Production of succinic acid from Basfia succiniciproducens up to the pilot scale from Arundo donax hydrolysate.

Abstract In the present work the recently isolated strain Basfia succiniciproducens BPP7 was evaluated for the production of succinic acid up to the pilot fermentation scale in separate hydrolysis and fermentation experiments on Arundo donax, a non-food dedicated energy crop. An average concentration of about 17 g/L of succinic acid and a yield on consumed sugars of 0.75 mol/mol were obtained demonstrating strain potential for further process improvement. Small scale experiments indicated that the concentration of acetic acid in the medium is crucial to improve productivity; on the other hand, interestingly, short-term (24 h) adaptation to higher acetic acid concentrations, and strain recovery, were also observed

Antioxidant and Hypolipidemic Activity of Açai Fruit Makes It a Valuable Functional Food

Several plant extracts are acquiring increasing value because of their antioxidant activity and hypolipidemic properties. Among them, great interest has been recently paid to acai fruit as a functional food. The aim of this study was to test the ability of acai extract in reducing oxidative stress and modulating lipid metabolism in vitro using different cell models and different types of stress. In fact, lipid peroxidation as evaluated in a HepG2 model was reduced five-fold when using 0.25 mu g/mL of extract, and it was further reduced (20-fold) with the concentration increase up to 2.5 mu g/mL. With the nonalcoholic fatty liver disease (NAFLD)in vitro model, all concentrations tested showed at least a two-fold reduced fat deposit. In addition, primary adipocytes challenged with TNF-alpha under hypoxic conditions to mimic the persistent subcutaneous fat, treated with acai extract showed an approximately 40% reduction of fat deposit. Overall, our results show that acai is able to counteract oxidative states in all the cell models analysed and to prevent the accumulation of lipid droplets. No toxic effects and high stability overtime were highlighted at the concentrations tested. Therefore, acai can be considered a suitable support in the prevention of different alterations of lipid and oxidative metabolism responsible for fat deposition and metabolic pathological conditions

A multi-analytical approach to better assess the keratan sulfate contamination in animal origin chondroitin sulfate

Abstract Chondroitin sulfate is a glycosaminoglycan widely used as active principle of anti-osteoarthritis drugs and nutraceuticals, manufactured by extraction from animal cartilaginous tissues. During the manufacturing procedures, another glycosaminoglycan, the keratan sulfate, might be contemporarily withdrawn, thus eventually constituting a contaminant difficult to be determined because of its structural similarity. Considering the strict regulatory rules on the pureness of pharmaceutical grade chondrotin sulfate there is an urgent need and interest to determine the residual keratan sulfate with specific, sensitive and reliable methods. To pursue this aim, in this paper, for the first time, we set up a multi-analytical and preparative approach based on: i) a newly developed method by high performance anion-exchange chromatography with pulsed amperometric detection, ii) gas chromatography-mass spectrometry analyses, iii) size exclusion chromatography analyses coupled with triple detector array module and on iv) strong anion exchange chromatography separation. Varied KS percentages, in the range from 0.1 to 19.0% (w/w), were determined in seven pharmacopeia and commercial standards and nine commercial samples of different animal origin and manufacturers. Strong anion exchange chromatography profiles of the samples showed three or four different peaks. These peaks analyzed by high performance anion-exchange with pulsed amperometric detection and size exclusion chromatography with triple detector array, ion chromatography and by mono- or two-dimensional nuclear magnetic resonance revealed a heterogeneous composition of both glycosaminoglycans in terms of sulfation grade and molecular weight. High molecular weight species (>100 KDa) were also present in the samples that counted for chains still partially linked to a proteoglycan core

In vitro model based on human keratinocyte to evaluate relaxin activity on wound healing exploiting time-lapse video microscopy

Wound repair is a well-ordered but complex process involving many cellular activities including inflammation, growth factor and cytokine production, and thus cell migration and proliferation. In vitro wound healing assays have been used and are well accepted, for the discovery and validation of biomolecules that may influence cell migration and improve repair (Liu et al. 2009). The aim of our research was to exploit time-lapse video-microscopy (Okolab, Italy) to observe and analyse scratched monolayers of keratinocyte cell line (HaCat), with specific interest in characterizing different relaxin preparations

Hyaluronan Hydrogels: Rheology and Stability in Relation to the Type/Level of Biopolymer Chemical Modification

BDDE (1,4-butanediol-diglycidylether)-crosslinked hyaluronan (HA) hydrogels are widely used for dermo-aesthetic purposes. The rheology and stability of the gels under physiological conditions greatly affect their clinical indications and outcomes. To date, no studies investigating how these features are related to the chemistry of the polymeric network have been reported. Here, four available HA-BDDE hydrogels were studied to determine how and to what extent their rheology and stability with respect to enzymatic hydrolysis relate to the type and degree of HA structural modification. H-1-/C-13-NMR analyses were associated for the quantification of the "true" HA chemical derivatization level, discriminating between HA that was effectively crosslinked by BDDE, and branched HA with BDDE that was anchored on one side. The rheology was measured conventionally and during hydration in a physiological medium. Sensitivity to bovine testicular hyaluronidase was quantified. The correlation between NMR data and gel rheology/stability was evaluated. The study indicated that (1) the gels greatly differed in the amounts of branched, crosslinked, and overall modified HA, with most of the HA being branched; (2) unexpectedly, the conventionally measured rheological properties did not correlate with the chemical data; (3) the gels' ranking in terms of rheology was greatly affected by hydration; (4) the rheology of the hydrated gels was quantitatively correlated with the amount of crosslinked HA, whereas the correlations with the total HA modification level and with the degree of branched HA were less significant; (5) increasing HA derivatization/crosslinking over 9/3 mol% did not enhance the stability with respect to hyaluronidases. These results broaden our knowledge of these gels and provide valuable information for improving their design and characterization

No Waste from Waste: Membrane-Based Fractionation of Second Cheese Whey for Potential Nutraceutical and Cosmeceutical Applications, and as Renewable Substrate for Fermentation Processes Development

Second cheese whey (SCW) derived from buffalo milk is the main by-product of the mozzarella cheese dairy industry. The objective of this study was to develop a membrane-based purification procedure to obtain specific fractions from SCW and assess their biological and applicative potential. Special interest was paid to the proteins and newly identified health-promoting compounds that could be recovered and used as value-added products in different sectors of food and pharmaceutical industries. SCW has been treated, sequentially, with microfiltration (MF), ultrafiltration (UF), and nanofiltration (NF) membranes giving the possibility to obtain three different fractions, namely retentates recovered on 100 and 10 kDa (R100 and R10) and a nanofiltration retentate (RNF). These retentates were compared for their ability to preserve human keratinocytes from dehydration, to form protein-based films by casting, and finally they were used for probiotic cultivations as the main substrate. Results showed that Lactobacillus rhamnosus could grow without any further additional nutrient up to 2.2 ± 0.3 × 109 CFU/mL in the RNF medium. Dehydration tests on HaCat cells proved R100 as the most efficient fraction in preserving cell viability from this specific stress. R10, after diafiltrations, formed transparent films with improved features when glycerol was added as a plasticizer

Molecular Mechanisms at the Basis of Pharmaceutical Grade Triticum vulgare Extract Efficacy in Prompting Keratinocytes Healing

Background: It has been shown that many plant- or microbial-derived oligos and polysaccharides may prompt tissue repair. Among the different extracts that have been studied, the aqueous one of Triticum vulgare (TVE) that was obtained from a whole germinated plant has been proven to have different biological properties that are useful in the process of wound healing. Nevertheless, with the long tradition of its use in pharmaceutical cream and ointments, especially in Italy, a new protocol was recently proposed (and patented) to improve the extraction process. Methods: In a simplified in vitro model, human keratinocyte monolayers were scratched and used to run time lapse experiments by using time lapse video microscopy (TLVM) to quantify reparation rate while considering a dose–response effect. Contemporarily, the molecular mechanisms that are involved in tissue repair were studied. In fact, key biomarkers that are involved in remodeling, such as MMP-2 and MMP-9, and in matrix structure assembly, such as collagen I, elastin, integrin αV and aquaporin 3, were evaluated with gene expression analyses (RT-PCR) and protein quantification in western blotting. Results: All TVE doses tested on the HaCat-supported cell proliferation. TVE also prompted cell migration in respect to the control, correctly modulating the timing of metalloproteases expression toward a consistent and well-assessed matrix remodeling. Furthermore, TVE treatments upregulated and positively modulated the expression of the analyzed biomarkers, thus resulting in a better remodeling of dermal tissue during healing. Conclusions: The in vitro results on the beneficial effects of TVE on tissue elasticity and regeneration may support a better understanding of the action mechanism of TVE as active principles in pharmaceutical preparation in wound treatment

Chondroitin Sulfate in USA Dietary Supplements in Comparison to Pharma Grade Products: Analytical Fingerprint and Potential Anti-Inflammatory Effect on Human Osteoartritic Chondrocytes and Synoviocytes

The biological activity of chondroitin sulfate (CS) and glucosamine (GlcN) food supplements (FS), sold in USA against osteoarthritis, might depend on the effective CS and GlcN contents and on the CS structural characteristics. In this paper three USA FS were compared to two pharmaceutical products (Ph). Analyses performed by HPAE-PAD, by HPCE and by SEC-TDA revealed that the CS and GlcN titers were up to −68.8% lower than the contents declared on the labels and that CS of mixed animal origin and variable molecular weights was present together with undesired keratan sulfate. Simulated gastric and intestinal digestions were performed in vitro to evaluate the real CS amount that may reach the gut as biopolymer. Chondrocytes and synoviocytes primary cells derived from human pathological joints were used to assess: cell viability, modulation of the NF-κB, quantification of cartilage oligomeric matrix protein (COMP-2), hyaluronate synthase enzyme (HAS-1), pentraxin (PTX-3) and the secreted IL-6 and IL-8 to assess inflammation. Of the three FS tested only one (US FS1) enhanced chondrocytes viability, while all of them supported synoviocytes growth. Although US FS1 proved to be less effective than Ph as it reduced NF-kB, it could not down-regulate COMP-2; HAS-1 was up-regulated but with a lower efficacy. Inflammatory cytokines were markedly reduced by Ph while a slight decrease was only found for US-FS1

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

Glycosaminoglycans, such as hyaluronic acid and chondroitin sulphate, are not only more and more required as main ingredients in cosmeceutical and nutraceutical preparations, but also as active principles in medical devices and pharmaceutical products. However, while biotechnological production of hyaluronic acid is industrially established through fermentation of Streptococcus spp. and recently Bacillus subtilis, biotechnological chondroitin is not yet on the market. A non-hemolytic and hyaluronidase negative S. equi subsp. zooepidemicus mutant strain was engineered in this work by the addition of two E. coli K4 genes, namely kfoA and kfoC, involved in the biosynthesis of chondroitinlike polysaccharide. Chondroitin is the precursor of chondroitin sulphate, a nutraceutical present on the market as anti-arthritic drug, that is lately being studied for its intrinsic bioactivity. In small scale bioreactor batch experiments the production of about 1.46 ± 0.38 g/L hyaluronic acid and 300 ± 28 mg/L of chondroitin with an average molecular weight of 1750 and 25 kDa, respectively, was demonstrated, providing an approach to the concurrent production of both biopolymers in a single fermentation

Cellular proliferation and wound healing activity induced by purified fractions from Sempervivum tectorum L.

In recent years, interest has increased in obtaining drugs from those plants with high content of bioactive substances like triterpenes, alkaloids, tannins and flavonoids for their potential use in promoting wound healing. Sempervivum tectorum L. (Crassulaceae), known as houseleek, is a evergreen plant which grows in central and southern Europe. Fresh juice or squeezed leaves are used in folk medicine exclusively for external purposes in the treatment of the ear inflammation, for wound, sores and abscesses as refrigerant and astringent [1]. Antinociceptive and antioxidant [2] activity, in vivo and in vitro model, was also investigated. The present study was undertaken with the aim to explore the biological activity of the extracts and of the purified fractions of Sempervivum tectorum L. leaves. The extract is rich in free sugars with the rare sedoheptulose, organic acids such as malic acid, flavonoids and 2-C-methyerithritol. We analyzed the effects of Sempervivum tectorum purified fractions on cellular proliferation and migration [3] in HCT cells. We observed that treatment with the fractions with high level of sedoheptulose and malic acid of Sempervivum tectorum (SVT high fraction), significantly improved cellular migration and proliferation. We next investigated on the intracellular signaling cascades triggered by the incubation with the SVT high fraction and we observed that incubation with the SVT high fraction induces a significant phosphorylation of EGFR and Src. Furthermore, HCT cells stimulated with the SVT high fraction show a significant increase of STAT3 phosphorylation and this event was prevented by AG1478, an EGFR selective inhibitor. Taken together these data strongly suggest that the SVT high fraction significantly improve cellular proliferation and migration by inducing a Src-dependent EGFR activation and might represent a new proliferative and new wound healing therapeutic approaches