Effect of Sourdough Fermentation Parameters On Bread Properties
Effect of Sourdough Fermentation Parameters On Bread Properties
Effect of Sourdough Fermentation Parameters On Bread Properties
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12-2016
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EFFECT OF SOURDOUGH FERMENTATION PARAMETERS
ON BREAD PROPERTIES
A Thesis
Presented to
the Graduate School of
Clemson University
In Partial Fulfillment
of the Requirements for the Degree
Master of Science
Food, Nutrition, & Packaging Sciences
by
Grace W. Couch
December 2016
Accepted by:
Paul Dawson
Michelle Parisi
Julie Northcutt
ABSTRACT
Sourdough is the oldest form of leavening which many believe was invented by the
Egyptians. Bread leavened with a sourdough culture relies on the metabolism of naturally
occurring lactic acid bacteria and wild yeasts. Historically there were many ties between
beer brewing and bread baking. In the fourteen and fifteen hundreds, it was discovered
that brewers yeast could also be used to leaven bread. Up until the invention of
commercial yeast in the nineteenth century, sourdough cultures and brewers yeast where
the only bread leavening methods. By 1910, traditional sourdough was much less
common because bread made with commercial yeast was much faster and easier, and
produced a more consistent product.1 The positive qualities of sourdough bread were
Phytic acid makes up about 1% of wheat and rye flours, and reduces the bioavailability of
calcium, magnesium, and iron by forming complexes with the divalent cations. Phytic
acid also inhibits enzymes in the digestive system needed to breakdown starch and
protein.2 This explains why some people experience discomfort from eating whole grain
wheat products. Sourdough bacteria breakdown phytic acid and “predigest” the grain
during the proofing process which releases easy to digest micronutrients.3 Specific
sourdough lactic acid bacteria breaks down sucrose to form exopolysaccharides that
contributes to bread volume, texture, and dietary fiber content. This increase in fiber
slows the digestion of the sourdough bread and does not cause rapid blood sugar spikes
ii
The objective of this study was to gain a better understanding of how fermentation time
and temperature affect sourdough production and give insight to why it is sometimes
more acceptable than non-fermented breads to the human digestive system. Three
identical batches of sourdough bread, 9 samples per batch, were produced and analyzed.
Samples 26-2, 26-4, 26-8, and 26-12 were fermented at 26°C and samples 4-14, 4-26, 4-
38, and 4-50 were fermented at 4°C to observe the affect of temperature on fermentation.
Bread samples were analyzed for moisture, loaf height, and protein content, and parallel
dough samples where analyzed for volatiles. This experiment shows evidence of protein
REFERENCES
[2] Vaintraub, I. A. & Bulmaga, V. P. (1991). Effect of phytate on the in vitro activity of
digestive proteinases. Journal of Agricultural and Food Chemistry 39 (5), 859-861 DOI:
10.1021/jf00005a008
iii
ACKNOWLEDGMENTS
This research would not have been possible without the aid of several individuals.
I would like to thank my advisor, Dr. Paul Dawson for his support, motivation, and
essential guidance. I would like to give my utmost gratitude to Dr. Inyee Han for her
endless encouragement and knowledge throughout my research project. I would also like
to thank my committee members, Dr. Julie Northcutt and Dr. Michelle Parisi for their
iv
TABLE OF CONTENTS
Page
ABSTRACT.....................................................................................................................ii
DEDICATION................................................................................................................iv
ACKNOWLEDGMENTS ............................................................................................... v
LIST OF TABLES..........................................................................................................ix
Introduction……..............................................................………………..…1
History of Sourdough.....................................................................................2
Volatile Analysis..........................................................................................10
Conclusion ...................................................................................................11
REFERENCES ............................................................................................12
Abstract ........................................................................................................14
v
Table of Contents (Continued) Page
1. Introduction.............................................................................................. 15
3. Analysis Methods.....................................................................................21
5. Conclusion ............................................................................................... 32
vi
Table of Contents (Continued) Page
5. Conclusion ...............................................................................................32
REFERENCES ............................................................................................35
vii
LIST OF TABLES
Table Page
Table 9. Headspace gas average volatile percentages for room temperature (70°F)
and refrigerated temperature (40°F) dough samples ...................................43
viii
LIST OF FIGURES
Figure Page
ix
CHAPTER ONE
INTRODUCTION
In recent years, the identification of individuals with celiac disease has been increasingly
more prevalent. Celiac disease is defined as an autoimmune disease of the small intestine
that is triggered by the ingestions of gluten proteins from wheat, barley, and rye. When
celiac patients ingest gluten proteins their immune cells, T and B, produce antibodies that
attack the villi in the small intestine and cause inflammation and damage. This causes
inability of the villi to absorb nutrients properly (Darewicz et al., 2008). Some of the
common symptoms of this disease include abdominal pain, diarrhea, fatigue, headaches,
and irritability (Silvester et al., 2016). Today the only proven cure for this disease is to
simply avoid foods containing gluten. If celiac patients continue to consume gluten they
are at risk of serious health problems like anemia (iron deficiency), early onset
Some celiac disease individuals have discovered they have no negative reactions after
consuming traditional wheat sourdough bread (Cagno et al., 2008) but very few studies
have looked into the reason behind this tolerance. Initially, the assumptions focused on
the gluten protein reduction occurring in the culture by microbial and enzymatic
reactions, but preliminary studies suggest that active probiotics may also be assisting in
1
the ease of digestion (Cagno et al., 2008)(Caputo et al., 2010). This chapter will review
HISTORY OF SOURDOUGH
Sourdough cultures are the oldest form of leavening, aging back to more than 5,000 years
ago. Initially, sourdough batters were a simple mixture of flour and water that were
fermented, and were then used as leavening to make bread rise. It was observed that this
process could be expedited by keeping the starter culture alive by continuously “feeding
it,” adding flour and water, and only taking a portion of it when it was time to make
bread. The starter, also known as a levain, is a mixture of flour, water, and naturally
occurring bacteria and yeast. The starter may be kept indefinitely if it is properly stored
and fed. Only a small portion of the starter is used to make bread by mixing with a large
portion of flour and a little bit of water. Before the mechanism was understood, the
unknown gas producers were called “seeds”. In the mid-1800’s Louis Pasteur discovered
the process was a result of living microorganisms feeding off the slurry. This discovery
Sourdough culture production requires only two ingredients: flour and water. Cultures are
not limited to wheat flour, as other types of flours used include rice, rye, spelt, barley,
and amaranth (Cagno et al., 2008). Part of the culture includes a third component of wild
yeasts and lactic acid bacteria (LAB). These microorganisms are included in the culture
2
from the environment and different species of yeast and bacteria are introduced in the
culture depending on what region of the world it is produced and what types of flours it
contains. Every region contains its own unique cocktail of bacteria so no two sourdough
cultures are the same. This results in regional flavor profiles (Gadsby, 2003). Traditional
San Francisco Sourdough is famous for its unique bacterial and flavor profile. Specific
maltose which is to their advantage because of the lack of competition with yeast for
glucose (Salim-ur-Rehman et al., 2006). A sensory study done by the Swiss Society of
Food Science and Technology found that sourdough bread made with heterofermentative
Lb. sanfranciscensis had a pleasant, mild, sour taste and odor, whereas homofermentative
Lb. plantarum fermented bread had an unpleasant metallic sour taste (Katina et al., 2006).
Flavor volatiles that have been correlated to with pleasant flavors of wheat bread crumb
In the artisanal baking world, sourdough cultures are kept alive for decades and passed
down through generations like a family memento. One of the oldest sourdough cultures
on record is 126 years old, owned by Lucille Clarke Dumbrill of Newcastle, Wyoming.
She got the starter from her mother, who got it from one of her husband’s students at the
3
University of Wyoming. The culture was traced back to a sheepherder’s wagon near
Kaycee, Wyoming in1889 (Matray, 2011). Like many owners of sourdough culture,
Lucille stores her culture in the refrigerator where is remains in a dormant state. To make
a batch of sourdough bread, a portion of the culture is removed from the fridge and
Food for the bacteria and yeasts consists of an even mixture, by weight, of flour and
water that is incorporated into the culture. To keep a sourdough culture active at room
on the flour type and maturity of the culture. When making a new wheat culture for
example, it is suggested that you feed the culture every 12 hours for at least the first three
days. When activating a dormant culture, the process could take anywhere from 1 to 3
days. Often a portion of the fed culture is discarded after every couple feedings because
otherwise there would be an excessive amount of culture. Often cultures are produced in
large mason jars covered lightly with a mesh-like fabric or cheesecloth when it is being
respire.
Once the culture is active and mature, at least two weeks old, it is ready to produce
sourdough bread. This requires about three parts flour, two parts sourdough culture, and
one part water by volume. After mixing and kneading, the dough is allowed to proof
4
punching down after four to twelve hours and then the dough is allowed to proof a second
time. The dough is then baked at around 400°F until the internal temperature reaches
Lactic acid bacteria (LAB) are the primary bacteria utilized to produce sourdough bread.
Other types of bacteria may be present in the bread but are not critical to the process.
Wild yeast works synergistically with LAB because yeast do not metabolize maltose and
compete with the bacteria like baker’s yeast would. Many untraditional sourdough breads
on the market today utilize baker’s yeast to expedite the proofing process. When the
artificial yeast is added they compete with the LAB for nutrients and some nutritional
benefits of a traditional sourdough are diminished (Lhomme et al., 2014). Lactic acid
pentosus, and Pediococcus pentosaceus. These species of bacteria are responsible for the
fermentation, while heterofermentative LAB produce lactic acid, carbon dioxide, and
bacteria use the glycolitic pathway to produce lactic acid, but are able to use the
heterolactic fermentation process when there is limited glucose (Dairy Foods Science
5
Notes, Cornell University). Common wild yeast often captured in the culture include
2008). A study done by the Department of Agricultural and Forest Science at the
Università degli Studi di Palermo in Italy looked at the affects of individual lactic acid
bacteria on the bread quality and characteristics. When produced with non-sterile flour,
bread made with Lb. sanfranciscensis showed the greatest loaf height. In the experiment
with sterile flour, Ln. citreum and W. cibaria consistently produced bread with higher
The fermentation process in sourdough leads to the activation of naturally occurring grain
enzymes. Studies have shown this activity increases nutrient bioavailability that may also
facilitate the ease of digestion of the bread. Starch degradation is the main source of
in the early stages of fermentation. Once the pH is reduced to 4.5 or below, the
maltogenic amylases are inhibited, but glucose from starch and maltodextrins are still
released by glucoamylase activity. A specific sourdough lactic acid bacteria breaks down
sucrose to form exopolysaccharides that contribute to bread volume, texture, and dietary
6
Proteolysis is the breakdown of proteins to peptides. In sourdough, this process is
dependent on metabolic activity of bacteria that lowers the pH, which leads to the
activation of endogenous proteases, the primary source of protein metabolism. The acidic
proteins and enzymatic degradation of gluten. When the pH of the culture drops due to
fermentation there is an accumulation of low molecular weight thiols, which increase the
them more prone to breakdown. Lactic acid bacteria help to increase the amount of free
2014).
Lipid oxidation begins when active endogenous lipoxygenase consume oxygen during
mixing of the dough. This enzyme oxidizes linoleic acid to form hydroxyperoxy acid.
7
Flavor active aldehydes are produced by both enzymatic and non-enzymatic degradation
Soaking and sprouting grains has been an increasingly popular preparation method
because of the nutritional advantages. Much of the increased nutrient value is associated
with the enzymatic degradation of phytate. Phytate makes up 1% of the wheat grain and it
forms complexes with divalent cations of calcium, magnesium, and iron. The complexes
with these nutrients make them unavailable to human digestion. Through enzymatic
(Gänzle, 2014). Vinegar is often added to the soaking liquid to lower the pH and
expedite the activation of phytases. Sourdough cultures have the same effect when the
2014).
Gluten is 75% protein based on dry weight, with the remainder being mostly starch and
lipid. The protein portion is responsible for the autoimmune response by celiac disease
patients. Majority of the proteins are prolamins, classified by their solubility in alcohol
and characterized by their high glutamine and proline content. Gliadins are monomeric
prolamins, and glutenins are polymeric prolamins (Shewry et al., 2002). Previously,
gliadins were believed to cause the autoimmune response in celiac patients, but more
recent research has concluded that glutenins also contribute to the autoimmune response.
8
The fermentation by bacteria and yeasts cause acidification and production of alcohols
including thiols. Cereal grain enzymes that participate in gluten degradation depend on
both the presence of thiols and the decreased oxidation-reduction potential caused by
the culture microbes and cereal grain enzymes. Thiols increase efficiency of enzymatic
degradation of the alcohol soluble gluten proteins. LAB is responsible for the conversion
of peptides to amino acids and amino acids to metabolites while the cereal grain enzymes
are important to convert gliadins to peptides. In sourdough cultures the rate of gliadin
hydrolysis is greater than glutenin hydrolysis mainly due to a more complex glutenin
A study supported by The Research Association of the German Food Industry (FEI)
analyzed the individual protein fractions and their extent of degradation during
albumins and globulins with NaCl and HKNaPO4, then extracting the gliadin subunits
with 60% (v/v) ethanol, and finally the glutenin fractions with a mixture of 1-propanol
containing urea, DTT, and Tris-HCl. Data indicated that sourdough fermentation caused
the greater decrease in glutenin fractions compared to other breadmaking methods. This
leads to an increase in alcohol soluble oligomeric proteins, which reside in the gliadin
fraction. This research also discovered that different (homo- or hetero-) fermentative
9
VOLATILE ANALYSIS
Sourdough can be characterized by its unique aroma and flavor profile produced during
fermentation. These aromas are dependant on volatile compounds produced from the
interaction between the flour and microorganisms present in the culture. These aroma
(GC-MS). This analysis has given deeper insight into the microbial and enzymatic
Association of Cereal Chemists analyzed the proteolysis and liberation of amino acids by
cereal and microbial enzymes in sourdough fermentation. This study proved that specific
bacteria species cause an accumulation of certain free amino acids that convert to
recognizable volitiles (Thiele et al., 2002). Therefore, specific bacteria and yeasts can
often be identified knowing that certain volatiles are specific byproducts of that species
metabolic process. A study published in the Journal of Cereal Science analyzed the
relationship between bacteria species in sourdough and volatiles produced and reported
influenced by the lactic and acetic acid content. Homofermentative lactic acid bacteria
(LAB) convert over 85% of hexoses into lactic acid, but heterofermentative LAB use
hexoses to produce lactic acid, acetic acid/ethanol, and CO2. Lactic and acetic acid ratios
have also been shown to be affected by environmental temperature which would in turn
10
CONCLUSION
One objective of this research was to analyze the extent of protein hydrolysis within the
cultures is of great interest because of the potential positive impact for celiac and gluten
sensitive patients. Some bacteria have shown to break down gluten content more
With the increasing prevalence and recognition of gluten related disorders, research on
reducing this autoimmune response is of great interest. This research will provide an
additional understanding of the rate at which sourdough cultures degrade protein. The
hope is that these findings will contribute to future discovery of the digestion mechanism
11
REFERENCES
Cagno, R.D., Rizzello, C.G., Angelis, M., Cassone, A., Giuliani, G., Benedusi, A., et al.
(2008). Use of selected sourdough strains of lactobacillus for removing gluten and
enhancing the nutritional properties of gluten-free bread. Journal of Food Protection,
71(7), 1491.
Caputo, I., Lepretti, M., Martucciello, S., & Esposito, C. (2010). Enzymatic strategies to
detoxify gluten: Implications for celiac disease. Enzyme Research, , 1-9.
doi:10.4061/2010/174354
Dairy Foods Science Notes, Milk Quality Improvement Program, Department of Food
Science, Cornell University.
doi:https://foodsafety.foodscience.cornell.edu/sites/foodsafety.foodscience.cornell.edu/fil
es/shared/documents/CU-DFScience-Notes-Dairy-Cultures-HomoHeteroferm-10-08.pdf
Gadsby, P. (2003). San fran's mighty microbes. Discover, 24(9), 26. Retrieved
from
http://search.ebscohost.com.libproxy.clemson.edu/login.aspx?direct=true&db=a9h&AN=
10511584
Katina, K., Heiniö, R.L., Autio, K., Poutanen, K. (2006). Optimization of sourdough
process for improved sensory profiles and texture of wheat bread. LWT-Food Science
and Technology, 39, 1189–1202.
12
Lhomme, E., Mezaize, S., Ducasse, M. B., Chiron, H., Champomier-Vergès, M. C.,
Chaillou, S., et al. (2014). A polyphasic approach to study the dynamics of microbial
population of an organic wheat sourdough during its conversion to gluten-free sourdough.
International Microbiology, 17, 1.
Settanni, L., Ventimiglia, G., Alfonzo, A., Corona, O., Miceli, A., Moschetti, G. (2013).
An integrated technological approach to the selection of lactic acid bacteria of flour
origin for sourdough production. Food Res Int, 12;54(2):1569-78.
Shewry, P., Halford, N., Belton, P., & Tatham, A. (2002). The structure and properties of
gluten: An elastic protein from wheat grain. Philos Trans R Soc Lond B Biol Sci., 357,
133-142.
Silvester, J.A., Graff, L.A., Rigaux, L., Walker, J.R., & Duerksen, D.R. (2016).
Symptomatic suspected gluten exposure is common among patients with coeliac disease
on a gluten-free diet.Alimentary Pharmacology & Therapeutics, 44(6), 612-619.
doi:10.1111/apt.13725
Thiele, C., Gänzle, M.G., Vogel, R.F. (2002). Contribution of sourdough lactobacilli,
yeast, and cereal enzymes to the generation of amino acids in dough relevant for bread
flavor. Cereal Chemistry Journal, 79:1:45.
Vaintraub, I.A. & Bulmaga, V.P. (1991). Effect of phytate on the in vitro activity of
digestive proteinases. Journal of Agricultural and Food Chemistry 39 (5), 859-861 DOI:
10.1021/jf00005a008
Vermeulen, N., Czerny, M., Gänzle, M.G., Schieberle, P., Vogel, R.F. (2007). Reduction
of (E)-2-nonenal and (E,E)-2,4-decadienal during sourdough fermentation. J Cereal Sci,
1;45(1):78-87.
Wieser, H., Vermeulen, N., Gaertner, F., Vogel, R. (2008). Effects of different
lactobacillus and enterococcus strains and chemical acidification regarding degradation
of gluten proteins during sourdough fermentation. European Food Research &
Technology, 04/29;226(6):1495-502.
13
CHAPTER TWO
ABSTRACT
The effects of fermentation duration at two different temperatures on the quality and
gluten content of sourdough bread were analyzed. A sourdough culture was used to
produce 3 separate batches of bread, of which there was 1 Control (0) and 8 fermentation
treatments. The Control was baked at time 0:00, not allowed fermentation time. Samples
26-2, 26-4, 26-8, and 26-12 were fermented at 26°C for 2, 4, 8, & 12 hours, respectively
and samples 4-14, 4-26, 4-38, and 4-50 were fermented at 4°C for 12, 24, 36, & 48 hours,
respectively. Loaf volume, bread moisture, protein content, and volatiles were analyzed
for each sample. Bread moisture increased as fermentation time increased for both the 26-
samples 4-14 and 4-50. Ethanol extractable protein consistently increased with
fermentation time when comparing the Control (0) with sample 26-12 or 4-14 with 4-50
within batches. This increase was at a significant (p<0.05) level when comparing the
mean of the Control and 26-12. Analysis on dough samples with a gas chromatograph
increase in alcohol. An increase in 1-hexanol was observed from the Control-d to samples
26-4d and 26-12d. Control-d, 26-4d, and 26-12d also showed a decrease in hexanal as
samples, 1-hexanol increased but not as significantly as the room temperature samples.
14
1. Introduction
Sourdough is the oldest and most traditional form of leavened bread (Kiple and Ornelas,
2000). Before the invention of Bakers Yeast, sourdough was the only option to create
leavened bread. Because sourdough is a natural process that relies on the metabolism of
bakers yeast became available in the United States in the 1860s then quickly became the
primary leavening method due to its convenience and consistency (Smith, 2004). It
appears that the health benefits and higher bread quality associated with sourdough
Although sourdough is more time-consuming than other bread-making methods, there are
a number of beneficial reactions that occur during the process. The lactic acid bacteria
and yeasts along with the endogenous enzymes are responsible for the microbial
ability, shelf life, nutritional factors, and overall taste of the bread. The production of
dietary fiber and prebiotic affects (Gänzle, 2014). In the small intestine the
oligosaccharides cannot be broken down therefore acting as a dietary fiber, but in the
15
heterofermentative lactobacilli. These bacteria produce exopolysaccharides to form
biofilms for protection against environmental factor, but in bread these polymers are
beneficial for there water binding capacity which helps prevent bread staling (Gänzle,
2014). The bacteria break down these carbohydrates into short chain fatty acids (SCFA)
that can then be absorbed as nutrients in the colon (Gibson et al., 1996). The
transformation of phenolic compounds adds both a nutritional and flavor benefit to the
bread. Without being broken down, phenolic compounds are considered an antinutrient,
they interfere with the absorption of starch and protein, and they also impart a bitter taste.
Once the phenols are metabolized by lactic acid bacteria and cereal enzymes the nutrient
bioavailability, ratio of absorbed nutrients, is increased and the bitter taste is eliminated.
Wheat flour contains about 1% phytate, a saturated cyclic acid that reduces the
cations. Phytate hydrolysis, by phytases, is dependent on a low pH, between 3.5-5, which
soluble in an acidic pH, 5 or below (Leenhardt et al., 2005). The complexes between
phytate and vitamins or minerals are weakened as sourdough ferments, making the
nutrients available for digestion. Soaking and sprouting grains is a process in which
nutrients and aid in digestion of the grain by activating cereal enzymes. The metabolism
compounds. Lipid oxidation begins in sourdough bread making during the mixing
16
acid is oxidized to hydroxyperoxy acids. Hydroperoxydes are degraded into flavor active
derived from peroxides, has potent anti-fungal property and can increase shelf life over
molecular weight thiols. Here, the pH shifts to the optimum level for aspartic proteases,
the main proteinase in the wheat grain. These factors increase both the solubility of
gluten proteins and the susceptibility to enzymatic break down (Gänzle et al., 2008). This
recent years have been recognized for there antioxidant, anti-cancer, or antihypertensive
activities (Gänzle, 2014). The protein transformations that occur in sourdough have
gained a great deal of attention over the past few decades due to reduced toxicity to some
patients with gluten related disorders. It is estimated that about 1% of the worlds
population has celiacs disease. In the United State only about 5% of the population
affected by celiacs disease have been diagnosed (Celiac Disease Center). It is important
to gain a deeper understanding of the disease and the tolerance of sourdough bread so that
The objective of this research was to contribute insight on the protein transformations and
quality changes that occur resulting from sourdough fermentation, specifically, due to
17
2. Materials and Bread Preparation
Unbleached wheat bread flour (King Author Flour, Norwich, Vermont) was used to make
the sourdough samples because it is the most common flour used in sourdough bread.
The sourdough culture was donated from Chef Cicely Austin, Executive Pastry Chef, of
Aramark Food Service at Clemson University. The culture was stored in a covered
container in a refrigerator at 4°C (40°F). For the duration of the experiment, 25 weeks,
the culture was feed once a week with 100 mL of spring water (Poland Spring, Poland,
Sourdoughs International, Inc (Cascade, Idaho). This culture arrived in a lyophilized form
and was fully activated by numerous feedings and close temperature monitoring.
The entire sourdough sample preparation process, parts 2.2 and 2.3, was done 3 separate
times for the production of 3 sourdough batches (Figure 2 & Figure 3).
Thirty-six hours prior to using the culture to make dough, the culture was fully activated
by removing 40 g of the culture from the refrigerator and mixing with 40 mL of spring
water (Poland Spring, Poland, Maine) and 40 g of bread flour. During this 36-hour
18
activation process the mixture was covered loosely and kept at room temperature, 21°C
(70°F), between each feeding. After 12 hours the mixture was fed for a second time with
120 mL of spring water and 120 g of bread flour. The third feeding was 8 hours later with
360 ml of water and 360 g of bread flour, and the fourth feeding was 8 hours after the
third and required 1080 mL of water and 1080 g of bread flour. At this point the culture
was active and doubled in size in less than 8 hours (Table 1 & Figure 2).
The bread dough was prepared 8 hours after the forth feeding with 3000 g of the active
starter mixed with 2400 g of bread flour, 670 mL of spring water and 40 g of fine sea salt.
Ingredients were mixed by hand and then kneaded in a planetary mixer (Model PM10,
Berkel Company, South Bend, Indiana) with a Spiral Dough Hook attachment for 10
minutes. The dough was then portioned into 205 g loaves that were formed into oblong
balls and placed into loaf pan (5.75” L x 3.25” W x 2.25” H). Each pan was prepared
with nonstick cooking spray (Pam Original, ConAgra Foods, Omaha, Nebraska) and 4 g
of cornmeal (Quaker, Chicago, Illinois). Nine dough samples, 25 g each, were made in
parallel with the 9 bread samples following all the steps except for the baking process.
The dough samples (d) were each stored in 2-oz plastic portion cups with lids. Instead of
being baked at the time of the loaf sample, this dough sample was frozen in order to
terminate further fermentation. These frozen dough samples were used for volatile
19
Charlotte, North Carolina). Samples 26-2, 26-4, 26-8, and 26-12 were held in a proofing
system, rolling rack covered with a large plastic bag, at 26°C and each taken out to be
baked at their designated times (2, 4, 8, & 12 hours, respectively). Temperatures of the
loaf samples were monitored with probe thermometers. Samples 4-14, 4-26, 4-38, and 4-
50 were wrapped in plastic film to hold in moisture and refrigerated at 4°C (40°F) for
their allotted time (12, 24, 36, & 48 hours, respectively). Before baking the 4- samples,
they were placed in the proofing system, at 26°C for 2 hours prior to baking (Table 2 &
Figure 3). A probe thermometer indicated that the 4- samples had an internal temperature
of 4°C when they were removed from the refrigerator and an internal temperature of
26°C after their 2 hours in the proofing system. All samples were baked at 204°C (400°F)
until the internal temperature reached 93°C (200°F). After each sample was baked, it was
taken out of the loaf pan and placed on a drying rack for 2 hours before being tightly
wrapped in plastic film, labeled, and stored in the freezer at -18 to -15°C (0-4°F).
Bread samples were removed from the freezer. The outer crust was removed and only the
inner portions were used for analysis. The samples were thinly sliced and broken into
small pieces not greater than ½ inch. Each sample was weighed (approximately 20 g) and
placed in a labeled tin tray. The samples were then placed in a Mechanical Convection
Oven (Blue M Electric Company, Blue Island, Illinois) for 24 hours at 100°C. Once
removed from the oven, the samples were weighed again for moisture analysis (Table 1).
20
Samples were then individually pulverized in a blender (Oster Osterizer with 8 oz jar,
John Oster MFG. CO, Milwaukee, Wisconsin) and stored in 50 mL centrifuge tubes at
3. Analysis Methods
The maximum height, highest point, of each bread loaf sample was measured in
centimeters to the nearest tenth with a standard ruler. Measurements were recorded for
further analysis to determine possible correlations between loaf height and extent of
fermentation.
The weight of each sample was taken before and after being dried in the Mechanical
Convection Oven (Blue M Electric Company, Blue Island, Illinois) for 24 hours at
100°C. The difference in the original and dried weight was divided by the original weight
immunoassay kit that quantitatively detects gliadin and gliadin fragments and was used
for gliadin detection for this research. The gliadin antibody used is 100% specific for a T
cell stimulatory peptide on the gliadin molecule of wheat, hordein in barley, and secalin
21
in rye. These epitopes are known to play a major role in eliciting symptoms of celiac
disease. The Gluten-Tec ELISA kit is sensitive enough to detect levels a low as 10 parts
The Gluten-Tec kit is an indirect ELISA method, meaning that the more gliadin detected
will result in less color. This test uses a substrate/chromogen solution that contains
horseradish peroxidase (HRP), an enzyme that catalyzes the oxidation of the substrate
TetraMethylBenzidine (TMB), which makes the HRP visual with color. The HRP binds
to the unoccupied sights (where gliadin binds when present) so more color will seen
when there are more open binding sites for HRP or less gliadin (Gluten-Tec® ELISA,
2011).
Buffer A was made by adding 154 mg of DL-Dithiothreitol (Sigma D0632) and 303 mg
tube and dissolved with 10 ml of distilled water. Thirty milliliters of 60% ethanol
solution was then added and the mixture was carefully blended. The pH of the solution
was adjusted to 8.0 with 1 M HCl using a pH meter (Orion model 420A and Orion
9157BN Triode Refillable pH, Orion Research Inc., Boston, Massachusetts). This
solution was then placed in a volumetric flask and filled to 50 ml with distilled water.
22
Buffer B was made by dissolving 1017 mg of iodoacetamide (Sigma ref. 16125-259,
SIGMA-ALDRICH Co., St. Louis, Missouri) and 303 mg Trisma base (Sigma T1503) in
40 ml of distilled water. The pH of this solution was then adjusted to 8.0 with 1 M HCl.
This mixture was then placed in a volumetric flask and filled to 50 ml with distilled
water.
Each pulverized bread sample was weighed out, 0.25 g, into a 15 ml screw cap Greiner.
Four point seven five mL of Buffer A was added to each and vortexed for 1 minute and
then incubated in a water bath for 30 minutes at 60°C. Samples were then vortexed for
temperature (70°F). Two hundred and fifty µl of the clear supernatant was transferred to
a tube and neutralized with 250 µl of Buffer B and vortexed for 1 minute. The neutralized
samples were then incubated at room temperature in the dark for 30 minutes before being
diluted 1:500 with the Sample Dilution Buffer (ready-to-use from kit) (Gluten-Tec®
ELISA, 2011).
Reagents were prepared and used within the same day. Prior to use, reagents were
brought to room temperature (1 hr). Microtiter and plate strips were also brought to room
temperature before each use. The unneeded strips were kept in refrigerator in the
resealable bag until further use. The Gluten-Tec kit Rinsing Buffer was diluted by a
23
water. The dilution buffers, both the conjugate and antibody dilution buffers, were
supplied in a bottle ready-to-use in the kit (Gluten-Tec® ELISA, 2011). To prepare the
conjugate solution the vial of conjugate was spun down in the centrifuge for 1 minute at
1000 × g (RPM) before 20 µl was mixed with 2 ml of conjugate dilution buffer. The
antibody, biotinylated anti-alpha-20, vial was spun down with a short centrifuge step for
buffer. The substrate solution was delivered ready-to-use. It was mixed well and brought
to room temperature before each use. The ethanol solution, 60%, was prepared by mixing
One hundred µl of the zero standard was pipetted in duplicate in wells A1 and A2
(blanks). Fifty µl of the zero standard were pipetted into wells B1 and B2 (maximal
signal). Each remaining standard solution, 0.25, 0.5, 1, 2, 4, and 8 ng/ml, were pipetted in
duplicate, 50 µl each, in wells C1, 2 to H1, 2 respectively. Fifty µl of each sample was
pipetted in duplicate in the microtiter plate. Then 50 µl of antibody was added to each
standard and sample well except the blank wells, A1 and A2. The plate was then sealed
and gently mixed for 30 seconds on a microtiter plate shaker before being incubated in
the dark for 1 hour at 4°C. Then the solution in the microtiter plate was discarded and
washed 3 times with the rinsing buffer. The conjugate was then pipetted into the wells,
24
100 µl in each, except for wells A1 and A2. The plate was sealed once again, shaken on
the plate shaker, and incubated for another hour in the dark at 4°C. After the incubation,
the solution was discarded once again and washed 3 times with rinsing buffer. Then 100
µl of the substrate solution was added into each well including the blank wells, A1 and
A2. At this point the plate was incubated for 30 minutes at room temperature (20°C -
25°C) before adding 100 µl of stop solution into each well. The absorbance values were
then read immediately by an Epoch BioTek plate reader (BioTek Instruments, Inc.,
Highland Park, Winooski, Vermont) at 450 nm and recorded for further calculation
The optical density (O.D.) of the wells containing the standards and the samples were
obtained by subtracting the mean O.D. of the blank wells, A1 and A2. The mean O.D.
values for each of the duplicates were divided by the mean O.D. value of the zero
standard (wells B1 and B2) and then multiplies by 100. This makes the zero standard
equal 100% or maximum absorbance while the other O.D. values are a percentage out of
25
The calculated values were then plotted on the Y-axis versus the alpha-20-peptide
In order to calculate the α20 peptide concentrations of the samples, the α20 peptide read
from the curve was multiplied by 1000. The conversion factor for peptide to gliadin is
100. This is based off the correlation of peptide content to gliadin found in wheat
Dried, pulverized bread samples (prepared in section 2.4) were used for determining
protein content. Buffer A was used to extract the protein from the samples and the
supernatant was taken after centrifugation for analysis. The Coomassie Brilliant Blue G-
250 binds to the polypeptide backbone electrostatically and with hydrophobic interactions
(Nielsen, 2010).
Bovine serum albumin (BSA) solutions were used as the reference standard. The standard
curve consisted of protein concentrations of 0.0 (blank), 0.125, 0.25, 0.50, 0.75, 1.0, 1.25,
26
Ten µL of each standard or sample was pipetted in duplicate into the appropriate wells of
a microplate. Then 300µL of Coomassie Plus Reagent was added to each well and mixed
on a plate shaker for 30 seconds. The plate was then incubated for 10 minutes at room
temperature before measuring the absorbance at 595nm in an Epoch BioTek plate reader
(Nielsen, 2010).
3.4.3. Calculations
The average for the Blank read was subtracted from all standard and sample reads. The
standard curve was drawn by plotting the Blank-corrected measurements for each BSA
standard vs. its concentration in µg/mL. The linear fitted calibration curve was then used
Prolamins (mostly gliadin and some glutenins) from the samples were extracted by
mixing 0.125 g of the pulverized bread with 5 mL 60% (v/v) ethanol in a rotary shaker
for 1 hour at room temperature (21°C, 70°F). The samples were then centrifuged at
10,000 x g for 10 minutes and the supernatant was separated. The precipitate was then
extracted for a second time by adding 2.5 mL of 60% (v/v) ethanol, mixed in the rotary
shaker for another hour at room temperature (21°C, 70°F), and then separated by
centrifuge at 5000 rpm for 10 minutes before removing the supernatant for further
27
analysis (De Angelis et al., 2006). The first and second extractions were measured for
Gas chromatography was used to analyze the volatiles associated with the various
sourdough samples, which help determine the extent of fermentation. In order to prevent
further fermentation prior to the analysis, dough samples were taken out of the freezer no
more than 30 minutes before running the test. This allowed the sample to thaw such that
Samples were analyzed with a gas chromatograph mass selective detector system
(Hewlett Packard HP 6890 Series gas chromatograph and Hewlett Packard 5973 mass
Technologies 7697A, 30.0 m × 250 µm × 0.25 µm). Prior to analysis, the sealed vials
containing the dough samples were heated to 80°C for 20 minutes in the HP 7694. For
each sample run the GC oven started at 35°C for 5 minutes, then increased 7°C per
minute until it reached 100°C, followed by an increase of 10°C per minute until 230°C
was reached. The total runtime was 32.29 minutes (Paucean et al., 2013). The plotted
28
The experiment was replicated 3 times using 3 different batches of sourdough. A general
linear model was used to compare all holding time treatments of room temperature (0, 4
and 12 hours) and refrigerated temperature (14, 26 and 50 hours). For parameters that
were found to be significantly affected by the treatments (P≤0.05) the means were
separated using the least significant difference command of SAS (SAS, 2016).
Measuring the height of each bread loaf can indicate peak fermentation time, a reflection
of maximum gas retention in the bread. Over fermentation can be identified by a loss of
volume and reduced gas production due to inactivity of microbes, often caused by a lack
of adequate nutrients but can also be affected by hold temperature. In Batch #1 the peak
fermentation time for 26°C samples was 8 hours, in Batch #2 the peak was closer to 4
hours, and in Batch #3 the peak was around 2 hours. In the samples held at 4°C, the peak
was at 48 hours for Batch #1, 36 hours for Batch #2, and 48 hours for Batch #3. One
factor that may have affected these observations is the scoring before baking. The loaves
must be scored to allow steam to escape during baking in order to prevent large air
pockets. Although all samples were scored in the same manner, some lost more volume
than others. The greatest decrease in volume caused by scoring was seen in the samples
29
In general, as the fermentation time increased, the bread moisture increased regardless of
hold temperature (Table 5). At 26°C fermentation the difference in bread moisture for the
0 and 12-hour treatments was not significant (p>0.05), although there was an average
where the moisture increased by an average of 1.87% from 12 to 48 hours. This increase
that result in water as a byproduct. Fermentation also increases water binding compounds
that contribute to dough hydration and prevent staling (Gänzle, 2014). This may prevent
the dough from losing water via evaporation during the baking process.
There was no consistent data indicating the reduction of wheat proteins throughout the
fermentation process. The large variation within batches indicated the assay was not well
suited for sourdough bread as three dilutions were needed to reduce the protein content
into the testing range. This process may have greatly reduced the accuracy and precision
of the test.
This analysis technique quantifies the gliadin and gliadin fragments of a sample through a
correlation factor to the amount of peptide that is detected. The protocol multiplies the
conversion factor, research has found an accumulation of peptides and amino acid
30
4.4. Bradford Assay Protein Quantification
No statistically significant results were found from the protein quantification using the
Bradford method. The Bradford method quantifies the presence of three basic amino acid
residues, arginine, lysine and histidine. Under acidic conditions the Coomassie dye binds
the protein residues and forms a complex (He, 2011). The disadvantage of the Bradford
method is that it is only testing for 3 of the 20 amino acids. Depending on the samples
being analyzed, the results could be very misleading because different protein sources
have different concentrations and combinations of these amino acids. That being said, the
concentration of these three amino acid residues, arginine, lysine and histidine,
When comparing samples, with the same treatment temperature but different
fermentation periods, there was a common trend throughout the data set. Within each
batch and treatment temperature, the protein extracted with ethanol increased with
fermentation. The difference in ethanol extractible protein between the Control and 26-12
were significantly different (p≤0.05) based on the sample means from the 3 batches
(Table 4). Although the trend was consistent throughout the 3 batches for samples 4-14
31
Research supported by the FEI (Forschungskreis der Ernährungsindustrie e. V., Bonn) of
Germany has shown that sourdough fermentation degrades gluten proteins. A decrease in
the glutenin fractions will generate an increase in alcohol soluble oligomeric proteins, or
gliadins (Wieser et al., 2008). When extracting with ethanol, the supermatant will contain
trends and these were chosen based on their presence in all of the dough samples
analyzed. 3-methyl-1-butanol and 1-hexanol increased with fermentation time in both the
room temperature and refrigerator fermented samples (Tables 7, 8, & 9). The increase in
these alcohol volatiles is consistent with fermentation yielding alcohol (Liu et al., 2014).
5. Conclusion
Sourdough cultures are a traditional leavening method that in recent years have been
Sourdough samples were allowed to ferment for various time durations. The height of
each loaf was measured and recorded. As a preparation method and for analysis purposes
the samples were dried in a Mechanical Convection Oven. A Gluten-Tec® ELISA kit was
used to quantify gliadin and gliadin fragments in the bread samples. This did not show any
32
different temperatures. In order for the samples to be in the gliadin detection range of the
kit, many dilutions of 1:1000 had to be made. Each dilution step can contribute to
inaccuracy of the results. Bradford assay was used to determine protein content although
lysine, and histidine. These particular residues are not found in any of the four confirmed
Tyr, and Gln-Pro-Tyr-Pro) so we cannot conclude that toxicity was not reduced with this
test result (Darewicz et al., 2008). Lastly, ethanol (60%) extraction was used to quantify
prolamins (mostly gliadin and some glutenins). Analysis showed that as fermentation time
increased, ethanol extractable prolamins increased. This data was significant (p<0.05)
when comparing the Control and 26-12. A study supported by the European Food
Research & Technology program found similar results by discovering a decrease in the
glutenin fraction that lead to an increase in the alcohol soluble gliadin fractions (Weiser et
al., 2008). Flavor volatiles were collected on the dough samples to analyze sourdough
quality and treads. An increase in 1-hexanol was observed as fermentation time increased
within the Control, samples held at 26°C and samples held at 4°C. This change was
significant (p<0.05) when comparing the Control, 0, and the 26°C treated samples, 26-4
and 26-12. Research done in The Netherlands on white bread volatiles and enzyme active
soya flour has suggested that an increase in 1-hexanol is a byproduct of lipid oxidation
caused by the addition of enzymes (Luning et al., 1991). For these same samples, hexanal
decreased as fermentation time increased. This change was significant (p<0.05) when
comparing the control and 26-12. A study done by the Dipartimento di Scienze Agrarie in
33
Italy analyzed the volatile profiles of white wheat sourdough bread and discovered that
hexanal was negatively correlated to the content of ethanol (Ripari et al., 2016). An
increases.
Further research needs to be done to be able to fully understand the extent of protein
hydrolysis, as well as the safety of sourdough bread for people sensitive to certain wheat
proteins. It would also be beneficial to pin point the sourdough bacteria with the highest
34
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36
Table 1. Sourdough culture activation schedule and dough sample preparation
procedure
ferment time
Feeding # culture (g) flour (g) water (g) sea salt (g)
(hr)
1 40 40 40 0 12
Figure 2.
Flow chart of sourdough culture activation process
37
Table 2.
Sourdough loaf sample fermentation schedule
38
Figure 3.
Flow chart of loaf and dough sample preparation
39
Table 3. Table 4.
Loaf height of samples fermented Loaf height of samples fermented
at 26°C, n=3 at 4°C, n=3
Table 5.
Percent Moisture in Sourdough Bread at minimum and maximum fermentation
times at both 26°C and 4°C
40
Table 6.
Ethanol extracted protein in sourdough bread at minimum and maximum
fermentation times at both 26°C and 4°C
41
Table 7.
Headspace gas volatiles for dough samples held at 26°C
Table 8.
Headspace gas volatiles for dough samples held at 4°C
42
Table 9.
Headspace gas average volatile percentages for dough samples held at 26°C and
dough samples held at 4°C
43