Expression of leakage and cell injury
Lou et al. (1994) measured the rate of DNA hydrolysis after dehydration of Lact. plantarum. By using a method where DNase diffuses into cells with injured cell envelopes and hydrolyses the intracellular DNA they showed that dehydration inactivation was associated with evidence of membrane damage.
We assumed that varying degrees of membrane damage might be associated with differences in survival between the strains. Different growth optima for the two strains of Lact. helveticus and Lact. delbrückii ssp. bulgaricus compared with the other strains may relate to different melting points of the phospholipids in the membranes. Based on this assumption, the drying of lactobacilli can have the potential to be more successfully conducted at temperatures as high as that for optimum growth. This point was substantiated by Valdéz et al. (1985b) who showed that rehydration at 0 °C gave less survival for freeze-dried Lact. helveticus than rehydration at higher temperatures for other lactobacilli. However, when protective agents are used at such temperatures the effect of the metabolic activity of the cell and the subsequent pH reduction are factors to consider.
In Figs 1 and 2, it can be seen that the survival of thermophilic Lact. helveticus CNRZ 303 was nearly zero at low temperatures without protective agents. Therefore, we chose 30 °C, in an attempt to reduce membrane changes during dehydration.
Significantly more LDH leaked out during lysozyme treatment of whole dried cells compared with undried thermophilic lactobacilli. The fraction of sublethally injured cells among the survivors was higher for thermophilic than for mesophilic lactobacilli which was confirmed by results on leakage and cell injury. Cytoplasmic membrane damage and release of LDH from injured cells could occur during dehydration, rehydration or through autolysis of cells. The protective effect of agents tested (particularly for strains Lact. helveticus and Lact. delbrückii ssp. bulgaricus) appears to be accompanied by less membrane injury as indicated also by the low values of the LDH analyses.
Effect of protective solutes
Adonitol, which cannot be metabolized by most lactobacilli, has shown a protective effect during freeze-drying by controlling the water content (Valdéz et al. 1985a; Kearney et al. 1990). This may be due to the steric conformations of its hydroxyl groups which may replace water molecules in protein structures (Valdéz et al. 1983b).
Lactic acid bacteria confronted with a lowered aw (osmotic stress) over a long period respond by accumulation of compatible solutes such as betaine and carnitine (Hutkins et al. 1987; Kets et al. 1996). A strain of Lactococcus lactis ssp. lactis contained large pools of proline or glycine betaine as a result of specific transport, when grown under conditions of high osmotic strength (Molenaar et al. 1993). The quaternary amine N,N,N-trimethylglycine (betaine) has been reported to be an important osmoprotective molecule in several groups of Gram-negative bacteria (Cairney et al. 1985; Perroud & Le Rudulier 1985; Hutkins et al. 1987). Betaine is a metabolically inert compound, has no net charge, does not inhibit cytosolic enzymes and, because of its dipolar nature, no counterion accumulation is necessary to maintain electroneutrality (Hutkins et al. 1987; Kashket 1987).
Glycerol has the ability to maintain the available water at the optimal level for the cell during dehydration (Lievense and van’t Riet 1994).
Summing up, the different protectants have high water-binding potentials together with other mechanisms for stabilizing proteins as well as mechanisms to stabilize membrane structures by hydrogen bonding with the phosphate of the phospholipid when water is removed and replaced by sugar molecules, for example.
Low air temperatures should help to avoid thermal inactivation and keep the metabolic activity at a low level. This was the reason for choosing 0–5 °C during preparation for drying and storage. The Ca-alginate beads were quite shrunk after dehydration and the diffusion of oxygen was thought to be limited. Further, storage stability increases with decreasing temperature (Lievense and van’t Riet 1994).
The osmolarity of the rehydration solution, the pH, rehydration temperature and rehydration volume are all factors expected to have some influence on survival rates (Mäyrä-Mäkinen & Bigret 1993). An appropriate nutritional energy source in the rehydration medium may also be important for cell recovery.
Concerning the survival rate, the time interval between rehydration with repair (recovery) in a rich medium and measuring can influence the result. Some divergence was observed between survival, as determined by cfu, and rate of lactic acid production (Fig. 2). Injured cells can repair during plate incubation (MRSA) (Lievense and van’t Riet 1994; Sørhaug 1992). However, only a limited time of 2 h was available for repair during activity measurements. The plate count method can also be misleading, since disaggregation of the chains of bacteria during the dissolving of beads can differ and influence the apparent numbers of survivors. In contrast to the drying conditions chosen for expression of cell injury, both fluidized-bed drying temperature and level of water content (Fig. 2) were not optimal, as indicated by cell leakage and survival during storage. The effect of protective solutes was shown under the unfavourable conditions chosen. In fact, suspension of Lactobacillus strains encapsulated in alginate beads in the presence of respective protective solutes appeared to be a possible strategy for a short period of storage with potential for further survival improvements.
It is important to be aware that the best protective agents during dehydration are not necessarily optimal for protection during storage of the cells (Crowe et al. 1987). Nevertheless, this was the situation for the solutes used in this study (Figs 1 and 2).
The choice of optimal water content for storage of cells is dependent on whether the goal is high survival rates immediately after drying or a low inactivation rate during storage.
The lactobacilli are probably not able to accumulate compatible solutes during a short drying process and therefore such compounds should be accumulated prior to drying, either by simple uptake of added agents or by activating the accumulation mechanisms.