Comparative analysis of the effect of Ca and Mg ions on antibacterial activity of lactic acid bacteria isolates and their associations depending on cultivation conditions

The reduction of pathogens growth in food and feed production and storage is very important, and the creation of new effective strategies for this purpose becomes more and more prospective. It has been known that lactic acid bacteria (LAB) produce several antimicrobial substances, including organic acids, other organic compounds, carbon dioxide, diacetyl, hydrogen peroxide and bacteriocins (Nes et al. 2011). It is also significant to define the role of metals in production of antimicrobial components and differentiate the metals that are essential for cells and included in composition of important enzymes (proteases, DNA polymerases, dipeptidases, tripeptidases, etc.). Such metals are calcium (Ca²⁺) and magnesium (Mg²⁺).

Investigations of the role of metals in microbial growth delayed because procedures for purification and detection were not sensitive enough to measure the small amounts of ions generally required by bacteria. Besides these technical troubles, circumstantial studies on the mineral demands for the growth of organisms are complicated because of the following (Boyaval 1989): metals replace each other; some metals adsorb others; some metals interact differently in the presence of others; many organic substances can combine with metals and render them unavailable for growth; colloidal suspensions of metals can precipitate because of shifts in pH before or during growth.

The great multiplicity of ecological-geographic conditions of Armenia with its precisely designated vertical zoning contributes the development of unique associations of LAB in traditional dairy products. During centuries Armenians have prepared traditional protein-enriched dairy products such as yoghurt, sour cream and different types of cheeses, having substantial physiological, antagonistic, antioxidant and antiallergenic activity (Bazukyan et al. 2010; Movsesyan et al. 2010). Antibacterial activity is connected to a synthesis of special substances-bacteriocins (Nes et al. 2012). Bacteriocin producing LAB can be applied as a starter cultures in food fermentation or added to a fresh food as bio-preservatives.

The investigation of different metals’ role in bacteria growth and antagonistic activity becomes more and more popular and required in recent years. It is interesting to study the change of inhibitory activity of bacteria by addition to their growth medium ions of different metals and compare the results. It has been showed that the antibacterial activity of LAB pure cultures could be differing from the same activity of the LAB associations, which is connected to the types of cultivation.

Consequently, the aim of this work was to compare the effects of Ca²⁺ and Mg²⁺, as well as their combined mixture on antibacterial activity of LAB isolates and their associations depending on cultivation conditions.

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.