Abstract
Yeasts can produce toxins in protein or glycoprotein structures that can act as an inhibitor on some bacteria and yeast species. The effects of those toxins on the growth of pathogenic and food spoilage microorganisms are subject to various studies. Metschnikowia pulcherrima was determined to be a killer toxin-producing yeast that was tested against three selected microorganisms, namely Escherichia coli Type-I, Micrococcus luteus and Candida albicans. The killer toxin only showed inhibitory activity against M. luteus. Different pH (5–6–7–8), temperature (20–25–30–35 °C) and carbon source (glucose–glycerol–ethanol–acetate) combinations were applied to stimulate the growth and toxin production of the killer yeast. The greatest increase among the different combinations was obtained at 20 °C and pH 7 when glycerol was used as the main carbon source. It was then also tested against other pathogen indicators or pathogens under these conditions. The killer toxin was partially purified by ethanol precipitation and showed inhibitory activity against M. luteus (36 mm). According to the protein profile obtained by SDS-PAGE, the molecular weight of the inhibitor toxin was measured about 7.4 kDa. The molecular weight with amino acid sequence of the killer toxin was 10.3 kDa and determined by MALDI-TOF mass spectrometry.
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Data availability
GenBank accession number for Metschnikowia pulcherrima TB26 isolate is MN622823 using the BLAST program.
References
Abu-Mejdad NMJA, Al-Badran AI, Al-Saadoon AH (2020) Purification and characterization of two killer toxins originated from Torulaspora delbrueckii (Lindner) and Wickerhamomyces anomalus (E.C. Hansen) Kurtzman, Robnett, and Basehoar-Powers. Bull Natl Res Cent 44:48. https://doi.org/10.1186/s42269-020-00308-w
Al-Qaysi SAS, Al-Haideri H, Thabit TA, Al-Razzaq Al-Kubaisy WH, Al-Rahman Ibrahim J (2017) Production, characterization, and antimicrobial activity of mycocin produced by Debaryomyces hansenii DSMZ70238. Int J Microbiol 2017:1–9. https://doi.org/10.1155/2017/2605382
Belgacem O, Pittenauer E, Openshaw ME, Hart PJ, Bowdler A, Allmaier G (2016) Axial spatial distribution focusing: improving MALDI-TOF/RTOF mass spectrometric performance for high-energy collisioninduced dissociation of biomolecules. Rapid Commun Mass Spectrom 30:343–351. https://doi.org/10.1002/rcm.7458
Bevan EA, Makower M (1963) The physiological basis of the killer character in yeast. In: Proceedings of the 11th international congress of genetics, The Hague, The Netherlands, September 1963, vol 1, pp 202–203
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72(1–2):248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Büyüksırıt Bedir T (2019) Determination of changes in antimicrobial production of yeasts under various carbon source and growth temperatures. Süleyman Demirel University, PhD Thesis, 145 p, Isparta
Büyüksırıt Bedir T, Kuleaşan H (2021) A natural approach, the use of killer toxin produced by Metschnikowia pulcherrima in fresh ground beef patties for shelf life extention. Int J Food Microbiol 345:109154. https://doi.org/10.1016/j.ijfoodmicro.2021.109154
Cannella V, Altomare R, Chiaramonte G, Di Bella S, Mira F, Russotto L, Pisano P, Guercio A (2019) Cytotoxicity evaluation of endodontic pins on L929 cell line. Biomed Res Int 2019:1–5. https://doi.org/10.1155/2019/3469525
Castillo A, Liebana J, Lopez E, Baca P, Liebana MJ, Castillo F (2006) Interference of antibiotics in the growth curves of oral Streptococci. Int J Antimicrob Agents 27:263–266. https://doi.org/10.1016/j.ijantimicag.2005.10.017
Chen WB, Han YF, Jong SC, Chang SC (2000) Isolation Purification and characterization of a killer protein from Schwanniomyces occidentalis. Appl Environ Microbiol 66:1029–1035. https://doi.org/10.1128/aem.66.12.5348-5352.2000
Csutak O, Vassu T, Sarbu I, Stoica I, Cornea P (2013) Antagonistic activity of three newly isolated yeast strains from the surface of fruits. Food Technol Biotechnol 51(1):70–77
El-Housseiny GS, Nagy GN, Ghobashi ZT, Mostafa RM, Abd-Allah IM, Saleh SE (2021) Optimization of antifungal activity by Bacillus subtilis isolate CCASU 2021–4 using response surface methodology. Arch Pharm Sci ASU 5(1):171–183. https://doi.org/10.21608/aps.2021.80383.1063
Ferrari M, Fornasiero MC, Isetta AM (1990) MTT colorimetric assay for testing macrophage cytotoxic activity in vitro. J Immunol Methods 131(2):165–172. https://doi.org/10.1016/0022-1759(90)90187-z
Fredericks LR, Lee MD, Eckert HR, Li S, Shipley MA, Roslund CR, Boikov DA, Kizer EA, Sobel JD, Rowley PA (2021) Vaginal isolates of Candida glabrata are uniquely susceptible to ionophoric killer toxins produced by Saccharomyces cerevisiae. Antimicrob Agents Chemother 65(7):e02450-e2520. https://doi.org/10.1128/AAC.02450-20
Golubev WI (2006) Antagonistic interactions among yeasts. In: Péter G, Rosa C (eds) Bio-diversity and ecophysiology of yeasts. Springer, Berlin, pp 197–219
Goto K, Iwase T, Kichise K, Kitano K, Totuka A, Obata T, Hara S (1990) Isolation and properties of a chromosome-dependent KHR killer toxin in Saccharomyces cerevisiae. Agric Biol Chem 54(2):505–509. https://doi.org/10.1271/bbb1961.54.505
Grahovac J, Pajčin I, Vlajkov V, Rončević Z, Dodić J, Cvetković D, Jokić A (2021) Xanthomonas campestris biocontrol agent: selection, medium formulation and bioprocess kinetic analysis. Chem Ind Chem Eng Q 27(2):131–142. https://doi.org/10.2298/CICEQ200508032G
Hicks RH, Moreno-Beltrán M, Gore-Lloyd D, Chuck CJ, Henk DA (2021) The oleaginous yeast Metschnikowia pulcherrima displays killer activity against avian-derived pathogenic bacteria. Biology 10:1227. https://doi.org/10.3390/biology10121227
Izgu F, Altinbay D (2004) Isolation and characterization of the K5-type yeast killer protein and its homology with an exo-β-1,3-glucanase. Biosci Biotechnol Biochem 68:685–693. https://doi.org/10.1271/bbb.68.685
Janisiewicz WJ, Tworkoski TJ, Kurtzman CP (2001) Biocontrol potential of Metchnikowia pulcherrima strains against blue mold of apple. Phytopathology 91:1098–1108. https://doi.org/10.1094/PHYTO.2001.91.11.1098
Kántor A, Hutková J, Petrová J, Hleba L, Kačániová M (2015/16) Antimicrobial activity of pulcherrimin pigment produced by Metschnikowia pulcherrima against various yeast species. J Microbiol Biotech Food Sci 5(3):282–285
Kim YS, Balaraju K, Jeon YH (2017) Biological characteristics of Bacillus amyloliquefaciens AK-0 and suppression of ginseng root rotcaused by Cylindrocarpon destructans. J Appl Microbiol 122:166–179. https://doi.org/10.1111/jam.13325
Klassen R, Meinhardt F (2005) Induction of DNA damage and apoptosis in Saccharomyces cerevisiae by a yeast killer toxin. Cell Microbiol 7:393–401. https://doi.org/10.1111/j.1462-5822.2004.00469.x
Kuchen B, Maturano YP, Mestre MV, Combina M, Toro ME, Vazquez F (2019) Selection of native non-Saccharomyces yeasts with biocontrol activity against spoilage yeasts in order to produce healthy regional wines. Fermentation 5(3):60. https://doi.org/10.3390/fermentation5030060
Kurtzman CP, Droby S (2001) Metschnikowia fructicola, a new ascosporic yeast with potential for biocontrol of postharvest fruit rots. Syst Appl Microbiol 24(3):395–399. https://doi.org/10.1078/0723-2020-00045
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685. https://doi.org/10.1038/227680a0
Łepecka A, Szymański P, Rutkowska S, Iwanowska K, Kołożyn-Krajewska D (2021) The influence of environmental conditions on the antagonistic activity of lactic acid bacteria isolated from fermented meat products. Foods 10(10):2267. https://doi.org/10.3390/foods10102267
Lopes CA, Sangorrin MP (2010) Optimization of killer assays for yeast selection protocols. Rev Argen Microbiol 42:298–306. https://doi.org/10.1590/S0325-75412010000400011
Magaldi S, Mata-Essayag S, Hartung de Capriles C (2004) Well diffusion for antifungal susceptibility testing. Int J Infect Dis 8:39–45. https://doi.org/10.1016/j.ijid.2003.03.002
Mai-Prochnow A, Clauson M, Hong J, Murphy AB (2016) Gram positive and Gram negative bacteria differ in their sensitivity to cold plasma. Sci Rep 6:38610. https://doi.org/10.1038/srep38610
Mamphogoro TP, Kamutando CN, Maboko MM, Aiyegoro OA, Babalola OO (2021) Epiphytic bacteria from sweet pepper antagonistic ın vitro to Ralstonia solanacearum BD 261, a causative agent of bacterial wilt. Microorganisms 9(9):1947. https://doi.org/10.3390/microorganisms9091947
Moura VS, Pollettini FL, Ferraz LP, Mazzi MV, Kupper KC (2021) Purification of a killer toxin from Aureobasidium pullulans for the biocontrol of phytopathogens. J Basic Microbiol 61:77–87. https://doi.org/10.1002/jobm.202000164
Oro L, Ciani M, Comitini F (2014) Antimicrobial activity of Metschnikowia pulcherrima on wine yeasts. J Appl Microbiol 116:1209–1217. https://doi.org/10.1111/jam.12446
Peña A, Sãnchez NS, Álvarez H, Calahorra M, Ramírez J (2015) Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae. FEMS Yeast Res 15(2):1–13. https://doi.org/10.1093/femsyr/fou005
Peng Y (2010) The studies on killer toxin and β-1,3-glucanase produced by the marine yeast Williopsis Saturnus WC91–2. Ocean University of China, Ph.D. Thesis. Key Laboratory of Marine Genetics and Gene Resource Exploitation
Piano S, Neyrotti V, Migheli Q, Gullino ML (1997) Biocontrol capability of Metschnikowia pulcherrima against Botrytis postharvest rot of apple. Postharvest Biol Technol 11(3):131–140
Radler F, Schmitt MJ, Meyer B (1990) Killer toxin of Hanseniaspora uvarum. Arch Microbiol 154:175–178
Ragavan ML, Das N (2020/21) Production and purification of killer toxin from probiotic yeasts and its effect on foodborne pathogens. J Microbiol Biotech Food Sci 10(3):350–353
Roostita LB, Fleet GH, Wendry SP, Apon ZM, Gemilang LU (2011) Determination of yeasts antimicrobial activity in milk and meat products. Adv J Food Sci Technol 3(6):442–445
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York, p 1546
Saravanakumar D, Ciavorella A, Spadaro D, Garibaldi A, Gullino ML (2008) Metschnikowia pulcherrima strain MACH1 outcompetes Botrytis cinerea, Alternaria alternata and Penicillium expansum in apples through iron depletion. Postharvest Biol Technol 49:121–128. https://doi.org/10.1016/j.postharvbio.2007.11.006
Schmitt MJ, Breinig F (2002) The viral killer system in yeast: from molecular biology to application. FEMS Microbiol Rev 26:257–276. https://doi.org/10.1111/j.1574-6976.2002.tb00614.x
Soares GAM, Sato HH (2000) Characterization of the Saccharomyces cerevisae Y500–4L killer toxin. Braz J Microbiol 31(4):291–297. https://doi.org/10.1590/S1517-83822000000400010
Suzuki C, Ando Y, Machida S (2001) Interaction of SMKT, a killer toxin produced by Pichia farinosa, with the yeast cell membranes. Yeast 18(16):1471–1478. https://doi.org/10.1002/yea.791
Türkel S, Korukluoğlu M, Yavuz M (2014) Biocontrol activity of the local strain of Metschnikowia pulcherrima on different postharvest pathogens. Biotechnol Res Int 2014:1–6. https://doi.org/10.1155/2014/397167
Vadkertiová R, Sláviková E (2007) Killer activity of yeasts isolated from natural environments against some medically important Candida species. Pol J Microbiol 56(1):39–43
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The authors thank to Scientific Research Projects Unit of Süleyman Demirel University (4848-D1-17), Isparta, Turkey, for providing financial support.
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TBB collected and analyzed the data, followed the analyses and evaluated the results. HK provided the planning and execution of the research. They did the writing of the article and the final checks together. All authors read and approved the final version of the manuscript.
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Büyüksırıt-Bedir, T., Kuleaşan, H. Purification and characterization of a Metschnikowia pulcherrima killer toxin with antagonistic activity against pathogenic microorganisms. Arch Microbiol 204, 337 (2022). https://doi.org/10.1007/s00203-022-02940-8
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DOI: https://doi.org/10.1007/s00203-022-02940-8