Synthesis of Lactose Phosphate and its Conjugation with Whey Proteins

Abstract

The aim of this study was to investigate the conjugation of whey proteins (WPs) with lactose phosphate (LP) and to compare the functional properties of these conjugates with either non-conjugated whey protein or whey protein conjugates using lactose. Lactose-6’-phosphate (Lact-6’P) was prepared chemically via two chemical pathways, either using unprotected lactose or with protected lactose via a pertrimethylsilyated derivative. The most successful production of Lact-6’P was via a mono-deprotected pertrimethylsilyated derivative of lactose. This compound reacted with diphenylphosphoryl chloride by hydrogenation before hydrolyzed to remove trimethylsilyl protecting groups. Purification and neutralisation, followed by lyophilisation, produced the pure dipotassium salt of Lact-6’P. This material gave LP ions at m/z = 421+ when analysed using High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) and had one triplet peak pattern around 4 ppm in the 31P NMR spectrum. Mixed regiosomers of unprotected LP were prepared by reaction of lactose with sodium hexametaphosphate at pH 5.5, 80°C for 5 days. Products from the latter preparation were purified by anion exchange chromatography monitored by p-hydroxybenzoic acid hydrazide (PAHBAH) analysis. This LP preparation contained Lact-6P and Lact-6’P, and other regioisomers. The regioisomeric mixture of protected LP (LP 1) and the 6’ regiospecific LP (LP 2) were conjugated to whey proteins using incubation time varying from 0 to 10 days, at 40°C and at water activity Aw = 0.79. SDS-PAGE analysis, visible browning, absorbance at 294 nm and 420 nm, OPA assay and mass spectrometry were employed to monitor the extent of conjugation. An incubation time of 3 days was selected as the standard conjugation time based on conjugation rates and the degree of Maillard browning. Mass spectrometric analysis of the modified whey proteins at 3 days indicated a maximum of 3 LP moieties were conjugated to α-Lac, in comparison to 8 lactose moieties for α-Lac-Lactose. Meanwhile, 5 LP moieties conjugated to β-Lg-LP in contrast to 10 lactose moieties. Functional studies using the conjugated LP-whey proteins showed that they possessed improved protein heat stability, better emulsifying properties, and also had good solubility compared to non-conjugated and lactose-conjugated whey proteins. Thus, a new approach of LP conjugation to whey proteins has shown to be a useful method for altering the functional properties of proteins.