Sociedade Brasileira de Espectrometria de Massas – BrMASS
Análise de Alimentos (FOO)
Milk adulteration analysis of cheese whey addition using tryptic
digestion and LC-MS/MS.
Tanara Magalhães Campos Motta1,2, Leonardo Meneghini2, Daniel Lorenzini2,
Rodrigo Hoff2, Tânia Mara Pizzolato1
[email protected]
1. Programa de Pós-Graduação em Química da Universidade Federal do Rio
Grande do Sul – PPGQ/UFRGS
2. Laboratório Nacional Agropecuário – LANAGRO/RS, Porto Alegre, RS.
Ministério da Agricultura, Pecuária e Abastecimento.
Milk adulteration by addition of cheese whey, a by-product of cheese production , is a
sanitary and economic concern in Brazil. This type of fraud does not require corrections
in pH, sugars, protein and other parameters, due to the similarity between these
matrices. Quantification of caseinomacropeptide (CMP), a peptide fragment from
chymosin proteolysis of k-casein (residues 106-169) that remains soluble in cheese
whey, is the most adequate target for the identification of this adulteration. However,
the proteolytic activity of psychrotrophic bacteria in milk, in inadequate conditions of
storage, produces by k-casein proteolysis a similar peptide, the pseudocaseinomacropetídeo (P-CMP), which differs, from the CMP, in length by only in one
amino acid residue.
A confirmatory method, developed at LANAGRO/RS (Ministry of Agriculture, Livestock
and Food Supply), using liquid chromatography-electrospray ionization tandem mass
spectrometry (LC–ESI-MS/MS) and tryptic digestion, was able to identify milk
adulteration, because tryptic digestion of CMPs produce two short peptides, MAIPPKK
and AIPPKK from CMP and P-CMP respectively, which are easily detected and
separated.
The method consists in protein precipitation with trichloracetic acid, solid phase
extraction using polystyrene-divinylbenzene (polymeric phase), tryptic digestion and
peptides analysis by LC-ESI-MS/MS. For the optimization of ion transfer and
fragmentation parameters in mass spectrometer and chromatographic separation,
synthetic standards of MAIPPKK and AIPPKK peptides were used.
A satisfactory confirmatory method was achieved. Through an adapted targeted
proteomics approach it is possible identify and differentiate bacterial contamination
from cheese whey addition in milk samples.
References
[1] Brasil, Ministério da Agricultura, Pecuária e Abastecimento, 2010. Instrução Normativa 07
de 03 de março de 2010.
[2] Mollé, D; Léonil, J. International Dairy J. 2005, 15, 419-428.
[3] Minkiewitz P; Slangen C. J; Lagerwerf F. M; Haverkamp J; Rollema H. S; Visser S. J
Chromatographic A 1996; 743:123-135.
[4] Fiat, A. M; Jolles, P. Mol Cell Biochem. 1989, 87, 5-30.
4º Congresso BrMass – 10 a 13 de Dezembro de 2011