Optimization of killer assays for yeast selection protocols

International Journal of Food Microbiology, 2008

In this work 51 yeasts strains isolated from seasoned green table olives and belonging to the Candida, Debaryomyces, Kluyveromyces, Pichia, and Saccharomyces genera were characterized by their killer activity in different conditions. Killer activity of isolates was analyzed in a medium with different pH's (3.5 to 8.5) and NaCl concentrations (5, 8, and 10%). At every pH tested, all the genera studied had killer strains, although the smallest percentages of killer yeasts were found at the highest pH (8.5). The presence of 5 and 8% NaCl increased the detected killer percentage, but the highest salt concentration (10%) decreased it. The interaction between the reference killer yeasts and yeasts isolated from olives was analyzed. Most isolates were killer-sensitive to one or more killer reference strains. Only 2 of the 51 strains tested were considered killer-neutral. Crossreaction trials between isolates and spoilage yeasts showed that, of the isolates, nine killer strains, belonging to Debaryomyces hansenii, Kluyveromyces marxianus, Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae, had the broadest spectra of action against yeasts that cause spoilage. These killer yeasts and the toxins that they produce are candidates for further investigation as suppressors of indigenous olive table yeast growth. The results confirmed the highly polymorphic expression of the killing activity, with each strain showing different killer activities. This method may thus be very useful for simple and rapid characterization of yeast strains of industrial interest.

Using sensitive Pichia strains viz., Y246-P. fabianii, YF42-P. heimii, Y206-P. jadinii, Y18-P. strasburgensis, Killer and Sensitive Pattern (KSP) was screened in 553 isolates of yeasts belonging to 89 species and 31 genera by cross reactions. Apart from ascomycetous yeasts killer phenomenon was significantly observed in certain basidiomycetous yeast species such as Bullera pseudoalba, B. pyricola, Crytococcus albidus, C. laurentii, C. macerans, Fibulobasidium inconspicuum and Sporidiobolus ruineniae against these sensitive Pichia strains. Among all the tested sensitive strains, Y18-Pichia strasburgensis appeared as the most sensitive strain. Calculation of diversity in percentage clearly showed the occurrence of sensitivity of this strain in all cases. The phenomenon of killer activity appeared as strain character. It is inferred that the use of killer toxins as a taxonomic tool should be preceded by a careful study of their KSP.

Biotechnology Techniques, 1994

A method, based on the reaction of a sensitive strain being transferred to medium prefermented by a killer strain, is proposed for the quantitative determination of the "killer" activity in fermentation media by Saccharomyces cerevisiae K1. This technique enables killer activity to be closely followed throughout the duration of batch fermentation. The killer activity in the culture medium is represented by the percentage decrease in viable biornass in comparison with the viable biornass of a reference culture.

World Journal of Microbiology and Biotechnology, 2012

Killer yeasts are frequently used to combat and prevent contamination by wild-type yeasts during wine production and they can even dominate the wine fermentation. Stuck and sluggish fermentations can be caused by an unbalanced ratio of killer to sensitive yeasts in the bioreactor, and therefore it is important to determine the proportion of both populations. The aim of this study was to provide a simple tool to monitor killer yeast populations during controlled mixed microvinifications of killer and sensitive Saccharomyces cerevisiae.

Killer yeasts present in musts may dominate wine fermentations initially inoculated with sensitive wine yeasts. Such contaminations can lead to the premature cessation of alcoholic fermentation, high volatile acidity, H2S production, and off-flavors caused by fusel oils, acetaldehyde, and lactic acid. All of these factors can result in serious financial losses to the wine industry. However, the use of killer yeasts as starter cultures in the wine industry to prevent growth of sensitive wild yeast strains has been proposed. In this review paper, the killer phenomenon in Saccharomyces cerevisiae is discussed with emphasis on the significance of K 2 killer yeasts in winemaking. The seemingly conflicting reports on the interaction of killer and sensitive yeasts during grape must fermentations, are critically evaluated.

Spoilage yeasts generate considerable economic losses in the wine industry, and although sulphur dioxide (SO 2) is traditionally used for control, its use has become controversial because of its negative effects on health. Biocontrol has emerged as a partial alternative to SO 2 , and most research has focused on the selection of biocontrol yeasts and/or the mechanisms involved, while little research has been directed to the environmental conditions that make biocontrol effective for application. When there are two or more interacting yeasts, the physicochemical factors that affect their antagonism are many and therefore the application of biocontrol is complex. To reduce SO 2 , the present study aimed to elucidate biocontrol mechanisms of two yeast interactions and to establish optimal physicochemical conditions for biocontrol of the spoilage yeast during grape must fermentation. Through the use of statistical design, it was possible to find relevant physicochemical factors and optimise them. Wickerhamomyces anomalus "BWa156" developed an active supernatant against Zygosaccharomyces rouxii "BZr6" while supernatant from Metschnikowia pulcherrima "BMp29" was ineffective. In mixed must fermentations, the first interaction (BWa156 vs. BZr6) showed fewer physicochemical factors impacting biocontrol compared to the second interaction (BMp29 vs. BZr6). However, the fewer factors of the first interaction had a stronger effect on the decline in the spoilage population. Validations showed that the optimal conditions for biocontrol with the first interaction could be predicted. Analysis of the results with BWa156 vs. BZr6 and BMp29 vs. BZr6 suggests that the first interaction is a competition that includes a killer toxin, while the second interaction involves competition for iron resources. Response surface methodology (RSM) allowed a reduction in the number of experiments and permitted to find the optimal biocontrol conditions (SO 2 : 0 mg mL-1 ; pH: 3.7; Reducing sugars: 23 °Brix) for the interaction between BWa156 and BZr6. k e y w o r d s Zygosaccharomyces rouxii, Brettanomyces bruxellensis, Metschnikowia pulcherrima, Wickerhamomyces anomalus, Grape must fermentation, SO 2 reduction, Healthy wines.

Brazilian Archives of Biology and Technology, 2004

Twenty-four yeasts out of 342 isolated from the fermentative process showed killer activity and three of them were selected for the fermentative efficiency evaluation in batch system with cell recycle, flask and fermentor experiments. The selected three killer strains did not present similar results to those of pressed (baking) yeast concerning ethanol (0. 07-0.18; 0.12-0.20; 0.10-0.13; 0.22-0.25 g/g, respectively) and biomass (0.19-0.26; 0.33-0.39; 0.13-0.27; 0.47-0.61 g/g, respectively) yields and fermentative efficiency (12.3-36.3; 21.0-40.0; 19.3-26.3; 47.6-54.0 %, respectively) in sugarcane juice, in flasks. In fermentor, similar behaviour was observed. However, the selected strains showed high cellular viability a nd killer activity (using cell-free filtrate) along the fermentative cycles, in spite of the unfavourable conditions of the medium, like high pH variation of the medium (from 5.5-6.0 to 3.0-4.0), low aeration and higher temperature (30 0 C), which were not the ideal ones for the production/activity of killer toxins. A Pichia strain (CCA 510) showed the best results among the killer yeasts tested, exhibiting a killer activity against 92% of isolated fermentative yeasts of the process and against the pressed (baking) ferment. It also demonstrated killer activity (using crude toxin preparation) at higher temperatures (38 0 C) and low pH (4.0) after 72 hours of incubation, under proliferative and non-proliferative conditions. The results indicated that the killer activity should be a characteristic to be looked for in the strain selection for ethanolic fermentation, beside other important productivity-based characteristics, since it assure the permanence of the selected strain during the process.

Killer toxins are proteinaceous compounds that could be considered as biological alternative to 23 sulphur dioxide for the prevention of wine spoilage by undesirable wild yeast. The current study 24 investigated the influence of crude killer toxins secreted by Wickerhamomyces anomalus and Pichia 25 membranifaciens strains on the fermentation process and chemical composition of apple wines. The 26 main oenological parameters (ethanol, extract, total sugars, reducing sugars, titratable acidity) of 27 obtained apple wines as well as selected volatile compounds and organic acids were analysed. It was 28 revealed that the application of crude killer toxins to apple juices inoculated with Saccharomyces 29 strains did not significantly change the fermentation kinetics, however, in most of the cases, the apple 30 wines were distinguished by slightly higher concentration of ethyl alcohol compared to the control 31 samples fermented without killer toxins. The volatile acidity in analysed wines depended on yeast 32 strain used in fermentation rather than the type of killer toxin. It was also found that the addition of 33 crude toxins slightly changed levels of several aroma components, however, the yeast strains used for 34 fermentation process contributed considerably to variations in profiles and concentrations of volatile 35 compounds. 36 37 Keywords 38 killer toxin, fermentation, apple wine, volatile compounds, Saccharomyces cerevisiae 39 40 The uncontrolled growth of undesirable microorganisms can change chemical composition of 41 wine, disrupting the taste and aroma. Low pH, high ethanol concentration and oxygen deficiency 42 during fermentation process lead to the reduction or elimination of a population of certain 43 microorganisms [1, 2]. However, the growth inhibition of undesirable spoilage microflora is mainly 44 achieved by the addition of SO 2 to grape or apple musts. High doses of sulfur dioxide should be 45 limited primarily for health reasons, but also because of their effect on the taste and aroma of wine. On 46 the other hand, low levels of SO 2 do not ensure the stability of wine [3]. Excessive oxidation and 47 microbial growth may adversely influence the quality of wine. Application of killer yeasts and their 48 toxins seems to be one of possible ways to prevent the development of undesired microorganisms [4, 49 5]. Killer yeasts secrete toxins (usually proteins or glycoproteins) which could substitute SO 2 in wine 50 production. The use of killer toxins during the pre-fermentation step could reduce SO 2 addition during 51 fermentation, and thus limit the quantity of this compound in the final product. Killer strains have been 52 regarded useful in biological control of spoilage yeasts and food preservation [4]. Starter cultures with 53 3 killer activity could be used to combat contaminating wild-type yeasts and filamentous fungi during 54 the production of wine, beer and bread [4]. 55 Killer toxins of Pichia spp. have broad spectrum of antifungal activity against wine spoilage 56 yeasts such as Brettanomyces bruxellensis, Hanseniaspora uvarum and Zygosaccharomyces [6, 7, 8, 57 9]. In this context, it is interesting to investigate whether the use of killer toxins against wild-type 58 yeast can negatively influence alcoholic fermentation and metabolic activity of killer-resistant starter 59 cultures. The aim of this study was to determine the impact of Wickerhamomyces anomalus (formerly 60 Pichia anomala) and Pichia membranifaciens crude toxin preparations on killer-resistant strains of 61 Saccharomyces cerevisiae during the fermentation of high-sugar apple juice and on the chemical 62 composition of obtained apple wines. 63 64 MATERIALS AND METHODS 65 Microorganisms and media 66 Killer yeast strains of W. anomalus CBS 1982, W. anomalus CBS 5759 and P. membranifaciens 67 CBS 7373 were provided from the CBS-KNAW Fungal Biodiversity Centre (Utrecht, the 68 Netherlands). Hanseniaspora uvarum DSM 2768 was purchased from the Leibniz Institute DSMZ-69 German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Yeast cultures 70 were maintained at 4 °C on yeast extract-peptone-dextrose (YEPD) growth medium containing 1% 71 yeast extract, 2% peptone, 2% glucose and 2% agar. 72 Dry active wine yeasts (Challenge Aroma White, ES 181 and Challenge Vintage White supplied by 73 Enartis (Novara, Italy) were used for the fermentation process. Yeast were weighed and suspended in 74 ten volumes of sterile water, and then the suspension was added to the apple juice. 75 76 Preparations of crude extracts of killer toxins 77 Killer yeast strains were cultured on YEPD agar for 24 h at 25 °C. Next, they were transferred into 78 50 ml of YEPD liquid medium and incubated at 25 °C for 24 h. At the next stage 125 ml of YEPD 79 liquid medium adjusted to pH 4.5 with 100 mmol·l -1 citrate-phosphate buffer was inoculated with the 80 killer strain to the final yeast cells concentration (dry matter) of 2 g⋅l -1 . Killer cultures were cultivated as described previously [10, 11] at 20-22 °C with shaking at 2 Hz on a laboratory shaker (Elpin Plus 82 358A, Lubawa, Poland). When yeast cells reached the stationary phase they were removed by 83 centrifugation (1400 ×g, 15 min, 4 °C) and the culture supernatant was filtered through and 0.2 µm 84 cellulose acetate membranes (Sartorius, Goettingen, Germany). The filtrate was concentrated about 85 100-fold by using the Centricon Plus-70 centrifugal filter device (Merck Millipore, Darmstadt, 86 Germany) with a molecular weight cutoff 10 kDa and 30 kDa. 87 88 Killer activity assay 89 Killer activity of crude toxin preparations was checked by the agar diffusion well method. Cells of 90 sensitive yeast strain were grown for 24 h at 25 °C on YEPD agar slants. Next, the suspension of 91 sensitive yeast in sterile water was mixed with molten YEPD-MB medium containing 1% yeast 92 extract, 2% peptone, 2% glucose, 3% sodium chloride, 2% agar, 0.003% methylene blue and adjusted 93 to pH 4.5 with 100 mmol·l -1 citrate-phosphate buffer. The plates were seeded with the sensitive yeast 94 strain to a final concentration of approximately 2 × 10 5 cells per millilitre of the assay medium. Toxin 95 samples of 70 µl were put into wells (5-mm diameter) and inoculated plates were incubated at 20 °C 96 for 72 h. The appearance of a clear zone of growth inhibition bounded by bluish-stained cells was 97 recorded as the presence of killer activity. 98 99 Determination of protein concentration 100 Protein concentration was determined according to the method of BRADFORD [12] using bovine 101 serum albumin as standard. 102 103 Fermentation conditions

Brazilian Archives of Biology and Technology, 2010

The aim of this work was to study the in vitro antibacterial activity possessed by killer yeast strains against bacteria contaminating alcoholic fermentation (Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides), in cell X cell and cell X crude toxin preparations. The bacteria were not inhibited by any S. cerevisiae killer strains (5 out of 11). The inhibition caused by two crude toxin preparations (Trichosporon figueirae and Candida sp) against L. plantarum was surprisingly high but not in the same extent for B. subtilis, especially with three killer strains (Candida glabrata, Pichia anomala and Candida sp). L. mesenteroides and L. fermentum strains were neither inhibited in cell X cell nor crude toxin X cell tests. The results suggested that killer activity of yeasts might operate over bacteria and it could be used for the biocontrol of contaminating bacteria from alcoholic fermentation if additional tests on toxin application in fermentation shown to be successful. A wider panel of S. cerevisiae killer strains should be used to confirm that they were really unable to control the growth of these Gram-positive bacteria.

OENO One

SO2 is traditionally used to limit or nullify the proliferation of spoilage yeasts in must/wine, but its use has become controversial due to its negative effects on human health. An alternative strategy for the control of spoilage yeasts is the use of biocontrol yeasts. Wickerhamomyces anomalus is an outstanding biocontroller yeast that has been examined by our group and that is a good biocontrol agent against Brettanomyces bruxellensis and Zygosaccharomyces rouxii. The challenge regarding the application of the results of microbial biocontrol studies lies in exploring technologically simple and economical ways to make use of microbial biocontrol activity. The introduction of foreign microbiota adds complexity to the fermentation medium and can cause technological difficulties and have oenological consequences inherent to the implementation of mixed cultures. Therefore, the objective of this study was to characterise the concentrated culture supernatant of W. anomalus and evaluate i...