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RESEARCH ARTICLE
ISSN 1679-9216
Pub. 1213
Deteco de fraude em leite e queijo de coalho caprino por adio de leite bovino
Detection of Bovine Milk Contaminants in Adulterated Milk and Curd Goat Cheese
Maria Rociene Abrantes1, Adriene Rosceli Menezes de Oliveira1, Manuella de Oliveira Cabral Rocha1,
Gisele Oliveira de Souza2, Evelise Oliveira Telles2, Sidnei Miyoshi Sakamoto1 & Jean Berg Alves da Silva1
ABSTRACT
Background: Goat milk is quite useful as an alternative for feeding children and adults who are sensitive or allergic to
bovine milk because of its peculiar characteristics, and goat milk ensures the subsistence of small producers. Therefore,
ensuring the purity and satisfactory quality of dairy goat products is essential for consumer safety. Accordingly, the present study aimed to detect adulteration of goat milk and curd goat cheese with bovine milk contaminants using polymerase
chain reaction (PCR).
Materials, Methods & Results: For this purpose, the detection limit of the PCR assay was tested using bovine milk added
to goat milk at concentrations of 0.5%, 1.0%, 5.0%, 10.0%, 25.0%, 50.0%, and 100%. Subsequently, goat milk from 79
producers and 43 samples collected from community cooling tanks was tested. Curd goat cheese was subsequently manufactured in the laboratory according to technical recommendations. The quality of the curd cheese was assessed by adding
the aforementioned concentrations of bovine milk to goat milk, and PCR was performed to detect contaminants. Genomic
DNA was extracted from milk and curd cheese samples and subjected to amplification by PCR using specific primers.
After amplification, PCR products were analyzed in 1.5% agarose gel, stained with ethidium bromide, and visualized using a UV transilluminator. The quality of milk DNA was evaluated using a spectrophotometer, and it corresponded to an
average value of 1.76. With regard to the detection limit, bovine DNA was detected using the smallest concentration of
bovine milk (0.5%) added to goat milk. Using these same experimental conditions for the curd cheese samples, a detection
limit of 5.0% of bovine milk was established. When evaluating goat milk samples from producers and cooling tanks, the
bovine gene was detected in 82.27% and 97.67% samples, respectively.
Discussion: DNA quality was determined on the basis of the amount of DNA and protein. The value obtained was close to
the minimum limit of 1.8, probably because of the high protein concentration in the milk. The high rate of contamination
of goat milk with bovine milk is concerning, taking into consideration that this contamination can cause serious health
problems to consumers who are allergic to bovine milk. In addition, government regulations and ethical or cultural objections are compromised. Some factors may contribute to goat milk contamination, such as the lack of practice in routine
inspection and the lack of rigorous supervision. Another factor is the lack of technical support for milk producers because
inadequate cleaning of equipment at milk processing facilities may contribute to the high levels of contamination. In the
present study, the threshold for detection of bovine DNA in curd cheese was high, which can be explained by product
characteristics such as compact texture and semi-solid consistency. Therefore, an extraction protocol capable of efficiently
recovering the maximum amount of DNA from this type of sample is required. The protocol used in the present study can
be used to detect bovine milk contaminants during the preparation of goat curd cheese, because it enables the extraction
and detection of bovine DNA. This methodology pioneers the use of PCR to check the authenticity of milk products;
therefore, it can be efficiently used to identify this type of adulteration.
Keywords: fraud, goat milk, coalho cheese, PCR.
Descritores: fraude, leite de cabra, queijo de coalho, PCR.
Laboratrio de Inspeo de Produtos de Origem Animal (LIPOA), Programa de Ps-graduao em Cincia Animal (PPGCA), Universidade Federal Rural
do Semi-rido (UFERSA), Mossor, RN, Brazil. 2Faculdade de Medicina Veterinria e Zootecnia, Departamento de Medicina Veterinria Preventiva e
Sade Animal, Universidade de So Paulo (USP), So Paulo, SP, Brazil. CORRESPONDENCE: M.R. Abrantes [rocienevet3@hotmail.com - Tel.: +55
(84) 3317-8381]. DCAN, UFERSA. BR 110 - Km 47 - Presidente Costa e Silva. CEP 59625-900 Mossor, RN, Brazil.
M.R. Abrantes, A.R.M. Oliveira, M.O.C. Rocha, et al. 2014. Deteco de fraude em leite e queijo de coalho caprino por
adio de leite bovino.
Acta Scientiae Veterinariae. 42: 1213.
INTRODUO
Extrao de DNA
Delineamento experimental
Inicialmente foi determinado o limite de deteco do ensaio de PCR utilizando leite de cabra SRD
(Sem Raa Definida) misturado nas concentraes de 0;
0,5; 1; 5; 10; 25; 50 e 100% com leite de vaca mestias
(girolanda), obtidas de rebanhos leiteiros da regio,
avaliando-se em seguida amostras de leite de cabras
de produtores e tanques de resfriamento comunitrios.
Posteriormente foram elaborados queijos de coalho
utilizando leite de cabra misturado com leite de vaca de
mesma procedncia e concentraes j citadas, sendo
utilizado o mtodo de PCR na deteco de fraude.
M.R. Abrantes, A.R.M. Oliveira, M.O.C. Rocha, et al. 2014. Deteco de fraude em leite e queijo de coalho caprino por
adio de leite bovino.
Acta Scientiae Veterinariae. 42: 1213.
A qualidade do DNA extrado do leite obteve valor mdio de 1,76. O limite de deteco alcanado neste
trabalho, utilizando o mtodo PCR, foi considerado a
menor porcentagem de mistura de leite bovino (0,5%)
[Figura 1] em leite caprino. Ao avaliar as amostras de
leite de cabra foi diagnosticada presena do gene bovino
em 82,27 (65) [Figura 2] e 97,67% (42) [Figura 3] do leite de produtores individuais e de tanques de resfriamento
comunitrios, respectivamente. Foi observado que em
87,7% (107) das amostras de leite de cabra analisadas
foi detectado fragmentos de DNA bovino.
J em relao as amostras de queijos de coalho elaborados a partir de misturas de leite, o teste foi
sensvel na deteco de 5% do leite bovino em leite
caprino (Figura 4).
Espcie
Bos taurus e Bos indicus
J01394a
Capra hircus M55541a
a
Sequncia 5 3
Senso 14814-GGCTTATATTACGGGTCTTACACT
Antisenso15092-GGCAATTGCTATGATGATAAATGGA
Senso 144-CGCCCTCCAAATCAATAAG
Antisenso 469-GTGTATCAGCTGCAGTAGGGTT
Figura 1. Amplificao de DNA por PCR com primers especficos para espcie bovina. M, 100 bp ladder; L1, leite bovino; L2,
0,5% de leite bovino; L3, 1% de leite bovino; L4, controle de reagentes; L5, 5% de leite bovino; L6, 10% de leite de bovino;
L7, 25% de leite bovino; L8, controle de reagentes; L9, 50% de leite bovino; L10, controle de reagentes; L11, leite de cabra.
Fragmento (pb)
279
326
M.R. Abrantes, A.R.M. Oliveira, M.O.C. Rocha, et al. 2014. Deteco de fraude em leite e queijo de coalho caprino por
adio de leite bovino.
Acta Scientiae Veterinariae. 42: 1213.
Figura 2. Amplificao de DNA por PCR com primers especficos para espcie bovina. M, 100 bp ladder; L1, leite bovino; L2 - L13, amostras de leite caprino de produtores; L14, controle dos reagentes; L15
leite caprino.
Figura 3. Amplificao de DNA por PCR com primers especficos para espcie bovina. M, 100 bp ladder; L1, leite bovino; L2 - L16, amostras de leite caprino de tanques de resfriamento; L17, controle dos
reagentes; L18, leite caprino.
Figura 4. Amplificao de DNA por PCR com primers especficos para espcie bovina. M, 100 bp ladder;
L1, queijo bovino; L2, 0,5% de leite bovino; L3, 1% de leite bovino; L4, controle de reagentes; L5, 5%
de leite bovino; L6, 10% de leite de bovino; L7, 25% de leite bovino; L8, controle de reagentes; L9, 50%
de leite bovino; L10, controle de reagentes; L11, queijo de cabra.
M.R. Abrantes, A.R.M. Oliveira, M.O.C. Rocha, et al. 2014. Deteco de fraude em leite e queijo de coalho caprino por
adio de leite bovino.
Acta Scientiae Veterinariae. 42: 1213.
DISCUSSO
CONCLUSO
M.R. Abrantes, A.R.M. Oliveira, M.O.C. Rocha, et al. 2014. Deteco de fraude em leite e queijo de coalho caprino por
adio de leite bovino.
Acta Scientiae Veterinariae. 42: 1213.
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