Study on the interactions between milk protein and digestive enzymes (pepsin) : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatū, New Zealand
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Date
2023-11-22
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Massey University
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Abstract
Pepsin, as a main digestive enzyme in the gastric environment, plays a key role in milk protein digestion. Pepsin and κ-casein at pH > 5, leading to coagulation of casein micelles. The hydrolysis of κ-casein in skim milk was investigated using reverse phase high performance liquid chromatography (RP-HPLC). The coagulation behavior and coagulum structure were observed using oscillatory rheology, microscopy, small and ultra-small angle neutron scattering. The effects of important variables, including temperature, pH, divalent cations, ionic strength, whey protein concentration, and pre-heat treatment of milk, were studied on the hydrolysis kinetics and coagulation process.
The pepsin-induced hydrolysis of κ-casein was described using a combined kinetic model of first-order hydrolysis and pepsin denaturation. The hydrolysis rate was independent of the divalent cations, ionic strength, and whey protein concentration, but was highly dependent on the temperature, pH, pre-heat treatment, and milk species.
Coagulation of the casein micelles occurred when a critical amount of κ-casein had been hydrolyzed (Hct). The Hct was independent of pepsin concentration (ranging from 0.275 to 5.50 U/mL milk) at a given pH, but it depended on the pH, temperature and calcium concentration: a lower Hct was observed at lower pH (5.3); at higher temperature (43 °C); and at a higher addition of CaCl₂ 17.5 (mM). Sheep milk coagulated at a lower Hct and goat milk coagulated at higher Hct compared to cow milk. In addition, the rheological properties and the microstructures of the curd were significantly different depending on all the variables.
A human gastric simulator (HGS) was used to investigate the in vitro dynamic gastric digestion of different processed milk. The 4 °C milk exhibited slower coagulation with a faster breakdown and hydrolysis of the caseins by pepsin, when compared to that at 37 °C and 50 °C. Addition of CaCl₂ enhanced coagulation of casein micelles and resulted in more cohesive curd, which decreased the emptying rate of caseins.
Overall, this research provides fundamental information on the kinetics of hydrolysis of κ-casein and how it affects the coagulation behavior of casein micelles under various conditions in milk systems. The different gastric digestion behaviors of different processed milks are explained. The outcomes from this study have the potential to design dairy products with specific coagulation and digestion behaviors to meet the specific digestion requirements of consumers.
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milk, protein, pepsin, digestion, hydrolysis, coagulation