Todor Vasiljevic

Bovine β-casein (β-CN) is a major milk protein that exists as multiple genetic variants, distinguished by differences in their amino acid sequences. Among these, the A1 and A2 variants are the most extensively studied. They differ at position 67, where β-CN A2 contains a proline residue, whereas β-CN A1 has a histidine. This single amino acid substitution may influence the enzymatic hydrolysis of β-CN, potentially affecting bioactive peptide release during cheese ripening or human digestion, of most interest are the β-casomorphins (BCMs). These BCMs have opioid-like activity that could impact gastrointestinal signaling. There have been a couple of studies with small-scale experimental cheeses made from milk differing in the β-CN A1 or A2 variant, which have investigated a few types of BCMs. The cheese ripening process is very complicated and many factors influence proteolysis, which could impact the release of BCMs. Exploring commercially manufactured cheeses made from different variants of β-CN that had different levels of maturity could help clarify if there are significant differences in the types and concentrations of a wide range of BCMs. We investigated whether differences in the β-CN polymorphism influenced the proteolytic patterns of a wide range of commercial A1/A2 and A2/A2 Cheddar cheeses. A total of 26 Cheddar cheese samples, comprising β-CN A1/A2 (n = 16) and A2/A2 (n = 10) of different ages were sourced from individual US cheese manufacturers. The β-CN variant present in each sample was confirmed via capillary electrophoresis (CE). pH 4.6-soluble nitrogen (pH 4.6-SN/TN), levels of free amino acids (FAAs), and various types of BCMs were analyzed to assess proteolysis. No significant differences were observed in the composition of the 2 types of Cheddar except for fat levels. Cheeses made with A2/A2 milks tended to have higher fat levels; most current manufacturers of these cheeses tended to be smaller plants where milk standardization facilities may be limited. Although lab studies have suggested that milk from the A2 variant has poorer rennet coagulation properties, we did not observe any difference in moisture contents between the 2 cheese types. The moisture content of cheese can be impacted by alterations in the coagulation behavior. The A2/A2 cheeses exhibited increased levels of total FAAs, which was likely due to having several raw milk cheeses within that group. There were no significant differences in levels of total BCMs between cheese groups (P > 0.05). However, BCM-7 was only detected in A1/A2 cheeses (≤9.75 mg/kg). High levels of BCM-9 were found in both types of cheeses (≤127 mg/kg). The type of β-CN variant in milk impacted the release of individual BCMs in Cheddar cheese but did not alter the total concentration of BCMs.

Commercially relevant processing conditions, including protein concentration, pH and shearing and their impact on the solubility, heat stability, and secondary structure of faba bean proteins (FBPIs), were studied. Most of the examined properties, including protein solubility and heat stability, were due to the simultaneous effects of pH and concentration. The shearing rate played a crucial role in determining the heat stability of FBPI during thermal processing through protein molecular activities, such as inter- and/or intramolecular force interactions. Under the heat treatment conditions (temperature of 95 °C and time of 30 min), the shearing rate of 1000 s⁻¹ enhanced the heat stability, compared to 100 s⁻¹. Meanwhile, concentration and pH shift contributed to the conformation of various protein structures of faba bean protein isolates. This study revealed that these structural changes involve the unfolding of the protein’s native tertiary structure, which likely exposes hydrophobic and sulfhydryl (–SH) groups, ultimately leading to protein aggregation. It also provided a comprehensive understanding of faba bean protein functionality by studying various interactions of FBPI proteins under thermal processing systems.

Electrostatic spray drying (ESD) of a milk protein matrix comprising whey protein isolate (WPI), skim milk powder (SMP), and lactose was compared to conventional spray drying (CSD) and freeze-drying (FD). ESD and CSD were used to produce powders at low (0.12–0.14), medium (0.16–0.17), and high (0.31–0.36) levels of water activity (a_w), while FD powders targeted low a_w (0.12). Maillard reaction indicators were studied after drying and during storage for up to 28 days at 20, 40, or 60 °C by measuring free -NH₂ groups, as an indicator of available lysine, and 5-hydroxymethylfurfural (HMF). After drying, levels of residual free -NH₂ groups were ~15% higher in ESD and FD powders than in their CSD counterparts. CSD powders also had ~14% higher HMF concentrations compared to their ESD and FD counterparts. Storage led to reductions in free -NH₂ groups and increases in HMF content in all powders, the extent of which increased with increasing storage temperature. Reductions in free -NH₂ groups followed first-order reaction kinetics at 20 and 40 °C but second-order reaction kinetics at 60 °C. Lactose crystallization was detected in high-a_w CSD powders after 14 d at 40 °C and in both CSD and ESD powders after 7 d at 60 °C. Overall, we found that ESD is a gentle drying technology which enables production of powders with lower Maillard reaction markers.

Faba bean protein isolate (FBPI) is emerging as a promising protein ingredient in the food industry. However, a lack of comprehensive scientific understanding of its functional properties, particularly solubility, limits broader application. This study investigated the reconstitution behaviour of FBPI under different conditions. For this purpose, FBPI dispersions (5% w/w protein) were prepared with varying pH (6.8 or 7.5), temperature (15, 40, or 65 °C), duration of stirring (30, 60, or 90 min), stirring intensity (1000 or 1500 rpm), and water hardness (0, 200, or 400 ppm). Low reconstitution temperature resulted in greater particle size and lower solubility, while elevated temperature minimised intermolecular attractions, improving solubility. Higher pH increased the net-negative charge and thus enhanced the repulsion between the proteins, leading to greater solubility. Water hardness was another important parameter, as greater hardness generally resulted in greater particle size and lower solubility, likely due to calcium bridging. The selection of conditions for the hydration of faba bean protein isolate is important to produce high-quality and high-stability suspensions.

Lactoferrin (LF) is one of the minor milk proteins that has been gaining attention for its beneficial physiological functions to human health, as an antioxidant, antimicrobial, anti-inflammatory, immunomodulatory, anti-viral and bone growth agent. These characteristics are partly attributed to its ability to bind iron, which results in regulating the sequestration and release of iron in the body and partly due to its ability to interact with the molecular and cellular components of pathogens and their hosts. For this reason, LF is marketed as a functional component in various types of products, including infant formula, and in tablets or other types of supplements for children and adults. Bovine LF (BLF) is the main commercial protein ingredient from large-scale extraction using various technologies. The use of cationic exchange chromatography is the most common technology, and allows BLF to be extracted with around 95% purity and 87–93% of the isolated protein. Due to its characteristics and its various health benefits, BLF has also been studied as an additional component in new therapeutic applications in serious diseases such as upper and lower respiratory tract infections and COVID-19, cancers, for use in dermatology and regenerative medicinal engineering.

Micellar calcium phosphate (MCP) plays an important role in maintaining the structure and stability of the casein micelle and its properties during processing. The objective of this study was to investigate how heating (10 min at 80 or 90 °C) at different pH levels (6.3, 6.6, 6.9, or 7.2) impacted the acid-induced gelation of MCP-adjusted milk, containing 67 (MCP₆₇), 100 (MCP₁₀₀), or 113 (MCP₁₁₃) % of the original MCP content. The unheated sample MCP₁₀₀ at pH 6.6 was considered the control. pH acidification to pH 4.5 at 30 °C was achieved with glucono delta-lactone while monitoring viscoelastic behaviour by small-amplitude oscillatory rheology. The partitioning of calcium and proteins between colloidal and soluble phases was also examined. In MCP-depleted skim milk samples, the concentrations of non-sedimentable caseins and whey proteins were higher compared to the control and MCP-enriched skim milk samples. The influence of MCP adjustment on gelation was dependent on pH. Acid gels from sample MCP₆₇ exhibited the highest storage modulus (G′). At other pH levels, MCP₁₀₀ resulted in the greatest G′. The pH of MCP-adjusted skim milk also impacted the gel properties after heating. Overall, this study highlights the substantial impact of MCP content on the acid gelation of milk, with a pronounced dependency of the MCP adjustment effect on pH variations.

The effect of shear on heat-induced changes in milk protein concentrate suspensions was examined at different pH levels, revealing novel insights into micellar dissociation and protein aggregation dynamics. Milk protein concentrate suspensions, adjusted to pH of 6.1, 6.4, 6.8, or 7.5, underwent combined heat (90 °C for 5 min or 121 °C for 2.6 min) and shear (0, 100, or 1000 s⁻¹) treatment. The fragmentation of protein aggregates induced by shear was evident in the control MPC suspensions at pH 6.8, irrespective of the temperature. At pH 7.5, shear increased the heat-induced micellar dissociation. This effect was particularly pronounced at 121 °C and 1000 s⁻¹, resulting in reduced particle size and an elevated concentration of κ-casein (κ-CN) in the non-sedimentable phase. At pH 6.1 or 6.4, shear effects were dependent on sample pH, thereby modifying electrostatic interactions and the extent of whey protein association with the micelles. At pH 6.1, shear promoted heat-induced aggregation, evidenced by an increase in particle size and a significant decline in both whey proteins and caseins in the non-sedimentable phase. At pH 6.4, shear-induced fragmentation of aggregates was observed, prominently due to comparatively higher electrostatic repulsions and fewer protein interactions. The influence of shear on heat-induced changes was considerably impacted by initial pH.