Evaluation of autochthonous Lactococcus lactis subsp. lactis strain as a candidate starter culture in white-brined cheese.

Lactococcus lactis subsp. lactis NCCB100539 isolated from an artisanal raw ewe's milk cheese was evaluated as a potential starter culture in white-brined cheese. As a safety criteria, the cytotoxicity of the viable and heat-killed cells and CFE of this strain were determined on Caco-2 cell line by MTT assay. The antibiotic sensitivity of the strain to nine different antibiotics was also investigated. Cheeses produced using this strain were compared with control cheese in terms of physicochemical, microbiological, sensory properties as well as the peptide and volatile profiles during the 90-days of ripening period. Experimental cheeses had more extensive proteolysis as well as higher sensory scores. Incorporated L. lactis also led to an improvement in the microbial cheese quality. Neither living nor the dead cells and CFE of the strain showed cytotoxicity on Caco-2 cells. Therefore, L. lactis NCCB100539 strain could be a good starter candidate for the industrial white-brined cheeses. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-023-01332-y.

Metabolic profiles of Weissella spp. postbiotics with anti-microbial and anti-oxidant effects.

INTRODUCTION: This present study aimed to isolate beneficial bacteria from honey bee pollen microbiota and to investigate the metabolite profiles of postbiotics exhibiting anti-microbial and anti-oxidant properties. METHODOLOGY: Pour plate technique was used to isolate bacteria from honey bee (Apis mellifera L.) pollen samples. Different colonies grown on agar plates were selected and screened for their anti-microbial activity against important pathogens using agar well diffusion assay. The isolates that exhibited remarkable inhibitory effects against all tested pathogens were identified by 16S rRNA sequence analysis. DPPH (2, 2-diphenyl-1-picrylhydrazil) free radical scavenging assays were used to assess the antioxidant capacity of their postbiotics. Besides, the total phenolic and total flavonoid compounds in postbiotics were determined as gallic acid and quercetin equivalents, respectively. The valuable metabolites in postbiotics were also profiled using chromatographic tools and Mass Spectrophotometry (MS) analysis. RESULTS: Twenty-seven strains were isolated from different honey bee pollen samples. 16 out of the 27 strains exhibited antagonistic activity against at least one reference strain of pathogens, tested. The most effective strains belonging to the genus Weissella were identified as W. cibaria and W. confusa. Postbiotics above 10 mg/mL exhibited higher radical scavenging activity and high total phenolic and total flavonoid contents. MS analysis demonstrated that metabolites in postbiotics derived from Weissella spp. were found very similar to the metabolites found in honeybee pollen. CONCLUSIONS: The outcomes of this study revealed that honey bee pollen could be considered a potential source for the bacteria which produce anti-microbial and anti-oxidant agents. The similarity with the nutritional dynamics of honey bee pollen also indicated that postbiotics could also be used as novel and sustainable food supplements.

Biocontrol potential of Apilactobacillus kunkeei EIR/BG-1 against infectious diseases in honey bees (Apis mellifera L.).

The significant reduction of honey bee colonies due to the various infectious agents highlights the need for the development of new alternatives and integrated management strategies to keep a hive strong and healthy. The main purpose of this study was to develop an environmentally and friendly microbial feed supplements to prevent honey bee mortality and keep the bee colony population healthy and productive. For this aim, Apilactobacillus kunkeei EIR/BG-1 isolated from honey bee gut microbiota was evaluated for its preventive role against American Foulbrood disease and nosemosis. To test the ability of the strain EIR/BG-1 for suppressing Paenibacillus larvae growth under in vitro conditions, the agar well diffusion method was used and viable cells of the strain EIR/BG-1 inhibited the growth of P. larvae with an efficient inhibition zone (24 ± 0.8 mm) similar to tetracycline antibiotic (30 µg). To determine the preventive role of the strain EIR/BG-1 on infection progression, its viable cells were applied against nosemosis in a laboratory experimental setting. Our results showed that prophylactic supplementation of Al. kunkeei EIR/BG-1 (106 cfu/bee) significantly reduced the spore load (66 ± 6.1%). Besides, gene expression of antimicrobial peptides in gut tissue has been up-regulated and infected midgut epithelium integrity and peritrophic membrane production were improved. In conclusion, our findings suggest that prophylactic supplementation of Al. kunkeei EIR/BG-1 as a natural strategy may enhance the honey bee's response when challenged by pathogens. Field applications towards gaining a better understanding of its biocontrol role will be the main goal of our future researches.

Analysis of Chemical Structure and Antibiofilm Properties of Exopolysaccharides from Lactiplantibacillus plantarum EIR/IF-1 Postbiotics.

Previous studies have indicated that the exopolysaccharides of lactic acid bacteria exhibit antibiofilm activity against non-oral bacteria by preventing their initial adhesion to surfaces and by downregulating the expression of genes responsible for their biofilm formation. The aims of this study were to (1) characterize the exopolysaccharides (EPSs) of Lactobacillus plantarum EIR/IF-1 postbiotics, (2) test their antibiofilm effect on dual biofilms, and (3) evaluate their bacterial auto-aggregation, co-aggregation, and hydrocarbon-binding inhibitory activity. The EPSs were characterized by FTIR, HPLC, and thermogravimetric analysis. Bacterial auto- and co-aggregation were tested by Kolenbrander's method and hydrocarbon binding was tested by Rosenberg's method. Dual biofilms were formed by culturing Fusobacterium nucleatum ATCC 25586 with one of the following bacteria: Prevotella denticola ATCC 33185, P. denticola AHN 33266, Porphyromonas gingivalis ATCC 33277, P. gingivalis AHN 24155, and Filifactor alocis ATCC 35896. The EPSs contained fractions with different molecular weights (51 and 841 kDa) and monosaccharides of glucose, galactose, and fructose. The EPSs showed antibiofilm activity in all the biofilm models tested. The EPSs may have inhibited bacterial aggregation and binding to hydrocarbons by reducing bacterial hydrophobicity. In conclusion, the EPSs of L. plantarum EIR/IF-1, which consists of two major fractions, exhibited antibiofilm activity against oral bacteria, which can be explained by the inhibitory effect of EPSs on the auto-aggregation and co-aggregation of bacteria and their binding to hydrocarbons.

Xenogenic Neural Stem Cell-Derived Extracellular Nanovesicles Modulate Human Mesenchymal Stem Cell Fate and Reconstruct Metabolomic Structure.

Extracellular nanovesicles, particularly exosomes, can deliver their diverse bioactive biomolecular content, including miRNAs, proteins, and lipids, thus providing a context for investigating the capability of exosomes to induce stem cells toward lineage-specific cells and tissue regeneration. In this study, it is demonstrated that rat subventricular zone neural stem cell-derived exosomes (rSVZ-NSCExo) can control neural-lineage specification of human mesenchymal stem cells (hMSCs). Microarray analysis shows that the miRNA content of rSVZ-NSCExo is a faithful representation of rSVZ tissue. Through immunocytochemistry, gene expression, and multi-omics analyses, the capability to use rSVZ-NSCExo to induce hMSCs into a neuroglial or neural stem cell phenotype and genotype in a temporal and dose-dependent manner via multiple signaling pathways is demonstrated. The current study presents a new and innovative strategy to modulate hMSCs fate by harnessing the molecular content of exosomes, thus suggesting future opportunities for rSVZ-NSCExo in nerve tissue regeneration.

The role of microbiota-derived postbiotic mediators on biofilm formation and quorum sensing-mediated virulence of Streptococcus mutans: A perspective on preventing dental caries.

Dental caries is not only one of the most prevalent diseases worldwide, but also a public health problem, undoubtedly. Among the various species of cariogenic bacteria, Streptococcus mutans is considered to be the major etiological pathogen of dental caries. The present study aimed to assess the influence of microbiota-derived postbiotic mediators (PMs) on the pathogenesis of dental caries. Within this aim, the antibacterial (agar diffusion method) and antibiofilm (crystal violet assay) characteristics of PMs derived from Lactiplantibacillus plantarum EIR/IF-1, Lactiplantibacillus curvatus EIR/DG-1, and Lactiplantibacillus curvatus EIR/BG-2 were analyzed against S. mutans (ATCC 25175). According to the results, PMs of the strain EIR/IF-1, isolated from infant feces showed the highest inhibitory effect (pH-dependent). Besides, sub-MIC doses of all PMs eliminated the biofilm formation following the co-treatment and pre-treatment assays. The reduction of cell viability and notable changes in biofilm formation were also confirmed both on glass coverslips and ex vivo human tooth surfaces by confocal laser scanning microscopy and scanning electron microscopy. Moreover, sub-MIC values of PMs down-regulated the expression of gtfC, comA, and comX, without any significant growth inhibition. Organic acids, fatty acids, and vitamins in PMs were also reported. Overall, these findings indicated the possible preventive role of microbiota-derived PMs in the pathogenesis of dental caries.

Nano-hydroxyapatite incorporated quince seed mucilage bioscaffolds for osteogenic differentiation of human adipose-derived mesenchymal stem cells.

In this study, the therapeutic hydrocolloid quince seed mucilage (QSM) from Cydonia oblonga Miller fruit is enriched with needle-like nano-hydroxyapatite (nHAp) crystals to fabricate a novel biomimetic osteogenic bioscaffold. The molecular weight (Mw) of water-based extracted QSM was measured with GPC (8.67 × 105 g/mol), and the composite blend was prepared at a ratio of 1:1 (w/w) QSMaq and nHAp. The porous bioscaffolds were manufactured by the freeze-drying method, and evaluated in-depth with advanced analyses. The XRD, ATR-FTIR, SEM-EDX, and elemental mapping analyses revealed a uniform coated semi-crystalline structure with no covalent bindings between QSM and nHAp. Moreover, due to the hydrocolloid backbone, a supreme swelling ratio (w/w, 6523 ± 190%) with suitable pore size (208.12 ± 99.22 µm) for osteogenic development was obtained. Further, the cytocompatible bioscaffolds were evaluated for osteogenic differentiation in vitro using human adipose-derived mesenchymal stem cells (hAMSCs). The immuno/histochemical (I/HC) staining revealed that the cells with the spherical morphology invaded the pores of the prepared bioscaffolds. Also, relatively early up-regulated osteogenic markers were observed by the qRT-PCR analyses. Overall, it is believed that the QSM-nHAp bioscaffolds might be favorable in non-load bearing applications, especially in the cranio-maxillofacial region, due to their regenerative, bendable, and durable features.

From a plant secretion to the promising bone grafts: Cryogels of silicon-integrated quince seed mucilage by microwave-assisted sol-gel reaction.

Design and fabrication of biologically active cryogels using novel biopolymer(s) are still of great importance at regenerating bone defects such as traumatic bone injuries, maxillofacial surgery, osteomyelitis, and osteoporosis. Nowadays, plant mucilage, an herbal biomaterial, has been drawn attention by scientists due to their marvelous potential to fabricate 3-dimensional (3D) physical constructs for the field of regenerative medicine. Herein, a 3D cryogel from silicon-integrated quince seed mucilage (QSM) is constructed using microwave-assisted sol-gel reaction, characterized in-depth by attenuated total reflectance Fourier transform-infrared spectroscopy (ATR-FTIR), solid-state silicon cross-polarization magic-angle nuclear magnetic resonance (29Si-CP-MAS NMR), X-ray diffraction (XRD), thermogravimetric analysis (TGA), differential scanning calorimeter (DSC), micro-mechanical testing, porosity, and swelling tests, contact angle measurements, Brunauer-Emmet-Teller and Barret-Joyner-Halenda (BET-BJH) analysis, enzymatic biodegradation test, and field emission-scanning electron microscopy-energy dispersive X-ray spectroscopy (FE-SEM-EDX) mapping. The osteobiologic capacity of the cryogels is determined using human adipose-derived mesenchymal stem cells (hAMSCs) under in vitro conditions. Osteogenic differentiation of hAMSCs on both QSM and silica-modified QSM (Si-QSM) cryogels is analyzed by histochemistry, immunohistochemistry, and quantitative-real time (q-RT) PCR techniques. The results obtained from in vitro experiments demonstrate that the upregulation of osteogenesis-related genes in Si-QSM cryogels presents a stronger and earlier development over QSM cryogels throughout the culture period, which in turn reveals the great potential of this novel Si-incorporated QSM cryogels for bone tissue engineering applications.

Synthesis of Silica-Based Boron-Incorporated Collagen/Human Hair Keratin Hybrid Cryogels with the Potential Bone Formation Capability.

Tissue engineering and regenerative medicine have evolved into a different concept, the so-called clinical tissue engineering. Within this context, the synthesis of next-generation inorganic-organic hybrid constructs without the use of chemical crosslinkers emerges with a great potential for treating bone defects. Here, we propose a sophisticated approach for synthesizing cost-effective boron (B)- and silicon (Si)-incorporated collagen/hair keratin (B-Si-Col-HK) cryogels with the help of sol-gel reactions. In this approach, collagen and hair keratin were engaged with a B-Si network using tetraethyl orthosilicate as a silica precursor, and the obtained cryogels were characterized in depth with attenuated total reflectance-Fourier transform infrared spectroscopy, solid-state NMR, X-ray diffraction, thermogravimetric analysis, porosity and swelling tests, Brunauer-Emmett-Teller and Barrett-Joyner-Halenda analyses, frequency sweep and temperature-dependent rheology, contact angle analysis, micromechanical tests, and scanning electron microscopy with energy dispersive X-ray analysis. In addition, the cell survival and osteogenic features of the cryogels were evaluated by the MTS test, live/dead assay, immuno/histochemistry, and quantitative real-time polymerase chain reaction analyses. We conclude that the B-Si-networked Col-HK cryogels having good mechanical durability and osteoinductive features would have the potential bone formation capability.

Weissella cibaria EIR/P2-derived exopolysaccharide: A novel alternative to conventional biomaterials targeting periodontal regeneration.

Healing and regeneration of periodontium are considered as a complex physiological process. Therefore, treatments need to be addressed with highly effective components modulating the multiple pathways. In this study, exopolysaccharide (EPS) produced by Weissella cibaria EIR/P2, was partially purified from the culture supernatant and subjected to characterization within the aim of evaluating its potential for periodontal regeneration. High-Performance Liquid Chromatography analysis revealed a single-peak corresponding to the glucose which identified the EPS as dextran. Fourier transform-infrared spectra were also displayed characteristic peaks for polysaccharides. According to the results of gel permeation/size exclusion-chromatography, the molecular mass was determined to be 8 × 106 Da. To clarify its anti-bacterial activity on Streptococcus mutans, effects on viability and biofilm formation was evaluated. At 50 mg/mL, dextran exhibited a bactericidal effect with 70% inhibition on biofilm formation. Besides, dose-dependent antioxidant effects were also detected. The efficacy of dextran in enhancing the viability of human periodontal ligament fibroblast cells (hPDLFCs) was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide (MTT) assay, and an increase was observed in the viability of hPDLFCs. In conclusion, dextran derived from W. cibaria can be potentially used as a multi-functional bioactive polymer in the design of new therapeutic strategies to promote healing and regeneration of periodontium.

Potential Properties of Lactobacillus plantarum F-10 as a Bio-control Strategy for Wound Infections.

In this study, Lactobacillus plantarum F-10, a promising probiotic strain isolated from fecal microbiota of healthy breastfed infant, was assessed as a bio-control strategy for wound infections. Pseudomonas aeruginosa PAO1/ATCC 27853, methicillin-resistant Staphylococcus aureus ATCC 43300, and their hospital-derived strains isolated from skin chronic wound samples were used as important skin pathogens. The cell-free extract (CFE) of the strain F-10 was shown to inhibit the growth of all pathogens tested, while no inhibition was observed when CFE was neutralized. The strain displayed no hemolysis and exhibited a strong auto-aggregating phenotype (51.48 ± 1.45%, 5 h) as well as co-aggregation. Antibiotic resistance profile was found to be safe according to the European Food Safety Authority. Biofilm formation was measured by crystal violet assay and visualized with scanning electron microscopy and confocal laser scanning microscopy. One hundred percent reduction in biofilm formation of all pathogens tested was obtained by sub-MIC value (12.5 mg/ml) of CFE following 24-h co-incubation. Inhibition of quorum sensing-controlled virulence factors (motility, protease and elastase activity, production of pyocyanin and rhamnolipid) of P. aeruginosa strains was also observed. DPPH radical scavenging activity of the CFE was determined as 88.57 ± 0.49%. In conclusion, our results suggest that L. plantarum F-10 may represent an alternative bio-control strategy against skin infections with its antimicrobial, anti-biofilm, anti-quorum sensing, and antioxidant activity.

Effect of Encapsulation on Viability of Pediococcus pentosaceus OZF During Its Passage Through the Gastrointestinal Tract Model.

The main goal of this study was to develop an improved oral delivery system for Pediococcus pentosaceus OZF, a promising probiotic bacterium, and to assess its viability under simulated gastrointestinal (GI) tract model by comparing the efficiency of microbiological and molecular approaches. Encapsulation was carried out using extrusion method and as a result, encapsulation system including 0.75 % lactulose, 1.8 % sodium alginate, 0.1 M CaCl2, and 5 min gelling time was shown to have a significantly protective effect against pH 2.0 acid stress over 3 h. However, completely loss of viability was exhibited by free OZF cells under similar conditions. To provide an additional barrier for capsules, coating process was investigated using different biopolymers, and the survival rates of free and encapsulated OZF cells upon expose to simulated GI conditions were detected by conventional culture techniques and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR) method. No significant differences between the biopolymers were detected, except the chitosan which leads totally 85 % protection and extra 25 % improvement in the survival of OZF cells compared to uncoated capsules. In conclusion, our findings indicated that chitosan-coated capsules provided an important protective effect on the viability of OZF cells against the GI system conditions encountered during the transit of food. In addition, this study was found successful in monitoring the viable OZF cells in capsules using PMA-qPCR method.

Effects of ELF magnetic field in combination with Iron(III) chloride (FeCl3) on cellular growth and surface morphology of Escherichia coli (E. coli).

This study investigated the effects of extremely low frequency (ELF) magnetic field with/without iron(III) chloride (FeCl3) on bacterial growth and morphology. The ELF exposures were carried out using a pair of Helmholtz coil-based ELF exposure system which was designed to generate 50 Hz sinusoidal magnetic field. The field was approximately uniform throughout the axis of the coil pair. The samples which were treated or non-treated with different concentrations FeCl3 were exposed to 50 Hz, 2 millitesla (mT) magnetic field for 24 h. ELF effect on viability was assessed in terms of viable colony counts (in colony-forming unit per milliliter) with the standard plate count technique. Scanning electron microscopy was used to investigate the magnetic field effect on surface morphology of Escherichia coli. No significant results were seen in terms of cell viability between ELF and sham-exposed bacterial strains. Similarly, FeCl3 treatment did not change cell viability of E. coli samples. However, we observed some morphological changes on E. coli cell surfaces. Pore formations and membrane destruction were seen on the surface of 24 h ELF field-exposed cells. We concluded that ELF magnetic field exposure at 2 mT does not affect cell viability; however, it may affect bacterial surface morphology.

Evidence for a chromosomally determined mesenterocin, a bacteriocin produced by Leuconostoc mesenteroides subsp. mesenteroides OZ.

Mesenterocin, a small anti-listerial peptide of 3.5 kDa produced by Leuconostoc mesenteroides subsp. mesenteroides OZ at the end of exponential growth was inactivated by proteolytic enymes, stable to cold storage (4 °C for 3 d), heat, organic solvents and surfactants, and exhibited maximum bactericidal mode of activity in the pH range 3 to 10. Although Leu. mesenteroides subsp. mesenteroides OZ harboured plasmids ranging in size from 3.6 to 9.7 kbp, no evidence was obtained indicating that mesenterocin was under the control of extrachromosomal plasmids because loss of bacteriocin production following plasmid curing experiments could not be correlated with plasmid loss and the lack of detectable plasmids suggested a chromosomal location for the genetic determinants of mesenterocin. To determine its chromosomal location, genetic determinants of the bacteriocin of Leu. mesenteroides subsp. mesenteroides OZ were tested with previously described bacteriocins of the Leuconostocs through PCR and results of PCR indicated that the gene for mesenterocin activity is located on the chromosome. No papers have been published, to the best of our knowledge, on chromosomal location of bacteriocin of Leuconostoc species. Association of meat spoilage with bacteriocin producing heterofermentative Leu. mesenteroides subsp. mesenteroides OZ is also suggested.

Evaluation of in vitro Probiotic Potential of Pediococcus pentosaceus OZF Isolated from Human Breast Milk.

This study was conducted to evaluate the probiotic properties of Pediococcus pentosaceus OZF isolated from human breast milk. The results obtained so far suggest that the strain is resistant to low pH, bile salt, pepsin and pancreatin, so it could survive while passing through the upper part of the gastrointestinal tract and reveal its potential probiotic action on host organism. The strain was non-pathogenic (γ-hemolytic), produced anti-Listerial bacteriocin, exhibited a strong autoaggregating phenotype (85.71%) and demonstrated 6.26 and 12.99% coaggregation with Salmonella enterica serotype Typhimurium SL 1344 and Escherichia coli LMG 3083 (ETEC), respectively. The degree of adhesion of Ped. pentosaceus OZF to the human Caco-2 cell line was investigated and when compared to the adhesion of pathogenic strains tested, it was shown to inhibit the growth of human enterotoxigenic E. coli LMG 3083 (ETEC) and of Salm. Typhimurium SL 1344. Ped. pentosaceus OZF seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and lipid factors on the bacteria and eukaryotic cell surface. The percentage of adhesion to n-hexadecane was 34% showing that the surface was rather hydrophilic. Higher affinity displayed by Ped. pentosaceus OZF for chloroform demonstrates the basic property of a cell, which may be due to the presence of carboxylic groups on the cell surface.