity in the development of future prebiotics.
The oximes were formed following the method of Brobst and Lott.26
A volume of 7ꢀ40 μL of sample constituted of approximately 4 mg of
sugars was added to 0.4 mL of internal standard (IS) solution containing
0.5 mg/mL phenyl-β-glucoside. The mixture was dried at 38ꢀ40 °C in a
rotary evaporator (B€uchi Labortechnik AG, Flawil, Switzerland). Sugar
oximes were formed by adding 200 μL of hydroxylamine chloride
(2.5%) in pyridine and heating the mixture at 70 °C for 30 min.
Subsequently, the oximes obtained in this step were silylated with
hexamethyldisilazane (200 μL) and trifluoroacetic acid (20 μL) and
kept at 50 °C for 30 min. Reaction mixtures were centrifuged at 7000g
for 5 min at 5 °C. Supernatants were injected in the GC or stored at 4 °C
prior to analysis.
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Figure 3. Effect of pH on stachyose hydrolysis and oligosaccharide production during the enzymatic treatment of stachyose (300 mg/mL) with
Pectinex Ultra SP-L (34 U/mL) at 60 °C in 0.1 M sodium acetate buffer: ([) pH 4.5; (9) pH 5.5; (2) pH 6.5.
Study of the Formation of Oligosaccharides from Hydro-
lysis of Stachyose. A series of experiments were performed in
which temperature, pH, and substrate and enzyme concentra-
tions were varied to select the optimum conditions for tri- and
oligosaccharide synthesis.
galactosyl-melibiose formation (Figure 3c) were attained at pH
5.5. At pH 4.5 the lowest fructosidase activity was observed.
The maximum formation of DP5 (Figure 3d) was observed at
pH 5.5 and 6.5 at 3 h of reaction, reaching a yield of 15%, and
then slowly decreased to 11% after 24 h. However, a marked
decrease to 1% was observed at pH 5.5, probably due to a higher
fructosidase activity of the enzyme under these conditions.
The highest amount of DP6 formed was 2%, at pH 5.5 after 6 h
of reaction, whereas yields at pH 4.5 and 6.5 were negligible.
These results are consistent with previous studies of Ghazi et al.28
showing that the optimal activity/stability of purified fructosyl-
transferase from Pectinex was in the pH range of 5.0ꢀ6.0, similar
to those of other fructosyltransferases derived from fungi.29,30
Effect of Enzyme Concentration. The effect of enzyme con-
centration (17, 34, and 68 U/mL) was studied using reaction
mixtures containing 300 mg/mL stachyose at pH 5.5 and 60 °C
(Figure 4). Loss of stachyose and formation of galactosyl-
melibiose noticeably increased when enzyme concentration
was raised to 34 U/mL, whereas a further increase of the enzyme
concentration did not result in a significant improve of stachyose
conversion and trisaccharide yield (Figure 4a,c). With regard to
DP5 formation, enzyme concentration did not exert any influ-
ence on maximum yields attained, although the formation rate of
this pentasaccharide increased with enzyme concentration
(Figure 4d). DP6 yields decreased with enzyme concentration,
the highest yield (2%) being observed at an enzyme concentra-
tion of 17 U/mL after 24 h (Figure 4d). With stachyose, the most
important effect observed was an increase of hydrolysis rate with
enzyme concentration. Also, the hydrolysis rate of the DP5 and
DP6 formed during the first 3ꢀ6 h increased with the enzyme
concentration (Figure 4d). This was consistent with the mono-
saccharide concentrations observed (Figure 4b). The highest
enzyme concentration tested (68 U/mL) gave rise to the highest
fructose (13%) and galactose + glucose (5%) concentrations
after 24 h of reaction. However, in all conditions it was observed
Effect of Temperature. To determine the influence of tempera-
ture on oligosaccharide synthesis, reaction was performed in a
temperature range of 50ꢀ70 °C, 300 mg/mL stachyose, 34 U/mL
enzyme, and pH 5.5, for 24 h (Figure 2). The loss of stachyose
(Figure 2a) increased with temperature from 50 to 60 °C but
declined with further increase of temperature to 70 °C. This fact
could be attributed to a thermal denaturation of enzyme at 70 °C.
During the first 6 h of the reaction, fructose release (Figure 2b)
was favored at 60 °C; however, after 24 h, when most of the
stachyose was consumed, the amount of fructose reached at
50 °C was similar to that observed at 60 °C. Galactosyl-melibiose
production (Figure 2c) reached maximum yields after 24 h either
at 50 or at 60 °C (59 and 63%, respectively).
The transferase/hydrolase ratio of the enzyme was optimal at
60 °C, the highest DP5 oligosaccharide yield (15%) being
observed after 3 h (Figure 2d). There is a sharp decrease of this
pentasaccharide at 50 and 60 °C after reaching maximum yield,
whereas larger amounts of this α-galactoside remained after 24 h
at 70 °C. DP6 oligosaccharide production was possible only at
60 °C, reaching the highest concentration (2%) after 6 h. Our
results are in agreement with previous works which reported that
the optimal temperature for fructooligosaccharide production
from sucrose by fructosyltransferase activity of Pectinex was
around 60 °C.21,28
Effect of pH. Different experiments were carried out at pH
values from 3.5 to 7.5 at 60 °C using reaction mixtures of
stachyose (300 mg/mL) and 34 U/mL enzyme. In assays
carried out at pH 3.5 and 7.5 hardly any stachyose hydrolysis
was observed (data not shown). The highest rates of sta-
chyose decrease (Figure 3a), fructose release (Figure 3b), and
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Figure 4. Effect of enzyme concentration on stachyose hydrolysis and oligosaccharide production during the enzymatic treatment of stachyose
(300 mg/mL) at 60 °C and pH 5.5 with Pectinex Ultra SP-L: ([) 17 U/mL; (9) 34 U/mL; (2) 68 U/mL.
Figure 5. Effect of stachyose concentration on hydrolysis and oligosaccharide production from reaction mixtures of stachyose [([) 100 mg/mL;
(9) 300 mg/mL; (2) 600 mg/mL] and Pectinex Ultra SP-L (34 U/mL) at pH 5.5 and 60 °C. (/) Data from Montilla et al.22
that galactosyl-melibiose was stable to hydrolysis; the highest
melibiose formation was only 3% after 24 h with 68 U/mL
enzyme concentration.
Effect of Initial Stachyose Concentration. To investigate the
influence of substrate concentration on the composition of
synthesis mixtures of oligosaccharides derived from stachyose,
different assays werecarried out in a rangefrom 100 to 600 mg/mL
(Figure 5). Reactions were performed at 60 °C, pH 5.5, and 34
U/mL enzyme for 24 h. During the first 6 h of reaction, stachyose
decreased rapidly (80% of hydrolysis), especially at the highest
concentration tested, 600 mg/mL (Figure 5a). At 24 h of
reaction, similar levels of stachyose were found at three studied
concentrations. The fructosidase activity of Pectinex was favored
with low stachyose concentrations (100 mg/mL) and longer
reaction time; thus, increasing amounts of free fructose were
released, reaching 14% after 24 h of reaction (Figure 5b).
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The rate of galactosyl-melibiose formation increased with
initial stachyose concentration during the first 6 h of reaction,
reaching levels of 313 mg/mL (corresponding to a yield of 52%)
(Figure 5b). However, the highest yield of galactosyl-melibiose
(67%) was achieved when the low stachyose amount was assayed
(100 mg/mL). The galactosyl-melibiose yield obtained using
β-galactosidase from A. aculeatus (Pectinex) and stachyose as
substrate was much higher than that obtained by Van Laere
et al.,31 who achieved 33% using α-galactosidase from Bifidobac-
terium adolescentis and 103 mg/mL melibiose. Tzortzis et al.32
and Goulas et al.33 obtained 26 and 21% of galactosyl melibiose,
respectively. The former used α-galactosidase from Lactobacillus
reuteri and 230 mg/mL melibiose and the latter, α-galactosidase
from B. bifidus and 400 mg/mL melibiose.
Galactosyl-melibiose is an interesting compound with prebio-
tic and symbiotic properties when used along with L. acidophilus
and L. reuteri. This has been proven using galactosyl-melibiose
mixtures, which produced higher increases in bifidobacterium
and lactobacillus populations and higher decreases of clostridia
and Escherichia coli count than carbohydrates such as FOS,
melibiose, and raffinose used as reference.34
Transfructosylation reaction mainly occurred at reaction times
of <3 h and increased with initial substrate concentrations to 600
mg/mL (Figure 5d). The highest yields of transfructosylation
products, 20% for DP5 (120 mg/mL) and 4% DP6 (23 mg/mL),
were found at the highest initial stachyose concentration (600
mg/mL) after 1 and 3 h, respectively. Our research group used
previously this concentration to obtain and characterize DP5 and
DP6 compounds.22 These results are consistent with previous studies
showing that a high substrate concentration exerts a noticeable
influence on the formation of stachyose-derived oligosaccharides by
fructosyltransferases due to an increase in transferase/hydrolysis
ratio.13,35 Ghazi et al.28 reported that the fructosyltransferase activity
in a purified enzyme from A. aculeatus is approximately 20-fold higher
than hydrolysis activity at sucrose concentrations >342 g/L.
These results show the noticeable effect of reaction conditions
on oligosaccharide formation during the enzymatic treatment
of stachyose with Pectinex. The optimal reaction conditions for
the synthesis of DP5 and DP6 were 60 °C, pH 5.5, 600 mg/mL
stachyose, and 34 U/mL enzyme. Under these conditions the time
of reaction may noticeably influence the composition of α-galacto-
side mixtures; thus, 0.7% DP6, 20% DP5, 55% stachyose, 21%
galactosyl-melibiose, and 1% monosaccharides were found after 1 h
of reaction and 4% DP6, 16% DP5, 27% stachyose, 44% galactosyl-
melibiose, and 2% monosaccharides after 3 h of reaction. However,
to obtain the maximum yield of galactosyl-melibiose (67%), the
assays should be carried out at 60 °C and pH 5.5, using 100 mg/mL
stachyose and 34 U/mL enzyme during 24 h.
Funding Sources
This work has been financed under a R+D program of the
Spanish Ministry of Science and Innovation Science, Projects
AGL-2008-00941/ALI and Consolider Ingenio 2010 (FUN-C-
FOOD) CSD 2007-00063; a R+D program of the Comunidad
de Madrid, Project ALIBIRD P2009/AGR-1469; and as a R+D
program of the Comunidad de Castilla-La Mancha, POII10-
0178-4685.
’ ACKNOWLEDGMENT
We are grateful to Ramiro Martinez (Novozymes A/S, Spain)
for providing us with Pectinex Ultra SP-L and to Plꢀacido Galindo
for his help in acquiring the MALDI-TOF-MS spectral data.
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α-Galactosides, which are industrially available in large amounts
as a byproduct from the production of soy protein isolate, seem to
be a promising raw material for the production of new oligosac-
charides through hydrolysis and transglycosylation using a GRAS
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mixtures that may be used to influence the microbial composition
in the distal colon, where most gut disorders occur.
’ AUTHOR INFORMATION
(14) Martínez-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 Kluyver-
omyces lactis (Lactozym 3000 L HP G). Food Chem. 2008, 107, 258–264.
Corresponding Author
*Phone: + 34 91 0017 954. Fax: + 34 91 0017 905. E-mail: nieves.
corzo@csic.es.
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