Marine-Derived Peptides with Anti-Hypertensive Properties: Prospects for Pharmaceuticals, Supplements, and Functional Food.

Hypertension, a major health concern linked to heart disease and premature mortality, has prompted a search for alternative treatments due to side effects of existing medications. Sustainable harvesting of low-trophic marine organisms not only enhances food security but also provides a variety of bioactive molecules, including peptides. Despite comprising only a fraction of active natural compounds, peptides are ideal for drug development due to their size, stability, and resistance to degradation. Our review evaluates the anti-hypertensive properties of peptides and proteins derived from selected marine invertebrate phyla, examining the various methodologies used and their application in pharmaceuticals, supplements, and functional food. A considerable body of research exists on the anti-hypertensive effects of certain marine invertebrates, yet many species remain unexamined. The array of assessments methods, particularly for ACE inhibition, complicates the comparison of results. The dominance of in vitro and animal in vivo studies indicates a need for more clinical research in order to transition peptides into pharmaceuticals. Our findings lay the groundwork for further exploration of these promising marine invertebrates, emphasizing the need to balance scientific discovery and marine conservation for sustainable resource use.

Comparative study on composition and functional properties of brewer's spent grain proteins precipitated by citric acid and hydrochloric acid.

Brewer's spent grain (BSG) is an abundant agro-industrial residue and a sustainable low-cost source for extracting proteins. The composition and functionality of BSG protein concentrates are affected by extraction conditions. This study examined the use of citric acid (CA) and HCl to precipitate BSG proteins. The resultant protein concentrates were compared in terms of their composition and functional properties. The BSG protein concentrate precipitated by CA had 10% lower protein content, 5.8% higher carbohydrate, and 5.4% higher lipid content than the sample precipitated by HCl. Hydrophilic/hydrophobic protein and saturated/unsaturated fatty acid ratios increased by 16.9% and 26.5% respectively, in the sample precipitated by CA. The formation of CA-cross-linkages was verified using shotgun proteomics and Fourier transform infrared spectroscopy. Precipitation by CA adversely affected protein solubility and emulsifying properties, while improving foaming properties. This study provides insights into the role of precipitants in modulating the properties of protein concentrates.

Enzymatic Hydrolysis of Orange-Footed Sea Cucumber (Cucumaria frondosa)-Effect of Different Enzymes on Protein Yield and Bioactivity.

While sea cucumber is a food delicacy in Asia, these food resources are less exploited in Europe. The aim of this study was to determine the chemical composition and potential food applications of the less exploited orange-footed sea cucumber (Cucumaria frondosa). In particular, the antioxidative capacity and free amino acids associated with the umami flavor released by enzymatic hydrolyses by either Bromelain + Papain (0.36%, 1:1) or Alcalase (0.36%) were studied. Fresh C. frondosa contained approximately 86% water, and low levels of ash (<1%) and lipids (<0.5%). The protein content was 5%, with a high proportion of essential amino acids (43%) and thus comparable to the FAO reference protein. The high concentration of free amino acids associated with umami, sour, sweet, and bitter may contribute to flavor enhancement. Hydrolysis by Bromelain + Papain resulted in the highest protein yield, and the greatest concentration of free amino acids associated with umami and sour taste. All samples showed promising antioxidant capacity measured by FRAP, ABTS, DPPH and ORAC compared to previous reports. The inorganic arsenic concentration of fresh C. frondosa ranged from 2 to 8 mg/kg wet weight and was not affected by processing. This is comparable to other seafood and may exceed regulatory limits of consumption.

The potential of anthocyanin-loaded alginate hydrogel beads for intelligent packaging applications: Stability and sensitivity to volatile amines.

pH indicators have emerged as promising tools for real-time monitoring of product freshness and quality in intelligent food packaging applications. However, ensuring the stability of these indicators is critical for practical use. This study aims to evaluate the stability of anthocyanins-loaded alginate hydrogel beads of varying sizes at different temperatures under accelerated light conditions and relative humidity (RH) levels of 53% and 97% during 21 days of storage. Moreover, their sensitivity to the principal spoilage volatiles of muscle food products such as ammonia (NH3), dimethylamine (DMA) and trimethylamine (TMA) was investigated. The half-life of cyanidin-3-glucoside in small hydrogel beads was roughly twice as long as that of the larger beads under accelerated light exposure at 4 °C and they were less likely to undergo noticeable color changes over time. Both sizes of hydrogel beads stored at 97% RH and 4 °C showed color stability over the 21-day period with minimal color variation (|ΔE| ≤ 3). The UV-vis spectra of the purple corn extract exhibited changes across pH 2 to 12, as evidenced by the visible color variations, ranging from pink to green. The limit of detection (LOD) for NH3 was 25 ppm for small beads and 15 ppm for large ones. Both types of beads exhibited similar LOD for DMA and TMA, around 48 ppm. This research showed that alginate hydrogel beads containing anthocyanins from purple corn are a viable option for developing intelligent packaging of muscle foods. Furthermore, the use of hydrogel beads of different sizes can be customized to specific muscle foods based on the primary spoilage compound generated during spoilage.

Alginate microbeads incorporated with anthocyanins from purple corn (Zea mays L.) using electrostatic extrusion: Microencapsulation optimization, characterization, and stability studies.

Microencapsulation of purple corn anthocyanins was carried out via an electrostatic extruder using alginate as a wall material. The influence of alginate concentration (1-2 %), extract concentration (20-30 %), and extrusion voltage (3-5 kV) on encapsulation efficiency and mean particle size was evaluated using response surface methodology. Optimal conditions were obtained to produce two different extract-loaded microbeads. Microbeads with the highest encapsulation efficiency (EE) and minimum particle size were achieved at 1 % alginate, 20 % extract, and 5 kV extrusion voltage (EEC3G = 70.26 %, EETPC = 91.59 %, particle size = 1.29 mm). In comparison, the microbeads with the efficient entrapment and maximum particle size were obtained at 1 % alginate, 26 % extract, and 3 kV (EEC3G = 81.15 %, EETPC = 91.01 %, particle size = 1.87 mm). Brunauer-Emmett-Teller (BET) surface area, pore size, and pore volume decreased after the inclusion of extract, with the lowest values reported for the smallest microbeads containing the extract. Scanning electron microscopy confirmed the results obtained by BET method and demonstrated fewer cracks and lower shrinkage of encapsulated samples. Fourier-transform infrared results proved the presence of anthocyanins and further possible interactions between phenolics and alginate. Stability studies revealed the color maintenance of anthocyanins-loaded microbeads during 4 weeks of storage at 4 °C and 8 °C. Moreover, the small and large particles showed a 7.6 and 3.4-fold reduction in degradation rate at 4 °C compared to their unencapsulated counterparts. Anthocyanins-loaded alginate microbeads retained over 80 % of cyanidin-3-glucoside at 4 °C and 8 °C, suggesting a promising potential of optimized microbeads for intelligent packaging applications.

Marine Capture Fisheries from Western Indian Ocean: An Excellent Source of Proteins and Essential Amino Acids.

The Republic of Seychelles is located in Western-Central Indian Ocean, and marine capture fisheries play a key role in the country's economic and social life in terms of food security, employment, and cultural identity. The Seychellois are among the highest per capita fish-consuming people in the world, with a high reliance on fish for protein. However, the diet is in transition, moving towards a Western-style diet lower in fish and higher in animal meat and easily available, highly processed foods. The aim of this study was to examine and evaluate the protein content and quality of a wide range of marine species exploited by the Seychelles industrial and artisanal fisheries, as well as to further to assess the contribution of these species to the daily intake recommended by the World Health Organization (WHO). A total of 230 individuals from 33 marine species, including 3 crustaceans, 1 shark, and 29 teleost fish, were collected from the Seychelles waters during 2014-2016. All analyzed species had a high content of high-quality protein, with all indispensable amino acids above the reference value pattern for adults and children. As seafood comprises almost 50% of the consumed animal protein in the Seychelles, it is of particular importance as a source of essential amino acids and associated nutrients, and as such every effort to sustain the consumption of regional seafood should be encouraged.

Food Safety Risks Posed by Heavy Metals and Persistent Organic Pollutants (POPs) related to Consumption of Sea Cucumbers.

The global production of sea cucumbers was 245 thousand tons in 2020. Sea cucumbers are important food items in Asian and Pacific cuisines, the highest proportion being consumed in China as "bêche-de-mer" dried, gutted, boiled and salted body wall. However, consumption of sea cucumbers is expanding in China and globally, and the high demand has led to decline in populations of sea cucumbers, due to overexploitation. Aquaculture, together with novel fisheries on new species in new regions is easing the demand. Thus, an assessment of food safety is warranted. A literature search on food hazards was performed. A high proportion of the selected papers concerned heavy metals and metalloid hazards, such as mercury (Hg), cadmium (Cd), lead (Pb), and arsenic (As). No specific maximum limits (MLs) have been set for contents of these in sea cucumbers. Thus, the contents were compared with maximum limits set for aquatic animals in general or bivalve molluscs if available. With regard to Hg and Cd levels, none of the samples exceeded limits set by the European Commission or the National Standard of China, while for Pb, samples from highly industrialised areas exceeded the limits. Surprisingly, data on contaminants such as POPs, including dioxins and dl-PCB, PAH and PFAS as well as microbial hazards were scarce. The availability of fresh sea cucumber has increased due to aquaculture. To preserve the original flavour some consumers are reported to prefer to eat raw sea cucumber products, sashimi and sushi, which inevitably causes challenges from the microbial food safety perspective. Altogether, this paper highlights specific needs for knowledge, in particular when harvesting new species of sea cucumbers or in industrialized regions. Systematic monitoring activities, appropriate guidelines and regulations are highly warranted to guide the utilization of sea cucumbers.

Climate Performance, Environmental Toxins and Nutrient Density of the Underutilized Norwegian Orange-Footed Sea Cucumber (Cucumaria frondosa).

Low trophic species are often mentioned as additional food sources to achieve broader and more sustainable utilisation of the ocean. The aim of this study was to map the food potential of Norwegian orange-footed sea cucumber (Cucumaria frondosa). C. frondosa contained 7% protein, 1% lipids with a high proportion of polyunsaturated fatty acids, and a variety of micronutrients. The nutrient density scores (NDS) of C. frondosa were above average compared towards daily recommended intakes (DRI) for men and women (age 31-60) but below when capped at 100% of DRI. The concentrations of persistent organic pollutants and trace elements were in general low, except for inorganic arsenic (iAs) (0.73 mg per kg) which exceeded the limits deemed safe by food authorities. However, the small number of samples analysed for iAs lowers the ability to draw a firm conclusion. The carbon footprint from a value chain with a dredge fishery, processing in Norway and retail in Asia was assessed to 8 kg carbon dioxide equivalent (CO2eq.) per kg C. frondosa, the fishery causing 90%. Although, C. frondosa has some nutritional benefits, the carbon footprint or possible content of iAs may restrict the consumption.

Suggestions for a Systematic Regulatory Approach to Ocean Plastics.

The research investigates the problems and maps the solutions to the serious threat that plastics pose to the oceans, food safety, and human health, with more than eight million tons of plastic debris dumped in the sea every year. The aim of this study is to explore how to better improve the regulatory process of ocean plastics by integrating scientific results, regulatory strategies and action plans so as to limit the impact of plastics at sea. Adopting a problem-solving approach and identifying four areas of intervention enable the establishment of a regulatory framework from a multi-actor, multi-issue, and multi-level perspective. The research methodology consists of a two-pronged approach: 1. An analysis of the state-of-the-art definition of plastics, micro-, and nanoplastics (respectively, MPs and NPs), and 2. The identification and discussion of loopholes in the current regulation, suggesting key actions to be taken at a global, regional and national level. In particular, the study proposes a systemic integration of scientific and regulatory advancements towards the construction of an interconnected multi-tiered (MT) plastic governance framework. The milestones reached by the project SECURE at UiT - The Arctic University of Norway provide evidence of the strength of the theory of integration and rights-based approaches. The suggested model holds substantial significance for the fields of environmental protection, food security, food safety, and human health. This proposed MT plastic governance framework allows for the holistic and effective organization of complex information and scenarios concerning plastics regulation. Containing a clear definition of plastics, grounded on the precautionary principle, the MT plastic framework should provide detailed mitigation measures, with a clear indication of rights and duties, and in coordination with an effective reparatory justice system.

An Update on the Content of Fatty Acids, Dioxins, PCBs and Heavy Metals in Farmed, Escaped and Wild Atlantic Salmon (Salmo salar L.) in Norway.

In this paper, we present updated data on proximate composition, amino acid, and fatty acid composition, as well as concentrations of dioxins, polychlorinated biphenyls (PCBs), and selected heavy metals, in fillets from farmed (n = 20), escaped (n = 17), and wild (n = 23) Atlantic salmon (Salmo salar L.). The concentrations of dioxins (0.53 ± 0.12 pg toxic equivalents (TEQ)/g), dioxin-like PCBs (0.95 ± 0.48 pg TEQ/g), mercury (56.3 ± 12.9 µg/kg) and arsenic (2.56 ± 0.87 mg/kg) were three times higher in wild compared to farmed salmon, but all well below EU-uniform maximum levels for contaminants in food. The six ICES (International Council for the Exploration of the Sea) PCBs concentrations (5.09 ± 0.83 ng/g) in wild salmon were higher than in the farmed fish (3.34 ± 0.46 ng/g). The protein content was slightly higher in wild salmon (16%) compared to the farmed fish (15%), and the amount of essential amino acids were similar. The fat content of farmed salmon (18%) was three times that of the wild fish, and the proportion of marine long-chain omega-3 fatty acids was a substantially lower (8.9 vs. 24.1%). The omega-6 to omega-3 fatty acid ratio was higher in farmed than wild salmon (0.7 vs. 0.05). Both farmed and wild Atlantic salmon are still valuable sources of eicosapentaenoic acid and docosahexaenoic acid. One 150 g portion per week will contribute to more (2.1 g and 1.8 g) than the recommended weekly intake for adults.

Protein Determination-Method Matters.

The reported protein content of foods depends on the analytical method used for determination, making a direct comparison between studies difficult. The aim of this study was to examine and compare protein analytical methods. Some of these methods require extraction preceding analysis. The efficacy of protein extraction differs depending on food matrices and thus extraction yield was determined. Overall, most analytical methods overestimated the protein contents. The inaccuracies were linked to indirect measurements, i.e. nitrogen determination and subsequent conversion to protein, or interference from other chemical substances. Amino acid analysis is the only protein analysis method where interfering substances do not affect the results. Although there is potential for improvement in regards to the hydrolysis method, we recommend that this method should be the preferred for food protein determination.

Antioxidant and Anti-Inflammatory Activities in Extracts from Minke Whale (Balaenoptera acutorostrata) Blubber.

Intake of long-chain omega-3 polyunsaturated fatty acids (LC-n3-PUFA) is commonly recognized to reduce cardiovascular disease (CVD). In previous studies, cold-pressed whale oil (CWO) and cod liver oil (CLO) were given as a dietary supplement to healthy volunteers. Even though CWO contains less than half the amount of LC-n3-PUFA of CLO, CWO supplement resulted in beneficial effects on anti-inflammatory and CVD risk markers compared to CLO. In the present study, we prepared virtually lipid-free extracts from CWO and CLO and evaluated the antioxidative capacity (AOC) and anti-inflammatory effects. Oxygen radical absorbance capacity (ORAC) and ferric reducing antioxidant power (FRAP) assays were used to test the AOC, and the results indicated high levels of antioxidants present in all extracts. The anti-inflammatory effects of the extracts were tested with lipopolysaccharide- (LPS-) treated THP-1 cells, measuring its ability to reduce cytokine and chemokine secretion. Several CWO extracts displayed anti-inflammatory activity, and a butyl alcohol extract of CWO most effectively reduced TNF-α (50%, p < 0.05) and MCP-1 (85%, p < 0.001) secretion. This extract maintained a stable effect of reducing MCP-1 secretion (60%, p < 0.05) even after long-term storage. In conclusion, CWO has antioxidant and anti-inflammatory activities that may act in addition to its well-known LC-n3-PUFA effects.

Preclinical and Clinical Studies on Antioxidative, Antihypertensive and Cardioprotective Effect of Marine Proteins and Peptides-A Review.

High seafood consumption has traditionally been linked to a reduced risk of cardiovascular diseases, mainly due to the lipid lowering effects of the long chained omega 3 fatty acids. However, fish and seafood are also excellent sources of good quality proteins and emerging documentation show that, upon digestion, these proteins are sources for bioactive peptides with documented favorable physiological effects such as antioxidative, antihypertensive and other cardioprotective effects. This documentation is mainly from in vitro studies, but also animal studies are arising. Evidence from human studies evaluating the positive health effects of marine proteins and peptides are scarce. In one study, a reduction in oxidative stress after intake of cod has been documented and a few human clinical trials have been performed evaluating the effect on blood pressure. The results are, however, inconclusive. The majority of the human clinical trials performed to investigate positive health effects of marine protein and lean fish intake, has focused on blood lipids. While some studies have documented a reduction in triglycerides after intake of lean fish, others have documented no effects.

Enzymatic Pre-Treatment Increases the Protein Bioaccessibility and Extractability in Dulse (Palmaria palmata).

Several common protein extraction protocols have been applied on seaweeds, but extraction yields have been limited. The aims of this study were to further develop and optimize existing extraction protocols and to examine the effect of enzymatic pre-treatment on bioaccessibility and extractability of seaweed proteins. Enzymatic pre-treatment of seaweed samples resulted in a three-fold increase in amino acids available for extraction. Combining enzymatic pre-treatment with alkaline extraction resulted in a 1.6-fold increase in the protein extraction yield compared to a standard alkaline extraction protocol. A simulated in vitro gastrointestinal digestion model showed that enzymatic pre-treatment of seaweed increased the amount of amino acids available for intestinal absorption 3.2-fold. In conclusion, enzymatic pre-treatment of seaweeds is effective for increasing the amount of amino acids available for utilization and may thus be an effective means for increasing the utilization potential of seaweed proteins. However, both the enzymatic pre-treatment protocol and the protein extraction protocol need further optimization in order to obtain optimal cost-benefit and results from the in vitro gastrointestinal digestion model need to be confirmed in clinical models.

Dietary intake of cod and scallop reduces atherosclerotic burden in female apolipoprotein E-deficient mice fed a Western-type high fat diet for 13 weeks.

BACKGROUND: It is now increasingly recognized that the beneficial effects of seafood consumption is not limited to lipids and fatty acid, but that the protein part, i.e., peptides and amino acids, together with vitamins and even unknown bioactive constituents also are important for disease prevention. This study was designed to evaluate the putative anti-atherogenic effects of different protein sources (a lean seafood and a nonseafood) in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS: Twenty-four 5-week-old female apoE(-/-) mice were fed Western type diets containing chicken or a combination of cod and scallops as dietary protein sources for 13 weeks. Atherosclerotic plaque burden, weight, serum levels of leptin, glucose and LDL cholesterol as well as gene expressions from liver and heart were evaluated. Statistical analyses were performed using SPSS. Differences between the variables were evaluated using independent t-test or Mann-Whitney U test for normally and non-normally distributed variables, respectively. Normality was defined by the Shapiro-Wilk test. RESULTS: The mice fed cod-scallop had a 24 % (p < 0.05) reduced total aorta atherosclerotic plaque burden compared to the chicken fed group, whereas the reduction in plaque in the less lesion prone thoracic and abdominal parts of the descending aorta were 46 % (p < 0.05) and 56 % (p < 0.05), respectively. In addition, mice fed cod-scallop gained less weight, and had lower serum levels of leptin, glucose and LDL cholesterol, compared to those fed chicken. Analysis of expression of the genes from liver and heart showed that hepatic endogenous antioxidant paraoxonase 2 (Pon2 gene) and the vascular cell adhesion molecule VCAM-1 (Vcam1 gene) were down regulated in mice fed cod-scallop compared to mice fed chicken. CONCLUSION: The present study revealed a metabolic beneficial effect of lean seafood compared to chicken, as atherosclerotic plaque burden, serum glucose, leptin and LDL cholesterol levels were reduced in mice fed cod-scallop.

ω-3 Fatty Acids and Cardiovascular Diseases: Effects, Mechanisms and Dietary Relevance.

ω-3 fatty acids (n-3 FA) have, since the 1970s, been associated with beneficial health effects. They are, however, prone to lipid peroxidation due to their many double bonds. Lipid peroxidation is a process that may lead to increased oxidative stress, a condition associated with adverse health effects. Recently, conflicting evidence regarding the health benefits of intake of n-3 from seafood or n-3 supplements has emerged. The aim of this review was thus to examine recent literature regarding health aspects of n-3 FA intake from fish or n-3 supplements, and to discuss possible reasons for the conflicting findings. There is a broad consensus that fish and seafood are the optimal sources of n-3 FA and consumption of approximately 2-3 servings per week is recommended. The scientific evidence of benefits from n-3 supplementation has diminished over time, probably due to a general increase in seafood consumption and better pharmacological intervention and acute treatment of patients with cardiovascular diseases (CVD).

The potential of cod hydrolyzate to inhibit blood pressure in spontaneously hypertensive rats.

Hypertension is an independent yet controllable risk factor for cardiovascular diseases. Synthetic angiotensin-converting enzyme (ACE) inhibitors used to treat hypertension are often associated with adverse effects, and the interest in diet-related inhibitors is increasing. We hypothesized that North Atlantic fish hydrolysate might inhibit ACE, thus preventing hypertension. We assessed the ACE inhibitory potential of various North Atlantic fish species and evaluated the effect of dietary supplementation of fish hydrolysates on the blood pressure of spontaneously hypertensive rats. Fish samples were hydrolyzed using simulated gastrointestinal digestion, and ACE inhibitory activity was evaluated using an ACE inhibitory activity assay. In vivo anti-hypertensive effects were evaluated by administering hydrolysates of wild Atlantic cod (Gadus morhua L.), haddock (Melanogrammus aeglefinus L.), or farmed Atlantic salmon (Salmo salar L.) to 10-week-old male, spontaneously hypertensive rats for 4 weeks. The dosing was 200 mg/kg body weight for 21 days, followed by 500 mg/kg body weight for 7 days. Water and Captopril (20 mg/kg body weight) were administered as the negative and positive controls, respectively. The analyzed fish hydrolysates exhibited a 50% ACE inhibition coefficient (IC50) of 1 to 2.7 µg/mU ACE. Fish hydrolysate supplements did not significantly inhibit the increase in blood pressure during the experimental period. The group receiving cod supplement had a lower (not significant) increase in blood pressure compared to the other groups. Although further studies are necessary to verify the antihypertensive effect of cod, the results obtained in this study indicate the potential that cod hydrolysate may have in inhibiting hypertension.

Proximate composition, antihypertensive and antioxidative properties of the semimembranosus muscle from pork and beef after cooking and in vitro digestion.

The aims of this study were to evaluate and compare proximate composition, antihypertensive activity and antioxidative capacity of the semimembranosus muscle from pork and beef and to study how these characteristics were affected by household preparation and subsequent digestion. The proximate composition was similar between pork and beef. Both pork and beef contained protein with the essential amino acids. Cooking in a heated pan did not affect the retention of lipid or sum of amino acids, but reduced the amount of the free amino acid taurine. The antihypertensive effect did not differ significantly between pork and beef, whereas the antioxidative capacity did. Cooking affected the antioxidative capacity negatively. The results from this study show that pork and beef are equally good sources of protein and bioactive properties, and whereas the nutritional composition is not affected, bioactive properties may be reduced after household preparations.

Changes in antioxidative capacity of saithe (Pollachius virens) and shrimp (Pandalus borealis) during in vitro digestion.

The health effects of seafood have primarily been linked to eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). These omega-3 fatty acids are highly susceptible to oxidation. Peptides exhibiting antioxidative capacity (AOC) are encrypted in seafood muscle proteins. Such components may protect the fatty acids during digestion and uptake, as well as exhibit antioxidative effects in general. The oxygen radical absorbance capacity (ORAC) assay and the ferric reducing antioxidant power (FRAP) assay were used to study the changes in AOC of fish and shrimp muscle and their aqueous fractions, referred to as press juice (PJ), during a simulated in vitro gastrointestinal (GI) digestion. Blueberry (Vaccinium myrtillus), well-known for its AOC, was included for comparison. During digestion the AOC increased in all samples. After digestion the AOC of muscle of both autumn saithe and shrimp were higher (130-165 micromol/g), and winter saithe comparable (110 micromol/g) to digested blueberry when measured by ORAC. The AOC of PJ was low in general (5-20 micromol/g). When measured by FRAP, blueberry exhibited ten times the AOC of seafood muscle. Antioxidative compounds in seafood increased significantly during digestion, and may offer protection of other beneficial food components such as EPA and DHA.