Characterization of probiotics as active agent in PLA packaging for counteracting spoilage in ricotta cheese

A probiotic strains, Lactobacillus plantarum IMC 509 (Synbiotec Srl, Camerino, Italy), and its cell-free supernatant (CFS) [1] were investigated as promising anti-spoilage components incorporated in wrapping polylactic acid (PLA) bags, in addition with graphene composite, to extend fresh ricotta cheese shelf-life in fridge. Lyophilized powder of L. plantarum IMC 509 (LP) was suspended in PBS buffer solution and the inner surface of a sterile PLA (under UV light) film was deliberately sprinkled with the homogeneous suspension (PLA + LP, PL as sample code), another sprinkled sample was added defined amount of graphene composite (PLA + GN + LP, PGL). The CFS was extracted from a 24-hours L. plantarum IMC 509 culture and then also sprinkled homogenously over a sterile PLA film, graphene composite was also added on this type of PLA film (PLA + GN + CFS, PGC). As control sample, simple wrapping PLA (under UV sterilization) bag have been used (PLA), second control sample was wrapped by using graphene composite incorporated on sterile PLA film (PLA + GN, PG). At each time point, during the 14-day storage, the total mesophilic aerobic bacteria, total lactic acid bacteria (LAB) and Pseudomonas spp. counts, the pH values, the sensory test and the free fatty acids contents were measured. In addition, the L. plantarum IMC 509’s viability on PLA packaging material was also monitored during time. PLA film was able to maintain low mesophilic bacteria counts up to 3 days storage, while the other packaging had little effects in limiting mesophilic bacteria increment during storage time. As for the total LAB, sample in PLA film demonstrated lower counts during experiment time, with only around 2.2 log increment. The total LAB have increased dramatically after 3 days in PL and PGL sample, due to the fact that these two packaging containing live L. plantarum IMC 509 on film surface, and their LAB concentrations kept quite high on the following days. Pseudomonas spp. is the most common and well known indicator of food spoilage, the results showed that the excellent capability of PGL film in limiting the increment of Pseudomonas spp., with only 1 log increment appeared after 9 days stored in fridge, therefore retarding the spoilage process of ricotta cheese. The pH values of ricotta cheeses stored in PLA and PLA sprinkled with L. plantarum IMC 509 did not change for 9 days in fridge, while a little drop was observed after 14 days. However, for the other samples, their pH values were dropped after 3 days, and this phenomenon kept for the following 9 days and increased a bit at the last time point. The sensorial characteristics of cheese in PGL-package had the best results among all samples, it kept its typical characteristics of fresh ricotta cheese even for 14-day of storage, while for the rest of samples, they lost their aspect, color, smell and texture dramatically. The investigated chemical markers (free fatty acid content), showed the biggest increment in PLA film while PG packaging film seemed the most effective compared to the other types of film. Finally, L. plantarum IMC 509 was able to maintain alive on PLA film and its viability slowing decreased to 80% (compared to the initial time point) after 50 days of storage. In the presence of graphene composite on film, the probiotics also survived up to 14 days. These data shows that L. plantarum IMC 509 could be alive and effective on PLA film for longer storage period in the presence of graphene composite or not, as an ideal natural substance to be applied on packaging. In conclusion, PLA film showed constrain ability in limiting the growth of mesophilic aerobic and total LAB, without changing cheese pH value. While PGL film demonstrated attractive properties in inhibit the growing of Pseudomonas spp. and conserving the characteristics of fresh ricotta cheese up to 14 days. However, PG film could maintain the level of free fatty acids. Previous works have illustrated the in vitro antimicrobial abilities of L. plantarum IMC 509 and its CFS, graphene composite [2] on some spoilage bacteria. In the present study, the application of this probiotic strain in combination with graphene composite effectively controlled Pseudomonas spp., further inhibiting the deterioration of cheese main characteristics for at least 14 days of refrigerated storage. To have a better understanding of their effects on other parameters, further tests are needed to conduct. This new approach is a promising alternative to replace chemical preservatives for fresh ricotta cheese preservation.