Investigation of Non-Saccharomyces Yeast Strains for Their Suitability for the Production of Non-Alcoholic Beers with Novel Flavor Profiles

Figures & data

Table 1. Yeast species and strain numbers with corresponding abbreviations used in this study.

Yeast strain number	Yeast strain abbreviation	Yeast species
CBS 5640	C. fab 5640	Cyberlindnera fabianii^a
CBS 5650	C. fab 5650	Cyberlindnera fabianii^a
TUM 238	C. mis 238	Cyberlindnera misumaiensis^b
YH837A-3D4	C. mis CM1	Cyberlindnera misumaiensis^b
TUM 247	C. sat 247	Cyberlindnera saturnus^b
CBS 4549	C. sat 4549	Cyberlindnera saturnus^a
YHMH22AA-3H1	C. sat CSa1	Cyberlindnera saturnus^c
YHMH47B-3C4	K. ser 3C4	Kazachstania servazzii^b
TUM G9K	K. lac G9K	Kluyveromyces lactis^b
TUM 653	K. mar 653	Kluyveromyces marxianus^b
CBS 3082 T	L. klu 3082	Lachancea kluyveri^a
YHAK1A-3I1	P. klu PK1	Pichia kluyveri^b
TUM SL17	S. lud SL17	Saccharomycodes ludwigii^b
PI S 6; Lu27	S. fib Lu27	Saccharomycopsis fibuligera^b
TUM G10S	S. pom G10S	Schizosaccharomyces pombe^b
YH824A-1I6	T. del 1I6	Torulaspora delbrueckii^b

a Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands; ^b Research Center Weihenstephan (BLQ), Freising, Germany; ^c Private collection

Table 2. Analytical methods of the wort and the non-alcoholic beers after MEBAK^a and Donhauser et al.^b

Analysis	Method	Device
Sugar composition (glucose, fructose, sucrose, maltose, maltotriose)	Donhauser et al.^b LS-HPLC 002_2	HPLC UltiMate 3000 (Thermo Fisher Scientific, Waltham, MA, U.S.A.)
Original wort, ethanol content, apparent attenuation	MEBAK^a WBBM 2.9.6.3	Bending vibration and NIR spectroscopy, Alcolyzer Plus with DMA 5000 X sample 122 (Anton-Paar GmbH, Ostfildern, Germany)
pH value	MEBAK^a WBBM 2.13	pH meter with pH electrode, ProfiLine pH3210 pH meter (Xylem Inc., New York, NY, U.S.A.)
Dimethyl sulfide	MEBAK^a WBBM 2.23.1	GC-FID Clarus 580 (Perkin Elmer, Waltham, MA, U.S.A.), Column: 50 m 0.32 mm Phenomenex FFAP, 0.25 μm
Fatty acid esters, fatty acids, 2-phenylethanol	MEBAK^a WBBM 2.23.6
Acetaldehyde, ethyl acetate, isoamyl acetate, higher alcohols (n-propanol, i-Butanol, amyl alcohols)	MEBAK^a WBBM 2.21.1	GC-FID Clarus 580, Turbo Matrix 40, Head Space (Perkin Elmer, Waltham, MA, U.S.A.), Column: INNOWAX cross-linked polyethylene glycol, 60 m × 0.32 mm 0.5 μm
4-Vinylguaiacol	MEBAK^a WBBM 2.21.3.3
Diacetyl, 2,3-pentanedione	MEBAK^a WBBM 2.21.5.1

a MEBAK® (2012), Editor: Dr. F. Jacob: The MEBAK collection of brewing analysis methods: Wort, beer, and beer-based beverages. Collection of methods of the Mitteleuropäischen Brauchtechnischen Analysenkommission. Self-published by MEBAK.

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

Figure 1. Chemical modification of glucose for HPLC visualization using UV/Vis detector. (Endres, F.; Prowald, A.; Fittschen, U. E. A.; Hampel, S.; Oppermann, S.; Jacob, F.; Zarnkow, M.; Hutzler, M.; Methner, Y.; Laus, A. Vergleichendes isothermes Maischen bei 72 °C, BRAUWELT 2021, 9-10, 223-227.)

Figure 2. Mean values (n = 3) with standard deviations of the fermentation samples’ total weight loss in grams of the investigated yeast strains during the fermentation process over a fermentation period of 144 h (6 days) at 20 °C.

Table 3. Regression equations and corresponding coefficients of determination R².

Yeast strain abbreviation	Regression equation	Coefficient of determination R^2
C. fab 5640	y = 0.0074 x	99.1%
C. fab 5650	y = 0.0102 x	99.7%
C. mis 238	y = 0.0096 x	97.9%
C. mis CM1	y = 0.0078 x	98.9%
C. sat 247	y = 0.0085 x	99.7%
C. sat 4549	y = 0.0072 x	97.4%
C. sat CSa1	y = 0.0121 x	98.9%
K. ser 3C4	y = 0.0081 x	97.0%
K. lac G9K	y = 0.0147 x	97.7%
K. mar 653	y = 0.0106 x	99.5%
L. klu 3082	y = 0.0166 x	96.1%
P. klu PK1	y = 0.0067 x	97.9%
S. lud SL17	y = 0.0149 x	96.4%
S. fib Lu27	y = 0.0126 x	97.7%
S. pom G10S	y = 0.0108 x	99.8%
T. del 1I6	y = 0.0196 x	94.8%

Table 4. Mean values and standard deviations (n = 3) of original wort (°P), apparent attenuation (%), ethanol content (% v/v) and pH value in the beers fermented by the 16 selected yeast strains.

Yeast strain abbreviation	Original wort (°P)	Apparent attenuation (%)	Ethanol content (% v/v)	pH value
C. fab 5640^a	7.05 ± 0.00	9.07 ± 0.21	0.33 ± 0.00	4.83 ± 0.00
C. fab 5650^a	7.02 ± 0.01	12.00 ± 0.00	0.43 ± 0.00	4.79 ± 0.01
C. mis 238^a	7.03 ± 0.00	8.93 ± 0.12	0.32 ± 0.01	4.55 ± 0.00
C. mis CM1^a	7.03 ± 0.02	8.60 ± 0.08	0.31 ± 0.00	4.62 ± 0.00
C. sat 247^a	7.02 ± 0.01	10.00 ± 0.00	0.36 ± 0.00	4.76 ± 0.01
C. sat 4549^a	7.03 ± 0.00	7.73 ± 0.05	0.28 ± 0.00	4.84 ± 0.00
C. sat CSa1^a	7.03 ± 0.01	12.77 ± 0.05	0.46 ± 0.00	4.83 ± 0.00
K. ser 3C4^b	6.94 ± 0.01	9.23 ± 0.09	0.33 ± 0.00	4.88 ± 0.01
K. lac G9K^b	6.85 ± 0.09	13.97 ± 0.09	0.49 ± 0.00	5.08 ± 0.01
K. mar 653^b	6.88 ± 0.00	10.53 ± 0.12	0.37 ± 0.00	4.60 ± 0.00
L. klu 3082^b	6.92 ± 0.00	15.73 ± 0.05	0.55 ± 0.00	4.69 ± 0.00
P. klu PK1^a	7.02 ± 0.01	7.33 ± 0.17	0.26 ± 0.01	4.82 ± 0.00
S. lud SL17^b	6.95 ± 0.05	14.02 ± 0.10	0.50 ± 0.00	4.92 ± 0.04
S. fib Lu27^b	6.91 ± 0.01	13.20 ± 0.29	0.47 ± 0.01	4.60 ± 0.01
S. pom G10S^b	6.90 ± 0.00	11.30 ± 0.16	0.40 ± 0.00	4.77 ± 0.00
T. del 1I6^b	6.89 ± 0.00	15.50 ± 0.00	0.54 ± 0.00	4.78 ± 0.01

a Wort batch 1; ^b Wort batch 2

Table 5. Sugar composition of the wort used for fermentation trials.

Sugar composition of wort in g/L
Fructose	0.6
Glucose	4.4
Sucrose	1.6
Maltose	31.7
Maltotriose	7.3

Figure 3. Mean values (n = 3) with standard deviations of wort sugar utilization in % of the 16 investigated yeast strains during fermentation from worts to non-alcoholic beers.

Figure 4. Mean values (n = 3) with standard deviations of yeast growth at 20 °C of ten yeast strains metabolizing more than 5% maltose in the HPLC analysis according to Table 2 (C. fabianii 5640, C. saturnus 247 and CSa1, K. servazzii 3C4, K. lactis G9K, L. kluyveri 3082, P. kluyveri PK1, S. fibuligera Lu27, S. pombe G10S, T. delbrueckii 1I6) measured at an extinction of 600 nm in YP-media with 1% maltose over 168 h (7 days). S. ludwigii SL17 was included as reference yeast strain.

Figure 5. Mean values (n = 3) with standard deviations of yeast growth at 20 °C of seven strains (C. fabianii 5640 and 5650, C. misumaiensis 238 and CM1, C. saturnus 247 and CSa1, P. kluyveri PK1) metabolizing more than 5% maltotriose in the HPLC analysis according to Table 2 and measured at an extinction of 600 nm in YP-media with 1% maltotriose for 168 h (7 days). S. ludwigii SL17 was included as a reference yeast strain.

Figure 6. Chromatograms (HPLC) of the modified sugars glucose, maltose, maltotriose and maltotetraose of the 7°P wort and beer fermented with the yeast strain C. saturnus 247. The sugar molecules contain one molecule of 3-methyl-1-phenyl-5-pyrazolone (fructose) and two molecules of 3-methyl-1-phenyl-5-pyrazolone (glucose, maltose, maltotriose and maltotetraose).

Figure 7. Heat map of volatile compounds in final non-alcoholic beers produced by the 16 investigated yeast strains during fermentation of brewer’s wort at 20 °C for 144 h and detected by headspace gas chromatography measurement. The 100% value represents the threshold of volatile compounds in beer, which is based on relative values from Meilgaard, Sannino et al., and Schieberle.^[56–58]

Table 6. Number of sensory assessors (n = 10) identifying the beers produced with the 16 different non-Saccharomyces yeast strains as “fruity”.

Yeast strain abbreviation	Number of assessors identifying the beer as “fruity”
C. fab 5640	10*
C. fab 5650	10*
C. mis 238	10*
C. mis CM1	9*
C. sat 247	10*
C. sat 4549	9*
C. sat CSa1	10*
K. ser 3C4	8
K. lac G9K	9*
K. mar 653	9*
L. klu 3082	9*
P. klu PK1	8
S. lud SL17	8
S. fib Lu27	9*
S. pom G10S	9*
T. del 1I6	7

* Significant fruitiness (α = 0.05) according to MEBAK sensory analysis 3.1.1. and DIN EN ISO 5495:2007

Figure 8. Aroma profiles of the beers fermented with the seven different Cyberlindnera yeast strains C. saturnus CSa1, 247, 4549, C. misumaiensis CM1, 238 and C. fabianii 5650, 5640.

Figure 9. Aroma profiles of the beers fermented with the reference yeast strain S. ludwigii SL17 and the investigated yeast strains K. servazzii 3C4, K. lactis G9K, K. marxianus 653, L. kluyveri 3082, P. kluyveri PK1, S. fibuligera Lu27, S. pombe G10S, and T. delbrueckii 1I6.

Figure 10. Principal Component Analysis (PCA) biplot with two principal components PC 1 and PC 2 showing the correlation of the beers produced with the 16 investigated yeast strains and the specific volatile compounds measured analytically as well as the individual flavor characteristics described by the tasters.

Table 7. Total DLG score according to the DLG evaluation scheme excluding the quality of bitterness as well as carbonation of the 15 investigated yeast strains and the reference yeast strain S. ludwigii SL17.

Yeast strain abbreviation	Total DLG score
C. fab 5640	4.16
C. fab 5650	4.44
C. mis 238	4.14
C. mis CM1	4.34
C. sat 247	4.54
C. sat 4549	4.54
C. sat CSa1	4.44
K. ser 3C4	4.16
K. lac G9K	4.00
K. mar 653	4.38
L. klu 3082	4.10
P. klu PK1	4.02
S. lud SL17	4.16
S. fib Lu27	4.32
S. pom G10S	4.22
T. del 1I6	4.08

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