CADERNO DE CIÊNCIAS AGRÁRIAS
Agrarian Sciences Journal
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 Acurcio
5
*
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 (10
4
to 10
6
CFU/g) suggest the
presence of Staphylococcus aureus above the maximum established limits (10
2
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 (10
4
to 10
6
UFC/g), ultrapassando os limites máximos permitidos estabelecidos (10
2
UFC/g). Pode-se concluir
1
Centro Universitário de Formiga (UNIFOR-MG) . Formiga, MG. Brasil.
https://orcid.org/0000-0003-0902-0000
2
Centro Universitário de Formiga (UNIFOR-MG). Formiga, MG. Brasil.
https://orcid.org/0000-0001-7814-8257
3
Centro Universitário de Formiga (UNIFOR-MG). Formiga, MG. Brasil.
https://orcid.org/0000-0002-4647-9294
4
Universidade Federal de Minas Gerais (UFMG) .Belo Horizonte, MG. Brasil.
https://orcid.org/0000-0002-0537-1666
5
Centro Universitário de Formiga (UNIFOR-MG) . Formiga, MG. Brasil.
https://orcid.org/0000-0002-2981-5479
*Autor para correspondência: leoacurcio@uniformg.edu.br
Recebido para publicação em 08 de abril de 2021. Aceito para publicação 07 de julho de 2021
e-ISSN: 2447-6218 / ISSN: 2447-6218 / © 2009, Universidade Federal de Minas Gerais, Todos os direitos reservados.
Campos, J. V. F. et al.
2
Cad. Ciênc. Agrá., v. 13, p. 01–05, https://doi.org/10.35699/2447-6218.2021.32993
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).
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).
“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).
According to Decree-Law 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).
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).
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,
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 10
3
CFU/gram
of coagulase positive Staphylococcus as a microbiologi-
cal standard for this category of microorganism (Brasil,
1996).
Normative Instruction 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).
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).
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
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.
The methodology used to obtain the results was
an adaptation of the technique described by Zurita et al.
(2010).
Search for Staphylococcus spp. in “Minas Frescal” cheeses made with raw milk and commercialized in the city of Formiga - MG
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Cad. Ciênc. Agrá., v. 13, p. 01–05, https://doi.org/10.35699/2447-6218.2021.32993
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.
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.
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
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).
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.
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
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 Log
10
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
Campos, J. V. F. et al.
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Cad. Ciênc. Agrá., v. 13, p. 01–05, https://doi.org/10.35699/2447-6218.2021.32993
able to accumulate at levels that cause food poisoning,
approximately 10
6
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).
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.
According to Ordinance 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-
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 10
5
(5 Log
10
) or 1 x
10
6
(6 Log
10
) 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 10
4
(4 Log
10
) CFU/g in Staphylococcus aureus
count.
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.
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