Evolution of Aromatic Proﬁle of Torulaspora delbrueckii Mixed Fermentation at Microbrewery Plant

: 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.


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][2][3][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.
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.

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.

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.

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.

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.

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.

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.

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.

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. 10 7 CFU/mL at 3 days of fermentation and decreased at 10 6 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 5 CFU/mL, while T. delbrueckii, started at a concentration >10 6 CFU/mL, achieved the maximum cell concentration at 3 days of fermentation (107 CFU/mL), and decreased at the end of fermentation (10 6 CFU/mL). The results for mixed fermentation indicated that T. delbrueckii at Fermentation 2020, 6, 7 4 of 9 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. The growth kinetics of the S. cerevisiae US-05 pure cultures achieved ca. 10 7 CFU/mL at 3 days of fermentation and decreased at 10 6 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 5 CFU/mL, while T. delbrueckii, started at a concentration >10 6 CFU/mL, achieved the maximum cell concentration at 3 days of fermentation (107 CFU/mL), and decreased at the end of fermentation (10 6 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.

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

By-Products and Volatile Compounds
The main volatile compound by-products are reported in Table 2. The growth kinetics of the S. cerevisiae US-05 pure cultures achieved ca. 10 7 CFU/mL at 3 days of fermentation and decreased at 10 6 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 5 CFU/mL, while T. delbrueckii, started at a concentration >10 6 CFU/mL, achieved the maximum cell concentration at 3 days of fermentation (107 CFU/mL), and decreased at the end of fermentation (10 6 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.

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

By-Products and Volatile Compounds
The main volatile compound by-products are reported in Table 2. The growth kinetics of the S. cerevisiae US-05 pure cultures achieved ca. 10 7 CFU/mL at 3 days of fermentation and decreased at 10 6 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 5 CFU/mL, while T. delbrueckii, started at a concentration >10 6 CFU/mL, achieved the maximum cell concentration at 3 days of fermentation (107 CFU/mL), and decreased at the end of fermentation (10 6 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.

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

By-Products and Volatile Compounds
The main volatile compound by-products are reported in Table 2. The growth kinetics of the S. cerevisiae US-05 pure cultures achieved ca. 10 7 CFU/mL at 3 days of fermentation and decreased at 10 6 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 5 CFU/mL, while T. delbrueckii, started at a concentration >10 6 CFU/mL, achieved the maximum cell concentration at 3 days of fermentation (107 CFU/mL), and decreased at the end of fermentation (10 6 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.

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

By-Products and Volatile Compounds
The main volatile compound by-products are reported in Table 2.
) individually for the mixed fermentation.

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

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). Table 2. Evolution of the main volatile compounds (mg/L) in the beer produced by pure (S. cerevisiae) and mixed fermentation (S. cerevisiae/T. delbrueckii) during the fermentation process. Data are means ± standard deviation. Data with different superscript letters ( a,b,c,d, )  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.

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

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

Sensory Analysis
The beers obtained by pure and mixed fermentations underwent sensory analysis, and the results were illustrated in Figure 2.
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.
) and mixed cultures of S. cerevisiae/T. delbrueckii ( 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).
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.

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

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