{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,24]],"date-time":"2026-07-24T04:49:07Z","timestamp":1784868547398,"version":"3.55.0"},"reference-count":45,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2025,12,15]],"date-time":"2025-12-15T00:00:00Z","timestamp":1765756800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Research Council of Thailand (NRCT), The Kasetsart University Research and Development Institute","award":["FF(KU)51.67"],"award-info":[{"award-number":["FF(KU)51.67"]}]},{"name":"Kasetsart University Institute for Advanced Studies (KUIAS) through the Reinventing University Program; and the Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["JoF"],"abstract":"<jats:p>This study applied a guided culturomics workflow to isolate and characterize gut-associated yeasts as probiotic candidates. Culture conditions were optimized using Dixon agar and Modified Sch\u00e4dler Agar with a moderate antibiotic cocktail (colistin, chloramphenicol, vancomycin) to suppress bacteria without impairing yeast growth, combined with incubation at 37 \u00b0C under anaerobic conditions to mimic the intestinal environment. From three healthy donors, 305 isolates were recovered (MSA: 193; Dixon: 112). After excluding pseudohyphal morphotypes and PCR positive Candida isolates, 127 non-Candida strains remained. Safety screening (hemolysis, DNase, coagulase) reduced the pool to 26 safe isolates. Simulated gastrointestinal stress tests showed that 20 out of 26 exhibited at least 50 percent survival under pH 2.0 or 0.5% bile salts. Functional assays revealed strain-specific antimicrobial activity against Staphylococcus aureus, Escherichia coli including O157:H7 and Salmonella species, with several isolates (Y6, Y22, Y42, Y48, Y55, Y56, Y73, Y105, Y127) showing broad and strong inhibition. Two isolates Y44 and Y55 further demonstrated robust bile and acid tolerance (&gt;50% survivability) in both conditions. All isolates displayed intracellular but not extracellular bile salt hydrolase activity, indicating a viability dependent cholesterol lowering potential. Overall, the workflow minimized bacterial interference while enriching for safe and functional yeasts.<\/jats:p>","DOI":"10.3390\/jof11120885","type":"journal-article","created":{"date-parts":[[2025,12,16]],"date-time":"2025-12-16T09:57:24Z","timestamp":1765879044000},"page":"885","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Optimized Selective Media Enhance the Isolation and Characterization of Gut-Derived Probiotic Yeasts"],"prefix":"10.3390","volume":"11","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4706-2578","authenticated-orcid":false,"given":"Kevin","family":"Mok","sequence":"first","affiliation":[{"name":"Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"},{"name":"Center of Excellence for Microbiota Innovation, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kwantida","family":"Popitool","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"},{"name":"Center of Excellence for Microbiota Innovation, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Areerat","family":"Songla","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tawisa","family":"Pongsuwanporn","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"},{"name":"Center of Excellence for Microbiota Innovation, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pitchsupang","family":"Torrungruang","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"},{"name":"Center of Excellence for Microbiota Innovation, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sunee","family":"Nitisinprasert","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"},{"name":"Center of Excellence for Microbiota Innovation, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiro","family":"Nakayama","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University, Fukuoka 819-0395, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1277-9299","authenticated-orcid":false,"given":"Massalin","family":"Nakphaichit","sequence":"additional","affiliation":[{"name":"Department of Biotechnology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"},{"name":"Center of Excellence for Microbiota Innovation, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1093\/femsre\/fuw047","article-title":"Forgotten fungi-the gut mycobiome in human health and disease","volume":"41","author":"Huseyin","year":"2017","journal-title":"FEMS Microbiol. Rev."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Garcia-Bonete, M.J., Rajan, A., Suriano, F., and Layunta, E. (2023). The Underrated Gut Microbiota Helminths, Bacteriophages, Fungi, and Archaea. Life, 13.","DOI":"10.3390\/life13081765"},{"key":"ref_3","first-page":"10","article-title":"State of the Art in the Culture of the Human Microbiota: New Interests and Strategies","volume":"34","author":"Naud","year":"2020","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"726","DOI":"10.1007\/s00284-018-1438-8","article-title":"Fungal Diversity of Human Gut Microbiota Among Eutrophic, Overweight, and Obese Individuals Based on Aerobic Culture-Dependent Approach","volume":"75","author":"Borges","year":"2018","journal-title":"Curr. Microbiol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/j.funeco.2015.01.006","article-title":"Fungi inhabiting the healthy human gastrointestinal tract: A diverse and dynamic community","volume":"15","author":"Kachman","year":"2015","journal-title":"Fungal Ecol."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Mok, K., Suratanon, N., Roytrakul, S., Charoenlappanit, S., Patumcharoenpol, P., Chatchatee, P., and Nakphaichit, M. (2021). ITS2 Sequencing and Targeted Meta-Proteomics of Infant Gut Mycobiome Reveal the Functional Role of Rhodotorula sp. during Atopic Dermatitis Manifestation. J. Fungi, 7.","DOI":"10.3390\/jof7090748"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Mar Rodr\u00edguez, M., P\u00e9rez, D., Javier Chaves, F., Esteve, E., Marin-Garcia, P., Xifra, G., and Fernandez Real, J.M. (2015). Obesity changes the human gut mycobiome. Sci. Rep., 5.","DOI":"10.1038\/srep14600"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Hoffmann, C., Dollive, S., Grunberg, S., Chen, J., Li, H., Wu, G.D., and Bushman, F.D. (2013). Archaea and fungi of the human gut microbiome: Correlations with diet and bacterial residents. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0066019"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Pais, P., Almeida, V., Y\u0131lmaz, M., and Teixeira, M.C. (2020). Saccharomyces boulardii: What Makes It Tick as Successful Probiotic?. J. Fungi, 6.","DOI":"10.3390\/jof6020078"},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Liu, S., Yang, L., Zhang, Y., Chen, H., Li, X., Xu, Z., and Liu, D. (2024). Review of yeast culture concerning the interactions between gut microbiota and young ruminant animals. Front. Vet. Sci., 11.","DOI":"10.3389\/fvets.2024.1335765"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1185","DOI":"10.1111\/1469-0691.12023","article-title":"Microbial culturomics: Paradigm shift in the human gut microbiome study","volume":"18","author":"Lagier","year":"2012","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1424","DOI":"10.1038\/s41587-023-01674-2","article-title":"High-throughput microbial culturomics using automation and machine learning","volume":"41","author":"Huang","year":"2023","journal-title":"Nat. Biotechnol."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Das, T.K., Pradhan, S., Chakrabarti, S., Mondal, K.C., and Ghosh, K. (2022). Current status of probiotic and related health benefits. Appl. Food Res., 2.","DOI":"10.1016\/j.afres.2022.100185"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/j.ijfoodmicro.2016.01.016","article-title":"Killer toxin from several food-derived Debaryomyces hansenii strains effective against pathogenic Candida yeasts","volume":"222","author":"Banjara","year":"2016","journal-title":"Int. J. Food Microbiol."},{"key":"ref_15","doi-asserted-by":"crossref","unstructured":"Shruthi, B., Deepa, N., Somashekaraiah, R., Adithi, G., Divyashree, S., and Sreenivasa, M.Y. (2022). Exploring biotechnological and functional characteristics of probiotic yeasts: A review. Biotechnol. Rep., 34.","DOI":"10.1016\/j.btre.2022.e00716"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Alkalbani, N.S., Osaili, T.M., Al-Nabulsi, A.A., Olaimat, A.N., Liu, S.Q., Shah, N.P., and Ayyash, M.M. (2022). Assessment of Yeasts as Potential Probiotics: A Review of Gastrointestinal Tract Conditions and Investigation Methods. J. Fungi, 8.","DOI":"10.3390\/jof8040365"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Fan, Y., Yin, C., Xu, L., Bai, R., Wei, Z., Gao, G., and Pi, Y. (2025). The Biological Functions of Yeast and Yeast Derivatives and Their Application in Swine Production: A Review. Microorganisms, 13.","DOI":"10.3390\/microorganisms13071669"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"164","DOI":"10.4161\/gmic.1.3.12127","article-title":"Safety assessment of probiotics for human use","volume":"1","author":"Sanders","year":"2010","journal-title":"Gut Microbes"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Xu, R., Yu, Y., and Chen, T. (2025). Exploring the dark side of probiotics to pursue light: Intrinsic and extrinsic risks to be opportunistic pathogens. Curr. Res. Food Sci., 10.","DOI":"10.1016\/j.crfs.2025.101044"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"315","DOI":"10.1016\/j.ijmmb.2021.05.004","article-title":"Comparison of antimicrobial susceptibility interpretation among Enterobacteriaceae using CLSI and EUCAST breakpoints","volume":"39","author":"Suravaram","year":"2021","journal-title":"Indian J. Med. Microbiol."},{"key":"ref_21","unstructured":"CLSI (2020). Clinical and Laboratory Standards Institute, CLSI. Available online: https:\/\/www.nih.org.pk\/wp-content\/uploads\/2021\/02\/CLSI-2020.pdf."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Angchuan, J., Khunnamwong, P., Wongpanit, K., Limtong, S., and Srisuk, N. (2021). Yeasts Associated with the Small-Intestinal Contents and Epithelium of Pon Yang Kham (Charolais Crossbred) Fattening Beef Cattle. Microorganisms, 9.","DOI":"10.3390\/microorganisms9071444"},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Mok, K., Honwichit, O., Funnuam, T., Charoensiddhi, S., Nitisinprasert, S., Nielsen, D.S., and Nakphaichit, M. (2024). Synergistic activity of Limosilactobacillus reuteri KUB-AC5 and water-based plants against Salmonella challenge in a human in vitro gut model. Sci. Rep., 14.","DOI":"10.1038\/s41598-024-53912-5"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Huseyin, C.E., Rubio, R.C., O\u2019Sullivan, O., Cotter, P.D., and Scanlan, P.D. (2017). The Fungal Frontier: A Comparative Analysis of Methods Used in the Study of the Human Gut Mycobiome. Front. Microbiol., 8.","DOI":"10.3389\/fmicb.2017.01432"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.cmi.2015.10.032","article-title":"A quasi-universal medium to break the aerobic\/anaerobic bacterial culture dichotomy in clinical microbiology","volume":"22","author":"Dione","year":"2016","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"191","DOI":"10.1016\/j.mimet.2007.08.013","article-title":"Quantitative real time PCR assays for the enumeration of Saccharomyces cerevisiae and the Saccharomyces sensu stricto complex in human feces","volume":"71","author":"Chang","year":"2007","journal-title":"J. Microbiol. Methods"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"21","DOI":"10.4137\/MBI.S38517","article-title":"Development of Candida-Specific Real-Time PCR Assays for the Detection and Identification of Eight Medically Important Candida Species","volume":"9","author":"Zhang","year":"2016","journal-title":"Microbiol. Insights"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.riam.2013.11.003","article-title":"Enzymatic and hemolytic activity in different Candida species","volume":"32","author":"Penatti","year":"2015","journal-title":"Rev. Iberoam. De Micol."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"848","DOI":"10.1128\/jcm.15.5.848-851.1982","article-title":"Microtube coagulase test for detection of coagulase-positive staphylococci","volume":"15","author":"Goldstein","year":"1982","journal-title":"J. Clin. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"244","DOI":"10.1136\/mp.54.4.244","article-title":"Identification of slide coagulase positive, tube coagulase negative Staphylococcus aureus by 16S ribosomal RNA gene sequencing","volume":"54","author":"Woo","year":"2001","journal-title":"Mol. Pathol."},{"key":"ref_31","first-page":"181","article-title":"Investigation of antibacterial, acid and bile tolerance properties of lactobacilli isolated from Koozeh cheese","volume":"3","author":"Hassanzadazar","year":"2012","journal-title":"Vet. Res. Forum"},{"key":"ref_32","unstructured":"Dhanasekaran, D., and Sankaranarayanan, A. (2021). Chapter 21\u2014Prospective Approaches of Pseudonocardia alaniniphila Hydrobionts for Litopenaeus vannamei. Advances in Probiotics, Academic Press."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"353","DOI":"10.1177\/10820132211012252","article-title":"Selection of potential probiotics with cholesterol-lowering properties for probiotic yoghurt production","volume":"28","author":"Wongrattanapipat","year":"2022","journal-title":"Food Sci. Technol. Int."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Seelbinder, B., Chen, J., Brunke, S., Vazquez-Uribe, R., Santhaman, R., Meyer, A.C., and Panagiotou, G. (2020). Antibiotics create a shift from mutualism to competition in human gut communities with a longer-lasting impact on fungi than bacteria. Microbiome, 8.","DOI":"10.1186\/s40168-020-00899-6"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Mart\u00ednez, J.L. (2017). Effect of antibiotics on bacterial populations: A multi-hierachical selection process. F1000Research, 6.","DOI":"10.12688\/f1000research.9685.1"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Abid, R., Waseem, H., Ali, J., Ghazanfar, S., Muhammad Ali, G., Elasbali, A.M., and Alharethi, S.H. (2022). Probiotic Yeast Saccharomyces: Back to Nature to Improve Human Health. J. Fungi, 8.","DOI":"10.3390\/jof8050444"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Hossain, M.N., Afrin, S., Humayun, S., Ahmed, M.M., and Saha, B.K. (2020). Identification and Growth Characterization of a Novel Strain of Saccharomyces boulardii Isolated from Soya Paste. Front. Nutr., 7.","DOI":"10.3389\/fnut.2020.00027"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"520","DOI":"10.1038\/s41551-019-0397-0","article-title":"A complex human gut microbiome cultured in an anaerobic intestine-on-a-chip","volume":"3","author":"Gazzaniga","year":"2019","journal-title":"Nat. Biomed. Eng."},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"de Carvalho, B.T., Suboti\u0107, A., Vandecruys, P., Deleu, S., Vermeire, S., and Thevelein, J.M. (2024). Enhancing probiotic impact: Engineering Saccharomyces boulardii for optimal acetic acid production and gastric passage tolerance. Appl. Environ. Microbiol., 90.","DOI":"10.1128\/aem.00325-24"},{"key":"ref_40","first-page":"313","article-title":"Fungi isolated from the stool in patients with gastrointestinal disorders in 2005\u20132009","volume":"64","author":"Macura","year":"2010","journal-title":"Prz. Epidemiol."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"3416","DOI":"10.3168\/jds.S0022-0302(03)73945-9","article-title":"Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese","volume":"86","author":"Psomas","year":"2003","journal-title":"J. Dairy Sci."},{"key":"ref_42","unstructured":"Donnenberg, M.S. (2002). Chapter 1\u2014The Genomes of Escherichia coli K-12 and Pathogenic E. coli. Escherichia Coli, Academic Press."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Im, E.J., Lee, H.H., Kim, M., and Kim, M.K. (2023). Evaluation of Enterococcal Probiotic Usage and Review of Potential Health Benefits, Safety, and Risk of Antibiotic-Resistant Strain Emergence. Antibiotics, 12.","DOI":"10.3390\/antibiotics12081327"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Vep\u0161tait\u0117-Monstavi\u010d\u0117, I., Luk\u0161a-\u017debelovi\u010d, J., Ap\u0161egait\u0117, V., Moz\u016braitis, R., Lisicinas, R., Stanevi\u010dien\u0117, R., and Servien\u0117, E. (2025). Profiles of Killer Systems and Volatile Organic Compounds of Rowanberry and Rosehip-Inhabiting Yeasts Substantiate Implications for Biocontrol. Foods, 14.","DOI":"10.3390\/foods14020288"},{"key":"ref_45","doi-asserted-by":"crossref","unstructured":"Hatoum, R., Labrie, S., and Fliss, I. (2012). Antimicrobial and probiotic properties of yeasts: From fundamental to novel applications. Front. Microbiol., 3.","DOI":"10.3389\/fmicb.2012.00421"}],"container-title":["Journal of Fungi"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2309-608X\/11\/12\/885\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,16]],"date-time":"2025-12-16T10:23:10Z","timestamp":1765880590000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2309-608X\/11\/12\/885"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,15]]},"references-count":45,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2025,12]]}},"alternative-id":["jof11120885"],"URL":"https:\/\/doi.org\/10.3390\/jof11120885","relation":{},"ISSN":["2309-608X"],"issn-type":[{"value":"2309-608X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,12,15]]}}}