Nutritional Enhancement of Farmed Salmon Meat via Non-GMO Nannochloropsis Gaditana: Eicosapentaenoic Acid (EPA, 20:5 n-3), Docosapentaenoic Acid (DPA, 22:5 n-3) and Vitamin D3 for Human Health

Omega-3 long-chain polyunsaturated fatty acids (n-3 LC PUFAs) and vitamin D3 are essential components of human nutrition. A regular human diet is highly deficient in n-3 LC PUFAs. Fish like salmon are highly recommended in the human diet as they are a major source of high-value n-3 LC PUFAs and vitamin D3. The levels of these nutrients have been decreasing over the last few years in farmed salmon, whose production urgently needs sustainable sources of these nutrients. The microalga Nannochloropsis gaditana (NG) is known for its naturally high potential for the production of eicosapentaenoic (EPA, 20:5 n-3) fatty acid. A commercial diet for Atlantic salmon was supplemented with 1% and 10% of spray-dried NG grown under controlled conditions for a high EPA content. Salmon were harvested on day 49, following which, boneless and skinless salmon meat was recovered from fish and analyzed for the fatty acid profile, total fat, and vitamin D3. Vitamin D3, EPA, and docosapentaenoic fatty acid (DPA, 22:5 n-3) levels were significantly increased (p < 0.05) by supplementing the basal diet with 10% NG, thus, NG represents a novel, functional, natural ingredient and a sustainable source of n-3 LC-PUFAs that can raise the levels of healthy fats and vitamin D3 in farmed salmon meat.


Introduction
Polyunsaturated fatty acids (PUFAs) are essential components of all cell membranes. They influence membrane fluidity and modulate a wide range of functions in the body [1]. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA, 22:6 n-3) have many health benefits; they are useful against hypertension and Crohn's disease, and reduce the risk of coronary artery disease and high serum triglycerides [2]. They also have beneficial effects on depression, bipolar disorder, schizophrenia, and dementia [2,3]. n-3 LC PUFAs play an important role in brain function and formation of the structure of the neuronal cell membranes. They are necessary for neurological potential in the trend of using natural additives. They are predicted to play important roles as nutritional enhancers in the food industry and as nutraceuticals in the pharmaceutical industry [32]. Algae oils have been recognized as rich sources of EPA and DHA, and have been used in diets for Atlantic salmon [28]. Transgenic camelina expressing algal genes have been tested to produce oil containing n-3 LC-PUFA to replace marine fish oil in salmon feed with no harmful effects on fish performance and no change in the nutritional quality of the meat [33]. However, genetically modified organisms (GMOs) have been linked to causing detrimental effects on human health including horizontal gene transfer [34][35][36] and allergenicity [34,35,37,38], and on animal health with respect to safety concerns on nutritional parameters, digestibility, herbicide and insecticide tolerance [34,35], and chemical composition of GM feed [35].
The urgent need for natural high-quality ingredients will increase with the growth of the aquaculture industry. Therefore, in the future, feed ingredients should be derived from sustainable sources [39]. Among the organisms present in marine and aquatic food webs, algae possess the highest ability to synthesize long-chain PUFAs; unlike, most animals are not able to synthesize essential fatty acids that can be converted into long-chain PUFAs. The fatty acids EPA and arachidonic (20:4 n-6) of the membrane phospholipids are precursors in prostaglandin synthesis. Prostaglandins are precursors to compounds known as tissue hormones [40]. As microalgae are primary producers of EPA and DHA in the marine and aquatic food web, there is increasing interest in their use as additives and supplements in fish feeds [39,41]. The biochemical composition of microalgae can be modulated by altering certain nutrients, environmental stress and culture conditions to induce the microorganisms to produce high concentrations of the desired nutrient [42]. Lipid metabolism is strongly influenced by environmental factors, especially nutrition, and limitation of nitrogen and phosphorus [31]. Although strain-and species-specific variations in fatty acid composition are evident, some microalgae are promising sources of PUFA, especially EPA and DHA [39,43]. The genus Nannochloropsis is more widely distributed in the marine ecosystems than that of freshwater ecosystems. Nannochloropsis is successfully used as feed in aquaculture due to the high content of long-chain PUFAs, especially eicosapentaenoic acid [40]. Nannochloropsis gaditana is a promising marine microalga species for its role in the food web due to its calcium content [44] and its ability to accumulate large amounts of the high-value n-3 fatty acid EPA during nitrogen starvation [45,46]. The aim of this study was to improve the nutritional quality of farmed salmon meat by enhancing the levels of long-chain PUFAs, such as EPA, and vitamin D 3 by using a natural source like the microalga Nannochloropsis gaditana (NG), prepared by spray drying and concentrating NG cultivated under controlled conditions.

NG EPA Induced Growth Conditions
Nannochloropsis gaditana strain (Lubián CCMP 527) was maintained under controlled conditions at 20 • C, in 250 mL flasks, with constant aeration at 0.1 v/v/min with no CO 2 supply, under constant illumination at 200 µE m −2 s −1 provided by fluorescent lamps, in "UMA 5" culture medium prepared from fertilizers instead of pure chemicals [47]. The culture medium was prepared using natural seawater and nutrients of analytical grade, and was autoclaved for 15 min. at 121 • C. The strain was sub-cultured every ten days by adding 10% of the old culture medium into 90% of fresh culture medium. The cultures were monitored by microscopic observation using a Leica CME microscope 40X/0.65 to verify the non-occurrence of contamination issues. The culture was scaled up to 1 L using spherical flasks under the same conditions. Nannochloropsis gaditana was cultured (December 2018) in an open raceway type system with a capacity of 14.4 m 3 under outdoor conditions, grown in batch mode and maintained for 15 days, and subsequently harvested at a dilution rate of 10% per day. Cultures were hatched at 20 • C. A UMA 5 culture medium was used (NaNO 3 : 0.4 g/L, NaH 2 PO 4 : 0.034 g/L, NaHCO 3 : 0.168 g/L and trace elements µmol/L, Zn 0.08, Mn 0.90, Mo 0.030, Co 0.050 and Fe 11.70) [47]. The culture was initiated in a semi-continuous mode, in which 75% of the culture medium and vitamin supplementation (thiamine, biotin, and cyanocobalamin) were deprived. Semi-continuous culturing in outdoor conditions allowed volumetric productivity of 46-56 mg/L/day and 1.6-2.2 g EPA/100 g biomass.

NG Spray Dryer Concentrate
NG algal cells were harvested by centrifugation using a model AS 1936076 continuous centrifuge (GEA Westfalia, Oelde, Germany) at a working flow rate of 2 m 3 /h and a maximum pressure of 3 bar. Once centrifuged, they were dehydrated and concentrated by spray drying (LPG-25 high speed centrifugal spray dryer, (Changzhou Yibu Drying Equipment C0 Ltd., Zhengzhou, Henan, China) using an initial inlet air temperature of 185 • C, a maximum drying chamber temperature of 90 • C and a final temperature of 80 • C for 5-15 s and a working flow rate of 4 L/h.

Nutritional Characterization of NG Concentrate
The proximate analyses, zinc and calcium as well as fatty-acid profiles, were determined using the official methods of analysis of the Association of Official Analytical Chemists (AOAC): crude protein combustion analysis [48] utilizing the calculation 6.25× nitrogen value; sodium and potassium determination [49]; zinc and calcium determination [50]; ash determination [51]; crude fat [52]; moisture content [53]; crude fiber [54] and fatty acid profile [55]. Vitamin D 3 (colecalciferol) was determined according to EN standard test method [56] All analyses were accredited according to ISO17025. The limit of quantification (LOQ) for vitamin D 3 was 0.25 µg/100 g.

Experimental Diets
Three different diets were produced: one control diet and two NG supplemented diets (formulated using two different levels of NG: 1% and 10%). NG powder was mixed with the base of a feed formulation in the following proportions to produce three experimental diets: 1.0%, 10.0% and a control diet with no supplemental NG (Table 1). Diets were analyzed for proximate composition sodium, calcium and fatty acid profile according to AOAC standard methods: crude protein combustion analysis [48] utilizing the calculation 6.25× nitrogen value; sodium [49]; calcium [50]; ash determination [51]; crude fat [52,57]; moisture content [53]; crude fiber [54] and fatty acid profile [55]. Vitamin D 3 (colecalciferol) was determined according to EN standard test method [56] All analyses were accredited according to ISO17025. The limit of quantification (LOQ) for vitamin D 3 was 0.25 µg/100 g.

Experimental Fish and Feeding
Atlantic salmon (Salmo salar) from a single family SNAQ16LSSCO were obtained from AquaGen Chile S.A., Piscicultura Ignao SA, Lago Ranco, Chile). Fish were maintained at 8.6 ± 1 • C, pH 7.11 ± 0.04, 8.51 ± 0.14 dissolved oxygen concentration and 24 h light photoperiod in a flow-through freshwater system in Piscicultura Iculpe-Ilihue, Lago Ranco, Chile. Two hundred and twenty-five fish (104.52 ± 1.29 g each) were distributed randomly in nine tanks (200 L, three tanks per diet). The fish were acclimatized to the tanks for 15 days prior to the start of the trial. The fish were hand-fed 3 mm experimental pellets to satiation twice a day for 49 days (March-May 2018). Environmental parameters (dissolved oxygen concentration, pH and temperature) and feed consumption were measured daily, and fish length and weight were recorded at the beginning and at the end of the experiment. The production parameters feed conversion ratio (FCR) and specific growth rate (SGR) were calculated using the following formulae: FCR = feed consumed/biomass increase SGR = 100 × (lnW 2 − lnW 1 )/feeding days, where ln is the natural logarithm and W 1 and W 2 are the initial and final weights of fish, respectively.

Tissue Sampling
Fifteen fish per treatment (five fish per tank) were randomly sampled on day 59 for proximate, sodium, potassium, and vitamin D 3 analyses and three fish per tank were randomly sampled for fatty acid profile. The fish were euthanized by cervical dislocation, their liver, gut, and skin were removed, and the obtained meat was weighed, lyophilized in an FDT 8632 model freeze dryer and stored for further analysis. Initial moisture, moisture, and A w (water activity) of the lyophilized meat were determined. Moisture was determined according to AOAC standard method [53]. Aw was determined using a HygroPalm 23-Aw-A digital meter (Rotronic, Hauppauge, NY, USA) All procedures, including handling, treatment, and euthanasia, were performed according to the guidelines provided by the University of Chile animal welfare committee.

Statistical Analysis
To test for differences in the nutrient composition of salmon farmed meat among the dietary treatment groups, the data were subjected to a one-way analysis of variance (ANOVA) using SPSS Statistics version 25 (IBM Corporation, Armonk, NY, USA). All data were checked for homogeneity of variance prior to the ANOVA. When differences were identified among the groups, multiple comparisons to the control were made using Dunnett's post-hoc test. The difference was considered significant if p was < 0.05. All results are presented as mean ± standard deviation (SD).

Fatty Acid and Vitamin D 3 Composition of the Experimental Diets
The fatty acid profile and vitamin D 3 content of the experimental diet groups are presented in Table 4. The values of crude fat, myristic fatty acid (14:0), palmitic acid (16:0), palmitoleic acid (9c-16:1), and arachidonic acid (20:4 n-6) increased in both the groups receiving diets supplemented with NG. The value of EPA (20:5 n-3) increased in the group receiving diet supplemented with 10% NG. The control diet group showed higher values of vitamin D 3 and DHA than the experimental diet groups supplemented with NG (Table 4).

Experimental Fish and Feeding
All experimental diets were received well by the animals, and no pathological or toxic signs were observed. At the end of the forty-nine-day feeding study, no significant differences were observed in feed intake across the three groups. Consumption of the diet supplemented with 10% NG resulted in a significant increase in weight gain and SGR (%) compared to the control diet (Table 5). Table 5. Effect of NG concentrate inclusion level on weight-gain, Specific growth rate-SGR and Feed conversion ratio-FCR animals feed experimental diets.

Salmon Meat: Proximate, Fatty Acid Profile, Minerals, and Vitamin D
In the lyophilization process, fish from the three dietary treatments had no significant differences in initial moisture and water activity of lyophilized meat. However, the fish treated with 10% NG showed significant differences in the final moisture compared to the control treatment (Table 6). Proximate analysis of Salmon meat from all dietary treatments showed similar dry matter, ash, protein and lipid compositions, no significant differences were observed (Table 7).   (Table 8).
Molecules 2020, 25, x FOR PEER REVIEW 9 of 16 treatment diet showed significant differences only in palmitic (16:0) and stearic (18:0) fatty acids (Table 8).   Values are based on the mean ± S.D. n = 3.     Values are based on the mean ± S.D. n = 5 for vitamin D 3 and n = 3 for fatty acid profile.

Discussion
Microalgae are a potential source of food and energy due to their high nutritional value and photosynthetic efficiency [58,59]. They are a good source of protein, energy, vitamins, essential fatty acids, pigments, and sterols [58][59][60]. The use of a microalgal strains such as Spirulina sp., Chlorella sp. or Scenedesmus sp. has proven to be a sustainable alternative for the complete replacement of fishmeal in aquaculture [59,61]. Dried microalgae have been used in foods formulated for fish and shrimp as a sustainable substitute for fishmeal protein [58,59]. Cultivation conditions such as CO 2 concentration, photoperiod, and light intensity favor the production of biomass and lipids in the marine strain Nannochloropsis sp. [62,63]. Our study showed that spray-dried NG produced under controlled culture conditions using UMA 5 culture medium is an important source of ash (21.26%), sodium (5.40%), and calcium (4.11%), and a major source of the polyunsaturated fatty acid EPA (26.73% expressed as a percent of total fatty acids). The high EPA values and the absence of DHA obtained with NG treatment are consistent with the values of these fatty acids reported previously for Nannochloropsis gaditana [31]. NG concentrate showed no presence of vitamin D 3 . Under natural conditions, planktonic vitamin D accumulates in the marine and aquatic food chain as zooplankton and phytoplankton have high concentrations of D 2 and D 3 . Fish accumulate large quantities of vitamin D 3 in their fat tissues, including fat associated with the muscle, but vitamin D 2 is almost absent in fish tissues. Fish are fully dependent on dietary sources to meet their requirements of vitamin D and do not synthesize this vitamin [64,65]. Although the synthesis of vitamin D 3 induced by ultraviolet light from 7-dehydrocholesterol (7-DH) has been demonstrated in fish, including rainbow trout, this mode of synthesis does not have a significant contribution (at least for marine fish) in their natural habitat, since most of the UVB irradiation is absorbed in the first few meters of the water column. In rainbow trout, it has been shown that the values of vitamin D metabolites in plasma depend on the environmental concentrations of calcium present in both freshwater and saltwater. Increased environmental calcium is associated with higher transformation to the compound 25,26-dihydroxycholecalciferol, whereas, lower environmental calcium concentrations induce higher conversion to 1,25-dihydroxycholecalciferol-like compound. In Atlantic salmon during the smoltification process and migrating from fresh water to sea water, vitamin D 3 is regulated by water Calcium (Ca 2+ ) concentrations [65]. In our study, vitamin D 3 levels were increased in Salmon meat enriched with NG. D 3 concentrations were not detected in the NG concentrate, but nevertheless, NG concentrate showed a high presence of EPA and minerals, with a high content of calcium. This could explain the increase in the concentration of vitamin D 3 in the treated meat.
Inclusion of the EPA-rich NG concentrate characterized by the presence of 26% EPA in diets for Atlantic salmon had a good effect on fish growth, showed a significant increase in EPA, DPA and a decrease in the n-6/EPA+DHA index, and had a significant effect on fatty acid deposition improving the levels of EPA + DHA (25%) and vitamin D 3 (106%) in fish meat, thus, ultimately resulting in an improvement in the nutritional quality of fish meat for human consumption (Table 9).  [68]. 5 Mg EPA + DHA per day adults should be consuming through oily fish consumption. 6 Minimum daily intake for cardiovascular health.

Conclusions
This study has shown that the inclusion of dietary spray-dried NG concentrate in the fish diet resulted in enhancement of EPA (20:5 n-3), DPA (22:5 n-3), and vitamin D 3 levels of salmon farmed meat without compromising the feed conversion rate or fish growth. Increasing these bioactive compounds could help attenuate or prevent diseases or disorders associated with its deficiency. The dietary value of salmon farmed meat is effectively improved by spray-dried NG concentrate prepared from non-GMO Nannochloropsis gaditana grown under controlled culture conditions to include health benefits for consumer populations.