Comparison of amino acid release between enzymatic hydrolysis and acid autolysis of rainbow trout viscera.

Fish protein hydrolysates were obtained from cultured rainbow trout (Oncorhynchus mykiss) viscera using commercial and endogenous enzymes. Two methods were employed for hydrolysis: acid autolysis (also known as silage) at room temperature for 10 days in acidic conditions, until total solubilisation, and enzymatic hydrolysis using Alcalase 2.4 LFG, Protana Prime, and the endogenous enzymes in the viscera. The effectiveness of both methods in releasing free amino acids (FAA) was assessed. After evaluating the results, the most effective enzymatic hydrolysis was optimized. The findings indicated that enzymatic hydrolysis with Alcalase, Protana Prime and endogenous enzymes combined for 7 h at a dose of 1% of protein, and a 7-day acid autolysis yielded the highest degree of hydrolysis (83.8% and 75.8%), a yield of FAA from viscera of 5.9% and 3.2%, and a yield of FAA from total protein of 71.3% and 52.5%, respectively. In conclusion, the use of commercial enzymes was more efficient in releasing amino acids, but endogenous enzymes showed a strong proteolytic capacity during acid autolysis, suggesting it also as a promising method to produce FAA-rich hydrolysates.

Determination of fluoroquinolones in fish tissues, biological fluids, and environmental waters by liquid chromatography tandem mass spectrometry.

This work describes the optimization, validation, and application in real samples of accurate and precise analytical methods to determine ten fluoroquinolones (FQs) (norfloxacin, enoxacin, pefloxacin, ofloxacin, levofloxacin, ciprofloxacin, danofloxacin, lomefloxacin, enrofloxacin, and sparfloxacin) in different environmental matrices, such as water (estuarine, seawater, and wastewater treatment plant effluent), fish tissues (muscle and liver), and fish biofluids (plasma and bile). The analysis step performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was fully optimized to improve the separation and detection steps. The extraction of analytes from fish tissues was accomplished using focused ultrasound solid-liquid extraction using methanol/acetic acid (95:5 v/v) as extractant. The preconcentration and clean-up steps were optimized in terms of extraction efficiency and cleanliness and the best strategy for each matrix was selected: (i) Oasis HLB for seawater and muscle, (ii) liquid-liquid extraction combined with Oasis HLB for the lipid-rich liver, (iii) the combination of Evolute-WAX and Oasis HLB for estuarine water and wastewater treatment plant effluent, and (iv) molecular imprinted polymers for biofluids. The methods afforded satisfactory apparent recoveries (80-126%) and repeatability (RSD < 15%), except for sparfloxacin, which showed a lack of correction with the available isotopically labeled surrogates ([2H8]-ciprofloxacin and [2H5]-enrofloxacin). Ciprofloxacin, norfloxacin, and ofloxacin were detected in both water and fish liver samples from the Biscay Coast at concentrations up to 278 ng/L and 4 ng/g, respectively. To the best of our knowledge, this work is one of the few analyzing up to ten FQs and in so many fish tissues and biofluids. Graphical abstract Determination of fluoroquinolones in different environmental matrices, such as water (estuarine, seawater, and wastewater treatment plant effluent), fish tissues (muscle and liver), and fish biofluids (plasma and bile).