The effect of heating processes on milk whey protein denaturation and rennet coagulation properties

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1 Master Thesis The effect of heating processes on milk whey protein denaturation and rennet coagulation properties Effekt af varmebehandling af mælk på valleprotein denaturering og koaguleringsegenskaber med chymosin Marije Akkerman Department of Food Science, Aarhus University Student number

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3 Preface The present master thesis, The effect of heating processes on milk whey protein denaturation and rennet coagulation properties of 60 ECTS was part of the education Molecular Nutrition and Food Technology at Aarhus University and was performed in the period October 2013 to October2014. The thesis was carried out at Arla Strategic Innovation Centre, Brabrand, and department of Food science, Aarhus University. The project was done with supervision from Lotte Bach Larsen from Aarhus University, and Mette Christensen from Arla Foods. Acknowledgement First, I would like to thank my supervisors Lotte Bach Larsen and Mette Christensen for good and helpful supervision. Furthermore, I would like to thank Dairy technician Kent Matzen for assistance in the pilot plant and especially Lene Buhelt Johansen, Per N. Andersen, Betina Hansen and Hanne Søndergaard for experimental guidance in the laboratories at Arla Strategic Innovation Centre, Brabrand, and Department of Food Science, Aarhus University, and for help with data analysis. l would also like to thank Valentin Rauh for help with data analysis and good discussions of results throughout the project and Eva Hansen for professional revision of the final report. At last, special thanks to my family and friends for moral support. Aarhus University, Department of Food Science, October

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5 Abstract Whey protein denaturation as a cause of heat treatment has been investigated by various authors with the study by Dannenberg and Kessler (1988) being the most acknowledged. Skim milk with various contents of whey proteins and caseins were heat treated using three different heating processes, namely Plate Heat Exchanger (PHE), Tubular Heat Exchanger (THE) and Direct Steam Injection (DSI) to provide further insight into how heat treatment affects whey protein denaturation and rennet coagulation. The samples were subjected to heating from 80 C to 145 C and holding times from 2s to300s. The milk samples were analysed on liquid chromatography (LC) to analyse the degree of whey protein denaturation, while rennet coagulation analysis were made with ReoRox rheometry. Heat induced aggregates were analysed at 1D- and 2D gel electrophoresis and size exclusion chromatography. Heat treatment increased the degree of whey protein denaturation as the holding time increases for all temperatures. Heat treatment using DSI gave the smallest increase in denaturation at all temperatures, with a degree of denaturation of 40 % of β-lactoglobulin B (β-lg B) heating at 145 C, while heating using THE and PHE showed denaturation degrees above 95 % heating at 130 C. Temperatures below 100 C resulted in higher degree of denaturation for heat treatment using THE compared to PHE, while at 130 C more than 90 % of β-lg B was denatured for both methods. A reaction order of 1.5 was found for β-lg and 1 for α-lactalbumin, with reaction kinetics showing similar pattern compared to previous findings. The variations are caused by different heating systems and heating profiles which have great impact on the whey protein denaturation. Rennet coagulation properties were impaired as the holding time increases for all temperatures. Heating using DSI resulted in the least impairments. Heat treatment using PHE gave better rennet coagulation properties when heating at temperatures below 100 C, compared to THE, while THE reached Rennet coagulation time(rct) within two hours at temperatures of 130 C which was not observed for heat treatment using PHE. The differences in heating systems using PHE and THE were primarily caused by variations in formation of heat induced aggregates. THE was found to result in a higher degree of large whey protein and κ-casein complexes, while a higher proportion of smaller whey protein aggregates were found when heating using PHE. Milk with reduced whey protein content showed improved rennet coagulation properties for all heating methods compared to skim milk. Increasing the casein level and reducing the whey protein content gave further improvements of rennet coagulation properties. Heating these at high temperatures, however, resulted in impairments of rennet coagulation properties which can be caused by a change in mineral solubility, casein dissociation and degradation of lactose which decrease the ability for rennet cleavage.

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7 Resume Denaturering af valleproteiner pga. varmebehandling er blevet undersøgt af forskellige forskere, hvor de mest anerkendte undersøgelser er foretaget af Dannenberg og Kessler (1988). For at få indsigt i forskellige varmebehandlingers påvirkning af denaturering af valleprotein og koaguleringsegenskaberne med chymosin, blev skummetmælk med forskellige indhold at valleproteiner og kaseiner udsat for tre typer varmebehandling: plade varmeveksler (Plate Heat Exchanger, PHE), rør varmeveksler (Tubular Heat Exchanger, THE) og injektion af damp (Direct steam injection, DSI). Temperaturer fra 80 C til145 C og holdetider fra 2 til 300 sekunder blev anvendt. Mælkeprøverne blev analyseret på væskekromatografi (liquid chromatographt,lc), for at undersøge denatureringen af valleproteiner, mens koagulerings-egenskaberne blev analyseret med ReoRox rheometer. Proteinaggregater dannet ved varmebehandling blev analyseret ved 1D og 2D gel-elektroforese og størrelseskromatografi. Varmebehandling øgede denatureringsgraden af valleprotein når holdetiderne øgedes for alle temperaturer. Varmebehandling med DSI gav mindst denaturering for alle undersøgte temperaturer, hvor 40 % af β-lactoglobulin B (β-lg B) var denatureret ved en varmebehandling på 145 C. Varmebehandling med THE og PHE resulterede i over 90 % denaturering af β-lg B ved varmebehandlinger på 130 C. Temperaturer under 100 C gav højere denaturering af valleproteiner ved brug af THE i forhold til PHE, hvor der ved de højere temperaturer var denatureringsgrader over 90 % for begge metoder. En reaktionsorden på 1,5 blev fundet for β-lg og 1 for α-lactalbumin (α-la). Variationer i aktiveringsenergi, sammenlignet med tidligere studier skyldes forskelle i varmebehandlingssystemer og varmeprofilen, hvilket har stor indflydelse på denatureringsgraden af valleproteinerne. Koaguleringsegenskaberne for koagulering med chymosin blev forværret når holdertiden blev øget for alle undersøgte temperaturer, hvor varmebehandling med DSI gav færrest ændringer. Varmebehandling med PHE viste bedre koagulering i forhold til THE for temperaturer under 100 C, hvorimod dette var modsat ved varmebehandlinger ved 130 C. Koaguleringspunktet blev opnået indenfor to timer ved THE, men ikke ved PHE. Forskellene i denaturering og koagulering med chymosin mellem THE og PHE skylles primært dannelsen af proteinaggregater som konsekvens af varmebehandlingen. THE havde en større andel af store aggregater bestående af κ-kasein og valleproteiner, hvorimod PHE havde en større andel mindre aggregater bestående af κ-kasein og valleproteiner. Varmebehandling af mælk med reduceret indhold af valleproteiner gav forbedringer af koaguleringsegenskaberne ved alle varmebehandlingsmetoder, i forhold til skummetmælk. Mælk med øget kasein indhold og reduceret valleprotein indhold gav yderligere forbedringer. Varmebehandling af disse med ved høje temperaturer gav fald i koaguleringsegenskaberne i forhold til kontrolmælken, hvilket kan skyldes en lavere opløselighed af mineraler, at kaseiner forlader kaseinmicellen, og degradering af laktose pga. varmebehandlingern og disse har alle negativ indflydelse på chymosins kløvning af κ-kasein.

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9 Abbreviations 1DGE 2DGE BSA CFR Cys DSI DTE E a FTIC Gly HCl HTST IG LC LTLT LVR MCI MCIc MF MS OHTC PHE pi RCT RP-HPLC RT SDS SDS-PAGE SEC TFA THE UHT α-la β-lg 1-dimensional gel electrophoresis 2-dimensional gel electrophoresis Bovine Serum Albumin Curd firming rate Cysteine Direct Steam Injection Dithiothreitol Activation energy Flouroscein-5-isothiocyanate Glycine Hydrochloric acid High Temperature, short-time heating Immunoglobulin Liquid chromatography Low temperature, long time Linear viscoelastic region Micellar Casein Isolate milk, total protein adjusted Micellar Casein Isolate milk, casein adjusted Microfiltration Mass spectrometry Overall heat transfer coefficient Plate Heat Exchanger Isoelectric point Rennet coagulation time Reverse Phase High Performance liquid chromatography Retention time Sodium dodecyl sulphate Sodium dodecyl sulphate polyacrylamide gel electrophoresis Size exclusion chromatography Triflouroacetic acid Tubular Heat Exchanger Ultra High Temperature α-lactalbumin β-lactoglobulin

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11 Contents 1 Objective Background Milk proteins Casein Whey proteins β-lactoglobulin α-lactalbumin Heat treatment of milk Heat treatment methods Plate Heat Exchanger Tubular Heat Exchanger Direct Steam Injection Heat induced denaturation of milk proteins Protein separation methods Liquid Chromatography Gel electrophoresis Protein identification Rennet coagulation of milk proteins Measurement of coagulation properties Materials and methods Milk types Heat treatments Protein analysis Analysis of total protein Analysis of ph 4.5 soluble protein Measurement of rennet coagulation Analysis of protein structure Analysis of protein aggregates DGE DGE Analysis of protein aggregate composition Statistical analysis

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13 4 Results Variation in milk protein composition Effect of indirect heating on skim milk Effect of indirect and direct heating systems on heat treatment of skim milk Effect of whey protein denaturation on rennet coagulation Effect of heat treatment of MCI milk samples on rennet coagulation Effect of milk type on rennet coagulation Formation of protein network Heat induced protein aggregation DGE DGE Size exclusion chromatography Identification of aggregates Kinetics of denaturation of whey proteins Discussion Conclusion Perspectives Future research References Appendix Appendix

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15 1 Objective For decades, the whey fraction was considered a waste product from the cheese production but today it is used as functional ingredient in a wide variety of food products. Some important functions of whey proteins in foods are water binding capacity, emulsification, foaming, whipping and gelation properties (Singh, 2011). To utilize these functionalities of the whey proteins optimal it is crucial to know the physical and chemical properties of each of the components in the product, as well as the interactions between the different components. The physical properties of whey proteins have been investigated for many years, both in purified form and in the milk matrix. One of the most dominant features is the heat denaturation of the proteins which has great impact on the usage and functionality of the proteins. The most acknowledged studies on the heat induced whey protein denaturation in skim milk are done by Dannenberg and Kessler (1988). Their results have been supported by various authors since, but given that the industry uses several types of heating protocols and new analysing methods, it is of interest to be able to verify these results and to be able to use this knowledge in the manufacturing of dairy products. The objective of this master thesis is to: Study the effect of three heating processes on milk whey protein denaturation and rennet coagulation properties on milk with various whey protein and casein content. Study how whey proteins influence the formation of heat induced protein aggregations. The hypotheses are: Increase in heating temperature and holding time will lead to an increase in whey protein denaturation which will have a negative effect on the coagulation properties of the milk in cheese production. Direct steam injection is the most gentle heat treatment, leading to least whey protein denaturation, while heat treatment using tubular heat exchanger and plate heat exchanger results in higher degrees of whey protein denaturation. Removal of whey proteins from milk will improve coagulation properties compared to skimmed milk. Increasing the heating temperature and holding time will lead to formation of large protein aggregates due to unfolding and denaturation of whey proteins

16 To test the hypotheses, skimmed milk with various whey protein and casein content were heated treated using three different heating methods, namely Plate Heat Exchanger, Tubular Heat Exchanger and Direct Steam Injection, at varying temperatures and holding times and combinations to investigate rennet coagulation properties and protein composition. 2 Background Milk and its individual compounds are of major importance in relation to human nutrition throughout millennia. The oldest evidence for consumption of milk from domesticated animals dates back to 4000 BC (Schmid, 2003). Bovine milk is the main type of milk used for human consumption. Various other species such as goats, sheep and horses also yield milk that is used in the food industry but these play a minor role (Belitz et al., 2004). Throughout this report, the term milk refers to bovine milk. Milk composition consists of approximately 87 % water, 3.5 % protein, 4.4 % fat, 4.5 % lactose, 0.7 % minerals with a ph of The composition of milk is not completely constant. There are variations in relation to breed, genetic variation, lactation stage, health, feed composition, climate and season (Heck et al., 2009). 2.1 Milk proteins Bovine milk contains between % proteins and variations are mostly caused by breed and genetic variation. More than 200 types of protein have been found in milk but only few groups of proteins are present in large quantities. The two major groups of proteins are caseins, representing 80 % of the protein in milk, and whey proteins which represent 20% of the milk protein. More than 60 different enzymes are found in milk, but they represent less than 1 % of the total protein content in milk (Farrell Jr. et al., 2004). The casein and whey protein composition is shown in Table 1 and key points from this table will be used in the following sections

17 Table 1. Distribution of the major proteins in milk and their main characteristics. Modified according to (DaLgleish, 2014; Farrell Jr. et al., 2004). Protein Amino acid residues Molecular mass (kda) Proline residues Phosphoseryl residues S-S linkages Approx. amount in milk (g/l) % of protein Disulphidresidues Total casein α S1 -Casein α S2 -Casein β-casein κ-casein Total whey β-lactoglobulin α-lactalbumin Bovine Serum Albumin Immunoglobulin Casein Casein is the protein fraction in milk which is characterized by precipitating at ph 4.6 at a temperature of 20 C. The caseins are divided into four major components, -, -, β- and κ-casein which are generally distributed in the proportions 40 %, 10 %, 40 %, 10 %, respectively, of the total casein (Dalgleish, 1993). They all contain high amounts of ester bound phosphate, proline residues and contain no or very few disulphide bonds, which is shown in Table 1. This results in very little secondary structure and random coiling of the primary chain. The structure makes the casein flexible, and the lack of secondary structure and thus an open structure exposes the hydrophobic parts of the amino acid chain which gives a higher surface hydrophobicity (Fox and Kelly, 2006). This open structure also makes caseins more vulnerable to proteolysis by various enzymes, especially pepsin, as they have easy access to the protein backbone and allowing them to cleave the protein into various peptides(swaisgood, 2003). Caseins belong to the group of phosphoproteins which have phosphoric acid groups bound to the amino acid backbone. The phosphate groups are located in clusters bound to Serine resides. The phosphate groups have, because of their negative charge, the ability to bind ions, especially Ca 2+. The binding of ions is essential for the transport of calcium and phosphate to the neonate. Caseins are sensitive to change in the calcium level in milk, and increasing calcium level can induce precipitation of these (Considine and Flanagan, 2008). -casein is the most calcium sensitive type of casein while κ-casein with the lowest amount of phosphate groups is not affected by the calcium concentrations normally present in milk (Ginger and Grigor, 1999)

18 Caseins have a tendency to associate with each other in casein micelles through hydrogen bonds to stabilize the structures. Around 95 % of the native casein is bound in casein micelles. 94 % of the casein micelles are protein and the remaining 6 % are referred to as colloidal calcium phosphate consisting of calcium, phosphate and small amounts of magnesium, citrate and other trace elements (Walstra, 1990). Casein micelles vary in size with an average diameter of 200 nm. There are several models trying to explain the composition of the casein micelle structure, e.g. the nanocluster model by Holt and Horne (Farrell Jr. et al., 2006) and the sub-micelle model by Walstra (1999) which are shown in Figure 1. Until now, none of the models are completely verified. It is surely known that the most hydrophobic and most calcium sensitive caseins, α- and β-casein, are primarily found in the core of the casein micelle while the more hydrophilic and calcium insensitive κ-casein is situated at the surface with its polar C-terminal outside the micelle core, making the casein micelle soluble in solution. Figure 1. Two models of the casein micelle structure. A: Nanocluster model with tread-like casein monomers and calcium phosphate nanoclusters (Farrell Jr. et al., 2006). B: casein sub-micelle structure with caseins bound in small sub-micelles in corporation with calcium phosphate which are bound to each other (Walstra, 1999) Whey proteins Whey proteins are the proteins in milk, which are soluble in solution at ph 4.6. Whey can be separated from the casein faction of milk during coagulation processes of the casein, such as rennet or acid coagulated cheese (Singh and Havea, 2003). The whey proteins contribute about 20 % of the total protein content in milk. The heterogeneity is large among the whey proteins and they share only few characteristics such as they are all soluble at ph 4..6 and at a temperature of20 C, at which the caseins are precipitated (Farrell Jr. et al., 2004). The four major whey proteins represent 90 % of all whey proteins. These are β-lactoglobulin (β-lg), α-lactalbumin (α-la), - 4 -

19 Bovine Blood Serum Albumin (BSA) and Immunoglobulins (Ig s). The remaining 10 % are different proteins such as lactoperoxidase, serum transferrin, enzymes and milk fat globular membrane proteins (Fox and Kelly, 2006).The main characteristics for the major whey protein are shown in Table 1. The whey proteins are highly structured proteins with stable secondary and tertiary structures. The major forces responsible for maintaining their globular structure are disulphide bonds, hydrophobic interactions, hydrogen bonding, ion-pair interactions and van der Waal s interactions (Singh and Havea, 2003). The native composition of the whey proteins makes them highly soluble in the milk over a broad range of ph. This is due to the large proportion of hydrophilic residues on the surface of the globular structure and the large amount of disulphide bonds (Dissanayake and Vasiljevic, 2009). The globular structure also makes the proteins resistant to proteolysis. They function as carrier proteins for different molecules, partly responsible to maintain the osmotic balance and immune responses, but not all functions are yet fully understood (de Wit, 1998). Pressure has also been shown to have an impact on whey protein stability. Pressure changes the globular structure which can lead to denaturation (Mozhaev et al., 1996). β-lg is the most pressure sensitive and can withhold pressure up to 200 Mpa, while the other whey proteins are stable for pressures up to 400 MPa (Considine et al., 2007). In this project, the focus will be on the two main whey proteins, namely β-lg and α-la, which contributes to 70 % of the total whey protein content and will be discussed below β-lactoglobulin β-lg is the major whey protein in milk, representing around 50 % of the whey proteins, and 12 % of the total protein content. β-lg consists of 162 residues per monomer with a molecular mass of 18,3 kda. These main characteristics are also shown in table 1.The structure of β-lg is shown in figure 2. Five of the residues which are highlighted in figure 2, are cysteine (Cys). Cys forms intermolecular disulphide bonds at Cys66-Cys160 and cys106-cys119, whereas Cys121, which does not form intermolecular disulphide bonds, is buried within the native structure. β-lg is a highly structured protein with anti-parallel β-sheets formed by nine β-strands and one α-helix (Kontopidis et al., 2004)

20 Figure 2. Structure of β-lg with the 5 cysteine residues and disulphide bonds highlighted. 13 different genetic variants of β-lg are identified, but the most common variants in Western dairy cattle are variant A and B which differ at position 64 (Glycine Aspartic acid) and 118 (Alanine Valine) in the amino acid composition. Variant A has shown to yield smaller aggregates than variant B which can be caused by variant B being less heat stable than variant A (Jakob and Puhan, 1992). β-lg belongs to the protein family of lipocalins, which act as transport proteins for small hydrophobic molecules, such as retinols (vitamin A) and lipids. The biological function of β-lg in milk is not fully understood, but it is believed to have other functions than delivering large quantities of amino acids to the neonate. It is known that β-lg is involved in the retinol transport from the mother to the neonate as it is resistant to proteolysis and acids in the stomach (Miranda and Pelissier, 1983). Palmitate has also shown to bind to β-lg and as vitamin A and it derivates are fat soluble, it can also be reasonable to believe that β-lg is related to fatty acid metabolism in milk (Pérez and Calvo, 1995). Under physiological conditions, β-lg is found as a dimer of two β-lg proteins in equilibrium with the monomer and can rapidly be transformed to native monomers (Croguennec et al., 2004; Papiz et al., 1986). The native monomer-dimer equilibrium shifts towards the monomer state when the β-lg concentration is low, the ionic strength is low or ph is increased above 7 (Reithel and Kelly, 1971) α-lactalbumin α-la represents 20 % of the whey protein in bovine milk. It is a small protein with 123 residues and a molecular weight of 14 kda. These main characteristics are also shown in table 1. 8 of these residues are cysteine which makes four intermolecular disulphide bonds at Cys6-Cys-120, Cys28-Cys111, Cys61-Cys77 and Cys 73-Cys91(Belitz et al., 2004)..There are several different genetic variants of α-la, but only variant B is present in western dairy cattle

21 α-la is the regulatory protein of the lactose synthase enzyme system and the concentration of lactose in milk is directly related to the concentration of α-la (Caffin et al., 1985). α-la enhances the binding of glucose to galactosyl transferase which is the limiting step in the lactose synthesis. Lactose is a very important component in milk and is responsible for maintaining 50 % of the osmotic pressure in milk which is therefore also an indirect physiological role of α-la (Brew, 2003). α-la is a metallo protein and can bind one Ca 2+ molecule per molecule. α-la has a strong calcium binding site which is important for the stability of α-la during heating as calcium increases the stability of α-la. 2.2 Heat treatment of milk Heat treatment is one of the major processing steps of milk. Regardless of its final use, the majority of milk is heat treated at least once. The most used heat treatments of milk are pasteurization and sterilization. Heat treatment of milk is preformed to limit bacterial load and enzyme activity to secure the safety of the dairy product for human consumption and for extending the shelf life of the final product. Heat treatment also gives rise to different chemical changes in milk, such as non-enzymatic browning reactions involving lactose and especially lysine residues in the protein. This gives rise to off-flavours, change in colour and loss of available lysine which has high nutritive value (Singh and Waungana, 2001). Furthermore, when heating above 100 C can cause decrease in ph, which is caused by formation of organic acids from lactose degradation and precipitation of calcium phosphate (Martinez-Castro et al., 1986). Pasteurization is a heat treatment which aims to reduce the number of pathogens to such an extent that it is does not constitute any health hazard. There are different pasteurization methods but one of the most used is high-temperature, short-time heating (HTST) where the milk is heated to C for s. Pasteurization at low temperature, long time (LTLT) at 63 C for 30 min is still used, but not to the same extent as HTST due to longer processing time and it has been shown that HTST gives less chemical changes than LTLT heat treatment (Lewis and Deeth, 2008). Ultra high temperature (UHT) heating is a sterilization process of milk which is used to destroy all microorganisms and spores in the milk and many enzymes are also inactivated. This is done by heating temperatures of C for 1-10 seconds (Fox and Kelly, 2006). The design of heat treatment and combination of temperature and heating time depends on the desired approach with least undesirable changes. High heat treatment and heating time gives most significant changes. Heat treatment causes whey protein denaturation which is an irreversible process. The mineral balance also changes during heat treatment. Calcium and phosphate becomes more insoluble and binds to the casein micellar structure. This is reversible for temperatures below 100 C, while severe heating can - 7 -

22 cause hydrolysis of phosphorserine of caseins and calcium phosphate can precipitate out of solution which are irreversible processes (Gaucheron, 2011) Heat treatment methods Various systems and technologies associated with different time temperature profiles are developed to obtain pasteurized and UHT milk, and the effect on the nutritional and sensory quality of commercial milk samples may vary substantially depending on the process. All heating processes of milk is divided in three steps; a preheating period, a heating period and a cooling period. In all steps heat must be transferred from one material to another. The heat exchange between two materials can be classified according to various parameters such as construction of the heat exchangers, flow arrangements, and phase of the process fluid (Thulukkanam, 2013). Here, the heat exchange is classified according to the heat transferring process. The preheating and cooling steps in all heating processes are always done indirectly, but the heating to the desired temperature can be done either directly or indirectly. In the indirect heating system, the heat transfer to milk is done via a medium, separating milk and the heating fluid which is normally hot water or steam. The temperature difference between the two fluids or steam facilitates the heat transfer between the fluids. This type of heat exchange requires large surface areas for heat exchange to get a sufficient heat transfer and an equal heat distribution in milk. In the direct Figure 3. Heating profiles for indirect and direct heating systems. Modified from Deeth and Datta (2002)

23 methods, the heating medium is steam which is in direct contact with the milk and as the steam condensates the heat is transferred to the milk (Lewis and Deeth, 2008). Indirect continuous heating systems are most commonly used. One of the main advantages of the indirect systems is the regeneration of heat which is possible by using counter-current heat exchange. The fluids flow in reverse directions of each other to minimize the temperature gradient between the two fluids and thereby reducing the fouling and minimize the energy used in the heating and cooling processes. In cocurrent heat exchange the fluids flow parallel to each other in the same direction. This gives a large temperature gradient when starting the heating process which can cause thermal stress in the exchanger material and gives rise to larger loss of heat as it cannot be utilized to the same extent as for counter current heat exchange (Visser and Jeurnink, 1997). Fouling can be a major issue in indirect heating systems. Fouling can be protein denaturation, which is mostly seen at temperatures below 110 C, while mineral fouling is seen at higher temperatures. Fouling is seen as deposits layer on the surface of the heat exchanger. Fouling can cause decrease in milk flow, increase in pressure through the system and reduces the heat rate transfer(boxler et al., 2014). The indirect heating systems can be used in a temperature range from 0 C up to 150 C while the direct heating system is only used for high temperature treatments due to milk being mixed with steam under pressure which forces the temperature to rise above 100 C ( Bansal and Chen, 2006). The direct heating system has a lower heat load than indirect heating when comparing these at equivalent bacterial effectiveness due to fast heating and cooling rates. This is shown in figure 3 at which the temperature-time profiles for UHT treatment using indirect and direct heating systems is displayed (Deeth and Datta, 2002). The difference in heating time and temperature can have a great impact on the denaturation of whey proteins. Preheating is always done indirectly by heating milk up to C. This step is also called protein stabilization step as the preheating temperature is often held from 15 seconds to a few minutes to denature the whey proteins and thereby reduce their ability to foul the heating system at higher temperatures in indirect systems. The holding period at preheat temperature is normally not used for direct systems as fouling is not a general problem due to the fast heating rate (Lewis and Deeth, 2008). The preheating step is also beneficial to minimize the energy used for heating, as it requires large amounts of energy to reach desired temperatures above 100 C in one step. The holding time at heating temperatures is easily changed for the heating systems. When the fluid has reached the desired temperature, the fluid is held at the desired temperature for a fixed time. The holding time can be varied from a few seconds up to several minutes (Edgerton et al., 1970). After passing the holding tubes the fluid goes into indirect system for cooling for both direct and indirect heating systems

24 Plate Heat Exchanger Plate heat exchangers (PHE) are widely used in the dairy industry for heat treatment of milk. It is an indirect heating system consisting of metal plates placed closely together in a frame. The formation and number of plates varies according to purpose of the heating. Gasketed plates has a maximum temperature around 150 C and a maximum operating pressure of 20.4 bar while welded plates can withstand temperatures from -50 C to 350 C and a pressure from vacuum to 40 bar (Abu-Khader, 2012). Each plate has orifices in the corners going through the whole plate frame. If the orifices are closed the fluid is forced through small channels which are made by the space between the plates. Wide flow channels give lower heating rates and a more non-uniform heating. The number of plates and channels vary according to the aim of the heating process, but the number of channels and plates are the same for both hot and cold fluids (Gut and Pinto, 2003). Hot and cold fluid flow counter current on each side of the plates and exchange heat through the plates. This is shown in figure 4. The surface of the plates is often corrugated in various degrees which causes turbulence in the milk while passing through the channels and thereby increasing the heat transfer and giving a more uniform heating of the milk. The corrugating depends on the fluid and heat transfer rate. Low corrugation and chevron angles on the surface of the plates decreases the heat transfer (Abu-Khader, 2012). The flow pattern can be parallel where you have a single pass between the plates. Here the first and last plates do not transfer heat. The flow pattern can also be a multi pass flow distribution where the fluid passes through multiple channels on its way through the plate heat exchanger. This is shown in figure 4. Figure 4. Schematic representation of plate heat exchanger with counter current heat exchange. (Karami, 2014)

25 The flow through the channels is arranged in series. The multi pass flow distribution gives the opportunity of longer and slower heat transition (Gutierrez et al., 2011). PHE is a compact system with a relative large surface area for heat transfer compared to the volume of the system. This gives a high overall heat transfer coefficient (OHTC) and minimum loss of heat throughout the system (Edmond, 2001). On the other hand, the large surface area makes the system more prone to fouling as the plates are heated to a higher temperature than the desired temperature for the milk and deposits are building up on the surface of the plates. This requires more often cleaning especially when more corrugated plates are used (Deeth and Datta, 2002; Visser and Jeurnink, 1997) Tubular Heat Exchanger Tubular heat exchangers (THE) are indirect heat exchangers, build up by one or more circular tubes surrounded by a larger pipe. Milk flows inside the tubes while water or steam is flowing outside the tubes. Heat transfer is done between the two fluids across the tube material. The heat exchange is often operated in counter current mode. The tubes can have different shapes, but they are relative smooth and there are few seals which gives less resistance and a smooth passage through the tubes and this system can tolerate high pressure compared to PHE (Deeth and Datta, 2002). The overall heat transfer rate is lower for THE compared with PHE and direct heating systems due to a lower surface area for heat exchange, a larger diameter of the fluid flowing through the system and less turbulence within the tubes (Edmond, 2001). The slower heating and cooling rates gives longer transit time through the heat exchanger which makes the fluid more prone to chemical changes and fouling (Lewis and Deeth, 2008). Figure 5. Schematic overview of a tube in tube heat exchanger with countercurrent heat exchange. (Thermaline Inc., 2014)

26 THE is a considerable flexible system as the core geometry is quite easily changed according to tube diameter, tube length and arrangement. This also makes the system more suitable for fluids with a higher viscosity even though the heat transfer to the core of the tube decreases (Ditchfield et al., 2007). The three main tubular heat exchange systems are tube in tube, shell-and-tube and spiral tube heat exchangers. Tube in tube, which is shown in figure 5, consists of one or more tubes surrounded by a pipe. It is the simplest of the tubular heating systems but is not very efficient in heat transferring and it occupies large space. Shell-and-tube is an advanced version of double pipe heating system. Shell-and-tube is the most common used heat exchanger in the industry. It consists of multiple tubes in which the product is flowing, surrounded by a shell with the heating or cooling material. The shell and tubes can be twisted or baffles can be placed inside the shell to direct the flow of the fluid outside the tubes and give a greater turbulence in the fluid to get a more efficient and equally distributed heat transfer (Shah and Sekulić, 2007) Direct Steam Injection Direct steam injection (DSI) and direct steam infusion are the two main direct heating methods used in the dairy industry. Direct steam infusion is also called product into steam heating system and is the opposite of DSI which is called steam into product (Campell, 2013). Further on, DSI will be described. DSI is a good method when a very accurate temperature is needed as no heat is lost in transferring material during transfer of energy from steam to milk. The heat transfer is also much more rapid as steam is mixed with the milk and gives a more uniform heating. DSI can only be used for temperature above 100 C as the water has to be steam which is capable of reaching temperatures above 150 C (Campell, 2013). Figure 6. Schematic overview of a direct steam injection system with THE as preheating and cooling system. (Powerpoint international, 2014)

27 Figure 6 shows a schematic overview of a DSI system. Milk is first preheated to temperatures from 75 C- 85 C by using an indirect heat exchanger before the steam can be injected (Schroyer, 1997). This preheating step is used to minimize the energy use, as it requires large amount of energy to heat the milk to temperatures above 100 C in one step. Furthermore, heating in one step is found to apply great stress to the milk (Deeth and Datta, 2002). During stream injection, the steam is injected, under pressure, into the milk as small bubbles which collapse rapidly as they are mixed with the milk and in this process transfer heat. The milk is heated but it also becomes diluted with water as the steam condensates due to release of energy. After heating, the water added as steam is removed from the milk in a vacuum chamber which also flash cools the milk to temperatures near preheating temperature at the same time. To ensure that the fluid is not diluted or concentrated during the heat treatment, the total solid content before and after heating is monitored. An indirect system is used for further cooling to the desired temperature (Lewis and Deeth, 2008). Limitations of the DSI system are that it cannot be used if the product is sensitive to mixing with steam and the following condensation. Furthermore, the water used to generate steam must be free of organic constituents to avoid an incorporation of these in the milk when it is mixed with steam and thereby contaminate the fluid. DSI is not very prone to fouling due to shorter total heating time and less surface area is available to initiate the fouling process. This short heating time compared to indirect methods also gives less chemical changes and protein denaturation(deeth and Datta, 2002; Lorenzen et al., 2011; Oldfield et al., 1998a). 2.3 Heat induced denaturation of milk proteins Caseins are very heat resistant due to their loose structure and it is generally accepted that they can withstand heating at 140 C for min as the random coiling of the primary chain is generally hard to destroy compared to secondary and tertiary structures. To some extent both dephosphorylation and hydrolysis of the caseins has been found in heat treated milk (Belitz et al., 2004; Farrell Jr. et al., 2004; Fox, 1980). Heat treatment above 100 C gives a decrease in micellar size due to increase in colloidal phosphate and dissociation of κ-casein from the micelle surface (Singh and Waungana, 2001). The globular structure of whey protein makes them heat labile. Heat treatment of the whey proteins above 60 C, results in unfolding of the globular structure and the proteins thereby denature. The denaturation of whey proteins is generally considered involving two steps. The first step is an unfolding of the native globular structure, which leads to exposure of hydrophobic residues and disulphide bonds. If the heat treatment is minimal, the unfolded protein can refold into native structure. At high temperatures, the unfolded proteins will form new hydrophobic interactions and disulphide bridges which can result in

28 refolding the protein, but this is often disordered and gives rise to a random structure. The increase in the reactivity of the unfolded protein heated at high temperatures can also lead to the second step of the denaturation process. The unfolded whey proteins can form aggregates with other molecules, mostly through disulphide bonding and covalent bonds (Singh and Latham, 1993). Immunoglobulin s and BSA are the least stable whey proteins, β-lg is intermediate and α-la is the most resistant protein to heat denaturation. These differences in extent of heat denaturation are caused by the differences in structure and strength of intermolecular bonds (Anema, 2008; Corredig and DaLgleish, 1996). The dimer of β-lg dissociates between 30 and 55 C, but these changes are reversible and the monomers can rebound by cooling if the temperature does not exceed 60 C. When heating to temperatures above C, the tertiary- and also partly secondary structure of the monomer starts to unfold, leading to exposure of the free thiol group (Cys121) and hydrophobic parts of the residues chain, resulting in a reactive monomer. (Iametti et al., 1995and Iametti et al. 1996). The formation of these monomers is irreversible and they cannot refold to native state. Instead there will be formed non-native monomers, which can form aggregates with other monomers but also aggregates with other types of proteins can be formed (Tolkach and Kulozik, 2007). α-la is the least heat resistant whey protein with a denaturation temperature around 62 C, but the unfolding at this temperature is reversible. It does not form aggregates or modified monomers at heating temperature below 80 C, at neutral ph (ph ). This is due to α-la having no free thiol groups which can change the reactivity of α-la (Eigel et al., 1984). α-la is capable of refolding to its native state in presence of calcium if the disulphide bonds are still intact (Brew, 2003). The binding of calcium is very ph dependent and calcium dissociates from the α-la binding site at ph below 5 which makes α-la lose the ability to refold to its native structure after heat treatment. Severe heating conditions with temperatures above 100 C for several minutes disrupt the disulphide bonds and formation disulphide linked aggregates of denatured α-la occurs (Singh and Havea, 2003). The main aggregates formed as a consequence of heat treatment of milk, are complexes formed by aggregation of denatured whey proteins and complexes between β-lg and κ-casein on the surface of the casein micelles via disulphide bonds and hydrophobic interactions. At temperatures below 70 C the interaction is mostly caused by hydrophobic interactions while at higher temperatures it is mostly caused by disulphide bonds (Corredig and DaLgleish, 1996; O Connell and Fox, 2011).The κ-casein and β-lg interactions are most pronounced when the κ-casein is placed on the surface of casein micelles as the association between β-lg and κ-casein is less favourable when κ-casein is dissolved in serum. This can be caused by κ-casein is present in a more compact structure when dissolved in serum whereas placed on the surface of casein micelles

29 the structure of κ-casein is more loose (Donato et al., 2007). The formation of these complexes may be altered by a slow heating rate or heating for a long time at lower temperatures. This gives longer time for the β-lg to unfold and associate with the casein micelles. In contrast, a rapid heating rate to the required temperature gives a shorter time for unfolding and it is more likely that β-lg refolds in a non-native structure or forms aggregates with other unfolded monomers instead of associating with κ-casein (Oldfield et al., 1998b). α-la does not associate with the casein micelles on its own like β-lg; it has to form complexes with β-lg which then associates with the casein micelle and it requires a prolonged heating period to start associating with the casein micelle (Oldfield et al., 1998b; Oldfield et al., 2000). The rate of denaturation is mainly controlled by heating temperature, heating time and ph but also protein concentration and ionic strength have been proved to have some effect (McSwiney et al., 1994; Oldfield et al., 2000; Qi et al., 1995). At neutral ph the free disulphide group of β-lg is very reactive and this is the main mechanism for aggregation and gives a faster aggregation of β-lg. Dissanayake et al. (2013b) have shown that the denaturation rate is significantly lower at ph 3 compared to ph 6 and the aggregates formed at ph 3 are caused by non-covalent bonding. This is consistent with free disulphide groups being inactivated at acidic conditions. At neutral ph most whey protein complexes formed by denaturation are soluble. A decrease in ph below 6.2 followed by heating gives a faster formation of whey protein/κ-casein complexes and they were often associated with the casein micelles. Heating at a ph above 6.8 leads to dissociation of the whey protein/κ-casein complexes from the micelle surface (Zúñiga et al., 2010). The reaction kinetics of the denaturation of β-lg has been widely investigated in order to predict the extent of denaturation according to different heat treatments. Dannenberg and Kessler (1988) determined the reaction order for the denaturation of β-lg for each temperature by using the model (1) where n is reaction order different from 1 and is the ratio of denatured β-lg at holding time t and k is the rate constant at a given temperature. They found that a reaction order of 1.5 gave a linear correlation between denaturation of β-lg and holding time in skim milk for various temperatures having the rate constant defined as the slope of the linear graph. This has been verified since, i.e. Zúñiga et al. (2010) and it is now widely used to report denaturation of β- Lg in skim milk. Kessler and Beyer (1991) have shown that the reaction number varies between 1.5 and 2 according to the casein/whey protein ratio in the milk with skim milk having a reaction order of 1.5 while sweet whey has a reaction order of

30 Factors such as the methods used to detect differences in β-lg denaturation, lack of enough data to make an accurate determination, the heating methods and how the samples are heated, including preheat time, temperature and cooling rate, can have a great impact on the denaturation of β-lg (Oldfield et al., 1998a). Dannenberg and Kessler (1988) also investigated the effect of temperature on the rate constant k for denaturation of β-lg in skim milk. The rate constants for various temperatures can med used to make an Arrhenius plot, equation 2. (2) This is visualised plotting the logarithm of rate constants against the inverse temperature in kelvin. From linear regression of the data points, is it possible to calculate the activation energy for the denaturation process. The Arrhenius plots are used to detect the effect of temperature on a specific chemical reaction. Figure 7 shows the Arrhenius plots for β-lg B, β-lg A and α-la, which can be used to determine the rate of denaturation of a given temperature. Figure 7. The effect of temperature on rate constant for β-lg A, β-lg B and α-la of skim milk. Remodeled from Dannenberg and Kessler (1988) From figure 7 it is observed that the relationship is linear, but only for given temperature ranges, namely C and C. The graph shows a bend around 95 C where the activation energy above 95 C decreases significantly. This change in rate constant and activation energy indicates that two reactions are taking place. At lower temperatures, denaturation and unfolding is the dominating reaction which requires large quantities of energy, while at higher temperatures aggregation dominates which does not

31 require same amount of energy. The kinetics of α-la has been investigated in similar way and a reaction order of 1 was found and verified since experiments made by Dannenberg and Kessler (1988) (Oldfield et al., 1998a). As seen in figure 7, α-la also shows a change in activation energy when looking at the temperature effect on the rate constant, but this change occurs abound 80 C compared to 95 C for β-lg. The activation energy for α-la in the temperature range 85 C-150 C is higher than the activation energy for β-lg which supports the theory of α-la being less heat stable but less prone to heat denaturation. However, this pattern for α-la is only seen when it is solution with other denatured whey proteins. Calvo et al. (1993) found no thermal aggregation of α-la in absence of other whey proteins and heat treated at 90 C for 24 min. Addition of β-lg caused aggregation of α-la which was depended on the content of free thiol groups present on β-lg. 2.4 Protein separation methods Protein separation and identification can be done in various ways, depending on purpose of the separation. In the following, two often used techniques will be described Liquid Chromatography A widely used method for separation is Liquid Chromatography (LC). LC comprises separation techniques that can be used separate various compounds, such as proteins, carbohydrates, fatty acids and amino acids, according to the desired characteristics, such as size and mass (Size Exclusion Chromatography (SEC)), hydrophobicity or biological function (affinity chromatography). There are many different LC systems referring to the pressure and phase used, such as e.g. Reverse Phased High Performance Liquid Chromatography (RP-HPLC) and Ultra High Performance Liquid Chromatography (UPLC). RP-HPLC is a fast an accurate method for separation and quantification of milk proteins separating according to hydrophobicity (Aguilar, 2004). It contains a column with a stationary phase which is non-polar and hydrophobic while the mobile phase is polar, which is the opposite of normal phased LC. The length of the carbon chain bound to the stationary phase depends on the sample to be separated. The longer carbon chain, the more hydrophobic is the stationary phase. The molecules in a sample mixture binds to the stationary phase if they are non-polar and hydrophobic. Molecules that have hydrophobic groups and longer alkyl chains will bind stronger to the stationary phase than molecules having polar groups in their structure. The more hydrophobic solute, the higher is the affinity of the solute to the stationary phase and the more time the solute spends in the stationary phase and the later it leaves the column. On the other hand, polar molecules will not bind to the column to the same extent and will leave the column earlier. As a result,

32 Figure 8. An example of a chromatogram separating proteins from milk according to their hydrophobicity. Peak 1-3: κ-casein, peak 4: α s2 -casein, peak 5: α s1 -casein with 8 phosphorylations, peak 6: α s1 -casein with 9 phosphorylations, peak 7: β-casein variant A 1, peak 8: β-casein variant A 2, peak 9: α-lactalbumin, peak 10: β-lactoglobulin B, peak 11: β-lactoglobulin A (Rauh et al., 2014). molecules with different hydrophobicity elute at different times and are thereby separated (Giacometti and Josić, 2013). In order to be able to separate all proteins and having no proteins bound to the column at the end of analysis, a gradient of organic solvent is used. By use of a gradient, it is possible to control the separation to certain extent in a specific area by changing the solvent from being non-polar to be more polar over time, and more proteins will be detach the stationary phase and thereby be eluted and detected. Even small changes in the gradient will change the separation and thereby the elution time. For detection, for example a UV detector can be used at 214 nm to detect peptide bonds or 280 nm to detect aromatic residues (Berg et al., 2006). The detection is visualized as a peak in a chromatogram showing detection time since starting the analysis and the intensity of the detection. The time from start of analysis until maximum detected of a fragment by the detector is called retention time (RT). If retention time is used to characterize molecules and peptides fragments, the analysis conditions have to be carefully controlled to be able to compare fragments from different samples. These conditions are; the pressure used, the column dimensions and stationary phase, the composition of solvent and temperature (Aguilar, 2004; Schlüter, 1999). Even small changes in these parameters will give a shift in retention time. The separation of milk proteins in skimmed milk can be seen in figure 8. The area of each peak on the chromatogram refers to the intensity of peptide bonds detected at a given time. The more intense peak, the more peptide bonds are detected. These chromatograms and peaks for each protein indicate the ratio between the different proteins, but it does not give a quantification of the concentration of each protein present in milk

33 Due to high resolution, RP-HPLC is capable of differentiating between the various genetic variants of both casein and whey proteins. Different structural isomers on the other hand will not be separated as they have same hydrophobicity. During irreversible denaturation of whey proteins, the globular structure is changed due to refolding in a non-native structure. The denatured whey proteins are said to sediment when acidifying the solution to ph 4.6 mainly due to aggregation, and they will therefore not occur when analysing the soluble phase from the acidification. By investigating the protein composition, it is possible to observe the amount of each protein that is in it native form from RP-HPLC data. The amount of denatured protein can be calculated by comparing the amount of native protein in non-heat treated samples with native protein in heat treated samples Gel electrophoresis Protein separation using one dimensional gel electrophoresis (1DGE) is another widely used method. One of the common gels used contains polymerization of acrylamide and bis-acrylamide. Polymerization of these introduces crosslinking and pores are formed between the crosslinks. The size of the pores is determined by the ratio between bisacrylamide and the concentration of acrylamide. This is used when preparing the gel, by making a gradient of acrylamide to make a more specific separation in a certain protein size area (Berg et al., 2006). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) is a commonly used method due to its high resolution of protein separating by size and charge which was firstly published by Laemmli, (1970) and this is still the basis of SDS-PAGE used today (Jones, 1993). SDS denatures the non-disulphide tertiary and secondary structure and covers the protein in negative charges corresponding to the length of the protein chain. If proteins in a protein mixture have to be separated completely, the disulphide bridges have to be reduced to separate eventually protein aggregates and unfold the proteins (Berg et al., 2006). The proteins migrate differently in an electric field due to the different charges and this migration is thus proportional with the molecular mass of the protein. Proteins can also be separated according to their isoelectric point (pi), called isoelectric focussing (Radola, 1984). The protein mixture is loaded into a ph gradient in an electric field. The electric field makes the proteins migrate towards their isoelectric point, pi, as a cause of their own charge and the migration stops when reaching the pi and they have no net charge. Isoelectric focusing has also become widely used as the first dimension of two-dimensional gel electrophoresis (2DGE), which is then followed by SDS-PAGE in second dimension. This technique is a powerfull

34 tool for protein separation and commonly used in proteomic profiling (Rabilloud et al., 2010). A major advantage of 2DGE is the opportunity to separate different modifications of a protein from each other, as these often vary in pi while the difference in mass can be difficult to separate as done in 1DGE. 2DGE also gives the possibility to separate many different proteins and peptides in a complex matrix as milk, in one single run. Each spot on the gel can be identified by different types of mass spectrometry (MS) (Jensen et al., 2012a; O Donnell et al., 2004). Limitations on separation with both gel electrophoresis methods, is the limited amount of sample loading, and the gels are often not capable to separate molecules with a mass above 250 kda. Furthermore is gel electrophoresis time consuming, especially for 2DGE where only one sample can be analysed at one gel. 2.5 Protein identification Protein separation described in section 2.4 can be coupled to MS analysis to identify the separated fractions. This coupling is also useful for identifying unknown peaks and to show how purified the peaks from LC and gel electrophoresis are (Lacorte and Fernandez-Alba, 2006). The principle of MS is to separate protein fractions according to their mass by use of an electric field. The proteins become ionised in an electric field and the movements in this electric field depends on the molecular weight of the protein (Berg et al., 2006). Small molecules move faster and are detected earlier by a UV-detector compared to the heavy molecules. Different molecular weights are often found when looking at the mass spectrometer of one fraction separated with LC or gel electrophoresis, which can correspond to genetic variants or attachment of e.g. one or more lactose units (Czerwenka et al., 2006). 2.6 Rennet coagulation of milk proteins Raw milk is very prone to spoilage during storage and it is therefore necessary to process the milk to prolong shelf life. Cheese is one of many processing opportunities of milk. The first step in the cheese production is the coagulation of casein micelles. This can be done enzymatic by adding different kinds of enzymes which causes the caseins to clot. One of the most used enzymes is the enzyme Chymosin, also called rennet, which is a milk clotting enzyme originally obtained from calf stomach. Alternatively, ph can be lowered to the isoelectric point for casein which also makes the caseins coagulate. Chymosin cleaves κ-casein between amino acid residue 105 and 106, resulting in a hydrophilic part, caseinomacropeptide, and a hydrophobic part, para-κ-casein (Belitz et al., 2004).By removing the hydrophilic part of κ-casein, the ca

35 sein micelles lose their solubility and when approximately 85 % of κ-casein is hydrolyzed, the colloidal stability of micelles is reduced in such an extent that they will start coagulate (Fox and McSweeney, 1998). It is widely known that milk treated at high temperatures has longer coagulation times, reduces the firmness and forms weaker gels when manufacturing cheese (Waungana et al., 1996a). This can be caused by complexes of denatured whey protein and κ-casein, leading to the formation of appendages on the micelle surface which makes the Phenylalanine 105-Metionine 106 bond of κ-casein less susceptible to hydrolysis by rennet and thereby decreases the coagulation abilities according to cheese production (Tolkach and Kulozik, 2007). Singh and Waungana (2001) observed that heat treatments which resulted in denaturation degrees of less than 60 % of β-lg had little effect on gelation time of skim milk, while gel strength decreased for all detected denaturation degrees. These impairments in rennet coagulation properties can be restored in some extent after severe heating by addition of CaCl 2 and lowering ph (Hougaard et al., 2010; Waungana et al., 1996b). Several modifications of milk have been investigated to reduce rennet coagulation time and improve texture. One method is to concentrate milk to achieve higher protein content. This gives firmer gels compared to skim milk and reduced decrease in coagulation properties when heated at high temperatures, while having the same degree of whey protein denaturation. This is due to the increase of caseins which are placed closer because of reduced volume and thereby can the coagulation be altered (McMahon et al., 1993). Another method is microfiltration of milk to reduce the amount of whey proteins, such as Micellar Casein Isolate (MCI). By removal of the whey proteins, a reduction in coagulation time and an increase in gel firmness can be observed, compared to skim milk, also after heating (Pierre et al., 1992; Wang et al., 2007). This supports the theory of whey proteins have a negative effect on the rennet coagulation abilities of milk Measurement of coagulation properties Knowledge about milk coagulation is of great importance to be able to make the best quality cheese. Throughout time many different methods used to investigate rennet coagulation of milk, like oscillation rheometry (Bohlin et al., 1984), dynamic light scattering (Horne and Davidson, 1990) and ultrasound (Beguigui et al., 1994). One of the recently new methods to measure the coagulation of the milk is by a ReoRox rheometer which is based on free oscillation rheometry. The ReoRox system is formerly used only for determining blood coagulation properties but is has been shown that the results from milk coagulation analysis on the ReoRox are consistent with analysis made on a rheometer which is well known and commonly used for milk coagulation in literature (Andersen, 2013; Frederiksen et al., 2011). The ReoRox con

36 tinuously measures the elasticity and viscosity of a sample throughout the sampling time. Figure 9 shows an example of a coagulation analysis of pasteurized skim milk with the four main characteristics shown. These are the rennet coagulation time (RCT) which is the point of which the milk goes from fluid like to solid like characteristics, the curd firming rate (CFR) which describes the speed of gel formation, and time at which the gel contains gel strength of 200 Pa. It is possible to measure multiple samples at the same time which makes it less time consuming when having many samples. The ReoRox is a single frequency oscillation test which operates with a fixed strain which is expected to be within the linear viscoelastic region for the given product. The ReoRox also uses a fixed frequency at 10 Hz. These two parameters fixed to fit standard milk, which makes it is possible to determine when the milk goes from fluid to more solid during time with the least variables to take into account (Andersen, 2013). Figure 9. Rennet coagulation analysis of low pasteurized skim milk on ReoRox 4.Clot onset time: time at which first clots are detected. Rennet coagulation time: time at which the milk goes from being more solid than fluid. Curd firming rate: the rate of curd formation, measured in Pa/min. Time at 200 Pa: the time when the milk gel reaches strength of 200 Pa. (Andersen, 2013.)

37 3 Materials and methods 3.1 Milk types Milk used in the trials was skim milk delivered from Brabrand Dairy (Brabrand, Denmark) and micellar casein isolate milk (MCI) from Arla Foods Innovation Centre Nr. Vium (Nr. Vium, Denmark). Skim milk from Brabrand Dairy contained % protein, % lactose, % total solid and less than 0.08 % fat. MCI was produced from skim milk which was microfiltered (MF) to remove whey proteins. Less than 4 % of the total protein content in MCI was whey protein. One portion MCI was adjusted to a total protein content equal to skim milk (referred to as MCI) and one portion MCI was adjusted to a casein content equal to skim milk (referred to as MCIc).The mineral, lactose and total solid content was equal in all three milk types. Pasteurised milk was used each trial day and delivered to Arla Strategic Innovation Centre (ASIC) (Brabrand, Denmark). Milk which did not receive further heat treatment is referred to as control milk. The overall milk composition of the control milk was measured by FT120 Milkoscan (Foss, Denmark). 3.2 Heat treatments A laboratory scale UHT heat exchanger (Powerpoint International Ltd, Japan) with 3 different possibilities to make heat treatments with both direct and indirect heating systems was used for heat treatments in the pilot plant at ASIC. The three heating methods were tubular heat exchanger (THE), plate heat exchanger (PHE) and direct stream injection (DSI). The plates and tubes are composed of two heating units and three cooling units. For temperatures above 100 C, backpressure is used to avoid milk evaporating. The pressure increases with increasing temperature and milk flow through the system and varied from 0.5 bar at 80 C to 4.2 bar at 140 C. The DSI system also uses vacuum down to 400 mbar, to be able to withdraw the injected water from milk after heating. The preheating is always indirect, while the second stage can be direct or indirect heating up to 150 C. The three cooling units are indirect where the first cooling unit uses tap water for cooling while second and third cooling unit uses ice water for cooling. The Plate heat exchanger contains corrugated flow plates, specialized for this heat exchanger. The tubes in the tubular heat exchanger are corrugated tubes in tubes made of stainless steel with an internal finish of 1 micron in a soft spiral pattern with a tube diameter of 8 mm. The passage through one plate or tube unit is approximately 20 s with a flow of 20 L/h. For DSI, PHE was used for preheating to 75 C and cooling

38 was done with flash cooling to 65 C followed by plate cooling system to 4 C. Preheat temperature for trials with an end temperature below 95 C was 60 C while a preheat temperature of 75 C was used for trials with an end heating temperatures of 95 C and above. Temperatures between 80 C and 145 C were used with a desired holding time between 2 s and 300 s. After heat treatment the samples were immediately cooled to the end temperature of 4 C. Table 2 shows the various temperature and holding time combinations for skim milk samples. MCI was not heated at 85 C and 105 C while the other combinations were equal. MCIc was only heated with PHE at temperatures of 80 C 115 C and 130 C with equal holding times compared to skim milk. For all skim milk samples, and MCI heated with PHE, the temperature and holding time combination was performed as biological duplicates at different trial days. For MCI heated with THE and DSI, each temperature and holding time was preformed once and for MCIc temperature and holding time was preformed was performed once using PHE. In total 135 skim milk samples, 68 MCI milk samples and 18 MCIc milk samples were produced and analysed. Table 2. Temperature and holding time combinations used for heat treatment of skim milk. The method is stated if the combination has been used for the particular method. The milk flow through the system for each holding time is given. Temperature ( C) Holding time (s) Flow (L/h) 4 20 DSI DSI DSI DSI 5 20 PHE PHE PHE PHE PHE PHE PHE PHE THE THE THE PHE PHE PHE PHE PHE PHE PHE PHE THE THE THE PHE PHE PHE PHE PHE PHE PHE PHE THE THE THE PHE THE PHE PHE THE PHE PHE PHE THE PHE THE PHE PHE PHE THE PHE

39 3.3 Protein analysis Protein analysis was performed by a LC-MS system consisting of Agilent 1290 LC infinity system with Agilent 6530 Accurate-Mass Q-TOF system (Agilent Technologies, USA). The MS detection was equal for all LC-MS analyses. Mass scans of the protein fractions separated on the LC system were continuously recorded to detect fractions with a mass to charge ratio between 300 and Data analysis of MS data was performed by using Mass Hunter (Agilent Technologies, USA) to identify the content of the peaks found through the LC analysis of the milk samples. Sample vials were kept 4 C and injected via an auto-sampler. The analysis method was a modification of the model used by Bonfatti et al. (2008) Analysis of total protein content For each milk sample, 200 mg milk was frozen at -20 C until further use. Prior to analysis samples were defrosted and added a reduction buffer containing 6 M urea, 0.1 M trisodium citrate and 0.5 M dithiothreitol (DTE). The samples were incubated at 30 C for 60 min while stirring to reduce non-covalent bonds, crosslinks and disulphide bonds. Hereafter the samples were centrifuged for 10 min at 4 C, 9300 g, and collected for analysis. A volume of 5 μl sample was injected into the LC-MS system. A Biosuite column C18 PA-B 2.1x250 mm, particle size of 3.5 μm and pore size of 300 Å was used (Waters, USA) is used. Buffer A contained 0.05 % triflouroacetic acid (TFA) in milliq water and buffer B contained 0.1 % (TFA) in acetonitrile. The column temperature was 40 C. A linear gradient of buffer B from 34.8 % to 46.5 % from 2 to 16.5 min was applied with a flow rate of 0.35 ml/min and total analysis time was 21 min. UV detection of 214 nm was used to detect the protein fractions Analysis of ph 4.5 soluble protein 20 ml of milk sample was adjusted to ph with 1M hydrochloric acid (HCl) to sediment the caseins and denatured whey proteins completely due to reaching their isoelectric point. This was done using a ph meter (Knick GmbH,Germany). ph was measured before adding HCl and the ph was lowered while stirring at room temperature. After reaching the ph the samples were stirred for at least 15 min to make sure that the buffer effect of milk was removed. The samples were then centrifuged 10 min, 4 C at g to separate the precipitated caseins and denatured whey proteins in the pellet and the soluble whey proteins in the supernatant. The supernatant

40 was collected in eppendorf tubes and frozen at -20 C until further analysis. Duplicates ph adjustments were made for each milk sample collected. Prior to LC analysis, one of each duplicate from all milk samples, ph 4.5 soluble protein solutions were centrifuged for 10 min at 4 C and 9300 g to separate eventually precipitated casein and fat particles from the supernatant. Approximately 1 ml of the supernatant was transferred to vials and analysed μl of each sample was injected into the LC-MS system. For milk samples heated less than 100 C, 5 μl was injected, for samples heated at C, 10 μl was injected and for samples heated at C, 20 μl was injected. These differences in injection volumes were caused by differences in content of protein left in the supernatant after adjusting the ph to 4.5. An Xbridge BEH300 C18, 2.1x250 mm column with diameter of 5 μm, particle size of 3.5 μm and pore size of 300 Å was used (Waters, USA). Buffer A contained 0.05 % TFA in milliq water and buffer B contained 0.1 % TFA in acetonitrile. A gradient was applied with 17 % buffer B at2 min, 40 % buffer B at min 8, 44 % buffer B in min 14. The column temperature was 45 C. The flow rate was 0.35 ml/min and total time for each analysis was 17 min. The UV detection of 214 nm was used to detect protein fractions. Peak areas for β-lg B, β-lg A and α-la was calculated for each sample. All peak areas were adjusted to same injection volume. The degree of denaturation of the whey proteins was calculated as the difference in peak area between reference sample and heat treated sample. 3.4 Measurement of rennet coagulation The coagulation properties of milk were performed using a ReoRox G2 Rheometer (Medirox, Sweden) one day after heat treatment. The oscillation frequency is fixed at 10 Hz and amplitude of 2 which means that the sample cup is swung 2 for each 2.5 seconds. This gives a corresponding strain of 0.07 which is the strain within the LVR for standard milk. The analysis was performed as described in Frederiksen et al. (2011). 103 g milk, corresponding to 100 ml milk, was adjusted to ph 6.5 with 10 % lactic acid while stirring. The milk samples were incubated in water bath at 33 C for 30 min. Chy-Max Extra was addition to the milk to a final concentration of (Chr. Hansen, Denmark) was made. The Chy-Max Extra used for all samples, was from the same batch. The addition of Chymosin defines starting point for the ReoRox analysis. 1 ml of the milk sample was transferred to a sample cup placed in the ReoRox while the remaining sample was placed in water bath for visual analysis and confocal laser scanning microscopy (CLSM).The ReoRox measures the rennet coagulation time, gelation point, curd firming rate, gel strength at 45 min analysis and time for reaching gel

41 strength of 200 Pa. The rheological measurements were performed for 2 hours at 33 C. Duplicates of all milk samples were measured. To avoid the impact of variation between the milk batches used, the coagulation properties for all heat treated milk samples were calculated as the relative difference to the corresponding control sample. 3.5 Analysis of protein structure For each milk type, three heat treated samples with a holding time of 10 s, and the matching control samples were analysed for protein structure 2 hours after addition of rennet while incubated at 33 C (see section 3.4 for renneting procedure). A thin slice of milk coagulate was placed on a microscope slide and added a droplet 0.02 % (v/v) fluorescein -5-isothiocyanate (FITC) solution in acetone. When the acetone was fully evaporated, the sample was analysed using a 40x and 100x oil immersion objective coupled to a Leica DMIRE2 inverted confocal laser scanning microscope (CLSM) (Leica Microsystems GmbH, Germany). The laser used was an Argon/Krypton ion laser. FITC excite UV light at 485 nm and the emission UV light is recorded in the green area, between 500 and 545 nm. Triplicates of each sample were performed. 3.6 Analysis of protein aggregates Heat induced protein aggregates were analysed by 1DEG, 2DGE and size exclusion chromatography (SEC). Table 3 shows the detailed description of the samples used for analysis of protein aggregates. 4 selected heat treated samples and 2 control samples of skim milk and MCI were analysed under reducing and nonreducing conditions. The non-reducing conditions did not contain DTE to maintain the disulphide bonds, while the reducing conditions contained DTE in the analysis process to break disulphide bonds

42 Table 3. Samples used for analysis of heat induced protein aggregates ID # Milk type Method Temperature ( C) Holding time (s) Protein % (milkoscan) 105 Skim Control Skim DSI MCI PHE Skim THE MCI Control Skim PHE DGE In 1-D analysis, the samples were analysed on a Criterion TGX any kda 1-D gel containing μl, 1.0 mm wells, (Biorad,USA). The milk was diluted to three different concentrations with MilliQ water. These dilutions were mixed with Laemmli sample buffer (65.8 mm Tris-HCl, ph 6.8, 2.1% SDS, 26.3% (w/v) glycerol, 0.01% pyronin Y) in the ratio 1:1. For each dilution, one fraction was reduced by adding 20mM DTE while the other fraction remained unreduced and was only added Laemmli sample buffer All sample solutions were incubated at 70 C for 1 min. 20 μl sample solution was loaded at the gel in two protein concentrations, 20 μg, 10 μg. 10 μl Spectra multicolour broad range protein ladder marker was loaded as marker (Pierce Biotechnology, USA). The gel was placed in criterion Dedeca cell system (Biorad, USA) with 1M running buffer (19.2 mm glycin, 0.01 % SDS, Tris-base) and migration of the proteins was achieved by applying 200 V for 35 min. The gel was fixed in Fix solution with 50 % ethanol and 8 % phosphoric acid for at least 2 hours while stirring. The gel was coloured according to the colloid coomassie staining method (Kang et al. 2002). Gel images were captured using an ImageScanner (Amersham Biosciences, Sweden). The different protein bands were identified according to (Souza et al., 2000) DGE The 2-D gel analysis was done according to (Jensen et al., 2012). For isoelectric focusing in 1 st dimension, samples were diluted 10 times with immobilised ph gradient (IPG) lysis buffer (7M urea, 2 M Thiurea, 40 mm Trisbase). For reducing conditions, 1 % DTE was added to the IPG lysis. Precast 11 cm strips, ph 4-7 (Biorad, USA), loaded with an amount of dilution corresponding to 100 μg protein and left for rehydration

43 for h at room temperature in darkness. Isoelectric focusing was carried out using an electrophoresis chamber (Biorad, USA) with an increasing volt gradient with a maximum of 57.6 kv hours. After focusing, the strips were incubated in equilibration buffer 1 (6M Urea, 30 % (v/v) glycerol, 2% SDS and 0.05 % Tri-HCl) for 15 min. 65mM DTE was added reduced samples. The strips were incubated in equilibration buffer 2 (6M Urea, 30 % (v/v) glycerol, 2% SDS and 0.05 % Tri-HCl) and for reduced samples 270 mm Iodoacetamide, for 15 min. The strips were then placed on top of a Criterion Precast gel, 8-16 % tris-hcl, 1.0 mm IPG+1 well comb (Biorad, USA) using 0.5% agarose coloured with bromophenolblue to bind the strip. The gels were electrophoresed, stained and coloured in same procedure as 1-dimensional gels (section 3.6.1). The different protein bands were identified according to Jensen et al. (2012b) and Larsen et al, (2010) Analysis of protein aggregate composition Size exclusion chromatography (SEC) was performed on a 1260-infinity semi-preparative LC system (Agilent Technologies, USA). Reduction of samples was performed as described in section was used. For the non-reduced samples, same procedure was used, but no DTE was added to the buffer. 150 μl of each sample was injected on a Bio SEC-3 Column (3μm, mm, 300 Å, Agilent Technologies, USA) at 30 C. The mobile phase contains of 0.2M sodium phosphate ph 7 and 0.2 M Sodium chloride. This column separates in the molecular range of to 5000 Da. A flow rate of 1 ml/min was applied and UV signals were detected at 214 nm. For 2 skim milk samples, respectively heated at 130 C for 5 s at PHE and THE, the protein aggregations were analysed. 1 ml milk sample was added 5 ml buffer containing 6 M urea and 0.1 M trisodium citrate. The samples were incubated at 30 C for 60 min while stirring followed by centrifugation for 10 min at 4 C, g and analysed as described above. 1 ml of four fractions in the time frame 8-12 min was collected by a fraction collector from each milk sample. The fractions were freeze dried under vacuum (0.1 bar) overnight in a Hetosicc freeze-dryer (Pfeiffer Vacuum GmbH, Germany). The freeze dried fractions were dissolved in 100 μl reduction buffer containing 6 M urea, 0.1 M trisodium citrate and 20 mm DTE. These samples were incubated at 30 C for 60 min while stirring. 20 μl of each sample was injected and analysed on LC as described in section

44 3.7 Statistical analysis One way-anova analysis was performed to determine significant differences (p<0.05) among milk types and heating methods, carried out using MATLAB (Mathworks, USA)

45 4 Results 4.1 Variation in milk protein composition Milkoscan was used for a fast determination of milk components. There were small variations in the amount of protein, lactose, dry matter and fat between the milk batches, but these differences were not significant and no pattern for the variation in composition was found. Further analysis of total protein was made of all control samples for all milk types, to investigate differences in the protein content between trial days. Figure 10A shows the analysis of total protein content of three Figure 10. Total protein analysis of control samples of the three milk types. A: three skim milk control samples from three different batches. B: Total protein analysis of control samples of skim milk, MCI and MCIc. Peak 1-3: κ-casein, peak 4: α s2 -casein, peak 5: α s1 -casein with 8 phosphorylations, peak 6: α s1 -casein with 9 phosphorylations, peak 7: β-casein variant A 1, peak 8: β-casein variant A 2, peak 9: α-la, peak 10: β-lg B, peak 11: β-lg A

46 control skim milks from three different batches from three different weeks, analysed on LC-MS. The peaks on the chromatogram were analysed by use of the MS data for each fraction. From figure 10A It is clear that there are small variations in the protein content, especially in the casein composition, which are observed in fraction 1-8. The total protein content measured on the Milkoscan also showed these variations, respectively %, % and 3,663 % protein for samples from sampling week 2, 14 and 21. Figure 10B shows analysis of total protein of control samples of the three milk types, from the same trail week but from different batches. Small variations were found for the caseins fractions, which are seen in fraction 1-8. The content for casein is higher in the MCI milk, while MCIc and skim milk are equal in casein content. There are only small amounts of whey protein present in MCI and MCIc compared to skim milk. The amount of β-lg B, β-lg A and α-la, shown in fraction 9-11, represent 2-4 % of the total protein content in MCI while it was around 2 % of total protein content for MCIc. Due to this low content for whey proteins in MCI and MCIc, the denaturation degree of whey protein was only analysed for skim milk samples. Variations in the milk composition also gave rise to variations in enzymatic coagulation. Table 4 shows the average relative standard deviation, minimum and maximum for the four measured parameters from 38 coagulation analyses on control skim milk from 19 different trial days. A large variation was found in the measured properties between the control skim milk samples. A variation of RCT from 11.6 min to 19.6 min and CFR varying from 10 Pa/min to 24 Pa/min had great influence on gel strength at 45 min and also on the time reaching gel strength of 200 Pa. The relative change in coagulation properties for heat treated samples according to the control sample was therefore calculated and used throughout the report, unless otherwise it stated. Table 4 Rennet coagulation properties of 38 control skim milk samples. Rennet coagulation time, curd firming rate, gel strength at 45 min and time at gel strength of 200 Pa is shown. Rennet coagulation time (min) Curd firming rate (Pa/min) Gel strength, 45 min (Pa) Time at 200 Pa gel strength (min) Average Relative standard deviation (%) Minimum Maximum

47 4.2 Effect of indirect heating on skim milk For each skim milk sample, ph 4.5 soluble protein fractions were analysed on LC MS in duplicates and integration of peak areas of each whey protein fraction was used to calculate the percentage of whey protein in the heat treated samples compared to the control sample. The denaturation degree is defined as the percentage of whey protein not appearing in ph 4.5 soluble protein analysis compared to the control sample, which is stated to have a native whey protein percentage of 100 % and thereby no denaturation. Heat treatment using THE and PHE were compared according to temperature and holding time to investigate differences in denaturation degree of whey proteins and coagulation properties between the two heating methods. Figure 11 shows the denaturation degree of β-lg B and α-la in skim milk heat treated using PHE and THE. The denaturation pattern of β-lg A is shown in Appendix 1 which shows similar results as denaturation of β-lg B. The denaturation of β-lg B is shown in figure 11A, for heating temperatures Figure 11. Denaturation degrees of β-lg B and α-la for skim milk heated at PHE and THE. A: Denaturation of β-lg B heated at temperatures below 100 C. B: Denaturation of β-lg B heated at temperatures above 100 C. C: Denaturation of α-la heated at temperatures below 100 C D: Denaturation of α-la heated at temperatures above 100 C Different letters indicate significant differences were found between heating methods for each given temperature (p<0.05)

48 below 100 C and in figure11b for heating temperatures above 100 C. For all temperatures, an increase in denaturation degree was observed as the holding time increases. However, the greatest effect was seen by increase in temperature, especially when temperatures above 85 C are used. A heating temperature of 85 C resulted in denaturation degree of 25 % of β-lg B with a holding time of 5 s, while heating at 130 C and 140 C resulted in denaturation degrees above 90 % even at very low holding times. This was applicable for both heating methods. Comparing the denaturation degrees for heating temperatures at 80 C and 95 C using PHE and THE from figure 11A, it is clear that THE results in significant higher denaturation degree than PHE for holding times above 10 s for 80 C (p<0.04) and for holding times below 120 s for 95 C (p< 0.009) as both methods have denaturation degrees above 90 % at holding times of 120 s and 300 s for heating temperature of 95 C. Figure 11B shows heating temperatures above 115 C, which results in denaturation degrees of β-lg B above 90 % for both heating methods. The denaturation of α-la is shown in figure 11C and figure 11D. It is observed that the denaturation of α-la is also affected by temperature and holding time. The degree of denaturation of α-la is however lower than the degree of denaturation of β-lg B, which is shown in figure11 A and figure 11B. Figure 11C shows the denaturation degree of α-la for temperatures below 100 C. From this, it is observed that the denaturation degree does not exceed 50 % at any of the investigated holding times for both PHE and THE. No significant difference in denaturation of α-la was found when heating at 80 C between methods. Heating temperature at 95 C results in significant higher denaturation degree was found for heat treatment using THE, compared to PHE, with a holding time of 60 s (p<0.002). Figure 11D shows the denaturation degree of α-la for heating at temperatures above 100 C. Heating at 130 C and 140 C gave the highest denaturation degree, but no denaturation degrees above 90 % were found for any of the investigated temperature and holding time combinations. Heating temperatures of 130 C results in significant higher denaturation degree for heat treatment using THE compared to PHE for all holding times (p<0.001). Rennet coagulation analysis of skim milk heat treated using PHE and THE are shown in figure 12. Figure 12A and figure 12B shows the relative RCT of heat treated skim milk samples, heat treated below and above 100 C, respectively. From figure 12A, it is observed that heat treatment at temperatures below 85 C only have slight increase in coagulation time at all holding times. Heating temperatures of 95 C had a significant increase in relative RCT, even at the short holding times for both heating methods (p< 0.006). Comparing the two methods, there is a tendency towards PHE having less increase in relative RCT for temperatures of 80 C and 95 C, but there is no significantly difference between the two methods at these temperatures for all holding time (p = [ ]). Heating at temperatures above 115 C shown in figure 12B, resulted in very long RCT and samples heated at 140 C using PHE did not reach RCT within 2 hours for any of the in

49 vestigated holding times. Samples heated at 130 C resulted in large increase in relative RCT and there is a significant difference between the methods for all holding times (p <0.03]). THE had less increase in relative RCT compared to PHE and RCT was detected within two hours with use of holding times of 30 and 60 s, which was not observed for heat treatment using PHE. The relative CFR for skim milk samples heated at PHE and THE is shown in figure 12C and figure 12D. The relative CFR decreases with increasing temperature and holding time. Comparing relative CFR for the two heating methods at temperatures below 100 C from figure 12C, there is a significantly lower CFR for THE heated at 80 C with holding times above 120 s (p<0.025). This is also observed for heating at 95 C with holding time less than 60 s (p<0.048). Holding times above 60 s results in very low relative CFR for both methods, with a CFR corresponding to less than 5% of control samples. Comparing heating at 80 C and 85 C at PHE, small differences in relative RCT was found in figure 12A, but when comparing CFR, the de- Figure 12. The relative Rennet coagulation time and relative curd firming rate for skim milk samples heated at PHE and THE. A: Relative RCT of skim milk heated at PHE and THE at temperatures below 100 C. B: Relative RCT of skim milk heated at PHE and THE at temperatures above 100 C. Graphical lines continuing out of the visualized graph indicates that the RCT is not reached within two hours of measurements. C: Relative CFR for skim milk heated at PHE and THE for temperatures below 100 C. D: Relative CFR for skim milk heated at PHE and THE for temperatures above 100 C. Graphical lines going out of the visual range indicates that no CFR was detected within two hours of measurement. Different letters indicate significant differences were found between heating methods for each given temperature, at a given holding time (p <0.05)

50 crease in relative CFR is significantly lower for 85 C compared to 80 C, at holding times of 30 s (p=0.002) and 60 s (p=0.003). Heating at temperatures above 100 C is shown in figure 12D had strong pronounced decrease in relative CFR, with only low CFR detected, even though the RCT was reached within two hours of measurement, as observed in figure 12B. The UHT treated samples did not reach RCT within two hours, and therefore no curd firming rate was detected. PHE had significant lower CFR when heating at 130 C for all holding times (p<0.03), compared to THE. 4.3 Effect of indirect and direct heating systems on heat treatment of skim milk To investigate how different heating method affects the milk properties, skim milk samples heated at DSI, PHE and THE were compared. Milk samples heated using DSI were only heated with one holding time, namely 4s. Milk samples heated indirectly used a holding time of 5 s. When comparing the three methods, the variation in holding time should be kept in mind. Figure 13. Denaturation degrees of whey protein in skim milk heated with DSI for 4 s and skim milk heated with PHE and THE for 5 s. A: denaturation degree of β-lg B. B: denaturation degree of α-la. Significant differences were found between heating methods for each given temperature. Different letters indicate significant differences were found between heating methods for each given temperature (p <0.05). Figure 13A shows the denaturation degree of β-lg B in skim milk heated at DSI, PHE and THE at various temperatures. Heating at 105 C, no denaturation was found when heating at DSI while heating at PHE was significantly higher (p<0.0001), which has a denaturation degree of 70 %. For indirect heating, a denaturation degree above 90 % was observed for heating temperatures of 115 C or more. There is significant difference between the two indirect heating methods and DSI (p<0.002) with DSI having lowest denaturation degree for all comparable temperatures. The denaturation of α-la is seen in figure 13B. No denaturation

51 was detected for heating at 105 C using DSI and a low increase in denaturation is observed as temperature increases. The denaturation of α-la is significantly lower for DSI compared to the indirect heating at all temperatures (p<0.0001). These variations in denaturation degrees between indirect heating and DSI are large, which makes it reasonable to say these large differences would also be present if the indirect heating samples had a holding time of 4 s. The rennet coagulation properties were compared for the three heating methods. Figure 14A shows the relative RCT for skim milk samples using PHE and THE heated for 5 s and DSI heated for 4 s. There is a clear significant difference between PHE and DSI for all temperatures (p<0.002), with DSI having the lowest increase in RCT. Skim milk was heated at 140 C using PHE and this did not reach RCT within two hours. DSI samples heated at 145 C reached RCT within two hours and the relative increase in RCT was lower than samples heated at 130 C using PHE. THE and DSI can only be compared at heating of 130 C but here is the difference also significant. In figure 14B, the relative CFR is shown. Both PHE and THE have significantly lower CFR for all temperatures compared to DSI (p<0.024). The DSI sample heated at 145 C reached RCT within two hours, but no CFR was detected. The differences in RCT and CFR between the indirect and direct methods are large, which makes it reasonable to say that there would still be a significant difference between the methods if they had the same holding time. Figure 14. Relative RCT and CFR of skim milk heated with PHE for 5 s and DSI for 4 s. A: relative RCT for skim milk heated at DSI, PHE and THE. PHE heated 140 C is not within two hours, which is indicted with a bar going out of scaled area. B: relative CFR for skim milk heated at DSI, PHE and THE. Samples with negative CFR indicate that RCT was not detected within two hours of measurement. Different letters indicate significant differences were found between heating methods for each given temperature (p <0.05)

52 4.4 Effect of whey protein denaturation on rennet coagulation The relative RCT and denaturation degree of β-lg B of heated skim milk was compared to investigate the correlations between degree of denaturation and relative RCT. Figure15 shows the relative RCT as a function of denaturation degree of β-lg B for skim milk samples heat treated using PHE, THE and DSI. Figure 15. The denaturation degree of β-lg B shown as a function of relative RCT of skim milk heated at DSI, PHE and THE. Data points exceeding relative RCT of 800 % did not reach RCT within two hours of measurement. It is observed that the denaturation degree of β-lg B in some extent explain the increase in relative RCT. Denaturation degrees of 50 % or less gives only small changes in relative RCT for the two indirect heating methods, while it is seen that THE has lower relative RCT as the denaturation degrees of β-lg B exceeds 70 %. Heat treatment using PHE at temperatures of 130 C and 140 C did not reach RCT within two hours of measurement and these samples had denaturation degrees of 92 % or more. These are imagined in figure 15 by exceeding relative RCT of 800 %. Heat treatment using THE can be described as a two phased linear correlation with a break at denaturation degrees around 95 %, while heat treatment using PHE can be explained exponentially. Heat treatment using DSI is also shown in figure 15. It is seen that a denaturation degree for β-lg B around 40 % resulted in significantly longer relative RCT compared to samples heated indirectly with same denaturation degree (p<0.001). From section 4.3, it is clear that this denaturation degree is observed at low temperatures for heat treatment using PHE and THE, while DSI was heated at 145 C to obtain same denaturation degree. This indicates that it is not only the denaturation degree of whey proteins that has an impact

53 on the RCT, but that the temperature has a great impact, as it gives rise to other chemical changes in the milk which has an impact on coagulation. 4.5 Effect of heat treatment of MCI milk samples on rennet coagulation The rennet coagulation properties of MCI milk samples heat treated using PHE and THE were analysed and compared to investigate the effect of heating method. Only one trial day was used to collect MCI samples heated using THE and thereby only temperatures with holding times of 5 and 10 s, and for 80 C also one sample with a holding time of 120 s, were collected. The data points for heat treatment using THE are therefore an average of two rennet coagulation analysis while milk heated at PHE is an average of four rennet coagulation analysis from two trial days. The relative RCT and relative CFR for MCI samples heat treated using PHE and THE are shown in figure 16. Figure 16. RCT and relative CFR for MCI samples heated at PHE and THE. A: Relative RCT of MCI samples heated at PHE and THE at temperatures below 100 C. B: Relative RCT of MCI samples heated at PHE and THE at temperatures above 100 C. C: Relative CFR for MCI samples heated at PHE and THE below 100 C. D: Relative CFR for MCI samples heated at PHE and THE above 100 C. Graphical lines going out of the visual range indicates that no CFR was detected within two hours of measurement. Different letters indicate significant differences were found between heating methods for each given temperature, at a given holding time (p <0.05)

54 Figure 16A shows the relative RCT for MCI samples heated at temperatures below 100 C. The relative RCT follows a parable formation for heat treatment using PHE at temperatures 100 C. The relative RCT increases for holding times up to 60s and then decreases when a holding time of 120 s is used. Here, the relative RCT was less than RCT of control samples. As not all holding times were measured for heat treatment using THE, it is not possible to say if heat treatment using THE follows the same pattern. The relative RCT is significantly larger for heating at THE than PHE when comparing each holding time between the methods (p<0.03). MCI samples heat treated at temperatures above 100 C are shown in figure 16B. Increases in relative RCT was observed for all temperatures and holding time combination, but all measured samples reached RCT within two hours of measurement. Heating at PHE was observed to give significant lower relative RCT compared to heating at THE (p<0.04). Figure 16C and figure 16D shows the relative CFR for MCI samples heat treated at temperatures below and above 100 C, respectively. A decrease in relative CFR is observed for all MCI samples heat treated using PHE at temperatures below 115 C with a holding time of 5s, but the relative CFR then increases as the holding time is increased from 5s up to 30 s. Holding times above 60s resulted in a decrease in the relative CFR. Heat treatment using PHE has higher relative CFR compared to the corresponding control sample. Comparing the two heating methods, there is significant lower relative CFR for heat treatment using THE compared to PHE at temperatures below 100 C (p<0.03). Heating at temperatures of 130 C and 140 C resulted in a fast decrease in relative CFR for heat treatment using PHE, but a CFR was detected for all MCI samples measured. No CFR was detected for heat treatment at 130 C using THE. Table 5. Relative RCT and relative CFR for MCI samples heated at PHE, 5s and THE, 5 s and DSI, 4s. PHE samples are heated at 140 C and not at 145 C ad DSI. *: No CFR was detected within two hours of measurement. Different letters indicate significant differences were found between heating methods for each given temperature (p <0.05). Relative RCT (%) Relative CFR (%) Temperature ( C) DSI, 4 s PHE, 5 s THE, 5 s DSI, 4 s PHE, 5 s THE, 5 s a c a b c a b 0*

55 The two indirect heating methods with a holding time of 5 s were compared with direct heat treatment using DSI with a holding time of 4s, which is shown in table 5. It is observed that the relative RCT increases slightly as the temperature increases for heat treatment using DSI. Only two temperatures could be compared directly between the heating methods, namely temperatures of 115 C and 130 C using PHE and 130 C using THE. There is a significantly difference between DSI and the two indirect methods for heat treatments at 130 C (p<0.002). MCI samples were heated at 140 C using PHE while samples using DSI were heated at 145 C. Comparing the relative RCT of these, there are significant differences between PHE at 140 C and DSI at 145 C (p<0,003) and it is therefore reasonable to state that a MCI sample heat treated at 145 C using PHE would have an even longer relative RCT and thereby also be significant different from MCI samples heat treated using DSI. The relative CFR for MCI heated using DSI decreased slightly as temperature increased. Heating at 145 C had a relative CFR of % while heating at 130 C using THE, no CFR was detected within two hours of measurement. There is significant larger decrease in CFR for the indirect methods compared to DSI for all comparable temperatures (p<0.02). 4.6 Effect of milk type on rennet coagulation Control milk samples from the three milk types were compared to investigate the differences in coagulation in relation to milk type. The three milk samples are from the same trial week, but from different milk batches, to avoid seasonal variation. From figure 17A, it is observed that the RCT for the three milk types were similar and no significant variation was found, even though skim milk as a tendency to have a longer RCT. Figure 17B shows the curd firming rate. From this, is it clear that MCI, with a casein content of 3.5 %, had faster gel formation compared to skim milk and MCIc which have casein contents of 3.05 % (p<0.04). The CFR of MCIc milk is larger than skim milk but this difference was not found to be significant. These results indicate that the removal of whey proteins from low pasteurized skim milk gives a slight faster gel formation, while removal of whey proteins and increasing the casein content increases the gel formation significantly. This is important to have in mind, as the relative difference of control samples can be equal for the milk types, but it does not consider MCI having faster curd firming rate

56 Figure 17. RCT and CFR of control milk samples for the three milk types, from the same trial week. A: RCT of skim milk, MCI and MCIc. Skim milk has the greatest RCT, but no significant differences between the three milk types. B: CFR of skim milk, MCI and MCIc. MCI has the CFR rate, but no significant differences between skim milk and MCIc. Different letters indicate significantly different values (p<0.05) The effects of heat treatment on the three milk types at PHE were analysed. Heat treatment of MCIc milk samples was only performed using PHE and each temperature and holding time combination was only measured once due to lack of time. These data points are therefore only the average of two ReoRox measurements while skim milk and MCI samples are an average of four ReoRox measurements from two trial days. Figure 17A and figure 17B shows the relative RCT for skim milk, MCI and MCIc samples heat treated using PHE. Significant variations were observed between the three milk types heated at 80 C (p<0.001). The MCI and MCIc heated at temperatures of 115 C and 130 C reached RCT within two hours, while skim milk samples with a holding time exceeding 30 s did not reach RCT within two hours for the same temperatures. No significant difference was found between MCI and MCIc but skim milk increased significantly in relative RCT compared to MCI and MCIc heat treated at temperatures of 115 C and 130 C (p<0.03). The relative CFR of heat treatment of the three milk types are shown in figure 18C and figure 18D. MCIc heated at 80 C resulted in large increase in CFR with a holding time of 5s and afterwards decreased until a holding time of 30 s from where it kept fairly constant. MCI and skim milk decreased in CFR with holding times below 10 s while a slight increase was observed for longer holding times. Overall, significant differences in relative CFR between all three milk types was found (p<0.03). Heat treatments at temperatures of 130 C resulted in significant difference between the three milk types (p<0.02). MCI had the least decrease in relative CFR which was significantly lower than for MCIc, even though CFR was detected for both milk types within two hours of measurement. CFR was only observed for heating at 130 C for 5 s for skim milk and this is significantly different from MCI and MCIc (p<0,001). Sig

57 nificant difference between the milk types heated at 115 C was found, with skim milk having the most intense decrease in CFR and MCI having the least decrease (p<0,01). Figure 18. Relative rennet coagulation time and curd firming rate for skim milk, MCI and MCIc samples heated at PHE. A: Relative RCT for the three milk types heated at 80 C. B: Relative RCT for the three milk types heated at 115 C and 130 C. Graphical lines continuing out of the visualized graph indicates that the RCT is not reached within two hours of measurements C. Relative CFR for the three milk samples heated at 80 C. D: Relative CFR for the three milk samples heated at 115 C and 130 C. Graphical lines going out of the visual range indicates that no CFR was detected within two hours of measurement. Different letters indicate significant differences were found between the milk types for each given temperature (p <0.05) Formation of protein network The formation of protein network induced by rennet was investigated for six samples three skim milk and three MCI samples. The skim milk samples are from same batch, which is also valid for the MCI samples. Figure 19 shows images of the protein structure of the six samples captured with CLSM. FTIC is bound to the proteins and this gives the green emission of the proteins. The heat treated samples were all heated using PHE with a holding time of 10 s. For all MCI samples and for control skim milk, it is seen that a strong and compact protein network was formed, which indicates that the majority of the caseins are bound in the network. This can be seen by the clear separation of the proteins coloured green and the dark background. The dark background indicates that the most protein detected on the images, are bound in protein network

58 The heat treated MCI samples shown in figure 19B and figure 19C form dense networks compared with the control sample, shown in figure 19A, having very compact and strong network. This is observed, as strong networks are compact and little space is found in between the protein networks and less protein not bound in the network. The skim milk sample heated at 80 C for 10 s shown in figure 19E, has no large contrast between the green colour between the network and background. This indicates that there are proteins not bound in the network. Comparing the sample heated treated at a temperature of 80 C with the corresponding control sample, shown in figure 19D, t is clear that the control sample has more compact network. The skim milk sample heated at 130 C, shown in figure 19F, contains many small networks, but the formation of one large protein gel has not occurred yet. This can also be seen as the solution surrounding the small networks contains large amounts of proteins due to the background is quite green. These results are consistent with the results shown in figure 17 and figure 18. An increase in heating temperature gives decrease in RCT and CFR, which therefore gives prolonged protein network formation. This is more pronounced in skim milk compared to MCI. Figure 19. CLSM images of the protein network in three skim milk samples and three MCI samples heated at PHE. The proteins are colored green. A: MCI control sample. B: MCI sample heated at 80 C, 10 s. C: MCI sample heat at 130 C, 10 s. D: Skim milk control sample. E: skim milk sample heated 80 C, 10 s. F: skim milk sample heated 130 C, 10 s

59 4.7 Heat induced protein aggregation Protein aggregation was investigated with three different analytical techniques, namely 1DGE and 2DGE and SEC. The main focus is on the skim milk samples, investigating the aggregate size and composition, mainly for indirect heating DGE Two 1-D gels with four skim milk samples and two MCI samples, analysed reduced and non-reduced, are shown in figure 20. The milk samples were analysed under reducing conditions to disrupt the S-S bridges within the proteins structure and between various proteins. Milk samples analysed under non-reducing conditions have intact S-S bridges and differences between the reduced and nonreduced analysis can be used to identify protein bands containing S-S bridges in the structure. The protein bands were identified according to (Souza et al., 2000). The whey protein bands of heat treated skim milk, which are observed at the low molecular mass range of the gels in figure 20 are of low intensity, both under reduced and non-reduced conditions. These bands are less intensive than the whey protein bands for the control skim milk. Decreases in intensity of these bands were most pronounced in heat treated samples using PHE while heat treatment using DSI having the least reduction. This is consistent with results presented in section 4.2 and section 4.3. No protein bands were found in the top of the gel, which indicates that there are no large protein aggregates present in the reduced samples. For all non-reduced fractions, there is a clear band at the top of the gels above the marker band of 300 kda. This indicates there are protein complexes that do not migrate on the gel. These are various large aggregates bound together with disulphide bonds as these do not appear in the reduced samples. The non-reduced samples of skim milk heated using the three different methods, shown in figure 20B, indicate that there are more complexes reaching the gel in the sample heat treated using DSI compared to the indirect methods, while there is a tendency toward more protein complexes that are not migrating at the gel for the indirect methods. The band for κ-cn is not very pronounced in the non-reduced samples for all milk samples but becomes clearer in the reduced samples for all six samples. The same tendency is seen for β-lg even though the amount of β-lg is low in the heat treated samples due to denaturation. This indicates that κ-cn and β-lg is present in the large complexes. For the heat treatment of the MCI sample, shown in figure 20A, the complexes not migrating on the gel in the non-reduced sample may also contain other caseins as the fraction of whey protein is very small and it seems like the casein bands become slightly more intense

60 Figure 20. 1DGE of six milk samples in reduced and non-reduced form, visualized by colloid Coomassie Brilliant Blue G-250 staining. The molar masses of the marker used are given, as well as the amount of protein loaded in each well. The most intense bands are identified. A: three samples; control skim milk, control MCI and MCI heated at 130 C, 5 s using PHE are shown in a reduced and non-reduced form. B: three skim milk samples; 130 C, 5 s at PHE, 130 C, 5 s using THE and 130 C, 4 s using DSI are shown in a reduced and non-reduced form

61 DGE 2DGE was performed on same samples as for 1D gel electrophoresis, section The protein sport on the gel were identified according to Jensen et al. (2012b) and Larsen et al, (2010). Four 2D gels are shown for in figure 21. These gels contain skim milk samples heat treated using PHE and THE, and analysed under reducing in both dimensions and under non-reducing conditions in both dimensions. The 2D gels that are nonreduced in both dimensions were less clear in the separation between protein bands compared to the recued 2D gels. It is observed for both milk samples that the spots of β-lg and α-la were moved towards a higher pi in the non-reduced samples compared to the reduced samples. This can be due to refolding of the denatured protein into a non-native structure and thereby changing the pi. β-casein was not affected by electrophoresis method, while some α s1 -casein multimers (sport 6) and α s2 -casein dimmers (spot 7) were observed on the non-reduced 2D gels. The 2D gels of the remaining milk samples are shown in Appendix 2. For the skim milk sample heat treated using PHE, figure 21A and figure 21B, there is a clear difference in Figure 21. 2D gel electrophoresis on skim milk samples heated with PHE and THE run with and without DTE in both dimensions. A: Reduced skim milk sample heated with PHE at 130 C for 5 s. B: Non-reduced skim milk sample heated with PHE at 130 C for 5 s. C: Reduced skim milk sample heated with THE at 130 C for 5 s. D: Non-reduced skim milk sample heated with THE at 130 C for 5 s. 1: genetic variants of κ-cn. 2:β-Lg. 3: α-la. 4: β-cn. 5: α s1 -CN. 6: α s1 -CN aggregates. 7: α s2 -CN dimers

62 intensity of the whey protein bands and also κ-casein bands at the non-reduced and reduced 2D gels. The κ-casein bands are not visible on the non-reduced 2D gel and the whey protein spots are weak. This indicates that the whey proteins and κ-casein are bound in various complexes which are not seen on the nonreduced 2D gels, while these complexes are broken down in reduced 2DGE analysis where each protein fragment is seen individually on the gel. Furthermore can the low intensity of κ-casein be caused by κ-casein fund as multimers in milk. The skim milk sample heat treated using THE, shown in figure 21C and figure 21D, shows the same tendency as the skim milk samples heated using PHE. One major difference though, is that THE skim milk sample has visible κ-casein bands in the non-reduced sample but the same intensity pattern for whey protein bands. This indicates that the complexes made of THE skim milk contain less κ-casein but the same amount of whey protein, as the protein content is the two samples are similar, shown in table 3, and it is therefore reasonable to believe that β-lg forms aggregates with other β-lg proteins Size exclusion chromatography Size exclusion chromatography was performed on milk samples in reduced and non-reduced form to investigate the amount of aggregates formed in each sample. Figure 22 shows the chromatograms of the reduced and non-reduced samples. The reduced samples in figure 22A results in peaks observed at retention time 7-9 min for all skim milk samples. The MCI samples have very low intensity of this peak indicating that there are some aggregates in this area containing whey proteins which are not S-S bound. For all samples under non-reduced conditions samples shown in figure 22B, large peaks are observed with retention times of min. This indicates a high amount of disulphide bound aggregates which contain caseins are present in this area. Compared to the reduced samples, no peaks at retention time min can be observed in the non-reduced samples. The control skim milk has a higher overall absorbance in the non-reduced form, figure 22B, compared to the heat treated samples which could indicate that there are even larger aggregates present in the heat treated milk which cannot be detected by the used column. Comparing the three heat treated skim milk samples, it is seen that THE has larger amount of the large aggregates and also a lower amount of the intermediate size proteins for reducing and non-reducing conditions while the amount of small peptides are equal for all heating methods. DSI has more intermediate aggregates, retention time 7-9 min, compared heat treatment using PHE and THE

63 Figure 22. Chromatograms for milk samples analyzed with SEC under reduced and non-reduced conditions. A: Reduced milk samples analyzed on SEC. B: non-reduced milk samples analyzed on SEC Identification of aggregates For two of the samples heat treated using PHE and THE at 130 C for 5 s, the large aggregates were collected and analysed to identify the content of the aggregates containing disulphide bonds. Figure 23 shows the chromatogram from SEC analysis. The total protein content measured on Milkoscan was 3.64% and 3.66% for the heat treated sample using THE and PHE, respectively. The chromatograms for the two samples show similar pattern, but differences in intensities. Heat treatment using PHE had higher absorbance and more protein is thereby detected by the use of the specific column compared to heat treatment using THE. This could indicate that there is a higher content of large protein complexes that cannot be detected with the used column for heat treatment using THE, as the total protein content in the two samples are similar. The used column separates molecules in the weight range from 5kDa to 1200 kda. From the results obtained from 1DGE shown in section 4.7.1, could it be concluded that protein aggregates above 300 kda were present in heat treated milk. Results presented in this section show that protein aggregates exceeding a molecular mass 1200 kda are present

64 Figure 23. Chromatograms for skim milk samples analysed with SEC in non-reduced conditions. The peak area from 8-12 min was collected into four fragments for further analysis. Figure 24. Total protein analysis of skim milk fractions collected at SEC. Peak 1-3: κ-casein, peak 4: α s2 - casein, peak 5: α s1 -casein, peak 6: β-casein, peak 7 : α-la, peak 8: β-lg B, peak 9: β-lg A

65 The protein aggregates appeared on the chromatogram with RT 8-12 min were analysed on LC-MC. Figure 24 shows the chromatogram of UV detection from LC analysis of the four fractions collected for both heat treatments. The four fractions all show similar pattern but are of different intensities. This is caused by the amount of protein in each fragment is not equal due to differences in intensity of the SEC analysis of which the four fragments were collected from. Comparing the chromatograms of the two heating methods, there were found variations in the protein content in the aggregates. The aggregates formed by heat treatment using PHE, contained large amounts of glycosylated κ-casein (peak 1-3), β-casein (peak 6) and whey proteins (peak 7-9). The aggregates formed by heat treatment using THE, contained large amount of α s1 -casein (peak 5) while almost no κ-casein and whey proteins are present compared to heating using PHE

66 4.8 Kinetics of denaturation of whey proteins The effect of heating temperatures and holding times were investigated to determine the rate of denaturation of β-lg and α-la. The order of reaction used for β-lg was 1.5 and for α-la it was 1 according to previous studies (Dannenberg and Kessler, 1988; Kessler and Beyer, 1991; Oldfield et al., 1998a; Zúñiga et al., 2010), using the rate equations, for n = 1.5 and, for n = 1 Figure 25. Denaturation degree of β-lg B and α-la for skim milk samples heat treated using PHE at temperatures from 80 C to 140 C at various holding times. For each temperature, a linear regression is fitted and these data are shown in table 5. A: denaturation of β-lg with a reaction order of 1.5. B: Denaturation of α-la with a reaction order of

67 Figure 25A shows graphical representation of the denaturation of β-lg B and figure 25B shows denaturation of α-la for skim milk samples heated at PHE. For each temperature, the best fitted straight line was plotted. This line indicates the kinetic fit which is used to calculate the rate constants shown in table 6. β-lg A follows same pattern as β-lg B and is therefore not shown graphically. The rate constant k was calculated from the slope of regression from the best fitted straight line for each temperature. The slope is, for a reaction order of 1.5 and k, for a reaction order of 1. Table 6. Rate ate constant k and correlation coefficient of reaction kinetics on denaturation of β-lg B, β-lg A and α-la in skim milk heated at PHE and THE. The values for β-lg B and α-la heated at PHE are obtained from figure 25. Values for β-lg A heated with PHE and all values heated at THE are obtained from graphical analysis, which are not shown. Temperature ( C) k 10 3 (S -1 ) R 2 k 10 3 (S -1 ) R 2 PHE THE β-lg B n = β-lg A n = , α-la n =

68 Table shows the achieved data from linear regression and the correlation coefficient for each temperature of β-lg B and α-la from figure 25The values for β-lg A heated at PHE and all values for heat treatment using THE were obtained in similar way. The reaction order of 1.5 for β-lg B and β-lg A and reaction order of 1 for α-la obtained good correlation for all measured temperatures, with R 2 from Comparing the reaction constants for heating at PHE and THE, it is observed that the reaction constant is larger for β-lg B and β-lg A when heating using THE which indicates that the denaturation of β-lg B and β-lg A is faster when heating using THE. The obtained rate constants were plotted against the reciprocal of the absolute temperature. Figure 26 shows the effect of temperature on the rate constant of denaturation of β-lg B, β-lg A and α-la for skim milk samples heated at PHE. For β-lg B and β-lg A heated at PHE, it is possible to make linear regression in the temperature range from 80 C to 95 C and again from 95 C to 140 C. For α-la, this break is found at 85 C. From the linear regression of each temperature range, the activation energy is calculated from the Arrhenius equation which is shown in table 7. Figure 26. The effect of temperature on rate constant for denaturation of β-lg B, β-lg A and α-la for skim milk heated at PHE. Linear regressions are made by fitting rate constants for heating at PHE

69 The activation energy for β-lg at heating temperatures below 95 C obtained activation energies of 250, while heating temperatures above 95 C obtained activation energies of similar shift in activation energy is observed for α-la, in the temperature ranges C and C. As can be seen in figure 26, the measured rate constants are fitted to a linear regression, which is stated in table 7. The calculation of reaction kinetics for α-la at the low temperature range, only two rate constants were available and thereby could the uncertainty of the fit not be given. It was not possible to calculate the reaction kinetics for heat treatment using THE as only three heating temperatures were used and the obtained kinetic result would be very uncertain. Table 7. Reaction kinetic for denaturation of β-lg B, β-lg A and α-la for skim milk heated at PHE. The values are calculated from data obtained from figure 26. Order (n) Temperature range C ln(k 0 ) E a R 2 β-lg B 1., β-lg A 1., α-la

70 5 Discussion In this study, three different types of heat treatments were performed on milk with various whey protein and casein content, and these were examined for coagulation properties, whey protein denaturation and formation of heat induced aggregates. The denaturation degree of whey proteins in skim milk increased with increasing holding time for all investigated temperatures. β-lg B showed a higher degree of denaturation compared to β-lg A, while α-la had the least denaturation at all measured temperature and holding time combinations and heating methods. This is consistent with theory, as β-lg A has a slightly lower denaturation temperature, but is less reactive due to a higher negative charge compared to β-lg (O Connell and Fox, 2011). α-la has a tendency to reform into native structure at low temperatures and it higher temperatures are required to form aggregates with other proteins which can explain the lower degree of denaturation observed for α-la. The reaction kinetics of denaturation of whey proteins was extensively investigated by Dannenberg and Kessler (1988) and their results are widely accepted and commonly used as reference for the effect of heat treatment on whey protein denaturation. Since the publication of their study, various different heating systems and other analytical methods have been used to analyse the denaturation degrees of whey protein. Figure 27. Effect of heat treatment with PHE on the denaturation of β-lg B in skim milk. The lines represent the calculated rate of denaturation measured by Dannenberg and Kessler (1988) and each point represents the measured denaturation degrees in the present study

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