*3.2. Application as Potential Active Packaging*

Once ascertained the effects on pathogens growth, the research focused on the formation of the probiotic biofilm on different packaging materials, in order to individuate an innovative packaging system.

The results obtained are shown in Table 3; after only 2 h, the probiotic biofilm was successfully formed on all tested materials, except for waxed paper. The sessile cellular load ranged from 5.77 log CFU/cm<sup>2</sup> (grease-proof paper) to 6.94 log CFU/cm<sup>2</sup> (polyethylene). After 96 h, polyethylene and ceramic resulted the materials on which the highest adhesion was recorded (6.54 log CFU/cm2). In general, any surface (plastic, rubber, glass, metal, paper, cement, stainless steel or wood, or food products themselves) are vulnerable to biofilm development and each biofilm is different, thus suggesting that every situation should be analysed individually and specifically [38].


**Table 3.** Cellular probiotic load in sessile form (log CFU/cm2) observed on common packaging materials used in the food industry and on ceramic.

A, B, C, Values in the same columns with different letters are significantly different (one-way ANOVA and Tukey s test) (*p* < 0.05).

Once individuated in polyethylene (PE) the material able to ensure the greatest adhesion of probiotics, in a second step, the attention was focused only on this material and it was used to test the potential for probiotic biofilms to control the growth of microorganisms in soft cheeses. The products were inoculated with *L. monocytogenes* (challenge test A) and *Ps. fluorescens* (challenge test B), wrapped in PE pellicles with pre-formed probiotic biofim, packed both in air and under vacuum, and stored at 4 and 15 ◦C. These model bacteria were chosen as main representatives of pathogen and spoilage bacteria naturally contaminating soft cheese [39,40].

At 4 ◦C, the cellular load of *L. monocytogenes* remained lower than 3 log CFU/g for the entire observation period (28 days), regardless the presence of the probiotic biofilm or the packaging. On the other hand, at 15 ◦C (simulated thermal abuse), the λ length was always longer in samples containing probiotic biofilms (EXP samples), if compared to CNT samples (without probiotic biofilms) (Table 4): its value increased from 0.04 to 3.37 days (in air packaging, Figure 1A) and from 0.00 to 2.40 days (under vacuum, Figure 1B). The growth rate (μmax) was also influenced by the presence

of probiotic biofilms, recording a decrease from about 0.7 to 0.4 Log(CFU/g)/day, in both packaging conditions. The maximum cell load reached in the stationary phase (A + N0) was not influenced, reaching approximately 5.6–5.7 log CFU/g, regardless of the presence or absence of probiotic biofilms. The cellular load of lactic bacteria (LAB) was also monitored, as well as pH and water activity. At 4 ◦C, the initial LAB count was 5.75 ± 0.18 log CFU/g in the control samples against 8.32 ± 0.20 log CFU/g in the experimental cheeses; after 28 days, there were no statistically significant differences between the samples (regardless of the presence of probiotic biofilms and the type of packaging), recording cellular loads between 7 and 8 log CFU/g (data not shown).

**Table 4.** Kinetic parameters calculated by fitting Gompertz equation to the experimental data by *L. monocytogenes* and *Ps. fluorescens* during their growth in soft cheeses with (EXP) or without (CNT) probiotic biofilms, packed in AIR o under vacuum (UV) and stored at 15 ◦C. (A + No) is the maximum bacterial load attained at the stationary phase, μmax is the maximal growth rate, λ is the lag time, TRS is the sanitary risk time, ST (stability time) is the maximum acceleration of microbial growth.


A, B, Values in the same columns with different letters are significantly different (one-way ANOVA and Tukey s test) (*p* < 0.05). \*, TRS, sanitary risk time, i.e., the time required (in days) to observe an increase of 2 log CFU/g in *L. monocytogenes* count [30]. \*\*, stability time, i.e., the maximum acceleration of microbial growth [dy2/dt<sup>2</sup> (day)] [31].

**Figure 1.** Evolution of *L. monocytogenes* during the challenge test at 15 ◦C. EXP, cheeses stored with probiotic biofilms; CNT, cheeses stored without probiotic biofilm. (**A**), AIR packaging; (**B**), under vacuum packaging (UV).

Additionally, for the pH, no significant differences between the samples were observed; this parameter decreased from 5.31–5.39 to 4.70–4.84 at the end of storage. In all samples, the value of water activity remained constant (0.99–1.00) for the entire duration of the experimentation (data not shown). Similar results were observed at 15 ◦C.

During the experimentation, both at 4 and 15 ◦C, a gradual decrease of the score from 10 to about 5.5–6 was recorded (end of storage), regardless of the presence of probiotic biofilms and the type of packaging applied, showing that the probiotic microorganisms had no impact on the sensory characteristics of cheeses; as an example, Figure 2 shows the sensorial scores for colour, odour, texture and overall acceptability of cheeses recovered during storage at 4 ◦C.

**Figure 2.** Sensorial scores for colour (**A**), odour (**B**), texture (**C**) and overall acceptability (**D**) of cheeses inoculated with *L. monocytogenes* stored at 4 ◦C. Mean values ± standard deviation. EXP, cheeses stored with probiotic biofilms; CNT, cheeses stored without probiotic biofilm.

Figure 3; Figure 4 show the evolution of *Ps. fluorescens* during the growth on EXP and CNT cheeses stored at 4 and 15 ◦C, respectively. The target microorganism was able to grow under all tested conditions, regardless of the presence of probiotic biofilms and the type of packaging. At 4 ◦C (Figure 3), the presence of the probiotic biofilm was able to influence the maximum cellular load reached in the stationary phase (A + N0), which was significantly lower in the EXP samples (5.59–5.72 log CFU/ g) compared to the CNT samples (6.36–6.39 log CFU/g). No influence was observed about the λ length and the maximum growth rate (μmax).

**Figure 3.** Evolution of *Ps. fluorescens* during the challenge test at 4 ◦C. EXP, cheeses stored with probiotic biofilms; CNT, cheeses stored without probiotic biofilm. (**A**), AIR packaging; (**B**), under vacuum packaging (UV).

**Figure 4.** Evolution of *Ps. fluorescens* during the challenge test at 15 ◦C. EXP, cheeses stored with probiotic biofilms; CNT, cheeses stored without probiotic biofilm. (**A**), AIR packaging; (**B**), under vacuum packaging (UV).

During storage at 15 ◦C (Figure 4), the presence of probiotic biofilms significantly slowed the growth of the target microorganism: λ increased from 0.54 to 4.40 days and from 0.01 to 3.30 days, in air and vacuum packaging, respectively. The maximum growth rate and the maximum cell load reached in the stationary phase were also lower in the EXP samples (probiotic biofilms) than the control samples, regardless of the packaging applied.

At both 4 and 15 ◦C, data on LAB, pH and water activity were similar to those observed in the challenge test with *L. monocytogenes* (data not shown). Results of sensory analyses confirmed that the probiotic microorganisms had no impact on the organoleptic characteristics of cheeses (data not shown).

To highlight the effectiveness of probiotic biofilms to slow the decay of the microbiological quality of soft cheeses at 15 ◦C, Table 4 shows the kinetic parameters of Gompertz equation accompained by two other parameters (TRS and stability time). In a well-known study on the growth of *L. monocytogenes* in food, Castillejo Rodriguez et al. [30] have proposed the sanitary risk time (TRS) for this pathogen as the time required (in days) to observe an increase of 2 log CFU/g in its count, considering that, under normal conditions, such a microorganism is present in foods in very low concentrations. As can be seen, for soft cheeses wrapped in probiotic biofilms and packaged both in air and under vacuum, the TRS was equal to 4.40–4.60 days; on the contrary, the same methods of packaging, applied to the control samples, allowed *L. monocytogenes* to reach risky cell counts in shorter times (2.88–2.95 days) (*p* < 0.05).

For the tests conducted with *Ps. fluorescens* at 15 ◦C, Table 4 also shows the stability time [31,41] which represents the maximum acceleration of microbial growth and indicates how long the product remains stable: after this time, an irreversible decay of the product begins. This parameter is generally used as an alternative to shelf life: the underlying principle implies that microbial degradation has to show a rate of the same order of magnitude as at the shelf life zero time. This condition is no longer met when microbial growth attains its maximum acceleration, because beyond such a threshold the system undergoes very fast changes with a rapid loss of the generally accepted safety or quality requirements. This principle seems more reliable than the current practice that defines food stability according to the ratio between attained and starting microbial population levels. The stability time increased by more than 3 and 4 days, in vacuum packaging and in air, respectively, highlighting the effectiveness of biofilms in slowing the decay of the microbiological quality of soft cheese.

Regarding the inhibitory effect of LAB against *L. monocytogenes*, some studies have already explored the possibility to use a preformed biofilm to inhibit the pathogen growth [15,16,18,42]. Namely, Guerrieri et al. [15] showed the potential of a *Lactobacillus plantarum* strain to reduce the pathogen growth over a 10-day period (about 4-log reduction). Mariani et al. [42] used the native

biofilm microflora of wooden cheese ripening shelves to achieve a 1- to 2-log reduction over a 12-day period. In previous studies, we have evaluated the use of LAB biofilms as a means to control the growth of *L. monocytogenes* in soft cheeses [16] and in laboratory media [18], finding that sessile LAB biofilms were able to delay the growth of *L. monocytogenes.* An anti-listerial activity was also observed by Léonard et al. [43] during their studies on biopolymeric matrices based on alginate and alginate-caseinate (an aqueous two-phase system) entrapping *Lactococcus lactis* subsp. *lactis* LAB3 cells and by Barbosa et al. [44] who entrapped *Lactobacillus curvatus* in calcium alginate: the effect against the pathogen was correlated to antimicrobial metabolites of proteinaceous nature.
