Dietary Administration of Probiotic Aeromonas veronii V03 on the Modulation of Innate Immunity, Expression of Immune-Related Genes and Disease Resistance Against Aeromonas hydrophila Infection in Common Carp (Cyprinus carpio).

This study investigates the effects of dietary Aeromonas veronii V03 supplementation on growth performances, innate immunity, and expression of immune-related genes in lymphoid organs of Cyprinus carpio and resistance to Aeromonas hydrophila infection. Fish were fed for 4 weeks with basal diet (BD; without probiotic), and experiment diet containing different doses of A. veronii V03 at 3.2 × 107 (DI) and 3.5 × 109 (DII) CFU g-1 of diet. At the end of the probiotic feeding trial, fish were challenged with A. hydrophila, and the percentage of survival rates was recorded over 7 days. Results revealed that fish fed with A. veronii V03 demonstrated a significant improvement in growth and enhancement of innate immunity, including respiratory burst, myeloperoxidase, and lysozyme activities, and total immunoglobulin level compared with BD fed to fish. Relatively, expression of cytokines (MyD88, IL-1ß1, IL-8, and IL-10) and c- and g-type lysozymes were significantly up- and downregulated in lymphoid organs of fish. Moreover, dietary supplementation of A. veronii V03 exhibited significantly (p < 0.001) higher survival rates of DI (90%) and DII (96.66%) compared with BD (53.33%) fed fish against A. hydrophila infection. These findings help to understand the effects of probiotic A. veronii V03 administrated feed influences on growth and ailment resistance to A. hydrophila infection by regulating innate and systemic immunity in common carp fish.

Production and characterization of exopolysaccharide from the sponge-associated Bacillus subtilis MKU SERB2 and its in-vitro biological properties.

In this study, the sponge-associated a potential endosymbiotic bacterium, Bacillus subtilis MKU SERB2 was identified and optimized the production of exopolysaccharide (EPS) by using response surface methodology (RSM). The central composite rotatable design (CCRD) exhibited the highest yield of EPS (617.81 µg/mL) obtained from the optimized medium containing 11.5 g/L of sucrose, 3.5 g/L of yeast extract, 3.0 g/L of peptone, and 2.5 g/L of calcium chloride. Fourier transform infrared (FTIR) spectrum of purified EPS indicated that the presence of carboxyl, hydroxyl, and amide as functional groups, and their structural composition was confirmed by 1H and 13C nuclear magnetic resonance (NMR) analysis. Moreover, the fibrous, porous and semi-crystalline nature of EPS was confirmed by SEM and X-ray powder diffraction (XRD) analysis and the EDX inferred demonstrated the presence of C, Na, O, N, S, and Cl respectively. Further, the isolated EPS exhibited potent antioxidant activity and moderate anticoagulant efficacy whereas there was no hemolytic and lymphocytes toxicity. Overall, our result suggests that the functional and biological properties of the EPS imply the potential applications in food and pharmaceutical industries in the future.

Induction of mitochondria-mediated apoptosis and suppression of tumor growth in zebrafish xenograft model by cyclic dipeptides identified from Exiguobacterium acetylicum.

Colorectal cancer is the most common type of gastrointestinal cancers with poor survival and limited therapeutic options. In this study, four structurally different cyclic dipeptides (or diketopiperazine) were isolated and identified as cyclo (L-Pro-L-Leu), cyclo (L-Pro-L-Val), cyclo (L-Pro-L-Phe) and cyclo (L-Pro-L-Tyr) from the ethyl acetate extract in the cell-free filtrate of Exiguobacterium acetylicum S01. The anticancer potential of identified DKPs on colorectal cancer HT-29 cells in vitro and in vivo zebrafish xenograft model was evaluated. The MTT (3-(4, 5-dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide)) assay showed that four DKPs exhibited significant inhibition of HT-29 cells viability in a dose-dependent manner whereas there were no cytotoxic effects on normal mouse fibroblast 3T3 cells. Also, we observed that all DKPs induce early and late apoptotic cell death in HT-29 cells. Moreover, the expression levels of apoptotic (cytochrome-c, caspase-3 and Bid) and anti-apoptotic (Bcl-2) markers were up- and down-regulated in HT-29 cells in response to DKPs treatments. Furthermore, these four DKPs remarkably inhibited the tumor progression in a zebrafish xenograft model within a nonlethal dose range. Overall, our findings suggest that cyclic dipeptides derived from E. acetylicum S01 could be promising chemopreventive/ therapeutic candidates against cancer.

Diapolycopenedioic-acid-diglucosyl ester and keto-myxocoxanthin glucoside ester: Novel carotenoids derived from Exiguobacterium acetylicum S01 and evaluation of their anticancer and anti-inflammatory activities.

Inflammation is pivotal for the development of gastrointestinal cancer and linked to poor survival and limited therapeutic options. In this study, six structurally different carotenoids were isolated and identified from the methanolic extract of Exiguobacterium acetylicum S01 namely lycopene (Car-I), diapolycopenedioic-acid-diglucosyl-ester (Car-II), ß-carotene (Car-III), zeaxanthin (Car-IV), astaxanthin (Car-V), and keto-myxocoxanthin glucoside-ester (Car-VI). Further, their anti-cancer, anti-inflammatory, and antioxidant potentials were evaluated. The MTT assay was used to determine the effect of carotenoids on viability of colorectal cancer (HT-29) as well as peripheral blood mononuclear cells (PBMCs). Results revealed that all the six carotenoids were demonstrated a significant inhibition of HT-29 cells viability in a dose-dependent manner whereas there was no cytotoxic effect in PBMCs. The study also recorded that six carotenoids considerably inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO) production, tumor necrosis factor-alpha (TNF-α), and lipid peroxidation in PBMCs. Moreover, antioxidant potentials of Car-II and Car-VI were significantly (p = 0.001) higher than ascorbic acid as determined by a 2,2-diphenyl-1-picrylhydrazyl radical scavenging assay. Therefore, our results ascertained the role of carotenoids derived from E. acetylicum S01 in developing potential therapeutic agents for inflammation-associated cancer.

Optimization of submerged fermentation process for improved production of ß-carotene by Exiguobacterium acetylicum S01.

Carotenoids are natural pigments with substantial applications in nutraceutical, pharmaceutical, and food industries. In this study, optimization of the fermentation process for enhancement of ß-carotene and biomass production by Exiguobacterium acetylicum S01 was achieved by employing statistical designs including the Placket-Burman design (PBD) and response surface methodology (RSM). Among the seven variables investigated by two levels in PBD, glucose, peptone, pH and temperature were indicated as crucial variables (p < 0.0001) for ß-carotene and biomass productivity. Response surface methodology was further applied to evaluate the optimal concentrations of these four variables for maximum ß-carotene and biomass productivity. The optimized medium contained glucose 1.4 g/L, peptone 26.5 g/L, pH 8.5, and temperature 30 °C, respectively. A significant increase in ß-carotene (40.32 ± 2.55 mg/L) and biomass (2.19 ± 0.10 g/L) productivities in E. acetylicum S01 were achieved by using RSM, which was 3.47-fold and 2.36-fold higher in the optimized medium compared to the un-optimized medium. Further, the optimum fermentation condition in the 5-L bioreactor was achieved a maximal ß-carotene yield of 107.22 ± 5.78 mg/L within 96 h. Moreover, the expression levels of carotenoid biosynthetic genes (phytoene desaturase (CrtI) and phytoene synthase (CrtB)) were up-regulated (2.89-fold and 3.71-fold) in E. acetylicum under the optimized medium conditions. Overall, these results suggest that E. acetylicum S01 can be used as a promising microorganism for the commercial production of ß-carotene.

Functional Characterization of Probiotic Potential of Novel Pigmented Bacterial Strains for Aquaculture Applications.

The bioprospecting proficient of novel pigmented probiotic strains with respect to aquaculture industry was unexplored hitherto. In this study, we investigated the probiotic potential of novel pigmented bacterial strains isolated from the indigenous soil sediments in their vicinal habitats, which were screened for their antimicrobial activity against aquatic pathogens using agar well diffusion assay. The strains namely Exiguobacterium acetylicum (S01), Aeromonas veronii (V03), and Chryseobacterium joostei (V04) were phenotypically identified and confirmed by 16S rRNA gene sequence analysis. Further characterization revealed that strains S01 and V03 survive relatively in lower pH and higher bile salt concentrations and possess good adherence ability and broad-spectrum antibiotic susceptibility. The isolate S01 exhibited the higher adhesion ability to hydrocarbons (82%) and mannose-specific adhesion (msa) gene expression. Additionally, the probiotic effects were evaluated in Artemia nauplii fed with algae supplemented with S01, V03, and V04 strains (2.7 × 107 cfu/mL) for 3 days under axenic environment. We observed a significant increase (p < 0.05) in the survival rate of Artemia nauplii treated with S01 (83 ± 5%) and V03 (55 ± 5%), whereas the survival rate was only 30 ± 0% in the untreated group. Moreover, the individual length (IL) was increased in treated group S01 (156.7 ± 2.2 µm), V03 (146.1 ± 3.4 µm), and V04 (134.4 ± 2.5 µm) compared with untreated group (116.0 ± 4.8 µm). Our results revealed that E. acetylicum S01 exhibits desirable functional probiotic attributes compared to A. veronii and C. joostei and it would be a promising probiotic strain, which can be efficiently used in the aquaculture applications.

Modulation of innate immunity, expression of cytokine genes and disease resistance against Aeromonas hydrophila infection in goldfish (Carassius auratus) by dietary supplementation with Exiguobacterium acetylicum S01.

Probiotic strains play an increasing role in the production of healthy animals used as a food source. Elucidating the mechanisms of action that allow probiotic-driven immunomodulation may facilitate different applications such as the prevention of infectious diseases in food organisms. This study elucidates the probiotic effects of Exiguobacterium acetylicum S01 on the growth, haematological profile, innate immune capacity, expression of cytokine genes, and resistance to diseases of Carassius auratus caused by Aeromonas hydrophila infection. Three fish groups were fed with the following diets containing different doses of E. acetylicum S01 (CFU g-1): basal diet 0 (BD, without probiotic), 2.5 × 107 (DI) and 2.7 × 109 (DII)-CFU g-1 for 4 weeks. After 4 weeks, the fish were injected intraperitoneally with A. hydrophila and the percentage of survival was recorded over 21 days of post-challenge. Results revealed that dietary supplementation of E. acetylicum S01 significantly (P < 0.05) enhanced the growth, haematological profile and cellular immune responses including respiratory burst, phagocytic activities and antimicrobial enzymes (myeloperoxidase and lysozyme) and total immunoglobulin levels were improved by probiotic feeding at both occasions. Comparatively, expression of c- and g-type lysozyme followed by pro- and anti-inflammatory cytokines (IL-1ß, IL-10 and TGFß) was up-regulated in kidney, head-kidney and spleen. Moreover, fish fed with diet DII had a significantly higher (P < 0.05) survival rate (73.2%) after challenging. The survival rate was only 33.2% of the BD group against A. hydrophila infection. Our results revealed that E. acetylicum S01 delivered probiotic in feed exerts its influence on growth performance and provides disease resistance by stimulating the immune system at the cellular and molecular levels in C. auratus.