Goldbeating is the ancient craft of thinning bulk gold (Au) into gossamer leaves. Pioneered by ancient Egyptian craftsmen, modern mechanized iterations of this technique can fabricate sheets as thin as ∼100 nm. We take inspiration from this millennia-old craft and adapt it to the nanoscale regime, using colloidally synthesized 0D/1D Au nanoparticles (AuNPs) as highly ductile and malleable nanoscopic Au ingots and subjecting them to solid-state, uniaxial compression. The applied stress induces anisotropic morphological transformation of AuNPs into 2D leaf form and elucidates insights into metal nanocrystal deformation at the extreme length scales. The induced 2D morphology is found to be dependent on the precursor 0D/1D NP morphology, size (0D nanosphere diameter and 1D nanorod diameter and length), and their on-substrate arrangement  prior to compression. Overall, this versatile and generalizable solid-state compression technique enables new pathways to synthesize and investigate the anisotropic morphological transformation of arbitrary NPs and their resultant emergent phenomena. Inspired by the ancient craft of mechanically beating bulk gold ingots into ultrathin leaves/sheets, we adopt this technique at the nanoscale regime, using gold nanocrystals to gain insights into the deformation of metallic nanoparticles at extreme length scales. Our uniaxial compression technique transforms arbitrary 0D and 1D metal nanoparticles into anisotropic 2D leaf-like morphologies, which are dependent on the precursor nanoparticles' original morphology, size, and particle arrangement. This opens new routes toward understanding anisotropic 2D transformation and the concomitant emergent material properties, generalizable to arbitrary nanocrystals.The process of thinning bulk gold (Au) into gossamer, leaf-like sheets—known as goldbeating—is an ancient handicraft. Adopted by the Egyptians more than five millennia ago, the unparalleled inertness, malleability, and durability of Au uniquely enable their transformation via manual pounding from bulk ingots to thin leaf form, becoming indispensable for myriad applications in art and adornments, such as on the tomb of Thebes and Saqqara . As the Au, as the canonical precious metal with superlative material properties, has been instrumental in shaping the modern world, from microelectronics to (nano)medicine . In nanoIn contrast, top-down, solid-state techniques remain underexplored as an alternative route to achieving morphological control and thus far have mainly entailed lithographic patterning   and compOverall, shape anisotropy in AuNPs induced by uniaxial compression remains underexplored. Further understanding of this severe plastic deformation process akin to nanoscale goldbeating lends insights into the deformation of metal NPs at extreme length scales and the associated parameters. These anisotropic transformations into 2D Au leaf morphologies are investigated experimentally and computationally. The top-down, uniaxial compression-based nanoscale goldbeating technique demonstrated here is versatile and generalizable to other metallic, polymeric, or ceramic NPs. The observed plasticity and transformed 2D morphologies depend on the precursor AuNP size (diameter and AR) and the on-substrate assembly/interparticle separation.2). A custom-built hard-contact compression setup was used to apply uniaxial compressive stress via direct contact between the top silicon wafer surface and the assembled AuNP on the bottom silicon substrate (AuNP/Si)  and applied normalized stress (σnorm) .AuNSs and AuNRs were colloidally synthesized and subsequently functionalized with thiolated methoxyl polyethylene glycol (mPEG-SH). The hydrophilic capping polymer brushes improve AuNP colloidal stability and reduce agglomeration in solution via steric isolation, thus improving deposition and assembly uniformity on the substrate for the compression process . The AuNS and AuNR colloidal solutions were drop-casted and assembled in (sub)monolayers onto polished silicon substrates (2 × 2 mmSi) Fig. A. The denormalized stress of ∼3.6 GPa would be sufficient to initiate dislocation nucleation and induce severe plastic deformation to study the anisotropic 2D morphological transformation. Our empirical results indeed show that AuNPs of different sizes, morphologies, and assemblies all exhibit significant plastic deformation under ∼3.6 GPa applied uniaxial normalized compressive stress.To determine the appropriate amount of stress to apply and induce severe plastic deformation and 2D transformation of the AuNPs, we refer to the strengths of Au micro/nanocrystals documented in the existing literature Table  51–58)..51–58). normalized compressive stress, with anisotropic expansion on the substrate in the lateral in-plane directions and concomitant thickness reduction in the vertical out-of-plane direction. As a result, the AuNSs deform into various disk-like, oblong, and oval shapes due to compressive stress-induced dislocation nucleation and mobility  of AuNSs, small ∼18 nm), medium (∼59 nm), and large (∼103 nm), were studied to understand the effect of uniaxial compressive stress on different-sized AuNSs and their resultant solid-state transformation to 2D morphology. These AuNSs were coated with 5k Da PEG  capping layer to provide a steric hindrance to maintain interparticle separation and stability in the monolayer assembly, which is estimated as ∼4.5 ± 4.7 nm , ∼7.9 ± 11.9 nm (medium AuNSs), and ∼7.7 ± 12.7 nm (large AuNSs) Fig. . The as-8 nm, medity Fig.  of Au exThe deformation of bulk  Au is dictated by the dislocation mobility, dislocation hindrance, dislocation entanglement, dislocation bowing (Orowan strengthening), dislocation pile-up , etc. , 60. At postmortem SEM images where shear bands are prominent . While the characterization of the relatively smaller AuNSs was elusive due to limitations in imaging resolution, the crystallographic orientation of the large AuNSs was resolvable both in their as-assembled and compressed 2D morphologies Fig. . The facion Fig. . After cion Fig. . This diεZ) of different-sized AuNSs are statistically quantified from AFM mapping by comparing the thicknesses of initial as-assembled (tas-assembled) versus the compressed NPs (tcompressed) /(tas-assembled). The effective thicknesses of the as-assembled AuNSs are determined to be 18.8 ± 0.4 nm , 51.6 ± 0.6 nm (medium AuNS), and 83.6 ± 0.7 nm (large AuNS)  of 38.12 ± 0.1% for small AuNS, 57.06 ± 0.1% for medium AuNS, and 55.71 ± 0.1% for large AuNS. Given the same applied normalized stress for all three AuNS sizes, the smaller AuNSs appear to be relatively less compressible.The morphological evolution and induced z-strain (ed) Fig. . Here, tS) Figs. . In compS) Figs. . This reIn addition to the NP size, the as-assembled interparticle separation and adjacent NP interactions also affect the transformed 2D morphology. More significant interparticle separation can be achieved by (i) reducing the drop-casted AuNS colloidal concentration and/or solution volume and (ii) attaching PEG capping layers of different (longer) lengths. However, just lowering the deposited colloidal concentration and/or solution volume alone is insufficient to effectively modulate the as-assembled interparticle separation, as it yields sparse but agglomerated (closed-packed) patches of AuNSs on the substrate surface. Therefore, the length of the PEG brushes grafted is varied for this study: shorter PEG—molecular weight (MW) 5k Da with a hydrodynamic length of ∼10–18 nm–and longer PEG—MW 40k Da with a hydrodynamic length of ∼34–39 nm Fig. . Along wz-direction  compared with the closed-packed small AuNSs with 5k PEG (38.12 ± 0.1%)   Fig. . This la1%) Fig. . AFM mea%) Figs.  compared%) Figs. .σnorm. Close-packed AuNSs with 5k PEG can be progressively compressed to ever-thinner 2D morphologies by applying progressively larger normalized compressive stress   induces a higher degree of 2D transformation and z-strain to the isolated AuNSs. Here, a higher εZ for isolated large AuNSs with 40k PEG (80.16 ± 0.1%) was observed under σnorm = 6 GPa compared with εZ = 75.54 ± 0.01% under σnorm = 3.6 GPa  Fig. . In addiPa Figs. . It was true stress is calculated based on the instantaneous contact surface area  cross-sectional area of the AuNS, similar to the empirical applied normalized compressive stress defined earlier. When compression starts, the isolated AuNS presents a linear elastic behavior  simulations were adopted to simulate the compression of isolated spherical AuNSs Fig. A at roomior Fig. C with zeior Fig. D. When tior Fig. , 67–72, As the deformation continues, the top and bottom contact surfaces act as stress concentrators, causing glissile Shockley partial dislocations to form at these contact surfaces on the [111] slip planes as the compression depth reaches 0.6 nm, resulting in a huge increment in dislocation density Fig. D. These normalized compressive stress. The transformed 2D morphologies of both the short , and their dependent properties. To demonstrate the generalizability of nanoscale goldbeating, two different-sized AuNRs were transformed into 2D morphologies: short AuNR  and long AuNR . The as-assembled short AuNRs Fig.  and longort Fig.  and longort Fig.  exhibit ort Fig. .Statistical metrology analysis of individual AuNRs was performed to gain further insight into the morphological transformation. The projected lateral areas for the as-assembled and compressed AuNRs were quantified from SEM images Fig. . Metrolo2) as a result of compression   Fig. . Due to  2) Fig. . Similar 2) Fig. .εZ, short AuNR of 49.33 ± 0.1% (from ∼18.1 ± 2.4 to ∼9.2 ± 0.7 nm)   Fig. , whereasnm) Fig. . Conside studies . Previou studies , 76–79. The ancient craft of goldbeating is extended by several orders of magnitude down to the single-digit nanometer scale via solid-state, uniaxial compression of 0D AuNSs and 1D AuNRs, transforming them into anisotropic 2D Au leaf morphologies. The resultant 2D morphologies are found to be influenced by the precursor Au nanocrystal morphology, dimensions , and interparticle interactions. In addition, the ability to induce additional NP shape anisotropy enables control of the leaf-like 2D Au's shape, lateral size, thickness, and crystallinity. We postulate that this nanoscale goldbeating process is potentially compatible with nanoimprinting or nanoembossing techniques to induce various hierarchical morphologies. Such versatility and generalizability of this solid-state compression methodology could open new pathways to investigate interesting morphological transformations and strain-induced emergent phenomena across a broad palette of nanocrystals.2O and 250 µL of 100 mM HAuCl4 were added to a 125-mL flask. The solution was stirred at 600 rpm and brought to a rapid boil before adding 7 mL of 1% sodium citrate (m/v). The solution was then stirred for 30 min at just below boiling. An additional 2.5 mL of 1% sodium citrate (m/v) was added, and the reaction was stirred while heating for 10 min. The heat was then turned off, and the particles were cooled to near-room temperature with continued stirring (60 min). The solution was centrifuged at 9,000 RCF for 20 min, the supernatant was removed, and the pellet was resuspended in 10 mL nanopure H2O.Small AuNSs (∼18 nm) were prepared via the Turkevich method  Fig. S4. One hun2O, 0.1524 mL of 100 mM HAuCl4, and 1.8 mL of 1% sodium citrate (m/v). The reaction was stirred for 10 min just below boiling, and the second sodium citrate addition was omitted. After cooling the particles to near-room temperature, the solution was centrifuged at 11,000 RCF for 20 min, the supernatant was removed, and the pellet was resuspended in 10 mL nanopure H2O. For the growth of the 59 and 103 nm AuNSs, 480 mL nanopure H2O and 1.27 mL of 100 mM HAuCl4 were added to two 500-mL flasks. While stirring at 800 rpm, Au seed solution was added to each flask to reach a final seed concentration of 0.0353 nM (for 59 nm AuNS) and 0.0127 nM (for 103 nm AuNS). 1.1 mL 1% sodium citrate (m/v) was added to each flask. The reactions were stirred 5 s, followed by rapidly adding 5 mL of 30 mM hydroquinone. The reactions were stirred at 800 rpm at 25°C for 60 min. Particles were then centrifuged at 4,000 RCF (59 nm AuNS) and 2,000 RCF (103 nm AuNS) for 15 min, supernatants were removed, and pellets were resuspended in 10 mL nanopure H2O.Medium (∼59 nm) and large (∼103 nm) AuNSs were prepared via the seed-mediated synthesis by Chan et al.  Fig. S4. As outl4. The reaction was stirred rapidly for 10 min and then left still for 1 h. For NR growth, 475 mL 100 mM aqueous CTAB, 1.25 mL 100 mM aqueous AgNO3, and 25 mL 10 mM HAuCl4 were added to a 1-L flask. Next, 2.75 mL 0.1 M ascorbic acid was added, which caused the solution to turn colorless. Finally, 600 μL of the Au seed solution was added, and after thorough mixing, the reaction was kept still at 27°C overnight. The solution was centrifuged at 4,500 RCF for 15 min, and the particles were washed twice in 1 mM aqueous CTAB.Short AuNRs  were prepared via our previously reported method . Single-4. The reaction was stirred rapidly for 10 min and then left still for 1 h. For NR growth, 475 mL 100 mM aqueous CTAB, 1.5 mL 100 mM aqueous AgNO3, and 25 mL 10 mM HAuCl4 were added to a 1-L flask. Next, 25 mL of 100 mM hydroquinone was added, which caused the solution to turn colorless. Finally, 8 mL of the Au seed solution was added, and after thorough mixing, the reaction was kept still at 27°C overnight. The solution was centrifuged at 4,500 RCF for 15 min, and the particles were washed twice in 1 mM aqueous CTAB.Long AuNRs  were prepared via the synthesis method of Vigderman and Zubarev et al. . Single-All particles were functionalized with mPEG-SH to reduce NP aggregation upon deposition. Particles were diluted to 1 nM in 0.2 mM mPEG-SH MW 5k (shorter) PEG or 0.1 mM mPEG-SH MW 40k (longer) PEG. Excess capping ligands were removed from the particles by washing them six times with nanopure water, followed by dispersion in nanopure water to the target particle concentration.2 plasma to increase hydrophilicity. The concentration and volume of the colloidal AuNP solutions were tailored to the desired as-assembled area coverage and then drop-casted onto the silicon wafers and left to dry slowly to ensure monolayer assembly of the AuNPs. The as-assembled AuNP/Si was characterized via SEM and AFM to statistically quantify the area of coverage and confirm monolayer closed-packed assembly.Polished silicon wafers  were used for assembling AuNS and AuNR. The silicon wafers were diced into 2 mm × 2 mm square substrates and cleaned using deionized (DI) water, acetone, and isopropyl alcohol (IPA) via ultrasonication, followed by piranha cleaning (3:1 sulfuric acid and 30% hydrogen peroxide) and DI water rinsing. Silicon substrates were further undergone through OUniaxial compression was applied to the AuNPs via a custom-built hydraulic press setup ensuring solid-state, hard-contact compression. This study uses this custom technique to ensure displacive motion-mediated structural transformation, in contrast to in situ deformation within transmission electron microscopy (TEM) setups, which is often dominated by diffusion-mediated structural reconstruction due to electron beam–induced sample heating and atomic diffusion . Siliconnominal stress (σnom) is calculated by considering the nominal force (Fnom) applied over the total nominal contact area (Anom) between the top compression silicon wafer and the bottom substrate silicon wafer (Anom = ∼1 mm2). The applied normalized stress (σnorm) is calculated by considering the nominal force (Fnom) acting on the area covered by the as-assembled AuNPs on the substrate  = (Fnom)/(Anom × area coverage of AuNP). Evenly spaced, 13 positions (with polar symmetry) are statistically sampled across each of the as-assembled AuNP/Si wafers to determine the area coverage of AuNP  potential  were collected using EDAX TEAM software with a 6-nm step size, the “Medium-Large” Hough mask setting, 8 × 8 EBSD pattern binning, and the “Enhanced” EBSD pattern digital processing setting. The EBSD pattern at each point in the map was simultaneously recorded during the collection of the map data set. Subsequently, EDAX OIM 8 software was used to generate the inverse pole figure maps and pole figures for each data set. Finally, neighbor pattern averaging and reindexing was performed to improve indexing accuracy using the acquired EBSD patterns for each map data set.pgad267_Supplementary_DataClick here for additional data file.


Eryngium foetidum L. is an edible plant widespread in Amazonian cuisine and its leaves have high levels of promising phenolic compounds for the production of extracts to be used as natural antioxidant additives. In this study, the in vitro scavenging capacity of three freeze-dried extracts of E. foetidum leaves, obtained by ultrasound-assisted extraction using green solvents , was investigated against the most common reactive oxygen species (ROS) and reactive nitrogen species (RNS) generated in both physiological and food systems. Six phenolic compounds were identified, chlorogenic acid  being the major compound for EtOH/H2O, H2O, and EtOH extracts, respectively. All E. foetidum extracts were efficient in scavenging all the ROS and RNS (IC50 = 45–1000 µg/mL), especially ROS. The EtOH/H2O extract showed the highest contents of phenolic compounds (5781 μg/g) and showed the highest efficiency in scavenging all the reactive species, with high efficiency for O2•− (IC50 = 45 μg/mL), except for ROO•, for which EtOH extract was the most efficient. Therefore, E. foetidum leaf extracts, especially EtOH/H2O, showed high antioxidant potential to be used as natural antioxidants in food formulations and are promising for nutraceuticals products. Eryngium foetidum L., one of the main non-conventional vegetables grown in Brazil [Plantas Alimentícias Não-Convencionais, PANCs, in Portuguese), is native to the Amazonia biome and Central America [n Brazil   and reactive nitrogen species (RNS). Although ROS and RNS have beneficial physiological effects, such as cell signaling, ROS and RNS can impair the normal functioning of cells when overproduced in the physiological system as a result of oxidative stress . AlthougThe understanding of the action of ROS and RNS in food systems is of great importance to the food industry, since these reactive species are responsible for initiating oxidative reactions, altering the nutritional, chemical, and sensory characteristics of foods during their processing, storage, and commercialization, making them less acceptable or even unacceptable to consumers .Due to the biological nature of foods, ROS and RNS are frequently formed. Studies on the chemistry of ROS and RNS and their effects on foods have been reported, since ROS are primarily responsible for initiating oxidative reaction in foods, interacting mainly with lipids, proteins, sugars, and vitamins, producing undesirable volatile compounds, degrading essential fatty acids, amino acids, and vitamins, and forming carcinogenic compounds ,15. AntiFrom this perspective, to support the application of natural extracts from plants as exogenous sources of antioxidants, it is necessary to carry out systematic investigations, from initial in vitro to in vivo studies, to determine their scavenging capacities against ROS and RNS of physiological importance. As widely reported in the literature, the frequent intake of bioactive compounds with antioxidant properties demonstrates a positive association with a decrease in the risk of developing chronic-degenerative diseases, such as cardiovascular diseases , atherosE. foetidum leaves (35–140 µg/mL) were shown to decrease intracellular ROS formation as induced by exposure to lipopolysaccharide for 24 h in a dose-dependent manner [E. foetidum leaves were able to scavenge DPPH radicals, while the essential oil of its leaves exhibited the highest reducing potential (FRAP assay). According to the literature, E. foetidum leaves were reported to contain phenolic acids in their composition, such as ferulic, syringic, galic, p-coumaric, protocatechuic, and sinapic acids [Extracts of t manner  reportedic acids  along wiic acids .E. foetidum showed high contents of bioactive compounds, especially phenolic compounds and carotenoids, and high in vitro antioxidant capacities against non-physiological and stable free radicals (ABTS and DPPH) and a highly reactive ROS named singlet oxygen (1O2) [E. foetidum to scavenge other relevant ROS: superoxide radical (O2•−), hypochlorous acid (HOCl), hydrogen peroxide (H2O2), and peroxyl radicals (ROO•); but the phenolic composition was not assessed to help us understand the compounds responsible for the claimed properties.In a previous study carried out by our research group, the leaves of en (1O2) . Anotheren (1O2)  determinE. foetidum cultivated in the Amazonia to scavenge RNS, such peroxynitrite anion (ONOO−). Therefore, this work aimed to report the production of green extracts of E. foetidum leaves from the Amazonia with high contents of phenolic compounds, followed by the determination of their phenolic compounds composition and antioxidant capacity to scavenge relevant ROS and RNS to further suggest them as natural food additives or nutraceutical products.Furthermore, systematic studies examining the antioxidant potential of plant extracts are of paramount importance to stimulate deeper future investigations to increase sustainable exploitation of underexploited sources of bioactive compounds. Moreover, as far as our knowledge is concerned, no information has been found concerning the antioxidant effects of characterized extracts of tert-butyl ether (MTBE), potassium persulfate methylene blue (MB), L-tryptophan, acetonitrile, formic acid, dihydrorhodamine 123 (DHR 123), 4,5-diaminofluorescein (DAF-2), 3-(aminopropyl)-1-hydroxy-3-isopropyl-2-oxo-1-triazene (NOC-5), hydrogen peroxide (H2O2), 30%, sodium hypochlorite solution with 4% available chlorine, β-nicotinamide adenine dinucleotide (NADH), phenazine methosulfate (PMS), nitroblue tetrazolium chloride (NBT), lucigenin, and all other analytical grade salts and solvents were purchased from Sigma-Aldrich . Ultrapure water was obtained from the Milli-Q System . For chromatographic analysis, samples and solvents were filtered using 0.22 and 0.45 µm nylon filtration membranes .Quercetin, chlorogenic acid, ascorbic acid, ethanol, methanol, methyl E. foetidum were obtained from a vegetable farm in Santa Izabel do Pará, Brazil  in July, 2017. The access to the selected plants was registered in the Brazilian National System for the Management of Genetic Heritage and Associated Traditional Knowledge . The leaves (approximately 500 g) were washed with distilled water, left to dry at room temperature (25 °C) for 2 h, frozen at −18 °C and freeze-dried  at −60 °C in continuous vacuum for 24 h. The freeze-dried leaves were crushed, packaged in plastic bags under vacuum, and stored at −18 °C, protected from light exposure.The leaves of E. foetidum leaves was carried out using three green solvents, as suggested elsewhere [w/v) (EtOH/H2O), and water (H2O), and they were chosen considering the permissibility of residue in extracts after evaporation, following the Commission Directive 95/45/EC of the European Communities [w/v), and the phenolic compounds were extracted during 5 min in an ultrasound bath  with a fixed frequency of 25 KHz, at room temperature. After the time in the ultrasonic bath, the extracts were centrifuged  at 11,627.2× g for 5 min at room temperature, and the supernatant fraction was collected. The solid residues were subjected to seven more extractions under the same conditions and all centrifuged liquid fractions were combined to compose the extract. Then, the extracts containing EtOH and EtOH/H2O were dried and concentrated, respectively, under reduced pressure in a rotary evaporator (T < 38 °C), and the ethanol free extracts were frozen at −18 °C followed by freeze-drying . All the freeze-dried extracts were stored at −18 °C until analysis, in amber flasks saturated with N2 flow. The extraction procedure was performed in triplicate (n = 3) for each solvent.The ultrasound-assisted method used to produce three extracts of lsewhere , with momunities . For eacE. foetidum leaves were solubilized in methanol/water  and injected into an Agilent HPLC  equipped with a quaternary pump (G1311C), an automatic injector (G7129), an oven (G1316A), and a DAD detector (G1328C). The phenolic compounds were separated on a Synergi Hydro C18 column , using a linear gradient of water/formic acid  and acetonitrile/formic acid (99.5: 0.5 v/v) as mobile phase, according to the procedure described in detail by [2 = 0.99, LOD = 0.15 μg/mL and LOQ = 0.62 μg/mL), luteolin , and chlorogenic acid . The contents of phenolic compounds were expressed in μg/g of extract (dry basis), considering three independent extraction procedures (n = 3). The phenolic compounds were tentatively identified by combining the following information: elution order and retention time on the C18 column, comparison with authentic standards analyzed under the same conditions, and UV-Visible spectra in comparison with the same compounds previously identified and quantified by our research group by mass spectrometry using electrospray as ionization source and sequential fragmentation data (HPLC-DAD-ESI-MSn) [The freeze-dried extracts of ol/water 0:20, v/v1O2-quenching assay, all the other assays were carried out in a microplate reader , equipped with a thermostat, by fluorescence, absorbance, or chemiluminescence measurements. No interference were observed among the extracts at the highest tested concentration for each assay with the specific probes. Chlorogenic acid, quercetin, and ascorbic acid were used as positive controls. Each ROS/RNS-scavenging assay corresponds to four independent experiments, each performed in triplicate (n = 3), at six different concentrations of E. foetidum extracts.Except for the 50) were obtained from the curves of the percentage of inhibition versus antioxidant concentration using Origin Pro v8 software .The results were expressed as percentage of inhibition  and the extract concentrations that were able to decrease the effect of each ROS and RNS by 50% (IC1O2-quenching capacity of E. foetidum extracts was determined by monitoring the inhibition of L-tryptophan degradation during 1O2 generation by photosensitization of MB [E. foetidum extracts (125 to 1000 µg/mL) were tested, along with quercetin (0.78 to 4.75 µg/mL) and chlorogenic acid (0.78 to 4.75 µg/mL) as positive controls. Kinetic data obtained from the intensity of tryptophan absorbance decay (219 nm) were fitted to a first-order reaction (Equation (1)), using the Origin Pro 8 software and the rate constants were calculated (Equation (2)). The percentage of protection that E. foetidum extract (EXT) (or positive controls) offered to tryptophan (TRP) was calculated using Equation (3).k is the pseudo-first order rate constant; x is the reaction time; t1⁄2 is the half-life time (min); E. foetidum extracts.The on of MB . Five co2•− was generated by the NADH/PMS/O2 system and the O2•−-scavenging capacity was determined by monitoring the effect of the E. foetidum extracts (or positive controls) on the NBT reduction induced by O2•− at 560 nm, after 10 min of incubation at room temperature [E. foetidum extracts varied from 3.91 to 125 µg/mL; for quercetin it was from 0.001 to 30 µg/mL, and for chlorogenic acid it was from 12.5 to 250 µg/mL. The results were expressed as percentage inhibition of the reduction of NBT to formazan.Operature . The conE. foetidum extracts was measured by monitoring the inhibition of the increase in fluorescence resulting from the oxidation of DHR 123 to rhodamine 123 as induced by HOCl [w/v) to 6.2 with the dropwise addition of 10% H2SO4, and the concentration was determined by spectrophotometry at 235 nm (ε = 100 M−1·cm−1). The E. foetidum extracts were tested from 31.5 to 650 µg/mL, quercetin from 0.001 to 30 µg/mL, and chlorogenic acid from 0.24 to 7.81 µg/mL.The HOCl-scavenging capacity of 2O2-scavenging capacity was determined by monitoring the effect of E. foetidum extracts on the inhibition of chemiluminescence observed during the H2O2-induced oxidation of lucigenin [E. foetidum extract (3.91 to 125 µg/mL), quercetin (0.001 to 30 µg/mL), or chlorogenic acid (12.50 to 250 µg/mL).The Hucigenin . The rea•-scavenging capacity was determined as described elsewhere [E. foetidum extracts on the inhibition of fluorescence decay of fluorescein resulting from ROO•-induced oxidation. The reaction media was pre-incubated at 37 °C for 15 min. The fluorescence signal was monitored every minute at the emission wavelength at 528 ± 20 nm, with excitation at 485 ± 20 nm, until total fluorescence decay. Trolox was used as positive control. The protection provided by the antioxidants was calculated using the difference between the area under the fluorescence decay curve in the presence of antioxidants and in its absence. The ROO•-scavenging capacity was expressed as the ratio between the slope of each extract (or positive control) and the slopes obtained for Trolox curves, as previously described [The evaluation of ROOlsewhere  and measescribed .−-scavenging capacity was determined by monitoring the effect of E. foetidum extracts on the inhibition of the increase in fluorescence resulting from the oxidation of DHR 123 to non-fluorescent rhodamine 123 as induced by ONOO− [− was chemically synthesized as described elsewhere [3 to simulate physiological conditions. The E. foetidum extracts were tested from 400 to 900 µg/mL, and chlorogenic acid from 0.06 to 1.95 µg/mL.The ONOOlsewhere . The flup < 0.05) with Statistica 8.1(Statsoft) software.All the results (mean ± standard deviation or standard error of the mean) were calculated and submitted to analysis of variance (one-way ANOVA) and the means were compared by Tukey’s test, or Student’s test, at the significance level of 95% , 45% (EtOH) and 53% (H2O) of the identified compounds, and confirming another previous report for E. foetidum leaves [The phenolic compounds composition showed that chlorogenic acid was the major phenolic compound in all the extracts , accountm leaves .Camelia sinensis, also being one of the main phenolic compounds of coffee. Chlorogenic acid has versatile properties as it acts both as a nutraceutical and food additive. As a nutraceutical, it exhibits antioxidant, anti-inflammatory and anti-hypertensive properties, among others, which can contribute to the prevention and treatment of several associated pathologies [Chlorogenic acid is a biologically active phenolic compound and is one of the phenolic acids widely found naturally in certain plant species, such as hologies ,30,31,32hologies ,29.2O extract presented the highest contents of phenolic compounds (5581 µg/g extract), followed by the H2O extract (3416 µg/g extract). The sum of the identified phenolic compounds in the EtOH extract (1104 µg/g extract) was three and five times lower than the total contents observed for the H2O and EtOH/H2O extracts, respectively , and the addition of water decreased the ability of ethanol to solubilize this type of phenolic compounds. Interestingly, the mixture of water and ethanol yielded the highest contents of flavonols, being represented in our study by quercetin glucuronide .In general, all the extracts of  species  in a con2O presented the highest antioxidant efficiency, probably due to its high contents of phenolic compounds, mainly phenolic acids   . Recentl2O extract showed superior efficiency in quenching 1O2 (IC50 at 76 μg/mL) as compared to the EtOH extract (IC50 at 1000 μg/mL), while the H2O extract did not show any antioxidant activity at the highest tested concentration (1000 µg/mL). The quenching efficiency observed for the EtOH/H2O extract of E. foetidum was 3.5 times higher than S. diploconos extract (IC50 = 269 μg/mL) [Caesalpinia crista leaf extracts (IC50 = 61 μg/mL) [1O2 can generate alkyl radicals (R•), alkoxyl radicals (RO•), ROO•, organic hydroperoxides (ROOH), O2•−, H2O2, hydroxyl radical (OH•), and ONOO− [1O2 oxidizes amino acids and vitamins, yet it reacts very quickly with polyunsaturated fatty acids and aromatic amino acids due to the high reactivity of π electrons of double bonds; the greater the unsaturation of fatty acids, the higher the reaction rate with 1O2 [1O2-quenching capacity are highly desirable as promising natural ingredients for food formulations to increase the stability of foods.The EtOH/H9 μg/mL)  and clos1 μg/mL) , but mucnd ONOO− ,52,53,54nd ONOO− ,56,57. Iwith 1O2 . Thus, n2•−, the EtOH/H2O extract of E. foetidum leaves presented the highest antioxidant capacity (IC50 = 45 μg/mL)  and quercetin (12.9 μg/mL). Conversely, Souza et al. [E. foetidum at the highest tested concentration of 4 mg/mL. However, this extract exhibited similar antioxidant efficiency to walnut leaf extracts (Ruglans regia), whose IC50 was 47 μg/mL [Vismia cauliflora leaf extract (51 μg/mL) [Regarding O5 μg/mL) , which wa et al.  reported47 μg/mL , and sup1 μg/mL) .2•− is considered an important precursor of a variety of other highly reactive pro-oxidant species [2•− can undergo dismutation, spontaneously generating H2O2 at low pH, or through enzymatic catalysis , and most of the H2O2 generated is used in the formation of HOCl, which is 100 to 1000 times more toxic than O2•− [2•− can be used to delay oxidative processes, including lipid peroxidation, which is one of the main pathways to food deterioration during processing and storage [Among the ROS, O species . O2•− cahan O2•− . Thus, b storage .2O extract was about twice as effective as compared to the EtOH and H2O extracts. When compared to the literature, this E. foetidum extract was more efficient than Caesalpinia crista leaf extracts (IC50 at 170 μg/mL) [E. foetidum (13 μg/mL) than the extracts obtained in our study (IC50 from 118 to 222 μg/mL). The importance of being an efficient HOCl-scavenger is mainly due to the fact that this ROS, by itself, is considered a strong oxidant that reacts with O2•− to generate •OH, another highly reactive ROS known as a potent pro-inflammatory agent [Concerning HOCl, the EtOH/H0 μg/mL) . However0 μg/mL)  reportedry agent , and whiry agent .2O2, statistic differences (p > 0.05) were observed among the three extracts, and the EtOH/H2O extract showed once more its high scavenging capacity, at approximately 2.5 to 3.5 times higher than the EtOH and H2O extracts, respectively. Importantly, the EtOH/H2O extract (IC50 at 110 μg/mL) was as effective as chlorogenic acid (IC50 at 100 μg/mL) in scavenging H2O2, yet almost three times less efficient than ascorbic acid  [Vismia cauliflora (IC50 at 289 μg/mL), a medicinal plant species abundant in the Amazonia [Meyna spinosa, a medicine plant from India (IC50 at 127 μg/mL) [E. foetidum at the highest tested concentration of 4 mg/mL. H2O2 is a reactive species capable of crossing cell membranes and oxidizing various cellular compartments, the source of many of its toxic effects [2O2 can indirectly cause lipid peroxidation, because H2O2 is also a precursor of •OH [In relation to Hbic acid . The EtOctively) , leaf exAmazonia , and lea7 μg/mL) . Contras7 μg/mL)  stated t effects . In foodr of •OH .• radicals, almost twice as efficient as the H2O extract and trolox. The H2O extract presented the same capacity to scavenge ROO• as observed for trolox, while the EtOH/H2O extract showed the least scavenging efficiency. As ROO• are widely known to be generated during lipid peroxidation of unsaturated fatty acids, independently of their localization, either as part of cell membranes or in food sources and formulations, these ROS have high physiological and food relevance. When compared to the literature, the EtOH extract showed higher antioxidant capacity (1.75) than hydroalcoholic extracts of artichoke leaves (Cynara cardunculus L.) (0.31) [Bactris setosa fruits [E. foetidum extracts to be added in food formulations to extend the shelf life of processed products by delaying lipid peroxidation.Contrasting the results, the EtOH extract was the most efficient extract for scavenging ROO) (0.31) , and exta fruits . Our res2O2, ¹O2, and O2•−, clearly demonstrating its ability to scavenge ROS [E. foetidum plants as a bioactive compound source.Previous studies have already pointed to the high efficiency of chlorogenic acid in protecting cells against oxidative damages caused by Henge ROS ,68, whicE. foetidum leaves were able to inhibit the oxidative/nitrosative effects of the tested RNS in a concentration-dependent manner, but with much lower efficiency than the positive controls. To the best of our knowledge, this is the first study reporting the RNS-scavenging capacity of E. foetidum extracts.Concerning RNS, 3, the EtOH/H2O extract presented the highest ONOO--scavenging capacity, while EtOH and H2O extracts showed similar IC50 values with no statistical difference  [E. foetidum extracts, while gallic acid (IC50 at 876 μg/mL) was only 1.6 times more efficient [50 at 0.23 μg/mL), either in the presence or absence of NaHCO3, it is probable that other non-identified compounds in the E. foetidum extracts might have interfered with the observed antioxidant effect.By comparison, the extracts of 8 μg/mL)  than thefficient . As chlo•NO) with O2•−. ONOO− can undergo homolysis of the O−O bond, generating extremely reactive •NO2 and •OH [•OH by ONOO− homolysis is approximately one million times faster than the metal-catalyzed generation of •OH from H2O2 via the Fenton reaction [− participates in one- and two-electron oxidation reactions with lipids, proteins and DNA, playing a key role in the process of cell/tissue injury under pathological conditions [Peroxynitrite is one of the most potentially harmful RNS, and it is generated in vivo by the reaction of nitric oxide ( and •OH ,70. The reaction . Anothernditions . These fE. foetidum leaves showed efficient antioxidant capacity to scavenge biologically relevant ROS and RNS. The EtOH/H2O extract presented the highest phenolic compound contents, highlighting chlorogenic acid as the major compound, and, in general, the higher antioxidant performance. Our results suggested that the E. foetidum extracts have promising compounds to be used in food formulations to delay lipid peroxidation, or even for drug design purposes to efficiently scavenging ROS during oxidative stress.The study clearly demonstrated that extracts of E. foetidum, which can be seen as a promising Amazonian plant to be rationally exploited by sustainable means for obtaining natural antioxidants.Therefore, this study stated the importance of further systematic research on the biological properties of


P) of 193.4 mW m−2 and P/(ΔT)2 of 0.236 mW m−2 K−2 were achieved at ΔT of 28.6 K. However, KOH-activated electrodes showed no performance enhancement regardless of activating agent/carbon precursor ratio. The electrode material developed here worked well under different temperature differences, proving its feasibility in harvesting electrical energy from various types of waste heat sources.The ever-increasing energy demand and global warming caused by fossil fuels push for the exploration of sustainable and eco-friendly energy sources. Waste thermal energy has been considered as one of the promising candidates for sustainable power generation as it is abundantly available everywhere in our daily lives. Recently, thermo-electrochemical cells based on the temperature-dependent redox potential have been intensely studied for efficiently harnessing low-grade waste heat. Despite considerable progress in improving thermocell performance, no attempt was made to develop electrode materials from renewable precursors. In this work, we report the synthesis of a porous carbon electrode from mandarin peel waste through carbonization and activation processes. The influence of carbonization temperature and activating agent/carbon precursor ratio on the performance of thermocell was studied to optimize the microstructure and elemental composition of electrode materials. Due to its well-developed pore structure and nitrogen doping, the mandarin peel-derived electrodes carbonized at 800 °C delivered the maximum power density. The areal power density ( Our society needs substantial quantities of energy for manufacturing, transportation, and living. However, unfortunately, most energy has been based on fossil fuels that are not replenished. Furthermore, the release of greenhouse gases from burning fossil fuels is responsible for rising global warming and climate change . To addr63−/Fe(CN)64− [2+/Fe3+ [−/I3− [Voc) of a thermocell relies on the temperature coefficient of redox potential (α), also known as ionic Seebeck coefficient, which can be written as [E is the redox potential of a given redox couple, ΔSrxn is the entropy change in the reaction, n is the number of electrons transferred, F is Faraday’s constant, T is the temperature at which the reaction occurs, and ΔT denotes the temperature difference between the two electrodes. In particular, thermocells have outstanding α values (−1.4 mV K−1 and 1.76 mV K−1 for Fe(CN)63−/Fe(CN)64− [2+/Fe3+ [−1) [Recently, thermocells , featuring low-cost, simple components, and direct heat-to-electricity conversion, have been considered as one of the promising technologies for waste heat harvesting ,4,5,6,7.e(CN)64− , Fe2+/Fe[2+/Fe3+ , or I−/I+ [−/I3− , is invoitten as :(1)α=∂E∂e(CN)64− ,13 and F[2+/Fe3+ , respecte3+ [−1) , which me3+ [−1) .3Fe(CN)6/K4Fe(CN)6 electrolyte under alkaline conditions, which enables the use of non-noble metals such as copper and nickel that have not been used due to the corrosion with the electrolyte of thermocells [Given the wide distribution and small temperature difference with the environment of low-grade heat, it is essential to make a large-scale device that requires inexpensive electrodes from abundant and cheap sources. Efforts have been dedicated to preparing inexpensive and high-performance electrode materials such as graphene ,18, carbrmocells ,24. Consrmocells , it is urmocells ,31,32,33rmocells ,35,36,37rmocells ,39 owingrmocells ,33, jujurmocells , tea wasrmocells , coffee rmocells , bagassermocells ,34, leafrmocells , and conrmocells  have beeThe research presented in this paper aims to develop a biomass-derived electrode for cost-effective and high-performance thermocells. We proposed a facile and environmentally friendly approach for synthesizing a porous carbon electrode prepared by carbonization and activation treatments of mandarin peel waste. A large number of mandarin peel waste generated from juice production are discarded, and their high biological oxygen demand and chemical oxygen demand lead to soil degradation and environmental pollution . Therefo2) flow of 100 sccm for 2 h in a quartz tube furnace at a heating rate of 5 °C min−1. Subsequently, the carbonized DMPs were immersed in 0.1 M hydrochloric acid (HCl) solution , stirred for 30 min, and washed with DI water several times to neutralize them and remove any impurities. Finally, the products were dried in a vacuum oven at 80 °C for 12 h. Based on their carbonization temperature, the obtained products were denoted as CMP-600, CMP-700, CMP-800, CMP-900 and CMP-1000.This work used Satsuma mandarin peel waste as a carbon precursor to prepare electrode materials through carbonization and activation processes. First, the mandarin peels were washed several times with deionized (DI) water to remove impurities, followed by drying in a forced air oven at 80 °C for 12 h. Afterward, the dried peels were ground and sieved to obtain mandarin peel powder with a particle size between 75 μm and 120 μm, named DMP. Next, the DMP was carbonized at 600, 700, 800, 900 and 1000 °C at low pressure with a nitrogen  were denoted as AMP-800-1, AMP-800-2, and AMP-800-3, respectively.The mixtures with different weight ratios of KOH and CMP-800  were prepared by mixing and grinding CMP and KOH flakes (Daejung) using an agate mortar and pestle to ensure homogeneity. The obtained mixtures were heated at 800 °C at low pressure with a Nθ range of 10–80° with an X-ray diffractometer  using Cu Kβ radiation (λ = 1.3922 Å) to identify the crystalline structures of CMP and AMP. Raman spectroscopy was performed at an excitation wavelength of 532 nm using a micro-Raman spectrometer . The specific surface areas (SSA) of CMP and AMP samples were measured using the methylene blue adsorption method [The surface morphology and microstructure of CMP and AMP samples were observed using field emission scanning microscopes . The surface elemental composition of the samples was determined by energy dispersive X-ray spectroscopy (EDS) with an analyzer  attached to the FESEM. X-ray photoelectron spectroscopy (XPS) was performed to investigate the surface functional groups and the chemical composition of CMP and AMP using a PHI 5000 Versa Probe Ⅱ . X-ray diffraction (XRD) spectra were measured in the 2n method . Approxi3Fe(CN)6/K4Fe(CN)6 electrolyte for thermocell performance tests. A thermocell was assembled with two identical electrodes, stainless steel current collectors, and a 5 mm thick spacer containing an electrolyte (CMP (or AMP), Super P, and polyvinylidene fluoride (Sigma-Aldrich) were mixed in N-Methyl-2-pyrrolidone (Daejung) with a weight ratio of 80:15:5 using a ball miller  to prepare a homogeneous slurry. It was then coated onto a stainless steel sheet  using an adjustable film applicator, followed by a drying process under vacuum at 40 °C for 12 h. The obtained sample was cut into several pieces with a 1 cm × 1 cm dimension for use as electrodes. The potassium hexacyanoferrate  and potassium hexacyanoferrate(II) trihydrate (Daejung) were dissolved in DI water to prepare a Kctrolyte b.α of the redox potential of Fe(CN)63−/Fe(CN)64− was determined using a U-shaped cell comprising two half-cells to prevent heat conduction between them (The een them . The tem3Fe(CN)6 and 0.1 M of KCl solution.The thermocell was placed between hot and cold blocks to make a temperature difference between the electrodes. These blocks were connected to refrigerated/heated bath circulators , and their temperatures were controlled with an accuracy of ±0.05 °C. The output electrical power of the thermocell was measured by the digital multimeter with different load resistances. The load resistances were controlled using a scanner card  mounted in the multimeter considering the internal resistance of the thermocell. The cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were conducted using a potentiostat/galvanostat  to examine the electrochemical properties of the electrodes. The cyclic voltammetry was performed with a three-electrode setup consisting of a CMP (or AMP) working electrode, a platinum counter electrode, and an Ag/AgCl reference electrode with 3.5 M KCl. The electrolyte was 5 mM of K−1 as the temperature increased from 20 to 40 °C. The thermal conductivity of the electrolyte was ~0.55 W m−1 K−1 [The ionic conductivity of the electrolyte was measured at different temperatures  using a  m−1 K−1 .2 + 2K2CO3). K2CO3 decomposes into K2O and CO2 at a temperature above 700 °C. K2CO3/K2O/CO2 further reacts with carbon atoms, introducing pores into carbon materials [aterials ,33.2O, MgO, and CaO in the washing process. Salt particles of various sizes were observed in as-synthesized CMP that was not washed after the carbonization  demonstrates the degree of defect and disorder of carbon materials. The ratio ID/IG of CMP increased from 0.88 to 1.09 as the carbonization temperature changed from 600 to 1000 °C. During the carbonization process, heteroatoms such as oxygen and nitrogen abundant in biomass feedstocks are eliminated, and the residual carbon atoms form a turbostratic structure consisting of nanometer-sized polyaromatic layers [ID/IG of Raman spectra of AMP, as presented in The crystal structures of the prepared samples were analyzed by XRD, and the results are shown in on atoms . The intc layers ,48. The CMP and AMP were analyzed by EDS in order to determine their elemental composition and distribution. The SEM and EDS mapping images shown in XPS was also examined to probe the elemental composition of the as-synthesized CMP-800 sample, as shown in 2 g−1 as the carbonization temperature raised to 700 °C and decreased to 60.0 m2 g−1 with the further temperature increase due to the pore generation and widening. Cyclic voltammograms 63−, n is the number of electrons transferred in the redox reaction, v is the potential scan rate, and C is the concentration of the reaction species (Fe(CN)63−) in the electrolyte. Therefore, the ESA is proportional to the peak current. The ESA of CMP normalized by that of CMP-600 is plotted in mmograms b show thThe cyclic voltammogram of AMP-800-3 showed a smaller peak current than CMP-800 despite its higher SSA c. Therefα of Fe(CN)63−/Fe(CN)64− redox couple was measured using a U-shaped thermocell comprising CMP-800 electrodes and 0.4 M K3Fe(CN)6/K4Fe(CN)6 electrolyte 63−/Fe(CN)64− redox couple [α indicates that the cold electrode has a higher potential than the hot electrode, and ΔSrxn of the redox couple is negative.The ctrolyte . For comx couple ,13. The Voc of CMP electrodes was 24.4 ± 0.18 mV, indicating that the ΔT between the electrodes was about 17.4 °C which was calculated from the Voc and α measured above. A temperature drop can arise from the thermal interface resistances at hot (cold) block/collecting electrode/CMP electrode interfaces and their thermal resistances, which reduces the ΔT between the electrodes.The power generation performance of CMP and AMP electrodes was investigated by measuring the voltage between the hot and cold electrodes under different external resistances. The temperatures of the hot and cold blocks were controlled at 45 °C and 15 °C. As plotted in CMP-800 showed the lowest internal resistance of 27.0 Ω obtained from the voltage–current curve slope. The corresponding areal power density–current curve plotted in The cell was repeatedly assembled and disassembled to prove the robustness of CMP electrodes. No delamination and crack were observed after ten assemblies and performance tests, as shown in Rs), charge transfer resistance (Rct), and mass transport resistance (Rmt) [Rs and Rct were calculated from EIS results using an equivalent circuit model  . EIS wasit model . Nyquist CMP-800 f. Althou CMP-800 c. Given T. The open-circuit voltage increased with increasing ΔT, and the slope of the voltage–current line declined, revealing that the internal resistance decreased as ΔT increased. The maximum power of thermocell can be expressed asRint denotes the internal resistance of thermocell. It can be found from T)2, which is attributed to the decrease in Rint. The three primary internal resistances are displayed in Rint. The resistances were estimated from the voltage–current curves in Rmt that can be reduced by the temperature gradient-induced convection flow of electrolyte. P) was 193.4 mW m−2 and P/(ΔT)2 was 0.236 mW m−2 K−2 for ΔT of 28.6 K (−2 K−2 at ΔT of 40 K) [−2 K−2 at ΔT of 50 K) [−2 K−2 at ΔT of 50 K) [−2 K−2 at ΔT of 50 K) [−2 K−2 at ΔT of 50 K) [The areal power density  , CNT (0.of 50 K) , CNT/graof 50 K) , N-dopedof 50 K) , and carof 50 K) .The mandarin peel waste was successfully converted into carbonaceous materials for thermocell electrodes. The influence of carbonization temperature on the structural, chemical properties, and thermocell performance of CMP was explored, and CMP-800 carbonized at 800 °C generated the maximum electrical power due to the combined contributions from highly porous structure and nitrogen doping. However, no enhancement of performance by KOH activation was observed. Harvesting electrical energy from various low-grade heat sources using sustainable electrode materials would provide a promising way to address global warming and energy crisis.


Lacticaseibacillus rhamnosus GG (LGG) is a Gram-positive beneficial bacterium that resides in the human intestinal tract and belongs to the family of lactic acid bacteria (LAB). This bacterium is a widely used probiotic and was suggested to provide numerous benefits for human health. However, as in most LAB strains, the molecular mechanisms that mediate the competitiveness of probiotics under different diets remain unknown. Fermentation is a fundamental process in LAB, allowing the oxidation of simple carbohydrates  for energy production under oxygen limitation, as in the human gut. Our results indicate that fermentation reshapes the metabolome, volatilome, and proteome architecture of LGG. Furthermore, fermentation alters cell envelope remodeling and peptidoglycan biosynthesis, which leads to altered cell wall thickness, aggregation properties, and cell wall composition. In addition, fermentable sugars induced the secretion of known and novel metabolites and proteins targeting the enteric pathogens Enterococcus faecalis and Salmonella enterica Serovar Typhimurium. Overall, our results link simple carbohydrates with cell wall remodeling, aggregation to host tissues, and biofilm formation in probiotic strains and connect them with the production of broad-spectrum antimicrobial effectors. Lactobacillaceae and Bifidobacteria, are widely recognized as the most frequently used genera probiotics2. This industry heavily relies on strains initially isolated from humans or fermented products5. This industry is growing exponentially, but the most consumed products fail to meet the criteria for biotherapeutics. One overarching hypothesis is that uncovering mechanisms that contribute to the competitiveness of probiotic strains and their response to different diet components may improve the compatibility and predictability of these strains as live therapeutic agents6.Probiotic strains are defined as strains that confer health benefits to their consumers and can be consumed as fresh fermentation products or as dried bacterial supplements8. Members of this family are core members of the microbiota residing in the gastrointestinal tract (GIT) of humans and animals9. Lactobacilli are widely used as probiotics and are frequently applied as a part of fermented foods and food supplements. The performance of Lactobacillaceae, as well as the performance of other probiotic strains in the gut, is heavily influenced by nutrient composition and availability in this habitat10.Lactobacillaceae are Gram-positive rod-shaped, facultative anaerobes that belong to the lactic acid bacteria (LAB) group. For LAB, the main end-product of carbohydrate metabolism is lactic acid11. While it was generally believed that fermentation across LAB strains occurs uniformly in direct correlation with the capacity to utilize glucose2, we recently found the repertoire of the response of LAB strains to glucose varies and that the overall growth of the culture also reflects complex adaptation to this carbohydrate12.Efficient fermentation plays a fundamental role in LAB physiology as these bacteria rely mainly on this core pathway to produce energy under anaerobic conditions or oxygen limitation. During fermentation, organic acids, alcohol, and carbon dioxide are generated via the oxidation of carbohydrates12. Specifically, the proficient probiotic bacterium Lacticaseibacillus rhamnosus GG (LGG) demonstrated the clearest alterations in cell shape and colony morphology that did not directly reflect cell growth12.One general feature significantly affected by fermentation in tested LAB strains was that glucose utilization shaped the colony morphology under static conditions, as all species exhibited morphological colony changes upon glucose treatmentHere we utilized LGG as our lens to unravel the molecular mechanisms that act downstream to glucose metabolism and contribute to altered fitness upon glucose utilization. We used an unbiased approach, mapping the overall changes in the proteome, metabolome, and volatilome of LGG in the presence and absence of fermented sugars. We aimed to determine the extent to which this model probiotic strain alters features that could contribute to the success of probiotics in the GIT, which of these differences relate to the growth of the bacteria, and whether acid stress contributes to these alterations. Our results indicate that fermented sugar regulates peptidoglycan homeostasis, biofilm formation, and antimicrobial production and, therefore, could alter the competitiveness of LGG in the competitive GIT.12. The application of glucose, galactose, and mannose, which can be fermented by LGG13, induced growth compared to the tryptic soy broth (TSB) control medium. Here, we extended the repertoire of consumed carbohydrates in LGG. The application of starch increased the growth of LGG to a lesser extent compared with glucose, galactose, and mannose. Starch is composed of glucose units joined by the glycosidic bond, and this result indicates that LGG can only partially degrade starch into glucose. However, non-fermentable sugars such as sucrose, xylose, and raffinose did not induce the growth of LGG in shaking cultures  in the headspace above the cultures using a GC-TOF-MS. Consistent with the metabolic rewiring of the metabolome and proteome, fermentation altered the VC profile overall and induced the production of organic volatiles Fig. . In the 12. Our proteome analysis also indicated global changes in peptidoglycan. About 195 out of 1800 proteins were significantly more abundant when the fermentable sugars glucose and mannose were applied, and about 140 proteins were significantly less abundant , which allowed us to resolve cellular structures by performing a cross-section of the bacterial cells. The cells grown without glucose had a rod shape with smooth edges. In contrast, cells grown on glucose had a rounder, atypical shape, consistent with their appearance in the light microscope. The edges were not smooth but rather laced , that binds N‐acetylglucosamine, and subtoxic concentration of vancomycin-BODIPY FL21 labeling the newly synthesized muramopeptide also indicated significant alterations in the cellular envelope , was significantly altered by glucose prior to detectable changes in cell growth  inhibited the growth of all three pathogens  can mimic the effects of fermentable glucose and mannose, we repeated our proteome analysis with subtoxic levels of acetate. While our data support the notion that the graduated accumulation of organic acids are slightly toxic to LGG  and a transcriptional repressor (WP_005687049.1) from the TetR/AcrR family. TetRs act as chemical sensors to both monitor the cellular environmental dynamics and regulate genes responsible for antibiotic production, osmotic stress, efflux pumps, multidrug resistance, and metabolic modulation34. This result indicates that subtoxic acetate levels may be sufficient to trigger cellular stress. Acetate also induces a phage protein, a tail assembly chaperon (WP_014569494.1), which may indicate intracellular stress as phages are often induced by damaging essential cellular components such as DNA integrity35. In addition, acetate significantly altered the levels of multiple transporters, including citrate sympoter (WP_005686012.1) and PTS sugar transporters (WP_014569015.1), and induced GNAT family N-acetyltransferase (WP_005714043.1), from the FabZ superfamily (WP_005684766.1), involved in maltose catabolism36.Indeed, subtoxic acetic acid levels altered the proteome of LGG Fig.  and signFirmicutes  with an unknown function. In all cases, these proteins were not induced by glucose. Interestingly, acetyl-CoA repressed a histidine kinase (WP_014569412.1), previously reported as responsible for the modulation of quorum sensing by acetic acid37. Acetyl-CoA was also associated with an altered metal homeostasis as both a heavy metal translocating P-type ATPase (WP_014568961.1) and a putative metal homeostasis regulator (WP_015765004.1) were significantly repressed.In addition to acetate, the levels of acetyl-CoA were elevated in the presence of glucose and mannose but not raffinose Fig. , and theOverall, our results linked organic acids to altered cell wall homeostasis, altered antimicrobial activity, and acetyl-CoA with specific pathways whose role in biofilm formation and cell wall homeostasis remains to be determined. A direct assessment of biofilm formation with pure acetic acid, or acetyl-CoA revealed little or no effect of these metabolites Fig. . HoweverOverall, these results indicate that the metabolic response to fermentable sugars may be intertwined and cannot be replicated with continuous growth in the presence of a single product of fermentation.38. The subsequent analysis of alterations in the gut microbiota, comparing the effects of multiple environmental factors, including nutrients, suggests that the altered microbial communities influence the liver and adipose tissues, altering their metabolic state39. The effects of the human microbiome on the host’s metabolism are extensive and include but are not limited to food intake, digestion, absorption, and the production of a broad repertoire of metabolites. These metabolites include bile acids, amino acids and their derivatives, and short-chain fatty acids, which were shown to play a fundamental part in GIT homeostasis41. Microbiota-derived metabolites also regulate the intestinal absorption and lipid metabolism within the intestine, and this effect is attributed mainly to organic acids42.The structure and function of microbiome communities are greatly affected by the availability of trace nutrients, carbon, and nitrogen sources. The gastrointestinal tract primarily provides these nutrients from the host’s diet29. By linking fermentable sugars with LGG performance in vitro, we can generate focused predictions regarding specific dietary-associated probiotic behavior. First, probiotics can exclude or inhibit pathogens through direct action or influence on the commensal microbiota45.Probiotic bacteria are a subset of GIT microbiota proposed to benefit human health. Here we use the probiotic bacterium LGG to link between simple fermentable carbohydrates, microbial physiology, and microbial metabolites. The health benefits of LGG include preventing and treating gastrointestinal infections and diarrhea and stimulating immune responses that promote vaccination or even prevent specific allergic symptoms. However, only some intervention studies have demonstrated the clinical benefit of probiotics, and even for the same conditions, the results are only sometimes consistent12 triggers the cell wall remodeling. Also supporting this notion, is that one of the antimicrobial proteins induced during fermentation  zero growth rates affect the proteome and metabolome46. However, carefully examining the curves suggests that the growth is also feeble, with glucose and mannose from the 12th hour of growth. Therefore, all cultures were analyzed for their proteome and metabolome of prolonged stationary phase. This status best reflects the growth in the GIT, where many beneficial bacteria are in a biofilm state and thereby are slowly replicating or at the stationary stage47. An additional consideration is the toxicity of glucose catabolites when these acidic catabolites are provided, even at sub-physiological concentrations before the gradual adaptation. A gradual pH drop could significantly differ from acute acid stress, allowing the fundamental adaptation of multiple processes reflected by the proteome remodeling. In addition, biofilm formation could not be restored to the basal levels by buffering  activates the biosynthesis of the bacteriocin rhamnosin B in LGG when applied as acetic acid salt37. Consistently, our data provide strong evidence that cell wall remodeling precedes an induced biofilm formation, which does not depend on organic acids  through specific pattern recognition receptors, including toll-like receptors (TLRs). These receptors mediate many of the interactions between probiotic bacteria, the immune cells, and the intestinal epithelium53. The behaviors of microbiome members greatly depend on the capacity of a bacterium to adhere to host tissues manifested in enhanced biofilm formation, which occurs downstream to fermentation  ATCC 53103 was the probiotic strain used in this study. Enterococcus faecalis ATCC 29212142, Salmonella enterica Serovar Typhimurium [kindly provided by Dr. Roi Avraham’s Lab] and Pseudomonas aeruginosa PA14 were used for competition assay. A single colony of L. rhamnosus GG was isolated on a solid deMan, Rogosa, Sharpe (MRS) plate (1.5% agar), inoculated into 5 mL MRS broth , and grown at 37 °C, without shaking overnight. A single colony of E. faecalis, S. typhimurium, and P. aeruginosa, respectively, isolated on a solid LB agar plate was inoculated into 3 mL Luria-Bertani (LB) (Difco) and grown at 37 °C, with shaking overnight. For biofilm colonies, these cultures were inoculated into a solid medium (1.5% agar) containing 50% tryptic soy broth (TSB), TSB supplemented with (1% w/v) D-(+)-glucose, (1% w/v) D-(+)-raffinose, (1% w/v) D- (+)-mannose or (1% w/v) D-(+)-glucose buffered with MOPS (3-(N-morpholino)propane-sulfonic acid) and potassium phosphate buffer. The bacteria were incubated in a BD GasPak EZ-Incubation Container with BD GasPak EZ CO2 Container System Sachets (260679) , for 72 h or 7 days, at 37 °C. The colony images were taken using a Stereo Discovery V20” microscope  with objectives Plan Apo S ×1.0 FWD 60 mm  attached to a high-resolution microscopy Axiocam camera. Data were generated and processed using Axiovision suite software (Zeiss). For planktonic growth, the bacterial cultures were inoculated into a liquid medium of 50% TSB with different sugars as described above, incubated for 24 h, with no shaking, at 37 °C.S. typhimurium, E. faecalis, and P. aeruginosa cultured cells grown overnight were diluted 1:100 in 200 μL liquid medium containing TSB supplemented with (1% w/v) and with 30% conditioned medium derived from LGG grown with and without sugars, as mentioned in the first section. For LGG growth with metabolites, acetic acid, acetyl-coA and pyruvate were added to TSB liquid medium in in different concentrations. Cells were grown with agitation at 37 °C for 16–20 h in a microplate reader , and the optical density at 600 nm (OD600) was measured every 30 min.LGG cultured cells grown overnight were diluted 1:100 in 200 μL liquid medium containing 50% TSB (BD) or TSB supplemented with (1% w/v) different sugars in a 96-well microplate . A bacterial biofilm colony of LGG grown as described above was suspended in 200 μL 1× phosphate-buffered saline (PBS), and dispersed by pipetting. Samples were centrifuged briefly, pelleted, and re-suspended in 5 μL of 1× PBS supplemented with the membrane stain FM1-43  at 1 μg/mL. The cells were then placed on a microscope slide and covered with a poly-L-Lysine (Sigma) treated coverslip. The cells were observed by Axio microscope . Images were analyzed by Zen-10 software .600 of the cultures was compared. The cell pellets were subjected to in-solution tryptic digestion using the suspension trapping (S-trap) method as previously described54. Briefly, bacterial cell pellets were homogenized in the presence of lysis buffer containing 5% SDS in 50 mM Tris-HCl, pH 7.4. Lysates were incubated at 96°C for 5 min, followed by six cycles of 30 s of sonication . Protein concentration was measured using a BCA assay . 50 ug of total protein was reduced with 5 mM dithiothreitol and alkylated with 10 mM iodoacetamide in the dark. Each sample was loaded onto S-trap microcolumns  according to the manufacturer’s instructions. After loading, samples were washed with 90:10% methanol/50 mM ammonium bicarbonate. Samples were then digested with trypsin (1:50 trypsin/protein) for 1.5 h at 47 °C. The digested peptides were eluted using 50 mM ammonium bicarbonate. Trypsin was added to this fraction and incubated overnight at 37 °C. Two more elutions were made using 0.2% formic acid and 0.2% formic acid in 50% acetonitrile. The three elutions were pooled together and vacuum-centrifuged to dry. Samples were resuspended in H2O with 0.1% formic acid and subjected to solid phase extraction  according to manufacturer instructions and vacuum-centrifuged to dryness. Samples were kept at −80 °C until further analysis.LGG was grown in TSB or TSB with 1% sugar for 24 h at 37 °C. For proteomics with metabolites, LGG was grown in TSB or TSB with 1% glucose or 20 µM acetic acid or 10 µM acetyl-CoA for 24 h in 37 °C. To process an equal number of bacteria, the ODULC/MS grade solvents were used for all chromatographic steps. Dry digested samples were dissolved in 97:3% H2O/acetonitrile + 0.1% formic acid. Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography . The mobile phase was: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase Symmetry C18 trapping column . The peptides were then separated using a self-packed analytic column containing ReproSil-Pur 120 C18-AQ resin  at 0.35 µL/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 30%B in 155 min, 30% to 90%B in 5 min, maintained at 90% for 5 min and then back to initial conditions.m/z), with a mass range of 375–1650 m/z, AGC of 3e6, and maximum injection time of 60 msec. MS2 resolution was set to 15,000, quadrupole isolation, 1.7 m/z, AGC of 1e5, dynamic exclusion of 50 sec, and maximum injection time of 60 msec.The nanoUPLC was coupled online through a nanoESI emitter  to a quadrupole orbitrap mass spectrometer  using a FlexIon nanospray apparatus (Proxeon). Data were acquired in data dependent acquisition (DDA) mode, using a Top10 method. MS1 resolution was set to 120,000 . The minimal peptide length was 7 amino acids and the minimum Andromeda score for modified peptides was 40. Peptide identifications were propagated across samples using the match-between-runs option. Searches were performed with the label-free quantification option selected. The quantitative comparisons were calculated using Perseus v1.6.0.7. Decoy hits were filtered out and only proteins that had at least 2 valid values after logarithmic transformation in at least one experimental group were kept. Principle component analysis (PCA) of the log intensity values and heat maps were performed/generated in R. Student’s t tests followed by FDR correction, after logarithmic transformation, were used to identify significant differences between the experimental groups, across the biological replica. Fold changes were calculated based on the ratio of geometric means of the different experimental groups. GO-term annotation of NC_017482.1 proteins was performed with Blast2Go tool. GO-terms over representation analysis (ORA) was performed with the WebGestalt 15 on the upregulated set of proteins  and using a custom database of the genome’s GO annotation performed with the Blast2Go tool. The cellular location for upregulated proteins  was predicted using PSORT Server v. 3.0 at https://www.psort.org/psortb/56.Raw data were processed with MaxQuant v1.6.6.0g, 5 min) and kept at −20 °C. Frozen cells pellets were washed with 1 M NaCl, resuspended in 8% SDS in 0.1 M Tris/HCl pH 6.8, and boiled for 30 min. The suspension was then centrifuged  to collect the pellet, and washed five times with dH2O to remove SDS. Then the samples entered a sonifier water bath for 30 min and centrifuged. The pellet was then suspended in 15 µg/mL DNAse, 60 µg/mL RNAse in 0.1 M Tris/HCl pH 6.8 and incubated for 60 min at 37 °C, with gentle shaking. This was followed by treatment with 50 μg/mL and incubated at 37 °C for an additional 60 min, with gentle shaking. To inactivate the enzymes, the suspension was boiled for 3 min and then centrifuged  and washed once with dH2O. Samples were then boiled again in 4% SDS (Sodium dodecyl sulfate) for 30 min, and washed five times with dH2O to remove SDS. The PG pellet was lyophilized, weighed, and stored at −20 until preparation for MS.Peptidoglycan (PG) sacculi were purified from planktonic cultures and biofilm colonies. Cells were collected by centrifugation  with mutanolysin solution (5.000 U/mL) for 16 h at 37 °C with gentle shaking. To reduce muropeptides, equal volume of the solution of disaccharide peptides and of borate buffer  were mixed and incubated for 20 min at room temperature. pH was adjusted to <4 with (1:5) phosphoric acid and filtered through 0.22 µm.57.An UltiMate 3000 UHPLC+ focused LC-MS system (Thermo Scientific™) coupled with a Q Exactive™ Focus Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo Scientific™) was used for LC/HRMS analysis. Muropeptides were separated using a C18 analytical column , column temperature at 50 °C. The flow rate was 0.2 mL/min when solvent A was 100% water with 0.1% formic acid, and solvent B was 100% acetonitrile, and 0.1% formic acid. 10 µl sample injected; MS/MS data were acquired over 40 min with a gradient of 0–12.5% B for 25 min, 12.5–20% B for 5 min, and held at 20% B for 5 min, and the column was re-equilibrated for 10 min under the initial conditions. The first 2 and last 5 min were excluded. The Q Exactive Focus was operated under positive ionization mode. The measurement was set to top 3 MS/MS fragmentations. The NCE was set with collision energy of 10,17.5,25. All MS spectra were analyzed by Xcalibur and freestyle software (Thermo Scientific™). PCA analysis was performed using MetaboAnalyst5.0A LGG biofilm colony was grown on solid medium (1.5% agar) containing 50% tryptic soy broth (TSB) with or without (1% w/v) D-(+) - glucose, for 7 days. Cells were fixed with 3% paraformaldehyde and 2% glutaraldehyde in 0.1 M cacodylate buffer containing 5 mM CaCl2 (pH 7.4), then post-fixed in 1% osmium tetroxide supplemented with 0.5% potassium hexacyanoferrate trihydrate and potassium dichromate in 0.1 M cacodylate (1 h), stained with 2% uranyl acetate in water (1 h), dehydrated in graded ethanol solutions and embedded in agar 100 epoxy resin . Ultrathin sections (70–90 nm) were viewed and photographed with a FEI Tecnai SPIRIT  transmission electron microscope operated at 120 kV and equipped with an EAGLE CCD Camera.58 and the boundaries were saved as regions of interest (ROIs) files. Automatic quantification was done using a Fiji macro that reads the TEM image together with the matching ROI file. Color-coded visualization of the results were created using the MorphoLibJ59 plugin. The macro is available at: https://github.com/WIS-MICC-CellObservatory/BacteriaCellWallThickness. All the images were manually calibrated using the scale bar, in order to get the measurements with proper calibration.We quantified the mean and standard deviation of bacteria cell wall thickness from TEM images, by manually drawing the inner and outer cell wall borders, and then automatically matching the pairs of inner and outer boundaries to create a ring-like region of interest and quantifying the local thickness along the centerline (skeleton) of each cell wall. Manual drawing was done in FijiBiofilm colonies were cultured and incubated as mentioned in the first section. Colonies were harvested after 72 h and separated with mild sonication. For cell wall labeling, cells were gently centrifuged, resuspended in 100 µl of PBS supplemented with WGA-FITC  or BODIPY™ FL vancomycin , incubated for 15 min at room temperature, and washed twice with PBS before imaging. Data were acquired by ImageStreamX Mark II  using a 60× lens (NA = 0.9). The laser used was at 785 nm (5 mW) for side scatter measurement. During acquisition, bacterial cells were gated according to their area (in square microns) and side scatter, which excluded the calibration beads . For each sample, 100,000 events were collected. Data were analyzed using IDEAS 6.2 (AMNIS). Focused events were selected by the Gradient RMS, a measurement of image contrast. Cells stained with WGA-FITCH or BODIPY™ FL vancomycin were selected using the intensity  and max pixel values  of the green channel (Ch02). Cell wall intensity was quantified using the mean pixel feature (the mean of the background-subtracted pixels contained in the input mask).21. Mounted samples were coated with a 3 nm thick Ir layer (Safematick). The imaging by secondary electron (SE) detector was preformed using a high-resolution Carl Zeiss Ultra 55 or Sigma scanning electron microscope.LGG biofilm colonies were grown on solid medium (1.5% agar) containing 50% tryptic soy broth (TSB) with or without (1% w/v) D-(+)-glucose, for 3 days. Then the biofilms were fixed overnight at 4 °C with 2% glutaraldehyde, 3% paraformaldehyde, 0.1 M sodium cacodylate (pH 7.4) and 5 mM CaCl2, dehydrated, and dried as described by Bucher et al.600 measured. The adherent cells were resuspended in PBS (2 mL) and their OD600 measured. The ratio calculated was adherent cell OD600/planktonic cell OD600.For biofilm growth, 1 µL of LGG starter culture was diluted (1:100) in 200 µL 50% TSB, TSB supplemented with (1% w/v) D-(+)-glucose, (1% w/v) D-(+)-raffinose, (1% w/v) D- (+)- mannose or TSB supplemented with (1% w/v) D-(+) – glucose + buffer, in 96-well polystyrene plates and incubated for 72 h at 37 °C. For biofilm formation with metabolites, acetic acid, acetyl-coA, pyruvate, and lactic acid were added to TSB liquid medium in different concentrations. Biofilm formation was assessed by crystal violet staining. After 72 h, planktonic cells were removed by pipetting, and wells were washed with DDW (Deuterium-depleted water). The adherent cells were stained with 0.05% crystal violet stain for 30 min. The stain was then removed, and the wells were washed with DDW. 100% ethanol was added to the wells for 15 min. Crystal violet intensity was determined by a spectrophotometer (OD 575 nm). For calculating the ratio between planktonic cells and attached cells, 2 µL of LGG starter culture was diluted (1:100) in 2 mL of 50% TSB, TSB supplemented with (1% w/v) D-(+)-glucose, (1% w/v) D-(+)- raffinose, (1% w/v) D- (+)-mannose or TSB supplemented with (1% w/v) D-(+) – glucose + buffer in 12-well polystyrene plates and incubated for 24 h at 37 °C. The planktonic cells were removed and their ODS. typhimurium, E. faecalis, and P. aeruginosa biofilm colonies were grown as mentioned in the first section. These bacteria were inoculated on solid medium (1.5% agar) containing 50% tryptic soy broth (TSB) with or without (1% w/v) D-(+) - glucose or D-(+)- raffinose next to each other at a distance of 0.2 mm for 7 days. As a control, each bacterium was inoculated separately. All images were taken with a Stereo Discovery V20” microscope  with objectives Plan Apo S ×0.5 FWD 134 mm or Apo S ×1.0 FWD 60 mm  attached to a high-resolution microscopy Axiocam camera. Data were generated and processed using Axiovision suite software (Zeiss).LGG, g to remove cells. The supernatant was than filtered through a 0.22 µm filter . Then the supernatant was separated by size to a large (>3 kDa) and small (<3 kDa) fraction using an Amicon ultrafiltration system with 3 kDa filter . The fraction was then filtered again through a 0.22 µm filter (Millipore).LGG planktonic cultures, incubated for 24 h, at 37 °C were spun for 20 min at 4 °C at 4000 S. typhimurium, E. faecalis, and P. aeruginosa bacterial cultures were inoculated into a liquid medium of 50% TSB supplemented with (1% w/v) D-(+) - glucose with 30% conditioned media (CM), incubated for 24 h, without shaking, at 37 °C. Then, the samples were serially diluted x10 into 96 well plates and 20 µL from each sample was plated on solid LB agar (1.5 % agar) using a multichannel pipette with the dot-spot technique. CFU enumeration was carried out following overnight incubation at 37 °C.Tritirachium album, Sigma) for 2 h in 37 °C with shaking. To remove the enzyme, the treated CM was filtered using an Amicon ultrafiltration system with 10 kDa filter . Then S. typhimurium and E. faecalis cultured cells grown overnight were diluted 1:100 in 200 μL liquid medium containing 30% treated conditioned medium and non-treated conditioned medium of large fraction (>3 kDa) and small fraction (<3 kDa), respectively. Cells were grown with agitation at 37 °C for 20 h in a microplate reader (Thremo Fisher), and the optical density at 600 nm (OD600) was measured every 30 min.Large (>3 kDa) and small (<3 kDa) fractions of conditioned media (CM) from LGG grown with TSB supplemented with (1% w/v) D-(+) – glucose treated with 200 µg/ml proteinase K , centrifuged twice (at maximum speed) to remove possible precipitants, and were injected into LC-MS system. Polar analysis in the polar phase was done as following: Analysis was performed using Waters Acquity I class UPLC System combined with a mass spectrometer (Thermo Exactive Plus Orbitrap) operated in a negative ionization mode. The LC separation was done using the SeQuant Zic-pHilic (150 mm × 2.1 mm) with the SeQuant guard column (20 mm × 2.1 mm) (Merck). The Mobile phase B: acetonitrile and Mobile phase A: 20 mM ammonium carbonate with 0.1% ammonia hydroxide in water: acetonitrile . The flow rate was kept at 200 μL min−1 and gradient as follows: 0–2 min 75% of B, 17 min 12.5% of B, 17.1 min 25% of B, 19 min 25% of B, 19.1 min 75% of B, 23 min 75% of B.LGG planktonic cultures grown in 50% TSB, TSB supplemented with (1% w/v) D-(+) - glucose, (1% w/v) D-(+)- raffinose or (1% w/v) D- (+)- mannose for 24 h at 37 °C. To get an equal number of bacteria the ODThe data processing was done using TraceFinder Thermo Fisher software. Polar metabolites were identified by accurate mass, retention time, and isotope pattern, and verified using an in-house mass spectra library.m/z.The samples were prepared for analysis in 20 mL headspace vials. The headspace (300 mL) above cultures was actively sampled. Volatile compound (VC) analysis was conducted on a thermal desorption-gas chromatography time-of-flight mass spectrometer (GC-TOF-MS) platform  combined with a Gerstel MPS autosampler (Germany). The VOCs (volatile organic compounds) were collected using SPME PDMS/DVB (pink) fiber at 30 °C for 15 min and desorbed for 3 min using temperature 220 °C. The GC column  was held at an initial temperature of 40 °C for 3 min, ramped to 205 °C at 7 °C min and held at 205 °C for 1 min. The GC runtime was 28 min. The TOF-MS was in electron ionization mode set at 70 eV. The source temperature was set to 250 °C, and spectra were acquired in dynamic range extension mode at 10 scans s−1 over a range of 39–500 GC-TOF-MS data were acquired and analyzed using ChromaTof . Chromatographic peaks and mass spectra were cross-referenced with National Institute of Standards and Technology (NIST 17) and Wiley libraries for putative identification purposes (matching factor >750 match).Statistical analyses were performed with GraphPad Prism 9.0  unless explicitly stated otherwise. Relevant statistical tests are mentioned in the indicated legends of the figures.Further information on research design is available in the Supporting InformationSupplementary Table 1Supplementary table 2Supplementary Table 3Supplementary Table 4Reporting Summary


Environmental pollutants have become quite ubiquitous over the past two centuries; of those, plastics, and in particular, microplastics (<5 mm), are among the most pervasive pollutants. Microplastics (MPs) have found their way into the air, water system, and food chain and are either purposely produced or are derived from the breakdown of larger plastic materials. Despite the societal advancements that plastics have allowed, the mismanagement of plastic waste has become a pressing global issue. Pioneering studies on MPs toxicity have shown that exposure to MPs induces oxidative stress, inflammation, and decreased cell viability in marine organisms. Current research suggests that these MPs are transported throughout the environment and can accumulate in human tissues; however, research on the health effects of MPs, especially in mammals, is still very limited. This has led our group to explore the biological and cognitive consequences of exposure to MPs in a rodent model. Following a three-week exposure to water treated with fluorescently-labeled pristine polystyrene MPs, young and old C57BL/6J mice were assessed using behavioral assays, such as open-field and light–dark preference, followed by tissue analyses using fluorescent immunohistochemistry, Western blot, and qPCR. Data from these assays suggest that short-term exposure to MPs induces both behavioral changes as well as alterations in immune markers in liver and brain tissues. Additionally, we noted that these changes differed depending on age, indicating a possible age-dependent effect. These findings suggest the need for further research to better understand the mechanisms by which microplastics may induce physiological and cognitive changes. Plastics, which are durable, low-cost, and rapidly produced , have coIn addition to these direct harmful impacts, plastics exposed to environmental factors, such as UV radiation, oxidation, and physical abrasion, have been shown to result in the formation of microplastics (MPs) . MicroplMore recently, MPs have been detected in human feces ,28, cirrIn order to study the effect that MPs have in vivo, we first tested cellular uptake and viability in vitro after exposure to commercially available pristine fluorescent polystyrene particles (PS-MPs). U-2 OS cells were cultured and subsequently treated with 0.1 and 2 μm PS-MPs at concentrations ranging from 0.01 to 1000 μg/mL. Following exposure times of 24, 48, and 72 h, cell viability was assessed via MTT assay. After 48 and 72 h, PS-MPs of both sizes induced a dramatic decrease in cell viability, which became more exaggerated with increased concentrations A. Microsn = 40) and old  C57BL/6J female mice. Animals were divided into four exposure groups (n = 10 per group): normal drinking water (control), 0.0025 mg/mL (low-dose), 0.025 mg/mL (medium-dose), and 0.125 mg/mL (high-dose) water treated with a 1:1 mixture of 0.1 and 2 μm PS-MPs , a marker of glial cells, which include activated astrocytes. We found decreased GFAP expression in brains from both young and old mice exposed to PS-MPs, as compared to age-matched controls A. A WestTNF-α (tumor necrosis factor) in liver from young and old mice exposed to PS-MPs , a major intermediate filament protein found in mature astrocytes that is involved in many cell processes such as autophagy, neurotransmitter uptake, and astrocyte development , can be TNF-α in both young and old PS-MP-exposed mice, as compared to controls  via drinking water in female C57BL/6J mice. There are still many questions that remain, including but not limited to how sex, MP delivery method, length of exposure to MPs, and MP surface chemistry impact exposure outcome. While these variations were not explored in this study, future work should examine these factors in order to understand the mechanisms by which MPs exert these effects and how these mechanisms are altered with age.g at 4 °C for 1 h 45 min, and the supernatant was discarded. The PS-MPs were resuspended using sterile distilled water. This process was repeated 3 times to remove trace amounts of DMSO and sodium azide.Dyed red aqueous pristine fluorescent polystyrene particles  were obtained at diameters of 0.1 and 2 μm. MPs in this size range were selected because 0.1 μm represents the smallest end of the microplastics spectrum, bordering on nanoplastics, and 2 μm nears the upper limit of what may enter into cells ,48. MPs Cell viability was assessed in vitro using an MTT assay. U-2 OS cells were seeded  in 24-well plates  and allowed 24 h to attach. Following this, cells were exposed to 0.01, 0.1, 1, 10, 100, and 1000 μg/mL concentrations of 0.1 and 2 μm PS-MPs for exposure times of 24, 48, and 72 h. At the end of exposure, 200 μL of MTT solution -2,5-Diphenyltetrazolium Bromide ) in 0.01 M sterile PBS  diluted 1:6 in Dulbecco’s Modified Eagle Medium  was added to each treated well. Negative controls received 200 μL of MTT solution, and positive controls received 180 μL of MTT solution and 20 μL of DMSO . Following this, plates were incubated at 37 °C for 2.5 h. After completion of the MTT reaction, cells were washed with 0.01 M PBS, and 200 μL of DMSO was added to each well. The plates were placed back in the incubator for 15 min before being placed on an orbital shaker at RT for 15 min. A total of 150 μL from each well was transferred to a 96-well plate, and absorbance was measured at 485 nm.g at 4 °C for 5 min to remove excess PS-MPs, and re-plated on 13 mm coverslips in a 6-well plate. After 24 h, the cells were washed 3 times with PBS, fixed for 10 min with 4% formaldehyde at RT in PBS, permeabilized with 0.1% Triton X-100  in PBS at RT for 15 min, stained with Hoescht  and Phalloidin  for 5 min at RT, washed 3 times in PBS, and mounted onto glass slides with an aqueous mounting medium. Fluorescence imaging  was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.U-2 OS cells were cultured and plated in a 6-well plate . Once adherent, cells were treated with 5 μg/mL of 0.1 μm PS-MPs or 20 μg/mL of 2 μm PS-MPs for 24 h. After exposure, the cells were washed with sterile PBS , detached, centrifuged in DMEM  at 1000× n = 40) and old  female C57BL/6J mice were obtained from the National Institute of Aging (NIA) aged rodent colony . Female mice were selected based on their ability to be re-housed after the mice were delivered and acclimated in our animal facility in order to minimize weight discrepancies between treatment groups. All mice were acclimated for at least 2 weeks in our animal facility prior to testing. Within each age cohort, four exposure groups (n = 10 per group) were established to receive 1:1 ratios of 0.1 and 2 μm PS-MPs via drinking water: normal drinking water (control group), 0.0025 mg/mL (low-dose group), 0.025 mg/mL (medium-dose group), and 0.125 mg/mL (high-dose group). Drinking water was selected as the delivery vehicle over oral gavage in order to allow for continuous exposure as opposed to timed bursts, as well as to minimize external stress that may impact behavior performance. Water consumption and body weights were monitored throughout exposure to ensure comparable exposure between groups  by the same researcher, with care taken to stagger the testing of mice from the different exposure and age cohorts. The mice were transported to and from the apparatus in a non-transparent plastic container cleaned with 70% ethanol after each use.A multi-cage infrared-sensitive motion detection system  was used to assess exploratory behavior and spontaneous locomotion. During this assay, the mice were placed in darkened transparent locomotor chambers (40 × 40 × 30 cm) equipped with a grid of infrared beams at floor level and 7.5 cm above floor level for 90 min while their movements were monitored in 5 min intervals in the x-, y-, and z-planes. All horizontal and vertical movements were recorded, and data were analyzed using the Fusion v6.5 software system. Locomotor boxes were cleaned with 70% ethanol after each test period.To assess exploratory and anxiety-related behaviors, all mice were placed in locomotor chambers (40 × 40 × 30 cm) divided into light and dark zones that were equipped with a grid of infrared beams at floor level and 7.5 cm above floor level. All mice were tested for 30 min with their movements being monitored in the x-, y-, and z-planes using an infrared-sensitive activity-monitoring cage system . All movements were recorded, and data were analyzed using the Fusion v6.5 software system. Locomotor boxes and inserts were cleaned with 70% ethanol after each test period.w/v) in 1X PBS, or rapidly frozen on dry ice, embedded , and stored at −80 °C.All mice were anesthetized with sodium pentobarbital (200 mg/kg) intraperitoneally and were killed by cervical dislocation; brain, lungs, heart, liver, kidneys, gastrointestinal tract (GI), spleen, and gastrocnemius muscle tissues were either post-fixed in 10% formalin  for 24 h at 4 °C, followed by 30% sucrose . The sections were allowed to dry at 30 °C for 5–10 min before being circled using a wax pen. The sections were washed for 5 min in TBS, permeabilized with 0.3% Triton X-100  in TBS for 30 min, and stained with DAPI  for 10 min. Sections were washed again in TBS for 5 min and coverslipped  with aqueous anti-fading mounting medium . Fluorescence imaging  was used to identify the dyed red fluorescent polystyrene particles using a TRITC (550 nm) filter.For detection of MPs, representative post-fixed brain, lung, heart, liver, kidney, GI, and spleen samples from control and high-dose groups from both young and old cohorts were examined. Frozen embedded tissues were sectioned at 20 μm using a cryostat  taken at −21 °C and collected onto glass slides , coverslipped with aqueous mounting medium, and dried at RT for 15–20 min protected from light. Fluorescence imaging  was used to evaluate GFAP expression.For determining GFAP expression, frozen and embedded post-fixed brain samples from control and high-dose groups from both the young and old cohorts were cryosectioned  at 30 μm taken at −21 °C; free-floating sections were collected into 12-well plate netwells  filled with PBS using previously reported methodology . Brieflyg for 10 min. Protein concentration was determined using a BCA protein assay kit  according to the manufacturer’s instructions. Lysates were mixed with loading buffer  plus 100 mM DTT and incubated at 98 °C for 10 min. Samples were fractionated in an SDS-PAGE precast 8–16% gradient gel  and blotted on a 0.2 μm nitrocellulose membrane  using transfer buffer . The membrane was blocked (0.1% TBS-Tween with 5% skim milk) and probed with rabbit anti-GFAP primary  and goat anti-rabbit-HRP secondary antibodies in 0.1% TBS-Tween ; then, immunocomplexes were detected by chemiluminescence  and visualized . Images were processed and quantified using appropriate software  [Brain samples from young and old control and high-dose cohorts were lysed in RIPA buffer  containing a proteinase  and phosphatase  inhibitor cocktail. Samples were incubated on ice for 30 min and then sonicated  for 3 min (30–30 pulse) at 4 °C with 30% amplitude and centrifuged at 10,000× WI, USA) .® RT Supermix Kit E3010, New England BioLabs Inc., Ipswich, MA, USA) according to the manufacturer’s protocol. Once the cDNA was synthesized, qPCR reactions were run using a SYBR green-based master mix  according to the manufacturer’s instructions using appropriate instrumentation  and the following conditions: 95 °C for 15 s, 95 °C for 15 s with optimal annealing temperature for each gene of interest (varied by gene) for 30 s × 40, and melt curve of 65–90 °C. The results were analyzed using the appropriate software . The primer sequences were as follows: TNFαFor: GGTGCCTATGTCTCAGCCTCTT, TNFαRev: GCCATAGAACTGATGAGAGGGAG; s100a8For: CCTTTGTCAGCTCCGTCTTCA, s100a8Rev: TCCAGTTCAGACGGCATTGT; s100a9For: AATGGTGGAAGCACAGTTGG, s100a9Rev: CTGGTTTGTGTCCAGGTCCTC. Samples of liver tissue (30–50 mg) from young and old control and high-dose groups were lysed, and RNA was extracted and purified  according to the manufacturer’s instructions. RNA concentration was determined using a spectrophotometer , and reverse transcription was run . Images were processed using the appropriate software  .p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, and trending with α p < 0.10.Data are presented to three significant digits as mean values (Ms) with SEM and are indicated in the figure legend together with sample size (N). Statistical analyses, unpaired t-tests, one-way ANOVAs, or two-way ANOVAs with Tukey posthoc multiple comparisons were performed with an α level of 0.05 using the appropriate software . Significances are denoted in figures with *