Importance and efficiency of in-depth antimicrobial activity for the control of listeria development with nisin-incorporated sodium caseinate films
Introduction
Antimicrobial agents have received a great deal of attention in recent years both from scientists and food industries due to their potential application as food preservatives. To inhibit growth of undesirable microorganisms in food during storage, antimicrobials can be incorporated directly into the product formulation, coated onto the surface of food or incorporated into packaging materials. Direct incorporation of antimicrobials into food results in an immediate and short-term reduction of bacterial populations, while antimicrobial films could maintain their activity, thus slowing down bacterial growth during extended storage after packaging (Hoffman, Han, & Dawson, 2001).
Among available commercial antimicrobials, chitosan and nisin are probably the most used in the food industry as food biopreservative worldwide. Nisin is a peptide bacteriocin produced by certain strains of Lactococcus lactis, and is the only bacteriocin recognized as safe by the World Health Organization for the food industry. It exhibits antimicrobial activity toward a wide range of Gram-positive bacteria, in particular against foodborne pathogens such as Listeria mococytogenes, Staphyloccocus aureus or Bacillus cereus (Brewer et al., 2002, Lopez-Pedemonte et al., 2003, Sobrino-Lopez and Martin-Belloso, 2006). It shows, however, little or no activity against Gram negative bacteria, yeasts and moulds.
Nisin incorporation into various kinds of films such as cellulose, nylon, whey protein isolate, hydroxypropyl methylcellulose and zein films. and the use of packaging films as nisin delivery systems to reduce undesirable bacteria in foodstuff has been the objective of many studies (Chollet et al., 2008, Coma et al., 2001, Gadang et al., 2008, Ko et al., 2001, Kristo et al., 2008, Natrajan and Sheldon, 2000, Neetoo et al., 2008, Nguyen et al., 2008, Teerakarn et al., 2002). Nisin effectiveness in food product is based on its diffusion throughout the food matrix, which depends on several parameters such as composition and physico-chemical properties of food and storage temperature (Carnet Ripoche, Chollet, Peyrol, & Sebti, 2006).
Cheese is one of ready-to-eat products which is considered as “potentially hazardous food”. The probability of consuming a contaminated cheese was 65.3% (Bemrah, Sanaa, Cassin, Griffiths, & Cerf, 1998). Depending on the type of cheese (density of the gel, moisture … ), of the origin of contamination (from raw materials, during the cheese making or ripening), and of the contaminant species (L. monocytogenes, Salmonella spp., Escherichia coli, S. aureus … ), pathogenic bacteria can multiply more or less towards the interior of the product. Among pathogenic microorganisms of most concern to cheese makers as Listeria or Samonella spp., etc., L. monocytogenes represents the highest risk because both the severity and the probability of listeriosis are high relatively to other foodborne diseases associated with cheese. Furthermore, it survives if not thrives under the conditions of cheese making and it is able to grow at refrigeration temperatures (i.e., cheese ripening and storage temperatures). It is thus of great importance to control cheese storage. To our knowledge, relatively little work has been done regarding the utilization of antimicrobial films for the inhibition of undesirable microorganisms in cheese during storage. Zottola, Yezzi, Ajao, and Roberts (1994) demonstrated that addition of nisin in Cheddar cheese resulted in significant reductions on the numbers of pathogenic bacteria in cheese after a 8-week storage at 23 °C and 37 °C. Scannell et al. (2000) investigated the immobilization of nisin and lacticin 3147 within packaging materials. The authors reported that the combination of antimicrobial packaging with modified atmosphere conditions and refrigeration temperatures reduced the levels of Listeria innocua and S. aureus by more than 2 log and 1.5 log units, respectively. Chollet et al. (2008) pointed out interactions between nisin and Emmental cheese slurry with antimicrobial activity when adding nisin directly to cheese. However, information on the effectiveness of nisin-coated films in inhibiting bacteria in cheese and the migration of nisin from active films to cheese matrix is still lacking. In dairy products, contamination can occur in the liquid milk, resulting in the inoculation of the whole product, or after milk gelation, in this case, contaminating cells remain in the vicinity of the contact area (Ly, Le, Belin, & Waché, submitted for publication). However, cell movement can depend upon the structure of the food matrix although this parameter has not been much investigated in real food products. Depending on the cell movement, a surface antimicrobial treatment can be more or less efficient. It is thus particularly important to evaluate the depth of both the efficiency of the treatment and the cell development. In the present study, we investigated the effect of films of sodium caseinate containing nisin on the inhibition of L. innocua from artificially contaminated cheese. Mini red Babybel® cheese was chosen as a model of semi-soft and non cooked cheese. This cheese has been used as a model of food mixture for development of microorganisms such as Listeria, Salmonella (Hallier-Soulier, Batisse-Debitte, & Badiche, 2005) or Staphyloccocus (Meyrand, Giraudon, Cavaud, & Rozand, 2000).
The antimicrobial activity produced by the films against evolution of L. innocua in experimentally contaminated cheese was determined during a 7-day storage at 4 °C under aerobic conditions. The influence of the food matrix on the growth of microorganism and also on the antimicrobial properties of the nisin film has been evaluated. For this purpose, two methods of inoculation, surface and in-depth inoculation, were developed in order to assess the effectiveness of the active films in controlling L. innocua present on the surface and in the interior of the packaged product.
Section snippets
Nisin solution preparation
Nisin was purchased from Sigma–Aldrich (Sigma Chemical, St. Louis, MO, USA). Nisin solution was prepared by dissolving 0.1 g of a commercial 2.5% nisin powder in 2 ml of 0.01 M HCl solution (pH = 2). This solution was then filtered through 0.2 μm-pore-size Millipore filters (Nalgene, Rochester, New York, USA) and stocked at 4 °C until use.
Preparation of nisin-coated sodium caseinate films
Films were prepared using a protocol described previously by Kristo et al. (2008). Solutions (6% w/v) of sodium caseinate were prepared by dissolving sodium
Zone inhibition assay
The antimicrobial properties of the films were evaluated by the apparition of inhibition or clear zones around the films after incubation. While sodium caseinate films with no added nisin exhibited no inhibition, an inhibitory zone of 13 mm diameter was observed around the nisin-coated film disc, corresponding to a total diameter of inhibition of 17 mm for an initial disc diameter of 4 mm. This result indicated diffusion of nisin from the films to the solid medium and subsequent growth inhibition
Discussion
Previous studies have shown that nisin may be efficiently incorporated into various kinds of packaging films and used for controlling pathogens in food products (Chollet et al., 2008, Coma et al., 2001, Gadang et al., 2008, Ko et al., 2001, Kristo et al., 2008, Natrajan and Sheldon, 2000, Neetoo et al., 2008, Nguyen et al., 2008, Teerakarn et al., 2002). Kristo et al. (2008) showed that among several antimicrobial films tested, nisin-containing sodium caseinate films were the most effective in
Conclusions
Nisin-containing sodium caseinate films were shown to be effective against L. innocua in cheese during refrigerated storage. Our study demonstrates the potential application of antimicrobial films as a promising method to overcome problems associated with post-process contamination, thus enhancing the safety and extending the shelf life of food products. Particularly, it was shown that although nisin did not migrate much inside the cheese matrix, its effect was sufficient for
Acknowledgments
We acknowledge the financial support from Bourgogne region (France). We also thank Frédéric Debeaufort, Pierre-André Marechal and Jean François Cavin for their scientific support, Christine Rojas for her technique assistance and Julia Hauck Tiburski for editing the English.
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2021, Carbohydrate Polymer Technologies and ApplicationsCitation Excerpt :Among the various dairy products, cheese has been most widely studied for shelf life enhancement through the application of edible antimicrobial films (Chawla, Sivakumar, Bansal & Singh, 2019). Application of nisin-supplemented sodium caseinate films in cheese stored at refrigeration temperature exhibited strong antimicrobial activity against L. monocytogenes (Cao-hoang, Grégoire, Chaine & Waché, 2010). Likewise, Cui, Wu, Li and Lin (2016) indicated effective inhibition of L. monocytogenes growth in fresh cheddar cheese at different storage temperatures (25 °C for 7 days and 4 °C for 15 days) due to the presence of chitosan coatings embedded with nisin-silica liposomes on cheese samples.