Sustainable Exploitation of Posidonia oceanica Sea Balls (Egagropili): A Review.

Posidonia oceanica (L.) Delile is the main seagrass plant in the Mediterranean basin that forms huge underwater meadows. Its leaves, when decomposed, are transported to the coasts, where they create huge banquettes that protect the beaches from sea erosion. Its roots and rhizome fragments, instead, aggregate into fibrous sea balls, called egagropili, that are shaped and accumulated by the waves along the shoreline. Their presence on the beach is generally disliked by tourists, and, thus, local communities commonly treat them as waste to remove and discard. Posidonia oceanica egagropili might represent a vegetable lignocellulose biomass to be valorized as a renewable substrate to produce added value molecules in biotechnological processes, as bio-absorbents in environmental decontamination, to prepare new bioplastics and biocomposites, or as insulating and reinforcement materials for construction and building. In this review, the structural characteristics, and the biological role of Posidonia oceanica egagropili are described, as well as their applications in different fields as reported in scientific papers published in recent years.

Enhanced Streptomyces roseochromogenes melanin production by using the marine renewable source Posidonia oceanica egagropili.

Since the possibility to biotechnologically produce melanin by Streptomycetes using plant biomass has been so far poorly investigated, Posidonia oceanica egagropili, a marine waste accumulating along the Mediterranean Sea coasts, was explored as a renewable source to enhance extracellular melanin production by Streptomyces roseochromogenes ATCC 13400. Therefore, different amounts of egagropili powder were added to a culture medium containing glucose, malt extract, and yeast extract, and their effect on the melanin biosynthesis was evaluated. A 2.5 g·L-1 supplementation in 120-h shake flask growths at 26 °C, at pH 6.0 and 250 rpm, was found to enhance the melanin production up to 3.94 ± 0.12 g·L-1, a value 7.4-fold higher than the control. Moreover, 2-L batches allowed to reach a concentration of 9.20 ± 0.12 g·L-1 in 96 h with a productivity of 0.098 g·L-1·h-1. Further studies also demonstrated that the melanin production enhancement was due to the synergistic effect of both the lignin carbohydrate complex and the holocellulose components of the egagropili. Finally, the pigment was purified from the broth supernatant by acidic precipitation and reversed-phase chromatography, characterized by UV absorbance and one- and two-dimensional NMR, and also tested for its chemical, antioxidant, and photo-protective properties. KEY POINTS: • S. roseochromogenes ATCC 13400 produces extracellular soluble melanin. • Egagropili added to the growth medium enhances melanin production and productivity. • Both the lignin carbohydrate complex and the holocellulose egagropili components influence the melanin biosynthesis.

Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair.

Chondroitin obtained through biotechnological processes (BC) shares similarities with both chondroitin sulfate (CS), due to the dimeric repetitive unit, and hyaluronic acid (HA), as it is unsulfated. In the framework of this experimental research, formulations containing BC with an average molecular size of about 35 KDa and high molecular weight HA (HHA) were characterized with respect to their rheological behavior, stability to enzymatic hydrolysis and they were evaluated in different skin damage models. The rheological characterization of the HHA/BC formulation revealed a G' of 92 ± 3 Pa and a G″ of 116 ± 5 Pa and supported an easy injectability even at a concentration of 40 mg/mL. HA/BC preserved the HHA fraction better than HHA alone. BTH was active on BC alone only at high concentration. Assays on scratched keratinocytes (HaCaT) monolayers showed that all the glycosaminoglycan formulations accelerated cell migration, with HA/BC fastening healing 2-fold compared to the control. In addition, in 2D HaCaT cultures, as well as in a 3D skin tissue model HHA/BC efficiently modulated mRNA and protein levels of different types of collagens and elastin remarking a functional tissue physiology. Finally, immortalized human fibroblasts were challenged with TNF-α to obtain an in vitro model of inflammation. Upon HHA/BC addition, secreted IL-6 level was lower and efficient ECM biosynthesis was re-established. Finally, co-cultures of HaCaT and melanocytes were established, showing the ability of HHA/BC to modulate melanin release, suggesting a possible effect of this specific formulation on the reduction of stretch marks. Overall, besides demonstrating the safety of BC, the present study highlights the potential beneficial effect of HHA/BC formulation in different damage dermal models.

Argan (Argania spinosa L.) Seed Oil Cake as a Potential Source of Protein-Based Film Matrix for Pharmaco-Cosmetic Applications.

Various different agri-food biomasses might be turned into renewable sources for producing biodegradable and edible plastics, potentially attractive for food, agricultural and cosmeceutical sectors. In this regard, different seeds utilized for edible and non-edible oil extraction give rise to high amounts of organic by-products, known as seed oil cakes (SOCs), potentially able to become protein-rich resources useful for the manufacturing of biodegradable films. This study reports the potential of SOC derived from Argania spinosa (argan), a well-known plant containing valuable non-refined oil suitable for food or cosmetic use, to be a promising valuable source for production of a protein-based matrix of biomaterials to be used in the pharmaco-cosmetic sector. Thus, glycerol-plasticized films were prepared by casting and drying using different amounts of argan seed protein concentrate, in the presence of increasing glycerol concentrations, and characterized for their morphological, mechanical, barrier, and hydrophilicity properties. In addition, their antioxidant activity and effects on cell viability and wound healing were investigated. The hydrophobic nature of the argan protein-based films, and their satisfying physicochemical and biological properties, suggest a biorefinery approach for the recycling of argan SOC as valuable raw material for manufacturing new products to be used in the cosmeceutical and food industries.

Unsulfated biotechnological chondroitin by itself as well as in combination with high molecular weight hyaluronan improves the inflammation profile in osteoarthritis in vitro model.

Several studies suggest that inflammation has a pivotal role during the progression of osteoarthritis (OA) and cytokines have been identified as the main process mediators. This study aimed to explore the ability to modulate the main OA pro-inflammatory biomarkers of novel gels (H-HA/BC) based on high molecular weight hyaluronan (H-HA) and unsulfated biotechnological chondroitin (BC). For the first time, BC was tested also in combination with H-HA on human primary cells isolated from pathological knee joints. Specifically, the experiments were performed using an OA in vitro model based on human chondrocytes and synoviocytes. To evaluate the anti-inflammatory effects of H-HA/BC in comparison with H-HA and BC single gels, NF-kB, COMP-2, MyD88, MMP-13 and a wide range of cytokines, known to be specific biomarkers in OA (e.g., IL-6, IL-8, and TNF-α), were evaluated. In addition, cell morphology and proliferation occurring in the presence of either H-HA/BC or single components were assessed using time-lapse video microscopy. It was shown that synovial fluids and cells isolated from OA suffering patients, presented a cytokine pattern respondent to an ongoing inflammation status. H-HA and BC significantly reduced the levels of 23 biomarkers associated with cartilage damage. However, H-HA/BC decreased significantly 24 biological mediators and downregulated 19 of them more efficiently than the single components. In synoviocytes cultures, cytokine analyses proved that H-HA/BC gels re-established an extracellular environment more similar to a healthy condition reducing considerably the concentration of 11 analytes. Instead, H-HA and BC significantly modulated 7 (5 only with a longer treatment) and 8 biological cytokines, respectively. Our results suggest that H-HA/BC beyond the viscosupplementation effect typical for HA-based gels, can improve the inflammation status in joints and thus could be introduced as a valid protective and anti-inflammatory intraarticular device in the field of Class III medical devices for OA treatments.

Streptomycetes as platform for biotechnological production processes of drugs.

Streptomyces is one of the most versatile genera for biotechnological applications, widely employed as platform in the production of drugs. Although streptomycetes have a complex life cycle and metabolism that would need multidisciplinary approaches, review papers have generally reported only studies on single aspects like the isolation of new strains and metabolites, morphology investigations, and genetic or metabolic studies. Besides, even if streptomycetes are extensively used in industry, very few review papers have focused their attention on the technical aspects of biotechnological processes of drug production and bioconversion and on the key parameters that have to be set up. This mini-review extensively illustrates the most innovative developments and progresses in biotechnological production and bioconversion processes of antibiotics, immunosuppressant, anticancer, steroidal drugs, and anthelmintic agents by streptomycetes, focusing on the process development aspects, describing the different approaches and technologies used in order to improve the production yields. The influence of nutrients and oxygen on streptomycetes metabolism, new fed-batch fermentation strategies, innovative precursor supplementation approaches, and specific bioreactor design as well as biotechnological strategies coupled with metabolic engineering and genetic tools for strain improvement is described. The use of whole, free, and immobilized cells on unusual supports was also reported for bioconversion processes of drugs. The most outstanding thirty investigations published in the last 8 years are here reported while future trends and perspectives of biotechnological research in the field have been illustrated. KEY POINTS: • Updated Streptomyces biotechnological processes for drug production are reported. • Innovative approaches for Streptomyces-based biotransformation of drugs are reviewed. • News about fermentation and genome systems to enhance secondary metabolite production.

Hard-to-heal wound treated with Integra Flowable Wound Matrix: analysis and clinical observations.

Skin healing defects severely impair the quality of life of millions of people and burden healthcare systems globally. The therapeutic approach to these pathologies still represents a challenge. Novel scaffolds, used as dermal substitutes, possibly represent a promising strategy in complex wound management. Integra Flowable Wound Matrix (IFWM) is composed of a lyophilised, micronised form of collagen/chondroitin sulphate matrix, already used in regenerative medicine and endorsed in the therapy of diabetic foot lesions. In this paper, IFWM was applied to a tunnelling hard-to-heal skin lesion in order to restore tissue integrity. Although the different phases of skin wound healing are well established, the molecular mechanism underpinning IFWM-induced tissue repair are almost unknown. Here, we report, for the first time, the comparative analysis of molecular, histological and clinical observations of the healing process of a hard-to-heal tunnelling skin wound. The therapeutic success of this clinical case allowed us to recommend the use of IFWM as a tissue substitute in this rare type of hard-to-heal wound in which the high inflammatory status hampered the natural healing process.

Acellular Dermal Matrix Used in Diabetic Foot Ulcers: Clinical Outcomes Supported by Biochemical and Histological Analyses.

Diabetic foot ulcer (DFU) is a diabetes complication which greatly impacts the patient's quality of life, often leading to amputation of the affected limb unless there is a timely and adequate management of the patient. DFUs have a high economic impact for the national health system. Data have indeed shown that DFUs are a major cause of hospitalization for patients with diabetes. Based on that, DFUs represent a very important challenge for the national health system. Especially in developed countries diabetic patients are increasing at a very high rate and as expected, also the incidence of DFUs is increasing due to longevity of diabetic patients in the western population. Herein, the surgical approach focused on the targeted use of the acellular dermal matrix has been integrated with biochemical and morphological/histological analyses to obtain evidence-based information on the mechanisms underlying tissue regeneration. In this research report, the clinical results indicated decreased postoperative wound infection levels and a short healing time, with a sound regeneration of tissues. Here we demonstrate that the key biomarkers of wound healing process are activated at gene expression level and also synthesis of collagen I, collagen III and elastin is prompted and modulated within the 28-day period of observation. These analyses were run on five patients treated with Integra® sheet and five treated with the injectable matrix Integra® Flowable, for cavitary lesions. In fact, clinical evaluation of improved healing was, for the first time, supported by biochemical and histological analyses. For these reasons, the present work opens a new scenario in DFUs treatment and follow-up, laying the foundation for a tailored protocol towards complete healing in severe pathological conditions.

Black Edible Films from Protein-Containing Defatted Cake of Nigella sativa Seeds.

Black biodegradable/edible protein-based films were prepared from defatted cake waste obtained from Nigella sativa (black cumin) seeds as by-product of oil extraction process. The effects of pH, glycerol concentrations, and transglutaminase-catalyzed protein cross-linking activity on the stability of film-forming solutions were studied to determine the best experimental conditions to produce handleable films. Proteins contained in the analyzed defatted cake were shown to be able to act as transglutaminase acyl donor and acceptor substrates being polymerized when incubated in vitro in the presence of the enzyme. Film-forming solutions containing 20% glycerol and casted at pH 8.0 after treatment with the enzyme gave rise to morphologically more homogeneous films possessing mechanical and barrier properties, as well as antimicrobial activity, compatible with their possible applications as food packaging materials and mulching sheets. These findings confirm the validity of the strategy to consider the seed oil processed cakes as protein-based renewable sources to produce not only fertilizers, animal feed, or culinary food but also further valuable products such as bioplastics.

The PI3K/AKT Pathway Is Activated by HGF in NT2D1 Non-Seminoma Cells and Has a Role in the Modulation of Their Malignant Behavior.

Overactivation of the c-MET/HGF system is a feature of many cancers. We previously reported that type II testicular germ cell tumor (TGCT) cells express the c-MET receptor, forming non-seminomatous lesions that are more positive compared with seminomatous ones. Notably, we also demonstrated that NT2D1 non-seminomatous cells (derived from an embryonal carcinoma lesion) increase their proliferation, migration, and invasion in response to HGF. Herein, we report that HGF immunoreactivity is more evident in the microenvironment of embryonal carcinoma biopsies with respect to seminomatous ones, indicating a tumor-dependent modulation of the testicular niche. PI3K/AKT is one of the signaling pathways triggered by HGF through the c-MET activation cascade. Herein, we demonstrated that phospho-AKT increases in NT2D1 cells after HGF stimulation. Moreover, we found that this pathway is involved in HGF-dependent NT2D1 cell proliferation, migration, and invasion, since the co-administration of the PI3K inhibitor LY294002 together with HGF abrogates these responses. Notably, the inhibition of endogenous PI3K affects collective cell migration but does not influence proliferation or chemotactic activity. Surprisingly, LY294002 administered without the co-administration of HGF increases cell invasion at levels comparable to the HGF-administered samples. This paradoxical result highlights the role of the testicular microenvironment in the modulation of cellular responses and stimulates the study of the testicular secretome in cancer lesions.

Biotechnological Transformation of Hydrocortisone into 16α-Hydroxyprednisolone by Coupling Arthrobacter simplex and Streptomyces roseochromogenes.

16α-Hydroxyprednisolone, an anti-inflammatory drug, could be potentially obtained from hydrocortisone bioconversion by combining a 1,2-dehydrogenation reaction performed by Arthrobacter simplexATCC31652 with a 16α-hydroxylation reaction by Streptomyces roseochromogenes ATCC13400. In this study we tested, for the first time, potential approaches to couple the two reactions using similar pH and temperature conditions for hydrocortisone bioconversion by the two strains. The A. simplex capability to 1,2-dehydrogenate the 16α-hydroxyhydrocortisone, the product of S. roseochromogenes transformation of hydrocortisone, and vice versa the capability of S. roseochromogenes to 16α-hydroxylate the prednisolone were assessed. Bioconversions were studied in shake flasks and strain morphology changes were observed by SEM. Whole cell experiments were set up to perform the two reactions in a sequential mode in alternate order or contemporarily at diverse temperature conditions. A. simplex catalyzed either the dehydrogenation of hydrocortisone into prednisolone efficiently or of 16α-hydroxyhydrocortisone into 16α-hydroxyprednisolone in 24 h (up to 93.9%). Surprisingly S. roseochromogenes partially converted prednisolone back to hydrocortisone. A 68.8% maximum of 16α-hydroxyprednisolone was obtained in 120-h bioconversion by coupling whole cells of the two strains at pH 6.0 and 26 °C. High bioconversion of hydrocortisone into 16α-hydroxyprednisolone was obtained for the first time by coupling A. simplex and S. roseochromogenes.

Isolation, characterization and analysis of pro-inflammatory potential of Klebsiella pneumoniae outer membrane vesicles.

Outer membrane vesicles (OMVs) are potent virulence factors, naturally secreted by gram-negative bacteria. Since Klebsiella pneumoniae has emerged as an important nosocomial pathogen, because of resistance to a wide spectrum of antibiotics, it is crucial to investigate its pathogenetic mechanism microorganism secretes outer membrane vesicles (OMVs), but the pathogenesis of Klebsiella pneumoniae as it relates to OMVs has not been well elucidated. In this study we focused on the isolation, characterization and evaluation of the virulence potential of OMVs obtained from Klebsiella pneumoniae. Our data demonstrate that Klebsiella pneumoniae OMVs are important secretory nanocomplexes that elicit a potent inflammatory response. Since OMVs are clearly involved in the pathogenesis of this bacterium during infection, further studies are required to determine whether they could be future targets for novel therapy and potential vaccine against Klebsiella pneumoniae.

In vitro genotoxic effects of titanium dioxide nanoparticles (n-TiO2 ) in human sperm cells.

Titanium dioxide nanoparticles (TiO2 -NPs) are one of the most widely engineered nanoparticles used. The study has been focused on TiO 2 -NPs genotoxic effects on human spermatozoa in vitro. TiO 2 -NPs are able to cross the blood-testis barrier induced inflammation, cytotoxicity, and gene expression changes that lead to impairment of the male reproductive system. This study presents new data about DNA damage in human sperms exposed in vitro to two n-TiO 2 concentrations (1 µg/L and 10 µg/L) for different times and the putative role of reactive oxygen species (ROS) as mediators of n-TiO 2 genotoxicity. Primary n-TiO 2 characterization was performed by transmission electron microscopy. The dispersed state of the n-TiO 2 in media was spectrophotometrically determined at 0, 24, 48, and 72 hr from the initial exposure. The genotoxicity has been highlighted by different experimental approaches (comet assay, terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL] test, DCF assay, random amplification of polymorphic DNA polymerase chain reaction [RAPD-PCR]). The comet assay showed a statistically significant loss of sperm DNA integrity after 30 min of exposure. Increased threshold of sperm DNA fragmentation was highlighted after 30 min of exposure by the TUNEL Test. Also, the RAPD-PCR analysis showed a variation in the polymorphic profiles of the sperm DNA exposed to n-TiO 2 . The evidence from the DCF assay showed a statistically significant increase in intracellular ROS linked to n-TiO 2 exposure. This research provides the evaluation of n-TiO 2 potential genotoxicity on human sperm that probably occurs through the production of intracellular ROS.

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.

Structural characterization of the lipid A from the LPS of the haloalkaliphilic bacterium Halomonas pantelleriensis.

Halomonas pantelleriensis DSM9661(Τ) is a Gram-negative haloalkaliphilic bacterium isolated from the sand of the volcanic Venus mirror lake, closed to seashore in the Pantelleria Island in the south of Italy. It is able to optimally grow in media containing 3-15 % (w/v) total salt and at pH between 9 and 10. To survive in these harsh conditions, the bacterium has developed several strategies that probably concern the bacteria outer membrane, a barrier regulating the exchange with the environment. In such a context, the lipopolysaccharides (LPSs), which are among the major constituent of the Gram-negative outer membrane, are thought to contribute to the restrictive membrane permeability properties. The structure of the lipid A family derived from the LPS of Halomonas pantelleriensis DSM 9661(T) is reported herein. The lipid A was obtained from the purified LPS by mild acid hydrolysis. The lipid A, which contains different numbers of fatty acids residues, and its partially deacylated derivatives were completely characterized by means of ESI FT-ICR mass spectrometry and chemical analysis. Preliminary immunological assays were performed, and a comparison with the lipid A structure of the phylogenetic proximal Halomonas magadiensis is also reported.

Enteroglial-derived S100B protein integrates bacteria-induced Toll-like receptor signalling in human enteric glial cells.

OBJECTIVE: Enteric glial cells (EGC) have been suggested to participate in host-bacteria cross-talk, playing a protective role within the gut. The way EGC interact with microorganisms is still poorly understood. We aimed to evaluate whether: EGC participate in host-bacteria interaction; S100B and Toll-like receptor (TLR) signalling converge in a common pathway leading to nitric oxide (NO) production. DESIGN: Primary cultures of human EGC were exposed to pathogenic (enteroinvasive Escherichia coli; EIEC) and probiotic (Lactobacillus paracasei F19) bacteria. Cell activation was assessed by evaluating the expression of cFos and major histocompatibility complex (MHC) class II molecules. TLR expression in EGC was evaluated at both baseline and after exposure to bacteria by real-time PCR, fluorescence microscopy and western blot analysis. S100B expression and NO release from EGC, following exposure to bacteria, were measured in the presence or absence of specific TLR and S100B pathway inhibitors. RESULTS: EIEC activated EGC by inducing the expression of cFos and MHC II. EGC expressed TLR at baseline. Pathogens and probiotics differentially modulated TLR expression in EGC. Pathogens, but not probiotics, significantly induced S100B protein overexpression and NO release from EGC. Pretreatment with specific inhibitors of TLR and S100B pathways abolished bacterial-induced NO release from EGC. CONCLUSIONS: Human EGC interact with bacteria and discriminate between pathogens and probiotics via a different TLR expression and NO production. In EGC, NO release is impaired in the presence of specific inhibitors of the TLR and S100B pathways, suggesting the presence of a novel common pathway involving both TLR stimulation and S100B protein upregulation.

Serogroup-specific interaction of Neisseria meningitidis capsular polysaccharide with host cell microtubules and effects on tubulin polymerization.

We have previously shown that during late stages of the infectious process, serogroup B meningococci (MenB) are able to escape the phagosome of in vitro-infected human epithelial cells. They then multiply in the cytosolic environment and spread intracellularly and to surrounding cells by exploiting the microtubule cytoskeleton, as suggested by results of infections in the presence of microtubule inhibitors and evidence of nanotubes connecting neighboring cells. In this study, by using microtubule binding assays with purified microtubule asters and bundles and microtubule bundles synthesized in vitro, we demonstrate that the MenB capsule directly mediates the interaction between bacteria and microtubules. The direct interaction between the microtubules and the MenB capsular polysaccharide was confirmed by coimmunoprecipitation experiments. Unexpectedly, serogroup C meningococci (MenC), which have a capsular polysaccharide that differs from that of MenB only by its anomeric linkage, α(2→9) instead of α(2→8), were not able to interact with the microtubules, and the lack of interaction was not due to capsular polysaccharide O-acetylation that takes place in most MenC strains but not in MenB strains. Moreover, we demonstrate that the MenB capsular polysaccharide inhibits tubulin polymerization in vitro. Thus, at variance with MenC, MenB may interfere with microtubule dynamics during cell infection.

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.

Self-esterified hyaluronan hydrogels: Advancements in the production with positive implications in tissue healing.

Hyaluronan-(HA) short half-life in vivo limits its benefits in tissue repair. Self-esterified-HA is of great interest because it progressively releases HA, promoting tissue-regeneration longer than the unmodified-polymer. Here, the 1-ethyl-3-(3-diethylaminopropyl)carbodiimide(EDC)-hydroxybenzotriazole(HOBt) carboxyl-activating-system was evaluated for self-esterifying HA in the solid state. The aim was to propose an alternative to the time-consuming, conventional reaction of quaternary-ammonium-salts of HA with hydrophobic activating-systems in organic media, and to the EDC-mediated reaction, limited by by-product formation. Additionally, we aimed to obtain derivatives releasing defined molecular-weight(MW)-HA that would be valuable for tissue renewal. A 250 kDa-HA(powder/sponge) was reacted with increasing EDC/HOBt amounts. HA-modification was investigated through Size-Exclusion-Chromatography-Triple-Detector-Array-analyses, FT-IR/1H NMR and the products(XHAs) extensively characterized. Compared to conventional protocols, the set procedure is more efficient, avoids side-reactions, allows for an easier processing to diverse clinically-usable 3D-forms, leads to products gradually releasing HA under physiological conditions with the possibility to tune the MW of the biopolymer-released. Finally, the XHAs exhibit sound stability to Bovine-Testicular-Hyaluronidase, hydration/mechanical properties suitable for wound-dressings, with improvements over available matrices, and prompt in vitro wound-regeneration, comparably to linear-HA. To the best of our knowledge, the procedure is the first valid alternative to conventional protocols for HA self-esterification with advances in the process itself and in product performance.

Limosilactobacillus fermentum from buffalo milk is suitable for potential biotechnological process development and inhibits Helicobacter pylori in a gastric epithelial cell model.

Probiotics are living microorganisms that give beneficial health effects while consumed, and each strain possesses diverse and unique properties and also different technological characteristics that affect its ability to be produced at large scale. Limosilactobacillus fermentum is a widely studied member of probiotics, however, few data are available on the development of fermentation and downstream processes for the production of viable biomasses for potential industrial applications. In the present study a novel L. fermentum strain was isolated from buffalo milk and used as test example for biotechnological process development. The strain was able to produce up to 109 CFU/mL on a (glucose based) semi-defined medium deprived of animal-derived raw materials up to the pilot scale (150 L), demonstrating improved results compared to commonly used, although industrially not suitable, media rich of casein and beef extract. The study of strain behavior in batch experiments indicated that the highest concentration of viable cells was reached after only 8 h of growth, greatly shortening the process. Moreover, initial concentrations of glucose in the medium above 30 g/L, if not supported by higher nitrogen concentrations, reduced the yield of biomass and increased production of heterolactic fermentation by-products. Biomass concentration via microfiltration on hollow fibers, and subsequent spray-drying allowed to recover about 5.7 × 1010CFU/gpowder of viable cells, indicating strain resistance to harsh processing conditions. Overall, these data demonstrate the possibility to obtain and maintain adequate levels of viable L. fermentum cells by using a simple approach that is potentially suitable for industrial development. Moreover, since often exopolysaccharides produced by lactobacilli contribute to the strain's functionality, a partial characterization of the EPS produced by the newly identified L. fermentum strain was carried out. Finally, the effect of L. fermentum versus H. pylori in a gastric epithelial cell model was evaluated demonstrating its ability to stimulate the response of the immune system and displace the infective agent.