The stress conditions i.e. 3% w/v NaCl for prior salt exposure and no lactose for lactose starvation were created for 24 hours at 30˚C in M17 broth. The control did not have any stress condition. After subjection to stress conditions or no stress condition (control) the cultures were transferred to five NaCl concentrations (0, 1, 3, 5 and 7% w/v). Bacterial counts were determined every 24 hours for 5 consecutive days. The experiments were conducted in duplicate and repeated three times. Data were analyzed as a complete randomized split plot design with repeated measures over time.

The M17 broth was used for all samples and it was prepared according to manufacturer’s specifications as follows: 37.25 grams of M17 broth powder (Fisher Scientific, Fair Lawn, NJ) was diluted in 1L of distilled water. Then 94 mL of the solution was poured into different bottles and sterilized at 121˚C for 15 min at 15 psi. A 10% w/v lactose solution (Fisher Scientific, Fair Lawn, NJ) was prepared separately and sterilized under the same conditions. After autoclaving, 5 mL of the sterile lactose solution was aseptically added into the sterile M17 bottles except for lactose starvation. Then the bottles were tempered at 30˚C in an aerobic incubator until temperature of 30˚C was reached. M17 agar supplemented with 0.5% lactose was used for all samples.

Pure culture of Lactococcus lactis DVS culture R-604 (CHR HANSEN, Milwaukee, WI) was thawed and inoculated in M17 broth supplemented with 0.5% w/v lactose and immediately plated (Day 0) and then incubated aerobically for 24 hours at 30˚C. Then 1 mL sample of this dilution was transferred to five different bottles containing sterile M17 broth with NaCl (Fisher Scientific, Fair Lawn, NJ) at 0, 1, 3, 5, and 7% w/v. The inoculated M17 broths with the five salt concentrations were incubated aerobically at 30˚C [18] for 5 days. Samples were drawn immediately after inoculation and every 24 hours for 5 days. Samples were plated using M17 agar with 0.5% w/v lactose. Pour plates were aerobically incubated at 30˚C for 3 days. Colonies were counted after the 3 days incubation.

Pure culture of Lactococcus lactis R-604 was thawed and inoculated in M17 broth containing 3% w/v NaCl and 0.5% lactose and immediately plated (Day 0) and then incubated aerobically for 24 hours at 30˚C. After this exposure to salt for 24 hours, which was the prior salt exposure condition, 1mL of sample was transferred to five different bottles containing sterile M17 broth supplemented with 0.5% lactose and five concentrations of NaCl at 0, 1, 3, 5, and 7% w/v. The inoculated broths with the five salt concentrations were incubated aerobically at 30˚C for 5 days. Samples were taken immediately after inoculation and every 24 hours for 5 days. Bacterial counts (CFU/mL) were determined by plate counting in M17 agar supplemented with 0.5% w/v lactose. Pour plates were incubated aerobically at 30˚C and counted after 72 hours.

Pure culture of Lactococcus lactis R-604 was thawed and inoculated in M17 broth containing no lactose and immediately plated (Day 0) and then incubated aerobically for 24 hours at 30˚C. After this deprivation of lactose for 24 hours, which was the lactose starvation stress condition, 1mL of sample was transferred to five different bottles containing sterile M17 broth supplemented with 0.5% lactose and five concentrations of NaCl at 0, 1, 3, 5, and 7% w/v. The inoculated broths with the five salt concentrations were incubated aerobically at 30˚C for 5 days. Samples were taken immediately after inoculation and every 24 hours for 5 days. Bacterial counts (CFU/mL) were determined by plate counting in M17 agar supplemented with 0.5% w/v lactose. Pour plates were incubated aerobically at 30°C and counted after 3 days.

Data were analyzed as a complete randomized design split plot with repeated measures over time using Proc Glimix of SAS (version 9.3 SAS Institute Inc., Cary, NC). Stress conditions, salt concentrations, and time of exposure were fixed effects, whereas replicates were random effects. Statistical analysis was done separately for each group of experiments. Significant differences were tested with least square means at P < 0.05 for main effects (stress conditions, time and salt concentration) and the interaction effect (stress conditions × salt concentrations × time of exposure). Significant differences (P < 0.05) between pair comparisons of interest were analyzed using a LS Means.

The growth of L. lactic R-604 under different salt concentration after 24 hours of incubation under ideal conditions (M17 broth supplemented with 0.5% lactose at 30˚C) is shown in Table 1. All samples had an exponential growth of 2 logs during the 24 hours (1 day) incubation period. For 0, 1%, 3% and 5% an exponential phase was observed during days 1 and 2, while for 7% the exponential phase was observed only at day 1. Stationary phase was observed at day 3 for 0, 1 and 5%, while their decline phases were observed at days 4, 5, 6. For the 3% the decline phase started at day 3 and continued to day 6. On day 6 the bacterial counts at 5% were significantly the lowest compared to 0, 1%, 3% and 7%.After day 1, the cells at 7% w/v NaCl did not grow as they did not show any significant change in counts over the 6 days. The growth inhibition produced by the sudden osmotic stress could cause the loss of cell turgor pressure and therefore its dehydration. Thus affecting the bacteria’s primary functions and reducing the reproduction rate [4] . The accumulation of compatible solutes has been proposed as a mechanism to overcome hyperosmotic conditions for many microorganisms [19] , particularly glycine betaine and proline for lactic acid bacteria [20] [21] .

There were no significant (P > 0.05) differences in bacterial counts between the cells prior exposed to NaCl and the cells that were not (control) (Table 1). According to Kilstrup et al. [22] , cells usually reduce metabolic activity in order to start an adaptation process. Kim et al. [18] showed that cells in stationary phase are more resistant to stress than cells in exponential phase. Cells in this study were in stationary phase at the time of transfer to the different salt concentrations. Kim et al. [18] stated that in order to generate an adaptation response cells must be subjected to a mild stress for an adequate amount of time, thus allowing them to synthetize necessary proteins for stress adaptation. These data show that prior exposure to 3% NaCl for 24 hours was not detrimental for the growth of L. lactis R-604.

Lactococcus lactis R-604 cells after lactose starvation grew at the same rate as Control regardless of the salt concentration. The only significant (P < 0.05) difference found was on day 3, at 3% w/v NaCl (Table 1), where the lactose deprived cells showed 1 log higher counts than the Control, at the same salt concentration on the same day (Table 1). The main proposed explanation for the cross-protection that starvation stress can confer to bacteria is the pattern change in gene expression. This leads to different polypeptides synthesis, stress induced proteins, that are produced under more than one environmental stressor [16] . However, a study involving glucose starved organisms failed to improve resistance to hydrogen peroxide stress, since the specific KatA and KatE catalase enzymes were lower than the catalase activity generated after an oxidative adaptation [23] .

Hartke et al. [14] showed that resistance to heat, ethanol, low acid and hydrogen peroxide can be achieved by stationary phase cells of L. lactis grown under lactose starvation. Other microorganisms such as Enterococcus faecalis and Listeria monocytogenes, after complete glucose starvation, can develop resistance to heat, hydrogen peroxide, bile salt and sodium chloride [17] [24] . Jenkins et al. [25] showed that E. coli can develop resistance to osmotic shock at 2.5% w/v NaCl after 4 hours of glucose starvation. Other ways to induce starvation stress include, growing the cells in suspension media such as saline solution (0.08% w/v NaCl), peptone water, or Phosphate Buffer Saline solution (PBS). However, Hartke et al. [14] stated that small amounts of carbohydrates (0.1% w/v glucose) in media can be used rapidly by cells and trigger stationary phase quicker and therefore helping the cells to adapt better to further stress conditions.

It must also be considered, that during starvation treatments amino acids must be present in media as a precursor for protein synthesis to obtain better stress adaptation [7] . Prior exposure of L. lactis R-604 in 3% w/v NaCl or lactose starvation and subsequent use of treated cells in up to 5% w/v NaCl did not have a negative influence on its growth which could be industrially feasible for high salt (5% w/v NaCl) foods.