Antimicrobial activity and partial characterization of bacteriocin-like inhibitory substances (BLIS) produced by Bifidobacterium infantis BCRC 14602

Abstract

Bifidobacterium infantis BCRC 14602 was found to produce a bacteriocin-like inhibitory substance (BLIS) with inhibitory activities against a wide range of Gram- positive and Gram-negative bacteria. An activity level of 400 AU/ml in the middle of the exponential phase (i.e. 6 h) and maximum activity (1600 AU/ml) at the beginning of the stationary phase (i.e. 16 h) was recorded in MRS broth at 37 °C. BLIS was partially purified by a two-step purification protocol resulting in a specific activity of 31,605 AU/mg and a purification fold of 120. Based on Tricine–SDS–PAGE, the BLIS is approximately 3.0 kDa in size. Complete inactivation of BLIS activity was observed after treatment with proteolytic enzymes, but not with catalase, α-amylase and lipase. The adsorption of the BLIS to the producer cells was strongly affected by the pH of the broth culture of which 100% adsorption to the killed cells occured between pH 6.0 and 7.0, whereas at pH values below 6.0 and above 7.0, the adsorption ratio decreased to 43 and 60%, respectively. BLIS showed high temperature stability up to 121 °C for 15 min with no loss in its activity, and had pH stability in the range of 4–10. The temperature and heat stability of BLIS makes it useful for applications in food processing technologies and food safety control applications.

Introduction

Bacteriocins are ribosomally synthesized, extracellularly released low-molecular-mass peptides or proteins (usually 30–60 amino acids) which have a bactericidal or bacteriostatic effect on other bacteria such as closely related bacteriocin producing species, food spoilage bacteria and food-borne pathogens (Klaenhammer, 1988). Bacteriocins of lactic acid bacteria (LAB) are classified into three classes (Drider, Fimland, Héchard, McMullen, & Prévost, 2006). Class I includes small (<5 kDa) ribosomally produced peptides that undergo extensive post-translational modification. This class is further divided into two subgroups. Class Ia includes elongated, amphipathic, pore-forming lantibiotics (e.g. nisin) and class Ib includes the rigid and globular antimicrobial peptides (e.g. mersacidin). Class II peptides are small (<10 kDa), membrane-active, unmodified peptides. This class is subdivided into three subgroups. Class IIa are pediocin- like anti-Listeria peptides with their N-terminal part characterized by a consensus YGNGVXC motif. Class IIb bacteriocins require a combination of two polypeptides for full antimicrobial activity (e.g. enterocin L50), while class IIc are other bacteriocins (e.g. acidocin B). Class IId; which has recently been proposed by Gray et al. (2006a) and includes thuricin 17 (Gray et al., 2006b) and bacthuricin F4 (Kamoun et al., 2005). Class III comprises of large, heat labile protein (>30 kDa) such as helveticin J (Drider et al., 2006).

Bifidobacterium is one of the most common genuses in the human intestinal microbiota. They represent up to 91% of the total population of the intestinal tract in newborns and 3–7% of the population in adults (Biavati, Vescovo, Torriani, & Bottazzi, 2000). Bifidobacteria play a major role in promoting health and preventing disease, and affect human physiology in a multiplicity of ways such inhibiting or excluding of undesirable microorganisms, immunomodulation, and synthesis of vitamins such B₆, B₁₂ and folic acid (Leahy et al., 2005, Bhunia and Johnson, 1992, Rioradan and Fitzgerald, 1998, Pompei et al., 2007, Servin, 2004).

Most research has been focused on the production of bacteriocins from members of lactic acid bacteria (LAB) in particular, Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Enterococcus sp., and Pediococcus sp. (Ghrairi et al., 2005, Ghrairi et al., 2008, Anand et al., 1985, Millette et al., 2007, Millette et al., 2008, Osmanagouglu, 2007, Todorov et al., 2007). Very few reports exist on the production of bacteriocins from Bifidobacterium sp. (Abd El-Salam et al., 2004, Anand et al., 1984, Anand et al., 1985, Kang et al., 1989, Uelivon, 2006, Yildirim and Johnson, 1998, Yildirim et al., 1999) and therefore their antimicrobial effects have not been characterized yet to the extent as for those of lactic acid bacteria. Only one review article is found in the literature on the antimicrobial proteinaceous substances produced by Bifidobacterium recently published (Cheikhyoussef, Pogori, Chen, & Zhang, 2008). Nevertheless, further studies focused on the isolation of the proteinaceous inhibitory substances of the selected bifidobacteria should be carried out to evaluate their possible prophylactic and therapeutic use.

The first reported bacteriocin produced by B. bifidum 1452 was bifidin (Anand et al., 1984, Anand et al., 1985). Batdorj et al., 2006, Kang et al., 1989 reported that B. longum can produce bifilong. Yildirim and Johnson (1998) described a bacteriocin produced by B. bifidum NCFB 1454 designated as bifidocin B. The bacteriocin was then purified to homogeneity by Yildirim et al. (1999). Bifidocin B is active against some food-borne pathogens and food spoilage bacteria such as Listeria, Enterococcus, Bacillus, Lactobacillus, Leuconostoc, and Pediococcus. B. lactis Bb-12 and B. longum Bb-46 produced bacteriocins designated as bifilact Bb-12 and bifilong Bb-46, respectively, in MRS broth with 0.05% L-cystein HCl (Abd El-Salam et al., 2004). Uelivon (2006) partially purified a bacteriocin produced by B. thermophilum RBL67 termed thermophilicin B67 using methanol acetone extraction and reversed phase HPLC. Table 1. summarizes the named bacteriocins which have been purified or partially purified from bifidobacteria. Bifidocin B is the only bacteriocin that has been purified to homogeneity and which has been sequenced Yildirim et al. (1999).

The prevalence of bifidobacteria as probiotics in many fermented dairy products makes the bacteriocins produced by them of interest as natural food preservatives. 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 in large intestine, and could be exploited to enhance the competitiveness of bifidobacteria in diverse ecosystems and their roles in food preservation.

This is the first report on a BLIS partially purified and characterized from B. infantis BCRC 14602. In this study the antimicrobial activity of B. infantis BCRC 14602, its production of a bacteriocin-like inhibitory substance (BLIS), some physiochemical properties of BLIS and its antimicrobial spectrum is reported.