Lactobacilli strains for use in a method of metals gut microbial bioremediation, mixture and composition comprising them

Abstract

The present invention is related to at least one isolated bacterial strain selected from at least one of the species selected from the group comprising or, alternatively, consisting of: i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus, or mixtures of said at least one isolated bacterial strain thereof, for use in a method of metals gut microbial bioremediation. Preferably, said metals involved in the gut microbial bioremediation are heavy metals, which are selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof. In particular, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is selected from the group comprising or, alternatively, consisting of Lactiplantibacillus plantarum 476LL 20 bi LP01 (LMG P- 21021), Lactiplantibacillus plantarum 776 / 1 bi LP02 (LMG P-21020), and Lactiplantibacillus plantarum LP14 (DSM 33401) or mixtures thereof, said at least one isolated bacterial strain belonging to the specie ii) Lactobacillus acidophilus is selected from the group comprising or, alternatively, consisting of Lactobacillus acidophilus LA02 (DSM 21717), and Lactobacillus acidophilus LA06 (DSM 23033), and said at least one isolated bacterial strain belonging to the specie iii) Lactobacillus crispatus is identified as Lactobacillus crispatus LCR04 (DSM 33487). Furthermore, the present invention regards a mixture comprising at least one isolated bacterial strain as above indicated, as well as a composition comprising said mixture, for use in a method of metals gut microbial bioremediation, wherein said metals involved in the gut microbial bioremediation are heavy metals, which are selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof and in the prevention and or reduction of the harmful effects of heavy metals on gut health.

Description

[0001]P240048 LACTOBACILLI STRAINS FOR USE IN A METHOD OF METALS GUT MICROBIAL BIOREMEDIATION, MIXTURE AND COMPOSITION COMPRISING THEM *** The present invention is related to at least one isolated bacterial strain selected from at least one of the species 5 selected from the group comprising or, alternatively, consisting of: i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus, or mixtures of said at least one isolated bacterial strain thereof, for use in a method of metals gut microbial bioremediation. Preferably, said metals involved in the gut microbial bioremediation are metals, which are selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof (in short, HMs). 10 In particular, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is selected from the group comprising or, alternatively, consisting of Lactiplantibacillus plantarum 476LL 20 bi LP01 (LMG P- 21021), Lactiplantibacillus plantarum 776 / 1 bi LP02 (LMG P-21020), and Lactiplantibacillus plantarum LP14 (DSM 33401) or mixtures thereof, said at least one isolated bacterial strain belonging to the specie ii) Lactobacillus acidophilus is selected from the group comprising or, alternatively, consisting of Lactobacillus acidophilus LA02 15 (DSM 21717), and Lactobacillus acidophilus LA06 (DSM 23033), and said at least one isolated bacterial strain belonging to the specie iii) Lactobacillus crispatus is identified as Lactobacillus crispatus LCR04 (DSM 33487). Furthermore, the present invention regards a mixture comprising at least one isolated bacterial strain as above indicated, as well as a composition comprising said mixture, for use in a method of metals gut microbial bioremediation, wherein said metals involved in the gut microbial bioremediation are metals, which are selected 20 from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof (in short, HMs), and in the prevention and or reduction of the harmful effects of said metals on gut health. BACKGROUND OF THE INVENTION 25 Contamination of the environment and agricultural products by chemicals is a severe problem perceived at global level. Nowadays, soil and water can get contaminated by the accumulation of several classes of chemicals as consequence of rapidly expanding industrial areas, emissions, mining activities, farming with application of fertilizers and pesticides, animal husbandry, manures, wastewater irrigation and several other modern human practices. 30 Under certain conditions and dosages, these chemical agents allowing modern farming or constructions techniques might be regarded as pollutants. Indeed, they can easily contaminate soil and water in first instance and then enter the food chain due to production, processing, handling, or transport, or as a result of environmental contamination of air, soil and water thus reaching humans. As described in literature, chemicals are supposed to meet three requirements to contaminate and accumulate in the environment (in particular water) to get to the food chain; i) 35 octanol-water partition; ii) stability in water and aqueous system; iii) low toxicity allowing to persist and not being 1 P240048 eliminated by the species they contaminate first. This statement is particularly valid for water-soluble chemicals that meet these requirements; however, lipid-soluble molecules tend to accumulate in animal feed and so reaching fatty tissues of animals and then humans. Bioaccumulation of chemical contaminants might also lead to biomagnification, meaning an increase in their concentration while being transferred from a species to another (e.g. 5 from pray to predator). Heavy metals (HMs) naturally occur in nature in the Earth’s crust and are difficult to decompose. HMs can be then introduced in enormous magnitude at various environmental levels due to human activities. Once in the environment at certain concentration, they persist for longer spreading up to the food chain and getting toxic for humans. HMs exist as inorganic elements (minerals) in rocks and comprise minerals with a specific density of more 10 than 5 g / cm3.The main threats to human health from HMs are associated with exposure particularly to Lead (Pb), Cadmium (Cd), Mercury (Hg) and Arsenic (As), and also to silver (Ag), Beryllium (Be), Barium (Ba), Chromium (Cr), Manganese (Mn) and Vanadium (V). However, even essential components such as iron (Fe), zinc (Zn), copper (Cu), and calcium (Ca) that are cofactors in various biological processes might lead to harmful effects when under excessive accumulation in the human body. Järup summarizes the adverse effects linked to human to HMs which 15 range from renal damages, bone issues and fractures, high blood levels, neurological disorders, and cancer. Furthermore, a recent issue has described dealing with adverse effects of heavy metals explicated at the intestinal level and specifically on the gut microbiota. The urgency of addressing HMs stems from their pervasive nature and the severe health consequences stemming from their accumulation and magnification in the environment, ultimately infiltrating the food chain and affecting human health through systemic adverse effects, including renal damage and 20 neurological disorders. Bioremediation is regarded as research largely focused on bacterial processes, which have numerous applications, applied with the specific aim of detoxifying pollutants. Bioremediation roots in the perspective of a sustainable environmental remediation by the implementation of rapid restoration strategies at the contaminated sites in an eco-friendly and economically viable framework. In respect to HMs, contaminated soils are commonly remedied 25 using chemical (e.g. precipitation, coagulation, chelation, electrodialysis), biological (e.g. biosorption by live / dead bacteria, bioaccumulation), and lastly physical (e.g. soil isolation, vitrification, nano-remediation, ion-exchange, particle trapping) methods which are the most implemented. However, the different methods for heavy metal remediation have pros and cons; some physic-chemical methods are expensive, require high amounts of chemicals, generate wastes, implies soil relocation, and do not fully solve the problem of a total bioremediation of 30 soils and other sites. Consequently, the research for novel sustainable remediation strategies finds a large field of applications. Phytoremediation, meaning the cultivation in soil dedicated to food production of plants adsorbing HMs, is listed as one the environmentally friendly strategies next to the use of microorganisms. Different groups of bacteria, fungi and algae of different extraction or microbial consortia have been applied in soil bioremediation: rhizobiont and soil 35 microbiota microorganism such as, among others, Lysobacter spp., Kaistobacter spp., Pseudomonas putida, 2 P240048 Cyanobacteria, Bacillus spp., the group of extremophiles such as halophiles, psychrophiles or acidophiles. Recently, genetically modified microorganisms (GMO) to enhance their bioremediation performances are also getting implemented in HMs detoxification; in this field, gene editing is used for the manipulation of genes that can convert toxic chemicals into less toxic compounds and for the remediation of xenobiotic compounds. However, 5 GMO implementation is allowed in soil remediation practices while it is not contemplated by any regulation for use in humans. The intestinal microbiota represents the complex and dynamic population of microorganisms localized in the small and large intestine which exert a marked influence on the host during homeostasis and disease. The study about the impact of HMs on the gut microbiota traces its origins back to the late 70s, when researchers found that rumen 10 microbiota populations could be inhibited by Hg, Cr and other HMs. Over the years many mechanisms in the complex relationship between gut microbiota and HMs have been elucidated. More in details, HMs create gut homeostasis disturbance via two main events: i) the modification of gut microbiome phylogenetic diversity and ii) the resulting alteration of metabolic activities (metabolites production and equilibrium). The consistent body of knowledge on the effects of HMs on gut microbiota composition depicts a general indication where the abundance 15 of Firmicutes and Proteobacteria decreases while Bacteroidetes populations rise. On the other side, changes in specific groups of metabolites such as i) Short Chain Fatty Acids (SCFA), ii) metabolites derived by the biotransformation of primary bile salts, iii) compounds derived by the metabolism of amino acids (e.g. p-cresol) and iv) lipids emerged in turn as importantly correlated to the structural modification of the gut populations as consequence of HMs exposure. However, the chances in the gut microbiota function and structure seem to be 20 metal dependent. It was demonstrated that Pb exposure in mice led to a decrease of Akkermansia spp. and an increase in Desulfovibrio spp. in concomitance with a body weight gain. But Desulfovibrio spp. have been also associated to Hg methylation thus facilitating Hg absorption at intestinal level. Desulfovibrio species members have been found to degrade mucin and to promote the metabolism of choline to trimethylamine-TMA (further oxidized trimethylamine oxide-TMAO in the liver). Consequently, an increase of Desulfovibrio spp. in the gut is thought to 25 compromise the gut barrier function and in turn exacerbate the absorption and impact at systemic level of the dietary HMs. What is emerging by this overview is that the intestine and the gut microbiota represent a target for HMs able to determine alterations in alfa- and beta-diversity, loss in the intestinal barrier function and perturbation in metabolites homeostasis thus setting the base for consequent impairment at a more systemic level. Heightened levels of environmental heavy metals (HMs) have been implicated as substantive contributory factors to adverse 30 pregnancy outcomes and developmental anomalies among vulnerable demographics, including infants, pregnant women, and the elderly. Moreover, coastal pollution and related heavy metal contamination carry implications for child health and future developmental outcomes. Given the rapid physiological development in children and foetuses, their heightened susceptibility to the toxic effects of HMs is particularly concerning. In view of the above, it is immediately apparent that the increasing presence of HMs in the environment constitutes a tangible threat to 35 human health, notably for those populations with inherent physiological vulnerabilities. 3 P240048 Document US 2023 / 0293604 A1 discloses a mixture comprising or, alternatively, consisting of at least one isolated bacterial strain belonging to the species Lactobacillus brevis, optionally combined with at least one isolated bacterial strain belonging to the species Lactobacillus plantarum and / or Lactobacillus pentosus. Said mixture is for use in a method for the preventive or curative treatment of a disorder or an ailment or a disease associated with an ingestion 5 or an accumulation -through food - of phosphonates in the human or animal microbiota. Gut bioremediation of heavy metals is never disclosed. Phosphonates and heavy metals are distinct chemical compounds with different properties and applications. Phosphonates are organic compounds containing a phosphorus-carbon bond, while heavy metals are a group of metals with high densities and toxicities. 10 Indeed, the toxicity of phosphonate is low to moderate, whereas the toxicity of heavy metals is high, even at low concentrations. Document US 2020 / 0164004 Al discloses a composition comprising a mixture which comprises or, alternatively, consists of at least one bacterial strain belonging to the genus Lactobacillus, Bifidobacterium, Lactococcus and Streptococcus, and / or at least one derivative thereof foruse in a method for the curative and / or preventive treatment 15 of (i) a pathology due to an imbalance in the intestinal microbiota, (ii) a neurodegenerative pathology, (iii) a cardiovascular pathology, (iv) a pathology linked to an immune system deficit, (v) a pathology linked to biological processes of physical aging and aging of the skin or cutis, (vi) a pathology linked to biological aging processes which lead to a progressive loss of memory and / or of the ability to concentrate, (vii) a pathology caused by oxidative stress, (viii) an autoimmune pathology, (ix) an inflammatory pathology and (x) a pathology due to an 20 altered intestinal permeability. Gut bioremediation of heavy metals is never disclosed. Document CN 111500505 A discloses a method for isolating probiotics capable of reducing heavy metal residues in the body, characterized in that it comprises: (1) isolating microorganisms from yogurt from Qinghai-Tibet Plateau pastures; (2) preparing MRS culture medium and PBS solution; (3) inoculation and cultivation; (4) bacterial strain 25 detection; (5) antioxidant capacity analysis of probiotics; (6) heavy metal resistance analysis of probiotics; and (7) heavy metal adsorption capacity analysis of probiotics. Document CN 111500505 A discloses Pediococcus acidilactici QZ-01, which was deposited at CCTCC on November 14, 2019, with a deposit number M2019930, is suggested to be used in the heavy metal residues in the body. 30 The present invention solves the technical problems related to the increasing presence of HMs by providing a novel bioremediation approach that leverages the health-promoting capabilities of specific lactobacilli strains to mitigate HMs' adverse effects in the gut, therefore presenting a viable and rapid strategy for intestinal HMs bioremediation. Gut microbiota can be regarded as source of bacteria potentially implemented in HMs detoxification. Up today, the data collected on regulatory framework, the safety of use and technological attitude are still scarce limiting the 4 P240048 possibility to market these bacteria in the coming future. Therefore, there is a need to provide probiotic strains capable of HMs detoxification which are safe to use. The inventors have carried out an intensive and prolonged research and development program with, among the others, the commercially available probiotic Lactobacilli derived from Probiotical S.P.A. bacteria strains collection. 5 All the bacteria strains disclosed and claimed in the present application have been deposited under the Budapest Treaty. A portion of the above research and development program was focused on a big group of bacteria for their ability to detoxify heavy metals, biogenic amines, and phosphonates. The project was then dedicated to the assessment of the best probiotics candidates strains to reduce the HM content in a complex gastro-intestinal model (SHIME). 10 SUMMARY OF THE INVENTION The present patent application outlines a methodology to select probiotic strains capable of HMs detoxification and assess their efficacy under simulated gastro-intestinal conditions. Through the innovative use of the simulation model SHIME®, the Applicant has validated the selected lactobacilli strains' capacity to reduce HMs' bioavailability. 15 Importantly, findings emphasize the strain- and HM-specific nature of this detoxification capability, underscoring the critical importance of targeted bacterial applications in effective bioremediation strategies. Lactobacilli are probiotics traditionally implemented in the large-scale development of fermented food and food supplements. Lactobacilli are members of the well-known Lactic Acid Bacteria (LAB) group and have a long history of use that routs its origin in the consumption of spontaneously fermented vegetable and animal foods. 20 Consequently, Lactobacilli in the human gut are constituted by adapted and resident members of the microbiota and by / plus fermented food- and food supplements associated transient lactobacilli. A first aspect of the present invention is related to at least one isolated bacterial strain selected from at least one of the species selected from the group comprising or, alternatively, consisting of: i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus, or mixtures of said at least one isolated bacterial strain 25 thereof, for use in a method of metals gut microbial bioremediation. The term “bioremediation” generally indicates the use of biological agents, such as bacteria, to detoxify pollutants i.e. to detoxify the gut microbiota from pollutants, such as HMs. This “bioremediation” provides a sustainable solution to environmental contamination that is increasingly critical in today's polluted world. In the context of the present invention, the term “bioremediation” indicates the reduction, removal or degradation 30 preferably of heavy metals (HMs) from the gut microbiota or intestinal microbiota through biological activity of microorganisms, in particular of lactobacilli bacterial strains. It is known that the gut is part of the digestive system — it's where food goes after eating it, to get broken down into nutrients and waste, and the intestine is the tubular part of the digestive tract that extends from the stomach to the anus (large intestine and small intestine). 5 P240048 In the context of the present invention, the term “gut microbial bioremediation” indicates the reduction, removal or degradation preferably of heavy metals at the intestinal level (for example, at the intestinal barrier) through biological activity of microorganisms, in particular of lactobacilli bacterial strains. The intestinal barrier is a semipermeable structure that allows the uptake of essential nutrients and immune 5 sensing, while being restrictive against pathogenic molecules and bacteria. Both structural and molecular components act together to fulfil this complex, but essential function of the gastrointestinal tract. The mucus layer forms a sieve-like structure overlying the intestinal epithelium. Antimicrobial peptides (AMPs) and secretory IgA molecules (sIgA) are secreted in the mucus layer as immune-sensing and regulatory proteins. The intestinal epithelial cells (IECs) form a continuous monolayer and are tightly attached to each other by junctional complexes. 10 The tight junctions (TJs) are located at the apical side of the cells and regulate the transport of small molecules and ions. The adherens junctions (AJs) and desmosomes provide strict cell-adhesion bonds and aid in the maintenance of the integrity of the intestinal barrier. The lamina propria contains immune cells (e.g. T cells, B cells, macrophages and dendritic cells) from the adaptive and innate immune system that take part in the immunological defence mechanisms of the intestinal barrier. 15 Preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is selected from the group comprising or, alternatively, consisting of Lactiplantibacillus plantarum 476LL 20 bi LP01 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P-21021, Lactiplantibacillus plantarum 776 / 1 bi LP02 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P-21020 and Lactiplantibacillus plantarum LP14 deposited on 16.01.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH 20 (DSMZ) and having accession number DSM 33401, or mixtures thereof; said at least one isolated bacterial strain belonging to the specie ii) Lactobacillus acidophilus is selected from the group comprising or, alternatively, consisting of Lactobacillus acidophilus LA02 deposited on 06.08.2008 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 21717, Lactobacillus acidophilus LA06 deposited on 13.10.2009 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH 25 (DSMZ) and having accession number DSM 23033, or mixtures thereof; said at least one isolated bacterial strain belonging to the specie iii) Lactobacillus crispatus is identified as Lactobacillus crispatus LCR04 deposited on 02.04.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) having accession number DSM 33487. Preferably, the at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum, is used in 30 mixture with at least one of ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. Preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum, preferably selected from LP01, LP02 and LP14, is used in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain 35 thereof. 6 P240048 Even more preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is LP14 and is used in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. The bacterial strains according to the present invention were selected because of their ability to detoxify the human 5 body from at least one heavy metal or a pool of heavy metals, biogenic amines and phosphonates. Specifically, they are able to detoxify the intestinal microbiota from pollutants, such as HMs, through the reduction, removal, or degradation of heavy metals (HMs) at the intestinal level (e.g., at the intestinal barrier level) due to their exerted biological activity once they have been administered into a subject's human body. The bacteria strains according to the present invention have been studied to determine their capacity to bind and 10 remove heavy metals (HMs) from the gastrointestinal environment (heavy metals detoxification by bacteria strains). Advantageously, the selected bacteria strains in accordance with the present invention show the ability to detoxify a pool of heavy metals (not only one heavy metal, but more than one at the same time) in the human’s GI. Further, they show the ability to favour HM segregation in the intestinal environment. Advantageously, the bacteria strains Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02, and 15 Lactobacillus crispatus LCR04, or mixtures thereof show their ability to detoxify heavy metals (HMs) and prevent resulting impairments on the intestine. In the context of the present invention, bacterial strains can also be indicated only with their trade name (for example, LP01, LP04, LP14, LA02, LA06, LCR04) and / or with their accession number. Preferably, said metals involved in the gut microbial bioremediation are heavy metals, which are selected from the group comprising or, 20 alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof. A second aspect of the present invention is related to a mixture comprising at least one isolated bacterial strain selected from at least one of the species selected from the group comprising or, alternatively, consisting of: i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, iii) Lactobacillus crispatus or mixtures of said at least one 25 isolated bacterial strain thereof, for use in a method of metals gut microbial bioremediation. Preferably, the mixture comprises or, alternatively, consists of at least one of the above-mentioned isolated bacterial strains. Preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum, is used in mixture with at least one of ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. 30 Preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum, preferably selected from LP01, LP02 and LP14, is used in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. 7 P240048 Even more preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is LP14 and is used in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. A third aspect of the present invention regards a composition comprising a mixture as above defined for use in a 5 method of metals gut microbial bioremediation, wherein said metals involved in the gut microbial bioremediation are heavy metals, which are selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof. According to the present invention, the method of gut microbial bioremediation is a method of detoxifying the human’s intestine from at least one pollutant, wherein said at least one pollutant is selected from a pool of heavy 10 metals, biogenic amines and phosphonates, or mixtures thereof. Biogenic amines, phosphonates and heavy metals are distinct chemical compounds. Biogenic amines are nitrogen-containing compounds classified into aromatic, aliphatic, and heterocyclic groups based on their chemical structure. For example, spermine and spermidine are biogenic amines naturally present in foods, whereas others, such as histamine, tyramine, putrescine, and cadaverine, are produced primarily through 15 microbial activity. The accumulation of biogenic amine in food is often associated with food spoilage and inadequate hygiene practices. Phosphonates are organic compounds containing a phosphorus-carbon bond, while heavy metals are a group of metals with high densities and toxicities. According to the present invention, said at least one isolated bacterial strain selected from at least one of the species selected from the group comprising or, alternatively, consisting of: 20 i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus, as above described, or mixtures thereof, is for use in a method of detoxifying the human’s intestine from heavy metals, preferably selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof. A detailed description of the above-mentioned aspects is below reported. 25 LIST OF FIGURES The present invention will now be described based on the attached figures, provided by way of non-limiting examples. Fig.1: Heavy metals (ions) reduction (%) in cell-free supernatant upon strain LP01 (a), LP02 (b), LP14(c), LA02 30 (d), LA06 (e), LCR04 (f) activity during co-culturing and exposition in post growth phase with heavy metals. Fig.2: Average viable population density (log count / reactor) ± stdev (n=3) of Lactiplantibacillus plantarum LP14 at different stages during small intestinal incubation (i.e., SI start, DUO end, JEJ end, and ILE end) and in the colonic environment (i.e., colon 0h and colon 24h). For clarification purposes, also the non-logarithmically transformed viable population densities (count / reactor) were included. Statistically significant differences from the preceding 35 time point were indicated by means of ‘∗’ (p<0.05). 8 P240048 Fig.3: Average viable population density (log count / reactor) ± stdev (n=3) of Lactobacillus crispatus LCR04 at different stages during small intestinal incubation (i.e., SI start, DUO end, JEJ end, and ILE end) and in the colonic environment (i.e., colon 0h and colon 24h). For clarification purposes, also the non-logarithmically transformed viable population densities (count / reactor) were included. Statistically significant differences from the preceding 5 time point were indicated by means of ‘∗’ (p<0.05). Fig.4: Average viable population density (log count / reactor) ± stdev (n=3) of Lactobacillus acidophilus LA02 at different stages during small intestinal incubation (i.e., SI start, DUO end, JEJ end, and ILE end) and in the colonic environment (i.e., colon 0h and colon 24h). For clarification purposes, also the non-logarithmically transformed viable population densities (count / reactor) were included. Statistically significant differences from the preceding 10 time point were indicated by means of ‘∗’ (p<0.05). Fig.5: Recovery (R) of Cadmium, Chromium, Mercury and Lead in cell free- supernatants for samples collected after small intestine incubation phase (ILE End) of strains LA02, LP14 and LCR04 in co-culture with heavy metals. HMs contents were measured by ICP-MS. In each strain, R is expressed with respect to the HMs content at ST start by adjusting results on volume changes along the reactors. Differences between cell pellets and cell free- 15 supernatant compared to pellets and supernatant in the blank sample were analyzed by t-test. Significance was declared as follows: *= 0.01 < p-value ≤ 0.05; **= 0.01 < p-value ≤ 0.001; ***= 0.001 < p-value ≤ 0.0001 Fig.6: Differential recovery (R) of Cadmium determined by ICP-MS in cell pellets and cell free- supernatants for samples collected after short term colonic incubation (C24h) of strains LA02, LP14 and LCR04 in co-culture with 20 heavy metals. Data are reported as mean values (n=3) and the error bars represent standard deviations. In each strain, R is expressed with respect to the HMs content at ST start by adjusting results on volume changes along the reactors. Differences between cell pellets and cell free- supernatant compared to pellets and supernatant in the blank sample were analyzed by t-test. Significance was declared as follows: *= 0.05 < p-value ≤ 0.01; **= 0.01 < p-value ≤ 0.001; ***= 0.001 < p-value ≤ 0.0001 25 Fig.7: Differential recovery (R) of Chromium determined by ICP-MS in cell pellets and cell free- supernatants for samples collected after short term colonic incubation (C24h) of strains LA02, LP14 and LCR04 in co-culture with heavy metals. Data are reported as mean values (n=3) and the error bars represent standard deviations. In each strain, R is expressed with respect to the HMs content at ST start by adjusting results on volume changes along the reactors. Differences between cell pellets and cell free- supernatant compared to pellets and supernatant in the 30 blank sample were analyzed by t-test. Significance was declared as follows: *= 0.05 < p-value ≤ 0.01; **= 0.01 < p-value ≤ 0.001; ***= 0.001 < p-value ≤ 0.0001 Fig.8: Differential recovery (R) of Mercury determined by ICP-MS in cell pellets and cell free-supernatants for samples collected after short term colonic incubation (C24h) of strains LA02, LP14 and LCR04 in co-culture with heavy metals. Data are reported as mean values (n=3) and the error bars represent standard deviations. In each 35 strain, R is expressed with respect to the HMs content at ST start by adjusting results on volume changes along 9 P240048 the reactors. Differences between cell pellets and cell free- supernatant compared to pellets and supernatant in the blank sample were analyzed by t-test. Significance was declared as follows: *= 0.05 < p-value ≤ 0.01; **= 0.01 < p-value ≤ 0.001; ***= 0.001 < p-value ≤ 0.0001 Fig.9: Differential recovery (R) of Lead determined by ICP-MS in cell pellets and cell free- supernatants for samples 5 collected after short term colonic incubation (C24h) of strains LA02, LP14 and LCR04 in co-culture with heavy metals. Data are reported as mean values (n=3), and the error bars represent standard deviations. In each strain, R is expressed with respect to the HMs content at ST start by adjusting results on volume changes along the reactors. Differences between cell pellets and cell free- supernatant compared to pellets and supernatant in the blank sample were analyzed by t-test. Significance was declared as follows: 10 *= 0.05 < p-value ≤ 0.01; **= 0.01 < p-value ≤ 0.001; ***= 0.001 < p-value ≤ 0.0001 Fig.10: Cell number (AFU / ml) and heat flow (µW / sec) of strain L. plantarum LP14 grown under different conditions; i) LP14 in MRS medium (optimal condition), ii) LP14 in MRS supplemented with HM mix in ratio 1:100 (cells plus HM mix 1:100), iii) LP14 in MRS supplemented with HM mix in ratio 1:1000 (cells plus HM mix 1:1000), iv) LP14 in MRS supplemented with HM mix in ratio 1:10000 (cells plus HM mix 1:10000, v) MRS plus HM mix (Blank with only 15 HM mix). Solid lines of thermographs represent the averaged values in heat production while halos surrounding solid lines indicate the standard deviation from mean values calculated with the calView 2.0 software. Enumeration data are shown in the boxes; initial (T=0) cell inoculum was standardized by flow-cytometric enumeration at 6.0 log / ml. Boxes of the same color indicates the AFU / ml recorded for the same sample at timepoint (h) 4, 22 and 48. Standard deviations were always below 0.4. 20 Fig.11: Cell number (AFU / ml) and heat flow (µW / sec) of strain L. crispatus LCR04 grown under different conditions; i) LCR04 in MRS medium (optimal condition), ii) LCR04 in MRS supplemented with HM mix in ratio 1:100 (cells plus HM mix 1:100), iii) LCR04 in MRS supplemented with HM mix in ratio 1:1000 (cells plus HM mix 1:1000), iv) LCR04 in MRS supplemented with HM mix in ratio 1:10000 (cells plus HM mix 1:10000, v) MRS plus HM mix (Blank with only HM mix). Experiments were conducted in triplicate. Solid lines of thermographs represent the averaged 25 values in heat production while halos surrounding solid lines indicate the standard deviation from mean values calculated with the calView 2.0 software (Symcel AB, 2023). Enumeration data are shown in the boxes; initial (T=0) cell inoculum was standardized by flow-cytometric enumeration at 6.0 log / ml. Boxes of the same color indicates the AFU / ml recorded for the same sample at timepoint (h) 4, 22 and 48. Standard deviations were always below 0.4. Fig. 12: Cell number (AFU / ml) and heat flow (µW / sec) of strain L. acidophilus LA02 grown under different 30 conditions; i) LA02 in MRS medium (optimal condition), ii) LA02 in MRS supplemented with HM mix in ratio 1:100 (cells plus HM mix 1:100), iii) LA02 in MRS supplemented with HM mix in ratio 1:1000 (cells plus HM mix 1:1000), iv) LA02 in MRS supplemented with HM mix in ratio 1:10000 (cells plus HM mix 1:10000, v) MRS plus HM mix (Blank with only HM mix). Experiments were conducted in triplicate. Solid lines of thermographs represent the averaged values in heat production while halos surrounding solid lines indicate the standard deviation from mean 35 values calculated with the calView 2.0 software (Symcel AB, 2023). Enumeration data are shown in the boxes; 10 P240048 initial (T=0) cell inoculum was standardized by flow-cytometric enumeration at 6.0 log / ml. Boxes of the same color indicates the AFU / ml recorded for the same sample at timepoint (h) 4, 22 and 48. Standard deviations were always below 0.4. Fig.13: Acidification kinetics of strain L. plantarum LP14 during culturing at 30°C in pure MRS and MRS and heavy 5 metals added in different concentrations (1:100, 1:1000 and 1:10000). Initial cell inoculum initial (T=0) cell inoculum was standardized by flow-cytometric enumeration at 6.0 log / ml. pH was monitored in continuous on the instrument iCinac L.A.B. Fermentation Monitor Series. Fig.14: Acidification kinetics of strain L. crispatus LCR04 during culturing at 30°C in pure MRS and MRS and heavy metals added in different concentrations (1:100, 1:1000 and 1:10000). Initial cell inoculum initial (T=0) cell inoculum 10 was standardized by flow-cytometric enumeration at 6.0 log / ml. pH was monitored in continuous on the instrument iCinac L.A.B. Fermentation Monitor Series. Fig.15: Acidification kinetics of strain L. acidophilus LA02 during culturing at 30°C in pure MRS and MRS and heavy metals added in different concentrations (1:100, 1:1000 and 1:10000). Initial cell inoculum initial (T=0) cell inoculum was standardized by flow-cytometric enumeration at 6.0 log / ml. pH was monitored in continuous on the 15 instrument iCinac L.A.B. Fermentation Monitor Series. Fig.16: Schematic representation of Gut Ex Vivo System (GEVS). The device consists of a silicon support with 6 independent chambers where small intestines are inserted. Each intestine is connected to two syringe systems allowing the complete flow of the medium in the inner intestinal compartment (luminal flow). Also, the chambers are imbibed with the medium to sustain the full viability of tissues. The medium flow system is allowed by two 20 synchronized pumps which inject (Pi, INPUT) and suck (Po, OUTPUT) the culture medium. Representation by Gagliardi et al., 2021. Fig.17: Small intestine from C57BL / 6J mice were cultivated in GEVS and exposed (5 h) to complete Iscove’s medium (CTRL) or heavy metal mix (MIX; Table 8), and tissue viability (A) or permeability (B) were evaluated. Experiments were performed twice and in technical duplicate. Histograms represent mean ± SD; ***p<0.001. 25 Fig.18: Small intestine from C57BL / 6J mice were cultivated in GEVS (5h) and treated with complete Iscove’s medium (CTRL), with heavy metal mix (MIX), and with mixture of cell free supernatant (CFS) obtained after co- culturing step of strain L. plantarum LP14 with HM mix (MIX+LP14). Intestinal permeability was evaluated by FITC- Dextran assay (A) and by measuring the expression levels of genes encoding for i) tight junction proteins (B) Claudin-2 (CLD-2), Claudin-15 (CLD-15) and Occludin (OCL); ii) pro inflammatory cytokines IFNγ and TNFα and 30 the anti-inflammatory cytokine IL-10 (C) by qPCR analysis. The expression (mRNA) levels of the genes were evaluated in the same experimental conditions. Histograms represent mean ± SD of duplicate sample; **** p < 0.0001; ***p<0.001; ** p < 0.01; * p <0.1. Fig.19: Small intestine from C57BL / 6J mice were cultivated in GEVS (5h) and treated with complete Iscove’s medium (CTRL), with heavy metal mix (MIX), and with mixture of cell free supernatant (CFS) obtained after co- 35 culturing step of strain L. crispatus LCR04 with HM mix (MIX+ LCR04). Intestinal permeability was evaluated by 11 P240048 FITC-Dextran assay (A) and by measuring the expression levels of genes encoding for i) tight junction proteins (B) Claudin-2 (CLD-2), Claudin-15 (CLD-15) and Occludin (OCL); ii) pro inflammatory cytokines IFNγ and TNFα and the anti-inflammatory cytokine IL-10 (C) by qPCR analysis. The expression (mRNA) levels of the genes were evaluated in the same experimental conditions. Histograms represent mean ± SD of duplicate sample; **** p < 5 0.0001; ***p<0.001; ** p < 0.01; * p <0.1. Fig.20: Small intestine from C57BL / 6J mice were cultivated in GEVS (5h) and treated with complete Iscove’s medium (CTRL), with heavy metal mix (MIX), and with mixture of cell free supernatant (CFS) obtained after co- culturing step of strain L. acidophilus LA02 with HM mix (MIX+ LA02). Intestinal permeability was evaluated by FITC-Dextran assay (A) and by measuring the expression levels of genes encoding for i) tight junction proteins (B) 10 Claudin-2 (CLD-2), Claudin-15 (CLD-15) and Occludin (OCL); ii) pro inflammatory cytokines IFNγ and TNFα and the anti-inflammatory cytokine IL-10 (C) by qPCR analysis. The expression (mRNA) levels of the genes were evaluated in the same experimental conditions. Histograms represent mean ± SD of duplicate sample; **** p < 0.0001; ***p<0.001; ** p < 0.01; * p <0.1. 15 DETAILED DESCRIPTION OF THE INVENTION The Applicant has been able to design a specific methodology in order to select probiotic strains capable of metal microbial bioremediation, in particular heavy metals gut microbial bioremediation, and to study and assess their efficacy under simulated gastro-intestinal conditions. The first aspect of the present invention is related to at least one isolated bacterial strain selected from at least one 20 of the species selected from the group comprising or, alternatively, consisting of: i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus, or mixtures of said at least one isolated bacterial strain thereof, for use in a method of metals gut microbial bioremediation. Preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum, is used in mixture with at least one of ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus as above identified, or 25 mixtures of said at least one isolated bacterial strain thereof. Preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is selected from the group comprising or, alternatively, consisting of Lactiplantibacillus plantarum 476LL 20 bi LP01 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P-21021, Lactiplantibacillus plantarum 776 / 1 bi LP02 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P-21020, and Lactiplantibacillus plantarum LP14 deposited on 16.01.2020 at 30 Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 33401, or mixtures thereof. Preferably, said at least one isolated bacterial strain belonging to the specie ii) Lactobacillus acidophilus is selected from the group comprising or, alternatively, consisting of Lactobacillus acidophilus LA02 deposited on 06.08.2008 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 12 P240048 21717, Lactobacillus acidophilus LA06 deposited on 13.10.2009 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 23033, or mixtures thereof. Preferably, said at least one isolated bacterial strain belonging to the specie iii) Lactobacillus crispatus is identified as Lactobacillus crispatus LCR04 deposited on 02.04.2020 at Deutsche Sammlung von Mikroorganismen und 5 Zellkulturen GmbH (DSMZ) having accession number DSM 33487. Preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum, preferably selected from LP01, LP02 and LP14, is used in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. 10 Even more preferably, said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is LP14 and is used in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. The bacterial strains according to the present invention have been selected because of their ability to detoxify the human body from at least one heavy metal or a pool of heavy metals, biogenic amines and phosphonates. Specifically, 15 they are able to detoxify the intestinal microbiota from pollutants, such as HMs, through the reduction, removal, or degradation of heavy metals (HMs) at the intestinal level (e.g., at the intestinal barrier level) due to their exerted biological activity once they have been administered into a subject's human body. The bacteria strains according to the present invention have been studied to determine their capacity to bind and remove heavy metals (HMs) from the gastrointestinal environment (heavy metals detoxification by bacteria strains). 20 Advantageously, the selected bacteria strains in accordance with the present invention show the ability to detoxify a pool of heavy metals (not only one heavy metal, but more than one at the same time) in the human’s GI. Further, they show the ability to favour HM segregation in the intestinal environment. More preferably, said isolated bacterial strain is selected from the group comprising or, alternatively, consisting of LP14 (DSM 33401), LA02 (DSM 21717) and LCR04 (DSM 33487) or mixtures thereof. Even more preferably, said 25 isolated bacterial strain is selected from LP14 (DSM 33401) and / or LCR04 (DSM 33487). Advantageously, the bacteria strains Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02, and Lactobacillus crispatus LCR04 or mixtures thereof show their ability to detoxify heavy metals (HMs) and prevent resulting impairments on the intestine. In fact, as reported in Example 2, among the strains examined, LP14 and LCR04, upon incubation with HMs, led 30 to a general increase of HMs found in the cell pellets. Preferably, said at least one isolated bacterial strain is advantageously used for gut microbial bioremediation of metals, wherein said metals are heavy metals (HMs), which are selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof (in short, HMs). 13 P240048 More preferably, said at least one isolated bacterial strain is advantageously used for heavy metals gut microbial bioremediation selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As even more preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof (in short, HMs). A second aspect of the present invention is a mixture comprising at least one isolated bacterial strain selected from 5 at least one of the species selected from the group comprising or, alternatively, consisting of: i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, iii) Lactobacillus crispatus or mixtures of said at least one isolated bacterial strain thereof, for use in a method of metals gut microbial bioremediation, wherein said method of gut microbial bioremediation is a method of detoxifying the human’s intestine from at least one pollutant, preferably selected from a pool of heavy metals, biogenic amines and phosphonates, or mixtures thereof. More preferably, the mixture 10 according the second aspect of the present invention is for use in a method of detoxifying the human’s intestine from heavy metals. Preferably, said mixture comprises or, alternatively, consists of: - at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is selected from the group comprising or, alternatively, consisting of Lactiplantibacillus plantarum 476LL 20 bi LP01 deposited on 16.10.2001 15 at BCCM / LMG and having accession number LMG P-21021, Lactiplantibacillus plantarum 776 / 1 bi LP02 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P-21020, and Lactiplantibacillus plantarum LP14 deposited on 16.01.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 33401, or mixtures thereof; - said at least one isolated bacterial strain belonging to the specie ii) Lactobacillus acidophilus is selected from the 20 group comprising or, alternatively, consisting of Lactobacillus acidophilus LA02 deposited on 06.08.2008 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 21717, Lactobacillus acidophilus LA06 deposited on 13.10.2009 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 23033, or mixtures thereof; - said at least one isolated bacterial strain belonging to the specie iii) Lactobacillus crispatus is identified as 25 Lactobacillus crispatus LCR04 deposited on 02.04.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) having accession number DSM 33487. Preferably, the mixture according to the second aspect of the present invention comprises at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum always in mixture with at least one of ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus. 30 Preferably, the mixture according to the second aspect of the present invention comprises at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum, preferably selected from LP01, LP02 and LP14, always in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified. 14 P240048 Even more preferably, the at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is LP14 and is used in mixture with at least one of ii) Lactobacillus acidophilus as above identified, and iii) Lactobacillus crispatus as above identified, or mixtures of said at least one isolated bacterial strain thereof. Said mixture according to the second aspect of the present invention is preferably for use in a method of detoxifying 5 the human’s intestine from heavy metals. The bacterial strains according to the present invention were selected because of their ability to detoxify the human body from at least one heavy metal or a pool of heavy metals, biogenic amines and phosphonates. Specifically, they are able to detoxify the intestinal microbiota from pollutants, such as HMs, through the reduction, removal, or degradation of heavy metals (HMs) at the intestinal level (e.g., at the intestinal barrier level) due to their exerted 10 biological activity once they have been administered into a subject's human body. The bacteria strains according to the present invention have been studied to determine their capacity to bind and remove heavy metals (HMs) from the gastrointestinal environment (heavy metals detoxification by bacteria strains). Advantageously, the selected bacteria strains in accordance with the present invention show the ability to detoxify a pool of heavy metals (not only one heavy metal, but more than one at the same time) in the human’s GI. Further, 15 they show the ability to favour HM segregation in the intestinal environment. More preferably, the at least one isolated bacterial strain in the mixture is preferably selected from the group comprising or, alternatively, consisting of LP14 (DSM 33401), LA02 (DSM 21717) and LCR04 (DSM 33487) or mixtures thereof, even more preferably from LP14 (DSM 33401) and / or LCR04 (DSM 33487). Advantageously, the bacteria strains Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02, and 20 Lactobacillus crispatus LCR04 or mixtures thereof show their ability to detoxify heavy metals (HMs) and prevent resulting impairments on the intestine. Preferably, said mixture is advantageously used in a method for gut microbial bioremediation of metals selected from heavy metals, said heavy metals being preferably selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As more preferably from Cd, Cr, Hg, and Pb or mixtures 25 thereof. A third aspect is related to a composition comprising a mixture as above described, and one or more additive and / or excipient of pharmaceutical or food grade, for use in a method of metals gut microbial bioremediation. Preferably, the composition according to the third aspect of the invention is for use in a method of gut microbial bioremediation of metals selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, 30 Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof. More preferably, the composition according to the third aspect of the invention is for use in a method of gut microbial bioremediation of heavy metals, preferably selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or mixtures thereof. Preferably, the composition further comprises at least one active ingredient having detoxifying activity. Preferably, 35 said at least one active ingredient having detoxifying activity is an ingredient for dietary supplement and / or a 15 P240048 botanical\active ingredient. In the context of the present invention, the term “ingredient(s) for dietary supplement” is meant to indicate an ingredient of a dietary nature or rather allowed in dietary supplementation. Preferably, the composition comprises at least one ingredient for dietary supplement selected from the group 5 comprising or, alternatively, consisting of curcumin, quercetin, melatonin, vitamin C, lutein and myo-inositol. Preferably, the composition comprises at least one botanical / active ingredient selected from the group comprising or, alternatively, consisting of rosmarinic acid, apigenin, epigallocatechin gallate and ellagic acid. Even more preferably, the composition comprises at least one ingredient for dietary supplement selected from the 10 group comprising or, alternatively, consisting of curcumin, quercetin, melatonin, vitamin C, lutein and myo-inositol and / or at least one botanical\active ingredient selected from the group comprising or, alternatively, consisting of rosmarinic acid, apigenin, epigallocatechin gallate and ellagic acid. Preferably, said composition is formulated for oral use, preferably in the form of granulate, a tablet, a gel or a capsule. 15 “Solid state” means that the composition may exist in the form of granules or powder. The granular or powder compositions are mixed with pharmacologically acceptable additives and excipients to provide an end product such as, for example, a dietary supplement, a medical device or a pharmaceutical composition. The end product may be in pharmaceutical dosing units, such as, for example, granulate in a sachet, tablet, gel or capsule. However, if desired or necessary, the composition may be formulated in liquid form, for example through dissolving 20 or suspension in water. Given the detoxifying ability of the bacterial strains described in the present invention, the mixture according to the invention or a composition comprising it may be advantageously used for the prevention and / or reduction of the harmful effects of heavy metals on gut health and / or for the treatment of diseases or disorders or reduction of risk factors caused by heavy metal. 25 In view of this, the mixture according to the invention and / or a composition comprising it can be used in a method of treatment for the prevention and / or reduction of the harmful effects of heavy metals on gut health and / or for the treatment of diseases or disorders or reduction of risk factors caused by metals, wherein said metals are selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As preferably from Cd, Cr, Hg, and Pb; or Al, or mixtures thereof. 30 Preferably, the mixture according to the invention and / or a composition comprising it can be used in a method of treatment for the prevention and / or reduction of the harmful effects of heavy metals on gut health and / or for the treatment of diseases or disorders or reduction of risk factors caused by heavy metals preferably selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As more preferably from Cd, Cr, Hg, and Pb, or mixtures thereof. Said method of treatment includes the administration of the mixture 35 according to the invention and / or a composition comprising it to a subject in need thereof. 16 P240048 In this regard, the methods of administration, the interval between treatment doses and the duration of the treatment will be established by the doctors as needed. EXPERIMENTAL SECTION 5 Example 1 - Screening for best candidate probiotic strains for heavy metals detoxification Probiotic strains included in the study were all belonging to lactobacilli and selection criteria included i) the selection of strains from different species, ii) literature reports on their presence as members of autochthonous microbiota in environmental or vegetable food matrices, iii) scientific evidence on the capability of these species to detoxify chemical agents. 10 Heavy metals (HMs) were selected as environmental chemical agents with an associated risk to contaminate food chains, followed by penetration of the human body via ingestion. Their persistence and difficulty in being remediated by the food chain make the assessment of new detoxification strategies for this contaminant category worth to be investigated. 15 Materials and Methods Bacterial strains Lactobacillus strains were all belonging to Probiotical strains collection and already registered at international depository Institutes at their use in this study. The strains used in the experiments were the following: - Lactiplantibacillus plantarum 476LL 20 bi LP01 deposited on 16.10.2001 at BCCM / LMG and 20 having accession number LMG P-21021, - Lactiplantibacillus plantarum 776 / 1 bi LP02 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P-21020, - Lactiplantibacillus plantarum LP14 deposited on 16.01.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 33401, 25 - Lactobacillus acidophilus LA02 deposited on 06.08.2008 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 21717, - Lactobacillus acidophilus LA06 deposited on 13.10.2009 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 23033, - Lactobacillus crispatus LCR04 deposited on 02.04.2020 at Deutsche Sammlung von 30 Mikroorganismen und Zellkulturen GmbH (DSMZ) having accession number DSM 33487. All the strains were previously characterized by Probiotical Research s. r. l. team for paramount features linked to their safety and functional activities including i) antibiotic resistance (genotypic and phenotypic analysis), ii) identification on Whole Genome Sequencing, iii) bio typing, iv) antimicrobial activity spectrum, v) modulation of immune system and vi) protection and restoring of human intestinal barrier function (data not shown). 17 P240048 Furthermore, all the strains are currently included in Probiotical finished products pipeline so their use and acceptance in humans has been extensively consolidated. Heavy metals solutions preparation 5 HMs included in this study were Cadmium (Cd), Chromium (Cr), Mercury (Hg) and Lead (Pb). All HMs were used in their inorganic form as CdS04, CrCl3, Hg(NO3)2 and PbCl2 for Cd, Cr, Hg and Pb respectively. It is known that in biological systems, for example, in the human biological system, metals are present in their stable ionic form, for example, in their stable cationic form. Salts of metals were purchased from Sigma Aldrich: CdS04 code: 481882-5G; CrCl3 code: 230723; Hg(NO3)2 10 code: 104439 PbCl2 code:268690; FeCl3 code 157740). HMs were mixed to demineralized water to obtain stock solutions at different concentration of each metal. HM solutions were then sterilized by filtration on 0.22 µm filters. HM stock solutions were then added to bacterial growth media to reach the final concentrations described in table 1. 15 Table 1 - Heavy Metals solutions and final concentrations used in challenge test against probiotic strains. Bacterial culture preparation Bacterial cultures were originally stored at Probiotical S.P.A as frozen Master Cell Bank (MCB) and Working Cell Bank (WCB), routinely checked for stability and purity by Quality Control Division of Probiotical S.P.A. Bacterial 20 cultures used in this study were activated by inoculating one glycerol cell stock at 10% in fresh growing medium, further proceeding with 24h-anaerobic incubation at 37°C. Growing medium was the de Man Rogosa Sharpe-MRS (Oxoid™) supplemented with 0.05% L-Cysteine-hydrochloride (Sigma Aldrich, code:168149) for strains LA02, LA06 and LCR04. Bacterial cultures were sub-cultured (2%) in MRS twice according to the procedure described above before their use in challenge tests against HMs. 25 Assessment of heavy metals detoxification activity by Lactobacilli Lactobacilli strains LP01, LP02, LP14, LA06, LA02 and LCR04 were studied for their potential ability to detoxify HMs. Bacterial cultures were inoculated with HM solutions (Table 1) to start two different challenge tests. According 18 P240048 to the first test, HM solutions were previously added to growing media to reach final concentrations of HM mix1 and HM mix2 (Table 1). Bacterial cultures were then inoculated to reach an OD of 0.2 (600 nm) in HM-containing media, followed by incubation for 24h at 37°C in anaerobic conditions. Differently, bacterial cultures were previously inoculated to reach an OD of 0.2 (600 nm) in growing media and incubated according to times and temperatures 5 reported above to allow bacterial growth; afterwards, overnight bacterial cultures were exposed to HM (for 24H, 37°C) solutions added to reach final concentrations of HM mix1 and HM mix2. Heavy Metals quantification HMs quantification was performed by Inductively Coupled Plasma Mass Spectroscopy (ICP-MS). ICP-MS was 10 performed on instrument iCAP RQ. Plasma vector was created by heating Argon to allow its passage from gas into plasma state. After ionization, the molecules in each sample were separated based on mass-to-charge ratio and quantified using a mass spectrometer based on AOAC 2015.06, EN 15763:2010, EPA 6020 B 2014. Bacterial cultures were separated in the fractions of cell-free supernatant and cell pellets by centrifugation (6000 rpm x 6 min). Cell free supernatants were analyzed for HMs detection and quantification before (T0=0h) and after bacterial 15 growth (T1=24h) in the case co-culturing with HMs mix; in case of biomass accumulation during growth followed by further exposition to MHs Mix, samples were analyzed at (T0=0h) and post HM exposition (T2=+24h) on cell free supernatants. All the culturing experiments and quantifications were run in triplicates. Results are calculated as reduction (%) of HMs by comparing levels obtained at T1 and T2 of the single HM with its initial concentration at T0. Results are 20 expressed as mean values ± standard deviations. Results and Discussion Lactobacilli strains activity on Heavy Metals Probiotic strains activity on HMs was characterized by HMs quantification during co-culturing experiment or during 25 HM exposition in the post-growth phase. HMs ions were quantified by ICP-MS at timing T0, T1 and T2 reported above. HMs were quantified at T0 to check that their concentrations (Table 2) were in line with the expected HMs levels in Mix1 and Mix2 (Table 1). Table 2 - Heavy metals (as salts and metallic ions) final concentration (mg / L) added to bacterial growth 30 medium and tested against probiotic strains. 19 P240048 When bacterial strains were co-cultured (24h) in growing medium supplemented with HMs or were exposed (24h) to HMs in post growth-phase, differences in HMs levels were detected (Table 3). Results suggested that HMs 5 reduction effects were strain-, dose- and metal specific, as reported in Table 3, and Fig.1. Table 3 - Heavy metals (ions) reduction (%) in cell-free supernatant upon strain LP01 (a), LP02 (b), LP14(c), LA02 (d), LA06 (e), LCR04 (f) activity during co-culturing and exposition in post growth phase with heavy metals. Heavy metals were tested at two different concentrations indicated as 1 and 2. 10 20 P240048 HM Reduction (%) Strai HM Co-culturing condition Post growth-exposition n Mix Cd2+Cr3+Hg2+Pb2+Cd2+Cr3+Hg2+Pb2+ 21 P240048 5 HM quantifications were carried out on cell-free supernatant after cell culture centrifugation for cell pellets separation in the hypothesis of HM removal by bacteria via cell binding / internalization phenomenon. It is worth mentioning that, since we dedicated this preliminary investigation to the pure assessment of the 10 differences in metal concentration before and after the bacterial intervention by direct analysis of bacterial cell-free supernatant after metal contact, we can argue that a reduction in HMs concentration might be mostly ascribed to binding or internalization activity by bacterial cells. Accordingly, we can ascribe a reduction of HM concentration in the supernatants because of the hypothetical metal-trapping activity operated by the cells. Then it is also due to mention that all the metals implemented in this study were used in their inorganic form (CdS04, CrCl3, Hg(NO3)2 15 and PbCl2) as metal form is a factor influencing the detoxification activity by bacteria. It is known that in biological systems, for example, in the human biological system, metals are present in their stable ionic form, for example, in their stable cationic form. Although with the observed differences, all the probiotic strains tested showed a shared general trend in the detoxification for the specific metals. Indeed, most of the strains proved to be able to reduce the amount of Cd, Hg 20 and finally Cr, while an activity on Pb was demonstrated only by LA02, LCR04 and LA06. Example 2 - Heavy metals detoxification by probiotic strains in a gut model system Contamination of the environment and agricultural products by chemicals is a severe problem impacting ecosystems globally. Nowadays, because of rapidly expanding industrial areas, emissions, farming with application of fertilizers and pesticides, animal breeding, wastewater irrigation and several other modern human practices, soils 25 and water can get contaminated. Under certain conditions and dosages, these chemical agents are regarded as pollutants. Indeed, they can initially contaminate soil, water, and air and then enter the food chain due to production, processing, handling, transport, or because of environmental contamination, thus reaching humans. Overall, HM exposure is correlated with unbalances at the systemic level (neurological and kidney damages, alteration of immune system functioning and metabolic pathways) but specific heavy metals are also responsible for targeted 30 disturbances. Accordingly, Mercury (Hg) is correlated to an exacerbation of oxidative stress by the inactivation of antioxidant enzymes and, in its form Hg (II) this metal is corrosive, provoking damage to the gastrointestinal layer. Lead can block calcium-dependent voltage channels, impair DNA and compromise transcription processes. On one hand HMs so extensively affect the gut microbiome, on the other hand the gut microbiota has been demonstrated to help the host in the detoxification of HM by i) modulating the absorption and metabolism of HMs, 22 P240048 ii) generating a suitable intestinal environment in terms of pH and oxidative balance, influencing the expression of detoxifying enzymes and proteins and iii) by maintaining an optimal intestinal barrier function so thus limiting HM systemic circulation. Considering these two-face and mutual aspects, the relationship between HM and gut microbiota can be interpreted as bidirectional and interactive. Lactobacilli in the human gut are constituted by 5 adapted and resident members of the microbiota and by / plus fermented food- and food supplements associated transient lactobacilli. Overall, chemicals direct detoxification by Lactobacilli can be sustained by chemical-cell wall interactions (e.g. sequestration, binding) without the involvement of bacterial metabolism or, differently, by complex processes (e.g. internalization, biotransformation) that rely on active metabolic mechanisms exerted on the given heavy metal. Among these mechanisms of action (MoA), some are generally explicated against HM while others 10 are HM-specific namely the reduction by L. plantarum TW1-1 of Cr (VI) to the less soluble and toxic form Cr (III) thus promoting Cr precipitation. The present experiment aimed at assessing whether selected lactobacilli from previous findings were able to operate HM gut microbial bioremediation. To investigate this feature, strains were tested in the Human Simulator of the Intestinal Ecosystem against HM solution prepared as bland of Mercury (Hg), Cadmium (Cd), Lead (Pb) and Chromium (Cr). SHIME reactor was set in the configuration for dynamic upper 15 gastrointestinal tract (GIT) simulation and short-term colonic simulation under fed conditions. Materials and Methods Bacterial strains The present experiment was carried out on Lactiplantibacillus plantarum LP14 (DSM 33401), Lactobacillus 20 acidophilus LA02 (DSM 21717), and Lactobacillus crispatus LCR04 (DSM 33487), Lactobacilli strains all belonging to Probiotical strains collection, and already registered at international depository Institutes at their use in this study. All the strains were previously characterized by Probiotical Research s. r. l. team for paramount features linked to their safety and functional activities including i) absence of antibiotic resistance (genotypic and phenotypic analysis), ii) identification by Whole Genome Sequencing, iii) biotyping, iv) antimicrobial activity spectrum, v) modulation of 25 immune system and vi) protection and restoring of human intestinal barrier function (data not shown). Bacterial culture preparation Bacterial cultures were originally stored at Probiotical S.P.A. as frozen and glycerol protected Cell Bank, routinely checked for stability and purity. Bacterial cultures used in this study were activated by inoculating glycerol cell 30 stocks at 10% in fresh growing medium, further proceeding with 16h- incubation at 37°C. Growth medium was de Man Rogosa Sharpe-MRS. All strains were sub-cultivated twice, then bacterial cultures were centrifuged (6000 rpm x 6 min) and cell pellets were washed with sterile water and subsequently resuspended in cryo-protectant solution (composition not disclosed) to be finally lyophilized (R&D lab scale freeze drier) in mono-dose glass vials to be used as inoculum in 35 the SHIME system. Before their use, lyophilized bacteria were checked for viable counts by flow-cytometry and 23 P240048 absence of contaminants. For their use in SHIME, lyophilized probiotic cultures were resuspended in sterile dH2O, let to rehydrate (10 min at room temperature) and inoculated in the SHIME reactors to ensure a concentration of approximately 1010 viable cell / reactor at the moment of inoculation. 5 Heavy Metals (HM) solution preparation HM included in this study were Cadmium (Cd), Chromium (Cr), Mercury (Hg) and Lead (Pb). All the HMs used in this study were purchased from Sigma-Aldrich. HMs were all tested in their inorganic form (Table 4) and were mixed to prepare a 10X HM stock solution in Phosphate-buffered saline solution- PBS (8.8 g / L K2HPO4, 6.4 g / L KH2PO4, 8.5 g / L NaCl, 0.5 g / L L-Cystein HCl). HM stock solution was used in the SHIME system to reach the final 10 concentration at the inoculum described at Table 4. Table 4 - Details of Heavy Metals form and concentration at the inoculum in SHIME System (starting point, stomach compartment) tested against probiotic strains. 15 Experimental Design in SHIME reactor Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02 or Lactobacillus crispatus LCR04 were tested for their ability to reduce the HM content of a contaminated food matrix during passage through the complete gastrointestinal tract (GIT). For this purpose, the Simulator of Human Intestinal Microbial Ecosystem in the configuration for dynamic upper GIT simulation and short-term colonic simulation under fed condition was used. In 20 detail, the technology platform was used to assess the impact of the probiotic strains on the bioaccessibility of HMs during digestion in the stomach and the small intestinal phase. The upper gastrointestinal tract simulation (i.e., stomach and small intestine) was extended with a sterile short-term colonic simulation to study the impact of the strains on bioaccessibility in the colon environment. Samples were collected at different time points during passage through the gastrointestinal tract to monitor survival of the probiotic bacteria and the bioaccessible HM 25 concentration. Upper gastrointestinal tract simulation under fed conditions and sample inoculum The upper GIT simulation was performed in double-jacketed reactors simulating the gastric and small intestinal digestion conditions in a sequential order. The temperature was maintained at 37°C during the entire upper GIT 30 simulation and magnetic stirring (300 rpm) was applied to homogenize the reactor content. Continuous pH control 24 P240048 was implemented by using a Senseline pH meter F410 (ProSense, Oosterhout, The Netherlands) and an automatic pump dosage of HCl (0.5 M; Chem-lab, Zedelgem, Belgium) or NaOH (0.5 M; Chem-lab). To mimic fed conditions (i.e., administration of the test product during or immediately after consumption of a meal), specific pH profiles, enzyme levels, and retention times were set accordingly. In addition, the gastric and small intestinal juices were 5 specifically designed to mimic fed environmental conditions. The stomach digestion had a total duration time of 120 min; during this timeframe the pH decreased from 4.6 to 3.0 in a sigmoidal way by the controlled addition of HCl (0.5 M) at established time points. Pepsin was supplied with the activity being standardized by measuring absorbance increase at 280 nm of trichloroacetic acid -soluble products upon digestion of haemoglobin (reference protein). Gastric simulating juice (222.4 ml) containing the SHIME® nutritional medium (23.64 g / L of product 10 PDNM001B from ProDigest), 4.18 g / L NaCl (VWR, Leuven, Belgium) and 0.76 g / L KCl (Chem-lab) was initially mixed with 1.3 mL lecithin (13.5 g / L; Carl Roth GmbH + Co. KG, Karlsruhe, Germany) and 12.1 mL pepsin (40 g / L; Chem-lab) at the incubation (starting point). The pH of this mixture was adapted to 4.6 prior to the addition of 33.7 mL HM stock solution (Table 4). After 120 min of gastric incubation, environmental conditions were adapted to mimic small intestinal (SI) conditions. 15 Pancreatic juice (99.6 ml) was constituted by 8.16 g / L NaHCO3 (Chem-lab), 15.9 g / l oxgall (Becton-Dickinson, Erembodegem, Belgium), 10.6 g / L pancreatin (Merck Life Science, Hoeilaart, Belgium), 6.45 mL trypsin solution (10 g / L; Carl Roth GmbH + Co. KG) and 8.1 mL chymotrypsin solution (10 g / L; Carl Roth GmbH + Co. KG). Next, sterile dH2O was added to reach a total volume of 451.05 mL. Environmental pH in this phase increased from 3.0 to 5.5. Cell inoculum was injected in the small intestinal simulator reactor. Lyophilized Lactiplantibacillus plantarum 20 LP14, Lactobacillus acidophilus LA02 and Lactobacillus crispatus LCR04 were resuspended in sterile dH2O up to a volume of 6 ml with a standardized cellular concentration of 1 x 10^9 viable cells\ml (AFU\g). After probiotic acclimatation, samples of each strain were added separately to the reactor (test condition). In the same way, 6 ml of pure sterile dH2O were added to a reactor to simulate the blank condition (no cell inoculum). After sample inoculum the small intestinal incubation was initiated. The environmental pH was increased from 5.5 to 6.5 and 25 maintained at this pH over a 27 min period, simulating the duodenal incubation (DUO). This phase was then followed by a stepwise pH increase (i.e., 0.1 pH units every 7 min) to 7.5 within a 63 min-period, mimicking the jejunal environment (JEJ). Finally, the pH remained constant at 7.5 during 90 min simulating the ileal incubation. The pH increase was achieved by the addition of NaHCO3 solution (4.8 g / L) following 60, 90, and 120 min of small intestinal incubation. As such, the dilution of the intestinal contents was simulated. The entire small intestinal 30 incubation was performed under anaerobic conditions. Short term colonic tract simulation At the end of the SI incubation, the colonic simulation (proximal colon) was started in a subsequent reactor. Colonic environment (Colon) was resembled by the addition of 40 mL upper GIT suspension to 86 mL fresh colonic medium 35 constituted by 23.89 g / L KH2PO4 (Chem-lab), 7.61 g / L K2HPO4 (Chem-lab), 2.93 g / L NaHCO3, 2.93 g / L yeast 25 P240048 extract (Oxoid, Basingstoke, GB), 2.93 g / L peptone (Oxoid), 1.16 g / L glucose (Merck Millipore, Massachusetts, USA), 2.32 g / L starch (Sourby, Roeselare, Belgium), 1.46 g / L mucin (Carl Roth GmbH + Co. KG), 0.74 g / L L- cysteine HCl (Merck, Overijse, Belgium), and 2.92 mL Tween® 80 (Sigma-Aldrich, Overijse, Belgium) and 14 mL filter-sterilized fecal inoculum. For this specific investigation, a sterile colon environment was simulated. 5 Accordingly, fecal inoculum was derived from a healthy adult human donor and prepared as described by Van den Abbeele, et al. and by Ghyselinck, et al.. Briefly, a mixture of 1:10 (w / v) of fecal sample and anaerobic phosphate buffer (8.8 g / L K2HPO4, 6.8 g / L KH2PO4, 0.01 g / L sodium thioglycolate) and 0.015 g / L sodium dithionite (Merck) was homogenized for 10 min (BagMixer 400, Interscience, Louvain-La-Neuve, Belgium). After centrifugation for 2 min at 500 ×g (Centrifuge 5417C, Eppendorf, VWR), large particles were removed, and the remaining suspension 10 was further processed via two subsequent centrifugation steps (same operative condition as above). The remaining supernatants was finally filtrated in two different steps, using a 0.45 µm bottle-top-filter (VWR) and a subsequent 0.22 µm bottle-top-filter (Carl Roth GmbH + Co. KG) in combination with a vacuum pump (N8163KN, KNF lab, Aartselaar, Belgium) to discard the microbial fraction. Filter-sterilization of the inoculum was performed to allow studying the fate of the supplemented probiotic strains in the colon in terms of viability and HM detoxification. The 15 environmental pH of the colonic incubation at starting point was equal to 6.5. The buffering capacity of the colonic medium maintained the environmental pH between 6.5 and 5.8 during the entire colonic simulation in order to mimic the proximal colon. The entire colonic incubation had a total duration of 24h and was performed under anaerobic conditions at 37 °C and 90 rpm agitation (MaxQ 4000 Benchtop Orbital Shaker, Thermo Fisher Scientific, Belgium). 20 Sample collection Samples (2 ml) were collected during the upper GIT and short-term colonic simulation at different time points; at the beginning of the stomach incubation (= ST start), at the beginning of the small intestinal incubation (= SI start, immediately after addition of the activated culture and / or sterile dH2O), at the end of the duodenal incubation (= DUO end), at the end of the jejunal incubation (= JEJ end), at the end of the ileal incubation (= ILE end), and at the 25 beginning and at the end of the colonic incubation (C=0h and C=24h, respectively). In particular, at timepoints ST start and ILE end, reactor contents was sampled to be analysed while at timepoints C 24h the reactor content was centrifuged to obtain two fractions namely cell-free supernatants and cell palettes that were both analysed for determination of the HMs content and distribution. Sampling times are resumed in Table 5. At the given timepoints, samples were processed for cell enumeration and were treated by centrifugation (6000 rpm x 6 min) for phase 30 separation in cell pellets and cell free supernatants. Cell pellets and / or cell free supernatants were analysed for HMs quantification. 26 P240048 Table 5 - Sampling times during experiment in SHIME System with co-inoculum of probiotic Lactobacillus strains and heavy metal solution. Bacterial cells enumeration 5 For each sample, a ten-fold dilution series was initially prepared in anaerobic PBS. Cell enumeration of probiotic bacteria was done by staining the appropriate dilutions with SYTO 24 (final concentration of 1 µM; Life Technologies Europe, Merelbeke, Belgium) and propidium iodide (1.33 µM final concentration; Thermo Fisher Scientific, Merelbeke, Belgium) for 15’ at 37° C of incubation in the dark. Samples were analyzed on a BD Accuri C6 Plus (BD Biosciences, Vianen, The Netherlands) using the high flow rate. Bacterial cells were separated from medium 10 debris and signal noise by applying two threshold values, i.e., a primary FSC-H threshold of 500 and a secondary FL-1 threshold of 700. Flow cytometry data were analyzed using FlowJo, version 10.5.2 and results were reported as average log (counts / reactor) ± st.dev. (n=3). Results were expressed as Active Fluorescent Unit (AFU) / ml taking into account the changes in volume occurring during the experiments through the reactors and so by applying the correction factors (see Table 6). 15 Quantification of Heavy Metals Samples were analysed for heavy metal content by Inductively Coupled Plasma mass spectrometry (ICP-MS) technique after acid digestion. The applied method consists of a hot acid digestion of the aqueous samples to dissolve metals associated with the particulate matter or present in colloidal and organic form. The aqueous sample 20 was transferred to an Erlenmeyer flask and added of concentrated nitric acid and H2O2 (Sigma Aldrich, high purity for ICP-MS) on a hot plate until complete mineralization, achieved when the solution is clear and transparent. After cooling to room temperature, samples were transferred to a volumetric calibrated flask and brought to volume with ultrapure water. Samples were then analysed using a Thermo Scientific™ iCAP™ TQe ICP-MS with a RF power of 1550W and a collision gas flow of 4-4.6 l / min. 25 27 P240048 Data analysis Three independent trials were carried out for each strain. Cell density data measured at the different timepoints (Table 5) are reported as mean values (n=3) ± standard deviation. Variations in HMs concentration for samples collected at ILE end (end of incubation in small intestinal simulated conditions) and C 24h (end of incubation in 5 colonic simulated conditions) were expressed as relative to the HMs concentration at ST start (beginning of gastric simulated phase). Results were adjusted considering the changes in volume occurring during the experiments through the reactors and so by applying the correction factors (see Table 6) and are expressed as mean values (n=3) ± standard deviations. A t-test was performed to analyse the differences found in HMs concentrations detected in the lactobacilli-HMs co-incubated samples vs the Blank sample (condition where only HMs were10 inoculated in the SHIME reactors) to determine the statistical significance. Significance was declared at 0.01 < p- value ≤ 0.05 (*), 0.01 < p-value ≤ 0.001 (**) and 0.001 < p-value ≤ 0.0001 (***). For each probiotic strain, statistically significant differences between the viable population densities (within the same strain) were determined between each sampling point and its preceding one to demonstrate changes in viable population. Significance was studied by t-test and was declared at 0.01 < p-value ≤ 0.05 (*), 0.01 < p-value ≤ 0.001 (**) and 0.001 < p-value ≤ 0.0001 15 Table 6: List of samples, timepoints and volume correction factors recorded during heavy metals and strains LP14, LA02 and LCR04 experiments in SHIME system. Volume correction factors were applied in data calculation (% recovery of heavy metals and viable cells). 28 P240048 Results and Discussion Lactobacillus strains viability and adaptation under simulated gastro-intestinal conditions in SHIME system 5 Lactiplantibacillus and Lactobacillus strains (LP14, LA02 and LCR04) were inoculated at T=120 min in the small intestine (SI start) simulating reactor (Duodenal simulating phase). According to sample preparation and standardization procedure, the average concentration of each strain / reactor measured on AFU (Log Count / reactor) was about 10.15 (with a standard deviation always below 0.1). All the strains showed good performances under 32 P240048 intestinal (small and large) simulated conditions; they indeed retained their viability indicating they were not negatively affected by the relatively harsh small intestinal environmental conditions (e.g., high bile salt concentrations, proteolytic enzymes, shift in pH) and showed good adaptation to colonic environment. Cell enumeration in AFU (expressed as Log Count / reactor ± sd) for strains LP14, LCR04 and LA02 are reported in Fig. 5 2, 3 and 4, respectively. In particular, strains LP14 and LCR04 showed a noteworthy adaptation in colonic condition as suggested by a cell load increase of about 1.5 log cycle under 24h incubation in the colonic environment. As shown in fig.4, strain LA02 was also able to survive the passage through the small intestinal phase but no bacterial growth was recorded under colonic incubation. Probiotic strains tested showed good viability performances under intestinal simulated conditions. This parameter 10 guaranteed a proper cell density to investigate bacterial activity on heavy metals. If the HM reduction would be an active process where bacterial growth and metabolism (e.g., Exopolysaccharide-EPS production) is involved, we could argue a prominent involvement of strains LP14 and LCR04 among the ones tested; in the case of non- metabolic mediated HM detoxification by cells (e.g., binding process), these experimental conditions ensured that all the strains were in the conditions to operate HM detoxification since a good biomass level was retained by LP14, 15 LA02 and LCR04. Heavy metal concentrations after co-incubation with probiotic strains during small intestine simulated conditions The influence of the different Lactobacillus strains on the HM content after coincubation in small intestine simulating conditions is shown in Figure 5. HM quantifications were conducted on the cell free supernatants obtained after 20 sample centrifugation of samples collected at the end-of-Ileum-simulating incubation (ILE end). HMs recovery-R (%) was calculated with respect to the initial concentration of HMs at timepoint ST start and by the relative volume correction factors (Table 6) for the small intestine. Most of the conditions analyzed revealed HM recoveries of around 100%, demonstrating the technical soundness of our approach. Moreover, the fact that no significant modification (p-value < 0.05) in the HMs concentration was typically detected after co-inoculum with 25 probiotic strains in small intestinal simulated reactors as compared to the blank conditions, suggests an inability of the strains to act as HM scavengers within the timeframe of this experimental set-up. A notable exception was the sample with HMs co-incubated with strain LP14, which displayed a reduction in Cd concentration (p-value < 0.05). Most likely, the short-term incubation (180 min) in simulated small intestinal conditions offered a limited reaction time to favor additional interactions among HMs and bacterial cells. 30 Heavy metals concentration after co-incubation with probiotic strains during large intestine simulated conditions The effect of probiotic Lactiplantibacillus and Lactobacillus strains on the HMs content determined after the short- term colonic simulation is represented in Figure 6-9. For all the conditions (including Blank sample) samples were pretreated to obtain the separation of the pellet fraction and the cell-free supernatant. Figure 6-9 show the effects 35 of strains LA02, LCR04 and LP14 on Cadmium, Chromium, Mercury and Lead respectively. Specifically, the graphs 33 P240048 illustrate the % of accumulation for the specific HM and for the specific strain in the cell pellet fraction and in the cell free supernatant under 24h of incubation in colonic simulated environment relative to the HM concentration detected at the inoculum phase (t0= ST start) in the SHIME system. Similarly, same data are reported for the Blank condition, meaning the sample where any bacteria was co-culture with HMs mix during the SHIME experiment. 5 Furthermore, HM recovery (%) data were adjusted by the relative volume correction factors (Table 6). The combined recovery for the supernatant and cell pellet fractions was close to 100% for each of the vessels, independent of the addition of probiotic strains, reiterating the technical feasibility of our experimental set-up. Among the strains examined, LA02 did not promote a significant variation in the HMs content detected in the supernatant and in the pellet (p-value < 0.05) relative to the blank condition; interestingly LA02 was the only strain 10 unable to growth during the colonic phase suggesting that the HM concentration within the SHIME ecosystem did prevent HM detoxification. By contrast, incubation of HMs with strains LP14 and LCR04 led to a general increase of HMs found in the cell pellets. Indeed, higher accumulation of HMs for strain LP14 and LCR04 was found in the cell pellet after centrifugation and results suggest that the effect was strain- and metal-specific. Lead was predominantly accumulated in LCR04 biomass by about 45% compared to the content detected in the Blank 15 condition. Cadmium (Fig. 6) was markedly accumulated in LP14 and LCR04 biomass with a recovery of approximately 20 and 40% higher with the respect to the Blank sample (p-value < 0.05). Similarly, both the strains LP14 and LCR04 led to an accumulation of Chromium (Fig.7) in the cell pellet fraction with a % of recovery of about 40% higher than the one detected in the Blank sample (p-value < 0.05). Mercury (Fig.8) was the metal less abundantly accumulated in cell pellet with an increase of 10% in metal associated with cell fraction in a strains 20 LCR04 and LP14 compared to the Blank condition. Finally, also Lead was significantly (p-value < 0.05) associated with the cell pellet fraction with an accumulation of about 30% higher than in the Blank condition. The longer exposure of HMs to cells in the colon compartment (T=24h) can explain the accumulation of HM in the cell pellet compared to the reduced co-incubation time under small intestinal simulated conditions (T=180 min). This investigation aimed at testing the potential ability of probiotic strains to remove / detoxify heavy metals (HM). 25 The strains Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02 and Lactobacillus crispatus LCR04 included in the study have been selected on the base of several criteria namely i) scientific evidence about the capability of these species to operate a detoxification of chemical agents and ii) previous findings obtained by this research group on the ability of specific strains to detoxify phosphonates, biogenic amines, and heavy metals (data not shown). Furthermore, all the strains have a long history of presence on the market as food supplements so their 30 use and acceptance in humans has been extensively consolidated. Previously in the text we mentioned the concept of bio-accessibility / bioavailability of HM in the intestinal environment. Bio-accessibility is defined as the percentage of the heavy metals that is transformed into absorbable forms and that remain soluble in the human gastrointestinal environment, whereas bioavailability is determined by the fraction of HM that is absorbed into the systemic circulation. HM are reported to exert great activity when in the gut by provoking a structural and functional alteration 35 of the gut microbiota and by directly affecting the intestinal epithelium. On this base, this work aimed at studying 34 P240048 the possibility to implement probiotic lactobacilli to subtract (e.g. via binding, biotransformation, bioaccumulation) available HM in the intestinal environment so thus potentially limiting their bio-accessibility / bioavailability and interaction with and uptake by the host. The intestinal environment was resembled by the implementation of the SHIME system (ProDigest). SHIME is an 5 in vitro model that accurately simulates the physiological conditions of the complete gastro-intestinal tract and, possibly, of the colonic microbiota allowing temporal sampling from each region (e.g. Duodenum, Ileum) under analysis. Our results demonstrate that, with different extent among the strains tested and with a metal-specific activity, lactobacilli mediate a trapping effect on HM as demonstrated by that fraction of HM detected in association to the 10 cell pellets. Therefore, strains LP14, LA02 and LCR04 promoted the removal of the HM tested by the aqueous fraction in the colon-simulating vessel so thus potentially limiting their bioaccessibility. Conclusions According to our knowledge, this is the first time that the ability of probiotics to detoxify a pool of heavy metals is 15 studied in a complex GI simulating system. Data presented here demonstrate the ability of probiotics to favour HM segregation in the intestinal environment. Example 3: Evaluating Probiotic Responses to Heavy Metal Stress: A Multifaceted Approach Using Isothermal Microcalorimetry, Flow-Cytometry, and pH Monitoring 20 So far, we have demonstrated the potential of probiotic strains Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02, and Lactobacillus crispatus LCR04 to detoxify heavy metals (HMs) and prevent resulting impairments on the intestine. As previously mentioned, detoxification by lactobacilli can be sustained by chemical-cell wall interactions without the involvement of bacterial metabolism or, alternatively, by complex processes that rely on active metabolic 25 mechanisms exerted on the given heavy metal. We decided to investigate the resistance of tested probiotic strains in the presence of HMs in terms of metabolic performance. To assess these features, we implemented a combined approach based on i) cell enumeration by Flow-cytometry, ii) study of acidification performances and iii) the monitoring of bacterial metabolism by measuring heat production. Our research group can claim a long history of use, optimization data interpretation of flow cytometric analysis for a better assessment of bacterial population 30 density. This also depends on mastering the interpretation and comparison of data derived by parallel enumeration of bacterial cultures in different status (liquid and dehydrate cultures) by Plate Counts and flow cytometric methodology coupled to the study of acidification kinetics. Next to this, we recently internalised the microcalorimetry technique with the scope of further improving our assessment in probiotic bacteria physiology. Isothermal microcalorimetry (IMC) found applications in the monitoring of bacterial activity and growth. It became a valid 35 P240048 methodology for obtaining real-time information on yield, growth, and stoichiometric evaluation of several biological processes. Noteworthy, IMC is not affected by factors limiting a precise quantification of bacteria (e.g. co- aggregation, turbidity) embodying a clear advantage when analysing and enumerating microorganisms of clinical (pathogens) or commercial (starter cultures, probiotics) interest. Example 3 presents a pilot study investigating 5 Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02, and Lactobacillus crispatus LCR04 metabolism with a combined approach based on Isothermal Microcalorimetry (IMC), Flow-cytometry and pH monitoring with the scope to better elucidate the response of probiotics in presence of HMs as stressing agents. Materials and Methods 10 Bacterial strains Lactobacillus strains were derived from Probiotical strain collection, and they were previously registered at international depository Institutes before their use in this study. Strains included here were Lactiplantibacillus plantarum LP14 (DSM 33401), Lactobacillus acidophilus LA02 (DSM 21717), and Lactobacillus crispatus LCR04 (DSM 33487). 15 Bacterial culture preparation Bacterial cultures were originally stored in seed-lot at Probiotical S.P.A. as frozen and glycerol protected Cell Bank, routinely checked for stability and purity. Bacterial cultures used in this study were activated by inoculating glycerol- cell stock at 10% in fresh growing medium, further proceeding with 16h- incubation at 37°C. Growing medium was 20 the de Man Rogosa Sharpe-MRS (BD Difco™). All the strains were sub-cultivated at least twice before co-incubation experiments with HMs solution and the evaluation of growth, acidification and metabolic (heat production) performances. Heavy Metals (HM) solution preparation 25 HM included in the study were Cadmium (Cd), Chromium (Cr), Mercury (Hg) and Lead (Pb). All HMs used in this study were purchased from Sigma-Aldrich. HMs were all tested in their inorganic form and were mixed to prepare a stock solution in Phosphate-buffered saline solution- PBS. HMs concentration in stock solution was equal to HM mix 2 used for experiments at Example 1 and is reported at Table 7. Before its use in the experiments, the stock solution was further diluted in ratio 1:100, 1:1000 and 1:10000 in PBS to study the effects of different HM 30 concentrations on bacterial performances. 36 P240048 Table 7 - Details of Heavy Metals concentration in stock solution. Stock solution was freshly used and diluted in media specific for each different test. Concentration of metal ions Heavy Metal (salt) (mg / ml) CdS040.22 CrCl3hexahydrate 3.5 Hg(NO3)2monohydrate 1.8 PbCl2 3.0 Sample preparation 5 Probiotic bacterial cultures were activated from glycerol cell stock and sub-cultured twice in MRS medium as previously described. Strains LP14, LC04 and LA02 were then cultured in MRS for a short incubation (3h) at 30 (LP14) and 37°C to ensure a proper cell activation before their use in the experimental tests. Cell cultures were previously checked and enumerated by flow cytometry and then centrifuged (6000 rpm x 6 min), washed once in PBS solution for cell standardization. At this scope, cell pellets were resuspended in a) pure MRS (positive control), 10 b) MRS plus HMs solution in ratio 1:100, c) MRS plus HMs solution c) MRS plus HMs solution in ratio 1:1000 and d) MRS plus HMs solution in ratio 1:10000 at a final cell density of 7.1±0.03 log AFU (Active Fluorescent Unit) / ml and 6.0±0.05 log AFU (Active Fluorescent Unit) / ml for all the strains. Resulting cell cultures were immediately incubated at 37°C for growth and acidification kinetics and for heat production assessment as followed described. 15 Enumeration of cell population by Flow-cytometry (FC) Probiotic cell samples prepared in MRS or MRS + HMs were incubated at 30 (for LP14) and 37°C (for X and Y) up to 48 h and were checked for cell counts by FC after 4, 22 and 48 h. FC analyses were performed using the BD Cell Viability Kit with liquid counting beads (distributed by BD Bioscience, San Josè, CA). The kit includes an easy- to-use dye combination to distinguish between live and dead cells based on assessment of cell membrane integrity. 20 Thiazole Orange (TO) solution allows the staining of all cells while Propidium Iodide (PI) targeted damaged and dead cells on the base of cell membrane damage. BD Liquid Counting Beads were always used as reference for the cell enumeration. Cell staining and analysis were performed according to the ISO 19344:IDF 232 protocol with custom integration described by the method reported by Foglia et al.. Briefly, bacterial cultures were serially diluted (1:10) in PBS solution and the optimal cell dilution was stained with 10 μL PI solution (in water at 0.2 mmol / L) and 25 5 μL of TO solution (in water at 42 μmol / L). Samples were homogenized, followed by incubation for 15 minutes at 37 ° C in the dark, ensuring proper cell staining. Prior to analysis, the bead suspension was gently vortexed for 30 seconds and then 50 μL was added to the cell suspension for a final volume of 1 ml. Samples were then analyzed on a FACScan FACSCalibur cytometer (BD FACSCalibur Software; Becton Dickinson, San Jose, CA) equipped 37 P240048 with 488 nm argon laser excitation and CellQuest software. An SSC-H (Side Scatter) threshold was used for microbial cells. Cells were gated using forward versus side scatter (FSC-H vs SSC-H). In order to guarantee result accuracy during all the experimental sets, the gating procedure was kept constant. Flow Cytometry results were expressed as Active Fluorescent Unit (AFU / g), non-Active Fluorescent Unit (n-AFU / g) and Total Fluorescent Unit 5 (TFU / g). AFU sub-population represents the cells with an intact cell membrane hence assumed to be alive and metabolically active. n-AFU represents the damaged and dead cells stained with PI, the non-permeant dye which only enters cells with a non-intact membrane and binds to DNA. The Total Fluorescent Unit (TFU) represents the total number of cells obtained by the sum of AFU and n-AFU cells. Damaged and dead cells (n-AFU) can be calculated also as TFU – AFU. For this experiment, TFU / ml and AFU / ml units have been exclusively used for data 10 representation. Study of bacteria acidification activity Probiotic cell samples prepared in MRS or MRS + HMs were all incubated in a thermostatic bath at 30 (for LP14) or 37°C (for X and Y) and acidification kinetics were observed in real time by a using an iCinac L.A.B. Fermentation 15 Monitor Series (AMS Alliance) offering the parallel monitoring of up to 12 samples. The production of metabolites during acid fermentation was interpreted as indicator of metabolic activity showed in optimal culturing condition (pure MRS) or retained by cells population during challenging incubation (MRS + HMs). Measurements of pH were recorded at the starting point (T0) and during 48 h incubation. 20 Heat production monitoring by Isothermal Microcalorimetry (IMC) Probiotic cell samples prepared in MRS or MRS + HMs were assessed by IMC implemented on the calScreenerTM isothermal microcalorimeter (Symcel AB, Sweden). The device is equipped with the calPlate system holding 48 titanium sealed vials developed to ensure an optimal heat transfer and maintenance during sample incubation. Titanium vials were completed with plastic disposable inserts before sample incubation (400 µm / vial) carried out in 25 sterile conditions. The vials were then sealed and placed into a calScreenerTM device. The upper and lower lanes of vials were filled with only pure MRS as they served as thermodynamic references. Before the incubation at 37°C was started, the calPlate was inserted stepwise in the calScreenerTM for temperature stabilization according to the Manufacturer’s instructions. After this acclimatization step (30 min) sample incubation was started for experimental data collection. Isothermal calorimetry with the calScreener allowed the evaluation of overall metabolic activity of 30 a sample by continuous measuring the heat flow (HF) generating thermograms in the form of heat-flow curves (µW / sec) during incubation at 30 (for LP14) and 37 °C (for X and Y) for 48 h. Since the microcalorimeter allows a real time detection of heat production, incubation time was prolonged for those samples showing a signal from heat flow still going after 48 h. Kinetic heat flow curves were monitored using the calView 2.0 software. Additional parameters (e.g. total heat produced during the experiment) were extracted using the online analysis tool calData. 38 P240048 For a detailed protocol description of calScreenerTM experiments, including sample preparation, data normalization (baseline correction), data export and analysis we refer to the paper by Beilharz et al., 2023. Data analysis Three independent trials were carried out for each strain. Cell density data measured at the different time point are 5 reported as mean values (n=3) ±standard deviation. Acidification kinetics are reported as mean curve obtained during 3 independent trials. Thermographs of microcalorimetric analysis are generated as mean heat flow (n=3) for samples at each condition tested, and background halos of the same colour represents the standard deviations. Results and Discussions 10 Viable counts and heat flow measurements strains- HMs co-culturing Probiotic strains LP14, LA02 and LCR04 were analyzed for metabolic performances in parallel experiments monitoring heat release, growth, and acidification kinetics when in presence of HMs compared to basal growth condition (MRS). At this scope bacterial cultures were prepared in a) pure MRS (positive control), b) MRS plus HMs solution in ratio 1:100, c) MRS plus HMs solution in ratio 1:1000 and d) MRS plus HMs solution in ratio 1:10000 15 with an initial cell concentration of 6.0 and 7.0 log AFU / ml. For better illustrating the experimental results, data from FC enumeration and heat production are represented in one figure for each of the strains. Accordingly, Figures 10, 11 and 12 illustrate the response of strains LP14, LCR04 and LA02, respectively, to different concentrations of HM in terms of heat production (sec / h) and cell counts (AFU / ml). Although with strain-specific differences, probiotic strains LP14, LR04 and LA02 showed a similar response to increased concentration of HMs. As general trend, HM 20 diluted in MRS in concentration 1:10000 did not affect cell performances, neither in terms of heat production nor in terms of cell counts (Figures 10-12) and therefore we can hypothesize that such HM concentration does not interfere with cell metabolic functions. By contrast, increasing HM concentration to 1:1000 in MRS, we progressively observed a clear extent of bacterial lag phase, delayed signal by the heat flow and a diminished heat production. In microcalorimetry, the part of the thermograph showing the highest value of heat plotted on time is referred as to 25 the Maximum Metabolic Rate (µW) while the time (h) required by cell population to give rise to heat production is referred to as Time to Activity (TTA); more in details, moving from 1:10000 to 1:1000 HM concentration, the maximum metabolic rate and TTA went from approximately a) 180 µW and 6 h to 80 µW and 8 h for strain LP14 (Fig.10), b) 85 µW and 8 h to 95 µW and 36 h for strain LCR04 (Fig.11), and c) 110 µW and 4 h to 90 µW and 24 h for strain LA02 (Fig.12). Finally, for all the strains, HMs mix diluted in ratio 1:100 in MRS led to a drastic 30 reduction of bacterial heat production and cell duplication throughout the experiment. For all the strains the strongest attempt to activate cell metabolism is observed in the first hours (0-10h) when cells were cultivated in MRS alone or MRS with the most diluted HMs mix concentration (1:10000) as shown by cell counts and Maximum Metabolic Rate (µW). Interestingly, also during co-cultivation of strains and HMs 1:100 the highest cell duplication and heat production occurred in the first hour of incubation, possibly suggesting the cellular effort in coping with the 35 stressor agents. After this first reaction to this condition, all the strains underwent a metabolic stasis as suggested 39 P240048 by heat flow and AFU values, or a cell population decay as suggested by an increase in TFU / ml and so by the AFU / TFU ratio (data not shown). Finally, in the last condition of incubation (MRS with HMs 1:1000) the Maximum Metabolic Rate (µW) was observed between the timepoint 20-40h, suggesting a preliminary adaptation phase of the strains to the HMs mix. The exposure of bacteria to antimicrobials influences their metabolism and physiology, 5 thereby affecting the energy released. Isothermal microcalorimetry (IMC) is gaining much interest in the study of microorganisms susceptibility to antimicrobials / antibiotics. As a matter of fact, IMC shows high sensitivity in detecting even low numbers of bacteria (approximately 104) and accuracy in maintaining the temperature set and recording with high precision heat fluctuation generated by bacterial metabolism. Isothermal microcalorimetry (IMC) measures dissipated energy form biochemical reactions within cells at microwatt levels by monitoring the released 10 heat which is a direct measure of metabolic activity. The heat recorded is to be interpreted as the balance by biological reactions releasing or absorbing heat. By IMC is possible to retrieve information on the total enthalpy changes over time meaning the heat flow. Indeed, cell growth is associated with an enthalpy change of the system and it is shown that, both in fermentation or respiration process, the heat flow is stoichiometrically connected to the net specific rate of substrates, byproducts (metabolites) and growth rate. The heat produced is for example 15 associated to the breakdown of carbon source operated by microbial cultures. Accordingly, considering only the growth-associated metabolism, metabolically active bacteria generate energy which is proportional to their growth rate in a cell culture. Thus, IMC represents a valid tool to monitor (mammalian and bacterial) cell growth and even to retrieve information about cell physiology in given conditions. Since the heat flow is influenced not only by metabolic reactions related to biomass formation but also to other physiological chances, IMC is getting 20 implemented for the assessment of various microbial features. In our opinion, one of the most proper studies comes from Robador et al. which applied the microcalorimetric approach next to protein quantification, oxygen consumption, cell size measurement and enumeration to study the energy partition of Shewanella oneidensis MR- 1 during growth in order to uncover the functional relationship between microbial activity and the physiological state of the population. Other prominent research demonstrates that, due to its high detection potency even with low 25 cellular amounts, IMC can be implemented to better investigate the metabolic pattern of cells in presence of antimicrobial compounds, as mentioned above, or during biofilm production. In the latter case, IMC is indeed considered a good proxy for biofilm or EPS production-associated heat. Based on the approach we set in place, we can conclude that the main heat production is also ascribed to highest momentum / capacity for cell replication. We can speculate on this after having compared in parallel experiment the 30 microbial response in terms of cell duplication and heat production. Furthermore, we can assume that the prolonged lag phase in heat production observed when samples were cultivated on intermediate concentration of HMs is ascribable to metabolism redirection or to adaptative phenomena such as HMs detoxification. With this experimental set we cannot, indeed, speculate on energy partition set in place by the stains during co-culturing experiments in terms of energy linked to cell duplication, physiological rearrangements, response to detrimental 35 agents or much interesting, biofilm formation which represents a HM detoxification route via HM trapping and a 40 P240048 physiological process releasing heat. As mentioned above, the latter feature might indeed be possible as literature data describe biofilm formation-associated heat. Furthermore, our previous findings on the ability of strains LP14, LCR04 and LA02 to detoxify HMs and the functional genomic analysis carried out on these strains to predict their ability in producing EPS (Example 2) might suggest an implication of extracellular matrix production in the 5 interpretation of heat rate data. Acidification profile during strains- HMs co-culturing To have a better picture of the impact of HMs on probiotic strains metabolism, microcalorimetric and viable cells measurements have been associated to the assessment of acidification ability during co-culturing experiments. 10 This parallel observation was possible since experiments were conducted on bacteria of well-known acidifying metabolism, thus allowing the correlation between pH reduction, cell growth and heat production. Acidification kinetics are depicted in Figures 13, 14 and 15 for strains LP14, LCR04 and LA02, respectively. In basal condition (MRS), strains LP14 and LCR04 showed the fastest acidification that occurred approximately between the second and the 9th hour of cultivation, while LA02 demonstrated the acidification activity approximately 15 in the range 4-12 hour. As observed for cell viable counts and heat production, co-cultivation with the lowest concentration of HMs (1:10000) did not affect the acidification profile. By contrast, in line with our ICM observations, a HM concentration of 1:1000 led to a distinct delay in the acidification, whereas a 1:100 dilution prevented the strains to produce acid metabolites altogether. To the extent of our knowledge, a comparison between heat flow and acidification profile in microorganisms of technological interest has not yet been reported. 20 Conclusion This study shows our initial attempt to compare the metabolic response of probiotic microorganisms cultivated in harsh environment due to the presence of chemicals. The approach was based on cell enumeration (Flow cytometry), acidification profile and heat production (Isothermal Microcalorimetry) which generated consistent 25 results; all the microbiological measurements showed that above a threshold concentration, the presence of heavy metals led to a remarkable delay or even a knockdown of all the parameters associated with cell metabolism. The experimental foundation of the present patent application was based on the selection and characterization of probiotic strains capable of heavy metal detoxification. From Probiotical S.P.A. microbial collection, strains were chosen based on a set of rigorous criteria: their natural presence in the human gut or common food sources, a 30 proven safety profile and a validated industrial production feasibility. Among the selected strains were Lactiplantibacillus plantarum LP14, Lactobacillus acidophilus LA02, and Lactobacillus crispatus LCR04. These strains were assessed in a series of controlled experiments to determine their capacity to bind and remove heavy metals from the gastrointestinal environment. Utilizing the Simulated Human Intestinal Microbial Ecosystem (SHIME) the present patent application provided 35 robust data supporting the efficacy of the selected probiotic strains in reducing heavy metal content. The SHIME 41 P240048 model, simulating the complex dynamics of the human gut, revealed that probiotics could effectively segregate heavy metals, thereby preventing their absorption. Techniques such as flow cytometry, isothermal microcalorimetry, and acidification profiling were used to analyse the metabolic response of probiotic strains. The findings revealed that high concentrations of heavy metals could block bacterial growth and metabolic activity; however, below a 5 certain threshold of metal concentration, the probiotics managed to sustain their viability and continue producing metabolic by-products, potentially beneficial for gut health. This resilience suggests the potential of probiotics not only as detoxifying agents but also as stabilizers of gut microbiota under toxic stress. The implications of probiotic use extend beyond the detoxification of heavy metals. These microorganisms may play a crucial role in maintaining gut barrier integrity, regulating immune responses, and mitigating oxidative stress. The antioxidant properties of 10 probiotics can counteract the oxidative damage typically induced by heavy metals, thereby preserving cellular health, and preventing chronic conditions. Moreover, by stabilizing the gut microbiota, probiotics help maintain an environment that supports nutrient absorption and immune modulation, thereby enhancing overall health resilience against environmental pollutants. Integrating probiotics into public health strategies could represent a non-invasive, cost-effective, and accessible 15 solution facilitating the access to detoxification strategies, particularly in underserved or poor communities where industrial pollution often has the most severe impacts. Example 4 - Impact of Environmental Heavy Metal Contamination on Intestinal Health: Investigating Gut Homeostasis Disruption and Probiotic Interventions Using Gut-Ex Vivo System (GEVS) 20 So far, animal models have represented an actual in vivo model to investigate and discriminate several adverse effects linked to the administration of different HMs highlighting the onset of, among others, metabolic disorders (e.g. lipids management, fatty liver disease, diabetes), kidney and liver malfunctioning and perturbation in gene expression and enzyme functioning. A cutting-edge cell model representing an evolution of Organ-on-Chip model has been recently described: the Gut-Ex-Vivo System (GEVS). 25 The Gut-Ex-Vivo System (GEVS) consist of a microfluidic system that supports the organ intestinal tissue which is kept alive for a given period, enabling the culture of large, intact intestinal tissues for ex-vivo experimentation; this model recapitulates the intestinal structure (relevant cells and micro-architectures) and physiology allowing the dissection of host-microbe interaction in a highly controlled fashion ex-vivo and with high temporal resolution. In this experiment we assess the potential ability of 3 probiotic lactobacillus 30 The experiments disclosed in Example 4 have been performed in collaboration with the research group led by Prof. Marco Corazzari, Cell Stress & Death Laboratory, Center for Translational Research on Autoimmune and Allergic Disease (CAAD), Department of Life Sciences, University of Piemonte Orientale (NO). 35 42 P240048 Materials and Methods Bacterial strains Lactobacillus strains were all belonging to Probiotical strains collection, and they were already registered at international depository Institutes at their use in this study. Strains were Lactiplantibacillus plantarum LP14 (DSM 5 33401), Lactobacillus acidophilus LA02 (DSM 21717), and Lactobacillus crispatus LCR04 (33487). Bacterial culture preparation Bacterial cultures were originally stored as seed lot at Probiotical S.P.A. in frozen Cell Bank, routinely checked for stability and purity. Bacterial cultures used in this study were activated by inoculating glycerol cells at 10% in fresh 10 growing medium, further proceeding with 16h- incubation at 37°C. Growing medium was the de Man Rogosa Sharpe-MRS (BD Difco™). All the strains were sub-cultivated at least twice before co-incubation experiments with HMs solution as described at “Gut Ex Vivo System (GEVS) and tissue cultures” below reported. Heavy Metals (HM) solution preparation 15 HM included in the study were Cadmium (Cd), Chromium (Cr), Mercury (Hg) and Lead (Pb). All the HMs used in this study were purchased from Sigma-Aldrich. HMs were all tested in their inorganic form and were mixed to prepare a HMs stock solution (Table 8) in Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, CA, USA). The stock solution was diluted 1:100 in IMDM medium supplemented as indicated in paragraph “Preparation of cell free supernatant (CFS) after co-incubation of probiotics and HM media” below reported, to be directly used in tissue 20 culture experiments. Table 8: Details of Heavy Metals concentration in stock solution. Stock solution was freshly used and diluted in media specific for each different test 25 43 P240048 The stock solution was diluted in ratio 1:100 in IMDM medium and supplemented to be directly used in experiments on tissue culture. Preparation of cell free supernatant (CFS) after co-incubation of probiotics and HM media 5 Bacterial cultures were all cultivated in MRS medium through incubation at 37°C for 6-8 hours. Initial bacterial cell inoculum was measured by flow cytometry to ensure an initial bacterial concentration standardized at 1×10^9 viable cells Active Fluorescent Unit (AFU) / ml. After incubation time (6-8h) bacterial cell cultures were checked again for cell enumeration by flow cytometry and cell concentration for all the strains was attested at 9.0 ± 0.05 log AFU / ml. After bacterial growth, HM stock solutions (Table 8) were added in ratio 1:10 to bacterial cultures for overnight (16h) 10 co-incubation at 37°C. Bacterial cultures were then centrifuged (4000 rpm x 10 min) to collect the cell-free supernatant (CFS) to use in tissue culture experiments. CFS is from now referred to as the component derived from the probiotic strains and HM mix co-culturing phase and used in ex-vivo experimentation. Gut Ex Vivo System (GEVS) and tissue cultures 15 Small intestines from 13 days-old C57BL / 6J mice were freshly resected and cultivated in a silicone based Gut Ex Vivo System-GEVS (see Fig.16) with serum free tissue culture medium containing Iscove’s Modified Dulbecco’s Medium (IMDM, Gibco, CA, USA) supplemented with 20% KnockOut serum replacement (Gibco), 2% B-27 and 1% N-2 supplements (Gibco), 1% L glutamine, 1% non-essential amino acids (NEAA) and 1% HEPES (complete Iscove’s medium). GEVS was set for the experiments according to the procedure reported by Gagliardi et al. (2021 20 a, b). Briefly, the model consists of six independent chambers for the insertion of the resected intestines. Each chamber is connected to two needles (input / output syringes) allowing the flow of complete nourishing medium in the inner intestinal compartment (luminal flow; flow rate of 99 ^^l / h), while tissues are imbibed with complete Iscove’s medium (outer medium) to keep the intestine alive. A schematic representation of GEVS is depicted in Fig.16. During the experiments, the temperature of GEVS was kept at 37°C using a standard laboratory warming plate and 25 heat spreader to ensure an optimal and constant heat transfer. A proper tissue oxygenation was allowed by humidified O2 (95%) and CO2 (5%) gas mixture injection into the device chamber. Gut Ex Vivo System (GEVS) treatment The long stimulation with a) complete Iscove’s medium alone (control intestines (GEVS) were treated for 5 hours 30 condition, Ctrl), b) complete Iscove’s medium added (ratio 1:100) of the HM stock solution (test condition 1, MIX) and c) complete Iscove’s medium added (ratio 1:100) of CFS recovered after preliminary probiotics-HMs co- incubation (MIX+ CFS LP14, MIX+ CFS LCR04 and MIX+ CFS LA02). Sample and relative code are presented in Table 9. Tissue samples were then checked for tissue integrity / permeability and inflammation markers as described below. 35 44 P240048 Table 9 - List of samples and relative codes implemented for treatments on Gut Ex Vivo System (GEVS) FITC-dextran permeability assay Tissue permeability was evaluated by measuring FITC-Dextran (FITC D4000; Merck) release into external medium. 5 Briefly, FITC-Dextran medium was administered into an input syringe connected with each intestinal lumen, at a concentration of 0.1 mg / ml in a final volume of 5 ml, using a stock solution of 100 mg / ml. After stimulation, the outer medium in contact with the intestinal tracts was collected. Fluorescence was measured by spectrophotometric assay (SPARK Multimode Microplate Reader TECAN) in 96 well plates (excitation: 485 nm, emission: 528 nm). Before sample analysis, a calibration curve for FITC- Dextran was obtained by measuring serially diluted FITC- 10 Dextran stock solutions (0, 125, 250, 500, 1000, 2000, 4000 and 8000 ng / mL). Subsequently, 100 μL of each sample was measured in 96-well microplate and FITC-Dextran concentrations were obtained using standard curve interpolation. RNA extraction and quantitative real-time PCR (qRT-PCR) assay 15 TripleXtractor reagent (Grisp) was used to isolate total RNA from tissues. ExcelRT Reverse Transcription Kit was used to produce cDNA according to the Manufacturer’s recommendations (Grisp). Quantitative PCR reaction was performed on a CFX96 BioRad thermocycler. ExcelTaqTM 2X Fast Q-PCR Master Mix (SMOBIO) was used to produce amplicon products during repetitive cycling and the melting curve protocol was used to check for probe 45 P240048 specificity. Primers were designed using the IDT PrimerQuest Tool software (IDT, Integrated DNA Technologies Inc., IA, USA). Primer sequences are reported in Table 10. Results were normalized using L34 as internal control and comparative Ct method (ΔΔCt) was used for relative quantification of gene expression. 5 Table 10 - List of primer sequences implemented in quantitative PCR (qPCR) assay Tissue viability Tissue viability was evaluated through AlamarBlue staining (Thermo). Briefly, tissues were recovered from GEVS, 10 weighed, placed in a 48-well plate, and incubated with DMEM supplemented with 10% FBS, 2mM L-glutamine, 100U / mL Penicillin, 0.1mg / mL Streptomycin (Euroclone) and 100g / mL AlamarBlue. After 2h, the absorbance of 100μL of medium from each sample was analyzed by a SPARK Multimode Microplate Reader (TECAN). Measures were normalized by dividing the absorbance by tissue weight. Viability of untreated tissue (CTRL) was arbitrarily set to 100%. 15 Statistical analysis All experiments were performed as technical duplicates and repeated twice, while statistical analysis was performed using GraphPad Prism 7. The student t-test or ANOVA were used to determine statistical significance. A p-value equal to or less than 0.05 was considered significant. mRNA expression levels were represented as ‘fold 46 P240048 change over control’, r.l. relative levels. Histograms represent mean ± SD; **** p < 0.0001; *** p < 0.001; ** p < 0.01; * p < 0.05; ns non-significant. Results and Discussion 5 Ability of Lactobacillus strains to mitigate HM effects on intestinal tissue model The exposure of the intestinal compartment to HMs can negatively impact maintenance of intestinal homeostasis. Depending on heavy metal type and concentration, HMs are in general known to have an impact on the gut, thus inducing tissue stress, and resulting in tissue inflammation. In this study, small intestines of 13 days-old mice were exposed (5 h) to a mixture of heavy metals used at a concentration (Table 8) able to induce a mild dysregulation of 10 tissue permeability while not compromising tissue viability, in our Gut Ex-Vivo System (Fig.17). Notably, the HM concentration was based on a pre-screening dose-response experiment performed in the same experimental conditions (data not shown). To assess the role of probiotics in mitigating the detrimental effects of HMs, the mix of HMs was pre-incubated with probiotic cells with the hypothesis of removing HMs (by binding / chelation / biotransformation / precipitation) and, thus, reducing HMs availability which interfere with intestinal 15 physiology. Results derived from experiments performed by using the probiotic strains LP14, LCR04 and LA02 are shown in figures 18, 19 and 20, respectively. Collectively, our data show a clear, mild dysregulation of the intestinal barrier functions by HMs, as evidenced by increased tissue permeability evaluated by the FITC-Dextran permeability assay (Fig.18-20 A), and confirmed by dysregulated expression of CLD-2, CLD-15 and OCL (Fig. 18-20 B), and ignition of tissue inflammation, as 20 evidenced by increased expression of pro-inflammatory IFNγ and TNFα, and down-regulation of anti-inflammatory IL-10 cytokines (Fig.18-20 C). Importantly, when HMs mix was previously incubated with probiotics (MIX+ LCR04, MIX+ LP01 or MIX+ LA02), the physiological permeability to FITC-dextran was restored (Fig.18-20 A). Similar results were observed in the gene expression levels of CLD-2 and CLD-15. Regarding OCL levels, pre-incubation of HMs with probiotics restored 25 physiological levels of the TJ protein, although the results were statistically significant only with LA02 (Fig.18-20 B). In parallel, our data also show that all probiotic tested efficiently abrogated the HMs-induced increased expression of the pro-inflammatory IFNγ and TNFα cytokines, at least at mRNA level, while restored the physiological level of the anti-inflammatory cytokine IL-10 (Fig.18-20 C). 30 Overall, our results confirmed a detrimental impact of heavy metals on intestinal functions, while lactobacilli-based probiotic strains can efficiently mitigate the adverse effects driven by HMs. Therefore, we can speculate that lactobacilli actively remove HMs from intestinal luminal fluids, potentially creating HMs / cell-associated precipitates, as widely discussed in the previous chapter of this thesis. Briefly, lactobacilli can implement different mechanisms based on i) direct (specific or non-specific) interactions with a heavy metal and implying mechanisms of trapping 35 (biosorption and complexation), or ii) exerting a metabolic action on HM, such as transformation of the given metal 47 P240048 to a less toxic form, or iii) by bioaccumulation. Furthermore, lactobacilli can promote HMs precipitation when in liquid matrices by modifying HMs chemical status or modifying environmental pH, thus causing HMs precipitation. They can finally reduce HMs toxicity even indirectly, for example by improving the intestinal barrier function thus resulting in a decreased intestinal uptake of HMs 5 These hypotheses are also sustained by our previous findings obtained by using the Simulator of Human Intestinal Ecosystem- SHIME. Accordingly, simulated intestinal conditions (small and large) allow HMs removal by binding / trapping / precipitation mechanisms as suggested by improved cell-associated HMs, thus preserving HM segregation in the intestinal environment. It is also important to note that both LP01 and LA02 increased the expression of IL-10, compared to untreated controls (CTRL), suggesting a potential anti-inflammatory activity of 10 bacterial postbiotic fractions in the cell-free supernatants. Conclusions Taken together, the evidence reported here confirms detrimental effects of HMs on intestinal cells. Although with a strain specific effect, all tested probiotic strains were able to mitigate HMs-induced negative outcomes, i.e. loss of 15 integrity, barrier function dysregulation, and ignition of tissue inflammation. 48 P240048 LIST OF ABBREVIATION It is known that in biological systems, for example, in the human biological system, metals are present in their stable 5 ionic form, for example, in their stable cationic form. 49

Claims

AMENDED CLAIMS received by the International Bureau on 29 December 2025 (29.12.2025)CLAIMS(amended under Art. 19 PCT)1 . At least one isolated bacterial strain selected from at least one of the species selected from the group comprising or, alternatively, consisting of: i) Lactobacillus plantarum, ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus, or mixtures of said at least one isolated bacterial strain thereof, for use in a method of gut microbial bioremediation, wherein the bacterial strain belonging to the specie i) Lactobacillus plantarum, is used in mixture with at least one of ii) Lactobacillus acidophilus, and iii) Lactobacillus crispatus or mixtures of said at least one isolated bacterial strain thereof.

2. The at least one isolated bacterial strain for use according to claim 1 , wherein said method of gut microbial bioremediation is a method of detoxifying the human's intestine from at least one pollutant.

3. The at least one isolated bacterial strain for use according to claim 1 or 2, wherein said method of gut microbial bioremediation is a method of detoxifying the human's intestine from at least one pollutant selected from a pool of heavy metals, biogenic amines and phosphonates, or mixtures thereof.

4. The at least one isolated bacterial strain for use according to any one of claims 1-3, wherein said at least one pollutant preferably is a pool of heavy metals, preferably selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As more preferably from Cd, Cr, Hg, and Pb; or mixtures thereof.

5. The at least one isolated bacterial strain for use according to any one of claims 1-4, wherein:- said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is selected from the group comprising or, alternatively, consisting of Lactiplantibacillus plantarum 476LL 20 bi LP01 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P -21021 , Lactiplantibacillus plantarum 776 / 1 bi LP02 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P -21020, and Lactiplantibacillus plantarum LP14 deposited on 16.01.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 33401 , or mixtures thereof;- said at least one isolated bacterial strain belonging to the specie ii) Lactobacillus acidophilus is selected from the group comprising or, alternatively, consisting of Lactobacillus acidophilus LA02 deposited on 06.08.2008 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 21717, Lactobacillus acidophilus LA06 deposited on 13.10.2009 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 23033, or mixtures thereof;- said at least one isolated bacterial strain belonging to the specie iii) Lactobacillus crispatus is identified as Lactobacillus crispatus LCR04 deposited on 02.04.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) having accession number DSM 33487.

6. The at least one isolated bacterial strain for use according to claim 5, wherein said isolated bacterial strain is preferably selected from the group comprising or, alternatively, consisting of LP14 (DSM 33401), LA02 (DSM21717) and LCR04 (DSM 33487), or mixtures thereof, more preferably it is selected from LP14 (DSM 33401) and / or LCR04 (DSM 33487).

7. A mixture comprising at least one isolated bacterial strain selected from at least one of the species selected from the group comprising or, alternatively, consisting of: I) Lactobacillus plantarum, II) Lactobacillus acidophilus, ill) Lactobacillus crispatus or mixtures of said at least one isolated bacterial strain thereof, for use in a method of metals gut microbial bioremediation, wherein the mixture comprises at least one isolated bacterial strain belonging to the specie I) Lactobacillus plantarum in mixture with at least one of II) Lactobacillus acidophilus, and ill) Lactobacillus crispatus, or mixture thereof.

8. The mixture for use according to claim 7, wherein for use in a method of gut microbial bioremediation which is a method of detoxifying the human's intestine from at least one pollutant.

9. The mixture for use according to any one of claims 7-8, wherein said method of gut microbial bioremediation is a method of detoxifying the human's intestine from at least one pollutant is selected from a pool of heavy metals, biogenic amines and phosphonates, or mixtures thereof.

10. The mixture for use according to any one of claims 7-9, wherein said at least one pollutant preferably is a pool of heavy metals, preferably selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As; or mixtures thereof.

11. The mixture for use according to claim 10, wherein said at least one pollutant is a pool of heavy metals, more preferably selected from Cd, Cr, Hg, and Pb; or mixtures thereof.

12. The mixture for use according to anyone claims 7-11 , wherein said mixture comprises or, alternatively, consists of:- said at least one isolated bacterial strain belonging to the specie I) Lactobacillus plantarum is selected from the group comprising or, alternatively, consisting of Lactiplantibacillus plantarum 476LL 20 bi LP01 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P -21021 , Lactiplantibacillus plantarum 776 / 1 bi LP02 deposited on 16.10.2001 at BCCM / LMG and having accession number LMG P -21020, and Lactiplantibacillus plantarum LP14 deposited on 16.01.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 33401 , or mixtures thereof;- said at least one isolated bacterial strain belonging to the specie ii) Lactobacillus acidophilus is selected from the group comprising or, alternatively, consisting of LA02 deposited on 06.08.2008 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 21717, Lactobacillus acidophilus LA06 deposited on 13.10.2009 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) and having accession number DSM 23033 or mixtures thereof;- said at least one isolated bacterial strain belonging to the specie ill) Lactobacillus crispatus is identified as Lactobacillus crispatus LCR04 deposited on 02.04.2020 at Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) having accession number DSM 33487.

13. The mixture for use according to claim 12, wherein the mixture comprises at least one isolated bacterial strain belonging to the specie I) Lactobacillus plantarum, preferably selected from LP01 , LP02 and LP14, in mixture withat least one of ii) Lactobacillus acidophilus as identified in claim 12, and iii) Lactobacillus crispatus as identified in claim 12.

14. The mixture for use according to claim 13, wherein said at least one isolated bacterial strain belonging to the specie i) Lactobacillus plantarum is LP14.

15. The mixture for use according to claim any one of claims 7-14, wherein said at least one isolated bacterial strain is selected from the group comprising or, alternatively, consisting of LP14 (DSM 33401), LA02(DSM 21717) and LCR04 (DSM 33487), or mixtures thereof, even more preferably from LP14 (DSM 33401) and / or LCR04 (DSM 33487).

16. A composition comprising a mixture according to any one of claims 7-15, and one or more additive and / or excipient of pharmaceutical or food grade, for use in a method of metals gut microbial bioremediation, wherein said metals involved in the gut microbial bioremediation are heavy metals, which are selected from the group comprising or, alternatively, consisting of Ag, Be, Ba, Cd, Cr, Mn, Hg, Pb, Cu, V, and As more preferably from Cd, Cr, Hg, and Pb; or mixtures thereof.

17. The composition for use according to claim 16, further comprising at least an active ingredient, preferably said at least one active ingredient is an ingredient for dietary supplement and / or a botanical\active ingredient, wherein said ingredient for dietary supplement is selected from the group comprising or, alternatively, consisting of curcumin, quercetin, melatonin, vitamin C, lutein and myo-inositol; and said botanicaIXactive ingredient is selected from the group comprising or, alternatively, consisting of rosmarinic acid, apigenin, epigallocatechin gallate and ellagic acid.

18. The composition for use according to claim 16 or 17, wherein said composition is formulated for oral use, preferably in the form of granulate, a tablet, a gel or a capsule.

19. The mixture according to any one of claims 7-15 or the composition according to any one of claims 16-18 for use in the prevention and / or reduction of the harmful effects of heavy metals on gut health and gut microbiota, and / or for the treatment of diseases or disorders or reduction of risk factors caused by heavy metals at the gastrointestinal environment, preferably at the large and small intestine level.

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