Hydrolysis of gluten and the formation of flavor precursors during sourdough fermentation [Elektronische Ressource] / Claudia Thiele

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Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 34
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

TECHNISCHE UNIVERSITÄT MÜNCHEN
LEHRSTUHL FÜR TECHNISCHE MIKROBIOLOGIE




Hydrolysis of gluten and the formation of flavor precursors during
sourdough fermentation


Claudia Thiele



Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung,
Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen
Grades eines



Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr.-Ing. Eberhard Geiger
Prüfer der Dissertation: 1. Univ.-Prof. Dr. rer. nat. habil. Rudi F. Vogel
2. Univ.-Prof. Dr. rer. nat. Henning Klostermeyer, em.


Die Dissertation wurde am 23.07.2003 bei der Technischen Universität München eingereicht und
durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt
am 15.09.2003 angenommen. Abbreviations


AP Aminopeptidases
AU Absorption unit
B. Bacillus
BC beforechrist
C. Candida
cfu Cell forming unit
CIEX Cation exchange chromatography
D Dough
D/TP Di- and tripeptidase
Da Dalton
DTT Dithiothreitol
EP Endopeptidases
FD Flavor dilution
FITC Fluorescenceisothiocyanate
GMP Gluten macropolymer
GPC Gelpermeation chromatography
GSH Glutathione
HMW High molecular weight
HPLC High performance liquid chromatography
IEX Ion exhange chromatography
IPAD Integrated pulsed amperometric detection
LAB Lactic acid bacteria
L. Lactobacillus
Lc. Lactococcus
Leuc. Leuconostoc LMW Low molecular weight
LTH Lebensmitteltechnologie Hohenheim
M Molar
MALLS Multiangle laserlight scattering
MW Molecular weight
n.g. Not grown
OD Optical density
PAGE Polyacrylamid gelelectrophoresis
PP Prolinepeptidase
rfu Relative fluorescence unit
RP Reversed phase
S. Saccharomyces
SDS Sodium dodecylsulfate
SEC Size exclusion chromatography
SPE Solid phase extraction
TFA Trifluoracetic acid
TMW Technische Mikrobiologie Weihenstephan
1. Introduction........................................................................................................................7
1.1 Technology of sourdough.........................................................................................................7
1.2 Microflora of sourdough fermentations.....................................................................................8
1.3 Flavor of wheat bread...............................................................................................................9
1.4 Proteolytic activity and amino nitrogen demand of lactic acid bacteria....................................13
1.5 Wheat proteins and gluten macropolymer...............................................................................16
1.6 Determination of amino acids in complex media by anion exchange chromatography and
integrated pulsed amperometric detection .............................................................................17
1.7 Aim of this work ....................................................................................................................18
2. Materials and Methods......................................................................................................20
2.1 General Methods....................................................................................................................20
2.1.1 Strains and culture conditions.......................................................................................20
2.1.2 Media ..........................................................................................................................20
2.1.3 Determination of viable cell counts, and pH. ................................................................21
2.1.4 Determination of carbohydrates and organic acids in dough. ........................................21
2.1.5 Determination of total amino nitrogen content..............................................................21
2.1.6 Size exclusion chromatography (SEC) of SDS-soluble proteins and SDS-DTT soluble
proteins. ........................................................................................................................22
2.1.7 SDS-polyacrylamide gel electrophoresis (SDS-PAGE).................................................23
2.2 Generation of flavor precursor compounds during sourdough fermentation.............................23
2.2.1 Sourdough fermentation...............................................................................................23
2.2.2 Baking experiments. ....................................................................................................24
2.2.3 Amino acid analysis by post column derivatization ......................................................26
2.3 Screening for proteolytic active microorganisms.....................................................................27
2.3.1 Strains and culture conditions.......................................................................................27
2.3.2 Media ..........................................................................................................................27
2.3.3 Screening on agar plates...............................................................................................28
2.3.4 Determination of proteolytic activity by fluorescent substrates .....................................28
2.4 Nitrogen demand and peptide utilization by L. sanfranciscensis..............................................29
2.4.1 Amino nitrogen free media...........................................................................................29
2.4.3 Doughs for peptide extraction ......................................................................................30
2.4.4 Preparative size exclusion chromatography ..................................................................30
2.4.5 Cation exchange chromatography ................................................................................31
2.4.6 Reverse-phase chromatography....................................................................................31
2.4.7 Growth experiments with dough fractions ....................................................................32
2.5 De-/polymerization and proteolysis of gluten proteins in sourdough .......................................32
2.5.1 Sourdough fermentation...............................................................................................32
2.5.2 Extraction of doughs and reversed-phase (RP)-HPLC analysis of dough extracts..........33
2.6 De-/Polymerization and proteolysis determined with FITC labeled wheat proteins..................34
2.6.1 Extraction of wheat proteins.........................................................................................34
2.6.2 Labeling of wheat proteins with FITC..........................................................................34
2.6.3 Purification of labeled proteins.....................................................................................34
2.6.4 Reconstitution of wheat doughs with fluorescent labeled proteins and dough
fermentation..................................................................................................................35
2.7 Amino Acid Analysis from Food by IPAD .............................................................................36
2.7.1 Chemicals and reagents................................................................................................36
2.7.2 Liquid chromatography37
2.7.3 Solid Phase Extraction .................................................................................................39
2.7.4 Recovery of amino acids from food samples. ...............................................................40
3. Results..............................................................................................................................42
3.1 Contribution of microorganisms and wheat enzymes on the flavor of rolls..............................42
3.1.1 Effect of salt, acid and dithiothreitol (DTT) on the total amino nitrogen content in sterile
wheat doughs. ...............................................................................................................42
3.1.2 Total amino nitrogen content in fermented wheat doughs .............................................43
3.1.3 Changes in individual amino acid concentration...........................................................46
3.1.4 Sensory evaluation of rolls ...........................................................................................49
3.2 Screening of proteolytic active lactic acid bacteria..................................................................50
3.3 Nitrogen demand and peptide utilization by L. sanfranciscensis..............................................56
3.3.1 Dough extraction and preparative SEC.........................................................................56
3.3.2 Dough fractionation with cation exchange chromatography..........................................59
3.3.3 Fractionation by reversed phase chromatography ................................................