Manufacture of some cheeses involves the use of Lactococcus lactis. During processing bacteria are subjected to environmental stresses such as acid and salt osmotic conditions. Identifying procedures and mechanisms to develop resistance to these environmental stresses, specifically salt, is important to improve the culture’s growth and performance during food manufacture and storage. Hypothesis was whether salt tolerance of Lactococcus lactis R-604 can be enhanced. Objective was to study the influence of prior salt exposure and lactose deprivation on salt tolerance of L. lactis. The culture was subjected to mild stress induced by lactose starvation or prior salt exposure for 24 hours aerobically at 30 ° C. A control was conducted without any stress. Cells that were prior mild stressed by lactose starvation or prior salt exposure were transferred to M17 broth with 5 concentrations of NaCl (0, 1, 3, 5 and 7% w/v) and incubated aerobically at 30 ° C. Plating was conducted immediately after inoculation and every 24 hours for 5 days in M17 agar supplemented with 0.5% of lactose and incubated aerobically at 30 ° C for 48 hours. Three replications were conducted. There was no need for lactose to grow this culture in M17 broth and it could tolerate prior exposure to 3% w/v NaCl in media without affecting its growth at different salt concentrations. Culture that was exposed to lactose starvation and then subsequently grown in 3% w/v NaCl, at day 3 showed enhanced salt tolerance which can be recommended as a pretreatment condition for obtaining daughter cells with improved salt tolerance.
In many dairy products salt is used as a preserving agent, as a proteolysis enhancer, and a desirable flavor contributor. Salt can also affect the quality and flavor development in the ripening process, since it inhibits microbial activity of both spoilage and beneficial bacteria such as lactic acid bacteria and probiotics [
Higher concentration of salt in a medium will cause an osmotic stress in bacteria, since it creates an imbalance of ion (K+, Na+ and Cl−) concentrations in the cytoplasm of the cell and its environment [
Resistance to stress can also be achieved by adaptation which is an evolutionary process whereby an organism becomes better able to live under certain conditions [
Another way to increase stress resistance is by subjecting bacterial cells into a medium with low or no nutrients, or where their availability is reduced due to modifications in environment such as extreme pH or low water activity which leads to a cell starvation [
The influence of prior salt (NaCl) exposure and lactose deprivation on salt tolerance of the cheese bacterium Lactococcus lactis R-604 is not well understood. The hypothesis was whether the subjection to prior mild stress conditions would enhance salt tolerance of Lactococcus lactis R-604. The objective was to study the effect of two different stress exposures (prior NaCl exposure and lactose deprivation) on the growth of Lactococcus lactis R-604 in five levels of NaCl (0, 1, 3, 5 and 7% w/v).
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 [
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 effect of each stress condition on the salt tolerance of Lactococcus lactis R-604 was evaluated by bacterial counts and is presented in
Days | |||||||||||||||||
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0 | 1 | 2 | 3 | 4 | 5 | 6 | |||||||||||
Control | Salt (% w/v NaCl) | 0 | 5.16 | Ea | 7.81 | BCa | 9.27 | Aa | 8.53 | ABabc | 7.14 | CDbc | 6.71 | Dab | 6.43 | Dabc | |
1 | 5.16 | Ca | 7.32 | Ba | 9.32 | Aa | 8.63 | Aab | 7.12 | BCbc | 6.42 | Cab | 6.62 | BCabc | |||
3 | 5.16 | Da | 7.31 | Ba | 9.28 | Aa | 7.77 | Bbcd | 6.49 | Cc | 5.78 | CDb | 5.78 | CDcd | |||
5 | 5.16 | CDa | 7.43 | Ba | 9.15 | Aa | 9.04 | Aa | 7.76 | Bab | 5.72 | Cb | 4.82 | De | |||
7 | 5.16 | Ba | 7.38 | Aa | 6.72 | Ab | 6.95 | Ade | 7.01 | Abc | 6.88 | Aab | 6.88 | Aab | |||
Prior salt exposure | Salt (% w/v NaCl) | 0 | 5.02 | Ea | 8.0 | Ba | 9.23 | Aa | 7.66 | BCcd | 7.04 | CDbc | 6.66 | Dab | 5.97 | Dbcd | |
1 | 5.02 | Da | 8.0 | Ba | 9.18 | Aa | 8.36 | ABabc | 6.98 | Cbc | 6.77 | Cab | 6.14 | Cbcd | |||
3 | 5.02 | Fa | 7.96 | Ba | 9.25 | Aa | 8.42 | ABabc | 6.63 | CDc | 5.96 | DEb | 5.29 | EFde | |||
5 | 5.03 | CDa | 7.84 | Ba | 9.2 | Aa | 8.98 | Aa | 8.08 | Ba | 5.62 | Cb | 4.76 | De | |||
7 | 5.03 | Ca | 7.64 | Aa | 7.01 | Ab | 7.41 | Acde | 7.38 | Aabc | 7.25 | ABa | 6.5 | Babc | |||
Lactose deprivation | Salt (% w/v NaCl) | 0 | 5.10 | Da | 7.49 | Ba | 9.31 | Aa | 8.89 | Aa | 7.34 | BCabc | 6.83 | BCab | 6.65 | Cabc | |
1 | 5.10 | Ca | 7.42 | Ba | 9.32 | Aa | 8.67 | Aab | 6.98 | Bbc | 6.76 | Bab | 6.73 | Bab | |||
3 | 5.10 | Da | 7.45 | Ba | 9.34 | Aa | 8.73 | Aa | 6.66 | BCc | 6.11 | Cb | 6.16 | Cbcd | |||
5 | 5.10 | Da | 7.44 | Ba | 9.18 | Aa | 9.06 | Aa | 8.11 | Ba | 6.03 | Cb | 4.65 | De | |||
7 | 5.10 | Ca | 7.38 | Aa | 6.21 | Bb | 6.53 | Be | 6.58 | ABc | 7.15 | Aa | 7.23 | Aa | |||
ABCLS Means with different capital letters within a row are significantly different (P < 0.05). abcLS Means with different lowercase letters within a column to include all treatments are significantly different. (P < 0.05).
Effect | P > F |
---|---|
Stress conditions | 0.9002 |
Salt Concentration | 0.0010 |
Stress conditions*Salt concentrations | 0.6911 |
Days | <0.0001 |
Stress conditions*Days | 0.0823 |
Salt concentrations*Days | <0.0001 |
Stress conditions*Salt concentrations*Days | 0.9984 |
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
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) (
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 (
Hartke et al. [
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 [
Prior salt exposure to 3% w/v or lactose starvation was not detrimental to this culture and even under lactose starvation conditions it performed similar to control maintaining comparable counts in salt concentrations as high as 7% w/v. On day 3 at 3% w/v NaCl, the lactose deprivation resulted in enhanced salt tolerance and this can be recommended as a pretreatment condition for obtaining daughter cells with improved salt tolerance.
This projected was funded by USDA Hatch funds and the Louisiana State University Agricultural Center.
Gonzalez, E.E. and Aryana, K. (2018) Salt Tolerance of Cheese Culture Lactococcus lactis R-604 as Influenced by Prior Salt Exposure and Lactose Deprivation. Food and Nutrition Sciences, 9, 633-641. https://doi.org/10.4236/fns.2018.96048