Purification and characterization of bacteriocin produced by Lactobacillus rhamnosus zrx01

Abstract

The bacteriocin-zrx01 produced by Lactobacillus rhamnosus zrx01 was purified, its basic biochemical characteristics and bacterial antibacterial sensitivity were studied. The bacteriocin-zrx01 was separated and purified using ethyl acetate, AKTA purifier™ 10 protein purifier with HiTrap™ Capto™ Q anion exchange chromatography and Superdex™ 75 10/300 GL gel chromatography. The purity of bacteriocin-zrx01 was analyzed using high performance liquid chromatography. The sensitivity to temperature, pH and enzymes of the purified bacteriocin-zrx01 was measured as the diameter of the antimicrobial ring. The results showed that the purified bacteriocin-zrx01 bacterial antibacterial activity was good, and the specific vitality was raised from the original fermentation broth at 625 to 5330 AU/mg. The bacteriocin-zrx01 had a higher stability at pH 3–9, and the antibacterial activity was the strongest at pH 4. When pH was 9, it still retained 86% of the antibacterial activity of the control group. Bacteriocin-zrx01 was cold-stable at −70 and −20 °C for 30 min. It still retained 57% antimicrobial activity compared with the control after being autoclaved at 121 °C for 30 min. Some enzymes, such as protease K, neutral protease, trypsin, thrombin, alkaline protease and papain, affected its antibacterial activity, but it was sensitive to pepsin, and the diameter of the antimicrobial ring decreased by 50% after 2 h of pepsin treatment. These results showed that the bacteriocin-zrx01 produced by Lactobacillus rhamnosus zrx01 might have a potential as a natural food preservative, although in vivo studies are needed.

Introduction

Some chemically synthesized preservative may have some toxicity, which can accumulate in the human body causing some effects on human health (Gokoglu, 2018). When it reaches a certain concentration or dose level, it will show toxic effects on the human body (Anita et al., 2014). Antibiotics are usually banned in food, so finding a natural, safe and efficient biological preservative to replace chemical preservative is potentially beneficial (Xiang, Li, et al., 2019, 2019c, 2019d, 2019a; Campêlo et al., 2017; Fei et al., 2018; Sun et al., 2017; Zhang et al., 2009). Lactic acid bacteria (LAB) have been thought to be having health-promoting effects with humans and are use in products such as yoghurt (Yang et al., 2014). Bacteriocins are ribosome-synthesized by LAB and they can inhibit or even kill bacteria that are closely related to producer strains, food-borne pathogens and spoilage bacteria. Bacteriocins are a natural food preservative (An et al., 2017). They may be able to partially or fully replace chemical preservatives and antibiotics (Cotter et al., 2005; Drider et al., 2006).

LAB bacteriocins are a bacterial polypeptide, protein or protein complex which are post-translationally modified or untreated, and usually released into the cytoplasm or extracellular media (Fimland et al., 2010). They are easily degraded by proteases in the human digestive tract, preventing adverse reactions. The nisin produced by Streptococcus lactis is the only FDA (USA) approved bacteriocin (Delves-Broughton., 1996). Recently, a number of different bacteriocins have been described and their basic biochemical characteristics and antimicrobial mechanisms were characterized for organisms including Lactobacillus plantarum, L. garvieae, L. paracasei, L. fermentum and L. alimentarius (Fernandes et al., 2017; Gao et al., 2015; Ge et al., 2016; Hu et al., 2017; Pascual et al., 2008). For example, bacteriocin XH1 from L. acidophilus XH1, can penetrate and inhibit Escherichia coli growth. It is heat-stable and active as an antimicrobial in acidic conditions (Zhao et al., 2015). Lactocin 160 isolated from L. rhamnosus has antibacterial effects on Micrococcus luteus and other pathogenic bacteria (Li et al., 2005). The bactericidal mode of lactocin 160 is typical of bacteriocins mode of action. It can disturb cell membranes and induce ATP release. The bacteriocin produced by L. alimentarius FM-MM4 still retained 84.7% antimicrobial activity at 121 °C for 15 min. It had active antibacterial activity from pH 3 to 8 (Hu et al., 2017). Bacteriocins produced by Gram-positive bacteria generally have broad-spectrum antibacterial properties. Ahn et al. (2017) observed that bacteriocins with a broad spectrum had good thermal stability, and were hydrophobic cationic peptides. The aim of the experiment was to isolated and purify the bacteriocin produced by L. rhamnosus zrx01 with a broad-spectrum, and to study its antimicrobial activity against E. coli.