Alim26760 Vol. 25 No. 2 (2026)

Vol. 25, No. 2 (2026), Alim26760 https://doi.org/10.24275/rmiq/Alim26760

Detection of diastatic potential in beer by a culture-assisted end-point PCR assay targeting STA genes

Authors

V.D. Hernández-Gutiérrez, E. Arellano-Ordoñez, E. Villegas-Aviles, A. Álvarez-Rivera, R. Guzmán-Cruz, A. Godínez-Oviedo, B. Martínez-Cano, C.A. Cedillo-Jiménez

Abstract

The presence of diastatic yeast strains in beer poses a recognized spoilage risk, as their ability to hydrolyze dextrins through extracellular glucoamylase activity may lead to secondary fermentation, package instability, and economic losses. Although commercial molecular diagnostic systems are available for brewery quality control, their adoption is often limited by high costs, dependence on proprietary reagents, and logistical constraints in certain regions. In this study, a culture-assisted in-house end-point PCR method was developed and evaluated for the detection of diastatic potential in finished craft beer. The workflow integrated a 48-hour enrichment step in YPD medium, an optimized DNA extraction protocol, and primers targeting conserved regions of the STA gene family. Sequence analysis of the amplified product revealed that the assay detects a conserved functional region shared by STA1, STA2, and STA2K, variants associated with a complete glycoside hydrolase family 15 (GH15) domain and functional glucoamylase activity, suggesting a broader detection scope than previously reported for these primers. The method demonstrated 94% accuracy, 100% sensitivity, and 88% specificity under controlled conditions. Compared with a commercial detection system, an overall agreement of 82.9% was observed across finished beer samples. Implementation of this non-proprietary approach reduced consumable costs by 74% and increased thermocycler throughput by 3.2-fold relative to the commercial system. These results suggest that reliable detection of diastatic potential is achievable using standard laboratory infrastructure, offering a technically transferable alternative for brewery quality assurance.

Keywords

beer quality control, diastatic spoilage, starch hydrolysis, molecular diagnostics, technological independence.

References
bioMérieux S.A. (2023). BREWSTAT®: Detection of spoilage yeasts in beer and beer-based beverages [Product sheet]. Available at: https://www.biomerieux.com/content/dam/biomerieux-com/03----our-offer/global-resources/food-resources/BREWSTAT%20GB_9318323.pdf.coredownload.pdf . Accessed: February 15, 2025. Edwards, K., Johnstone, C. and Thompson, C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19, 1349–1349. https://doi.org/10.1093/nar/19.6.1349 Gallardo-Martínez, D., Viniegra-González, G., Figueroa-Martínez, F., Rocha, J. and Cruz-Guerrero, A. (2024). Isolation and characterization of epiphytic, fructanolytic, homofermentative lactic acid bacteria from Agave salmiana. Revista Mexicana de Ingeniería Química 23, Alim24142. https://doi.org/10.24275/rmiq/Alim24142 Garza-Garza, C. L., Olguin-Maciel, E., Valdez-Ojeda, R., Huchin-Poot, E., Toledano-Thompson, T., Azcorra-May, K. J., Alzate-Gaviria, L., Dominguez-Maldonado, J., Lappe-Oliveras, P. and Tapia-Tussell, R. (2025). Bio-saccharification and fermentation process of a non-conventional starchy material with isolates of autochthonous strains. Revista Mexicana de Ingeniería Química 24, Bio25497. https://doi.org/10.24275/rmiq/Bio25497 Hellborg, L. and Piškur, J. (2009). Yeast diversity in the brewing industry. In: Beer in Health and Disease Prevention, (V.R. Preedy, ed.), Pp. 77–88. Academic Press, New York. https://doi.org/10.1016/B978-0-12-373891-2.00007-9 Kim, K., Bajszár, G., Lee, S.Y., Knudsen, F. and Mattoon, J.R. (1994). Cloning of a new allelic variant of a Saccharomyces diastaticus glucoamylase gene and its introduction into industrial yeasts. Applied Biochemistry and Biotechnology 44, 161–185. https://doi.org/10.1007/BF02921653 Meier-Dörnberg, T., Kory, O.I., Jacob, F., Michel, M. and Hutzler, M. (2018). Saccharomyces cerevisiae variety diastaticus friend or foe? Spoilage potential and brewing ability of different Saccharomyces cerevisiae variety diastaticus yeast isolates by genetic, phenotypic and physiological characterization. FEMS Yeast Research 18, foy023. https://doi.org/10.1093/femsyr/foy023 Oldham, R.C. and Held, M.A. (2023). Methods for detection and identification of beer-spoilage microbes. Frontiers in Microbiology 14, 1217704. https://doi.org/10.3389/fmicb.2023.1217704 Sakamoto, K. and Konings, W.N. (2003). Beer spoilage bacteria and hop resistance. International Journal of Food Microbiology 89, 105–124. https://doi.org/10.1016/S0168-1605(03)00153-3 Shankar, V., Mahboob, S., Al-Ghanim, K.A., Ahmed, Z., Al-Mulhm, N. and Govindarajan, M. (2021). A review on microbial degradation of drinks and infectious diseases: A perspective of human well-being and capabilities. Journal of King Saud University Science 33, 101293. https://doi.org/10.1016/j.jksus.2020.101293 Shima, H., Inui, M., Akada, R. and Yamashita, I. (1989). Upstream regions of the yeast glucoamylase gene which are required for efficient transcription. Agricultural and Biological Chemistry 53, 749–755. https://doi.org/10.1080/00021369.1989.10869353 Suiker, I.M., Arkesteijn, G.J.A., Zeegers, P.J. and Wösten, H.A.B. (2021). Presence of Saccharomyces cerevisiae subsp. diastaticus in industry and nature and spoilage capacity of its vegetative cells and ascospores. International Journal of Food Microbiology 347, 109173. https://doi.org/10.1016/j.ijfoodmicro.2021.109173 Suzuki, K. (2020). Emergence of new spoilage microorganisms in the brewing industry and development of microbiological quality control methods to cope with this phenomenon: A review. Journal of the American Society of Brewing Chemists 78, 245–259. https://doi.org/10.1080/03610470.2020.1782101 Vega-Ramírez, G., Valle-Guadarrama, S., Hernández-Rodríguez, B.E. and Sandoval-Castilla, O. (2026). Amylases from malted barley (Hordeum vulgare) seeds separated by aqueous two-phase extraction. Revista Mexicana de Ingeniería Química 25, Alim26667. https://doi.org/10.24275/rmiq/Alim26667 Vuillemin, M., Moreno Prieto, E.S., Pilgaard, B., Siebenhaar, S., Holck, J., Henrissat, B., Bahieldin, A., Hakeem, K.R. and Alghamdi, K.M. (2024). Biochemical exploration of family GH119 reveals a single α-amylase specificity and confirms shared catalytic machinery with GH57 enzymes. International Journal of Biological Macromolecules 262, 129783. https://doi.org/10.1016/j.ijbiomac.2024.129783 Watanabe, M., Lee, K., Goto, K., Kumagai, S., Sugita-Konishi, Y. and Hara-Kudo, Y. (2010). Rapid and effective DNA extraction method with bead grinding for a large amount of fungal DNA. Journal of Food Protection 73, 1077–1084. https://doi.org/10.4315/0362-028X-73.6.1077

References

bioMérieux S.A. (2023). BREWSTAT®: Detection of spoilage yeasts in beer and beer-based beverages [Product sheet]. Available at: https://www.biomerieux.com/content/dam/biomerieux-com/03----our-offer/global-resources/food-resources/BREWSTAT%20GB_9318323.pdf.coredownload.pdf . Accessed: February 15, 2025.
Edwards, K., Johnstone, C. and Thompson, C. (1991). A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucleic Acids Research 19, 1349–1349. https://doi.org/10.1093/nar/19.6.1349
Gallardo-Martínez, D., Viniegra-González, G., Figueroa-Martínez, F., Rocha, J. and Cruz-Guerrero, A. (2024). Isolation and characterization of epiphytic, fructanolytic, homofermentative lactic acid bacteria from Agave salmiana. Revista Mexicana de Ingeniería Química 23, Alim24142. https://doi.org/10.24275/rmiq/Alim24142
Garza-Garza, C. L., Olguin-Maciel, E., Valdez-Ojeda, R., Huchin-Poot, E., Toledano-Thompson, T., Azcorra-May, K. J., Alzate-Gaviria, L., Dominguez-Maldonado, J., Lappe-Oliveras, P. and Tapia-Tussell, R. (2025). Bio-saccharification and fermentation process of a non-conventional starchy material with isolates of autochthonous strains. Revista Mexicana de Ingeniería Química 24, Bio25497. https://doi.org/10.24275/rmiq/Bio25497
Hellborg, L. and Piškur, J. (2009). Yeast diversity in the brewing industry. In: Beer in Health and Disease Prevention, (V.R. Preedy, ed.), Pp. 77–88. Academic Press, New York. https://doi.org/10.1016/B978-0-12-373891-2.00007-9
Kim, K., Bajszár, G., Lee, S.Y., Knudsen, F. and Mattoon, J.R. (1994). Cloning of a new allelic variant of a Saccharomyces diastaticus glucoamylase gene and its introduction into industrial yeasts. Applied Biochemistry and Biotechnology 44, 161–185. https://doi.org/10.1007/BF02921653
Meier-Dörnberg, T., Kory, O.I., Jacob, F., Michel, M. and Hutzler, M. (2018). Saccharomyces cerevisiae variety diastaticus friend or foe? Spoilage potential and brewing ability of different Saccharomyces cerevisiae variety diastaticus yeast isolates by genetic, phenotypic and physiological characterization. FEMS Yeast Research 18, foy023. https://doi.org/10.1093/femsyr/foy023
Oldham, R.C. and Held, M.A. (2023). Methods for detection and identification of beer-spoilage microbes. Frontiers in Microbiology 14, 1217704. https://doi.org/10.3389/fmicb.2023.1217704
Sakamoto, K. and Konings, W.N. (2003). Beer spoilage bacteria and hop resistance. International Journal of Food Microbiology 89, 105–124. https://doi.org/10.1016/S0168-1605(03)00153-3
Shankar, V., Mahboob, S., Al-Ghanim, K.A., Ahmed, Z., Al-Mulhm, N. and Govindarajan, M. (2021). A review on microbial degradation of drinks and infectious diseases: A perspective of human well-being and capabilities. Journal of King Saud University Science 33, 101293. https://doi.org/10.1016/j.jksus.2020.101293
Shima, H., Inui, M., Akada, R. and Yamashita, I. (1989). Upstream regions of the yeast glucoamylase gene which are required for efficient transcription. Agricultural and Biological Chemistry 53, 749–755. https://doi.org/10.1080/00021369.1989.10869353
Suiker, I.M., Arkesteijn, G.J.A., Zeegers, P.J. and Wösten, H.A.B. (2021). Presence of Saccharomyces cerevisiae subsp. diastaticus in industry and nature and spoilage capacity of its vegetative cells and ascospores. International Journal of Food Microbiology 347, 109173. https://doi.org/10.1016/j.ijfoodmicro.2021.109173
Suzuki, K. (2020). Emergence of new spoilage microorganisms in the brewing industry and development of microbiological quality control methods to cope with this phenomenon: A review. Journal of the American Society of Brewing Chemists 78, 245–259. https://doi.org/10.1080/03610470.2020.1782101
Vega-Ramírez, G., Valle-Guadarrama, S., Hernández-Rodríguez, B.E. and Sandoval-Castilla, O. (2026). Amylases from malted barley (Hordeum vulgare) seeds separated by aqueous two-phase extraction. Revista Mexicana de Ingeniería Química 25, Alim26667. https://doi.org/10.24275/rmiq/Alim26667
Vuillemin, M., Moreno Prieto, E.S., Pilgaard, B., Siebenhaar, S., Holck, J., Henrissat, B., Bahieldin, A., Hakeem, K.R. and Alghamdi, K.M. (2024). Biochemical exploration of family GH119 reveals a single α-amylase specificity and confirms shared catalytic machinery with GH57 enzymes. International Journal of Biological Macromolecules 262, 129783. https://doi.org/10.1016/j.ijbiomac.2024.129783
Watanabe, M., Lee, K., Goto, K., Kumagai, S., Sugita-Konishi, Y. and Hara-Kudo, Y. (2010). Rapid and effective DNA extraction method with bead grinding for a large amount of fungal DNA. Journal of Food Protection 73, 1077–1084. https://doi.org/10.4315/0362-028X-73.6.1077