The phenolic compounds hydroxycinnamates, anthocyanins, flavonols, and flavan-3-ols of sweet cherry cultivars Burlat, Saco, Summit, and Van harvested in 2001 and 2002 were quantified by HPLC-DAD. Phenolics were analyzed at partially ripe and ripe stages and during storage at 15 +/- 5 degrees C (room temperature) and 1-2 degrees C (cool temperature). Neochlorogenic and p-coumaroylquinic acids were the main hydroxycinnamic acid derivatives, but chlorogenic acid was also identified in all cultivars. The 3-glucoside and 3-rutinoside of cyanidin were the major anthocyanins. Peonidin and pelargonidin 3-rutinosides were the minor anthocyanins, and peonidin 3-glucoside was also present in cvs. Burlat and Van. Epicatechin was the main monomeric flavan-3-ol with catechin present in smaller amounts in all cultivars. The flavonol rutin was also detected. Cultivar Saco contained the highest amounts of phenolics [227 mg/100 g of fresh weight (fw)] and cv. Van the lowest (124 mg/100 g of fw). Phenolic acid contents generally decreased with storage at 1-2 degrees C and increased with storage at 15 +/- 5 degrees C. Anthocyanin levels increased at both storage temperatures. In cv. Van the anthocyanins increased up to 5-fold during storage at 15 +/- 5 degrees C (from 47 to 230 mg/100 g of fw). Flavonol and flavan-3-ol contents remained quite constant. For all cultivars the levels of phenolic acids were higher in 2001 and the anthocyanin levels were higher in 2002, which suggest a significant influence of climatic conditions on these compounds.

Saponins are naturally occurring amphiphilic molecules and have been associated with many biological activities. The aim of the present study was to investigate whether soya saponins trigger the onset of soyabean-induced enteritis in Atlantic salmon (Salmo salar L.), and to examine if dietary soya saponins increase the epithelial permeability of the distal intestine in Atlantic salmon. Seven experimental diets containing different levels of soya saponins were fed to seawater-adapted Atlantic salmon for 53 d. The diets included a fishmeal-based control diet, two fishmeal-based diets with different levels of added soya saponins, one diet containing 25 % lupin kernel meal, two diets based on 25 % lupin kernel meal with different levels of added soya saponins, and one diet containing 25 % defatted soyabean meal. The effect on intestinal morphology, intestinal epithelial permeability and faecal DM content was examined. Fish fed 25 % defatted soyabean meal displayed severe enteritis, whereas fish fed 25 % lupin kernel meal had normal intestinal morphology. The combination of soya saponins and fishmeal did not induce morphological changes but fish fed soya saponins in combination with lupin kernel meal displayed significant enteritis. Increased epithelial permeability was observed in fish fed 25 % defatted soyabean meal and in fish fed soya saponin concentrate independent of the protein source in the feed. The study demonstrates that soya saponins, in combination with one or several unidentified components present in legumes, induce an inflammatory reaction in the distal intestine of Atlantic salmon. Soya saponins increase the intestinal epithelial permeability but do not, per se, induce enteritis.

The current work aimed at tracing the causative components for soybean-induced enteritis in Atlantic salmon (Salmo salar L.). Soybean molasses was subjected to phase separation using n-butanol. Three subfractions were obtained as follows: butanol phase, precipitate, and water phase. The biochemical composition of soybean molasses and the obtained subfractions were analyzed in detail: Protein, fat, and ash were quantified according to standard methods. Sucrose, raffinose, and stachyose were quantified using high-performance anion-exchange chromatography. Soyasaponins were quantified using reverse-phase high-performance liquid chromatography. Finally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to evaluate the size distribution of the proteins present in each fraction. Molasses and the different subfractions were thereafter fed to Atlantic salmon in two successive fish trials. The level of intestinal inflammation was evaluated by light microscopy using a semiquantitative scoring system. Histological assessments revealed that Atlantic salmon fed a combination of butanol phase and precipitate displayed significant enteritis. Atlantic salmon fed the water phase displayed normal intestinal morphology. The causative components for soybean-induced enteritis withstand butanol treatment and prolonged heating at 70 degrees C. Sucrose, raffinose, stachyose, nor soybean proteins larger than 10 kDa induce enteritis alone. Soyasaponins, or components that follow the same solubility pattern, trigger the inflammatory reaction. We therefore suggest that soybean-induced enteritis in Atlantic salmon is induced by soyasaponins alone or by soyasaponins in combination with other factors, e.g., antigenic soybean proteins or the intestinal microflora.

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The stability of soyasaponins in fish feed formulations was investigated. The level of soyasaponin Ab, Bb, Bc, Ba-2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (Ba-DDMP), Bb-DDMP, and Bc-DDMP was quantified in 15 samples of defatted soybean meal, two full fat soybean meals, and two soybean protein concentrates by reverse phase high-performance liquid chromatography. The total level of saponins in the 15 samples of commercial defatted soybean meal ranged from 4.8-6.8 micromol/g (5.1-7.0 g/kg). The two full fat meals contained 4.4 and 4.7 micromol/g whereas no saponins could be detected in the alcohol-extracted soybean protein concentrates. Fifteen batches of fish feed containing 20% defatted soybean meal were produced by twin-screw extrusion from the 15 different samples of defatted soybean meal. Extrusion did not reduce the total level of group B saponins, but the ratio between DDMP-conjugated group B saponins and non-DDMP-conjugated group B saponins was slightly reduced. A soybean-containing diet was fed to seawater adapted Atlantic salmon for 9 weeks. Yttrium oxide was included in the feed as an inert marker in order to estimate the disappearance of saponins during gut passage. High levels of intact non-DDMP-conjugated group B soyasaponins were found in feces whereas only low levels of DDMP-conjugated saponins could be detected. The overall disappearance of saponins was close to zero, and the concentration of intact saponins in dry feces reached levels several fold higher than dietary levels. The present work demonstrates that non-DDMP-conjugated group B soyasaponins resist extrusion cooking and remain intact during gut passage in Atlantic salmon. The latter is contrary to earlier findings in endothermic animals.

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The aquaculture industry has become a sustainable source of food for humans. Remaining challenges include disease issues and ethical concerns for the discomfort and stress of farmed fish. There is a need for reliable biomarkers to monitor welfare in fish, and the stress hormone cortisol has been suggested as a good candidate. This study presents a novel method for measurement of cortisol in fish feces based on enzymatic hydrolysis, liquid–liquid extraction, derivatization, and finally instrumental analysis by liquid chromatography coupled with tandem mass spectrometry. Hydrolysis and extraction conditions were optimized. Cortisol appeared to be mostly conjugated to sulfate and less conjugated to glucuronic acid in the studied samples of feces from farmed Atlantic salmon. The method was suitable for quantification of cortisol after enzymatic deconjugation by either combined glucuronidase and sulfatase activity, or by glucuronidase activity alone. The limit of detection was 0.15 ng/g, the limit of quantification was 0.34 ng/g, and the method was linear (R2 > 0.997) up to 380 ng/g, for measurement of cortisol in wet feces. Method repeatability and intermediate precision were acceptable, both with a coefficient of variation (CV) of 11%. Stress level was high in fish released into seawater, and significantly reduced after eight days.

Measuring the level of steroid and thyroxine hormones is key to understanding organism health conditions. Liquid chromatography coupled with tandem mass spectrometry has become the method of choice for such hormone analyses in clinical laboratories. Detection of hormones at low levels typically requires a time-consuming sample preparation, such as liquid-liquid extraction followed by solvent evaporation and re-solubilization of the sample extract. Instead, we applied salting-out assisted liquid-liquid extraction (SALLE) for the extraction of thyroxine, testosterone, cortisone, and cortisol from human serum and fish plasma samples. SALLE allowed direct injection of sample extracts. Sodium chloride and ammonium sulfate were evaluated as salting-out reagents together with four different organic solvents. High extraction recovery and reduced matrix interference were achieved by using ammonium sulfate together with 10% methanol in acetonitrile. Limits of quantification were in the range of 0.1–0.2 ng/mL and signal responses were linear (R2 > 0.997) up to at least 100 ng/mL for all hormones. The method was applied for hormone measurements in fish plasma. In conclusion, SALLE combines the simplicity of crude protein precipitation with the high analyte enrichment of a liquid-liquid extraction. Here we have presented it as a novel sample preparation method for clinical laboratories where mass spectrometry is utilized in the field of endocrinology.