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The aim of this study was to improve the acid tolerance of Lactobacillus acidophilus by combining atmospheric and room temperature plasma (ARTP) mutation with adaptive laboratory evolution (ALE). To achieve a high mutation efficiency, 60 s was determined as the ideal exposure time for ARTP mutation of L. acidophilus with a survival rate of 5.91%. The ARTP-ALE mutant strain LAartp-ale2 displayed increased lactic acid stress tolerance with survival rates of 75.67% and 25.78% when cultured in pH 3.0 and 2.5, respectively, for 3 h. Physiolog