World-class publication “Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese” from TFTAK
Feb 14, 2018
Center of Food and Fermentation Technologies scientists Anastassia Taivosalo alongside with T. Kriščiunaite, A. Seiman, N. Part, I. Stulova and R. Vilu published a high-level article in the Journal of Dairy Science. Comprehensive analysis of proteolysis during 8 months of ripening of high-cooked Old Saare cheese was carried out.
We applied capillary electrophoresis, liquid chromatography coupled with tandem mass-spectrometry (MS/MS), and ultra-performance liquid chromatography to determine the composition of water-insoluble and water-soluble proteinaceous fractions of the cheese and to study in detail the degradation of caseins during 8 mo of ripening of Estonian high-temperature cooked hard cheese Old Saare. The application of high-resolution and high-accuracy MS/MS enabled identification of more than three thousand small peptides, representing fairly full casein peptidome containing peptides of 4 to 25 amino acids in length – 1,049 from β-, 944 from αs1-, 813 from αs2-, and 234 from κ-CN. The majority of β- and αs1-CN derived peptides originated from the N-terminal parts of the molecule, f6-93 and f1-124, respectively; peptides from αs2-CN arose predominantly from the C-terminal end f100-162. At the beginning of ripening, we found a relatively high amount of peptides originating from the glycomacropeptide part of κ-CN, while peptides from para-κ-CN prevailed during the later stages of ripening of the cheese. The cleavage patterns of β-, αs2-, as well as αs1-CN showed that primary proteolysis was started mainly by plasmin, although a low proteolytic activity of chymosin was also evident. Based on the analysis of cleavage sites, we observed a significant participation of proteolytic enzymes, including amino- and carboxypeptidases, of both mesophilic and thermophilic starter bacteria in further hydrolysis of oligopeptides during the ripening. Several new phosphopeptides were detected in the result of MS/MS data analysis. The profiles of the estimated concentrations of phosphopeptides revealed that those originating from β- and αs1-CN accumulated during the cheese maturation. In contrast, we did not notice any generation of phosphopeptides from highly phosphorylated part of αs2-CN, f25-80, presumably due to the inaccessibility of this region to the action of plasmin and chymosin.
The analysis of cleavage sites and the combination of principal component and clustering analyses provided a characterization of the complex dynamics of formation and degradation of peptides during the cheese maturation. We made an attempt to obtain a comprehensive picture of proteolysis during the Old Saare cheese ripening on the basis of the detailed peptidomic data, including also the less abundant peptides determined by MS/MS, and complemented by the data on intact caseins and free amino acids and reported the results in the paper.
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