Food Bioscience 37 (2020) 100704

Contents lists available at ScienceDirect

Food Bioscience
journal homepage: www.elsevier.com/locate/fbio

Improving bread aroma using low-temperature sourdough fermentation

Dan Xu a, b, c, Huang Zhang a, b, d, Jinzhong Xi a, b, c, Yamei Jin a, b, c, Yisheng Chen a, b, c,
Lunan Guo a, b, c, Zhengyu Jin a, b, c, Xueming Xu a, b, c, *
a
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China
b
School of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China
c
International Joint Laboratory on Food Safety, Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu, 214122, PR China
d
Henan University of Animal Husbandry and Economy, Zhengzhou, Henan, 450046, PR China

A R T I C L E I N F O A B S T R A C T

Keywords: To improve the aroma of bread, two aroma-producing yeast isolates from the Chinese liquor starter culture called
Meyerozyma guilliermondii Daqu, Meyerozyma guilliermondii CGMCC 17606 and Pichia kudriavzevii CGMCC 17607, were combined with
Pichia kudriavzevii Lactobacillus sanfranciscensis DSM20451T, were used for sourdough fermentation at 10, 20, and 30 ◦ C. Volatile
Lactobacillus sanfranciscensis
aroma compounds in bread crumb were extracted using headspace solid-phase micro-extraction and analyzed
Sourdough bread
Low-temperature fermentation
using gas chromatography-mass spectrometry and gas chromatography-olfactometry-mass spectrometry. The
Volatile aroma compounds quality of the final bread was analyzed using sensory evaluation. Addition of sourdough fermented with mixed
starters at 10 ◦ C promoted bread aroma, showed increased sensorial qualities, and was preferred by panelists.
Acetic acid, 1-octen-3-one, and dimethyl trisulfide, were the most aroma-active compounds in sourdough bread
fermented with M. guilliermondii and L. sanfranciscensis, whereas those in sourdough bread fermented with
P. kudriavzevii and L. sanfranciscensis were acetic acid, 1-octen-3-one, 2-methoxy-4-vinylphenol, 3-methylthio-
propanal, furfural, and 1-(2-furanyl)-ethanone. Reducing the fermentation temperature to 10 ◦ C increased the
formation of these key volatiles, as well as other aroma-active compounds, such as 3-methyl-1-butanol, 3-
methylbutanal, 2-methyl-1-propanol, 1-octen-3-ol, isogeraniol, benzaldehyde, benzene acetaldehyde, ethyl
octanoate, ethyl decanoate, phenylethyl acetate, furfural, and 2-pentylfurane. The amount of furans, pyrazines,
and volatiles converted from amino acids in bread crumb also increased with the protein hydrolyzation and
amino acid formation in sourdough fermented at 10 ◦ C.

1. Introduction trend is to use aroma-producing microorganisms and then select a

Sourdough is a mixture of cereal flour, water, and microorganisms,
such as lactic acid bacteria (LAB) and yeast, which has been traditionally
used as a leavening agent for bread making (Gänzle & Ripari, 2016).
Sourdough can improve the properties of bread dough, increasing the
overall quality of bread (bread texture, flavor, and shelf life), delaying
the staling process, and protecting bread from spoilage (Gänzle, 2014;
Poutanen et al., 2009). The sourdough microbiota consists of yeasts and
LAB, which produce abundant organic acids and special flavoring sub­
stances such as alcohols, aldehydes, and esters during fermentation,
imparting a distinct flavor to sourdough bread (Gänzle & Ripari, 2016;
Hansen & Hansen, 1996). Traditional sourdough relies on the sponta­
neous fermentation and frequent backslopping to maintain the survival
of microorganisms in sourdough (Tang et al., 2017). Modern sourdough
fermentation requires a stable composition of starter cultures, and the
suitable fermentation method, which can ensure the improved quality of
products, particularly bread aroma (Plessas et al., 2008).
Daqu, a traditional starter culture for brewing Chinese liquor (bai­
jiu), is made using cereal and contains various molds, yeasts, and bac­
teria (Wang et al., 2008; Zou et al., 2018). Daqu has an important role in
the aroma development of baijiu (Zhang et al., 2012), and the
aroma-producing yeast in Daqu improves the fragrance of baijiu (Fan
et al., 2018; Wang et al., 2014). Most aroma-producing yeasts are
non-Saccharomyces yeasts, which produce high levels of volatile aroma
compounds and increase the perceived aroma complexity in baijiu and
wine (Kurita, 2010; Strauss et al., 2010; Wang et al., 2018). Thus, the
application of aroma-producing yeasts from Daqu might improve the
overall aroma characteristics of sourdough bread. In addition, yeast cells
can produce various higher alcohols, carbonyl compounds, phenolic
compounds, fatty acid derivatives and sulfur compounds, which are

* Corresponding author. State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, PR China.
E-mail address: xmxu@jiangnan.edu.cn (X. Xu).

https://doi.org/10.1016/j.fbio.2020.100704
Received 24 June 2019; Received in revised form 8 July 2020; Accepted 9 July 2020
Available online 5 August 2020
2212-4292/© 2020 Elsevier Ltd. All rights reserved.
D. Xu et al. Food Bioscience 37 (2020) 100704

volatile and emit pungent aromas that are often important for bread
quality (Dzialo et al., 2017; Hansen & Schieberle, 2005). This secondary
metabolite formation by yeasts is species- or strain-specific and may be a
response to the different preferences of insect vectors (Dzialo et al.,
2017). Therefore, Daqu is also a good source of diverse yeasts that have
distinct flavor production potential.
Temperature, a vital parameter for sourdough fermentation, affects
the growth of Lactobacilli and yeasts and changes the microbial me­
tabolites in sourdough (Gänzle et al., 1998). Previous studies showed
that the optimum temperature for fermentation or yeast growth (28–30
◦
C) ensures efficient fermentation; however, low-temperature fermen­
tation may increase the synthesis of secondary aromas (Birch et al.,
2013). Low-temperature fermentation is preferred by numerous wine­
makers to obtain enhanced taste and aroma (Redón et al., 2011). For
bread making, several studies have shown that longer time and lower
temperature of straight dough fermentation can increase the formation
of aroma compounds (Birch et al., 2013; Maeda et al., 2009). However,
information on the use of low fermentation temperature in bread aroma
development is limited despite studies on the use of low fermentation
temperature to enhance yeast-related flavors in sourdough bread
(Gänzle et al., 1998). The influence of variations in sourdough fermen­
tation temperature on bread crumb aroma led to an interest in product
aroma enhancement for the bread industry.
Yeast strains Pichia kudriavzevii CGMCC 17607 and Meyerozyma
guilliermondii CGMCC 17606 isolated from Chinese liquor Daqu were
used as mixed starter cultures for preparing sourdough bread and
improving bread aroma (Xu et al., 2019). However, the effect of sour­
dough on bread quality, particularly bread aroma, has not been linked to
fermentation temperature. Thus, this study aimed to evaluate the effects
of sourdough fermented with mixed starters at different temperatures
(10, 20, and 30 ◦ C) on the aroma profile of bread crumb. This study also
aimed to identify the key odorants in bread crumb and determine the
relationship between temperature and aroma formation. P. kudriavzevii
CGMCC 17607 and M. guilliermondii CGMCC 17606 were combined with
Lactobacillus sanfranciscensis DSM20451T for a one-stage sourdough
fermentation. The breads were assessed using sensory evaluation. The
volatile aroma compounds in bread crumbs were extracted using
headspace solid-phase micro-extraction (HS-SPME) and further
analyzed using gas chromatography–mass spectrometry (GC-MS) and
gas chromatography-olfactometry-mass spectrometry (GC-O-MS).
2. Materials and methods
2.1. Materials
High-gluten wheat flour purchased from Yihai Kerry Food Industry
Co., Ltd. (Shanghai, China) with 72.0% starch, 12.8% protein, 1.6% fat,
and 13% moisture was used. Baker’s yeast was supplied by Angel Yeast
Co., Ltd. (Wuhan, Hubei, China). Sugar and salt were purchased from a
local store in Wuxi, Jiangsu, China. The peptone, yeast extract, and beef
extract were obtained from Oxoid Ltd. (Basingstoke, Hampshire, UK).
All other chemicals and analytical standards were obtained from Sino­
pharm Chemical Reagent Co., Ltd. (Suzhou, Jiangsu, China) and were of
analytical grade unless otherwise stated.
(Gänzle et al., 1998) for 16–20 h. The yeast strains M. guilliermondii
CGMCC 17606 and P. kudriavzevii CGMCC 17607 were isolated from
Daqu (Xu et al., 2019), and cultivated aerobically (180 rpm) at 30 ◦ C in
yeast extract peptone dextrose medium (YPD, 2% peptone, 2% glucose,
1% yeast extract, with 1.5% (w/v) agar included for solid medium)
(Taylor-Mayer et al., 1988) for 20–24 h.
2.3. Sourdough fermentation and bread making
Sourdough fermentations were prepared as following: The strains
were harvested using centrifugation (ST16R, Thermo Fisher Scientific
Inc., San Jose, CA, USA) at 5000 × g, 20 ◦ C for 10 min, then washed
twice and resuspended in sterile tap water with the absorbance at 600
nm (P7 Double Beam UV–Visible spectrophotometer, Mapada,
Shanghai, China) of 0.2 for L. sanfranciscensis, 0.01 for P. kudriavzevii,
and 0.005 for M. guilliermondii. Wheat flour (200 g) was mixed with the
cell suspensions (200 g) and the initial amounts of microorganisms were
around 1 × 107 cfu/g (on a flour basis) of L. sanfranciscensis and 1 × 105
cfu/g of yeast of each species. The sourdough was fermented in a beaker
covered with plastic wrap at 10, 20, and 30 ◦ C. The chemically acidified
sourdough was prepared by adding a mixed acids solution (acetic acids
(100% w/w) and lactic acid (85% w/w) in a ratio of 1:4 (v/v)) to give a
pH value of 3.8 ± 0.1, then incubated using the same condition as
described.
The bread formula is shown in Table 1. To make sourdough bread
dough, 400 g of flour, 200 g of sourdough, 10 g of table salt, 10 g of
sugar, 2.5 g of baker’s yeast, and 200 g of tap water were mixed with a
kneader (SM-25, Sinmag Equipment Corp., Wuxi, Jiangsu, China) for 6
min, then rested for 10 min. Dough were scaled to 80 g, shaped and
proofed in a proofer (SM-40SP, Sinmag Equipment) at 37 ◦ C, 85%
relative humidity for 80 min, then baked at 220 ◦ C for 15 min in a
convection oven (SM2-523, Sinmag Equipment). The regular straight
dough bread (without sourdough), and chemically acidified bread were
prepared as controls (Xu et al., 2018). After baking, breads were cooled
down at room temperature (20 ± 2 ◦ C) for 2 h, then sealed in airtight
bags for further analysis.
2.4. Determination of pH and cell count CFU/g during fermentation
To determine the pH, 10 g of fresh sourdough sample was homoge­
nized at full speed with 90 mL of deionized water (Milli-Q Millipore
system, Bedford, MA, USA) using a homogenizer (T18, IKA-
Labortechnik, Dottingen, Germany), and the value obtained with a pH
meter (FE28-Standard, Mettler-Toledo, Greifensee, Switzerland). For
cell counts, sourdough samples were prepared with serial dilutions of
sterile 0.9% saline solution by plating them on mMRS and YPD agar, and
incubated anaerobically and aerobically at 30 ◦ C for 2 days,
respectively.
2.5. HS-SPME of volatiles
The HS-SPME was done to extract the volatile compounds in the
bread crumb. Before extraction, 2 g of each entirely crushed bread
crumb sample (through a 1.5 mm sieve) was placed in a headspace vial

2.2. Strains, media, and growth conditions Table 1

Ingredients of bread. (wheat flour weight basis, g).
The Lactobacilli used in the study was Lactobacillus sanfranciscensis Wheat flour Sourdough Water Baker’s yeast Sugar Salt
DSM20451T (supplied by the Agricultural Food and Nutritional Science
LS 400 200 200 2.5 10 10
Department, University of Alberta, Edmonton, Alberta, Canada), a strain LS + MG 400 200 200 2.5 10 10
originally isolated from sourdough, grown anaerobically at 30 ◦ C in LS + PK 400 200 200 2.5 10 10
modified (with extra maltose) De Man, Rogosa, and Sharp medium CA 400 200 200 2.5 10 10
(mMRS, 1% maltose, 0.5% glucose, 0.5% fructose, 1% peptone, 0.5% RC 500 – 300 2.5 10 10
yeast extract, 0.5% beef extract, 0.4% K2HPO4⋅3H2O, 0.26% KH2PO4, Note: LS: L. sanfranciscensis; LS + MG: L. sanfranciscensis and M. guilliermondii; LS
0.3% NH4Cl, 0.05% L-Cys HCl⋅H2O, 0.1% Tween 80, 0.005% + PK: L. sanfranciscensis and P. kudriavzevii; CA: chemically acidified control; RC:
MnSO4⋅H2O, and 0.02% MgSO4⋅7H2O, and the pH was adjusted to 6.2) regular bread control.

2
D. Xu et al.
(20 mL) and sealed with a Teflon cap, then incubated at 60 ◦ C for 20 min
with agitation. Extraction was achieved using a SPME fibre (2 cm–50/
30 mm DVB/Carboxen/PDMS Stable Flex, Supelco, Bellefonte, PA, USA)
which was placed inside the vial and exposed to the headspace for 30
min at 60 ◦ C for adsorption. After HS-SPME, the fiber was inserted into
the injection port of the GC (250 ◦ C) for 7 min to desorb the analytes.
2.6. GC-MS and GC-O-MS analysis of bread crumb
The GC-MS was used to determine the bread crumb volatile com­
pounds. Headspace volatile compounds were analyzed using a Shimadzu
model SPL-2010 PLUS gas chromatograph coupled to a GCMS-QP2010
Ultra mass spectrometer (Shimadzu, Kyoto, Japan). The column used
was DB-WAX122-7032 (30 m × 0.25 mm × 0.25 μm, Agilent Technol­
ogies, Santa Clara, CA, USA). The program was set as following: 40 ◦ C for
3.5 min, then increased by 5 ◦ C/min to 90 ◦ C and held for 5 min, and
then increased by 12 ◦ C/min to 220 ◦ C and held for 7 min. The injector
temperature was 280 ◦ C. The flow rate of ultrahigh-purity helium was
0.8 mL/min. The mass spectra were measured using electronic impact
(EI) at 70 eV, and the electron source temperature was 200 ◦ C. The mode
was used to scan all the compounds in the m/z range of 33–495. The
volatile compounds identification was done by comparing with standard
compounds and MS data obtained from NIST (National Institute of
Standards and Technology, Gaithersburg, MD, USA) library (https
://webbook.nist.gov). Semi-quantification of the volatiles was done
using ethyl hexanoate as the internal standard. Data were collected in
total ion mode for all samples and standards. Semi-quantitative data of
those volatiles were obtained using the following formula:
C (μg/L) = Ac/Ais × Cis (μg/L)
where C is the relative concentration of analyte; Cis is the final con­
centration of the internal standard in the bread sample; Ac is the peak
area of analyte; Ais is the peak area of internal standard as calculated by
the software with the instrument.
The active aroma compounds were analyzed using GC-MS, equipped
with an ODP-2 olfactory detector port (Gerstel, Mulheim an der Ruhr,
Germany) using an aroma extract dilution assay (AEDA). The odor
characteristics of each compound could be detected at the sniffing port,
and the flame ionization detector signal was obtained simultaneously.
To determine the aroma dilution factor (FD), the SPME extracted vola­
tile compounds were diluted by nitrogen with the split ratio (by volume)
of 1:3, 1:9, 1:27, until no further aroma was detected. Moist air was
pumped into the sniffing port at the rate of 40 mL/min to quickly remove
the odorant residual from the sniffing port. The time for onset and end
while sniffing the effluent from the sniffing mask, perceived odor
characteristic, and aroma intensity were obtained using three trained
panelists (2 males and 1 female, aged between 25 and 30 years). Aroma
descriptors were obtained from “Flavornet and human odor space” (htt
p://www.flavornet.org/flavornet.html) and the odor database (http://
www.thegoodscentscompany.com/).
2.7. Determination of metabolites in sourdough
2.7.1. Total α-amino nitrogen
The method of Lie was used with some modification (Lie, 1973). The
fermented sourdough was freeze dried using a laboratory scale freeze
dryer (18N, Scientz Biotechnology Co., Ltd., Ningbo, Zhejiang, China).
The freeze dried sample (1.0 g) was mixed thoroughly with 2.5 mL of 7%
perchloric acid with a Vortex Mixer (MX-S, DLAB Scientific Inc., Beijing,
China), then stored at 4 ◦ C for 16 h. The samples were centrifuged at 10,
000 × g for 15 min at 4 ◦ C, and 600 μL potassium hydroxide (0.43 M)
was added to 800 μL supernatant. The reagents 1 (10% Na2HPO4⋅12H2O,
6% KH2PO4, 0.5% ninhydrin, 0.3% fructose, and the pH was 6.6–6.8)
and 2 (2 g KIO3 dissolved in 600 mL distilled water and 400 mL of 96%
ethanol) were prepared as described by Lie (1973). The reagent 1 (100
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Food Bioscience 37 (2020) 100704
μL) and sterilized distilled water (190 μL) were mixed with 10 μL treated
supernatant, which had been incubated at 20 ◦ C for 1 h, and incubated in
boiling water for 16 min, then cooled down at 20 ◦ C for 20 min. Reagent
2 (500 μL) was added into the samples and mixed for measurement, and
the absorbance was measured at 570 nm (UV–Visible spectrophotom­
eter, Mapada). Glycine dissolved in distilled water was used to establish
a standard curve.
2.7.2. Free amino acids
For amino acids detection, 10 g of lyophilized dough sample was
mixed with 25 mL of 5% (w/w) trichloroacetic acid, and treated in a 20
◦
C ultrasound water bath (40 kHz, 0.06 W/cm3, KQ300DE, Kunshan
Shumei Ultrasonic Equipment Corp., Suzhou, Jiangsu, China) for 5 min.
Samples were centrifuged at 10,000 × g for 10 min at 20 ◦ C, and the
supernatant was collected and filtered using a 0.22 μm Millipore filter
(MCE, Navigator Lab Instrument Co., Ltd., Tianjin, China). The filtrate
was then analyzed using an Agilent 1100 high-performance liquid
chromatograph (HPLC) system (Agilent Technologies) equipped with a
UV detector and ODS-2 Hypersil capillary column (Thermo Fisher Sci­
entific). The mobil phase was methanol-acetonitrile solution with a ratio
of 1:2 (v/v), containing 20 mM sodium acetate. The flow rate was 1.0
mL/min, the column temperature was 40 ◦ C, the injection volume of all
samples was 10 μL, and the UV detector was set at 338 nm for amino
acids detection. Glutamate, lysine, methionine, isoleucine, leucine,
valine, and phenylalanine standards were prepared for quantification of
each amino acid.
2.8. Sensory evaluation
The sensory sessions were carried out in panel booths at the Sensory
Analysis Laboratory of Jiangnan University (Jiangsu, Wuxi, China).
Each bread sample was sliced into 10 mm thick pieces using a slicer (SM-
302N, Sinmag Equipment), placed in a plate covered with plastic food
wrap and labeled with random 3-digit numbers. The order of the pre­
sentation of all samples was randomized to prevent carryover effects.
Water and plain crackers were provided to cleanse the palate between
samples. All samples were examined by 30 consumer panelists at Jian­
gnan University (15 males and 15 females, aged between 20 and 35
years), who were asked to give scores on a 1 (disliked extremely) to 10
(liked extremely) for appearance, aroma, taste, texture and overall
acceptability.
2.9. Loaf specific volume and crumb hardness
The loaves were weighed and loaf volume was measured using the
rapeseed displacement method (AACC Method 10–05.01). The specific
volume of bread was calculated as mL/g.
Bread crumb hardness was determined using a texture analyzer (TA.
XT Plus, Stable Micro System Co. Ltd., Surrey, England). The breads
were sliced into 10 mm thick pieces using a slicer (SM-302N, Sinmag
Equipment), and the parameters used were set as: the probe type was P/
25 with a diameter of 25 mm; the pre-test speed was 3.00 mm/s; testing
speed was 1.00 mm/s; post-test speed was 5.00 mm/s; compressed twice
with 40% compression, and the interval time was 10 s.
2.10. Statistical analysis of data
Sourdough fermentations and bread making were carried out in
triplicate independent experiments. HS-SPME and GC-MS analyses were
done with triplicate samples while the GC-O-MS analyses were done
once with a split ratio of 4. Data were analyzed using the Statistical
Package for the Social Sciences (SPSS) software (version 20 for Win­
dows, SPSS Inc. Chicago, IL, USA). One-way analysis of variance
(ANOVA) and Duncan’s multiple range tests were used to determine
significant (p < 0.05) differences among the results. Principal compo­
nent analysis (PCA) and partial least squares regression (PLSR) were
D. Xu et al. Food Bioscience 37 (2020) 100704

done using Soft Independent Modeling of Class Analogy (SIMCA-P)
software (11.5 Demo, Umetrics, Umea, Sweden). Heatmaps were done
using the website MetaboAnalyst (http://www.metaboanalyst.ca) to
analyze dissimilarities among the samples in terms of their volatile
components.
3. Results and discussion
3.1. Cell count and pH during sourdough fermentation
Temperature is one of the important physical parameters that sub­
stantially influence the growth of LAB and yeast cells, as well as the
fermentation process (Gänzle et al., 1998). The viable cell counts and pH
levels were determined to monitor the fermentation microbiota of
sourdoughs at different temperatures. The growth curves of
L. sanfranciscensis and each yeast in sourdough fermented at different
temperatures are shown in Fig. 1. The variation in fermentation tem­
perature ranging from 10 to 30 ◦ C induced no major shifts in the
yeast-to-LAB ratio and the maximum population density of the strains in
all groups. Acidification of sourdough is also essential for baking. The
pH of the sourdough decreased with the prolongation of fermentation
time, and the acidification rate was affected by temperature. With strain
growth activity and acid accumulation in sourdough considered, the end
points selected were 20, 36, and 72 h when the fermentation tempera­
tures were set to 30, 20, and 10 ◦ C, respectively.

Fig. 1. Cell counts and pH of sourdough during fermentation at different temperatures. Results are presented as means ± standard deviation of triplicate independent
fermentation trails. LS L. sanfranciscensis; PK P. kudriavzevii; MG M. guilliermondii.

4
D. Xu et al.
3.2. Effect of temperature on volatile aroma compounds in bread crumb
Bread aroma is an important parameter for bread quality (Birch
et al.,. 2014), and volatile compounds are derived from dough fermen­
tation, lipid oxidation, enzymatic reactions, and reactions in microbial
cells (Pico et al., 2015). To explore the potential relationship between
sourdough fermentation temperatures and volatiles produced in breads
fermented with these starter cultures. Volatile compounds in all breads
were extracted and determined using SPME–GC–MS. The GC–MS results
showed that 56 compounds with odor or potential odorants (17 alcohols,
11 esters, 12 aldehydes, 7 ketones, 3 acids, and 6 other compounds)
were identified in the bread crumbs. PCA was done on the relative
content of volatiles identified in different types of sourdough bread. The
Fig. 2. PCA of volatile compounds of bread crumb. LS L. sanfranciscensis; PK P. kudriavzevii; MG M. guilliermondii; CA Chemically acidified; RC Regular bread.
5
Food Bioscience 37 (2020) 100704
loading plot in Fig. 2 shows the effects of different starter cultures and
temperatures on the volatile compounds of sourdough bread. The breads
appear well separated on the loading plot, indicating that products
fermented with different starters at different temperatures showed
different volatile composition patterns.
PCA plots and heatmaps in Figs. 2 and 3 show that compared with
other groups, the regular bread contains less acid, 2-methylpropanal,
furfural, isoamyl acetate, and (E)-2-octenal. However, the concentra­
tions of (E,E)-2,4-decadienal in the regular bread and the chemically
acidified bread were higher than that in the inoculated sourdough bread.
The aldehyde concentration was reduced by L. sanfranciscensis to those
of the corresponding unsaturated alcohols, which was consistent with a
previous study (Vermeulen et al., 2007). With the addition of inoculated
D. Xu et al. Food Bioscience 37 (2020) 100704

Fig. 3. Heatmap showing the relative abundance and distribution of volatiles in bread. The color code indicates relative abundance, ranging from blue (low
abundance) to white to red (high abundance). LS L. sanfranciscensis; PK P. kudriavzevii; MG M. guilliermondii; CA Chemically acidified; RC Regular bread. (For
interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

sourdough, the types and concentrations of flavor substances were
significantly improved, particularly the sourdough fermented with a
mixed starter at reduced temperatures. Sourdough breads fermented
with selected yeasts showed higher relative concentrations of specific
alcohols (1-pentanol, 1-octanol, and 2,3-butanediol), acids (acetic acid,
2-methylpropanoic acid, and hexanoic acid), esters (ethyl acetate, iso­
amyl acetate, propyl lactate, isoamyl lactate, and ethyl decanoate), al­
dehydes (2-methylpropanal, (E)-2-octenal, and benzeneacetaldehyde),
and other volatiles (2,3-pentanedione, furfural, and 2-furanmethanol),
compared with products fermented by sourdough inoculated with the
L. sanfranciscensis single strain. Compared with other bread groups, the
LS + MG bread showed higher contents of 2-methoxy-4-vinylphenol and
(Z)-3,7-dimethyl-3,6-octadien-1-ol (isogeraniol), which were also iden­
tified as aroma-active compounds.
More aroma compounds were produced in the inoculated sour­
doughs fermented at 10 ◦ C. The left side of the heatmaps shows that low-
temperature (10 ◦ C) fermentation resulted in the accumulation of most
aroma compounds. The concentrations of octanal, nonanal, 3-methylbu­
tanal, and hexanal were higher in the 10 ◦ C LS group than in the 10 ◦ C
LS + MG and 10 ◦ C LS + PK groups, and these concentrations could be
reduced by yeast to that of the corresponding alcohol during sourdough
fermentation (Vermeulen et al., 2007). Yeast metabolism may be the

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D. Xu et al. Food Bioscience 37 (2020) 100704

main reason for the increased concentrations of 1-octanol, 1-nonanol, Table 2

3-methyl-1-butanol, and 1-hexanol in the products fermented with Odor-active compounds identified in bread crumb. The bread was made with
mixed starters. Acidification by LAB during sourdough fermentation sourdough fermented at 10 ◦ C.
activates proteinases for the production of amino acids, which serve as No. Compound Odor at the sniffer FD factor
flavor precursor (Birch et al.,. 2014). Other accumulated aldehydes and port
LS + LS +
alcohols (benzene acetaldehyde, 2-methylpropanal, 2-methyl-1-­ PK MG
propanol, and 2-methylbutanal), which further enhance bread aroma,
Alcohols
may be derived from phenylalanine, isoleucine, and valine using the 1 3-Methylthio-1-propanol Cooked potato 3 0
Ehrlich pathway in yeast cells (Pétel et al., 2016). Esters contribute to 2 2-Methyl-1-propanol Sweet, wine 1 3
the fruity flavor in fermented food (Pico et al., 2015). Compared with 3 3-Methyl-1-butanol Floral 1 1
other bread types, the bread prepared with mixed starters showed a 4 1-Octen-3-ol Mushroom 0 3
5 Benzyl alcohol Floral, 3 3
significantly higher ester content, indicating that the sources of these 6 Phenylethyl alcohol Honey, sweet 3 3
aromatic compounds were the reaction products in yeast cells. More­ 7 (E)-3,7-Dimethyl-2,6-octadien- Floral 3 3
over, ester accumulation increased when the sourdoughs were fer­ 1-ol
mented at 10 ◦ C. Meanwhile, 10 ◦ C LS + PK bread contained markedly (Isogeraniol)
8 2-Methoxy-4-vinylphenol Fruity, clove 9 9
large amounts of 3-ethyl-cyclobutanol acetate. Relatively higher con­
Aldehydes
tents of ethyl acetate, phenylethyl acetate, and ethyl 2-hydroxypropa­ 9 3-Methylthio-propanal Malty 9 27
noate also accumulated. In 10 ◦ C LS + MG bread, isoamyl acetate, 10 3-Methyl-butanal Sweet 9 9
isoamyl lactate, ethyl octanoate, and ethyl decanoate were significantly 11 Hexanal Fruity 1 3
higher than those in other groups. Strain-specific ester formation by 12 Octanal Honey, sweet 1 3
13 Nonanal Citrus 3 3
yeasts is not generally used to improve the quality of bread; however, 14 Benzaldehyde Almond, caramel 3 3
ester formation by brewers’ yeasts contributes to the distinct flavor of 15 Benzeneacetaldehyde Honey, sweet 9 9
individual products and ultimately determine its final quality (Pires Acids
et al., 2014). 16 Acetic acid Sour 27 27
17 3-Methylbutanoic acid Sour, cheesy 3 9
Ketones
18 1-Octen-3-one Mushroom 27 27
3.3. Identification of key odorants in sourdough bread fermented with 19 3-Octen-2-one Mushroom 0 1
mixed culture Esters
20 Ethyl lactate Light fruity 0 1
21 Ethyl octanoate Fruity 1 3
To investigate the important aroma compounds of bread fermented 22 Ethyl decanoate Sweet, fruity 0 9
with Daqu-originated yeast, GC–O analysis, combined with AEDA, was 23 Phenylethyl acetate Rosey, sweet wine 9 9
done. More types and higher contents of volatiles were found in the 24 Isopropyl myristate Floral 9 9
bread crumb made with sourdough fermented at 10 ◦ C. The results of the Heterocyclic compounds
25 2-Pentylfurane Floral, fruity 1 3
olfactometric analysis of these breads are shown in Table 2. GC–O
26 2-Methylpyrazine Burnt, boiled rice 9 0
analysis of bread crumb identified 26 and 27 volatile compounds in the 27 Furfural Roasted, almond 9 27
LS + PK and LS + MG groups, respectively. The highest FD factor of 27 28 1-(2-Furanyl)-ethanone Burnt 3 9
was obtained for 6 odorants among the aforementioned samples. The 29 2-Furanmethanol Caramel, burnt 9 3
Others
results showed that the qualitative pattern of important odor-active
30 Dimethyl trisulfide Cabbage 27 0
volatiles was similar between the breads fermented with
M. guilliermondii and those fermented with P. kudriavzevii; regardless, Note: LS + MG: L. sanfranciscensis and M. guilliermondii; LS + PK:
their content and aroma intensity were affected by the yeasts. L. sanfranciscensis and P. kudriavzevii.
In the LS + PK bread, the compounds acetic acid, 1-octen-3-one, and
dimethyl trisulfide (FD = 27) showed sour, mushroom-, and cabbage- 3.4. Effect of temperature on total α-amino nitrogen in sourdough and
like attributes, respectively. A rather lower FD factor (FD = 9) was related aroma compounds in bread
determined for 2-methoxy-4-vinylphenol (clove), 3-methylthio-propa­
nal (malty), 3-methyl-butanal (sweet), benzene acetaldehyde (sweet, During sourdough fermentation, proteins are partly degraded and
honey-like), phenylethyl acetate (rosey, sweet wine), isopropyl myr­ proteolytic events, such as the formation of peptides and amino acids,
istate (floral), 2-methylpyrazine (burnt, boiled rice), furfural (roasted, which can contribute to the taste, flavor, and texture of bread (Thiele
almond), and 2-furanmethanol (caramel, burnt). In the LS + MG bread, a et al., 2004). To identify the relationship between protein hydrolysis and
high FD factor (FD = 27) was also measured for acetic acid and 1-octen- the formation of bread aroma, overall proteolysis in sourdough was
3-one; in addition, a high FD factor (FD = 27) was determined for other quantified by determining total α-amino nitrogen (Table 3) (Lie, 1973).
compounds, such as 2-methoxy-4-vinylphenol (clove), 3-methylthio- After chemical acidification for 20 h, the concentration of amino ni­
propanal (malty), and furfural (roasted, almond). trogen in the dough doubled from the initial value, which further
Several other odorants also showed large differences in FD factor. increased with the inoculation of microorganisms. Significant differ­
The FD factors of 2-methyl-1-propanol (sweet, wine), hexanal (fruity), ences were not observed among inoculated sourdoughs, except for 10 ◦ C
octanal (honey-like, sweet), 3-methylbutanoic acid (sour, cheesy), ethyl LS + MG and 20 ◦ C LS + MG. The results suggested that the acids
octanoate (fruity), and 2-pentylfurane (floral, fruity) were higher in the increased the proteinase activity of the flour and led to an increase in
LS + MG bread. 3-Methylthio-1-propanol (cooked potato) was smelled amino nitrogen. The concentration of amino nitrogen also showed a
only in LS + PK bread. Meanwhile, 1-octen-3-ol (mushroom), 3-octen-2- strain-dependent contribution to proteolytic activity and varied with
one (mushroom), ethyl lactate (fruity), and ethyl decanoate (sweet, fermentation temperature and time. M. guilliermondii might have
fruity) were found only in bread fermented with M. guilliermondii. absorbed more amino acids at 20 ◦ C; however, the accumulated amino
Among these odorants, 2-methoxy-4-vinylphenol and isopropyl myr­ nitrogen in sourdough was higher than that absorbed by yeast when
istate were rarely reported in bread flavor, which had a distinctive fermented at 10 ◦ C. Therefore, the low temperature contributed more to
fragrance, as determined using an olfactometric detector; these odorants the accumulation of amino nitrogen by M. guilliermondii at the same
were also speculated to be among the most important compounds that yeast growth stage, which showed the potential to provide larger
contribute to the aroma of the two sourdough bread samples. amounts of flavor volatiles or flavor precursors. In Fig. 3, several key

7
D. Xu et al. Food Bioscience 37 (2020) 100704

Table 3
Concentration of total amino nitrogen and amino acids in fermented sourdoughs and volatiles related to amino acid metabolism in bread crumb. (n = 3).
RC CA 30 ◦ C 30 ◦ C 30 ◦ C 20 ◦ C 20 ◦ C 20 ◦ C 10 ◦ C 10 ◦ C 10 ◦ C
LS LS + MG LS + PK LS LS + MG LS + PK LS LS + MG LS + PK

Total amino nitrogen 0.6 ± 1.3 ± 0.1d 2.0 ± 1.9 ± 0.1b 2.0 ± 1.9 ± 1.5 ± 1.9 ± 1.9 ± 2.2 ± 1.9 ±
(mg/g) 0.1e 0.1b 0.1b 0.1b 0.1c 0.1b 0.1b 0.1a 0.1b
free amino acids ( £ 10¡2 mg/g) and related volatiles (μg/kg)
Glutamate 10.4 ± 5.9 ± 0.3f 16.3 ± 15.8 ± 15.6 ± 16.3 ± 13.6 ± 13.8 ± 16.8 ± 17.5 ± 14.1 ±
0.2e 0.3bc 0.3c 0.1c 0.3bc 0.3d 0.4d 0.3b 0.2a 0.4d
Lysine 2.2 ± 6.6 ± 0.1f 11.2 ± 11.7 ± 10.4 ± 11.5 ± 10.6 ± 9.1 ± 0.2e 11.7 ± 12.9 ± 9.7 ±
0.1g 0.4b 0.2b 0.3c 0.2b 0.2c 0.2b 0.1a 0.2d
Methionine 4.65 ± 3.53 ± 3.17 ± 4.56 ± 3.2 ± 0.1 3.22 ± 4.68 ± 3.50 ± 3.29 ± 5.16 ± 3.95 ±
0.02b 0.05e 0.02g 0.03c fg 0.04 fg 0.05b 0.03e 0.05f 0.05a 0.04d
Isoleucine 0.96 ± 2.50 ± 5.27 ± 5.42 ± 4.30 ± 5.21 ± 4.6 ± 3.71 ± 5.40 ± 5.55 ± 4.01 ±
0.03h 0.01g 0.04c 0.03b 0.04d 0.03c 0.1a 0.03f 0.04b 0.04a 0.03e
Leucine 4.1 ± 11.9 ± 16.3 ± 16.9 ± 14.6 ± 16.4 ± 13.0 ± 10.7 ± 16.9 ± 15.6 ± 12.0 ±
0.1g 0.2e 0.3a 0.3a 0.2c 0.4a 0.4d 0.3f 0.5a 0.2b 0.4e
3-Methyl-1-butanol 1.27 ± 1.1 ± 0.1 1.04 ± 1.23 ± 1.17 ± 1.18 ± 1.2 ± 0.1 1.21 ± 1.48 ± 1.72 ± 1.99 ±
4
0.03 ( × ( × 10 )f 0.04 ( × 0.02 ( × 0.04 ( × 0.02 ( × ( × 104)e 0.03 ( × 0.03 ( × 0.02 ( × 0.02 ( ×
104)d 104)f 104)de 104)e 104)e 104)de 104)c 104)b 104)a
3-Methylbutanal 34±3h 39±2gh 110 ± 61±4g 84±3f 330 ± 20c 120 ± 260 ± 640 ± 310 ± 44 ± 20b
10ef 10e 10d 30a 10c
Isoamyl acetate 40±3h 220 ± 20g 240 ± 20 430 ± 30 340 ± 270 ± 450 ± 390 ± 10c 290 ± 470 ± 410 ±
fg ab 10d 10ef 20a 10e 40a 10bc
Valine 4.4 ± 6.7 ± 0.1d 11.3 ± 11.5 ± 10.1 ± 11.5 ± 10.5 ± 9.6 ± 0.2c 11.8 ± 11.9 ± 9.8 ±
0.1e 0.3a 0.2a 0.4bc 0.2a 0.2b 0.3a 0.4a 0.1c
2-Methyl-1-propanol 1.4 ± 0.1 1.3 ± 0.1 0.8 ± 0.1 1.05 ± 1.1 ± 0.1 1.15 ± 1.2 ± 0.1 1.4 ± 0.1 1.6 ± 0.1 2.0 ± 0.1 1.6 ± 0.1
3 3
( × 10 )c ( × 10 )d ( × 103)f 0.03 ( × ( × 103)e 0.03 ( × ( × 103) ( × 103)c ( × 103) ( × 103)a ( × 103)b
103)e 103)de de bc
2-Methylpropanal – 6.6 ± 0.7 7.2 ± 0.3 8.6 ± 0.4 ( 8.4 ± 0.3 7.8 ± 0.2 1.10 ± 1.1 ± 0.1 8.4 ± 0.3 1.10 ± 1.3 ± 0.1
( × 102)e ( × 102) × 102)c ( × 102)c ( × 102) 0.05 ( × ( × 103)b ( × 102)c 0.05 ( × ( × 103)a
de cd 103)b 103)b
2-Methylpropanoic 8±3e 21±5d 44±4c 94±8a 97 ± 12a – – 65±6b – – 61±6b
acid
Phenylalanine 1.37 ± 4.84 ± 8.28 ± 8.8 ± 0.1b 7.14 ± 8.35 ± 7.8 ± 6.76 ± 8.7 ± 9.6 ± 7.57 ±
0.02j 0.03i 0.04d 0.03g 0.05d 0.1e 0.05h 0.1c 0.1a 0.05f
Phenylethanol 5.2 ± 0.1 6.2 ± 0.2 4.6 ± 0.2 5.7 ± 0.2 ( 7.2 ± 0.3 7.7 ± 0.2 7.2 ± 0.2 7.0 ± 0.1 1.5 ± 0.1 1.5 ± 0.1 1.3 ± 0.1
( × 102)e ( × 102)d ( × 102)e × 102)de ( × 102)c ( × 102)c ( × 102)c ( × 102)c ( × 103)a ( × 103)a ( × 103)b
Benzeneacetaldehyde 2± < 0.5e 3± < 0.5e 6±1e 52±6c 36±4d 9±2e 60 ± 65±9b 12±3e 69±6b 83 ± 11a
12bc
Phenylethyl acetate 10±2f 12±2f 18±3f 31±4cd 15±3f 18±5e 26±3d 33±2c 44±5b 46±5b 62±4a

Note: LS: L. sanfranciscensis; LS + MG: L. sanfranciscensis and M. guilliermondii; LS + PK: L. sanfranciscensis and P. kudriavzevii; CA: chemically acidified control; RC:
wheat flour or regular bread control. –: Not detected. Data are shown as means ± standard deviation of triplicate independent fermentation. Values labeled with
different letters are significantly different in the same row (p < 0.05).

aroma-active compounds from the Maillard reaction, such as 2-pentyl­
furane, furfural, and 2-furanmethanol, were found in bread crumbs,
and their contents increased with the sourdough fermented at 10 ◦ C. The
accumulated amino nitrogen might have reacted with the sugars in
sourdough, increasing the amounts of furans and pyrazines even in
bread crumbs.
3.5. Correlation of free amino acids in sourdough to the important aroma
compounds in bread
Microbial modification of amino acids during fermentation and
thermal degradation during baking can influence the flavor of bread
(Salim-ur-Rehman et al., 2006). This study also investigated the effects
of microbiota and temperature on the accumulation of free amino acids
in fermented sourdoughs. The concentrations of the free amino acids are
shown in Table 3. The relationships between free amino acids in sour­
dough fermented with different starter cultures and fermentation tem­
perature were then assessed by PCA (Fig. 4). The contents and types of
amino acids were evidently affected by starter cultures and fermentation
temperatures. The conversion of amino acids to flavor volatiles using the
Ehrlich pathway in yeast cells is one of the main metabolic pathways for
flavor formation. Methionine, isoleucine, leucine, valine, and phenyl­
alanine are precursors for flavor active compounds (Pétel et al.,. 2016).
The relative contents of volatiles related to amino acid metabolism are
also shown in Table 3. PLSR was used to evaluate the effect of amino
acids on the formation of volatile compounds.
The accumulation of free amino acids in sourdough was strongly
influenced by the decrease in pH. The contents of most amino acids,
except for glutamate and methionine, increased significantly. In sour­
dough fermented with L. sanfranciscensis, all amino acids, except for
methionine, further accumulated and were comparable at different
temperatures. These results indicated that microbial peptidases facili­
tate the release of amino acids during fermentation (Yazar 2012). The
concentration of individual amino acids varied between sourdoughs
inoculated with different mixed starter cultures, which can be attributed
to the strain-specific proteinase activity and amino acid utilization of
yeast. The overall trend of proteolysis was consistent with the effect of
respective cultures on the accumulation of amino nitrogen. As shown in
Table 3, although the two yeast strains showed different changes in
amino acids in fermented sourdough, the corresponding products to the
amino acids were consistent with the Ehrlich pathway. Sourdoughs
fermented with L. sanfranciscensis and P. kudriavzevii accelerated the
utilization of valine, phenylalanine, isoleucine, leucine, and lysine,
which had to be converted to corresponding aldehydes, alcohols, and
acids using the Ehrlich pathway (Hazelwood et al., 2008). For example,
leucine content in the 30 ◦ C LS + PK group was about 0.146 mg/g, which
was lower than that in the 30 ◦ C LS group; meanwhile, the corre­
sponding products of 3-methylbutanal were consumed, and 3-methyl-1-­
butanol accumulated, compared with the 30 ◦ C LS group. Similar
changes related to the Ehrlich pathway were also observed in the
sourdough fermented with M. guilliermondii at different temperatures.
With a decrease in temperature to 10 ◦ C, the amino acid metabolism

8
D. Xu et al. Food Bioscience 37 (2020) 100704

Fig. 4. PCA of free amino acid concentrations in fermented sourdough (Panel A) and PLSR correlation loadings plot of volatile compounds in the X-matrix for bread
and amino acid in the Y-matrix for sourdough (Panel B). Ellipses represent r2 = 0.5 and 1.0, respectively.

correlated products, such as 3-methyl-1-butanol, 3-methylbutanal, 3.6. Bread sensory evaluation

2-methyl-1-propanol, 2-methylpropanal, phenylethanol, and benzene
acetaldehyde, were increased. This effect may be attributed to the
accumulation of amino acids during fermentation. Glutamate, which
imparts umami or a savory flavor (Tang et al., 2017), also accumulated
at 10 ◦ C.
To compare the sensory quality of breads made with sourdough
fermented at different temperatures, the appearance, aroma, taste,
texture, and overall quality of the breads were evaluated by a consumer
panel. No significant difference in bread appearance was observed

9
D. Xu et al. Food Bioscience 37 (2020) 100704

among all samples; moreover, chemically acidified bread had the lowest
scores in the other aspects (Fig. 5). These results indicated that microbial
metabolism, rather than simple acidification, is the main reason for
bread quality improvement. Breads made with mixed culture achieved
the highest scores for aroma and taste. The 10 ◦ C LS + MG and 10 ◦ C LS
+ PK groups received the highest scores (>7.2) for aroma, indicating
that the sourdough fermented at low temperatures for a long time
contributed more to aroma formation. The result was consistent with
GC-MS. With regard to taste, the 10 ◦ C LS + MG and 10 ◦ C LS + PK
sourdough breads still achieved higher scores, compared with the other
groups. The sourdough bread fermented with P. kudriavzevii showed no
advantages in volume and bread crumb hardness, which may be

Fig. 5. Sensory evaluation of breads. (n = 30) LS L. sanfranciscensis; PK P. kudriavzevii; MG M. guilliermondii; CA Chemically acidified; RC Regular bread.

10
D. Xu et al.
attributed to gluten proteolysis; however, it was comparable to bread
prepared using the straight-dough method (additional data on bread
volume and crumb hardness are shown in Fig. S1). The texture of the
breads fermented with mixed starters at 10 ◦ C improved, which may be
due to the compensation of extracellular polysaccharides formed by LAB
during low-temperature fermentation over an extended period (Korakli
et al.,. 2001). For overall quality, the sourdough bread with mixed
cultures fermented at 10 ◦ C achieved the highest scores, indicating that
the bread prepared with sourdough fermented at low temperatures was
preferred by the panelists.
4. Conclusions
Improvements with low-temperature sourdough fermentation using
mixed starters (M. guilliermondii and P. kudriavzevii combined with
L. sanfranciscensis) to fermentation done at 30 ◦ C, as evidenced by the
enriched aroma compounds of the sourdough bread. The variation in
yeast in the starter culture differentiated the products based on the
production of volatiles, which contributed to the variations in bread
aroma profile. The most important compounds affecting the sensory
qualities of the sourdough bread fermented with M. guilliermondii were
acetic acid, 1-octen-3-one, and dimethyl trisulfide. Those affecting the
sensory qualities of sourdough bread fermented with P. kudriavzevii
were acetic acid, 1-octen-3-one, 2-methoxy-4-vinylphenol, 3-methyl­
thio-propanal, and furfural. Bread produced with sourdoughs fer­
mented at different temperatures varied in aromatic volatile profiles.
Reducing the fermentation temperature to 10 ◦ C increased the formation
of these important volatile compounds and other aroma-active com­
pounds, such as 3-methyl-1-butanol, 3-methylbutanal, 2-methyl-1-prop­
anol, 1-octen-3-ol, isogeraniol, benzaldehyde, benzene acetaldehyde,
ethyl octanoate, ethyl decanoate, phenylethyl acetate, furfural, and 2-
pentylfurane. The addition of sourdough fermented with mixed start­
ers at 10 ◦ C promoted bread aroma and generally improved their sensory
qualities; as such, they were preferred by panelists. This study developed
a different concept for industrial sourdough production as well as
traditional fermented foods. Low-temperature fermentation for an
extended period may also help achieve sourdough fermentation during
transport from factory to bakery, thus reducing production time and
energy consumption.
CRediT authorship contribution statement
Dan Xu: Conceptualization, Methodology, Investigation, Writing -
original draft. Huang Zhang: Supervision, Writing - review & editing.
Jinzhong Xi: Software, Formal analysis. Yamei Jin: Software, Valida­
tion. Yisheng Chen: Funding acquisition. Lunan Guo: Writing - review
& editing. Zhengyu Jin: Supervision, Funding acquisition. Xueming
Xu: Supervision, Funding acquisition, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The National “Thirteenth Five-Year” Plan for Science & Technology
Support of China (No. 2018YFD0400604), the China Postdoctoral Sci­
ence Foundation (No. 2020M671344), the National Natural Science
Support of China (No. 31872905), the Development and Research
Center of Sichuan Cuisine Foundation (CC19Z06), the Natural Science
Foundation of Jiangsu Province (BK20170177), and the Key Scientific
and Technological Innovation Project of Shandong Province (No.
2019JZZY010722), and the foundation (No. KF201910) of State Key
Labortary of Biobased Materials and Green Papermaking, Qilu
11
Food Bioscience 37 (2020) 100704
University of Technology, Shandong Acadamy of Science.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.fbio.2020.100704.
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