sensitive yeast interactions

JOURNAL OF FERMENTATION AND BIOENGINEERING

Vol. 76, No. l, 67-69. 1993

Stuck Wine Fermentations: Effect of Killer/Sensitive Yeast Interactions FRANCISCO M. CARRAU, 1. EDISON NEIROTTI, 2 ~a~D OCTAVIO G I O I A l

Laboratory of Research & Development, Bodegas Castel Pujol, Cesar M. Gutierrez 2556, 12.400,1 and Laboratory of Microbiology, Faculty of Science, University of the Republic, Tristan Narvaja 1674,2 Montevideo, Uruguay Received 28 September 1992/Accepted 21 April 1993 Mixed-strain fermentations were carried out with different proportions of killer/sensitive yeasts. It was observed that stuck and sluggish fermentations due to killer/sensitive strain interaction depended on: a. the killer/sensitive yeast proportion at the start of fermentation, b. the concentration of nitrogen source in the grape must, and c. the presence of bentonite during fermentation. The effect of the addition of active carbon and commercial yeast cell walls to sluggish fermentation media was also studied.

Killer yeasts strains (phenotype K+R +) produce an extracellular toxin that kills other yeasts, called sensitive strains (phenotype K - R - ) . Neutral yeasts (phenotype K - R +) that are resistant to the killer toxin but do not produce it also exist (killer yeast and its relationship to winemaking are described in detail by Shimizu (1). The killer phenomenon in yeast was discovered in 1963 by Bevan and Makower (Proc. Intl. Congr. Genet. XI, 1, 202-203, 1963). In the genus Saccharomyces, killer strains (types K1, K2 and K3), harbor virus-based killer systems. These yeast viruses contain double-stranded RNA genomes (M1, M2 and M3), which encode both immunity and toxin precursors (2). Usually these viruses are advantageous to the cell, although in some special conditions they could be pathogenic (3). In the last decade genetic and molecular approaches have been applied to an elucidation of these systems in yeasts (4, 5). The killer type K2 is common in winery ecosystems (6), with its occurrence being now well confirmed in several regions (7-14). However, it has been suggested that the killer factor which is present in oenologicai strains may not be expressed efficiently in the pH conditions of wine fermentation (15, 16). The optimum pH for the yeast killer toxins is generally between 4.0 and 4.5, a characteristic that may not represent enough of an advantage for the dominance of killer strains in wine fermentations. In spite of these suggestions, several researchers have proved the dominance of killer strains in laboratory wine fermentations by mixed cultures at pH 3.0 to 3.5, using indirect methods (17-20). Recently, direct approaches were developed for quantitative measurement of the interactions between killer and sensitive yeasts, using differences in colony morphology (12) and colony colours (13), and genetically marked strains (21). Stuck wine fermentation is one of the most important problems in the wine industry (15, 22). Several causes of stuck and sluggish wine fermentation have been described (16, 22-26). As expected, killer toxins inhibit wine fermentations of sensitive yeasts (I 1, 27). In our laboratory, we have observed that a certain killer/sensitive yeast proportion at the start of fermentation may cause stuck wine fermentations (Carrau, F. et al., Abstract, 6th. Intl. Symp. Genet. Industr. Microorg. Strasbourg, France, p . 6 , 1990), but the characteristics of this phenomenon have

not been well understood. In this work we examined this problem more closely, studying mixed-culture wine fermentations. The competition between selected killer strains (type K2) and sensitive commercial strains was studied under different conditions to elucidate the possible causes of stuck and sluggish wine fermentations. Saccharomyces cerevisiae, wine strain Montrachet 522 (University of California) and OC 2, IAM 4274 (Japan), were used as sensitive strains for mixed cultures. Wine Yeasts, S. cerevisiae KU1 (our laboratory) and K1 (Montpellier, ICV) were used as K2 type killer strains. Semillon grape juice (pH3.2, total sugar content: 156 g//) sterilized by filtration through a membrane filter (pore size 0.45 pro) was used as a fermentation medium. Killer/sensitive yeast proportions were confirmed in solid medium WL Nutrient (Difco), on which OC 2 and KU1 formed white colonies, while Montrachet and K1 formed green ones. All fermentations were conducted at 20°C in 50ml (stopped with Muller valves) of the fermentation medium inoculated with 105 cells/ml of killer/sensitive strains in the exponential phase. The killer/sensitive proportions in the inoculated strains varied from 1 : 1 to 1 : 10,000. Single-strain fermentations were also carried out as control tests. Each fermentation was carried out in duplicate. Residual sugars were analyzed (28) throughout the fermentations and 5 d after the end of fermentation. Loss of carbon dioxide was measured by weight loss and the numbers of dead cells were counted by the methylene blue-staining technique. A proportion of 1 : 100 was selected to study the effect of different nutrients, growth factors and adsorbant substances. The following concentrations were used for the investigation: ammonium sulfate 100mg/l, peptone 100 mg/l, Roviferm (nitrogen source with vitamins of Roche) 200 mg/l, activated carbon 2 g/l, bentonite 200 mg/l and yeast cell walls (Springer-Fould, France) 200 mg/l. Competition between killer and sensitive (K : S) strains in mixed-strain fermentations is shown in Fig. 1. In the case of a K : S proportion of 1 : 1 , sensitive ceils were killed by KU1 within 24 h (Fig. l a) while approximately 5% of the population of Montrachet remained viable with a K : S inoculum proportion of 1 : 10 (Fig. lb). Figure lc shows that in the case of a K : S proportion of 1 : 100, approximately 75% of the total cell population was sensitive strains at the end of fermentation. However, these

* Corresponding author. 67

68

CARRAU ET AL.

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cells were not active enough to finish the fermentation (Fig. 2). Occurrence o f stuck fermentations was investigated under different inoculum conditions. Figure 2 shows that the highest residual sugar concentration was obtained with a K : S proportion o f 1 : 100 and that the residual sugar concentration decreased not only with K/S ratios higher than 1 : 100 but also with lower ones. It was also found that a higher percentage o f dead cells was observed with a proportion of 1 : 100 (82%) than with proportions of 1 : 1 (22%) and 1 : 10,000 (21°/~), respectively (data not shown). These observations, and the effect o f a m m o n i u m addition as shown in Table 1, led to the suggestion that dead cells m a y n o t release some nutrients into the m e d i u m and that nutrient depletion in the m e d i u m m a y not permit killer cells to finish the fermentation. This suggestion was also s u p p o r t e d by the facts that sensitive cells that were killed b y K1 killer toxin did not lyse and that m a c r o m o lecules were not released from the killed cells (29). Table 1 shows that stuck fermentations were avoided by the a d d i t i o n o f either a m m o n i u m sulfate or Roviferm, especially in the exponential phase, whereas the a d d i t i o n o f p e p t o n e exhibited little effect. Practically the same results were obtained b y the a d d i t i o n o f either bentonite or activated c a r b o n in the stationary phase, in which stuck or sluggish fermentations could generally be foreseen (Table 1). Since bentonite, a p r o t e i n - a d s o r b i n g material comm o n l y used in the wine industry, is k n o w n to inhibit killer toxin action (27, 30), it is suggested that the a b o v e effect is exhibited by a d s o r b a n c e o f the killer toxin. This suggestion is s u p p o r t e d by the fact that bentonite a d d i t i o n brought a b o u t an increased p r o p o r t i o n o f viable sensitive

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Product No addition (Control) Peptone 0.1 g/l Rf R 0.2 g/l Ammonium 0.1 g/l Bentonite 0.2 g/l YCW 0.2 g/l A. Carbon 2 g/l

Added in: exponential phase-stationary phase residual sugar level (g//) 4.5 4.5 3.6 3.2 1.2 2.0 1.0 1.7 NT 1.2 NT 2.1 NT 1.1

Mixed-strain fermentations with a K : S proportion of 1 : 100 were carried out at 20°C either in the presence or absence of the various materials. Rf R (Roviferm, Roche); YCW (Yeast Cell Walls, Springer-Fould); NT (not tested); A. Carbon (activated carbon).

cells at the end of fermentation (data not shown). The addition of yeast cell walls in the stationary phase did not give as g o o d a result as those obtained with bentonite and a m m o n i u m (Table 1). A f t e r the a d d i t i o n o f a m m o n i u m , a p p r o x i m a t e l y 100% o f the viable cell p o p u l a t i o n was c o m p o s e d o f killer strains (data not shown). This observation suggests that the nitrogen source could have been a limiting factor for killer strain growth in these experiments. A m o r e detailed analysis o f the nitrogen levels and p H variation during ferm e n t a t i o n is now in progress in our l a b o r a t o r y for a better understanding o f this p h e n o m e n o n . In conclusion, K : S yeast interaction caused stuck or sluggish fermentation; this p h e n o m e n o n was mainly influenced by the following factors: (i) the K : S yeast p r o p o r t i o n at the start o f fermentation, (ii) a d d i t i o n o f a m m o n i u m or Roviferm into the grape must, and (iii) the presence o f bentonite during fermentation. In several situations, the a d d i t i o n o f substances comm o n l y used for sluggish fermentations is avoiding problems caused by K / S cell interaction. We believe that most o f these situations could be prevented b y studying the natural yeast communities present in the winery ecosystem and careful selection o f the yeast strains used. The results described above will be useful to avoid stuck or sluggish fermentation in industrial winemaking. The authors would like to thank Prof. Howard Bussey (McGill University, Montreal), Prof. Juan L. Carrau (IB, University of Caxias, Brazil) and Dra. Rosario Lagunas (IIB, CSIC, Madrid) for their critical comments on the manuscript. This research was supported by the Programme of R & D on "Native killer yeasts for wine fermentation" of Castel Pujol Winery. REFERENCES 1. Shimizu, K.: Killer yeasts, p. 243-264. In Fleet, G. H. (ed.), Wine microbiology and biotechnology. Harwood Academic Publishers, Switzerland 0993). 2. Wicimer, R.B.: Double-stranded RNA replication in yeast: the killer system. Annu. Rev. Biochem., 55, 373-395 (1986). 3. Esteban, R. and Wickner, R.B.: A new non-Mendelian genetic element of yeast that increases cytopathology produced by M1 double-stranded RNA in ski strains. Genetics, 117, 399-408 (1987). 4. Wickaer, R. B.: Yeast virology. FASEB J., 3, 2257-2265 (1989). 5. Polonelli, L., Coati, S., Gefloal, M., Chezzi, C., and Moraee, G.: Interfaces of the yeast killer phenomenon. Crit. Rev.

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7. 8. 9. 10. 11. 12. 13.

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NOTES

20.

21.

22. 23. 24.

25.

26. 27. 28. 29. 30.

69

cerevisiae sur une souche de levure sensible de la m~me esp~ce, non productrice de H2S et selection6e pour la vinification dans un milieu de culture mixte. Bull. O.I.V., 648--649, 214-217 (1985). Seki, T., Choi, E. H., and Ryu, D.: Construction of killer wine yeast strain. Appl. Environ. Microbiol., 49, 1211-1215 (1985). Petering, J.E., Symons, M.R., Langridge, P., and Hensehke, A.: Determination of killer activity in fermenting grape juice by using a marked Saccharomyces wine yeast strain. Appl. Environ. Microbiol., 57, 3232-3236 (1991). Lafon-Lafoureade, S.: Souches de levures. Bull. O.I.V., 57, 185203 (1984). Lafon-Lafourcade, S. and Joyeux, A.: Les bact~ries ac~tiques du vin. Bull. O.I.V., 608, 803-829 (1981). Lafon-Lafourcade, S., Lame, F., and Rih~reau-Gayon, P.: Evidence for the existence of (survival factors) as an explanation for some peculiarities of yeast growth, especially in grape must of high sugar concentration. Appl. Environ. Microbiol., 38, 10691073 (1979). Larue, F., Lafon-Lafonrcade, S., and Rib~reau-Gayon, P.: Relationship between the sterol content of yeast cells and their fermentation activity in grape must. Appl. Environ. Microbiol., 39, 808-811 (1980). Rib~reau-Gayon, P., Lafon-Lafoureade, S., and Bertrand, A.: Le debourbage de mouts de vendange blanche. Conn. Vigne. Vin., 9, 117-139 (1975). Radler, F. and Schmitt, M.: Killer toxins of yeasts: Inhibitors of fermentation and their adsorption. J. Food Protect., 50,234--238 (1987). Schales, O. and Schales, S. S.: A simple method for the determination of glucose in blood. Arch. Biochem., 8, 285-292 (1945). Bussey, H.: Physiology of killer factor in yeast. Adv. Microb. Physiol., 22, 93-122 (1981). Tiourina, L. V., Bourjan, N. I., and Skarikova: Emploi des cultures pures du ph~notype killer duns la fermentation des mouts de raisin. Bull. O.I.V., 53, 573-576 (1980).