The main goal of 'A to omega-3' project was to safely include high long chain omega-3 polyunsaturated fatty acids (LCn-3PUFA) from heterotrophic microalgae (HM) (Schizochytrium sp., Alltech Ltd) in salmon feeds. The specific objectives were to: 1) Develop downstream processing technologies improving HM dewatering cost-efficiency and digestibility of the saturated fatty acid fraction; 2) Develop protocols for the use of algae-rich feed formulations in extruded high-quality fish feeds; 3) Screen major metabolic effects of HM lipids in salmon cell cultures and study biological effects of using HM instead of Fish oil in salmon feeds with focus on fish welfare and health, growth performance, feed efficiency, filet quality, and 4) Establish the economic feasibility of using HM as sources of omega-3 fatty acids in salmon feeds.
Processing: We assessed the rheological properties of HM and evaluated dewatering efficiency by microfiltration, which can be a more cost efficient technology than centrifugation, and which was proven feasible up to 14% dry matter. We compared HM oil extraction efficiency and DHA stability between different technologies e.g. heat pre-treatment or mechanical cell wall disruption followed by different extraction methods (supercritical CO2, Bligh & Dyer, Soxhlet) and solvents. HM biomass contains 60-65% lipids in triglycerides (TAG), composed of saturated fatty acids (FA) (mainly 16:0) and a mixture of saturated FA and highly unsaturated fatty acids (DHA, DPA and less EPA). Fully saturated TAGs reduced FA digestibility. This may be improved by TAG modification. Interesterification is a method of FA enzymatic (lipase) hydrolysis and re-esterification to new positions in TAG molecules. Commercial Lipozyme RM IM and Lipozyme 435 with immobilized lipases (Novozymes AS) were used as catalysts for interesterification of HM oil/rapeseed oil blends. TAG FA composition results analysed by lipidomics showed significant effects of enzyme load and incubation time at 60 or 80°C.
Extrusion: Is a technology enabling production of feed of a consistent and high physical quality and is thus the dominating technology used in commercial salmon feed production. It involves use of moisture and high temperature achieved by water/steam injection and mechanical energy dissipation to obtain acceptable physical product quality and density specifications. Lipid levels above 13-15% in the feed mass will lubricate the system and reduce heat from friction, resulting in poor physical feed quality. Thus, most of the lipids have to be vacuum coated onto dried feed pellets. We studied how increasing HM levels affect viscous dissipation in the extrusion process and physical feed quality, under standardized extrusion and drying conditions. The experiment was based on a 3-component mixture design with commercially relevant feed blends varying wheat gluten, soy protein concentrate and HM. Increased inclusion level of HM and the ratio of wheat gluten and soy protein concentrate affected all measured responses (mechanical energy, pellet hardness, fracturability, sectional and longitudinal expansion, bulk density, maximum fat adsorption, fat leakage). The optimal HM inclusion level was determined.
Fish physiology: Cell culture trial with adipocytes isolated from salmon was realised to investigate how FA composition in HM, i.e. high content of DHA and 16:0 and low EPA, influences immune responses compared to a FA composition with EPA instead of DHA. The results showed that cultivating cells in media with either 16:0+DHA or 16:0+EPA gives reduced inflammation response in adipocytes compared to cells cultivated in only 16:0. FA composition in the growth medium influenced also genes involved in the biosynthesis of omega-3 FA. Three extruded diets (FO, FO/AO, AO) with graded levels HM (0-2.75-5.5%) and fish oil 3.7-1.85-0%) were used in two continuous feeding trials with pit tagged salmon smolt and post smolt. The fish were previously fed similar diets from parr stage in fresh water and were further fed to slaughter (3kg) in cages with feeds FO and AO. Up to 1kg body weight HM had positive effects on slaughter yield, protein efficiency ratio, protein digestibility and retention efficiency of several FA also that of EPA+DHA. We saw no effects on growth, feed efficiency and liver size. Haematology showed no effects on ALAT, ASAT or CK. Global transcriptomics (15.000 genes) in the intestine showed significant responses on immune, reparation/growth, protein and long chain FA and sphingosin metabolism of fish fed AO and AO/FO compared to FO. There was no effect on stress, cell death, proliferation or toxicity. Histology and immunohistochemistry (PCNA, iNOS, MHC II, CD8) showed no differences among the different groups. Organoleptic evaluators evaluated smell, colour, taste and consistency on fillets and found differences in colour tone, strength and whiteness. Fish fed AO had redder fillet compared to the other treatments.
More than 70% of the world's fish species are about to be fully exploited, and the salmon feed industry utilizes more than 50% of the global fish oil production today. Microalgae, being the primary producers of marine omega-3, are recognized as among the most prominent alternatives to fish oil in fish feed. Heterotrophic microalgae (HM) do not compete for light and can be produced on inexpensive low value raw materials in any part of the world, still the methods used for processing are too costly for bulk raw material production. Large scale production of HM (e.g. Schizochytrium sp.) with high lipid content (55-75% in dry matter) and as much as 30% DHA of total lipids is a reality today using well established fermentation technologies. It is thus possible to supplement salmon diets with marine omega-3 fatty acids from these algae and support further salmon production growth. To reach this goal, further improvements in cost efficiency and end product quality in the downstream processing of HM are needed as well as convincing studies on the lifelong metabolic effects in fish fed HM rich diets. Provision of adequate quantities of wet and dry raw material for pilot scale processing, feed technology and fish feeding trials are thus necessary. In this project A lltech Inc holds this role, rendering the risks of not achieving the set goals very low. The project works include i) development of downstream processing technologies to improve HM dewatering cost and saturated lipid digestibility, ii) development of ing redient specific extrusion protocols, iii) screening of main metabolic effects of HM oils in salmon cell cultures, iv) studies on the biological effects of dietary HM on farmed salmon focusing on fish welfare and health, growth performance, product qualit y and metabolic effects, and v) establishment of the economic feasibility of using HM as sources of omega-3 fatty acids in salmon feeds.