Increasing Higher Alcohols and Acetates in Low-Alcohol Beer by Proteases
Abstract
:1. Introduction
2. Results and Discussion
2.1. Design of Experiments and Mashing with Proteases
2.1.1. Design of Experiments to Maximize the Leucine Molar Fraction in Wort
Effect Screening of Endoprotease, Exoprotease, and Temperature
Response Surface Model of Endoprotease Endo1 and Temperature
2.1.2. Fermenting Wort Production and Amino Acid Molar Fraction
2.2. Fermentation and Aroma Output
3. Materials and Methods
3.1. Mashing and Wort Production
3.2. Fermentation and Volatile Measurement
3.3. Design of Experiments and Statistical Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Sample Availability
Appendix A
Yeast | Control | Endo1 + Exo3 | ||
---|---|---|---|---|
Attenuation 1 | ABV% 2 | Attenuation | ABV% | |
S. pastorianus | 60.9% | 2.59 ± 0.05 | 61.2% | 2.57 ± 0.02 |
P. kluyveri | 8.2% | 0.426 ± 0.002 | 7.6% | 0.386 ± 0.002 |
S. ludwigii | 8.3% | 0.395 ± 0.002 | 7.7% | 0.360 ± 0.004 |
Compounds | Saccharomyces pastorianus | Pichia kluyveri | Saccharomycodes ludwigii | ||||
---|---|---|---|---|---|---|---|
Ctrl | Protease | Ctrl | Protease | Ctrl | Protease | ||
Higher alcohol * | 3-methylbutan-1-ol | 27.18 ± 2.40 b | 33.48 ± 1.02 a | 10.71 ± 0.70 c | 4.47 ± 1.03 d | 14.18 ± 0.89 c | 26.47 ± 0.69 b |
2-methylbutan-1-ol | 7.58 ± 0.79 a | 7.39 ± 0.31 a | 2.56 ± 0.12 c | 4.27 ± 0.76 b | 1.39 ± 0.11 c | 1.52 ± 0.03 c | |
2-methylpropan-1-ol | 2.33 ± 0.14 a | 1.61 ± 0.12 b | 1.44 ± 0.08 b | 1.51 ± 0.32 b | 0.39 ± 0.03 c | 0.39 ± 0.01 c | |
2-phenylethanol | 36.61 ± 4.52 b | 24.62 ± 0.37 c | 13.61 ± 1.50 d | 4.02 ± 1.13 e | 40.94 ± 2.22 ab | 46.14 ± 0.96 a | |
Higher acetate * | 3-methylbutyl acetate | 5.92 ± 0.65 b | 13.84 ± 1.96 b | 131.37 ± 16.81 a | 125.11 ± 23.32 a | 0.33 ± 0.05 b | 0.55 ± 0.06 b |
2-methylbutyl acetate | 3.34 ± 0.31 d | 6.40 ± 0.80 c | 28.07 ± 1.06 b | 34.93 ± 1.90 a | 0.21 ± 0.02 e | 0.31 ± 0.02 e | |
2-methylpropyl acetate | 0.51 ± 0.04 c | 0.49 ± 0.07 c | 0.88 ± 0.13 b | 1.39 ± 0.27 a | 0.02 ±0.01 d | 0.02 ± 0.01 d | |
2-phenylethyl acetate | 18.07 ± 2.48 c | 16.80 ± 1.12 c | 206.19 ± 18.52 a | 143.53 ± 14.92 b | 21.04 ± 1.40 c | 21.90 ± 1.37 c | |
Medium-chain fatty acid ethyl esters | ethyl hexanoate | 6.90 ± 0.60 a | 7.30 ± 1.11 a | 0.34 ± 0.15 b | 0.43 ± 0.10 b | 0.12 ± 0.02 b | 0.16 ± 0.02 b |
ethyl octanoate | 44.37 ± 10.82 b | 78.21 ± 14.92 a | 0.38 ± 0.18 c | 0.34 ± 0.08 c | 0.02 ± 0.04 c | 0.08 ± 0.07 c | |
ethyl decanoate | 18.51 ± 1.97 b | 36.84 ± 9.80 a | 0.44 ± 0.06 c | 0.52 ± 0.20 c | 0.02 ± 0.04 c | 0.13 ± 0.05 c | |
Other esters | ethyl acetate | 5.66 ± 0.40 b | 7.93 ± 1.08 ab | 14.07 ± 5.09 a | 13.61 ± 4.13 a | 1.05 ± 0.04 b | 0.98 ± 0.04 b |
3-methylpropanoate | 0.028 ± 0.02 c | 0.03 ± 0.01 c | 2.2 ± 0.5 a | 1.0 ± 0.2 b | 0.0172 ± 0.0006 c | 0.019 ± 0.001 c | |
Aldehyde off-flavor | 3-methylbutanal | 0.19 ± 0.06 b | 0.95 ± 0.27 a | 0.023 ± 0.004 b | 0.027 ± 0.002 b | 0.028 ± 0.002 b | 0.04 ± 0.01 b |
2-methylbutanal | 0.27 ± 0.09 b | 1.18 ± 0.05 a | 0.03 ± 0.01 c | 0.031 ± 0.04 c | 0.027 ± 0.002 c | 0.09 ± 0.01 c | |
2-phenylethanal | 0.04 ± 0.01 b | 0.24 ± 0.04 a | 0.04 ± 0.02 b | 0.018 ± 0.003 b | 0.04 ± 0.01 b | 0.065 ± 0.003 b | |
Alcohol by volume (ABV%) | - | 2.59 ± 0.05 | 2.57 ± 0.02 | 0.426 ± 0.02 | 0.386 ± 0.002 | 0.395 ± 0.002 | 0.360 ± 0.004 |
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Run | Endoprotease (mg EP/kg-Grist) | Exoprotease (mg EP/kg-Grist) | Temperature (°C) | Endo1 + Exo1 Leu% | Endo1 + Exo3 Leu% |
---|---|---|---|---|---|
1 | 0 | 0 | 52 | 7.54 | 7.68 |
2 | 0 | 0 | 52 | 7.66 | 7.57 |
3 | 100 | 0 | 52 | 10.38 | 10.72 |
4 | 0 | 100 | 52 | 7.47 | 7.86 |
5 | 100 | 100 | 52 | 10.17 | 12.90 |
6 | 100 | 100 | 52 | 10.04 | 12.99 |
7 | 0 | 0 | 63 | 7.28 | 7.42 |
8 | 100 | 0 | 63 | 7.97 | 8.50 |
9 | 100 | 0 | 63 | 8.24 | 8.61 |
10 | 0 | 100 | 63 | 7.27 | 7.65 |
11 | 0 | 100 | 63 | 7.32 | 7.68 |
12 | 100 | 100 | 63 | 9.50 | 11.41 |
Term | Parameter Estimate | p Value | Term | Parameter Estimate | p Value |
---|---|---|---|---|---|
Endo1(0, 100) | 1.055625 | 0.00001 | Endo1(0, 100) | 1.6325 | 0.00000 |
Exo1(0, 100) | 0.125625 | 0.02374 | Exo3(0, 100) | 0.695 | 0.00000 |
Temp(52, 63) | −0.421875 | 0.00028 | Temp(52, 63) | −0.5125 | 0.00001 |
Endo1 × Exo1 | 0.154375 | 0.01201 | Endo1 × Exo3 | 0.575 | 0.00001 |
Endo1 × Temp | −0.298125 | 0.00108 | Endo1 × Temp | −0.4125 | 0.00003 |
Exo1 × Temp | 0.226875 | 0.00303 | Exo3 × Temp | 0.08 | 0.01540 |
Endo1 × Exo1 × Temp | 0.190625 | 0.00573 | Endo1 × Exo3 × Temp | 0.0775 | 0.01712 |
Run | Endo1 (mg EP/kg-Grist) | Temperature (°C) | Leu% | Predicted Leu% |
---|---|---|---|---|
1 | 83.5 | 52 | 10.18 | 10.18 |
2 | 0 | 52 | 7.67 | 7.67 |
3 | 39.5 | 57 | 9.32 | 9.36 |
4 | 39.5 | 57 | 9.39 | 9.36 |
5 | 100 | 59 | 9.58 | 9.58 |
6 | 100 | 59 | 9.59 | 9.58 |
7 | 0 | 62 | 7.50 | 7.50 |
8 | 63.5 | 63 | 7.97 | 7.97 |
Term | Parameter Estimate | p Value |
---|---|---|
Endo1(0, 100) | 0.8660797 | 0.0004 |
Temp(52, 63) | −0.762476 | 0.0006 |
Temp × Temp | −0.842294 | 0.0012 |
Endo1 × Temp | −0.515997 | 0.0021 |
Endo1 × Endo1 | −0.375853 | 0.0056 |
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Lin, C.L.; Petersen, M.A.; Gottlieb, A. Increasing Higher Alcohols and Acetates in Low-Alcohol Beer by Proteases. Molecules 2023, 28, 4419. https://doi.org/10.3390/molecules28114419
Lin CL, Petersen MA, Gottlieb A. Increasing Higher Alcohols and Acetates in Low-Alcohol Beer by Proteases. Molecules. 2023; 28(11):4419. https://doi.org/10.3390/molecules28114419
Chicago/Turabian StyleLin, Claire Lin, Mikael Agerlin Petersen, and Andrea Gottlieb. 2023. "Increasing Higher Alcohols and Acetates in Low-Alcohol Beer by Proteases" Molecules 28, no. 11: 4419. https://doi.org/10.3390/molecules28114419