Antilisterial and physical properties of polysaccharide-collagen films embedded with cell-free supernatant of Lactococcus lactis

Abstract

The physical and antimicrobial properties of sodium alginate or sodium carboxymethylcellulose films containing cell-free supernatant of Lactococcus lactis ATCC 11454 were evaluated in this study. The antimicrobial activity of the films against Staphylococcus aureus ATCC 6538, and Escherichia coli ATCC 25922 was evaluated. Cell-free supernatants were obtained by centrifugation and filtration of Lactococcus lactis cultures in MRS broth; the filtrates were freeze-dried, and rehydrated (4, 8, or 12 mg/mL) to produce collagen film. The thickness, tensile strength, color, moisture content, solubility, and water vapor permeability of the films were measured to examine the effects of cell-free supernatants on film matrices. Noticeable antimicrobial activity was observed against Staphylococcus aureus, and Escherichia coli upon the addition of 12 mg/mL cell-free supernatants. Our results indicate that the incorporation of cell-free supernatant changed the physical properties of the edible films. Gradual and significant (p < 0.05) increase in color difference and solubility were observed with the addition of increasing concentrations of cell-free supernatant. Antimicrobial films were brown in color and with variable moisture contents. Sodium alginate films were better than sodium carboxymethylcellulose films for most of the evaluated parameters. In summary, collagen films containing cell-free supernatants are interesting alternative natural antimicrobial films for functional food packaging.

Introduction

There is currently an urgent need to replace petroleum-based polymeric materials which represent a big environmental concern due to their non-renewability and poor biodegradability [1]. Current packaging materials not only consume our limited natural resources, but also lead to different kind of waste. About 8 million metric tons of plastic end up in the ocean every year, threatening not only the surrounding environment, but also human health [2]. The development of renewable materials that conform to the principles of sustainability has become a necessity [3]. On the other hand, pathogenic microbial contamination and infections through surface contacts of solid materials is a major public health concerns. In these contexts, there has been a growing interest in active packaging of food including the search for biopolymers and natural antimicrobial compound in recent years [4]. Antimicrobial and bioactive edible films represent eco-friendly food packaging because they are constructed with natural polymeric ingredients such as sodium alginate (SA), sodium carboxymethyl cellulose (CMC), and collagen (COL). SA, a natural polysaccharide, is usually extracted from brown algae, but it is also found in some bacteria. SA can be used as a thickening, emulsifying, or stabilizing agent in suspensions, gels, films, and textile fibers [5]. Additionally, SA is widely used in flocculation, and in the manufacturing of detergents, paper, foods, and drugs. Biotechnological applications of SA are based on its instantaneous and almost temperature-independent physical cross-linking and its sol/gel transition in the presence of multivalent cations in aqueous medium [6]. The process is simple and cost-effective, resulting in a physical gel with highly tunable mechanical properties [7]. CMC, an important industrial biopolymer obtained from cellulose, is highly viscous, non-toxic, and non-allergenic. CMC is used in the manufacturing of foods, papers, cosmetics, and drugs. COL (pI = 6–7) is a structural protein and component of the extracellular matrix [8]. It possesses good biocompatibility, low antigenicity, and high film-forming ability, and is widely used in edible-protein membranes.

Lactic acid bacteria (LAB) typically produce a wide range of antimicrobial substances such as lactic acids, acetic acids, hydrogen peroxide, reuterin, nisin, pediocin, or other bacteriocin-like substances [9], [10]. Lactococcus lactis (L. lactis) is one of the important model bacteria of lactobacillus, it is widely used in the food industry and generally regards as safe (GRAS) [11]. Cell-free supernatants (CFS) could act as appropriate antimicrobial agents as they contain a wide variety of antimicrobials. Potential uses of CFS of LAB have been suggested. Beristain-Bauza et al. reported the anticancer activity of three Lactobacillus rhamnosus strains SHA111, SHA112, and SHA113 isolated from human breast milk [12]. De Marco et al demonstrated the possible use of probiotic metabolites as adjuvant in anti-inflammatory therapy for the prevention and amelioration of intestinal inflammation [13]. As reported by Quinteiro-Filho et al, the CFS of Lactobacillus acidophilus, Lactobacillus reuteri, and Lactobacillus salivarius increased macrophage NO production without affecting the phagocytic activity [14]. LAB are potential antimicrobial agents based on the diversity and amounts of metabolites present in their CFS. Moreover, CFS has been considered a natural antimicrobial agent [15]. Poornachandra Rao et al evaluated the CFS of Lactobacillus plantarum strain MYS44 against the growth and aflatoxin production of Aspergillus parasiticus MTCC 411, and the findings of the study suggest thatthe CFS of LpMYS44 has potential antifungal activity [16]. Rishi et al. evaluated the effect of the CFS of Lactobacillus paraplantarum combination with conventional antibiotics against ampicillin- and oxacillin-resistant Staphylococcus aureus (S. aureus) strain. Furthermore, Aminnezha et al developed the CFS of Lactobacillus casei into a promising prophylactic agent against various pseudomonal infections [17], [18]. Beristain-Bauza et al. evaluated the antimicrobial activity of whey protein isolate films containing the CFS of Lactobacillus sakei on beef inoculated with Escherichia coli (E. coli) and Listeria monocytogenes (L. monocytogenes). In the study, the antimicrobial films reduced 1.4 log10 CFU/g of L. monocytogenes after 120 h, while E. coli decreased the microbial load of L. monocytogenes to 2.3 log10 CFU/g after 36 h. Grilled beef wrapped with antimicrobial film was well accepted by panelists; however, scores indicated no significant differences between wrapped and unwrapped beef [19].

The development and application of antimicrobial and bioactive edible films for food packaging is a growing research area. Based on the results of previous studies, the CFS of LAB could inhibit the growth of harmful microorganisms, and therefore, it could be incorporated into packaging films. The aim of this work was to evaluate the antimicrobial activity and physical properties of SA and CMC films containing CFS of L. lactis. The antimicrobial activity of the films was evaluated against the target bacteria S. aureus, and E. coli.