fmic b-11-01291 June 18, 2020 T ime: 14:55 # 1
ORIGINAL RESEARCH
published: 16 June 2020
doi: 10.3389/fmicb.2020.01291
Edited by:
Victor Ladero,
Consejo Superior de Investigaciones
Científicas, Spain
Reviewed by:
Karina T eixeira
Magalhães-Guedes,
Universidade Federal da Bahia, Brazil
Cintia Ramos,
Universidade Federal dos V ales do
Jequitinhonha e Mucuri, Brazil
Elisardo C. V asquez,
Federal University of Espirito Santo,
Brazil
*Correspondence:
Fatemeh Nejati
[email protected]
† ORCID:
Fatemeh Nejati
orcid.org/0000-0002-8991-7442
Specialty section:
This article was submitted to
Food Microbiology ,
a section of the journal
Frontiers in Microbiology
Received: 13 April 2020
Accepted: 20 May 2020
Published: 16 June 2020
Citation:
Nejati F , Junne S, Kurreck J and
Neubauer P (2020) Quantification
of Major Bacteria and Y east Species
in Kefir Consortia by Multiplex T aqMan
qPCR. Front. Microbiol. 11:1291.
doi: 10.3389/fmicb.2020.01291
Quantification of Major Bacteria and
Y east Species in Kefir Consortia by
Multiplex T aqMan qPCR
Fatemeh Nejati 1 * † , Stefan Junne 1 , Jens Kurreck 2 and Peter Neubauer 1
1 Department of Bioprocess Engineering, Faculty III Process Sciences, Institute of Biotechnology , T echnische Universität
Berlin, Berlin, Germany, 2 Department of Applied Biochemistry , Faculty III Process Sciences, Institute of Biotechnology ,
T echnische Universität Berlin, Berlin, Germany
Kefir grains ar e complex microbial systems of several gr oups of micr oorganisms.
The identification and quantification of the micr obial composition of milk kefirs was
described in several studies, which provided an insight into the micr obial consortia
in this complex ecosystem. Nevertheless, the current methods for identification and
quantification ar e not appropriate for deeper studies on kefir consortia, e.g., population
dynamics and micr obial interactions in kefir grains. This requir es another sensitive and
r eliable quantitative method. Therefor e, this study aims to develop multiplexed qPCR
assays to specifically detect and quantify , as an example, several micr oorganisms of the
milk kefir micr obial community . Primer -probe sets, which target species-specific genes
in six bacteria and five yeasts, wer e designed, and their sensitivity and specificity to
the target species was analyzed in simplex as well as four multiplex qPCR assays.
The self-designed multiplex assays wer e applied for the detection of target bacteria
and yeast species in milk kefirs, in both, grain and beverage fractions. Detection of all
target micr oorganisms in simplex and multiplex qPCR was achieved by good linearity ,
ef ficiency , repeatability and r epr oducibility in all assays. When the designed assays wer e
applied on six kefirs, all target micr oorganisms were detected in dif fer ent samples, but
not all in one kefir sample. The two ubiquitous lactobacilli Lactobacillus kefiranofaciens
and Lb. kefiri wer e present in all six kefirs studied, but wer e associated with dif ferent
other yeasts and bacteria. Especially on the yeast community a significant diversity was
observed. In general, multiplex T aqMan qPCR as developed her e was proven to have
high potential for specific identification of target microorganisms in kefir samples and for
the first time, eleven target bacteria and yeasts of kefir micr obiota were rapidly detected
and quantified. This study , thus, pr ovides a fast and reliable pr otocol for futur e studies
on kefir and other similar micr obial ecosystems.
Keywords: T aqMan qPCR, milk kefir , quantification, microbial community , lactic acid bacteria, yeast, Acetobacter
INTRODUCTION
Kefir is an ancient fermented milk beverage, which became very popular recently. Traditional kefir
is produced by fermentation of milk with kefir grains, which is a protein-polysaccharide structure
that contains a complex mixture of lactic acid bacteria (LAB), acetic acid bacteria (AAB), and
yeasts. More than 50 microbial species were identified in different milk kefirs ( Bourrie et al., 2016 ;
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Nejati et al. Kefir Microbiota Detection by qPCR
Liu et al., 2019 ). It is supposed that several inter-microbial actions
are responsible for the maintenance of the kefir grains ’ integrity as
well as their bio-functionalities as robust starter cultures ( Nejati
et al., 2020 ). The microbial composition of kefir grains can
change due to several factors, such as the cultivation conditions
(e.g., temperature, grain to milk ratio, milk sour ce) and the
geographical origin of kefir ( Prado et al., 2015 ).
Several culture-dependent and -independent methods were
applied to gain a deeper view into the microbial composition
of kefir in the last decade. Accurate identification is es sential to
thoroughly understand the community functions and its proper
industrial application. Culture-dependent methods enable the
quantification of microbial species on the basis of colony forming
units (CFU), although this method has se veral drawbacks; a
long duration (especially for several slow growing spe cies of
lactobacilli such as Lactobacillus (Lb.) kefir anofaciens in kefir),
a rather low reproducibility and an inability to differentiate
accurately between bacterial strains ( H ansen et al., 2018 ).
Additionally, the accuracy is low since viable-but-non-culturable
cells (VBNC) cannot be counted ( Liu et al., 2018 ). PCR -
denaturing gradient gel electrophoresis (PCR -DGGE) was the
most common culture-independent method for the identification
of kefir microbiota for many years ( Chen et al., 2008 ). L ater ,
however , studies proved that this met hod has a low a ccuracy
when the composition of complex microbial ecosystems need to
be investigated ( Leite et al., 2012 ; Garofalo et al., 2015 ). Although
next generation sequencing (NGS) techniques are powerful
approaches when studying microbial community composition,
they still suffer from misclassification at the spe cies level ( W inand
et al., 2019 ). This might lead to a wrong identification of bacteria
and yeasts that belong to a same genus. Additionally, concerns
about technical pitfalls and potential biases were raised in
literature when results were interpreted ( Nilsson et al., 2019 ; Jian
et al., 2020 ). Nevertheless, these techniques provided successfully
a more detailed picture on the microbial composition of kefir ,
especially for low abundant species ( Z amberi et al., 2016 ; Gao
and Zhang, 2019 ). In order to study kefir in different aspects
apart from the pure microbial composition, for example the
generation of grains and the population changes in response to
environmental conditions, the identification te chnique s, which
ha ve been applied so far are insufficient. The authors thus
suppose that the development of a spe cies-specific assay is a
requirement to unra vel the mysteries on the formation, integrity
and functionality of kefir grain generation and knowledge-driven
co-cultivation of species, which were isolated from kefir.
Quantitative real time polymerase chain reaction (qPCR),
due to its high specificity and sele ctivity, is a qualified method
for the detection of a specific microorganism in a microbial
matrix. For qPCR, two reporter systems are commonly used:
(i) intercalating dyes such a s SYBR Green and (ii) fluorogenic
hybridization oligoprobes, particularly 5 0 exonuclease assays (also
called T aqMan TM ) ( Smith and Osborn, 2009 ). While intercalating
dyes bind non-specifically to all generated amplicons, dual-
labeled oligoprobes of T aqMan assay anneal specifically to a target
region, which guarantees specific detection ( Smith and Osborn,
2009 ). In addition, application of T aqMan probes facilitates
multiplexing as several differently labeled probes can be used
simultaneously in the same assay ( Duffy et al., 2013 ). High costs
for labeling of each end of the probes with different dyes is
in comparison to the use of intercalating dyes is compensated
by higher selectivity and a reduced process time in multiplexed
assays. Multiplex T aqMan qPCR recently appeared to be a very
useful tool for the identification and quantification of different
microbial communities ( F arkas et al., 2017 ; Baudy et al., 2019 ;
Enora et al., 2019 ; Lochman et al., 2019 ). A ccordingly, this study
aims to establish a multiplex qPCR approac h for the simultaneous
detection and quantification of eleven frequently reported
bacteria and yeast species of milk kefirs. In order to achieve
this, accuracy and precision of multiplexed qPCR assays were
assessed toward target and non-tar get microorganisms, while
the applicability was evaluated by quantification of the t ar geted
species in six milk kefirs, in both grain and beverage fractions.
MA TERIALS AND METHODS
Micr obial Strains and Genetic Materials
The bacterial and yeast strains used in this study are shown
in T able 1 . LAB strains were cultivated in De Man-Rogosa-
Sharpe (MRS) broth (Carl Roth, Germany). The medium was
supplemented with 10% filter -sterilized white table wine for
cultivation of two strains of Lb. kefir anofaciens 5016 and 10550.
T ABLE 1 | Microorganisms, which were used in this study .
Microorganism Species Strain name Function
Bacteria Lb. kefiranofaciens
ssp.
kefiranofaciens
DSM 5016 T T arget
Lb. kefiranofaciens
ssp. kefirgranum
DSM 10550 T T arget
Lb. kefiri DSM 20587 T T arget
A. orientalis FKG1 a T arget
Ln. mesenteroides LG2-A a T arget
A. fabarum P3S1 a T arget
Lc. lactis ssp.
cremoris
NZ9000 T arget
Lc. lactis ssp.
lactis
IL1403 T arget
Lb. helveticus DSM 20075 T Non-target
Lb. reutrei DSM 20016 T Non-target
Lb. paracasei DSM 20008 Non-target
Lb. parakefiri DSM 10551 T Non-target
Lb. plantarum SZ5 b Non-target
Weissella cibaria HY21 b Non-target
Y easts Kz. turicensis DBVPG 7206 T T arget
Kz. unispora DBVPG 6429 T arget
Kl. marxianus DBVPG 6141 T arget
S. cerevisiae DBVPG 10191 T arget
D. anomalus DBVPG 10201 T arget
Kz. exigua DBVPG 3191 Non-target
T T ype strain. a Strains were isolated from kefir and identified by 16S sequencing,
unpublished data. b Strains were isolated and identified by 16S sequencing in a
previous study ( T aghi-Zadeh and Nejati, 2017 ).
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Acetobacter (A.) or ient alis and A. fa barum were cultivated in
AAB broth [5 g L − 1 yeast extract (Merck, Germany), 5 g L − 1
bacto peptone (Merck, Germany), 5 g L − 1 glucose (Mer ck,
Germany), 1 g L − 1 MgSO 4 × 7 H 2 O (Sigma, Germany)] and
yeasts were cultivated in YPG broth [10 g L − 1 yeast extract, 20 g
L − 1 peptone (Merck, Germany), 20 g L − 1 glucose]. All strains
were incubated at 30 ◦ C under static aerobic conditions, except
Lb. kefiranof aciens ssp., which were cultivated under severe
oxygen-limited conditions (Anaerocult A R
 ; Merck, Germany).
The genomic DN A (gDN A) of bacteria and yeast species was
extracted from exponentially growing cultures [24 h for all
strains except Lb. kefir anofaciens ssp. and K azach tania (Kz.)
turicensis, which were cultivated between 4 and 5 days] by
using a NucleoSpin R
 Microbial DN A kit (MA CHEREY -N AGEL,
Germany). Quality and concentration of extracted gDN A
was measured with a NanoDrop ND-1000 spectrophotometer
(Peqlab Biotechnologie, Germany).
Primers and T aqMan Pr obes Design and
V erification
Primers and probes for multiplexed assays were designed using
the Beacon Designer TM software (v. 8.21, PREMIER Biosoft
International, United States) after specific genes for each target
species were selected pre viously. The criteria for the selection
of target genes were: (i) species-specificity of the chosen
gene/sequence as investigated via NCBI’ s BL AS T algorithm 1 ,
(ii) existence of only one copy of selected gene/sequence in
each target species, and (iii) suitability of the selected sequence
as provided by the Beacon Designer TM software for designing
of primers and probes (e.g., GC content of the sequence) to
achieve higher design success rates, respectively. Parameter s
for the design of primer -probe sets in multiplexed assays
(e.g., length, melting temperature and GC content of primers,
probes and amplicons) were applied as defined by the default
settings of the software. In order to increase the stability,
specificity and sensitivity of the T aqMan probes, each probe
contained four Locked Nucleic A cid TM (LN A) bases. In T able 2 ,
information is summarized about the target species, target genes
in each species and all primer -probe set sequences, which were
generated in this study.
Firstly, the specificity of primers, probes and amplicons was
verified by performing a NCBI’ s BL AS T. Then, the amplicon
sequences were checked against the whole corresponding
microorganism genome using BLAST sear ch. Next and prior to
probe synthesis, the specificity of all primer pairs (synthesized by
Sigma-Aldrich, Germany) was tested in simplex or multiplexed
reactions using conventional PCR in order to investigate the
production of expected amplicons of target species, in addition to
the verification of absence of the products in non-target species.
For this purpose, 10 µ L (final volume) of a PCR mixture consisted
of 2 µ L Green GoT aq reaction buffer , 0.05 µ L GoT aq DN A
polymerase (5 U µ L − 1 ; Promega, Germany), 0.2 µ L of dNTP s
mix (each 10 mM), appropriate amounts of each primer to reach a
final concentration of 1.2 µ M in simplex PCR reactions or 0.8 µ M
in multiplex PCR, and 0.7 µ L DN A template. Both, simplex and
1 https://blast.ncbi.nlm.nih.gov/Blast.cgi
multiplex PCR were performed in an Eppendorf MasterCycler
under the following cycling conditions: 95 ◦ C for 5 min; 35 cycles
of 95 ◦ C for 30 s, 45 ◦ C for 30 s, and 72 ◦ C for 20 s; and a final
extension of 5 min at 72 ◦ C. PCR products were analyzed on
2% agarose gels (Roth, Germany) containing the GelRed TM dye
(Biotium, Inc., United States).
Then, each pair of primers was used separately in qPCR
runs with or without DN A template, in order to investigate the
amplification of a single product in target microorganisms and
the absence of primer -dimers. For this experiment, 2x SYBR R

Green Master Mix (Bio-Rad, Germany) was used in a 20 µ L
batch containing 400 nM of each of the primers. The qPCR
program was initialized by 95 ◦ C for 5 min, continued by 40
cycles of 95 ◦ C for 15 s and 60 ◦ C for 30 s, followed by a melting
cur ve to verify amplification specificity and the absence of primer
dimers. qPCR was performed with a Bio-Rad CFX96 re al-time
PCR thermocycler by applying the “all channels ” reading mode
in 96-microwell clear unskirted plates (Biozym, Germany) that
were sealed using optical adhesive films (Bio-R ad, Germany).
LN A TM substituted T aqMan R
 probes were obtained from
Merck (H averhill, United Kingdom); they contained of the
fluorescent reporter dye on the 5 0 - end and the non-fluores cent
quencher on the 3 0 -end. Probes for six bacteria and five
yeast species were designed for the application in two distinct
multiplexed assays, due to the limitation of qPCR thermocycler
channels. These four experimental designs are presented in
T able 2 , assays 1 and 2 as fiveplex and assays 3 and 4 as fourplex
set up. A ccording to this, bacterial species Lb. kefiranofaciens,
Lb. kefiri, Leuconostoc (Ln.) mesenteroides and A. orientalis were
analyzed in assays 1 and 2, while Lacococcus ( Lc) lactis and
A. fa barum were analyzed only in assay 1 and assay 2. Similarly,
yeast species Kazac hst ania ( Kz.) turicensis, Kluyveromayces (Kl.)
marxianus and Dekkera (D.) anomalus were analyzed in assays 3
and 4, while Kz. unispor a and Saccharomyce s (S.) cerev isiae were
analyzed only in assay 3 and assay 4. Reporter dyes of each probe
are shown in T able 2 .
Evaluation of Primers-Pr obe Sets and
Assays
Analysis of Primers’ and Pr obes’ Specificity in
Simplex qPCR
For analyzing the specificity of primer-probe sets in
simplex reactions, each test comprised of six DN A standard
concentrations from the target strain in addition to obligatory
no-template controls (NTC). All analyses were performed in
triplicate. The total reaction volume of 20 µ L consisted of 2 µ L
gDN A (or ddH 2 O), 10 µ L 2 × SsoAdvanced TM Universal Probes
Supermix (Bio-R ad , Germany), primers and probe, in order to
reach a final concentration of 400 nM for each primer and 200 nM
for the probe, and 7.52 µ L of RN ase-free water , respectively. The
qPCR program covered an initial DN A polymerase activation
step of 3 min at 95 ◦ C, 40 cycles of denaturation for 15 s at 95 ◦ C,
and hybridization and extension for 30 s at 60 ◦ C, respectively.
Fluorescence intensity was measured at the end of e ac h cycle
with the CFX Manager T M software v3.1 (Bio-R ad). Finally,
standard cur ves were created.
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T ABLE 2 | Information on target species and their primer -probe set sequences generated in this study in addition to qPCR assay set up.
Organism Genom accession
number
T arget gene Primer/
probe
Sequence (5 0 -3 0 ) Melting
T em. ( ◦ C)
Amplicon
size (bp)
Assay (s)
Lb. kefiranofaciens AZGG00000000 DNA helicase RecG Forward
Reverse
Probe a
GCAACAACCAAAGT A TTGT A
T AGCCGAAGAGGA TCT AA
Q705 -ACC[+A]CA[+T]CA[+C]CA[+A]CTCT AA- BHQ3
60.1
59.8
69.0
118 1 and 2
Lb. kefiri A YYV00000000 β -glucuronidase Forwar d
Reverse
Probe
TCGCTTTCAAGCA TTGAA
CGAACTTCCCA TT A TCCA T A
Cy5 -CA T[+C]AA[+G]CC[+A]AC[+A]GCAG- BHQ3
61.4
60.4
68.7
134 1 and 2
A. orientalis AP018515.1 Copr oporphyrinogen III oxidase Forward
Reverse
Probe
CAGAGT A TT ACCCWCGCTTT A
GGTGAAGGCAAAGTCTTG
TxRed -CAA[+T]GT[+T]GC[+C]AC[+T]A TCGAG- BHQ2
62.7
62.1
68.0
137 1 and 2
Ln. mesentreoides LA YU00000000 Mannose-6-phosphate isomerase Forward
Reverse
Probe
CGAACCAACAACTT A TCT A TG
CCTCA TGT AGA TCCT ACCTT A
HEX -T AC[+C]AC[+G]A T[+T]GT[+T]GACCA- BHQ1
60.3
61.2
69.4
107 1 and 2
Lc. lactis NC_002662 Phosphotransferase system
cellobiose-specific component IIC
Forward
Reverse
Probe
ACCTCTTGGACTT AA T AACC
CACGT ACCCAAAGGTT AA
F AM -CAC[+T]CA[+C]AA[+G]GT[+A]TCCGT - BHQ1
60.2
60.3
69.4
131 1
A. fabarum NCXK01000006 Glutamate racemase Forward
Reverse
Probe
CAGGCA TTGGTGGA TT AG
TGGGAAAGAAGGGAGA T AA
F AM -A TC[+A]TC[+C]TG[+C]TC[+A]CCGT A- BHQ1
61.3
60.9
71.2
148 2
Kz. turicensis PPOO01000000 Threonylcarbamoyl-AMP synthase Forward
Reverse
Probe
CAAGGTT AAACCAGAA TCAA
GCAACTGT A TCGTCTGT A
Cy5 -TTC[+T]CG[+C]CT[+A]AC[+T]CCGT A- BHQ3
59.5
59.8
69.4
138 3 and 4
Kl. marxianus CM004405 Golgi apparatus membrane protein
TVP38
Forward
Reverse
Probe
TCCTCGACAGT AA TGA T AA
AGCACTCAA TTCA TCGT A
TxRed -CTC[+C]TG[+A]T A[+G]AC[+C]GCTT - BHQ2
58.4
59.4
69.1
140 3 and 4
D. anomalus MDSB00000000 Phosphoglycerate kinase Forward
Reverse
Probe
GAGCAGACTGAGAAGTTC
CGACCA T AGAAGAGTGAG
F AM -A TT[+G]AC[+C]GC[+T]CT[+T]GCT - BHQ1
60.5
59.6
68.9
100 3 and 4
Kz. unispora PPON00000000 RNA polymerase II Forward
Reverse
Probe
GTTGCA TGGCAA TCAAAA
CGAAGACGCTCAAGAA T A
HEX -TCT[+T]CT[+T]CT[+T]CG[+G]CA TCAA- BHQ1
60.7
60.2
68.4
101 3
S. cerevisiae GCA_000146045 Golgi transport complex subunit
COG6
Forward
Reverse
Probe
CGACAACAAA TTGCTGAA
CTCTCGAACA T AACTCTGT A
HEX -CA T[+C]CA[+G]TC[+G]CT[+A]TCCAA T - BHQ1
60.0
59.7
68.8
147 4
a Each oligoprobe was labeled with a fluorescent dye (Q705, Quasar 705; Cy5, Cyanine 5; HEX, Hexachloro-fluorescein; TxRed, T exas red and F AM, 6-carboxyfluorescein) at 5 0 end and corresponding quencher (Black
Hole Quencher: BHQ3, BHQ2, and BHQ1) at 3 0 end.
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Evaluation of Multiplexed Assays
A complete microbial genomic (CMG) pool was prepared
by mixing equal quantities of gDN A of all ele ven target
microorganisms. This CMG pool was then serially diluted 10-
fold for the use as DN A template during the generation of dat a
for standard cur ves in multiplexed qPCR assays. As outlined
in Section “Evaluation of Primer s-Probe Sets and Assays, ”
eleven target microorganisms were analyzed in four multiplexed
assays ( T able 2 ). In each assay, CMG was applied as DN A
template, however , only primers and probes of the distinct target
microorganism of that assay were added to the qPCR reaction.
The total reaction volume of 20 µ L consisted of 2 µ L template (or
ddH 2 O) and 18 µ L master mix that contained 10 µ L of 2 × iQ TM
Multiplex Powermix (Bio-Rad, Germany), and primer-probe sets
to reach the final concentration of 300 nM of each primer , and
200 nM of each probe, respectively. The qPCR program covered
an initial DN A polymerase activation step of 3 min at 95 ◦ C, 45
cycles of denaturation for 12 s at 95 ◦ C, and hybridization and
extension for 45 s at 60 ◦ C, respectively. Fluorescence signals were
detected in all channels; standard cur ves were plotted with the
CFX Mana ger TM software v3.1 (Bio-R ad).
Cr eation of Standards Curves
Standard cur ves were created by plotting the Cq against the log 10
input genome copy number. The copy number of gDN A was
calculated with formula (1) 2 :
Number of copies = (amount of DN A, ng × 6.022 × 10 23 )/
(genome size, bp × 10 9 × 660) (1)
The genome sizes used for calculations were 2257141,
2501983, 2036093, 3214967, 3095430, 2365589, 12134345,
12784682, 10776003, 14202998, 12323254, and 14202998 bp
for Lb. kefiranof acins 5016 , Lb. kefiri 20587 , Ln. mesenteroides
LBE16 , A. orientalis F AN1 , A. fa barum OG2 , Lc. lactis IL1406 ,
S. cerev is iae ySR128 , D. anomalous Y V396 , Kl. marxianus LHW-
O , Kz. unispor a NRRL Y -1556 and Kz. turicensis NRRL Y -
48834, respectively 3 . In simplex qPCR runs, gDN A of e ach
microorganism was serially diluted 10-fold in water to final
concentrations between 10 7 and 10 1 genome copies per µ L. In
multiplexed assays, CMG was serially diluted 10-fold to final
concentrations between 10 5 and 10 1 genome copies per µ L.
The simplex and multiplex assays were evaluated based on their
correlation coefficient (R 2 ), slope and efficiency [ E = (10 ( − 1 / slope )
− 1)] for standard cur ves, which were calculated by the software.
Intra-assay repeatability was evaluated with the coefficient of
variations (CV%) based on Cq-values; it was calculated for
various concentrations from standard cur ves in replicate samples
in the same PCR run. The CV% was also used to estimate
inter -assay reproducibility when calculated for at least three
independent PCR runs. The limit of detection (LOD) was
defined as the Cq-value on the standard cur ve corresponding
to 3 PFU s, while t he limit of quantification (LOQ) was the
2 http://scienceprimer.com/copy- number - calculator- for- realtime- pcr
3 www.ncbi.nlm.nih.gov
concentration (copies reaction − 1 ) corresponding to the LOD
( Baudy et al., 2019 ).
Quantification of T argeted Bacteria and
Y easts in Kefir Samples
Evaluation of DNA Extraction Kits
As DN A extraction can be a critical step in studies of microbial
communities, we first compared the yield and quality of
the DN A extracted from grains with two DN A extraction
kits, either DNeasy PowerSoil Pro or DNeasy PowerBiofilm
(Qiagen, Germany), each containing different bead systems.
Two hundred milligram of grains from each of the two
different home-made kefirs (PN and FN) were used for this
investigation. DN A extraction was performed according to the
manufacturer’ s guidelines except that a MM400 Mixer Mill
(Retsch, Germany) was applied for bead beating (10 min at
30 Hz) instead of T issueLyser. The quality of extracted DN A
was measured using a spectrophotometer (N anoDrop ND-1000,
Peqlab Biotechnologie, Germany).
Analysis of T argeted Species in Kefir Samples
Samples from six milk kefirs were analyzed in this study; four
kefirs from home-made kefirs in Berlin (Germany, denoted as
PN, FN, and BK) and Umbria (Italy, denoted as EK), as well
as two samples from commercial kefirs (Primal Life UG, Berlin,
Germany, denoted as CKM and CMC). Before analysis, 10 g
grains of each kefir were added to 40 mL cow milk (Arla, 1.5%
fat) and after 24 h fermentation at 25 ◦ C, with few occasionally
shaking, samples were applied to DN A extraction. In all cases,
the DN A was extracted from kefir beverages and kefir grains
separately with the DNeasy PowerSoil Pro kit. In case of t he
beverage fraction, 1.8 mL of 24 h-fermented kefir was used
as sample, which was treated according to the manufacturer’ s
protocol. Two grams of grains were washed by stirring 3 times
in 50 mL of 0.85% N aCl, each for 15 min. Then, 0.2 g of grains
were applied to DN A extraction.
As template , 2 µ L of extracted DN A were separately
used in qPCR analysis (as described in “Analysis of T argeted
Species in Kefir Samples ”). In each qPCR plate, three different
concentrations of ten-fold serially diluted CMG (as described
in “Analysis of T argeted Species in Kefir Samples ”) in addition
to NTC controls were applied. Reactions were rated as positive
if all triplicates showed a Cq and if the a verage Cq was
below the LOD of the respective microorganisms, otherwise
it was rated as negative. After analysis, the number of each
target species was extrapolated to 1 g of kefir grains or 1 mL
of kefir beverage.
RESUL TS AND DISCUSSION
Specific detection of Lb. kefiranofac iens in kefir with 16s rDN A -
based T aqMan probes was reported by Kim et al. (2015b) ;
however , this report des cribes the simultaneous analysis of
eleven microorganisms in kefir samples for first time. These
eleven microbial targets were chosen on the basis of several
previous reports about the microbial composition of milk kefir
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Nejati et al. Kefir Microbiota Detection by qPCR
( Garofalo et al., 2015 ; Blasche et al., 2019 ; Gao and Zhang,
2019 ). As an example , in kefirs studied by Blasche et al.
(2019) , Lb. Kefir anofaciens, Lb. kefiri, Lc. lactis, Ln. mesenteroide s
and Acetobacter spp. accounted for more than 95% of the
kefir community.
Identification and quantification of different microbial
communities is an important topic in microbiology.
A development of sensitive and reliable quantification
methods are needed in order to answer questions about
microbial communities, for example how a population will
respond to intrinsic or extrinsic factors and/or how these
changes a ffect t he quality of the final products. Kefir grains
are complex microbial communities, hence, so far , artificially
developed kefir starters for industrial applications did not
result in the production of authentic final products. This is
mainly due to a lack of knowledge on the exact microbial
composition of natural starters, their functions, and their
interaction with each other , respectively. Kim et al. (2015a)
developed group-specific primers to quantify several groups of
bacteria (e.g., Lactobacilli, Lactococci, Streptococci, Enterococci )
and yeasts (e.g., Candida, Sacch aromyces ) in milk kefir with
qPCR, howe ver , a specific detection of defined target species
in kefir has rarely been performed. The main goals of the
current study were to highlight the potential of qPCR for
fast detection and multiplex quantification of several t ar get
microorganisms in milk kefir as an example of a complex
microbial community and to provide a sensitive and fast method
for further studies on milk kefir.
T arget Genes Selection
A 16S rDN A in bacteria and 26S rDN A and ITS region in ye asts
were commonly used for the identification and differentiation of
microbial species in DGGE-PCR , high throughput sequencing
(HTS) and qPCR techniques ( Kim et al., 2015b ; Korsak et al.,
2015 ; Gao and Zhang, 2019 ; Okai et al., 2019 ). Nevertheless,
as the varying parts of these regions are not long enough for
generating high-rank primer-probe sets for multiple xing, other
specific genes in each species were selected in this study.
For some of the target species, either more than one subspecies
was found in kefir microbiota or subspecies were not clarified. In
order to be more specific, for Lb. kefiranof aciens, two subspecies
Lb. kefir anofaciens and Lb. kefirgr anums were isolated from
milk kefir samples ( V ancanneyt et al., 2004 ; Ma galhães et al.,
2010b ), while Lc. lactis and Ln. mesenteroides subspecies were not
defined in many studies. This can be possibly due to the poor
discrimination power of 16S rDN A for subspe cies differentiation.
A ccordingly, here we attempted to generate primer -probe sets
that are able to be amplified in all subspecies for the target
species. The attempt only failed for Ln. mesenteroides , as no
region was found, which was long enough and suitable for the
design of primer -probe sets that included all subspecies. Thus, Ln.
mesenteroides s sp. suionicum was excluded ( Figure 1 ). However ,
FIGURE 1 | Primer -probe stets for panel (A) Ln. mesenteroides (ssp. cremoris strain A TCC 19254, ssp. dextranicum strain DSM20484, ssp. mesenteroides A TCC
8293, ssp. jonggajibkimchii strain DRC1506 and ssp. suionicum strain L T -38) and (B) Lc. lactis (ssp. cremoris strain MG1363, ssp. lactic strain IL1403 and ssp.
lactis bv . diacetylactis strain FM03).
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Jeon et al. (2017) proposed that Ln. mesenteroides ssp. suionicum
should be reclassified as a novel species of the genus Leuconostoc ,
and not as a subspecies of Ln. leuconostoc , as it is genetically
distant to other subspecies of Ln. mesenteroides a ccording to
the results of whole-genome-based taxonomic methods. Lc. lactis
has two subspecies; Lc. lactis ssp. lactis and Lc. lactis ssp.
cremoris , and one biovar; Lc. lactis ssp. lactis bv. d iacetlactis ; all
of them are dairy products-related. The designed primer -probe
set detect all of them ( Figure 1 ). In Acetobacter group, the species
A. fa barum and A. lovaniensis ha ve a 99.79% identity in their 16S
rDN A sequence (NR_042678 and NR_040832), which makes it
difficult to discriminate them based on 16S rDN A sequencing. In
several kefir studies, the detection of one of them was reported
( Ma galhães et al., 2010a ; Blasche et al., 2019 ). The primers-probe
set designed in this study can specifically dete ct A. fa barum, but
does not recognize A. lovaniensis .
Evaluation of Primer -Pr obe Sets
Specificity
The specificity of designed primer -probe sets toward the target
species was confirmed by bioinformatic analysis (NCBI’ s BLAST).
Next, the production of amplicons with the expected sizes was
shown by conventional PCR experiments. No cross-reactivity was
obser ved with non-target templates ( Supplementary Figure S1 ).
In addition to experiments with the target strains shown in
T able 1 , the specificity of primer sets was tested against several
isolates, including two of Lb. kfir anofaciens , five of Lb. kefiri ,
four of Ln. mesenteroides and four of A. orientalis (data not
shown). Lb. helvet icus, Lb. reuteri, Lb. paracasei, Lb. par akefiri,
Lb. plantarum and Kz. exigua, which were indicated as kefir
microbiota in different studies ( Bourrie et al., 2016 ). The absence
of the amplification product in multiplex conventional PCR
verified the specificity of the designed primer sets to t he target
microorganisms ( Supplementary F igure S1 ).
Formation of primer dimers or unwanted PCR products
through a non-specific binding of primers reduced significantly
the sensitivity of qPCR analysis. Therefore, the formation of
such products was investigated in this study. The melting cur ve
analysis showed no primer dimer formation in all primer pairs
( Supplementary Figure S2A ). In addition, production of a single
melting peak for each primer set verified the high specificity of
the designed primer pairs toward target species ( Supplementary
Figures S2B ,C for b acteria and yeast target species).
Performance of Simplex and Multiplexed
qPCR Assays
For all primer -probe sets, the fluorescence signal reached the
threshold line only when the target microorganism was included
in the reaction, which indicate all newly designed primer -probe
sets ha ve a high sele ctivity to the target species, both in simplex
and multiplex qPCR assays. The correlation coefficient (R 2 ), slop
and efficiency (E) of standard cur ves in simplex and multiplex
assays for the target microorganisms are shown in T able 3
(Additional Information in Supplementary Figures S3, S4
for simplex qPCR, and Supplementary Figures S5, S6 for
multiplexed assays). The R 2 -values, ranging from 0.984 to
1.000, indicate a linear correspondence between the logarithmic
genome copy number and their Cq-values. The efficiency (E),
as one of the most important indicators of the performance of
a qPCR assay, was between 86.4 and 104.7% in simplex, and
between 90.6 and 103.4% in multiplex assays, which proves a
good performance of the primer-probe sets ( Baudy et al., 2019 ;
Lochman et al., 2019 ). In all four assays, PCR probes appeared
to be very specific and no loss of activity was obser ved upon
multiplexing. The difference in R 2 , E and the slope for each target
microorganism was negligible between simplex and multiplex
assays. The intra-assay repeatability, inter -assay reproducibility,
LOD and LOQ for each primer set are summarized in T able 4 .
The coefficients of variation (CV) were less than 3% and 4%
for intra and inter -assay, respectively. A ccording to the dilutions
used in qPCR assays, the defined LOQs in multiplex assays were
from 6 copies for S. cerev is iae to 75 copies for Lb. kefiri per qPCR
T ABLE 3 | Performance of primer -probe sets designed in this study in simplex and multiplex assays for target micr oorganisms.
T arget microorganism Simplex qPCR Multiplex qPCR
Assay 1 Assay 2 Assay 3 Assay 4
R 2 E (%) Slope a R 2 E (%) Slope a R 2 E (%) Slope a R 2 E (%) Slope a R 2 E (%) Slope a
Lb. kefiranofaciens 0.999 94.9 3.456 0.998 99.0 3.346 0.997 103.4 3.242 − − − − − −
Lb. kefiri 1.000 102.2 3.270 0.998 99.4 3.337 0.999 99.2 3.341 − − − − − −
A. orientalis 0.999 89.1 3.613 0.999 100.0 3.322 0.997 97.1 3.394 − − − − − −
Ln. mesenteroides 1.000 95.5 3.409 0.993 100.2 3.316 0.989 98.6 3.356 − − − − − −
Lc. lactis 0.997 86.4 3.696 0.990 98.9 3.347 − − − − − − − − −
A. fabarum 0.997 93.0 3.502 − − − 0.995 101.7 3.281 − − − − − −
Kz. turicensis 0.994 91.3 3.549 − − − − − − 0.997 91.3 3.550 0.997 90.6 3.570
Kz. unispora 0.996 93.9 3.477 − − − − − − 0.990 94.6 3.458 − − −
Kl. marxianus 0.996 88.4 3.636 − − − − − − 0.997 100.1 3.319 0.998 96.5 3.408
S. cervisiae 0.997 104.7 3.214 − − − − − − − − − 0.997 98.1 3.369
D. anomalus 0.998 90.7 3.568 − − − − − − 0.994 102.0 3.276 0.997 102.3 3.267
a The slopes are negative values.
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T ABLE 4 | Intra- and inter -assay repeatability and r eproducibility , LOD and LOQ of the quantification of eleven kefir -related micr oorganisms using
multiplexed qPCR assays.
Primer -probe set DNA (copies reaction − 1 ) Intra-assay r epeatability Inter -assay-repr oducibility LOD (Cq) LOQ (copies reaction − 1 )
Mean-crossing
point (Cq ± SD)
CV (%) Mean-crossing
point (Cq ± SD)
CV (%)
Lb. kefiranofaciens 5.44E + 05
5.44E + 03
5.44E + 02
5.44E + 01
18.71
25.17
28.63
32.11
0.26
0.09
0.36
0.17
1.40
0.36
1.25
0.54
18.46
25.07
28.26
31.92
0.29
0.32
0.52
0.54
1.56
1.27
1.85
1.68
32.11 5.44E + 01
Lb. kefiri 7.44E + 05
7.44E + 03
7.44E + 02
7.44E + 01
18.21
24.73
28.15
31.45
0.05
0.11
0.30
0.28
0.27
0.46
1.05
0.89
18.37
25.06
28.37
31.53
0.57
0.68
0.30
0.38
3.11
2.73
1.07
1.19
31.45 7.44E + 01
A. orientalis 2.76E + 04
2.76E + 02
2.76E + 01
22.32
29.02
32.33
0.17
0.41
0.10
0.76
1.41
0.31
23.07
29.70
32.98
0.61
0.55
0.92
2.63
1.84
2.78
32.33 2.76E + 01
Ln. mesentreoides 1.92E + 04
1.92E + 03
1.92E + 02
26.11
29.69
33.13
0.14
0.21
0.01
0.54
0.72
0.05
26.56
33.42
36.72
0.44
0.34
0.74
1.66
1.03
2.01
36.19 1.92E + 01
Lc. lactis 5.51E + 04
5.51E + 02
5.51E + 01
21.79
28.52
31.28
0.15
0.10
0.47
0.69
0.35
1.51
21.63
28.22
31.21
0.22
0.27
0.35
1.00
0.97
1.11
31.28 2.75E + 01
A. fabarum 1.73E + 05
1.73E + 03
1.73E + 02
20.01
26.18
29.30
0.26
0.23
0.07
1.30
0.88
0.25
20.35
27.06
29.87
0.44
0.88
0.81
2.18
3.25
2.70
33.33 1.73E + 01
Kz. turicensis 1.75E + 05
1.75E + 03
1.75E + 02
21.87
28.95
32.41
0.19
0.32
0.35
0.89
1.11
1.09
21.57
28.57
32.10
0.38
0.59
0.35
1.76
2.08
1.10
35.21 1.75E + 01
Kl. marxianus 5.25E + 04
5.25E + 02
5.25E + 01
22.42
29.22
32.27
0.07
0.03
0.15
0.31
0.11
0.46
22.82
29.67
32.29
0.40
0.36
0.02
1.77
1.22
0.07
32.27 5.25E + 01
D. anomalus 1.21E + 05
1.21E + 03
1.21E + 02
19.44
26.21
28.98
0.16
0.05
0.17
0.80
0.20
0.58
19.49
26.31
29.50
0.11
0.24
0.73
0.58
0.91
2.47
33.02 1.21E + 01
Kz. unispora 3.08E + 04
3.08E + 02
3.08E + 01
25.68
32.71
35.96
0.21
0.48
0.76
0.82
1.46
2.13
24.79
31.43
34.86
0.56
0.75
0.96
2.26
2.39
2.74
35.96 3.08E + 01
S. cerevisiae 6.02E + 04
6.02E + 02
6.02E + 01
22.01
28.84
32.35
0.04
0.05
0.42
0.20
0.18
1.32
22.79
29.86
32.70
0.68
0.88
0.68
2.99
2.95
2.08
35.42 6.02E + 00
Cq, Cycle of quantification; CV , Coefficient of variation; SD, Standard deviation.
reaction. These data demonstrate that the all qPCR assays had
very good performances for the target species. Thus, multiplex
qPCR was used in subsequent analysis on real kefir samples.
Analysis of Kefir Samples
Ef fect of DNA Extraction Kit
DN A extraction methods have s hown to play a substantial role
when the microbial composition of different communities were
investigated ( Ketchum et al., 2018 ; V aidya et al., 2018 ). Here,
gDN A from two kefir grain samples (PN and FN) was extracted
by using the DNeasy PowerSoil Pro or DNeasy PowerBiofilm kits.
These two kits benefit from different bead materials with various
sizes (proprietary to manufacturer), which might impact DN A
extraction yields. It was obser ved that the DN A extracted with
the DNeasy PowerSoil Pro kit had a higher yield (21.1 ng µ L − 1 )
in comparison to the DNeasy PowerBiofilm kit (7.9 ng µ L − 1 )
for PN grain and 16.5 ng µ L − 1 compared to 5.1 ng µ L − 1 for
FN grain, and relatively better purity (A260/230; 1.89 compared
to 1.92 for PN grain, and 2.28 compared to 2.62 for FN grain)
( Supplementary T able S1 ). Therefore, the DNeasy PowerSoil Pro
kit was used for extraction of DN A.
Abundance of T arget Bacteria and Y easts Species in
Kefir Samples
The abundance of eleven bacteria and yeast species in six milk
kefirs was quantified by the previously described assays. The
findings of this analysis are shown in Figures 2 , 3 for bacteria
and yeast communities. In general, all the kefir samples contained
a minimum 2.17E + 09 and 1.63E + 08 number of identified
bacteria, and 9.86E + 06 and 9.38E + 05 number of identified
yeasts per unit of grain and beverage fraction (g or mL),
respectively. These findings confirm data from previous studies
( Guzel-Seydim et al., 2005 ), although it may be considered that
not all microorganisms of the kefir microbiota were quantified in
the present study.
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FIGURE 2 | Number of each six bacteria species per g or mL of kefir grain (purple bar) or beverage fraction (green bar) of six milk kefirs. Numbers r epresent the
mean of triplicate measurements. Err or bars repr esent the standard deviation (SD).
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FIGURE 3 | Number of each five yeast species per g or mL of kefir grain (red bar) or beverage fraction (blue bar) of six milk kefirs. Numbers r epresent the mean of
triplicate measurements. Err or bars repr esent the standard deviation (SD).
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Lactobacillus kefiranof aciens and Lb. kefir i strains were
detected in all kefir samples. They constituted the major number
of bacteria in grains and appeared in high numbers in kefir
beverages (a minimum of 1.0E + 08 of Lb. kefiranof aciens and
1.0E + 07 of Lb. kefiri in mL of kefir be verage). The pre sence of
the other four bacteria, i.e., Lc. lactis, A. or ient alis, A. fa barum
and Ln. mesenteroides , varied among the different kefir s. They
were detected in the be vera ge fraction and not in the grain
fraction in some samples ( A. orientalis in FN, A. fa barum in EK,
Lc. lactis in CK C and Ln. mesenteroides in CKM). Occasionally,
in some beverage fractions, Lc. lactis appeared as dominant
species, such as in kefirs EK and BK: the portion of identified
bacteria constituted of Lc. lactis by 93.2% and 97.1%, while Lb.
kefir anofaciens and Lb. kefiri were still dominant in the be verage
fractions of FM and CKM kefirs, in which they represented more
than 98% of the identified bacteria. Ln. mesenteroides was only
detected in one of the kefir grains (EK) with a low percentage
(0.02%) and did not exceed more that 2.2% of the total identified
bacteria in the beverage fraction. Kefirs ’ microbiota can differ
with respect to Acetobacter species: although present in most
samples, they were abundant in kefir BK. As it is believed
that Acetobacter spp. support Lactobacilli growth by oxygen
consumption and acetic acid release ( Dong et al., 2018 ), it is
possible that other Acetobacter species like A. okinawens is and
A. syzygii are active with a similar function in BK kefir , which
requires further investigations.
Results concerning the presence and abundance of specific
species in kefir differ a lot among various studies, which is due
to either differences in kefir samples or the role of different
methods for the identification of microbiota ( Nejati et al., 2020 ).
As a re sult, a meaning ful comparison of results among different
studies are not easily feasible. For example, as mentioned above,
Lb. kefiranof aciens and Lb. kefiri appe ared to high amounts in
all kefir beverage fractions in the current study, while Gao and
Zhang (2019) reported them as a minority in five kefir beverage
fractions using high-throughput Illumina sequencing te c hnique.
In general, our obser vations on the diversity of dominant bacteria
in kefir beverages are closer to the findings of Korsak et al.
(2015) who investigated microbial diversity of five kefirs by 16S
rDN A pyrosequencing.
Regarding the yeast composition, a high variation among the
five analyzed species was found within the six kefirs ( Figure 3 ).
A ccording to the obser vations in this study, not all five yeast
species were detected in one kefir sample, but it appeared
that each kefir sample hosted between two and three of them.
Among the five yeast species, Kz. tur icenis, Kl. marxianus,
S. cerev is iae and D. anomalus , were always detected in both,
the grain and beverage fractions. This characteristic, however ,
seems to be strain specific for Kz. unispor a , as well as for non-
lactobacilli species, i.e., Lc. lactis, A. fa barum, A. or ient alis and
Ln. mesenteroides .
The yeast community of kefir has been found to be as
complex as the bacterial community ( Liu et al., 2019 ; Zhimo
et al., 2020 ). Gao and Zhang (2019) analyzed five kefirs and
found K azachs tania , Kluyveromyces , and Sacch aromyces as the
ma jor genera, and Kazac hst ania species were found to be as
most abundant in both grains and beverages. In K azachst ania ,
it seems that at least three species ( Kz. tur icens is, Kz. Unispor a ,
and Kz. exigua) contribute to the ye ast community of many
milk kefirs ( Garofalo et al., 2015 ; Blasche et al., 2019 ; Gao and
Zhang, 2019 ). Kz. unispor a has been isolated from kefir more
often than the other two species. Reports on isolation of Kz.
turicensis from kefir was not as frequent as Kz. unispor a , until
next generation sequencing methods were applied recently. In
this study, Kz. turicensis and Kz. unispor a each were found in
four out of six kefirs. Garofalo et al. (2015) obser ved that both
species were found as a minority in milk kefirs. Kl. marxianus and
S. cerev is iae , individually or together , were frequently identified
as absolute dominant yeasts in milk kefir samples ( Leite et al.,
2012 ; Kalamaki and Angelidis, 2017 ; Zhimo et al., 2020 ); however ,
in this study, K azachst ania species appeared in higher numbers
than Kl. marxianus and S. cerev isiae in three kefir s PN, FN and
CK C and in kefir EK none of the Kl. marxianus and S. cerevisiae
were found. D. anomalus has been rarely reported as a ma jor
yeast in milk kefir. The absolute contribution of this species
to milk kefir microbial community was reported by Garofalo
et al. (2015) when studied six Italian milk kefirs. Interestingly,
D. anomalus was only detected in the Italian kefir s ample (EK)
and in none of the German samples. It seems t hat all samples
contain lactose fermenting and non-lactose fermenting yeasts, a
factor that probably stabilizes yeast communities in kefir. This
character of yeast communities was found in all kefir samples in
this study, and was also reported by Garofalo et al. (2015) .
In some of the grain fractions, the detection of some spe cies,
i.e., Ln. mesenteroides , Lc. lact is , A. fa barum and A. orientalis
among bacteria and Kz. unispor a among yeast species, either was
not achieved at all or they were lower than LOQ ( T able 4 ), and
accordingly rated as being negative. While these species have
been identified in a high number in be vera ge fractions, their low
number in grain fractions is probably due to their release from
the grain structure during the washing steps. This might also
indicate that they do not bind strongly to the grain structure
or other microbial consortia ’ s members. As the absence of these
species in kefir grain vs. kefir beverage was only obser ved in
some kefirs and not in all cases (e.g., detection of A. orientalis
in PN vs. FN kefirs as an example), this may imply that binding
properties is strain-specific. Cell surface-related phenomena like
cell-cell and cell-matrix adhesion, flocculation, auto- and co-
aggregation are not only strain-specific ( Soares, 2011 ; Wang et al.,
2012 ), but also strongly correlated to medium composition, e.g.,
bivalent ions strength and the pH-value ( Wang et al., 2019 ).
A high concentration of calcium and a low pH-value have been
related with higher flocculation of different bacteria and ye asts
( Soares, 2011 ; Wang et al., 2017, 2019 ). A c cordingly, it can be
implied that a natural pH-value of our washing solution and
the presence of monovalent ions (N a + ) instead of divalent ions
(which are naturally provided by milk) can lead to resolve some
cell-cell attachments. Although the effect of medium composition
and pH-value on cell attachments ha ve been studied for a few
numbers of Lactobacills species ( Wang et al., 2019 ), no kefir -
related microorganisms were a study target though. It is also
worthwhile to mention that the arrangement of microbial species
on or in a grain structure is still a matter of research. Although
some studies show that the microorganisms occupy all interior
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Nejati et al. Kefir Microbiota Detection by qPCR
and exterior surface of grains ( Ma galhães et al., 2010a , Ma galhães
et al., 2011 ; Wang et al., 2012 ), microorganisms are hardly
obser ved on the outer surface of the grains, but only embedded
in the fibrillar matrix near the surface in anot her study ( Ismaiel
et al., 2011 ). There is also the possibility that this property
depends on the microbial community composition and varies
among different kefirs harboring different species and strains,
which open a topic for further studies.
CONCLUSION
This study presents new multiplex T aqMan qPCR assays that
can detect and quantify ele ven frequently reported bacteria and
yeast species of milk kefir microbiota. Due to its relatively
fast nature, the method can be an advantageous tool when
the dynamics of these species in microbial communities
shall be monitored. Furthermore, by ad justment of the
reporter dyes, it is possible to adapt different custom-based
multiplex assays. Based on the results of this study, Lb.
kefir anofaciens and Lb. kefiri are ubiquitous in milk kefirs,
though the presence of other nine spe cies varied among
different kefirs.
DA T A A V AILABILITY ST A TEMENT
The datasets presented in this study can be found in
online repositories. The names of the repository/repositories
and accession number(s) can be found in the article/
Supplementary Material .
AUTHOR CONTRIBUTIONS
FN contributed to design of the experiment and analysis, and
writing the original draft. PN, JK, and S J contributed to the
writing, reviewing, and editing t he manuscript.
FUNDING
FN was supported by the Einstein foundation under the program
Einstein Junior Scholarships; MICROB ALANCE project.
ACKNOWLEDGMENTS
The authors thank V iola Röhrs for technical assistance
with qPCR machine. We acknowledge support by the German
Research Foundation and the Open A ccess Publication Fund of
TU Berlin.
SUPPLEMENT AR Y MA TERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmic b.
2020.01291/full#supplementary- material
REFERENCES
Baudy, P., Zubrod, J. P., Röder , N., B aschien, C., Feckler , A., S chulz, R., et al. (2019).
A glance into the black box: novel species-specific quantitative re al-T ime PCR
assays to disentangle aquatic hyphomycete community composition. Fungal
Ecol. 42:100858. doi: 10.1016/J.FUNECO.2019.08.002
Blasche, S., Kim, Y., Mar s, R., Kafkia, E., M aansson, M., Mac hado, D., et al.
(2019). Emergence of stable coexistence in a complex microbial community
through metabolic cooperation and spatio-temporal niche partitioning. BioRxiv
[Preprint], doi: 10.1101/541870
Bourrie, B. C. T., W illing, B. P., and Cotter , P. D. (2016). The microbiot a and health
promoting characteristics of the fermented beverage kefir. Front. M icrobiol.
7:647. doi: 10.3389/fmicb.2016.00647
Chen, H.-C., Wang, S.-Y., and Chen, M.-J. (2008). Microbiological study of lactic
acid bacteria in kefir grains by culture-dependent and culture-independent
methods. Food M icrobiol. 25, 492–501. doi: 10.1016/J.FM.2008.01.003
Dong, J., Liu, B., Jiang, T., Liu, Y., and Chen, L. (2018). The biofilm hypothesis: the
formation mechanism of tibetan kefir grains. Int. J. Dairy Tec hnol. 71, 44–50.
doi: 10.1111/1471- 0307.12473
Duffy, T., Cura, C. I., Ramire z, J. C., A bate, T., C ayo, N. M., P arrado, R. et al. (2013).
Analytical performance of a multiplex real-time PCR assay using T aqMan
probes for quantification of Trypanosoma cruzi satellite DN A in blood samples.
PLoS Negl. Trop. Dis. 7:e2000. doi: 10.1371/journal.pntd.0002000
Enora, D., Martial, B., M arie-Agnès, J., and Sophie, C. (2019). Novel tetraple x
QPCR assays for simultaneous detection and identification of Xylella
fast idiosa subspecies in plant tissues. BioRxiv [Preprint], doi: 10.1101/
699371
Farkas, K., Peter s, D. E., McDonald , J. E., de Rougemont, A., Malham, S. K., and
Jones, D. L. (2017). Evaluation of two triplex one-step QR T -PCR assays for
the quantification of human enteric viruses in environmental samples. Food
Environ. V irol. 9, 342–349. doi: 10.1007/s12560- 017- 9293- 9295
Gao, W., and Zhang, L. (2019). Comparative analysis of the microbial community
composition between tibetan kefir grains and milks. Food Res. Int. 116, 137–144.
doi: 10.1016/J.FOODRES.2018.11.056
Garofalo, C., Osimani, A., Milanovi ´
c, V., Aquilanti, L., De Filippis, F., Stellato, G.,
et al. (2015). Bacteria and yeast microbiota in milk kefir grains from different
italian regions. Food Microb iol. 49, 123–133. doi: 10.1016/J.FM.2015.01.017
Guzel-Seydim, Z., Wyffels, J. T., Seydim, A. C., and Greene, A. K. (2005).
Turkish kefir and kefir grains: microbial enumeration and electron microscobic
obser vation. Int. J. Da iry Technol. 58, 25–29. doi: 10.1111/j.1471- 0307.2005.
00177.x
H ansen, S. J. Z., Morovic, W., DeMeules, M., Sta hl, B., and Sindelar , C. W. (2018).
A bsolute enumeration of probiotic strains Lactobacillus acidophilus NCFM§and
Bifidobacterium animalis Subsp. Lactis Bl- 04 R
 via chip-based digital PCR.
Front. M icrobiol. 9:704. doi: 10.3389/fmicb.2018.00704
Ismaiel, A. A., Ghaly, M. F., and El-N aggar , A. K. (2011). Milk kefir : ultrastructure,
antimicrobial activity and efficacy on aflatoxin B1 production by As pergillus
flavus . Curr. M icrobiol. 62, 1602–1609. doi: 10.1007/s00284- 011- 9901- 9909
Jeon, H. H., Kim, K. H., Chun, B. H., Ryu, B. H., Han, N. S., and Jeon, C. O. (2017).
A Proposal of Leuconostoc mesenteroides subsp. Jonggajibk imch ii subsp. nov.
and Reclassification of Leuconostoc mesenteroides subsp. suionicum (Gu et al.,
2012) as Leuconostoc suionicum sp. nov. based on complete genome. Int. J. Syst.
E vol. M icrobiol. 67, 2225–2230. doi: 10.1099/ijsem.0.001930
Jian, C., Luukkonen, P., Yki-J är vinen, H., Salonen, A., and Korpela, K. (2020).
Quantitative PCR provides a simple and accessible method for quantitative
microbiota profiling. Ed. Ivone V az-Moreira . PLoS One 15:e0227285. doi: 10.
1371/journal.pone.0227285
Kalamaki, M. S., and Angelidis, A. S. (2017). Isolation and molecular identification
of yeasts in greek kefir. Int. J. Dairy Technol. 70, 261–268. doi: 10.1111/1471-
0307.12329
Ketchum, R. N., Smith, E. G., V aughan, G. O., Phippen, B. L., McP arland, D., Al-
Mansoori, N., et al. (2018). DN A extraction method plays a significant role
Frontiers in Micr obiology | www .frontiersin.org 12 June 2020 | V olume 11 | Article 1291

fmic b-11-01291 June 18, 2020 T ime: 14:55 # 13
Nejati et al. Kefir Microbiota Detection by qPCR
when defining bacterial community composition in the marine invertebrate
echinometra mathaei. Front. M ar. Sci. 5:255. doi: 10.3389/fmars.2018.00255
Kim, D.-H., Chon, J.-W., Kim, H., Kim, H.-S., Choi, D., Hwang, D.-G., et al.
(2015a). Detection and enumeration of lactic acid bacteria, acetic acid bacteria
and yeast in kefir grain and milk using quantitative real-time PCR. J. Food Saf.
35, 102–107. doi: 10.1111/jfs.12153
Kim, D.-H., Chon, J. -W., Kim, H. S., Y im, J. H., Kim, H., and Seo, K. H. (2015b).
Rapid dete ction of Lactobacillus kefir anofaciens in kefir grain and kefir milk
using newly developed real-time PCR. J. Food Prot. 78, 855–858. doi: 10.4315/
0362- 028X.JFP - 14- 329
Korsak, N., T aminiau, B., Leclercq, M., Nezer , C., Creve coeur , S., Ferauche, C., et al.
(2015). Short communication: evaluation of the microbiota of kefir samples
using metagenetic analysis targeting the 16S and 26S ribosomal DN A fragments.
J. Dairy Sci. 98, 3684–3689. doi: 10.3168/JDS.2014- 9065
Leite, A. M. O., M ayo, B., R achid , C. T. C. C., Peixoto, R. S., Silva, J. T., Paschoalin,
V. M. F., et al. (2012). Asse ssment of the microbial diversity of brazilian
kefir grains by PCR -DGGE and pyrosequencing analysis. Food Microbiol. 31,
215–221. doi: 10.1016/J.FM.2012.03.011
Liu, J., Deng, Y., Li, L., Li, B., Li, Y., Zhou, S., et al. (2018). Discovery and control
of culturable and viable but non-cultura ble cells of a distinctive Lactobacillus
harb inensis strain from spoiled beer. Sci. Rep. 8:11446. doi: 10.1038/s41598-
018- 28949- y
Liu, W., Zhang, M., Xie, J., Wang, H., Zhao, X., Chen, B., et al. (2019). Comparative
analyses of microbial community diversities of tibetan kefir grains from three
geographic regions. Int. J. Dairy Technol. 72, 536–544. doi: 10.1111/1471- 0307.
12616
Lochman, J., Zapletalova, M., Poskerova, H., Izakovicova Holla, L., and Borilova
Linhartova, P. (2019). Rapid multiplex real-time PCR method for t he detection
and quantification of selected cariogenic and periodontal b acteria. Dia gnostics
10:8. doi: 10.3390/diagnostics10010008
Mag alhães, K. T., de Melo Pereira, G. V., Campos, C. R., Dragone, G., and
Schwan, R. F. (2011). Brazilian kefir : structure, microbial communities and
chemical composition. Br azilian J. Microb iol. 42, 693–702. doi: 10.1590/S1517-
83822011000200034
Mag alhães, K. T., de Pereira, G. V. M., Dias, D. R., and Schwan, R. F. (2010a).
Microbial communities and chemical changes during fermentation of sugary
brazilian kefir. World J. M icrobiol. B iotechnol. 26, 1241–1250. doi: 10.1007/
s11274- 009- 0294- x
Mag alhães, K. T., Pereira, M. A., Nicolau, A., Dragone, G., Domingues, L., Teixeira,
J. A., et al. (2010b). Production of fermented cheese whey-based beverage
using kefir grains as starter culture: evaluation of morphological and microbial
variations. Biore sour. Technol. 101, 8843–8850. doi: 10.1016/j.biortech.2010.06.
083
Nejati, F., Junne, S., and Neubauer , P. (2020). A big world in small grain:
a review of natural milk kefir starters. Microor ganisms 8:192. doi: 10.3390/
microorganisms8020192
Nilsson, R. H., Anslan, S., Bahram, M., Wurzbacher , C., Baldrian, P., and Tedersoo,
L. (2019). Mycobiome diversity: high-throughput sequencing and identification
of fungi. N at. Rev. M icrobiol. 17, 95–109. doi: 10.1038/s41579- 018- 0116- y
Okai, C., Itani, Y., Furuta, A., Mizunoe, Y., and Iwase, T. (2019). R apid
identification and quantification of Lactobacillus Rhamnosus by real-time PCR
using a T aqMan probe. Jpn. J. Infect. Dis. 72, 323–325. doi: 10.7883/yoken.JJID.
2019.102
Prado, M. R., Blandón, L. M., V andenberghe, L. P. S., Rodrigues, C., Castro,
G. R., Thomaz-Soccol, V., et al. (2015). Milk kefir : composition, microbial
cultures, biological activities, and related products. Front. M icrobiol. 6:1177.
doi: 10.3389/fmicb.2015.01177
Smith, C. J., and Osborn, A. M. (2009). Advantages and limitations of quantitative
PCR (Q-PCR)-based approaches in microbial ecology. FEMS M icrobiol. Ecol.
67, 6–20. doi: 10.1111/j.1574- 6941.2008.00629.x
Soares, E. V. (2011). Flocculation in Saccharomyces cerevisiae : a review. J. A ppl.
M icrobiol. 110, 1–18. doi: 10.1111/j.1365- 2672.2010.04897.x
T aghi-Zadeh, A., and Nejati, F. (2017). S creening of lactic acid bacteria isolated
from iranian sourdoughs for antifungal activity: Enterococcus faecium showed
the most potent antifungal activity in bread. A ppl. Food Biotechnol. 4:16560.
doi: 10.22037/afb.v4i4.16560
V aidya, J. D., van den Bogert, B., Edwards, J. E., Boek horst, J., van Gastelen, S.,
Saccenti, E., et al. (2018). The effect of DN A extraction methods on obser ved
microbial communities from fibrous and liquid rumen fractions of dairy cows.
Front. M icrobiol. 9:92. doi: 10.3389/fmicb.2018.00092
V anc anneyt, M., Mengaud , J., Cleenwerck, I., V anhonacker , K., Hoste, B., Dawyndt,
P., et al. (2004). Reclassification of Lactobacillus kefirgranum T akizawa et al.
1994 as Lactobacillus kefiranof aciens subsp. kefirgranum subsp. nov. and
emended description of L. kefiranof aciens Fujisawa et al. 1988. Int. J. Syst. E vol.
M icrobiol. 54, 551–556. doi: 10.1099/ijs.0.02912- 2910
Wang, S. Y., Chen, K. N., Lo, Y. M., Chiang, M. L., Chen, H. C., Liu,
J. R., et al. (2012). Investigation of microorganisms involved in biosynthesis
of the kefir grain. Food Microb iol. 32, 274–285. doi: 10.1016/j.fm.2012.
07.001
Wang, T., Flint, S., and Palmer , J. (2019). Magnesium and calcium ions: roles
in bacterial cell attachment and biofilm structure maturation. Biofouling 35,
959–974. doi: 10.1080/08927014.2019.1674811
Wang, Z., Chen, Z., Y ang, L., T an, F., Wang, Y., Li, Q., et al. (2017).
Effect of surface physicochemical properties on the flocculation behavior
of Bacillus licheniformis . RSC Adv. 7, 16049–16056. doi: 10.1039/c6ra
28057a
W inand, R., Bogaerts, B., Hoffman, S., Lefe vre , L., Delvoye, M., V an Braekel, J., et al.
(2019). T argeting the 16S RRN A gene for bacterial identification in complex
mixed samples: comparative evaluation of second (Illumina) and third (Oxford
nanopore technologies) generation sequencing technologies. Int. J. Mol. Sci.
21:298. doi: 10.3390/ijms21010298
Zamberi, N. R., Mohamad, N. E., Yeap, S. K., Ky, H., Beh, B. K., Liew, W. C., et al.
(2016). 16S metagenomic microbial composition analysis of kefir grain using
MEGAN and BaseSpace. Food Biotechnol. 30, 219–230. doi: 10.1080/08905436.
2016.1200987
Zhimo, V. Y., Biasi, A., Kumar , A., Feygenberg, O., Salim, S., Vero, S., et al.
(2020). Yeasts and bacterial consortia from kefir grains are effective biocontrol
agents of posthar vest diseases of fruits. M icroorganisms 8:428. doi: 10.3390/
microorganisms8030428
Conflict of Interest: The authors declare that the research was conducted in the
absence of any commer cial or financial relationships that could be construed as a
potential conflict of interest.
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Why organizations use Identific for document trust, entry 48

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