Microbiology in Dairy Processing Challenges and Opportunities Institute of Food Technologists Series

An authoritative guide to microbiological solutions to common challenges encountered in the industrial processing of milk and the production of milk products

Microbiology in Dairy Processing offers a comprehensive introduction to the most current knowledge and research in dairy technologies and lactic acid bacteria (LAB) and dairy associated species in the fermentation of dairy products. The text deals with the industrial processing of milk, the problems solved in the industry, and those still affecting the processes. The authors explore culture methods and species selective growth media, to grow, separate, and characterize LAB and dairy associated species, molecular methods for species identification and strains characterization, Next Generation Sequencing for genome characterization, comparative genomics, phenotyping, and current applications in dairy and non-dairy productions.

In addition, Microbiology in Dairy Processing covers the Lactic Acid Bacteria and dairy associated species (the beneficial microorganisms used in food fermentation processes): culture methods, phenotyping, and proven applications in dairy and non-dairy productions. The text also reviews the potential future exploitation of the culture of novel strains with useful traits such as probiotics, fermentation of sugars, metabolites produced, bacteriocins. This important resource:

• Offers solutions both established and novel to the numerous challenges commonly encountered in the industrial processing of milk and the production of milk products
• Takes a highly practical approach, tackling the problems faced in the workplace by dairy technologists
• Covers the whole chain of dairy processing from milk collection and storage though processing and the production of various cheese types

Written for laboratory technicians and researchers, students learning the protocols for LAB isolation and characterisation, Microbiology in Dairy Processing is the authoritative reference for professionals and students. 

List of contributors xv

Foreword xix

Preface xxi

Acknowledgements xxiii

1 Milk fat components and milk quality 1
Iolanda Altomonte, Federica Salari and Mina Martini

1.1 Introduction 1

1.1.1 Milk fat globules 2

1.1.2 Milk fat and fatty acid composition 4

1.2 Conclusions 7

References 7

2 Spore]forming bacteria in dairy products 11
Sonia Garde Lopez]Brea, Natalia Gomez]Torres and Marta Avila Arribas

2.1 Introduction 11

2.2 The bacterial spore 13

2.2.1 Structure and chemical composition of bacterial spores 14

2.2.1.1 Exosporium 14

2.2.1.2 Spore coat 14

2.2.1.3 Outer spore membrane 15

2.2.1.4 Cortex and germ cell wall 15

2.2.1.5 Inner spore membrane 15

2.2.1.6 The core spore 15

2.2.2 Spore resistance 16

2.2.3 Life cycle of spore]forming bacteria 17

2.3 Spore]forming bacteria important for the dairy industry 18

2.3.1 Class Bacilli 18

2.3.1.1 Bacillus genus 19

2.3.1.1.1 Bacillus cereus 19

2.3.1.1.2 Other Bacillus species 20

2.3.1.1.3 Importance of Bacillus spp. in the dairy industry 21

2.3.1.2 Geobacillus and Anoxybacillus genera 24

2.3.1.3 Paenibacillus genus 25

2.3.2 Class Clostridia 25

2.3.2.1 Clostridium botulinum 26

2.3.2.2 Clostridium perfringens 28

2.3.2.3 Clostridium tyrobutyricum and related species 28

2.4 Control strategies to prevent poisoning and spoilage of milk and dairy products by spore]forming bacteria 30

2.5 Conclusions 31

References 32

3 Psychrotrophic bacteria 37
Milena Brasca, Marilu Decimo, Stefano Morandi, Solimar Goncalves Machado, FrancoisBagliniere and Maria Cristina Dantas Vanetti

3.1 Introduction 37

3.2 Sources of psychrotrophic bacteria contamination of milk 38

3.3 Important spoilage psychrotrophic bacteria in milk 42

3.4 Molecular tools to characterize psychrotrophic bacteria 43

3.5 Influence of psychrotrophic contamination of raw milk on dairy product quality 45

3.5.1 Bacterial proteases and proteolytic changes in milk 46

3.5.2 Bacterial lipases and phospholipases and their significance in milk 49

3.6 Regulation of extracellular enzymes 52

3.7 Control of psychrotrophic bacteria and related enzymes 53

3.8 Conclusions 54

References 54

4 Stabilization of milk quality by heat treatments 63
Palmiro Poltronieri and Franca Rossi

4.1 Introduction 63

4.2 Thermal treatments of milk 63

4.2.1 Thermization 63

4.2.2 Pasteurization 64

4.2.3 Grade A pasteurized milk 66

4.3 Milk sterilization 67

4.3.1 Control of proper time/temperature setting for safety of milk and milk products 67

4.4 Diseases associated with unpasteurized milk, or post]pasteurization dairy]processing contamination 68

4.5 Conclusions 68

References 68

5 Genomics of LAB and dairy]associated species 71
Palmiro Poltronieri, Franca Rossi, Cesare Camma, Francesco Pomilio and Cinzia Randazzo

5.1 Introduction 71

5.2 Genomics of lab and dairy]associated species 71

5.2.1 Next]generation sequencing of strains, dairy starter genomics and metagenomics 72

5.2.2 Pacific Bioscience single]molecule real]time sequencing technology 73

5.2.3 Illumina MySeq and HiSeq 2000 73

5.2.4 Ion Torrent platform 73

5.3 NGS platform applied to sequencing of microbial communities 74

5.3.1 Pangenomics 74

5.3.2 Omic technologies: transcriptomics, proteomics, functional genomics, systems biology 75

5.4 Metabolomics and proteomics 76

5.4.1 Subcellular localisation (SLC): secretion systems for secreted proteins 77

5.4.2 Interactome for cell adhesion and pathogen exclusion 78

5.4.3 Lab peptidome 79

5.5 Comparative genomics of dairy]associated bacteria: the lactobacillus genus complex, streptococci/lactococci, enterococci, propionibacteria and bifidobacteria 79

5.5.1 Comparative genomics of Lb. rhamnosus and Lb. casei 83

5.5.2 Lb. casei core genome and ecotype differences in dairy adapted strains 84

5.6 Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in adaptive immunity 84

5.7 Regulation in carbon metabolism 85

5.7.1 Transcriptional and posttranscriptional regulation in carbon metabolism 85

5.7.2 Two]component systems and phosphorylation in sugar substrate regulation 86

5.7.3 Regulatory RNAs and alternative sigma factors in gene expression 87

5.8 Conclusions 88

References 88

6 Metabolism and biochemistry of LAB and dairy]associated species 97
Palmiro Poltronieri, Giovanna Battelli and Nicoletta Pasqualina Mangia

6.1 Introduction 97

6.2 Carbohydrate substrates, glycolysis and energy production 98

6.2.1 Pentose phosphate pathway 99

6.2.2 Citrate fermentation 99

6.3 Proteolysis, protein substrates and amino acid availability influencing gene expression 100

6.3.1 Cell]envelope proteinases: the Prt system 101

6.3.2 Oligopeptide permeases and other transporters for peptides and amino acids 101

6.3.3 Peptidolysis and free amino acids 102

6.3.4 Peptidolysis and catabolite repression 105

6.3.5 Amino acid biosynthesis and auxotrophy 105

6.4 Lipolysis, lipases, esterases 106

6.5 Aroma and flavour products of metabolism 107

6.5.1 Aldehydes, alcohols and carboxylic acids 110

6.5.2 Amino acids as precursor flavour compounds 112

6.6 Nonenzymatic production of flavours 113

6.7 Methods of analysis of flavours in dairy products: HPLC, gas chromatography/ mass analysis (GC/MS) 114

6.8 Natural biodiversity of strains in dairy productions 115

6.9 Conclusions 116

References 117

7 Growth needs and culture media for LAB and dairy]associated species 123
Giuseppe Blaiotta, Maria Aponte and Palmiro Poltronieri

7.1 Introduction 123

7.2 Established culture media for lactobacilli 123

7.2.1 Rogosa agar 124

7.2.2 MRS medium 125

7.2.3 Skim milk and whey agar 125

7.3 M17 medium for selection and enumeration of lactococci and streptococci 126

7.3.1 St. thermophilus agar 126

7.4 Selective media for lactobacilli 127

7.4.1 MRS vancomycin 127

7.4.2 Additional selective agents 128

7.4.3 MRSV plus selective agents for Lb. casei group enumeration 129

7.4.4 MRS]salicin, MRS]sorbitol, MRS]ribose, MRS gluconate agar 129

7.4.5 MRS]clindamycin]ciprofloxacin agar 129

7.4.6 MMV medium for Lb. casei group enumeration 130

7.4.7 MRS containing fructose (MRSF) 130

7.4.8 mMRS]BPB 131

7.4.9 MRS]NNLP agar and chromogenic agars for complex communities 131

7.4.10 Homofermentative]heterofermentative differential medium 131

7.5 Media for the isolation of bifidobacteria 132

7.5.1 MRS]NNLP agar 133

7.5.2 BSM, WSP, TOS]MUP 133

7.5.3 MRS]ABC 134

7.6 Phenotyping 134

7.7 Conclusions 135

References 135

8 LAB species and strain identification 139
Cinzia Randazzo, Alessandra Pino, Koenraad Van Hoorde and Cinzia Caggia

8.1 Introduction 139

8.2 Genotypic fingerprinting methods 140

8.3 Culture]dependent approaches 142

8.3.1 Random amplification of polymorphic DNA 142

8.3.2 ARDRA and RFLP 143

8.3.3 Ribotyping 143

8.3.4 Repetitive element sequence]based PCR 144

8.3.5 Amplified fragment length polymorphism 145

8.3.6 Pulsed field gel electrophoresis 145

8.4 Non]genotypic fingerprinting methods 146

8.5 Culture]independent approaches 147

8.5.1 Culture]independent methods for qualitative analysis of dairy foods microbiota 147

8.5.2 Culture]independent methods for quantitative analysis of dairy foods microbiota 150

8.6 Novel high]throughput techniques: sequencing and metagenomics 151

8.7 Conclusions 152

References 152

9 LAB strains with bacteriocin synthesis genes and their applications 161
Lorena Sacchini, Giacomo Migliorati, Elisabetta Di Giannatale, Francesco Pomilio andFranca Rossi

9.1 Introduction 161

9.2 Bacteriocins from lab 161

9.3 Potential for use of lab bacteriocins as food preservatives 164

9.4 Bacteriocins produced by dairy lab 165

9.5 Identification of lab]producing bacteriocins 168

9.6 A novel approach for screening lab bacteriocins 170

9.7 Biotechnological interventions for bacteriocin engineering 171

9.8 Conclusions 172

References 172

10 Starter strains and adjunct non]starter lactic acid bacteria (NSLAB) in dairy products 177
Paola Dolci and Luca Cocolin

10.1 Introduction 177

10.2 Controlled fermentation 177

10.2.1 Natural versus selected lactic acid bacteria starters 178

10.2.2 Starter strains: selection parameter approaches and strain concept 179

10.2.3 Starter culture formulation 180

10.3 Adjunct non]starter lactic acid bacteria 181

10.3.1 Biodiversity and adaptation to cheese environment 181

10.3.2 Prospective in industrial application 182

10.3.3 Biopreservation and health benefits 183

10.4 Conclusions 185

References 185

11 Milk Fat: stability, separation and technological transformation 191
Gianluigi Scolari

11.1 Introduction 191

11.1.1 Composition and physical state of milk fat 192

11.1.2 Melting point of milk fat 194

11.2 Physical instability of milk fat 194

11.3 Milk fat separation 195

11.3.1 Flocculation or natural creaming 195

11.3.2 Milk fat separation by centrifugation 197

11.4 Partial coalescence 199

11.4.1 General aspects 199

11.4.2 Barrier against coalescence 201

11.4.2.1 Low molecular mass surfactants 201

11.4.2.2 Large sized surfactants (casein micelle) 202

11.4.2.3 Polymeric surfactants (proteins and polysaccharides) 203

11.4.2.4 Mixed films 203

11.5 Foam in milk and cream 204

11.5.1 General aspects 204

11.5.1.1 Foam formation without surfactants 204

11.5.1.2 Foam formation with surfactants 205

11.5.1.3 Drainage of dispersion liquid in foam 206

11.5.2 Foam from cream containing more than 30% milk fat 207

11.6 Whipped cream and butter 209

11.6.1 Technological factors affecting whipped cream and butter production 209

11.7 Churning process 210

11.7.1 Type of cream 210

11.7.2 Physical (crystallization) and biological maturation of cream before churning 212

11.7.3 Churning technology 215

11.7.4 Continuous churning 216

11.7.5 Moulding and packaging 217

11.8 Conclusions 217

References 218

12 Biological traits of lactic acid bacteria: industrial relevance and new perspectivesin dairy applications 219
Diego Mora, Fabio Dal Bello and Stefania Arioli

12.1 Introduction 219

12.2 Selecting fermenting bacteria for their ability to have a respiratory metabolism 220

12.3 Selecting galactose]positive yogurt cultures: working “against the natural evolution of the species” 221

12.4 Accelerating the milk acidification process by selecting proteinase]positive strains 222

12.5 Accelerating the milk acidification process by selecting urease]negative S. thermophilus strains 224

12.6 Protective cultures for dairy applications: “work but please do not grow and do not modify the sensory profile of the product” 225

12.7 Selection of starter culture free of transferable antibiotic]resistance mechanisms 227

12.8 Conclusions 228

References 229

13 Lactic acid bacteria bacteriophages in dairy products: problems

and solutions 233
Giorgio Giraffa, Miriam Zago and Domenico Carminati

13.1 Introduction 233

13.2 Phage classification 234

13.3 Phage]host interactions 236

13.4 Sources of contamination 238

13.4.1 Milk and cheese whey 238

13.4.2 Dairy cultures 239

13.4.2.1 The lysogenic state 239

13.5 Phage detection and quantification 240

13.6 Methods to control phage contamination 242

13.6.1 Phage inactivation by physical treatments 242

13.6.2 Phage inactivation by chemical treatments 244

13.6.3 Phage control by biological approaches 245

13.7 Conclusions 246

14 Lactic acid bacteria: a cell factory for delivering functional biomolecules in dairyproducts 251
Tiziana Silvetti, Stefano Morandi and Milena Brasca

14.1 Introduction 251

14.2 Vitamins 253

14.2.1 Vitamin B2 or Riboflavin 254

14.2.2 Vitamin B9 or Folate 255

14.2.3 Vitamin B12 or cobalamin 256

14.2.4 Vitamin K: menaquinone 257

14.2.5 Other B]group vitamins 258

14.3 Minerals 258

14.4 Bioactive compounds 261

14.4.1 Anti]hypertensive peptides 262

14.4.2 Antioxidative peptides 263

14.4.3 Bioactive amines 265

14.4.4 Immune system affecting peptides 267

14.4.5 Opioid peptides 267

14.4.6 Metal]binding peptides 268

14.4.7 Conjugated linoleic acid and conjugated linolenic acid 268

14.5 Low]calorie sweeteners 269

14.6 Exopolysaccharides (EPS) 271

14.7 Conclusions 273

References 273

15 Dairy technologies in yogurt production 279
Panagiotis Sfakianakis and Constantina Tzia

15.1 Introduction 279

15.2 Yogurt types 280

15.3 Yogurt manufacturing process 281

15.3.1 Initial treatment of milk 281

15.3.2 Standardization of milk components – fat and SNF content 283

15.3.3 Homogenization 284

15.3.4 Heat treatment 286

15.3.5 Fermentation process 288

15.3.5.1 Monitoring of fermentation process – prediction of fermentation evolution 290

15.3.6 Post]fermentation processing 292

15.3.6.1 Cooling – addition of additives 292

15.3.6.2 Addition of fruit 292

15.3.6.3 Packaging 294

15.3.7 Quality control of yogurt production 294

15.4 Conclusions 295

References 295

16 Milk protein composition and sequence differences in milk and fermented dairy products affecting digestion and tolerance to dairy products 299
Maria Gabriella Giuffrida, Marzia Giribaldi, Laura Cavallarin and Palmiro Poltronieri

16.1 Introduction 299

16.2 Caseins 301

16.2.1 Gene polymorphisms in κ]casein genes 302

16.2.2 Gene polymorphisms in β]casein gene 303

16.3 Proteolytic release of bioactive peptides in fermented milk and cheese 304

16.4 Minor milk proteins 305

16.4.1 Lactoferrin 305

16.4.2 β]Lactoglobulin (β]LG) 306

16.4.3 α]Lactalbumin (α]LA) 306

16.5 Proteins with bioactive roles 307

16.6 MFGM-associated proteins 308

16.7 Cow’s milk protein allergy (CMPA) 308

16.8 Conclusions 309

References 309

Index 315

Palmiro Poltronieri, PhD, is a Researcher at the Institute of the Sciences of Food Productions (CNR-ISPA), National Research Council of Italy. He obtained his Ph.D. in Cellular and Molecular Biology and Pathology in 1995 at the Institute of Chemical Biology, Medical Faculty of Verona University. Working in the Microbiology laboratory since 1999, he has established collaboration with the principal laboratories working in the field of food microbiology.