International Journal of Food Science and Technology 2016 1

Original article
Inferring the role of microorganisms in water kefir fermentations

Abigail Mart
ınez-Torres, Sandra Guti
errez-Ambrocio, Pamela Heredia-del-Orbe, Lourdes Villa-Tanaca &

C
esar Hern

andez-Rodr
ıguez*
Departamento de Microbiolog
ıa, Escuela Nacional de Ciencias Biol

ogicas, Instituto Polit
ecnico Nacional, Prol. Carpio y Plan de Ayala, Col.
Casco de Santo Tom

as. Distrito Federal, CP 11340, Mexico, D.F., Mexico
(Received 5 August 2016; Accepted in revised form 25 October 2016)

Summary Water kefir is a slightly alcoholic, lactic and acetic beverage fermented by yeasts, lactic acid bacteria and
acetic acid bacteria that are associated with the polysaccharide of the water kefir grains. In this study, the
three main metabolic products of microorganisms were evaluated during a traditional 192-h water kefir
fermentation and also after inoculating the microorganisms in fresh medium or sterilised broth from dif-
ferent fermentation stages. The first process to occur was alcoholic fermentation, carried out in particular
by Saccharomyces cerevisiae. After 24 h, lactic and acetic acid accumulation was generated by Lactobacil-
lus hilgardii and Acetobacter tropicalis. By the end of fermentation, ethanol had been almost entirely con-
sumed and oxidised to acetic acid, possibly by a dissimilatory route of Acetobacter species. An original
hypothetical diagram is proposed for the carbon flux from sucrose, and the metabolic role of the main
yeasts and bacteria is assigned for the distinct stages of water kefir fermentation.
Keywords Acetobacter tropicalis, Lactobacillus hilgardii, Saccharomyces cerevisiae, water kefir fermentation.

1989; Bergmann et al., 2010; Waldherr et al., 2010;

Introduction
Water kefir (WK) (also known as sugary kefir or
‘tepache de tibicos’) is a fermented beverage made with
panela, a sugar cane product mainly composed of 80–
89% sucrose, 10% reducing sugars and about 0.4%
protein (Guerra & Mujica, 2010). Some fruits (e.g. figs
or lemons) are usually added to the WK fermentation
broth to enhance flavour (Pidoux et al., 1988; Reiß,
1990). Fermentation results in WK vinegar after
2 weeks.
The starter culture to produce the beverage is made
of amorphous, compact, translucent grains, named
‘tibicos’ or WK grains (WKGs). These grains are
mainly composed of water and a gelling polysaccha-
ride, in which different bacteria and yeasts are embed-
ded. The particular microbiota depends on the
location of sample collection along with the specific
procedure for preparing the beverage (Gulitz et al.,
2011; Miguel et al., 2011). Nevertheless, Saccha-
romyces cerevisiae yeasts as well as certain species of
Lactobacillus and acetic acid bacteria have been
repeatedly observed (Moinas et al., 1980; Pidoux,
*Correspondent: Fax: +52 55 57 29 62 07;
e-mail: chdez38@hotmail.com
Gulitz et al., 2011; Miguel et al., 2011).
There have been some attempts to understand the
interactions of the microorganisms in WK fermenta-
tions. In particular, two studies suggest that different
symbiotic relationships can be established between
yeasts and lactic acid fermenters isolated from different
WK fermentations. Firstly, the interaction of a
polysaccharide producer (Lactobacillus hilgardii) with
a yeast (Saccharomyces florentinus) enhances the sur-
vival of bacteria in glucose yeast extract medium and
increases their growth and lactic acid production.
Meanwhile, bacteria affect the growth and ethanol
production of the yeasts (Leroi & Pidoux, 1993). Sec-
ondly, a mutualistic relationship was also established
in another report on the enhanced cell yields of the
two partners of a co-culture of Lactobacillus nagelii
and Lactobacillus hordei with Zygotorulaspora flo-
rentina (formerly S. florentinus) (Stadie et al., 2013).
To our knowledge, only one study has explored the
microbial dynamics in a WK fermentation during
192 h, in that case using panela and figs as the sub-
strate at an initial pH of 4.6. The resulting microbial
diversity in grains and supernatant included five Lacto-
bacillus species, Acetobacter lovaniensis/fabarum and
two yeast species (S. cerevisiae and Dekkera bruxellen-
sis). The entire sugar source was consumed after 96 h

doi:10.1111/ijfs.13312
© 2016 Institute of Food Science and Technology
2 Fermenting microorganism of water kefir A. Mart
ınez-Torres et al.

of fermentation, and the first to be completely
exhausted was sucrose at 24 h. The most representa-
tive metabolites were ethanol and lactic acid, although
some acetic acid appeared at 144 h (Laureys & De
Vuyst, 2014).
The aim of the present study was to assign a role,
during WK fermentation, to the main microbial guilds
– yeasts, lactic acid bacteria and acetic acid bacteria.
A general characterisation was formulated of the order
of primary metabolite accumulation as fermentation
progressed. Then, an evaluation was made of the
microbial consumption of carbon sources as well as
the production by specific microorganisms of meta-
bolic products in fresh medium and in sterilised broth
from different stages of WK fermentation. In this way,
it was possible to establish the main producers of
metabolites in the system. The current results represent
an advance in the understanding of microbial succes-
sion and dynamics during WK fermentation.
Materials and methods
Conditions and sampling time of WKGs
WKGs were obtained from a traditional Mexican cul-
ture and propagated in a 5% panela solution with tap
water containing 0.2–1.5 mg L
1 sodium hypochlorite,
incubated at 20 °C for 96 h. This was repeated ten
times before utilising the grains for further analyses.
and the pressure 10 PSI. A sample of 3 mL was intro-
duced into a 20-mL Pyrex glass vial, which was sealed
with a silicon/Teflon septum and an Al tap. Operating
conditions for the HS-GC-FID are specified in Table 1.
Lactic acid was determined using HPLC, as previ-
ously described (Gezginc et al., 2015), on a Varian
920-LC HPLC apparatus and a C18 column. The
absorbance was detected at a wavelength of 214 nm
and a temperature of 30 °C.
Microbial isolation
For microbial isolation, samples were taken every 6 h
during 72 h of fermentation. WKGs were strained and
1 g was crushed with a pestle and suspended in 9 mL
of distilled water. Decimal dilutions were carried out
until reaching 10
6 and then were inoculated on five
different media: MRS medium (De Man et al., 1960),
MRS modified medium with sucrose instead of glu-
cose, GYC medium [10% wt/vol dextrose, 1% wt/vol
yeast extract, 2% wt/vol calcium carbonate], YPD
medium [2% wt/vol dextrose, 1% wt/vol yeast extract,
2% wt/vol peptone] and sucrose–casein peptone med-
ium [5% wt/vol sucrose, 1% wt/vol casein peptone].
Colonies having different macroscopic and microscopic
morphologies were isolated and preserved in 25% glyc-
erol at
72 °C. Similar decimal dilutions were per-
formed with the fermentation broth or supernatant of
each sample until reaching 10
3.

Physical and chemical properties of WK fermentation DNA extraction, amplification and sequencing of the 16S
rRNA bacterial gene and the ITS1-5.8S rRNA-ITS2
Fermentation was performed in triplicate using
region of yeasts
100 mL of 5% panela solution in flasks, inoculated
with 1 g of WKGs and incubated at 26 °C for 240 h. DNA extraction was performed according to the
Samples of grains and supernatant, obtained every Hoffman & Winston (1987). PCR amplifications of
24 h, were conserved at 4 °C to measure wet weight 16S rRNA bacterial genes were performed utilising a
and pH as well as to determine the concentration of
sugars and the quantity of metabolites. The weight Table 1 HS-GC-FID conditions
increase curve was adjusted with the Gompertz equa-
tion, a sigmoid function widely used to describe micro- Headspace parameters
bial growth (Zwietering et al., 1990).
Headspace temperature 80 °C
The sucrose, fructose and glucose concentrations
Thermostatic heating time 5 min
were determined by Sucrose, D-Fructose and D-Glu- Pressurisation time 0.2 min
cose assay procedures of the K-SUFRG 06/14 Kit Injection time 0.12 min
(Megazyme, Ireland) according to the manufacturer’s Withdrawal time 0.5 min
instructions. Absorbance was measured with a micro- Needle temperature 110°C
plate spectrophotometer at 540 nm (Multiskan GO, Transfer line temperature 115°C
Thermo scientific, USA). Sample volume 3 mL
Ethanol and acetic acid were estimated by headspace GC parameters
gas chromatography (HS-GC-FID) on a Perkin-Elmer Temperature/time 1 55 °C/7 min
AutoSystem XL Gas Chromatograph fitted with a Temperature (oven)/time 2 160 °C/0 min
Temperature/time 3 220 °C/2.5 min
flame ionisation detector (FID) and a Perkin Elmer HS
Heating speed 1 10 °C/min
40 XL Automatic Headspace Sampler, using a CAR- Heating speed 2 20 °C/min
BOWAX column (Polyethylene glycol, 30 m; 32 mm Injector and detector temperature 150 °C
i.d.; film thickness 0.5 lm). The carrier gas was nitrogen

International Journal of Food Science and Technology 2016 © 2016 Institute of Food Science and Technology

reaction mixture containing 100 ng of template DNA,
1X Green Go TaqÒFlexi Buffer, 10 pM of dNTPs
mixture, 1X GoTaqÒFlexi DNA, 10 pM of each pri-
mer and 3.5 mM of MgCl2 in a final volume of 25 lL.
The universal bacterial primers 8F (50 GCG GAT
CCG CGG CGG CTG CAG AGT TTG ATC CTG
GCT 30 ) and 1492R (50 GGC TCG AGC GGC CGC
CCG GGT TAC CTT GTT ACG ACT T 30 ) were
used (Relman, 1993). The PCR procedure included an
initial denaturation step at 94 °C for 5 min, followed
by 35 cycles at 94 °C for 1 min, 55 °C for 1 min,
72 °C for 2.5 min and a final extension step at 72 °C
for 10 min. For PCR amplification of the ITS1-5.8S
rRNA-ITS2 region of yeasts, the aforementioned
reaction mixture and PCR conditions were utilised
and the primers ITS1 (50 -TCCGTAGGTGAACC
TGCGG-30 ) and ITS 4 (50 -TCCTCCGCTTATTGAT
ATGC-30 ) were included in the reaction mix (White
et al., 1990). PCR products were purified using the
ZymocleanTM Gel DNA Recovery Kit (ZYMO
RESEARCH, USA) and sequenced by a DNA
sequencing service (Macrogen Inc., Korea).
Phylogenetic analysis for microbial identification
Sequences were compared using BLAST (Altschul
et al., 1990) and taxonomically related species were
collected from GenBank at the National Center for
Biotechnology Information (NCBI; http://www.ncbi.
nlm.nih.gov/Taxonomy/taxonomyhome.html/). All the
sequences were aligned and manually edited with SEA-
VIEW software (Galtier et al., 1996). The most appro-
priate model of nucleotide substitution was selected
with the jModelTest 2.0 program (Darriba et al.,
2012). Phylogenetic trees were constructed using
MEGA6 software (Tamura et al., 2013; http://
www.megasoftware.net/), with 1000 bootstrap replica-
tions to assess nodal support in the tree. All sequences
were submitted to GenBank under accession numbers
KX214626-KX214656 for bacteria and KX279834-
KX279858 for yeasts
Carbon assimilation profiles of bacteria and yeasts
The carbon assimilation profiles of ten bacterial and
twenty-two yeast isolates were determined in 96-well
plates with 100 lL of modified MES-buffered minimal
medium (MBMM) (Rathnayake et al., 2013), using
ammonium nitrate instead ammonium chloride and
each carbon source at 0.5% (glucose, fructose, sucrose,
lactic acid, acetic acid and ethanol). Additionally, both
organic acids and ethanol were evaluated at 0.25% as
sole carbon sources. The plates were incubated at
28 °C for 168 h and absorbance was measured each
24 h, with a microplate spectrophotometer at 620 nm
(Multiskan FC, Thermo scientific, USA).
© 2016 Institute of Food Science and Technology
Fermenting microorganism of water kefir A. Mart
ınez-Torres et al. 3
Growth assays and metabolite production/consumption on
fresh and conditioned WK media
Fresh medium (FM, corresponding to 5% panela solu-
tion) and supernatants from four different fermentation
times, consisting of 24 h (24FWK), 48 h (48FWK),
72 h (72FWK) and 96 h (96FWK), were centrifuged at
18 516 g for 30 min and successively filtered through
membranes of different pore sizes (0.8, 0.6 and 0.2 lm).
These sterilised samples of FM and FWK were used as
culture media for previously isolated bacteria (Aceto-
bacter orientalis, Acetobacter okinawensis, Acetobacter
tropicalis, Pseudoarthrobacter chlorophenolicus, Lacto-
bacillus casei and L. hilgardii) and yeasts (S. cerevisiae,
Candida californica and Pichia membranifaciens).
Microbial growth was determined by absorbance
(620 nm) every 24 h for a total of 96 h, except that the
growth of L. hilgardii was measured by wet weight.
Metabolite production was analysed by HS-GC-FID
and HPLC at each fermentation stage. The relevant fer-
mentations in the process were confirmed in modified
MBMM supplemented with each carbon source. The
production or consumption of ethanol, lactic acid and
acetic acid was determined after 96 h of incubation in
each condition, using HS-GC-FID and HPLC as
described previously. For controls, FM and FWK
(from each time) were employed without inoculation.
Results
Isolation and identification of microorganisms
Microbial identification by the phylogenetic approach
indicated the presence of seven bacteria, including Lac-
tobacillus ghanensis, L. casei/paracasei, L. hilgardii,
P. chlorophenolicus (formerly Arthrobacter chlorophe-
nolicus), A. orientalis, A. tropicalis and A. okinawensis
(Fig. 1), as well as three yeasts, S. cerevisiae, C. cali-
fornica and P. membranifaciens (Fig. 2).
Lactobacillus spp., Acetobacter spp. and S. cerevisiae
populations were estimated at approximately
107–108 UFC g
1 in grain. Meanwhile, L. casei/para-
casei, the three aforementioned Acetobacter species
and S. cerevisiae were detected in the supernatant with
populations of approximately 105–106 UFC mL
1 at
24 h of incubation. L. hilgardii (WKGPB13) was only
isolated from WKGs, in sucrose MRS medium, panela
medium and sucrose–casein peptone medium, produc-
ing irregular translucent jelly colonies with a hard con-
sistency (Fig. 3).
Characterisation of WK fermentation
The increase in wet weight and pH was determined
during 216 h of fermentation using 5% panela medium
inoculated with 1 g of WKGs per 100 mL. Grain
International Journal of Food Science and Technology 2016
4 Fermenting microorganism of water kefir A. Mart
ınez-Torres et al.

Figure 1 Maximum-likelihood phylogenetic

tree of a partial sequence of the 16S rRNA
gene in microorganisms isolated from water
kefir. With the Jukes–Cantor substitution
model, the sequence of Bacteroides fragilis
(NR119164) was used as the external group.
The bar indicates the number of substitu-
tions per site. The numbers in the nodes rep-
resent the bootstrap value of 1000 replicates.
The numbers in brackets are the GenBank
accession numbers of each sequence.

biomass increased 2.7 times and the initial pH of 7 The main metabolites in WK were quantified and
decreased to 3.5 (Fig. 4a). The biomass of WKGs kept their production was determined during 192 h of fer-
accumulating even after a pH of 4 was reached in the mentation (Fig. 4b). Ethanol started to accumulate at
supernatant. the onset of fermentation and reached its maximum

International Journal of Food Science and Technology 2016 © 2016 Institute of Food Science and Technology

Figure 2 Maximum-likelihood phylogenetic
tree of a partial sequence of the ITS1-5.8S
rRNA-ITS2 gene in microorganisms isolated
from water kefir. The Tamura 3 substitution
model was used, with the sequence of
Yarrowia lipolytica (DQ683010) as the
external group. The bar indicates the
number of substitutions per site. The num-
bers in the nodes represent the bootstrap
value of 1000 replicates. The numbers in
brackets are the GenBank accession numbers
of each sequence.
(a)
Figure 3 (a) Plate with L. hilgardii colonies
in sucrose–casein peptone solid medium. (b)
Image of a single colony of L. hilgardii in
sucrose–casein peptone solid medium.
concentration of 4.29 g L
1 at 96 h, and then gradu-
ally diminished from 144 h to 192 h when it reached
0.7 g L
1. Lactic acid accumulation was detected as of
24 h, reached 1.38 g L
1 at 48 h and then stabilised
between 1.29 and 1.79 g L
1 (with no reduction
observed during fermentation). The accumulation of
acetic acid began at 24 h and rose to a maximum con-
centration of 28.35 g L
1 at 192 h.
© 2016 Institute of Food Science and Technology
Fermenting microorganism of water kefir A. Mart
ınez-Torres et al. 5
Saccharomyces cerevisiae (KC588952)
WK11B, WK49, WK50, WK26, WK57, WK4B, WK13C
Saccharomyces cerevisiae (DQ167469)
Saccharomyces cerevisiae (KC254075)
99 WK146, WK90A, WK33B, WK45, WK33D, WK17C, WK209
Saccharomyces cerevisiae (KC515364)
97 WK79, WK44, WK165, WK93A, WK110, WK1, WK49
Saccharomyces cerevisiae (CP006468)
Saccharomyces yakushimaensis (AB097400)
Candida californica (JX188105)
Candida californica (JX188102)
WK64, WK172, WK214
100Candida californica (JX188103)
WK205
71
Candida californica (DQ104728)
Candida ethanolica (FJ662418)
Pichia membranifaciens (AF270935)
96 WK199
Pichia membranifaciens (DQ198954)
99
Pichia membranifaciens (KP132516)
70
91 Pichia membranifaciens (DQ104715)
Pichia manshurica (FM199959)
99
Pichia fermentans (KF646196)
Candida inconspicua (KP131717)
Yarrowia lipolytica (DQ683010)
0.05
(b)
The consumption of sucrose and the presence of glu-
cose and fructose in the fermentation of WK were
quantified as well (Fig. 4c). Sucrose was gradually
assimilated from 46.61 to 4.76 g L
1 during the fer-
mentation. The end of sucrose assimilation was not
associated with a decrease in pH, evidenced by the fact
that a pH of 3.6 was detected as of 72 h. The initial
concentration of glucose and fructose in the panela
International Journal of Food Science and Technology 2016
6 Fermenting microorganism of water kefir A. Mart
ınez-Torres et al.
medium was 0.83 and 2.04 g L
1, respectively. The
level of both monosaccharides increased slightly by
24 h, to 1.5 and 3.04 g L
1, respectively. This may
have been due to the presence of extracellular invertase
activity, as occurred with S. cerevisiae (data not
shown). After 48 h, the values of glucose and fructose
oscillated from 0.0–0.74 to 0.0–0.54 g L
1, respec-
tively.
Figure 4 (a) pH variations and kinetics of the biomass of water
kefir grains during 216 h of fermentation (the kinetics data were
adjusted with the Gompertz equation). (b) The concentration of
ethanol, lactic acid and acetic acid during 192 h of WK fermenta-
tion. (c) The concentration of sucrose, glucose and fructose during
192 h of WK fermentation.
International Journal of Food Science and Technology 2016
Carbon assimilation profiles of bacteria and yeasts
Carbon utilisation tests were performed with ten bacte-
ria (six Acetobacter spp., three Lactobacillus spp. and
P. chlorophenolicus) and twenty-two yeasts (nineteen
S. cerevisiae, two C. californica and one P. mem-
braneafaciens). All bacteria were able to use sucrose,
fructose or glucose, but not acetic acid as a sole car-
bon source (Fig. 5a). Acetobacter spp. grew better in
lactic acid (Fig. S1). As expected, yeasts could also
grow with glucose, fructose or sucrose as the sole car-
bon source. The majority of the S. cerevisiae, P. mem-
branifaciens and C. californica strains grew in ethanol
and a relatively low lactic acid concentration (0.25%)
(Fig. 5b). Only P. membranifaciens grew in acetic acid
(Fig. 6).
Figure 5 Carbon source utilization by (a) bacteria and (b) yeasts
isolated from WK. Glucose, fructose and sucrose were at 0.5 w/v.
© 2016 Institute of Food Science and Technology

Microbial role in WK fermentation
This assay was carried out to explore the environmen-
tal changes that occurred during fermentation when
using each of the following microorganisms: L. casei,
L. hilgardii, A. tropicalis, A. okinawensis, A. orientalis,
P. chlorophenolicus, S. cerevisiae, P. membranifaciens
and C. californica. Five fermentation stages were
assayed, including FM, 24FWK, 48FWK, 72FWK
and 96FWK.
After inoculating microorganisms in FM, the growth
of yeasts and P. chlorophenolicus was significant and
that of L. casei slight (Fig. 7a). However, none of the
acetic acid bacteria grew in this condition. Growth
was noted in the different fermentation stages as fol-
lows: the yeasts and A. okinawensis at 24FWK
(Fig. 7b), the yeasts at 48FWK (Fig. 7c), S. cerevisiae
and P. membranifaciens at 72FWK (Fig. 7d) and none
of the microorganisms at 96FWK (Fig. 7e). L. hilgar-
dii showed increased biomass in FM, 24FWK and
48FWK.
At the early stages of fermentation, the majority of
microorganisms reduced the pH of the medium com-
pared to the internal control (Fig. 8a). The decrease in
pH was particularly evident with acetic acid bacteria,
reaching pH <5.3.
Saccharomyces cerevisiae was the main ethanol pro-
ducer in all stages, particularly early in fermentation
(FM, 24FWK and 48FWK). Ethanol was also gener-
ated by L. hilgardii in the late stages (72FWK and
96FWK), but not by C. californica or P. membranefa-
ciens at any stage (8B). On the other hand, A. tropi-
calis consumed ethanol and transformed it into acetic
acid during the late stages (72FWK and 96FWK),
causing almost complete depletion (Fig. 8b and d).
Lactobacillus hilgardii induced the accumulation of a
moderate amount of lactic acid, mainly in FM,
24FWK and 48FWK, while L. casei and P. chlorophe-
nolicus produced scarce quantities of the same
Figure 6 Kinetics of Pichia membranifaciens growing in different
carbon sources.
© 2016 Institute of Food Science and Technology
Fermenting microorganism of water kefir A. Mart
ınez-Torres et al. 7
(Fig. 8c). In some stages, it seemed that the produc-
tion and consumption of lactic acid occurred simulta-
neously, although this effect was not confirmed in
modified MBMM supplemented with glucose, fructose,
sucrose or lactic acid (data not shown).
Acetic acid was generated in FM and 24FWK,
mainly by L. hilgardii. In the later stages, it was pro-
duced mainly by A. tropicalis, and to a lesser extent by
A. orientalis, A. okinawensis, L. casei and
P. chlorophenolicus (Fig. 8d).
Discussion
WK should not be confused with milk kefir (MK),
another fermented beverage that needs polysaccharide
grains having microbes embedded as starters. MK uses
milk and its by-products as the substrate for fermenta-
tion to yield kefiran grains, a glucogalactan mainly
synthetised by Lactobacillus kefiranofaciens (Wszolek
et al., 2001 Piermaria et al., 2008; Magalh~aes et al.,
2010a, 2011; Zajsek et al., 2011; Rodrigues et al.,
2016). Meanwhile, WKGs employ sucrose as the sub-
strate and produce an a 1–6 glucose dextran syn-
thetised by L. hilgardii (Pidoux et al., 1990; Waldherr
et al., 2010). Although both beverages harbour micro-
bial communities that include yeast, lactic acid bacte-
ria and acetic acid bacteria, the relative abundance
and composition of each of these are very different
(Gulitz et al., 2013; Zamberi et al., 2016). The fermen-
tation and growth of WKGs have been reported in
milk (Hsieh et al., 2012), and the particular WK
employed in the current study can ferment milk. How-
ever, no growth of WKGs was observed, mainly
because L. hilgardii does not assimilate or produce
polysaccharides from lactose (data not shown).
Some reports have explored the microbial complexity
in WK (Moinas et al., 1980; Pidoux, 1989; Gulitz et al.,
2011, 2013; Miguel et al., 2011; Laureys & De Vuyst,
2014). In this study, seven bacteria (L. ghanensis, L. ca-
sei/paracasei, L. hilgardii, A. orientalis, A. tropicalis,
A. okinawensis and P. chlorophenolicus) and three
yeasts (S. cerevisiae, C. californica and P. membranifa-
ciens) were isolated and identified. To our knowledge,
this is the first report of A. okinawensis and
P. chlorophenolicus isolated in this environment.
Lactobacillus hilgardii has been widely reported in
WK studies (Pidoux, 1989; Magalh~aes et al., 2010b;
Gulitz et al., 2011). It was presently isolated and cul-
tured in medium containing only sucrose, producing
colonies very similar to the WKGs found in panela
medium. Hence, this bacterium is probably responsible
for inducing the formation of these grains. Indeed,
L. hilgardii, previously known as Bacterium vermi-
forme, was reported as the polysaccharide producer in
WK in 1892 (Ward, 1892). Another study (Pidoux,
1989), almost 100 years later, reported a colonial
International Journal of Food Science and Technology 2016
8 Fermenting microorganism of water kefir A. Mart
ınez-Torres et al.
Figure 7 During 96 h of incubation, kinetics of the growth of bacteria and yeasts isolated from WK in the different fermentation stages: (a)
FM, (b) 24FWK, (c) 48FWK, (d) 72FWK and (e) 96FWK.
morphology of pure cultures of L. hilgardii similar to
that observed presently. It must be mentioned that
there are other reports of the same species forming
slimy or mucoid colonies in sucrose MRS medium
(Waldherr et al., 2010; Davidovi
c et al., 2015). More-
over, other WK inhabitants, such as Lactobacillus bre-
vis, Leuconostoc mesenteroides, L. nagelii and
International Journal of Food Science and Technology 2016
L. hordei, can produce polysaccharides and have been
related to WKG formation (Pidoux et al., 1988; Gulitz
et al., 2011). Nonetheless, the current results are in
agreement with previous reports in the sense that that
L. hilgardii is the microbe mainly responsible for the
production of polysaccharides to form the grains of
WK fermentation (Pidoux, 1989).
© 2016 Institute of Food Science and Technology

Figure 8 (a) pH variation produced by microorganisms isolated from WK in each fermentation stage. Concentration of (b) ethanol, (c) lactic
acid and (d) acetic acid in different media with microorganisms isolated from WK.
Although about twenty Lactobacillus and ten Aceto-
bacter species have been reported in WK beverages, in
many studies, these species were identified only through
the use of biochemical and physiological tests (Pidoux,
1989; Rubio et al., 1993). Due to the phenotypic plastic-
ity of bacteria and the limitations inherent in commer-
cial identification tests, species are frequently
misidentified, particularly those in the Lactobacillus
genus. DNA hybridisation analyses have proven that
some biochemically identified species were misidentified,
such as confusing L. hilgardii with L. brevis (Lonvaud-
Funel et al., 1991). The usefulness of API 50 CH carbo-
hydrate fermentation strips and other phenotype identi-
fication methods is greatly diminished by a high level of
phenotype variability (as in the case of the commensal
Lactobacillus bacterium from the human vagina) and
limited databases. Hence, 16S rRNA sequencing may be
preferable (Boyd et al., 2005).
Indeed, bacterial 16S rRNA or the 18S or 26S
rRNA partial gene sequences of yeasts obtained from
PCR-DGGE amplicons and pyrosequencing have been
used for molecular identification of WK microbiota
© 2016 Institute of Food Science and Technology
Fermenting microorganism of water kefir A. Mart
ınez-Torres et al. 9
(Magalh~aes et al., 2010b; Gulitz et al., 2011, 2013;
Laureys & De Vuyst, 2014). It turns out that the short
length of the sequences (200–300 nt) obtained with
these methods are not suitable for species-level identifi-
cation by the phylogenetic approach and similitude.
Additionally, only BLAST analyses have been per-
formed without taking into account any formal taxo-
nomic criterion. As no accession numbers were
provided, confirmation, taxonomical reclassification
and phylogenetic comparisons are not possible. More-
over, no phylogenetic trees were included to show
formally described species, much less several strains
of the identified species. Given that no reliable
species-level identifications have been performed, the
variety of microorganisms in WK may not be as broad
as supposed.
The grain biomass herein exhibited a 2.7-fold
increase, which is different from other reports on the
quantification of grain biomass. Nevertheless, these
results must be carefully compared because of differ-
ences in the grains and the media employed, as well as
in the initial pH and incubation temperature. In a
International Journal of Food Science and Technology 2016
10 Fermenting microorganism of water kefir A. Mart
ınez-Torres et al.
previous study using 6% panela medium inoculated
with 3 g of grains per 100 mL, there was only a 1.7-
fold increase in the wet weight of WKGs, although the
decrease in pH was similar to that found presently
(pH 3.4) (Rubio et al., 1993). Another fermentation of
kefir, performed with brown sugar and figs, started at
a pH of 4.8 and obtained only a small increase in the
biomass of the grains, approximately 1.7 fold, verify-
ing the relevance of pH in this process (Laureys & De
Vuyst, 2014). Furthermore, in the present study, the
biomass kept accumulating even after a pH of 4 was
reached in the supernatant, which confirms that an ini-
tial alkaline pH leads to higher biomass yields
(Fig. S2). It is also possible that the polysaccharide
biomass of WKGs buffers the environment inside the
biofilm and temporally protects L. hilgardii and the
other microorganisms from the acidic pH of the super-
natant. Other bacterial biofilm polysaccharides gener-
ate an internal environment protected from external
acidic conditions, such as the biofilms of Lactobacillus
plantarum and Streptococcus mutans (Zhu et al., 2001;
Kubota et al., 2008).
Quantification of the main metabolites in WK dur-
ing 192 h demonstrated that the first fermentation was
alcoholic, as has been reported previously with similar
WK fermentations (Reiß, 1990; Rubio et al., 1993;
Magalh~ aes et al., 2010b; Laureys & De Vuyst, 2014).
The maximum ethanol concentration was 4.29 g L
1,
a moderate value considering the broad range
obtained in other studies (0.18–20.3 g L
1) (Rubio
et al., 1993; Laureys & De Vuyst, 2014). After 144 h,
the ethanol content gradually declined until reaching
0.7 g L
1, an effect not previously reported for any
other WK fermentation, although it is known to occur
in apple cider vinegar production and spontaneous
cocoa bean fermentation (De Vuyst & Weckx, 2016;

Stornik et al., 2016).
The second fermentation was lactic, and the maxi-
mum concentration of this metabolite was 1.70 g L
1,
less than that previously reported (Laureys & De
Vuyst, 2014). Even during a fermentation of only
24 h, lactic acid reached a concentration up to
2.5 mg mL
1 (Magalh~ aes et al., 2010b). On the other
hand, no decrease in accumulated lactic acid was
herein observed during fermentation, which is in agree-
ment with previous reports on WK fermentation
(Rubio et al., 1993; Laureys & De Vuyst, 2014).
Acetic fermentation occurred as of 24 h, resulting in
an acetic acid concentration above 28 g L
1 by the
end of fermentation. This represents the highest level
reported for WK, in which concentrations have ranged
between 1 and 7 g L
1 (Rubio et al., 1993; Laureys &
De Vuyst, 2014). In Mexico, it is common for WK or
‘tepache de tibicos’ to be fermented for 2–3 weeks to
obtain homemade vinegar, a product with an acetic
acid content similar to other commercial or traditional
International Journal of Food Science and Technology 2016
vinegars, which contain from 10.8 to 111.7 g L
1 (Li
et al., 2015).
The content of sugars was also measured in the pre-
sent study, finding that during fermentation, the level
of sucrose diminished, although this sugar was not
completely consumed. In other reports, sucrose was
exhausted and grain biomass growth was associated
with disaccharide availability (Laureys & De Vuyst,
2014). Another study reported that the supply of
sucrose was almost exhausted within 24 h, the level
dropping from approximately 47.5 to 1.2 g L
1, while
the level of fructose rose above 20 g L
1. In the cur-
rent contribution, neither glucose nor fructose under-
went any marked increase, their values remaining
below 0.74 and 0.54 g L
1, respectively, for most of
the process. According to previous reports, the three
main sugars are totally consumed before 96 h
(Magalh~aes et al., 2010b; Laureys & De Vuyst, 2014).
The carbon assimilation profiles of the microorgan-
isms evidenced that all bacteria were able to use
sucrose, glucose or fructose as a carbon source. Aceto-
bacter spp. grew better in lactic acid, a common fea-
ture in this genus because it can oxidise lactate and
acetate to carbon dioxide and water (Lisdiyanti et al.,
2000, 2001; Iino et al., 2012). The yeasts could utilise
most of the carbon sources, except that only P. mem-
branifaciens was able to proliferate in the presence of
acetic acid. The Pichia genus (particularly P. membran-
ifaciens) is polyphyletic. This species is associated with
the Pichia clade that includes some Issatchenkia spp.
as well as Candida species, such as C. ethanolica and
C. californica (Kurtzman et al., 2008). P. membranifa-
ciens is highly distributed in fermentations, as are
other related species in the same clade, presenting a
predilection for ethanol and simple organic acids as
carbon sources and having poor fermentative power
(Suh et al., 2006).
To study their metabolic capacity and possible role
during WK production, microorganisms were inocu-
lated in sterilised broth taken from WK at different
fermentation stages. Overall, yeasts were able to grow
in most of the fermentation stages, while P. chlorophe-
nolicus and L. casei grew in FM and A. okinawensis in
24FWK. Although the other bacteria did not prolifer-
ate, they were metabolically active at the different
stages of fermentation. The pH was reduced by all
bacteria in FM (except P. chlorophenolicus), possibly
because even a very low amount of lactic acid or acetic
acid, when excreted in a panela medium with any
buffering capacity, is capable of acidifying it. In the
other fermentation stages, no pH decrease was
observed compared to the internal control.
Previous reports on WK have assumed that ethanol
and acetic acid are afforded by any yeast or acetic acid
bacterium, respectively, present in the consortium
(Magalh~aes et al., 2010b; Laureys & De Vuyst, 2014).
© 2016 Institute of Food Science and Technology
 Fermenting microorganism of water kefir A. Mart
ınez-Torres et al. 11

The current results are not in agreement with this The present identification of the most metabolically
assumption. Indeed, ethanol was mainly yielded by active species during WK fermentation has provided
S. cerevisiae, a traditional ethanol producing yeast further insights into the dynamics of this process,
related to the production of alcoholic beverages (Fay allowing for the proposal of a minimal and efficient
& Benavides, 2005). The two other yeasts identified by consortium for WK production, formed by L. hilgar-
the phylogenetic approach herein employed, C. califor- dii, S. cerevisiae and A. tropicalis. This simplified con-
nica and P. membranifaciens, were unable to produce sortium could be used in a new fermentation process
ethanol in any fermentation stage. for beverage development, focusing on the examina-
Acetic acid, on the other hand, was initially gener- tion of microbial interaction and expression patterns
ated by L. hilgardii, and later by Acetobacter spp., as well as in vitro evolution, among other experiments.
mostly A. tropicalis. The latter species consumed etha- In previous reports on WK fermentations, the
nol and oxidised it to acetic acid. After 96 h, other assignment of the fermentation process to particular
microorganisms also produced acetic acid, which yeasts and bacteria was based on previous knowledge
explains both the decrease in ethanol at 144 h and the of the fermentation abilities of each microbial group.
high concentration of acetic acid at 192 h of fermenta- To our knowledge, the current contribution demon-
tion. In a previous study, acetic acid bacteria appeared strates for the first time the production of the main
after 144 h and therefore were not an important part primary metabolites by specific microorganisms after
of the WK ecosystem (Laureys & De Vuyst, 2014). It inoculating these into fermentation broths from differ-
is evident that kefir beverages are very different ent stages (thus having distinct chemical and physico-
according to the particular region (even within the chemical compositions).
same country), as evidenced by a Brazilian report In conclusion, the present WK preparation resulted
(Miguel et al., 2011). in an initial increase (appearing as of 0 h) in alcohol,

Sucrose
(a) (b) 0h
Extracellular invertase

Intracellular invertase

pH 7
Glucansucrase

Biomass 1g
g L–1
–1
S. cerevisiae A. tropicalis L. hilgardii Sugars Metabolites gL
A. orientalis Sucrose 46.6 Ethanol 0
P. membranifaciens Glucose 0.83 Lactic acid 0
Fructose 2.04 Acetic acid 0

24 h
pH 4.97
Fructose Glucose Fructose Glucose Fructose Dextran Biomass 1.52 g
g L–1
–1
(Polysaccharide grain) Sugars Metabolites gL
Sucrose 36.06 Ethanol 1.62
Glycolytic enzymes

Glucose 1.5 Lactic acid 0.09

Fructose 3.04 Acetic acid 0.51

All species 48 h
pH 4.02
Piruvate
Biomass 2.34 g
Sugars g L–1 Metabolites gL
–1

Sucrose 17.6 Ethanol 4.11

dehydrogenase
dehydrogenase
decarboxylase

Glucose 0.7 Lactic acid 1.37

Pyruvate
Pyruvate
Pyruvate

Fructose 0.54 Acetic acid 2.97

Lactate dehydrogenase

72 h
Aldehyde pH 3.61
dehydrogenase Biomass 2.75 g
g L–1
–1
Acetaldehyde Acetyl CoA Sugars Metabolites gL
A. tropicalis Sucrose 15.56 Ethanol 4.2
CoA transferase
CoA transferase

L. hilgardii S. cerevisiae L. hilgardii

A. tropicalis A. orientalis A. tropicalis Glucose 0 Lactic acid 1.12
A. orientalis A. orientalis Fructose 0.24 Acetic acid 8.99
Alcohol dehydrogenase
Alcohol dehydrogenase

96 h
pH 3.59
Biomass 3.13 g
Acetyl-P
g L–1
–1
Sugars Metabolites gL
Acetate kinase

Acetate kinase

Sucrose 13.56 Ethanol 4.23

Glucose 0.17 Lactic acid 1.44
Fructose 0.31 Acetic acid 13.29

Lactic acid Ethanol Acetic acid

Figure 9 (a) Hypothetical flux of carbon during a WK fermentation from sucrose to the main metabolic products. The alcoholic, lactic acid
and acetic acid fermentations and production of acetic acid from ethanol are indicated. WK microorganisms are located in each relevant meta-
bolic pathway of sucrose hydrolysis and the various fermentations. The hypothetical enzymes in each reaction are shown next to the arrows.
(b) The substrate consumption, pH evolution, biomass accumulation and fermentation progress from 0 to 96 h.

© 2016 Institute of Food Science and Technology International Journal of Food Science and Technology 2016
12 Fermenting microorganism of water kefir A. Mart
ınez-Torres et al.
this being most abundantly yielded by S. cerevisiae.
Lactic and acetic acid began to accumulate after 24 h,
mainly produced by L. hilgardii (the producer of
polysaccharide in WKG) and A. tropicalis, respec-
tively. Finally, the fermentation process generated an
acetic acid accumulation and WK vinegar as of 192 h.
By the end of fermentation, ethanol had almost been
entirely consumed and oxidised to acetic acid, possibly
by a dissimilatory route of Acetobacter spp. Based on
the present results, we suggest an original hypothetical
scheme of the carbon flow in WK production from
sucrose to metabolite products (Fig. 9a), and provide
an image outlining the changes occurring in WK dur-
ing 96 h of fermentation (Fig. 9b).
Acknowledgments
We give thanks to Bruce Allan Larsen for reviewing
the English version of this manuscript and to Jesus
Luna for photos of L. hilgardii. We are grateful for
the support provided by the Central de Instru-
mentaci
on de Espectroscop
ıa and the Departamento
de Graduados en Alimentos, both of the ENCB-IPN,
and are beholden to Dr. Juan Carlos Villalobos Rocha
for his critical review. AMT, SGA and PHO thank
CONACyT and BEIFI-IPN for fellowships. This work
was supported by the Secretar
ıa de Ciencia y Tec-
nolog
ıa del DF (PICSO12-136, ICYTDF/206/2012)
and the Instituto Polit
ecnico Nacional, SIP 20151102
and SIP 20161850. CHHR and LVT are thankful for
COFFAA, SNI and EDI fellowships.
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Supporting Information
Additional Supporting Information may be found in
the online version of this article:
Figure S1. Kinetics of Acetobacter spp. growing in
different carbon sources; A. tropicalis, A, okinawensis
and A. orientalis
Figure S2. Water kefir biomass production in differ-
ent pH values.
International Journal of Food Science and Technology 2016