INTRODUCTION
The concept of "Integrated Multi-Trophic Aquaculture" (IMTA) has been given great interest in recent decades and is one of several strategies to increase biomass production from the sea by reducing the environmental impact of fish farming and by increased production of low-level trophic organisms such as macroalgae. However, due to lack of data and scientific evidence, appropriate food regulations are lacking for IMTA to guide the production of macroalgae and their application as food products. Macroalgae are however recognized to have valuable bioactivities including antioxidative activity. Due to variations in bioactivities between species, geographic location and season, characterization of the quality of specific IMTA products will be important for further marketing purposes. Besides valuable compounds, macroalgae can also contain elements with unwanted effects such as allergens. Thus, there is a growing concern from the food industry and food safety authorities regarding the potential occurrence of contaminating marine allergens in seaweed food products. A s seaweed is produced in the ocean, allergens of marine species, such as crustaceans, molluscs or even fish might find their way into the production line and these hazards must be monitored to evaluate the risk for allergenic incidents and the need for appropriate labelling for future food products. Here, the occurrence of marine food allergens as well as total phenolic compounds and antioxidant activity have been assessed in S. latissima produced in three different locations in 2020 in the vicinity of salmonids farms in Norway.
MATERIALS AND METHODS
In 2020, 10 kg of seaweed was collected at three different Norwegian IMTA farms located at three different sites (Bjønnspjotneset, Dyrholmen Vest, Furholmen) owned respectively by Osland Havbruk AS, Sulefisk AS and Engesund Fiskeoppdrett. The seaweed was washed, freeze dried and ground. Extractions and analyses were conducted in triplicates.
For analysis of phenolic compounds and antioxidants, seaweed samples were dissolved at 25 mg/ml in dH2O or MeOH:dH2O (4:1) , incubated in an ultrasonic water bath (Branson 2200, 40kHz) for 30 min where indicated, and filtered. Phenolic compounds were measured by the Folin-Ciocalteu method
with g allic acid (Sigma Aldrich G7384) standard curve and undiluted samples. Antioxidative activity was assessed by ORAC assay, according to the BioTek Application Note 2006
. Samples were diluted to 25 µg/ml (1:1000) for MeOH extracts and to 5 mg/ml (1:5) for water extracts.
For allergen detection, ELISA kits for the antigens fish parvalbumin, mollusc tropomyosin or crustacean tropomyosin (Demeditec DEFISE1, DEMOLE1 and DECRUE1) were used. Dried and grinded seaweed (0.2 g, exact weight) was dissolved in 20 ml extraction buffer , filtered, and diluted 2-times to limit matrix effects.
RESULTS
The content of phenolic compounds was in a range of 1-2 mg GAE/g dry weight for all three locations and all three extraction methods . Antioxidative capacity was significantly increased when extracted in aqueous methanol as compared to water extracts in all samples. In the methanol extracts, t he seaweed from Osland contained the significantly highest antioxidative activity with 1665.1 ± 39.4 µmol TE/g, followed by Sulefisk with 1243.2 ± 68.9 and Engesund with 630.9 ± 31.5 µmol TE/g. The difference in ORAC values between Osland and Sulefisk was not consistent for all extraction methods, while the sample from Engesund had the overall significantly lowest values. The treatment in an ultrasonic water bath did not have a significant effect on the content of phenolic compounds or antioxidant capacity but decreased the viscosity of water extracts.
Further, the seaweed samples were tested for the established food allergens crustacean, mollusc and fish. Values for crustacean tropomyosin were for all samples above the lower detection limit of 20 ppm in the assay (corresponding to 0.2 mg/kg sample) and ranged from 0.442 ± 0.05 mg/kg for Osland to 1.005 ± 0.39 mg/kg at Sulefisk. The measured values were not significantly different between the three IMTA sites, although the lowest values were all detected in the samples from Osland. The m ollusc tropomyosin concentrations in the seaweed samples were numerically above, but very near the lower detection limit of 10 ppm in the assay or 0.1 mg/kg sample. Fish allergens, provided as cod concentrations, were below the lower detection limit of 4 ppm in the assay (or 40 mg/kg sample) for the seaweed samples from Sulefisk and Engesund. The concentration measured in the samples from Osland was above, but near the detection limit. Osland was also the location with the highest fish production in 2020. According to literature, cod contains parvalbumin at 2 mg/g
with higher concentrations in muscle
. Based on this, 40 mg cod per kg as the detection limit would translate to 0.08 mg parvalbumin per kg.
CONCLUSIONS
Products with relatively high market value such as food and feed ingredients are predicted to play an important role in creating value from Norwegian-grown seaweed and kelp
. This work confirmed the presence of phenolic compounds and antioxidative activity in S. latissima, produced in IMTA systems, in a similar range for all assessed locations and comparable to previous studies, indicating that this species and production method can play a role for the development of natural food additives, functional food or health products from seaweed. The here reported bioactivities should be further investigating to fully understand the potential and limit of using S. latissima as functional ingredients in food or feed formulas.
Our analyses of S. latissima bulk samples with commercial sandwich ELISA kits for known food allergens have shown acceptable detection limits, linearity and recovery. Only crustacean allergens could be detected in all tested locations in this study, while mollusc tropomyosin was very near the detection limit, thus assumed as not detectable. Cod parvalbumin was detected slightly above detection limit only in one location. In the context of realistic incorporation in final food products and resulting daily ingestion, the presence of contaminating marine species in seaweed raw products does not seem to be critical, but should be further followed through seasons, locations and productions as more processed products are developed.
REFERENCES
1. Agregán et al. Medicines 5, 33 (2018).
2. Aslan et al. Mar. Pollut. Bull. 141, 313–317 (2019).
3. Held. Appl. Note, Biotek 9 (2006).
4. Kuehn et al. Allergol. Sel. 1, 120–126 (2017).
5. Kuehn et al. Int. Arch. Allergy Immunol. 153, 359–366 (2010).
6. Koppelman et al. Food Addit. Contam. - Part A Chem. Anal. Control. Expo. Risk Assess. 29, 1347–1355 (2012).
7. Stévant et al. Aquaculture International 25, 1373–1390 (2017).