Operating conditions that affect the resistance of lactic acid bacteria to freezing and frozen storage

Thermophilic lactic acid bacteria exhibit different survival rates during freezing and frozen storage, depending on the processing conditions. We used a Plackett and Burman experimental design to study the effects of 13 experimental factors, at two levels, on the resistance of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus to freezing and frozen storage. The resistance was evaluated by quantifying the decrease of acidification activity during freezing and throughout 8 weeks of storage. Acidification activity after freezing and frozen storage was affected by 12 experimental factors. Only the thawing temperature did not show any significant effect. S. thermophilus was more resistant than L. bulgaricus and the cryoprotective effect of glycerol during freezing and storage was confirmed. The temperature and duration of the cryoprotection step influenced acidification activity following the freezing step: the lower the temperature and the shorter the duration, the higher the activity. Acidification activity after storage was affected by several experimental factors involved in the fermentation stage: use of NaOH instead of NH4OH for pH control, addition of Tween 80 in the culture medium, and faster cooling led to better cryotolerance. Resistance to freezing and frozen storage was improved by using a high freezing rate and a low storage temperature. Finally, this study revealed that the conditions under which lactic acid bacteria are prepared should be well controlled to improve their preservation and to limit the variability between batches and between species.