Effectiveness of the guava leaf extracts against lipopolysaccharide-induced oxidative stress and immune responses in Cyprinus carpio.

The anti-inflammatory activity of the guava leaf extracts (GLE) against LPS-induced inflammatory responses in fish macrophage cell lines is well documented. Here, we evaluated the effects of dietary GLE on LPS-induced oxidative stress, immune responses, and glucocorticoid receptor-related gene expression in Cyprinus carpio. Basal diet was supplemented with 0 (control), 100, 150, 200, or 250 mg kg-1 GLE for eight weeks. Highest (p < 0.05) weight gain rate was obtained in fish group supplemented with 200 mg kg-1 of GLE. The results showed that superoxide dismutase, glutathione-S-transferase, glutathione peroxidase, glutathione reductase, lysozyme, and complement C3 decreased, while malondialdehyde level increased in the liver and spleen upon LPS-challenge. Dietary GLE supplementation (especially 200 or 250 mg kg-1) alleviated LPS-induced changes. Similarly, GLE (150-250 mg kg-1) reversed LPS-induced alteration of serum biochemical parameters such as alkaline phosphatase, aspartate transaminase, alanine transaminase, and myeloperoxidase. LPS treatment markedly induced increased the mRNA levels of TNF-α, IL-1ß, and NF-κB p65 in both the liver and kidney tissues; however, GLE pre-treatment attenuated LPS-induced elicitation of TNF-α, IL-ß, and NF-κB p65. Moreover, dietary GLE supplementation significantly increased the expression of HSP70 and HSP90, and glucocorticoid receptor in the liver and kidney after LPS challenge. Thus, GLE attenuated LPS-induced inflammation response by up-regulating glucocorticoid receptor-related gene expression in carp. Finally, GLE supplementation reduced carp mortality after LPS-challenge. These results suggest that dietary supplementation with 200 mg kg-1 GLE is adequate for effectively attenuating LPS-induced oxidative stress and immune-suppressive effects in C. carpio.

Antioxidant, antibacterial, and anti-adhesive activities of biosurfactants isolated from Bacillus strains.

Biosurfactants are surface-active compounds that display a range of physiological functions. The present study investigated the antioxidant, antimicrobial, and anti-adhesive or anti-biofilm potential of biosurfactants isolated from Bacillus subtilis VSG4 and Bacillus licheniformis VS16. The antioxidant activity of the biosurfactants was studied in vitro using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl radicals. At 5 mg/mL of the biosurfactant concentration, the scavenging of DPPH and hydroxyl radicals was found to be between 69.1-73.5% and 63.3-69.8%, respectively. The biosurfactants also displayed significant antibacterial activities against both Gram-positive and Gram-negative bacteria. The anti-adhesive activities of the biosurfactants were evaluated against Staphylococcus aureus ATCC 29523, Salmonella typhimurium ATCC 19430, and Bacillus cereus ATCC 11778. The biosurfactants exhibited anti-adhesive activity, even at concentrations of 3-5 mg/mL. Moreover, both biosurfactants displayed notable anti-biofilm activities with a biofilm eradication percentage ranging from 63.9 to 80.03% for VSG4 biosurfactant, and from 61.1-68.4% for VS16 biosurfactant. Furthermore, VSG4 biosurfactant exhibited emulsification and surface tension stability over a wide range of pH (4-10) and temperature up to 100 °C. These results show that VSG4 and VS16 biosurfactants can be potentially used as natural antioxidants, antimicrobials, and/or anti-adhesive agents for food and biomedical applications.

Effects of bioactive substance from turmeric on growth, skin mucosal immunity and antioxidant factors in common carp, Cyprinus carpio.

Present study evaluated the effects of curcumin, the principal curcuminoid of turmeric, on Cyprinus carpio growth, skin mucosal immune parameters, immune-related gene expression, and susceptibility to pathogen challenge. Diets containing four various concentrations of curcumin (g Kg-1): 0 g [basal diet], 5 g [T5], 10 g [T10], and 15 g [T15] were fed to the carp (average weight: 16.37 g) for 8 weeks. Growth parameters were analysed at 4 and 8 weeks post-feeding. Skin mucosal immune responses and expression were examined in 8 weeks post-feeding. Growth performance was significantly higher in T10 and T15, with final weight gain of 102.26 ±â€¯2.31 g and specific growth rate of 3.24 ±â€¯0.37, respectively. The lowest feed conversion ratio (2.35 ±â€¯0.16) was recorded in T15 than in the control (P < 0.05). Among the skin mucosal immune parameters examined, lysozyme (36.8 ±â€¯4.03 U mL-1), total immunoglobulin (6.74 ±â€¯0.5 mg mL-1), protein level (18.7 ±â€¯1.62 mg mL-1), alkaline phosphatase (96.37 ±â€¯6.3 IU L-1), and protease activity (9.47 ±â€¯0.82%) were significantly higher in T15, while the peroxidase activity was higher in T10 (10.24 ±â€¯0.9 U mg-1 protein). Further, lysozyme, superoxide dismutase (SOD) and catalase (CAT) activities were measured in serum and found to be higher in T10 or T15 than in the control (P < 0.05). However, malondialdehyde level decreased significantly in T10 and T15. Furthermore, antioxidant genes (SOD, CAT, nuclear factor erythroid 2-related factor 2) and anti-inflammatory cytokine Interleukin-10 were upregulated in the head kidney, intestine, and hepatopancreas of fish in T10 and T15. Conversely, expression of pro-inflammatory cytokines (IL-1ß, tumour necrosis factor-alpha), signalling molecule NF-κBp65 were down-regulated in the tested tissues of T10 and T15. Expression of Toll-like receptor 22 (TLR22) was down regulated in head-kidney and intestine of T15. Fish from T15 exhibited significantly higher relative post-challenge survival (69.70%) against Aeromonas hydrophila challenge. Results of the present study suggest that dietary supplements of curcumin at 15 g Kg-1can significantly improve the growth performance, skin mucosal and serum antioxidant parameters, and strengthen the immunity of C. carpio. Therefore, curcumin represents a promising food additive for carps in aquaculture.

Heat-killed whole-cell products of the probiotic Pseudomonas aeruginosa VSG2 strain affect in vitro cytokine expression in head kidney macrophages of Labeo rohita.

Present study was undertaken to investigate the efficiency of heat-killed whole-cell products (HKWCPs) of probiotic Pseudomonas aeruginosa VSG2 strain in stimulating the cytokine responses in the head kidney (HK) macrophages of Labeo rohita. The HK macrophages were incubated with HKWCPs or lipopolysaccharide (LPS), and the responses of cytokine genes, namely interleukin-10 (IL-10), IL-1ß, IL-p35, IL-12p40, tumour necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), cyclooxygenase-2 (COX-2), interferon-alpha (IFN-α), and interferon-gamma (IFN-γ) were assessed by quantitative real-time PCR (qRT-PCR) at 2, 8, 16, 24, 48, 72 h post-stimulation (hps). Among proinflammatory cytokines, significantly higher expression of IL-1ß and TNF-α was observed at 8-24 hps, and 2-16 hps with HKWCPs, respectively, as compared to controls. However, COX-2 and NF-κB displayed strong expression (P < 0.05) at 2-8 hps, and 8, 16 and 72 hps with HKWCPs, respectively. Antiviral cytokines IFN-α and IFN-γ displayed strong expression (P < 0.05) at 8-24 hps, and 2, 24 and 48 hps with HKWCPs, respectively. Expressions of cell-mediated immune factor genes (IL-12p35 and IL-12p40) were also significantly upregulated at various time points, except IL-12p40 at 72 hps, in HK macrophages stimulated with HKWCPs. Expression of the anti-inflammatory cytokine IL-10 was upregulated (P < 0.05) at 2-24 hps HKWCPs, compared to controls. Enhanced cellular (phagocytic activity and superoxide anion production) and humoral (lysozyme) immune parameters of treated HK macrophages confirmed the induction of inflammatory response. Thus, our results indicated that HKWCPs of probiotic P. aeruginosa VSG2 had greater potential for stimulating the in vitro expression of cytokines in fish and that these HKWCPs may be used as vaccine adjuvants in aquaculture.

Flavonoid fraction of guava leaf extract attenuates lipopolysaccharide-induced inflammatory response via blocking of NF-κB signalling pathway in Labeo rohita macrophages.

Psidium guajava L. is a well-known traditional medicinal plant widely used in folk medicine. To explore the anti-inflammatory activity of the flavonoid fraction of guava leaf extract (FGLE), we investigated its ability to suppress the levels of inflammatory mediators elevated by lipopolysaccharide (LPS) in Labeo rohita head-kidney (HK) macrophages. HK macrophages of L. rohita were treated with LPS in the presence or absence of the FGLE. We examined the inhibitory effect of FGLE on LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production. The inhibitory effect of FGLE on nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) were investigated by RT-PCR and western blot. The effect of FGLE on proinflammatory cytokines tumour necrosis factor alpha (TNF-α) or interleukin-1ß (IL-1ß) was also investigated by ELISA and RT-PCR. The phosphorylation of three mitogen activated protein kinases (MAPK) molecules ERK, JNK and p38 was analysed by western blot analysis. FGLE inhibited LPS-induced NO and PGE2 production. It also effectively inhibited TNF-α, IL-1ß, IL-10, iNOS, and COX-2 production in a concentration-dependent manner. In addition, FGLE suppressed the mRNA expression levels of TNF-α and IL-1ß in LPS-stimulated HK macrophages. RT-PCR and western blot analysis showed that FGLE decreased both the mRNA and protein expression levels of LPS-induced iNOS and COX-2 in HK macrophages. FGLE suppresses the phosphorylation of MAPK molecules in LPS-stimulated HK macrophages. FGLE also significantly inhibited LPS-induced NF-κB transcriptional activity. The molecular mechanism by which FGLE suppresses the expression of inflammatory mediators appears to involve the inhibition of NF-κB activation, through the suppression of LPS-induced IκB-α degradation. Together these results suggest that FGLE contains potential therapeutic agent(s), which regulate NF-κB activation, for the treatment of inflammatory conditions in L. rohita macrophages.

Effects of intracellular products of Bacillus subtilis VSG1 and Lactobacillus plantarum VSG3 on cytokine responses in the head kidney macrophages of Labeo rohita.

The efficiency of intracellular products (ICPs) of the probiotics Bacillus subtilis VSG1 and Lactobacillus plantarum VSG3 in stimulating cytokine responses in the head kidney (HK) macrophages of Labeo rohita was investigated. The HK macrophages were incubated with ICPs and lipopolysaccharide (LPS), and the responses of cytokine genes, namely interleukin-10 (IL-10), IL-1ß, IL-12p35, IL-12p40, IL-18, tumour necrosis factor-α (TNF-α), nuclear factor kappa B (NF-κB), cyclo-oxygenase-2 (COX-2), interferon-1 (IFN-1), and IFN-γ were assessed by quantitative real-time PCR (qRT-PCR) at 2, 6, 12, 24, and 48 h post-stimulation (hps). Among the proinflammatory cytokines, a strong increase in the gene expression of IL-1ß and TNF-α was displayed mainly at 2-6 hps with ICPs, as compared to that of the positive control (LPS) or the negative control (PBS) (P < 0.05). However, COX-2 and NF-κB showed higher expression at 2 and 24 hps, and 6-24 hps with ICPs, respectively. Antiviral cytokines IFN-1 and IFN-γ displayed strong expressions (P < 0.05) at 6-12 hps, and 12-24 hps with ICPs, respectively. Upregulation of the anti-inflammatory cytokine, IL-10, was recorded at 6-24 hps with ICPs, as compared to that controls. Expressions of cell-mediated immune factor genes (IL-12p35, IL-12p40, and IL-18) were also significantly upregulated at different time points, except 48 hps, in HK macrophages stimulated with ICPs. Furthermore, enhanced cellular (phagocytic activity and nitroblue tetrazolium assay) and humoral (lysozyme) immune parameters in stimulated cells confirmed the induction of the inflammatory response. Therefore, the results of this in vitro study indicate that the ICPs of B. subtilis VSG1 or L. plantarum VSG3 have great potential for stimulating the cytokine responses in fish, and are thereby potential immunostimulants to fish. Further studies could be conducted to explore its suitability as an adjuvant vaccine in aquaculture.

Effect of cellular products of potential probiotic bacteria on the immune response of Labeo rohita and susceptibility to Aeromonas hydrophila infection.

In the present study, the immunological efficacy of cellular components from the potential probiotic bacteria Bacillus subtilis VSG1, Pseudomonas aeruginosa VSG2, and Lactobacillus plantarum VSG3 was evaluated in Labeo rohita fingerlings. Fish were immunized intraperitoneally with 0.1 mL phosphate-buffer solution (PBS) containing 0.1 mg of any of the following cellular components: intercellular products (ICPs) of VSG1 (BS-ICPs), heat-killed whole cell products of VSG2 (PA-HKWCPs), or ICPs of VSG3 (LP-ICPs). Fish injected with 0.1 mL PBS served as the control. Various immunological parameters, including the expression of immune-related genes, were measured 14 and 21 days post-immunization. The fish were challenged with Aeromonas hydrophila and mortality was recorded up to 21 days post-infection. The results revealed that administration of cellular components significantly increased the activity of serum lysozyme and the alternative complement pathway, phagocytosis, and respiratory bursts throughout the experimental period. Total serum protein, albumin, and globulin levels were significantly higher in experimental groups than in the control up to 14 days post-immunization, and decreased thereafter. With respect to immune-related genes, IL-1ß, COX-2, iNOS, and IL-10 were highly (P < 0.05) up-regulated in fish immunized with cellular components, compared to the control. The expression of TNF-α and NF-κB was up-regulated in immunized fish up to 14 days post-immunization. Interestingly, fish immunized with LP-ICPs exhibited a significantly higher post-challenge relative percent survival (83.32%), followed by PA-HKWCPs (66.66%), and BS-ICPs (50%). These results indicate that cellular components of probiotic bacteria can influence immune responses, enhance disease protection, and stimulate immune-related gene expression in rohu. Hence, these cellular components may be useful as adjuvants for vaccines in aquaculture.

Effect of guava leaves on the growth performance and cytokine gene expression of Labeo rohita and its susceptibility to Aeromonas hydrophila infection.

The present study aimed to evaluate the effects of Psidium guajava L. (guava) leaves on the growth and immune response of the fish species Labeo rohita and its susceptibility to Aeromonas hydrophila infection. Diets containing five different concentrations of guava leaves (0% [basal diet], 0.1% [G1], 0.5% [G2], 1% [G3], and 1.5% [G4]) were fed to fish (average weight: 11.1 g) for 60 days. Various growth and immune parameters were examined 60 days post-feeding. Fish were challenged with A. hydrophila at the end of the trial, and mortalities were recorded over 15 days post-infection. We found that growth parameters such as percent weight gain (657.61 ± 9.74) and specific growth rate (3.37 ± 0.021) were significantly higher in G2 group than in the control (P < 0.05). Among the immune parameters examined, lysozyme levels (79.5 ± 5.1 U mL(-1)), leukocyte phagocytic activity (52 ± 4.3%), and alternative complement pathway activity (ACP) (186.1 ± 8.3 U mL(-1)) were significantly high (P < 0.05) in G2 fed group; there was, however, no significant effect of guava leaves at any concentration on plasma IgM level. Of the cytokine-related genes examined, interleukin-1beta (IL-1ß) and tumour necrosis factor-alpha (TNF-α) were up-regulated in the head-kidney, intestine, and hepatopancreas of fish fed experimental diets, and expression was significantly higher in G2 and G3 than in the control group. In contrast, gene expression of IL-10, transforming growth factor-beta (TGF-ß), inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and transcription factor nuclear factor-κB (NF-κB) were down-regulated in the treatment groups. Moreover, fish fed the G2 diet exhibited a significantly higher post-challenge survival rate (66.66%). Collectively, these results suggest that dietary supplementation with guava leaves (at 0.5% concentration) could promote growth performance and strengthen immunity of L. rohita. Guava leaves therefore represent a promising feed additive for carps in aquaculture.

Production and characterization of a thermostable bioflocculant from Bacillus subtilis F9, isolated from wastewater sludge.

A bacterium isolated from wastewater sludge, identified as Bacillus subtilis F9, was confirmed to produce bioflocculant with excellent flocculation activity. The effects of culture conditions such as initial pH, temperature, carbon source, nitrogen source, and inoculum size on bioflocculant production were studied here. The results indicated that 2.32g/L of purified bioflocculant could be extracted with the following optimized conditions: 20gL(-1) sucrose as the carbon source, 3.5gL(-1) peptone as the nitrogen source, an initial pH of 7.0, and a temperature of 40°C. The purified bioflocculant consisted of 10.1% protein and 88.3% sugar, including 38.4% neutral sugar, 2.86% uronic acid, and 2.1% amino sugar. The neutral sugar consisted of sucrose, glucose, lactose, galactose, and mannose at a molar ratio of 2.7:4.7:3.2:9.1:0.8. Elemental analysis of the purified bioflocculant revealed that the weight fractions of carbon, hydrogen, oxygen, nitrogen, and sulfur were 30.8%, 5.3%, 54.7%, 6.4%, and 2.9%, respectively. Furthermore, the purified bioflocculant was pH tolerant within the range of 2-8 and thermotolerant from 10°C to 100°C, with optimal activity at pH 7.0 and at a temperature of 40°C. The purified bioflocculant showed industrial potential for the treatment of drinking water. Considering these properties, especially its low molecular weight (5.3×10(4)Da), this bioflocculant with excellent solubility and favorable flocculation activity is particularly suited for flocculating small particles.

Role of HSP70 in cytoplasm protection against thermal stress in rohu, Labeo rohita.

To understand the function of HSP70 of Labeo rohita (LrHSP70) in cellular protection, LrHSP70 ORF cDNA was inserted into the plasmid of pET-32a(+) or pEGFP-L1. Then, the recombinant plasmids were transformed or transfected into Escherichia coli cells, mouse myeloma cells (MPC-11) or fish hepatoma cells (PLHC-1). Western blot results revealed that LrHSP70 was expressed in E. coli cells and molecular weight was estimated to be 70 kDa. In cells, LrHSP70 was over-expressed following thermal or cold stress. Results revealed that LrHSP70 protected prokaryotic cells against thermal or cold extremes as well as played the same role in MPC-11 and PLHC-1 cells. After heat treatment at 42 °C for 1 h, the viability of the cell was declined considerably. PLHC-1 cells with pEGFP-L1/LrHSP70 exhibited a higher survival rate (50%) than wild-type cells (18%) or cells with only pEGFP-L1 (21.2%). When the time lag extended to 2 h, the survival rates were 30%, 3.4% and 5.3% respectively. The present study revealed that LrHSP70 plays an important role in response to thermal and cold stress in fish.

Potential probiotic Lactobacillus plantarum VSG3 improves the growth, immunity, and disease resistance of tropical freshwater fish, Labeo rohita.

The study evaluated the effects of dietary doses of Lactobacillus plantarum VSG3 on the growth performance, immunity, and disease resistance of Labeo rohita juveniles against Aeromonas hydrophila infection. Fish (mean body weight 60 g) were fed with diet containing 0 (control), 10(6), 10(8), 10(10) cfu g(-1)L. plantarum VSG3 for 60 days. Various growth and immune parameters were examined at 30 and 60 days of post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 10 days post-infection. Results showed that administration of VSG3 for 60 days had significant effects (P < 0.05) on the specific growth rate (SGR) and feed utilization efficiency of L. rohita. Dietary administration of L. plantarum VSG3 significantly increased the serum lysozyme and alternative complement pathway (ACP) activities, phagocytosis and respiratory burst activity in L. rohita throughout the experimental period. The highest superoxide dismutase (SOD) activity (P < 0.05) was observed in the fish group fed diet containing VSG3 at 10(8) cfu g(-1). The serum IgM levels were significantly higher in the experimental groups compared to the control group after 30 days of feeding; but, the result was opposite after 60 days of feeding. Further, fish fed the diet containing 10(8) cfu g(-1)L. plantarum VSG3 had significantly higher (P < 0.05) post-challenge survival rate (77.7%). These results collectively suggest that dietary supplementation of L. plantarum VSG3 at 10(8) cfu g(-1) to L. rohita is optimal for enhancing the growth, immunity, and disease resistance against A. hydrophila infection.

Immobilization and characterization of carbonic anhydrase purified from E. coli MO1 and its influence on CO2 sequestration.

The present investigation entails the immobilisation and characterisation of Escherichia coli MO1-derived carbonic anhydrase (CA) and its influence on the transformation of CO2 to CaCO3. CA was purified from MO1 using a combination of Sephadex G-75 and DEAE cellulose column chromatography, resulting in 4.64-fold purification. The purified CA was immobilised in chitosan-alginate polyelectrolyte complex (C-A PEC) with an immobilisation potential of 94.5 %. Both the immobilised and free forms of the enzyme were most active and stable at pH 8.2 and at 37 °C. The K(m) and V(max) of the immobilised enzyme were found to be 19.12 mM and 416.66 µmol min⁻¹ mg⁻¹, respectively; whereas, the K(m) and V(max) of free enzyme were 18.26 mM and 434.78 µmol min⁻¹ mg⁻¹, respectively. The presence of metal ions such as Cu²âº, Fe²âº, and Mg²âº stimulated the enzyme activity. Immobilised CA showed higher storage stability and maintained its catalytic efficiency after repeated operational cycles. Furthermore, both forms of the enzyme were tested for targeted application of the carbonation reaction to convert CO2 to CaCO3. The amounts of CaCO3 precipitated over free and immobilised CA were 267 and 253 mg/mg of enzyme, respectively. The results of this study show that immobilised CA in chitosan-alginate beads can be useful for CO2 sequestration by the biomimetic route.

Contact angle measurement system with liquid sodium in inert atmosphere.

Liquid sodium is the heat transport medium in fast breeder reactors due to its favorable chemical, physical, and nuclear properties. Wetting of liquid sodium with the material of reactor components is essential for the effective functioning of the reactor. Liquid sodium wetting plays a crucial role, such as in (i) heat transfer and (ii) image reconstruction under sodium ultrasonic scanners. Contact angle is the inverse measure of the wetting of liquids over solid surfaces. A contact angle measurement system was set up in an inert atmosphere glow box to study the wetting behavior of liquid sodium on reactor materials. The contact angle of liquid sodium on nickel surface was measured as a function of temperature, and the results obtained were found to be in good agreement with the literature values. The contact angle of liquid sodium on SS316LN as a function of temperature was measured for the first time using this facility.

Effects of dietary supplementation of potential probiotic Pseudomonas aeruginosa VSG-2 on the innate immunity and disease resistance of tropical freshwater fish, Labeo rohita.

The effects of dietary Pseudomonas aeruginosa VSG-2 supplementation on innate immunity and protection against Aeromonas hydrophila infection were evaluated in Labeo rohita. Fish were fed for 60 days with control diet or 3 experimental diets containing P. aeruginosa VSG-2 at 10(5), 10(7), and 10(9) cfu g(-l), respectively. Various innate immune parameters were examined at 30 and 60 days post-feeding. Fish were challenged with A. hydrophila 60 days post-feeding and mortalities were recorded over 10 days post-infection. Dietary supplementation of P. aeruginosa VSG-2 significantly increased serum lysozyme and alternative complement pathway (ACP) activities, phagocytosis, and respiratory burst activity in head kidney macrophages of L. rohita throughout the experimental period. Superoxide dismutase (SOD) activity significantly increased after 60 days in the groups fed diets containing 10(7) and 10(9) cfu g(-1) P aeruginosa. Serum IgM levels were significantly higher in the treatment groups than in the control group after 30 days of feeding; however, the opposite result was observed at 60 days. Moreover, fish fed diets containing 10(7) and 10(9) cfu g(-1)P. aeruginosa had significantly higher post-challenge survival rates against A. hydrophila infection. Further, P. aeruginosa VSG-2 was found to be safe for mammals. These results indicate that dietary P. aeruginosa VSG-2 supplementation at 10(7) cfu g(-1) can effectively improve innate immunity and disease resistance in L. rohita.

Immobilization of carbonic anhydrase enzyme purified from Bacillus subtilis VSG-4 and its application as CO(2) sequesterer.

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan-alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co(2+), Cu(2+), and Fe(3+), increased the enzyme activity, whereas CA activity was inhibited by Pb(2+), Hg(2+), ethylenediamine tetraacetic acid (EDTA), 5,5'-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO(2) to CaCO(3). The maximum CO(2) sequestration potential was achieved with immobilized CA (480 mg CaCO(3)/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO(2) sequestration.

Characterisation of Lactic Acid Bacteria Isolated from the Gut of Cyprinus carpio That May Be Effective Against Lead Toxicity.

The present study was conducted to isolate and characterise Pb-resistant lactic acid bacteria (LAB), and thus determine their potential for use as probiotics against Pb toxicity. A total of 107 Pb-resistant LAB strains were isolated from the gut content of Cyprinus carpio, of which 41 were established to be gram-positive and catalase-negative. Investigation of the Pb-binding ability of these isolated LAB identified seven strains (P2, P6, P7, P9, P16, P19 and P22) with comparatively high Pb-binding activities. These were selected for further screening to establish their Pb tolerance, anti-oxidative capacity and in vitro probiotic characteristics. Strain P16 exhibited both the highest Pb-binding and a relatively good antioxidant capacity. Furthermore, P16 displayed a high survival rate during 4 h of exposure to both low-pH (2.5-3.5) conditions and 10.0% fish bile, and a strong capacity to adhere to fish intestinal mucus (62.4%). Furthermore, P16 showed strong antibacterial activities against all tested fish pathogens. Strains P6, P9, P16, P19 and P22 were susceptible to a range of tested antibiotics, but not to vancomycin. Thus, of the isolated lactobacilli, strain P16 exhibited the best Pb-binding ability, a high level of antioxidant activity and satisfactory in vitro probiotic properties. Biochemical and 16S-rRNA gene analyses identified P16 as Lactobacillus reuteri. Thus, the results of the conducted in vitro tests suggest that the fish-associated P16 Lact. reuteri strain is a promising candidate probiotic, which should undergo further investigation to assess its suitability for use in protecting against lead-exposure-induced toxicities in aquaculture.

Corrigendum: Therapeutic Effect of Intestinal Autochthonous Lactobacillus reuteri P16 Against Waterborne Lead Toxicity in Cyprinus carpio.

[This corrects the article DOI: 10.3389/fimmu.2018.01824.].

Therapeutic Effect of Intestinal Autochthonous Lactobacillus reuteri P16 Against Waterborne Lead Toxicity in Cyprinus carpio.

Harmful effects of heavy metals are myriad. Lead (Pb) from soil and atmosphere contaminates water bodies and affects the aquatic animals. Our previous study confirmed the in vitro probiotic potential of Lactobacillus reuteri against Pb toxicity, but further investigation is necessary for gaining insights into the related protection mode. Therefore, in this study, we investigated the protective effects of the potential probiotic L. reuteri P16 against waterborne Pb exposure-induced toxicity in the freshwater fish Cyprinus carpio. Fish (average weight: 23.16 ± 0.73 g) were allocated to four groups (control, Pb only, Pb + L. reuteri P16, and L. reuteri P16 only) and Pb groups were exposed to waterborne Pb (1 mg L-1) for 6 weeks. L. reuteri P16 (108 CFU g-1) supplemented diet was provided twice daily. Growth performances, hemato-biochemical parameters, innate immune responses, intestinal microbiota, and Pb accumulation in tissues were measured at the end of the trial. When the fish were exposed to Pb, dietary supplementation of L. reuteri P16 effectively decreased mortality and accumulation of Pb in tissues, and improved the growth performance. Co-treatment with Pb and L. reuteri P16 alleviated Pb exposure-induced oxidative stress, reversed alterations in hemato-biochemical parameters, improved innate immune parameters, and restored intestinal enzymatic activities. Moreover, L. reuteri P16 supplementation reversed the changes in intestinal microbiota in Pb-exposed fish. Furthermore, Pb exposure decreased the expressions of pro-inflammatory cytokines (TNF-α, IL-1ß). However, the expression of heat shock proteins (HSP70 and HSP90) increased, which might have increased the cellular stress. Interestingly, the Pb-induced alterations of gene expressions were reversed by L. reuteri P16 supplementation. Thus, dietary administration of the potential probiotic L. reuteri P16 had several beneficial effects on growth performance and immune responses, decreased Pb accumulation in tissues, and reversed alterations in hematological responses of C. carpio. Furthermore, it offered direct protection against Pb-induced oxidative stress. Therefore, L. reuteri P16 may be a novel dietary supplement for enhancing growth performance and preventing Pb-exposure-induced toxicity in fish in aquaculture and aquatic products.

Role of Bacillus licheniformis VS16-Derived Biosurfactant in Mediating Immune Responses in Carp Rohu and its Application to the Food Industry.

Multifarious applications of Bacillus licheniformis VS16-derived biosurfactant were explored. Labeo rohita fingerlings were injected intraperitoneally with 0.1 mL of phosphate-buffered saline (PBS) containing purified biosurfactant at 0 (control), 55 (S55), 110 (S110), 220 (S220), or 330 (S330) µg mL-1 concentrations. Various immunological parameters and the expression of immune-related genes were measured at 7, 14, and 21 days post-administration (dpa). At 21 dpa, fish were challenged with Aeromonas hydrophila and mortality was recorded for 14 days. Immune parameters such as lysozyme levels (39.29 ± 2.14 U mL-1), alternative complement pathway (61.21 ± 2.38 U mL-1), and phagocytic activities (33.37 ± 1.2%) were maximum (P < 0.05) in the S220 group at 14 dpa; but immunoglobulin levels (11.07 ± 0.83 mg mL-1) were highest in the S220 group at 7 dpa, compared to that in controls. Activities of digestive enzymes (amylase, protease, and lipase) were higher (P < 0.05) in the S220 and S330 groups than in the control group. Regarding cytokine gene expression, pro-inflammatory cytokines (TNF-α and IL-1ß) were down-regulated (P < 0.05) in the S220 and S330 groups. Expression of IL-10, TGF-ß, and IKB-α were up-regulated in the S220 and S330 groups at 14 dpa, with the highest levels in the S220 group. The expression of NF-κB p65 and IKK-ß were down-regulated in treatment groups, and were lowest (P < 0.05) in the S220 group. The highest post-challenge survival rate (72.7%) was recorded in S220 group. Further, the potential of this substance to inhibit biofilm formation, and heavy metal removal from vegetables were also evaluated. Biosurfactant was effective in inhibiting biofilm formation up to 54.71 ± 1.27%. Moreover, it efficiently removed cadmium (Cd) from tested vegetables such as carrot, radish, ginger, and potato, with the highest removal efficiency (60.98 ± 1.29%) recorded in ginger contaminated with Cd. Collectively, these results suggest that isolated biosurfactant could be used in the aquaculture industry, in addition to its potential application to the food industry.

Spectral characteristics and nonlinear studies of crystal violet dye.

Solid-state dye-doped polymer is an attractive alternative to the conventional liquid dye solution. In this paper, the spectral characteristics and the nonlinear optical properties of the dye crystal violet are studied. The spectral characteristics of crystal violet dye doped poly(methylmethacrylate) modified with additive n-butyl acetate (nBA) are studied by recording its absorption and fluorescence spectra and the results are compared with the corresponding liquid mixture. The nonlinear refractive index of the dye in nBA and dye doped polymer film were measured using z-scan technique, by exciting with He-Ne laser. The results obtained are intercompared. Both the samples of dye crystal violet show a negative nonlinear refractive index. The origin of optical nonlinearity in the dye may be attributed due to laser-heating induced nonlinear effect.