Search for
Staphylococcus
spp. in “Minas Frescal” cheeses made with raw milk and
commercialized in
the city of Formiga - MG
João Victor Ferreira Campos
1
, Luiza Camattari Resende
2
, Acácio Freire Bastos
3
, Mariana Oliveira Silva
4
,
Leonardo Borges
Acurcio5*
DOI: https://doi.org/10.35699/2447-6218.2021.32993
Abstract
“Minas Frescal” cheese is defined by legislation as a fresh cheese obtained by enzymatic coagulation of the milk with rennet
and/or other appropriate coagulant enzymes, supplemented or not with the action of specific lactic bacteria. Brazilian law
requires cheese to be made from pasteurized milk and prohibits marketing when made with raw milk, considering the risk of
being an important vehicle of pathogenic microorganisms. The objective of this work was to analyze the presence of
Staphylococcus spp. in artisanal “Minas Frescal” type cheeses made with raw milk and sold locally in the municipality of
Formiga, Minas Gerais. The microbiological standard for this microorganism is defined in technical regulation. Plating in
Mannitol Salt Agar was conducted in order to search for Staphylococcus spp. and differentiate S. aureus from non S. aureus.
Results obtained from analyzed samples (104 to 106 CFU/g) suggest the presence of Staphylococcus aureus above the
maximum established limits (102 CFU/g). It can be concluded that sam- pled cheeses were not suitable for consumption due to
high counts of Staphylococcus spp., which could be due to the fact of their production with raw milk.
Keywords
: Contamination. Foodborne illness. Pathogenic Microganism.
Pesquisa de
Staphylococcus
spp. em queijos “Minas Frescal” feitos a partir de leite cru e
comercializados
no município de Formiga-MG
Resumo
O queijo “Minas Frescal”, de acordo com a legislação, é definido como o queijo fresco obtido por coagulação enzimá- tica do
leite com coalho e/ou outras enzimas coagulantes apropriadas, complementada ou não com ação de bactérias lácticas
específicas. A legislação brasileira exige que estes sejam elaborados a partir de leites pasteurizados e proíbe a comercialização
quando elaborados com leite cru pois, assim, podem ser importantes veículos de micro-organismos patogênicos. O objetivo
deste trabalho foi analisar a presença de Staphylococcus spp. em queijos do tipo “Minas Fres- cal” elaborados de forma
artesanal (com leite cru) e comercializados em mercados locais do município de Formiga
- MG. O padrão microbiológico para este micro-organismo está definido em seu regulamento técnico de identidade e qualidade.
O plaqueamento em Ágar Sal Manitol foi realizado em busca de Staphylococcus spp. e afim de diferenciar
S. aureus de S. não aureus. Os resultados das amostras analisadas foram sugestivos para a presença de Staphylococcus aureus
(104 to 106 UFC/g), ultrapassando os limites máximos permitidos estabelecidos (102 UFC/g). Pode-se concluir
1Centro Universitário de Formiga (UNIFOR-MG) . Formiga, MG. Brasil.
https://orcid.org/0000-0003-0902-0000
2Centro Universitário de Formiga (UNIFOR-MG). Formiga, MG. Brasil.
https://orcid.org/0000-0001-7814-8257
3Centro Universitário de Formiga (UNIFOR-MG). Formiga, MG. Brasil.
https://orcid.org/0000-0002-4647-9294
4Universidade Federal de Minas Gerais (UFMG) .Belo Horizonte, MG. Brasil.
https://orcid.org/0000-0002-0537-1666
5Centro Universitário de Formiga (UNIFOR-MG) . Formiga, MG. Brasil.
https://orcid.org/0000-0002-2981-5479
*Autor para correspondência: leoacurcio@uniformg.edu.br
CADERNO DE CIÊNCIAS AGRÁRIAS
Agrarian Sciences Journal
2
Campos, J. V. F. et al.
que os queijos não estavam aptos para consumo pela elevada contagem de Staphylococcus spp., que pode se dar pelo fato destes
serem elaborados a partir de leite cru.
Palavras-chave
: Contaminação. Doenças transmitidas por Alimentos. Micro- organismo Patogênico.
Introduction
Milk is the ideal growth medium for the deve-
lopment of several microorganisms, especially bacteria,
which are the main causes of diseases transmitted by dairy
products worldwide (Grace; Haveelar, 2020).
as they produce thermoresistant enterotoxins in foods,
which make them important sources of food poisoning
(Rosa et al., 2015).
To guarantee food security in the consumption of
“Minas Frescal” cheese, Ordinance No. 146 of March 7,
1996, defined the maximum limit of 103 CFU/gram of
coagulase positive Staphylococcus as a microbiologi- cal
standard for this category of microorganism (Brasil, 1996).
One of the most popular dairy products in Brazil
is
“Minas Frescal” cheese, being made from enzymatic
coagulation of milk (Martins, 2012).
Brazilian law requires that “Minas Frescal” chee-
ses
be produced from pasteurized milk and, in addition, prohibits
their legal commercialization when made with raw milk.
However, this commercialization has been carried out
openly, in several Brazilian states (Lima; Cardoso, 2019;
Vinha; Pinto; Chaves, 2018).
Normative Instruction Nº 57 points out that
artisanal cheese that is made with raw milk undergo a
maturation period of 60 days or less, when technical-s-
cientific studies prove that the reduced maturation time does
not imply in reduced quality and/or no product safeness.
However, the only cheese made by hand, with raw milk and
matured for varying periods, is “Minas Artisanal” cheese,
which has guaranteed food security when maturation is
carried out for the correct time and form (Brasil, 2013).
“Minas Frescal” cheese flowchart is basically
composed of: pasteurization of milk, addition of bacterial
culture, calcium chloride and rennet, cutting and stirring
the
mass, hanging, draining, salting and packaging the cheese.
Pasteurization can be done by two different pro- cesses,
HTST and LTLT. HTST (high temperature, short time) is
conducted by heating the milk at 72–75°C for 15 to 20
seconds and the LTLT (low temperature, long
time), at 65°C
for 30 minutes and, in both cases, the milk
must be
immediately cooled to 35°C, to add the next ingredients
(Santos et al., 2019).
Given the illegality of the production of “Minas
Frescal” cheese made from raw milk, the importance of
the
veterinarian role in inspecting this food is highlighted.
Therefore, it is worth mentioning that in order to produce
a
milk or any dairy product with good quality and a high
level of
safety for human consumption, it is extremely im- portant that
the veterinarians involved in the production
chain inspection
perform their role (Santos; Carvalho, 2013).
According to Decree-Law Nº 923, of October 10,
1969, the sale of raw milk for direct consumption by the
population is prohibited (Brasil, 1969). Although it is
prohibited by law to sell it, it still occurs in small towns,
where the inhabitants maintain the habit of consuming raw
milk due to a belief that this product is purer and healthier.
In addition, it has lower cost per liter and its
purchase is more
comfortable (door to door) (Raymundo;
Bersot; Osaki, 2017).
The objective of this work was to research, quan- tify
and differentiate bacteria of the genus Staphylococcus in
“Minas Frescais” cheeses made with raw milk and sold
in the
municipality of Formiga - Minas Gerais.
Material and Methods
There are many factors that are related to food
illness, such as erroneous temperature during cooking,
cooling and/or food storage; poor personal hygiene from
manufacturers; cross-contamination between raw and
processed products; as well as improper monitoring of
processes (Lindsay et al., 2021).
The present experiment was carried out at the
Microbiology Laboratory of the Centro Universitário de
Formiga - Minas Gerais. For this, “Minas Frescal” cheeses
from five producers, sold in local markets (in Formiga -
MG), were used. The samples were purchased and kept
under refrigeration until the experiment was carried out.
One of the major problems with the consumption
of
these cheeses made with raw milk is the presence of
bacteria
of the genus Staphylococcus. This is due to the fact that these
microorganisms are among the most present in
artisanal
cheeses (especially with regard to the absence of
pasteurization) and have high pathogenic potential,
The methodology used to obtain the results was an
adaptation of the technique described by Zurita et al. (2010).
Cad. Ciênc. Agrá., v. 13, p. 01–05, https://doi.org/10.35699/2447-6218.2021.32993
3
Search for
Staphylococcus
spp. in “Minas Frescal” cheeses made with raw milk and commercialized in the city of Formiga - MG
From the samples obtained, one gram of each
was
weighed, under sterile conditions, for further dilution
in sterile
saline (0.9% NaCl). Five serial decimal dilu- tions of each
sample were plated in sterile Petri dishes, using the pour
plate method, containing mannitol salt agar (Kasvi, São
José dos Pinhas, Brasil). This method consists of adding 1
mL of the selected dilutions in Petri dishes, adding the
culture medium and homogenizing. After plating, the plates
were incubated in temperatures between 35-37°C, for at least
48 hours.
tube, the colonies are striated and the plates incubated again,
for a period of 48 hours, at temperatures of 35- 37°C.
Finally, the differentiation of the colonies on the plates was
done in S. aureus and not aureus, observing
the
fermentation of mannitol present in the medium and
a change
in color (or not) for this differentiation. The experiment was
done in duplicate, with one repetition.
Data comparison was performed with GraphPad Prism
6.0 (GraphPad Software, San Diego, USA) using One-way
ANOVA with Tukey post-hoc test, at a 5% significance
level (p<0.05).
After incubation, the colonies were counted and
differentiated. The differentiation was made through the
colony staining and they were divided into white and
yellow. To differentiate the colonies’ morphology, they were
visualized in immersion, under optical microscopy, at 100x
magnification, after GRAM staining.
Results and discussion
The results of the “Minas Frescal” cheese samples
from the Formiga region - Minas Gerais can be seen in
Figures 1 and 2.
The differentiated colonies were later added in
test
tubes with Brain Heart Infusion Broth (BHI, Himedia, Mumbai,
India) and incubated at 35-37°C for a minimum
period of 48
hours. After cultivation in tubes containing BHI broth, each
tube was plated again, using Mannitol Salt Agar (Kasvi).
However, using the striation method, which consists of
adding the medium to the Petri dishes and waiting for their
solidification and, with the aid of platinum handles,
flambéed and submerged in each
Figure 1 shows the results of the total count of
Staphylococcus spp. and it is possible to observe that all
samples obtained a very high count of this bacterium, a
worrying fact, since this genus of bacteria can be trans-
mitted through the hands of manipulators and become an
important source of food contamination. In addition,
several
species of this genus can cause food related disease
in humans
(Dittmann et al., 2017).
Figure 1 – Average results of the total counts of Staphylococcus spp. in mannitol salt agar of “Minas Frescal” cheeses made
from raw milk, sold in Formiga - MG
Caption: different letters represent different results (p <0.05) by One-way ANOVA with Turkey post-hoc test.
In Figure 2, the results obtained are suggestive of
Staphylococcus aureus, which are coagulase positive, since
there was fermentation of the mannitol from the mannitol
salt agar and color change to yellow. It is pos- sible to
observe that 100% of the samples, in addition to obtaining a
high count of S. aureus, do not attend the microbiological
standard provided for in Ordinance Nº 146 from 1996, from
MAPA (Agricultural and Livestock Ministry and Supply)
(Brasil, 1996). This Ordinance
recommends that the samples obtain a maximum count of
1.000 CFU/g (or 1x10³ CFU/g or 3 Log10 CFU/g) of
coagulase positive Staphylococcus. A worrying result, since
high counts of this microorganism suggest failures
in
processing, such as absence or deficiency in pasteuriza-
tion,
inadequate hygiene conditions, as well as incorrect
storage
temperature (Lindsay et al., 2021). Another wor- rying factor
is that this agent can produce staphylococcal
toxins, which
cause food poisoning. For the toxin to be
Cad. Ciênc. Agrá., v. 13, p. 01–05, https://doi.org/10.35699/2447-6218.2021.32993
4
Campos, J. V. F. et al.
able to accumulate at levels that cause food poisoning,
approximately 106 CFU/g of food is needed (Forsythe,
2013). This demonstrates that two of the analyzed sam- ples
have the potential to cause food poisoning.
Figure 2 – Average results of total colony counts suggestive of Staphylococcus aureus on mannitol salt agar of “Minas Frescal”
cheeses made from raw milk, sold in Formiga - MG
Caption: different letters represent different results (p <0.05) by One-way ANOVA with Turkey post-hoc test. Dashed line represents the maxi- mum limit
defined by legislation (3 Log10 CFU/g).
From the visualization of the Figures 1 and 2, it
was possible to observe that sample 2 had a lower (p
<0.05) count of microorganisms, when compared to
samples 3 and 5, but still not in compliance with the
legislation. This result was possibly due to the fact that it
was made from a better quality milk, with lower load of
microorganisms and/or in better hygiene conditions,
which
are points closely linked to the quality of the “Minas
Frescal”
cheese (Filho e Filho, 2000).
tions of unsatisfactory health ”and, when they present
S. aureus in a value greater than 10 times the maximum limit
established, they are considered“ Products poten- tially
capable of causing foodborne illness”. Therefore, almost all
samples approached 1 x 105 (5 Log10) or 1 x 106 (6 Log10)
CFU/g, or even passed, except for sample 2, showing that
they are products potentially capable of causing diseases
transmitted by foods. Sample 2, even though it is of better
quality, is considered a product in unsatisfactory hygiene
and sanitary conditions, as it is close to 1 x 104 (4 Log10)
CFU/g in Staphylococcus aureus count.
The legislation does not have microbiological
standards for other species of the Staphylococcus genus,
such
as coagulase negative. This demonstrates that there
is no
agreement with what has already been reported by
Heilmann; Ziebuhr; Becker (2019) about the patho- genicity
of coagulase negative Staphylococcus, since it is proven it
can also cause foodborne poisonings.
Conclusion
According to the results obtained after conducting the
research on “Minas Frescal” cheese samples in Formiga
- MG, it was found that all samples had high counts of
positive coagulase Staphylococcus (aureus), well above
that
established by therefore, unfit for consumption. This
represents a public health concern, as these cheeses are not
inspected and their raw material does not undergo heat
treatment.
According to Ordinance Nº 451 of 1997 of the
National Health Surveillance Agency (ANVISA) (Brasil,
1997), products that present S. aureus in a value of up to 10
times the maximum limit established in microbio- logical
standards, are considered “Products in condi-
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