Synthesis and physic-chemical characterization of phytate compounds
The synthetic procedure followed for the preparation of SrPhy and ZnPhy derivatives is displayed in Fig. 1. Both compounds were synthesized by the hydrothermal reaction of the commercial sodium salt of PA with the respective chloride metal salt, SrCl2 and ZnCl2, and obtained in high yields (> 90%). Precipitation of metal complexes was carried out by dropping each chloride metal solution over the respective PA solutions at a pH value of 7.4 in a final proportion M2+:PA of 6:1. The pure phytate complexes obtained were used in their solid form for all the physic-chemical characterization. The reaction scheme showed in Fig. 1 represents a conformational change of the phytate molecule when the metallic complexes are formed as it has been previously reported by other authors38 and it will be discussed below in this work. Figure 1 shows a generic formula for both complexes. Nevertheless, based on reported works regarding the coordination bonds formed between the cations and the corresponding phosphate group coordination modes, and attending to the charge and the number of cations, it can be assumed that divalent cations were bonded preferably as a bridge between adjacent phosphate groups39.
Compositional analysis and thermal degradation
The empiric molecular formula of the precursor and the metallic phytates are shown in Table 1. The amount of sodium, strontium and zinc coordinated to phosphate anions was quantified by ICP, and the content of water molecules was calculated from the HRES-TGA analysis. EDS spectroscopy confirmed the presence of characteristic peaks for Sr (1.80 keV), and Zn (1.01 keV), in the SrPhy and ZnPhy derivatives respectively, and for P (2.01 keV) in all compounds; EDS results also revealed that phytate complexes were obtained purely without chloride impurities, Fig. 2A. The compositional analysis of phytate complexes made by ICP and elemental microanalysis reveals that 4 and 6 metal atoms of Sr2+ and Zn2+ respectively were coordinated to phytate rings reaching P/M2+ molar ratios of ≈ 1.6 and 1.09 for SrPhy and ZnPhy and a P/Na+ molar ratio of ≈ 1 for PA. Atomic content in C and H were found as: PA 8.1%C, 2.6%H; SrPhy 6.1%C, 2.2%H; ZnPhy 6.2%C, 2.3%H. These results indicate the presence of C6 in the molecular formula of all compounds, and H12, H10 and H6 for PA, SrPhy and ZnPhy respectively. Besides, the addition of Sr2+ to the PA occurs in conjunction with the precipitation of Sr4Phy at pH 7.4 in a similar manner that has been described for Ca-Phy40, while Zn2+ forms Zn6Phy precipitates22,33.
When the metallic complexes were formed, the results of the HRES-TGA curves obtained in inert atmosphere exhibited different degradation profiles respect to that of PA, Fig. 2B. HRES-TGA thermograms obtained under air atmosphere are shown in Fig. S1. Up to 150 °C, PA suffered a weight loss of ≈ 5% due to the release of water molecules (TMAX 92 °C). In contrast, water loss from SrPhy and ZnPhy started at early times (TMAX 82 °C and 75 °C respectively) and it was faster, displaying more pronounced mass decrease compared to PA, due to their higher water content, finally resulting in 3, 5 and 6 units of water molecules coordinated to PA, SrPhy and ZnPhy structures respectively. The main degradation step has been associated with the carbonization and dehydration of hydroxyl groups33,41. The decomposition of phytate anions in the metal complexes (130–290 °C for both compounds) took place at lower temperature than for PA (190–380 °C). In the final degradation step, further decomposition of carbon structure occurred at ≈ 380 °C for PA and it was close to 300 °C for the phytate complexes. Once again, the presence of divalent cations coordinated with the phosphate groups produces a decrease in the thermal stability of the compounds. As expected, the residue obtained at the maximum temperature evaluated (600 °C) had a greater mass for SrPhy (80%) and ZnPhy (81%) than for PA (77%) due to the presence of the non-degradable metallic components.
Some authors have determined the complexation ability of PA with several transitions metals and found a different binding capacity attending to the phase state analysed, the solution complexation or the solid formation19,20,22. Overall results exhibited that under PA excess conditions, soluble species with 1:1 stoichiometry (M2+:P) predominated at low pH values. However, when metal cations were in excess, the precipitation of solid phytate complexes took place for which there is some diversity in the final content of water molecules and cations coordinated to phytate anion. Ermanno et al. reported that zinc-phytate compounds synthesized under cation excess conditions are composed of one water molecule, deduced by TGA, and six coordinated cations, calculated via ICP22. Comel et al. found that zinc complexes synthesized in the same conditions and bearing the same amount of cations per molecule, lead to six coordinated water molecules33, while Champagne et al. informed that Zn/phytate ratio was initially 4, and decreased to 3.5 after 24 h, monitored by31P NMR32. For the strontium complex, Gancheff et al. found a 5:1 stoichiometry between cation and phytate anion determined by elemental analysis, and owing 16 water units per molecule, for an initial mixture 5:1 (M2+:PA)21. Interestingly, the methodologies followed for the synthesis of Zn-phytate complexes employed an acidified PA solution contrary to the methodology described in this work, which may influence the coordination number of the isolated solids.
Raman and ATR-FTIR characterization
The conformational state 5-axial/1-equatorial of the inositol ring displayed in the reaction scheme of Fig. 1 is in agreement with the work published by Isbrandt et al.42. To evaluate this, Isbrant and coworkers performed a combined analysis of Raman spectroscopy, 31P-NMR and 13C-NMR with sodium phytate complexes. Raman results (Fig. 3A) have demonstrated that C–C–H and O–C–H bending vibrational bands found in the range of 1250 and 1400 cm−1 have a maximum intensity at 1380 cm−1 when PA has a 5-axial/1-equatorial arrangement, and thus, it has been assumed that all phytate compounds have the same conformation as is represented in Fig. 1. Detailed bands assignment of Raman spectra is collected in Table 2 and it is in accordance with previous reports of other authors39,42,43. Signals of SrPhy and ZnPhy shifted to greater or lower wavenumber when compared to those of PA bands attending to the coordinated cation. Interestingly, numerous reports have demonstrated that in the liquid equilibrium of phytic acid, the conformational state adopted below pH values of 9 corresponds to the 1-axial/5-equatorial form, conversely to the evidences obtained for the solid-state of the phytate complexes explored in this study and for other solid phytate salts38,42.
ATR-FTIR spectroscopy analysis provided confirmation of the metal complexation of each compound. Expanded regions of the spectra obtained for each compound are shown in Fig. 3B and the assignment of the main vibrational mode bands in the whole spectra is collected in Table 343,44. All compounds showed a broad band around 3300 cm−1 and a single peak centered in 1640 cm−1 which were attributed to the stretching and bending vibrational modes of O–H bonds respectively, coming either from coordinated water molecules or unbounded P–O–H bonds39. The main vibrational modes of the C(O)PO3 groups are found in the region of 750–1300 cm−1. The spectra obtained in this region for each compound are shown in Fig. 3B. SrPhy and ZnPhy spectra displayed peak shifts to higher wavenumber in all these bands in comparison to those of PA. This effect has been reported for similar metallic phytate complexes and it was attributed to a change in the P-O strength bond due to the modification of the chemical structure of PA in which the formation of the coordination bond takes place. The spectrum of the PA sodium salt employed in this study (C6H12O24P6·6Na.3H2O) exhibited a peak at 1190 cm−1 that corresponds to the asymmetric stretching of P–O bonds in protonated HPO3− groups 39. Nevertheless, in the spectra obtained for the metallic compounds this band is overlapped with the symmetric stretching vibrational mode of P-O bonds. This behavior is explained by the disappearance of protonated HPO3− groups due to the establishment of the coordination bond with the divalent cation39,44. Besides, the band centred at 917 cm−1 in PA spectra splits into a double peak for ZnPhy (924—980 cm−1), and a triplet peak in the case of SrPhy (1000–960–926 cm−1) as expected since the conjugation of the metal cation is different in each complex34,39.
31P and 13C NMR analysis
The coordination bond between zinc or strontium with phytate anion was analyzed by solid 31P NMR and 13C spectroscopy, and the results are shown in Fig. 4. For the three compounds, each 31P NMR spectrum presented a broad peak for all the phosphorus atoms and two symmetrical spinning sidebands (Fig. 3A). It can be observed that signals obtained for SrPhy and ZnPhy compounds moved towards lower chemical shift when compared to those of the precursor PA. This migration can be explained by the formation of a coordination bond created between Sr2+ or Zn2+, and phosphate groups, leading to a decrease in the electric dipole moment of the bridge oxygen atom, and the consequent displacement of the signals to lower chemical shifts. Similar effect has been previously reported for metallic phytate compounds in the solid state finding that both the type of metal and the number of metal-phytate bonds influence the chemical shift of the phosphorous atoms and the spinning sidebands45. In the solid 13C NMR spectra of PA, SrPhy and ZnPhy (Fig. 3B) a single peak centered at 75.7 ppm for each compounds was displayed, denoting that there is not a direct interaction between the respective metals with the carbon atoms of the molecule42.
In overall, at sight of the published results and those obtained in the present work regarding the structural characterization of the metallic phytates, it can be concluded that there exists a relationship between the synthetic procedure and the coordination number of the as-obtained metallic complex. The complexation ability of PA highly depends on the nature of the cation, the ratio PA:M2+, the pH reaction values and the ionic strength of the medium. The variability of these parameters leads to the obtaining of solid phytate complexes with different number of coordinated cations. Physical and spectroscopic data obtained for SrPhy and ZnPhy are comparable to those previously reported for similar complexes differing mainly just in the molecular formulas33,40,43.
Biofilm inhibition ability
The antimicrobial potential of phytate compounds was assessed as a function of their capability to impair the growth and production of biofilm by S. mutans cultures. This strain has been described as one of the main cariogenic bacterial species of the oral microbiota and it is considered as an important risk factor in the development of dental caries46,47,48. S. mutans synthesizes glucans that promote the biofilm formation and the acidification of the buccal environment47,48,49,50,51,52 which leads to the proliferation of other biofilm bacterial species. Therefore, strategies to inhibit the proliferation and formation of S. mutans in dental plaque are key for cariogenic prevention49,53. In this vein, biofilm formed was studied by means of CV staining and colony forming unit counts (CFU) in agar-BHI solid plates. CV is a protein dye commonly used for the identification of all biofilms that stains the extracellular matrix of polysaccharides and negatively charged surface molecules54. This method implies an improvement in the determination of total biofilm and not just functional biofilm, since CV can dye both viable and dead cells together with extracellular matrix55. Phytate compounds were dissolved in a mixture of BHI:Tris–HCl 50 mM (1:1) at 100 µg/mL, and tested at a final concentration of 50 µg/mL. As control sample, bacteria were treated with a mixture of BHI:Tris–HCl 50 mM (1:1) emulating the same culture conditions established for the experimental samples but without phytate supplementation. The concentration of the Tris–HCl buffer employed was reported to not affect bacterial growth56. Moreover, based on the thermodynamics equilibriums previously established for both phytate complexes, the expected phytate species found in solution at the experiment pH value (≈7) are Zn2+ (83%) and ZnH3Phy7− (17%) for Zn-containing complexes; and Sr2+ (≈71%) and SrH5Phy5 (≈29%)for strontium phytate compounds21,33.
The quantification of CV found in the biofilms formed is represented as the optical density in Fig. 5. Inhibition capacity was composition-dependent regarding the cation bonded in each PA-complex. All phytate compounds were able to significantly reduce the biofilm produced by S. mutans (***p < 0.001), and interestingly, cells treated with ZnPhy exhibited a remarkably improved anti-biofilm activity with respect to PA and SrPhy samples (**p < 0.005). In parallel, the count of viable bacteria found in both the biofilm matrix and the planktonic supernatant were determined. There is only a significant bactericidal effect in the resuspended biofilm matrix (nearly 1 log) for ZnPhy sample (*p < 0.005) (see Supplementary Fig. S2), which is in accordance with the biofilm disruption assessment (Fig. 5). On the contrary, the CFU counts found in the planktonic solution did not show significant differences, suggesting that the antimicrobial activity when phytate treatment was applied is mainly effective for the biofilm formation. Therefore, our results demonstrate the effectiveness of two metallic phytate-complexes bearing Sr2+ and Zn2+ (SrPhy and ZnPhy) to prevent the S. mutans biofilm.
However, the metabolic role of PA in the disaggregation of bacterial biofilms remains poorly understood. It is believed that the antibacterial mechanism of PA is based in the disruption of the cell membrane integrity due to the high negative charges of its chemical structure, and thus, cellular morphology and intracellular ATP levels may be affected8,11,12. This theory is supported by the broad spectrum of both gram-positive and gram-negative bacteria for which PA has demonstrated antibacterial potential, and also by the rapid action required to achieve antibacterial activity15. Another explanation of the mechanism of action could be associated with the iron-chelating properties of PA since there are some reports that support the anti-biofilm ability for other iron-chelating agents43,44,45.
The greater disaggregation of the polymeric biofilm observed for Sr/Zn-bearing phytate complexes can be understood as a possible synergic effect between the positive cation and the negative charges of PA that may hinder the aggregation of proteins required for the adhesion of biofilm-forming polysaccharides presumably by the establishment of a ternary protein-metal-phytate complex46,47. For its part, other authors have explored the combined effect exhibited by antibiotic-based systems including Zn2+. The formation of Zn2+ complexes with kefzol (a commercial antibiotic) has been reported to remarkably improve the antibacterial activity exhibited by kefzol treatment alone48. The system zinc citrate/triclosan was also analysed and a similar synergic antimicrobial effect against S. mutans was detected, attributed to the presence of Zn2+49, which agrees with our findings. Zinc has been reported for affecting S. mutans metabolism, at mM concentration, via multiple inhibitory actions comprising the modulation of oxoenzymes, the inhibition of glycolysis, alkali production, the function of the phosphoenolpyruvate system (sugar phosphotransferase, PTS) and F-ATPase49,50,51,52, which allow biofilm growth to be controlled. However, the antimicrobial effect of Zn2+ by itself seems to be bacteriostatic since the inhibition of glycolysis, PTS and F-ATPase were reversible processes50. Thus, Zn2+ is expected to only may enable bacteria killing in combination with other bactericidal agents50, though an improvement of their intrinsic potential was noticed as described above, and also supported by our results48,49. On the contrary, a recent study found equal bacteriostatic and bactericidal properties for zinc sulphate and zinc acetate salts (tested in the range of µg/mL) against S. mutans cultures53. In our work, a low concentration of Zn2+ (≈18 µg/mL) enabled to reduce the production of biofilm by S. mutans.
Growth curves of S. mutans cultures under PA-compounds treatment were recorded by automatic measurement of OD600 each 20 min during 16.5 h, and the results obtained are displayed in Fig. 6. Ten-fold serial dilutions (10−1–10−4) were obtained from an initial culture at OD600 0.2. Diluted samples were growth under constant phytate compounds treatment at a fixed final concentration (50 µg/mL), due to the limited solubility of phytate complexes (Fig. 6A–D). The profile of the growth curves obtained for 10−1 diluted samples from cultures treated with different PA-compounds did not show any significative difference when compare to the untreated control sample (Fig. 6A). Nevertheless, we detected an increase in the duration of the lag phase in cultures treated with PA compounds at dilution factors 10−2–10−4 (Fig. 6B–D). Precisely, the most significant effects on the lag phase of growth were observed for cultures treated with ZnPhy and SrPhy complexes (Fig. 6D). These results could suggest a bacteriostatic effect of the PA-compounds based on their ability to increasing the time needed for bacterial population to adapt to a new environment as reported57,58. Interestingly, SrPhy and ZnPhy treated samples also exhibited this effect when 10−2 and 10−3 sample dilutions were tested, which suppose an enhancement of the intrinsic bacteriostatic properties registered for PA.
Bacteria induce the biofilm formation in response to environmental signals. The processes by which S. mutans undergoes the formation of biofilms are highly conditioned by the quorum sensing (QS) system59. QS is activated in response to the release of autoinducer molecules or pheromones in a cell density-dependent manner and confers a bacteria population the ability to alter their physiology and behaviour as a group unit instead of single entities59,60. In this sense, QS enables a collective response of bacterial populations when they are exposed to any environmental stress by the regulation of different physiological processes including sporulation, antibiotic production, competence development and biofilm differentiation, among others60,61. In our experiments, phytate supplementation altered the levels of biofilm production and the number of viable bacteria embedded in polymeric biofilm, perhaps associated by their role in the modulation of the QS transduction system. In fact, the significantly decreased of CFU found in the biofilm of ZnPhy samples (Supplementary Fig S2) is in accordance to its higher biofilm disaggregation ability (Fig. 5). Furthermore, it could be speculated that the higher OD600 values obtained for ZnPhy in Fig. 6 are tentatively attributed to the synergic role between the cation and phytate in S. mutans metabolism, which drives the proliferation of viable CFU to the planktonic solution since biofilm formation is expected to be unfavoured as was highly inhibited in Fig. 5. This work demonstrates the possibilities of applying these type of formulations in cariogenic prevention strategies. In fact, the inhibition of the proliferation of key strains such as S. mutants in dental plaque, supports further validation for testing these compounds in vivo.