Letters in Applied Microbiology 1995, 21, 1 15-1 16

Isolation and identification of killer yeasts from sugar cane molasses

M. Bonilla-Sallnas, P. Lappel, M. Ulloal, M. Garcia-Garibay and L. Gomez-Ruiz Departamento de Biotecnologia, Universidad Autonoma Metropolitana, lztapalapa, Mexico, and ’ Laboratorio de Micologia, lnstituto de Biologia, Universidad Nacional Autonoma de Mexico, Mexico DF, Mexico

MOMJ132: received and accepted 14 March 1995

M. BONILLA-SALINAS. P . LAPPE, M. U L L O A , M . G A R C ~ A - G A R I B A Y AND L. G O M E Z - R U I Z . 1995. A total of 13 yeast strains were isolated from 10 samples of sugar cane molasses obtained from sugar mills in Mexico; nine of them were killer strains. The re was a t least one killer strain in every sample. T h e resulting species were the following : Torulaspora delbrueckii (2), Schizosaccharomyces pombe (7), Cryptococcus albidus var. albidus ( 1) and Saccharomyces

I . 1 . cerevisiae (3). A new killer species is reported Schixosaccharomyces pombe.

INTRODUCTION

Wild killer yeasts have been identified as inhibitory to strains used as starters in the production of alcoholic bever- ages such as beer and wine (Stewart and Russell 1985; Young 1987; van Vuuren and Jacobs 1992; Carrau et al. 1993). Even though sugar cane molasses are widely used as raw material in the fermentation industry, particularly to produce rum, ethanol and bakers’ yeast, as far as is known there are no studies concerning the occurrence of killer strains in this material. It is not unlikely that wild killer yeasts naturally present in sugar cane molasses could inhibit the growth of sensitive strains used as starters in the manu- facture of these fermented products.

The current paper deals with the isolation and identifica- tion of wild yeasts from sugar cane molasses from different sugar mills in Mexico, and the incidence of killer yeasts among these wild strains.

MATERIALS AND METHODS

Molasses

Ten samples from different sugar mills in the Mexican States of Morelos and Veracruz were analysed. The names are listed in Table 1.

Isolation

Cultures were enriched diluting 10 g of molasses in 90 ml of medium containing 0.5% (NH,),SO, (J. T. Baker, Xalostoc, Mexico), 0.5% KH,PO, (J.T. Baker, Xalostoc, Mexico) and 0.2% yeast extract (Bioxon, Mexico City). The cultures were incubated in 500 ml Erlenmeyer flasks in

Correspondence t o : Dr Lorena Gbmez-Rurz, Departamento de Bzoternologia, linrversrdad Autinoma Metropolrtana, Iztapalapa, Apartado Postal 55-53.r, Miwrro DF 09340, M e m o .

$3 1995 The Society for Applied Bacteriology

a New Brunswick G-24 rotatory shaker (New Brunswick, NJ, USA) (200 rev min-I, 25°C) during 48 h. Liquid cul- tures were plated on Petri dishes with potato dextrose agar (Bioxon, Mexico City). Isolated colonies were maintained on potato dextrose agar slants at 5°C.

ldentificatlon

Isolated yeasts were identified by means of morphological, biochemical and physiological procedures, and the results were assessed using taxonomic keys according to Kreger- van Rij (1984).

Killer activity

Activity tests were performed as reported by Farris et al. (1991), using as sensitive strains Candida glabrata Y55 (kindly given by Dr Marc Andrk Lachance from the Uni- versity of Western Ontario, London, Ontario, Canada) and Saccharomyces cerevisiae NCYC 1006 (National Collection of Yeast Cultures, Norwich, UK), which were spread as a lawn on the agar. The pH of the medium was buffered to 4.2, 4.6 and 4.8 (assessing killer activity at every pH).

RESULTS AND DISCUSSION

From the 10 samples of sugar cane molasses, 13 strains were isolated. Frequencies, identity and killer activity of the strains are shown in Table 1. The 13 strains were identified to species and the following resulted : Saccharomyces cerevi- siae (3), Torulaspora delbrueckii (2), Schizosaccharomyces pombe (7) and Cryptococcus albidus var. albidus (1).

As can be seen in Table 1, only four of the 13 strains were not killer yeasts when tested against the two sensitive strains. That means that 69% of the isolated strains from sugar cane molasses were confirmed as killer yeasts.

116 M. BONILLA-SALINAS E T A L .

Table 1 Yeast species isolated from the different sugar cane molasses samples and their killer activity against two sensitive strains

Sugar mill Isolated strains Species

Killer Killer activity us activity us Saccharomyces cerevisiae Candida glabrata

+ +

San Nicolas 1 Torulaspora delbrueckii - Central Progreso 1 Saccharomyces cerevisiae -

Motzorongo 1 T . delbrueckii + + San Nicolas I1 1 Schizosaccharomyces pombe + + Sayula 3 Sch. pombe +

Sch. pombe - - Sch. pombe - -

San Miguelito 3 Sch. pombe - Sch. pombe - - Sch. pombe - -

-

+

El Naranjal 0 El Potrero 1 Cryptococcus albidus var. albidus - Granja Santa Ana 1 S. cerevisiae + Atzacan Cuautlapan 1 S. cerevisiae + +

+ -

Yeast species found in molasses and raw cane sugar reported elsewhere (Tilbury 1980) include Saccharomyces heterogenicus, Torulopsis holmii, Saccharomyces rouxii and Torulopsis candida. I n a study done in Trinidad and Tobago (Nagassar-Mohit and Ali 1989), with samples from sugar mills collected from processing lines the major species found were : Cryptococcus laurentii, Pichia membranaefa- ciens, Rhodotorula glutinis, R h . rubra and Zygosaccharomyces bailli. Therefore, there is no coincidence between the species found in sugar cane molasses from Mexican sugar mills and those reported elsewhere.

Most of the killer strains (Table 1) showed activity against C. glabrata, but only two strains had activity against S. cerevisiae but not against the former. T h a t was due to the fact that C . glabrata Y55 is a strain with a broad sensi- tivity to killer toxins (Starmer et a l . 1987, 1992).

T h e widely recognized yeast genera in which killer species have been found are Saccharomyces, Kluyveromyces, Debaryomyces, Hansenula, Pichia, Cryptococcus and Candida (including the former genus Torulopsis) (Young 1987). According to Goto et al. (1992) one out of three strains of Torulaspora delbrueckii isolated from grape musts had killer activity, while neither of two strains of Schizosaccharomyces pombe was a killer strain. Radler et a l . (1993) also reported a killer strain of T. delbrueckii. There are no previous reports of killer strains of the species Schizosaccharomyces pombe; in this study three out of seven isolated strains of this species showed zymocidal activity.

A high incidence of killer strains was demonstrated in sugar cane molasses; practically there was at least one killer strain in every sample. These strains could eventually inhibit industrial starters used in the production of alcohol or bakers’ yeast. A new killer species is reported: Schizo- saccharomyces pombe.

REFERENCES

Carrau, F.M., Neirotti, E. and Gioia, 0. (1993) Stuck wine fer- mentations: effect of killer/sensitive yeast interactions. Journal of Fermentation and Bioengineering 76, 6749.

Farris, G.A., Mannazzu, I. and Budroni, M. (1991) Identification of killer factor in the yeast genus Metschnikowia. Biotechnology Letters 13, 297-298.

Goto, S., Kitano, K. and Shinohara, T. (1992) Utilization of KHR killer as genetic marker for purity test of starter yeast during fermentation of grape musts. Journal of Fermentation and Bioengineering 73, 7&72.

Kreger-van Rij, N.J.W. (1984) The Yeasts. A Taxonomic Study. Amsterdam : Elsevier.

Nagassar-Mohit, G. and Ali, D.A. (1989) Studies on Yeasts in the Sugar Cane Industry in Trinidad and Tobago. Presented at the Seminar on Biotechnology, Havana, Cuba.

Radler, F., Herzberger, S., Schonig, I. and Schwarz, P. (1993) Investigation of a killer strain of Zygosaccharomyces bailii. Journal of General Microbiology 139, 495-500.

Starmer, W.T., Ganter, P.F., Aberdeen, V., Lachance, M.A. and Phaff H.J. (1987) The ecological role of killer yeasts in natural communities of yeasts. Canadian Journal of Microbiology 33,

Starmer, W.T., Ganter, P.F. and Aberdeen, V. (1992) Geographic distribution and genetics of killer phenotypes for the yeast Pichia kluyveri across the United States. Applied and Environ- mental Microbiology 58, 99C997.

Stewart, G.G. and Russell, I. (1985) Tradition meets innovation in brewing. BiolTechnology 3,791-798.

Tilbury, R.H. (1980). Xerotolerant (osmophilic) yeasts. In Biology and Activities of Yeasts ed. Skinner, F.A., Passmore, S.M. and Davenport, R. R. pp. 153-179. London: Academic Press.

van Vuuren, H.J.J. and Jacobs, C.J. (1992) Killer yeasts in the wine industry: a review. American Journal of Enology and Viti- culture 43, 119-128.

Young, T.W. (1987) Killer yeasts. In The Yeasts, 2nd edn, ed. Rose, A. H. and Harrison, J.S. Vol. 2, pp. 131-164. London: Academic Press.

0 1995 The Society for Applied Bacteriology, Letters in Applied Microbiology 21. 115-116