Preparation of anti-Listeria films by using methyl cellulose, sodium caseinate biopolymers and Lactobacillus casei PTCC 1608

The environmental problems of plastic materials have increased the importance of renewable edible films. In the manufacture of edible films, hydrocolloid compounds such as methyl cellulose and sodium caseinate are usually employed. Methyl cellulose is a biopolymer, soluble in water, and has substantial inhibitory properties against oxygen and lipids. Sodium caseinate has favorable mechanical properties and a high inhibitory effect on air transmission. Furthermore, it possesses good nutritional value. One of the most notorious pathogenic bacteria in food is the Listeria spp. which hastens food decay. Listeria monocytogenes is one of the most harmful species of bacteria that causes disease in humans. Lactic acid bacteria can inhibit the growth of cells that cause decay in food. The use of lactic acid bacteria in food has been made legal by the Food and Drug Administration of the United States of America (FDA). In this study, Lactobacillus casei PTCC1608 (L. casei 1608) was added directly to the film forming solution. The primary effect of L. casei 1608 and film type, i.e. sodium caseinate and methylcellulose polymers, were evaluated based on mechanical properties, water vapor permeability (WVP) and the opacity of films. Anti-listeria features of the films were studied under experimental conditions as the films were prepared against the growth of Listeria innocua IBRC-M10799 (L. innocua 10799). The L. innocua 10799 was selected because of its non-pathogenicity and its physiological similarity with Listeria monocytogenes.
L. casei 1608 and L. innocua 10799 were purchased from credible organizations. The bacteria were prepared as lyophilization form. In order to propagate and prepare bacterial suspensions of L. casei 1608 and L. innocua 10799, the cells were inoculated in the de Man Rogosa Sharpe Broth (MRS Broth, Liofilchem, Italy) and Blood Heat Infusion broth medium (BHI, Liofilchem, Italy), respectively. The cultures were then incubated at temperatures between 30 and 37°C, respectively, for 24 hour. The cells were harvested after centrifugation for 15 minutes at 3500 rpm (Eppendorf 5810 model, Germany). The collected medium was washed twice with a saline buffer solution. The population growth curve of L. casei 1608 and L. innocua 10799 were obtained using the pour plate method and by using the UV-VIS spectrophotometer device (Cecil model, England) at OD= 600nm. In order to prepare the film formation solution of methyl cellulose (CAS 9004-67-5, Sigma-Aldrich, Madrid, Spain), four grams of powder and 1 g of glycerol were mixed with 100 ml sterilized distilled water. After homogenization and degassing, the film forming solution was transferred to the plastic container. To prepare the film formation solution of sodium caseinate, 5 g of sodium caseinate powder (CAS 9005-46-3, Sigma-Aldrich, Madrid, Spain) was added to distilled water along with 1.5 g of glycerol. The solution was planned to reach 100 ml and, after complete dissolution, the film formation solution warmed up to 85°C and then cooled. To prepare the bio-film, L. casei 1608 was added to the film forming solution until the number of L. casei 1608 in the dried film reached 106 CFU/cm2. The film formation solutions were transferred to plastic containers and were dried at 25°C for 24 hours. All films were placed in a desiccator at 25°C and 75% RH for one week. In order to evaluate the anti-listeria activity, the bio-films were put on tryptic soya agar (TSA) medium, inoculated with L. casei 1608 (102 CFU / cm2) for 12 days at 5°C. The number of pathogenic bacteria and L. casei 1608 were counted on days 0, 4, 8 and 12. Also, the prepared films were evaluated according to the ASTM D-882 (ASTM, 2001) as per the ASTM D-882 instruction manual (Testometric, Model M350-10CT, UK). WVP was determined according to the ASTM-96-95. The opacity of the films were assessed by using the spectrophotometric apparatus according to the method described by Nu nez-Flores et al. (2012). Comparing the mean values of data was based on a factorial experiment in a completely randomized design (CRD) with three replications, by the SAS software version 9.1. The chemical and microbial mean values were calculated using Duncan’s and Tukey’s tests, respectively.
Data analysis showed that the bio-caseinate film hosted the highest survival rate of L. casei 1608 on the eighth day of culture (123.8%) when L. casei was concurrently present with L. innocua 10799. Analysis of data by the bio-methyl cellulose film showed that the strongest inhibitory effect on the growth rate of L. innocua 10799 was observed on the fourth day of the 12-day experimental duration (P0.05). Laboratory evaluations showed that the presence of lactic acid bacteria in films significantly increased the transparency, WVP and amount of elasticity. Nonetheless, a significant decrease in tensile strength and modulus of elasticity was observed. The type of matrix had a significant effect on mechanical properties. By comparing the methyl cellulose matrix and the sodium caseinate, it was observed that the methyl cellulose matrix showed significantly greater elasticity, tensile strength and higher modulus of elasticity while having less WVP and less opacity (p