Separation and Purification Technology 288 (2022) 120678

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Separation and Purification Technology

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

From sucrose to fructo-oligosaccharides: Production and purification of

fructo-oligosaccharides by an integrated enzymatic catalysis and membrane
separation process
Weilei Cao a, b, Tingting Deng c, Weifeng Cao a, b, *, Fei Shen a, b, Yinhua Wan a, b, *
a
State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, People’s Republic of China
b
School of Chemical Engineering, University of Chinese Academy of Sciences, Beijing 100049, People’s Republic of China
c
Agro-product Safety Research Center, Chinese Academy of Inspection and Quarantine, Beijing 100123, People’s Republic of China

A R T I C L E I N F O A B S T R A C T

Keywords: Fructo-oligosaccharides (FOS) are a group of linear fructose oligomers with numerous health benefits, which can
Fructo-oligosaccharides be synthesized by the transfructosylation of sucrose using fructosyltransferases (FTases). In this study, it was
Membrane found that the selected FTases showed both hydrolytic and transfructosylating activities. For FOS production, the
Fructosyltransferases
hydrolytic activities of FTases became significant when the reaction time was above 1 h. Thus, FTases needed to
Diafiltration
be inactivated or removed from the reactor after achieving the highest FOS conversion. We therefore developed
an integrated ultrafiltration-diafiltration- concentration process for FTases reuse and FOS purification. Because of
the 100% rejection of FTases activity and a higher average flux, a PES-10 ultrafiltration membrane was selected
for FTases recovery, where the cake layer formation was the main membrane fouling mechanism. Then, a fed-
batch reaction process was developed for FOS production, where the hydrolytic behavior of FTases was negli­
gible, and the FTases dosage was reduced by 75% compared to single batch operation (four batches). Moreover,
the FOS production was comparable, and 95.5% FTases was recovered by the PES10 membrane after the four
fed-batches processes. Subsequently, a NF5 nanofiltration membrane was selected to further separate FOS from
the FTases-free FOS solution by a constant volume diafiltration process under 45 ◦ C, and the monosaccharides in
the reaction mixture (glucose and fructose) were completely removed, increasing the FOS purity from 55.8% to
92.3%. Finally, the FOS and sucrose in the diafiltration permeate was recovered by a DL nanofiltration mem­
brane under 60 ◦ C. The outcome of this work offers a complete technical route for FOS production using sucrose
as substrate.

1. Introduction either by the enzymatic catalysis of sucrose using FOS synthase, or by

Fructo-oligosaccharides (FOS), such as 1-kestose (GF2), nystose
(GF3), 1-fructofuranosyl nystose (GF4) and 1,1,1,1-kestohexose (GF5),
have attracted high interest in the last decades due to their different
health-promoting properties (prebiotic, dietary fibre source, increase
mineral absorption, anticarcinogenic, etc.), and their industrial interest
as sweeteners with low calories [1–4]. Moreover, FOS are considered as
a non-digestible, non-cariogenic and low-caloric fibre, which are able to
improve taste, mouthfeel, texture and shelf-life of food products [5].
Nowadays, they are considered as natural food ingredients in most
countries [5,6].
Conventionally, commercial FOS production is mainly achieved
chemical or enzymatic hydrolysis of raw inulin [6–8]. However, the
production of food-grade FOS is preferred using biocatalysts (enzymes
obtained from fungi or bacteria), since the chemical hydrolysis of inulin
uses chemicals with high toxicity and lacks in specificity and these
synthetic sugars may be rejected by the consumer [5]. Compared to
inulin, sucrose is an abundant feedstock in many countries, and FOS can
be produced readily from sucrose by fermentation or enzymatic catalysis
[7,9–11]. Owing to the abundance and easy availability of the substrate
(i.e., sucrose), transfructosylation of sucrose using fructosyltransferases
(FTases) appears to be the first of choice. In addition, during the FOS
production process, FOS synthase showed both hydrolytic and trans­
fructosylating activities [12,13]. Thus, it needed to timely separate or

* Corresponding authors at: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190,
People’s Republic of China.
E-mail addresses: wfcao@ipe.ac.cn (W. Cao), yhwan@ipe.ac.cn (Y. Wan).

https://doi.org/10.1016/j.seppur.2022.120678
Received 6 December 2021; Received in revised form 9 February 2022; Accepted 13 February 2022
Available online 17 February 2022
1383-5866/© 2022 Elsevier B.V. All rights reserved.
W. Cao et al.
inactivate the synthase in the reaction system for high FOS production,
since the synthase activity was high even at room temperature [7].
However, the inactivation of FOS synthase activity during the catalysis
or purification process resulted in high cost of FOS production. In an
ideal FOS production system, enzymes would be harvested or recycled
readily with a minimal processing. Though enzyme immobilization
circumvents the requirement to separate soluble enzymes from their
products, immobilization limits the spatial orientation of the enzyme,
which can increase mass transfer resistance, and change the catalytic
properties of the enzyme [8,14,15]. Recently, it was reported that
membrane technology was suitable to achieve invertase reuse [16], and
an integrated membrane process was a good choice for refining sugars
[17,18]. Therefore, an integrated enzymatic catalysis and membrane
separation process may achieve FTases reuse and high FOS production.
During FOS production by enzymatic catalysis of sucrose, the FOS
mixtures are only around 60% purity, containing FOS, sucrose, fructose,
and glucose, which prevents their inclusion in diabetic and dietetic food
[7,19], because the unwanted monosaccharides and disaccharide are
not prebiotic and thus increase blood sugar levels after absorption [20].
Recently, microbial treatment using successive purification fermenta­
tions has attracted great attention [19,21,22]. In such process, a mi­
crobial treatment was proposed to reduce the amount of small
saccharides in the mixture, where the microorganisms mainly include
Saccharomyces cerevisiae [19] and Bacillus coagulans [21]. The highest of
FOS mixtures with 81.6% (w/w) purity can be obtained using a two-step
fermentation in which FOS were first synthesized by Aureobasidium
pullulans and then the small saccharides were metabolized by
S. cerevisiae [23]. Moreover, a purity of 96.6% was achieved using an
enzyme membrane bioreactor and subsequent fermentation with pro­
biotic B. coagulans [21]. However, though the used microorganisms can
selectively metabolize the small sugars, some by-products, such as lactic
acid, ethanol, biomass, and bad smell, can also be produced, which
should be separated in the next step. Meanwhile, the nutrient medium
for the cells’ growth would also pay negative effects on downstream
processing of FOS [21,23]. Recently, it was reported that ultrafiltration
(UF) membrane can improve reusability of invertases with a molecular
weight of 135–270 kDa [10]. Moreover, nanofiltration (NF) was used for
the separation of sucrose and reducing sugar (i.e., fructose and glucose)
in cane molasses by a cascade diafiltration-concentration process [24].
Thus, an integrated enzymatic catalysis and membrane separation pro­
cess may be suitable for achieving a high purity of FOS. However,
though ultrafiltration membranes and nanofiltration membranes have
been reported to improve the reusability of enzyme and separate su­
crose/reducing sugars, respectively, the integrated enzymatic catalysis
and the separation process using ultrafiltration and nanofiltration
membrane has not been carried out in FOS production. That is, the
invertase with a higher molecular weight (135–270 kDa) [10,16] is
different from FTases with a dimeric structure of an estimated molecular
weight 65 kDa/monomer [13]. Moreover, the FOS system is more
complicated which contains sucrose, glucose, fructose, and FOS (i.e., a
mixture of GF2, GF3, GF4, and GF5), where the FOS are the main mate­
rials. However, the system of sucrose, glucose, and fructose [24], where
sucrose is the main material. Therefore, the integrated membrane pro­
cess for FOS production can not be simply deduced from the references
[10,16,24], which should be in-depth studied.
Herein, a novel integrated enzymatic catalysis and membrane sep­
aration process was proposed for the production and purification of FOS.
Firstly, a suitable UF membrane for FTases separation and reuse from
the crude FOS solution was selected. Secondly, a diafiltration was pro­
posed with a NF membrane of NF5, aiming at removing sucrose and
reducing sugar from the crude FOS solution. Thirdly, another concen­
tration process was proposed with a NF membrane of DL for recovering
FOS and sucrose.
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Separation and Purification Technology 288 (2022) 120678
2. Materials and methods
2.1. Experimental setup and procedure
The dead-end filtration experiments at constant flux (Fig. S1) or
pressure (Fig. S2) were the same as described in in our previous work
[24,25] (see Supplementary information), respectively. A new mem­
brane was used for each series of experiments, and each membrane disc
was firstly dipped in 50% ethanol solution for about 5 s to remove
manufacturing residues from the membrane and then soaked in deion­
ized water for at least 12 h prior to use. In our previous work [16], a PES
membrane was suitable to recycle invertase because it enabled complete
invertase retention and relatively high permeate flux, and when L-
cysteine (Cys) was grafted on the polydopamine-coated membrane, it
could reduce invertase adsorption on the membrane. Thus, we selected
the different PES membranes for FTases recovery without comparing the
different membrane materials for FTases recovery in this work. For
selecting a suitable membrane for the enzyme recovery, 50 mL deion­
ized water was pre-filtrated, and then 50 mL model solution of the
FTases was filtrated at 40 ◦ C, pH 5.5 and 0.3 Mpa. The diafiltration
processes with the NF5 membrane for FOS recovery and the DL mem­
brane for sucrose and FOS recovery were the same as described in our
previous work [24]. After each test, the used membrane module was
rinsed using hot deionized water with the same temperature as feed
temperature. Membrane permeability was examined before and after
the filtration to monitor the irreversible fouling. The Characteristics of
tested UF membranes (Table S1) and NF membranes (Table S2) were
listed in the Supplementary information.
2.2. Enzymatic synthesis of crude FOS
FTases, with an enzyme activity of 2600 U/mL, an optimum tem­
perature of 65 ◦ C and an optimum pH of 5.2–5.4, were purchased from
Beijing Solarbio Science & Technology Co., LTD (Beijing, China), which
were appropriately diluted according to the demand with 0.1 M citrate
buffer. The reaction mixture consists the following compositions: 5 mL
of sucrose 100 g/L, 4 mL of 0.1 M citrate buffer, and 1 mL diluted
enzyme, where the sucrose and FTases solutions were respectively pre­
pared by 0.1 M citrate buffer. To investigate the effect of enzyme dosage
on FOS production, FTases was diluted to 300, 400, 500, 600, 700, 800
and 900 U/mL with 0.1 M citrate buffer. The reaction was carried in the
following conditions: (a) pH 5.5, (b) temperature 55 ◦ C and (c) reaction
time 1 h. To investigate the effect of pH on FOS production, 0.1 M citrate
buffer with different pH (4.0, 4.5, 5.0, 5.5, 6.0 and 6.5) was used to
prepare the corresponding FTases and sucrose solutions. The reaction
was carried in the following conditions: (a) final concentration of
enzyme activity 80 U, (b) temperature 55 ◦ C, and (c) reaction time 1 h.
To investigate the effect of temperature on FOS production, the reaction
temperature was controlled at 25, 30, 35, 40, 45, 50, 55, 60, and 65 ◦ C.
The reaction was carried in the following conditions: (a) final concen­
tration of enzyme activity 80 U, (b) pH 5.5, and (c) reaction time 1 h.
After optimizing the enzyme reaction conditions, FOS production
was further carried by fed-batch reaction to study the enzymatic catal­
ysis mechanism in 1-L scale bioreactor. Then, the obtained solution was
used to recover FTases and purify the crude FOS by an integrated
membrane process.
2.3. Analytical methods
The FTases activity, and the concentrations of the FOS, sucrose,
glucose, and fructose were measured as described in our previous work
[7] (see Supplementary information). After enzymatic catalysis process,
the enzyme reaction was stopped by heating at 100 ◦ C for 15 min. The
concentrations of the sugars were measured using a HPLC apparatus
(LC-20AT, Shimadzu, Japan) equipped with an RI-detector using the
Asahipak NH2P-50 4E column (Shodex, Japan). The column
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temperature was set to 30 ◦ C, and a mixture of acetonitrile/distilled
water (7:3, v/v) was applied as the mobile phase at a flow rate of 1 mL/
min. The observed retention (Robs) of sucrose and reducing sugar, was
calculated as described in the Supplementary information [17,24]. The
proteins in the crude enzyme were measured with a Pierce™ BCA pro­
tein assay kit (Thermo scientific, USA). Statistical analysis of the
different experimental groups was conducted by subjecting the experi­
mental data to one-way analysis of variance (ANOVA) using the Ori­
ginPro 2018 software (Origin Lab Corporation, Northampton, MA, USA)
at a 95% confidence level. The data presented in Figs. 1–10 are the
average values with error bars.
FOS(g/L) = GF2 (g/L) + GF3 (g/L) + GF4 (g/L) + GF5 (g/L)
Fig. 1. Effect of enzyme dosage (a and b), pH (c and d), and temperature (e and f) on FOS production. Data are given as mean ± SD, n = 3.
Separation and Purification Technology 288 (2022) 120678
GFn (g/L)
PercentageofGFn (%, n = 2, 3, 4, or5) = × 100 (2)
FOS(g/L)
3. Results and discussion
3.1. Optimization of catalytic conditions for the selected FTases
3.1.1. Effect of FTases dosage on FOS production
FOS production was mainly affected by the substrate (i.e. sucrose)
concentration and the FTases activity and dosage, while FTases activity
is strongly affected by environmental pH and temperature. Thus, the
(1) effect of FTases dosage on FOS production was first evaluated at the
fixed sucrose concentration (i.e. 50 g/L) (Fig. 1 a and b). As shown in
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Fig. 2. Effect of enzymatic catalysis time on FOS production under pH 5.5,
45 ◦ C, 50 g/L sucrose, and 80 U/mL FTases. Data are given as mean ± SD, n
= 3.
Fig. 1a, the FOS concentration increased with FTases dosage until above
80 U/mL as the produced GF2 began to decrease. Moreover, the ratio of
GF2 in the FOS decreased with the increased FTases dosage, and the ratio
of GF3 showed a reverse trend. Meanwhile, the fructose concentration
kept constant in all the FTases dosages tested (Fig. 1b), which indicated
that the hydrolysis reaction of FOS was not significant (the concentra­
tion of fructose in the reaction medium would increase if the hydrolysis
of FOS happened [26]). Therefore, the suitable FTases dosage for FOS
production was 80 U/mL.
3.1.2. Effect of pH on FOS production
Most of the FTases displayed maximal activity in the slightly acidic
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Separation and Purification Technology 288 (2022) 120678
Fig. 3. Effect of excessive amounts of enzymes on FOS production. The enzy­
matic catalysis time was 24 h. Data are given as mean ± SD, n = 3.
pH range of 4.5–6.0, while the transfructosylating versus hydrolytic
behavior varied depending upon the optimal conditions [6]. Thus, the
effect of pH on the FOS production was evaluated at a fixed sucrose
concentration of 50 g/L and a FTases dosage of 80 U/mL. As shown in
the Fig. 1c, the maximal concentrations of FOS, GF2 and GF3 were ob­
tained at pH5.5, and their concentrations decreased when the pH was
above pH5.5. Moreover, when pH value was below 5.5, the ratio of GF3
in the FOS increased with the increased pH and its maximal ratio was
obtained at pH 5.5, while the the ratio of GF2 showed a reverse trend.
When pH value was above pH5.5, the ratio of GF3 in the FOS decreased,
and the ratio of GF2 showed a reverse trend. Meanwhile, the fructose
began to appear when the pH was above 5.0 (Fig. 1d), which suggested
that the hydrolysis reaction of FOS appeared when the pH was above pH
5.0. In addition, when the pH was above 5.5, the residual sucrose began
to increase, which indicated that the transfructosylating activity of the
FTases began to be inactivated. Therefore, the favorable pH value for
FOS production was 5.5, considering the maximal FOS concentration
25.1 g/L with a FOS yield of 50.2% obtained at pH 5.5.
3.1.3. Effect of temperature on FOS production
The optimum temperature of FTases usually ranges from 50 ◦ C to
70 ◦ C [6], while they did not report whether the transfructosylating
versus hydrolytic behavior was affected by the temperature. Thus, the
effect of temperature on the FOS production was evaluated at pH 5.5
under a fixed sucrose concentration of 50 g/L and a FTases dosage of 80
U/mL. For GF2, its concentration was not affected significantly by the
temperature when the temperature was below 55 ◦ C, while its
W. Cao et al.
Fig. 4. Effect of 800 U/mL FTases and 500 g/L sucrose on FOS production. The
concentrations of FTases and sucrose were 10 times of those under the opti­
mization condition (i.e., 80 U/mL FTases and 50 g/L sucrose), and the enzy­
matic catalysis time was 10 h. Data are given as mean ± SD, n = 3.
concentration decreased sharply when the temperature was above
55 ◦ C. For GF3, its concentration increased when the temperature was
below 50 ◦ C, while its concentration began to decrease when the tem­
perature was above 50 ◦ C. As a result, the maximal FOS concentration
28.9 g/L with a FOS yield of 57.8% was obtained at 45 ◦ C which was
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Separation and Purification Technology 288 (2022) 120678
much lower than that in the previous work reported by Lateef et al. [27].
Moreover, the ratio of GF2 in the FOS decreased with the increased
temperature when the temperature was below 50 ◦ C, then its ratio
started to decrease when the temperature was above 50 ◦ C, while the
ratio of GF3 in FOS showed a reverse trend compared with that of GF2.
Meanwhile, the residual sucrose began to increase when the tempera­
ture was above 55 ◦ C (Fig. 1f), indicating that the transfructosylating
activity of the FTases began to be inactivated when the temperature was
above 55 ◦ C. In addition, the fructose concentration was obviously
appeared between the temperature 40 and 60 ◦ C (Fig. 1f), which sug­
gested that the hydrolysis reaction of FOS appeared among the tem­
peratures. Therefore, the transfructosylating and hydrolytic behaviors
were both affected by the temperature, and the favorable temperature
value for FOS production was 45 ◦ C, considering that the maximal FOS
concentration was obtained at 45 ◦ C.
3.1.4. Effect of enzymatic catalysis process on FOS production
Above sections showed that the FTases possessed both the trans­
fructosylating and hydrolytic behaviors, which were affected by the
FTases dosage, pH, and temperature (Fig. 1). Meanwhile, the ratios of
the components in the FOS varied in the reaction system, which was
similar to that reported in our previous work [7]. Thus, we are interested
in exploring whether an equilibrium among the FOS products, enzyme,
residual sucrose, and byproducts (i.e. glucose and fructose) could be
achieved at certain time. To reveal this issue, effect of the reaction time
on FOS production was firstly evaluated under pH 5.5, 45 ◦ C, 50 g/L
sucrose, and 80 U/mL FTases. As showed in Fig. 2a, the concentrations
of FOS and GF2 decreased with the increased reaction time in general.
Similarly, the maximum FOS concentration also occurred quite early
[26]. For GF2, its concentration and ratios in the FOS decreased in
general. For GF3, its concentration increased at the initial stage, then
began to decrease obviously after 8 h. Similarly, the ratio of GF3 in the
FOS increased in the initial stage, then began to decrease after 8 h. For
GF4, its concentration increased up to 10 h, then kept about constant,
while its ratios increased with the increased reaction time in general
(Fig. 2b). Similarly, GF2 and GF3 also acted as both fructosyl donor and
acceptor to produce GF4 [26]. Surprisingly, the residual sucrose was not
converted to FOS completely, which kept around 6 g/L after 2 h
(Fig. 2c). This phenomenon was different from that in the previous work
reported by Fan et al.[8], that is the residual sucrose could not be con­
verted to FOS using the FTases. Moreover, the concentration of glucose
and fructose increased with the increased reaction time, which indicated
that the hydrolytic activities of the FTases were strong. As a result, most
of the produced FOS were hydrolyzed by the FTases after 24 h though
the substrate (i.e. sucrose) still existed. It also suggested that the enzyme
had no obvious substrate specificity, and it can simultaneously use su­
crose, dimers and trimers as substrates.
Furthermore, the above phenomenon was further verified using
excessive amounts of FTases on FOS production under pH 5.5, 45 ◦ C, 50
g/L sucrose, and enzymatic catalysis for 24 h. As shown in Fig. 3a, little
FOS exited in the reaction system using excessive amounts of FTases,
and the main products were glucose and fructose. This phenomenon was
similar to that in Fig. 2. Then, the above phenomena were further
verified using 10 times of the substrate and FTases (i.e. 800 U/mL FTases
and 500 g/L sucrose) in the FOS production for 10 h under pH 5.5, 45 ◦ C.
As shown in Fig. 4a, the concentrations of FOS, GF2 and GF3 decreased
with the increased reaction time in general. For GF4, its concentration
increased with the the increased reaction time up to 6 h, then kept
almost constant. Surprisingly, the GF5 was detected in the reaction
system, and its concentration increased with reaction time. Moreover,
the ratios of GF2 and GF3 decreased with reaction time in general
(Fig. 4b), while the ratios of GF4 and GF5 showed a reverse trend. In
addition, it was amazing that the residual sucrose kept at a high con­
centration (i.e. 230 g/L) up to 10 h (Fig. 4c), and it could not be further
converted into FOS, which was also different from that in another work
reported by Fan et al. [8]. However, the fructose began to be detected
W. Cao et al. Separation and Purification Technology 288 (2022) 120678

Fig. 5. FTases recovery by different UF membranes. Data are given as mean ± SD, n = 3.

Fig. 6. Filtration characteristics of the model enzyme solution using the PES 10 membrane in four batches. In Figs. (a), (b) and (c), the filtration characteristics of the
selected membrane PES10 were evaluated using a 80 U/mL model FTases solution in the first batch, then the permeate was recycled in the cell and another batch was
restarted. In Figs. (d), (e), and (f), the filtration characteristics were evaluated using a new 80 U/mL model FTases solution in each batch without considering the
residual enzyme activity in the previous batch, where the permeate was not recycled in the cell. Data are given as mean ± SD, n = 3.

after reacting for 4 h which indicated that the transfructosylating and
hydrolytic behaviors simultaneously took place. Fig. 4 also suggested
that a high initial concentration of substrate (i.e. sucrose) would pro­
duce high concentration of FOS with a higher polymerization degree,
while high polymerization degree of FOS would decrease the FOS yield,
as the FOS was the total concentration of all the components. This results
were different from those in another work reported by Zhang et al. [28],
which may be caused by the different sources of enzymes. Thus, lower
initial sugar concentration (i.e., 50 g/L) was more suitable for FOS
production using the selected FTases.
3.2. Optimization of the membrane processes for the FTases recovery
3.2.1. Membranes selection for FTases recovery using model FTases
solutions
The selected commercial FTases were preliminarily analyzed by SDS-
PAGE (Fig. S3), revealing that the FTases were an enzyme mixture with a
molecular weight range of 30–115 kDa, and the main part was around
65 kDa which was similar to that in another work reported by Ghazi
et al. [13]. In the previous section, the FTases were used only once,
increasing the cost of the FOS production. In order to reuse the FTases,
different polyether sulfone (PES) membranes (i.e., PES8, PES10, PES20,
PES50 and PES100 membrane) were used to recover the FTases using
the model FTases solutions (i.e., final concentration of 80 U/mL FTases
in 0.1 M pH5.5 citrate buffer). As shown in Fig. 5a and b, about 100%
FTases activity was recovered by the PES8, PES10 or PES20 membrane,
and some FTases activity lost in the permeate of PES50 and PES100
membranes since the molecular cut off (MWCO) of the PES50 or PES100
was too large, and their pore size was not small enough to retain the
enzyme molecules. It was also amazing that the average flux 185.5 L/
(h⋅m2) of the PES10 was higher than that of PES20, though the MWCO of
the PES10 was smaller than that of PES20 (Fig. 5a). Moreover, Fig. 5b
indicated that the irreversible fouling of the PES20 was 1.84 times of the
PES10 membrane. Thus, the severer fouling of the PES 20 resulted in the

6
W. Cao et al.
Fig. 7. Filtration of the crude FOS solution using the PES 10 membrane in four
batches. Data are given as mean ± SD, n = 3.
lower average flux than that of PES10. However, the less irreversible
fouling on the PES50 and PES100 membranes can be explained by the
lower retention of FTases. Although high flux and minimal fouling are
advantageous properties for a membrane, the undesired low retention of
FTases by the PES50 and PES100 membranes indicated that they were
unsuitable for recovering FTases. Thus, the PES10 membrane was suit­
able for FTases recovery, due to a higher average flux of 185.5 L/(h⋅m2),
100% FTases activity recovery, and a relative lower irreversible fouling
of 39.6%.
3.2.2. Filtration behavior of PES10 membrane for FTases recovery using
model FTases solution in four batches
Filtration behaviors were primarily investigated by membranes se­
lection, the feed solution, and the interactions between them. The
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Separation and Purification Technology 288 (2022) 120678
Fig. 8. Effect of fed-batch processes on FOS production. The fed-batch pro­
cesses were carried out under 45 ◦ C and pH 5.5. The FTases activity and sucrose
concentration were 80 U/mL and 50 g/L, respectively, and new finial concen­
tration 50 g/L solid sucrose was fed into the crude FOS solution every 2 h. Data
are given as mean ± SD, n = 2.
reaction system of FOS production consisted of several sugar compo­
nents (i.e., FOS, sucrose, glucose, and fructose) and FTases. Each
component affected the filtration performance in a different manner.
Thus, since the FTases activity was not detected in the permeate of
PES10 membrane (Fig. 5b) and a relative high average flux was obtained
with the membrane, the filtration behaviors of the selected PES10
membrane were evaluated using a 80 U/mL model FTases solution in the
first batch, then the permeate was recycled in the cell and another batch
was restarted. As shown in Fig. 6a, the average flux decreased sharply in
the second batch, and then it decreased slightly. Compared with the
W. Cao et al.
Fig. 9. The solutes concentration in the retentate during diafiltration step using
a dead-end filtration cell under constant flux mode at 13.26 Lm− 2h− 1. V0 and V
are the cell volume and diafiltration solvent volume, respectively. Data are
given as mean ± SD, n = 3.
previous batch (Fig. 6a), the average fluxes were decreased by 19.92%,
6.68% and 5.73% in batch 2, batch 3 and batch 4, respectively. Mean­
while, the activity of the FTases can not be detected in the permeate
among all the batches (Fig. 6b), and the new loss of the FTases activity in
the retention was 0.53, 9.53, 12.56, and 8.33 U/mL in the batch 1, batch
2, batch 3 and batch 4, respectively, which was 0.66%, 11.82%, 17.66%,
and 14.22% of the initial FTases activity in the batch 1, batch 2, batch 3,
and batch 4, respectively. The lower newly lost FTases activity in the
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Separation and Purification Technology 288 (2022) 120678
Fig. 10. The solutes concentration in the retentate during the concentration
step with a DL membrane under 60 ◦ C using a dead-end filtration cell under
constant flux mode. The permeate flux was 39.8 Lm− 2h− 1. The initial concen­
trations of FOS, sucrose, fructose and glucose in the solution were 5.5, 5.2, 0.2
and 3.7 g/L, respectively. V0 and V are the cell volume and crude FOS volume,
respectively.
batch 4 was caused by the lower FTases activity in the retention of
batches 4, which was only 47.67 U/mL. In addition, since the FTases
activity could not be detected in the permeate and the FTases activity
was stable in the control, it indicated that the undetected FTases were
mainly adsorbed in/on the PES membrane, which also resulted in the
irreversible fouling of the membrane (Fig. 5b). The similar phenomenon
was also reported in another work reported by Wang et al. [16].
Moreover, to further evaluate the adsorption characteristics of the
FTases in /on the PES membrane, new 80 U/mL mode FTases solution
was added in each batch without considering the residual enzyme ac­
tivity in the previous batch, and the permeate was not recycled in the
cell. As shown in Fig. 6d, the average flux decreased sharply in the
second batch, and then it decreased slightly, which was similar to that in
the Fig. 6a. Compared with the previous batch (Fig. 6d), the average
fluxes were decreased by 29.68%, 21.99% and 14.76% in batch 2, batch
3 and batch 4, respectively. Similarly to that in the Fig. 6b, the activity of
the FTases was not detected in the permeate among all the batches
(Fig. 6e). Meanwhile, the new loss of the FTases activity in the retention
was 0.53, 17.56, 22.97, and 23.08 U/mL in the batch 1, batch 2, batch 3
and batch 4, respectively, which was 0.66%, 11.01%, 10.60%, and
8.43% of the initial FTases activity in the batch 1, batch 2, batch 3, and
batch 4, respectively. Compared with that in the Fig. 6c, the adsorption
W. Cao et al. Separation and Purification Technology 288 (2022) 120678

quantity of FTases by the PES 10 membrane was lower than that from the sucrose, fructose or glucose was not the prebiotics [20], the sepa­
the second batches in Fig. 6c. In view of the higher decrease of the ration of them form the crude FOS solution was necessary.
average flux in Fig. 6d than in Fig. 6a from the second batch, it indicated
that the membrane fouling was much more serious when the FTases 3.3.2. Purification of FOS from the crude FOS solution in the fed-reaction
concentration was increased from second batch. That is, filter cake were process with a NF5 membrane
more easy to form on the membrane, since the main mechanism of In order to improve the FOS purification, an integrated ultrafiltration
membrane fouling is cake layer formation (Table 1). Once the filter cake -diafiltration-concentration process was proposed. Fist, the crude FOS
was formed, other substances were hard to enter the pores of the solution from the fed-batch reaction was treated by the PES 10 mem­
membrane. Thus, the absorption ratio of FTases by the PES10 membrane brane to recover the FTases. After filtering 50 mL of the crude solution,
in Fig. 6e was much lower than that in Fig. 6b, which was caused by the about 76.4 U/mL FTases was recovered by washing the cell with 50 mL
high concentration of FTases from second batches, and the high mo­ 0.1 M pH 5.5 citrate buffer, and the filtration characteristics were similar
lecular weight of the FTases which were higher enough than the MWCO to those in Fig. 7 (Data were not shown here). Then, compared the
of the PES 10 membrane (Fig. S3). molecular weights of FOS, sucrose, fructose or glucose with the MWCO
Furthermore, the filtration characteristics of the selected membrane of membranes [29,30], a NF5 membrane was selected to further sepa­
PES10 were evaluated using a crude FOS solution, which was obtained rate FOS from the FTases-free crude FOS solution by a constant volume
under 50 g/L sucrose, pH 5.5, 80 U/mL FTases, 45 ◦ C, and 2 h. As shown diafiltration process under 45 ◦ C. As shown in Fig. 9a, the FOS con­
in Fig. 7a, the average flux was 173.2 L/(h⋅m2) in the first batch, which centration in the retentate is decreasing, and the GF2 concentration
was a little lower than that in the Fig. 6a since the formed sugars decreased much faster than FOS, while GF3 concentration kept almost
increased the viscosity of the system. Meanwhile, the average fluxes constant. When V/V0 = 5, the fructose and glucose can not be detected
decreased sharply in the second batch, and they kept about constant 100 in the retentate (Fig. 9b), and the FOS purity reached 92.3% (Fig. 9c)
L/(h⋅m2) in the subsequent three batches, which were similar to that in which was comparable to that in another work reported by Fan et al.[8]
the Fig. 5a. In the permeate, the FOS concentration and the ratios of each and Nobre et al. [19], while no by-products was produced in this pro­
components showed no much difference in the four batches (Fig. 7b and cess. Thus, the diafiltration process with the NF5 membrane was more
c), since the MWCO of the PES10 membrane was larger enough that the favorable for FOS purification.
FOS.
3.3.3. Recovery of sucrose and FOS with a DL membrane from NF5
3.3. Production and purification of FOS from the crude FOS solution permeate
After diafiltration, the ratio of FOS in total sugars increased from
3.3.1. Production of the FOS in a fed-reaction process 51.4 to 92.3%, while the total FOS and sucrose lost by 20.4% and 82.6%
The preliminary study indicated that a lower initial sucrose con­ in the permeate, respectively. Thus, the FOS and sucrose needed to be
centration (i.e., 50 g/L) was favorable for the FOS production, and the recovered from the diafiltration permeate. As reported in our previous
enzyme retained a stable activity. Meanwhile, the PES 10 membrane can work [24], a sucrose retention of 96% and a reducing sugar (i.e. fructose
absorb some FTases. In order to maximize use of the enzymes, a new fed- or glucose) retention of 5% was obtained with a DL membrane under
batch reaction was carried out to produce FOS. As shown in Fig. 8a, FOS 60 ◦ C. Since the molecular weight of the smallest FOS (i.e., GF2) was 162
productions were 28.9, 27.0, 27.6 and 28.2 g/L in the batch 1, batch 2, Da larger than sucrose, the FOS retention should be higher than 96%.
batch 3 and batch 4, respectively. Though the total concentration of Thus, the DL membrane was further selected to recover FOS and sucrose
FOS, GF2 and GF3 increased with the batches, the ratios of GF2 or GF3 in from the diafiltration permeate of NF5. As shown in Fig. 10, the final
the FOS varied little (Fig. 8b), which were different from those in the concentrations of GF2, GF3 and sucrose were 20.1, 5.98, and 23.0 g/L
batch processes in the Fig. 3 and Fig 0.4. Meanwhile, the fructose con­ after 6 times concentration, respectively, where the retentions of GF2,
centrations showed no much difference in the four batches (Fig. 8c), GF3 and sucrose were 78, 81 and 73.3%, respectively. Here, the reten­
which indicated that the hydrolytic behavior was not significant. tion of sucrose was much lower, since the permeate flux was only 13.27
Moreover, after 4 batches, the FTases activity was 76.9 U/mL, and the Lm− 2h− 1 in the literature [31], which was only one third of the flux in
total FOS production was 111.7 g/L with a yield of 55.8%. In the fed- this experiment. Since the solute concentration in permeate decreased at
reaction process, the total FOS production was comparable to four sin­ a higher solvent flux due to the “dilution effect” [32], the recovered FOS
gle batches, while the FTases dosage was only one quarter of the four and sucrose could be used as the washing water in the initial stage of the
single batches. Meanwhile, the residual FTases also could be recovered diafiltration using the NF5 membrane. In addition, a preliminary com­
with the PES 10 membrane according to results in the Figs. 4 and 5. parison of operation costs of the FOS production process with and
Moreover, the remained sucrose increased linearly with the increased without the integrated process is listed in Table 2 and Table 3. It can be
batches, which could not be further converted to FOS. Thus, the fed-
batch process were more suitable for FOS production. Moreover, since Table 2
Comparison of FOS production with different process.
Table 1
Strategy Yield Purity Drawback Ref.
Coefficient R2 of linear regression of fitting experimental data according to (%) (%)
Hermia’s model linear equations.
Enzymatic catalysis 59 59 Low purity [27]
1
Strategy Complete Standard Intermediate Caker Whole-cell catalysis 60 60 Low purity [28]
blocking blocking blocking layer Combining – 92 By-products [8]
Strategy I 0.99264 0.99483 0.99649 0.99822* fermentation with production, and
Strategy II 0.99394 0.99753 0.99944* 0.99825 immobilized FTase introduced ingredients
Strategy 0.95384 0.96089 0.96726 0.97791* Two-step fermentation – 81.6 for cells’ growth [23]
III Enzyme membrane – 96.6 [21]
bioreactor and
1
Strategy I means that the PES 10 membrane was used to recover the FTases subsequent
from the model FTases solutions in one batch of Fig. 4; Strategy II means that the fermentation
PES 10 membrane was used to recover the FTases from crude FOS solution in the Integrated enzymatic 58.8 92.3 Membrane separation This
first batch of Fig. 7; Strategy III means that the PES 10 membrane was used to catalysis and cost study
membrane separation
recover the FTases from crude FOS solution after the fed-batch reaction in the
process
first batch.

9
W. Cao et al. Separation and Purification Technology 288 (2022) 120678

Table 3 Acknowledgement
Calculation details of operation cost of the integrated process for FTases reuse
and FOS purification (100 tons crude FOS solution per day). The authors thank the Beijing Natural Science Foundation, China
Items Fed-batch Fed-batch reaction The increment (+) or (No. 5182025), the Science and Technology Service Network Program of
reaction with the integrated reduction (-) in operation Chinese Academy of Sciences (No.KFJ-STS-QYZX-096), the National
process cost (USD/ day) Natural Science Foundation of China, China (No. 21406240) and the
Membrane – 22.41 +22.41 National High Technology Research and Development Program of China
costs a (Nos. 2015AA021002 and 2014AA021005) for the financial supports.
Enzyme cost 4615 1281.0 − 3334
Energy – 36.3 +36.3
consumption Appendix A. Supplementary data
Chemical usage – 53.94 +53.94
Wastewater – 72.0 +72.0 Supplementary data to this article can be found online at https://doi.
treatment org/10.1016/j.seppur.2022.120678.
Water – 2.88 +2.88
consumption
Total operation 4615 1468.53 − 3146.47 References
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