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

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. The most commonly used protein preparations are derived from wheat gluten and legume plants, mostly soy and pea.

This complete change in fish diets 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), however, has pointed out 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 health.

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 have been used in studies in a zebrafish model and in on-growing salmon. Fish muscle and organs are currently analysed with a multitude of analytical methods (proteomics, chemical analysis, fish nutrigenomics, fish transcriptomics, immunochemical assays including screening tests with food allergic patients) to study effects on fish physiology and substance carry-over, and finally to access consumer health risk.

The SafeFish project is a collaboration of the Norwegian Veterinary Institute, Nofima, the University of Oslo, the Nord University in Bodø, Haukeland University Hospital in Bergen and the Mass Spectrometry Center of the University of Colorado in Denver, USA. Amritha Johny is PhD student on the project. Additionally, the PostDoc project (EpiGreen) of Anusha Dhanasiri is associated with the SafeFish.

Funding: NFR Havbruk2 2016-2020

Current status:

Custom-made fish feed has been prepared and the feeding studies in zebrafish and salmon are completed. A multi-analyte LC-HRMS/MS method for the simultaneous determination of relevant mycotoxins and isoflavones in feed and fish matrices has been developed and validated.

Proteomic analysis of wheat gluten peptides in fish matrix has been generated, including an innovative approach to sample preparation and the detection of suitable gluten peptides. Additionally, a new quantitative AQUA-LC-MS/MS has been tested with samples from the salmon feeding trial.

We were successful in developing an in vitro assay for the biotransformation of contaminants in fish using salmon liver microsomes or hepatocytes. The method is used for in vitro studies of mycotoxin and phytoestrogen in salmon.

Tissues and organs from zebrafish and salmon fed with the different diets are analysed for epigenetic changes indicating differences between the groups.

Sera of patients with immunoglobulin E is used for immunological screening of extracts from wheat-gluten exposed salmon.

Presentations and publications

2017:
Fæste CK, Johny A, Ivanova L. In vitro metabolism of enniatin B1 and deoxynivalenol by salmon liver microsomes. 1st MYCOKEY International Conference, 11.-14.9.2017, Ghent, Belgium.

Dhanasiri AKS, Johny A, Fæste CK, Bogevik AS, Fernandes JMO. Nutrigenomic effects of plant protein-based aquafeeds on fish. Aquaculture Europe, 16.-20.10.2017, Dubrovnik, Croatia.

2018:
Dhanasiri AKS, Chen X, Dahle D, Siriyappagouder P, Fæste CK, Fernandes JMO. Nutriepigenomic effects of plant protein-based aquafeeds on zebrafish intestine. GIA2018 5th International Symposium on genomics in aquaculture. 21.-23.3.2018, Albufeira, Portugal.

Dhanasiri AKS, Johny A, Fæste CK, Bogevik AS, Zhang Q, Fernandes JMO. Nutrigenomic effects of plant protein-based aquafeeds on fish muscle and spleen. GIA2018 5th International Symposium on genomics in aquaculture. 21.-23.3.2018, Albufeira, Portugal.

Dhanasiri AKS, Johny A, Fæste CK, Bogevik AS, Fernandes JMO. Nutrigenomic effects of plant-based aquafeeds on fish muscle and spleen. Havbruk 2018, 18.-20.4.2018, Oslo, Norway

Johny A, Ivanova L, Fæste CK. In vitro metabolism and phase II biotransformation of isoflavones diadzein, genistein and glycitein in salmon liver microsomes. International Conference on Aquaculture, 4.-6.6.2018, Copenhagen, Denmark.

Johny A, Fæste CK, Bogevik A, Berge GM, Fernandes JMO, Ivanova L (2018). Development and validation of a liquid chromatography high-resolution mass spectrometry method for the simultaneous determination of 25 mycotoxins and phytoestrogens in plant-based fish feed and exposed fish. J Chromatogr A (submitted).

2019:

Dhanasiri AKS, Chen X, Berge GM, Bogevik AS, Fæste CK, Fernandes JMO (2019). Nutriepigenomic effects of wheat gluten-based diet on liver of Atlantic salmon. Aquaculture 2019, 7.-11.3.2019, New Orleans, USA.