Alberto Alfano

Application of a 22L scale membrane bioreactor and cross-flow ultrafiltration to obtain purified chondroitin

Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed‐batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale‐up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨︁KTA cross‐flow instrument, was translated to a UNIFLUX‐10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C‐3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled‐up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild‐type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity.

Purification of chondroitin precursor from Escherichia coli K4 fermentation broth using membrane processing.

Recently the possibility of producing the capsular polysaccharide K4, a fructosylated chondroitin, in fed‐batch experiments was assessed. In the present study, a novel downstream process to obtain chondroitin from Escherichia coli K4 fermentation broth was developed. The process is simple, scalable and economical. In particular, downstream procedures were optimized with a particular aim of purifying a product suitable for further chemical modifications, in an attempt to develop a biotechnological platform for chondroitin sulfate production. During process development, membrane devices (ultrafiltration/diafiltration) were exploited, selecting the right cassette cut‐offs for different phases of purification. The operational conditions (cross‐flow rate and transmembrane pressure) used for the process were determined on an ÄKTA cross‐flow instrument (GE Healthcare, USA), a lab‐scale automatic tangential flow filtration system. In addition, parameters such as selectivity and throughput were calculated based on the analytical quantification of K4 and defructosylated K4, as well as the major contaminants. The complete downstream procedure yielded about 75% chondroitin with a purity higher than 90%.

A combined fermentative-chemical approach for the scalable production of pure E. coli monophosphoryl lipid A

Lipid A is the lipophilic region of lipopolysaccharides and lipooligosaccharides, the major components of the outer leaflet of most part of Gram-negative bacteria. Some lipid As are very promising immunoadjuvants. They are obtained by extraction from bacterial cells or through total chemical synthesis. A novel, semisynthetic approach to lipid As is ongoing in our laboratories, relying upon the chemical modification of a natural lipid A scaffold for the fast obtainment of several other lipid As and derivatives thereof. The first requisite for this strategy is to have this scaffold available in large quantities through a scalable process. Here, we present an optimized fed-batch fermentation procedure for the gram-scale production of lipid A from Escherichia coli K4 and a suitable phenol-free protocol for its purification. A study for regioselective de-O-phosphorylation reaction was then performed to afford pure monophosphoryl lipid A with an attenuated endotoxic activity, as evaluated by cytokine production in human monocytic cell line THP-1 in vitro. The reported method for the large-scale obtainment of monophoshoryl lipid A from the fed-batch fermentation broth of a recombinant strain of E. coli may permit the access to novel semisynthetic lipid A immunoadjuvant candidates.

A Semisynthetic Approach to New Immunoadjuvant Candidates: Site‐Selective Chemical Manipulation of Escherichia coli Monophosphoryl Lipid A

A semisynthetic approach to novel lipid A derivatives from Escherichia coli (E. coli) lipid A is reported. This methodology stands as an alternative to common approaches based exclusively on either total synthesis or extraction from bacterial sources. It relies upon the purification of the lipid A fraction from fed-batch fermentation of E. coli, followed by its structural modification through tailored, site-selective chemical reactions. In particular, modification of the lipid pattern and functionalization of the phosphate group as well as of the sole primary hydroxyl group were accomplished, highlighting the unusual reactivity of the molecule. Preliminary investigations of the immunostimulating activity of the new semisynthetic lipid A derivatives show that some of them stand out as promising, new immunoadjuvant candidates.

Lactobacillus plantarum: Microfiltration experiments for the production of probiotic biomass to be used in food and nutraceutical preparations

Several studies have focused their attention on increasing the production of lactobacillus ssp. (LAB) biomass via‐fermentation, in particular exploiting novel in situ product removal bioreactors that prevent accumulation of lactic acid, and therefore growth inhibition. Lactobacillus plantarum is one of the most studied species, used in nutritional supplements and in food processing. This research aimed to obtain high cell densities of L. plantarum, through fed‐batch and microfiltration experiments. The latter achieved a 5‐fold higher biomass density compared with batch experiments. Furthermore, the L. plantarum strain, isolated from Portoguese chorizo, was characterized for its ability to survive simulated digestion in vitro and competition potential toward certain common pathogens. Finally, the possibility of exploiting dairy liquid wastes (whey) as medium components was also explored demonstrating the strains capability of metabolizing bovine‐ovine whey. This finding might be relevant in liquid waste treatments of diary industries that are well distributed in our region.

Beta-Defensin-2 and Beta-Defensin-3 Reduce Intestinal Damage Caused by Salmonella typhimurium Modulating the Expression of Cytokines and Enhancing the Probiotic Activity of Enterococcus faecium

The intestinal microbiota is a major factor in human health and disease. This microbial community includes autochthonous (permanent inhabitants) and allochthonous (transient inhabitants) microorganisms that contribute to maintaining the integrity of the intestinal wall, modulating responses to pathogenic noxae and representing a key factor in the maturation of the immune system. If this healthy microbiota is disrupted by antibiotics, chemotherapy, or a change in diet, intestinal colonization by pathogenic bacteria or viruses may occur, leading to disease. To manage substantial microbial exposure, epithelial surfaces of the intestinal tract produce a diverse arsenal of antimicrobial peptides (AMPs), including, of considerable importance, the β-defensins, which directly kill or inhibit the growth of microorganisms. Based on the literature data, the purpose of this work was to create a line of intestinal epithelial cells able to stably express gene encoding human β-defensin-2 (hBD-2) and human β-defensin-3 (hBD-3), in order to test their role in S. typhimurium infections and their interaction with the bacteria of the gut microbiota.

Valorization of Olive Mill Wastewater by Membrane Processes to Recover Natural Antioxidant Compounds for Cosmeceutical and Nutraceutical Applications or Functional Foods

Olive oil boasts numerous health benefits due to the high content of the monounsaturated fatty acid (MUFA) and functional bioactives including tocopherols, carotenoids, phospholipids, and polyphenolics with multiple biological activities. Polyphenolic components present antioxidant properties by scavenging free radicals and eliminating metabolic byproducts of metabolism. The objective of this research project was to recover the biologically active components rich in polyphenols, which include treatment of olive oil mills wastewater, and, at the same time, to remove the pollutant waste component resulting from the olive oil manufacturing processes. With specific focus on using technologies based on the application of ultra and nanofiltration membranes, the polyphenols fraction was extracted after an initial flocculation step. The nano-filtration permeate showed a reduction of about 95% of the organic load. The polyphenols recovery after two filtration steps was about 65% w/v. The nanofiltration retentate, dried using the spray dryer technique, was tested for cell viability after oxidative stress induction on human keratinocytes model in vitro and an improved cell reparation in the presence of this polyphenolic compound was demonstrated in scratch assays assisted through time lapse video-microscopy. The polyphenols recovered from these treatments may be suitable ingredients in cosmeceuticals and possibly nutraceutical preparations or functional foods.