Amino Acid Profile and Protein Quality Assessment of Macroalgae Produced in an Integrated Multi-Trophic Aquaculture System

Seaweeds are a recognized source of bioactive compounds and techno-functional ingredients. However, its protein fraction is still underexplored. The aim of this study was to determine the total and free amino acid profile and protein content of four seaweeds species (Porphyra dioica, Porphyra umbilicalis, Gracilaria vermiculophylla, and Ulva rigida) produced in an integrated multi-trophic aquaculture system, while assessing their protein quality. Samples were submitted to acid and alkaline hydrolysis (total amino acids) and to an aqueous extraction (free amino acids) followed by an automated online derivatization procedure, and analyzed by reverse phase-high performance liquid chromatography. Protein-, non-protein and total-nitrogen were quantified by the Kjeldahl method. Crude and true protein contents were estimated based on the nitrogen and amino acid composition. Protein quality was assessed based on the amino acids profile. Porphyra species presented the highest protein content compared to the remaining three seaweed species tested. All samples presented a complete profile of essential amino acids and a high quality protein profile, according to World Health Organization and Food and Agriculture Organization standards. Methionine and tryptophan were the first limiting amino acids in all species. Red species (Porphyra and Gracilaria) presented high levels of free alanine, glutamic, and aspartic acids. The results highlight the potential of using seaweeds as an alternative and sustainable source of protein and amino acids for human nutrition and industrial food processing.


Introduction
Seaweeds are a diverse group of marine species that have been part of the human diet for thousands of years, especially in Asian countries. In western countries, however, algae are mainly exploited as a source of techno-functional polysaccharides (carrageenan, agar, and alginates), used as technological aids (such as texturizing and stabilizing agents in foods, for example) [1,2]. Besides, seaweeds may also have agriculture application. The development of novel, sustainable, and eco-friendly solutions is an extremely relevant topic in this field. For instance, some authors have reported that the use of a small quantity of different seaweed liquid extracts efficiently enhanced the in vitro mass propagation of an important crop of Solanum melongena L. This suggests the possibility of using it as an alternative to commercially available plant growth regulators that can be toxic and costly when compared with natural extracts [3]. The antimicrobial activity of seaweeds is also often reported. Recently, a study in a screw-cap tube. The reaction tubes were flushed under an N2 stream during 5 min in order to minimize oxidation and immediately closed prior to being placed in a Thermo block (SBH130D/3, Stuart, Stafford, UK) at 110 • C for 24 h. The hydrolyzed samples were centrifuged (3000 rpm, 10 min, Megafuge 16 centrifuge, Heraeus, Germany) and 50 µL of supernatant was collected and neutralized with 940 µL of borate buffer (pH 10.2). The internal standard (10 µL norvaline 2 mg/mL) was added to each sample. The homogenized mixtures were centrifuged for 10 min at 13,000 rpm (Biofuge pico Heraeus, Hanau, Germany) and supernatants were finally transferred into injection vials for amino acid analysis. Alkaline hydrolysis was used to determine the content of tryptophan as it degrades under acidic conditions. Alkaline hydrolysis was performed using a similar protocol, with modifications in the following steps: 150 mg of each sample were weighed and mixed with 3 mL of 4 M KOH; the tubes containing the samples were put in a Thermo block (4 h, 110 • C); following hydrolysis, the samples were neutralized with 0.1 M HCl. The extractions were performed in triplicate for each sample.

Free Amino Acids Extraction
The extraction of free amino acids was based on the study by Machado et al. [25]. Briefly, samples were prepared by dispersing the milled sample in deionized water (1:25, w/v). The extraction was performed under shaking in a multi-rotator (Multi RS-60, Biosan, Latvia) at room temperature for 30 min. After centrifugation (3000 rpm, 10 min, Megafuge 16 centrifuge, Heraeus, Hanau, Germany) the supernatants were collected. The residue was re-extracted with 5 mL of deionized water for 15 min. After re-extraction, the samples were centrifuged again and the supernatants were finally combined. Then, 990 µL of the supernatant were mixed with of the internal standard (10 µL norvaline 2 mg/mL) in an injection vial for amino acid analysis. The procedure was carried out in triplicate.

Chromatographic Analysis
The total and free amino acids contents were analyzed by RP-HPLC (Reverse Phase-High Performance Liquid Chromatography) using an integrated system from Jasco (Jasco, Japan) equipped with two high-pressure pumps (PU-980), an automatic injector (AS-4150), a fluorescence detector (FP-2020 Plus), and a UV/Vis absorption detector (MD-2015 Plus). The samples (extracts of total and free amino acids) were derivatized online with OPA/3-MPA and FMOC as described by Machado et al. [25]. Amino acids were identified based on the retention time of the corresponding standards. The quantification of each amino acid was based on the response of the fluorescence signal of each standard, converted into units of concentration through calibration curves obtained for each compound, using the internal standard method. The determination of the total and free amino acid contents was performed in triplicate.

Evaluation of Protein Quality
The AAS and the EAAI were used to assess the protein quality of the analyzed seaweeds. These parameters were calculated as follows (Equations (1) and (2)) [26,27]: AAS (%) = [mg AA in 1 g of the protein tested/mg AA in 1 g reference protein] × 100 (1) where log EAA = [1/n] × [log (100 a1/a1R) + . . . + log (100 an/anR)]; a in mg of amino acid in 1 g of tested protein; aR in mg of amino acid in 1 g of reference protein; n is the number of amino acids considered for the calculation (the pair methionine-cysteine count as 1). AA and EAA stand for amino acid and essential amino acids in Equations (1) and (2), respectively. The reference protein used was the amino acid pattern defined by the World Health Organization and the Food and Agriculture Organization (FAO/WHO/UNU) [27]. The AAS of protein was considered the lowest AAS value within essential amino acids.

Determination of Total Nitrogen, Protein Nitrogen, and Non-protein Nitrogen
Total nitrogen (TN) was estimated by Kjeldahl analysis (AOAC, method 984.13) [28]. Protein nitrogen (PN) was determined as described by Machado et al. [25]. Each sample was precipitated with 15% (w/v) trichloroacetic acid prior filtering the solution using a low N content filter paper (GPL, Porto, Portugal). The residue was then digested, distilled, and titrated under the above-mentioned conditions. Non-protein nitrogen (NPN) was estimated from the difference between TN and PN (insoluble nitrogen in 15% (w/v) trichloroacetic acid). The protein content was estimated using the conversion factor 5.00 [19]. Analyses were performed in duplicate and the results expressed as g/100 g dried sample (ds).

Statistical Analysis
Statistical analysis was performed using SPSS software, v. 26 (IBM Corporation, Armonk, NY, USA). Data were analyzed by one-way analysis of variance (ANOVA) at a significance level of p < 0.05. Where applicable, multiple comparisons were performed using Tukey s post-hoc test. Data are presented as mean ± standard deviation (SD) of triplicate extractions.

Results and Discussion
In this study, the total and free amino acid profiles of four seaweed species (Porphyra dioica, Porphyra umbilicalis, Gracilaria vermiculophylla, and Ulva rigida) produced in an IMTA system were assessed. Their protein content and protein quality based on the corresponding amino acid score (AAS) and essential amino acid index (EAAI) were also determined.

Total Amino Acids
The composition in total amino acids of the four different species of seaweeds is presented in Table 1. For both species of Porphyra it was possible to quantify the amino acids of the sporophyte (conchocelis) and gametophyte (blades) life cycle stages. In general, the analyzed seaweeds presented a similar amino acid profile, although showing significant differences (p < 0.05) in the amounts of some compounds.
The sum of total amino acid residues ( TAA) refers to the true protein content of seaweeds. Porphyra species (193.34-203.99 ds and 230.34-286.56 mg/g ds, for blades and conchocelis, respectively) showed a significantly higher (p < 0.05) TAA compared to Gracilaria vermiculophylla (106.62 mg/g ds) and Ulva rigida (92.22 mg/g ds). The TAA in the Porphyra dioica blades (203.99 ± 8.20 mg/g ds) and Porphyra umbilicalis blades (193.34 ± 2.16 mg/g ds) was consistent with the values reported by Biancarosa et al. [29] (242 and 177 mg/g dw, respectively). The lowest levels of TAA were found in Gracilaria vermiculophylla and Ulva rigida but with no significant (p < 0.05) differences between these samples. Higher values have been described by Shuuluka et al. [30] for Ulva rigida (152 mg/g dw). The values obtained for Gracilaria vermiculophylla TAA were comparable with other species of Gracilaria, namely Gracilaria changii (91.90 mg/g dw) [31] and Gracilaria birdiae (91 mg/g dw) [32]. This may reflect differences in the chemical composition of those samples related with the species itself, harvest season, geographic location, and diverse environmental conditions [33,34].
For the Porphyra species, the TAA in the conchocelis stage (286.56-230.34 mg/g ds for Porphyra dioica and Porphyra umbilicalis, respectively) was significantly higher (p < 0.05) compared to the corresponding blades stage (203.99-193.34 mg/g ds, by the same order). As described for phycobiliproteins [35], the higher content of amino acids in the conchocelis stage might be related to the pathways of nitrogen uptake, utilization, and storage. It should also be pointed out that cell walls present a different chemical structure at the different life cycle stages. In blades, these present more than twice of the fiber content compared to that found in conchocelis. Hence, the latter are reported to present significantly higher protein values compared to blades [36]. Amino acids are represented by the 3-letter abbreviation code. In each row, different superscript letters represent significant differences between samples (p < 0.05), while the same superscript letters denote no significant differences (p > 0.05). n.d.: not detected.

Essential Amino Acids
Despite the quantitative differences observed between the different species for the individual compounds, all samples presented a complete profile of EAA (Table 1). The most abundant EAA in red species (Porphyra dioica, Porphyra umbilicalis, and Gracilaria vermiculophylla) were leucine, valine, and threonine. The leucine values ranged from 9.01 to 16.63 mg/g ds; valine from 6.84 to 12.79 mg/g ds; and threonine from 6.21 to 12.27 mg/g ds. Significant differences were observed (p < 0.05) between the Porphyra species and Gracilaria vermiculophylla, which presented the lowest values for the aforementioned compounds. These results are consistent with other studies [33,37,38]. The amino acid composition of proteins is frequently used to determine its nutritional quality [6]. High quality protein presents a complete and optimal EAA composition for human needs. Most dietary protein of animal origin (meat, fish, milk, and egg) present higher levels of EAA being considered as high quality protein [39,40]. In contrast, some plant-based dietary proteins can be seen as being of lower nutritional quality due to their low content in one or several EAA [40]. EAA have important roles in the body, namely branched-chain amino acids, which are essential for the proliferation of lymphocytes and maturation of dendritic cells, and some inhibitory effect in cancer cell proliferation [41]. From another perspective, the incorporation of Porphyra umbilicalis in pork products promoted a significant increase in serine, glycine, alanine, valine, tyrosine, phenylalanine, and arginine levels of the formulations [42]. The high lysine concentrations found in the analyzed samples may be interesting, as seaweeds may be used to balance the amino acids composition of cereal-based products, which often contain a low lysine content, also considered the limiting amino acid in plant-based proteins [43].
The % EAA for Porphyra dioica and Porphyra umbilicalis blades was similar to the values presented by Biancarosa et al. [29] (38.7 and 38.5% dry weight (dw), respectively). However, the values obtained herein were lower than those presented by Vieira et al. [8] (39.9-44.3% dw for Porphyra spp). The % EAA presented for Ulva rigida was higher than that presented by Shuuluka et al. [28] (30.8% dw), and comparable to what Lourenço et al. [47] have reported for Ulva fasciata (41.4% dw). The % EAA in Gracilaria vermiculophylla was lower than the values previously described for other species of Gracilaria, which ranged from 50-61% dw [31,32].

Non-Essential Amino Acids
Acid aspartic was the most abundant non-essential amino acid (NEAA) in all samples, ranging from 12.05 to 24.15 mg/g ds in adult seaweeds, reaching 32.49 ± 0.21 mg/g ds in Porphyra dioica conchocelis. Glutamic acid was the second most abundant amino acid in all species ranging from 9.47 to 31.48 mg/g ds, except in blade stage of Porphyra species, which was richer in alanine. The content of acidic amino acids ranged from 22.33 ± 0.07% to 23.60 ± 0.28% of the total amino acids obtained in Porphyra dioica conchocelis and in Gracilaria vermiculophylla, respectively. Astorga-España et al. [33] also found high levels of these amino acids for Porphyra spp. and Ulva spp. (22.98 and 21.79%, respectively). The levels of acidic amino acids include the simultaneous quantification of the amides (glutamine and asparagine), which are converted to the corresponding acids during acid hydrolysis [45].
All samples have also presented high concentrations of other NEAA such as alanine, arginine, serine, and glycine. The presence of these amino acids is interesting due to their important biological functions. For instance, arginine supplementation improves the function of the intestinal barrier and vascular development [49]; glycine helps in the regulation of the immune response and helps to prevent rejections after organ transplantation [50]; alanine is used to treat muscle degeneration [9]; serine supplementation may be useful in the treatment of hereditary sensory and autonomic neuropathy type 1 [51]. From another perspective, high levels of glutamic acid, aspartic acid, alanine, and glycine have been described as responsible for the characteristic umami flavor of seaweeds [34,45]. Hydroxyproline was the NEAA that presented the lowest concentration, reaching the maximum of 1.06 ± 0.02 mg/g ds in the green algae Ulva rigida. All other samples presented hydroxyproline values ≤0.26 mg/g ds. Porphyra blades had a similar profile of NEAA (p > 0.05). Significant differences were only observed in glycine content (16.75 ± 0.74 and 13.61 ± 0.09 ±, for Porphyra dioica and Porphyra umbilicalis blades, respectively). Overall, Porphyra dioica blades had higher levels of NEAA (p < 0.05) than those found in Porphyra umbilicalis (63.16 ± 0.44 and 61.55 ± 0.10 mg/g ds). Porphyra dioica conchocelis had a significantly higher (p < 0.05) content of aspartic acid, glutamic acid, serine, arginine, alanine, tyrosine (32.49 ± 0.21 > 31.48 ± 0.43 > 30.25 ± 0.29 > 23.68 ± 0.24 > 16.42 ± 0.20 > 10.17 ± 0.16 mg/g ds, respectively) than the blades and conchocelis of Porphyra umbilicalis. Proline content was similar in both stages of the analyzed Porphyra species.

Free Amino Acids
Free amino acids are not linked to other amino acids, peptides, or proteins, usually contributing to the food taste [6]. These amino acids act as one of the main reservoirs for storing nitrogen in both green and red seaweeds [52].
The free amino acid composition of the four different seaweeds species, including the two life cycle stages of Porphyra, is shown in Table 2 in which it is possible to observe significant differences (p < 0.05) in the amino acid composition between the analyzed species, even between the life stages of the same Porphyra species.
The free amino acids composition was mainly represented by alanine, glutamic acid, and aspartic acid in both Porphyra species and Gracilaria Vermiculophylla, presenting concentrations ranging from 0.26 to 5.50, 1.14 to 4.77, and 0.53 to 3.53 mg/g ds, respectively. These amino acids play an important role in the umami flavor, characteristic of seaweeds [45]. Noda et al. [53] also found that the predominant free amino acids in Porphyra spp. were aspartic acid, glutamic acid, alanine, and taurine. Admassu et al. [54] found that in addition to the above-mentioned amino acids, arginine was also one of the main amino acids in Porphyra spp. In Ulva rigida, the most abundant amino acid was histidine (1.96 ± 0.09 mg/g ds) and asparagine (1.45 ± 0.06 mg/g ds), presenting significantly higher (p < 0.05) concentrations compared to the remaining seaweeds. Methionine and tryptophan were only detected in Porphyra dioica conchocelis. In the red seaweeds species, the free amino acid composition was represented in over 80% by NEAA. Ulva rigida presented a different proportion between EAA and NEAA, corresponding to approximately 45 and 55%, respectively. Table 2. Free amino acids composition expressed in mg/g of dry sample (ds) of Porphyra dioica (blades and conchocelis), Porphyra umbilicalis (blades and conchocelis), Gracilaria vermiculophylla, and Ulva rigida.

Evaluation of Protein Quality Based on the Amino Acids Profile
The composition, proportion, and availability of essential amino acids in proteins contribute to determine the nutritional quality of food [33]. The EAA/NEAA ratio was used to evaluate the distribution of those amino acids in proteins from the analyzed seaweeds. The results ranged from 0.58 to 0.67 within the red species and reached 0.69 in the green algae Ulva rigida. These values indicate that EAA were in lower concentrations compared to NEAA in all samples. Higher values have reported for Gracilaria spp., Porphyra spp. and Ulva spp., which have presented an EAA/NEAA ratio of 1.74, 1.32, and 1.32, respectively [8] determined an EAA/NEAA ratio of 1.74, 1.32, and 1.32 for the Gracilaria sp., Porphyra sp., and Ulva sp., respectively. Nevertheless, the results obtained herein are similar to those reported by other authors: 0.63 in both Porphyra umbilicalis and Porphyra dioica [29]; 0.54 and 0.56 for Porphyra spp. and Ulva spp. [33]. Once again, several factors (seasonality, place of production...) influence the composition of seaweeds, namely their composition in amino acids, which may be the cause of the differences observed [29].
In this study, two important chemical parameters were used to evaluate protein quality: the AAS and the EAAI. The first is intended to predict protein quality relying on the potential ability of a food protein to provide the appropriate pattern of dietary essential amino acids [55]. The EAAI compares the protein quality through the geometric mean value of the essential amino acid in relation to a reference protein [37]. EAAI reflects more the biological quality of a protein than AAS [37]. Table 3 presents the AAS and EAAI values for all samples, based on the recommended amino acid pattern for adults, according to FAO/WHO/UNU [27]. As tyrosine can substitute phenylalanine, through metabolic processes, these amino acids were combined (tyrosine + phenylalanine) for the calculation of AAS [33]. From the AAS it was possible to determine the limiting amino acid, which corresponds to the essential amino acid that presented the greatest difference in concentration compared with the same amino acid in the reference protein [8]. Table 3. Amino acid score (AAS) and essential amino acid index (EAAI) values for Porphyra dioica (blades and conchocelis), Porphyra umbilicalis (blades and conchocelis), Gracilaria vermiculophylla, and Ulva rigida. LAA: limiting amino acid; AAS: amino acid score; EAAI: essential amino acid index. * Met + Cys. Amino acids represented using the 3-letter abbreviation code. In each row, different superscript letters represent significant differences between samples (p < 0.05) while the same superscript letters denote no significant differences (p > 0.05).

Essential
The AAS was 48.7% for Porphyra dioica blades, 56.5% for Porphyra umbilicalis blades, 58.4% for Gracilaria vermiculophylla, and 90.8% for Ulva rigida. Conchocelis had a higher AAS than the corresponding blades (87.1 and 93.4%, for Porphyra dioica and Porphyra umbilicalis, respectively). The concentrations of isoleucine, leucine, lysine, threonine, phenylalanine + tyrosine were higher than the FAO/WHO/UNU standard [27] for all samples, meaning that the corresponding AAS values exceeded 100%. Methionine and tryptophan were the first limiting amino acids in all red seaweeds species. For Porphyra umbilicalis blades histidine was the second limiting amino acid. These results are in agreement with other authors [8,31,34,37] which reported that sulfur-containing amino acids, tryptophan, and histidine are the main limiting amino acids. Benjama and Masniyom [56] and Mišurcová et al. [45] found lysine as the main limiting amino acid in Gracilaria and Porphyra, with AAS ranging from 41.6 to 82.2% in Gracilaria. However, AAS values obtained herein for lysine were relatively high, ranging from 109.72% to 173.20% for the Ulva rigida and Porphyra dioica conchocelis, respectively. These differences may be related to the reference protein used, species, geographical origin, season of harvesting, environmental conditions, and the physiology of each species [33]. The EAAI ranged from 90.77% to 115.74% in red species, reaching 123.38% in Ulva rigida. The protein quality of the Ulva rigida exceeded that of the other seaweeds, that is, it exhibited an amino acid profile closer to the reference protein. A protein with high quality and efficiency is generally characterized by a high EAAI value. According to Brown and Jeffrey [57], a protein has high quality when the EAAI value is greater than 90%, moderate quality when the EAAI is between 70-89% and low quality when the EAAI is less than 70%. Based on the reference standard [27], the proteins of the analyzed seaweeds can be considered of high quality. Therefore, based on the results, these seaweed species could be used as a source of high quality protein, or as ingredients to improve the amino acid profile of food formulations.

Nitrogen and Protein Content
Three different approaches were used to assess the protein content samples, which comprised: (i) the conversion of total nitrogen (determined by the Kjeldahl method) to protein by the conversion factor 5.00 [19]; (ii) the conversion of protein nitrogen (following precipitation with trichloroacetic acid) to protein using the conversion factor 5.00 [19]; (iii) the sum of the total amino acids. Method (i) estimates the crude protein, while the latter (ii and iii) estimates the "true protein." Table 4 shows the nitrogen content (non-protein, protein, and total) and the estimated protein content based on the above-mentioned approaches. The main disadvantage of the Kjeldahl method is the quantification of non-protein nitrogen, which includes chlorophyll, nucleic acids, free amino acids, and inorganic nitrogen [19]. The use of the universal nitrogen-to-protein conversion factor of 5.00, proposed by Angell et al. [19] allows for estimating the protein content of seaweeds more accurately, based on the protein nitrogen fraction. However, it should be noted that this proposed factor is a median value and that seaweeds have a variable amino acid composition and amount of non-protein nitrogen. The protein content estimation according to approach (iii) is widely accepted since the 1970s, although it presents some disadvantages [33]. Amino acid analysis can underestimate protein content due to partial or total destruction of some amino acids during hydrolysis (in particular, cysteine, tryptophan, methionine, and serine); furthermore, the use of a single hydrolysis time may not assure the complete hydrolysis of certain amino acids without destroying others [19]. However, Lourenço et al. [47] stated that if a sample contains 10% free amino acids, the typical loss during acid hydrolysis might compensate for the influence of free amino acids in the protein quantification using the sum of the total amino acid residues. In this study, the free fraction was less than 10% of the total amino acids, thus there might have occurred an underestimation when assessing the true protein values using the sum of total amino acid residues.
In general, the genus Porphyra had a significantly higher (p < 0.05) protein content compared to Gracilaria vermiculophylla and Ulva rigida, regardless of the approach used. The crude protein content was slightly higher than the true protein content (estimated by the conversion NP × 5.00, and obtained by the TAA), except for Porphyra dioica conchocelis. Notwithstanding, the obtained values were within a similar range.
Ulva rigida showed a significantly lower (p < 0.05) protein nitrogen content (0.22 ± 0.17 g/100 g ds) compared with the Porphyra dioica blades (1.12 ± 0.04 g/100 g ds). No significant differences (p > 0.05) were observed for the protein nitrogen content of the red seaweed species. In fact, Lourenço et al. [47] reported that the latter present higher amounts of protein nitrogen than green and brown seaweeds, corroborating the results obtained herein.
The protein content of the analyzed seaweeds (9.62-28.66 g/100 g ds) was comparable to that of protein-rich plant-based foods, such as beans (20.9%), lupine (30.5%), chickpeas (24.7%), linseed (26.35%), peanuts (29.59%), and rice (9.57%) [43]. The used algal species have great potential to be used in human nutrition as a source of protein. However, the digestibility of these proteins must be evaluated, as it tends to be limited by the non-protein fraction [37].
There are other concerns regarding the consumption of seaweeds, namely those related with the excessive intake of iodine. The recommended daily allowance (RDA) by the World Health Organization is 150 µg for adults [58]. A small amount of seaweeds can exceed the tolerable intake limit for humans (1100 µg), and consequently affect thyroid health [6]. For example, a portion of 0.3 g (dw) of Laminaria spp. may exceed the tolerable intake limit. On the other hand, it is reported that 5 g (dw) of Porphyra tenera or Ulva rigida contain 80 µg (53% of RDA) and 40 µg (27% of RDA) of iodine, respectively [6], is what can be considered as safe. The concentration of iodine in different seaweed species is highly variable; therefore, the content of this micronutrient must be accurately labelled to avoid excessive intake [6]. Generally, Porphyra and Ulva species have lower levels of iodine compared to brown algae [6]. The consumption of a portion of 5 g (dw) of Porphyra or Ulva may be the most adequate, in order to maintain a safety margin. However, elements were not analyzed in this study.
Based on the results of this study, the consumption of a 5 g portion (ds) of Porphyra dioica conchocelis, Porphyra umbilicalis conchocelis, Porphyra dioica blades, Porphyra umbilicalis blades, Gracilaria vermiculophylla, and Ulva rigida provides an average of 1.43, 1.15, 1.02, 0,97, 0.53, and 0.48 g of protein, respectively. Considering that the RDA of protein intake for an adult of 70 kg is equivalent to 58 g [25] the intake of 5 g (ds) of the Porphyra dioica conchocelis, Porphyra umbilicalis conchocelis, Porphyra dioica blades, Porphyra umbilicalis blades, Gracilaria vermiculophylla, and Ulva rigida contributes to 2.47, 1.99, 1.76, 1.67, 0.92, and 0.83% of RDA, respectively. Based on these observations, seaweeds can be a sustainable alternative to diversify or complement the diet. Extracting proteins from seaweeds may also be a strategy to take advantage of their high protein content, as the presence of iodine or even heavy metals restricts the intake of larger seaweed portions. Seaweed-derived protein isolates or hydrolysates may be used as food ingredients, contributing, for example, to improve the composition of high protein food formulations. The inclusion of seaweeds (protein extracts) in food products can bring added-value for the food industry. However, efforts are needed in the food biotechnology field in order to make this process viable and accessible.

Conclusions
Results obtained in this study demonstrated that the analyzed seaweeds, which were produced in an IMTA system, presented a complete EAA profile and, consequently, a high quality protein profile, according to the FAO/WHO/UNU standards. Porphyra species (both life cycle stages) were characterized by a higher protein content compared to Gracilaria vermiculophylla and Ulva rigida. Conchocelis, in particular, had the highest protein content. As the free amino acid fraction in red seaweeds was characterized by a high content in the alanine, glutamic acid, and aspartic acid (amino acids responsible for umami flavor), these may possibly be extracted and used as flavor enhancers. These results highlight the potential of using seaweeds as an alternative and sustainable source of protein and amino acids for human nutrition and industrial food processing.
Overall, it appears that the production of these seaweed species in an IMTA system contributed to the production of high protein quality biomass.