Improved detection of beer-spoilage bacteria: validation of the NBB-C®-enrichment method
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Keywords
Abstract
Beer spoilage is mainly caused by a limited group of bacteria, including Levilactobacillus brevis, Fructilactobacillus lindneri, and Pediococcus damnosus. Detection is essential for beer quality control. Among the microbiological tools used by breweries, enrichment in Nutrient Media for Beer-Spoiling Bacteria (NBB-C®) is based on European Brewery Convention analytical procedures, EBC ANALYTICA analytical methods; however, in our accredited method (MI LMB 01-2021), the standard 14-day incubation is modified to 7 days to provide faster results suitable for routine brewery monitoring. This study aimed to validate the modified EBC ANALYTICA 4.3.1.4 Enrichment, 2011 method using NBB-C® media by determining its probability of detection (POD) and limit of detection using pasteurized lager beer as a test matrix. For the first time, POD curves with 95% confidence intervals were estimated for L. brevis, F. lindneri, and P. damnosus. The results provided an objective and quantitative performance evaluation of the modified NBB-C®-enrichment procedure, supporting its suitability for International Organization for Standardization/International Electrotechnical Commission (ISO/IEC) 17025-compliant testing in brewing laboratories.
Riferimenti bibliografici
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Back, W., Leibhard, M. and BohakI. 1992. Flash pasteurization – membrane filtration. Comparative biological safety. Brauwelt Int. 1:42.
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). 2025, July 21. Deutsche Sammlung von Mikroorganismen und Zellkulturen protocol. https://www.dsmz.de/collection/catalogue (Accessed on: 30 Mar 26).
Duygu, D. 2017. Mikrobiyolojik test Metotlarinin Doğrulanmasi (Vali̇dasyonu). Trakya Univ J Nat Sci 2017;18(1):65-69. https://doi.org/10.23902/trkjnat.271725
EBC ANALYTICA. European Brewery Convention (EBC). https://brewup.eu/ebc-analytica (Accessed on: 30 Mar 26).
Eleftheriadou, M. and Tsimillis, K.C. 2023. Eurachem Guide: Accreditation for Microbiological Laboratories. Eurachem, Middlesex, UK. www.eurachem.org. (Accessed on 30 Mar 26). ISBN: 978-91-87017-92-6
Hill, A.E. 2015. Brewing Microbiology: Managing Microbes, Ensuring Quality, and Valorising Waste. Wood Head Publication, Cambridge, UK.
Iijima, K., Suzuki, K., Asano, S., Kuriyama, H. and Kitagawa, Y. 2007. Isolation and identification of potential beer-spoilage Pediococcus inopinatus and beer-spoilage Lactobacillus backi strains carrying the horA and horC gene clusters. J Inst Brew. 113:96. https://doi.org/10.1002/j.2050-0416.2007.tb00262
International Organization for Standardization/International Electrotechnical Commission (ISO/IEC). 2017. ISO/IEC 17025: General Requirements for the Competence of Testing and Calibration Laboratories. ISO/IEC, Geneva, Switzerland.
International Organization for Standardization/International Electrotechnical Commission (ISO/IEC). 2016. ISO/IEC 17034: General Requirements for the Competence of Reference Material Producers. ISO/IEC, Geneva, Switzerland.
Jespersen, L. and Jakobsen, M. 1996. Specific spoilage organisms in breweries and laboratory media for their detection. Int J Food Microbiol. 33(1):139–155. https://doi.org/10.1016/0168-1605(96)01154-3
Liu, J., Deng, Y., Soteyome, T., Li, Y., Su, J., Li, L., Li B., Shirtliff, M.E., Xu, Z. and Peters, B.M. 2018. Induction and recovery of the viable but nonculturable state of hop-resistance Lactobacillus brevis. Front Microbiol. 9:2076.
Magnusson, B. and Örnemark, U. 2024. Eurachem Guide: The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics, 3rd edn. Eurachem, Middlesex, UK. www.eurachem.org. (Accessed on: 30 Mar 26). ISBN 978-91-87461-59-0.
Magnusson, B. and Tsimillis, K.C. 2023. Accreditation for Microbiological Laboratories, 3rd edn. Eurachem, Middlesex, UK. www.eurachem.org. (Accessed on: 30 Mar 26). ISBN 978-91-519-6581-9.
• Matoulková, D., and Kubizniaková, P. 2015. Microbiology of brewing – lactic acid bacteria and cultivation methods of their detection – part I. Kvasnýprůmysl. 61:76. https://doi.org/ 10.18832/kp2015012
Michel, M., Cocuzza, S., Biendl, M., Peifer, F., Hans, S., Methner, Y., Pehl, F., Back, W., Jacob, F. and Hutzler, M. 2020. The impact of different hop compounds on the growth of selected beer spoilage bacteria in beer. J Inst Brew. 126(4):330-336. https://doi.org/10.1002/jib.624
Priest, F.G. 2003. Gram-positive brewery bacteria . In: Brewing Microbiology Priest, F.G. and Campbell, I. (Eds.).Springer, Boston, MA, p. 181. https://doi.org/10.1007/978-1-4419-9250-5_5
Priest, F.G. 2006. Microbiology and microbiological control in the brewery. In: Priest, F.G. and Stewart, G. G. (Eds.) Handbook of Brewing. CRC Press, Boca Raton, FL, 622 p. https://doi.org/10.1201/9781420015171-20
Pringle A.T. 2020. A brewer’s guide to applying PCR in a quality assurance program. MBAA TQ. 57(3):190. https://doi.org/10.1094/TQ-57-3-1011-01
Riedl, R., Dünzer, N., Michel, M., Jacob, F. and Hutzler, M. 2019. Beer enemy number one: genetic diversity, physiology and biofilm formation of Lactobacillus brevis. J Inst Brew. 125:250. https://doi.org/10.1002/jib.553
Rodríguez-Saavedra, M., González de Llano, D. and Moreno-Arribas, M.V. 2020. Beer spoilage lactic acid bacteria from craft brewery microbiota: Microbiological quality and food safety. Food Res Int. 138:109762. https://doi.org/10.1016/j.foodres.2020.109762
Sakamoto, K. and Konings, W.N. 2003. Beer spoilage bacteria and hop resistance. Int J Food Microbiol. 89(2–3):105. https://doi.org/10.1016/s0168-1605(03)00153-3
Schneiderbanger, J., Grammer, M., Jacob, F. and Hutzler, M. 2018. Statistical evaluation of beer spoilage bacteria by real-time PCR analyses from 2010 to 2016. J Inst Brew. 124(2):173. https://doi.org/10.1002/jib.486
Storgårds, E. and Suihko, M.L. 1998. Detection and identification of Lactobacillus lindneri from brewery environments. J Inst Brew. 104(January–February):47–54.
Suzuki, K. 2011. 125th Anniversary review: microbiological instability of beer caused by spoilage bacteria. J Inst Brew. 117(2):131. https://doi.org/10.1002/j.2050-0416.2011.tb00454.x
Suzuki, K., Asano, S., Iijima, K. and Kitamoto, K. 2008. Sake and beer spoilage lactic acid bacteria – a review. J Inst Brew. 114(3):209. https://doi.org/10.1002/j.2050-0416.2008.tb00331.x
Taskila, S., Juuti, T., Kylä-Nikkilä, K. and Ojamo, H. (2011). Improved enrichment cultivation of beer-spoiling lactic acid bacteria by continuous glucose addition. J Inst Brew. 117(2):264–271.
Thelen, K., Beimfohr, C., and Snaidr, J. 2006. Evaluation study of the frequency of different beer-spoiling bacteria using the VIT analysis. Tech Q Master Brew Assoc Am. 43:31.
Vaughan, A., O’Sullivan, T., Sinderen, D., Van Sinderen, D. and Sinderen, D. 2005. Enhancing the microbiological stability of malt and beer – a review. J Inst Brew. 111(4):355. https://doi.org/10.1002/j.2050-0416.2005.tb00221.x
Wilrich, C. and Wilrich, P.T. 2009. Estimation of the POD function and the LOD of a qualitative microbiological measurement method. J AOAC Int. 92(6):1763. https://doi.org/10.1093/jaoac/92.6.1763
Zheng, J., Wittouck, S., Salvetti, E., Franz, C.M.A.P., Harris, H.M.B., Mattarelli, P., O'Toole, P.W., Pot, B., Vandamme, P., Walter, J., Watanabe, K., Wuyts, S., Felis, G.E., Gänzle, M.G. and Lebeer, S. 2020. A taxonomic note on the genus Lactobacillus: description of 23 novel genera, emended description of the genus Lactobacillus beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol. 70(4):2782. https://doi.org/10.1099/ijsem.0.004107
Back, W. 2009. Microbiology. In: Eßlinger, H.M. (Ed.), Handbook of Brewing: Process, Technology, Markets. Wiley VCH Verlag GmbH, Weinheim, Germany, Chap. 18, p. 477.
Back, W., Leibhard, M. and BohakI. 1992. Flash pasteurization – membrane filtration. Comparative biological safety. Brauwelt Int. 1:42.
Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). 2025, July 21. Deutsche Sammlung von Mikroorganismen und Zellkulturen protocol. https://www.dsmz.de/collection/catalogue (Accessed on: 30 Mar 26).
Duygu, D. 2017. Mikrobiyolojik test Metotlarinin Doğrulanmasi (Vali̇dasyonu). Trakya Univ J Nat Sci 2017;18(1):65-69. https://doi.org/10.23902/trkjnat.271725
EBC ANALYTICA. European Brewery Convention (EBC). https://brewup.eu/ebc-analytica (Accessed on: 30 Mar 26).
Eleftheriadou, M. and Tsimillis, K.C. 2023. Eurachem Guide: Accreditation for Microbiological Laboratories. Eurachem, Middlesex, UK. www.eurachem.org. (Accessed on 30 Mar 26). ISBN: 978-91-87017-92-6
Hill, A.E. 2015. Brewing Microbiology: Managing Microbes, Ensuring Quality, and Valorising Waste. Wood Head Publication, Cambridge, UK.
Iijima, K., Suzuki, K., Asano, S., Kuriyama, H. and Kitagawa, Y. 2007. Isolation and identification of potential beer-spoilage Pediococcus inopinatus and beer-spoilage Lactobacillus backi strains carrying the horA and horC gene clusters. J Inst Brew. 113:96. https://doi.org/10.1002/j.2050-0416.2007.tb00262
International Organization for Standardization/International Electrotechnical Commission (ISO/IEC). 2017. ISO/IEC 17025: General Requirements for the Competence of Testing and Calibration Laboratories. ISO/IEC, Geneva, Switzerland.
International Organization for Standardization/International Electrotechnical Commission (ISO/IEC). 2016. ISO/IEC 17034: General Requirements for the Competence of Reference Material Producers. ISO/IEC, Geneva, Switzerland.
Jespersen, L. and Jakobsen, M. 1996. Specific spoilage organisms in breweries and laboratory media for their detection. Int J Food Microbiol. 33(1):139–155. https://doi.org/10.1016/0168-1605(96)01154-3
Liu, J., Deng, Y., Soteyome, T., Li, Y., Su, J., Li, L., Li B., Shirtliff, M.E., Xu, Z. and Peters, B.M. 2018. Induction and recovery of the viable but nonculturable state of hop-resistance Lactobacillus brevis. Front Microbiol. 9:2076.
Magnusson, B. and Örnemark, U. 2024. Eurachem Guide: The Fitness for Purpose of Analytical Methods – A Laboratory Guide to Method Validation and Related Topics, 3rd edn. Eurachem, Middlesex, UK. www.eurachem.org. (Accessed on: 30 Mar 26). ISBN 978-91-87461-59-0.
Magnusson, B. and Tsimillis, K.C. 2023. Accreditation for Microbiological Laboratories, 3rd edn. Eurachem, Middlesex, UK. www.eurachem.org. (Accessed on: 30 Mar 26). ISBN 978-91-519-6581-9.
• Matoulková, D., and Kubizniaková, P. 2015. Microbiology of brewing – lactic acid bacteria and cultivation methods of their detection – part I. Kvasnýprůmysl. 61:76. https://doi.org/ 10.18832/kp2015012
Michel, M., Cocuzza, S., Biendl, M., Peifer, F., Hans, S., Methner, Y., Pehl, F., Back, W., Jacob, F. and Hutzler, M. 2020. The impact of different hop compounds on the growth of selected beer spoilage bacteria in beer. J Inst Brew. 126(4):330-336. https://doi.org/10.1002/jib.624
Priest, F.G. 2003. Gram-positive brewery bacteria . In: Brewing Microbiology Priest, F.G. and Campbell, I. (Eds.).Springer, Boston, MA, p. 181. https://doi.org/10.1007/978-1-4419-9250-5_5
Priest, F.G. 2006. Microbiology and microbiological control in the brewery. In: Priest, F.G. and Stewart, G. G. (Eds.) Handbook of Brewing. CRC Press, Boca Raton, FL, 622 p. https://doi.org/10.1201/9781420015171-20
Pringle A.T. 2020. A brewer’s guide to applying PCR in a quality assurance program. MBAA TQ. 57(3):190. https://doi.org/10.1094/TQ-57-3-1011-01
Riedl, R., Dünzer, N., Michel, M., Jacob, F. and Hutzler, M. 2019. Beer enemy number one: genetic diversity, physiology and biofilm formation of Lactobacillus brevis. J Inst Brew. 125:250. https://doi.org/10.1002/jib.553
Rodríguez-Saavedra, M., González de Llano, D. and Moreno-Arribas, M.V. 2020. Beer spoilage lactic acid bacteria from craft brewery microbiota: Microbiological quality and food safety. Food Res Int. 138:109762. https://doi.org/10.1016/j.foodres.2020.109762
Sakamoto, K. and Konings, W.N. 2003. Beer spoilage bacteria and hop resistance. Int J Food Microbiol. 89(2–3):105. https://doi.org/10.1016/s0168-1605(03)00153-3
Schneiderbanger, J., Grammer, M., Jacob, F. and Hutzler, M. 2018. Statistical evaluation of beer spoilage bacteria by real-time PCR analyses from 2010 to 2016. J Inst Brew. 124(2):173. https://doi.org/10.1002/jib.486
Storgårds, E. and Suihko, M.L. 1998. Detection and identification of Lactobacillus lindneri from brewery environments. J Inst Brew. 104(January–February):47–54.
Suzuki, K. 2011. 125th Anniversary review: microbiological instability of beer caused by spoilage bacteria. J Inst Brew. 117(2):131. https://doi.org/10.1002/j.2050-0416.2011.tb00454.x
Suzuki, K., Asano, S., Iijima, K. and Kitamoto, K. 2008. Sake and beer spoilage lactic acid bacteria – a review. J Inst Brew. 114(3):209. https://doi.org/10.1002/j.2050-0416.2008.tb00331.x
Taskila, S., Juuti, T., Kylä-Nikkilä, K. and Ojamo, H. (2011). Improved enrichment cultivation of beer-spoiling lactic acid bacteria by continuous glucose addition. J Inst Brew. 117(2):264–271.
Thelen, K., Beimfohr, C., and Snaidr, J. 2006. Evaluation study of the frequency of different beer-spoiling bacteria using the VIT analysis. Tech Q Master Brew Assoc Am. 43:31.
Vaughan, A., O’Sullivan, T., Sinderen, D., Van Sinderen, D. and Sinderen, D. 2005. Enhancing the microbiological stability of malt and beer – a review. J Inst Brew. 111(4):355. https://doi.org/10.1002/j.2050-0416.2005.tb00221.x
Wilrich, C. and Wilrich, P.T. 2009. Estimation of the POD function and the LOD of a qualitative microbiological measurement method. J AOAC Int. 92(6):1763. https://doi.org/10.1093/jaoac/92.6.1763
Zheng, J., Wittouck, S., Salvetti, E., Franz, C.M.A.P., Harris, H.M.B., Mattarelli, P., O'Toole, P.W., Pot, B., Vandamme, P., Walter, J., Watanabe, K., Wuyts, S., Felis, G.E., Gänzle, M.G. and Lebeer, S. 2020. A taxonomic note on the genus Lactobacillus: description of 23 novel genera, emended description of the genus Lactobacillus beijerinck 1901, and union of Lactobacillaceae and Leuconostocaceae. Int J Syst Evol Microbiol. 70(4):2782. https://doi.org/10.1099/ijsem.0.004107
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Apr 18, 2026
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Bravi, E., De Francesco, G., Moretti, E., Floridi, S., & Marconi, O. (2026). Improved detection of beer-spoilage bacteria: validation of the NBB-C®-enrichment method. Italian Journal of Food Science, 38(2), 136–147. https://doi.org/10.15586/ijfs.v38i2.3418
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Come citare
Bravi, E., De Francesco, G., Moretti, E., Floridi, S., & Marconi, O. (2026). Improved detection of beer-spoilage bacteria: validation of the NBB-C®-enrichment method. Italian Journal of Food Science, 38(2), 136–147. https://doi.org/10.15586/ijfs.v38i2.3418