238 International Journal of Food Science and Technology 2014, 49, 238–245

Original article
Antipathogenic activity and preservative effect of levan
(b-2,6-fructan), a multifunctional polysaccharide

Bo Young Byun,1 Su-Jin Lee2 & Jae-Hyung Mah1*

1 Department of Food and Biotechnology, Korea University, Sejong, 339-700, Korea
2 RealBioTech Co., Ltd., Sejong 339-802, Korea
(Received 3 March 2013; Accepted in revised form 11 July 2013)

Summary The objective of this study was to investigate the antibacterial activity of levan compounds, including
high-molecular-weight levan, low-molecular-weight levan and difructose dianhydride IV (DFA IV). The
levans exhibited broad antibacterial spectra against foodborne pathogenic bacteria, in a concentration-
dependent manner. From comparison with simple saccharides, often regarded as by-products of levan
production, it turned out that the antibacterial activity of levans was primarily caused by themselves. The
strongest effect was observed with low-molecular-weight levan as compared to the others, and oligosac-
charides as well. The low-molecular-weight levan was therefore applied to bread making. The bread sam-
ples inoculated with pathogenic bacteria were divided into two groups: bread with sugar alone (control)
and bread with both sugar and levan (treatment). It was found from the in situ test that the viability of
pathogenic bacteria in bread was reduced by the addition of low-molecular-weight levan. Therefore, levan
compounds have potential as alternative sweeteners for reduction in pathogenic contamination.
Keywords Antimicrobial spectrum, bread, foodborne pathogenic bacteria, growth inhibition, levan, MIC, b-2,6-fructan.

Meanwhile, major foodborne pathogenic bacteria

Introduction
Levan is a homopolysaccharide which is composed of
D-fructofuranosyl residues joined by 2,6 with multiple
branches by 2,1 linkages. Microbial levan is extracellu-
larly produced from sucrose-based substrates by levan-
sucrase that catalyses levan biosynthesis by
transferring fructose on sucrose donor to acceptor
molecule. Although various microorganisms have the
enzyme activity, Zymomonas mobilis is considered a
suitable microorganism for large-scale production of
levan (Han, 1990; Hendry & Wallace, 1993). Levan
has been known to be beneficial for improving health
by reducing body fat and cholesterol (Yamamoto
et al., 1999), stimulating immunity (Xu et al., 2006),
and inhibiting cancer (Yoo et al., 2004). Thus, levan
has received attention due to its multifunctional prop-
erties. Furthermore, levan is converted to difructose
dianhydride IV (DFA IV) by bacterial levan fructo-
transferase (Tanaka et al., 1983). This difructose also
has various functional features. Regarding the benefits
of DFA IV, Mellet & Fern
andez (2010) reviewed that
this compound is low-caloric and acariogenic and pro-
motes the growth of beneficial microflora in the gut.
*Correspondent: Fax: (82) 44 860 1586; e-mail: nextbio@korea.ac.kr
include salmonellae, listeriae, pseudomonads, and cer-
tain strains of Escherichia coli. Salmonella spp., Staphy-
lococcus aureus, Bacillus cereus and B. subtilis are the
major microbial hazards of frozen and refrigerated
dough, refrigerated or dry pasta and noodles and baked
goods, including breads, muffins, cakes, pastries, cook-
ies, biscuits and bagels (Rosenkvist & Hansen, 1995;
Tauxe, 2002; Institute of Food Technologists, 2003).
Baked goods in general contain sugar to confer sweet-
ness and reduce water activity, which in part reduces
hazardous microbial growth, but mainly serves as a
carbon source for microbial growth (Glass & Doyle,
1991). In the past decades, several oligosaccharides have
been of particular interest to food scientists and dentists
because they have potentialities as acariogenic and
nondigestible sweeteners (Whistler & BeMiller, 1997).
Unfortunately, however, there have been no sweeteners
showing broad antibacterial spectra against various
Gram-positive and Gram-negative spoilage and patho-
genic bacteria.
The objective of this study was therefore to deter-
mine the antibacterial activity of levans, including
high-molecular-weight levan, low-molecular-weight
levan and DFA IV, against various spoilage and path-
ogenic bacteria. Furthermore, the antibacterial effects
of levans were compared not only with those of

doi:10.1111/ijfs.12304
© 2013 The Authors. International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

by-products of levan production, but also with oligo-
saccharides. Finally, low-molecular-weight levan,
which was found to have the strongest antibacterial
activity, was applied to bread making to confirm its in
situ activity against pathogenic bacteria and potential
as an alternative sweetener. To the best of our knowl-
edge, there have been no reports available in literature
on the antibacterial activity of levans, especially
against foodborne pathogenic bacteria. Therefore, this
is the first study describing the antipathogenic activity
of levans, particularly low-molecular-weight levan.
Materials and methods
Samples and strains used
High-molecular-weight levan (MW 3 9 106 Da; purity,
>95%) and low-molecular-weight levan (MW
5 9 104 Da; purity, >95%) that had been synthesised
by the levansucrase from Zymomonas mobilis, and di-
fructose dianhydride IV (DFA IV) synthesised by a
levan fructotransferase from Arthrobacter ureafaciens
were obtained from RealBioTech Co. Ltd. (Sejong,
Korea). The purity and composition of levan com-
pounds were preliminarily determined by HPLC, and
by-products (<5% of each levan compound) of levan
biosynthesis were composed of sucrose, fructose and
glucose. DFA IV commercial sample contained 54% of
DFA IV, 21% of low-molecular-weight levan and 25%
of by-products such as sucrose (3%), fructo-oligosac-
charide (8%) and fructose (14%). In addition, sucrose,
fructose and glucose were purchased from Junsei
Chemical Co. (Tokyo, Japan). Fructo-oligosaccharide,
galacto-oligosaccharide, isomalto-oligosaccharide and
malto-oligosaccharide were gifted by Samyang Co.
(Seoul, Korea).
The original stocks of strains used in this study,
including Pseudomonas aeruginosa KCTC 1750, Ente-
robacter aerogenes KCTC 2190, Salmonella Typhimuri-
um KCTC 1925, Escherichia coli O157:H7 KCCM
40406 (Gram-negative bacteria), Staphylococcus aureus
KCTC 1916, Listeria monocytogenes KCTC 3710,
Bacillus cereus KCTC 1012 and B. subtilis KCTC 1021
(Gram-positive bacteria), were purchased from the
Korean collection for type cultures (KCTC; Daejeon,
Korea) and the Korean Culture Center of Microor-
ganisms (KCCM; Seoul, Korea). The strains were
grown in tryptic soy broth (TSB; Becton Dickinson,
Detroit, MI, USA) for 24 h at 37 °C and then stored
in a freezer by using glycerol (60%, v/v).
Determination of in vitro antibacterial activity
To determine the antibacterial activity and estimate
the minimal inhibitory concentration (MIC) of levan
compounds and other saccharides, the growth curves
© 2013 The Authors
International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology
Antibacterial activity of levan B. Y. Byun et al. 239
of spoilage and pathogenic bacteria in TSB containing
levan compounds were monitored by measuring the
optical density at 600 nm of bacterial cultures in a
Bioscreen apparatus (Labsystems, Helsinki, Finland).
Briefly, each well of the plate was filled with 340 lL of
fresh TSB containing levan compounds and other sac-
charides at a given final concentration (1%, 3% and
5%, w/v) and inoculated with 10 lL of a 24-h TSB
culture (OD600 of 1.0  0.1) of each strain. The plate
was then incubated at 37 °C for 48 h with continuous
shaking and absorbance reading at 600 nm every 3 h.
The values from Bioscreen apparatus were analysed
using spreadsheet software (Excel 2003; Microsoft Co.,
Redmond, WA, USA). The MIC was defined as the
lowest concentration of test substance inhibiting the
visible growth of each bacterium.
In addition to the turbidity method described above,
each test bacterium was grown at 37 °C in 5 mL TSB
containing low-molecular-weight levan (1%, 3% and
5%, w/v), and the number of viable cells after 48-h
incubation was measured by plating an aliquot of the
culture on plate count agar (PCA; Becton Dickinson)
to confirm the OD data.
Preparation of bread specimens and determination of in
situ antibacterial activity
Formulations presented in Table 1 were used to
prepare sponge bread, a food model system. Sponge
bread samples sweetened with sugar alone (34.5%,
w/w) served as a control, while other sponge bread
samples in which 5% of sugar was replaced by the
same weight of levan (consequently, sweetened with
32.78% sugar and 1.72% levan, w/w) served as levan-
treated groups. To prepare sponge bread, briefly, egg
white was whipped with a hand eggbeater until a
creamy suspension formed (approximately 5 min). Egg
yolk, sugar (alternatively sugar and levan) and weak
wheat flour (100 meshes) were then added to the
creamy suspension, and the mixture was whipped with
a hand eggbeater until the dough developed (approxi-
mately 3 min). Finally, the dough was poured into a
paper baking cup and baked at 200 °C for 10 min in
Table 1 Formulation of bread dough
Ingredients (%) Control Levan treatment
Flour 27.10 27.10
Egg white 26.10 26.10
Egg yolk 12.30 12.30
Sugar 34.50 32.78
Levan – 1.72*
*Approximately 5% of sugar was replaced by the same weight of
low-molecular weight levan.
International Journal of Food Science and Technology 2014
240 Antibacterial activity of levan B. Y. Byun et al.
an oven (NOVA ETO-405; Green Hill Co., Gimpo,
Korea). The baked sponge bread was allowed to cool
at room temperature and aseptically cut into bread
pieces (1 9 1 9 1 cm; 1 g). The procedures were
adapted from the work of Ronda et al. (2005).
One hundred microlitres of a 24-h TSB culture
(OD600 of 1.0  0.1) of each strain were inoculated
onto the surface of sponge bread piece, and the inocu-
lated samples were held for a storage test at 37 °C for
24 h. One gram of the inoculated sponge bread sam-
ples was homogenised in a stomacher with 9 mL of
dilution solution before and after the incubation per-
iod, and serial dilutions were prepared for bacterial
counting. One hundred microlitres of dilutions were
spread onto PCA and incubated at 37 °C for 24 h,
and then colony counts were carried out. The in situ
experiments with different strains were performed
independently.
Statistical analysis
The data were presented as means and standard devia-
tions of triplicate determinations. The significance of
differences was determined by one-way analysis of var-
iance (ANOVA) with Tukey’s pairwise comparison mod-
ule of the Minitab statistical software, version 12.11
(Minitab Inc., State College, PA, USA), and differ-
ences with P-values of <0.05 were considered statisti-
cally significant.
Results and discussion
Antibacterial activity of different levan compounds
The antibacterial activity of three different levan
compounds was tested against various spoilage and
pathogenic bacteria frequently involved in food spoil-
age and foodborne diseases worldwide. As shown in
Fig. 1 (also see supplementary data in Table S1), low-
molecular-weight levan strongly inhibited the growth
of all tested bacteria including, Bacillus subtilis,
B. cereus, Staphylococcus aureus, Listeria monocytoge-
nes, Escherichia coli O157:H7, Salmonella Typhimuri-
um, Psedomonas aeruginosa and Enterobacter
aerogenes, at concentrations above 3%. Even at a con-
centration of 1%, the low-molecular-weight levan
effectively inhibited the growth of B. cereus, S. aureus,
E. coli O157:H7 and P. aeruginosa. The levan com-
pound also slightly, but not significantly, inhibited the
growth of B. subtilis, L. monocytogenes and E. aeroge-
nes, however, not that of Salmonella Typhimurium at
the same concentration. It is worth mentioning that
while B. subtilis, Salmonella Typhimurium and E. aer-
ogenes were less sensitive to the antibacterial effect of
low-molecular-weight levan, and S. aureus, L. mono-
cytogenes and P. aeruginosa were more sensitive to the
International Journal of Food Science and Technology 2014
International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology
levan (Table S1), results obtained from viable cell
counts were in accordance with the turbidity data
obtained using Bioscreen apparatus. High-molecular-
weight levan effectively inhibited the growth of most
tested spoilage and pathogenic bacteria, except for
B. subtilis, Salmonella Typhimurium and E. aerogenes,
at 5% level. This levan compound also slightly and
significantly inhibited the growth of B. cereus,
S. aureus and P. aeruginosa and strongly inhibited that
of E. coli O157:H7 at 1% level. The other bacteria,
such as B. subtilis, L. monocytogenes, Salmonella
Typhimurium and E. aerogenes, were not inhibited by
high-molecular-weight levan at concentrations below
5% (see supplementary data in Fig. S1). DFA IV
strongly (or at least effectively) inhibited the growth of
most tested pathogenic bacteria, except for E. aeroge-
nes, at concentrations above 3%. Even at a concentra-
tion of 1%, the compound significantly inhibited the
growth of L. monocytogenes, however, not that of the
other pathogenic bacteria (see supplementary data in
Fig. S2).
Meanwhile, the antibacterial activity of three differ-
ent levan compounds was directly compared at a con-
centration of 5%. In most cases, as shown in Fig. 2,
low-molecular-weight levan showed the strongest anti-
bacterial activity among the levan compounds tested.
This might be due to different molecular weights of
levan compounds, allowing distinct antibacterial
behaviours. DFA IV exhibited a stronger or similar
inhibitory activity against the growth of E. coli O157:
H7 depending on incubation time, as compared to
low-molecular-weight levan. High-molecular-weight
levan revealed a stronger inhibitory activity against
the growth of L. monocytogenes, while a weaker or
similar activity against the other bacteria, as compared
to DFA IV. The MICs of different levans against all
tested bacteria were also estimated based on the bacte-
rial growth inhibition observed above. As compiled in
Table 2, the MICs of low-molecular-weight levan
against most tested bacteria were found to be less than
or around 1%, which were lower than those of high-
molecular-weight levan and DFA IV. Only for Salmo-
nella Typhimurium, the low-molecular-weight levan
had a higher MIC (1–3%) than high-molecular-weight
levan (MIC, 1%). In addition, S. aureus and E. coli
O157:H7 seemed to be more sensitive to the antibacte-
rial effects of levan compounds than the other tested
bacteria. Taken together, levan compounds were
found to have antibacterial activity against all tested
bacteria in a concentration-dependent manner. It is
not clear yet whether the inhibitory action of levans is
bactericidal or bacteriostatic because the levans did
not extend the lag phase of the tested bacteria
although they reduced both the growth rate and the
maximum population density of the bacteria at given
concentrations.
© 2013 The Authors
 Antibacterial activity of levan B. Y. Byun et al. 241

2.5 2.5
(a) (b)

Optical density (600 nm)

Optical density (600 nm)
2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)

2.5 (c) 2.5 (d)

Optical density (600 nm)

Optical density (600 nm)

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)

2.5 (e) 2.5 (f)

Optical density (600 nm)

Optical density (600 nm)

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)
Figure 1 Inhibitory effect of low-molecular-
weight levan on the growth of spoilage 2.5 2.5
(g) (h)
Optical density (600 nm)

Optical density (600 nm)

and pathogenic bacteria. (a) Bacillus subtilis,
2.0 2.0
(b) Bacillus cereus, (c) Staphylococcus aureus,
(d) Listeria monocytogenes, (e) Escherichia 1.5 1.5
coli O157:H7, (f) Salmonella Typhimurium, 1.0 1.0
(g) Psedomonas aeruginosa, (h) Enterobacter
aerogenes. ●, control; ○, low-molecular- 0.5 0.5

weight levan at 1%; △, low-molecular-weight 0.0 0.0

levan at 3%; □, low-molecular-weight levan 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
at 5%. Incubation time (h) Incubation time (h)

fructose showed an effective inhibitory activity against

Comparison of antibacterial activity of levan with other
S. aureus and E. coli O157:H7, while other saccharide
saccharides including possible by-products of levan
mixtures containing only one of the monosaccharides
biosynthesis
were less effective (see supplementary data in Fig. S4).
Because sucrose, fructose and glucose are often It is worth reminding that one of these monosaccha-
by-products of levan production, the antibacterial rides and mixtures thereof effectively inhibited the
activity of levan compounds was compared with that growth of only S. aureus and E. coli O157:H7 but
of sucrose, fructose or glucose alone and mixtures of stimulated that of the other tested bacteria. Therefore,
two or three of these saccharides mixed in the same it can be suggested that levan compounds may have
ratios. At a given final concentration of 5% (w/v), at least two different modes of antibacterial action:
none of the mono- and disaccharides and their mix- (i) induction of osmotic stress and/or reduction in
tures showed antibacterial activity against most tested water activity by levan compounds and degradation
bacteria, except for S. aureus and E. coli O157:H7 (see products (glucose and fructose) thereof and (ii)
supplementary data in Figs S3 and S4). However, both competitive interference of levan molecules themselves
glucose and fructose inhibited the growth of S. aureus with bacterial absorption of an essential nutrient(s).
and E. coli O157:H7, showing a slightly stronger or Because a high concentration of mono- and disaccha-
similar activity as compared to low-molecular-weight rides causes osmotic stress and low water activity that
levan (see supplementary data in Fig. S3). Similarly, prevents bacterial spoilage of foods (Peddie & Cham-
saccharide mixtures containing both glucose and bers, 1993; Kwakman & Zaat, 2012), the former

© 2013 The Authors International Journal of Food Science and Technology 2014
International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology
242 Antibacterial activity of levan B. Y. Byun et al.

2.5 2.5
(a) (b)
Optical density (600 nm)

Optical density (600 nm)

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)

2.5 (c) 2.5 (d)

Optical density (600 nm)

Optical density (600 nm)

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)

2.5 (e) 2.5 (f)

Optical density (600 nm)

Optical density (600 nm)

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)
Figure 2 Comparison of inhibitory effects
2.5
of different levan compounds on the
(g) 2.5 (h) growth of spoilage and pathogenic bacteria.
Optical density (600 nm)

Optical density (600 nm)

2.0 2.0 (a) Bacillus subtilis, (b) Bacillus cereus,

1.5 1.5
(c) Staphylococcus aureus, (d) Listeria
monocytogenes, (e) Escherichia coli O157:H7,
1.0 1.0
(f) Salmonella Typhimurium, (g) Psedomon-
0.5 0.5 as aeruginosa, (h) Enterobacter aerogenes.
●, control; ○, DFA IV at 5%; △, low-molec-
0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48 ular-weight levan at 5%; □, high-molecular-
Incubation time (h) Incubation time (h) weight levan at 5%.

Table 2 Minimal inhibitory concentration (MIC) of levan compounds and oligosaccharides

Levans (%) Oligosaccharide (%)

Microorganisms High-molecular-weight levan Low-molecular-weight levan Levan DFA IV Fructo- Galacto- Isomalto- Malto-

Bacillus subtilis 1 1 1< – <3 5 5 5 5

Bacillus cereus 1 <1 1< – <3 5 5 5< 5<
Staphylococcus aureus 1 <1 <1 <1 <1 <1 <1
Listeria monocytogenes 1 1 1< – <3 5 3 5< 5<
Escherichia coli O157:H7 <1 <1 1 <1 <1 <1 <1
Salmonella Typhimurium 1 1< – <3 1< – <3 5 3< – <5 1< – <3 3
Pseudomonas aeruginosa 1< – <3 <1 1< – <3 5 5 5< 5<
Enterobacter aerogenes 1 1 1< – <3 5 5 5 5

speculation seems to be valid, which is again sup- dom et al., 2011). Thus, it is likely that levan com-
ported by current observations described above. More- pounds may have a similar inhibitory mechanism to
over, some researchers have reported that chitosan chitosan and lectin. The latter speculation may be
and lectin form a layer around spores and disturb the clarified by isotopic and electron microscopic studies
absorption of nutrients (Paiva et al., 2010; Singburau- in the near future.

International Journal of Food Science and Technology 2014 © 2013 The Authors
International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology
 Antibacterial activity of levan B. Y. Byun et al. 243

2.5 2.5
(a) (b)

Optical density (600 nm)

Optical density (600 nm)
2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)

2.5 2.5
(c) (d)

Optical density (600 nm)

Optical density (600 nm)

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
Incubation time (h) Incubation time (h)

2.5
(e) 2.5 (f)
Optical density (600 nm)

Optical density (600 nm)

2.0 2.0

1.5 1.5

1.0 1.0

0.5 0.5

0.0 0.0
Figure 3 Comparison of inhibitory effects 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48
of low-molecular-weight levan and different Incubation time (h) Incubation time (h)
oligosaccharides on the growth of spoilage
and pathogenic bacteria. (a) Bacillus subtil- 2.5 (g) 2.5 (h)
Optical density (600 nm)

Optical density (600 nm)

is, (b) Bacillus cereus, (c) Staphylococcus 2.0 2.0
aureus, (d) Listeria monocytogenes, (e)
1.5 1.5
Escherichia coli O157:H7, (f) Salmonella
Typhimurium, (g) Psedomonas aeruginosa, 1.0 1.0
(h) Enterobacter aerogenes. ■, low-molecu-
0.5 0.5
lar-weight levan at 5%; ▲, fructo-oligosac-
charide at 5%; ○, galacto-oligosaccharide 0.0 0.0
at 5%; △, isomalto-oligosaccharide at 5%; 0 6 12 18 24 30 36 42 48 0 6 12 18 24 30 36 42 48

□, malto-oligosaccharide at 5%. Incubation time (h) Incubation time (h)

Meanwhile, the antibacterial activity of low-molec- effects of levan samples were mostly attributed to
ular-weight levan was also compared with that of levan compounds themselves because none of
oligosaccharides, including fructo-oligosaccharide, possible by-products and combinations thereof
galacto-oligosaccharide, isomalto-oligosaccharide and showed stronger or similar inhibitory activity, as
malto-oligosaccharide. As shown in Fig. 3, it was compared to levan compounds, even at the same
observed that, while none of the oligosaccharides concentration (Fig. 3; also see supplementary data in
displayed significant antibacterial activity against Figs S3 and S4).
most tested bacteria, malto-oligosaccharide seemed to
even stimulate the growth of the bacteria. Consider-
Application of levan to control of spoilage and pathogenic
ing both health-promoting and antibacterial effects
bacteria in bread, a food model system
of levan compounds, therefore, it can be suggested
that levans are preferable as alternative sweeteners to To further investigate the inhibitory effect of low-
the oligosaccharides. In addition, the oligosaccharides molecular-weight levan on the growth of spoilage and
somehow exhibited inhibitory effect on the growth of pathogenic bacteria in situ, sponge bread samples with
only E. coli O157:H7. This might be due to the sugar alone (control; 34.5%, w/w) and sponge bread
osmotic sensitivity of E. coli O157:H7 as described samples with both sugar and levan (treatment; 32.78%
above. It is also worth mentioning that antibacterial and 1.72%, respectively) were prepared and inoculated

© 2013 The Authors International Journal of Food Science and Technology 2014
International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology
244 Antibacterial activity of levan B. Y. Byun et al.

with spoilage and pathogenic bacteria. As shown in
Fig. 4 (upper panel), it was observed that levan
treatment immediately (just after inoculation) and
significantly reduced the counts of S. aureus, L. mono-
cytogenes, E. coli O157:H7 and P. aeruginosa inocu-
lated onto the sponge bread samples (P < 0.05) and
slightly, but not significantly, reduced those of B. subtil-
is, B. cereus, Salmonella Typhimurium and E. aeroge-
nes (P > 0.05), as compared to control, which were in
strong agreement with our observation that the latter
bacteria were more tolerant to levan treatments in vitro,
as described above. Meanwhile, significant reduction in
bacterial counts was observed for only L. monocytoge-
nes and P. aeruginosa (P < 0.05), as compared to con-
trol, when the inoculated sponge bread samples were
stored at 37 °C for 24 h (Fig. 4, lower panel). The
levan treatment also slightly, but not significantly,
reduced the counts of B. subtilis, B. cereus, S. aureus,
E. coli O157:H7 and Salmonella Typhimurium under
the same condition (P > 0.05), however, not that of
P. aeruginosa. Considering that bread is usually stored
at room temperature, it is expected that the achievable
4.0
Control
3.5
$ $
$ $
Survivors (Log CFU mL–1)
safety improvement of bread with levan treatment
would be much more successful. Regarding the
improvement of bread quality, some researchers have
suggested that organic acids, nisin, essential oils and
other natural compounds are effective in reducing
putrefactive microorganisms (Rosenquist & Hansen,
1998; Suhr & Nielsen, 2003). Likely, levan treatment
would be an effective way not only to reduce putrefac-
tive and pathogenic microorganisms in bread but also
to confer health-promoting benefits on bread.
In addition, both S. aureus and E. coli O157:H7
that are sensitive to the antibacterial effects of levans,
and Salmonella Typhimurium as well, have been fre-
quently detected in raw and ground beef (Ingham
et al., 2005). Due to this, various intervention methods
have been used to reduce these foodborne pathogenic
bacteria in raw and processed beef, which involve
chemical (Hajmeer et al., 2004; Lim & Mustapha,
2004, 2007) and physical treatments (Thayer & Boyd,
2001; Patel et al., 2012). However, some of these treat-
ments may cause changes in organoleptic properties of
beef and raise new safety concerns for the treated
foods. Thus, greater consumers’ awareness and con-
cerns have led researchers and food manufacturers to
Levan
look for harmless food additives with a broad spec-
$ $ $ trum of antibacterial activity (Marino et al., 2001).

$ $ % $ $
3.0 $ % %
%
Furthermore, there has been growing interest in the
2.5 use of natural food additives and incorporation of
2.0 health-promoting bioactive compounds into foods
1.5
(Varga, 2006). Considering that levan compounds are
not only beneficial for improving health as described
1.0
in the beginning of this paper, but are also effective in
0.5 inhibiting the growth of pathogenic bacteria frequently
0.0 detected in beef, it is expected that the compounds
a b c d e f g h
Food-borne pathogens
have potential as alternative sweetening ingredients of
4.0
seasonings and sauces for ready-to-cook beef, such as
Control Levan beef steak, beef burger and Bulgogi.
3.5
$
Survivors (Log CFU mL–1)

3.0 $
$ $ $ Conclusions
$ $ $
2.5 $ % $ %
$

2.0
$ $ $ This study was conducted to determine antibacterial
activity of levan compounds, including high-molecular-
1.5
weight levan, low-molecular-weight levan and difructose
1.0 dianhydride IV (DFA IV). The strongest in vitro inhibi-
0.5
tory effect was observed with low-molecular-weight
levan and followed by the other tested levan com-
0.0
a b c d e f g h pounds. Although the underlying mechanism by which
Food-borne pathogens levan compounds inhibit the growth of spoilage and
pathogenic bacteria is still veiled, it was assumed that
Figure 4 Ability of low-molecular-weight levan to reduce bacterial
levan may have two different modes of inhibitory action:
population in sponge bread before (upper) and after storage (lower).
Error bars indicate standard deviations calculated from three inde-
induction of osmotic stress and/or reduction in water
pendent experiments. Mean values with different letters are signifi- activity and competitive interference with bacterial
cantly different (P < 0.05). (a) Bacillus subtilis, (b) Bacillus cereus, absorption of an essential nutrient(s). Meanwhile, low-
(c) Staphylococcus aureus, (d) Listeria monocytogenes, (e) Escherichia molecular-weight levan was found to significantly
coli O157:H7, (f) Salmonella Typhimurium, (g) Psedomonas reduce the viability of spoilage and pathogenic bacteria
aeruginosa, (h) Enterobacter aerogenes. in bread when applied to bread making to confirm its in

International Journal of Food Science and Technology 2014 © 2013 The Authors
International Journal of Food Science and Technology © 2013 Institute of Food Science and Technology

situ activity against the bacteria. Taken together, this
study suggests that levan compounds have potential as
alternative sweeteners not only for promoting health but
also for reducing the chances of pathogenic outbreaks.
Acknowledgment
This work was supported by a Korea University Grant.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Table S1. The viable cell counts of spoilage and
pathogenic bacteria grown in the presence of low-
molecular-weight levan.
Figure S1. Inhibitory effect of high-molecular-weight
levan on the growth of spoilage and pathogenic bacteria.
Figure S2. Inhibitory effect of DFA-IV on the growth
of spoilage and pathogenic bacteria.
Figure S3. Comparison of inhibitory effects of levan
compounds and mono- and disaccharides on the growth
of spoilage and pathogenic bacteria.
Figure S4. Comparison of inhibitory effects of low-
molecular-weight levan and mixtures of mono- and
disaccharides on the growth of spoilage and pathogenic
bacteria.
International Journal of Food Science and Technology 2014