Master of science department of Food Science University of Manitoba Winnipeg © 2009 by Blanca Gómez Guerrero




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Effects of Amino Acid Profile, Endoprotease Activities and Wort Quality on

Fermentability under Different Malting and Brewing Conditions


by




Blanca Gómez Guerrero


A Thesis submitted to the Faculty of Graduate Studies of


The University of Manitoba


in partial fulfilment of the requirements of the degree of


MASTER OF SCIENCE







Department of Food Science


University of Manitoba


Winnipeg


Copyright © 2009 by Blanca Gómez Guerrero




ACKNOWLEDGEMENTS


My sincere appreciation is extended to Dr. Michael Edney for all his support during my time in Winnipeg at the Grain Research Laboratory and the online assistance when I was in Uruguay. His guidance, advice, support and research enjoyment had made a significant contribution to my academic, professional and personal development. I also would like to acknowledge Dr. Marta Izydorczyk, Basic Barley Research at the Grain Research Laboratory, Dr. Fawzi Razem and Dr. Murray Ballance Department of Plant Science, University of Manitoba for serving on my graduate committee.

I am absolutely grateful to the Technological Laboratory of Uruguay (LATU) and Uruguayan Government for their financial support.

I extend my sincere thanks to my sister María Eugenia (mujer águila de las estrellas) for her unconditional support and love, and to Dr. Edney’s family (Franziska, Katya and Alexander) to make me feel at home in Canada. My sincere thanks to all the staff of Barley Applied Research, Basic Barley Research, and CMBTC at the Canadian Grain Commission for allowing me to use their facilities during my research, and share their time and knowledge. Thank you to all the staff of Malting Unit, Cereals Department, Fermented Beverages, Administration and Human Resources at LATU for their constant support and trust. Thank you to Patricia Suárez for her guidance and support. Also thank you to all the Staff and Graduate Students at the Food Science Department, and my Winnipeggers friends for your support and friendship. Especial thanks to Lorena Cammarota, my selected sister and Luis Ordeig for his Office Excel assistance.

I am completely grateful to my family, my mother Blanca Guerrero, my father Carlos Gómez, my sisters, brothers, nieces, nephews, aunts, uncles, cousins and my Uruguayan friends, for all their love, trust, inspiration and support through out the years. MUCHAS GRACIAS, hoy estoy y soy gracias a todo el amor que me brindaron, los quiero pila pila, chi!!


To all the beings of light


(A todos los seres de luz)


TABLE OF CONTENTS

Acknowledgements............................................................................................................ii

Dedication……..................................................................................................................iii

Table of Contents..............................................................................................................iv

List of Tables.....................................................................................................................ix

List of Figures...................................................................................................................xii

List of Abbreviations.......................................................................................................xvii

Abstract.............................................................................................................................

Chapter I: Introduction....................................................................................................1

1.1General Introduction....................................................................................................2

Chapter II: Literature Review..........................................................................................6

2.1 Overview of the malting and brewing processes............................................................7

2.1.1 Malting process.................................................................................................7

2.1.2 Brewing process................................................................................................9

2.2 Malt quality ………………..............................................................................................11

2.2.1 Malt extract........................................................................................................12

2.2.2 Cytolysis...........................................................................................................14

2.2.3 Wort clarity and colour.............................................................................................16

2.2.4 Proteolysis..........................................................................................................17

2.2.5 Wort pH.............................................................................................................19

2.2.6 Fermentability........................................................................................................19

2.2.7 Starch-degrading enzymes......................................................................................20

2.3 Processing conditions........................................................................................................23

2.3.1 Barley varieties........................................................................................................23

2.3.2 Steeping............................................................................................................24

2.3.3 Germination........................................................................................................26

2.3.4 Additives...........................................................................................................27

2.3.5 Kilning..............................................................................................................32

2.4 Free amino acids in barley, malt and wort.......................................................................36

2.4.1 Effects of barley variety & barley quality on levels of free amino acids...................38

2.4.2 Movement of amino acids within the kernel during germination..............................42

2.4.3 Effect of kilning on free amino acids......................................................................44

2.4.4 Effect of mashing on free amino acids...................................................................46

2.4.5 Effect of wort boiling on free amino acids................................................................48

2.4.6 Free amino acids and their role in yeast fermentation.............................................48

2.4.7 Absorption of amino acids by yeast......................................................................51

2.4.8 Amino acid turnover by yeast..............................................................................54

2.4.9 Amino acids and higher alcohols production.........................................................55

2.4.10 Fermentability of amino acid supplemented wort.................................................56

2.4.11 Methodologies for amino acid quantification..........................................................57

2.5 Endoproteases..............................................................................................................62

2.5.1 Proteases in barley, malt and wort......................................................................62

2.5.2 Endoproteases classes......................................................................................66

2.5.3 Endoproteases assays........................................................................................71

2.5.4 Effect of processing conditions on endoproteases activities....................................77

2.5.5 Endogenous endoprotease inhibitors......................................................................85

2.6 Fermentability...............................................................................................................88

2.6.1 Brewing fermentation..........................................................................................88

2.6.2 Wort composition..............................................................................................92

2.6.3 Yeast genotype.................................................................................................107

2.6.4 Phenotypic yeast expression - formation of flavours compounds..........................113

2.6.5 Factors that affect wort composition and fermentability........................................121

2.6.6 Malt fermentability assays.................................................................................133

Chapter III: Materials & Methods...................................................................................137

3.1 Barley samples...........................................................................................................137

3.2 Malting methods and schedules....................................................................................138

3.2.1 Automatic Phoenix micromalting........................................................................139

3.2.2 Pilot plant.........................................................................................................141

3.3 Barley and malt analyses..............................................................................................141

3.3.1 Barley analysis...................................................................................................142

3.3.2 Malt analysis......................................................................................................143

3.4 Enzyme analysis...........................................................................................................145

3.5 Amino acid analysis.....................................................................................................146

3.5.1 Setting conditions for the UPLC system................................................................147

3.5.2 Preparing mobile phases and wash solutions........................................................149

3.5.3 Sample preparation...............................................................................................151

3.5.4 Derivatization procedure.......................................................................................153

3.5.5 Setting the sample set table................................................................................154

3.6 Endoprotease analysis..................................................................................................155

3.6.1 Solutions..........................................................................................................155

3.6.2 Pure enzymes...................................................................................................156

3.6.3 Azogelatin assay................................................................................................156

3.6.4 Azo-casein assay.................................................................................................158

3.7 Fermentability analysis.....................................................................................................160

3.7.1 AAL EBC method...................................................................................................161

3.7.2 Micro-brewing.....................................................................................................161

3.7.3 Broth preparation and AAL measurement............................................................164

3.8 Experimental design and statistical analysis....................................................................165

Chapter IV: Results & Discussion.................................................................................167

4.1 Introduction..................................................................................................................168

4.2 General presentation on UPLC amino acid analysis........................................................168

4.2.1 Wort amino acid analysis....................................................................................170

4.2.2 Barley and malt amino acid analyses...................................................................172

4.3 General presentation on endoprotease enzymatic method..............................................174

4.3.1 Azogelatin substrate..........................................................................................174

4.3.2 Azo-casein substrate...........................................................................................179

4.4 Differences in malt quality among barley samples...........................................................188

4.5 Differences in malt quality among processing conditions.................................................190

4.6 Individual wort amino acid profiles....................................................................................193

4.6.1 Individual wort amino acid profiles among malt barley varieties............................193

4.6.2 Individual wort amino acid profiles among malt processing treatments.................195

4.6.3 Individual wort amino acid profiles versus FAN.....................................................201

4.7 Endoprotease activities among malt barley varieties and processing conditions.............205

4.8 Changes in endoprotease activities & amino acid profiles during malting process..........208

4.8.1 Endoproteases activities during malting.............................................................208

4.8.2 Amino acids during malting....................................................................................209

4.9 Relationships between levels of endoproteases and amino acids...................................218

4.10 Changes in the amino acid profile during brewing process............................................220

4.10.1 Effects of mashing and wort boiling on amino acid profile...................................220

4.10.2 Changes in amino acid profiles during fermentation............................................223

4.11 Relationships between fermentability and amino acids..................................................228

4.11.1 Broth experiment..................................................................................................229

4.11.2 Amino acid contents in broths with varying ratios................................................231

4.11.3 Using the broth method to investigate effects of amino acids on fermentability..234

4.11.4 Different pitching rates experiment......................................................................239

4.12 Relative importance of amino acids, starch-degrading enzymes and endosperm

modification in explaining fermentability............................................................................242

Chapter V: Conclusions..................................................................................................245

5.1 Conclusions.................................................................................................................246

References.......................................................................................................................248

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