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Natural Product Research
Formerly Natural Product Letters
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Quantitative insights on the interaction between
metal ions and water kefir grains: kinetics studies
and EPR investigations
Giulia Costamagna , Giorgio Volpi , Elena Ghibaudi & Marco Ginepro
To cite this article: Giulia Costamagna , Giorgio Volpi , Elena Ghibaudi & Marco Ginepro (2020): Quantitative insights on the interaction between metal ions and water kefir grains: kinetics studies and EPR investigations, Natural Product Research, DOI: 10.1080/14786419.2020.1855164
To link to this article: https://doi.org/10.1080/14786419.2020.1855164
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Published online: 07 Dec 2020.
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SHORT COMMUNICATION
Quantitative insights on the interaction between metal
ions and water kefir grains: kinetics studies and EPR
investigations
Giulia Costamagna, Giorgio Volpi , Elena Ghibaudi and Marco Ginepro
Department of Chemistry, University of Turin, Torino, Italy
ABSTRACT
Water kefir is an acid, softly alcoholic and fragrant beverage fer-mented by a stable consortium of multispecies microbial commu-nity. Aim of this study was to investigate the ability of water kefir grains to abate significant amounts of heavy metal ions during the preparation of the water kefir beverage and to set up an experimental and analytical methodology based on ICP-OES spec-troscopy and ionic chromatography for the evaluation of heavy metal bioaccumulation by water kefir grains. We investigated the absorption kinetics of the process. The use of EPR spectroscopy enabled us to characterize the interaction between water kefir grains and paramagnetic metal ions from the structural viewpoint. Our results highlight significant differences in both the kinetics and the structural aspects of the interaction between distinct metal ions and water kefir grains. They concur clarifying the potential role of water kefir grains as detoxifying agents towards heavy metal ions.
ARTICLE HISTORY Received 27 July 2020 Accepted 20 November 2020 KEYWORDS
Water kefir; fermentation; biosorption; bioremediation; yeasts;
pollu-tants; beverages
CONTACTGiorgio Volpi giorgio.volpi@unito.it
Supplemental data for this article can be accessed online athttps://doi.org/10.1080/14786419.2020.1855164.
1. Introduction
Water kefir is an acid, softly alcoholic and fragrant drink produced by fermentation of sugar–water solutions. The water kefir fermentation process starts by adding solid water kefir grains to a sugar–water solution and (dried) fruits; the mixture is generally prepared at room temperature under anaerobic conditions upon 24- to 48-h incuba-tion. A slightly sweet, softly alcoholic and acidic beverage with a yellowish colour and a fruity flavour is subsequently obtained by filtration (Corona et al. 2016; Sabokbar and Khodaiyan2016; Karaca et al. 2018; Koh et al. 2019). The solid water kefir grains contain an insoluble glucan-type exopolysaccharides, different kinds of bacteria and yeasts (Laureys and De Vuyst 2014; Arslan 2015; Corona et al. 2016; Randazzo et al.2016).
In a previous work, we have demonstrated the ability of water kefir grain to absorb many kinds of metal ions during the fermentation process (Volpi et al. 2019). Significant bioaccumulation rates by water kefir grains have been reported for Cu(II), Mn(II), Ni(II), Pb(II), Cr(III), Cr(VI) ions. Gerbino et al. (2012, 2014, 2015) demonstrated the involvement of the S-layer proteins (SLP) in the interaction of water kefir grains with metal ions [such as Pb(II), Cd(II), Zn(II) and Ni(II)], by using FTIR and Raman spectroscopy.
In the present work, we have focused our attention on a set of metal ions [Cu(II), Pb(II), Mn(II), Ni(II), Cr(III–VI)] characterised by high toxicity levels, in order to shed light on the kinetics and molecular aspects of their interaction with water kefir grains and to assess the ability of water kefir grains to abate heavy metal ions. We investigated the kinetics of the biosorption processes occurring at the interface between water kefir grains and aqueous solutions containing metal ions and we set up an experimen-tal and analytical method, based on ICP-OES spectroscopy and ionic chromatography, for the evaluation of heavy metal bioaccumulation during the fermentation time. In the peculiar case of chromium, to distinguish the two chromium species [Cr(III) and Cr(VI)], chromatography was used for the determination of Cr(VI) whereas total chro-mium was determined by atomic emission ICP-OES and Cr(III) for difference (Bianco Prevot et al.2018). Moreover, EPR studies allowed gathering information on the speci-ficity of the interaction with paramagnetic ions. Cu(II) ions turned out to interact spe-cifically with water kefir grains, whereas Mn(II) ions did not.
2. Results and discussion
In the present study, we set up an effective protocol for assessing the concentrations and abatement rates of metal ions in fermented drinks, whose fermentation is brought about by solid water kefir grains. The protocol is based on the concerted use of ICP-OES spectroscopy and ionic chromatography. We also show that EPR spectroscopy may provide valuable information on the interaction between paramagnetic metal ions and water kefir grains, at the structural and molecular level.
We collected evidence that water kefir grains are able to retain heavy metal ions dissolved in sugared aqueous solution during the fermentation time. As previously reported (Fiorda et al.2017; Bengoa et al.2018; Volpi et al.2019), the metabolic activ-ity of water kefir grains is influenced by the environmental conditions, i.e. sugar
amount, temperature, pH (buffer), water kefir grains/metal ions ratio. In the present work, tests on commercial water kefir grains have been performed by soaking them in sugar solutions (commercial 5% wt/vol sucrose) and by adding standard solutions of several metal ions at 2 mg L1and 20 mg L1 concentration.
In all cases, the most significant abatement rate of metals in the supernatant was achieved in the presence of low concentrations (2 mg L1), whereas higher concen-trations (20 mg L1) likely imply saturation mechanisms. In all cases, a variably pro-nounced drop of metal concentration was observed immediately after the addition of water kefir grains, followed by a slower decrease phase (see Figure 1 for Cu(II) ions and Supporting Information for Pb(II), Mn(II), Ni(II), Cr(III–VI) kinetics).
This effect can be interpreted as the consequence of two distinct mechanism of metal interaction with water kefir grains: the initial, quicker decrease is likely due to superficial complexation phenomena, where protons found on the water kefir grains surface are replaced by positive metal ions (as suggested by Gerbino et al. 2011,
2012). In the subsequent phase, slow metabolic phenomena can occur: these may cause the pH decrease, due to formation of acids, such as lactic and acetic acid, metabolites of the fermentation process. Precipitation, adsorption and complexation equilibria are controlled by pH. During the fermentation process, a pH shift towards more acidic conditions was established (see Supporting Information Figures S1–S5). Consequently, metals that were formerly complexed were partially released into the solution where lactic or acetic acid (as well as other metabolites) may have acted as free ligands towards metal ions. The highest metal abatement rate varied from species to species; nevertheless, it occurred within the 2–12 h range in all cases (see Figure 1
and Supporting InformationFigures S6–S9). Afterwards, the pH decrease affected the retention of metal ions by the colonies to a significant extent (see Supporting InformationFigures S1–S5). This process generally implied the release of the adsorbed metal ions, resulting in a gradual increase of metal concentration in the supernatant. However, the metal release was never complete. A fraction of metal remained bio-sorbed, even after long incubation times and very low pH values.
EPR spectroscopy provided evidence of the existence of specific interactions between Cu(II) ions and water kefir grains, due to the formation of metal complexes (see Supporting InformationFigure S10).
Figure 1. Concentration trend of Cu(II) ions (2.33 mg L1 left and 20.7 mg L1 right) vs. time in the supernatant solutions after incubation with water kefir grains.
EPR data unequivocally show that Cu(II) absorbs on water kefir grains to a signifi-cant extent. Spectral parameters proved that the chemical environment and the coordination geometry of copper ions in the presence of water kefir grains were radic-ally distinct from those typical of the aqueous copper complex [Cu(H2O)6]2þ. Hence,
Cu(II) forms specific complexes within the water kefir grains (see Supporting InformationFigure S10).
Unlike copper, Mn(II) does not prove to interact with water kefir grains to a mean-ingful extent (see Supporting Information Figure S11). The distinct behaviour of Cu(II) and Mn(II) ions is likely related with the type of ligands available for metal coordin-ation in water kefir. It can be justified by the presence of specific uptake mechanisms only for those metal ions that play relevant physiological roles.
These EPR results are consistent with the hypothesis– by Gerbino et al. (2015)– on the role played by S-layer proteins (SLP) in the interaction between metal ions and water kefir grains. It must also be borne in mind that other chemical species (contain-ing carboxylate, phosphate or other chemical groups) found in the multispecies micro-bial community or in the water kefir grain matrix may also be involved in complexation processes and play a relevant role in the interaction of metal ions with water kefir grains.
We have thus shown that the combined use of ICP-OES spectroscopy, ionic chroma-tography and EPR may help getting a deeper understanding of the net of complex-ation equilibria found in kefir suspensions during the fermentcomplex-ation process.
Acknowledgements
A special thanks to the Company Kefiring of Fabio Marcolongo, Via Genova, 11.37069 Villafranca di Verona (VR).
Disclosure statement
No potential conflict of interest was reported by the authors.
Funding
Authors gratefully acknowledge the financial support from University of Torino (Ricerca Locale ex-60%, Bando 2018 (CDD 29/05/2018)).
ORCID
Giorgio Volpi http://orcid.org/0000-0002-9695-9202 Marco Ginepro http://orcid.org/0000-0003-2856-5233
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