Thick juice degradation: study of the microbial population dynamics and control of the causative flora during storage

Sugar thick juice degradation: study of the microbial commuity dynamics and control of the causative microflora during storage Storing sugar extracts as thick juice, a form of sucrose syrup, is widely practiced in the sugar industry because it allows to spread sugar production over an entire season. However, thick juice storage as currently practiced commonly faces problems due to juice degradation. The precise cause for this problem was ill defined but believed to be of microbial origin. The major challenge at the start of the research described inthis thesis was to describe the microbial population dynamics during thick juice storage, in order to identify the causative degradation flora and to define improved good storage practices with the ultimate goal of preventing thick juice degradation. In the last two decades, major changes have occurred in how microbial ecologists study microbial communities. Limitations associated with traditional culture-based methods have driven the development of culture-independent techniques, which are primary based on the analysis of nucleic acids. In this research, thick juice microflora has been thoroughly studied with molecular tools, encompassing the application of 16S rRNA gene clone libraries and Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis, providing a more comprehensive representation of the thick juice microflora than the previous studies. The initial, heterogeneous microflora in freshly produced thick juice evolved to the dominance (>99%) of Tetragenococcus halophilus during storage. Basedon its high population density (10*6 10*7 cfu/ml), the ability to consume sucrose and the similar acidification pattern of artificially infested sterile thick juice, T. halophilus is proposed to be the key player in thick juice degradation. Remarkably, T. halophilus has thus far been associated only with high salt food products and ourwork is the first to associate it with high sugar matrices. Therefore, different T. halophilus strains either from thick juice or from high salt environments were compared in depth. Using a range of genetic typing methods and physiological tests including repetitive extragenic palindrome analysis (REP-PCR), random amplification of polymorphic DNA(RAPD), and determination of carbon utilization patterns (Biolog), clear differences were found between T. halophilus strains isolated from salt and sugar rich environments. Irrespective these differences,DNA-DNA hybridization grouped all strains within the species T.halophilus , except two isolates from sugar thick juice that appear to represent two new species of Tetragenococcus .In addition to T. halophilus , other bacteria such as Staphylococcus and Bacillus species were consistently present, though in lower steady concentrations of 10*3 cfu/ml. In order to simultaneously detect the different bacteria that may occur in thick juice, a DNA array was developed containing detector oligonucleotides for the genera Bacillus , Kocuria , Staphylococccus and Tetragenococcus , and the species Aerococcus viridans , Leuconostoc mesenteroides and T. halophilus . The developed macroarray was shown reliable and sensitive and has potential for monitoring the thick juice microflora during storage as an early warning system. Finally, best available storage practiceswere defined based on laboratory and pilot scale storage experiments using the independent variables solids content, pH, storage temperature and biocide concentration.In conclusion, this work has contributed to a better description of themicrobial population dynamics during thick juice storage and degradation, and to the definition of improved storage practices that will be useful for the sugar industry.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 136status: publishe

Tetragenococcus

In 1990 the genus Tetragenococcus was created after reclassification of the halophilic lactic acid bacterium (LAB) Pediococcus halophilus as T. halophilus. Tetragenococci are typical LAB in that they are Gram-positive, catalase negative, and oxidase negative. Physiologically, tetragenococci are distinguished from other LAB mainly by their high salt tolerance and ability to grow at high pH values. Presently, the genus comprises a limited number of species, including T. halophilus, T. koreensis, T. muriaticus, T. osmosphilus and T. solitarius. Based on both physiological and genetic characteristics as well as on the origin of the strains, the species T. halophilus was further subdivided into two subspecies, including T. halophilus subsp. halophilus and T. halophilus subsp. flandriensis for strains isolated from salt-rich and sugar-rich environments, respectively. In this chapter, both phenotypical and genotypical characteristics of the genus are outlined, with a detailed description of each species comprising the genus. In addition, emphasis is put on the industrial relevance of the genus.status: publishe

Shifts in microbial community structure during malting with emphasis on xylanase producing bacteria

status: publishe

Xylanase producing bacteria during malting

status: publishe

Fundamental research enables process optimization in the sugar Industry

Storing sugar extracts as thick juice, a form of sucrose syrup, is common practice in the sugar industry. However, this thick juice storage commonly faces problems due to juice degradation. The precise cause for this problem was ill defined but believed to be of microbial origin. In this research, the microbial population dynamics during thick juice storage was described and we identified the causative degradation flora. Finally, optimal good storage practices were defined with the ultimate goal of preventing thick juice degradation. The thick juice microflora has been thoroughly studied with both culture-based and culture-independent techniques, encompassing the application of 16S rRNA gene clone libraries and T-RFLP analysis, providing a more comprehensive representation of the thick juice microflora than the previous studies. The initial, heterogeneous microflora in freshly produced thick juice evolved to the dominance (>99%) of Tetragenococcus halophilus during storage. Based on its high population density (106–107 cfu/ml), the ability to consume sucrose and the similar acidification pattern of experimentally infested thick juice, T. halophilus is proposed to be the key player in thick juice degradation. Remarkably, T. halophilus has thus far been associated only with high salt food products and our work is the first to associate it with high sugar matrices. In addition to T. halophilus, other bacteria such as Staphylococcus and Bacillus species were consistently present, though in lower steady concentrations of 103 cfu/ml. In order to be able to detect the different bacteria that may occur in thick juice, a DNA array was developed containing detector oligonucleotides for the genera Bacillus, Kocuria, Staphylococccus and Tetragenococcus, and the species Aerococcus viridans, Leuconostoc mesenteroides and T. halophilus. The developed macroarray was shown reliable and sensitive (up to 102 cfu/ml) and has potential for monitoring the thick juice microflora during storage as an early warning system. Finally, best available storage practices were defined based on laboratory and pilot scale storage experiments using the independent variables solids content, pH, storage temperature and biocide concentration. In conclusion, this work has contributed to a better description of the microbial population dynamics during thick juice storage and degradation, and to the definition of improved storage practices that will be useful for the sugar industry.status: publishe

Fundamental research enables process optimization in the sugar industry

Storing sugar extracts as thick juice, a form of sucrose syrup, is common practice in the sugar industry. However, this thick juice storage commonly faces problems due to juice degradation. The precise cause for this problem was ill defined but believed to be of microbial origin. In this research, the microbial population dynamics during thick juice storage was described and we identified the causative degradation flora. Finally, optimal good storage practices were defined with the ultimate goal of preventing thick juice degradation. The thick juice microflora has been thoroughly studied with both culture-based and culture-independent techniques, encompassing the application of 16S rRNA gene clone libraries and T-RFLP analysis, providing a more comprehensive representation of the thick juice microflora than the previous studies. The initial, heterogeneous microflora in freshly produced thick juice evolved to the dominance (>99%) of Tetragenococcus halophilus during storage. Based on its high population density (106–107 cfu/ml), the ability to consume sucrose and the similar acidification pattern of experimentally infested thick juice, T. halophilus is proposed to be the key player in thick juice degradation. Remarkably, T. halophilus has thus far been associated only with high salt food products and our work is the first to associate it with high sugar matrices. In addition to T. halophilus, other bacteria such as Staphylococcus and Bacillus species were consistently present in lower steady concentrations of 103 cfu/ml. In order to be able to detect the different bacteria that may occur in thick juice, a DNA array was developed containing detector oligonucleotides for the genera Bacillus, Kocuria, Staphylococccus, and Tetragenococcus, and the species Aerococcus viridans, Leuconostoc mesenteroides and T. halophilus. The developed macroarray was shown reliable and sensitive (up to 102 cfu) and has potential for monitoring the thick juice microflora during storage as an early warning system. Finally, best available storage practices were defined based on laboratory and pilot scale storage experiments using the independent variables solids content, pH, storage temperature and biocide concentration. In conclusion, this work has contributed to a better description of the microbial population dynamics during thick juice storage and degradation, and to the definition of improved storage practices that will be useful for the sugar industry.status: publishe

Protective effect of hop ß-acids on microbial degradation of thick juice during storage

Aims: This study assessed the value of a commercial alkaline solution of hop beta-acids (HBA) for prevention of microbial degradation of thick juice, a concentrated intermediate product in the production of beet sugar.status: publishe

Development of a DNA array for the simultaneous detection and identification of sugar thick juice bacterial contaminants

Despite the use of generally accepted good storage practices, sugar thick juice degradation caused by microbiological contamination occasionally occurs, causing considerable financial loss. In this study, a DNA array was developed for simultaneous detection and identification of the most prominent microflora present during thick juice storage, which may cause degradation of the thick juice. Specific oligonucleotides were developed for several bacterial taxa, including the genera Bacillus, Kocuria, Staphylococcus and Tetragenococcus and the species Aerococcus viridans, Leuconostoc mesenteroides and Tetragenococcus halophilus. The DNA array was validated using both pure cultures and industrial samples. In addition, comparisons were made between the developed array, PCR assays specifically targeting the thick juice contaminants and classical microbial platings. The array was found to be reliable and sensitive enough to detect and identify the target bacteria. In addition, the array was used to monitor the target microbial populations in thick juice during longterm storage and degradation. Results are discussed in relation to DNA stability in thick juice.status: publishe

Predominance of Tetragenococcus halophilus as the cause of sugar thick juice degradation

The industrial storage of sugar thick juice was simulated on a laboratory scale. Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis and the application of Clone Libraries in parallel with classical microbiology were used to study the bacterial diversity and all revealed a dominance (>99%) of Tetragenococcus halophilus during storage. The degradation of thick juice correlated with the appearance of L-lactic acid and high concentrations of T. halophilus. In addition, pure cultures of T. halophilus induced degradation of sterile thick juice. A specific PCR was developed to detect T. halophilus and industrial thick juice samples from Belgium, Germany and France all contained T. halophilus, suggesting a consistent association of this organism with thick juice. T. halophilus has been known only as a halophile thus far, and this report is the first to show an association of this organism with a sugar-rich environment.status: publishe

Protective effect of hop β-acids on microbial degradation of thick juice during storage

Storing sugar extracts as thick juice, a form of sucrose syrup, is common practice in the sugar industry. However, thick juice storage commonly faces problems due to microbial degradation. In this study the value of a commercial alkaline solution of hop β-acids (HBA) was assessed for prevention of microbial degradation of thick juice. The antimicrobial effect of different concentrations of HBA against juice degradation was tested in a pilot-scale thick juice storage experiment. Thick juice degradation, indicated as a decrease in pH, was generally accompanied by an increase in the count of fastidious bacteria (FB) on Columbia Agar with Sheep Blood (CAwSB), which were mainly identified as Tetragenococcus halophilus. Addition of HBA delayed juice acidification and the development of T. halophilus in a concentration-dependent manner. The susceptibility of T. halophilus to HBA was determined by plating degraded thick juice (> 105 CFU/ml) on CAwSB plates with different concentrations of HBA (0–160 ppm). None of the HBA concentrations tested reduced the number of FB colonies formed, but increasing HBA concentrations extended the lag time of colony formation. In conclusion, HBA can prolong the storage life of thick juice in the sugar industry, although degradation cannot be eliminated.status: publishe