LYSINE PRODUCTION BY USING ACETYLENOTROPHIC BACTERIA

ΜΕΛΕΤΗΘΗΚΕ Η ΦΥΣΙΟΛΟΓΙΑ ΣΤΕΛΕΧΩΝ ΤΟΥ ΓΕΝΟΥΣ RHODOCOCCUS ΤΑ ΟΠΟΙΑ ΧΡΗΣΙΜΟΠΟΙΟΥΝ ΑΚΕΤΥΛΕΝΙΟ ΩΣ ΜΟΝΗ ΠΗΓΗ ΑΝΘΡΑΚΟΣ. ΤΑ ΣΤΕΛΕΧΗ ΑΠΟΜΟΝΩΘΗΚΑΝ ΑΡΧΙΚΑ ΑΠΟ ΤΟ ΕΔΑΦΟΣΚΑΙ ΕΞΕΤΑΣΤΗΚΑΝ ΩΣ ΠΡΟΣ ΤΗΝ ΙΚΑΝΟΤΗΤΑ ΤΟΥΣ ΝΑ ΠΑΡΑΓΟΥΝ ΕΞΩΚΥΤΤΑΡΙΚΑ ΛΥΣΙΝΗ. ΠΕΡΑΙΤΕΡΩ ΕΓΙΝΕ ΜΙΑ ΠΡΟΣΠΑΘΕΙΑ ΔΗΜΙΟΥΡΓΙΑΣ ΕΞΕΙΔΙΚΕΥΜΕΝΩΝ ΜΕΤΑΛΛΑΓΩΝ ΜΕ ΣΤΟΧΟ ΤΗΝ ΑΥΞΑΝΟΜΕΝΗ ΠΑΡΑΓΩΓΗ ΛΥΣΙΝΗΣ. ΓΙΑ ΤΟ ΣΚΟΠΟ ΑΥΤΟ ΧΑΡΑΧΘΗΚΑΝ 3 ΔΙΑΦΟΡΕΤΙΚΟΙ ΤΡΟΠΟΙ ΠΡΟΣΕΓΓΙΣΗΣ: 1) ΕΛΕΓΧΟΣ ΕΠΙΔΡΑΣΗΣ ΧΗΜΙΚΩΝ ΑΝΑΛΟΓΩΝ ΑΜΙΝΟΞΕΩΝ [S-(Β- ΑΜΙΝΟΑΙΘΥΛ)-L-ΚΥΣΤΕΙΝΗ (AEC), ΥΔΡΟΞΑΜΙΚΟ ΑΣΠΑΡΤΙΚΟ ΟΞΥ (ΑΑΗ), ΥΔΡΟΞΑΜΙΚΗ ΛΥΣΙΝΗ (LHXX), Β-ΦΘΟΡΙΟΠΥΡΟΣΤΑΦΥΛΙΚΟ ΟΞΥ (FP)], ΣΤΟ ΣΤΕΛΕΧΟΣ ΦΥΣΙΚΟΥ ΤΥΠΟΥ ΚΑΙ ΑΠΟΜΟΝΩΣΗ ΑΝΘΕΚΤΙΚΩΝ ΣΤΕΛΕΧΩΝ ΠΟΥ ΠΡΟΕΡΧΟΝΤΑΙ ΑΠΟ ΦΥΣΙΚΗ ΜΕΤΑΛΛΑΞΟΓΕΝΕΣΗ. 2) ΕΠΙΔΡΑΣΗ ΕΙΤΕ ΜΕ ΥΠΕΡΙΩΔΗ ΑΚΤΙΝΟΒΟΛΙΑ (UV), ΕΙΤΕ ΜΕ ΝΙΤΡΟΖΟΓΟΥΑΝΙΔΙΝΗ Η ΜΕ ΜΕΤΑΘΕΤΑ ΣΤΟΙΧΕΙΑ ΚΑΙ ΑΠΟΜΟΝΩΣΗ ΑΥΞΟΤΡΟΦΩΝ ΜΕΤΑΛΛΑΓΜΕΝΩΝ ΣΤΕΛΕΧΩΝ. 3) ΜΕΤΑΛΛΑΞΟΓΕΝΕΣΗ ΜΕ ΝΙΤΡΟΖΟΓΟΥΑΝΙΔΙΝΗ ΚΑΙ ΑΠΟΜΟΝΩΣΗ ΜΕΤΑΛΛΑΓΜΕΝΩΝ ΥΠΕΡ-ΕΥΑΙΣΘΗΤΩΝ ΣΤΕΛΕΧΩΝ ΣΤΟ ΠΑΡΕΜΠΟΔΙΣΤΗ FP. Η ΜΕΓΑΛΥΤΕΡΗ ΠΑΡΑΓΩΓΗ ΛΥΣΙΝΗΣ ΜΕΤΑΞΥ ΤΩΝ ΑΥΞΟΤΡΟΦΩΝ (ΑΥΞΗΣΗ ΠΑΝΩ ΑΠΟ 70% ΣΕ ΣΧΕΣΗ ΜΕ ΤΟ ΣΤΕΛΕΧΟΣ ΦΥΣΙΚΟΥ ΤΥΠΟΥ) ΠΑΡΑΤΗΡΗΘΗΚΕ ΑΠΟ ΕΝΑ ΠΟΛΛΑΠΛΑ ΑΥΞΟΤΡΟΦΟ ΣΤΕΛΕΧΟΣ, ΤΟ ΟΠΟΙΟ ΠΑΡΟΥΣΙΑΖΕΙ ΤΗ ΜΙΑ ΤΟΥΛΑΧΙΣΤΟΝ ΑΥΞΟΤΡΟΦΙΑ ΤΟΥΣΤΗΝ ΟΜΑΔΑ ΤΟΥ ΑΣΠΑΡΤΙΚΟΥ ΟΞΕΟΣ ΚΑΙ ΣΥΓΧΡΟΝΩΣ ΕΙΝΑΙ ΑΝΘΕΚΤΙΚΟ ΣΤΟΝ ΠΑΡΕΜΠΟΔΙΣΤΗ ΑΑΗ. ΕΠΙΣΗΣ ΜΕΤΑΞΥ ΤΩΝ ΜΕΤΑΛΛΑΓΜΕΝΩΝ ΕΥΑΙΣΘΗΤΩΝ ΣΤΕΛΕΧΩΝ ΣΤΟ FP, ΤΡΙΑ ΣΤΕΛΕΧΗ (5, 14, 18 FPS) ΠΑΡΟΥΣΙΑΖΟΥΝ ΥΠΕΡ-ΔΙΠΛΑΣΙΑΣΜΟ ΤΗΣ ΠΑΡΑΓΩΓΗΣ ΤΗΣ ΛΥΣΙΝΗΣ, ΤΟΣΤΕΛΕΧΟΣ 30 FPS ΤΕΤΡΑΠΛΑΣΙΑΣΜΟ, ΤΟ 32 FPS ΠΕΝΤΑΠΛΑΣΙΑΣΜΟ, ΕΝΩ ΤΟ 28 FPS ΠΑΡΑΓΕΙ ΠΕΡΙΠΟΥ 1,29/L ΕΞΩΚΥΤΤΑΡΙΚΗ ΛΥΣΙΝΗ.A FIRST STUDY OF A RHODOCOCCUS SPP. ISOLATE (WILD TYPE STRAIN ISOLATED FROM SOIL), WHICH CAN CONSUME ACETYLENE AS THE SOLE CARBON SOURCE, HAS BEEN ATTEMPTED. ONCE, THE ROLE OF SEVERAL PHYSIOLOGICAL PARAMETERS IN THE PRODUCTION OF EXTRACELLULAR LYSINE WAS EXAMINED, ATTEMPTS TO ISOLATE MUTANTS WITH IMPROVED L-LYSINE PRODUCTIVITY FOLLOWED. THE MUTAGENESIS EXPERIMENTS WERE BASED ON THREE DIFFERENT APPROACHES: 1) BY CHECKING THE EFFECT OF AMINO ACID ANALOGS ON THE WILD STRAIN AND BY ISOLATING RESISTANT MUTANTS ON THESE ANALOGS [S-(Β- AMINOETHYL)-L-CYSTEINE (AEC), ASPARTIC ACID HYDROXAMATE (AAH), LYSINE HYDROXAMATE (LHX)AND Β-FLUOROPYRUVATE (FP)]. 2) BY INDUCED MUTAGENESIS WITH ULTRA VIOLET IRRADIATION (UV), NITROSOGOUANIDINE (MNNG), OR TRANSPOSONS AND THE SUBSEQUENT ISOLATION OF AUXOTROPHIC MUTANTS. 3) BY MUTAGENESIS WITH MNNG AND ISOLATION OF SENSITIVE MUTANTS TO THE ANALOG FP. THE HIGHEST LYSINE ACCUMULATION OF ALL AUXOTROPHS EXAMINED (ENHANCEMENT OVER THE 70% OF THE WILD STRAIN'S EXTRACELLULAR LYSINE) WAS NOTICED IN A MULTIPLE AUXOTROPH WITH AT LEAST ONE AUXOTROPHY BETWEEN THE AMINO ACIDS OF ASPARATE FAMILY AND RESISTANT TO AAH ANALOG. ALSO, FROM A LARGE NUMBER OF MUTANTS SENSITIVE TO FP TASTED, THREE DOUBLED THEIR PRODUCTION IN EXTRACELLULAR LYSINE, THE 30 FPS AND 32 FPS MUTANT SHOWED A 4-FOLD AND 5-FOLD LYSINE PRODUCTION LEVEL RESPECTIVELY WHILST THE 28 FPS MUTANT PRODUCED ABOUT 1.2 G/L OF EXTRACELLULAR LYSINE, USING AS SOLE SOURCE OF CARBON ACETYLENE

The Evaluation of Hazards to Man and the Environment during the Composting of Sewage Sludge

Composting is considered an effective treatment option to eliminate or substantially reduce potential hazards relating to the recycling of sewage sludge (SS) on land. The variation of four major types of hazards (heavy metals, instability, pathogenic potential and antibiotic resistance) was studied during laboratory-scale composting of two mixtures of sludge and green waste (1:1 and 1:2 v/v). The heavy metal content of the final compost was governed by the initial contamination of SS, with the bulking agent ratio having practically no effect. The composts would meet the heavy metal standards of the United States of America (USA) and the European Union member states, but would fail the most stringent of them. A higher ratio of bulking agent led to a higher stabilisation rate, nitrogen retention and final degree of stability. A good level of sanitisation was achieved for both mixtures, despite the relatively low temperatures attained in the laboratory system. The antibiotic resistance was limited among the E. coli strains examined, but its occurrence was more frequent among the Enterococcus spp. strains. The type of antibiotics against which resistance was mainly detected indicates that this might not be acquired, thus, not posing a serious epidemiological risk through the land application of the SS derived composts

PCR detection of Salmonella spp. using primers targeting the quorum sensing gene sdiA

Bacteria communicate with one another and with their host using chemical signalling molecules. This phenomenon is generally described as quorum sensing. A set of primers for PCR detection of Salmonella spp. has been designed using as target the sdiA gene which encodes a signal receptor of the LuxR family. The PCR product (274 bp) was confirmed by sequencing. A number of 81 non-Salmonella strains (representing 24 different species) were tested and gave negative results, while a total of 101 different serotypes of Salmonella (155 strains) tested positive for the presence of the sdiA gene. The sensitivity and specificity of the sdiA-based PCR assay were also checked in artificially contaminated human faecal samples. In this study, we demonstrate that quorum sensing genes can be successfully exploited as diagnostic markers

Inhibition of Listeria monocytogenes Growth, Adherence and Invasion in Caco-2 Cells by Potential Probiotic Lactic Acid Bacteria Isolated from Fecal Samples of Healthy Neonates

Lactic acid bacteria (LAB) isolated from healthy humans may prove an effective tool against pathogen growth, adherence and invasion in intestinal epithelial cells. This study aimed to evaluate the antilisterial properties of LAB isolated from fecal samples of healthy neonates. Forty-five LAB strains were tested for their antimicrobial activity against ten Listeria monocytogenes strains with spot-on-lawn and agar-well diffusion assays, and ten lactobacilli strains were further assessed for their inhibitory effect against adherence and invasion of Caco-2 cells by L. monocytogenes EGDe. Inhibition was estimated in competition, exclusion or displacement assays, where lactobacilli and L. monocytogenes were added to Caco-2 monolayers simultaneously or 1 h apart from each other. Inhibition of L. monocytogenes growth was only displayed with the spot-on-lawn assay; cell-free supernatants of lactobacilli were not effective against the pathogen. Lactobacillus (L.) paragasseri LDD-C1 and L. crispatus LCR-A21 were able to adhere to Caco-2 cells at significantly higher levels than the reference strain L. rhamnosus GG. The adherence of L. monocytogenes to Caco-2 cells was reduced by 20.8% to 62.1% and invasion by 33.5% to 63.1% during competition, which was more effective compared to the exclusion and displacement assays. These findings demonstrate that lactobacilli isolated from neonatal feces could be considered a good candidate against L. monocytogenes

Inhibition of <i>Listeria monocytogenes</i> Growth, Adherence and Invasion in Caco-2 Cells by Potential Probiotic Lactic Acid Bacteria Isolated from Fecal Samples of Healthy Neonates

Lactic acid bacteria (LAB) isolated from healthy humans may prove an effective tool against pathogen growth, adherence and invasion in intestinal epithelial cells. This study aimed to evaluate the antilisterial properties of LAB isolated from fecal samples of healthy neonates. Forty-five LAB strains were tested for their antimicrobial activity against ten Listeria monocytogenes strains with spot-on-lawn and agar-well diffusion assays, and ten lactobacilli strains were further assessed for their inhibitory effect against adherence and invasion of Caco-2 cells by L. monocytogenes EGDe. Inhibition was estimated in competition, exclusion or displacement assays, where lactobacilli and L. monocytogenes were added to Caco-2 monolayers simultaneously or 1 h apart from each other. Inhibition of L. monocytogenes growth was only displayed with the spot-on-lawn assay; cell-free supernatants of lactobacilli were not effective against the pathogen. Lactobacillus (L.) paragasseri LDD-C1 and L. crispatus LCR-A21 were able to adhere to Caco-2 cells at significantly higher levels than the reference strain L. rhamnosus GG. The adherence of L. monocytogenes to Caco-2 cells was reduced by 20.8% to 62.1% and invasion by 33.5% to 63.1% during competition, which was more effective compared to the exclusion and displacement assays. These findings demonstrate that lactobacilli isolated from neonatal feces could be considered a good candidate against L. monocytogenes

Fermentation Supernatants of Pleurotus eryngii Mushroom Ameliorate Intestinal Epithelial Barrier Dysfunction in Lipopolysaccharide-Induced Caco-2 Cells via Upregulation of Tight Junctions

In recent years, modulation of gut microbiota through prebiotics has garnered interest as a potential to ameliorate intestinal barrier dysfunction. The aim of the study was to examine the in vitro effect of fermentation supernatants (FSs) from rich in β-glucan Pleurotus eryngii mushrooms on the expression levels of tight junctions (TJs) genes in Caco-2 cells stimulated by bacterial lipopolysaccharides (LPS). Mushrooms were fermented using fecal inocula in an in vitro batch culture model. Caco-2 cells were subjected to LPS and FS treatment under three different conditions: pre-incubation with FS, co- and post-incubation. Reverse transcription PCR was applied to measure the expression levels of zonulin-1, occludin and claudin-1 genes. FSs from P. eryngii mushrooms led to a significant upregulation of the TJs gene expression in pre-incubation state, indicating potential preventive action. Down-regulation of all TJs gene expression levels was observed when the cells were challenged with LPS. The FS negative control (gut microbiota of each donor with no carbohydrate source) exhibited a significant upregulation of TJs expression levels compared to the cells that were challenged with LPS, for all three conditions. Overall, our data highlighted the positive and potential protective effects of P. eryngii mushrooms in upregulation of TJs’ genes.Funding agency:Greek national funds through the Operational Program Competitiveness, Entrepreneurship and Innovation under the call RESEARCH-CREATE-INNOVATE T1EDK-03404 </p

In Vitro Fermentation of Edible Mushrooms: Effects on Faecal Microbiota Characteristics of Autistic and Neurotypical Children

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD

In Vitro Fermentation of Edible Mushrooms: Effects on Faecal Microbiota Characteristics of Autistic and Neurotypical Children

Children with autism spectrum disorder (ASD) often suffer gastrointestinal disturbances consistent with gut microbiota (GM) alterations. Treatment with pro/prebiotics may potentially alleviate gut symptoms, but the evidence for prebiotics is scarce. This study aims to evaluate the effects of edible mushrooms (Pleurotus, Basidiomycota) and prebiotic compounds on GM composition and metabolite production in vitro, using faecal samples from autistic and non-autistic children. Specific microbial populations were enumerated after 24 h of fermentation by quantitative PCR, and the metabolic production was determined by gas chromatography. Higher levels of Prevotella spp. and Bifidobacterium spp. were measured in neurotypical children compared to ASD children. A total of 24 h fermentation of Pleurotus eryngii and P. ostreatus mushroom powder increased the levels of Bifidobacterium, while known prebiotics increased the levels of total bacteria and Bacteroides in both groups. Only P. eryngii mushrooms resulted in significantly elevated levels of total bacteria Bacteroides and Feacalibacterium prausnitzii compared to the negative control (NC) in the ASD group. Both mushrooms induced elevated levels of butyrate after 24 h of fermentation, while short-chain fructooligosaccharides induced increased levels of acetate in the ASD group, compared to NC. Overall, this study highlights the positive effect of edible mushrooms on the GM and metabolic activity of children with ASD

Genoprotective Properties and Metabolites of β-Glucan-Rich Edible Mushrooms Following Their In Vitro Fermentation by Human Faecal Microbiota

A variety of bioactive compounds, constituents of edible mushrooms, in particular &beta;-glucans, i.e., a group of &beta;-d-glucose polysaccharides abundant in the fungal cell walls, have been linked to immunomodulating, anticancer and prebiotic activities. The aim of the study was the investigation of the genoprotective effects of edible mushrooms produced by Pleurotus eryngii, Pleurotus ostreatus and Cyclocybe cylindracea (Basidiomycota). Mushrooms from selected strains of the species mentioned above were fermented in vitro using faecal inocula from healthy volunteers. The cytotoxic and anti-genotoxic properties of the fermentation supernatants (FSs) were investigated in Caco-2 human colon adenocarcinoma cells. The FSs were cytotoxic in a dose-dependent manner. Non-cytotoxic concentrations were used for the genotoxicity studies, which revealed that mushrooms&rsquo; FSs have the ability to protect Caco-2 cells against tert-butyl hydroperoxide (t-BOOH), a known genotoxic agent. Their global metabolic profiling was assessed by 1H-NMR spectroscopy. A total of 37 metabolites were identified with the use of two-dimensional (2D) homo- and hetero-nuclear NMR experiments. Multivariate data analysis monitored the metabolic variability of gut microbiota and probed to biomarkers potentially associated with the health-promoting effects of edible mushrooms