2.2.1. Probiotic Biofilm Formation on Different Materials

The materials assayed were polypropylene (PP), polyvinyl chloride (PVC), greaseproof paper (GP), waxed paper (WP), polyethylene (PE) and ceramic; all materials were cut in rectangles of 2.5 × 5.0 cm and cleaned by immersion in ethanol. Each individual chip was well rinsed with ultrapure water and dried at room temperature. Probiotic biofilms were left to form for 96 h by simultaneously inoculating the cocktail of probiotics (*B. infantis* DSM20088 and *L. reuteri* DSM20016, about ~108 CFU/mL) on chips of different materials, left at room temperature (20 ◦C) for 2 h. After this time interval, the chips were transferred to aliquots of peptone water (1% bacteriological peptone) and incubated at 15 ◦C for 96 h [19].

Biofilm cells were enumerated at 2, 24, and 96 h after inoculation. At these times, chips were aseptically removed and rinsed with sterile distilled water, in order to eliminate the unattached cells. Sessile cells were detached from chips in a sterile test tube containing 45 mL of sterile saline with a "Vibra Cell" sonicator (SONICS, Newcastle, Conn., USA) at 20 kHz for 3 min. Viable and cultivable cells were enumerated by serial dilutions in 0.9% NaCl solution and plating on MRS Agar (Oxoid).

#### 2.2.2. Challenge Tests

Inoculations for experiments were prepared by centrifugation of the 24-h microbial cultures in an ALC 4239R centrifuge (ALC, Milan, Italy) at 3000× *g* for 15 min at 4 ◦C. For the inoculations of challenge tests, after centrifugation the pellets were resuspended in sterile isotonic solution (0.9% NaCl) at a temperature of 4 ◦C and serial dilutions were made to obtain approximately 104 CFU/mL for each microorganism. For biofilm formation, the probiotic pellet was resuspended in sterile isotonic solution at a temperature of 4 ◦C and used on polyethylene films to form experimental pellicles with pre-formed probiotic biofilm (EXP).

Miniature soft cheeses were made using pasteurized, whole and homogenized milk, purchased in a local market. The milk had the following characteristics: lactose 5.0%, protein 3.2%, fat 3.6%, pH 6.6. The cheeses were produced using a domestic cheese-maker ("Casaro", Philips, Milan, Italy) by pouring the milk into the single-wall cheese-maker vessel and heating to 85 ◦C. As soon as the temperature reached 85 ◦C (after a few minutes), 4 g/L of sodium chloride was added and the salted milk was immediately left to cool to 30 ◦C. Renneting was performed with 3 mL/L of liquid calf rennet (concentrate extract of Liquid Rennet, CHR. Hansen s.p.a., Milan, Italy). After coagulation and curd strengthening (approximately 40 min), the curd was cut and the whey discarded. Finally, miniature soft cheeses of a round shape (25 g, 6 cm diameter) and regular smooth surfaces were made by hand and placed in sterile boxes fitted with a grid to facilitate whey draining. The boxes were kept at room temperature for 6 h until packaging.

To test the potential for probiotic biofilms to control the growth of microorganisms in soft cheese, they were inoculated with *L. monocytogenes* (challenge test A) and *Ps. fluorescens* (challenge test B). The inoculation (about 10<sup>2</sup> CFU/g) was carried out in the most homogeneous way possible, spreading 0.2 mL of the prepared microbial suspension across the entire surface of miniature cheese by means of a

sterile spatula. After inoculation, all cheeses were wrapped in polyethylene films (EXP) and packed in high-barrier plastic bags (Nylon/Polyethylene, 102 μm (Tecnovac, San Paolo D'Argon, Bergamo, Italy)) by means of S100-Tecnovac equipment. Control batches were prepared by wrapping cheeses in pellicles without pre-formed biofilm (CNT). All samples were packaged in air and under vacuum. During the storage at 4 and 15 ◦C for 28 and 14 days, respectively, microbiological analyses, determination of pH and measurements of *aw* were made, details of which are given below.
