Activité Killer chez des levures isolées des sols du Nord-Est Algérien

Abstract

The general purpose of this thesis is to search for killer activity in yeast strains isolated from agricultural and forest soil in the region of Constantine for biotechnological applications.The isolation of yeasts allows to list 15 strains. The molecular taxonomy based on the sequences of D1/D2 domain of the 26S ribosomal ARN gene groups these isolates into 6 different species: Cryptococcus aerius, Hanseniaspora opuntiae, Hanseniaspora uvarum, Pichia kluyveri, Meyerozyma guilliermondii and Saccharomyces cerevisiae. Morphological, physiological and biochemical characteristics of the strains are in agreement with those cited in literature. Preliminary testing for killer activity reveals that only the strain L5 is able to produce killer activity. The killer strain is belonging to the species P. kluyveri. The crude killer protein produced by the strain P. kluyveri reveals that the toxin is active against food and beverage spoilage yeast strains belonging to the genera: Dekkera, Kluyveromyces, Pichia, Saccharomyces, Torulaspora, Wickerhamomyces and Zygosaccharomyces. The highest killer activity is obtained against the strain Dekkera bruxellensis DBVPG 6706. Both the heat shock (30 min at 100°C) and the enzymatic treatment with pronase demonstrate the complete loss of the initial killer activity of the toxin, and hence its proteinaceous nature is apparently confirmed. The active protein migrates as a single band in SDS-PAGE and has a molecular mass of 54 kDa. The purified killer toxin has an optimal pH between 4.0-4.5, while the optimal of temperature is 25°C.The determination of minimum inhibitory concentrations (MICs) shows that the purified killer protein has a high in vitro activity against D.bruxellensis (MICs from 64 000- to 256 000-fold lower than that exhibited by potassium metabisulphite) and Saccharomyces cerevisiae (MICs from 32 000- to 64 000-fold lower than potassium sorbate). No in vitro synergistic interactions (calculated by FIC index -Σ FIC) are observed when killer protein is used in combination with the active compounds : potassium metabisulphite, potassium sorbate, or ethanol. The killer protein exhibited a dose–response effect against D. bruxellensis and S. cerevisiae in a soft drink « Campari MIXX » (Milan, Italy) and pear juice « Santal » (Parma, Italy), respectively. The results of the present study suggest that the killer protein could be proposed as a novel food-grade compound useful for the control of food and beverage spoilage yeasts. Additionally, the purified killer protein maintains its killing action at least for 3 days against S. cerevisiae DBVPG 6500 inoculated in pear juice and for 10 days against D. bruxellensis DBVPG 6706 in soft drink. The results of the present study suggest that the killer protein produced by the strain P. kluyveri (L5) could be proposed as a novel natural agent for the biocontrole of beverage spoilage yeasts.