LWT - Food Science and Technology 72 (2016) 81e89

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LWT - Food Science and Technology

journal homepage: www.elsevier.com/locate/lwt

Simultaneous synthesis and purication (SSP) of galacto-

oligosaccharides in batch operation

s Illanes*
Carla Aburto, Cecilia Guerrero, Carlos Vera, Lorena Wilson, Andre

lica de Valparaso, Avenida Brasil, 2085, Valparaso, Chile
School of Biochemical Engineering, Ponticia Universidad Cato

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 galacto-oligosaccharides (GOS) considers the enzymatic synthesis by lactose
Received 5 February 2016 transgalactosylation and the purication from the reacted mixture (raw GOS). Simultaneous synthesis
Received in revised form and purication of GOS (SSP) was conducted in a one-pot operation using b-galactosidase from Asper-
5 April 2016
gillus oryzae and Saccharomyces cerevisiae or Kluyveromyces marxianus cells, allowing the selective
Accepted 16 April 2016
removal of unwanted carbohydrates (monosaccharides and lactose). The reaction was carried out in a
Available online 19 April 2016
stirred reactor at 40
C, pH 4.5 and 200 rpm, using lactose without additional supplements, evaluating
the effect of yeast genus (S. cerevisiae or K. marxianus), enzyme-substrate ratio (20e400 IUH/g lactose),
Keywords:
Prebiotic
initial lactose concentration (20e50% w/w) and substrate mass-biomass ratio (0.05e0.4). The best GOS
Simultaneous synthesis and purication yield was obtained with S. cerevisiae at 50 IUH/g lactose and 0.25 g cell biomass/g carbohydrates. These conditions
Galacto-oligosaccharides were used for scaling-up into a bioreactor (1000 g of 40% w/w lactose) operated under temperature and
b-galactosidase pH control at an aeration rate of 5 vvm, obtaining a GOS yield of 40% at 24 h of reaction, which is
Yeasts signicantly higher than obtained in the conventional system of GOS synthesis. SSP may represent a
technological advantage in terms of productivity and yield of GOS production.
2016 Published by Elsevier Ltd.

1. Introduction
Non-digestible oligosaccharides are important ingredients of
functional foods. A few of them, namely fructo-oligosaccharides
(FOS), inulin, lactulose and galacto-oligosaccharides (GOS) are
properly considered as prebiotics (Kova
cs et al., 2013). GOS are
particularly attractive for being derived from surplus industrial by-
products (whey or whey permeate) and having salient properties as
human milk oligosaccharide (HMOS) replacers (Ben et al., 2008;
Venema, 2012). GOS are composed of a variable number of galac-
tose units linked to a terminal glucose, the number of galactose
units usually varying from 2 to 5 (Mussatto & Mancilha, 2007).
Galactose units are linked to each other by b-(1e3), b-(1e6) and b-
(1e4) bonds, while galactose-glucose linkage is in most cases b-
(1e4). Prebiotic effect is for the most part associated to the tri-
saccharides (GOS-3), mostly 4'- and 6'-galactosyl-lactose (GOS-3),
and tetrasaccharides (GOS-4), mostly 6'-digalactosyl-lactose (GOS-
4) (Gopal, Sullivan, & Smart, 2001; Panesar, Panesar, Singh,
Kennedy, & Kumar, 2006). Besides, the disaccharides allolactose
* Corresponding author.
E-mail address: aillanes@ucv.cl (A. Illanes).
and galactobiose may also be present, being its contribution to the
prebiotic effect a matter of debate (Tzortzis & Vulevic, 2009).
GOS are synthesized by the kinetically controlled reaction of
lactose transgalactosylation catalyzed by b-galactosidase (EC
3.2.1.23). Most reported b-galactosidases for GOS production
belong to the genera Kluyveromyces (Kim, Lee, & Lee, 2001;
Martinez-Villaluenga, Cardelle-Cobas, Corzo, Olano, & Villamiel,
2008), Aspergillus (Albayrak & Yang, 2002; Gaur, Pant, Jain, & Khare,
2006) and Bacillus (Boon, Janssen, & Vant Riet, 2000; Gosling et al.,
2009). The enzyme from Aspergillus oryzae is particularly attractive
for being a robust, reasonably priced and readily available catalyst
recognized as safe for food and pharmaceutical use, producing
mostly GOS-3 and GOS-4 (Albayrak & Yang, 2002; Huerta, Vera,
Guerrero, Wilson, & Illanes, 2011). Main drawback is the rather
low GOS yields obtained (30% or less) so that a major issue in GOS
production is downstream processing to remove the undesirable
sugars (residual lactose, glucose and galactose) from the raw GOS
product of the enzymatic reaction, that may affect the quality of the
food matrices to which GOS are added, precluding its use for per-
sons suffering diabetes or lactose intolerance (Hern
andez, Ruiz-
Matute, Olano, Moreno, & Sanz, 2009). Most GOS products avail-
able are partially puried preparations containing 50e60% GOS
with signicant differences in the type of linkage and molecular

http://dx.doi.org/10.1016/j.lwt.2016.04.029
0023-6438/ 2016 Published by Elsevier Ltd.
82 C. Aburto et al. / LWT - Food Science and Technology 72 (2016) 81e89
size distribution according to the origin of the enzyme (Crittenden
& Playne, 2009; Rastall, 2006). Current technology for GOS puri-
cation is simulated moving bed chromatography, which is quite
effective but complex and costly, representing a signicant fraction
of the total operation cost (Illanes, 2011). Several alternative stra-
tegies have been proposed, like selective adsorption (Herna
ndez
et al., 2009), differential precipitation (Sen et al., 2011), supercriti-
cal extraction (Montan ~e

s, Olano, Reglero, Iba

n~ ez, & Fornari, 2009)
and nanoltration (Co
rdova et al., 2016; Michelon, Manera,
Limoeiro Carvalho, & Maugeri Filho, 2014). Most of them are
energy-intensive operations and levels of purity attained are in
several cases limited, so its adoption at industrial level is doubtful.
Selective fermentation has been proposed as a promising strategy
to remove unwanted sugars from the raw GOS in terms of cost,
environmental sustainability and quality of product for GOS puri-
cation using yeasts from the genera Kluyveromyces and Saccha-
romyces (Cheng et al., 2006; Guerrero et al., 2014; Li, Xiao, Lu, & Li,
2008). Kluyveromyces ferment lactose and monosaccharides due to
the presence of the LAC/GAL regulon (Rubio-Texeira, 2006) con-
taining the genes involved in lactose internalization and hydrolysis.
Purities of 95% have been obtained with diluted GOS (Gosling,
Stevens, Barber, Kentish, & Gras, 2010; Li et al., 2008) and close to
100% with undiluted raw GOS at high biomass to carbohydrate mas
ratios by selective fermentation (bioconversion) with Kluyver-
omyces marxianus (Guerrero et al., 2014). Saccharomyces cerevisiae
has also been used for GOS purication (Li et al., 2008) despite that
only monosaccharides can be removed (Yoon, Mukerjea, & Robyt,
2003), so that maximum GOS purity attainable is below 50%;
advantage is that low cost bakers' yeast or even spent brewers
yeast can be used.
Based on that background, this work presents results on the
simultaneous synthesis and purication (SSP) of GOS by selective
fermentation with A. oryzae b-galactosidase and yeasts cells as a
strategy to obtain partially puried GOS in a one-pot one-step
operation with the consequent benets in terms of productivity
and production cost with respect to conventional sequential op-
erations. This strategy has been previously applied to the simulta-
neous saccharication and fermentation process (SSF) for
producing galactose (Grosova
, Rosenberg, Gdovin, Sla
vikova
, &
Rebros, 2009) and bioethanol from the cellulosic (Olofsson,
Bertilsson, & Lide
n, 2008) and hemicellulosic fractions of ligno-
cellulose biomass (Silva et al., 2012).
2. Materials and methods
2.1. Materials
D () lactose monohydrate, D () galactose and D () glucose
were products of Sigma (St. Louis, MO, USA). The enzyme used is
Enzeco Fungal Lactase, a b-galactosidase preparation from A.
oryzae that was kindly donated by Enzyme Development Corpo-
ration, EDC (New York, NY, USA). Specic hydrolytic activity of the
b-galactosidase was 186,000 IUH$g1, determined according to
Vera, Guerrero, and Illanes (2011). One international unit of hy-
drolytic activity (IUH) was dened as the amount of b-galactosidase
that hydrolyzes 1 mmole of o-NPG per min at 50 mM o-NPG, pH 4.5
and 40
C. Hydrolysis rates were determined by measuring o-NP
release during the rst 90 s of reaction. o-NP was determined by
absorbance at 420 nm. All other reagents were analytical grade
products from Sigma or Merck (Darmstadt, Germany).
2.2. Analysis of carbohydrates
Substrates and products of SSP were analyzed in a Jasco RI 2031
HPLC delivery system (Jasco, Easton, USA) provided with a
refractive index detector, a isocratic pump (Jasco PU2080) and
autosampler (Jasco AS 2055). BP-100 Ca columns
(300 mm  7.8 mm) provided by Benson Polymerics (Sparks, NV,
USA) were used for carbohydrate analysis following the procedure
reported by Vera et al. (2011). Standards of tri- (GOS-3) and tetra-
saccharides (GOS-4), lactose, glucose and galactose were used for
the determination of retention times and for checking the linear
range of the measurements.
2.3. Yeasts and culture conditions
K. marxianus var marxianus NRLLY-1109 was cultured in 1 L
Erlenmeyer asks with 200 mL of working volume. Incubation was
done at 30
C and 200 rpm in an orbital shaker (Shin Saeng Skir-
601; Gyeonggi-do Korea). Medium was carbon limited with the
following composition (in g L1): carbon source: 20; yeast extract:
5.5; ammonium sulfate: 3.3; monobasic potassium phosphate: 0.8;
magnesium sulfate: 0.5. Cells were grown for 24 h and then
recovered by centrifugation at 13,300 g for 10 min at 5
C in a
Sorvall RC 2-B centrifuge (Wilmington, DE, USA). The cell pellet was
washed twice with distilled water and subjected again to centri-
fugation at the same conditions as above and then used as inoc-
ulum for the fermentation of raw GOS. Dry cell weight was
determined after drying the samples for 24 h at 105
C.
S. cerevisiae cells were obtained from commercial bakers yeast
(Lefersa S.A., Santiago, Chile). Yeast cells were reactivated with
20 g L1 of sucrose for 8 h at 40
C, pH 4.5 and 200 rpm before use.
The cell pellet was obtained as above described for K. marxianus.
2.4. Simultaneous synthesis and purication (SSP) of GOS
The experiments of SSP were carried out in 50 mL Erlenmeyer
asks with 20 mL of lactose medium to which the corresponding
mass of K. marxianus or S. cerevisiae cells were added. Samples
(0.1 mL each) were taken at regular time intervals and cells were
removed by centrifugation.
One-pot synthesis and purication of GOS was evaluated in
terms of the following parameters:
- GOS yield (YGOS):
MGOS
YGOS $100 (1)
MLac
where MGOS is the mass of total GOS produced and MLac is the initial
mass of lactose.
- Initial cell to carbohydrates mass ratio (R):
Mcell
R (2)
Mcarbohyd
where Mcell is the initial dry cell mass and Mcarbohyd is the mass of
total initial carbohydrates.
- Specic productivity of GOS (pGOS):
MGOS
pGOS (3)
ME $t
where MGOS is the masses of GOS, ME is the mass of enzyme added
and t is the nal time of fermentation.
- Final Purity (Pnal):
 C. Aburto et al. / LWT - Food Science and Technology 72 (2016) 81e89
Fig. 1. Comparison of the effect of R on YGOS (a), pGOS (b), Pnal (c) and pP (d) during the SSP of GOS with A. oryzae b-galactosidase and S. cerevisiae cells ( ) and with A. oryzae b-
galactosidase and K. marxianus cells after 24 h of reaction ( ) at 40
C, 200 rpm and pH 4.5. Nomenclature is in the text.
MGOS
Pfinal
Mcarbohyd;final
where MGOS and Mcarbohyd, nal are the masses of GOS and carbo-
hydrates at the end of fermentation respectively:
- Specic productivity of purication (pP):
MF
pP
Mcell $t
where MF represents the amount of unwanted carbohydrates
(glucose, galactose and lactose) removed per unit of cell mass
(Mcell) and unit time (t) at the end of fermentation.
The effect of the yeast genus (S. cerevisiae or K. marxianus),
enzyme-substrate ratio, initial lactose concentration and biomass-
substrate mass ratio on the parameters above described, were
determined during SSP at the time in which the concentration of
GOS was at its maximum. Experiments were conducted by moving
one variable at the time. 200 IUH/g lactose, 40% w/w lactose, R 0.1,
pH 4.5, 40
C and 200 rpm were considered as standard operation
conditions. Analysis of variance of one factor was done to evaluate
statistically signicant differences between the evaluated effects on
the considered parameters at a signicance level of 0.05.
2.4.1. Effect of yeast genus on SSP
K. marxianus and S. cerevisiae cells were added at R of 0.1, 0.2 and
83
0.3 g biomass/g lactose. Standard conditions of GOS synthesis (40% w/w
lactose initial concentration, 200 IUH/g lactose, 40
C and pH 4.5)
(4)
were evaluated for selecting the yeast strain to be used in SSP. The
reaction was carried out for 24 h and YGOS was used as selection
criterion. The samples were taken in duplicate at the conditions
stated above and quantied twice.
2.4.2. Effect of initial carbohydrates concentration on SSP
SSP with S. cerevisiae were conducted at 40
C, pH 4.5 and with
(5) lactose concentrations of 50, 40, 30 and 20% (w/w), keeping con-
stant the enzyme-substrate ratio in 200 IU/g lactose and the biomass
to lactose mass ratio (R) in 0.1 g biomass/g lactose (the biomass was
increased with the concentration of lactose to keep a constant R of
0.1). The samples were taken in duplicate at the conditions stated
above and quantied twice.
2.4.3. Effect of dry cell to initial carbohydrates mass ratio (R) on SSP
SSP with S. cerevisiae were conducted with 40% w/w of lactose at
40
C, pH 4.5 and 200 IU/g lactose with dry cell to total carbohydrate
mass ratios in the range from 0.05 to 0.4. The samples were taken in
duplicate at the conditions stated above and quantied twice.
2.4.4. Effect of enzyme-substrate ratio on SSP
SSP with S. cerevisiae were conducted with 40% w/w of lactose at
40
C, pH 4.5 with dry cell to total carbohydrate mass ratios (R) of
0.1. Enzyme-substrate ratios evaluated were 10, 25, 50, 100, 200 and
400 IU/g lactose. The samples were taken in duplicate at the
84 C. Aburto et al. / LWT - Food Science and Technology 72 (2016) 81e89

30 5
a) b)
25
4

GOS (gmg-1h-1)
20

YGOS (% w/w)
3
15
2
10

5 1

0 0
20 30 40 50 20 30 40 50
Carbohydrate (% w/w) Carbohydrate (% w/w)

40 5
c) d)
4
30
P (gg-1h-1)
Pfinal (%)

3
20
2

10
1

0 0
20 30 40 50 20 30 40 50
Carbohydrate (% w/w) Carbohydrate (% w/w)

Fig. 2. Effect of carbohydrate concentration on YGOS (a), pGOS (b), Pnal (c) and pP (d) during the SSP of GOS with A. oryzae b-galactosidase and S. cerevisiae cells at 40
C, 200 rpm and
pH 4.5 after 24 of reaction. Nomenclature is in the text.

conditions stated above and quantied twice.
2.4.5. Scale-up of SSP
At the operational conditions at which the best values of the
above described parameters were obtained, the SSP process was
scaled-up to 1 L in a reactor provided with temperature, pH and
agitation speed controls. These experiments were conducted with
40% w/w of lactose. The samples were taken in duplicate at the
conditions stated above and quantied twice.
3. Results and discussion
3.1. Effect of yeast genus on SSP
Values of YGOS, pGOS, Pnal and pP obtained in the SSP of GOS
with A. oryzae b-galactosidase and S. cerevisiae and K. marxianus
cells after 24 h of reaction are compared in Fig. 1. As seen in Fig. 1a
and b, YGOS and pGOS were reduced with the increase in R (initial
cell to carbohydrates mass ratio) in the SSP with both yeasts.
However, values obtained with K. marxianus cells were lower than
obtained with S. cerevisiae cells which is due to the capacity of K.
marxianus to consume lactose, where two genes are involved:
LAC12 that allows the internalization of lactose and LAC4 which
hydrolyzes it into glucose and galactose (Rubio-Texeira, 2006).
Therefore, only a fraction of it is being used as substrate for trans-
galactosylation reactions while the other fraction is being
consumed by the yeast cells. b-galactosidases are endowed with
hydrolase and transferase activities (Mahoney, 1998), and both
capacities have been exploited by the industry: their trans-
galactosylation activity is used for the production of oligosaccha-
rides, while their hydrolytic activity is employed essentially for
reducing the lactose concentration in milk and dairy products
(Rhimi et al., 2010). The predominant activity of b-galactosidases
will depend on reaction conditions, i.e. when lactose concentration
in the reaction medium is decreased, in this case by the con-
sumption of yeast cells, hydrolysis is favored over trans-
galactosylation with the consequent decrease in YGOS. The Pnal
using S. cerevisiae increases when R is higher, while with K. marx-
ianus occurs otherwise (Fig. 1c). It is likely that at low concentra-
tions of lactose, the enzyme hydrolyzes it and also some of the
synthesized GOS, to their monosaccharide units reducing the purity
of the product. It is important to recall that K. marxianus cells are
bigger than S. cerevisiae cells so a higher number of the latter were
present since experiments were conducted at equal mass of cells;
this may be reected in a higher rate of sugars consumption by S.
cerevisiae, obtaining higher purities. This is clearly reected in the
values of pP shown in Fig. 1d.
Based on these results and despite the fact that the purity
 C. Aburto et al. / LWT - Food Science and Technology 72 (2016) 81e89 85

25 4
a) b)
20
3

GOS (gmg-1h-1)
YGOS (% w/w)
15
2
10

1
5

0 0
0.05 0.11 0.20 0.30 0.39 0.05 0.11 0.20 0.30 0.39

R (gg-1) R (gg-1)

40 5
c) d)
4
30

P (gg-1h-1)
Pfinal (%)

3
20
2

10
1

0 0
0.05 0.11 0.20 0.30 0.39 0.05 0.11 0.20 0.30 0.39

R (gg-1) R (gg-1)

Fig. 3. Effect of R on YGOS (a), pGOS (b), Pnal (c) and pP (d) during the SSP of GOS with A. oryzae b-galactosidase and S. cerevisiae cells at 40
C, 200 rpm and pH 4.5. Nomenclature is
in the text.

attainable is modest because lactose will not be degraded, S. cer-
evisiae was selected for further studies of SSP of raw GOS, consid-
ering that the above problem can be circumvented by including a
pre-hydrolysis step of raw GOS with a highly hydrolytic b-galac-
tosidase. Additionally, S. cerevisiae has several advantages that
make it convenient: i) a generally regarded as safe status, which
eases waste disposal and product and process approval; ii) good
tolerance to low pH, which means low risk of bacterial contami-
nation and no need of neutralization of acidic products; and iii)
good tolerance to fermentation inhibitors (Borodina & Nielsen,
2014).
It should be emphasized that in a conventional system of two
sequential steps of synthesis and purication, K. marxianus is the
best alternative since purities close to 100% have been reached; this
is because in raw GOS, lactose, glucose and galactose are consumed
by the yeast without interfering with the synthesis of GOS (Gosling
et al., 2010; Guerrero et al., 2014; Li et al., 2008).
3.2. Effect of initial carbohydrates concentration on SSP
Fig. 2 shows the effect of carbohydrates concentration on the
SSP of GOS with A. oryzae b-galactosidase and S. cerevisiae cells,
expressed in terms of YGOS, pGOS, Pnal and pP. As seen in Fig. 2a,
YGOS increases with carbohydrate concentration because of the
higher availability of lactose for transgalactosylation. Initial
carbohydrates concentration showed a statistically signicant ef-
fect on YGOS (p-value of 0.002). These results are consistent with
those reported in the literature, where lactose initial concentration
is a most important variable in GOS synthesis, so a high concen-
tration is necessary to depress the hydrolytic potential and promote
synthesis, leading to high YGOS (Huerta et al., 2011; Torres,
Gonalves, Teixeira, & Rodrigues, 2010; Vera, Guerrero, Conejeros,
& Illanes, 2012). However, the values of YGOS shown in Fig. 2a, are
lower than reported for the conventional synthesis of GOS with A.
oryzae b-galactosidase where YGOS was 0.28 (Vera et al., 2012). As
seen in Fig. 2b, pGOS decreases with carbohydrate concentration;
this is because all experiments were contacted with the same
amount of wet biomass (3.5 g), so that R was different in each
experiment. It is worthwhile evaluating the results obtained at
different lactose concentrations and constant R. Initial carbohy-
drates concentration showed a statistical signicant effect on pGOS
(p-value of 0.003). As seen in Fig. 2c, Pnal increases with carbo-
hydrate concentration; however, it did not show a statistically
signicant effect (p-value of 0.33). As previously mentioned, the
higher transgalactosylation activity at high carbohydrates concen-
tration reduces the lactose concentration allowing a higher avail-
ability of monosaccharides for yeast consumption. Fig. 2d shows
the decrease of pP with increasing lactose concentration due to the
lower amount of biomass provided, showing a statistically signi-
cant effect (p-value of 0.02). This effect is similar to that reported by
86 C. Aburto et al. / LWT - Food Science and Technology 72 (2016) 81e89

30 25
a) b)
25
20

GOS (gmg -1h-1)

YGOS (% w/w)
20
15
15
10
10

5 5

0 0
10 25 50 100 200 400 10 25 50 100 200 400
E/S ratio (IU/g lactose) E/S ratio (IU/g lactose)

35 5
c) d)
30
4
25
P (gg-1h-1)
Pfinal (%)

20 3

15 2
10
1
5

0 0
10 25 50 100 200 400 10 25 50 100 200 400

E/S ratio (IU/g lactose) E/S ratio (IU/g lactose)

Fig. 4. Effect of enzyme-substrate ratio (E/S) on YGOS (a), pGOS (b), Pnal (c) and pP (d) during the SSP of GOS with A. oryzae b-galactosidase and S. cerevisiae cells at 40
C, 200 rpm
and pH 4.5. Nomenclature is in the text.

Guerrero et al. (2014) for the selective purication of raw GOS with
K. marxianus, where an increase in initial concentration caused a
decrease in pP.
3.3. Effect of dry cell biomass to initial carbohydrates mass ratio (R)
on SSP
Fig. 3 shows the effect of (dry) cell biomass to carbohydrates
mass ratio on the SSP of GOS with A. oryzae b-galactosidase and S.
cerevisiae cells, expressed in terms of YGOS, pGOS, Pnal and pP, at 40%
(w/w) lactose, 200 IUH/g lactose and 40
C. As seen in Fig. 3a, no
signicant differences were observed in YGOS around 21% (p-value
of 0.99), which is lower than obtained in conventional GOS syn-
thesis with A. oryzae b-galactosidase. Fig. 3b shows that pGOS
decreased with R, which may be due to the dilution produced when
adding different cell biomasses. R showed a statistically signicant
effect on pGOS (p-value of 0.001). Fig. 3c shows that R had no sig-
nicant effect on Pnal which was in all cases around 30%. This
suggests that the R values evaluated were not high enough to
consume all sugars; in fact only glucose was consumed while
galactose consumption was nil. While it has been reported that S.
cerevisiae completely metabolizes galactose (Berry & Brown, 1987;
Yoon et al., 2003), other results show only a slight consumption
(Goulas, Tzortzis, & Gibson, 2007). These authors propose a strat-
egy where the yeast is grown in a medium containing galactose as
the sole carbohydrate source, considering that the mechanism that
controls galactose uptake is inducible. Theoretical maximum value
for Pnal with S. cerevisiae is 71%, so the value obtained corresponds
to 44% of such value. Fig. 3d shows the sharp decrease in pP with R;
however, the effect was not statistically signicant (p-value of
0.22).
3.4. Effect of enzyme-substrate ratio on SSP
Fig. 4 shows the effect of the enzyme-substrate ratio E/S (in IUH
per unit mass of lactose) on the SSP of GOS with A. oryzae b-
galactosidase and S. cerevisiae cells, expressed in terms of YGOS,
pGOS, Pnal and pP, at 40% (w/w) lactose and 40
C. Fig. 4a shows that
E/S has a positive effect on YGOS in the range from 10 to 50 IUH/g
lactose, but slightly negative over it. Maximum YGOS of 0.25 ob-
tained at 50 IUH/g lactose was slightly lower than obtained in
conventional GOS synthesis. Fig. 4b shows that pGOS decreases with
E/S, which differs from results reported for the conventional syn-
thesis of GOS where no signicant differences were observed in the
range from 50 to 300 IUH/g lactose (Vera et al., 2012). Fig. 4c shows
that the effect of E/S on Pnal is similar than shown in Fig. 4a for the
effect on YGOS, with maximum Pnal of 31% obtained at 50 IUH/g
lactose. This effect may occur when the lactose transgalactosylation
rate of the enzyme-catalyzed reaction equals the monosaccharide
consumption rate by S. cerevisiae; it is possible that at higher IUH/g
lactose, the monosaccharides will increase and instead of being
consumed by the cells, they will inactivate the enzyme and GOS
 C. Aburto et al. / LWT - Food Science and Technology 72 (2016) 81e89 87

100 100
a) b)

Carbohydrates (% w/w)

Carbohhydrates (% w/w)
80 80

60 60

40 40

20 20

0 0
0 2 4 6 0 10 20 30 40 50
Time (h) Time (h)

100 40
c) d)
Carbohydrates (% w/w)

80
30

GOS (% w/w)
60
20
40

10
20

0 0
0 20 40 60 0 20 40 60
Time (h) Time (h)

Fig. 5. Carbohydrates proles (% of total) during SSP of raw GOS with A. oryzae b-galactosidase at 40
C, 200 rpm y pH 4.5. a) Control: conventional synthesis of GOS at 50 IUH/g
lactose); b) SSP at 50 IUH/g lactose and 0.25 g cell biomass/g carbohydrates in10 g of lactose at 40% w/w, c) SSP in bioreactor at 50 IUH/g lactose, 0.25 g cell biomass/g carbohydrates
in 1000 g of 40% w/w lactose at 5 vvm. : Lactose, - Glucose, A Galactose, C GOS; d) proles of GOS produced (% of total carbohydrates) in (a) ( ), (b) ( ), and (c) (C).

yield will be decreased. Fig. 4d shows the increase in pP with E/S;
this can be explained because at higher E/S the production of
monosaccharides increases to the point in which the yeast is no
longer capable of consuming them and start accumulating them in
the reaction medium with the consequent decrease in product
purity. Enzyme-substrate ratio did not show a statistically signi-
cant effect on YGOS (p-value of 0.06), pGOS (p-value of 0.07), Pnal (p-
value of 1.12) and pP (p-value of 0.06) for the signicance level
chosen.
3.5. Scale-up of SSP
Scale-up is a very important issue in process development.
Expanding a fermentation process from a lab-scale unit to a com-
mercial one is a challenge due to the difculty in assessing the
biological, chemical and physical factors involved in scale up (Hsu &
Wu, 2002; Junker, 2004). This study considered mainly physical
factors such as reactor conguration, aeration, agitation and tem-
perature control.
Fig. 5 shows the scale-up of the system to a 1 L reaction volumen
operation at the conditions where YGOS was maximum, this is E/
S 50 IUH/g lactose, R 0.25, 40
C, pH 4.5 and 40% w/w lactose.
The reactor was operated under temperature and pH control at an
aeration rate of 5vvm (volumes of air per volume of reaction me-
dium per minute). Fig. 5a shows the control (conventional GOS
synthesis) with a YGOS of 28% at 4 h of reaction, corresponding to
53% lactose conversion, the product having 15% glucose and 4%
galactose. Fig. 5b shows the results of SSP at small scale, where a
higher YGOS was obtained (33.6%) at 24 h of reaction, corresponding
to 39.9% lactose conversion, the product having 14.2% glucose and
12.3% galactose.
S. cerevisiae is a facultative fermenting yeast, whose metabolic
behaviour depends on the oxygen and glucose concentrations in
the medium. Thus, under anaerobic or oxygen-limited conditions it
exhibits alcoholic fermentation, but under fully aerobic conditions
a mixed respiratory-fermentative metabolism is observed (Crab-
tree-positive effect) when the sugar concentration exceeds a
certain threshold value (approximately 1 mM). Conditions leading
to sugar fermentation results in the formation of ethanol, acetic
acid, glycerol and other compounds and consequently it reduces
the biomass yield (Beudeker, van Dam, van der Plaat, & Vellenga,
1990).
Fig. 5c shows the results in the scaled-up SSP process where also
a 40% YGOS was obtained at 24 h of reaction, corresponding to 40.3%
lactose conversion, the product having 0.8% glucose and 19.2%
galactose. In the latter case, aeration allowed maintaining a high
cell viability with a much higher glucose consumption, whose level
was below 1% during the whole experiment. Galactose % increased
steadily, which may be due to the early and preferential glucose
consumption that impedes galactose to be consumed until most of
the glucose has disappeared. However, glucose acts as a repressor
even at low concentrations (lower than 1% of total carbohydrates).
In fact, glucose is the main carbon source for S. cerevisiae; its
presence triggers a signaling cascade that, among other effects,
causes the repression of the GAL genes and others genes involved in
the utilization of alternative carbon sources (Nehlin, Carlberg, &
88 C. Aburto et al. / LWT - Food Science and Technology 72 (2016) 81e89
Ronne, 1991; Rubio-Texeira, 2005; Schuller, 2003; Verstrepen et al.,
2004).
Galactose increase also coincides with GOS decay after 30 h of
reaction, since a fraction of them is being hydrolyzed along with
lactose hydrolysis that occurs because its concentration has drop-
ped so that hydrolysis prevails over transgalactosylation. A. oryzae
b-galactosidase is reportedly inhibited by galactose so that the
enzyme is progressively being inhibited (Vera et al., 2011).
The YGOS obtained (40% w/w) is in agreement with the values
published by Li et al. (2008) for the selective fermentation with S.
cerevisiae of a raw GOS diluted to 20% w/w total carbohydrates,
where nal purity obtained was 39%. However, the values of YGOS
obtained are higher than reported for the conventional synthesis of
GOS with A. oryzae b-galactosidase where YGOS was 28% (Vera et al.,
2012).
4. Conclusions
SSP of GOS was conveniently performed by transgalactosylation
of lactose with A. oryzae b-galactosidase and partial purication by
sugars removal with S. cerevisiae cells. GOS yields were higher than
obtained in a conventional system when the one-pot operation is
carried out in a temperature, pH and aeration rate-controlled
bioreactor; in this system, reduction of monosaccharides is main-
tained along the operation, being glucose almost completely
depleted. Even though not removed in SSP when using S. cerevisiae
cells, lactose removal is attainable by pre-hydrolysis of lactose in
raw GOS. SSP may represent a technological advantage with respect
to conventional GOS synthesis in terms of productivity, reaching
levels of purities and yields already reported but in a shorter re-
action time and in a one-pot operation.
Acknowledgements
This work was nanced by Chilean Fondecyt Grant 1130059 and
Grant DI 203.797/2013 from the Ponticia Universidad Cato
lica de
Valparaiso. We acknowledge the generous donations of A. oryzae b-
galactosidase by Enzyme Development Corporation (New York,
USA).
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