A novel thermo-responsive phospholipase A1 with high selectivity and efficiency in enzymatic oil degumming.

The limited availability of phospholipase A1 (PLA1) has posed significant challenges in enzymatic degumming. In this study, a novel PLA1 (UM2) was introduced to address this limitation, which had a unique thermo-responsive ability to switch phospholipase and lipase activities in response to temperature variations. Remarkably, UM2 displayed an unprecedented selectivity under optimized conditions, preferentially hydrolyzing phospholipids over triacylglycerols-a specificity superior to that of commercial PLA1. Moreover, UM2 demonstrated high efficiency in hydrolyzing phospholipids with a predilection for phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A practical application of UM2 on crude flaxseed oil led to a dramatic reduction in phosphorus content, plummeting from an initial 384.06 mg/kg to 4.38 mg/kg. Broadening its industrial applicability, UM2 effectively performed enzymatic degumming for other distinct crude vegetable oils with a unique phospholipid composition. Collectively, these results highlighted the promising application of UM2 in the field of oil degumming.

Enhancement of resistant starch content in ethyl cellulose-based oleogels cakes with the incorporation of glycerol monostearate.

The objective of this work was to completely replace margarine with peanut diacylglycerol oil/ethyl cellulose-glycerol monostearate oleogel (DEC/GMS) oleogel, and evaluate its effect on starch digestibility of cakes. The in vitro digestibility analysis demonstrated that the DEC/GMS-6 cake exhibited a 26.36% increase in slowly digestible starch (SDS) and resistant starch (RS) contents, compared to cakes formulated with margarine. The increased SDS and RS contents might mainly be due to the hydrophobic nature of OSA-wheat flour, which could promote the formation of lipid-amylose complexes with GMS and peanut diacylglycerol oil. XRD pattern suggested that the presence of GMS in DEC-based oleogels facilitated the formation of lipid-amylose complexes. The DSC analysis revealed that the addition of GMS resulted in a significant increase in gelatinization enthalpy, rising from 249.7 to 551.9 J/g, which indicates an improved resistance to gelatinization. The FTIR spectra indicated that the combination of GMS could enhance the hydrogen bonding forces and short-range ordered structure in DEC-based cakes. The rheological analysis revealed that an increase in GMS concentration resulted in enhanced viscoelasticity of DEC-based cake compared to TEC-based cakes. The DEC-based cakes exhibited a more satisfactory texture profile and higher overall acceptability than those of TEC-based cakes. Overall, these findings demonstrated that the utilization of DEC-based oleogel presented a viable alternative to commercial margarine in the development of cakes with reduced starch digestibility.

Efficient Enzymatic Enrichment of High-purity Nervonic Acid from Malania oleifera Seed Oil.

Nervonic acid (NA) is a monounsaturated fatty acid vital for brain health and is of emerging importance in various industrial applications, including therapeutics, food, and cosmetics. Given the growing demands of the food and pharmaceutical industries, there's a pressing need for high-purity NA. Previously, NA constituents in plant seed oils were chemically transformed into nervonic acid ethyl ester (NAEE) to facilitate extraction from seed oils. In this study, we present an enzymatic approach to convert NA constituents in Malania oleifera seed oil to NAEE. Combined with the utilization of the semi-preparative chromatography, we achieved a remarkable purity of 97.52% NAEE. Compared to conventional chemical preparations characterized by multiple steps, prolonged processing times, and low yields and purities, our enzymatic method stands out as a more efficient and advantageous alternative. On top of that, this innovative approach is environmentally friendly and circumvents health and safety issues associated with chemical processes.

Physicochemical properties and feasibility of coconut oil-based diacylglycerol as an alternative fat for healthy non-dairy creamer.

Non-dairy creamers have been widely used for coffee whitening and texture improvement. To avoid the intake of trans fatty acids from partially hydrogenated oil, coconut oil-based diacylglycerol (CO-DAG) was applied in non-dairy creamer as core material. In this study, effects of DAG content (30, 50, 70, 90%) on the characteristics of CO-DAG were evaluated, including rheological and thermodynamic properties. The CO-DAG with a content of 50% exhibited a wide plastic range and contained mixture of ß and ß' polymorphic forms. Using CO-DAG (50%) as core material, the physicochemical properties of non-dairy creamer were characterized and compared with commercial products. The results indicated that CO-DAG-based non-dairy creamers showed similar encapsulation efficiency (92.74%) and thermal stability to commercial products. Furthermore, CO-DAG-based non-dairy creamer showed higher whiteness index (54.20) than commercial non-dairy creamers (50.22) when applied to black coffee. Overall, it is anticipated that CO-DAG-based non-dairy creamers have great potentials in coffee whitening.

Quality characteristics of peanut protein-based patties produced with pre-emulsified olive oil as a fat replacer: Influence of acylglycerol type.

The emulsion (O/W) may be used as a fat replacer to develop healthier meat analogs. The purpose of this work was to evaluate the effects of oil incorporation methods (direct oil addition and emulsion addition) and oil types [triacylglycerol (TAG) and diacylglycerol (DAG)] on the quality characteristics of peanut protein-based patties crosslinked by transglutaminase (TGase). The patties formulated with emulsions showed larger texture parameters (springiness, cohesiveness and gumminess), lower cooking loss and higher acceptability compared with directly adding oil. The rheological results confirmed that the presence of emulsions strengthened the gel structure in patties, which allowed the patties containing emulsions to stabilize free water. Whereas, TAG-based emulsion was more effective than DAG-based emulsion in improving quality of products, possibly because the competitive adsorption at oil-water interface of DAG reduced the crosslinking between the interfacial protein and adjacent protein molecules. This study revealed the relationship between the acylglycerol type in emulsion and the patty quality, providing a reference for the development of plant-based patties.

Deep-fat Frying Using Soybean Oil-based Diacylglycerol-Palm Olein Oil Blends: Thermo-oxidative Stability, 3-MCPDE and Glycidyl Ester Formation.

Diacylglycerol (DAG) is commonly known as one of the precursors for the 3-monochloro-1,2-propanediol esters (3-MCPDE) and glycidyl esters (GE) formation. However, due to its health-promoting effects, its potential as alternative frying medium was examined. This study aimed to assess the frying performance of soybean oil-based diacylglycerol oil (DO) and its oil blends with palm olein (PO), in comparison with PO. Four different oil types (DO, PO, OB I (DO:PO, 1:1, w/w) and OB II (DO:PO, 1:2, w/w)) were used to fry potato chips for five consecutive days at 180℃. The formation of oxidation compounds, acylglycerol composition, 3-MCPDE and GE changes throughout the frying study were investigated. Both OB I and OB II exhibited lower oxidation compounds' formation rates than PO. Besides, significant (p < 0.05) reductions of 3-MCPDE and increments of GE levels were observed in all frying systems throughout the frying study. After 25 frying cycles, the 3-MCPDE levels in all frying oils were below 0.13 mg/kg, while the GE levels ranged from 1.51 mg/kg to 1.89 mg/kg. Despite the poorer oxidative stability of DO, its 3-MCPDE and GE levels were much lower compared to PO. In comparison to DO, the 3-MCPDE degradation and GE formation rates were enhanced and reduced, respectively with the blending of PO and DO. This study showed the potential of DO:PO oil blend in deep-fat frying application. With appropriate blending ratio of DO and PO, an alternative frying medium with enhanced nutritional value and oxidative stability could be developed.

Monoacylglycerol lipase from marine Geobacillus sp. showing lysophospholipase activity and its application in efficient soybean oil degumming.

Enzymatic degumming is an essential refining process to improve oil quality. In this study, a monoacylglycerol lipase GMGL was derived from marine Geobacillus sp., and was found that not only took monoacylglycerol (MAG) as substrate, but also had activity toward lysophosphatidylcholine (LPC), lysophosphatidylethanolamine (LPE) and glycerolphosphatidylcholine (GPC). Binding free energy showed LPC and LPE could bind with enzyme stably as MAG. It presented great potential in the field of enzymatic degumming. The phosphorus content in crude soybean oil decreased from 680.50 to 2.01 mg/kg and the yield of oil reached to 98.80 % after treating with phospholipase A1 (Lecitase Ultra) combined with lipase GMGL. An ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed to identify 21 differential phospholipids between crude soybean oil and enzymatic treatment. This work might shed some light on understanding the catalytic mechanism of monoacylglycerol lipase and provide an effective strategy for enzymatic degumming.

Soy lecithin increases the stability and lipolysis of encapsulated algal oil and probiotics complex coacervates.

BACKGROUND: Co-encapsulation of probiotics and omega-3 oil using complex coacervation is an effective method for enhancing the tolerance of probiotics under adverse conditions, whereas complex coacervation of omega-3 oil was found to have low lipid digestibility. In the present study, gelatin (GE, 30 g kg-1 ) and gum arabic (GA, 30 g kg-1 ) were used to encapsulate Lactobacillus plantarum WCFS1 and algal oil by complex coacervation to produce microcapsules containing probiotics (GE-P-GA) and co-microcapsules containing probiotics and algal oil (GE-P-O-GA), and soy lecithin (SL) was added to probiotics-algal oil complex coacervates [GE-P-O(SL)-GA] to enhance its stability and lipolysis. Then, we evaluated the viability of different microencapsulated probiotics exposed to freeze-drying and long-term storage, as well as the survival rate and release performance of encapsulated probiotics and algal oil during in vitro digestion. RESULTS: GE-P-O(SL)-GA had a smaller particle size (51.20 µm), as well as higher freeze-drying survival (90.06%) of probiotics and encapsulation efficiency of algal oil (75.74%). Moreover, GE-P-O(SL)-GA showed a higher algal oil release rate (79.54%), lipolysis degree (74.63%) and docosahexaenoic acid lipolysis efficiency (64.8%) in the in vitro digestion model. The viability of microencapsulated probiotics after simulated digestion and long-term storage at -18,4 and 25 °C was in the order: GE-P-O(SL)-GA > GE-P-O-GA > GE-P-GA. CONCLUSION: As a result of its amphiphilic properties, SL strongly affected the physicochemical properties of probiotics and algal oil complex coacervates, resulting in higher stability and more effective lipolysis. Thus, the GE-P-O(SL)-GA can more effectively deliver probiotics and docosahexaenoic acid to the intestine, which provides a reference for the preparation of high-viability and high-lipolysis probiotics-algal oil microcapsules. © 2022 Society of Chemical Industry.

An ultra-rapid and eco-friendly method for determination of loganic acid and gentiopicroside from Gentianae Macrophyllae Radix by vortex-assisted matrix solid-phase dispersion extraction and LC-MS.

An ultra-rapid and eco-friendly method for the determination of loganic acid and gentiopicroside in Gentianae Macrophyllae Radix (GMR) was developed by vortex-assisted matrix solid-phase dispersion extraction (VAMSPD) and liquid chromatography with mass spectrometry (LC-MS). The optimized VAMSPD parameters are as follow: sample-dispersant (diatomaceous earth) ratio is 1:5, grinding for 0.5 min and whirling with 0.5 mL 15 % ethanol for 0.5 min. The LC separation is performed on a Poroshell 120 EC-C18 column (30 ×2.1 mm, 2.7 µm) and eluted by an eco-friendly mobile phase (14 % ethanol containing 0.1 % formic acid) at a flow rate of 0.5 mL min-1 in isocratic mode, and detected by mass spectrometry (MS). The developed method exhibits a good linearity for the analytes (r > 0.9990). The RSDs of precision and repeatability are less than 4.0 %, the recoveries for loganic acid and gentiopicroside are 106.5 % (RSD=3.6 %) and 95.7 % (RSD=8.0 %), respectively. The developed method was successfully applied in the analysis of loganic acid and gentiopicroside in GMR samples. The total analysis time is 2 min, including 1 min for sample extraction and 1 min for LC-MS analysis. In addition, the method only requires 0.3 mL of ethanol.

Formation of 3-MCPD and glycidyl esters in biscuits produced using soybean oil-based diacylglycerol stearin-shortening blends: Impacts of different baking temperatures and blending ratios.

Diacylglycerol (DAG) is commonly known as one of the precursors for 3-monochloropropane-1,2-diol esters (3-MCPDE) and glycidyl esters (GE) formation. Besides, 3-MCPDE and GE are heat-induced contaminants which can be formed in fat-containing baked products during the baking process. This study attempted to replace the conventional palm-based shortening (SH) with a healthier fat, namely soybean oil-based diacylglycerol stearin (SDAG) in producing biscuits. The effects of different baking temperatures (200, 210 and 220 °C) and SDAG:SH fat blend ratios (0:100, 60:40 (D64S), 80:20 (D82S), 100:0, w/w) towards the biscuits' physical properties were evaluated. Moreover, the oxidative stability, 3-MCPDPE and GE formation in the fats extracted from the biscuits were also investigated. SDAG-produced biscuit showed slight reductions in the spread ratio compared to the SH-produced biscuit. The elevated baking temperatures resulted in biscuits with increased hardness and low moisture content. Pure SDAG and the other fat blends exhibited significant (p < 0.05) poorer oxidative stability than SH. However, D64S was found to be more oxidative stable compared to SDAG and D82S. The D64S fat blend exhibited the lowest 3-MCPDE and GE formation rates among all fat samples with the increasing baking temperatures. Furthermore, the amount of 3-MCPDE and GE detected in the fats extracted from the biscuits baked at highest temperature (220 °C) were still within the safety limit. In overall, better quality biscuits were produced when lower baking temperature (200 °C) was used as all biscuits baked with different fats showed similar textural properties (hardness and cohesiveness), higher oxidative stability and lower formation of 3-MCPDE and GE compared to biscuits baked at higher temperatures. The findings justified the potential of D64S fat blend in replacing the conventional SH in producing healthier biscuits.

The Formation of 3-Monochloropropanediol Esters and Glycidyl Esters during Heat-Induced Processing Using an Olive-Based Edible Oil.

With the prevalence of edible diacylglycerol (DAG) oil, which is beneficial to human, the generation of 3-monochloropropanediol esters (3-MCPDE) and glycidyl esters (GE) as well as the stability of physical properties during heat-induced processing still need to be explored. In this study, the experiment used olive-based edible oil with different contents of DAG (40, 60, and 80%) to make crackers and fry chicken. They were heated at 160 and 180 °C to determine the changes in 3-MCPDE and GE, the crackers' hardness and gumminess, and the physical properties of the oil. During baking and frying, 3-MCPDE decreased, while the content of GE slightly increased with the prolonged heating duration. Finally, 3-MCPDE and GE were lower than 1.25 mg/kg and 1.00 mg/kg, respectively. The AV increased proportionally as duration increased and POV was below 0.30 g/100 g. In general, the changes in 3-MCPDE and GE were related to the heating temperature and duration, and not significantly (p > 0.05) related to the content of DAG.

Typical Characterization of Commercial Camellia Oil Products Using Different Processing Techniques: Triacylglycerol Profile, Bioactive Compounds, Oxidative Stability, Antioxidant Activity and Volatile Compounds.

The processing technique is one of the key factors affecting the quality of camellia oil. In this study, camellia oils were obtained using four different processing techniques (cold-pressed, roast-pressed, fresh-pressed, and refined), and their triacylglycerols (TAGs) profile, bioactive compound (tocopherols, sterols, squalene, and polyphenols) level, oxidative stability, and volatile compounds were analyzed and compared. To further identify characteristic components in four camellia oil products, the TAG profile was analyzed using UPLC-QTOF-MSE. Five characteristic markers were identified, including OOO (m/z 902.8151), POL (m/z 874.7850), SOO (m/z 904.8296), PPL (m/z 848.7693), PPS (m/z 852.7987). Regarding the bioactive compound level and antioxidant capacity, the fresh-pressed technique provided higher α-tocopherols (143.15 mg/kg), ß-sitosterol (93.20 mg/kg), squalene (102.08 mg/kg), and polyphenols (35.38 mg/kg) and showed stronger overall oxidation stability and antioxidant capacity. Moreover, a total of 65 volatile compounds were detected and identified in four camellia oil products, namely esters (23), aldehydes (19), acids (8), hydrocarbons (3), ketones (3), and others (9), among which pressed oil was dominated by aldehydes, acid, and esters, while refined oil had few aroma components. This study provided a comprehensive comparative perspective for revealing the significant influence of the processing technique on the camellia oil quality and its significance for producing camellia oil of high quality and with high nutritional value.

Water-in-oil emulsions enriched with alpha-linolenic acid in diacylglycerol form: Stability, formation mechanism and in vitro digestion analysis.

This study developed an alpha-linolenic acid (ALA) supplement with emulsion form using ALA-rich diacylglycerol (ALA-DAG) and ALA-DAG stearin (DAG-SF) as a new source of ALA and emulsifier. Stable, commercial surfactant-free W/O emulsions with 90 wt% oil phase (including DAG-SF and ALA-DAG with 10:90 - 20:80 wt ratio) was fabricated. Microstructure and Raman spectra revealed that the compact crystal networks and high amounts of solid acyl chains were responsible for high emulsion stability. These emulsions exhibited good potential in improving the ALA nutritional status (with ALA release level of 60.49% - 62.98%). Furthermore, the emulsifier-to-oil ratio greatly impacted the emulsion texture (solid-like or liquid-like) and emulsions showed great oxidation stability (2.80 - 3.09 meq/kg lipid of peroxide value at 6th week). The tunable texture and high oxidation stability make this emulsion system useful for a wide range of food products. This developed emulsion system could provide valuable information for other important fatty acids supplement.

Characteristics and feasibility of olive oil-based diacylglycerol plastic fat for use in compound chocolate.

With an aim to prepare the healthier functional chocolate, olive oil-based diacylglycerol plastic fat (ODAGP) was prepared by mixing olive oil-based diacylglycerol stearin (O-DAGS) and olive oil-based diacylglycerol olein (O-DAGO) as confectionery fat in compound chocolate. We reported the physicochemical properties of ODAGP and ODAGP-CB blends, and then evaluated their application potential in compound chocolate based on sensory, blooming property and polymorphic transition. ODAGP (40% O-DAGS) showed a wide plastic range (7.1-45.2%) and high component compatibility. The crystal properties results indicated that ODAGP mainly existed in stable ß-forms (ß1 and ß2). On the other hand, the ODAGP-CB binary system containing 10-40% ODAGP displayed similar melt-in-mouth property and crystal polymorphism (Form V) to natural CB. Compound chocolate prepared with ODAGP-CB blends obtained satisfactory overall acceptability (score > 7.8) and showed stronger fat bloom resistance.

Production of Cocoa Butter Substitute via Enzymatic Interesterification of Fully Hydrogenated Palm Kernel Oil, Coconut Oil and Fully Hydrogenated Palm Stearin Blends.

This research synthesized structure lipids (SL) from blends of fully hydrogenated palm kernel oil (FHPKO), coconut oil (CNO) and fully hydrogenated palm stearin (FHPS) by enzymatic interesterification (EIE)using rProROL, an sn-1,3-specific lipase from Rhizopus oryzae, as a catalyst. Five physical blends of FHPKO:CNO:FHPS were prepared with the following wt. ratios: 40:10:50, 50:10:40, 60:10:30, 70:10:20 and 80:10:10. The EIE reactions were carried out at 60℃ for 6 h in a batch-type reactor using rProROL 10% wt. of the substrate. It was found that EIE significantly modified the triacylglycerol compositions of the fat blends resulting in changes in the crystallization and melting behavior. In particular, SL obtained from EIE of blend 70:10:20 exhibited high potential to be used as a cocoa butter substitute (CBS) because it showed similar solid fat content curve to the commercial CBS and crystallized into fine spherulites and desirable ß' polymorph.

Synthesis and Characterization of Epoxidized Silkworm Pupae Oil and Its Application as Polyvinyl Chloride.

More and more industries demand environmental friendliness. Silkworm pupae oil (SPO), extracted from the desilked silkworm pupae, can serve as a promising substrate alternative to use in plasticization. This study aimed to prepare epoxidized silkworm pupae oil (ESPO) and investigate their effects on the thermal stability and plasticization of polyvinyl chloride (PVC) films. A chemo-enzymatic method of ESPO was developed in the presence of Lipase SMG1-F278N and H2O2 in natural deep eutectic solvents (DESs). Lipase SMG1-F278N could initiate the epoxidation reaction effectively at room temperature with a negligible loss of activities 10 batches. A maximum oxirane value of 6.94% was obtained. The formation of oxirane ring in ESPO was confirmed by FTIR and 13C NMR spectra. Moreover, ESPO showed a better thermal stability and lower freezing point than epoxidized soybean oil (ESO). It was demonstrated that ESPO had a good frost resistance. In addition, ESPO showed a significantly improved plasticizing effect on flexible polyvinyl chloride (PVC). Compared with ESO, ESPO could increase the tensile elongation at break effectively. A significantly lower migration rate of plasticizer was observed in PVC plasticized with ESPO.

Two-step enzymatic synthesis of α-linolenic acid-enriched diacylglycerols with high purities from silkworm pupae oil.

In this study, α-linolenic acid-enriched diacylglycerols (ALA-DAGs) were prepared via a two-step enzymatic way by combi-lipase using silkworm pupae oils as substrates. Firstly, several factors including temperature, mass ratio of water to oil, pH and enzyme loading were optimized for the hydrolysis of silkworm pupae oil. The maximum fatty acid content (96.51%) was obtained under the conditions: temperature 40 °C, water/oil 3:2 (w/w), pH 7, lipase TL100L loading 400 U/g, lipase PCL loading 30 U/g. Then, ALA was enriched by urea inclusion, with an increased ALA content of 82.50% being obtained. Secondly, the ALA-enriched silkworm pupae DAG oil (SPDO) was prepared by lipase PCL-catalyzed esterification reaction. After molecular distillation, the final SPDO product contained contents of DAGs (97.01%) and ALA (82.50%). This two-step enzymatic way for production of ALA-DAGs was successfully applied in a 100-fold scale-up reaction. Overall, our study provides a promising way for the preparation of ALA-DAGs.

Enhancing production of lipase MAS1 from marine Streptomyces sp. strain in Pichia pastoris by chaperones co-expression

Background: A thermostable lipase MAS1 from marine Streptomyces sp. strain was considered as a potential biocatalyst for industrial application, but its production level was relatively low. Here, the effect of chaperones co-expression on the secretory expression of lipase MAS1 in Pichia pastoris was investigated. Result: Co-expression of protein disulfide isomerase (PDI), HAC1 and immunoglobulin binding protein could increase the expression level of lipase MAS1, whereas co-expression of Vitreoscilla hemoglobin showed a negative effect to the lipase MAS1 production. Among them, PDI co-expression increased lipase MAS1 expression level by 1.7-fold compared to the control strain harboring only the MAS1 gene. Furthermore, optimizing production of lipase MAS1 with Pichia pastoris strain X-33/MAS1-PDI in a 30-L bioreactor were conducted. Lower induction temperature was found to have a benefit effect for lipase MAS1 production. Lipase activity at 24 and 22°C showed 1.7 and 2.1-fold to that at 30°C, respectively. Among the induction pH tested, the highest lipase activity was obtained at pH 6.0 with activity of 440 U/mL after 144 h fermentation. Conclusion: Our work showed a good example for improving the production of recombinant enzymes in Pichia pastoris via chaperon co-expression and fermentation condition optimization.

An improved nonchromatographic method for the purification of recombinant proteins using elastin-like polypeptide-tagged proteases.

Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.

A zebrafish (Danio rerio) model of infectious spleen and kidney necrosis virus (ISKNV) infection.

Zebrafish is a model animal for studies of genetics, development, toxicology, oncology, and immunology. In this study, infectious spleen and kidney necrosis virus (ISKNV) was used to establish an infection in zebrafish, and the experimental conditions were established and characterized. Mortality of adult zebrafish infected with ISKNV by intraperitoneal (i.p.) injection exceeded 60%. ISKNV can be passed stably in zebrafish for over ten passages. The ailing zebrafish displayed petechial hemorrhaging and scale protrusion. Histological analysis of moribund fish revealed necrosis of tissue and enlarged cells in kidney and spleen. The real-time RT-PCR analysis of mRNA level confirmed that ISKNV was replicated in zebrafish. Immunohistochemistry and immunofluorescence analyses further confirmed the presence of ISKNV-infected cells in almost all organs of the infected fish. Electron microscope analyses showed that the ISKNV particle was present in the infected tissues. The establishment of zebrafish infection model of ISKNV can offer a valuable tool for studying the interactions between ISKNV and its host.