Abstract
Non-conventional yeasts can be isolated from a wide range of environmental sources and are often found in the beverage industry in mixed fermentations, in which the microbial community is usually not fully known. However, it is important to know the compositions of these starter cultures because in addition to enabling reproducibility during fermentation, other properties can be discovered. Thus, the objective of this work was to identify and characterize non-conventional yeasts isolated from the environment, evaluating their probiotic potential and possible use in beer brewing. Isolates were obtained from flowers, fruits, leaves and mixed-fermentation beers, with the species being identified by PCR. Yeasts with promising activity were evaluated regarding their growth under different pHs, temperature and the presence of organic acids. To explore probiotic potential, in vitro tests were performed for antimicrobial activity and co-aggregation with food-spoiling microorganisms, auto-aggregation and survival in simulated gastrointestinal tract conditions. In this study, Pichia kluyveri (LAR001), Hanseniaspora uvarum (PIT001) and Candida intermedia (ORQ001) were selected among 20 isolates for further study. P. kluyveri was the only strain that tolerated pH 2.5. Lactic acid was not inhibitory, but acetic acid and incubation at 37 °C had partially inhibitory effects on yeast growth. All yeasts tolerated α-acids from hops and up to 1% NaCl. Our results also suggest that these isolates are able to adhere to intestinal cells and positively influence the host to combat pathogens, as they showed auto-aggregation rates > 99% and antagonistic activity to pathogenic bacteria. The yeasts tolerated gastric environment conditions, but were more sensitive to pancreatic conditions. We conclude that these non-conventional yeasts have probiotic potential and promising application in beer fermentation.
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Tikka C, Osuru HP, Atluri N et al (2013) Isolation and characterization of ethanol tolerant yeast strains. Bioinformation 9:421–425. https://doi.org/10.6026/97320630009421
Steensels J, Verstrepen KJ (2014) Taming wild yeast: potential of conventional and nonconventional yeasts in industrial fermentations. Annu Rev Microbiol 68:61–80. https://doi.org/10.1146/annurev-micro-091213-113025
Holt S, Mukherjee V, Lievens B et al (2018) Bioflavoring by non-conventional yeasts in sequential beer fermentations. Food Microbiol 72:55–66. https://doi.org/10.1016/j.fm.2017.11.008
Binati RL, Salvetti E, Bzducha-Wróbel A, Bašinskienė L, Čižeikienė D, Bolzonella D, Felis GE (2021) Non-conventional yeasts for food and additives production in a circular economy perspective. FEMS Yeast Res. https://doi.org/10.1093/femsyr/foab052
Michel M, Meier-Dörnberg T, Jacob F et al (2016) Review: Pure non-Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications. J Inst Brew 122:569–587. https://doi.org/10.1002/jib.381
Postigo V, Sánchez A, Cabellos JM, Arroyo T (2022) New approaches for the fermentation of beer: non-Saccharomyces yeasts from wine. Fermentation 8:280–302. https://doi.org/10.3390/fermentation8060280
Martin V, Valera M, Medina K et al (2018) Oenological impact of the Hanseniaspora/Kloeckera yeast genus on wines—a review. Fermentation 4:76. https://doi.org/10.3390/fermentation4030076
Bellut K, Michel M, Zarnkow M et al (2018) Application of non-saccharomyces yeasts isolated from kombucha in the production of alcohol-free beer. Fermentation 4:66. https://doi.org/10.3390/fermentation4030066
Methner Y, Hutzler M, Zarnkow M, Prowald A, Endres F, Jacob F (2022) Investigation of non-Saccharomyces yeast strains for their suitability for the production of non-alcoholic beers with novel flavor profiles. J Am Soc Brew. https://doi.org/10.1080/03610470.2021.2012747
Gutiérrez A, Boekhout T, Gojkovic Z, Katz M (2018) Evaluation of non-Saccharomyces yeasts in the fermentation of wine, beer and cider for the development of new beverages. J Inst Brew 124:389–402. https://doi.org/10.1002/jib.512
Vicente J, Calderón F, Santos A et al (2021) High potential of Pichia kluyveri and other Pichia species in wine technology. Int J Mol Sci 22:1–15. https://doi.org/10.3390/ijms22031196
Matraxia M, Alfonzo A, Prestianni R et al (2021) Non-conventional yeasts from fermented honey by-products: Focus on Hanseniaspora uvarum strains for craft beer production. Food Microbiol 99:103806. https://doi.org/10.1016/j.fm.2021.103806
Pham T, Wimalasena T, Box WG et al (2011) Evaluation of ITS PCR and RFLP for differentiation and identification of brewing yeast and brewery ‘wild’ yeast contaminants. J Inst Brew 117:556–568. https://doi.org/10.1002/j.2050-0416.2011.tb00504.x
Corbett KM, de Smidt O (2019) Culture-dependent diversity profiling of spoilage yeasts species by PCR-RFLP comparative analysis. Food Sci Technol Int 25:671–679. https://doi.org/10.1177/1082013219856779
Cassanego D, Richards N, Valente P et al (2017) Identification by PCR and evaluation of probiotic potential in yeast strains found in kefir samples in the city of Santa Maria, RS, Brazil. Food Sci Technol 38:59–65. https://doi.org/10.1590/1678-457x.13617
Pinto FO, Lopes T, Vieira AM, Oliveira RO, Gomes FF, Fabricio MF, Ayub MAZ, Mendes SDC, Pagani DM, Valente P (2022) Isolation, selection and characterization of wild yeasts with potential for brewing. J Am Soc Brew. https://doi.org/10.1080/03610470.2022.2031777
Younis G, Awad A, Dawod RE, Yousef NE (2017) Antimicrobial activity of yeasts against some pathogenic bacteria. Vet World 10:979–983. https://doi.org/10.14202/vetworld.2017.979-983
Sampaolesi S, Briand LE, Antoni G, Peláez AL. (2022) The synthesis of soluble and volatile bioactive compounds by selected brewer’s yeasts: antagonistic effect against enteropathogenic bacteria and food spoiler – toxigenic Aspergillus sp. 13:100193. https://doi.org/10.1016/j.fochx.2021.100193
FAO/WHO (2001) Health and nutritional properties of probiotics in food including power milk with live lactic acid bacteria. Amerian Córdoba Park Hotel, Cordoba, Argentina. Accessed at: http://www.who.int/foodsafety/publications/fs_ management/en/probiotics.pdf?ua¼1
Bevilacqua A, Perricone M, Cannarsi M et al (2009) Technological and spoiling characteristics of the yeast microflora isolated from Bella di Cerignola table olives. Int J Food Sci Technol 44:2198–2207. https://doi.org/10.1111/j.1365-2621.2009.02060.x
Fakruddin M, Hossain MN, Ahmed MM (2017) Antimicrobial and antioxidant activities of Saccharomyces cerevisiae IFST062013, a potential probiotic. BMC Complement Altern Med 17:64. https://doi.org/10.1186/s12906-017-1591-9
Labbani FZK, Turchetti B, Bennamoun L et al (2015) A novel killer protein from Pichia kluyveri isolated from an Algerian soil: purification and characterization of its in vitro activity against food and beverage spoilage yeasts. Antonie van Leeuwenhoek Int J Gen Mol Microbiol 107:961–970. https://doi.org/10.1007/s10482-015-0388-4
Goerges S, Aigner U, Silakowski B, Scherer S (2006) Inhibition of Listeria monocytogenes by food-borne yeasts. Appl Environ Microbiol 72:313–318. https://doi.org/10.1128/AEM.72.1.313-318.2006
Tiago FCP, Martins FS, Rosa CA et al (2009) Physiological characterization of non-Saccharomyces yeasts from agro-industrial and environmental origins with possible probiotic function. World J Microbiol Biotechnol 25:657–666. https://doi.org/10.1007/s11274-008-9934-9
Ogunremi OR, Sanni AI, Agrawal R (2015) Probiotic potentials of yeasts isolated from some cereal-based Nigerian traditional fermented food products. J Appl Microbiol 119:797–808. https://doi.org/10.1111/jam.12875
Yildiran H, Başyiğit Kiliç G, Karahan Çakmakçi AG (2019) Characterization and comparison of yeasts from different sources for some probiotic properties and exopolysaccharide production. Food Sci Technol 39:646–653. https://doi.org/10.1590/fst.29818
Staniszewski A, Kordowska-Wiater M (2021) Probiotic and potentially probiotic yeasts— characteristics and food application. Foods 10:1306–1319. https://doi.org/10.3390/foods10061306
Amorim JC, Piccoli RH, Duarte WF (2018) Probiotic potential of yeasts isolated from pineapple and their use in the elaboration of potentially functional fermented beverages. Food Res Int 107:518–527. https://doi.org/10.1016/j.foodres.2018.02.054
Zivkovic M, Cadez N, Uroic K et al (2014) Evaluation of probiotic potential of yeasts isolated from traditional cheeses manufactured in Serbia and Croatia. J Intercult Ethnopharmacol 4:12. https://doi.org/10.5455/jice.20141128051842
Osburn K, Ahmad NN, Bochman ML (2016) Bio-prospecting, selection, and analysis of wild yeasts for ethanol fermentation. Zymurgy 39:81–89. https://doi.org/10.13140/RG.2.2.16952.14080
Preiss R, Tyrawa C, Krogerus K et al (2018) Traditional Norwegian Kveik are a genetically distinct group of domesticated Saccharomyces cerevisiae brewing yeasts. Front Microbiol. https://doi.org/10.3389/fmicb.2018.02137
Zeng X, Fan J, He L et al (2019) Technological properties and probiotic potential of yeasts isolated from traditional low-salt fermented Chinese fish Suan yu. J Food Biochem 43:1–14. https://doi.org/10.1111/jfbc.12865
Samanfar B, Shostak K, Moteshareie H et al (2017) The sensitivity of the yeast, Saccharomyces cerevisiae, to acetic acid is influenced by DOM34 and RPL36A. PeerJ. https://doi.org/10.7717/peerj.4037
Pereira V, Lopes C, Castro A et al (2009) Characterization for enterotoxin production, virulence factors, and antibiotic susceptibility of Staphylococcus aureus isolates from various foods in Portugal. Food Microbiol 26:278–282. https://doi.org/10.1016/j.fm.2008.12.008
Collado MC, Meriluoto J, Salminen S (2008) Adhesion and aggregation properties of probiotic and pathogen strains. Eur Food Res Technol 226:1065–1073. https://doi.org/10.1007/s00217-007-0632-x
Bonatsou S, Benítez A, Rodríguez-Gómez F et al (2015) Selection of yeasts with multifunctional features for application as starters in natural black table olive processing. Food Microbiol 46:66–73. https://doi.org/10.1016/j.fm.2014.07.011
Schneiderbanger H, Koob J, Poltinger S et al (2016) Gene expression in wheat beer yeast strains and the synthesis of acetate esters. J Inst Brew 122:403–411. https://doi.org/10.1002/jib.337
Holt S, Miks MH, De Carvalho BT et al (2019) The molecular biology of fruity and floral aromas in beer and other alcoholic beverages. FEMS Microbiol Rev 43:193–222. https://doi.org/10.1093/femsre/fuy041
Menoncin M, Bonatto D (2019) Molecular and biochemical aspects of Brettanomyces in brewing. J Inst Brew 125(4):402–411. https://doi.org/10.1002/jib.580
Piraine RE, Nickens DG, Sun DJ et al (2022) Isolation of wild yeasts from Olympic National Park and Moniliella megachiliensis ONP131 physiological characterization for beer fermentation. Food Microbiol 104:103974. https://doi.org/10.1016/j.fm.2021.103974
Bokulich NA, Bamforth CW (2013) The microbiology of malting and brewing. Microbiol Mol Biol Rev 77:157–172. https://doi.org/10.1128/mmbr.00060-12
Hansen B, Wasdovitch B (2005) Malt ingredients in baked goods. Cereal Foods World 50:18–22
Michel M, Kopecká J, Meier-Dörnberg T et al (2016) Screening for new brewing yeasts in the non- Saccharomyces sector with Torulaspora delbrueckii as model. Yeast 33:129–144. https://doi.org/10.1002/yea.3146
Methner Y, Hutzler M, Zarnkow M et al (2022) Investigation of non- saccharomyces yeast strains for their suitability for the production of non-alcoholic beers with novel flavor profiles. J Am Soc Brew Chem. https://doi.org/10.1080/03610470.2021.2012747
Saerens SMG, Swiegers JH (2017) Production of low-alcohol or alcohol-free beer with Pichia kluyveri yeast strains. Patent, US009580675B2.
Lu Y, Voon MKW, Chua JY et al (2017) The effects of co- and sequential inoculation of Torulaspora delbrueckii and Pichia kluyveri on chemical compositions of durian wine. Appl Microbiol Biotechnol 101:7853–7863. https://doi.org/10.1007/s00253-017-8527-7
Capece A, Romaniello R, Siesto G, Romano P (2018) Conventional and non-conventional yeasts in beer production. Fermentation 4:38. https://doi.org/10.3390/fermentation4020038
Fai AEC, da Silva JB, de Andrade CJ et al (2014) Production of prebiotic galactooligosaccharides from lactose by Pseudozyma tsukubaensis and Pichia kluyveri. Biocatal Agric Biotechnol 3:343–350. https://doi.org/10.1016/j.bcab.2014.04.005
López S, Mateo JJ, Maicas SM (2016) Characterisation of Hanseniaspora Isolates with Potential Aroma-enhancing Properties in Muscat Wines. South African J Enol Vitic 35:292–303. https://doi.org/10.21548/35-2-1018
Bamforth C (2001) pH in brewing: an overview. Mbaa Tq 38:1–8
Vriesekoop F, Krahl M, Hucker B, Menz G (2012) 125th Anniversary review: bacteria in brewing: The good, the bad and the ugly. J Inst Brew 118:335–345. https://doi.org/10.1002/jib.49
Riedl R, Fütterer J, Goderbauer P et al (2019) Combined yeast biofilm screening-characterization and validation of yeast related biofilms in a brewing environment with combined cultivation and specific real-time PCR screening of selected indicator species. J Am Soc Brew Chem 77:99–112. https://doi.org/10.1080/03610470.2019.1579036
Branda SS, Vik Å, Friedman L, Kolter R (2005) Biofilms: The matrix revisited. Trends Microbiol 13:20–26. https://doi.org/10.1016/j.tim.2004.11.006
Deng Z, Luo XM, Liu J, Wang H (2020) Quorum sensing, biofilm, and intestinal mucosal barrier: involvement the role of probiotic. Front Cell Infect Microbiol 10:1–10. https://doi.org/10.3389/fcimb.2020.538077
Zeng X, Xia W, Jiang Q, Yang F (2013) Effect of autochthonous starter cultures on microbiological and physico-chemical characteristics of Suan yu, a traditional Chinese low salt fermented fish. Food Control 33:344–351. https://doi.org/10.1016/j.foodcont.2013.03.001
Priest F, Campbell I (1996) Brewing Microbiology. Springer US, Boston, MA. https://doi.org/10.1007/978-1-4757-4679-2
Rogers CM, Veatch D, Covey A et al (2016) Terminal acidic shock inhibits sour beer bottle conditioning by Saccharomyces cerevisiae. Food Microbiol 57:151–158. https://doi.org/10.1016/j.fm.2016.02.012
Murakami CJ, Wall V, Basisty N, Kaeberlein M (2011) Composition and acidification of the culture medium influences chronological aging similarly in vineyard and laboratory yeast. PLoS ONE. https://doi.org/10.1371/journal.pone.0024530
Reis VR, Bassi APG, da Silva JCG, Ceccato-Antonini SR (2013) Characteristics of Saccharomyces cerevisiae yeasts exhibiting rough colonies and pseudohyphal morphology with respect to alcoholic fermentation. Brazilian J Microbiol 44:1121–1131. https://doi.org/10.1590/S1517-83822014005000020
Munna MS, Humayun S, Noor R (2015) Influence of heat shock and osmotic stresses on the growth and viability of Saccharomyces cerevisiae SUBSC01 Microbiology. BMC Res Notes 8:1–8. https://doi.org/10.1186/s13104-015-1355-x
Czerucka D, Piche T, Rampal P (2007) Review article: Yeast as probiotics - Saccharomyces boulardii. Aliment Pharmacol Ther 26:767–778. https://doi.org/10.1111/j.1365-2036.2007.03442.x
Ludovico JMP, Rodrigues F, et al (2012) Stress and Cell Death in Yeast Induced by Acetic Acid. In: Cell Metabolism - Cell Homeostasis and Stress Response. InTech https://doi.org/10.5772/27726
Narendranath NV, Thomas KC, Ingledew WM (2001) Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium. J Ind Microbiol Biotechnol 26:171–177. https://doi.org/10.1038/sj.jim.7000090
Corte L, Rellini P, Lattanzi M et al (2006) Diversity of salt response among yeasts. Ann Microbiol 56:363–368. https://doi.org/10.1007/BF03175033
Stratford M, Steels H, Novodvorska M et al (2019) Extreme osmotolerance and halotolerance in food-relevant yeasts and the role of glycerol-dependent cell individuality. Front Microbiol 10:1–14. https://doi.org/10.3389/fmicb.2018.03238
Osburn K, Amaral J, Metcalf SR et al (2018) Primary souring: A novel bacteria-free method for sour beer production. Food Microbiol 70:76–84. https://doi.org/10.1016/j.fm.2017.09.007
Methner Y, Hutzler M, Matoulková D et al (2019) Screening for the brewing ability of different non-Saccharomyces yeasts. Fermentation. https://doi.org/10.3390/fermentation5040101
Syal P, Vohra A (2013) Probiotic potential of yeasts isolated from traditional indian fermented foods. Int J Microbiol Res 5:390–398. https://doi.org/10.9735/0975-5276.5.2.390-398
Ashtavinayak P, Elizabeth HA (2016) Review: gram negative bacteria in brewing. Adv Microbiol 06:195–209. https://doi.org/10.4236/aim.2016.63020
Piraine REA, Leite FPL, Bochman ML (2021) Mixed-culture metagenomics of the microbes making sour beer. Fermentation 7:174. https://doi.org/10.3390/fermentation7030174
Rodhouse L, Carbonero F (2019) Overview of craft brewing specificities and potentially associated microbiota. Crit Rev Food Sci Nutr 59:462–473. https://doi.org/10.1080/10408398.2017.1378616
Panel EB, Koutsoumanis K, Allende A, et al (2021) Updated list of QPS-recommended biological agents for safety risk assessments carried out by EFSA. https://doi.org/10.5281/ZENODO.4917383
Minelli EB, Benini A (2008) Relationship between number of bacteria and their probiotic effects. Microb Ecol Health Dis 20:180–183. https://doi.org/10.1080/08910600802408095
Habschied K, Živković A, Krstanović V, Mastanjević K (2020) Functional beer—a review on possibilities. Beverages 6:1–15. https://doi.org/10.3390/beverages6030051
Calumba KF, Vondel R, Bonilla F et al (2021) Ale beer containing free and immobilized Lactobacillus brevis, a potential delivery system for probiotics. Food Produc Process and Nutr 3:8–24. https://doi.org/10.1186/s43014-021-00051-32
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The present work was carried out with the support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—Brazil—financial code 001, and support from Conselho Nacional de Desenvolvimento Científico (CNPq). We thank all students involved directly or indirectly with this study.
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The present work was carried out with the support from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—Brazil—financial code 001, and support from Conselho Nacional de Desenvolvimento Científico (CNPq).
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Piraine, R.E.A., Retzlaf, G.M., Gonçalves, V.S. et al. Brewing and probiotic potential activity of wild yeasts Hanseniaspora uvarum PIT001, Pichia kluyveri LAR001 and Candida intermedia ORQ001. Eur Food Res Technol 249, 133–148 (2023). https://doi.org/10.1007/s00217-022-04139-z
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DOI: https://doi.org/10.1007/s00217-022-04139-z