CRISPR/Cas9-based gene knockout in animal cells, particularly in teleosts, has proven to be very efficient with regards to mutation rates, but the precise insertion of exogenous DNA or gene knock-in via the homology-directed repair (HDR) pathway has seldom been achieved outside of the model organisms. Here, we succeeded in integrating with high efficiency an exogenous alligator cathelicidin gene into a targeted non-coding region of channel catfish (Ictalurus punctatus) chromosome 1 using two different donor templates (synthesized linear dsDNA and cloned plasmid DNA constructs). We also tested two different promoters for driving the gene, zebrafish ubiquitin promoter and common carp β-actin promoter, harboring a 250-bp homologous region flanking both sides of the genomic target locus. Integration rates were found higher in dead fry than in live fingerlings, indicating either off-target effects or pleiotropic effects. Furthermore, low levels of mosaicism were detected in the tissues of P₁individuals harboring the transgene, and high transgene expression was observed in the blood of some P₁fish. This can be an indication of the localization of cathelicidin in neutrophils and macrophage granules as also observed in most antimicrobial peptides. This study marks the first use of CRISPR/Cas9 HDR for gene integration in channel catfish and may contribute to the generation of a more efficient system for precise gene integration in catfish and other aquaculture species, and the development of gene-edited, disease-resistant fish.

Seaweeds are increasingly valued in the food industry for their bioactive compounds, mainly hydrocolloids like carrageenan. This study investigates E. perplexum, a red seaweed, as a unique and sustainable source of carrageenan with promising functional properties. Using response surface methodology (RSM), the extraction process was optimized through alkaline extraction, identifying optimal conditions of 85 °C for 3 h with 2.58 M KOH, which yielded 77.10% carrageenan. The extracted carrageenan exhibited strong emulsifying activity (71.53 ± 2.41) and color properties comparable to commercial carrageenan, highlighting its viability for food applications. Chemical evaluation revealed a higher sulfate content (8.45 ± 0.16) and slightly reduced carbohydrate levels, which may influence its gelling and stabilizing abilities. Structural examination through ATR-FTIR spectroscopy corroborates the presence of key functional groups, including sulfate esters and galactose derivatives, inferring molecular integrity. These results emphasize the importance of RSM in optimizing extraction and underscore the ability of E. perplexum as a promising source of the derived carrageenan, which is a high-performance additive in food systems. Further research on purification, functional enhancement, and safety assessment is recommended to facilitate its integration into commercial food systems.

Lactic acid bacteria from a fermented rice-shrimp mixture traditionally known in the Philippines as Balao-balao were isolated in the present study. Screening of lactic acidproducing strains revealed that ten isolates showed promising inhibitory activities against test microorganisms namely Staphylococcus aureus BIOTECH 1634, Escherichia coli BIOTECH 1582, Bacillus subtilis BIOTECH 1679 and Vibrio harveyi SEAFDEC 010. Of interest was isolate PL12, a bacteriocin-producing strain which showed the highest inhibitory activity against the pathogens tested. Isolate PL12 was identified as Pediococcus pentosaceus (GenBank accession number MF353992) with 100% similarity by 16S rDNA sequence analyses. Excluding the effects of organic acids and hydrogen peroxide, the cell-free supernatant (CFS) of PL12 isolate exhibited strong antagonistic activities against test pathogens in an agar well diffusion assay. These results confirmed the isolate’s proteinaceous nature and are indicative of typical characteristics of bacteriocins. To further concentrate the proteins present in the CFS, ammonium sulfate precipitation followed by column purification (Sep-Pak C18 cartridge column) was performed. Positive antagonism of PL12 bacteriocin was observed in both Gram-positive and Gram-negative bacteria tested. The highest inhibitory activity was found against E. coli in every purification step. These results suggested that bacteriocin-producing PL12 isolate can be a promising preservative in the food industry and as probiotics in aquaculture since it possesses antagonistic activities against V. harveyi.