This article was downloaded by: [Kakatiya University]

On: 01 October 2012, At: 05:52

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Archives Of Phytopathology And Plant

Protection
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/gapp20

Influence of carbon and nitrogen

sources on ochratoxin A production by
two species of Penicillium isolated from
poultry feeds
a

V. Koteswara Rao , M. Venkata Ramana , S. Girisham & S.

Madhusudhan Reddy

Department of Microbiology, Kakatiya University, Warangal,

Andhra Pradesh, India
b

Defence Food Research Laboratory, Division of Microbiology,

Mysore, Karnataka, India
Version of record first published: 31 Aug 2012.

To cite this article: V. Koteswara Rao, M. Venkata Ramana, S. Girisham & S. Madhusudhan Reddy
(2012): Influence of carbon and nitrogen sources on ochratoxin A production by two species of
Penicillium isolated from poultry feeds, Archives Of Phytopathology And Plant Protection, 45:16,
1917-1927
To link to this article: http://dx.doi.org/10.1080/03235408.2012.718221

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-andconditions
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden.
The publisher does not give any warranty express or implied or make any representation
that the contents will be complete or accurate or up to date. The accuracy of any
instructions, formulae, and drug doses should be independently verified with primary
sources. The publisher shall not be liable for any loss, actions, claims, proceedings,

Downloaded by [Kakatiya University] at 05:52 01 October 2012

demand, or costs or damages whatsoever or howsoever caused arising directly or

indirectly in connection with or arising out of the use of this material.

Archives of Phytopathology and Plant Protection

Vol. 45, No. 16, October 2012, 19171927

Inuence of carbon and nitrogen sources on ochratoxin A production

by two species of Penicillium isolated from poultry feeds
V. Koteswara Raoa*, M. Venkata Ramanab, S. Girishama and
S. Madhusudhan Reddya

Downloaded by [Kakatiya University] at 05:52 01 October 2012

a
Department of Microbiology, Kakatiya University, Warangal, Andhra Pradesh, India; bDefence
Food Research Laboratory, Division of Microbiology, Mysore, Karnataka, India

(Received 26 April 2012; nal version received 3 May 2012)

Mycotoxins are natural, secondary metabolites produced by fungi on agricultural
commodities in the eld and during storage under a wide range of climatic conditions. The ochratoxin A (OTA) is a nephrotoxic mycotoxin produced by several
species of Aspergillus and Penicillium. In this study, the inuence of carbon and
nitrogen sources on ochratoxigenic Penicillium species was assessed. The ochratoxigenic Penicillium species were isolated from poultry feed samples of Andhra
Pradesh, India. The isolated ochratoxigenic Penicillium species were identied,
screened and characterised as OTA producers by high performance thin layer
chromatography (HPTLC) and conrmed by high performance liquid chromatography (HPLC). This experiment was carried out using Czapak yeast Autolysate
(CYA) medium with dierent carbon (C) and nitrogen (N) sources at pH 6.5 and
incubated at 25 + 28C under dark condition. Maximum OTA production was
recorded in the presence of D-glucose followed by D-galactose and D-lactose as
carbon sources. Similarly, the maximum amount of OTA production was observed
in thiourea followed by potassium nitrate as nitrogen source. However, OTA
production, nal pH of the medium, and mycelial yield and OTA production of
both the species of Penicillium varied with C and N present in the medium. The
kinetics of the both species of Penicillium was observed for 30 days at an interval of
three days. The maximum amount of OTA was detected by 12 and 15 days incubation periods for Penicillium nordicum and Penicillium verrucosum, respectively.
Keywords: poultry feed; Penicillium verrucosum; P. nordicum; HPTLC; carbon;
nitrogen sources; ochratoxin A; HPLC

Introduction
The major species of fungi responsible for ochratoxin A (OTA) production in
dierent agricultural commodities by moulds Penicillium and Aspergillus, especially
the genus Penicillium are Penicillium verrucosum and Penicillium nordicum.
Ochratoxin A can be found in cereal grains and a variety of food products such
as coee or grapes. Research has shown that OTA can cause health problems in
humans and animals which include cancer, immunosuppressive disorders and
teratogenic disorders (IARC 1993; Schlatter et al. 1996). Ochratoxin A contamination in food and feeds was until recently believed to be produced only by Aspergillus
ochraceus and P. verrucosum, which aect mainly dried stored foods and cereals,
*Corresponding author. Email: koteswararaovankudoth@gmail.com
ISSN 0323-5408 print/ISSN 1477-2906 online
2012 Taylor & Francis
http://dx.doi.org/10.1080/03235408.2012.718221
http://www.tandfonline.com

Downloaded by [Kakatiya University] at 05:52 01 October 2012

1918

V. Koteswara Rao et al.

respectively, in dierent regions of the world. However, some recent surveys have
clearly shown that certain species belonging to the black aspergilli and Penicillium are
the sources of OTA in food commodities and poultry feeds worldwide (Abarca et al.
2003; Rosa et al. 2006; Fraga et al. 2007; Heperkan et al. 2009). The composition of
the nutrient medium and other extrinsic factors such as temperature, water activity
(aw) and oxygen are the important factors governing the growth and mycotoxin
production by fungi (Filtenborg et al. 2000). Penicillium nordicum and P. verrucosum
are regarded as food-borne mycotoxigenic fungal species of great importance in the
temperate regions of the world (Frisvad and Thrane 2000). Although it is a recognised
fact that the mycotoxin prole of a fungal species is dependent on the growth media
and environmental conditions, very limited information is available on OTA
production by two species of Penicillium. Competitive endogenous oras of foods
are also reported to inuence OTA production (Marquardt and Frohlich 1992).
Therefore, the determination of the environmental domain and food commodities
that support OTA production by above two species was considered to be great
signicant for food safety. Several studies have been carried out to estimate the
combined eect of OTA production by P. verrucosum and P. nordicum on culture
media. Garies and Garies (2007) and Munoz et al. (2011) have studied the eect of
water activity and incubation temperature. In this study, we have examined the
inuence of carbon and nitrogen source on growth and OTA accumulation by two
ochratoxigenic isolates of P. verrucosum and P. nordicum associated with poultry
feeds. The isolates were assessed with the help of high performance liquid
chromatography (HPLC) and the results are discussed.
Materials and methods
Standards and reagents
Acetonitrile, acetic acid, water, HPLC grade and reference compound OTA (OTA
purity 98%) were obtained from Sigma Aldrich. Toluene, ethyl acetate, formic acid,
chloroform, o-phosphoric acid, sodium hydroxide, all the media, carbon and
nitrogen sources were purchased from Merck manufacturers.
Media
A basal medium containing sucrose (30 g/l), NaNO3 (3 g/l), K2HPO4
3H2O (1.3 g/
l), MgSO4.7H2O (0.5 g/l), KCl (0.5 g/l), FeSO4.7H2O (0.01 g/l), ZnSO4 (0.01 g/l);
CuSO4 (0.005 g/l); yeast extract (10 g/l) was used; pH was adjusted with 1 M
phosphoric acid/0.1 N sodium hydroxide.
Isolation and identication of ochratoxigenic Penicillium species
Poultry feed samples from dierent poultry farms located in dierent geographical
regions of Andhra Pradesh, India were analysed for the presence of Penicillium spp.,
by dilution plate technique (Waksman 1922) using Czapek Yeast Autolysate (CYA)
agar medium (Pitt 1979) and incubated at 25 + 28C for 57 days under dark
condition. The plates were examined and colonies of Penicillium spp. were picked
and transferred to malt extract agar slants for further identication. The subgenus
Penicillium species were identied by a key proposed by Samson and Pitt (1990) and
a previous report by Koteswara Rao et al. (2011).

Archives of Phytopathology and Plant Protection

1919

Downloaded by [Kakatiya University] at 05:52 01 October 2012

HPTLC analysis
Fungal isolates were characterised as OTA producers as reported by Koteswara Rao
et al. (2011) and conrmed by high performance thin layer chromatography
(HPTLC) (Vega and Castillo 2006) with minor modications. Single spore cultures
of Penicillium species were inoculated into CYA medium and incubated at 25 + 28C
on rotary shaker at 150 rpm for toxin production. At the end of incubation period,
the biomass was separated by sterile lter. Twenty-ve millilitre of chloroform was
added to 25 ml of culture ltrate, shaken properly and extracted twice with 25 ml of
chloroform. After extraction, the chloroform was evaporated by rotary evaporator
and the compound was resuspended in 0.5 ml of chloroform. These extracts were
used for toxin detection by HPTLC. Chromatography was carried out on
10 6 10 cm precoated silica gel HPTLC plates (Merck). Test samples and
standards were applied with automatic TLC sampler (ATS III) from CAMAG
(Muttenz, Switzerland), using the spray-on technique with the following setting:
band length 3 mm; rst application X-axis 14 mm, Y-axis 9 mm for 20 tracks
per plate. The standard volumes applied were between 0.25 and 3 ml and the
sample volumes were adjusted to be always near to 10 ml per band. The
chromatography was carried out in a 20 6 10 cm twin trough chamber
(CAMAG) with a 50-mm-run length using toluene:ethyl acetate:formic acid
(6:3:1 v/v/v) solvent system. Fluorescence detection was carried out by postchromatographic derivatisation with 10% aluminium chloride in methanolwater
mixture, 2, 4-Dinitrophenylhydrazine (2, 4-DNP), FeCl3 and CeSo4. The plate
was immersed in the derivatisation solution using a dipping device III (CAMAG)
and heated for 20 min at 1108C. The plate was then scanned at 333/4400 nm
with a densitometer TLC scanner 3 (CAMAG) using a slit dimension of
5.0 6 0.5 mm and scanning speed of 40 mm/s.
Optimisation of carbon source for OTA production
The sucrose of the medium was replaced by dierent C sources, (D-glucose, Dmaltose, D-fructose, D-mannose, D-galactose, D-xylose, D-mannitol, D-lactose, citric
acid, tartaric acid, succinic acid and tannic acid) so as to supply same amount of
carbon and sucrose served as control. The pH was adjusted to 6.5 and the nal
volume of medium in each 250-ml Ehrlenmeyer ask was 100 ml. The medium was
inoculated with seven-days-old fungal culture and incubated at 25 + 28C for 12 and
15 days for P. verrucosum and P. nordicum, respectively, and the amount of OTA
produced was estimated with the help of HPLC.
Optimisation of nitrogen source for OTA production
Sodium nitrate of the medium was replaced by dierent inorganic N sources
(ammonium chloride, ammonium molybdate, ammonium nitrate, ammonium
sulphate, potassium nitrate and thiourea) and organic N sources (L-glutamic acid,
L-glycine, L-alanine, L-arginine, L-asparagine, L-asparticacid, L-cystine, L-histidine, Lleucine, L-lysin, L-tyrosin, L-tryptophan, p-amino benzoic acid, L-methionine and
urea) and sodium nitrate alone served as control. The pH was adjusted to 6.5 and the
nal volume of medium in 250-ml Erlenmeyer asks was 100 ml. The asks were
inoculated and incubated as indicated earlier and analysed for the amount of OTA
produced.

1920

V. Koteswara Rao et al.

Analysis of OTA production

Downloaded by [Kakatiya University] at 05:52 01 October 2012

At the end of incubation period, the asks were harvested on Whatman No.42 lter
paper for assessing biomass production, while the ltrate was employed for the
extraction of OTA with chloroform twice. Water phase was discarded and the
organic phase was collected and concentrated by evaporation. The residue was
redissolved in 1 ml of acetonitrile and 20 ml of extract was injected into the HPLC
system for the determination of OTA.
The standard OTA (100 mg) was dissolved in 1 ml of acetonitrile and dierent
concentrations (5, 10, 15, 20, 25, 30, 35 and 40 mg/ml) were made for the preparation
of a standard graph (Figure 1).
HPLC system
The HPLC analysis was performed using Jasco 975 model (Japan) by injecting 20 ml
of extract. The C18 isocratic reversed-phase column Luna (250 6 4.6 mm internal
diameter, 5 mM particle size) was used for the analysis of OTA. The mobile phase
composition was acetonitrile:water:acetic acid (99:99:2 v/v/v), the degassing was
adjusted using bath sonicator (Elma Ultrasonicator, Germany) and the ow rate was
1 ml/min. The analysate used for OTA using UV detector at 333 nm and the column
was kept at 25 + 28C.
Kinetics of OTA production
To study the dynamics of OTA production by two species of Penicillium, CYA
medium was selected and supplemented with yeast extract (1%). Four hundred
millilitres of the medium was taken in a 1000 ml Erlenmeyer ask and inoculated
with both the species of Penicillium and incubated at 25 + 28C for 30 days. The
amount of OTA production was assessed on 3, 6, 9, 12, 15, 18, 21, 24, 27 and 30 days
of incubation by employing 25 ml culture ltrate.
Results
Inuence of C source on OTA production
Inuence of dierent C sources on growth and OTA production by P. verrucosum
and P. nordicum was studied and the results were presented (Table 1). Table 1 reveals

Figure 1.

Preparation of standard graph of OTA under HPLC.

Downloaded by [Kakatiya University] at 05:52 01 October 2012

Archives of Phytopathology and Plant Protection

1921

a denite inuence on OTA production and growth of both the species of Penicillium
under study. Penicillium verrucosum produced maximum OTA when sucrose was
served as C source followed by D-glucose, D-galactose and D-mannitol, while least
OTA production was recorded on citric acid followed by succinic acid, tartaric acid,
tannic acid and D-xylose, but the rest of C sources favoured the intermediate
amount of OTA production. The OTA production was low on all carbon sources
tried in comparison to that produced on control (sucrose). The nal pH of the broth
recorded was near neutral in the medium containing tannic acid, D-mannose and Dmannitol, while it was acidic in rest of the carbon sources tried. D-Glucose, Dgalactose, D-mannitol and D-mannose supported maximum growth of P. verrucosum,
while succinic acid followed by citric acid, tartaric acid and tannic acid supported
least mycelial growth. The rest of the C sources supported intermediate degree of
mycelial growth.
Penicillium nordicum produced maximum amount of OTA during its growth on
D-glucose followed by D-galactose, while in the medium containing succinic acid
followed by tannic acid, citric acid and tartaric acid, OTA production was
intermediate (Table 2). The nal pH of the broth was near neutral in tannic acid
followed by D-mannose, while it was acidic in rest of the C sources tried. Maximum
mycelial growth of P. nordicum was recorded on D-galactose followed by mannitol
and maltose, while citric acid, tartaric acid and tannic acid supported least amount
of biomass of P. nordicum. The rest of the carbon sources supported intermediate
amount of mycelial growth.
Inuence of nitrogen source on OTA production
The inuence of N sources on growth and OTA production by P. verrucosum and P.
nordicum was also studied by replacing sodium nitrate of the medium with dierent
N source so as to supply same amount nitrogen and the results were presented.
Penicillium verrucosum produced maximum OTA on thiourea followed by L-glycine,
L-glutamic acid, L-asparagine and L-alanine, while on inorganic N sources such as

Table 1. Inuence of carbon sources on OTA production by Penicillium verrucosum isolated

from poultry feeds.

Name of the carbon source

Sucrose (control)
D-Glucose
D-Fructose
D-Mannose
Galactose
D-Xylose
Mannitol
Lactose
Maltose
Citric acid
Succinic acid
Tartaric acid
Tannic acid

Final pH

Dry weight (mg/ml)

OTA (mg/ml)

X + SD

X + SD

X + SD

4.88
4.90
4.88
4.44
4.52
4.67
4.86
4.66
4.46
4.73
4.99
4.65
5.55

+
+
+
+
+
+
+
+
+
+
+
+
+

0.45
48
0.61
0.29
0.45
0.67
0.36
0.45
0.13
0.57
0.44
0.02
0.50

5.75
6.91
5.61
6.74
6.28
5.97
6.19
4.62
5.66
3.48
2.78
3.30
3.63

+
+
+
+
+
+
+
+
+
+
+
+
+

Results are mean of duplicates; X + SD: mean + standard deviation.

0.15
0.77
0.38
0.57
0.23
0.52
0.32
0.41
0.63
0.09
0.50
0.51
0.09

22.03
19.07
08.40
12.12
17.10
07.10
13.57
14.21
10.10
01.87
01.15
01.19
01.27

+
+
+
+
+
+
+
+
+
+
+
+
+

3.77
3.86
3.95
1.80
1.40
1.12
1.59
1.42
1.11
1.08
0.34
0.28
0.52

1922

V. Koteswara Rao et al.

Table 2. Inuence of carbon sources on OTA production by Penicillium nordicum isolated

from poultry feeds.

Downloaded by [Kakatiya University] at 05:52 01 October 2012

Name of the carbon source

Sucrose (control)
D-Glucose
D-Fructose
D-Mannose
Galactose
D-Xylose
Mannitol
Lactose
Maltose
Citric acid
Succinic acid
Tartaric acid
Tannic acid

Final pH

Dry weight (mg/ml)

OTA (mg/ml)

X + SD

X + SD

X + SD

4.33
4.94
4.44
5.28
4.74
4.38
4.54
4.60
4.47
4.36
4.27
4.86
5.36

+
+
+
+
+
+
+
+
+
+
+
+
+

0.30
0.41
0.29
0.10
0.31
0.37
0.31
0.05
0.53
0.22
0.07
0.36
0.10

6.23
5.16
6.41
4.78
6.91
5.78
6.59
5.85
6.35
2.90
3.68
4.27
3.86

+
+
+
+
+
+
+
+
+
+
+
+
+

0.41
0.38
0.67
0.51
0.61
0.28
0.59
0.38
0.30
0.77
0.17
0.99
0.36

21.47
18.82
05.81
08.34
19.83
14.93
12.32
15.31
13.72
02.92
00.96
02.07
2.30

+
+
+
+
+
+
+
+
+
+
+
+
+

1.88
0.68
1.01
0.64
0.03
0.61
0.01
1.53
1.95
0.74
0.65
0.88
0.71

Results are mean of duplicates; X + SD: mean + standard deviation.

zirconyl nitrate and bismuth nitrate supported minimal fungal growth and trace
amount of OTA production (Table 3). The other inorganic sources such as
ammonium chloride, ammonium molybdate, ammonium sulphate, potassium nitrate
and organic N sources such as L-arginine, L-asparatic acid, L-cystine, L-histidine and
L-lysine were of intermediate nutritive value for the production of OTA. The nal
pH of the broth was near neutral in media containing urea and zirconyl nitrate, while
it was acidic in the rest of the N sources tried. The maximum dry weight of P.
verrucosum was recorded on L-tyrosine as N source followed by ammonium
molybdate, L-glutamic acid potassium nitrate and thiourea. On the other hand,
zirconyl nitrate, p-amino benzoic acid and bismuth nitrate supported the least
mycelial growth. The rest of the N sources tried were of intermediate nutritive value
for the growth and OTA production.
Penicillium nordicum produced maximum OTA in the medium containing
potassium nitrate followed by thiourea, L-leucine, L-lysine, L-asparatic acid and Lglycine, while the rest of the N sources supported the least amount OTA production
(Table 4). Ammonium chloride, p-amino benzoic acid, bismuth nitrate and zirconyl
nitrate were unfavourable for the production of OTA. The nal pH of the broth
recorded was near neutral in the medium containing zirconyl nitrate, L-asparagine, Lalanine, ammonium molybdate, urea and L-glutamic acid. Penicillium nordicum
achieved maximum biomass on L-cystine followed by bismuth nitrate, ammonium
chloride, L-histidine and ammonium molybdate. Inorganic salts were comparatively
good N source for P. verrucosum, while for P. nordicum, they were interior sources of
N for OTA production.
Kinetics of OTA production
To perform this experiment, yeast extract in combination with sodium nitrate and
sucrose was chosen. In general, more OTA was produced by both the species of
Penicillium under study. OTA production by each species of Penicillium over a

Archives of Phytopathology and Plant Protection

1923

Table 3. Inuence of nitrogen sources on OTA production by Penicillium verrucosum

isolated from poultry feeds.

Downloaded by [Kakatiya University] at 05:52 01 October 2012

Name of the nitrogen source

Sodium nitrate (control)
Ammonium chloride
Ammonium molybdate
Ammonium nitrate
Ammonium sulphate
L-Glutamic acid
L-Glycine
L-Alanine
L-Arginine
L-Asparagine
L-Aspartic acid
L-Cystine
L-Histidine
L-Lysine
L-Leucine
L-Tryptophan
L-Tyrosine
L-Methionine
p-Aminobenzoic acid
Potassium nitrate
Thiourea
Urea
Zirconyl nitrate
Bismuth nitrate

Final pH

Dry weight (mg/ml)

OTA (mg/ml)

X + SD

X + SD

X + SD

4.56
4.54
3.93
3.55
3.69
4.29
4.22
3.52
2.80
4.35
4.38
4.47
4.26
4.68
3.33
3.52
3.01
2.79
4.48
3.64
3.58
5.69
5.19
2.72

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

0.47
0.31
0.39
0.42
0.07
0.11
0.14
0.10
0.43
0.21
0.40
0.04
0.15
0.45
0.17
0.10
0.15
0.26
0.25
0.19
0.02
0.41
0.12
0.17

6.57
6.99
7.58
5.49
6.27
7.22
6.51
7.23
6.72
6.47
6.68
6.75
7.08
6.48
5.35
6.58
8.46
6.74
1.21
7.87
7.92
5.49
0.74
2.38

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

0.29
0.68
0.46
0.53
0.22
0.25
0.40
0.17
0.27
0.32
0.32
0.02
0.27
0.76
0.01
0.19
1.27
0.27
0.01
0.77
0.71
0.44
0.41
0.38

16.47
04.18
12.76
12.08
04.73
11.37
12.54
10.80
01.87
07.84
12.87
10.64
11.13
11.92
05.89
06.47
06.20
02.17
01.21
02.14
20.40
04.00
00.96
01.09

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

1.59
0.87
2.17
0.30
0.51
1.46
1.56
0.79
0.89
0.65
0.74
1.77
0.94
0.96
0.70
1.44
0.13
0.32
0.28
1.14
1.60
3.24
0.52
0.22

Results are mean of duplicates; X + SD: mean + standard deviation.

period of 30 days is shown in Figures 2 and 3, where it is clear that the kinetics of
OTA production varied with the species. Penicillium verrucosum produced maximum
OTA on the third day of incubation, while P. nordicum did the same on the sixth day.
The dynamics of OTA production by Penicillium species were quite similar but
diered in the dynamics of its production. Ochratoxin A maximum production was
recorded on the twelfth and fteenth days by P. verrucosum and P. nordicum,
respectively. In both the cases, OTA progressively increased with the adjacent
incubation period. In general, the OTA production was higher in P. verrucosum
when compared to P. nordicum at dierent incubation periods (Chromatogram of
OTA through HPLC; Figure 4).
Discussion
Although the incidence of P. verrucosum and P. nordicum was high in poultry feeds
(Koteswara Rao et al. 2011), their OTA-producing capacity was high, among isolates
of Penicillium on other substrates such as cereals (Mateo et al. 2004), roasted coee
(Patel et al. 1997), soluble coee (Pittet et al. 1996), grapes (Medina et al. 2008), cattle
feeds, (Rosa et al. 2007), rabbit feed (Rosa et al. 2006) and pig feed (Fraga et al. 2007).
Ferreira and Pitout (1969) have recorded highest yield of OTA on sucrose and
considerably low on D-glucose; these results are in agreement with our observations
on P. nordicum and P. verrucosum. However, when D-lactose or D-fructose were

1924

V. Koteswara Rao et al.

Table 4. Inuence of nitrogen sources on OTA production by Penicillium nordicum isolated

from poultry feeds.

Downloaded by [Kakatiya University] at 05:52 01 October 2012

Name of the nitrogen source

Sodium nitrate (control)
Ammonium chloride
Ammonium molybdate
Ammonium nitrate
Ammonium sulphate
L-Glutamic acid
L-Glycine
L-Alanine
L-Arginine
L-Asparagine
L-Aspartic acid
L-Cystine
L-Histidine
L-Lysine
L-Leucine
L-Tryptophan
L-Tyrosine
L-Methionine
p-Aminobenzoic acid
Potassium nitrate
Thiourea
Urea
Zirconyl nitrate
Bismuth nitrate

Final pH

Dry weight (mg/ml)

OTA (mg/ml)

X + SD

X + SD

X + SD

5.16
4.44
5.16
4.35
4.43
4.72
5.41
5.32
5.79
4.67
4.47
3.91
4.36
4.47
4.27
4.95
4.46
4.67
4.61
4.22
4.09
5.51
4.33
4.17

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

0.72
0.16
0.07
0.00
0.26
0.72
0.38
0.02
0.26
0.24
0.24
0.57
0.22
0.24
0.24
0.58
0.30
0.62
0.07
0.14
0.79
0.52
0.02
0.07

7.17
7.67
6.82
7.21
5.92
7.42
6.19
6.67
6.15
6.13
6.57
8.07
7.27
6.01
5.62
6.25
5.98
6.24
1.99
6.61
4.72
6.82
1.23
7.27

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

0.67
0.60
0.82
0.02
0.45
0.24
0.29
0.18
0.06
0.37
0.24
0.60
0.88
0.46
0.10
0.20
0.16
0.36
0.91
0.41
0.87
0.67
0.16
1.31

13.32
01.29
02.93
02.30
01.73
07.74
13.24
10.41
06.35
06.70
11.87
05.12
12.12
08.97
13.08
12.22
10.33
04.99
00.90
20.92
15.29
03.85
00.14
00.50

+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+

1.72
0.07
0.37
1.56
0.19
0.50
1.61
1.43
1.35
0.55
0.88
1.09
1.52
0.89
2.14
1.44
1.36
0.85
0.47
2.33
1.28
0.91
0.14
0.28

Results are mean of duplicates; X + SD: mean + standard deviation.

Figure 2.

Kinetics of OTA production in Penicillium verrucosum in dierent incubation days.

served as C sources, no detection of OTA was recorded; these results are in

disagreement with Ferreira and Pitout (1969) studied on two species of Aspergillus.
This may be attributed to the preference of Penicillium species for carbon sources. In
most of the cases, a positive correlation was observed between OTA and biomass
production by both the species under investigation. Penicillium verrucosum preferred
L-glutamic acid, while for P. nordicum inferior as observed (Ferreira 1967) for

Downloaded by [Kakatiya University] at 05:52 01 October 2012

Archives of Phytopathology and Plant Protection

1925

Figure 3.

Kinetics of OTA production in Penicillium nordicum in dierent incubation days.

Figure 4.

HPLC detection of OTA extracted from Penicillium species.

A. ochraceus. Further, our observations are in agreement with Davis et al. (1969)
who recorded increased production of OTA by A. ochraceus.
Inhibition of Penicillium nordicum for OTA production by KNO3 and D-glucose
was studied by Farber and Geisen (2004), but in our investigation P. verrucosum
produced a good amount of OTA, while P. nordicum produced only limited amount
when potassium nitrate was used as an N source and sucrose was used as a C source.

1926

V. Koteswara Rao et al.

Downloaded by [Kakatiya University] at 05:52 01 October 2012

Two species of Penicillium under investigation diered signicantly in their degree of

OTA production. Similarly, Rosa et al. (2007) reported that A. ochraceus and P.
verrucosum produced maximum amount of OTA on yeast extract sucrose medium
followed by Aspergillus carbonarius and Aspergillus niger.
The kinetics observation of OTA production by two species of Penicillium is
dierent (Llorens et al. 2004). In the present investigations, P. verrucosum produced
maximum amount of OTA on the third day, whilst on the sixth day for P. nordicum.
These results are in agreement with Medina et al. (2008) reports on OTA production
by Aspergillus tubingensis. Signicant dierences were observed in OTA production
not only between the isolates of dierent species but also between the isolates of
same species, which may be attributed to the gene expression or degradation by the
fungi (Bejaoui et al. 2006; Medina et al. 2008).
Conclusion
High levels of OTA found in many poultry industries reveal the need for better
surveillance and monitoring by policy makers or food toxicologists and physicians to
reduce the exposure of human and animals to this toxic compound produced by P.
verrucosum and P. nordicum. It is essential that suitable management strategies with
good post-harvest practices should be developed to avoid fungal infestation.
Observation on OTA production under the inuence of dierent C and N sources
may be useful in determining dierences in gene expression in response to C and N
sources and identifying biosynthetic and regulatory genes for OTA production.
Altering of these conditions can be applied to food industries for the safety of food.
Acknowledgements
This research has been nancially supported by UGC.F .N.36-129/2008 (SR) MRP, New
Delhi and the authors are grateful for all the facilities provided by the Head of the Department
of Microbiology, Kakatiya University. The authors also thank Dr H.V. Batra (Additional
Director) and Dr H.S. Murali (HOD, Microbiology), Defence Food Research Laboratory,
Mysore, Karnataka, India, for their valuable support and suggestions.

References
Abarca ML, Accensi F, Bragulat MR, Castella G, Cabanes FJ. 2003. Aspergillus carbonarius
as the main source of ochratoxin A contamination in dried vine fruits from the Spanish
market. J Food Prot. 66:504506.
Bejaoui H, Mathieu F, Taillandier P, Lebrihi A. 2006. Biodegradation of ochratoxin A by
Aspergillus section nigri species isolated from French grapes: a potential means of ochratoxin
A decontamination in grape juices and musts. FEMS Microbiol Lett. 5:203208.
Davis ND, Searcy JW, Diener UL. 1969. Production of ochratoxin A by Aspergillus ochraceus
in a semi synthetic medium. Appl Microbiol. 17:742744.
Farber P, Geisen R. 2004. Analysis of dierentially expressed ochratoxin A biosynthesis genes
of Penicillium nordicum. Eur J Plant Pathol. 110:661669.
Ferreira NP. 1967. Recent advances in research on ochratoxin. In: Mateles RI, Wogen GN,
editors. Biochemistry of some food borne microbial toxins. Cambridge (MA): MIT Press.
p. 157168.
Ferreira NP, Pitout MJ. 1969. The biosynthesis of ochratoxin. J South Afr Chem Inst. 22:S1.
Filtenborg O, Frisvad TC, Samson RA. 2000. Specic association of fungi to foods and
inuence of physical environmental factors. In: Samson RA, Hoekstra ES, Frisvad JC,
Filtenborg O, editors. Introduction to food- and airborne fungi. Utrecht: Centraalbureau
voor Schimmelcultures. p. 306320.

Downloaded by [Kakatiya University] at 05:52 01 October 2012

Archives of Phytopathology and Plant Protection

1927

Fraga ME, Curvello F, Gatti MJ, Cavaglieri LR, Dalcero AM, Rosa CAR. 2007. Potential
aatoxins and ochratoxin A production by Aspergillus species in poultry feed processing.
Vet Res Commun. 31:343353.
Frisvad JC, Thrane U. 2000. Mycotoxin production by common lamentous fungi. In:
Samson RA, Hoekstra ES, Frisvad JC, Filtenborg O, editors. Introduction to food- and
air-borne fungi. Utrecht: Centraalbureau voor Schimmelcultures. p. 321331.
Garies M, Garies EM. 2007. Guttation droplets of Penicillium nordicum and Penicillium
verrucosum contain high concentration of ochratoxin A and B. Mycopathologia. 163:207
214.
Heperkan D, Dazkir DS, Kansu DZ, Guler FK. 2009. Inuence of temperature on citrinin
accumulation by Penicillium citrinum and Penicillium verrucosum in black table olives.
Toxin Rev. 8:180186.
International Agency for Research on Cancer (IARC). 1993. Monographs on the evaluation
of carcinogenic risks to humans, some naturally occurring substances: food items and
constituents, heterocyclic aromatic amines and mycotoxins (vol. 56). Lyon (France):
World Health Organization. p. 489521.
Koteswara Rao V, Shilpa P, Girisham S, Reddy SM. 2011. Incidence of mycotoxigenic
Penicillia in feeds of Andhra Pradesh, India. Int J Mol Biol Biotechnol. 2:4650.
Llorens A, Mateo R, Hinojo MJ, Valle-Algarra FM, Jimenez M. 2004. Inuence of
environmental factors on the biosynthesis of type B trichothecenes by isolates of Fusarium
spp. from Spanish crops. Int J Food Microbiol. 94:4354.
Marquardt RR, Frohlich AA. 1992. A review of recent advances in understanding
ochratoxicosis. J Anim Sci. 70:39683988.
Mateo R, Medina A, Gimeno-Adelantado JV, Jimenez M. 2004. An overview on the status of
toxigenic fungi and mycotoxins in Spain. In: Visconti A, Logrieco A, editors. Advances on
the occurrence of toxigenic fungi and mycotoxins in Europe. Dordrecht (the Netherlands):
Kluwer. p. 219235.
Medina A, Mateo EM, Valle Algarra FM, Mateo F, Mateo R, Jimenez M. 2008. Inuence of
nitrogen and carbon sources on the production of ochratoxin A by ochratoxigenic strains
of Aspergillus spp. isolated from grapes. Int J Food Microbiol. 122:9399.
Munoz, K, Vega M, Rios G, Geisen R, Degen GH. 2011. Mycotoxins production by dierent
ochratoxigenic Aspergillus and Penicillium species on coee and wheat based media.
Mycotoxin Res. 27:239247.
Patel S, Hazel CM, Winterton AGM and Gleadle AE. 1997. Survey of ochratoxin A in UK
retail coees. Food Addit Contam. 14:217222.
Pitt JL. 1979. The genus Penicillium and its teleomorphic states Eupenicillium and
Talaromyces. 1st ed. London: Academic Press. p. 634.
Pittet A, Tornare D, Huggett A, Viani R. 1996. Liquid chromatographic determination of
ochratoxin A in pure and adulterated soluble coee using an immuno anity column
cleanup procedure. J Agric Food Chem. 44:35643569.
Rosa CAR, Cavaglieri LR, Ribeiro JMM, Keller KM, Alonso VA, Chiacchiera MM, Dalcero
AM, Lopes CWG. 2007. Mycobiota ad naturally occurring ochratoxin A in dairy cattle
feed from Rio de Janeiro state, Brazil. World Mycotoxin. 1:195201.
Rosa CAR, Ribeiro JMM, Fraga ME, Gatti MJ, Cavaglieri LR, Magnoli CE, Dalcero AM,
Lopes CWG. 2006. Mycobiota and ochratoxin-producing ability by Aspergillus and
Penicillium species isolated from poultry feed in Brazil. Vet Microbiol. 113:8996.
Samson RA, Pitt JI. 1990. Modern concepts in Penicillium and Aspergillus classication.
New York (NY): Plenum Press.
Schlatter CH, Struder-Rohr J, Rasonyi TH. 1996. Carcinogenicity and kinetic aspects of
ochratoxin A. Food Addit Contam. 13:4344.
Vega M, Castillo D. 2006. Determination of deoxynivalenol in wheat by validated GC/ECD
method comparison with HPTLC/FLD. Electron J Food Plants Chem. 1:1620.
Waksman, SA. 1922. A method for counting the number of fungi in the soil. J Bot. 7:339341.