A contribution to the ecology of fission yeasts on grapes (1977)

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This beautiful paper just crossed my desk, found by a colleague in New Zealand who has amassed quite a collection of fission yeasts. The translation was actually provided by another colleague and fission yeasts collector who is also among the very most knowledgable of their use in alcoholic fermentation.

The paper is quite interesting and shows that Schizosaccharomyces pombe is not just confined to the tropics but is basically in our backyard. What we see is that when fission yeasts were used to de-acidify wines by metabolizing malic acid, it wasn’t just a novel foreign yeast brought to wine but rather a hidden voice that was in many terroirs all along. The opinion is presented that little attention has been given to these yeasts because of the difficulty of isolation which relies on selective media constructed from exclusive ingredients. The authors propose a selecctive media, but others are known that are more accessible, however they may only work for certain substrates.

The paper contains one incredible detail not mentioned in other works, which we will not point out, but does offer a host of unique possibilities for beverage use. Fission yeasts have a lot of special metabolic features not yet fully elucidated that will open a lot of doors in industrial practice.

The authors use an older dialect and words like schizolytes basically mean fission yeasts but were not worth changing in the translation. Blastomycetic may simply imply yeast on grape skins although the term is not common anymore outside of dermatology.


Vitis 16, 38-44 (1977)

Istituto di Microbiologia Agraria e Tecnica dell’Università di Firenze, Italia
Direttore: Prof. G. FLORENZANO

A contribution to the ecology of fission yeasts on grapes
by
G. FLORENZANO, W. BALLONI and R. MATERASSI

Summary.—Results of a preliminary investigation on the distribution of fission yeasts on grapes in western Sicily, carried out with a new selective enrichment method for Schizosaccharomyces pombe are reported.

Fission yeasts have been found on 26 out of the 68 samples examined, belonging chiefly to the Zibibbo, Catarratto, Inzolia, Grillo and Grecanico cultivars. It has been shown that on Zibibbo grapes these yeasts are the main group among the epiphytic blastoflora.

Most of the 42 strains isolated from Sicilian grapes have been classified as Schizosaccharomyces pombe. Some of them show remarkable physiological and biochemical properties from the oenological point of view.

Results reported here show that, in the region considered in this work, fission yeasts must be included among the normal components of the microflora of grapes. On the other hand, it is reasonable to assume that the failure in the detection of Schizosaccharomyces on grapes by previous investigators should be ascribed to an inadequate experimental procedure.

Introduction

Most of the research on the zymology of grapes and musts has been driven by the need to acquire basic knowledge on the nature of the main agents responsible for wine fermentation in the various winemaking areas both in Italy and elsewhere (CASTELLI 1948, FLORENZANO 1949, VERONA 1951, PEYNAUD and DOMERCQ 1953, CASTELLI and IÑIGO LEAL 1958, MINÁRIK 1965, etc.). This required application in the isolation of the criterion of dominance and frequency of the species capable of rapid growth on ordinary yeast culture media. [This sentence needs better phrasing, but basically these early researchers could only describe what grew on ordinary media. If something had a low frequency of occurrence, they would miss it.]

Survey techniques used were consequently inadequate to provide a comprehensive picture of the complex ecology of yeasts on grapes and in the environment of the vineyard, such as that outlined in the detailed studies by DAVENPORT (1969, 1972, 1974) and in the researches of BELIN (1972). The latter has identified, by means of scanning electron microscopy, in lenticels, peduncles, stomata, etc., as many niches of elective settlement of certain blastomycetic forms, in correspondence of particular exudates. L’A. has formulated a critique of the traditional zymological study methods, as they are unable to highlight the presence of many species, which, even if secondary, can nevertheless constitute a significant portion of the entire blastomycetic population of grapes. [The singling out of lenticels, peduncles, stomata, imply that certain parts of the plant may harbor certain yeast.]

Among the blastomycetic species escaped from conventional zymological research, noteworthy are those of the genera Brettanomyces and Schizosaccharomyces. The yeasts Brettanomyces, reported on grapes from VERONA and FLORENZANO (1947) and FLORENZANO (1951, 1952), they were, in the following years, found in musts and wines from other AA [as above?]. (SCHANDERL and DRACZYNSKI 1952, PEYNAUD and DOMERCQ 1956, VAN DER WALT and VAN KERKEN 1958 etc.). But only with the development of a selective means by VAN DERWALT et al. (see PHAFF et al. 1966, p. 104-105) it became possible to evaluate the real diffusion of these yeasts in musts and wines.

A similar case occurred for the yeasts of the genus Schizosaccharomyces, which have aroused the oenological interest for the ability to ferment malic acid (PEYNAUD and SUDRAUD 1964, CASTELLI and HASNEDARI 1968, 1971, BALLONI et al. 1970, FLORENZANO 1972, BENDA 1974, BIDAN et al. 1974, HASNEDARI 1976). Such yeasts did not appear among the normal components of the blastomycetic microflora of grapes and musts, as their distribution seemed limited to very sugary substrates and tropical habitats, until the predominance of Schizosaccharomyces pombe was reported on Venezuelan grapes (BALLONI et al. 1966). Subsequently schizolievites (fission yeasts) were isolated from grape musts of Castelli Romani (BALLONI et al. 1970), from sediments of Tuscan wines of the Certaldo-San Gimignano area (FLORENZANO 1972) and, more recently, from wine samples from the Modena area (1975, unpublished finds).

Other reports have been made by BRUGIRARD (see BIDAN et al. 1974, p. 684) on French grape musts from the Eastern Pyrenees area, from RELAN and VYAS (1971) on grapes from the Indian region of Haryana and from DAVENPORT (1974) in the aforementioned ecological study on a vineyard in Long Ashton in England.

Repeated findings of schizolievites [fssion yeasts] on grapes and wines in temperate regions postulate a radical revision of the concepts on the ecology of Schizosaccharomyces yeasts and they pose the problem of identifying the causes that have made this blastomycetic group go unnoticed in zymology research for so long. In this respect it is important to note that schizolytes (fission yeasts) have a growth rate much lower than that of common wine yeasts and that the experiences of mixed cultures have shown how they are particularly affected unfavorably by competition with other blastomycetic species (FLORENZANO 1972). It follows that the highlighting of schizolytes requires adoption of culture media and clearly selective incubation conditions which give these yeasts a clear advantage over the associated blastomycetic microflora.

Results of a distribution survey are presented in this note of Schizosaccharomyces yeasts on grapes from western Sicily, which demonstrate how the highlighting of such a blastomycetic group is linked to a large extent to the use of an appropriate zymological research technique.

Materials and Methods

The survey was conducted in the years 1974 and 1975 and involved a whole 68 samples (66 of grapes and 2 of concentrated musts) taken from farms and wineries in the provinces of Palermo, Trapani and Agrigento.

Once the samples arrived in the laboratory, they were mashed in the same containers, transferring the must obtained, after separation of the stalks, into sterile flasks.

Schizolytes research was conducted, as well as with the usual technique based on sowing appropriate serial dilutions of the sample to be tested on malt agar in Petri dishes, with a selective method for Schizosaccharomyces pombe recently developed by BALLONI and PELOSI, (1977). Selectivity was obtained by taking into account some distinctive physiological and biochemical properties of schizolytes, such as:

  1. Ability to use malic acid in anaerobiosis
  2. Prototrophy for sterols in anaerobiosis
  3. Relative resistance to the combined action of S02 and actidione [Cycloheximide]

Consequently the culture medium has the following composition: Bacto vitamin-free yeast base (Difco) 16.7 g; glucose 90 g; L-malic acid 10 g; actidione 10 ppm; K2S2O3 50 ppm; thiamine hydrochloride 1 ppm; nicotinamide 0.5 ppm; Ca-pantothenate 0.5 ppm; p-aminobenzoic acid 0.5 ppm; biotin 0.05 ppm; inositol 7 ppm; agar 20 g; distilled water 1,000 ml; pH 4. Petri dishes, after sowing, come incubated in anaerobiosis, carried out in the manner described by KOCH (see MOSSEL et al. 1962). [anaerobic incubation here is novel]

TABLE 1
Characteristics of the samples examined and distribution of fission yeasts on grapes of western Sicily

Presence of schizolytes was determined by microscopic examination of colonies developed on plates after incubation at 25° C for 3-5 days (malt agar) and 8-12 days for the selective medium. Particular morphology of the Schizosaccharomyces yeasts genus allows, as is well known, their discrimination from other blastomycetic forms. It should be noted that on the selective medium for schizolytes the colonies of these are, after 10 days, larger than those of the other yeasts and they have a central area of ​​a reddish-brown color. All this makes it easier for their identification. For the identification of isolated Schizosaccharomyces strains the methods proposed by LODDER (1970) were followed from the various grape samples.

Results

Table 1 shows the results of the zymological survey on the distribution of Schizosaccharomyces yeasts on grapes from western Sicily.

Using the selective method, schizolytes were found in 26 of 68 samples examined, 66 of which consisted of grapes and the remaining 2 from concentrated musts of Zibibbo grapes. This finding is noteworthy, considering that sampling on malt agar generally received a negative response. In fact, with this substrate the schizolievites were found only in two samples of Zibibbo grapes and in one of concentrated must from the same grapes. It should be noted that in the three samples tested positive on malt agar, microscopic examination had already shown the prevalence of schizolievites on the other components of the natural blastomycetic microflora.

On the contrary, microscopic examination of the other samples had not allowed detection of the presence of schizolytes, which indicates that the selectivity of the method of search for such organisms is sufficient to allow their discovery, even when they constitute a secondary component of the blastomycetic microflora.

Table 2
Summary of the data on the distribution of fission yeasts on the Sicilian grape cultivars.

As for the distribution of Schizosaccharomyces on the grapes, there are noticeably some evident differences between the different varieties examined (Table 2). In general the classic Sicilian varieties Catarratto, Inzolia, Grillo, Grecanico, Zibibbo include schizolievites among the normal components of the epiphytic microflora. This is true especially for grapes with a higher sugar content such as Zibibbo and Grillo.

The absence of schizolievites on the 11 Tuscan Trebbiano samples must be underlined, given that this vine is recently introduced in Sicilian viticulture.

Micro-photography of schizosaccharomyces pombe, strain 4Z; left, dividing vegetative cells; right, typical asci with four ascospores (X750).

During the ecological investigation, 62 sample areas for schizolytes were chosen in such a way as to constitute a representative sample of the different “cultivars” and areas of origin of the samples examined.

From a taxonomic point of view, all the strains refer to the species Schizosaccharomyces pombe (Figure), however, differences in capacity were found to assimilate some carbon compounds in different isolates and this requires a more careful evaluation of their systematic position.

Defining the main physiological and biochemical properties in oenological function for the strains isolated from Sicilian grapes is still in progress. However it can so far be claimed that such organisms exhibit a distinct set of properties different from those reported by literature as characteristics of the species Schizosaccharomyces pombe (PEYNAUD and SUDRAUD 1964, CASTELLI and HASNEDARI 1968, FLORENZANO 1972). By way of example, the main properties found are reported for the 4Z strain of Schizosaccharomyces pombe isolated from Zibibbo grapes: 17.2% alcohol; glycerin 5.5 g / L; 2,3-butylene glycol 0.75 g / L; succinic acid 1.6 g / L; volatile acids 0.4 g / L. Furthermore, this strain has the unusual property of fermenting more actively at 18 °C instead of 24 °C or 30 °C as happens for the other strains of the same species.

Conclusion

The data briefly exposed, despite being the result of a first summary zymological recognition, as well as constituting the first report of the presence of yeast of the genus Schizosaccharomyces on Sicilian grapes, show that schizolytes represent one of the normal components of the blastomycetic microflora of grapes from western Sicily, which in some vines (Zibibbo di Pantelleria grapes) have a predominant character.

Apart from the mentioned case, schizolytes are to be counted among the secondary components of the blastomycetic microflora of grapes. This explains the failure to find these yeasts in previous investigations (CASTELLI and DEL GIUDICE 1953) and confirms the validity of the selective method proposed for the research of Schizosaccharomyces. The fact that these yeasts, although widespread on grapes, are not predominant in the natural fermentation of musts, depends on their inability to compete with saccharomycetes (BUJAK and DABKOWSKI, see Rose and HARRISON 1970, p. 45; FLORENZANO 1972).

The presence, among the isolated strains, of biochemically interesting styles for oenological purposes (high fermentation power with over 17% alcohol, low production of volatile acids, excellent fermentation temperature between 18 and 20°C), indicates the great variability of biochemical behavior of schizolytes and demonstrates that thoroughly conducted ecological research is not only of theoretical but also applied interest.

Results

Results of a preliminary investigation on the distribution of yeasts of the genus Schizosaccharomyces on grapes from western Sicily, conducted with a new selective enrichment method for Schizosaccharomyces pombe.

Schizolytes were found to be present in 26 of 68 grape samples examined, belonging mainly to the cultivars Zibibbo, Catarratto, Inzolia, Grillo and Grecanico. On the Zibibbo di Pantelleria grapes these yeasts make up the dominant group in the blastomycetic microflora.

Among the 62 isolated fission yeast strains, mostly referable to Schizosaccharomyces pombe, some exhibit interesting physiological and biochemical properties for oenological purposes.

Results obtained indicate that, in the area under consideration, schizolytes represent a normal component of the epiphytic microflora of grapes and suggest that the sporadic reporting of this blastomycetic group in the grape zymology research is to be attributed to inadequate methods of investigation.

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Received on 8. 12. 1976

Prof. G. FLORENZANO
Università di Firenze
Istituto di Microbiologia
Agraria e Tecnica
Firenze
Italia

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