Lactiplantibacillus plantarum from Unexplored Tunisian Ecological Niches: Antimicrobial Potential, Probiotic and Food Applications.

The continued exploration of the diversity of lactic acid bacteria in little-studied ecological niches represents a fundamental activity to understand the diffusion and biotechnological significance of this heterogeneous class of prokaryotes. In this study, Lactiplantibacillus plantarum (Lpb. plantarum) strains were isolated from Tunisian vegetable sources, including fermented olive and fermented pepper, and from dead locust intestines, which were subsequently evaluated for their antimicrobial activity against foodborne pathogenic bacteria, including Escherichia coli O157:H7 CECT 4267 and Listeria monocytogenes CECT 4031, as well as against some fungi, including Penicillium expansum, Aspergilus niger, and Botrytis cinerea. In addition, their resistance to oro-gastro-intestinal transit, aggregation capabilities, biofilm production capacity, adhesion to human enterocyte-like cells, and cytotoxicity to colorectal adenocarcinoma cell line were determined. Further, adhesion to tomatoes and the biocontrol potential of this model food matrix were analyzed. It was found that all the strains were able to inhibit the indicator growth, mostly through organic acid production. Furthermore, these strains showed promising probiotic traits, including in vitro tolerance to oro-gastrointestinal conditions, and adhesion to abiotic surfaces and Caco-2 cells. Moreover, all tested Lpb. plantarum strains were able to adhere to tomatoes with similar rates (4.0-6.0 LogCFU/g tomato). The co-culture of LAB strains with pathogens on tomatoes showed that Lpb. plantarum could be a good candidate to control pathogen growth. Nonetheless, further studies are needed to guarantee their use as probiotic strains for biocontrol on food matrices.

Multi-Functional Potential of Lactic Acid Bacteria Strains and Antimicrobial Effects in Minimally Processed Pomegranate (Punica granatum L. cv Jolly Red) Arils.

This study aimed to evaluate the antimicrobial activity of both cells, and cell-free supernatants (CFS) of 7 selected lactic acid bacteria (LAB) strains belonging to Limosilactobacillus fermentum (4 strains), Lacticaseibacillus paracasei (1 strain), Lacticaseibacillus rhamnosus (1 strain), and Enterococcus faecium (1 strain) species, against Listeria monocytogenes, Escherichia coli, Salmonella Typhimurium, Pseudomonas aeruginosa and Staphylococcus aureus, by both the agar-well diffusion and co-culture methods. In addition, probiotic and safety traits were also detected. Great variability was detected on antimicrobial effects, whereas all tested strains were found sensitive to most of the tested antibiotics, and without any DNase, gelatinase, or hemolytic activity. Moreover, strains showed excellent survival in acidic conditions and exhibited tolerance to pepsin and bile salts. Based on the in vitro results, the CFSs of two selected L. fermentum strains were applied, in a mixed solution, as bio-preservative into minimally processed pomegranate arils, inoculated with a cocktail of L. monocytogenes and E. coli. Samples, packaged in an ordinary atmosphere, were analyzed during refrigerated storage, for up to 12 days, for physicochemical (as weight loss, texture, color, pH, total soluble solids and organic acid content) and for microbiological traits. Results revealed the effectiveness of CFS, up to 12 days, in reducing weight loss and microbial growth, without any significant effect on texture, total soluble solid content and color, found comparable to the acid citric treatment, highlighting the multi-functional potential of selected probiotic strains.

Correlation between metabolites of lactic acid bacteria isolated from dairy traditional fermented Tunisian products and antifungal and antioxidant activities.

AIMS: The objective of this study is to identify and investigate the antifungal and antioxidant potential of lactic acid bacteria (LAB) isolated from traditional fermented products. METHODS AND RESULTS: In this work, a collection of LAB was isolated from traditional fermented products collected in four Tunisian regions. After first screening using the overlay method, seven bacterial strains were retained due to their high antifungal effect. Four strains of Limosilactobacillus fermentum were identified, one strain of Lacticaseibacillus paracasei, one strain of Lacticaseibacillus rhamnosus and one strain of Enterococcus faecium. The antifungal and the antioxidant potential of these bacteria were then evaluated. Bacterial strains were effective against six fungal strains with minimum inhibitory concentrations ranging from 25 to 100 mg/ml and minimum fungicidal concentrations ranging from 50 to 200 mg/ml. Cell-free supernatants of LAB were analysed by HPLC-DAD and LC-MS-qTOF-MS analysis. Results showed significant production of organic acids as well as several phenolic compounds. Correlation analysis confirmed that PLA and 1,2-dihydroxybenzene were positively correlated with antifungal potential. The results of the antioxidant activity highlighted an ABTS radical cation scavenging activity ranging from 49% to 57% and a DPPH trapping percentage ranging from 80% to 97%. CONCLUSIONS: Therefore, due to these characteristics, identified lactic acid bacteria strains have shown their effectiveness to perform as antifungal and antioxidant agents. SIGNIFICANCE AND IMPACT OF THE STUDY: Since microbial contamination is at the root of extensive losses in the food sector, the identified strains or their metabolites can potentially be used as additives to limit micro-organism spoilage in food products and increase their shelf life.

Garlic, Onion, and Cinnamon Essential Oil Anti-biofilms' Effect against Listeria monocytogenes.

Biofilms represent a serious problem for food industries due to their persistence in processing surfaces, from which they can cause food spoilage or, even worse, lead to foodborne diseases. Microorganisms immersed in biofilms are more resistant to biocides. The search for natural effective alternatives for the prevention and the control of biofilms has increased lately. The aim of this research was to test the antibacterial and the anti-biofilm activities of cinnamon, onion, and garlic essential oils against Listeria monocytogenes. The methodology highlighted first the effect of these essential oils on L. monocytogenes using disc diffusion and minimum inhibitory concentration (MIC) methods and then on initial cell attachment and six hours preformed biofilms. The inhibition of biofilms was assessed by crystal violet assay. Sulfides were the most abundant compounds present in onion and garlic essential oils, while cinnamaldehyde was predominant in cinnamon essential oil. MIC values were of 0.025 mg mL-1 for onion essential oil and 0.100 mg mL-1 for cinnamon and garlic. Onion essential oil inhibited initial cell attachment by 77% at 0.5 of the MIC dose, while at MIC, cinnamon and garlic essential oils inhibited the initial microbial adhesion completely. All three essential oils completely inhibited initial cell attachment when applied at 2 MIC. On the contrary, preformed biofilms were more resistant, and the inhibition rate ranged from 33% to 78%. In summary, this investigation revealed that the essential oils of garlic, onion, and cinnamon show an effective antibiofilm activity against L. monocytogenes and are promising natural antimicrobial alternatives for food processing facilities.

Draft Genome Sequence of Enterococcus faecalis Strain OB15, a Probiotic Strain Recently Isolated from Tunisian Rigouta Cheese.

Enterococcus faecalis OB15 is a probiotic strain that was isolated from rigouta, a popular traditional Tunisian fermented cheese. We report here the draft genome sequence of this strain, consisting of 2,912,159 bp, with an average G+C content of 37.49%.

Probiotic Potential and Safety Evaluation of Enterococcus faecalis OB14 and OB15, Isolated From Traditional Tunisian Testouri Cheese and Rigouta, Using Physiological and Genomic Analysis.

Lactic acid bacteria (LAB) strains OB14 and OB15 were isolated from traditional Tunisian fermented dairy products, Testouri cheese and Rigouta, respectively. They were identified as Enterococcus faecalis by the MALDI TOF-MS (matrix assisted laser desorption-ionization time of flight mass spectrometry) biotyper system and molecular assays (species-specific PCR). These new isolates were evaluated for probiotic properties, compared to E. faecalis Symbioflor 1 clone DSM 16431, as reference. The bacteria were found to be tolerant to the harsh conditions of the gastrointestinal tract (acidity and bile salt). They were low to moderate biofilm producers, can adhere to Caco-2/TC7 intestinal cells and strengthen the intestinal barrier through the increase of the transepithelial electrical resistance (TER). Susceptibility to ampicillin, vancomycin, gentamicin and erythromycin has been tested using the broth microdilutions method. The results demonstrated that E. faecalis OB14 and OB15 were sensitive to the clinically important ampicillin (MIC = 1 µg/mL) and vancomycin (MIC = 2 µg/mL) antibiotics. However, Whole Genome Sequencing (WGS) showed the presence of tetracycline resistance and cytolysin genes in E. faecalis OB14, and this led to high mortality of Galleria Mellonella larvae in the virulence test. Hierarchical cluster analysis by MALDI TOF-MS biotyper showed that E. faecalis OB15 was closely related to the E. faecalis Symbioflor 1 probiotic strain than to OB14, and this has been confirmed by WGS using the average nucleotide identity (ANI) and Genome-to-Genome Hybridization similarity methods. According to these results, E. faecalis OB15 seems to be reliable for future development as probiotic, in food or feed industry.

Two novel peptides with angiotensin I converting enzyme inhibitory and antioxidative activities from Scorpaena notata muscle protein hydrolysate.

Fish protein hydrolysate was prepared from muscle of small red scorpionfish (Scorpaena notata) by treatment with a protease from the fungus Penicillium digitatum. Protein hydrolysate was found to strongly inhibit the angiotensin I converting enzyme and exhibited high antioxidative activity through 1,1-diphenyl-2-picrylhydrazyl free radical scavenging assay. After ultrafiltration, peptides were isolated by a two-step procedure: size exclusion chromatography on a Toyopearl HW-40 followed by reversed-phase high-performance liquid chromatography with a high purification yield of 2.5 mg of peptide per gram of initial protein. Two major peptides were then identified by nanoscale liquid chromatography coupled to tandem mass spectrometry (nano-LC-MS/MS), corresponding to the following sequences: Leu-Val-Thr-Gly-Asp-Asp-Lys-Thr-Asn-Leu-Lys (1,204.665 Da) and Asp-Thr-Gly-Ser-Asp-Lys-Lys-Gln-Leu (992.511 Da). These peptides, mainly composed of hydrophilic amino acids, showed high antioxidative and angiotensin I converting enzyme inhibitory activities. These data suggest that the two novel peptides isolated from the muscle hydrolysate of small red scorpionfish can be a beneficial ingredient for functional foods or pharmaceuticals against hypertension and oxidative stress.

Purification and Biochemical Characterization of a Neutral Serine Protease from Trichoderma harzianum. Use in Antibacterial Peptide Production from a Fish By-Product Hydrolysate.

This study reports the purification and biochemical characterization of an extracellular neutral protease from the fungus Trichoderma harzianum. The protease (Th-Protease) was purified from the culture supernatant to homogeneity by a three-step procedure with 14.2% recovery and 9.06-fold increase in specific activity. The purified enzyme appeared as a single protein band after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a molecular mass of about 20 kDa. The optimum pH and temperature for the proteolytic activity were pH 7.0 and 40 °C, respectively. The enzyme was then investigated for its potential application in the production of antibacterial peptides. Interestingly, Scorpaena notata viscera protein hydrolysate prepared using the purified serine protease (Th-Protease) showed remarkable in vitro antibacterial activities. A peptide with a high antibacterial activity was further purified by a three-step procedure, and its sequence was identified as FPIGMGHGSRPA. The result of this study offers a promising alternative to produce natural antibacterial peptides from fish protein hydrolysate.

An improved method for the production of fructooligosaccharides by immobilized ß-fructofuranosidase from Sclerotinia sclerotiorum.

This work is focused on the prebiotic synthesis by a purified immobilized ß-fructofuranosidase (FFase) using a by-product molasses as a substrate. When cultivated on wheat bran, the fungus Sclerotinia sclerotiorum produces FFase with interesting transfructosylating activity. The enzyme was purified by gel filtration and anion exchange chromatography to homogeneity. It showed a specific activity of 66.06 U/mg and a molecular mass of 50 kDa. The FFase was immobilized covalently on alginate and chitosan, and the immobilization yield was 90% and 81% respectively, yet the immobilization efficiency was 52% and 93% in that order. The fixed enzymes were stable at a pH varying from 4.0 to 7.0 and at a temperature ranging from 4 to 70 °C. Yet, kinetic parameters and catalytic efficiency were determined for both immobilized and free FFases. Interestingly, chitosan cross-linked enzyme activity was maintained at 89.24% level after 50 reuses during 1 week. Continuous production of fructooligosaccharides (FOS) from beet molasses in chitosan enzyme reactor was improved. The maximum production yield obtained in 12 H was 72.2% (g FOS/g Sucrose). Thin-layer chromatography analysis showed that the major products are kestose and nystose.

Neutral serine protease from Penicillium italicum. Purification, biochemical characterization, and use for antioxidative peptide preparation from Scorpaena notata muscle.

In the present study, purification and properties of an extracellular neutral serine protease from the fungus Penicillium italicum and its potential application as an antioxidant peptides producer are reported. The protease was purified to homogeneity using ammonium sulfate precipitation, Sephacryl S-200 gel filtration, diethylaminoethanol (DEAE)-Sepharose ion exchange chromatography, and TSK-HPLC gel filtration with a 10.2-fold increase in specific activity and 25.8 % recovery. The purified enzyme appeared as single protein band with a molecular mass of 24 kDa in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the proteolytic activity were pH 7.0 and 50 °C, respectively. The enzyme was stable in the pH range of 6.0-9.0. The protease was activated by divalent cations such as Ca(2+) and Mg(2+). Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and relatively broad specificity. Scorpaena notata muscle protein hydrolysates prepared using purified serine protease (protease from P. italicum (Prot-Pen)) showed good in vitro antioxidative activities. The antioxidant activities of Scorpaena muscle protein hydrolyzed by Prot-Pen (SMPH-PP) were evaluated using various antioxidant assays: 1, 1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, reducing power, ferrous chelating activity, and DNA nicking assay. SMPH-PP showed varying degrees of antioxidant activity and almost the same strongest protection against hydroxyl radical induced DNA breakage.

Purification and biochemical characterization of a novel protease from Penicillium digitatum - Use in bioactive peptides production.

This work reports the production of a novel serine protease enzyme (P. dig-protease) from the fungus Penicillium digitatum. The protease was purified from the culture supernatant to homogeneity using ammonium sulfate precipitation, Sephadex G-150 gel filtration and carboxymethyl-sepharose ion exchange chromatography with a 13-fold increase in specific activity. The apparent molecular weight of P.dig-protease was estimated to be 120 kDa by native high performance liquid chromatography (HPLC), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a single polypeptide at about 30 kDa that indicates a tetrameric protein. The proteolytic activity was inhibited by phenylmethylsulfonyl fluoride suggesting a serine-protease enzyme. P.dig-protease stability was investigated over broad range of pH, temperature, salt concentrations, surfactants and metal ions. The purified P.dig-protease was used for the production of bioactive peptides. Red scorpionfish (Scorpaena notata) muscle was hydrolyzed with P.dig-protease in order to obtain peptides with biological activities. Interestingly, the hydrolysate revealed the presence of antioxidant and angiotensin-I converting enzyme inhibitor peptides.

Monoolein production by triglycerides hydrolysis using immobilized Rhizopus oryzae lipase.

Lipase extracted from Rhizopus oryzae was immobilized in alginate gel beads. The effects of the immobilization conditions, such as, alginate concentration, CaCl2 concentration and amount of initial enzyme on retained activity (specific activity ratio of entrapped active lipase to free lipase) were investigated. The optimal conditions for lipase entrapment were determined: 2% (w/v) alginate concentration, 100mM CaCl2 and enzyme ratio of 2000IU/mL.In such conditions, immobilized lipase by inclusion in alginate showed a highest stability and activity, on olive oil hydrolysis reaction where it could be reused for 10 cycles. After 15min of hydrolysis reaction, the mass composition of monoolein, diolein and triolein were about 78%, 10% and 12%. Hydrolysis' products purification by column chromatography lead to a successful separation of reaction compounds and provide a pure fraction of monoolein which is considered as the widest used emulsifier in food and pharmaceutical industries.

Immobilized Sclerotinia sclerotiorum invertase to produce invert sugar syrup from industrial beet molasses by-product.

The fungus Sclerotinia sclerotiorum produces invertase activity during cultivation on many agroindustrial residues. The molasses induced invertase was purified by DEAE-cellulose chromatography. The molecular mass of the purified enzyme was estimated at 48 kDa. Optimal temperature was determined at 60 °C and thermal stability up to 65 °C. The enzyme was stable between pH 2.0 and 8.0; optimum pH was about 5.5. Apparent K(m) and V(max) for sucrose were estimated to be respectively 5.8 mM and 0.11 µmol/min. The invertase was activated by ß-mercaptoethanol. Free enzyme exhibited 80 % of its original activity after two month's storage at 4 °C and 50 % after 1 week at 25 °C. In order to investigate an industrial application, the enzyme was immobilized on alginate and examined for invert sugar production by molasses hydrolysis in a continuous bioreactor. The yield of immobilized invertase was about 78 % and the activity yield was 59 %. Interestingly the immobilized enzyme hydrolyzed beet molasses consuming nearly all sucrose. It retained all of its initial activity after being used for 4 cycles and about 65 % at the sixth cycle. Regarding productivity; 20 g/l of molasses by-product gave the best invert sugar production 46.21 g/day/100 g substrate related to optimal sucrose conversion of 41.6 %.

MS analysis and molecular characterization of Botrytis cinerea protease Prot-2. Use in bioactive peptides production.

Prot-2 protease previously purified to homogeneity from Botrytis cinerea showed potentiality to be used in detergency and for production of bioactive peptides. To extend the characterization of Prot-2 protease, antifungal and antibacterial assays were performed in vitro using protein hydrolysates prepared from muscle of mackerel (Scomber scomborus) treated with this enzyme. The most active hydrolysate (degree of hydrolysis of 8 %) exhibited inhibition effect towards bacteria and phytopathogenic fungi, demonstrating that Prot-2 proteolysis generated bioactive peptides. Biochemical and molecular characterization of the purified Prot-2, by SDS-PAGE/Tryptic in gel-digestion and LC-MS/MS analysis, was investigated. The peptide amino acid sequence alignment search in database revealed a moderate homology between the determined amino acid sequence of Prot-2 protease and the known fungal trypsin/chymotrypsin in particular from Glomerella, Metarhizium and Streptomyces. From peptide sequence data obtained by mass spectrometry and sequences homologies, primers were defined and a cDNA fragment of 786 bp was amplified by RT-PCR. The cDNA nucleotide sequence analysis revealed an open reading frame coding for 262 amino acid residues. The deduced amino acid sequence of Prot-2 showed moderate identity with trypsin of Glomerella graminicola (74 %) and with chymotrypsin from Metarhizium anisopliae (71 %). Prot-2 exhibited a Ser protease homology and showed in addition the specific His motif of trypsin/chymotrypsin family.

Purification and characterization of an alkaline protease Prot 1 from Botrytis cinerea : biodetergent catalyst assay.

Alkaline thiol protease named Prot 1 was isolated from a culture filtrate of Botrytis cinerea. The enzyme was purified by ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Thus, the enzyme was purified to homogeneity with specific activity of 30-fold higher than that of the crude broth. The purified alkaline protease has an apparent molecular mass of 43 kDa under denaturing conditions as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native molecular mass (45 kDa), determined by gel filtration, indicated that the alkaline protease has a monomeric form. The purified protease was biochemically characterized. The enzyme is active at alkaline pH and has a suitable and high thermostability. The optimal pH and temperature for activity were 9.0-10.0 and 60 degrees C, respectively. This protease was stable between pH 5.0 and 12.0. The enzyme retained 85% of its activity by treatment at 50 degrees C over 120 min; it maintained 50% of activity after 60 min of heating at 60 degrees C. Furthermore, the protease retained almost complete activity after 4 wk storage at 25 degrees C. The activity was significantly affected by thiol protease inhibitors, suggesting that the enzyme belongs to the alkaline thiol protease family. With the aim on industrial applications, we focused on studying the stability of the protease in several conditions. Prot 1 activity was not affected by ionic strength and different detergent additives, and, thus, the protease shows remarkable properties as a biodetergent catalyst.