Markoska, Tatijana ORCID: 0000-0003-1235-6365 (2023) Probing β-casein structure using Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared (FTIR) spectroscopy. PhD thesis, Victoria University.

Abstract

Milk proteins are essential nutritive components and have great impact on the quality of dairy processing. Milk proteins are classified into two groups or caseins and whey proteins. Among the caseins, the second most abundant casein is β-casein. In the recent years, the difference in the genetic variants of β-casein and its impact on human health and milk processing has led to an increased interest among researchers. Thus, individual genetic variants have shown dissimilarities in the physicochemical behaviour during processing. The commercially most common β-casein milk variants are A1 and A2 milk. The difference between these variants is in the amino acid position 67 in the β-casein polypeptide chain with histidine in A1 milk and proline in A2 milk. Concerning the milk processing, A1 casein have shown to have better coagulation characteristics compared to A2 milk which is important for production of fermented products like cheese or yoghurt. However, in regards to the human consumption epidemiological evidence debate that consumption of A1 β-casein may be a risk factor for many diseases although the literature does not support these claims. Moreover, this appears to be due to the proteolysis of β-casein into smaller peptides in the human gut or bioactive peptides known as β-casomorphins (BCM). The most common peptides are BCM5, BCM7, BCM11 and BCM15. The most studied peptide to date is BCM7 that have been characterised to have opioid activity by binding to the μ-receptors in the human body and leads to a range of physiological responses. However, the structure of the peptide in conditions that have importance for its cleavage in human gut or during milk processing is lacking in the literature. In the current study, we observed that BCM7 shown structural difference under modified pH conditions including pH 2.3 and pH 7.0 that are native environmental condition of human gut and thus gives maximum activity for enzymatic cleavage in the digestive track. These structural differences were observed to exist due to the cis-trans isomerisam of the X-Pro bond. The finding shown pH dependence of the peptides bond accessibility for cleavage into smaller particles. The most important peptide concerning the difference between A1 and A2 β casein is BCM11. BCM11 from A1 milk have histidine in position 8 of the polypeptide chain and BCM11 from A2 milk have proline in the same position of the polypeptide chain. These amino acids are the major factor contributing to the different properties of β casein molecule originating from A1 or A2 milk. By observing the structure, we verified that the peptides adapt different conformers that further rearrange when pH and temperature conditions are modified. This was complemented with higher structural flexibility for BCM11 peptide from A1 β-casein due to loose conformation and tautomeric behaviour of imidazole ring of histidine that was dependent on the applied conditions. These structural differences have great effect on the accessibility of peptide bonds for cleavage into smaller peptides. Further computational studies of these peptides confirmed that the imidazole ring of the histidine have important role on the structural stability of the molecule involving changed charge transfer and affecting the acid-base chemistry of the molecule. In addition, the accessibility of the peptide bond for proteolysis can be dependent on the structural orientation of the β-casein. The proline in position 67 in A2 β-casein can be the factor for low accessibility of the bond 66-67 for cleavage. This is due to greater involvement of proline in turns formation in molecules. Moreover, the structural conformation of the molecules are also dependent of the environment. The predominant factor is the pH since the enzymes have optimal activity on defined pH values. Thus, the structural conformation of the β-casomorphin peptides can be pH dependent. The effect of the temperature and pH on the β-casein molecule was observed to have large effect on the secondary structure of the molecule. The findings confirmed loss of well defined structural components (α-helix) and formation of random structures when pH was increased to neutral and temperature lowered to 4 ᵒC. The modified conditions that are essential for milk processing disturbed the native hydrogen bonds and promoted formation of new structures. The changed conditions were also observed to have different effect on the β-casein genotype. β-Casein from A1 and A2 milk adapt different conformational states. Hence, β-casein from A2 have open and porous protein network with native aggregated β-sheets, however, β-casein from A1 milk adapts more dense structure with small network pores. The effect of pH largely affects the conformational orientation of both genotypes with formation of large irregular aggregates in A2 β-casein and more dense aggregated β-sheets in A1 β-casein. Observing the structural features of the peptides and β-casein can assist in understanding the reasons for their specific behaviour during milk processing or human digestion.

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