The International Society for Cutaneous Lymphoma (ISCL) proposes a diagnostic algorithm for early mycosis fungoides (MF) that includes clinical, histological, immunophenotypical, and molecular criteria. Here, we analyzed the immunologic markers and features of T-cell clonality in 38 early MF cases and 22 non-MF cases to validate the ISCL algorithm. We found that CD5 and CD7 expression differed significantly between early MF and non-MF cases, with epidermal discordance of CD7 expression more frequently identified in early MF. Notably, increasing the cut-off value for CD7 expression from 10% to 22.5% improved its sensitivity. Furthermore, TCR-γ and β chain rearrangements were more frequently detected in early MF than in non-MF cases. Based on these findings, we propose CD5 and CD7 deficiency as mandatory immunopathologic criteria and PCR-based testing for TCR-γ and β chains as required molecular/biologic criteria to improve the efficiency of early MF diagnosis using the ISCL algorithm. Mycosis fungoides (MF) is the most common type of primary cutaneous T-cell lymphoma and is characterized by the evolution of patches, plaques, and tumors on the skin . Most caThe International Society for Cutaneous Lymphoma (ISCL) proposes an algorithm for the diagnosis of early MF and Sézary syndrome based on clinical, histologic, immunohistochemical (IHC), and molecular features, which are most important for recognizing classic MF at its initial presentation ,6. AccorSeveral previous studies validated the ISCL diagnostic algorithm to demonstrate its effectiveness and accuracy ,8. TheseIn this study, we classified cases into early MF or non-MF groups according to the ISCL diagnostic algorithm. By comparing each parameter of the algorithm between groups, we identified the most reliable biomarkers that can play a determinant role in efficiently diagnosing early MF and established new cut-off values that differentiate early MF from its mimickers.We retrospectively obtained specimens from 60 patients who were biopsied with clinical suspicion of MF from 2008 to 2021 at Severance Hospital, Yonsei University College of Medicine, and reviewed their associated clinical presentation, microscopic findings, IHC staining results, and molecular studies. Slides stained with hematoxylin and eosin were reviewed by two pathologists (HJR and SKK). Ancillary studies were performed upon diagnosis. All methods and experimental protocols using human tissue were carried out in accordance with relevant guidelines and regulations as approved by the Institutional Review Board of Severance Hospital, Yonsei University Health System (IRB no. 4-2021-0462). The ISCL algorithm for the selection of eligible cases of early MF is shown in The mimickers were clinically diagnosed as annular erythema, drug eruption, parapsoriasis, chronic eczema, pseudolymphoma, psoriasiform dermatitis, and lupus panniculitis after ICH and molecular work-up, although eight cases were still clinically suspicious for early MF .® XT, Ventana Medical System, Inc., Oro Valley, AZ, USA). Antibodies against CD2 , CD3 , CD5 , and CD7  were utilized. Three pathologists  blinded to the pathologic information independently evaluated IHC staining.IHC staining was performed at diagnosis. Formalin-fixed paraffin-embedded (FFPE) blocks were cut into 4-μm-thick sections and processed using heat-induced epitope retrieval. IHC staining was performed in an automated immunostainer  and the Maxwell® CSC instrument. Polymerization was performed with identiClone TCR-γ, β, and δ gene clonality assay kits according to the manufacturer’s instructions . After purification by electrophoresis and dying in 6% polyacrylamide for 2 h, analysis was conducted using the gel imaging system.TCR gene rearrangement analysis was performed at diagnosis to evaluate TCR clonality. Genomic DNA was extracted from 10-μm sections cut from FFPE tissue blocks using the Maxwell2 tests, Fisher’s exact tests, or unpaired t-tests. Receiver operator characteristic (ROC) curves were constructed for IHC results, and the area under the ROC curve (AUC) was calculated. To choose optimal cut-off values, Youden’s index [p-values <0.05 were considered statistically significant. P-values are indicated as follows: * p < 0.05; ** p < 0.005; *** p < 0.0005.All statistical analyses were performed using SPSS version 26.0  and GraphPad Prism version 7.04 . Categorical data are reported as frequencies and percentages, and continuous data are reported as mean and standard deviation (SD) or standard error of the mean (SEM). Differences between groups were compared using χ’s index  for each’s index . Two-sidn = 38) and its mimickers  according to the ISCL algorithm for the diagnosis of early MF and Sézary syndrome , although there was no significant difference in epidermal discordance in CD5 expression by dermal T-cells. CD7 expression was also significantly lower in early MF (17.45 ± 2.648%) than in non-MF , and epidermal discordance in CD7 expression by dermal T-cells was more frequently identified in early MF  than in non-MF . There were no significant differences in CD2 and CD3 expression between groups.We found that CD5 expression was significantly lower in early MF (81.18 ± 3.438%) than in non-MF . CD3 also had a high cut-off value (92.5%) with very low sensitivity . CD5 and CD7 showed fair and good discriminatory abilities, respectively. CD5 had a high cut-off value (92.5%) with 65.8% sensitivity and 72.7% specificity (p = 0.005). CD7 had a cut-off value of 17.5% at the highest Youden’s index with 55.3% sensitivity and 100.0% specificity. However, if we chose 22.5% as the cut-off value for CD7, it had 73.7% sensitivity and 77.3% specificity (p < 0.001).According to Muller’s value, the AUCs of CD2 and CD3 showed poor and failing discriminatory abilities, respectively, at the highest Youden’s index. CD2 had a high cut-off value (92.5%) with low sensitivity (60.5%) and specificity (68.2%), although it was statistically significant (p < 0.0001). TCR-γ and β gene rearrangements were more frequently identified in early MF than in non-MF (p > 0.9999).To specify the features of clonal TCR gene rearrangement in early MF, we performed PCR-based detection of TCR-γ, δ, and β chain genes a. We foun non-MF b. TCR-β We classified early MF and non-MF cases according to the ISCL diagnostic algorithm and analyzed each parameter in the algorithm to improve the accuracy and reliability of ancillary findings. We found that total, clinical, pathologic, molecular, and immunopathologic scores differed significantly between groups. Therefore, benign mimickers should easily be able to be excluded according to the ISCL diagnostic algorithm. However, a diagnostic problem still remains because the clinical features of early MF vary greatly across patients ,15, and Although the ISCL algorithm clearly specifies the antibodies that must be stained, their cut-off values are undependable due to their low sensitivities ,10,11,12The ISCL diagnostic algorithm for early MF clearly documents the methodology for PCR-based analysis of TCR gene rearrangement. However, critical clones and features of TCR genes are not included. To evaluate these molecular features, we performed PCR-based testing to detect the monoclonality or oligoclonality of TCR-γ, δ, and β chains. We found that TCR-γ and β chains were frequently mutated in early MF but that there was no difference in TCR-δ mutation between early MF and non-MF groups. Notably, TCR-β clonality was not identified in any non-MF cases, therefore we believe that TCR-β clonality has 100% specificity in early MF. TCR rearrangements were also detected in 4 out of 22 non-MF cases in this study, which are presumed to be persistent cutaneous inflammatory infiltrates of monoclonal or oligoclonal T-cells due to conditions secondary to autoimmune or iatrogenic immune dysregulation and chronic idiopathic dermatoses ,19,20.In this study, after making pathologic diagnoses, dermatologists changed the clinical diagnosis from mycosis fungoides to parapsoriasis in 7 out of 22 non-MF cases. Parapsoriasis describes a group of cutaneous diseases that can be characterized by scaly patches or slightly elevated papules and/or plaques dispersed on the trunk or proximal extremities that have a resemblance to psoriasis . HistoloIn conclusion, we propose CD5 and CD7 deficiency as mandatory immunopathologic criteria and PCR-based testing for TCR-γ and β chains as required molecular/biologic criteria to improve the efficiency of diagnosing early MF using the ISCL algorithm. In particular, the cut-off value of CD7 expression should be increased from <10% to <22.5% to improve the discriminatory ability of this criterion.


Botrytis elliptica, the causal agent of gray mold disease, poses a major threat to commercial Lilium production, limiting its ornamental value and yield. The molecular and metabolic regulation mechanisms of Lilium's defense response to B. elliptica infection have not been completely elucidated. Here, we performed transcriptomic and metabolomic analyses of B. elliptica resistant Lilium oriental hybrid “Sorbonne” to understand the molecular basis of gray mold disease resistance in gray mold disease. A total of 115 differentially accumulated metabolites (DAMs) were detected by comparing the different temporal stages of pathogen infection. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed the differentially expressed genes (DEGs) and DAMs were enriched in the phenylpropanoid and flavonoid pathways at all stages of infection, demonstrating the prominence of these pathways in the defense response of “Sorbonne” to B. elliptica. Network analysis revealed high interconnectivity of the induced defense response. Furthermore, time-course analysis of the transcriptome and a weighted gene coexpression network analysis (WGCNA) led to the identification of a number of hub genes at different stages, revealing that jasmonic acid (JA), salicylic acid (SA), brassinolide (BR), and calcium ions (Ca2+) play a crucial role in the response of “Sorbonne” to fungal infection. Our work provides a comprehensive perspective on the defense response of Lilium to B. elliptica infection, along with a potential transcriptional regulatory network underlying the defense response, thereby offering gene candidates for resistance breeding and metabolic engineering of Lilium. Lilium is one of the most economically important genera of ornamental monocots, whose species are used worldwide as cut flowers, garden plants, and potted plants. However, both the ornamental value and yield of commercial Lilium are often restricted by gray mold  genes, whose encoded products can specifically recognize the cognate effector or pathogen avirulence proteins, function to regulate ETI  signaling -triggered immunity (PTI) and effector-triggered immunity (ETI)  were identified in B. elliptica-infected L. regale through RNA-seq and miRNA-seq  has accelerated the pace of genetic studies by facilitating de novo genome assemblies from sequence reads obtained using the Illumina technology . The mycelium was cultured on potato dextrose agar  medium in Petri dishes (9 cm diameter) at 25°C in the dark for 1 week.Lilium plants at the flower bud stage, and inoculated with B. elliptica, according to the “detached leaves inoculation methods” of Gao et al. (B. elliptica mycelium discs (5-mm diameter) were collected from the PDA plates using a sterilizing puncher, and then used to inoculate the abaxial surface of the detached lily leaves in vitro. Each leaf was inoculated with six mycelium discs (inoculated treatment), while those uninoculated with B. elliptica served as the control treatment. The inoculated and uninoculated leaves were placed in Petri dishes (15 cm diameter) lined with moist filter paper. The filter paper and cotton surrounding the leaf petiole were soaked with sterile water to maintain humidity within a range of 90–100%.Fully expanded, but not senescent, leaves were collected from o et al. . Before https://imagej.nih.gov/ij/). Superoxide dismutase (SOD) activity was assayed by monitoring the photoreduction of nitro blue tetrazolium (NBT), as describe previously . The area of each lesion was measured with the ImageJ software , and stored in sample bottles for ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis.The leaf samples were freeze-dried under vacuum using the Scientz-100F lyophilizer , and then crushed at 30 Hz for 1.5 min using a grinder  to obtain a fine powder. Then, 100 mg of each powdered sample was extracted in 0.6 ml of 70% methanol. The samples were stored at 4°C overnight, during which time they were vortexed six times to hasten the extraction. Then, each sample was centrifuged at 10 000 × https://www.shimadzu.com.cn/) and MS/MS  were performed using a C18 chromatographic column  with solvent A (0.04% acetic acid in ultrapure water) and solvent B (0.04% acetic acid in acetonitrile) as the mobile phase. A 4-μl aliquot of each sample was injected into the column, and eluted using the following gradient program: 0 min with 95% A and 5% B; 0–10 min with 95–5% A and 5–95% B; 10–11 min with 5% A and 95% B; 11–11.1 min with 5–95% A and 95–5% B; and 11.1–14 min, 95% A and 5% B. The flow rate and column temperature were maintained at 0.35 mL/min and 40°C, respectively. The mass spectrometer parameters were set as follows: temperature of electrospray ionization: 550°C; voltage: 5.5 kV; curtain gas: 30 psi; collision-activated dissociation: high. Each ion pair was scanned and detected based on optimized declustering potential and collision energy in the triple quadrupole system.To analyze the extracts, UPLC  fragments, MS2 fragments isotope distribution, and retention time (RT). Through the MetWare self-developed intelligent secondary spectrum matching method, the secondary spectrum and RT of the metabolites in the project samples are intelligently matched one by one with the MetWare database. The MS tolerance and MS2 tolerance are set to 2 and 5 ppm, respectively. The peak area integral of all the mass spectrum peaks was derived after obtaining the metabolic substance spectrum analysis data of different samples, followed by an integral correction performed for the mass spectrum peak of the same metabolite occurring in different samples , KEGG compound database, and MetWare database. Metabolites were identified based on their molecular weight, Mass Spectrometry (MS2fold change (FC) ≥ 1. Pathways with significantly regulated metabolites mapped to it were then subjected to metabolite set enrichment analysis; their respective statistical significance was determined using the hypergeometric test and its p-value.The “MetaboAnalystR 1.0.1” package in the R computing platform v 3.5.0  platform  and TransDecoder  software, respectively. Differentially expressed genes (DEGs) were analyzed using the DESeq2 (v1.22.2) software package in R, and subjected to the Benjamini–Hochberg method for multiple hypothesis testing  .A total of 18 cDNA libraries were sequenced using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® . Clean reads obtained from each cDNA library were assembled Actin (ACT) and elongation factor 1 (EF1) served as reference genes  as the plug used for inoculation were observed at 12 hpi . Over tiB. elliptica infection, we performed non-targeted metabolome analysis using control_6 h, control_24 h, control_48 h, inoculated_6 h, inoculated_24 h, and inoculated_48 h samples. A total of 524 metabolites common to all samples were identified based on their chromatographic and mass spectrometric parameters (r > 0.819) across all three biological replicates , Elaeis guineensis (8.72%), Phoenix dactylifera (7.39%), Vitis vinifera (3.63%), and Cajanus cajan (3.45%) gave the top BLASTx hits  ≥ 1 and Padj ≤ 0.05. All DEGs identified at the three temporal stages were analyzed using the K-means clustering algorithm. The DEGs could be grouped into six clusters and classified into four types  with those of control leaf samples  using cutoff values of logur types : up-reguur types . Gene onur types . This sutabolism . Taken tB. elliptica infection at the transcript level.A Venn diagram was used to demonstrate the DEGs identified at all three stages. Overall, 5,295 DEGs were detected, of which 125, 434, and 3,089 were uniquely expressed at 6, 24, and 48 hpi, respectively, and 72 DEGs were common to all three stages . Kyoto ELilium hybrid “Sorbonne” responds to B. elliptica infection. All the DEGs and DAMs participating in the phenylpropanoid and flavonoid biosynthesis pathways that exhibited up- or down-regulated trends in the different infection stages were selected and mapped to this network , a structural gene that catalyzes phenylalanine, clustering at 24 hpi. The expression of PAL (DN129152_c1_g2) in the inoculated treatment was 3.5- and 6.5-fold higher at 24 and 48 hpi, respectively, compared with the control  that catalyzes 4-hydroxybenzoic acid. The accumulated level of caffeic acid, a precursor of the lignin pathway, was 1.6-, 3.8-, and 11-fold higher in inoculated leaves at 6, 24, and 48 hpi, respectively, compared with the control , a structural gene that promotes the synthesis of the three abovementioned metabolites, was induced at all three stages (UDP-glycosyltransferase (DN130728_c1_g2) in the inoculated treatment increased significantly at 6 hpi, which could potentially explain the accumulation of kaempferol derivatives at 24 and 48 hpi. The accumulation of four downstream metabolites of kaempferol, namely kaempferol 3-O-β-d-glucopyranoside, kaempferol 3-O-β-glucosyl(1 → 2)(6′-O-acetyl)-β-D-galactoside, kaempferol 7-O-rhamnoside, and kaempferol 3-O-β-(2″-O-acetyl-β-D-glucuronide), was significantly increased in the inoculated treatment at 48 hpi compared with the corresponding control , one MYB30 homolog (DN134583_c1_g1), and one AS1 homolog (DN125418_c0_g1). Among these, DOF5.5 (DN129414_c0_g2) showed a regulatory relationship with the three defense response-related hub genes. In addition, one WRKY70 gene (DN139965_C0_G2) showed significant difference in expression between inoculated and control treatments at 6 hpi. Additionally, an ERF homolog (DN131819_c0_g10) and a HCT homolog (DN141355_c0_g2) were up-regulated at the early stage of infection. In the dark-green module (PBL19 homolog (DN136771_c2_g1), a CRK2 homolog (DN142167_c0_g1), and a WAK5 homolog (DN136340_c0_g1). Notably, PBL19 showed a strong regulatory relationship with other two hub genes, CRK2 and WAK5. A CKX9 homolog (DN129547_c0_g1), a I2′H homolog (DN140336_c1_g1), and a CAD homolog (DN127300_c1_g1) were significantly up-regulated in the dark-green module. At the late stage of the infection (WRKY22 homolog (DN139438_c8_g1), a WRKY30 homolog (DN126506_c0_g1), three JAZ homologs , a DELLA homolog (DN126417_c0_g11), a MAPK17/18 homolog (DN137625_c0_g), and a CML27 homolog (DN125403_c0_g1). Furthermore, both an RPS2 homolog (DN131875_c0_g1) and an RPM1 homolog (DN143855_c2_g1) displayed strong regulatory relationships with other genes in the green module. Genes in the dark-gray module  were up-regulated at all three infection stages (MYB61 homolog (DN137495_c4_g1), a WRKY33 homolog (DN123783_c0_g1), MYC2 homolog (DN143930_c9_g2), and a BRI1 homolog (DN141312_c0_g1), among others , which encoded an AP2-EREBP family TF, also exerted a great influence on plant–pathogen interactions.In the black module , seven hn module , we idennfection , 11 hub n stages . Analysig others . A numbeHCT and CSE) were significantly up-regulated in the inoculated treatment  enhances host plant resistance against invading pathogens  and conjugated compounds, which can be stored in vacuoles for long periods, increased with time post-inoculation. FLS and F3′H are responsible for the synthesis of kaempferol and quercetin, and were recently shown to be critical for disease resistance in plants  , genes including PBL19, CRK2, and WAK5, which are all members of the Interleukin-1 Receptor-Associated Kinase (IRAK) gene family, were instrumental in the defense against B. elliptica , necrotic lesions on leaves expanded, and JAZ, WRKY30, and WRKY22 genes played central roles in plant defense  receptor BRI1 , the SA and JA signaling pathways play a crucial role in the response of “Sorbonne” to fungal infection  and greater flagellin-induced activation of mitogen-activated protein kinases (MAPKs) than the wild type  for mediating bifurcate immune responses .The original contributions presented in the study are publicly available. This data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number DL and NC: conceptualization. ML and NC: methodology. NC, JX, and RZ: formal analysis and investigation. JX, NC, and ZS: writing—original draft preparation. DL, YC, and SS: writing—review and editing. YC: funding acquisition. ML: resources. DL and SS: supervision. All authors contributed to the article and approved the submitted version.This work was supported by the National Key Research and Development Program of China (2018YFD1000407).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Despite high vaccination rates in high-income countries, resurgence of pertussis cases is an occurring problem that urges the necessity of developing an improved vaccine. Likewise, the efficacy of vaccines for Bordetella bronchiseptica, which causes similar disease in pigs and companion animals, is debatable. A promising approach for novel vaccines is the use of outer membrane vesicles (OMVs). However, spontaneous OMV (sOMV) release by Bordetella spp. is too low for cost-effective vaccine production. Therefore, we investigated the influence of growth in various media commonly used for culturing Bordetella in the Bvg+, i.e. virulent, phase and of a heat shock applied to inactivate the cells on OMV production. Inactivation of the bacterial cells at 56 °C before OMV isolation greatly enhanced OMV release in both Bordetella spp. without causing significant cell lysis. The growth medium used barely affected the efficiency of OMV release but did affect the protein pattern of the OMVs. Differences were found to be related, at least in part, to different availability of the nutrient metals iron and zinc in the media and include expression of potentially relevant vaccine antigens, such as the receptors FauA and ZnuD. The protein content of OMVs released by heat shock was comparable to that of sOMVs as determined by SDS-PAGE and Western blot analysis, and their heat-modifiable electrophoretic mobility suggests that also protein conformation is unaffected. However, significant differences were noticed between the protein content of OMVs and that of a purified outer membrane fraction, with two major outer membrane proteins, porin OmpP and the peptidoglycan-associated RmpM, being underrepresented in the OMVs. Altogether, these results indicate that the application of a heat shock is potentially an important step in the development of cost-effective, OMV-based vaccines for both Bordetella spp.Pertussis, also known as whooping cough, is caused by the Gram-negative bacterium   OMVs are non-replicative blebs of 10–300 nm in size naturally released from the outer membrane (OM) of Gram-negative bacteria . A chall species . OMV pro species , and cel species . Periplahia coli . The inc in OMVs . Furtherngitidis .B. pertussis and B. bronchiseptica as a one-health approach. Various media have been described for the growth of B. pertussis. The first serum-free liquid medium for Bvg+, i.e. virulent-phase, B. pertussis was described by Hornibrook  derivative of Tohama I , were grown on Bordet-Gengou (BG) agar (Difco) plates supplemented with 15% (v/v) defibrinated sheep blood (bioTRADING) at 35 °C. For liquid cultures, bacteria were scraped from BG plates and pre-grown for two days in Verwey medium at 35 °C while shaking at 175 rpm. Cells were harvested, washed with physiological salt solution, and diluted to an optical density at 600 nm (OD600) of 0.05–0.1 in Verwey, SS, or THIJS medium . When indicated, media were supplemented with 1 g/L of heptakis -β-cyclodextrin (heptakis) (Sigma-Aldrich) to protect B. pertussis against fatty acids that are produced during growth (600 with a Novaspec III+ spectrophotometer (Biochrom). To kill bacterial cells, cultures were incubated for 1 h (unless otherwise notified) at 56 °C in a water bath. Viability of the cells was determined by spotting 10 µL of 10-fold serial dilutions of the cultures in physiological salt solution on BG agar plates. E. coli strains DH5α, BL21(DE3) and MG1655 were grown at 37 °C on lysogeny broth (LB) agar plates or in liquid LB while shaking at 200 rpm. For selection or plasmid maintenance, 100 µg/mL of ampicillin was added to the medium.Tohama I , and B. g growth . In some2.2g for 10 min, and supernatants were passed through 0.45-µm pore-size filters (Sarstedt). In the case of 100-mL cultures, cell-free filtrates were concentrated using 100-kDa cutoff centrifugal filter units (Amicon). OMVs were pelleted by ultracentrifugation for 2 h at 40,000 rpm and 4 °C , and resuspended in phosphate-buffered saline (PBS) or 2 mM Tris–HCl (pH 7.5).Bacterial cultures were grown for one day in conical tubes with an air:liquid ratio of 3.33:1 or, alternatively, for two days in baffled flasks with an air:liquid ratio of 5:1. Subsequently, cultures were incubated or not at 56 °C, as indicated. Bacterial cells were pelleted by centrifugation at 5000 x OMVs were quantified based on protein content using a bicinchoninic acid (BCA) assay (Pierce) or a Lowry DC protein assay (Bio-Rad) according to the manufacturers’ instructions or based on lipopolysaccharide (LPS) content using the purpald assay . For the2.3B. pertussis was grown for two days in SS medium and subsequently inactivated by incubation for 30 min at 56 °C. In the case of E. coli, an overnight culture of strain MG1655 was diluted 1:50 in fresh LB and grown for 3 h, after which the cells were either inactivated by incubation for 30 min at 56 °C or incubated for 30 min on ice. Cells were harvested by centrifugation at 10,000 rpm  for 10 min and washed with physiological salt solution. Spheroplasts were made as previously described . Then, 35 µL of 40 mg/mL lysozyme were added, followed by 14 mL of 1.5 mM EDTA (pH 7.5). The suspension was incubated for 30 min at RT. Spheroplasts were frozen at −80 °C, thawed, and 20 µg/mL of DNase and of RNase were added. Subsequently, spheroplasts were lysed by ultrasonication. Unbroken cells were removed via centrifugation for 10 min at 10,000 rpm for B. pertussis or at 2700 rpm for E. coli in an Eppendorf 5920-R centrifuge (FA-6 × 50 rotor). The supernatant was then centrifuged for 90 min at 40,000 rpm , and the resulting pellet was resuspended in 25 mM Tris–HCl, 1.25 mM EDTA (pH 7.5). Cell envelopes were loaded onto a discontinuous sucrose gradient consisting of a 3-mL cushion of 55%, four 2.5-mL layers of 50, 45, 40, and 35%, and a 1.8-mL top layer of 30% (w/w) sucrose, all in 25 mM Tris–HCl, 1.25 mM EDTA (pH 7.5). The sucrose gradient was centrifuged for 16 h at 25,000 rpm  at 4 °C and fractions were collected. Lactate dehydrogenase activity in the gradient fractions was determined as described  and mouse antiserum directed against BrkA were kindly provided by Nathalie Devos , and rabbit antisera directed against GroEL and against SecA were from our laboratory stocks.B. pertussis strain B213 using the primers listed in Table S2. Amplicons were cloned into pET16b (Novagen) after NdeI/BamHI restriction digestion, and the resulting plasmids were introduced in E. coli BL21(DE3). Strains containing the pET16b derivates were grown to an OD600 of 0.6, after which 0.5 (OmpP and ZnuD) or 1 mM (FauA) isopropyl β-d-1-thiogalactopyranoside was added, and gene expression was induced for 2 h. The recombinant proteins, containing an N-terminal six-His-tag, accumulated in inclusion bodies. Harvested cells were washed twice in 10 mM Tris–HCl (pH 8) and disrupted by sonication. Inclusion bodies were pelleted by centrifugation  and solubilized in 8 M urea, 100 mM glycine, 20 mM Tris–HCl (pH 8). Residual membrane fragments were pelleted by ultracentrifugation for 1 h at 100,000 x g, 4 °C. His-tagged proteins were purified by binding to Ni-NTA agarose beads (Qiagen), washed with a buffer containing 100 mM NaH2PO4, 10 mM Tris–HCl, 8 M urea, 20 mM imidazole, pH 8, and eluted with the same buffer containing 300 mM imidazole. Purified proteins were used to immunize rabbits at Eurogentec .To obtain antisera directed against the major porin OmpP (BP0840), and the TonB-dependent receptors BP3077 (ZnuD) and FauA, the corresponding genes were amplified without their signal sequence-encoding parts by PCR from chromosomal DNA of To obtain antisera directed against the IM protein FtsH (BP1077) and OM protein (OMP) RmpM (BP0943), peptides CLPETDRYSMDKERL and CASNKTREGRAQNRR, respectively, were designed with the OptimumAntigen design tool and used to immunize rabbits at GenScript . The N-terminal cysteine in both peptides was introduced for coupling to keyhole limpet hemocyanin as a carrier protein for immunization.2.5in situ prior to blotting. Membranes were developed with the Clarity Western ECL Blotting Substrate (Bio-Rad). The intensity of band signals was determined using Image Lab v5.2.1 (Bio-Rad).Bacterial cells, harvested by low-speed centrifugation, or isolated OMVs were mixed with sample buffer . Protein2.6Siderophores secreted by bacteria were detected by the chrome azurol S assay . Superna2.7Bacterial cells and OMVs were adsorbed to Formvar/carbon-coated copper grids for 10 min and washed three times with PBS. They were then fixed on the grids with 1% (v/v) glutaraldehyde in PBS for 10 min, washed twice with PBS and subsequently four times with Ultrapure water (Milli-Q). Samples were negatively stained by shortly rinsing the grids and subsequently incubating the grids for 5 min with methylcellulose/uranyl acetate (pH 4) on ice. Excess liquid was removed, and grids were air-dried. Samples were imaged using a FEI Tecnai 12 transmission electron microscope. OMV diameters were determined using ImageJ software.33.1B. pertussis strain B213 and B. bronchiseptica strain BB-D09-SR were grown in Verwey, SS or THIJS medium. It is noteworthy that, although SS and THIJS medium were successively developed as optimized media compared to Verwey medium, the final growth yield was generally even higher in the latter medium . After growth, the bacteria were either killed for 1 h at 56 °C or not. Subsequently, OMVs were isolated from equal amounts of bacterial cells, based on OD600, and analyzed by SDS-PAGE. For B. pertussis, OMV release was greatly enhanced by heat shock, and the highest amounts of OMVs appeared to be released by cells grown in Verwey medium  were isolated. Also under these conditions, heat shock enhanced OMV production by both species, but the yield of hOMVs appeared rather independent of the growth medium used  and BrkA, in the OMVs was investigated by Western blotting. BrkA was equally detectable in sOMVs and hOMVs, but Prn, although present in whole-cell lysates, was not detectable in either OMV preparation B. In anyExposing the cells for 1 h to 56 °C may denature OMPs. To evaluate the effect of incubation at 56 °C on protein conformation on a macroscopic scale, the heat modifiability of OMPs in the OMV preparations was assessed by semi-native SDS-PAGE. OMPs are generally β-barrels that retain their native conformation when not heated in sample buffer before SDS-PAGE, and their heat denaturation results in a different electrophoretic mobility . OMV sus3.3B. pertussis cultures showed ∼104-fold reduction in colony-forming units after 15 min of incubation at 56 °C . Similarly, ZnuD was repressed in SS medium supplemented with zinc . We considered the possibility that this deviation was due to the 30-min incubation at 56 °C used to inactivate the bacteria. For safety reasons, this step was unfortunately unavoidable as, after spheroplast formation and freezing of the spheroplasts at −80 °C, viable cells were still detected in the suspension, which is a risk in the subsequent sonication step because of aerosol formation. Hence, we turned to E. coli to investigate the effect of heat inactivation on membrane separation. Heat inactivation did not affect the buoyant density of the OM, as can be concluded by the presence of the highest amounts of the OM markers, the porins OmpF/C and OmpA, in fractions 5 and 6 in either case . In contrast, the IM marker SecA was found, as expected, in lower density fractions if the cells were not heat inactivated, but it shifted to the high-density fractions after heat inactivation . In conclusion, although heat inactivation dramatically affects the buoyant density of the IM, we succeeded to separate IM and OM of B. pertussis and to obtain purified OM fractions.To be able to directly compare the protein content of isolated OMVs and the bacterial OM, we wished to isolate a pure OM fraction. Therefore, the IM and OM were separated via isopycnic sucrose-gradient density centrifugation. Remarkably, whereas the bacterial OM usually has a higher buoyant density than the IM , the IM N. meningitidis  has been reported to result in cell death with concomitant OM blebbing in E. coli  of The Netherlands Organization for Scientific Research (NWO) with financial aid from GlaxoSmithKline Biologicals SA and PULIKE Biological Engineering Inc.This work was supported by a Eline F. de Jonge: Conceptualization, Investigation, Writing - original draft. Melanie D. Balhuizen: Investigation, Writing - review & editing. Ria van Boxtel: Investigation. Jianjun Wu: Investigation. Henk P. Haagsman: Supervision, Writing - review & editing. Jan Tommassen: Conceptualization, Supervision, Funding acquisition, Writing - original draft.Part of this work is included in a European patent application (EP20187477.3) with EFdJ, MDB, HPH, and JT as inventors. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


PLA2G6 is the causative gene for a group of autosomal recessive neurodegenerative disorders known as PLA2G6-associated neurodegeneration (PLAN). We present a case with early-onset parkinsonism, ataxia, cognitive decline, cerebellar atrophy, and brain iron accumulation. Sequencing of PLA2G6 coding regions identified only a heterozygous nonsense variant, but mRNA analysis revealed the presence of an aberrant transcript isoform due to a novel deep intronic variant (c.2035-274G > A) leading to activation of an intronic pseudo-exon. These results expand the genotypic spectrum of PLAN, showing the paramount importance of detecting possible pathogenic variants in deep intronic regions in undiagnosed patients. PLA2G6-associated neurodegeneration (PLAN) is a heterogeneous group of rare autosomal recessive neurodegenerative disorders caused by mutations in the PLA2G6 gene [2G6 gene . This ge2G6 gene . However2G6 gene –6.In general, genetic disorders causative mutations have been prevalently identified in exons and in RNA donor or acceptor splice sites. However, despite next generation sequencing (NGS) has revolutionized genetic testing, a considerable proportion of patients with a clinical diagnosis for a recessive condition have only one heterozygous mutation, suggesting the presence of not detected deep intronic variations. Intronic point mutations can activate pseudo-exons, such as intronic sequences flanked by apparently good-to-consensus acceptor and donor-site signals that are never recognized by the splicing machinery .PLA2G6 gene.Here, we report the first case of PLAN caused by a combination of nonsense and deep intronic variants in PLA2G6 transcript, PCR products were processed with Nextera XT DNA sample kit (Illumina) [This study was approved by the Ethics Committee of the Besta Institute with an informed, written consent. For sequencing of llumina) . Real-tillumina) . The c.2The patient is a 43-year-old lady born to non-consanguineous parents and with no family history of neurological diseases. Her symptoms started at age 32 years with gait ataxia and scanned speech, and, since age 36, a progressive asymmetric parkinsonian syndrome, with rigidity, hypokinesia, resting and postural tremor predominantly on the right, and cognitive decline. Response to L-Dopa was poor. She later developed behavioral abnormalities—irritability and occasional aggressiveness—treated with quetiapine. She progressively lost walking ability and developed dysphagia and severe akinesia.At 43 years, examination revealed severe cognitive decline in memory, linguistic, frontal-executive, and visual-spatial skills; pursuit saccadization, vertical upward gaze palsy, marked hypomimia, almost absence of spontaneous speech, which was scanned and monotonous, dysphagia, and drooling; severely hypokinetic gait, with double support; marked axial and limb rigidity, with trochlea sign, and generalized akinesia; oromandibular and bilateral upper limb resting tremor; increased deep tendon reflexes; and bilateral Babinski sign.Brain MRI showed T2 GRE hypointensity of the pallida, substantia nigra and head of the left caudate nucleus, cerebellar atrophy, T2 hyperintensity of cerebellar cortex and dentate nuclei, and clava hypertrophy, and diffuse cerebral atrophy with hyperintensity of the hippocampi Fig. .Fig. 1NeEEG showed some slow abnormalities in the temporal regions, more pronounced on the left side, photosensitivity, and photomyoclonic response, without clinical evidence of seizures. There was latency prolongation of the central components of somatosensory evoked potentials.PLA2G6 coding and flanking intron sequences revealed heterozygosity for the nonsense variant c.109C > T in exon 2 of the NM_003560.4 transcript (Fig. PLA2G6 by MLPA analysis. Therefore, we isolated RNA from patient’s fibroblasts and retrotranscribed it into cDNA to search for a second variant. Using intron-spanning primers pairs, we amplified an abnormal band of higher molecular weight in the patient that was not detected in controls (Fig. PLA2G6 transcript revealed the inclusion of a 118 nt region belonging to intron 14 (Fig. PLA2G6 transcript (Fig. PLA2G6 transcript (Fig. Sequencing of ipt Fig. , which iols Fig. . Sequenc 14 Fig. . Then, w 14 Fig. , which w 14 Fig. , Table 1 14 Fig. . Sequenc 14 Fig. . By NGS,ipt Fig. . Likewisipt Fig. , suggestPLA2G6 gene, causing the creation of a new donor splice site leading to a pseudo-exon inclusion by activating a preexisting cryptic acceptor splice site. This case, in which only one coding variant was detected at first, highlights that the existence of putative dominant variants in PLA2G6 should be reexamined [PLA2G6 mutant allele, missing the second mutation [It has been estimated that approximately half of the patients affected by rare genetic diseases remains without a definite molecular diagnosis, about 10% of which is due to pathogenic variants located deep within introns . Here, wexamined . Moreovemutation –15. Our


Hermetia illucens larvae (HIL) extract from crude-oil-extracted crushed powder prepared by using a developed automatic oil extractor for biodiesel production. We found the extract effectively reduced the survival of pathogens and antimicrobial-peptide-resistant bacteria. The results demonstrate that defatted HIL extract from crude-oil-extracted crushed powder prepared by using a developed automatic oil extractor should be used as a feed additive having antimicrobial activity with low production cost.We assessed the antimicrobial activity of Hermetia illucens (black soldier fly) larvae (HIL) are considered useful industrial insects for the production of feed for livestock, eco-friendly fertilizer from organic wastes, and biodiesel. Therefore, we evaluated the antimicrobial activity in the extract of crude-oil-extracted crushed HIL powder prepared from HIL fed organic waste containing fermented effective micro-organisms for biodiesel production. The result showed that antimicrobial activity was not fully induced in HIL fed L. casei-containing feed. In contrast, increased antimicrobial activity was observed in defatted HIL extract prepared from crude-oil-extracted crushed HIL powder. We found that the extract effectively inhibited the growth of pathogens and antimicrobial-peptide-resistant bacteria, such as three kinds of Salmonella species, and Enterococcus faecalis, Streptococcus mutans, Candida albicans, Serratia marcescens, and Pseudomonas tolaasii, with a minimum inhibitory concentration of 200–1000 µg/100 µL. Furthermore, no cytotoxicity to CaCO-2 human intestinal cells was observed in the extract. We also found that the production fee of extract equivalent to the antimicrobial activity of melittin was approximately 25-fold less than the production fee of melittin. Therefore, the results demonstrate that crude-oil-extracted crushed HIL powder prepared from HIL fed organic waste containing fermented effective micro-organisms for biodiesel production should be used as the feedstock for synthetic, preservative-free livestock feed and food additives. Taken together, the present study supports the usefulness of HIL as an eco-friendly feedstock in the biodiesel, agricultural, food, and feed industries. Hermetia illucens, in the production of fertilizer, livestock feed, and fishery feed [H. illucens larvae (HIL) can be used as protein-rich food for humans [Hermetia illucens is a useful insect for agricultural and eco-friendly industries.Many investigators have reported on the use of black soldier fly larvae, ery feed ,2,3,4. Fr humans . HIL werr humans ,7,8. TheIn modern times, the industrial farming of livestock has been established to improve livestock productivity because meat consumption has increased. Therefore, to improve productivity, intensive farming of livestock and animal growth promotion was required. The requirements were achieved by using antibiotics in animal feed. However, although antibiotics effectively prevent infectious diseases from developing, many problems, such as antibiotic resistance, antibiotic residues in livestock products, ecosystem pollution from livestock manure, and weak disease resistance in livestock, were caused by the abuse of in-feed antibiotics ,10,11. TMany researchers have investigated and searched for natural antibiotic substances and suggested that insects are potent natural antibiotic substances because insects produce antimicrobial peptides (AMPs) via activation of their innate immune system to prevent pathogen invasion ,13,14,15Lactobacillus casei using a fine needle [Salmonella species in L. casei-immunized HIL extract with increased expression of AMPs genes, such as cecropin 1 and defensin 1 [Organic wastes are colonized by micro-organisms. Therefore, with HIL intake organic waste, microbes can be ingested into HIL. As a result, the innate immune response in HIL should be activated and can produce AMPs ,20. In pe needle . Moreovee needle , and some needle ,24. We afensin 1 . The invHIL have also been studied as a source of biodiesel because they contain a high content of lipids . SeveralEscherichia coli (KCCM 11234) and three Lactobacillus strains, namely Lactobacillus brevis (KCCM 10553), Lactobacillus casei (KCCM 12413), and Lactobacillus fermentum (KCCM 11441), were obtained from the Korean Collection for Type Culture . The yeast Candida albicans (KACC 30071), the pathogenic bacteria Enterococcus faecalis (KACC 11859), Streptococcus mutans (KACC16833), Salmonella pullorum (KVCC-BA0702509), Salmonella typhimurium (KCCM 40406), and Salmonella enteritidis (KCCM 12021), and the AMP-resistant bacteria Serratia marcescens (KACC11961) and Pseudomonas tolaasii (KACC15293) were bought from the National Institute of Animal Science of the Rural Development Administration to assess the antibiotic activity of HIL extracts . All experiments were performed in Biosafety Level-2 laboratories approved by the Korean Disease Control and Prevention Agency.HIL were gifted from the Department of Agricultural Biology at the National Institute of Agricultural Sciences of the Rural Development Administration . HIL were routinely incubated at room temperature (26 ± 1 °C) in relative humidity of 60%. We purchased various bacteria strains to assess the antimicrobial activity of HIL extract. Six-day-old HIL were inoculated into feed prepared using a mixture of dried food waste (60 g/100 g), chicken manure (40 g/100 g), and waste cooking oil (2 mL/100 g) containing a fermented effective microbe (F-EM) solution (0.8%). F-EM is a fermented solution made by mixing cultures containing lactic-acid bacteria, photosynthesis bacteria, and yeast. HIL were grown for 11 days and harvested. The live HIL were put in a 700-W microwave and dried for 8.5 min.Dried HIL were defatted by expeller press, and crushed HIL powder was collected and stored at −20 °C until use. Defatted, crushed HIL powder (50 g) was suspended in 500 mL of 0–30% acetic acid solution and boiled at 100 °C for 10, 30, or 60 min. Then, the crushed HIL powder suspension was centrifuged at 4500 rpm for 30 min at 4 °C, and then the supernatant was removed and 0.45 µm filtered. The filtered supernatant was collected and dried for 16 h in a vacuum-spin drier. The dried supernatant was resuspended in sterilized 0.05% acetic acid solution and centrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was transferred to a new tube and held as defatted HIL extract. The extract was stored at −20 °C.E. coli and S. aureus. Underlay gel  was sterilized and liquefied by autoclave, mixed with each type of bacteria after cooling to 50–55 °C and poured onto a 100-mm square plate. After the underlay gel hardened, 3.5-mm diameter wells were prepared on the underlay gel, and then the defatted HIL extract was placed into the wells and held at 37 °C. After 3-h incubation, a sterilized overlay gel (6% tryptic soy broth and 1% low-electroendosmosis agar) was used to cover the underlay gel, and the gel plates were incubated for 18 h at 37 °C. Then, we determined antimicrobial activity of defatted HIL extract by measuring clear zone widths. We used melittin  as a positive control.A radial-diffusion assay (RDA) was used to evaluate the antimicrobial activity of the defatted HIL extract against 6 cfu/mL at 37 °C with 200 rpm shaking. Then, bacterial cultures were diluted to 1 × 106 cfu/mL, and 90 µL of the diluent was added to each well of a 96-well plate. The diluted bacterial cultures were incubated for 18 h at 37 °C after treatment with serially diluted extracts (10 µL). MICs were determined by normalized absorbance of bacterial cultures at 600 nm. We measured the absorbance of diluted extract solutions for each concentration to normalize absorbance in bacterial cultures treated at the same concentration.Antimicrobial activity of defatted HIL extract against bacteria was measured as MICs. To assess MICs, bacteria were grown in liquid media to 4 × 104 cells into 96-well plates and incubated cells to attach for 24 h in a 5% CO2 atmosphere at 37 °C. Then, culture medium was replaced with conditioned media containing various concentrations of defatted, crushed HIL powder extract (0–5000 µg/100 µL) and further incubated for 24 h. After the incubation, 20 µL of 5 mg/mL MTT reagent was administrated to each well and left for 4 h in the dark. Then, 200 µL of dimethyl sulfoxide was added to each well to dissolve the formazan after removing the conditioned media. The absorbance of formazan dissolved in dimethyl sulfoxide was measured at 540-nm wavelength using an ELISA reader.We analyzed the cytotoxicity of defatted, crushed HIL powder extract using Caco-2 human intestinal cells using 3--2,5-diphenyltetrazolium bromide (MTT) reagent. We plated 1 × 10t-test using SPSS Ver. 20.0 . A significant difference for each experimental group against control was considered when the p value was less than 0.05. All values were represented as means ± standard deviations (SD).Statistical analysis of data was performed by the one-way analysis of variance followed by Fisher’s LSD test or Student Firstly, we assessed the nutritional composition of the HIL and crushed HIL powder . The resE. coli and S. aureus. Then, we obtained the standard curve of the inhibitory zone of melittin to determine the relative content of the antimicrobial substance in HIL extract compared with melittin  was measured by RDA and compared to the activities of melittin using melittin A,B. The E. coli, a Gram-negative bacterium, and S. aureus, a Gram-positive bacterium. The result showed that the MIC for E. coli and S. aureus was 300 µg/100 µL (Enteroccocus faecalis, Streptococcus mutans, Salmonella pullorum, Salmonella typhimurium, Salmonella enteritidis, and Candida albicans. HIL extract effectively inhibited the survival of S. marcescens, S. pullorum, and S. typhimurium in a MIC range of 200–300 µg/100 µL (E. faecalis, S. mutans, and C. albicans were 200 µg/100 µL, 1000 µg/100 µL, and 1000 µg/100 µL, respectively (C. albicans.Based on the above results, the antimicrobial activity of defatted HIL extract on pathogenic bacteria survival was estimated by determining the MICs. Firstly, we evaluated the effect on the survival of g/100 µL . Next, wg/100 µL A, and thectively B. These S. marcescens and P. tolaasii, AMP-resistant bacteria, by determining the MIC. The result showed that 1–10 µg/100 µL of melittin did not affect the survival of S. marcescens and P. tolaasii (P. tolaasii with a MIC of 200 µg/100 µL, and the viability of S. marcescens was significantly decreased at a MIC of 300 µg/100 µL (AMP is a strong candidate to overcome antibiotic-resistant bacteria. However, some bacteria can recover from the cytotoxicity of AMPs. Therefore, we evaluated the antimicrobial activity of defatted HIL extract against tolaasii . In contg/100 µL . This reLactobacillus species, is higher, it cannot be used as a feed additive. Therefore, we assessed the cytotoxicity of defatted HIL extract to Lactobacillus species such as L. brevis, L. casei, and L. fermentum. As shown in Lactobacillus species were killed by 1 µg/100 µL of melittin. In contrast, the MICs of defatted HIL against Lactobacillus species were 1000 or 2000 µg/100 µL (Although defatted HIL extract has strong antimicrobial activity against pathogens, if the cytotoxicity to probiotics, such as g/100 µL A. The MIg/100 µL A. We alsg/100 µL B. IntereL. casei infection [Here, we showed the highest antimicrobial activity was observed in defatted HIL extract manufactured with a 20% aqueous acetic acid solution . In contnfection . TherefoL. casei using a fine needle against pathogens [Lactobacillus species (We showed antimicrobial activity of the extract of HIL infected by athogens ,25. In t species A. In pre species B. ConseqE. coli and 21.3 µg of melittin against S. aureus. This means that defatted, crushed HIL powder extracts equivalent to the antimicrobial activity of melittin can be produced at approximately 25-fold-lower production fees.The industrial use of HIL as an eco-friendly fertilizer, animal feed, and protein source has been suggested by many investigators ,34,35,36We found that defatted HIL extract has antimicrobial activities against pathogens and AMP-resistant bacteria with lower toxicity to probiotics and animal cells. The present investigation demonstrates that crude-oil-extracted crushed HIL powder can be used as an eco-friendly feedstock for feed and food additives having antimicrobial activity. Taken together, the present study supports the usefulness of HIL as an eco-friendly feedstock in the biodiesel, agricultural, food, and feed industries.


All the water atomized powders were sieved with different aperture sizes, of 32–75 μm, 75–125 μm, 125–200 μm, and <200 μm, and subsequently consolidated using hot pressing at 490 °C. The grain sizes were found to increase with increasing powder particle size, which also increased carrier mobility due to improved carrier transport. The maximum electrical conductivity of 1457.33 Ω−1 cm−1 was obtained for the 125–200 μm samples due to their large grain sizes and subsequent high mobility. The Seebeck coefficient slightly increased with decreasing particle size due to scattering of carriers at fine grain boundaries. The higher power factor values of 4.20, 4.22 × 10−3 W/mk2 were, respectively, obtained for large powder specimens, such as 125–200 μm and 75–125 μm, due to their higher electrical conductivity. In addition, thermal conductivity increased with increasing particle size due to the improvement in carriers and phonons transport. The 75–125 μm powder specimen exhibited a relatively high thermoelectric figure of merit, ZT of 1.257 due to this higher electric conductivity.Developing inexpensive and rapid fabrication methods for high efficiency thermoelectric alloys is a crucial challenge for the thermoelectric industry, especially for energy conversion applications. Here, we fabricated large amounts of p-type Cu The power generation mode is based on the Seebeck effect, where a temperature gradient between two different materials can develop a voltage. On the other hand, thermoelectric cooling is based on the Peltier effect, where cooling and heat can be generated at the junctions of two different materials by maintaining an electrical current ,2,3. Genin W/m-K ,5,6. Matin W/m-K . Many rein W/m-K ,8, Pb-Tein W/m-K , clathrain W/m-K , skutter [4Sb12) , Ag-Pb b [4Sb12)  in effor0.5Sb1.5Te3+x alloys at the grain boundaries, and achieved a ZT of 1.8 at 320 K [Among them, the binary and ternary compounds of bismuth-telluride (Bi-Te) based alloys exhibit excellent room temperature thermoelectric performance due to their unique properties, including a narrow band gap and ease of doping with metallic/semiconductor elements ,8. In that 320 K . They arat 320 K  at the iat 320 K . Cao et at 320 K .2Te3 + 75%Sb2Te3 alloys using GA followed by a spark plasma sintering process, and investigated the influence of powder size on thermoelectric properties. In this report, the grain size increased with increasing powder size and, as a result, electrical conductivity was increased, due to increasing carrier mobility values. We obtained a maximum ZT of 1.2 with a 32–75 μm specimen due to the high power factor and reasonable thermal conductivity [Despite these improvements in thermoelectric and mechanical properties, the ascribed powder metallurgical processes have low production rates and are expensive and time-consuming methods. In comparison, the preparation of thermoelectric alloys using gas atomization (GA) process is an excellent alternative. GA can be used to fabricate desired powders at a large scale through a rapid solidification process. It can produce 2–3 kgs/min of desired powders with a homogeneous chemical composition and fine-grained alloys . Recentluctivity . However2Te3-based pellets due to its simple procedure, and produces a high density [0.07Bi0.5Sb1.5Te3 alloys were fabricated in large-scale quantity using water atomization followed by hot pressing, rather than the un-doped BiSbTe and SPS in the previous work. In the present work, we have mainly focused on the effect of different WA particle sizes on microstructural features, and their subsequent effect on thermoelectric properties and mechanical properties were systematically investigated. In addition, the suppression of intrinsic/bipolar conduction due to the Cu content in vBi0.5Sb1.5Te3 alloys was also explored at high temperatures.In the WA process, it is not necessary to maintain a vacuum or have inert gas in the chamber, as in the GA process, which significantly reduces production time and cost. Hot pressing is a traditional sintering method used to make Bi density . Cu is c0.07Bi0.5Sb1.5Te3 alloys. The atomization chamber was initially filled with up to 60% water instead of inert gas. All of the elements were placed into a graphite crucible inside an induction furnace and maintained 100 °C above the melting point, as monitored by a thermocouple. Later, the melt solution was poured into a boron nitrate-based nozzle set up, of 5 mm in diameter, and disintegrated with the water stream at 40 bar pressure. The fabricated wet powder was collected and dried in a vacuum chamber at 180 °C for 8 h. The fabricated powders had a wide range of particle sizes due to the different in cooling rates during the process. A high cooling rate resulted in a fine powder size, while the slow cooling rate resulted in coarse powders [High purity (5N) of Bi, Sb, Cu, and Te were used  according to the stoichiometric portions of the Cu powders ,22. To i3) for Seebeck coefficient and electrical conductivity, and into square pieces  for measurement of thermal conductivity. The electrical conductivity and Seebeck coefficient were measured by thermoelectric power measurement system  from room temperature to 500 K. The thermal diffusivity (D), and specific heat (Cp) were measured using the laser flash method (Netzsch-LFA-467 system) and a differential scanning calorimeter (PerkinElmer DSC8000). The Hall mobility and carrier concentrator were measured at room temperature using a four probe-hall system (Ecopia HMS-3000). The thermoelectric figure of merit, ZT was calculated using the Seebeck coefficient, electrical conductivity, and thermal conductivity (D × Cp × d). All thermoelectric transport properties were measured in the direction perpendicular to the pressing.The powder morphology, microstructure and bulk fracture surface analysis were characterized using scanning electron microscopy (SEM- MIRA-LMH II (TESKAN)). The particle size was measured using laser diffraction particle size analyzer . The density (d), was measured 10 times using the Archimedes principal, and we averaged its value. Micro-Vickers hardness was obtained using a Vickers indenter measurement system with an indenting load of 300 gF. The oxygen contamination was analyzed using an Oxygen Nitrogen Hydrogen—2000 analyzer (ELTRA GmbH). The crystal structures of the powders and bulk samples were examined using an X-ray diffractometer at room temperature . The hot-pressed samples were cut into small rectangular pieces about  32–75 μm, (b) 75–125 μm, (c) 125–200 μm, and (d) <200 μm. The insets in (a)–(d) show their corresponding high magnification images. All the water atomized (WA) powder particles exhibited irregular/polygonal shapes. The rapid cooling rate due to the water stream during the atomization process was responsible for the formation of the irregular shapes, unlike the spherical powders produced by gas atomizer. The powders are clearly distinguishable according to their powder size, and the highly magnified images revealed that the water atomized powder were highly compacted. 0.07Bi0.5Sb1.5Te3 powders fabricated by water atomization. Average particle sizes of about 38.84 μm, 106.09 μm, 169.50 μm, and 72.24 μm were obtained for the 32–75 μm, 75–125 μm, 125–200 μm, <200 μm powders, respectively. This PSA data is consistent with the SEM powder morphology in 0.07Bi0.5Sb1.5Te3 samples, sintered using hot pressing. Laminar structures can be clearly observed on the fracture surface [ surface ,20. In a0.07Bi0.5Sb1.5Te3 alloys. All specimens showed a relative density of over 98% of their theoretical density. This confirms that combining water atomization and hot pressing can successfully provide higher density values. The mechanical properties of the TE materials are crucial to reduce wastage during TE module preparation. The micro-Vickers hardness gradually decreased with increasing powder size, mainly due to increased grain size. The 32–75 μm specimen showed a peak hardness of 53.92 Hv, which is higher than reported for single crystal samples [ 49-1713 . The Cu  samples .0.07Bi0.5Sb1.5Te3 alloys as a function of temperature. The electrical conductivity of all samples decreased quasi-linearly with increasing temperature, reflecting their highly degenerate semiconducting nature. The electrical conductivity (σ) can be expressed in terms of carrier concentration (n), and mobility (μ) as: σ =neμ, where e is the charge of the carrier [n and μ values were measured and are listed in 2, 3.1 × 102, and 3.3 × 102 cm2/Vs were measured for the 32–75 μm, 75–125 μm, and 125–200 μm specimens, respectively. It was noted that the μ values gradually increased with increasing powder size, from the 32–75 μm specimen to the 125–200 μm specimen, due to increasing grain sizes, which is consistent with the earlier report [2Te3 crystal system exhibits anisotropic electronic properties. Recently, Manzano et al. prepared highly oriented Bi2Te3 films by pulsed electrodeposition, and achieved high electronic transport properties perpendicular to the c-axis that were about 5 times greater than parallel to the c-axis [n and μ are inversely proportional to each other. It is evident from −1 cm−1 due to high mobility. The electrical conductivity values gradually decreased with decreasing powder size, due to reduced grain sizes. Fine grains can severely scatter carriers due to the high density of grain boundaries, which causes a decrease in mobility, and which can be solely responsible for the decreasing electrical conductivity. carrier . The n ae c-axis . This oce c-axis ,20. In aα) of the p-type Cu0.07Bi0.5Sb1.5Te3 alloys as a function of temperature. The positive sign confirms the p-type semiconducting nature of all specimens [α values of all samples increased with temperature up to 425 K, then decreased. The decreasing α values were mainly due to the excitation of minority carriers which contributed to the intrinsic conduction at high temperature [0.5Sb1.5Te3 WA powder specimens exhibited a more feeble excitation of minority carriers than previously reported pure Bi-Sb-Te alloys prepared by GA + SPS, as the addition of Cu controls the intrinsic conduction [−3 W/mK2 for the larger powder size samples, 125–200 μm and 75–125 μm, respectively, due to their higher electrical conductivity values. The present specimens prepared with water atomization powders revealed a higher power factor than previously reported gas atomized specimens, due to their high electrical conductivity, resulting from the 0.07 wt% Cu content in the Bi0.5Sb1.5Te3 matrix [pecimens ,23. Irrenduction ,18. From3 matrix ,20.0.07Bi0.5Sb1.5Te3 alloys as a function of temperature. The total thermal conductivity (κ) is the sum of the electronic constituent (κe) and phonon constituent (κph) [e was calculated using the Lorenz number, and is shown in 0.5Sb1.5Te3 matrix [nt (κph) ,18,19. T3 matrix . Among a3 matrix . If the 0.5Sb1.5Te3 matrix is credited for the higher values of ZT, due to high electric conductivity, compared to pure BiSbTe from the gas atomization process [The figure of merit ZT for the different investigated powder size samples are presented in  process . The hig0.07Bi0.5Sb1.5Te3 alloy powders using an inexpensive and rapid solidification process, water atomization, and sintering by hot pressing. The 32–75 μm powders contained high oxygen contamination due to their high surface to volume ratio. It was determined that the grain size gradually increased with increasing particle size, which severely impacted the mechanical and transport properties of the thermoelectric materials. The 32–75 μm specimen displayed the highest Vickers hardness of 53.92 Hv compared to other specimens, due to its fine grain structure. A noticeable increase in carrier mobility of about 330 cm2 V−1 s−1 was observed for the 125–200 μm specimen. This was also responsible for the increase in electrical conductivity, to 1457.33 Ω−1 cm−1, reflecting the higher carrier transport due to coarse grain size. The peak Seebeck coefficient of 208.52 μV/K as well as the lowest thermal conductivity of 1.17 W/m-K were exhibited by the 32–75 μm specimen, due to the severe scattering of carriers (low carrier mobility) and phonons at the fine grain boundaries. The maximum figure of merit, ZT of 1.257 was exhibited by the 75–125 μm powder specimen, due to the high electrical conductivity values.We fabricated a large quantity (2–3 kgs) of p-type Cu


Triticum aestívum L. and Secale cereále L. are widely used in the production of bakery products. From the total volume of grain cultivated, roughly 85% is used for the manufacturing of flour, while the remaining part is discarded or utilized rather inefficiently. The limited value attached to bran is associated with their structural complexity, i.e., the presence of cellulose, hemicellulose, and lignin, which makes this material suitable mostly as a feed supplement, while in food production its use presents a challenge. To valorize these materials to food and pharmaceutical applications, additional pre-treatment is required. In the present study, an effective, sustainable, and eco-friendly approach to ferulic acid (FA) production was demonstrated through the biorefining process accomplished by non-starch polysaccharides degrading enzymes. Up to 11.3 and 8.6 g kg−1 of FA was released from rye and wheat bran upon 24 h enzymatic hydrolysis with multi-enzyme complex Viscozyme® L, respectively.Historically  Triticum aestívum L.) and rye  in the last five decades has increased by 190.5% from 257.4 million tons (Mt) in 1961 to 747.9 Mt in 2018. From the total volume of wheat and rye cultivated, roughly 85% or 635.7 Mt is utilized for flour production, while 10–15% or 74.8–112.1 Mt is converted into by-products, i.e., wheat and rye bran. These by-products are discarded or utilized rather inefficiently [Bifidobacteria in the human intestinal tract [One of the challenges facing food technology specialists at present is the utilization of agro-industrial by-products and food waste materials. Burgeoning demand for plant-made food products has resulted in a sharp increase in food processing waste, including cereal bran . Accorditrans- and cis-isomers, so additional purification process needs to be performed. Therefore, it may be beneficial to identify alternative sources and develop technologies for the release of FA that can be cost-effective and environmentally friendly. It has been proposed that under the proper conditions, hydrolysates which arise during EH of by-products might be purified and used for the production of FA that afterward could be utilized for the manufacturing of natural and inexpensive HPLC standards (a mixture of FA isomers or pure trans-FA), lipid oxidation inhibitors, agents with antimicrobial activities as medicines, and food additives [Ferulic acid (FA) 3-(4-hydroxy-3-methoxyphenyl)acrylic acid is hydroxycinnamic acid representative that is produced mostly by chemical synthesis at industrial-scale. The manufacturing of FA by this way is reported to be a laborious and time-consuming process (up to three weeks) that requires significant input of conditionally toxic reagents , solvents (piperidine), and catalysts (benzylamines) . Moreovedditives ,14,15.AspergiIlus niger CBS 120.49 and the endo-1,4-β-xylanase (EC 3.2.1.8) from Trichoderma viride [−1. Even though promising yields of FA were obtained, it should be mentioned that the production  of FE is a labor and material-costly process that makes this product applicable exclusively for research purposes and in vitro diagnostics.In nature, FA appears as a component of plant cell-walls and forms a three-dimensional structure with cellulose, hemicellulose, and lignin . The rola viride . This asa viride  who intrEC 3.2.1. from TriThe limited information on the recovery of FA from other cereal by-products, in particular from rye bran as well as for valorization purposes of this material, promoted the design of this study addressing the evaluation of the yield of FA from wheat and rye bran through biorefining process accomplished by C-DEs. The efficiency of FA release through hydrolytic enzymes will be compared with the conventional approach utilizing either alkali-assisted hydrolysis or micro-saponification. Triticum aestívum L.) and rye . Both brans consisted of outer pericarps, inner pericarps , seed coats (testa), hyaline, and aleurone layers with attached starch granules. The proximal analysis of the bran samples is shown in Two types of commercial food-grade bran samples harvested in 2019 were retrieved from the Latvian Mill Joint Stock Company (JSC) “Rigas dzirnavnieks” , separated as summer wheat (w/v), loaded into 25 mL reagent bottles with screw caps , and subjected to autoclaving using a digital autoclave with counter-pressure  for 10 min at 121 ± 1 °C temperature and counter pressure 2.0 Pa. Upon thermal processing, the liquid fraction was collected for further chromatographic analysis , while the solids were freeze-dried using a FreeZone freeze-dry system  at −51 ± 1 °C under a vacuum of 0.065–0.070 mbar for 72 h. Dried samples were packed in polypropylene zip-lock silver bags  (200 g in each) and stored at a temperature of −18 ± 1 °C until further analysis and use, a maximum of 4 wk. Moisture content was analyzed gravimetrically as proposed by Ruiz [Each bran sample before EH was defatted following the methodology described by Radenkovs et al.  and grou by Ruiz .trans- and cis-isomers of ferulic acid (FA), vanillic acid (VA), and p-coumaric acid (p-CA) were purchased from Sigma-Aldrich Chemie Ltd., . Ethylenediaminetetraacetic acid disodium salt 2-hydrate (Na2EDTA) was acquired from Panreac Quimica S.L.U. . Hydrochloric acid (HCl), formic acid (CH2O2) (puriss r.a.), sodium hydroxide (NaOH), potassium hydroxide (KOH), citric acid (C6H8O7), sodium citrate dihydrate (C6H5Na3O7·2H2O) of reagent grade, methanol (MeOH), ethanol (EtOH), and acetonitrile (MeCN) of UHPLC grade were purchased from Merck KGaA . The ultrapure water was produced using the reverse osmosis technique Mili-Q® Direct water purification system . Phenolic compound commercial standards, i.e., caffeic acid (CA), a mixture of ®”  for laboratory purposes. Three types of starch and N-SPs degrading enzymes, i.e., Amylase® AG XXL (AMY), Celluclast® 1.5 L (CELLU), Viscozyme® L (VISCO) were used to hydrolyze carbohydrates. High purity recombinant FE from rumen microorganism was obtained from Megazyme™  to liberate esterified FA forms from plant material matrix during an additional hydrolysis process. In this work, enzymes were tested both independently and in combination, in order to establish the synergetic effect. The list of enzymes used is given in Industrial starch and N-SPs degrading enzymes preparations have been kindly provided by the company “Novozymes®, Büchenbach, Germany). Afterward, the mixture was subjected to ultrasound treatment at 42 kHz with output wattage of 100 W for 10 min at 25 ± 1 °C using a “Branson 3510” ultrasonic bath , followed by pH adjustment with 4 M HCl to reach pH 4.0 ± 0.2 and centrifuged at 20,160× g at in an “EBA 21 Hettich” centrifuge  for 10 min at 25 ± 1 °C. Following centrifugation, the supernatants were decanted and 3 times extracted with 3 volumes of ethyl acetate:diethyl ether . The organic layer was collected in a pear-shaped flask and vacuum dried using a Laborota 4000 vacuum rotary evaporator . The yellowish powder was then re-dissolved in 10 mL of 70% acidified MeOH (methanol:DDW:formic acid ratio 70:24:1 v/v/v). The collected extracts were filtered through a polyvinylidene fluoride membrane filter (PVDF) with a pore size of 0.22 µm  before analysis. Filtered samples were diluted appropriately before HPLC-DAD-ESI/MSn and UPLC-ESI-QTOF/MS analysis. To estimate the total amount of bound FA (ester-linked), the alkali-assisted hydrolysis of bran samples was done according to the method proposed by Zhang et al.  as a conv/v) in 25 mL reagent bottles with screw caps (VWR™) followed by 1 min intensive Vortexing using an “IKA TTS2” mixer  and incubation in a water bath at 60 ± 1 °C for 1 h at 85 rpm. After incubation, the samples were subjected to ultrasonication for 10 min at 25 ± 1 °C, followed by pH adjustment with 4 M HCl to reach pH 4.0 ± 0.2 and centrifugation at 25 ± 1 °C for 10 min at 20,160× g. The obtained hydrolysates before chromatographic analysis were filtered using 0.22 µm PVDF.In this experiment, a micro-saponification of wheat and rye bran by methanolic KOH was performed for de-esterification of bound FA. Briefly, 1 g of bran was mixed with 10 mL 1 M KOH in 80% MeOH , the process of EH was done within the range of 4–24 h depending on the enzyme used. The optimal conditions for each enzyme were selected individually based on the Novozymesribed by ,29. The −1 of endo-1,4-β-xylanase (VISCO) or 10 EGU mL−1 of 1,4- β-d-endoglucanase (CELLU) was added to 100 µg of bran sample. The mixture was then Vortexed for 2 min and incubated in a water bath at 44 ± 0 °C for 24 h, at 85 rpm, respectively. The release of FA at different time points, i.e., 4, 8, 12, and 24 h of enzymatic hydrolysis, was ascertained chromatographically. For this purpose, 1 mL 0.5 M sodium citrate buffer containing either 6 FBG mLg for 10 min at 25 ± 1 °C. Before analysis, the supernatants were filtrated through 0.22 µm PVDF membrane filters. The kinetic of hydrolytic enzymes was estimated by the selective measurement of glucose level in hydrolysates using a commercial blood glucose meter  as proposed by Heinzerling et al. [Before chromatographic analysis to terminate the reaction, the obtained hydrolysates were subjected to ultrasonication at 42 kHz for 10 min at 25 ± 1 °C, followed by centrifugation of 20,160× g et al. .Step II. A set of experiments was done to estimate the synergistic effect of sequential hydrolysis of bran samples utilizing either amylolytic enzymes alone or in combination with N-SPs degrading enzymes.Aspergillus niger was used. The selected enzyme does not require additional alkalization of the material, which makes it attenable to maintain constant pH of substrate for both cellulolytic and amylolytic enzymes. In brief, 100 µg of bran sample was mixed with 1.0 mL 0.1 M sodium citrate buffer (pH 4.6), containing 10 AGU mL−1 of glucan-1,4-α-glucosidase (AMY) and incubated either for 1 or 4 h in a water bath at 55 ± 1 °C under constant agitation of 85 rpm. To terminate the reaction, the slurry was then subjected to ultrasonication at 42 kHz for 10 min at 25 ± 1 °C using an ultrasonic bath. Thereafter, an aliquot of 300 µL was collected and centrifuged at 20,160× g for 10 min at 25 ± 1 °C. The supernatants collected were then filtrated through 0.22 µm PVDF membrane filter. The amount of FA and other hydroxycinnamates was quantitatively and qualitatively evaluated by using the above-mentioned systems. The following EH process for 4 and 24 h was done using destarched slurry as a substrate for endo-1,4-β-xylanase (VISCO) and 1,4- β-d-endoglucanase (CELLU). The amount of enzymes added corresponded to 6 FBG mL−1 and 10 EGU mL−1 in 0.1 M sodium citrate buffer.For destarching purposes, AMY  isolated from w/v) Na2EDTA, 0.02% (w/v) sodium azide, and 7.5 U mL−1 (20 µL) of FE [Step III. To test the synergistic effect of endo-1,4-β-xylanase (VISCO) and FE (Megazyme™), the sequential EH of wheat and rye bran was accomplished with the use of defatted, destarched slurry with the following determination of FA in hydrolysates . The relL) of FE . The yiep-CA. All calibration curves had R2 > 0.98.Quantification of compounds was done using calibration curves of the following standards: CA, FA, VA, −1 at room temperature (25 °C), with the elution gradient being 1.0% formic acid (solvent A) and pure ACN (solvent B). A stepwise gradient elution program from 15% solvent B up to 20% in 25 min, from 25 to 55 min solvent B concentration was increased to 70% and held for 5 min. The injection volume was 5 μL. Spectral data from all peaks were accumulated in the range of 240–400 nm, and chromatograms were recorded at 320 and 280 nm. For the identification of hydroxycinnamates, a Bruker series LC/MSD HCT Ultra ion trap detector with electrospray ionization (ESI) interface  was used. The ionization conditions were adjusted at 350 °C and 4.0 kV for capillary temperature and voltage, respectively. The nebulizer pressure and flow rate of nitrogen (N2) (purity–99.0%) were 65.0 psi and 11 L min−1, respectively. The full scan mass covered the range from 100 up to 1200 atomic mass units (AMU), with target mass of 400 AMU. Collision-induced fragmentation was performed in the ion trap using helium  as the collision gas, with voltage ramping cycles from 0.3 up to 2 V. Mass spectrometry data were acquired in the negative ionization mode. MS2 was carried out in the automatic mode on the more abundant fragment ion in MS.Quantitative analysis of FA was done using an Agilent HPLC 1200 series system  as previously described by Ferreres et al. . The HPLm/z ratios. Sample of 1 μL was injected onto a reversed-phase Luna Omega column  with SecurityGuard ULTRA cartridges of the same material operating at 30 °C and a flow rate of 0.5 mL min−1. The mobile phases used were acidified DDW (0.1% formic acid) (A) and acidified ACN (0.1% formic acid) (B). Separation of compounds was done using the following gradient conditions: elution started with 1% B to obtain 10% B at 8 min, 30% B at 14 min, 50% B at 16 min, and 1% B at 20 min. Furthermore, MeOH injections were included every three samples as a blank run to avoid the carry-over effect.The analyses were carried out using an Agilent 1290 Infinity series LC system coupled to a 6550 I-Funnel Accurate-Mass QTOF  with a dual electrospray ionization interface (ESI-Jet Stream Technology) for simultaneous spraying of a mass reference solution that enabled continuous calibration of detected −1, nebulizer pressure of 45 psi, sheath gas temperature of 400 °C, sheath gas flow of 12 L min−1, a capillary voltage of 3500 V, nozzle voltage of 500 V, fragmentor voltage of 100 V, skimmer of 65 V and octopole radiofrequency voltage of 750 V. TOF spectra acquisition rate/time was 1.5 spectra s−1 and 666.7 ms/spectrum, respectively, and transients/spectrum were 5484. The mass range was between 50 and 1100 m/z. At the beginning of the batch, the instrument was calibrated to assure mass accuracy during the MS analysis using a mixture of reference compounds (Tuning Mix). Furthermore, continuous internal calibration was performed during analyses using signals m/z 112.9855 and m/z 1033.9881 in negative polarity and m/z 121.0509 and m/z 922.0098 in positive polarity. Auto recalibration reference mass parameters were a detection window of 100 ppm and a minimum height of 1000 counts. Data were processed using the Mass Hunter Qualitative Analysis software .Data were acquired using the Mass Hunter Workstation software . The system was operated using both negative and positive ion polarity, and data were acquired in centroid and profile mode, with a data storage threshold of 5000 absorbance for MS. The operating conditions were as follows: gas temperature of 280 °C, drying nitrogen gas of 9 L min2O = 1.0–4.8 torr) (PH2O = 0.1–1.0 torr) using a large field detector (LFD). For ESEM analysis, samples were mounted onto SEM stubs using double-sided adhesive carbon discs and structural changes were observed at 10–20 kV acceleration voltage. To improve sample electrical conductivity and ability to reflect electrons and hence ensure higher resolution of images, all samples were covered with a 15 nm thick gold layer before analysis.The morphology of untreated control and EH bran was analyzed by SEM using a Tescan Mira/LMU scanning electron microscope  according to the method proposed by Rahman et al. . The conn = 3). The p-value < 0.05 was used to denote significant differences between mean values determined using one-way analysis of variance (ANOVA) and the Duncan’s multiple range test done using the assistance of IBM® SPSS® Statistics program 20.0 .The results obtained are shown as means ± standard error of the mean from three replicates  higher than that reported by Ideia et al. [p-vinylphenol (p-VP)  from agro-industrial by-products, including wheat bran ,20,23,32ectively A. The yia et al. . It is wl (p-VP) C as the p-CA to p-VP corresponds to 81.0:19.0 and to 45.2:54.8, respectively. This finding supports the conclusion that the formation of 4-VG  and p-VP  might take place not only during the pyrolysis and microbiological conversion via decarboxylation pathway as reported by Ohra-Aho et al. [The intensities of peaks indicate the relative abundance of these two compounds in alkali-hydrolyzed samples, the percentage ratio of FA to 4-VG and o et al.  and Salgo et al. , but als® (protease) Termamyl® (α-amylase), and Peptopan® . The yield of FA using this approach was somehow similar to that found in this study applying alkaline-hydrolysis with 2 M NaOH for 1 h. The amount of FA recovered was 0.82–1.05 g kg−1 of bran. Very recently, in an attempt to effectively recover FA, the sequential hydrolysis of wheat bran biorefining has been developed by an Italian research group . An obse−1, respectively  leaves and their hydrodistilled residue by-products. To validate the ability of N-SPs degrading enzymes alone or in combination with glycoside hydrolase (AMY) and FE (Megazyme™) to release the bound FA forms, a serial EH of wheat and rye bran was applied. In this set of experiments, the wheat and rye bran samples underwent EH for 4 to 24 h though N-PSs, i.e., CELLU and VISCO to solubilize cellulose and hemicellulose. As is seen, the highest concentration of FA in both cases using either CELLU or VISCO yielded after 24 h with enzyme doses of 10 EGU and 6 FBG mLectively . Considei et al. , indicat−1 for wheat and rye bran hydrolysates after EH with CELLU, with rye bran having the highest glucose level, with wheat bran the lowest  > VISCO 3 FBG (r = 0.9117) > CELLU 5 EGU (r = 0.8357) > VISCO 6 FBG (r = 0.7642). Similar results were obtained carrying out correlation analyses of glucose level with FA in rye bran hydrolysates, although with a different order of correlations: CELLU 10 EGU (r = 0.9804) > VISCO 6 FBG (r = 0.9744) > VISCO 3 FBG (r = 0.9524) > CELLU 5 EGU (r = 0.8017). The results show that since VISCO is a multi-enzyme complex containing a wide range of carbohydrases, i.e., cellulases, arabanases, β-glucanase, hemicellulase, and xylanases where the end products of synergistic action could be glucose, arabinose, mannose, galactose, and xylose, the release of FA is to a lesser extent associated with the accumulation of glucose. This finding is reinforced by report of Shin et al. [p < 0.05) lower in hydrolysates after EH with CELLU, while the strongest correlation between FA and glucose indicates that partial solubilization of arabinoxylans could be achieved even through the application of cellulose-degrading enzyme CELLU as a sole enzyme [The main substrates for the cellulolytic enzymes used in this study were those non-starch polysaccharides present in wheat and rye bran. Since the main activity of hydrolytic enzymes used is cellulolytic, in addition to the FA content, the efficiency of hydrolysis was estimated by measuring the amount of glucose in hydrolysates. The results of glucose content show values ranging between 11.23 and 31.20 mmol Le lowest . The maxe enzyme .d-glycosidic linkages in cellulose and on 1,2-α-, 1,3-α-, and 1,5-α-L-arabinofuranosidic and 1,4-β-d-xylosidic linkages in arabinoxylan, arabinan, and other arabinose-containing hemicelluloses, releasing relatively shorter polysaccharides or oligosaccharides composed either of glucose or xylose [The decomposition of bran N-SPs caused by EH was confirmed by SEM . After 2In general, EH has led to a decrease of total dietary fiber combined with a shift from water-insoluble fiber (W-I) to water-soluble (W-S). The results are consistent with those observed by Arrigoni et al. , noting −1 of AMY at 55 °C. At the end of the process, destrached slurry was subjected to ultrasonication for 30 min to terminate the catalytic reaction. Afterward, the CELLU or VISCO in the dose of 10 EGU and 6 FBG mL−1, respectively, was separately added followed by EH for 4 or 24 h. Both combinations were assayed for FA release.For the specific release of FA from wheat and rye bran cell-wall matrix, commercial FE and AMY were tested alone or in combination with CELLU and VISCO enzymes. Before EH with cellulolytic enzymes, wheat and rye bran samples were destarched for 1 or 4 h with 10 AGU mLAn earlier study of Yu et al.  showed td-glycosidic linkages between glucose monomers in amylose and amylopectin, converting these polymers into simple sugars such as glucose, maltose, and maltotriose [This was not surprising, since α-amylase acts specifically on the 1,4-α-totriose . Since btotriose . A combitotriose .−1  from lignocellulosic biomass. For these purposes, two commercial food-grade types of grain by-products, i.e., wheat and rye bran, were used as substrates for hydrolytic enzymes. The process of enzymatic hydrolysis (EH) was done by sequential biorefining, applying four hydrolytic enzymes. The EH was performed under optimal conditions for each enzyme and the release of FA was monitored over 24 h. Among the enzymes tested, the superiority of multi-enzyme complex Viscozyme® L has been highlighted, as the maximal yield in the amount of 11.3 and 8.6 g kg−1 FA from rye and wheat bran was obtained upon 24 h of EH, respectively. Up to 75.4 and 135.6% release of the total alkali-extractable FA was achieved from wheat and rye bran, respectively. No synergistic effect was observed combining Viscozyme® L either with feruloyl esterase or Amylase® AG XXL. When combining Celluclast® with Amylase® AG XXL up to the 1.1-fold and 0.9-fold increase in the yield of FA was found as compared to Celluclast® when used alone.The present study was undertaken to establish the ability of hydrolytic enzymes to act on 1,4-β-® L confirmed the presence of degradation products of FA and p-coumaric acid to a lesser extent than that of alkaline-hydrolysis, indicating gentler decomposition of bran matrix and fewer negative impact on compounds of interest. In the process of enzymatic hydrolysis, the formation of glucose as the final product of cellulose depolymerization was observed in this study. In the context of the zero-waste biotechnological process, the glucose arisen could be further exploited for bioethanol production, while solid residues may find applicability within the food and agricultural sector as a material suitable for synbiotic food products and compost production.The UPLC-ESI-QTOF/MS analysis of enzymatically obtained hydrolysates with ViscozymeTo summarize, the introduced approach is an environmentally friendly alternative with a safer profile than a conventional one and could represent the future for sustainable industrial-scale ferulic acid production. However, the issue of efficient and environmentally friendly purification of ferulic acid remains relevant and needs to be addressed. Part II of this series of studies will discuss the purification of ferulic acid taking advantage of solid-phase extraction techniques by commercially available polymers such as AmberLite™, Sephadex™, and polyvinylpyrrolidone (PVP).