Methods: Aquaculture allows fish to be farmed under strict controlled conditions. However, knowledge on how farming practices can be used to modulate fish allergenicity is inexistent. Creatine is a nitrogenous organic acid that occurs naturally in vertebrates and helps to supply energy to cells, primarily muscle. Creatine was verified to reduce in 75% the expression of parvalbumin in rat skeletal muscle . A trial was performed with S. aurata using 3 different concentrations of creatine (2%, 5% and 8%) that were added to a control diet (no creatine supplementation) and tested in tanks of 25 individuals in triplicate. At the end of the trial, plasma and muscle were individually collected for further analysis. Creatine levels in muscle samples were analysed. Cortisol was measured in plasma to address fish welfare/stress levels. Parvalbumin detection in muscle samples was studied using both proteomics and Western Blot techniques and an ELISA commercial kit (Bio-Check, UK). Comparative proteomics was performed on muscle samples to study differences in protein expression between the various treatments and further understand the effect of creatine in fish metabolism. To address this we used 2D Difference Gel Electrophoresis (DIGE). Differences in expression were analysed using the Samespots software (Totallab). Proteins with significant differences (P<0.05) were excised manually and identified by MALDI-TOF/TOF.
Results: Cortisol levels are similar to basal levels reported for S. aurata, although the treatment with 8% creatine shows a significant reduction compared with the control diet (p=0.03). Parvalbumin concentrations show no significant differences between the treatments. Creatine concentrations in muscle show a slight decrease in fish fed with supplemented diets. Comparative proteomics in muscle show some differences in protein expression submitted to diets supplemented with creatine. The four protein spots identified as parvalbumins show no significant differences in expression. Proteins differentially expressed are currently being identified by MALDI-TOF/TOF.
Conclusion and current work: No accumulation of creatine was found in muscle of fish fed with diets supplemented with creatine. The tested creatine percentages in fish diets did not significantly affect S. aurata allergenic potential. Current work involving new formulated diets and/or different fish species is underway. We expect in a near future to develop a specific fish diet that will target the expression of fish parvalbumins in order to reduced its allergenic potential.
 Gallo, M., et al. 2008. Am J physiol, 294, R1319–R1328.