Vegan grade medium component screening and concentration optimization for the fermentation of the probiotic strain Lactobacillus paracasei IMC 502® using Design of Experiments.

Lactobacillus paracasei IMC502® is a commercially successful probiotic strain, however, there are no reports that investigate growth medium composition in relation to improved biomass production for this strain. The major outcome of the present study is the design and optimization of a growth medium based on vegan components to be used in the cultivation of Lactobacillus paracasei IMC502®, by using Design of Experiments (DoE). Besides comparing different carbon sources, the use of plant-based peptones as nitrogen sources was considered. In particular, the use of guar peptone as the main nitrogen source, in the optimization of fermentation media for the production of probiotics, could replace other plant peptones (e.g. potato, rice, wheat and soy) which are part of the human diet, thereby avoiding an increase in product and process prices. A model with R2 and adjusted R2 values higher than 95% was obtained. Model accuracy was equal to 94.11%. The vegan-optimized culture medium described in this study increased biomass production by about 65% compared to growth on De Man-Rogosa-Sharpe (MRS) medium. Moreover, this approach showed that most of the salts and trace elements generally present in MRS are not affecting biomass production, thus a simplified medium preparation can be proposed with higher probiotic biomass yield and titer. The possibility to obtain viable lactic acid bacteria at high density from vegetable derived nutrients will be of great interest for specific consumer communities, opening the way to follow this approach with other probiotics of impact for human health.

Biosynthesis of Hesperetin, Homoeriodictyol, and Homohesperetin in a Transcriptomics-Driven Engineered Strain of Streptomyces albidoflavus.

Flavonoids exhibit various bioactivities including anti-oxidant, anti-tumor, anti-inflammatory, and anti-viral properties. Methylated flavonoids are particularly significant due to their enhanced oral bioavailability, improved intestinal absorption, and greater stability. The heterologous production of plant flavonoids in bacterial factories involves the need for enough biosynthetic precursors to allow for high production levels. These biosynthetic precursors are malonyl-CoA and l-tyrosine. In this work, to enhance flavonoid biosynthesis in Streptomyces albidoflavus, we conducted a transcriptomics study for the identification of candidate genes involved in l-tyrosine catabolism. The hypothesis was that the bacterial metabolic machinery would detect an excess of this amino acid if supplemented with the conventional culture medium and would activate the genes involved in its catabolism towards energy production. Then, by inactivating those overexpressed genes (under an excess of l-tyrosine), it would be possible to increase the intracellular pools of this precursor amino acid and eventually the final flavonoid titers in this bacterial factory. The RNAseq data analysis in the S. albidoflavus wild-type strain highlighted the hppD gene encoding 4-hydroxyphenylpyruvate dioxygenase as a promising target for knock-out, exhibiting a 23.2-fold change (FC) in expression upon l-tyrosine supplementation in comparison to control cultivation conditions. The subsequent knock-out of the hppD gene in S. albidoflavus resulted in a 1.66-fold increase in the naringenin titer, indicating enhanced flavonoid biosynthesis. Leveraging the improved strain of S. albidoflavus, we successfully synthesized the methylated flavanones hesperetin, homoeriodictyol, and homohesperetin, achieving titers of 2.52 mg/L, 1.34 mg/L, and 0.43 mg/L, respectively. In addition, the dimethoxy flavanone homohesperetin was produced as a byproduct of the endogenous metabolism of S. albidoflavus. To our knowledge, this is the first time that hppD deletion was utilized as a strategy to augment the biosynthesis of flavonoids. Furthermore, this is the first report where hesperetin and homoeriodictyol have been synthesized from l-tyrosine as a precursor. Therefore, transcriptomics is, in this case, a successful approach for the identification of catabolism reactions affecting key precursors during flavonoid biosynthesis, allowing the generation of enhanced production strains.

Highly Concentrated Stabilized Hybrid Complexes of Hyaluronic Acid: Rheological and Biological Assessment of Compatibility with Adipose Tissue and Derived Stromal Cells towards Regenerative Medicine.

Cells and extracts derived from adipose tissue are gaining increasing attention not only in plastic surgery and for aesthetic purposes but also in regenerative medicine. The ability of hyaluronan (HA) to support human adipose stromal cell (hASC) viability and differentiation has been investigated. However, the compatibility of adipose tissue with HA-based formulation in terms of biophysical and rheological properties has not been fully addressed, although it is a key feature for tissue integration and in vivo performance. In this study, the biophysical and biochemical properties of highly concentrated (45 mg/mL) high/low-molecular-weight HA hybrid cooperative complex were assessed with a further focus on the potential application in adipose tissue augmentation/regeneration. Specifically, HA hybrid complex rheological behavior was observed in combination with different adipose tissue ratios, and hyaluronidase-catalyzed degradation was compared to that of a high-molecular-weight HA (HHA). Moreover, the HA hybrid complex's ability to induce in vitro hASCs differentiation towards adipose phenotype was evaluated in comparison to HHA, performing Oil Red O staining and analyzing gene/protein expression of PPAR-γ, adiponectin, and leptin. Both treatments supported hASCs differentiation, with the HA hybrid complex showing better results. These outcomes may open new frontiers in regenerative medicine, supporting the injection of highly concentrated hybrid formulations in fat compartments, eventually enhancing residing staminal cell differentiation and improving cell/growth factor persistence towards tissue regeneration districts.

Thermophilic biocatalysts for one-step conversion of citrus waste into lactic acid.

Agri-food residues offer significant potential as a raw material for the production of L-lactic acid through microbial fermentation. Weizmannia coagulans, previously known as Bacillus coagulans, is a spore-forming, lactic acid-producing, gram-positive, with known probiotic and prebiotic properties. This study aimed to evaluate the feasibility of utilizing untreated citrus waste as a sustainable feedstock for the production of L-lactic acid in a one-step process, by using the strain W. coagulans MA-13. By employing a thermophilic enzymatic cocktail (Cellic CTec2) in conjunction with the hydrolytic capabilities of MA-13, biomass degradation was enhanced by up to 62%. Moreover, batch and fed-batch fermentation experiments demonstrated the complete fermentation of glucose into L-lactic acid, achieving a concentration of up to 44.8 g/L. These results point to MA-13 as a microbial cell factory for one-step production of L-lactic acid, by combining cost-effective saccharification with MA-13 fermentative performance, on agri-food wastes. Moreover, the potential of this approach for sustainable valorization of agricultural waste streams is successfully proven. KEY POINTS: • Valorization of citrus waste, an abundant residue in Mediterranean countries. • Sustainable production of the L-( +)-lactic acid in one-step process. • Enzymatic pretreatment is a valuable alternative to the use of chemical.

Correction: Vassallo et al. Hyaluronic Acid-Based Injective Medical Devices: In Vitro Characterization of Novel Formulations Containing Biofermentative Unsulfated Chondroitin or Extractive Sulfated One with Cyclodextrins. Pharmaceuticals 2023, 16, 1429.

In the original publication [...].

Hyaluronic Acid-Based Injective Medical Devices: In Vitro Characterization of Novel Formulations Containing Biofermentative Unsulfated Chondroitin or Extractive Sulfated One with Cyclodextrins.

Currently, chondroitin sulfate (CS) and hyaluronic acid (HA) pharma-grade forms are used for osteoarthritis (OA) management, CS as an oral formulations component, and HA as intra-articular injective medical devices. Recently, unsulfated chondroitin, obtained through biofermentative (BC) manufacturing, has been proposed for thermally stabilized injective preparation with HA. This study aimed to highlight the specific properties of two commercial injective medical devices, one based on HA/BC complexes and the other containing HA, extractive CS, and cyclodextrins, in order to provide valuable information for joint disease treatments. Their biophysical and biomechanical features were assayed; in addition, biological tests were performed on human pathological chondrocytes. Rheological measurements displayed similar behavior, with a slightly higher G' for HA/BC, which also proved superior stability to the hyaluronidase attack. Both samples reduced the expression of specific OA-related biomarkers such as NF-kB, interleukin 6 (IL-6), and metalloprotease-13 (MMP-13). Moreover, HA/BC better ensured chondrocyte phenotype maintenance by up-regulating collagen type 2A1 (COLII) and aggrecan (AGN). Notwithstanding, the similarity of biomolecule components, the manufacturing process, raw materials characteristics, and specific concentration resulted in affecting the biomechanical and, more interestingly, the biochemical properties, suggesting potential better performances of HA/BC in joint disease treatment.

Restoring Adipose Tissue Homeostasis in Response to Aging: Initial Clinical Experience with Profhilo Structura®.

The aim of the case series was to determine the efficacy of a new medical device developed for adipose tissue restoration in the face. The medical device used the patented NAHYCO® Hybrid Technology to deliver 45 mg of high- (1400 ± 200 kDa) and 45 mg of low- (100 ± 20 kDa) molecular-weight hyaluronan, in 2 mL. Patients and methods: Twenty-two volunteers, aged 36-60 years. Two mL of Profhilo® Structura was injected using a 25 G cannula for each hemiface, into superficial fat compartment along the line from the preauricular area to the mandibular angle. Two injections were performed, and Profhilo Structura's effect on restoring adipose tissue was evaluated immediately after treatment, and over a 6-month follow-up. The studied medical device revealed a pseudoplastic behavior and consistency that allowed easy extrusion from a syringe. It showed a lower viscosity compared to dermal fillers, based on crosslinked HA. Clinically, the soft tissue thickness increased immediately after injection, and the clinical improvement persisted across a 6-month follow-up. The self-reported satisfaction with the treatment showed an amelioration in the midface of all the subjects enrolled, with no adverse effects. Profhilo® Structura demonstrated a peculiar fat compartment integration, with a regenerating effect on adipose tissue senescence. The skin thickening and compaction effects were similar to those obtained using chemically crosslinked dermal fillers, while a natural look was preserved, and the use of crosslinking agents was avoided.

Physicochemical Characterization of Chitosan/Poly-γ-Glutamic Acid Glass-like Materials.

This paper sets up a new route for producing non-covalently crosslinked bio-composites by blending poly-γ-glutamic acid (γ-PGA) of microbial origin and chitosan (CH) through poly-electrolyte complexation under specific experimental conditions. CH and two different molecular weight γ-PGA fractions have been blended at different mass ratios (1/9, 2/8 and 3/7) under acidic pH. The developed materials seemed to behave like moldable hydrogels with a soft rubbery consistency. However, after dehydration, they became exceedingly hard, glass-like materials completely insoluble in water and organic solvents. The native biopolymers and their blends underwent comprehensive structural, physicochemical, and thermal analyses. The study confirmed strong physical interactions between polysaccharide and polyamide chains, facilitated by electrostatic attraction and hydrogen bonding. The materials exhibited both crystalline and amorphous structures and demonstrated good thermal stability and degradability. Described as thermoplastic and saloplastic, these bio-composites offer vast opportunities in the realm of polyelectrolyte complexes (PECs). This unique combination of properties allowed the bio-composites to function as glass-like materials, making them highly versatile for potential applications in various fields. They hold potential for use in regenerative medicine, biomedical devices, food packaging, and 3D printing. Their environmentally friendly properties make them attractive candidates for sustainable material development in various industries.

ERK Signaling Pathway Is Constitutively Active in NT2D1 Non-Seminoma Cells and Its Inhibition Impairs Basal and HGF-Activated Cell Proliferation.

c-MET/hepatocyte growth factor (HGF) system deregulation is a well-known feature of malignancy in several solid tumors, and for this reason this system and its pathway have been considered as potential targets for therapeutic purposes. In previous manuscripts we reported c-MET/HGF expression and the role in testicular germ cell tumors (TGCTs) derived cell lines. We demonstrated the key role of c-Src and phosphatidylinositol 3-kinase (PI3K)/AKT adaptors in the HGF-dependent malignant behavior of the embryonal carcinoma cell line NT2D1, finding that the inhibition of these onco-adaptor proteins abrogates HGF triggered responses such as proliferation, migration, and invasion. Expanding on these previous studies, herein we investigated the role of mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK) pathways in the HGF-dependent and HGF-independent NT2D1 cells biological responses. To inhibit MAPK/ERK pathways we chose a pharmacological approach, by using U0126 inhibitor, and we analyzed cell proliferation, collective migration, and chemotaxis. The administration of U0126 together with HGF reverts the HGF-dependent activation of cell proliferation but, surprisingly, does not exert the same effect on NT2D1 cell migration. In addition, we found that the use of U0126 alone significantly promotes the acquisition of NT2D1 «migrating phenotype¼, while collective migration of NT2D1 cells was stimulated. Notably, the inhibition of ERK activation in the absence of HGF stimulation resulted in the activation of the AKT-mediated pathway, and this let us speculate that the paradoxical effects obtained by using U0126, which are the increase of collective migration and the acquisition of partial epithelium-mesenchyme transition (pEMT), are the result of compensatory pathways activation. These data highlight how the specific response to pathway inhibitors, should be investigated in depth before setting up therapy.

Biotechnological advances in the synthesis of modified chondroitin towards novel biomedical applications.

Chondroitin sulfate (CS) is a well-known glycosaminoglycan present in a large variety of animal tissues, with an outstanding structural heterogeneity mainly related to molecular weight and sulfation pattern. Recently, few microorganisms, eventually engineered, proved able to synthesize the CS biopolymer backbone, composed of d-glucuronic acid and N-acetyl-d-galactosamine linked through alternating ß-(1-3)- and ß-(1-4)-glycosidic bonds, and secrete the biopolymers generally unsulfated and possibly decorated with other carbohydrates/molecules. Enzyme catalyzed/assisted methods and chemical tailored protocols allowed to obtain a variety of macromolecules not only resembling the natural extractive ones, but even enlarging the access to unnatural structural features. These macromolecules have been investigated for their bioactivity in vitro and in vivo establishing their potentialities in an array of novel applications in the biomedical field. This review aims to present an overview of the advancements in: i) the metabolic engineering strategies and the biotechnological processes towards chondroitin manufacturing; ii) the chemical approaches applied to obtain specific structural features and targeted decoration of the chondroitin backbone; iii) the biochemical and biological properties of the diverse biotechnological-sourced chondroitin polysaccharides reported so far, unraveling novel fields of applications.

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.

Multi-step Strategies Toward Regioselectively Sulfated M-Rich Alginates.

Sulfated alginates (ASs), as well as several artificially sulfated polysaccharides, show interesting bioactivities. The key factors for structure-activity relationships studies are the degree of sulfation and the distribution of the sulfate groups along the polysaccharide backbone (sulfation pattern). The former parameter can often be controlled through stoichiometry, while the latter requires the development of suitable chemical or enzymatic, regioselective methods and is still missing for ASs. In this work, a study on the regioselective installation of several different protecting groups on a d-mannuronic acid enriched (M-rich) alginate is reported in order to develop a semi-synthetic access to regioselectively sulfated AS derivatives. A detailed structural characterization of the obtained ASs revealed that the regioselective sulfation could be achieved complementarily at the O-2 or O-3 positions of M units through multi-step sequences relying upon a silylating or benzoylating reagent for the regioselective protection of M-rich alginic acid, followed by sulfation and deprotection.

Novel Insights into circRNA Saga Coming from Spermatozoa and Epididymis of HFD Mice.

Obesity is a pathophysiological disorder associated with adiposity accumulation, oxidative stress, and chronic inflammation state that is progressively increasing in younger population worldwide, negatively affecting male reproductive skills. An emerging topic in the field of male reproduction is circRNAs, covalently closed RNA molecules produced by backsplicing, actively involved in a successful spermatogenesis and in establishing high-quality sperm parameters. However, a direct correlation between obesity and impaired circRNA cargo in spermatozoa (SPZ) remains unclear. In the current work, using C57BL6/J male mice fed with a high-fat diet (HFD, 60% fat) as experimental model of oxidative stress, we investigated the impact of HFD on sperm morphology and motility as well as on spermatic circRNAs. We performed a complete dataset of spermatic circRNA content by a microarray strategy, and differentially expressed (DE)-circRNAs were identified. Using a circRNA/miRNA/target network (ceRNET) analysis, we identified circRNAs potentially involved in oxidative stress and sperm motility pathways. Interestingly, we demonstrated an enhanced skill of HFD sperm in backsplicing activity together with an inefficient epididymal circRNA biogenesis. Fused protein in sarcoma (FUS) and its ability to recruit quaking (QKI) could be involved in orchestrating such mechanism.

Preparation and Characterization of New Bioplastics Based on Polybutylene Succinate (PBS).

Sea and environmental pollution due to microplastics are global problems that in recent years have attracted particular interest in the scientific community. The increase in the world population and the consequent consumerism of non-reusable materials are amplifying these problems. In this manuscript, we present novel bioplastics, which are completely biodegradable, for their potential use in food packaging, to replace fossil-fuel-derived plastic films and slow food degradation due to oxidative processes or microbial contamination. In this study, thin films based on polybutylene succinate (PBS) were prepared to reduce pollution, and different percentages by weight (1, 2 and 3 wt%) of extra virgin olive oil (EVO) and coconut oil (CO) were included to improve the chemico-physical properties of the polymer and possibly improve the functionality of the films in terms of prolonged food preservation. Attenuated total reflectance Fourier transform infrared (ATR/FTIR) spectroscopy was used to evaluate the interactions between the polymer and the oil. Furthermore, the mechanical properties and thermal behavior of the films were evaluated as a function of the oil content. A scanning electron microscopy (SEM) micrograph showed the surface morphology and the thickness of the materials. Finally, apple and kiwi were selected for a food-contact test, and the wrapped sliced fruit was monitored and evaluated for 12 days to macroscopically evaluate the oxidative process and/or eventually occurring contamination. The films were shown to reduce the browning of sliced fruit due to oxidation, and no molds were evidenced up to 10/12 days of observation with the addition of PBS, with 3 wt% of EVO achieving the best outcomes.

In Vitro Evaluation of the Most Active Probiotic Strains Able to Improve the Intestinal Barrier Functions and to Prevent Inflammatory Diseases of the Gastrointestinal System.

Background: The integrity of the intestinal barrier is fundamental to gut health and homeostasis; its damage can increase intestinal permeability, with translocation of bacteria and/or endotoxins from gut, and the onset of various intestinal diseases. Lactobacillus spp. is one of the most common probiotics normally found in fermented foods and dairy products and is known for its anti-inflammatory and immunomodulatory properties and for its ability to protect and enhance the intestinal barrier functions. The aim of this work was to evaluate the ability of different strains of Lactobacillus spp. to improve in vitro the integrity of the intestinal barrier, to exert anti-inflammatory and immunomodulatory activity and to prevent Salmonella Typhimurium and enteroinvasive Escherichia coli (EIEC) infections. Methods: We analyzed the cellular expression of tight junctions, antimicrobial peptide HBD-2, pro-inflammatory cytokines and the inhibition of pathogens adhesion and invasion in a model of co-cultured epithelial cells treated with Lactobacillus spp. Results: L. brevis, L. reuteri and L. rhamnosus proved to be more effective in protecting the intestinal epithelium. Conclusions: These in vitro studies can help select strains particularly active in their intended use to obtain consortia formulations that can have as much maximum yield as possible in terms of patient benefit.

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.

Antioxidants positively regulate obesity dependent circRNAs - sperm quality - functional axis.

Obesity is a pathophysiological condition, dependent on body fat accumulation, that progressively induces systemic oxidative stress/inflammation leading to a set of associated clinical manifestations, including male infertility. CircRNAs, covalently closed RNA molecules, are key regulators of sperm quality. Recently, we have characterized a complete profile of high-fat diet (HFD) spermatic circRNA cargo, predicting paternal circRNA dependent networks (ceRNETs), potentially involved in sperm oxidative stress and motility anomalies. In the current work, using HFD C57BL6/J male mice, orally treated with a mix of bioactive molecules (vitamin C; vitamin B12; vitamin E; selenium-L-methionine; glutathione-GSH) for 4 weeks, a reversion of HFD phenotype was observed. In addition, the functional action of the proposed formulations on circRNA biogenesis was evaluated by assessing the endogenous spermatic FUS-dependent backsplicing machinery and related circRNA cargo. After that, spermatic viability and motility were also analyzed. Paternal ceRNETs, potentially involved in oxidative stress regulation and sperm motility defects, were identified and used to suggest that the beneficial action of the food supplements here conveniently formulated on sperm motility was likely due to the recovery of circRNA profile. Such a hypothesis was, then, verified by an in vitro assay.

Evaluation of unsulfated biotechnological chondroitin in a knee osteoarthritis mouse model as a potential novel functional ingredient in nutraceuticals and pharmaceuticals.

Osteoarthritis is a very disabling disease that can be treated with both non-pharmacological and pharmacological approaches. In the last years, pharmaceutical-grade chondroitin sulfate (CS) and glucosamine emerged as symptomatic slow-acting molecules, effective in pain reduction and improved function in patients affected by osteoarthritis. CS is a sulfated glycosaminoglycan that is currently produced mainly by extraction from animal tissues, and it is commercialized as a pharmaceutical-grade ingredient and/or food supplement. However, public concern on animal product derivatives has prompted the search for alternative non-extractive production routes. Thus, different approaches were established to obtain animal-free natural identical CS. On the other hand, the unsulfated chondroitin, which can be obtained via biotechnological processes, demonstrated promising anti-inflammatory properties in vitro, in chondrocytes isolated from osteoarthritic patients. Therefore, the aim of this study was to explore the potential of chondroitin, with respect to the better-known CS, in an in vivo mouse model of knee osteoarthritis. Results indicate that the treatment with biotechnological chondroitin (BC), similarly to CS, significantly reduced the severity of mechanical allodynia in an MIA-induced osteoarthritic mouse model. Decreased cartilage damage and a reduction of inflammation- and pain-related biochemical markers were also observed. Overall, our data support a beneficial activity of biotechnological unsulfated chondroitin in the osteoarthritis model tested, thus suggesting BC as a potential functional ingredient in pharmaceuticals and nutraceuticals with the advantage of avoiding animal tissue extraction.

Molecular Fingerprint of Human Pathological Synoviocytes in Response to Extractive Sulfated and Biofermentative Unsulfated Chondroitins.

Pharma-grade extractive chondroitin sulfate (CS) is widely used for osteoarthritis (OA) treatment. Recently, unsulfated biofermentative chondroitin (BC) proved positive effects in OA in vitro model. This study, based on primary pathological human synoviocytes, aimed to analyze, by a multiplex assay, a panel of OA-related biomarkers in response to short-term treatments with bovine (CSb), pig (CSp) and fish (CSf) chondroitins, in comparison to BC. As expected, all samples had anti-inflammatory properties, however CSb, CSf and especially BC affected more cytokines and chemokines. Based on these results and molecular weight similarity, CSf and BC were selected to further explore the synoviocytes' response. In fact, Western blot analyses showed CSf and BC were comparable, downregulating OA-related biomarkers such as the proteins mTOR, NF-kB, PTX-3 and COMP-2. Proteomic analyses, performed by applying a nano-LC-MS/MS TMT isobaric labelling-based approach, displayed the modulation of both common and distinct molecules to chondroitin treatments. Thus, CSf and BC modulated the biological mediators involved in the inflammation cascade, matrix degradation/remodeling, glycosaminoglycans' synthesis and cellular homeostasis. This study helps in shedding light on different molecular mechanisms related to OA disease that may be potentially affected not only by animal-source chondroitin sulfate but also by unsulfated biofermentative chondroitin.

Optimization of growth of Levilactobacillus brevis SP 48 and in vitro evaluation of the effect of viable cells and high molecular weight potential postbiotics on Helicobacter pylori.

Several Levilactobacillus brevis strains have the potential to be used as probiotics since they provide health benefits due to the interaction of live cells, and of their secreted products, with the host (tissues). Therefore, the development of simple fermentation processes that improve cell viability to reduce industrial production costs, and at the same time the characterization and biological evaluation of cell-free postbiotics that can further promote application, are of great interest. In the present study, small scale batch fermentations on semi defined media, deprived of animal derived raw materials, were used to optimize growth of L. brevis SP48, reaching 1.2 ± 0.4 × 1010 CFU/ml of viable cells after 16 h of growth. Displacement, competition, and inhibition assays compared the effect, on Helicobacter pylori, of L. brevis cells to that of its partially purified potentially postbiotic fraction rich in exopolysaccharides and proteins. The expression of pro and anti-inflammatory biochemical markers indicated that both samples activated antimicrobial defenses and innate immunity in a gastric model. Moreover, these compounds also acted as modulators of the inflammatory response in a gut in vitro model. These data demonstrate that the high molecular weight compounds secreted by L. brevis SP48 can contrast H. pylori and reduce inflammation related to intestinal bowel disease, potentially overcoming issues related to the preservation of probiotic viability.