Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Efficacy of natamycin for control of growth and

ochratoxin A production by Aspergillus carbonarius strains
under different environmental conditions
A. Medina1,2, M. Jimenez2, R. Mateo3 and N. Magan1
1 Applied Mycology Group, IBST, Cranfield University, Silsoe, UK
2 Department of Microbiology and Ecology, Faculty of Biology, University of Valencia, Burjassot, Spain
3 Department of Analytical Chemistry, Faculty of Chemistry, University of Valencia, Burjassot, Spain

Keywords
Aspergillus carbonarius, environmental
factors, grape-based products, mould growth,
mycotoxigenic fungi, natural preservatives.
Correspondence
N. Magan, Applied Mycology Group,
Cranfield Health, Cranfield University, Silsoe,
MK45 4DT UK.
E-mail: n.magan@cranfield.ac.uk
2007 0221: received 13 February 2007,
revised 17 April 2007 and accepted 4 May
2007
doi:10.1111/j.1365-2672.2007.03462.x
Abstract
Aims: To examine the efficacy of natamycin produced by Streptomyces natalen-
sis against strains of Aspergillus carbonarius growth and ochratoxin A (OTA)
production under different environmental factors on a grape juice-based
medium.
Methods and Results: Detailed studies in the range 020 ng ml)1 for control
of growth and ochratoxin production by strains of A. carbonarius at 098, 096
and 094 water availabilities (aw) and 1525C on a fresh red grape extract
medium were examined. Inhibition of growth was depending on temperature
and aw level. At 15C, 510 ng ml)1 natamycin was effective in reducing
growth almost completely. However, at 2025C and all the three aw levels,
growth was only slightly inhibited by 510 ng ml)1 natamycin. There were
strain differences with regard to inhibition of OTA production. At 15C and
098 aw, 10 ng ml)1 was required to inhibit production by >90%. However, at
096 and 094 aw, almost complete inhibition occurred. At 20C, OTA produc-
tion was only significantly inhibited by 10 ng ml)1 natamycin at 094 aw. At
096 and 098 aw, some inhibition occurred with 510 ng ml)1, but greater
concentrations would be required for effective inhibition. At 25C, 5 ng ml)1
was effective at all aw levels. However, at 15C and 25C and a wide range of
aw levels, natamycin effectively controlled OTA production.
Conclusions: Natamycin appears to be a very effective for controlling growth
and OTA production by strains of A. carbonarius over a range of aw and
temperature conditions on grape-based media.
Significance and Impact of the Study: This is the first detailed study to demon-
strate the impact of natamycin against A. carbonarius. This study suggests that
use of natamycin at 50100 ng ml)1 can give complete inhibition of growth of
A. carbonarius and OTA production over a range of environmental conditions.
Natamycin could be an important component of a system to prevent OTA
contamination of wine as well during the drying and production of vine fruits.

2003, 2006). Studies have demonstrated that using less

Introduction
There has been interest in reducing the amounts of
preservatives based on propionate and sorbate used in
intermediate moisture bakery products (Magan et al.
than the recommended amounts (<03%) of these
products could result in a stimulation of growth by
xerotolerant mycotoxigenic moulds and in some cases
also of the associated mycotoxins (Arroyo et al. 2005).

2007 The Authors

2234 Journal compilation 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 22342239
A. Medina et al.
There has thus been interest in examining alternative
anti-fungals for controlling growth of mycotoxigenic
moulds.
Natamycin is a fungicide produced by Streptomyces
natalensis and commonly used in dairy-based food prod-
ucts for controlling spoilage by moulds, especially cheese
(de Ruig and van den Berg 1985; Reps et al. 2002).
Studies by Basilico et al. (2001) have shown that nata-
mycin at 2% (v v) and the parabens (05% w v, anti-
oxidants) effectively controlled thread mould growth in
vacuum-packed hard cheeses. It has a broad spectrum of
activity against spoilage moulds and is considered to be
a very stable product with efficacy against Aspergillus
flavus and aflatoxin production (Rusul and Marth
1988), A. niger, A. versicolor, Penicillium chrysogenum,
P. glabrum, P. brevicompactum, P. cammenbertii, P. com-
mune, P. corylophilum, P. verrucosum, Cladosporium cla-
dosporiodes, C. tenuissimum, Byssochlamys nivea, Mucor
racemosus, Eurotium herbariorum, Alternaria alternata
and others (Stark 2003), although interaction with dif-
ferent environmental factors has not been studied in
detail. It is also effective against yeast and fungal patho-
gens like Candida albicans, Trichophyton spp., Epidermo-
phyton floccosum, Acremonium spp., Cunninghamella
spp., Fusarium spp. and Pseudallescheria boydii (Raab
1972; Reuben et al. 1989; Rotowa et al. 1990).
There has been interest in using mild treatments of
fruit juices with pulsed electric fields and integrating this
with natamycin to inhibit mould spores while maintain-
ing organoleptic properties. Recently, A. carbonarius has
been demonstrated to be the mould responsible for col-
onization and contamination of wine, grapes, grape juice
and vine fruits with ochratoxin (Magan and Aldred
2005). Indeed, the EU has implemented legislative limits
to prevent OTA contaminated foods from reaching the
food chain. No studies have previously examined the
efficacy of natamycin for control of OTA production by
A. carbonarius strains.
The objective of this study was to examine the efficacy
of natamycin against (i) mycelial extension and (ii) och-
ratoxin A (OTA) production by A. carbonarius strains on
a freshly made grape juice medium under different inter-
acting temperatures (15C, 20C and 25C) and water
availabilities (aw, 098094).
Materials and methods
Isolates
Three isolates of A. carbonarius isolated from wine grapes
(refs 1102, 263 and 476) were used. These isolates are
kept in the fungus collection of the Applied Mycology
Group, (IBST, Cranfield University, Silsoe, UK).
2007 The Authors
Journal compilation 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 22342239
Efficacy of natamycin and OTA production
Formulation of natamycin
The formulation used in these studies was Delvocid (50%
a.i.). It was diluted in methanol and a stock solution con-
taining 100 lg ml)1 of natamycin was made. This solu-
tion was diluted to screen a range of concentrations in
the range 1 ng ml)1 to 20 lg ml)1.
Growth studies
A freshly prepared red grape extract from organic table
grapes (grape extractwater 20 : 80 v v), which was
modified with glycerol to the required aw levels (098,
096 and 094), was used. The pH of the medium was
adjusted to 41. The culture medium was prepared and
once autoclaved, the proper volume of natamycin stock
solution was added (around 45C) to get the desired con-
centration in the medium. The medium was vigorously
shaken and poured into Petri dishes. Incubation tempera-
tures were 15C, 20C and 25C based on the informa-
tion available that optimum conditions for OTA
production are 1520C while for growth they are about
3035C (Mitchell et al. 2004). Mycelial growth rates were
determined by daily measurement of two randomly cho-
sen right-angled diameters of the colonies over periods of
12 days. Linear regression of colony radius (mm) against
time (days elapsed from the day the colony was 5-mm
diameter wide) was used to determine the growth rates
(mm day)1), which are the slopes of these lines.
Ochratoxin extraction and analysis
The method used was adapted from Bragulat et al.
(2001). Twenty grams of each fungal culture
(agar + fungal biomass) was cut into small pieces and
extracted with 50 ml methanol (Sigma-Aldrich, Alco-
bendas, Spain) by shaking at 110 rev min)1 for 1 h in
orbital shaker at 25C in the dark. The extracts were
filtered through filter paper (Whatman No. 4) containing
510 g of Celite 545 (Sigma-Aldrich). One millilitre of
each filtered extract was removed and centrifuged at
1150 g for 15 min for further purification. The super-
natant of each tube was removed and placed in an
amber vial for LC analysis.
Analysis of the amount of OTA accumulated in the
culture media was carried out 12 days after inoculation.
This protocol was used on three dishes which had under-
gone the same treatment.
The LC system used consisted of a Millipore Waters
600E system controller, a Millipore 712 WISP autosam-
pler and a Millipore Waters 470 scanning fluorescence
detector (Millipore Corporation, MA, USA) (excitation
and emission wavelengths were 330 and 460 nm, respect-
2235
Efficacy of natamycin and OTA production A. Medina et al.

ively). The samples were separated using a C18 Luna 60 (a)

Spherisorb ODS2 column (150 46 mm, 5 lm) (Phe- 50
nomenex, Macclesfield, UK), with a guard column of the 40
same material. 30
Run time for samples was 12 min with OTA being 20

Radial growth (mm)

detected at about 575 min. The flow rate of the mobile 10
phase (acetonitrilewateracetic acid; 57 : 41 : 2 v v v) 0
was 1 ml min)1. Standards used were in the 50 0 2 4 6 8 10
1200 ng ml)1 range. The recovery rate was 88% from
50 (b)
the agar-based medium with a limit of detection of
<001 lg OTA g)1 medium, based on a signal-to-noise 40
ratio of 3 : 1. Analysis of the results was carried out on a 30
computer with Kroma systems 2000 software (Bio-Tek 20
Instruments, Milan, Italy). 10
0
0 2 4 6 8 10 12
Statistical analysis Time (days)
The statistical analysis was performed using Statgraphics Figure 1 Comparisons of effect of natamycin (a) without natamycin,
5.1 Plus Professional Edition (Statistical Graphics Corp., (b) 10 ng ml)1 of natamycin, on mycelial radial extension of Aspergil-
Herndon, USA). lus carbonarius (isolate 476) at 25C on a red grape juice extract
medium at three water activity levels ( = 094; = 096; r = 098).
Bars indicate SE of means.
Results
Initial studies were carried out on Malt extract agar and mycin and water availability conditions for a strain. The
grape juice-based medium at 25C and approx. 099 aw. growth rates obtained in the different experiments are
This showed that on the former medium no growth shown in Table 1. The effect of aw, temperature, isolate
occurred at >05 lg ml)1, while on grape juice-based and the concentration of natamycin added to the medium
medium no growth occurred at >025 lg ml)1. Thus can be seen.
detailed studies were subsequently carried out over a nar- The statistical analysis of the data (multifactor anova)
rower range of concentrations of natamycin to examine revealed that the four factors assayed and aw-natamycin
efficacy on growth and OTA production. concentration, awtemperature and natamycin concentra-
tiontemperature interactions had significant effects on
growth rate (P < 005).
Effect of natamycin and environmental factors
Using Tukey-honestly significant difference (Tukey-
on mycelial growth
HSD) multiple range test at 95% confidence level, the
Figure 1 shows an example of the linear regression of the cases were grouped into homogeneous groups with
temporal mycelial extension in relation to different nata- regard to the different parameters used. The aw level

Table 1 Effect of different concentrations of natamycin (ng ml)1) on growth (mm day)1) of different strains of Aspergillus carbonarius on a
grape-juice based medium modified with glycerol to give 098, 096 and 094 aw. The pH of the medium was 41

Natamycin at 15C (ng ml)1) Natamycin at 20C (ng ml)1) Natamycin at 25C (ng ml)1)

Strain aw 0 1 5 10 20 0 1 5 10 20 0 1 5 10 20

263 098 31 30 NG NG NG 34 44 39 NG NG 136 149 77 58 NG

096 18 21 03 NG NG 54 47 38 NG NG 112 114 82 50 NG
094 NG NG NG NG NG 25 23 NG NG NG 57 38 39 23 NG
476 098 15 24 08 NG NG 39 48 40 NG NG 110 109 92 71 NG
096 18 19 NG NG NG 36 32 28 NG NG 91 92 69 51 NG
094 NG NG NG NG NG 17 15 NG NG NG 55 36 29 31 NG
1102 098 25 19 NG NG NG 41 51 56 075 NG 135 143 97 67 NG
096 10 19 NG NG NG 59 49 39 04 NG 101 114 83 59 NG
094 NG NG NG NG NG 3 06 01 NG NG 50 40 33 25 NG

NG, no growth.

2007 The Authors

2236 Journal compilation 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 22342239
A. Medina et al. Efficacy of natamycin and OTA production

provided three different clusters (098, 096 and 094

aw). Mould growth rate was higher when the aw used
was higher. The study of the temperature provided
three different clusters (15C, 20C and 25C). Thus,
the lowest temperature assayed (15C) produced a sig-
nificant reduction in growth rate with respect to 20C
and 25C.
The concentration of natamycin split the cases into
four homogeneous nonoverlapping groups, corresponding
to 20, 10, 5 and 10 ng ml)1 (0 and 1 ng ml)1 were
included in the same group). The growth rate reduced as
the natamycin concentration was increased. This decrease
was significant in all cases except when 1 ng ml)1 was
added when there was no appreciable difference with the
blank.
The Tukey-HDS test (P = 95%) displayed only one
homogeneous group with regard to the isolate tested.
Although anova showed that all the factors were statisti-
cally significant, the P value for the isolate tested was
Effect of natamycin and environmental factors
on ochratoxin production
Accumulation of OTA was studied as a function of four
factors (aw, natamycin concentration, temperature and
isolate). Figure 3 displays the effect of natamycin (0
10 ng ml)1) on OTA production at 098094 aw by two
strains of A. carbonarius after 12 days of incubation on a
red grape juice medium. The statistical treatment of the
data by multifactor anova showed that factors aw, nata-
mycin and temperature had significant effects on OTA
accumulation (P < 005). There were also two significant
2nd-order interactions (awtemperature and awisolate).
The P value for the factor isolate was 01223, as this P
15C
25

0086, which indicates that this factor was the least influ- 20
ential.
15
Figure 2 shows the effect of natamycin level, aw and
10
temperature on the growth rate of a A. carbonarius strain.
It can be seen that the efficacy of very low concentrations 5
of this fungicide on fungal growth depends on aw and 0 Str 2
Str 2
temperature. At 20 ng ml)1, there was inhibition of my- Str 2
98

Wa Str 1 +1
96
0

ter Str 1 +5
0

celial growth and it can be seen that natamycin efficacy

94

act Str 1 +1
0

ivit +5 20C
was influenced by aw and temperature. y
OTA ng g1

The results show that inhibition of growth was depend- 25

ing on temperature and aw level. At 15C, 510 ng ml)1 20
natamycin was effective reducing growth almost com-
15
pletely. However, at 2025C fungal growth was only
slightly inhibited by 510 ng ml)1 natamycin regardless of 10

the aw level tested. 5

tr 2
tr 2 S
0
tr 2 Str 2 S 1
tr 1 S +
98 Str 1 S +5
Wa 0 96 4 Str 1 +1 +10
20 ter 0 Str 1
act 09 + 5
18 ivit
y +10 25C
16
Growth rate (mm day1)

25
14
20
12
15
10
8 10
6
5 2
4 2 Str
2 Str 1 1
1 Str 5 + l )
0
tr 1 S tr
+10
+ gm
2 tr 1 S t io n (n
98

S 1 tr a
Str 1 +5 + cen
96
0

con
94

0
0

Wat +10 ycin

0

0 5 10 15 20 25 er a m
ctivit
y nata
in and
Natamycin (ng ml1) Stra

Figure 2 Effect of natamycin (ng ml)1) on growth rate of Aspergillus Figure 3 Effect of natamycin (010 ng g)1) on OTA production at
carbonarius (isolate 1102) on a red grape juice extract medium at 098094 aw by two strains of Aspergillus carbonarius after 12 days
1525C and 098094 aw. r, 098 aw; n, 096 aw; m, 094 aw;






, of incubation on a red grape juice medium at 15C, 20C and 25C.
15C; , 20C; , 25C. Key to strains: Str1, 476; Str2, 1102.

2007 The Authors

Journal compilation 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 22342239 2237
Efficacy of natamycin and OTA production
value is higher than 005 this factor has no statistically
significant effect on OTA accumulation.
The Tukey-HSD test at the 95% confidence level pro-
duced several groupings. The aw-level led to three homo-
geneous nonoverlapping groups. The highest levels of
OTA were found at 098 aw and decreased when aw
decreased. Thus, the amount of available water positively
influences the accumulation (and production) of OTA.
The same test applied to the natamycin level in the
medium gave rise to two homogeneous groups although
there was overlapping between them. One group corres-
ponded to cultures that contained 01 ng ml)1 of nata-
mycin in the medium and the other to cultures where
natamycin concentrations were 120 ng ml)1. The con-
centration of this fungicide negatively influenced OTA
accumulation so that the higher the natamycin level, the
lower the OTA accumulation in the medium.
The temperature split the data into two homogeneous
groups; one for 25C and 15C and the other for 20C. It
should be noted that optimum growth of A. carbonarius
occurs at 3035C, while OTA production is optimum at
20C (Mitchell et al. 2004). Because of this, a higher con-
centration of natamycin was required at this temperature.
Thus, at 15C, 10 ng ml)1 was required to inhibit OTA
production by >90% at 098 aw. However, at 096 and
094 aw, almost complete inhibition occurred. In contrast,
at 20C, where the highest OTA levels were produced,
inhibition was statistically significant at 10 ng ml)1 nata-
mycin at 094 aw. At 096 and 098 aw, some inhibition
occurred at 510 ng ml)1, but greater concentrations
would be required for effective inhibition. At 25C,
5 ng ml)1 was effective at all aw levels. This suggests that
under optimum temperature and aw conditions for OTA
production, >10 ng ml)1 natamycin was required. How-
ever, at 15C and 25C and over a wide range of aw
levels, natamycin effectively controlled OTA production.
Discussion
The use of natamycin to control A. carbonarius growth
and OTA production has not been previously tested. This
makes comparison of our results with those of other
authors very difficult. Natamycin is a polyene and the
toxicity of polyenes to fungal cells is thought to occur as
a result of their binding to sterols in cell membranes, thus
changing the membranes physical state and impairing
membrane functions, resulting in enhanced permeability
to protons and leakage of internal constituents such as
K+, Ca2+ and PO43) (Bolard 1986; Vanden Bossche et al.
1987; Van den Bossche et al. 1995).
Although natamycin is a natural substance with
efficacy against pathogenic fungi of animals and plants,
and important in the food industry as a preservative to
2238 Journal compilation 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 22342239
A. Medina et al.
prevent mould contamination of foods, especially in
cheese, its relative efficiency in controlling growth and
OTA production on A. carbonarius isolates under differ-
ent aw temperature conditions had not been previously
tested.
The use of other fungicides of chemical origin such as
thiabendazole, carbendazim, carboxincaptan and mycon-
azole on growth of A. flavus and A. ochraceus and afla-
toxin and OTA production has been studied by other
authors (Rama Devi and Polasa 1984; Gabal 1987). In
some cases, toxin production was controlled with or
without affecting fungus growth (Rama Devi and Polasa
1984; Gabal 1987). In other cases, the addition of these
agents increased the production of these secondary
metabolites (Buchanan et al. 1987; Badii and Moss 1988).
In our study, the presence of natamycin prevented
A. carbonarius growth when applied to the medium at a
concentration of 20 ng ml)1 and it has also been shown
that natamycin is able to control OTA production as well.
When OTA production was optimum (20C) and lower
concentrations of natamycin were used, both aw and tem-
perature modulated the effect of natamycin. Our results
agree with those obtained by Battilani et al. (2003), who
found some fungicides able to reduce both growth and
OTA production. Natamycin has a powerful action at not
very high concentrations against typical OTA producers
such as A. carbonarius which has been identified as the
main fungus responsible for contaminating wine, grapes,
grape juice and vine fruits (Magan and Aldred 2005).
This study suggests that natamycin at 50100 ng ml)1 is
an effective potential compound for complete inhibition
of A. carbonarius growth and OTA production over a
range of environmental conditions in these and other
products which may be colonized and thus minimize
exposure of consumers to this important toxin.
Acknowledgements
The authors are grateful for the financial the Valencia
Regional Government (Conselleria dEmpresa, Universitat
i Ciencia) for financing a research grant.
References
Arroyo, M., Aldred, D. and Magan, N. (2005) Environmental
factors and weak organic acid interactions have differential
effects on control of growth and ochratoxin A production
by Penicillium verrucosum isolates in bread. Int J Food
Microbiol 98, 223231.
Badii, F. and Moss, M.O. (1988) The effect of the fungicides
tridemorph, fenpropimorph and fenarimol on growth and
aflatoxin production by Aspergillus parasiticus Speare. Lett
Appl Microbiol 7, 3739.
2007 The Authors
A. Medina et al.
Basilico, J.C., Debasilico, M.Z., Chiericatti, C. and Vinderola,
C.G. (2001) Characterization and control of thread mould
in cheese. Lett Appl Microbiol 32, 419423.
Battilani, P., Pietri, A., Bertuzzi, T., Formenti, S., Barbano, C.
and Languasco, L. (2003) Effect of fungicides on ochratox-
in producing black aspergilli. J Plant Pathol 85, 285.
Bolard, J. (1986) How do the polyene macrolide antibiotics
affect the cellular membrane properties? Biochim Biophys
Acta 864, 257304.
Bragulat, M.R., Abarca, M.L. and Cabanes, F.J. (2001) An easy
screening method for fungi producing ochratoxin A in
pure culture. Int J Food Microbiol 71, 139144.
Buchanan, R.L., Jones, S.B. and Stahl, H.G. (1987) Effect of
miconazole on growth and aflatoxin production by Asperg-
illus parasiticus. Mycopathologia 100, 135144.
Gabal, M.A. (1987) Preliminary study on the use of thiaben-
dazole in the control of common toxigenic fungi in grain
feed. Vet Hum Toxicol 29, 217221.
Magan, N. and Aldred, D. (2005) Conditions of formation of
ochratoxin A in drying, transport and in different com-
modities. Food Addit Contam 22, S10S16.
Magan, N., Arroyo, M. and Aldred, D. (2003) Mould preven-
tion in bread, Chap. 24. In Bread Making: Improving
Quality ed. Cauvain, S.P. Cambridge, UK: Woodhead
Publishing.
Magan, N. and Aldred, D. (2006) Managing microbial spoilage
in cereal and bakery products, Chap. 8. In Food Spoilage
Microorganisms ed. Blackburn, C.W. Cambridge, UK:
Woodhead Publishing.
Mitchell, D., Parra, R., Aldred, D. and Magan, N. (2004)
Water and temperature relations of growth and ochratoxin
A production by Aspergillus carbonarius strains from
grapes in Europe and Israel. J Appl Microbiol 97, 439445.
Raab, W.P. (1972) Natamycin (pimaricin) its Properties and Pos-
sibilities in Medicine. Stuttgart: George Thieme Publishers.
2007 The Authors
Journal compilation 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 22342239
Efficacy of natamycin and OTA production
Rama Devi, G. and Polasa, H. (1984) Inhibitory effects of
bavistin (carbendazim) on growth and mycotoxin
production by Aspergilli grown on maize. Pesticides 18,
3234.
Reps, A., Jedrychowski, L., Tomasik, J. and Wisniewska, K.
(2002) Natamycin in ripening cheeses. Pak J Nutrition 1,
243247.
Reuben, A., Anaissie, E., Nelson, P.E., Hashem, R., Legrand,
C., Ho, D.H. and Bodey, G.P. (1989) Antifungal suscepti-
bility of 44 clinical isolates of Fusarium species determined
by using a broth microdilution method. Antimicrob Agents
Chemother 33, 16471649.
Rotowa, N.A., Shadomy, H.J. and Shadomy, S. (1990) In vitro
activities of polyene and imidazole antifungal agents
against unusual opportunistic fungal pathogens. Mycoses
33, 203211.
de Ruig, W.G. and van den Berg, G. (1985) Influence of the
fungicides sorbate and natamycin in cheese coatings on
the quality of the cheese. Nederlands Instituut voor
Zuivelonderzoek.
Rusul, G. and Marth, E.H. (1988) Growth and aflatoxin pro-
duction by Aspergillus parasiticus in a medium at different
pH values and with or without pimaricin. Eur Food Res
Technol 187, 436439.
Stark, J. (2003) Natamycin: an effective fungicide for food and
beverages. In Natural Antimicrobials for the Minimal
Processing of Foods. ed. Roller, S. pp. 8297. Cambridge,
UK: Woodhead Publishing Limited.
Van den Bossche, H., Willemsens, G. and Marichal, P. (1987)
Anti-candida drugs the biochemical basis for their activ-
ity. Crit Rev Microbiol 15, 5772.
Van den Bossche, H., Koymans, L. and Moereels, H. (1995)
P450 inhibitors of use in medical treatment: focus
on mechanisms of action. Pharmacol Ther 67,
79100.
2239