Interactions between metals and microorganisms are diverse, but can be divided into 3 major categories: metals essential for metabolism; metals which are accumulated; metals which undergo biochemical transformation (including leaching). Three individual functions were presented: metal ions act as catalytic centers of enzymes; metal ions, not primarily involved in the catalysis, act as binding groups to bring enzyme and substrate together; metal ions maintain physiological control (antagonism with other metals). More recently, other aspects of the role of metal ions in metabolism have been investigated, e.g. the involvement of metal ions in the reactivation of EDTA inhibited proteolytic enzymes from LAB and the narrow tolerance for specific metals in the synthesis of secondary metabolites (Weinberg 1970, 1978). The ionic environment may interfere with bacterial cell walls, especially in Gram-positive bacteria such as Lactobacillus and Streptococcus which contain teichoic and teichuronic acids (Ellwood and Tempest 1972). The relative affinities of various cations for Gram-positive bacterial cell walls have been reported by Marquis et al. (Marquis et al. 1976).
Calcium is a very important metal for growth and activity of LAB. As a rule, concentration of calcium in milk is approximately 15 mM (Boyaval 1989). But it enhances the growth of LAB depending on their genera and the composition of their growth medium. Particularly, during studies with L. casei, L. arabinosus, Leuconostoc mesenteroides and S. faecalis, only L. casei growth was enhanced by calcium addition. Ca2+ stimulated early growth of L. casei in an amino acid medium and in media containing limiting amounts of serine (Boyaval 1989). It is interesting to note that in the presence of Ca2+ short chains were formed, while in the absence of this ion the cells were in longer chains. The results of our experiments showed that Ca2+ induced the antibacterial activity of LAB isolates, as well as LAB associations. Interestingly, Ca2+ also stimulated the proteolytic activity of some our investigated LAB strains, as it was shown in previous work (Keryan et al. 2014). The proteolytic activity of MDC9632 and MDC9633 strains was detected previously (Keryan et al. 2017). So, we can consider that there is an interesting correlation between stimulation of antibacterial and proteolytic activity by Ca2+. So we can speculate that the antibacterial components were produced after proteolysis, i.e. have the proteinaceous nature. But for confirmation of this hypothesis the further detailed investigations are required. Wright and Klaenhammer (1981) showed that Ca2+ supplementation of MRS resulted in a morphological transition of L. acidophilus from filamentous to bacilloid rods, which were more resistant to freezing. It was also known that Ca2+ plays essential role in the cell wall but it is not clear yet. Mills and Thomas (1978) showed that the liberation of proteinase from cell walls of S. lactis and S. cremoris stopped when CaCl2 was added to the buffer, or when the temperature was raised or when the pH reached 5.5. Thomas et al. (1974) thought that Ca2+ linked the cell wall and the enzyme, while Exterkate (1979) showed that Ca2+ stabilized proteinase activity.
Magnesium is the major divalent cation in all living cells. In bacterial cells, the intracellular Mg2+ content is equivalent to 20–40 mM Mg2+ (Silver and Clark 1971). In milk, the Mg2+ concentration varies from 4.2 to 6.25 mM, depending on the geographic region (Veisseyre 1975). Interestingly, supplementation of milk with 1-2.1 mM Mg2+ permitted both a stimulation of growth of S. thermophilus and S. lactis and a better survival rate of the lactic streptococci (Amouzou et al. 1985). Interesting results about antibacterial properties of Mg2+ were obtained by Duane and coauthors (Robinson et al. 2010). Particularly, they investigated antibacterial properties of magnesium against Gram-positive (S. aureus) and Gram-negative (E. coli and P. aeruginosa) bacteria by addition of Mg2+ to their growth medium. Authors indicated that Mg metal has a significant effect on CFUs of both Gram-positive and Gram-negative bacteria. Added to the growth media Mg2+ corrosion products would inhibit the growth of E. coli, P. aeruginosa and S. aureus but this is only a hypothesis which needs confirmation. In our experiments the increasing of antibacterial activity after addition of Mg ions was approximately as much as after Ca ions addition.
Ca2+ and Mg2+ have other amazing properties. Particularly, it was shown that these cations can influence on bacterial biofilm formation (Guvensen et al. 2012; Song and Leff 2006). These ions can also interact with antibiotics and protect the bacterial outer membrane from damage (Sahalan et al. 2013).
There are some interesting data about the effects of other metal ions on bacterial cell growth. Particularly, the authors investigated the growth and oxidation–reduction potential of Enterococcus hirae in the presence of Mn2+ (Vardanyan and Trchounian 2013). They showed that addition of Mn2+ (MnCl2) within the range of 0.01 to 1 mM affected E. hirae growth by decreasing lag phase duration and increasing specific growth rate. Another work was devoted to study of various heavy metal ions effects on bio-hydrogen production and the FoF1-ATPase activity of Rhodobacter sphaeroides (Hakobyan et al. 2012). It was shown that Fe2+ plays a very important role for growth, hydrogen production and ATPase activity of R. sphaeroides.
There is limited data about the effect of ions on the microbial antibacterial activity. Our data showed that some investigated mixed cultures of LAB revealed strong inhibitory effects against pathogenic test-organisms. These associations were Mixes 1, 2, 4, 8, 10, 19 and 20 (see Table 1). Particularly, in case of separated cultivation Mixes 1, 2, 10 and 19 (see Table 1) showed the strongest antibacterial activity against P. aeruginosa and S. aureus. In case of simultaneous cultivation, the growth of mentioned test-organisms was inhibited by Mixes 4, 8, 10 and 20 (see Table 1). Interestingly, in case of time-spaced cultivation inhibitory effect of mixed cultures was induced by Ca2+ and their mixture with Mg2+. Antibacterial effect was mostly stimulated by combined mixture of Ca2+ and Mg2+ in case of simultaneous cultivation. The biggest diameter of growth inhibition zone of P. aeruginosa was 18 mm and 23 mm for S. aureus caused by Mixes 10 and 20, respectively.
The investigation of effect of Ca2+ and Mg2+ on growth and biological properties of LAB becomes more prospective. The fact that Ca2+ and Mg2+ and their mixture stimulated the inhibitory effect of investigated LAB isolates and their associations can play a role for creation of new effective antimicrobial drugs for prevention of pathogens growth. Anyway, the further more detailed investigations are required.