Copyright is owned by the Author of the thesis.  Permission is given for 
a copy to be downloaded by an individual for the purpose of research and 
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DEVELOPMENT OF LOW FODMAP GLUTEN FREE 

SOURDOUGH BREAD 

 

 

A thesis presented in partial fulfillment of requirements for the degree of 

Master of Food Technology 

 

 

 

Massey University 

Albany, New Zealand 

 

 

 

Haojing Tian 

 

 

 

May 2021 

  



ii 
 

 

Dedication 

This work is dedicated to my parents: my mother June Liu and my father Shaolei Tian, 

who both taught me everything I need to know in my life, who always love and support me 

with their endless encouragement and kindness. 

  



iii 
 

ABSTRACT 

Fermentable oligosaccharides, disaccharides, monosaccharides and polyols (FODMAPs) comprise 

a group of indigestible short-chain carbohydrates that have gained attention due to their potential to 

trigger gastrointestinal (GI) symptoms such as abdominal pain, bloating and diarrhea in patients with 

GI-related disorders (e.g., Crohn’s disease, Coeliac disease and Irritable Bowel Syndrome). Certain GI-

related disorders are triggered by consuming gluten-containing products, including bread. Gluten-

related GI disorders can be prevented by strict adherence to a lifelong gluten-free (GF) diet. Although 

GF breads are generally low in FODMAPs (total FODMAPs < 0.5 g), consuming quantities that exceed 

the threshold (0.5 g/meal) may cause symptoms. 

Sourdough fermentation has been applied in GF bread for improving its overall quality. Sourdough 

culture is a complex microbial ecosystem dominated by lactic acid bacteria (LAB) and yeast which can 

degrade FODMAP sugars during fermentation. This study quantified FODMAPs in seven commercial 

GF breads (three conventional GF breads and four GF sourdough breads) in New Zealand by HPAEC-

PAD and enzymatic assay. This study also reformulated existing formulation and applied retard 

proofing (fermentation at refrigeration temperature) to reduce the FODMAPs in the GF sourdough bread.  

Results showed that the GF sweet potato sourdough bread (GFSD1) formulation (Control) 

contained the highest FODMAP levels. Three strategies were applied to reformulate the existing 

formulation and investigate the effects on the reduction of FODMAPs in GF sweet potato sourdough 

bread as follows: replacing coconut sugar with rice syrup (low FODMAP ingredient) (RS); reducing 

kumara flour (potentially high in mannitol) from 2.81 % to 1.50 % (RS/RK); increasing the percentage 

of sourdough starter from 24.31 % to 35.00 % (RS/ISD). Retard proofing (12 h at 8 ℃/87 % RH) was 

applied to the Control formulation (Control/RP) instead of existing proofing conditions (1 h at 37 ℃/87 % 

RH) to investigate the effect of different fermentation processes on reduction of FODMAPs in GF 

sourdough bread. The microbiological and physical-chemical characteristics and FODMAP content of 

GF sourdough starter during propagation were determined. The bread dough samples before and after 

proofing were collected for measurement of microbiological and physical-chemical characteristics as 

well as FODMAP analysis. The baking characteristics, sensory properties and FODMAP content of GF 

sweet potato sourdough bread samples were also analysed. 

The pH of the GF sourdough starter decreased during propagation of the sourdough starter, while 

the LAB and yeast cell density, total titratable acid (TTA), lactic and acetic acids increased (p < 0.05). 

GF sourdough starter contained very low amounts of FODMAPs, and the overall FODMAP content 

decreased during propagation for 72 h. Significant growth of the LAB and yeast cell density were 

observed in all the bread dough samples during proofing (p < 0.05). After proofing, the pH of all the 

bread doughs decreased (p < 0.05), while the TTA increased due to increasing concentrations of lactic 

and acetic acids. Addition of a high percentage of sourdough starter enhanced sourdough acidification 

reflected by the lowest pH, and the highest TTA and concentrations of organic acids of the RS/ISD 

bread dough samples. The concentrations of lactic and acetic acids of bread doughs made with rice 

syrup (RS, RS/RK) were lower than that made with coconut sugar (Control and Control/RP) (p < 0.05), 

indicating the impact of the type of sugar on the carbohydrate metabolism of LAB and sourdough 

acidification. The fermentation quotient of the Control/RP (2.74) was close to the optimum FQ range 

(2.00 - 2.70) recommended for producing superior sourdough bread. 



iv 
 

Fructose, glucose and total fructans were reduced in all the bread dough samples during proofing. 

Only trace amounts of sugar polyols (sorbitol and mannitol), galacto-oligosaccharides (raffinose and 

stachyose) and fructo-oligosaccharides (nystose) were detected in the bread dough and bread samples. 

Replacing coconut sugar with rice syrup in the bread formulation significantly reduced the amounts of 

excess fructose, mannitol and total fructans in both bread dough and bread samples (p < 0.05). 

Control/RP bread produced by retard proofing contained lower total fructans than the Control bread (p 

< 0.05). However, the RS/RK and RS/ISD formulations had negligible effects on further reducing 

FODMAPs in GF sweet potato sourdough bread. 

Although GF sweet potato sourdough breads made with rice syrup (RS, RS/RK and RS/ISD) 

contained lower levels of FODMAPs, the overall baking characteristics and consumer acceptability 

were lower than the Control and the Control/RP breads made with coconut sugar. Reducing kumara 

flour and increasing the percentage of sourdough starter negatively impacted on the crumb loaf and 

crumb texture, resulting in lower sensory scores for the aroma, taste and overall acceptability. However, 

retard proofing not only reduced the total FODMAPs in GF sweet potato sourdough breads, but it also 

increased loaf volume, reduced baking weight loss, enhanced crumb softness, cohesiveness and 

resilience (p < 0.05). The Control/RP bread received the highest sensory scores for all the attributes (p 

< 0.05), and it was well-accepted by sensory panelists. Therefore, retard proofing could be considered 

as a low-cost application in the development of low FODMAP GF sourdough breads. 

  



v 
 

ACKNOWLEDGEMENTS 

I would first like to thank Venerdi (NZ) Ltd. for providing funding, supplies, and technical 

support for this study. I greatly appreciate this precious opportunity of exploring the science 

and food technology of the gluten-free and bakery industry. 

I would like to thank my main supervisor, Dr. Tony Mutukumira, for his contribution and 

advice, continuous support and guidance throughout my Masters study and life. I would say 

working with him was really enjoyable, because his enthusiasm and encouragement always 

motivated me. Thanks go to my co-supervisor, Associate Prof. Kay Rutherfurd-Markwick, for 

her patience, constant support and guidance throughout this research. I also want to extend my 

gratitude to Prof. Marie Wong, for her invaluable knowledge and advice on sugars and organic 

acids analysis, and HPLC training. 

I would like to thank all the School of Food and Advanced Technology staff, who gave me 

advice and assistance during the time I conducted my experiments. John Sykes at Palmerston 

North campus, for his expertise in HPLC instruments and advice on HPLC column selection. 

Dr Nuri Begum and Negah Nikanjam, for their training in the chemistry and food product 

development laboratory, technical support and general assistance for analytical instruments. 

Rachel Liu, for her training as well as guidance in the microbiology laboratory. 

I would like to acknowledge Dr. Chuanfeng Huang and Dr. Haiyan Wang from the National 

Institutes for Food and Drug Control (Beijing, China), for providing HPAEC-PAD instruments 

for this study, and technical support and guidance for FODMAP analysis. 

Finally, I would like to thank my parents, for their endless love, support and encouragement. 

My husband, Bo Cao, for his support and encouragement throughout the days I studied for my 

research project and lived in NZ. Without the inspiration of my family, I could not have 

completed this study. 



vi 
 

TABLE OF CONTENTS 

ABSTRACT .............................................................................................................................. iii 

ACKNOWLEDGEMENTS ....................................................................................................... v 

LIST OF TABLES .................................................................................................................... ix 

LIST OF FIGURES ................................................................................................................... x 

LIST OF ABBREVIATIONS .................................................................................................. xii 

CHAPTER 1  INTRODUCTION ............................................................................................ 1 

CHAPTER 2  LITERATURE REVIEW ................................................................................. 5 

2.1 FODMAPs ....................................................................................................................... 5 

2.2 Types of FODMAPs ........................................................................................................ 6 

2.3 Dietary Strategies for the Management of GI-related Diseases ....................................... 7 

2.3.1 Gluten-free diet ....................................................................................................... 7 

2.3.2 Low FODMAP-diet ................................................................................................ 8 

2.3.3 Combination of Gluten-free diet and low FODMAP-diet ...................................... 9 

2.4 Gluten-free Bread ........................................................................................................... 10 

2.4.1 Gluten ................................................................................................................... 10 

2.4.2 The role of gluten in bread-making ...................................................................... 12 

2.4.3 The challenge of making GF bread ...................................................................... 13 

2.5 Sourdough Bread ........................................................................................................... 14 

2.5.1 Classification of sourdough fermentation ............................................................ 15 

2.5.2 Sourdough microbiota .......................................................................................... 16 

2.5.3 Metabolism of sourdough microorganisms .......................................................... 17 

2.5.4 Carbohydrate metabolism by sourdough lactic acid bacteria ............................. 18 

2.5.5 Yeast carbohydrate metabolism ........................................................................... 23 

2.6 Characterisation of Sourdough Parameters .................................................................... 25 

2.6.1 pH and total titratable acid (TTA) ........................................................................ 26 

2.6.2 Fermentation quotient (FQ) ................................................................................. 26 

2.6.3 Flour type ............................................................................................................. 27 

2.6.4 Dough yield .......................................................................................................... 27 

2.6.5 Fermentation time and temperature ..................................................................... 28 

2.7 Sourdough in GF Bread-making .................................................................................... 29 

2.7.1 Production of exopolysaccharides (EPS) ............................................................. 29 

2.7.2 Production of aroma compounds ......................................................................... 30 

2.7.3 Control of staling in GF bread ............................................................................. 32 

2.7.4 Improvement of shelf-life ...................................................................................... 33 

2.7.5 Improvement of nutritional value and health benefits .......................................... 34 

2.8 FODMAP Content of Bread .......................................................................................... 36 

2.9 Development of Low FODMAP Bread ......................................................................... 36 

2.9.1 Low FODMAP ingredients ................................................................................... 37 

2.9.2 Yeast fermentation ................................................................................................ 38 

2.9.3 Sourdough fermentation ....................................................................................... 39 

2.9.4 Prolonged sourdough fermentation ...................................................................... 40 

2.9.5 Specific sourdough starter cultures ...................................................................... 41 

2.10 Quantification of FODMAPs in Foods ........................................................................ 42 



vii 
 

2.10.1 Conventional and recent techniques .................................................................. 42 

2.10.2 Separation mechanism........................................................................................ 43 

2.10.3 Pulsed amperometric detection .......................................................................... 43 

2.10.4 Stationary and mobile phase .............................................................................. 44 

CHAPTER 3  MATERIALS AND METHODS ................................................................... 46 

3.1 Experimental Design ...................................................................................................... 46 

3.1.1 Commercial GF breads used for FODMAP analysis ........................................... 48 

3.1.2 Reformulation of the existing GF sweet potato sourdough bread ........................ 49 

3.1.3 Production of GF sweet potato sourdough bread by retard proofing .................. 51 

3.1.4 Summary of the testing parameters in this study .................................................. 52 

3.2 Phase 1: Quantification of FODMAPs in Commercial GF Breads ................................ 53 

3.2.1 Samples, reagents, and standards ........................................................................ 53 

3.2.2 Preparation of bread samples for FODMAP quantitation ................................... 53 

3.2.3 Extraction of FODMAPs from the bread samples ................................................ 54 

3.2.4 Quantification of FODMAPs in GF bread samples by HPAEC-PAD ................. 54 

3.2.5 Measurement of total fructans in GF bread samples by enzymatic assay ............ 56 

3.3 Phase 2: Reduction of FODMAP Levels in Existing GF Sweet Potato Sourdough Bread59 

3.3.1 Overview of GF sweet potato sourdough bread production ................................ 59 

3.3.2 Propagation of the GF sourdough starter ............................................................ 61 

3.3.3 Bread production .................................................................................................. 61 

3.3.4 Sampling ............................................................................................................... 62 

3.4 Phases 3 and 4: Characterisation of GF Sourdough During Propagation, Bread Dough and 

Bread Samples of the Control and the Reformulated GF Sweet Potato Sourdough Breads 62 

3.4.1 Enumeration of LAB and yeast ............................................................................. 62 

3.4.2 Measurement of pH .............................................................................................. 63 

3.4.3 Determination of total titratable acidity (TTA) .................................................... 63 

3.4.4 Analysis of organic acids in GF sourdough starter, bread dough, and bread samples by 

HPLC ............................................................................................................................. 64 

3.4.5 Quantification of FODMAPs in GF sourdough starter, bread dough, and bread samples 

by HPAEC-PAD and enzymatic assay .......................................................................... 66 

3.5 Phase 4: Characterisation of the Control and the Reformulated GF Sweet Potato Sourdough 

Breads .................................................................................................................................. 67 

3.5.1 Loaf volume .......................................................................................................... 67 

3.5.2 Baking weight loss ................................................................................................ 67 

3.5.3 Water activity ....................................................................................................... 67 

3.5.4 Texture profile analysis (TPA) ............................................................................. 68 

3.5.5 Crumb and crust colour........................................................................................ 68 

3.5.6 Microbiological analysis of bread samples .......................................................... 69 

3.5.7 Sensory evaluation................................................................................................ 69 

3.6 Data Analysis ................................................................................................................. 70 

CHAPTER 4  RESULTS AND DISCUSSION ..................................................................... 71 

4.1 Quantification of FODMAPs in Commercial Gluten-free Breads ................................. 71 

4.1.1 Introduction .......................................................................................................... 71 

4.1.2 Chromatogram profiles of FODMAP standards and bread sample .................... 71 



viii 
 

4.1.3 Calibration curves, detection and quantification limits, and repeatability of the method 73 

4.1.4 FODMAP content of commercial gluten-free breads ........................................... 74 

4.1.5 Summary of Phase 1 ............................................................................................. 80 

4.2 Characterisation of GF Sourdough Starter During Propagation .................................... 81 

4.2.1 Introduction .......................................................................................................... 81 

4.2.2 Characteristics of the GF sourdough starter ....................................................... 81 

4.2.3 LAB and yeast counts ........................................................................................... 82 

4.2.4 pH and total titratable acidity .............................................................................. 84 

4.2.5 Concentration of organic acids ............................................................................ 85 

4.2.6 FODMAP content ................................................................................................. 87 

4.2.7 Summary of Phase 2 and Phase 3 ........................................................................ 92 

4.3 Characterisation of Bread Dough and Bread Samples of the Control and the Reformulated GF 

Sweet Potato Sourdough Breads .......................................................................................... 93 

4.3.1 Introduction .......................................................................................................... 93 

4.3.2 LAB and yeast counts ........................................................................................... 93 

4.3.3 pH and total titratable acidity .............................................................................. 96 

4.3.4 Concentration of organic acids ............................................................................ 98 

4.3.5 FODMAPs .......................................................................................................... 101 

4.3.6 Summary of Phase 4 ........................................................................................... 113 

4.4 Characterisation of the Control and the Reformulated GF Sweet Potato Sourdough Breads

 ........................................................................................................................................... 115 

4.4.1 Introduction ........................................................................................................ 115 

4.4.2 Bread volume ...................................................................................................... 116 

4.4.3 Baking weight loss .............................................................................................. 118 

4.4.4 Water activity ..................................................................................................... 119 

4.4.5 Crumb texture ..................................................................................................... 121 

4.4.6 Colour of bread crumb and crust ....................................................................... 125 

4.4.7 Sensory evaluation.............................................................................................. 129 

4.4.8 Summary of Phase 5 ........................................................................................... 131 

CHAPTER 5  OVERALL CONCLUSIONS AND RECOMMENDATIONS ................... 133 

REFERENENCES ................................................................................................................. 134 

APPENDIX ............................................................................................................................ 177 

A. Composition of the Media Used for Microbiological Analyses ................................... 177 

B. Sensory Evaluation Questionnaire ................................................................................ 178 

C. Data Analysis ................................................................................................................ 182 

D. Statistic Output .............................................................................................................. 205 

 

  



ix 
 

LIST OF TABLES 

Table 1 Cut-off levels of FODMAPs (per single serve) ........................................................................ 9 

Table 2 Homofermentative, heterofermentative and facultative heterofermentative LAB in sourdough 

bread ........................................................................................................................................... 19 

Table 3 Most common columns used for carbohydrate HPAEC-PAC analysis .................................. 45 

Table 4 Description of commercial GF breads used for FODMAP analysis ....................................... 49 

Table 5 Basic formulations of the Control and the reformulated GF sweet potato sourdough breads 50 

Table 6 Comparison of the proofing conditions of the Control and the Control/RP GF sweet potato 

sourdough breads ........................................................................................................................ 51 

Table 7 Parameters that were determined for the GF sourdough starter samples during propagation, the 

bread dough samples (before and after proofing), and the bread samples (after baking) of the GF 

sweet potato sourdough breads ................................................................................................... 52 

Table 8 Gradient programme for carbohydrate separation by HPAEC-PAD on the CarboPac PA20 

column ........................................................................................................................................ 56 

Table 9 Calibration curve, linearity, limit of detection (LOD) and limit of quantification (LOQ), and 

repeatability (% RSD) of the major FODMAP sugars ............................................................... 74 

Table 10 FODMAP content of seven commercial GF breads ............................................................. 75 

Table 11 FODMAP content of GF sourdough starter during 72 h propagation at 8 ℃/87 % RH in the 

chiller .......................................................................................................................................... 89 

Table 12 Fermentation quotient of GF sweet potato sourdough bread samples made using different 

formulations and fermentation processes ................................................................................. 101 

Table 13 FODMAP content (monosaccharides and sugar polyols) of GF sweet potato sourdough bread 

dough samples (before and after proofing) by standard fermentation or retard proofing ......... 102 

Table 14 FODMAP content (GOS, FOS, total fructans) of GF sweet potato sourdough bread dough 

samples (before and after proofing) by standard fermentation or retard proofing .................... 103 

Table 15 FODMAP content of GF sweet potato sourdough bread samples made using different 

formulations and fermentation processes ................................................................................. 108 

Table 16 Loaf weight, loaf volume and specific volume of baked GF sweet potato sourdough breads 

made using different formulations and fermentation processes................................................ 116 

Table 17 Baking weight loss of baked GF sweet potato sourdough breads made using different 

formulations and fermentation processes ................................................................................. 118 

Table 18 Water activity (Day 0 and Day 7) of baked GF sweet potato sourdough breads made using 

different formulations and fermentation processes ................................................................... 119 

Table 19 Textural parameters of baked GF sweet potato sourdough breads made using different 

formulations and fermentation processes ................................................................................. 121 

Table 20 Crumb and crust colour parameters of baked GF sweet potato sourdough breads made using 

different formulations and fermentation processes ................................................................... 125 

 

 

  



x 
 

LIST OF FIGURES 

Figure 1 General production steps of sourdough bread ....................................................................... 15 

Figure 2 Homolactic fermentation (glycolysis, or EMP pathway) by homofermentative LAB .......... 20 

Figure 3 Heterolactic fermentation (phosphoketolase pathway) by heterofermentative LAB ............ 22 

Figure 4 Carbohydrate metabolism by yeast ....................................................................................... 23 

Figure 5 Factors influencing the composition and metabolite production of sourdough microbiota, 

characteristics of sourdough, and product quality ...................................................................... 25 

Figure 6 The five experimental phases of this study ........................................................................... 47 

Figure 7 Control/RP bread doughs during retard proofing in a chiller at 8 ℃/87 % RH .................... 51 

Figure 8 The principles of Megazyme K-FRUCHK Assay Kit ........................................................... 57 

Figure 9 Production procedure of GF sweet potato sourdough bread ................................................. 60 

Figure 10 HPAEC-PAD chromatogram profile of a mixture of FODMAP sugar standards separated 

using a Dionex CarboPac PA20 column ..................................................................................... 72 

Figure 11 HPAEC-PAD chromatogram profile of FODMAP sugars from the gluten-free sourdough 

bread sample 1 (GFSD1) separated using a Dionex CarboPac PA20 column ............................ 73 

Figure 12 Comparison between the levels of individual groups of FODMAPs in commercial GF breads 

and the recommended cut-off levels (g per serve) for a low FODMAP-diet .............................. 79 

Figure 13 Appearance of GF sourdough starter after 48 h propagation at 8 ℃/87 % RH in the chiller

 .................................................................................................................................................... 82 

Figure 14 Lactic acid bacteria (LAB) and yeast counts of GF sourdough starter during 72 h propagation 

at 8 ℃/87 % RH in the chiller .................................................................................................... 83 

Figure 15 pH and total titratable acidity (TTA) of GF sourdough starter during 72 h propagation at 

8 ℃/87 % RH in the chiller ........................................................................................................ 84 

Figure 16 Lactic and acetic acid concentrations in GF sourdough starter during 72 h propagation at 

8 ℃/87 % RH in the chiller ........................................................................................................ 86 

Figure 17 HPAEC-PAD chromatogram profile of FODMAPs from unfermented sourdough starter (UD 

0 h) separated using a Dionex CarboPac PA20 column ............................................................. 90 

Figure 18 HPAEC-PAD chromatogram profile of FODMAPs from fermented sourdough starter (FD 

72 h) separated using a Dionex CarboPac PA20 column ........................................................... 90 

Figure 19 Lactic acid bacteria (LAB) counts of bread dough samples (before proofing and after 

proofing) of GF sweet potato sourdough breads by standard fermentation or retard proofing .. 94 

Figure 20 Yeast counts of bread dough samples (before proofing and after proofing) of GF sweet potato 

sourdough breads by standard fermentation or retard proofing .................................................. 95 

Figure 21 pH and total titratable acidity (TTA) of bread dough samples (before proofing and after 

proofing) by standard fermentation or retard proofing, and bread samples of GF sweet potato 

sourdough breads ........................................................................................................................ 96 

Figure 22 Lactic acid concentration in bread dough samples (before proofing and after proofing) by 

standard fermentation or retard proofing, and bread samples of GF sweet potato sourdough breads

 .................................................................................................................................................... 99 

Figure 23 Acetic acid concentration in bread dough samples (before proofing and after proofing) by 

standard fermentation or retard proofing, and bread samples of GF sweet potato sourdough breads

 .................................................................................................................................................... 99 



xi 
 

Figure 24 HPAEC-PAD chromatogram profile of FODMAPs in the Control bread dough sample before 

proofing (BP) separated using a Dionex CarboPac PA20 column ........................................... 104 

Figure 25 HPAEC-PAD chromatogram profile of FODMAPs in the Control bread dough sample after 

proofing (AP) separated using a Dionex CarboPac PA20 column ........................................... 104 

Figure 26 HPAEC-PAD chromatogram profile of FODMAPs in the Control bread sample (GF sweet 

potato sourdough bread with coconut sugar) separated using a Dionex CarboPac PA20 column

 .................................................................................................................................................. 109 

Figure 27 HPAEC-PAD chromatogram profile of FODMAPs in the RS bread sample (GF sweet 

potato sourdough bread with rice syrup) separated using a Dionex CarboPac PA20 column .. 109 

Figure 28 Total FODMAPs in bread dough samples (before proofing and after proofing) by standard 

fermentation or retard proofing, and bread samples of GF sweet potato sourdough breads ..... 112 

Figure 29 Whole loaves and cross sections of baked GF sweet potato sourdough breads made using 

different formulations and fermentation processes ................................................................... 115 

Figure 30 Lightness (L*) of bread crumb and crust of baked GF sweet potato sourdough breads made 

using different formulations and fermentation processes ......................................................... 126 

Figure 31 Redness (a*) of bread crumb and crust of baked GF sweet potato sourdough breads made 

using different formulations and fermentation processes ......................................................... 127 

Figure 32 Yellowness (b*) of bread crumb and crust of baked GF sweet potato sourdough breads made 

using different formulations and fermentation processes ......................................................... 128 

Figure 33 Mean sensory attribute scores of baked GF sweet potato sourdough breads made using 

different formulations and fermentation processes ................................................................... 130 

 

  



xii 
 

LIST OF ABBREVIATIONS 

AACC American Association of Cereal Chemists 

Ac- acetate ion 

acetyl-CoA acetyl coenzyme A 

acetyl-P acetyl phosphate 

AcOH glacial acetic acid 

AEX 

ANOVA 

anion-exchange 

analysis of variance 

APC total aerobic plate counts 

ATIs amylase-trypsin-inhibitors 

ATP adenosine triphosphate 

Aw water activity 

CD coeliac disease 

CFU/g colony-forming units per gram 

CH3CN acetonitrile 

CIE International Commission on Illumination 

CO2 carbon dioxide 

DHAP dihydroxyacetone phosphate 

DP degrees of polymerisation 

DY dough yield 

EMP Embden-Mayerhoff Parnas 

EPS exopolysaccharides 

FODMAPs fermentable oligo-, di-, and monosaccharides, and polyols 

FOS fructo-oligosaccharides 

FQ fermentation quotient 

G-6-P glucose 6-phosphate 

G-6-PDH glucose 6-phosphate dehydrogenase 

GABA γ-aminobutyric acid 

GAP glyceraldehyde-3-phosphate 

GC-MS gas chromatography coupled with mass spectrometry 

GF gluten-free 

GI                        gastrointestinal 

GOS galacto-oligosaccharides 

HePS heteropolysaccharides 

HK hexokinase 

HMW-GS high-molecular-weight glutenin subunits 

HoPS homopolysaccharides 

HPAEC-PAD high-performance anion-exchange chromatography with pulse 

amperometric detector 

HPLC high-performance liquid chromatography 

HPLC-ELSD high-performance liquid chromatography equipped with evaporative 

light scattering detection 



xiii 
 

HPLC-RI high-performance liquid chromatography equipped with refractive 

index detection 

IBD inflammatory bowel disease 

IBS irritable bowel syndrome 

IBS-D diarrhea predominant IBS 

KHP potassium hydrogen phthalate 

LAB lactic acid bacteria 

LDH lactate dehydrogenase 

LMW-GS low-molecular-weight glutenin subunits 

MeOH methanol 

MRS De Man, Rogosa and Sharpe agar 

MW molecular weights 

N2 nitrogen 

NAD+ nicotinamide adenine dinucleotide 

NADH nicotinamide adenine dinucleotide + hydrogen 

NADPH nicotinamide adenine dinucleotide phosphate + hydrogen 

NaOAc sodium acetate 

NaOH sodium hydroxide 

NCGS non-coeliac gluten sensitivity 

NCWS non-coeliac wheat sensitivity 

NZ 

OH- 

New Zealand 

hydroxide ions 

PAD pulsed amperometric detection 

PCA Plate Count Agar 

PGI phosphoglucose isomerase 

RH relative humidity 

RSD relative standard deviation 

SCFAs short-chain fatty acids 

SD standard deviation 

sp. species 

spp. several species, indicate many species of the same genus 

TCA tricarboxylic acid 

TPA texture profile analysis 

TTA total titratable acid 

w/v weight by volume 

w/w weight by weight 

YGC Yeast Extract Glucose Chloramphenicol Agar 

 

 

 

 

 

 



1 
 

CHAPTER 1   INTRODUCTION 

In recent years, an increasing number of people have been diagnosed with gastrointestinal (GI) 

related diseases associated with the ingestion of foods containing gluten, including bread (Muir 

et al., 2019). Coeliac disease (CD) and non-coeliac gluten or wheat sensitivity (NCGS/NCWS) 

are frequently diagnosed GI immune-mediated systemic disorders triggered by gluten ingestion, 

which affect about 1 % and 6 % of the global population, respectively (Dickey & Kearney, 

2006; Picarelli et al., 2013). Irritable bowel syndrome (IBS) is another group of functional 

bowel disorder induced by altered bowel habits. IBS constitutes the majority of health problems 

associated with GI symptoms, affecting approximately 11 % of the general population (Lovell 

& Ford, 2012). Crohn’s disease is an inflammatory bowel disease (IBD) that can affect any part 

of GI tract and may cause symptoms similar to IBS such as diarrhea, abdominal pain and 

cramping (Gibson & Shepherd, 2005).  

Gluten is the major storage protein found in cereal grains (e.g., wheat, barley, rye) (Arendt et 

al., 2008), but it contains toxic gliadin peptides fraction that trigger the T cell-mediated immune 

response of patients with CD (Sollid & Khosla, 2005), and the innate immune response of 

patients with NCGS (Sapone et al., 2009). Fermentable oligosaccharides, disaccharides, 

monosaccharides and polyols (FODMAPs) which are naturally present in daily food sources, 

are also dietary triggers of GI symptoms for patients with GI-related disorders (Muir et al., 

2019). As FODMAPs are indigestible and unabsorbed, they can reach the colon and cause water 

to be dragged into the gut lumen due to osmotic stress (Gibson et al., 2007). The carbohydrates 

are rapidly fermented by the gut bacteria and produce substantial amounts of gas resulting in 

luminal distension, which is the major cause of many IBS symptoms (Barrett et al., 2010; 

Halmos et al., 2014).  



2 
 

The only available treatment for people suffering from CD and NCGS or NCWS is strict 

adherence to a lifelong gluten-free (GF) diet (Bascuñán et al., 2017). A low FODMAP-diet has 

been effectively used to reduce the GI symptoms of IBS and Crohn’s disease patients (Gearry 

et al., 2009; Gibson & Shepherd, 2009; Staudacher et al., 2011). Recent studies started to 

combine a GF-diet with a low FODMAP-diet to help patients with gluten-related GI disorders 

gain better control of their symptoms and avoid complications associated with these disorders 

(Roncoroni et al., 2018; Roncoroni et al., 2019). Thus, there is an opportunity to develop well-

tolerated low FODMAP GF products suitable for affected patients, which can be used as part 

of a holistic dietary therapy for management of these GI-related diseases. 

Bread is one of the most frequently consumed food products, but it is also the major dietary 

source of disease-activating components, such as gluten and FODMAPs (Laatikainen et al., 

2016). Rising consumer demands for GF breads is correlated with the increase of people 

diagnosed with GI-related disorders. However, GF breads usually have low crumb volume and 

have a rapid staling rate due to the absence of gluten (Gallagher, Gormley & Arendt, 2003; 

Naqash et al., 2017). Gluten is the major factor contributing to the structural properties of baked 

products and provides viscoelastic properties to the dough and unique features for baking 

performance (Arendt et al., 2008). For this reason, numerous studies have focused on the 

development of GF formulations to improve the overall quality and nutritional characteristics 

of GF breads (Gallagher et al., 2004; Moroni, Dal Bello & Arendt, 2009; Arendt & Moroni, 

2013; Oshea et al., 2014; Nionelli & Rizzello, 2016).  

Sourdough fermentation is the oldest leavening technology dating back to ancient Egypt 

(Chavan & Chavan, 2011). It is still widely applied in modern baking industries due to its 

effectiveness in improving the quality of bread products, and unique sourdough aroma and 

flavour (Gobbetti, 1998; Arendt et al., 2007; Corsetti & Settanni, 2007). In recent decades, 

particular interest has been given to using sourdough to produce palatable GF breads, as 

sourdough fermentation can improve textural properties, generate aromatic and flavour 



3 
 

compounds, retard bread staling, extend shelf life and enhance the nutritional values of GF 

breads (Moroni, Dal Bello & Arendt, 2009; Vogelmann et al., 2009; Nionelli & Rizzello, 2016).  

The FODMAP content of bread depend on the types of ingredients and grains used within the 

bread formulation (Biesiekierski et al., 2011). Development of low FODMAP bread can be 

achieved by using low FODMAP ingredients (such as GF flours and ingredients) (Ispiryan, 

Zannini & Arendt, 2020), and through longer yeast fermentation to degrade fructans into 

glucose and fructose by the enzyme invertase (Lammens et al., 2009). Additionally, sourdough 

fermentation represents a novel technology for making low FODMAP bread as it contains 

specific strains of Lactobacillus, utilises a prolonged fermentation time and creates a favorable 

acidic environment for enzymatic activities of yeasts and lactic acid bacteria (LAB), which can 

promote the degradation of FODMAPs within the breads (Teixeira et al., 2012; Knez, Abbott 

& Stangoulis, 2014; Gänzle, 2015; Ziegler et al., 2016; Struyf et al., 2017; Loponen & Gänzle, 

2018).  

To the authors knowledge, no previous study has investigated the effects of sourdough 

fermentation on FODMAPs in GF breads and the quality characteristics of low FODMAP GF 

sourdough bread. Therefore, this research was aimed to quantify FODMAPs in seven 

commercial GF breads (three conventional GF breads and four GF sourdough breads) produced 

by a local company in New Zealand, and to reduce the levels of FODMAPs in existing GF 

sourdough bread containing the highest levels of FODMAPs by reformulating and using retard 

proofing (fermentation at refrigeration temperature). The effects of sourdough fermentation on 

the FODMAP degradation and the characteristics of low FODMAP GF sourdough breads were 

studied with the following objectives. 

  



4 
 

Objectives: 

1. To quantify FODMAPs (including total fructans) in seven commercial GF breads (three 

conventional GF breads and four GF sourdough breads) produced by a local company 

in New Zealand using high-performance anion-exchange chromatography with pulse 

amperometric detector (HPAEC-PAD) and enzymatic assay; 

2. To reduce the levels of FODMAPs in existing GF sweet potato sourdough bread through 

reformulation and using retard proofing (fermentation at refrigeration temperature); 

3. To investigate the effects of sourdough fermentation on the microbiological and 

physico-chemical characteristics, and the FODMAPs of GF sourdough starter during 

propagation; 

4. To investigate the effects of different formulations and fermentation processes on the 

microbiological and physico-chemical characteristics, and the FODMAPs of bread 

dough samples (before and after proofing) and bread samples of GF sweet potato 

sourdough breads; And, 

5. To investigate the effects of different formulations and fermentation processes on the 

baking characteristics and sensory properties of GF sweet potato sourdough breads. 

  



5 
 

CHAPTER 2   LITERATURE REVIEW 

2.1 FODMAPs 

FODMAPs is an acronym that stands for fermentable oligosaccharides, disaccharides, 

monosaccharides, and polyols (Gibson et al., 2007). It is used to describe a group of fermentable 

and indigestible short-chain carbohydrates that are naturally present in foods in variable 

quantities, including excess fructose (apple, pear, corn syrup, etc.), lactose (milk, yoghurt, 

cheese, etc.), fructans and fructo-oligosaccharides (FOS) (wheat, rye, cereal, etc.), galacto-

oligosaccharides (GOS) (beans, lentils, chickpeas, etc.), and sugar polyols (apple, peach, 

artificial sweeteners, etc.) (Muir et al., 2009). In the last decade, this group of carbohydrates 

has gained more attention due to its potential risks in triggering GI symptoms in patients with 

Crohn’s disease and IBS (Gibson & Shepherd, 2005; Gibson & Shepherd, 2009). 

There is evidence linking FODMAPs and development of GI symptoms (Halmos et al., 2014; 

Shepherd et al., 2013; Muir et al, 2019). Typical GI symptoms such as luminal distention and 

abdominal pain are due to increased fluid in the lumen due to osmotic effects and visceral 

hypersensitivity which occurs when poorly absorbed FODMAPs pass through the small 

intestine (Barrett et al., 2010). Bacterial fermentation of FODMAPs in the colon produces 

hydrogen and methane gasses, which contribute to flatulence and bloating symptoms (Dugum, 

Barco & Garg, 2016). Increased fluid delivery to the bowel due to the osmotic effects of 

FODMAPs may result in a laxative effect and induce diarrhea (Gibson & Shepherd, 2009). 

Malabsorption of higher levels of FODMAPs can cause greater amounts of water to be 

delivered to the lumen and production of more gasses by bacterial fermentation. The level of 

malabsorption is dependent on the amounts of the FODMAPs ingested and the polymerisation 

of the saccharides (Gibson & Shepherd, 2012). 



6 
 

2.2 Types of FODMAPs 

Fructose is a hexose sugar that can be ingested into small intestine in three different forms, free 

monosaccharide, disaccharide (sucrose) and polymerised forms (fructans, fructo-

oligosaccharides and inulins) (Hayes, Fraher & Quigley, 2014). It is estimated that around 50 % 

of the general population affected by fructose malabsorption is unable to absorb 25 g of fructose 

(Gibson et al., 2007). Absorption of fructose is more rapid when equal or higher levels of 

glucose are present through co-transportation via the GLUT-2 glucose transporter (Rumessen 

& Gudmand-Høyer, 1988). However, if fructose is in excess of glucose, fructose malabsorption 

will occur due to the low capacity of absorption through the GLUT-5 fructose transporter 

(Putkonen, Yao & Gibson, 2013). Fructose malabsorption can cause intestinal distention and 

visceral hypersensitivity that can further induce and exacerbate GI symptoms (Barrett & Gibson, 

2007; Shepherd et al., 2008).  

Lactose is a disaccharide found in milk and other dairy products, consisting of glucose and 

galactose, which can potentially trigger GI symptoms such as diarrhea which is predominant in 

IBS (IBS-D) (Böhmer & Tuynman, 2001). The absorption of lactose occurs through enzymatic 

hydrolysis by lactase. Partial hydrolysis of lactose due to lack of the enzyme lactase in 

susceptible individuals can cause lactose malabsorption (Vernia et al., 1995). Gas production 

through bacterial fermentation of unabsorbed lactose in the colon can cause abdominal pain, 

bloating and diarrhea (Hayes, Fraher & Quigley, 2014).  

Fructans are oligo- and polysaccharides consisting of fructose molecules joined by β-(2->1) 

linkages with a D-glucosyl residue at the end. They are mainly found in grains and cereals such 

as wheat, rye and barley. Fructo-oligosaccharides (FOS) are short-chain fructans made up of 2-

9 fructose molecules, and inulins are longer-chain fructans normally containing more than ten 

fructose molecules (Shepherd & Gibson, 2006). Two major FOS commonly found in food 

products are kestose and nystose. Kestose is a trisaccharide, composed of two fructose 



7 
 

molecules and one glucose molecule. Nystose is a tetrasaccharide consisting of two fructose 

molecules linked by β-(2->1) bonds to the fructosyl moiety of sucrose (BeMiller, 2007).  

Galacto-oligosaccharides (GOS) usually contain 2-5 galactose molecules and one linked 

glucose molecule, and are primarily found in legumes, beans and some nuts. Raffinose and 

stachyose are the most common GOS from dietary sources. Raffinose is made up of one 

fructose, one glucose and one galactose unit linked together. Stachyose comprises raffinose 

with an additional galactose unit (Gibson & Shepherd, 2005). The human body does not have 

digestive enzymes to digest FOS or GOS, so they are not digested in the small intestine and 

reach the colon. They are fermented by colonic bacteria into gases and short-chain fatty acids 

(SCFAs), which may induce GI symptoms such as severe pain, diarrhea, and constipation in 

IBS patients (Hayes, Fraher & Quigley, 2014).  

Polyols including sorbitol, mannitol, maltitol and xylitol are poorly absorbed sugar-alcohols, 

due to their lack of specific transporter mechanisms. These sugar-alcohols are absorbed very 

slowly and incompletely through passive diffusion, as they are too large to pass through the 

pores in the small intestinal epithelial cell wall (Barrett & Gibson, 2007; Wilson & Hill, 2014). 

Unabsorbed polyols can lead to laxative effects in the colon and may cause bloating, flatulence 

and diarrhea (Khan et al., 2015).  

2.3 Dietary Strategies for the Management of GI-related Diseases 

2.3.1 Gluten-free diet  

Strict lifelong adherence to a gluten-free (GF) diet is the only available therapy for patients 

with GI-related diseases such as CD and NCGS (Vici et al., 2016). All foods made from grains 

containing gluten such as wheat, rye and barley must be eliminated from the diet (Catassi et al., 



8 
 

2007). Even ingestion of trace amounts of contaminating gluten can induce severe damage to 

the lining of the small intestine of CD patients (Catassi & Fasano, 2008).  

Strict omission of gluten from diet results in healing of the small bowel mucosa and remission 

of the inflammatory GI symptoms (Lanzini et al., 2009). Improved symptomatic and intestinal 

function as well as intestinal permeability have been observed for patients after being 

introduced to a GF-diet for two months (Cummins et al., 2001). However, the complete 

restoration of the small bowel mucosal needs at least a year or more after treatment with a GF-

diet (Tuire et al., 2012). GF-diets can also be used as a treatment for subjects who suffer from 

IBS and NCGS symptoms (Aziz et al., 2016; Niland & Cash, 2018; Volta Volta et al., 2019). 

2.3.2 Low FODMAP-diet 

The low FODMAP-diet was developed by a group of gastroenterology researchers at Monash 

University in Australia to manage IBS symptoms (Gibson et al., 2007). Dietary restrictions of 

fermentable short-chain carbohydrates effectively reduced the absorption of osmotically active 

short-chain carbohydrates in the small intestine, thus preventing the accumulation of luminal 

water and luminal gas production by colonic fermentation (Gibson & Shepherd, 2009). Strong 

evidence has supported the efficacy of a low FODMAP-diet for the management of GI-related 

diseases (e.g., IBS, fructose malabsorption) with positive effects in relieving overall GI 

symptoms (Shepherd & Gibson, 2006; Gearry et al., 2009; Staudacher et al., 2011; Halmos et 

al., 2014). The effect is dose-dependent; that is, highly associated with the amounts of 

FODMAPs ingested, especially fructose or fructans (Shepherd et al., 2008). 

The cut-off levels of different subgroups of FODMAPs and the main food sources (Table 1) 

provided a dietary guideline for IBS patients and adherence produced significant improvements 

in GI symptoms (Varney et al., 2017). A low FODMAP-diet should contain less than 0.5 g total 

FODMAPs per meal (Halmos et al., 2014). 



9 
 

Table 1 Cut-off levels of FODMAPs (per single serve) 

Individual 

FODMAPs 
Main Sources 

Grams per serve 

(Individual food) 

Fructose Apple, pear, watermelon, asparagus, honey, corn syrup < 0.15 g* 

Lactose Milk, yoghurt, cheese, ice cream < 1.0 g 

Fructans, FOS Wheat, rye, barley, garlic, onion < 0.2 g 

GOS Beans, legumes, lentils < 0.3 g 

Polyols Blackberry, peach, apricot, apple, sorbitol, mannitol, xylitol < 0.4 g 

*Fructose in excess of glucose = fructose - glucose; FOS = fructo-oligosaccharides; GOS = galacto-oligosaccharides 

(Varney et al., 2017). 

2.3.3 Combination of Gluten-free diet and low FODMAP-diet 

Methods have been developed to quantify and characterise the contents of FODMAPs in 

different types of foods to provide effective dietary guidelines for affected patients to manage 

GI symptoms (Biesiekierski et al., 2011; Prichard et al., 2016; Tuck et al., 2018). The results 

revealed that GF products (e.g., bread, pasta) generally have reduced amounts of fructans and 

oligosaccharides and low levels of FODMAPs compared to gluten-containing products (Muir 

& Gibson, 2013). This is probably due to the co-existence of gluten and fructans in grains and 

cereals (Muir et al., 2019).  

Greater symptomatic improvements can be achieved by a combination of a GF-diet and low 

FODMAP-diet for patients with functional GI diseases (Piacentino et al., 2014; Roncoroni et 

al., 2018; Usai-Satta et al., 2020). Roncoroni et al. (2019) found that nutritional intervention 

with a low FODMAP-diet for CD patients already on a GF-diet significantly reduced typical 

GI symptoms compared with patients who only followed a regular GF-diet. GF-diets containing 

high levels of FODMAPs can induce GI symptoms in treated CD patients (Roncoroni et al., 

2018). Therefore, a low FODMAP-diet can have beneficial effects for patients with GI-related 

diseases who are on a GF-diet but still present with persistent GI symptoms. 



10 
 

2.4 Gluten-free Bread 

Demand for GF products continues to increase because of improved diagnostic methods for 

CD and NCGS, and health concerns of consumers (Gallagher, 2008). However, due to the 

absence of the viscoelastic properties of gluten, GF breads are generally poor in overall quality 

with low crumb volume, rapid staling rate, and not well accepted by the majority of the 

consumers (Gallagher, Gormley & Arendt, 2003). Hence, GF formulations and technologies to 

improve bread quality, fortified ingredients or nutrients to enhance the nutritional values of 

bread, or addition of natural preservatives to increase the shelf-life of GF bread products are 

now available (Gallagher et al., 2004; Moroni, Dal Bello & Arendt, 2009; Arendt & Moroni, 

2013; Oshea et al., 2014; Nionelli & Rizzello, 2016). However, production of GF breads of 

good quality still remains the biggest challenge for bread manufacturers due to a lack of low-

cost alternatives and applications (Arendt & Moroni, 2013). 

2.4.1 Gluten 

Gluten is the major storage protein composite of wheat grain constituting approximately 80-85 

% of the total proteins in wheat (Arendt et al., 2008). Gluten proteins play a crucial role in 

forming the strong, cohesive dough capable of trapping gas and production of palatable bread 

products. They are found in the starchy endosperm of wheat grain and their function is to 

nourish the embryo by providing nitrogen and amino acids for germination (Van Der Borght et 

al., 2005; Wieser et al., 2014). When the grain matures, the gluten protein bodies become 

disrupted and coalesce to form a continuous matrix surrounding the starch granules (Shewry et 

al., 1995). The matrix contributes to the development of the continuous gluten network when 

wheat flour is mixed with water and kneaded mechanically.  

Gluten proteins are insoluble in water or dilute salt solutions and can be classified into two 

functional proteins, gliadins (wheat prolamins) and glutenins (wheat glutelins) (Lindsay 



11 
 

Lindsay & Skerritt, 1999). Gliadins are monomeric proteins comprising 40-50 % of total flour 

protein content, which contain single-chain polypeptides with intra-chain cysteine disulphide 

bonds resulting in the globular nature of gliadins (Cornell, 2003). The molecular weights (MW) 

of gliadins vary from 30,000 to 55,000, and they can be distinguished into four groups: α-, β-, 

γ-, and ω-gliadins based on their electrophoretic mobility at low pH (Khatkar et al., 2002). The 

amino acid composition of gliadins contains extremely high levels of glutamine (~ 35 %), high 

levels of proline (~ 20 %), low levels of charged amino acids (arginine, lysine, histidine, 

aspartic acid and glutamic acid), as well as low amounts of cysteine (~ 3 %) (Cornell, 2003). 

Upon hydration, gliadins impart the viscous component to the viscoelastic characteristics of 

gluten, contributing to the extensibility and viscosity of the dough (Pomeranz, 1988; Don et al., 

2003).  

Glutenins are heterogeneous polymeric proteins linked with inter- and intra-molecular 

disulphide bonds, making up 30-45 % of wheat flour proteins. Glutenins can form the elastic 

backbone of gluten with a high MW of between 50,000 to 10 million (Wieser, 2007). Two 

classes of glutenin subunits, high-molecular-weight glutenin subunits (HMW-GS; MW 65,000 

to 90,000) and low-molecular-weight glutenin subunits (LMW-GS; MW 30,000 to 60,000), are 

present in wheat when using reducing agents which break down the glutenin disulphide bonds 

(Goesaert et al., 2005). Glutenins have a similar amino acid composition to gliadins, comprising 

high glutamine (~ 30 %) in addition to proline (~ 13 %), and lower amounts of charged amino 

acids. Higher levels of glycine, but lower levels of isoleucine, phenylalanine, and valine have 

been reported in HMW-GS compared with LMW-GS (Cornell, 2003). When glutenins are 

hydrated, they are predominantly responsible for forming a continuous polymeric network 

structure, which provides elasticity, tenacity and strength (resistance to deformation) to the 

dough (MacRitchie, 1980). 



12 
 

2.4.2 The role of gluten in bread-making 

During dough mixing, wheat flour is mixed with water and mechanically kneaded to form a 

dough, which disrupts the discrete masses of gluten proteins resulting in a continuous cohesive 

and viscoelastic gluten network (Singh & MacRitchie, 2001). Gliadin proteins act as 

plasticisers and interact with glutenin’s polymeric network through hydrophobic and hydrogen 

bond linkages, providing plasticity and viscosity to the dough system (Van Der Borght et al., 

2005). The gluten network is capable of encapsulating gas or carbon dioxide (CO2) (produced 

during fermentation and oven-rise), starch granules and other filler materials such as bran, 

contributing to loaf volume and crumb structure of the breads (Arendt et al., 2008; Scanlon & 

Zghal, 2001).  

The physical characteristics of the dough (extensibility, mixing sensitivity, gas holding ability) 

are influenced by the relative proportions of gliadins and glutenins present in the dough. A 

higher glutenin content, imparting greater dough strength allows the dough to rise and improves 

the loaf volume (MacRitchie, 1987). In contrast, a higher proportion of gliadins, results in 

higher viscosity and an increased degree of softening of the dough, leading to poor performance 

during baking (Barak et al., 2014). The correct balance of extensibility and elasticity of the 

dough is necessary to produce bread of good quality (MacRitchie, 1987). Insufficient elasticity 

of the dough impairs gas retention, whereas excessive elasticity of the dough limits the dough 

expansion (Shewry et al., 1995).  

During baking, coagulation of gluten induced by heating causes denaturation of gliadins and 

glutenins (Wang, Jin & Xu, 2015). This dramatically changes the hydrophobicity of the protein 

surface, increases the cross-linking and polymerisation of gluten polymers, and interchange 

reactions of sulphydryl/disulphide bonds (Schofield et al., 1983; Lefebvre, Popineau & Cornec, 

1993; Guerrieri et al., 1996). This results in the typical porous, spongy and elastic crumb 



13 
 

structure of breads with gelatinised starch granules and enclosed fibre fragments (Durrenberger 

et al., 2001; Ortolan & Steel, 2017).  

2.4.3 The challenge of making GF bread 

Absence of gluten significantly impairs the quality of the breads, as GF dough is more similar 

to a batter than dough, which cannot retain gas produced during fermentation due to its lack of 

cohesiveness and elasticity (Arendt & Moroni, 2013). This results in high permeation of gas 

and failure to maintain the bread structure (Goesaert et al., 2005). Removal of gluten also 

impairs the water-holding capacity of the breads, leading to early crumbly structure and rapid 

staling. Thus, GF breads are generally Characterised with low specific volume, high crumb 

hardness, pale light crust, poor taste and flavour, and short shelf life (Gallagher, Gormley & 

Arendt, 2003; Gallagher et al., 2004; Hager et al., 2012; Matos & Rosell, 2012). Furthermore, 

as most GF breads are made with starch and are not fortified, they do not have adequate amount 

of essential nutrients, such as dietary fibre, minerals, folate, and vitamins B (Arendt et al., 2008), 

which contributes to nutritionally unbalanced diets of patients with CD or other gluten-related 

GI disorders who are on a strict GF-diet (Bardella et al., 2000; Thompson, 2000; Yazynina et 

al., 2008).  

To produce GF breads with good baking quality and sensory characteristics, alternative flours, 

ingredients or additives which mimic the functionalities of wheat flour for bread-making have 

been studied (Gallagher et al., 2004). In the last decades, promising improvements in the quality 

of GF breads have been achieved by utilisation of permitted flours and legumes (e.g., rice, 

maize, sorghum, oat flours; chickpea, soya, and lentil), incorporation of nutrient dense 

ingredients such as whole grain and pseudocereals (e.g., buckwheat, amaranth, and quinoa), 

addition of starches, hydrocolloids (e.g., carboxymethyl cellulose, guar gum, and xanthan gum), 

emulsifiers, shortenings (e.g., butter and vegetable oil), proteins (e.g., egg proteins and dairy 

proteins) or enzymes (e.g., transglutaminase and glucose oxidase), and use of novel 



14 
 

technologies (e.g., sourdough fermentation and high hydrostatic pressure) (Anton & Artfield, 

2008; Kohajdová, Karovičová & Juszczak, 2009; Moroni, Dal Bello & Arendt, 2009; Zannini 

et al., 2012; Oshea et al., 2014; Foschia et al., 2016). However, these alternatives inevitably 

increase the cost of GF bread production and the analysis of additives of the products. Thus, 

production of GF breads of superior quality and with good consumer acceptability still presents 

a challenge to bread manufacturers (Arendt & Moroni, 2013). 

2.5 Sourdough Bread 

The use of sourdough as a natural starter for bread leavening is considered the oldest form of 

biotechnological process (Röcken & Voysey, 1995; Chavan & Chavan, 2011). Sourdough is 

made from a mixture of flour (e.g., wheat, rye, rice, etc.), water, and possibly with other 

ingredients (e.g., salt) that are fermented by spontaneous LAB and yeasts introduced from the 

flour or baking environment (Corsetti & Settanni, 2007; Nionelli & Rizzello, 2016). During 

sourdough fermentation, LAB are mainly responsible for acidification through production of 

lactic and acetic acids, which confer the unique flavour and sour taste to the bread (De Vuyst 

& Neysens, 2005). Whereas yeasts primarily contribute to leavening through production of CO2 

from alcoholic fermentation, resulting in increased bread volume (Corsetti, 2013; De Vuyst et 

al., 2014). A general procedure for sourdough bread production is outlined in Figure 1.  



15 
 

 

Figure 1 General production steps of sourdough bread (Hansen & Schieberle, 2005) 

2.5.1 Classification of sourdough fermentation 

Sourdough fermentation can be classified into three types, type I, II, and III (De Vuyst & 

Neysens, 2005; Corsetti, 2013; Siepmann et al., 2018). Type I sourdough represents the 

traditional sourdough technique that involves refreshing starter culture by addition of fresh 

flour and water at ambient temperatures (< 30 °C) used for dough leavening, without addition 

of baker’s yeast Saccharomyces cerevisiae (S. cerevisiae). The repeated reinoculation process 

from a previous batch is known as backslopping, which keeps the sourdough microorganisms 

in a continuous state of growth with high metabolic activities (De Vuyst & Vancanneyt, 2007). 

At present, type I sourdough is still the most widely used method for traditional bread 



16 
 

production, because it improves the texture, flavour and shelf life of the bread, and also 

enhances the nutritional and functional properties of the bread products (Gobbetti et al., 2018).  

Type II, or industrial liquid sourdoughs are generally used as dough acidifiers during bread-

making to enrich the aroma and flavour of the breads, characterised by long fermentation 

periods (up to 5 days) with high concentrations of microbial inoculation produced in large-scale 

silos or tanks at a higher fermentation temperature (> 30 °C) to shorten the fermentation process 

(Decock & Cappelle, 2005). Addition of baker’s yeast is required for type II sourdoughs to 

leaven the final dough, as strong sourdough acidification may inhibit the growth of yeast 

(Corsetti & Settanni, 2007). 

Type III, or industrial dried sourdoughs are similar to type II sourdoughs with the exception 

that are dried or stabilised by pasteurisation after preparation. Yeasts are rarely found in this 

type of sourdough due to the high drying temperature, so the addition of baker’s yeast is also 

required. Type III sourdoughs are mainly used for industrial baking as their quality is more 

consistent compared to other types of sourdoughs and convenient for commercialisation, 

producing breads with more flavour properties and consistent quality (Chavan & Chavan, 2011).  

2.5.2 Sourdough microbiota 

The microbiota of sourdough is determined by endogenous and exogenous ecological factors 

(Vogelmann & Hertel, 2011). Endogenous factors are mainly associated with the microbial and 

chemical composition of the dough, including the original microbial ecology of the flour, 

endogenous enzyme activities, carbohydrates and nitrogen sources within the flour, and 

presence of growth inhibitors (Gobbetti et al., 2008). Exogenous factors are determined by the 

process parameters applied during fermentation, such as dough yield, fermentation time, 

fermentation temperature, oxygen level, salt level, the composition of the starter cultures, and 

the number of propagation steps (Hammes & Gänzle, 1998). If these factors are kept constant 



17 
 

for a long period, their interplay will create a distinct selection of different genera and species 

of LAB and yeasts that are derived from the flour and adapted to the environment (Gobbetti, 

1998). In order to achieve optimum activities and maintenance, the ratio of LAB to yeasts 

should be around 100:1, with higher cell densities of LAB (107 to 109 CFU/g) than yeasts (105 

to 107 CFU/g) (De Vuyst & Neysens, 2005). 

More than 60 species of LAB and over 25 species of yeasts have been previously isolated from 

sourdoughs made from different origins (Gobbetti et al., 2008; De Vuyst et al., 2014). The main 

species of LAB (more than 23 species) isolated from sourdoughs belong to the genus 

Lactobacillus, but species from the Leuconostoc, Pediococcus and Weissella are also frequently 

found in sourdoughs (De Vuyst & Neysens, 2005). The predominant species of LAB found in 

cereal and pseudocereal sourdoughs are L. sanfranciscensis, L. fermentum, L. plantarum, L. 

pontis, L. plaralimentarius, L. spicheri, L. fructivorans, L. helveticus, and L. delbrueckii (De 

Vuyst & Vancanneyt, 2007).  

The adaptability of yeasts within the sourdough ecosystem depends on the type of grain used, 

dough hydration, dough temperature, and the extent of acidification (Emerald et al., 2016). The 

majority of yeasts isolated from mature sourdough belong to the genera of Saccharomyces and 

Candida (Hammes & Gänzle, 1998). The typical yeasts found in sourdough include S. 

cerevisiae, S. exiguous, C. milleri, C. holmii, and Issatchenkia orientalis (Vernocchi et al., 

2004), as well as other species such as Debaryomyces hansenii, Torulaspora delbrueckii, 

Pichia anomala, P. membranifaciens, and P. saitoi (Gobbetti et al., 1994; Succi et al., 2003; 

Emerald et al., 2016). 

2.5.3 Metabolism of sourdough microorganisms 

Sourdough fermentation is a complex process based on the metabolic activities of sourdough 

microbial communities (LAB and yeasts), which directly or indirectly affect the organoleptic 



18 
 

and technological characteristics of the bread products (Gobbetti et al., 2008). Different species 

of LAB and yeasts utilise different carbohydrate and nitrogen substrates as their energy source 

for growth and metabolism, resulting in the production of different metabolites and aromatic 

compounds (Diowksz & Ambroziak, 2006). The performance of sourdough is also influenced 

by the interaction between LAB and yeasts (De Vuyst et al., 2014). Therefore, high sourdough 

acidification and production of full aroma can be achieved by using sourdough starters 

containing a mixture of selected strains of LAB and yeasts (Chavan & Chavan, 2011; Corsetti, 

2013; De vuyst, Van Kerrebroeck & Leroy, 2017).  

2.5.4 Carbohydrate metabolism by sourdough lactic acid bacteria 

Lactic acid bacteria are characterised by two main hexose monosaccharide fermentation 

pathways, which are known as homolactic and heterolactic pathways (Axelsson, 2004; Gänzle, 

Vermenulen & Vogel, 2007). Based on these two pathways, LAB have been divided into three 

categories according to their different capabilities to utilise sugars: homofermentative, 

heterofermentative and facultative heterofermentative (Axelsson, 2004). Homofermentative 

LAB (Lactococcus sp., Pediococcus sp., Streptococcus sp., and certain Lactobacillus sp.) 

ferment hexoses via the glycolytic Embden-Mayerhoff Parnas (EMP) pathway, to produce 

mainly lactic acid by anaerobic metabolism. Heterofermentative LAB (Leuconostoc sp., 

Weisella sp., Oenococcus sp., and some Lactobacillus sp.) metabolise hexoses using the 

phosphoketolase pathway (also known as the pentose phosphate pathway), produce lactic acid 

as well as significant amounts of CO2, acetic acid and/or ethanol as end-products due to the 

presence of additional substrates acting as electron-acceptors. Facultative heterofermentative 

LAB can ferment sugars through both pathways, predominantly the pentose phosphate pathway 

in the absence of fermentable carbon sources (Hutkins, 2006). Each metabolic category of LAB 

predominantly present in sourdough breads are shown in Table 2.  

  



19 
 

Table 2 Homofermentative, heterofermentative and facultative heterofermentative LAB in sourdough 

bread* 

*Source: De Vuyst and Neysens (2005); Zhou and Therdthai (2012). 

In homofermentative pathways (glycolysis or EMP pathway), homofermentative LAB convert 

one glucose molecule to two lactic acid molecules to release two molecules of ATP (Figure 2) 

(Aarnikunnas, 2006; Von Wright & Axelsson, 2011). Glycolysis starts from the 

phosphorylation of glucose to glucose-6-phosphate by glucokinase. Glucose-6-phospate is 

converted to fructose-6-phosphate by phosphoglucose isomerase through isomerisation. 

Fructose-6-phosphate is then phosphorylated by phosphofructokinase into fructose-1,6-

diphosphate, which further splits into glyceraldehyde-3-phosphate (GAP) and 

dihydroxyacetone phosphate (DHAP) (also converted into GAP) by the catalysation of 

fructose-1,6-diphosphate aldolase. Glyceraldehyde-3-phosphate is oxidised to pyruvate in two 

steps of substrate-level phosphorylation, then pyruvate is finally reduced to lactic acid by 

lactate dehydrogenase (LDH), coupled by the oxidation of NADH to NAD+ (Axelsson, 2004). 

Homofermentative LAB Heterofermentative LAB Facultative heterofermentative LAB 

L. amylovorus 

L. acidophilus 

L. delbrueckii 

L. farciminis 

L. mindensis 

L. johnsonii 

L. acidifarinae 

L. brevis 

L. fermentum 

L. frumenti 

L. reuteri 

L. rossiae 

L. sanfranciscensis 

L. zymae 

L. plantarum 

L. pentosus 

L. casei 

L. paralimentarius 

L. alimentarius 



20 
 

 

Figure 2 Homolactic fermentation (glycolysis, or EMP pathway) by homofermentative LAB 

(Aarnikunnas, 2006) 

Enzymes: 1. glucokinase; 2. fructose-1,6-diphosphate aldolase; 3. pyruvate kinase; 4. Lactate dehydrogenase. 

 



21 
 

In heterofermentative pathways (phosphoketolase pathway, or pentose phosphate pathway) 

(Figure 3), heterofermentative LAB also produce CO2, acetic acid or ethanol through 

conversion of glucose by glucose-6-phosphate dehydrogenase and 6-phosphogluconate 

dehydrogenase. The phosphoketolase pathway initiated from phosphorylation of glucose, then 

one glucose-6-phosphate molecule is dehydrogenated by the enzyme glucose-6-phosphate 

dehydrogenase to 6-phosphogluconate, which is further decarboxylated to ribulose-5-

phosphate and to produce CO2. Ribulose-5-phosphate is isomerised to xylulose-5-phosphate, 

then cleaved into GAP and acetyl-P by phosphoketolase. Subsequently, GAP is metabolised by 

LAB to produce lactate by homolactic fermentation, whereas acetyl-P is converted to acetate 

or dephosphorylated to acetyl coenzyme A (acetyl-CoA). Acetyl-CoA is further converted to 

acetaldehyde by acetaldehyde dehydrogenase, and finally reduced to ethanol by alcohol 

dehydrogenase (Von Wright & Axelsson, 2011). 

Most sourdough Lactobacillus spp. can ferment sucrose, maltose, hexoses, and pentoses, 

although some species show different preferences (Martínez-Anaya, 2003). For example, L. 

plantarum is fructose-negative and therefore it grows more rapidly in glucose and maltose than 

in fructose. Sucrose metabolism is based on the permease system of the cells and is initiated by 

the enzyme sucrose hydrolase to split sucrose into glucose and fructose, or in certain LAB 

related to the enzyme sucrose-6-phosphate hydrolase which cleaves sucrose-6-phosphate into 

fructose and glucose-6-phosphate, which can then enter the metabolic pathway (Thompson & 

Chassy, 1981). Metabolism of maltose is also mediated by specific enzymes, such as the 

enzyme maltose phosphorylase which enables L. sanfranciscensis to convert maltose to 

glucose-1-phosphate and glucose (Stolz et al., 1993). Metabolism of pentoses only occurs by 

heterolactic fermentation; pentoses are transported into the cells by specific permeases and 

form ribulose-5-phosphate or xylulose-5-phosphate by phosphorylases and isomerases, then 

enter the phosphoketolase pathway without production of CO2 with no dehydrogenation steps. 

Acetyl-P is only converted to acetate and forms ATP by acetate kinase, therefore ethanol is not 

produced (Kandler, 1983; Von Wright & Axelsson, 2011).  



22 
 

 

Figure 3 Heterolactic fermentation (phosphoketolase pathway) by heterofermentative LAB 

(Aarnikunnas, 2006) 

Enzymes: 1. 6-phosphogluconate dehydrogenase; 2. phosphoketolase; 3. Alcohol dehydrogenase. 



23 
 

2.5.5 Yeast carbohydrate metabolism 

The main carbohydrate substrates in flour that can be metabolised by yeast are sucrose, glucose, 

fructose, and maltose (De Vuyst et al., 2016). Invertase, an enzyme secreted by yeast cells, can 

rapidly break down sucrose into glucose and fructose during the initial dough mixing and the 

early stages of fermentation (Rodrigues, Ludovico & Leão, 2006). Yeast invertase is also 

capable of hydrolysing short-chain oligosaccharides (e.g., fructans, composed of fructose and 

glucose molecules) originating from flour (Bacon, 1954; Koch, Smith & Geddes, 1954). Most 

of the yeasts identified in typical sourdough breads (e.g., S. cerevisiae) are facultative-

fermentative yeasts, which are capable of both aerobic respiratory metabolism and anaerobic 

fermentative metabolism (Figure 4) (De Vuyst et al., 2016).  

Figure 4 Carbohydrate metabolism by yeast (García et al., 2016) 

TCA cycle = tricarboxylic acid cycle; OXPHOS = oxidative phosphorylation; Enzymes: Pdh = pyruvate 

dehydrogenase; Pdc = pyruvate decarboxylase; Ald = aldehyde dehydrogenase; Acs = acetyl-CoA synthetase; Adh 

= alcohol dehydrogenase.  



24 
 

The primary carbohydrate metabolism of yeast is glycolysis, where one molecule of glucose is 

converted into pyruvate, ATP and NADH (Pronk, Yde Steensma & Van Dijken, 1996). When 

oxygen is available, pyruvate produced from glycolysis is converted into acetyl-CoA through 

decarboxylation catalysed by pyruvate dehydrogenase (Kresze & Ronft, 1981). The acetyl-CoA 

is then oxidised by transferring the acetyl group into CO2 through the tricarboxylic acid (TCA) 

cycle and oxidative phosphorylation (Pronk, Yde Steensma & Van Dijken, 1996).  

In the absence of oxygen, pyruvate enters the alcoholic fermentation pathway and is 

decarboxylated by pyruvate decarboxylase to acetaldehyde, which then reduced into ethanol by 

alcohol dehydrogenase through oxidation of NADH released from glycolysis (Rodrigues, 

Ludovico & Leão, 2006). Acetyl-CoA is the major metabolite within the TCA cycle, and it can 

also be formed through oxidation of acetaldehyde that is produced from alcoholic fermentation 

to acetate catalysed by acetaldehyde dehydrogenase (García et al., 2016). Acetate can be 

converted into acetyl-CoA by acetyl-CoA synthetase (Pronk, Yde Steensma & Van Dijken, 

1996).  

When sufficient carbohydrate substrates and oxygen are present, alcoholic fermentation is the 

predominant metabolic pathway of yeast over respiration (De Deken, 1966). When 

carbohydrate substrates are exhausted, yeast switches alcoholic fermentation to full respiration, 

which slows the growth rate by aerobic respiration using ethanol (Heitmann, Zannini & Arendt, 

2018). CO2 and ethanol are the dominant metabolic products produced by yeast metabolism, 

which have the greatest impact on the quality characteristics of bread (Ali et al., 2012). The 

CO2 produced by yeast leads to dough leavening and contributes to bread volume, and the 

ethanol results in a decrease in extensibility and extensional viscosity of dough and 

subsequently evaporates during baking (Horstmann et al., 2018).  



25 
 

2.6 Characterisation of Sourdough Parameters 

The composition of sourdough microbiota determines metabolite production and sourdough 

characteristics, which are reflected in the quality of bread product (Figure 5). The performance 

of sourdough is influenced by a series of factors, such as acidification properties of LAB 

(production of lactic and acetic acid), microbial composition of the starter cultures (species and 

number of LAB and yeast, ratio, performances), and the interactive effects determined by the 

process conditions (flour type, fermentation time, temperature, dough yield, oxygen, number 

of refreshments) (Hansen, 2004; Arendt et al., 2007; Chavan & Chavan, 2011). 

Figure 5 Factors influencing the composition and metabolite production of sourdough microbiota, 

characteristics of sourdough, and product quality (Hammes & Gänzle, 1998; De Vuyst et al., 2017) 

 

 

 

Endogenous factors  

Carbohydrates 

Nitrogen sources 

Minerals 

Lipids, free fatty acids 

Enzymatic activities 

(e.g. amylases, proteases) 

 

Flour, raw materials, 

Environment 

 

 

 

 

Exogenous factors 

Fermentation time 

Temperature 

Dough yield 

Oxygen (redox potential) 

Number of refreshments 

Process 

conditions 

Physico-chemical characteristics of 

sourdough 

pH, total titratable acidity, 

fermentation quotient 

Substrates  

Maltose, sucrose, glucose, fructose, 

free amino acids... 

Product quality 

Volume 

Flavour 

Texture 

Shelf-life 

Nutritional value 

Lactic acid bacteria 

Microbial 

composition, ratio, 

performances 

Yeasts Contaminating 

microbiota 

Metabolites 

Lactic acid, acetic acid, carbon dioxide 

ethanol, aromatic compounds… 



26 
 

2.6.1 pH and total titratable acid (TTA) 

The pH and total titratable acid (TTA) are important sourdough parameters, which can be used 

as indices for evaluating the development of sourdough fermentation. The pH of typical 

sourdoughs made from wheat and rye is in the range of 3.5-3.8 (Hansen, 2004). In contrast the 

TTA for sourdoughs varies between 8-26 depending on the type of flours used for bread 

production, as the amount of carbohydrate substrates are different. For example, sourdoughs 

made with low extraction flour have a lower TTA (8-11), sourdoughs made with whole meal 

flour have a higher TTA (16-22), and rye sourdoughs generally have a TTA of 15-26 (Lund, 

Hansen & Lewis, 1989; Brümmer & Lorenz, 1991; Hansen & Hansen, 1994; Spicher & Stephan, 

1999). During sourdough fermentation, pH decreases and the TTA of sourdough increases as 

the amount of organic acids increase (Hansen, 2004). The decrease in pH is mainly due to the 

lactic acid produced by sourdough LAB (Leenhardt et al., 2005). The acetic acid generated 

during sourdough fermentation only slightly impacts on the pH of sourdough but affects the 

sensory acidity and structure formation of the final bread (Valjakka et al., 2003; Corsetti & 

Settanni, 2007). 

2.6.2 Fermentation quotient (FQ) 

The fermentation quotient (FQ), which is defined as the molar ratio of lactic acid and acetic 

acid produced during fermentation, indicates the metabolite profile of LAB that are mainly 

determined by the composition of the starter cultures (Corsetti, 2013). Sourdoughs with 

obligate homofermentative and facultative heterofermentative LAB as predominant species 

produce relatively lower levels of acetic acid, resulting in a higher FQ (Loenner & Ahrne, 1995). 

Endogenous (e.g., flour type, substrate concentration, etc.) and exogenous factors (e.g., 

fermentation temperature, dough yield, etc.) also affect the FQ (Corsetti, 2013). The FQ 

decreases with decreased dough yield and fermentation temperature, as these factors influence 

the acidification rate of the sourdough (Spicher & Rabe, 1980; Spicher & Stephan, 1999). The 



27 
 

lower the dough yield or temperature, the slower the acidification, resulting in lower levels of 

lactic acid and more acetic acid being produced (Chavan & Chavan, 2011). The correct FQ 

value is very important for the flavour development of the bread and an FQ in the range of 4-9 

is preferred as the bread will have a mild acid taste and flavour, whereas bread with a low FQ 

will have a strong acid flavour, which is not considered desirable for most bread products 

(Hansen, 2006). 

2.6.3 Flour type 

Flour type markedly influences the progress and outcome of sourdough acidification, as the 

production of desirable metabolites by sourdough LAB and yeasts depend on the metabolisable 

substrates, nutrients and the extraction rate of the initial flours (Chavan & Chavan, 2011). 

Higher amounts of acids are produced in wholemeal flour sourdough than low extraction flour 

(Hansen, 2004), as wholemeal flour has higher α-amylase activity especially in the bran fraction 

that hydrolyses starch into maltose, which is then converted to glucose providing more 

carbohydrate substrates for LAB metabolism (Martínez-Anaya, 2003). The microbial 

composition and diversity of the sourdough microbiota is influenced by the amount of 

substrates that can be utilised, which varies with the type of cereal (Hammes & Gänzle, 1998). 

Endogenous enzymes within the flour, such as α-amylase and peptidase, also influence the 

growth of sourdough microbiota as they play an important role in the production of fermentable 

carbohydrates and free amino acids for microbial metabolism (Hansen, 2006; De vuyst et al., 

2017). 

2.6.4 Dough yield 

The dough yield (DY) is the proportion between flour and water used for baking. Dough yield 

= (flour weight + water weight) × 100 / flour weight (Chavan & Chavan, 2011). The DY 

indicates the consistency of sourdough and the amount of water added in the dough formula 



28 
 

(Gianotti et al., 1997). The DY is highly correlated with the sourdough flavour profile 

(Martínez-Anaya, de Barber & Esteve, 1994), with a lower DY having a firmer texture and 

producing more acetic acid and less lactic acid (Chavan & Chavan, 2011). The DY also 

influences the acidification rate of the sourdough, with a higher DY having a faster acidification 

rate, because of better diffusion of the organic acids produced during sourdough fermentation 

(Spicher & Stephan, 1999).  

2.6.5 Fermentation time and temperature 

Typical sourdough fermentation time is around 8-24 hours at 25-35 ℃ (Hansen, 2004; Corsetti, 

2013). The temperature of sourdough fermentation affects the propagation of LAB and yeasts 

and the acidification rate, as the optimum temperature for microbial growth and acidification 

is different (Hansen, 2006), being around 30-35 ℃ for LAB (Spicher & Stephan, 1999), and 

between 26-32 ℃ for yeast (Diowksz & Ambroziak, 2006). Sourdough fermentation at higher 

temperatures (28-35 ℃) accelerates the activities of sourdough LAB, producing more lactic 

acid than acetic acid, but inhibits the development of yeast. Therefore, the final bread is 

characterised by a relatively mild acidity flavour and lower volume (Corsetti, 2013; De vuyst 

et al., 2017). However, prolonged fermentation times at low temperatures between 20-27 ℃ is 

recommended as this will promote the metabolic activities of yeasts as well as maintain the 

development of LAB, thus not only producing CO2 for rising the dough but also generate 

aromatic precursors for flavour development, resulting in bread with increased volume and 

flavour (Poitrenaud, 2003). While the amount of lactic acid produced during sourdough 

increases with increasing temperature, however, the influence of temperature on the production 

of acetic acid is negligible (Hansen, Lund & Lewis, 1989).  



29 
 

2.7 Sourdough in GF Bread-making 

The exploitation of sourdough for improving the quality of wheat-containing breads is well-

established and applied in the baking industry (Clarke & Arendt, 2005; Chavan & Chavan, 

2011). Similarly, sourdough microorganisms possess unique metabolic and enzymatic 

activities capable of improving the quality of GF breads (Arendt et al., 2007). They can produce 

exopolysaccharides (EPS) which improve bread structure, generate aroma compounds for 

bread flavour enhancement, hydrolyse starch to retard bread staling, produce anti-fungal 

compounds to improve the shelf life of breads, and generate bioactive compounds that enhance 

nutritional value and confer health benefits to breads (Gobbetti, 1998; Corsetti & Settanni, 2007; 

De Vuyst & Vancanneyt, 2007; Moore et al., 2007; Schober et al., 2007; Poutanen, Flander & 

Katina, 2009).  

2.7.1 Production of exopolysaccharides (EPS) 

LAB produce high molecular weight polymer EPS with physicochemical characteristics similar 

to commercial hydrocolloids that are externally added into bread formulations (Arendt & 

Moroni, 2013). EPS can be classified into two types based on their main-chain polymer 

composition: homopolysaccharides (HoPS) called glucans and fructans that have a single 

monosaccharide (glucose or fructose), and heteropolysaccharides (HePS) that consist of more 

than one type of monosaccharide (De Vuyst & Degeest, 1999). HePS are formed in very low 

amounts with minor effects in cereal and bakery products, therefore they are the dominant EPS 

useful in bread-making (Moroni, Dal Bello & Arendt, 2009). HoPS are synthesised by the 

action of extracellular enzymes glucansucrases or fructansucrase produced by sourdough LAB 

(Lacaze, Wick & Cappelle, 2007). 

Application of sourdough fermentation with HoPS-producing LAB strains can be used to 

produce GF breads with improved structural properties (Waldherr & Vogel, 2009). Dough 



30 
 

acidification enhances the swelling of the polysaccharides, which mimic the structure-forming 

function of gluten network (Galle, 2013). The EPS produced by LAB during sourdough 

fermentation are capable of binding water and retaining gas, thereby increasing the specific 

volume of bread and inducing softness to the crumb (Moore, Dal Bello & Arendt, 2008). 

Sourdough fermented with EPS-producing Weissella strains can be used as hydrocolloid 

replacers to improve the rheology of GF dough (Galle et al., 2010). These strains also produce 

small amounts of acetate, which can balance the negative effects of acidification and produce 

breads with favorable flavours and improved bread texture (Schwab et al., 2008). This is due 

to dextran produced by Weissella strains, which is capable of binding water and influencing the 

firmness of the bread (Wolter et al., 2014). The isomaltooligosaccharides and dextran produced 

by Weissella strains may be retained in the bread and provide additional nutritional value to the 

GF breads, by increasing the levels of prebiotic GOS (Schwab et al., 2008).  

2.7.2 Production of aroma compounds 

The highly appreciated flavour of sourdough breads is mainly attributed to the microbial and 

enzymatic activities of the yeast and LAB that convert carbohydrates, amino acids, and lipids 

into volatile compounds during fermentation (Mugula, Sorhaug & Stepaniak, 2003; Edema & 

Sanni, 2008; Moroni, Dal Bello & Arendt, 2009; Arendt & Moroni, 2013). Sourdough 

fermentation and lipid oxidation produce volatile metabolites such as acids, alcohols, esters, 

ketones and aldehydes responsible for the typical bread crumb odour. Whereas other flavour 

compounds such as pyrazines, pyridines, pyrroles, furan, acetaldehyde, generated during 

baking and caused by the Maillard reaction, caramelisation of sugars, and thermal degradation 

reactions, are particularly important for the crust odour of bread (Pico, Bernal & Gomez, 2015; 

Pétel, Onno & Prost, 2017). During sourdough fermentation, LAB-induced acidification creates 

favorable acidic conditions for endogenous protease activation that degrade the proteins in 

flours (Thiele, Gänzle & Vogel, 2002). The peptides liberated by proteolysis are further 



31 
 

hydrolysed into free amino acids through the activities of intracellular peptidases produced by 

LAB (Gobbetti et al., 2005; Gänzle et al., 2008). The large amounts of free amino acids 

produced by proteolysis can either contribute to flavour compounds directly or undergo further 

enzymatic catabolism or thermal degradation during baking, resulting in the production of 

volatile compounds (Nionelli & Rizzello, 2016).  

The flavour development of sourdough bread is influenced by the microbial composition 

(Gobbetti, 1998). Homofermentative and heterofermentative LAB can be differentiated based 

on the non-volatile flavour compounds produced during fermentation. Both strains can 

metabolise hexoses, but homofermentative LAB produce lactic acid, whereas 

heterofermentative LAB produce lactic and acetic acids, CO2 and ethanol (Hansen & Schieberle, 

2005). With regards to the volatile metabolites produced during fermentation, 

homofermentative strains mainly produce diacetyl, hexanal and acetaldehyde, while 

heterofermentative strains synthesise aldehydes, ethyl acetate, and hexyl acetate (Damiani et 

al., 1996). The fermentation quotient is the most important factor affecting the aroma of 

sourdough breads (Corsetti & Settanni, 2007). The addition of S. cerevisiae with sourdough 

cultures containing L. sanfranciscensis, L. plantarum, L. sanfrancisco or L. delbrueckii can 

modify the volatile profiles of breads and produce more yeast-derived fermentation products, 

such as ethanol, 2- and 3-methyl-1-butanol and methylpropanol (Gobbetti, 1995a; Hansen & 

Hansen, 1996).  

The types of flour used for GF bread-making affect the aromatic flavour of the final breads 

(Gobbetti et al., 1995), and along with starches (teff, rice, buckwheat, amaranth, quinoa, and 

corn starch) result in different aroma profiles (Pico et al., 2017). Higher levels of nonanal and 

2,4-decadienal are present in rice and teff breads due to ethyl hexanoate, ethyl nonanoate, 

aldehydes as well as alcohols produced by fatty acid oxidation. Quinoa and amaranth breads 

are characterised by higher levels of aldehydes and alcohols (Pico et al., 2017).  



32 
 

Processing parameters, such as fermentation temperature and time also influence the 

development of volatile compounds (Hansen & Schieberle, 2005). The production of lactic acid 

increases with increased fermentation temperature, thereby affecting the fermentation quotient, 

and volatile profiles. The production of ethanol, 1-propanol, 2-methyl-1-propanol, and 3-

methyl-1-propanol markedly increase when the fermentation temperature rises 25 to 30 °C, 

indicating that yeast fermentation is more favorable at a slightly higher temperature of around 

30 °C (Gobbetti et al., 1995). In addition, production of sufficient volatile compounds requires 

a longer fermentation time (12-24 h), whereas with short fermentation periods (under 3 h), iso-

alcohols produced by yeast fermentation account for the dominant volatile.  

2.7.3 Control of staling in GF bread 

Bread staling involves changes that occur in the crumb and crust of the bread after baking 

including changes to crumb firmness, loss of flavour, loss of bread freshness and crust crispness 

(D'appolonia & Morad, 1981). The cause of bread staling is not a single effect and has been 

attributed to a series of physico-chemical events, including amylopectin retrogradation, 

moisture migration and redistribution, and interactions between protein and starch (Gray & 

Bemiller, 2003). GF breads stale quickly, as most are made from starch-based ingredients, and 

migration of moisture is easier in the absence of a gluten network (Gallagher et al., 2003). 

During cooling and storage, the amylose and amylopectin in bread realign themselves to form 

a semi-crystalline structure. Retrogradation of starch causes water to be expelled from the bread 

and the crumb becomes firm and stale, which is unacceptable to the consumers (Goesaert et al., 

2009). Amylases are normally used in bread-making to reduce staling rate of bread and 

contribute to the production of dextrins that can interfere with starch retrogradation, thus 

prevent bread firmness (Schultz et al., 1952; Zobel & Senti, 1959). 

Incorporation of sourdough in GF bread-making can retard the firming of bread and reduce the 

rate of staling (Corsetti et al., 1998a; Arendt et al., 2007; Moore et al., 2007). Production of 



33 
 

organic acids during sourdough fermentation accelerates the amylase and protease activities of 

the flour changing the starch and protein fractions, thus increasing bread volume and softness 

(Barber et al., 1992; Dal Bello et al., 2007; Tamani et al., 2013; Cappa et al., 2016). Additionally, 

proteolysis has a synergetic effect, increasing α-amylase activity by liberating water from the 

protein network, therefore reducing bread firmness and dryness (Schwimmer, 1981). 

2.7.4 Improvement of shelf-life 

Mould growth represents the major cause of bread spoilage which results in serious economic 

losses for the manufacturer as well as health concerns for the consumers due to the possibility 

of mycotoxin contamination (Legan, 1993). The development of mould is caused by post-

processing contamination, from dust and mould spores distributed in the environment. 

Sourdough fermentation is known to prolong the shelf life of bakery products because of the 

production of anti-mould and anti-fungal substances by sourdough LAB. Sourdough-associated 

strains possess various anti-mould activities, mainly attributed to the presence of obligately 

heterofermentative Lactobacillus spp. (Corsetti et al., 1998b). The inhibitory substances 

produced by heterofermentative LAB during sourdough fermentation such as organic acids, 

ethanol, hydro peroxide, CO2, diacetyl, and other substances (e.g., bacteriocins and antibiotic) 

can prevent the growth of spoilage mould (Röcken, 1996; Messens & De Vuyst, 2002; De 

Vuyst & Leroy, 2007). 

The organic acids (acetic, caproic, formic, butyric, propionic, and n-valeric acids) produced by 

L. sanfrancisco can inhibit most common types of spoilage fungi Penicillium, Fusarium, 

Aspergillus and Monilia present in spoiled breads (Corsetti et al., 1998b). Strains of L. 

plantarum and L. amylovorous can produce antifungal compounds such as 4-

hydroxyphenyllactic, 3-phenyllactic, hydroferulic and phloretic acids that act against the 

growth of several species of Fusarium, Penicillium, and Aspergillus (Lavermicocca et al., 2000; 

Lavermicocca et al., 2003; Dal Bello et al., 2007; Ryan et al., 2009; Axel et al., 2015). Strains 



34 
 

of L. reuteri can produce antibiotic reutericyclin which inhibit gram-positive LAB and yeast 

by releasing reuterin (3-hydroxypropionaldehyde), a compound with broad anti-microbial 

activity (Gänzle et al., 2000; Gänzle, 2004). Sourdough fermentation can prevent rope spoilage 

in bread induced by Bacillus spp. (Katina et al., 2002; Valerio et al., 2008). The production of 

organic acids and other effective anti-bacterial compounds by LAB strains L. plantarum and P. 

pentosaceus are capable of inhibiting the germination of Bacillus spores (Pepe et al., 2003). 

Application of sourdough provides potential for natural biopreservative ingredients that can 

replace the use of additives in the production of GF breads, thus prolonging shelf life and 

improving quality (Garofalo et al., 2012). The addition of sourdough containing anti-fungal 

strain L. plantarum in GF breads significantly retarded the growth of pathogenic fungi 

Fusarium culmorum for up to 3 days (Moore, Dal Bello & Arendt, 2008).  

2.7.5 Improvement of nutritional value and health benefits 

Sourdough has been used to leaven GF breads made from amaranth, buckwheat, maize, millet, 

quinoa, and rice and it has gained popularity due to its effectiveness in improving the nutritional 

value and health benefits of GF breads (Moroni, Dal Bello & Arendt, 2008; Vogelmann et al., 

2009; Nionelli & Rizzello, 2016). Sourdough fermentation does this by improving the 

bioavailability of minerals, regulating the levels of bioactive compounds, and reducing the 

digestibility of starch (Poutanen, Flander & Katina, 2009).  

Cereal foods, especially bread, provide a good source of minerals, such as magnesium, calcium, 

zinc, iron, and phosphorus that are essential for human health. However, the absorption of 

minerals may be limited by the presence of anti-nutritional phytic acid in cereal grains. Phytic 

acid has a strong chelating capacity, which interferes with mineral absorption through forming 

insoluble phytate complexes with dietary cations (Bohn, Meyer & Rasmussen, 2008). Phytases 

are enzymes that can dephosphorylate phytate into lower inositol phosphate esters and free 



35 
 

inorganic phosphorus. Sourdough fermentation produces a favorable acidic environment 

(optimal pH for phytase activity is 4.5) that accelerates the activation of endogenous phytases 

in cereal flours (Fretzdorff & Brummer, 1992). Slight acidification (pH decrease to 5.5) induced 

by sourdo