ITEM METADATA RECORD
Title: Thick juice degradation: study of the microbial population dynamics and control of the causative flora during storage
Other Titles: Diksapdegradatie: studie van de microbiële populatiedynamiek en beheersing van de causale flora tijdens opslag
Authors: Justé, Annelies
Issue Date: 14-Jul-2008
Table of Contents: Table of contents


Abstract i

Samenvatting iii

Publications v

List of abbreviations vii

1. CURRENT KNOWLEDGE ABOUT SUGAR THICK JUICE PRODUCTION 1
1.1 SUGAR PRODUCTION FROM SUGAR BEETS 2
1.2 THICK JUICE DEGRADATION DURING STORAGE 4
1.3 MICROBIAL COMMUNITY ANALYSIS TECHNIQUES IN FOOD AND FOOD- ASSOCIATED MATRICES 9
1.3.1 Choice of target genes 10
1.3.1.1. Ubiquitously conserved genes 10
1.3.1.2. Functional genes 11
1.3.2 Microbial community analysis techniques 12
1.3.2.1. Microbial diversity 12
1.3.2.2. Identity 17
1.3.2.3. Quantification 21
1.3.3 General pitfalls and limitations 26
1.3.3.1. Sampling 26
1.3.3.2. DNA extraction and PCR amplification 26
1.3.3.3. Viable versus non-viable 28
1.3.3.4. Active versus non-active 29
1.3.3.5. Sequence databases: availability and quality 29
1.3.4 Automation 29
1.3.5 Concluding Remarks 30
1.4 AIM AND OUTLINE OF THE THESIS 32

2. EFFECT OF HOP EXTRACT, SOLIDS CONTENT AND STORAGE TEMPERATURE ON MICROBIAL DEGRADATION OF THICK JUICE 33
2.1 INTRODUCTION 33
2.2 MATERIALS AND METHODS 35
2.2.1 Thick juice storage experiments and sampling 35
2.2.2 Microbiological analysis 38
2.2.3 Biochemical and chemical analyses 39
2.2.4 Sequencing of bacterial 16S rRNA genes 39
2.2.5 Susceptibility of fastidious bacteria in thick juice to hop ß-acids 39
2.2.6 Data analysis 40
2.3 RESULTS 40
2.3.1 Effect of total soluble solids (°Bx) on thick juice degradation 40
2.3.2 Stability of hop ß-acids during thick juice storage 41
2.3.3 Effect of hop ß-acids and temperature on thick juice degradation 42
2.3.4 Effect of hop ß-acids on thick juice microflora 43
2.3.5 Preliminary identification of the Fastidious Bacteria (FB) 44
2.3.6 Sensitivity of fastidious bacteria to hop ß-acids 46
2.4 DISCUSSION 47

3. DOMINANCE OF TETRAGENOCOCCUS HALOPHILUS DURING THICK JUICE DEGRADATION 51
3.1 INTRODUCTION 51
3.2 MATERIALS AND METHODS 52
3.2.1 Thick juice storage experiment and sampling 52
3.2.2 Viable counts 53
3.2.3 Acid analysis 53
3.2.4 DNA extraction 54
3.2.5 Analysis of 16S rDNA T-RFLP 54
3.2.6 Application of clone libraries 54
3.2.7 Sequencing of bacterial 16S rRNA gene fragments 55
3.2.8 Specific PCR assay development and verification 55
3.3 RESULTS AND DISCUSSION 56
3.3.1 Culturable microflora: viable counts and identification 56
3.3.2 Acids Analysis 61
3.3.3 T-RFLP analyses and application of clone libraries 63
3.3.4 Tetragenococcus halophilus can cause thick juice degradation 68
3.3.5 Specific PCR for Tetragenococcus halophilus 70

4. GENETIC AND PHYSIOLOGICAL DIVERSITY OF TETRAGENOCOCCUS HALOPHILUS STRAINS ISOLATED FROM SUGAR- AND SALT-RICH ENVIRONMENTS 75
4.1 INTRODUCTION 75
4.2 MATERIALS AND METHODS 76
4.2.1 Bacterial strains 76
4.2.2 DNA extraction 76
4.2.3 RAPD fingerprinting 78
4.2.4 16S rRNA gene sequence analysis 78
4.2.5 REP-PCR fingerprinting 79
4.2.6 DNA-DNA hybridization 79
4.2.7 Carbon source metabolic fingerprint 79
4.2.8 Salt and sucrose tolerance 80
4.3 RESULTS 80
4.3.1 RAPD fingerprinting 80
4.3.2 16S rRNA gene sequencing and phylogenetic analysis 82
4.3.3 REP-PCR fingerprinting 85
4.3.4 DNA-DNA hybridization 86
4.3.5 Biochemical characterization 86
4.3.6 Physiological characterization: salt and sucrose tolerance 88
4.4 DISCUSSION 91

5. DEVELOPMENT OF A DNA ARRAY FOR DETECTION AND IDENTIFICATION OF THICK JUICE CONTAMINANTS DURING THICK JUICE STORAGE 95
5.1 INTRODUCTION 95
5.2 MATERIALS AND METHODS 96
5.2.1 Bacterial isolates and DNA extraction 96
5.2.2 Selection of oligonucleotides 96
5.2.3 DNA array production 98
5.2.4 PCR amplification, labeling and hybridization 98
5.2.4.1. Validation of the DNA array 100
5.2.4.2. Monitoring thick juice contaminants during storage and degradation 101
5.2.4.3. Stability of Recovery of hybridized sequences 102
5.2.4.4. Stability of DNA in thick juice 102
5.3 RESULTS AND DISCUSSION 103
5.3.1 Design of the DNA array 103
5.3.2 Evaluation of the specificity and sensitivity of the DNA array 106
5.3.3 Validation of the DNA array 110
5.3.4 Monitoring thick juice contaminants 111
5.3.5 Stability of DNA in thick juice 116

6. INFLUENCE OF INDUSTRIALLY RELEVANT PROCESS AND STORAGE PARAMETERS ON MICROBIAL THICK JUICE DEGRADATION BY TETRAGENOCOCCUS HALOPHILUS 119
6.1 INTRODUCTION 119
6.2 MATERIAL AND METHODS 120
6.2.1 Thick juice storage experiments and sampling 120
6.2.2 Microbiological analyses 121
6.2.3 Biochemical and chemical analyses 121
6.2.4 Data analysis 121
6.3 RESULTS 122
6.3.1 Evolution of Tetragenococcus halophilus counts 122
6.3.2 pH evolution 122
6.3.3 Evolution of other degradation parameters 124
6.3.4 Case study: pilot scale storage experiment 127
6.4 DISCUSSION 130

7. GENERAL CONCLUSIONS 131

References 136
URI: 
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Centre for Food and Microbial Technology
Microbial and Molecular Systems - miscellaneous

Files in This Item:
File Description Status SizeFormat
PhD.pdf Published 1578KbAdobe PDFView/Open

 


This item is licensed under a Creative Commons License
Creative Commons

All items in Lirias are protected by copyright, with all rights reserved.