Effect of cryopreservation on motility, DNA integrity and gene expression in grey mullet, Mugil cephalus sperm.

This study documents the effect of cryopreservation on motility, DNA integrity, and gene expression in Mugil cephalus sperm. Fresh sperm were cryopreserved using V2 extender (V2E) or 0.3 M glucose, each in combination with one of three cryoprotective agents (CPAs), i.e., 10 % of dimethylsulfoxide, ethylene glycol, or glycerol, all at once. After two different storage (7- vs 60- day) periods in liquid nitrogen, sperm samples were thawed. Single-cell gel electrophoresis was used to detect the DNA integrity. Heat shock proteins (HSPs), HSP70, HSP90 and glutathione peroxidase (GPx2) genes mRNA expression levels was documented using qRT-PCR. The results demonstrated that among 0.3 M glucose + CPAs combinations, EG recorded higher frozen-thawed motility 69 % (7- day) and 59 % (60- day). Similarly, in V2E + CPAs combinations, EG recorded higher frozen-thawed motility 31 % (7- day) and 26 % (60- day). The DNA integrity of all thawed sperm (both periods) did not differ from that of fresh sperm. The qRT-PCR results revealed that in the combination of 0.3 M glucose + CPAs, the level of HSP90 and GPx2 gene expression was found to be upregulated in frozen-thawed sperm on both periods. Whereas, the expression level of the HSP70 gene was down-regulated. On the contrary, in the combination of V2E + CPAs, the expression levels of HSP70, HSP90 and GPx2 genes could not be detected on both periods. Overall, the findings of this study demonstrate that the cryomedium (extender + cryoprotectant) has a more influential role in the motility and levels of gene expression in the frozen-thawed sperm of M. cephalus.

Preparation and functional characterization of the bio-composite film based on chitosan/polyvinyl alcohol blended with bacterial exopolysaccharide EPS MC-5 having antioxidant activities.

In this study, the plate casting method was successfully used to prepare biocomposite films containing EPS from probiotic Enterococcus faecium MC-5 in combination with PVA and chitosan. The findings demonstrated that EPS was uniformly distributed in the film matrices and significantly improved the physicochemical properties of the resulting composite films. The development of intermolecular connections between the polymers was detected by high tensile strength and low water vapour transmission rate. EPS plays an important role in limiting the passage of UV- and visible light radiations through the films. FT-IR analysis was used to determine the molecular compatibility between the functional groups of the blended films made up of chitosan-EPS and PVA-EPS. The TGA results showed that composite films have a significant degree of thermal stability. The presence of amorphous peaks in the composite film was confirmed by XRD analysis. The EPS blended films displayed a greater antioxidant property than the PVA and chitosan films, as determined by DPPH and hydroxyl radical scavenging activities. Interestingly, the EPS-derived films showed enhanced metal chelation activity and strong antibacterial properties against Listeria monocytogenes and Staphylococcus aureus. EPS-based composite films performed better than chitosan and PVA films in terms of degradation rate. The overall functional characteristics of the EPS blended films suggested that they could be used as a packaging material to replace or reduce the use of conventional petroleum-based packaging materials.

Effect of cryopreservation on post-thaw motility and viability of Grey mullet, Mugil cephalus sperm (Linnaeus, 1758).

In this study, we optimized a simple method of cryopreservation for Mugil cephalus sperm based on post-thaw motility and viability. A series of experiments were conducted by changing the extender, cryoprotectant and freezing height above the liquid nitrogen (LN) surface. First, we carried out the cryopreservation using the extender V2E and cryoprotective agents (CPAs) namely, propylene glycol (PG), methanol (MeOH), glycerol (GLY), ethylene glycol (EG), dimethylsulfoxide (Me2SO) and dimethylacetamide (DMA) at a final concentration of 5% and 10%. We found that 10% of GLY, EG and Me2SO were more suitable compared to other CPAs. Then, different freezing heights (6, 8, 10 and 12 cm) above the LN surface were experimented with extender V2E and optimized CPAs. Then, 0.3 M of glucose, sucrose and trehalose were tested as extender along with optimized CPAs and freezing height. Additionally, the effect of fast-rate freezing and storage days (7, 30 and 180) on post-thaw sperm quality was documented using the factors optimized in earlier experiments. For all experiments, the fresh sperm was diluted at a ratio of 1:1 with cryomedium (CPA + extender), loaded into cryovials (2.0 mL) and frozen. The cryopreserved sperm was thawed at 30 °C for 90-120 s and their quality was evaluated. Among the experimented factors, sperm diluted in cryomedium (0.3 M glucose + 10% EG) and frozen at 4 cm above the LN surface registered significantly (P < 0.05) highest post-thaw motility (73 ± 2%) and (71 ± 1%) viability. Fast-rate freezing has resulted in lower (about 30%) post-thaw motility and viability of sperm. The storage days (7, 30 and 180) did not have a significant effect on post-thaw sperm quality. Overall results show that using the factors optimized through this study, high-quality sperm can be obtained after cryopreservation.

Optimization and Physicochemical Characterization of Polysaccharide Purified from Sonneratia caseolaris Mangrove Leaves: a Potential Antioxidant and Antibiofilm Agent.

The Box-Behnken design was applied to determine the optimal parameters of the extraction condition by using the response surface methodology (RSM) from the leaves of Sonneratia caseolaris L. The result indicates the best-optimized conditions used for the extraction of polysaccharides at 84.02 °C temperature, 3.12 h time, and 27.31 mL/g for the water-to-material ratio. The maximum experimental yield of 8.81 ± 0.09% was obtained which is in agreement with the predicted value of 8.79%. Thereafter, low molecular weight polysaccharide (SCLP) was separated after sequentially being purified through column chromatography with a relative molecular weight of 3.74 kDa. The physicochemical properties were evaluated by characterization techniques such as FT-IR spectra, NMR spectrum, and SEM analysis. RP-HPLC analysis confirmed that SCLP was a heteropolysaccharide, majorly comprising rhamnose (28.25%), and xylose (27.17%) residues, followed by mannose (18.90%), and galactose (17.17%), respectively. Thermal analysis (TGA-DSC) results showed that SCLP is a highly thermostable polymer with a degradation temperature of 361.63 °C. X-ray diffraction patterns and tertiary structure analyses indicate that SCLP had a semi-crystalline polymer having a triple-helical configuration. Moreover, SCLP displayed potential antibiofilm ability for all the tested pathogens while stronger activity against Klebsiella pneumoniae and Pseudomonas aeruginosa. In addition, SCLP has potential in vitro antioxidant activity on DPPH, ABTS radical, superoxide, and Fe2+ chelating. These findings indicate that the polysaccharide has potentially been used in functional food, cosmetics, and pharmacological industries.

Effects of polysaccharide-based silver and selenium nanoparticles on growth performance, biochemical parameters, and immune response of Cyprinus carpio.

Avicennia marina mangrove leaves polysaccharide (AMLP) was used for the synthesis of polysaccharide-based selenium (AMLP-SeNPs) and silver nanoparticles (AMLP-AgNPs). The synthesized nanoparticles were further characterized by UV-Vis, DLS, FT-IR, X-ray diffraction, and HR-TEM analysis. A 60-day (8 weeks) feeding trial experiment was conducted to investigate the effects of AMLP, AMLP-SeNPs, and AMLP-AgNPs dietary supplementation on growth performance parameters, blood parameters, immunological and enzymatic profiles in Cyprinus carpio. The characterization results of AMLP-SeNPs and AMLP-AgNPs confirmed the formation of well-stabilized spherical nanoparticles with a mean particle size of 37.25 and 72.40 nm, respectively having a crystalline structure. The feeding experiment results demonstrated that 2 mg/kg of AMLP-SeNPs followed by 0.2 mg/kg of AMLP-AgNPs showed significantly (p Ë‚ 0.05) higher final weight, weight gain (WG), specific growth rate (SGR%), protein and lipid efficiency, and lower food conversion ratio as compared to other groups. The catalase, superoxidase dismutase, and glutathione peroxidase activity were significantly (p Ë‚ 0.05) higher in the group fed 2 mg/kg supplemented AMLP-SeNPs. Total protein and globulin contents were significantly (p Ë‚ 0.05) higher and albumin concentration was significantly lower in fish that received 2 mg/kg of AMLP- SeNPs as compared to control. A significant increase in serum HDL and decrease in LDL and MDA concentrations were observed in the group supplemented with 2 mg/kg of nano selenium. The body's crude lipid, protein, moisture, and ash were not significantly different from the control. The AMLP-SeNPs showed significantly (p Ë‚ 0.05) lower aspartate aminotransferase (AST), alanine aminotransferase (ALT), and higher alkaline phosphatase (ALP) activities compared to other test groups. The relative percentage survivability (RPS%) was higher in AMLP-SeNPs (84.6%) followed by AMLP-AgNPs (76.7%) after 8th weeks of supplementary diets as compared to control groups. Overall, the finding of these studies revealed that the inclusion of AMLP-SeNPs improved the growth performance and antioxidant defense system, enhance immune response, and provide resistance against Aeromonas hydrophila in Common carp.

Enhancement of growth, innate immunity, and disease resistance by probiotic Enterococcus faecium MC-5 against Aeromonas hydrophila in Indian major carp Cirrhinus mrigala.

E. faecium MC-5 was isolated and characterized from the gut content of C. carpio and supplemented with a pellet diet at the ratio of 1.0 × 106, 1.0 × 109, and 1.0 × 1012 cfu g-1. Then it was fed to Cirrhinus mrigala for 42 days and the growth performances were evaluated. The growth performance of experimental groups was significantly (P < 0.05) increased than the control group. During 21 and 42 days of the feeding experiment, all the tested immunological parameters such as phagocytic, respiratory burst, lysozyme, acid phosphatase (ACP), superoxide dismutase (SOD), myeloperoxidase activities, and IgM level of experimental groups were significantly (P < 0.05) increased than a control group. After 21 and 42 days of feeding experiment, the fishes were challenged with A. hydrophila, and the relative percentage of survival of experimental groups (PT-I to PT-III) over the control group was recorded from 30.94 to 61.53 and 38.04-77.52 %, respectively. During the challenge experiment, A. hydrophila load was enumerated from dead fish at every 7-day interval. The A. hydrophila load in both the tissues of experimental groups decreased positively from the 7th to 14th day of challenge duration. Overall, the results suggested that the addition of E. faecium MC-5 enhanced the growth performance, immunological response, disease resistance to A. hydrophila, and antioxidant defense system in C. mrigala. Hence these results suggest that E. faecium MC-5 could be used as an advantageous probiotic supplement in aquaculture production.

Characterization of potential probiotic bacteria Enterococcus faecium MC-5 isolated from the gut content of Cyprinus carpio specularis.

The goal of this study was to determine the unique characteristics of Enterococcus faecium MC-5, a probiotic bacteria isolated from the intestine of a fish, Cyprinus carpio specularis, collected from Dal Lake in Srinagar, Kashmir, India. For this, the important valuable probiotic attributes, some functional properties, and safety assessments were analyzed in-vitro for the strain MC-5. The strain E. faecium MC-5 exhibited high resistance to low pH, high bile salt, lysozyme, and phenol. The strain MC-5 showed excellent auto- and co-aggregation properties and displayed remarkable hydrophobicity towards various tested hydrocarbons which suggested that the strain possesses venerable adhesion properties. Apart from these, the cell-free supernatant (CFS) of strain MC-5 exhibited phenomenal antimicrobial activity against the tested pathogens. A scanning electron microscope (SEM) image revealed strain MC-5 finely adhered to human colon adenocarcinoma cells (HCT-15 cells). The strain MC-5 showed high bile salt hydrolase activity and excellent cholesterol removal ability of 70.27%. The intact cells of strain MC-5 also showed strong DPPH scavenging activity. The EPS produced by E. faecium MC-5 inhibited the adhesion of Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica on HCT-15 cells with maximum inhibition rates of 41.82, 40.34, and 55.51%, respectively for displacement assay, which was higher as compared to exclusion (26.06, 26.11, and 39.23%) and competition assays (30.06, 26.7, and 41.20%). Strain MC-5 did not exhibit hemolysis and was also found susceptible to vancomycin and other clinically important antibiotics. When evaluating all the results from the present study, it is propounded that strain MC-5 has enviable probiotic characteristics and thus can be used as bio-protective cultures and/or bio-shield in food and pharmaceutical industries.

Functional and molecular characterization of a cold-active lipase from Psychrobacter celer PU3 with potential antibiofilm property.

The lipase gene from Psychrobacter celer PU3 was cloned into pET-28a(+) expression vector and overexpressed in E. coli BL21 (DE3) pLysS cells. The purified Psychrobacter celer lipase (PCL) was characterized as an alkaline active enzyme and has a molecular mass of around 30 kDa. The PCL was active even at a low temperature and the optimum range was observed between 10 and 40 °C temperatures. MALDI-TOF and phylogenetic analysis ensured that Psychrobacter celer PU3 lipase (PCL) was closely related to P. aureginosa lipase (PAL). MD simulation results suggest that temperature change did not affect the overall structure of PCL, but it might altered the temperature-dependent PCL functional changes. R1 (129-135 AA) and R2 (187-191 AA) regions could be important for temperature-dependent PCL function and they fluctuated much at 35 °C temperature. PMSF completely inhibited PCL lipase activity and it demonstrates the presence of serine residues in the active site of PCL. PCL is moderately halophilic and most of the tested organic solvents found to be inhibiting the lipase activity except the solvents ethanol and methanol. PCL activity was increased with surfactants (SDS and CTAB) and bleaching agents (hydrogen peroxide). The effect of different metal ions on PCL resulted that only mercuric chloride was found as the enhancer of the lipase activity. Antibiofilm property of PCL was evaluated against pathogenic Vibrio parahaemolyticus isolated from the diseased shrimp and MIC value was 500 U. PCL significantly altered the morphology and biofilm density of V. parahaemolyticus and the same was observed through scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM) imaging. RT-PCR analysis revealed that the mRNA expression level of biofilm, colony morphology and major toxin-related (aphA, luxS, opaR, tolC, toxR) genes of V. parahaemolyticus were significantly downregulated with PCL treatment.

Physico-chemical characterization of biosurfactant from Pseudomonas aeruginosa PU1 and its application in microbial enhance oil recovery.

Biosurfactant-producing bacteria were isolated from transformer oil-contaminated soil. The strain was identified as Pseudomonas aeruginosa PU1 based on its molecular characterization by 16S rRNA gene sequencing. The extraction of biosurfactant was done by acid precipitation method using 2 N hydrochloric acid and further purified by silica-gel column chromatography. The highest rhamnolipid biosurfactant production (8.92 ± 0.08 g/L) was obtained using molasses (6%, w/v) and ammonium nitrate (5%, w/v). The purified biosurfactant showed a reduction of surface tension of water from 70.23 mN/m to 29.77 mN/m at a concentration of 30 mg/L. The functional groups were characterized by fourier transform infrared spectroscopy (FT-IR) and proton nuclear magnetic resonance (1H NMR). The liquid chromatography-mass spectrometry (LC-MS) analysis showed six different rhamnolipid congeners with psuedomolecular ions (m/z) of 305, 361, 451, 505, 532 and 621. X-ray diffraction (XRD) analysis and the thermal analysis showed crystalline nature and thermal stability of the biosurfactant. The stability study of the biosurfactant reveals that the emulsifying activity was much stable at various ranges of temperature (4-120 °C), pH (2-12), and NaCl concentration (2-10%, w/v) even on the 7th day. The purified biosurfactant PU1 was found to be effective in oil recovery and showed 68.53 ± 3.07% of oil recovery in the sand pack column under saline condition, which was higher than anionic surfactant sodium dodecyl sulphate (SDS). The physico-chemical properties and the stability studies of the biosurfactant suggested that it has enormous potential in oil recovery in the soil contaminated with the oils.

Physicochemical and functional characterization of mannan exopolysaccharide from Weissella confusa MD1 with bioactivities.

The present study focused on production, optimization and characterization of exopolysaccharide (EPS) from Weissella confusa MD1. The purified EPS MD1 was also evaluated for in vitro biological activities. The maximum yield of EPS (10.07 ± 0.32 g/L) was obtained with optimized culture conditions of 35 °C at pH 6.5 for 36 h with 4% (w/v) galactose and 1% (w/v) ammonium nitrate supplemented in MRS broth. The crude EPS was purified with diethylamino ethanol-Sepharose Fast Flow column and Sephadex-G 75 column. The physicochemical and functional characterization of the EPS was done by high-performance thin-layer chromatography (HPTLC), high-performance gel permeation chromatography (HPGPC), fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR), thermo gravimetric and differential scanning calorimetric (TG-DSC) analysis, x-ray diffraction (XRD) and scanning electron microscopy (SEM). HPGPC analysis showed molecular weight of purified EPS MD1 as 2.909 KDa. Monosaccharide analysis showed that EPS was a novel mannan with mannose as the only monomeric unit present, suggesting EPS to be a homopolysaccharide containing â†’ 6) α-Man p (1 â†’ linkages. Microstructure studies by SEM showed MD1 EPS has globular and porous structure. The purified EPS MD1 exhibited higher thermal stability having degradation temperature around 267.74 °C with melting enthalpy value (Δ H) of 337.7 J/g. The EPS showed promising antioxidant activities with excellent antibiofilm activity against Staphylococcus aureus, Listeria monocytogenes, Salmonella enterica and Salmonella typhi. These striking physicochemical characteristics features and bioactivities of EPS would serve as potential candidate in food processing industry to be used as food adjunct in foods with health benefits. This is the first study reporting a mannan homopolysaccharide from Weissella sp. to be structurally characterized.

Cloning, overexpression, purification of bacteriocin enterocin-B and structural analysis, interaction determination of enterocin-A, B against pathogenic bacteria and human cancer cells.

In this present study, a gene (ent-B) encoding the bacteriocin enterocin-B was cloned, overexpressed and purified from Enterococcus faecium por1. The molecular weight of the bacteriocin enterocin-B was observed around 7.2 kDa and exhibited antimicrobial activity against several human pathogenic bacteria. The antimicrobial activity of cloned enterocin-B was increased effectively by combining with another bacteriocin enterocin-A from the same microorganism. Protein-protein docking and molecular dynamics simulation studies revealed that the bacteriocin enterocin-B is interacting with enterocin-A and formation of a heterodimer (enterocin A + B). The heterodimer of bacteriocin enterocin-A + B exhibited potential anti-bacterial, anti-biofilm activity against Staphylococcus aureus, Acinetobacter baumannii, Listeria monocytogenes and Escherichia coli. The bacteriocin enterocin-B, A and heterodimer of bacteriocin enterocin A + B showed no haemolysis on human RBC cells. This is the first report that the cell growth inhibitory activity of the bacteriocin enterocin B against HeLa, HT-29 and AGS human cancer cells and this cell growth inhibitory activity was significantly increased when cancer cells treated with the heterodimer of bacteriocins enterocin-A + B. The cell growth inhibitory activity of the bacteriocin enterocin-B and the heterodimer of bacteriocin enterocin-A + B were not observed in non-cancerous INT-407 cells (intestinal epithelial cells).

Anti-inflammatory and Antioxidant Properties of Probiotic Bacterium Lactobacillus mucosae AN1 and Lactobacillus fermentum SNR1 in Wistar Albino Rats.

The potent antioxidant probiotic strains Lactobacillus mucosae AN1 and Lactobacillus fermentum SNR1 were assessed for anti-inflammatory properties in carrageenan (acute) and complete Freund's adjuvant-induced inflammation (chronic) models in the present study. The two probiotic strains were administered orally along with feed to the Wistar albino male rats as whole cell as well as microencapsulated form. The following experiments were performed to evaluate the anti-inflammatory properties of probiotic strains and the results were observed that the encapsulated and unencapsulated probiotic strains have exhibited statistically significant decrease in paw thickness. Percentage of inhibition in paw thickness of microencapsulated probiotic bacteria (Group VIII), unencapsulated strains (Group IX) were revealed 85 ± 13% and 77 ± 25%, respectively. In Hematoxylin and Eosin staining, results were revealed that the probiotic strains were exhibited anti-inflammatory effects on inflammation-induced paw tissues. qRT-PCR studies revealed upregulation of anti-inflammatory cytokine genes and down-regulation pro-inflammatory cytokine genes in probiotic-treated rat paw tissues. Further, the expression of anti-inflammatory and pro-inflammatory cytokines were examined using immunohistochemistry and ELISA methods. The probiotic administered rat paw tissue in different groups have exhibited the low level of lipid peroxides formation and higher anti-oxidant activities when compared to the control and inflammation control tissues.

Sulfated polysaccharides of seagrass Halophila ovalis suppresses tumor necrosis factor-α-induced chemokine interleukin-8 secretion in HT-29 cell line.

OBJECTIVES: The present study aims to investigate the anti-oxidant and anti-inflammatory properties of seagrass Halophila ovalis sulfated polysaccharide on HT-29 cell line. SUBJECTS AND METHODS: Monosaccharides composition was identified using liquid chromatography-mass spectrometry (LC-MS) and the functional groups were analyzed using Fourier transform-infrared (FT-IR) spectroscopy. The antioxidant and anti-inflammatory potential of crude extract and purified fractions was investigated in vitro. RESULTS: FT-IR spectra revealed that the presence of different functional groups and the presence of galactose (82.4%), xylose (7.6%), fructose (4.0%), mannose (2.0%), fucose (1.6%), glucose (1.2%), and arabinose (1.0%) was observed using LC-MS. Ho-SP and its fractions showed radical scavenging activity in hydroxyl, 2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid, and ferric reducing antioxidant power assay in a dose-dependent manner. Noticeable anti-inflammatory activity of purified fraction Ho FrIV (IC 50= 43.85 µg/ml) was observed in a noncytotoxic range of concentrations and inhibited the tumor necrosis factor-α (TNF-α)-induced interleukin-8 (IL-8) secretion (0.27 ng/ml) in HT-29 cell line. CONCLUSION: Overall, the results presented in this study suggest that purified fraction Ho FrIV of Ho-SP could suppress the TNF-α-induced secretion of IL-8 in HT-29 and thus could be used as a promising antioxidant and anti-inflammatory candidate with potential benefits.

Molecular characterization and application of lipase from Bacillus sp. PU1 and investigation of structural changes based on pH and temperature using MD simulation.

A gene coding lipase from Bacillus sp. PU1 was cloned and expressed in E. coli BL21(DE3) pLysS. The purified lipase has a molecular weight of 23kDa, is highly alkaline (pH range 8-10) and mesophilic (20-50°C). Three dimensional structure of the lipase was modeled by comparative homology and identified as a typical serine lipase by the presence of conserved Ser77, Asp133, His156. The molecular stability and behavior of the lipase was carried out using MD simulation studies at different pH and temperature was performed in comparison with biochemical analysis. Structural modifications of the lipase under these conditions were trapped by dihedral based FEL analysis and the functional loops (loop-H5/B4 and loop-H6/B5 of lipase) are identified which would cause the catalytic behavior of the lipase by high flexibility. Further characteristic feature of lipase are observed as follows; SDS completely inhibits the lipase activity and enzyme activity is enhanced with non-ionic surfactants. The lipase was highly stable in different organic solvents and also it could tolerate NaCl (0.4-0.8M). This enzyme was found to disrupt the biofilm of tested pathogenic bacterial strains.

Antinociceptive and anti-inflammatory activities of Sargassum wightii and Halophila ovalis sulfated polysaccharides in experimental animal models.

The present study investigated the effects of sulfated polysaccharides from brown seaweed Sargassum wightii (Sw-SP) and seagrass Halophila ovalis (Ho-SP) in nociceptive and inflammatory models. In the formalin test, Sw-SP and Ho-SP significantly reduced licking time in both phases of the test at a dose of 10 mg/kg. In the hot plate test, the antinociceptive effect was observed only in animals treated with 10 mg/kg of Sw-SP and 5, 10 mg/kg of Ho-SP, suggesting that the analgesic effect occurs through a central action mechanism at the higher dose. Sw-SP and Ho-SP (10 mg/kg) significantly inhibited paw edema induced by carrageenan, especially at 3 h after treatment and potentially decreased neutrophil migration by 53% and 52%, respectively. In Freund's adjuvant-induced arthritic rats, there was a significant increase in the rat paw volume and decrease in body weight, but in Sw-SP- and Ho-SP-treated groups (10 mg/kg), a significant reduction in paw volume and a normal gain in body weight were observed. The present results indicate that Sw-SP and Ho-SP possess antinociceptive and anti-inflammatory effects and have potential usefulness for development as therapeutic agents.

Probiotics and its functionally valuable products-a review.

During the past two decades probiotic bacteria have been increasingly proposed as health promoting bacteria in variety of food system, because of its safety, functional, and technological characteristics. Commonly, Lactobacillus spp., Bifidobacterium spp., Saccharomyces boulardii, and some other microorganisms have been considered as probiotic strains. Possibly these bacterial strains exerted several beneficial effects into gastrointestinal tract of host while administered with variety of food system. Lactic acid bacteria (LAB) usually produce antimicrobial substances like bacteriocin which have broad spectrum of antagonist effect against closely related Gram positive and Gram negative pathogens. LAB strains often produce polymeric substances such as exopolysaccharides (EPS) which increase the colonization of probiotic bacteria by cell-cell interactions in gastrointestinal tract. LAB also produces biosurfactant which showed that the wide range of antimicrobial activity against bacterial pathogen as well as its antiadhesive properties reduces the adhesion of pathogens into gastric wall membrane. Furthermore, LAB strains have also been reported for production of antioxidants which are ability to scavenge the free radicals such as superoxide anions and hydroxyl radicals. For this sense, this review article is mainly focused on the ecology, biosynthesis, genetics, target sites, and applications of bacteriocins and EPS from LAB strains. Moreover, this review discusses about the production and functions of nutritive essential element folate and iron chelating agent such as siderophores from LAB.

Synthesis and functional characterization of antibiofilm exopolysaccharide produced by Enterococcus faecium MC13 isolated from the gut of fish.

The synthesis and functional characterization of an antibiofilm exopolysaccharide (EPS) from a probiotic Enterococcus faecium MC13 were investigated. The temperature of 35 °C, pH of 6.5, and salinity of 1-2% were found to be optimum for EPS production. The sucrose (30 g l⁻¹) and yeast extract (20 g l⁻¹) acted as suitable carbon and nitrogen sources, respectively, which strongly influenced EPS production with yield of 11.33 and 11.91 g l⁻¹. Based on the thin layer chromatography, EPS of E. faecium MC13 was found to be a heteropolysaccharide, composed of galactose and glucose sugar units with a molecular mass of 2.0 × 105 Da. Fourier transform infrared spectrum analysis of the EPS revealed many predominant functional groups including hydroxyl, carboxyl, and amide groups. EPS exhibited better emulsifying and flocculating activities which is relatively similar to those of commercial polysaccharides. In vitro antioxidant inspect of EPS showed lesser antioxidant activity than that of the control ascorbic acid. Thermal behavior of EPS was different from the other EPS produced by other lactic acid bacteria. In vitro antibiofilm assay of EPS exhibited significant biofilm inhibition, especially with Listeria monocytogenes. To the best of our knowledge, this is the first report on EPS of E. faecium with strong emulsifying and flocculating activities.

Application of response surface methodology in the optimisation of a growth medium for enhanced natural preservative bacteriocin production by a probiotic bacterium.

In this study, a statistics-based experimental design was utilised for the optimisation of a growth medium which possibly enhanced bacteriocin production by Streptococcus phocae PI80. Carbon, nitrogen sources and a bio-surfactant were first screened using a one variable at a time technique and scored for increasing yield production. The selected variables were further statistically optimised using response surface methodology with a central composite design. The high- and low-level limits of the selected variables were determined, and a set of 34 experimental runs were performed. The concentration of each medium ingredient influenced the bacteriocin activity to about 22,500 AU mL⁻¹. The carbon and nitrogen sources were identified as significant factors in restraining the bacteriocin activity produced by S. phocae PI80. The statistics-based experimental design was found to be very efficient in optimising the media components in a number of experimental runs, with a three-fold increase in bacteriocin activity compared to the un-optimised medium. The optimum medium composition was found to be sodium succinate (10.0 g L⁻¹), yeast extract (4.0 g L⁻¹), glucose (9.0 g L⁻¹), NaCl (10.0 g L⁻¹), Tween 80 (6.0 g L⁻¹) and K2HPO4 (1.0 g L⁻¹). This optimised medium is two-fold more cost effective than the commercial Lactobacillus MRS medium.

Production and purification of a novel exopolysaccharide from lactic acid bacterium Streptococcus phocae PI80 and its functional characteristics activity in vitro.

Optimum culture conditions which ease the synthesis of a novel exopolysaccharide (EPS) from a potent marine strain Streptococcus phocae was proposed in this study. The strain grows well at 35 °C, pH 7.0 and NaCl (2%) with lactose and yeast extract as best carbon and nitrogen sources. The maximum yield of EPS (11.75 and 12.14 g/L) was obtained in the presence of lactose and yeast extract at a concentration of 20 g/L respectively. EPS was refined by gel filtration chromatography using phenyl Sepharose column which revealed the presence of arabinose, fructose and galactose sugar units with molecular mass about 2.8 × 10(5) Da. Emulsifying and flocculating stability of EPS compared with three commercial hydrocolloids. EPS exhibited better activities which are similar to that of commercial hydrocolloids. Both crude and purified EPS exhibited strong antioxidant potential by quenching hydroxyl and superoxide anion radicals. Antibiofilm activity by inhibition of Gram positive and Gram negative biofilm forming bacteria was evident in our studies. Potential antioxidant activity and biofilm inhibiting property of EPS may lead to the development of novel food grade adjuncts.

Design and optimization of fermentation medium for enhanced bacteriocin production by probiotic bacterium Enterococcus faecium MC13.

Statistics-based experimental designs were used to develop a cost-effective medium for enhanced production of viable cells and bacteriocin by probiotic Enterococcus faecium MC13. Carbon, nitrogen, and mineral sources were first screened by one-variable-at-a-time (OVAT) methods. In order to increase yield production, the selected variables were further statistically optimized using response-surface methodology (RSM) with central composite design (CCD). The maximum and minimum levels of the selected variables were determined and a set of 34 experimental runs was performed. The optimum concentrations of the tested variables for production of viable cells (12.24 log CFU mL(-1)) and bacteriocin activity (25,600 AU mL(-1)) were tryptone (10.0 g/L), peptone (6.0 g/L), maltose (3.0 g/L), glucose (9.0 g/L), NaCl (15.0 g/L), sodium citrate (2.5 g/L), sodium acetate (1.0 g/L), and dipotassium PO(4) (0.1 g/L). Threefold increased yield of bacteriocin was achieved in optimized medium compared to the unoptimized counterpart, and this was two times less cost than commercial MRS medium.