In the present study, we aimed to develop a novel isotretinoin delivery model for treating skin diseases, revealing its potential advantages in drug delivery and targeted therapy.Using a self-assembly strategy, we grafted a dendrimer, based on a well-defined branched structure for nanomedical devices, with a well-defined nanoarchitecture, yielding spherical, highly homogeneous molecules with multiple surface functionalities. Accordingly, a self-assembled dendrimer-conjugated system was developed to achieve the transdermal delivery of isotretinoin (13cRA-D).Herein, 13cRA-D showed remarkable controlled release, characterized by slow release in normal tissues but accelerated release in tissues with low pH, such as sites of inflammation. These release characteristics could abrogate the nonteratogenic side effects of isotretinoin and allow efficient skin permeation. Moreover, 13cRA-D exhibited high therapeutic efficacy in acne models. Based on in vitro and in vivo experimental results, 13cRA-D afforded better skin penetration than isotretinoin and allowed lesion targeting. Additionally, 13cRA-D induced minimal skin irritation.Our findings suggest that 13cRA-D is a safe and effective isotretinoin formulation for treating patients with skin disorders. Isotretinoin  is the first-choice treatment for severe acne . HoweverRecently, transdermal delivery approaches have substantially contributed to the field of medicine . HoweverDendrimers are hyperbranched materials that belong to a new class of polymers with unique properties –22. DendIn the present study, we synthesized dendrimer-conjugated isotretinoin (13cRA-D) and evaluated its ability to reduce side effects, enhance transdermal drug delivery and improve the therapeutic effect of isotretinoin.2. Imiquimod cream was purchased from Sichuan Mingxin Pharmaceutical . The hydrophilic oligoether dendrimer was synthesized as reported previously [Isotretinoin was obtained from APExBIO Technology  and applied to cells under dimmed yellow light. Advanced MEM, phosphate-buffered saline , antibiotic–antimycotic solution, fetal bovine serum (FBS), and l-glutamine solution (100 mM) were purchased from Sigma-Aldrich . The SZ95 human sebaceous gland cells  were culeviously –29.1H NMR spectra at 500 MHz. We used parts per million (ppm) to express the chemical shifts compared to the tetramethylsilane or residual solvent peaks . The indices s (singlet), d (doublet), and t (triplet) represent the multiplicity. Hz indicated the coupling constants.An FT-NMR spectrometer was used to obtain 4 vapor, generated by reacting a mixture of RuCl3⋅3 H2O (0.2 g) and 5% NaClO (10 mL) for 5 min. A JEOL JEM HR2100 instrument operating at 120 kV was used to observe the dried specimens.To examine the self-assembled constructions, one drop of each sample solution was placed on a 200 mesh carbon-coated copper grid with Formvar  and then evaporated under surrounding conditions. We then performed negative staining of samples by depositing a drop of uranyl acetate aqueous solution (0.2–0.4 wt %) on the grid surface of loaded samples. For positive staining, the sample-loaded grids were exposed to RuO2 atmosphere. Finally, absorbance was measured at 450 nm using a plate reader.Dendrimer toxicity was evaluated using Cell Counting Kit-8 (CCK-8). Prepared SZ95 sebocytes were treated with gradient concentrations of the dendrimer  for 24 h. Next, CCK-8 reagent was added to the plates and incubated with cells for 1 – 2 h at 37 ℃ and under a humidified 5% CO4. After the filtrate was condensed under reduced pressure, the crude product was purified by silica gel flash column chromatography using methanol:ethyl acetate (1:50) as the eluent to yield a 26% (100 mg) yellow liquid.A mixture of 300 mg isotretinoin , 290 mg dendrimer (0.3 mmol), 45 mg 4-dimethylaminopyridine (0.37 mmol), and 82 mg dicyclohexylcarbodiimide (0.4 mmol) was maintained under stirring in dry toluene (8 mL) at 0 ℃ for 1 h. The reaction was performed for 3 days. After completion of the reaction, the reaction mixture was quenched with water and extracted using ethyl acetate following thin-layer chromatography. The brine used to wash the organic layer was then dried over anhydrous MgSOWe analyzed 13cRA-D release in vitro using a dialysis membrane (molecular weight cutoff = 1000 kDa). 13cRA-D was sealed in a dialysis bag and immersed in methanol (pH 5.0 or 7.0) at 37 ℃. Methanol was collected at 0.5, 1, 2, 4, 8, 12, 24 and 48 h. Then, methanol was measured using an ultraviolet–visible spectrophotometer to assess drug release. After the removal of each sample, 1 mL of fresh solvent was added to maintain a constant volume.Animal experiments were performed according to the guidelines for the Care and Use of Experimental Animals of Jilin University and were approved by the Animal Experiment Ethics Committee of Jilin University.2) and soaked in PBS at 37 ℃ for 1 h before use. Experiments were performed using a Franz diffusion cell at 37 ℃ and 100 rpm, avoiding light exposure. Then, 30% isopropanol and 70% PBS were added to receptor cells. The donor cells contained PBS, 0.25 mg/mL 13cRA, an equal weight of 13cRA-D comprising 0.25 mg/mL 13cRA or an equal weight of dendrimer. The prepared skin was placed between the receptor and donor cells. Samples were collected from the side arms at 2, 4, 6, 8, and 10 h to perform high-performance liquid chromatography (HPLC) measurements. All experiments were performed in triplicate.For transdermal testing, we employed the skin of Yorkshire pigs (1-month-old). We carefully excised the subcutaneous tissue to obtain an intact epidermis and then stored the skin at − 80 ℃ until use. The skin was cut into slices  and homogenized in a 20 mL mixture of chloroform:methanol (1:2) to extract the retained drug, which was measured using HPLC.Following the skin penetration study, skin samples were cut into small pieces (~1 mm × 1 mm) and fixed in 4% paraformaldehyde overnight. Next, the samples were embedded in paraffin and sliced into approximately 4 μm tissue sections. Finally, mounted sections were observed under a fluorescence microscope, and images were analyzed using ImageJ software .The notum hair of 12 BALB/c mice  was carefully removed, ensuring the epidermis remained intact. Mice were randomly divided into four groups treated with PBS, dendrimer, 13cRA or 13cRA-D, applied to the exposed skin; the applied substance was carefully removed with cotton after 6 h. Finally, the skin was scored (0–4) according to a published standard for evaluation [To construct the Kligman acne model , we applWe randomly divided 20 female BALB/c mice (20–30 g) into four groups and removed notum hair using a depilatory cream. Imiquimod cream  was evenly applied daily to 15 of the 20 mice for seven days. We applied gentle pressure for 30 min until the cream was fully absorbed. Six hours after each application, PBS, 13cRA or 13cRA-D was applied for 30 s. Subsequently, we assessed the skin for gross morphological and histopathological changes. Skin inflammation severity was assessed using the Psoriasis Area Severity Index (PASI) . Subsequt-tests, followed by the least significant difference post hoc test. A p-value < 0.05 indicated a statistically significant difference between groups.Data are presented as mean ± standard deviation (SD). Statistical analyses were performed using analysis of variance (ANOVA) or two-sample Student’s 1H-NMR and was in full agreement with the expected chemical structure. 13cRA-D is amphiphilic, given the presence of hydrophobic isotretinoin and a hydrophilic dendrimer, and is capable of self-assembling into nanostructures in an aqueous solution comprising a hydrophobic isotretinoin core surrounded by hydrophilic dendrimer segments. The ester linkage grafting of isotretinoin and the hydrophilic dendrimer can be cleaved by altering pH from neutral pH, thereby facilitating isotretinoin release after tissue delivery at low pH.Figure 1H NMR  δ 7.66 , 7.07 , 6.34–6.16 , 5.69 , 4.07 , 3.54–3.34 , 3.23 , 2.15 , 2.09–1.95 , 1.69 , 1.62–1.53 , 1.48–1.41 , 1.02 .13C NMR  δ 140.55, 137.60, 137.42, 134.47, 133.57, 133.11, 131.19, 130.11, 129.39, 128.90, 126.86, 116.45, 71.77, 70.45, 70.33, 70.28, 70.14, 70.09, 69.27, 69.02, 58.52, 40.65, 40.45, 40.24, 40.03, 39.82, 39.61, 39.40, 34.33, 33.11, 29.27, 21.98, 20.94, 19.19, 13.12.The formation of self-assembled isotretinoin amphiphile nanostructures was examined using negatively-stained TEM images Fig. . The imaAs assessed using the CCK-8 assay, the dendrimer mildly promoted the proliferation of sebaceous gland cells at concentrations ranging from 0.025 to 0.4 mg/mL, with the most notable effect observed at 0.025 mg/mL (p = 0.0400, Fig. Given that acids can affect ester hydrolysis, we performed drug release studies at two physiologically relevant pH levels. We found that 13cRA-D exhibited excellent controlled release properties Fig. A, with iAs shown in Fig. Isotretinoin accumulation in the skin is crucial to treat skin diseases. Based on fluorescence microscopy assessments Fig. A, B, 13cTo evaluate the degree of irritation caused by PBS, dendrimers, 13cRA and 13cRA-D, we compared the severity of skin erythema after the application of respective compounds. Six hours post-application, we observed that 13cRA-D induced less skin irritation than free isotretinoin Fig. . No erytAfter 14 days of treatment, the 13cRA-D group showed almost no comedones, whereas 13cRA and untreated groups showed slight to moderate comedones Fig. A. Based As shown in Fig. Dendrimers can reportedly increase transdermal permeation and specific targeting of drugs . The tetOur skin permeation analysis demonstrated that 13cRA-D had better transdermal penetration than free isotretinoin, which poorly permeates the stratum corneum. 13cRA-D was present on the exterior surface of the dendrimer as a lipid-like hydrophilic structure, which is ideal for use as a drug delivery vehicle. The physical characteristics of 13cRA-D enabled it to reach the desired target more quickly and efficiently than free isotretinoin. In addition, 13cRA-D facilitated isotretinoin retention in the skin. The permeation and retention of a high drug concentration in the skin can be beneficial for treating several skin disorders. Importantly, uniform distribution may reduce side effects and enhance treatment efficacy.In vivo, 13cRA-D effectively reduced skin irritation, indicating its potential as a topical formulation that minimizes isotretinoin-related side effects. In acne and psoriasis models, 13cRA-D significantly improved treatment efficacy. Accordingly, 13cRA-D may represent a new therapeutic option for multiple skin disorders, including acne and psoriasis.The 13cRA-D formulation could control isotretinoin release, which is an ideal feature of a drug carrier. The dendrimer and isotretinoin are connected by a hydrolyzable ester bond, which is easier to break in an acidic environment than in a neutral one. Typically, the inflammatory environment is acidic owing to the accumulation of inflammatory factors ; therefo13cRA-D can efficiently permeate the skin and accelerate isotretinoin release at the site of inflammation.In recent years, considerable efforts have been made to develop topical delivery systems for nano- and microparticles. Wang et al. loaded isotretinoin nanoparticles into human umbilical cord mesenchymal stem cells and showed that these nanoparticles efficiently controlled isotretinoin release and reduced side effects . Future Collectively, we demonstrated the benefits of coating isotretinoin with dendrimers for drug delivery and disease treatment. 13cRA-D controlled isotretinoin release, improved skin distribution, reduced side effects, and permeated the skin efficiently, positively impacting therapeutic efficacy. Accordingly, we demonstrated a new approach for isotretinoin application that could enhance the biological characteristics of isotretinoin and provide a more precise treatment of acne and psoriasis.


However, the anisotropic ion transport in layered structures and the poor intrinsic conductivity of MoS2 lead to unacceptable ion transport capability. Here, we propose in-situ construction of interlayer electrostatic repulsion caused by Co2+ substituting Mo4+ between MoS2 layers, which can break the limitation of interlayer van der Waals forces to fabricate monolayer MoS2, thus establishing isotropic ion transport paths. Simultaneously, the doped Co atoms change the electronic structure of monolayer MoS2, thus improving its intrinsic conductivity. Importantly, the doped Co atoms can be converted into Co nanoparticles to create a space charge region to accelerate ion transport. Hence, the Co-doped monolayer MoS2 shows ultrafast lithium ion transport capability in half/full cells. This work presents a novel route for the preparation of monolayer MoS2 and demonstrates its potential for application in fast-charging lithium-ion batteries.High theoretical capacity and unique layered structures make MoSThe online version contains supplementary material available at 10.1007/s40820-023-01042-4. Over th2, etc.) –9, trans2, etc.) , metal s3, etc.) , SiOx (x (x < 2) –14, and  (x < 2)  have bee2 with interlayer spacing of 0.62 nm and lithium-ion storage capacity of 670 mAh g−1 looks very attractive [2 nanostructure [2 interlayer spacing [2 [2 layers number [2/carbon [2, the anisotropic ion transport features due to the native layered structure of MoS2 are not fundamentally changed, which inevitably leads to the same ion transport capability. The fabrication of monolayer MoS2 can be considered as the most effective route to circumvent the tricky problem due to the completely open ion transport pathway owing to the disappearance of layered structure [2 can be divided into two categories: (i) substrate growth, including chemical vapor deposition [2 membrane, not powder, which is inappropriate as LIB anode. The latter can achieve monolayer MoS2 powder but has low monolayer performance. Furthermore, the obtained monolayer MoS2 powder, which suffers from severe agglomeration, must be dispersed in solution and their poor EC is not improved, which is disadvantageous for application in fast-charging LIBs. In order to handle this tricky problem, the interoverlapped structure of monolayer MoS2 and monolayer carbon has been successfully constructed by solvothermal [2 agglomeration and improve its EC. However, such interoverlapping structure reintroduces anisotropic ion transport features between monolayer MoS2 and monolayer carbon, which is unfavorable for the full reflection of intrinsic ion transport capability of monolayer MoS2. With the aim of solving this problem, monolayer MoS2/carbon composites, in which monolayer MoS2 is uniformly dispersed into carbon matrix, have been synthesized by electrospinning [−1 at 0.1 A g−1) and good rate capability (60.6% capacity retention at 10 A g−1 relative to the capacity obtained at 0.1 A g−1). Although the electrochemical performances of MoS2 have been improved in these researches, some problems are still waiting to be solved. Generally, under the same areal capacity, higher specific capacity makes active materials with less mass loading, thus resulting in thinner electrodes, which certainly increases the ion transport rate throughout the electrode. However, the addition of low-capacity carbon materials inevitably reduces the capacity of the composites, which has an adverse effect on rate performances. The introduction of active materials with high capacity has been the key to solving this problem. Additionally, the intrinsic semiconductor properties of monolayer MoS2 have not been fundamentally changed, which certainly decreases ion transport capability in basal plane of monolayer MoS2. For these problems, it is vital and urgent to seek a new preparative technology of monolayer MoS2 to spur monolayer MoS2 application in fast-charging LIBs.MoStractive . Based otructure , 17, enl spacing , doping acing [2 , reducins number , and fab2/carbon  have beetructure . The curposition , epitaxyposition ,magnetroposition , etc.; . As LIB anode, the CoMoS2/C has the following advantages: i) monolayer MoS2 with fully exposed basal planes has an open Li+ transport path owing to the disappearance of anisotropic ion transport path in layered structure, thus ensuring ultrafast lithium-ion transport capability; ii) a maximized contact area of monolayer MoS2 and C can sufficiently improve the EC of MoS2, thus accelerating the charge transfer rate; iii) Co doping fundamentally changes the electronic structure of monolayer MoS2 to enhance its intrinsic EC, thereby remarkably accelerating the charge transport; iv) Co-doped monolayer MoS2 can be transformed into small superparamagnetic Co and Mo nanoparticles (~ 2 nm) during the conversion reaction, which can create a space charge region to accelerate charge transfer. The i-iv superiorities enable CoMoS2/C with ultrahigh capacity (1512.9 mAh g−1 at 0.1 A g−1) and ultrafast ion transport capability (1063.3 mAh g−1 at 20 A g−1) in half cells. Moreover, an energy density of 136.2 Wh kg−1 is acquired at 4 C in full cells with 76.6% retention corresponding to 0.1 C. Our work indicates that the in situ construction of electrostatic repulsion in the interlayer is a very effective route for the fabrication of monolayer MoS2 and reveals the application potential of Co-doped monolayer MoS2 in fast-charging LIBs.Herein, we report a facile route that Co4)2MoS4, and N,N-dimethylformamide (DMF) in a fixed total mass of 1.6 g with different mass ratio of 0:1:0, 0:1:3, 3:4:9, 5:4:7, and 7:4:5 to synthesize MoS2, MoS2/C, CoMoS2/C-I, CoMoS2/C-II, CoMoS2/C-III, respectively, in a self-made device that can be sealed. The devices were heated to 520 °C for 20 min in a tube furnace with an Ar flow and then experienced natural cooling.The samples were fabricated using mixed precursor of Cobalt bis (2-ethylhexanoate), (NH+), Raman spectroscopy (Horiba LabRam HR Evolution), electron spin resonance spectroscopy , photoluminescence spectra , Brunner–Emmet–Teller method , transmission electron microscopy  with an energy-dispersive spectroscopy (EDS), and physical property measurement system .The obtained materials were tested by scanning electron microscopy , thermogravimetric analysis , elemental analyzer , X-ray diffraction , X-ray photoelectron spectroscopy , and polyvinylidene fluoride (PVDF) with a mass ratio of 8:1:1 on Cu foil and dried at 100 °C under vacuum with a time of 12 h. Working electrode, counter/reference electrode (lithium foil), separator (Celgard 2400), and electrolyte were assembled in 2032 coin-type cells, with the active mass of 1.5 mg cm−2. The lithium-ion storage performances were tested by Land CT2001A battery system at 0.1–20 A g−1 with a voltage window of 0.01 to 3 V (vs. Li/Li+). A CHI 760D electrochemical workstation was used to test cyclic voltammetry (CV) at 0.1–1 mV s−1 with the same voltage window and electrochemical impedance spectroscopy (EIS) from 105 to 10−2 Hz with 5 mV amplitude.The working electrodes were made by spreading slurries of MoS4 cathode made by spreading the slurries of 95.0 wt% LiFePO4, 2.5 wt% PVDF, and 2.5 wt% AB on Al foil and CoMoS2/C-II anode with a N/P ratio of ~ 1.06, in which the active mass loading of anodes and cathodes are 2.0 and 16.4 mg cm−2, respectively. Other components are the same as those of half cells. Before packaging, the anode was pre-cycled (3 cycles at 0.1 A g−1) to ameliorate its first Coulombic efficiency (CE). The performances were tested at 0.1–4 C (1 C = 170 mA g−1) with a voltage window of 1.0 to 4.0 V at 30 °C.Coin-type full cells were assembled with LiFePO− and Li+ are detailed by Projected Augmented-Wave potentials, meanwhile exchange–correlation interactions are calculated by executing Perdew–Burke–Ernzerhof pseudopotentials of Generalized Gradient Approximation. DFT-D3 method is applied to determine the van der Waals (vdW) interaction. Plane-wave energy cutoff and convergence threshold are considered as 450 eV, and 1.0 × 10−5 eV in energy and 0.02 eV per Angstrom in force, respectively. Brillouin zone is sampled with 4 × 4 × 1 k-points. A vacuum space of 3 nm is inserted in z direction to prevent interactions between periodic images. Migration energy barrier of Li+ is calculated by executing climbing image nudged elastic band (CI-NEB) method, and the force is approximate to 0.3 eV nm−1.Density functional theory (DFT) is executed in view of Vienna Ab-initio Simulation Package. Interactions between e2 exhibits bulk morphology with size of over ten microns . Clearly, MoS2/C exhibits spherical shape with sub-micron size and maintains the morphology of nanosheets . This indicates that MoS2 is not completely embedded in carbonaceous materials due to the low mass of carbon materials arising from DMF [2/C samples show no obvious flaky morphology  due to the increase in mass of carbon materials (confirmed by TGA curves below) arising from cobalt bis (2-ethylhexanoate) and DMF, which fully wraps the MoS2 nanosheets. Clearly, the CoMoS2/C-III also exhibits the appearance of a peculiar bulk with a size of ~ 200 nm marked with yellow circles , which is attributed to the formation of Co3S4 (confirmed below) due to the excessive addition of C16H30CoO4. The microstructures are observed via TEM, in which the pure MoS2 is multilayered (~ 15 layers) with a d-spacing of 0.62 nm . After bringing in DMF, few-layered MoS2 (~ 5 layers) with an enlarged d-spacing of ~ 0.96 nm and distributed into the carbon substrate is obtained in MoS2/C . After drawing into a small amount of C16H30CoO4, few-layered MoS2 (~ 5 layers) with a larger d-spacing (1.16 nm) and distributed into the carbon substrate are attained in CoMoS2/C-I . As the amount of C16H30CoO4 continues to increase, the intrinsic layered structure of MoS2 completely disappears, so monolayer MoS2 is formed in CoMoS2/C-II . The results indicate that monolayer MoS2 can be successfully fabricated by varying the amount of organic ionic liquid (C16H30CoO4 containing one positively charged Co2+ and two negatively charged (CH3)2C(CH2)4COOH−) in the solution of (NH4)2MoS4 and DMF. It should be noted that the source mass of Co and DMF is simultaneously changed, which is mainly from the perspective of ensuring the experimental safety and the success of monolayer MoS2 synthesis. Since the maximum capacity of our reaction device is 5 mL, according to our experience in vapor-phase high-pressure reaction, the total volume of added liquid cannot exceed 30% of the total capacity. Otherwise, the pressure of the vapor phase generated by liquid pyrolysis would exceed the maximum resistance pressure of the device, which causes safety hazards. In addition, the pressure of the vapor phase generated in the reaction is also important for the synthesis of monolayer MoS2. Although the mass ratio of cobalt bis (2-ethylhexanoate)/(NH4)2MoS4/DMF remains unchanged, the decrease in total mass leads to a low vapor pressure that is insufficient to form monolayer MoS2 . Therefore, appropriate total precursor mass and liquid volume are very important for experimental safety and synthesis of monolayer MoS2. Based on the above discussion, the samples are synthesized with the same total mass of the precursors, and with the increase in the mass of Co source, and the mass of DMF decreases.Figure S1 displays the morphology of the obtained samples, where pure MoSfrom DMF . Differe2 should be explored, as shown in Fig. 4)2MoS4, DMF, and C16H30CoO4 starts 2MoS4 can be decomposed into NH3, H2S, and MoS2 [3, etc. [16H30CoO4 can be decomposed into OHC·, ·CH3, etc. The production of vast gases and gaseous groups leads to high vapor pressure in the sealed vessel [2+ into the nucleating and growing MoS2 interlayers ) of one Co2+ provides two negative charges in the interlayer and two loads on MoS2 layers, leaving four charges for the model. The electrostatic repulsions are triggered via the accumulated negative charges to augment d-spacing of MoS2. MoS2 retains an interlayer spacing of 0.62 nm  is slightly higher than 1.16 nm of CoMoS2/C-II under a similar amount of Co doping (based on XPS results below). This may be due to the volume shrinkage arising from the conversion from gas to solid phase under high vapor-phase pressure and the incomplete insertion of negatively charged gaseous groups in the interlayer. Continuing to increase the amount of Co atoms and negative charges in the model , the interlayer distance is further increased to 2.36 nm , where the diffraction peaks at ~ 14.2, 33.5, and 58.5° correspond to (002), (100), and (110) of MoS2 crystal planes, respectively. Clearly, the diffraction peak of the (002) crystal plane for MoS2/C shifts to 9.0°, which corresponds to d-spacings of ~ 0.96 nm calculated by Bragg’s Law (2d sinθ = nλ), further confirming the enlargement of the interlayer spacing. Obviously, compared with the MoS2/C, the diffraction peak of the (002) plane of the CoMoS2/C-I shifts to 7.8°. This indicates a larger interlayer spacing (1.16 nm), confirming the role of Co doping enhancement in increasing the d-spacings. Interestingly, the (002) plane of CoMoS2/C-II and CoMoS2/C-III completely disappears, which belongs to the feature of monolayer MoS2 [2. Besides, the CoMoS2/C-III shows the peaks of Co3S4 (JCPDS No. 02-1338) to further testify the formation of Co3S4. Clearly, no obvious diffraction peaks of carbon materials appear in the XRD pattern, which indicates that the carbon materials are amorphous. Figure E12g (~ 378 cm−1) and A1g (~ 401 cm−1) Raman peaks of MoS2 [2. Besides the pure MoS2, all other samples show the peaks of disordered carbon  and ordered graphitic carbon  of carbon materials [E12g and A1g vibrations are related to the layer number of MoS2, which are 27.2, 24.6, 23.7, 20.6, and 20.6 cm−1 for pure MoS2, MoS2/C, CoMoS2/C-I, CoMoS2/C-II, and CoMoS2/C-III, respectively . In previous reports [−1 corresponding to the multilayered, few-layered (3–6 layers), bilayered, and monolayer MoS2, respectively. The results further demonstrate the presence of multilayered MoS2 in pure MoS2, few-layered MoS2 in MoS2/C and CoMoS2/C-I, and monolayer MoS2 in CoMoS2/C-II and CoMoS2/C-III. Moreover, the disorder degree of graphitic structure can be validated by the intensity ratios of D and G peaks (ID/IG) [2/C, CoMoS2/C-I, CoMoS2/C-II, and CoMoS2/C-III, respectively. Noticeably, the ID/IG ratio shows an upward trend with increasing C16H30CoO4 amount. This may be because the carbon-containing gaseous groups arising from the pyrolysis of C16H30CoO4 have a greater tendency to transform into disordered carbon under high vapor pressure, thus increasing the ratio ID/IG. So high ID/IG values suggest the presence of extensive defects in carbon materials. Moreover, the defects in MoS2 can be evidenced by the ESR  and PL spectra . Compared with pure MoS2, the ESR spectra of MoS2/C and CoMoS2/C show a stronger characteristic peak intensity (g = 2.003). This confirms the presence of abundant non-intrinsic defects in MoS2 [2/C and CoMoS2/C have a lower emission peak intensity at ~ 625.0 nm compared to pristine MoS2, which is related to bandgap transition of MoS2. This indicates the effective prevention of free electron-hole pairs due to the presence of defects [2/C samples than MoS2/C are owing to Co doping. The above results suggest that compared with pure MoS2, vast defects exit in MoS2/C and CoMoS2/C samples, which is advantageous for charge transfer and storage.Figure yer MoS2 , 32 and JCPDS:37–92, where reports –38, the JCPDS:37–92, where (ID/IG) , 40, whi in MoS2 . Moreove defects . Especia2, in which the presence of C and O should be due to adventitious impurities [2 and the amount of N,O doping in carbon materials are also calculated and are shown in Tables S2 and S3, respectively. Apparently, the amount of Co doping in MoS2 increases  (Table S2) due to the increase in the addition amount of C16H30CoO4, while the amount of N,O doping in C decreases (Table S3), which is attributed to the decrease in the addition amount of DMF. The spectra of Mo 3d , ~ 229.3 (Mo 3d5/2), ~ 162.3 (S 2p1/2) ~ 161.2 (S 2p3/2) eV [2/C samples show clear Co 2p XPS spectra  [3S4. The C 1s spectra shows peaks at ~ 284.5 (C–C), 286.1 (C-N/C-O), and 288.4 (C=O) eV , all samples show their proper element signals besides MoS, all samig. S10) , further2/C, CoMoS2/C-I, CoMoS2/C-II, and CoMoS2/C-III, respectively. After heating theses samples in air atmosphere, Mo, Co, C, and N can be oxidated into MoO3, Co2O3, CO2, and NO2, respectively, in which the front two result in the increase in the mass; the latter two, together with the loss of O, lead to the decrease in mass. Therefore, the final residues after heating these samples are MoO3 and/or Co2O3. According to the EA results (Table S4), final residues mass for MoS2, MoS2/C, CoMoS2/C-I, CoMoS2/C-II, and CoMoS2/C-III are 88.9, 73.4, 69.6, 66.2, and 60.1 wt%, respectively, which shows a similar trend with the results in TGA curves . Clearly, carbon mass fraction values are 18.1, 19.2, 20.4, and 21.7 wt%, corresponding to MoS2/C-II shows a higher first capacity (1512.9 mAh g−1) than the others  due to its successful construction of Co-doped monolayer MoS2 and the absence of large-size Co3S4. Figure 2/C-II shows the highest capacity (1504.3 mAh g−1) after 100 cycles and delivers almost 100% capacity retention similar to MoS2/C and CoMoS2/C-I. This is due to the integrity of electrode structure after cycling  and the low volume expansion in the electrode thickness  after 100 cycles. Obviously, the pure MoS2 shows the worst cyclability with a retention of 41.5% after 100 cycles due to its severe structural deterioration  and large volume expansion in the electrode thickness  after 100 cycles, which is attributed to the absence of few-layered or single-layered MoS2 and N,O codoped carbon matrix. Besides, the CoMoS2/C-III also shows poor cyclability with a retention of 83.6% after 100 cycles due to electrode cracking  caused by the presence of large-sized Co3S4. Figure 2/C-II obviously exhibits superior Li+ transport rate (1063.6 mAh g−1 at 20 A g−1) compared to other samples, which is ascribed to its lowest charge transfer resistance  [ct and ion diffusion impedance can accelerate charge transfer, thus strengthening rate capability. Higher specific surface area can ensure more complete contact of active materials and electrolyte to boost ion transport and thus improve the rate performance. Besides, a higher EC can cause electrons to transfer faster, thus enhancing the rate capability.Figure 2/C-II are further tested. A first charge capacity of 1512.9 mAh g−1 with a CE of 75.3% is achieved  and scan rate (v) [+ storage. In addition, the equation i(V), k1V, and k2V1/2 represent the total current at a fixed voltage (V), pseudocapacitance ratio, and diffusion-controlled ratio, respectively. Obviously, the pseudocapacitance contribution increases from 55.3 to 81.3% , 28.3–50.3% of MoS2/C , 39.3–65.3% of CoMoS2/C-I , and 43.2–69.0% of CoMoS2/C-III . The higher pseudocapacitance ratio for CoMoS2/C-II results in faster ion transport and greater ion storage than other samples. The detailed pseudocapacitance contribution (81.3%) at 1 mV s−1 is depicted in red region  [+ diffusion kinetics, in which ip, v, A, n, and CLi+ represent peak current, scan rate, contact area of materials/electrolyte, electrons number involved in reaction, and Li+ bulk concentration, respectively. According to linear relationship of DLi+ of CoMoS2/C-II is in the range of 7.05 × 10−10–1.67 × 10−9 cm2 s−1 with an average value of 1.19 × 10−9 cm2 s−1. This value is higher than 2.21 × 10−12, 1.58 × 10−11, 2.43 × 10−10, and 1.72 × 10−10 cm2 s−1, corresponding to MoS2, MoS2/C, CoMoS2/C-I, and CoMoS2/C-III, respectively. The results fully demonstrate that monolayer MoS2 has faster lithium-ion diffusion capability than multilayered or few-layered MoS2. The DLi+ of CoMoS2/C-II is higher than the value reported for MoS2-based anodes  . Generalehaviors , where i.3% Fig. c, corresd orbits of Mo and Co under an electric field to trigger spin-polarized surface capacitance, thus creating a space charge region  [d (229.3 eV) and Co 2p (778.5 eV), Mo 3d and Co 2p  and 3d (Co) energy levels, thereby causing the magnetization reduction. When Li ions are removed from the surface of Mo and Co, the electrons also are transferred out from the interior of Mo and Co, thus bringing about the increase in magnetization. After charging up to 3 V, the lower binding energy of Mo0 and Co0 and the higher electrode magnetization are attributed to the irreversibility of Co and Mo. Generally, the first discharging process involves phase transformation reactions of xMoS2 to Li2S and + is removed from Li2S to produce Space charge zone is confirmed in detail. TEM images Fig. a, b, SAEig. S23) . Compareo 2p 778. eV, Mo 3o and Co , 50, whio and Co . Additioo and Co . Detaill23) [d 22.3 eV and→Mo+Li2S , 21, 32.23) [d 22.3 eV and2Li++2e- , 21, 32.2e-→Li2S , 21, 32.2 on lithium-ion storage, ex situ TEM observations are performed after discharging the MoS2, MoS2/C, CoMoS2/C-I, and CoMoS2/C-III electrodes to 0.01 V, where nanoparticles with sizes of ~ 5.4, 4.0, 3.0, and 3.5 nm are formed for pure MoS2 , MoS2/C , CoMoS2/C-I , and CoMoS2/C-III  electrodes, respectively. Obviously, the size (~ 2 nm) of nanoparticles obtained for CoMoS2/C-II electrode after discharging to 0.01 V  enormous Co doping sites to enhance surface energy of MoS2 to store extra Li+ [2 [2 and C to increase Li+ active sites [+ active sites [+ storage mechanism of traditional MoS2 materials.Considering the above results, the capacity of CoMoSxtra Li+ , 19; ii)a Li+ [2 , 32; iiive sites , 51; iv)ve sites , 52. Amo4 cathode are tested to evaluate the application potential of CoMoS2/C-II anode. The charge/discharge profiles of cycles 1 and 100 , where Cc, V, mactive, and minactive denote the cell capacity (2.7 mAh), nominal voltage, active mass of cathode and anode (17.8 mg), and inactive mass (15.6 mg), respectively. The EG at 0.1 C can be calculated as 177.8 Wh kg−1 and remains 136.2 Wh kg−1 (76.6% retention relative to 177.8 Wh kg−1) at 4 C within a charging time of 11.5 min . Clearly, a light-emitting-diode array with 59 unit elements is cushily lighted by a single full cell . Figure + migration paths in monolayer MoS2 and Co-doped monolayer MoS2, respectively. It can be seen that the Li-ion diffusion energy barrier (0.19 eV) of Co-doped monolayer MoS2 is lower than 0.28 eV of monolayer MoS2. This implies that Co doping can greatly improve the Li-ion diffusion kinetics, thus causing fast Li+ transport. Besides, the doped Co atoms can significantly reduce the bandgap of monolayer MoS2 to enable high carrier transport, as validated by calculated density of states (DOS)  Fig. d. Monola of MoS2 , 54.Fig.2 can make lithium ions have isotropic transport behavior ; path II is to bypass layer A and diffuse to the interlayer to react with layers B and C . Through DFT calculation, it is found that path I is impossible because forcing lithium-ion diffusion through layer A makes the model collapse. In other words, the diffusion energy barrier that lithium ion in path I needs to overcome is extremely high. Path II is feasible, and DFT calculation results show that the diffusion energy barrier of lithium ion between layers is only 0.23 eV  during conversion reaction, thus creating strong surface-capacitance effects to accelerate lithium-ion transport Below is the link to the electronic supplementary material.


Neocallimastix cameroonii strain G341 and Clostridium kluyveri DSM 555 in one-pot two-step fermentation.Lignocellulosic biomass plays a crucial role in creating a circular bioeconomy and minimizing environmental impact. Enset biomass is a byproduct of traditional Ethiopian Enset food processing that is thrown away in huge quantities. This study aimed to produce caproate from Enset fiber using N. cameroonii on Enset fiber as a carbon source for 7 days. Subsequently, the fungal culture was inoculated with active C. kluyveri preculture and further incubated. The results showed that N. cameroonii grew on 0.25 g untreated Enset fiber as the sole carbon source and produced 1.16 mmol acetate, 0.51 mmol hydrogen, and 1.34 mmol formate. In addition, lactate, succinate, and ethanol were detected in small amounts, 0.17 mmol, 0.08 mmol, and 0.7 mmol, respectively. After inoculating with C. kluyveri, 0.3 mmol of caproate and 0.48 mmol of butyrate were produced, and hydrogen production also increased to 0.95 mmol compared to sole N. cameroonii fermentation. Moreover, after the culture was supplemented with 2.18 mmol of ethanol during C. kluyveri inoculation, caproate, and hydrogen production was further increased to 1.2 and 1.36 mmol, respectively, and the consumption of acetate also increased.The process started by growing 2 by a co-culture of the anaerobic fungi N. cameroonii and C. kluyveri. This opens a new value chain for Enset farmers, as the process requires only locally available raw materials and low-price fermenters. As the caproate production was mainly limited by the available ethanol, the addition of locally produced ethanol-containing fermentation broth (“beer”) would further increase the titer.A novel microbial cell factory was developed to convert untreated lignocellulosic Enset fiber into the medium chain carboxylic acid caproate and HThe online version contains supplementary material available at 10.1186/s12934-023-02224-w. International Energy Agency (IEA) bioenergy task 42 defines a biorefinery concept as a process that converts biomass into a variety of valuable products such as chemicals, materials, energy, food, and feed. The goal is to expand the range of products made from biomass to create other valuable products , 2. The Neocallimastix cameroonii, Pecoramyces ruminantium, Caecomyces spec., Khoyollomyces ramosus, Orpinomyces joyonii, and Aestipascuomyces dubliciliberans. According to the study, of all isolates Neocallimastix cameroonii strain G341 proved to be the most efficient organism for utilizing wheat straw and was capable of producing acetate, succinate, ethanol, hydrogen, formate and lactate [Research showed that anaerobic fungi can utilize lignocellulosic biomass without any pretreatment process and produce valuable products. As they grow, anaerobic fungi penetrate lignocellulosic biomass with their hyphal tips and degrade it with the enzyme complexes they produce , 12. Ana lactate . These p lactate , however lactate , 18. MorClostridium kluyveri is capable of utilizing acetate, ethanol, succinate, and hydrogen to produce caproate and other medium chain fatty acids [N. cameroonii strain G341 from lignocellulosic biomass could be a suitable substrate for C. kluyveri fermentation. The objectives of this study were to evaluate the growth of N. cameroonii on Enset fiber, and to develop and characterize a one-pot two-step fermentation process for caproate production using N. cameroonii strain G341 and C. kluyveri DSM 555 from Enset fiber. To date, no research has been conducted on the growth of N. cameroonii on Enset fiber as well as the production of caproate from Enset fiber using N. cameroonii and C. kluyveri in a single pot two-step fermentation.In chain elongation process, volatile fatty acids are changed to more valuable medium chain fatty acids through anaerobic fermentation . A mediuty acids , 24. TheN. cameroonii on Enset fiber, and to analyze the fermentation products, the fungi were grown initially on 0.25 g Enset fiber as sole carbon source. Before fermentation, the bottles were filled with 8.06 mmol CO2 gas at atmospheric pressure (≈1.00 bar). As shown earlier by Theodorou M. et al. [N. cameroonii along with the absolute pressure built up. Within 24 h, an absolute pressure of 1.2 bar was developed and 0.97 mmol of CO2 were produced. As fermentation progressed, the gas composition in the bottle headspace changed to hydrogen and CO2, and a maximum pressure of 1.39 bar was generated on the fourth day. After 5 days, 0.51 mmol of hydrogen was produced with no significant changes in hydrogen quantity in the following days. However, there was a slight decrease in the amount of CO2 gas as well as the pressure. In the control experiments with 0.25 g wheat straw as substrate, the gas profiles were similar and the fungi developed a maximum pressure of 1.25 bar and 0.39 mmol hydrogen, which was lower than with Enset fiber  of Enset fiber were tested. Figure N. cameroonii growth at different substrate loading of Enset fiber. The highest pressure of 1.72 bar was recorded with 1% (w/v) of Enset fiber, but the pressure decreased with increasing substrate loading. Statistical analysis with p < 0.05 showed a significant difference between different substrate loadings of Enset fiber on pressure development by N. cameroonii  when the substrate loading was low, however, the acetate percentages increased to 53% (mmol/mmol) when substrate loading was high. Additionally, as substrate loading increased, the hydrogen percentage tended to decrease from 14 to 8% (mmol/mmol) as well  was produced compared to monosaccharide substrates, which was less than when Enset fibers were used as the substrate. Furthermore, in our study, the amount of CO2 increased until the fourth day, similar to what Borneman W. et al. [Neocallimastix sp. Strain RI grown on Italian ryegrass hay. However, it was not sure whether it came from bicarbonate buffer or anaerobic fungal growth [2 sources.Anaerobic fungi release carbohydrate-active enzymes (CAZymes) such as cellulases and hemicellulases that can degrade lignocellulosic biomass, and produce several organic acids, and hydrogen as byproducts of its growth process . In thisng place . Growth hin 24 h , 27. Stahin 24 h  characte. et al.  observedl growth . It is nN. cameroonii is the type of substrate. N. cameroonii grew better on Enset fiber and produced more metabolites than with wheat straw, compared to the control experiment shown in Fig. Neocallimastix sp. (strain LM-1), Sphaeromonas sp. (strain NM-1) and Piromonas sp. (strain SM-1), and the results showed that the lignin component of straw was not degraded. However, other researchers observed a high percentage of dry weight and lignin loss in perennial ryegrass stem cell walls [N. cameroonii growth.One of the factors that affect the metabolite production and the growth of ll walls , Bermudall walls , wood [3ll walls , and Itall walls , when trll walls . In addill walls . HoweverN. cameroonii growth on Enset fiber was observed at higher substrate loading. Even though there was a mixing problem due to the high amount of solid content in the bottles, at 7% (w/v) substrate loading of the Enset fiber a pressure of 1.53 bar was developed. However, the carbon balance calculation showed that at maximum substrate loading, the fungi’s carbon conversion efficiency was minimized from 98.8 to 16.5%  such as caproate and butyrate. In addition, it was observed that hydrogen production was maximized in a two-step fermentation process compared to a single fermentation of anaerobic fungi Fig. A. In thi. et al. . Furtherconsumed . In addiPecoramyces species) and a methanogen (Methanobrevibacter species), and the results showed that methane can be produced from various parts of corn stover through coculturing. However, in another study, Dollhofer V. et al. [C. kluyveri for the availability of nutrients and substrates, since the strains were added sequentially and the nature of metabolic pathways between them were different. In the two-step fermentation process, N. cameroonii degraded lignocellulosic biomass by carbohydrate-active enzymes (CAZymes) to monosaccharides such as glucose and xylose, which are then metabolized through the Embden-Meyerhof pathway to generate energy carrier compounds. After that, pyruvate metabolism takes place in the cytosol and hydrogenosomes, producing various metabolic end products like acetate, succinate, ethanol, hydrogen, formate, and lactate [C. kluyveri metabolism and are involved in energy production and biosynthesis of cellular components [C. kluyveri can break down fatty acids into medium-chain carboxylates (MCC) through the reverse β-oxidation pathway [Numerous studies have been conducted to evaluate the potential of anaerobic fungi for co-cultivation with other strains. Li Y. et al.  studied . et al.  showed t. et al. . In this lactate , 47. Themponents . C. kluy pathway . Despite pathway .C. kluyveri [C. kluyveri and concluded that caproate is not formed through hydrogenotrophic processes but by hydrogenogenic processes. Moreover, we observed that metabolite production ceased after ethanol was consumed completely, and 0.76 mmol of acetate produced by anaerobic fungi remained unconsumed  as a carbon source corresponding to a substrate loading of 0.5% (w/v). 1 L minimal medium consisted of 150 mL of salt solution I (K2HPO4 (3.0 g/L)), 150 mL of salt solution II (KH2PO4 (3.0 g/L), (NH4)2SO4 (6.0 g/L), NaCl (6.0 g/L), MgSO4.7H2O (0.6 g/L), CaCl2.2H2O (0.6 g/L)), 2 mL of 0.05% (w/v) hemin solution , 2 mL of 0.1% (w/v) resazurin solution, NaHCO3 (6 g/L), cysteine-HCl·H2O (1 g/L) and 10 mL of trace elements solution . The media was anaerobized by flashing with 100% CO2 until the color turned yellow, and the pH was corrected to 6.9 with 5 M NaOH. During anaerobization, a serum bottle containing 0.25 g of Enset fiber was filled with 49.5 mL of medium, enclosed with a rubber stopper and aluminum cap, and autoclaved. After autoclaving, 0.5 mL filter-sterilized vitamin solution  was added [N. cameroonii preculture propagated every seven days for two consecutive cultivations before being used as a preculture. The bottle was incubated at 39 °C for 7 days in the dark in an incubator  without shaking. The pressure of the bottle was measured with manometer GMH 3100 Series  at 39 °C, and 5 mL gas and 1.5 mL liquid samples were taken with needles every day and centrifuged at 10,000 rpm for 10 min [In a previous study, Stabel M. et al.  isolatedas added , 55. Ther 10 min . 0.25 g N. cameroonii to investigate the effects of substrate loading. A total of 50 mL of media was prepared with Enset fiber at different substrate loadings ) as carbon source, inoculated with 5 mL of anaerobic fungal culture, and incubated at 39 °C for 7 days [The same conditions as mentioned above were used to cultivate r 7 days . At the Clostridium kluyveri DSM 555 culture was acquired from the German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany (DSMZ), and revived according to DSMZ protocol. The DSM-52 medium contained buffer stock solution (K2HPO4 (31 g/L), KH2PO4 (23 g/L), NH4Cl (25 g/L)), vitamin stock solution (B12 (0.1 g/L), p-aminobenzoic acid (0.08 g/L), biotin (0.02 g/L), nicotinic acid (0.2 g/L), D-Ca-pantothenate (0.1 g/L), pyridoxine-HCl (0.3 g/L), thiamine-HCl.2H2O (0.2 g/L)), yeast extract stock solution (100 g/L), Na2CO3 stock solution (100 g/L), Na2S.9H2O stock solution (100 g/L), L-Cysteine-HCl.H2O stock solution (100 g/L), and mineral stock solution (MgSO4.7 H2O (20 g/L), 100 mL of trace element solution, 100 mL of selenite-tungstate solution. The trace element solution SL-10 consisted of 10 mL of (25%) HCl in 1 L, FeCl2.4H2O (1.5 g/L), ZnCl2 (0.07 g/L), MnCl2.4H2O (0.1 g/L), H3BO3 (0.006 g/L), CoCl2.6H2O (0.19 g/L), CuCl2.2H2O (0.002 g/L), NiCl2.6H2O (0.024 g/L), Na2MoO4.2H2O (0.036 g/L). The selenite-tungstate solution was prepared by mixing NaOH (0.5 g/L), Na2SeO3.5H2O (0.003 g/L), and Na2WO4.2H2O (0.004 g/L). All the stock solutions were filter sterilized and anaerobized with 100% N2 gas. For the cultivation experiment, 10 g/L CH3CO2K and 1 g/L resazurin were added into 47.75 mL distilled water. The pH was corrected to 6.9 with 1 M NaOH/1 M HCl and filled into 250 mL serum bottles, enclosed with an aluminum cap and rubber stopper. Each bottle was flashed with a mixture of 80% N2 and 20% CO2 gases using needles and autoclaved. Then, after autoclaving, 1 mL of (99.5%) ethanol was injected with a needle and 1% (v/v) of each stock solution was added. 10% (v/v) of active culture with an OD of 0.8-1.0 was inoculated into bottles and incubated at 37 °C and 150 rpm. The culture was transferred three times before being used as a preculture [Freeze-dried O4.7 H2O  g/L, 100N. cameroonii was first grown on 0.25 g Enset fiber at 39 °C for 7 days in 250 mL serum bottles containing 50 mL of the basal medium described above. Subsequently, the fungal culture was supplemented with 1% (v/v) sterilized DSM-52 stock medium without acetate, ethanol, and reducing agents. The bottle containing the grown fungal culture was then inoculated with 10% (v/v) of the actively growing C. kluyveri culture with an OD of 0.8-1.0 and incubated at 37 °C and 130 rpm for 7 days. During the second fermentation, the pressure was measured, and the 5 mL gas and 1.5 mL liquid samples were taken using needles  of bacterial cells. For liquid samples, the pH was measured by Profilab pH 597  and metabolites were determined by high-performance liquid chromatography (HPLC) in a 1100 Series System . HPLC was performed with a Rezex ROA Organic Acid H+ (8%) column at 55 °C, and 5 mM H2SO4 eluent with a flow rate of 0.6 mL/min [After inoculation, the gas pressure inside the bottle was monitored using a manometer with a needle. As anaerobic fungi grew and produced gases, the manometer recorded changes in pressure over time. By correlating gas pressure measurements with fungal growth, this method provided insights into anaerobic fungal growth . UltraspBelow is the link to the electronic supplementary material.Additional file 1: Table S1. Output from ANOVA analysis in OriginPro 2021; the pressure developed by N. cameroonii on different substrate loading of Enset fiber. Figure S1. Percentage of all metabolites for N. cameroonii growth on different substrate loading of Enset fiber at the end of fermentation. Table S2. Carbon balance calculation and conversion factor for N. cameroonii growth on Enset fiber


Aspergillus niger A8 + Trichoderma viride co-cultures utilizing pomegranate peels. The partially purified enzyme showed maximal relative activity at 37–65 °C for 10 min and kinetics of thermal inactivation energy at a high point at 60 °C for 0.040/min. The half-life was 37 °C for 58.6 min, temperature coefficient Q10 of tannase was maximal for 1.38 between 40 and 50 °C, and the activation energy was 17.42 kJ/mol. The enzyme activity peaked in the pH range of 4–8, and the maximum relative activity (100.6%) for tannase was achieved at pH 6. The Km and Vmax values for purified enzymes using tannic acid were 7.3 mg/mL and 3333.33 U/mL, respectively. The enzyme reduced the total tannin content in all tannin-rich substrates after 12h. The gallic acid (GA) had total phenols of 77.75 ppm and antioxidant activity of 82.91%. It was observed that the GA as antimicrobial influencer exhibited the largest inhibitory zone diameter (IZD) of 31 ± 1.0 mm against Pseudomonas aeruginosa ATCC27853. The GA minimum inhibitory concentration value was ranged from 7770.0–121.41 µg/mL. The obtained GA showed a bactericidal effect against all bacterial strains except Shigella sonnei DSM5570 and Salmonella typhi DSM17058, which showed bacteriostatic behavior.Bioconversion using fungi, as natural factory of many applicable bioactive compounds, as enzymes utilizing agro-residue substrates as a solid, abundant, low-cost growth and enzyme production media. This study characterized and applied a tannase enzyme (308 U/mg) from  There are several uses for various plants' peel, leaves, oil, and juice, including pomegranate, guava, banana, grapes, and orange. Due to their abundance and rich content of antioxidants, including flavones, anthocyanidins, alkaloids, luteolin, tannins, and others4, they are also high in luteolin. Pomegranate peel wastes generate bioactive compounds from plants among the fruits described because they are particularly rich in polyphenolic compounds. This fruit has antioxidant effects and slows the oxidation of vitamin C due to its high tannin content, namely gallotannic acid, which produces gallic acid when hydrolyzed6.Gallic acid (GA), one of the crucial industrial and therapeutic significant molecules with a demand of over 10,000 tons per year, can be produced by tannase in various ways. Tannins are present in angiosperms, gymnosperms, and pteridophytes and are primarily found in plant leaves, roots, bark, and fruit7. GA is a common secondary metabolite found in most plants . A slightly colorless or slightly yellow crystalline solid, it is. Its chemical formula is C7H6O5, with a molecular weight of 170.11954 g/mol. Additionally, it has a 210 °C melting point and a 235 °C to 240 °C decomposition range, which results in the production of carbon dioxide and carbon monoxide, 1.69 kg/L (20 °C) is its density. It is insoluble in benzene, chloroform, ether petroleum, and alcohol but soluble in water, ethanol, glycerol, and ether8.A significant amount of tannic acid is also frequently present in tannery effluents. The toxic tannery effluent's tannin load can be reduced with the help of tannase, allowing for the possibility of lowering operating costs. The accessibility of various agro-industrial residues paves the way for the most significant possible utilization of tannase production for tannin degradation and, ultimately the output of GA1. Tannin bioconversion by microbial tannase can efficiently produce significant amounts of GA6.On a large scale, GA is created by tannase, a glycoprotein esterase breaking down tannic acid. Tannase from microbial sources has been suggested for the degradation of natural tannins over the past three decadesAspergillus sp., Rhizopus sp., and Penicillium sp. have been the most common filamentous fungi involved in tannin bioconversion, even individually or in co-cultures. Along with them, various other fungi from the genera Trichoderma sp., Fusarium sp., Chaetomium sp., Rhizoctonia sp., and others have been reported for their ability to degrade tannins, particularly hydrolyzable tannins. Aspergillus sp. has been the most potent and extensively studied tannase producer among the existing fungal sources9.GA and tannase production are linked because tannase catalyzes the depolymerization of hydrolyzable tannins, resulting in the release of GA. 10. Aspergillus sp. and Penicillium sp. fungi, in particular, primarily through fermentation11. In a solid medium (SSF) and submerged (SMF), Costa, et al.12 obtained tannase and GA from Aspergillus tamarin. Better results were obtained when A. tamarin and A. japonicus were combined to produce tannase and GA13. Also, tannase from co-culture of Penicillium chrysogenum + Trichoderma viride was used to hydrolyze tannic acid for the production of gallic acid (84 ± 2 U/g/min) using grape's peel as a substrate14.Fungal co-culture has important roles in increasing the activity of valuable enzymes such as cellulase, laccase, pectinase, tannase, etc., and improving the yield of essential products such as gallic, botulinic, and oleanolic acids. Also, It offers the unique advantages of low cost and simple operation and does not require expensive chemicals and complex gene-level manipulation15. Recently, some studies have been published relating the effect of GA before the formation of amyloid plaques, considered the initial step in Alzheimer's disease16. In addition to medicinal aspects, GA is applied in other areas. Its first application was in the skin and leather industry as a chelating agent12. The first photographs used GA as a developer17. GA is used to synthesize trimethoprim, an antimicrobial agent, and as a preservative in food and beverages, primarily because of its power to kidnap free radicals18.This metabolite exhibits a range of bioactivities, including antioxidant, antimicrobial, anti-inflammatory, anti-ulcerogenic, and anticancerA. niger A8 + T. viride from pomegranate peels using the SSF technique. ii) utilize the gallic acid end product's antibacterial and antioxidant properties as well as its ability to remove tannin from agricultural residues.Additionally, the goal of the current study was to i) partially purify and characterize the tannase produced by combining the cultures of Bacillus cereus ATCC11778, Escherichia coli ATCC 8739, Enterococcus faecalis ATCC 7080, Klebsiella pneumonia ATCC00607, Pseudomonas aeruginosa ATCC27853, Salmonella typhi DSM 17,058, and Shigella sonnei DSM5570. These strains were taken from Microbiological Resource Centers (MIRCEN), Cairo, Egypt. Pathogenic bacterial strains were maintained on glucose agar19 and kept in the refrigerator at 4 °C.Seven pathogenic bacterial strains were used to investigate the antibacterial activity, namely Aspergillus niger A8 + Trichoderma viride (5:5% v/v inoculum size) utilizing pomegranate peels with SSF technique through Ahmed and Abou-Taleb20.Extracellular fungal tannase crude enzyme with 12.93 U/mg specific activity was obtained from the cultural consortium 21, different amounts of solid ammonium sulfate were separately added to 1L of a crude enzyme to attain 0–80% saturation at 4 °C and stirred. The crude enzyme was placed in an ice bath, and solid ammonium sulfate was gradually dissolved to achieve an initial 20% saturation at 4 °C. The mixture was centrifuged  at 4°C for 10 min at 10,000 rpm. The precipitated protein pellet was thrown away. More solid ammonium sulfate was added to the supernatant to reach 40% saturation at 4 °C. It was centrifuged after another night at 4 °C. More ammonium sulfate was added to the supernatant to achieve 60% saturation at 4 °C. It was centrifuged after another night at 4 °C. The precipitated protein pellet was then collected, and additional ammonium sulfate was added to the supernatant to achieve 80% saturation at 4 °C. The supernatant was discarded in the same manner as previously mentioned. The precipitate from each source was dissolved individually in a small amount of 0.1M phosphate buffer pH 7.0 and then dialyzed  overnight at 4 °C with twice changes of the buffer to remove ammonium sulfate, which inhibits the catalytic function of an enzyme. Total proteins and enzyme activity were measured in the method described below.Fractional precipitation with ammonium sulfate followed by dialysis partially purified crude tannase. According to Kumar, et al.20. One milliliter of the crude enzyme (filtrate) was added to a 4 mL solution (consisting of 0.35 g tannic acid dissolved in 100 mL citrate buffer (0.05M and pH 5.5) and mixed well. The mixture reaction was incubated in a water bath  at 37 °C, and 0.2 mL of the reacting compound was withdrawn at zero time (t0) and after 30 min of incubation time (t1). The enzyme reaction was stopped by adding ethanol 95% (2 mL v/v). The absorbance of the t0 and t1 was measured using a UV spectrophotometer (Chrom Tech CT-2200 UV/Vis) at 310 nm. The absorbance of the t1 and t2 were scored at 310 nm using a UV spectrophotometer (Chrom Tech CT-2200 UV/Vis). One unit (U) of tannase activity was considered the enzyme needed to hydrolyze 1μmol of ester per 1 min per mL. The enzyme activity was calculated according to the following Eq. , the Gallic acid (GA) was measured in culture filtrate and recorded at 255 nm and 294 nm using a UV spectrophotometer (Chrom Tech CT-2200 UV/Vis). The following Eq.  for 10 min. The enzyme activity and kinetics of the activation energy (EEA) and thermal deactivation (Kd) and the temperature coefficient (Q10) were calculated as mentioned below in Eqs. The Arrhenius equation is rearranged as above it is a linear Eq.  with thed constant at each temperature and the half-life (T1/2) were calculated using Eqs. ,  of a reaction increases for every 10-degree rise in the temperature (T). The Qwing Eq. 26:9\docuThe optimum pH was determined using different buffers (0.1 M) with different pH values: glycine—HCl buffer (pH 1.5–2.0), sodium acetate buffer (pH 3.0–5.0), sodium phosphate buffer (pH 6.0–8.0), and borate buffer (pH 9.0–10.0) for 10 min at optimum temperature. To investigate pH stability, the enzyme solution was dissolved in 0.05 M buffer solutions (1:1), citrate buffer (pH 3.0–6.0), phosphate buffer (pH 7.0–8.0), and Tris–HCl (pH 9.0–10.0), and then incubated for 15, 30, 45, and 60 min.m and Vmax) was investigated. The partially purified enzyme was incubated with tannic acid at various concentrations  in the reaction mixture. Under standard assay conditions, each substrate concentration determined the activity per unit of time. The obtained enzyme kinetics data were plotted using the Michaelis–Menten, Lineweaver–Burk, and Hanse-Woolf plots to calculate Km and Vmax values following Eqs. , , which was assumed to be 100%.28. In brief, 2.5 mL of Folin-Ciocalteu's phenol reagent and 0.5 mL of a tenfold diluted sample were combined, incubated for 5 min, and 2 mL of 20% (w/v) Na2CO3 was added. At room temperature, the mixture was incubated for 60 min. At 760 nm, the absorbance concerning the prepared blank was observed. The mg of tannin was calculated using a tannic acid standard. To calculate the percentage of tannin degradation hydrolysis, use Eq. , guava leaves (GLS), and pomegranate peel (PPS) collected from Cairo, Egypt's local markets. Partially purified enzyme (0.5% v/v) was added to the waste (0.5 g), thoroughly mixed, and incubated at 55 °C for 24 h. GAC (mg/mL) released following enzymatic breakdown was measured using the method previously mentioned. The total tannin content of each material was estimated using a procedure outlined in Pinto, et al. use Eq.  below. E use Eq.  and 15)28. In br30 with some modifications. A mixture of 2 mL of 160-fold diluted fungal GA and 2 mL of 0.1 mmol/L 2,2-Diphenyl-1-picrylhydrazyl (DPPH) methanolic solution was prepared. After vigorously shaking the mixture and leaving it in the dark for 20 min, the absorbance at 517 nm was measured with a UV spectrophotometer (Chrom Tech CT-2200 UV/Vis). According to Folin-Ciocalteu's method, the total phenol content of fungal GA was determined31. The test sample was transferred to an Erlenmeyer flask (100 mL) in a GA amount of about 0.1 mL, and the final volume was raised by adding distilled water to reach 46 mL. After that, 1.0 mL of the Folin-Ciocalteu's reactive solution was added, and the mixture was left to sit for 3 min at room temperature. The previously mentioned solution was combined with 3 mL of sodium carbonate (2% w/v). After 30 min, the absorbance at 760 nm was then determined. The total phenol was expressed as GA equivalents based on the calibration curve.The free radical scavenging ability of fungal GA was measured using the method described by Marques et al.32, standard inoculums for pathogenic bacterial strains were produced. A loop of the active bacterial growth was used to make the bacterial fresh inoculant, which was then shaken at 150 rpm in flasks for 24 h at 37 °C until it reached the 0.5 MacFarland standard for turbidity. The tested bacteria had a standard inoculum of 1 × 105 colony-forming units (CFU)/ mL. The agar well diffusion method was used to evaluate the antibacterial activity of the fungal GA against the tested pathogenic bacterial strains22. Wells of 8 mm in diameter were established on the inoculated plates with a sterile cork borer. Then 100 μL of standard inoculum of tested pathogen and 100 μL GAC (77.7 mg/mL) were added into the established wells. The plates remained standing until the samples were fully absorbed and incubated at 37° C for 24 h. The antibacterial activity was assayed by measuring the inhibition zone diameter (IZD) around the colonies. Ampicillin  and Streptomycin  were positive controls. Activity index (AI) was calculated (Eq. (33.According to the technique described by Sen and Batrated (Eq. ) accordi34, a dilution method was used to determine fungal GA minimum inhibitory concentration (MIC). GA was serially diluted twice to achieve final concentrations of 7.77 mg/mL (control), 3.88, 1.94, 0.97, 0.485, 0.242, and 0.121 mg/mL by first adding 1 mL of stock to the first tube that contained 1 mL of distilled water and then adding 1 mL to each of the following tubes. As previously mentioned, these dilutions were put into the wells created in the inoculated plates.According to the Clinical and laboratory standard Institute35. The MBC value was calculated by sub-culturing from the MIC-tested plates that did not grow. GA effect on pathogenic bacterial strains was calculated as the MBC/MIC ratio. When the MBC/MIC ratio was 2 or 4, the action was classified as bactericidal or bacteriostatic36.The minimum bactericidal concentration (MBC) of fungal GA: the lowest GA concentration inhibiting the growth of pathogenic bacterial strains® SPSS® Statistics Server Version 23.0. (2015), as Muijs37 suggested at the 5% confidence level.The obtained data was analyzed using IBMWith the permission of the local market, samples of tannin-rich raw plant materials were taken. The authors indicate that all actions were taken under the regulations and rules that were in effect.38.Enzyme purification is typically unnecessary for commercial enzyme use, but high-purity enzymes are needed for pharmaceutical and clinical applications. Tannase from microbial sources has frequently been purified using conventional purification methods. These methods include separating the culture from the fermentation medium and selective concentration by precipitation using ammonium sulfate or organic solvents, according to Gupta et al.The fungal crude enzyme was partially purified by ammonium sulfate precipitation at a range of concentrations  to determine the optimum concentration for partial purification. According to Table 39 reported that tannase of Rhodococcus NCIM 2891 had 0.295 U/mg specific activity after purification. In addition, Ma et al.40 found that A. ficuum Gim 3.6 tannase-specific activity after purification was 2.74-fold, with an enzyme activity recovery of 77.1%. In addition, Wan et al.24 mentioned that A. niger NL112 tannase specific activity of 57.96 U/mg proteins with a purification of 5.1-Fold. While tannase-specific activity was found to be 9.55 U/mg protein of the pure tannase from A. awamori41 and 10.22 U/mg protein of the pure tannase from A. niger42.Nadaf and Ghosh24, The optimal temperature of tannase from K. pneumoniae MTCC 7162 and A. niger NL112 was found to be 40 °C and 45 °C, respectively.The activity was estimated by operating the assay for 10 min at various temperatures between 25 and 85 °C. The results in Fig. 25. Data in Fig. 43 revealed that the Ea for tannase produced by Aspergillus niger was 21.38 kJ/mol at 4.4 mM of methyl gallate. Moreover, Valera et al.25 reported that the optimal temperature for extracellular tannase II activity from A. carbonarius was 60 °C, and the Ea was 28.93 kJ/mol. Whereas de Sena, et al.44 found Ea for hydrolysis of tannic acid by tannase produced from A. tamarii URM7115 was 34.92 kJ/mol.The activation energy (Ea) is an important aspect from an industrial point of view, as it is relevant to know the Ea required for tannic acid hydrolysis by tannases for efficient reduction45 revealed that tannases were stable at a temperature ranging from 30 °C to 60 °C. As well as, Valera et al.25 reported that A. carbonarius tannase II was stable at a temperature range of 20–60 °C for 120 min.Regarding the enzyme exposed to temperatures ranging from 37 to 85 °C for 10–60 min to check its thermal stability. Results in Fig. d at different temperatures, the half-life t1/2, and the thermal inactivation rate constant k. Tannase produced from Aspergillus niger A8 + Trichoderma viride thermal deactivation energy Kd where the native (active) form is transformed in the denaturated (inactive) form by a first-order unimolecular irreversible reaction. It is known that the inactivation of the enzyme is a first-order reaction24. The Kd was at a high point at 60 °C for 0.040/min, and the lowest was at 37 °C for 0.012/min, which means the Kd increased by 30% from 37 °C and 60 °C  refers to the time needed to halve the activity of the enzyme. The half-life of tannase also followed the first-order kinetics  of the tannase was determined at various temperatures, and it was found to decrease rapidly and be maximum at 37 °C for 58.6 min while the lowest was scored at 60 °C for 17.3 min. Moreover, the tannase from A. aculeatus with a t1/2 of 17.5 min and kd 0.039/min at 80 °C) and from A. carbonarius t1/2 of 62 min at 75 °C46. Whereas, the A. tamarii URM7115 tannase t1/2 of 247.55 min (4.13 h) at 40 °C and t1/2 of 106.54 min at 80 °C and the kd increased from 0.13 to 0.39/min as the temperature increased from 40 to 80 °C44. While the t1/2 of tannase from A. niger NL112 decreased rapidly with increasing temperature and maintained the greatest t1/2 at 40 ~ 50 °C. It only remained 15% of the original activity after being incubated at 70 °C for 15 min24.The thermal stability of tannase is characterized by three parameters: the thermal inactivation energy K10, which is the factor by which the velocity is measured on raising the temperature by 10 °C47. The temperature coefficient of enzyme reactions usually lies between 1 and 2. Results recorded in Fig. 10 of tannase was maximal for 1.38 between 40 and 50 °C, whereas the Q10 value was at the lowest rank by 0.35 between 75 and 65 °C. According to de Sena, et al.44, the Q10 value of tannase from A. tamarii URM 7115 was 1.24. Whereas Jana, et al.46 calculated the Q10 value for tannase from Bacillus subtilis PAB 2 was 2.08. In another study by Cavalcanti, et al.48, the maximal tannase activity was achieved at 40–60 ºC for 120 min. Also, the soluble tannase produced by A. fumigatus CAS-21 presented an optimum activity at temperatures ranging from 30 to 40 °C for 60 min. As well as they showed a half-life (t50) of 60 min at 45 and 50 °C for 3 h49.The effect of temperature on enzyme reaction is usually given in terms of the temperature coefficient Q48.From the previous results, it can be concluded that 37–55 °C were suitable for tannase storage and stability for 10 min, it might be due to no auto-digestion of tannase occurring during the incubation period of 10 min. Temperatures above 55 °C promoted a reduction in enzymatic activity because the weak bonds in the three-dimensional structure of the protein break under high temperatures, causing its denaturation and loss of activityThe tannase activity was found to have a wide pH range from 3 to 10. Results shown in Fig. 50, the amino acid properties of the active site control the pH-dependent enzyme activity. While bacterial activity ranged from pH 4.5 to 5.5, most optimal pH values reported for fungal tannase activity ranged from pH 2.0 to 8.051. As Wan et al.24 stated, the optimal A. niger NL112 tannase pH for the purified tannase active in an acidic environment was 5.0. Over the pH range of 4.0–6.0, it retained more than 80% relative activity. Whereas the maximal tannase activity was achieved at pH 5.0–6.048.Additionally, the results showed that tannase could maintain activity over 50% at pH level 5 for 40 min, reaching 55.6% of relative activity. Further, this enzyme was maintained at a high pH of 8 for 40 min with 57.8% relative activity Fig. b. From tm and Vmax27. To simplify the evaluation of Km and Vmax, the Michaelis–Menten equation, as 1/V = (Km/Vmax) X (1/[S]) + 1/Vmax. The enzyme activity was evaluated by varying its substrate concentration from 0.175 to 1.225 mg/mL. The results presented in Fig. max being 0.0003, The slope represents Km/Vmax being 0.0022 for partially purified tannase. The constant Km for tannic acid was calculated to be 7.3 mg/ mL, while its maximum velocity, Vmax was 3333.33 U/mL, as presented in Table max being 0.0019, and the slope represents Km/Vmax being 0.0012 for partially purified tannase. The constant Km for tannic acid was calculated to be 0.63 mg/ mL, while its maximum velocity, Vmax, was 526.32 U/mL, as presented in Table 52 revealed that Km and Vmax values for Paecilomyces variation tannase were 0.61 µmol and 0.55 U/mL. Whereas Farias et al.53 reported that the Km value was 7.49 mM for Cryptomeria parasiticus tannases using methyl gallate as substrate.The enzyme source and particular substrate may define the values of kinetic constants of K54.Tannase activity decreased when the tannic acid concentration was increased by more than 0.70%. Furthermore, data in Fig. 24, A. niger NL112 tannase Km increased with temperature. It could be due to the changes in the molecular space structure of the tannase protein and the loss of some activity caused by excessively increased substrate43.In the same line Wan et al.A. niger A8 + T. viride crude enzyme and partially purified enzyme of tannase. As shown in Fig. Conversion of tannin-rich wastes of PPS, BPS, and GLS into GA by Moreover, the maximum conversion coefficient of wastes was 58.91, 85.56, and 60.39%, with GA productivity of 1.03, 0.73, and 0.35 mg/mL/h after 9, 12, and 9 h for PPS, BPS, and GLS, respectively. Whereas with partially purified enzyme treatment, it was found that the tannin hydrolysis was 46.9, 40.8, and 39.4%, and GAC was 7.77, 6.52, and 3.16 mg/mL for PPS, BPS, and GLS after 12 h, respectively. For partially purified enzyme treatment, the efficiency of tannin content hydrolysis increased by 6.1% and 7.5% in the case of PPS and BPS compared to GLS. For the GAC, it was found that the formation efficiency increased using PPS with 1.2-folds and 2.5-folds compared with BPS and GLS. In addition, the maximum conversion coefficient of wastes was 36.19, 53.75, and 59.96%, with productivity of 0.65, 0.54, and 0.26 mg/mL/h after 12 h for PPS, BPS, and GLS, respectively.55. A. niger tannase removed 73.6% of the tannin from fresh amla (Phyllanthus emblica) juice after 3 h of processing56. Tannase from Penicillium atramentosum has been shown to reduce the tannin content of tea extract by 74%, grape wine by 43.5%, and jamum wine by 38% after 3 h57. In a related study, after 3 h of treatment with T. harzianum crude tannase, the maximum tannin reduction was attained, and it was around 57%58. When A. niger MTCC 2425 was used to declare pomegranate, it was discovered that after 2 h with partially purified tannase (173 U/mg), tannin content had decreased by 56%59. Additionally, after being incubated for 2 h with crude tannase from T. harzianum, the tannin degradation efficiency of various agro-residues did not increase the production of GA58. Additionally, they showed that some tannin wastes and sources, such as amla fruits, jamun leaves, tamarind seeds, keekar leaves, and mulberry leaves, proved to be better substrates for tannase than tannic acid. The enzyme also demonstrated notable activity of 97, 74, 55, 66, and 88%, as well as tannin content of 1.22, 0.74, 0.66, 0.22, and 0.6 mg/mL, respectively, with pomegranate peels, guava leaves, guava park, eucalyptus bark, and mango leaves.In comparison with crude enzyme and partially purified enzyme treatment for PPS, it was found that the tannin hydrolysis decreased by 14.1%, and for gallic concentration, it was reduced by 17% (− 1.19-fold reduction). When comparing the crude enzyme and partially purified enzyme treatments for BPS, the loss for tannin hydrolysis and GAC were 9.2% and 26% (− 1.43-fold reduction). On the other hand, the GLS was the lowest affected in tannin hydrolysis and gallic concentration compared with the other peels (PPS and BPE), as the reduction only scored 1.4% and 1.6% (− 1.01-Fold reduction). So, after all, the PPS was selected as the source for GA formation in the antimicrobial investigation. In a similar vein, many researchers have used tannase to reduce tannin content in various applications, such as pomegranate juice, where it was reported that 40% of the tannin had been removed61. Without tannase enzyme treatment, the antioxidant activity of P. charlesii crude tannase for the tea leaves was 35%62. The Red grape (RGW) had the highest total phenols and antioxidants by 36.5 mg/L (ppm) and it was 166.5% larger than the White grape (WGW) and Moscato grapes (MGW) in the crude without tannase Paecilomyces variotii treatment of various grape wastes63. Additionally, it was found that tannase was most effective in the hydrolysis of polymeric polyphenolics as it increased total phenol content by 61.1% in RGW, 21.5% in WGW, and 33.2% in MGW compared to the corresponding untreated grapes water. The antioxidant activity of tannase from A. nidulans FT10 was 60%, in contrast64. Tannase was also added to green tea leaves, which resulted in a 27% increase in the antioxidant content60. As shown in Fig. 61. Additionally, it was noted that the total phenolic content had increased to 12.48 mg GAE/g (dry weight). In a different study, the Saccharomyces cerevisiae CCMB 520 tannase was used to treat pitanga (Eugenia uniflora) and reach tannins juice. The total phenolic content was found to be 4855 mg/L, and the antioxidant activity was 69.41%65.Furthermore, after exposure to the unprocessed enzyme tannase, black tea's antioxidant activity increased by 52.31% from its baseline activity22. The pathogenic bacterial strains were B. cereus ATCC11788 and E. faecalis ATCC7080 (as G+ve bacteria) and E. coli ATCC8739, K. pneumonia ATCC00607, P. aeruginosa ATCC27853, S. typhi DSM17058 and S. sonnei DSM5570 (as G-ve bacteria). Results in Fig. -ve bacteria, compared with recommended antibiotics, namely streptomycin for G+ve and ampicillin for G−ve, with a concentration of 1000 µg/mL. GA (at 7770.0 μg/mL) achieved an inhibition zone diameter (IZD) ranging from 18 ± 0.37 mm to 31 ± 1.00 mm. In comparison, antibiotics gave IZD ranging from 17 ± 0.40 mm to 35 ± 077 mm. The largest significant IZD (31 ± 1.00 mm) was recorded for P. aeruginosa ATCC27853 followed by 28 ± 1.36, 24 ± 0.51, 23 ± 1.05, 22 ± 0.66 and 21 ± 0.86 mm IZD for E. coli ATCC8739, E. faecalis ATCC7080, S. sonnei DSM5570, and K. pneumonia ATCC00607, respectively. In contrast, the lowest IZD was 18 ± 0.37 mm for B. cereus ATCC11788. The highest activity index (AI) was recorded for S. sonnei DSM5570, K. pneumonia ATCC00607, and E. coli ATCC8739 at 1.35, 1.24, and 1.08, respectively. The lowest activity was 0.77 and 0.78 for E. faecalis ATCC7080 and B. cereus ATCC11788, respectively. Various foodborne pathogens and bacteria, including P. aeruginosa, Helicobacter pylori, E. coli, S. aureus, S. mutans, and L. monocytogenes are susceptible to GA's antibacterial properties66. The antimicrobial effectiveness of tannase enzyme against Streptococcus agalactiae, S. aureus, P. aeruginosa, S. flexneri, and K. pneumonia increased with lower MIC and increased diameter of inhibition for all tested bacteria when combined with other antibiotics67. Additionally, they stated that the proper antibacterial mechanism for the tannase enzyme and its GA end product is linked to the prevention of bacterial surface attachment, the disruption of quorum sensing, decreased cell viability, the destruction of cell membranes, and changes in the morphology of the bacteria. With 500, 250, 125, 62.5, 31.25, 15.63, 7.18, and 3.91 µg GA /mL, GA inhibited Mannheimia haemolytica, bovine respiratory disease associated-pathogens by 84, 78, 43, 38, 20, 20 and 3%, while Pasteurella multocida inhibited by 90, 79, 55, 40, 30, 20 and 6%, respectively68. Using the agar disc diffusion technique, the antibacterial activity of the GA from co-cultivated Bacillus gottheilii M2S2 and Bacillus cereus M1GT against food-borne pathogenic E. coli, S. aureus, and Serratia marcescens and showed a zone of inhibition of 20.0 , 16.0, and 13.0 mm, respectively69. It was thought that GA caused irreversible changes in membrane properties  by changing hydrophobicity, decreasing negative surface charge, and causing local rupture or pore formation in cell membranes, resulting in leakage of essential intracellular71. TGA inhibited efflux pumps, which are a key mechanism in the development of AMR in S. aureus strains and Pseudomonas sp. and multidrug resistant E. coli strains72. It further emerged that GA antimicrobial activities were associated with various pathways within the cytoplasmatic membrane through destabilization, permeabilization, and inhibitory enzyme by oxidized products, possibly through reaction with sulfhydryl groups or more nonspecific interactions with proteins and inhibition of nucleic acid synthesis for both Gram-negative and Gram-positive bacteria73. Previously, the study reported that Gram-positive bacteria were more resistant than Gram-negative bacteria due to the addition of hydroxyl groups, followed by the substitution of hydroxyl groups to methoxy groups, resulting in increased GA activity. GA's antibacterial efficacy is associated with a mechanism that alters bacterial hydrophobicity, which is facilitated by its physicochemical surface properties. Gallic acid caused changes in the polar, nonpolar, and electron acceptor (c +) components of bacterial cells. It resulted in differential ability for increased electron acceptor, as seen in Gram-positive bacteria, and decreased electron acceptor, as seen in Gram-negative bacteria74. It was also electrophilic and had a strong reliance on bacterial surface components68.The antibacterial activity of the tannase enzyme end product (GA) from PPS with a concentration of 7.77 mg/mL was detected by the agar well-diffusion method, as recommended by Balouiri et al.P. aeruginosa ATCC27853 was 1942.5 µg/ mL, while it was 971.25µg/ mL for B. cereus ATCC11788, and the rest bacterial strains scored MIC for 242.81 µg/ mL with E. coli ATCC8739, E. faecalis ATCC7080, S. sonnei DSM5570, S. typhi DSM17058, and K. pneumonia ATCC00607. Results also show that the antibacterial potential at the tested concentrations that ranged from 3885.0 to 1942.5 µg/ mL had 100% of spectrum activity, which inhibited 7 tested strains, whereas, at concentrations of 971.25, 485.63, and 242.81 µg/mL, the GA recorded 85.7, 71.4, and 71.4% of spectrum activity which inhibited 6, 5, and 5 strains out of 7 tested pathogenic strains, respectively.Results in Table L. monocytogenes, E. coli, and P. aeruginosa, respectively70. In contrast, K. pneumoniae has a higher MIC (9.75 μg/mL) than S. epidermidis (9.8 μg/mL) and S. aureus (19.5 μg/mL)73. The MIC for S. flexneri was at 2 mg/mL67. GA was active against M. haemolytica and P. multocida with MIC values of 250 and 500 μg/mL, respectively68. In contrast, the GA from the Altingia excelsa Noronha leaf extract had an inhibitory effect on E. faecalis with a MIC value of 12.25 µg/mL74.GA exhibited antimicrobial activity against the bacteria tested, with MIC values of 100, 1100, and 1250 µg/mL for P. aeruginosa ATCC27853 and B. cereus ATCC11788 was 971.25 µg/ mL. In comparison, it was 485.63 µg/mL for both S. typhi DSM17058 and S. sonnei DSM5570 and 242.81 µg/ mL for E. coli ATCC8739. At the same time, the MBC score for the rest of the bacterial strains scored MBC for 121.41 µg/ mL with E. faecalis ATCC7080 and K. pneumonia ATCC00607 (Table +ve bacteria and 5250 and 500 g/mL for Gr−ve bacteria E. coli and P. aeruginosa70. GA MBC value against S. flexneri was 8 mg/mL67.Using the GA, the maximum obtained MBC value for S. sonnei DSM5570 and S. typhi DSM17058, which had a bacteriostatic impact with MBC\MIC ≥ 2.The observed action of GA against the pathogenic bacterial strains is presented in Table A. niger A8 + T. viride gradually increased to 14.78 U/mg protein at a 40–60% saturation fraction with a purification fold of 1.14. The enzyme's thermal and pH stability parameters were 37–65 °C and 4–6 for 10 min, and its thermal kinetic parameters were Kd, t1/2, Q10, and Ea. They were 60 °C for 0.040/min, 37 °C for 58.6 min, 1.38 between 40 and 50 °C, and 17.42 kJ/mol, respectively. The Km and Vmax values were 7.3 mg/mL and 3333.33 U/mL, respectively. This enzyme was used to reduce tannins in three agricultural wastes, including PPS, BPS, and GLS, with hydrolysis rates ranging from 40.8 to 61.0% for the crude enzyme and from 39.4 to 46.9% for the partially purified enzyme. The GA end product was an antioxidant and antibacterial agent against foodborne pathogens. As a result, this enzyme and end product will be produced in a bioreactor and used on a large scale in the food industry.The specific activity of partially purified tannase from co-cultures of


DBT) and 4-amino-5,7-dinitrobenzotriazole (ADBT). The pressure differential scanning calorimetry was employed to study the decomposition kinetics of DBT experimentally because the measurements under atmospheric pressure are disturbed by competing evaporation. The thermolysis of DBT in the melt is described by a kinetic scheme with two global reactions. The first stage is a strong autocatalytic process that includes the first-order reaction  = 12.82 ± 0.09) and the catalytic reaction of the second order with Ea2I = 136.5 ± 0.8 kJ mol−1, log(A2I/s−1) = 11.04 ± 0.07. The experimental study was complemented by predictive quantum chemical calculations (DLPNO-CCSD(T)). The calculations reveal that the 1H tautomer is the most energetically preferable form for both DBT and ADBT. Theory suggests the same decomposition mechanisms for DBT and ADBT, with the most favorable channels being nitro-nitrite isomerization and C–NO2 bond cleavage. The former channel has lower activation barriers  and dominates at lower temperatures. At the same time, due to the higher preexponential factor, the radical bond cleavage, with reaction enthalpies of 298 and 320 kJ mol−1, dominates in the experimental temperature range for both DBT and ADBT. In line with the theoretical predictions of C–NO2 bond energies, ADBT is more thermally stable than DBT. We also determined a reliable and mutually consistent set of thermochemical values for DBT and ADBT by combining the theoretically calculated (W1-F12 multilevel procedure) gas-phase enthalpies of formation and experimentally measured sublimation enthalpies.Nitro derivatives of benzotriazoles are safe energetic materials with remarkable thermal stability. In the present study, we report on the kinetics and mechanism of thermal decomposition for 5,7-dinitrobenzotriazole ( DBT) and 4-amino-5,7-dinitrobenzotriazole (ADBT), are studied in the present work  and then decomposes with the extrapolated onset of 564 K (291 °C), while ADBT melts with a subsequent decomposition at 610 K (337 °C) ; calculated for C6H3N5O4: 210.0258. Elemental analysis calculated (%) for C6H3N5O4: C, 34.46; H, 1.45; N, 33.49; found: C, 34.57; H, 1.39; N, 33.60.5,7-Dinitrobenzotriazole , which combines thermogravimetry (TG) and differential scanning calorimetry (DSC). The samples of ca. 1.8 mg weight were placed in open aluminum crucibles and heated at a rate β = 10 K min−1 under an argon flow of 50 mL min−1.The thermal behavior of DBT was assessed using the pressure differential scanning calorimeter DSC 204 HP (Netzsch). Samples were poured into closed aluminum crucibles with pierced lids and heated with the rates β of 0.5, 1, 2, 5, and 10 K min−1 up to 500 °C. The instrument has been calibrated with respect to heat flow and temperature using highly-pure metal calibrants, and all measurements were performed under a nitrogen flow of 150 mL min−1. The sample mass of DBT was varied from 0.2 to 3.9 mg with the heating rate to keep the DSC signal magnitude below 8 mW to minimize the detrimental self-heating phenomenon  = 216.04 kJ mol−1, ΔfHm0K(g) [C] = 711.19 kJ mol−1, ΔfHm0K(g) [N] = 470.82 kJ mol−1, and ΔfHm0K(g) [O] = 246.79 kJ mol−1 were taken from the NIST-JANAF tables [To calculate the standard state enthalpies of formation in the gas phase, Δrocedure  along wirocedure ,70 was erocedure . Note throcedure ,73, and rocedure . In the rocedure . The heaF tables .DBT) and 4-amino-5,7-dinitrobenzotriazole (ADBT) using several complementary thermal analysis techniques and high-level quantum chemical calculations. Thermoanalytical experiments under elevated pressure allowed tracking of the thermolysis of DBT in the melt free of the concomitant vaporization. Model-fitting thermokinetic analysis shows that the kinetic scheme with two global consecutive steps provides the best fit for DBT decomposition. The first global stage (I) is a strong autocatalytic process that includes the noncatalytic  = 12.82 ± 0.09) and catalytic reaction of the second order with Ea2I = 136.5 ± 0.8 kJ mol−1, log(A2I/s−1) = 11.04 ± 0.07. The second global stage (II) is of non-integer order and corresponds to the secondary processes with the effective kinetic parameters EaII = 134.5 ± 1.8 kJ mol−1, log(AII/s−1) = 8.7 ± 0.2.In the present work, we studied thermochemistry, tautomerism, and thermal stability of 5,7-dinitrobenzotriazole , the C–NO2 cleavage  becomes the dominant channel at the experimental temperatures.Mechanistic insights into the decomposition process were obtained using quantum chemical calculations. We show that the


Renal ischemia/reperfusion (I/R) injury is a major cause of acute kidney injury (AKI). Dysfunction of E74-like ETS transcription factor 4 (ELF4) leads to inflammation. This research intended to look into the function and mechanisms of ELF4 in I/R and oxygen–glucose deprivation/reperfusion (OGD/R) model.In I/R and OGD/R model, ELF4 expression was downregulated. ELF4 knockout aggravated I/R-induced kidney injury, oxidative stress (OS), endoplasmic reticulum stress (ERS), apoptosis, inflammation, and pyroptosis in mice. In HK-2 cells treated with OGD/R, suppression of ELF4 expression inhibited cell proliferation and promoted cell apoptosis, OS, ERS, inflammation, and pyroptosis. Moreover, ELF4 overexpression led to the opposite results.ELF4 deficiency aggravated I/R induced AKI, which was involved in apoptosis, OS, ERS, inflammation, and pyroptosis. Targeting ELF4 may be a promising new therapeutic strategy for preventing inflammation after IR-AKI.The online version contains supplementary material available at 10.1186/s12860-023-00485-2. As a major kidney disease, acute kidney injury (AKI) is a clinical syndrome characterized by sudden loss of renal function with sublethal renal tubular injury . ClinicaThe E-Twenty-Six (ETS) transcription factor family is composed of 29 members in human and 28 members in mouse, which participates in various signaling pathways –13. E74 At the present study, in vivo model induced by I/R and in vitro model induced by oxygen–glucose deprivation/reperfusion (OGD/R) were used for determining the influences and mechanisms of ELF4 in AKI. Mice lacking ELF4 showed worsened kidney structure and function after I/R. Subsequent studies revealed a direct effect of ELF4 in protecting HK-2 cells from apoptosis, OS, ERS, inflammation, and pyroptosis. A new target for AKI treatment may obtain from our findings.−/− mice were applied to investigate the function of ELF4 in I/R mice , OS related proteins , and ERS related proteins . Figure Subsequently, the influence of ELF4 on inflammatory cytokines was investigated. Figure In clinic, renal ischemia that occurs in kidney surgery, transplantation, hemorrhagic, cardiogenic, septic shock, and other clinical environments is the most common cause of AKI , 23. RenIn I/R and OGD/R model, we restrained ELF4 expression to explore the effects of ELF4 in AKI. The most commonly used markers for renal function assessment including Cr and BUN . The levThe pathogenesis of AKI associated with I/R is involved in tubular damage and inflammatory response . More anThe accumulation of ROS during I/R process is vital in the course of AKI . After rIn summary, inhibition of ELF4 aggravated renal injury in I/R treated mice by promoting OS, ERS, inflammation, and pyroptosis. In future studies, ELF4 supported the transcription of OS, ERS, inflammation, and pyroptosis-related gene will be studied in I/R-induced AKI. Our findings may provide a novel mechanistic insight into I/R-induced AKI.−/−) and wild-type (WT) C57BL/6 mice were provided by Beijing Vital River Laboratory Animal Technology Co., Ltd. (China). In a 12 h light/12 h dark cycle, eating and drinking could be freely obtained by mice. The animal care and use committee of our hospital authorized all experiments.The male ELF4 knockout  with 1% penicillin/streptomycin and 10% foetal bovine serum.Human proximal tubular epithelial cells (HK-2) were provided by Procell Life Science&Technology Co.,Ltd . Under condition of 37 °C and 5% CO2, 5% CO2, and 94% N2 for 4 h at 37 °C [2 was used for maintaining cells for 6 h. In control group, cells were maintained in complete medium with 21% O2.Small interference RNA targeting ELF4 (si-ELF4), overexpression plasmid of ELF4 (ov-ELF4), and negative control (si-NC and ov-NC) were transfected into HK-2 cells by way of Lipofectamine 2000 reagents . After transfection for 48 h, cells in OGD/R + si-NC, OGD/R + si-ELF4, OGD/R + ov-NC, and OGD/R + ov-ELF4 group were treated with OGD/R. To establish OGD/R model, HK-2 cells were exposed to glucose-free medium supplemented with 1% Oat 37 °C . Next, tIn kidney tissues and HK-2 cells, total RNA was extracted by means of TRIzol reagent . The PrimeScript™ RT Reagent Kit  was made use of synthesizing complementary DNA. The SYBR Premix Ex Tag Kit  was made use of performing qRT-PCR. The sequences of primers were listed in Supplementary table In kidney tissues and HK-2 cells, RIPA lysis buffer  was used for extracting proteins. An equal quantity of protein was electrophoresed using SDS-PAGE and then transferred onto PVDF membranes. At 4 °C, primary antibodies against ELF4, Nrf2, NQO-1, GRP78, KIM-1, and IL-6 ; IL-1β ; IL-18 and N-GSDMD ; Bax, Bcl-2, TNF-α, caspase-4, caspase-11, Caspase-12, CHOP, HO-1, and GSDMD , and GAPDH  were deal with membranes overnight. After that, secondary antibodies  were handled with membranes for 1 h. At last, protein bands were visualized by way of an enhanced chemiluminescence detection kit . Image J software (USA) was used to quantify protein expression.The kidney tissues of mice were fixed in 10% formalin . After that, paraffin was taken to embed kidney samples. After dewaxing and rehydrating, Sects. (4 μm-thick) were incubated with 3% hydrogen peroxide  for 10 min. Next, sections were blocked with 5% bovine serum albumin  for 1 h and incubated with antibody against ELF4  4˚C. Nexy day, sections were incubated with secondary antibody . At last, sections were developed color using 3,3’-diaminobenzidine . Under a microscopy , ELF4 positive cells were quantified.After dewaxing and rehydrating, the renal Sects. (4 μm-thick) were stained with haematoxylin  for 5 min and stained with eosin  for 1 min. Under an optical microscope (Olympus), renal tissue damage was assessed in a blinded manner.In kidney tissues, cell apoptosis was ascertained by way of the TUNEL apoptosis detection kit . Briefly, after dewaxing and rehydrating, the renal sections were treated with proteinase K without DNase (20 µg/ml) for 20 min. Next, sections were stained with the TUNEL reaction mixture, followed by staining with DAPI. Eventually, under an optical microscope, apoptotic cells were observed and counted.For the detection of serum creatinine , blood urea nitrogen , superoxide dismutase , catalase , and glutathione peroxidase , the serum was collected from mice blood through centrifuging at 3,000 rpm for 10 min. Nanjing jiancheng bioengineering institute (China) provided corresponding kits.After indicated treatment, cell counting kit 8 (CCK-8) reagents  were applied to handle HK-2 cells. After handling for 2 h, at 450 nm, a BioTek ELX-800 microplate reader (USA) was utilized to gauge the optical density (OD) value.After indicated treatment, 200 µl Annexin V-FITC  and 5 µl PI  were handled with HK-2 cells for 15 min. Finally, the apoptotic cells were gauged using FACS Calibre flow cytometry .After indicated treatment, DCFH-DA  was applied to handle with HK-2 cells for 20 min. Next, serum free medium was used to wash cells for three times, and then cells were analyzed by way of flow cytometry at 480 nm excitation wavelength and 525 nm emission wavelength.P < 0.05 was supposed to statistical significant. All data were statistically compared using t-test or one-way analysis of variance (ANOVA).Data analyses were conducted by means of GraphPad Prism software (USA). Additional file 1.