Food Chemistry 121 (2010) 307318

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Recent advances rening galactooligosaccharide production from lactose

Aaron Gosling a,b, Geoff W. Stevens a, Andrew R. Barber c, Sandra E. Kentish a, Sally L. Gras a,b,*
a
The Department of Chemical and Biomolecular Engineering, The University of Melbourne, Victoria 3010, Australia
b
The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010, Australia
c
Innovative Food and Plants Division, South Australian Research and Development Institute, Regency International Centre, Days Road, Regency Park, SA 5010, Australia

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

Article history: The production of prebiotic galactooligosaccharides (GOS) by the b-galactosidase catalysed conversion of
Received 26 June 2009 lactose has become commercially important. Yet it remains a challenge to sufciently understand the
Received in revised form 21 October 2009 structure and activity of b-galactosidase, to increase the efciency of transgalactosylation and GOS pro-
Accepted 14 December 2009
duction and to improve the quality of GOS products in a rational way. This review covers the broad but
related aspects of GOS synthesis including: the structure and reaction mechanism of b-galactosidase, fac-
tors effecting yield and productivity of GOS synthesis systems, the structure of GOS products, models for
Keywords:
the kinetics of GOS synthesis and reactor congurations for GOS synthesis. It aims to couple recent dis-
Galactooligosaccharide
Galactosyl oligosaccharide
coveries with established knowledge to enhance understanding of the complex biochemistry of GOS
Galacto-oligosaccharide synthesis.
GOS 2009 Elsevier Ltd. All rights reserved.
Prebiotic
Galactosyl transfer
b-galactosidase
Lactose hydrolysis
Review

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
2. Reaction mechanism and structure of b-galactosidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 308
3. Factors effecting GOS yields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
4. Structure of GOS products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
5. Modelling of the kinetics of GOS synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313
6. Reactor configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
6.1. Whole cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
6.2. Membrane processing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314
7. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315

1. Introduction as galactooligosaccharides (GOS) are an example of functional

Functional foods are generally dened as foods or food ingredi-
ents which impart a health benet above and beyond the nutri-
tional value expected from food. The group of foods referred to
* Corresponding author. Address: The Department of Chemical and Biomolecular
Engineering, The University of Melbourne, Victoria 3010, Australia. Tel.: +61 3 8344
6281; fax: +61 3 8344 4153.
E-mail address: sgras@unimelb.edu.au (S.L. Gras).
foods, as they are prebiotic (Van Loo et al., 1999). A prebiotic has
been dened as being a selectively fermented ingredient that al-
lows specic changes, both in the composition and/or activity in
the gastrointestinal microora that confers benets upon host well
being and health (Roberfroid, 2007). Simply put, GOS are not di-
gested by humans or other animals, and selectively increase the
benecial microora of the intestine, leading to health benets
that are extensively recognised (Macfarlane, Steed, & Macfarlane,
2008).

0308-8146/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2009.12.063
308 A. Gosling et al. / Food Chemistry 121 (2010) 307318
GOS molecules (for example, Gal (b 1 ? 4) Gal (b 1 ? 4) Glc) are
typically synthesised by the enzymatic activity of b-galactosidase
on lactose in a reaction known as transgalactosylation. Other car-
bohydrate-modifying enzymes, such as b-glucosidases and b-gly-
cosidases, can also catalyse this chemistry. b-Galactosidase
belongs to a class of hydrolytic enzymes and has long been used
in the dairy industry to hydrolyse lactose, producing glucose and
galactose. This hydrolytic activity increases the sweetness of a
dairy product, and also lowers lactose concentration, which can
be benecial to lactose intolerant consumers (Lomer, Parkes, &
Sanderson, 2008). The competing transgalactosylation reaction
and formation of GOS during b-galactosidase-catalysed conversion
of lactose was rst observed in the early 1950s (Wallenfels, 1951).
Since this time, GOS have become a commercially important prod-
uct manufactured in Asia and Europe (Crittenden & Playne, 1996),
with a manufacturing facility also opening recently in Australia. Six
thousand tonnes of GOS were manufactured in 2005 in Japan alone
(Taniguchi, 2005).
With increasing public awareness of nutrition (Cowburn &
Stockley, 2005) there has been increased demand for foods with
demonstrable health benets (Borra & Bouchoux, 2009). An exam-
ple of this can be found with yoghurts, where consumers put a high
preference on products perceived to be low in fat (Valli & Traill,
2005). Dairy products processed exploiting the GOS-forming activ-
ity of b-galactosidase could contain lower sugar content (e.g., lac-
tose) and higher soluble bre (e.g., GOS) and therefore have a
competitive edge. This makes further increases in GOS manufac-
ture likely.
This review provides an overview of the recent advances in GOS
production from lactose for food applications. The importance of
the eld is emphasised by the literature activity surrounding it,
with recent reviews published on the health benets and charac-
teristics of prebiotic oligosaccharides (including GOS) (Barreteau,
Delattre, & Michaud, 2006; Gnzle, Haase, & Jelen, 2008; Macfar-
lane et al., 2008) and b-galactosidase enzymology (Mlichova &
Rosenberg, 2006; Panesar, Panesar, Singh, Kennedy, & Kumar,
2006; Trincone & Giordano, 2006). Here, the focus is on recent ad-
vances concerning the reaction mechanism and structure of b-
galactosidase, factors effecting GOS yields the structure of GOS
products, models for the kinetics of GOS synthesis, and reactor con-
gurations for GOS synthesis. Specically, this review aims to in-
crease understanding of scientic issues related to production of
GOS on a commercial scale for sale as a food product.
2. Reaction mechanism and structure of b-galactosidase
The biochemical mechanism for b-galactosidase activity is
highly relevant to the synthesis of GOS products as it explains
how b-galactosidase, renowned for its hydrolytic activity, can olig-
omerise galactosides under kinetic control. This mechanism also
offers insight into some of the environmental factors which inu-
ence GOS synthesis and yield.
A brief summary of the critical catalytic events and the struc-
tural features of the enzyme important for these events will be pre-
sented here, as previous reviews are available on the hydrolysis
reaction mechanism of both b-galactosidases specically (Mahon-
ey, 1998), and glycoside hydrolases more generally (Vocadlo & Da-
vies, 2008). Recent studies on the structure of b-galactosidase with
substrates and/or inhibitors bound in the active site are particu-
larly relevant and will form the emphasis of this section.
The rst event during b-galactosidase activity is the docking of a
substrate molecule. When the substrate is lactose the glycan moi-
ety is galactosyl and the aglycan moiety is glucosyl (represented as
the R1 group in Fig. 1). While b-galactosidase shows high substrate
specicity requiring substrates with galactosyl glycans linked by a
glycosidic bond of beta stereochemistry, there is broad specicity
for the aglycan moiety. Consequently, the R1 group (Fig. 1) could
equally represent an oligosaccharide, when the galactoside sub-
strate is a GOS molecule. Structural features of b-galactosidase that
are important in substrate docking interactions within the active
site have been reported and include groups participating in hydro-
gen bonding, an electrostatically bound sodium ion and aromatic
amino acid side chains forming a platform essential for binding
hexose substrates (Gloster et al., 2004; Hidaka et al., 2002; Juers
et al., 2001; Nerinckx, Desmet, & Claeyssens, 2003).
After the substrate has docked in the active site of the b-galac-
tosidase, catalysis can occur. It is currently accepted that the reac-
tion occurs with a retaining mechanism. The details of the catalysis
are well described (Brs, Moura-Tamames, Fernandes, & Ramos,
2008; Juers et al., 2001; Vasella, Davies, & Bhm, 2002; Zechel &
Withers, 2000). A covalent bond forms between the galactosyl
moiety and the enzyme, followed by galactosyl transfer to an
acceptor nucleophile (Fig. 1).
The reaction pathway diverges at this point. The result of the
reaction depends on the identity of the galactosyl acceptor, for
which there is broad specicity. When the galactosyl acceptor is
water (i.e., R2 = H in Fig. 1), free galactose is released. The result
of the reaction is hydrolysis of the galactoside, leading to degrada-
tion of both lactose and GOS.
When the reaction pathway diverges towards galactosyl trans-
fer and GOS synthesis, the galactosyl acceptor is another saccha-
ride (i.e., R2 = glucose, galactose, lactose, or a GOS molecule). The
mechanism of transgalactosylation by b-galactosidase has received
less attention compared to that of hydrolysis. To our knowledge, all
currently published data on the mechanism of transgalactosylation
draws inferences from the hydrolytic reaction, rather than directly
observing transgalactosylation. It is not known if the interactions
in the active site differ when the acceptor is a saccharide, com-
pared to water. However, it is well known that different enzymes
display different selectivities for water and saccharides, as differ-
ent enzymes yield varying amounts of GOS at the same lactose
concentration (Prenosil, Stuker, & Bourne, 1987b; Zarate & Lopez-
Leiva, 1990). It seems likely that this difference in the GOS yields
is a result of structural and/or mechanistic differences between
b-galactosidases from different sources. The structures of GOS
products may also vary due to differences in the structure and/or
mechanism of different b-galactosidases.
Mutagenesis studies have suggested, if not demonstrated, some
of the structural features which are important in inuencing the
propensity of an enzyme for transferring galactosyl moieties to
saccharides rather than water. Deleting the sequence coding for
580 amino acid residues at the C-terminal of the b-galactosidase
gene encoding BIF3 from Bidobacterium bidum yielded an en-
zyme that produced GOS but had little or no hydrolytic activity
(Jrgensen, Hansen, & Stougaard, 2001). This C-terminal section
of the protein contained a domain with homology to known galact-
ose binding domains. It was hypothesised that deletion of this do-
main allowed greater steric access to the active site, favouring
saccharide acceptors. A patent on this truncated enzyme has been
led for applications including production of dairy foods and die-
tary supplements (Jrgensen, Hansen, & Stougaard, 2007).
Mutagenesis of single amino acids has also proved informative.
The GOS yield and pH optima of the b-glucosidase from Pyrococcus
furiosus were both increased by single amino acid substitutions
and a mutant enzyme carrying both changes gave a GOS yield of
approximately double the native enzyme (Hansson, Kaper, van
der Oost, de Vos, & Adlercreutz, 2000). It seems likely that negative
public opinion towards the inclusion of genetically modied ingre-
dients in food may render this interesting approach unsuitable for
dairy processing. Perhaps the true value lies in the deeper under-
standing of the structural features of the enzyme which impact
 A. Gosling et al. / Food Chemistry 121 (2010) 307318 309

Fig. 1. The reaction mechanism for b-galactosidase activity on galactosides (adapted from Juers et al. (2001)). R1 represents the aglycan moiety of the galactoside substrate;
R2 represents the nucleophile galactosyl acceptor.

on the maximum possible GOS yield, and this could aid in vitro and vant GOS producing b-galactosidases becomes available there will
in silico screening to identify novel native b-galactosidases with be opportunities for informative comparison with this model
higher GOS-producing characteristics. enzyme.
To aid the development of bioinformatic tools, more structural Another data set that could prove useful for building bioinfor-
data on the enzymes currently employed in GOS production is re- matic tools to identify optimum b-galactosidases exists in the Car-
quired. The X-ray crystal structures of b-galactosidase from sev- bohydrate-Active Enzymes database (http://www.cazy.org
eral microbial sources have been deposited in the Protein Data (Cantarel et al., 2009)). This database classies enzymes which
Bank (http://www.rcsb.org (Berman et al., 2000)) (Table 1). How- act on carbohydrates according to their primary amino acid se-
ever, none of the enzymes with solved structures are known to be quence (Henrissat, 1991). b-Galactosidases are placed in a broad
used in food processing. The structure of the Escherichia coli classication of glycoside hydrolases, which are further organised
enzyme is well characterised (Matthews, 2005) and the transga- into glycoside hydrolase families (GHF). Enzymes designated as
lactosylation characteristics of this enzyme have also been thor- b-galactosidases (E.C. 3.2.1.23) fall into four GHFs: 1, 2, 35 and
oughly described (Chen, Ou-Yang, & Yeh, 2003; Huber, Kurz, & 42. GHF 1, 2 and 35 contain enzymes other than b-galactosidases,
Wallenfels, 1976; Juers, Hakda, Matthews, & Huber, 2003; while only b-galactosidase has currently been classied into GHF
Mladenoska, Winkelhausen, & Kuzmanova, 2008; Richard, Wester- 42. It has been stated that b-galactosidases from GHF 2 generally
feld, Lin, & Beard, 1995). As structural data from industrially rele- have higher activity for both hydrolysis and transfer (van den
Broek, Hinz, Beldman, Vincken, & Voragen, 2008) but a systematic
study investigating correlations between amino acid sequence
homology and enzyme characteristics has not yet been reported.
Table 1
b-Galactosidases with crystal structures deposited in the Protein Databank (Berman Such a study is hampered by a lack of both published amino acid
et al., 2000). sequences for the commonly-used, commercially-available GOS
producing b-galactosidases, and standard reaction conditions to
Microbial Source Reference
compare the GOS-producing characteristics of different enzymes
Escherichia coli Juers et al. (2001)
(e.g., maximum GOS yield at a standard lactose concentration with
Thermus thermophilus A4 Hidaka et al. (2002)
Sulfolobus solfataricus Gloster et al. (2004) other dened reaction parameters).
Arthrobacter sp. C2-2 Sklov et al. (2005)
Bacteroides fragilis Ramagopal et al. (2009) 3. Factors effecting GOS yields
Bacteroides Palani, Kumaran, Burley, and Swaminathan
thetaiotamicron (2009)
Penicillium sp. Rojas et al. (2004) The maximum concentration of GOS typically obtained in a
reaction varies widely, depending on reaction conditions, as does
310 A. Gosling et al. / Food Chemistry 121 (2010) 307318

Table 2
Maximum GOS concentrations achieved in different reaction conditions.

Enzyme sourcea Reaction conditions Lactose Maximum Reference

conversion GOS yieldb
Initial lactose Temperature pH Buffer composition
(%)
concentration (C)
Sulfolobus 600 g/l 80 6.0 50 mM potassium phosphate 70 52.5% (w/w) Park et al., (2008)
solfataricus
Aspergillus oryzae 500 g/l 40 4.5 20 mM citrate phosphate 56 52% (w/w) Neri et al. (2009)
Bullera singularis 180 g/l 50 6.0 50 mM sodium phosphate 72 50% (w/w) Cho, Shin, and Bucke (2003)
Aspergillus oryzae 1.67 M 40 4.5 pH adjusted with 1 M HCl 55 31% (mol/ Iwasaki et al. (1996)
mol)
Saccharopolyspora 600 g/l 70 7.0 pH maintained by addition of 1 M 75 41% (w/w) Nakao et al. (1994)
rectivirgula NaOH, 10 lM MnCl2
Sterigmatomyces 200 g/l 60 5.0 100 mM sodium acetate 63 39% (w/w) Onishi and Tanaka (1995)
elviae
Lactobacillus 205 g/l 37 6.5 50 mM phosphate containing 80 38% (w/w) Splechtna et al. (2006)
reuteri 1 mM MgCl2
Pyrococcus 270 g/l 70 5.5 20 mM sodium citrate 80 33% (w/w) Petzelbauer, Reiter, Splechtna,
furiosus Kosma, and Nidetzky (2000)
Bidobacterium 400 g/l 45 6.8 30 mM phosphate 60 32.5% (w/w) Hsu et al. (2007)
longum
Sulfolobus 270 g/l 70 5.5 20 mM sodium citrate 72 26% (w/w) Petzelbauer et al. (2000)
solfataricus
Kluyveromyces 400 g/l 40 7.0 200 mM potassium phosphate 92 24.8% (w/w) Chockchaisawasdee et al. (2005)
lactis containing 2 mM MgCl2
Thermotoga 500 g/l 80 6.0 50 mM potassium phosphate 50 19% (w/w) Ji et al. (2005)
maritima
Pyrococcus 745 mmol/kg 80 5.0 0.2 mol/kg citrate 73 18.1% (w/w) Bruins et al. (2003a)
furiosus
a
All enzymes are b-galactosidases, except the Sulfolobus solfataricus and Pyrococcus furiosus derived b-glycosidases.
b
GOS concentration as a percentage of initial lactose concentration.

the GOS yield (as a percentage of the initial lactose). This is true
even when using b-galactosidases recognised for their propensity
to form GOS, as shown in Table 2 (see Prenosil et al. (1987b) and
Zarate et al. (1990), for past compilations). A yield of 315 g/l GOS
from 600 g/l lactose (52.5% (w/w) yield) has been reported (Park,
Kim, Lee, Kim, & Oh, 2008); it appears that yields over 50% are
not often exceeded. More typical optimized yields are between
30% and 40% (w/w).
The time at which a reaction is harvested has an inuence on
the maximum GOS concentration (and therefore yield on initial
lactose) because GOS are simultaneously synthesised and degraded
by b-galactosidase. The concentration of GOS at its peak is deter-
mined by the relative kinetics of synthesis and degradation (Buch-
holz, Kasche, & Bornscheuer, 2005). As such, factors which increase
the maximum concentration of GOS can be thought of as factors
which increase the rate of GOS synthesis or decrease the rate of
degradation.
With this in mind, it is not surprising that the initial lactose con-
centration is well accepted as one of the key factors inuencing the
maximum GOS concentration achieved (Mahoney, 1998). This
dependence on initial lactose concentration has at least two con-
tributing factors: increased availability of saccharide galactosyl
acceptors, and decreased availability of water. The former would
be expected to increase the rate of GOS synthesis, and the latter
would be expected to decrease both the rate of GOS degradation
and lactose hydrolysis. However, the relative importance of these
two contributions is unclear and will be discussed here.
It is difcult to quantitatively assess the contribution of saccha-
ride acceptor concentration on the maximum GOS concentration.
Traditional MichaelisMenten enzyme kinetic constants, and espe-
cially the rate constant at saturating substrate concentrations kcat,
are rarely reported for GOS formation. This is perhaps due to the
difculties in accurately quantifying GOS formation rates. When
the Michaelis constant (Km) is quoted for transgalactosylation, it
is typically orders of magnitude higher than for lactose hydrolysis;
e.g., 800 mM lactose for transgalactosylation compared to 13 mM
for lactose hydrolysis for a B. bidum b-galactosidase (Dumortier,
Brassart, & Bouquelet, 1994), demonstrating the need for high lac-
tose concentrations to achieve the maximum transgalactosylation
velocity.
By comparison, the contribution of water activity to transgalac-
tosylation is easier to probe. Yields of GOS have been found to be
higher when using organic solvent systems that lower the water
activity of the reaction media (Cruz-Guerrero, Gmez-Ruiz, Vinie-
gra-Gonzlez, Brzana, & Garca-Garibay, 2006). In some instances,
this increased yield was shown to be due to reduced hydrolysis
(Chen, Wei, & Hu, 2001; Maugard, Gaunt, Legoy, & Besson, 2003),
consistent with the commonly accepted theory that decreasing
the water activity of reaction media increases the yields of enzy-
matic transfer reactions by decreasing competing hydrolysis reac-
tions (Trincone & Giordano, 2006). The toxicity of organic solvents
limits their use in food applications but these studies do demon-
strate that decreasing water activity contributes to increasing
GOS yields. Therefore, the increase in GOS yield noted with in-
creased initial lactose concentration is likely to result in part from
a decreased water activity within the reaction media due to high
saccharide concentrations.
Temperature can increase the solubility of lactose, which is rel-
atively low at ambient temperatures (c.a. 220 g/l at 25 C (White,
2000). Much recent research has therefore been directed towards
the discovery and characterisation of thermostable enzymes (Bru-
ins, Strubel, van Lieshout, Janssen, & Boom, 2003a; Chen et al.,
2008; Hatzinikolaou et al., 2005; Ji, Park, & Oh, 2005; Park et al.,
2008). These enzymes allow processes to be operated at increased
temperatures and therefore with higher initial lactose concentra-
tions, which in turn can deliver increases in the maximum GOS
concentration achieved. For example, 315 g/l GOS was produced
from a solution of 600 g/l initial lactose compared to 150 g/l GOS
produced from a solution of 300 g/l initial lactose when both solu-
tions were at 80 C (Park et al., 2008). Interestingly, while the max-
imum GOS concentration increased in this instance, the yield of
GOS per gram of initial lactose did not vary across this wide range
 A. Gosling et al. / Food Chemistry 121 (2010) 307318
of lactose concentrations. The possibility of operating at higher
temperature using thermostable enzymes also offers the advan-
tage of limiting potential for microbial contamination within the
process. Increases in yields will, however, need to be balanced with
the increased loss of enzyme activity at higher temperatures. Mail-
lard reactions between amino side-chains on the enzyme and sug-
ars have been shown to become signicant in inactivating a
thermostable enzyme at and above 80 C (Bruins, Van Hellemond,
Janssen, & Boom, 2003b).
Temperature seems to further affect GOS synthesis indepen-
dently of lactose concentration. There are several examples of
studies which conclude that GOS yields increase with increasing
temperature (Boon, Janssen, & vant Riet, 2000; Hsu, Lee, & Chou,
2007). However, individual enzymes clearly display different char-
acteristics in their response to temperature, as there are also exam-
ples of GOS yield remaining constant with varied temperature
(Iwasaki, Nakajima, & Nakao, 1996; Prenosil, Stuker, & Bourne,
1987a; Splechtna et al., 2006).
It has long been recognised that pH affects the kinetics of lac-
tose hydrolysis and GOS synthesis by the E. coli b-galactosidase dif-
ferently (Huber et al., 1976). This suggests that it may be possible
to selectively control the rates of GOS synthesis and degradation by
varying the pH of the reaction medium, thereby increasing GOS
yields. However, this may be a characteristic which varies between
individual enzymes, similar to the effect of temperature on yield.
Many recent studies have also found similar pH optima for GOS
synthesis and galactoside hydrolysis (Hsu, Yu, & Chou, 2006; Hsu
et al., 2007; Ji et al., 2005; Park et al., 2008). This was the case with
b-galactosidase from Sulfolobus solfataricus, which showed an opti-
mal pH for GOS synthesis at 6.0, close to the optimal for hydrolysis
of the model colorimetric substrate ortho-nitro phenol galactoside
at pH 6.5 (Park et al., 2008).
Galactose and/or glucose are commonly recognised as inhibi-
tors of lactose hydrolysis for many b-galactosidases, with galactose
typically exerting a greater effect (Hatzinikolaou et al., 2005; Jura-
do, Camacho, Luzn, & Vicaria, 2004). Galactose inhibition of lac-
tose hydrolysis is generally competitive, as galactose can form
galactosylenzyme intermediates with b-galactosidase, precluding
lactose from the active site. Glucose inhibition of lactose hydrolysis
appears more complex, as it inhibits some b-galactosidases com-
petitively (Boon, Janssen, & van der Padt, 1999) and some uncom-
petitively (Demirhan, Apar, & zbek, 2008). Inhibition by these
monosaccharides has also been recently shown to affect the
amount of GOS produced. The presence of galactose reduced the
rate at which the b-galactosidase from Aspergillus oryzae consumed
lactose more than the presence of glucose, and both glucose and
galactose, separately and in combination, appeared to lower the
Fig. 2. Examples of how GOS structure can vary. (A) Composition, (B) regiochemistry and (C) degree of polymerisation. Abbreviations are Gal for galactose and Glc for glucose.
311
maximum amount of GOS formed (Neri et al., 2009). Enzymes from
different sources again display different characteristics. For exam-
ple, lactose utilisation by b-galactosidase from Kluyveromyces lactis
was inhibited by both glucose and galactose but only galactose re-
duced the maximum GOS concentration achieved with this en-
zyme (Chockchaisawasdee, Athanasopoulos, Niranjan, & Rastall,
2005).
4. Structure of GOS products
GOS structures can differ in saccharide composition, regiochem-
istry of the glycosidic linkages and the degree of polymerisation, as
illustrated in Fig. 2. There are a number of important consequences
arising from these structural differences: GOS structures give dif-
ferent chemical properties, in vitro evidence suggests that prebiotic
microbes grow differently on oligosaccharides with different struc-
tures (Rycroft, Jones, Gibson, & Rastall, 2001; Sanz, Cote, Gibson, &
Rastall, 2006a, 2006b; Sanz, Gibson, & Rastall, 2005) and GOS
structure also affects characteristics relevant to their use in food
applications. Understanding the factors which inuence the struc-
ture of the GOS products may therefore help to design processes
that produce oligosaccharide motifs with better properties and
higher yield. Some of the vast number of structurally different
GOS species produced by a variety of enzymes acting on lactose
have been compiled previously (Prenosil et al., 1987b; Zarate
et al., 1990; Mahoney, 1998). In this section, recent research and
generalisations about the structures of GOS produced by industri-
ally relevant enzymes are highlighted.
The composition of GOS produced from lactose by b-galactosi-
dase usually has the structure GalnGlc, where n indicates the de-
gree of polymerisation (DP), and is typically 15 (Mussatto &
Mancilha, 2007). However, Galn Gal structures are also known
(Sanz, Sanz, & Martinez-Castro, 2002), as well as branched struc-
tures, which contain multiple galactose residues attached to a sin-
gle glucose moiety at the reducing terminal of the oligosaccharide
(Yanahira et al., 1995).
It has been well established that the regiochemistry of the GOS
product is primarily controlled by the identity of the enzyme used
(Wallenfels & Weil, 1972), and it seems likely this is related to the
structure and/or reaction mechanism of the enzyme. For example,
the galactosyl moiety moves deeper into the active site of the E. coli
b-galactosidase when galactosyl-enzyme intermediates forms
(Juers et al., 2001). This makes the galactosyl-like moiety less ste-
rically accessible to a nucleophilic acceptor. This inaccessibility
would be expected to favour galactosyl transfer, resulting in the
formation of a 1 ? 6 bond with the most sterically open alcohol
312 A. Gosling et al. / Food Chemistry 121 (2010) 307318

group (C6OH) of a saccharide acceptor. This is consistent with the lus circulans b-galactosidase, the abundance of Gal (b1 ? 4) Gal
b1 ? 6 linkage in allolactose (Gal b1 ? 6 Glc), the major transga- (b1 ? 4) Glc decreased from 95% to 35% of the total trisaccharides
lactosylation product of the activity of the E. coli enzyme on lactose between 1 and 24 h, respectively, while Gal (b1 ? 4) Gal (b1 ? 3)
(Jobe & Bourgeoi, 1972). This 1 ? 6 linkage also appears to be the Glc increased its abundance from 4% to 18% of the total trisaccha-
preferred regiochemistry produced between dimers of galactose by rides over the same time interval (Yanahira et al., 1995). This
the E. coli b-galactosidase, as Gal (b1 ? 6) Gal was the only regio- highlights the opportunities for more selective production of spe-
isomer of di-galactose detected after reaction of a synthetic Gal do- cic structural motifs in GOS products through reaction
nor (o-nitrophenyl galactoside) with a synthetic Gal acceptor (3-O- engineering.
methyl Glc) (Yoon & Mckenzie, 2005). Many of the industrially rel- The degree of polymerisation (DP) may also affect the prebi-
evant b-galactosidases also preferentially produce b1 ? 6 linkages otic efcacy of GOS. There is in vitro evidence that some Bidobac-
between saccharides (Table 3), suggesting similar interactions be- terium species preferentially catabolise trimeric and tetrameric
tween the enzyme and galactosyl moieties in these systems. To our GOS over disaccharides (Gopal, Sullivan, & Smart, 2001) or lactose
knowledge, no mechanistic explanation has been offered to date to (Amaretti et al., 2007). It also seems likely that higher DP GOS
explain the preferential production of other linkages, such as would be more resistant to fermentation in the gut, and would
b1 ? 4 or b1 ? 3. therefore deliver a prebiotic effect in more distal locations in
It seems that b-galactosidases from prebiotic microbes produce the intestine. It has been suggested that controlling the DP can re-
distinctly structured GOS mixtures and grow more rapidly on the duce possible unpleasant side-effects linked to GOS consumption,
GOS which were produced by their own b-galactosidases (Rabiu, such as atulence and a laxative effect, due to osmotic pressure in
Jay, Gibson, & Rastall, 2001). This can translate to greater prebiotic the intestinal lumen (Topping & Clifton, 2001). DP also affects the
efcacy. It has been demonstrated in a human clinical trail that characteristics of GOS relevant to its incorporation into food
GOS produced by b-galactosidase from the prebiotic microbe B. products, such as sweetness and gelling properties (Delzenne,
bidum had different structure and signicantly greater prebiotic 2003).
potency compared to a commercial GOS mixture (Vivinal GOS As with regiochemistry, the typical DP of a GOS product is char-
produced by b-galactosidases from A. oryzae) (Depeint, Tzortzis, acteristic of the b-galactosidase used (Prenosil et al., 1987a) and is
Vulevic, IAnson & Gibson, 2008). The GOS produced by the B. bi- also a function of the reaction time. For example, trimeric GOS
dum b-galactosidase for this trial contained a majority of b1 ? 3 need to be present to act as galactosyl acceptors, before tetrameric
linkages, with a lesser amount of b1 ? 4 and b1 ? 6 linkages, GOS can be formed. In reactions catalysed by the b-galactosidase
while the commercial GOS contained primarily b1 ? 4 and from A. oryzae, the maximal concentration peak of the tetrameric
b1 ? 6 linkages. GOS occurs later than that of trimeric GOS (Neri et al., 2009). There
While the b-galactosidase directs GOS regiochemistry, this reg- has also been reports showing that penta- and hexasaccharides
iospecicity is not absolute, and GOS are inevitably produced as reach a maximum concentration well after the maximum concen-
mixtures of regioisomers (Mahoney, 1998). The linkages between tration of GOS has been reached (Albayrak & Yang, 2002), indicat-
sugars in these mixtures are formed and hydrolysed at different ing sacrices in total yield may be required to produce elevated
rates, so the reaction time inuences the structures present in a levels of high-DP GOS within the product mixture. This approach
GOS mixture. For example, during lactose conversion by the Bacil- may also lead to a decrease in the average DP of the mixture, as di-
meric GOS species can become dominant after the maximum con-
centration of GOS is reached and the degradation of higher DP GOS
Table 3 becomes increasingly important (Smart, 1991).
GOS-producing characteristics of commercially important b-galactosidases. Examples where the DP of the GOS product has been controlled
Microbial enzyme Yields with 5% Typical GOS Predominant by process parameters such as temperature, pH and lactose con-
source (w/v) initial degree of linkages centration have also been recently reported. A commercial prepa-
lactose polymerisation formed ration of Aspergillus aculeatus b-galactosidase produced higher
concentration maximum concentrations of high DP and trimeric GOS and de-
(%)
creased maximum concentrations of dimeric GOS with an increase
Cryptococcus laurentii 43b Mostly b1 ? 6 in reaction temperature. The same enzyme also produced higher
Ohtsuka et al. trisaccharides
maximum concentrations of trimeric GOS at pH 6.5 than at pH
(1990)
Bacillus circulans 41b Di- to b1 ? 4 Usui, 4.5, 5.5 or 7.5 without producing a signicant difference in dimeric
Mozaffar, pentasaccharides Kubota, and or high-DP GOS (Cardelle-Cobas, Villamiel, Olano, & Corzo, 2008).
Nakanishi, Ohi (1993) The maximum amount of tri- and tetrameric GOS and the ratio
Matsuno, and
of trimers to tetramers, produced by A. oryzae b-galactosidase
Kamikubo (1984)
Streptococcus 25m Disaccharides b1 ? 6 Toba,
operating in milk, could also be varied by altering the reaction tem-
thermophilus Tomita, Itoh, perature and the initial lactose concentration in this system (Chen,
Greenberg and and Adachi Hsu, & Chiang, 2002).
Mahoney (1983) (1981) Much potential for optimising the structure of a GOS product
Kluyveromyces fragilis 22b Roberts and Di- and b1 ? 6
exists, particularly for improvements in prebiotic efcacy. How-
Pazur, Tipton, Pettinati (1957) trisaccharides
Budovich, and ever, identifying structural motifs which considerably enhance
Marsh (1958) prebiotic bioactivity remains a challenge. Commercially-produced
Aspergillus oryzae 15b Iwasaki Di- to b1 ? 6 GOS are typically complex mixtures containing glucose, galactose
Toba, Yokota, and et al. (1996) hexasaccharides
and lactose, as well as many GOS species (Crittenden & Playne,
Adachi (1985)
Kluyveromyces lactis 14m Mozaffar, Di- to b1 ? 6
1996). The difculties in attributing increased prebiotic potency
Asp, Burvall, Nakanishi, and tetrasaccharides to a single structural feature of a saccharide in such mixtures is
Dahlqvist, Matsuno (1985) perhaps best exemplied by lactose itself being dubbed a
Hallgren, and conditional prebiotic (Adamczak, Charubin, & Bednarski, 2009;
Lundblad (1980)
Schaafsma, 2008; Szilagyi, 2004). More research is therefore
b
Lactose and buffer based reaction medium. required to purify and characterise the prebiotic qualities of indi-
m
Milk-based reaction medium. vidual GOS species.
 A. Gosling et al. / Food Chemistry 121 (2010) 307318 313

5. Modelling of the kinetics of GOS synthesis
GOS synthesis is complex. Complexity is generated through dif-
ferent reactions (transfer and hydrolysis) acting on different sub-
strates (differently structured GOS species and lactose) at
different rates. Even the stereochemistry of the anomeric carbon
changes the kinetics of b-galactosidase-catalysed hydrolysis of lac-
tose (Huber, Hurlburt, & Turner, 1981) and the stereochemistry of
GOS molecules could also be expected to similarly alter the kinetics
of b-galactosidase catalysis. Despite these complexities, there have
been many recent publications that model the formation of GOS
over time (Table 4). These models can be categorised as either
mechanistic or empirical.
Mechanistic models are built by initially proposing a reaction
pathway. Rate constants are allocated to each step during catalysis,
and then experimentally estimated. The detailed knowledge of the
reaction mechanism of b-galactosidase makes this approach
attractive. However, to limit the number of rate constants which
need experimental estimation, simplications are inevitably made
about the reaction system. For example, it has been common for
studies to lump GOS molecules into DP classes (e.g., trimers, tetra-
mers, etc.), rather than attempt to study the rates of formation and
degradation for all regioisomers within a DP class. When more
simplications are made, the model can be less accurate, but more
convenient to construct, so a balance between the accuracy and the
feasibility of making the model needs to be found.
An example of a mechanistic model describing the change in
lactose, glucose, galactose, GOS disaccharides and GOS trisaccha-
rides formed over time was proposed for the activity of b-galacto-
sidase from K. lactis on lactose (Kim, Ji, & Oh, 2004). This model
included complex considerations, such as competitive galactose
inhibition and a preference for this enzyme to use glucose as the
galactosyl acceptor over lactose. The model also included simpli-
cations, such as assuming that trimers were the largest GOS species
formed and that all trimers had a Gal2Glc composition. The t of
the data to the model was reasonable but nine constants required
estimation.
Simpler mechanistic models do exist. A model has been re-
ported which uses only six constants (one set to unity) to describe
the concentrations of trisaccharide, lactose, glucose and galactose
during the course of the reaction catalysed by the b-galactosidase
preparation from B. circulans (Boon et al., 1999). Although the sim-
plicity of this model is attractive, it was less accurate for the lowest
and highest initial lactose concentration reaction courses reported
and appeared to become less accurate the longer the reaction pro-
gressed. Perhaps these deciencies were due to the simplication
that all oligosaccharides were considered to be trimeric species
containing two galactose and one glucose residue. It is known that
the GOS products of B. circulans b-galactosidase activity frequently
do not have this composition or DP (Yanahira et al., 1995).
Thus it seems that available mechanistic models strike a bal-
ance between a poor t of the model to experimental data due to
oversimplication and inappropriate estimation of a large number
of rate constants. Models including more terms are generally able
to t experimental data more closely but the greater number of de-
grees of freedom in such a model can produce very large error mar-
gins around individual terms.
Mechanistic models can offer insights into which process
parameters can be usefully manipulated to optimise the reaction.
The MichaelisMenten equation is a classic model most commonly
used for this purpose. This equation describes the reaction velocity
as a function of substrate concentration, with the initial reaction
velocity asymptotically approaching a theoretical maximum as
substrate concentration increases. For the b-glycosidase from P.
furiosus, the initial rate of galactose formation obeys this Michae-
lisMenten saturation behaviour but initial rates for the formation

Table 4
Kinetic models of GOS formation by b-galactosidase proposed in the literature.

Model description Number of Enzyme source Simplicationsa/variables with no signicant effect Complexitiesa/variables Reference
constantsa/ on GOS yieldb with signicant effect on
variables modelledb GOS yieldb
Mechanistic, saccharide 6 Bacillus GOS dened as only GalGalGlc. Gal inhibition Glc inhibition Boon et al.
concentration over circulans noted, but not modelled. GOS dened as tri and (1999)
time tetrasaccharides.
14 Aspergillus Hydrolysis of tetrasaccharides not included. No Viscosity of medium Iwasaki
oryzae distinction made between Gal and Glc. included et al.
(1996)
9 Kluyveromyces GOS dened as GalGlc dimers and trisaccharides. Glc and Gal inhibition Kim et al.
lactis (2004)
5 Pyrococcus. GOS dened as trisaccharides. Competitive Glc inhibition Bruins
furiosus et al.
(2003a)
16 Immobilised GOS dened as only 2 species; trimers and GalGal. Inhibition and mutarotation Bakken
Bacillus of Glc and Hill
circulans (1992)
Empirical, separate [L]i, T, R.T., [E]: [L]i Aspergillus [L]i, T [E]: [L]i, R.T. Chen et al.
yield of different DP oryzae (2002)
saccharides
Empirical, GOS [L]i, T, R.T., cell Bidobacterium T, R.T., cell concentration [L]i Roy et al.
concentration and concentration infantis whole (2002)
yield cells
Aspergillus [E], [L]i pH, T
oryzae
[E], [L]i, pH, T Kluyveromyces T [E], [L]i, pH Rustom
lactis et al.
Kluyveromyces [E], [L]i pH, T (1998)
fragilis

[L]i-Initial lactose concentration; [E]-enzyme concentration; T-temperature; and RT-reaction time.

a
Mechanistic models.
b
Empirical models.
314 A. Gosling et al. / Food Chemistry 121 (2010) 307318
of trisaccharides, glucose and the initial rate of lactose consump-
tion continued to increase with increasing lactose concentration
(Bruins et al., 2003a). As the difference between galactosyl transfer
and hydrolysis rates appears to become greater at increasing lac-
tose concentrations, the results suggest that the optimal initial lac-
tose concentration for GOS synthesis in this system is saturation.
Further complexity is encountered at high saccharide concen-
trations, as the high concentrations of lactose which favour GOS
formation also increase the viscosity of the reaction solution,
changing mass transfer rates and enzyme dynamics. The rate con-
stants in a mechanistic model have been found to correlate to the
viscosity of the reaction solution (Iwasaki et al., 1996). It can be
extrapolated from this nding that the mixing efciency and vig-
our are important effectors of the reaction rate at conditions of
high saccharide concentration.
Empirical models can also be used to mathematically describe
the production of GOS. This approach involves tting equations
to experimentally measured reaction time courses, in order to
accurately determine and optimise GOS yields. Empirical ap-
proaches have the advantage of being able to include multiple pro-
cess parameters (such as initial lactose concentration, temperature
and pH) in a single model without the need for detailed reaction
mechanisms. This approach is well suited to more complex reac-
tion systems, such as milk or other dairy products, which have var-
iable composition.
Several recent studies have applied a popular form of empirical
optimisation, known as response surface methodology (Bas & Boy-
aci, 2007) to optimise GOS production (Table 4). These examples
include modelling GOS synthesis by whole live Bidobacterium
infantis RW-8120 cells (Roy, Daoudi, & Azaola, 2002). In such a sys-
tem, detailed knowledge of important reaction parameters, such as
the rate of lactose uptake into the cell, as well as the intracellular
concentration of lactose, GOS and b-galactosidase, are difcult to
determine. The effect of cell concentration, initial lactose concen-
tration, reaction time and temperature were more easily examined
and the model found that the initial lactose concentration was the
only signicant variable affecting GOS yield.
Interestingly, other empirical models (Chen et al., 2002; Rus-
tom, Foda, & Lpez-Leiva, 1998) found no signicant effect of initial
lactose concentration on GOS yield. Given the wide acceptance that
initial lactose concentration is one of the major effectors of GOS
yields, these ndings may point to deciencies in these models.
6. Reactor congurations
As for most reactions catalysed by a biological entity, there are
many modes in which GOS producing reactions can be performed.
The b-galactosidase can be soluble or immobilised. It can be iso-
lated after intra- or extracellular expression, or provided within
whole cells. The mode in which the process is undertaken may
change the behaviour of the enzyme, the concentrations of product
and substrate around the enzyme and will certainly inuence the
performance and economics of the process. A recent publication
reviewed the immobilisation of b-galactosidase for GOS production
(Panesar et al., 2006). Here, we present a review of the literature
using two alternative approaches: (1) whole cells as biocatalysts
and (2) membrane reactors, emphasising the differences in the
outcomes of the process due to these alternative reactor formats.
6.1. Whole cells
Using resting or living whole cells removes the need for the iso-
lation of the b-galactosidase enzyme. Protein isolation can be
demanding and costly and requires additional processing steps
after the culture of the b-galactosidase producing organism. Using
the organism directly can circumvent these problems (Fukuda,
Hama, Tamalampudi, & Noda, 2008). GOS yields of 36% (w/v) to
43% (w/w) have been achieved in a whole cell system, demonstrat-
ing comparable yields to those achieved with isolated enzymes
(Goulas, Tzortzis, & Gibson, 2007).
There have been studies describing examples of GOS production
employing probiotic microbial cells as catalysts (Roy et al., 2002,
Tzortzis, Goulas, & Gibson, 2005). A further report describes adding
a pre-incubation step during yoghurt making, that allows the star-
ter culture sufcient time to act as GOS producing biocatalysts
(Lamoureux, Roy, & Gauthier, 2002). Given the argument that b-
galactosidase from probiotic organisms produce GOS with an en-
hanced prebiotic effect (Rastall, 2004) (also see Structure of GOS
products section), this approach may provide an opportunity to
produce GOS with superior prebiotic qualities without the need
for the manufacturer to purchase b-galactosidase.
An advantage of whole cells over isolated enzymes during the
development of an optimal biocatalyst is the ability to evolve.
Changing an isolated protein typically involves manipulation at
the DNA level. Regulation of genetically-modied food ingredients
makes this approach inappropriate and removes the most powerful
tool for optimising an isolated enzyme. However, standard breed-
ing and selection techniques offer options for improving food-
grade whole cell biocatalysts. Chemical mutagenesis followed by
screening, to select for strains with increased b-galactosidase activ-
ity, has been applied to probiotic strains, leading to increases of
70222% in b-galactosidase activity (Ibrahim & OSullivan, 2000).
Screening of isolates of healthy intestinal microora without muta-
genesis also offers an opportunity for discovering novel whole cell
biocatalysts for GOS production (OSullivan, 2001).
It is generally believed that the added complexity of using
whole cells for biocatalysis can be a disadvantage compared to iso-
lated enzymes (Woodley, 2006). However, whole cells also provide
the opportunity to include additional metabolic functions such as
the consumption of the monosaccharides glucose and galactose
from a GOS product. These monosaccharides do not show a prebi-
otic effect, raise the caloric value of food containing unpuried
GOS preparations and can be considered as undesirable. For exam-
ple, mixtures of bacterial and yeast strains (B. bidum and Saccha-
romyces cerevisiae) have been combined to produce GOS and to
preferentially consume 92% of the glucose, respectively, thereby
purifying the prebiotic carbohydrates of unwanted monosaccha-
rides (Goulas et al., 2007). A similar approach employed immobi-
lised Zymomonas mobilis cells (Crittenden & Playne, 2002) and
the selective monosaccharide catabolism of these cells may be
benecial. A study comparing monosaccharide removal from Vivi-
nal GOS using two chromatographic techniques (size exclusion or
activated charcoal) and the addition of S. cerevisiae cells found that
cellular treatment gave the highest GOS yields (Hernandez, Ruiz-
Matute, Olano, Moreno, & Sanz, 2009). However, it should be noted
that the metabolic end products, such as ethanol, lactic acid or
other metabolites, will affect taste and other characteristics of
the nal product when this approach is employed.
6.2. Membrane processing
The cost associated with using isolated enzymes can also be
ameliorated by recycling the biocatalyst. One means for enzyme
recycling involves ultraltration (UF). In this approach, a semi-per-
meable membrane is used to retain the larger enzyme within the
bioreactor while allowing the passage of smaller molecules, such
as substrate and product. Continuous removal of product saccha-
rides and replacement with fresh substrate allows the reaction to
be continued indenitely while the integrity of the bioreactor is
maintained.
 A. Gosling et al. / Food Chemistry 121 (2010) 307318
For example, a commercial preparation of the K. lactis b-galacto-
sidase reached its maximum GOS yield in a 1 h batch reaction,
yielding 7.6 mg GOS per unit enzyme activity. GOS yields de-
creased after this time. When the reaction was performed in a UF
membrane reactor, 6.4 mg GOS were produced per unit enzyme
activity per hour for 4 h (Chockchaisawasdee et al., 2005) repre-
senting a total increase in yield of 240%.
UF membranes can also give control over the average amount of
time that lactose is exposed to the b-galactosidase. This is an
important parameter to be able to control, given that it inuences
GOS yield and structure. It has been found that shorter average res-
idence times in UF reactors gave higher GOS yields for both the K.
lactis and A. oryzae enzymes (Czermak et al., 2004).
A recognised caveat to performing enzymatic reactions in UF
reactors is that the shear forces and/or higher pressures present
can cause enzyme denaturation (Bowen, 1993). However, there is
evidence that the benets of UF can outweigh this limitation. An
increased transmembrane pressure in a UF unit gave higher rate
constants than an unpressured batch reaction using b-galactosi-
dase from A. oryzae (Matella, Dolan, & Lee, 2006). This study con-
cluded that the positive effects on reaction rates from increased
mixing at higher uid pressures was greater than the negative ef-
fects due to shear-induced enzyme denaturation.
The structure of the GOS product can also be inuenced by
using a continuous UF reactor. This was illustrated in a study com-
paring the behaviour of the b-galactosidase from Lactobacillus reu-
teri in both a continuous UF reactor and a batch reactor (Splechtna,
Nguyen, & Haltrich, 2007). It was reported that the GOS product
from the continuous system had a higher percentage of disaccha-
rides. This was explained by the higher concentration of glucose
in the continuous system at steady state compared to the batch
system. Glucose was fourfold preferred as an acceptor for transga-
lactosylation than lactose for this enzyme, so the higher glucose
concentration in the UF system led to more GalGlc dimers. It
was also found that the continuous system produced a higher ratio
of b1- > 6 to b1- > 3 glycosidic linkages as the b1- > 3 bonds were
hydrolysed more rapidly than b1- > 6 bonds.
Membranes with smaller pore sizes can also be used for purify-
ing GOS products from the monosaccharide hydrolysis products,
glucose and galactose. In one example, up to 88% of the di- and oli-
gosaccharides were recovered from a commercial GOS mixture
using a nanoltration membrane with only 19% of the monosac-
charides remaining in the retentate stream (Goulas, Grandison, &
Rastall, 2003). Other literature on the benets of cost-effective
membrane separation of monosaccharides from oligosaccharides
(Catarino, Minhalma, Beal, Mateus, & de Pinho, 2008; Feng, Chang,
Wang, & Ma, 2009; Goulas, Kapasakalidis, Sinclair, Rastall, &
Grandison, 2002) is available as a comparison to industrial chro-
matography processes (Playne & Crittenden, 1996).
7. Outlook
Few enzymes are as well described as b-galactosidase. Yet gaps
still exist in our knowledge of b-galactosidase-catalysed GOS syn-
thesis. This review has highlighted important recent research that
contributes to our understanding of b-galactosidase structure and
activity, and drawn attention to areas where greater insight would
be useful for the future rational improvement of GOS production
processes at a commercial scale.
Opportunities exist to optimise features of b-galactosidase. For
example, systematic studies of different enzymes and their resul-
tant GOS structures could be used to tailor desired characteristics
in commercially applicable enzymes. Further enzyme engineering
could be directed towards enzymes that produce GOS with higher
prebiotic efcacy. The next generation of optimised b-galactosi-
315
dases are also likely to include thermotolerant enzymes, as in-
creased lactose solubility at higher temperatures generally leads
to increased GOS.
Solid inroads are being made into the historically challenging
task of mathematically modelling this complex system. Effective
models for GOS synthesis will allow GOS production to be opti-
mised. A careful choice of reactor conguration can further mini-
mise production costs and promote desired product
characteristics.
Increases in GOS yield delivered by enhancing the transgalac-
tosylation reaction, through advances in reaction and enzyme
engineering, will result in higher concentrations of GOS, alleviating
some of the need for further separation of GOS from other carbohy-
drates. This will have an impact on both the economics of process-
ing and the potential utilisation of GOS.
Together these advances will lower the cost of GOS production
and increase prebiotic potency. This should decrease the price of
GOS ingredients which will also be effective at lower concentra-
tions, leading to health benets across society.
Acknowledgements
This research was funded by an Australia-India Strategic Re-
search Fund Program BF01-0024 from The Department of Innova-
tion, Industry and Science Research. We acknowledge Daniel
Otieno for preliminary work leading to this study and Professor
Chiranjib Bhattacharjee from Jadavpur University Kolkata and Pro-
fessor P.K. Bhattacharya from the Indian Institute of Technology
Kanpur India for useful discussions. We would also like to
acknowledge the Particulate Fluids Processing Centre, Special Re-
search Centre of the ARC, for access to equipment.
References
Adamczak, M., Charubin, D., & Bednarski, W. (2009). Inuence of reaction medium
composition on enzymatic synthesis of galactooligosaccharides and lactulose
from lactose concentrates prepared from whey permeate. Chemical Papers,
63(2), 111116.
Albayrak, N., & Yang, S.-T. (2002). Production of galacto-oligosaccharides from
lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth.
Biotechnology and Bioengineering, 77(1), 819.
Amaretti, A., Bernardi, T., Tamburini, E., Zanoni, S., Lomma, M., Matteuzzi, D., et al.
(2007). Kinetics and metabolism of Bidobacterium adolescentis MB 239 growing
on glucose, galactose, lactose, and galactooligosaccharides. Applied and
Environmental Microbiology, 73(11), 36373644.
Asp, N. G., Burvall, A., Dahlqvist, A., Hallgren, P., & Lundblad, A. (1980).
Oligosaccharide formation during hydrolysis of lactose with Saccharomyces
Lactis lactase (Maxilact). 2 Oligosaccharide structures. Food Chemistry, 5(2),
147153.
Bakken, A. P., & Hill, C. G. (1992). Hydrolysis of lactose in skim milk by immobilised
beta-galactosidase (Bacillus circulans). Biotechnology and Bioengineering, 39(4),
408417.
Barreteau, H., Delattre, C., & Michaud, P. (2006). Production of oligosaccharides as
promising new food additive generation. Food Technology and Biotechnology,
44(3), 323333.
Bas, D., & Boyaci, I. H. (2007). Modeling and optimization I: Usability of response
surface methodology. Journal of Food Engineering, 78(3), 836845.
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., et al.
(2000). The protein data bank. Nucleic Acids Research, 28(Database issue),
235242.
Boon, M. A., Janssen, A. E. M., & van der Padt, A. (1999). Modelling and parameter
estimation of the enzymatic synthesis of oligosaccharides by beta-galactosidase
from Bacillus circulans. Biotechnology and Bioengineering, 64(5), 558567.
Boon, M. A., Janssen, A. E. M., & vant Riet, K. (2000). Effect of temperature and
enzyme origin on the enzymatic synthesis of oligosaccharides. Enzyme and
Microbial Technology, 26(24), 271281.
Borra, S. T., & Bouchoux, A. (2009). Effects of science and the media on consumer
perceptions about dietary sugars. Journal of Nutrition, 139(6), 1214S1218S.
Bowen, R. (1993). Understanding ux patterns in membrane processing of protein
solutions and suspensions. Trends in Biotechnology, 11(11), 451460.
Brs, N. F., Moura-Tamames, S. A., Fernandes, P. A., & Ramos, M. J. (2008).
Mechanistic studies on the formation of glycosidase-substrate and glycosidase-
inhibitor covalent intermediates. Journal of Computational Chemistry, 29(15),
25652574.
Bruins, M. E., Strubel, M., van Lieshout, J. F. T., Janssen, A. E. M., & Boom, R. M.
(2003a). Oligosaccharide synthesis by the hyperthermostable beta-glucosidase
316 A. Gosling et al. / Food Chemistry 121 (2010) 307318
from Pyrococcus furiosus: Kinetics and modelling. Enzyme and Microbial
Technology, 33(1), 311.
Bruins, M. E., Van Hellemond, E. W., Janssen, A. E. M., & Boom, R. M. (2003b).
Maillard reactions and increased enzyme inactivation during oligosaccharide
synthesis by a hyperthermophilic glycosidase. Biotechnology and Bioengineering,
81(5), 546552.
Buchholz, K., Kasche, V., & Bornscheuer, U. T. (2005). Equilibrium and kinetically
controlled reactions catalysed by enzymes. In Biocatalysts and enzyme
technology (pp. 4246). Weinheim: Wiley-VCH Verlag GmbH& Co.
Cantarel, B. L., Coutinho, P. M., Rancurel, C., Bernard, T., Lombard, V., & Henrissat, B.
(2009). The carbohydrate-active enzymes database (CAZy): An expert resource
for glycogenomics. Nucleic Acids Research, 37(Database issue), D233238.
Cardelle-Cobas, A., Villamiel, M., Olano, A., & Corzo, N. (2008). Study of galacto-
oligosaccharide formation from lactose using Pectinex Ultra SP-L. Journal of the
Science of Food and Agriculture, 88(6), 954961.
Catarino, I., Minhalma, M., Beal, L. L., Mateus, M., & de Pinho, M. N. (2008).
Assessment of saccharide fractionation by ultraltration and nanoltration.
Journal of Membrane Science, 312(12), 3440.
Chen, C. S., Hsu, C. K., & Chiang, B. H. (2002). Optimization of the enzymic process
for manufacturing low-lactose milk containing oligosaccharides. Process
Biochemistry, 38(5), 801808.
Chen, C. W., Ou-Yang, C. C., & Yeh, C. W. (2003). Synthesis of galactooligosaccharides
and transgalactosylation modeling in reverse micelles. Enzyme and Microbial
Technology, 33(4), 497507.
Chen, S.-X., Wei, D.-Z., & Hu, Z.-H. (2001). Synthesis of galacto-oligosaccharides in
AOT/isooctane reverse micelles by beta-galactosidase. Journal of Molecular
Catalysis B: Enzymatic, 16(2), 109114.
Chen, W., Chen, H., Xia, Y., Zhao, J., Tian, F., & Zhang, H. (2008). Production,
purication, and characterization of a potential thermostable galactosidase for
milk lactose hydrolysis from Bacillus stearothermophilus. Journal of Dairy Science,
91(5), 17511758.
Cho, Y.-J., Shin, H.-J., & Bucke, C. (2003). Purication and biochemical properties of a
galactooligosaccharide producing beta-galactosidase from Bullera singularis.
Biotechnology Letters, 25(24), 21072111.
Chockchaisawasdee, S., Athanasopoulos, V. I., Niranjan, K., & Rastall, R. A. (2005).
Synthesis of galacto-oligosaccharide from lactose using beta-galactosidase from
Kluyveromyces lactis: Studies on batch and continuous UF membrane-tted
bioreactors. Biotechnology and Bioengineering, 89(4), 434443.
Cowburn, G., & Stockley, L. (2005). Consumer understanding and use of nutrition
labelling: A systematic review. Public Health Nutrition, 8(1), 2128.
Crittenden, R. G., & Playne, M. J. (1996). Production, properties and applications of
food-grade oligosaccharides. Trends in Food Science and Technology, 7(11),
353361.
Crittenden, R. G., & Playne, M. J. (2002). Purication of food-grade oligosaccharides
using immobilised cells of Zymomonas mobilis. Applied Microbiology and
Biotechnology, 58(3), 297302.
Cruz-Guerrero, A. E., Gmez-Ruiz, L., Viniegra-Gonzlez, G., Brzana, E., & Garca-
Garibay, M. (2006). Inuence of water activity in the synthesis of
galactooligosaccharides produced by a hyperthermophilic beta-glycosidase in
an organic medium. Biotechnology and Bioengineering, 93(6), 11231129.
Czermak, P., Ebrahimi, M., Grau, K., Netz, S., Sawatzki, G., & Pfromm, P. H. (2004).
Membrane-assisted enzymatic production of galactosyl-oligosaccharides from
lactose in a continuous process. Journal of Membrane Science, 232(12),
8591.
Delzenne, N. M. (2003). Oligosaccharides: State of the art. Proceedings of the
Nutrition Society, 62(1), 177182.
Demirhan, E., Apar, D. K., & zbek, B. (2008). Product inhibition of whey lactose
hydrolysis. Chemical Engineering Communications, 195(3), 293304.
Depeint, F., Tzortzis, G., Vulevic, J., IAnson, K., & Gibson, G. R. (2008). Prebiotic
evaluation of a novel galactooligosaccharide mixture produced by the
enzymatic activity of Bidobacterium bidum NCIMB 41171, in healthy
humans: A randomized, double-blind, crossover, placebo-controlled
intervention study. American Journal of Clinical Nutrition, 87(3), 785791.
Dumortier, V., Brassart, C., & Bouquelet, S. (1994). Purication and properties of a
beta-D-galactosidase from Bidobacterium bidum exhibiting a galactosidase from Kluyveromyces lactis for both hydrolysis and
transgalactosylation reaction. Biotechnology and Applied Biochemistry, 19(3),
341354.
Feng, Y. M., Chang, X. L., Wang, W. H., & Ma, R. Y. (2009). Separation of galacto-
oligosaccharides mixture by nanoltration. Journal of the Taiwan Institute of
Chemical Engineers, 40(3), 326332.
Fukuda, H., Hama, S., Tamalampudi, S., & Noda, H. (2008). Whole-cell biocatalysts
for biodiesel fuel production. Trends in Biotechnology, 26(12), 668673.
Gnzle, M. G., Haase, G., & Jelen, P. (2008). Lactose: Crystallization, hydrolysis and
value-added derivatives. International Dairy Journal, 18(7), 685694.
Gloster, T. M., Roberts, S., Ducros, V. M.-A., Perugino, G., Rossi, M., Hoos, R., et al.
(2004). Structural Studies of the beta-glycosidase from Sulfolobus solfataricus in
complex with covalently and noncovalently bound inhibitors. Biochemistry,
43(20), 61016109.
Gopal, P. K., Sullivan, P. A., & Smart, J. B. (2001). Utilisation of galacto-
oligosaccharides as selective substrates for growth by lactic acid bacteria
including Bidobacterium lactis DR10 and Lactobacillus rhamnosus DR20.
International Dairy Journal, 11(12), 1925.
Goulas, A., Tzortzis, G., & Gibson, G. R. (2007). Development of a process for the
production and purication of alpha- and beta-galactooligosaccharides from
Bidobacterium bidum NCIMB 41171. International Dairy Journal, 17(6),
648656.
Goulas, A. K., Grandison, A. S., & Rastall, R. A. (2003). Fractionation of
oligosaccharides by nanoltration. Journal of the Science of Food and
Agriculture, 83(7), 675680.
Goulas, A. K., Kapasakalidis, P. G., Sinclair, H. R., Rastall, R. A., & Grandison, A. S.
(2002). Purication of oligosaccharides by nanoltration. Journal of Membrane
Science, 209(1), 321335.
Greenberg, N. A., & Mahoney, R. R. (1983). Formation of oligosaccharides by beta-
galactosidase from Streptococcus thermophilus. Food Chemistry, 10(3), 195204.
Hansson, T., Kaper, T., van der Oost, J., de Vos, W. M., & Adlercreutz, P. (2000).
Improved oligosaccharide synthesis by protein engineering of beta-glucosidase
CelB from hyperthermophilic Pyrococcus furiosus. Biotechnology and
Bioengineering, 73(3), 203210.
Hatzinikolaou, D. G., Katsifas, E., Mamma, D., Karagouni, A. D., Christakopoulos, P., &
Kekos, D. (2005). Modeling of the simultaneous hydrolysis-ultraltration of
whey permeate by a thermostable beta-galactosidase from Aspergillus niger.
Biochemical Engineering Journal, 24(2), 161172.
Henrissat, B. (1991). A classication of glycosyl hydrolases based on amino acid
sequence similarities. Biochemical Journal, 280(2), 309316.
Hernandez, O., Ruiz-Matute, A. I., Olano, A., Moreno, F. J., & Sanz, M. L. (2009).
Comparison of fractionation techniques to obtain prebiotic
galactooligosaccharides. International Dairy Journal, 19(9), 531536.
Hidaka, M., Fushinobu, S., Ohtsu, N., Motoshima, H., Matsuzawa, H., Shoun, H., et al.
(2002). Trimeric crystal structure of the glycoside hydrolase family 42 beta-
galactosidase from Thermus thermophilus A4 and the structure of its complex
with galactose. Journal of Molecular Biology, 322(1), 7991.
Hsu, C. A., Lee, S. L., & Chou, C. C. (2007). Enzymatic production of
galactooligosaccharides by beta-galactosidase from bidobacterium longum
BCRC 15708. Journal of Agricultural and Food Chemistry, 55(6), 22252230.
Hsu, C. A., Yu, R. C., & Chou, C. C. (2006). Purication and characterization of a
sodium-stimulated beta-galactosidase from Bidobacterium longum CCRC
15708. World Journal of Microbiology and Biotechnology, 22(4), 355361.
Huber, R. E., Hurlburt, K. L., & Turner, C. L. (1981). The anomeric specicity of beta-
galactosidase and lac permease from Escherichia coli. Canadian Journal of
Biochemistry, 59(2), 100105.
Huber, R. E., Kurz, G., & Wallenfels, K. (1976). A quantitation of the factors which
affect the hydrolase and transgalactosylase activities of beta-galactosidase
(E. coli) on lactose. Biochemistry, 15(9), 19942001.
Ibrahim, S. A., & OSullivan, D. J. (2000). Use of chemical mutagenesis for the
isolation of food grade beta-galactosidase overproducing mutants of
Bidobacteria, Lactobacilli and Streptococcus thermophilus. Journal of Dairy
Science, 83(5), 923930.
Iwasaki, K.-i., Nakajima, M., & Nakao, S.-i. (1996). Galacto-oligosaccharide
production from lactose by an enzymic batch reaction using beta-
galactosidase. Process Biochemistry, 31(1), 6976.
Ji, E.-S., Park, N.-H., & Oh, D.-K. (2005). Galacto-oligosaccharide production by a
thermostable recombinant beta-galactosidase from Thermotoga maritima. World
Journal of Microbiology and Biotechnology, 21(5), 759764.
Jobe, A., & Bourgeoi, S. (1972). Lac repressoroperator interaction 6. Natural inducer
of Lac Operon. Journal of Molecular Biology, 69(3), 397404.
Jrgensen, F., Hansen, O. C., & Stougaard, P. (2001). High-efciency synthesis of
oligosaccharides with a truncated beta-galactosidase from Bidobacterium
bidum. Applied Microbiology and Biotechnology, 57(56), 647652.
Jrgensen, F., Hansen, O. C. & Stougaard, P. (2007). Beta-galactosidase isolated from
bidobacterium. Patent no. EP1283876.
Juers, D. H., Hakda, S., Matthews, B. W., & Huber, R. E. (2003). Structural basis for the
altered activity of Gly794 variants of Escherichia coli beta-galactosidase.
Biochemistry, 42(46), 1350513511.
Juers, D. H., Heightman, T. D., Vasella, A., McCarter, J. D., Mackenzie, L., Withers, S. G.,
et al. (2001). A structural view of the action of Escherichia coli (lacZ) beta-
galactosidase. Biochemistry, 40(49), 1478114794.
Jurado, E., Camacho, F., Luzn, G., & Vicaria, J. M. (2004). Kinetic models of activity
for beta-galactosidases: inuence of pH, ionic concentration and temperature.
Enzyme and Microbial Technology, 34(1), 3340.
Kim, C. S., Ji, E.-S., & Oh, D.-K. (2004). A new kinetic model of recombinant beta-
transgalactosylation reactions. Biochemical and Biophysical Research
Communications, 316(3), 738743.
Lamoureux, L., Roy, D., & Gauthier, S. F. (2002). Production of oligosaccharides in
Yogurt containing bidobacteria and Yogurt cultures. Journal of Dairy Science,
85(5), 10581069.
Lomer, M. C. E., Parkes, G. C., & Sanderson, J. D. (2008). Review article: Lactose
intolerance in clinical practice Myths and realities. Alimentary Pharmacology
and Therapeutics, 27(2), 93103.
Macfarlane, G. T., Steed, H., & Macfarlane, S. (2008). Bacterial metabolism and
health-related effects of galacto-oligosaccharides and other prebiotics. Journal
of Applied Microbiology, 104(2), 305344.
Mahoney, R. R. (1998). Galactosyl-oligosaccharide formation during lactose
hydrolysis: A review. Food Chemistry, 63(2), 147154.
Matella, N. J., Dolan, K. D., & Lee, Y. S. (2006). Comparison of galactooligosaccharide
production in free-enzyme ultraltration and in immobilized-enzyme systems.
Journal of Food Science, 71(7), C363C368.
Matthews, B. W. (2005). The structure of E Coli beta-galactosidase. Comptes Rendus
Biologies, 328(6), 549556.
Maugard, T., Gaunt, D., Legoy, M. D., & Besson, T. (2003). Microwave-assisted
synthesis of galacto-oligosaccharides from lactose with immobilized beta-
galactosidase from Kluyveromyces lactis. Biotechnology Letters, 25(8), 623629.
 A. Gosling et al. / Food Chemistry 121 (2010) 307318
Mladenoska, I., Winkelhausen, E., & Kuzmanova, S. (2008). Transgalactosylation /
hydrolysis ratios of various beta-galactosidases catalyzing alkyl-beta-
galactoside synthesis in single-phased alcohol media. Food Technology and
Biotechnology, 46(3), 311316.
Mlichova, Z., & Rosenberg, M. (2006). Current trends of beta-galactosidase
application in food technology. Journal of Food and Nutrition Research, 45(2),
4754.
Mozaffar, Z., Nakanishi, K., & Matsuno, R. (1985). Formation of oligosaccharides
during hydrolysis of lactose in milk using beta-galactosidase from Bacillus
circulans. Journal of Food Science, 50(6), 16021606.
Mozaffar, Z., Nakanishi, K., Matsuno, R., & Kamikubo, T. (1984). Purication and
properties of beta-galactosidases from Bacillus circulans. Agricultural and
Biological Chemistry, 48(12), 30533061.
Mussatto, S. I., & Mancilha, I. M. (2007). Non-digestible oligosaccharides: A review.
Carbohydrate Polymers, 68(3), 587597.
Nakao, M., Harada, M., Kodama, Y., Nakayama, T., Shibano, Y., & Amachi, T. (1994).
Purication and characterization of a thermostable beta-galactosidase with
high transgalactosylation activity from Saccharopolyspora rectivirgula. Applied
Microbiology and Biotechnology, 40(5), 657663.
Neri, D. F. M., Balcao, V. M., Costa, R. S., Rocha, I., Ferreira, E., Torres, D. P. M., et al.
(2009). Galacto-oligosaccharides production during lactose hydrolysis by free
Aspergillus oryzae beta-galactosidase and immobilized on magnetic
polysiloxane-polyvinyl alcohol. Food Chemistry, 115(1), 9299.
Nerinckx, W., Desmet, T., & Claeyssens, M. (2003). A hydrophobic platform as a
mechanistically relevant transition state stabilising factor appears to be present
in the active centre of all glycoside hydrolases. FEBS Letters, 538(13), 17.
Ohtsuka, K., Tanoh, A., Ozawa, O., Kanematsu, T., Uchida, T., & Shinke, R. (1990).
Purication and properties of a beta-galactosidase with high galactosyl transfer
activity from Cryptococcus laurentii Okn-4. Journal of Fermentation and
Bioengineering, 70(5), 301307.
Onishi, N., & Tanaka, T. (1995). Purication and properties of a novel thermostable
galacto-oligosaccharide-producing beta-galactosidase from Sterigmatomyces
elviae CBS8119. Applied and Environmental Microbiology, 61(11), 40264030.
OSullivan, D. J. (2001). Screening of intestinal microora for effective probiotic
bacteria. Journal of Agricultural and Food Chemistry, 49(4), 17511760.
Palani, K., Kumaran, D., Burley, S. K., & Swaminathan, S. (2009). Crystal structure of a
beta-galactosidase from Bacteroides thetaiotaomicron. http://www.rcsb.org/pdb/
explore.do?structureId=3D3A Accessed 23.01.09.
Panesar, P. S., Panesar, R., Singh, R. S., Kennedy, J. F., & Kumar, H. (2006). Microbial
production, immobilization and applications of beta-D-galactosidase. Journal of
Chemical Technology and Biotechnology, 81(4), 530543.
Park, H. Y., Kim, H. J., Lee, J. K., Kim, D., & Oh, D. K. (2008). Galactooligosaccharide
production by a thermostable beta-galactosidase from Sulfolobus solfataricus.
World Journal of Microbiology and Biotechnology, 24(8), 15531558.
Pazur, J. H., Tipton, C. L., Budovich, T., & Marsh, J. M. (1958). Structural
characterization of products of enzymatic disproportionation of lactose.
Journal of the American Chemical Society, 80(1), 119121.
Petzelbauer, I., Reiter, A., Splechtna, B., Kosma, P., & Nidetzky, B. (2000).
Transgalactosylation by thermostable beta-glycosidases from Pyrococcus
furiosus and Sulfolobus solfataricus. European Journal of Biochemistry, 267(16),
50555066.
Playne, M., & Crittenden, R. (1996). Commercially available oligosaccharides.
Bulletin of the International Dairy Federation, 313, 1022.
Prenosil, J. E., Stuker, E., & Bourne, J. R. (1987a). Formation of oligosaccharides
during enzymatic lactose hydrolysis and their importance in a whey hydrolysis
process: Part II: Experimental. Biotechnology and Bioengineering, 30(9),
10261031.
Prenosil, J. E., Stuker, E., & Bourne, J. R. (1987b). Formation of oligosaccharides
during enzymatic lactose: Part I: State of Art. Biotechnology and Bioengineering,
30(9), 10191025.
Rabiu, B. A., Jay, A. J., Gibson, G. R., & Rastall, R. A. (2001). Synthesis and
fermentation properties of novel galacto-oligosaccharides by beta-
galactosidases from Bidobacterium species. Applied and Environmental
Microbiology, 67(6), 25262530.
Ramagopal, U. A., Rutter, M., Toro, R., Hu, S., Maletic, M., Gheyi, T., et al. (2009).
Crystal structure of putative beta-galactosidase from Bacteroides fragilis. http://
www.rcsb.org/pdb/explore.do?structureId=3CMG. Accessed 16.06.09.
Rastall, R. A. (2004). Bacteria in the gut: Friends and foes and how to alter the
balance. Journal of Nutrition, 134(8), 2022S2026.
Richard, J. P., Westerfeld, J. G., Lin, S., & Beard, J. (1995). Structure-reactivity
relationships for beta-galactosidase (Escherichia coli, lac Z). 2. Reactions of the
galactosyl-enzyme intermediate with alcohols and azide ion. Biochemistry,
34(37), 1171311724.
Roberfroid, M. (2007). Prebiotics: The concept revisited. Journal of Nutrition, 137(3),
830s837s.
Roberts, H. R., & Pettinati, J. D. (1957). Oligosaccharide production, concentration
effects in the enzymatic conversion of lactose to oligosaccharides. Journal of
Agricultural and Food Chemistry, 5(2), 130134.
Rojas, A. L., Nagem, R. A. P., Neustroev, K. N., Arand, M., Adamska, M., Eneyskaya, E.
V., et al. (2004). Crystal structures of beta-galactosidase from Penicillium sp. and
its complex with galactose. Journal of Molecular Biology, 343(5), 12811292.
Roy, D., Daoudi, L., & Azaola, A. (2002). Optimization of galacto-oligosaccharide
production by Bidobacterium infantis RW-8120 using response surface
methodology. Journal of Industrial Microbiology and Biotechnology, 29(5),
281285.
317
Rustom, I. Y. S., Foda, M. I., & Lpez-Leiva, M. H. (1998). Formation of
oligosaccharides from whey UF-permeate by enzymatic hydrolysis Analysis
of factors. Food Chemistry, 62(2), 141147.
Rycroft, C. E., Jones, M. R., Gibson, G. R., & Rastall, R. A. (2001). A comparative in vitro
evaluation of the fermentation properties of prebiotic oligosaccharides. Journal
of Applied Microbiology, 91(5), 878887.
Sanz, M. L., Cote, G. L., Gibson, G. R., & Rastall, R. A. (2006a). Inuence of glycosidic
linkages and molecular weight on the fermentation of maltose-based
oligosaccharides by human gut bacteria. Journal of Agricultural and Food
Chemistry, 54(26), 97799784.
Sanz, M. L., Cote, G. L., Gibson, G. R., & Rastall, R. A. (2006b). Selective fermentation
of gentiobiose-derived oligosaccharides by human gut bacteria and inuence of
molecular weight. FEMS Microbiology Ecology, 56(3), 383388.
Sanz, M. L., Gibson, G. R., & Rastall, R. A. (2005). Inuence of disaccharide structure
on prebiotic selectivity in vitro. Journal of Agricultural and Food Chemistry,
53(13), 51925199.
Sanz, M. L., Sanz, J., & Martinez-Castro, I. (2002). Characterization of O-trimethylsilyl
oximes of disaccharides by gas chromatographymass spectrometry.
Chromatographia, 56(910), 617622.
Schaafsma, G. (2008). Lactose and lactose derivatives as bioactive ingredients in
human nutrition. International Dairy Journal, 18(5), 458465.
Sklov, T., Dohnlek, J., Spiwok, V., Lipovov, P., Vondrckov, E., Petrokov, H.,
et al. (2005). Cold-active beta-galactosidase from Arthrobacter sp. C2-2 forms
compact 660 kDa hexamers: Crystal structure at 1.9 resolution. Journal of
Molecular Biology, 353(2), 282294.
Smart, J. B. (1991). Transferase reactions of the beta-galactosidase from
Streptococcus Thermophilus. Applied Microbiology and Biotechnology, 34(4),
495501.
Splechtna, B., Nguyen, T.-H., & Haltrich, D. (2007). Comparison between
discontinuous and continuous lactose conversion processes for the production
of prebiotic galacto-oligosaccharides using beta-galactosidase from
Lactobacillus reuteri. Journal of Agricultural and Food Chemistry, 55(16),
67726777.
Splechtna, B., Nguyen, T.-h., Steinbock, M., Kulbe, K. D., Lorenz, W., & Haltrich, D.
(2006). Production of prebiotic galacto-oligosaccharides from lactose using
beta-galactosidases from Lactobacillus reuteri. Journal of Agricultural and Food
Chemistry, 54(14), 49995006.
Szilagyi, A. (2004). Redening lactose as a conditional prebiotic. Canadian Journal of
Gastroenterology, 18(3), 163167.
Taniguchi, H. (2005). Carbohydrate active enzymes for the production of
oligosaccharides. In C.-T. Hou (Ed.), Handbook of industrial biocatalysis. Boca
Raton: CRC Press. pp. 20-120-23.
Toba, T., Tomita, Y., Itoh, T., & Adachi, S. (1981). Beta-galactosidases of lactic acid
bacteria: Characterization by oligosaccharides formed during hydrolysis of
lactose. Journal of Dairy Science, 64(2), 185192.
Toba, T., Yokota, A., & Adachi, S. (1985). Oligosaccharide structures formed during
the hydrolysis of lactose by Aspergillus oryzae beta-galactosidase. Food
Chemistry, 16(2), 147162.
Topping, D. L., & Clifton, P. M. (2001). Short-chain fatty acids and human colonic
function: Roles of resistant starch and nonstarch polysaccharides. Physiology
Reviews, 81(3), 10311064.
Trincone, A., & Giordano, A. (2006). Glycosyl hydrolases and glycosyltransferases in
the synthesis of oligosaccharides. Current Organic Chemistry, 10(10), 11631193.
Tzortzis, G., Goulas, A. K., & Gibson, G. R. (2005). Synthesis of prebiotic
galactooligosaccharides using whole cells of a novel strain, Bidobacterium
bidum NCIMB 41171. Applied Microbiology and Biotechnology, 68(3), 412416.
Usui, T., Kubota, S., & Ohi, H. (1993). A convenient synthesis of beta-galactosyl
disaccharide derivatives using the beta-galactosidase from Bacillus circulans.
Carbohydrate Research, 244(2), 315323.
Valli, C., & Traill, W. B. (2005). Culture and food: A model of yoghurt consumption in
the EU. Food Quality and Preference, 16(4), 291304.
van den Broek, L. A. M., Hinz, S. W. A., Beldman, G., Vincken, J. P., & Voragen, A. G.
J. (2008). Bidobacterium carbohydrases Their role in breakdown and
synthesis of (potential) prebiotics. Molecular Nutrition and Food Research, 52(1),
146163.
Van Loo, J., Cummings, J., Delzenne, N., Englyst, H., Franck, A., Hopkins, M., et al.
(1999). Functional food properties of non-digestible oligosaccharides: A
consensus report from the ENDO project (DGXII AIRII-CT94-1095). British
Journal of Nutrition, 81(2), 121132.
Vasella, A., Davies, G. J., & Bhm, M. (2002). Glycosidase mechanisms. Current
Opinion in Chemical Biology, 6(5), 619629.
Vocadlo, D. J., & Davies, G. J. (2008). Mechanistic insights into glycosidase chemistry.
Current Opinion in Chemical Biology, 12(5), 539555.
Wallenfels, K. (1951). Enzymatische synthese von oligosacchariden aus
disacchariden. Naturwissenschaften, 38(13), 306.
Wallenfels, K., & Weil, R. (1972). In P. D. Boyer (Ed.). The enzymes (vol. 7,
pp. 617663). New York: Academic Press.
White, J. S. (2000). Sugar, special sugars. In Kirk-othmer encyclopedia of chemical
technology. doi:10.1002/0471238961.1916050323080920.a01. Accessed
16.06.09.
Woodley, J. M. (2006). Choice of biocatalyst form for scalable processes. Biochemical
Society Transactions, 34(2), 301303.
Yanahira, S., Kobayashi, T., Suguri, T., Nakajima, M., Miura, S., Ishikawa, H., et al.
(1995). Formation of oligosaccharides from lactose by Bacillus circulans beta-
galactosidase. Bioscience Biotechnology and Biochemistry, 59(6), 10211026.
318 A. Gosling et al. / Food Chemistry 121 (2010) 307318

Yoon, J. H., & Mckenzie, D. (2005). A comparison of the activities of three beta- Zechel, D. L., & Withers, S. G. (2000). Glycosidase mechanisms: Anatomy of a nely
galactosidases in aqueous-organic solvent mixtures. Enzyme and Microbial tuned catalyst. Accounts of Chemical Research, 33(1), 1118.
Technology, 36(4), 439446.
Zarate, S., & Lopez-Leiva, M. H. (1990). Oligosaccharide formation during enzymatic
lactose hydrolysis: A literature review. Journal of Food Protection, 53(3),
262268.