Applied Microbiology and Biotechnology

https://doi.org/10.1007/s00253-017-8733-3

BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING

Succession sequence of lactic acid bacteria driven by environmental

factors and substrates throughout the brewing process of Shanxi
aged vinegar
Yu Zheng 1 & Jun Mou 1 & Jiwei Niu 1 & Shuai Yang 1 & Lin Chen 1 & Menglei Xia 1 & Min Wang 1

Received: 10 August 2017 / Revised: 2 December 2017 / Accepted: 11 December 2017

# Springer-Verlag GmbH Germany, part of Springer Nature 2018

Abstract
Lactic acid bacteria (LAB) are essential microbiota for the fermentation and flavor formation of Shanxi aged vinegar, a famous
Chinese traditional cereal vinegar that is manufactured using open solid-state fermentation (SSF) technology. However, the dy-
namics of LAB in this SSF process and the underlying mechanism remain poorly understood. Here, the diversity of LAB and the
potential driving factors of the entire process were analyzed by combining culture-independent and culture-dependent methods.
Canonical correlation analysis indicated that ethanol, acetic acid, and temperature that result from the metabolism of microorgan-
isms serve as potential driving factors for LAB succession. LAB strains were periodically isolated, and the characteristics of 57
isolates on environmental factor tolerance and substrate utilization were analyzed to understand the succession sequence. The
environmental tolerance of LAB from different stages was in accordance with their fermentation conditions. Remarkable correla-
tions were identified between LAB growth and environmental factors with 0.866 of ethanol (70 g/L), 0.756 of acetic acid (10 g/L),
and 0.803 of temperature (47 °C). More gentle or harsh environments (less or more than 60 or 80 g/L of ethanol, 5 or 20 g/L of acetic
acid, and 30 or 55 °C temperature) did not affect the LAB succession. The utilization capability evaluation of the 57 isolates for 95
compounds proved that strains from different fermentation stages exhibited different predilections on substrates to contribute to the
fermentation at different stages. Results demonstrated that LAB succession in the SSF process was driven by the capabilities of
environmental tolerance and substrate utilization.

Keywords Lactic acid bacteria . Succession sequence . Shanxi aged vinegar . Environmental tolerance . Substrate utilization

Introduction
Solid-state fermentation (SSF) is one of the oldest and most
economical ways of producing and preserving foods; more-
over, SSF may improve the nutritional values, sensory proper-
ties, and functional qualities of raw materials (Lu et al. 2016).
The SSF processes of most known natural and industrial
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00253-017-8733-3) contains supplementary
material, which is available to authorized users.
* Min Wang
minw@tust.edu.cn
1 cereals, such as sorghum, sticky rice, and wheat bran (Wu et al.
State Key Laboratory of Food Nutrition and Safety. Key Laboratory
of Industrial Fermentation Microbiology, Ministry of Education.
College of Biotechnology, Tianjin University of Science &
Technology, Tianjin 300457, People’s Republic of China
fermented foods, such as vinegars (Li et al. 2016b) and cheese
(Wolfe et al. 2014), are driven by multispecies microbial com-
munities. In an open work environment, succession of micro-
bial communities is always consecutive and can affect the char-
acteristics and quality of the final fermented food (Li et al.
2016b). Many microbiological studies have been conducted
to reveal the diversity and succession of microbial communities
during these open-SSF processes (Nie et al. 2015; Wu et al.
2012); however, gaps remain between the community forma-
tion and function of these complex microbial communities dur-
ing the open-SSF processes.
Vinegars have been used worldwide as acerbic table condi-
ments, preservatives, health products, and medicine for thou-
sands of years. In China, vinegars are traditionally made from
2010; Li et al. 2016b). Shanxi aged vinegar (SAV) is one of the
famous Chinese vinegars, which is produced with a typical SSF
technology involving Daqu, alcohol fermentation (AF), acetic

acid fermentation (AAF), smoking pei, and aging stages (Liu
et al. 2012; Wu et al. 2012). Various microorganisms, including
molds, yeasts, and bacteria, are spontaneously enriched and
disappeared in the SSF process (Nie et al. 2015; Solieri and
Giudici 2009) (Fig. 1). Such microorganisms are significant
in the production and characteristics of SAV. Smoking
pei, in which microorganisms scarcely remain due to the
high-temperature environment (80 to 90 °C), is a special tech-
nique for SAV that is performed after AAF stage. Daqu, a type
of mixed-culture starter, is used as inoculum for the SSF, and
the dominant beneficial microorganisms in Daqu are molds, of
the genera Aspergillus, Rhizopus, and Mucor, and yeasts of the
genus Saccharomyces (Nie et al. 2013). Yeasts of the genera
Saccharomyces and Saccharomycopsis typically dominate the
AF stage because they can rapidly consume low-molecular-
weight sugar and thus increase ethanol concentration (Li et al.
2015b). Acetic acid bacteria (AAB) and lactic acid bacteria
(LAB) are crucial to the success of SAV. AAB, especially
Acetobacter, dominate the AAF stage because of their excel-
lent capability of rapid conversion of ethanol into acetic acid,
which is the major source of total acid and dominant flavor
components of vinegars (Nie et al. 2017; Solieri and Giudici
2009). LAB, particularly Lactobacillus, are the most relevant
bacteria throughout the SSF process of SAV and greatly con-
tribute to the production of organic acids, which are the main
flavor substances of SAV and contribute a soft taste (Li et al.
2016b; Nie et al. 2015, 2017), as well as other flavor sub-
stances. The composition of LAB constantly changes during
the fermentation process, from Daqu to AF, AAF, and aging
stages due to the open-SSF technology and varied conditions
(Nie et al. 2015; Wu et al. 2012). Thus, studies on how highly
variable environmental conditions affect LAB succession in
the SSF process of SAV are becoming increasingly important.
Recent studies have suggested that bacterial communi-
ties are often formed under controlled conditions in dis-
crete units, which allow the measurement and manipula-
tion of migration into the community, environmental con-
ditions, and growth substrates (Li et al. 2015a, 2016a;
Wang and Xu 2015; Wolfe et al. 2014; Zheng et al.
2014). Others have argued that bacterial dynamics is large-
ly driven by individual traits (Nemergut et al. 2016). These
studies generally have implied a degree of mathematical
analysis in the driving factors of bacterial community suc-
cession. However, knowledge on how environmental con-
ditions, substrates, and individual traits affect the microbial
community structure has not been identified yet. The suc-
cessional trajectory of LAB communities and the mecha-
nism controlling the dynamics at a scale that is relevant for
these organisms are significant to understand considering
the importance of LAB for the SAV SSF process.
In this work, the succession of LAB and its potential driv-
ing factors in the open-SSF process of SAV were revealed by
using high-throughput sequencing and canonical correlation
Appl Microbiol Biotechnol
analysis (CCA) methods. LAB strains were periodically iso-
lated according to the brewing process to understand the suc-
cession sequence of LAB. Characteristics of LAB strains on
environmental adaption and substrate utilization were ex-
plored to reveal the succession sequence of LAB throughout
the fermentation process. To our knowledge, this is the first
report to perform comparative studies to investigate the suc-
cession sequence of the LAB community by combining
culture-independent and culture-dependent methods. The re-
sults will greatly increase the scientific understanding of the
brewing mechanism in the open-SSF process and may render
rational strategies for regulating the SSF process.
Materials and methods
Samples
Samples of Daqu, Jiulao (samples from AF stage with mi-
crobes and substrates), Cupei (samples from AAF stage with
microbes and substrates), and aging stages were collected
from a SAV factory (Shanxi Zilin Vinegar Industry Co.,
Ltd., Qingxu, China) according to the production process.
Daqu was collected from a mature Daqu storage room. Four
Jiulao samples were collected at different times (1st, 4th, 7th,
and 10th day), along with the AF stage. Five Cupei samples
were collected at the 0th, 1st, 3rd, 5th, and 7th day, along with
the AAF stage. Aging samples were collected at the aging
stage of SAV. Parts of these samples were used to determine
physicochemical indictors and were dealt with as follows:
Samples of Jiulao were centrifuged at 4629×g for
10 min after homogenization, and the supernatant was
used for detection. For Cupei pretreatment, 5 g of samples was
diluted into 45 mL of distilled water, shaken for 30 min
completely at room temperature, and centrifuged. The super-
natant was collected for analysis.
Analyses of the diversity and succession of LAB
community
The succession of LAB along with the entire fermentation
process was analyzed using high-throughput sequencing. For
DNA extraction, each sample was homogenized using liquid
nitrogen (flash-frozen in liquid nitrogen and then rapidly
thawed in a water bath at 65 °C for 2 min, these procedures
were repeated three times), and approximately 500 mg of each
sample was used for genomic DNA extraction which was
performed according to a previous method (Nie et al. 2015).
Bacterial 16S rDNA genes (V3–V5 variable regions) were
amplified from the total genomic DNA by using primers 341
F and 926 R, which incorporated 454 FLX titanium adapters
and a sample barcode sequence (Nie et al. 2015). Sequencing
was performed using a 454 GS FLX titanium system (Roche,
Appl Microbiol Biotechnol
America) at The Beijing Genomics Institute (BGI, Shenzhen,
China). 16S rDNA gene sequences were processed and mod-
ified following previously described methods (Jeong et al.
2013; Kostic et al. 2012). Pyrosequencing data were proc-
essed using a data curation pipeline implemented in Mothur
(Schloss et al. 2009). Sequencing reads were sorted to specific
samples on the basis of unique barcodes, and the barcodes
were subsequently trimmed. Sequencing reads were removed
if the reads were < 200 or > 1000 bp, had a non-exact barcode
match, exceeded two ambiguous bases (N), exceeded eight
consecutive identical bases, or had average quality scores <
25. A nearest-alignment space termination-based sequence
aligner was used to align sequences to a custom reference on
the basis of SILVA alignment (Kostic et al. 2012; Schloss et al.
2009). Chimeric sequences were identified and filtered by
quality. Chimera-free sequences were clustered in operational
taxonomic units (OTUs) defined by 97% similarity using a
complete-linkage clustering tool. Representative sequences
per OTU were classified according to previously described
methods (Kostic et al. 2012). Ribosomal Database Project
naïve Bayesian rDNA classifier was used to hierarchically
classify high-quality sequencing reads at the genus level with
a confidence threshold of 80% (Wang et al. 2007). The OTUs
that belonged to LAB were selected for the biodiversity anal-
ysis of LAB in the SAV-brewing process. LAB biodiversity
indices, including Berger–Parker (d), Margalef (d Ma ),
Simpson index (D), Shannon index (H′), and Pielou evenness
index (Je), which represent species dominance, richness, di-
versity probability, diversity level, and uniformity, respective-
ly, were calculated from data of relative quantities of OTUs
that belong to LAB (Nie et al. 2015).
CCA was performed to determine the relationship between
LAB community and fermentation parameters. Nest to
Fig. 1 Fermentation process of SAV
metadata obtained by high-throughput sequencing (relative
abundance), main fermentation parameters (pH, total acid,
reducing sugar, and temperature), and metabolites (ethanol,
lactic acid, and acetic acid) were taken. The analysis
was conducted using Canoco for Windows v4.51 and
Canodraw (Wageningen UR, Netherlands) (Nie et al. 2017).
Data standardization was performed using IBM SPSS 19.0
(http://www.spss.com.hk/).
Isolation of LAB
A total of 5 g of each sample was added to 45 mL of physio-
logic saline solution and homogenized in an incubator with
shaking at 200 rpm for 5 min. The blending suspensions were
serially diluted with a sterilized physiologic saline solution,
and 150 μL of appropriate diluted solution was spread onto
Man–Rogosa–Sharpe (MRS) plates (Beijing land and
bridge, China) with 0.1% (w/v) nystatin dihydrate for
inhibiting fungus. Plates were incubated at 37 °C for 2
to 3 days, and colonies with different phenotypes were select-
ed and purified. All isolates were submitted to Gram staining,
spore staining, catalase activity testing, and acid production
testing according to LAB definition (Waters et al. 2015) and
were stored at 4 °C. Sequence accession numbers for the iso-
lates used in the following experiments are provided in
Supplemental Table S1.
Genotype comparison
The LAB isolates were cultured in MRS media for 12 to 18 h.
Genome was extracted with MiniBEST Bacterial Genomic
DNA Extraction Kit (Takara, Dalian, China) according to
the instruction manual.

All LAB isolates in this study were subjected to enterobac-
terial repetitive intergenic consensus (ERIC)–polymerase
chain reaction (PCR) to dislodge the potentially same strain
by comparing the fingerprints. Amplifications were conducted
in 25 μL of reaction mixture containing 0.6 μL of Premix Ex
Taq (Takara, Dalian, China) and 1 μM of each primer of
ERIC1 (5′-ATGTAAGCTCCTGGGGATTCAC-3′) and
ERIC2 (5′-AAGTAAGTGACTGGGGTGAGCG-3′) (Wu
et al. 2011). PCR was performed as follows: an initial dena-
turation at 94 °C for 5 min; 35 cycles consisting of denatur-
ation at 94 °C for 30 s; annealing at 52 °C for 90 s, with
extension at 72 °C for 3 min; and a final extension at 72 °C
for 10 min. PCR products were examined by 1.5% agarose gel
electrophoresis, and the amplicon fingerprint was analyzed
using Quantity One 4.6.2 (Bio-Rad, Hercules, CA).
Cluster analysis was performed with Minitab 16 software
(http://www.minitab.com/en-us/) (Minitab, America) using
the unweighted pair–group method with arithmetic means,
and a dendrogram was produced on the basis of each simple
matching matrix.
Identification of isolates
All LAB isolates with different ERIC-PCR fingerprints were
identified by 16S rDNA sequencing. Amplification of 16S
rDNA genes was assessed from the protocol using universal
primers 27 F and 1492 R (Oguntoyinbo and Narbad 2012).
Samples were submitted to GENEWIZ Company (Suzhou,
China) for sequencing after purification. The obtained nearly
full-length 16S rDNA sequences were compared with those
deposited in GenBank using BLAST search. Sequences
that showed more than 98% similarity were considered
to belong to the same OTU (Altschul et al. 1997). Consensus
sequences were imported into MEGA 5.1 (http://www.
megasoftware.net/), where a similarity matrix and a
dendrogram were created on the basis of maximum
composite likelihood method.
Characteristics of LAB regarding environment
Ethanol and acetic acid tolerance assays were conducted in-
dependently in fresh MRS media that contained 10 g/L of
acetic acid or 70 g/L of ethanol. All LAB isolates were culti-
vated to mid-logarithmic phase and inoculated into fresh MRS
media that contained acetic acid or ethanol with 5% (v/v)
inoculum volume. Cells were incubated statically at 37 °C
for 24 h and shaken for 20 s with an interval of 4 h. Cell
growth was monitored by measuring optical density at
600 nm (OD600). In temperature tolerance assays, all trans-
ferred LAB were cultured at 47 °C for 24 h, and cell growth
was detected.
Principal component analysis (PCA) was performed to ex-
plore the correlation between environmental tolerance and cell
Appl Microbiol Biotechnol
growth in different stresses of acetic acid, ethanol, and tem-
perature. Correlation analysis between cell growth of LAB
from each stage under environmental stress and the environ-
mental parameters that varied along with fermentation was
performed using SPSS.
Characteristics of LAB regarding substrate
The capability of LAB to utilize substrates was assessed with a
Gram-positive (GP) plate (GEN III Microstation, Biology,
Hayward CA) that contained 95 carbon sources, among which
nearly 60 were detected in SAV fermentation in a pre-
vious study (Li et al. 2016b). The capability of LAB to
decompose a particular substrate was evaluated accord-
ing to the color of the GP plate. Data standardization
was performed using SPSS. PCA was used to evaluate
the metabolic characteristics of 57 isolates. The hierarchical
cluster analysis (HCA) managed by software HCE3 (http://
www.cs.umd.edu/hcil/hce/) was performed to explore
various LAB catabolism capabilities. A heat map was drawn
according to HCA data.
Results
Fermentation process of SAV
Dynamics of physicochemical parameters throughout the fer-
mentation are shown in Fig. 2a. Ethanol, total acid, and tem-
perature were the universal indicators used to monitor the SSF
process of SAV. Ethanol accumulated up to 6.9 g/100 mL in
the AF stage, which was then constantly consumed in the
AAF stage. Total acid, which mainly resulted from acetic acid,
and lactic acid increased to a non-significant level at the AF
stage but rapidly increased to 5.7 g/100 mL in the AAF stage.
Lactic acid was mainly converted from glucose by LAB (Wu
et al. 2011). Lactic acid constantly accumulated from AF1d to
AAF5d but decreased from AAF5d to AAF7d, which was
probably consumed by microorganisms. AAB could convert
lactic acid to generate acetone, which is a flavor compound
(Moens et al. 2014). Acetic acid was mainly converted from
ethanol, through AAB constantly accumulating from AF1d to
AAF5d, and then stabilized from AAF5d to AAF7d.
Reducing sugar content decreased continuously, especially
in the process of AF4d to AF7d. The content modestly
increased in the AFF stage, which suggested that part of
the macromolecules (e.g., starch) were degraded to pro-
duce small-molecule reducing sugar (Cocchi et al.
2006). Fermentation temperature remained at approxi-
mately 30 °C in the AF stage, whereas it rapidly increased to
47 °C in the AAF stage. The temperature increase mainly
resulted from the exothermic reaction of microorganism
metabolism.
Appl Microbiol Biotechnol
Composition and succession of LAB communities
High-throughput sequencing was used to investigate the com-
position and succession of bacterial communities during SAV
fermentation. At least 12 OTUs in 4 genera, including
Lactobacillus, Weissella, Pediococcus, and others, were iden-
tified as LAB (shown in Fig. 2b). The relative abundance of
Lactobacillus reached 98.7%, in addition to small amounts of
Weissella (0.8%), Pediococcus (0.4%), and others (0.1%) of
LAB. OTUs of LAB in the AF stage (11 OTUs) were richer
than those in Daqu (3 OTUs) and AAF (5 OTUs) stages,
which conformed to the results of LAB diversity indices
(shown in Table 1). Species dominance (d), richness (dMa),
Simpson (D), and Shannon (H′) indices of the AF stage were
Fig. 2 Dynamics of physicochemical indicator and LAB in SAV
fermentation and their correlation analysis. a Time curves of SAV
fermentation. b Dynamics of relative abundance of LAB analyzed by
high-throughput sequencing. c CCA analysis of the correlation of LAB
higher than those of the Daqu and AAF stages. OTU 6, OTU
9, OTU 11, and OTU 12, which belonged to Lactobacillus,
existed in the AF and AAF stages. Relative abundances of
OTU 9 and OTU 12 constantly increased from the AF to the
AAF stages. OTUs of LAB decreased from 11 to 5 with fer-
mentation from the AF stage to the AAF stage, which indicat-
ed the vanishing of LAB species during SAV fermentation.
However, the succession sequence of LAB remained unclear.
Relationships between environmental factors
and LAB community succession
LAB existing in the brewing process of SAV spontaneously
propagated to form a particular LAB community under the
community structure with environmental factors. Arrows indicate the
direction and magnitude of environmental factors associated with LAB
community structure
 Appl Microbiol Biotechnol

effects of nutrition and environmental factor changes, espe- (Nie et al. 2015, 2017). In the present study, LAB strains were
cially temperature, pH, total acid, and reducing sugar (Li et al. isolated according to the brewing process to determine the real
2017). Effects of these environmental factors and dominant characteristics of LAB from different stages. LAB are a group
metabolites (ethanol, lactic acid, and acetic acid) on the LAB of GP, catalase-negative, non-motile, non-respiring, and non-
community succession were evaluated using the CCA meth- spore bacteria that can make bromocresol turn green yellow
od, as shown in Fig. 2c. The two axes explained 86.4% of the (Wu et al. 2011; Waters et al. 2015). A total of 85 strains of
variation in LAB community differentiation, which suggested LAB were isolated from different brewing stages of SAV ac-
a remarkable correlation between LAB structure and these cording to their morphology on plates and under an optical
factors. Reducing sugar, ethanol, pH, and lactic acid showed microscope. All isolates were screened using the ERIC-PCR
strongly positive correlation with LAB distribution in the AF method, which has been proven to be an effective method for
stage but showed negative correlation in the AAF stage. differentiating LAB at the species, subspecies, and strain
Ethanol showed a stronger correlation than those of reducing levels because of its high repeatability and accuracy to char-
sugar, pH, and lactic acid. The remarkable correlation of eth- acterize and differentiate each strain (Gevers et al. 2001;
anol with LAB community indicated that ethanol, as the main Masco et al. 2003; Wu et al. 2012). The ERIC-PCR molecular
product in the AF stage, potentially played a major role in the fingerprint clustering of all strains to reflect microbial diver-
diversity and succession of LAB and corresponded to a pre- sity is shown in Fig. 3 (Yuan et al. 2010). Strains isolated from
vious report (Li et al. 2015b). Total acid and temperature, as the same sample with an identical genotype were considered
the most important physiochemical indicators for AAF, the same strain, and only one was selected for further study.
showed positive correlation with LAB composition in the For instance, among the AAF3-2, AAF3-4, AAF3-6, AAF3-
AAF stage (Li et al. 2016b). By contrast, they were negatively 7, and AAF3-8 that were isolated from sample AAF3d, only
correlated with LAB in the AF stage. The same result was AAF3-2 was selected. However, AAF7-4, AAF3-2, AAF1-4,
observed for the correlation of acetic acid with LAB compo- and AAF0-4 that were identified as Lactobacillus casei in
sition. Acetic acid and temperature showed an almost equal different samples were selected. A total of 57 isolates (listed
correlation with LAB distribution in the AAF stage, which in Supplemental Table S1) were accordingly selected and
indicated that acetic acid and temperature might play impor- identified by 16S rDNA combined with morphological phys-
tant roles in LAB structure in the AAF stage. Thus, exploring iological analysis. A phylogenetic tree was constructed on the
the environmental factor tolerance of LAB, including ethanol, basis of 16S rDNA sequences (Supplemental Fig. S1).
temperature, and acetic acid, would be a rational direction to ERIC-PCR fingerprints demonstrated the diversity of LAB
reveal the succession sequence of LAB throughout the strains throughout the fermentation process. The LAB com-
brewing process of SAV. position changed along with the fermentation process due to
distinct predilections to circumstance. Ethanol concentration
Isolation and identification of LAB increased in the AF stage, and ethanol concentration de-
along with fermentation process creased and acidity and temperature became the main envi-
ronmental stressors in the AAF stage according to the CCA
In previous reports, the succession of microbial community result (Fig. 2c). Thus, some LAB could exist continuously
was mainly analyzed using a culture-independent method throughout the fermentation process; for example,
Lactobacillus helveticus appeared almost in the entire fermen-
tation process. By contrast, Pediococcus acidilactici was iso-
Table 1 Microbial diversity indices of LAB communities. d: Berger-
Parker index, dMa: Margalef index, D: Simpson index, H′: Shannon lated only from samples of Daqu and AAF stages, and
index, Je: Pielou index Lactobacillus fermentum was isolated from the AF stage.
This result might be due to their different adaptability to en-
Sample number d dMa D H′ Je
vironmental stresses that were detected. Furthermore,
Daqu 2.45 0.43 0.66 1.55 0.98 Lactobacillus plantarum and Lactobacillus coryniformis were
AF1d 3.91 1.95 0.85 2.93 0.88 only detected only in AF1d, and Lactobacillus rhamnosus
AF4d 4.34 2.17 0.86 2.97 0.86 was detected only in the aging sample due to its extremely
AF7d 4.45 2.17 0.85 2.89 0.84 acidic and irradiant environment.
AF10d 4.10 1.52 0.82 2.59 0.86
AAF0d 2.87 0.87 0.74 2.06 0.89 Characteristics of LAB regarding environment
AAF1d 2.02 0.87 0.65 1.75 0.75
AAF3d 1.84 0.65 0.61 1.54 0.77 Ethanol and acetic acid, as the most significant products of AF
AAF5d 1.62 0.65 0.52 1.27 0.63
and AAF stages, respectively, were mainly converted by
AAF7d 1.54 0.43 0.47 1.00 0.63
yeasts from sugars and by AAB from ethanol (Nie et al.
2015). In addition to ethanol and acidity, high temperature
Appl Microbiol Biotechnol
also affected the LAB distribution, which was mainly due to
microorganism metabolism. The most adaptive temperatures
of LAB were 30 to 40 °C (Waters et al. 2015), and the peak
temperature of AAF reached 47 °C (Fig. 2a). Therefore, the
characters of LAB on environmental tolerance were studied
with acetic acid, ethanol, and temperature as indicators to
reveal the succession sequence of LAB throughout the
brewing process of SAV.
As shown in Fig. 4a–c, cell growth under environmental
stress was distinguished along with the fermentation process.
The growth capacity of LAB from different stages against
environmental stress was in accordance with the trend of en-
vironmental factors throughout the fermentation. Remarkable
Fig. 3 ERIC-PCR clustering
analysis of LAB strains
correlations were determined between the LAB growth at
each stage and environmental factors that varied with fermen-
tation. These findings agreed with CCA results. The correla-
tion coefficients between cell growth and ethanol, acetic acid,
and temperature were 0.866, 0.756, and 0.803, respectively.
Results demonstrated that ethanol, acetic acid, and tempera-
ture are important environmental factors that drive the succes-
sion of the LAB community and other microorganisms. The
growth of some LAB strains with a low tolerance against
environmental factors was inhibited, whereas LAB strains
with a high environmental tolerance grew properly and be-
came the predominant LAB community for respective stages.
LAB from AAF grew better than those from AF stages under

10 g/L of acetic acid or 47 °C stress, whereas LAB from AF
grew better than those from AAF stages under 70 g/L of eth-
anol stress. Moreover, we compared the growths of LAB un-
der gentle and harsh environments, including acidity stress of
5 or 20 g/L, ethanol stress of 60 or 80 g/L, and thermal stress
of 30 or 55 °C. However, no distinct differences in growths
were observed among the LAB strains from different stages
due to low inhibition conditions (5 g/L of acetic acid, 60 g/L of
ethanol, and 30 °C) or excessive inhibition conditions (20 g/L
of acetic acid, 80 g/L of ethanol, and 55 °C) (data not shown).
Tolerance variations of LAB against environmental factors
were roughly matched with the variations in physiochemical
indicators (Fig. 4a–c).
PCA was used to evaluate LAB tolerance against environ-
mental stresses. In Fig. 4d, the two axes explain 88.5% of the
overall environmental effect on LAB growth. LAB in AF1d,
AF4d, AF7d, and AF10d were greatly influenced by ethanol.
LAB in AAF1d, AAF3d, AAF5d, and AAF7d were greatly
Fig. 4 Characteristics of LAB tolerance against environmental factors. a
Environmental tolerance of LAB against acetic acid (10 g/L) throughout
the fermentation process. b Environmental tolerance of LAB against
ethanol (70 g/L) throughout the fermentation process. c Environmental
tolerance of LAB against temperature (47 °C) throughout the
Appl Microbiol Biotechnol
influenced by acetic acid and temperature. LAB in AAF0d
were influenced by ethanol, acetic acid, and temperature.
Acetic acid showed almost the same inhibition effect com-
pared with that of temperature, which agreed with the CCA
results.
Characteristics of LAB regarding substrate
Nutrition was another factor that affected the succession of
LAB community in addition to other environmental factors.
For SAV fermentation, Jiulao and Cupei samples contained
abundant sugars, alcohols, amino acids, organic acids, and
other materials (Li et al. 2016b). Their changes in composition
were closely related to microbial metabolism. A GP plate was
used to analyze the diversity of LAB in substrate metabolism,
which could indicate the substrate utilization capabilities of
LAB strains. Figure 5 describes the heat map of the
substrate-decomposing capability of LAB strains. More
fermentation process; solid curves indicate the average growth of LAB
under different environmental stresses; dashed curves indicate the
parameter change throughout the fermentation process. d PCA of LAB
tolerance on environmental factors; arrows indicate the direction and
magnitude of environmental factors associated with the LAB tolerance
Appl Microbiol Biotechnol

substrates could be catalyzed by the strains from the AAF substrate catalytic activity. Substrates in group A showed
stage than those from AF, which indicated higher substrate high-catalysis activities by LAB from AF4d and AAF7d.
utilization by LAB in AAF than that in the AF stage. Moreover, lactic acid could be utilized by most LAB. This
Substrates were clustered into four groups on the basis of result was consistent with the concentration change of lactic

Fig. 5 Heat map analysis for LAB characteristic on substrate metabolism


acid in the fermentation process, which first increased and
then decreased (as shown in Fig. 2a). The concentration of
acetic acid continuously increased; therefore, the total acid
consistently increased (Li et al. 2016b). Sugars, lactulose, ino-
sine, uridine, and D-galactose exhibited strong positive reac-
tions in AF4d. In the AF stage, sugars were metabolized into
ethanol by yeasts, but they could also be metabolized by LAB
into multiple organic acids and other metabolites (Nie et al.
2015). Substrates in group B were mainly catalyzed by LAB
from AAF7d and the aging stage, including lactulose that was
utilized by many LAB (Devos and Vaughan 1994). For group
C, LAB from AAF5d could metabolize most substrates. L-
Arabinose, glycerin, pyruvic acid, and α-methyl-D-galactose
glucoside showed strongly positive reactions from LAB in
AF10d. D-Cellobiose, 5′-uridine phosphate, and D-sorbitol
showed strongly positive reactions from LAB in AAF7d.
Substrates in group D exhibited strongly positive reac-
tions from LAB in Daqu, AAF7d, and the aging stages.
Similar reaction profiles between LAB in AAF7d and
the aging stage were mainly because AAF7d was affected
by the aging process.
Principal component comprehensive score was used to
evaluate the substrate utilization capability of LAB according
to Biology system data (GEN III Microstatian, Biolog,
Hayward CA). Table 2 shows that the scores of the first 18
compounds are mainly influenced by sugars, in addition to D-
lactic acid methyl ester, lactic acid, and pyruvic acid, which
indicated that those compounds could be utilized by most
isolates. Sugars, including glucose, mannose, melibiose, su-
crose, and maltose, are considered important factors that affect
LAB succession and are the predominant in carbon nutrition
in SAV fermentation for cell growth and metabolism (Vos
et al. 1998). Dextrin is a polycarbohydrate, is detected in
SAV fermentation, and is the product of starch hydrolyses
from raw materials, sorghum, and bran (Bernal et al. 2002).
L-Lactic acid is a common metabolic product of LAB, but it
could also be utilized by LAB under the catalysis of lactate
dehydrogenase (Koo et al. 2005). D-Lactic acid methyl ester
could be hydrolyzed by methyl lactate hydrolase to produce
lactate and methyl groups (Murphy et al. 2016) and then be
utilized by LAB. Pyruvic acid is an important intermediate
metabolite, which has been proven important for LAB acidity
resistance (Viana et al. 2005). 2,3-Butanediol could be cata-
lyzed by some LAB strains, especially from AAF7d, which
could be catalyzed into acetoin, a flavor compound in SAV
fermentation, by dehydrogenation of LAB (Moens et al.
2014). Acetoin could then react with amino compounds to
form tetramethylpyrazine (Lu et al. 2016), a physiological
activity ingredient.
PCA was employed to assess the metabolic characteristics
of 57 isolates (Fig. 6a) and LAB from different fermentation
stages (Fig. 6b). Figure 6a shows that the PCA for the propor-
tion of total variance could explain 63.7% of the information.
Appl Microbiol Biotechnol
A scatter plot was created with the PCA results (Fig. 6a),
stating that 72.0% (18 over 25) of LAB in the first group were
mainly isolated from AF, and 59.3% (16 over 27) of LAB in
the second group were mainly isolated from AAF stages. The
plot suggested similar metabolic characteristics of LAB from
each fermentation stage. Particularly, group 3 was isolated
from AAF7d, the later period of AAF with high acidity.
Calculation of the average overall metabolism of LAB for
each day indicted that PCA could explain 73.9% of the infor-
mation, as shown in Fig. 6b. LAB from AF and AAF stages
were divided into two groups, except LAB from AAF7d that
were far from others due to the high acidity and low sugar
concentration at the seventh day of AAF (as shown in Fig. 2a).
Discussion
The importance of microbial communities for the fermenta-
tion and flavor formation of SAV SSF process is becoming
increasingly clear (Li et al. 2016b); however, dissecting the
formation mechanisms of these communities remains poorly
identified due to their high species diversity, low cultivability,
and incapability to easily simulate their natural environment
(Wolfe et al. 2014). Recent studies have applied culture-
independent methods, including denaturing gradient gel elec-
trophoresis and high-throughput sequencing, to evaluate the
microbial ecology of traditional fermented food, including
vinegars, with advantages of high speed, accuracy, and ability
to determine uncultured microorganisms (Li et al. 2015a, b).
However, results of such culture-independent methods are
sometimes influenced by some strains that are inactivated or
even dead. Therefore, in addition to advances in the direct
study of complex microbial communities, culture-dependent
methods can help in the identification and characterization of
functional strains and further facilitate works toward under-
standing the succession mechanism (Wolfe et al. 2014; Wu
et al. 2011). This research revealed the diversity and succes-
sion sequence by combining culture-dependent and culture-
independent methods.
High-throughput sequencing described the succession of
LAB community. CCA indicated that ethanol, acidity, and
temperature, which are natural products from microorganism
metabolism accumulated from the fermentation process, are
potential important factors that influence the succession
of LAB community, as shown in Fig. 2c. Our findings
are consistent with those of recent studies that indicated
that microbial communities are largely affected by envi-
ronmental variables and substrates (i.e., moisture is the best
predictor of cheese rind community composition) (Li et al.
2016a; Wolfe et al. 2014).
LAB strains were isolated periodically according to the
fermentation process to reveal the real diversity of LAB in
different fermentation stages. After repeated strains were
Appl Microbiol Biotechnol

Table 2 Principal component

comprehensive score Rank Substrate Scores Rank Substrate Scores

1 D-Lactic acid methyl ester 8.027 49 Thymidine − 0.572

2 Dextrin 7.806 50 Water − 0.608
3 α-D-Glucose 7.161 51 L-Fucose − 0.616
4 L-Lactic acid 6.719 52 Succinic acid − 0.620
5 D-Mannose 4.381 53 Mannan − 0.637
6 D-Melibiose 4.204 54 Acetic acid − 0.676
7 D-Ribose 3.649 55 p-Hydroxy-phenylacetic acid − 0.682
8 Sucrose 2.796 56 L-Serine − 0.692
9 Maltose 2.349 57 Turanose − 0.709
10 D-Galactose 1.977 58 Palatinose − 0.724
11 Adenosine 1.963 59 Glycerol − 0.725
12 Maltotriose 1.960 60 D-Cellobiose − 0.734
13 L-Arabinose 1.727 61 D-Mannitol − 0.737
14 Pyruvic acid 1.708 62 Lactamide − 0.739
15 D-Xylose 1.523 63 Salicin − 0.744
16 Amygdalin 1.115 64 D-Fructose-6-phosphate − 0.842
17 D-Raffinose 0.820 65 β-Methyl-D-glucoside − 0.852
18 D-Tagatose 0.804 66 D-Psicose − 0.854
19 N-Acetyl-L-glutamic acid 0.533 67 D-Arabitol − 0.854
20 α-Hydroxybutyric acid 0.384 68 γ-Hydroxybutyric acid − 0.881
21 D-Gluconic acid 0.380 69 Propionic acid − 0.896
22 β-Cyclodextrin 0.330 70 2′-Deoxy adenosine − 0.917
23 α-Methyl-D-glucoside 0.310 71 Stachyose − 0.962
24 α-D-Lactose 0.233 72 3-Methyl-D-glucose − 0.970
25 Xylitol 0.140 73 α-Ketoglutaric acid − 1.006
26 M-Inositol 0.093 74 Tween 40 − 1.084
27 β-Methyl-D-galactoside 0.091 75 α-Methyl-D-glucoside − 1.143
28 D-Melezitose 0.091 76 L-Pyroglutamic acid − 1.161
29 D-Malic acid 0.065 77 L-Glutamic acid − 1.168
30 β-Hydroxybutyric acid 0.040 78 N-Acetyl-D-glucosamine − 1.215
31 D-Trehalose − 0.064 79 N-Acetyl-β-D-mannosamine − 1.295
32 Succinamic acid − 0.068 80 Thymidine-5′-monophosphate − 1.325
33 D-Sorbitol − 0.142 81 Inosine − 1.347
34 Pyruvic acid methyl ester − 0.155 82 α-Cyclodextrin − 1.483
35 D-Fructose − 0.156 83 Succinic acid mono-methyl ester − 1.518
36 D-Galacturonic acid − 0.178 84 L-Asparagine − 1.521
37 L-Alaninamide − 0.240 85 D-Glucose-6-phosphate − 1.542
38 Sedoheptulosan − 0.243 86 Uridine − 1.628
39 Lactulose − 0.244 87 Uridine-5′-monophosphate − 1.649
40 2,3-Butanediol − 0.244 88 α-D-glucose-1-phosphate − 1.760
41 Putrescine − 0.274 89 D-L-α-glycerol phosphate − 1.841
42 L-Malic acid − 0.310 90 Glycyl-L-glutamic acid − 1.931
43 Gentiobiose − 0.329 91 L-Alanyl-glycine − 2.001
44 α-Ketovaleric acid − 0.329 92 Adenosine-5′-monophosphate − 2.385
45 Glycogen − 0.355 93 L-Alanine − 2.392
46 α-Methyl-D-galactoside − 0.369 94 D-Alanine − 2.397
47 L-Rhamnose − 0.371 95 Arbutin − 2.559
48 Tween 80 − 0.434 96 Inulin − 3.281

Fig. 6 PCA of the metabolic characteristic of LAB. a PCA metabolic
characteristics of 57 isolates. b PCA of the metabolic characteristic of
every fermentation stage
removed, 57 strains that belonged to 10 genera were obtained,
which presented 83.3% (10 over 12) of the information on
LAB in the SAV-brewing process. LAB from different stages
were divided and grouped depending on their environmental
tolerance (Fig. 4) and substrate utilization characteristics
(Figs. 5 and 6). Composition and diversity of LAB demon-
strated a succession along with the brewing process of SAV.
The succession schematic according to these results is shown
Fig. 7 Succession schematic
of LAB in the brewing
process of SAV
Appl Microbiol Biotechnol
in Fig. 7. Substrates promoted the succession at the initial AF
stage, and then, substrates and ethanol drove the succession at
the stages of later AF and initial AAF. At the middle and later
stages of AAF, moreover, acetic acid and temperature drove
the succession.
The results on substrate utilization demonstrated that most
sugars, including glucose, lactose, and mannose, could be
consumed by LAB for growth and for producing organic
acids. As in the study of Li et al. (2015b), the current research
determined that microorganisms could decompose starch, pro-
tein, cellulose, and other macromolecules from raw materials
and could produce rich sugars, alcohols, organic acids, and
amino acids that are the main compositions and flavor
sources of SAV. Moreover, we also found that lactic
acid and lactic acid methyl ester could be utilized by many
LAB strains. This finding could explain the decrease in lactic
acid at the later period of AAF, considering LAB are the
prominent bacteria in AAF.
L. rhamnosus and L. coryniformis were isolated from SAV
for the first time (Wu et al. 2011). Several strains could resist
environmental stresses and catalyze different substances,
which have potential applications in many ways (Li et al.
2017). Aging or maturation is an important step in the tech-
nique for vinegar production to improve the quality and could
last from 1 to 10 years (Liang et al. 2016; Solieri and Giudici
2009). During the aging stage, some complex physical and
chemical reactions occur, and the amounts of aromatic and
function compounds, such as esters, Maillard reaction prod-
ucts, and polyphenol compounds, and pyrazizes (e.g.,
tetramethylpyzazine), greatly increase (Liang et al. 2016; Li
et al. 2015b). Most researchers paid considerable attention to
changes in physicochemical indexes and flavor substances but
rarely considered the presence of microorganisms in the aging
stage in view of the extremely acidic environment (Liang et al.
2016; Li et al. 2015b; Wu et al. 2011; Solieri and Giudici
2009). This study for the first time isolated LAB strains, such
as Staphylococcus capitis, L. rhamnosus, P. acidilactici, and
Appl Microbiol Biotechnol
Pediccoccus pentosaceus, from aging SAV samples with ap-
proximately 8% (w/v) acidity (Wu et al. 2011). Therefore, the
action of microbial metabolism on SAV aging should be fur-
ther studied.
Moreover, we focused on the LAB community succession
sequence driven by environmental variables. The environ-
mental tolerance experiment proved that strains from the AF
stage showed better tolerance against ethanol, which was the
main product in AF (Solieri and Giudici 2009), and LAB from
the AAF stage showed better tolerance against acetic acid and
temperature, which are the main products in AAF. These re-
sults indicated that environmental disturbances leave strains
with excellent environmental tolerance and became dominant
in each stage, to result in the succession of the LAB commu-
nity in SAV fermentation. Only appropriate environmental
stresses could drive the succession of the LAB community.
More gentle or harsh environments (less or more than 60 or
80 g/L of ethanol, 5 or 20 g/L of acetic acid, and 30 °C or
55 °C temperature) would not affect the LAB succession.
Although individual traits were previously determined to scale
up the bacterial community succession (Salles et al. 2009), our
results provided insights into the environmental- and
metabolic-driven succession sequence of the LAB com-
munity assemblies during the open-SSF process. Such
environmental- and metabolic-driven succession changes
might also apply to other microorganisms in SAV fer-
mentation. As reported previously, LAB are confronted
with abiotic and biotic stresses during food fermentation
and in complex ecosystems (Papadimitriou et al. 2016). In the
majority of studies on LAB stress physiology, strains could
adapt to stresses by appropriate molecular responses, includ-
ing those of genes and proteins, to ameliorate negative effects
and restore growth or survival potential (Papadimitriou et al.
2016). Thus, unraveling gene expression, which is related to
the succession of LAB community, via metatranscriptomic
approaches is ongoing in further studies to understand the
mechanism.
Theoretically, the thermo-, acid-, or temperature-tolerant
LAB obtained in this research might improve SAV fermenta-
tion due to their increased metabolism activity under environ-
mental stresses (Li et al. 2017). Therefore, a bioaugmenting
fermentation technology using environment-tolerant LAB as
inoculum is being performed in our laboratory to improve the
substrate utilization and production of SAV.
Acknowledgments We appreciate the Shanxi Zilin Vinegar Industry Co.
for providing samples.
Funding information This work was supported by the National Key
R&D Program of China (2016YFD0400505), the National Natural
Science Foundation of China (31671722), the Tianjin Municipal
Science and Technology Commission (16YFZCNC00650), and the
Program for Changjiang Scholars and Innovative Research Team in
University (IRT15R49).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Ethical statement This article does not contain any studies with human
participants or animals performed by any of the authors.
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