Journal of’ Applied Bacferioloyy 1985, 59, 299-302 2022/11/84

Sensitivity of Saccharomyces cerevisiae vegetative cells and spores to antimicrobial compounds

P A T R I Z I A R O M A N O & G . S U Z Z I Dipartimento di Protezione e Valorizzazione Ayroalimentare, Sezione Microbioloyica, Universith di Bologna, Coviolo, 42100 R e y y i o Emilia, I t a l y

Received 5 November 1984, revised 9 M a r c h 1985 and accepted 26 April 1985

R O M A N O , P. & SUZZI , G . 1985. Sensitivity of Saccharomyces cereuisiae vegetative cells and spores to antimicrobial compounds. Journal of Applied Bacteriology 59, 299-302.

The sensitivity of Saccharamyces cereuisiae spores and vegetative cells to various antimicrobial compounds was compared. Sulphur dioxide, benzoic acid, potassium sorbate, salicylic acid, nystatin, actidione and pimaricin were tested. Generally, the Saccharomyces spores were more resistant than the corresponding vegetative cells. I t was also observed that this greater resistance shown by the spores varied with the antimicrobial compound used. Only potassium sorbate was not selective and killed both vegetative cells and spores at about the same rate.

In nature, production of spores by yeasts prob- ably plays a part in adaptation and survival under changing environmental conditions. It is often assumed that yeasts are able to survive unfavourable conditions in the form of asco- spores, but, unlike bacterial spores, these are only slightly more resistant than vegetative cells (Fowell 1969). The resistance of spores to heat, alcohol, diethyl ether, and other physical- chemical agents formed the basis of some suc- cessful methods of isolating spores from vegetative cells and simplifying genetic manipu- lation (Fowell 1969; Rousseau & Halvorson 1969; Dawes & Hardie 1974; Romano et al. 1983).

Put et al. (1976) investigated yeasts causing spoilage in soft drinks, and found that asporo- genous yeasts were generally less heat resistant than were ascomycetous types ; Saccharomyces cerevisiae, and Sacch. chevalieri (now Sacch. cerevisiae showed the highest heat resistance. These authors (Put et al. 1976) considered that Sacch. cerevisiae and Sacch. bailii (now Zygosac- charomyces bailii) are the dominant spoilage yeasts. Recently, it was observed that the heat resistance (D6,,Lc values) of the Sacch. cerevisiae

and Zygosacch. bailii ascospores were 50-1 50- fold higher than the D,,o, values of the corre- sponding vegetative cells (Put & De Jong 1982). Other species of Saccharomyces have been implicated in fermentative spoilage of carbonate beverages and other food products (Pitt 1974). Zygosaccharomyces bailii affects products which have a high sugar content, a low pH and contain benzoic, sorbic or acetic acid, as pre- servatives (Warth 1977).

It is not known if spores of Saccharomyces spp. and strains generally show high resistance to food preservatives and other compounds compared with that shown by their vegetative cells. This paper describes a study of the sensi- tivity of Saccharomyces cerevisiae spores com- pared with that of vegetative cells to various compounds commonly used in food preser- vation.

Materials and Methods

O R G A N I S M S

Saccharomyces cerevisiae strains 694-1A, 129- 1 C and 742-1A, from the collection of Microbio- logical section of our Department, isolated from

Patrizia Romano and G. Suzzi spoiled soft drinks were used. These are homo- thallic with a high sporulation frequency. The approximate sporulation rates are 90%, 78% and 70Y0 for strains 694-1A, 129-1C and 742-1A, respectively.

P R E P A R A T I O N OF V E G E T A T I V E C E L L S A N D

S P O R E S

For vegetative cell production from the stock cultures, YPD medium was used. This con- tained (g/l) yeast extract, 10; peptone, 20; glucose, 20. After inoculation it was incubated at 25°C for 24 h and then used for the sensitivity tests.

For spore production and separation of spores from diploids, the method suggested by Romano et al. (1983) was used. A culture of cells grown on YPD medium for 24 h was plated on sporulation medium, containing (g/l) sodium acetate, 1.0; agar, 20. This was incubated at 25°C for 24 h. The culture then contained a mixture of two-three-, and four-spored asci (90%) as well as unsporulated cells (10%). Approximately lo7 spores from the sporulation medium were suspended in 0.5 ml of Glusulase (IBF) (Johnston & Mortimer 1959) and diluted with 9.5 ml of sterile water and incubated at 30°C for 18 h. Most of the vegetative cells were killed by this procedure and living spores were liberated from the asci. After repeated washing, the sample was suspended in a suitable buffer and treated with an antimicrobial compound. Since the spores tend to adhere together after treatment they were separated by an oil-water phase before plating, according to the method of Siddiqi (1971) to obtain colonies from single spores.

P R E P A R A T I O N OF A N T I M I C R O B I A L S T O C K S O L U T I O N

Benzoic acid and salicylic acid were dissolved in 95% ethanol to give 10% (w/v) stock solution. Potassium sorbate and sulphur dioxide were dissolved in citrate buffer pH 3.6 to give 10% (w/v) stock solution.

Nystatin (Calbiochem) and actidione (BDH) were respectively dissolved in 95% ethanol and citrate buffer pH 3.6 to give 0.01% (w/v) stock solutions. Pimaricin (Gist-Brocades) containing 50% active pimaricin, was dissolved in citrate buffer pH 3.6 to give 0.01% (w/v) stock solu-

tion. These solutions were filter-sterilized using the Millipore filters (0.22 pm exclusion limits) and used immediately. Dilutions of each anti- microbial solution were made in sterile citrate buffer.

D E T E R M I N A T I O N O F S E N S I T I V I T Y T O A N T I M I C R O B I A L C O M P O U N D S

Vegetative cells and spores of 694-1A, 129-1C, 742-1A were suspended in 100 ml of citrate buffer (pH 3.6) and counted microscopically. Serial dilutions in the same buffer were then made to give a final concentration of 200 vege- tative cells or spores per ml. Immediately after preparing the dilution and just before the anti- microbial treatment, viable cell-counts were made using poured plates of YPD agar. To the cell and spore suspensions 1.5 ml of the suitable dilution of each antimicrobial compound were added. The mixture was then incubated at 25°C with shaking. Samples ( 1 ml) from each test were plated on YPD agar at the following contact times: 1, 2, 3, 4, 8, 16 and 24 h. After incubation at 25°C for 48 h the number of colony-forming units was counted. Each test was repeated three times and the results are expressed as average.

Results

When treated with the antimicrobial com- pounds, the three strains behaved similarly. Table 1 shows all the results for the tests carried out on strain 694-1A which gave the most con- sistent results. The spores and vegetative cells of the strains were treated with 100, 200 and 400 mg/l sulphur dioxide. The vegetative cells did not survive SO, treatment up to 200 mg/l after exposure for 2 h and up to 100 mg/l after 3 h. A higher resistance was observed in the spores than in the vegetative cells. In fact the spores were little affected during 24 h treatment with 100 and 200 mg/l sulphur dioxide.

Vegetative cells and spores treated with 500, 1000, 1500 mg/l of potassium sorbate behaved in the same way, both at different doses and during 24 h treatment, showing a high sorbate resistance.

The benzoic acid treatment with 500 mg/l did not affect spore viability, whereas vegetative cells were gradually killed during 24 h of expo- sure. After 2 h treatment, the 1000 mg/l dose of

Ascospore resistance 30 1 Table 1. Viable counts* of cells and spores of Saccharomyces cereuisiae 694-1A remaining after treatment with anti-

microbial compounds Time of treatment (h)

1 2 3 4 8 16 24 - ~ ~ ~ ~ - ~ 0

c s c s c s c s c s c s c s c s Sulphur dioxide (mg/l)

100 197 202 96 198 50 196 0 192 0 186 0 170 0 130 0 94 200 201 204 0 197 0 192 0 182 0 170 0 130 0 80 0 30 400 200 198 0 150 0 104 0 66 0 44 0 16 0 0 0 0

500 204 205 192 204 190 202 182 196 174 184 156 168 132 152 116 144 I000 201 201 196 198 184 198 166 184 148 164 IOX 152 94 136 84 112 1500 204 194 152 172 128 144 108 130 96 102 68 72 28 42 3 22

500 198 202 168 198 140 198 122 198 110 198 64 190 20 190 9 190 1000 198 202 26 195 0 184 0 162 0 150 0 106 0 90 0 76 1500 200 199 2 134 0 98 0 62 0 38 0 12 0 0 0 0

500 204 200 192 198 192 195 174 196 160 192 121 180 96 160 73 116 1000 202 207 134 198 98 194 68 150 24 106 14 64 0 32 0 14 I500 200 202 11 130 5 108 2 88 0 64 0 26 0 3 0 0

0.25 198 202 130 196 122 194 108 194 102 192 88 186 72 180 46 176 0.50 198 197 11 178 0 168 0 162 0 162 0 154 0 150 0 146 I .oo 200 202 0 63 0 55 0 52 0 48 0 17 0 0 0 0

20 201 200 0 100 0 104 0 106 0 98 0 90 0 92 0 85 40 201 205 0 85 0 82 0 87 0 82 0 78 0 65 0 51 60 198 199 0 52 0 48 0 51 0 48 0 42 0 38 0 29

10 199 205 123 188 102 182 84 174 52 172 34 166 5 158 0 125 15 202 202 110 160 68 158 12 154 0 148 0 126 0 110 0 88 20 200 195 42 132 28 126 0 112 0 102 0 80 0 58 0 36

Potassium sorbate (mg/l)

Benzoic acid (mg/l)

Salicylic acid (mg/l)

Actidione (pgiml)

Nystatin (pg/ml)

Pimaricin (pg/ml)

* Means of three determinations. C, Cells; S, spores.

benzoic acid killed all the vegetative cells and did not affect the spores. The 1500 mg/l dose of benzoic acid reduced spore viability, killing spores after 16 h of exposure.

Salicylic acid treatment both at different doses and during 24 h affected cell and spore viability in a similar way to the benzoic acid treatment.

The vegetative cells were very sensitive to actidione and nystatin; after 2 h treatment with 0.5 pg/ml actidione as after 1 h treatment with 20 pg/ml nystatin they were totally eliminated. The spores, on the other hand, were still less sensitive; after 3 h treatment, in fact, about 80% of the spores survived the actidione dose and about 50% the nystatin dose. Vegetative cells treated with pimaricin showed higher survival than celb treated with other antibiotics. Spore survival in response to pimaricin, however, was similar to that resulting from nystatin and acti- dione treatment.

Discussion

The antimicrobial compound resistance of the Saccharomyces cerevisiae spores proved to be higher than that of the corresponding vegetative cells. Our results are in agreement with those of Put & De Jong (1982) on the heat resistance of ascospores of Sacch. cerevisiae and Zygosucch. bailii. Nevertheless, it may be interesting to observe that the highest resistance of the spores varied in level in relation to the antimicrobial compound used. The three antibiotics tested, actidione, nystatin and pimaricin, emphasized a very different type of behaviour between vegeta- tive cells and spores, from that to sulphur dioxide. Benzoic and salicylic acid still exercised considerably more antimicrobial action on the vegetative cells than on the spores. Potassium sorbate, on the contrary, did not have any selec- tive action and killed the vegetative cells and spores a t about the same rate.

302 Patrizia Romano and G. Suzzi These results suggest that, as well as playing

the primary role in sexual reproduction, the Saccharomyces spores play an important role in species conservation, as d o the bacterial spores though to a lesser extent or in a different way.

The different sensitivity to the compounds tested between vegetative cells and spores sug- gests that the latter can be considered a pre- dominant cause of beverage spoilage by Saccharomyces cerevisiae.

This work was supported by the Minister0 Pub- blica Istruzione (Roma).

References

DAWES, I.W. & HARDIE, I.D. 1974 Selective killing of vegetative cells in sporulated yeast cultures by exposure to diethyl ether. Molecular and General Generics 131, 281-289.

FOWELL, R.R. 1969 Sporulation and hybridization of yeasts. In The Yeasts ed. Rose, A.H. & Harrison, J. S., Vol. 1 pp. 303-383. London : Academic Press.

JOHNSTON, J.R. & MORTIMER, R.K. 1959 Use of snail digestive juice in isolation of yeast spore tetrads. Journal of Bacteriology 78, 292.

PITT, J.I. 1974 Resistance of some food spoilage yeasts to preservatives. Food Technology in Australia 26, 238-241.

PUT, H.M.C., DE JONG, J., SAND, F.E.M.J. & VAN GRINSVEN, A.M. 1976 Heat resistance studies on yeasts causing spoilage in soft drinks. Journul 01 Applied Bacteriology 40, 135-1 52.

PUT, H.M.C. & DE JONG, J. 1982 The heat resistance of ascospores of four Saccharomyces spp. isolated from spoiled heat-processed soft drinks and fruit products. Journal of Applied Btrcterioloyy 52, 235- 243.

ROMANO, P., SOLI, M.G. & SUZZI, G. 1983 Procedure for mutagenizing spores of Saccharomyces cereoi- siae. Journal of Bacteriology 156, 907-908.

ROUSSEAU, P. & HALVORSON, H.O. 1969 Preparation and storage of single spores of Saccharomyces cere- uisiae. Journal of Bacteriology 100, 1426-1427.

SIDDIQI, B.A. 1971 Random spore analysis in Suc- charornyces cerevisiae. Hereditas 69, 67-76.

WARTH, A.D. 1977 Mechanism of resistance of Sac- charomyces bailii to benzoic, sorbic and other weak acids used as food preservatives. Journul of Applied Bacteriology 43, 215-230.