Epsilon Open Archive

Abstract

Freeze-drying is a commonly used drying technique for sensitive biologicals, such as lactic acid bacteria. Freeze-drying survival and storage viability of freeze-dried lactic acid bacteria have been shown to depend upon many factors including species, fermentation and formulation procedure, freeze-drying process, storage conditions and rehydration conditions. Lactobacillus (Lb.) coryniformis strain Si3 was selected as the model strain for this thesis work mainly due to its low freeze-drying survival but also potential usefulness as biopreservative, i.e. broad antifungal activity. Preconditioning Lb. coryniformis Si3 with mild stress during fermentation induced changes in the lipid membrane (increased the degree of unsaturation) or increased the uptake of the solute betaine. However, freeze-drying survival was significantly lowered after preconditioning Lb. coryniformis Si3 with cold, base, acid or salt stress prior to freeze-drying. After either optimal growth or preconditioning by mild heat stress, Lb. coryniformis Si3 survived to approximately 70% in a skim milk and sucrose formulation. Betaine was shown to be a poor cryoprotective and lyoprotective agent compared to sucrose. Betaine crystallised upon drying resulting in survival rates of below 3%. By adding small amounts of sucrose to the betaine, crystallisation was inhibited and a 10-fold increase in survival was achieved. Betaine was an effective plasticiser for sucrose, lowering the Tg both in the freeze-concentrate and the freeze-dried product. Our design of experiments approach revealed interactions between the formulation (cell density and sucrose concentration) and freeze-drying process (i.e. cooling rate) with regard to survival of Lb. coryniformis Si3. It was possible to vary the survival rate from 6% to ~70% by varying the different parameters. Storage stability was dependent on the formulation, humidity and temperature. At low temperatures, the matrix stability and cell viability of freeze-dried Lb. coryniformis Si3 in sucrose could be increased by addition of polymers. The suggested 'rule of thumb' of keeping the product 50 degrees below the Tg for retained stability of the amorphous matrix was applicable in our system to maintain high cell viability and technical quality of the products. By combining this 'rule' with the effect of moisture on the Tg, it was possible to determine product-specific storage conditions to ensure high viability and good technical product quality.