High-throughput characterization of the effect of sodium chloride and potassium chloride on 31 lactic acid bacteria and their co-cultures

Abstract

Salt (NaCl) is associated with a risk of hypertension and the development of coronary heart disease, so its consumption should be limited. However, salt plays a key role in the quality and safety of food by controlling undesirable microorganisms. Since studies have focused primarily on the effect of salts on the overall counts of the lactic acid bacteria (LAB) group, we have not yet understood how salt stress individually affects the strains and the interactions between them. In this study, we characterized the effect of sodium chloride (NaCl) and potassium chloride (KCl) on the growth and acidification of 31 LAB strains. In addition, we evaluated the effect of salts on a total of 93 random pairwise strain combinations. Strains and co-cultures were tested at 3% NaCl, 5% NaCl, and 3% KCl on solid medium using an automated approach and image analysis. The results showed that the growth of LAB was significantly reduced by up to 68% at 5% NaCl (p < 0.0001). For the co-cultures, a reduction of up to 57% was observed at 5% NaCl (p < 0.0001). However, acidification was less affected by salt stress, whether for monocultures or co-cultures. Furthermore, KCl had a lesser impact on both growth and acidification compared to NaCl. Indeed, some strains showed a significant increase in growth at 3% KCl, such as Lactococcus lactis subsp. lactis 74310 (23%, p = 0.01). More importantly, co-cultures appeared to be more resilient and had more varied responses to salt stress than the monocultures, as several cases of suppression of the significant effect of salts on acidification and growth were detected. Our results highlight that while salts can modulate microbial interactions, these latter can also attenuate the effect of salts on LAB.

Keywords: acidification; bacterial co-culture; high-throughput culturing; image analysis; lactic acid bacteria; microbial interactions; salt.

Figure 1

Measuring growth and acidification of pure cultures and their binary co-cultures using a high-throughput approach. (A) Schematic setup of the experiment: two 96-spot plates, each containing pure cultures, are combined by the robot to form a 96-spot plate with co-cultures at each spot (93 co-cultures and three negative controls). (B) Growth and acidification are quantified using image analysis (Coltapp). For instance, tracking the growth and acidification of Lactococcus cremoris 7311, Lactococcus lactis subsp. diacetylactis UL 719, and their co-culture is performed over a period of 9 days. The light gray curve represents the control, while the light blue, dark blue, and red curves represent 3% NaCl, 5% NaCl, and 3% KCl, respectively. The growth score reflects the cumulative biomass based on the size and opacity of the colony. The acidity score corresponds to the cumulative red intensity on the RGB scale.

Figure 2

Difference of growth and acidification scores between each salt condition (3% NaCl, 5% NaCl, and 3% KCl) relative to the control (PCASL). Difference of growth and acidification score for each salt condition. The growth score reflects the cumulative biomass based on the size and opacity of the colony. The acidity score corresponds to the cumulative red intensity on the RGB scale. Light blue, dark blue, and red shapes represent differences at 3% NaCl, 5% NaCl, and 3% KCl concentrations, respectively. Filled shapes indicate statistically significant differences (FDR adjusted P-value < 0.05), while hollow shapes were non-significant.

Figure 3

Correlation between acidification and growth scores for LAB strains in different salt conditions. (A) Control (PCASL media) (Pearson correlation r = 0.7394, p < 0.0001, N = 29). (B) 3% NaCl (Pearson correlation r = 0.2266, p = 0.25, N = 27). (C) 5% NaCl (r = 0.2061, p = 0.31, N = 26). (D) 3% KCl (Pearson correlation r = 0.7694, p < 0.0001, N = 29). Note that Lactiplantibacillus pentosus ATCC 8041 and Lactoccocus cremoris 7,311 were identified as growth score outliers and excluded from these analysis.

Figure 4

Overall effects of salts on growth and acidification in monoculture and co-culture. Difference of growth and acidification score for each salt condition. Light blue shapes represent the effect at 3% NaCl, dark blue shapes represent the effect at 5% NaCl, and red shapes represent the effect at 3% KCl. The asterisk symbolizes a significant difference between growth at 5% NaCl for co-cultures and growth for other conditions in monocultures.

Figure 5

Volcano plot of the difference in growth and acidification score of co-cultures as a function of salts. The dashed line represents the significance threshold (FDR adjusted p = 0.05). Difference of growth and acidification score for each salt condition. Light blue circles represent the difference at 3% NaCl, the dark blue circles represent the difference at 5% NaCl, while the red circles represent the difference at 3% KCl.

Figure 6

Microbial interactions measured on growth and acidification scores between lactic acid bacteria across salt conditions. (A) Overview of the intersection of detected effects on growth and acidification scores. Hollow circles represent either non-significant interactions for growth and acidification (black) or for one of these two measured parameters (blue and red). Solid circle points showed stable interactions for both growth and acidification (blue) or for one of these parameters (black). Diamonds represent condition-dependent interactions for growth and acidification (red) or for one of these parameters (black). (B) This graph shows the type of interactions between our LAB forming binary co-cultures. Colors and shapes match with those in (A).