The impact of genetic variation on functional, technological and digestive properties of β-lactoglobulin

Kelly, Liam M.

The impact of genetic variation on functional, technological and digestive properties of β-lactoglobulin

cb

Files

PhD Thesis Liam Kelly 116309156.pdf(5.26 MB)

Full Text E-thesis

Date

2025

Authors

Kelly, Liam M.

Publisher

University College Cork

Abstract

Genetic variations of milk proteins have been studied since their discovery in 1946. Since then, over 50 variants have been found among the four caseins (αs1-, αs2-, β-, and κ-casein) and the two main whey proteins, β-lactoglobulin (β-lg) and α-lactalbumin. In the early 1990’s, it was speculated that certain genetic variants of β-casein could be associated with negative health effects, including heart disease, diabetes and schizophrenia. While no scientific evidence has since been published in support of these health claims, these reports did stimulate a renewed scientific and commercial interest in genetic variants of milk proteins, with some companies even structuring their business models around them, such as the a2 Milk Company (Auckland, New Zealand). Undoubtedly, there is potential for genetic variation among the milk proteins to alter functional, technological and possibly nutritional and digestive properties of dairy products. Thus, research is required to define potential beneficial and deleterious effects of specific protein variants on the processing and nutritional quality of milk. Irish herds generally do not select for specific genetic variants as part of the national breeding programme, resulting in a diverse pool of variants among Irish cows. Initially, to support understanding of the distribution of genetic variants within an Irish herd, the protein profiles of 48 cows was assessed; of these cows, 17 genetic variants were identified among β-casein, κ-casein and β-lg. The A2A2 β-casein genotype had the highest frequency in Holstein Friesian (HF) cows; however, the A1I and the A2B genotypes were associated with higher levels of true protein and casein. In addition, it has also been found that milk with the A variant of β-lg was associated with higher levels of total whey proteins compared to milk with the B variant. This difference in β-lg concentration has been linked to the DNA mutations that cause the genetic variation being located close to the promoter region for gene expression. The differences in the macro composition of milk as a result of genetic variation could be of benefit to both the farmer and milk processor, in terms of farm gate milk price and differentiated added value products, respectively. Additionally there may also be a benefit to consumers who seek milk products with designated milk genetic variants. Identification of the genetic variants among 48 cows, using high performance liquid chromatography analysis, indicated that the AA, AB and BB genotypes of β-lg were commonly distributed among the cows tested. As β-lg is the principal whey protein on a mass basis, it determines the nutritional and functional quality of subsequent whey protein ingredients, such as concentrates and isolates. These whey protein ingredients, produced from pre-treated cheese whey, or isolated directly from milk, through a combination of microfiltration (MF), ultrafiltration (UF)/nanofiltration and spray drying, often find application in high value, life-stage nutritional products. Thus, the primary whey protein β-lg, its genetic variants and ingredients/products produced therefrom, are the primary research focus of this thesis. The primary structural differences between the A and B variants of β-lg are due to amino acid substitutions that occur at positions 64 and 118. At position 64, the A variant has an aspartic acid, whereas the B variant has a glycine. Aspartic acid contains a charged functional group, while the glycine functional group is not charged. This substitution in particular causes the A variant of β-lg to have a greater net negative charge. The substitution that occurs at position 118, in which the A variant has a valine and the B variant has an alanine is not thought to significantly impact the structure, and thus functionality, of β-lg. Most studies to date have assessed the functional properties of β-lg in milk, a complex system in which other components may present confounding factors which mask differences in functionality attributable to the protein variant. By purifying the whey protein fraction of milk into a whey protein isolate (WPI) containing either the A or B variant of β-lg, the true effects of genetic variation can be assessed with limited interference from other components of milk. As this thesis had already identified cows that produced only the homozygous AA or BB genotype of β-lg, their milk was segregated for separation of the individual β-lg variants using a combination of MF and UF. MF is widely used in the manufacture of WPIs from pre-treated cheese whey, and also in the separation of caseins and whey proteins from skim milk. While separation and purification of the β-lg variants was the primary goal, their impact on the efficiency of whey protein separation was also assessed. The results of this work demonstrated that the accumulation of reversible and irreversible foulants, rejection of whey proteins and overall process efficiency were determined by the β-lg variant present in the skim milk. This is not surprising, considering that fouling of the membrane plays a critical role in both flux decline, and by proxy, process efficiency, with β-lg reported to be a major contributor to fouling. It was found that transmission of whey proteins during MF of skim milk was higher for the skim milk containing the A variant than the B variant of β-lg, and while the skim milk containing the A variant had higher accumulation of both total and reversible fouling than the skim milk with the B variant, it was not rate limiting relative to whey protein permeation. These results indicate that skim milk with the A variant of β-lg could be advantageous to whey processors in terms of process efficiency and yield. Analysis of the WPIs produced by a combination of MF and UF described in this thesis identified unique functional properties specific to the β-lg variants. The thermal denaturation and gelation onset temperatures were higher for the WPI with the B variant of β-lg. However, despite the A variant having a lower denaturation/gelation temperature, it had better stability against heat-induced gel formation in the pH range 6.6-7.2. These differences in functionality, specifically in terms of denaturation and aggregation processes, presented additional research questions surrounding behaviour of the proteins in more complex products. By manufacturing WPI containing either β-lg A or B genetic variants, and observing unique functional properties between the variants, the research question was proposed as to whether the differences between the variants would also be evident in a more complex dairy product, and whether the variants had a significant effect on in vitro digestion behaviour. Infant milk formula (IMF) was thus chosen as a suitable system to evaluate the β-lg variants, as it constitutes approximately 50% of the protein in a 0-6 month formula. By creating IMF with different heat treatments of either high temperature short time (HTST) or ultra-high temperature (UHT), containing either the A or B variants of β-lg, the impact of the genetic variation of β-lg on digestion was assessed using a semi-dynamic infant digestion model. While the effect of heat treatment between the samples was very clear, surprisingly, there was little difference between the β-lg variants during simulated digestion, in terms of curd formation, protein breakdown and free amine concentrations at the end of digestion. Overall, the original research presented in this thesis provides new and important insights into the impact of genetic variation on the functional, technological and digestive properties of β-lg, by producing purified whey ingredients for evaluation at semi-commercial scale. Overall, there does appear to be a case for selection of the A variant of β-lg in the dairy herd, especially for dairy processors who specialize in whey protein ingredients.

Keywords

Genetic variation , Protein chemistry , β-lactoglobulin

Citation

Kelly, L. M. 2025. The impact of genetic variation on functional, technological and digestive properties of β-lactoglobulin. PhD Thesis, University College Cork.