The Use of Candida pyralidae and Pichia kluyveri to Control Spoilage Microorganisms of Raw Fruits Used for Beverage Production.

Undesired fermentation of fruit-derived beverages by fungal, yeast and bacterial spoilage organisms are among the major contributors of product losses in the food industry. As an alternative to chemical preservatives, the use of Candida pyralidae and Pichia kluyveri was assessed for antimicrobial activity against several yeasts (Dekkera bruxellensis, Dekkera anomala, Zygosaccharomyces bailii) and fungi (Botrytis cinerea, Colletotrichum acutatum and Rhizopus stolonifer) associated with spoilage of fruit and fruit-derived beverages. The antagonistic properties of C. pyralidae and P. kluyveri were evaluated on cheap solidified medium (grape pomace extract) as well as on fruits (grapes and apples). Volatile organic compounds (VOCs) from C. pyralidae and P. kluyveri deemed to have antimicrobial activity were identified by gas chromatography-mass spectrometry (GC-MS). A cell suspension of C. pyralidae and P. kluyveri showed growth inhibition activity against all spoilage microorganisms studied. Direct contact and extracellular VOCs were two of the mechanisms of inhibition. Twenty-five VOCs belonging to the categories of alcohols, organic acids and esters were identified as potential sources for the biocontrol activity observed in this study. This study reports, for the first time, the ability of C. pyralidae to inhibit fungal growth and also for P. kluyveri to show growth inhibition activity against spoilage organisms (n = 6) in a single study.

Grape Pomace Extracts as Fermentation Medium for the Production of Potential Biopreservation Compounds.

Microbial spoilage causes food losses in the food industry and as such, the use of synthetic chemical preservatives is still required. The current study proposes the use of agro-waste, i.e., grape pomace extracts (GPE), as production medium for biopreservation compounds. Production kinetics, subsequent to optimization using response surface methodology (RSM) for biopreservation compounds production was studied for three yeasts using GPE broth as a fermentation medium. The results showed that the highest volumetric zone of inhibition (VZI) was 1.24 L contaminated solidified media (CSM) per mL biopreservation compounds used (BCU) when Candida pyralidae Y1117 was inoculated in a pH 3-diluted GPE broth (150 g L-1) incubated at 25 °C for 24 h. Similar conditions were applied for Pichia kluyveri Y1125 and P. kluyveri Y1164, albeit under slightly elongated fermentation periods (up to 28 h), prior to the attainment of a maximum VZI of only 0.72 and 0.76 L CSM mL-1 ACU, respectively. The potential biopreservation compounds produced were identified to be isoamyl acetate, isoamyl alcohol, 2-phenyl ethylacetate and 2-phenyl ethanol.

Kinetic modelling and optimisation of antimicrobial compound production by Candida pyralidae KU736785 for control of Candida guilliermondii.

Biological antimicrobial compounds from yeast can be used to address the critical need for safer preservatives in food, fruit and beverages. The inhibition of Candida guilliermondii, a common fermented beverage spoilage organism, was achieved using antimicrobial compounds produced by Candida pyralidae KU736785. The antimicrobial production system was modelled and optimised using response surface methodology, with 22.5 ℃ and pH of 5.0 being the optimum conditions. A new concept for quantifying spoilage organism inhibition was developed. The inhibition activity of the antimicrobial compounds was observed to be at a maximum after 17-23 h of fermentation, with C. pyralidae concentration being between 0.40 and 1.25 × 109 CFU ml-1, while its maximum specific growth rate was 0.31-0.54 h-1. The maximum inhibitory activity was between 0.19 and 1.08 l contaminated solidified media per millilitre of antimicrobial compound used. Furthermore, the antimicrobial compound formation rate was 0.037-0.086 l VZI ml-1 ACU h-1, respectively. The response surface methodology analysis showed that the model developed sufficiently described the antimicrobial compound formation rate 1.08 l VZI ml-1 ACU, as 1.17 l VZI ml-1 ACU, predicted under the optimum production conditions.

Candida pyralidae killer toxin disrupts the cell wall of Brettanomyces bruxellensis in red grape juice.

AIMS: The control of the wine spoilage yeast Brettanomyces bruxellensis using biological methods such as killer toxins (instead of the traditional chemical methods, e.g. SO2 ) has been the focus of several studies within the last decade. Our previous research demonstrated that the killer toxins CpKT1 and CpKT2 isolated from the wine yeast Candida pyralidae were active and stable under winemaking conditions. In this study, we report the possible mode of action of CpKT1 on B. bruxellensis cells in red grape juice. METHODS AND RESULTS: Brettanomyces bruxellensis cells were exposed to CpKT1 either directly or through co-inoculation with C. pyralidae. This exposure yielded a temporary or permanent decline of the spoilage yeast population depending on the initial cell concentration. Scanning electron microscopy revealed cell surface abrasion while propidium iodide viability staining showed that CpKT1 caused plasma membrane damage on B. bruxellensis cells. Our data show that the exposure to CpKT1 resulted in increased levels of ß-glucan, suggesting a compensatory response of the sensitive cells. CONCLUSIONS: The toxin CpKT1 causes cell membrane and cell wall damage in B. bruxellensis. SIGNIFICANCE AND IMPACT OF THE STUDY: Candida pyralidae shows potential to be used as a biocontrol agent against B. bruxellensis in grape juice/wine.

Characterization of novel killer toxins secreted by wine-related non-Saccharomyces yeasts and their action on Brettanomyces spp.

Wine spoilage associated with Brettanomyces bruxellensis is a major concern for winemakers. An effective and reliable method to control the proliferation of this yeast is therefore of utmost importance. To achieve this purpose, sulphur dioxide (SO2) is commonly employed but the efficiency of this chemical compound is subject to wine composition and it can elicit allergic reactions in some consumers. Biological alternatives are therefore actively sought. The current study focused on identifying and characterizing killer toxins which are antimicrobial compounds that show potential in inhibiting B. bruxellensis in wine. Two killer toxins, CpKT1 and CpKT2, from the wine isolated yeast Candida pyralidae were identified and partially characterized. The two proteins had a molecular mass above 50kDa and exhibited killer activity against several B. bruxellensis strains especially in grape juice. They were active and stable at pH3.5-4.5, and temperatures between 15 and 25°C which are compatible with winemaking conditions. Furthermore, the activity of these killer toxins was not affected by the ethanol and sugar concentrations typically found in grape juice and wine. In addition, these killer toxins inhibited neither the Saccharomyces cerevisiae nor the lactic acid bacteria strains tested. These preliminary results indicated that the application of these toxins will have no effect on the main microbial agents that drive alcoholic and malolactic fermentations and further highlight the potential of using these toxins as agents to control the development of B. bruxellensis in grape juice or wine.