Beer fermentation performance and sugar uptake of Saccharomycopsis fibuligera–A novel option for low-alcohol beer [original]

Frontiers in Microbiology 01 frontiersin.org

Beer fermentation performance

and sugar uptake of

Saccharomycopsis fibuligera–A

novel option for low-alcohol

beer

YvonneMethner

1, FredericoMagalhães

2,3, LuisRaihofer

MartinZarnkow

1, FritzJacob

1 and MathiasHutzler

1 Research Center Weihenstephan for Brewing and Food Quality, Technical University of Munich,

Freising, Germany, 2 Chair of Brewing and Beverage Technology, Technical University of Berlin,

Berlin, Germany, 3 VTT Technical Research Centre of Finland Ltd., Espoo, Finland

There is a growing trend for beers with novel flavor profiles, as consumers

demand a more diversified product range. Such beers can beproduced by

using non-Saccharomyces yeasts. The yeast species Saccharomycopsis

fibuligera is known to produce exceptionally pleasant plum and berry flavors

during brewer’s wort fermentation while its mycelia growth is most likely a

technological challenge in industrial-scale brewing. To better understand and

optimize the physiological properties of this yeast species during the brewing

process, maltose and maltotriose uptake activity trials were performed. These

revealed the existence of active transmembrane transporters for maltose in

addition to the known extracellular amylase system. Furthermore, a single cell

isolate of S. fibuligera was cultured, which showed significantly less mycelial

growth during propagation and fermentation compared to the mother culture

and would therefore bemuch more suitable for application on an industrial

scale due to its better flocculation and clarification properties. Genetic

differences between the two cultures could not be detected in a (GTG)5

rep-PCR fingerprint and there was hardly any difference in the fermentation

process, sugar utilization and flavor profiles of the beers. Accordingly, the

characteristic plum and berry flavor could also be perceived by using the

culture from the single cell isolate, which was complemented by a dried

fruit flavor. A fermentation temperature of 20°C at an original gravity of 10 °P

proved to beoptimal for producing a low-alcohol beer at around 0.8% (v/v) by

applying the S. fibuligera yeast culture from the single cell isolate.

KEYWORDS

Saccharomycopsis fibuligera, non-Saccharomyces yeasts, fermentation, brewing,

low-alcohol beer, sugar uptake, micromanipulation, flavor

TYPE Original Research

PUBLISHED 05 October 2022

DOI 10.3389/fmicb.2022.1011155

OPEN ACCESS

EDITED BY

Haifeng Zhao,

South China University of Technology,

China

REVIEWED BY

Diego Bonatto,

UFRGS, Brazil

Olimpio Montero,

Spanish National Research Council (CSIC),

Spain

André Alcarde,

University of São Paulo, Brazil

*CORRESPONDENCE

Mathias Hutzler

[email protected]

SPECIALTY SECTION

This article was submitted to

Food Microbiology,

a section of the journal

Frontiers in Microbiology

RECEIVED 03 August 2022

ACCEPTED 06 September 2022

PUBLISHED 0 October 20225

CITATION

Methner Y, Magalhães F, Raihofer L,

Zarnkow M, Jacob F and Hutzler M (2022)

Beer fermentation performance and sugar

uptake of Saccharomycopsis fibuligera–A

novel option for low-alcohol beer.

Front. Microbiol. 13:1011155.

doi: 10.3389/fmicb.2022.1011155

Zarnkow, Jacob and Hutzler. 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

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.

Methner et al. 10.3389/fmicb.2022.1011155

Frontiers in Microbiology 02 frontiersin.org

Introduction

The yeast species Saccharomycopsis fibuligera (synonym:

Endomyces fibuliger) is known from food fermentations,

especially from liqueur production (Aidoo etal., 2006; Xie

etal., 2021). It was isolated from traditional fermented foods,

such as Indonesian tapé made from glutinous rice or cassava

tuber (Djien, 1972). In Southeast Asia in particular, the yeast

species is used in rice wine fermentation and is known to

produce desirable floral, fruity, and honey-like flavors during

fermentation as a result of high production of esters and

higher alcohols (Xie et al., 2021; Yang et al., 2021).

Characteristic fruity, flowery, and honey flavors were also

noted when S. fibuligera was cultured in selective media

containing different carbohydrate sources (Lee etal., 2018).

Due to its positive flavor properties, it was also tested for its

suitability to ferment brewer’s wort, where there is a demand

for diversification of the product range. As a result,

S. fibuligera produced exceptionally promising plum-like, and

also berry-like flavors in the beers (Srithamma, 2009;

Methner etal., 2019, 2022). It was noticeable that, depending

on the yeast strain, different sugar utilization patterns took

place in brewer’s wort, so it was possible to produce both

non-alcoholic and alcoholic beers. Lindner, who isolated the

yeast species in the context of bread spoilage in 1907,

described it as strongly utilizing sucrose during fermentation,

weakly utilizing glucose, and unable to utilize maltose

(Lindner, 1907). In contrast, Kreger-van-Rij stated in 1984

that the yeast species is capable of fermenting glucose as well

as sucrose and maltose (Kreger-Van Rij, 1984). Nevertheless,

existing literature explains that maltose utilization is slow

(Kurtzman and Smith, 2011). Generally, S. fibuligera is known

to possess amylolytic activity and therefore has the ability to

degrade starch. In 1944, Wickerham observed the presence of

an extracellular amylase system for S. fibuligera (Wickerham

et al., 1944). Through several studies, the two enzymes

α-amylase and glucoamylase from S. fibuligera were isolated

and characterized (Futatsugi etal., 1993; Chen etal., 2010).

The endoamylase α-amylase is capable of randomly cleaving

α-1,4 glycosidic bonds and thus reducing amylose to

oligosaccharides such as maltotriose and maltose.

Glucoamylase (synonym: amyloglucosidase) yields individual

glucose units from the hydrolysis of non-reducing ends of

amylose and amylopectin and additionally cleaves maltose

and maltotriose (Janeček and Baláž, 1992; Pandey, 1995;

Tiwari etal., 2015). Hostinová observed that S. fibuligera can

synthesize either both enzymes or only one type of amylase

in a strain-specific manner (Hostinová, 2002). In a study by

Methner etal. the glucoamylase activity gave an indication of

why maltose from selective media could befully metabolized

by the yeast strain S. fibuligera S. fib Lu27 although hardly any

maltose and maltotriose could beutilized by this strain from

brewer’s wort (Methner et al., 2022). In contrast, the

S. fibuligera yeast strain S. fib SF4 was able to partially

metabolize maltose and maltotriose from brewer’s wort in

another study (Methner etal., 2019). To date, it has not yet

been investigated whether this yeast species utilized maltose

due to extracellular amylase activity and subsequent passive

glucose transport into the cell or due to transmembrane

transporters (permeases) which actively transport maltose

and/or maltotriose into the cell. Since the fermentation trials

with S. fib Lu27 and S. fib SF4 were performed at different

temperatures in the two aforementioned studies by Methner

et al., a possible temperature dependence on the sugar

utilization needs to betaken into consideration.

A distinctive characteristic of the yeast species S. fibuligera is

its morphology, as it is dimorphic and forms individual round or

oval budding cells as well as mycelia (Nga etal., 1995; Xie etal.,

2021). The mycelium represents a challenge in terms of its

suitability for brewing, since it does not flocculate, triggering a

restriction of clarification of the beer. For this reason, single cells

were isolated and recultivated in this study to establish in a direct

comparison with the mother culture, whether the single cell

isolation would influence the morphology during propagation and

fermentation. To ensure that the culture of the isolated single cell

had no genetic differences from the mother culture, additional

(GTG)5 rep-PCR fingerprints were applied. Furthermore, since

the yeast should retain the ability to produce exceptionally fruity

flavors during the fermentation of brewer’s wort as a consequence

of the single cell isolation, the sensory properties of the final beers

were studied.

The objective of this study was to elucidate whether the yeast

species S. fibuligera possesses an active transport system for

maltose and maltotriose, which was examined by maltose and

maltotriose transport assays. Furthermore, it was investigated –

exemplified by the yeast strain S. fib SF4 – whether it was possible

to reduce the characteristic mycelial formation by means of

micromanipulation and recultivation in order to increase the

suitability for brewing on an industrial scale. Possible influences

of micromanipulation on brewing potential including sugar

utilization and beer flavor profiles were studied and compared

with the mother culture and the domesticated reference lager yeast

strain Saccharomyces pastorianus TUM 34/70 at fermentation

temperatures of 20 and 28°C.

Materials and methods

Yeast strains

Table 1 lists the yeast strains with the corresponding

abbreviations that were investigated or used as reference yeast

strains in this study. While the two S. fibuligera yeast strains

represented the yeasts under investigation, S. pastorianus S. pas

34/70 served as the reference yeast strain for all conducted

experiments. S. eubayanus S. eub 12357 and S. ludwigii S. lud SL17

were used as additional reference yeasts for the maltose and

maltotriose transport assays.

Methner et al. 10.3389/fmicb.2022.1011155

Frontiers in Microbiology 03 frontiersin.org

Maltose and maltotriose uptake activity

S. lud SL17 was used as a negative control as the yeast strain is

unable to take up maltose or maltotriose during fermentation

(Boundy-Mills etal., 2011) whereas S. eub 12357 is known to

bemaltose-positive but maltotriose-negative during fermentation

and thus represented the maltotriose-negative control (Gibson

etal., 2013). S. pas 34/70, which represents a traditional group II

lager yeast can utilize both maltose and maltotriose and was

therefore used as the positive control. The maltose and maltotriose

uptake activity of the two yeast strains S. fib Lu27 and S. fib SF4

were to be determined. For maltose and maltotriose uptake

measurement, S. lud SL17 was grown in YP medium prepared

from 1.0% yeast extract (Sigma-Aldrich, St. Louis, MO, U.S.A.),

2.0% peptone from casein, pancreatic digest (Sigma-Aldrich, St.

Louis, MO, U.S.A.) and 1.0% glucose. S. eub 12357 was grown in

YP containing 1.0% maltose and the remaining three strains

(S. pas 34/70 and the two S. fibuligera S. fib Lu27 and S. fib SF4)

were grown in YP medium containing maltose (1% w/v) or

maltotriose (1% w/v). All strains were propagated at 20°C in liquid

medium to an OD600nm between 4 and 8. The cells were harvested

by centrifugation (10 min, 2,968 g, 0°C), washed twice with

ice-cold water and once with 0.1 M tartrate-Tris (pH 4.2) before

being re-suspended in the same buffer to a concentration of

200 mg/ml fresh yeast. Zero-trans rates of [U-14C]-maltotriose

uptake were measured at 20°C essentially as described by Lucero

etal. (1997). Briefly, aliquots of 40 μl of yeast suspension were

added to 20 μl of 15 mM labeled maltotriose (for a final

concentration of 5 mM [U-14C]-maltotriose) and incubated for

60 s at 20°C. The reaction was stopped by adding 5 ml ice-cold

water. The suspension was immediately filtered and washed with

an additional 5 ml ice-cold water. The filter was submerged in

3.5 ml of Optiphase HiSafe 3 scintillation cocktail (Perkin Elmer,

MA, UnitedStates) and the radioactivity measured in a Perkin

Elmer Tri-carb 2,810 TR scintillation counter. [U-

C]-maltotriose

(ARC 627) was obtained from American Radiolabeled Chemicals

(St. Louis, MO, United States) and re-purified before use as

described by Dietvorst etal. (2005). Maltose (minimum purity,

99%) and maltotriose (minimum purity, 95%) were from Sigma-

Aldrich (St. Louis, MO). The cell washing was expected to remove

any potential extracellular carbohydrate-hydrolase that could

interfere with the results. In addition, keeping cells on ice until the

uptake assay, the short incubation time in maltotriose, and subpar

temperature and pH conditions for known extracellular

glucoamylases of the Saccharomyces genus (pHOpt = 4.5–6,

Opt

= 40–60°C; (Hostinová and Gašperík, 2010)) were expected

to limit the activity of any residual carbohydrate hydrolase that

might bepresent.

Micromanipulation and yeast

propagation

For micromanipulation, a TransferMan® 4r micromanipulator

with DualSpeed™ Joystic and CellTram® 4r Air, a pneumatic

manual microinjector, were used (Eppendorf SE, Hamburg,

Germany). The capillary with an inner diameter of 10 μm

(BioMedical Instruments, Zöllnitz, Germany) was connected to a

Nikon eclipse Ti-e inverse microscope (Nikon, Tokyo, Japan)

using an adapter. For micromanipulation, 3 ml of sterilized wort

(100°C, 45 min) with an original gravity of 10 °P was pipetted into

three sterile cell culture dishes (35 × 10 mm; Greiner Bio-One

GmbH, Frickenhausen, Germany). Wort was prepared from

unhopped malt extract (Weyermann®, Bamberg, Germany) by

re-dilution with distilled water. The yeast strain S. fib SF4 was

transferred from a wort slant agar (mother culture) into the wort

of one cell culture dish and was distributed using a sterile

inoculation loop. Using the micromanipulator, two single cells of

the yeast strain S. fib SF4 were isolated and one cell per cell culture

dish was transferred into the wort. Due to the filamentous growth

of S. fibuligera, care was taken not to isolate filaments from the

mother culture along with the single cells. One cell culture dish

was incubated at 20°C for 72 h, the other sample was incubated at

28°C for 72 h. Temperatures at 20 and 28°C were selected, since

there are already existing studies with S. fibuligera yeast strains in

brewer’s wort on these two approximate temperatures (Methner

etal., 2019, 2022). The yeast cultures grown in the cell culture

dishes were each transferred to 50 ml sterile wort in 100 ml flasks

sealed with cotton plugs and propagated for another 72 h at

the respective temperatures on a WiseShake orbital shaker

(Witeg Labortechnik GmbH, Wertheim, Germany) at 80 rpm.

Simultaneously, S. fib SF4 mother culture from wort slant agar was

incubated under sterile conditions into 2 × 50 ml of the identical

wort in 100 ml flasks. Here as well, one sample was propagated at

20°C, while the second sample was propagated at 28°C. At the end

of the 72 h propagation, 1 ml material of the four different samples

was removed in each case into sterile 1.5 ml SafeSeal micro tubes

(Sarstedt AG & Co. KG, Nümbrecht, Germany) for (GTG)5

rep-PCR fingerprint analysis. The remaining propagation yeasts

were transferred to 250 ml fresh, sterile 10 °P wort in 500 ml flasks

and propagated for an additional 72 h at the respective parameters

selected at the outset. In a final propagation step, the four samples

were transferred to 1800 ml sterile wort in 2500 ml flasks and

propagated for another 72 h before the yeasts were further

processed for fermentation. Since a reference beer was to

be produced for the fermentations in addition to the four

experimental beers, the yeast strain S. pas 34/70 was also

TABLE1 Yeast species and strain numbers with corresponding

abbreviations used in this study.

Yeast strain number Yeast strain

abbreviation Yeast species

TUM SL17 S. lud SL17 Saccharomycodes ludwigii

VTT C-12902/CBS12357 S. eub 12357 Saccharomyces eubayanus

VTT A-13220/TUM 34/70 S. pas 34/70 Saccharomyces pastorianus

PI S 6; Lu27 S. fib Lu27 Saccharomycopsis fibuligera

PI S 7; Lu 26/SF4 S. fib SF4 Saccharomycopsis fibuligera

Methner et al. 10.3389/fmicb.2022.1011155

Frontiers in Microbiology 04 frontiersin.org

propagated as described as a commercial domesticated lager yeast

strain for brewing. Additionally, comparative microscopic images

were taken of the mother culture S. fib SF4 from the wort slant

agar and the two yeasts propagated at 28°C (mother culture and

micromanipulated culture) to compare the morphologies using

the aforementioned brightfield Nikon inverse microscope at a

magnification of × 40 (objective) plus digital zoom (scale is visible

in microscopic pictures).

(GTG)5 rep-PCR fingerprint

(GTG)

rep-PCR fingerprint system was applied to determine

if the four differently propagated S. fibuligera S. fib SF4 yeast

cultures had identical or different genetic fingerprints. According

to Versalovic etal. (1994), the primer (GTG)

(5′-GTG GTG GTG

GTG GTG-3′) was originally developed for bacteria and was

successfully transferred to differentiate various non-Saccharomyces

yeasts (Meyer et al., 1993; Erdem et al., 2016). After sample

preparation, DNA fingerprint amplification was performed,

followed by capillary gel electrophoresis and the data processing

of the generated fingerprints. Table 2 lists the four different

propagation approaches of the S. fibuligera yeast strain S. fib SF4

with their corresponding varying parameters and abbreviations,

which were subsequently investigated.

For sample preparation, DNA was first isolated from the

liquid samples. For this purpose, the yeast-wort suspensions were

centrifuged (Mikro 200, Andreas Hettich GmbH & Co. KG,

Tuttlingen, Germany) in sterile 1.5 ml SafeSeal microtubes

(Sarstedt AG & Co. KG, Nümbrecht, Germany) at 16,000 g for

2 min and the supernatant was discarded. 200 μl Insta-Gene

Matrix was added to the samples before they were incubated in the

thermomixer preheated to 56°C for 30 min. Samples were then

vortexed in the tubes and placed in the 95°C preheated

thermomixer for an additional 8 min. After a further

centrifugation step at 16,000 g for 2 min, 100 μl of the supernatant

was transferred to fresh sterile tubes. In the next step, DNA

concentrations were measured using NanoDrop1000

spectrophotometer (Peqlab Biotechnologie GmbH, Erlangen,

Germany) and adjusted to 25 ng/μL, containing 12.5 μl RedTaq

Mastermix (2×) 2-fold (Genaxxon bioscience, Ulm, Germany),

10 μl Primer Solution (50 pmol/l), 5 μl PCR-clean double distilled

water (ddH2O) and 2.5 μl sample DNA. The PCR temperature

protocol of the DNA fingerprint amplification and subsequent

capillary gel electrophoresis including data processing were

described according to Riedl etal. (2019).

Fermentation and beer analysis

Before starting the small-scale fermentation trials in triplicate,

the yeast cell counts for the reference strain S. pas 34/70 and the

four individually propagated S. fib SF4 cultures were determined.

The Cellometer® Vision (Nexcelom Bioscience LLC, Lawrence,

MA, UnitedStates) was used to determine cell counts. The pitching

rate for the fermentation experiments was set at 10 × 106 cells/mL

(± = 1 × 106 cells/mL). After cell counting, the corresponding

calculated propagation yeast volumes were centrifuged (Roto

Super 40, Andreas Hettich GmbH & Co. KG, Tuttlingen, Germany)

at 750 g for 5 min in sterilized 500 ml PPCO centrifuge bottles

(Nalgene, Thermo Fisher Scientific, Waltham, MA, USA).

Subsequently, the wort supernatant was discarded and the yeast

samples were pitched into 1800 ml unhopped sterilized wort

(10.2 °P, pH 5.3) prepared from malt extract (Weyermann®,

Bamberg, Germany) in 2000 ml sterile Duran glass bottles (Schott

AG, Mainz, Germany). The fermentation bottles were closed with

glass fermentation airlocks on top. While yeast cultures already

propagated at 28°C were also fermented at 28°C, yeast cultures

propagated at 20°C were fermented at 20°C accordingly. By

weighing the samples every 24 h, the fermentation progress was

monitored by weight loss, which was mainly due to escaping

carbon dioxide and based on Balling’s assumption that during

fermentation, an average of 2.0665 g of extract is converted into 1 g

alcohol, 0.9565 g carbon dioxide and 0.11 g yeast (Esslinger, 2009).

Following the main fermentation, which lasted for 480 h (20 days),

the samples were sealed with sterile screw caps and cooled to 2°C

for an additional 168 h (7 days) before the analyses shown in

Table 3 were performed. For the sugar utilization results,

one-sample t-tests were performed using OriginPro 2020 as

statistical software to evaluate if the mean values of carbohydrate

utilizations of the different fermentations varied significantly.

Sensory evaluation

The beer samples were tempered to 12°C and were profiled

at 20°C room temperature by a sensory panel of eight

DLG (Deutsche Landwirtschafts-Gesellschaft e.V., Frankfurt,

Germany)-certified assessors. The accredited sensory evaluations

were conducted according to DIN EN 17025. To exclude external

interferences during the tastings, they were held in an

appropriately neutral, white-colored room with individual tasting

chambers. A sensory test was carried out according to the DLG

evaluation scheme, which comprises a rating scale from zero to

five. While zero is considered the lowest score and represents

insufficient product quality, a score of five points fully meets the

quality expectations of the product and matches the quality

TABLE2 Four different propagation approaches with the

corresponding varying parameters of the yeast strain

Saccharomycopsis fibuligera SF4.

Abbreviation Yeast culture Propagation and

fermentation temperature

SF4-MI-20 Micromanipulated 20°C

SF4-MK-20 Mother culture 20°C

SF4-MI-28 Micromanipulated 28°C

SF4-MK-28 Mother culture 28°C

Methner et al. 10.3389/fmicb.2022.1011155

Frontiers in Microbiology 05 frontiersin.org

description very well (Hildebrandt and Schneider-Haeder, 2009).

The odor, purity of taste and body of the beers were evaluated,

while the quality of bitterness as well as carbonation were

neglected as the beers were produced from unhopped malt extract

and the fermentations were conducted without pressure. For the

tasting, the samples were assigned randomized three-digit

numbers and 50 ml of each sample was served in brown 200 ml

tasting glasses. There was also an additional sensory test method

– a descriptive tasting. The method was based on the descriptive

sensory evaluation of Meier-Dörnberg etal. (2017) with the seven

main categories of tropical fruity, fruity (other fruits), citrus, spicy,

floral, malty, and other flavors. The sensory assessors were asked

separately about beer odor and taste, which they scored from 0

(not noticeable) to 5 (extremely noticeable). The results were

statistically analyzed by first determining the interquartile ranges

and removing extreme outliers (3 × IQR) to obtain only statistically

significant values. For significant results, the mean values of odor

and taste were calculated before the beer samples fermented with

the S. fibuligera yeast strain S. fib SF4 were compared with the

reference beers fermented with S. pastorianus S. pas 34/70.

Results

Maltose and maltotriose uptake activity

Prior to the uptake activity assessment, the strains were

propagated when possible in both maltose and maltotriose as sole

carbon sources. Due to the inability of S. eub 12357 to grow on

maltotriose, it was propagated only on maltose. S. lud SL17 cannot

grow on maltose nor maltotriose and was therefore only

propagated in glucose. The results of the maltose and maltotriose

uptake activity are presented in Figure1, respectively. Raw data

can befound in the Supplementary material Table1.

S. ludwigii S. lud SL17 maltose uptake activity was just above

the limit at 0.8 μmol min−1g−1 DY but not significantly (standard

deviation of ±0.51; cf. Figure1). This strain is known to bemaltose

negative and the higher measured value is likely a result of

experimental variability. Maltotriose activity was, however, clearly

below the minimum activity required (cf. Figure1). S. eubayanus

S. eub 12357, as expected, showed high maltose uptake activity

and no maltotriose uptake activity. The strain S. pas 34/70 had

maltose uptake activity comparable to that of S. eub 12357 and it

was the only strain with maltotriose uptake activity significantly

above 0.5 μmol min−1g−1 DY. Growth on maltose or maltotriose

generally did not seem to affect the uptake activity of either sugar.

The only exception was S. fib Lu27, which had 50% higher maltose

uptake activity when grown on maltotriose. Otherwise, the

S. fibuligera strains S. fib Lu27 and S. fib SF4 showed very similar

behavior. Maltose uptake activity was lower but still comparable

to that of S. eub 12357 and S. pas 34/70. Maltotriose activity was

below 0.5 μmol min−1g−1 DY for both strains irrespective of the

sugar used. However, the error is quite high, particularly for S. fib

Lu27. This can partly be explained by the difficulty of

homogenizing cells with filamentous growth. This created

challenges for the uptake experiments and the measurement of

cell mass. Regardless, the results strongly suggest that S. fibuligera

does not have any significantly active transporters able to take up

maltotriose into the cells. Maltotriose utilization is therefore

TABLE3 Analytical methods of the wort and the beers according to MEBAKa and Donhauser etal.b.

Analysis Method Device

Original gravity, apparent attenuation, ethanol content MEBAKa WBBM 2.9.6.3 Bending vibration and NIR spectroscopy, Alcolyzer Plus with DMA 5000 X

sample 122 (Anton-Paar GmbH, Ostfildern, Germany)

pH value MEBAKa WBBM 2.13 pH meter with pH electrode, ProfiLine pH3210 pH meter (Xylem Inc.,

NewYork, NY, UnitedStates)

Sugar composition (glucose, fructose, sucrose, maltose,

maltotriose)

Donhauser etal.b LS-HPLC 002_2 HPLC UltiMate 3000 (Thermo Fisher Scientific, Waltham, MA, United

States)

aMEBAK® (2012), Editor: Dr. F. Jacob: The MEBAK collection of brewing analysis methods: Wort, beer and beer-based beverages. Collection of methods of the Mitteleuropäischen

Brautechnischen Analysenkommission. Self-published by MEBAK.

bDonhauser, S.; Wagner, D. (1990): Zucker- und Endvergärungsgradbestimmung mittels der HPLC, 9:306–309. Monatsschrift für Brauwissenschaft.

FIGURE1

Zero-trans rates of maltose (Mal) and maltotriose (Malt) uptake

activity (μmol min−1g−1 dry yeast (DY)) measured at 20°C for cells

propagated on glucose (white), maltose (light grey) or maltotriose

(dark grey) with n = 4. An uptake activity equal or below 0.5 μmol

min−1g−1 DY is considered negligible. Yeast strain abbreviations

according to Table1.

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