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Applied and Environmental Microbiology, October 2006, p. 6474-6482, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00998-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Stabilization of Frozen Lactobacillus delbrueckii subsp. bulgaricus in Glycerol Suspensions: Freezing Kinetics and Storage Temperature Effects

F. Fonseca,1* M. Marin,1 and G. J. Morris2

UMR Génie et Microbiologie des Procédés Alimentaires, Institut National de la Recherche Agronomique, Institut National Agronomique Paris-Grignon, F-78850 Thiverval-Grignon, France,1 Asymptote Ltd., St. John's Innovation Centre, Cowley Road, Cambridge CB4 0WS, United Kingdom2

Received 28 April 2006/ Accepted 20 July 2006

The interactions between freezing kinetics and subsequent storage temperatures and their effects on the biological activity of lactic acid bacteria have not been examined in studies to date. This paper investigates the effects of three freezing protocols and two storage temperatures on the viability and acidification activity of Lactobacillus delbrueckii subsp. bulgaricus CFL1 in the presence of glycerol. Samples were examined at –196°C and –20°C by freeze fracture and freeze substitution electron microscopy. Differential scanning calorimetry was used to measure proportions of ice and glass transition temperatures for each freezing condition tested. Following storage at low temperatures (–196°C and –80°C), the viability and acidification activity of L. delbrueckii subsp. bulgaricus decreased after freezing and were strongly dependent on freezing kinetics. High cooling rates obtained by direct immersion in liquid nitrogen resulted in the minimum loss of acidification activity and viability. The amount of ice formed in the freeze-concentrated matrix was determined by the freezing protocol, but no intracellular ice was observed in cells suspended in glycerol at any cooling rate. For samples stored at –20°C, the maximum loss of viability and acidification activity was observed with rapidly cooled cells. By scanning electron microscopy, these cells were not observed to contain intracellular ice, and they were observed to be plasmolyzed. It is suggested that the cell damage which occurs in rapidly cooled cells during storage at high subzero temperatures is caused by an osmotic imbalance during warming, not the formation of intracellular ice.

* Corresponding author. Mailing address: UMR Génie et Microbiologie des Procédés Alimentaires, Institut National de la Recherche Agronomique, F-78850 Thiverval-Grignon, France. Phone: (33) 1 30 81 59 40. Fax: (33) 1 30 81 55 97. E-mail: fonseca{at}grignon.inra.fr.

Applied and Environmental Microbiology, October 2006, p. 6474-6482, Vol. 72, No. 10
0099-2240/06/$08.00+0     doi:10.1128/AEM.00998-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.





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