Skim milk powder

After treatment, cells were collected and resuspended in PBS, and mixed with isometric RIPA buffer containing 1% protease inhibitor at 4 °C for 30 min. After centrifuge at 10000 g for 5 min, the supernatant was mixed with equal volume of 2 × loading buffer and heated to 95 °C for 5 min. Proteins were loaded on polyacrylamide gel and run at stock gel (5%) for 15 min (90 V) and at separated gel (8%) for 70 min (130 V). Proteins were resolved on an 8% SDS/PAGE gel and subsequently transferred to a PVDF membrane (Millipore). After blocked with TBS buffer containing 5% skim milk powder (Oxoid) for 30 min, the membrane was incubated in TBS buffer containing 5% milk and primary antibodies against P-gp (1:2500, abcam), PARP-1 (1:1000, abcam), PAR (1:2000, abcam), β-tubulin (1:5000, abcam) for 2 h, respectively, and washed with TBS buffer (5 min × 5). The membrane was further incubated in TBS buffer containing 5% milk and secondary antibodies (1:5000, goat anti-rabbit, Pierce) for 1 h, and washed with TBS buffer (5 min × 5). Bands were visualized by WesternBright ECL (Advansta) and pictures were taken by an imaging system (GE Healthcare Bio-sciences AB). Multi-exposure images were obtained. The expression level of β-tubulin was used as a standard to normalize the expression of other proteins.

Proteolytic and lipolytic activities were evaluated according to Meng et al. [1 (link)] using Plate Count Agar supplemented with 1% (w/v) skim milk powder (Oxoid, Milan, Italy) and Tributyrin Agar (Merck, France) media, respectively. Exopolysaccharides (EPS) production was tested on both MRS and mMRS, in which glucose was replaced by 10% of sucrose, following the method described by Meng and co-workers [1 (link)]. The NSLAB strains’ ability to produce diacetyl was evaluated according to Ribeiro et al. [23 (link)]. Diacetyl production was indicated by the formation of a red ring at the top of the tubes. Based on the presence and intensity of the red colour, the strains were scored as no (−), moderate (+), high (++), or strong (+++) diacetyl producers.

Eighty strains representing four starter lactic acid bacteria species, Lactobacillus helveticus, Lactobacillus delbrueckii subsp. bulgaricus, Lactococcus lactis, and Streptococcus thermophilus (Table 1), were analyzed by impedance measurements. The strains, belonging to the collection of the Laboratory of Food Microbiology of the Department of Food Science of University of Parma, have been previously isolated from dairy matrixes and identified by16S rRNA sequencing.
Strains, maintained as frozen stocks cultures in MRS (Oxoid, Ltd., Basingstoke, United Kingdom) (Lactobacillus), or M17 (Oxoid Ltd.) (Lactococcus and Streptococcus) broth containing 20% (v/v) glycerol at −80°C, were recovered in MRS or M17 broth by two overnight sub-culturing (5% v/v) at 42°C for Lactobacillus and Streptococcus, and 30°C for Lc. lactis. Then, other 28 h sub-culturing (5% v/v) of each strain in skim milk powder (Oxoid Ltd.), reconstituted to 10% (w/v) and sterilized at 110°C for 30 min (SSM), were performed before use.