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Intraspecific diversity and fermentative properties of Saccharomyces cerevisiae from Chinese traditional sourdough
Journal Pre-proof Intraspecific diversity and fermentative properties of Saccharomyces cerevisiae from Chinese traditional sourdough Huanyi Yang, Tongjie Liu, Guohua Zhang, Guoqing He PII:
S0023-6438(20)30183-3
DOI:
https://doi.org/10.1016/j.lwt.2020.109195
Reference:
YFSTL 109195
To appear in:
LWT - Food Science and Technology
Received Date: 24 October 2019 Revised Date:
25 January 2020
Accepted Date: 21 February 2020
Please cite this article as: Yang, H., Liu, T., Zhang, G., He, G., Intraspecific diversity and fermentative properties of Saccharomyces cerevisiae from Chinese traditional sourdough, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109195. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Credit Author Statement Huanyi Yang: Investigation, Methodology, Data curation, Writing-original draft. Tongjie Liu: Validation, Writing-review & editing. Guohua Zhang: Funding acquisition. Guoqing He: Supervision, Project administration.
1
Intraspecific diversity and fermentative properties of Saccharomyces cerevisiae from
2
Chinese traditional sourdough
3 4
Huanyi Yang a, Tongjie Liu b, Guohua Zhang d ∗∗, Guoqing He c ∗
5
a
College of Life Science, Shaoxing University, Shaoxing, 312000, China
6
b
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
7
c
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058,
8
China
9
d
College of Life Science, Shanxi University, Taiyuan, 030006, China
10 11
Abstract
12
One hundred and fifty isolates of Saccharomyces cerevisiae from thirteen Chinese traditional
13
sourdoughs of different regions were obtained and typed into 5 clusters by random amplified
14
polymorphic DNA-polymerase chain reaction (RAPD) analysis. Then twenty-two S. cerevisiae
15
strains were selected, according to the RAPD profiles, to evaluate their fermentation properties in
16
dough fermentation, and to assess their influences on the texture and flavor of steamed bread. The
17
results showed that the S. cerevisiae strains greatly varied in fermentation capacity and influence
18
on organoleptic property of steamed bread. However, the variation was not apparently related to
19
their genetic diversity distinguished by RAPD. A total of 19 volatile compounds were identified in
∗
Corresponding author. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China. E-mail address: [email protected] (G. He) ∗∗ Co-corresponding author. College of Life Science, Shanxi University, Taiyuan, 030006, China. E-mail address: [email protected] 1
20
the crumb of the steamed breads fermented by the 22 S. cerevisiae strains with ethanol and
21
3-methyl-1-butanol being the most encountered compounds. S. cerevisiae fermentation had a
22
significant effect on textural properties of steamed bread, especially hardness, chewiness and
23
gumminess.
24
Keywords: sourdough; S. cerevisiae; RAPD; leavening ability; volatile compounds
25 26
1. Introduction
27
Sourdough, a mixture of flour and water fermented by lactic acid bacteria (LAB) and yeast,
28
has been used in making cereal-based fermented products for thousands of years around the world
29
(De Vuyst and Neysens, 2005). In China, sourdough has traditionally served as the starter for the
30
preparation of the staple food Chinese steamed bread (CSB) for more than one thousand years
31
(Wu et al., 2012). Sourdough fermentation confers positive effects to breads by improving dough
32
properties such as volume, texture and flavor, increasing nutritional values and prolonging its
33
shelf life (Vigentini et al., 2014; Zotta et al., 2006) ascribed to the metabolic activities of inherent
34
LAB and yeasts (Zotta et al., 2006).
35
Although the main contribution of yeasts in sourdough fermentation is leavening, they play
36
an important role in aroma formation and texture development (Chavan and Chavan, 2011; Katina
37
et al., 2006). Particularly, Saccharomyces cerevisiae has been considered the predominant yeast in
38
sourdough ecosystems (Minervini et al., 2012; Zhang, Sadiq, et al., 2015; Zhang et al., 2011). S.
39
cerevisiae can rapidly generate CO2 from sugars during fermentation, which results in dough
40
expanding (Heitmann et al., 2016). Moreover, S. cerevisiae improves the flavor of breads by
2
41
producing a wide range of flavor compounds, such as alcohols, aldehydes, acetoin and esters
42
(Birch et al., 2013; Frasse et al., 1992). In addition, the production of glutathione, glycerol and
43
pyruvic acid by S. cerevisiae helps to develop and strengthen gluten network in dough, and leads
44
to a positive effect on the texture of bread (Corsetti et al., 2000; Lampignano et al., 2013;
45
Verheyen et al., 2015).
46
It has been reported that S. cerevisiae showed a phenotypic and genotypic intra-species
47
diversity in sourdoughs (Landry et al., 2006). Thus, the possibility of discriminating at strain level
48
is of great importance for a successful culture selection and starter development. To date, several
49
studies have been conducted to investigate the intra-species diversity of S. cerevisiae (Ayoub et al.,
50
2006; Bogusławska-Wąs et al., 2007; Munoz et al., 2009). However, in most cases, no further
51
information was available regarding the correlation between genetic diversity and fermentative
52
properties in dough fermentation.
53
To the best of the authors' knowledge, the genetic and fermentative diversities of S.
54
cerevisiae in Chinese traditional sourdough have never been collectively investigated and
55
analyzed. This study aimed at delineating the genetic diversity and fermentative properties of S.
56
cerevisiae strains isolated from Chinese traditional sourdoughs of different regions.
57
2. Materials and methods
58
2.1. Sourdough collection
59
Thirteen home-made sourdoughs from ten different provinces in China were sampled in this
60
study. Samples were chosen for their specific origin and were stored immediately at 4 °C once in
61
the laboratory.
3
62
2.2. Yeasts enumeration and isolation
63
Five grams of sourdough was homogenized with 45 mL of sterile peptone water (peptone 1
64
g/L and NaCl 8.5 g/L) solution and decimally diluted. An aliquot of 100 µl of the 10-3 to 10-5
65
dilutions was plated onto yeast extract peptone dextrose (YPD) agar medium (1% yeast extract,
66
2% peptone, 2% dextrose, and 2% agar) supplemented with chloramphenicol (0.1 g/L). Colonies
67
were counted after incubation at 28 °C for 48 h. The plate counts were performed in triplicate. For
68
each sourdough sample, fifteen colonies were randomly picked from the plates of the three
69
dilutions, then purified by successive streak plate method.
70
2.3. Identification of S. cerevisiae strains
71
Based on the colony morphology (Casalone et al., 2005), certain isolates were considered
72
presumptive strains of S. cerevisiae and further identified by 26S rRNA gene sequencing.
73
Genomic DNA from presumptive S. cerevisiae isolate was extracted using a DNA Extraction Kit
74
(Axygen, Hangzhou, China), following the manufacturer’s instructions. The primers, NL1 and
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NL4 (Di Cagno et al. 2014), were used to amplify the D1/D2 region of the 26S rRNA gene. The
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PCR products were sequenced by a company (Shanghai Sangon Biotech, Shanghai, China), and
77
the results were compared with those deposited in the GenBank using the BLAST algorithm.
78
Strains showing ≥98% identity with S. cerevisiae were selected for further characterization.
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2.4. RAPD-PCR analysis
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Genotypic diversity of S. cerevisiae strains was assessed by RAPD analysis using two primers
81
separately, namely M13 (5’-GAGGGTGGCGGTTCT-3’) (Stenlid et al., 1994) and MV1
82
(5’-GGACGCTTCTG-3’) (Venturi, Guerrini and Vincenzini, 2012). The amplification conditions
4
83
with primer M13 were as follows: preliminary denaturation for 5 min at 94 °C, followed by 34
84
cycles of 94 °C for 1 min, 45 °C for 2 min, 72 °C for 1.5 min, and terminated with an extension at
85
72 °C for 10 min (Liu et al., 2018). The conditions with primer MV1 were set according to
86
Venturi, Guerrini, Granchi, et al. (2012). Breifly, preliminary denaturation for 5 min at 94 °C,
87
followed by 30 cyceles of 94 °C for 1 min, 40 °C for 1 min and 72 °C for 2 min, and terminated
88
with an extension at 72 °C for 10 min. The amplicons were electrophoresed on 1.5% agarose gel
89
supplemented with the DNA dye GelRed Nucleic Acid Gel Stain (Biotium Hayward, Ca, USA)
90
for 5.5 h at a constant voltage of 70 V in 1×TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0)
91
buffer and the gel images were captured in a Gel Imaging System (ChampGel 500,
92
SAGECREATION, Beijing, China). BioNumerics version 6.6 software package (Applied Maths,
93
Sint-Martens-Latem, Belgium) was used to convert and analyze the resulting fingerprints. RAPD
94
profiles generated with M13 and MV1 primers were integrated to increase the typing efficiency
95
and discriminatory power. According to the RAPD profiles, representative strains were selected
96
and inoculated in a rotary shaking incubator with 180 rpm at 28 °C for 48 h. The cells were
97
collected by centrifugation at 3800×g for 10 min and washed twice with sterile distilled water for
98
subsequent experiments.
99
2.5. Leavening ability determination
100
The leavening ability of the selected S. cerevisiae isolates was measured according to the
101
methods described by Liu et al. (2018). The dough was inoculated with S. cerevisiae at an
102
inoculum size of 107 cfu/g. Leavening ability was expressed by volume (milliliter) of CO2
5
103
produced at 30 °C in one hour. Experiments were performed in triplicate and dough without
104
inoculation was set as a negative control.
105
2.6. Glucose fermentation determination
106
Wheat flour, water and yeast cells (inoculum size of 107 cfu/g dough) were mixed to obtain a
107
dough with a dough yield (dough weight × 100/flour weight) of 150 and placed in an incubator at
108
30 °C and 80% RH for 2 h (STIK (Shanghai) Co., Ltd, China). Ten grams of dough was
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homogenized with 90 mL of distilled water for 15 min using a magnetic stirrer (IKA basic 2 RH,
110
Germany) until the sample was thoroughly suspended. Then, the suspension was centrifuged at
111
3800 × g for 10 min. The concentration of glucose in supernatant was measured using a glucose
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content assay kit (Shanghai Rongsheng Biotech Co., Ltd.). Experiments were performed in
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triplicate and the dough without inoculation was used as a control.
114
2.7. Steamed bread manufacturing
115
The steamed bread was made according to the method given by Liu et al. (2018). Wheat flour,
116
water and the cells of S. cerevisiae (inoculum size of 107 cfu/g dough) were thoroughly mixed in a
117
mixer (HMJ-D3826, Guangdong bear electric Co., Ltd. China) for 10 min to form a dough with a
118
dough yield of 150. Afterwards, the dough was shaped manually and placed in an incubator at
119
30 °C and 80% RH for 2 h. Finally, the dough was steamed for 20 min in a steamer. The controls
120
were prepared using the same procedures but were not inoculated with S. cerevisiae strains.
121
2.8. Texture analysis of the steamed bread
122
The textural properties of the steamed bread were investigated using a TA-XT2i Texture
123
Analyzer (Stable Micro System, Ltd., Godalming, UK) with a Texture Profile Analysis (TPA)
6
124
mode (Caine et al., 2003). After cooling for 5 min, the samples were uniformly sliced with a
125
thickness of 10 mm and cut into cylindrical slices (35 mm diameter) from the center of each loaf.
126
To measure the textural properties of the samples, a 100 mm diameter cylindrical probe was used
127
to compress each slice with 40% deformation at a speed of 1 mm/s. At least 6 replicates were
128
performed for each sample.
129
2.9. Volatile compounds of steamed bread
130
Volatile compounds of the steamed breads fermented by different S. cerevisiae strains were
131
determined according to Liu et al. (2018). Solid phase microextration (SPME) was used for the
132
extraction of the volatile compounds and gas chromatography-mass spectrometry (GC-MS) was
133
used for the separation and identification. The flavor extraction and subsequent injection processes
134
were automatically performed using the MPS autosampler (Gerstel, Mülheim, Germany). The
135
fiber 75 µm Carboxen/polydimethylsiloxane (CAR/PDMS; Supelco, Bellefonte, PA) was
136
employed for the extraction of volatile compounds at 60 °C for 30 min and then inserted into the
137
injection port of the GC (7890B; Agilent Technologies), equipped with a DB-WAX capillary
138
column (J&W Scientific, 30 m long × 0.25 mm internal diameter, 0.25 µm film thickness), to
139
desorb the extracted volatiles at 250 °C for 4 min in splitless mode. The column temperature was
140
set at 40 °C for 2 min, increased to 230 °C at a rate of 5 °C/min. The carrier gas was helium with a
141
flow rate of 1.0 mL/min. The GC was coupled to an MS detector (5977A; Agilent Technologies)
142
used in scan mode (35 to 500 m/z) with an electronic impact of 70 eV. The identification of
143
volatile compounds was carried out by comparison of the mass spectral data obtained with those
7
144
in a commercial database (NIST 11). The peak area of each volatile compound was integrated by
145
selecting a specific ion as described by Di Cagno et al. (2014).
146
2.10. Statistical analysis
147
Data from yeast enumeration, leavening ability and glucose fermentation were subjected to
148
one-way ANOVA analysis and were expressed as mean values ± standard deviations and the
149
multiple comparisons was performed using Tukey’s test (SPSS Statistics). Principal components
150
analysis (PCA) was performed using the software XLSTAT (version 2014.5.03) to compare the
151
steamed breads fermented with different S. cerevisiae strains based on their volatile profiles.
152
3. Results and discussion
153
3.1. Isolation of S. cerevisiae from Chinese sourdoughs
154
The results of yeast enumeration of the sourdoughs were given in Table 1. Cell density of
155
yeasts varied from 5.63 ± 0.02 (Xj) to 8.46 ± 0.01 (Pl) log cfu/g, and the median value was 7.08
156
log cfu/g. Similar results can be found in the previous studies performed by Lhomme et al. (2015)
157
and Minervini et al. (2012). Noticeably, S. cerevisiae was not found in the sourdough sample Ts.
158
According to the colony morphology, a total of 178 presumptive S. cerevisiae strains were
159
subjected to 26S rRNA sequencing analysis. 150 of the 178 strains exhibited ≥98% identity with
160
S. cerevisiae. Thus, 76.9% of the total strains isolated from the sourdough samples in this study
161
were S. cerevisiae (Table 1), illustrating the predominance of this species in Chinese traditional
162
sourdough, in accordance with previous studies (Valmorri et al., 2010; Zhang, Sadiq, et al., 2015).
163
3.2. Genotypic characterization by RAPD analysis
8
164
RAPD fingerprinting is a simple, rapid and sensitive technique and it has been widely used to
165
differentiate strains of yeast species (Bogusławska-Wąs et al., 2007; Di Cagno et al., 2014;
166
El-Fiky et al., 2012; Pfliegler et al., 2014). In order to increase the typing efficiency and
167
discriminatory power, RAPD patterns generated with M13 and MV1 primers individually were
168
integrated into a unique clustering profile as shown in Fig. 1. Based on the similarity level of 75%,
169
at which level isolates in different types differed by no less than three bands, the 150 S. cerevisiae
170
isolates from Chinese traditional sourdoughs were grouped into 5 clusters. Cluster 3 was the most
171
represented cluster with 116 strains isolated from all sourdough samples except Zj, accounting for
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77.3% of total isolates, while cluster 5 included all the S. cerevisiae strains isolated from
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sourdough Zj. Meanwhile, isolates from the same sourdough could be divided into different
174
clusters. For instance, strains isolated from sourdough Sx, Ah, Xj, Wf and Gm were separately
175
divided into at least two clusters. Particularly, strains from sourdough Xj, allocated into four
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clusters, showed the most diversity. The genetic characterization of this study showed the
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intraspecific variability of S. cerevisiae isolates from traditional sourdoughs, in accordance with
178
the finding of De Vuyst et al. (2016). Regarding this observation, the technological factors, the
179
manufacturing environment as well as geographical origins may explain the genotypic diversity of
180
these populations (Legras et al., 2007; Tapsoba et al., 2015), but it should be confirmed in further
181
studies through a much wider sampling.
182
3.3 Leavening ability and glucose fermentation
183
In order to describe the fermentative properties, a total of 22 isolates (Table 2) were selected
184
on the basis of the genotypic differentiation by RAPD analysis. Leavening ability is one of the
9
185
properties required for the commercial applications of yeast. A high leavening ability of S.
186
cerevisiae strains is apparently economically beneficial for bread producers. S. cerevisiae use
187
alcoholic fermentation to convert sugar into ethanol, carbon dioxide and glycerol. Carbon dioxide
188
causes the leavening of the dough. As shown in Table 3, the CO2 production of the selected strains
189
varied in an extensive range from 28.33 ± 4.04 mL (Wf4) to 220.67 ± 2.81 mL (Sx5), indicating
190
significant differences in the leavening abilities of these S. cerevisiae strains. Sx5, Ah1, Gs10 and
191
Hb3 were the strains possessing the highest leavening ability (220.67 ± 2.81, 217.33 ± 7.06,
192
211.80 ± 2.46 and 211.70 ± 4.19 mL CO2, respectively). S. cerevisiae ferment the flour
193
carbohydrates present in the dough to carbon dioxide and ethanol through glycolysis and glucose
194
is the preferentially utilized sugar (Heitmann et al., 2016). The glucose fermentation
195
characteristics of the 22 S. cerevisiae strains were also measured (Table 3). Interestingly, not all
196
strains with high leavening ability correlated with high ability of glucose utilization. For instance,
197
strain Xj7 and Gs6 produced more amount of CO2 than other strains, yet they showed an inferior
198
ability in glucose fermentation. It seems that some other factors apart from the ability of glucose
199
fermentation could affect the leavening ability of S. cerevisiae strains. Although S. cerevisiae
200
mainly ferments glucose in the beginning of fermentation, it also uses maltose and fructose as
201
carbohydrate sources for metabolism in later stages (Gabriela and Daniela, 2010). Moreover, the
202
leavening ability of S. cerevisiae has been proven to be influenced not only by glycolytic activity,
203
but also by sucrase activity and osmotolerance (Tokashiki et al., 2011). The mode of regulation of
204
maltose might affect its leavening ability as well (Birch et al., 2013; Zhang, Bai, et al., 2015).
205
Hence, the difference of these factors among S. cerevisiae strains might lead to the variations in
10
206
leavening ability. However, the underlying mechanism that can explain the variation in leavening
207
ability needs further investigation.
208
According to the results in this study, no obvious relationship could be found between
209
genetic typing by the two primers and the abilities of leavening and glucose fermentation. Strains
210
belonging to the same cluster based on RAPD analysis differed in the leavening and glucose
211
fermentation abilities, such as Gm4 and Xj7. On the other hand, some strains divided into
212
different clusters had similar abilities of leavening and glucose fermentation, such as Sx5 and Ah1.
213
Therefore, it indicated that the genetic diversity delineated by RAPD in this study may not reflect
214
the heterogeneity of the glycolytic pathway genes or maltose degradation operon, which was in
215
according to the previous study of De Angelis et al. (2007).
216
3.4 Textural properties of steamed bread
217
Texture of steamed bread is an important organoleptic property affecting consumer’s
218
acceptability (Arendt et al., 2007). The hardness, gumminess, chewiness, springiness,
219
cohesiveness, and resilience of steamed bread crumb fermented by different S. cerevisiae strains
220
were obtained from texture profile analysis (TPA). Relationships between textural properties and
221
S. cerevisiae strains were analyzed by PCA (Fig. 2). The first two components of the PCA
222
explained 82.81% of the total variance (58.78% and 24.03%, respectively). As shown in Fig. 2, all
223
samples fermented with S. cerevisiae strains were separated considerably from the blank,
224
indicating that S. cerevisiae fermentation had a significant effect on textural properties (especially
225
hardness, chewiness and gumminess) of steamed bread. In addition, the effect seemed to vary
226
among different strains, for most of the samples fermented with different S. cerevisiae strains
11
227
were separated from each other on the PCA map, including the strains belonged to the same
228
cluster by RAPD analysis such as Gs6 and Xj4, Gm4 and Xj7 and Wf4 and Hb11. During dough
229
fermentation, CO2 is produced by yeast through glycolysis route and is entrapped in the gluten
230
matrix of dough generating numerous gas cells, which leads to a lower extensibility and a higher
231
specific volume of steamed bread (Heitmann et al., 2016; Verheyen et al., 2014). Apart from
232
leavening dough, yeast also helps to develop and strengthen the gluten network. Glutathione,
233
which could be released by some nonviable yeast cells as a stress response (Penninckx, 2002;
234
Verheyen et al., 2015), modifies the viscoelastic gluten network of dough by increasing the rate of
235
thiol-disulfide interchange reactions because of its strong reducing effect (Verheyen et al., 2015).
236
Moreover, yeast is able to produce glycerol and pyruvic acid in the early stage of fermentation
237
(Heitmann et al., 2016), which also has a positive effect on the texture of steamed bread (Corsetti
238
et al., 2000). Therefore, the variety of leavening abilities and the production of glutathione,
239
glycerol and pyruvic acid in the yeasts might result in the different texture characteristics of the
240
steamed breads fermented with the 22 S. cerevisiae strains in this study.
241
3.5 Volatile compounds profiles
242
In this study, a total of 19 volatile compounds were identified in the crumb of steamed breads
243
fermented with the 22 S. cerevisiae strains, including 9 alcohols, 5 aldehydes, 2 ketones, 1 acid, 1
244
ester and 1 furan. Compounds that have a score < 90 when compared with NIST 11 database were
245
discarded. A PCA was performed to provide an overview of the differences in flavor of the
246
steamed bread samples (Fig. 3). The first two principal components (PCs) were extracted with a
247
cumulative explained variance of 61.32%. As shown in Fig. 3, the samples were divided into five
12
248
groups. The samples fermented with Ah1 and Hb3 were grouped together and featured a relatively
249
high content of ethanol, 3-methyl-1-butanol, 2-methyl-1-propanol, phenylethyl alcohol, acetoin,
250
2,3-pentanedione and octanoic acid ethyl ester. Ethanol was found to be a predominant compound
251
in steamed bread (Kim et al., 2009). It has been reported that 95% of fermentable glucides present
252
in flour can be transformed into ethanol and carbon dioxide by S. cerevisiae through glycolysis
253
route (Pico et al., 2015). 3-methyl-1-butanol, the concentration of which was highest in the
254
steamed bread fermented with strain Hb3, was one of the most important aroma active compounds
255
in bread (Aponte et al., 2014) with a balsamic or alcoholic odor (Lee and Noble, 2003). This
256
compound was formed via the Ehrlich pathway by conversion of leucine present in flour
257
(Hazelwood et al., 2008). Likewise, phenylethyl alcohol, another typical Ehrlich volatile
258
compound, was derived from phenylalanine (Pico et al., 2015) and also correlates positively with
259
the aroma of bread. The content of octanoic acid ethyl ester was highest in the samples fermented
260
with Hb3. Generally, esters are supposed to be important flavor ingredients since they are often
261
characterized as having a fruity and pleasant aroma (Lee and Noble, 2003).
262
The samples fermented with the strains Ah13, Hr1, Xj4, Pl2, Xj7, Xj3, Hb11 and Pl6 were
263
grouped together and possessed a relatively high amount of 2-pentyl-furan, heptanal,
264
2-butoxy-ethanol,
265
2-nonen-1-ol. Among these compounds, nonanal and 2-pentyl-furan correlate positively with the
266
aroma of steamed bread like, while benzaldehyde and 1-octen-3-ol correlate negatively (Pico et al.,
267
2015). In addition, heptanal, 1-hexanol, nonanal and 1-octen-3-ol were the compounds deriving
268
from lipid oxidation (Pico et al., 2015). Generally, no significant differences of the aroma
1-octen-3-ol,
1-hexanol,
nonanal, benzaldehyde,
13
hexanoic acid
and
269
compounds originating from lipid oxidation could be found between bread samples (Birch et al.,
270
2013). However, in our study, the concentrations of these compounds were significantly different
271
between the samples (Fig. 3). This could be explained by that S. cerevisiae utilizes the oxygen
272
available during kneading to grow (Pico et al., 2015) and the diversity of their growth abilities
273
may affect the access of oxygen to lipoxygenase enzymes in generating these compounds.
274
As shown in Fig. 3, the volatile profiles of the samples fermented by S. cerevisiae strains
275
were different in comparison with the control dough and control steamed bread. Actually, flavor
276
profile of steamed bread is more complex than that of dough, since it could be influenced by
277
thermal processing (Paterson and Piggott, 2006). Therefore, some compounds which appeared
278
after steaming, such as heptanal, 2-pentyl-furan, nonanal, 2-(E)-octenal, 2-nonen-1-ol and
279
benzaldehyde (Fig. 3), may lead to the differentiation of the aroma profiles between dough and
280
steamed bread. In addition, the steaming process could also influence the concentration of some
281
volatile compounds and result in the difference of the two samples.
282
According to the results, it seems that the differences in aroma profiles of steamed bread
283
fermented with S. cerevisiae strains have no obvious correlation with the genotypic variations
284
showed by RAPD based on the two primers. Although strains Ah13, Hr1, Xj4 and Pl2, which
285
were grouped together based on aroma profiles of steamed bread, belonged to the same cluster by
286
RAPD analysis, other strains sharing a similar volatile profile were classified into different types
287
by RAPD, like Xj7, Xj3 and Pl6 (Table 2 and Fig. 3). On the other hand, the aroma profiles of
288
steamed bread fermented with strains of the same RAPD types could be quite different, such as
289
Hb3, Ah13 and Gs6 from the cluster 3. The differences in the aroma profile between the breads
14
290
have been reported to be due to the differences in the gene-regulating mechanisms and
291
biosynthetic pathways of aroma compound formation (Birch et al., 2013). Thus, to obtain a better
292
correlation between genotypic characterization and aroma profile, analysis of the relevant gene
293
regions of the S. cerevisiae genome, such as the glycolysis route and Ehrlich pathway, could be
294
promising in further studies.
295
4. Conclusions
296
The use of RAPD analysis was helpful in discriminating S. cerevisiae strains isolated from
297
the Chinese traditional sourdoughs. Differentiation was found in the leavening and glucose
298
fermentation abilities among S. cerevisiae strains, as well as the texture and volatiles of the
299
steamed bread fermented with the S. cerevisiae strains. The combined analyses of fermentative
300
traits are desirable for higher diversity of S. cerevisiae strains and starter culture selection. In this
301
study, no obvious relationship could be found between genetic diversity and fermentative
302
properties of S. cerevisiae strains, indicating the molecular characterization obtained by RAPD
303
analysis might not reflect the heterogeneity of some specific regions of the genome which are
304
more correlated with phenotypic traits, such as glycolysis route and Ehrlich pathway.
305
Acknowledgments
306
This research was supported by the National Natural Science Foundation of China (Grant
307
Number 31601461).
308
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309
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22
Table 1 Yeast enumeration of sourdough samples Tot number of Sourdough
Sources
Yeast (log cfu/g) isolated S. cerevisiae
Wf
Shandong province
6.45 ± 0.07f
15
Gm
Shandong province
6.98 ± 0.08e
8
Pl
Shandong province
8.46 ± 0.01a
10
Bj
Shannxi province
7.82 ± 0.06b
15
Sx
Shanxi province
7.37 ± 0.02c
15
Hr
Heilongjiang province
7.23 ± 0.02d
2
Ah
Anhui province
6.51 ± 0.03f
15
Sq
Henan province
7.91 ± 0.02b
15
Gs
Gansu province
7.31 ± 0.02cd
15
Ts
Hebei province
7.30 ± 0.03cd
0
Hb
Hebei province
5.73 ± 0.08g
15
Zj
Zhejiang province
7.33 ± 0.02cd
15
Xj
Xinjiang province
5.63 ± 0.02g
10
Values are means of triplicates ± SD. Values with different superscript letters within a column are significantly different (p<0.05). The total number of isolates for each sample is 15.
Table 2 List of the selected S. cerevisiae isolates for further technologically characterization. Isolates
RAPD type
Isolates
RAPD type
Sx5
1
Sq7
3
Wf5
1
Gs6
3
Xj3
1
Gs10
3
Gm4
2
Pl2
3
Xj7
2
Pl9
3
Bj1
3
Xj4
3
Bj8
3
Hr1
3
Sx2
3
Wf4
3
Ah13
3
Gm2
3
Hb3
3
Ah1
4
Hb11
3
Zj8
5
Table 3 Leavening ability and glucose fermentation characteristics of the 22 S. cerevisiae strains. Strains
CO2 production (mL)
Glucose in dough (mg/g)
Blank
0
2.01 ± 0.09a
Hb3
211.70 ± 4.19b
1.17 ± 0.05ij
Hb11
160.30 ± 1.75g
1.40 ± 0.11fgh
Xj3
180.70 ± 3.74e
1.38 ± 0.15fgh
Xj4
133.47 ± 1.50i
1.47 ± 0.16defg
Xj7
199.77 ± 4.86c
1.91 ± 0.17ab
Wf4
28.33 ± 4.04n
1.34 ± 0.05fghi
Wf5
54.90 ± 3.47m
1.66 ± 0.15cd
Pl2
202.63 ± 3.02
c
1.50 ± 0.10defg
Pl6
167.33 ± 2.63f
1.64 ± 0.06cde
Hr1
178.73 ± 2.48e
1.08 ± 0.06jk
Ah1
217.33 ± 7.06ab
1.49 ± 0.09defg
Ah13
147.07 ± 1.76h
0.97 ± 0.05k
Gm2
57.57 ± 3.13m
1.55 ± 0.13def
Gm4
70.33 ± 2.39l
1.26 ± 0.10hij
Sx2
190.67 ± 1.66d
1.64 ± 0.14cde
Sx5
220.67 ± 2.81a
1.45 ± 0.14efgh
Bj1
131.60 ± 3.00i
1.77 ± 0.09bc
Bj8
172.03 ± 1.61
f
1.50 ± 0.13defg
Sq7
123.80 ± 3.60j
1.32 ± 0.06ghi
Gs6
188.70 ± 3.87d
1.87 ± 0.06ab
Gs10
211.80 ± 2.46b
1.12 ± 0.03jk
Zj8
89.67 ± 4.18k
1.36 ± 0.12fghi
Values are means of triplicates ± SD. Values with different superscript letters within a column are significantly different (p<0.05).
Figure Captions
Fig.1. Dendrogram of the RAPD-PCR analysis of 150 S. cerevisiae strains isolated from Chinese traditional sourdough obtained with the primers M13 and MV1. The clustering was based on correlation levels expressed as percentage values of the Pearson correlation coefficient using the unweighted pair-group algorithm with arithmetic averages (UPGMA) analyses. A similarity level of 75% is used to group them into clusters.
Fig. 2. Loading (A) and score (B) biplot of PCA showing the influence of S. cerevisiae strains on the textural properties of steamed bread. The first two components explained 82.81% of the total variance.
Fig. 3. PCA biplot showing the aroma compounds formed in the steamed bread samples fermented with 22 S. cerevisiae strains and the control. Samples divided into the same group are marked with circles.
Fig. 1.
Wf 5 Xj 3 Sx5 Wf 8 Gm 4 Xj 7 Wf 10 Wf 2 Xj 4 Hr 2 Wf 11 Hr 1 Wf 3 Wf 4 Wf 9 Xj 10 Xj 8 Xj 9 Xj 1 Ah13 Ah5 Xj 2 Xj 6 Pl 9 Pl 10 Pl 8 Pl 7 Pl 1 Pl 2 Pl 3 Pl 4 Pl 5 Pl 6 Sq14 Sq2 Sq15 Sq1 Sq10 Sq11 Sq12 Sq13 Sq3 Sq5 Sq6 Sq7 Sq8 Sq9 Wf 1 Wf 6 Sq4 Bj 12 Sx15 Hb11 Hb2 Sx13 Sx14 Bj 1 Bj 10 Bj 11 Bj 13 Bj 14 Bj 15 Bj 2 Bj 4 Bj 6 Bj 7 Gs 1 Gs 10 Gs 11 Gs 2 Gs 3 Gs 4 Gs 5 Gs 7 Gs 8 Gs 9 Hb1 Hb10 Hb3 Hb4 Hb5 Hb7 Hb9 Sx1 Sx10 Sx11 Sx2 Sx3 Sx4 Sx8 Sx12 Sx6 Sx7 Sx9 Bj 3 Bj 5 Gs 12 Hb8 Hb14 Bj 9 Hb6 Hb13 Hb15 Gs 6 Bj 8 Gm 5 Gm 6 Gm 8 Gm 1 Gm 2 Gm 3 Gm 7 Gs 13 Xj 5 Gs 15 Gs 14 Hb12 Wf 12 Wf 14 Wf 15 Wf 13 Ah3 Ah6 Ah10 Ah11 Ah15 Ah7 Ah8 Ah9 Ah1 Ah2 Ah4 Ah12 Ah14 Zj 10 Zj 11 Zj 12 Zj 13 Zj 14 Zj 9 Zj 15 Zj 2 Zj 4 Zj 1 Zj 3 Zj 5 Zj 7 Zj 8 Zj 6
Fig. 2.
Fig. 3.
Highlights •
150 S. cerevisiae isolates were obtained and genotypically characterized
•
5 clusters were revealed by RAPD
•
22 S. cerevisiae strains were selected to evaluate their fermentative properties
•
Differentiation was found in fermentative properties among S. cerevisiae strains
•
Fermentative properties seemed not linked to genotypic diversity showed by RAPD
Conflict of interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.
S0023-6438(20)30183-3
DOI:
https://doi.org/10.1016/j.lwt.2020.109195
Reference:
YFSTL 109195
To appear in:
LWT - Food Science and Technology
Received Date: 24 October 2019 Revised Date:
25 January 2020
Accepted Date: 21 February 2020
Please cite this article as: Yang, H., Liu, T., Zhang, G., He, G., Intraspecific diversity and fermentative properties of Saccharomyces cerevisiae from Chinese traditional sourdough, LWT - Food Science and Technology (2020), doi: https://doi.org/10.1016/j.lwt.2020.109195. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Credit Author Statement Huanyi Yang: Investigation, Methodology, Data curation, Writing-original draft. Tongjie Liu: Validation, Writing-review & editing. Guohua Zhang: Funding acquisition. Guoqing He: Supervision, Project administration.
1
Intraspecific diversity and fermentative properties of Saccharomyces cerevisiae from
2
Chinese traditional sourdough
3 4
Huanyi Yang a, Tongjie Liu b, Guohua Zhang d ∗∗, Guoqing He c ∗
5
a
College of Life Science, Shaoxing University, Shaoxing, 312000, China
6
b
College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China
7
c
College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058,
8
China
9
d
College of Life Science, Shanxi University, Taiyuan, 030006, China
10 11
Abstract
12
One hundred and fifty isolates of Saccharomyces cerevisiae from thirteen Chinese traditional
13
sourdoughs of different regions were obtained and typed into 5 clusters by random amplified
14
polymorphic DNA-polymerase chain reaction (RAPD) analysis. Then twenty-two S. cerevisiae
15
strains were selected, according to the RAPD profiles, to evaluate their fermentation properties in
16
dough fermentation, and to assess their influences on the texture and flavor of steamed bread. The
17
results showed that the S. cerevisiae strains greatly varied in fermentation capacity and influence
18
on organoleptic property of steamed bread. However, the variation was not apparently related to
19
their genetic diversity distinguished by RAPD. A total of 19 volatile compounds were identified in
∗
Corresponding author. College of Biosystems Engineering and Food Science, Zhejiang University, Hangzhou, 310058, China. E-mail address: [email protected] (G. He) ∗∗ Co-corresponding author. College of Life Science, Shanxi University, Taiyuan, 030006, China. E-mail address: [email protected] 1
20
the crumb of the steamed breads fermented by the 22 S. cerevisiae strains with ethanol and
21
3-methyl-1-butanol being the most encountered compounds. S. cerevisiae fermentation had a
22
significant effect on textural properties of steamed bread, especially hardness, chewiness and
23
gumminess.
24
Keywords: sourdough; S. cerevisiae; RAPD; leavening ability; volatile compounds
25 26
1. Introduction
27
Sourdough, a mixture of flour and water fermented by lactic acid bacteria (LAB) and yeast,
28
has been used in making cereal-based fermented products for thousands of years around the world
29
(De Vuyst and Neysens, 2005). In China, sourdough has traditionally served as the starter for the
30
preparation of the staple food Chinese steamed bread (CSB) for more than one thousand years
31
(Wu et al., 2012). Sourdough fermentation confers positive effects to breads by improving dough
32
properties such as volume, texture and flavor, increasing nutritional values and prolonging its
33
shelf life (Vigentini et al., 2014; Zotta et al., 2006) ascribed to the metabolic activities of inherent
34
LAB and yeasts (Zotta et al., 2006).
35
Although the main contribution of yeasts in sourdough fermentation is leavening, they play
36
an important role in aroma formation and texture development (Chavan and Chavan, 2011; Katina
37
et al., 2006). Particularly, Saccharomyces cerevisiae has been considered the predominant yeast in
38
sourdough ecosystems (Minervini et al., 2012; Zhang, Sadiq, et al., 2015; Zhang et al., 2011). S.
39
cerevisiae can rapidly generate CO2 from sugars during fermentation, which results in dough
40
expanding (Heitmann et al., 2016). Moreover, S. cerevisiae improves the flavor of breads by
2
41
producing a wide range of flavor compounds, such as alcohols, aldehydes, acetoin and esters
42
(Birch et al., 2013; Frasse et al., 1992). In addition, the production of glutathione, glycerol and
43
pyruvic acid by S. cerevisiae helps to develop and strengthen gluten network in dough, and leads
44
to a positive effect on the texture of bread (Corsetti et al., 2000; Lampignano et al., 2013;
45
Verheyen et al., 2015).
46
It has been reported that S. cerevisiae showed a phenotypic and genotypic intra-species
47
diversity in sourdoughs (Landry et al., 2006). Thus, the possibility of discriminating at strain level
48
is of great importance for a successful culture selection and starter development. To date, several
49
studies have been conducted to investigate the intra-species diversity of S. cerevisiae (Ayoub et al.,
50
2006; Bogusławska-Wąs et al., 2007; Munoz et al., 2009). However, in most cases, no further
51
information was available regarding the correlation between genetic diversity and fermentative
52
properties in dough fermentation.
53
To the best of the authors' knowledge, the genetic and fermentative diversities of S.
54
cerevisiae in Chinese traditional sourdough have never been collectively investigated and
55
analyzed. This study aimed at delineating the genetic diversity and fermentative properties of S.
56
cerevisiae strains isolated from Chinese traditional sourdoughs of different regions.
57
2. Materials and methods
58
2.1. Sourdough collection
59
Thirteen home-made sourdoughs from ten different provinces in China were sampled in this
60
study. Samples were chosen for their specific origin and were stored immediately at 4 °C once in
61
the laboratory.
3
62
2.2. Yeasts enumeration and isolation
63
Five grams of sourdough was homogenized with 45 mL of sterile peptone water (peptone 1
64
g/L and NaCl 8.5 g/L) solution and decimally diluted. An aliquot of 100 µl of the 10-3 to 10-5
65
dilutions was plated onto yeast extract peptone dextrose (YPD) agar medium (1% yeast extract,
66
2% peptone, 2% dextrose, and 2% agar) supplemented with chloramphenicol (0.1 g/L). Colonies
67
were counted after incubation at 28 °C for 48 h. The plate counts were performed in triplicate. For
68
each sourdough sample, fifteen colonies were randomly picked from the plates of the three
69
dilutions, then purified by successive streak plate method.
70
2.3. Identification of S. cerevisiae strains
71
Based on the colony morphology (Casalone et al., 2005), certain isolates were considered
72
presumptive strains of S. cerevisiae and further identified by 26S rRNA gene sequencing.
73
Genomic DNA from presumptive S. cerevisiae isolate was extracted using a DNA Extraction Kit
74
(Axygen, Hangzhou, China), following the manufacturer’s instructions. The primers, NL1 and
75
NL4 (Di Cagno et al. 2014), were used to amplify the D1/D2 region of the 26S rRNA gene. The
76
PCR products were sequenced by a company (Shanghai Sangon Biotech, Shanghai, China), and
77
the results were compared with those deposited in the GenBank using the BLAST algorithm.
78
Strains showing ≥98% identity with S. cerevisiae were selected for further characterization.
79
2.4. RAPD-PCR analysis
80
Genotypic diversity of S. cerevisiae strains was assessed by RAPD analysis using two primers
81
separately, namely M13 (5’-GAGGGTGGCGGTTCT-3’) (Stenlid et al., 1994) and MV1
82
(5’-GGACGCTTCTG-3’) (Venturi, Guerrini and Vincenzini, 2012). The amplification conditions
4
83
with primer M13 were as follows: preliminary denaturation for 5 min at 94 °C, followed by 34
84
cycles of 94 °C for 1 min, 45 °C for 2 min, 72 °C for 1.5 min, and terminated with an extension at
85
72 °C for 10 min (Liu et al., 2018). The conditions with primer MV1 were set according to
86
Venturi, Guerrini, Granchi, et al. (2012). Breifly, preliminary denaturation for 5 min at 94 °C,
87
followed by 30 cyceles of 94 °C for 1 min, 40 °C for 1 min and 72 °C for 2 min, and terminated
88
with an extension at 72 °C for 10 min. The amplicons were electrophoresed on 1.5% agarose gel
89
supplemented with the DNA dye GelRed Nucleic Acid Gel Stain (Biotium Hayward, Ca, USA)
90
for 5.5 h at a constant voltage of 70 V in 1×TAE (40 mM Tris-acetate, 1 mM EDTA, pH 8.0)
91
buffer and the gel images were captured in a Gel Imaging System (ChampGel 500,
92
SAGECREATION, Beijing, China). BioNumerics version 6.6 software package (Applied Maths,
93
Sint-Martens-Latem, Belgium) was used to convert and analyze the resulting fingerprints. RAPD
94
profiles generated with M13 and MV1 primers were integrated to increase the typing efficiency
95
and discriminatory power. According to the RAPD profiles, representative strains were selected
96
and inoculated in a rotary shaking incubator with 180 rpm at 28 °C for 48 h. The cells were
97
collected by centrifugation at 3800×g for 10 min and washed twice with sterile distilled water for
98
subsequent experiments.
99
2.5. Leavening ability determination
100
The leavening ability of the selected S. cerevisiae isolates was measured according to the
101
methods described by Liu et al. (2018). The dough was inoculated with S. cerevisiae at an
102
inoculum size of 107 cfu/g. Leavening ability was expressed by volume (milliliter) of CO2
5
103
produced at 30 °C in one hour. Experiments were performed in triplicate and dough without
104
inoculation was set as a negative control.
105
2.6. Glucose fermentation determination
106
Wheat flour, water and yeast cells (inoculum size of 107 cfu/g dough) were mixed to obtain a
107
dough with a dough yield (dough weight × 100/flour weight) of 150 and placed in an incubator at
108
30 °C and 80% RH for 2 h (STIK (Shanghai) Co., Ltd, China). Ten grams of dough was
109
homogenized with 90 mL of distilled water for 15 min using a magnetic stirrer (IKA basic 2 RH,
110
Germany) until the sample was thoroughly suspended. Then, the suspension was centrifuged at
111
3800 × g for 10 min. The concentration of glucose in supernatant was measured using a glucose
112
content assay kit (Shanghai Rongsheng Biotech Co., Ltd.). Experiments were performed in
113
triplicate and the dough without inoculation was used as a control.
114
2.7. Steamed bread manufacturing
115
The steamed bread was made according to the method given by Liu et al. (2018). Wheat flour,
116
water and the cells of S. cerevisiae (inoculum size of 107 cfu/g dough) were thoroughly mixed in a
117
mixer (HMJ-D3826, Guangdong bear electric Co., Ltd. China) for 10 min to form a dough with a
118
dough yield of 150. Afterwards, the dough was shaped manually and placed in an incubator at
119
30 °C and 80% RH for 2 h. Finally, the dough was steamed for 20 min in a steamer. The controls
120
were prepared using the same procedures but were not inoculated with S. cerevisiae strains.
121
2.8. Texture analysis of the steamed bread
122
The textural properties of the steamed bread were investigated using a TA-XT2i Texture
123
Analyzer (Stable Micro System, Ltd., Godalming, UK) with a Texture Profile Analysis (TPA)
6
124
mode (Caine et al., 2003). After cooling for 5 min, the samples were uniformly sliced with a
125
thickness of 10 mm and cut into cylindrical slices (35 mm diameter) from the center of each loaf.
126
To measure the textural properties of the samples, a 100 mm diameter cylindrical probe was used
127
to compress each slice with 40% deformation at a speed of 1 mm/s. At least 6 replicates were
128
performed for each sample.
129
2.9. Volatile compounds of steamed bread
130
Volatile compounds of the steamed breads fermented by different S. cerevisiae strains were
131
determined according to Liu et al. (2018). Solid phase microextration (SPME) was used for the
132
extraction of the volatile compounds and gas chromatography-mass spectrometry (GC-MS) was
133
used for the separation and identification. The flavor extraction and subsequent injection processes
134
were automatically performed using the MPS autosampler (Gerstel, Mülheim, Germany). The
135
fiber 75 µm Carboxen/polydimethylsiloxane (CAR/PDMS; Supelco, Bellefonte, PA) was
136
employed for the extraction of volatile compounds at 60 °C for 30 min and then inserted into the
137
injection port of the GC (7890B; Agilent Technologies), equipped with a DB-WAX capillary
138
column (J&W Scientific, 30 m long × 0.25 mm internal diameter, 0.25 µm film thickness), to
139
desorb the extracted volatiles at 250 °C for 4 min in splitless mode. The column temperature was
140
set at 40 °C for 2 min, increased to 230 °C at a rate of 5 °C/min. The carrier gas was helium with a
141
flow rate of 1.0 mL/min. The GC was coupled to an MS detector (5977A; Agilent Technologies)
142
used in scan mode (35 to 500 m/z) with an electronic impact of 70 eV. The identification of
143
volatile compounds was carried out by comparison of the mass spectral data obtained with those
7
144
in a commercial database (NIST 11). The peak area of each volatile compound was integrated by
145
selecting a specific ion as described by Di Cagno et al. (2014).
146
2.10. Statistical analysis
147
Data from yeast enumeration, leavening ability and glucose fermentation were subjected to
148
one-way ANOVA analysis and were expressed as mean values ± standard deviations and the
149
multiple comparisons was performed using Tukey’s test (SPSS Statistics). Principal components
150
analysis (PCA) was performed using the software XLSTAT (version 2014.5.03) to compare the
151
steamed breads fermented with different S. cerevisiae strains based on their volatile profiles.
152
3. Results and discussion
153
3.1. Isolation of S. cerevisiae from Chinese sourdoughs
154
The results of yeast enumeration of the sourdoughs were given in Table 1. Cell density of
155
yeasts varied from 5.63 ± 0.02 (Xj) to 8.46 ± 0.01 (Pl) log cfu/g, and the median value was 7.08
156
log cfu/g. Similar results can be found in the previous studies performed by Lhomme et al. (2015)
157
and Minervini et al. (2012). Noticeably, S. cerevisiae was not found in the sourdough sample Ts.
158
According to the colony morphology, a total of 178 presumptive S. cerevisiae strains were
159
subjected to 26S rRNA sequencing analysis. 150 of the 178 strains exhibited ≥98% identity with
160
S. cerevisiae. Thus, 76.9% of the total strains isolated from the sourdough samples in this study
161
were S. cerevisiae (Table 1), illustrating the predominance of this species in Chinese traditional
162
sourdough, in accordance with previous studies (Valmorri et al., 2010; Zhang, Sadiq, et al., 2015).
163
3.2. Genotypic characterization by RAPD analysis
8
164
RAPD fingerprinting is a simple, rapid and sensitive technique and it has been widely used to
165
differentiate strains of yeast species (Bogusławska-Wąs et al., 2007; Di Cagno et al., 2014;
166
El-Fiky et al., 2012; Pfliegler et al., 2014). In order to increase the typing efficiency and
167
discriminatory power, RAPD patterns generated with M13 and MV1 primers individually were
168
integrated into a unique clustering profile as shown in Fig. 1. Based on the similarity level of 75%,
169
at which level isolates in different types differed by no less than three bands, the 150 S. cerevisiae
170
isolates from Chinese traditional sourdoughs were grouped into 5 clusters. Cluster 3 was the most
171
represented cluster with 116 strains isolated from all sourdough samples except Zj, accounting for
172
77.3% of total isolates, while cluster 5 included all the S. cerevisiae strains isolated from
173
sourdough Zj. Meanwhile, isolates from the same sourdough could be divided into different
174
clusters. For instance, strains isolated from sourdough Sx, Ah, Xj, Wf and Gm were separately
175
divided into at least two clusters. Particularly, strains from sourdough Xj, allocated into four
176
clusters, showed the most diversity. The genetic characterization of this study showed the
177
intraspecific variability of S. cerevisiae isolates from traditional sourdoughs, in accordance with
178
the finding of De Vuyst et al. (2016). Regarding this observation, the technological factors, the
179
manufacturing environment as well as geographical origins may explain the genotypic diversity of
180
these populations (Legras et al., 2007; Tapsoba et al., 2015), but it should be confirmed in further
181
studies through a much wider sampling.
182
3.3 Leavening ability and glucose fermentation
183
In order to describe the fermentative properties, a total of 22 isolates (Table 2) were selected
184
on the basis of the genotypic differentiation by RAPD analysis. Leavening ability is one of the
9
185
properties required for the commercial applications of yeast. A high leavening ability of S.
186
cerevisiae strains is apparently economically beneficial for bread producers. S. cerevisiae use
187
alcoholic fermentation to convert sugar into ethanol, carbon dioxide and glycerol. Carbon dioxide
188
causes the leavening of the dough. As shown in Table 3, the CO2 production of the selected strains
189
varied in an extensive range from 28.33 ± 4.04 mL (Wf4) to 220.67 ± 2.81 mL (Sx5), indicating
190
significant differences in the leavening abilities of these S. cerevisiae strains. Sx5, Ah1, Gs10 and
191
Hb3 were the strains possessing the highest leavening ability (220.67 ± 2.81, 217.33 ± 7.06,
192
211.80 ± 2.46 and 211.70 ± 4.19 mL CO2, respectively). S. cerevisiae ferment the flour
193
carbohydrates present in the dough to carbon dioxide and ethanol through glycolysis and glucose
194
is the preferentially utilized sugar (Heitmann et al., 2016). The glucose fermentation
195
characteristics of the 22 S. cerevisiae strains were also measured (Table 3). Interestingly, not all
196
strains with high leavening ability correlated with high ability of glucose utilization. For instance,
197
strain Xj7 and Gs6 produced more amount of CO2 than other strains, yet they showed an inferior
198
ability in glucose fermentation. It seems that some other factors apart from the ability of glucose
199
fermentation could affect the leavening ability of S. cerevisiae strains. Although S. cerevisiae
200
mainly ferments glucose in the beginning of fermentation, it also uses maltose and fructose as
201
carbohydrate sources for metabolism in later stages (Gabriela and Daniela, 2010). Moreover, the
202
leavening ability of S. cerevisiae has been proven to be influenced not only by glycolytic activity,
203
but also by sucrase activity and osmotolerance (Tokashiki et al., 2011). The mode of regulation of
204
maltose might affect its leavening ability as well (Birch et al., 2013; Zhang, Bai, et al., 2015).
205
Hence, the difference of these factors among S. cerevisiae strains might lead to the variations in
10
206
leavening ability. However, the underlying mechanism that can explain the variation in leavening
207
ability needs further investigation.
208
According to the results in this study, no obvious relationship could be found between
209
genetic typing by the two primers and the abilities of leavening and glucose fermentation. Strains
210
belonging to the same cluster based on RAPD analysis differed in the leavening and glucose
211
fermentation abilities, such as Gm4 and Xj7. On the other hand, some strains divided into
212
different clusters had similar abilities of leavening and glucose fermentation, such as Sx5 and Ah1.
213
Therefore, it indicated that the genetic diversity delineated by RAPD in this study may not reflect
214
the heterogeneity of the glycolytic pathway genes or maltose degradation operon, which was in
215
according to the previous study of De Angelis et al. (2007).
216
3.4 Textural properties of steamed bread
217
Texture of steamed bread is an important organoleptic property affecting consumer’s
218
acceptability (Arendt et al., 2007). The hardness, gumminess, chewiness, springiness,
219
cohesiveness, and resilience of steamed bread crumb fermented by different S. cerevisiae strains
220
were obtained from texture profile analysis (TPA). Relationships between textural properties and
221
S. cerevisiae strains were analyzed by PCA (Fig. 2). The first two components of the PCA
222
explained 82.81% of the total variance (58.78% and 24.03%, respectively). As shown in Fig. 2, all
223
samples fermented with S. cerevisiae strains were separated considerably from the blank,
224
indicating that S. cerevisiae fermentation had a significant effect on textural properties (especially
225
hardness, chewiness and gumminess) of steamed bread. In addition, the effect seemed to vary
226
among different strains, for most of the samples fermented with different S. cerevisiae strains
11
227
were separated from each other on the PCA map, including the strains belonged to the same
228
cluster by RAPD analysis such as Gs6 and Xj4, Gm4 and Xj7 and Wf4 and Hb11. During dough
229
fermentation, CO2 is produced by yeast through glycolysis route and is entrapped in the gluten
230
matrix of dough generating numerous gas cells, which leads to a lower extensibility and a higher
231
specific volume of steamed bread (Heitmann et al., 2016; Verheyen et al., 2014). Apart from
232
leavening dough, yeast also helps to develop and strengthen the gluten network. Glutathione,
233
which could be released by some nonviable yeast cells as a stress response (Penninckx, 2002;
234
Verheyen et al., 2015), modifies the viscoelastic gluten network of dough by increasing the rate of
235
thiol-disulfide interchange reactions because of its strong reducing effect (Verheyen et al., 2015).
236
Moreover, yeast is able to produce glycerol and pyruvic acid in the early stage of fermentation
237
(Heitmann et al., 2016), which also has a positive effect on the texture of steamed bread (Corsetti
238
et al., 2000). Therefore, the variety of leavening abilities and the production of glutathione,
239
glycerol and pyruvic acid in the yeasts might result in the different texture characteristics of the
240
steamed breads fermented with the 22 S. cerevisiae strains in this study.
241
3.5 Volatile compounds profiles
242
In this study, a total of 19 volatile compounds were identified in the crumb of steamed breads
243
fermented with the 22 S. cerevisiae strains, including 9 alcohols, 5 aldehydes, 2 ketones, 1 acid, 1
244
ester and 1 furan. Compounds that have a score < 90 when compared with NIST 11 database were
245
discarded. A PCA was performed to provide an overview of the differences in flavor of the
246
steamed bread samples (Fig. 3). The first two principal components (PCs) were extracted with a
247
cumulative explained variance of 61.32%. As shown in Fig. 3, the samples were divided into five
12
248
groups. The samples fermented with Ah1 and Hb3 were grouped together and featured a relatively
249
high content of ethanol, 3-methyl-1-butanol, 2-methyl-1-propanol, phenylethyl alcohol, acetoin,
250
2,3-pentanedione and octanoic acid ethyl ester. Ethanol was found to be a predominant compound
251
in steamed bread (Kim et al., 2009). It has been reported that 95% of fermentable glucides present
252
in flour can be transformed into ethanol and carbon dioxide by S. cerevisiae through glycolysis
253
route (Pico et al., 2015). 3-methyl-1-butanol, the concentration of which was highest in the
254
steamed bread fermented with strain Hb3, was one of the most important aroma active compounds
255
in bread (Aponte et al., 2014) with a balsamic or alcoholic odor (Lee and Noble, 2003). This
256
compound was formed via the Ehrlich pathway by conversion of leucine present in flour
257
(Hazelwood et al., 2008). Likewise, phenylethyl alcohol, another typical Ehrlich volatile
258
compound, was derived from phenylalanine (Pico et al., 2015) and also correlates positively with
259
the aroma of bread. The content of octanoic acid ethyl ester was highest in the samples fermented
260
with Hb3. Generally, esters are supposed to be important flavor ingredients since they are often
261
characterized as having a fruity and pleasant aroma (Lee and Noble, 2003).
262
The samples fermented with the strains Ah13, Hr1, Xj4, Pl2, Xj7, Xj3, Hb11 and Pl6 were
263
grouped together and possessed a relatively high amount of 2-pentyl-furan, heptanal,
264
2-butoxy-ethanol,
265
2-nonen-1-ol. Among these compounds, nonanal and 2-pentyl-furan correlate positively with the
266
aroma of steamed bread like, while benzaldehyde and 1-octen-3-ol correlate negatively (Pico et al.,
267
2015). In addition, heptanal, 1-hexanol, nonanal and 1-octen-3-ol were the compounds deriving
268
from lipid oxidation (Pico et al., 2015). Generally, no significant differences of the aroma
1-octen-3-ol,
1-hexanol,
nonanal, benzaldehyde,
13
hexanoic acid
and
269
compounds originating from lipid oxidation could be found between bread samples (Birch et al.,
270
2013). However, in our study, the concentrations of these compounds were significantly different
271
between the samples (Fig. 3). This could be explained by that S. cerevisiae utilizes the oxygen
272
available during kneading to grow (Pico et al., 2015) and the diversity of their growth abilities
273
may affect the access of oxygen to lipoxygenase enzymes in generating these compounds.
274
As shown in Fig. 3, the volatile profiles of the samples fermented by S. cerevisiae strains
275
were different in comparison with the control dough and control steamed bread. Actually, flavor
276
profile of steamed bread is more complex than that of dough, since it could be influenced by
277
thermal processing (Paterson and Piggott, 2006). Therefore, some compounds which appeared
278
after steaming, such as heptanal, 2-pentyl-furan, nonanal, 2-(E)-octenal, 2-nonen-1-ol and
279
benzaldehyde (Fig. 3), may lead to the differentiation of the aroma profiles between dough and
280
steamed bread. In addition, the steaming process could also influence the concentration of some
281
volatile compounds and result in the difference of the two samples.
282
According to the results, it seems that the differences in aroma profiles of steamed bread
283
fermented with S. cerevisiae strains have no obvious correlation with the genotypic variations
284
showed by RAPD based on the two primers. Although strains Ah13, Hr1, Xj4 and Pl2, which
285
were grouped together based on aroma profiles of steamed bread, belonged to the same cluster by
286
RAPD analysis, other strains sharing a similar volatile profile were classified into different types
287
by RAPD, like Xj7, Xj3 and Pl6 (Table 2 and Fig. 3). On the other hand, the aroma profiles of
288
steamed bread fermented with strains of the same RAPD types could be quite different, such as
289
Hb3, Ah13 and Gs6 from the cluster 3. The differences in the aroma profile between the breads
14
290
have been reported to be due to the differences in the gene-regulating mechanisms and
291
biosynthetic pathways of aroma compound formation (Birch et al., 2013). Thus, to obtain a better
292
correlation between genotypic characterization and aroma profile, analysis of the relevant gene
293
regions of the S. cerevisiae genome, such as the glycolysis route and Ehrlich pathway, could be
294
promising in further studies.
295
4. Conclusions
296
The use of RAPD analysis was helpful in discriminating S. cerevisiae strains isolated from
297
the Chinese traditional sourdoughs. Differentiation was found in the leavening and glucose
298
fermentation abilities among S. cerevisiae strains, as well as the texture and volatiles of the
299
steamed bread fermented with the S. cerevisiae strains. The combined analyses of fermentative
300
traits are desirable for higher diversity of S. cerevisiae strains and starter culture selection. In this
301
study, no obvious relationship could be found between genetic diversity and fermentative
302
properties of S. cerevisiae strains, indicating the molecular characterization obtained by RAPD
303
analysis might not reflect the heterogeneity of some specific regions of the genome which are
304
more correlated with phenotypic traits, such as glycolysis route and Ehrlich pathway.
305
Acknowledgments
306
This research was supported by the National Natural Science Foundation of China (Grant
307
Number 31601461).
308
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Table 1 Yeast enumeration of sourdough samples Tot number of Sourdough
Sources
Yeast (log cfu/g) isolated S. cerevisiae
Wf
Shandong province
6.45 ± 0.07f
15
Gm
Shandong province
6.98 ± 0.08e
8
Pl
Shandong province
8.46 ± 0.01a
10
Bj
Shannxi province
7.82 ± 0.06b
15
Sx
Shanxi province
7.37 ± 0.02c
15
Hr
Heilongjiang province
7.23 ± 0.02d
2
Ah
Anhui province
6.51 ± 0.03f
15
Sq
Henan province
7.91 ± 0.02b
15
Gs
Gansu province
7.31 ± 0.02cd
15
Ts
Hebei province
7.30 ± 0.03cd
0
Hb
Hebei province
5.73 ± 0.08g
15
Zj
Zhejiang province
7.33 ± 0.02cd
15
Xj
Xinjiang province
5.63 ± 0.02g
10
Values are means of triplicates ± SD. Values with different superscript letters within a column are significantly different (p<0.05). The total number of isolates for each sample is 15.
Table 2 List of the selected S. cerevisiae isolates for further technologically characterization. Isolates
RAPD type
Isolates
RAPD type
Sx5
1
Sq7
3
Wf5
1
Gs6
3
Xj3
1
Gs10
3
Gm4
2
Pl2
3
Xj7
2
Pl9
3
Bj1
3
Xj4
3
Bj8
3
Hr1
3
Sx2
3
Wf4
3
Ah13
3
Gm2
3
Hb3
3
Ah1
4
Hb11
3
Zj8
5
Table 3 Leavening ability and glucose fermentation characteristics of the 22 S. cerevisiae strains. Strains
CO2 production (mL)
Glucose in dough (mg/g)
Blank
0
2.01 ± 0.09a
Hb3
211.70 ± 4.19b
1.17 ± 0.05ij
Hb11
160.30 ± 1.75g
1.40 ± 0.11fgh
Xj3
180.70 ± 3.74e
1.38 ± 0.15fgh
Xj4
133.47 ± 1.50i
1.47 ± 0.16defg
Xj7
199.77 ± 4.86c
1.91 ± 0.17ab
Wf4
28.33 ± 4.04n
1.34 ± 0.05fghi
Wf5
54.90 ± 3.47m
1.66 ± 0.15cd
Pl2
202.63 ± 3.02
c
1.50 ± 0.10defg
Pl6
167.33 ± 2.63f
1.64 ± 0.06cde
Hr1
178.73 ± 2.48e
1.08 ± 0.06jk
Ah1
217.33 ± 7.06ab
1.49 ± 0.09defg
Ah13
147.07 ± 1.76h
0.97 ± 0.05k
Gm2
57.57 ± 3.13m
1.55 ± 0.13def
Gm4
70.33 ± 2.39l
1.26 ± 0.10hij
Sx2
190.67 ± 1.66d
1.64 ± 0.14cde
Sx5
220.67 ± 2.81a
1.45 ± 0.14efgh
Bj1
131.60 ± 3.00i
1.77 ± 0.09bc
Bj8
172.03 ± 1.61
f
1.50 ± 0.13defg
Sq7
123.80 ± 3.60j
1.32 ± 0.06ghi
Gs6
188.70 ± 3.87d
1.87 ± 0.06ab
Gs10
211.80 ± 2.46b
1.12 ± 0.03jk
Zj8
89.67 ± 4.18k
1.36 ± 0.12fghi
Values are means of triplicates ± SD. Values with different superscript letters within a column are significantly different (p<0.05).
Figure Captions
Fig.1. Dendrogram of the RAPD-PCR analysis of 150 S. cerevisiae strains isolated from Chinese traditional sourdough obtained with the primers M13 and MV1. The clustering was based on correlation levels expressed as percentage values of the Pearson correlation coefficient using the unweighted pair-group algorithm with arithmetic averages (UPGMA) analyses. A similarity level of 75% is used to group them into clusters.
Fig. 2. Loading (A) and score (B) biplot of PCA showing the influence of S. cerevisiae strains on the textural properties of steamed bread. The first two components explained 82.81% of the total variance.
Fig. 3. PCA biplot showing the aroma compounds formed in the steamed bread samples fermented with 22 S. cerevisiae strains and the control. Samples divided into the same group are marked with circles.
Fig. 1.
Wf 5 Xj 3 Sx5 Wf 8 Gm 4 Xj 7 Wf 10 Wf 2 Xj 4 Hr 2 Wf 11 Hr 1 Wf 3 Wf 4 Wf 9 Xj 10 Xj 8 Xj 9 Xj 1 Ah13 Ah5 Xj 2 Xj 6 Pl 9 Pl 10 Pl 8 Pl 7 Pl 1 Pl 2 Pl 3 Pl 4 Pl 5 Pl 6 Sq14 Sq2 Sq15 Sq1 Sq10 Sq11 Sq12 Sq13 Sq3 Sq5 Sq6 Sq7 Sq8 Sq9 Wf 1 Wf 6 Sq4 Bj 12 Sx15 Hb11 Hb2 Sx13 Sx14 Bj 1 Bj 10 Bj 11 Bj 13 Bj 14 Bj 15 Bj 2 Bj 4 Bj 6 Bj 7 Gs 1 Gs 10 Gs 11 Gs 2 Gs 3 Gs 4 Gs 5 Gs 7 Gs 8 Gs 9 Hb1 Hb10 Hb3 Hb4 Hb5 Hb7 Hb9 Sx1 Sx10 Sx11 Sx2 Sx3 Sx4 Sx8 Sx12 Sx6 Sx7 Sx9 Bj 3 Bj 5 Gs 12 Hb8 Hb14 Bj 9 Hb6 Hb13 Hb15 Gs 6 Bj 8 Gm 5 Gm 6 Gm 8 Gm 1 Gm 2 Gm 3 Gm 7 Gs 13 Xj 5 Gs 15 Gs 14 Hb12 Wf 12 Wf 14 Wf 15 Wf 13 Ah3 Ah6 Ah10 Ah11 Ah15 Ah7 Ah8 Ah9 Ah1 Ah2 Ah4 Ah12 Ah14 Zj 10 Zj 11 Zj 12 Zj 13 Zj 14 Zj 9 Zj 15 Zj 2 Zj 4 Zj 1 Zj 3 Zj 5 Zj 7 Zj 8 Zj 6
Fig. 2.
Fig. 3.
Highlights •
150 S. cerevisiae isolates were obtained and genotypically characterized
•
5 clusters were revealed by RAPD
•
22 S. cerevisiae strains were selected to evaluate their fermentative properties
•
Differentiation was found in fermentative properties among S. cerevisiae strains
•
Fermentative properties seemed not linked to genotypic diversity showed by RAPD
Conflict of interest The authors declared that they have no conflicts of interest to this work. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.