Salt induced stress responses in dairy bacteria

Abstract

Excessive sodium intake increases the risk of hypertension, osteoporosis, and kidney stones. Sodium chloride (NaCl; common salt) is the major source of sodium in our food; cheese, for instance, may contain high levels of salt. Owing to the increasing awareness about healthy food, there has been increasing interest to produce low-salt cheeses. Although there have been several attempts to reduce sodium in cheeses, there is limited information on effects of NaCl reduction and substitution with other salts, such as potassium chloride (KCl) on dairy bacteria, which is critical to understand their activity in dairy products. This study thus aimed to investigate salt induced changes in structure and function of dairy bacteria.

The effects of NaCl (0-10%) and pH (4.0-6.0) on membrane surface of Lactobacillus acidophilus were investigated using Fourier transform infrared spectroscopy (FT-IR). Since salt had greater impact than pH, further studies focused on effects of NaCl reduction, and substitution with KCl on Lb. acidophilus, Lb. casei, and Bifidobacterium longum. Changes in functional groups on bacterial membrane surface were evaluated using FT-IR. Critical NaCl concentration and an optimum KCl substitution level that inhibited the pathogenic bacteria (Escherichia coli) without affecting the potential probiotic bacteria were determined. The changes occurring in membrane integrity and dye-extrusion ability were evaluated using flow cytometry, in bacterial structure using transmission electron microscopy (TEM), and in membrane composition using gas-chromatography (fatty acids, FA) and thin-layer chromatography (phospholipids). Adhesion ability of bacteria to Caco-2 cells was also evaluated. Lastly, the effects of NaCl/KCl on Akawi cheese were investigated. Angiotensin-converting enzyme (ACE)-inhibitory and antioxidant activities, and amino acids (AA) released, during storage in different brine solutions (5-10% NaCl, with 50% KCl substitution) were studied. Viability of normal and cancerous human colon cells as affected by the cheese extract showing improved peptide profiles and highest AA (i.e. cheese brined in 7.5% NaCl+KCl) was evaluated. Whole-genome sequencing was also performed for the bacterium which was most robust to salt stress (Lb. casei).

Salt-induced changes occurred mainly in amide regions of FT-IR spectra in most bacteria. The critical NaCl concentration was 2.5% (wt./vol.) and 50% substitution with KCl was found to be optimal. Escherichia coli and B. longum were more sensitive to substitution compared with other bacteria. Flow cytometric analysis showed that salt stress reduced dye-extrusion activity, and salt tolerance of Lb. casei was highest among the bacteria studied. Bacteria appeared elongated, intracellular content appeared contracted when subjected to salt stress, and unsaturated to saturated FA ratio increased. Among phospholipids, phosphatidylglycerol was reduced, whereas phosphatidylinositol and cardioplipin were increased due to salt stress. Adhesion of bacteria to Caco-2 cells was reduced on exposure to NaCl; however, it improved on substitution with KCl. Essential AA (leucine, phenylalanine, tryptophan, and valine) content increased in cheese brined in 7.5% salt. Cheese extracts improved the growth of normal colon cells, and reduced the growth of cancerous cells. Overall, the study provided insights into damages to bacterial cells caused due to high salt, and reducing/replacing salt with KCl may be beneficial from food safety and health aspects.