Adaptation of Lactobacillus acidophilus to Thermal Stress Yields a Thermotolerant Variant Which Also Exhibits Improved Survival at pH 2 Sonia Kulkarni 1 & Saiful F. Haq 1 & Shalaka Samant 1 & Sunilkumar Sukumaran 1 # Springer Science+Business Media, LLC 2017 Abstract Loss in probiotic viability upon exposure to stress- ful storage and transport conditions has plagued the probiotic market worldwide. Lactobacillus acidophilus is an important probiotic that is added to various functional foods. It is known to be fairly labile and susceptible to temperature variations that it encounters during processing and storage which in- creases production cost. It has been repeatedly demonstrated that pre-exposure to sub-lethal doses of stress, particularly, temperature and pH, leads to improved survival of various probiotics when they subsequently encounter the same stress of a much greater magnitude. Attempts to adapt L. acidophilus to temperatures as high as 65 °C to arrive at a thermotolerant variant have not been reported previously. To improve viabil- ity at elevated temperatures, we gradually adapted the L. acidophilus NCFM strain to survival at 65 °C for 40 min. Following adaptation, the variant showed a 2-log greater sur- vival compared to wild-type at 65 °C. Interestingly, this thermotolerant variant also demonstrated a 2-log greater sta- bility compared to wild-type at pH 2.0. The improved pH and temperature stress tolerance exhibited by this variant remained unaltered even when the strain was lyophilized. Moreover, the thermotolerant variant demonstrated improved stability com- pared to wild-type when stored for up to a week at 37 and 42 °C. Probiotic properties of the variant such as adherence to epithelial cells and antibacterial activity remained unaltered. This strain can potentially help address the issue of significant loss in viable cell counts of L. acidophilus which is typically encountered during probiotic manufacture and storage. Keywords Lactobacillus acidophilus . Thermotolerant . Adaptation . Cross-tolerance . Heat stress Introduction There has been an increasing demand for probiotic reinforced food and therapeutic products in the past several years [1]. Probiotics exert their beneficial effects in the gut through var- ious actions such as immunomodulation to production of bio- active molecules which play an important role in disease eva- sion, reduction in duration of infectious diarrhea, reduction in serum cholesterol, reduced lactose intolerance, reduction of allergic responses, and management of inflammatory bowel disease [2–5]. In order to exert their beneficial effects in the gut, probiotics must withstand two kinds of stresses: those encoun- tered during processing and storage, and those encountered during transit through the gastrointestinal tract (GIT). Manufacture and storage of probiotic products involve pro- cesses which are detrimental to the viability of the microor- ganisms. Probiotics are typically grown to high numbers and then dried by a suitable process to generate a high-cell density probiotic powder. Probiotic organisms encounter temperature extremes depending on the drying process used. Temperatures can reach as high as 200 °C during spray-drying and while the exposure time for the cells is extremely short, the integrity of viable bacterial cells can be severely compromised [6]. On the other hand, temperatures as low as - 196 °C are encountered during freeze-drying. Although freeze-drying is less harsh on cells than spray drying and results in higher cell numbers, the low temperatures still compromise cellular integrity [7–11]. Also, probiotic stability during powder storage can be ad- versely affected by storage temperature [12]. Oxidative stress and reduced water activity (a w ) are the other major stresses * Shalaka Samant shalaka.s@anthembio.com 1 Anthem Biosciences Pvt. Ltd., Bommasandra Industrial Area Phase-I, Hosur Road, Bangalore 560099, India Probiotics & Antimicro. Prot. DOI 10.1007/s12602-017-9321-7