Caffeine-milk protein interactions in coffee brews : effects on the in vitro bioaccessibility of caffeine : a thesis submitted in partial fulfilment of the requirements for the degree of Master of Science in Food Technology, Massey University, Palmerston North, New Zealand
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Date
2025
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Massey University
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Abstract
Coffee is one of the most consumed beverages around the world and is known for a myriad of health benefits. Black coffee is a rich source of bioactive compounds and has high antioxidant activity. Coffee has been consumed in various forms with and without the addition of milk. The impact of milk addition on the phenolic properties, antioxidant activity, and functionality of bioactive components has been an interesting topic among researchers for the past few decades. But to the best of our knowledge, the effect of milk addition to coffee brew on caffeine bioaccessibility has not yet been addressed in the literature. Accordingly, this study aimed to investigate the interactions between caffeine and milk proteins in coffee and the effect on in vitro bioaccessibility of caffeine, as well as the antioxidant properties. A range of analytical techniques was employed to assess these interactions. Ultraviolet (UV) spectroscopy and high-performance liquid chromatography (HPLC) were used to quantify caffeine content in black coffee and milk-based coffee samples at various stages of digestion. Fourier transform infrared (FTIR) spectroscopy was applied to examine the molecular interactions between caffeine and milk proteins, focusing on non-covalent binding mechanisms such as hydrogen bonding and Van der Waals forces. Total phenolic content (TPC) and antioxidant capacity were measured using the Folin-Ciocâlteu assay and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, respectively. Additionally, particle size distribution and ζ-potential analysis were performed to determine the stability and colloidal properties of the coffee samples. In vitro digestion was conducted to simulate gastrointestinal conditions, including the gastric and intestinal phases, to evaluate changes in caffeine bioaccessibility over digestion. The results demonstrate a significant reduction in caffeine content upon milk addition at all digestion stages, indicating strong interactions between caffeine and milk proteins. UV spectroscopy and HPLC analyses revealed that in black coffee, the initial free caffeine content before digestion was 173.1 µg/mL, which decreased to 90.7 µg/mL post-gastric digestion but increased to 147.9 µg/mL after the intestinal phase, suggesting increased free caffeine concentration at the end of digestion. The free caffeine concentration obtained for milk-based coffee was 142 µg/mL before digestion, and at the end of digestion, it was 108 µg/mL, which indicates that milk addition reduced caffeine recovery. Similarly, the addition of milk reduced the TPC and DPPH radical scavenging activity of pure caffeine and coffee brews. The TPC values for black coffee and milk-based coffee were 133.4 and 94.7 μg gallic acid equivalent (GAE)/mL, respectively, at the end of digestion. The DPPH antioxidant activity values were 27.42 and 3.54 µg/mL, respectively, for black coffee and milk-based coffee after intestinal digestion. The data from FTIR analysis suggest that non-covalent interactions, such as hydrogen bonding and Van der Waals forces, exist between milk proteins and caffeine, which may affect the functionality of caffeine. Shifts at the major caffeine peak regions (2950-2930 cm-¹) indicate the C-H stretching vibrations, suggesting a possibility for hydrogen bonding and Van der Waals interactions. The results from the particle size data are inconclusive in determining the extent of caffeine interactions or bioaccessibility, necessitating further molecular-level and bioavailability studies. Data from ζ-potential measurement reveals that milk-based coffee has a more stable system compared to black coffee after intestinal digestion. This complements other results as higher stability for a colloidal system could be an indication of less availability for further interactions or release of free caffeine. The in vitro digestion studies reveal that the overall bioaccessibility of caffeine decreases when milk is added to coffee brew. The percentage bioaccessibility of caffeine in black coffee and milk-based coffee was 85.8 and 77%, respectively. The findings of this research contribute to the understanding of coffee as a functional beverage and provide insights into how the addition of milk to the coffee brew may affect the functionality.
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Figure 2.2 is re-used with permission.