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

During the winemaking process Saccharomyces cerevisiae is the main yeast species but other yeasts called non-Saccharomyces as well as different species of lactic acid bacteria (LAB) are also present. Then, one strategy to prevent or reduce microbial contamination during the winemaking process is the use of killer yeasts. The aim of this study was to evaluate the killer activity (KA) of autochthonous yeasts from Northwest region of Argentine (S. cerevisiae Cf8 and Wickerhamomyces anomalus Cf20) on spoilage yeasts and in LAB of the wine. The KA was evaluated using cell-free supernatants obtained from pure and mixed cultures of strains Cf8-Cf20. S. cerevisiae Cf8 showed a growth reduction between 7 and 48% on D. anomala BDa15, P. membranifaciens BPm481 and Z. bailii Bzb317 while W. anomalus Cf20 exhibited KA of 20, 61, 91 and 92% against B. bruxellensis Ld1, D. anomala BDa15, P. membranifaciens BPm481 and P. guilliermondii Cd6, respectively. Killer mixed supernatants showed growth inhibition similar to strain Cf20. Screening against LAB showed that both killer toxins were able to inhibit the growth of L. hilgardii 5w as well as to reduce a 16–31% histamine production by this LAB strain. These results confirm the potential of autochthonous killer yeasts as biocontrol agents in winemaking process. The mixed culture S. cerevisiae Cf8-W. anomalus Cf20 presented a wide range of KA on spoilage yeasts as well as on L. hilgardii. Therefore, the use of killer yeasts as starter cultures would allow producing wines with controlled quality.


Introduction
During the winemaking process there is a predominance of the fermentative yeast Saccharomyces cerevisiae, which is used as starter culture of the alcoholic fermentation.However, during wine fermentation other yeasts called non-Saccharomyces as well as different species of lactic acid bacteria (LAB) are also present [1].Yeasts of the genera Dekkera/Brettanomyces as well as Pichia guilliermondii and P. membranifaciens are generally considered wine contaminants during the fermentation stage or post-fermentation due to the production of phenolic aromas [2].On the other hand, among wine LAB some species as Lactobacillus hilgardii and Pediococcus pentosaceus are also considered undesirable bacteria mainly for its ability to produce biogenic amines, compounds that affect the sanitary and sensory quality of wines [1].One strategy to prevent or reduce microbial contamination during the winemaking process is the use of killer yeasts as starter cultures.These yeasts produce proteic toxins that inhibit the growth of unwanted yeasts and fungi, but its effect on wine LAB has not been reported yet [1,3,4].The aim of this study was to evaluate the killer activity (KA) of autochthonous yeasts from Northwest region of Argentine, S. cerevisiae Cf8 and Wickerhamomyces anomalus Cf20 on different strains of spoilage yeasts and LAB of wine.

Strains and growth media
Yeasts and bacteria used in this study are listed in Tables 1 and 2, respectively.The autochthonous killer strains S. cerevisiae Cf8 and W. anomalus Cf20 were isolated from wineries of Northwestern region of Argentina.Spoilage yeasts were gently provided to us from Yeast collection of San Juan University (Argentina).LAB strains were obtained from Microbiology Institute of Tucuman National University (Argentina).
Depending on the experiments, the yeasts were cultured in: YPD broth (10 g/L yeast extract, 20 g/L peptone, 20 g/L glucose) buffered at different pH with 0.1 M citric acid/dibasic sodium phosphate; YPD-MB agar (YPD supplemented with 30 mg/L methylene blue, 20 g/L agar).Depending on the experiments bacteria were cultured in MRS broth, MRS agar or basal medium supplemented with histidine 1% (BM-Histidine) described by Joosten and Northolt [5].Media were sterilized by filtering or autoclaving at 121 • C for 20 min.Microorganisms were incubated at 28 • C.

Production of killer cell-free supernatants
Killer strains S. cerevisiae Cf8 and W. anomalus Cf20 of were inoculated at 1 × 107 cells/mL in pure and mixed cultures into YPD broth pH 4.0 and incubated for 96 h at 20 • C. Yeast cells were separated from the supernatants by

L.hi l gar di i Cf8 Cf20 P. pent osaceus
centrifugation at 10,000 g for 10 min.Control supernatant was obtained treating Cf20 cell-free supernatant at 100 • C for 1 h to denature killer toxin.

Inhibitory effect on wine spoilage yeasts
Spoilage yeasts were inoculated at 1 × 10 6 cell/mL in the killer-and control-cell-free supernatants.Cultures were incubated at 20 • C for 48 h and absorbance at 600 nm (A 600 ) was measured.Killer activity (KA) was expressed as % reduction of absorbance according to the following formula: Where A x is the absorbance of cultures in killer supernatants, and A C is the absorbance of cultures in control supernatants.

Inhibitory screening assay on wine lactic acid bacteria (LAB)
LAB strains (Table 2) were cultured as a lawn in MRS agar plates and 50 µL of killer cell-free supernatants concentrated 10× by ultrafiltration (30 kDa, Amicon) were spotted on the agar.Control supernatants (heat treated) were also spotted on the agar.Plates were incubated at 20 • C for 5-7 days.Inhibitory activity was considered positive when a clear zone with no growth was detected in the agar.

Effect of killer supernatants on growth and histamine production of Lactobacillus hilgardii 5w
Cultures of L. hilgardii 5w in MB-histidine supplemented with 10× killer-and control-cell-free supernantants (final 1× ) were carried out and incubated at 20

Inhibitory effect on wine spoilage yeasts
S. cerevisiae Cf8 supernatant produced a growth reduction between 7 and 48% on D. anomala BDa15, P. membranifaciens BPm481 and Z. bailii Bzb317 (Fig. 1A) while W. anomalus Cf20 exhibited KA of 20, 61, 91 and 92% against B. bruxellensis Ld1, D. anomala BDa15, P. membranifaciens BPm481 and P. guilliermondii Cd6, respectively (Fig. 1B).Killer mixed supernatants showed growth inhibition similar to strain Cf20 (data not shown).These results demonstrate the biocontrol potential of these killer strains on spoilage yeasts, noting that P. guilliermondii Cd6 is an autochthonous strain from northwestern region of Argentina as Cf8 and Cf20 strains.These findings are in concordance with the literature, which express that killer toxins from S. cerevisiae have narrow inhibition spectra, while those belonging from W. anomalus often present broad inhibition spectra [8][9][10].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.

Inhibitory effect on wine LAB
As shown in Table 2, screening assay of concentrated killer cell-free supernatants (> 30kDa) against LAB showed that Cf8 and Cf20 supernatants were able to inhibit 02001-p.2 the growth of L. hilgardii N4L and 5w, the latter a histamine-producing strain.When the inhibitory effect of Cf8 and Cf20 supernatants was evaluated on L. hilgardii 5w in liquid cultures, it was observed a longer lag phase and lower final growth (Fig. 2A) as well as a 16-31% decrease in biogenic amine production, being higher the inhibitory effect in the coculture supernatant (Fig. 2B).Several authors have reported the inhibitory effect of non-Saccharomyces killer yeast supernatants on different bacterial groups, mainly gram positive bacteria [14][15][16].
No studies about the mechanism of action of the killer toxins on prokaryotic cells have been performed.Although the toxic mechanisms remain unknown, the decrease on histamine production may be due to the growth inhibition of L. hilgardii.This is the first report of the effect of killer yeasts on wine-relevant LAB and the first report of the inhibitory effect of a S. cerevisiae killer strain on bacteria.The literature has extensively reported studies about the use of simultaneous and sequential starter cultures of Saccharomyces and non-Saccharomyces strains in order to improve the wine flavor and avoid the excessive production of aroma compounds [8,17,18].In contrast, this work propose the use a combination of a S. cerevisiae killer strain with remarkable enological properties as Cf8 [7] and a potent non-Saccharomyces killer strain to optimize The results of this work confirm the potential of autochthonous killer yeasts as biocontrol agents in winemaking process.The mixed culture S. cerevisiae Cf8-W.anomalus Cf20 presented a wide range of KA on spoilage yeasts as well as on L. hilgardii.Therefore, the use of killer yeasts as starter cultures would permit to produce wines with controlled quality.

Figure 2 .
Figure 2. Effect of killer supernatants of pure and mixed cultures of S. cerevisiae Cf8 and W. anomalus Cf20 on growth (A) and histamine production (B) of L. hilgardii 5w.

Table 1 .
Killer and sensitive yeast strains used in this study.
[7]enotype. a Strains isolated from Northwestern wineries[7].b Strains provided from Yeast Collection of San Juan University.

Table 2 .
Inhibitory effect of Cf8 and Cf20 killer supernatants on wine lactic acid bacteria.