Understanding the bioaccessibility of grape phenolics in association with milk proteins : A thesis submitted in partial fulfilment of the requirements for the Degree of Doctor of Philosophy at Lincoln University

Abstract

Phenolics are compounds that are widespread in plants and abundant in many fruits. Grapes and their products contain high concentrations of phenolics, particularly anthocyanins, and flavanols such as catechins and epicatechins. phenolics have been reported to have various health benefits, but these may be limited by their low uptake, low bioavailability, and rapid degradation owing to enzymatic and non-enzymatic reactions during digestion. Research has explored several protected delivery options to increase the bioavailability of phenolics in different fruits. Milk proteins have been identified as effective delivery agents for several bioactive compounds including phenolics. Although milk proteins are effective delivery agents for phenolic compounds, the specific interactions of the phenolics with the milk proteins, and factors affecting phenolic-protein interactions require to be understood to facilitate the utilisation of these delivery systems in food, nutraceutical, and pharmaceutical applications. Commercially available extracts of red grape skin from Pinot Noir, grape seed from Sauvignon Blanc, and sodium caseinate 180 were used in this study. Grape extracts and Na-casein protein stock solutions were separately prepared and mixed at different phenolic-to-protein weight ratios, 5:40, 10:40, and 15:40, phenolic-alone controls (5:0, 10:0, and 15:0) and casein-alone control (0:40) in phosphate-buffered saline (PBS). The protein phenolic complex samples were then freeze-dried. In the casein-based grape samples, the total extractable phenolic contents increased from a phenolic-toprotein ratio of 5:40 to 15:40. The percentages were 47.0% in CSK5 to 69.1% in CSK15 and 41.3% in CSE5 to 54.8% in CSE15 in casein-based skin and seed FD samples respectively. Further analysis by HPLC found that catechin, epicatechin, epigallocatechin gallate, gallic acid, and quercetin-3-glucoside were the abundant phenolics in casein-based skin whereas in casein-based seed samples, procyanidin B1, procyanidin B2, catechin, epicatechin and epigallocatechin gallate were the major phenolics. The antioxidant capacity was reflected in the total extractable phenolic content and strong positive linear correlations were found between the total extractable phenolic content and the antioxidant activity and between ABTS and DPPH assays in FD samples. The free phenolic results from the loading efficiency study and total extractable phenolic contents revealed that grape phenolics are more likely to bind with casein when a sample contains a higher protein concentration (e.g. 5:40 phenolic-to-protein ratio containing sample). In the same way, DH% results displayed that protein hydrolysis of casein-based grape skin FD samples was significantly increased during in-vitro digestion, and the increasing order being CSK0 < CSK5 < CSK10 < CSK15 shows that protein hydrolysis is more favourable with the sample having a lower proportion of protein. Significant increases (p<0.05) in phenolic release were noted in three different phenolic-to-protein ratios after one hour of pepsin addition. In contrast, phenolic-alone skin samples showed significant reductions (p<0.05) in phenolic release under acidic pH conditions one hour after adding pepsin indicating rapid phenolic degradation. Even though, both casein-based and phenolic-alone grape skin samples showed a notable increase in phenolic content one hour after pancreatin addition, again it declined during the final two hours in the phenolic-alone skin samples. No differences were found in the phenolic content of three casein-based samples after five hours of digestion resulting in a slower release of phenolics during in-vitro digestion even with the sample having a lower phenolic-to-protein ratio. Reliable with the trends noted in DH% and phenolic release, the antioxidant activity was increased in casein-based grape samples. FT-IR results discovered that grape phenolics interact with casein mainly via non-covalent bonding like hydrogen bonding, hydrophobic, and electrostatic interactions, and no covalent bonds were formed during the formation of casein-grape phenolic complexes. In addition, grape phenolics appear to alter casein's secondary structure by decreasing β-sheet and random coil contents and increasing β-turn and α-helix structures, reflecting increased hydrogen bonding and protein conformational changes, leading to flexible and disordered protein structure and antioxidant activity of casein. The findings of the fluorescence quenching study revealed that adding different concentrations of grape skin and seed phenolics caused to significant decrease in fluorescence intensity of the main emission band of Nacaseinate and the shift of the emission peak slightly toward a higher wavelength. Moreover, both dynamic and static quenching mechanisms were involved with samples at a higher phenolic-to-protein ratio. The highest modified Stern–Volmer quenching constant (K), 4.6 x 10³ M⁻¹ and quenching constant (Kq), 1.5 x 10¹² M⁻¹s⁻¹ at 280 nm excitation wavelengths at room temperature of grape skin phenolics showed stronger quenching interactions and greater binding affinity towards casein protein indicating higher radical scavenging activity in casein. Furthermore, the binding constants (KA) for casein-based skin, 3.49 x 10⁷ M⁻¹ at 290 nm excitation wavelength revealed stronger static quenching interactions between skin phenolics and casein protein. Overall, findings based on protein hydrolysis, phenolic release, antioxidant activity during in-vitro digestion, and the interactions between grape phenolics and casein protein, which together enhance bioaccessibility during digestion, this study confirmed that casein-based grape FD powders are an effective food complex, beneficial for the nutraceutical and food industries.