COMPARISON OF THREE DIFFERENT METHODS FOR THE ISOLATION OF BACTERIOCIN‐LIKE INHIBITORY SUBSTANCES FROMBIFIDOBACTERIUM INFANTISBCRC 14602

Abstract

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.

Taxonomy, Physiology, and Natural Products of Actinobacteria

“…Among the many probiotic features that have been attributed to bifidobacteria are (i) the induction of immunoglobulin production, (ii) improvement of a food's nutritional value by assimilation of substrates not metabolized by the host, (iii) anticarcinogenic activity, and (iv) folic acid synthesis (152)(153)(154). Some bifidobacteria produce antimicrobials (155) and notably, also bacteriocins (156,157).…”

Production of bacteriocin-like inhibitory substance byBifidobacterium lactisin skim milk supplemented with additives

“…However, these interesting reports have mainly focused on the separation and purification processes, or on the influence of pH and temperature, inhibitory compounds and bacteriocin structure stability rather than on the optimisation of medium composition for Bifidobacterium sp. cultivations (Yildirim & Johnson, 1998; Yildirim et al 1999; Von Ah, 2006; Cheikhyoussef et al 2009b, 2010; Lee et al 2011). Additional significant issues that must be considered in bacteriocin production are the high cost of complex media, normally used in large scale fermentation processes such as Man, Rogosa and Sharpe (MRS) and Bifidus selective medium (BSM), as well as the low reproducibility and sensitivity of the agar diffusion method for the determination of bacteriocin activity (Pongtharangkul & Demirci, 2004).…”

Produção de bacteriocina por Bifidobacterium lactis a partir de leite desnatado

“…Recentemente, Cheikhyoussef et al, (2009a;2009b; descobriram uma nova bacteriocina produzida por Bifidobacterium infantis em meio sintético MRS suplementado com cisteína. Essa nova bacteriocina, denominada Bifidin I, mostrou amplo espectro de ação, incluindo bactérias Gram-negativas e Gram-positivas, além de clara inibição com relação à L. monocytogenes.…”