Microstructure of Dairy Products

Mamdouh Mahmoud Abdel-Rahman El-Bakry is a Postdoctoral Researcher in the Department of Chemical Engineering at the Universitat Autònoma of Barcelona, Spain. Antoni Sanchez is a Professor in the Department of Chemical Engineering at the Universitat Autònoma of Barcelona, Spain. Bhavbhuti M. Mehta is an Associate Professor in the Dairy Chemistry Department, SMC College of Dairy Science, Anand Agricultural University, Gujarat, India.

List of Contributors xiii

Preface xv

1 Microscopy Techniques for Dairy Products – An Introduction 1
Mark A.E. Auty

1.1 Introduction 1

1.1.1 Brief History and Background 1

1.2 Conventional Optical Microscopy Techniques 4

1.2.1 Conventional Light Microscopy – Optical Contrast 4

1.2.1.1 Bright Field 4

1.2.1.2 Polarized Light 4

1.2.1.3 Phase Contrast 4

1.2.1.4 Differential Interference Contrast 5

1.2.1.5 Fluorescence 5

1.2.2 Chemical Contrast Techniques in Light Microscopy 5

1.3 Confocal Scanning Laser Microscopy 6

1.3.1 Confocal Principle 6

1.3.2 Identifying Dairy Primary Components in CSLM: Labeling Strategies 8

1.3.2.1 Generic Labeling 8

1.3.2.2 Specific Labeling 10

1.3.2.3 Covalent Labeling 11

1.3.3 Some Applications of Confocal Microscopy to Dairy Products and Ingredients 12

1.3.3.1 Spreads 12

1.3.3.2 Emulsions and Foams 12

1.3.3.3 Fermented Milks 12

1.3.3.4 Cheese 13

1.3.3.5 Dairy Powders 13

1.3.3.6 Milk Protein Gel Systems 14

1.3.3.7 Dynamic CSLM Techniques 14

1.4 Electron Microscopy (EM) Techniques 16

1.4.1 Transmission Electron Microscopy 16

1.4.2 Scanning Electron Microscopy 18

1.4.3 Other EM Techniques 18

1.4.3.1 X‐ray Microanalysis 18

1.4.3.2 Cryo‐electron Microscopy 19

1.4.3.3 Environmental and Variable Pressure SEM 20

1.5 Emerging Microscopy Techniques 20

1.5.1 Atomic Force Microscopy 20

1.5.2 Advanced Fluorescence Microscopy Techniques 22

1.5.3 Confocal Raman Microscopy 22

1.5.4 X‐ray Nano/Microtomography 22

1.5.5 Super‐Resolution Microscopy 23

1.6 Image Analysis 23

1.7 Conclusions 24

References 24

2 Light Microscopy and CSLM Techniques, Principles and Applications 33
Johan Hazekamp

2.1 Introduction 33

2.1.1 The History of Microscopy 33

2.1.2 Evolution of Confocal Microscopy 34

2.1.3 Food Microscopy 35

2.1.4 Wide Field Microscopy 36

2.1.5 Confocal Scanning Laser Microscopy (CSLM) 38

2.2 Sample Preparation and Specific Staining and Labeling 41

2.2.1 Specific Labeling 44

2.2.2 Dynamic Imaging 46

2.2.3 Future Perspectives 46

References 47

3 Electron Microscopy Techniques 51
Semih Otles and Vasfiye Hazal Ozyurt

3.1 Introduction 51

3.2 Types of EM 51

3.2.1 Scanning Electron Microscopy (SEM) 51

3.2.2 Transmission Electron Microscopy (TEM) 52

3.2.3 Cryo‐SEM 52

3.2.4 Cryo‐TEM 53

3.2.5 Environmental Scanning Electron Microscopy (ESEM) 53

3.3 Sample Preparation for EMs 53

3.3.1 Scanning Electron Microscopy 53

3.3.2 Transmission Electron Microscopy 53

3.3.3 Cryo‐Scanning Electron Microscopy 53

3.4 Dairy Microstructure 54

3.5 Electron Microscopy for the Dairy Product 54

3.6 Summary 60

References 64

4 Emerging Techniques for Microstructural Analysis 67
I. Hernando, E. Llorca, and A. Quiles

4.1 Introduction 67

4.2 Scanning Probe Microscopy 67

4.2.1 Scanning Tunneling Microscope (STM) 69

4.2.2 Atomic Force Microscope (AFM) 70

4.2.3 Applications of the Main Probe Microscopes 71

4.3 X‐Ray Tomography 72

4.4 Small‐Angle‐Scattering (SAS) Methods: SAXS and SANS 74

4.4.1 Small‐Angle X‐Ray Scattering (SAXS) 74

4.4.2 Small‐Angle Neutron Scattering (SANS) 75

4.4.3 Applications of Small‐Angle‐Scattering Methods 75

4.5 Vibrational Spectroscopies (Fourier Transform Infrared‐FTIR and Raman Microscopy) 75

4.5.1 Fourier Transform Infrared (FTIR) Spectroscopy 76

4.5.2 Raman Spectroscopy 78

4.6 Magnetic Resonance: NMR and MRI 80

4.7 Conclusions 82

References 82

5 Quantitative Image Analysis in Microscopy 89
Gaetano Impoco

5.1 Aim and Scope 89

5.2 Image Analysis Software 90

5.3 Applications to Microscopy for Dairy Science 97

5.3.1 Porosity 98

5.3.2 Fat Globules 99

5.3.3 Microbial Cells 100

5.4 Image Analysis and Quantitative Measurement 100

5.4.1 Image Analysis Basics 101

5.4.1.1 Feature Detection 102

5.4.1.2 Quantitative Analysis 103

5.4.2 Common Pitfalls 105

5.4.3 Misuse and Wrong Interpretation of Image Analysis Results 114

5.4.4 Good Practices 116

5.4.5 Image Analysis in your Lab 119

5.5 Conclusions 122

Acknowledgments 123

References 123

6 Microstructure of Milk 127
Michael H. Tunick

6.1 Components of Milk 127

6.2 Fat 127

6.2.1 Fat Globules 127

6.2.2 Milkfat Globule Membrane 128

6.2.3 Cream 129

6.3 Protein 133

6.3.1 Types of Protein 133

6.3.2 Casein Micelles in Bovine Milk 133

6.3.3 Casein Micelles in Caprine Milk 133

6.3.4 Casein Micelles in Milk of Other Species 136

6.3.5 Micelle Structure 136

6.4 Bacteria and Somatic Cells 137

6.5 Concentrated Milk 138

6.6 Digested Milk 140

6.7 Conclusion 142

Acknowledgments 142

References 142

7 Microstructure of Cheese Products 145
Bhavbhuti M. Mehta

7.1 Introduction 145

7.2 Factors Affecting the Development of Microstructures in Cheeses 146

7.2.1 Addition of Calcium Chloride 148

7.2.2 Rennet Coagulation 149

7.2.3 Acid‐Coagulation 150

7.2.4 Coagulation Temperature 150

7.2.5 Syneresis 151

7.2.6 Salting 151

7.2.7 Ripening 152

7.2.8 Homogenization and High Pressure Treatments 153

7.2.9 Evaporation and Ultrafiltration Treatments 155

7.2.10 Freezing 156

7.2.11 Fat Replacers 156

7.3 Microstructures of Various Components in Cheese Matrix 158

7.3.1 Protein in Cheese Matrix 158

7.3.2 Fat Globule in Cheese Matrix 159

7.3.3 Calcium in Cheese Matrix 162

7.4 Crystals in Cheese Matrix 162

7.5 Starter Bacteria in Cheese Matrix 163

7.6 Microstructure of Selected Varieties of Cheeses 164

7.6.1 Processed Cheese 164

7.6.1.1 Curd Granules and Fat 166

7.6.1.2 Occurrence of Crystals 166

7.6.2 Cheese Analogs 166

7.6.3 Feta Cheese 167

7.6.4 Domiati Cheese 167

7.6.5 Fresh Cheese 167

7.6.6 Cream Cheese 168

7.6.7 Mold‐Ripened Cheeses 169

7.6.8 Cheese Powder 169

7.7 Cheese Matrix and Digestion 170

7.8 Conclusions 171

References 171

8 Microstructural Aspects of Yogurt and Fermented Milk 181
P.H.P. Prasanna, C.S. Ranadheera, and J.K. Vidanarachchi

8.1 Yogurt and Fermented Milk: An Overview 181

8.2 Yogurt and Fermented Milk: Production Technologies 184

8.3 Microstructure of Yogurt and Fermented Milk 187

8.4 Factors Influencing Microstructure of Yogurt and Fermented Milk 188

8.4.1 Effects of Type of Milk on Structure 188

8.4.2 Rate of Inoculation Level and Starter Culture Composition on Microstructure of Yogurt and Fermented Milk 189

8.4.2.1 Rate of Inoculation 189

8.4.2.2 Culture Composition 189

8.4.3 Effect of Exopolysaccharide Producing Starter Culture on Microstructure 190

8.4.4 Incubation Temperature on Structure 191

8.4.5 Effect of Different Processing Steps 191

8.4.5.1 Homogenization of Milk 191

8.4.5.2 Heat Treatment of Milk 192

8.4.5.3 Effect of Stirring 192

8.4.6 Effect of Addition of Different Hydrocolloids and Fibers on Microstructure 193

8.5 Microscopy Methods Used for Analyzing Microstructure of Fermented Milk 194

8.5.1 Light Microscopy 194

8.5.1.1 Bright Field Light Microscopy 194

8.5.1.2 Polarized Light Microscopy 194

8.5.1.3 Fluorescence Microscopy 195

8.5.1.4 Confocal Laser Scanning Microscopy 197

8.5.2 Electron Microscopy 198

8.5.2.1 Scanning Electron Microscopy 198

8.5.2.2 Transmission Electron Microscopy 200

8.6 Conclusions 201

References 202

9 Microstructure of Milk Fat and its Products 209
Pere Randy R. Ramel and Alejandro G. Marangoni

9.1 Introduction 209

9.2 Milk Fat Crystal Structure 211

9.2.1 Mesoscale Structure of Milk Fat 211

9.2.1.1 Polymorphism 211

9.2.1.2 Phase Behavior and Fractionation 213

9.2.1.3 Solid Fat Content and Crystallization/Melting Behavior 214

9.2.2 Nanoscale Structure of Fat Crystal Networks 216

9.3 Effect of Different Factors on the Crystallization Behavior and Microstructure of Milk Fat 218

9.3.1 Processing Conditions 218

9.3.1.1 Different Crystallization Mechanisms 218

9.3.1.2 Crystallization Temperature and Cooling Rate 218

9.3.1.3 Agitation, Shear and Ultrasound 219

9.3.2 Composition 220

9.3.2.1 Minor Components 220

9.3.2.2 Blending with Different Fats and Oils, and Waxes 220

9.3.3 In a Dispersed State (Emulsion) 221

9.3.3.1 Emulsified State (Cream) vs Bulk State or Anhydrous Milk Fat (AMF) 222

9.3.3.2 Emulsion Droplet Size 222

9.3.3.3 Addition of Emulsifiers 223

9.3.4 In Food Matrices 223

9.3.4.1 Water‐in‐Oil Emulsion 223

9.3.4.2 Foamed Emulsions 224

9.3.4.3 Chocolate 225

9.3.4.4 Cheese 226

9.4 Impact of Resulting Microstructure on the Properties of Different Milk Fat Products 226

9.4.1 Rheology 226

9.4.2 Thermal Stability 229

9.4.3 Sensory Qualities 229

9.5 Conclusions 229

References 230

10 Microstructure of Ice Cream and Frozen Dairy Desserts 237
Samantha R. VanWees and Richard W. Hartel

10.1 Overview of Frozen Desserts 237

10.1.1 Ingredients 238

10.1.2 Processing 239

10.2 Frozen Dessert Structure 240

10.2.1 Serum Phase 240

10.2.2 Ice Crystals 242

10.2.3 Fat Phase 245

10.2.4 Air Cells 247

10.2.5 Proteins and Hydrocolloids 250

10.3 Storage 251

10.3.1 Recrystallization 251

10.3.2 Sugar Crystallization 253

10.3.3 Air Coarsening 254

10.3.4 Shrinkage 255

10.4 Conclusion 256

References 256

11 Whey Wastes and Powders 261
J. Chandrapala

11.1 Whey 261

11.2 Current Whey Uses 263

11.3 Processing of Liquid Whey 263

11.3.1 Recovery of Casein Fines and the Separation of Fat 264

11.3.2 Concentration of Total Solids 265

11.3.3 Drying 266

11.3.4 Fractionation of Total Solids 270

11.4 Whey Powders 274

11.4.1 Whey Protein Concentrates 275

11.4.2 Whey Protein Isolates 277

11.4.3 Whey Protein Hydroxylates 279

11.4.4 Other Whey Powders 280

11.4.4.1 Defatted Whey Protein Concentrates 280

11.4.4.2 Demineralized Whey Protein Concentrates 280

11.4.4.3 Delactosed Whey Powders 283

11.4.4.4 Acid Whey Powders 283

11.4.4.5 Salty Whey Powders 284

11.5 Utilization and Applications of Whey Powders 285

11.6 Conclusion 287

References 287

12 Microstructure of Selected Traditional Indian Dairy Products 293
Bhavbhuti M. Mehta

12.1 Introduction 293

12.2 Heat Desiccated Dairy Products 294

12.2.1 Khoa and Khoa‐Based Sweets 294

12.2.1.1 Microstructure of Khoa 294

12.2.1.2 Microstructure of Gulabjamun 295

12.2.1.3 Microstructure of Burfi and Kalakand 298

12.3 Heat‐Desiccated Milk Cereal Based Desserts 299

12.3.1 Microstructure of Kheer 299

12.4 Heat‐Acid Coagulated Dairy Products 300

12.4.1 Microstructure of Paneer 300

12.4.1.1 Fried Paneer 300

12.4.2 Microstructure of Chhana and Chhana Based Sweets 302

12.4.2.1 Microstructure of Rasogolla 302

12.4.2.2 Microstructure of Chhana Podo 305

12.5 Fermented Dairy Products 306

12.5.1 Microstructure of Dahi 306

12.5.2 Microstructure of Shrikhand 306

12.6 Conclusion 307

References 307

13 Using Microscopy for Microorganism Localization within Dairy Products 311
I.T. Smykov

13.1 Introduction 311

PART 1 312

13.1.1 Microorganisms and Starters 312

13.1.2 Techniques Used in the Microstructure Analyses 313

13.1.3 Interactions Occurring in the Microstructure 315

PART 2 318

13.2 Materials and Methods 318

13.2.1 Bacterial Strains and Dairy Products 318

13.2.2 Electron Microscopy 318

13.2.2.1 Surface Topography Heavy Metal Shadowing 319

13.2.2.2 Negative Staining Transmission Electron Microscopy 319

13.2.3 Freeze‐Fracture Replication 319

13.3 Results and Discussion 320

13.3.1 Casein Micelle 320

13.3.2 Bacteria 324

13.3.3 Bacteria in a Protein Matrix 327

13.3.4 Bacteria in Cheese Eyes 331

13.3.5 Bacteria in Yoghurt 333

13.3.6 Bacteriophages 336

13.4 Conclusions 338

Acknowledgment 339

References 339

14 Microstructure of Dairy Products: Challenges and Future Trends 345
Maricê Nogueira de Oliveira

14.1 Introducing Microstructure of Dairy Products 345

14.2 Microstructure of Fermented Milks 346

14.3 Microstructure of Yogurt and Milk Drinks 347

14.3.1 Yogurt 347

14.3.2 Milk Drinks or Lactic Beverages 354

14.4 Microstructure of Cheeses 356

14.5 Conclusion 359

References 359

Index 363