Biochemical and Biophysical Research Communications xxx (2018) 1e5

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Biochemical and Biophysical Research Communications

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Combination of two b-galactosidases during the synthesis of

galactooligosaccharides may enhance yield and structural diversity
Christin Fischer*, Thomas Kleinschmidt
€then, Germany
Anhalt University of Applied Sciences, Department of Applied Biosciences and Process Engineering, Bernburger Str. 55, 06366, Ko

a r t i c l e i n f o a b s t r a c t

Article history: In this study, the combination of two b-galactosidases to synthesize prebiotic galactooligosaccharides
Received 8 October 2018 (GOS) was evaluated in terms of total GOS yield as well as GOS structures (chain length). Two different
Accepted 15 October 2018 combinations of either Aspergillus oryzae and Cryptococcus laurentii or Aspergillus oryzae and Kluyver-
Available online xxx
omyces lactis were tested to examine the influence of enzyme origin. Neither consecutive nor simulta-
neous synthesis with A. oryzae and C. laurentii led to an increased GOS yield. However, with the latter,
Keywords:
synthesis of higher GOS (
3 monomer units) was enhanced from 38.5% to 40% with special emphasis of
Galactooligosaccharide
tetra- and pentasaccharides, which increased from 6.7% to 12.8% and from 0.4% to 3.3%, respectively.
GOS
Prebiotics
Additionally, due to the different preferences of the two b-galactosidases in terms of types of glycosidic
Beta-galactosidase linkages, the structural diversity of the final GOS product could be increased. Using K. lactis following the
Transgalactosylation synthesis with A. oryzae increased the yield of total GOS from 24.6% to 33.1%, which was mainly due to
Combination the formation of GOS disaccharides. On the other hand, applying A. oryzae as the second enzyme led to a
degradation of di- and trisaccharides, and thus total GOS yield was diminished, although the yield of
tetrasaccharides could be enhanced. In conclusion, with both studied enzyme combinations it was
possible to increase the percentage of higher GOS and reduce the residual lactose content of the final
mixture, which is beneficial for subsequent purification processes. Thus, using more than one b-galac-
tosidase during the synthesis of GOS represents an interesting research area, which should be explored in
more detail in the future.
© 2018 Elsevier Inc. All rights reserved.

1. Introduction
The research in the optimization of the synthesis of gal-
actooligosaccharides (GOS) is of ongoing interest as they exhibit
prebiotic properties, and thus are beneficial compounds for infants
[1e3] and adults [4e6] alike. GOS are generated in a side reaction
called transgalactosylation during the hydrolysis of lactose by b-
galactosidase (EC 3.2.1.23), if the acceptor molecule is another sugar
instead of water [7]. As this sugar molecule can be any sugar in the
reaction mixture, that is glucose, galactose, lactose, or GOS itself,
the chain length (or degree of polymerization (DP)) of the gener-
ated GOS will increase with ongoing reaction time. However, GOS
are also possible substrates for the enzyme and their hydrolysis will
exceed their synthesis after a certain degree of lactose conversion is
reached [8].
* Corresponding author.
E-mail addresses: christin.fischer@hs-anhalt.de (C. Fischer),
kleinschmidt@hs-anhalt.de (T. Kleinschmidt).
With the enzyme source being one of the most important fac-
tors influencing GOS yield [9], as well as GOS structure in terms of
b-glycosidic linkage [10], we propose that the combination of two
b-galactosidases from different origins, which thus have different
preferences for transgalactosylation and hydrolysis, might lead to
an enhanced GOS yield. However, research on this topic is very rare
with only a handful of publications during a period of more than 30
years of GOS research. While Yakult Pharmaceutical Industry Co.,
Ltd uses two enzymes from Sporobolomyces singularis and Kluy-
veromyces lactis during the production of Oligomate® [11], with the
second enzyme aiming mainly for hydrolysis of unreacted lactose, it
is not clear whether the second enzyme also contributes to the total
GOS yield. Similar, Vitalus Nutrition Inc. [12] submitted a patent
application for a consecutive combination of b-galactosidases from
Aspergillus oryzae and K. lactis, which led to an increased GOS yield
from about 32% to 41%. On the other hand, Moon et al. [13] reported
a reduced synthesis of GOS tri- and tetrasaccharides (disaccharides
were unchanged), when A. oryzae and Kluyveromyces fragilis were
thomas. used simultaneously in comparison to K. fragilis alone, ultimately

https://doi.org/10.1016/j.bbrc.2018.10.091
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Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
2 C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5
resulting in a decreased total GOS yield (37% compared to 27%). A
consecutive application of A. oryzae followed by Bacillus circulans
could increase GOS yield (DP3-DP6) from 26% to 34% [14]. However,
this was considerably lower compared to using only the B. circulans
enzyme (40% GOS yield). Investigating the consecutive (but
without inactivation of the first enzyme) and simultaneous
coupling of b-galactosidases from B. circulans with those from
A. oryzae or K. lactis, also showed no increase in GOS yield (exem-
plified by the two main trisaccharides) compared to B. circulans
alone [15]. This study aims to investigate the consecutive and
simultaneous combination of different b-galactosidase enzymes
and its effect on total GOS yield, as well as potential compositional
changes.
2. Materials and methods
2.1. Enzymes
The b-galactosidase enzymes were from Aspergillus oryzae
(Maxilact A4 from DSM Food Specialties B.V., The Netherlands) and
Kluyveromyces lactis (optilactase LX2 from optiferm GmbH, Ger-
many). Additionally, whole cells of Cryptococcus laurentii (strain
DSM 27153 from Leibniz Institut DSMZ - Deutsche Sammlung von
Mikroorganismen und Zellkulturen GmbH, Germany) were used.
Cells were cultivated in an enzyme production medium proposed
by Ohtsuka et al. [16]. A preculture was grown in 15 mL medium
(100 mL Erlenmeyer flask) at 30  C and 160 rpm in a rotary shaker
(model KS 4000 i control, IKA®-Werke GmbH & Co. KG, Germany)
for 24 h. The main culture (100 mL in a 500 mL Erlenmeyer flask)
was inoculated with 2% of the preculture and incubated (30  C,
160 rpm) for 78e96 h until the stationary growth phase was
reached (data not shown). Cells were harvested by centrifugation
(25,000 g, 10 min, 4  C, Sigma 3K30 from Sigma Laborzentrifugen
GmbH, Germany), washed once with buffer (50 mM sodium
phosphate pH 5.0), resuspended in the same buffer, and stored
at 20  C until use.
2.2. Enzyme activity assay
b-Galactosidase activity was measured using o-nitrophenyl-b-D-
galactopyranoside (oNPG) as the substrate. For the enzymes Max-
ilact A4 and optilactase LX2, 480 mL buffered substrate solution
(30 mM oNPG) was tempered at least 10 min in a thermo shaker
(model TS-100C from Biosan, Lativa), then 20 mL of appropriately
diluted enzyme solution were added. The reaction was stopped
after 10 min by addition of 0.4 M Na2CO3 and absorbance was
measured at 420 nm. For the C. laurentii cell solution, 700 mL of a
65 mM oNPG solution was used, 50 mL of the cell solution was
added and the reaction was stopped after 10 min by adding 750 mL
of 1 M Na2CO3. Samples were centrifuged for 10 min (model VR-1
from Heraeus Instruments GmbH, Germany) to remove the cells.
The supernatant was transferred into cuvettes and then measured
at 420 nm. Assay buffers and temperatures were selected according
to the GOS synthesis parameters, that is PEM buffer pH 6.5 (50 mM
potassium phosphate containing 1 mM MgSO4 * 7 H2O and 0.056 mM
EDTA * 2 H2O according to the FCC method [17]) at 45  C for opti-
lactase LX2, 50 mM McIlvaine buffer pH 4.5 and PEM buffer pH
6.5 at 55  C and 45  C for Maxilact A4, and 50 mM McIlvaine buffer
pH 4.5 at 55  C for C. laurentii. One unit was defined as the amount
of enzyme that releases 1 mmol of o-nitrophenol per minute under
the respective assay conditions.
2.3. Galactooligosaccharide synthesis
Two experimental approaches were carried out studying either
Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
the consecutive (with inactivation of the first enzyme by heat
treatment for 5 min at 95  C) or simultaneous combination of
A. oryzae and K. lactis as well as A. oryzae and C. laurentii. Lactose
was dissolved in PEM buffer pH 6.5 for the former two enzymes and
McIlvaine buffer pH 4.5 for the latter two enzymes at a concen-
tration of 200 g/L for all experiments. Enzyme/substrate ratios were
based on the initial lactose concentration and set to 50 UONPG/
gLactose for A. oryzae and K. lactis and to 1 UONPG/gLactose for
C. laurentii. In the simultaneous approach, both enzymes were used
in a ratio of 1:1. Reaction temperature was set to 45  C for K. lactis
and the simultaneous A. oryzae/K. lactis experiments. All other re-
actions were carried out at 55  C. Samples were withdrawn at
different time intervals and enzymes were inactivated by heating at
95  C for 5 min in a thermo shaker (model 5436 from Eppendorf
Vertrieb Deutschland GmbH, Germany).
2.4. Galactooligosaccharide analysis
GOS were analyzed on a LaChrom Elite HPLC system (VWR In-
ternational GmbH, Germany). For the determination of the sugar
classes according to chain length, a Hi-Plex Na column
(300 mm  7.7 mm, Agilent Technologies Sales & Services GmbH &
Co. KG, Germany) was used at 80  C with 0.2% sodium azide in
water as an eluent and a flow rate of 0.3 mL/min. GOS-disaccharides
were separated from lactose either on a Zorbax Carbohydrate col-
umn (250 mm  4.6 mm, Agilent Technologies Sales & Services
GmbH & Co. KG, Germany; eluent 75:25 acetonitrile:water, 1.4 mL/
min, 35  C) or a Microsorb-MV 100 NH2 column (250 mm  4.6 mm
with guard column Polaris 5 NH2 MetaGuard, Agilent Technologies
Sales & Services GmbH & Co. KG, Germany; eluent 70:30 acetoni-
trile:water, 1.3 mL/min, 40  C). Both columns had a very similar
peak pattern and could therefore be used equally (see
supplemental Fig. S1). Sugars were detected by refractive index and
concentrations were determined using external standard calibra-
tions. For the GOS structures, lactose was used for disaccharides,
raffinose for trisaccharides and maltotetraose for tetrasaccharides
and higher oligosaccharides. Samples were subjected to protein
precipitation using Carrez reagent, diluted appropriately and
filtered through a PES 0.22 mm syringe filter (Carl Roth GmbH,
Germany).
3. Results and discussion
3.1. Combination of A. oryzae and C. laurentii
Fig. 1 displays the total GOS yield as a function of lactose con-
version obtained by various combinations of b-galactosidases from
A. oryzae and C. laurentii. In general, C. laurentii exhibits a much
higher transfer activity compared to A. oryzae with the maximum
GOS yield being about twice as high (50% compared to 24%). At the
same time, the former has a very low hydrolytic activity with a GOS/
galactose ratio of 55.6 compared to 2.5 for A. oryzae (see also
supplemental Table S1). In the consecutive combination with
A. oryzae, the enzyme from C. laurentii was able to continue GOS
synthesis, and thus increased GOS yield by 20%. This was mainly
because of the formation of new disaccharide structures (increase
from 2.6% to 12%), but the concentration of tri- and tetrasaccharides
was also enhanced by 5.9% and 3.9%, respectively (see
supplemental Fig. S2). Probably because of an inhibiting effect of
glucose and/or galactose, which were present at the start of the
reaction due to the hydrolysis activity of the first enzyme
(A. oryzae), the total GOS yield (44%) compared to the single
enzyme approach was diminished. Despite this inhibiting effect,
the fact that the free galactose concentration was decreased from
9.2% (3 h with A. oryzae) to 8.1% (another 168 h with C. laurentii,
 C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5
Fig. 1. Total GOS yield as a function of lactose consumption for different combinations
of A. oryzae and C. laurentii in McIlvaine buffer pH 4.5 at 55  C. Symbols: A A. oryzae,
: C. laurentii, ◊ A. oryzae after 96 h synthesis with C. laurentii, D C. laurentii after 3 h
synthesis with A. oryzae, B simultaneous synthesis with C. laurentii and A. oryzae
(ratio 1:1).
data not shown) suggests that galactose can also be used as an
acceptor molecule, and thus, di-galactose derivatives are formed.
This thesis is backed by the HPLC chromatograms (see
supplemental Fig. S4B), where a second disaccharide peak appears
only when C. laurentii is used as the second enzyme (i.e. when a
higher amount of free galactose is available at the beginning of the
reaction), but not when using C. laurentii or A. oryzae alone.
A simultaneous application did not lead to an increase in total
GOS yield compared to the single C. laurentii enzyme, but
maximum yield was obtained at a higher lactose conversion (82.8%
compared to 67.4%, see Fig. 1). The reported yields with C. laurentii
can be considered to be the maximum achievable yield under the
described conditions as the reaction reached equilibrium state (see
supplemental Fig. S3). A sequential combination with C. laurentii as
the first and A. oryzae as the second enzyme led to a degradation of
total GOS.
However, taking a closer look at the synthesized GOS structures
(Fig. 2), the A. oryzae b-galactosidase not only hydrolyzed GOS
synthesized by C. laurentii, but also led to an increased share of
GOS
DP4 (Fig. 2A). After the addition of A. oryzae, an abrupt
depletion of di- and trisaccharides was noted, but concentration of
tetrasaccharides rose from 4.6% to a maximum of 12.3% after
another 0.5 h. Similar, the concentration of pentasaccharides
Fig. 2. GOS composition expressed as percent of total sugars as a function of lactose conversion. A: Consecutive coupling of C. laurentii (96 h, 64% hydrolysis) and A. oryzae. B:
Simultaneous combination of C. laurentii and A. oryzae (open symbols) and synthesis with C. laurentii only (closed symbols). Symbols: C/B disaccharides other than lactose, :/D
⋄
trisaccharides, A/ tetrasaccharides, -/, pentasaccharides, þ hexasaccharides (not detected if C. laurentii was used alone).
Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
3
increased from 0.2% to 3.4% (after another 2 h) and also a small
amount of hexasaccharides (0.7% after another 2 h) was detected.
Therefore, the galactosyl moiety from the hydrolyzed trisaccharides
was at least partially used to build new GOS structures. Because of
the different preferences for elongation, with C. laurentii synthe-
sizing predominantly 4’-galactosyllactose [18] and A. oryzae
preferring b1/6 linkages [10], the structural diversity of the GOS
mixture was enhanced. This can also be depicted from the HPLC
chromatogram with slightly different retention times of the tri- and
tetrasaccharide peaks, respectively (see supplemental Fig. S4A). If
only GOS with a DP
4 are of interest, the yield obtained by the
consecutive synthesis compared to the synthesis with C. laurentii
alone was actually enhanced (15.7% after 97 h with 71% lactose
conversion compared to 7.0% after 168 h with 67% lactose conver-
sion). Considering trisaccharide-GOS as well, the yield was slightly
diminished (35.3% after 97 h compared to 38.5% after 168 h,
respectively).
Anyway, the highest GOS yield (about 50%) was obtained using
C. laurentii alone or C. laurentii and A. oryzae simultaneously. With
trisaccharides being the main fraction in both cases, the latter
supports the synthesis of higher GOS, namely tetra-, penta-, and
hexasaccharides (see Fig. 2B), which increased from 6.7%, 0.4%, and
0% to 12.8%, 3.3%, and 0.4%, respectively (see also supplemental
Table S1). Using C. laurentii alone led to a higher formation of di-
saccharides (12.1% compared to 10.4% with the simultaneous sys-
tem). However, if the synthesis of higher GOS is of predominantly
interest, the costs for applying a second enzyme could still be
worthwhile, despite total GOS yield was not increased. Addition-
ally, similar to the discussion before, the structural diversity of the
GOS mixture was enhanced (see supplemental Fig. S4C).
3.2. Combination of A. oryzae and K. lactis
Fig. 3 shows the GOS yield gained by different combinations of
the K. lactis and A. oryzae b-galactosidases. Despite the enzyme
from A. oryzae has the highest activity at acid pH [19], GOS syn-
thesis was just as well at pH 6.5, which is in accordance with the
findings of Albayrak and Yang [20].
Using the K. lactis enzyme resulted in a GOS yield of 24.6%,
which is the same as with the A. oryzae b-galactosidase. If the latter
was used as the second enzyme in a consecutive reaction, GOS yield
was considerably diminished to 20.4% after another 2 h of treat-
ment. Unexpectedly, the degree of lactose conversion was also
slightly declining from 88.7% to 86.3%. This can be explained by the
hydrolysis of galactosyl-lactose derivatives, which were
4 C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5
Fig. 3. Total GOS yield as a function of lactose consumption for different combinations
of A. oryzae and K. lactis in PEM buffer pH 6.5 at 45  C (K. lactis) and 55  C (A. oryzae).
Symbols: A A. oryzae, : K. lactis, ◊ A. oryzae after 30 min synthesis with K. lactis, D
K. lactis after 3 h synthesis with A. oryzae, B simultaneous synthesis with K. lactis and
A. oryzae (at 45  C, ratio 1:1).
synthesized by K. lactis [10], thus resulting in more free lactose,
which in turn lowers the degree of lactose conversion. However,
the glucose/galactose ratio was remaining constant at about 1.4
(data not shown), suggesting that the galactosyl moiety was not
released, but rather transferred to another sugar molecule. This is
supported by Fig. 4A, which shows that trisaccharides were rapidly
declining, while tetrasaccharides increased from 0.7% (30 min with
K. lactis) to 1.5% (another 2 h with A. oryzae). Hence, trisaccharides
formed by K. lactis were likewise used as substrate and acceptor
molecules. Due to the rising concentration of tetrasaccharides, a
small amount of pentasaccharides (0.07%) could also be detected.
Because neither the galactose nor the glucose concentration was
changing with ongoing reaction time (data not shown), the distinct
decline in GOS disaccharides is believed to be because of them
being also used as acceptor molecules, instead of being hydrolyzed
into monosaccharides.
On the contrary, using A. oryzae as the first and K. lactis as the
second enzyme resulted in an increase of total GOS from 22.7% (3 h
with A. oryzae) to 33.1% (another 20 min with K. lactis). Compared to
the maximum yield with either enzyme alone (24.6%), the total GOS
yield could be increased by 8.5%. This was mainly due to the for-
mation of GOS disaccharides by K. lactis (see Fig. 4B), because
glucose and galactose, which are already present in the mixture
Fig. 4. GOS composition expressed as percent of total sugars as a function of lactose conversion. A: Consecutive coupling of K. lactis (0.5 h, 90% hydrolysis) and A. oryzae. B:
Consecutive coupling of A. oryzae (3 h, 45% hydrolysis) and K. lactis (closed symbols) and synthesis with K. lactis only (open symbols). Symbols: C/B disaccharides other than
⋄
lactose, :/D trisaccharides, A/ tetrasaccharides, -/, pentasaccharides (not detected if K. lactis was used alone).
Please cite this article in press as: C. Fischer, T. Kleinschmidt, Combination of two b-galactosidases during the synthesis of
galactooligosaccharides may enhance yield and structural diversity, Biochemical and Biophysical Research Communications (2018), https://
doi.org/10.1016/j.bbrc.2018.10.091
due to the hydrolytic activity of A. oryzae, can both be used as
acceptor molecules [10]. However, the concentration was lower
compared to using K. lactis alone (10.3% compared to 13%, respec-
tively). Whereas a slight increase in trisaccharides could be noted
with the sequential synthesis (from 16.6% to max. 18.3% or rather
17.1% at maximum total GOS yield), the concentration of tetra- and
pentasaccharides remained the same. Thus, the K. lactis b-galacto-
sidase is not able to use the trisaccharides formed by A. oryzae as
acceptor molecules to synthesize higher GOS. On the other hand,
they are also unsuitable substrates and are thus maintained in the
final product. In summary, using the consecutive approach led to a
higher percentage of GOS
DP3 (22.8%) compared to using the
A. oryzae (21.6%) or K. lactis (11.6%) enzymes alone (see also
supplemental Table S1). Additionally, due to its high hydrolytic
activity, using K. lactis as a second enzyme also reduced the residual
lactose content (degree of lactose conversion doubles from 45% to
90%, see also Fig. 3). If both enzymes were used simultaneously, no
effect on total GOS yield could be observed, indicating that K. lactis
dominated the reaction. Further studies with different enzyme
ratios (that is, reducing the amount of K. lactis) might be needed to
evaluate this process in more detail.
Regarding the structural diversity of the GOS mixture obtained
by the combined approach, no differences were observed in the
HPLC chromatograms (data not shown). This is not surprising since
both enzymes exhibit a preference for b1/6 linkages, thus the
synthesized structures are similar [10] and no shift of retention
times occurs. As a result, combining A. oryzae and K. lactis does not
enhance the structural diversity, but total GOS yield.
3.3. Comparison of both combinations, general conclusions and
further research
From the results presented, it can be inferred, that the synthesis
of galactooligosaccharides using more than one b-galactosidase
enzyme has a high research potential. In principal, an increase in
GOS yield is possible, but strongly dependent on the used enzyme
sources, as well as process design (consecutive or simultaneous
approach). As a general rule, with the consecutive approach it
seems reasonable to use a b-galactosidase, that is rarely inhibited
by monosaccharides and is also able to use them as acceptor mol-
ecules, as the second enzyme. It should then be possible to increase
total GOS yield (including disaccharides) compared to only using
the first enzyme. This is valid for the b-galactosidases from
C. laurentii and K. lactis, which both were able to utilize the sugar
 C. Fischer, T. Kleinschmidt / Biochemical and Biophysical Research Communications xxx (2018) 1e5
mixture prepared with A. oryzae, and synthesize new GOS struc-
tures, of which a large share were disaccharides. On the other hand,
both consecutive approaches, which used A. oryzae as the second
enzyme led to a degradation of GOS and thus diminished GOS yield.
b-Galactosidase from A. oryzae is known to hardly produce any
disaccharides [10,21], hence, the monosaccharides generated by
C. laurentii or K. lactis could not be used as acceptor molecules. But,
depending on the extent of monosaccharide inhibition, total GOS
yield might still be lower compared to using only one enzyme (as
was shown for the consecutive utilization of A. oryzae and
C. laurentii compared to C. laurentii alone). However, with both
studied enzyme combinations (A. oryzae and K. lactis in consecutive
mode, C. laurentii and A. oryzae in simultaneous mode), it was
possible to increase the percentage of higher GOS (DP
3). This can
be beneficial, if a subsequently purification, that is the removal of
glucose, galactose, and lactose, of the mixture is desired, because
the main loss during this step is caused by GOS disaccharides
[14,22,23]. Also, the residual lactose content is reduced (for a
detailed composition of the final reaction mixture see
supplemental Table S1), which is favorable for food applications for
lactose maldigestors. Additionally, as could be shown for the
C. laurentii/A. oryzae combinations, it is possible to enhance the
structural diversity of the final product, but further research is
needed to provide a detailed analysis of the change in GOS struc-
tures (types of glycosidic linkages). Considering the complexity of
human milk oligosaccharides (HMOS), with about 130 different
structures identified so far [24], increasing the diversity of GOS
mixtures might be beneficial for its prebiotic properties, and thus,
its intended use in infant formula as a HMOS substitute. However,
further studies need to be carried out to verify this thesis.
Funding sources
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.bbrc.2018.10.091.
Transparency document
Transparency document related to this article can be found
online at https://doi.org/10.1016/j.bbrc.2018.10.091.
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