International Journal of Food Microbiology 204 (2015) 916

Contents lists available at ScienceDirect

International Journal of Food Microbiology

journal homepage: www.elsevier.com/locate/ijfoodmicro

Identication of acetic acid bacteria in traditionally produced vinegar and

mother of vinegar by using different molecular techniques
Ahmet E. Yetiman, Zlal Kesmen
Erciyes University, Faculty of Engineering, Food Engineering Department, Kayseri, Turkey

a r t i c l e

i n f o

Article history:
Received 15 July 2014
Received in revised form 24 February 2015
Accepted 12 March 2015
Available online 21 March 2015
Keywords:
Acetic acid bacteria
Vinegar
PCR-DGGE
Rep-PCR
Intercalating dye assays

a b s t r a c t
Culture-dependent and culture-independent methods were combined for the investigation of acetic acid bacteria
(AAB) populations in traditionally produced vinegars and mother of vinegar samples obtained from apple and
grape. The culture-independent denaturing gradient gel electrophoresis (DGGE) analysis, which targeted the
V7V8 regions of the 16S rRNA gene, showed that Komagataeibacter hansenii and Komagataeibacter europaeus/
Komagataeibacter xylinus were the most dominant species in almost all of the samples analyzed directly. The
culture-independent GTG5-rep PCR ngerprinting was used in the preliminary characterization of AAB isolates
and species-level identication was carried out by sequencing of the 16S rRNA gene, 16S23S rDNA internally
transcribed to the spacer (ITS) region and tuf gene. Acetobacter okinawensis was frequently isolated from samples
obtained from apple while K. europaeus was identied as the dominant species, followed by Acetobacter
indonesiensis in the samples originating from grape. In addition to common molecular techniques, real-time
PCR intercalating dye assays, including DNA melting temperature (Tm) and high resolution melting analysis
(HRM), were applied to acetic acid bacterial isolates for the rst time. The target sequence of ITS region generated
species-specic HRM proles and Tm values allowed discrimination at species level.
2015 Elsevier B.V. All rights reserved.

1. Introduction
Vinegar is the product of a two-step fermentation process involving
the alcoholic fermentation of sugars into ethanol by yeasts, and, subsequently, the oxidation of ethanol into acetic acid by acetic acid bacteria
(AAB). Vinegar has been renowned as a food preservative since ancient
times and is still used today in the food industry as a highly sought-after
condiment and a multi-functional product utilized for pickling, preserving and avor-balancing purposes (Sengun and Karapinar, 2004;
Steinkraus, 2002). All raw materials containing sugar or fruits can be
employed as starting substances for producing vinegar. Several different
types of vinegars are produced around the world by using diverse
manufacturing techniques and raw materials native to specic areas.
In general, two well-dened methods, namely, slow surface culture fermentation and fast submerged fermentation, are used in vinegar
manufacturing (Tesfaye et al., 2002). The slow method is also called traditional vinegar production and the AAB grow on the surface of the liquid during the fermentation process, which may take from several
weeks up to a few months (Nanda et al., 2001). A non-toxic lm composed of AAB and cellulose accumulates on the surface of the vinegar
throughout the oxidation process. This lm, called mother of vinegar,
is used in the back-slopping practice as an indigenous starter culture to

Corresponding author. Tel.: +90 352 2076666/32729.

E-mail address: zkesmen@erciyes.edu.tr (Z. Kesmen).

http://dx.doi.org/10.1016/j.ijfoodmicro.2015.03.013
0168-1605/ 2015 Elsevier B.V. All rights reserved.

trigger acetication in the production of traditional vinegar (Holzapfel,

2002; Hidalgo et al., 2010).
As a result of the two stage fermentation, several microbial species
show competitive activity in the vinegar throughout the production
process (Holzapfel, 2002). The microbial transformations give the
characteristic taste and fragrance to vinegar and the microbial metabolites have benecial health effects (Shimoji et al., 2002; Stasiak and
Blaejak, 2009). Acetic acid bacteria, which carry out the second transformation, namely oxidative fermentation, are one of the main microbial populations in vinegar.
The three genera of acetic acid bacteria, Acetobacter (A.),
Gluconacetobacter (Ga.) and Komagataeibacter (K.) are mainly responsible for acetic fermentation in vinegar. The species of
Acetobacter aceti, Acetobacter malorum, Acetobacter pasteurianus,
Acetobacter pomorum, Komagataeibacter europaeus, Komagataeibacter
hansenii, Komagataeibacter intermedius, Komagataeibacter oboediens,
Komagataeibacter xylinus, Komagataeibacter medellinensis and
Komagataeibacter maltiaceti were previously detected in the vinegar
ecosystem (Sievers et al., 1992; Trcek et al., 1997; Sokollek et al.,
1998; Boesch et al., 1998; Trcek et al., 2000; De Vero et al., 2006;
Gullo et al., 2006; Yamada et al., 2012; Castro et al., 2013; Slapsak
et al., 2013). However, the proles of AAB are unstable and show particular diversity in accordance with the raw material characteristics and
production process features (Gullo et al., 2009).
Identication of AAB based only on morphological, biochemical, and
physiological characteristics is not reliable and, therefore, is insufcient

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A.E. Yetiman, Z. Kesmen / International Journal of Food Microbiology 204 (2015) 916

because of the poor reproducibility and discriminatory power of these

phenotypic tests (Cleenwerck and De Vos, 2008; Papalexandratou
et al., 2009). For this reason, nucleic acid-based molecular methods
are now used to characterize and identify isolates of AAB from wine
and vinegar ecosystems. These have included Enterobacterial Repetitive
Intergenic Consensus-PCR (ERIC-PCR) (Gonzalez et al., 2004; Gullo
et al., 2009; Nanda et al., 2001), Repetitive Extragenic PalindromicPCR (REP-PCR) (Gonzlez et al., 2004), (GTG)5-rep-PCR ngerprinting
(De Vuyst et al., 2008) and RAPD-PCR (Trcek et al., 1997; Bartowsky
et al., 2003; Nanda et al., 2001). A reliable taxonomic identication is obtained when these techniques are combined with the sequencing of 16S
rDNA genes (Gonzalez, 2005) and internal transcribed spacer sequences
(ITS) of the 16S23S rDNA genes (Gonzalez and Mas, 2011). Restriction
fragment length polymorphism (RFLP) analysis of the ribosomal genes
or their spacer regions has also been used for the identication of AAB
present in food-related ecosystems (Gullo et al., 2006; Ruiz et al.,
2000; Trcek and Teuber, 2002; Trcek, 2005; Vegas et al., 2010).
The combination of culture-independent methods with culturedependent methods in a polyphasic system is recommended as an effective approach to overcome the difculties regarding the isolation
and cultivation of AAB strains (Cleenwerck and De Vos, 2008; Gullo
et al., 2009; Sengun and Karapinar, 2011). In the last decade, several
studies using the culture-independent DGGE and Temporal Temperature Gradient Gel Electrophoresis (TTGE) techniques were reported
for the characterization of the microbial community in vinegar and the
determination of population dynamics of AAB during fermentation
(De Vero et al., 2006; De Vero and Giudici, 2008; Ilabaca et al., 2008).
In addition, the real-time PCR technique has also been proposed for
culture-independent detection of different genera, or species, of AAB
(Andorra et al., 2008; Torija et al., 2010; Valera et al., 2013). Recently,
the intercalating dye-based real-time PCR analysis involved in specic
melting temperature (Tm) and high-resolution melting analysis were
applied as a highly promising new approach for conrming the identication and grouping of the culturable strains belonging to different bacterial species (Kao et al., 2007; Juvonen et al., 2008; Kesmen et al., 2014),
but not yet to AAB. These new approaches provide a rapid and reliable
tool for the detection of small differences in the target DNA sequences
of closely related species.
The identication of indigenous AAB has critical importance to improve the process control, overcome unpredictable fermentation problems and select the most suitable strains as the potential starter
culture. Therefore, in this study, we aimed to detect and compare AAB
populations in grape and apple vinegar and in mother of vinegar
samples obtained from different regions of Turkey. Thus the cultureindependent PCR-DGGE technique was combined with culturedependent molecular techniques, including (GTG)5-rep-PCR and
sequence analysis of the 16S rRNA gene, 16S23S rRNA internal transcribed sequences (ITS) region and tuf gene for identication and characterization of AAB isolated from analyzed samples. Furthermore, realtime PCR intercalating dye-based analysis was applied to AAB isolates
to obtain species-level discrimination.

2. Materials and methods

2.1. Vinegar and mother of vinegar samples
Vinegar and mother of vinegar samples produced by the spontaneous fermentation method were obtained from three different local producers in Kayseri and Dzce, in Turkey. The samples, consisting of 2
grape vinegars (bg and eg) and their mothers (BG and EG), 2 apple vinegars (da and ha) and their mothers (DA and HA), were collected at the
end of the acetic fermentation from wood barrels in which traditional
surface fermentation is carried out. The acetic acid content of the vinegar samples were reported by producers as 3.96, 4.08, 4.28 and 4.37%
for the samples ha, da, bg and eg respectively. After the sampling

process, the vinegar and mother of vinegar samples were analyzed for
AAB using cultural and molecular methods.

2.2. Isolation of acetic acid bacteria (AAB)

10 ml of each of the 5 vinegar samples were diluted with 90 ml of
Maximum Recovery Diluent (Merck, GmbH, Darmstadt, Germany),
and homogenized for 2 min with a shaker (IKA, Germany). Serial decimal dilutions were prepared with the same diluent and subjected to
the agar plate method for the isolation of AAB on GYC (5% D-glucose,
1% ethanol, 1% yeast extract, 1% CaCO3, 0.05% bromocresol purple, 2%
agar) and MYP (1% mannitol, 1% yeast extract, 0.3% peptone, 2% agar)
agar plates. To inhibit yeast growth, 100 ppm cycloheximide (Sigma)
was added to both agars. All plates were incubated at 30 C for 5 days.
For each sample, 1015 catalase-positive, oxidase-negative, and Gramnegative colonies, showing different morphological characteristics
were puried by streak-plate technique and subjected to further characterization. All isolates selected from samples were stored at 80 C.
2.3. DNA extraction from pure cultures, vinegar and mothers of vinegar
samples
Bacterial cells harvested from GYC and MYP agar were washed in
1 ml TE buffer (50 mM TrisHCl, pH 8.0, 1 mM EDTA) and resuspended
in a 300 l lysis buffer (100 mM TrisHCl, pH 8.0, 10 mM EDTA, 2% SDS,
1% PVP, 0.15% NaCl). After homogenization, the bacterial DNA was extracted with the method described by Gullo et al. (2006). The total
DNA, which was extracted directly from each vinegar and mother of
vinegar sample, was used for PCR-DGGE analysis. The mother of vinegar
samples were powdered under liquid nitrogen and vinegar samples
were pelleted by centrifugation at 10 000 g for 10 min before
performing DNA extraction as described by De Vero et al., 2006. The
quantity and purity of the extracted DNA from the pure cultures and
the samples were measured by using a microvolume UV/Vis spectrophotometer (UVS-99, ACTGene, USA) at 260 nm and standardized at
the nal concentration of 100 ng/l.
2.4. PCR-DGGE analysis of vinegar and mother of vinegar samples
The V7V8 region of the 16S rDNA was amplied by using DNA isolated from each vinegar and mother of vinegar sample. The primers
WBAC1 (5-GTC GTC AGC TCG TGT CGT GAG A-3) and WBAC2 (5CCC GGG AAC GTA TTC ACC GCG-3) were used to amplify an approximately 328 bp fragment of the target region (Lopez et al., 2003). A GC
clamp (5-CGC CCG CCG CGC CCC GCG CCC GGC CCG CCG CCC CCG
CCC C-3) was attached to the WBAC1 primer, according to Lopez et al.
(2003). All of the PCR amplications were performed in a nal volume
of 50 l, containing 25 l of commercial PCR master mix (Dream
Taq, Fermentas, USA), 40 pmol of forward primer with a GC clamp,
20 pmol of reverse primer and 100 ng sample DNA. The thermal cycler
(TC-5000 gradient thermal cycler, Techne, UK) conditions were programmed in accordance with De Vero et al. (2006). The amplication
products were checked by electrophoresis in 2% (w/v) agarose gel containing ethidium bromide and visualized under UV light. The sequence
specic separation of the PCR products was performed on the Dcode
TM Universal Mutation Detection System (BioRad, Hercules, USA) by
using 1 mm polyacrylamide gel (8% [wt/vol] acrylamidebisacrylamide
37.5:1), containing 30% to 60% ureaformamide denaturing gradient
(100% corresponds to 7 m urea and 40% [w/v] formamide). Electrophoresis was performed at 60 C in TAE buffer 1 (40 mM Tris base, 20 mM
acetic acid glacial, 1 mM EDTA 0.5 M, pH 8.0 and dH2O) with a constant
voltage of 150 V at 60 C for about 4 h. After electrophoresis, the DGGE
gels were viewed under UV transillumination (Gel Doc XR, BioRad) after
being stained with ethidium bromide solution (50 g/ml) for 20 min.

A.E. Yetiman, Z. Kesmen / International Journal of Food Microbiology 204 (2015) 916

2.5. Sequencing of DGGE bands

All of the DGGE bands were cut from the gels and the DNA fragments
were eluted overnight in 30 l of sterile water at 4 C. Two microliters of
the eluted DNA were used for re-amplication of the target region with
the WBAC1 and WBAC2 primers without GC clamps. PCR products were
puried by using a commercial kit (QIAquick, Qiagen GmbH, Germany)
and then sequenced by Macrogen Inc. (the Netherlands). The research
on GenBank was performed by using the Blast program to determine
the closest known relatives of the partial 16S rDNA sequence obtained.
2.6. (GTG)5-rep-PCR ngerprinting
Genomic DNA extracted from bacterial isolates was used as a template to obtain rep-PCR ngerprintings. The amplication reactions
were carried out in 30 l reaction volumes, containing 15 l commercial
PCR master mix (Dream Taq, Fermentas), 75 pmol of primer and 100 ng
template DNA. The GTG5 (5-GTG GTG GTG GTG GTG-3) primer (Gevers
et al., 2001) was used for (GTG)5-rep-PCR. PCR amplications were
carried out with a Techne gradient thermal cycler (TC-5000 gradient
thermal cycler, Techne, UK) using the following program: initial denaturation at 94 C, for 5 min, and 35 cycles included denaturation at
94 C for 1 min, annealing at 40 C for 1 min and nal extension at
65 C for 8 min. The program nished with an additional nal extension
at 65 C for 5 min.
2.7. Gel electrophoresis and analysis of banding patterns
The PCR products obtained from GTG5-rep-PCR were electrophoretically separated on ethidium bromide-stained 1.5% agarose gels, in 0.5
TBE (Tris-borate-EDTA) buffer for 20 h at 50 V. The banding patterns
were visualized on a UV transilluminator (Biorad) and analyzed with
Bionumerics software (version 6.5; Applied Maths, Kortrijk, Belgium).
Dendrograms were constructed by using the unweighted pair group
method with the arithmetic averages (UPGMA) clustering algorithm
and similarities were expressed as percentage values of the Pearson correlation coefcient.
2.8. Intercalating dye-based analyses with real-time PCR
A common primer pair for AAB (Ruiz et al., 2000; Gonzalez and Mas,
2011) was used to amplify an approximately 700800 bp fragment of
the ITS region for the real-time PCR melting curve and HRM analysis.
Real-time PCR amplications were performed in a total volume of
10 l, 5 l commercial real-time PCR master mix, 10 pmol forward primer, ITS1 (5-ACC TGC GGC TGG ATC ACC TCC-3) and reverse primer, ITS2
(5-CCG AAT GCC CTT ATC GCG CTC-3), and 50 ng template DNA. Two
different real-time PCR master mixes, one containing SYBR Green
(FastStart SYBR Green Master, Roche, Germany) and the other containing ResoLight (LightCycler 480 High Resolution Melting Master), were
used for the melting curve and HRM analysis, respectively. PCR amplications were carried out under the following conditions: an initial denaturation of DNA at 94 C for 5 min was followed by 35 cycles of
denaturation at 94 C for 30 s, annealing at 65 C for 30 s and extension
at 72 C for 60 s, a nal extension of incomplete products at 72 C for
7 min and a hold stage for melting curve analysis at 40 C for 4 min. A
melting curve was programmed at the end of amplication by slowly
heating the amplication products at 0.1 C/s increments from 60
95 C and was measured by the uorescence accumulation in the
LightCycler Nano System (Roche Diagnostics, Germany). The Tm
value of each product was calculated from the plot, which constructed
the negative rst derivative of the change in uorescence (-d(RFU)/
dT, the rate of change of uorescence) versus temperature by using
LightCycler Nano software version 1.0.3. HRM proles obtained by
software analysis included normalizing the raw melting curve data,

11

shifting the normalized curves along a temperature axis and plotting

the melting curve differences between various samples.
2.9. Sequencing of the 16S rRNA gene, ITS region and tuf gene
The full sequence of the 16S rRNA gene of the strains was amplied by
PCR with the forward primer 16Sd (5-GCTGGCGGCATGCTTAACACAT-3)
and the reverse primer 16Sr (5-GGAGGTGATCCAGCCGCAGGT-3) described by Ruiz et al. (2000). The primers ITS1 and ITS2 were used for amplication of the ITS region. We also designed a forward (tuf-F, 5-GTG
TGC CGG CTC TGG TTG-3) and a reverse primer (tuf-R 5-GTG AAG AAC
GGC GTA TGA C-3) to amplify an approximately 600 bp fragment of tuf
gene for sequence analysis. All amplication reactions were carried out
in a nal volume of 50 l, 25 l commercial PCR mater mix (DreamTaq,
Fermentas), 10 pmol of each primer and 100 ng DNA template by using
the following amplication conditions: the initial denaturation of DNA
for 10 min at 94 C was followed by 35 cycles of denaturation at 94 C
for 50 s, annealing at 65 C (for amplication of the 16S rRNA gene and
ITS region) and 54 C (for amplication of the tuf gene) for 45 s, extension
at 72 C for 120 s, and a nal extension of incomplete products at 72 C for
10 min. Amplication products were checked by 1.5% (w/v) agarose gel
electrophoresis. The PCR products were sequenced (Macrogen Inc., the
Netherlands) and then the obtained sequences were aligned with
known sequences in the database (http://www.ncbi.nlm.nih.gov/BLAST)
using the algorithm.
3. Results and discussion
In this study, AAB populations in vinegar and mother of vinegar samples collected from 4 different small-scale producers in Turkey were investigated by combining culture-dependent and culture-independent
molecular methods.
3.1. Culture-independent analysis of vinegar and mothers of vinegar
samples
All of the bands in the DGGE patterns that belong to each sample
were sequenced and at least a 98% sequence similarity was obtained
with their respective species when searched in the GenBank nucleotide
database. The AAB species identied in vinegar and mother of vinegar
samples were mainly distributed within two genera, namely
Acetobacter and Komagataeibacter (Fig. 1). The Komagataeibacter genus
was more dominant than the Acetobacter genus and K. hansenii and
K. europaeus/K. xylinus group were identied as co-dominant species
followed by the Acetobacter tropicalis/Acetobacter indonesiensis group.
The band of K. hansenii clearly separated from other Komagataeibacter
species and it was detected in all samples except da.
The A. tropicalis/A. indonesiensis, Acetobacter nitrogenigens/
Acetobacter aceti and Acetobacter syzygii/Acetobacter lovaniensis species
that belong to the Acetobacter genus showed the same migration properties and could not be clearly separated in the gradient gel. PCR-DGGE
is useful to test the diversity of the bacterial populations without cultivation but the co-migration of closely related species is a major drawback of this method, as reported in previous studies (Ercolini 2004;
Cocolin et al., 2001; Kesmen et al., 2012). Although the WBAC primer
set which was originally developed for 16S rDNA gene sequence of Lactobacillus plantarum allows the differentiation of some of the AAB species. Therefore, Garcia-Armisen et al. (2010) and Papalexandratou
et al. (2011) reported that a new primer set targeting only AAB is required to obtain a higher resolution.
On the other hand, some species, namely Oenococcus oeni, produced
multiple banding patterns in DGGE gel, probably resulting from
intragenomic heterogeneity (Dahllf et al., 2000; Case et al., 2007;
Snchez and Dorado, 2008). Some of the previous authors have shown
similar, multiple banding patterns produced by O. oeni in DGGE gel
(Kato et al., 2011; Prez-Martn et al., 2014).

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A.E. Yetiman, Z. Kesmen / International Journal of Food Microbiology 204 (2015) 916

which belong to lactic acid bacteria, were also found. Lactic acid bacteria
have been previously detected in vinegar samples analyzed with DGGE.
These bacteria are probably contaminated by the fruits used as raw materials and manufacturing equipments (Mamlouk et al., 2011).
3.2. Culture-dependent analysis of acetic acid bacteria

Fig. 1. DGGE proles of bacterial 16S rDNA gene V7V8 region amplied from Vinegar and
vinegar mother (all of the bands were identied with sequencing). Bands: bg-1, da-1, DA1, eg-1, EG-1, ha-1, HA-1: K. hansenii, bg-2, BG-1, DA-3, eg-3, EG-3, ha-2, HA-2:
K. europaeus/K. xylinus, da-2, DA-2, eg-2, EG-2: K. europaeus/K. xylinus, bg-3, BG-3, eg-4,
ha-3, HA-3: A. tropicalis/A. indonesiensis, bg-2, bg-5, EG-4, EG-5, EG-6, EG-7:
Komagataeibacter sp., bg-4: Lb. ghanensis, eg-5, eg-6, eg-7, eg-8: O. oeni, da-5, DA-4:
A. nitrogenigens/A. aceti, da-6, ha-4, HA-4: A. syzygii/A. lovaniensis, ha-5, HA-5: Lactobacillus sp., ha-6: Lb. mali, da-3, da-4,: uncultured acetic acid bacterium.

The diversity of AAB species between vinegar samples and their

mothers were partly similar but not completely identical. The number
of different species detected in all samples ranged from 3 to 6. Apart
from acetic acid bacteria, the bands of Lb. ghanensis and Lb. mali,

100

90

80

70

60

50

40

30

GTG5
20

10

GTG5

AAB isolates puried from the vinegar and mother of vinegar samples were initially grouped by using (GTG)5-rep-PCR ngerprinting
and the respective isolates (45 isolates in total) selected from each cluster were identied through the full-length sequence analysis of 16S
rRNA gene, the ITS region and tuf gene. (GTG)5 rep-PCR ngerprints obtained from 87 isolates in total were analyzed by using BioNumerics 6.5
software and a dendogram representing the relationship between AAB
strains was constructed (Fig. 2). It was detected that 59.1% of the
isolates recovered from the vinegar and mother of vinegar samples
belonged to the Acetobacter genus, while 37.5% of them belonged to
the Komagataeibacter genus. Acetobacter okinawensis, representing
51.9% of all Acetobacter isolates was the dominant species, followed by
A. indonesiensis (25.0%), the second dominant species in the Acetobacter
genus. Other Acetobacter species, namely Acetobacter ghanensis (9.6%),
Acetobacter persici (7.7%), A. tropicalis (3.9%) and A. syzygii (1.9%),
were detected in numbers varying between 15 isolates in all samples.
In the Komagataeibacter genus, K. europaeus was found as the most dominant species representing half (50.0%) of the Komagataeibacter population. Other Komagataeibacter species, namely K. intermedius, K. xylinus,
K. hansenii, Komagataeibacter nataicola, Komagataeibacter saccharivorans
and K. oboediens, were detected at minor levels. The Komagataeibacter
genus was recently introduced to the classication of AAB as the result

Isolates

Species

TmSD

eg 4-5

K. europaeus

88.8470.008

ha 6-5

A. okinawensis

86.6920.020

BG 1-21

K. europaeus

88.7460.007

HA 7-3

A. okinawensis

86.6740.006

bg 2-26

K. europaeus

88.8200.010

DA 6-7

A. okinawensis

86.6110.021

EG 3-4

K. europaeus

88.7590.008

BG 7-1

A. okinawensis

86.6960.010

bg 1-23

K. europaeus

88.8600.018

ha 5-1

A. okinawensis

86.6410.021

BG 1-27

K. europaeus

88.7930.015

HA 6-9

K. hansenii

88.7020.014

DA 5-2

A. okinawensis

86.6960.010

bg 2-11

K. hansenii

88.6920.007

DA 7-4

A. okinawensis

86.5810.008

bg 2-12

K. hansenii

88.6910.002

DA 6-10

A. okinawensis

86.5790.022

DA 7-3

A. okinawensis

86.6370.007

HA 7-1

A. okinawensis

86.5930.017

HA 4-7

A. okinawensis

86.6190.004

da 6-1

A. okinawensis

86.5880.001

bg 3-15

K. saccharivorans

89.2800.003

DA 6-4

A. okinawensis

86.6600.006

bg 3-12

K. saccharivorans

89.2830.002

ha 4-8

A. okinawensis

86.6190.004

bg 3-13

K. saccharivorans

89.2880.003

ha 6-7

A. tropicalis

87.5280.001

A. tropicalis

87.5110.012

da 5-2

A. okinawensis

86.6690.010

DA 6-8

A. okinawensis

86.6300.008

ha 6-6

da 5-3

A. okinawensis

86.6960.010

DA 6-11

A. persici

88.2790.010

ha 2-6

A. okinawensis

86.6740.006

HA 7-2

A. persici

88.2240.025

DA 4-1

A. okinawensis

86.6210.003

HA 4-2

A. persici

88.2790.010

BG 7-4

A. okinawensis

86.5810.008

ha 6-3

A. persici

88.2860.015

da 4-5

A. okinawensis

86.6300.008

da 1-8

As. krungthepensis 87.5650.002

ha 5-2

A. okinawensis

86.6580.023

da 2-1

As. krungthepensis 87.5660.022

HA 4-4

A. okinawensis

86.6300.008

da 1-7

As. krungthepensis 87.5630.002

BG 5-5

A. okinawensis

86.6730.005

ha 6-2

K. xylinus

88.9330.023

HA 4-3

A. ghanaensis

86.7350.001

HA 6-4

K. xylinus

89.0240.014

HA 6-2

A. ghanaensis

86.7380.005

HA 7-7

K. xylinus

89.0480.002

ha 5-4

A. ghanaensis

86.7400.005

ha 5-3

A. ghanaensis

86.7410.005

da 5-1

A. okinawensis

86.6740.005

ha 6-6

A. ghanaensis

86.7420.001

BG 4-7

A. okinawensis

86.6730.004

EG 3-2

K. nataicola

86.9100.005

DA 4-5

A. okinawensis

86.6260.006

ha 6-8

A. syzygii

86.4990.003

HA 7-9

K. obediens

87.4860.013

da 3-1

K. intermedius

87.7270.004

HA 7-23

K. obediens

87.4720.024

da 3-2

K. intermedius

87.7320.009

BG 5-1

A. indonesiensis

87.7120.003

da 2-2

K. intermedius

87.7270.001

eg 5-8

A. indonesiensis

87.6890.023

da 2-3

K. intermedius

87.7290.010

EG 5-6

A. indonesiensis

87.6880.005

eg 4-2

K. europaeus

88.7220.011

eg 7-3

A. indonesiensis

87.6470.003

BG 1-21

K. europaeus

88.7250.014

BG 5-7

A. indonesiensis

87.6890.004

bg 1-25

K. europaeus

88.8070.016

BG 5-6

A. indonesiensis

87.6790.025

bg 2-23

K. europaeus

88.7140.019

bg 5-7

A. indonesiensis

87.6520.004

eg 3-4

K. europaeus

88.7460.007

eg 5-7

A. indonesiensis

87.6650.003

BG 1-22

K. europaeus

88.7970.015

eg 4-8

A. indonesiensis

87.6520.004

EG 4-3

K. europaeus

88.7640.011

BG 5-2

A. indonesiensis

87.6690.009

bg 2-22

K. europaeus

88.7660.014

eg 3-2

K. europaeus

88.8540.004

EG 4-9

A. indonesiensis

87.7270.006

EG 3-7

K. europaeus

88.8550.015

BG 7-6

A. indonesiensis

87.6920.021

eg 4-5

K. europaeus

88.8470.008

EG 7-2

A. indonesiensis

87.6900.023

Fig. 2. Rep-PCR ngerprinting patterns of DNA from bacterial isolates originating from vinegar and mother of vinegar samples (dendogram derived from rep-PCRngerprints).

A.E. Yetiman, Z. Kesmen / International Journal of Food Microbiology 204 (2015) 916

of transferring a signicant number of Gluconacetobacter species to this

novel genus (Yamada et al., 2012). Apart from these two dominant genera, 3.4% of total isolates were detected as Asaia krungthepensis, which
belongs to the Asaia genus.
In the last decade, several molecular procedures based on the analysis of the 16S rDNA region have been widely used as phylogenetic
markers for the characterization and classication of AAB (Sievers
et al., 1995; Yamada et al., 1997; Lisdiyanti et al., 2000; Trek and
Teuber, 2002). However, the 16S23S rDNA internally transcribed spacer (ITS) region showing higher variation in sequence and length was recently proven to be more useful for the phylogenetic studies of AAB
(Gonzalez and Mas, 2011). In recent years, several studies have demonstrated that the sequencing of the elongation factor Tu gene (tuf) has
greater discriminatory power than 16S rRNA gene for Lactobacillus,
Bidobacterium, Enterobacteriaceae and coagulase-negative staphylococci (Ventura et al., 2003; Paradis et al., 2005; Hwang et al., 2011).
The tuf gene was also used by Huang et al. (2014) as an alternative molecular target for identication of species of Acetobacter. In this study, tuf
gene-based sequence analysis was performed for further conrmation
of Acetobacter species. The nucleotide sequences of tuf gene produced
by tuf-F and tuf-R primers showed that all Acetobacter species had a
high degree of similarity (98%) with corresponding species in GenBank.
However, there was no information about the genus Komagataeibacter, as
the database for these species is yet to become available.
The composition of the AAB population was considerably inuenced
by the original raw material used in the production of the samples. It
was determined that Acetobacter genus were dominant in the samples
originating from apple, in contrast to the samples originating from
grape in which Komagataeibacter was found as the dominant genus. Although A. okinawensis was found to be the most dominant species in
apple vinegar and mother of vinegar samples, K. europaeus was the
most frequently isolated species in the grape vinegar and mothers of
grape vinegar samples.

13

In this study, real-time PCR intercalating dye analysis was applied to

AAB strains for the rst time. The highly variable ITS region generated
species-specic Tm values and HRM proles allowed discrimination at
species level (Fig. 3). The Tm values of all isolates ranged from 86.50
to 89.29 and the mean of Tm values for each species was signicantly
different from that of other species (Table 1). Tm and HRM analysis
based on the gradual denaturation (melting) of amplicons and then
detection of changes in uorescence intensity by using a intercalating
uorescent dye present in the PCR mixture. Tm analysis allows differentiation of DNA fragments which have small nucleotide variations while
HRM analysis provides more sensitive characterization and theoretically permits the discrimination of DNA molecules even in single base
differences. Tm values and HRM proles have been previously used in
discrimination for spoilage species in beer (Juvonen et al., 2008),
lactobacilli in probiotic products (Kao et al., 2007) and catalasepositive cocci in traditional fermented sausage (Kesmen et al., 2014).
In this study A. okinawensis was found as the most abundant species
by the culture-dependent method. A. okinawensis was isolated in all
samples originating from apple (da, DA, ha and HA) and in only one
mother of grape vinegar sample (BG). Phylogenetic analysis performed
with (GTG)5-rep-PCR primers, demonstrated that a total of 27 isolates
identied as A. okinawensis were divided into 4 clearly separate clusters.
The Tm values of A. okinawensis isolates ranged from 86.58 to 86.70 and
the average Tm value of intraspecies clusters were not signicantly
different from each other. Similarly, although the HRM proles of
A. okinawensis strains showed a species-specic characterization
discriminative enough to differentiate it from other species, identical
proles were obtained for all A. okinawensis isolates in different clusters.
A novel acetic acid bacterium, A. okinawensis, has so far been isolated
from the fruits of grape (Vitis vinifera) and stems of sugarcane
(Saccharum ofcinarum) samples (Lino et al., 2012). The presence of
A. okinawensis in vinegar has not been previously reported, probably
due to the use of inadequate classication tools in earlier studies or to

Fig. 3. Normalized HRM curves of AAB species obtained from HRM analysis [1- K. oboediens(HA 7-23), 2- As. krungthepensis(da 2-1), 3- K. europaeus(BG 1-22), 4- K. hansenii (HA 6-9), 5K. xylinus(HA 6-4), 6- A. persici (HA 4-2), 7- K. saccharivorans(bg 3-12), 8- K. Intermedius(da 2-3), 9- K. nataicola(EG 3-2), 10- A. indonesiensis(eg 5-7), 11-A. syzygii (ha 6-8), 12A. tropicalis(ha 6-7), 13- A. okinawensis(da 4-5), 14- K. ghanaensis(HA 6-2)].

14

A.E. Yetiman, Z. Kesmen / International Journal of Food Microbiology 204 (2015) 916

Table 1
Prevalence of AAB species and results of sequencing and intercalating dye analyses.
Species

A. okinawensis
A. indonesiensis
A. ghanensis
A. syzygii
A. tropicalis
A. persici
K. intermedius
K. hansenii
K. nataicola
K. europaeus
K. saccharivorans
K. xylinus
K. oboediens
As. krungthepensis

Bg

5
3

BG

4
5

da

DA

eg

EG

1
4

ha

3
1

HA

2
2

2
2

16S rRNA gene

ITS region

Accession
Number

Percentage
Similarity
(%)

AB_665075.1
NR_113847.1
NR_113555.1
NR_113850.1
NR_113846.1
AB_665071.1
NR_113394.1
NR_118177.1
NR_113395.1
NR_026513.1
NR_113398.1
NR_036787.1
NR_118186.1
AB_682130.1

99
99
99
99
99
99
98
99
100
99
98
99
98
97

Tuf gene

Accession
number

Percentage
Similarity
(%)

Accession
number

Percentage
Similarity
(%)

99

94
97

99
99
92
97
93
94
99
ND

KC505728.1
KC505684.1
KC505716.1
KC505707.1
KC505692.1
KC505703.1

99
99
98
97
98
99

ND

FR716479.1
1

FR716487.1
FR716480.1
1

KC478454.1
FR716497.1
KC478461.1
HE802685.1
FR716491.1
KC478459.1
HE861937.1
ND

1
1
1
1
1
1
1

ND

The means of
Tm values SD
(C)

Number of
strains
identied

86.65l 0.02
87.71g 0.02
86.77k 0.00
86.54m 0.03
87.56hi 0.01
88.31e 0.02
87.77f 0.01
88.74d 0.01
86.95j 0.01
88.80c 0.01
89.33a 0.00
89.05b 0.01
87.52i 0.02
87.61h 0.01

27
13
5
1
2
4
4
3
1
16
3
3
2
3

Means in the same column that are followed by the same letter are not signicantly different (P N 0.05), in terms of the mean value.
ND: Not detected.
1
Not avaliable in Gen Bank.

the lack of sequence information in the GenBank nucleotide database

about this novel bacterium until recent years.
Although in the rep-PCR analysis K. europaeus was found as the second dominant species, it was only detected in samples originating from
grape, but not in any of the samples originating from apple. Phylogenetic analysis of rep-PCR ngerprints revealed that K. europaeus strains
formed a large cluster which was clearly distinguished from other
species. The Tm values of K. europaeus isolates ranged from 88.71 to
88.86. The average Tm value (88.80) was signicantly different from
other species and the HRM proles showed species-specic characterization. On the other hand, although K. hansenii was detected as a codominant species by DGGE analysis, it was found in only two samples
(bg and HA) with lower percentages through culture-dependent
methods. The mean of Tm values (88.74) of K. hansenii strains was
also signicantly different from the other identied species and the
clearly characteristic HRM prole allowed a successful discrimination.
A. indonesiensis, representing 14.8% of all isolates, was determined as
the third dominant species. While A. indonesiensis was detected both in
grape vinegar and in mother of grape vinegar samples, it was not found
in any samples originating from apple. The Tm values of A. indonesiensis
isolates ranged from 87.65 to 87.75 and the average Tm value (87.71)
was statistically different from all of the other identied species. In
DGGE analysis, A. indonesiensis co-migrated with A. tropicalis and
A. tropicalis/A. indonesiensis groups were identied in all samples originating from grape and apple except for da and EG. However,
A. tropicalis was identied in only one sample (HA) (2 isolates) by
using culture-dependent methods. The average Tm value (87.56) of
A. tropicalis strains was signicantly different from other Acetobacter
and Komagataeibacter species, except for K. oboediens.
Other species in the Acetobacter genus, namely A. ghanensis,
A. persici, A. tropicalis and A. syzygii, were characterized at minor levels
representing between 5.71.1% of all isolates and only isolated from
apple vinegar and mother of apple vinegar samples. Intercalating dye
analysis of these species showed that HRM proles were speciesspecic and Tm values were signicantly different from other species.
In grape vinegar and mother of grape vinegar samples, the
Komagataeibacter genus was dominant. K. europaeus was the most
frequently isolated species, while others, such as K. intermedius,
K. nataicola, K. hansenii and K. saccharivorans were detected at
minor levels in these samples. However, A. indonesiensis was the
only Acetobacter species found in all samples originating from
grape and was not detected in any sample originating from apple.
A. okinawensis was found in only one mother of grape vinegar sample
(4 isolates), while it was determined as the dominant species in all

apple vinegar and mother of apple vinegar samples. Furthermore,

A. ghanensis and A. persici were also detected at minor levels in all
samples obtained from apple (Fig. 2). On the other hand, only 10 isolates
obtained from apple vinegar and mother of apple vinegar samples were of
the Komagataeibacter genus. These Komagataeibacter species including
K. xylinus, K. oboediens, K. intermedius and K. hansenii accounted for
15.6% of all samples originating from apple. Comparing the distribution
of AAB species between the samples of vinegar and their mothers, many
species such as A. okinawensis, K. europaeus, A. indonesiensis, K. hansenii,
A. ghanensis and K. xylinus were identied in both sample groups.
A comparison of the results of culture-dependent and cultureindependent methods revealed that the species of K. hansenii,
K. europaeus/K. xylinus, A. syzygii/A. lovaniensis and A. tropicalis/
A. indonesiensis groups could be identied through both methods.
However, A. okinawensis, A. persici, K. nataicola, K. saccharivorans,
K. oboediens and As. krungthepensis were only detected by means
of the culture-dependent method, while they were not detected by
PCR-DGGE. The reason for the missing bands in PCR-DGGE analysis was
previously explained by authors who obtained similar results by the
poor quality or low quantity of initial templates in the PCR mixture in
which templates compete for primers (Ercolini, 2004; Wilson, 1997;
Cocolin et al., 2011; Kesmen et al., 2012). The other reason for undetectable species may be due to the inadequate specicity of the WBAC primer
set for each AAB (Lopez et al., 2003; De Vero and Giudici, 2008).
In this study, vinegar samples had an acetic acid content varying
from 3.96 to 4.37%, which can be characterized to be low-acidity vinegar, as the common feature of the traditional vinegars (Vegas et al.,
2010). Only one sample showed a slightly lower acidity than the 4%
(w/v), that is the minimum acetic acid concentration that must occur
in vinegar, in accordance with the Turkish Standard (TS 1880 EN
13188). High acetic acid concentration is one of the major limitation
factors that inuence the growth rates of the AAB in the vinegar process.
K. europaeus had the highest acetic acid resistance that was detected in
products showing acetic acid content higher than 4.0% (Mamlouk et al.,
2011) and followed by other members of Komagataeibacter genus
(K. xylinus, K. oboediens and K. intermedius) (Schller et al., 2000;
Sievers and Teuber, 1995; Sokollek et al., 1998). Previous studies
showed that the acetic acid tolerance of A. aceti was much lower than
genus Komagataeibacter and A. pasteurianus was mostly detected in vinegars with a low concentration of acetic acid (Haruta et al., 2006; Treck
et al., 2006; Ilabaca et al., 2008; Nanda et al., 2001).
In this study, the acetic acid contents of the vinegar samples were
not high enough to restrict bacterial activity for the most of AAB species
and it rendered possible the growth of 6 different species of Acetobacter

A.E. Yetiman, Z. Kesmen / International Journal of Food Microbiology 204 (2015) 916

of which acetic acid resistance have not been previously studied. However, it was considered that the diversity of AAB proles between vinegar samples was probably more inuenced by raw materials and other
manufacturing features than acetic acid content, since the total acidity
of the samples was very close to each other.
4. Conclusion
In the last two decades, the PCR-DGGE technique has been a commonly used method for the investigation of a complex microbial population,
especially that of viable but non-culturable microorganisms. However,
the discriminatory ability of PCR-DGGE analysis is limited by the high sequence similarity of 16S rDNA between AAB species. Therefore different
regions with higher heterogeneity should be dened for the purpose of
the analysis of AAB. On the other hand, a complementary analysis based
on culture-dependent methods should also be employed to obtain a detailed knowledge of the AAB population in vinegar.
In this study, intercalating dye analysis was applied to AAB species
for the rst time. The Tm values and HRM proles of AAB isolates
showed that the practice of targeting the ITS region by intercalating
dye-based method can be successfully used as a classication system.
On the other hand, we applied Tm and HRM analyses to a limited number of AAB strains isolated from only 8 samples. Therefore, the success of
the method should be tested on a greater number of strains isolated
from different environments. Since Tm and HRM assays provide the simultaneous analysis of many DNA fragments having sufcient heterogeneity, they can be used not only for the characterization of AAB but
also for that of other bacteria. However, in order to obtain reliable and
reproducible results in real-time PCR HRM and/or Tm analysis, it is important to standardize the quantity of the DNA used in the reaction to
ensure DNA purity and quality.
Acknowledgment
This work was supported by a Research Fund of the Erciyes University, Turkey (Project Nr: FBY-10-3229).
References
Andorra, I., Landi, S., Mas, A., Guillamo'n, J.M., Esteve-Zarzoso, B., 2008. Effect of oenological practices on microbial populations using culture-independent techniques. Food
Microbiol. 25, 849856.
Bartowsky, E.J., Xia, D., Gibson, R.L., Fleet, G.H., Henschke, P.A., 2003. Spoilage of bottled
red wine by acetic acid bacteria. Lett. Appl. Microbiol. 36, 307314.
Boesch, C., Trcek, J., Sievers, M., Teuber, M., 1998. Acetobacter intermedius, sp. nov. Syst.
Appl. Microbiol. 21, 220229.
Case, R.J., Boucher, Y., Dahllf, I., Holmstrm, C., Doolittle, W.F., Kjelleberg, S., 2007. Use of
16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl.
Environ. Microbiol. 73, 278288.
Castro, C., Cleenwerck, I., Trcek, J., Zuluaga, R., De Vos, P., Caro, G., Aguirre, R., Pataux, J.L.,
Ganan, P., 2013. Gluconacetobacter medellinensis sp. nov., cellulose- and non-cellulose
producing acetic acid bacteria isolated from vinegar. Int. J. Syst. Evol. Microbiol. 63,
11191125.
Cleenwerck, I., De Vos, P., 2008. Polyphasic taxonomy of acetic acid bacteria: an overview
of the currently applied methodology. Int. J. Food Microbiol. 125, 214.
Cocolin, L., Manzano, M., Cantoni, C., Corni, G., 2001. Denaturing gradient gel electrophoresis analysis of the 16S rRNA gene V1 region to monitor dynamic changes in the bacterial population during fermentation of Italian sausages. Appl. Environ. Microbiol.
67, 51135121.
Cocolin, L., Dolci, P., Rantsiou, K., 2011. Biodiversity and dynamics of meat fermentations:
the contribution of molecular methods for a better comprehension of complex ecosystems. Meat Sci. 89, 296302.
Dahllf, I., Baillie, H., Kjelleberg, S., 2000. rpoB-Based microbial community analysis avoids
limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl. Environ.
Microbiol. 66, 33763380.
De Vero, L., Giudici, P., 2008. Genus-specic prole of acetic acid bacteria by 16S rDNA
PCR-DGGE. Int. J. Food Microbiol. 125, 96101.
De Vero, L., Gala, E., Gullo, M., Solieri, L., Landi, S., Giudici, P., 2006. Application of denaturing gradient gel electrophoresis (DGGE) analysis to evaluate acetic acid bacteria in
traditional balsamic vinegar. Food Microbiol. 23, 809813.
De Vuyst, L., Camu, N., De Winter, T., Vandemeulebroecke, K., Van de Perre, V., Vancanneyt,
M., et al., 2008. Validation of the (GTG)5-rep-PCR ngerprinting technique for rapid
classication and identication of acetic acid bacteria, with a focus on isolates from
Ghanaian fermented cocoa beans. Int. J. Food Microbiol. 125, 7990.

15

Ercolini, D., 2004. PCR-DGGE ngerprinting: novel strategies for detection of microbes in
food. J. Microbiol. Methods 56, 297314.
Garcia-Armisen, T., Papalexandratou, Z., Hendryckx, H., Camu, N., Vrancken, G., De Vuyst,
L., 2010. Diversity of the total bacterial community associated with Ghanaian and
Brazilian cacoa bean fermentation samples as revealed by a 16S rRNA gene clone library. App. Microbiol. Biotechnol. 87, 22812292.
Gevers, D., Huys, G., Swings, J., 2001. Applicability of rep-PCR ngerprinting for differentiation of Lactobacillus species. FEMS Microbiol. Lett. 205, 3136.
Gonzlez, A., 2005. Application of molecular techniques for identication of acetic acid
bacteria. Universitat Rovira I Virgili, Tarragona, Spain (PhD thesis).
Gonzalez, A., Mas, A., 2011. Differentiation of acetic acid bacteria based on sequence analysis of 16S23S rRNA gene internal transcribed spacer sequences. Int. J. Food
Microbiol. 147, 217222.
Gonzlez, A., Hierro, N., Poblet, M., Rozs, N., Mas, A., Guillamn, J.M., 2004. Application of
molecular methods for the differentiation of acetic acid bacteria in a red wine fermentation. J. Appl. Microbiol. 96, 853860.
Gullo, M., Caggia, C., De Vero, L., Giudici, P., 2006. Characterization of acetic acid bacteria
from traditional balsamic vinegar. Int. J. Food Microbiol. 106, 209212.
Gullo, M., De Vero, L., Giudici, P., 2009. Succession of selected strains of acetobacter
pasteurianus and other acetic acid bacteria in traditional balsamic vinegar. Appl. Environ. Microbiol. 75 (8), 25852589.
Haruta, S., Ueno, S., Egawa, I., Hashiguchi, K., Fujii, A., Nagano, M., Ishii, M., Igarashi, Y., 2006.
Succession of bacterial and fungal communities during a traditional potfermentation of
rice vinegar assessed by PCR-mediated denaturing gradient gelelectrophoresis. Int.
J. Food Microbiol. 109, 7989.
Hidalgo, C., Vegas, C., Mateo, E., Tesfaye, W., Cerezo, A.B., Callejn, R.M., et al., 2010. Effect
of barrel design and the inoculation of Acetobacter pasteurianus in wine vinegar production. Int. J. Food Microbiol. 141, 5662.
Holzapfel, W.H., 2002. Appropriate starter culture technologies for small-scale fermentation in developing countries. Int. J. Food Microbiol. 75, 197212.
Huang, C.-H., Chang, M.-T., Huanga, L., Chua, W.-S., 2014. Utilization of elongation factor
Tu gene (tuf) sequencing and species-specic PCR (SS-PCR) for the molecular identication of Acetobacter species complex. Mol. Cell. Probes 28, 3133.
Hwang, S.M., Kim, M.S., Park, K.U., Song, J., Kim, E.C., 2011. Tuf gene sequence analysis has greater discriminatory power than 16S rRNA sequence analysis in identication of clinical isolates of coagulase-negative staphylococci. J. Clin. Microbiol.
49, 41424149.
Ilabaca, C., Navarrete, P., Mardones, P., Romero, J., Mas, A., 2008. Application of culture
culture-independent molecular biology based methods to evaluate acetic acid bacteria diversity during vinegar processing. Int. J. Food Microbiol. 126, 245249.
Juvonen, R., Koivula, T., Haikara, A., 2008. Group-specic PCR-RFLP and real-time PCR
methods for detection and tentative discrimination of strictly anaerobic beerspoilage bacteria of the class Clostridia. Int. J. Food Microbiol. 125, 162169.
Kao, Y.-T., Liu, Y.-S., Shyu, Y.-T., 2007. Identication of Lactobacillus spp. in probiotic products by real-time PCR and melting curve analysis. Food Res. Int. 40, 7179.
Kato, S., Ishihara, T., Hemmi, H., Kobayashi, H., Yoshimura, T., 2011. Alterations in D-amino
acid concentrations and microbial community structures during the fermentation of
red and white wines. J. Biosci. Bioeng. 111, 104108.
Kesmen, Z., Yetiman, A.E., Gulluce, A., Kacmaz, N., Sagdic, O., Cetin, B., Adiguzel, A., Sahin,
F., Yetim, H., 2012. Combination of culture-dependent and culture-independent molecular methods for the determination of lactic microbiota in sucuk. Int. J. Food
Microbiol. 153, 428435.
Kesmen, Z., Yarimcam, B., Aslan, H., Ozbekar, H., Yetim, H., 2014. Application of different
molecular techniques for characterization of catalase-positive cocci isolated from
sucuk. J. Food Sci. 79 (2), M222M229.
Lino, T., Suzuki, R., Kosako, Y., Ohkuma, M., Komagata, K., Uchimura, T., 2012. Acetobacter
okinawensis sp. nov., Acetobacter papayae sp. nov., and Acetobacter persicus sp. nov.;
novel acetic acid bacteria isolated from stems of sugarcane, fruits, and a ower in
Japan. J. Gen. Appl. Microbiol. 58, 235243.
Lisdiyanti, P., Kawasaki, H., Seki, T., Yamada, Y., Uchimura, T., Komagata, K., 2000. Systematic study of the genus Acetobacter with descriptions of Acetobacter indonesiensis sp.
nov., Acetobacter tropicalis sp. nov., Acetobacter orleanensis (Henneberg 1906) comb.
Nov., Acetobacter lovaniensis (Frateur 1950) comb. nov., and Acetobacter estunensis
(Carr 1958) comb. nov. J. Gen. Appl. Microbiol. 46, 147165.
Lopez, I., Ruiz Larrea, F., Cocolin, L., Orr, E., Phister, T., Marshall, M., et al., 2003. Design and
evaluation of PCR primers for analysis of bacterial populations in wine by denaturing
gradient gel electrophoresis. Appl. Environ. Microbiol. 69, 68016807.
Mamlouk, D., Hidalgo, C., Torijab, M.-J., Gullo, M., 2011. Evaluation and optimisation of
bacterial genomic DNA extraction for no-culture techniques applied to vinegars.
Food Microbiol. 28, 13741379.
Nanda, K., Taniguchi, M., Ujike, S., Ishihara, N., Mori, H., Ono, H., Murooka, Y., 2001. Characterization of acetic acid bacteria in traditional acetic acid fermentation of rice vinegar (komesu) and unpolished rice vinegar (kurosu) produced in Japan. Appl. Environ.
Microbiol. 67, 986990.
Papalexandratou, Z., Cleenwerck, I., De Vos, P., De Vuyst, L., 2009. (GTG)5-PCR reference
framework for acetic acid bacteria. FEMS Microbiol. Lett. 301, 4449.
Papalexandratou, Z., Camu, N., Falony, G., De Vuyst, L., 2011. Comparison of the bacterial
species diversity of spontaneous cocoa bean fermentations carried out at selected
farms in Ivory Coast and Brazil. Food Microbiology 28, 964973.
Paradis, S., Boissinot, M., Paquette, N., Belanger, S.D., Martel, E.A., Boudreau, D.K., Picard,
F.J., Ouellette, M., Roy, P.H., Bergeron, M.G., 2005. Phylogeny of the Enterobacteriaceae
based on genes encoding elongation factor Tu and F-ATPase beta-subunit. Int. J. Syst.
Evol. Microbiol. 55, 20132025.
Prez-Martn, F., Sesea, S., Fernndez-Gonzlez, M., Arvalo, M., Palop, M.L., 2014. Microbial communities in air and wine of a winery at two consecutive vintages. Int. J. Food
Microbiol. 190, 4453.

16

A.E. Yetiman, Z. Kesmen / International Journal of Food Microbiology 204 (2015) 916

Ruiz, A., Poblet, M., Mas, A., Guillamon, J.M., 2000. Identication of acetic acid bacteria by
RFLP of PCR-amplied 16S rDNA and 16S23S rDNA intergenic spacer. Int. J. Syst.
Evol. Microbiol. 50, 19811987.
Snchez, J.A., Dorado, D., 2008. Intragenomic ITS2 variation in Caribbean seafans. Proceedings of the 11th International Coral Reef Symposium, Ft. Lauderdale, Florida, 711
July 2008pp. 13831387.
Schller, G., Hertel, C., Hammes, W.P., 2000. Gluconacetobacter entanii sp. nov., isolated
from submerged high-acid industrial vinegar fermentations. Int. J. Syst. Evol.
Microbiol. 50, 20132020.
Sengun, I.Y., Karapinar, M., 2004. Effectiveness of lemon juice, vinegar and their mixture
in the elimination of Salmonella typhimurium carrots (Daucus carota L.). Int. J. Food
Microbiol. 96, 301305.
Sengun, I.Y., Karapinar, M., 2011. Importance of acetic acid bacteria in food industry. Food
Control 22, 647656.
Shimoji, Y., Tamura, Y., Nakamura, Y., Nanda, K., Nishidai, S., Nishikawa, Y., et al., 2002. Isolation and identication of DPPH radical scavenging compounds in Kurosu (Japanase
unpolished rice vinegar). J. Agric. Food Chem. 50, 65016503.
Sievers, M., Teuber, M., 1995. The microbiology and taxonomy of Acetobacter europaeus in
commercial vinegar production. J. Appl. Bacteriol. Symp. Suppl. 79, 8495.
Sievers, M., Sellmer, S., Teuber, M., 1992. Acetobacter europaeus sp. nov., a main component
of industrial vinegar fermenters in Central Europe. Syst. Appl. Microbiol. 15, 386392.
Slapsak, N., Cleenwerck, I., De Vos, P., Trcek, J., 2013. Gluconacetobacter maltiaceti sp. nov.,
a novel vinegar producing acetic acid bacterium. Syst. Appl. Microbiol. 36, 1721.
Sokollek, S.J., Hertel, C., Hammes, W.P., 1998. Description of Acetobacter oboediens sp. nov.
and Acetobacter pomorumsp. nov., two new species isolated from industrial vinegar
fermentation. Int. J. Syst. Evol. Microbiol. 48, 935940.
Stasiak, L., Blaejak, S., 2009. Acetic acid bacteria-perspectives of application in biotechnology a review. Pol. J Food Nutr. Sci. 59 (1), 1723.
Steinkraus, K.H., 2002. Fermentations in World food processing. Compr. Rev. Food Sci.
Food Saf. 1, 2332.
Tesfaye, W., Morales, M.L., Garca-Parrilla, M.C., Troncoso, A.M., 2002. Wine vinegar: technology, authenticity and quality evaluation. Trends Food Sci. Technol. 13, 1221.
Torija, C., Mateo, E., Guillamon, J.M., Mas, A., 2010. Identication and quantication of
acetic acid bacteria in wine and vinegar by TaqMan-MGB probes. Food Microbiol.
27, 257265.

Trcek, J., 2005. Quick identication of acetic acid bacteria based on nucleotide sequences
of the 16S23S rDNA internal transcribed spacer region and of the PQQ-dependent
alcohol dehydrogenase gene. Syst. Appl. Microbiol. 28, 735-4.
Trcek, J., Teuber, M., 2002. Genetic and restriction analysis of the 16S23S rDNA internal
transcribed spacer regions of the acetic acid bacteria. FEMS Microbiol. Lett. 208, 6975.
Trcek, J., Ramus, J., Raspor, P., 1997. Phenotypic characterization and RAPD-PCR prolling of
Acetobacter sp. isolated from spirit vinegar production. Food Technol. Biotechnol. 35,
6367.
Trcek, J., Raspor, P., Teuber, M., 2000. Molecular identication of Acetobacter isolates from
submerged vinegar production, sequence analysis of plasmid pJK2-1 and application
in development of a cloning vector. Appl. Microbiol. Biotechnol. 53, 289295.
Trcek, J., Toyama, H., Czuba, J., Misiewitz, A., Matsushita, K., 2006. Correlation between
acetic acid resistance and characteristics of PQQ-dependent ADH in acetic acid bacteria. Appl. Microbiol. Biotechnol. 70, 366373.
Valera, M.J., Torija, M.J., Mas, A., Mateo, E., 2013. Acetobacter malorum and Acetobacter
cerevisiae identication and quantication by Real-Time PCR with TaqMan-MGB
probes. Food Microbiol. 36, 3039.
Vegas, C, Mateo, E., Gonzlez, ., Jara, C., Guillamn, J.M., Poblet, M., et al., 2010. Population dynamics of acetic acid bacteria during traditional wine vinegar production.
Int. J. Food Microbiol. 138 (1-2), 130136.
Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T.R., Zink, R., 2003. Analysis, characterization, and loci of the tuf genes in Lactobacillus and Bidobacteriumspecies and their direct application for species identication. Appl. Environ. Microbiol. 69, 69086922.
Wilson, I.G., 1997. Inhibition and Facilitation of Nucleic acid amplication. Appl. Environ.
Microbiol. 63, 37413751.
Yamada, Y., Hoshino, K.-I., Ishikawa, T., 1997. The Phylogeny of Acetic Acid Bacteria Based
on the Partial Sequences of 16S Ribosomal RNA: The Elevation of the Subgenus
Gluconoacetobacter to the Generic Level. Biosci. Biotechnol. Biochem. 61, 12441251.
Yamada, Y., Yukphan, P., Lan Vu, H.T., Muramatsu, Y., Ochaikul, D., Tanasupawat, S.,
Nakagawa, Y., 2012. Description of Komagataeibacter gen. nov., with proposals of
new combinations (Acetobacteraceae). J. Gen. Appl. Microbiol. 58, 397404.