Acute Toxicity Evaluation of Phosphatidylcholine Nanoliposomes Containing Nisin in Caenorhabditis elegans.

Liposomes are among the most studied nanostructures. They are effective carriers of active substances both in the clinical field, such as delivering genes and drugs, and in the food industry, such as promoting the controlled release of bioactive substances, including food preservatives. However, toxicological screenings must be performed to ensure the safety of nanoformulations. In this study, the nematode Caenorhabditis elegans was used as an alternative model to investigate the potential in vivo toxicity of nanoliposomes encapsulating the antimicrobial peptide nisin. The effects of liposomes containing nisin, control liposomes, and free nisin were evaluated through the survival rate, lethal dose (LD50), nematode development rate, and oxidative stress status by performing mutant strain, TBARS, and ROS analyses. Due to its low toxicity, it was not possible to experimentally determine the LD50 of liposomes. The survival rates of control liposomes and nisin-loaded liposomes were 94.3 and 73.6%, respectively. The LD50 of free nisin was calculated as 0.239 mg mL-1. Free nisin at a concentration of 0.2 mg mL-1 significantly affected the development of C. elegans, which was 25% smaller than the control and liposome-treated samples. A significant increase in ROS levels was observed after exposure to the highest concentrations of liposomes and free nisin, coinciding with a significant increase in catalase levels. The treatments induced lipid peroxidation as evaluated by TBARS assay. Liposome encapsulation reduces the deleterious effect on C. elegans and can be considered a nontoxic delivery system for nisin.

Structural features of myofibrillar fish protein interacting with phosphatidylcholine liposomes.

The interactions between liposomes and fish myofibrillar protein (surimi ground salted protein, SURP) were evaluated. Liposomes prepared with ultrapure phosphatidylcholine (UPC) or partially purified phosphatidylcholine (PPC) were dispersed at different weight ratio on SURP. Changes in protein stability and structure were evaluated using FTIR, intrinsic fluorescence and free sulfhydryl groups, and changes in liposome properties were studied by dynamic light scattering and electron microscopy. PPC promoted denaturation and aggregation of SURP, reflected in secondary structure loss, exposure of tyrosine residues and increment of free sulfhydryl. UPC produced partial unfolding and changes in the secondary structure of SURP from α-helical to ß-strand. Liposome size increased by about 40% and showed modified surface charge after SURP exposure, indicating the formation of protein corona. Surface charge and composition of liposomes influence SURP stability and could exert different effects on the myofibrillar protein network, which is important for liposome applications in surimi products.

Antifungal properties of phosphatidylcholine-oleic acid liposomes encapsulating garlic against environmental fungal in wheat bread.

Liposomes have gained great interest in the food and pharmaceutical industry as colloidal carriers of bioactive compounds. In this work, liposomes of phosphatidylcholine (PC) and oleic acid (OA) encapsulating garlic extract (GE) were developed to determine its aptitude as antifungal agent in wheat bread. The influence of GE on the properties of liposomes were followed by determination of size, Zeta potential, Fourier transform infrared patterns (FTIR), morphology, differential scanning calorimetry (DSC) and thermogravimetric (TGA) techniques. The produced PC-OA-GE liposomes showed spherical morphology with narrow size distribution, entrapment efficiency of 79.7% and zeta potential of -27.9 mV. In vitro antifungal test showed noticeable inhibitory activities for free and encapsulated GE against selected fungal strains. TGA analysis revealed that the presence of OA and GE in the formulation retards the liposomal thermal decomposition, as compared with the pure PC liposomes and the DSC enthalpy and main transition temperature variation in PC-OA-GE liposomes suggested a strong heat-induced rigidifying effect that could be attributed to the presence of garlic polysaccharides in the liposome surface, observed by FTIR. In the in situ test, the bread formulations with free or liposome-encapsulated GE (0.65 mL/100 g of dough) were microbiologically more stable as compared with the controls, showing mold inhibition for five days. Therefore, liposomes formulated with OA and GE showed potential as natural antifungal agent in bakery products.

Antimicrobial activity of some natural extracts encapsulated within silica matrices.

Natural extracts (anthocyanins, tannin, anatto, curcuma and olive leaf extracts) were encapsulated within a silica network by acid or base-catalyzed sol-gel methods. The nominal encapsulated contents were between 2.5 and 50wt.-%. The resulting materials were characterized by Fourier transform infrared spectroscopy, diffuse reflectance in the UV region, nitrogen adsorption/desorption porosimetry and small angle X-ray scattering. Encapsulated anthocyanin and tannin afforded inhibition zones between 9-21mm towards Staphylococcus aureus, Escherichia coli, Bacillus cereus, Candida sp. and Aspergillus niger, which was comparable to the free bioactive material in which the inhibition zones were between 10 and 22mm. Anthocyanin exhibited high antioxidant activity in the free state, while tannin showed good antioxidant activity both in its free state and in its encapsulated form. Antimicrobial and antioxidant activities were shown to be dependent on the textural characteristics of the encapsulated materials.

Bioaccumulation and distribution of selenium in Enterococcus durans.

Selenium is an essential nutrient for all living organisms. Under appropriate conditions lactic acid bacteria (LAB) are capable for accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. In this study, the capacity of selenium bioaccumulation by Enterococcus durans LAB18s was evaluated. The distribution of organic selenium in selenium-enriched E. durans LAB18s biomass was analyzed, and the highest percentage of organic selenium was found in the fraction of total protein, followed by the fractions of polysaccharides and nucleic acids. When the protein fraction was obtained by different extractions (water, NaCl, ethanol and NaOH) it was demonstrated that alkali-soluble protein showed the higher Selenium content. Analysis of protein fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that selenium was present in the proteins ranging from 23 to 100kDa. The cells were analyzed by scanning electron microscopy (SEM); scanning electron microscopy/energy dispersive spectrometry (SEM/EDS) and transmission electron microscopy (TEM). SEM, TEM and SEM/EDS showed the morphology, the selenium particles bioaccumulated into and on the cells and the amounts of selenium present into the cells, respectively. Thus, the isolate E. durans LAB18s can be a promising probiotic to be used as selenium-enriched biomass in feed trials.

Effects of Achyrocline satureioides Inflorescence Extracts against Pathogenic Intestinal Bacteria: Chemical Characterization, In Vitro Tests, and In Vivo Evaluation.

Three Achyrocline satureioides (AS) inflorescences extracts were characterized: (i) a freeze-dried extract prepared from the aqueous extractive solution and (ii) a freeze-dried and (iii) a spray-dried extract prepared from hydroethanol extractive solution (80% ethanol). The chemical profile, antioxidant potential, and antimicrobial activity against intestinal pathogenic bacteria of AS extracts were evaluated. In vitro antioxidant activity was determined by the total reactive antioxidant potential (TRAP) assay. In vivo analysis and characterization of intestinal microbiota were performed in male Wistar rats (saline versus treated animals with AS dried extracts) by high-throughput sequencing analysis: metabarcoding. Antimicrobial activity was tested in vitro by the disc diffusion tests. Moisture content of the extracts ranged from 10 to 15% and 5.7 to 17 mg kg-1 of fluorine. AS exhibited antioxidant activity, especially in its freeze-dried form which also exhibited a wide spectrum of antimicrobial activity against intestinal pathogenic bacteria greater than those observed by the antibiotic, amoxicillin, when tested against Bacillus cereus and Staphylococcus aureus. Antioxidant and antimicrobial activities of AS extracts seemed to be positively correlated with the present amount of flavonoids. These findings suggest a potential use of AS as a coadjuvant agent for treating bacterial-induced intestinal diseases with high rates of antibiotic resistance.

Development and characterization of phosphatidylcholine nanovesicles, containing garlic extract, with antilisterial activity in milk.

Phospholipid nanovesicles were developed to improve the stability of garlic (Allium sativum L.) extract. Electron microscopy of liposomes revealed nanometric and spherical-shaped vesicles with a mean particle size of 174.6±17.3nm and polydispersity index of 0.26±0.02. The entrapment efficiency was 47.5±7.3% and the nanoliposomes had a zeta potential of -16.2±5.5mV. The antimicrobial activity of free and encapsulated garlic extract was evaluated against different strains of Listeria spp. in milk at 37°C for 24h. For free and encapsulated garlic extracts at 5% concentration, a decrease of 4log cycles in viable cell counts was observed at 10h, against four of the five strains of Listeria spp. tested. The results indicate that liposomes constitute a suitable system for encapsulation of unstable garlic active compounds and the encapsulation of garlic extract proves to be a promising technology for multiple applications, including antimicrobial agents.

Sulphate production by Paracoccus pantotrophus ATCC 35512 from different sulphur substrates: sodium thiosulphate, sulphite and sulphide.

One of the problems in waste water treatment plants (WWTPs) is the increase in emissions of hydrogen sulphide (H2S), which can cause damage to the health of human populations and ecosystems. To control emissions of this gas, sulphur-oxidizing bacteria can be used to convert H2S to sulphate. In this work, sulphate detection was performed by spectrophotometry, ion chromatography and atomic absorption spectrometry, using Paracoccus pantotrophus ATCC 35512 as a reference strain growing in an inorganic broth supplemented with sodium thiosulphate (Na2S2O3·5H2O), sodium sulphide (Na2S) or sodium sulphite (Na2SO3), separately. The strain was metabolically competent in sulphate production. However, it was only possible to observe significant differences in sulphate production compared to abiotic control when the inorganic medium was supplemented with sodium thiosulphate. The three methods for sulphate detection showed similar patterns, although the chromatographic method was the most sensitive for this study. This strain can be used as a reference for sulphate production in studies with sulphur-oxidizing bacteria originating from environmental samples of WWTPs.

Production of selenium-enriched biomass by Enterococcus durans.

Selenium (Se) is an essential micronutrient for several organisms, and there is an increased interest about adequate sources for dietary selenium supplementation. The aim of this study was to evaluate the selenium bioaccumulation capacity of an Enterococcus strain. The isolate LAB18s was identified as Enterococcus durans by the VITEK® 2 system and analysis of both 16S rDNA gene sequence (JX503528) and the 16S-23S rDNA intergenic spacer (ITS). After 24-h incubation, E. durans LAB18s bioaccumulated elevated Se(IV) concentrations, reaching 2.60 and 176.97 mg/g in media containing initial amounts of 15 and 240 mg/l sodium selenite, respectively. The isolate grew optimally and had high selenium bioaccumulation at initial pH of 7.0 and 30 °C. Time course studies showed that E. durans LAB18s displayed the highest bioaccumulation of Se(IV) after 6 h of incubation. Analyses from scanning electron microscopy (SEM) demonstrated the presence of filaments connecting the cells of E. durans LAB18s cultivated in the presence of sodium selenite. It was demonstrated that a considerable amount of Se(IV) was absorbed by E. durans LAB18s. Therefore, this strain may represent an alternative source of organic dietary selenium.

Effect of cis-9,trans-11 and trans-10,cis-12 isomers of conjugated linoleic acid on the integrity and functionality of cryopreserved bovine spermatozoa.

Plasma membranes of sperm subjected to low temperatures undergo changes in their structure and permeability. The addition of fatty acids in semen cryopreservation media may influence the sperm motility after thawing, possibly by maintaining the membrane fluidity due to their incorporation in lipid bilayers. In this work, different concentrations of the isomers cis-9,trans-11 and trans-10,cis-12 of conjugated linoleic acid (CLA) were added in the cryopreservation medium of bovine sperm. Four Jersey bulls were used, and the ejaculates were processed as a pool. The Tris-based extender (Dilutris®) was supplemented with 20% egg yolk (MB). The treatments with CLA (Luta-CLA®), which had oily presentation, were prepared from MB with addition of 1% sodium lauryl sulfate, and denominated MBL. The concentrations of CLA tested were 50, 100, and 150 µM. The motility characteristics of the post-thaw semen were analyzed by computerized analysis system (CASA), and plasma membrane integrity and acrosomal and mitochondrial function assessed by the association of the fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA), JC-1 and Hoechst 33342. No significant differences were observed among treatments, excepting for a decreased mitochondrial potential of cells treated with 150 µM CLA. The addition of CLA, at the concentrations used, showed no advantages on the integrity and functionality of bovine sperm submitted to cryopreservation.

Bacteriocin-like substance inhibits potato soft rot caused by Erwinia carotovora.

Soft rot is a major problem encountered in potatoes during postharvest storage. The soft rot bacterium Erwinia carotovora was inhibited by a novel bacteriocin-like substance (BLS) produced by Bacillus licheniformis P40. The BLS caused a bactericidal effect on E. carotovora cells at 30 microg mL(-1). Transmission electron microscopy showed that BLS-treated cells presented wrinkled bacterial surfaces and shrinkage of the whole cell, indicating plasmolysis. Erwinia carotovora cells treated with BLS were analyzed by FTIR showing differences in the 1390 cm(-1) and 1250-1220 cm(-1) bands, corresponding to assignments of membrane lipids. BLS was effective in preventing E. carotovora spoilage on potato tubers, reducing the symptoms of soft rot at 240 microg mL(-1) and higher concentrations. Soft rot development was completely blocked at 3.7 mg mL(-1). This BLS showed potential to protect potato tubers during storage.

Use of 1,4-naphthoquinones for control of Erwinia carotovora.

The antimicrobial effect of 5 naphthoquinones was tested against the phytopathogenic bacteria Erwinia carotovora. Disk diffusion tests and determination of minimal inhibitory concentrations (MIC) indicate that the compound naphthazarin (NTZ) has the best antibacterial activity among the naphthoquinones tested. Studies on the mode of action indicate the effect of NTZ was bactericidal at 10 microg/mL. When cultivation was done in the presence of sodium ascorbate, the restoration of E. carotovora growth was observed with 3 microg/mL NTZ, but not when a 10 microg/mL dose was used. The incubation of NTZ with bacterial suspension of E. carotovora resulted in important changes in the absorption spectra of this naphthoquinone, indicating that a redox reaction takes place. These results may suggest that NTZ induces an increase of reactive oxygen species that are toxic to the cell. The compound NTZ was also effective in preventing E. carotovora growth on potato tubers, inhibiting the soft rot development at a concentration of 2 mg/mL.