**Screening and Application of** *Cyberlindnera* **Yeasts to Produce a Fruity, Non-Alcoholic Beer**

#### **Konstantin Bellut 1, Maximilian Michel 2, Martin Zarnkow 2, Mathias Hutzler 2, Fritz Jacob 2, Jonas J. Atzler 1, Andrea Hoehnel 1, Kieran M. Lynch <sup>1</sup> and Elke K. Arendt 1,3,\***


Received: 14 November 2019; Accepted: 13 December 2019; Published: 17 December 2019

**Abstract:** Non-alcoholic beer (NAB) is enjoying growing demand and popularity due to consumer lifestyle trends and improved production methods. In recent years in particular, research into the application of non-*Saccharomyces* yeasts to produce NAB via limited fermentation has gained momentum. Non-*Saccharomyces* yeasts are known to produce fruity aromas, owing to a high ester production. This trait could be harnessed to mask the often-criticized wort-like off-flavor of NAB produced via limited fermentation. Six *Cyberlindnera* strains were characterized and screened in wort extract. Four of the six strains produced a pleasant, fruity aroma while exhibiting low ethanol production. The strain *Cyberlindnera subsu*ffi*ciens* C6.1 was chosen for fermentation optimization via response surface methodology (RSM) and a pilot-scale (60 L) brewing trial with subsequent sensory evaluation. A low fermentation temperature and low pitching rate enhanced the fruitiness and overall acceptance of the NAB. The NAB (0.36% ABV) produced on pilot-scale was significantly more fruity and exhibited a significantly reduced wort-like off-flavor compared to two commercial NABs. This study demonstrated the suitability of *Cyberlindnera subsu*ffi*ciens* to produce a fruity NAB, which can compete with commercial NABs. The outcome strengthens the position of non-*Saccharomyces* yeasts as a serious and applicable alternative to established methods in NAB brewing.

**Keywords:** brewing; *Cyberlindnera*; NABLAB; non-alcoholic beer; non-conventional yeast; non-*Saccharomyces* yeast; response surface methodology

#### **1. Introduction**

While the overall market growth of beer is slowing down, non-alcoholic and low alcohol beer (NABLAB) is growing in volume and popularity, owed to stricter legislation, lifestyle trends and improved production methods [1]. The increasing interest has fueled research in NABLAB production methods, especially in recent years, aimed at overcoming taste deficits compared to regular beer and consequently improving consumer acceptance. The two major production methods, physical dealcoholization and limited fermentation, both compromise the taste of the beer. Dealcoholized beer is often criticized for its lack of body and aromatic profile, a consequence of the removal of volatile esters and higher alcohols in conjunction with ethanol. Apart from a sweet taste due to residual sugars, one of the main points of criticism of NAB produced by limited fermentation is its wort-like off-flavor caused by aldehydes present in the wort [2]. In regular beer, ethanol significantly increases aldehyde retention, reducing the perceptibility of the wort-like flavor. However, in NAB produced by limited fermentation, the low ethanol content and higher levels of mono- and disaccharides intensify this undesired off-flavor [3].

It is known that esters, which yeast produce as a by-product of alcoholic fermentation, are extremely important for the flavor profile of beer [4,5]. The lack thereof, as well as their overproduction, can significantly compromise the flavor. Aside from strain-specific differences, the process parameters such as the fermentation temperature, pitching rate and wort gravity have been shown to have a significant influence on ester formation [4,6]. In non-alcoholic beers, ester concentrations are lower compared to regular beer, independent of the production method [7,8]. While physical dealcoholization removes esters that were previously produced, limited fermentation adversely affects the production of substantial amounts in the first place.

Non-*Saccharomyces* yeasts are known for their important contribution to the flavor profile of fermented foods and beverages and have therefore been investigated for their targeted application in bioflavoring and, not least, NABLAB brewing [1,9,10]. Species that have been mentioned in the context of NABLAB production belong to the genera *Cyberlindnera*, *Hanseniaspora*, *Lachancea*, *Mrakia*, *Pichia*, *Torulaspora*, *Saccharomycodes*, *Sche*ff*ersomyces* and *Zygosaccharomyces* [1,11–16]. In particular, the *Cyberlindnera* species are known for their high ester production, which was shown in studies with *Cyberlindnera saturnus* (formerly *Williopsis saturnus*), *C. mrakii* (formerly *Williopsis saturnus* var. *mrakii*) and *C. subsu*ffi*ciens* (formerly *Williopsis saturnus* var. *subsu*ffi*ciens*) [17–20]. Furthermore, it has been proposed to use yeasts with high production of flavor compounds (i.e., esters, higher alcohols) to mask the wort-like flavor of NAB produced by limited fermentation. However, research in that direction is sparse [21,22]. In addition, such yeasts are capable of reducing aldehydes to their correspondent alcohol, which can also enhance the reduction of the often-criticized wort-like off-flavor [23,24].

In this study, six strains of the genus *Cyberlindnera* were investigated to create a fruity NAB. After identification, the strains were characterized for their substrate utilization, flocculation behavior and stress responses. A screening in diluted wort extract was performed to investigate the strains' potential to produce a pronounced fruity flavor without the production of high concentrations of ethanol. Interspecific differences in sugar consumption and the production of volatile fermentation by-products was investigated by means of high-performance liquid chromatography (HPLC) and gas chromatography (GC). The most promising strain was studied further to determine the optimal fermentation conditions to enhance the fruity flavor, which was performed by means of response surface methodology (RSM). Finally, a non-alcoholic beer was produced on pilot-scale (60 L), and its analytical attributes, aroma, and taste compared to two commercial NABs were examined.

#### **2. Materials and Methods**

#### *2.1. Materials*

All reagents used in this study were at least analytical grade from Sigma-Aldrich (St Louis MO, USA) unless stated otherwise. The wort extract applied in this study was spray-dried wort from 100% barley malt (Spraymalt Light, Muntons plc, Suffolk, UK). For the pilot-scale brewing, pilsner malt and acidulated malt were sourced from Weyermann (Malzfabrik Weyermann, Bamberg, Germany).

#### *2.2. Yeast Strains*

#### Strain Origin and Identification

Strain 837A was isolated from a brewery cellar, NT Cyb originates from a dried fermentation starter for rice wine, strain C6.1 originates from a coconut, and L1 from "Lulo", the fruit of *Solanum quitoense*. The type strains CBS 1707 and CBS 5763 originate from soil samples. For identification, the D1/D2 domain of the 26S rRNA gene was amplified, sequenced and compared to publicly available sequences in the National Center for Biotechnology Information (NCBI) database using the Basic Local Alignment Search Tool (BLAST; https://blast.ncbi.nlm.nih.gov/Blast.cgi).

The DNA of the yeast isolates was extracted using an extraction kit (Yeast DNA Extraction Kit, Thermo Fisher Scientific, Waltham MA, USA). To amplify the D1/D2 domain

of the 26S rRNA gene, the primers NL1 (5 -GCATATCAATAAGCGGAGGAAAAG-3 ) and NL4 (5 -GGTCCGTGTTTCAAGACGG-3 ) were used. Polymerase chain reaction (PCR) was performed using the temperature protocol: 95 ◦C/2 min; 30 cycles of 95 ◦C/30 s, 56 ◦C/15 s; 72 ◦C/60 s; 72 ◦C/5 min. Stock cultures were kept in 50% (*v*/*v*) glycerol at −80 ◦C.

#### *2.3. Yeast Characterization*

#### 2.3.1. Flocculation Assay and Phenolic Off-Flavor (POF) Test

The flocculation test was performed using a slightly modified Helm's assay [25,26]. Essentially, all cells were washed in ethylenediaminetetraacetic acid (EDTA) and the sedimentation period was extended to 10 min. Wort was composed of 75 g/L spray-dried malt extract (Spraymalt Light, Muntons plc, Suffolk, UK) adjusted to 15 International Bitterness Units (IBU) (15 mg/mL iso-α-acids; from 30% stock solution; Barth-Haas Group, Nürnberg, Germany).

The phenolic off-flavor test was performed according to Meier-Dörnberg et al. [27]. In short, yeast strains were spread on yeast and mold agar plates (YM-agar) containing only one of the following precursors: either ferulic acid, cinnamic acid or coumaric acid. After three days of incubation at 25 ◦C, plates were evaluated by a trained panel by sniffing to detect any of the following aromas: clove-like (4-vinylguajacol), Styrofoam-like (4-vinylstyrene) and medicinal-like (4-vinylphenol). *Saccharomyces cerevisiae* LeoBavaricus—TUM 68®(Research Center Weihenstephan for Brewing and Food Quality, Freising-Weihenstephan, Germany) was used as a positive control.

#### 2.3.2. Substrate Utilization

To analyze substrate utilization by the *Cyberlindnera* strains, the test kit API ID 32C (BioMérieux, Marcy-l'Étoile, France) was used. Preparation of the inoculum and inoculation of the strips were performed according to the manufacturers' instructions. Colonies for the inoculum were grown on yeast extract peptone dextrose (YPD) agar plates for 48 h at 27 ◦C. After inoculation, API ID 32C strips were incubated for 2 days at 28 ◦C. The samples were evaluated visually for turbidity in the wells, differentiating positive (+), negative (−), and weak (w) growth.

#### 2.3.3. Stress Tests

Stress tests were performed via the measurement of yeast growth in a microplate, through the repeated measurement of absorbance over a time period of 96 h (Multiskan FC, Thermo Scientific, Waltham, MA, USA). The substrate for the hop sensitivity test was sterile-filtered wort extract (75 g/L Muntons Spraymalt Light) adjusted to 0, 50 and 100 mg/L iso-α-acids (1 mg/L = 1 International Bitterness Unit, IBU), respectively, by using an aliquot of a stock solution of 3% iso-α-acids in 96% (*v*/*v*) ethanol (Barth-Haas Group, Nürnberg, Germany). For testing ethanol sensitivity, the sterile-filtered wort extract was adjusted to 0%, 2.5%, 5% and 7.5% ABV with an aliquot of 100% (*v*/*v*) ethanol. For testing pH sensitivity, the sterile-filtered wort extract was adjusted to the following pHs with 2 M HCl: 5.5 (control without addition of HCl): 5.0, 4.0 and 3.0. For inoculation, strains were grown in sterilized wort extract for 24 h at 25 ◦C under aerobic conditions. The microtiter plate wells were inoculated with a concentration of 105 cells/mL. The wells contained 200 μL of the respective wort substrates. Plates were incubated at 25 ◦C, and absorbance was measured every 30 min at 600 nm without shaking over a time period of 96 h (Multiskan FC, Thermo Scientific, Waltham, MA, USA). Stress tests were performed in triplicate.

#### *2.4. Yeast Screening*

#### 2.4.1. Propagation

Single colonies of the respective strains were taken from yeast extract peptone dextrose (YPD) agar plates after 72 h growth at 25 ◦C and transferred into a 250 mL sterile Duran glass bottle (Lennox

Laboratory Supplies Ltd, Dublin, Ireland) containing 150 mL propagation wort consisting of 75 g/L spray-dried malt (Spraymalt light, Muntons plc, Suffolk, UK) and 30 g/L glucose (Gem Pack Foods Ltd., Dublin, Ireland), sterilized at 121 ◦C for 15 min. The bottles were covered with sterile cotton and placed in an incubator with orbital shaker (ES-80 shaker-incubator, Grant Instruments (Cambridge) Ltd, Shepreth, UK) and incubated for 24 h at an orbital agitation of 170 rpm at 25 ◦C (Strain 837A was incubated for 48 h). Cell count was performed using a Thoma Hemocytometer with a depth of 0.1 mm (Blaubrand, Sigma-Aldrich, St. Louis, MO, USA).

#### 2.4.2. Fermentation

Fermentation wort was prepared by dissolving 75 g/L spray-dried malt extract (Munton Spraymalt light) in 1 L of brewing water and sterilizing at 121 ◦C for 15 min, followed by filtration through a sterile grade 1V Whatman filter (Whatman plc, Maidstone, UK) to remove hot trub formed during sterilization. The analytical attributes of the fermentation wort for the yeast screening trial and RSM trial is shown in Table 1.


**Table 1.** Attributes of screening wort from wort extract.

Fermentation trials were carried out in 1 L sterile Duran glass bottles, equipped with an air lock. Per yeast strain, triplicate bottles were filled with 400 mL of wort and left untouched throughout the fermentation. Yeast cells for pitching were washed by centrifugation at 900 *g* for 5 min and resuspended in sterile water to ensure no carryover of sugars from the propagation wort into the fermentation wort. Pitching rate was 3 <sup>×</sup> 107 cells/mL. Fermentation temperature was 25 ◦C. Fermentation was performed until no change in extract could be measured for two consecutive days.

#### *2.5. Scanning Electron Microscopy (SEM)*

Yeast cultures for scanning electron microscopy (SEM) were prepared following the protocol for cultured microorganisms by Das Murtey and Ramasamy [28]. Single colonies were taken from a YPD agar plate and grown in YPD broth for 24 h at 25 ◦C. One milliliter of sample was centrifuged at 900 g for 2 min for pellet formation and resuspended in 5% glutaraldehyde solution prepared in 0.1 M phosphate buffer (pH 7.2) for fixation. After 30 min, the sample was centrifuged, the supernatant was discarded, and the pellet was washed twice in 0.1 M phosphate buffer. Consequently, the pellet was resuspended in 1% osmium tetroxide prepared in 0.1 M phosphate buffer. After 1 h, cells were again washed twice in 0.1 M phosphate buffer. The sample was then dehydrated through an ethanol series of 35%, 50%, 75%, 95%, absolute ethanol, and hexamethyldisilazane (HDMS), with 30 min per step (last two ethanol steps twice), centrifuging and discarding the supernatant at each change. Lastly, the second HDMS was discarded and the sample left drying overnight in a desiccator.

The dehydrated yeast sample was mounted onto plain aluminum stubs using carbon double surface adhesive and coated with a 5 nm gold-palladium (80:20) layer using a Gold Sputter Coater (BIO-RAD Polaron Division, SEM coating system, England), then observed under a constant accelerating voltage of 5 kV under a JEOL scanning electron microscope type 5510 (JEOL, Tokyo, Japan).

#### *2.6. Response Surface Modeling (RSM)*

To investigate optimal fermentation conditions for C6.1 to produce a fruity, non-alcoholic beer, response surface methodology (RSM) was performed using DesignExpert 9 software (StatEase, Minneapolis, MN, USA). A two-factorial, face-centered, central composite design with single factorial points and 5 replications of the center point was chosen. The predictor factors were temperature (17, 22, 27 ◦C), and pitching rate (10, 35, 60 <sup>×</sup> 106 cells/mL).

Spray-dried malt extract (Spraymalt light, Muntons plc, Suffolk, UK) served as the substrate. Wort preparation, propagation and inoculation were carried out as outlined in 2.4.1. The wort used was the same as in the screening (Table 1). Fermentation volume was 150 mL in 250 mL Duran glass bottles equipped with an air lock. Fermentation was performed until no change in extract could be measured for two consecutive days. Table 2 shows the experimental design.

**Table 2.** Response surface methodology (RSM) experimental design: Two-factorial, face-centered, central composite design with five repetitions of the center point. Factor 1, A: temperature, range 17, 22, 27 ◦C. Factor 2, B: pitching rate, range 10, 35, 60 <sup>×</sup> 106 cells/mL.


\* Center point.

Models were produced applying backward elimination regression of insignificant model terms with α to exit of 0.1 (detailed report in supplementary Data Sheet S1). For significant models with insignificant lack of fit (LOF), 3D response surface plots were produced. Fermentations for model validation were performed in the same wort with propagation as outlined in 2.4.1 and fermentation as outlined above.

#### *2.7. Pilot-Scale Brewing*

#### 2.7.1. Wort Production

Wort for the pilot brew was produced in a 60 L pilot-scale brewing plant consisting of a combined mash-boiling vessel, a lauter tun and whirlpool (FOODING Nahrungsmitteltechnik GmbH, Stuttgart, Germany). The grain bill comprised 6.65 kg Weyermann Pilsner Malt and 0.35 kg Weyermann Acidulated Malt (Malzfabrik Weyermann, Bamberg, Germany). Grains were milled with a two-roller mill ("Derby", Engl Maschinen, Schwebheim, Germany) at a 0.8 mm gap size. The crushed malt was mashed-in with 30 L of brewing water at 50 ◦C. The following mashing regime was employed: 20 min at 50 ◦C, 20 min at 62 ◦C, 10 min at 72 ◦C and mashing out at 78 ◦C. The mash was pumped into the lauter tun, and lautering was performed after a 15 min lauter rest, employing four sparging steps of 5 L hot brewing water each. Boil volume was 50 L at a gravity of 1.030 (7.0 ◦P), and total boiling time was 60 min. Thirty minutes into the boil, 15 g of Magnum hop pellets (14% iso-α-acids) were added for

a calculated IBU content of 9. After boiling, gravity was readjusted to 1.030 (7.0 ◦P) with hot brewing water, and hot trub precipitates and hop residue were removed in the whirlpool with a rest of 20 min. Clear wort was pumped through a heat exchanger and filled into 60 L cylindroconical fermentation vessels at a temperature of 17 ◦C.

#### 2.7.2. Propagation, Fermentation and Aftercare

A first propagation step was employed as described in 2.4.1. A second propagation step was performed by transferring the small-scale propagated wort into a 5 L carboy filled with 2 L of sterile wort extract at 7 ◦P and closed with sterile cotton. The second propagation step was conducted for 24 h under constant agitation at ambient temperature (20 ± 2 ◦C).

Yeast was pitched into the fermenter at a pitching rate of 10<sup>7</sup> cells/mL. Fermentation was carried out in cylindroconical fermentation vessels with a capacity of 60 L, at ambient pressure and at a glycol-controlled fermentation temperature of 17 ◦C. Samples were withdrawn every day. Fermentation was carried out until no change in extract could be measured for two consecutive days. The beer was then filled into a 50 L keg and carbonated by repeated pressurization with CO2 to 1 bar at 2 ◦C. After 5 days, the carbonated beer was filled into 330 mL brown glass bottles with a counter-pressure hand-filler (TOPINCN, Shenzen, China) and capped. Bottles were pasteurized in a pilot retort (APR-95; Surdry, Abadiano, Vizcaya, Spain) with spray water at 65 ◦C for 10 min resulting in approximately 23 pasteurization units (PU). The successful pasteurization was confirmed by plating the pasteurized NAB on agar plates. Beer bottles were stored at 2 ◦C in a dark place for further analysis and sensory evaluation.

#### *2.8. Sensory Evaluation*

The sensory evaluation of the samples produced during yeast screening and RSM trial were judged by a panel of 12–15 experienced tasters. Samples were given at ambient temperature (20 ◦C) with a three-digit code. Each panelist evaluated the samples in an individual booth at ambient temperature (20 ◦C). The tasters were asked to desribe the sample in their own words, followed by evaluation of the intensity of a fruity smell and the overall acceptance of the smell of the sample on a hedonic scale from 0 ("not fruity"/"dislike extremely") to 5 ("extremely fruity"/"like extremely") according to MEBAK Sensory Analysis 3.2.1 "Simple Descriptive Test" and 3.2.2 "Profile Test", respectively.

The non-alcoholic beer samples (C6.1 pilot scale and commercial samples) were tasted and judged by a sensory panel of ten experienced and certified (DLG International Certificate for Sensory Analysis—beer and beer-based mixed drinks; Deutsche Landwirtschafts-Gesellschaft e.V.) panelists. A "Simple Descriptive Test" and "Profile Test" were performed according to MEBAK Sensory Analysis 3.2.1 and 3.2.2, respectively. Attributes for the aroma were "wort-like", "floral", "fruity", "citrus-like" and "tropical". A taste attribute "sweet taste" was also included. Panelists were asked to evaluate the attributes in their intensity on a line-marking scale from 0, "not perceptible", to 5, "strongly perceptible". Before the evaluation of the intensity, a descriptive sensory was performed, where the panelists were asked to describe the aroma of the samples in their own words. Samples were provided in dark glasses with a three-digit code and evaluated at a temperature of 20 ◦C in order to evaluate the full flavor profile (following DLG guidelines). The commercial samples NAB A and NAB B were non-alcoholic beers produced by limited fermentation [29] and "dialysis technology" [30], respectively. Each panelist tasted the samples in an individual booth at ambient temperature (20 ◦C). The amount of sample tasted was 50 mL per sample.

#### *2.9. Wort and Beer Analyses*

#### 2.9.1. HPLC Analyses

Sugars and ethanol were determined by HPLC Agilent 1260 Infinity (Agilent Technologies, Santa Clara CA, USA) equipped with a refractive index detector (RID) and a Sugar-Pak I 10 μm, 6.5 mm × 300 mm column (Waters, Milford MA, USA), with 50 mg/L Ca-EDTA as mobile phase and a flow rate of 0.5 mL/min at 80 ◦C. Differentiation of maltose and sucrose was achieved with a Nova-Pak 4 μm, 4.6 mm × 250 mm column (Waters, Milford MA, USA), with acetonitrile/water 78:22 (*v*/*v*) as mobile phase and a flow rate of 1.0 mL/min. Quantification was achieved by external standards in a calibration range of 0.5 to 30 mM.

#### 2.9.2. GC Analyses

Free vicinal diketones were quantified by a Clarus 500 gas chromatograph (Perkin-Elmer, Waltham MA, USA) with a headspace unit and Elite-5 60 m × 0.25 mm, 0.5 μm column using a 2,3-hexandione internal standard. Fermentation by products (esters, higher alcohols) was quantified using a Clarus 580 (Perkin-Elmer, Waltham MA, USA) gas chromatograph with a headspace unit and INNOWAX cross-linked polyethylene-glycol 60 m × 0.32 mm, 0.5 μm column (Perkin-Elmer, Waltham MA, USA). Vials containing beer samples were equilibrated for 25 min at 60 ◦C. The samples were injected at 50 ◦C, rising to 85 ◦C after one minute by heating at 7 ◦C/min. A temperature of 85 ◦C was maintained for one minute and then elevated to 190 ◦C at a heating rate of 25 ◦C/min.

#### 2.9.3. Other

Glycerol was determined via enzymatic assay kit (glucokinase method), following the recommended procedure (K-GCROLGK, Megazyme, Bray Co. Wicklow, Ireland). The method is based on the use of ADP-glucokinase and an increase in absorbance on conversion of NAD<sup>+</sup> to NADH, and is performed at ambient temperature at a sample volume of 2 mL.

Free amino nitrogen (FAN) was measured using a ninhydrin-based dying method, where absorbance is measured at 570 nm against a glycine standard (ASBC Method Wort-12 A). The method is performed at a total volume of 10 mL. Following the color reaction at 95 ◦C, the samples are measured at ambient temperature.

Extract (apparent and real) and ethanol (for fermentation monitoring) were analyzed via density meter DMA 4500M with Alcolyzer Beer ME (Anton-Paar GmbH, Graz, Austria) at 20 ◦C and a sample volume of 30 mL.

The pH was determined using a digital pH meter (Mettler Toledo LLC, Columbus, OH, USA).

#### *2.10. Statistical Analyses*

Screening fermentations and analyses were carried out in triplicate. Statistical analysis was performed using RStudio, Version 1.1.463 with R version 3.5.2 (RStudio Inc, Boston, MA, USA; R Core Team, r-project). One-way analysis of variance (ANOVA) was used to compare means, and Tukey's post hoc test with 95% confidence intervals was applied for the pairwise comparison of means. When available, values are given as the mean ± standard deviation. Statistical analyses during the RSM trials were performed using the DesignExpert 9 software (StatEase, Minneapolis, MN, USA).

#### **3. Results and Discussion**

#### *3.1. Yeast Strain Characterization*

To identify the species of the yeast strains, amplification of the D1/D2 domain via PCR was performed and sequenced. The obtained sequences were compared to publicly available sequences in the NCBI nucleotide database via BLAST. The results of the strain identification are shown in Table 3.


**Table 3.** Yeast strain designation, species and origin of yeast strains used in this study.

<sup>1</sup> Research Centre Weihenstephan for Brewing and Food Quality, Technische Universität München; <sup>T</sup> Type strain.

The yeast strains were found to belong to the species *Cyberlindnera misumaiensis* (837A), *C. fabianii* (NT Cyb), *C. jadinii* (L1), and *C. subsu*ffi*ciens* (C6.1). The *Cyberlindnera mrakii* type strain CBS 1707 (former *Williopsis saturnus* var. *mrakii*; synonym NCYC 500) was included in this study as a strain that has previously been investigated for the production of a low alcohol beer with high levels of esters [20]. The *Cyberlindnera subsu*ffi*ciens* type strain CBS 5763 was included as an example to investigate potential intraspecific differences from C6.1.

#### *3.2. API Substrate Utilization*

Before considering non-conventional yeasts for NABLAB brewing, their behavior regarding utilization of important wort sugars like maltose and sucrose should be investigated. An API ID 32C test was performed to investigate the utilization of those sugars and to show general, interspecific differences between the strains. The results of the API test are shown in Table 4.

**Table 4.** Results of the API ID 32C substrate utilization test of the individual strains. Substrates without brewing relevance, which were negative for all strains, are not shown. "+" positive, "−" negative, "w" weak.


<sup>1</sup> Growth "variable" according to Kurtzman et al. [31].

Maltose utilization was positive for NT Cyb, L1 and CBS 1707, in accordance with the reported literature, although assimilation of maltose by CBS 1707 is classified as "variable" [31]. Sucrose utilization was positive for four of the six strains and negative for 837A and CBS1707. The results suggest that in brewers' wort, where maltose is the most abundant fermentable sugar, only NT Cyb, L1 and CBS 1707 have the capability to achieve high attenuations. However, the API test investigates substrate utilization under aerobic conditions. Sugar consumption during fermentation, under anaerobic conditions, can differ significantly [31], which is also known as the Kluyver effect [32]. Due to the inability of 837A and CBS 1707 to utilize sucrose, lower attenuations in fermentations in wort could be expected.

#### *3.3. Stress Tests*

When considering non-*Saccharomyces* yeast strains for brewing purposes, several brewing-relevant parameters such as flocculation behavior, POF production and stress responses should be investigated [33]. The flocculation behavior can give initial indications regarding yeast handling in terms of potential bottom cropping. POF behavior is important because in most beer styles, POF is not desired. Substances like hop-derived iso-α-acids, ethanol content, or the pH value of the wort can have significant influences on yeast activity, manifesting mainly in a prolonged lag time, and even complete growth inhibition [33–35]. With the investigated yeast strains, iso-α-acid concentrations of up to 100 IBU had no significant effect on the yeast growth (data not shown), which is in accordance with previous reports on seven different non-*Saccharomyces* species [34,35]. However, Michel et al. [33] reported a minor prolongation in the lag time of *Torulaspora delbrueckii* strains in concentrations of up to 90 IBU. The results of the investigated characterization attributes are shown in Table 5.


**Table 5.** Characterization of yeast strains for flocculation behavior, phenolic off-flavor (POF) production and lag time in wort with and without a stressor at different concentrations. "—" no growth.

CBS 1707 exhibited the strongest flocculation behavior, at 85%, followed by 837A and CBS 5763, at 78% and 51%, respectively. NT Cyb, L1 and C6.1 exhibited very low flocculation of below 35%. All strains were negative for POF behavior. NT Cyb and C6.1 exhibited the fastest growth in wort (without a stress factor), overcoming the lag time after only 6 hours, followed by L1 and the CBS strains after 9 hours. Strain 837A exhibited a long lag phase of 18 hours (Figure 1). Concentrations of 2.5% ABV ethanol in the wort affected the lag time of all investigated strains. 837A was especially susceptible, with a prolonged lag phase of 120 hours. The remainder of the strains showed an extension of the lag phase of 3 to 12 hours. At 5% ABV, growth was fully inhibited for 837A and CBS 5763, while the other strains again exhibited an extension of the lag phase, of up to a maximum of 48 hours in CBS 1707. Complete growth inhibition was observed for L1 and C6.1 at 7.5% ABV, while the lag phase of NT Cyb and CBS 1707 was prolonged to 42 and 126 hours, respectively. All strains except 837A, which showed a significant extension of the lag phase to 66 hours, remained unaffected by a lower pH of 4. Only at pH 3 were lag times affected, while 837A was fully inhibited. Growth at low pH is important when

considering the yeast for sour beer production, where the yeast must withstand pH values of below 4 [36]. However, it has been shown that organic acids like lactic acid can have a stronger inhibitory effect on yeasts and other microorganisms than HCl, which is caused by its chemical properties as a weak acid [35,37]. Inhibition by lactic acid could therefore be more pronounced than the HCl inhibition observed in this study. Figure 1 shows the growth of the investigated yeast strains in wort without the addition of a stressor.

**Figure 1.** Growth of yeast strains in 7 ◦P wort extract at 25 ◦C without a stressor. Growth curves shown are the mean of a triplicate.

#### *3.4. Screening*

To investigate interspecific differences in the fermentation of wort, fermentation trials were performed in a diluted wort extract of 7 ◦P. Previous studies have shown that extract contents of around 7 ◦P will yield ethanol concentrations of around 0.5% ABV, a popular legal limit for NAB [7], in fermentations with maltose-negative yeast strains [1,14,34,38]. After aerobic propagation for 24 hours, NT Cyb exhibited the highest number of cells, at 2 <sup>×</sup> <sup>10</sup><sup>9</sup> cells/mL, more than four-fold the amount of cells compared to L1, C6.1, and the CBS strains with counts between 3.4 and 4.9 <sup>×</sup> <sup>10</sup><sup>8</sup> cells/mL (Table 6). Due to a delayed growth (compare Figure 1), 837A had to be propagated for 48 hours, reaching a cell count of 6.1 <sup>×</sup> 108 cells/mL. For the screening in wort, yeast cells were added at a concentration of 3 <sup>×</sup> 10<sup>7</sup> cells/mL, after a gentle washing step in water to prevent carry-over of propagation wort sugars. The results from the yeast screening are shown in Table 6. The fermentations were carried out until no change in extract could be measured for two consecutive days.



Cell count after 48 h due to delayed growth compared to other strains (compare Figure 1). LOD 'limit of detection'. Different superscripts of values within a row indicate a significant difference (*<sup>p</sup>*≤0.05).

1 Strains 837A and CBS 1707 exhibited the lowest attenuation of only 18% and 17%, respectively, owing to their inability to utilize sucrose (Table 4), which was confirmed by the lack of sucrose consumption. Liu and Quek [20] also reported the absence of sucrose utilization by CBS 1707. The other strains, which depleted sucrose completely, reached attenuations of 21% to 24%. Consequently, 837A and CBS 1707 also produced, at 0.55% and 0.56% ABV, the lowest amounts of ethanol (*p* ≤ 0.05) compared to the remaining strains, where ethanol concentrations ranged from 0.63% to 0.67% ABV. The final pH of the fermented samples ranged from 4.33 (CBS 5763) to 4.51 (NT Cyb). Residual FAN ranged from 78 (CBS 1707) to 88 mg/L (837A). As expected, none of the strains consumed maltotriose. Maltose consumption was also neglectable in all strains, although the species *Cyberlindnera fabianii* (like NT Cyb) has been reported to be able to ferment maltose [31,39]. The observations also underlined that results from the API substrate utilization test (where NT Cyb, L1 and CBS 1707 were positive for maltose) are not necessarily reflected in practice, especially since sugar utilization during respiration and fermentation can differ [31,32,40]. While glucose was depleted by all strains, fructose was only fully depleted by L1. The remaining strains exhibited glucophilic behavior and consumed only 73% to 83% of fructose during fermentation. Regarding fermentation by-products, glycerol concentrations were low, ranging from 0.18 to 0.36 g/L. The strains 837A and NT Cyb accumulated significantly higher amounts of acetaldehyde, at 9.7 and 8.1 mg/L, respectively, compared to 2.6 to 3.8 mg/L in the remaining samples. The sample fermented with *Cyberlindnera misumaiensis* 837A exhibited extremely high values of ethyl acetate, at 65.7 mg/L, twice the flavor threshold concentration in beer [2,41]. Ethyl acetate is described to have a fruity, estery character but also solvent-like, especially in high concentrations. The remaining strains exhibited ethyl acetate production between 4.9 (C6.1) and 22.6 mg/L (NT Cyb). Isoamyl acetate, which is predominantly described as having a fruity, banana-like aroma, has a much lower flavor threshold of only 1.4–1.6 mg/L [2,41]. The strains C6.1 and CBS 1707 produced the highest amounts of isoamyl acetate, at 1.67 and 1.60 mg/L, followed by CBS 5763, 837A and L1, at 1.03, 0.90 and 0.15 mg/L, respectively. NT Cyb did not produce detectable amounts of isoamyl acetate. Concentrations of ethyl formate and ethyl propionate in the fermented samples were low, ranging from undetectable to 2.7 mg/L. Ethyl butyrate and ethyl caproate were not detected in either of the samples (data not shown). The strain L1 produced a significantly higher amount of higher alcohols, at 35.8 mg/L, followed by NT Cyb, at 27.8 mg/L, and the remaining strains at 20–23 mg/L. During sensory evaluation, the high ethyl acetate concentration in the sample fermented with 837A was indeed perceptible and described as an unpleasant, solvent-like aroma. The sample fermented with NT Cyb was described as having an unpleasant, cabbage-like aroma. The remaining samples were characterized by a pleasant, fruity aroma.

The unpleasant, solvent-like aroma in the sample fermented with 837A was attributed to the very high ethyl acetate concentration, well above the flavor threshold. However, the cabbage-like aroma, which is generally associated with sulfides or thiol compounds [41], that was detected in the sample fermented with NT Cyb could not be linked to the volatile by-products that were measured. Interestingly, ethyl acetate concentrations in the remaining samples, characterized by a pleasant, fruity aroma, were low, at only 2.6–3.8 mg/L. However, C6.1, CBS 1707 and CBS 5763 exhibited higher amounts of isoamyl acetate, a desired ester in beer (particularly ales) [42], when compared to the samples with unpleasant aroma. The concentrations of 1.0–1.6 mg/L are within the reported flavor threshold in beer of 0.5–2.0 mg/L [43]. Additionally, it is well known that synergistic effects between esters occur that can push the concentration of perception below their individual flavor thresholds [42,44,45]. Isoamyl acetate could therefore have been a cause of the fruity aroma in the samples fermented with C6.1, CBS 1707 and CBS 5763. However, the sample fermented with L1, which was also characterized by a fruity aroma, only contained a very low isoamyl acetate concentration of 0.15 mg/L. It is noteworthy, however, that the L1 sample contained a significantly higher amount of isoamyl alcohol, at 23.2 mg/L, which is described as having an alcoholic, fruity and banana-like flavor [2]. The results have confirmed that not a high amount of esters, but rather a balanced profile will lead to a pleasant, fruity aroma [5].

Based on the results from the screening, *Cyberlindnera subsu*ffi*ciens* C6.1 was chosen for optimization of fermentation conditions by means of response surface methodology, followed by an up-scaled brewing trial at 60 L to create a fruity, non-alcoholic beer (≤0.5% ABV). Strains 837A and NT Cyb were eliminated because of their poor flavor characteristics. CBS 1707 was eliminated due to its inability to ferment sucrose, which apart from the lower attenuation, would remain in the wort after fermentation, acting as an additional sweetening agent and potential contamination risk. *Cyberlindnera jadinii* strain L1 was eliminated due to its very low isoamyl acetate production (Table 6) and due to its maltose utilization when oxygen was present (Table 4). The decision between the two similarly performing *Cyberlindnera subsu*ffi*ciens* strains C6.1 and CBS 5763 was made in favor of C6.1 due to a more pleasant fruitiness. In addition, C6.1 showed increased tolerance towards stress caused by ethanol or low pH (Table 5).

#### *3.5. Response Surface Methodology (RSM)*

To find the optimal fermentation conditions for C6.1 for an up-scaled application to produce a fruity, non-alcoholic beer, RSM was performed. Michel et al. [46] applied RSM to optimize the fermentation conditions of a *Torulaspora delbrueckii* strain for brewing purposes. They found that the pitching rate and fermentation temperature were crucial parameters, which influenced the flavor character of the final beer. The optimal fermentation conditions were shown to be at 21 ◦C with a high pitching rate of 60 <sup>×</sup> 10<sup>6</sup> cells/mL. Especially for non-*Saccharomyces* yeasts, the pitching rate can be crucial since most non-*Saccharomyces* species have comparably smaller cell sizes [46]. Figure 2 shows an example of the differing cell size between *Cyberlindnera subsu*ffi*ciens* strain C6.1 (A) and the brewers' yeast strain *Saccharomyces cerevisiae* WLP001 (B) at identical magnification.

**Figure 2.** Scanning electron microscopy (SEM) picture of *Cyberlindnera subsu*ffi*ciens* strain C6.1 (**A**) and the brewers' yeast strain *Saccharomyces cerevisiae* WLP001 (**B**) at a magnification of × 3700. Size of bar: 5 μm.

It is also known that temperature and pitching rate have an influence on ester production, though strain-specific differences also play a role [4,6]. Previously reported fermentation temperatures of *Cyberlindnera subsu*ffi*ciens* and other *Cyberlindnera* spp. range from 20 to 25 ◦C [12,17,19,20,47]. Consequently, a two-factorial, face-centered central composite design was chosen with Factor A: fermentation temperature (17, 22, 27 ◦C), and Factor B: pitching rate (10, 35, 60 <sup>×</sup> 106 cells/mL). The individual experiment runs are listed in Table 2. The wort extract applied in the RSM trial was the same as that used for the screening, at an extract content of 7 ◦P (Table 1). Fermentation was conducted until no change in extract could be measured for two consecutive days. With the measured response values, significant models could be produced. The significant response models, with their respective minima and maxima and a summary of the model statistics, are shown in Table 7. Insignificant response models are not shown, and response models with a significant lack of fit will not be discussed in this

study but are included in the visualized data for the sake of a complete picture. For a full report on model statistics and response values, refer to the supplementary Data Sheet S1.

**Table 7.** Analysis of variance (ANOVA) results for response models of the response surface methodology (RSM) trial.


Model terminology: "RQuadratic" Reduced Quadratic; "2FI" Two-Factor Interaction; "RLinear" Reduced Linear. "LOF" Lack of Fit. ANOVA significance codes: \*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01, \* *p* ≤ 0.05.

It was possible to create significant models for 12 responses (Table 7). However, five also exhibited significant lack of fit (LOF), rendering them unusable for predictions. The aim of the RSM was to investigate the optimal fermentation conditions to create a fruity, non-alcoholic beer. The three-dimensional response surface plots of the interactive effects of temperature and pitching rate on the final ethanol content and the fruitiness of the produced NAB are shown in Figures 3 and 4.

**Figure 3.** Three-dimensional response surface plot of the interactive effects of temperature and pitching rate on the ethanol content of the produced non-alcoholic beer (*p* < 0.01).

**Figure 4.** Three-dimensional response surface plot of the effects of temperature and pitching rate on the fruitiness of the produced non-alcoholic beer (*p* < 0.01).

Ethanol content was lowest at a low temperature of 17 ◦C and low pitching rate (107 cells/mL), and it went up with increasing temperature and pitching rate, but lowered again at a high pitching rate combined with a high fermentation temperature (Figure 3). The minium and maximum values were 0.41% and 0.60% ABV. Sugar analysis revealed that at 17 ◦C and 10<sup>7</sup> cells/mL, about 0.5 g/L of glucose was remaining after fermentation, while it was fully depleted in worts fermented at higher pitching rates and higher temperatures (data not shown). The residual sugar explained the lower final ethanol concentration. Fructose was only fully depleted in the samples that were fermented at 27 ◦C. At 22 ◦C, fermented samples exhibited residual fructose concentrations between 0.2 and 0.5 g/L, and at 17 ◦C, fermented samples showed remaining fructose concentrations between 0.2 and 0.7 g/L. Acetaldehyde concentrations were only dependent on the pitching rate, with increasing amounts of acetaldehyde found at lower pitching rates (Figure A1). This result correlates with other studies that found a decrease in acetaldehyde with increasing pitching rate in wort fermentations with brewers' yeasts [48,49]. However, overdosing yeast (><sup>5</sup> <sup>×</sup> <sup>10</sup><sup>7</sup> cells/mL) can lead to an increase in acetaldehyde again, as observed by Erten et al. [50]. The temperature did not have a significant effect on the acetaldehyde concentration and was therefore excluded from the model (*p* = 0.39; supplementary Data Sheet S1). However, regarding higher alcohols, the fermentation temperature had a stronger effect, with increasing amounts of higher alcohols found at higher temperatures (Figures 5 and A2), which is consistent with the literature [4,5]. Isoamyl acetate concentrations were generally high and ranged from 0.8 to 2.2 mg/L. Although the model was significant (*p* < 0.05), it was unsuitable for value prediction due to a significant lack of fit (*p* = 0.046).

Interestingly, the production of the esters ethyl acetate and isoamyl acetate did not show a clear correlation to temperature, which underlines that the general rule of thumb, that higher fermentation temperatures lead to increased ester production, is not valid for all yeast strains (Figure 5) [4]. Furthermore, the amount of esters that were quantified in this study did not correlate with the perceived fruitiness of the NAB, which tentatively suggests that the fruity flavor profile was caused by yet unidentified compounds (Figure 5).


**Figure 5.** Map visualizing correlations of response surface methodology (RSM) factors and responses based on the Pearson Correlation Coefficient. 1 signifies strong positive correlation, 0 signifies no correlation, and −1 signifies a strong negative correlation.

In terms of fruitiness, a low fermentation temperature paired with a low pitching rate led to the highest perceived fruitiness. Indeed, the highest fruitiness was recorded at 17 ◦C and 1 <sup>×</sup> <sup>10</sup><sup>7</sup> cells/mL and the lowest at 27 ◦C and 6 <sup>×</sup> 10<sup>7</sup> cells/mL, following a linear model. General acceptance showed a strong positive correlation with the fruitiness, indicating that the panel preferred fruity samples (Figures 5 and A3).

Due to the ideal combination of lowest ethanol content and highest fruitiness and acceptance, the fermentation temperature of 17 ◦C and pitching rate of 1 <sup>×</sup> 10<sup>7</sup> cells/mL were chosen as the optimal fermentation conditions for application to produce a fruity, non-alcoholic beer.

A small-scale fermentation at the optimal conditions (17 ◦C, 107 cells/mL) was conducted to validate the RSM model. Table 8 shows the predicted mean including 95% prediction intervals (PI) and the measured ("observed") mean with standard deviation.

Although predicted by a significant model, the observed means for ethanol, acetaldehyde and isobutanol values were not within the 95% prediction interval. Sugar analysis revealed the complete depletion of glucose in the experimental fermentation trial at optimal conditions compared to the RSM model prediction, which explained the increased ethanol production (data not shown). The moderate success in model validation demonstrates the limitations in the application of RSM to optimize fermentations, where small differences in substrate and process conditions can have significant influences on the outcome. Because wort is a very complex substrate, comprising a complex mixture of different sugars, nitrogen sources, minerals and vitamins, among others, any interpretation or the transfer of the RSM results to other substrates (even different wort substrates) should be made with caution. In particular, a different sugar composition will have a significant effect on the responses when applying maltose-negative yeasts. However, the improved fruitiness and therefore higher acceptance of the NAB produced at low temperature and low pitching rate, the main goal from the optimization, was significant and reproducable (Table 8).


**Table 8.** Response surface methodology (RSM) model validation via predicted value vs. observed value.

\* Significant model with insignificant lack of fit. 'PI' Prediction interval.

#### *3.6. Pilot-Scale Brewing*

Despite the limited model validation, the fermentation parameters were successfully optimized to enhance the fruity character of the NAB. Therefore, the pilot-scale brewing trial was conducted with the optimized conditions of 17 ◦C fermentation temperature and a pitching rate of 107 cells/mL.

The grain bill of the wort for the pilot-scale brewing trial consisted of 95% pilsner malt and 5% acidulated malt to lower the starting pH of the wort, to account for the reduced pH drop during fermentations with non-*Saccharomyces* yeasts compared to brewers' yeast. A low beer pH is desired to prevent microbial spoilage and to ensure good liveliness of the beer [51,52]. The analytical attributes of the wort produced at pilot-scale are shown in Table 9.


**Table 9.** Attributes of the wort produced on pilot-scale.

To assess the suitability of *Cyberlindnera subsu*ffi*ciens* C6.1 to produce a fruity NAB, it was compared to two commercial NABs. NAB A was a commercial non-alcoholic beer produced by limited fermentation [29], and NAB B was a non-alcoholic beer produced by "dialysis technology" [30]. The NABs were analyzed for their extract, ethanol, FAN and glycerol content as well as their sugar composition and concentration of volatile fermentation by-products. The results are shown in Table 10.


**Table 10.** Attributes of the non-alcoholic beer (NAB) produced with C6.1 compared to two commercial NABs, NAB A and NAB B.

The C6.1 NAB reached final attenuation after 13 days of fermentation at 17 ◦C, at an ethanol content of 0.36% ABV. At the end of fermentation, 2.77 g/L glucose was remaining in the wort and sucrose was fully depleted. Compared to the initial sugar concentration of the wort (Table 9), fructose concentrations in the final beer were significantly higher, at 1.65 g/L, twice as high as the starting concentration in the wort. Since sucrose was fully depleted, it can be assumed that it was converted to glucose and fructose by the yeast's invertase. The high residual fructose could therefore be attributed to the previously observed glucophilic character of the C6.1 strain in the screening and RSM trial. As a result, fructose was not consumed by the yeast due to the permanent presence of glucose until fermentation came to a halt. As expected, maltose and maltotriose consumption was negligible. Despite the limited fermentation, C6.1 produced a relatively high amount of esters, at 12.8 mg/L, the majority of which was ethyl acetate (12 mg/L). NAB A had an ethanol content of 0.50% ABV. Interestingly, the sugar composition was very similar to that of the C6.1 NAB. Regarding fermentation by-products, however, NAB A exhibited very low concentrations, at about half the amount of higher alcohols and a total lack of the esters ethyl acetate and isoamyl acetate. NAB B had an ethanol content of 0.49% ABV. Owing to its fundamentally different production method, the analyzed attributes were very different from those of the two NABs produced solely by limited fermentation. The low FAN content together with a high glycerol content compared to the other NABs were indicators of a more extensive fermentation, with subsequent removal of ethanol. However, NAB B still exhibited high amounts of monosaccharides, which suggested that the production of the NAB either also entailed a limited fermentation, or the dealcoholized beer was blended with wort (or other means of sugar addition). The increased amounts of higher alcohols in NAB B, at 24.8 mg/L, are uncommon for beers dealcoholized via dialysis, since the process commonly reduces their content in the final NAB by 90%–95% [7]. Despite the addition of acid malt during the wort production for the C6.1 NAB, the final pH after fermentation was, at 4.45, higher compared to 4.29 in the commercial NABs.

Due to the high amounts of residual sugars, proper pasteurization is essential for non-alcoholic beers produced by limited fermentation to avoid microbial spoilage [1,38,53]. After bottling, C6.1 NAB was therefore pasteurized with approximately 23 PU, and the successful pasteurization was confirmed by plating the pasteurized NAB on agar to check for microorganism growth, which was found to be negative.

#### *3.7. Sensory Evaluation*

For a holistic evaluation of the C6.1 NAB compared to the two commercial NABs, a sensory trial was conducted with 10 trained and experienced panelists. The panel was asked to describe the flavor of the beer in their own words, followed by an assessment of several intensity attributes. The mean score values of the parameters wort-like, floral, fruity, citrus-like and tropical aroma, as well as sweet taste, of the NABs are shown in Figure 6.

**Figure 6.** Spider web with the means of the descriptors from the sensory trial of the NAB produced with *Cyberlindnera subsu*ffi*ciens* C6.1 and the two commercial NABs. Different letters next to data points indicate a significant difference as per Tukey's *post hoc* test. Significance codes: \*\*\* *p* ≤ 0.001, \*\* *p* ≤ 0.01.

The NAB produced with C6.1 was described as very fruity with aromas of pear, banana, mango and maracuja together with a slightly wort-like character. NAB A was described as malty, wort-like and hoppy, while NAB B was described as wort-like and caramel-like. The C6.1 NAB was indeed evaluated as being significantly more fruity than the commercial NABs (*p* ≤ 0.01), at an average of 3.6 out of 5 compared to 2.1 and 2.2 out of 5, scoring also higher in citrus-like and tropical aromas. Consequently, the wort-like aroma, one of the most criticized flaws of NABs produced by limited fermentation [1,2,52], was least pronounced in the NAB produced with C6.1 with an average of 1 out of 5, followed by NAB B with 1.8 out of 5. NAB A exhibited, at an average of 3.2, a significantly more pronounced wort-like aroma (*p* ≤ 0.001). A sweet taste, caused by a high amount of residual sugars, is another major point of criticism for NABs produced by limited fermentation [1,2,52]. All NABs scored similarly in sweet taste without significant differences. NAB B scored lower for "floral" compared to the other NABs. However, the difference was not statistically significant. When the panelists were asked for their favorite sample, 40% chose C6.1 NAB, 40% chose NAB A, and 20% chose NAB B. Similarly, Strejc et al. [3] investigated the production of a non-alcoholic beer (0.5% ABV) by a cold contact process (characterized by a low temperature and high pitching rate) with a mutated lager yeast strain (*Saccharomyces pastorianus*). The strain's targeted mutation resulted in an overproduction of isoamyl acetate and isoamyl alcohols. The authors reported that the fruity flavour of the NAB produced with the mutated strain was "partially able to disguise" the typical wort-like off-flavor [21]. However, the isoamyl acetate concentration of the resulting NAB was, at 0.5 mg/L, lower than the concentration in the C6.1 NAB in this study (Table 10). Furthermore, the complex mutation and isolation procedure paired with a potentially limited stability of the mutation limits its applicability in practice. Saerens and Swiegers [22] reported the successful production of a NAB at 1000 L scale with a *Pichia kluyveri* strain, owing to its high production of isoamyl acetate (2–5 mg/L), which reportedly gave the NAB a fruity flavor that was more like that of a regular beer than commercial NABs. In accordance, the results of the sensory indicated that a strong fruity aroma can mask the wort-like off flavor, and that

the non-*Saccharomyces* yeasts, which produce a pronounced fruity character, can therefore be a means to produce NAB with improved flavor characteristics.

#### **4. Conclusions**

The *Cyberlindnera* genus was found to be a promising non-*Saccharomyces* genus for application in the production of a fruity, non-alcoholic beer. Four of the six investigated species produced a fruity character, despite the limited fermentative capacity, which resulted in a low ethanol concentration. It was shown that through optimization of the fermentation parameters of temperature and pitching rate, the fruity character could be enhanced. Process up-scaling with *Cyberlindnera subsu*ffi*ciens* strain C6.1 produced a NAB that was significantly more fruity compared to two commercial NABs. Owing to the strong fruity aroma, the often-criticized wort-like aroma could successfully be masked. Yeast handling throughout the process (i.e., propagation, yeast pitching, fermentation) proved to be suitable for pilot-scale brewing, with potential for application at industrial scale. Further studies should investigate if the masking effect was enhanced by a reduction of wort aldehydes via yeast metabolism.

This study demonstrated the suitability of the non-*Saccharomyces* species *Cyberlindnera subsu*ffi*ciens* for the production of non-alcoholic beer (<0.5% ABV) with novel flavor characteristics that can compete with commercial NABs. The successful pilot-scale (60 L) brewing trial gives prospect to future studies with diverse non-*Saccharomyces* yeasts and strengthens their position as a serious and applicable alternative to established methods in non-alcoholic and low alcohol beer brewing.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2311-5637/5/4/103/s1, Data Sheet S1: RSM response values and model statistics.

**Author Contributions:** Conceptualization, K.B., M.M., M.H., M.Z. and E.K.A.; methodology, K.B. and M.M.; investigation, K.B., J.J.A., and A.H.; resources, M.H., F.J. and E.K.A.; formal analysis, K.B.; writing—original draft preparation, K.B.; writing—review and editing, K.B., M.M., M.Z., M.H., K.M.L. and E.K.A.; visualization, K.B.; supervision, E.K.A.; project administration, E.K.A.; funding acquisition, E.K.A.

**Funding:** This research was supported by the Baillet Latour Fund within the framework of a scholarship for doctoral students.

**Acknowledgments:** The authors would like to thank Philippe Lösel for his valuable contribution to this study, Muntons plc for supplying the sprayed wort extract, the Barth-Haas Group for supplying the iso-α-acids extract, and David De Schutter and Luk Daenen for their review.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A**

**Figure A1.** Three-dimensional response surface plot of the effect of pitching rate on the acetaldehyde content of the produced NAB (*p* < 0.01). The factor temperature was excluded from the model due to insignificance (*p* = 0.39; supplementary Data Sheet 1).

**Figure A2.** Three-dimensional response surface plot of the interactive effects of temperature and pitching rate on the sum of higher alcohols of the produced NAB (*p* < 0.001).

**Figure A3.** Three-dimensional response surface plot of the effects of temperature and pitching rate on the overall acceptance of the produced NAB (*p* < 0.05).

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Evolution of Aromatic Profile of** *Torulaspora delbrueckii* **Mixed Fermentation at Microbrewery Plant**

#### **Laura Canonico 1,\*, Enrico Ciani 2, Edoardo Galli 1, Francesca Comitini <sup>1</sup> and Maurizio Ciani 1,\***


Received: 10 December 2019; Accepted: 6 January 2020; Published: 8 January 2020

**Abstract:** Nowadays, consumers require quality beer with peculiar organoleptic characteristics and fermentation management has a fundamental role in the production of aromatic compounds and in the overall beer quality. A strategy to achieve this goal is the use of non-conventional yeasts. In this context, the use of *Torulaspora delbrueckii* was proposed in the brewing process as a suitable strain to obtain a product with a distinctive aromatic taste. In the present work, *Saccharomyces cerevisiae*/*T. delbrueckii* mixed fermentation was investigated at a microbrewery plant monitoring the evolution of the main aromatic compounds. The results indicated a suitable behavior of this non-conventional yeast in a production plant. Indeed, the duration of the process was very closed to that exhibited by *S. cerevisiae* pure fermentation. Moreover, mixed fermentation showed an increase of some aromatic compounds as ethyl hexanoate, α-terpineol, and β-phenyl ethanol. The enhancement of aromatic compounds was confirmed by the sensory evaluation carried out by trained testers. Indeed, the beers produced by mixed fermentation showed an emphasized note of fruity/citric and fruity/esters notes and did not show aroma defects.

**Keywords:** *Torulaspora delbrueckii*; craft beer; microbrewery plant; mixed fermentation; aroma profile

#### **1. Introduction**

In the last years, there has been a worldwide growth in microbreweries, which leads to competition in the beer market to find new beers and also those that are characterized by peculiar aroma taste. To achieve this, the brewers paid attention to the ingredients which are water, malts, hops, and yeast [1–4]. In particular, the brewers focused their attention on the yeast strains to use in brewing fermentation which are selected not only for their good fermentation efficiency but also for their characteristic aroma and flavors.

In this regard, several recent investigations were focused on the selection of non-conventional yeasts [5–8]. Non-*Saccharomyces* yeasts represent a large source of biodiversity to produce new beer styles. In the last years, different non-*Saccharomyces* yeasts were proposed in brewing, such as *Brettanomyces bruxellensis*, *Torulaspora delbrueckii*, *Candida shehatae*, *Candida tropicalis*, *Zygosaccharomyces rouxii*, *Lachancea thermotolerans*, *Saccharomycodes ludwigii*, and *Pichia kluyveri* [9–13]. *T. delbrueckii* is one of the most well-known non-*Saccharomyces* yeasts and it can be found in wild environments such as plants and soils as well as in wine or in fermented food processes. In the brewing process, *T. delbrueckii* received particular attention due to its ability to ferment maltose, produce ester compounds, and biotransform the monoterpenoid flavor compounds of hops [12,14–16]. In particular, *T. delbrueckii* can

improve the amount of different fruity aromas, such as β-phenyl ethanol ("rose" flavors), n-propanol, iso-butanol, amyl alcohol ("solvent brandy" aroma), and ethyl acetate [17–19].

Canonico et al. [6,12] evaluated the use of *T delbrueckii* for beer production, both pure and in mixed cultures with different *S. cerevisiae* starter strains. *T. delbrueckii* in mixed fermentation with different *S. cerevisiae* starter strains showed different behavior and resulting in beers with distinctive flavors. Generally, the main aromatic compounds that were affected by *T. delbrueckii* are some fruity esters. Furthermore, in mixed fermentation, *T. delbrueckii* provided higher levels of higher alcohols, in contrast to data obtained in winemaking, where higher alcohols had lower levels. Moreover, beers obtained with *T. delbrueckii* pure cultures were characterized by a distinctive analytical, aromatic profile, and a low alcohol content (2.66% *v*/*v*) [12].

Michel et al. [16] investigated different *T. delbrueckii* strains coming from different habitats. One strain was able to produce a fruity and floral aroma (β-phenyl ethanol) and amyl alcohols. Furthermore, two strains were found to be suitable for producing low-alcohol beer owing to their inability to ferment maltose and maltotriose but still produced good flavor. However, investigation into the use of non-conventional yeasts in the brewing process has been performed at a laboratory scale or at a pilot scale while validation trials are lacking at the industrial level, which would give a more accurate assessment of their brewing ability. For this reason and based on the results of previous investigations [6,12] in this study, the contribution of *T. delbreuckii* in mixed fermentation with *S. cerevisiae* starter strain at inoculum ratio 1:20 was assessed at the microbrewery plant. The effect of this non-conventional yeast in mixed fermentation on the evolution of biomass and aroma profile as well as on the final beer composition was evaluated. The sensorial profile of the final beers was also tested.

#### **2. Materials and Methods**

#### *2.1. Yeast Strains*

*T. delbrueckii* DiSVA 254 comes from the Yeast Collection of the Department of Life and Environmental Sciences (DiSVA) of the Polytechnic University of Marche (Italy). *T. delbrueckii* strain DiSVA 254 and *S. cerevisiae* commercial strain US-05 (Fermentis, Lesaffre, Marcq En Baroeul, France) were used in mixed fermentation at inoculum ratio 20:1 as reported in a previous study [12]. The US-05 was rehydrated following the manufacturer's instructions and was plated on YPD agar medium at 25 ◦C, by spreading 0.1 mL yeast suspension onto the surface of the medium.

The yeast strains were maintained on yeast extract (10 g/L), peptone (20 g/L), dextrose (20 g/L), (YPD) agar (18 g/L) at 4 ◦C, for short-term storage, and in YPD liquid with 80% (*w*/*v*) glycerol at −80 ◦C for long-term storage.

#### *2.2. Wort Production and Fermentation Condition*

The wort used for the trials was produced at Birra dell'Eremo Microbrewery (Assisi, Italy) from a batch of 1500 L in duplicate fermentations. The wort was made with pilsner malt (100%), the Cascade hop variety, and produced according to the scheme reported by Canonico et al. [6]. The main analytical characters of this wort were pH 5.5, specific gravity 12.3◦ GPlato, and 20 IBU. The fermentation process was carried out in 2 different batches of 1500 L at 20 ◦C.

#### *2.3. Growth Kinetics*

The biomass evolution was monitored during the fermentation process using viable cell counts on WL Nutrient Agar (Oxoid, Hampshire, UK) and Lysine Agar (Oxoid, Hampshire, UK). Lysine Agar is a medium unable to support the growth of *S. cerevisiae* [20] for the differentiation of *T. delbrueckii* yeast from *S. cerevisiae* US-05 starter strain.

#### *2.4. Bottle Conditioning*

At the end of the fermentation process, the beers obtained were transferred into 500-mL bottles, adding 5.5 g/L of sucrose. The secondary fermentation in the bottle was carried out at 18–20 ◦C for 7–10 days.

#### *2.5. Analytical Procedures*

The contents of acetaldehyde, ethyl acetate, higher alcohols (n-propanol, isobutanol, amyl alcohol, isoamyl alcohol) were determined by direct injection into a gas–liquid chromatography system. The volatile compounds were determined by the solid-phase microextraction (HS-SPME) method. Five ml of each sample was placed in a vial containing 1g NaCl closed with a septum-type cap. HS-SPME was carried out under magnetic stirring for 10 min at 25 ◦C. After this period, an amount of 3-octanol as the internal standard (1.6 mg/L) was added and the solution was heated to 40 ◦C and extracted with a fiber Divinylbenzene/Carboxen/Polydimethylsiloxane (DVB/CAR/PDMS) for 30 min by insertion into the vial headspace. The compounds were desorbed by inserting the fiber into the Shimadzu gas chromatograph GC injector for 5 min. A glass capillary column was used: 0.25 μm Supelcowax 10 (length, 60 m; internal diameter, 0.32 mm). The fiber was inserted in split–splitless mode: 60 s splitless; the temperature of injection, 220 ◦C; the temperature of detector, 250 ◦C; carrier gas, with nitrogen; flow rate, 2.5 mL/min. The temperature program was 50 ◦C for 5 min, 3 ◦C/min to 220 ◦C, and then 220 ◦C for 20 min. The compounds were identified and quantified by comparisons with external calibration curves for each compound.

#### *2.6. Sensorial Analysis*

At the end of the fermentation process, the beers obtained were transferred into 330-mL bottles, adding 5.5 g/L sucrose. The secondary fermentation in the bottle was carried out at 18–20 ◦C for 7–10 days. After this period, the beers were stored at 4 ◦C underwent sensory analysis using a scale from 1 to 10 (Analytica EBC, 1997). This was carried out by a group of 14 trained testers, that evaluated the main aromatic notes regarding the olfactory and gustatory perception and structural features. The data were elaborated with statistical analyses to obtained information about the contribution of each descriptor on the organoleptic quality of beer.

#### *2.7. Statistical Analysis*

Analysis of variance (ANOVA) was applied to the main characteristics of the beers. The means were analyzed using the STATISTICA 7 software. The significant differences were determined by the means of Duncan tests, and the results were considered significant if the associated *p*-Values were < 0.05. The results of the sensory analysis were also subjected to Fisher ANOVA, to determine the significant differences with a *p*-Value < 0.05.

#### **3. Results**

#### *3.1. Yeast Species Evolution*

The growth kinetics of *T. delbrueckii* in mixed fermentations and *S. cerevisiae* pure culture were reported in Figure 1.

The growth kinetics of the *S. cerevisiae* US-05 pure cultures achieved ca. 107 CFU/mL at 3 days of fermentation and decreased at 10<sup>6</sup> CFU/mL until the end of fermentation. Regarding the mixed fermentation, *S. cerevisiae* reached cell concentrations <106 CFU/mL at 3 days of fermentation and decreased at 10<sup>5</sup> CFU/mL, while *T. delbrueckii*, started at a concentration >106 CFU/mL, achieved the maximum cell concentration at 3 days of fermentation (107 CFU/mL), and decreased at the end of fermentation (10<sup>6</sup> CFU/mL). The results for mixed fermentation indicated that *T. delbrueckii* at 20-folds higher than *S. cerevisiae* dominated the fermentation process and highlighted a high level of competitiveness of *T. delbrueckii* towards *S. cerevisiae* commercial strain.

**Figure 1.** Growth kinetics of pure and mixed fermentation. Pure culture of *S. cerevisiae* ( ), *S. cerevisiae* ( ), and *T. delbrueckii* ( ) individually for the mixed fermentation.

#### *3.2. Main Analytical Profile*

The analytical compositions of the beers are reported in Table 1.


**Table 1.** The main analytical characteristics of the beer produced by pure and mixed fermentations.

Data are means ± standard deviation. The initial composition of the sugars in the wort was sucrose 5.9 g/L; glucose 8.2 g/L; maltose 61.76 g/L. The wort gravity at the start was 12.3 ◦P.

Both trials finished the process on the 10th day of fermentation highlighting that *T. delbrueckii* in the condition used at the microbrewery plant did not influence the time of the fermentation process.

*S. cerevisiae*/*T. delbrueckii* mixed fermentation and *S. cerevisiae* pure fermentation, produce beer with a comparable amount of ethanol content and final values of ◦P. Regarding the residual sugar, both fermentation trials consumed all sucrose and glucose content, while beer brewed by *S. cerevisiae*/*T. delbrueckii* mixed fermentation exhibited a slightly higher amount of maltose.

#### *3.3. By-Products and Volatile Compounds*

The main volatile compound by-products are reported in Table 2.

For the main volatile compound by-products, *S. cerevisiae* US-05/*T. delbrueckii* mixed fermentations showed different profiles to those produced by *S. cerevisiae* US-05 pure fermentation. In particular, the evolution of the main aroma compounds during the fermentation process showed that β-phenyl ethanol significantly increases in mixed fermentation in all steps of the fermentation process if compared with *S. cerevisiae* starter strain pure culture. Differently, there were no significant differences between the trials for amyl and isoamyl alcohol content with the exception of *S. cerevisiae* pure culture trials, which exhibited a lower amount of these two alcohols at the first step of fermentation (after one day).



#### *Fermentation* **2020**, *6*, 7

Acetaldehyde content showed a different trend: pure culture trials showed a progressive reduction of this carbonyl compound during the fermentation, while the *S. cerevisiae*/*T. delbrueckii* mixed fermentation exhibited the same acetaldehyde content during the process. Ethyl acetate and ethyl hexanoate were detected only in mixed fermentation until the beginning of fermentation. The same trend was also exhibited by α-terpineol. Moreover, regarding ethyl hexanoate and α-terpineol, there was a significant increase at the end of fermentation. For isoamyl acetate content, the results did not show a significant difference between the two fermentations.

#### *3.4. Sensory Analysis*

The beers obtained by pure and mixed fermentations underwent sensory analysis, and the results were illustrated in Figure 2.

**Figure 2.** Sensory analysis of beer produced in the microbrewery plant by the *T. delbrueckii* mixed fermentation. From pure cultures of *S. cerevisiae* ( ) and mixed cultures of *S. cerevisiae*/*T. delbrueckii* ( ). (**A**) Olfactory analysis; (**B**) gustatory analysis. DMS, dimethyl sulfide. \* = Significantly different (Fisher ANOVA).

All of the beers analyzed showed significant differences for their main aromatic notes regarding the olfactory and gustatory analysis. In particular, for the main sensorial descriptors, the data showed that the beer obtained with the mixed fermentation was significantly different from that of the *S. cerevisiae* US-05 starter strain for a variety of the sensorial characteristics. Regarding olfactory analysis (Figure 2A), beers brewed with *S. cerevisiae*/*T. delbrueckii* mixed fermentation showed a bouquet with notes that emphasized the fruity/esters, fruity/citric, and caramel. Moreover, the perception of DMS (dimethyl sulfide) and other sulfide compounds shows they are less well perceived than beers obtained by *S. cerevisiae* pure culture. However, the only significant difference between the two beers was exhibited by the cereal note, which resulted in the emphasis of the product brewed with the *S. cerevisiae* starter strain.

Regarding gustatory analysis (Figure 2B), the beers obtained by mixed fermentation are characterized by the significant perception of fruity/esters notes. The beers obtained with *S. cerevisiae* pure culture were significantly characterized by hop and cereal notes.

In addition, the beers produced by *T. delbrueckii* mixed fermentation were characterized by a pale yellow color, clarity, and persistent and compact foam, which are very important features in the assessment of the quality of a beer (data not shown).

#### **4. Discussion**

The use of non-conventional yeasts in the brewing process was recently proposed with the aim to produce beers with distinctive aromatics note or to develop a new technology to increase the typicity of specialty beers such as low-calorie beer, low alcohol beer, novel flavored beer, and gluten-free beer [8,12,13,16,21–24]. In previous studies, the use of *T. delbrueckii* (strain DiSVA 254) in mixed fermentation with *S. cerevisiae* starter strains was investigated at a laboratory scale [6,12]. The results indicated a promising behavior of this yeast for use at microbrewery plants. Indeed, the interactions between *S. cerevisiae* and *T. delbrueckii* produced beers characterized by a distinctive aromatic profile (fruity/citric notes, fruity/esters notes, and full-bodied attributes) [12]. For these reasons, the application of *S. cerevisiae*/*T. delbrueckii* was assessed at the microbrewery plant evaluating the evolution of the volatile compounds during the fermentation process. The first relevant aspect for its application at the industrial level was the duration of fermentation. Similarly, to the laboratory-scale trials, the brewing process carried out with *T. delbrueckii* mixed fermentation showed a comparable fermentation time to that exhibited by the *S. cerevisiae* starter strain showing good competitiveness with *S. cerevisiae* in the co-culture. This aspect is crucial for its application in a microbrewery where for economic reasons the fermentation process should not exceed 10–15 days.

Regarding the evolution of aroma compounds, generally higher alcohols did not show a significant difference between mixed and pure fermentations. Regarding β-phenyl ethanol content, known for the rose and floral aroma with an odor threshold of 10 mg/L [18], the results showed an increase in mixed fermentation exhibiting a different trend by a previous study [6,12]. An increase of β-phenyl ethanol was observed by Toh et al. [25] and Drosou et al. [26] highlighted that the production of this compound was determined by *T. delbrueckii* and *S. cerevisiae* strains used in fermentation but also by the fermentation condition.

Esters compounds produced by esterification between alcohol and short- or long-chain fatty acids are important compounds that can affect the aroma of the beer [27]. Phenyl ethyl acetate is known for the floral, sweet, honey, and fruity aroma with a threshold of 3.8 mg/L [16] was significantly affected by the presence of *T. delbrueckii* as previously reported [6,12,26].

This study, confirming a previous study [12], showed the significant increase of ethyl hexanoate, fruity esters associated with apple flavor [27], when *T. delbrueckii* was used in mixed fermentation while a different trend was observed with different *T. delbrueckii* and *S. cerevisiae* strains [25,26]. αterpineol, the terpene responsible for balsamic/fruit notes, was detected only in mixed fermentation and highlighted that these aroma compounds were related to *T. delbrueckii*.

Regarding the evolution of the main aroma compounds during fermentation, the *S. cerevisiae* pure culture and mixed fermentation exhibited a different trend. In particular, the evolution of acetaldehyde content is related to a different metabolic pathway of *S. cerevisiae* and *T. delbrueckii*. Indeed, in *S. cerevisiae* fermentation the acetaldehyde content decreased during the fermentation process, while in mixed fermentation the content of this carbonyl compound remains similar from beginning to end. The same trend was also exhibited for the main alcohol compounds.

Few works are present in the literature regarding the application of *T. delbrueckii* in the brewing process and there are no data about its use at the industrial level. These results confirming the fermentation behavior of *T. delbrueckii* in mixed fermentation, emphasize and reinforce its possible use at the industrial level allowing one to obtain beers with characteristics different from those obtained with *S. cerevisiae* starter strains and with a sensory profile appreciated by tasters.

**Author Contributions:** L.C., E.C., E.G., F.C., and M.C. contributed equally to this manuscript. All authors participated in the design and discussion of the research. L.C. carried out the experimental part of the work. E.C. carried out the fermentation in the microbrewery. L.C., E.C., E.G., F.C., and M.C. carried out the analysis of the data and wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** The authors wish to thank the Birra dell'Eremo Microbrewery (Assisi, Italy) for making the microbrewery available and for supporting the technical experimental design. Moreover, thanks go to the UNIONBIRRAI association (Milano, Italy) and the trained testers belonging to UNINOBIRRAI BEER TASTERS (UBT Marche region) for helping the authors to complete the study for an industrial application.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Communication* **Adaptive Evolution of Industrial Brewer's Yeast Strains towards a Snowflake Phenotype**

**Yeseren Kayacan 1,2,**†**, Thijs Van Mieghem 1,2,**†**, Filip Delvaux 3, Freddy R. Delvaux <sup>3</sup> and Ronnie Willaert 1,2,4,\***


Received: 10 January 2020; Accepted: 3 February 2020; Published: 5 February 2020

**Abstract:** Flocculation or cell aggregation is a well-appreciated characteristic of industrial brewer's strains, since it allows removal of the cells from the beer in a cost-efficient and environmentally-friendly manner. However, many industrial strains are non-flocculent and genetic interference to increase the flocculation characteristics are not appreciated by the consumers. We applied adaptive laboratory evolution (ALE) to three non-flocculent, industrial *Saccharomyces cerevisiae* brewer's strains using small continuous bioreactors (ministats) to obtain an aggregative phenotype, i.e., the "snowflake" phenotype. These aggregates could increase yeast sedimentation considerably. We evaluated the performance of these evolved strains and their produced flavor during lab scale beer fermentations. The small aggregates did not result in a premature sedimentation during the fermentation and did not result in major flavor changes of the produced beer. These results show that ALE could be used to increase the sedimentation behavior of non-flocculent brewer's strains.

**Keywords:** *Saccharomyces cerevisiae*; industrial brewer's strains; adaptive laboratory evolution (ALE); snowflake phenotype; beer fermentation

#### **1. Introduction**

Bulk sedimentation of yeast cells during fermentation is a crucial part of the brewing process. At the end of the fermentation, single yeast cells aggregate and form macroscopic "flocs" [1,2]. These clumps of cells then rapidly sediment from the beer and can be harvested from the bottom (lager fermentation) or float and can be harvested from the top (open ale fermentation) at the end of the primary fermentation. This phenomenon allows the brewer to separate the yeast from the beer in an effective, cost-efficient, and environmentally-friendly way, leaving only the clear and almost cell-free product. The neatly harvested yeast can also be "repitched" into the next fermentation. The timing of sedimentation is of considerable importance to the process. Sedimentation should not take place prematurely and cause stuck fermentation leading to beers with low quality flavor profiles. Complete sedimentation at the end of fermentation is preferred by the brewer, which provides the opportunity for a neat separation of the yeast cells from the beer [3].

Flocculation is the reversible, asexual self-adhesion of yeast cells which leads to their sedimentation [4]. Although single yeast cells do sediment, the large clumps formed by flocculating cells sediment at a much higher rate. The flocculation capacity of yeast is highly strain-dependent, influenced mainly

by the genetic background since expressed cell wall flocculins that are lectins, effectuate cell-cell binding [5–7]. However, several environmental factors can affect flocculation too. Calcium availability, pH, temperature, ethanol concentration and oxygen concentration are some of the physiological factors that influence flocculation while physical factors such as cell surface hydrophobicity and favorable hydrodynamic conditions can also affect formation of flocs [8–10]. The sedimentation rate is dependent on the size, shape and density of these flocs.

Optimization of the brewer yeast towards a more flocculating phenotype can lead to a more efficient beer production and a higher final beer quality. Recent advances in DNA sequencing, high-throughput technologies and genetic manipulation methods have led to the molecular and genomic characterization of the brewer's yeast. However, the exponential increase in knowledge generated in the field of functional genomics of yeast can only facilitate strain improvement efforts to some degree. Procedures to obtain approval for modified GMO yeasts are complicated and consumer acceptance for a GMO-produced beer is lacking [11,12]. These hurdles have guided researchers to look elsewhere to generate strains with desired properties.

One attractive more "natural" approach to enhance the attributes of microorganisms is the adaptive laboratory evolution (ALE) approach [13]. In ALE, microorganisms are cultivated under clearly defined conditions for long periods of time, allowing metabolic engineering of microorganisms utilizing genetic variation and selection for beneficial mutations [14,15]. Already a more-and-more used tool in microbial strain improvement, ALE has been applied for improving yeast strains such as for the utilization of alternative sugars by *S. cerevisiae*[16], increasing tolerance of *S. cerevisiae*to environmental conditions [17], for increasing the fermentation capacity of a lager *S. pastorianus* brewing strain under hyperosmotic conditions [18], modifying the production of flavor compounds by *S. pastorianus* for alcohol-free beer production [19], the adaptation of lager strains to very high-gravity brewing conditions [20,21], and for enhancing the fermentation rate with decreased formation of acetate and greater production of fermentative aroma of *S. cerevisiae* wine strains [22,23].

Microbial cells can be cultivated in parallel serial cultures for ALE but varying population densities, fluctuating growth rate, nutrient supply, and environmental conditions characterize this batch cultivation. Continuous (chemostat) cultures, however, ensure more stable conditions such as constant growth rate, tightly controlled nutrient supply and stable pH and oxygen availability [24–26].

Previously, we performed ALE of a *S. cerevisiae* strain in a 3D-printed continuous mini tower fermentor using gravity as a selective pressure to obtain a snowflake phenotype [27]. In this work, we've used the ALE approach for the continuous cultivation of three non-flocculating industrial *S. cerevisiae* brewing strains in miniature chemostats (ministats) [28]. This simple and low-cost setup was used to carry out adaptive evolution experiments where gravity is also the selective pressure on the planktonic cells, which are continuously removed while aggregating cells' sediment are retained. Stable aggregating cells were observed during continuous cultivation, showing a "snowflake" phenotype of unseparated daughter and mother cells. Finally, we have also demonstrated the beer fermentation performance of these evolved strains in lab scale tall tubes fermentors.

#### **2. Materials and Methods**

#### *2.1. Yeast Strains and Media*

The industrial *Saccharomyces cerevisiae* brewer's strains BCD1, BCD2, BCD3, and BCD4 were provided by Biercentrum Delvaux (Neerijse, Belgium). The lab strains BY4742 [29], BY4742 [*FLO1*], and BY4742::*FLO8* [5] and the strong flocculating industrial strain BCD4 were used as control strains in the flocculation assay. All strains were precultured in YPD (Yeast extract–peptone–dextrose) medium (1% *m*/*v* yeast extract, 2% *m*/*v* peptone, 4% *m*/*v* glucose) overnight at 30 ◦C. For the continuous ALE in ministats, a high-glucose medium (100 g/L D-glucose, 4 g/L (NH4)SO4, 1.5 g/L KH2PO4,1g/L MgSO4·7H2O, and 5 g/L yeast extract) was used. The yeast cells and aggregates were visualized by microscopy (Nikon Eclipse Ti2, Tokyo, Japan).

#### *2.2. Flocculation Assay*

The assay described by D'Hautcourt and Smart [30] was used with minor modifications. Cells were cultivated for 24 h in YPD, harvested by centrifugation and resuspended in EDTA buffer (50 mM EDTA, pH 7) to reach an OD600nm value of 10. A sample of 50 μL was taken at 0.5 mL below the meniscus, and the sample was diluted 20 times in a 1.5 mL cuvette with EDTA buffer (50 mM EDTA, pH 7). The tubes were centrifuged (4000 rpm, 3 min), and the supernatant was discarded. The cells were resuspended in 1 mL flocculation buffer A (3 mM CaSO4). The last step was repeated, but the cells were resuspended in flocculation buffer B (3 mM CaSO4, 83 mM CH3COONa, 4% *v*/*v* ethanol, pH 4.5). The tubes were shaken at 100 rpm for 10 min. Prior to taking 50 μL samples 0.5 mL below the meniscus, 3 min of sedimentation in a vertical position took place. The sample was diluted 20 times with EDTA buffer in a 1.5 mL cuvette. The absorbance of both suspensions in the cuvettes was determined, and the related flocculation percentage was calculated:

$$\text{Floculation percentage} \left( \% \right) = \frac{OD\_{EDTA} - OD\_{Focalation\ buffer}}{OD\_{EDTA}} \times 100$$

For the evolved strains, *ODEDTA* corresponded to the OD600nm value of the non-evolved reference strain.

#### *2.3. Experimental Setup with Ministats*

The continuous fermentation of industrial strains was carried out in a ministat set-up (Figure S1) [28]. Briefly, 15 mL test tubes were kept in an analog heat block (VWR®, Bridgeport, NJ, USA) at 30 ◦C and fed with high-glucose growth medium using a peristaltic pump (Type ISM833A, Ismatec®, Zurich, Switzerland). This high-glucose growth medium was previously also used in the adaptive evolution of *S. cerevisiae* strains towards a snowflake phenotype using a mini tower fermentor [27]. Medium was supplied at a flow rate of 30 μl/min. Air from a 4-port aquarium pump was fed to the medium in the test tube through an air filter (0.20 μm) and the needle was pushed to the bottom of the tube, in order to agitate the solution. During the experiment, a volume of 7–10 mL was maintained. The ministats were inoculated with 1 mL of an overnight culture. The pH, cell concentration (OD at 600 nm) and the glucose concentration (estimated using a refractometer (Brouwland, Belgium)) were measured during the ALE experiments.

#### *2.4. Wort Fermentations in Tall Tubes*

Laboratory-scale tall tubes, made from glass (75 cm high and 8-cm diameter), were used to assess beer fermentation with the evolved strains (Figure S2a). The tall tubes were filled with 2 L of wort with a density of 11 ◦P, which was provided by Biercentrum Delvaux (Neerijse, Belgium), and autoclaved before inoculation. The evolved strains from the ALE experiment and the original brewer's strains BCD1, BCD2, and BCD3 were added to the tall tubes at a cell concentration of 10 <sup>×</sup> 106 cells/mL. Fermentations were carried out in duplicates and sampled daily.

Alcolyzer Plus Beer Analyzing System (Anton Paar®, Graz, Austria) and headspace gas chromatography (GC) (Autosystem XL, Perkin Elmer®, Waltham, MA, USA) were used for the analyses of the fermentation process. For the GC analysis, the samples were filtered through a filter paper (Grade MN 713 <sup>1</sup> <sup>4</sup> , Macherey-Nagel®, Düren, Germany). During the experiment, the apparent extract (% *m*/*m*) and the ethanol content (% *v*/*v*) were measured. The apparent extract (Ea) is a direct measurement of the dissolved solids in brewer's wort, gauged according to specific gravity. During fermentation, the fermentable carbohydrates (glucose, maltose, and maltotriose) are consumed by the yeast and the progress of the fermentation is monitored by measuring the disappearance of these solids [31].

Concentrations of the volatile compounds (acetaldehyde, ethyl acetate, diacetyl, propanol, 2,3-pentanedione, isobutanol, isoamyl acetate (3-methyl-1-butylacetate), isoamyl alcohol (3-methyl1-butanol), and ethyl caproate) in the beer samples were determined by headspace gas chromatography (HS-GC FID/ECD) as previously described [32,33]. Shortly, collected samples were cooled on ice and after centrifugation, 5 mL of the cooled supernatant was transferred to a vial. The vials were analyzed with a calibrated Autosystem XL gas chromatograph with a headspace autosampler (HS40; Perkin Elmer, Wellesley, MA, USA), equipped with a Chrompack-Wax 52 CB column (length 50 m, 0.32 mm internal diameter, 1.2 μm layer thickness; Varian, Palo Alto, CA, USA). Samples were heated for 16 min at 60 ◦C in the headspace autosampler before injection (needle temperature 70 ◦C). Helium was used as the carrier gas. The oven temperature was kept at 50 ◦C for 7.5 min, increased to 110 ◦C at 25 ◦C/min, and was held at that temperature for 3.5 min. Detection of esters, and higher alcohols was established with a flame ionization detector (FID); diacetyl was detected with an electron capture detector (ECD). The FID and ECD temperatures were kept constant at 250 ◦C and 200 ◦C, respectively.

#### **3. Results**

#### *3.1. Adaptive Evolution in Ministats*

Adaptive evolution experiments were performed with the three selected non-flocculating industrial strains using continuous cultivation in the ministats. The flocculation behavior before evolution was assessed and compared to the non-flocculating haploid lab strain BY4742, the strongly flocculating BY4742 [*FLO1*] (constitutively overexpressed *FLO1*), the naturally flocculating BY4742::*FLO8* (functional Flo8p) and the strongly flocculating industrial strain BCD4 (Figure 1a). The flocculation percentages of the three industrial strains were low (<21% ± 2%), ranking them below the natural flocculating BY4742::*FLO8* reference lab strain (46% ± 4%). The strongly flocculating industrial brewer's strain BCD4 shows the same flocculation capacity as the BY4742 lab strain with constitutively overexpressed *FLO1*.

For the ALE experiments, cultivation with the three strains was initiated with a dilution rate of approximately 0.2 h−<sup>1</sup> using a medium feeding flow rate of 30 μL/min. The cultivation was monitored daily by measuring the glucose concentration, pH, and cell density (Figure 2). Between days 5–7, steady-state was reached and over time, and the dilution rate was increased gradually to 0.35 h-1 (BCD1) and 0.45 h−<sup>1</sup> (BCD2, BCD3) to avoid wash-out and to select for larger aggregates. The continuous cultivation was stopped after 45 days, and the yeast populations were examined by microscopy (Figure 3). The evolved BCD2 aggregates were smaller than the BCD1 and BCD3 aggregates. The nature of the cell clusters was determined by resuspending the cells in EDTA-buffer, which chelates Ca+<sup>2</sup> ions and disrupts yeast cell clusters if they are formed via flocculin-dependent adhesion. The aggregates persisted for all three strains. This indicated that the clusters are not the result of flocculin interactions and are likely due to failure in separation of the mother and daughter cells, described previously as the "snowflake" phenotype [27,34–36]. The evolved strains were subsequently cultivated in batch cultures and the aggregating phenotype was found to be stable.

The flocculation assay was repeated with the evolved strains to estimate and compare their sedimentation velocity to that of the BCD1, BCD2, and BCD3 strains before evolution (Figure 1). All three evolved strains showed an increase in "flocculation" percentage. Even though none of the strains evolved towards a real flocculating phenotype, their multicellular aggregates contributed to a significant larger sedimentation velocity.

**Figure 1.** Flocculation and sedimentation behavior of laboratory and industrial strains. (**a**) Flocculation percentages determined for the control strains (BY4742, BY4742::*FLO8*, BY4742 (*FLO1*), the industrial BCD4 strain) and for the three industrial non-flocculating strains BCD1, BCD2, and BCD3 before (-) and after (-) ALE in ministats. All measurements were performed in triplicates. (**b**) The sedimentation behavior of the three industrial brewer's strains before and after adaptive laboratory evolution (ALE) in the ministats.

**Figure 2.** Cultivation of the industrial strains in the ministats: (**a**) BCD1, (**b**) BCD2, and (**c**) BCD3. Glucose content (•), pH (-), OD600 nm (), and the dilution rate (1/h) (**—**).

**Figure 3.** Microscopic observations of the industrial strains before and after the evolution in the ministats. Snowflake clumps are observed for each strain and could not be disrupted by treatment with 50 mM EDTA.

#### *3.2. Performance of Evolved Strains During Beer Fermentation*

The performance and behavior of the evolved yeast strains, compared to their reference strains, were evaluated during wort fermentations in tall tubes (Figure S2a). The fermentations were monitored by sampling and measuring cell concentration and beer characteristics such as apparent extract (Ea), ethanol content (Table S1), and the concentration of several flavor compounds (Figure S3 and S4).

The reference strain BCD1 showed a faster fermentation capacity than the evolved strain: The fermentation was almost completed after only 1 day as observed from the evolution of the apparent extract and ethanol concentration (Table S1) as well as from the suspended cell concentration (Figure S2b). The fermentation capacity of the evolved and reference BCD2 strains were similar (Table S1). In contrast, the evolved BCD3 strain showed a faster fermentation than the reference strain (Table S1). The evolution of the suspended cell concentrations during the fermentations for the evolved and reference strains is shown in Figure S1b. There are no large differences observable in the number of suspended cells between the evolved and the reference strains, except for the evolved BCD1 at the second day of fermentation where a much lower cell concentration of the evolved strain was present.

Flavor compounds in the beer were quantified by headspace gas chromatography (Figure S3 and S4). In general, no major influence of the evolved yeast strains on the development of the flavor profile was observed. Some remarkable observations include an increased content of vicinal diketones diacetyl (up to 2.1 ppm) and 2,3-pentanedione (up to 0.9 ppm) during the initial stages of the fermentation for the evolved BCD1 strain, which was decreased by the third day for both compounds to 0.1 ppm. Also, the aliphatic higher alcohol isobutanol concentration increased to 178 ppm (compared to 53 ppm for the reference strain) at the third day of the fermentation. The isobutanol concentration of the evolved BCD2 strain fermentation was doubled at the second day compared to the reference strain, but was still below the isobutanol flavor threshold of 100–200 ppm [37]. By the third day, the evolved BCD3 gave a lower concentration of acetaldehyde than its reference, but a higher concentration of the higher alcohol propanol and isobutanol, and the esters ethylacetate and isoamylacetate.

#### **4. Discussion**

Miniature, low-cost chemostats (ministats) were used for adaptive laboratory evolution (ALE) of three industrial *S. cerevisiae* brewer's strains towards a more favorable, aggregating phenotype. The three strains—BCD1, BCD2, and BCD3—were characterized with a low flocculation ability and were continuously cultivated with high-glucose medium for 45 days. Small clusters of cells were observed around day 15, corresponding to approximately 110 generations, which is comparable to other *S. cerevisiae* ALE experiments [27,38,39]. The yeast cell clusters were not disrupted by treatment by EDTA, which indicated that the cell-cell interactions are not based on flocculins. Microscopy showed that the multicellular clusters look like "snowflakes". This "snowflake" phenotype was previously described as the result of the failed separation of daughter cells from mother cells. This phenotype is caused by a frameshift mutation in the transcription factor *ACE2*, which is responsible for the activation of the *CTS1* gene encoding the chitinase necessary to break down the septum between the mother and the daughter cells [34,36,40]. The clusters of cells formed in this way are unlike flocs in that they consist entirely of genetically identical cells and surrounding cells can not adhere to the cluster [36].

The performance of the evolved strains was compared to the reference strains in beer fermentations using tall tubes fermentors. During the fermentation only small aggregates of the evolved strains were observed. These aggregates were kept in suspension during the convective mixing by the CO2 release by the fermenting yeast cells as was clear from the evolution of the suspended cell concentration (Figure S2b). Apparently, shear stress by convective mixing will break up large aggregates and the presence of these small aggregates will not lead to premature sedimentation during fermentation. The CO2 production stops at the end of the fermentation and convective currents are reduced significantly. At this moment, the sedimentation of the snowflake aggregates will be significantly faster (Figure 1b) than the reference strains.

To assess the effect of adapted evolution of the 3 strains on the beer flavor, the evolution of a few flavor compounds was determined during the first 3 days of the fermentation. The evolved BCD1 strain showed an increased production of the vicinal diketones and the higher alcohol isobutanol. The synthesis of these compounds is linked to the isoleucine–leucine–valine (ILV) pathway. Although the flavor threshold of diacetyl (0.1–0.15 ppm [41]) was exceeded and the 2,3-pentanedione concentration was close to the flavor threshold (1.0–1.5 ppm [41]) during the first 2 days of fermentation, these concentrations were reduced significantly below the flavor threshold at the third day. Also, the isobutanol content in the beer fermented by the evolved BCD1 strain was much higher than for the reference strain, but did not exceed the flavor threshold of 100–200 ppm [37]. After 7 days, the green beer from the evolved and the reference BCD1 strain both tasted fruity (isoamyl acetate and acetaldehyde). Although the isobutanol concentration was larger in the beer from the evolved BCD2 strain, no difference was tasted. In both beers, the apple flavor (acetaldehyde) could be recognized. The beer from the evolved BCD3 strain contained a higher concentration of the ester isoamyl acetate (banana aroma) and ethyl acetate ester (fruity, solvent-like aroma), which presence could be tasted in the green beer.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2311-5637/6/1/20/s1, Figure S1: The experimental setup of the ministats. Figure S2: Beer fermentations in tall tube fermenters, Figure S3 and Figure S4: Comparison of the evolution of some flavor compounds during the tall tubes' fermentations; Table S1: Evolution of the apparent extract and ethanol content during the tall tube fermentations.

**Author Contributions:** Conceptualization, R.W. and F.R.D.; methodology, R.W., T.V.M.; formal analysis, T.V.M.; investigation, T.V.M., Y.K., F.D., F.R.D., and R.W..; resources, R.W.; writing—original draft preparation, Y.K., T.V.M.; writing—review and editing, Y.K., R.W., F.R., and F.R.D.; supervision, R.W., F.R.D., and F.D.; project administration, R.W.; funding acquisition, R.W. All authors have read and agree to the published version of the manuscript.

**Funding:** This research was funded by ESA-Belspo, grant PRODEX "Yeast Bioreactor".

**Acknowledgments:** The Belgian Federal Science Policy Office (Belspo) and the European Space Agency (ESA) PRODEX program supported this work. The Research Council of the Vrije Universiteit Brussel (Belgium) and the University of Ghent (Belgium) are acknowledged to support the Alliance Research Group VUB-UGent NanoMicrobiology (NAMI), and the International Joint Research Group (IJRG) VUB-EPFL BioNanotechnology & NanoMedicine (NANO).

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Characterization of Old Wine Yeasts Kept for Decades under a Zero-Emission Maintenance Regime**

#### **Katrin Matti 1, Beatrice Bernardi 1,2, Silvia Brezina 1, Heike Semmler 1, Christian von Wallbrunn 1, Doris Rauhut <sup>1</sup> and Jürgen Wendland 1,\***


Received: 14 December 2019; Accepted: 9 January 2020; Published: 11 January 2020

**Abstract:** All laboratories dealing with microbes have to develop a strain maintenance regime. While lyophilization based on freeze-drying may be feasible for large stock centers, laboratories around the world rely on cryopreservation and freezing of stocks at −80 ◦C. Keeping stocks at these low temperatures requires investments of several thousand kW/h per year. We have kept yeast stocks for several decades at room temperature on agar slants in glass reagent tubes covered with vaspar and sealed with cotton plugs. They were part of the Geisenheim Yeast Breeding Center stock collection that was started in the 19th century, well before −80 ◦C refrigeration technology was invented. Of these stocks, 60 tubes were analyzed and around one-third of them could be regrown. The strains were typed by sequencing of rDNA PCR fragments. Based on BlastN analyses, twelve of the strains could be assigned to *Saccharomyces cerevisiae*, two to *S. kudriavzevii*, and the others to *Meyerozyma* and *Candida*. The strains were used in white wine fermentations and compared to standard wine yeasts Uvaferm/GHM (Geisenheim) and Lalvin EC1118. Even with added nitrogen, the strains exhibited diverse fermentation curves. Post-fermentation aroma analyses and the determination of residual sugar and organic acid concentrations indicated that some strains harbor interesting flavor characteristics, surpassing current standard yeast strains. Thus, old strain collections bear treasures for direct use either in wine fermentations or for incorporation in yeast breeding programs aimed at improving modern wine yeasts. Furthermore, this provides evidence that low-cost/long-term culture maintenance at zero-emission levels is feasible.

**Keywords:** strain collection; aroma profiling; gas chromatography; wine yeast; *Saccharomyces*; fermentation; volatile aroma compounds

#### **1. Introduction**

At the end of the 19th century, Emil Christian Hansen at the Carlsberg Laboratory in Copenhagen, Denmark, established the first pure culture lager yeast strain, Unterhefe No. 1 [1]. This strain then became known as *Saccharomyces carlsbergensis*. The finding that one yeast strain was sufficient to generate a fermented beverage of high quality started a new era and lead to new developments in the beer and dairy industry. It was soon recognized by Julius Wortmann at the Geisenheim Research Center in Germany that Hansen's findings were also applicable to wine making [2]. This started efforts in collecting wine yeast strains from different vineyards and wineries in the Rheingau area. These strains were characterized for their fermentation capacity and flavor attributes. At the "Geisenheimer

Hefe-Reinzuchtstation" (Geisenheim Yeast Breeding Center), founded in 1894, these strains were produced as liquid starter cultures and dispatched to the wineries upon request.

With the isolation of pure yeast cultures came the responsibility to maintain stocks of these cultures. Wine making requires yeast starter cultures only once a year just after the grape harvest. By contrast, yeasts for beer production are in constant use throughout the year. Even before the isolation of pure cultures, there was an interest in generating dry yeast cakes for longer term storage. The history of both the patents and literature in this field has been covered in depth by a recent excellent review [3]. A solid supply of dehydrated yeast became a necessity for long distance shipments, which came around 1940 when the Fleischman Co. produced active dry yeast. This yeast required 'reactivation', i.e., rehydration prior to use. In the 1970s, Lesaffre introduced an instant dry yeast which could be used directly without reactivation. In microbiological laboratories, however, bacterial and yeast cultures are nowadays generally preserved by storing at −80 ◦C which, since the 1970s, has become technically feasible on a larger scale [4].

Thus, for decades after the 1880s, yeast cultures had to be kept by other means. Two techniques used were water stocks and yeast slants covered with vaspar (a mix of paraffin and Vaseline) [5,6]. The method of storing yeasts in distilled water at room temperature, as proposed by Castellani, is not only a cheap way of preserving cultures but is also a very effective way of culturing a collection over many years without the need for constant propagation [7]. This method is particularly useful for yeasts [8]. Storage of fungal cells in distilled water can be extended for 20 years [9]. Therefore, it was stated that storing yeast cultures in distilled water may reach similar efficiencies as freezing at −80 ◦C [10]. The use of a paraffin or vaspar overlay is also a very cheap way of yeast culturing, although the viability may be reduced when compared to the other methods.

There are only a few long-term studies describing yeast viability, one of which used the traditional method of yeasts grown on slants and covered by paraffin oil and found cells to be viable after a seven-year incubation period [11]. At the Geisenheim Yeast Breeding Center, we have a large collection of wine yeasts and non-conventional yeasts dating back to the 1890s. Samples were routinely stored with a vaspar overlay. Of course, over time, the strain collection was transferred to either storage in liquid nitrogen or in freezers at −80 ◦C. Nevertheless, we still stored a few samples for over 30 years at room temperature in the old way. In this study, we examined 60 tubes containing these decade-old samples. The yeast were restreaked, and those strains that could be regrown were subjected to fermentation studies and volatile aroma analyses. Our results show that strain collections can safely be stored at room temperature. Such a strain maintenance regime could contribute to energy conservation and the reduction of CO2 emissions.

#### **2. Materials and Methods**

#### *2.1. Strains and Media*

The yeast strains used in this study are shown in Table 1, including standard wine yeast strains used for comparison. Yeast strains were subcultured in YPD (1% yeast extract, 2% peptone, 2% glucose).


**Table 1.** Strains used in this study.

Bold: to indicate true *S. cerevisiae* strains. These strains will be important for winemaking. The others are non-conventional yeasts.

#### *2.2. Molecular Analysis of Yeast Strains*

Typing of the strains was determined by performing ITS-PCR (ITS, internal transcribed spacer) using standard ITS1F-fungal specific-(5 -CTTGGTCATTTAGAGGAAGTAA-3 ) and ITS4-universal-(5 -TCCTCCGCTTATTGATATGC-3 ) primers and sequencing of the PCR products as previously described [12]. Sequencing was conducted by Starseq, Mainz, Germany.

#### *2.3. Fermentation Conditions*

Lab-scale fermentations were carried out in duplicate with a standard pasteurized white wine must with a sugar concentration of 72 ◦Oechsle. The must was supplemented by the addition of Fermaid E (inactivated yeast product; according to the supplier's instructions; Lallemand, Vienna, Austria). Cells were inoculated at a density of OD600 = 0.5. The fermentation temperature was set to 18 ◦C and cultures were incubated with constant stirring at 300 rpm.

#### *2.4. Analytical Methods*

At the end of the fermentations, several compounds including fructose, glucose, ethanol, and organic acids were analyzed by high-performance liquid chromatography (HPLC) with an Agilent 1100 Series (Agilent Technologies, Waldbronn, Germany). Quantitative analyses were done as described in [13]. The HPLC equipment was equipped with a variable wavelength detector (UV/VIS) and a refractive index detector (RID). The column for separation is an Allure Organic Acids (Restek GmbH, Bad Homburg, Germany) with a 5 μm particle size, 60 A pore size and dimensions of 250 mm x i.d. 4.6 mm. A water-based solution of sulfuric acid (0.0139% *v*/*v*) and ethanol (0.5% *v*/*v*) was used as eluent. Gas chromatography was conducted using a GC 7890A (Agilent, Santa Clara, CA, USA), coupled with a MSD 5977B mass spectrometer (Agilent, Santa Clara, CA, USA). The determination of aroma

compounds followed the analytical approach described in Belda et al. [14] according to the method of Camara et al. [15].

#### **3. Results**

#### *3.1. Regrowing of Dormant Strains Kept under Vaspar for Decades*

At the Geisenheim Yeast Breeding Center (GYBC), we stored two racks containing 60 reagent tubes with yeast strains. The tubes contained slants on which yeast strains were spread and grown and then overlaid with vaspar (Figure 1). This was the standard procedure for maintaining strains at the GYBC. The collections were generated before the introduction of −80 ◦C refrigeration. These samples represent an even older stock as younger samples (20+ years of age) were kept in reagent tubes with screw cap closures. The apparent age of the old samples is, therefore, estimated to be over 30 years but less than 60 years, as one isolate carried a label with the year 1959 indicative of the year of isolation (Table 1).

**Figure 1.** (**A**,**B**). Old samples from the Geisenheimer Yeast Breeding Center. Samples were generated as agar slants with yeasts grown and covered in vaspar. Tubes were plugged by a cotton ball. Samples were labeled according to the location of the isolate and the year of isolation (see Table 1).

We wanted to find out if these strains were still alive and, once propagated, what their fermentation behavior would be like. To this end, we either took samples with an inoculation loop and restreaked them on full medium YPD or inoculated them in liquid YPD. Astonishingly, about one-third (18 out of 60) of the strains could be regrown and cultivated under these conditions.

The strain labels often indicated the area where these strains had been isolated and did not necessarily identified the species. Thus, we went on to type the strains by PCR amplification of a region of the ribosomal DNA using a standard primer pair designed for fungal species (ITS1 and ITS4). These primers are located at the end of the 18S and start of the 28S rDNA and thus amplify the internal transcribed spacer (ITS) region including the 5.8S rDNA. This region is highly variable and allows strain determination to the species level. PCR products were sequenced, and the sequences compared to the NCBI non-redundant database using BlastN. The sequence comparisons indicated that most strains could indeed be assigned to *Saccharomyces cerevisiae*. Two of the strains were found to be *S. kudriavzevii* while three strains matched *Meyerozyma guilliermondii* and one strain could be assigned to a newly described species of *Candida sanyaensis* (Table 1).

#### *3.2. Fermentation Performance*

We went on to study the fermentation characteristics of the *Saccharomyces* strains. To this end, strains were used to ferment a standard white wine must of 72 ◦Oechsle to which additional amino

nitrogen was added via an inactivated yeast product (Fermaid E). Fermentations were carried out at 18 ◦C with stirring over a period of 12 days and fermentation rates were followed by daily measurements of CO2 release. Strains were compared to the standard wine strain Lalvin EC1118 (Figure 2). It turned out that half of the strains generated a weight loss slightly larger than Lalvin EC1118, while the other half performed less well than Lalvin EC1118 in this respect. The largest weight loss was found with *S. cerevisiae* strain Steinberg 1893 and the *S. kudriavzevii* strain Würzburg (Stein). Lalvin EC1118 required a short lag phase of one day to enter alcoholic fermentation. Several of the tested yeast strains exhibited an extended lag phase of 3–4 days, particularly the strains that later showed the greatest weight loss and also Geisenheimer Mäuerchen, Winningen 1892, and Bordeaux 1892. Thus, even given the extended lag phase, these strains managed a complete fermentation within the 12-day fermentation window.

**Figure 2.** Fermentation curves based on CO2 release/weight loss of *Saccharomyces* cultures derived from isolates of the old collection. Release of CO2 was measured daily. (**A**) Strains are shown that released more CO2 than the EC1118 control wine yeast strain. (**B**) Strains are shown that released less CO2 than the EC1118 control wine yeast strain.

To analyze the fermented liquids in more detail, the residual sugars, organic acids, and final ethanol content were determined (Table 2). Most of the *Saccharomyces* strains reached complete fermentation with around 7% alcohol content. Two glucophilic strains, however, failed to utilize all of the fructose in the 12-day fermentation time. The *S. kudriavzevii* strains produced less alcohol than the *S. cerevisiae* strains. The two *S. cerevisiae* strains Rüdesheimer Hinterhaus 1893 and Heimersheimer Ruth, while using up glucose, did not utilize fructose completely during the 12-day fermentation. All strains showed a similar organic acid profile, with malate being the pronounced acid. Rüdesheimer Hinterhaus 1893, on the other hand, showed a surprising amount of shikimic acid (Table 2).



#### *Fermentation* **2020**, *6*, 9

#### *3.3. Production of Aroma Compounds*

We routinely examined 28 aroma compounds, specifically alcohols and esters (Table S1). A comparison within strains using a selection of eight major compounds is shown in Figure 3 and Table 3. While all species produced a range of compounds, it was interesting to see that a major current wine production strain, EC1118, was actually not the highest producer of certain aroma compounds in our assay. The three strains that produced most fruity esters were Rüdesheimer Hinterhaus 1893, Alpiarca 1896, and Valdepenas Criptana 1909. Additionally, Rüdesheimer Hinterhaus 1893 championed the production of isoamyl acetate (acetic acid 3-methyl butyl ester) and 2-phenylethyl acetate (acetic acid 2-phenylethylester), which is the acetate ester of 2-phenylethanol. This is apparently a consequence of Ehrlich pathway output as regarding the production of alcohols, particularly i-butanol, isoamylalcohol (3-methyl-butanol), and 2-phenlyethanol, the strain that came on top for each of the compounds was also the Rüdesheimer Hinterhaus 1893 yeast (see Table S1).

**Figure 3.** (**A**–**C**). Bar charts with selected alcohol (in mg/L) and ester (in μg/L) aroma compounds of *Saccharomyces* strains compared to the EC1118 wine yeast. Flavor compounds were measured using gas chromatography at the end of fermentation. The full list of aroma compounds is shown in Table S1.


 **3.** Aroma compound generation determined at the end of fermentation.\*

**Table**

82

*Fermentation* **2020**, *6*, 9

What was interesting to note is that some strains produced either high levels of hexanoic acid ethylester (ethyl hexanoate) or high levels of acetic acid phenylethylester, but not of both substances. This was observed when comparing Rüdesheimer Hinterhaus 1893 with Zell 1895 and Alpiarca 1896. The first was low on hexanoic acid ethylester but produced high levels of 2-phenylethyl acetate, while the latter strains produced high levels of hexanoic acid ethylester and much lower levels of 2-phenylethyl acetate (Table 3, Figure 3; see Section 4).

#### **4. Discussion**

Alcoholic beverage production has been carried out by spontaneous fermentation throughout the ages, and today, often still the preferred method of fermentation for some. In beer and baking enterprises, it was already realized in the middle ages that there are special properties in the slurry that leavens bread and makes beer ferment and foam. It was the traditional occupation of a 'Hefner' in Germany to maintain and provide sufficient supplies of this leavening activity [16]. Yet, as it was not clear what the causal activity in the slurry was, the German 'Reinheitsgebot' (purity law) from 1516 only stated that beer should be brewed using barley malt, hops, and water. With the work of Pasteur, published in his Etudes sur le vin and Etudes sur la biere, and the work of others, it became evident that yeast, *Saccharomyces cerevisiae*, was responsible for the observed fermenting power.

The pure yeast strain isolation procedure worked out by Hansen was transferred to the wine industry by the German Julius Wortmann, who founded the 'Geisenheim Yeast Breeding Center' in 1894 [1,2]. The isolation of pure yeast strains in Geisenheim had been initiated in the early 1890s and these strains are still preserved today. Today's strain maintenance relies on deep freezing of culture collections at −80 ◦C, while supplies for the industry are generally provided as instant dry yeasts [17,18].

Yet, at the Geisenheim Yeast Breeding Center, stocks were originally maintained as slants with a paraffin overlay. Cultures were stored either in this manner or, even simpler, in plain water [5,6]. A recent report analyzed long-term storage (12 years) of >1000 stocks and provided evidence that water storage yielded survival rates of 98.9%, closely resembling that of frozen stocks (99.5%), while survival rates under a mineral oil layer were a bit less with 88.2% [10]. Two racks of Geisenheim yeast stocks were kept over the years, more as display items than out of necessity. Such a long-term storage has not been previously reported. In fact, it is not entirely clear anymore when exactly these stocks were generated (besides the fact that they are very old and between 30 and 60 years of age). Collectively, these studies show, however, that yeast strain collections can be routinely kept under a zero-emission regime, which could be used as an incentive to reduce the energy-demanding storage of cultures at −80 ◦C.

The 'old' Geisenheim yeast strains can be both a heritage and a source of new strains for yeast breeding programs. As a heritage, they could be used to strengthen the local character of wines produced with them and thus contribute to the terroir of these wines [19–21]. To be useful as a breeding stock, these old strains need to be characterized in more detail, preferably including their genomes [22,23]. In our study, the yeast with the most pronounced flavor production capability of alcohols and esters was the *S. cerevisiae* strain Rüdesheimer Hinterhaus 1893 ('Hinterhaus' refers to backyard). It was apparent that this strain is a remarkable producer of acetate esters, but not so of medium chain fatty esters. The former are produced by the acetyl transferases Atf1 and Atf2, while the latter are generated by the acyl-coenzymeA:ethanol O-acyltransferases Eeb1 and Eht1 [24,25]. This suggests strong activity of the ATF alcohol acetyl-coA transferases in the Rüdesheimer Hinterhaus strain isolated already in 1893, with reduced formation of medium chain fatty acids. It will be interesting to explore these properties through a full-scale wine fermentation trial and use in-depth genomics and transcriptomics to generate molecular markers for yeast breeding.

The strive for more volatile aromas has spurred the search for alternative yeasts and renewed the interest in spontaneous fermentations [26]. Yet, due to the unpredictable and inconsistent outcomes of spontaneous fermentations, the development of improved starter cultures or consortia may provide better alternatives [27,28].

#### **5. Conclusions**

In conclusion, our study of yeast isolates stored for a very long period opens several new research avenues in the use of these strains, either directly as starter cultures or as stocks for the Geisenheim Yeast Breeding Center; both will be interesting to exploit in the future. For general use, our data and previous work, e.g., on water cultures show that zero-emission strain-keeping of yeast cultures is feasible and should be more generally exploited.

**SupplementaryMaterials:** The following are available online at http://www.mdpi.com/2311-5637/6/1/9/s1, Table S1. Full list of aroma compounds.

**Author Contributions:** Conceptualization, J.W., D.R., and C.v.W.; methodology, K.M., B.B., S.B., H.S.; validation, J.W., K.M., B.B., S.B., H.S.; formal analysis, J.W., K.M., S.B., H.S.; investigation, J.W., K.M., B.B., S.B., H.S.; resources, J.W.; D.R., C.v.W. data curation, J.W., K.M., S.B., H.S.; writing—original draft preparation, J.W.; writing—review and editing, J.W., D.R., C.v.W., K.M., B.B., S.B., H.S.; visualization, K.M. and J.W. supervision, J.W. and D.R.; project administration, J.W. and D.R.; funding acquisition, J.W. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded in part by the European Union Marie Curie Initial Training Network Aromagenesis 764364 (http://www.aromagenesis.eu).

**Acknowledgments:** We thank Judith Muno-Bender for taking photographs of the old yeast samples; Stefanie Fritsch for calibrations of the analytical equipment and Bettina Mattner for excellent technical service and all Department members for input and support of this project.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Identification of Yeasts with Mass Spectrometry during Wine Production**

#### **Miroslava Kaˇcániová 1,2,\*, Simona Kunová 3, Jozef Sabo 1, Eva Ivanišová 4, Jana Žiarovská 5, So ˇna Felsöciová <sup>6</sup> and Margarita Terentjeva <sup>7</sup>**


Received: 16 November 2019; Accepted: 3 January 2020; Published: 7 January 2020

**Abstract:** The aim of the present study was to identify yeasts in grape, new wine "federweisser" and unfiltered wine samples. A total amount of 30 grapes, 30 new wine samples and 30 wine samples (15 white and 15 red) were collected from August until September, 2018, from a local Slovak winemaker, including Green Veltliner (3), Muller Thurgau (3), Palava (3), Rhein Riesling ¯ (3), Sauvignon Blanc (3), Alibernet (3), André (3), Blue Frankish (3), Cabernet Sauvignon (3), and Dornfelder (3) grapes; federweisser and unfiltered wine samples were also used in our study. Wort agar (WA), yeast extract peptone dextrose agar (YPDA), malt extract agar (MEA) and Sabouraud dextrose agar (SDA) were used for microbiological testing of yeasts. MALDI-TOF Mass Spectrometry (Microflex LT/SH) (Bruker Daltonics, Germany) was used for the identification of yeasts. A total of 1668 isolates were identified with mass spectrometry. The most isolated species from the grapes was *Hanseniaspora uvarum*, and from federweisser and the wine—*Saccharomyces cerevisiae*.

**Keywords:** yeasts; grape; federweisser; wine; microbiota identification; MALDI-TOF MS Biotyper

#### **1. Introduction**

Yeasts naturally occur in wines and vineyards and are especially common on the grapes. Population of yeast species on the grape is not constant and increases during the ripening process. *Kloeckera apiculata* is a lemon-like cell shape yeast, which colonizes the grape surface [1]. *Kloeckera apiculata* comprises more than 50% of the total healthy grape microbiota. Other yeasts like *Kloeckera* were isolated from the surface of the grapes, which included mainly genera *Metschnikowia*, *Candida*, *Cryptococcus*, *Pichia*, *Rhodotorula*, *Zygosaccharomyces* or *Kluyveromyces* [2]. The presence of yeasts of the genus *Aureobasidium* attracted attention as a transitional genus between yeast and microscopic fungi. All the yeasts associated with natural microbiota of grapes are wild yeast strains or non-saccharomyces. Despite the presence of those yeasts on the surface of grapes, the wine production consists of subsequent fermentation stages, which are typical for only particular yeast genera [3]. The *Saccharomyces* genus is the most important

for the wine making process; however, this yeast is found on the grapes only in very small amounts. Previous studies that counted *Saccharomyces* on grapes found as little as 50 CFU/g. Mostly wild yeasts cultures could be found on the grapes and in freshly pressed must with colonization rates of 103 to 10<sup>5</sup> CFU/mL. During alcoholic fermentation, *Saccharomyces cerevisiae* is dominant, while yeasts in the *Pichia* and *Candida* genera are widespread in finished wine. The osmotolerant yeasts *Zygosaccharomyces* were reported in wines with higher content of residual sugar; yeasts of the *Brettanomyces* genus were common for wines in barrels [4,5].

The most important yeasts associated with wine production were Hanseniaspora uvarum (anamorph Kloeckera apiculata), Metschnikowia pulcherrima, Rhodotorula mucilaginosa, Rhodotorula glutinis, Aureobasidium pullulans, Cryptococcus magnus, Pichia manshurica, Pichia membranifaciens (anamorph Candida valida), Pichia fermentans, Pichia kluyveri, Pichia occidentalis (anamorph Candida sorbosa), Wickerhamomyces anomalus (anamorph Candida pelliculosa; Pichia anomala is synonymous), Cyberlindnera jadinii (Pichia jadinii is synonymous), Kregervanrija fluxuum (anamorph Candida vini), Candida stellata, Candida inconspicua, Meyerozyma guilliermondii, Zygosaccharomyces bailii, Brettanomyces bruxellensis (teleomorph Dekkera bruxellensis), Saccharomycodes ludwigii, Torulaspora delbrueckii and Saccharomyces cerevisiae. Kluyveromyces marxianus and Debaryomyces hansenii associated with grapes and are known as a contaminant in wine production. The microbiota of grapes creates better conditions for the growth of yeasts rather than bacteria. Low pH (pH 3–3.3), high content of sugars (mainly glucose) in grapes, and an anaerobic environment in must are necessary for ethanol fermentation of sugars, converting them into alcohol (ethanol) and CO2 [5–8].

The aim of this study was to identify yeasts in grapes, federweisser and wine samples.

#### **2. Materials and Methods**

#### *2.1. Collection of Grape, Federweisser and Wine Samples*

An amount of 90 samples, including grape berries (*n* = 30), federweisser (*n* = 30) and wine (*n* = 30) of *Vitis vinifera* were collected aseptically in the viticultural area of Vrbové (approximately 48◦37 12" N and 017◦43 25" E) in 2018. The grape berry samples were transported on ice and stored at −20 ◦C until processing. The white grape varieties Green Veltliner, Muller Thurgau, Palava, Rhein Riesling and ¯ Sauvignon Blanc as well as red grape varieties Alibernet, André, Blue Frankish, Cabernet Sauvignon and Dornfelder were collected. Three sampling points in distal spatial points of different rows were used for sampling of grape berries. Grape samples were collected in August, and processed independently.

Samples of "federweisser" were collected at the end of August 2018 and in the middle of September 2018 from the same winery as the grapes. Samples were collected into 200 mL sterile plastic bottles and stored at 8 ± 1 ◦C in a refrigerator. Before testing, the samples (*n* = 30) were diluted with sterile physiological saline (0.85%). A total of 100 μL of each dilution (10−<sup>1</sup> to 10−5) was used for microbiological testing.

An amount of 200 mL of each unfiltered wine (before microfiltration) and immediately after were stored at 6–8 ◦C in a refrigerator. Collected wine samples were fermented with *Saccharomyces cerevisiae* in the producing process. The samples were later incubated in the laboratory at room temperature (25 ± 2 ◦C) for one week until the laboratory testing was initiated.

#### *2.2. Cultivation Media*

Wort agar (WA) (HiMedia, Mumbai, India), yeast extract peptone dextrose agar (YPDA) (Conda, Madrid, Spain), malt extract agar (MEA) (Biomark, Maharashtra, India) and Sabouraud dextrose agar (SDA) (Conda, Madrid, Spain) were used for identification of yeasts. All media were supplemented with chloramphenicol (100 mg/L) to inhibit bacterial growth. Chloramphenicol (Biolife, Monza, Italy) was added into cultivation media before sterilization by autoclaving at 115–121 ◦C for 15 min. The acid base indicator bromocresol green (BG, Biolofe, Monza, Italy) (20 mg/L) (pH range: 3.8–5.4) was added into the MEA and WA cultivation media before sterilization. Media for yeast cultivation were

inoculated with 100 μL of the sample suspension. Inoculated agars were incubated at 25 ◦C for 3–5 days and the yeasts were identified by colony morphology (colour, surface, edge and elevation) and reinoculated onto trypton soya agar (TSA) (Oxoid, Basingstoke, UK). Yeast species were identified with a MALDI-TOF MS Biotyper.

#### *2.3. Identification of Isolates with Mass Spectrometry*

Qualitative analysis of yeasts isolates was performed with MALDI-TOF mass spectrometry (Bruker Daltonics, Bremen, Germany). Isolates were put in 300 μL of distilled water and 900 μL of ethanol, and the suspension centrifuged for 2 min at 14,000 rpm. The pellet was centrifuged repeatedly and allowed to dry. An amount of 30 μL of 70% formic acid was added to the pellet and 30 μL of acetonitrile. Tubes were centrifuged for 2 min at 14,000 rpm and 1 μL of the supernatant was used for MALDI identification. Once dry, every spot was overlaid with 1 μL of an HCCA matrix and left to dry at room temperature before analysis. Generated spectra were analyzed on a MALDI-TOF Microflex LT (Bruker Daltonics, Bremen, Germany) instrument using Flex Control 3.4 software and Biotyper Realtime Classification 3.1 with BC-specific software. Criteria for reliable identification were a score of ≥2.0 at species level [9].

#### *2.4. Statistical Analysis*

The statistical processing of the data obtained from each evaluation was done with Statgraphics Plus version 5.1 (AV Trading, Umex, Dresden, Germany). For each replication the mean was calculated, and the data set were log transformed. Descriptive statistics and logical-cognitive methods and one-way analysis ANOVA were used in the evaluation and statistical analysis.

#### **3. Results and Discussion**

Grapes are inhabited by versatile microbial groups and have a complex microbial ecology, including filamentous fungi, yeasts and bacteria. These microorganisms pose different physiological characteristics and may affect the wine quality. Some species of parasitic fungi or environmental bacteria might be only found in grapes, while other microorganisms like yeast, lactic acid and acetic acid bacteria occur during the winemaking process [10].

The yeast count in grape ranged from 2.34 (Greener Veltliner) to 2.67 (Dornfelder) log CFU/g on MEA, from 2.19 (Muller Thurgau) to 2.38 (Dornfelder) log CFU ¯ /g on WA, from 2.46 (Greener Veltliner) to 2.66 (Dornfelder) log CFU/g on YPDA, and from 1.55 (Greener Veltliner) to 1.88 (Dornfelder) log CFU/g on SDA. The colonization of grapes with yeasts is shown in Table 1.


**Table 1.** Yeasts counts in grape berries on different media.

WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

ANOVA analysis was performed to inspect the significant differences among the microbial count for individual wine varieties when different cultivation media were used (Table 2).


**Table 2.** One-way ANOVA for analyzed wine varieties—grapes.

WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

Statistically significant differences among microbial counts for individual cultivation media were found in three of the four cultivation media used (Table 3).

**Table 3.** Significant differences among analyzed grape varieties for individual cultivation media.



**Table 3.** *Cont*.

A—Green Veltliner; B—Muller Thurgau; C—Palava; D—Rhein Riesling; E—Sauvignon Blanc; F—Alibernet; ¯ G—André; H—Blue Frankish; I—Cabernet Sauvignon; J—Dornfelder; WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

Different studies have evaluated the surface microbiota of grape berries due to a possible impact on the hygienic state of the grapes and the direct influence on the winemaking process and wine quality [11–18].

The yeasts count in "federweisser" ranged from 3.51 in Greener Veltliner and Palava to 3.80 log CFU/mL in Dornfelder on MEA. On WA, the yeasts count from 3.30 in Palava to 3.53 log CFU/mL in Dornfelder were observed. On YPDA, the yeasts count varied from 3.24 in Rhein Riesling to 3.45 log CFU/mL in Dornfelder, and from 3.13 (Sauvignon Blanc) to 3.33 (Dornfelder) log CFU/mL on SDA. Yeasts counts in federweisser are summarized in Table 4.


**Table 4.** Yeast counts in "federweisser" on different media.

WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

In study in Slovakia [19], the highest yeasts counts were on MEA for Pinot Noir—6.43 log CFU/mL and the lowest for Moravian Muscat—4.62 log CFU/mL. The highest yeasts count on WA were in Pinot Noir—6.39 log CFU/mL, but the lowest in Irsai Oliver—5.38 log CFU/mL. The highest count of yeasts on wild yeast medium (WYM) was in Blue Frankish 6.33 log CFU/mL and the lowest in Dornfelder 4.20 log CFU/mL [19].

As the results show, a higher number of yeasts were detected in "federweisser" than in grape. The young wine is a product of fermentation where *S. cerevisiae* was mostly found. Other species like *Hanseniaspora uvarum*, *Metschnikowia pulcherrima* or the genera *Pichia* or *Candida* may be present during the individual fermentation stages when the alcohol content do not exceed 4–6% [5,20]. The main microbiota of the grape is the yeast *Hanseniaspora uvarum* followed by *Metschnikowia pulcherrima* [4]. These species also initiate the pre-alcoholic fermentation but are being replaced by the dominant *S. cerevisiae* 3–4 days after fermentation. *Saccharomyces cerevisiae* starts to multiply within 20 days after inoculation into the must [21].

ANOVA analysis was performed to inspect the significant differences among the microbial count for individual wine varieties when different cultivation media were used (Table 5).


**Table 5.** One-way ANOVA results for the analyzed wine varieties—federweisser.

WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

Statistically significant differences among microbial counts for individual cultivation media were found in three of the four cultivation media used (Table 6).

**Table 6.** Significant differences among analyzed federweisser samples for individual cultivation media.



**Table 6.** *Cont*.

A—Green Veltliner; B—Muller Thurgau; C—Palava; D—Rhein Riesling; E—Sauvignon Blanc; F—Alibernet; ¯ G—André; H—Blue Frankish; I—Cabernet Sauvignon; J—Dornfelder; WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

The yeast counts in the unfiltered wines are summarized in Table 7. The yeast counts in wine ranged from 1.51 (Greener Veltliner) to 3.23 (Dornfelder) log CFU/mL on MEA, from 1.43 (Greener Veltliner) to 2.89 (Dornfelder) log CFU/mL on WA, from 1.18 (Greener Veltliner) to 2.65 (Dornfelder) log CFU/mL on YPDA and from 1.09 (Rhein Riesling) to 2.21 (Dornfelder) log CFU/mL on SDA.


**Table 7.** Yeast counts in wine on different media.

WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

ANOVA analysis was performed to inspect the significant differences among the microbial count for individual wine varieties when different cultivation media were used (Table 8).

**Table 8.** One-way ANOVA results for analyzed wine varieties—unfiltered wine.


WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

Statistically significant differences among microbial count for individual cultivation media were found in three of the four cultivation media used (Table 9).


**Table 9.** Significant differences among unfiltered wine samples for individual cultivation media.


**Table 9.** *Cont*.

A—Green Veltliner; B—Muller Thurgau; C—Palava; D—Rhein Riesling; E—Sauvignon Blanc; F—Alibernet; ¯ G—André; H—Blue Frankish; I—Cabernet Sauvignon; J—Dornfelder; WA—wort agar; YPDA—yeast extract peptone dextrose agar; MEA—malt extract agar; SDA—Sabouraud dextrose agar.

Altogether, 1668 isolates were identified with mass spectrometry with a score of ≥2.0 (Table 10). The most isolated species from grape was *Hanseniaspora uvarum* (70 isolates), and from "federweisser" and wine *S. cerevisiae* (85 and 120 isolates, respectively). Yeasts species of grape, "frederweisser" and wine are shown in Figures 1–3.


**Table 10.** Yeasts species in grape, "federweisser" and wine.

*Brettanomyces bruxellensis*, *Candida stellata*, *Saccharomyces cerevisiae* and *Zygosaccharomyces bailii* were the yeasts identified in wine [22–25]. In our study, *Pichia mandshurica*—the main contaminant of wines—was present in 66% samples of white wines (10 out of 15) and in seven samples of red wines (46%). *Pichia membranifaciens* was isolated from five samples of white (33%) and five samples of red wines (33%). *Saccharomyces cerevisiae* was isolated from all white and red wines (100%). *Zygosaccharomyces bailii* was found in 14 samples of white (93%) and two samples of red (13%) wines. Our study shows that *Z. bailii* and *P. mandshurica* were isolated more frequently from white than from red wines, while *S. cerevisiae* was identified in white and red wines. The occurrence of *Pichia manshurica* and *S. cerevisiae* was different between the wine samples. According to Thomas [26], the presence of *Zygosaccharomyces* in wine is unacceptable in terms of wine quality. The author has stated that the minimum number of yeast present in wine spoils the product under appropriate conditions [26]. *Saccharomyces cerevisiae*, *Debaryomyces hansenii*, *Wickerhamomyces anomalus (Pichia anomala)*, *Pichia membranifaciens*, *Rhodotorula glutinis*, *Rhodotorula mucilaginosa*, *Torulaspora delbrueckii*, *Kluyveromyces marxianus*, *Issatchenkia orientalis*, *Zygosaccharomyces bailii parapsilosis*, *Pichia fermentans* and *Hanseniaspora uvarum* are frequent contaminants of wines as well [27,28]. However, Renous [29] did not describe associations between wine and *Pichia manshurica*, *Kregervanrija fluxuum (Candida vini)*, *Candida inconspicua* and *Zygotorulaspora florentina*. Saez [30] found that *S. cerevisiae* (13.93%),

*Wickerhamomyces anomalus* (8.72%), *Pichia fermentans* (6.74%) and *Metschnikowia pulcherrima* (6.39%) were the most abundant in wine. *Pichia* (*Pichia manshurica*, *P. membranifaciens*) and *Brettanomyces* are producing volatile phenols, thereby affecting the quality of the wine [30].

**Figure 1.** Yeasts isolated from the grapes.

**Figure 2.** Yeasts isolated from the "federweisser".

**Figure 3.** Yeasts isolated from the wine.

Sporadically, *Candida inconspicua* (5 isolates, 0.58%), *Candida saitoana* (5 isolates, 0.58%), *Candida sake* (5 isolates, 0.58%), *Pichia norvegensis* (5 isolates, 0.58%) and other species were isolated. Jolly et al. [31] noticed the importance of *Candida*, *Cryptococcus*, *Kloeckera* and *Rhodotorula* species in the wine making process. *Candida* was considered as the dominant genus, including their teleomorphic stages—*Candida pulcherrima* (*Metschnikowia pulcherrima*), *Candida vini* (*Kregervanrija fluxuum*) and *Candida valida* (*Pichia membranifaciens*) [31].

#### **4. Conclusions**

A total of 90 samples (30 from grapes, 30 of "federweisser" and 30 of wine) was studied for characterization of the yeast species. The mass spectrometry method was used for identification of 1668 grape, "federweisser" and wine isolates. From grape, 26 species of 17 genera within 9 families, and in "federweisser" 4 species of 3 genera and families were found. In wine, 26 species of 17 genera within 6 families were identified. *Rhodoturulla* species were not included in any family and they were classified as incertae sedis (not belonging anywhere).

**Author Contributions:** M.K., M.T., J.Ž. were responsible for the design of the study; M.K., S.K., J.S., S.F., conducted the study and collected the samples; M.K., S.K., J.S., J.Ž. performed the laboratory analysis; M.K., S.K., E.I., M.T. were responsible for writing and editing the manuscript; all authors have carefully revised and approved the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work has been supported by the grants of the Slovak Research and Development Agency No. VEGA 1/0411/17.

**Acknowledgments:** The Paper was supported by the project: The research leading to these results has received funding from the European Community under project no. 26220220180: Building Research Centre "AgroBioTech".

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Review* **Modulating Wine Pleasantness Throughout Wine-Yeast Co-Inoculation or Sequential Inoculation**

#### **Alice Vilela**

Department of Biology and Environment, School of Life Sciences and Environment, University of Trás-os-Montes and Alto Douro, CQ-VR, Chemistry Research Centre, 5001-801 Vila Real, Portugal; avimoura@utad.pt; Tel.: +351-259-350-973; Fax: +351-259-350-480

Received: 17 November 2019; Accepted: 5 February 2020; Published: 9 February 2020

**Abstract:** Wine sensory experience includes flavor, aroma, color, and (for some) even acoustic traits, which impact consumer acceptance. The quality of the wine can be negatively impacted by the presence of off-flavors and aromas, or dubious colors, or sediments present in the bottle or glass, after pouring (coloring matter that precipitates or calcium bitartrate crystals). Flavor profiles of wines are the result of a vast number of variations in vineyard and winery production, including grape selection, winemaker's knowledge and technique, and tools used to produce wines with a specific flavor. Wine color, besides being provided by the grape varieties, can also be manipulated during the winemaking. One of the most important "tools" for modulating flavor and color in wines is the choice of the yeasts. During alcoholic fermentation, the wine yeasts extract and metabolize compounds from the grape must by modifying grape-derived molecules, producing flavor-active compounds, and promoting the formation of stable pigments by the production and release of fermentative metabolites that affect the formation of vitisin A and B type pyranoanthocyanins. This review covers the role of *Saccharomyces* and non-*Saccharomyces* yeasts, as well as lactic acid bacteria, on the perceived flavor and color of wines and the choice that winemakers can make by choosing to perform co-inoculation or sequential inoculation, a choice that will help them to achieve the best performance in enhancing these wine sensory qualities, avoiding spoilage and the production of defective flavor or color compounds.

**Keywords:** wine yeasts; lactic acid bacteria; co-inoculation; sequence inoculation; flavor compounds; color pigments

#### **1. Introduction**

#### *1.1. The Human Senses in Wine Evaluation*

Five senses are involved in perceiving wine sensory quality: sight, taste, hearing, touch, and smell. Color perception results from the stimulus of the retina by light (wavelengths 380 to 760 nm). In wine, color and appearance are the first attributes by which quality is assessed. According to Spence [1], color is the most important product-intrinsic indicator used by consumers when searching, purchasing, and subsequently consuming food or a libation. Color, clarity, and hue affect the perception of other attributes such as flavor due to the association with color. For example, a yellow/green beverage is expected to have a lemon flavor and an acidic taste.

Taste is a chemical sense and happens when taste stimuli contact with the taste receptors located on the tongue, called taste buds. Humans can distinguish six basic tastes: sweet, sour, salty, bitter, umami, and fatty [2,3]. Between 20 and 30 levels of intensity can be distinguished for each taste, and each taste quality represents different nutritional or physiological requirements, or a potential dietary risk [4].

Sound (waves which strike the eardrum, causing it to vibrate [5]) is also important when judging a wine. For instances, when we hear a champagne cork popping, it is a sign that the wine has an enjoyable gas.

Texture in wine can be defined as the total sum of kinesthetic sensations derived from oral manipulation. It encompasses mouthfeel, masticatory properties, residual properties, and even visual and auditory properties [6].

Aroma and flavor are chemical senses stimulated by the chemical properties of odor molecules which must reach the olfactory bulb to interact with olfactory cells in the olfactory mucosa [7]; therefore, to smell, molecules must be airborne (i.e., volatile). The sensory term which we call "flavor" is a mingled experience based on human judgment, built on personal differences in perception thresholds.

In conclusion, and as reported by Swiegers et al. [8], all of the senses play a key role in wine/flavor development—color, aroma, mouthfeel, sound, and, ultimately, taste. Altogether, these sensory perceptions are very complex. Wine contains many flavor and aroma-active compounds. Terpenes, methoxypyrazines, esters, ethanol and other alcohols and aldehydes impart distinct flavors and aromas (floral, pepper, fruit, woody and vinylic flavors, among others) to wine [9–11]. The taste of wine can be described as sweet, sour, salty, umami, bitter, and, to a lesser extent, fat [12]. These properties are the result of the presence of sugars, polyols, salts, polyphenols, flavonoid compounds, amino acids, and fatty acids. Compounds such as glycerol, polysaccharides, and mannoproteins contribute to the viscosity and mouthfeel of wines [13]; grape anthocyanins contribute to the color [14], and ethanol, by sheer mass, also carries other alcohols along, promoting a mouth-warming effect [15].

#### *1.2. Main Wine Aroma and Flavor Compounds from the Fermentative Origin*

Yeast and bacteria are vital to the development of wine flavor. Many biosynthetic pathways, in wine yeast and malolactic bacteria, are responsible for the formation of wine aroma and flavor. However, we cannot discard the other factors that can also influence the wine chemical composition, such as viticultural practices, grape-must composition, pH, fermentation temperature, and technological aspects associated with the vinification process [8]. So, depending on their origin, wine aroma and flavor compounds can be named varietal aromas (originating from the grapes), fermentative aromas (originating during alcoholic and malolactic fermentations), and aging aromas (developed during the reductive or oxidative wine-aging that depends on storage conditions) [16].

Most of the wine aroma and flavor compounds are produced or released during wine fermentation due to microbial activities of *Saccharomyces* and non-*Saccharomyces* yeast genera (*Brettanomyces, Candida, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Kluyveromyces, Lachancea, Metschnikowia, Pichia, Saccharomycodes, Schizosaccharomyces, Torulaspora,* and *Zygosaccharomyces*). Both in spontaneous and inoculated wine fermentations, non-*Saccharomyces* are important in early stages of the fermentation, before *Saccharomyces* becomes dominant in the culture, and contribute meaningfully to the global aroma profile of wines by producing flavor-active compounds [17,18].

A group of aroma compounds has been directly linked to specific varietal flavors and aromas in wines [19,20]. Most of these compounds are present at low concentrations in both grapes and fermented wine. These aroma compounds are found in grapes in the form of non-odorant precursors that, due to the metabolic activity of *Saccharomyces* and non-*Saccharomyces* yeast during fermentation, are transformed to aromas and flavor that are of great relevance in the sensory perception of wines [20] (Table 1).


**Table 1.** Main odorants contributing to varietal aromas of some monovarietal wines.

During alcoholic fermentation, some yeast, mainly non-*Saccharomyces* yeasts, can release β-glucosidases that hydrolyze the glycosidic bonds of the odorless non-volatile glycoside linked forms of monoterpenes (geraniol, linalool, nerol, among others), releasing the odor compounds to the wine [29]. Volatile thiols that give Sauvignon blanc wines their characteristic aroma (bell pepper, black currant, grapefruit, and citrus peel) are not present in grape juice but occur in grape must as odorless, non-volatile, cysteine-bound conjugates. During fermentation, the wine yeasts are responsible for the cleaving of the thiol from the precursor [30].

However, the major groups of aromas and flavor compounds from the fermentative origin are ethanol, higher alcohols or fusel alcohols, and esters. The biosynthetic pathways responsible for the formation of higher alcohols, the Ehrlich pathway, or the enzymes responsible for the formation of esters, have been studied in wine yeasts [31].

Higher alcohols are derived from amino acid catabolism via a pathway that was first described by Ehrlich [32] and later revised by Neubauer and Fromherz in 1911 [33]. Amino acids that are assimilated by the Ehrlich pathway (valine, leucine, isoleucine, methionine, and phenylalanine), present in grape must are metabolized by yeasts, sequentially, throughout the fermentation. Figure 1 shows the metabolism of phenylalanine with the production of 2-phenylethanol and, after oxidation of phenylacetaldehyde, the formation of phenylacetate. Both compounds possess a pleasant rose-like aroma/flavor.

**Figure 1.** Schematic representation of the Ehrlich pathway for the catabolism of the aromatic amino acid, phenylalanine leading to the formation of 2-phenylethanol [34]. This biosynthetic pathway consists of three steps (reactions 1, 2 and 3): first, amino acids are deaminated to the corresponding α-ketoacids, in reactions catalyzed by transaminases. In a second step, α-ketoacids are decarboxylated and converted to their corresponding aldehydes (five decarboxylases are involved in this process), in a third step, alcohol dehydrogenases (Adh1p to Adh6p and Sfa1p) catalyze the reduction of aldehydes to their corresponding higher alcohols [35].

Studies have shown that profiles and concentrations of higher alcohols produced vary by yeast species, even when the fermentation conditions are similar, which indicates that the mechanisms that regulate the Ehrlich pathway are diverse in non-*Saccharomyces* yeasts compared to *Saccharomyces* [16,36]. So, Ehrlich pathway mechanisms should be explored in detail in non-*Saccharomyces* yeasts as it contributes to the formation of important and flavorful wine aromas [36].

The most important esters are synthetized by yeasts during alcoholic fermentation as a detoxification mechanism since they are less toxic than their correspondent alcohol or acidic precursors. Moreover, their synthesis serves as a mechanism for the regeneration of free CoA from its conjugates [16,37].

Esters (Figure 2) that contribute to wine aroma, derived from fermentation, belong to two categories: the acetate esters of higher alcohols and the ethyl esters of medium-chain fatty acids (MCFA). Acetate esters are formed inside the yeast cell, and in *S. cerevisiae* the reaction is metabolized by two alcohol acetyltransferases, AATase I and AATase II (encoded by genes *ATF1* and *ATF2* [35,38]). Eat1p is responsible for the production of acetate and propanoate esters [39,40]. Most medium-chain fatty acid ethyl ester biosynthesis during fermentation is catalyzed by two enzymes, Eht1p and Eeb1p [38,41].

**Figure 2.** Schematic representation of the most important wine esters: ethyl acetate (glue-like aroma), isoamyl acetate (banana aroma), 2-phenylethyl acetate (roses and honey aromas), isobutyl acetate (sweet-fruits aromas), and ethyl caproate and ethyl caprylate with a sour-apple aroma [38].

Volatile fatty acids also contribute to the flavor and aroma of the wine. During yeast fermentation, long-chain fatty acids (LCFAs) are also formed via the fatty-acid synthesis pathway from acetyl-CoA in concentrations varying from ng/L to g/L [42]. Medium-chain fatty acids (MCFAs (C6 to C12)) are produced primarily by yeasts as intermediates in the biosynthesis of LCFAs that are prematurely released from the fatty acid synthase complex. These acids (Table 2) directly contribute to the flavor of wine or serve as substrates that participate in the formation of ethyl acetates [43]. As most have unpleasant aromas (see Table 2), their formation should be minimized.


**Table 2.** Main medium-chain fatty acids (MCFAs (C6 to C12)), produced by yeasts during alcoholic fermentation.

<sup>1</sup> Measured in model wine, water/ethanol (90 + 10, *w*/*w*) [44].

Sulfur-containing compounds can also be formed by yeasts during alcoholic fermentation. They are usually perceived as off-flavors. The sulfur-containing compounds can be derived from the grape and the metabolic activities of yeast and bacteria. They can also occur due to the chemical reactions during the wine aging and storage and also due to environmental contamination [45]. They can be formed by enzymatic mechanism as the products of metabolic and fermentative pathways whose substrates are both amino acids and some sulfur-containing pesticides. When wine microorganisms metabolize these thiols, the sulfur compounds formed are considered off-flavors [46] which convey negative notes such as cabbage, garlic, onion, rotten eggs, rubber, and sulfur to wines [47]. However, there are some volatile thiols that may confer enjoyable aromatic notes at trace levels, such as 4-mercapto-4-methylpentan-2-one (4MMP), 3-mercaptohexan-1-ol (3MH), already mentioned in Table 1, and 3-mercaptohexyl acetate (3MHA), important for the characterization of the typical Sauvignon Blanc wine aroma [24,25,48].

Finally, another important family of aromatic compounds present in wines are the carbonyl compounds. In this group we may include acetaldehyde, acrolein, ethyl carbamate, formaldehyde, and furfural [49]. Several factors may contribute to the presence of carbonyl compounds in wines, including the fermentation of over-ripe grapes and increasing the maceration time, probably due to increased concentration of the precursors like amino acids and glucose in the must [50]. Due to their carbonyl group, carbonyl compounds present a high reactivity with the nucleophile's cellular constituents [51] and may cause cell damage. So, these compounds are toxic, and their formation should be avoided.

#### **2. Yeast Modulation of Wine Aroma and Flavor Compounds**

#### *2.1. Non-Saccharomyces and Saccharomyces Co-inoculation vs. Sequential Inoculation*

The wine industry attempts to diversify producing wines with distinctive characteristics and creating high-quality new products. A true test for winemakers is to blend several grapes, grown on different soil and climate conditions (terroir), with a developing science of yeast and bacterial metabolism, to produce the most enjoyable wine [52]. Many winemakers today use commercial yeast and bacteria starter cultures for alcoholic and malolactic fermentation, respectively. The selection of a "fit-for-purpose" starter strain has a pivotal role in optimizing flavor and aroma.

There is no consensus on the impact of indigenous yeasts on wine sensory properties; while some researchers show a positive effect, others show negative effects on the wine chemical composition and sensory properties [53,54]. For example, Varela et al. [55] showed an increase in the concentration of some higher alcohols and esters in wines produced with autochthonous yeasts compared to the wines produced with commercial yeasts. Moreover, some non-*Saccharomyces* yeast may increase the concentration of biogenic amines in wines [56].

In red winemaking, significant increases in the concentrations of desirable compounds such as ethyl lactate (sweet, fruity, acidic, ethereal with a brown nuance aroma), 2,3-butanediol (buttery aroma), 2-phenylethanol, and 2-phenylethyl acetate (both with a rose-like scent) can be obtained when non-*Saccharomyces* yeasts are introduced into the fermentation process [57,58].

Another selection criterion for non-*Saccharomyces* yeasts, when aiming to improve the wine aroma, is the presence of β-glucosidase activity that favors the hydrolysis of the non-volatile aromatic precursors from the grape [59,60]. Non-*Saccharomyces* species display superior β-glucosidase activity to that of *Saccharomyces* species, which has been defined as intracellular and strain-dependent [23].

Budic-Leto et al. [61] found that Prosek, a traditional Croatian dessert wine, produced with native and inoculated yeasts differed in its volatile compounds. Using descriptive sensory analysis, it was shown that the sensory properties of the wines were significantly different depending on the type of fermentation, namely determined for the attributes strawberry jam aroma, and fullness. So, recently, non-*Saccharomyces* yeast species have been suggested for winemaking as they could contribute to the improvement of wine quality mostly in terms of aromatic characteristics [62,63]. Thus, starter cultures composed of non-*Saccharomyces* yeasts together with *S. cerevisiae* have been used for co-, or sequential wine fermentations [64].

Co-inoculation involving *S. cerevisiae* and non-*Saccharomyces* yeasts species typically results in the disappearance (or the presence in relative low amounts) and loss of viability of non-*Saccharomyces*[65,66]. Though *S. cerevisiae* dominance can be explained by the depletion of sugar and nutrients from the grape must followed by ethanol production and lack of oxygen, some direct mechanisms for yeast species antagonism have also been described: (i) killer factors (so-called killer toxins or killer proteins), which are secreted peptides, encoded by extrachromosomal elements of *S. cerevisiae* that affect other yeast species [67]; (ii) similar compounds have also been described for *Torulaspora delbrueckii* species [68] and for the genera *Pichia, Kluyveromyces, Lachancea, Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Hansenula, Kluyveromyces, Metschnikowia, Torulopsis, Ustilago, Williopsis*, and *Zygosaccharomyces,* indicating that the killer phenomenon is indeed widespread among yeasts. [69]; (iii) *S. cerevisiae* are also able to secret antimicrobial peptides (AMPs) during alcoholic fermentation that are active against wine-related yeasts (e.g., *Dekkera bruxellensis*) and bacteria (e.g., *Oenococcus oeni*). These AMPs correspond to fragments of the *S. cerevisiae* glyceraldehyde 3-phosphate dehydrogenase (GAPDH) protein [70].

Several authors agree that the sequential culture is better than the mixed culture, especially because it allows for a greater expression of the metabolism of non-*Saccharomyces* yeasts at the beginning of fermentation [71,72]. However, as recently reported by Loira et al. [73], the winemaker selection criteria for performing co-inoculation or sequential inoculation with the appropriate non-*Saccharomyces* is dependent on the characteristics of the wine to be produced, including the desired sensory properties. The ratio of inoculation (non-*Saccharomyces* vs. *Saccharomyces*) is also a subject that must be considered. Moreover, the contribution of the inoculation of non-*Saccharomyces* strains to wine fermentation can be direct or indirect, through biological interactions with *S. cerevisiae*. Recently, Renault et al. [74,75] described a synergic interaction between *S. cerevisiae* and *T. delbrueckii* resulting in increased levels of 3-sulfanylhexan-1-ol a compound that presents a sulphurous aroma and an initially fruity flavor. However, with over-aging, the aroma/flavor evolves to savory and chicken meaty with roasted coffee shades and a hint of fruitiness [76].

Not long ago, García et al. [63] performed small-scale fermentations where they studied the oenological characterization of five non-*Saccharomyces* native yeast species under several co-culture conditions in combination with a selected strain of *S. cerevisiae*, aiming to improve the sensory characteristics of the Malvar wines. Sequential inoculations were elaborated with *S. cerevisiae* CLI 889 in combination with several non-*Saccharomyces*: (i) *T. delbrueckii* CLI 918, which produced wines with a lower ethanol content and higher fruity and floral aroma; (ii) *C. stellata* CLI 920, which augmented the aroma complexity and glycerol content; (iii) *L. thermotolerans* 9-6C, which produced an increase in acidity and floral and ripe-fruit aroma and a lower volatile acidity; (iv) *Schizosaccharomyces pombe*, which produced wines with fruity aromas; and (v) *M. pulcherrima*, which produced wines with lower volatile acidity and an increase of glycerol and ripe-fruit aroma.

Continuing their work on Malvar wines, García et al. [77] performed fermentations at the pilot scale, using sequential-inoculation strategies which resulted in wines that tasters were able to distinguish

from the controls. Moreover, the wines were most appreciated, namely, those produced in sequential cultures with *T. delbrueckii* CLI 918/*S. cerevisiae* CLI 889 and *C. stellata* CLI 920/*S. cerevisiae* CLI 889 and, also, with mixed and sequential cultures of *L. thermotolerans* 9-6C/*S. cerevisiae* CLI 889 strains. Studies have shown that sequential cultures can produce more different wines, when compared with the controls, providing sensory properties associated with the non-*Saccharomyces* strains. Some strains of *T. delbrueckii* in sequential fermentation with *S. cerevisiae* can produce significant amounts of 3-ethoxy-1-propanol [78], with a fruity-like aroma with a low perception threshold, 0.1 mg/L [79].

However, sequential inoculation is not only favorable for positive aromas sequential. It is a fermentation technique that can be used to prevent or diminish the production of some undesirable compounds, augmenting the production of others. Viana et al. [80] reported a decrease in the higher alcohol's concentration (considered as possessing a fuel-like aroma) from 452.5 mg/L (control) to 306.2 mg/L when carrying out mixed fermentations with *H. osmophila* and *S. cerevisiae*. Moreover, higher concentrations of 2-phenylethyl acetate (rose-like aroma) were obtained. A higher intensity of fruitiness was also detected in these wines when compared to the control wine, obtained throughout the fermentation of a pure *S. cerevisiae* culture.

#### *2.2. Saccharomyces and Lactic Acid Bacteria co inoculation vs. Sequential Inoculation*

The vinification involves different microbiological processes, mainly alcoholic fermentation (AF) conducted by *Saccharomyces cerevisiae* and malolactic fermentation (MLF) conducted by lactic acid bacteria (*Oenococcus oeni*). These two distinctive fermentation processes represent an essential step in the improvement of the quality of red wines [81].

MLF naturally occurs after AF, however, the timing of the start of MLF depends on several parameters like temperature, pH, alcoholic degree and the concentration of sulfur dioxide (SO2), as well as on certain yeast metabolites available, such as medium-chain fatty acids and peptidic fractions [82,83]. The success of spontaneous MLF is not always guaranteed, and the addition of starter culture can improve its viability. To overcome this issue, the winemakers may carry out traditional LAB inoculation after alcoholic fermentation (sequential inoculation), or simultaneous inoculation in the must with yeast (co-inoculation). The co-inoculation has, in the last several years, been adopted by some winemakers, particularly in warm climates with higher temperatures, where high concentrations of ethanol can inhibit LAB growth [84].

There are not many studies focusing on the impact of the co-inoculation technique on the aromatic and biochemical profile of wines. Abrahamse and Bartowsky [85] and Knoll et al. [86] have shown that the timing of inoculation with LAB in both white and red wines could influence the profile of aromatic yeast-derived compounds such as higher alcohols, terpene, esters, and fatty acids. However, these works were performed at the lab scale, and no sensory evaluation was performed on the wines.

Antalick and collaborators [87] demonstrated the impact of the timing of inoculation with LAB on the metabolic profile of wines manufactured at production scale. They have clearly shown that this technique has an impact on the aromatic profile of the wines, mainly in the presence of lactic and fruity notes. Co-inoculation can modulate the intensity of these descriptors, due to the production/degradation of metabolites or by the development of an aromatic mask over the short and long term. Moreover, they discuss that the metabolic and aromatic changes that occur with co-inoculation depend strongly on the yeast and LAB strains, as well as on the composition of the must. Co-inoculation of musts at the beginning of vinification can also lead to a faster vinification process without an excessive increase in volatile acidity [88].

Due to the importance of the interaction between yeasts (*Saccharomyces* and non-*Saccharomyces*) and bacteria during wine processing, Berbegal et al. [89] applied a next-generation sequencing (NGS) analysis to several fermentation modalities: uninoculated must, pied-de-cuve, *S. cerevisiae*, *S. cerevisiae,* and *Torulaspora delbrueckii* co-inoculated and sequentially inoculated, along with *S. cerevisiae* and *Metschnikowia pulcherrima* co-inoculated and sequentially inoculated, continued by spontaneous malolactic consortium to perform MLF. Each experimental trial led to the different

taxonomic composition of the bacterial communities of the malolactic consortia, in terms of prokaryotic phyla and genera. Among other interesting findings, they found that MLF was delayed when *M. pulcherrima* was inoculated and was even inhibited when the inoculated yeast strain was *T. delbrueckii* [89]. Thus, an antagonistic effect of *M. pulcherrima* and especially *T. delbrueckii* on lactic bacteria population has been proven, which may be due to the ability of *T. delbrueckii* to produce "toxins" or killer factors that prevent bacteria growth [68] and to the ability of *M. pulcherrima* to produce high amounts of pulcherrimin (an iron chelator) that inhibits the growth of bacteria [90].

#### **3. Yeast Modulation of Wine Color and Pigment Formation**

Anthocyanins and their derivatives, originating in the grapes, are the main pigments responsible for the red wine color, and their structural modifications result in a characteristic variation of color in red wines, from pale ruby (young red wine) to deep purple-red color (aged red wine) [91,92]. Such variations can also result in changes in wine mouthfeel and flavor.

*Saccharomyces* yeasts can directly or indirectly contribute to wine color, altering color parameters such as intensity and tonality by: (i) increasing the formation of stable pigment precursors (vinyl phenols, acetaldehyde, and pyruvic acid); and (ii) modifying the pH due to organic acid metabolism (production or consumption) [73].

Pyruvic acid and acetaldehyde promote the formation of vitisins of types A and B, respectively [93, 94], during must fermentation, (Figure 3A). Vitisins contribute more to wine color parameters than unmodified anthocyanins and exhibit a hypsochromic shift, i.e., a change of spectral band position (in the absorption, reflectance, transmittance, or emission spectrum) to a shorter wavelength corresponding to a higher frequency. Vitisins can change towards an orange-red hue due to the long conjugation afforded by pyran ring [95]. Moreover, their color expression remains stable against discoloration due to the presence of sulfur dioxide (bleaching capacity) or changes in pH [95] (Figure 4).

**Figure 3.** (**A**) Structures of vitisin A and vitisin B generated from malvidin-3-*O*-glucoside. (**B**) The formation mechanism of vitisin A produced by condensation of an anthocyanin (malvidin-3-*O*-glucoside) with pyruvic acid [92,96,97].

**Figure 4.** UV-visible spectra of malvidin-3-*O*-glucoside and vitisins A and B. Adapted from [98].

During the fermentation process, practices like pellicular maceration that increase the extraction of anthocyanins from the skins of grape berries will promote the formation of vitisins [14]; also, acetaldehyde and pyruvic acid, as mentioned before, are important intermediate compounds in yeast metabolism and influence wine vitisin content [93,99,100].

During sugars' fermentation by yeasts, pyruvate is metabolized into acetaldehyde, with the latter being the terminal electron acceptor in the formation of ethanol. Acetaldehyde and pyruvate, formed in yeast cytoplasm, are rapidly metabolized (the first is reduced to ethanol, and the second is either decarboxylated to acetaldehyde or used in the formation of acetyl-CoA). However, some of the acetaldehyde and pyruvate molecules, through cell lysis, pass to the wine medium and are sufficiently reactive to attack other molecules, enabling the transformation of anthocyanins into compounds such as pyranoanthocyanins and their secondary generated pigments (anthocyanin oligomers and polymeric anthocyanin) [92,97]. Pyroanthocyanins are the most important group of anthocyanin derivatives present in fermented beverages, including wine, and the A-type vitisins or carboxy-pyroanthocyanins are produced, as mentioned before, by condensation of anthocyanin with pyruvic acid [97] (Figure 3B).

In terms of vitisin kinetic formation, during *S. cerevisiae* fermentation, type-A vitisins are produced in the first six days of fermentation (when pyruvic acid is available). At the end of fermentation, when nutrients are limited, the amount of acetaldehyde is high enough to lead to the formation of type-B vitisins [93,99]. So, to generate a more pleasant red wine color, before fermentation the winemaker must select the wine yeast strains that will be able to increase anthocyanin extraction and/or can produce more pyruvic acid and acetaldehyde. Postponing or starting MLF early can prevent the consumption of pyruvic acid and acetaldehyde by lactic acid bacteria [101], increasing the possibility for vitisin synthesis.

Oxidative fermentation (fermentation in barrels or with micro-oxygenation) and wine aging in wood give rise to pyruvic acid and acetaldehyde, increasing the vitisin levels and, consequently, color intensity and stability [97]. Yellowish α-pyranone-anthocyanins called oxovitisins were also described by He et al. [102] in aged red wines derived from the direct oxidation of A-type vitisins. Moreover, A-type vitisins are the pyranoanthocyanins detected in higher concentrations in port wines. Port wine is made by stopping the fermentation process with the addition of wine spirit "aguardente", leaving the wine with a high concentration of sugars. So, when fermentation is stopped, the pyruvic acid concentration is relatively high and increases after wine fortification [97].

Vinylphenolic pyroanthocyanin adducts result from the condensation between vinyl phenols and anthocyanins. These color compounds also show high color stability [94]. Yeast strains are also able to affect the concentration and the composition of wine tannins as well as the degree of tannin polymerization [103], thus, indirectly throughout yeast actions affecting stabilization of anthocyanins, and consequently, stabilization of color can occur due to reaction between anthocyanins and tannins forming pigmented tannins and through copigmentation of anthocyanins [104].

Additionally, what about non-*Saccharomyces* wine yeasts? Well, we have already mentioned that an improvement in fermentation quality, efficiency, and wine pleasantness is obtained when sequential or co-inoculation of non-*Saccharomyces* and *Saccharomyces* yeast is performed.

*Torulaspora delbrueckii* is one non-*Saccharomyces* species available commercially (Viniflora® Harmony.nsac and Viniflora® Melody.nsac, Zymaflore® Alpha, BIODIVA®, and Viniferm NS-TD® are some commercial examples), therefore, this yeast could be a good candidate for wine color improvement as it is reported to have a positive influence on the taste and aroma of wines. For instance, Pinotage grape must inoculate with *T. delbrueckii* originated red wines improved in color intensity (anthocyanins) and mouthfeel (flavanols) when compared to the control (musts inoculated with *S. cerevisiae*) [105]. However, *T. delbrueckii* has poor production of acetaldehyde [106], thus being a poor candidate for wine color improvement in the context of B-type vitisins.

Other non-*Saccharomyces* yeasts, not yet available as commercial products, but studied in the academic community, could be good candidates for wine color improvement. Medina et al. [107] reported that in the case of co-fermentation of *Metschnikowia* and *Hanseniaspora* with *S. cerevisiae*, only an increase in the content of B-type vitisins occurred, probably due to the enhanced acetaldehyde formation.

Further experiments performed by sequential inoculation of *Schizosaccharomyces pombe* and *Lachancea thermotolerans* exposed an increase of type-A vitisins when compared with the control *S. cerevisiae* [108]. Also, several authors detected interesting features in non-*Saccharomyces* yeasts. *P. guilliermondii* strains presenting a high hydroxycinnamate decarboxylase activity may improve the formation of vinylphenolic pyranoanthocyanins; non-*Saccharomyces* yeasts, such as *Candida valida, Metschnikowia pulcherrima, Kloeckera apiculata* and *Starmerella bombicola*, which synthesize and release pectolytic enzymes, can improve wine color due to the extraction of a greater amount of polyphenolic compounds during fermentation and by facilitating clarification and filtration processes [73,98,109,110].

#### **4. The Role of** *Saccharomyces* **and non-***Saccharomyces* **Mannoproteins in Aroma and Color of Wines**

Mannoproteins are highly glycosylated glycoproteins located on the external layer of the yeast cell wall, representing 35% to 40% of the *S. cerevisiae* cell wall (Figure 5) [111]. Mannoproteins are composed of 10% to 20% protein and 80% to 90% d-mannose associated with residues of d-glucose and N-acetylglucosamine [112].

**Figure 5.** Schematic representation of the yeast cell wall. The yeast cell wall is composed of mannan–oligosaccharide (mannoproteins), complex polymers of β-(1,3)/(1,6) glucan, and chitin. As shown in Figure 5, mannoproteins are located on the surface of the cell wall.

In wine, we can find two groups of mannoproteins: one made up of those secreted into wine by yeast during alcoholic fermentation (100–150 mg/L) with molecular weights from 5000 to more than 800,000 Da [113], the other one composed by those that are released into the wine due to the autolysis of yeasts during aging on lees, probably through the cleavage of linkages between mannoproteins, glucans, and chitin [113,114].

The presence of these mannoproteins in wines has many positive consequences, from the reduction of the protein haze in white wines [115] to decreasing astringency of red wines, by increased inhibition of tannin aggregation [116,117]. Among other positive factors, mannoproteins also interact with wine volatile compounds [118]. So, these yeast-derived glycoprotein complexes can have positive effects on the technological and sensorial properties of wines [119].

In terms of improving wine palatability and mouth feel, yeast mannoproteins promote the increase of wine sweetness [120] and improve the aroma persistence and complexity [114,121]. However, the number of mannoproteins released by yeast into wine can vary concerning the strain and the chemical–physical and compositional conditions of the wine system [121,122].

Their presence can also affect the release of volatile compounds, affecting the final perception of the wine [121]. The physicochemical interactions between aroma compounds and mannoproteins depends on the nature of the volatile, since a greater degree of interaction is often observed with hydrophobic compounds [123], as well as the conformational structure of the mannoproteins [114]; moreover, Chalier et al. [114] demonstrated that both the glycosidic and peptidic parts of the mannoproteins may interact well with the aroma compounds.

It has been shown that the use of mannoproteins (in low amounts) or the contact of the wine with fine lees increases the levels of esters (ethyl hexanoate, methyl, and ethyl hexadecanoate, which present fruity aromas), due to the esterification of fatty acids released during yeast fermentation or yeast autolysis [121,124], while higher amounts increase, in excess, fatty acid content, producing yeasty, herbaceous, and cheese-like smells [125]. However, it has been suggested that mannoproteins can be used to remove or reduce the incidence of wine off-flavors—4-ethylguaiacol and 4-ethylphenol. The sorption of these compounds to the yeast walls could be due to their interactions with the functional groups of the mannoproteins and the free amino acids on the surface of the cell walls [126].

The release of mannoproteins into the wine is not a physiological characteristic of just *S. cerevisiae* yeast strains. In 2014, Domizio and collaborators [127], studied eight non-*Saccharomyces* wine strains (*H. osmophila*, *L. thermotolerans, M. pulcherrima*, *P. fermentans*, *S. ludwigii*, *S. bacillaris*, *T. delbrueckii*, and *Z. florentinus*) in mixed inocula fermentations of a synthetic polysaccharide-free grape juice for their ability to release mannoproteins. The eight non-*Saccharomyces* yeasts confirmed a higher capacity to release polysaccharides when compared to *S. cerevisiae*. Moreover, Pérez-Través et al. [128] also studied the ability of natural hybrids of *S. cerevisiae* × *S. krudriavzevii* to release mannoproteins. Interestingly, they found that this strain, in the fermentation conditions studied, was able to produce a higher quantity of mannoproteins when compared with the sample in which only *S. cerevisiae* was used. Furthermore, the authors also found that the genome interaction in hybrids creates a biological ecosystem that boosts the release of mannoproteins.

As mentioned before, the presence of mannoproteins in wines, namely red wines, promotes tannin stability and reduction of astringency [116,117]. The interaction between mannoproteins and wine phenolic compounds is a matter of interest, as studies show that they may have an impact on color stability [129,130]. However, results are contradictory, as some authors state that there was no positive interaction between mannoproteins and color compounds and that the interaction between mannoproteins and tannins results in a decrease of wine tannin content due to the precipitation of tannin and mannoprotein [112,120,131,132], thus being responsible for a decrease in wine color intensity or lower filterability [133], whereas, others state that mannoproteins appear to stabilize anthocyanin-derived pigments, from a colloidal point of view, avoiding their aggregation and further precipitation [134]. The study of the exhaustive pigment composition of wines has shown that the addition of mannoproteins can stabilize type-A vitisins and other derivative pigments [134].

#### **5. Final Remarks**

Wines are complex and evolve physiochemically and sensorially through time. Most of the wine aroma and flavor compounds are produced or released during wine fermentation due to microbial activity of *Saccharomyces*, non-*Saccharomyces* yeast genera, and lactic acid bacteria. A true challenge for winemakers is the selection of a "fit-for-purpose" microbial starter culture or culture strains that can have a crucial role in optimizing flavor, aroma, and color of wines, among other sensory properties.

Co-inoculation involving *S. cerevisiae* and non-*Saccharomyces* yeasts species may result in the death or loss of variability of non-*Saccharomyces*, once *S. cerevisiae* dominates the fermentation and is stress-resistant to the inhibitory ethanol effect. So, several authors suggest that the sequential inoculation (non-*Saccharomyces* followed by *S. cerevisiae*) is a better technique than the mixed culture, allowing a higher expression of the metabolism of non-*Saccharomyces.* Nevertheless, the ratio of inoculation (non-*Saccharomyces* vs. *Saccharomyces*) must be taken into account, especially if the wine should present a special and desirable characteristic such as the expression of a peculiar aroma-flavor, or even the inhibition of the production of a specific family of compounds like, for instance, higher alcohols.

Concerning the MLF, the co-inoculation process has been adopted by some winemakers, in warm climates, where high concentrations of ethanol can inhibit lactic acid bacteria (LAB) growth. Co-inoculation of musts at the beginning of vinification can also accelerate the process without an excessive increase in volatile acidity. However, winemakers must be aware of the possible interactions between yeasts (*Saccharomyces* and non-*Saccharomyces*) and LAB during wine processing. LAB feed on dead and lysed yeast cells but some non-*Saccharomyces* may delay (*M. pulcherrima*) or inhibit (*T. delbrueckii*) bacterial growth, thus inhibiting the occurrence of MLF.

Regarding wine color characteristics, especially red wine, *Saccharomyces* and non-*Saccharomyces* yeasts can directly or indirectly contribute to wine intensity and tonality, by increasing the formation of stable pigment precursors (vinyl phenols, acetaldehyde, and pyruvic acid) and by modifying the pH due to organic acid metabolism. Pyruvic acid is necessary for vitisin synthesis; these important pigments contribute more to wine color parameters than unmodified anthocyanins. Concerning acetaldehyde, this compound has several negative impacts (one on health, the other as a potent binder of SO2), so its formation, although beneficial to wine color, should be avoided. Also, it is important to choose the right time for promoting MLF, either spontaneously or by inoculation of a commercial LAB strain, to prevent consumption of pyruvic acid by lactic acid bacteria and to promote vitisin synthesis.

Finally, mannoproteins may have a positive effect on sensory perception of red wine, reducing astringency and bitterness and encouraging aroma revelation and odor complexity, but further studies are necessary in order to unravel the possible stabilization mechanism and the relationship between *Saccharomyces* and non-*Saccharomyces* mannoprotein characteristics and their ability to stabilize wine color.

So, in conclusion, knowledge and control of yeast and bacteria can help winemakers enhance the sensory quality of their wines for flavor and color.

**Funding:** We appreciate the financial support provided to the Research Unit in Vila Real [grant number UID/QUI/00616/2019] by FCT-Portugal and COMPETE

**Acknowledgments:** The author wants to acknowledge Interreg Program for the financial support of the Project IBERPHENOL, Project Number 0377\_IBERPHENOL\_6\_E, co-financed by European Regional Development Fund (ERDF) through POCTEP 2014-2020.

**Conflicts of Interest:** The author declares no conflict of interest

#### **References**


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