P. TEIXEIRA, H. CASTRO AND R. KIRBY. 1995. Spray drying and freeze drying as methods for concentration of Lactobacillus bulgaricus starter cultures were compared in terms of viability, lag phase until onset of pH decrease and total acid production. For the experimental conditions used, no significant differences were detected between the methods. The effect of spray drying on the cell membrane of Lactobacillus bulgaricus was studied. Five separate methods were used to study the theory that spray drying causes cell membrane damage; three relating to leakage of intracellular components from the cell into the surrounding environment (260 and 280 nm absorbing materials, potassium ions and proteins); and two relating to increased cell permeability (increased sensitivity to NaCl and increased permeability to o‐nitrophenyl‐β‐D‐galactopyranoside (ONPG). Partial loss of some cytoplasmic material from the damaged cells was observed. The dried cells also became sensitive to NaCl and permeable to ONPG. Heat shock increased the survival of exponential cells as compared to controls but did not result in normal levels found with unshocked stationary phase cells. Heat shock had no effect on stationary phase cells. Different rehydration methods and media were investigated: slow rehydration increased survival.

P. TEIXEIRA, H. CASTRO, C. MOHÁ CS I-F AR KA S AN D R . KI RB Y . 1997. Heat resistance of Lactobacillus bulgaricus in skimmed milk at 62°, 64°, 65°and 66°C was studied. The response to increasing temperatures in this range was not linear, with temperatures at 65°C and above giving a lower survival rate than would be predicted from experiments at lower temperatures. To identify sites of injury at these temperatures, chemical markers were used. Heating at 64°C and below resulted in damage to the cytoplasmic membrane. At temperatures of 65°C and above chemical markers also indicated damage in the cell wall and proteins. Using differential scanning calorimetry analysis of whole cells of Lact. bulgaricus seven main peaks were observed (l-51, m 1 -61, m 2 -73, n-80, p-89, q-100, r-112°C). Three of these peaks (l r , m r and p r ) were the result of reversible reactions. Analysis of cell fractions identified the cell structure involved in giving rise to each of the three reversible peaks; l r , cell membrane lipids, m r , ribosomes, and p r , DNA. The evidence presented in this paper shows that irreversible reactions in the cell ribosomes are a critical site of damage in Lact. bulgaricus during heat stress in liquid media at 65°C and above.

P. TEIXEIRA, H. CASTRO AND R. KIRBY. 1996. Membrane fatty acids of Lactobacillus bulgaricus were analysed by gas‐liquid chromatography before and after spray drying. The ratio unsaturated/saturated fatty acids decreased following spray drying, indicating the formation of lesions in cellular lipid‐containing structures. The same method was used to analyse membrane lipids of Lact. bulgaricus during storage. Similarly the ratio of unsaturated/saturated fatty acids in dried cells decreased further during storage in air, presenting evidence of lipid oxidation after prolonged storage. The mechanisms of cell death during storage in the dried state are still unknown, but from these results and those presented in the literature, it seems evident that lipid oxidation and survival during storage may be related.

The viability of lyophilized cultures of Lactobacillus bulgaricus in skim milk, during storage at different temperatures, relative humidities, and atmospheres was investigated. Survival was greatest at 11% relative humidity and at 5°C. Indirect and direct evidence is presented supporting the hypothesis that membrane damage occurs during storage. Experiments on the lipid composition of the cell membrane demonstrate that changes occur with time that are probably the result of oxidation. A study on the li!pid composition of the cell membrane by gas chromatography showed that the unsaturated/saturated fatty acid index changes with time during storage.

H.P. CASTRO, P.M. TEIXEIRA AND R. KIRBY. 1997. T h e mechanism of inactivation of Lactobacillus bulgaricus due to freeze drying was investigated. Cells were freeze-dried i n skim milk powder, maltodextrin, glycerol, trehalose and water. Results are presented confirming previous authors' observations regarding membrane damage during freeze drying.I n an attempt to define more clearly the nature of this damage, further experiments were carried out. Results show that following freeze drying changes occur in the unsaturated : saturated fatty acid ratio, a decrease in the activity of the membrane-bound enzyme ATPase and a loss of ApH.