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Background: High chitosanase-producing microorganisms from natural sources have extensive applications in food and agriculture. This study aimed to optimal conditions for high-activity chitosanase production. A named CGMCC21422 chitosanase-producing strain -2T-2 was isolated from soil and identified named as Aspergillus fumigatus chitosanase (A. fumigatus chitosanase). This enzymatic activity was validated in various culture conditions. It is stored in the China General Microbiological Culture Collection Center. The efficacy of A. fumigatus chitosanase in the degradation of chitosan was validated. Results: In this study, we determined that the optimal fermentation conditions of stain-2T-2 were 1.0% powered chitosan, 0.8% ammonium nitrate, 37°C culture temperature, initial pH 5.0, culture time 6 d, bottle volume 50 mL, and 2% inoculation dosage. Under these culture conditions, the highest enzyme activity of fermentation broth in the shaker flask reached 827.53 U/mL. The optimal reactive conditions of A. fumigatus-produced chitosanase are 55–60°C and pH 4.5. When the reactive temperature was over 60°C, the A. fumigatus chitosanase was easily inactivated. The chitosanase catalyzed substrate chitosan to produced ≈20% chito-oligosaccharide and ≥80% glucosamine salt samples in a variety of acidic solutions. These reactive products are not cytotoxic or mild to MH7A cells. Conclusions: A. fumigatus chitosanase strain -2T-2 is a strain with high chitosanase and can catalyze chitosan into chito-oligosaccharide in acidic solutions.How to cite: Yang, H., Wang, L., Xu, C, et al. Screening for Aspergillus fumigatus strain-2T-2 with high chitosanase production activity and its application in chitosan degradation. Electron J Biotechnol 2024; 68. https://doi.org/10.1016/j.ejbt.2024.01.003.

The escalation of jellyfish stings has drawn attention to severe skin reactions, underscoring the necessity for novel treatments. This investigation assesses the potential of hydroxybenzoic acid derivatives, specifically protocatechuic acid (PCA) and gentisic acid (DHB), for alleviating Nemopilema nomurai Nematocyst Venom (NnNV)-induced injuries. By employing an in vivo mouse model, the study delves into the therapeutic efficacy of these compounds. Through a combination of ELISA and Western blot analyses, histological examinations, and molecular assays, the study scrutinizes the inflammatory response, assesses skin damage and repair mechanisms, and investigates the compounds’ ability to counteract venom effects. Our findings indicate that PCA and DHB significantly mitigate inflammation by modulating critical cytokines and pathways, altering collagen ratios through topical application, and enhancing VEGF and bFGF levels. Furthermore, both compounds demonstrate potential in neutralizing NnNV toxicity by inhibiting metalloproteinases and phospholipase-A2, showcasing the viability of small-molecule compounds in managing toxin-induced injuries.

Monkfish (Lophius litulon) is a nutritious marine product that has attracted increasing attention. In this study, we demonstrated that the monkfish protein hydrolysate (MPH) administration could significantly prolonged the exhaustive swimming time of mice, and increased the hepatic glycogen (HG) level, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxides (GSH-PX) activities; in addition, markedly decreased the blood urea nitrogen (BUN), blood lactic acid (BLA), malonaldehyde (MDA) levels and lactate dehydrogenase (LDH) activity. Furthermore, the MPH administration also enhanced the proliferation of spleen lymphocytes and the activity of natural killer (NK) cells in mice. Besides, 43 peptides from MPH that rich in Phe, His, Ile, Leu and Thr were identified. Finally, 1245 of differentially expressed genes (DEGs) in NC vs MPH-H groups were obtained by transcriptome analysis, and GO classification and enrichment of KEGG pathway were studied. In conclusion, MPH treatment had the anti-fatigue and immunological effects in mice.

Chitin is a valuable and renewable carbohydrate. It is the second abundant but underexploited carbohydrate polymer. Its monomers, glucosamine (GlcN) and N-acetyl-glucosamine (NAG), are widely used in food, cosmetic, and pharmaceutical fields owing to their high biocompatibility and other properties. Therefore, conversion of chitin to highly hydrophilic and active GlcN and NAG can enhance its utility. In this review, we summarize the design strategies, reaction conditions for the preparation of GlcN and NAG from chitin and the synthesis of derivatives from GlcN/NAG. Moreover, we discuss the advantages of physicochemical properties and active advantages of GlcN derivatives formed by active compounds when they are conjugated with GlcN/NAG. Based on these experiences, we finally proposed a novel strategy for GlcN derivatization, which is expected to yield highly active GlcN derivatives that can be applied in pharmaceutical or food fields.

Jellyfish peptides are of significant interest, due to their potential application as a source of bioactive peptides in pharmaceutical domains. Here, we investigated the action of Nemopilema nomurai peptides (NNPs) on lipids reduction in 3 T3-L1 adipocytes. We demonstrated for the first time that NNPs prepared by papain enzymolysis were able to reduce lipid accumulation in 3 T3-L1cells. Differentially expressed metabolites among control group, MDI group, GW9662 treatment group, and NNPs treatment group were identified using non-targeted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics. A total of 105 differentially expressed metabolites were identified between MDI group and NNPs treatment group. These metabolites were predominantly enriched in amino acid, carbohydrate and lipid metabolism. Metabolomics analysis indicated that NNPs leads to imbalance of lipid, which represent as down-regulated glycerolipid and glycerophospholipid, up-regulated sphingolipid metabolism and sphingolipid signaling pathway. Additionally, ATP level was significantly decreased in TCA cycle and oxidative phosphorylation pathway, which correlated with glucagon signaling pathway and fatty acid synthesis. The lipid-lowering activity of NNPs may associate with lipid and energy-related metabolism in 3 T3-T1 cell. ATP may be a potential biomarker for lipid-lowering, with a fold change of 0.534.

Jellyfish is a valuable biological resource in marine ecosystems, and blooms been observed in numerous coastal regions. However, their utility is limited by their high water content. Recent research has focused on extracting antioxidants from marine sources. In this study, we obtained jellyfish peptides (JPHT-2) through enzymatic hydrolysis of lyophilized jellyfish powder under optimal conditions and measured their antioxidant activity. Our findings indicate that JPHT-2 possesses significant radical-scavenging activity and reducing power. At a concentration of 0.74 mg/mL, JPHT-2 exhibited a remarkable ability to scavenge hydroxyl radicals, with a rate of up to 50%. The EC50 values for scavenging superoxide anion and DPPH radical were 1.55 mg/mL and 1.99 mg/mL, respectively. At the cellular level, JPHT-2 was able to protect HaCaT cells from H2O2-induced oxidative damage by increasing the level of superoxide dismutase (SOD) in cells. In conclusion, jellyfish peptides with low molecular weight can be easily obtained through hydrolysis with three enzymes and exhibit excellent antioxidant activity and safety. Jellyfish can serve as a promising source of antioxidants.

Context: Compounds to treat hypothyroidism in the absence of cardiac side effects are urgently required. In this regard, γ-aminobutyric acid (GABA) has gained interest due to its anti-anxiolytic, antihypertensive and antioxidant properties, and reported benefits to the thyroid system. Objective: We investigated the ability of GABA to ameliorate fluoride-induced thyroid injury in mice, and investigated the mechanism(s) associated with GABA-induced protection. Materials and methods: Adult male Kumning mice (N = 90) were exposed to NaF (50 mg/kg) for 30 days as a model of hypothyroidism. To evaluate the effects of GABA administration, fluoride-exposed mice received either thyroid tablets, or low (25 mg/kg), medium (50 mg/kg) or high (75 mg/kg) concentrations of pure GABA orally for 14 days groups (N = 10 each). The effects of low (50 mg/kg); medium (75 mg/kg) and high (100 mg/kg) concentrations of laboratory-separated GABA were assessed for comparison. Effects on thyroid hormone production, oxidative stress, thyroid function-associated genes, and side-effects during therapy were measured. Results: GABA supplementation in fluoride-exposed mice significantly increased the expression of thyroid TG, TPO, and NIS (P

Drug carrier nanoparticles (NPs) were prepared by the polyelectrolyte method, with chitosan sulfate, with different substituents and quaternary ammonium chitosan, including C236-HACC NPs, C36-HACC NPs, and C6-HACC NPs. To evaluate whether the NPs are suitable for loading different antigens, we chose bovine serum albumin (BSA), ovalbumin (OVA), and myoglobin (Mb) as model antigens to investigate the encapsulation effect of the NPs. The characteristics (size, potential, and encapsulation efficiency) of the NPs were measured. Moreover, the NPs with higher encapsulation efficiency were selected for the immunological activity research. The results showed that chitosan derivative NPs with different substitution sites had different loading effects on the three antigens, and the encapsulation rate of BSA and OVA was significantly better than that of Mb. Moreover, the NPs encapsulated with different antigens have different immune stimulating abilities to DCS cells, the immune effect of OVA-coated NPs was significantly better than that of BSA-coated NPs and blank NPs, especially C236-HACC-OVA NPs. Furthermore, we found that C236-HACC-OVA NPs could increase the phosphorylation level of intracellular proteins to activate cell pathways. Therefore, C236-HACC NPs are more suitable for the loading of antigens similar to the OVA structure.

As a popular marine saccharide, chitooligosaccharides (COS) has been proven to have good antioxidant activity. Its antioxidant effect is closely related to its degree of polymerization, degree of acetylation and sequence. However, the specific structure–activity relationship remains unclear. In this study, three chitosan dimers with different sequences were obtained by the separation and enzymatic method, and the antioxidant activity of all four chitosan dimers were studied. The effect of COS sequence on its antioxidant activity was revealed for the first time. The amino group at the reducing end plays a vital role in scavenging superoxide radicals and in the reducing power of the chitosan dimer. At the same time, we found that the fully deacetylated chitosan dimer DD showed the strongest DPPH scavenging activity. When the amino groups of the chitosan dimer were acetylated, it showed better activity in scavenging hydroxyl radicals. Research on COS sequences opens up a new path for the study of COS, and is more conducive to the investigation of its mechanism.

Jellyfish are rich in resources and widely distributed along coastal areas. As a potential approach to respond to jellyfish blooms, the use of jellyfish-derived products is increasing. The citrus spider mite (Panonychus citri) is one of the key citrus pests, negatively impacting the quality and quantity of oranges. Due to the resistance and residue of chemical acaricides, it is important to seek natural substitutes that are environmentally friendly. The field efficacy of the venom from the jellyfish Nemopilema nomurai against P. citri was assayed in a citrus garden. The frozen N. nomurai tentacles were sonicated in different buffers to isolate the venom. The venom isolated by PBS buffer (10 mM, pH 6.0) had the strongest acaricidal activity of the four samples, and the corrected field efficacy 7 days after treatment was up to 95.21%. This study demonstrated that jellyfish has potential use in agriculture.

In this paper, chitooligosaccharides in different salt forms, such as chitooligosaccharide lactate, citrate, adipate, etc., were prepared by the microwave method. They were characterized by SEM, FTIR, NMR, etc., and the nitric oxide (NO) expression was determined in RAW 264.7 cells. The results showed that pure chitooligosaccharide was an irregular spherical shape with rough surface, and its different salt type products are amorphous solid with different honeycomb sizes. In addition to the characteristic absorption peaks of chitooligosaccharides, in FTIR, the characteristic absorption of carboxyl group, methylene group, and aromatic group in corresponding acid appeared. The characteristic absorption peaks of carbon in carboxyl group, hydrogen and carbon in methyl, methylene group, and aromatic group in corresponding acid also appeared in NMR. Therefore, the sugar ring structure and linking mode of chitooligosaccharides did not change after salt formation of chitooligosaccharides. Different salt chitooligosaccharides are completely different in promoting NO secretion by macrophages, and pure chitooligosaccharides are the best.

Chitosan’s activities are significantly affected by degree of deacetylation (DDA), while fully deacetylated chitosan is difficult to produce in a large scale. Therefore, this paper introduces a compression method for preparing 100% deacetylated chitosan with less environmental pollution. The product is characterized by XRD, FT-IR, UV and HPLC. The 100% fully deacetylated chitosan is produced in low-concentration alkali and high-pressure conditions, which only requires 15% alkali solution and 1:10 chitosan powder to NaOH solution ratio under 0.11–0.12 MPa for 120 min. When the alkali concentration varied from 5% to 15%, the chitosan with ultra-high DDA value (up to 95%) is produced.

Oyster (Crassostrea talienwhanensis) protein was hydrolyzed by trypsin to produce peptides with different response values, and response surface methodology (RSM) was applied to optimize the hydrolysis conditions. The highest degree of hydrolysis (DH) of the oyster peptide (OP) was obtained at an enzyme concentration of 1593.2 U/g, a pH of 8.2, a hydrolysis temperature of 40.1 °C, a hydrolysis time of 6.0 h, and a water/material ratio of 8.2. The greatest hydroxyl-radical-scavenging activity of OP was obtained at an enzyme concentration of 1546.3 U/g, a pH of 9.0, a hydrolysis temperature of 50.2 °C, a hydrolysis time of 5.1 h, and a water/material ratio of 5.6. The largest branched-chain amino acid (BCAA) content of OP was obtained at an enzyme concentration of 1323.8 U/g, a pH of 8.3, a hydrolysis temperature of 41.7 °C, a hydrolysis time of 6.7 h, and a water/material ratio of 4.8. The three experimental values were significantly in agreement with the predicted values within the 95% confidence interval. Furthermore, ultrafiltration and chromatographic methods were used to purify the OP, and 13 peptides that were rich in Lys, Arg, His, and Thr were identified by LC-MS/MS. The results of this study offer different optimum hydrolysis conditions to produce target peptides from oyster protein by using RSM, and this study provide a theoretical basis for the high-value utilization of oyster protein.

Plant root-knot nematode disease is a great agricultural problem and commercially available nematicides have the disadvantages of high toxicity and limited usage; thus, it is urgent to develop new nematicides derived from nature substances. In this study, a novel fluorinated derivative was synthesized by modifying chitosan oligosaccharide (COS) using the strategy of multiple functions. The derivatives were characterized by FTIR, NMR, elemental analysis, and TG/DTG. The activity assays show that the derivatives can effectively kill the second instar larvae of Meloidogyne incognita in vitro, among them, chitosan-thiadiazole-trifluorobutene (COSSZFB) perform high eggs hatching inhibitory activity. The derivatives can regulate plant growth (photosynthetic pigment), improve immunity (chitinase and β-1,3-glucanase), and show low cytotoxicity and phytotoxicity. According to the multi-functional activity, the derivatives exhibit a good control effect on plant root-knot nematode disease in vivo. The results demonstrate that the COS derivatives (especially fluorinated derivative) perform multiple activities and show the potential to be further evaluated as nematicides.

Mackerel (Pneumatophorus japonicus) viscera contain large amount of protein. We used five proteases to hydrolyze the viscera, and the hydrolysate treated by neutrase exhibited the highest nitrogen recovery (NR). Then we optimized the preparation conditions for mackerel viscera hydrolysate (MVH) by response surface methodology and investigated the antioxidant activity of MVH. The optimal conditions were as follows: enzyme concentration of 1,762.87 U/g, pH of 6.76, temperature of 43.75 °C, extraction time of 6.0 h and water/material ratio of 20.37 (v/w), and the maximum NR was 37.84%. Furthermore, the molecular weight distribution of MVH was almost below 3,000 Da determined by TSK G2000 SWXL gel filtration chromatography, and the MVH exhibited good antioxidant activities in various in vitro assays, including DPPH radical, hydroxyl radical and superoxide anion scavenging activities, reducing power and similar effectivelness as butylated hydroxytoluene and Vitamin E to inhibit lipid peroxidation. The results suggested that MVH could be used as a potential source of antioxidant peptide in food industries.

The structure modification of chitosan has great application potential. 6-Oxychitosan was prepared by specially oxidizing the C6-OH of chitosan, then 6-oxychitosan was reacted with three kinds of aldehydes to prepare N-quaternized 6-oxychitosan derivatives in this paper. The derivatives were characterized by FT-IR, NMR, and elemental analysis. The antimicrobial activity of these derivatives was tested against two common plant-threatening fungi and three plant disease bacteria. The results showed that N-quaternized 6-oxychitosan derivatives had good water-solubility and excellent antimicrobial activity. Moreover, derivative 3 which connected 8-hydroxyquinolines had the highest antimicrobial activity than the other derivatives. The inhibitory indices of derivative 3 against V. albo-atrum and P. hibernalis are 89.1% and 72.8% at 0.4 mg/ml. The MICs of 3 against X. oryzae, P. syringae, and E. rhapontici were 625, 625, and 156 mg/l, respectively. All the results indicate that derivative 3 has the potential of becoming an alternative to harmful agricultural chemicals.

The silkworm Bombyx mori L. (B. mori) has a significant impact on the economy by producing more than 80% of the globally produced raw silk. The exposure of silkworm to pesticides may cause adverse effects on B. mori, such as a reduction in the production and quality of silk. This study aims to assay the effect of venom from the jellyfish Nemopilema nomurai on growth, cuticle and acetylcholinesterase (AChE) activity of the silkworm B. mori by the leaf dipping method. The experimental results revealed that the four samples caused neither antifeeding nor a lethal effect on B. mori. The sample SFV inhibited B. mori growth after 6 days of treatment in a dose-dependent manner. The samples SFV, DSFV and Fr-1 inhibited the precipitation and synthesis of chitin in the cuticle after 12 and 14 days of treatment. In the case of the four samples, the AChE was significantly improved after 14 days of treatment.

The present study investigated and compared the hypoglycemic activity of differently regioselective chitosan sulfates in alloxan-induced diabetic rats. Compared with the normal control rats, significantly higher blood glucose levels were observed in the alloxan-induced diabetic rats. The differently regioselective chitosan sulfates exhibited hypoglycemic activities at different doses and intervals, especially 3-O-sulfochitosan (3-S). The major results are as follows. First, 3,6-di-O-sulfochitosan and 3-O-sulfochitosan exhibited more significant hypoglycemic activities than 2-N-3, 6-di-O-sulfochitosan and 6-O-sulfochitosan. Moreover, 3-S-treated rats showed a more significant reduction of blood glucose levels than those treated by 3,6-di-O-sulfochitosan. These results indicated that –OSO3− at the C3-position of chitosan is a key active site. Second, 3-S significantly reduced the blood glucose levels and regulated the glucose tolerance effect in the experimental rats. Third, treatment with 3-S significantly increased the plasma insulin levels in the experimental diabetic rats. A noticeable hypoglycemic activity of 3-S in the alloxan-induced diabetic rats was shown. Clinical trials are required in the future to confirm the utility of 3-S.

Chitosan plays an important role in regulating growth and eliciting defense in many plant species. However, the exact metabolic response of plants to chitosan is still not clear. The present study performed an integrative analysis of metabolite profiles in chitosan-treated wheat seedlings and further investigated the response of enzyme activities and transcript expression related to the primary carbon (C) and nitrogen (N) metabolism. Metabolite profiling revealed that chitosan could induce significant difference of organic acids, sugars and amino acids in leaves of wheat seedlings. A higher accumulation of sucrose content was observed after chitosan treatment, accompanied by an increase in sucrose phosphate synthase (SPS) and fructose 1, 6-2 phosphatase (FBPase) activities as well as an up-regulation of relative expression level. Several metabolites associated with tricarboxylic acid (TCA) cycle, including oxaloacetate and malate, were also improved along with an elevation of phosphoenolpyruvate carboxylase (PEPC) and pyruvate dehydrogenase (PDH) activities. On the other hand, chitosan could also enhance the N reduction and N assimilation. Glutamate, aspartate and some other amino acids were higher in chitosan-treated plants, accompanied by the activation of key enzymes of N reduction and glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. Together, these results suggested a pleiotropic modulation of carbon and nitrogen metabolism in wheat seedlings induced by chitosan and provided a significant insight into the metabolic mechanism of plants in response to chitosan for the first time, and it would give a basic guidance for the future application of chitosan in agriculture.

The aim of this study was to isolate and purify antioxidative peptides from Pacific herring (Clupea pallasii) protein. Five enzymes (pepsin, trypsin, papain, flavourzyme, and neutrase) were used for protein hydrolysis, and Pacific herring protein hydrolysates (PHPH) were separated by ultrafiltration. The fraction with the molecular weight below 3500 Da exhibited the highest in vitro antioxidant activities and cellular antioxidant activity. The PHPH was isolated and purified by consecutive chromatographic methods including gel filtration chromatography and reverse high-performance liquid chromatography (RP-HPLC). The purified antioxidant peptides were identified as Leu-His-Asp-Glu-Leu-Thr (MW = 726.35 Da) and Lys-Glu-Glu-Lys-Phe-Glu (MW = 808.40 Da), and the IC50 values of cellular antioxidant activity were 1.19 ± 0.05 mg/mL and 1.04 ± 0.06 mg/mL. The results demonstrate that is possible to produce natural antioxidative peptides from Pacific herring protein via enzymatic hydrolysis and purification.

Jellyfish envenomations result in extensive dermatological symptoms, clinically named as jellyfish dermatitis, which can seriously affect the daily activities and physical health of people. Inflammatory response accompanies the whole process of jellyfish dermatitis and the complexity of jellyfish venom components makes it difficult to treat jellyfish dermatitis symptoms effectively. Moreover, inhibiting inflammation is essential for the treatment of jellyfish stings and exploring the main components of jellyfish venom that cause inflammation is an urgent research area. In this study, the inhibitory effects of matrix metalloproteinase (MMP) inhibitors for venom-induced inflammation were explored at a cellular level. The expression of the three inflammatory factors, IL-6, TNF-α and MCP-1 in two skin cell lines, human keratinocyte cells (HaCaT) and human embryonic skin fibroblasts cells (CCC-ESF-1), at the cellular level, after treatment with the inhibitors of jellyfish Nemopilema nomurai (N. nomurai) nematocyst venom (NnNV-I), were determined. The results showed that inhibitors of MMP can significantly reduce the toxic effects of jellyfish Nemopilema nomurai nematocyst venom (NnNV) to skin cells. The expression levels of the three inflammatory factors IL-6, MCP-1, and TNF-α in the cells were also significantly decreased, indicating that MMPs in jellyfish venom are probably vital factors leading to jellyfish dermatitis. This study is beneficial in the prevention and treatment of jellyfish stings.

Chitooligosaccharides (COS), the hydrolyzed products of chitin and chitosan, can be obtained by various methods. In this study, water-soluble COS were prepared from α- and β-chitosan by microwave-assisted degradation and their immunostimulatory effects were investigated in RAW 264.7 macrophages. The results indicated that α-COS were more active than β-COS in promoting the production of nitric oxide (NO) and cytokines, such as tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). Quantitative real-time reverse transcription polymerase chain reaction and Western blotting indicated that COS also enhanced the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α. Further analyses demonstrated that COS induced the phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38, p85 and Akt, and the nuclear translocation of p65, indicating that they are able to activate the mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinases (PI3K)/Akt signaling pathways dependent on nuclear factor (NF)-κB activation. In conclusion, COS activate RAW 264.7 cells via the MAPK and PI3K/Akt signaling pathways and are potential novel immune potentiators.

In this study, 3-methyl-1,2,4-triazolyl chitosan (MTACS) and 3-chloromethyl-1,2,4-triazolyl chitosan (CMTACS) were prepared via cyclization of acyl thiourea chitosan (TUCS). Their structures were confirmed by FT-IR, 1H-NMR, elemental analysis, DSC, XRD, and SEM. The conformations were predicted using the Gaussian 09 program. Additionally, the antifungal properties of MTACS and CMTACS against Stemphylium solani weber (S. solani), Alternaria porri (A. porri), and Gloeosporium theae-sinensis (G. theae-sinensis) were assayed in vitro and ranged from 250 μg/mL to 1000 μg/mL. The results showed that MTACS and CMTACS exhibited enhanced inhibitory effect on the selected fungi compared to the original chitosan and TUCS. In particular, they displayed better antifungal activities against A. porri and G. theae-sinensis than that of the positive control, Triadimefon. The findings described here may lead to them being used as antifungal agents for crop protection.

Fungi isolated from marine invertebrates are of considerable importance as new promising sources of unique secondary metabolites with significant biomedical potential. However, the cultivable fungal community harbored in jellyfish was less investigated. In this work, we seek to recover symbiotic fungi from different tissues of jellyfish Nemopilema nomurai. A total of seven morphotypes were isolated, which were assigned into four genera (Aspergillus, Cladosporium, Purpureocillium, and Tilletiopsis) from two phyla (Ascomycota and Basidiomycota) by comparing the rDNA-ITS sequences with the reference sequences in GenBank. The most fungi were found in the inner tissues of subumbrella. Two of the cultivation-independent procedures, changing media type and co-cultivation, were employed to maximize the complexity of metabolites. Thus, thirteen EtOAc gum were obtained and fingerprinted by High Performance Liquid Chromatography (HPLC) equipped with a photodiode array (PDA) detector. Antibacterial and antifungal activities of these complex mixtures were tested against a panel of bacterial and fungal pathogens. The antimicrobial results showed that all of the 13 EtOAc extracts displayed different levels of antibacterial activity, three of which exhibited strong to significant antibacterial activity to the bacterial pathogens Staphylococcus aureus and Salmonella entrica. Antifungal activity indicated that the EtOAc extracts from pure culture of Aspergillus versicolor and co-culture of A. versicolor and Tilletiopsis sp. in rice media were promising for searching new compounds, with the maximal mycelial growth inhibition of 82.32% ± 0.61% for Rhizoctonia solani and 48.41% ± 11.02% for Botrytis cinerea at 200 μg/ml, respectively. This study is the first report on the antibacterial and antifungal activity of jellyfish-associated fungi and allows the first sight into cultivable fungal community residing in jellyfish. Induced metabolites by cultivation-dependent approaches provides a new reservoir for drug discovery from jellyfish-derived fungi.

Background: Medusozoans utilize explosively discharging penetrant nematocysts to inject venom into prey. These venoms are composed of highly complex proteins and peptides with extensive bioactivities, as observed in vitro. Diverse enzymatic toxins have been putatively identified in the venom of jellyfish, Nemopilema nomurai and Cyanea nozakii, through examination of their proteomes and transcriptomes. However, functional examination of putative enzymatic components identified in proteomic approaches to elucidate potential bioactivities is critically needed. Methods: In this study, enzymatic toxins were functionally identified using a combined approach consisting of in gel zymography and liquid chromatography tandem mass spectrometry (LC-MS/MS). The potential roles of metalloproteinases and lipases in hemolytic activity were explored using specific inhibitors. Results: Zymography indicated that nematocyst venom possessed protease-, lipase- and hyaluronidase-class activities. Further, proteomic approaches using LC-MS/MS indicated sequence homology of proteolytic bands observed in zymography to extant zinc metalloproteinase-disintegrins and astacin metalloproteinases. Moreover, pre-incubation of the metalloproteinase inhibitor batimastat with N. nomurai nematocyst venom resulted in an approximate 62% reduction of hemolysis compared to venom exposed sheep erythrocytes, suggesting that metalloproteinases contribute to hemolytic activity. Additionally, species within the molecular mass range of 14–18 kDa exhibited both egg yolk and erythrocyte lytic activities in gel overlay assays. Conclusion: For the first time, our findings demonstrate the contribution of jellyfish venom metalloproteinase and suggest the involvement of lipase species to hemolytic activity. Investigations of this relationship will facilitate a better understanding of the constituents and toxicity of jellyfish venom.