17Tolerance to freeze-thaw stress is an important characteristic in recent fermentation 18 processes. To gain insight into the freeze-thaw tolerance of lactic acid bacteria (LAB), we 19performed screening experiments and observed that several Lactobacillus curvatus strains 20 showed high freeze-tolerance even in the absence of cryoprotectants. These Lb. curvatus 21 strains also showed high levels of freeze tolerance in a milk fermentation process. 22Lactobacillus sakei (closely related to Lb. curvatus) was not revealed to be a freeze-thaw 23 tolerant strain. These data indicate that Lb. curvatus has specific mechanisms underlying its 24 tolerance to freeze-thaw stress. 25 Importance: Our findings demonstrate that Lb. curvatus strains frequently show high levels 26of freeze-thaw tolerance in both culture and milk and that Lb. curvatus strains are suitable as 27 a model species for investigations of the molecular mechanisms underlying freeze-thaw 28 tolerance in LAB and for applications in fermentation industries. 29 30 KEYWORDS: lactic acid bacteria, freeze-thaw stress, stress tolerance 31. 32 IR1 strains in milk that contained a potential cryoprotectant, because LAB strains are often 69 used to produce fermented foods derived from milk products such as yogurt. After the 70 addition of cells of Lb. curvatus, we observed strong coagulation of the milk (data not 71 shown). In contrast, partial milk coagulation observed when W. cibaria IR1 was used under 72 the same conditions. As shown in Figure 2, Lb. curvatus IR8 showed much higher 73 survivability compared to W. cibaria IR1 and Lb. delbrueckii subsp. bulgaricus. These data 74 suggested that Lb. curvatus strains may be useful in the production of fermented foods 75 derived from milk. 76Lb. curvatus, which are closely related to Lactobacillus sakei (7), is detected in a 77 variety of resources including fermented foods and animal intestine, and Lb. curvatus has 78shown genomic diversity (8, 9). Our present analyses identified three Lb. curvatus strains 79 with freeze-thaw tolerance. The three strains were isolated from different isolation resources 80 6 Although Lb. sakei was included in our screening resources (data not shown), no Lb. sakei 81 strain was detected as a freeze-thaw-tolerant strain. 82The Lb. curvatus strains screened in this study may produce extracellular 83 polysaccharide (EPS) (data not shown), and it is possible that EPS functions in cell 84 protectants under freezing conditions (10). Although the molecular mechanisms underlying 85 tolerance to freeze-thaw stress are not yet known, it is possible that Lb. curvatus has specific 86 genes and/or cellular systems that contribute to the tolerance observed in three strains. 87 88 Strains and medium 89The LAB strains (~70 strains) had been preserved in our laboratory. They were obtained 90 from natural resources in Japan including fermented foods. Lb. delbrueckii subsp. 91 bulgaricus isolated from a commercial yogurt product (Meiji, Tokyo) was used as a control 92 strain. All LAB strains were cultivate...