Antibacterial Activity of Licochalcone A against Spore-Forming Bacteria

Abstract

The bacterial spores of the genera Bacillus and Clostridium act as a survival stage, which is characterized by a high level of resistance to heat and other adverse conditions typically used to kill vegetative cells and other microorganisms (2). In food, the spores themselves do not represent a hazard. However, despite being metabolically dormant, the spores have a functional environmental sensory mechanism that can trigger germination under favorable conditions (22). Thus, the processes of germination, outgrowth (vegetative growth after germination), and/or toxin formation can result in spoilage and/or food poisoning (17). In practice, conditions for outgrowth in many foods are suboptimal due to the presence of a combination of factors, such as water activity, reduced pH, preservatives, organic acids or salt, and pasteurization steps (2).

Generally, the surface charge of microbial cells is anionic at a neutral pH because of the dissociation of acidic groups, such as phosphate and carboxylate, on the cell surface (16). Recently, a great deal of attention has been paid to cationic antimicrobial peptides, such as protamine and ɛ-poly-l-lysine, as natural food preservatives with a low toxicity. Protamine found in salmon spermatozoan nuclei (salmine) is a basic peptide of 32 amino acids, of which 21 are arginine (6, 10). Protamine has activity against a broad spectrum of bacteria, yeasts, and fungi, and this activity is considered to be the result of its polycationic nature (6, 10, 16). The broad antimicrobial spectrum of protamine and the fact that protamine is naturally occurring and nontoxic to humans make it a promising biological alternative to chemical preservatives and disinfectants (6, 10, 16). Alternatively, ɛ-poly-l-lysine from culture filtrates of Streptomyces albulus also shows antimicrobial activity with a broad spectrum (14, 16, 20, 23). These peptides have the common features of being highly basic due to the presence of multiple arginine and lysine residues and of forming amphipathic structures (14, 16). However, the growth inhibitory effects of these peptides against food contaminant microorganisms are repressed by the presence of a high concentration of salt, which could interfere with the binding of cationic peptides to the cell surface (10, 16). Although these peptides carry many cationic residues required for antimicrobial activity, peptides bound to the cell surface are subject to protease digestion (16). In terms of practical applications, cationic antimicrobial peptides would be less effective in the preservation of salted foods and are not available for the preservation of foods with protease activity.

Licorice, the root and rhizome of the Glycyrrhiza spp. Glycyrrhiza uralensis (9, 19), Glycyrrhiza glabra (3, 5, 13, 15, 18, 19), and Glycyrrhiza inflata (7, 8, 11, 18, 19), is currently used in the tobacco, food, and pharmaceutical industries. It has been used for centuries as a medicine because of its wide-ranging therapeutic properties, including relief of rheumatic and other pain, and its healing effect on gastric ulcers (19). The crude extract of licorice has also found commercial use as a food additive in Japan, as it contains the sweetening principal glycyrrhizin (19). Chemical investigations have revealed the presence of a wide variety of bioactive phenolic constituents in licorice; these have attracted attention as a potential source of chemical leads (19). G. inflata is one of the main botanical sources of licorice and is chemically characterized by the presence of retrochalcones, which are distinguished from ordinary chalcones by the absence of oxygen functionality at position 2 (7, 8, 11, 18, 19). Five retrochalcones, licochalcones A, B, C, and D and echinatin, were isolated from G. inflata roots and characterized (7, 8, 11, 18); the content of licochalcone A (Fig. ​(Fig.1)1) was found to be very high (19). We chose to investigate licochalcone A simply because it was the most abundant component of the naturally occurring mixture.

FIG. 1.

Structure of licochalcone A.

Recently, from among the five retrochalcones, various biological activities of licochalcone A were reported, e.g., antiprotozoal (1), anti-inflammatory (19), anti-tumor promoting (19), antioxidative (7, 18), and antimicrobial (8, 18) activities, but little information has been reported for the other chalcones. Furthermore, the antimicrobial activity of licochalcone A has not been thoroughly investigated, and little is known about its activity against food contaminant bacteria, including spore-forming bacteria, such as the genera Bacillus and Clostridium, and toxin-producing bacteria, such as Bacillus cereus and Staphylococcus aureus. In this study, we purified licochalcone A from the roots of G. inflata and evaluated its in vitro activities against some food contaminant microorganisms and its effects in the presence of a combination of factors, such as pH, salt, protease, and heat, as food preservation conditions.

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