Effect of Storage on Lactase-Treated β-Casein and β-Lactoglobulin with Respect to Bitter Peptide Formation and Subsequent in Vitro Digestibility

Abstract

Using active lactose to hydrolyze lactose during storage is a common process to produce lactose-hydrolyzed (LH) milk. Proteolysis induced by residual proteases in commercial lactase was studied in a system using purified β-casein or β-lactoglobulin during a 60-day storage period at 22 or 38 °C. The proteolysis of β-casein by residual proteases occurred more extensively than that of β-lactoglobulin. Peptidomic analysis by LC-ESI-MS/MS revealed that Ile, Leu, Tyr, and Phe residues near the C-terminus of β-casein were the main sites of cleavage by the residual proteases, generating assumed bitter peptides. In the subsequent in vitro digestion study, proteolysis during storage was shown to greatly affect the subsequent digestibility of β-casein, leading to an elevated degree of hydrolysis and the formation of new digested peptides. This study highlights the potential influence of residual proteases in commercial lactase on the storage quality and digestibility of LH milk containing active lactase.

Interplay between Residual Protease Activity in Commercial Lactases and the Subsequent Digestibility of β-Casein in a Model System

“…Comparatively, the addition of either Lactases A or B resulted in a more pronounced increase in DH values during 60-day storage, observed to increase (for example) from 0.24% to 5.72% in the sample incubated with Lactase A, and from 0.2% to 8.1% in the Lactase B sample. These results confirm that proteolysis of β-CN induced by proteases activity in the tested commercial lactase preparations occurred during storage, but also that the hydrolysis varies with lactase preparation [4,5,7,8,9].…”

“…Notably, all the representative cleavage sites, including Val (170), Lys (176), Ala (177), Ala (189), Phe (190), Leu (191), Tyr (193), Pro (204), Phe (205), Pro (206), Ile (207), and Ile (208), were found to be located near the hydrophobic C-terminus of β-CN. The hydrophobic C-terminus of β-CN was shown earlier to be more vulnerable to cleavage by proteolytic side-activities in both Lactases A or B, corresponding to the earlier observations of Zhao and Rauh [5,13]. Apart from sharing some common cleavage sites for both Lactase A and Lactase B, in Lactase B, some proteotytic side activity further acted before or after the Gln (194), Leu (192), Pro (204) sites.…”

“…β-Casein (β-CN) was purified from the milk obtained from the research herd at Aarhus University (Tjele, Denmark) using the cooling and centrifugation methods published earlier [5]. Commercial lactase preparations A and B were obtained from the DSM Company (Heerlen, Netherland).…”

“…β-CN was dissolved to 10 mg/mL in 0.025 M sodium phosphate buffer (pH 6.5, 0.05% sodium azide) and heated at 95 °C for 5 min in a water bath to inactivate the potential indigenous milk enzymes in the β-CN preparation. From this β-CN stock solution, samples containing an additional 4.8% ( w / v ) lactose were prepared as a model of conventional milk (control sample) [5]. Furthermore, β-CN samples containing 3.0% ( w / v ) lactose (corresponding to a situation where 40% of lactose was removed by filtration, as is normal practice in Northern European countries to avoid excessive sweetness) and 0.09% ( v / v ) of either Lactase A (β-CN + Lactase A) or B (β-CN + Lactase A), were prepared as models of the post-hydrolysis process in the production of LH milk [5].…”

“…The post-hydrolyzed LH milk contains active lactase, and the hydrolysis continues throughout the storage of LH milk. The production of high quality post-hydrolyzed LH milk in Northern Europe usually starts with filtration to remove 40% of lactose, which can avoid the excessive sweetness of the product [5]. After that, the product undergoes heat treatment before the addition of a commercial lactase preparation to hydrolyze lactose during storage.…”