Shib Sankar Sen

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.

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-1β, 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 dietary leucine on the growth parameters and expression of antioxidant, immune, and inflammatory genes in the head kidney of Labeo rohita fingerlings

The present investigation evaluated the effects of dietary leucine (Leu) on growth performance, head kidney antioxidant status, and gene expression in Labeo rohita juveniles. Fish were fed with six isonitrogenous diets containing graded levels of Leu at 0.75 (control), 1.7, 3.2, 4.6, 6.3, and 7.6 g Leu kg(-1) of feed for 8 weeks. Compared with the control group, appropriate Leu supplementation significantly enhanced the percent weight gain (PWG), feed intake (FI), and protein efficiency ratio (PER) (P<0.05) but decreased the plasma ammonia content (PAC) (P<0.05). Similarly, optimal Leu supplementation stimulated head kidney glutathione (GSH) content, and superoxide dismutase (SOD) and glutathione peroxidase (GPx) activities as compared to the control group; however, a reverse trend was observed in malondialdehyde (MDA) content. Further, relative gene-expression levels of lysozyme, complement C3, β-microglobulin, immunoglobulin-M, SOD, GPx, nuclear factor erythroid 2-related factor 2 (Nrf2), natural killer-cell enhancing factor β (NKEF-β), and toll-like receptor-22 (TLR22) in the head kidney were enhanced (P<0.05) at leucine levels of 4.6 g kg(-1) of feed. Conversely, the mRNA levels of tumour necrosis factor-α (TNF-α), Kelch-like-ECH-associated protein 1 (Keap1), and interleukin 1β (IL-1β) in head kidney were down-regulated by Leu supplementation. Collectively, our results revealed that appropriate Leu supplementation improved fish growth and antioxidant capacity, and regulated the mRNA levels of related signalling molecules in L. rohita juveniles. Based on the quadratic regression analysis of PWG, PER, and PAC, the optimum dietary leucine requirements of L. rohita juveniles were estimated to be 4.7, 4.5, and 4.8 g kg(-1) of feed.

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-1β, 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-1β 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.

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-1β, 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-1β 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.

Use of a Potential Probiotic, Lactobacillus plantarum L7, for the Preparation of a Rice-Based Fermented Beverage

This study aimed to isolate potential probiotic lactic acid bacteria from a traditional rice-based fermented beverage “bhaati jaanr” and to evaluate their role during preparation of the beverage. Among various isolates, Lactobacillus plantarum strain L7 exhibited satisfactory in vitro probiotic characteristics such as acid resistance and bile tolerance, cell surface hydrophobicity, auto-aggregation, antibiotic susceptibility, and antimicrobial activities. Therefore, performance of L7 as a starter culture in rice fermentation was determined during a 6-day rice fermentation study. L. plantarum L7 decreased the pH, associated with an increase in total titratable acidity and organic acid production up to the 4th day of fermentation. The highest concentrations of succinic acid (0.37 mg/g), lactic acid (4.95 mg/g), and acetic acid (0.36 mg/g) were recorded on the 3rd, 4th, and 5th days of fermentation, respectively. Saccharifying (148.13 μg/min g−1) and liquefying (89.47 μg/min g−1) activities were the highest on days 3 and 2, respectively, and thereafter, they decreased. Phytase activity and the cleavage of free minerals (sodium, calcium, magnesium, manganese, and ferrous) increased up to days 3–4. The concentration of various accumulated malto-oligosaccharides (glucose, fructose, maltotriose, and maltoterose) was noted to be the maximum on days 4 and 5. Furthermore, gas chromatography-mass spectrometry analysis indicated the presence of various volatile compounds. The fermented material also exhibited 1,1-diphenyl-2-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging activity. Therefore, the probiotic, L. plantarum L7, has a significant role in the fermentation of this beverage and enhances its functional properties.

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-1β) were down-regulated (P < 0.05) in the S220 and S330 groups. Expression of IL-10, TGF-β, 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-β 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.

Protective effects of leucine against lipopolysaccharide-induced inflammatory response in Labeo rohita fingerlings

The present study investigated the protective effects of leucine against lipopolysaccharide (LPS)-induced inflammatory responses in Labeo rohita (rohu) in vivo and in vitro. Primary hepatocytes, isolated from the hepatopancreas, were exposed to different concentrations of LPS for 24 h to induce an inflammatory response, and the protective effects of leucine against LPS-induced inflammation were studied. Finally, we investigated the efficiency of dietary leucine supplementation in attenuating an immune challenge induced by LPS in vivo. Exposure of cells to 10-25 μg mL(-1) of LPS for 24 h resulted in a significant production of nitric oxide and release of lactate dehydrogenase to the medium, whereas cell viability and protein content were reduced (p < 0.05). LPS exposure (10 μg mL(-1)) increased mRNA levels of the pro-inflammatory cytokines TNF-α, IL-1β and IL-8 in vitro (p < 0.05). However, pretreatment with leucine prevented the LPS-induced upregulation of TNF-α, IL-1β and IL-8 mRNAs by downregulating TLR4, MyD88, NF-κBp65, and MAPKp38 mRNA expression. Interestingly, mRNA expression of the anti-inflammatory cytokine, IL-10, which was increased by LPS treatment, was further enhanced (p < 0.05) by leucine pretreatment. The enhanced expression of IL-10 might inhibit the production of other pro-inflammatory cytokines. It was found that leucine pretreatment attenuated the excessive activation of LPS-induced TLR4-MyD88 signaling as manifested by lower level of TLR4, MyD88, MAPKp38, NF-κBp65 and increased level of IκB-α protein in leucine pre-treatment group. In vivo experiments demonstrated that leucine pre-supplementation could protect fish against LPS-induced inflammation through an attenuation of TLR4-MyD88 signaling pathway. Taken together, we propose that leucine pre-supplementation decreases LPS-induced immune damage in rohu by enhancing the expression of IL-10 and by regulating the TLR4-MyD88 signaling pathways.

Use of a Potential Probiotic, Lactobacillus casei L4, in the Preparation of Fermented Coconut Water Beverage

Coconut water (CW) is a clear, nutritive liquid found as the coconut endosperm of green coconuts such as Cocos nucifera L., and its widespread consumption owes to its unique composition of sugars, minerals, vitamins, enzymes, and hormones. Probiotic fermentation of CW may facilitate the development of an improved functional beverage with probiotic benefits; therefore, we aimed to produce a fermented CW beverage using the potential probiotic Lactobacillus casei L4. CW was fermented with L. casei L4 for 48 h at 35°C, and the pH, organic acid-production rate, antioxidant activity, antibacterial activity, sugar, mineral, vitamin B12 levels, and total viable bacteria counts were investigated at 24 and 48 h. We demonstrated that the fermentation of CW with probiotic lactobacilli increased the cell viability count. Vitamin B12 production was highest in the extracellular environment at 48 h (11.47 μg/mL), while the total phenolic content was significantly (p < 0.05) higher in the fermented CW at 48 h (72.1 μg/mL gallic acid equivalents) than observed with the other investigated groups or time points. The fermented materials exhibited the highest 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) radical-scavenging activities at 48 h (58.4 and 69.2%, respectively). The levels of most minerals remained unchanged in the fermented CW, except for calcium, manganese, phosphorus, and sodium. Furthermore, the culture supernatant from fermented CW inhibited the growth of foodborne pathogens such as Bacillus cereus, Listeria monocytogenes, Staphylococcus aureus, and Salmonella typhi, although the degree of inhibition varied between the species. Moreover, adding 15% honey and artificial coconut flavor to the fermented CW resulted in a better-tasting product, as demonstrated by a sensory-evaluation test. The obtained results indicated that the CW product fermented by L. casei L4 may be used as a novel functional beverage containing both electrolytes and probiotics, and can serve as a good vehicle for preparing a wider range of novel products.

Physiological Role of Heat Shock Proteins, Molecular Function and Stress Removal in Fishes

Depending on the size, the specific locations and physiological roles of molecular chaperones differ within the cell. However, to tolerate stress HSP drives molecular mechanisms in animals. These proteins interact with multiple systems in diverse ways regulated by the endocrine system. HSP have crucial physiological roles in fish. The important role of HSP is in thermo tolerance as well as tolerance to cytotoxic effects of environmental contaminants and other stressors which are non thermal. HSP enhances immune response through both intra- and extra-cellular activities. Similarly, many studies have proved that most HSP are molecular chaperones which funtion for other cell proteins, and have better cytoprotective effects. To know more about factors, both biotic and abiotic regulating heat shock proteins data has been collected rapidly. However, earlier reports are focused on the role of HSP in development and their importance in fish in nature. Functional genomic approaches will provide the tools necessary to gain a comprehensive understanding of the significance of heat shock proteins in the cellular stress response, in the physiological processes. Heat shock proteins are considerably adaptable and potent molecules, the significance of which to biological procedures is highlighted by the high degree to which their structure and function are phylogenetically preserved. Our knowledge regarding physiological role of heat shock proteins is presently partial; though, a better understanding of their function and thus the skills of the capacity to control their power may lead to their use as therapeutic agents.