fmicb-12-732019 October 29, 2021 Time: 14:18 # 1
ORIGINAL RESEARCH
published: 04 November 2021
doi: 10.3389/fmicb.2021.732019
Edited by:
Jian Zhao,
University of New South Wales,
Australia
Reviewed by:
Stefan Junne,
Technical University of Berlin,
Germany
Graciela Liliana Garrote,
National University of La Plata,
Argentina
Volkmar Passoth,
Swedish University of Agricultural
Sciences, Sweden
*Correspondence:
Maximilian Schmacht
Martin Senz
†
†
†ORCID:
Sarah Köhler
orcid.org/0000-0003-3522-6512
Maximilian Schmacht
orcid.org/0000-0003-2033-5775
Marie Ludszuweit
orcid.org/0000-0001-5982-3501
Nils Rettberg
orcid.org/0000-0002-3667-2007
Martin Senz
orcid.org/0000-0002-3997-5011
Specialty section:
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
Received: 28 June 2021
Accepted: 11 October 2021
Published: 04 November 2021
Citation:
Köhler S, Schmacht M,
Troubounis AHL, Ludszuweit M,
Rettberg N and Senz M (2021)
Tradition as a Stepping Stone
for a Microbial Defined Water Kefir
Fermentation Process: Insights in Cell
Growth, Bioflavoring, and Sensory
Perception.
Front. Microbiol. 12:732019.
doi: 10.3389/fmicb.2021.732019
Tradition as a Stepping Stone for a
Microbial Defined Water Kefir
Fermentation Process: Insights in
Cell Growth, Bioflavoring, and
Sensory Perception
Sarah Köhler1†, Maximilian Schmacht1,2*†, Aktino H. L. Troubounis1, Marie Ludszuweit1†,
Nils Rettberg3†and Martin Senz1*†
1Department Bioprocess Engineering and Applied Microbiology, Research and Teaching Institute for Brewing (VLB) in Berlin,
Berlin, Germany, 2Technische Universität Berlin, Faculty III Process Sciences, Chair of Bioprocess Engineering, Institute
of Biotechnology, Berlin, Germany, 3Research Institute for Beer and Beverage Analysis, Research and Teaching Institute for
Brewing (VLB) in Berlin, Berlin, Germany
A process development from a traditional grain-based fermentation to a defined
water kefir fermentation using a co-culture of one lactic acid bacterium and one
yeast was elaborated as a prerequisite for an industrially scalable, controllable, and
reproducible process. Further, to meet a healthy lifestyle, a low ethanol-containing
product was aimed for. Five microbial strains—Hanseniaspora valbyensis,Dekkera
bruxellensis,Saccharomyces cerevisiae,Liquorilactobacillus nagelii, and Leuconostoc
mesenteroides—were used in pairs in order to examine their influence on the
fermentation progress and the properties of the resulting water kefir products against
grains as a control. Thereby, the combination of H. valbyensis and L. mesenteroides
provided the best-rated water kefir beverage in terms of taste and low ethanol
concentrations at the same time. As a further contribution to harmonization and
reduction of complexity, the usage of dried figs in the medium was replaced by fig
syrup, which could have been proven as an adequate substitute. However, nutritional
limitations were faced afterward, and thus, an appropriate supplementation strategy
for yeast extract was established. Finally, comparative trials in 5-L scale applying
grains as well as a defined microbial consortium showed both water kefir beverages
characterized by a pH of 3.14, and lactic acid and aromatic sensory properties. The
product resulting from co-culturing outperformed the grain-based one, as the ethanol
level was considerably lower in favor of an increased amount of lactic acid. The
possibility of achieving a water kefir product by using only two species shows high
potential for further detailed research of microbial interactions and thus functionality of
water kefir.
Keywords: water kefir, co-culture, lactic acid bacteria (LAB), yeast, bioflavoring, process development, yeast
extract
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Köhler et al. Microbiology and Bioflavoring of Water Kefir
INTRODUCTION
Traditionally fermented beverages enjoy increasing popularity
against the background of a conscious and healthy lifestyle
(Kandylis et al., 2016). Especially sour fermented beverages, such
as kombucha or kefir, promise health benefits, e.g., antimicrobial,
anticancer, or probiotic, by their indigenous microorganisms
(Prado et al., 2015;Tamang et al., 2016;Ghosh et al., 2019).
Thereby, water kefir (WK), which is based on sugary water
(usually sucrose is used) and a fruit component, offers a vegan
and lactose-free alternative. The characteristic microorganisms
of WK are lactic acid bacteria (LAB) and different yeasts,
but also acetic acid bacteria can be involved (Bourrie et al.,
2016). The microorganisms are organized in so-called grains that
are based on exopolysaccharides mainly produced by the LAB
(Nielsen et al., 2014).
Generally, the so-called cross-feeding occurs when LAB and
yeast are fermented in co-culture meaning that both groups of
organisms can profit from the metabolic products of one another
(Bader et al., 2010;Smid and Lacroix, 2013;Stadie et al., 2013;
Zhang et al., 2017). This is especially important in the case of
WK, where the nutrients in general and nitrogen in particular
are limited and may induce sulfurous off-flavors (Vardjan et al.,
2013;Laureys et al., 2018). Lactobacilli produce lactate in the
course of their carbohydrate metabolism, which yeast can absorb
and break down. This prevents a sharp drop in pH and aids
the lactobacilli in continuing their usual metabolic activities.
The yeasts in turn produce a large number of amino acids and
also some vitamins, such as vitamin B6 (Stadie et al., 2013),
which are essential nutrients for lactobacilli (Ponomarova et al.,
2017;Zhang et al., 2017). Due to this interaction, stimulated
growth of lactobacilli is often observed in a co-culture compared
with the pure culture (Stadie et al., 2013;Vardjan et al., 2013;
Bechtner et al., 2019). Further studies describe culture-related
differences in the expression of genes that affect the metabolism
of carbohydrates and amino acids (Yamasaki-Yashiki et al., 2017;
Bechtner et al., 2019), as well as exopolysaccharide formation
(Yamasaki-Yashiki et al., 2017) and aggregation factors (Bechtner
et al., 2019) involved. In addition, during co-cultivation, the
aerobic metabolism of the yeasts reduces the oxygen content
of the nutrient medium, which in turn can be beneficial for
the growth of lactobacilli (Mendes et al., 2013;Nejati et al.,
2020). Although some general modes of interactions between
different groups of microorganisms are known (Nejati et al.,
2020), the relationship between different strains and whether
the interactions are synergistic or counteractive is still not fully
understood and remains a topic of current research. Additionally,
the mixture of the specifically involved organisms results in a
distinct profile of organic acids and exopolysaccharides that have
an important influence on the sensory properties of the final
beverage (Bader et al., 2010;Bertsch et al., 2019;Jin et al., 2019).
Besides the consortium of microorganisms, also the fruit
component is rather complex. Traditionally, e.g., figs, dates,
raisins, and/or lemons, were used to produce WK (Açik et al.,
2020). However, as with every natural ingredient, quality can
vary; and there may occur seasonal effects that influence
the industrial production. Moreover, the choice of the fruit
component significantly influences the fermentation process by
the provision of nutrients for the involved microorganisms as
well as the final sensory properties of the beverage.
In summary, industrializing the production of WK with a
complex mixture of raw materials and microorganisms is very
difficult in terms of final product properties and especially
reproducibility (Laureys and de Vuyst, 2017;Nejati et al., 2020).
To the knowledge of the authors, successful industry-scale
water kefir products are scarce because the management of the
fermentation process to obtain reproducible products is similarly
complex as the microbial consortia themselves. Therefore, the
companies keep their recipes and control strategies guarded.
A systematic understanding of mutual interactions aids in the
specific control of the production process resulting in safe,
defined, and reproducible products. Consequently, efforts to
develop defined fermentations applying specific starter cultures
are undertaken, whereby a reasoned selection of production
strains is of utmost importance. Thereby, one can assume that
the level of interactions in a defined co-culture is lower than a
complex microbial community, posing the risk of also resulting
in a less complex product. However, the application of defined co-
cultures has the potential to be better controllable due to its lower
level of complexity resulting in less “adjustment screws” and thus
lower product deviations.
In order to get a deeper insight which microorganisms
originating from natural WK grains contribute synergistically
to the fermentation progress resulting in an organoleptic
characteristic beverage, systematic experiments to reduce the
microbial complexity were conducted. Furthermore, the main
focus was the production of a lactic acidic beverage by co-
culture fermentation, which should preferably contain a low
amount of alcohol meeting the consumer demands for a healthy
drink. Thereby, the influence of the available nutrients, such as
amino acids and trace elements, on the microbial interactions
and the sensory properties represented by different microbially
built chemical compounds as well as possible limitations were
considered. This paper finally presents a comparison of a
traditionally produced complex WK and a fermented beverage
product produced with specific starter cultures mimicking WK
regarding the fermentation parameters as well as the final
product properties.
MATERIALS AND METHODS
Microorganisms
In the present studies, WK grains were used. These have been
used successfully for many years for the traditional production
of WK via back-slopping processes. The microbial strains used
as defined cultures were isolated and identified from these
mentioned grains. Therefore, grains were homogenized by
using the ULTRA-TURRAXR
T25 basic (IKAR
-Werke GmbH
& CO. KG, Staufen, Germany), and different dilutions of the
homogenized liquid were plated on yeast extract (YE) dextrose
agar as well as MRS agar according to De Man et al. (1960).
Incubation took place at 26, 30, and 37◦C under aerobic and
anaerobic conditions. Pure cultures were gained by repeating the
plating step of single colonies several times. From those single
colonies, material was taken for DNA extraction (PhireTM Plant
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Direct PCR Master Mix, Thermo Fisher Scientific Inc., Waltham,
MA, United States) followed by PCR amplification using primers
8(F) (50-AGAGTTTGATCCTGGCTCAG-30) and 1492(R)
(50-GGTTACCTTGTTACGACTT-30) for bacteria (Turner et al.,
1999) and ITS1(F) (50)-TCCGTAGGTGAACCTGCGG-30)
ITS4(R) (50-TCCTCCGCTTATTGATATGC-30) for yeast (Op
De Beeck et al., 2014) followed by sequencing and blasting the
resulting PCR products for species identification. Selected isolates
of LAB and yeast were transferred in the VLB strain collection
and further examined in fermentation studies: Hanseniaspora
valbyensis Hs-0302 (Han), Dekkera bruxellensis Br-0115 (Dek),
Saccharomyces cerevisiae Sa-07366 (Sac), Liquorilactobacillus
nagelii La-3804 (formerly Lactobacillus nagelii; Liq), and
Leuconostoc mesenteroides Le-0304 (Leu). Every yeast strain was
combined with every bacterium in co-culture, which led to six
applied combinations.
Media Composition and Preparation
Two different media, herein referred to as basis medium and
modified medium, were used for the fermentation studies. The
basis medium was established on a common recipe for WK
production. In detail, it was composed of the following (per
liter): 1 dry fig (Alesto, Lidl Dienstleistung GmbH & Co. KG,
Neckarsulm, Germany), 60 g of brown cane sugar (Fairglobe, Lidl
Dienstleistung GmbH & Co. KG), and 7.5 mL of lemon juice
concentrate (Solevita Zitrone, Lidl Dienstleistung GmbH & Co.
KG). The modified medium was composed of the following (per
liter): 9 mL of fig syrup (Schoeneberger, Salus Pharma GmbH,
Bruckmühl, Germany), 60 g of brown cane sugar, 7.5 mL of lemon
juice concentrate, and a defined concentration of either X-SEED
KAT YE or X-SEED Peptone (Ohly GmbH, Hamburg, Germany),
a yeast peptone. Both yeast derivatives were of food grade quality.
For media preparation, sugar, dry fig/fig syrup, and YE when
applicable were dissolved in boiling tap water (80% of needed
volume). After being cooled down to room temperature (20◦C),
lemon juice concentrate was added, and batches were filled up to
desired volume with lukewarm tap water.
Experiments
Pre-cultivation
The strains were pre-cultured in two steps. In both steps, yeast
strains were pre-cultured in 250-mL shaking flask at 130 rpm
and 30◦C for 72 h in YE dextrose (YED) medium containing
50 g/L of glucose (AppliChem GmbH, Darmstadt, Germany)
and 10 g/L of YE (SERVABACTERR
, SERVA Electrophoresis
GmbH, Heidelberg, Germany), pH 5.5. LAB were pre-cultured
as stand culture in closed 250-mL bottles at 30◦C for 72 h in MRS
medium according to De Man et al. (1960) from DifcoTM (Becton
Dickinson GmbH, Franklin Lakes, NJ, United States). Pre-culture
1 was inoculated with 1% (v/v) of cryo-culture stocks, and pre-
culture 2 was inoculated with 1% (v/v) of pre-culture 1. Before
the main cultures were inoculated, the microbial suspensions
were washed in 0.9% sodium chloride solution to reduce the
influence of media components. WK grains were cultivated in a
typical matrix [60 g of sucrose (AppliChem GmbH, Darmstadt,
Germany), 100 g of pre-washed grains, and two dried figs per liter,
26◦C] over several 3-day cycles in the back-slopping process.
Testing of Different Combinations of Microbial Strains
In the first part of the studies, fermentations with different
combinations of two microbial strains in co-culture were
investigated. Thereby, 1 ·106yeast cells/mL and 1 ·107LAB/mL
were applied in co-culture. The ratio yeast/LAB (1:10) was
derived from the respective cell concentrations in the supernatant
of WK fermented by grains. For comparison, fermentations
with WK grains were investigated simultaneously. Thereby,
100 g of WK grains per liter was used for the fermentation.
For the investigations, the basis medium, which contained
dried fruits, was used. The fermentations were conducted in
1-L glass bottles with loosely sealed caps at 22◦C (±2◦C)
(room temperature) as stand cultures. At the start of the
fermentation and before sampling, the samples were mixed
by mild horizontal shaking. The sampling was executed once
per day for offline analyses, which included determination of
cell count, high-performance liquid chromatography (HPLC)
analysis, pH measurement, and sensory analysis. After 7 days
of fermentation, the products showed the best obtainable
characteristics and were analyzed toward a wide range of volatile
components (see section “Analyses of Volatile Components”).
The fermentation studies were executed in biological duplicates.
The most appropriate combination of microorganisms was then
applied in further trials.
Testing the Impact of Different Yeast Extracts and
Concentrations During Fermentations With
Leu +Han
In further experiments, the components of the basis medium
were adapted to make the composition more defined,
further called modified medium. Thereby, the impact of
the supplementation of either X-SEED KAT YE or X-SEED
Peptone (Ohly GmbH), a yeast peptone, was tested in the
concentration of 0.2 and 1.0 g/L each. Roughly, these two yeast
supplements differed in their protein content (75% in X-SEED
KAT vs. 67% in X-SEED Peptone) and composition [higher
proportion of short-chain peptides and amino acids in X-SEED
KAT (available at X-SEED product sheets)]. Furthermore, the
modified medium contained fig syrup in a defined volume (cf.
section “Media Composition and Preparation”) to avoid the
usage of whole dried fruits. The sampling was executed once
per day for offline analyses, which included determination
of cell count, HPLC analysis, pH measurement, and sensory
analysis. After 7 days of fermentation, aroma analyses of the final
products took place. The fermentation studies were executed in
biological duplicates.
Five-Liters Fermentation With Water Kefir Grains or
the Co-culture of Leu +Han Under Defined
Conditions
Finally, fermentations in 5-L bioreactors with WK grains as
well as the co-culture combination Leu +Han with the
modified medium were performed. For this purpose, 4.5 L
of modified medium including 1.3 g/L of X-SEED KAT and
4.5 L of modified medium without YE was used in the case
of the co-culture Leu +Han and WK grains, respectively.
Fermentations took place in 5-L BiostatR
B Twin bioreactors
(Sartorius AG, Göttingen, Germany), at 26◦C with a low
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stirring rate of 20 rpm and no aeration. The pH value and
dissolved oxygen (pO2-%-saturation) were measured online via
EASYFERM PLUS VP PH/RX 325 and VISIFERM DO ECS 325
H0 (Hamilton Germany GmbH, Gräfelfing, Germany) probes.
The sampling was executed three times a day for offline analyses,
which included determination of cell count, HPLC analysis, pH
measurement, and once-per-day sensory analysis. Fermentations
were carried out in biological triplicates. Data from identical
sampling times were shown as the mean value of a triple, double,
or single sampling and were marked accordingly.
Analytical Methods
Determination of Total Cell Concentration by Coulter
Counter
The total cell concentration was analyzed by using impedance
measurement (MultisizerTM 3, Beckman Coulter GmbH, Brea,
CA, United States). In the case of samples including grains, only
the liquid fraction was analyzed. Ten microliters of the sample
was diluted in 10 mL of Isoton II, and 50 µL thereof was analyzed
using a 30 µL capillary. With the use of the MultisizerTM 3
Software Version 5.53, the pulse data were converted to size
features. In the case of co-culturing, LAB and yeast strains were
used, which differed in their cell sizes; and thus, a discrimination
of the respective populations was possible. Particles with a size
of 0.6–2.0 µm were considered as LAB cells, whereas particles
with a size of 2.0–10.0 µm were considered as yeast cells based
on cell size distribution in pure cultures, respectively. Their
concentration per mL was calculated. A potential overlap of the
LAB and yeast populations by crossing cell sizes was in neither
case significant and thus was neglected for the determination
of the real population distribution ratio, which was a good
approximation. For the strains used, a chain formation could not
have been microscopically verified in the conditions applied in
the experiments.
Determination of Low-Molecular Sugars, Ethanol,
and Organic Acids
The analyses of low-molecular sugars (in detail glucose, fructose,
and sucrose) and ethanol and organic acids (more detailed
lactic acid and acetic acid) were conducted via HPLC (Knauer
Wissenschaftliche Geräte GmbH, Berlin, Germany) applying
10 µL of sample on a NucleogelR
Ion 300 OA column (Macherey-
Nagel GmbH & Co. KG, Düren, Germany) at 40◦C column
temperature. The separation of the target compounds was
achieved using an isocratic elution with 5 mmol/L of H2SO4
at a flow rate of 0.4 mL/min. The sugar and ethanol detection
was performed in a refractive index detector. Organic acids were
detected via multiple wavelength detector at a wavelength of
210 nm. The residual sugar was defined as the sum of glucose,
fructose, and sucrose.
Analyses of Volatile Components
The final fermented products as well as the respective
unfermented beverage bases were tested for a number of
volatile components and fatty acids, which are described in
detail as follows.
Quantification of Acetaldehyde, Higher Alcohols, and
Acetate Esters
The quantification of acetaldehyde, higher aliphatic and
aromatic alcohols, and acetate esters was determined by
static headspace gas chromatography with flame ionization
detection (HS-GC-FID) according to method 9.39 outlined
in the European Brewery Convention (EBC) (Analytica-EBC,
1997). The GC-FID instrument used was a Shimadzu GC-2010
(Shimadzu Corp., Kyôto, Japan) equipped with a DB-Wax
(60 m ×0.32 mm ×0.5 µm film thickness) from Agilent
(Santa Clara, CA, United States). Quantification of higher
alcohols and acetate esters was reached using butan-1-ol and
phenol as internal standards added at a concentration of
15 mg/L. The calibration ranges of the compounds differed
and were as follows: acetaldehyde (1.3–75 mg/L), ethyl acetate
(1.3–75 mg/L), propanol (2.5–75 mg/L), 2-methylpropan-1-ol
(isobutanol) (2.5–75 mg/L), 3-methylbutyl acetate (isoamyl
acetate) (0.2–10 mg/L), 2-methyl-1-butanol (2.5–75 mg/L), 3-
methyl-1-butanol (2.5–100 mg/L), phenyl ethanol (2.5–75 mg/L),
and phenylethyl acetate (0.05–5 mg/L). The lowest concentration
of the calibration range was defined as limit of quantification
for each compound.
Determination of Ethyl Esters
Ethyl butanoate, ethyl hexanoate, ethyl octanoate, ethyl
decanoate, and ethyl dodecanoate were determined by
headspace–solid-phase microextraction (HS-SPME) gas
chromatography–mass spectrometry (GC-MS). The GC-MS
system consisted of a Shimadzu GC 2010 interfaced with a MS-
QP2010 Plus (Shimadzu Corp.) equipped with a Gerstel MPS 2XL
auto sampler (Gerstel, Linthicum Heights, MD, United States)
for automated HS-SPME sampling. Data evaluation was done
using the GCMSsolution software Version 4.45 SP1 (Shimadzu
Corp.). Esters were extracted from 2 mL of liquid sample using
a 50/30 µm divinylbenzene/carboxen/polydimethylsiloxane
(DVB/CAR/PDMS) fiber (Supelco, Bellefonte, PA, United States).
The column used for chromatographic separation was a HP-5MS
UI column [30 m ×0.25 mm i.d. ×0.25 µm film thickness
from Agilent (Santa Clara)]. The extraction and GC parameters
were used as described by Dennenlöhr et al. (2020a). The mass
spectrometer was operated in selected ion monitoring (SIM)
mode, using the following qualifier and quantifier ions: ethyl
butanoate (m/z= 71 and m/z= 60), ethyl hexanoate (m/z= 88
and m/z= 101), ethyl octanoate (m/z= 88 and m/z= 127),
ethyl decanoate (m/z= 88 and m/z= 73), and ethyl dodecanoate
(m/z= 88 and m/z= 101). The calibration ranges for all ethyl
esters were 5–1,000 µg/L. The lowest concentration of the
calibration range (5 µg/L) was defined as limit of quantification.
Isotopically labeled d5-ethyl hexanoate (m/z= 93 and m/z= 106)
was used as internal standard at a concentration of 100 µg/L.
Determination of Volatile Sulfur Components
The analyses of volatile sulfur components were accomplished
by application of two separate HS-SPME GC-MS/MS methods
that were both run on an Agilent Technologies 7890B gas
chromatograph interfaced to a 7000C Triple Quadrupole
mass spectrometer (Agilent). This GC-MS/MS setup was
equipped with a Gerstel MPS 2XL sampler (Gerstel) for
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automated HS-SPME sampling, and the column used for
chromatographic separation was a HP-5ms Ultra Inert GC
Column (30 m ×0.25 mm, ×0.25 µm film thickness
from Agilent). Agilent MassHunter Workstation Software—
Qualitative Analyzes (ver. B.07.00) was used for data analyses.
Method 1 covered methanethiol, ethanethiol, propane-1-thiol,
and butane-1-thiol. The second method covered the thioesters
and sulfides, namely, S-methyl thioacetate, ethyl thioacetate,
carbon disulfide, diethyl sulfide, dimethyl disulfide, and dimethyl
trisulfide. Sample preparation for the analyses of volatile
thiols was done using the on-fiber derivatization (OFD) assay
as published by Dennenlöhr et al. (2020b). The conditions
during sample preparation and the GC separation were also
as previously published. The mass spectrometer was operated
in electron impact ionization (70 eV) multiple-reaction ion
monitoring (MRM) mode using the following transitions for
quantification and qualification: methanethiol (m/z= 228 →181
and m/z= 181 →161), ethanethiol (m/z= 242 →181 and
m/z= 213 →45), propane-1-thiol (m/z= 256 →181 and
m/z= 214 →181), and butane-1-thiol (m/z= 270 →89
and m/z= 213 →181). Analytes were calibrated in a range
from 5 to 500 ng/L (methyl mercaptan: 100–10,000 ng/L) and
the lowest concentration of the calibration range (5 ng/L for
ethanethiol, propane-1-thiol, and butane-1-thiol and 100 ng/L
for methanethiol) was defined as limit of quantification. 1-
Hexanethiol was used as an internal standard (m/z= 117 →83
and m/z= 298 →117), and it was added to the samples
at a concentration of 100 ng/L at the very beginning of the
sample preparation. Sample preparation for the analysis of
thioesters and sulfides was done under similar conditions as
described for the thiols, while the OFD step was skipped. The
GC temperature program used to separate the components
of interest was as follows: start at 30◦C, ramp with 2◦C/min
to 41◦C, then ramp with 40–140◦C, then ramp with 60–
300◦C. The mass spectrometer was operated in MRM except
for carbon disulfide that was analyzed in SIM with selected
ions m/z= 76 (quantification) and m/z= 78 (qualification).
The MRM transitions used for quantification and qualification
of the remaining components were S-methyl thioacetate
(m/z= 90 →43, no qualifier transition), ethyl thioacetate
(m/z= 104 →43 and 43 →42), diethyl sulfide (m/z= 90 →62
and 75 →47), dimethyl disulfide (m/z= 122 →94 and 94 →66),
and dimethyl trisulfide (m/z= 126 →61 and 79 →45).
Isotopically labeled d6-dimethyl trisulfide (m/z= 132 →82 and
114 →50) was used as an internal standard and was added to the
samples at a concentration of 1 µg/L at the very beginning of the
sample preparation. The calibration ranges of the components
differed and were as follows: carbon disulfide (0.005–0.1 µg/L),
S-methyl thioacetate (0.025–20 µg/L), ethyl thioacetate (0.005–
4µg/L), diethyl sulfide (0.005–2 µg/L), dimethyl disulfide
(0.005–4 µg/L), and dimethyl trisulfide (0.005–1 µg/L). The
lowest concentration of the calibration range was defined as limit
of quantification for each component.
Quantification of Short- and Medium-Chain Fatty Acids
Short- and medium-chain fatty acids, herein referred to
as fatty acids, were determined by HS-SPME-GC-FID.
The GC-FID system used was a Shimadzu GC-2010
(Shimadzu Corp.) equipped with an Agilent CP Wax 58
FFAP (50 m ×0.32 mm ×0.32 µm). HS-SPME sampling was
done using a Gerstel MPS 2XL auto sampler (Gerstel) equipped
with an 85 µm polyacrylate fiber (Supelco). To extract the fatty
acids, 2 mL of aliquots of the liquid samples was acidified by
addition of 80 µL of 1 M HCl in 10-mL amber headspace vials.
To enrich components in the headspace above the sample, sealed
headspace vials we incubated for 15 min at 50◦C (500 rpm),
followed by an extraction for 15 min at 50◦C. The loaded fiber
was then desorbed for 1 min at 250◦C using a split ratio of 2.
A temperature program starting at 60◦C, followed by a ramp of
17◦C/min to 150◦C, and followed by a ramp of 8◦C/min to 220◦C
was used to separate the target analytes. The calibration ranges
were as follows: butanoic acid (0.15–12 mg/L), 3-methyl butanoic
acid (0.05–4 mg/L), pentanoic acid (0.03–2.4 mg/L), hexanoic
acid (0.05–4 mg/L), octanoic acid (0.1–8 mg/L), decanoic acid
(0.04–3.2 mg/L), and dodecanoic acid (0.03–2.4 mg/L). The
lowest concentration of the calibration range was defined as limit
of quantification for each component. 4-Methyl pentanoic acid
was used as internal standard; it was added to the samples at a
concentration of 3 mg/L prior to acidification with HCl.
Sensory Analyses
Three trained experts in tasting of sour fermented beverages
analyzed sensory properties of the fermentation products. For
this purpose, a descriptive and evaluative analysis scheme for
sour fermented beverages established at the VLB was used.
After sampling, all beverages were stored in closed screw-cap
tubes for a maximum of 2 days at 4◦C and brought to room
temperature shortly before tasting. Samples were tasted in a
non-blinded manner.
RESULTS
In the following, the fermentation progress of the trials
examining different microbial combinations (section “Different
Lactic Acid Bacteria–Yeast Combinations for Producing Water
Kefir Beverages”) as well as adapted media (sections “Using Fig
Syrup Compared With Dry Figs Based on Fermentation With
Water Kefir Grains” and “Effect of Different Yeast Extracts and
Concentrations on the Performance of Leu +Han During Water
Kefir Fermentation”) aiming for a more defined WK production
process is shown. Finally, 5-L bioreactor fermentations applying
the appropriate microorganism combination compared with
fermentations with original WK grains were performed (section
“Five-Liters Bioreactor Fermentation With Water Kefir Grains
and Defined Co-culture Leu +Han”).
Different Lactic Acid Bacteria–Yeast
Combinations for Producing Water Kefir
Beverages
Firstly, systematic experiments to reduce the microbial
complexity were conducted. Therefore, combinations of
one lactic acid bacterium and one yeast strain were applied in
basis medium against the usage of grains as a control. The focus
was on the production of a lactic acidic beverage, which should
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preferably contain a low amount of alcohol besides an overall
balanced sensory perception.
Figure 1 demonstrates the differences of cell growth and
pH changes during 7 days of fermentation caused by different
microorganism combinations. Firstly, the usage of WK grains
led to different courses in pH and the concentrations of cells in
the liquid phase, metabolites, and volatile components compared
with defined combinations (cf. Figures 2,3). After at least 4 days,
the residual sugar was close to zero, and the alcohol content
increased to a mean value of finally 24 g/L (3.0 Vol.-%) by the
usage of grains, which is shown in Figure 2. In mean, 2 g/L
of lactic acid and 1 g/L of acetic acid were produced. The acid
concentrations were higher than those produced by the co-
cultures. This led to the fastest pH decrease resulting in a final
pH value of 3.5 displayed in Figure 1C.
The grains showed the fastest rise of bacterial cell growth
compared with the defined co-cultured bacteria cells (1 vs. 2 days
to end exponential growth phase). However, at the end of the
fermentation, similar bacterial cell concentrations were reached
(∼1·108cells/mL). In contrast, lower final yeast cell counts
of 7.64 ·106cells/mL than in co-culture (∼2.5 ·107cells/mL
on average), which showed similar progress of the yeast cell
counts, were observed. In the co-culture, the bacteria cell
growth depended on the combined yeast. Consequently, the
combinations including Dek showed the lowest yeast cell growth
compared with the other combinations but the fastest bacteria
growth in case of co-culture, which resulted in an amount
of 1.8 g/L of lactic acid (cf. Figure 2) and led to low pH
values of 3.81. In conjunction with a low sugar consumption
(1.2% observed sugar reduction), a low amount of ethanol
was produced during the fermentation processes including Dek
(<2.0 g/L; ∼0.25 Vol.-%). The low cell growth of Dek in the
beginning allowed a faster growth of the LAB. These observations
indicate an interaction between the used LAB and Dek regarding
the competition for nutrients and differ from the interaction of
other used yeasts, Han and Sac.
Combinations with Han and Sac led to ethanol concentrations
of ∼6 g/L (∼0.76 Vol.-%), which was sensory recognizable in
the case of Liq +Han. The combination of Leu +Sac showed
the lowest pH decrease to finally 4.3 (cf. Figure 1). This is
in accordance with the lowest observed acid production of
∼1 g/L of lactic acid (cf. Figure 2), and the sensory evaluation,
in which only a weak acid was perceptible (cf. Figure 4).
Figure 4 demonstrates less pronounced typical WK-like sensory
attributes—low sourness, carbonization (in case of combinations
with Leu), and fruitiness for instance. Sac seemed to have
been inhibiting or have less promoting effects on Leu, which
could be concluded from the lower bacteria cell concentration
(6.57 ·107cells/mL) compared with that of the other co-cultures
(>1·108cells/mL) and the resulting lower acidity due to less
produced lactic acid.
Figure 3 shows the concentration of different volatile
components in the unfermented basis medium as well as the
final fermented WK products after 7 days. The unfermented basis
medium containing dried figs, sugar, and lemon juice concentrate
contained a few volatile components in a detectable range. These
were smaller fatty acids as well as low amounts of methanethiol
and related sulfurous components (cf. Figures 3C,E,F). In the
fermented products, concentrations of the volatile components
were generally higher but varied from product to product. The
volatile profile of WK fermented by usage of grains differed
from that observed for WK from co-cultures. Within the WK
resulting from co-cultures, volatile profile differed, and this is
attributed to the combination of different microorganisms. Ethyl
acetate was the most dominant form of volatile component
and was detected in the products containing the yeasts Han
and Sac in combination with Liq and Leu, respectively, but did
not occur in a detectable range in the product using grains.
The detected amounts of higher aliphatic alcohols and esters
differed depending on the used combination (cf. Figures 3A,B).
It is conspicuous, that either grains or microbial co-cultures
produced certain components of these higher aliphatic alcohols
and esters. The combination of Dek +Liq as well as Dek +Leu
led to no formation of those alcohols. It is of particular note
that the latter combination led to the highest amount of fatty
acid ethyl esters, which led to the fruity characteristics of the
products and which were produced in a much lower quantity
by the other combinations. Figure 3C displays a much lower
FIGURE 1 | Course of bacteria (A) and yeast (B) cell concentration and pH change (C) during WK fermentation with different starter culture combinations. Data are
the mean value of biological duplicates (LAB–yeast combinations) or triplicates (grains). WK, water kefir; LAB, lactic acid bacteria.
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FIGURE 2 | Course of produced ethanol, lactic, and acetic acid and consumed sugar during the WK fermentations with different LAB–yeast combinations (A–F) and
complex grains (G). Shown are the data of biological duplicates (LAB–yeast combinations) or biological triplicates (complex grains) and the corresponding trend
lines. WK, water kefir; LAB, lactic acid bacteria.
amount of fatty acids, especially hexanoic acid as well as octanoic
acid, in the fermented products after 7 days compared with
the unfermented basis medium. These components have been
metabolized by the cells. Although the components butanoic
acid and 3-methylbutanoic acid are the most dominant fatty
acids, their concentration was either lower than or the same as
concentration in the medium.
In contrast to fruity aroma components, thiols and
sulfurous components were found in a detectable range
in all combinations as well as in the basis medium, which
is displayed in Figures 3E,F. Thereby, the most abundant
component was methanethiol, which acts as a precursor for
further sulfurous components such as dimethyl disulfide and
dimethyl trisulfide (Kinzurik et al., 2020), and was formed by
the microbial combination consisting of Dek +Liq as well as
Dek +Leu mostly. Other sulfurous components were produced
by the usage of Liq in combination with the yeast Han as well as
Sac in the highest amount, e.g., ethanethiol as well as dimethyl
disulfide. The specific aroma profile of the products led to a
specific sensory taste as well, which is demonstrated in Figure 4.
Although the combinations of Dek with Liq and Leu (orange
lines in Figure 4) showed a high potential in the expression
of aimed WK-like attributes, the usage of these combinations
was evaluated as not appropriate for beverage production.
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FIGURE 3 | Concentrations of different selected volatile components in the unfermented basis medium and beverages produced with different LAB–yeast
combinations and WK grains after 7 and 4 days of fermentation, respectively. The quantified aroma-relevant analytes included higher aliphatic alcohols and esters
(A,B), fatty acids (C), fatty acid ethyl esters (D), and thiols and other sulfur components (E,F). The detection limit is specified for the components for which no
detection was possible. The analytes pentanoic acid, dodecanoic acid, ethyl hexanoate, and propane-1-thiol (all not shown) were not detected in any sample. Data
of the fermented products are the mean value of biological duplicates ±mean deviation. WK, water kefir; LAB, lactic acid bacteria.
After 7 days, the final products were lactic and citric acidic,
full-bodied, mildly fruity, and having a dominant fig flavor.
However, these products showed a strong off-flavor, which
was describable as “mustily,” “stuffy,” and “stale” (not shown
in the sensory panel). This can be attributed to the applied
yeast, Dek.
Further, the blue lines in Figure 4 display the well-
balanced composition between sourness and sweetness of the
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FIGURE 4 | Sensory properties of products fermented with different LAB–yeast combinations after 7 days of fermentation. Shown are the mean data of two samples
produced in biological duplicates. The rating scale of the descriptive analysis (A) ranges from 1 (imperceptible) to 5 (very pronounced). The overall rating of the taste
(B) ranges from 1 (very good) to 5 (bad taste). LAB, lactic acid bacteria.
final products fermented by Han +LAB after 7 days. These
results determined the final fermentation duration of 7 days.
In contrast, the fermented products by using the yeast Sac
showed both lactic and acetic acid taste represented by green
lines in Figure 4. Apart from the desired product properties,
a highly pronounced sulfurous taste and smell occurred during
fermentation, especially in combinations with Liq. The products
smelled and tasted like rotten eggs. The most promising products
were fermented by the yeast Han. Thereby, the combination with
Liq showed stronger alcoholic and sulfurous taste as well as less
sourness than in combination with Leu.
Summarizing, the fermentations with the co-cultivation of
LAB and yeasts lasted longer to get a final product than the
fermentations with grains. The fermentation with WK grains was
faster and led to a more complex taste and a higher diversity in
perceptible sensory properties but resulted in a higher content of
alcohol in the final product. By using defined starter cultures in a
co-cultivation, the formation of ethanol was lower and might be
more controllable than by fermentations with grains.
Sensory-wise, the combination of Leu +Han was preferred by
the panelists, which was based on a well-balanced characteristic
between sourness (lactic acid) and sweetness and the presence
of desired attributes such as fruity and carbonized. Based on the
results, the combination was chosen for further trials.
Using Fig Syrup Compared With Dry Figs
Based on Fermentation With Water Kefir
Grains
In order to implement more defined media components in the
production process, investigations with fig syrup were executed.
For this purpose, different volumes of fig syrup were tested to
get the most appropriate concentration equal to the usage of one
dry fig per liter. In a test row, basis media were investigated,
which differed in the volumes of liquid syrup. Sugar and lemon
juice concentrate, as was used above, were added, and sensorial
tests were performed. In conclusion, 9.0 mL of fig syrup per
liter corresponded to the usage of one dry fig per liter regarding
the taste as the basis. Based on this, fermentations with WK
grains with the chosen amount of fig syrup compared with
the usage of dry figs were performed, and results can be seen
in Supplementary Figure 1. In this context, the fermentations
showed similar behavior in cell growth, pH value decrease, and
metabolic activity as well as sensory properties. Thus, fig syrup
was used in following studies.
Effect of Different Yeast Extracts and
Concentrations on the Performance of
Leu +Han During Water Kefir
Fermentation
In order to avoid the noticeable presence of sulfurous
components in the final product when applying defined starter
cultures, two different YEs, developed for LAB cultivation
especially, were used as a sufficient supplement of nutrients. The
fermentation performance of the microorganism combination
Leu +Han without the supplementation of YE was not as good
as in the first study (cf. Figures 1,2). This might be because
of the replacement of dry fig (used in experiments shown in
Figure 1) with fig syrup (experiments shown in Figure 5) and
an associated lack of nutrients. It was concluded that fig syrup
was not sufficient as the only source of nutrition in the case of
the here-applied co-cultivation for the WK production. On the
contrary, a positive effect of adding YE to the medium for the WK
production with two microbial strains was apparent, which was
demonstrated by a higher concentration of LAB compared with
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FIGURE 5 | Course of cell concentrations (A,B), pH value (C), residual sugar (D), produced lactic acid (E), and ethanol (F) during the WK fermentation by Leu + Han
with different types and concentrations of supplemented YE. Data are the mean value of biological duplicates. WK, water kefir; YE, yeast extract.
the fermentation without YE (Figure 5A). Thereby, increasing
the YE concentration led to a faster growth of LAB. Accordingly,
Figure 5C shows a faster decrease of the pH based on the
added YE, especially 1 g/L of X-SEED KAT. Furthermore,
the final pH was lower than the values observed during the
fermentation with grains. These high pH decreases are because
of the high yield of lactic acid concentrations and can be seen
in Figure 5E. Lactic acid reached values higher than 7 g/L using
1 g/L of YE, whereas the application of 0.2 g/L of YE led to
∼2 g/L. Concluding, the amount of produced lactic acid differed
between different applied concentrations of YE. In contrast, the
yeast cell growth was not influenced by different applied YEs
and their concentrations (Figure 5B); thus, the yielded ethanol
concentration differed remarkably after 3 days of fermentation
except for the usage of 1 g/L of X-SEED KAT YE with a slightly
higher concentration (Figure 5F).
Like previously shown, the fermentations with the
combination Leu +Han without adding YE differed when
using fig syrup or dried fruits. These results (cf. Figure 5)
were confirmed by the analyses of the volatile components
(Figures 3,6). That is why further results are compared
with those of the previously described trials, in which
dried figs were used.
The comparison of the volatile components, displayed in
Figure 3 (basis medium) and Figure 6 (modified medium, 0 g/L
of YE), in the two main media largely showed similarities.
Differences occurred in the amount of thiols and sulfurous
components. Methanethiol was twice as high in the modified
medium with fig syrup regardless of the additional usage of
YE media compared with the medium with dried figs, and the
associated component ethanethiol was 10-fold higher, which is
displayed in Figures 6E,F, respectively.
The analyses of the volatile components of the products
fermented by Leu +Han with the usage of different
concentrations of YE and the replacement of dried figs by
fig syrup showed a smaller amount of esters and higher
aliphatic alcohols, e.g., acetaldehyde and ethyl acetate and can
be seen in Figure 6, compared with the former experiments
(cf. Figure 3). The formerly solely detected aliphatic alcohol—2-
phenylethyl acetate—was under the range of detection without
the supplementation of YE, whereas with the addition of YE,
half as much was formed in maximum than observed as shown
in section “Different Lactic Acid Bacteria–Yeast Combinations
for Producing Water Kefir Beverages.” Thereby, the higher
the YE concentration, the higher the formed amount was.
In contrast to the lower amount of some fermentation by-
products, in detail, higher aliphatic alcohols as well as esters
(part A and B), the amount of fatty acid ethyl esters was
similar or increased. This was associated with the usage of 1 g/L
of KAT YE, compared with the former experiments without
YE and dried figs, and comparable with the amounts in the
unfermented media.
The fatty acid concentration in the products during the
supplementation of YE decreased similarly to the product
previously described, and no influence of different YEs as well
as concentrations was observed. Interestingly, the concentration
of butanoic acid of the fermented product without YE was the
highest in all experiments at about 24 mg/L. The associated rancid
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FIGURE 6 | Concentrations of different volatile components in modified media (references) and in 7-day fermented beverages produced with the Leu + Han
combination and different types and amounts of YE. The quantified aroma-relevant analytes included higher aliphatic alcohols and esters (A,B), fatty acids (C), fatty
acid ethyl esters (D), and thiols and other sulfur components (E,F). The detection limit is specified for the components for which no detection was possible. The
analytes pentanoic acid, dodecanoic acid, ethyl hexanoate, and propane-1-thiol (all not shown) were not detected in any sample. Data are the mean value of
biological duplicates ±mean deviation. YE, yeast extract.
bad taste was clearly identified in the sensory evaluation as well
(cf. Figure 7).
Of particular note during these experiments was the
presence of sulfurous components, recognizable in detectable
volatile components (Figures 3E,F,6E,F) as well as smell
and taste (cf. Figures 4,7). Although most of the analyzed
sulfurous components in the unfermented modified media
were concentrated higher than in the basis medium, their
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FIGURE 7 | Sensory properties of the products fermented with the microbial combination Leu + Han under the investigation of 0, 0.2, and 1 g/L of two different YEs
after 7 days of fermentation. Shown are the mean data of two samples produced in biological duplicates. The rating scale of the descriptive analysis (A) ranges from
1 (imperceptible) to 5 (very pronounced). The overall rating of the taste (B) ranges from 1 (very good) to 5 (bad taste). YE, yeast extract.
amount was reduced by the addition of YE, except for
methanethiol, which was in the same range. The concentrations
of ethanethiol were 5- to 10-fold lower as well as the
formed amount of dimethyl disulfide, which was about 10-
fold lower, except for the addition of 0.2 g/L of X-SEED
Yeast Peptone. Thereby, the highest amount of dimethyl
disulfide was produced during the trials, which was still ∼30%
lower than those without YE. The detected concentration of
dimethyl trisulfide in the product without YE was similar
to the former experiments, and reduced to levels under
the range of detection with the addition of YE, except
for the addition of 0.2 g/L of X-SEED Yeast Peptone,
which led to 3-fold higher concentrations of this. This
reduction in sulfurous components was also observed in
the sensory analysis of the products and is displayed in
Figure 7.
The final products showed improved sensory properties,
recognizable in a higher pronounced sourness, more full-bodied
products, and more fruitiness as compared with the study
without YE in co-cultivation (dotted blue line in Figure 7).
Thereby, the expression depended on the applied YEs and
their concentration. The supplementation of the tested YEs
avoided the production of noticeable sulfurous components at
the end of the fermentation except for the usage of 0.2 g/L
of X-SEED Peptone Yeast Peptone, as shown in the dashed
yellow line in Figure 7. Concluding, the usage of 1 g/L
of YE of both X-SEED KAT and X-SEED Peptone showed
the best final products after 7 days of fermentation. That
is why the next investigations were performed under the
supplementation of X-SEED KAT YE with the microorganism
combination Leu +Han. Through the fact that no YE off-
flavor was detectable in taste and smell, a slightly higher
concentration amounting to 1.3 g/L of YE was used in
following studies.
Five-Liters Bioreactor Fermentation With
Water Kefir Grains and Defined
Co-culture Leu +Han
In order to estimate the reproducibility of the WK production
by co-cultivation compared with WK grains, fermentations in
5-L bioreactors were performed (Figure 8 and Supplementary
Figure 2). For the fermentation, fig syrup was used, and in case of
co-cultivation, 1.3 g/L of X-SEED KAT YE was used additionally
as tested before. Figure 8 shows the averaged values of triple,
double, or single sampling.
It is of particular note that the grain-based fermentations
gave desirable sensorially similar products after 3, 6, and 5 days,
whereas beverages produced by co-cultures were sensorially
similar after 7 days of fermentation each. This was not
obvious from the measured data but is of importance for
later industrial application. The reached cell concentrations
of yeast and bacteria were higher in co-cultivation than
during the fermentation with grains (∼3-fold for bacteria
and 10-fold for yeast). Incidentally, the yeast concentration
decreased after 3 days of fermentation by using grains, which
is most probably reasoned by the low stirring rate and the
impeded homogenization caused by an increasing grain mass
during fermentation. The gray lines in Figure 8 show a
similar pH progress in fermentation by using grains and co-
cultivation of the two microorganisms until the fourth day
of fermentation. Afterward, the pH of the grain fermentation
stagnated, whereas the pH in co-culture further decreased to a
final value of 3.14.
The usage of grains led to higher ethanol concentrations
(23 g/L at 3 days; ∼3 Vol.-%) than the co-cultivation of
Leu +Han, accompanied by a nearly complete consumption
of the sugar at the fourth day of fermentation, which is
comparable with the fermentations in section “Different Lactic
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FIGURE 8 | Off-line data of WK fermentations in 5-L bioreactors with grains (A,C) and the defined co-culture Leu + Han (B,D). Fermentations were conducted in
biological triplicates. Data from identical sampling times are shown as the mean value of a triple (full-filled mark), double (half-filled mark), or single (unfilled mark)
sampling. WK, water kefir.
Acid Bacteria–Yeast Combinations for Producing Water Kefir
Beverages” in 1-L scale. After 6 days of fermentation with
co-cultures, the amount of ethanol increased to 3.3 g/L
(0.42 Vol.-%), and to a final value of 7 g/L on average
after 1 week of fermentation rapidly. When comparing the
three fermentations with co-cultures, the sugar consumption
as well as the ethanol content in the end deviated (14.8 vs.
2.7 and 3.5 g/L), although staying below 3 g/L until 6 days
of fermentation, which is why the mean value of ethanol
was that high. The lactic acid concentration reached higher
values by co-cultivation than by the fermentation with grains
(4.98 vs. 1.33 g/L), which was recognizable in the taste of
the final products.
Additional online analyses of the pH, dissolved oxygen,
and redox value are illustrated in Supplementary Figure 2.
In summary, these data confirm the fermentation processes
shown in Figure 8. The essential differences, namely, that the
sugar is consumed faster when using grains and proportionally
fewer organic acids are formed from it, is confirmed by
the online curves. It also illustrates the rapid setting of
anaerobic conditions in both approaches. The dissolved oxygen
was consumed within the first 2.5 h when applying grains,
whereas the usage of Leu +Han resulted in full oxygen
consumption after 8 h of fermentation. This was largely
due to the activity of the yeasts in the beginning of
the fermentation, whereby the defined one only contained
one yeast strain.
Figure 9 shows the mean scores of sensory properties of
biological triplicates at the best time of each single fermentation
depending on the best achieved sensory properties as stated
above (3, 6, and 5 days for grain fermentations; 7 days for co-
culture fermentations). The final products of the fermentation
with the co-cultivation of Leu +Han (blue dashed line in
Figure 9) showed more desirable sensory properties in line
with the study aim of a balanced low-alcoholic beverage than
the product produced by the fermentation with grains (red
line in Figure 9). Thereby, a higher value of sourness as well
as a fruitier taste was observed (Figure 9). Furthermore, the
products were more full-bodied and not that much alcoholic
than the products fermented by using grains. Further, no off-
flavor based on sulfurous taste or smell was observed in any
of the products.
DISCUSSION
The aim of this study was to expand the knowledge about the
impact of different microbial consortia representing different
degrees of complexity on the characteristics of WK. Therefore,
LAB and yeasts isolated from WK grains were used for
fermentations as defined starter cultures in co-cultivation against
the original grains as a control. The focus of the trial was
firstly to reduce the complexity of WK fermentations in order
to provide a more controllable and reproducible process and
secondly to monitor differences in cell growth, the corresponding
metabolization of sugars, production of organic acids, and the
perceptible taste in combination with chemical analysis of the
microbial volatile components.
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FIGURE 9 | Sensory properties (A) and rating (B) of products fermented with the combination of Leu + Han as well as WK grains in 5-L bioreactors. Shown are the
mean data of biological triplicates at the best time of each single fermentation depending on the best achieved sensory properties: Leu + Han at 7 days of
fermentations. Grain trial 1 (f1) at 3 days, trial 2 (f2) at 6 days, and trial 3 (f3) at 5 days of fermentation. The rating scale of the descriptive analysis (A) ranges from 1
(imperceptible) to 5 (very pronounced). The overall rating of the taste (B) ranges from 1 (very good) to 5 (bad taste). WK, water kefir.
Water Kefir Production With Complex
Grains and Defined Starter Cultures
Based on previous internal studies regarding isolated defined
strains from traditional WK grains (data not shown),
combinations of two different LAB, L. nagelii (Liq) and
L. mesenteroides (Leu), as well as three different yeasts,
H. valbyensis (Han), D. bruxellensis (Dek), and S. cerevisiae
(Sac), were applied within the present study. Comparative
fermentations with six microorganism combinations in
co-cultivation as well as kefir grains were performed.
In summary, the fermentations with grains were much faster
than in co-cultivation, which could be explained by a high
adaptation of the higher number of microorganisms in grains
among one another in contrast to only two species that might
not be able to fully compensate for all microbial interactions
(Figure 1). This is supported by data from Laureys et al. (2021),
who also found that WK liquor as a fermentation starter to
progress slower as compared with the usage of grains. Metabolic
exchanges, which can act as a power source as well as a supply
of microbial growth factors, were reported before (Prado et al.,
2015), whereby the mutual interactions can appear directly as
well as indirectly. In a consortium of several different microbial
strains, a possible undersupply of nutrients can be compensated,
whereas in co-cultivation, the nutrition exchange is limited to
the present microbial strains. That is why the production of
WK by using a preferably small number of microbial strains is
possible but is less self-regulating and thus requires more process
knowledge. However, the conducted fermentations with WK
grains resulted in a final product characterized by ethanol levels
of around 20 g/L as well as higher amounts of higher alcohols
compared with co-culture trials, which could be observed by
Laureys and de Vuyst (2014) as well. In line with Chen et al.
(2009), who declared that the application of co-culture processes
for the WK production could sidestep the intense formation of
alcohols, the beverages produced with defined co-cultivations
showed significantly lower ethanol formation in the present
study. The resulting effects could not be traced back to the
performance of one single microorganism in the applied co-
cultivations, but rather to microbial interactions.
Combinations including S. cerevisiae showed the lowest
growth of the investigated LAB in co-cultivation, especially in
the case of co-cultivated L. mesenteroides, compared with the
other applied yeasts, which resulted in the lowest produced
amount of lactic acid in association with the lowest observed
pH drop (Figures 1C,2). This resulted in less pronounced WK
typical characteristics. In accordance with this, Stadie et al. (2013)
observed a poorer growth of L. nagelii in co-cultivation with
S. cerevisiae compared with the co-cultivation with the yeast
Zygotorulaspora florentina in their investigations of metabolic
activity and symbiotic interactions of LAB and yeasts isolated
from WK. Similar observations were described by Bechtner et al.
(2019). They investigated proteomic analyses of L. nagelii in
the presence of S. cerevisiae isolated from WK. In contrast,
the study of Jin et al. (2019) did not demonstrate a negatively
affected growth rate of Lactobacillus plantarum by the presence
of S. cerevisiae but a possible inhibition of yeast by L. plantarum,
which is recognizable in a lower growth rate of the population
compared with the single culture of the yeast. It is obvious
that a generalization of the microbial interactions during the
co-cultivation of a lactic acid bacterium with S. cerevisiae as
well as other yeast species is not possible and has to be
examined individually.
Different gas chromatographic methods were conducted
to quantify volatile components in the fermented products.
Laureys and de Vuyst (2014) found ethyl acetate, isoamyl
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acetate, ethyl hexanoate, ethyl octanoate, and ethyl decanoate as
the most prevalent volatile components in their investigations
of the microbial species diversity, community dynamics, and
metabolite kinetics of WK fermentation with the usage of grains.
Except for ethyl hexanoate, these components were produced
in different amounts during their fermentations with the tested
combinations, whereby ethyl acetate was the most prevalent
component (Laureys and de Vuyst, 2014). Walsh et al. (2016)
pointed out a correlation between Saccharomyces spp. and
esters’ production during their investigations regarding microbial
succession and flavor production in the fermented dairy beverage
kefir. In accordance with Walsh et al. (2016),S. cerevisiae
combined with L. mesenteroides led to the highest amount of
ethyl acetate in the present study. The usage of D. bruxellensis
showed a different behavior in the formation of ethyl acetate,
whereby in combination with L. nagelii no formation and in
combination with L. mesenteroides, a low formation of ethyl
acetate occurred. The presence of higher aliphatic alcohols
after 7 days of fermentation was observed, whereby a high
variety especially during the usage of grains occurred. The
combination of H. valbyensis and L. nagelii led to the highest
detectable amounts of these followed by grains and the usage of
S. cerevisiae. The combination containing Dek formed a lower
amount of higher aliphatic alcohols and no amount of esters in a
detectable range. Compared with the other co-cultures, the lowest
degradation of fatty acids occurred, which may be attributed to
a lower fermentation activity of Dek. In contrast, the associated
ethyl esters were present in the highest amount, which led to
fruity characteristics of the products. This could be explained by
a high noticeable fig taste after 7 days and, consequently, less
pronounced sensory properties of these products due to a lower
fermentation performance of D. bruxellensis. This could be due
to slightly lower growth rates of D. bruxellensis in the microbial
combinations with L. nagelii and L. mesenteroides compared
with the other tested combinations. Former studies including
D. bruxellensis showed a similar behavior regarding a slow growth
rate in the beginning of fermentation (e.g., Abbott et al., 2005),
which might promote the LAB growth rate and increase the
beginning acidification. Thus, the combinations including LAB
and Dek had a specific effect on the fermentation performance
favoring LAB growth and acidification. Furthermore, a strong-off
flavor, which was describable as “stuffy” and “stale,” was observed
in these products. In the wine industry, Dek is well-known for
the production of the observed flavors and thus considered as
a wine-spoilage yeast. Among other studies, this observation
was mentioned by Lambrechts and Pretorius (2019) in their
investigations of yeasts’ importance to wine aroma in context
of wine contamination with D. bruxellensis. Whereas in other
contexts the combination of Dekkera and LAB was advantageous
[e.g., in a commercial alcohol production process described by
Passoth et al. (2007)], in the case of the present study, the usage of
this yeast in co-culture was not appropriate for WK production.
In contrast to the presence of fruity notes, in all products
except for the combinations with D. bruxellensis, a sulfurous
off-flavor in taste and smell was observed. This was more
pronounced when using L. nagelii than L. mesenteroides, which
led to the conclusion that the production of these components
might have been influenced by the presence of the lactic
acid bacterium in co-culture. Accordingly, this observation was
confirmed by the volatile component analysis. The unfermented
medium already contained small amounts of methanethiol,
ethanethiol, diethyl sulfide, and dimethyl disulfide most probably
originating from the figs. In the fermented products, however,
high amounts of methanethiol as well as ethanethiol and
dimethyl disulfide, ethyl thioacetate, and dimethyl trisulfide
occurred, which share the thiol precursor methanethiol (Landaud
et al., 2008;Kinzurik et al., 2020). Landaud et al. (2008)
described the formation of these thiols due to the degradation of
sulfur/carbon bonds of methionine or cysteine derivates during
their investigation of the formation of volatile sulfur components
and metabolism of methionine and other sulfur components in
fermented food. Particularly hydrogen sulfide, methanethiol and
ethanethiol as volatile sulfurous components are often sensed
as garlic, rotten eggs, onion, and fermented cabbage (Moreira
et al., 2002;Landaud et al., 2008). These components have a
negative impact on the beverage aroma, when they are present
in an amount higher than their perception thresholds. This is
especially observed in wine and often is being traced back to
enzymatic formation resulting from yeast metabolism (Moreira
et al., 2002). The component H2S as an undesirable possible by-
product during the alcoholic fermentation of S. cerevisiae caused
by the conversion of cysteine, can further be converted from
ethanol to ethanethiol (Landaud et al., 2008), which was in a
detectable range during all co-cultivations as well as during the
usage of grains. The quantities of the components methanethiol,
ethanethiol, dimethyl disulfide, and dimethyl trisulfide were
the highest of all tested sulfurous components, whereby the
combination of L. nagelii and H. valbyensis as well as S. cerevisiae
showed the highest formation.
Irrespective of the occurred off-flavors, the combination of the
yeast H. valbyensis and the lactic acid bacterium L. mesenteroides
showed the highest potential in order to develop a water kefir
beverage based on the usage of defined microbial strains. This
combination in co-cultivation led to the best final product
with high-pronounced attributes, such as lactic acidic, full-
bodied, fruity, and little carbonized. In this context, the sensory
characteristic of the final products played a crucial role in the
evaluation of an appropriate combination of microorganisms.
Overall, no undesirable microbial interactions were evidenced
during this microbial combination, which affected the growth
rates as well as microbial metabolisms of these participating
microorganisms adversely; thus, this combination was used for
further investigations.
Adaption of the Fermentation Basis
Medium by Supplementation of Yeast
Extract and Substitution of Dried Figs by
Fig Syrup
Regardless of the type of the chosen starter cultures, in addition
to a carbon source (mostly raw cane sugar or pure sucrose), most
dried fruits (e.g., figs or cranberries) are used for the production
of WK. Although the latter serves as a source of amino acids,
vitamins, and minerals, the small applied amount of dried fruits
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Köhler et al. Microbiology and Bioflavoring of Water Kefir
compared with the richness in nutrients of fresh fruits leads to a
WK medium relatively poor in nutrients (Laureys et al., 2018).
One step in the process development in this study aiming for
a more defined product was the replacement of dried figs with
liquid fig syrup that is well-defined (see Manna-Feigen-Sirup
product sheets). Pretests were used to set the concentration in
such a way that it corresponded to the previously proven use of
dried figs in terms of sensorial and chemical parameters during
fermentation, in which 9 mL of fig syrup per liter sugar water
was considered as appropriate. The executed fermentations with
grains and syrup did not differ to the usage of dried figs in their
main performances (Supplementary Figure 1). However, the sole
use of sucrose and fig extract in co-cultivation led to a nutritional
undersupply of the starter cultures and in particular of the applied
lactic acid bacterium (L. mesenteroides), which was expressed in
a reduced bacterial growth rate and acid formation (see Figure 5;
0 g/L of YE). This led to an unbalanced product with poor sensory
ratings, for instance, high sweetness as well as a high-pronounced
characteristic fig flavor and low acidity, which were perceived
as unpleasant in this combination (Figure 7; 0 g/L of YE).
Consequently, in the course of the investigations, two essential
factors could be identified that are crucial for the production
process of this beverage under defined conditions: efficient
nutrient supply and prevention of the formation of sulfurous
metabolic products, which can be caused by yeast metabolism,
especially in the case of an undersupply of assimilable nitrogen
components (Song et al., 2020).
In order to overcome nutritional limitations in co-
culture approaches, the supplementation of YE in different
concentrations was investigated. Vardjan et al. (2013) described
a decreased biochemical activity as well as no growth of various
microorganisms, which were separated as pure cultures for
milk kefir production, in their studies on the characterization
and stability of lactobacilli and yeast microbiota in kefir
grains. In order to overcome these issues, they mentioned an
improvement by the addition of YE to the growth medium,
which was done in the present study as well. The X-SEED KAT
YE contains a high content of free amino acids and is rich in
B vitamins and minerals, whereas the X-SEED Peptone yeast
peptone contains almost 80% of the available amino acids in
the form of small peptides. The application of YEs as shown
here (Figures 5,6) promoted growth rates of LAB, which led
to higher acidity, and was associated with a higher lactic acid
formation and pH drop of the products as compared with
fermentations without the supplementation of YE. This was also
recognizable in well-balanced sensorial properties of the final
products, e.g., a higher acidity and more full-bodied profiles
(Figure 7). Thereby, the extent of the improved performances
was dependent on the concentration of the applied YE: the higher
the concentration, the better the fermentation performance of
the LAB as well as the yeast. However, there was no clear
preference in either of the yeast derivatives, concluding that
L. mesenteroides was able to use the nitrogen in the form of
free amino acids as well as in the form of peptides equally well.
Contrarily, no promotion of the growth rate of H. valbyensis by
adding YE during the co-cultivation with L. mesenteroides was
detectable at all.
Laureys et al. (2018) investigated the influence of different
fruits or YE/yeast peptone on the WK fermentation process
using grains. During their studies, different fermentation
characteristics, indicated by formation of ethanol (21.3 g/L
using figs; 26.1 g/L applying a mixture of YE and peptone)
and lactic acid production (2.83 g/L using figs; 2.06 g/L
applying a mixture of YE and peptone) among others were
observed (Laureys et al., 2018). Interestingly, within this
study, no excessive production of ethanol was observed by
the supplementation of YE applying the co-cultivation of
H. valbyensis and L. mesenteroides (e.g., ∼1.2 g/L of ethanol using
1 g/L of X-SEED KAT YE). A lower production of fermentation
by-products as well as sulfurous metabolic components was
observed, which is most likely based on the yeast metabolism.
The concentration of methanethiol was in the same range
comparing fermentations without YE and with the usage of
dried figs (Figures 3,6). However, the amount of the associated
metabolites, for instance, ethanethiol as well as dimethyl
disulfide, has been reduced by about 10-fold (Figures 3,6).
This was recognizable in smell and taste. The final products
including YE exhibited no sulfurous aroma in smell and taste,
except for the usage of 0.2 g/L of X-SEED Peptone yeast peptone.
Accordingly, this product showed the highest amount of thiols
and sulfurous components detected via gas chromatographic
analysis after fermentation.
Concluding, the addition of at least 1 g/L of YE, especially
the applied X-SEED KAT YE, promoted a nutritional supply
and might balance a lack of nutrition, which avoids the
production of high amounts of sulfurous components when
defined starter cultures are used. Furthermore, this effect allowed
the replacement of dried figs with fig syrup. These steps led to a
more defined process of the WK production.
However, the concentration of YE could be increased in
order to decrease the fermentation time and therefore the
production process bearing in mind avoiding noticeable yeasty
flavor components in smell or taste. The usage of X-SEED KAT
YE showed slightly higher lactic acid concentrations (Figure 5E)
in the development of a WK-like beverage by co-cultivation
consisting of one LAB and yeast, respectively, and thus, further
investigations with 1.3 g/L of X-SEED KAT YE were conducted.
Characterization of Water Kefir
Fermentation Based on Grains and
Defined Co-cultures in 5-L Bioreactors
The development of a defined process for WK production
instead of using complex grains enables the possibility to produce
a WK-like beverage by using the co-cultivation of one LAB
and yeast. In order to compare the production of WK with
grains with the usage of defined co-cultivation under well-
monitored and scalable conditions, 5-L bioreactor fermentations
were performed in triplicates (see Figure 8 and Supplementary
Figure 2). Fermentations performed by a small number of
participated microorganism cultures are aimed for because of
their higher opportunity to control and guide the process and
the properties of the final product (Chen et al., 2009). Further,
for a large-scale production, a stable microbial community,
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Köhler et al. Microbiology and Bioflavoring of Water Kefir
which does not change over time, is crucial (Vardjan et al.,
2013). Additionally, possible contaminations might be much
earlier detectable as compared with a non-defined complex
consortium, allowing for early counteractions as well. Although
the fermentation with two defined starter cultures is still a
complex process, and interactions within the processes are still
not fully understood, the fermentation of co-cultures showed
a better-defined and more controllable process. This led to
a more reproducible production process compared with the
fermentation with WK grains, which showed high fluctuation
regarding the time when the beverage had the best sensory
properties (3, 5, and 6 days; cf. section “Five-Liters Bioreactor
Fermentation With Water Kefir Grains and Defined Co-culture
Leu +Han”). The usage of WK grains has been a traditional
method for many years, which is similar to the milk kefir
production. However, the industrial production of WK by using
grains is challenging. There are only few opportunities to control
the process (Chen et al., 2009;Laureys and de Vuyst, 2017),
which leads to a lower reproducibility and might end in high
alcoholic products, which were observed in the present study.
The fermentations with grains showed a high formation of
ethanol during the first 3 days of the fermentation with a
final amount of 23 g/L. Laureys and de Vuyst (2014) also
noted a high production of ethanol in WK (around 20 g/L),
which was produced by using grains. In comparison, with the
co-culture, only 0.49 Vol.-% ethanol was produced until the
time the beverage was “ready,” which consequently complied
with the legislation in the regions of Germany, Austria, and
Switzerland, which permit an alcoholic content of 0.5 Vol.-
% as a maximum in beverages declared as “alcohol-free” (Das
Eidgenössische Department des Innern, 2005). In contrast, the
product fermented by Leu +Han showed a significantly higher
residual sugar content than did the grain fermentation. As
illustrated in Figure 8,∼80% of the originally inserted amount
of sugar (60 g/L) was found after 6 days of fermentation
corresponding to ∼48 g/L. The World Health Organization
recommends a sugar uptake of free sugars to <10% of total
energy intake corresponding to ∼50 g/L of free sugars for an
adult having a diet of 2,000 kcal (World Health Organization
[WHO], 2015). However, in comparison with common soft
drinks, such as Coca-Cola (14 FO bottle; 111 g/L of sugars),
Pepsi (20 FO bottle; 118 g/L of sugars), or Red Bull (8.4
FO can; 109 g/L of sugars) (Ventura et al., 2011), the WK
produced by co-culturing within the present study would be
considered as a low-sugar-containing beverage according to the
classification of Bandy et al. (2020). With regard to further
potential health benefits associated with fermented drinks in
general, the herein presented product would be competitive on
the beverage market.
Summarizing, the WK beverage gained by co-cultivation with
L. mesenteroides and H. valbyensis differed in the determined
cell count, acidity, fermentation duration, ethanol content,
and consequently its sensory profile as compared with the
grain-based WK. Wang et al. (2016) encapsulated yeast, LAB,
and acetic acid bacteria in liquid core capsules and achieved
a volatile aroma profile close to that of the grain-based
product in milk kefir. WK produced by Laureys and de
Vuyst (2014) by using grains contained 4.9 g/L of lactic acid
and 1 g/L of acetic acid, which is in accordance with the
metabolite concentrations of the developed fermented beverage
without using grains in this study. However, two different
products were gained in the present study, whereby both
products showed typical attributes of WK. In addition, the
co-cultivated products performed slightly better in sensory
terms than those made with grains (cf. Figure 9) regarding
a low alcoholic balanced sour and fruity drink. These first
results are therefore very promising for further beverage
developments to be based on. Under this aspect, the studies
carried out here can be understood as a piece of the puzzle
in order to establish knowledge-based production processes
for complex fermentation products such as WK, so that
these are more controllable and reproducible, especially for
industrial production. The first studies were conducted by
Stadie et al. (2013) as well as Bechtner et al. (2019), which
made symbiotic interactions of co-cultures for WK production
subjects of discussion.
CONCLUSION
The work presented herein described steps from a traditionally
produced process to a defined water kefir fermentation process
applying a co-culture of only one LAB and one yeast, respectively.
It could be shown that the main characteristics of WK—a
fruity, aromatic, and acidic beverage made by fermentation
of characteristic strains—were achieved by the use of two
microbes L. mesenteroides and H. valbyensis, although the
chemical analyses revealed differences compared with those of
the undefined grain origin. However, the defined consortium
outperformed the grains, as considerably lower levels of ethanol
were formed. These results proved the possibility to reduce
the complexity of the fermentation process by keeping the
aimed product characteristics at the same time. The here-
examined defined microbial consortia for the production of WK
caused different product characteristics, opening up further space
for detailed research and in which remaining challenges, e.g.,
a possible optimization of the fermentation time, should be
addressed. Especially, the usage of one LAB and one yeast each
is a promising approach in order to get deeper insights into
microbial interactions during fermentation and simultaneously
avoiding blurring effects due to an uncontrollable number of
associated actors.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
AUTHOR CONTRIBUTIONS
AT, MSc, ML, and SK conducted the experiments. NR was
responsible for the aroma analyses. MSe and SK performed the
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fmicb-12-732019 October 29, 2021 Time: 14:18 # 18
Köhler et al. Microbiology and Bioflavoring of Water Kefir
data analyses. MSe, MSc, and SK drafted the manuscript. ML
reviewed and contributed to the structure and content of the
manuscript. All authors contributed to the article and approved
the submitted version.
FUNDING
Parts of this research was funded by the German Federal
Ministry for Economic Affairs and Energy (program INNO-
KOM; research projects: CoKuFerm, funding no. 49VF200039
and sfAFG, funding no. 49MF190041).
ACKNOWLEDGMENTS
We thank Christian Schubert and Sarah Thörner for the analyses
of volatile components and data evaluation. We also acknowledge
support by the German Research Foundation and the Open
Access Publication Fund of TU Berlin.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fmicb.
2021.732019/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be construed as a
potential conflict of interest.
The reviewer SJ declared a shared affiliation with one of the authors MSc, to the
handling editor at time of review.
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Frontiers in Microbiology | www.frontiersin.org 19 November 2021 | Volume 12 | Article 732019
