Implications of green fish feed for consumer safety - carry-over of plant peptides, natural toxins and bioactive compounds

Feed for farmed fish consists nowadays mostly of plant ingredients because there are not enough marine raw materials to cover the growing demand. Plants commonly used in fish feed are wheat and legumes like soy and pea. The significant changes in the feed can have consequences for the fish and for the consumers of fish-based food. The provision of the fish with enough nutrients has been achieved through the production of innovative feed compositions. In contrast, potential effects on consumers have been less investigated. Thus, the Norwegian Scientific Committee for Food Safety (VKM) had pointed out in a benefit-risk assessment of fish and fish products that there was a need to find out if some plant ingredients might be transferred from the feed to the edible parts of fish, which could be a risk for human health. The SafeFish project examined if some typical plant ingredients can be carried over from feed to food. Three different substance groups were analysed: potentially allergenic fragments of plant proteins (peptides), toxins produced by plant-colonising fungi (mycotoxins) and plant-produced compounds that resemble human hormones (phytoestrogens). Fish feed containing specific amounts of wheat, soy or pea was produced and used in studies with zebrafish and on-growing farmed salmon under defined conditions in a research facility. Samples of the fish were analysed with different methods to search for transferred substances, to look for possible changes in the biological functions of the fish, and to evaluate the potential health risk of consumers. The analytical methods used ranged from the specific detection of mycotoxins, phytoestrogens and proteins by mass spectrometry to the analysis of physiological changes in the fish by DNA and RNA technologies, and testing of the allergenic potential of edible parts from plant-fed fish with sera of patients with food allergy. The project was a collaboration of specialised teams at two Norwegian research institutes, two Norwegian universities, a Norwegian university hospital and a US-university and included the training of a PhD student. The project started in July 2016. Each year, a project meeting was held to discuss strategies, progress and result dissemination between the partners. The project progressed according to the planned schedule. Project-specific chemical and proteomic analyses were developed for the targeted mycotoxins, soybean pseudo-hormones and wheat gluten peptides. Custom-made fish feed was prepared and feeding studies in zebrafish and salmon performed. All samples from the fish studies were analysed for mycotoxins and phytoestrogens showing that the marginal amounts detected are not of health concern to consumers. The results have been published in the journal "Toxins". We were successful in developing an in vitro assay for the biotransformation of contaminants using a preparation from salmon liver (first reported functional assay). The method was used to characterise the products of phytoestrogen metabolism in fish. We found that salmon can detoxify phytoestrogens by conjugation to certain sugar molecules (glucuronidation), and that therefore human exposure from the consumption of fish products is rather unlikely. The results have been published in the journal «Ecotoxicology and Environmental Safety». We also developed an innovative method for the digestion of gluten proteins with different enzyme, which was used to prepare samples of gluten-exposed fish for proteomic analyses by mass spectrometry. In parallel, we used sera of patients with wheat allergy in immunoblot analyses to detect traces of gluten proteins in exposed fish. The experiments showed that very small amounts of gluten peptides might be transferred into the fish. Additional experiments were performed for the verification of these findings, and the results published in 2021. DNA- and RNA technologies were applied for an extensive study of possible changes in salmon and zebrafish fed with plant-based diets. We found that in particular feed with a high percentage of gluten activated genes in zebrafish muscle that are connected to inflammation reactions. The results have been published in the journal «Frontiers in Genetics». Potential effects on the liver and intestine of salmon were also analysed. The results have been published in «Genes». Additional findings on gene expression changes in zebrafish milt and epigenetic changes in salmon liver were published in 2021. The project results were presented at several national (e.g. Havbruk) and international conferences. The PhD student connected to this project has defended her thesis on 4/12/2020 at NMBU and achieved a PhD. A final (digital) project seminar with all partners was held. The project has ended on 31.12.2020.

No risk for consumers of fish produced with three plant-based feeds was found regarding allergenic peptides, mycotoxins and phytoestrogens, but a negative impact on fish health is possible, especially from enteropathological inflammation. Specific outcomes: - Development of 25-in-1 LC-HRMS/MS suitable for analysis of mycotoxins and phytoestrogens in feed and fish. - Simultaneous characterisation of 5 major CYP P450 and 5 major UGT enzymes in liver fractions using specific substrates and LC-TripleQ-MS/MS analysis. - Establishment of generally applicable salmon in vitro biotransformation assays using primary hepatocytes or liver microsomes. - Novel sequential protease digestion method for wheat gluten sample preparation in proteomics. - Development of label-free semi-quantitative nLC-nESI-MS/MS and quantitative LC-TripleQ-MS/MS for proteomics of gluten peptides in feed and fish. - Workflows for the nutrigenomic and nutriepigenomic analysis of different fish tissues.

The continued growth of Norwegian aquaculture depends on the utilisation of new, sustainable protein resources for fish feed since marine reserves are limited. The last decade has seen a tremendous transition from marine to agricultural feed materials, which currently make up to 70 % of the aquafeeds used in Norwegian salmon farming. Commonly used are protein preparations derived from wheat and legume plants, mostly soy and pea. This complete change in the diet of fish has led to new challenges regarding fish health and welfare, and product quality. Many initial problems have been solved by extensive research on feed processing methodologies and feed compositions. The Norwegian Scientific Committee for Food Safety (VKM) has, however, recently pointed out in an updated benefit-risk assessment of fish and fish products that plant ingredients may introduce new contaminants into feed, which could be transferred into the eatable portion of fish and might be a risk for consumer safety. The SafeFish project attempts to fill this data gap by determining the carry-over potential of three typical constituents of plants: plant peptides (stable fragments of plant proteins), mycotoxins (products of fungi infections), and hormone-like compounds (phytoestrogens). Custom-made feedingstuffs containing different amounts of typical plant protein preparations will be used in studies in a zebrafish model and in on-growing salmon. Fish muscle and organs will be analysed with a multitude of analytical methods (proteomics, chemical analysis, fish nutrigenomics, fish transcriptomics, immunochemical assays including test with food allergic patients) to study effects on fish physiology and substance carry-over, and finally to access consumer health risk. This multi-disciplinary project involves the collaboration of two Norwegian research institutes, two Norwegian universities, a Norwegian university hospital and one university in the USA, and includes the training of one PhD student