Parental Social Isolation during Adolescence Alters Gut Microbiome in Rat Male Offspring.

Previous work from our laboratory demonstrated that parental stress, induced by social isolation starting at puberty, leads to behavioral, endocrine, and biochemical changes in the male, but not female, offspring (ISO-O) of Sprague-Dawley rats. Here, we report alterations in the gut microbiota composition of ISO-O vs. grouped-housed offspring (GH-O), although all animals received the same diet and were housed in the same conditions. Analysis of bacterial communities by next-generation sequencing (NGS) of 16S rRNA gene revealed alterations at family and order levels within the main phyla of Bacteroides, Proteobacteria, and Firmicutes, including an almost total deficit in Limosilactobacillus reuteri (formerly Lactobacillus reuteri) and a significant increase in Ligilactobacillus murinus (formerly Lactobacillus murinus). In addition, we found an increase in the relative abundance of Rhodospirillales and Clostridiales in the families of Lachnospiraceae and Ruminococcaceae, and Bacteroidales in the family of Prevotellaceae. Furthermore, we examined plasma levels of the proinflammatory cytokines interleukin-1-beta and tumor necrosis factor-alpha, which did not differ between the two groups, while corticosterone concentrations were significantly increased in ISO-O rats. Our findings suggest that adverse environmental conditions experienced by parents may have an impact on the likelihood of disease development in the subsequent generations.

Probiotic lactobacilli attenuate oxysterols-induced alteration of intestinal epithelial cell monolayer permeability: Focus on tight junction modulation.

Oxidative stress and inflammation lead by dietary oxidised lipids, as oxysterols, have been linked to the loss of intestinal barrier integrity, a crucial event in the initiation and progression of intestinal disorders. In the last decade, probiotic lactobacilli have emerged as an interesting tool to improve intestinal health, thanks to their antioxidant and anti-inflammatory properties. The aim of the present study was to evaluate the ability of two commercial probiotic strains of lactobacilli (Lactiplantibacillus plantarum 299v® (DMS 9843) and Lacticaseibacillus casei DG® (CNCMI-1572)), both as live bacteria and intracellular content, to attenuate the oxysterols-induced alteration of intestinal epithelial Caco-2 cell monolayer permeability. Our investigation was focused on the modulation of tight junctions (TJs) proteins, occludin, ZO-1 and JAM-A, in relation to redox-sensitive MAPK p38 activation. Obtained results provided evidence on the ability of the two probiotics to counteract the alteration of monolayer permeability and loss of TJs proteins, at least in part, through the modulation of p38 pathway. The protective action was exerted by live bacteria, whose adhesion to Caco-2 cells was not altered by oxysterols, and bacterial intracellular components equally able to interact with the signaling pathway.

Inhibitory Effect of Lactiplantibacillusplantarum and Lactococcus lactis Autochtonous Strains against Listeria monocytogenes in a Laboratory Cheese Model.

In the present study, six Lactococcus lactis and seven Lactiplantibacillus plantarum strains isolated from artisanal Sardinian dairy products were evaluated for their efficacy in controlling the growth of Listeria monocytogenes during the storage of miniature fresh cheese manufactured on a laboratory scale to exploit their possible use as biopreservatives. The strains were tested for antimicrobial activity and some technological characteristics before using them in miniature fresh cheese to evaluate their in situ antilisterial effect. Our results showed that five strains (L. lactis 16FS16-9/20234-11FS16 and Lpb. plantarum 1/14537-4A/20045) could be considered suitable candidates for use as protective cultures in fresh cheese manufacture since they significantly lowered the pathogen counts by 3-4 log units compared to the control; however, all strains tested were capable of decreasing L. monocytogenes numbers. Our results suggest that the single and combined action of the acidifying power and the production of bacteriocin of these strains was capable of controlling and/or reducing the growth of L. monocytogenes. Considering their technological characteristics, they might be used as starter/adjunct cultures to increase the safety of the products, perhaps in association with other antimicrobial hurdles.

Influence of Autochthonous Putative Probiotic Cultures on Microbiota, Lipid Components and Metabolome of Caciotta Cheese.

The present study was undertaken to produce probiotic Caciotta cheeses from pasteurized ewes' milk by using different combinations of autochthonous microbial cultures, containing putative probiotic strains, and evaluate their influence on gross composition, lipid components, sensory properties and microbiological and metabolite profiles of the cheeses throughout ripening process. A control cheese was produced using commercial starter cultures. The hydrophilic molecular pools (mainly composed by amino acids, organic acids, and carbohydrates) were characterized by means of 1H NMR spectroscopy, while the cholesterol, α-tocopherol and fatty acid composition by HPLC-DAD/ELSD techniques. Conventional culturing and a PCR-DGGE approach using total cheese DNA extracts were used to analyze cheese microbiota and monitor the presence and viability of starters and probiotic strains. Our findings showed no marked differences for gross composition, total lipids, total cholesterol, and fatty acid levels among all cheeses during ripening. Differently, the multivariate statistical analysis of NMR data highlighted significant variations in the cheese' profiles both in terms of maturation time and strains combination. The use of autochthonous cultures and adjunct probiotic strains did not adversely affect acceptability of the cheeses. Higher levels of lactobacilli (viability of 108-109 cfu/g of cheese) were detected in cheeses made with the addition of probiotic autochthonous strains with respect to control cheese during the whole ripening period, suggesting the adequacy of Caciotta cheese as a carrier for probiotic bacteria delivery.

Metal self-assembly mimosine peptides with enhanced antimicrobial activity: towards a new generation of multitasking chelating agents.

Mimosine is a non-protein amino acid with various properties, such as antibacterial, anti-inflammatory, anti-cancer and anti-virus among others. Due to its structural similarity with deferiprone (DFP), mimosine is a potential excellent metal chelator. In the present work, we combine experimental and theoretical (DFT) approaches in order to investigate the properties of mimosine peptides. Six different peptides were synthesized and their complex stoichiometry and stability were characterized by means of UV-Vis spectrophotometry. Then, the binding mode and self-assembly features of the peptides were evaluated using a DFT approach, taking into account different number of mimosine amino acids and varying the length of the spacer between the mimosine residues, and there was good agreement between experimental data and computational calculations. Further elucidations of the structural properties of these peptides allowed us to propose improvements in the structure of the mimosine moiety which can lead to enhanced affinity for high-valent metals. Moreover, we demonstrate that these peptides show an anti-microbial activity against Gram positive bacteria that is enhanced by the formation of a complex with iron(iii) ions. The mimosine peptides could be an alternative to antimicrobial peptides (AMPs), which are expensive and susceptible to proteolytic degradation. In summary, in the present work, we propose a new generation of multipurpose mimosine-based peptides as new metal self-assembly chelators which could be a turning point in biomedical and nanotechnological applications.

Antibacterial Activity and Molecular Docking Studies of a Selected Series of Hydroxy-3-arylcoumarins.

Antibiotic resistance is one of the main public health concerns of this century. This resistance is also associated with oxidative stress, which could contribute to the selection of resistant bacterial strains. Bearing this in mind, and considering that flavonoid compounds are well known for displaying both activities, we investigated a series of hydroxy-3-arylcoumarins with structural features of flavonoids for their antibacterial activity against different bacterial strains. Active compounds showed selectivity against the studied Gram-positive bacteria compared to Gram-negative bacteria. 5,7-Dihydroxy-3-phenylcoumarin (compound 8) displayed the best antibacterial activity against Staphylococcus aureus and Bacillus cereus with minimum inhibitory concentrations (MICs) of 11 g/mL, followed by Staphylococcus aureus (MRSA strain) and Listeria monocytogenes with MICs of 22 and 44 g/mL, respectively. Moreover, molecular docking studies performed on the most active compounds against Staphylococcus aureus tyrosyl-tRNA synthetase and topoisomerase II DNA gyrase revealed the potential binding mode of the ligands to the site of the appropriate targets. Preliminary structure-activity relationship studies showed that the antibacterial activity can be modulated by the presence of the 3-phenyl ring and by the position of the hydroxyl groups at the coumarin scaffold.

Microbiota of Sardinian Goat's Milk and Preliminary Characterization of Prevalent LAB Species for Starter or Adjunct Cultures Development.

This work was performed to study the microbiota of raw goat's milk (67 samples) collected in different areas of Sardinia, in order to select autochthonous lactic acid bacteria (LAB) strains for use in goat cheese manufacturing. Total mesophilic bacteria ranged between 105 and 107 cfu/mL; mean counts of Enterobacteriaceae did not exceed 4 log cfu/mL whereas those of E. coli and coagulase-positive staphylococci were lower than 1.5 and 2 log ufc /ml, respectively. Neither Salmonella spp. nor Listeria monocytogenes were recovered. The numbers of total LAB were in the range from 104 to 107 cfu/mL and mean yeasts counts varied between 103 and 105 cfu/mL. The most frequently isolated LAB species were Lactococcus lactis subsp. lactis and Lactobacillus paracasei. The presence of Enterococcus faecium was also noteworthy. The in vitro study of some functional characteristics related to technological properties of the strains belonging to these species allowed to point out some strains possessing good potential for use as adjunct or starter cultures in the production of cheese.

Volatile Molecule Profiles and Anti-Listeria monocytogenes Activity of Nisin Producers Lactococcus lactis Strains in Vegetable Drinks.

This work aimed to evaluate the potential of 15 nisin producing Lactococcus lactis strains, isolated from dairy products, for the fermentation of soymilk and carrot juice. In particular, the acidification and the production of nisin in the food matrices were recorded. Moreover, three strains (LBG2, FBG1P, and 3LC39), that showed the most promising results were further scrutinized for their anti-Listeria monocytogenes activity and volatile molecules profile during fermentation of soymilk and carrot juice. Lactococcus lactis strains LBG2, FBG1P, and 3LC39 resulted the most interesting ones, showing rapid growth and acidification on both food matrices. The higher amounts of nisin were detected in soymilk samples fermented by the strain LBG2 after 24 and 48 h (26.4 mg/L). Furthermore, the rapid acidification combined with the production of nisin resulted in a strong anti-Listeria activity, reducing the pathogen loads below the detection limit, in carrot juice samples fermented by the strains LBG2 and FBG1P and in soymilk by the strain LBG2. The fermentation increased the presence of volatile molecules such as aldehydes and ketones with a positive impact on the organoleptic profile of both the fermented products. These results highlighted the interesting potential of three nisin producing L. lactis strains for the production of fermented carrot juice and soymilk. In fact, the fermentation by lactic acid bacteria, combined or not with other mild technologies, represents a good strategy for the microbiological stabilization of these products. Furthermore, the increase of molecules with a positive sensory impact, such as aldehydes and ketones, in the fermented products suggests a possible improvement of their organoleptic characteristics.

Application of Autochthonous Lactobacillus Strains as Biopreservatives to Control Fungal Spoilage in Caciotta Cheese.

Fungal spoilage is one of the main causes of economic losses worldwide in the food industry. In the last years, consumer's demands for preservative-free processed foods have increased as a result of growing awareness about the health hazards associated with chemicals. Lactic acid bacteria have been extensively studied for their antibacterial and antifungal potential in order to be used as biopreservatives. The first objective of this study was to investigate in vitro the antifungal activity of autochthonous Lactobacillus strains against moulds commonly associated with cheese spoilage. Then, the Lactobacillus strains with the highest inhibitory effect and broadest spectrum were tested in single or mixed cultures against Penicillium chrysogenum ATCC 9179 and Aspergillus flavus ATCC 46283 on miniature Caciotta cheese produced at laboratory scale to evaluate in situ their ability to prevent mould growth and to determine their impact on cheese organoleptic properties and starter culture activity. The growth of the starter lactococcal population exhibited similar trend and values during ripening, suggesting that the addition of lactobacilli did not influence its growth and survival. Inhibition of P. chrysogenum inoculated in the milk was determined in cheeses produced with single or mixed Lactobacillus adjuncts as compared to cheeses without adjunct. The mixed adjunct cultures resulted in more effective, significantly reducing mould counts of more than 2 log units at the end of ripening. The application of the adjunct cultures resulted in a delay in mycelial growth of P. chrysogenum and A. flavus inoculated on the cheese surface as well. Finally, we found no significant differences among samples for the sensory parameters evaluated that received similar ratings. Our results indicate that the selected Lactobacillus strains may have a potential effect in controlling mould contamination on cheeses. Further studies are currently being carried out to identify the molecules responsible for the antifungal activity.

Biological Activities of Aerial Parts Extracts of Euphorbia characias.

The aim of the present study was to evaluate antioxidant, antimicrobial, anti-HIV, and cholinesterase inhibitory activities of aqueous and alcoholic extracts from leaves, stems, and flowers of Euphorbia characias. The extracts showed a high antioxidant activity and were a good source of total polyphenols and flavonoids. Ethanolic extracts from leaves and flowers displayed the highest inhibitory activity against acetylcholinesterase and butyrylcholinesterase, showing potential properties against Alzheimer's disease. Antimicrobial assay showed that leaves and flowers extracts were active against all Gram-positive bacteria tested. The ethanolic leaves extract appeared to have the strongest antibacterial activity against Bacillus cereus with MIC value of 312.5 µg/mL followed by Listeria monocytogenes and Staphylococcus aureus that also exhibited good sensitivity with MIC values of 1250 µg/mL. Moreover, all the extracts possessed anti-HIV activity. The ethanolic flower extract was the most potent inhibitor of HIV-1 RT DNA polymerase RNA-dependent and Ribonuclease H with IC50 values of 0.26 and 0.33 µg/mL, respectively. The LC-DAD metabolic profile showed that ethanolic leaves extract contains high levels of quercetin derivatives. This study suggests that Euphorbia characias extracts represent a good source of natural bioactive compounds which could be useful for pharmaceutical application as well as in food system for the prevention of the growth of food-borne bacteria and to extend the shelf-life of processed foods.

Data on the changes of the mussels׳ metabolic profile under different cold storage conditions.

One of the main problems of seafood marketing is the ease with which fish and shellfish undergo deterioration after death. (1)H NMR spectroscopy and microbiological analysis were applied to get in depth insight into the effects of cold storage (4 °C and 0 °C) on the spoilage of the mussel Mytilus galloprovincialis. This data article provides information on the average distribution of the microbial loads in mussels׳ specimens and on the acquisition, processing, and multivariate analysis of the (1)H NMR spectra from the hydrosoluble phase of stored mussels. This data article is referred to the research article entitled "Metabolomics analysis of shucked mussels' freshness" (Aru et al., 2016) [1].

Metabolomics analysis of shucked mussels' freshness.

In this work a NMR metabolomics approach was applied to analyze changes in the metabolic profile of the bivalve mollusk Mytilus galloprovincialis upon storage at 0°C and 4°C for 10 and 6 days, respectively. The most significant microbial groups involved in spoilage of mussels were also investigated. The time-related metabolic signature of mussels was analysed by Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) which revealed a clear discrimination between the fresh samples and those stored at 0°C and 4°C. The results evidenced a noticeable increase in acetate, lactate, succinate, alanine, branched chain amino acids, trimethylamine and a progressive decline of osmolytes like betaine, homarine and taurine during storage. Exploration of the correlations of these metabolites with microbial counts suggested their use as potential biomarkers of spoilage. The results support the use of NMR metabolomics as a valuable tool to provide information on seafood freshness.

Metabolomics and microbiological profile of Italian mozzarella cheese produced with buffalo and cow milk.

Italian buffalo mozzarella (BM) cheese metabolite profile and microbial communities were characterised and compared to cow mozzarella (CM). Polar metabolite profiles were studied by gas-chromatography mass-spectrometry (GC-MS) and results elaborated by multivariate analysis (MVA). BM produced using natural whey starter cultures (NWS) exhibited a higher microbial diversity with less psychrotrophic bacteria. BM samples were higher in threonine, serine, valine, and lower in orotic acid and urea. CM produced with commercial starters (CMS) had the highest count of Streptococcus thermophilus and higher levels of galactose and phenylalanine. CM obtained by direct acidification (CMA) had lower microbial counts and higher levels of urea and sugars. Orotic acid was the only metabolite linked to milk animal origin. Results indicated that this metabolite pool well reflects the different production protocols and microbial complexity of these dairy products. This approach can help to protect the designation of origin of Italian buffalo mozzarella.

Chitinase III in Euphorbia characias latex: Purification and characterization.

This paper deals with the purification of a class III endochitinase from Euphorbia characias latex. Described purification method includes an effective novel separation step using magnetic chitin particles. Application of magnetic affinity adsorbent noticeably simplifies and shortens the purification procedure. This step and the subsequently DEAE-cellulose chromatography enable to obtain the chitinase in homogeneous form. One protein band is present on PAGE in non-denaturing conditions and SDS-PAGE profile reveals a unique protein band of 36.5 ± 2 kDa. The optimal chitinase activity is observed at 50 °C, pH 5.0. E. characias latex chitinase is able to hydrolyze colloidal chitin giving, as reaction products, N-acetyl-D-glucosamine, chitobiose and chitotriose. Moreover, we observed that calcium and magnesium ions enhance chitinase activity. Finally, we cloned the cDNA encoding the E. characias latex chitinase. The partial cDNA nucleotide sequence contains 762 bp, and the deduced amino acid sequence (254 amino acids) is homologous to the sequence of several plant class III endochitinases.

Preliminary evaluation of probiotic properties of Lactobacillus strains isolated from Sardinian dairy products.

Twenty-three Lactobacillus strains of dairy origin were evaluated for some functional properties relevant to their use as probiotics. A preliminary subtractive screening based on the abilities to inhibit the growth of microbial pathogens and hydrolyze conjugated bile salts was applied, and six strains were selected for further characterization including survival under gastrointestinal environmental conditions, adhesion to gut epithelial tissue, enzymatic activity, and some safety properties. All selected strains maintained elevated cell numbers under conditions simulating passage through the human gastrointestinal tract, well comparable to the values obtained for the probiotic strain Lactobacillus rhamnosus GG, and were able to adhere to Caco-2 cells to various extents (from 3 to 20%). All strains exhibited high aminopeptidase, and absent or very low proteolytic and strong ß-galactosidase activities; none was found to be haemolytic or to produce biogenic amines and all were susceptible to tetracycline, chloramphenicol, erythromycin, ampicillin, and amoxicillin/clavulanic acid. Our results indicate that the Lactobacillus strains analyzed could be considered appropriate probiotic candidates, due to resistance to GIT simulated conditions, antimicrobial activity, adhesion to Caco-2 cell-line, and absence of undesirable properties. They could be used as adjunct cultures for contributing to the quality and health related functional properties of dairy products.

A NMR metabolomics study of the ripening process of the Fiore Sardo cheese produced with autochthonous adjunct cultures.

Fiore Sardo (FS) is a traditional Italian raw ewe's milk cheese carrying a Protected Designation of Origin (PDO). This study investigated the kinetics of FS cheese ripening by physicochemical parameters, microbial counting, and NMR metabolomics using aqueous extracts. Four Fiore Sardo cheeses, manufactured from milk with deliberately added autochthonous lactic acid bacteria (LAB) or commercial starters were studied during a period of 90days of ripening. Major differences in the metabolic profiles were observed amongst the samples as a function of the adjunct culture utilised. (1)H NMR metabolomics in combination with multivariate data analysis was able to classify cheese samples on the basis of their maturation age and the type of added cultures. These findings lay the metabolic basis for the authentication of Fiore Sardo cheese produced in compliance with PDO specifications which allow the use of only native LAB cultures.

Antilisterial activity of nisin-like bacteriocin-producing Lactococcus lactis subsp. lactis isolated from traditional Sardinian dairy products.

With the aim of selecting LAB strains with antilisterial activity to be used as protective cultures to enhance the safety of dairy products, the antimicrobial properties of 117 Lactococcus lactis subsp. lactis isolated from artisanal Sardinian dairy products were evaluated, and six strains were found to produce bacteriocin-like substances. The capacity of these strains to antagonize Listeria monocytogenes during cocultivation in skimmed milk was evaluated, showing a reduction of L. monocytogenes counts of approximately 4 log units compared to the positive control after 24 h of incubation. In order for a strain to be used as bioprotective culture, it should be carefully evaluated for the presence of virulence factors, to determine what potential risks might be involved in its use. None of the strains tested was found to produce biogenic amines or to possess haemolytic activity. In addition, all strains were sensitive to clinically important antibiotics such as ampicillin, tetracycline, and vancomycin. Our results suggest that these bac+ strains could be potentially applied in cheese manufacturing to control the growth of L. monocytogenes.