Preparation of high-purity fructo-oligosaccharides by Aspergillus japonicus β-fructofuranosidase and successive cultivation with yeast

Article abstract of DOI:10.1021/jf703586q

The short-chain FOSs with high purity were prepared using a two-step strategy: Aspergillus japonicus extracellular β-fructofuranosidase- catalyzed synthesis of FOSs followed by cultivation with Pichia pastoris (P. pastoris). The higher FOSs content was obtained after 8 h under the catalysis of β-fructofuranosidase at pH 5.5 and 55°C. Successive P. pastoris cultivation exhausts almost all monosugars in 12 h at 30°C, which increases the purity of FOSs, and also recovers β-fructofuranosidase activity by ceasing the inhibition of glucose from catalysis of the enzyme, yielding more FOSs. Finally, the FOSs purity was increased from 56.55 to 84.45% (26.47% 1-kestose and 57.98% nystose).

Full text of DOI:10.1021/jf703586q

J. Agric. Food Chem. 2008, 56, 2805–2809 2805
Preparation of High-Purity Fructo-oligosaccharides
by Aspergillus japonicus ꢀ-Fructofuranosidase and
Successive Cultivation with Yeast
YA-LIN YANG, JIAN-HUA WANG,* DA TENG, AND FAN ZHANG
Gene Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences,
12 Zhongguancun Nandajie Street, Beijing 100081, People’s Republic of China
The short-chain FOSs with high purity were prepared using a two-step strategy: Aspergillus japonicus
extracellular ꢀ-fructofuranosidase-catalyzed synthesis of FOSs followed by cultivation with Pichia
pastoris (P. pastoris). The higher FOSs content was obtained after 8 h under the catalysis of
ꢀ-fructofuranosidase at pH 5.5 and 55 °C. Successive P. pastoris cultivation exhausts almost all
monosugars in 12 h at 30 °C, which increases the purity of FOSs, and also recovers ꢀ-fructofura-
nosidase activity by ceasing the inhibition of glucose from catalysis of the enzyme, yielding more
FOSs. Finally, the FOSs purity was increased from 56.55 to 84.45% (26.47% 1-kestose and 57.98%
nystose).
KEYWORDS: Fructo-oligosaccharides; Aspergillus japonicus; Pichia pastoris; ꢀ-fructofuranosidase
INTRODUCTION
above synthesis mixture to remove the feedback inhibition of
glucose, a byproduct of the ꢀ-fructofuranosidase reaction. The
amount and purity of FOSs had the potential to significantly
increase and showed the potential for industrial applications.
The efficiency and purity of fructo-oligosaccharides (FOSs)
synthesis from sucrose catalyzed by Aspergillus oryzae or
Aspergillus sydowi ꢀ-fructofuranosidase is very low. Glucose,
a byproduct of the reaction, inhibits the enzyme activity severely,
resulting in decreases of the quality and quantity of FOSs; at
least the bottleneck, a limitation of 60% of target FOS, cannot
be easily overcome (1–8). There are some strategies that have
been developed for obtaining FOSs with high purity: separating
FOS from mono- and disaccharides by chromatography (1, 8)
or converting mono- and disaccharides selectively to tolerable
forms by means of glucose oxidase (2–4). The above strategies
can improve the quality of FOS at some extent with high cost.
An alternative approach is extraction from the root or tube of
a crop such as chicory and Jerusalem artichoke, rich in inulin,
to get a mixture with a broad degree of polymerization (DPn)
from 3 to 100; similarly, its separation and purification processes
are cost-dependent (9–11), and it is not easy to get short-chain
FOS as 3 or 4 DPn as 1-kestose or nystose with special
pharmaceutical and nutrition functions on humans and animals
(12, 13).
MATERIALS AND METHODS
Chemicals, Strains, and Their Cultivation. The chemicals nystose
and 1-kestose were from Sigma Co., Ltd. The other chemicals were
analytical grade. A. japonicus 3.2769 was from China General
Microbiological Culture Collection Center (CGMCC) (Beijing). P.
pastoris GS115 was from Invitrogen (Beijing).
A. japonicus was cultured at 30 °C in the liquid medium (15). P.
pastoris GS115 was cultured at 30 °C in YPD medium containing 10
g/L yeast extract, 20 g/L peptone, and 20 g/L glucose. P. pastoris GS115
was deficient in protease activity.
Preparation of Extracellular ꢀ-Fructofuranosidase of A. japoni-
cus. One loop of mature spores of A. japonicus 3.2769 grown on
Czpeck’s slant at 30 °C for 4 days was inoculated into 100 mL of
medium with shaking at 250 rpm and 30 °C. After 96 h of incubation,
the cells were removed by centrifugation at 5000g for 5 min, and the
supernatant containing crude ꢀ-fructofuranosidase was used for its
catalysis synthesis and cocultivation with yeast.
The enzyme activity was assayed in a mixture consisting of 0.9 mL
of 55.6% sucrose dissolved in 0.05 mol/L sodium acetate buffer (pH
5.5) as the substrate and 0.1 mL of crude enzyme 100-fold diluted in
the same buffer. The reaction was run at 55 °C for 1 h and stopped by
adding 3,5-dinitrosalicylic acid solution (DNS), boiling at 100 °C for
5 min, and then cooling down to room temperature. The reducing sugar
was determined at 540 nm. One unit of ꢀ-fructosyltransferase activity
was defined as the enzyme amount producing one micromole of
reducing sugar per minute (16, 17).
By means of nutritional characteristics of Pichia pastoris (P.
pastoris), which can ferment monosaccharides but not disac-
charides and oligosaccharides (14), we established a “two-step
strategy” to improve the purity of FOSs: First, extracellular
ꢀ-fructofuranosidase of Aspergillus japonicus (A. japonicus) was
used to catalyze the FOS synthesis from sucrose, and then, P.
pastoris was cultivated to selectively use monosugars in the
Cultivation and Collection of P. pastoris Cells. One loop of P.
pastoris cells was inoculated on YPD agar (15 g/L) medium at 30 °C
for 3–5 days and was inoculated into 100 mL of YPD medium and
* To whom correspondence should be addressed. Tel/Fax: 0086-
10-62145735. E-mail: wangjianhua@mail.caas.net.cn or jhwangfribio@
yahoo.com.cn.
10.1021/jf703586q CCC: $40.75  2008 American Chemical Society
Published on Web 03/12/2008

2806 J. Agric. Food Chem., Vol. 56, No. 8, 2008
Yang et al.
Figure 1. Content of FOSs and other carbohydrates in a catalysis reaction
mixture by A. japonicus ꢀ-fructofuranosidase during biosynthesis.
incubated with shaking at 250 rpm and 30 °C for 24 h. A 1 mL culture
was centrifugated at 10000g at 4 °C for 10 min. The fresh weight (FW)
of cells was measured and used for successive cocultivation.
FOS Synthesis Catalyzed by ꢀ-Fructofuranosidase. The extra-
cellular ꢀ-fructofuranosidase was added into 100 mL of 50% sucrose
(W/V) dissolved in 0.05 mol/L sodium acetate buffer (pH 5.5) at the
ratio 5:1 of activity:sucrose (U:g) and incubated at 55 °C for 20 h. The
content of FOSs and other sugars synthesized in above solution was
determined by high-performance anion-exchange chromatography
(HPAEC) (18) when the reaction was run for 0, 0.5, 1, 2, 3, 4, 5, 6, 8,
12, 16, and 20 h, respectively.
Figure 2. Content of FOSs and other carbohydrates in the successive
cultivation product with P. pastoris.
hour, and decreases gradually later. This is because the
conversion of 1-kestose is higher than its formation (Figure
1). The content of nystose increases slowly and linearly during
the whole period (Figure 1). The equal amounts of 25%
1-kestose and nystose were observed at about 15 h, and the
highest content of 54.09% FOSs was reached after 8 h. Actually,
a desirable content of about 47% FOSs was reached at the fourth
hour in terms of economics (Figure 1). The content of total
FOSs remains almost at a stable level of 53–55% after 8 h.
The content of glucose increases from zero to 30% in the first
6 h, up to 35% at 12 h, and then decreases down back to a
level of below 30% later, while fructose is always about at 2.5%
or less in the first 12 h and increases up to a level of 12–13%
later. In fact, the glucose decreases significantly with the increase
of fructose during the late period from 16 to 20 h; this cha-
nge magnitude of glucose and fructose seems to be equal mole
(Figure 1). It might be deduced that glucose isomerase
coexisting in crude enzyme is activated when glucose ac-
cumulates up to some extent and the function of fructosyltrans-
ferase is weakened (20, 21). As result, a part of glucose is
transferred into fructose.
Biopurification of FOSs by Successive Cocultivation with P.
pastoris. FOS products from 10 h of enzymatic synthesis were diluted
to the concentration of 25% (w/v) with sterilized water and mixed with
P. pastoris cells at the ratio of 1:0.45 (DW:FW). A 100 mL amount of
such a mixture was incubated at 30 °C with agitation at 250 rpm for
24 h. The content of FOSs and other sugars was determined when
cocultivation was for 0, 2, 4, 6, 8, 10, 12, 16, 20, and 24 h, respectively
(18).
HPAEC Assay of Carbohydrate. One milliliter of cocultivation
mixture was centrifuged, and the supernatant was diluted with deionized
water and was analyzed with HPAEC assay (18) using a BioLC
Chromatography System. A 250 mm × 4 mm CarboPac PA100 anion-
exchange column and a 25 mm × 3 mm CarboPac PA guard column
(Dionex, Breda, The Netherlands) with a linear gradient ratio of 100
mmol/L NaOH and 500 mmol/L NaAc to 100 mmol/L NaOH in
deionized water from 99:1 to 80:20 (v:v) were used. The running rate
was 1 mL/min, and the running time was 1 h. The concentration
gradients of 1, 20, 40, 60, 80, and 100 µg/mL for standard glycerol,
glucose, fructose, sucrose, 1-kestose, and nystose were prepared,
respectively. The total FOSs were the sum of 1-kestose and nystose.
Higher FOSs (DPn, n > 4) less than 3% were ignored (3). All samples
with HPAEC assays were sent to the Analysis Center of our institute
for determination.
According to the FOSs synthesis described above (Figure 1), a
product of target sugars with a desirable composition and concen-
tration can be obtained at different periods. For example, 4 h is
enough to get the high content of 37.67% 1-kestose, while 16–20
h is needed to get the high content of near 30% nystose. Fifteen
hours is suitable to get a mixture with equal amounts of 25%
1-kestose and nystose. To obtain a high content of 50-55% of
total FOSs, 6-8 h can be chosen. We suggest that 4 h is a more
reasonable reaction time to get the higher content of 47.5% FOSs
(Figure 1). It is very interesting that almost only two types of short-
chain FOSs, 1-kestose and nystose, were detected in the synthesis
mixture (Figures 1 and 2); unlike in the similar process where
there is a broad distribution of DPn from 3 to 12 (8), it is very
helpful to get high-purity short-chain FOS without additional
RESULTS AND DISCUSSION
Enzymatic Synthesis of FOSs. As shown in Figure 1, the
substrate sucrose is rapidly converted into glucose and 1-kestose
upon the initiation of reaction, and a part of 1-kestose starts to
be converted into nystose a few hours later. The concentration
of the sucrose decreases rapidly from over 80% down to 20–30%
in the first 4 h. Then, the FOSs synthesis reaction rate drops
down after 4 h. After 8 h, the sucrose is always kept at a stable
level of about 11%. This is because the severe feedback
inhibition of glucose as a byproduct on ꢀ-fructofuranosidase
reduces and ceases the conversion of sucrose and finally results
in the low purity of FOSs (1–4).
The content of 1-kestose increases steadily up over 30% in
the first 3 h, reaches the highest level of 37.67% at the fourth

Preparation of Fructo-Oligosaccharides
J. Agric. Food Chem., Vol. 56, No. 8, 2008 2807
Table 1. Content of FOSs and Other Carbohydrates from Catalysis with
ꢀ-Fructofuranosidase and Successive 16 h Cultivation with Yeast
purification processes to separate other long-chain or higher FOSs,
and these short-chain FOSs have very important pharmaceutical
and nutrition values (12, 13).
product from biosynthesis
by enzyme
product after
Successive Cultivation of P. pastoris in FOS Mixture
Synthesized by the Enzyme. Change of Glucose and Fructose.
The FOS mixture from enzymatic synthesis at the 10th hour
was diluted to 25% (DW/V). As shown in Figure 2, when 100
mL of 25% FOSs mixture was incubated together with P.
pastoris cells at 30 °C and at the ratio of 0.45:1 (yeast cells:
FOSs solution, FW/DW), glucose is rapidly exhausted from
28.78% at the beginning to 1.65% after 16 h and to almost zero
at 24 h. At the same time, the content of fructose changes
between 1.8 and 3.1%. This is probably related to glucose
isomerase mentioned above (20, 21).
sugar
cultivation with yeast
glycerol
glucose
fructose
sucrose
2.47a
28.78
2.21
10.01
1.65
2.56
1.33
5.54
26.47
57.98
84.45
100.00
9.99
total mono- and disaccharidesb
40.98
30.06
26.49
56.55
100.00
1-kestose
nystose
total FOSc
total
a Percentage content (w/w). b The total amount of mono- and disaccharides is
assumed to be the sum of glucose, fructose, and sucrose. c The total FOS was
assumed to be the sum of 1-kestose and nystose.
Change of Sucrose Content and Total Content of G + F +
GF (Figure 2). As the content of glucose decreases gradually
and its feedback effect disappears quickly, the activity of
ꢀ-fructofuranosidase is recovered; thus, sucrose is again con-
verted into FOSs (Figure 2), which is similar to previous
reports (2–4). As a result, the content of sucrose decreases
gradually from 9.99% at the beginning to 1.32% at 16 h and to
almost zero at 20 h. What is more interesting is that the content
of both mono- and disaccharides decreases significantly from
40.98% at the beginning to 5.54% at 16 h and to 1.84% at 24 h;
this huge and quick utilization especially for mono sugars is
much more helpful to remove the feedback effect of glucose
and to recover the activity of ꢀ-fructofuranosidase (5–8, 19).
Change of 1-Kestose, Nystose, and Total FOS Content. In
the first 6 h, the content of 1-kestose increases slightly from
about 30 to 35% due to its synthesis from sucrose but then
decreases slowly and gradually down to less than 20% until
the end because its synthesis is less than its conversation into
sucrose and nystose. In comparison, the content of nystose is
significantly and linearly increased from about 27 to 70% during
the whole cultivation; obviously, this increase of nystose content
is not closely related to the change of 1-kestose but highly
related to the decreases of content of G + F + GF from about
40 to 2% and glucose from 30% to zero, respectively (Figure
2). It is exciting that 70% nystose is the highest level without
regular physical and chemical separation as previously reported
(18); so far as we know, the comparison advantage of this
biopurification is obvious in terms of safety, economics, and
efficiency.
In the first 12 h, the total amount of FOSs increases obviously
and linearly from about 57 to 83% (at the same time, mono-
and disaccharides are almost exhausted). To achieve both higher
yield and higher purity of 80% FOSs, the cultivation time of
12 h is enough with only trace amounts of sucrose and glucose
remaining (Figure 2), despite the fact that the content of 84.45%
FOSs can be reached later after 12 h of cultivation with a higher
cost. By fermenting FOSs syrup and the enzyme-synthesized
product, using P. pastoris, the content of FOSs is elevated from
56.55 to 84.45% (26.47% 1-kestose and 57.98% nystose) (Table
1). HPAEC chromatograms of mixture from P. pastoris cultiva-
tion at 0 and 16 h were assayed and compared as shown in
Figure 3; the peaks of glucose, fructose, and sucrose are
significantly lowered after 16 h of cultivation (Figure 3a,b)
because monosaccharide is fermented and the disaccharide is
converted greatly. In contrast, the higher peak of nystose and
the lower peak of 1-kestose peak imply that a trace of 1-kestose
was converted into nystose.
Figure 3. HPAEC chromatogram of FOSs and other sugars from catalysis
of ꢀ-fructofuranosidase (a) and successive 16 h cultivation product with
P. pastoris (b). The numbers are retention time of glycerol, glucose,
fructose, sucrose, 1-kestose, and nystose; they are 1.58, 4.42, 5.33, 7.67,
14.83, and 33.42th min, respectively.
Change of Glycerol Content. Glycerol will certainly ac-
cumulate in yeast cells to balance the high osmotic pressure
outside (15, 22) caused by a high concentration of sugars. As
expected, about 10% glycerol is produced in the cultivation
(Figure 3). The glycerol content increases significantly in the
successive cultures by three times from 0 to 16 h (Figure 3).

2808 J. Agric. Food Chem., Vol. 56, No. 8, 2008
Yang et al.
of ꢀ-fructofuranosidase activity to effectively transfer 1-kestose
and sucrose into nystose.
In summary, the two-step strategysenzymatic synthesis of
FOS followed by its successive cultivation with P. pastorissfor
the production of high-purity FOSs was established. The
synthesis time for the higher conversion of sucrose into FOS is
between 4 and 8 h at 55 °C, and the successive cultivation time
for removing the glucose feedback effect should be 12 h or
longer at 30 °C. In combination of two steps, a total of 84.45%
FOSs is achieved with only 1.65% glucose, 2.56% fructose,
and 1.33% sucrose remaining. Other characteristics include
almost only two types of short-chain FOSss1-ketose and
nystosesand exist in FOSs synthesis, and yeast cocultivation
mixtures, high contents near 70% nystose and near 90% total
FOSs, can be reached after successive cocultivation with yeast.
These results can contribute to novel approach developments
with a potential for industrial synthesis of high-purity FOSs.
ABBREVIATIONS USED
A. japonicus, Aspergillus japonicus; P. pastoris, Pichia
pastoris; DW, dry weight; DNS, 3,5-dinitrosalicylic acid; FOSs,
fructo-oligosaccharides; FW, fresh weight; Gpd, glycerol-3-
phosphate dehydrogenase; Gpp, glycerol-3-phosphatase; DPn,
degree of polymerization; n, moiety number of sugar; HPAEC,
high-performance anion-exchange chromatography.
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Preparation of Fructo-Oligosaccharides
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and 2004AA246040).
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