NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium isolated in cloud water

Article abstract of DOI:10.1016/j.carres.2010.12.012

Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate (RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exopolysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of polymerization (dp 2-7) and monosaccharides fructose:glucose = 9:1. Levan was composed of two components with molecular mass ～3500 and ～100 kDa in the ratio 2.3:1. Disaccharide fraction contained difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in the ratio 2:1:3. Fractions with dp 4-7 were mixtures of FOSs of levan (2,6-βFruf) and inulin (1,2-βFruf) type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio 1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kestose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type (2,6-βFruf) containing α-glucose. In the pentasaccharide fraction also the presence of a homopentasaccharide composed of 2,6-linked βFruf units only was identified. Nystose, inulin (1,2-βFruf) type, was identified as DIM tetrasaccharide. Identification of levan 2,6-βFruf and inulin 1,2-βFruf type oligosaccharides in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp. 3B6.

Full text of DOI:10.1016/j.carres.2010.12.012

Carbohydrate Research 346 (2011) 501–507
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
NMR structural study of fructans produced by Bacillus sp. 3B6, bacterium
isolated in cloud water
Mária Matulová a, , Slavomíra Husárová a, Peter Capek a, Martine Sancelme b, Anne-Marie Delort b
⇑
a Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia
b Laboratoire de Synthèse et Etude de Systèmes à Intérêt Biologique, UMR 6504, Université Blaise Pascal, CNRS, F-63177 Aubière, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 August 2010
Received in revised form 22 November 2010
Accepted 14 December 2010
Available online 21 December 2010
Bacillus sp. 3B6, bacterium isolated from cloud water, was incubated on sucrose for exopolysaccharide
production. Dialysis of the obtained mixture (MWCO 500) afforded dialyzate (DIM) and retentate
(RIM). Both were separated by size exclusion chromatography. RIM afforded eight fractions: levan exo-
polysaccharide (EPS), fructooligosaccharides (FOSs) of levan and inulin types with different degrees of
polymerization (dp 2–7) and monosaccharides fructose:glucose = 9:1. Levan was composed of two com-
ponents with molecular mass ꢀ3500 and ꢀ100 kDa in the ratio 2.3:1. Disaccharide fraction contained
difructose anhydride DFA IV. 1-Kestose, 6-kestose, and neokestose were identified as trisaccharides in
the ratio 2:1:3. Fractions with dp 4–7 were mixtures of FOSs of levan (2,6-bFruf) and inulin (1,2-bFruf)
type. DIM separation afforded two dominant fractions: monosaccharides with fructose: glucose ratio
1:3; disaccharide fraction contained sucrose only. DIM trisaccharide fraction contained 1-kestose, 6-kes-
tose, and neokestose in the ratio1.5:1:2, penta and hexasaccharide fractions contained FOSs of levan type
Keywords:
Levan
Inulin
FOSs
DFA IV
Bacillus
Cloud water
(2,6-bFruf) containing
a-glucose. In the pentasaccharide fraction also the presence of a homopentasac-
charide composed of 2,6-linked bFruf units only was identified. Nystose, inulin (1,2-bFruf) type, was iden-
tified as DIM tetrasaccharide. Identification of levan 2,6-bFruf and inulin 1,2-bFruf type oligosaccharides
in the incubation medium suggests both levansucrase and inulosucrase enzymes activity in Bacillus sp.
3B6.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
type of linkage between bFruf units FOSs of levan type (2,6-bFOSs),
inulin type (1,2-bFOSs) or mixed type may be formed. Low molec-
Microbial polysaccharides offer a potential new source of func-
tional biopolymers for food, industrial, cosmetic or medical appli-
cations. Depending on the carbon source used they could be
homopolymers (composed of one type of monosaccharide) or het-
eropolymers. Sucrose is the substrate for bacterial production of
fructans. Levan and inulin are fructans composed of 2,6-linked b-
fructofuranoses (2,6-bFruf) and 1,2-linked b-fructofuranoses (1,2-
bFruf), respectively. Strains producing fructans can be found in
many genera of bacteria.1 Fructans produced by bacteria are
mostly of levan type and they are synthetized from sucrose by lev-
ansucrase2,3 and only few bacterial species are equipped by inulo-
sucrase and excrete inulin.4,5 In Lactobacillus reuteri 121 both,
levansucrase and inulinsucrase were identified.6 Levan producing
capacity can be limited to a few strains of a particular bacterial
species.7 Besides levan polymer some bacterial levansucrases are
able produce also fructooligosaccharides (FOSs) with only few
bFruf units linked to the glucose molecule.8–11 Depending on the
ular mass FOSs have a sweet taste, while those of higher molecular
weight have a neutral one. They form emulsions, and due to their
properties to increase foam stability and a fat-like texture which
improves organoleptic characteristic, they are designated for their
application in food industry.12,13 Due to the absence of enzymes
that are able to degrade fructans in the upper human digestive
tract, fructans and FOSs make a part of non digestible fibers and
they act as prebiotics. In the digestive system they serve as a car-
bon source for colon bacteria, such as lactobacilli and bifidobacte-
ria, which are beneficial to human health. In this way fructans help
to modify the composition of the colon flora by stimulating bacte-
rial growth and/or activity. Besides their function as dietary fibers,
many other properties are ascribed to fructans. For example, pre-
vention of colon cancer due to stimulation of mineral absorption
from the colon or immune system stimulation.14,15 Linkage type
and molecular mass of fructan polysaccharide and/or FOSs depends
on enzymes involved in their synthesis, sucrose concentration, and
composition of the incubation medium. Difructose anhydrides
(DFAs), cyclic disaccharides composed of two fructose residues,
also belong to FOSs. Depending on the type of linkage between
⇑
Corresponding author. Tel.: +421 2 59410245; fax: +421 2 59410222.
E-mail addresses: Maria.Matulova@savba.sk, matulova@zoznam.sk (M. Matu-
lová).
two bFruf units four types of DFA were described: DFA I, a-D-Fruf
0008-6215/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2010.12.012

502
M. Matulová et al. / Carbohydrate Research 346 (2011) 501–507
-b-
dianhydride; DFA IV, b-
DFA V, -Fruf-b-
-Fruf-20,6:2,10-dianhydride. Production of DFA
D
-Fruf-20,1:2,10-dianhydride; DFA III,
a
-
D
-Fruf-b-
D
-Fruf-20,1:2,3-
2.2. Incubation conditions
D
-Fruf-b-
D
-Fruf-20,6:2,60-dianhydride and
a-D
D
Growth conditions: Bacillus sp. 3B6 strain was grown in 100 mL
portions of Trypcase-Soy broth (bioMérieux, France) in 500 ml
Erlenmeyer flasks incubated at 27 °C and 200 rpm for 24 h. Culture
(500 mL) were centrifuged at 8000ꢂg and 4 °C for 5 min. The bac-
terial pellet was twice washed with NaCl (ACROS) (8‰) and there-
after re-suspended in the medium for incubation with sucrose
(ACROS).
has been observed for different strains of Arthrobacter, Pseudomo-
nas, Aspergilus fumigatus, Streptomyces sp. MCI-2524 and Bacillus
sp. Snu-7 when they were grown on inulin or levan as substrates.
Enzymes involved in their synthesis are levan/inulin depolymeris-
ing fructosyltransferases.16–19 DFA due to their unique structures
and properties are expected to have novel physiological functions.
It was already shown that they increase mineral absorption (iron,
calcium).20–23 They have beneficial effect on colon microbiota.12
DFA has half of the sucrose sweetness, they have a stabile structure
and the fact that they are not hydrolyzed by invertase and inulin-
ase strongly suggests its use in the food industry for dietary pur-
poses as a low calorie sweetener.19,24
Besides the importance of oligo- and polysaccharides produced
by the microorganisms for their different applications in biotech-
nologies (particularly in nutraceutical, pharmaceutical and food
industry), these molecules are well known to play a role in ecolog-
ical niches. The precise role of EPS in EPS-producing bacteria is
dependent on the natural environment of the microorganism. Most
of functions ascribed to EPS are of a protective nature. Fructans
were already shown to protect microbial cells against an abiotic
and biotic stress, such as desiccation, freezing, antibiotics, or toxic
compounds, and attacks of parasites and predators.13,25
2.3. Incubation with sucrose
2.3.1. Incubation procedure No .1
Media for metabolism study were prepared by dissolution of su-
crose in VolvicÒ mineral water. The final concentration 20 mmol Lꢁ1
of saccharides and 109 cell mLꢁ1 of microorganisms was used. Bacil-
lus sp. 3B6 was grown in this medium for 5 days. Samples were taken
in regular intervals (0, 15, 19, 24, 39, 48, 63, 72, 96, 120, 144 h).
Extracellular incubation media were separated from cells by centri-
fugation (3 min, 12,000ꢂg).
2.3.2. Incubation procedure No. 2
For EPS production a medium proposed by Gandhi et al.27 was
used. Its composition was as follows: sucrose (30 g Lꢁ1) (ACROS),
ammonium nitrate (0.4 g Lꢁ1) (ACROS), potassium sulfate (1 g Lꢁ1
)
In this paper, we describe the production, purification, and de-
tailed NMR structural identification of the exopolysaccharide levan
and FOSs produced by Bacillus sp. 3B6 when it was grown on su-
crose. As Bacillus sp. 3B6 has been isolated in cloud water26 a dis-
cussion is given about the potential implication of EPS production
in such an atmospheric environment.
(ACROS), sodium chloride (1 g Lꢁ1) ACROS, potassium dihydrogen
phosphate (3 g Lꢁ1) (FLUKA), calcium chloride (0.02 g Lꢁ1) (SIGMA
A.), magnesium sulfate (0.001 g Lꢁ1) (SIGMA A.). Medium was auto-
claved for 15 min at 121 °C. Old inoculum (24-h) was used to seed
the sterile medium. The culture was incubated in a rotary shaker
200 rpm at 32 °C. After 8 days of incubation, the medium was centri-
fuged (20,000ꢂg, 25 °C, 30 min) and Bacillus sp. 3B6 cells were sep-
arated from the supernatant.
2. Material and methods
2.4. Isolation of carbohydrates from culture medium and gel
filtration chromatography
2.1. Isolation and identification of the bacterial strain
Cloudsamples weretakenatPuydeDômesummit(1465 m)inthe
Massif Central Region (France) as described by Amato et al.26 Briefly,
cloud droplets were collected with single-stage cloud collectors with
Total volume (1000 mL) of the incubation medium was reduced
by rotatory evaporator (ꢀ200 mL) and subjected to dialysis for
3 daysagainstdistilledwater(MWCO500). Dialyzate(DIM)contain-
ing salts, low molecular mass saccharides and amino acids was trea-
ted by cation and anion exchangers and further subjected to size
a cut-off droplet diameter of 7 lm. All material needed for sampling
was previously sterilized by autoclaving. A sterile mask and gloves
were worn during sample collection to avoid any contamination.
Samples were kept in cold (4 °C) for a few hours until laboratory
experiments. Microorganism cultures were made on solid nonselec-
tive media for bacteria already described26 by application of 0.1 mL
of 1 to 10ꢁ3 dilutions of cloud water. From these mixed cultures, pure
strains were isolated from individual colonies on Petri dishes.
First, the bacterial strain was cultivated in Trypcase-Soy broth
medium, 27 °C, 200 rpm. Cells were collected after 24 h of incuba-
tion (centrifugation, pellet re-suspended in phosphate-buffered
saline) for DNA extraction using Easy-DNA kit (InvitrogeneTM;
Invitrogen, Cergy Pontoise, France). Amplification of the 16S rRNA
gene was then performed by polymerase chain reaction (PCR), with
about 100 ng of genomic DNA for 0.3 U of Taq DNA Polymerase
(1.5 U; Qbiogene, Illkirch, France), and eubacterial universal prim-
ers (F8 50-AGA GTT TGA TCM TGG CTC-30 and 1492r 50-GNT ACC
TTG TTA CGA CTT-30, where M corresponds to A or C and N to
any of the four nucleotides. The PCR conditions were as follows:
94 °C for 5 min; 25 cycles of: 30 s at 94 °C (denaturation), 30 s at
55 °C (hybridization), and 15 min at 72 °C (elongation). It was ter-
minated by a 7 min step at 72 °C. The PCR products were purified
using Strataprep Purification kit (StratageneTM, Amsterdam, Neth-
erlands) and finally freeze-dried. The entire 16S rRNA gene se-
quence was obtained by capillary electrophoresis using the
previously described primers.
Figure 1. Elution profile on Bio-Gel P2 of the dialyzate (DIM) obtained after a
dialysis of a complex incubation medium (incubation procedure No. 2) after 3 days
dialysis against water (8 days of incubation of Bacillus sp. 3B6 on sucrose).

M. Matulová et al. / Carbohydrate Research 346 (2011) 501–507
503
3
exclusion chromatography (SEC). SEC of DIM afforded major
fractions: 6FD containing fructose and glucose (1:10) and 5FD (su-
crose); fractions 4-1FD contained traces of oligosaccharides (Fig. 1).
Retentante represented retained incubation medium (RIM). Carbo-
hydrates in RIM were recovered by precipitation with five volumes
ofacidified 96% ethanoland left overnightin a coldroom. The precip-
itate was removed by centrifugation (5000ꢂg, 25 °C, 10 min), dis-
solved in water and dialyzed another three days against distilled
water. It was concentrated and freeze-dried to give light brown
materialofa crudecarbohydratesmixture, whichwasseparatedinto
two parts. One part was directly analyzed by NMR and the second
one was, after dissolving in water, separated on Bio-Gel P2 column
(200 ꢂ 2.5 cm). Fractions (5 mL) were collected in which the carbo-
hydrate content was checked according the procedure of Dubois
et al., 1956.28 Separation of RIM afforded eight fractions: monosac-
charides 8FR, oligosaccharides 7-2FR and a polymeric residue 1FR
(Fig. 2).
at d 4.118 ppm with JH,H coupling constant 3.5 Hz revealed the
presence of Fru. In the course of the incubation, Glc signal intensi-
ties decreased and they disappeared after 24 h. 1H NMR spectra of
samples taken between 63 to 120 h suggested a complex mixtures
of metabolites.
3.2. EPS production by Bacillus sp. 3B6
The complexity of the in situ 1H NMR spectrum collected at the
end of the incubation of Bacillus sp. 3B6 on sucrose (not shown)
highly suggested a mixture of metabolites in which an EPS could
be present. In order to produce the medium with higher concentra-
tion of metabolites for structural analyses, Bacillus sp. 3B6 was
incubated during 8 days using the incubation procedure No. 2.
After 8 days a complex incubation medium of very high viscosity
was obtained. After dialysis, a retentate of the incubation medium
(RIM) and dialyzate (DIM) were obtained. Analysis of 1H, 13C, COSY
and HSQC spectra (not shown) showed that RIM was a mixture of
various compounds. In the anomeric region of the 1H NMR spec-
2.5. NMR spectroscopy
trum the presence of three signals at d 5.41, 5.39, and 5.17 ppm
3
NMR spectra were measured on 600 MHz VNMRS Varian
equipped with HCN 13C enhanced salt tolerant cold probe in D2O
at 25 °C using acetone as an external standard. Samples of oligosac-
charides were freeze-dried from D2O before dissolution in 99.98%
D2O. Shigemi NMR tubes or micro-capillaries were used in the case
of small quantity of FOS samples. Advanced techniques from Var-
ian pulse sequence library of 2D homo- and hetero-correlated
spectroscopy (gCOSY, gTOCSY, gHSQCAD, gHMBCAD, gH2BC)
including 1D sequences with selective excitations (1DNOESY,
1DTOCSY) were used for the signal assignments.
with small JH1,H2 coupling constants indicated
a
configuration of
these sugars, most probably of glucose (Glc) origin. Their intensity
was not in accordance with signal intensities of skeletal protons in
the region d 4.3–3.3. Comparison of RIM 1H and HSQC NMR spectra
with those due to sucrose and fructose showed the presence of a
small quantity of sucrose, not used by the bacterium, fructose re-
leased after sucrose hydrolysis and other not identified signals of
low intensities. However, dominant signals due to a new metabo-
lite were broader indicating its high molecular mass. The HSQC
spectrum revealed a huge amount of CH2 signals suggesting the
presence of a metabolite composed of fructose or fructose similar
sugar units (not shown).
3. Results
3.1. Monitoring of sucrose metabolism by in situ 1H NMR
spectroscopy
3.3. RIM separation into oligomeric fractions on Bio-Gel P2
RIM separation by size exclusion chromatography on Bio-Gel P2
afforded eight fractions that is, monosaccharides 8FR, oligosaccha-
rides fractions 7–2FR and polymeric residue 1FR. NMR analysis of
the lowest molecular mass fraction 8FR confirmed the presence
of fructose and glucose in the ratio 9:1. The comparison of spectral
patterns of 1H NMR spectra indicated that fractions 1–6FR belong
to a coherent series of oligo- and poly- saccharides, while the
one due to 7FR was different. Figure 3 shows 1H NMR spectra of
RIM fractions 1FR, 4–6FR ,and sucrose used as a standard.
Sucrose degradation by Bacillus sp. 3B6 was monitored by
in situ 1H NMR spectroscopy (spectra not shown) directly in sam-
ples of incubation media (Incubation procedure No. 1) taken at reg-
ular intervals without any treatment.29 At time zero only sucrose
1H NMR signals were present. After 15 h of incubation the first sig-
nals due to released metabolites appeared. Anomeric signals due to
free glucose were observed at d 5.234 ppm for
aGlc and d
4.647 ppm for bGlc (its H2 is present at d 3.246 ppm) and a doublet
3.4. FOSs identification in RIM
The 1H NMR spectrum (Fig. 3) of high molecular mass fraction 1FR
showed in the region of skeletal protons (d 4.25–3.10) seven signals
due to bFruf units, which were easily assigned by COSY and HSQC
spectra (Table 1). The ketoanomeric signal C2 due to bFruf appeared
at d 105.06 ppm, C1 and C6 signals were detected at d 60.74 and
64.25 ppm, respectively. 2,6-Linkages between bFruf units were
confirmed by C2/H6 cross peaks d 64.25 of bFruf in the HMBC spec-
trum (not shown) and confirmed the structure of levan. The absence
of the H1 signal due to a-glucose avoided a polymer molecular mass
estimation from signal intensities in the 1H NMR spectrum. By HPLC
it was found that 1FR has two components, one of molecular mass
ꢀ3500 and the other of ꢀ100 kDa in the ratio 2.3:1, respectively.
In the 1H NMR spectra of penta- (4FR) and tetrasaccharides (5FR)
(Fig. 3) some signals remained similar to those observed in the 1FR
suggesting thus the presence of oligomers of levan type (2,6-bFOSs).
However, besides them, in 13C NMR spectra also diagnostic signals of
inulin type FOSs with 1,2-linked bFruf could be detected; particu-
larly the ketoanomeric C2 at d 104.1, further C5 at d 82.2, C6 at d
63.0, and C1 at d 61.5.30,31 Because the detailed analysis of 4FR–6FR
Figure 2. Elution profile on Bio-Gel P2 of the retentate (RIM) obtained after a
dialysis of a complex incubation medium (incubation procedure No. 2) retained
after 3 days dialysis against water (8 days of incubation of Bacillus sp. 3B6 on
sucrose).

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M. Matulová et al. / Carbohydrate Research 346 (2011) 501–507
Figure 3. Series of 1H NMR spectra of fractions obtained by separation on Bio-Gel P2 of retentate (RIM) and dialyzate (DIM). Fractions 1FR and 4–6FR from RIM, fractions 1–
5FD from DIM, di—disaccharide, tri—trisaccharide, tetra—tetrasaccharide, penta—pentasaccharide, hexa—hexasaccharide, poly—polymer, HOD—residual signal of water.
Table 1
1H and 13C NMR data of levan, inulin, DFA IV compared with
a, bFruf ,and sucrose as standards
Compound
Fraction
Sugar unit
1H and/or 13C chemical shifts/d
1,10
2
3
4
5
6,60
Fructose
Sucrose
Standard
Standard
bFruf
C
C
63.09
61.81
102.2
105.2
76.1
82.02
75.17
76.6
81.39
82.68
63.38
63.63
a
Fruf
a
Glcp(1?
H
C
H
C
5.403
92.89
3.666
62.07
3.547
71.78
—
3.748
73.29
4.205
77.13
3.459
69.94
4.039
74.72
3.831
73.12
3.877
82.09
3.847
60.84
3.805
63.08
?2)bFruf
2,6-bFruf
104.4
Levan
Inulin
DFA IV
1FR
H
C
3.77, 3.670
60.75
—
105.00
4.178
77.15
4.096
76.00
3.958
81.07
3.898, 3.549
64.20
a
1,2-bFruf
H
C
3.79, 3.69
60.57
—
104.10
4.19
76.68
4.030
74.04
3.85
81.39
3.80, 3.635
62.36
7FR
[-6)bFruf(2-]2
H
C
3.747, 3.639
60.038
—
103.9
4.182
77.58
4.597
72.56
3.842
81.62
3.947, 3.640
61.09
a
Taken from HSQC spectrum of the 2FD fraction (Fig. 4).
proton spectra was not straightforward their 2D hetero-correlated
HSQC spectra (Fig. 4) were examined. Characteristic levan type cross
peaks were well separated in the spectrum of the levan polymer
(1FR). H5/C5 at d 3.958/81.07, and H6,H60/C6 at d 3.898, 3.545/64.2
were used as levan type diagnostic cross peaks. Thus in theRIM spec-
tra of lower molecular mass samples 4-6FR (Fig. 4) cross peaks due to
inulintype 1,2-bFOSs H5/C5 3.85/81.39, H6,H60/C6 at d ꢀ3.80, 3.635/
62.36, and H1,H10/C1 at d ꢀ3.79, 3.69/60.57 could be easily recog-
trisaccharides in 6FR. The spectral pattern was in strong accordance
with the one published by Ammar et al.32 for a mixture of 1-kestose,
6-kestose, and neokestose (Fig. 5) in which proton signals of identi-
cal chemical shifts were assigned as follows: 5.415 to 1-kestose,
5.386 to neokestose, and 5.362 to 6-kestose. Chemical shifts of C2
ketoanomeric and C1 anomeric signals in the 6FR in the 13C NMR
spectrum were found to be in accordance with the literature data
for these trisaccharides.33,34 The ratio of 1-kestose, 6-kestose, and
neokestose in the 6FR fraction was 2:1:3, respectively. Ascending
TLC afforded a qualitative proof of their presence in 6FR.35 6FR HSQC
spectrum is shown in Figure 4.
nized. They suggested the presence of nystose (
bFruf-1,2-bFruf) in the fraction 5FR and in 4FR a pentasaccharide
Glc-1,2-bFruf-[1,2-bFruf]2-1,2-bFruf) of inulin type. Sucrose
building block was confirmed in all oligosaccharides by the presence
aGlc-1,2-bFruf-1,2-
(a
of
a
-Glc cross peaks. Very high molecular weight of the polymer
3.5. FOSs identification in DIM
(1FR) may be the reason of their absencein the HSQC spectrum. Spec-
tral patternof the 1HNMR spectrum oftrisaccharidefraction 6FR was
the most complex (Fig. 3). It showed the presence of
signals at d 5.415; 5.386, and 5.362 suggesting at least three different
SEC of dialyzate (DIM) afforded two dominant fractions, 6FD con-
taining monosaccharides glucose and fructose (3:1) and a disaccha-
ride fraction (5FD) containingsucrose only. Fraction of trisaccharides
a-glucose unit

M. Matulová et al. / Carbohydrate Research 346 (2011) 501–507
505
Figure 4. Part of HSQC NMR spectra (cross peaks of skeletal protons and carbons) of chosen fractions obtained by separation on Bio-Gel P2 of retentate (RIM) and dialyzate
(DIM). 3FD tetrasaccharide DIM fraction, 1FR and 4–6FR—polymer and tri-, tetra-, and pentasaccharides of RIM, inu—inulin type 1, 2-bFOSs, lev—levan type 2,6-bFOSs, S—
sucrose, G—glucose, F—b-fructofuranose, I—cross peaks due to inulin type 1,2-bFOSs.
Figure 5. Structures of 1-kestose, 6-kestose, neokestose, and DFA IV.
4FD was identified as a mixture of 1-kestose, 6-kestose, and neokes-
tose. In fractions 1-3FD mixtures of higher molecular mass oligosac-
charides (dp 4–6) were present in trace amounts. DIM Elution profile
on the Bio-Gel P2 column and 1H NMR spectra of individual fractions
are shown in Figures 1 and 3, respectively.
bFruf]4), while the other is a homopentasaccharide composed of
five 2,6-linked bFruf units ([2,6-bFruf]5).
When signals in the 1H NMR spectra of 4FD and 3FD fractions
were compared with NMR data of inulin type 1,2-bFOSs published
by Fukushi et al. (2000)36 it was found that chemical shift of signals
in the 3FD spectrum are consistent with those of nystose. Spectral
pattern of its HSQC spectrum (Fig. 4) confirmed inulin type struc-
ture of this tetrasaccharide showing characteristic chemical shift
H5/C5 due to 1,2-linked bFruf units, while the cross peak due to
H5/C5 of 2,6-linked bFruf units was missing in the spectrum.
Spectral pattern of 1H NMR spectra due to the levan polymer in
1FR RIM was similar to those of 1FD and 2FD DIM fractions (Fig. 3)
suggesting the levan type FOSs with 2,6-linked Fruf residues in 1FD
and 2FD. However, in 2FD the integral ratio of the H3 bFruf signals
at d 4.20 and
a-Glc was 9:1 suggesting a decasaccharide in this
fraction. However, this conclusion was not in accordance with
the molecular mass of this fraction suggested by elution profile
(pentasaccharide) and the ratio of abovementioned signals in 1FD
fraction showing the presence of hexasaccharides there. These
facts indicated that in 2FD two types of pentasaccharides are pres-
3.6. Identification of difructose anhydride DFA IV
NMR spectra (Fig. 6) of the disaccharide fraction 7FR showed
that dominant signals belong to only one bFruf unit in which H4/
C4 was unusually downfield shifted to d 4.597/72.56 (Table 1).
ent. In the first one four bFruf units are linked to a-Glc (aGlc-[2,6-

506
M. Matulová et al. / Carbohydrate Research 346 (2011) 501–507
The HMBC of 7FR revealed cross peaks due to long range interac-
tions between ketoanomeric C2 signal present at d 103.90 and all
protons of both CH2 groups (H6, H60, H1, and H1’) of bFruf ring. This
fact confirmed that in this compound two molecules of bFruf are
linked by two 2,6-linkages forming thus a cyclic symmetric disac-
tion 1-kestose, 6-kestose, and neokestose were present in the ratio
2:1:3. In DIM their ratio was 1.5:2:1. DIM higher molecular mass
fractions contained only traces of tetra, penta, and hexasaccharides.
In penta and hexasaccharides 2,6-linked bFruf units were linked to
a-glucose. However, DIM pentasaccharide fraction contained also
charide molecule—difructose anhydride type DFA IV (Fig. 6). Its 13
chemical shifts agreed with literature data.17,18,37
C
homopentasaccharide composed of 2,6-linked bFruf units only. In
DIM tetrasaccharide fraction nystose was identified. 1-Kestose is a
precursor for its synthesis. Polymeric levan (RIM) was found to be
composed of two components, one of molecular mass ꢀ3500 and
the other of ꢀ100 kDa with the ratio 2.3:1. Identification of levan
2,6-bFruf and inulin 1,2-bFruf type oligosaccharides in the incuba-
tion medium suggests both levansucrase and inulosucrase enzymes
activity in Bacillus sp. 3B6 similarly to L. reuteri 121.6
In the 1H NMR spectrum of 7FR besides signals of DFA IV other
signals could be detected indicating the presence of low amounts
of further compound in this fraction. Previous NMR analysis of
complex RIM showed the presence of sucrose not metabolized by
the bacterium. Degree of polymerization of 7FR was dp = 2 and thus
it should contain sucrose. The signal at d 5.40 was identified as H1
due to
a
-Glc in sucrose molecule. The ratio of sucrose and DFA IV in
Depolymerising levan/inulin fructosyltransferase was found to
produce DFA when bacteria were grown on the polymer, inulin,
or levan.16–18 The high quantity of DFA IV identified in the incuba-
tion medium suggests that the activity of this enzyme in Bacillus
sp. 3B6 is very high. Further studies will be necessary to elucidate
enzyme activities in this bacterium.
7FR was found to be 1:5. The origin of other very low intensities
signals present in the spectra was not identified.
NMR spectra of 3F and 2F fractions were very complex showing
the presence of diagnostic signals of both, levan 2,6-bFOSs and inu-
lin 1,2-bFOSs together with signals due to DFA IV. Further studies
will be necessary to precise structures of compounds present in
these mixtures.
Identification of fructans production by Bacillus sp. 3B6, isolated
by Amato et al.26 from cloud water, raises questions about their po-
tential role in the natural environment where this strain was first
isolated. Clouds represent an aqueous environment of low temper-
ature (ꢁ10 till 15 °C) which transports material and energies to
long distances between ecosystems. It was admitted, that liquid
and super-cooled water represents better temporary environment
for living organisms transported by air than dry air. Thus droplets
in clouds can be considered as an environment where cells can
reproduce themselves.38,39 Transformation of sucrose by Bacillus
sp. 3B6 into EPS shown in this work might have some implications
considering cloud environment, because the presence of various
saccharides in atmosphere has been already proven. From identi-
fied saccharides, glucose and sucrose were the most abundant.40,41
It is known that EPS, and especially fructans, can protect microor-
ganisms against abiotic and biotic stress, such as desiccation and
4. Discussion
Monitoring the metabolism of sucrose at rather low concentra-
tions by Bacillus sp. 3B6 using in situ 1H NMR showed that this bac-
terium started to degrade sucrose to free glucose and fructose. While
glucose was used by the bacterium as a source of energy, fructose re-
mained in the medium. At higher sucrose concentration, fructose
accumulated in the medium and it served for the EPS synthesis. Dial-
ysis of complex incubation medium afforded retentate (RIM) and
dialyzate (DIM). Both, monosaccharide fractions of DIM and RIM
contained glucose and fructose. In the disaccharide fraction of RIM
the ratio of difructose dianhydride DFA IV and sucrose was 5:1, while
inthatoneofDIM onlysucrosewas found. InRIMtrisaccharidesfrac-
Figure 6. HSQC and HMBC spectra of the disaccharide difructose anhydride DFA IV present in the 7FR fraction obtained after the separation of the dialyzed complex
incubation medium RIM (incubation procedure No. 2) on Bio-Gel P2.

M. Matulová et al. / Carbohydrate Research 346 (2011) 501–507
507
freezing.12 Production of EPS could be an efficient way for this
strain to survive in the extreme environment of clouds, stay meta-
bolically active, and to contribute to atmospheric chemistry and
nucleation processes.42–44
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Obtained results clearly show the potential interest of using this
new strain, Bacillus sp. 3B6 in biotechnology processes in order to
obtain levan polysaccharide, levan and inulin type FOSs, and
difructose anhydride DFA IV. These compounds are particularly rel-
evant for applications in nutraceutical, pharmaceutical, and food
industry. However, these results also indicate a new direction of
the research about the potential role of bacteria producing EPS in
the atmosphere.
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Acknowledgements
This project has been supported by the French Government
scholarship (fellowship for S. Husárová), the Slovak Grant Agency
VEGA No. 2/0155/08 and 2/0116/10, APVV Grants No. 0030-08
and 0650-07, Slovak state program 2003SP200280203, Štefánik
project No. 17947UE (SK-FR-0009-07), BIOCLOUDS CNRS-DFG
French German bilateral project.
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