In milk thistle (Silybum marianum), commercial interest has focused almost exclusively on seeds and aerial parts, leaving behind substantial root biomass that remains outside current value chains despite its richness in bioactive polyphenols. This study evaluates the bioaccessibility, bioavailability, and antioxidant potential of milk thistle root infusion polyphenols using in vitro digestion, Caco-2 permeability assays, and in silico ADMET profiling. Digestion enhanced total phenolic content 6.89-fold (56.84 ± 1.10 mg GAE/g) and increased antioxidant activity by 11.88- and 1.58-fold for DPPH and ABTS, respectively. Caco-2 assays showed 57 Milk thistle roots are valorized as a sustainable source of antioxidants. Digestion increases root polyphenols bioaccessibility by 6.89-fold. Caco-2 assays reveal 57

Hepcidin and ferritin are key proteins of iron homeostasis in mammals. In this study, we characterize a chimera by fusing camel hepcidin to a human ferritin H-chain to verify if it retained the properties of the two proteins. The construct (HepcH) is expressed in E. coli in an insoluble and iron-containing form. To characterize it, the product was incubated with ascorbic acid and TCEP to reduce and solubilize the iron, which was quantified with ferrozine. HepcH bound approximately five times more iron than the wild type human ferritin, due to the presence of the hepcidin moiety. To obtain a soluble and stable product, the chimera was denatured and renatured together with different amounts of L-ferritin of the H-chain in order to produce 24-shell heteropolymers with different subunit proportions. They were analyzed by denaturing and non-denaturing PAGE and by mass spectroscopy. At the 1:5 ratio of HepcH to H- or L-ferritin, a stable and soluble molecule was obtained. Its biological activity was verified by its ability to both bind specifically cell lines that express ferroportin and to promote ferroportin degradation. This chimeric molecule showed the ability to bind both mouse J774 macrophage cells, as well as human HepG2 cells, via the hepcidin–ferroportin axis. We conclude that the chimera retains the properties of both hepcidin and ferritin and might be exploited for drug delivery.

Hepcidin a 25-amino-acid and highly disulfide bonded hormone, is the central regulator of iron homeostasis. In this chapter we propose ferritin as a peptide carrier to promote the association of the hybrid hepcidin/ferritin nanoparticle with a particular cell or tissue for therapeutic or diagnostic use. Indeed, human ferritin H-chain fused directly (on its 5’end) with camel mature hepcidin was cloned into the pASK-43 plus vector and expressed using BL21 (DE3) pLys E. coli strain. The transformed E.coli produced efficiently hepcidin-ferritin construct (hepcH), consisting of 213 amino acids with a molecular weight of 24 KDa. The recovered product is a ferritin exposing hepcidin on outer surface. The hepcH monomer was characterized by immunoblotting using a monoclonal antibody specific for human ferritin and a polyclonal antibody specific for hepcidin-25. The results were also confirmed by MALDI-TOF mass spectrometry. The recombinant native human ferritin and the commercial human hepcidin-25 were used as controls in this experiment. The assembly of hepcH, as an heteropolymer molecule, was performed in presence of denatured human ferritin-H and -L chains. After cysteine oxidation of the recombinant nanoparticles, cellular binding assays were performed on mammalian cells such as mouse monocyte–macrophage cell line J774, HepG2 and COS7.

Metabolic disorders, whether inherited or acquired, are currently considered as global health concerns that are in desperate need of rapid and efficient therapeutic solutions. Despite the tremendous progress of modern medicine in diagnosing and treating many metabolic diseases, a considerable number of illnesses are still hard to manage. The economic and health burden of these disorders is increasing progressively due to multiple factors encompassing genetic transmission, unbalanced diets, and unhealthy modern lifestyles. Through the history of stubborn diseases, whenever the conventional medicine resources seem insufficient to deal with a health problem, traditional medicine has always a valuable thing to offer. The majority of traditional medicine systems, namely, Chinese, Indian, and Greco-Arab old medicine, recognized the symptoms of many metabolic disorders mainly obesity and diabetes. Ancient physicians used mainly medicinal plants and herbal formulas to treat patients or to alleviate the symptoms of certain metabolic disorders. The encouraging reported testimonies inspired the researchers to unravel the compositions of many of the prescribed plants using analytical techniques and also to test their effects in vitro. Depending on the obtained results, some of these plant extracts were investigated using animal models. However, only a few number of plant extracts and/or compounds were administrated to human subjects in randomized clinical trials, where most of the gathered results appear to be conflicting yet promising. The aims of this chapter are to give an overview of the ancient use of traditional medicine in the treatment of metabolic diseases and to discuss the findings obtained from in vitro, preclinical and clinical trials, in an attempt to provide a realistic perspective on the contemporary medical role of medicinal plants and their phytochemicals in the field of metabolic disorders.

A specific macroalgal biomass decomposing fungus SL1 newly isolated and identified as Aspergillus fumigatus was found to be an efficient cellulases producer. This strain when cultivated on the whole green macroalgae as sole carbon source permitted the production of specific cellulases (specific activities of 30 and 33 U/mg of proteins for endoglucanase and β-glucosidase, respectively) different from commercial ones as shown by zymography. The application of the produced cellulases-based enzymatic cocktail for the saccharification of alkali pretreated Ulva sp. biomass yielded of 58%. This saccharification rate was optimized using response surface methodology (RSM). An increase of 36% in saccharification yield was obtained under optimized conditions (13 U CMCase, 4% substrate and 135 rpm agitation) which agreed with model predictions. The biocatalytic conversion using specific fungal cellulases may be a promising approach for the biodegradation of stranded macroalgae and its valorization mostly for bioethanol production.

Ferritin is a molecule with enormous potentiality in biotechnology that have been already used to encapsulate molecules, as contrast in magnetic resonance imaging and to carry epitopes. We proposed to use it to carry another key protein of iron metabolism, hepcidin that is a small hormone peptide that control systemic iron homeostasis. In this work, we purified the previously produced camel hepcidin and human H-ferritin heteropolymer (HepcH-FTH) and to monitor its binding capability toward J744 cell line in presence or absence of ferric ammonium citrate. Fused camel hepcidin and human H-ferritin monomer (HepcH) as well as the assembled HepcH-FTH heteropolymer (ratio 1:5) was easily purified by a one-step purification using size exclusion chromatography. SDS-PAGE electrophoresis of HepcH, purified from soluble and insoluble fractions, showed a single band of 24 kDa with an estimated purity of at least 90%. The purification yields of HepcH from the soluble and insoluble fractions was, respectively, of about 6.80 and 2 mg/L of bacterial culture. Time curse cellular binding assays of HepcH-FTH revealed its great potential to bind the J774 cells after 15 min of incubation. Furthermore, HepcH-FTH was able to degrade ferroportin, the unique hepcidin receptor, even after 30 min of incubation with J774 cells treated with 100 µM ferric ammonium citrate. In conclusion, we proposed ferritin as a peptide carrier to promote the association of the hybrid HepcH-FTH nanoparticle with a particular type of cell for therapeutic or diagnostic.

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 μg/mL) and vancomycin (MIC = 2 μg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry.

Summary In this report, we investigated for the first time the total polyphenols content ( TPC ) and antioxidant activity before and after digestion of Carapichea ipecacuanha root infusion, better known as ipecac, prepared at different concentrations. An in vitro digestion system coupled to a Caco‐2 cell model was applied to study the bioavailability of antioxidant compounds. The ability of ipecac bioaccessible fractions to inhibit reactive oxygen species ( ROS ) generation at cellular level was also evaluated. The findings revealed that water volume of 50 mL g −1 of sample provided the maximum yield of extraction of TPC and antioxidant activity. Polyphenols increased in content and activity after digestion and they were highly bioavailable (75% of intestinal absorption). Polyphenols were also present in the residual parts which indicate a possible local activity. Results also suggest that ipecac infusion could represent a promising source of effective bioavailable antioxidants to be exploited in functional foods field.

The main objective of the present study is to introduce a new and ecologically safe method for managing the rice weevil, Sitophilus oryzae. Therefore, the Agave americana leaf extract's phytochemical profile, and its insecticidal activity against the adults of S. oryzae were evaluated. The A. americana leaf extract was screened for the following phytochemicals: total phenolics (14.70 ± 0.31 mg GAE/g FW), total flavonoids (5.15 ± 0.18 mg RE/g FW) and saponins (10.32 ± 0.20 mg OAE/g FW). The HPLC-ESI/TOF-MS analysis results revealed that flavonoid glycosides (kaempferol, quercetin, and isorhamnetin derivates) were the major phenolic compounds of the A. americana leaf extract. In addition, the GC-MS analysis identified n-alkanes (77.77%) as significant compounds of the lipophilic fraction from the leaf extract. Moreover, the insecticidal potential was assessed through contact and repellent bioassays towards the rice weevil adults. The LD50, LC50, and RC50 values were 10.55 μg/insect, 8.99 μg/cm2, and 0.055 μg/cm2 for topical application method, treated filter-paper method, and repellent bioassay, respectively. Furthermore, the A. americana leaf extract inhibited digestive enzyme activities, and median inhibition concentrations IC50 were evaluated to be 146.06 ± 1.74 and 86.18 ± 1.08 μg/mL for α-amylase and protease, respectively. Overall, our results highlighted the promising potential of the leaf extract against S. oryzae adults, allowing us to recommend the extract under investigation as an ecofriendly alternative to synthetic insecticides.

Hepcidin, a liver‐expressed antimicrobial peptide, has been demonstrated to act as an iron regulatory hormone as well as to exert a wide spectrum of antimicrobial activity. The aim of this work was the expression, as secreted peptide, purification, and characterization of a new recombinant polyHis‐tagged camel hepcidin (HepcD‐His) in yeast Pichia pastoris . The use of this eukaryotic expression system, for the production of HepcD‐His, having 6 histidine residues at its C terminus, was simpler and more efficient compared with the use of the prokaryotic system Escherichia coli . Indeed, a single purification step was required to isolate the soluble hepcidin with purity estimated more that 94% and a yield of 2.8 against 0.2 mg/L for the E coli system. Matrix‐assisted laser desorption/ionization time‐of‐flight (TOF)/TOF mass spectrometry of the purified HepcD‐His showed 2 major peaks at m / z 4524.64 and 4634.56 corresponding to camel hepcidin with 39 and 40 amino acids. Evaluation of disulfide bond connectivity with the Ellman method showed an absence of free thiol groups, testifying that the 8 cysteine residues in the peptide are displayed, forming 4 disulfide bridges. Circular dichroism spectroscopy showed that camel hepcidin structure was significantly modified at high temperature of 90°C and returns to its original structure when incubation temperature drops back to 20°C. Interestingly, this peptide showed also a greater bactericidal activity, at low concentration of 9.5μM, against E coli , than the synthetic analog DH3. Thus, the production, at a large scale, of the recombinant camel hepcidin, HepcD‐His, may be helpful for future therapeutic applications including bacterial infection diseases.

PURPOSE:The transcription factor Krüppel-like factor 6 (KLF6) regulates various cellular functions, such as metabolism, cell proliferation, and differentiation. KLF6 plays a key role in the development and progression of multiple human cancers.METHODS:Fifty primary biopsies and 10 normal nasopharyngeal mucosae were used to analyze by RT-QPCR the expression and the copy number of wtKLF6 and the spliced variants (KLF6-SV1, KLF6-SV2, and KLF6-SV3) in Tunisian patients with nasopharyngeal carcinoma. The expression analysis of E-cadherin and cyclin D1 was conducted by RT-QPCR and Western blot, respectively.RESULTS:The wtKLF6 was significantly downexpressed in tumors compared to normal tissues (p = 0.0015), whereas KLF6-SV1 and KLF6-SV2 were overexpressed in tumors compared to wtKLF6 and KLF6-SV3 (p < 0.0001). Copy number variation was reduced in tumors compared to normal tissues (p = 0.0071). Interestingly, KLF6-SV1 is associated with the juvenile form (p = 0.0003) which is more aggressive than the adult form of NPC. Furthermore, the oncogenic variant KLF6-SV1 was overexpressed in tumors lacking the expression of E-cadherin (p = 0.0022) suggesting its role in metastasis and tumor progression. The wtKLF6 is associated negatively with cyclin D1 in tumor tissues (p = 0.048).CONCLUSION:The wtKLF6 was downexpressed in contrast with the oncogenic variants. Overexpression of KLF6-SV1 is associated with young patients, and loss of E-cadherin suggests that this variant correlated with the aggressiveness of NPC.

This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20 kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40 °C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

The present work aimed to determine the antioxidant and antiproliferative potential of Luffa cylindrica fruits collected at two different maturation stages and to identify and compare their functional components composition. The MeOH extracts of L. cylindrica fruits harvested at 60 - 65 days after seeding (S1) and 85 - 90 days after seeding (S2) were investigated for their antioxidant activity using various assays. Furthermore, the antiproliferative activity of the extracts against HeLa human cervical cancer cells was explored with xCELLigence real time cell analyzer, while the effect of the samples on the membrane integrity of the same cell line was assessed using LDH cytotoxicity leakage assay. Ultimately, the phytochemicals were analyzed using GC/MS and HPLC/TOF-MS. The S1 sample had higher contents and more diversity in the phenolic compounds composition than S2. Furthermore, the S1 extract showed the highest antioxidant and antiproliferative activity, while the S2 extract had higher cytotoxicity towards HeLa cells. The findings revealed that the time of harvest has a big impact on the phytochemicals content and activity and that harvesting L. cylindrica at an early stage before the beginning of the development of the cellulose fibrous system is recommended for a rich phytochemical composition and efficient antioxidant and antiproliferative activities.

Although fennel stems and leaves represent an abundant biomass, they remain poorly exploited in terms of extraction of biomolecules. This work aimed at the valorization of the non-edible parts of fennel by the extraction and the characterization of a new milk-clotting protease. Both, enzymatic extraction and milk-clotting conditions were optimized in the context of developing new aspects of cheese. Fennel stems and leaves were sorted and coarsely grounded. The extraction consists in an aqueous maceration and extracts were used to coagulate raw cow's milk. Maximum milk-clotting activity was obtained under a temperature of 20°C, extraction time of 12h and solid-to-solvent ratio of 150:100gmL−1. Besides, the impact of extract concentration, milk pH, calcium content and temperature on clotting-time were studied. The statistical analysis of the results confirmed significant effects of the four studied parameters. The use of specific inhibitors suggested the presence of a serine protease in the crude enzymatic extracts. The findings of this research confirmed that the use of fennel protease could be a new alternative for the commercial proteases in dairy products.

The green macroalgal biomass corresponds to an emerging and promising biofuel feedstock. Their biological pretreatment and energetic conversion to biomethane were investigated and the enhancement of biogas production using the solid-state fermentation (SSF) as an eco-friendly innovative pretreatment of Ulva sp. was precisely assessed. Compared to conventional acid and alkali pretreatments, the highest methane potential of 153±3mLCH4g−1VS with an anaerobic biodegradability of 57% was obtained using SSF pretreatment with a locally isolated Aspergillus fumigatus SL1 strain. It was 132±2mLCH4g−1VS for raw Ulva sp. with biodegradability of 49%. Acid pretreatment with 4% HCl at 150°C had a negative effect on Ulva sp.'s methane potential while alkali pretreatment with 4% NaOH at 20°C showed a significant effect. The proposed SSF-based pretreatment enhanced therefore biogas production of 21% and permitted an eco-friendly valorization of large amounts of abundant macroalgae.

Green macroalgae are an abundant and undervalued biomass with a specific cell wall structure. In this context, different pretreatments, namely ethanol organosolv (Org), alkaline, liquid hot water (LHW), and ionic liquid (IL) pretreatments, were applied to the green macroalgae Ulva lactuca biomass and then evaluated. Their effects on chemical composition, biomass crystallinity, enzymatic digestibility, and theoretical ethanol potential were studied. The chemical composition analysis showed that the Org and LHW pretreatments allowed the highest glucan recovery (80.8 ± 3.6 and 62.9 ± 4.4 g/100 g DM, respectively) with ulvan (80.0 and 99.1%) and hemicellulose (55.0 and 42.3%) removal. These findings were in agreement with both thermogravimetric analysis and scanning electron microscopy results that confirm significant structural changes of the pretreated biomasses. It was found that the employed pretreatments did not significantly affect the cellulose crystallinity; however, they both increased the whole crystallinity and the enzymatic digestibility. This later reached 97.5% in the case of LHW pretreatment. Our results showed high efficiency saccharification of Ulva lactuca biomass that will constitute the key step of the implementation of a biorefinery process.

Enzymatic saccharification of lignocellulosic biomass has been widely studied. Mainly endoglucanases were found to be a prerequisite for the quick initial biomass liquefaction. In the present study, Pichia pastoris was used as a host for the heterologous expression of a Sclerotinia sclerotiorum GH45 endoglucanase, Endo2. The recombinant plasmid pPICZαA was used to transform Pichia pastoris. Pichia culture supernatants expressing the recombinant Endo2 (rEndo2) were used for the purification and biochemical characterization of this enzyme. Therefore, rEndo2 was purified 6.7 fold to homogeneity with 34% yield and gave 19 U/mg specific activity. It also showed maximum activity at pH 7.0 and 60 °C (against pH 5.0 and 50 °C for the native enzyme) and was thermostable at relatively high temperatures. Furthermore, rEndo2 retained its activity in a wide pH range (from 5 to 8). Besides, the recombinant endoglucanase was produced as an active 47 kDa enzyme. This molecular weight differs from the one of the native enzyme (34 kDa), which suggested a potential glycosylation of the recombinant enzyme. Moreover, rEndo2 was able to produce fermentable sugars after enzymatic assay on various cellulosic substrates with an interesting yield. Therefore, all these features offer prospects for large-scale production and industrial application of the recombinant endoglucanase.

ABSTRACT A new protease was isolated from the intestine of small red scorpionfish. After ammonium sulfate precipitation, the enzyme was purified to homogeneity by a two-step chromatography with 6.69% recovery and fivefold increase in specific activity. The molecular weight of the protease was about 24 kDa as estimated by size exclusion chromatography and sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The enzyme optimum pH and temperature were pH 10.0 and 40°C, respectively. The protease was stable at temperatures below 40°C and over a broad pH range (8.0–11.0). The Km and Kcat values were 0.36 mmol L−1 and 1.61 s−1, respectively. Interestingly, the enzyme presented specificity for casein, egg albumin, bovine serum albumin, and gelatin with obtained degrees of hydrolysis ranging from 3.14% to 6.58%, after 2 h of hydrolysis. Moreover, gluten hydrolysate analysis confirmed protein hydrolysis and solubilization. These results suggested the potential use of the protease in the preparation of food protein hydrolysates with interesting properties.