A final purity of 87–90% in the MELs was obtained by filtering two diavolumes of methanol or ethyl acetate solutions through a PBI 26% membrane, resulting in MELs losses of 14.7 ± 6.1% and 15.3 ± 2.2%, respectively. Higher biosurfactant purities can be archived using the PBI 26% membrane at higher DV, but at the cost of higher product losses. Namely, in MeOH, the use of 6 DV leads to losses of 32% for MELs and 18% for sophorolipids. To obtain MELs at reagent grade with purities equal or higher than 97%, a two-sequential cascade filtration approach was implemented using the commercial membrane, GMT-oNF. In such a process, MELs with 98% purity was obtained at the cost of 11.6% MELs losses. Finally, decoloration, important in some applications, was successfully assessed using activated carbon. Overall, this study reports a unique solution for microbial biosurfactants production with minimal product losses, enabling solvent recycling and potentially reducing costs.Glycolipid biosurfactants are the most prominent group of microbial biosurfactants, comprising rhamnolipids, sophorolipids and mannosylerythritol lipids (MELs). Usually, large amounts of hydrophobic substrates  are used to achieve high titers (~200 g/L) of a crude product of low purity at values limited to 50–60%, contaminated with unconsumed triacylglycerol and residual free fatty acids and monoacylglycerides. The methods reported for the removal of these contaminants use a mixture of organic solvents, compromising solvent recyclability and increasing final process costs. This study reports, for the first time, an innovative downstream method for MELs, in which 90% of the triacylglycerols are separated from the crude MEL mixture in a first stage and the other lipid derivatives  are removed by organic solvent nanofiltration (OSN). Three commercially available membranes  and several homemade membranes, casted from 22, 24 or 26% ( Starmerella bombicola; mannosylerythritol lipids (MELs), produced by the yeasts of the Moesziomyces (formerly Pseudozyma) genus and Ustilago maydis fungus; and rhamnolipids (RMs), the most explored mBS, produced by Pseudomonas species [Microbial biosurfactants (mBS) are surfactants of microbial origin that are excreted extracellularly or incorporated as part of the cell membrane and are produced by various bacteria, yeasts and fungi ,2. When  species ,6,7. The species ,9,10,11.−1·day−1) [Nevertheless, the price of the cheapest mBs (34 USD/kg of SLs) is ten-fold more costly than chemical surfactants such as sodium lauryl sulphate (~1–2 USD/kg) . This in1·day−1) , but so 1·day−1) . Specifi1·day−1) .These types of glycolipids biosurfactants can be produced from a variety of substrates, including carbohydrate-based materials, lipids, as well as other compounds, such as hydrocarbons and glycerol . While tv/v of fermentation broth), but able to recover 8% of the MELs produced. The MTBE used in the initial extraction has the potential to be recycled by distillation, but the other solvents would require challenging separation and should be considered waste. In another study, Shen et al. [The processes reported for harvesting the biosurfactant fraction from the culture broth are based on organic solvent extraction, using tert-butyl methyl ether (MTBE), ethyl acetate (EtOAc) and other solvents, or sedimentation/decantation, often coupled with heating/boiling . Examplen et al.  achievedRegardless of the purity obtained, all these mBSs have a distinct color , which in some applications is not desired. The use of activated carbon is often adopted for product decolorization ,28. In tThe innovative proposed process in this study involves the use of organic solvent nanofiltration (OSN), with the goal of removing lipid derivatives  smaller than MELs, RMs and SLs molecules. The separation by OSN of molecules of similar molar mass is challenging . Therefore, prior to the OSN stage, the larger TAGs are removed by solvent-selective dissolution based on the solubility differences between MELs and TAGs in a selected solvent. Finally, AC was used to remove color impurities generated during the fermentation from the MELs, resulting in a pure product with light coloration. The downstream process is presented schematically in Moesziomyces antarcticus PYCC 5048T (CBS 5955) and M. aphidis PYCC 5535T, provided by the Portuguese Yeast Culture Collection (PYCC), CREM, FCT/UNL, Caparica, Portugal, following previously established fermentation medium and conditions [TM BiofloR/CelliGenR 115), using 1.5 L of working volume, with a medium composition described elsewhere [Mannosylerythritol lipids (MELs) used in this study were produced by nditions . Yeasts lsewhere . The temlsewhere ; (B) a nlsewhere , yieldinNote that such compositions are presented for indicative purposes, as the exact values are batch-dependent. Therefore, when crude MELs of different compositions are used, the initial MELs purity before the separation step is indicated in the respective data set. Sophorolipids (SLs) were provided by Holiferm, UK, and were also used to assess the separation strategies proposed in the current study.® 435, Novozymes, Copenhagen, Denmark) enzymes, diluted in MilliQ water, were used for the hydrolysis of lipids and their derivatives. This mixture was kept at 50 °C and mixed with a magnetic stirrer at 400 rpm for up to 24 h, when the organic phase was collected. This mixture of lipids and lipid derivatives was characterized, and mixed with biosurfactants in different ratios, to simulate the extracts collected from fermentations with residual lipid impurities.Commercial immobilized CAL-B (Novozymv/v).Fatty acids, mono-, di- and triacylglycerides contents were determined by high-performance liquid chromatography (HPLC), following a method developed by Badenes et al. . SamplesMELs and residual lipids from the feed, permeate and retentate solutions were quantified after solvent evaporation, following the methods previously described, through GC analysis of methyl esters generated by methanolysis of the solutes . SLs wer254) with 0.5–1.0 mg (dw) of the analyzed sample being placed for each lane on the TLC. To visualize the compounds, a developing solution of α-naphthol in sulfuric acid  was sprayed, and the plate was heated over 100 °C for 1 min. Further assessment of the separation of MELs from TAGs, based on the difference in their solubility in a selected solvent, is described in the Results and Discussion sections.Samples of crude MELs, MELs, lipids and other solutions were eluted using various solvents, such as isopropanol (IPA), chloroform, tert-butyl methyl ether (MTBE), methanol (MeOH), ethyl acetate (EtOAc), dichloromethane (DCM), n-hexane, water, ethanol (EtOH) and acetone, in a closed TLC development chamber. The standard solvent mixture used for the separation of different MELs homologues including the use of a solvent system of chloroform/MeOH/water (6.5:1.5:0.2) as an eluent. Precoated aluminum TLC sheets with a silica gel 60 coating were used , PBI Performance Products Inc., North Charleston, SC, USA) was diluted with DMAc  to 22, 24 and 26 wt% PBI concentration. The solution was mechanically stirred at 60 rpm overnight to obtain a homogeneous dope solution, and an additional 24 h unstirred for the removal of air bubbles. The resulting solution was first manually casted using a homemade casting knife, height of 250 µm, on the top of a non-woven Polyolefin Novatexx 2471 , selected to provide additional mechanic support of the PBI membranes, without significant addition of mass transfer resistance. The film obtained was then immersed in a distilled water precipitation bath , and then in an IPA  bath  for water removal. The obtained membranes were kept on IPA until further use. All the processes were performed at room temperature and 40% humidity.A homemade polybenzimidazole (PBI) organic solvent membrane (OSN) was manufactured by the phase inversion technique. CelazoleThe cross-linking of the above-described OSN membranes was also considered following a post-manufacture treatment of the membrane. A cross-linking solution was prepared by mixing 3% of α,α,-Dibromo-p-xylene  with acetonitrile. The membranes prepared and stored in IPA were immersed and agitated in the cross-linking solution for 24 h at 80 °C under reflux. After 24 h, the reaction was stopped and the membrane was washed three times with IPA, to remove residues of the cross-linker solution and solvent. All membranes were stored in IPA. The cross-linked PBI membranes were labeled as PBI-X.The commercially available OSN membranes assessed in this work were (i) the GMT-oNF-2 , DuraMem-500  and PuraMem-600 (Evonik), with molecular cut-off (MWCO) of about 600, 500 and 600 Da, respectively; and (ii) the homemade PBI and PBI-X OSN membranes, made as previously described.2. Duplicates were performed using different membrane samples, which were preconditioned by filtering pure solvent (~400 mL) until a constant solvent flux was obtained. A pressure of 15 bar was applied using pressurized nitrogen gas, and all experiments were performed under magnetic stirring of 300 rpm at room temperature. Concentrations of MELs and residual lipids on OSN studies were assessed by GC as previously described. The experimental strategy followed used crude MELs, with typical compositions based on the one described in The filtrations were performed on a dead-end Sterlitech HP 4750 Stirred Cell, fitted with a circular OSN membrane with an area of 14.6 cm(Am), filtration time (t) and permeate volume (VP). The membrane rejection (R) was estimated by Equation (2), considering solute concentrations in the feed (CF) and permeate (CP), after permeation of 50% of the feed volume in concentration mode.The solvent flux (ϕ) was estimated by Equation (1), based on membrane area R) and permeate (CP), after the addition of 2, 4 and 6 DV of solvent, i.e., after permeation of 100, 200 and 300 mL of permeate. MEL losses or contaminants removal can be calculated by Equation (3), according with membrane rejection to the solute and DV used.MELs purifications were assessed in diafiltration mode, allowing the retention of the product, as smaller lipidic molecules were pushed through the OSN membrane. An HPLC pump Series I, Scientific Systems Inc., was used to add fresh solvent (EtOAc or MeOH) as required to keep the retentate nanofiltration cell volume constant at a value of 50 mL, compensating for the volume leaving the system through the permeate. Diavolume (DV) is defined as the volume of fresh solvent added by the retentate constant volume . Samples were collected to measure the solute concentrations on the retentate (Cw/w) AC, vigorously mixed using a vortex mixer for 30 s, and then centrifuged for 8 min at 10,000 rpm to precipitate AC. The supernatant was separated, 200 μL samples of it were collected and measured in a spectrophotometer (Hitachi U-2000) for absorbance between 450 and 650 nm. To the absorbance value measured for each sample, we subtracted the value obtained by a blank measurement containing the solvent .Absorbance at 450–650 nm wavelength spectres were determined in freshly made samples, autoclaved samples, and autoclaved samples treated with 1% activated carbon. Autoclavation was carried out in an autoclave  for 20 min at 121 °C and 1 bar, following manufacturer instructions. For activated carbon (AC) treatment, the autoclaved samples were added to falcon tubes with 1% , and A novel multi-step downstream process, a first stage combining TAG selective removal from glycolipids and a second stage using OSN for molecular separation of the glycolipid from smaller lipidic impurities, is proposed here for the purification of glycolipids biosurfactants produced from lipid-based substrates. Finally, a stage of product decoloration using activated carbon was also assessed.OSN is assessed here with the aim of removing free fatty acids and monoacylglycerols of smaller molecule size. Those are predominant contaminants on longer fermentations, or the ones optimized to avoid overfeeding of vegetable oils used as substrates , the higher the differences in solvents’ affinity to MELs and TAGs, and thus, the higher potential of using such solvents in these compounds’ separation. Dichloromethane (DCM) and chloroform present the higher ΔRf, but given their poor environmental performance, the extracting solvent was selected from the best performing non-halogenated ones, i.e., ethanol (EtOH), methanol (MeOH) and tert-butyl methyl ether (MTBE). Ideally, the selected solvent should be possible to be produced from renewable resources in a sustainable manner, and, importantly to be able to separate MELs and TAGs by dissolving and eluting one of the compounds at high concentrations while not solvating the other one and thus promoting its precipitation as a separating layer. This would allow implementing a “washing” step, to remove TAGs from the crude MELs.MeOH was selected as the best candidate for separation of TAG and MELs, in spite of other solvents have presented a higher ΔRf, considering chloroform or DCM have been exclude due to their chlorohydrocarbons nature and volatility. MTBE and EtOH were not efficient on discriminating between TAG and MELs on their elution/precipitation ,40. On tThe removal of TAGs from MELs, exploring the different compound solubility in MeOH, was then studied. Crude MELs (feeding strategy B) and SBO, accounting for a total mass of 8.3 g, were vigorously mixed in 20 mL of MeOH. The mixture obtained represents a case study where MEL is highly contaminated with TAGs—indeed the solute mass fractions of MELs and TAG were similar (~39% each), and the remaining solute fraction of 22% accounts for ~7% free fatty acids (FFA), ~14% monacylglycerides (MAG), and ~1% diacylglycerides (DAG). The mixture was then submitted to a mild centrifugation (2 min at 4000 rpm) to speed up phase separation. The top solution, enriched in MELs, was separated and kept as a purified fraction, while TAGs were predominantly left in the bottom layer, a precipitate. Additional washings can be performed to increase triacylglycerol removal with additional MELs losses.Importantly, a single MeOH washing step allowed, by selective dissolution, to remove about 94% of TAG from the crude MELs, with a product loss of 12.3%. , step 1.On the other hand, MELs losses could be mitigated by using higher volumes of solvent or applying consecutive steps of MeOH washing to further retrieve the lost MELs from the bottom layer. Such a strategy was experimentally applied by redissolving in MeOH, the bottom layer obtained in the previous step. The phases were separated as previously described. This was repeated two times. The results of the analysis of the phases obtained in this three-step separation process are presented in The results obtained point out that it is possible to reduce MEL losses to values of 3.55%, maintaining MELs purity at values above 90%. Importantly, FFA and MAG are preferentially dissolved together with MELs on the top MeOH solution. In the case of the experiment performed here, after removal of the TAGs, 30% of the total weight of the product is comprised of FFA and MAG . Therefo2+) and of organic matter . On the other hand, for OSN the use of membranes stable on the solvents used, i.e., without swelling and able to maintain their performance, is of utmost importance. In the last two decades, the development of OSN membranes have been extensively evolved and have been assessed for the separation and purification of many compounds [The technological challenges for organic solvent nanofiltration (OSN) and aqueous stream nanofiltrations are slightly different. For aqueous systems, one of the main issues is fouling at the membrane surface, due to the combined action of ions , while smaller residual lipids, e.g., FA and MAG (280–350 Da) are pushed into the permeate. The diafiltration should be carried out in a solvent which, to facilitate its recycling, should be the same that is used in the previous step of the downstream process. Such solvents are either (i) MeOH, used for removal of TAGs , PuraMem 600  and the DuraMem-500  and homemade PBI membranes casted from solutions with 22, 24 and 26% PBI solutions, as prepared or cross-linked. These membranes have been previously characterized by Razali et al.  for theiIn the current study, the membranes were assessed by estimating solvent fluxes, for EtOAc and MeOH, through the membrane and the membrane rejections to MELs and lipids present on the crude MELs. Those values were then imputed for theoretical estimations of MELs losses and diavolumes needed to obtain MEL at a reagent grade of 97%, starting from a crude MEL with a purity of 85%. Finally, the more promising OSN membranes were experimentally assayed for crude MELs purification, removing the smaller lipidic derivatives by diafiltration in MeOH or EtOAc. A sequential cascade OSN system was also assessed, aiming at higher MELs purities, with the permeate of the first diafiltration fed to a second diafiltration to improve overall MEL recovery yields.−2·h−1) for the GMT-oNF-2 (based on polydimethylsiloxane) and the DuraMem-500 (cross-linked polyimide) membranes, respectively. This is explained by the interaction of the solvent with the membrane, as discussed by Razali et al. [−2·h−1 EtOAc flux was measured through PuraMem-600 (non-crossed linked polyimide membrane) under an applied pressure of 15 bar. The EtOAc fluxes of the homemade PBI membranes decrease, from 36 to 9 L·m−2·h−1, with increasing concentration of PBI casted solution from 22% to 26%. The observed MeOH fluxes are higher and more independent of PBI concentrations, ranging between 30.0 to 43.5 L·m−2·h−1, as PBI is a hydrophilic polymer, and so the interaction with MeOH is higher.The solvent fluxes through the membranes were estimated by Equation (2) . In the i et al. , showingRetention of the larger product to purify is typically highlighted as the main decision criteria for the selection of the membrane. However, as previously illustrated mathematically by Ferreira et al. , the higConcerning the use of commercially available membranes, the rejection of DuraMem-500 for residual lipids in MeOH is 77%, making this separation very challenging as it is difficult to push these solutes through the membrane. On the other hand, GMT-oNF-2 has a rejection of residual lipids of 32% in EtOAc, and thus it has the potential to separate them from MELs. Still, the forecast product losses may be significant as this membrane presents a rejection to MEL of only 87.1%.The PBI 22% membrane, with residual lipids rejection coefficients of 26.6% in MeOH and 32.6% in EtOAc has the potential to effectively remove these compounds’ residual lipids from MELs. However, again the rejection coefficients for MELs are low, at values of around 67.7% and 78.4%, respectively, for MeOH and EtOAc.In order to increase MEL retention, a decision was made to increase the percentage of PBI in the casting solution and to assess the cross-linking of the membrane after its manufacture. The intended effect was observed, with PBI 24 and 26% membranes revealing higher MELs rejection (>90%), with no significant differences between both solvents, EtOAc or MeOH . HoweverCross-linking PBI membranes typically leads to decreased membrane MWCO and increased robustness, as shown by Valtcheva et al. . As expeImportantly, note that average rejection coefficients are estimated with significant associated standard deviations , especiaIn this study, MELs purification will be assessed following a diavolume (DV) strategy, aimed at retaining and purifing MELs, while pushing the smaller lipids molecules through the OSN membrane. In this regard, the minimum theoretical DV and the MELs losses associated for each membrane were calculated to achieve (at least) 97% of MEL purity by considering the rejection coefficients values obtained for MELs and for residual lipids  as well In The poorest performances were estimated for filtrations using PBI 22% and GMT-oNF-2 membranes, with MELs losses ranging from 65–80%. Nevertheless, since these OSN membranes (PBI 22% and GMT-oNF-2) had a higher MWCO than PBI 26%, lipids permeation was less challenging and only 4 DV were necessary to achieve 97% purity. The use of such membranes could be interesting for a cascade membrane system, where MELs, lost to the permeate on the first step, would be recovered in the second step. Therefore, their performance, under diafiltration mode, was also experimentally assessed.The experimental results for MELs purity and losses obtained for each DV using for PBI 22, 26% and GMT-oNF2 membranes, are shown in When using PBI 26%, the best-performing membrane, there were no differences in the final MEL purity attained with MeOH and EtOAc, reaching more than 97% MELs purity and losses of 26.3% and 32.4% for EtOAc and MeOH, respectively, after 6 DV. These values are still high compared to the best value reported, 10% MELs losses, which used several solvent extractions to reach similar purities [Remarkably, after 2 DV of EtOAc, a diafiltration using GMT-oNF-2 or PBI 26% resulted in a purity higher than 90%, with losses of 15.08 ± 3.10% and 15.3 ± 2.2%, respectively. Again, a similar performance, corresponding to MELs losses of 14.7 ± 6.1%, could be achieved by diafiltration of 2 DV of MeOH through the PBI 26%.The strategy presented here can be an interesting purification platform for other biosurfactants, particularly glycolipids, that face similar challenges in the separation of lipidic fractions from the glycolipids. As a proof of concept for other microbial biosurfactants, a solution of 50 g/L of crude SLs with 87% of purity was diafiltrated using PBI 26% with MeOH, as SLs do not dissolve well in EtOAc. After 6 DV, it is possible to purify SLs , and witEnvisaging membrane cascade systems, when using GMT-oNF-2 or PBI 22% membrane, after diafiltrating 4 DV of EtOAc, 36.0 or 58.0% of the MELs will be in the permeate, while the MELs on the retentate will have purities of 99% or 95%, respectively. For MeOH-based diafiltrations, the PBI 22% with 2 DV could be used to obtain MELs in the retentate with 98% purity, while 62% of MELs will be in the permeate. MELs losses theoretically and experimentally estimated are quite similar for PBI 22% and GMT-oNF-2 (coefficients of variation of 3–12%) but more divergent for the PBI 26% (coefficients of variation between 20–26%). A possible explanation is related to the fact that rejection coefficients were obtained in concentration mode with a decrease of the retentate volume to half of its initial value, while in the diafiltration, the retentate volume was kept constant.As discussed, the use of the membranes assessed in this study were far from ideal for the separation intended. The membranes that provide high retentions to MELs (above 97%), also present high rejections for the lipids (above 70%), implying the need of using high DV, which over the extensive diafiltration leads to cumulative substantial losses in MELs to the permeate, as illustrated on the previous section for PBI 26%. A possible strategy to obtain high product purity with low losses would be the development of new membranes with better performance, i.e., lower MWCO able of higher retentions of MELs and sharper rejection curves with lower rejections for lipids.However, here we follow the non-obvious approach, and we suggest the use OSN membranes, such as GMT-oNF-2 and PBI 22%, that have lower MELs rejections (67.7 to 87.1%), but also with lower rejections to the lipids (26.6–32.6%). In other words, to facilitate the removal of lipids, we indulged in a separation with high MELs losses to the permeate. Such a strategy, if implemented on a single step, will lead to non-acceptable losses of MELs. Still, a two-step filtration cascade OSN membrane system, as described by Kim et al. , can be −2·h−1) for EtOAc permeation through GMT-oNF-2, pressurized at 15 bar. EtOAc is an appropriate solvent to extract crude MELs from the fermentation broth. Therefore, this approach is particularly adequate when coupled with our co-substrate feeding strategy A  have a distinct coloration ranging from yellow to brown. Although for some applications this is acceptable, in other cases, glycolipid decoloration would increase their value. To address this challenge, we considered possible contributions for MELs pigmentation to be associated with: (i) components present in the fermentation medium and/or formed during its sterilization by autoclave; (ii) components or interactions between components generated during the fermentation; and (iii) due to the MEL itself. To gain insight into the source of coloration, the spectra of the visible light wavelength (450–650 nm) range of the medium components and carbon sources  solutions were performed before and after sterilization by autoclave. Yeast extract (YE), showed a significant increase in coloration during sterilization, with absorbance increasing at similar wavelengths of the one’s characteristic for crude MELs. Therefore, to further investigate strategies for MEL decoloration, MELs were produced with medium formulated with YE, either as received (YE-non treated) or after to be treated with AC (YE-AC treated). Finally, the MELs, produced using the YE-AC treated, was further dissolved and treated with AC (MELs-AC treated). The MELs obtained were dissolved and the images of the samples and respective spectra obtained are shown in Interestingly, four distinct peaks are observed in the MELs visible light spectrum. They could probably be attributed to specific compounds that contribute to the coloration of MELs. The total absorption value, within the visible light spectrum, for MELs produced using AC-treated YE was comparable, with a decrease in absorbance of only 8.68%, to the one of produced from YE without any treatment . TherefoThe results of quantification of MEL before and after AC treatment of MELs, shown in (i)The type of solvent and volumes used, reported in relation to fermentation broth volume harvested;(ii)The solvent recyclability using a simple distillation was assessed as easy (labeled as “Y”) when no solvent mixtures are formed, the solvent used is of low toxicity and the solvent has an acceptable low boiling point; or difficult (labeled as “N”) when solvent mixtures used formed stable azeotropes, hindering solvent separation by simple distillation, which often ends up as waste streams;(iii)The number of solvent shifts, i.e., the number of times in the process that the total volume of the solvent is completely evaporated and replaced completely by another solvent, which are high energy-intensive steps; and(iv)Recovery efficiency, i.e., the weight of product isolated on the end of the downstream route per weight of product produced in the upstream states, in percentage; and(v)Final purity, i.e., the total weight of the product by the total weight of the product sample.The novel downstream process suggested here is compared with other reported approaches for microbial biosurfactant purification in The method proposed in the present article theoretically enables the complete reuse of solvent used in the downstream process, as mixing multiple solvents is avoided. In the case studies in which the TAGs are not present at the end of the fermentation, crude biosurfactant can be harvested by extraction from the fermentation broth with EtOAc  and then purified by OSN also in EtOAc, without any solvent shifts. For such case studies, we recommend the use of a cascade system (4 DV + 4 DV) using a GMT-oNF-2 membrane, followed by AC treatment if color removal is required. The whole process can take place on the same solvent, and if 90% of the solvent used is recycled, a ratio of 1.1:1 of the total volume of EtOAc to fermentation broth will be used.On the other hand, when residual TAGs are present at the end of the fermentation, TAGs are extracted together with the biosurfactant by EtOAc from the fermentation broth . Thus, for such cases, a single solvent shift is necessary from EtOAc to MeOH (0.12:1) and additional MeOH  will be used for removal of TAG by selective dissolution with minimal MELs losses. The three fractions of MELs in MeOH will be added together, with additional methanol to obtain a 50 g/L crude MELs solution that will be fed to OSN. Removal of small lipidic contaminants will be performed by a single diafiltration using a 6 DV using a PBI 26%, followed by AC treatment if color removal is required. This process uses two solvents, EtOAc for the initial harvesting extraction and MeOH for the other unit operations, but do not yield solvent mixtures. When 90% of the solvent used is recycled, a ratio of 1:1 of total solvent volume to fermentation broth will be used. Furthermore, in the suggested process, the use of non-sustainable and toxic solvents is fully avoided.For the first time, this study presents a unique method, based on OSN, for the purification of glycolipid biosurfactants that uses only one solvent and reaches a 98% purity, with product losses of about 11.6% and the possibility of solvent recycling.Different commercially available and homemade membranes (made of polybenzimidazole (PBI) were selected to retain MELs, while allowing permeation of smaller lipidic contaminants (FFA and MAG). None of the assessed OSN membranes presented an interesting performance for the targeted separation. While, some have low rejections for MELs (67.7 to 87.1%), the ones with high retentions to MELs (rejections above 97%) also present high rejections for the lipids (above 70%), such as the case of the PBI 26% membrane. According to the model presented in our previous study , such meDifferent case studies are evaluated. A first case study highlights the importance of optimizing fermentations to avoid the presence to triacylglycerols (TAGs) at the end of the fermentation. In such cases, only one solvent, EtOAc, was used for the whole process. The PBI 26% can effectively be used in diafiltrations with 2 DV or 6 DV, respectively, to reach purities of 90% or 97% with MELs losses of 15% or 58%. While such a strategy is interesting to reach the lower purity grade, the MELs losses that require attaining 97% purity are prohibitive. Therefore, a sequential two-filtration diafiltration, each with 4 DV, was implemented using a GMT-oNF-2. Such a membrane has lower rejections for the small lipids, so MELs purifications will be facilitated. However, its rejection of MELs is also quite low and, thus, the permeate of the first diafiltration is fed to a second OSN is to recover the MELs lost. Remarkably, for GMT-oNF-2, it is possible to recover 67.8% of MELs lost in the first filtration, achieving an overall MELs purity of 98% with losses of 11.6%. So far, this is one of the best values reported in the literature for downstream of microbial biosurfactants. It is noteworthy that as this downstream route only uses one solvent, its recycling by simple distillation will be facilitated.Further reduction of solvent volume used can be relatively easy and significantly optimized, against solvent flux against the membrane, by increasing the concentration of crude MELs used in the OSN to values above 50 g/L . Moreover, performing only two extractions of the fermentation broth will also reduce solvent use . However, the implementation of further solvent volume reduction in the harvesting extraction step is challenging and deserves dedicated research attention.A second interesting case study considers the presence of TAG on the end of the fermentation. This corresponds to many of the scenarios reported on the literature, as large amounts of vegetable oils are used as carbon sources to attain high glycolipids biosurfactant production. To address such separation requirements, an additional operation unit was successfully developed for the removal of 90% TAGs, with minimal losses of only 4.1% of MELs. In this process route, to avoid further solvent shifts, the process route for removal of other lipid derivatives  by OSN was processed in MeOH. Unfortunately, for diafiltrations in MeOH, the limitations posed by the sub-optimal performance of the membranes could not be circumvented using the two-filtration cascade system. Still, our homemade PBI 26% membrane could be used in MeOH to obtain, in a single diafiltration of 2 DV or of 6 DV, MELs purities of 90% or 97%, with losses of 15% and 32%, respectively. Note that the process described here can be further improved using membranes with rejections slightly higher MEL or lower for residual MELs, respectively, in the single diafiltration or cascade system modes.A final purification step with activated carbon for the removal of color was implemented. While, in the first instance, yeast extract drew our attention, we could not assert its responsibility. Instead, this study shows that other contaminants originated during fermentation could clearly be removed from the crude MELs samples without losses of this product.Overall, the proposed downstream process for glycolipid biosurfactants was tailored to reduce solvent waste streams and avoid or mitigate solvent shifts. This resulted in a downstream process that is potentially more sustainable, as solvent streams based on a single solvent facilitates their recyclability by distillation.


Glycine max during two seasons (2019/2020 and 2020/2021), to test the effectiveness of PSRB  compared with results observed for the inoculant BiomaPhos (mix of Bacillus subtilis and Bacillus megaterium). The present study was based on the evaluation of vegetative growth, nutritional and yield parameters, and microbial biomass carbon (MBC). PSRB were more effective than, or showed similar effectiveness to, BiomaPhos for most of the evaluated vegetative, nutritional, and yield characteristics. In the fields tested in the summer 2019/2020 crop, SAC36 and SAF9 strains stood out as growth promoters, whereas in the 2020/2021 crop, SAF11, SAC36, and BiomaPhos were notable. There did not seem to be a direct relationship between long histories of soybean cultivation as a monoculture and low yield in the field. However, yield seems to be associated with soil nutritional characters such as Ca, Mg, K, P, cation exchange capacity, and organic matter levels. PSRB inoculation positively affected nodulation (NN) and nodule dry mass (NDM) in the evaluated fields in the 2019/2020 crop, and the aerial part dry mass (APDM), NN, NDM, yield, and MBC of the evaluated fields in the 2020/2021 crop. In contrast, the inoculation method was observed to have a strong effect on APDM, NN, root dry mass, and MBC, as the plants inoculated via seed showed higher mean values than those in the plants inoculated via soil. This study demonstrated the growth-promoting potential of new phosphate-solubilizing strains, which may eventually be incorporated by the biostimulants market to freely compete with BiomaPhos.Field experiments testing the effect of phosphate-solubilizing rhizobacteria (PSRB) should consider the cropping history and the method used to inoculate the strains. We evaluated the hypothesis that PSRB previously isolated from soybean seedlings could be effective in promoting growth in this oilseed crop in soils with different cultivation periods. We also evaluated whether this growth promotion could be influenced by cultivation histories or the inoculation method (via seeds or soil). Thus, we conducted an experiment in five fields cultivating  Glycine max L.) stands out due to developments in technologies and soil management practices [The Cerrado is an important Brazilian grain- and fiber-producing region, which has seen a large increase in cultivable area during the last 30–40 years . Among tractices . In the Phosphorus is the nutrient that most limits the productivity of agricultural soils in the Cerrado Domain in Brazil. High investments in fertilization are required to ensure adequate P levels, as this is the most expensive nutrient in the fertilization of soybean crops . AlthougAzospirillum, Pseudomonas, Enterobacter, Bacillus, Paenibacillus, Serratia, Burkholderia, Brevibacillus, and Klebsiella [G. max, either under laboratory conditions or greenhouse trials [Plant-growth-promoting rhizobacteria have been associated with several bacterial genera, including ebsiella ,14,15,16e trials ,18,19. He trials ,21,22. Ue trials ,24,25.Experiments conducted in the field should consider potential adverse factors, such as the history of the cropping field and the initial nutritional status of the soil, because soil quality is profoundly dependent on management practices ,27. ThisInocula are differentially resistant to abiotic and biotic stresses when made available to soil, and seed inoculation techniques used for research purposes are often not feasible on a commercial scale. Furthermore, maintaining inoculum viability throughout commercial seed treatment and storage processes faces significant technical challenges . Thus, tG. max cultivation to evaluate the hypothesis that phosphate-solubilizing rhizobacteria, previously isolated from soybean seedlings, could effectively promote the growth of this species in soils with different soybean cropping histories. This study compared its results with those observed for the commercial product BiomaPhos to test the effectiveness of these rhizobacteria. We also investigated the hypothesis that the cultivation history of the planted fields can alter the effect of plant-growth-promoting rhizobacteria on soybean cultivation, and that inoculation via seeds is superior to inoculation via soil.Considering all these factors, we conducted an experiment in five fields of G. max seedlings grown on eutrophic red Latosol, sampled from an agricultural field with a 30-year history of soybean cultivation, located in the municipality of Indiara, in the southwest of the state of Goiás, Brazil. Three rhizobacteria were chosen from 139 strains that were previously isolated from G. max seedlings and tested in vitro for their multifunctional potential. These three rhizobacteria stood out when evaluated for efficiency in the solubilization of CaHPO4 and FePO4 (mg L−1 in GL culture medium), synthesis of the phytohormones indole acetic acid (IAA) and gibberellin (µg mL−1), production of siderophores, and antibiosis to the phytopathogenic fungi Fusarium sp. and Sclerotinia sclerotiorum  (data undergoing publication process)  .−1) at 28 °C for 48 h. The inocula were then prepared in 2 L nutrient broth medium . The cell concentration of each suspension was standardized with OD600  of 0.8, according to the correlation between optical density and the number of colony-forming units (CFU mL−1). The adjustment was made using saline solution, and the OD600 of 0.8 corresponded to approximately 109 CFU mL−1 of the suspensions. The inoculum suspensions were refrigerated until use.The isolated rhizobacteria were refrigerated at 12 °C and separately reactivated in nutrient agar culture medium , and the total area of each plot was 480 mBefore the installation of the experiments, three soil samples were collected from each field for chemical and granulometric characterization. The sampling was always performed in the 0–20 cm depth layer, and the soil was classified as eutrophic Red Latosol .9 CFU mL−1 using a backpack pump at a flow rate of 150 mL ha−1 (0.36 mL per plot). Seeds of the cultivar M7110 IPRO were used at a density of 40 seeds m2. The seeds were previously inoculated with Bradyrhizobium spp. and Azospirillum brasilense AbV5 and AbV6 (2 × 108 CFU mL−1), and treated with fungicide and insecticide. In this treatment, we used the insecticides Cruiser 350 FS (Tiametoxam 350 g L−1), at the recommended dose of 200 mL per 100 kg of seeds, and Avicta 500 FS (Abamectin 350 g L−1), at a dose of 100 mL per 100 kg of seeds. The product Maxin Advanced  was used as a fungicide at a dose of 100 mL per 100 kg of seeds. Sowing occurred on 6 December 2019, with fertilizer  applied directly in the planting furrow. Plants grown in soil without inoculum, and in soil treated with the commercial product BiomaPhos (mix of Bacillus subtilis CNPMS B2084-BRM034840 and Bacillus megaterium CNPMS B119-BRM033112), were used as control treatments.In the 2019/2020 summer crop, the experiments were conducted in Fields 1, 2, and 5. After sowing, each rhizobacteria inoculum was applied to the soil at a concentration of 109 CFU mL−1, diluted in 20 mL of sterile water. The material was agitated for 60 s to facilitate adherence of the inoculum to the seeds. The treated seeds were arranged on a layer of aluminum foil, in the shade, and were immediately planted when dry. Sowing was performed on 13 November 2020 at a density of 20 seeds linear m−1 with fertilizer  directly applied to the planting furrow. Plants from non-inoculated seeds and from seeds treated with BiomaPhos were used as control treatments.The experiments in Fields 3 and 4 were conducted during the 2020/2021 summer crop. In these tests, the rhizobacterial inocula were directly incorporated onto the soybean seeds. The seeds were initially treated with fungicide and insecticide, as mentioned above, at the same doses. The number of seeds required for each plot was calculated (192 g), and the seeds were placed in polyethylene bags and separately treated with 0.36 mL of each inoculum at a concentration of 10−1), mass of one thousand grains (g), and productivity (kg ha−1). Aerial height was evaluated by measuring the distance from the neck of the plant to the end of the canopy. The aerial part and roots were separated, conditioned in paper bags, placed to dry in an oven with forced air circulation under constant temperature (65 °C) for 72 h, and then weighed to determine their dry mass. Samples of the aerial part were ground in a Willey mill and stored in plastic bags for determination of the N and P levels, according to Malavolta [For each inoculation treatment, five soybean plants were randomly sampled per plot at the R2 reproductive stage and the following vegetative growth parameters were determined: plant height (cm), aerial part and root dry mass (APDM and RDM-g), number of nodules (NN), nodules’ dry mass (NDM-mg), total N and P levels in the aerial part and grains  × 0.4 (L2) m frame, while taking care to maintain the pre-established soil volume for each plant at approximately 0.032 m3, with dimensions of 0.4 (L1) × 0.4 (L2) × 0.2 (H). The roots, nodules, and soil were separated using a 3-mm mesh sieve and running water. The nodules were placed separately in paper bags, identified, and dried in an oven at 65 °C until constant weight. The dry masses were obtained and the NN per plant was counted.To evaluate root nodulation, plants were collected using a cutting spade, centering the plant in the 0.4  by uprooting them from each plot. They were threshed in a mechanical thresher and the grains were weighed to determine the productivity at 13% humidity (wet basis). A sample from each plot was separated for determination of the mass of 1000 grains on precision electronic scales (0.01 g), and for determination of the total P and N levels in the grains.MBC) was measured in soil samples from the different fields. These samples were collected at a depth of 10 cm, and the MBC was evaluated according to Silva et al. [MBC after applying chloroform to the samples, causing the death of the microorganisms and release of the cellular components. The calculation of the MBC content was performed using the following expression:MBC is the C of the microbial biomass (mg kg−1); F, the C of the fumigated sample (mg g−1); NF, the C of the non-fumigated sample (mg kg−1); and Kc the factor used to convert the extracted C into MBC. This study adopted the Kc value of 0.33 suggested by Silva et al. [At the end of the experiments, the microbial biomass carbon . As these variables had different measurement units, a correlation PCA was performed and constructed using data standardized to have a mean value of 0 and standard deviation of 1. The number of components was chosen according to the eigenvalues (>1.0) and explained variance (above 80%).Brevibacillus sp.), SAF11 (Brevibacillus sp.), and SAC36 (Bacillus velezensis), control without inoculation, and inoculation with the commercial product BiomaPhos. These treatments were evaluated in three fields in the 2019/2020 crop and two fields in the 2020/2021 crop. The experiments were conducted in a randomized block design with four replicates, with each replicate being considered a plot. The effect of the bacteria within each experimental field was evaluated for each individual strain within each crop, as was the effect of the cultivation history. We also evaluated the effect of the inoculation method (comparing different crops) for each tested strain.The study evaluated five treatments: inoculation with the strains SAF9  were analyzed using analysis of variance, and in cases of differences between the mean values of the treatments, these were compared using the Tukey test at a probability level of 5%. All statistical tests were performed in R software, version 4.0.4 .−1, respectively)  .−3, respectively), Mg , K , P , and the highest cation exchange capacity . In contrast, Field 4 contained high levels of organic matter (OM) (37.10 g dm−3) and high base saturation (86.07%). Field 3 was the most divergent, with acid soils (pH 4.83), low concentrations of the nutrients Ca, Mg, K, and P  and high concentrations of Fe (59.88 mg dm−3) and clay (52.00 g kg−1).Fields 1 and 2 contained the highest levels of Ca  compared to plants inoculated via seed (0.09 mg) e.G. max (−1), than in plants inoculated via seed (0.28 g kg−1)   b. The P ctively) d. The in−1 and 4629.80 kg ha−1, seed and soil inoculation, respectively) and BiomaPhos ; thus, higher mean values were observed in plants with inoculation via seed , isolated from corn rhizosphere, showed characteristics of plant growth promotion, such as N2 fixation, IAA production, ammonia production, and siderophore production.Bacteria of  studies ,49,50. W studies  and Ray  studies  discuss  studies , the strG. max plants with plant-growth-promoting rhizobacteria to ensure high levels of productivity [Similarly, the effectiveness of the BiomaPhos inoculant was confirmed for soybean and corn in several studies ,55, and uctivity ,57 and ductivity ,59.Bradyrhizobium (inoculant previously incorporated to the seeds used in the current experiment) and other rhizobacteria in nodulation and growth promotion in G. max [B. amyloliquefaciens and Bradyrhizobium japonicum inoculation in increasing nodulation, which may explain the efficient NN and NDM values observed in this study, in plants treated with BiomaPhos.The relationship between inoculation with strains of some rhizobacteria and the promotion of nodulation and nitrogen fixation in legumes is mediated through the bacterial production of flavonoid-type compounds or by stimulating the host legume to produce more flavonoid signaling molecules ,61. In cn G. max ,63,64,65n G. max , demonstBrachiaria ruziziensis. According to Singh and Gupta [In the 2019/2020 crop, Fields 1 and 2 stood out when the vegetative characters of growth and nutritional levels of plants were evaluated; however, Fields 1 and 5 were prominent with respect to the yield characteristics. Thus, although the mean values observed for various variables were dissimilar in fields with different cropping histories, there does not seem to be a direct relationship between longer periods of soybean cultivation as a monoculture and lower yield in the field. Yield, however, seems to be associated with the nutritional characteristics of the soil. Fields 1 and 2 concentrated the highest observed Ca, Mg, K, and P levels in the soil, and the highest cation exchange capacity, which is accompanied by the low leaching of cations. These characteristics may explain the development and accumulation of nutrients in plants grown in these fields. However, in Field 2, unlike in Field 1, the increased accumulation of N and P in the aerial part and grains observed in the plants treated with some of the rhizobacteria did not result in increased productivity. In Field 5, the cultivation history, namely, the first year of soybean planting, explains the high concentrations of MBC observed even in the soil where the control plants were grown. This MBC represents the remaining microbial biomass from previous plantations of the forage crop nd Gupta , soil miIn the 2020/2021 crop, compared to Field 3, plants grown in Field 4, exhibited excellent vegetative characteristics of development, mass of 1000 grains and productivity. Field 3 was the most dissimilar among the fields, with soils containing low amounts of Ca, Mg, K, and P, high levels of Fe, and a very acidic pH. Acidic pH interferes with the uptake of nutrients, with absorption of Fe, Cu, Mn, Zn, Co, and Ni being increased under this condition, and because pH needs to be close to neutral for the best uptake of essential nutrients such as N, P, S, K, Ca, and Mg . This maG. max with the tested rhizobacteria favors the MBC owing to nutrient cycling [The MBC was positively influenced by inoculation of SAC36 in Fields 1 and 2; by SAC36, SAF9, and BiomaPhos in Field 5; and by all inoculation treatments in Field 3. High MBC values indicate that nutrients are temporarily immobilized, which results in lower losses of these nutrients in the soil–plant system . The res cycling , mainly A strong effect of the inoculation method was observed on APDM, RDM, NN, and MBC, with the plants inoculated via seed showing higher means for these variables than plants inoculated via soil. This is because the survivability of the strains can be affected when bacterial inoculants are sprayed on the soil, as they become more susceptible to biotic and abiotic stresses . In the Rhizobium is accompanied by a clear laboratory demonstration of the ability of a wide range of other beneficial microorganisms to improve crop performance, very few microbial inoculants are commercially available [G. max, there is the possibility that these strains, especially SAC36, will eventually be incorporated into the biostimulants market, contributing to the technological development of agriculture and the consolidation of eco-friendly agricultural technologies.Although the long history of inoculating legume seed with vailable . As the G. max seedlings can be effective in promoting the growth of plants of this species in crops cultivated in soils with different oilseed cultivation histories. Thus, in the 2019/2020 summer crop, the strains SAC36 of Bacillus velezensis and SAF9 of Brevibacillus sp. were notable as growth promoters, whereas, in the 2020/2021 season, SAF11 of Brevibacillus sp., SAC36, and BiomaPhos were prominent. The tested rhizobacteria were sometimes more effective than, and sometimes as effective as, the commercial product BiomaPhos. In contrast, no direct relationship was observed between a longer periods of soybean cultivation as a monoculture and lower yield in the field, whereas yield was possibly associated with the nutritional conditions of the soils . The hypothesis of the superiority of the inoculation method via seeds was confirmed, as it ensured higher nodulation and a higher NDM than soil inoculation. This study confirmed the growth-promoting potential of new phosphate-solubilizing strains, which may eventually be incorporated by the biostimulants market to freely compete with BiomaPhos.This study confirmed the hypothesis that phosphate-solubilizing rhizobacteria previously isolated from


Obtained hydrogels can be used as intermediate products or ingredients (like fruit fillings or spreads) for the improvement or development of novel food products to increase their fiber content and antioxidant potential.The objective of this study was to prepare hydrogels based on pectin and blackberry juice and additionally to fortify those hydrogels with apple fiber. For that purpose, two types of pectin (low methoxylated and high methoxylated) were used, and fortification was conducted with the addition of 10% of apple fiber. The hydrogels were evaluated for phenolic compounds, antioxidant activity and inhibition of α-glucosidase. In addition, the stability of these parameters after 8 months of storage was evaluated. Pectin type and addition of apple fiber had an impact on investigated parameters. Low methoxylated pectin hydrogels had a higher concentration of anthocyanins than high methoxylated pectin hydrogels, while the addition of apple fibers caused a decrease in anthocyanin content. However, fortified hydrogels had higher antioxidant activity due to the presence of phenolics from apple fibers. The results showed that anthocyanins were more favorable in inhibiting α-glucosidase because samples with higher anthocyanins concentration had lower IC Phenolics, in the form of extracts and as pure substances, are being studied as potential alternatives to commercially available medicines for hyperglycemia because their application could reduce the likelihood of the occurrence of side effects [More than ever, nutritional guidelines are highlighting the importance of the consumption of fruits and their products in everyday life. In general, berry fruits are widely recommended for their high content of valuable bioactive compounds, especially flavonoids, phenolic acids, tannins, and anthocyanins. Those bioactive compounds possess a high antioxidant potential, and they can individually and/or synergistically help protect our organisms against cardiovascular disorders, cancer, inflammation, obesity, and other chronic diseases. Blackberries are very popular among consumers, known for their high content of bioactive compounds, especially anthocyanins and ellagitannins ,2,3, whi effects .Due to the high and rapid perishability of berries, as well as their very high nutritional value, bioactive compounds must be transferred from berries into products. Traditional products would include juices, jams, jellies, and canned fruits. However, there is a need for the development of novel formulations that would protect bioactive components and improve their functionality. Those novel formulations can be used as intermediate products or ingredients (like fruit fillings or spreads) for the improvement or development of novel food products.Like the phenolic compounds, dietary fibers are also well known for their positive influence on our health. They have an essential role in intestinal health and additionally, they are associated with a reduction of hypercholesterolemia and modification of the glycemic response . They arThe aim of this study was to prepare pectin/blackberry hydrogels unfortified or fortified with the addition of apple fibers. Obtained hydrogels were investigated for the stability of anthocyanins during the preparation of hydrogels as well as after 8 months of storage, depending on the composition of formulations. In addition, the antioxidant potential of prepared hydrogels and inhibition of α-glucosidase were monitored.Saccharomyces cerevisiae) and acarbose were obtained from Sigma-Aldrich . The 4-nitrophenyl-α-D-glucopyranoside was obtained from Alfa Aesar  and potassium dihydrogen phosphate was from BDH Prolabo . Potassium persulfate, Folin-Chiocalteu reagent and sodium carbonate were obtained from Kemika . Neocuproine, 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ) and copper (II) chloride were purchased from Acros Organics . Methanol (HPLC grade) was from J.T. Baker  and orthophosphoric acid (HPLC grade > 85%) was from Fisher Scientific . Iron (III) chloride hexahydrate, sodium acetate, ethanol, ammonium acetate and starch were bought from Gram-mol . Cyanidin 3-glucoside was from Extrasynthese .The apple fiber powder was supplied by Biesterfeld AG  and both types of pectin were the product of CP Kelco . The 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), trolox, 4-(dimethylamino)-cinnamaldehyde (DMAC), chlorogenic, caffeic, p-coumaric, gallic and ellagic acids, rutin, quercetin, phloretin and phlorizin were purchased from Sigma-Aldrich . The α-glucosidase (from 2 solution (100 mM) were added. For each type of pectin, one sample was enriched with the addition of apple fiber (10%). After homogenization for 20 min, the hot mixtures were poured into preheated glass jars. Hydration was completed overnight and then the samples were analyzed. The other set of samples was stored for 8 months at room temperature and in the presence of light.The juice was obtained by pressing blackberry fruits and filtering the resulting mass. First, blackberry juice was heated to 70 °C on a magnetic stirrer. In the next step, 3% of low methoxylated or high methoxylated pectin and 10% of CaClBefore the HPLC analysis and evaluation of antioxidant activity, extraction was carried out. For the extraction of hydrogels, 3 g of sample was mixed with 30 mL of acidified methanol (HCl:methanol ratio was 1:99). The obtained mixture was well homogenized and left for 24 h. After that time, the mixture was filtered to obtain the clear extract.A slightly modified Folin–Ciocalteu method was used to determine the total phenolics. This method had been previously described by Singleton and Rossi . First, Total proanthocyanidins were determined using a method by Prior et al. . BrieflyO-glucoside, quercetin, chlorogenic acid, ellagic acid, rutin, caffeic acid, gallic acid, phloretin and phlorizin hydrate. Cyanidin-3-O-dioxalylglucoside was tentatively identified by comparing its peak spectrum with standard and literature data and was quantified using cyanidin-3-glucoside. The UV/Vis spectra were recorded in the range of 190 to 600 nm and used for identification of peaks.First, extracts were filtered through 0.2 µm PTFE membranes. For the evaluation of phenolics, HPLC (High-performance liquid chromatography) system 1260 Infinity II  was applied. The method used was previously described by Ivić et al. . The sysFor the evaluation of antioxidant activity, FRAP, CUPRAC, DPPH and ABTS methods were used. The FRAP antioxidant capacity of samples was determined according to the Benzie and Strain method  with somAntioxidant activities evaluated by the mentioned methods were calculated from the calibration curve with Trolox as standard and expressed as µmol TE/100 g. For each method, all measurements were performed in triplicates and for the blank, the extract was replaced with water.For evaluation of additive, synergistic or antagonistic effects, experimental values of antioxidant activity of fortified hydrogels were compared to the theoretical ones. Theoretical antioxidant activity was obtained by calculating the sum of the antioxidant activity of blackberry juice and the antioxidant activity of apple fiber. These values were compared with experimentally obtained antioxidant activities of fortified hydrogels. Higher experimental values of fortified hydrogels than theoretical ones indicate a potentially synergistic effect. Lower experimental values of fortified hydrogels than theoretical ones indicate a potentially antagonistic effect. The numerically equal experimental and theoretical values of fortified hydrogels indicate an additive effect.Inhibition of α-glucosidase was determined spectrophotometrically using the method previously described in the study by Granados-Guzman et al.  with sli50) were determined using a graphical method and plots of inhibition percentage versus log inhibitor concentration. The IC50 values were then calculated by non-linear regression analysis from the mean inhibitory values.Concentrations of inhibitors that exhibited 50% inhibition of enzyme activity  on volatile compounds was conducted. The software program STATISTICA 13.1  was used for statistical analyses. The results were presented as the mean values ± standard deviation.Comparison of formulated complexes was conducted by analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) with the significance defined at The results for the concentration of total phenolics and proanthocyanidins determined in blackberry juice, apple fiber and hydrogels after preparation, as well as after storage, are presented in From the results for total proanthocyanidins content, it was concluded that apple fiber served as a great source of these bioactive compounds. Similar findings were observed in the study by Kołodziejczyk et al. . SamplesO-glucoside and cyanidin-3-O-dioxalylglucoside. The type of pectin used for hydrogels preparation as well as the addition of apple fiber had an impact on both anthocyanins. The LMP hydrogel had a higher amount of both anthocyanins  than HMP hydrogel . The addition of apple fiber negatively affected anthocyanins, probably due to interactions of apple fibers components with them. Their amounts in fortified hydrogels were 57.15 mg/kg and 20.56 mg/kg, respectively, for LMP hydrogel, and 55.61 mg/kg and 20.77 mg/kg, respectively, for HMP hydrogel. Calculating the retention of anthocyanins during the preparation of hydrogels, it was concluded that both anthocyanins were retained in quite high amounts in correspondence to blackberry juice, over 93.5%. However, higher retention of both anthocyanins was observed for LMP hydrogels. In blackberry juice, ellagic acid, chlorogenic acid, caffeic acid and gallic acid were detected, while in apple fiber, considerable amount of chlorogenic acid was found. From all these phenolic acids, ellagic and chlorogenic acids were detected in hydrogels. The type of pectin had an impact on the retention of ellagic acid, and a higher amount of this phenolic acid was detected in HMP hydrogels (5.6 mg/kg) regardless of fortification with apple fiber. While ellagic acid in hydrogels originated from blackberry juice, according to the results, chlorogenic acid originated from the apple fiber. Hydrogels prepared only with pectin did not contain chlorogenic acid, while in fortified hydrogels, this phenolic acid was determined. The higher amount of this phenolic acid was determined in LMP hydrogels (16.50 mg/kg). The same tendency was observed for rutin, i.e., it was determined in fortified hydrogels but in the same amount (20.4 mg/kg) regardless of pectin type. In addition to rutin, quercetin was detected in blackberry juice and was also determined in apple fibers. However, this flavonoid was not detected in hydrogels. In addition to quercetin in apple fibers, phloretin and phloridzin were also detected. Only phloridzin was determined in hydrogels fortified with apple fiber with a slightly higher amount in LMP hydrogels (95.29 mg/kg).Concentration values for individual phenolic compounds and anthocyanins detected in blackberry juice and apple fibers are presented in O-glucoside was determined in fortified LMP hydrogel and HMP hydrogel (11.35 mg/kg) with retention rates of 19.9% and 16%, respectively, while the lowest retention of this anthocyanin was determined in fortified HMP hydrogel (12.9%). The amount of cyanidin-3-O-dioxalylglucoside was similar (around 7.10 mg/kg) in all hydrogels except for fortified HMP hydrogel (6.45 mg/kg). Even though this anthocyanin was detected in higher amounts in unfortified hydrogels, after storage its retention was higher in fortified ones, indicating that fortification of hydrogels with apple fiber caused its protection through storage. Its retention was 34.8% in fortified LMP hydrogel and 31% in fortified HMP hydrogel, while in the unfortified one, the retention was around 26.5%. Interestingly, ellagic acid was determined in a slightly higher amount after storage in both LMP hydrogels, probably due to the conversion of other phenolics into it (6.88 mg/kg and 7.98 mg/kg for unfortified and fortified one). In HMP hydrogels, retention of this phenolic acid was 86.3% and 83.5% for unfortified and fortified hydrogels, respectively. Chlorogenic acid was determined in a higher amount in fortified LMP hydrogel, and its retention was 87.6% while in HMP hydrogels, its retention was 79.7%. Rutin was identified only in fortified HMP hydrogel with retention of 62.3%. Phloridzin was detected in a much higher concentration in fortified LMP hydrogel (77.6 mg/kg) than in fortified HMP hydrogel (59.18 mg/kg) with a retention of 81.4% and 62.4%, respectively. Generally, it was concluded that after storage the highest retention of phenolic compounds was observed for fortified LMP hydrogels, except for rutin. By comparing the total concentrations of phenolic compounds, it was concluded that fortified hydrogels retained higher amounts of these valuable compounds than the unfortified ones.The stability of phenolic compounds over 8 months of storage was also determined and their retention after storage was calculated. The highest amount of cyanidin-3-O-glucoside. The initial reaction of its degradation in an aqueous solution starts with the hydrolysis of glucosidic bonds and the opening of the pyrylium ring due to heating [O-quinones that generate polymerized products by quinine–phenol reactions [Elevated temperature, as was the case for the preparation of hydrogels, can cause various reactions like oxidation, polymerization and/or condensation reactions of phenolics, leading to loss and/or formation of different compounds. Thermal degradation is one of the most important mechanisms of degradation of anthocyanins and it includes two main pathways . One pat heating . The fureactions .In our study, high retention of anthocyanins was achieved in unfortified hydrogels, while the addition of apple fibers caused a substantial degradation of these pigments. This can be explained by the pectin structure, which contributed to ionic interactions between anthocyanin cations and the carboxylic groups of pectin . Higher Generally, phenolics and dietary fibers can create chemical interactions in foods . These iAntioxidant activities of blackberry juice, apple fibers and hydrogels (after preparation and after storage) are presented in Additionally, because blackberry juice and apple fiber possess antioxidant activity, the synergistic effects between phenolics in fortified hydrogels after preparation were evaluated. While the results for the DPPH and ABTS methods showed an antagonistic effect, for FRAP a slight antagonistic effect, for CUPRAC a quite high synergistic effect was observed. An antagonistic effect was expected as a high temperature was used during preparation causing various reactions on phenolics leading to loss and/or formation of different compounds with phenolic nature. However, these degradation products probably were responsible for the synergistic effect determined by the CUPRAC method. The results depended on the applied method ,56. Diff50 values) expressed as µg GAE/mL are presented. The results showed that all extracts had lower IC50 values than acarbose, which means that prepared samples were more effective than acarbose in the same assay system. The same effect was observed in the study by de Oliveira Raphaelli et al. [50 of blackberry juice was 0.87 µg GAE/mL and for apple fiber it was 22.23 µg GAE/mL). Blackberry juice showed the lowest IC50 value among all tested samples and could be used as a more potent inhibitor than acarbose. Samples after preparation and after storage showed the same trend, i.e., hydrogels fortified with apple fiber had higher IC50 values than those without apple fiber addition and ranged from 1.98 µg GAE/mL to 6.55 µg GAE/mL for samples after preparation, and from 3.86 µg GAE/mL to 10.47 µg GAE/mL for samples after storage. From the results obtained in this study, it was concluded that IC50 values of α-glucosidase inhibition after preparation depended on the concentration of individual phenolics, such as cyanidin-3-O-glucoside, rather than total phenolics determined spectrophotometrically. Higher concentration of this anthocyanin might cause lower IC50 values, which means that the lower concentration of this phenolic compound reduces the activity of α-glucosidase by 50%. It was previously reported that anthocyanins have a good potential for the prevention and treatment of diabetes mellitus [α-glucosidase is an enzyme that is related to treatment of disorders connected with carbohydrate metabolism like hyperglycemia and diabetes . In Tabli et al. , where amellitus ,67. Our mellitus . In the mellitus , inhibitmellitus . Althougmellitus , in our mellitus .The results of this research showed the importance of proper formulation of the products. After the preparation of hydrogels, fortification of pectin/blackberry hydrogels with the apple fiber caused a decrease of anthocyanins content and α-glucosidase inhibitory activity, while on the other hand, an increase of total phenolics, proanthocyanidins and antioxidant potential was achieved. High retention of anthocyanins was achieved in unfortified hydrogels, while the addition of apple fibers caused a substantial degradation of these pigments, proving that interactions between compounds play important roles in retention of this valuable pigment, which also has antioxidant activity. On the other hand, those interaction cause formation of other phenolic compounds improving antioxidant potential of fortified hydrogels. Obtained hydrogels can be used as intermediate products or ingredients (like fruit fillings or spreads) for the improvement or development of novel food products to increase their fiber content and antioxidant potential.


CF), geo-accumulation index (geoI), enrichment coefficients and transfer coefficient, and to determine the source of heavy metals pollution in the Wanjiang Economic Zone, Anhui Province, China. A total of 308 pairs of soil-crop samples were collected in this study, comprising 245 pairs of soil-rice samples, 53 pairs of soil-wheat samples, and 10 pairs of soil-corn samples. The concentrations of cadmium (Cd) and nickel (Ni) in the soil of the study area exceeded the national limitation of heavy metals in the soil of China . The concentrations of copper (Cu), zinc (Zn) and lead (Pb) were also above the national limits to a lesser extent. All eight heavy metals , chromium (Cr), and mercury (Hg)) exceeded the background values in the study area. The enrichment coefficients of rice, wheat and maize to Cd, Cu and Zn were higher than those to other elements. On the basis of geoI, it can be indicated that the rhizosphere soil of rice was slightly polluted by Cd and Hg, while the concentrations of the other heavy metals were below the safety limits. The CF and pollution load index (PLI) indicated that the soil in the study area was heavily contaminated with heavy metals. A principal component analysis identified different sources of soil heavy metal pollution, that is, Cu, Pb, Zn and Cd from industrial sources, Cr and Ni from natural sources, and As and Hg from agricultural sources. The carcinogenic risk of heavy metals was related to the intake of crops. Residents in the study area ingest rice, wheat, and corn on a daily basis. On the basis this study, it is suggested that local governments should pay attention to the carcinogenic risk of heavy metals in rice.Understanding the extent of contamination, sources and various carcinogenic and non-carcinogenic risks associated with different heavy metals in soil-crop systems is crucial for the prevention of heavy metal pollution. A survey was undertaken to determine heavy metal concentrations and degree of pollution in soil-crop systems  using various indices such as pollution factor ( Soil is a key element of life on earth. Heavy metals in soil are related to the quality and safety of agricultural products and directly or indirectly affect human health. As such, the study of heavy metal levels has attracted extensive attention. With the development of intensified agriculture and the excessive use of various chemical pesticides and fertilizers, the accumulation of heavy metals in farmland soil has become a serious problem . Heavy mIn recent years, a large number of studies ,16,17,18The Anhui section of the Yangtze River has superior agricultural resources, producing mainly rice, wheat, and corn ,20. Heav2 and includes 46 cities and counties. The main prefecture-level cities are Hefei, Wuhu, Anqing, Chizhou, Tongling, Maanshan, and Chuzhou. The study area has a subtropical, humid monsoon climate with an annual average temperature of 15.7~16.6 °C and annual average precipitation of 1500~1800 mm [The study area is located in the middle of Anhui Province, with geographic coordinates of 115°45′–119°23′ E and 29°34′–33°10′ N. The area comprises 75,800 km~1800 mm . The areMagmatic rocks, sedimentary rocks, and metamorphic rocks are distributed in the study area, with complex geological structures and favorable ore-forming conditions. Metal and nonmetal mineral resources are abundant in reserves. More than 1900 large-, medium- and small-sized deposits have been discovered, of which the Tongling ore concentration area is an important part of the iron, copper, sulfur, and gold metallogenic belt in the middle and lower reaches of the Yangtze River . The soiThe rational allocation of light-heat-water-soil resources in the study area is advantageous for agricultural production. The grain crops in the region are mainly rice and wheat, while the cash crops are mainly cotton, rape, tea, hickory nut, tree peony bark, and fresh ginger.The collection of all soil-crop system samples not only considered the actual planting situation of agricultural products but also met the uniformity requirements of the sampling space. We focused on collecting rhizosphere soil samples. From July to August 2017, 308 pairs of soil-crop samples were collected , comprisIn accordance with the methods for Regional Geochemical Sample Analysis (DZ/T0279-2016), a test protocol consisting of the following analytical methods was selected. The concentrations of six elements  were determined by X-ray fluorescence spectrometry (XRF). The concentration of Cd was determined by inductively coupled plasma mass spectrometry (ICP-MS). The concentrations of As and Hg were determined by atomic fluorescence spectrometry (AFS). The pH of the soil was determined by the ion selective electrode method (ISE).According to Analysis Methods of Animal and Plant Samples for Eco-geochemical Evaluation (DZ/T0253-2014), inductively coupled plasma spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS) are the main methods for the detection of heavy metals in crops. These methods may be supplemented by atomic fluorescence spectrometry (AFS) and colorimetry.In this study, the heavy metal pollution index, geo-accumulation index, and potential ecological risk index were used to evaluate the potential ecological risk level of heavy metal pollution in the soil-crop system. The enrichment coefficient method of heavy metals was used to evaluate the hazard of heavy metals to the ecological environment. Statistical methods such as principal component and correlation analyses were used to determine the sources of soil heavy metal pollution. The health risk assessment model was used to evaluate the health risk of heavy metals in the soil-crop system.EF is the enrichment factor of heavy metal elements in soil, iC is the concentration of heavy metals in soil, and refC is the concentration of reference elements in soil. More generally, elements with stable geochemical properties during the supergene process are selected as reference elements, while elements such as scandium (Sc), Mn, titanium (Ti), aluminum (Al), and Fe are often selected as reference elements in the crust [EF < 2 for low enrichment, 2 ≤ EF < 5 for moderate enrichment, 5 ≤ EF < 20 for significant enrichment, 20 ≤ EF < 40 for high enrichment, and EF ≥ 40 for extreme enrichment.Enrichment factors are often used to judge the enrichment levels and sources of heavy metals in soil ,25. The he crust . In thishe crust ,29. The he crust ,31: EF <geoI) was put forward by German scholar Muller. It reflects the potential pollution degree of heavy metals in sediment or soil [nC is the concentration of a heavy metals in soil, nB is the background value of a heavy metal element in local soil, and the constant 1.5 is the correction coefficient of natural fluctuation of heavy metal concentrations during diagenesis [geoI < 0 for no pollution; 0 ≤ geoI < 1 for no pollution to slight pollution; 1 ≤ geoI < 2 for moderate pollution; 2 ≤ geoI < 3 for moderate pollution to heavy pollution; 3 ≤ geoI < 4 for heavy pollution; 4 ≤ geoI < 5 for heavy pollution to extremely heavy pollution; geo ≥I 5 for extremely heavy pollution.Geo-accumulation index  and pollution load index (PLI) can be employed to characterize the pollution degree of soil. The formula of pollution factor (EF) is as follows:metalC is the concentration of the corresponding heavy metals in soil samples and backgroundC is the background value of heavy metals. The CF can be divided into four rankings: CF < 1 for low pollution; 1 ≤ geoI < 3 for moderate pollution; 3 ≤ geoI < 6 for serious pollution; CF ≥ 6 for extremely serious pollution.Pollution factor PLI=Cy metals .The single factor index method was used to evaluate the heavy metals pollution of crop seeds. The reference value is the safety value of the National Food Safety Standard Limits for Pollutants in Food (GB 2762-2017). The ratio of the concentration of elements in crop seed samples to the limits specified in the national food safety standards reflects the concentration of heavy metals.BCF), which represents the distribution law of element concentration between soil and crops, is a basic index to estimate the quality of the soil environment. It has direct significance in evaluations of the ecological safety of soil and agricultural products. The calculation formula is as follows [pC is the concentration of a heavy metal element in crops and sC is the concentration of heavy metal elements in the soil.The relationship between the concentration of elements in crops and the concentration of elements in the soil environment in which the crops live is an important factor by which to investigate whether crops can safely be grown in the soil. The biological concentration factor ( follows :(5)BCF=CRI is the comprehensive ecological risk index; RI < 150 for insignificant; 150 ≤ RI < 300 for moderate, 300 ≤ RI < 600 for severe, and RI ≥ 300 for serious.On the basis of the interaction between the physical and chemical properties of heavy metals and the environment, Hakanson  proposedectively . The potADI) of heavy metals in different exposure pathways is as follows:ADI of direct oral intake, respiratory inhalation, and skin contact, respectively, in mg/(kg·d), and iC is the concentration of heavy metals in mg/kg. The meanings and values of other parameters are listed in Human health risk assessments are commonly used to quantify potential human health risks from exposure to certain heavy metals. In general, heavy metals present carcinogenic and non-carcinogenic risks to humans through direct oral intake, skin contact, and inhalation. In this study, the human exposure risk assessment model recommended by the U.S. Environmental Protection Agency (USEPA) was used to evaluate the carcinogenic and non-carcinogenic risks to adults and children in the study area. The calculation formula for average daily intake , presented in ijHQ or HI < 1, the non-carcinogenic health risk is not significant, i.e., it presents an acceptable risk. When ijHQ or HI > 1, there is a non-carcinogenic risk.Health risks are divided into non-carcinogenic and carcinogenic, among which the non-carcinogenic health risk index ijCR is the single carcinogenic risk index, ijSF is the slope factor in mg/(kg·d), and TCR is the total carcinogenic risk of multiple heavy metals. When ijCR or ij <CR 10−6, there is no carcinogenic risk, and when ijCR or ijCR is between 1 × 106~1 × 104, there is an acceptable risk.The carcinogenic risk index is calculated as follows:The ArcGIS10.2 software  was employed to draw the sampling distribution map. Data processing and descriptive statistics were performed in the Origin 2021 software , and SPSS 20.0 software  was used for correlation and principal component analyses to determine the sources of heavy metals in soil.In comparison to the environmental background values of heavy metals in the soil in the study area , except The changing factors of heavy metal concentrations in rhizosphere soil of different crops were: Cu 194.33%) > Pb (92.44%) > Cd (91.72%) > As (75.53%) > Ni (59.63%) > Hg (51.05%) > Zn (43.56%) > Cr (35.55%) in rice rhizosphere soil, Ni (114.52%) > Hg 91.47%) > Cd 70.48%) > Cr (63.34%) > As (47.06%) > Cu (33.35%) > Zn (30.91%) > Pb (20.91%) in wheat rhizosphere soil and Cu (90.63%) > Zn (65.09%) > As (58.98%) > Pb (47.29%) > Hg (46.07%) > Cd (27.95%) > Cr (25.51%) > Ni (17.51%) in maize rhizosphere soil. The degrees of heavy metal concentrations in crop rhizosphere soil were as follows rice rhizosphere soil > wheat rhizosphere soil > maize rhizosphere soil. Among them, Cu in rice rhizosphere soil was abnormally strong, while Pb, Cd, As, Ni and Hg were strong variations; Ni in wheat rhizosphere soil was abnormally strong, while Hg, Cd, and Cr were strong variations. No abnormal variations were observed in maize rhizosphere soil, where only Cu, Zn and As were strong variations. This indicates that the change of heavy metal concentrations in crop rhizosphere soil in the study area were not only affected by geological background, but also by human activities, although needs to be further verified by source analysis [% > Cd (7.33% > Pb% > Cr (6geoI) of the study area are displayed in geoI) of other elements was significantly below 1. According to Muller’s grading standard [geoI of heavy metals in rhizosphere soil was much lower than stated in [The calculation results of the geo-accumulation index  of rhizosphere soil in the study area is displayed in EF < 2 indicates that soil elements are enriched compared with the reference background. In contrast, EF > 2 indicates that soil-forming elements are enriched or influenced by some human input [EF value may point to human influence [EF values of Cd, Hg, Zn, and Cu in rice rhizosphere soil and Cu in maize rhizosphere soil in the study area were greater than 2. The sources of these heavy metals can be attributed to human activities, while the rest of the elements in rhizosphere soil were likely of natural origin. The results show that the rhizosphere soil in the study area is polluted by heavy metals , mainly as a result of human activities.The enrichment factor (an input . The enrnfluence . The avePLI) of rice rhizosphere soil, wheat rhizosphere soil, and maize rhizosphere soil in the study area were 1.44, 1.21, and 1.23, respectively, and the pollution load index was greater than 1, which indicated that the soil in the study area had been polluted by heavy metals.The calculation results of contamination factors in the study area are displayed in RI) in the study area are displayed in RI) of rice rhizosphere soil, wheat rhizosphere soil, and maize rhizosphere soil in the study area were 220.28, 133.53, and 151.74, respectively, which indicated that the heavy metals in the study area were at moderate pollution levels. Relevant research has indicated that the use of chemical fertilizers and pesticides can present high ecological risks in agricultural soils [RI was due to the high Cd concentration in soil [The basic statistical data of potential ecological risk factor (ER) and ecological risk index  (p < 0.01), likewise indicating that they may have similar sources. There was a significant negative correlation among Hg, Cr and Ni (p < 0.01), which indicated that Hg had different sources from Cr and Ni. In addition, there were no correlations between (i) Cr, Cd, and Pb, (ii) Ni and Cu, and (iii) Pb and As. This may indicate that these heavy metals had similar or different sources.The relationships among heavy metals can provide important information about the sources of such pollutants . There w < 0.01) , with a p < 0.01). The data were therefore considered to be suitable for principal component analysis. As the characteristic value was greater than 1, three principal components were extracted , the loads of Cu, Pb, Zn, and Cd were the highest; the variance contribution rate was 28.4192%. According to the correlation analysis, there were significant correlations among four elements: Cu, Pb, Zn, and Cd. Relevant research has indicated that industrial waste gas and automobile exhaust emissions are the main sources of heavy metal pollution in the atmosphere. Heavy metal elements in the atmosphere enter agricultural soil by wet and dry deposition, leading to the increase of Cd, Cu, Pb, and Zn contents in soil . For exap < 0.01). Relevant research has also indicated that the concentrations of Cr and Ni in soil are similar to those in parent materials, indicating that their presence was related to diagenetic components as opposed to human activities [The loads of Cr and Ni were the largest for PC2, with a variance contribution rate of 26.028%. Cr in the study area was largely absent. A correlation analysis indicated that Ni and Cr had a significant correlation (0.96) , heavy metals in other crops were within the relevant limits. At a single sample point, 12.24%, 18.78%, 2.45%, and 1.22% of samples exceeded the national safety thresholds for Ni, Cd, As and Hg, respectively, in rice seeds. Additionally, Pb and Cd in wheat and maize also exceeded the standard.Except for the fact that the concentration of Pb in maize seeds slightly exceeded the limit of national food safety standard , while Ni (81.41%) and Hg (58.30%) showed strong variation and all other pollutants moderate variation. In wheat seeds, Ni (68.74%), Cd (69.97%) and Hg (71.52%) indicated strong variation, while other elements indicated moderate variation. All of the tested heavy metal elements showed moderately strong variation in maize seeds. The above results indicate that there may be abnormal Cd enrichment in rice seeds.BCFs) of rice, wheat, and maize relative to soil heavy metals are displayed in The bio-concentration factors (HQ) and the total non-carcinogenic risks (HI) of heavy metals in soil through oral intake, skin contact, and respiratory inhalation for adults and children are displayed in The single non-carcinogenic health risks index , all HIs were less than 1, indicating that there were no non-carcinogenic risks except with wheat rhizosphere soil. In addition, it was also found that the role of As in rice rhizosphere soil, wheat rhizosphere soil, and maize rhizosphere soil was the most obvious, accounting for 48.71%, 45.91%, and 50.90% of adults and 47.70%, 44.80%, and 50% of children, respectively.Except for children’s The carcinogenic risk indexes of As, Ni, Pb, Cd, and Cr in the rhizosphere soil of the study area are displayed in CR) of heavy metals for adults and children in different rhizosphere soils, from high to low, were both as follows As > Cd > Cr > Pb > Ni (TCR) for children and adults in three rhizosphere soils were as follows: 2.13 × 10−5, 1.45 × 10−5; 1.93 × 10−5, 1.36 × 10−5 and 1.73 × 10−5, 1.17 × 10−5 respectively. TCRs were within the maximum acceptable levels (10−6~10−4) recommended by USEPA. The results indicated that for all humans, considering single heavy metal or multiple heavy metal elements, the CR and TCR values of all samples were lower than the maximum acceptable levels as stated by USEPA, indicating that there was no obvious long-term health risk impact. Therefore, the lifetime carcinogenic risk of heavy metals in rhizosphere soil to children and adults was within the acceptable risk range.The carcinogenic risk indexes  of eight heavy metals in rice, wheat, and maize for adults and children were all greater than 1, i.e., 6.26, 8.72, 4.75, 7.04, 4.22, and 6.34, respectively. The results indicated that the intake of rice in the study area could cause great harm to residents. Compared with adults, the comprehensive non-carcinogenic hazard coefficient of heavy metals in children was higher, i.e., 1.50, 1.39, and 1.48 times those for adults, respectively. This indicated that the non-carcinogenic risk of intake by children of crops in the study area was more serious, which is in line with results reported from other areas [The comprehensive hazard coefficients  and total carcinogenic risk (TCR) of adults and children due to heavy metal intake resulting from the consumption of crops in the study area are displayed in The evaluation and calculation results of the individual carcinogenic risk index  recommended by USEPA. It can be seen that the daily intake of rice, wheat, and maize by residents in the study area poses certain carcinogenic risks, and thus, the carcinogenic effects of heavy metals in rice on human health should be carefully studied.The total carcinogenic risk to children and adults by ingesting rice, wheat, and maize were, respectively: 9.40 × 10In this paper, the pollution characteristics and ecological health risk posed by heavy metals in the soil-crop system in Wanjiang Economic Zone were studied. Compared with the concentrations of heavy metals in China’s soils elsewhere, Cd and Ni in the studied soils mainly exceeded the standard values. Cu, Zn and Pb also exceeded the standards to some extent, and all eight elements exceeded the background values for the study area. The concentrations of Ni, Cd, As and Hg in crop seeds exceeded the standard. The bio-concentration factors indicated that rice, wheat, and maize had higher enrichment coefficients for Cd, Cu and Zn, while other elements had lower bio-concentration factors.geoI) indicated slight pollution from Cd and Hg in rice rhizosphere soil and Cu, Cd, and Zn in maize rhizosphere soil, while other heavy metals were largely absent. The enrichment factor (EF) indicated that Cd, Hg, Zn, and Cu in rice rhizosphere soil and Cu in maize rhizosphere soil were significantly enriched. The contamination factor (CF) and pollution load index (PLI) indicated that the soil in the study area had been polluted by heavy metals. The potential ecological risk assessment (RI) for heavy metals indicated that the soil in the study area was at a moderate pollution level.The geo-eaccumulation index (A correlation analysis indicated that the heavy metals in the soil in the study area had similar or different sources. The results of a principal component analysis allowed us to identify three sources: Cu, Pb, Zn and Cd originated from “industrial sources”, Cr and Ni from “natural sources”, and As and Hg from “agricultural sources”.The USEPA model was used to evaluate the health risks of soil and crops. There was no non-carcinogenic risk of heavy metals in the rhizosphere soil in the study area. The TCR results also indicated that the lifelong carcinogenic risk of heavy metals in the rhizosphere soil of the study area to children and adults was within an acceptable risk range. However, consuming crops grown in the study are will pose a non-carcinogenic risk due to the presence of heavy metals. In addition, the daily intake of rice, wheat, and maize by residents in the study area poses certain carcinogenic risks. In particular, the study area needs to focus on the carcinogenic effects of heavy metals in rice on human health.