Identification of bacteriocins secreted by the probiotic Lactococcus lactis following microwave-assisted acid hydrolysis (MAAH), amino acid content analysis, and bioinformatics.

A novel, generally applicable method of identifying peptides using HPLC, microwave-assisted acid hydrolysis (MAAH), and bioinformatics is described. Method validation was performed on bacteriocins-antibacterial peptides produced by probiotic bacteria-using nine different bacteriocin isolates secreted by the probiotic Lactococcus lactis. Calibration curves were constructed for 23 amino acid PTH derivatives, and analysis was performed using norleucine as the internal standard. Validation of amino acid analysis performed in the range 2.5-100 nmol/mL indicated excellent method linearity, while the LODs ranged from 0.17 to 2.88 nmol/mL and the LOQs from 0.51 to 8.75 nmol/mL. The MAAH method was developed by irradiating nisaplin for various durations at 700 W, with 7 min providing the best results. The amino acid content of each sample was estimated following the application of MAAH to ten different samples. The bacteriocins in our samples were identified using the UniProt database. Eight of nine peptides were identified as UniProt entries: nisin A (P13068), nisin Z (P29559), I4DSZ9, OB7236, P36499, OB7237, A0A0M7BH60, and T2C9F0. The phylogenetic tree was constructed for nisin A and nisin Z using the multiple sequence aligning tool Clustal Ω. The identified nisin types presented excellent correlation with their ModBase-predicted structures. The present method gives true, precise, and rapid results, and requires only standard technical equipment. Our results suggest that the present approach can facilitate the discovery of novel bacteriocins and provide useful information on not only the amino acid contents of peptides but also the evolution of protein biology. Graphical abstract Identification of eight bacteriocins secreted by the probiotic L. lactis, following microwave assisted acid hydrolysis (MAAH), amino acid content analysis of each sample with HPLC-DAD and bioinformatics analysis using Uniprot, Clustal Ω and ModBase.

Defense systems in developing Artemia franciscana nauplii and their modulation by probiotic bacteria offer protection against a Vibrio anguillarum challenge.

The alterations of immune responses of Artemia franciscana nauplii as a function of culture time and after a challenge with the pathogen Vibrio anguillarum were studied. The effect of the administration of the probiotic bacteria Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis either alone or in combination with the pathogen was evaluated. The activity of the antioxidant enzymes superoxide dismutase (SOD), Glutathione reductase (GRed), Glutathione transferase (GST) and Phenoloxidase (PO) presented a significant increase as a function of culture time, appeared elevated following probiotic administration and were depleted 48 h following the experimental challenge. Lipid peroxidation reached peak levels at 48 h of culture, when nauplii start feeding and returned to lower values at 144 h, remaining however significantly higher than control (P < 0.05). The three probiotics significantly reduced lipid peroxidation in comparison with the corresponding control, while challenge with the pathogen resulted in its threefold increase. Survival of nauplii remained high throughout culture and was either increased or remained at control levels following the administration of the probiotics. The challenge with the pathogen resulted in a significantly decreased survival of 15.3% for the positive control, while in the probiotic treated series survival values were not significantly different from the negative control (P > 0.05). Following a combined administration of each probiotic and the pathogen the activities of all enzymes tested were significantly lower (P < 0.001) than the negative control (no treatment), but higher than the positive control (challenge, no probiotic). Lipid peroxidation was significantly lower in the probiotic treated series in comparison to the positive control (P < 0.001). The results of the present study provide evidence that major alterations take place as a function of culture time of Artemia nauplii. In addition the pathogen induces an oxidative stress response. The probiotics B. subtilis, L. plantarum and L. lactis protect Artemia against a V. anguillarum challenge by enhancing its immune responses thus contributing to reduced oxidative damage and increased survival.

Probiotic-Derived Bioactive Compounds in Colorectal Cancer Treatment.

Colorectal cancer (CRC) is a multifactorial disease with increased morbidity and mortality rates globally. Despite advanced chemotherapeutic approaches for the treatment of CRC, low survival rates due to the regular occurrence of drug resistance and deleterious side effects render the need for alternative anticancer agents imperative. Accumulating evidence supports that gut microbiota imbalance precedes the establishment of carcinogenesis, subsequently contributing to cancer progression and response to anticancer therapy. Manipulation of the gut microbiota composition via the administration of probiotic-derived bioactive compounds has gradually attained the interest of scientific communities as a novel therapeutic strategy for CRC. These compounds encompass miscellaneous metabolic secreted products of probiotics, including bacteriocins, short-chain fatty acids (SCFAs), lactate, exopolysaccharides (EPSs), biosurfactants, and bacterial peptides, with profound anti-inflammatory and antiproliferative properties. This review provides a classification of postbiotic types and a comprehensive summary of the current state of research on their biological role against CRC. It also describes how their intricate interaction with the gut microbiota regulates the proper function of the intestinal barrier, thus eliminating gut dysbiosis and CRC development. Finally, it discusses the future perspectives in precision-medicine approaches as well as the challenges of their synthesis and optimization of administration in clinical studies.

Interactions of Bisphenol A with Artemia franciscana and the ameliorative effect of probiotics.

In the present study, the bidirectional interactions of Artemia franciscana with BPA, administered either alone or following treatment with the probiotics Bacillus subtilis, Lactococcus lactis or Lactobacillus plantarum, were evaluated. A 24 h exposure to BPA below LC50 induced oxidative stress to Artemia, indicated by diminished activity of superoxide dismutase, glutathione reductase, glutathione transferase and phenoloxidase, increased lipid peroxidation and decreased survival. Probiotic treatment prior to BPA exposure, led to increased survival, reduced lipid peroxidation and increased enzyme activities. BPA quantification in Artemia and its culture medium, showed a time dependent reduction in its levels, more evident in probiotic series, indicating its biotransformation. ESI-MS analysis confirmed the presence of the tentative BPA metabolites hydroquinone and BPA-sulfate, while BPA-disulfate formation was confirmed in only in the probiotic series. Our results provide evidence that probiotics alleviate the oxidative stress response induced by BPA, by enhancing the BPA biotransformation ability of Artemia.

Administration of probiotics affects Artemia franciscana metanauplii intestinal ultrastructure and offers resistance against a Photobacterium damselae ssp. piscicida induced oxidative stress response.

The effects of Photobacterium damselae ssp. piscicida (Phdp) on immune responses and intestinal ultrastructure of Artemia franciscana following infection and their amelioration by the probiotic bacteria Bacillus subtilis, Lactobacillus plantarum and Lactococcus lactis were evaluated. Pathogen growth inhibition in coculture with each probiotic and its virulence against Artemia were confirmed with an LC50 of 105 CFU mL-1. Phdp administration to Artemia at sublethal levels resulted in depletion of superoxide dismutase, glutathione reductase, glutathione transferase and phenoloxidase activities, extensive lipid peroxidation and reduced survival. Following a combined administration of each probiotic and the pathogen, enzyme activities and survival were significantly higher, while lipid peroxidation was reduced, compared to the infected group with no probiotic treatment (P < 0.05). The transmission electron microscopy study revealed that pathogen infection resulted in disarranged and fragmented microvilli, formation of empty or pathogen containing cytoplasmic vacuoles and damaged mitochondria. In the probiotic-treated and Phdp-infected series, intestinal cells showed normal appearance, except for the presence of pathogen-containing vacuoles and highly ordered but laterally stacked microvilli. The results of the present study indicate that Phdp induces cell death through an oxidative stress response and probiotics enhance Artemia immune responses to protect it against the Phdp induced damage.

Evaluation of the probiotics Bacillus subtilis and Lactobacillus plantarum bioencapsulated in Artemia nauplii against vibriosis in European sea bass larvae (Dicentrarchus labrax, L.).

Two potential probiotics Bacillus subtilis and Lactobacillus plantarum were evaluated for use in aquaculture as preventive measures against vibriosis. In vitro evaluation of the probiotics using co-culture assays with the pathogen Vibrio anguillarum and testing for the production of antibacterial substances showed the presence of antagonism and confirmed the production of antibacterial substances. Both potential probiotics were administered to the live fish feed Artemia franciscana nauplii, offering protection against a subsequent challenge of the nauplii with the fish pathogen V. anguillarum, with best survival rates of the nauplii and the most efficient protection offered by B. subtilis. Nauplii enriched with B. subtilis were further used to evaluate the protection of sea bass larvae against vibriosis. The untreated group of fish challenged with V. anguillarum presented low survival of 36.7 %, while the fish treated with nauplii enriched with the probiotic B. subtilis showed significantly increased survival rates of 86.7 % after challenge with the pathogen. The survival of healthy unchallenged fish treated with the probiotic was not significantly different from control unchallenged fish (90-94 %). Our results indicate that B. subtilis is a probiotic suitable to be used for the prevention of vibriosis in fish larvae and can be safely administered through their live feed Artemia nauplii.

Simultaneous Quantification of Bisphenol-A and 4-Tert-Octylphenol in the Live Aquaculture Feed Artemia franciscana and in Its Culture Medium Using HPLC-DAD.

Aquaculture, a mass supplier of seafood, relies on plastic materials that may contain the endocrine disruptors bisphenol-A (BPA) and tert-octylphenol (t-OCT). These pollutants present toxicity to Artemia, the live aquaculture feed, and are transferred through it to the larval stages of the cultured organisms. The purpose of this work is the development and validation of an analytical method to determine BPA and t-OCT in Artemia and their culture medium, using n-octylphenol as the internal standard. Extraction of the samples was performed with H2O/TFA (0.08%)-methanol (3:1), followed by SPE. Analysis was performed in a Nucleosil column with mobile phases A (95:5, v/v, 0.1% TFA in H2O:CH3CN) and B (5:95, v/v, 0.08% TFA in H2O:CH3CN). Calibration curves were constructed in the range of concentrations expected following a 24 h administration of BPA (10 µg/mL) or t-OCT (0.5 µg/mL), below their respective LC50. At the end of exposure to the pollutants, their total levels appeared reduced by about 32% for BPA and 35% for t-OCT, and this reduction could not be accounted for by photodegradation (9-19%). The developed method was validated in terms of linearity, accuracy, and precision, demonstrating the uptake of BPA and t-OCT in Artemia.

Characterization and Quantitative Determination of a Diverse Group of Bacillus subtilis subsp. subtilis NCIB 3610 Antibacterial Peptides.

Five antibacterial peptides produced by Bacillus subtilis NCIB 3610 were purified, quantified, characterized, and identified in the present study. Cell-free extracts were subjected to three purification protocols employing ammonium sulfate or organic solvent precipitation and their combination, followed by ion-exchange chromatography, solid-phase extraction, and preparative high-performance liquid chromatography (HPLC). The combined ammonium sulfate and organic solvent precipitation extraction protocol presented the best results for peptide purification. In the five fractions that presented antimicrobial activity, antibacterial peptides were quantified by the turbidometric method and by HPLC using nisin for external calibration, with the second providing more accurate results. All peptides were pH- and temperature-resistant and their sensitivity to proteases treatment indicated their proteinic nature. The five peptides were subjected to microwave-assisted acid hydrolysis (MAAH) and following derivatization were analyzed using norleucine as the internal standard, to determine their amino acid content. The identification of the isolated peptides using the UniProt and PubChem databases indicated that the four peptides correspond to UniProt entries of the bacteriocins Subtilosin-A (Q1W152) Subtilosin-SbOX (H6D9P4), Ericin B (Q93GH3), Subtilin (P10946), and the fifth to the non-ribosomal antibacterial lipopeptide surfactin (CID:443592). The amino acid content determination and computational analyses, applied in the present work on the antimicrobial peptides of B. subtilis, proved an efficient screening and quantification method of bacteriocins that could potentially be applied in other bacterial strains. The constructed phylogenetic trees heterogeneity observed across the five peptides investigated might be indicative of competitive advantage of the strain.

Bisphenol A removal and degradation pathways in microorganisms with probiotic properties.

Bisphenol-A (BPA) is a constituent of polycarbonate plastics and epoxy resins, widely applied on food packaging materials. As BPA exposure results in health hazards, its efficient removal is of crucial importance. In our study five potentially probiotic microorganisms, namely Lactococcus lactis, Bacillus subtilis, Lactobacillus plantarum, Enterococcus faecalis, and Saccharomyces cerevisiae, were tested for their toxicity tolerance to BPA and their BPA removal ability. Although BPA toxicity, evident on all microorganisms, presented a correlation to both BPA addition time and its concentration, all strains exhibited BPA-removal ability with increased removal rate between 0 and 24 h of incubation. BPA degradation resulted in the formation of two dimer products in cells while the compounds Hydroquinone (HQ), 4-Hydroxyacetophenone (HAP), 4-Hydroxybenzoic acid (HBA) and 4-Isopropenylphenol (PP) were identified in the culture medium. In the proposed BPA degradation pathways BPA adducts formation appears as a common pattern, while BPA decomposition as well as the formation, and the levels of its end products present differences among microorganisms. The BPA degradation ability of the tested beneficial microorganisms demonstrates their potential application in the bioremediation of BPA contaminated foods and feeds and provides a means to suppress the adverse effects of BPA on human and animal health.

ß-Ν-Methylamino-L-alanine interferes with nitrogen assimilation in the cyanobacterium, non-BMAA producer, Synechococcus sp. TAU-MAC 0499.

The production of ß-Ν-methylamino-L-alanine (BMAA) in cyanobacteria is triggered by nitrogen-starvation conditions and its biological role, albeit unknown, is associated with nitrogen assimilation. In the present study, the effect of BMAA (773 µg L-1) on nitrogen metabolism and physiology of the non-diazotrophic cyanobacterium and non-BMAA producer, Synechococcus sp. TAU-MAC 0499, was investigated. In order to study the combined effect of nitrogen availability and BMAA, nitrogen-starvation conditions were induced by transferring cells in nitrogen-free medium and subsequently exposing the cultures to BMAA. After short-term treatment (180 min) and in the presence of nitrogen, BMAA inhibited glutamine synthetase, which resulted in low concentration of glutamine. In the absence of nitrogen, although there was no effect on glutamine synthetase, a possible perturbation in nitrogen assimilation is reflected on the significant decrease in glutamate levels. During the long-term exposure (24-96 h), growth, photosynthetic pigments and total protein were not affected by BMAA exposure, except for an increase in protein and phycocyanin levels at 48 h in nitrogen replete conditions. Results suggest that BMAA interferes with nitrogen assimilation, in a different way, depending on the presence or absence of combined nitrogen, providing novel data on the potential biological role of BMAA.

Monitoring of multiple bacteriocins through a developed dual extraction protocol and comparison of HPLC-DAD with turbidometry as their quantification system.

The present study describes the development of a simple and efficient screening system that allows identification and quantification of nine bacteriocins produced by Lactococcus lactis. Cell-free L. lactis extracts presented a broad spectrum of antibacterial activity, including Gram-negative bacteria, Gram-positive bacteria, and fungi. The characterization of their sensitivity to pH, and heat, showed that the extracts retained their antibacterial activity at extreme pH values and in a wide temperature range. The loss of antibacterial activity following treatment of the extracts with lipase or protease suggests a lipoproteinaceous nature of the produced antimicrobials. The extracts were subjected to a purification protocol that employs a two phase extraction using ammonium sulfate precipitation and organic solvent precipitation, followed by ion exchange chromatography, solid phase extraction and HPLC. In the nine fractions that presented antimicrobial activity, bacteriocins were quantified by the turbidometric method using a standard curve of nisin and by the HPLC method with nisin as the external standard, with both methods producing comparable results. Turbidometry appears to be unique in the qualitative determination of bacteriocins but the only method suitable to both separate and quantify the bacteriocins providing increased sensitivity, accuracy, and precision is HPLC.