Heat tolerance is one of the most important abilities of LABs necessary to survive during manufacturing processes, such as food fermentation or pasteurization, in which they can be exposed to high temperatures (up to 60°C) . One study showed that heat tolerant Escherichia coli were developed using ALE method by continuously cultivating the bacteria at 48.5°C . In another study, researchers were able to increase the survival temperature of Corynebacterium glutamicum from 33°C to 41.5°C . However, very few studies about manipulating tolerance of S. thermophilus strains through ALE exist, and no studies have been done dedicated to manipulating the bacteria’s heat tolerance in over 60°C.
In this study, bacterial strains with elevated heat tolerance threshold were developed using ALE method. Several probiotic strains primarily isolated from fermented dairy foods in South Korea and two S. thermophilus of LABthat were able to survive at 50°C for 24h were selected for this study. These two strains showed the difference that inherently high heat tolerance (BIOPOP–1) and those having low heat tolerance (BIOPOP–2) when cultured at 50°C. The growth of strain BIOPOP–1 was able to proliferate well, while BIOPOP–2 survived but hardly grew. This explains that strains of the same species can have different thresholds of heat tolerance. The adaptive laboratory evolution method was applied to the bacteria by gradually increasing the temperature and the final surviving bacteria were designated ALE strains.
Figure 3 shows that detectable changes in both strains started 72°C strains, and increased until achieving 84°C for BIOPOP–1, and 81°C for BIOPOP–2. Significant difference in the readings observed between start (60°C) and each end (BIOPOP–1: 84°C, BIOPOP–2: 81°C) strains, suggesting that bacteria increased heat tolerance to a greater extent. It is theorized that the evolutionary shifts of both strains were triggered around temperature points over 70°C.
Two types of heat treatment experiments to compare viability between WT and ALE strains conducted and the overall results matched with the hypothesis that the viabilities of ALE strains were relatively higher than those of WT strains (fig. 4 and 5). Also, WT strains were completely absent during the final stage of each experiment, whereas ALE strain cells remained alive. In case of BIOPOP–1, general viability of ALE strain was higher than that of WT strain, but there was no significant difference in the values between WT and ALE. However, in case of BIOPOP–2, the heat tolerance of ALE strain increased substantially, and the results being significantly different compared to WT cells. In addition, an interesting observation was revealed that a strain with lower basal heat tolerance (BIOPOP–2) could extend its upper threshold by a greater value, while strain with higher basal heat tolerance (BIOPOP–1) would raise its upper limit to a very marginal extent. It might be considered that all bacteria have certain capacity to increase their stress tolerance limit. The lower the base values, the higher will the increment be, and higher based values mean there is less room for expansion.
Cross-protection is based on mechanism that closely related responses are generated by different stress conditions . In other words, different types of stresses lead to a common or similar type of response, as well as specific response by some stresses . The strains in this study also expanded their cross-protection against multiple stress conditions such as high acidity, bile and salinity as a result of ALE compared to WT strains. Probiotics must withstand multiple stress conditions to be able to colonize a colon of human in abundant numbers . Before reaching the intestinal tract, probiotic bacteria must first survive acidic environment of the stomach generated by gastric juice . In this experiment, ALE strains exhibited higher level of acid tolerance than the control group. Upon reaching the intestine, probiotic bacteria face with another challenge, which is bile salts. It was confirmed that ALE strains grew better than WT cells when they were exposed to 0.5% and 1% bile salts for 3h. Lactic acid bacteria can also be exposed to osmotic pressure during manufacture processes when additives such as salt or sugar are added to the product. Osmotic changes in the environment could rapidly damage essential cell functions, and bacteria need to adapt to such a change in order to survive . They were exposed to 20% NaCl for 2h and 24h, and ALE strain again demonstrated higher level of stress tolerance than WT cells. Overall, the bacteria became more tolerate to the above mentioned stress conditions they might face during manufacturing and ingestion processes.
The analysis of fatty acid contents was carried out to determine the cause of increased heat tolerance. Constant heat shock to cells induced their heat tolerance enhancement, and this is clearly linked to modifications in membrane fatty acid composition . In other words, it suggests that composition of the cellular fatty acids plays an important role in the response to heat stress in these strains. The results clearly indicate an increase in saturation level of fatty acids (SFA) as a response to being exposed to high temperatures (Table 2.). The SFA enhances acyl-chain packing in the membrane, and thus increases van der Waal interactions between the chains, which consequently leads to decreased membrane fluidity . And this raises its ability to withstand multiple stresses. The amount of SFAs capable of increasing acyl-chain packing in the cell membrane is considered to be one of the most important factors for successful growth under various stress environments .
Lastly, lactic acid bacteria are exposed to low temperatures during industrial processes such as freezing and refrigerated storage , during which stabilized viability of LAB may contribute to the industries such as the storage of the products and the prolonged conservation conditions. One study identifies that heat tolerance of L. plantarum is also related to cold stress response . In order to identify whether tolerance of ALE strains during freezing storage was improved by heat induced ALE, both WT and ALE strains were stored at –80°C for 12 months. Figure 6 shows that ALE strains were more stable to freezing than WT. Through these results, increased heat tolerance also positively affected bacterial ability to be stored in frozen condition.