Journal of Culture Collections, Volume 3, 2000-2002, pp. 72-77

INFLUENCE OF THE GROWTH CONDITIONS ON THE RESISTANCE OF Saccharomyces cerevisiae, STRAIN NBIMCC 181, BY FREEZE-DRYING

Todor Donev and Tsonka Uzunova-Doneva*

National Bank for Industrial microorganisms and Cell Cultures, 1113 Sofia, P. O. Box 239, Bulgaria

Code Number: cc02011

Experiments were carried out for determination the influence of the growth conditions on the resistance of brewery yeast Saccharomyces cerevisiae, strain 181, to freeze-drying. Growth curves were studied in parallel with the viability of the strain by three variants of cultivation – in a liquid broth, in a solid agar medium and in combination of liquid and solid agar nutrition medium. Better preservation of the cell suspensions obtained in agar medium was determined, where the maximum value was about 8.3‑8.7 % after freeze-drying preservation at the 64^th and 104^th hour of cultivation, whereas in a liquid broth it was 0.28–0.25 % between the 18^th and 24^th hours. We propose a combinated two-step method for consecutive growing in liquid and agar medium, by which viability of 13.2 % from the yeast cells after lyophilization was reached.

The variety of results in the world literature, regarding the influence of the conditions of regeneration and cultivation on the resistance to cryoconservation of different microorganisms could be accepted more as a challenge than as an opportunity for applying the foreign experience. In the collection activity in the guidelines and instructions for work it is recommended that the microorganism must be inoculated on a solid nutrition medium [2, 3, 6, 8]. The well-grown culture is then washed with cryoprotecting medium and then freeze-dried in ampoules [5-7]. If the culture was received in the collection grown on a solid medium then the same procedure was followed – directly got to conservation. No comparative studies have been met, regarding the preservation of the microorganisms by this method to the method by which the culture was grown in a liquid broth. Besides, since the obtained cell suspension was extremely heterogeneous in relation with its age, the appropriate period for preservation has not been defined.

In the present study an attempt was made for finding out the conditions by which high cell concentration was reached before freeze-drying, which correlates with a high resistance of the cells after cryopreservation, in question with freeze-drying. The experiments were carried out with a strain 181, belonging to Saccharomyces cerevisiae, a species, which according to a number of studies was de-scribed as a low lyoresistant [1, 4, 7, 9, 10].

Microorganism. An industrial strain from the collection of National Bank for Industrial Microorganisms and Cell Cultures (NBIMCC), belonging to the species of S. cerevisiae with NBIMCC number 181, was studied, which is generally used in the brewery production [2].

Media and packaging.

Methods.

Cultivation in a liquid broth

The resistance to freeze-drying of the culture at different age was checked. The growth curve and the survival of the yeasts after freeze-drying were studied in dynamics. The results were presented graphically in Fig. 1.

It could be seen that the highest percentage of the viable cells (0.28 %) was achieved by preservation of the strain after the second third of the logarithmic phase (at about the 18^th hour of the cultivation). We estimate the result from the conservation as very low and unsatisfactory.

Cultivation on agar nutrition medium

For assessment the optimal period, during which the investigated strain S. cerevisiae showed maximal resistance to freeze-drying preservation, we carried out experiments by which the growth curve and the survival were also studied in dynamics (Fig. 2).

It was established that the logarithmic growth of the strain was to the 64^th hour (Fig. 2), as by the logarithmic phase of the submerge cultivation. The highest number of the CFUs was achieved at the 72^nd hour. In the following hours the cells were grown on the free agar nutrition medium, by which it was observed that the number of the viable ones was kept relatively constant till the 120^th hour. As the nutrition medium got poorer the balance between the dividing and dieing cells was disrupted.

The results from these investigations for preserved viability of the strain 181 after freeze-drying preservation correlated with the growth curve. The survived cells, conserved at age between the 64^th and 104^th hour were 4.7 x 10⁶ ±0.7 x 10⁶, by which the loss was about 1.07 log₁₀±0.01. The establishment of broad range in the course of time, with near values of the viable cell after lyophilic conservation, makes the work easier for preparation of the strains for lyophilization. The maximal survival of the strain (8.7 %) was achieved by its preservation at age about the 72^nd hour. Comparing the best results, established after the freeze-drying of the culture, obtained by cultivation in non-agar nutrition medium, by this method 30 fold increasing of the survival of the cells was achieved.

In the recent study it was experimental established, that for the aim of the collection work, when preparing the strain S. cerevisiae 181 for lyophilic conservation, the agar nutrition medium was more appropriate in comparison with the liquid one. We suppose that the higher survival after the freeze-drying preservation correlates with the age heterogenicity of the cells in the suspension.

A combinated method for cultivation

The method that we developed combines the good growth of the cultures by cultivation in liquid medium and following high-density growth on agar nutrition medium.

At the first stage, the inoculum, 1 ml cell suspension with concentration 3÷8 x 10⁶ cells/ ml, was homogenized with 9 ml of the liquid nutrition medium in a glass tube and then grown statically for 18÷20 hours at temperature 27 ± 0.5°C. At the second stage, 2 ml well grown cultural suspension, with concentration of 10⁸ cells/ml were poured out in glass tubes with slopping agar. The glass tubes were then left in a slopping state at angle 18^o, in order the cells to slayer equally on the agar medium, after which the remaining liquid was discarded. The strain was grown statically at temperature 27 ± 0.5°C. For the aim of the study samples from the culture were taken in every 8 hours for freeze-drying. The results were described in Fig. 3. The growth curve and the survival of the culture after freeze-drying were depicted.

By the so proposed two-stepped method for propagation, the culture from the second stage before washing differed visually in a significant extend from the culture grown by the classical surface cultivation. It represented a white pellicle, equally covered the agar nutrition medium. The obtained results showed increasing in concentration of the cells, both before and after freeze-drying of the strain, with one order, the maximum survival had been increased from 8.71 % to 13.54 %, i.e. above 55 %.

On the basis of the recent study we recommend when preparing the cell suspension of S. cerevisiae for freeze-drying a combinated two-stepped method for propagation of the strains to be applied. Similar experiments were carried out with other yeast strains belonging to this species and analogical results had been obtained.

References