The occurrence of killer, sensitive, and neutral yeasts in Brazilian Riesling Italico grape must and the effect of neutral strains on killing behaviour

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Abstract
The occurrence of killer toxins amongst yeasts in Brazilian Riesling Italico grape must was investigated by using the sensitive strain EMBRAPA-26B as a reference strain at 18°C and 28°C. From a total of 85 previously isolated yeasts, 21 strains showed ability to kill the sensitive strain on unbuffered grape must/agar (MA-MB) and 0.1 M citrate/phosphate-buffered yeast extract/peptone/dextrose/agar (YEPD-MB) media both supplemented with 30 mg/l methylene blue. The killer activity of only four yeasts depended on the incubation temperature rather than the medium used. At 28°C, the strains 11B and 53B were not able to show killer action. On the other hand, strains 49B and 84B did not kill the sensitive yeast at 18°C. The killer strain EMBRAPA-91B and a commercial wine killer yeast K-1 were employed to examine the sensitivity of the isolated yeasts on YEPD-MB and MA-MB at 18°C. The sensitivity and neutral characteristics of yeasts were shown to be dependent on the medium and the killer strain. Interactions, including K- R-, K- R+ and K+ R+ strains, simultaneously, have revealed that some K-R+ strains appear to protect the K- R- strain against the killer toxin. Sensitive dead cells, although to a less extent, also exhibited similar protection. Kinetic studies have shown that the maximum specific growth rates were higher for the 20B YEPD-MB-sensitive strain (μmax=0.517 h-1) than for both the 91B (μmax=0.428 h-1) and K-1 (μmax= 0.466 h-1) killer strains. The protective capacity of neutral or sensitive cells that contaminate a fermentation, as well as the higher maximum specific growth rate of sensitive yeasts, besides other factors, may preclude the dominance of a killer strain. This protective capacity may also reduce the risk of a sensitive inoculum being killed by wild-type killer yeasts in open non-sterile fermentation.

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