Fonterra is one of the leading manufacturers and exporters of casein-based ingredients such as milk protein concentrates (MPCs), caseins and caseinates. These casein-based ingredients have increasingly been used in various applications, including neutral-pH beverages, long-shelf-life ultra-high-temperature (UHT)- treated milks, yoghurts, cheeses and many other products. One of the inherent problems is that, during the manufacture and storage of casein-based ingredients such as MPC, the protease system (the plasmin system), which is naturally present in milk, is also concentrated. Therefore, the plasmin activities in such ingredients can be very high.
The protease system occurs in milk, is very complex and includes plasminogen, plasmin, plasminogen activators (PAs), plasminogen activator inhibitors (PAIs) and plasmin inhibitors (PIs). The components of this system have different heat labilities and, when considered as a whole, the system has a moderate resistance to heat inactivation. In products such as UHT milk reconstituted from low-heat skim milk powder or MPC, it is possible that the PAs are free to facilitate the conversion of plasminogen to plasmin during storage because of their survival during heat treatment. The presence of plasmin activity is dependent on favourable conditions for the activation of plasminogen to plasmin in the product and is controlled by and dependent on the presence and status of the PAs and PAIs. Plasmin activity leads to many undesirable flavour and texture defects in the product during storage.
Previous research has suggested that the plasmin in skim milk is largely unaffected by pasteurisation conditions and that, in many cases, 30–40% of its activity remains even after UHT processing. The protease system (plasminogen and its activators) is heat resistant and survives UHT treatments, but its role in the gelation of UHT- treated milk is not yet fully understood. This remains one of the long-standing unresolved problems and an important risk factor or challenge to be considered
when using casein-based ingredients in final applications, particularly neutral-pH applications.
1.2 COMMERCIAL SIGNIFICANCE
Many of the neutral-pH applications of our casein-based ingredients are constrained mainly by a significant gap in capability, knowledge and understanding. The consequence of not developing this capability and knowledge will be that Fonterra might miss out on important opportunities to promote its current ingredients or new ingredients into applications in which plasmin is considered to be a risk factor.
One means of overcoming such consequences is to develop greater fundamental understanding of this problem and to develop capabilities to provide unique, cost- effective solutions. Fonterra is a leading solution provider for dairy ingredient customers by virtue of its expert knowledge in the areas of milk protein functionality and by designing tailor-made value-added ingredients for specific product applications, and would like to remain ahead in this area by continuously developing new skills, providing unique solutions to solve problems and generating new knowledge and new research capabilities. Prevention of the plasmin-induced hydrolysis of casein in the milk system is likely to provide a competitive advantage to Fonterra and will be very beneficial to Fonterra in providing solutions to this complex problem. The increase in the scientific knowledge from the project through high-quality publications and conference presentations will be beneficial to the wider group.
1.3 THESIS OBJECTIVES
This proposed capability development project was aimed at addressing gaps in capability and knowledge in a systematic manner with underlying robust science. The main objectives of this research project were as follows.
Part A: Understanding the effect of substrate modification on plasmin-induced hydrolysis in a pure protein model system
o Effect of lactosylation on the plasmin-induced hydrolysis of β-casein
o Effect of transglutaminase cross-linking on plasmin-induced hydrolysis: a comparative study
Part B: Application of substrate modification in a real milk system to control plasmin-induced hydrolysis
o Succinylation of skim milk and its effect on plasmin-induced hydrolysis: effect of micellar casein structure along with substrate modification
o Plasmin resistance of high-heat-treated milk ‒ a sequential study: effect of lactosylation and the association of whey proteins with the casein micelle on plasmin-induced hydrolysis
Part C: Understanding the plasmin-induced hydrolysis of the milk system
o Plasmin-induced dissociation of the casein micelle
o Role of plasmin in sedimentation and gelation 1.4 THESIS STRUCTURE
The thesis starts with an overall introduction, followed by a literature review and different chapters that address the above objectives. Apart from the main literature review chapter, each chapter also begins with relevant background information that is not addressed in the main literature review. The chapters describing the experimental work are divided into three sections to show the progressive approach, starting from simple systems, progressing to complex systems and concluding with the role of plasmin in sedimentation and gelation. The thesis outline is briefly described below.
Chapter 1 aims to introduce the topic of the research and starts with the problem definition and the commercial significance of the research, followed by the thesis objectives and ends with a brief outline of the whole thesis.
Chapter 2 is a general review of the literature that is relevant to the research topic and discusses different components of the complex plasmin system, the action of plasmin on different milk proteins, factors that affect plasmin activity in milk and the presence of plasmin in milk products.
The second section consists of three chapters on the prevention of plasmin-induced hydrolysis in a pure protein model system. In this section, the mechanism of the prevention of plasmin action on proteins is established using a pure protein model system and a plasmin-resistant protein is developed.
Chapter 3 describes the development of the plasmin-resistant protein using a simple chemical modification, i.e. succinylation, to establish a mechanism for the resistance of protein to plasmin.
In Chapter 4, learnings and the mechanism from the succinylation work are applied using the food-grade modification reaction, lactosylation.
The Maillard reaction is a complex reaction and involves cross-linking along with substrate modification. Therefore, the decrease in plasmin-induced hydrolysis is also studied using the food-grade cross-linking reaction, transglutamination. The effect of cross-linking using the transglutamination reaction on plasmin-induced hydrolysis is discussed in Chapter 5 and the three different means of substrate modification (succinylation, lactosylation and transglutamination) are compared to understand the mechanism better.
Chapter 6 describes the role of the structure of the casein micelle in preventing plasmin action along with the effect of succinylation on plasmin-induced hydrolysis in skim milk.
Chapter 7 describes the effect of lactosylation and the association of whey proteins with the casein micelle on plasmin-induced hydrolysis in skim milk.
Chapter 8 describes the plasmin-induced dissociation of the casein micelle and the physicochemical changes in milk as a result of plasmin action to give further insight into the role of plasmin in sedimentation and gelation in heat-treated milk.
Chapter 9 summarises the key findings of the thesis and gives recommendations. Chapter 10 suggests future directions.