High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production

<p>Abstract</p> <p>Background</p> <p>The bacteria <it>Escherichia coli </it>K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from <it>Escherichia coli </it>K4 capsular polysaccharide was investigated and a 1.4 g·L^-1K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.</p> <p>Results</p> <p>The inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L^-1), with corresponding average yields (0.13 ± 0.006 g_{K4 CPS}·g_cdw^-1), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.</p> <p>Conclusion</p> <p>The increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.</p

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

Comparative Analyses of Pharmaceuticals or Food Supplements Containing Chondroitin Sulfate: Are Their Bioactivities Equivalent?

Abstract Introduction: Oral supplementation of chondroitin sulfate (CS) and glucosamine (GlcN), symptomatic slow-acting molecules, is recommended by European Society for Clinical and Economic Aspects of Osteoporosis and Osteoarthritis and Musculoskeletal Diseases (ESCEO) and other European Union (EU) guidelines for the restoration of the articular cartilage surface in patients affected by osteoarthritis (OA). They are commercialized as pharmaceutical grade products and as food supplements in combination with plant extracts hyaluronic acid, methylsulfonylmethane, and other components. Food supplements do not need to undergo the strict regulatory controls of pharmaceutical grade products; thus, composition and contaminants that could be present may not be evidenced before commercialization and these uncertainties may give rise to concerns about the bioactivity of these formulations. Methods: In this paper 10 different food supplements (FS) from diverse European countries were analyzed in comparison with two pharmaceutical grade products (Ph) using updated analytical approaches and biochemical cell-based assays. The purity, the titer, and the origin of CS in Ph and FS samples were initially assessed in order to successively compare the biological function. Both food supplements and pharmaceutical formulations were tested in vitro, using the same final CS concentration, on primary chondrocytes and synoviocytes in terms of (i) cell viability, (ii) activation of the NF-κB-mediated inflammation pathway, (iii) cartilage oligomeric matrix protein (COMP-2), IL-6, and IL-8 production. Results: All the FS presented a certain insoluble fraction; the CS and the GlcN contents were lower than the declared ones in 9/10 and 8/10 samples, respectively. All FS contained keratan sulfate (KS) at up to 50% of the total glycosaminoglycan amount declared on the label. Primary cells treated with the samples diluted to present the same CS concentration in the medium showed cytotoxicity in 7/10 FS while Ph preserved viability and reduced NF-κB, COMP-2, and secreted inflammatory cytokines. Conclusion: Among all samples tested, the pharmaceutical grade products demonstrated effective modulation of biomarkers counteracting the inflammation status and improving viability and the physiological condition of OA human primary chondrocyte and synoviocyte cells. In contrast to that, most FS were cytotoxic at the tested concentrations, and only 3/10 of them showed similarities to Ph sample behavior in vitro

European chondroitin sulfate and glucosamine food supplements: a systematic quality and quantity assessment compared to pharmaceuticals

Chondroitin sulfate and glucosamine, commercialized as anti-osteoarthritis food supplements, do not undergo the strict quality controls of pharmaceuticals. In this paper a systematic multi-analytical approach was designed to analyse 25 food supplements from 8 European countries compared to 2 pharmaceuticals by using high performance anion-exchange chromatography with pulsed amperometric detection, size exclusion chromatography with triple detector array, capillary electrophoresis, mono and bi-dimensional NMR. Furthermore the biological activity was assessed on in vitro human synoviocyte and chondrocyte primary cell models. Most of the samples (over 19 out of 25) showed lower condroitin sulfate and glucosamine contents than the declared ones (up to -60.3%) while all of them showed a KS contamination (up to 47.1%). Mixed animal origin chondroitin sulfate and multiple molecular weight species were determined in more than 32% of the samples. Only 1 on 5 biologically screened samples had an effective action in vitro almost comparable to the pharmaceuticals

Biotechnological transformation of hydrocortisone to 16?-hydroxy hydrocortisone by Streptomyces roseochromogenes

Streptomyces roseochromogenes is able to hydroxylate steroid compounds in different positions of their cycloalkane rings thanks to a cytochrome P-450 multienzyme complex. In this paper, the hydroxylation of the hydrocortisone in the 16α position, performed by bacterial whole cells, was investigated in both shake flask and fermentation conditions; the best settings for both cellular growth and transformation reaction were studied by investigating the optimal medium composition, the kinetic of conversion, the most suitable substrate concentration and the preferred addition timing. Using newly formulated malt extract- and yeast extract-based media, a 16α-hydrohydrocortisone concentration of 0.2±0.01 g L−1 was reached in shake flasks. Batch experiments in a 2-L fermentor established the reproducibility and robustness of the biotransformation, while a pulsed batch fermentation strategy allowed the production to increase up to 0.508±0.01 g L−1. By-product formation was investigated, and two new derivates of the hydrocortisone obtained during the bacterial transformation reaction and unknown so far, a C-20 hydroxy derivate and a C-21 N-acetamide one, were determined by NMR analyses

Exploiting Potential Biotechnological Applications of Poly-γ-glutamic Acid Low Molecular Weight Fractions Obtained by Membrane-Based Ultra-Filtration

Since the potentialities of applications of low molecular weight poly-γ-glutamic acid (γ-PGA) chains have been so far only partially explored, the separation of diverse molecular families of them, as well as their characterization for potential bioactivity and ability to form films, were investigated. Two different approaches based on organic solvent precipitation or on ultra- and nano-filtration membrane-based purification of inexpensive commercial material were employed to obtain size-specific γ-PGA fractions, further characterized by size exclusion chromatography equipped with a triple detector array and by ultra-high-performance liquid chromatography to assess their average molecular weight and their concentration. The γ-PGA low molecular weight fractions, purified by ultra-filtration, have been shown both to counteract the desiccation and the oxidative stress of keratinocyte monolayers. In addition, they were exploited to prepare novel hydrocolloid films by both solvent casting and thermal compression, in the presence of different concentrations of glycerol used as plasticizer. These biomaterials were characterized for their hydrophilicity, thermal and mechanical properties. The hot compression led to the attainment of less resistant but more extensible films. However, in all cases, an increase in elongation at break as a function of the glycerol content was observed. Besides, the thermal analyses of hot compressed materials demonstrated that thermal stability was increased with higher γ-PGA distribution po-lymer fractions. The obtained biomaterials might be potentially useful for applications in cosmetics and as vehicle of active molecules in the pharmaceutical field