Abstract
Kefir is a fermented probiotic drink obtained by placing kefir granules in a suitable substrate. The kefir granules are a consortium of bacteria and yeasts embedded in a exopolysaccharide matrix. The aim of this research was the isolation and identification of yeasts from kefir of different origin, the evaluation of their antifungal capacity against Aspergillus spp., and the characterization of virulence related traits. Using RFLP of ITS1/ITS4 region, D1/D2 region sequencing, and RAPD techniques, 20 kefir isolates were identified as Geotrichum candidum, Pichia kudriavzevii, Pichia membranifaciens, Saccharomyces cerevisiae, and Candida ethanolica. Their antifungal capacity was evaluated by their conidia germination reduction, which allowed the selection of eight isolates with high to moderate conidia germination reduction against Aspergillus flavus and Aspergillus parasiticus. Furthermore, these selected isolates showed growth inhibition on contact in the dual culture assay for both Aspergillus species and 3 of them—belonging to S. cerevisiae and P. kudriavzevii species—generated volatile organic compounds which significantly affected the growth of both fungi. For the evaluation of virulence-related traits, growth at high temperatures, enzymatic activities, and the adhesion to Caco-2 cells were analyzed. The isolates did not present more than one positive virulence-related trait simultaneously. In particular, it is important to highlight that the adhesion capacity to the model of intestinal barrier was extremely low for all of them. According to the results obtained, further studies would be of interest for the possible use of these promising yeasts as biocontrol agents against fungi in food.
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Abbreviations
- %GR:
-
Percentage of germination reduction
- CC:
-
Chancaca
- CFS:
-
Cell-free supernatant
- LAB:
-
Lactic acid bacteria
- M:
-
Milk
- MO:
-
Molasses
- MU:
-
Muscovado
- RAPD:
-
Randomly amplified polymorphic DNA
- RFLP:
-
Restriction fragment length polymorphism
- VOC:
-
Volatile organic compounds
- WP:
-
Whey permeate
References
Abdel-Kareem MM, Rasmey AM, Zohri AA (2019) The action mechanism and biocontrol potentiality of novel isolates of Saccharomyces cerevisiae against the aflatoxigenic Aspergillus flavus. Lett Appl Microbiol 68:104–111. https://doi.org/10.1111/lam.13105
Azi F, Tu C, Rasheed HA, Dong M (2020) Comparative study of the phenolics, antioxidant and metagenomic composition of novel soy whey-based beverages produced using three different water kefir microbiota. Int J Food Sci Technol 55:1689–1697. https://doi.org/10.1111/ijfs.14439
Basavaiah R, Nagesh M, Harishchandra Sripathy M, Vardhan Batra H (2019) In vitro screening and characterization of kefir yeast for probiotic attributes. Int J Nutr Sci Food Technol 5:1–11
Ben TF, Mansour C, Chaieb K (2020) Inhibitory effect of kefir on Aspergillus growth and mycotoxin production. Euro-Mediterranean J Environ Integr 5:1–8. https://doi.org/10.1007/s41207-020-0141-x
Van Burik JH, Magee PT (2001) Aspects of fungal pathogenesis in humans. Annu Rev Microbiol 55:743–772. https://doi.org/10.1146/annurev.micro.55.1.743
Chanchaichaovivat A, Ruenwongsa P, Panijpan B (2007) Screening and identification of yeast strains from fruits and vegetables: potential for biological control of postharvest chilli anthracnose (Colletotrichum capsici). Biol Control 42:326–335. https://doi.org/10.1016/j.biocontrol.2007.05.016
Chen H, Ju H, Wang Y, Du G, Yan X, Cui Y, Yuan Y, Yue T (2021) Antifungal activity and mode of action of lactic acid bacteria isolated from kefir against Penicillium expansum. Food Control 130:108274. https://doi.org/10.1016/j.foodcont.2021.108274
Choińska R, Piasecka-Jóźwiak K, Chabłowska B, Dumka J, Łukaszewicz A (2020) Biocontrol ability and volatile organic compounds production as a putative mode of action of yeast strains isolated from organic grapes and rye grains. Antonie Van Leeuwenhoek 113:1135–1146. https://doi.org/10.1007/s10482-020-01420-7
da Miguel CP, Cardoso PG, Magalhães KT, Schwan RF (2011) Profile of microbial communities present in tibico (sugary kefir) grains from different Brazilian States. World J Microbiol Biotechnol 27:1875–1884. https://doi.org/10.1007/s11274-010-0646-6
Di Canito A, Mateo-vargas MA, Mazzieri M, Cantoral J, Foschino R, Cordero-Bueso G, Vigentini I (2021) The role of yeasts as biocontrol agents for pathogenic fungi on postharvest grapes: a review. Foods 10:1–15. https://doi.org/10.3390/foods10071650
Diosma G, Romanin DE, Rey-Burusco MF, Londero A, Garrote GL (2014) Yeasts from kefir grains: isolation, identification, and probiotic characterization. World J Microbiol Biotechnol 30:43–53. https://doi.org/10.1007/s11274-013-1419-9
Esteve-Zarzoso B, Belloch C, Uruburu F, Querol A (1999) Identification of yeasts by RFLP analysis of the 5.8S rRNA gene and the two ribosomal internal transcribed spacers. Int J Syst Bacteriol 49:329–337. https://doi.org/10.1099/00207713-49-1-329
Freimoser FM, Rueda-Mejia MP, Tilocca B, Migheli Q (2019) Biocontrol yeasts: mechanisms and applications. World J Microbiol Biotechnol 35:1–19. https://doi.org/10.1007/s11274-019-2728-4
Gamba RR, Caro CA, Martínez OL, Moretti AF, Giannuzzi L, De Antoni GL, León Peláez A (2016) Antifungal effect of kefir fermented milk and shelf life improvement of corn arepas. Int J Food Microbiol 235:85–92. https://doi.org/10.1016/j.ijfoodmicro.2016.06.038
Gamba RR, Moure MC, Diosma G, Giannuzzi L, De Antoni GL, León Peláez ÁM (2016) Application of whey permeate fermented with kefir grains for the shelf-life improvement of food and feed. Adv Microbiol 6:650–661. https://doi.org/10.4236/aim.2016.69064
Gamba RR, Yamamoto S, Sasaki T, Michihata T, Mahmoud AH, Koyanagi T, Enomoto T (2019) Microbiological and functional characterization of kefir grown in different sugar solutions. Food Sci Technol Res 25:303–312. https://doi.org/10.3136/fstr.25.303
Gao J, Gu F, Abdella NH, Ruan H, He G (2012) Investigation on culturable microflora in Tibetan kefir grains from different areas of China. J Food Sci 77:M425–M433. https://doi.org/10.1111/j.1750-3841.2012.02805.x
Garrote GL, Abraham AG, DeAntoni GL (1997) Preservation of kefir grains, a comparative study. Food Sci Technol Technol 30:77–84. https://doi.org/10.1006/fstl.1996.0135
Garrote GL, Abraham AG, De Antoni GL (1998) Characteristics of kefir prepared with different grain:milk ratios. J Dairy Res 65:149–154. https://doi.org/10.1017/S0022029997002677
Gente S, Sohier D, Coton E, Duhamel C, Gueguen M (2006) Identification of Geotrichum candidum at the species and strain level: proposal for a standardized protocol. J Ind Microbiol Biotechnol 33:1019–1031. https://doi.org/10.1007/s10295-006-0130-3
Gerez CL, Torino MI, Rollán G, Font de Valdez G (2009) Prevention of bread mould spoilage by using lactic acid bacteria with antifungal properties. Food Control 20:144–148. https://doi.org/10.1016/j.foodcont.2008.03.005
Gonda M, Garmendia G, Rufo C, León Peláez Á, Wisniewski M, Droby S, Vero S (2019) Biocontrol of Aspergillus flavus in ensiled sorghum by water kefir microorganisms. Microorganisms 7:253. https://doi.org/10.3390/microorganisms7080253
Gulitz A, Stadie J, Wenning M, Ehrmann MA, Vogel RF (2011) The microbial diversity of water kefir. Int J Food Microbiol 151:284–288. https://doi.org/10.1016/j.ijfoodmicro.2011.09.016
Hernandez-Montiel LG, Droby S, Preciado-Rangel P, Rivas-García T, González-Estrada RR, Gutiérrez-Martínez P, Ávila-Quezada GD (2021) A sustainable alternative for postharvest disease management and phytopathogens biocontrol in fruit: antagonistic yeasts. Plants 10:2641. https://doi.org/10.3390/PLANTS10122641
Hsieh HH, Wang SY, Chen TL, Huang YL, Chen MJ (2012) Effects of cow’s and goat’s milk as fermentation media on the microbial ecology of sugary kefir grains. Int J Food Microbiol 157:73–81. https://doi.org/10.1016/j.ijfoodmicro.2012.04.014
Hua SST, Beck JJ, Sarreal SBL, Gee W (2014) The major volatile compound 2-phenylethanol from the biocontrol yeast, Pichia anomala, inhibits growth and expression of aflatoxin biosynthetic genes of Aspergillus flavus. Mycotoxin Res 30:71–78. https://doi.org/10.1007/s12550-014-0189-z
Ismaiel AA, Ghaly MF, El-Naggar AK (2011) Milk kefir: ultrastructure, antimicrobial activity and efficacy on aflatoxin b1 production by Aspergillus flavus. Curr Microbiol 62:1602–1609. https://doi.org/10.1007/s00284-011-9901-9
Kalamaki MS, Angelidis AS (2017) Isolation and molecular identification of yeasts in Greek kefir. Int J Dairy Technol 70:261–268. https://doi.org/10.1111/1471-0307.12329
Kawtharani H, Snini SP, Heang S, Bouajila J, Taillandier P, Mathieu F, Beaufort S (2020) Phenyllactic acid produced by Geotrichum candidum reduces Fusarium sporotrichioides and F. langsethiae growth and T-2 toxin concentration. Toxins (Basel) 12:209. https://doi.org/10.3390/toxins12040209
Kumar B, Kumar B, Bharti S, Kumar J (2017) Effect of co-existing filamentous fungi on growth inhibition and aflatoxin production by Aspergillus parasiticus. Int J Curr Microbiol Appl Sci 6:2789–2799. https://doi.org/10.20546/ijcmas.2017.608.332
Kurtzman CP, Robnett CJ (1997) Identification of clinically important ascomycetous yeasts based on nucleotide divergence in the 5’ end of the large-subunit (26S) ribosomal DNA gene. J Clin Microbiol 35:1216–1223. https://doi.org/10.1128/jcm.35.5.1216-1223.1997
Laureys D, De Vuyst L (2014) Microbial species diversity, community dynamics, and metabolite kinetics of water kefir fermentation. Appl Environ Microbiol 80:2564–2572. https://doi.org/10.1128/AEM.03978-13
Lavermicocca P, Valerio F, Visconti A (2003) Antifungal activity of phenyllactic acid against molds isolated from bakery products. Appl Environ Microbiol 69:634–640. https://doi.org/10.1128/AEM.69.1.634-640.2003
León Peláez AM, Serna Cataño CA, Quintero Yepes EA, Gamba Villarroel RR, De Antoni GL, Giannuzzi L (2012) Inhibitory activity of lactic and acetic acid on Aspergillus flavus growth for food preservation. Food Control 24:177–183. https://doi.org/10.1016/j.foodcont.2011.09.024
Liu J, Sui Y, Wisniewski M, Droby S, Liu Y (2013) Review: utilization of antagonistic yeasts to manage postharvest fungal diseases of fruit. Int J Food Microbiol 167:153–160. https://doi.org/10.1016/j.ijfoodmicro.2013.09.004
Llanos R, Fernández-Espinar MT, Querol A (2006) A comparison of clinical and food Saccharomyces cerevisiae isolates on the basis of potential virulence factors. Antonie Van Leeuwenhoek 90:221–231. https://doi.org/10.1007/s10482-006-9077-7
Lõoke M, Kristjuhan K, Kristjuhan A (2011) Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques 50:325–328. https://doi.org/10.2144/000113672
Macedo GA, Park YK, Pastore GM (1997) Partial purification and characterization of an extracellular lipase from a newly isolated strain of Geotrichum sp. Rev Microbiol 28:90–95
Magalhães KT, de Melo Pereira GV, Campos CR, Dragone G, Schwan RF (2011) Brazilian kefir: structure, microbial communities and chemical composition. Brazilian J Microbiol 42:693–702. https://doi.org/10.1590/S1517-83822011000200034
Masih E, Paul B (2002) Secretion of β-1,3-glucanases by the yeast Pichia membranifaciens and its possible role in the biocontrol of Botrytis cinerea causing grey mold disease of the grapevine. Curr Microbiol 44:391–395. https://doi.org/10.1007/s00284-001-0011-y
Mayer F, Wilson D, Hube B (2013) Candida Albicans Pathogenicity Mechanisms Virulence 4:119–128. https://doi.org/10.4161/viru.22913
Murphy A, Kavanagh K (1999) Emergence of Saccharomyces cerevisiae as a human pathogen implications for biotechnology. Enzyme Microb Technol 25:551–557. https://doi.org/10.1016/S0141-0229(99)00086-1
Nielsen B, Gürakan GC, Ünlü G (2014) Kefir: a multifaceted fermented dairy product. Probiotics Antimicrob Proteins 6:123–135. https://doi.org/10.1007/s12602-014-9168-0
Pailin T, Kang DH, Schmidt K, Fung DYC (2001) Detection of extracellular bound proteinase in EPS-producing lactic acid bacteria cultures on skim milk agar. Lett Appl Microbiol 33:45–49. https://doi.org/10.1046/j.1472-765X.2001.00954.x
Pantelides IS, Christou O, Tsolakidou MD, Tsaltas D, Ioannou N (2015) Isolation, identification and in vitro screening of grapevine yeasts for the control of black aspergilli on grapes. Biol Control 88:46–53. https://doi.org/10.1016/j.biocontrol.2015.04.021
Park M, Do E, Jung WH (2013) Lipolytic enzymes involved in the virulence of human pathogenic fungi. Mycobiology 41:67–72. https://doi.org/10.5941/MYCO.2013.41.2.67
Pérez-Torrado R, Llopis S, Jespersen L, Fernández-espinar T, Querol A (2012) Clinical Saccharomyces cerevisiae isolates cannot cross the epithelial barrier in vitro. Int J Food Microbiol 157:59–64. https://doi.org/10.1016/j.ijfoodmicro.2012.04.012
Pérez-Través L, Lopes CA, Querol A, Barrio E (2014) On the complexity of the Saccharomyces bayanus taxon: hybridization and potential hybrid speciation. PLoS One 9:e93729. https://doi.org/10.1371/journal.pone.0093729
Peréz-Través L, de Llanos R, Flockhart A, García-Domingo L, Groenewald M, Pérez-Torrado R, Querol A (2021) Virulence related traits in yeast species associated with food; Debaryomyces hansenii, Kluyveromyces marxianus, and Wickerhamomyces anomalus. Food Control 124. https://doi.org/10.1016/j.foodcont.2021.107901
Piasecka-Jozwiak K, Chablowska B (2017) Anti-mold properties of yeast strains as a biological agent. J Res Appl Agric Eng 62:84–89
Purutoğlu K, İspirli H, Yüzer MO, Serencam H, Dertli E (2020) Diversity and functional characteristics of lactic acid bacteria from traditional kefir grains. Int J Dairy Technol 73:57–66. https://doi.org/10.1111/1471-0307.12633
Sacristán N, González L, Castro JM, Fresno JM, Tornadijo ME (2012) Technological characterization of Geotrichum candidum strains isolated from a traditional Spanish goats’ milk cheese. Food Microbiol 30:260–266. https://doi.org/10.1016/j.fm.2011.10.003
Sarikkha P, Nitisoravut R, Poljungreed I (2015) Identification of bacteria and yeast communities in a Thai sugary kefir by Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis (PCR-DGGE) analyses. J Ind Technol 11:25–39
Sellitto VM, Zara S, Fracchetti F, Capozzi V, Nardi T (2021) Fermentation microbial biocontrol as an alternative to synthetic fungicides: boundaries between pre-and postharvest applications on vegetables and fruits. Fermentation 7:60. https://doi.org/10.3390/fermentation7020060
Singh R, Gupta N, Goswami VK, Gupta R (2006) A simple activity staining protocol for lipases and esterases. Appl Microbiol Biotechnol 70:679–682. https://doi.org/10.1007/s00253-005-0138-z
Timar AV (2010) Comparative study of kefir lactic microflora. An le Univ Ńii din Oradea Fascicu- la Ecotoxicologie, Zooteh si Tehnol Ind Aliment
Vero S, Garmendia G, González MB, Bentancur O, Wisniewski M (2013) Evaluation of yeasts obtained from Antarctic soil samples as biocontrol agents for the management of postharvest diseases of apple (Malus × domestica). FEMS Yeast Res 13:189–199. https://doi.org/10.1111/1567-1364.12021
Witthuhn RC, Schoeman T, Britz TJ (2005) Characterisation of the microbial population at different stages of Kefir production and Kefir grain mass cultivation. Int Dairy J 15:383–389. https://doi.org/10.1016/j.idairyj.2004.07.016
Zhimo VY, Biasi A, Kumar A, Feygenberg O, Salim S, Vero S, Wisniewski M, Droby S (2020) Yeasts and bacterial consortia from kefir grains are effective biocontrol agents of postharvest diseases of fruits. Microorganisms 8:428. https://doi.org/10.3390/microorganisms8030428
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This work was supported by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP: 0819), Universidad Nacional de La Plata (11 × 838), Agencia Nacional de Promoción Científica y Tecnológica (PICT 2018 03702) and Programa Iberoamericano de Ciencia y Tecnología para el desarrollo (CYTED 121RT0110).
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Moure, M.C., Pérez Torrado, R., Garmendia, G. et al. Characterization of kefir yeasts with antifungal capacity against Aspergillus species. Int Microbiol 26, 361–370 (2023). https://doi.org/10.1007/s10123-022-00296-z
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DOI: https://doi.org/10.1007/s10123-022-00296-z