The occurrence of molds, yeasts and mycotoxins in pre-cooked pizza dough sold in Southern Rio Grande do Sul

Abstract

The quality of pre-cooked pizza dough was investigated by assessing the occurrence of molds, yeasts and mycotoxins. Random sampling of commercial pre-cooked pizza cakes was done in different stores in the cities of Rio Grande and Pelotas, RS, between 1996 and 1997. The products were analysed on the sampling day and after storage at room (22-30ºC) or refrigerated temperature (7ºC) following the shelf life stated by the manufacturer (25,30 and 45 days). The results showed that mold and yeast contamination was frequently above the maximum limits (10³CFU/g-1) established by Brazilian guide lines, even in samples kept at refrigerated temperatures up to the end of shelf life. Although no mycotoxin contamination was detected, a strain of the Penicillium genus, isolated from various samples, produced ochratoxin A at refrigeration temperatures.

Full text

Molds, yeasts and mycotoxins in pizza dough
99
* Corresponding author. Mailing address: Fundação Universidade Federal do Rio Grande, Laboratório de Micotoxinas, Rua Alfredo Huch, 475, CEP 96201-
900, Rio Grande, RS, Brasil.
Brazilian Journal of Microbiology (2000) 31:99-102
ISSN 1517-8382
THE OCCURRENCE OF MOLDS, YEASTS AND MYCOTOXINS IN PRE-COOKED PIZZA DOUGH
SOLD IN SOUTHERN RIO GRANDE DO SUL
Beatriz Helena Pinho
1*
; Eliana Badiale Furlong
2
1
Departamento de Patologia and
2
Departamento de Química, Fundação Universidade Federal do Rio Grande,
Rio Grande, RS, Brasil
Submitted: June 07, 1999; Returned to authors for corrections: September 29, 1999; Approved: June 26, 2000
ABSTRACT
The quality of pre-cooked pizza dough was investigated by assessing the occurrence of molds, yeasts and
mycotoxins. Random sampling of commercial pre-cooked pizza cakes was done in different stores in the
cities of Rio Grande and Pelotas, RS, between 1996 and 1997. The products were analysed on the sampling
day and after storage at room (22-30ºC) or refrigerated temperature (7ºC) following the shelf life stated by
the manufacturer (25,30 and 45 days). The results showed that mold and yeast contamination was frequently
above the maximum limits (10
3
CFU/g
-1
) established by Brazilian guide lines, even in samples kept at
refrigerated temperatures up to the end of shelf life. Although no mycotoxin contamination was detected, a
strain of the Penicillium genus, isolated from various samples, produced ochratoxin A at refrigeration
temperatures.
Key words: pre-cooked pizza dough, fungi, mycotoxins
INTRODUCTION
The bakery industry has been increasing and varying its
production during the last few years, and pizza has become very
popular. They are sold ready to use, frozen, chilled or pre-cooked
in many different commercial establishments. The large
consumption of this food is not only due to its flavor but also
because it is practical, easy to prepare, has high nutritional value
and is cheap.
The dough of pre-cooked pizzas, being a product based on
cereals and having intermediary moisture content, is a potential
substrate for fungal development, caused by contamination
occurring after baking during packaging and inappropriate
storage (13).
These fungi can grow and affect the nutritional and sensory
properties of the product and if the species are toxigenic, they
may produce mycotoxins (4). The occurrence of mycotoxins
has been observed worldwide in wheat, peanut, corn, beans and
grains, besides meat and milk. In addition, mycotoxins are not
completely eliminated by processing (2,6, 10).
The aflatoxins B
1
, B
2
, G
1
, G
2
and ochratoxin A are the most
frequent mycotoxins present in grains and foods because they
are produced by ubiquitous fungal genera like Aspergillus and
Penicillium. The cancerous and nephrological effects of these
toxins reinforce the need for a regular search for their occurrence.
Due to their peculiar blue and green fluorescence, they can be
detected by relatively simple techniques (10,12,19)
In the specific case of pre-cooked pizza doughs, wich are
baked, the fungi may be inactivated, even though they can
contaminate the food again after processing. The mycotoxins
may come from the raw material or be produced in the food
when contaminated during post baking handling (4, 10, 13).
The aim of the present work was to study the contamination
levels in pre-cooked pizza dough by an assessment of the molds
and yeasts present and the identification of the more frequent
genera present. The ocurrence of the following mycotoxins:
aflatoxins B
1
, B
2
, G
1
, G
2,
ochratoxin A and zearalenone was also
determined.

100
B.H. Pinho and E.B. Furlong
MATERIALS AND METHODS
Sampling
The pre-cooked pizza doughs were acquired from
commercial establishments in the cities of Pelotas and Rio
Grande in the State of Rio Grande do Sul, Brazil. The three
brands chosen had expiration dates within 25,30 and 45 days,
according to the manufacturers. Sample collection was
performed in the fall, winter and spring of the year of 1996 and
the summer of 1997.
Sets of 8 discs of pre-cooked pizza crusts were formed at
each sample collection for brands A, B and C.This procedure
was repeated three times for each brand comprising 9
experiments and totalizing 72 discs, which constituted the
analytical samples. The determinations on the samples were
performed at time zero (To), a period between the first and the
fifth day after manufacture, and after storage at refrigerated
temperature (RFT) (7ºC) and at room temperature (RT) (22-
30ºC) for up to two days before the end of the corresponding
shelf life for to each brand.
Mycological Determinations
The enumeration of molds and yeasts was performed after
using
pour plate technique in acidified potato dextrose agar (PDA)
according to A.P.H.A(1984). After enumeration, the most
frequent fungal colonies found in the pre-cooked pizza crusts
were isolated and kept in Sabouraud agar until the identification
procedures were completed.
The results of the enumeration using PDA were compared
with those obtained using a Petrifilm YM 3M in one experiment
(15).
The identification of the most frequent genera in PDA was
effected from the macroscopical and microscopical
characteristics of the colonies and microcultive (11, 15,16).
Determination of Aflatoxins B
1
, B
2
, G
1
, G
2,
ochratoxin A and
zearalenone
A thin layer chromatographic method (19) was used for the
simultaneous detection of aflatoxins B1, B2, G1, G2, ochratoxin
A and zearalenone. The toxins were extracted using methanol
and 4% KCl (9+1), followed by clarification with ammonium
sulfate and partition with chloroform. Confirmation was
accomplished by chemical reaction (8,17), elution with different
solvents and co-chromatography.
The determinations were performed on the pre-cooked pizza
cakes at time zero and after storage at refrigerated temperature
(7ºC) and at room temperature (22 - 30ºC) for up to two days
before the corresponding expiration date of each brand and on
the isolated fungal cultures, incubated in Sabourand agar under
the same conditions as the pizza doughs.
The detection limits of the method were 1.7; 6.5 and 60 ng.g
-1
for aflatoxins, ochratoxin A and zearalenone respectively (3).
RESULTS AND DISCUSSION
The enumeration of mold and yeast was done using a PDA
media because it is convenient for screening a large number of
species, and the observations can be made over an extended
period of time on a single colony. (3,7). This was observed during
previous studies where bread was used as a model (3).
As for Beuchat et al. (16) no differences between the
enumeration on Petrifilm and on PDA during the test experiment.
The latter was chosen because of its lower cost and the ease of
recovering fungi for identification procedures. The average
results of the enumeration of molds and yeasts on PDA for the
three studied brands are presented in Table 1.
Table 1: Enumeration of molds and yeasts for pre-cooked pizza dough before
and after being stored
Samples TO RFT RT
CFU.g
-1
CFU.g
-1
CFU.g
-1
A1 1.3X10
4
7.2X10
4
2.9X10
6
A2 1.6X10
4
1.4X10
4
2.5X10
6
A3 4.3X10 9.6X10
3
2.8X10
6
B1 2.0X10
2
2.7X10
6
6.8X10
3
B2 9.2X10
2
4.1X10
3
1.51X10
4
B3 1.2X10
2
4.7X10
4
9.5X10
4
C1 7.9X10 4.8X10
3
Mold
C2 1.1X10
2
1.6X10
4
1.2X10
4
C3 1.4X10
2
2.1X10 Mold
TO: zero time
RFT: refrigerated temperature (7ºC)
RT: room temperature (22-30ºC)
A, B, C: brands of pre-cooked pizza cakes
1, 2, 3: periods of sample collection
According to the Brazilian guideline, the maximum tolerated
level for molds and yeasts in baked products is 5x10
3
CFU.g
-1
(Ministery of Health, SVS portaria 451, 19/09/97). According
to DINAL (Divisão Nacional de Alimentos), another national
organization food control, the maximum level for molds and
yeasts is 10
4
CFU.g
-1
(5).
The most contaminated sample was
brand A, with respect to molds and yeasts, at the moment of
sampling.
All the brands of pre-cooked pizza doughs showed values
higher than the limits established by the national control boards
to molds and yeasts, after storage at room and refrigerated
temperatures. In general, the samples stored at refrigerated
temperatures showed lower values of CFU.g
-1
. This was due to
refrigeration temperatures slowing down the exogenous or
endogenous degradation reactions.
An interesting proposal was made by Rayman et al. (18),
for the adequacy of baked products. According to their criteria,

Molds, yeasts and mycotoxins in pizza dough
101
in terms of molds and yeasts, the limit between acceptable quality
and marginal quality is 2 x 10
3
CFU. g
-1
and the limit separating
marginal quality from unacceptable quality is 5 x 10
4
CFU. g
-1.
According to the above mentioned proposal (18) the pre-
cooked pizzas doughs, stored at room temperature, were not
recommended for consumption. Storage at refrigeration
temperature was not sufficiently effective to avoid the samples
being considered of marginal quality with respect to their molds
and yeasts counts.
The identification of the genera was effected from the
macroscopical and microscopical characteristics of the colonies
and the microcultive showed that the fungi belonging to the
genera Penicillium and Aspergillus were more frequent. Others
genera present were identified as Mucor sp, Geotrichium sp
and yeasts. These fungal genera are present in the environment
and easily contaminated food.
These results suggest that more care should be taken in the
handling of these products after they are baked, that storage
should be done under refrigeration and also that the established
shelf life period should be reviewed. Another aspect that might
be studied is the type of package to be recommended for this
kind of product. All these parameters determine the fungal
recontamination of baked and cooked products.
The study of mycotoxins in pre-cooked pizza doughs is
justified due to their thermostability, allowing for their presence
in the raw material used in the pizza dough formulation. Although
the fungi and their spores may be destroyed by the thermal
treatment during baking, the product can be recontaminated in
the next steps by toxigenic fungi present in the environment (3,10,
14). In these specific cases the probabiliy of mycotoxin occurrence
was high because the number of forming colony units was high.
During screening for aflatoxin (B
1
, B
2
, G
1
, G
2
), ochratoxin
A and zearalenone it was shown that 22% of the samples were
probably contaminated, although this was not confirmed during
the confirmatory test. These results imply that the raw material
used in the manufacture of the products did not present at
detectable levels of toxins after formulation. The high number
of samples with false positive results may be caused by
fluorescent components in the formulation or have been formed
during baked. We cant discard the possibility of contamination
by some other mycotoxins not researched here.
A previous survey of mycotoxins in pre formed loaves
conducted in our laboratory showed the occurrence of ochratoxin
A in moldy pre-form loaves during the shelf life period (3).
This suggested the possibility that an extended storage under
unsuitable conditions could lead to the occurence of mycotoxins
if there were toxigenic fungal species present.
Considering the above mentioned information, three types
fungal colonies frequently found in pre-cooked pizza dough,
belonging to the Penicillium genera and denominated I, II and
III, were submitted to a screening chemical test for toxigenicities
(14). The isolated colonies were incubated in Sabourand agar
under the same conditions (time and temperature) as the
commercial products. They were then submitted to the
multimethods proposed by Soares, taking care to follow the
methods specified to determine mycotoxins (19). The results
are presented in Table 2.
Table 2. Screening of mycotoxins in fungal colonies isolated from pre-cooked
pizza cakes.
Colony RFT RFT RT
(25 days) (45 days) (25 days)
I *ochratoxin A *ochratoxin A *ochratoxin A
II - Aflatoxins B
1
e B
2
-
III *ochratoxin A *ochratoxin A and -
Aflatoxins B
1
e B
2
I, II, III: Penicillium fungal colonies
RFT: refrigerated temperature (7ºC)
RT: room temperature (22-30ºC)
* confirmed presence of ochratoxin A
The choice of the incubation conditions was according to
the shelf life of the brand from which the colonies were isolated.
Only one colony incubated at room temperature producted
ochratoxin A after 25 days, colonies II and III produced the
toxins at refrigerated temperatures after 45 and 25 days
respectively. This is a characteristic of many toxigenic
Penicillium species mencioned by other authors (7, 10, 15).
Unfortunately the species was not identified in this work.
CONCLUSIONS
The number of colony forming units of molds and yeasts
increased during storage at the temperatures studied, being more
intense when the samples were stored at room temperature.
The storage of the pre-cooked pizza dough at refrigerated
temperatures was not effective in keeping the number of colony
forming units in the samples within the limits established by the
legislation during the shelf life period.
The samples of pizza dough were not contaminated with
aflatoxins B
1,
B
2,
G
1
, G
2
, ochratoxin A or zearalenone at the time
of sample collection or after storage during the shelf life.
Fungal colonies belonging to the Penicillium genus produced
ochratoxin A under the time and temperature conditions
established in this work.
RESUMO
Ocorrência de bolores, leveduras e micotoxinas em
massa de pizza pré-fabricada comercializada no
Rio Grande do Sul
A qualidade de massa de pizza pré-fabricada foi avaliada
através da determinação de bolores, leveduras e micotoxinas.

102
B.H. Pinho and E.B. Furlong
Entre 1996 e 1997, fez-se uma amostragem ao acaso de discos
de pizza pré-cozidos, em diferentes estabelecimentos comerciais
das cidades de Pelotas e Rio Grande, RS, Brasil. Os produtos
foram analisados no dia da amostragem e após o armazenamento
em temperatura ambiente (22-30
o
C) ou de refrigeração (7
o
C),
seguida o prazo de validade indicado pelo fabricante (20, 30 e
45 dias). Os resultados indicaram que contaminação por bolores
e leveduras estavam frequentemente acima dos limites (10
3
UFC/
g), estabelecidos pelos padrões brasileiros, mesmo nas amostras
mantidas em refrigeração. Embora nenhuma micotoxina tenha
sido detectada, uma cepa do gênero Penicillium, isolado de
várias amostras, produziu ocratoxina A em refrigeração.
Palavras-chave: massa de pizza pré-cozida, fungos,
micotoxinas
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