Concentrated cultures of Lactobacillus bulgaricus were prepared by resuspending cells grown in semisynthetic media in sterile 10% non-fat milk solids. The concentrated cultures were frozen in liquid nitrogen for 24 h. The cell suspensions exhibited decreased viability after storage, and the amount of death varied among the different strains tested. Storage stability of all strains examined was improved by supplementing the growth medium with sodium oleate. Radioisotopes were used to study the fate of sodium oleate with L. bulgaricus NCS1. [1-"C sodium oleate was incorporated solely into the lipid portion of the cells, including both neutral and polar lipids. The fatty acid composition of L. bulgaricus NCS1, NCS2, NCS3, and NCS4 grown with and without sodium oleate was studied. The major fatty acids of strains NCS1, NCS2, and NCS3 grown without sodium oleate were dodecanoic, tetradecanoic, hexadecanoic, hexadecenoic, and octadecenoic acids. In addition to these, strain NCS4 contained C1 cyclopropane fatty acid. The major fatty acids of all strains grown with sodium oleate were tetradecanoic, hexadecanoic, hexadecenoic, octadecenoic, and Cl, cyclopropane fatty acids. All strains grown in broth containing sodium oleate contained larger amounts of octadecenoic and C1, cyclopropane fatty acid, and less saturated fatty acids than when grown without sodium oleate. Statistical analyses indicated that Cl, cyclopropane fatty acid was most closely related to stability of the lactobacilli in liquid nitrogen. A negative regression line that was significant at P < 0.001 was obtained when the cellular content of this fatty acid was plotted against death.

Electrohydraulic shock was shown to produce oxidation reactions which inactivated certain compounds important in cellular metabolism. Enzymes that were inactivated included lactic dehydrogenase, trypsin, and proteinases of Bacillus subtilis. Free sulfhydryl groups and reduced nicotinamide adenine dinucleotide were oxidized. Adenosine triphosphate was destroyed, but deoxyribonucleic acid was not affected. Intracellular material of Escherichia coli lost its ability to absorb at 260 m,u after electrohydraulic shock. The bactericidal mechanism involved appeared to be due to nonselective oxidation reactions produced by high-voltage discharges in water. These oxidation reactions were probably mediated by free radicals produced in the water. Electrohydraulic shock treatment is a process in which the high-voltage electricity stored in a capacitor(s) is rapidly discharged across an electrode gap beneath the surface of water or other liquid. When the high voltage is applied to the electrode, a certain amount of ionization must occur in the water at the electrode gap in order for the discharge to occur. The discharge results in the formation of highly ionized, high-pressure, gaseous plasma which tends to expand. The surrounding water resists the expansion and thus creates high transient pressure with each discharge, resulting in the production of shock waves. One of the resultant effects is marked bactericidal action (3, 9, 15; F. Fruengel, U.S. Patent 2,931,947, 1960). The bactericidal action of the submerged highvoltage sparks has been attributed to mechanical action in the form of shock waves and to chemical action on the bacterial cells (3; F. Fruengel, U.S. Patent 2,931,947, 1960). It has also been suggested that the bactericidal action was in part due to free radicals (3). Unstable molecules and free radicals can be formed by electrical discharges in various gaseous systems (8). Free radicals have been reported in corona formation, which is the result of incomplete breakdown of gases by an electric discharge (4).

Single-strain cultures of Streptococcus cremoris were grown in a semisynthetic medium with automatic pH control. After centrifugation, the cells were resuspended in sterile nonfat milk (2% of the original volume). There was no significant difference in the maximum population attained when cultures were grown at pH values of 5.5, 6.0, or 6.5 with sodium hydroxide as the neutralizer. With ammonium hydroxide as the neutralizer, maximum populations obtained were increased about twofold. In most cases, the acid-producing ability of the culture concentrates was comparable to that of fresh-milk cultures. There was some variation among strains of S. cremoris with respect to the effects of different neutralizers and levels of pH control on the biological activity of the culture concentrates. The culture concentrates were stored in liquid nitrogen for as long as 231 days without significant loss in biological activity.

Concentrated cultures of lactic streptococci varied with respect to survival at-17 C. Cells of each strain grown at pH 6.0 were more stable to freezing than were those grown statically. The lipid fraction of the cells from static cultures was important in preventing death during freezing. As the percentage of octadecenoic acid in the cellular lipids from different cultures increased, the percentage of survivors decreased. Capsular material associated with cells from cultures grown both statically and at pH 6.0 was also important in protecting the cells at-17 C. The amount of capsular material, measured as percentage of cellular glucose, varied among the cultures tested. Cultures containing larger amounts of the capsular material were more resistant to the stress of freezing than those containing low levels.

Acid production in milk by lactic streptococci was stimulated by added j3galactosidase. Both glucose and galactose accumulated rapidly in the presence of this enzyme. Glucose accumulation ceased as the culture entered the most rapid period of acid production, whereas galactose accumulation continued. In cultures without added f3-galactosidase, a low concentration of galactose accumulated in the milk, whereas glucose was not detected after 2 hr of incubation. Cultures grew and produced acid faster in broth containing glucose rather than galactose or lactose. These observations suggest that the lactic streptococci do not metabolize the lactose in milk efficiently enough to permit optimum acid production and that a phenomenon such as catabolite repression functions to allow for a preferential use of glucose over either galactose or lactose. In addition to providing the culture with a more readily available energy source, it is possible that the culture produced more acidic metabolites as a result of preferentially utilizing the glucose released by the action of the f,-galactosidase.