Culture medium optimization for osmotolerant yeasts by use of a parallel fermenter system and rapid microbiological testing

• A culture medium for osmotolerant yeasts (OM) was developed in a parallel fermenter.
• A significant increase of the maximum CO2 transfer (CTR) rate was achieved.
• The maximum CTR for Z. rouxii and T. delbrueckii increased by a factor ≥ 2.6.
• W. anomalus was detected 69 h faster in OM in relation to growth in YEG 50.
• Inoculation tests in OM proved that detection times of 3 days can be realized.

In the present study, a culture medium for qualitative detection of osmotolerant yeasts, named OM, was developed. For the development, culture media with different concentrations of glucose, fructose, potassium chloride and glycerin were analyzed in a Biolumix™ test incubator. Selectivity for osmotolerant yeasts was guaranteed by a water activity (aw)-value of 0.91. The best results regarding fast growth of Zygosaccharomyces rouxii (WH 1002) were achieved in a culture medium consisting of 45% glucose, 5% fructose and 0.5% yeast extract and in a medium with 30% glucose, 10% glycerin, 5% potassium chloride and 0.5% yeast extract. Substances to stimulate yeast fermentation rates were analyzed in a RAMOS® parallel fermenter system, enabling online measurement of the carbon dioxide transfer rate (CTR) in shaking flasks. Significant increases of the CTR was achieved by adding especially 0.1–0.2% ammonium salts ((NH4)2HPO4, (NH4)2SO4 or NH4NO3), 0.5% meat peptone and 1% malt extract. Detection times and the CTR of 23 food-borne yeast strains of the genera Zygosaccharomyces, Torulaspora, Schizosaccharomyces, Candida and Wickerhamomyces were analyzed in OM bouillon in comparison to the selective culture media YEG50, MYG50 and DG18 in the parallel fermenter system. The OM culture medium enabled the detection of 102 CFU/g within a time period of 2–3 days, depending on the analyzed yeast species. Compared with YEG50 and MYG50 the detection times could be reduced. As an example, W. anomalus (WH 1021) was detected after 124 h in YEG50, 95.5 h in MYG50 and 55 h in OM bouillon. Compared to YEG50 the maximum CO2 transfer rates for Z. rouxii (WH 1001), T. delbrueckii (DSM 70526), S. pombe (DSM 70576) and W. anomalus (WH 1016) increased by a factor ≥ 2.6. Furthermore, enrichment cultures of inoculated high-sugar products in OM culture medium were analyzed in the Biolumix™ system. The results proved that detection times of 3 days for Z. rouxii and T. delbrueckii can be realized by using OM in combination with the automated test system even if low initial counts (101 CFU/g) are present in the products. In conclusion, the presented data suggest that the OM culture medium is appropriate for the enrichment of osmotolerant yeasts from high-sugar food products.