PMKT2, a new killer toxin from Pichia membranifaciens, and its promising biotechnological properties for control of the spoilage yeast Brettanomyces bruxellensis

Abstract

Pichia membranifaciens CYC 1086 secretes a killer toxin (PMKT2) that is inhibitory to a variety of spoilage yeasts and fungi of agronomical interest. The killer toxin in the culture supernatant was concentrated by ultrafiltration and purified to homogeneity by two successive steps, including native electrophoresis and HPLC gel filtration. Biochemical characterization of the toxin showed it to be a protein with an apparent molecular mass of 30 kDa and an isoelectric point of 3.7. At pH 4.5, optimal killer activity was observed at temperatures up to 20 6C. Above approximately this pH, activity decreased sharply and was barely noticeable at pH 6. The toxin concentrations present in the supernatant during optimal production conditions exerted a fungicidal effect on a variety of fungal and yeast strains. The results obtained suggest that PMKT2 has different physico-chemical properties from PMKT as well as different potential uses in the biocontrol of spoilage yeasts. PMKT2 was able to inhibit Brettanomyces bruxellensis while Saccharomyces cerevisiae was fully resistant, indicating that PMKT2 could be used in wine fermentations to avoid the development of the spoilage yeast without deleterious effects on the fermentative strain. In small-scale fermentations, PMKT2, as well as P. membranifaciens CYC 1086, was able to inhibit B. bruxellensis, verifying the biocontrol activity of PMKT2 in simulated winemaking conditions. INTRODUCTIONWorldwide, microbial growth destroys large amounts of various products, causing yield losses in the agronomical and biotechnological industries. Traditionally, biocides have been used to deal with these problems but different disadvantages such as establishment of resistant strains and suppression of natural competitors have made alternatives such as biological control necessary (Beever et al., 1989;Raposo et al., 2000). Biological control strategies include natural plant-and animal-derived compounds, as well as antagonistic micro-organisms (Ciani & Fatichenti, 2001).During recent decades, microbiological control of spoilage micro-organisms has evolved as a possibility. Many yeast strains and other micro-organisms inhibiting plant pathogens have been reported, especially within the fruit-and vegetable-producing sector, and several new products have reached the commercial market (Janisiewicz & Korsten, 2002). The suggested modes of action of biocontrol yeasts are not likely to constitute any hazard for the consumer (Janisiewicz et al., 2001;Masih & Paul, 2002;Comitini et al., 2004;Santos & Marquina, 2004a).The food and beverage industries were among the first to explore the application of killer-toxin-producing yeasts to kill spoilage micro-organisms (Lowes et al., 2000). Yeast strains often achieve competitive advantage by producing killer toxins, which kill off competing sensitive cells belonging to either the same or a different species (Young, 1987;Ciani & Fatichenti, 2001). The most thoroughly studied examples are the Saccharomyces cerevisiae toxins K1, K2 and K28; producers of these toxi...

Mediated amperometry reveals two distinct modes of yeast responses to glucose

“…These strains secreted a red pigment (probably also pulcherrimin; the composition is now under examination) and produced as-yet unidentified substances with killer effect. Certain yeasts have the ability to secrete low molecular weight glycoproteins (killer toxins) [30][31][32][33], which are lethal to receptive cells. Therefore, yeasts with killer effect can prevent the proliferation of wild sensitive yeasts and could be useful for developing microbiological protection of food against spoilage microorganisms.…”

Leveduras no rúmen de caprinos e bovinos de corte criados em pastagem tropicais

“…Estudos devem avaliar a participação dessa levedura na microbiota autóctone ruminal de caprinos, considerando-se um possível potencial probiótico ou patogênico oportunista no ecossistema ruminal. Recentes estudos indicam que essa toxina killer tem atividade reduzida em pH abaixo de 6 e efeito fungicida contra determinados fungos, entretanto já foi constatada a inatividade contra a espécie Saccharomyces cerevisiae (Santos et al, 2009) .…”

“…Estudos devem avaliar a participação dessa levedura na microbiota autóctone ruminal de caprinos, considerando-se um possível potencial probiótico ou patogênico oportunista no ecossistema ruminal. Recentes estudos indicam que essa toxina killer tem atividade reduzida em pH abaixo de 6 e efeito fungicida contra determinados fungos, entretanto já foi constatada a inatividade contra a espécie Saccharomyces cerevisiae (Santos et al, 2009) .A espécie Candida tropicalis tem sido relatada na literatura como prevalente em quadros patogênicos em humanos imunocomprometidos (Lima et al, 2008). Com isso, evidencia-se ainda a importância desses microrganismos em casos de saúde pública.…”

Killer yeasts as biocontrol agents of spoilage yeasts and bacteria isolated from wine

“…Bibliographic reports frequently make reference to the inhibitory effect of one killer strain or its toxin acting on sensitive or spoilage yeasts [11][12][13]. This is the first report about the effect of combined killer strains and its toxins in the inhibitory activity against spoilage yeasts as a strategy to increase the inhibitory capacity of a starter culture.…”