fmicb-14-1108961 February 3, 2023 Time: 16:51 # 1
TYPE Original Research
PUBLISHED 09 February 2023
DOI 10.3389/fmicb.2023.1108961
OPEN ACCESS
EDITED BY
Shao Quan Liu,
National University of Singapore, Singapore
REVIEWED BY
Lisa Solieri,
University of Modena and Reggio Emilia, Italy
Graeme Walker,
Abertay University, United Kingdom
*CORRESPONDENCE
Riikka Linnakoski
†These authors have contributed equally
to this work
SPECIALTY SECTION
This article was submitted to
Food Microbiology,
a section of the journal
Frontiers in Microbiology
RECEIVED 26 November 2022
ACCEPTED 23 January 2023
PUBLISHED 09 February 2023
CITATION
Linnakoski R, Jyske T, Eerikäinen R, Veteli P,
Cortina-Escribano M, Magalhães F,
Järvenpää E, Heikkilä L, Hutzler M and
Gibson B (2023) Brewing potential of strains
of the boreal wild yeast Mrakia gelida.
Front. Microbiol. 14:1108961.
doi: 10.3389/fmicb.2023.1108961
COPYRIGHT
© 2023 Linnakoski, Jyske, Eerikäinen, Veteli,
Cortina-Escribano, Magalhães, Järvenpää,
Heikkilä, Hutzler and Gibson. This is an
open-access article distributed under the terms
of the Creative Commons Attribution License
(CC BY). The use, distribution or reproduction in
other forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in this
journal is cited, in accordance with accepted
academic practice. No use, distribution or
reproduction is permitted which does not
comply with these terms.
Brewing potential of strains of the
boreal wild yeast Mrakia gelida
Riikka Linnakoski1*†, Tuula Jyske1†, Ronja Eerikäinen2, Pyry Veteli1,
Marta Cortina-Escribano1, Frederico Magalhães2,3, Eila Järvenpää4,
Lotta Heikkilä1, Mathias Hutzler5and Brian Gibson2,3
1Natural Resources Institute Finland (Luke), Helsinki, Finland, 2VTT Technical Research Centre of Finland Ltd.,
Espoo, Finland, 3Institute of Food Technology and Food Chemistry, Chair of Brewing and Beverage
Technology, Technische Universität Berlin, Berlin, Germany, 4Natural Resources Institute Finland (Luke),
Jokioinen, Finland, 5Research Centre Weihenstephan for Brewing and Food Quality, Technical University of
Munich, Berlin, Germany
Demand for low- or non-alcoholic beers has been growing in recent years. Thus,
research is increasingly focusing on non-Saccharomyces species that typically are
only able to consume the simple sugars in wort, and therefore have a limited
production of alcohol. In this project, new species and strains of non-conventional
yeasts were sampled and identified from Finnish forest environments. From this wild
yeast collection, a number of Mrakia gelida strains were selected for small-scale
fermentation tests and compared with a reference strain, the low-alcohol brewing
yeast Saccharomycodes ludwigii. All the M. gelida strains were able to produce
beer with an average of 0.7% alcohol, similar to the control strain. One M. gelida
strain showing the most promising combination of good fermentation profile and
production of desirable flavor active compounds was selected for pilot-scale (40 L)
fermentation. The beers produced were matured, filtered, carbonated, and bottled.
The bottled beers were then directed for in-house evaluation, and further analyzed
for sensory profiles. The beers produced contained 0.6% Alcohol by volume (ABV).
According to the sensory analysis, the beers were comparable to those produced by
S. ludwigii, and contained detectable fruit notes (banana and plum). No distinct off-
flavors were noted. A comprehensive analysis of M. gelida’s resistance to temperature
extremes, disinfectant, common preservatives, and antifungal agents would suggest
that the strains pose little risk to either process hygiene or occupational safety.
KEYWORDS
Finnish forests, low-alcohol beer, tree, non-Saccharomyces, brewing yeast
1. Introduction
Beer is the most consumed alcoholic beverage in the world. The global beer market reached
US$ 554.65 billion in 2021 and it is expected to grow annually by compound annual growth
rate (CAGR) 10.15% from 2022 to 2025 (Statista,2022a). The global non-alcoholic beer market
is expected to grow even faster, annually by CAGR 13.44% during the same time. In 2021, the
non-alcoholic beer market generated revenue of roughly US$ 25.28 billion (Statista,2022b).
According to Eurostat (2021) in 2020 almost 32 billion liters of beer containing alcohol and
1.4 billion liters of beer containing less than 0.5% alcohol or no alcohol content at all were
produced in the EU. Non-alcoholic and low-alcoholic beers had a volume share of more than
10 percent in Spain and Germany in 2019, making them the countries where the beverages were
the most popular (Statista,2021b).
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The beer market is driven by the increasing demand for premium
and craft beers. Those are preferred because of their authenticity,
taste, and aroma (Morgan et al.,2020). At the same time, increasing
awareness of the negative health effects of alcohol consumption and
rising adoption of healthy lifestyles, including weight management,
are driving the growth of non- and low-alcohol beer market
(Chrysochou,2014;Gernat et al.,2020). Additionally, during the
last few years technological developments have improved the flavor
of non-alcoholic beer (Gernat et al.,2020;Melton,2022), further
increasing the market growth since many consumers are looking for
products that taste similarly to their alcoholic equivalents (Muller
et al.,2020).
The growing interest in low- and non-alcoholic beers has inspired
research into novel or improved methods of production (Bellut and
Arendt,2019). These methods can broadly be described as physical
or biological methods. Of these, the former approach, which includes
membrane filtration and vacuum distillation, requires considerable
investment in facilities. Biological methods, which include modified
mashing procedures or limited fermentation processes are generally
considered more cost effective and can also produce beers that more
closely match those produced in conventional brewing. From the
various methods that involve limiting fermentation, the use of non-
conventional yeasts is gaining considerable interest (Methner et al.,
2019;Statista,2021a). These yeasts often utilize only simple sugars
(glucose and fructose), but are unable to consume the dominant
sugars found in brewer’s wort (maltose and maltotriose). With such
yeasts there is therefore a natural limit to the extent of fermentation
that occurs, and consequently the amount of alcohol that forms.
Such yeasts have been isolated from multiple sources including other
fermentation systems like kombucha or sourdough (Bellut et al.,
2020), or even from the native brewery microbiota (Krogerus et al.,
2021).
Many different yeast species have been considered for the
production of low-alcohol beers, though they have invariably been
members of the Ascomycota (Yabaci Karaoglan et al.,2022),
presumably due to their ability to tolerate alcohol and anaerobic
conditions. One exception is the yeast species Mrakia gelida, a
member of the Basidiomycota. This species has shown potential for
brewing (De Francesco et al.,2018), with beers rated well by a sensory
panel compared to a reference brewing yeast, Saccharomycodes
ludwigii, a yeast which has been used for over a century for the
production of low-alcohol beers (Bellut and Arendt,2019).
When introducing new strains to the brewing process, it is
advisable to assess the potential risks to the process or to occupational
safety. Relevant parameters in this regard include potential for
production of yeast biofilm, resistance to disinfectants, preservatives
and antibiotics, and ability to grow at different temperatures. All
these properties affect the functionality and handling of yeasts in
production. Strains that are better able to produce biofilm, grow at
higher temperatures (e.g., at 37◦C), and tolerate preservatives as well
as disinfectants and antibiotics are potentially a greater risk to the
brewing process and to human health.
A particularly interesting feature of M. gelida is that the species is
psychrophilic. Low temperatures are typically employed in breweries
when producing lager beers. The low temperature tolerance of
M. gelida makes it compatible with current commercial brewing
processes. In contrast, many non-conventional yeasts tested for
low-alcohol beer production are mesophilic and require relatively
warm temperatures for fermentation. Maintaining low temperatures
during the whole brewing process also limits the likelihood of
contamination–a considerable risk with low-alcohol beers, which
contain relatively high levels of sugar as well as alcohol concentrations
that do not inhibit contaminant growth. The fact that M. gelida
does not grow at room temperature (i.e., above ca. 15◦C) is also
potentially an advantage in terms of brewery hygiene, i.e., simple
steam treatments are sufficient for cleaning vessels, and there is little
risk of cross-contamination within breweries.
The objectives of this study therefore were, firstly, to assess
the brewing potential of a range of M. gelida strains of Finnish
forest environment origin via small-scale fermentations and select
a strain suitable for scaled-up fermentations; and secondly, perform
a pilot-scale fermentation with the selected strain and a reference
strain (S. ludwigii), after which bottled beers would be prepared for
sensory evaluation.
2. Materials and methods
2.1. Collection and maintenance of yeast
isolates
The M. gelida strains were collected during a larger survey
conducted in Punkaharju experimental forests in Finland in late
March 2019. In total, nine hardwood tree species and 10 individuals
of each tree species were sampled. The yeasts were isolated using a
modified method by Sniegowski et al. (2002), which has been used
previously for the isolation of yeasts from tree bark. In the field, bark
samples approximately 0.5 ×0.5 cm from around the bases of trees
(at breast height, i.e., 1.3 m on the stem from tree stump) were cut
using a sterile scalpel and tweezers and placed in 2.5 mL Eppendorf
tubes containing sterile tap water.
The samples were stored in a freezer and transferred to laboratory
and stored for up to 3 days at 4◦C before enrichment culturing. In
the laboratory, the bark samples were transferred to 50 mL Falcon
tubes containing 5 mL of a sterile liquid enrichment medium [Yeast
Malt (YM) Broth] consisting of 3 g yeast extract, 3 g malt extract,
5 g peptone and 10 g dextrose per liter. The YM Broth was acidified
to pH 3.5 prior to autoclaving. The tubes were then incubated for
up to 3 weeks at 5◦C with shaking (80 rpm) and inspected for signs
of proliferation of yeasts or other cells. Aliquots of 10 µL from each
of these liquid enrichment cultures were then streaked on solid YM
agar medium containing the same ingredients as the YM Broth and
20 g agar per liter. Plates were incubated for several days at 5◦C and
examined regularly for any growth of yeast or other colonies. Purified
cultures were obtained by picking colonies and re-streaking them
onto to fresh YM Agar medium. The cultures are stored at the culture
collection of Natural Resources Institute Finland (Luke), Helsinki.
2.2. Identification of yeasts
The isolated strains were identified based on the internal
transcribed spacer (ITS) gene region sequences. The DNA was
extracted using PrepManTM Ultra Sample preparation reagent
(Applied Biosystems, Foster City, CA, USA) following the
manufacturer’s protocol. The ITS gene region was amplified,
and sequencing performed using a primer pair ITS1-F (Gardes
and Bruns,1993) and ITS4 (White et al.,1990). The PCR reaction
mixture (25 µL volume) contained 0.2 µL of Phusion High-Fidelity
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DNA Polymerase (2 U µL−1) (Thermo Fisher Scientific, Waltham,
MA, USA), 4 µL of Reaction Buffer (5 ×Phusion HF), 0.4 µL of
dNTPs (10 mM), and 0.5 µL of each primer. PCR reactions were
performed as follows: an initial denaturation step at 98◦C for 30 s,
followed by 35 cycles of 5 s at 98◦C, 10 s at 55◦C and 30 s min at 72◦C,
and a final chain elongation at 72◦C for 8 min. PCR products were
visualized under UV light after staining 5 µL aliquots with ethidium
bromide and separation on a 1% agarose gel. Successfully amplified
products were purified using the Exo-SAP protocol: 20 µL of the
PCR product was mixed with 8 µL of Exo-SAP [5 µL of Exonuclease
I (20 U µL−1) (Fermentas, Vilnius, Lithuania) and 100 µL of Shrimp
Alkaline Phosphatase (1 U µL−1) (Roche Diagnostics, Indianapolis,
USA) in a 1,000 µL reaction mixture] and incubated at 37◦C for
15 min and following immediate incubation at 80◦C for 15 min. The
sequencing was conducted at Macrogen Europe.
The forward and reverse sequences were assembled using
Geneious 10.2.6 (Biomatters Ltd., Auckland, New Zealand). The
obtained ITS sequences were initially identified using BLASTn
search1(National Center for Biotechnology Information, U.S.
National Library of Medicine, Bethesda MD, USA) in the NCBI
nucleotide (nt) database. The identification of the obtained Mrakia
isolates was further confirmed by three phylogenetic methods:
maximum likelihood (ML), maximum parsimony (MP), and
Bayesian inference (BI) as described earlier by Linnakoski et al.
(2021). The dataset including type sequences and sequences of closed
related sequences was compiled with MEGA v.7 (Kumar et al.,
2016) and aligned using the online version of MAFFT v.7 (Katoh
and Standley,2013) with the FFT-NS-i strategy. The sequence data
generated in this study has been deposited in GenBank (Table 1).
2.3. Selection of yeast strains for brewing
trials
Table 1 shows the M. gelida strains selected for the present study.
2.4. Brewing process
2.4.1. Mashing process
Wort was prepared using malt from Viking Malt Oy (Lahti,
Finland) (90% pilsner malt and 10% Vienna malt). 26 kg of malt
(hammer milled) was used to produce approx. 1 hL of 15◦P wort.
3 L of water were added per kg malt. The mash was supplemented
with 52 mL of 85% lactic acid [this is a standard adjustment for the
brews prepared at VTT. The pH was not adjusted to a level typical
for finished beers (<pH 4.5)], 30 g CaCl.2H2O, 10 g CaSO4.2H2O,
and 53 mg ZnSO4.7H2O. Mash profile was as follows: 48◦C, 30 min;
63◦C, 30 min; 72◦C, 30 min and 78◦C, 10 min. A Meura filter was
used for wort separation. Wort was boiled for 60 min with 113 g
Magnum hops (15% alpha acid, target IBU 45). Hot trub was removed
by whirlpool. The wort was then diluted with deaerated water 50:50
to achieve 7.5◦P prior to the fermentation.
2.4.2. Fermentation process
Strains of S. ludwigii (C-79089, cider isolate, NCYC collection)
and M. gelida were propagated in 50 mL YP medium containing 2%
1https://blast.ncbi.nlm.nih.gov/Blast.cgi
(w/v) glucose. S. ludwigii was grown for 24 h at room temperature (at
about 20◦C; also, from herein when referred to room temperature),
while M. gelida strains were grown for 48 h at 15◦C. 20% (w/v) yeast
slurries were prepared with distilled water for each strain and cell
numbers evaluated with the aid of the Chemometec Nucleocounter.
A total of 100 mL of 7.5◦P brewer’s wort in a 250 mL Erlenmeyer
flask was inoculated at a rate of 10 million cells mL−1. Airlocks were
attached to maintain anaerobic conditions. Fermentations proceeded
at 10◦C for 14 days and fermentation progress was monitored via%-
mass loss from the wort. The S. ludwigii fermentations were carried
out at 10◦C or 20◦C. For 40 L –scale fermentations, S. ludwigii C-
79089 and M. gelida YGW184 were propagated in 1 L Yeast extract,
Peptone, dextrose (YPD) and subsequently 10 L YPD at 15◦C. Yeast
slurries were prepared as before and inoculated into 40 L of 7.5◦P
wort. Fermentations proceeded at 10◦C for 6 days, before maturation
for 8 days at 4◦C.
After maturation, beers were depth-filtered (Seitz EK, Pall
Corporation, Port Washington, NY, USA) and were assessed for
alcohol content, wort density, pH, and yeast content (mass in
suspension). Filtered beers were carbonated to approx. 5 g L−1CO2.
Beers were then transferred to brown, 330 mL bottles and stored cold
(about +10◦C to +12◦C) until sensory evaluation.
2.5. Analytics
The collected samples of wort or beer were centrifuged, and
supernatants used for analyses after manual degassing. The specific
gravity, alcohol level (% v/v) and pH of samples were determined
from the centrifuged and degassed wort and fermentation samples
using an Anton Paar Density Meter DMA 5000 M with Alcolyzer Beer
ME and pH ME modules (Anton Paar GmbH, Austria).
Yeast-derived aroma compounds (acetaldehyde, higher alcohols,
and esters) were determined by headspace gas chromatography
with flame ionization detector (HS-GC-FID) analysis. 4 mL samples
were filtered (0.45 µm), incubated at 60◦C for 30 min and then
1 mL of gas phase was injected (split mode; 225◦C; split flow of
30 mL min−1) into a gas chromatograph equipped with an FID
detector and headspace autosampler (Agilent 7890 Series; Palo Alto,
CA, USA). Analytes were separated on a HP-5 capillary column
TABLE 1 Mrakia gelida strains included in the present study.
Luke
culture number
Species Host tree GenBank
Acc. no
YGW70 Mrakia gelida Quercus robur OM604737
YGW132 Mrakia gelida Sorbus aucuparia OM604738
YGW150 Mrakia gelida Tilia cordata OM604739
YGW172 Mrakia gelida Ulmus laevis OM604740
YGW180 Mrakia gelida U. laevis OM604741
YGW184 Mrakia gelida U. laevis OM604742
YGW279 Mrakia gelida Q. robur OM604743
YGW321 Mrakia gelida Q. robur OM604744
YGW322 Mrakia gelida Q. robur OM604745
YGW335 Mrakia gelida Populus tremula OM604746
YGW344 Mrakia gelida P. tremula OM604747
YGW352 Mrakia gelida P. tremula OM604748
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(50 m ×320 µm×1.05 µm column, Agilent, USA). The carrier
gas was helium (constant flow of 1.4 mL min−1). The temperature
program was 50◦C for 3 min, 10◦C min−1to 100◦C, 5◦C min−1
to 140◦C, 15◦C min−1to 260◦C and then isothermal for 1 min.
Compounds were identified by comparison with authentic standards
and were quantified using standard curves. 1-butanol was used as
internal standard.
Concentration of fermentable sugars (glucose, fructose, maltose,
and maltotriose) in bottled beers was measured by high-performance
liquid chromatography (HPLC) using a Waters 2,695 separation
module and Waters system interface module liquid chromatograph
coupled with a Waters 2,414 differential refractometer (Waters Co.,
Milford, MA, USA). An Aminex HPX-87H organic acid analysis
column (300 ×7.8 mm, Bio-Rad Inc., Hercules, CA, USA) was
equilibrated with 5 mM H2SO4(Titrisol, Merck, Germany) in water
at 55◦C, and samples were eluted with 5 mM H2SO4in water at a
0.3 mL min−1flow rate.
Aldehydes were analyzed as oximes by using a headspace
sampler (Agilent 7697A) coupled with gas chromatograph (Agilent
7890B) and compounds were detected using a Micro Electron
Capture Detector (HS-GC-ECD). Carbonyl compound standards
were 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, hexanal,
furfural, methional, phenylacetaldehyde, and (E)-2-non-enal (Sigma-
Aldrich, St. Louis, MO, USA). A stock solution containing a mixture
of the standard compounds in ethanol was prepared at 1,000 µg
L−1each. The calibration range was 0.5–40 µg L−1and dilutions
were prepared in 5% ethanol. The sum of the peak areas of the two
geometrical isomers (E and Z) was used for calculations. Correlation
coefficient (R2) values were 0.995–0.9999. An aqueous solution of
derivatization agent O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine
(PFBOA) (Sigma-Aldrich) was prepared at a concentration of 6 g
L−1. One hundred microliters of this solution and 5 mL of deionized
water or beer were placed in a 20-mL glass vial and sealed with a
crimp cap (Agilent Technologies Inc., Santa Clara, CA, USA). The
sample/standard vial was then placed in the headspace sampler with
following conditions: sample equilibrium in oven for 30 min at 60◦C,
after which 1 min of injection of sample fill pressurized at 25 psi.
Loop temperature was 100◦C and transfer line was held at 110◦C.
The following GC conditions were applied: HP-5 capillary column,
50 m ×0.32 mm ×1.05 µm (J&W Scientific, Folsom, CA, USA).
Helium was the carrier gas at a flow rate of 1.0 mL min−1and for
ECD, nitrogen make up gas was applied at a flow rate of 30 mL min−1.
The front inlet temperature was 250◦C. The injection was in the split
mode and the split ratio 10:1 was applied. The oven temperature
program used was 40◦C for 2 min, followed by an increase of 10◦C
min−1to 140◦C (held 5 min) and 7◦C min−1to 250◦C. The final
temperature was held for 3 min.
2.6. Sensory analysis
Bottled beer samples (matured as described in Section “2.4.2.
Fermentation process”) were tasted and judged by a trained sensory
panel of seven panelists certified by the Deutsche Landwirtschafts-
Gesellschaft (DLG, Frankfurt, Germany). Tasting was performed in
a dedicated tasting room (individual tasting chambers, white-colored
room, no distracting influences, and brown glasses with three-digit
number labels) to exclude all external misleading factors. The main
flavor impressions were determined at a range from 1 (almost no
perception) to 5 (very high perception). Flavor impressions were
chosen according to Meier-Dörnberg et al. (2017). In addition, a
tasting was performed under the same circumstances with the DLG
scheme, in which the beer is judged by its aroma, taste, carbonation,
body, and bitterness in a range of 1–5, 1 being the lowest value
(negative) and 5 being the highest value (positive).
2.7. Hygiene and safety of strains
These experiments tested the tolerance of M. gelida (YGW 184
strain) to common preservatives [isomerized hop extract IsoHopR
30 IBU (BarthHaas, Nuremberg, Germany), ethanol 5% (v/v) (AaS,
Rajamäki, Finland), benzoate 150 mg L−1(sodium benzoate, Sigma-
Aldrich, Darmstadt, Germany), sorbate 250 mg L−1(potassium
sorbate, Sigma-Aldrich, Darmstadt, Germany), and sulfite 200 mg
L−1(potassium metabisulphite, Brown, Poland)], antibiotics
(Anidulafungin, Amphotericin B, Micafungin, Caspofungin, 5-
Flucytosine, Posaconazole, Voriconazole, and Itraconazole and
Fluconazole) and disinfectant [P3-oxonia active (Oy Ecolab Ab,
Helsinki, Finland)], its ability to grow at different temperatures,
and its ability to form a biofilm in static and agitated conditions.
Known brewer’s yeasts Saccharomyces pastorianus (lager) and
S. ludwigii (non-alcoholic) strains were used as references. M. gelida
is a well-known psychrophile, so tests were carried out at suitable
temperatures (i.e., +1◦C, +4◦C, and +37◦C). At the same time, the
behavior of the reference yeasts under these conditions was tested.
2.7.1. Biofilm-forming potential
Yeast biofilm production was tested using 96-well microplates.
Strains were propagated by taking a loopful of fresh yeast mass
from YPD agar and inoculating into 25 mL of liquid YPD and
incubating for 1 day on a shaker (120 rpm) at room temperature.
Grown cell cultures’ optical densities (OD600) were measured with a
spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan).
Based on the lowest optical density, the yeasts were diluted for
growth. Most wells were filled with 250 µL of 10◦P inoculation
wort, including wells for yeasts and for blank samples. Remaining
wells were empty or filled with water. 2.5 µL of culture was used to
inoculate 10 replicate wells. Growing times were one or 4 days, with
either slight shaking or without agitation. Assays were performed
at 13◦C. After 24 h or 4 days, OD600 values were measured from
each yeast from one well, to be sure there has been growth. Biofilm-
forming potential was assessed by measuring attachment of the cells
to the walls of the wells. The plate was first rinsed with sterile Milli-
Q-filtered water. After which, 300 µL of 0.1% crystal violet solution
was placed in the wells for 5 min. It was then rinsed in the collection
vessel three times with sterile Milli-Q-filtered water. The plate was left
to air-dry for 15 min in a laminar flow cabinet. The remaining crystal
violet, which was still bound to the cells, was dissolved with 300 µL of
95% Etax B (AaS, Rajamäki, Finland). Absorbances of the wells were
measured at 595 nm with a Multiskan EX (Labsystems Oy, Finland).
2.7.2. Temperature tolerance
The ability of the yeasts to grow at different temperatures was
tested using a spot plate technique. Yeasts were propagated by
taking a loopful of fresh yeast mass from YPD agar and inoculating
into 25 mL of liquid YPD for 1 day on a shaker (120 rpm) at
room temperature. Each culture was centrifuged, washed, and re-
suspended to OD600 of 0.1 (2 ×106 cells mL−1) and further diluted
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to concentrations of 0.01, 0.001, and 0.0001 using sterile Milli-Q-
filtered water. 5 µL of each suspension was spotted onto the surface
of a YPD agar plate. The plates were incubated at 37◦C for 3 days, 4◦C
for 2 weeks, and 1◦C for 3 weeks.
2.7.3. Preservative tolerance
The tolerance of the yeasts to common food preservatives was
assessed in microplate cultivations using a Bioscreen C incubator and
plate reader (Labsystems Oy, Finland). Yeasts were propagated by
taking a loopful of fresh yeast mass from YPD agar and inoculating
into 25 mL of liquid YPD for 1 day on a shaker (120 rpm) at room
temperature. The culture was then centrifuged at 9,000 rpm for 5 min
at 4◦C. The pellet was washed with 10 mL of sterile physiological
saline solution (0.9% NaCl). A 20%-slurry was prepared, and cell
density was measured with a NucleoCounter YC-100 (Chemometec,
Denmark). YPD media were adjusted to pH 4 with hydrogen chloride
(HCl) before use. The microplate’s wells were filled with 150 µL YPD
(1% glucose w/v, pH 4) and with 140 µL of one of the following
preservatives to complete these final volumes: isomerized hop extract
IsoHopR
30 IBU (BarthHaas, Nuremberg, Germany), ethanol 5%
(v/v) (AaS, Rajamäki, Finland), benzoate 150 mg L−1(sodium
benzoate, Sigma-Aldrich, Darmstadt, Germany), sorbate 250 mg
L−1(potassium sorbate, Sigma-Aldrich, Darmstadt, Germany), and
sulfite 200 mg L−1(potassium metabisulfite, Brown, Poland).
A total of 20% yeast slurry was diluted to obtain approximately
2,000 cells per well (by using NucleoCounter YC-100 results and
adding dilution at 10 µL per well). The final volume was 300 µL
per well, and the cultivations were carried out at 13◦C with
moderate shaking. Each culture was prepared in triplicate for each
preservative.
2.7.4. Disinfectant tolerance
The tolerance of yeasts to the disinfectant P3-oxonia active
(Oy Ecolab Ab, Helsinki, Finland) was tested as follows. YPD
agar plates were prepared with suspensions with yeast and bovine
serum albumin (BSA) (Sigma-Aldrich, Missouri, United States). One
concentration of BSA represented “clean” conditions (0.3% BSA),
and one represented “unclean” condition (3% BSA). BSA dilutions
were made with sterile milli-Q-filtered water. Yeast suspensions were
prepared by taking a loopful of fresh yeast mass from a YPD agar plate
culture and inoculating into 5 mL of sterile 0.9% NaCl solution. The
yeast suspension was added at 60 µL for both conditions, to 3 mL of
0.3% BSA and to 3 mL of 3% BSA. Each yeast was added to test agar
plates in duplicate for each sample time. Before adding disinfectant,
both suspensions with yeast were spread on test plates for controls.
The disinfectant was added at 300 µL (at a final concentration of
0.3%) for both conditions, and timing started after addition. Times
were 2.5, 5, 10, and 20 min. At each time point, solutions were mixed
well by vortexing [to obtain a condition similar to the cleaning in
place (CIP) method] and transferred to an agar plate. The plates were
incubated at 25◦C, except for M. gelida at 10◦C, for three to 5 days,
depending on the growth yield. Resistance to the P3-oxonia was
assessed qualitatively depending on the relative growth of the yeasts.
2.7.5. Antibiotic resistance
Mrakia gelida is a psychrophile and does not therefore pose
a direct risk to human health. To further ensure handling safety,
the species’ resistance to antibiotics was assessed. Resistance
was evaluated using the YeastOne YO10 (Thermo Fischer)
test plate according to the manufacturer’s instructions. The
antifungal test included nine commonly used antifungal agents:
Anidulafungin, Amphotericin B, Micafungin, Caspofungin, 5-
Flucytosine, Posaconazole, Voriconazole, and Itraconazole and
Fluconazole. The test was performed at 13◦C for 3 days.
2.8. Statistical analysis
Linear mixed models were used to compare the mean
concentrations of key flavor active volatiles present in the beers
produced by using the M. gelida yeast strain YGW184 against those
produced by the other M. gelida strains and S. ludwigii by using
Dunnett’s pairwise comparison test. The assumption of unequal
variances of treatments was allowed when necessary (based on
a likelihood ratio test) and technical replicates were taken into
account through a random effect. The assumption of normality
of the residuals was studied graphically and was found to be
adequate for all models.
3. Results
3.1. Isolation and identification of the
yeasts
A total of 12 M. gelida strains isolated from the bark of five
different host trees collected in Punkaharju, Finland, were identified
by sequence similarity of the ITS gene region (Table 1). The accurate
identity was further confirmed by phylogenetic analysis. Based on the
phylogenetic analysis of the ITS region, the M. gelida strains collected
in this study formed a clade with high bootstrap support value
with the European isolate of M. gelida (GenBank acc. MK496827)
and the type strain from Antarctica (GenBank acc. NR_163504)
(Supplementary Figure 1).
3.2. Small-scale fermentation
performance
Fermentation progress, as measured by mass loss, was similar
for all M. gelida strains (Figure 1). There was no indication of
any substantial lag phase before fermentation started, as all strains
showed some evidence of fermentation after 20 h. Performance of
the reference S. ludwigii strain at 10◦C was marginally better than
the test strains in the first 72 h of fermentation. This advantage
was not, however, apparent after this time. When the reference
strain was incubated at 20◦C, fermentation was considerably faster
and more efficient. Test strains did not meet the same level of
fermentation despite their reported psychrophilic nature. For test
strains the% mass loss varied from 0.28 to 0.32%. Values were
considerably lower than would be expected with a production yeast
strain, where values of approx. 5% mass loss would be typical.
Results were consistent with only monosaccharide sugars being
utilized.
Alcohol levels were also correspondingly low, with values ranging
from 0.69 to 0.74% Alcohol by volume (ABV). Highest alcohol level
was seen with the reference strain fermentations at 20◦C (0.78%),
consistent with the higher mass loss values seen with this yeast
(Table 2 and Figure 1). pH of the beer was approx. 4.8 for all strains
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FIGURE 1
Small-scale fermentation progress as measured by mass loss from 7.5◦P wort fermented with strains of Mrakia gelida at 10◦C. Reference conditions
include Saccharomycodes ludwigii incubated at 10◦C and 20◦C. Values are means of two independent replicate fermentations (±standard deviation).
(Table 2). This relatively high value is typical for low-alcohol beers
and can readily be corrected to a normal beer pH value (approx. 4.4)
through addition of lactic acid to wort or beer.
Yeast fresh mass was consistent amongst the test strains, with
values all falling in the range of 13.5–16.1 g L−1(Table 2). By contrast,
the reference yeast produced 26 g L−1and 30 g L−1after incubation
at 10◦C and 20◦C, respectively.
All strains maintained a high viability (>90%) indicating, firstly,
that the fermentation conditions were not particularly stressful, and
secondly, that the yeast at the end of fermentation would be suitable
for repitching, i.e., re-use in subsequent fermentations–a standard
brewery practice (Table 2). The S. ludwigii yeast from the 20◦C
fermentation was in a flocculent state which prevented the viability
assay from being carried out in this case.
Analysis of flavor volatiles (acetaldehyde, higher alcohols,
and esters) showed a relatively low level of yeast-derived flavor
compounds in all beers (Table 3). No compounds were present
above normally recognized flavor threshold values (Meilgaard,1975).
Acetaldehyde levels in test beers were close to the flavor threshold
of 25 mg L−1, though the grassy flavor imparted by acetaldehyde
is not generally seen as a positive flavor attribute. Acetaldehyde
values were considerably lower in the reference beers (<1.0 mg
L−1). Based on its slightly higher production of flavor volatiles, the
M. gelida strain YGW 184 was chosen for scaled-up fermentations.
The statistically significant differences in concentrations of flavor
volatiles between the YGW 184 as compared to other strains are
shown in Table 3.
Many wort aldehydes were present at high levels prior to
fermentation, but most were reduced to levels lower than flavor
thresholds in the beers. This was particularly the case for the M. gelida
strains, which were more efficient reducers of aldehydes compared to
S. ludwigii. An important aldehyde, methional (cooked potato aroma)
still above the flavor threshold after fermentation with S. ludwigii but
was below the threshold in all the M. gelida beers (Table 4).
3.3. Pilot-scale fermentation performance
Pilot-scale (40 L) fermentations with M. gelida YGW 184 and
the control strain S. ludwigii were carried out for 6 days at 10◦C.
Fermentation progress was assessed by daily measurement of the
specific gravity, alcohol level (% v/v), pH, and yeast mass (Figure 2).
Fermentation performance was identical in the first 24 h but from
then on, the selected strain of M. gelida outperformed S. ludwigii,
reaching an alcohol concentration of 0.61 versus 0.36% ABV at the
TABLE 2 Alcohol content and pH of Mrakia gelida beers, and yeast fresh
mass and yeast viability after fermentation of 7.5◦P wort for 2 weeks
at 10◦C.
Yeast Alcohol (%
v/v)
pH Yeast fresh
mass (g L−1)
Yeast
viability (%)
YGW 70 0.75 (0.01) 4.81 (0.02) 14.5 (0.26) 94.9 (0.2)
YGW 132 0.71 (0.01) 4.76 (0.00) 14.2 (0.23) 93.0 (1.0)
YGW 150 0.70 (0.00) 4.78 (0.00) 15.6 (0.15) 93.0 (0.0)
YGW 172 0.75 (0.01) 4.78 (0.01) 14.5 (0.10) 93.9 (0.0)
YGW 180 0.73 (0.01) 4.76 (0.00) 15.2 (0.60) 95.2 (1.0)
YGW 184 0.70 (0.00) 4.75 (0.00) 15.6 (0.56) 92.7 (0.0)
YGW 279 0.72 (0.01) 4.96 (0.10) 16.1 (0.11) 91.1 (0.0)
YGW 321 0.71 (0.00) 4.75 (0.00) 14.0 (0.33) 92.4 (0.0)
YGW 322 0.75 (0.02) 4.77 (0.00) 16.1 (0.57) 94.7 (0.9)
YGW 335 0.69 (0.01) 4.77 (0.00) 15.8 (0.97) 92.9 (0.0)
YGW 344 0.71 (0.00) 4.75 (0.00) 13.5 (0.46) 97.2 (0.0)
YGW 352 0.73 (0.01) 4.75 (0.02) 15.9 (1.25) 89.0 (0.2)
S. ludwigii 10◦C 0.76 (0.02) 4.75 (0.00) 25.8 (0.87) 91.1 (0.0)
S. ludwigii 20◦C 0.78 (0.00) 4.82 (0.01) 29.8 (0.99) 94.9
Reference conditions include Saccharomycodes ludwigii incubated at 10◦C and 20◦C. Values are
means of two independent replicate fermentations (range in parenthesis). Viability value for
S. ludwigii at 20◦C could not be calculated due to formation of recalcitrant flocs. The strain in
bold refers to the one that was selected for the larger scale fermentation.
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TABLE 3 Mean concentrations (±range) of key flavor active volatiles present in the beers produced by using the Mrakia gelida yeast strains and
Saccharomycodes ludwigii.
Concentrations (mg L−1)
Yeast Acetaldehyde Propanol Ethyl acetate 2-Methyl-
propanol
3-Methyl-
butanol
2-Methyl-
butanol
YGW 70 20.76 (1.19) b7.68 (0.05) b0.04 (0.00) b9.67 (0.28) b5.35 (0.23) 1.31 (0.05)
YGW 132 19.26 (1.41) b6.95 (0.09) b0.03 (0.00) 6.10 (0.07) b6.47 (0.17) 1.40 (0.01)
YGW 150 19.42 (0.33) b5.92 (0.01) b0.06 (0.02) 6.14 (0.03) 7.57 (0.01) b1.59 (0.02)
YGW 172 b17.58 (0.46) b7.87 (0.01) 0.14 (0.01) 6.36 (0.09) b6.19 (0.09) 1.41 (0.01)
YGW 180 22.39 (1.13) b6.34 (0.01) 0.14 (0.00) 5.92 (0.14) b5.79 (0.13) 1.27 (0.03)
YGW 184 a22.24 (0.69) a8.82 (0.10) a0.20 (0.05) a6.38 (0.28) a8.34 (0.28) a1.33 (0.02)
YGW 279 22.53 (0.04) 8.26 (0.15) 0.14 (0.00) 6.84 (0.21) 9.34 (0.33) 1.49 (0.04)
YGW 321 b16.55 (0.28) b11.1 (0.12) 0.17 (0.03) b8.09 (0.13) 8.96 (0.05) b1.48 (0.02)
YGW 322 20.14 (0.86) 9.12 (0.04) 0.11 (0.06) b11.11 (0.05) b5.99 (0.09) b1.08 (0.02)
YGW 335 17.66 (2.57) 8.32 (0.02) b0.04 (0.01) 6.01 (0.12) 8.41 (0.33) b1.16 (0.02)
YGW 344 19.19 (1.96) b6.51 (0.11) b0.04 (0.00) 5.83 (0.02) b6.03 (0.02) b1.21 (0.00)
YGW 352 b13.91 (1.84) 7.57 (0.59) b0.04 (0.00) 6.81 (0.18) 8.57 (0.63) 1.35 (0.02)
S. ludwigii 10◦Cb0.56 (0.01) b2.25 (0.01) b0.54 (0.00) b10.93 (0.01) b27.63 (0.05) b3.68 (0.01)
S. ludwigii 20◦Cb0.76 (0.07) b2.36 (0.03) b0.59 (0.01) b12.51 (0.13) b20.77 (0.22) b4.63 (0.04)
The values are means of two independent replicates. Statistically significant differences (p<0.05) in concentrations between those of YGW 184 as compared to those of other strains are indicated
with the lower-case letter b. Lower-case letter a refers to the strain that was used in pair-wise comparisons. The strain in bold is the one selected for the larger scale fermentation.
TABLE 4 Concentrations of aldehydes (µg L−1) present in beers, and in the original wort.
Concentrations (µg L−1)
Yeast 2-Methyl-
propanal
2-Methyl
–butanal
3-Methyl-
butanal
Furfural Methional Phenyl
acetaldehyde
Benz-aldehyde
YGW 70 3.7 (0.1) 0.2 (0.0) 0.3 (0.0) 0.2 (0.0) 1.1 (0.0) 0.3 (0.0) nd
YGW 132 5.3 (0.4) 0.3 (0.0) 0.6 (0.0) 0.3 (0.0) 1.5 (0.3) 0.6 (0.2) nd
YGW 150 6.3 (0.5) 0.4 (0.0) 1.3 (0.2) 0.3 (0.1) 2.0 (0.5) 0.7 (0.1) nd
YGW 172 5.1 (0.3) 0.3 (0.0) 0.6 (0.1) 0.3 (0.0) 1.5 (0.1) 0.5 (0.1) nd
YGW 180 5.8 (0.5) 0.3 (0.0) 1.1 (0.0) 0.3 (0.0) 1.2 (0.3) 0.3 (0.1) nd
YGW 184 4.5 (1.0) 0.3 (0.1) 0.7 (0.4) 0.3 (0.1) 1.0 (0.2) 0.4 (0.1) nd
YGW 279 4.6 (0.7) 0.3 (0.1) 0.8 (0.5) 0.3 (0.0) 1.1 (0.2) 0.4 (0.0) nd
YGW 321 3.7 (0.3) 0.2 (0.1) 0.3 (0.2) 0.3 (0.0) 1.5 (0.5) 0.6 (0.3) nd
YGW 322 3.5 (0.9) 0.2 (0.0) 0.4 (0.2) 0.3 (0.1) 1.6 (0.4) 0.8 (0.4) nd
YGW 335 3.9 (1.5) 0.2 (0.1) 0.5 (0.4) 0.3 (0.0) 1.5 (0.5) 0.6 (0.3) nd
YGW 344 5.8 (0.0) 0.4 (0.0) 0.9 (0.1) 0.3 (0.0) 1.3 (0.1) 0.6 (0.2) nd
YGW 352 7.3 (1.4) 0.5 (0.1) 2.4 (0.6) 0.3 (0.0) 3.1 (0.2) 1.5 (0.3) nd
S. ludwigii 10◦C 12.7 (0.8) 1.7 (0.1) 4.7 (0.0) 2.2 (0.0) 6.2 (0.3) 6.9 (0.1) 0.6 (0.0)
S. ludwigii 20◦C 15.6 (2.3) 2.3 (0.0) 5.8 (0.1) 2.3 (0.1) 5.0 (0.2) 7.8 (0.5) 0.7 (0.1)
Wort 72.7 (15.4) 35.3 (1.6) 117.1 (23.2) 106.6 (8.0) 208.1 (6.2) 48.2 (4.0) 3.0
Values are means of two independent replicates. Values in bold are above flavor thresholds (Meilgaard,1975;Saison et al.,2009). Deviation from the mean is indicated by range in parenthesis. nd,
not detected.
end of the fermentation. Relative to the small-scale fermentation,
the performance difference between the two strains was greater.
S. ludwigii appeared to show relatively heavy sedimentation, as
demonstrated by the low yeast mass in suspension during the
fermentation (Figure 2).
3.4. Bottled beer analysis
The concentration of fermentable sugars remaining in the
beers was quantified by HPLC. Neither strain was expected to
consume any of the maltose and maltotriose present in the wort,
however, there were differences in the final concentration between
the strains (Table 5). This can be at least partially explained by
the lower cell count obtained for S. ludwigii during propagation
step (3.34 ×108cells mL−1for S. ludwigii and 13.2 ×108cells
mL−1for M. gelida). This difference reflects in the volume of cell
suspension needed for inoculation (1,198 mL for S. ludwigii and
304 mL for M. gelida). A higher inoculation volume diluted the sugar
concentration in the wort. Besides this, beers produced with M. gelida
beers had no residual glucose present (compared to 1.8 g L−1in
S. ludwigii beers) and half the concentration of fructose relative
to S. ludwigii, consistent with M. gelida’s superior fermentation
performance.
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FIGURE 2
Fermentation of 7.5◦P wort at 10◦CbyMrakia gelida YGW184 and Saccharomycodes ludwigii measured by alcohol formation (A), change in wort pH
during fermentation (B), and change in yeast dry mass in suspension during the fermentation (C).
The concentrations of flavor active aroma compounds in the
beers were generally lower that those obtained in the small-scale
fermentation (Table 6), with the exceptions being acetaldehyde and
phenylethyl acetate in S. ludwigii. However, the concentrations were
still lower than the flavor threshold (Meilgaard,1975). The lower
concentration of flavor compounds was likely explained by the
shorter fermentation time and low temperature.
3.5. Sensory analysis
Beer “S. ludwigii bottled” was evaluated with grades above four
according to the DLG tasting scheme (Table 7). Values below
4 indicate off-flavors. Hence no off-flavors were recognized and
described in beer “S. ludwigii bottled.” Beer “M. gelida YGW 184”
was evaluated with grades above four for the attribute’s purity of taste,
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body, carbonation. Beer “M. gelida YGW 184” had grades of 3.9 for
the attributes aroma (smell) and bitterness. For the attribute aroma
(smell) one taster out of eight tasters gave the grade 3 with the off-
flavor description diacetyl. Some tasters are very sensitive to diacetyl
and can even recognize this sweet-buttery-smelling substance below
the usual detection limit in beer (0.1 mg per liter). Overall, seven out
of eight tasters did not recognize diacetyl or other off-flavors. This
result indicates that consumers would not recognize an off-flavor
for this low alcohol beer “M. gelida YGW 184.” For the attribute,
quality of bitterness of the beer “M. gelida YGW 184” one taster
out of eight tasters gave the grade 3. The quality of bitterness with
a grade 3 means that the bitterness is not harmonic and persists at
the back of the mouth and the tongue. Seven out of eight tasters
rated the quality of bitterness with the grade 4 which means that the
bitterness was harmonious. This result indicates that the bitterness
for this low alcohol beer “M. gelida YGW 184” would be recognized
as harmonious for consumers. The overall sensorial rating of this low
alcohol beer “M. gelida YGW 184” can be regarded as an average
rating for a beer without having any special characteristics and
some minor negative characteristics that are not recognized by an
“untrained consumer in sensorial analysis.”
Results of a sensory scheme with a focus on yeast-derived aromas
according to Meier-Dörnberg et al. (2017) are shown in Figure 3. The
two beers produced with M. gelida YGW 184 (top) and S. ludwigii
(bottom) have similar aroma profiles but differ in certain categories.
Tasters recognized tropical fruity, sweet, and fruity attributes for both
beers. More tasters recognized citrus and floral attributes in beer
S. ludwigii than in beer M. gelida YGW 184. Only single tasters
recognized phenolic attributes and other attributes (e.g., fungoid,
forest floor). The significance level of the following main aroma
attributes was of α= 0.05 according to the method pairwise test
of MEBAK sensory 3.1.1 and DIN EN ISO 5495:2007: fruity, sweet
in beer M. gelida YGW 184 and sweet in beer S. ludwigii. The
presence of the aroma attributes wort-like and honey was tasted in
beer S. ludwigii with a significance level of 0.05, whereas the presence
of those aroma attributes was not significant for beer M. gelida.
An absence of the main flavor spicy (consisting of aroma attributes
clove, juniper, pepper, and cinnamon) was tasted for beer M. gelida
YGW 184 with a significance level 0.05. No other main flavors had
significant results in the sensory analysis according to the mentioned
method. In summary, both beers were very similar with a decent
fruitiness and a remaining sweetness. The aroma attributes honey
and wort-like within the main flavor category sweet were significantly
tasted in the beer produced with S. ludwigii, whereas not in the beer
produced with M. gelida, which indicates that reduction of sweet,
wort-like aromas is more pronounced in M. gelida wort fermentation
than in S. ludwigii wort fermentation. S. ludwigii had higher rates in
citric aroma attributes, which were statistically not significant.
3.6. Hygienic characteristics
3.6.1. Biofilm production
Almost all microbes produce biofilm, and the more complex
the forming biofilms are, the more difficult it is to eliminate them.
The tolerance of microbes to various stressors, such as disinfectants
and antibiotics, increases many times over in the form of biofilm,
which is why mechanical washing is the most effective way to remove
biofilms. Biofilms are viscoelastic and can move and divide, if the
situation so requires, for example, due to a lack of nutrients, and
proceed elsewhere. Thus, knowledge of microbial biofilm production
is important to ensure adequate hygiene management and to know
the risks of production.
After 1 day, M. gelida was better able to form a biofilm when
no agitation was applied during incubation relative to the reference
yeast (see Supplementary Table 1). When agitation was applied, the
strain did not have time to grow at all or the attachment was so
weak that it may have been removed during the rinsing step. Biofilm
TABLE 5 Alcohol, pH, and sugar concentration in the bottled beers
fermented with Saccharomycodes ludwigii and Mrakia gelida for 6 days
at 10◦C.
S. ludwigii M. gelida
YGW184
Alcohol (% v/v) 0.36 0.61
pH 5.1 4.9
Sugars (g/L)
Maltose 31.1 (1.3) 35.1 (1.3)
Maltotriose 11.3 (0.4) 13.0 (0.6)
Glucose 1.8 (0.0) ND
Fructose 1.8 (0.0) 0.8 (0.1)
The beers were matured for 8 days at 4◦C before the analysis. Sugar values are means of two
technical replicates (±standard deviation). ND, not detected.
TABLE 6 Concentrations of the flavor active volatiles present in
the bottled beers.
S. ludwigii M. gelida YGW
184
Acetaldehyde 2.72 (0.34) 1.17 (0.19)
Propanol 0.88 (0.04) 4.58 (0.12)
Ethyl acetate 0.29 (0.03)
2-Me-Propanol 5.10 (0.10) 3.38 (0.05)
3-Me-Butanol 13.50 (0.19) 5.16 (0.02)
2-Me-Butanol 2.06 (0.09) 0.70 (0.00)
3-Me-Butylacetate 0.01 (0.00) ND
Ethyl hexanoate ND ND
Phenyl ethanol 7.22 (0.42) ND
Ethyl octanoate ND ND
Phenylethylacetate 0.03 (0.05) ND
Ethyl decanoate ND ND
Values are means of two technical replicates (±standard deviation). ND, not detected.
TABLE 7 Sensory analysis results according to DLG scheme (2.6), in which
the beer is judged by its aroma, taste, carbonation, body, and bitterness in a
range of 1–5, 1 being the lowest value (negative), 5 being the highest value
(positive); arithmetic mean of results of eight tasters.
M. gelida YGW
184 bottled
S. ludwigii
bottled
Aroma (smell) 3.9 4.3
Purity of taste 4.1 4.5
Body 4.8 4.6
Carbonation 4.9 4.9
Quality of bitterness 3.9 4.0
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FIGURE 3
Comparison of the flavors grouped according to the main flavors (sum parameter) and the respective main aroma attribute for beer produced with the
yeast strains Mrakia gelida YGW 184 (top) and Saccharomycodes ludwigii (bottom).
production after 4 days was intermediate relative to the reference
yeasts under both conditions. Thus, the yield of M. gelida biofilm
does not differ significantly from that of the reference yeasts in these
circumstances. Compared to S. ludwigii,M. gelida poses a lower risk
for biofilm production.
3.6.2. Temperature
Temperature plays a major role in the ability of yeasts to grow
and maintain their function. Here, the yeasts’ ability to grow at low
temperatures was investigated, as well as their ability to grow at
37◦C, a property that influences the risk of infections in humans.
Temperature tests showed M. gelida’s relatively strong performance
in cold conditions. Compared to the reference yeasts, its growth was
significantly higher at 4◦C and 1◦C (refrigerator and cold storage
conditions). S. pastorianus growth at 4◦C was weak and S. ludwigii
did not grow at all. Reference strains showed no growth at 1◦C. Only
S. ludwigii showed slight growth at 37◦C (Supplementary Figure 2).
3.6.3. Preservatives
M. gelida growth was completely inhibited by the common
beverage preservatives sorbate and benzoate. Sulfite delayed growth
by 14.5 h and the growth rate decreased by only 14%. Ethanol
delayed growth by 48 h and the growth rate decreased by 57%. Thus,
M. gelida’s growth is moderately sensitive to ethanol (Figure 4).
M. gelida was clearly more sensitive to the preservatives than the
reference yeasts.
3.6.4. Disinfectant tolerance
S. pastorianus withstood the disinfectant only weakly in the
“unclean condition” at the beginning and was inhibited completely
after 5 min. S. ludwigii had weak growth at all times in unclean
conditions. Both reference yeasts failed to grow in “clean conditions.”
M. gelida showed no tolerance to the disinfectant in either the unclean
or clean conditions, indicating that its use in production does not
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FIGURE 4
Growth curves with IsoHop (IBU), benzoate, ethanol, sulfite, sorbate and control for Mrakia gelida (A),Saccharomycodes ludwigii (B), and S. pastorianus
(C). The x-axis indicates time (h), and the y-axis shows the yeast absorbance at 600 nm (au).
involve high risks of contamination if proper hygiene management
is maintained (Supplementary Figure 2).
3.6.5. Antibiotic resistance
All nine antifungals inhibited M. gelida’s growth completely, so
there are no clinical risks with M. gelida. Reference yeasts were not
tested in this research.
4. Discussion
Mrakia species have been found in arctic, glacial, and alpine
habitats such as soils of Antarctica (Di Menna,1966;Xin and Zhou,
2007), glaciers (Depriest et al.,2000;de García et al.,2007), ice and
melting waters (Margesin et al.,2002;Turchetti et al.,2008). In
Antarctica, Mrakia and closely related genera have been found as
dominant micro-organisms in the soils (Margesin and Miteva,2011).
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They have remarkable adaptation strategies to survive in the cold
environments, such as cold-active enzymes which are also of
biotechnological potential (Buzzini et al.,2012). In the context of
low-alcohol brewing, this psychrophilly is a particularly advantageous
property. Lowering temperature is a routine means to reduce
microbial spoilage, and M. gelida’s ability to actively ferment at low
temperature permits a continuously cool production process–from
yeast pitching through to packaging. In contrast, many maltose-
negative yeasts that have been used, or considered for use in brewing,
are mesophilic and require relatively high fermentation temperatures.
S. ludwigii, the yeast most commonly used for the production of low-
alcohol beers, is for example quite cold-sensitive and grows poorly
at 15◦C or below (Johansson et al.,2021). In the present study,
the cold sensitivity of S. ludwigii was particularly apparent in the
pilot-scale fermentation. At the relatively warm temperatures used
in breweries for fermentation with maltose-negative yeasts, there is a
distinct risk of contamination. This risk may be mitigated through
the use M. gelida or other cold-tolerant yeast at low fermentation
temperatures. Use of maltose-negative yeasts for low-alcohol beer
brewing is an approach that is feasible for smaller brewing companies
without the capacity to invest in dealcoholization equipment (Yabaci
Karaoglan et al.,2022). The approach is made even more attractive
through the use of a cold-tolerant yeast, as many smaller breweries are
capable of maintaining cold temperatures throughout fermentation
and downstream processing.
All M. gelida strains tested here were capable of fermentation in
7.5◦P wort. Results indicate that the yeast were only able to utilize the
monosaccharide sugars present, as shown previously be De Francesco
et al. (2018). This is supported by low alcohol levels of approx. 0.7%
ABV. Reaching a target value of 0.5% ABV can be readily achieved
by dilution or by using a weaker wort, e.g., 6.5◦P. Likewise, future
iterations of the process could involve lowering pH of the wort
through the use of lactic acid or similar process with the aim of
producing a beer with pH value of <4.5. In the current work, the pH
level of the wort was not adjusted to levels typical of finished beers
(<pH 4.5) and therefore beers had a quite high pH value of approx.
4.7.
The feasibility of using any new yeast strain for brewing is
dependent on the flavor profile of the beers produced. To this end,
both chemical and sensorial analyses were performed on the resultant
M. gelida beers. Levels of yeast-derived higher alcohols and esters
were low in both test and reference strains, indicating minimal
addition of fruit/floral flavors to beers. Yeast contributes to beer
flavor not only by producing volatile aroma compounds, but also
by removing the aldehydes that contribute to the worty or grainy
flavors, and which are common in beers generated using limited
fermentation. In this regard, all yeast strains tested were efficient
reducers of wort aldehydes. The test strains appeared to be more
efficient at aldehyde reduction than the reference yeast S. ludwigii.
Results indicate that the M. gelida isolates may be particularly
suited for the removal of the “worty” flavors. Of note here is that
concentrations of methional, which typically imparts a cooked potato
aroma, were above the flavor threshold in S. ludwigii beers, but below
this concentration in the M. gelida beers. The particularly “clean”
flavor profile of beer produced with M. gelida means that this could
serve as a base beer for further development. Recent research has
indicated that high-quality, low-alcohol beers can be created through
blending with full-strength beers, or the judicial use of dry hopping
(Rettberg et al.,2022). Such approaches could likewise be used to
further develop the taste profile of M. gelida beers.
Stress tolerance is an important characteristic of any strain
adopted for use in brewing. This is because, firstly, a batch of yeast
must be able to survive and function through multiple fermentations
and, secondly, because a yeast should be controllable and not pose
a risk to brewery operations or to the health of those handling the
yeast. Regarding the former case, there was no indication that the
fermentation conditions employed in these trials were stressful for
the yeast. All test strains were capable of growth and maintained a
high viability (>90%)–making them suitable for re-use in subsequent
fermentations. Regarding control M. gelida, the yeast was found to
be sensitive to the disinfectant Oxonia (peroxyacetic acid). Sensitivity
was greater than that of the reference yeasts S. pastorianus and
S. ludwigii.M. gelida was likewise more sensitive to a range of
preservative compounds relative to the reference yeasts. Sorbate
and benzoate prevented growth completely. Growth was possible
in the presence of sulfite and ethanol, but in both cases the onset
of growth was delayed significantly, and growth rate was reduced.
There is therefore no indication that preservative tolerance of
M. gelida would pose any risk to brewery operations. Additionally,
the biofilm-forming potential of M. gelida was found to be limited,
and lower than that of S. ludwigii. It can be assumed therefore
that no additional tolerance could be achieved through this means.
The strain’s tolerance of hop bitter acids is a potential advantage
if increased hop dosage, or dry-hopping, is considered as a way
to enhance the flavor profile of the beer. With respect to the risk
to human health, there is no known case of a Mrakia species
causing a disease in humans. Infection by M. gelida is in any
case precluded by the species’ inability to grow at human body
temperature. Furthermore, M. gelida strain showed no ability to grow
when challenged with common anti-mycotic agents, meaning that an
infection would be readily treatable. In the laboratory safety studies
with the beers produced with different Mrakia species (Mrakia
robertii sp. nov., Mrakia blollopis sp. nov., and Mrakiella niccombsii
sp. nov.), no abnormalities in the laboratory rats were observed
(Thomas-Hall et al.,2010). It can be said therefore that with this
yeast the risk to the brewery operations or operators is minimal.
On the contrary, the sensitivity of the yeast to even moderately high
temperatures may be a disadvantage as there is a risk to viability when
a cold-chain in the brewery cannot be maintained.
Mrakia gelida is not on the GRAS (i.e., Generally Recognized
As Safe) list of organisms updated by the United States Food and
Drug Administration (FDA), nor on the qualified presumption
of safety (QPS) list of the European Food Safety Authority
(EFSA) (last updated 7/2022). Earlier, there has not been enough
information about the characteristics of the species nor information
to offer because breweries have not been familiar with the
potential of M. gelida. If the consumer-safety/acceptability of
products fermented with M. gelida were to be assessed, the
following aspects will be considered: According to EFSA, the
species must not be resistant to antifungal substances. Another
aspect of acceptability is that the yeast can be used in the
production processes, if there are no longer any viable cells in
the final product. On the other hand, the number of living
cells in the final products made with M. gelida should not be
a problem as it cannot grow at the temperatures of the human
digestive tract.
Results of this study support the contention of De Francesco
et al. (2018) and Yabaci Karaoglan et al. (2022) that M. gelida
is a promising species with respect to low-alcohol brewing.
We further show that this potential does not appear to be
Frontiers in Microbiology 12 frontiersin.org
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Linnakoski et al. 10.3389/fmicb.2023.1108961
strain-specific, as all M. gelida strains tested here had similar
functional properties. Additional tests showed that any risk
contamination or infection was negligible. Results suggest that the
species is suitable for the production of clean tasting low-alcohol
beers at temperatures that would prohibit the growth of contaminant
yeasts or bacteria. Fermentation with M. gelida therefore represents
a reliable, safe, and relatively low-cost method for producing low-
alcohol beers, and may be particularly attractive for those brewers
that do not have access to dealcoholization equipment.
Data availability statement
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found in the article/Supplementary material.
Author contributions
RL and PV: conceptualization, strain isolations, and original draft
preparation. RL and MC-E: strain identifications. FM: fermentation
trials and analysis of fermentation data. BG: conceptualization,
original draft preparation, supervision, and methodology. RE:
hygienic assays, analysis of data, and original draft preparation.
MH: sensory and aroma analysis on the bottled beers, original
draft preparation, and conceptualization. EJ and LH: original draft
preparation. TJ: funding acquisition, project administration, and
editing and finalization of the manuscript. All authors contributed
to the article and approved the submitted version.
Funding
This project was funded by the LukeLEADS strategic research
funding (project “YeastsGoWild”).
Acknowledgments
Sirpa Tiikkanen, Juha Puranen, and Tuija Hytönen, and
Punkaharju staff at Luke are thanked for their skilful technical
assistance in the laboratory and field. Tuomo Tupasela, Juha-Matti
Pihlava, Susanne Heiska, and Henri Vanhanen are thanked for
expertise in in-house sensory analysis of beers at Luke. Eero Mattila
and Aila Siltala are thanked for their help with brewing and beer
analyses. Liisa Änäkäinen and Atte Mikkelson are thanked for their
assistance with beer analytics. Mr. Janne Kaseva is thanked for
designing and implementing the statistical analysis at Luke.
Conflict of interest
RE, FM, and BG were employed by VTT Technical Research
Centre of Finland Ltd.
The remaining 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.
Publisher’s note
All claims expressed in this article are solely those of the
authors and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the reviewers.
Any product that may be evaluated in this article, or claim that may
be made by its manufacturer, is not guaranteed or endorsed by the
publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fmicb.2023.1108961/
full#supplementary-material
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