Strawberries were inoculated at 10⁴ CFU mL^-1 with B. cinerea and 10⁵ CFU mL^-1 with R. stolonifer (Camacho and Nieto, 2017). Sodium hydroxide solutions pH of 11, 12 or 13 were prepared by adding and dissolving NaOH in distilled water, under constant stirring, until the desired pH was reached. Afterwards, the fruits were placed in NaOH solutions pH 11, 12 or 13 and immediately were rinsed with distilled water. Strawberry surface swabbing was performed for microbiological analysis. Microbial growth was reported as CFU of B. cinerea and R. stolonifer, according to the methodology described by "Norma Técnica Ecuatoriana" NTE INEN 1529-10:2013 (INEN, 2013). All analyses were performed in triplicate.

Strawberries were inoculated at 10⁶ CFU mL^-1 with both Salmonella and E. coli (Ledesma et al., 2018). Afterwards, the fruits were immersed in alkaline solutions pH 11, 12 or 13 during 20, 40 or 60 min. A strawberry surface sampling was performed (previously described) and microbial growth was reported as CFU.

Analysis of inhibition was determined according to EUCAST (2013) with slight modifications. Petri dishes were inoculated at 10⁴ CFU mL^-1 with B. cinerea and 10⁵ CFU mL^-1 with R. stolonifer (Camacho and Nieto, 2017) using Sabouraud dextrose culture medium, whereas Salmonella spp. and E. coli were inoculated both at 10⁶ CFU mL^-1 in Salmonella-Shigella agar (HiMedia Laboratories, India). Afterwards, Petri dishes were incubated at 37 °C for 2 days. An amount of 20 µL of alkaline solution was added to filter paper disks (Fisher Scientific Q2) of 5 mm diameter. The disks were placed in the centre of the Petri dish, previously prepared and incubated at 25 °C with both B. cinerea and R. stolonifer for 24 h and Salmonella spp. and E. coli at 37 °C for 24 h. The zones of inhibition of microorganisms growth were measured after incubation and reported as mm of inhibition. Analyses were performed in triplicate.

Data were subjected to ANOVA and the significance of the difference between means was determined by Tukey test (P<0.05) with InfoStat statistics software (Infostat version 2014, Argentina). All measurements were performed in triplicate.

Results of the effect of alkaline solutions against B. cinerea showed differences among the three pH (P<0.05). The smallest zone of inhibition was obtained by a pH 11 solution, with a diameter of 8 mm, whereas the largest zone by a pH of 13 with a diameter of 11.67 mm (Table 1). An increase of pH led to 1-log reduction from pH 11 to 12. Regarding R. stolonifer, there was no difference on the inhibition zone among the three pH (P<0.05) and additionally, the increase of pH did not cause a log reduction in CFU mL^-1.

Table 1

Similar results of inhibition were obtained by Ahlem et al. (2012), showing that an alkaline pH 10 gave a better inhibition of B. cinerea than a lower pH. Besides, Qin et al. (2010) showed the effectiveness of NaOH solution to control B. cinerea on table grapes.

Inhibition of B. cinerea and R. stolonifer in the presence of an alkaline solution could be due to a drying effect of the microorganism resulting from osmotic dehydration. In fact, salinity affects microbes via osmotic effect by drawing water out of cells which may kill microbes through plasmolysis (Oren, 1999).

Effect of pH. There were no differences on the zones of inhibition of Salmonella spp. (Table 2), whereas differences were found for E. coli (P<0.05), with zones of inhibition of 11.56 and 12.11 mm, when solutions at pH 11 and 12, respectively, were used. Smaller zones (10.33 mm) were obtained using solutions pH 13. The highest pH values led to a higher inhibition in Salmonella spp. In fact, salts have been used to control Salmonella spp. in food (Aspridou et al., 2018; Tiganitas et al., 2009). Zhou et al. (2011) observed that Salmonella suffers an initial decline in cell numbers when inoculated into a high salt concentration medium. However, when the stress is not lethal, the cells could adapt and subsequently grow under the new condition. Similar studies in sub-lethally stressful environments reported that cell populations suffered an initial loss followed by a recovery (Mellefont et al., 2005). Differences of zone of inhibition between pH 13 and lower pH (11 and 12) may not reflect real differences since longer times of analyses may be needed to guarantee a full recovery of cell population.

Table 2

Effect of immersion time. Table 3 shows that there was no growth of Salmonella spp. for the three immersion times in the pH 13 solution. The 20 min immersion in a pH 11 solution showed the highest CFU counting. Regarding E. coli, there were differences of CFU counting among different immersion times (P<0.05). Treatment of pH 13 for 20 min showed the highest CFU counting (5.92x10⁶ CFU mL^-1) and pH 13 for 60 min showed no growth. Sampathkumar et al. (2003) showed a reduction of CFU of Salmonella enterica when pH was increased of 10 to 11 within 20 min of exposure to alkaline solutions, whereas Gill et al. (2019) observed a reduction of Salmonella enterica population after exposure to NaOH solution pH 11 for 2 h. Different results may be due to the use of a different strain. The difference in growth among the bacterial species examined, could be due to different strategies to cope with osmotic stress (Wood, 2007).

Table 3