Influence of reaction conditions on the selectivity of the synthesis of lactulose with microbial β-galactosidases

Article abstract of DOI:10.1016/j.molcatb.2011.06.007

Commercial β-galactosidase preparations from Bacillus circulans, Kluyveromyces lactis and Aspergillus oryzae were evaluated as catalysts for the synthesis of lactulose. Among them, the enzyme from A. oryzae was selected for further studies. The effect of reaction conditions was then studied on product composition during the kinetically controlled synthesis of lactulose by transgalactosylation with A. oryzae β-galactosidase. Product composition was not affected by pH, temperature, total initial concentration of sugar (lactose plus fructose) and enzyme to substrate ratio within the ranges studied. However, lactose to fructose ratio strongly influenced product composition being then possible to control the lactulose to galacto-oligosaccharide ratio within ample margins. Maximum lactulose yield (0.282 g of lactulose per g initial lactose) was obtained using 1/8 lactose to fructose molar ratio, 50% (w/w) total initial sugars, 40 °C, pH 4.5 and enzyme to initial lactose ratio equivalent to 200 IU/g.

Full text of DOI:10.1016/j.molcatb.2011.06.007

Journal of Molecular Catalysis B: Enzymatic 72 (2011) 206–212
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Influence of reaction conditions on the selectivity of the synthesis of lactulose
with microbial ␤-galactosidases
a
a
b
a,∗
Cecilia Guerrero , Carlos Vera , Francisco Plou , Andrés Illanes
a
School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso, Avenida Brasil 2147, Valparaíso, Chile
Institute of Catalysis and Petroleum Chemistry, CSIC, 28049 Madrid, Spain
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Commercial ␤-galactosidase preparations from Bacillus circulans, Kluyveromyces lactis and Aspergillus
oryzae were evaluated as catalysts for the synthesis of lactulose. Among them, the enzyme from A.
oryzae was selected for further studies. The effect of reaction conditions was then studied on prod-
uct composition during the kinetically controlled synthesis of lactulose by transgalactosylation with
A. oryzae ␤-galactosidase. Product composition was not affected by pH, temperature, total initial
concentration of sugar (lactose plus fructose) and enzyme to substrate ratio within the ranges stud-
ied. However, lactose to fructose ratio strongly influenced product composition being then possible
to control the lactulose to galacto-oligosaccharide ratio within ample margins. Maximum lactulose
yield (0.282 g of lactulose per g initial lactose) was obtained using 1/8 lactose to fructose molar
Received 18 March 2011
Received in revised form 2 June 2011
Accepted 8 June 2011
Available online 15 June 2011
Keywords:
Lactulose
Prebiotic
␤
-Galactosidase
◦
Transgalactosylation
ratio, 50% (w/w) total initial sugars, 40 C, pH 4.5 and enzyme to initial lactose ratio equivalent
Galacto-oligosaccharides
to 200 IU/g.
©
2011 Elsevier B.V. All rights reserved.
1
. Introduction
conditions ␤-galactosidases can catalyze the galactosyl transfer
of a ␤-galactoside to a hydroxyl containing acceptor moiety dif-
ferent than water, so synthesis instead of hydrolysis occurs [15].
Since lactose and fructose are present in the reaction medium for
lactulose synthesis, both of them can act as acceptors rendering
galacto-oligosaccharides (GOS) and lactulose, respectively [11]. The
synthesis of lactulose is schematically represented in Fig. 1.
The purpose of this work is to evaluate the effect of selected
variables (source of ␤-galactosidase, total initial concentration of
sugars, lactose to fructose molar ratio, enzyme to initial lactose
ratio, temperature and pH) on product composition during the
kinetically controlled enzymatic synthesis of lactulose so that, by
controlling them, a certain pre-established product composition
(lactulose to GOS ratio) may be obtained at will. Even though
the simultaneous formation of GOS and lactulose may be con-
sidered as a disadvantage from the standpoint of product purity,
GOS are valuable because they are prebiotics as well and a mix-
ture of both may lead to the production of a NDO of increased
prebiotic index [16,17]. In fact, we have some evidences indicat-
ing that mixtures of those NDOs have a better prebiotic index
than lactulose or GOS alone, so for this reason the synthesis
will be assessed in terms of the selectivity of the reaction of
transgalactosylaction.
Lactulose (4-O-␤-d-galactopyranosyl-d-fructose) is
a non-
digestible oligosaccharide (NDO) widely recognized as prebiotic
1–3]. Lactulose was the first lactose derived NDO marketed and
[
sold as a laxative for the treatment of acute and chronic consti-
pation [4]; however, its potential as prebiotic has renewed the
interest in its production [5,6]. Lactulose is currently produced by
alkaline isomerization catalysis [7]; however, the chemical pro-
cess has several drawbacks since reaction is poorly specific, side
reactions occurring so that low product yields are obtained, col-
ored by-products are formed and intense purification is required
[
8,9]. Recently, the enzymatic synthesis of lactulose has been arisen
as an interesting technology for lactulose production, both in
terms of process and environmental considerations. However, the
enzymatic process requires further research to be assessed as an
industrially viable option.
Lactulose can be produced by a kinetically controlled reac-
tion of transglycosylation from lactose using fructose as galactosyl
acceptor and ␤-galactosidase [10–13] or ␤-glycosidase [14] as cata-
lysts. ␤-Galactosidase catalyzes the hydrolysis of lactose to glucose
and galactose and it is used extensively in food and pharmaceu-
tical products as lactose remover. However, under appropriate
Among ␤-galactosidases, that from Aspergillus oryzae outstands,
because it is a very active and stable commodity enzyme, having
GRAS status; moreover, this enzyme has a high transgalactosy-
lation activity [18]. However, enzymes from yeast and bacteria
∗ Corresponding author. Tel.: +56 32 2273642; fax: +56 32 2273803.
E-mail address: aillanes@ucv.cl (A. Illanes).
1
381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.06.007

C. Guerrero et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 206–212
207
E
Lactulose
Fructose
Lactulose
Fructose
H O
Glucose
E-Gal*
2
Lactose
Galactose
Lactose
GOS-3
Lactose
GOS-3
Galactose
E
E
Galactose
E
E-Gal
Fig. 1. Mechanism of synthesis of lactulose and GOS with ␤-galactosidase.
have been proven as lactulose producers as well and therefore
under the assay conditions. The assays were carried out in triplicate
with less than 5% difference among samples.
␤
-galactosidases from fungal, yeast and bacterial origin were con-
sidered in this study.
2.3. HPLC analysis of the reaction products
2
. Materials and methods
During the stage of selection of the enzyme source, glucose,
2
.1. Materials
galactose, fructose, lactulose, GOS and lactose were determined
by HPLC using an evaporative light scattering detector (ELSD)
model 2000 ES (Alltech) at 92 C and a nitrogen flow-rate of
◦
D(+) lactose monohydrate, o-nitrophenol (o-NP) and o-
nitrophenyl-␤-d-galactopyranoside (o-NPG) and GOS standards
were supplied by Sigma (St. Louis, MO, USA). Lactulose was pro-
vided by Discovery Fine Chemicals (Wimborne, UK). All other
reagents were analytical grade and provided either by Sigma or
Merck (Darmstadt, Germany).
3 L/min. Samples were eluted in a Luna-NH2 (Phenomenex) col-
umn (250 mm × 4.6 mm) at 40 C and flow-rate of 1 mL/min. The
◦
initial mobile phases was acetonitrile/water 80/20 during the first
25 min, a gradient to acetonitrile/water 70/30 was performed
for 10 min and finally a gradient to acetronitrile/water 80/20 for
the last 10 min. The processing and analysis of the results was
made in the software Varian Star LC, Workstaion 6.41. Standards
of galactose, glucose, fructose, lactose, lactulose, 4␤-galactobiose,
4␤-galactosyllactose and 3␣-4␤-3␣ galactotetraose were used to
determine their retention times and check the linear range of the
measurements.
Three commercial ␤-galactosidases from different origin were
initially tested. A commercial ␤-galactosidase preparation of A.
®
oryzae, marketed under the trade name Enzeco Fungal Lactase
Concentrate, was kindly donated by Enzyme Development Corpo-
ration, New York, USA and used in all experiments. The enzyme
preparation had a specific activity of 196,000 IU/g, being one inter-
national unit of activity (IU) equivalent to the amount of enzyme
In the following stages, substrates and products of synthe-
sis were analyzed in a Jasco RI 2031 HPLC equipment, provided
with refractive index detector, an isocratic pump (Jasco PU2080)
and autosampler (Jasco AS 2055), using BP-100 Ca++ columns
(300 mm × 7.8 mm) for carbohydrate analysis (Benson Polymerics,
Reno, USA). Samples were eluted with milli-Q water at a flow-
rate of 0.5 mL/min. Column and detector temperatures were 80 and
◦
hydrolyzing 1 ␮mol of o-NPG per minute at pH 4.5, 40 C and 30 mM
o-NPG. Lactozym 3000 L HP G from Kluyveromyces lactis was a prod-
uct from Novozymes kindly supplied by Novo Nordisk Brasil; the
enzyme has a specific activity of 3000 IU/mL, being in this case
one international unit of activity (IU) equivalent to the amount of
◦
enzyme hydrolyzing 1 ␮mol of o-NPG per minute at pH 6.5, 40 C
◦
and 15 mM o-NPG. Lactoles L3 from Bacillus circulans was a product
from Daiwa Kasei K.K (Amano, Japan); the enzyme has a specific
activity of 2420 IU/mL, being in this case one international unit
40 C respectively. During synthesis, 0.5 mL samples were taken at
regular intervals and the reaction was stopped by adding 0.5 mL
of 200 mM NaOH solution and then diluted and filtered through
0.22 ␮m Durapore membranes (Filterpore, Chile) prior to assay.
Chromatograms were integrated using the software ChromPass.
The composition of samples was determined by assuming that the
area of each peak is proportional to the weight percentage of the
respective sugar on the total sugar mass [18–21]. The accuracy
of this assumption was checked by a material balance accord-
ing to Boon et al. [19]. Standards of galactose, glucose, fructose,
lactose, lactulose, 4␤-galactobiose and 3␣-4␤-3␣ galactotetraose
were used to determine their retention times and check the linear
range of the measurements.
of activity (IU) equivalent to the amount of enzyme hydrolyzing
◦
1
␮mol of o-NPG per minute at pH 5.5, 45 C and 15 mM o-NPG.
2.2. Hydrolytic activity over o-NPG
Initial hydrolysis rates of o-NPG were determined by mea-
suring o-NP release during the first 5 min of reaction. o-NP was
determined by absorbance at 420 nm in a Jenway 6715 spectropho-
−
1
−1
tometer, using an extinction coefficient of 253.5 M cm for pH
4
− −1 −1 −1
1
.5, 628.35 M cm for pH 5.5 and 1640.16 M cm for pH 6.5

2
08
C. Guerrero et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 206–212
2
.4. Selection of the enzymatic source
a
25
Lactulose synthesis was initially performed with the three bio-
2
1
1
0
5
0
5
0
◦
catalysts at 40 C, 50% (w/w) total initial sugars concentration,
lactose to fructose molar ratio of 1200 IU/g lactose at the corre-
sponding pH optima (4.5 for the A. oryzae, 5.5 for the B. circulans
and 6.5 for the K. lactis ␤-galactosidase). Reactions were carried
out in 150 mL Erlenmeyer flasks by dissolving 32.76 g of lactose
and 17.4 g of fructose (molar ratio of 1/1) in 40 g of 100 mM McIl-
vaine citrate–phosphate buffer at the corresponding pH. Substrates
(
a)
◦
were dissolved by heating the solution at a temperature over 95 C
(
no degradation of sugars was observed) and then, after cooling to
the reaction temperature, 10 g of a properly diluted enzyme solu-
tion was added to start the reaction. Time course of the reaction
was followed to determine the maximum lactulose concentra-
tion attained. Product composition is expressed as the percentage
of each component in the reacted mixture. It was determined
by analyzing the amounts of lactulose, GOS-2, GOS-3 and GOS-4
synthesized, numbers meaning the monosaccharide units of each
galacto-oligosaccharide. The assays were carried out in duplicate,
with standard deviations never exceeding 5%. The quantification of
sugars was carried out in a HPLC with evaporative light scattering
detector, because ELSD detector is more sensitive than refractive
index detector and it allows the separation and quantification of
the different types of disaccharides (GOS-2) and lactulose present
in the reaction media, which is in this case required.
Lactulose
GOS-2 1 GOS-3
GOS-4
b
25
2
1
0
5
(b)
10
5
0
Lactulose
GOS-2 1GOS-3
GOS-4
c 25
2
.5. Effect of variables on the synthesis of lactulose with the
selected biocatalyst
(c)
2
0
Syntheses were carried out with A. oryzae ␤-galactosidase in
magnetically stirred 150 mL Erlenmeyer flasks. Sugars were dis-
solved in 100 mM citrate–phosphate buffer at the corresponding
15
10
5
◦
pH of reaction by previously heating over 95 C and then adjust-
ing to the corresponding reaction temperature. Afterwards, 10 g of
enzyme solution in 100 mM McIlvaine citrate–phosphate buffer at
the corresponding pH of reaction were added.
An experimental design was conducted to select the most rele-
vant variables in the synthesis of lactulose with ␤-galactosidase
from A. oryzae, which was the one selected as ␤-galactosidase
source. Temperature, pH, enzyme to initial lactose ratio, total initial
sugars and lactose to fructose molar ratio were chosen as variables
to be tested.
0
Lactulose GOS-2 1GOS-3 GOS-4
◦
Fig. 2. Product composition in the reaction of lactulose synthesis at 40 C, 50% (w/w)
total initial sugars concentration, lactose/fructose molar ratio of 1/1, enzyme to ini-
tial lactose ratio of 200 IU/g ␤-Galactosidase from: (a) A. oryzae at pH 4.5, (b) K. lactis
at pH 6.5 and (c) B. circulans at pH 5.5. Difference between duplicates was less than
5% in all cases.
In order to evaluate the effect of pH in the range from 2.5 to 8.5,
◦
the remaining variables were kept constant: 40 C, 200 IU/g, 50%
(
w/w) total initial sugars concentration and 1/1 lactose to fructose
molar ratio. To test the effect of temperature, syntheses were car-
ried out in the range from 40 to 60 C, the remaining variable being
-
Lactulose yield (YLactulose), representing the fraction of initial
lactose converted into lactulose at maximum concentration of
lactulose in the reaction mixture.
◦
kept constant in the values above at pH 4.5. To assess the effect
of total initial sugars concentrations, syntheses were conducted in
the range from 40 to 60% (w/w), other variables were kept con-
g lactulose
YLactulose
=
(2)
◦
stant as above at a temperature of 40 C. To determine the effect
g lactoseinital
of the enzyme to initial lactose ratio syntheses were conducted in
the range from 40 to 600 IU/g, other variables being kept constant
as above at 50% (w/w) total sugars concentration. Finally, the effect
of lactose to fructose molar ratio was evaluated in the range from
-
GOS yield (YGOS), representing the fraction of initial lactose con-
verted into GOS at maximum concentration of lactulose in the
reaction mixture.
4
/1 to 1/8, other operational variables being kept constant as above.
g GOS
g lactoseinital
YGOS
=
(3)
The assays were carried out in duplicate with less than 5% difference
among samples.
The following evaluation parameters were defined:
- Specific productivity of lactulose (ꢀLactulose), representing the
amount of lactulose produced per unit of biocatalyst mass
(mbiocat) and unit time at maximum concentration of lactulose
in the reaction mixture.
-
Lactose conversion (XLactose), representing the fraction of initial
lactose transformed during the reaction.
g lactoseinital − g lactose
g lactulose
XLactose =
(1)
ꢀLactulose = m
(4)
g lactose
· time
inital
biocat

C. Guerrero et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 206–212
209
6
5
4
3
2
1
0
0
0
0
0
0
0
20
1
1
6
2
8
4
0
0
150
300
Time (min)
450
600
0
0.2
0.4
0.6
0.8
1
X Lactose
Fig. 3. Time course of the synthesis of lactulose from lactose and fructose at molar
ratio of 1/1, 50% (w/w) total initial sugars concentration, enzyme to initial lactose
Fig. 4. Effect of pH in the synthesis of lactulose from lactose and fructose with A.
oryzae ␤-galactosidase. Lactose to fructose molar ratio of 1/1, 50% (w/w) total initial
sugars concentration, enzyme to initial lactose ratio of 200 IU/g and 50 C. GOS total
◦
ratio of 200 IU/g, 50 C and pH 4.5 in 100 mM McIlvaine citrate–phosphate buffer. ꢀ,
lactose; ♦, fructose; ꢀ, glucose; ꢁ, galactose; ꢀ, lactulose; ꢂ, trisaccharides (GOS-3);
◦
×
, tetrasaccharides (GOS-4). Difference between duplicates was less than 5% in all
cases.
ꢀ
, pH 2.5; ꢀ, pH 4.5; ꢂ, pH 6.5; ᭹, pH 8.5. Lactulose ꢁ, pH 2.5; ♦, pH 4.5; ꢀ, pH 6.5;
ꢁ
, pH 8.5. Difference between duplicates was less than 5% in all cases.
-
-
Specific productivity of GOS (ꢀGOS), representing the amount of
GOS produced per unit of biocatalyst mass (mbiocat) and unit time
at maximum concentration of lactulose in the reaction mixture.
reaction rate but not the oligosaccharide distribution according
to its chain length [20,21]. This effect has also been observed for
permeabilized cells of K. lactis, where the rate of synthesis of lactu-
lose decreased significantly at high pHs [10]. Furthermore, YLactulose
and YGOS were not significantly affected by pH as reported for the
synthesis of GOS [20,21] and lactulose using ␤-galactosidases from
other sources [10,11].
g GOS
mbiocat · time
ꢀGOS
=
(5)
-Selectivity of transgalactosylation reaction, representing the
molar ratio of lactulose to total GOS.
mole of lactulose
Selectivity =
(6)
mole of GOS
3.3. Effect of temperature
3
. Results and discussion
Effect of temperature in the synthesis of lactulose is presented
in Fig. 5. No effect of temperature on product composition at the
same lactose conversion was observed within the range studied.
A similar behavior has been reported for the synthesis of GOS
with lactose as substrate [15,20,21]. YLactulose and YGOS were not
significantly affected by temperature as reported for the synthe-
sis of GOS [20,21] and lactulose using ␤-galactosidases from other
3
.1. Selection of the biocatalyst
Results of the synthesis of lactulose with the three biocatalysts
under predetermined conditions are presented in Fig. 2. Product
composition was strongly dependent on the enzyme source. The
tendency to easily accept other nucleophiles than water in the
active center and to properly transfer the galactosyl moiety to fruc-
tose greatly differs among ␤-galactosidases from different origins
sources [10,11]. However, as in the case of pH, ꢀLactulose and ꢀGOS
were affected by temperature increasing in the whole range con-
sidered (see Table 1). Another important aspect is that the enzyme
[
22]. The ␤-galactosidase from A. oryzae produced both lactulose
◦
remained active during the synthesis at 60 C, which was unex-
and GOS, mostly GOS-3 and GOS-4 (Fig. 2a). The enzyme from
K. lactis produced mainly GOS-2 with lesser amounts of lactulose
pected considering the thermal stability of A. oryzae enzyme under
non-reactive conditions [18,21]. However, it is known that the
stability of A. oryzae ␤-galactosidase [18] and other glycosidases
(
Fig. 2b). The enzyme from B. circulans produced mainly GOS-3 with
small amounts of lactulose (Fig. 2c). Based on these results, the
enzyme form A. oryzae was selected for matching the conditions
required for this study.
A typical time course of the reaction of synthesis of lactulose
with A. oryzae ␤-galactosidase is presented in Fig. 3, where sub-
strates and products profiles are shown. A series of experimental
variables (pH, temperature, enzyme to substrate ratio, total initial
sugars concentration and lactose to fructose ratio) were further
examined in terms of their effect in the composition of the reaction
mixture.
2
0
16
12
8
3.2. Effect of pH
4
Fig. 4 shows the effect of pH on the synthesis of lactulose, where
0
the concentrations of lactulose and total GOS synthesized are plot-
ted against lactose conversion. As seen, product composition is not
affected by pH; however, reaction rate is and a significant decrease
is obtained at pH 6.5 or over. Table 1 shows this effect, expressed
as maximum specific lactulose productivity (ꢀLactulose) and spe-
cific GOS productivity (ꢀGOS). A similar behavior has been reported
for the synthesis of GOS with free and immobilized A. oryzae ␤-
galactosidase where, at same lactose conversion, pH affected the
0
0.2
0.4
0.6
0.8
1
X Lactose
Fig. 5. Effect of temperature in the synthesis of lactulose from lactose and fructose
with A. oryzae ␤-galactosidase. Lactose and fructose molar ratio of 1/1, 50% (w/w)
total initial sugars concentration, enzyme to initial lactose ratio of 200 IU/g and pH
◦
◦
4
5
.5 in 100 mM McIlvaine citrate–phosphate buffer. GOS total ꢀ, 40 C; ꢀ, 45 C; ꢂ,
0 C; ×, 55 C; ᭹, 60 C. Lactulose ♦, 40 C; ꢁ, 45 C;ꢀ, 50 C; ꢁ, 55 C; , 60 C.
◦
◦
◦
◦
◦
◦
◦
◦
Difference between duplicates was less than 5% in all cases.

2
10
C. Guerrero et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 206–212
Table 1
Effect of operational variables on specific productivity of lactulose (ꢀLactulose) and GOS (ꢀGOS) in the synthesis of lactulose with ␤-galactosidase from A. oryzae.
−
1
−1
ꢀGOSa (g h mg enz
−1
−1
)
Variable
Level
ꢀLactulose (g h mg enz
)
2.5
4.5
6.5
8.5
0.0088
0.0078
0.0040
0.0025
0.0148
0.0128
0.0101
0.0119
pH
4
4
50
0
5
0.0078
0.0107
0.0133
0.0 179
0. 0211
0.0128
0.0171
0.0242
0.0295
0.0322
◦
Temperature ( C)
5
6
5
0
4
50
0
0.0079
0.0078
0.0064
0.0110
0.0128
0.0126
Initial sugar concentration (% w/w)
Enzyme/lactose ratio (IU/g lactose)
60
4
2
4
6
0
0.0073
0.0078
0.0079
0.0070
0.0125
0.0128
0.0131
0.0124
00
00
00
4
2
1
/1
/1
/1
0.030
0.040
0.0183
0.0123
0.0128
0.0113
0.0092
0.0062
0.0085
0.0078
0.0128
0.0180
0.0234
0.0414
Lactose/fructose molar ratio
1/2
1
1
1
/4
/6
/8
a
At maximum concentration of lactulose on the reaction mixture.
[
23,24] is highly improved under reactive conditions as sugars are
3.5. Effect of the total initial concentration of sugars
frequently enzyme stabilizers.
As seen in Fig. 7, there was no effect of the total initial concentra-
tion of sugars on product composition in the reaction of synthesis
of lactulose. Despite this, the concentration of products increased
with the initial concentration of sugars, producing a maximum lac-
3
.4. Effect of the enzyme to substrate ratio
Fig. 6 shows that there is no effect of the enzyme to lactose
−
1
ratio on product composition along lactose conversion during the
synthesis of lactulose. As expected, reaction rate increased with
the increase in the enzyme to lactose ratio, as already reported for
the synthesis of lactulose with recombinant Escherichia coli bearing
the gene of the thermostable ␤-galactosidase from the hyperther-
mophilic bacterium Sulfolobus solfataricus [11]. Also YLactulose was
not significantly affected by the enzyme to substrate ratio, remain-
ing around 0.09 g lactulose/g initial lactose for the whole range
considered. This finding is in agreement with previously reported
results for the synthesis of GOS with ␤-galactosidase of A. oryzae
from lactose as substrate [25]. Furthermore, ꢀLactulose and ꢀGOS
were not significantly affected by increasing the enzyme to lactose
ratio (see Table 1).
tulose concentration of 39.2, 64.2 and 91.6 g kg of water at 40,
50 and 60% (w/w) of total initial concentration of sugars, respec-
tively. Lee et al. [10] reported an increase in the level of synthesis
of lactulose with permeabilized cells of K. lactis when increasing
the initial concentration of sugars at constant lactose to fructose
ratio, very much in the same way as in the case of the cloned ther-
mostable ␤-galactosidase from S. solfataricus already referred [11].
YLactulose and YGOS were slightly affected by the total initial concen-
tration of sugars, increasing at higher initial sugars concentration.
This behavior is similar than those previously reported for the syn-
thesis of GOS [15,20,21]. However, ꢀLactulose decreased and ꢀGOS
increased at higher initial sugars concentration (see Table 1). This
2
1
1
0
6
2
8
4
0
2
1
1
0
6
2
8
4
0
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
X Lactose
0.8
1
X Lactose
Fig. 6. Effect of the enzyme to lactose ratio in the synthesis of lactulose from lac-
tose and fructose with A. oryzae ␤-galactosidase. Lactose to fructose molar ratio
of 1/1, 50% (w/w) total initial sugars concentration, 40 C and pH 4.5 in 100 mM
Fig. 7. Effect of the total initial sugars concentration in the synthesis of lactulose
from lactose and fructose with A. oryzae ␤-galactosidase. Lactose and fructose at
molar ratio of 1/1, enzyme to initial lactose ratio of 200 IU/g, 40 C and pH 4.5 in
◦
◦
McIlvaine citrate–phosphate buffer. GOS total ꢀ, 40 IU/g; ᭹, 200 IU/g; ꢂ, 400 IU/g;
100 mM McIlvaine citrate–phosphate buffer. GOS total ꢂ, 40% (w/w); ꢀ, 50% (w/w);
ꢀ, 60% (w/w). Lactulose ꢀ, 40% (w/w); ♦, 50% (w/w); ꢁ, 60% (w/w). Difference
between duplicates was less than 5% in all cases.
ꢀ
, 600 IU/g. Lactulose ♦, 40 IU/g; ꢁ, 200 IU/g; ꢀ, 400 IU/g; ꢁ, 600 IU/g. Difference
between duplicates was less than 5% in all cases.

C. Guerrero et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 206–212
211
2
2
1
1
5
0
5
0
5
0
20
a
b
(
a)
(b)
1
1
5
0
5
0
0
0.2
0.4
0.6
0.8
0
0.2
0.4
0.6
0.8
X Lactose
X Lactose
8
6
4
2
0
6
c
d (d)
(
c)
4
2
0
0
0.2
0.4
0.6
0.8
1
0
0.2
0.4
0.6
0.8
X Lactose
X Lactose
Fig. 8. Effect of lactose to fructose molar ratio on product composition in the synthesis of lactulose from lactose and fructose with A. oryzae ␤-galactosidase at 50% (w/w)
◦
total initial sugars concentration, enzyme to initial lactose ratio of 200 IU/g, 40 C and pH 4.5 in 100 mM McIlvaine citrate–phosphate buffer. ♦, lactulose; ꢀ, total GOS. Lactose
to fructose molar ratio (a) 2/1; (b) 1/1; (c) 1/4; (d) 1/8. Difference between duplicates was less than 5% in all cases.
may be the consequence of an increase in the viscosity of the reac-
tion media which produces a decrease on the reaction rate due to
mass transfer limitations [20]; it may also be the consequence of a
decrease in the reaction rate produced by the low water activity at
high sugar concentrations [26].
1
0
8
6
4
2
0
3.6. Effect of the lactose to fructose ratio
Fig. 8 presents the results on the effect of lactose to fructose
ratio on product composition along the reaction of synthesis of lac-
tulose. Product composition was strongly affected by this variable
and, as shown in Fig. 8, there is an inverse correlation between the
lactose to fructose ratio and the lactulose to total GOS ratio in the
product. Lactulose/total GOS molar ratios four times higher than
those reported in the four previous sections were obtained. Fig. 9
shows that the effect of lactose to fructose ratio is stronger over GOS
than lactulose synthesis. When lactose to fructose ratio was higher
than 1/1, synthesis of GOS was favored in detriment of lactulose.
At ratios between 1/2 and 1/4 two distinct zones were observed:
at low substrate conversion synthesis of GOS was still predomi-
nant, while at higher conversions lactulose synthesis outweighed
GOS synthesis. At ratios below 1/6, lactulose synthesis prevailed
at all substrate conversions. This is consistent with the work by
Adamczak et al. [27] where they reported an increase in lactu-
lose concentration at higher fructose to lactose ratios. However
they merely increased fructose concentration while keeping lactose
concentration constant, so they actually increased the total initial
concentration of sugars. According to the work reported using ␤-
galactosidase from S. solfataricus, high concentration of lactulose
was obtained in the range of molar lactose to fructose ratio from
0
0.2
0.4
0.6
0.8
1
X Lactose
Fig. 9. Effect of lactose to fructose molar ratio on the selectivity of A. oryzae ␤-
galactosidase in the synthesis of lactulose from lactose and fructose at 50% (w/w)
total initial sugars concentration, enzyme to initial lactose ratio of 200 IU/g, 40 C
and pH 4.5 in 100 mM McIlvaine citrate–phosphate buffer. Lactose to fructose molar
ratio ꢀ, 4/1; ♦, 1/1; ꢀ, 1/2; ꢁ, 1/4; ꢂ, 1/6; ꢀ, 1/8. Difference between duplicates was
less than 5% in all cases.
◦
fructose ratios ꢀLactulose and YLactulose synthesis were increased and
ꢀGOS decreased, as shown in Table 1.
Table 2 summarizes the effect of the molar ratio of lactose to
fructose in YLactulose and YGOS (evaluated at the maximum concen-
tration of lactulose). As shown, the lactulose to total GOS molar
ratio can be varied within ample margins by tuning the initial lac-
tose to fructose molar concentrations. This allows controlling the
proportion of both components at will so as to obtain a mixture
of oligosaccharides of an increased prebiotic effect. Preliminary
results indicate that mixtures of lactulose and GOS in certain pro-
portions have a better prebiotic index than the oligosaccharides
alone.
2
/1 to 1/2, but at ratios over 4/1 and under 1/4 lactulose concen-
tration was severely reduced [11]. Furthermore, at lower lactose to

2
12
C. Guerrero et al. / Journal of Molecular Catalysis B: Enzymatic 72 (2011) 206–212
Table 2
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. Conclusions
9
Selectivity of the reaction of synthesis of lactulose with ␤-
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