Curr Microbiol
DOI 10.1007/s00284-017-1295-x
Production of Functional Inulin-Type Fructooligosaccharides
by an Enzyme from Penicillium citrinum
Yoshiya Tashiro1 • Hideo Ueno2 • Masakazu Takaba2 • Sachio Hayashi1
Received: 16 November 2016 / Accepted: 29 June 2017
 Springer Science+Business Media, LLC 2017
Abstract We report the production of functional inulin-
type fructooligosaccharides such as trisaccharide 1-kestose,
O–b-D-fructofuranosyl-(2?1)-b-D-fructofuranosyl a-D-glu-
copyranoside, and tetrasaccharide nystose, O-b-D-fructo-
furanosyl-(2?1)–b-D-fructofuranosyl-(2?1)–b-D-fructofu-
ranosyl a-D-glucopyranoside, from sucrose by an enzyme
from Penicillium citrinum. Sucrose acted as a fructosyl
donor and acceptor for the enzyme. The optimum pH and
temperature for the enzymatic reaction were 5 and 50 C,
respectively. The enzyme was stable in the pH range of
4.5–7 and at 50 C. The maximum concentration of
1-kestose obtained was 110 mg/ml, and the maximum
production efficiency was 37.3% after a 48-h reaction. The
maximum efficiency of combined fructooligosaccharide (1-
kestose and nystose) production was 47.1% after a 72-h
reaction. Fructooligosaccharides were therefore success-
fully produced via a fructosyl transfer reaction catalyzed by
an enzyme from P. citrinum.
Introduction
Inulin-type fructooligosaccharides are usually produced
industrially and used widely because they are low-calorie,
non-cariogenic sweeteners that exhibit interesting attri-
butes such as supporting large numbers of Bifidobacterium,
& Sachio Hayashi
shayashi@cc.miyazaki-u.ac.jp
1 Penicillium citrinum was cultivated in liquid culture
The Interdisciplinary Graduate School of Agriculture and
Engineering, University of Miyazaki, 1-1 Gakuen Kibanadai
Nishi, Miyazaki-Shi, Miyazaki 889-2192, Japan
2
Nippon Oligo Co. Ltd, Izumisawa, Nanto-Shi,
Toyama 447-8506, Japan
and so are useful in some health foods [1–4]. Enzymes
from a number of microorganisms, such as Aspergillus
japonicus [5], Aspergillus niger [3], Aspergillus oryzae [6],
Aureobasidium pullulans [7], Chrysonilia sitophila [8],
Fusarium oxysporum [9], and Penicillium expansum [10],
produce fructooligosaccharides from sucrose as the sole
fructose donor and acceptor via fructosyl transfer activity,
and simultaneously glucose is released into the reaction
mixture. While the production of fructooligosaccharides,
including neokestose (Frufb2?6Glca1?2bFruf) and
neonystose (Frufb2?6Glca1?2bFruf1?2bFruf), by sus-
pended cells [11] and immobilized cells [12, 13] of Peni-
cillium citrinum has been reported previously, there are no
reports on the production of only inulin-type fruc-
tooligosaccharides such as 1-kestose and nystose using
extracted intracellular enzymes from P. citrinum.
In the present report, we investigated the pH and tem-
perature profiles and the production of inulin-type func-
tional fructooligosaccharides using an enzyme from P.
citrinum to increase the value of sucrose as a low-cost
substrate, and herein describe the amount and efficiency of
fructooligosaccharide production. We confirmed the
potential of the present enzyme extracted from P. citrinum
for the industrial production of fructooligosaccharides.
Materials and Methods
Cell Cultivation and Enzyme Preparation
medium containing the following (% w/v): sucrose, 20;
yeast extract, 2; K2HPO4, 0.2; and NaCl, 0.1. Cultivation
was conducted at pH 6.5 and 30 C for 5 days. The enzyme
exhibiting fructose transfer activity was solubilized from
123
 Y. Tashiro et al.

5 g of dried cells using 20 mg of Kitalase (2000 U, endo-b- Results

1,3-glucanase; Wako Pure Chemical Industries Ltd.,
Osaka, Japan), which has no b-fructofuranosidase activity,
in 75 mmol/l McIlvaine buffer (pH 5) at 40 C for 2 h. The
supernatant obtained by centrifugation (130009g, 15 min)
was used as the obtained intracellular enzyme.
Enzyme Activity Assay
Reaction Conditions
The pH and temperature profiles of the Penicillium citri-
num enzyme were investigated for the production of fruc-
tooligosaccharides (Fig. 1). The optimum pH of the
enzyme was 5.0 as shown in Fig. 1a. The enzyme was

The enzymatic reaction was initiated by the addition of

0.2 ml of enzyme solution to a 0.8 ml of 37.5% (w/v) (A)
sucrose dissolved in 100 mmol/l McIlvaine buffer (pH 5).
The reaction was carried out at 50 C for 20 min and ter-
minated by boiling for 10 min.
The released glucose was assayed using a glucose oxi-
dase method (Glucose CII-test; Wako), because the amount
of glucose released is the same as that of fructose trans-
ferred. One unit of enzymatic activity was defined as the
quantity of enzyme required to transfer 1 lmol fructose in 2 3 4 5 6 7 8 9
1 min. pH
(B)
120
Enzymatic Reaction
100
Relative activity (%) 80
To determine the optimum pH of the enzyme, the reaction
was performed in solution buffered between pH 3 and 8. To 60
evaluate pH stability of the enzyme, the residual enzymatic 40
activity was determined under optimum reaction conditions 20
after the enzyme was kept in buffer with pH range of 4–8 0
for 3 h. 3 4 5 6 7 8 9
pH
To determine the optimum temperature of the enzyme,
the enzyme was measured at different temperatures
(C)
120
between 20 and 80 C and pH 5. To investigate the heat
Relative activity (%)

100
stability of the enzyme, the residual activity was measured
80
under optimum conditions after incubating the enzyme for
15 min at various temperatures between 20 and 80 C. 60

Enzymatic reactions for the production of fruc- 40

tooligosaccharides were conducted as follows. The reaction 20
mixture comprised 8 ml of 37.5% (w/v) sucrose dissolved 0
in 100 mmol/l McIlvaine buffer (pH 5) and 2 ml of 10 20 30 40 50 60 70 80 90
Temperature (
enzyme solution (10 U) and was incubated at 30 C for
72 h. (D)
120
The obtained oligosaccharides were measured by high-
Relative activity (%)

100
performance liquid chromatography (HPLC, Shimadzu
LC-10A, Shimadzu, Kyoto, Japan) using a Wakosil 5NH2 80
(4.6 9 250 mm; Wako) using the following conditions: 60
temperature, 40 C; mobile phase, acetonitrile/water, 40
80:20, v/v; flow rate, 1 ml/min; and RI detector (RID-6A, 20
Shimadzu). 0
The efficiency of oligosaccharide production is pre- 10 20 30 40 50 60 70 80 90
sented as the percentage of the concentration of produced
oligosaccharide compared to the initial sucrose concen-
Fig. 1 Effect of pH on the enzymatic activity (a) and stability (b),
tration in the reaction mixture. and effect of temperature on the enzymatic activity (c) and stability
(d)

123
Production of Functional Inulin-Type Fructooligosaccharides by an Enzyme from Penicillium…
stable in the pH range of 4.5–7, retaining 90% of its
activity after 3 h within this range (Fig. 1b).
The optimum reaction temperature of the enzyme was
50 C as shown in Fig. 1c. The enzyme was stable for
15 min at 50 C and retained 70% of activity at 60 C, but
was almost inactivated at 80 C (Fig. 1d).
Production of Fructooligosaccharides Using
Enzymatic Reaction
In Fig. 2, the time-course of the reaction catalyzed by the
enzyme from P. citrinum is shown. The enzyme produced
1-kestose from sucrose and released glucose in the reaction
mixture. Fructose was released into the reaction mixture
after prolonged incubation. The concentration of 1-kestose
was 110 mg/ml after a 48-h reaction as compared to the
initial sucrose concentration (295 mg/ml), providing a
conversion efficiency of 37.3%. Apparent nystose pro-
duction from 1-kestose began after a 24-h reaction and its
concentration was 34.2 mg/ml after 72 h. The maximum
concentration of fructooligosaccharides (1-kestose and
nystose) produced from the initial sucrose was 139 mg/ml
after a 72-h reaction, and the efficiency was 47.1%. The
concentration of glucose increased during 1-kestose pro-
duction, whereas the concentration of fructose remained
essentially unchanged during the enzymatic reaction.
Discussion
The present intracellular enzyme extracted from Penicil-
lium citrinum by Kitalase (endo-b-1,3-glucanase) produced
only inulin-type fructooligosaccharides such as 1-kestose
and nystose by fructosyl transfer activity using the
350
Fructose
300 Glucose
Sucrose
250
1-Kestose
Sugar conc. (%)
Nystose
200
150
100
50
0
0 10 20 30 40 50 60 70
Time ( h
Fig. 2 Time-course of fructooligosaccharide production using the
enzymatic reaction
substrate sucrose as a sole fructose donor and acceptor, and
simultaneously glucose was released into the reaction
mixture; cells of P. citrinum have been reported previously
to produce neo-fructooligosaccharides such as neokestose
and neonystose [11, 12].
The optimum pH value of 5 for the enzyme was similar to
that (pH 5) of Penicillium citreonigrum [14] and to those (pH
5.5) of the Penicillium purpurogenum [1] and Penicillium
oxalicum enzymes [15]. The enzyme was stable in the pH
range of 4.5–7, and it appeared that denaturation occurred
around pH 4 and 8, because the fluctuation range of residual
activity was large. While the stability of the enzyme in the pH
range of 2–4 and above 8 was lower than those of P. citre-
onigrum [14] and P. oxalicum [15], we consider it enough for
the industrial production of fructooligosaccharides.
The optimum reaction temperature of the enzyme (50 C)
was almost the same as that (55 C) of the P. purpurogenum
enzyme [1] and fairly lower than that (60 C) of the P.
oxalicum enzyme [15]. The thermal stability of the enzyme
at 50 C was similar to that of P. oxalicum [15] and is
considered to be favorable. We think that denaturation of the
enzyme began at around 60 C because the values of
residual activity were relatively variable. The pH and tem-
perature profiles of Penicillium enzymes thus far reported
are therefore not so different from each other.
The efficiency of inulin-type fructooligosaccharides
production by the enzyme (47.1%) was similar to that of
the P. chrysogenum enzyme (42.51%) and higher than
those of P. purpurogenum and Penicillium islandium
enzymes [16]. The efficiency of enzyme was also higher
than that of Penicillium sizovae cells [17].
The present enzyme from P. citrinum is thus considered
to be very useful for industrial production of functional
inulin-type fructooligosaccharides because of its favorable
characteristics. As the enzyme is considered to be a b-
fructofuranosidase, which has high fructosyl-transferring
activity, the purification and characterization of the enzyme
will be carried out in the near future.
In conclusion, inulin-type fructooligosaccharides were
efficiently produced from sucrose using an enzyme
extracted from P. citrinum. Scale-up and optimization of
the enzymatic reaction for the industrial production of
fructooligosaccharides will be carried out in the near
future. The enzymatic process demonstrated here increases
the value of sucrose and raises the prospect that it can be
used for the production of functional oligosaccharides.
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