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.

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. 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 to specific consumer communities, opening the way to follow this approach with other probiotics of impact for human health.

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.

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.

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.

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.

Differential Secretome Profiling of Human Osteoarthritic Synoviocytes Treated with Biotechnological Unsulfated and Marine Sulfated Chondroitins.

Symptomatic slow-acting drugs (SYSADOA) are increasingly used as effective therapies for osteoarthritis, representing an attractive alternative to analgesics or non-steroidal anti-inflammatory drugs to relieve disease symptoms. Pharmaceutical preparations of chondroitin sulfate, derived from animal sources, alone or in combination with glucosamine sulfate, are widely recognized for their beneficial effect on osteoarthritis treatment. A growing interest has also been devoted to understanding the molecular mechanisms modulated by SYSADOA using -omic strategies, most of which rely on chondrocytes as a model system. In this work, by using an integrated strategy based on unbiased proteomics and targeted cytokine profiling by a multiplexed protein array, we identified differences in the secretomes of human osteoarthritic synoviocytes in response to biotechnological unsulfated, and marine sulfated chondroitins treatments. The combined strategy allowed the identification of candidate proteins showing both common and distinct regulation responses to the two treatments of chondroitins. These molecules, mainly belonging to ECM proteins, enzymes, enzymatic inhibitors and cytokines, are potentially correlated to treatment outcomes. Overall, the present results provide an integrated overview of protein changes in human osteoarthritic synoviocytes secretome associated to different chondroitin treatments, thus improving current knowledge of the biochemical effects driven by these drugs potentially involved in pathways associated to osteoarthritis pathogenesis.

Protective effect of extractive and biotechnological chondroitin in insulin amyloid and advanced glycation end product-induced toxicity.

Glycosaminoglycans are extracellular matrix components related to several biological functions and diseases. Chondroitin sulfate is a sulphated glycosaminoglycan synthesized as part of proteoglycan molecules. They are frequently associated with amyloid deposits and possess an active role in amyloid fibril formation. Recently, a neuroprotective effect of extracellular matrix components against amyloid toxicity and oxidative stress has been reported. Advanced glycation end products (AGEs), the end products of the glycation reaction, have been linked to amyloid-based neurodegenerative disease as associated with oxidative stress and inflammation. In this study we have analyzed the effect of chondroitin sulfate isolated from different species, in comparison with a new biotechnological unsulfated chondroitin, in the amyloid aggregation process of insulin, as well as the ability to prevent the formation of AGEs and related toxicity. The results have showed a determining role of chondroitin sulfate groups in modulating insulin amyloid aggregation. In addition, both sulfated and unsulfated chondroitins have shown protective properties against amyloid and AGEs-induced toxicity. These data are very relevant as a protective effect of these glycosaminoglycans in the AGE-induced toxicity was never observed before. Moreover, considering the issues related to the purity and safety of chondroitin from natural sources, this study suggests a new potential application for the biotechnological chondroitin.

Development of Semisynthetic, Regioselective Pathways for Accessing the Missing Sulfation Patterns of Chondroitin Sulfate.

Chondroitin sulfate (CS) is a glycosaminoglycan playing several biological functions, which seem to be encoded through its sulfation pattern. This "sulfation code" is still to be deciphered. One of the barriers to this goal is the difficulty in achieving structurally well-defined CS polysaccharides since extraction from natural sources often leads to complex heterogeneous structures. Instead, an approach relying on chemical modification of a microbially sourced unsulfated chondroitin can allow access to semisynthetic CS polysaccharides with a well-defined sulfation pattern. We report herein some new, suitably developed chemical strategies affording CSs with unprecedented sulfation patterns, carrying a single sulfate group regioselectively placed at either C-2 or C-3 position of the glucuronic acid residues or at both sites. In this way, all the possible variants of CS sulfation patterns can be now accessed. This will allow more detailed and complete structure-activity relationship investigations of CS biological functions and applications.

Molecular weight determination of heparosan- and chondroitin-like capsular polysaccharides: figuring out differences between wild -type and engineered Escherichia coli strains.

Heparin and chondroitin sulfate are used as anti-thrombic and anti-osteoarthritis drugs, respectively, but their pharmacological actions depend on their structural characteristics such as their sulfation grade and their molecular weight. In the last years, new fermentation-based biotechnological approaches have tried to obtain heparin and chondroitin sulfate starting from the heparosan and chondroitin-like capsular polysaccharides produced by Escherichia coli K5 and K4. The study of the microbial capsular polysaccharide molecular weight is critical to obtain nature-like or structural tailor cut glycosaminoglycan homologues. However, so far, it has been scarcely investigated. In this paper, for the first time, a new protocol was set up to determine the molecular weights of the capsular polysaccharides of three wild-type and three engineered E. coli K5 and K4 strains. The protocol includes a small-scale downstream train to purify the intact polysaccharides, directly from the fermentation broth supernatants, by using ultrafiltration membranes and anion exchange chromatography, and it couples size exclusion chromatography analyses with triple detector array. In the purification high recovery (> 85.0%) and the removal of the main contaminant, the lipopolysaccharide, were obtained. The averaged molecular weights of the wild-type capsular polysaccharides ranged from 51.3 to 90.9 kDa, while the engineered strains produced polysaccharides with higher molecular weights, ranging from 68.4 to 130.6 kDa, but with similar polydispersity values between 1.1 and 1.5.

Physiological characterization and quantitative proteomic analyses of metabolically engineered E. coli K4 strains with improved pathways for capsular polysaccharide biosynthesis.

Among capsulated bacteria, some produce polysaccharides with unique properties that have been shown to possess relevant industrial applications and commercial value. The capsular polysaccharide (CPS) produced by Escherichia coli K4 is similar to chondroitin sulphate, and recent efforts focused on the development of genetic and fermentation strategies to increase its production titers up to technologically attractive levels. However, the control of the metabolic pathways leading to CPS synthesis together with the effect of varying the concentration of pathway intermediates on CPS final titers, is still quite unexplored, and not fully understood. In the present study four genes involved in the biosynthesis of UDP-sugar CPS precursors, namely kfoA, kfoF, pgm, and galU, were overexpressed in different combinations, and diversely affected the biosynthetic machinery. At the physiological level, results revealed a central role for kfoF, coding for UDP-glucose dehydrogenase, that increased CPS production mostly. In the attempt to unravel the molecular mechanisms regulating CPS biosynthesis, an in depth analysis of the proteome of the recombinant strains overexpressing respectively pgm and galU, and pgm, galU, and kfoF was performed and compared to the wild-type. Although, interestingly, in both strains the impact of the genetic manipulation seemed rather limited at the proteome level, results obtained from the triple mutant indicated a crosstalk between the two pathways leading to UDP-sugar precursors biosynthesis, and also an unexpected link with the purine biosynthetic pathway. Overall our results present new insights into the role of metabolic intermediates for the formation of capsular polysaccharides, utilizing a systematic approach of metabolic engineering, combined with state-of-the-art quantitative proteomic approaches, as well as genetic and physiological information.

Lactobacillus crispatus L1: high cell density cultivation and exopolysaccharide structure characterization to highlight potentially beneficial effects against vaginal pathogens.

BACKGROUND: Vaginal lactic acid bacteria defend the host against pathogens through a combination of competitive exclusion, competition for nutrients, production of antimicrobial substances and through the activation of the immune system. A new human isolate named Lactobacillus crispatus L1 was characterized in this work, and a preliminary evaluation of its probiotic potential is described together with a process to obtain a high productivity of viable biomass. RESULTS: In a simulated digestion process 1.8⋅10(10) cells∙ml(-1) survived the gastric environment with 80% viability, without being affected by small intestine juices. Experiments on six different C sources were performed to analyze growth and organic acids production and, glucose, provided the best performances. A microfiltration strategy was exploited to improve the cellular yield in 2 L-fermentation processes, reaching 27 g · l(-1) of dry biomass. Moreover, L. crispatus L1 demonstrated a greater stability to high concentrations of lactic acid, compared to other lactobacilli. The specific L. crispatus L1 exopolysaccharide was purified from the fermentation broth and characterized by NMR showing structural features and similarity to exopolysaccharides produced by pathogenic strains. Live L. crispatus L1 cells strongly reduced adhesion of a yeast pathogenic strain, Candida albicans in particular, in adherence assays. Interestingly a higher expression of the human defensin HBD-2 was also observed in vaginal cells treated with the purified exopolysaccharide, indicating a possible correlation with C. albicans growth inhibition. CONCLUSIONS: The paper describes the evaluation of L. crispatus L1 as potential vaginal probiotic and the fermentation processes to obtain high concentrations of viable cells.

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.

Homologous overexpression of RfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide.

BACKGROUND: Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation. RESULTS: E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g ∙ L(-1) of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis. CONCLUSIONS: The present study describes the identification of a gene target and the application of a successful metabolic engineering strategy to the unconventional host E. coli K4 demonstrating the feasibility of using the recombinant strain as stable cell factory for further process implementations.

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production.

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological-chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.

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.

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.

Evaluation of Different Activity of Lactobacillus spp. against Two Proteus mirabilis Isolated Clinical Strains in Different Anatomical Sites In Vitro: An Explorative Study to Improve the Therapeutic Approach.

Urinary tract infections (UTIs) and catheter-associated UTIs (CAUTIs) are the principal hospital-acquired infections. Between these, bacterial prostatitis is believed to be the leading cause of recurrent UTIs in men under 50 years of age and is often unresponsive to antibiotic treatment. Proteus mirabilis is more commonly associated with UTIs in these abnormalities, especially in patients undergoing catheterization. Lactobacillus spp. are an important component of the human microbiota and occur in large quantities in foods. Probiotics are proposed as an alternative to antibiotic therapy in the treatment of urinary tract infections. In addition to their ability to produce antimicrobial metabolites, they have immunomodulatory activity and do not cause side effects. For this reason, the combination of probiotic microorganisms and conventional drugs was considered. The aim of this work was to select the most active Lactobacillus strains against two clinical isolates of P. mirabilis on bladder and prostatic epithelium, potentially exploitable to improve the clinical management of UTIs.

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.

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.