Effect of heat treatment on physicochemical properties and immune functionality of bovine milk proteins

Milk proteins are one of the important nutritional components in bovine milk, besides  carbohydrates, lipids and vitamins. Bovine milk proteins can be classified into two groups, casein and whey proteins. Whey proteins, accounting for 20% of the amount of bovine milk proteins, include β-lactoglobulin, α-lactalbumin, bovine serum albumin, and hundreds of other less abundant proteins. The milk proteins not only provide amino acids as nutrients for the body, but also exhibit several biological functions like bacteriostatic activity and immunomodulation. Immune-active proteins in whey, like lactoferrin, immunoglobulin, and lactoperoxidase, are mainly responsible for these immune-modulating functionalities. A variety of pathogens can multiply in milk due to due to its richness in nutrients, which could poses serious health risks to consumers. Heat treatments, such as pasteurization and sterilization, are extensively used in dairy industry to ensure the safety of dairy products and to extend shelf life. However, heat treatment induced denaturation, aggregation and glycation of whey proteins result in the changes of physicochemical properties and biological activity. This thesis sought to investigate how different heat treatments within the pasteurization range induce structure changes of immune-active proteins, and subsequently change their physicochemical properties and immune functionality.

As one of the important immune-active proteins in milk, lactoferrin is often enriched in infant formula or functional food products, to provide immune benefits for consumer. The aggregation of lactoferrin with other whey proteins may occur during heating, which could reduce the immunological activity of lactoferrin. Therefore, the role of major whey proteins (β-lactoglobulin, α-lactalbumin and bovine serum albumin) in the disulphide linked aggregation of lactoferrin after heating at 55-90 °C for 30 min were investigated in Chapter 2. The disulphide bond interchange within lactoferrin as well as between β-lactoglobulin and lactoferrin were measured to explore the underlying mechanism for disulphide-linked aggregation of lactoferrin. Our results showed that β-lactoglobulin enhanced the disulphide linked-aggregation of lactoferrin the most among these three proteins β-lactoglobulin, α-lactalbumin and bovine serum albumin, which was attributed to the presence of the free thiol group within β-lactoglobulin. After heating at 85 °C for 30 min, 8 cysteines formed 36 different disulphide bonds in 47 different crosslinked peptides among lactoferrin molecules (inter- and intramolecular disulphide bonds) were found. Among these cysteines, 8 cysteines from lactoferrin formed 9 different disulphide bonds in 21 heterologous crosslinked peptides with 2 cysteines from β-lactoglobulin (intermolecular disulphide bonds).

Besides the major whey proteins studied in Chapter 2, whey proteins encompass many more hundreds of proteins that could possibly interact with lactoferrin during heating, with subsequent changes in physicochemical properties and biological activity. The aggregation of lactoferrin in milk serum after heating 65, 70, and 75 °C for 30 min was characterized, and the effect of such aggregation on bacteriostatic activity and in vitro digestion were further investigated in Chapter 3. The aggregation of lactoferrin was enhanced when LF was heated in milk serum, compared to it being heated alone. Lactoferrin heated alone at 70 °C or above led to self-aggregation via disulphide interchange, whereas heating lactoferrin in milk serum already led to the disulphide linked aggregation of lactoferrin at 65 °C and above. Lactoferrin formed disulphide-linked aggregates with whey protein and non-micellar casein, which involved β-lactoglobulin, α-lactalbumin, bovine serum albumin, immunoglobulin, κ-casein and many low abundant proteins. Lactoferrin also formed disulphide-linked aggregates when it was heated with β-lactoglobulin, indicating that the formation of lactoferrin-whey protein complexes was, at least partly, mediated by β-lactoglobulin. The more loss of bacteriostatic activity was observed when heating lactoferrin with whey protein, which was associated with their aggregation. Lactoferrin that was involved in whey protein aggregates formed at 65 °C and 70 °C were more susceptible to infant in vitro digestion, while those formed at 75 °C were much more resistant to digestion. In conclusion, whey protein affected the aggregation of lactoferrin, which subsequently changed both its bacteriostatic activity and digestion behaviour.

Not only lactoferrin is antibacterial, but a wider range of whey proteins its bacteriostatic activity, such as lactoperoxidase and immunoglobulins, as well as low abundant immune-active proteins. Different heat treatments can induce the denaturation of these proteins to a different degree, which could result in a gradual change of their bacteriostatic activity. The effect of different heat treatments on the proteome of whey proteins and their bacteriostatic activity were investigated in Chapter 4. Skim milk samples were heated at 65 °C, 70 °C, 75 °C, 80 °C, and 85 °C, for 30 minutes. Milk serum was isolated from the heat-treated skim milk and the bacteriostatic capacity of this milk serum was tested against Streptococcus thermophilus, Escherichia coli, Lactococcus lactis and Pseudomonas fluorescens. The bacteriostatic activity of milk serum negatively correlated with the intensity of heat treatment, which was also reflected in the reduced level of native antibacterial proteins. There is a slight decrease of bacteriostatic activity, which was associated with denaturation of low abundant immune-active proteins, such as complement C7, monocyte differentiation antigen CD 14 and polymeric immunoglobulin receptor. There is a significant difference between milk samples treated for 30 minutes at <75 °C and milk samples treated at ≥75 °C in both bacteriostatic capacity and native antibacterial proteins. Growth rates of Streptococcus thermophilus, Lactococcus lactis, and Pseudomonas fluorescens were negatively correlated with the retention of lactoferrin and lactoperoxidase. In conclusion, the bacteriostatic capacity of milk serum decreases with increasing heating intensity, which is strongly correlated with the denaturation of antibacterial proteins that extensively occurred after heating at 75 °C for 30 min.

In addition to bacteriostatic activity, immune-active proteins were indicated to be involved in the protection from the development of allergic diseases, but a direct link between these proteins and the protection against allergic diseases was missing, which therefore has been investigated in Chapter 5. Raw cow’s milk was heated at 50, 60, 65, 70, 75 and 80 °C for 30 min and the native whey protein profile of these differentially heated milk samples was determined using LC-MS/MS-based proteomics. The capacity of different heat treated milk samples to prevent the development of ovalbumin-induced food allergy was evaluated in a murine animal model. The allergy-protective effect of raw cow’s milk was lost after heating milk for 30 min at 65 °C or higher temperatures. Although a substantial loss of native whey proteins, as well as extensive protein aggregation, was observed from 75 °C, whey proteins with immune-related functionalities already started to denature from 65 °C, which coincided with the temperature at which a loss of allergy protection was observed in the murine model. Of the immune-active whey proteins, complement C7, monocyte differentiation antigen CD14, and polymeric immunoglobulin receptor concentrations decreased significantly at this temperature. In addition, several other immune-active whey proteins also showed a decrease around 65 °C. This chapter thus suggests that immune-active whey proteins that denature around 65 °C are probably mainly responsible for the allergy-protective capacity of raw cow’s milk, although  their specific role in the loss of the allergy-protective effect still needs to be confirmed.

Chapter 6 discussed how the obtained results in this thesis contribute to our understanding about how different heat treatments within the pasteurization temperature range affected the structure, digestion, and biological function of immune-active whey proteins. Comparison of the aggregation of lactoferrin in a whey protein model system, milk serum and skim milk suggested that heating lactoferrin in the presence of other proteins can increase the irreversible aggregation by sulfhydryl-disulphide interchange. It thereby provides evidence that heating lactoferrin and other protein ingredients in dairy products separately may decrease the disulphide-linked aggregation of lactoferrin with other proteins, which may ultimately help to retain protein bioactivity. The extensive aggregation of lactoferrin with whey proteins, partly via disulphide bond interchange, retards the in vitro gastric digestion of lactoferrin. Therefore, preventing disulphide bond interchange between proteins could be a way to prevent formation of large insoluble aggregates or produce smaller aggregates, leading to higher digestibility. The contribution of different whey proteins in the bacteriostatic and allergy protective activity of raw milk, and the appropriate heat treatment for preserving them, were discussed. A heat intensity below 75 °C/30 min would meet the requirement to maintain the bacteriostatic activity, whereas a heat intensity below 65 °C/30 min would be required to preserve the allergy protective activity. However, with heating below 65°C/30 min, the microbiological safety can’t be guaranteed, therefore, non-thermal methods may be used to inactivate pathogen or other time/temp combinations, other raw milk criteria, etc. could also be used to inactivate pathogen.