In the freezing of ice cream, cold mix is admitted to a freezing chamber and subjected to whipping in the presence of air while the ice crystals that form on the wall of the freezing cylinder are scraped from the wall. Temperature drops rapidly and ice forms quickly in continuous freezers, but the process takes several minutes in batch freezers. These conditions place severe stresses on microorganisms in the mix. Factors that affect survival of microorganisms during freezing and frozen storage include the type and physiological condition of the cells, composition of the food, treatment of the food before freezing, rate and method of freezing, and the temperature, time, and conditions of storage. Ice crystals that form outside the cells reduce the amount of free water in which solute can be dissolved. Those that form inside cells have the potential to puncture cell membranes. Mazur (1966) concluded that viabilities of microorganisms subjected to subzero temperatures are affected primarily by solute concentration and intracellular freezing. Water that freezes in the cell is free water, and this water forms ice crystals. Bound water remains unfrozen. As crystals form, the cytoplasm becomes more concentrated and viscous. Electrolytes and acids are concentrated. Colloidal constituents may be precipitated and proteins denatured. Intracellular ice is thought to be more harmful to microorganisms than extracellular ice. However, Ray and Speck (1973) concluded that, during freezing, formation of extracellular ice was the principal cause of bacterial death and that cells in the stationary phase of growth resist freezing better than those in the logarithmic phase.
The result is that many microorganisms die. Generally, gram-negative rods and the vegetative cells of yeasts and molds are more easily killed than grampositive bacteria, and bacterial and fungal spores are largely unaffected by subzero temperatures (Georgala and Hurst, 1963). Encapsulated bacteria survive freezing better than do the same strains that have lost the ability to express capsules because of mutations. The number of strains of encapsulated yogurt bacteria is limited, and it is important that yogurt bacteria survive freezing so they can deliver P-galactosidase to the human intestine of persons who are deficient in that enzyme and cannot, therefore, digest amounts of lactose they may ingest. During frozen storage at -20°C, the rate of death of yogurt bacteria in frozen yogurt was observed to be quite low. Ingram (1951) summarized the following effects of freezing on selected microorganisms: (a) many species experience an abrupt loss in viability on freezing and (b) cells left viable after freezing die slowly during frozen storage, with the death rate being highest when temperature approaches the melting point of the food and lowest at -20°C and below.
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