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“Mechanical properties” are crucial for biodegradable and/or non‐biodegradable materials used in tissue engineering applications. In this study, bio‐hybrid films were produced by using both Bifidobacterium animalis subsp. lactis BB‐12 probiotic strain and Bifidobacterium infantis in combination with sodium alginate (SA), which demonstrates biocompatibility and facilitated gelation properties. Bio‐hybrid films were characterized by using different methods. Based on the spectroscopic and mechanical analysis, it was found that mechanical strength increased in films produced by adding Bifidobacterium infantis in SA while this increase was relatively lower as compared to those containing Bifidobacterium animalis subsp. lactis BB‐12 as cross‐linking ratio increases. Besides, bacteria contained in bio‐hybrid films increased the percentage of amorphous zone of SA in SA/bacteria films, which reduced the crystallinity …