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The objective of this research was to compare three different methods for the isolation of bacteriocin‐like inhibitory substances (BLIS) from Bifidobacterium infantis BCRC 14602 (Bioresource Collection and Research Center) for their ease of use and reliability. Ammonium sulfate with 80% saturation of the neutralized cell‐free supernatant resulted in 80% of total activity and a 4.56‐fold increase in specific activity. After dialysis, the specific activity increased 76‐fold, but total activity recovered decreased to 8%. In the second method, adsorption and desorption of the BLIS onto/from the producer cells, the adsorption of the BLIS to the producer cells was strongly affected by the pH of the culture broth whereby 100% adsorption to the killed cells occurred between pH 6.0 and 7.0. Desorption of BLIS from the producer cells was achieved at pH 1.5–2.0. This procedure increased the purification fold 120 times, which was the highest among the three methods studied with a specific activity of 31,605 AU/mg and a yield of 64%. In the adsorption and desorption of BLIS onto/from silicic acid, it was confirmed that the adsorption of the BLIS to silicic acid (5%) was strongly affected by the pH of the broth culture of which 100% adsorption occurred between pH 5.0 and 7.0, whereas at pH values below 5.0 and above 7.0, the adsorption ratio decreased to 67% and 45%, respectively. The best method in terms of purification fold and yield was the adsorption and desorption of BLIS onto/from silicic acid. PRACTICAL APPLICATIONS Very low amounts of published data exist on the isolation of bacteriocins or bacteriocin‐like inhibitory substances (BLIS) from Bifidobacterium sp. To the best of our knowledge, this is the first report on a BLIS isolation from Bifidobacterium infantis BCRC 14602 (Bioresource Collection and Research Center) using different methods. The ability of the partially purified BLIS to inhibit the growth of gram‐negative bacteria, which cause food spoilage and foodborne diseases, makes the bacteriocins purified from bifidobacteria very useful in food safety applications. Further studies should focus on the isolation of the bacteriocins or BLIS of the selected bifidobacteria to evaluate their applications and roles in food preservation. The production of bacteriocins and BLIS can contribute to the elucidation of the mechanism used by bifidobacteria in host defense against potentially pathogenic bacteria dominating the gastrointestinal tract and to control the proliferation of harmful and pathogenic bacteria.

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