Genetic variation and posttranslational modification of bovine κ-casein: effects on caseino-macropeptide release during renneting.

Chymosin-induced cleavage of κ-casein (κ-CN) occurs during the first enzymatic phase in milk coagulation during cheese manufacturing, where the hydrophilic C-terminal peptide of κ-CN, named caseino-macropeptide (CMP), is released into the whey. The CMP peptide is known for its rather heterogeneous composition with respect to both genetic variation and multiple posttranslational modifications, including phosphorylation and O-linked glycosylation. An approach of liquid chromatography coupled with mass spectrometry was used to investigate (1) the overall protein profile and (2) the release of various forms of CMP after addition of chymosin to individual cow milk samples from 2 breeds, Danish Jersey (DJ) and Danish Holstein-Friesian (DH). The cows were selected to represent distinct homo- and heterozygous types of the κ-CN genetic variants A, B, and E (i.e., genotypes AA, BB, AB, EE, and AE). Initially, investigation of the protein profile showed milk with κ-CN BB exhibited the highest relative content of κ-CN, whereas AE milk exhibited the lowest, and after 40min of renneting >90% of intact κ-CN was hydrolyzed by chymosin in milk representing all κ-CN genotype. By in-depth analysis of the CMP chromatographic profile, multiple CMP isoforms with 1 to 3 O-linked glycans (1-3 G) and 1 to 3 phosphate groups (1-3 P) were identified, as well as nonmodified CMP isoforms. The number of identified CMP isoforms varied to some extent between breeds (21CMP isoforms identified in DJ, 26CMP isoforms in DH) and between κ-CN genetic variants (CMP variant A being the most heterogeneous compared with CMP B and E), as well as between individual samples within each breed. The predominant forms of glycans attached to CMP were found to be the acidic tetrasaccharide {N-acetyl-neuraminic acid α(2-3)galactose ß(1-3)[N-acetyl-neuraminic acid α(2-6)]N-acetyl galactose} or trisaccharides {N-acetyl-neuraminic acid α(2-3)galactose ß(1-3)N-acetyl galactose and galactose ß(1-3)[N-acetyl-neuraminic acid (α2-6)]N-acetyl galactose}. The CMP release was calculated to follow first-order kinetics and was determined by the measurement of CMP content during renneting. The highest rate of release for all CMP isoforms occurred from 0 to 2min after chymosin addition. Concurring results from both breeds showed that CMP variant A with 1-2 P had the highest reaction rate of CMP release, followed by CMP B 1-2 P and then by CMP E 1-2 P (only in DH). All the identified glycosylated CMP isoforms had lower reaction rates of release compared with that of nonglycosylated CMP, thus glycan modifications seemed to negatively influence the reaction rate of chymosin-induced hydrolysis of κ-CN.

Antioxidant properties of green tea extract protect reduced fat soft cheese against oxidation induced by light exposure.

The effect of two different antioxidants, EDTA and green tea extract (GTE), used individually or in combination, on the light-induced oxidation of reduced fat soft cheeses (0.2 and 6% fat) was investigated. In samples with 0.2% fat, lipid hydroperoxides as primary lipid oxidation products were not detected, but their interference was suggested from the formation of secondary lipid oxidation products such as hexanal and heptanal. The occurrence of these oxidation markers was inhibited by spiking with 50 ppm EDTA or 750 ppm GTE, or a combination of the two prior to irradiation. In contrast, addition of 50 ppm EDTA to samples with 6% fat was ineffective, but 750 ppm GTE (alone or in combination with EDTA) strongly reduced levels of hexanal and heptanal. Accumulation of primary lipid hydroperoxides was not affected by GTE, hence antioxidative activity was ascribed to scavenging of hexanal and heptanal precursors. These radical intermediates result from hydroperoxide disintegration, and subsequent scavenging by GTE, which acts as a radical sink, corroborates the intense signal observed by electron paramagnetic resonance (EPR) spectroscopy.