FACULTY OF BIOSCIENCE ENGINEERING. INTERUNIVERSITY PROGRAMME (IUPFOOD) MASTER OF SCIENCE IN FOOD TECHNOLOGY Option Food Science and Technology

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1 Katholieke Universiteit Leuven FACULTY OF BIOSCIENCE ENGINEERING INTERUNIVERSITY PROGRAMME (IUPFOOD) MASTER OF SCIENCE IN FOOD TECHNOLOGY Option Food Science and Technology Academic year Emulsifying Properties of Milk Fat Globule Fragment Prepared from Butter Milk by Microfiltration by Md. Asaduzzaman Promotor : Prof. Dr. Ir. Koen Dewettinck Tutor : Que Phan Thi Thanh Master's dissertation submitted in partial fulfilment of the requirements for the degree of Master of Science in Food Technology

2 The author and promoter give the permission to consult and copy parts of this work for personal use only. Any other use is under the limitations of copyrights laws, more specifically it is obligatory to specify the source when using results from this Master s Dissertation. Gent, August 2011 The promoter The author. Prof. dr. ir. K. Dewettinck.. Md. Asaduzzaman

3 ACKNOWLEDGMENTS In the name of Allah, the most gracious, the most merciful, all praise is God Lord of all creation. I would sincerely like to thank all those who helped and inspired me to complete this dissertation. It is my honour to express the most special thanks and gratitude to my promoter Prof. Dr. ir. Koen Dewettinck for providing me the invaluable opportunity to carry out the research and by giving continuous inspiration, suggestion, support and guidance during this research period. I am indebted to my supervisor Que Phan Thi Thanh for her wonderful guidance, support, intellectual guidance and company given to me to cope with everything throughout my research study. I am also grateful to Mrs. Kathleen Anthierens and Ruth Van den Driessche for their friendly assistance and generous help on every occasion. Your cordial guidance highly motivated me to continue no matter how difficult things were, being far from my home country. To all members and staff of the Laboratory of Food Technology and Engineering, I am grateful for their cooperation and warm friendship. Especially Eveline, Thein, Bart, Natalie, kim, Benny, Corine, and Bea for their invaluable technical assistance. I also like to thanks prof. dr. ir. Bruno De Meulenaer and prf. dr. ir. Paul Van der Meeren for giving me the access to the equipment at their laboratories. I am greatly indebted to VLIR for providing me the scholarship to pursue this master program, without which this study work would have not been possible and also wish to extend my sincere thanks to the VLIR staff for their cordial concern about the international student. I will always treasure the happy moments with my Bangla community family and my circle of friends and to express my thanks for their initiative to make of students feel secured and ever blessed.. My stay in Belgium became more exciting thanks to Kamrul, Banti, Akter, Ifty, Shaown, Salatul, Wahid, Nipa, Neelanjona, Robiul and the great Simum. To my fellow scholars Fahrizal, Puji, Joyce, Lily, Tarak, Thu, Bao, Biniam, Dian, Nathalia and Zahra, the fun times that we had, the tours we made and the crazy moments will always inspire me and I believe that friendship never ends I would like to express my special thanks to my beloved parents, sister, brother; they have given me all the necessary opportunities, and were there when needed. Tasmia, my beloved daughter, you're the very best that ever happened to me. I miss you a lot every day, every moment. Last but not least I am so indebted to my lovely wife who always shares with me love, happiness and sorrow. Md. Asaduzzaman Ghent, August 2011 ii

4 TABLE OF CONTENTS ACKNOWLEDGMENTS... ii LIST OF TABLES... v LIST OF FIGURES... vi LIST OF ABBREVIATION... vii LIST OF APPENDICES... viii ABSTRACT... x CHAPTER I: INTRODUCTION... 1 CHAPTER II: LITERATURE REVIEW Introduction of dairy by-products used in the emulsification process Milk fat globule membrane (MFGM) Origin Structure Composition of MFGM Lipids Proteins Separation and isolation of the MFGM materials Emulsifying properties of the MFGM Dairy emulsion system Definition Factors effect on stability of dairy emulsions Effect of proteins Effect of the MFGM Effect of homogenization Whipping properties Whipped cream Characterization of whipped cream CHAPTER III: MATERIALS AND METHODS Materials and chemicals Materials Chemicals Compositional analysis of the experimental materials Experiment 1: Characteristics of emulsions stabilized with milk fat globule membrane fragments iii

5 3.3.1 Emulsion preparation Determination of emulsion properties Microscopic observation Rheological characteristic Particle size distribution measurement Creaming stability Experiment 2: whipping properties of the MFGM fragments Experimental procedure Determination of whipping properties Overrun Serum loss Firmness Statistical analysis CHAPTER IV: RESULTS AND DISCUSSION Composition of the experimental materials Creaming stability Particle size distribution Microscopic observation Rheological characteristics Whipping properties of the recombined cream CHAPTER V: CONCLUSION FUTURE PERSPECTIVE LIST OF REFERENCES iv

6 LIST OF TABLES Table 1. Estimated average composition of the milk fat globule membrane 6 Table 2. Protein components of the MFGM.. 8 Table 3. Composition of the formulation materials Table 4. Average Sauter mean diameter D 3.2 of emulsions at different homogenization pressure. 32 Table 5. Average surface-weighted mean diameter D 3.2 of different emulsions at 90/20 bar. 33 Table 6. Rheological parameters of emulsions homogenized at 90/20 bar.. 37 Table 7. Rheological parameters of emulsions prepared with different dairy materials and homogenized at different pressures.. 39 Table 8. Whipping parameter of recombined cream containing BMP and MFGM-BMP v

7 LIST OF FIGURES Figure 1. Structure of the fat globule with detailed arrangement of the main MFGM 5 Figure 2. Schematic of the fluid mosaic membrane according to Singer and Nicolson... 5 Figure 3. SDS-PAGE of different dairy products... 9 Figure 4. Protein profiling of different dairy materials by SDS-PAGE.. 27 Figure 5. Creaming stability of emulsions prepared with different dairy materials upon storage at 4 C.. 28 Figure 6a. Particle size distribution of different emulsions homogenized at different pressures, after dilution in water and SDS. 30 Figure 6b. Particle size distribution of different emulsions homogenized at different pressures, after dilution in water and SDS. 31 Figure 7. Particle size distribution of emulsions prepared with different material at homogenization pressure 90/20 bar Figure 8. Microscopy images of emuslsions prepared with BMP, MFGM-BMP, MFGM-BMW, SC and SMP at different homogenization pressures (0/20, 90/20, and 210/20 bar).. 34 Figure 9. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM- BMP and SC) at different homogenization pressures, after 1 day of storage. 36 Figure 10. Flow curve for the emulsions (BMP, SMP, MFGM-BMW, MFGM- BMP and SC) homogenized at 90/20 bar. 37 Figure 11. Whipping properties of cream containing BMP and MFGM-BMP. 40 vi

8 LIST OF ABBREVIATION ADPH ANOVA AOAC BPM BSA BTN Adipophilin Analysis of variance Association of Official Analytical Chemists Buttermilk powder Bovine serum albumin Butyrophilin CD36 Cluster of differentiation 36 IEP Isoelectric point MFGM Milk fat globule membrane MFGM-BMP MFGM isolated from buttermilk MFGM-BMW MFGM isolated from buttermilk whey MUC 1 Mucin 1 O/W Oil-in-water PAS 6/7 Periodic acid Schiff 6/7 SC SDS SDS-PAGE SMP XO Sodium-Caseinate Sodium dodecylsulfate Sodium dodecylsulfate polyacrylamide gel electrophoresis Skim milk powder Xanthine oxidase vii

9 LIST OF APPENDICES Appendix 1 Appendix Table 1 Appendix Table 2 Appendix Table 3 Appendix Table 4 Appendix Table 5 Appendix Table 6 Appendix Table 7 Appendix Table 8 Appendix Table 9 Appendix Table 10 Appendix Table 11 Appendix Table 12 Appendix Table 13 Appendix Table 14 Appendix Table 15 Appendix Table 16 Appendix Table 17 Appendix Table 18 Appendix Table 19 Appendix Table 20 Appendix Table 21 Appendix Table 22 Flow curve for the emulsions prepared with BMP, SMP, MFGM- BMW, MFGM-BMP and SC at different homogenization pressures ANOVA on protein content of different dairy materials ANOVA on fat content of different dairy materials ANOVA on ash content of different dairy materials ANOVA on lactose content of different dairy materials ANOVA on polar lipids content of different dairy materials ANOVA on surface-weighted mean D 3,2 of BMP after dilution in SDS. ANOVA on surface-weighted mean D 3,2 of BMP after dilution in water ANOVA on surface-weighted mean D 3,2 of SMP after dilution in SDS ANOVA on surface-weighted mean D 3,2 of SMP after dilution in water ANOVA on surface-weighted mean D 3,2 of SC after dilution in SDS. ANOVA on surface-weighted mean D 3,2 of SC after dilution in water ANOVA on surface-weighted mean D 3,2 of MFGM-BMW after dilution in SDS. ANOVA on surface-weighted mean D 3,2 of MFGM-BMW after dilution in water. ANOVA on surface-weighted mean D 3,2 of MFGM-BMP after dilution in SDS. ANOVA on surface-weighted mean D 3,2 of MFGM-BMP after dilution in water. ANOVA on surface-weighted mean D 3,2 of different dairy materials at 90/20 bar after dilution in SDS ANOVA on surface-weighted mean D 3,2 of different dairy materials at 90/20 bar after dilution in water ANOVA on consistency index (K) of emulsions prepared with BMP after 1 day storage. ANOVA on consistency index (K) of emulsions prepared with BMP after 8 days storage. ANOVA on consistency index (K) of emulsions prepared with SMP after 1 day storage. ANOVA on consistency index (K) of emulsions prepared with SMP after 8 days storage. ANOVA on consistency index (K) of emulsions prepared with SC after 1 day storage. viii

10 Appendix Table 23 ANOVA on consistency index (K) of emulsions prepared with SC after 8 days storage. Appendix Table 24 ANOVA on consistency index (K) of emulsions prepared with MFGM- BMW after 1 day storage Appendix Table 25 ANOVA on consistency index (K) of emulsions prepared with MFGM- BMW after 8 days storage Appendix Table 26 ANOVA on consistency index (K) of emulsions prepared with MFGM- BMP after 1 day storage Appendix Table 27 ANOVA on consistency index (K) of emulsions prepared with MFGM- BMP after 8 days storage Appendix Table 28 ANOVA on consistency index (K) of emulsions prepared with different dairy materials after 1 day storage Appendix Table 29 ANOVA on consistency index (K) of emulsions prepared with different dairy materials after 8 days storage Appendix Table 30 ANOVA on Power law index (n) of emulsions prepared with BMP after 1 day of storage. Appendix Table 31 ANOVA on Power law index (n) of emulsions prepared with BMP after 8 days of storage. Appendix Table 32 ANOVA on Power law index (n) of emulsions prepared with SMP after 1 day of storage. Appendix Table 33 ANOVA on Power law index (n) of emulsions prepared with SMP after 8 days of storage. Appendix Table 34 ANOVA on Power law index (n) of emulsions prepared with SC after 1 day of storage. Appendix Table 35 ANOVA on Power law index (n) of emulsions prepared with SC after 8 days of storage. Appendix Table 36 ANOVA on Power law index (n) of emulsions prepared with MFGM- BMW after 1 day of storage Appendix Table 37 ANOVA on Power law index (n) of emulsions prepared with MFGM- BMW after 8 days of storage Appendix Table 38 ANOVA on Power law index (n) of emulsions prepared with MFGM- BMP after 1 day of storage Appendix Table 39 ANOVA on Power law index (n) of emulsions prepared with MFGM- BMP after 8 days of storage. Appendix Table 40 ANOVA on Power law index (n) of emulsions prepared with different dairy materials after 1 day of storage Appendix Table 41 ANOVA on Power law index (n) of emulsions prepared with different dairy materials after 8 days of storage ix

11 ABSTRACT The milk fat globule membrane (MFGM) consists of specific membrane proteins and phospholipids which possess nutritional and technological functionality. The MFGM materials produced from buttermilk and buttermilk whey by microfiltration were used to stabilize oil-in-water emulsions. The emulsifying properties of these materials were also compared with BMP, SMP and sodium-caseinate. Whipping properties of recombined cream enriched with MFGM-BMP was also investigated. The objective of this study was to investigate the potentiality of MFGM materials as food emulsifier and how this potentiality does influences the whipping properties of recombined dairy cream. Emulsions containing MFGM-BMP showed significantly smaller particle size distribution than MFGM-BMW, BMP and SMP. Microscopic observation was also in favor smaller particle size distribution of MFGM-BMP. In addition MFGM-BMP showed good creaming stability. Similar emulsions prepared with BMP, SMP, SC and MFGM-BMW showed extensive flocculation with the only exception of MFGM-BMW for which a good stability to creaming was found. Emulsions prepared with SC showed smaller particle size distribution but a poor stability compared to the other emulsions. The Newtonian flow behavior was observed for emulsions prepared with MFGM-BMP, whereas, shear thinning and thixotropic behaviors were exhibited by the other materials. These research results indicated that a selective concentration of MFGM isolated from buttermilk powder by microfiltration has the potential for the development of ingredients that differ substantially from that of MFGM- BMW and other dairy materials. In addition to the conclusions described above, it can be concluded from an additional study on the whipping properties of MFGM-BMP that recombined cream with MFGM-BMP has a characteristic longer whipping time, higher overrun, similar firmness and a relatively higher serum loss as compared to recombined cream with BMP. Therefore, more detailed work is needed on the quantification and functionality of MFGM materials in the development of microstructure and physical appearance of whipped cream. x

12 CHAPTER I: INTRODUCTION The fat globules in milk are surrounded by the MFGM, a true biological membrane, mainly composed of polar lipids and unique membrane specific proteins, which have an interesting nutritional functionality. The polar lipids are amphiphilic in nature; contain a hydrophobic tail and a hydrophilic head group, which mainly contribute to the emulsifying properties of the membrane. The MFGM materials can be used as emulsifiers or stabilizers, resulting in a combined technological and nutritional functionality (Dewettinck et al., 2008) Several side streams of the dairy industry such as buttermilk and buttermilk whey are wrongly considered as inferior products. The aqueous phase derived as by-product from the butter making process is known as buttermilk. It is rich in milk fat globule membrane (MFGM) fractions and also contains all water soluble material present in milk such as lactose, casein and whey protein. On the other hand, whey is the by-product of the cheese and casein manufacturing process. It still contains some residual fat which consists of lipoprotein particles, MFGM and small fat globules (Rombaut et al., 2007). It has been shown that dairy side streams can be an interesting source of functional compounds such as the MFGM. Although the MFGM concentration in whey is less than in buttermilk, the absence of casein makes it easier to separate and makes it an interesting source of MFGM materials (Morin et al., 2006). In recent years there has been increasing interest in accumulating knowledge on the composition and properties of the MFGM materials. The composition and properties of the MFGM is completely different from that of milk or plasma. The MFGM prevents flocculation and coalescence of milk fat globules as natural emulsifier and has a protective action against enzymatic action on the fat globules. The properties of the MFGM largely depend on the type of treatment of the milk during processing e.g. heating, cooling, homogenization, evaporation, drying etc. (Evers, 2004) and the method of isolation and separation of the MFGM from raw materials (Singh, 2006). A number of studies have been conducted to investigate the potentiality of MFGM materials in food and non- food applications. Lopez et al. (2007) and Michalski et al. (2007) investigated the influence of MFGM enrichment on the microstructure of different cheeses by using small fat globules; Thompson and Singh (2006) produced liposomes from the MFGM using the microfluidization technique. Also several applications of MFGM materials, based on their emulsifying properties (due to high polar lipid content), have been reported such as 1

13 their use as baking improver, their function as fat disperser and anti-staling agent in bakery goods; for the prevention of spattering, browning and dispersion of sediment in margarine; for the reduction of the viscosity and to prevent crystallization in chocolate and as wetting, dispersion and stabilization agent in instant products (Vanhoutte et al., 2004; Szuhaj, 1983; Vannieuwenhuyzen, 1981). The side streams of dairy processing still have a lower economical value compared with the mainstream products. Proper utilization of these sources to isolate the functional MFGM material and then apply it in the development of new products may bring great economical and technological success. Food processing industries have been emphasizing on the use of MFGM materials as natural components, to improve the nutritional value and create specific functionalities of food products. Although several workers (Roesch et al., 2004, Sodini et al., 2006)) found the MFGM isolated from buttermilk to be a good emulsifier but opposite results were also reported by others (Correding and Dalgleish, 1997; Wong and Kits, 2003). However, this study focuses on the characterization of oil-in-water emulsions prepared with MFGM materials produced by microfiltration from buttermilk and buttermilk whey. The properties of these materials will also be compared with buttermilk powder (BMP), skim milk powder (SMP) and sodium-caseinate. This comparative study will allow us to select MFGM material with unique interfacial functionality. Hence, the objective of this study is to investigate the potentiality of MFGM materials as food emulsifier and how this potential does influence the whipping properties of recombined dairy cream. 2

14 CHAPTER II: LITERATURE REVIEW 2.1 Introduction of dairy by-products used in the emulsification process Many common dairy products are emulsions such as creams, whipped cream, ice cream, yoghurt etc. Emulsifying properties of milk derived components influence the physical characteristic of dairy emulsions. Skim milk powder, sweet buttermilk powder, butter-derived aqueous phase, whey proteins, casein dispersions, phospholipids and purified milk fat globule membrane (MFGM) suspensions have been used successfully to make emulsions (Elling et al., 1996; Tomas et al., 1994; McCrae et al., 1999). Milk proteins are highly valued for their emulsifying and emulsion stabilizing properties. Skim milk powder is rich in protein e.g. caseins and whey proteins. Caseins from milk have high surface active properties and are adsorbed readily at the O/W interface. Whey proteins are good emulsifying and foaming agents. The lipid fractions of whey protein concentrate or whey protein isolate influence the whipping properties of cream (Ennis and Mulvihill, 1998). Buttermilk is rich in MFGM derived fractions. The use of buttermilk in food systems is closely related to its particular composition in emulsifying components such as phospholipids which can act as emulsifier in dairy emulsions. MFGM contains various proteins and phospholipids which are efficient natural surface-active materials for emulsion formation. Hence, MFGM materials should be considered as a potential emulsifying agent for certain foods and other emulsions (Kanno et al., 1991) 2.2 Milk fat globule membrane (MFGM) The dispersion of milk fat globules in milk is not a simple O/W emulsion. The fat globules are surrounded by a complicated membrane, which cannot be considered as a simple monomolecular film of surface active material. Instead, the membrane has several distinct layers that are laid down during synthesis in the secretory cells of the mammary gland. This membrane is well known as the milk fat globule membrane (MFGM). The membrane is different from that of either milk or plasma and represents a unique biophysical system (Singh, 2006). The MFGM acts as a natural emulsifying agent, protecting fat globules from enzymatic action and preventing flocculation and coalescence of fat globules in milk (McPherson & Kitchen, 1983; Walstra, 1995). 3

15 2.2.1 Origin The membrane surrounding milk lipid globules is essentially a tripartite structure that originates from the apical plasma membrane, the endoplasmic reticulum and possibly from other intracellular compartments. The MFGM fraction originating from the apical membranes has a typical bilayer appearance and is termed the primary membrane. The material derived from the endoplasmic reticulum has the appearance of a monolayer of proteins and polar lipids that covers the triacylglycerol-rich core lipids to the globules before secretions (Keenan and Mather, 2006). The triglycerols are synthesized in or on the surfaces of rough endoplasmic reticulum membranes and accumulate in the form of micro-lipid droplets in the cytoplasm. These intracellular droplets are covered by a diffuse interfacial layer, which consists of phospholipids, glycosphingolipids, cholesterol and proteins. Micro-lipid droplets grow in volume by fusion with each other to form cytoplasmic lipid droplets of various sizes, which are then transported to the apical pole of the cell through the cytoplasm and are secreted from the epithelial cell. During secretion the droplets are coated with the outer plasma membrane from the cell. The composition of the fat globule membrane is similar to that of the apical plasma membrane of secretory cells (Keenan & Dylewski, 1995; Keenan, 2001 and Heid & Keenan, 2005). Apart from the MFGM, secretory cell fragments can also be secreted into the lumen. They are rich in polar lipids, have a similar composition as the MFGM and comprise only 4% of the total milk fat (Deeth, 1997; Keenan et al., 1999) Structure The MFGM is highly structurized and contains unique polar lipids and membrane specific proteins. The natural MFGM consist of three distinct layers, viewed from the inside lipid core to outwards, - a monolayer of proteins and polar lipids surrounding the intracellular fat droplet, - an intermediate electron dense proteinaceous coat on the inner face of the bilayer and finally, - a true bilayer membrane of polar lipids and protein. The entrained cytoplasmic materials between the inner coat and outer bilayer membrane form the cytoplasmic crescents. (Danthine et al., 2000; Evers, 2004; Michalaski et al., 2002). The MFGM originates from several distinct layers with a total thickness varying from 10 to 20 nm (Wooding, 1971; Walstra et al., 1999). 4

16 Figure 1. Structure of the fat globule with detailed arrangement of the main MFGM. A double layer of polar lipids is placed on an inner monolayer of polar lipids. Membrane-specific proteins are distributed along the membrane. ADPH is located in the inner polar lipid layer, XDH/XO is located in between both layers. MUC1, BTN, CD36 and PASIII are located in the outer layer. PAS6/7 and PP3 are only loosely attached at the outside of the MFGM. The choline-containing phospholipids, PC and SM, and the glycolipids, cerebrosides and gangliosides, are largely located on the outside of the membrane, while PE, PS and PI are mainly concentrated on the inner surface of the membrane. (cited from Dewettinck et al., 2008) The bilayer membrane of the MFGM is derived from the apical plasma membrane of the secretory cell and the most widely accepted model for this type of membrane is the fluid mosaic model. This suggests that the phospholipid bilayer serves as backbone of the membrane, which exists in a fluid state. Peripheral membrane proteins are partially embedded or loosely attached to the bilayer. Trans-membrane proteins extend through the lipid bilayer. Carbohydrate moieties from glycolipids and glycoproteins are oriented outwards. Cholesterol is present in the polar lipid bilayer (Dewettinck et al., 2008) Figure 2. Schematic of the fluid mosaic membrane according to Singer and Nicolson (1972). 5

17 2.2.3 Composition of MFGM The amount and composition of the MFGM varies considerably depending on the fat globule size and the fat content of milk, which is in turn, is influenced by several others factors such as type of feed, breed, age, health and stage of lactation of cows (Keenan, 2001; Keenan & Dylewski, 1995). The major components of the MFGM comprise membrane specific proteins (mainly glycoproteins), phosphor- and sphingolipids. Protein and phospholipids together account for over 90% of the membrane dry weight but the relative proportions of lipids and proteins may vary widely (Singh, 2006). The average composition of the MFGM is given in the table 1. Table 1. Estimated average composition of the milk fat globule membrane (Walstra et al., 2006) Component mg/100gfat globules g/100g MFGM dry matter Protein Phospholipids Cerebrosides 80 3 Cholesterol 40 2 Monoglycerides + a? Water + - Carotenoids + Vit. A Fe Cu Total a +; present, but quantity unknown The composition of the milk fat globule membrane also changes when subjected to different processing treatment such as cooling, agitation, heating and aging. (Evers, 2004). Kirst, (1996) divided the factors that affect the composition of the MFGM into 3 groups, viz. physiological, chemical/enzymatic and physical/mechanical. Physical/mechanical factors are related to milk handling during and after milking. Pre-factory milk handling includes air inclusion, pumping and stirring of milk, changes in temperature and changes in time. Milk handling at the factory involves aging, agitation, heat treatment, separation, homogenization and changes in water content (McPherson and Kitchen, 1983). Sometimes the presence of air is wanted (e.g. butter making process) but the mixing of air or gas with milk or cream significantly reduces the stability of the fat globules. When milk fat globules and air come into contact with each other the milk fat globule membrane is disrupted. Consequently the 6

18 MFGM materials spread over the air/milk plasma interface and are released through the milk plasma when the air bubbles collapse (van Boekel and Walstra, 1989). Large fat globules are more susceptible to shear stress than the smaller ones. Wiking et al., (2003) reported that the resistance of the fat globules membrane against coalescence during agitation is determined by the size of the fat globule, the fat content, the milk temperature and the shear rate. Ye et al., (2002) showed that, when fat globules are heated in absence of serum proteins, there is a tendency to form a complex between butyrophilin and xanthine oxidoreductase by disulphide bonds at temperature as low as 60 C. At higher temperature (80 C) a significant amount of serum proteins, particularly β-lactoglobulin, interacts with the MFGM (Lee and Sherbon, 2002). Homogenization of the milk decreases the fat globule size and increases the milk fat surface area; consequently new materials from the milk serum (predominantly casein) come to cover the free surface and change the composition of the membrane (Darling and Butcher, 1978) Lipids Milk fat globule membranes consist mainly of polar lipids and a negligible amount of neutral lipids (triglycerides, diglycerides, monoglycerides, cholesterol and its esters). High melting triglycerides are the major fraction of the neutral lipids in the MFGM (Wooding & Kemp, 1975). However these triglycerides appear to originate from contamination by the core of the fat globules during the isolation process of the membrane (Walstra, 1985). Hence, the method of isolation has great influence on the triglyceride content of the MFGM. The MFGM associated triglycerides contain higher proportions of long chain saturated fatty acids than that of the globule core fat. Cholesterol amounts to 90% of the sterols content of the MFGM (Jensen and Newberg 1995) In bovine milk, about 60% of the phospholipids are associated with the milk fat globules and the rest is located in the membrane material of skim milk (Patton and Keenan, 1975). The major polar lipid fraction present in the MFGM consists of pholphatidylcholine (PC), 35%; phosphatidylethanolamine (PE), 30%; sphingomyelin (SM), 25%; phosphatidylinositol (PI), 5%; phosphatidylserine (PS), 3%. glucosylceramide (GluCer), lactosylceramide (LacCer). Gangliosides (Gang) are present in trace amounts (Danthine et al., 2000; Deeth, 1997) The MFGM phospholipids contain high levels of long chain (> C 14 ) fatty acids; short and medium chain (C 4 -C 14 ) fatty acids are virtually absent. Especially, PE is highly unsaturated followed by PI and PS. PC is rather saturated as compared to other glycerophospholipids. 7

19 Sphingomyelin (SM) is very uncommon in its fatty acid pattern and consists of around 70-97% of saturated FA and of a high proportion (40-50%) of fatty acids having a chain length of C 20 or more. These two physical features contribute to less fluidity and maintaining rigidity of the MFGM (Bitman and Wood, 1990) Proteins Depending on the isolation method, the protein content of the MFGM varies from 25-60% (Singh, 2006) and the type of proteins largely depends on the origin or source of the milk. It is well accepted that the MFGM protein fractions represent only 1-4% of the total milk proteins (Park, 2009). The major MFGM proteins are xanthine oxidase, butyrophilin (BTN), adidophilin (ADPH) and periodic acid Schiff (PAS) 6/7; the minors proteins are polymeric immunoglobulin receptor protein, apolipoprotein E, apolipoprotein A1, 71 kg/mol heat-shock cognate protein, clusterin, lactoperoxidase, immunoglobulin heavy chain and eptidylprolyl isomerase A, actin, fatty acid binding protein, cluster of differentiation 26 and mucin (Fong et al., 2007). Furthermore parts of the proteose peptone fraction like proteose peptone 3 (PP3) originate from the MFGM (Campagna et al., 2001). Table 2. Protein components of the MFGM (cited from Singh, 2006). Proteins Molecular weight (g/mol) Mucin I (MUC 1) 160, ,000 Xanthine oxidase 150,000 Pass III 95, ,000 CD36 or PAS IV 76,000 78,000 Butyrophilin 67,000 Adipophilin (ADPH) 52,000 PAS 6/7 48,000 52,000 Fatty acid binding protein (FABP) 13,000 BRCA 1 210,000 The protein composition is highly dependent on the isolation method and the analytical procedure. Moreover, all proteins are not equally connected with the MFGM. Some are integral proteins, some are peripheral proteins and others are only loosely attached. Upon separation by SDS-PAGE the MFGM material is resolved into 7-8 major bands. However, several minor proteins are still unidentified (Dewettinck, at al., 2008). 8

20 Figure 3. SDS-PAGE of different dairy products. Names of MFGM proteins are given at the right, the other proteins are named at the left. Separation is performed on gradient (4 12%) polyacrylamide gels with the Xcell Surelock system. Visualization was done with SilverXpress silver stain. On each lane, 250 ng of protein was loaded: (1) raw milk; (2) skimmed milk; (3) acid buttermilk; (4) butter serum, aqueous fraction obtained from churning of cream; (5) acid buttermilk whey, soluble fraction obtained from acidification of sweet-cream buttermilk; (6) acid buttermilk quark, coagulated fraction obtained from the acidification of the sweet-cream buttermilk; (7) MFGM-isolate; and (8) mark 12 molecular weight standard (Rombaut, 2006). (Cited from Dewettinck, 2008) 2.3 Separation and isolation of the MFGM materials Buttermilk is considered to be a suitable source of the MFGM for commercial-scale production, mainly because of its low cost and relatively high content of MFGM components. Buttermilk is a side stream of the butter making process and has limited commercial value. Food processing industries have been emphasizing on the utilization of natural components to improve the nutritional value and create specific functionalities of food products (Innocente et al., 1997). Many attempts have been made to separate and isolate MFGM material from commercial buttermilk. The similarity in size of casein micelles and MFGM components is the first major consideration taken into account to achieve an effective fractionation of buttermilk (Sachdeva and Buchhiem, 1997). Micro- and ultrafiltration of buttermilk depends on the filtration conditions such pore size and type of membrane material, temperature, ph, and the type of buttermilk (Morin et al., 2004; Rombaut et al., 2007). Correding et al. (2003) reported that increasing the number of diafiltration from 2 to 6 reduces casein contamination in the retentate from 30% to 6% but increasing the diafiltration number has the disadvantage of loss of MFGM material (Rombaut et al 2006). Correding et al. (2003) 9

21 use citrate to dissociate the casein micelles of buttermilk; the MFGM material is then recovered by high speed centrifugation. In later studies they observed that microfiltration of citrate treated buttermilk through a membrane of 0.1 µm nominal pore size is more suitable for isolating MFGM materials than high speed centrifugation. Rombaut et al. (2006) reported that the addition of sodium citrate dissociates not only the casein micelles but also the MFGM fragments and high citrate concentrations may result in loss of polar lipids (64% recovery) during filtration due to blocking and fouling of the filter membrane with MFGM particles. Whey contains 0.4 to 0.5% of residual fat which is the main source of small fat globules, lipoprotein particles and milk fat globule membrane fragments (Rombaut et al., 2007). Isolation of the MFGM from whey (whey buttermilk, acid buttermilk whey) by filtration is favorable because whey is free of caseins. Absence of casein facilitates to obtain concentrated MFGM material by filtration, although the MFGM concentration of whey is limited compared with other dairy fractions such as buttermilk or butter serum. Morin et al. (2006) reported that whey buttermilk is favorable for MFGM isolation by filtration due to absence of casein and the expected transmission of MFGM proteins through the membrane was lower when using whey buttermilk compared to regular buttermilk. 2.4 Emulsifying properties of the MFGM MFGM materials are found in quite significant amounts in different dairy products especially in cream, butter, buttermilk, cheese and cheese whey. The unique functionality of MFGM materials have led to research and developing technologies for isolation, separation and use in different food emulsions (Singh, 2006). The MFGM enriched dairy products are often preferred for their emulsifying properties and capacity to improve flavour and texture especially for reduced fat food products (Ward et al., 2006). Lopez et al. (2007) have discussed the influence of the MFGM on the microstructure and flavour development of Emmental cheese. Milk fat globule membranes are composed of mainly polar lipids and negligible amounts of neutral lipids. Polar lipids are amphiphilic molecules which contain a hydrophobic tail and a hydrophilic head group. This unique feature largely contributes to the emulsifying properties of the membrane. MFGM materials are considered to be an efficient and natural emulsifying agent due its amphiphilic nature and original function in stabilizing fat globules in whole milk. Corredig and Dalgleish (1998a) prepared a model soy oil-in-water emulsion with MFGM materials from fresh raw cream and found that the newly formed oil droplets covered 10

22 by the MFGM material, behave differently from emulsions stabilized by other milk proteins i.e. no displacement occurs on the addition of small molecular weight surfactant (Tweens and Triton X-100) and the droplets are not affected by the presence of proteins such as an addition of β-lactoglobulin or caseins. This is due to fact that strong interaction may exist at the interface and phospholipids component of the MFGM may lower the interfacial tension (Corredig & Dalgleish, 1998c). MFGM materials isolated from industrial buttermilk are poor emulsifiers of soy oil-in-water emulsions as compared to those isolated from fresh cream (Corredig & Dalgleish, 1997). This is related to the intensity of membrane protein denaturation and association with β-lactoglobulin during heat treatment and the churning process following the manufacturing of buttermilk. Type of raw material, pretreatment and method of separation have a significant effect on the composition of MFGM isolates and consequently on their emulsification properties. In reconstituted milk fat emulsion (20-80 mg MFGM/g fat), it was observed that MFGM can stabilize 25 times its mass of milk fat. Droplets size decreases linearly with increasing membrane concentration which is comparable to those of homogenized milk. The surface protein coverage also increases at acid ph with the increase of MFGM concentration in the emulsion; by using > 80mg MFGM material/g fat it is possible to prepare stable milk fat globules that have similar stability as the natural milk fat globules (Kanno, 1989; Kanno, et al., 1991). However, Wong & Kitts (2003); Corredig & Dalgleish (1997) found that commercial buttermilk has inferior emulsifying and stabilizing capacity than non-fat dried milk. This may be due to the fact that heat treatment (80 C for min.) and churning have an influence on the behavior of the membrane and could have caused excessive denaturation of membrane proteins which influences the association of whey protein with MFGM (Houlihan et al., 1992). Sodini et al. (2006) studied the compositional and functional properties of sweet, sour and whey buttermilk and reported that sweet and cultured buttermilk exhibit lower emulsifying properties and higher viscosity and lower ph whereas the functional properties of whey buttermilk were independent of ph. The emulsifying properties of those three types of buttermilk are better than milk and whey but have lower foaming capacity. However, among the above listed buttermilk samples, whey buttermilk was found to have the highest emulsifying properties and the lowest foaming capacity. Possible reasons for this could be the higher ratio of phospholipids to protein in whey buttermilk compared with sweet or cultured buttermilk (Sodini et al., 2006). Innocente et al. (1997) studied the change in surface 11

23 properties of the soluble fraction of the MFGM (SFMFGM) at different temperatures (4-40 C) and observed that during preparation of emulsions temperature affects their functional properties. A temperature of even as low as 65 C results in loss of emulsifying properties of the membrane fraction. The stability of oil-in-water emulsion prepared with MFGM material depends on the heat treatment of the cream (Corredig & Dalgleish, 1998b). Roesch et at. (2004) studied the emulsifying properties of the MFGM fraction obtained by microfiltration of commercial buttermilk and buttermilk concentrate (BMC). An emulsion prepared with 10% soybean oil and with >0.25% MFGM isolate (60% w/w proteins) from buttermilk, showed good stability against creaming and small particle size distribution increased with MFGM concentration, whereas a similar emulsion prepared with BMC showed extensive flocculation. These findings are in disagreement with the previous work by Corredig and Dalgleish (1997). These authors observed that the MFGM isolate is a poor emulsifier compared to a whole isolate of buttermilk (containing whey proteins, caseins, and protein of MFGM). Emulsions with 10% soybean oil needed much higher percentage (>8%) of MFGM isolate to produce a droplet size distribution similar to that found for the emulsions prepared with 1-2% (w/v) whole isolate. From the different emulsification studies it can be deduced that the source, the intensity and frequency of the heat treatment, the type of fractionation procedure as well as the preparation condition results in major differences in the efficiency of MFGM material in emulsion preparation. The solubility of MFGM materials is influenced by the degree of denaturation of the MFGM proteins, the intensity of complex formation between MFGM proteins and the lipid fractions and the association of whey proteins with the MFGM caused by the heat treatment (Corredig and Dalgleish, 1998b). Hence, one of the recommendations could be that MFGM isolate should hydrate completely before its use in emulsification. 2.5 Dairy emulsion system Definition Milk is a well known natural oil-in-water emulsion. The fat droplets in milk are surrounded and stabilized by their own membrane, the bilayer membrane of the MFGM. Dairy cream, the fat rich fraction of whole milk, is also an oil-in-water emulsion in which fat globules are dispersed in a continuous aqueous phase. According to Dewettinck and Fredrick (2009), the inner layer of this surrounding membrane, existing of globular protein and phospholipids, lies on a layer of high melting glycerides and has no enzymatic activity. The outer layer has 12

24 enzymatic activity and determines agglutination, adsorption phenomena and the stability of the fat globule. The fat globules range in diameter from approximately 0.1 to 20 µm with a mean value of 3.5 µm Factors effect on stability of dairy emulsions The stability of milk fat emulsions is related to the physical and chemical characteristic of milk and dairy products. Thermodynamically, emulsions are not stable. Stability is a time dependent kinetic phenomenon and is largely dependent on the size distribution of the globules (Dewettinck and Fredrick 2009). Fox and McSweeney, (1998) reported that emulsion stability strongly depends on the integrity of the MFGM. Lipid emulsions are inherently unstable systems due to the difference in density and the interfacial tension between the lipid and the aqueous phases Effect of proteins Milk proteins are well known for their emulsifying and emulsion stabilizing properties. Proteins play a major role in the stabilization of the fat globules against partial coalescence through adsorption on the interface which results in electrostatic repulsion and increased thickness and viscoelasticity of the interfacial layer (Dalgleish, 2006; Goff, 1997). During emulsification, milk proteins become rapidly adsorbed on the O/W interface as individual molecules or in the form of aggregates (Walstra and Smulders, 1997). The newly formed layer results in steric stabilization and protects finely dispersed droplets against recoalescence. It provides long term stability towards creaming and flocculation during subsequent storage (Dalgleish 1997). The type of proteins affects the degree of stabilization, for example caseins from milk are highly surface active and adsorb readily the whey proteins. In practice casein stabilized emulsions have fine, small and stable droplets (Graham and Philips 1979). The protein-fat ratio in emulsions also influences the degree of stabilization. At a lower ratio the amount of proteins is not enough to cover the overall O/W interface and as a result not all fat globules are stabilized by proteins. During emulsification these uncovered fat globules may aggregate and form clusters (Fredrick et al., 2010). Van Camp et al., (1996) observed the effect of the fat-protein ratio on emulsions stabilized by whey protein concentrate and sodium caseinate and reported that an increase in the fat-protein ratio resulted in a decreased whipping time, overrun and increased firmness and stability of the whipped emulsion. 13

25 In milk based emulsions, whey proteins adsorb on the interface and also act as bridging material in the homogenization induced flocculation of fat droplets. Heat denaturation after homogenization retards desorption of whey proteins and inhibit protein loss on washing (Dickinson, 1986). Shimizu et al., (1981) reported that the amount of whey proteins adsorbed on the fat globules is dependent on ph during emulsification and the gross adsorption features are as follows: at lower ph greater adsorption of α-lactalbumin and at IEP greater adsorption of serum albumin Effect of the MFGM The milk fat globule membrane is a natural surface active material used in emulsion preparation. The MFGM is closely involved in creaming and agglutination of milk and those processes are highly affected by the treatment of dairy products such as cooling, heating and homogenization (McPherson and Kitchen, 1983). The stability and acceptability of dairy products are determined by the response of MFGM materials to these treatments (Houlihan et al., 1992). Mechanical treatments during processing of milk cause damage of the native MFGM and reduce the fat globule size and increase the interfacial area. The MFGM can no longer entirely cover the newly formed fat globule, and as a result some caseins micelles and whey proteins adsorb onto the surface. Homogenization induces such a high shear force that the fat globules are disrupted and their average diameter falls below 1 µm. The new droplets are almost entirely composed of milk proteins. Since the interfacial and electrostatic properties are changed, the damage of the fat globule membrane can significantly affect the stability and properties of dairy products (Walstra, 1994). Kanno et al., (1991) concluded that the adsorption of protein on the milk fat surface is affected by ph, the MFGM concentration and the milk fat content and that the MFGM could be a potential emulsifier for certain food and other emulsions Effect of homogenization Homogenization is the most common method to improve the stability of a dairy and food emulsion. It results in smaller particle sizes with more uniform size distributions, hence limiting the rate of phase separation (Biasutti et al., 2010). Droplet size distribution is perhaps the most important factor in determining the emulsion properties like consistency, rheology, stability, colour and taste (Stang et al., 2001). During homogenization proteins are absorbed from the continuous phase into the newly created fat globule membrane. The composition and 14

26 the structure of this absorbed layer influence the stability and the rheological properties of emulsion (Dickinson, 1998). Phipps (1975) and Mulder & Walstra (1974) reported that, for homogenization pressures between MPa, the average fat globule diameter (d) of emulsions decreases with emulsification pressure (P) in a relation d~p (-0.6). Robin et al. (1992) used a microfluidizer to prepare butteroil-in-water emulsions and observed that the average size of the particles decreased with an increase in pressure and reached a minimum value at around 60 MPa. Homogenization could be a critical factor in the network formation process. San Martin- Gonzalez et al. (2009) reported that an emulsion containing 30% oil and 2-3.5% casein showed a gel like structure when homogenized at pressures between 20 to 100 MPa. The main effect of homogenization is the reduction in droplet size and consequently in the increase of the O/W surface area. The increase in homogenization pressure reduces the droplet size of the emulsions and thus improves the shelf life of the products by reducing the creaming rate. High pressure homogenization resulted in the increase of the surface activity of emulsifying molecules; it may improve the efficiency of the product e.g. coating ability or penetration action (Flouryu et at., 2000) 2.6 Whipping properties Whipped cream The fat rich fraction of whole milk is known as cream. Dairy cream can be mixed with air and the volume of the resulting colloid becomes roughly double of the original cream. Thus, the whipped cream can be defined as a foamed dairy product, consisting of a dispersion of gas bubbles, surrounded by partially coalesced fat at the air-serum interface and supported by a high viscosity serum phase (Smith et al., 2000). According to Hotrun et al. (2005) the whipping process consists of three stages: 1) introduction of air bubbles and simultaneous protein adsorption into their surfaces, 2) accumulation and adsorption of fat globules onto the surfaces and 3) formation of a fat globule aggregate network. The whipping of cream introduces air bubbles in the structure and fat globules become partially coalesced and form a network which stabilizes the incorporated air Characterization of whipped cream Whipped creams are thermodynamically unstable. Destabilization of whipped cream takes place over time and is undesired. It is difficult to control these physical properties and they 15

27 affect the characteristics of whipped cream such as flavour, appearance and mouth feeling (Walstra et al., 2006). Serum loss, Ostwald ripening and bubble coalescence are the three principle mechanism of instability of whipped cream. All of these mechanisms occur simultaneously and result in complete phase separation: aqueous, fat and the air phase (Fredrick et al., 2010). Ostwald ripening is the disappearance of small air cells, resulting in the formation of bigger air cells due to the difference in Laplace pressure. It largely depends on the solubility of the gaseous phase into the serum phase. On the other hand bubble coalescence is the merging of two neighboring air bubbles. Consumers and dairy industries expect whipping cream to have certain desirable qualities such as taste, shelf-life and whipping characteristics. The dairy industries express whipping characteristics in terms of shorter whipping time, high overrun, good firmness and high stability of whipped product (Bruhn and Bruhn, 1988). Whipping time is the time needed to whip cream until it reaches maximum volume. Depending on the rate of partial coalescence, the whipping time is changed and faster partial coalescence rate results in a shorter whipping time (Hotrun et al., 2005). Serum loss is the amount of aqueous phase that is released from the whipped cream after standing for a period of time. It is one of the indications of whipped cream instability. Serum loss should be as minimum as possible. Overrun is defined as the volume of air incorporated into the cream structure relative to the volume of the cream. For traditional dairy whipped cream the desirable overrun is around % (Graf and Muller, 1965). Firmness is the expression of structural rigidity of the whipped cream. The texture, stability and whipping properties of whipped cream depend on several factors. Whipping of cream is in fact a destabilization mechanism whereby milk fat globules partially coalesce so that the interfacial layer is not being too strong. A control destabilization or partial coalescence of the emulsion is needed during further processing to develop an internal structure of agglomerated fat which alters the texture and physical appearance of the product. When the milk fat is homogenized in the presence of proteins e.g. caseins and whey proteins, the resulting emulsion become too stable. When small molecular weight surfactants replace protein on the interface, due their high affinity to the surface and their capability to reduce surface tension, weaker spot on interface and partial coalescence are promoted (Goff, 1997). MFGM materials, isolated from native milk fat globules, have good surface active properties. It was observed that the reduction of interfacial tension of the surface covered by the MFGM is similar to that covered by caseins (Chazelas et al., 1995). The MFGM can be used as an emulsifier in reconstituted milk fat emulsions; early work was done by Kanno et al. (1991) 16

28 whereby they emulsified anhydrous milk fat (AMF) with MFGM fragments and investigated the emulsifying properties (emulsion capability, foam and emulsion stability, and whippability) of the MFGM fractions. They concluded that the amount of protein adsorbed onto the surface of the milk fat globules is affected by the ph, the concentration of the MFGM and the fat content. When emulsions are prepared with MFGM materials (derived from untreated cream) the newly formed oil droplets covered by the MFGM material behave differently from the emulsions stabilized by other milk proteins: no displacement occurs on the addition of small molecular weight surfactant. Also the addition of milk proteins (βlactoglobulin or caseins) after emulsification does not seem to have remarkable affect on the composition of the interface (Corredig and Dalgleish 1998c). 17

29 CHAPTER III: MATERIALS AND METHODS 3.1 Materials and chemicals Materials Buttermilk powder was purchased from Friesland-Campina (Lummen, Belgium); Buttermilk whey was obtained from a local dairy company (Bϋllinger Butterei Bϋllingen, Belgium). Skim milk powder was purchased from Hochdorf Swiss Milk AG (Hochdorf, Switzerland), sodium caseinate from Acros-Organic (Geel, Belgium),, Lummen, Belgium), soy oil from a local supermarket and milk fat from Friesland Campina. Deionized water from Millipore ultrapure water purification system (Millipore SA, Malsheim, France) was used to reconstitute the buttermilk powder. Buttermilk powder was reconstituted in deionized water with 4% buttermilk solids. Trisodium citrate, 1%, with > 99% purity was also added for dissociating the casein micelles into casein species which are small enough to permeate through the membrane during the filtration process (Roesch et al 2004, Rombaut et al 2006). After complete dissolution of the powder, the ph was obtained at 7.6 and the solution was allowed to stand overnight at 4 C to ensure complete hydration. Buttermilk whey was also stored at 4 C and before microfiltration; the ph was adjusted to 7.5 by adding (1N) KOH (Rombaut et al 2007). Isolation of the MFGM materials The cross-flow microfiltration (MF) method with four steps continuous diafiltration was used to isolate the MFGM materials from reconstituted buttermilk and buttermilk whey. The microfiltration unit consist of a Millipore frame with Pellicon 2 cassette filter (PVGVPPC05), a hydrophilic PVDF membrane Durapore with a pore size of 0.22 µm and a membrane surface of 0.5 m 2 (Screen type C). The feed-pump was a compressed air-operated diaphragm pump (Chemicor series of Almatec, Kamp-lintfort, Germany). The feed flow rate was adjusted at 200 L/h and the permeate flow rate was adjusted with a Millipore peristaltic pump at 15 L/h. The microfiltration process was carried out at C. The trans-membrane pressure was bar. The dry matter (DM) content of the final MFGM material was about 9% and the material was stored at < -20 C for further analysis and emulsion preparation. 18

30 3.1.2 Chemicals Chemicals for analysis were obtained from Chem-Lab (Zedelgem, Belgium) and Sigma- Aldrich (Steinheim, Germany). For the analysis of phospholipids, high-performance-liquidchromatography (HPLC)-grade dichloromethane, supra-gradient methanol and HPLC grade water were obtained from Biosolve (Valkenswaard, Netherland) 3.2 Compositional analysis of the experimental materials The dry matter content of the samples was determined by measuring the loss of weight after drying the samples to a constant weight at 105 C (Williams, 1984). The total protein content of the samples was determined by the Kjeldhal method (Egan et al, 1981) using 6.38 as conversion factor and the total fat content by gravimetric determination using the Röse- Gottlieb method (IDF, 1987). In case of the MFGM samples (lower in mineral content) 3 ml 10% (w/v) CaCl 2 solution was added before carrying out the extraction process. CaCl 2 was added to increase the ionic strength of the solution (MFGM) which then improves the phase separation process during fat extraction (Le at al., 2010). Total ash content was determined by heating and igniting the samples in an electric muffle furnace at 550 C (Williams, 1984) and the lactose content by subtraction methods. All chemical analyses were measured in duplicate with two replications. Phospholipids were extracted using a mixed solution of chloroform: methanol (2:1), chloroform: methanol (20:1), HPLC chloroform: methanol (88:12) and 10% (w/v) CaCl 2 solution and the extracted material was stored in the freezer for further analysis. Samples were analyzed using the HPLC method developed by Le et al., (2011). The Shimadzu HPLC System (Tokyo, Japan) consisting of a controller (CBM-20A), an online degasser (DGU- 20A 5 ), a solvent delivery module (LC-20AT), an autosampler (SIL-20AT) and a column oven (CTO-20AC), was connected to an evaporative light-scattering (ELSD) detector (Alltech- 3300, Alltech Associates Inc., Lokeren, Belgium). The separation column was 150 x 3.0 mm Prevail silica 3 µm, connected behind a 7.5 x 3.0 mm pre-column, and made of the same material, with 5µm packing particle size. The column oven and sample chamber of the autosampler was set at 40 C and 20 C respectively. Two solvent lines were used: line-a contained dichloromethane and line-d contained methanol and acetic acid/triethylamine buffer solution (with a ratio of 500:21 (v/v) and at ph 4.5). The ELSD parameters were set at 65 C for the tube temperature, 2.1 L/min for the nebulizer gas (N 2 ) and 1 L/min for the acquisition gain. The injection volume was 10 µl and each extract was injected twice. The 19

31 mobile phase pumping was in linear gradient program with volume ratios of A and D being as follows: 96:4 at start to 88:12 at t=4 min. and to 6:94 at t=12 min. and back to 96:4 at t= 17 min. respectively. The pumping was then maintained at this condition until t=22.5 min. before a new injection started. Total mobile phase flow rate was 0.5 ml/min. The buffer solution was prepared by adding 7.16 ml acetic acid (From Acros-Organics, Belgium) and 8.0 ml triethylamine (From Sigma-Aldrich, Belgium, ) to ml HPLC grade water (From Biosolve, Netherland). Protein profiling by SDS-PAGE In order to study the protein profiles of the experimental samples, reducing SDS-PAGE (Sodium dodecyl sulfate polyacrylamide gel electrophoresis) was used. The separation is based on the mobility of protein in the acrylamide matrix according to their molecular weight. The separation system, all reagents and the precast gels were obtained from Invitrogen (Carlsbad, USA). The sample preparation and reduction was according to method developed by Le et al., (2009). Approximately 16 µg of total protein was loaded on each well of a precast gel (NuPAGE NOVEX 4-12% Bis-Tris gel, 1.0 mm x 17 wells) and the same amount of Mark 12 solution was also added as molecular weight (MW) standard. Electrophoresis was performed in 50 min. at 200 V and 125 ma/gel. After separation, the gel was washed 3 times (for 5 min.) with deionized water and stained for one hour with Simply Blue Safe Stain (Coomassie blue).. After de-staining the wet gel was scanned at 400 dpi using a high-resolution transmission scanner (UMAX Powerlook III, Taipei, Taiwan) and was analyzed by Imagemaster Totallab Software (GE Healthcare, Diegem, Belgium). Different types of protein were identified by comparing their MW with the standard and named according to Mather, (2000). 3.3 Experiment 1: Characteristics of emulsions stabilized with milk fat globule membrane fragments Emulsion preparation Emulsions were prepared for buttermilk powder (BMP), skim milk powder (SMP), sodium caseinate, milk fat globule membrane from buttermilk powder (MFGM-BMP) and milk fat globule membrane from buttermilk whey (MFGM-BMW). Suspensions in deionized water were made by dispersing each of the materials, using a magnetic stirrer. Samples were stored overnight at 4 C for hydration. The final protein content in emulsions was standardized at 20

32 2.3g/100g. Emulsions were then prepared by adding soy oil at a concentration of 35% and the ph was adjusted at 7.0. Samples were then warmed at 50 C and pre-homogenized at rpm for 2 minutes with a Ultra-Turrax (IKA - Werke, Germany). The emulsions were then passed through a two steps lab scale high pressure homogenizer (APV Systems, Albertslund, Denmark) at 50 C. For each sample five different homogenization pressures (0/20, 30/20, 90/20, 150/20 and 210/20 bar) were applied to make five different emulsions. The emulsions were stored in a refrigerator at 4 C for further analysis Determination of emulsion properties Microscopic observation Microphotographs of the different emulsions were obtained with an optical microscope (Diaplan, Van Hopplynus Instrument, Germany) equipped with a Nikon Coolpix 4500 digital camera. Samples were first diluted 10x and a sample drop was carefully placed on a glass slide, covered with cover slip and observed with a plan fluor objective (10x magnifications). Although the resolution was not sufficient to distinguish individual droplet, the method gave a good approximation of the qualitative difference in the flocculation structures of the emulsions Rheological characteristic The rheological characteristics of the samples were measured at 1 st and 8 th days of storage using a TA instruments AR2000 controlled-stress rheometer, equipped with a conical concentric cylinder geometry (28 mm diameter) and a cup (30 mm diameter). For the characterization of the flow curves, emulsion samples were mixed gently and passed through a syringe to ensure homogeneity of the sample. After this step 20 g of each emulsion sample was transferred into the cup of the rheometer for testing. Flow curves were measured at a shear rate between 0.1 to 100 s -1 (31 measuring points) and at a temperature of 20 C. The experimental data were fitted to the power law equation. Where K is the consistency index and n the flow behavior index. For Newtonian fluid, n= 1 and for non-newtonian fluid n is far from Particle size distribution measurement The particle size distribution of the emulsions was determined with a long bench Malvern Mastersizer S (Malvern Instruments, Malvern, UK) with a MS17 automated sample 21

33 dispersion unit. The polydisperse analysis of the samples dissolved in water was performed using a 300RF lens and the standard presentation code (1.5295, 0.01, for the relative particle refractive index, particle absorption and dispersant refractive index respectively). Measurement of the emulsions was carried out by dispersing the samples both in water and in 1% SDS (Sodium Dodecyl Sulfate) solution. SDS solution facilitates the separation of flocculated fat particles. The Sauter mean diameter (D 3.2 ) and volume-surface average particle diameter (D 4.3 ) were measured. The samples were measured twice and average values were taken Creaming stability The creaming of emulsion was measured by placing the samples in graduated tubes and stored quiescently at 4 C for 8 days. Emulsion sample of 10 ml was placed in graduated tubes. The volume of serum layer (V s ) formed at the bottom of the tubes was recorded and expressed as a percentage of the total volume of emulsion (V t ) in the tube Experiment 2: whipping properties of the MFGM fragments Experimental procedure Materials used are milk fat, buttermilk powder and milk fat globule membrane from buttermilk powder. The milk fat was added to the water phase of reconstituted buttermilk (6.71%) and the MFGM-BMP (3.31%) in order to obtain 2.3% protein in the final emulsion. The ph was adjusted to 7.0, heated at 60 C and mixed using the Ultra-Turrax at rpm for 2 minutes. The mixtures were then homogenized using a two steps high pressure homogenizer at 30/20 bars at 55 C and subsequently cooled to 5 C. The recombined creams were then stored in refrigerator for 1 day at 5 C. The recombined cream was whipped using a Hobart mixer with D wire whip agitator at medium speed (about 250 rpm). In order to avoid heating of the cream, the bowl and the agitator were stored in the refrigerator at 5 C. Cream temperature was recorded before and after whipping to make sure that the temperature was within 5 C. Approximately 1 liter cream was whipped in each whipping experiment. Whipping continued until visually acceptable whipped cream is obtained (i.e. when the cream does not flow around the wire) and whipping time was recorded. 22

34 3.4.2 Determination of whipping properties Overrun To measure the overrun of the whipped cream, first an empty plastic cup was weighed, filled fully with un-whipped cream and weighed again to know the mass of the un-whipped cream. After filling the cup was tapped gently on the table to remove air bobbles and the surface was flattened by scraping off with a knife. The same cup was used to measure the weight of the whipped cream following the same procedure. The measurements of overrun were performed 6 times and the average values were used for comparison. The overrun of whipped creams was calculated using the following equation: % 100 Where M 1 = Mass of the un-whipped cream with a certain volume M 2 = Mass of the whipped cream with the same volume Serum loss The serum loss measurement was performed at 20 C for 1 hour and at 5 C for 24 hours by putting whipped cream into a funnel that was placed on top of a flask. The empty flask was weighed first and then approximately 30g of whipped cream was put into the funnel. The liquid that leaked from the whipped cream and dripped onto the flask through the funnel was weighed and the percentage serum loss was calculated using the following equation: % 100 Where M 1 = Mass of the liquid that comes out from whipped cream after storage. M 2 = Initial mass of the whipped cream Firmness The compression force was performed using a TA 500 texture analyzer (Lloyd Instrument, Bognor Regis, West Sussex, UK) to determine the firmness of the whipped cream. A cylindrical probe with 500N load was used for measurement. The probe penetrates into the sample to a depth of 20 mm at a rate of 1 mm/s with 0.2 N trigger values. The firmness of the whipped cream samples was measured after 1 hour and 24 hours of storage at 5 C. The sample cups were wrapped with aluminium foil to prevent drying during storage in the refrigerator. 23

35 3.5 Statistical analysis. All statistical analyses were performed using S-Plus 8.0 package for Windows (Tibco Software Inc., Palo Alto, CA, USA). Results of rheological and physical characteristics of the emulsions were statistically processed using analysis of variance (one-way ANOVA) and multiple comparisons of means were adjudged by the Tukey test when a significant difference was observed. Two sample-t tests were performed to compare the whipping properties of recombined creams. All the tests were performed at 5% level of significance. 24

36 CHAPTER IV: RESULTS AND DISCUSSION 4.1 Composition of the experimental materials The composition of different dairy materials (BMP, SMP, MFGM-BMP, MFGM-BMW and SC) is shown in table 3. The result of proximate analysis indicated that the composition of the different experimental materials is not the same at 5% level of significance. Table 3. Composition of the formulation materials Materials Dry matter (%) Total protein Composition on dry matter (g/100g) Total lipid ash lactose Polar lipids MFGM-BMP 8.61 b ± c ± c ± a ± a ± c ± 0.31 MFGM- BMW 7.34 a ± a ± d ± c ± b ± d ± 0.19 BMP d ± b ± b ± b ± c ± b ± 0.13 SMP d ± b ± a ± b ± d ± a ± 0.01 SC c ± d ± 0.01 _ 2.25 a ± 0.03 Values are mean ± standard deviation of proximate composition of different samples expressed in percentage. Means having different superscripts are significantly different at overall 5% level of significance. Reported data are means for duplicate batches - Not analyzed for composition Result of multiple comparisons of the means showed that the mean total protein and ash content of MFGM-BMP, MFGM-BMW and SC are significantly (p<0.05) different from that of BMP and SMP. No significant (p>0.05) difference in mean total protein and ash content were found between BMP and SMP. Significant (p<0.05) differences in mean total lipids, lactose and polar lipids content were observed among different dairy materials. MFGM-BMW revealed the lowest total protein (22.88 g/100g) content but the highest total lipids (39.17 g/100g), ash (28.34 g/100g) and polar lipids (12.14 g/100g) content among dairy materials. The low protein content is due to the fact that large amount of proteins mainly casein were already coagulated and precipitated during acid coagulation of butter milk and high ash content might be due to the addition of CaCl 2 as process additives during the coagulation process of buttermilk cheese. On the contrary, the MFGM-BMP contained 1.6 times lower fat, 8.7 times lower ash, 2.8 times lower lactose and 1.3 times lower polar lipids but 3 times more protein than MFGM-BMW. Similar results on the composition of MFGM-BMP and MFGM-BMW also reported by Le et al., (2011). The relatively higher protein content in MFGM-BMP could imply that some fractions of casein may contaminate the final product 25

37 due the similarity in size of casein micelles and MFGM fragments (Sachdeva and Buchheim, 1997). When the composition of BMP was compared with that of MFGM-BMP, it was found that MFGM-BMP concentration of polar lipids increased by a factor of 2.67 (table 3). Total protein and lipids level also increased 2 and 2.8 times respectively in MFGM fraction. Whereas, the lactose and ash content was found to decrease by a factor of 17 and 2.1 respectively in MFGM-BMP. These results are also in agreement with the data reported by Le et al., (2011). The lower lactose and ash content in MFGM-BMP was expected because during microfiltration the majority of lactose and minerals were drained out through permeate. On the contrary, damaged fat globule membrane fractions rich in polar lipids were isolated as retentate which contributed in the higher total lipids and polar lipids content of MFGM-BMP. The polar lipids fraction comprises 40-50% of total lipids of MFGM materials (Singh, 2006). Skim milk powder showed the lowest total lipids (1.93 g/100g) and polar lipids (0.16 g/100g) and the highest lactose (56.53 g/100g) content as compared to other materials. The lowest total lipids content in SMP is due the fact of cream separation from raw milk. However, BMP presented 4.3 times higher total lipids and 20 times more polar lipids then SMP. The result on the composition of BMP and SMP were comparable with the result of Elling et al., 1996; Trachoo (2003) and Remeuf et al., (2003). Sodium caseinate has the highest total protein content (96.46) among the materials but does not contain lipids, lactose and polar lipids. Protein profiling by SDS-PAGE The SDS-PAGE profile of different dairy materials used in this experiment is shown in Figure 4. The SDS-PAGE profile is used for qualitative interpretation of the composition of different materials. The major MFGM proteins are MUC1, XO, PAS III, CD36, BTN, PAS 6/7 and ADPH (Singh, 2006; Berglund et al., 1996). From the figure 4 it is observed that, in addition to standard proteins the MFGM-BMP (lane-4) contained considerable amount of non-mfgm protein such as caseins and whey proteins e.g. β-lactoglobulin and α-lactalbumin. XO and BTN was found to 1.2 and 1.4 times higher ratio in MFGM-BMP than BMP. Corredig and Dalgleish, 1998b, Ye et al., 2004 reported that heat treatment during buttermilk powder production may induce the interaction between β-lactoglobulin, α-lactalbumin, κ-casein and MFGM proteins which could be the reason for the presence of casein and whey proteins in the MFGM fraction. Moreover, the casein micelles are roughly similar in size as compared to the 26

38 MFGM fraction, which also makes it possible that some of the caseins micelles might be collected with the retentate during the isolation of MFGM from buttermilk powder. Figure 4. SDS-PAGE PAGE of different dairy materials. (1) Skimmed milk powder; (2) Buttermilk powder; (3) Sodium m caseinate; (4) MFGM isolated from buttermilk powder; (5) MFGM isolated from buttermilk whey and (6) mark 12 molecular weight standard. The load on each lane contained 16µg 16 total protein. Separation was performed on gradient (4-12%) (4 polyacrylamide crylamide gels with Xcell Surelock system. XO= xanthine ine oxidase; CD36=cluster of differentiation 36; BSA= bovine serum albumin; BTN=butyrophilin; PAS 6/7= periodic acid Schiff 6/7; ADPH= adipophilin. The he major MFGM proteins such as XO, Lactoferrin, CD36 and BTN were not observed in MFGM-BMW (lane-5). 5). But BSA, BSA β-lactoglobulin and α-lactalbumin lactalbumin were clearly distinguished. MFGM-BMW BMW contained a 1.3 and 1.6 times higher ratio of BSA and αlactalbumin than MFGM-BMP BMP but the amount of β-lactoglobulin was found to be b almost similar. High molecular weight caseins (> 32 kg/mol) are also not found in MFGM-BMW. MFGM Similar observations were also reported by Rombaut et al., (2007). Because, during the buttermilk cheese making process most of the caseins are separated out by coagulation, buttermilk whey is virtually free of caseins. caseins SMP (lane-1) and BMP (lane-2) showed an almost similar protein profile with the exception of PAS 6/7 and ADPH which was not found in SMP. But SMP showed a 2 times higher ratio of casein. The resultss were in agreement with the data published by Le et al., (2011). (2011). Sodium caseinate (SC) is a commercially purified form of casein. As it was expected, expected only the casein fractions were clearly distinguished in SC (lane-3). 27

39 4.2 Creaming stability Emulsions prepared with different dairy materials (BMP, SMP, SC, MFGM-BMP and MFGM-BMW) were studied for their stability. Figure 5 illustrate the creaming behavior of different emulsions. Separation (%) BMP Separaion (%) SMP 0/20 bar 30/20 bar 90/20 bar 150/20 bar 210/20 bar Storage time (day) Storage (d) SC MFGM-BMP 0/20 30/20 90/20 150/20 210/20 Separation (%) Separation (%) Storage time (day) Storage time (day) Figure 5. Creaming stability of emulsions prepared with different dairy materials upon storage at 4 C Emulsions prepared with BMP and SMP showed creaming behavior when lower homogenization pressure is applied but showed better stability only at higher homogenization pressure (>90/20 bar). SMP and BMP contain equal amounts of protein (table 3). The SDS- PAGE analysis also confirms the unique pattern of casein and whey protein distribution (see section 4.1). The casein and whey protein content of those materials play an important role in stabilizing the emulsion. High pressure homogenization results in further unfolding of the globular proteins and the exposure of hydrophobic sites. This influences the attachment of proteins at the interface, reduces interfacial tension and stabilizes the emulsion (Roesch and Corredig, 2003; San Martin-Gonzalez et al., 2009) 28

40 On the other hand emulsions prepared with MFGM-BMW showed good stability (no separation) irrespective of homogenization pressure. This could be due the highest polar lipid content of MFGM-BMW (Elling and Duncan, 1996). Emulsions prepared with MFGM-BMP also showed good stability at homogenization pressure higher than 30/20 bar. The better creaming stability of these emulsions can be attributed to the presence of more native milk fat membrane constituents, such as phospholipids materials (Scott et al., 2003). In general the higher homogenization pressure was found to be more efficient in yielding stable emulsions (Elling and Duncan, 1996). From a colloidal point of view, it is well known that the reduction of surface tension is an important factor in the formation and stabilization of emulsions. Although high homogenization pressure increases the surface area, hydrophobic proteins and a higher content of polar lipids of the MFGM materials facilitate emulsion stability (Kanno et al., 1991). Emulsions prepared with sodium caseinate showed greater instability irrespective of the homogenization pressure. Polar lipids are absent in sodium caseinate. The lower stability against creaming may be caused by flocculation of droplets or adsorption of proteins in an aggregated form because aggregated proteins are less effective emulsifiers (Roesch et al 2004). Dalgleish, (1997) reported that during homogenization casein micelles are disrupted and adsorb at the interface either as a whole or in fragments form. The presence of large amounts of unabsorbed proteins causes phase separation due to depletion flocculation (Dickinson and Golding, 1997). 4.3 Particle size distribution The particle size distribution of the emulsions prepared with different dairy materials (BMP, SMP, MFGM-BMP, MFGM-BMW and SC) and homogenized at different pressures (0/20, 30/20, 90/20, 150/20 and 210/20 bar) are shown in figure 6a and 6b. These results show that the average particle size of emulsions prepared with different materials decreases significantly (p<0.05) with the increase in homogenization pressure (table 4). The results are in agreement with the data published by Mulder & Walstra, (1974), Phipps, (1975) and Robin, et al., (1992). Emulsions prepared with SC at higher homogenization pressure (>150/20 bar ) showed a bimodal distribution with one population of about 0.5µm droplet diameter and the second larger group with an average droplet size of about µm. The presence of a bimodal distribution is undesirable because it cases aggregation and flocculation of particles and subsequently makes the emulsion unstable. 29

41 Volume (%) Volume (%) Volume (%) BMP 0/20 bar 30/20 bar 90/20 bar 150/20 bar 210/20 bar A 0,01 0, Diameter (µm) SMP 0/20 bar 30/20 bar 90/20 bar 150/20 bar 210/20 bar 0,01 0, Diameter (µm) MFGM-BMW 0/20 bar 30/20 bar 90/20 bar 150/20 bar 210/20 bar 0,01 0, Diameter (µm) Volume (%) Volume (%) Volume (%) B 16 BMP ,01 0, Diameter (µm) 16 SMP ,01 0, Diameter (µm) 16 MFGM-BMW ,01 0, Diameter (µm) Figure 6a. Particle size distribution of different emulsions homogenized at different pressures, after dilution in water and SDS. Lane-A after dilution in water and Lane-B after dilution in SDS. 30

42 Volume (%) Volume (%) MFGM-BMP 0/20 bar 30/20 bar 90/20 bar 150/20 bar 210/20 bar A 0,01 0, Diameter (µm) SC 0/20 bar 30/20 bar 90/20 bar 150/20 bar 210/20 bar 0,01 0, Diameter (µm) Volume (%) Volume (%) B MFGM-BMP ,01 0, Diameter (µm) 16 SC ,01 0, Diameter (µm) Figure 6b. Particle size distribution of different emulsions homogenized at different pressures, after dilution in water and SDS. Lane-A after dilution in water and Lane-B after dilution in SDS. The bimodal distribution of particles can be due to clustering by bridging flocculation of fat droplets. In case of bridging flocculation the surface of newly formed particles share a common protein at the interface. The result of particle size distribution was also confirmed by what we found in microscopic observation (figure 8). On the other hand a monomodal distribution was found for emulsions prepared with BMP, SMP and MFGM-BMW. But, the average particle diameters were found to be higher than that of MFGM-BMP and sodium caseinate (table 4). All emulsions after incubation with SDS showed a lower particle diameter as compared to those after dilution in water (table 4). Dilution with SDS caused disruption of particle aggregates and displacement of protein by a surfactant molecule. When measuring the particle size distribution in the presence of SDS, it was possible to determine the size distribution of 31

43 Table 4. Average Sauter mean diameter D 3.2 of emulsions at different homogenization pressure Pressure (bar) D 3.2 (µm) after dilution in water at different pressure BMP SMP MFGM-BMW MFGM-BMP SC 0/ a ± c ± a ± e ± d ± / a ± a ± b ± d ± c ± / a ± a ± c ± c ± b ± / a ± bc ± c ± b ± a ± / a ± bc ± a ± a ± a ±0.04 D 3.2 (µm) after dilution in SDS at different pressure 0/ e ± e ± d ± e ± e ± / d ± d ± c ± d ± d ± / c ± c ± b ± c ± c ± / b ± b ± a ± b ± b ± / a ± a ± a ± a ± a ±0.03 Data are expressed as sample means ± Standard deviation Means having different superscripts (by column) are significantly different at overall 5% level of significance. the individual fat droplet and not of the aggregated or flocculated ones. The droplet size distribution of emulsions prepared with SMP, BMP and MFGM-BMW diluted with water were found to be quite different from those diluted with 1% SDS (figure 6a, 6b and table 4), indicating the presence of flocculated oil droplets in the emulsions. In all cases the droplet size was smaller when incubated with SDS, indicating the fact that the oil surface was partially covered and therefore bridging between droplets in close proximity occurred. Volume (%) A BMP MFGM-BMP MFGM-BMW S C SMP 0,01 0, ,01 0, Diameter (µm) Diameter (µm) Figure 7. Particle size distribution of emulsions prepared with different material at homogenization pressure 90/20 bar. A) After dilution in water. B) After dilution in SDS. Volume (%) B 32

44 The effect of different materials on droplet size distribution is shown in figure 7. After dilution in water, the emulsions prepared with MFGM-BMP and SC have a smaller particle size compared to those of BMP, SMP and MFGM-BMW. Table 5. Average surface-weighted mean diameter D 3.2 of different emulsions at 90/20 bar Materials D 3.2 (µm) after dilution in water D 3.2 (µm) after dilution in SDS BMP 6.36 a ± a ±0.09 SMP 4.63 b ± a ±0.09 MFGM-BMW 6.28 a ± a ±0.18 MFGM-BMP 1.89 c ± b ±0.07 SC 2.00 c ± b ±0.15 Data are expressed as sample means ± Standard deviation Means having different superscripts (by column) are significantly different at overall 5% level of significance. The observation was confirmed by the results of the average Sauter mean diameter (D 3.2 ) of different materials (table 5). The emulsions prepared with MFGM-BMP and SC were characterized by a significantly lower (p<0.05) Sauter mean diameter as compared to BMP, SMP and MFGM-BMW. The creaming stability of SC was found to be quite lower than that of MFGM-BMP (see section 4.2). It can also be noted that, even at higher homogenization pressure (> 90/20 bar), the emulsions with BMP, SMP and MFGM-BMW showed larger value of the particle size distribution, in opposition with what was found in case of MFGM-BMP and SC (table 4). In the case of BMP and SMP a lack of surface active material could be a reason for the larger particle sizes. Toams et al. (1994) also reported that an inadequate amount of proteins could cause some aggregation of the fat droplets. On the other hand the chemical composition analysis showed that MFGM-BMW contains a higher level of polar lipids (table 3). Instead of a higher polar lipids content of MFGM-BMW, the larger diameter in water indicates that some other factors are interacting with the absorption process at the surface. This could be due to the higher ash content (minerals e.g., Ca) of MFGM-BMW which might have influence on the aggregation process. Because Ca +2 is a bivalent cation it can bind two MFGM particles so at a higher ph interaction between Ca +2 and MFGM fragments is favored which results in the formation of MFGM aggregates (Rombaut and Dewettinck, 2007). Among the materials, MFGM-BMP showed the smallest droplet size both in water and SDS solution. The higher phospholipids content of MFGM materials might be the cause of better emulsifying properties (Sodini, et al., 2006). 33

45 4.4 Microscopic observation Materials 0/20 bar 90/20 bar 210/20 bar BMP SMP MFGMBMW MFGMBMP SC Figure 8. Microscopy images of emuslsions prepared with BMP, MFGM-BMP, MFGM-BMW, SC and SMP at different homogenization pressures (0/20, 90/20, and 210/20 bar). 34