MALOLACTIC FERMENTATION AND BIOGENIC AMINE FORMATION. SCREENING FOR LOW ...

115 Pages·2012·1.71 MB·English

Universität für Bodenkultur, Wien

University of Natural Resources and Life Sciences, Vienna

Department of Food Sciences and Technology
Laboratory of Food Biotechnology

MALOLACTIC FERMENTATION AND BIOGENIC
AMINE FORMATION. SCREENING FOR LOW
BIOGENIC AMINE FORMING Oenococcus oeni STRAINS.

Master Thesis

Submitted by Nuno Miguel Barreira Braz da Silva Bakk. techn.

Supervised by o. Univ. Prof. Dipl.-Chem. Dr. rer. nat. Klaus D. Kulbe

Vienna, July 2012
Biogenic Amines Nuno Silva, July 2012
Acknowledgements
It is a pleasure to thank all the people who helped me during my studies at the
University of Natural Resources and Life Sciences, Vienna and made this thesis
possible.
First and foremost, I want to offer my sincere gratitude to Prof. Dr. Klaus Dieter Kulbe
for the assistance and for offering me the opportunity to join the FWF project which
provided me important financial support during my thesis. Very special thanks go to my
supervisor Dr. Andrés del Hierro for his expertise, his inspiration and his efforts to
explain things clearly and simply. He was more than a supervisor and became a really
good friend. Without him this thesis would have never been possible. I am also truly in
debt to Dr. DI Herbert Michlmayr for all the patience, the support and all the skills he
transmitted to me, that added considerably to my graduated experience.
Moreover, I wish to thank the other members of the wine group, DI Christina
Schümann, DI Kateryna Rogowicz and DI Veronika Rogl for the help and support but
more importantly for the friendship within this group that provided a funny and
stimulating environment in which to learn and grow. Furthermore, I would also like to
thank all the other student colleagues of the laboratory of Food and Biotechnology for
the help and for providing a good working climate.
Special thanks go to Dr. Reinhard Eder for the collaboration in this project. I wish to
thank all my working colleagues from the department of Chemistry, Biology and Wine
Cellar from the Federal College and Research Institute for Viticulture and Pomology,
Klosterneuburg, Austria, for the support and pleasant working atmosphere.
Despite the geographical distance my family was always nearby. I would like to thank
my parents, Felizmina Maria and Domingos Lima as well as my sister Maria José and
my brother Francisco for all the support they provided me in the course of my academic
career. It is to them that I dedicate this thesis. To my girlfriend Angelika, you were my
shadow and my light, you supported, encouraged, understood and loved me at every
moment during this process that seemed endless at times.
I finally thank my close friends of Wolfganggasse 7 and Dijkgraaf 5C who shared good
and bad moments with me during this entire journey.
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Biogenic Amines Nuno Silva, July 2012
Table of Contents
Index of Tables ................................................................................................................ 7

Index of Figures .............................................................................................................. 8

Abbreviations ................................................................................................................ 10

1. Introduction .............................................................................................................. 11
1.2. Hazard Identification ........................................................................................... 12
1.2.1. Background ................................................................................................... 12
1.2.2. Formation of Biogenic Amines ..................................................................... 14
1.2.3. Decarboxylases.............................................................................................. 15
1.2.3.1. Histidine decarboxylase ............................................................................. 16
1.2.3.2. Tyrosine decarboxylase .............................................................................. 17
1.2.3.3. Ornithine decarboxylase ............................................................................. 18
1.2.3.4. Lysine decarboxylase ................................................................................. 20
1.2.3.5. Phenylalanine decarboxylase ..................................................................... 20
1.2.4. Metabolic pathway of decarboxylation ......................................................... 21
1.2.5. Main scenarios leading to Biogenic Amine formation in wine ..................... 22
1.2.5.1. Substrate availability .................................................................................. 22
1.2.5.2. Viticulture conditions ................................................................................. 22
1.2.5.3. Fermentation............................................................................................... 23
1.2.5.3.1. Alcoholic fermentation ............................................................................ 23
1.2.5.3.2. Malolactic fermentation .......................................................................... 27
1.2.5.4. Aging and storage....................................................................................... 30
1.2.5.5. Wine preservatives ..................................................................................... 30
1.2.5.5.1. Sulphur dioxide ....................................................................................... 30
1.2.5.5.2. Dimethyl Dicarbonate ............................................................................. 33
1.2.5.6 Wine lees ..................................................................................................... 34
1.2.5.7 Time ............................................................................................................ 35
1.2.5.8 Filtration ...................................................................................................... 35
1.2.5.9. Type of barrel ............................................................................................. 36
1.2.5.10. Temperature ............................................................................................. 36
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1.2.6. Factors affecting growth of Biogenic Amine producers ............................... 36
1.2.6.1 Grape skin maceration ................................................................................. 36
1.2.6.2 Pectolytic enzymes ...................................................................................... 37
1.2.6.3. Phenolics .................................................................................................... 37
1.2.7. Wine physicochemical composition.............................................................. 38
1.2.7.1. Wine influence on the decarboxylase gene expression .............................. 38
1.2.7.2. Glucose and L-malic acid ........................................................................... 38
1.2.7.3. Lactic acid .................................................................................................. 38
1.2.7.4. Ethanol ....................................................................................................... 39
1.2.7.5. Sulphur dioxide and pH ............................................................................. 39
1.3. Toxicology of Biogenic Amines .......................................................................... 40
1.3.1. Histamine ...................................................................................................... 42
1.3.1.1. Synthesis and metabolism in humans ........................................................ 42
1.3.1.2. Physiological functions .............................................................................. 44
1.3.1.3. Toxic response............................................................................................ 44
1.3.1.4. Toxic dose .................................................................................................. 45
1.3.2. Tyramine and Phenylethylamine ................................................................... 46
1.3.2.1. Synthesis and metabolism in humans ........................................................ 46
1.3.2.2. Physiological functions .............................................................................. 46
1.3.2.3. Toxic response............................................................................................ 47
1.3.2.4. Toxic dose .................................................................................................. 47
1.3.3. Putrescine and Cadaverine ............................................................................ 47
1.3.3.1. Synthesis and metabolism in humans ........................................................ 47
1.3.3.2. Physiological functions .............................................................................. 48
1.3.3.3. Toxic response............................................................................................ 48
1.3.3.4. Toxic dose .................................................................................................. 48
1.4. Analytical tools for detection of Biogenic Amines in wine ................................. 49
1.4.1. Extraction techniques .................................................................................... 49
1.4.2. Rapid and semi-quantitative methods ........................................................... 49
1.4.2.1. Screening methods using selective media .................................................. 49
1.4.2.2. Enzymatic methods .................................................................................... 50
1.4.3. Quantitative methods..................................................................................... 50
1.4.3.1. Chromatography methods .......................................................................... 50
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1.4.3.1.1. Thin-layer Chromatography .................................................................... 50
1.4.3.1.2. Liquid Chromatography .......................................................................... 51
1.4.3.2. Capillary Electrophoresis ........................................................................... 52
1.4.4. Polymerase Chain Reaction .......................................................................... 52
1.5. Control of Biogenic Amines production .............................................................. 55

2. Experimental overview............................................................................................. 58

3. Materials and Methods ............................................................................................ 59
3.1 Material ................................................................................................................. 59
3.1.1. Media ............................................................................................................. 59
3.1.2. Buffers ........................................................................................................... 60
3.1.3. Isolation of Lactic Acid Bacteria .................................................................. 60
3.1.4. Inoculum preparation .................................................................................... 61
3.1.5. Wine preparation ........................................................................................... 61
3.1.6. Sampling........................................................................................................ 62
3.2. Methods ............................................................................................................... 63
3.2.1. Microscope .................................................................................................... 63
3.2.2. Molecular methods ........................................................................................ 63
3.2.3. Chemical methods ......................................................................................... 65
3.2.4. Panel tasting .................................................................................................. 67

4. Results ........................................................................................................................ 70
4.1. Bacterial growth ................................................................................................... 70
4.1.1. 16S Amplified Ribosomal DNA Restriction Analysis Method .................... 70
4.2. Wine parameters .................................................................................................. 73
4.3. Fourier Transform Infrared spectroscopy analysis .............................................. 74
4.3.1. Analyses from the Institute of Chemistry ..................................................... 75
4.3.2. Analyses from the Institute of Oenology ...................................................... 78
4.5. Chemical analyses ................................................................................................ 81
4.5.1. Biogenic Amine Production .......................................................................... 81
4.5.2. Alcohols and ethyl acetate............................................................................. 84
4.5.3. Phenolics ....................................................................................................... 85
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4.6. Wine aroma .......................................................................................................... 86

5. Discussion and Conclusion ....................................................................................... 89
5.1. Identification and characterization of Lactic Acid Bacteria ................................ 89
5.2. Comparison between the two cellars ................................................................... 89
5.3 Amino acids .......................................................................................................... 91
5.4. Lactic Acid Bacteria ............................................................................................ 91
5.5. Biogenic amines ................................................................................................... 92
5.6. Wine aroma .......................................................................................................... 93

6. Summary ................................................................................................................... 95

7. Zusammenfassung .................................................................................................... 97

8. References.................................................................................................................. 99

9. Annex ....................................................................................................................... 110

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Index of Tables
Table 1.1: Biogenic amines in foods and their pharmacological effects. 12
Table 1.2: Biogenic amine producing microorganisms found in wine. 15
Table 1.3: Wine pH determines the quantity of free sulphur dioxide needed
to produce 0.8 mg/L of molecular sulphur dioxide. 32
Table 1.4: Biogenic amine content in different food products. 40
Table 1.5: Dose response of histamine alcoholic drinks in patients after oral
administration. 46
Table 3.1: Different Man, Rogosa and Sharpe media used. 59
Table 3.2: Origin of the selected strains. 60
Table 4.1: Summary of the identification of Lactic Acid Bacteria isolates by
restriction DNA patterns after digestion with BFaI and MSeI. 72
Table 4.2: Control wine analyses. 73
Table 4.3: Amino acid concentrations (mg/L) of the wines in the different
cellars after malolactic fermentation. 82
Table 4.4: Biogenic amine content of the wines produced in two different
cellars after malolactic fermentation. 83
Table 4.5: Higher alcohols and esters (ethyl acetate) measured
in the final wines. 84
Table 4.6: Phenolic compounds measured in finished wines. 85
Table 4.7: Results of the wine descriptive analyses. 87

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Index of Figures
Figure 1.1: Chemical structure of some of the most important biogenic amines
from the oenological point of view. 13
Figure 1.2: Pathways of arginine metabolism in bacteria. 19
Figure 1.3: Biogenic amine biosynthesis pathway in bacteria. 21
Figure 1.4: Schematic representation of the main biochemical mechanisms
of yeast metabolism during alcoholic fermentation. 24
Figure 1.5: The average concentration for histamine, putrescine, tyramine, cadaverine
and phenylethylamine in the seven stages of industrial winemaking. 26
Figure 1.6: Schematic description of the major pathways of hexose (glucose)
fermentation by lactic acid bacteria. 28
Figure 1.7: Oenococcus oeni viewed with phase contrast microscopy at a
magnification of 1000x. 29
Figure 1.8: The different forms of sulphur dioxide according with the pH values. 31
Figure 1.9: Sulphur dioxide presented in the different forms. 31
Figure 1.10: Effect of biogenic amines in the human intestinal tract. 41
Figure 1.11: Schematic representation of the histamine metabolism. 43
Figure 1.12: Multiplex Polimerase Chain Reaction amplification of wine bacteria
producing histamine, tyramine and putrescine. 54
Figure 3.1: Cultured strains before inoculation into the wine. 61
Figure 3.2: The wineries where the experiments took place. 62
Figure 3.3: Statistical wine tasting tool in order to evaluate the wine according
with different defined aromas. 69
Figure 3.4: Sensory spider diagram for wine. 69
Figure 4.1: Restriction pattern after BfaI digestion isolated from Austrian wines. 71
Figure 4.2: Restriction pattern after MseI digestion isolated from Austrian wines. 71
Figure 4.3: FTIR analyses from strain K1. 75
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Figure 4.4: FTIR analyses from strain SK3. 75
Figure 4.5: Final sulphur dioxide concentrations of wines from the Institute
of Chemistry. 77
Figure 4.6: FTIR analyses from the strain 551. 78
Figure 4.7: FTIR analyses from the strain SK3. 78
Figure 4.8: Final sulphur dioxide concentrations from wines of the Institute
of Oenology. 80
Figure 4.9: Aroma spider plots of wines from the Institute of Oenology 88
Figure 4.10: Aroma spider plot of wines from the Institute of Chemistry. 88
Figure 9.1: FTIR analyses from strain 551 (Institute of Chemistry). 110
Figure 9.2: FTIR analyses from strain 433 (Institute of Chemistry). 111
Figure 9.3: FTIR analyses from strain 78 (Institute of Chemistry). 112
Figure 9.4: FTIR analyses from the strain K1 (Institute of Oenology). 113
Figure 9.5: FTIR analyses from the strain 433 (Institute of Oenology). 114
Figure 9.6: FTIR analyses from the strain 78 (Institute of Oenology). 115

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Abbreviations
aaDC Amino acid decarboxylase
AF Alcoholic fermentation
ADI Agmatine deiminase
ARDRA Amplified rDNA restriction analysis
BA Biogenic Amines
BOKU Universität für Bodenkultur, Wien
University of Natural Resources and Applied Life Sciences, Vienna
Bp base pair
CE Capillary electrophoresis
DAO Diamino oxidase
FTIR Fourier Transform Infrared Spectroscopy
GC Gas chromatography
HPLC High-performance liquid chromatography
HDC Histidine decarboxylase
KC Carbamate kinase
LAB Lactic Acid Bacteria
MAO Monoamino oxidase
MLF Malolactic fermentation
ODC Ornithine decarboxylase
OTC Ornithine transcarbomylase
PCR Polymerase Chain Reaction
PLP Pyridoxal-5-phosphate
TDC Tyrosine decarboxylase
TLC Thin-layer chromatography
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