Chromosome level genome assembly and transcriptome analysis of E11 cells infected by tilapia lake virus.

The E11 cell line, derived from striped snakehead fish (Channa striata), possesses a distinctive feature: it is persistently infected with a C-type retrovirus. Notably, it exhibits high permissiveness to piscine nodavirus and the emerging tilapia lake virus (TiLV). Despite its popularity in TiLV research, the absence of genome assembly for the E11 cell line and Channa striata has constrained research on host-virus interactions. This study aimed to fill this gap by sequencing, assembling, and annotating the E11 cell line genome. Our efforts yielded a 600.5 Mb genome including 24 chromosomes with a BUSCO score of 98.8%. In addition, the complete proviral DNA sequence of snakehead retrovirus (SnRV) was identified in the E11 cell genome. Comparative genomic analysis between the E11 cell line and another snakehead species Channa argus revealed the loss of many immune-related gene families in the E11 cell genome, indicating a compromised immune response. We also conducted transcriptome analysis of mock- and TiLV-infected E11 cells, unveiling new perspectives on virus-virus and host-virus interactions. The TiLV infection suppressed the high expression of SnRV in E11 cells, and activated some other endogenous retroviruses. The protein-coding gene comparison revealed a pronounced up-regulation of genes involved in immune response, alongside a down-regulation of genes associated with specific metabolic processes. In summary, the genome assembly and annotation of the E11 cell line provide valuable resources to understand the SnRV and facilitate further studies on nodavirus and TiLV. The RNA-seq profiles shed light on the cellular mechanisms employed by fish cells in response to viral challenges, potentially guiding the development of therapeutic strategies against TiLV in aquaculture. This study also provides the first insights into the viral transcriptome profiles of endogenous SnRV and evading TiLV, enhancing our understanding of host-virus interactions in fish.

Metabolomics captures the differential metabolites in the replication pathway of snakehead vesiculovirus regulated by glutamine.

Snakehead vesiculovirus (SHVV) is a negative-sense single-stranded RNA virus that infects snakehead fish. This virus leads to illness and mortality, causing significant economic losses in the snakehead aquaculture industry. The replication and spread of SHVV in cells, which requires glutamine as a nitrogen source, is accompanied by alterations in intracellular metabolites. However, the metabolic mechanisms underlying the inhibition of viral replication by glutamine deficiency are poorly understood. This study utilized liquid chromatography-mass spectrometry to measure the differential metabolites between the channel catfish Parasilurus asotus ovary cell line infected with SHVV under glutamine-containing and glutamine-deprived conditions. Results showed that the absence of glutamine regulated 4 distinct metabolic pathways and influenced 9 differential metabolites. The differential metabolites PS(16:0/16:0), 5,10-methylene-THF, and PS(18:0/18:1(9Z)) were involved in amino acid metabolism. In the nuclear metabolism functional pathway, differential metabolites of guanosine were observed. In the carbohydrate metabolism pathway, differential metabolites of UDP-d-galacturonate were detected. In the signal transduction pathway, differential metabolites of SM(d18:1/20:0), SM(d18:1/22:1(13Z)), SM(d18:1/24:1(15 Z)), and sphinganine were found. Among them, PS(18:0/18:1(9Z)), PS(16:0/16:0), and UDP-d-galacturonate were involved in the synthesis of phosphatidylserine and glycoprotein. The compound 5,10-methylene-THF provided raw materials for virus replication, and guanosine and sphingosine are related to virus virulence. The differential metabolites may collectively participate in the replication, packaging, and proliferation of SHVV under glutamine deficiency. This study provides new insights and potential metabolic targets for combating SHVV infection in aquaculture through metabolomics approaches.

Identification and characterization of immune-related microRNAs in hybrid snakehead(Channa maculata♀ × Channa argus♂)after treated by Echinacea purpurea (Linn.) Moench.

Echinacea purpurea (Linn.) Moench (EP) is a globally popular herbal medicine, which showed effects on growth promotion, antioxidant and immunomodulatory activities in fish culture world widely. However, there are few studies about the effects on miRNAs by EP in fish. The hybrid snakehead fish (Channa maculate♀ × Channa argus ♂) was new important economic specie of freshwater aquaculture in China with high market value and demand while there were only a few reports about its miRNAs. To overview immune-related miRNAs of the hybrid snakehead fish and to further understand the immune regulating mechanism of EP, we herein constructed and analyzed three small RNA libraries of immune tissues including liver, spleen and head kidney of the fish with or without EP treatment via Illumina high-throughput sequencing technology. Results showed that EP can affect the immune activities of fish by the miRNA-regulated ways. Totally, 67 (47 up and 20 down) miRNAs in liver, 138 (55 up and 83 down) miRNAs in spleen, and 251 (15 up and 236 down) miRNAs in spleen were detected, as well as 30, 60, 139 kinds of immune-related miRNAs belonging to 22, 35 and 66 families of the three tissues respectively. The expressions of 8 immune-related miRNA family members were found in all the three tissues, including miR-10, miR-133, miR-22 and etc. Some miRNAs have been identified involved in the innate and adaptive immune responses, such as the miR-125, miR-138, and miR-181 family. Ten miRNA families with antioxidant target genes were also discovered, including miR-125, miR-1306, and miR-138, etc. Results from Gene Ontology (GO) and KEGG pathway analysis further confirmed there are a majority immune response targets of the miRNAs involved in the EP treatment process. Our study deepened understanding roles of miRNAs in fish immune system and provides new ideas for the study of immune mechanism of EP.

Screening of genes encoding proteins that interact with ISG15: Probing a cDNA library from a snakehead fish cell line using a yeast two-hybrid system.

Interferon-stimulated gene 15 (ISG15) regulates cellular life processes, including defense responses against infection by a variety of viral pathogens, by binding to target proteins. At present, various fish ISG15s have been identified, but the biological function of ISG15 in snakehead fish is still unclear. In this study, total RNA was extracted from snakehead fish cell line E11, ds cDNA was synthesized and purified using SMART technology, and the resulting cDNA library was screened by co-transforming yeast cells. The library titer was 4.28 × 109 CFU/mL. Using snakehead ISG15 as the bait protein, the recombinant bait vector pGBKT7-ISG15 was constructed and transformed into the yeast strain Y2HGold. The toxicity and self-activation activity of the bait vector were detected on the deficient medium, and the prey proteins interacting with ISG15 were screened. In total, 19 interacting proteins of ISG15 were identified, including mitotic checkpoint protein BUB3, hypothetical protein SnRVgp6, elongation factor 1-beta, 60S ribosomal protein L9, dual specificity protein phosphatase 5-like, eukaryotic translation initiation factor 3 subunit I and ferritin. A yeast spotting assay further probed the interaction between ISG15 and DUSP5. These results increase our understanding of the interaction network of snakehead ISG15 and will aid in exploring the underlying mechanisms of snakehead ISG15 functions in the future.

Induction of apoptosis in SSN-1cells by Snakehead Fish Vesiculovirus (SHVV) via Matrix protein dependent intrinsic pathway.

An increasing important area in immunology is the process cell death mechanism, enabling the immune system triggered thru extrinsic or intrinsic signals to effectively remove unwanted or virus infected cells called apoptosis. A recently isolated infectious Snakehead fish vesiculovirus (SHVV), comprising negative strand RNA and encoded viral matrix (M) proteins, is responsible for causing cytopathic effects in infected fish cells. However, the mechanism by which viral M protein mediates apoptosis has not been elucidated. Therefore, in the present experiments, it was investigated the regulatory potential of apoptosis signals during SHVV infection. By employing the model of SHVV infection in SSN-1 cells, the accelerated apoptosis pathway involves an intrinsic pathway requiring the activation of caspase-9 but not caspase-3 or -8. In the groups of infection (SHVV) or treatment (hydrogen peroxide) were induced apoptotic morphological changes and indicated the activation of the main caspases, i.e.; executioner caspase-3, initiators caspase-8 and caspase-9 using colorimetric assays. Turning to the role of viral M protein when it was overexpressed in SSN-1 cells, it was indicated that the viral M gene alone has the ability to induce apoptosis. To elucidate the mechanism of apoptosis in SSN-1 cells, the activation inhibitors of main caspases were used showing that inhibiting of caspase-3 or caspase-8 activation did not seize induction of apoptosis in virus-infected SSN-1 cells. However, the inhibiting of caspase-9 activation reduced significantly the apoptosis initiation process and sharply the expression of viral M gene, suggesting that SHVV plays a major role in the early induction of apoptosis by caspase-9. Interestingly, there were also differences in the mitochondrial membrane potential after the apoptotic induction of caspases, which confirm that caspase-9 is primarily responsible for the cleavage of caspases during apoptosis. Taken together, these findings can therefore be assumed that viral M protein induces apoptosis via the intrinsic apoptotic pathway in SHVV infecting SSN-1 cells.

Bacillus velezensis WLYS23 strain possesses antagonistic activity against hybrid snakehead bacterial pathogens.

AIM: The aims of this study were to screen an antagonistic probiotic for the prevention and control of bacterial diseases in snakehead fish and to evaluate the antimicrobial activities, biosafety and biocontrol effect of the antagonistic strain. METHODS AND RESULTS: In this study, the WLYS23 strain exhibiting the most effective antagonistic properties against several fish pathogens was selected from the intestine of healthy snakehead fish. The strain was identified as Bacillus velezensis based on morphological, physiological and biochemical characteristics, and phylogenetic analysis. This strain showed amylase, cellulase, protease and lipase activities according to extracellular enzyme activity assays. Moreover, the cell-free extract of the WLYS23 strain exhibited strong antibacterial activity, with MICs of ≤64 µg ml-1 for most fish pathogens. Additionally, the cell-free extract was heat and pH stable, and resistant to protease, whereas their antimicrobial activities were significantly influenced by metal ions at high concentration. The WLYS23 strain and its cell-free extract were safe for snakehead and zebrafish. The disease resistance of snakehead fish was significantly increased following administration of the WLYS23 strain and its cell-free extract respectively. The complete genome of the WLYS23 strain was sequenced and found to harbour seven gene clusters encoding secondary metabolites with antimicrobial activity. CONCLUSION: The WLYS23 strain possesses great potential as a biocontrol agent, which can be commercially developed to improve disease control in freshwater aquaculture. SIGNIFICANCE AND IMPACT OF THE STUDY: Snakehead fish are important commercially farmed fish worldwide. However, the bacterial diseases caused by Aeromonas sp. and Nocardia seriolae in farmed snakehead fish lead to huge economic losses. Safe, economical and efficient probiotics are limited to prevent and control these diseases. Here, we provide a promising biocontrol agent with antagonistic activity against bacterial diseases of snakehead.

Pathogenicity of snakehead vesiculovirus in rice field eels (Monopterus albus).

Snakehead vesiculovirus (SHVV) has caused mass mortality to cultured snakehead fish in China, resulting in enormous economic losses in snakehead fish culture. In this report, the whole genome of SHVV was sequenced. Interestingly, it shared more than 94% nucleotide sequence identity with Monopterus albus rhabdovirus (MoARV), which has caused great economic loss to cultured rice field eel (Monopterus albus). Therefore, the concern of cross-species infection of these viruses prompted us to investigate the susceptibility of rice field eel to SHVV infection. The results showed that rice field eel was susceptible to SHVV in both intracoelomical injection and immersion routes. Severe hemorrhage was observed on the skin and visceral organs of SHVV-infected rice field eels. Histopathological examination showed vacuoles in the tissues of infected liver, kidney and heart. Viral RNA or protein was detected in the tissues of infected fish by reverse transcription polymerization chain reaction (RT-PCR), in situ hybridization (ISH), or immunohistochemistry assay (IHC). Investigation of the epidemic of vesiculovirus in rice field eel as well as other co-cultured fish is invaluable for the prevention of vesiculovirus infection.

Identification and Characterization of MicroRNAs in Snakehead Fish Cell Line upon Snakehead Fish Vesiculovirus Infection.

MicroRNAs (miRNAs) play important roles in mediating multiple biological processes in eukaryotes and are being increasingly studied to evaluate their roles associated with cellular changes following viral infection. Snakehead fish Vesiculovirus (SHVV) has caused mass mortality in snakehead fish during the past few years. To identify specific miRNAs involved in SHVV infection, we performed microRNA deep sequencing on a snakehead fish cell line (SSN-1) with or without SHVV infection. A total of 205 known miRNAs were identified when they were aligned with the known zebrafish miRNAs, and nine novel miRNAs were identified using MiRDeep2 software. Eighteen and 143 of the 205 known miRNAs were differentially expressed at three and 24 h post-infection (poi), respectively. From the differentially-expressed miRNAs, five were randomly selected to validate their expression profiles using quantitative reverse transcription polymerase chain reaction (qRT-PCR), and their expression profiles were consistent with the microRNA sequencing results. In addition, the target gene prediction of the SHVV genome was performed for the differentially-expressed host miRNAs, and a total of 10 and 58 differentially-expressed miRNAs were predicted to bind to the SHVV genome at three and 24 h poi, respectively. The effects of three selected miRNAs (miR-130-5p, miR-214 and miR-216b) on SHVV multiplication were evaluated using their mimics and inhibitors via qRT-PCR and Western blotting. The results showed that all three miRNAs were able to inhibit the multiplication of SHVV; whereas the mechanisms underlying the SHVV multiplication inhibited by the specific miRNAs need to be further characterized in the future.

Application of ultrasound-assisted cryoprotectant impregnation for improving the storage quality of snakehead fish fillets.

In aquatic products companies, saccharides and phosphates are often used to impregnate products to improve their quality. Insignificant impregnation effect is a main problem met in the impregnation process. The effect of ultrasonic-assisted impregnation on the storage quality of snakehead fish fillets at -18 °C was studied. For all samples, such parameters as water holding capacity, pH value, salt-soluble protein content, whiteness and cooking loss rate were examined periodically. Furthermore, the changes of moisture distribution and migration in snakehead fish fillets during storage were investigated using low-field nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). The results demonstrated that ultrasonic-assisted impregnation showed better anti-freeze and water holding capacity effects than that of cryoprotectant impregnation alone. Therefore, ultrasonic treatment could be used as an effective way to improve the quality of snakehead fish fillets during the frozen process. This study could provide a broad application prospect in aquatic product processing industry.

Population genetics of the African snakehead fish Parachanna obscura along West Africa's water networks: Implications for sustainable management and conservation.

An essential factor for aquatic conservation is genetic diversity or population divergence, which in natural populations reflects the interplay between geographical isolation with restricted gene flow and local evolution of populations. The long geological history of Africa may induce stronger among-population divergence and lower within-population divergence in fish populations of African watersheds. As an example, we studied population structure of the African snakehead fish Parachanna obscura. Our study aimed: (1) to develop a set of highly polymorphic microsatellite markers suitable for the analysis of genetic diversity among P. obscura and (2) to study the genetic diversity and structure of P. obscura populations from the West Africa region and mostly from Côte d'Ivoire, with respect to the effects of climate region and geographical distance on the genetic differentiation. A total of 259 specimens from 15 locations of P. obscura were collected over the West Africa region reflecting a high variability of pairwise geographical distances and variability of hydrological connectivity of the area. We developed a set of 21 polymorphic microsatellite markers for studying population genetics of the fish. The results showed relatively low intragenetic diversity for all the 15 locations, certainly attributable to confinement of fish in segregated catchments, resulting in limited gene flow. We also found evidence for local adaptation processes, suggested by five out of 21 microsatellite loci being under putative selection, according to BAYESCAN analysis. In turn, there was strong genetic differentiation (F ST > 0.5) among fish from most locations, reflecting the allopatric development in watersheds without hydraulic connectivity. Neighbor-joining dendrogram, Principal Coordinate Analysis, and analysis of ancestral groups by STRUCTURE suggested that the 15 locations can be divided into three clusters, mainly matching the dominant climate zones and the segregation of the watersheds in the region. The distinct genetic structure of the fish from the 15 locations obtained in this study suggests that conservation and sustainable management actions of this fish resource should avoid genetic mixing of potentially locally adapted populations.

The effects of trehalose synergy with NaCl on the textural, water distribution, and microstructure of snakehead fish filets induced by freeze-thaw cycles.

In this study, we investigated the effect of trehalose in concert with NaCl on the flesh of snakehead fish that had undergone freeze-thaw cycles. Four groups of treatments were compared in this study, including distilled water, 3% NaCl, antifreeze (4% wt/vol) with 3% NaCl, and 4% trehalose (wt/vol) with 3% (wt/vol) NaCl. The results showed that the addition of 4% trehalose (wt/vol) with 3% (wt/vol) NaCl reduced the cracks between muscle fibers and the pores on muscle fiber bundles caused by freeze-thaw cycles during frozen storage of snakehead fish, thus reducing mechanical damage to the fish tissue structure. Moreover, the treatment was able to reduce the thawing loss of snakehead fillets, reduce cooking loss, and help maintain the color of the fish. Further, 4% trehalose (wt/vol) + 3% (wt/vol) NaCl (T) could slow down the reduction of hardness, elasticity, and chewiness of fish fillets during frozen storage. This study provides a theoretical basis for reducing the freeze-thaw cycle on the quality changes of snakehead fish during transportation and marketing.

Effect of different cooking methods on proximate and mineral composition of striped snakehead fish (Channa striatus, Bloch).

The effects of different cooking methods (boiling, baking, frying and grilling) on proximate and mineral composition of snakehead fish were investigated. The mean content of moisture, protein, fat and ash of raw fish was found to be 77.2 ± 2.39, 13.9 ± 2.89, 5.9 ± 0.45 and 0.77 ± 0.12% respectively. The changes in the amount of protein and fat were found to be significantly higher in frying and grilling fish. The ash content increased significantly whereas that of the minerals (Na, K, Ca, Mg, Fe, Zn and Mn) was not affected in all cooking methods. Increased in Cu contents and decreased in P contents were observed in all cooking methods except grilling. In the present study, the grilling method of cooking is found to be the best for healthy eating.

Comparative Study of the Nutritional Composition and Antioxidant Ability of Soups Made from Wild and Farmed Snakehead Fish (Channa Argus).

In recent years, fish soup has become an important product for commercial processing of fish due to its health effects. In this study, nutritional composition and antioxidant ability of soups prepared from farmed and wild snakehead fish were analyzed (hereafter FS and WS soup, respectively). For the FS soup, the proximate composition of protein, fat, ash, free amino acids, and soluble peptides were 2.55%, 0.89%, 0.92%, 0.47%, and 0.62%, respectively. The total amino acid was 390.11 mg/ g, and the proportion of essential amino acid was 27.59%. The total fatty acid was 13.64 g/100 g, of which monounsaturated fatty acid was 5.78 g/100 g, n-6 polyunsaturated fatty acid 3.50 g/100 g, and n-3 polyunsaturated fatty acid 0.41 g/100 g, respectively. The contents of Zn and Ca were 9.04 mg/ kg and 1.13 mg/ g, respectively. The DPPH radical-scavenging ability, Fe2+ chelating ability, and hydroxyl radical-scavenging ability was 57.89%, 21.21%, and 25.61%, respectively. Overall, there was no obvious difference in the nutritional composition and antioxidant activity between the FS and WS soups. The protein content (1.90%) of the WS soup was relatively lower, but the total fatty acid (16.22 g/100 g), MUFA (7.17 g/100 g), and Zn (12.57 mg/ kg) contents were significantly higher.

Influence of composite flour constituents and extrusion temperature in the production of analog rice.

This study was conducted to determine the effect of composite flour (CF) constituents and different extrusion temperatures on the production of analog rice and community perceptions about the produced rice. CF was produced by mixing modified cassava flour (Mocaf) and snakehead fish flour (SF) in the following ratios: 100:0; 97:3; 94:6; 91:9; and 88:12. Analog rice was then extruded from CF at the following temperatures: 50, 70, and 90°C. The analog rice was tested for physical properties (bulk density and cooking time), chemical properties (nutrient content), and organoleptic properties. The results showed that CF and extrusion temperature affect the nutrient content of analog rice. The best analog rice formulation constituted of CF with Mocaf:SF ratio of 91:9 and extrusion temperature of 70°C, and contained 14.34% water, 0.85% ash, 71.83% carbohydrate, 11.24% protein, 1.12%, calcium 1,113 ppm, and 2.43% dietary fiber. This study included 42 respondents aged 20-23 years, including 20 males and 22 females. All the respondents showed acceptance for the analog rice, suggesting that it could substitute ordinary rice as a staple food.

Effect of combined chlorogenic acid and chitosan coating on antioxidant, antimicrobial, and sensory properties of snakehead fish in cold storage.

Degradation of meat quality has always been a burning issue in fish preservation. To maintain the quality, a novel combination of chlorogenic acid (CGA) and chitosan (CS) coating was applied to snakehead fish fillets. Fish fillets were soaked into 2% chitosan (2CS), 0.2% CGA in 2% chitosan (0.2CGA/2CS), 0.5% CGA in 2% chitosan (0.5CGA/2CS), or 1.0% CGA in 2% chitosan (1.0CGA/2CS) solution; and then, coated samples were vacuum-packaged and stored at 2 ± 0.5°C. pH values, color values, microbial loads, hardness, sensory qualities, and oxidization of lipids and proteins of stored fish fillets were investigated for 5 months. Antimicrobial activity was found to be nonsignificant (p ≤ .05) among different coated fish fillets, while color, antioxidant, and pH values were significantly (p ≤ .05) different. Lipid oxidation and protein oxidation were found to be inhibited in 2CS-, 0.5CGA/2CS- and 1.0CGA/2CS-coated fish fillet. All CGA/CS coating delayed increase in pH (p ≤ .05) and resulted brown color. However, only CS coating resulted in higher sensory scores (p ≤ .05) and controlled browning. Considering antioxidant properties and other quality parameters, CGA/CS coating might be applied commercially in fish preservation.

Gonadosomatic Index, Oocyte Development and Fecundity of the Snakehead Fish (Channa striata) in Natural River of Mae La, Singburi Province, Thailand.

BACKGROUND AND OBJECTIVE: Reproductive cycle of snakehead fish (Channa striata) is necessary to inform management plans of the natural fish. This work aimed to study the gonadosomatic index (GSI), oocyte development and fecundity of C. striata in the Mae La River, Singburi province, Thailand from November, 2017-October, 2018. MATERIALS AND METHODS: GSI was calculated from gonadal weight and body weight. Ovarian sections derived from standard histological methods were used to evaluate oocyte development, respectively. Fecundity was measured using the gravimetric method and the relationships of fecundity-total length, fecundity-body weight and fecundity-ovarian weight were evaluated. RESULTS: The highest average GSI values for females and males were found in July (6.15±0.20 and 0.14±0.12, respectively). Ovarian histology revealed 4 stages of ovarian development: (1) immature, (2) maturing, (3) mature and (4) spent. Stages 1, 2, 3 and 4 were observed for 11, 10, 6 and 2 months, respectively. The highest percentages of immature, mature and spent oocytes were found in October (93.33%), July (89.56%) and August (33.33%), respectively. Fecundity ranged from 4,160-46,890. Fecundity varied depending on total length, body weight and ovarian weight, with correlation coefficients (R) of 0.986, 0.960 and 0.989, respectively. CONCLUSION: Oocyte maturation period of C. striata occurs from April to August. The highest gonads development was in July. Fecundity exhibited linear relationships with body length, body weight and ovarian weight.

The effect of snakehead fish extract supplementation to first-line eradication regimen on macrophage migration inhibitory factor (MIF) expression in rats induced by Helicobacter pylori infection.

OBJECTIVE: This work was organized to assess macrophage migration inhibitory factor (MIF) expression in snakehead fish extract supplementation to first-line eradication regimen in rats induced by Helicobacter pylori infection. MATERIALS AND METHODS: A total of 28 manly rats were haphazardly isolated equally into four groups. Group-1 was the control negative, and groups-2-4 were H. pylori-infected groups. Group-2 was the control positive. Groups-3 and 4 were treated with first-line eradication regimen and first-line eradication regimen supplemented with snakehead fish extract, respectively. Immunoreactive scores (IRS) of MIF expression and eradication testing procedure were carried out. The comparison and difference between groups were analyzed by Kruskal-Wallis and post hoc Mann-Whitney U-test. A value of p < 0.05 was considered to be a limit of significance. RESULTS: The average IRS of MIF expression in group-2 was the highest among other groups (p < 0.05). Group-4 (supplemented by snakehead fish extract) had a lower median value IRS of MIF expression compared to group-3 [1.0 (0.0-2.0) vs. 3.5 (2.0-6.0), p = 0.004]. CONCLUSION: MIF expression was higher in rats induced by H. pylori infection. Snakehead fish extract supplementation to first-line eradication regimen significantly reduces more MIF expression compared to a single administration of first-line eradication regimen in rats induced by H. pylori infection.

Study of HMG-CoA Reductase Inhibition Activity of the Hydrolyzed Product of Snakehead Fish (Channa striata) Skin Collagen with 50 kDa Collagenase from Bacillus licheniformis F11.4.

Bioactive peptides produced from enzymatic hydrolysis fibrous protein have been proven to have several biological activities. Previous study showed that the hydrolysis product of snakehead fish skin collagen with 26 kDa collagenase from Bacillus licheniformis F11.4 showed HMG-CoA (HMGR) inhibition activity. The aim of this research was to determine the ability of the hydrolysis product produced from snakehead fish skin collagen hydrolysed by 50 kDa collagenase from B. licheniformis F11.4 in inhibiting HMGR activity. Snakehead fish skin collagen was extracted using an acid method and collagenase was produced from B. licheniformis F11.4 using half-strength Luria Bertani (LB) medium containing 5% collagen. Crude collagenase was concentrated and fractionated using the DEAE Sephadex A-25 column eluted with increasing gradient concentrations of NaCl. Collagen, collagenase, and fractions were analyzed using SDS-PAGE and collagenolytic activity was analyzed by the zymography method. Collagenase with 50 kDa molecular weight presented in fraction one was used to hydrolyze the collagen. The reaction was done in 18 hours at 50°C. The hydrolysis product using 3.51 µg collagen and 9 ng collagenase showed 25.8% inhibition activity against pravastatin. This work shows for the first time that the hydrolysis product of snakehead fish skin collagen and 50 kDa collagenase from B. licheniformis F11.4 has potential as an anticholesterol agent.

Formulation, optimization, and characterization of snakehead fish (Ophiocephalus Striatus) powder nanoemulgel

The aim of the present study was to characterize and evaluate nanoemulgel of snakehead fish powder (SFP) for the poorly water-soluble drug. SFP was formulated into nanoemulsion utilizing the best comparison of surfactant, co-surfactant, and oil. Diverse nanoemulsion components (oil, surfactant, and co-surfactant) were chosen based on solvency and emulsification capacity. SFP 0.1% loaded nanoemulsion which tested by stress-stability testing which carried out for all formulations and those that passed these tests were characterized for droplet size, polydispersity index (PDI), zeta potential, pH, viscosity, and transmittance. After that, nanoemulsion was added with 1.5%, 2.0%, and 2.5% of HPMC in different concentrations and mixed until nanoemulgel form and evaluated for pH, viscosity, spreadability, and extrudability measurement. The results of this research showed that SF nanoemulsion produced clear, stable, and transparent formula having the transmittance value 99.87%. Mean droplet size and zeta potential of the optimized nanoemulsion (NE4) were found to be 98.6±0.93 nm (PDI 0.1±0.20) and -57.5±0.3 MV respectively. Meanwhile, the evaluation results of nanoemulgel (NEG) showed NEG1.5 gave pH 6.0, viscosity 210 cP, spreadability 5.8 g cm/s and extrudability 1.4 g/cm2. Otherwise, NEG2.0 and NEG2.5 had high viscosity and pH generating low spreading on the skin i.e. 3.9 g cm/s and 2.8 g cm/s respectively. The results of the evaluation and preparation stability test showed a good level of stability of NEG1.5 with the viscosity and pH by one way ANOVA which did not change significantly.