Introduction
Marine ecosystems harbour a substantial proportion of the planet’s biomass and represent an immense reservoir of sustainable food, feed, energy, and bioactive compounds (Wells et al., Reference Wells, Potin, Craigie, Raven, Merchant, Helliwell and Brawley2017). The combined pressures of a growing global population and shifting sociodemographic patterns are expected to intensify demand on the world’s natural resources and necessitate the diversification of food systems. In this context, macroalgae (seaweeds), as marine primary producers, have been increasingly proposed as novel and sustainable food sources (Henchion et al, Reference Henchion, Hayes, Mullen, Fenelon and Tiwari2017). Unlike terrestrial plants, macroalgae are highly efficient biomass producers: they absorb nutrients directly from the surrounding water, exhibit rapid growth rates, and require neither arable land nor freshwater (Duarte et al., Reference Duarte, Wu, Xiao, Bruhn and Krause-jensen2017).
The nutritional composition of macroalgae, characterized by high protein, amino acid, vitamin, and mineral content alongside a low lipid fraction, makes them an attractive seafood alternative for health-conscious consumers, ranking second only to fish in nutritional relevance (Southgate, Reference Southgate, Southgate, Waldron, Johnson and Fenwick1990). The protein content of algae, which have been consumed as a food staple in East Asian countries for centuries, is broadly comparable to that of many terrestrial protein sources (Jeon et al., Reference Jeon, Lee and Ryu1980). Although the lipid fraction typically constitutes only a small proportion of the total dry weight (dw) of macroalgae, its qualitative composition is of considerable nutritional importance. Despite their overall low fat content, seaweeds are recognized as a valuable source of biologically active lipids, particularly unsaturated fatty acids (Jaworowska and Murtaza, Reference Jaworowska and Murtaza2023). Furthermore, seaweeds are known to selectively absorb and accumulate minerals from ambient seawater within their thalli, rendering them suitable as mineral-rich food or feed additives following appropriate processing (Rohani-Ghadikolaei et al., Reference Rohani-Ghadikolaei, Abdulalian and Ng2012).
Despite this general nutritional potential, the biochemical composition of macroalgae is not static. Algal metabolism is highly sensitive to fluctuations in environmental conditions, and abiotic factors such as light intensity, photoperiod, water temperature (Hu et al., Reference Hu, Zou, He, Shi and Liu2022), and the seasonal availability of nutrients, particularly nitrate and phosphate, directly influence the biosynthetic pathways governing protein, lipid, and carbohydrate production (Boyce et al., Reference Boyce, Lewis and Worm2010; Xu et al., Reference Xu, Zou and Gao2010). It is well established that ecological variables shape not only the reproduction, development, and morphology of algae, but also their biochemical composition (Lobban and Harrison, Reference Lobban and Harrison1994; Nascimento et al., Reference Nascimento, Coelho-gomes, Barbarino and Lourenço2014; Garcia-Vaquero et al., Reference Garcia-Vaquero, Rajauria, Miranda, Sweeney, Lopez-alonso and O’doherty2021).
The genus Ulva (commonly known as sea lettuce, and historically treated as synonymous with the genus Enteromorpha) is a cosmopolitan member of the Chlorophyta and represents an important model organism for investigating these seasonal biochemical dynamics. Ulva species are capable of forming dense accumulations known as green tides through rapid proliferation in coastal areas affected by eutrophication. While these blooms constitute an ecological concern, they simultaneously present an opportunity for the valorization of abundant, locally available biomass.
The Black Sea is a distinctive marine environment characterized by reduced salinity relative to the Mediterranean, pronounced seasonal temperature fluctuations, and elevated eutrophication potential driven largely by riverine inputs. These specific physicochemical conditions are expected to impart unique biochemical profiles to macroalgae inhabiting this region, potentially differing from those reported for populations in the Atlantic or Mediterranean. Türkiye’s Black Sea coastline supports a diverse macroalgal flora, including established populations of Ulva intestinalis (Aysel et al., Reference Aysel, Erdugan and Dural-tarakci2005, Reference Aysel, Dural, Şenkardeşler, Erduğan and Aysel2008; Karaçuha and Gönülol, Reference Karaçuha and Gönülol2007; Karaçuha et al., Reference Karaçuha, Yıldız and Karaçuha2018; Yıldız et al., Reference Yıldız, Karaçuha and Karaçuha2019). However, comprehensive seasonal investigations into the nutritional composition, specifically the fatty acid, amino acid, and mineral contents of U. intestinalis from this region remain limited.
Identifying the optimal harvest season, during which the biochemical content is maximized, is essential for the sustainable exploitation of this biomass as a functional food ingredient, dietary supplement, or animal feed additive. Accordingly, the present study aims to characterize the seasonal variation in the fatty acid, amino acid, and mineral element profiles of U. intestinalis L. naturally distributed along the southern Black Sea coast (Sinop), and to statistically evaluate the influence of season on these biochemical components.
Materials and methods
Sampling
The study was conducted at four distinct sampling locations (DSİ, Hamsilos, Karakum, and Akliman) along the coastline of the Sinop Peninsula (the map presented in Figure 1 was generated using the HGM Atlas application). Specimens of the chlorophyte U. intestinalis were collected seasonally from rocky substrates at depths of 0–1 m. No samples were obtained during autumn. Upon collection, specimens were immediately placed in polyethylene containers, appropriately labelled, and transported to the laboratory. Following arrival, samples were carefully cleaned to remove extraneous biotic and abiotic material, including epiphytic organisms, faunal components, and sedimentary particles, and subsequently rinsed with distilled water. After removal of excess surface water, the samples were oven-dried at 60°C for 48 hours (Moustafa and Batran, Reference Moustafa and Batran2014; Yeşilova et al., Reference Yeşilova, Balkis and Taşkın2017; Ersoy Karaçuha et al., Reference Ersoy Karaçuha, Karaçuha and Yıldız2023). The dried algal biomass was ground to a fine powder (<500 µm) and stored at −20°C in vacuum-sealed polyethylene bags until analysis. All analyses were performed promptly after the completion of the sampling period to minimize potential biochemical degradation. For elemental analysis, 0.5 g aliquots of each sample were digested with Suprapur® HNO₃ in a microwave digestion system (Milestone Systems, Start D 260), following the standardized Aquatic Plant HPR-FO-08 protocol (Milestone). Elemental concentrations in the resulting digests were determined by inductively coupled plasma mass spectrometry (ICP-MS; Agilent Technologies, 7700X) (SEM Laboratory, 2016).
Sampling areas.
Lipid content analysis
The lipid extracts were prepared according to the methodology established by Bligh and Dyer (Reference Bligh and Dyer1959), employing a mixture of chloroform and methanol as the extraction solvents. The extracted algal lipid was then subjected to transesterification to produce fatty acid methyl esters (FAME), as described by Ichihara et al. (Reference Ichihara, Shibahara, Yamamoto and Nakayama1996). Specifically, 250 mg of extracted oil was dissolved in 4 mL of n-heptane, followed by the addition of 400 μL of 2 N methanolic potassium hydroxide solution. The mixture was vortexed vigorously for 120 seconds to initiate methylation, then centrifuged at 5,000 rpm for 300 seconds to allow for phase separation. The upper heptane layer (approximately 1.5–2.0 mL), containing the FAMEs, was carefully aspirated and transferred into chromatographic vials for analysis. Gas chromatography-mass spectrometry (GC-MS) analysis was performed using an automated injection system (Autosampler AI 1310), with instrumental parameters standardized according to established protocols (Thermo Scientific) to ensure optimal separation and identification of individual fatty acids.
Fatty acid analysis
FAMEs were characterized using a GC-MS system (Thermo Scientific). Chromatographic separation was achieved on a high-resolution Trace Gold TG-WaxMS capillary column (60 m length, 0.25 μm internal diameter, 0.25 μm film thickness; Thermo Scientific, catalogue number: 26088-1540). The temperature programme consisted of an initial isothermal hold at 100°C for 3 minutes, followed by a linear temperature gradient of 4°C per minute until reaching a final temperature of 240°C. Sample injection was performed at a 1:20 split ratio, with helium as the carrier gas at a constant flow rate of 1 mL/min. Mass spectral data were collected using the ISQ LT (Intelligence Single Quadrupole Leading Technology) mass-selective detector in electron impact ionization mode (70 eV). Compound identification was achieved by comparing retention times with a standardized 37-component FAME reference mixture (Supelco 37 Component FAME Mix, Sigma-Aldrich). To ensure analytical rigour and reproducibility, all measurements were performed in triplicate.
Amino acid analysis
Amino acid profiles were determined using high-performance liquid chromatography (HPLC) with the AccQ · Tag method developed by Waters Corporation (Liu et al., Reference Liu, Chang, Yan, Yu and Liu1995; Waters Corporation, 1993). The samples were hydrolysed in sealed glass tubes with 6 N HCl containing 1% phenol under a nitrogen atmosphere at 110°C for 22 hours, following the protocols outlined in Yaich et al. (Reference Yaich, Garna, Besbes, Paquot, Blecker and Attia2011) and Benjama and Masniyom (Reference Benjama and Masniyom2011). After hydrolysis, the acidic solution was evaporated to dryness using rotary evaporation. The residue was reconstituted with an appropriate internal standard and diluted volumetrically with deionized water. Subsequently, a 10 μL aliquot of the filtered solution was mixed with 70 μL of AccQ fluor buffer and 20 μL of AccQ fluor reagent for derivatization, in line with procedures reported by Liu et al. (Reference Liu, Chang, Yan, Yu and Liu1995). The mixture was heated at 55°C for 10 minutes in a temperature-controlled block and allowed to cool before instrumental analysis. Chromatographic separation was performed using a Waters Alliance 2695 HPLC system coupled with a Waters 2475 Multi-λ fluorescence detector set at an excitation wavelength of 250 nm and an emission wavelength of 395 nm (Ortiz et al., Reference Ortiz, Romero, Robert, Araya, Lopez-hernández, Bozzo, Navarrete, Osorio and Rios2006; Ratana-arporn and Chirapart, Reference Ratana-arporn and Chirapart2006). Separation was achieved on a Hypersil Gold C18 analytical column (3.9 × 150 mm, 3 μm particle size). The mobile phase consisted of a binary gradient composed of AccQ · Tag eluent A and a mixture of acetonitrile and water (component B). Identification of amino acids was performed by comparing the retention times of the sample peaks with those of standard amino acids (Sigma Chemicals).
Mineral content analysis
Elemental analyses were conducted following Environmental Protection Agency Method 200.2 and Method 6020, governing sample preparation for total recoverable elements and multi-elemental determination by ICP-MS in environmental samples, respectively. Dried plant samples, homogenized by grinding, ranging from 0.2 to 0.5 g, were digested using 8 mL of concentrated nitric acid (Suprapur, 65% HNO₃) in accordance with the microwave digestion procedure HPR-FO-08, employing a microwave digestion system. The digestion programme consisted of a temperature ramp from ambient to 180°C over 15 minutes, followed by an isothermal hold at 180°C for 15 minutes, and a subsequent controlled cooling phase back to room temperature over approximately 1 hour. The resulting digest was quantitatively transferred into 50-mL polypropylene Falcon tubes and made up to a final volume of 50 mL with ultrapure water.
Elemental concentrations were determined by ICP-MS (Agilent 7700X). Calibration curves were constructed using certified multi-element standard solutions obtained from Agilent (27-element mix: 8500-6940 2A and 8500-6940 Hg). Quality assurance and quality control were implemented through triplicate measurements and the parallel analysis of certified reference material (CRM). BCR-279 (Ulva lactuca, sea lettuce) was employed as the CRM, and all target elements were recovered with an accuracy of ≥90%. Throughout the analysis, a 1 ppm internal standard solution (Agilent 5188-6525) was continuously co-analysed with all samples to monitor and compensate for instrumental drift (U.S. Environmental Protection Agency, 2007).
Statistical analyses
Statistical analyses were performed to evaluate differences between groups using one-way analysis of variance (ANOVA) when parametric assumptions were met, or the non-parametric Kruskal–Wallis H test when they were not. To identify specific significant differences, the Tukey HSD post hoc test was employed following ANOVA, while the Dunn–Bonferroni test was used following the Kruskal–Wallis H test. Correlations among the fatty acid, amino acid, and mineral contents of U. intestinalis were examined using Spearman’s rank correlation coefficient (ρ). Principal component analysis (PCA) was conducted to elucidate multivariate relationships regarding the temporal variations of these components and to visualize clustering patterns of sampling periods. All analyses were carried out using SPSS Statistics (Version 27) and OriginPro 2026 (64-bit), with statistical significance set at p < 0.05.
Results
Fatty acids
The seasonal variations in the fatty acid composition of U. intestinalis are presented in Table 1 and Figure 2. Total fatty acid content differed significantly across seasons, peaking in spring (2,235 ± 40.03 mg/100 g dw) and declining to its lowest value in summer (761 ± 9.56 mg/100 g dw), with winter yielding an intermediate value (1,817 ± 11.67 mg/100 g dw). Significant seasonal fluctuations were observed in the proportions of saturated fatty acids (SFAs), monounsaturated fatty acids (MUFAs), and polyunsaturated fatty acids (PUFAs) (p < 0.05).
Comparison of the total fatty acids, SFA, MUFA, PUFA, PUFA/SFA ratio, and ω-6/ω-3 ratio of U. intestinalis, seasonally.
Figure 2 Long description
Bar graph with six grouped categories on the horizontal axis: SFA percent, MUFA percent, PUFA percent, PUFA slash SFA, omega minus 6 slash omega minus 3 and Total left parenthesis mg over 100g right parenthesis. A legend lists Winter, Spring, Summer. Vertical axis shows two separated scale sections. Lower section tick labels: 0.0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8. Upper section tick labels: 0, 500, 1000, 1500, 2000, 2500. SFA percent: Winter 38.83, Spring 38.89, Summer 51.46. MUFA percent: Winter 40.82, Spring 37.08, Summer 39.47. PUFA percent: Winter 20.33, Spring 24.04, Summer 9.07. PUFA slash SFA: Winter 0.52, Spring 0.61, Summer 0.17. omega minus 6 slash omega minus 3: Winter 0.67, Spring 0.75, Summer 0.72. Total left parenthesis mg over 100g right parenthesis: Winter 1817, Spring 2235, Summer 761.
Fatty acid composition and content of U. intestinalis
Table 1 Long description
Seasonal fatty acid composition of Ulva intestinalis is reported for winter, spring, and summer as mean plus or minus standard deviation, with a significance column for seasonal differences. Saturated fats are similar in winter and spring (about 38.8) but rise in summer (about 51.5), driven mainly by palmitic acid increasing from about 24 to 35 and behenic acid from about 1.4 to 3.5. Monounsaturated fats are highest in winter (about 40.8), lowest in spring (about 37.1), and intermediate in summer (about 39.5); oleic acid drops from about 17.6 in winter to 13.8 in spring, while elaidic acid is slightly higher in spring than winter. Polyunsaturated fats peak in spring (about 24.0), are slightly lower in winter (about 20.3), and fall markedly in summer (about 9.1); linoleic acid declines from about 7 to 3, and key long-chain omega-3 and omega-6 acids such as EPA and DHA are highest in spring and lowest in summer. Total omega-6 and total omega-3 follow the same pattern, highest in spring and lowest in summer, while the omega-6 to omega-3 ratio stays below 1 across seasons and is highest in spring. The polyunsaturated-to-saturated ratio is highest in spring (about 0.61) and lowest in summer (about 0.17), indicating a shift toward more saturated profiles in summer. Total fatty acids (milligrams per 100 grams dry weight) are highest in spring (about 2235), lower in winter (about 1817), and much lower in summer (about 761). Some entries are not applicable in certain seasons (for example, eicosatrienoic acid appears only in spring), and statistical significance is indicated by p-values and letter groupings within each row.
Data are presented as mean values ± standard deviation (SD) of three replicates. N/A: not applicable; dw: dry weight. Values indicated by different superscript letters (a–c) in the same row represent statistically significant differences (p < 0.05).
Significant seasonal differences determined by Kruskal–Wallis H test followed by Dunn–Bonferroni correction.
SFAs constituted a major fraction of the total fatty acid pool and exhibited marked seasonal dependence. Total SFA content was lowest during the colder months (winter: 38.83% ± 0.19%; spring: 38.89% ± 0.12%) and increased significantly to a maximum of 51.46% ± 0.60% in summer (p = 0.061). Among individual SFAs, palmitic acid (C16:0) was the predominant component across all seasons, rising significantly from 24.30% ± 0.19% in winter to 35.24% ± 0.64% in summer (p = 0.027). Stearic acid (C18:0) was the second most abundant SFA, ranging from 6.15% to 7.94%, with the highest concentrations recorded in winter.
Total MUFA content remained relatively high throughout the year, reaching a maximum in winter (40.82% ± 0.12%) and declining slightly in spring (37.08% ± 0.15%) (p = 0.027). Oleic acid (C18:1ω9 cis) and elaidic acid (C18:1ω9 trans) were identified as the codominant MUFAs, with oleic acid concentrations ranging from 13.75% to 17.55% and the highest values recorded in winter. Eicosenoic acid (C20:1ω9) also declined significantly from winter (2.44%) to summer (1.79%).
PUFA exhibited the most pronounced seasonal variation among all fatty acid classes, reflecting a metabolic response to environmental change. Total PUFA content peaked in spring (24.04% ± 0.12%), coinciding with the active growth season, before declining sharply to 9.07% ± 0.31% in summer (p = 0.027). α-Linolenic acid (ALA, C18:3ω3) was the most abundant PUFA in both winter (10.62%) and spring (10.70%), decreasing markedly to 4.36% in summer. LA (C18:2ω6) and arachidonic acid (ARA, C20:4ω6) followed similar declining trends. Eicosapentaenoic acid (EPA, C20:5ω3) was significantly more abundant in spring (1.51%) than in summer (0.43%). Notably, docosahexaenoic acid (DHA, C22:6ω3) displayed a contrasting pattern: unlike total PUFAs, DHA concentrations were highest in summer (3.49 ± 0.06%) compared to winter (0.98%) and spring (1.45%) (p = 0.027).
The nutritional quality of the lipid fraction varied with the seasons. The ω-6/ω-3 ratio remained within a favourable range (0.67–0.75) throughout the year, with the lowest value recorded in winter. The PUFA/SFA ratio was significantly higher in spring (0.61) and winter (0.52) compared to the summer season (0.17) (p = 0.027), reflecting the saturation of lipids during the warmer period.
Amino acid profiling
The amino acid composition of U. intestinalis exhibited significant seasonal variation (p < 0.001), as presented in Table 2. The total content of both essential amino acids (EAAs) and non-essential amino acids (NEAAs) was highest in winter and declined progressively towards summer.
Amino acid composition (g/100 g dw) of U. intestinalis (significant seasonal differences determined by one-way ANOVA followed by Tukey’s test)
Table 2 Long description
Seasonal amino acid composition of U. intestinalis is reported in grams per 100 grams dry weight for winter, spring, and summer, with mean and standard deviation and a significance column for seasonal differences. Overall amino acids are highest in winter at 11.97 and lower in spring at 6.55 and summer at 5.83, with a significant seasonal effect. Essential amino acids total 4.45 in winter, 2.69 in spring, and 2.44 in summer, while non-essential amino acids total 7.28 in winter, 3.86 in spring, and 3.39 in summer; both totals decline from winter to summer with significant differences. The ratio of essential to non-essential amino acids increases across seasons, from 0.61 in winter to 0.70 in spring and 0.72 in summer, and is reported as significantly different. Many individual amino acids follow the same pattern of winter highest and summer lowest, including lysine (0.68 to 0.40), leucine (0.84 to 0.46), aspartic acid (1.39 to 0.56), and glutamic acid (1.41 to 0.51), with significant seasonal differences. Some values are not determined in spring and summer for cystine and glycine, so comparisons for those amino acids are incomplete.
Nd, not determined; EAAs, essential amino acids; NEAAs, non-essential amino acids; AAs, amino acids. Values are presented as mean ± SD. Means within the same row with distinct superscript letters are significantly different at p < 0.05.
Among EAAs, leucine (Leu) was the predominant amino acid across all seasons, ranging from 0.84 ± 0.00 g/100 g dw in winter to 0.46 ± 0.01 g/100 g dw in summer, followed by threonine (Thr) and phenylalanine (Phe). Methionine (Met) and histidine (His) were the least abundant EAAs. Notably, cystine (Cys) was detected exclusively in winter samples (0.24 ± 0.00 g/100 g dw) and was absent in spring and summer. Total EAA content decreased significantly from 4.70 ± 0.01 g/100 g dw in winter to 2.44 ± 0.01 g/100 g dw in summer (p < 0.001).
Among NEAAs, glycine (Gly) was the most abundant component in winter, reaching 7.44 ± 0.00 g/100 g dw. Glutamic acid (Glu), Aspartic acid (Asp), and alanine (Ala) were also present in substantial quantities, with Glu ranging from 1.41 g/100 g dw in winter to 0.51 g/100 g dw in summer. Total NEAA content declined sharply from 7.28 ± 0.02 g/100 g dw in winter to 3.39 ± 0.01 g/100 g dw in summer.
Despite the overall reduction in total amino acid content during the warmer months, the EAA/NEAA ratio increased slightly from 0.65 in winter to 0.72 in summer, indicating proportionally greater retention of EAAs relative to NEAAs during this period.
The annual mean distribution of amino acids in U. intestinalis is illustrated in Supplementary Figure S1. Analysis of the total amino acid pool revealed that NEAAs constituted the predominant fraction, accounting for 60.23% of total composition, while EAAs comprised 39.77%. Within the EAA profile, Leu was the most abundant component (19.2% of total EAAs), followed by Phe (18.0%), lysine (16.6%), and Thr (14.7%). Met (3.4%) and His (3.9%) represented the lowest proportions and were identified as the limiting amino acids. Among NEAAs, Ala exhibited the highest relative abundance at 23.3%, followed by Glu (17.6%) and Asp (17.3%), which together formed a substantial portion of the non-essential pool. Gly accounted for the lowest share among NEAAs (3.4%).
Seasonal changes in the relative proportions of EAAs and NEAAs are illustrated in Figure 3. The amino acid profile was dominated by NEAAs across all seasons, although the EAA proportion increased slightly during the warmer months, rising from 37.98% in winter to a peak of 41.50% in spring. Within the EAA fraction, Leu consistently remained the predominant component throughout the year, accounting for 18.8% of total EAAs in both winter and summer and peaking at 20.1% in spring. Phe and lysine also remained major contributors across all seasons. While Leu and Phe proportions were relatively stable or increased slightly in warmer seasons, Thr exhibited a declining trend, decreasing from 16.8% in winter to 13.2% in summer.
Seasonal distribution of the most abundant essential amino acids and non-essential amino acids in U. intestinalis (%). (a) EAAs in winter, (b) EAAs in spring, (c) EAAs in summer, (d) NEAAs in winter, (e) NEAAs in spring, and (f) NEAAs in summer.
Figure 3 Long description
Six pie charts display the seasonal distribution of essential amino acids (EAAs) and non-essential amino acids (NEAAs) in U. Intestinalis. Charts a, b and c represent EAAs for winter, spring and summer, with proportions of 37.98 percent, 41.50 percent and 40.68 percent respectively. Charts d, e and f depict NEAAs for winter, spring and summer, with proportions of 62.02 percent, 58.50 percent and 59.32 percent respectively. Each chart includes segments for amino acids such as histidine, leucine, phenylalanine, proline, alanine, glutamic acid and aspartic acid. Leucine is the largest segment in EAAs, peaking at 20.1 percent in spring. NEAAs are dominated by glutamic acid and aspartic acid. Legends indicate segment labels. Spring has the highest EAA share, while winter has the lowest. The charts highlight seasonal variations in amino acid composition.
The NEAA fraction displayed more pronounced seasonal shifts. In winter, the profile was relatively balanced, with Glu (19.4%), proline (19.1%), and Asp (18.6%) sharing dominance. A notable redistribution occurred in spring and summer, with Ala emerging as the clearly dominant NEAA, rising to 24.3% and 26.9%, respectively. Correspondingly, the relative proportions of proline and Glu declined during these warmer periods relative to winter values.
Mineral contents
The seasonal variations in the macro-, micro-, and trace element profiles of U. intestinalis are presented in Table 3 and Figure 4. The results of Kruskal–Wallis H test indicated that season significantly influenced the concentrations of calcium (Ca), potassium (K), and cadmium (Cd) (p < 0.05), while the levels of all other investigated elements remained statistically stable throughout the year.
Seasonal variation in mineral contents of U. intestinalis (mean ± SD, mg/kg dw). Significant differences (p < 0.05, Kruskal–Wallis with Dunn–Bonferroni) are indicated by asterisks.
Figure 4 Long description
Bar graph showing mineral contents in U. Intestinalis for Winter, Spring and Summer. Vertical bars are grouped by mineral: Ca, K, Mg, Na, Al, Cu, Si, Fe, Mn, Zn, Ni, Cd, Pb, As, Hg, Se. Y-axis: mg per kg DW, ranging from approximately 0.06 to 75,000. Winter is represented by one color, Spring by another and Summer by a third. Calcium is highest in Spring (~70,000 mg/kg), lowest in Winter (~36,000 mg/kg). Potassium is highest in Winter (~38,000 mg/kg), lowest in Spring (~19,000 mg/kg). Notable seasonal differences include higher calcium in Spring and higher potassium in Winter. Error bars indicate variability. The graph highlights significant seasonal variations in mineral content.
Seasonal variation in the mineral contents of U. intestinalis (mean ± SD mg/kg dw)
Table 3 Long description
Mineral concentrations in U. intestinalis (milligrams per kilogram dry weight) are reported for winter, spring, and summer, with means, standard deviations, and a seasonal significance value. Among macrominerals, calcium rises from about 35,963 in winter to 69,795 in spring, then 51,258 in summer, and this seasonal change is statistically significant. Potassium shows the opposite pattern, dropping from about 38,547 in winter to 19,803 in spring, then 29,593 in summer, also statistically significant. Magnesium and sodium vary less across seasons and are not statistically significant at the reported threshold. For microminerals, aluminum, copper, silicon, iron, manganese, and zinc show no statistically significant seasonal differences, though several peak in winter and are lower in spring. For trace elements, cadmium is the only one with a statistically significant seasonal difference, lowest in spring (about 0.115) compared with winter (about 0.167) and summer (about 0.150). Nickel, lead, arsenic, mercury, and selenium do not show statistically significant seasonal differences based on the provided significance values. Letter groupings next to seasonal means indicate which seasons differ from each other, and results should be interpreted with the reported variability.
Values indicated by different superscript letters (a–c) in the same row represent statistically significant differences (p < 0.05).
Significant seasonal differences are determined by Kruskal–Wallis H test, followed by Dunn–Bonferroni correction.
Among macroelements, calcium was the most abundant mineral in the biomass, increasing significantly from a minimum of 35,963 ± 2,426 mg/kg dw in winter to a peak of 69,795 ± 4,388 mg/kg dw in spring (p = 0.027). Potassium exhibited an inverse trend, with the highest concentrations recorded in winter (38,547 ± 2,098 mg/kg dw) and a significant decline observed in spring (p = 0.044). Magnesium also constituted a major component of the mineral contents with mean concentrations exceeding 42,000 mg/kg dw; however, seasonal fluctuations in magnesium were not statistically significant (p = 0.077).
Among microelements, aluminium (Al) and iron (Fe) were the predominant constituents, with annual mean concentrations of 2,678 ± 920 and 2,464 ± 625.6 mg/kg dw, respectively. Zinc (Zn) concentrations appeared higher in winter (626.9 ± 122.8 mg/kg dw) than in other seasons, although this difference did not reach statistical significance (p = 0.061). No significant seasonal differences were detected for Al, Cu, Si, Fe, Mn, or Zn (p > 0.05).
Among trace elements, arsenic (As) was detected at the highest concentrations, ranging from 4.06 to 6.44 mg/kg dw, with no significant seasonal dependence (p = 0.099). Cadmium (Cd) was the only trace element to exhibit significant seasonal variation (p = 0.027), with concentrations markedly higher in winter (0.167 ± 0.006 mg/kg dw) than in spring. Mercury (Hg) was consistently the least abundant element, with concentrations remaining below 0.015 mg/kg dw across all seasons.
Correlation analysis of biochemical composition and mineral contents
The relationships among macrominerals, trace elements, fatty acid groups, and amino acid categories in U. intestinalis were evaluated using Spearman’s rank correlation coefficients (Figure 5). A strong positive correlation was detected between micro- and trace elements (r = 0.833, p < 0.01), and a similarly strong positive correlation was observed between trace elements and MUFAs (r = 0.800, p < 0.01).
Interrelationships between fatty acids, amino acids, and mineral content in Ulva intestinalis.
Figure 5 Long description
The heatmap displays pairwise correlations between various nutritional components in Ulva intestinalis. Both axes list the same variables: Macro, Micro, Trace, SFA, MUFA, PUFA, PUFA over SFA, omega-6 plus omega-3, Total FA, EAA and NEAA. Each cell represents the correlation coefficient between the row and column variables, with values ranging from negative one to positive one. Red indicates positive correlation, while blue indicates negative correlation. The legend on the right maps the color gradient from negative one to positive one. Notable patterns include strong positive correlations between Micro and Trace elements and between Trace elements and MUFA, with coefficients of 0.833 and 0.800 respectively. Significance markers indicate p-values less than 0.05 and 0.01. The heatmap highlights key relationships among macro-minerals, trace elements, fatty acid groups and amino acid categories.
SFAs were negatively correlated with PUFA (r = −0.683, p < 0.05) and with the PUFA/SFA ratio (r = −0.717, p < 0.05). In contrast, PUFAs exhibited a strong positive correlation with both the PUFA/SFA ratio (r = 0.983, p < 0.01) and total fatty acid content (r = 0.850, p < 0.01). MUFA content showed a strong negative correlation with the ω-6/ω-3 ratio (r = −0.851, p < 0.01), and total fatty acid concentration was positively correlated with the PUFA/SFA ratio (r = 0.867, p < 0.01).
With respect to amino acid categories, a near-perfect negative correlation was identified between EAAs and NEAAs (r = −1.000, p < 0.01). SFAs displayed a moderate positive correlation with EAAs (r = 0.617) and a corresponding negative correlation with NEAAs (r = −0.617).
Collectively, these findings indicate that the mineral, fatty acid, and amino acid profiles of U. intestinalis are interconnected through statistically significant and multidimensional relationships.
Principal component analysis
PCA was performed to evaluate seasonal variation and the interrelationships among the biochemical parameters (minerals, fatty acids, and amino acids) of U. intestinalis. The first three principal components (PCs) with eigenvalues greater than 1 were retained (Supplementary Table S1 and Supplementary Fig. S2), collectively accounting for 95.08% of the total variance. For visualization purposes, the biplot was constructed using PC1 and PC2, which together explained 85.49% of the total variance (Figure 6).
PCA biplot of biochemical parameters and seasonal samples of U. intestinalis.
Figure 6 Long description
PC2 (37.4 percent) versus PC1 (48.1 percent) biplot of principal component scores (unitless). The horizontal axis is labeled PC1 (48.1 percent), with tick labels at minus 6, minus 4, minus 2, 0, 2 and 4. The vertical axis is labeled PC2 (37.4 percent), with tick labels at minus 10, minus 5, 0, 5 and 10. A legend lists Spring, Summer, Autumn, Winter and Loadings. Multiple loading vectors start at the origin and extend outward. Labels at vector tips include SFA, MUFA, PUFA, Total FA, NEAA, n-3, Micro, Trace and EA. The vectors for NEAA, Micro, Trace, MUFA, PUFA and Total FA extend into positive PC1 values. The vectors for SFA, EA and n-3 extend into negative PC1 values. PUFA and Total FA extend into negative PC2 values while remaining at positive PC1 values. SFA extends into positive PC2 values while at negative PC1 values. Seasonal sample points are plotted around the origin. Several points occur at negative PC1 values between about minus 5 and minus 2, with PC2 values between about minus 6 and plus 6. Several points occur at positive PC1 values between about 1 and 4, with PC2 values between about minus 6 and plus 6. A circular outline centered on the origin is drawn over the plot area.
PC1 accounted for 48.10% of the total variance and was characterized by high negative loadings for EAAs (−0.4056), SFAs (−0.3814), and the ω-6/ω-3 ratio (−0.2800), alongside high positive loadings for NEAAs (0.4056), the PUFA/SFA ratio (0.3317), and PUFA (0.3221). PC2 explained 37.39% of the total variance and was primarily influenced by positive loadings for MUFAs (0.4543) and trace elements (0.4263), with negative loadings for total fatty acids (−0.3429), PUFA (−0.3269), and the PUFA/SFA ratio (−0.3148). PC3 accounted for the remaining 9.59% of the variance and was dominated by macrominerals (0.6916) and microelements (0.5042) (Table S2).
The PCA biplot revealed a clear seasonal separation of U. intestinalis samples. Summer samples clustered in the negative region of PC1, associated with higher SFA and EAA proportions. Spring samples were positioned in the positive region of PC1 and the negative region of PC2, showing strong association with PUFA, total fatty acid content, and the PUFA/SFA ratio. Winter samples were distributed in the positive region of both PC1 and PC2, reflecting elevated MUFA concentrations and higher trace and microelement levels. The loading vectors confirmed a near-perfect inverse relationship between EAAs and NEAAs, projected in opposite directions along the same axis, and visually corroborated the strong negative relationship between SFAs and PUFAs.
Discussion
Fatty acids
The fatty acid profile of U. intestinalis demonstrated marked seasonal fluctuations throughout the study period. Total fatty acid concentrations peaked during the vernal season, reaching 2,235 mg/100 g dw, before precipitously declining to a basal level of 761 mg/100 g dw in the summer. This overarching trend underscores the acute sensitivity of macroalgal lipid metabolism to environmental determinants, most notably thermal conditions and solar irradiance. Congruent with these findings, antecedent research has established that lipid accumulation in chlorophytes undergoes substantial spatiotemporal variation across the growth season, driven by abiotic perturbations including temperature fluxes and light availability (Toth et al., Reference Toth, Harrysson, Wahlström, Olsson, Oerbekke, Steinhagen, Kinnby, White, Albers and Pavia2020; Pereira et al., Reference Pereira, Horta, Barroso, Mendes and Gil2021). The elevated fatty acid content observed in spring may be attributed to active photosynthetic activity under optimal growth conditions, as increased solar irradiance and mild temperatures promote algal membrane lipid synthesis and energy storage (Cardoso et al., Reference Cardoso, Meißner, Sawicki, Bartsch, Valentin, Steinhagen, Buck and Hofmann2023). Conversely, the attenuation of lipid concentrations during the summer aligns with documented paradigms indicating a widespread reduction in lipid ratios among macrophytes subjected to thermal stress, a phenomenon intrinsically linked to homeoviscous adaptation and the ontogenetic trajectories of these organisms (Kostetsky et al., Reference Kostetsky, Goncharova, Sanina and Shnyrov2004).
SFAs constituted a substantial proportion of the fatty acid pool throughout all seasons, reaching a maximum of 51.46% in summer. Palmitic acid (C16:0) was the dominant SFA in every season, increasing from 24.30% in winter to 35.24% in summer. Similarly, Jeliani et al. (Reference Jeliani, Fazelian and Yousefzadi2021) reported that palmitic acid was the predominant unsaturated fatty acid in U. intestinalis samples collected from the Persian Gulf; this finding confirms that this fatty acid serves as a consistent biochemical marker for this species across geographically distinct populations exposed to different environmental conditions. From an ecophysiological perspective, the pronounced accretion of SFAs under elevated thermal conditions is widely construed as a homeoviscous adaptive mechanism imperative for the modulation of membrane fluidity (Hazel, Reference Hazel1995; Ernst et al., Reference Ernst, Ejsing and Antonny2016). Analogous temperature-dependent SFA augmentations have been chronicled across diverse Ulva taxa and broader macroalgal assemblages, thereby substantiating the dictating role of ambient temperature on lipid metabolic routing (Rohani-Ghadikolaei et al., Reference Rohani-Ghadikolaei, Abdulalian and Ng2012; Kostetsky et al., Reference Kostetsky, Chopenko, Barkina, Velansky and Sanina2018; Jeliani et al., Reference Jeliani, Fazelian and Yousefzadi2021). By increasing membrane saturation at high temperatures, algae counteract excessive fluidity and thereby maintain membrane integrity. Stearic acid (C18:0) was the second most abundant SFA and reached its highest levels during winter, which may reflect shifts in lipid biosynthesis pathways aimed at preserving structural stability under cold conditions (Guschina and Harwood, Reference Guschina and Harwood2006; Pereira et al., Reference Pereira, Horta, Barroso, Mendes and Gil2021).
MUFAs remained at relatively high proportions throughout the year, peaking at 40.82% in winter. Oleic acid (C18:1ω9) and elaidic acid (C18:1ω9 trans) were identified as the dominant MUFAs. The elevated oleic acid concentration in winter is consistent with its established role in optimizing membrane fluidity during cold adaptation, as MUFAs are known to maintain membrane flexibility at low temperatures (Toth et al., Reference Toth, Harrysson, Wahlström, Olsson, Oerbekke, Steinhagen, Kinnby, White, Albers and Pavia2020).
PUFAs displayed the most pronounced seasonal variation among all fatty acid classes, reflecting the metabolic responsiveness of U. intestinalis to environmental change. The aggregate PUFA fraction culminated at 24.04% in the spring, synchronized with the phase of maximal vegetative growth, prior to a precipitous drop to 9.07% in the summer. Such biphasic dynamics suggest a tightly coupled regulatory network between PUFA biosynthesis and photosynthetic output, which is fundamentally governed by the interplay of irradiance and temperature (Cardoso et al., Reference Cardoso, Meißner, Sawicki, Bartsch, Valentin, Steinhagen, Buck and Hofmann2023). ALA was the most abundant PUFA in both winter and spring (10.62% and 10.70%, respectively), but decreased markedly to 4.36% in summer. LA and ARA exhibited similar declining trends. EPA levels were notably higher in spring than in summer (1.51% vs. 0.43%), a finding that reflects the limited capacity for EPA biosynthesis in green algae even under favourable growth conditions (Guschina and Harwood, Reference Guschina and Harwood2006; Kumari et al., Reference Kumari, Kumar, Reddy, Jha and Domínguez2013; Schmid et al., Reference Schmid, Guihéneuf and Stengel2014).
The nutritional quality of the lipid fraction was further assessed using the ω-6/ω-3 and PUFA/SFA ratios. The ω-6/ω-3 ratio remained within a favourable range throughout the year (0.67–0.75), consistently meeting the 1:1 to 5:1 range recommended for human health (Simopoulos, Reference Simopoulos2002; Gutierres et al., Reference Gutierres, Pacheco and Reis2025). The lowest ratio, recorded in winter (0.67), indicates a particularly ω-3-enriched profile. The PUFA/SFA ratio was markedly higher in spring (0.61) and winter (0.52) relative to summer (0.17), reflecting the progressive lipid saturation that occurs during the warm season. Given that high PUFA/SFA ratios are generally associated with more favourable lipid nutritional profiles (Kris-Etherton et al, Reference Kris-Etherton, Harris and Appel2002), biomass harvested in winter and spring may offer greater nutritional value for functional food applications. Consequently, the implementation of chronobiologically targeted harvesting paradigms can optimize nutritional yield while concomitantly advancing sustainable ecological management, particularly when integrated with rigorous spatial selection and purification protocols designed to sequester deleterious heavy metals.
Amino acid profiling
The amino acid composition of U. intestinalis showed pronounced seasonal variability, with both EAAs and NEAAs reaching their highest concentrations in winter and declining progressively toward summer. Specifically, the aggregate EAA fraction contracted from 4.70 g/100 g dw in the winter to 2.44 g/100 g dw during the summer. This temporal depletion is fundamentally consistent with the accelerated exhaustion of dissolved inorganic nitrogen reserves in the Black Sea epipelagic zone during the summer months, a deficit further exacerbated by the dilution of intracellular nitrogen pools consequent to rampant biomass expansion (Pedersen and Borum, Reference Pedersen and Borum1996; Naldi and Viaroli, Reference Naldi and Viaroli2002).
These empirical observations substantiate the premise that macroalgal protein metabolism and amino acid sequestration are profoundly contingent upon environmental matrices, aligning with established literature indicating that nitrogenous allocation in seaweeds is inextricably linked to thermal gradients, nutrient stoichiometry, and inherent growth kinetics (Fleurence, Reference Fleurence1999; Lourenço et al., Reference Lourenço, Barbarino, De-paula, Pereira and Marquez2002). The elevated amino acid concentrations observed in winter can be attributed to the slowing of protein catabolism under reduced metabolic activity at low temperatures, combined with the accumulation of dissolved nutrients in the water column (Marinho-Soriano et al., Reference Marinho-Soriano, Fonseca, Carneiro and Moreira2006; Unis et al., Reference Unis, Chemodanov, Gnayem, Gnaim, Israel, Palatnik, Gnaim and Golberg2023). Furthermore, the compounded ecophysiological stressors of low temperature and curtailed photoperiods may induce an elevated metabolic mandate for the biosynthesis and preservation of structural proteins, thereby upregulating nitrogen assimilation during the colder months. Analogous oscillatory paradigms in proteinaceous content have been extensively documented across various congeners within the genus Ulva (Benjama and Masniyom, Reference Benjama and Masniyom2011; Queirós et al., Reference Queirós, Circuncisão, Pereira, Válega, Abreu, Silva and Cardoso2021; Jansen et al., Reference Jansen, Bernard, Nederlof, van der Meer and van der Werf2022).
Among EAAs, Leu was the dominant amino acid across all seasons, ranging from 0.84 g/100 g in winter to 0.46 g/100 g in summer, followed by Thr and Phe. The consistent dominance of Leu as a branched-chain amino acid throughout the seasons not only serves as a direct energy substrate via oxidative metabolism but also functions as a signalling molecule activating protein synthesis pathways (Shimomura et al., Reference Shimomura, Murakami, Nakai, Nagasaki and Harris2004; Wu, Reference Wu2009; Tian et al., Reference Tian, Diao, Li and Zhang2025). Despite the overall decreases in total amino acid content during the summer months, its proportional preservation further supports its biochemical indispensability in U. intestinalis.
The subsequent suppression of Glu to 0.51 g/100 g during the summer is indicative of progressive protein turnover and systemic amino acid mobilization driven by seasonal exigencies. Physiologically, both Glu and Asp execute dual roles within algae; they are integral to the scaffolding of structural proteins while concurrently functioning as primordial nitrogen reservoirs (Young and Beardall, Reference Young and Beardall2003; Taylor et al., Reference Taylor, Barr, Grant and Rees2006). The predominance of Ala among NEAAs (23.3% of total NEAAs), followed by Glu (17.6%) and Asp (17.3%), is consistent with the typical amino acid distribution reported for macroalgae (Ortiz et al., Reference Ortiz, Romero, Robert, Araya, Lopez-hernández, Bozzo, Navarrete, Osorio and Rios2006; Benjama and Masniyom, Reference Benjama and Masniyom2011).
Despite the overall reduction in total amino acid content during warmer months, the ratio of essential to NEAAs (ΣEAA/ΣNEAA) increased slightly from 0.65 in winter to 0.72 in summer, indicating that EAAs were proportionally better preserved relative to NEAAs during this period. Consequently, while absolute amino acid quantities decline in summer, the relative amino acid profile becomes more balanced.
Assessing the annualized mean distribution, NEAAs comprised 60.23% of the overarching structural pool, with EAAs representing 39.77%. In accordance with the protein quality criteria promulgated by the FAO/WHO (Food and Agriculture Organization / World Health Organization), a nutritionally robust protein source necessitates an EAA fraction constituting a minimum of 40% of the total amino acid mass (FAO/WHO/UNU, 2007), equivalent to an intrinsic EAA/NEAA ratio of roughly 0.67. The empirical metrics acquired in this investigation (oscillating between 0.65 in winter and 0.72 in summer) align robustly with these established international benchmarks. Within the EAA profile, Leu accounted for the highest proportion (19.2%), followed by Phe (18.0%) and lysine (16.6%), further supporting the nutritional potential of Ulva as a protein source. These results suggest that algal proteins from U. intestinalis, when combined with complementary protein sources, hold potential for use as functional food and feed additives (Dawczynski et al., Reference Dawczynski, Schubert and Jahreis2007; Holdt and Kraan, Reference Holdt and Kraan2011). Nonetheless, the naturally occurring significant seasonal fluctuation in total amino acid biosynthesis presents important challenges for the widespread adoption of protein extraction on an industrial scale and the necessity of biochemical standardization. Consequently, winter and spring periods emerge as the most suitable chronobiological periods for harvesting the specified biomass for high-throughput proteomic extraction, while summer harvests, despite their quantitative deficiencies, maintain a highly stable and nutritionally favourable amino acid stoichiometry (Van der Spiegel et al., Reference Van der Spiegel, Noordam and der Fels-klerx HJ2013; Bikker et al., Reference Bikker, Krimpen, Wikselaar, Houweling-tan, Scaccia, Hal, Huijgen, Cone and López-contreras2016).
Mineral contents
A comprehensive mineral analysis of U. intestinalis was conducted to determine macro-, micro-, and trace element concentrations across seasons. Statistical analysis revealed that season significantly affected calcium (Ca), potassium (K), and cadmium (Cd) concentrations, while the levels of all other elements remained statistically stable throughout the year. Among macroelements, calcium was the most abundant mineral in the biomass, increasing from a minimum of 35,963 mg/kg in winter to a peak of 69,795 mg/kg in spring. This amplified sequestration of calcium in the vernal phase is physiologically symptomatic of an escalating biochemical requisite for calcium during the accelerated biosynthesis of the algal cell wall and the concomitant provision of structural integrity inherent to periods of exponential vegetative growth (Raven, Reference Raven1970; Lobban and Harrison, Reference Lobban and Harrison1994).
Potassium exhibited an inverse seasonal trend, with the highest concentrations recorded in winter (38,547 mg/kg) and a significant decrease observed in spring. As an essential macroelement involved in osmotic regulation, enzyme activation, and ionic balance (Lobban and Harrison, Reference Lobban and Harrison1994; Jacobsen et al., Reference Jacobsen, Boeckmann, Höper, Berge, Lock and Graeve2023), elevated potassium levels in winter are likely associated with the maintenance of cellular osmotic pressure and protection against cold stress at low temperatures (Reed et al., Reference Reed, Collins and Russell1985). Magnesium, which serves as the central coordinating atom of the chlorophyll molecule, also constituted a major component of the mineral contents, with annual mean values exceeding 42,000 mg/kg; however, seasonal fluctuations in magnesium were not statistically significant, consistent with Ulva’s high and sustained photosynthetic capacity (Geider and La Roche, Reference Geider and La Roche1994). For microelements, no significant seasonal differences were detected for Al, Cu, Si, Fe, Mn, or Zn, suggesting that these essential elements are maintained under tight homeostatic control regardless of seasonal environmental variation (Sunda, Reference Sunda2012).
Among trace elements, arsenic (As) was detected at the highest concentrations (4.06–6.44 mg/kg) but showed no significant seasonal dependence. Cadmium was the only trace element exhibiting significant seasonal variation, with concentrations markedly higher in winter compared to spring (0.167 mg/kg). This pattern may reflect seasonal differences in cadmium bioavailability in the water column (Ho, Reference Ho1981; Amado Filho et al., Reference Amado Filho, Karez, Andrade, Yoneshigue-valentin and Pfeiffer1997). The significantly higher concentration of cadmium (Cd) observed during the winter months can be attributed to the well-documented ‘growth dilution’ effect. Rapid biomass accumulation during the active spring growth phase effectively dilutes the tissue concentrations of trace metals. In contrast, the reduced growth rates in winter, potentially coupled with increased metal bioavailability from winter run-offs, lead to higher internal concentrations of heavy metals. Mercury (Hg) was consistently the least abundant element, with concentrations remaining below 0.015 mg/kg across all seasons, a favourable characteristic from a food safety perspective, given the well-documented toxicity of mercury to human health (Jacobsen et al., Reference Jacobsen, Boeckmann, Höper, Berge, Lock and Graeve2023).
Taken together, the mineral composition of U. intestinalis suggests that this species can make a valuable contribution to human nutrition, given its high concentrations of calcium, magnesium, and potassium. However, the presence of toxic elements such as arsenic and cadmium requires careful monitoring in the context of food applications. It is also known in the literature that green algae species such as Ulva show high affinity for metals such as Fe, Zn, Mn, Cd, and Pb, and are good biomarkers reflecting their environment. These changes in metal accumulation in tissues are explained by seasonal fluctuations in algal metabolism (Hernández-Almaraz et al., Reference Hernández-Almaraz, Mendez-rodriguez, Zenteno-savín, García-domínguez, Vázquez-botello and Serviere-zaragoza2014; Méndez-Rodríguez et al., Reference Méndez-Rodríguez, Piñón-gimate, Casas-valdez, Cervantes-duarte and Arreola-lizárraga2021). The accumulation of these metals in macroalgae, which are primary producers in coastal ecosystems, leads to their direct transfer in the food chain. Therefore, when harvesting U. intestinalis as a functional food raw material, knowing that its mineral content varies seasonally is critical for planning safe harvesting periods. Although the concentrations detected in this study are below current regulatory thresholds, the sustainable commercial and functional use of U. intestinalis requires careful selection of harvesting timing, preference for low-pollution collection areas, and the application of appropriate processing methods to minimize heavy metal content.
Correlation and PCA of biochemical components
Correlation analysis revealed statistically significant and multidimensional relationships among macrominerals, trace elements, fatty acid groups, and amino acid categories in U. intestinalis. The negative correlation between SFA and PUFA (r = −0.683, p < 0.05) indicates that these two fatty acid classes are subject to inverse metabolic regulation, supporting the hypothesis that algae modulate lipid desaturation activity in response to temperature and other environmental stressors (Guschina and Harwood, Reference Guschina and Harwood2006; Ernst et al., Reference Ernst, Ejsing and Antonny2016). The strong positive correlation between PUFA and the PUFA/SFA ratio (r = 0.983, p < 0.01) confirms that this ratio is a primary determinant of lipid quality, while the positive correlation between total fatty acid concentration and the PUFA/SFA ratio (r = 0.867, p < 0.01) indicates that PUFAs predominate in membrane structure during periods of high lipid accumulation. The strong negative correlation between MUFA content and the ω-6/ω-3 ratio (r = −0.851, p < 0.01) suggests that MUFA synthesis is closely associated with omega fatty acid metabolism, likely through competition for shared biosynthetic substrates (Cardoso et al., Reference Cardoso, Meißner, Sawicki, Bartsch, Valentin, Steinhagen, Buck and Hofmann2023).
The near-perfect negative correlation between EAAs and NEAAs (r = −1.000, p < 0.01) is particularly noteworthy. This result indicates that within a relatively stable total amino acid pool, an increase in one category is accompanied by a proportional decrease in the other, likely reflecting the capacity of U. intestinalis to optimize its protein composition under fluctuating environmental conditions (Lourenço et al., Reference Lourenço, Barbarino, De-paula, Pereira and Marquez2002; Marinho-Soriano et al., Reference Marinho-Soriano, Fonseca, Carneiro and Moreira2006). The strong positive correlation between micro- and trace elements (r = 0.833, p < 0.01) suggests shared environmental sources and coordinated physiological regulation, while the positive correlation of trace elements with MUFAs (r = 0.800, p < 0.01) may reflect the influence of metal ions on lipid peroxidation and membrane dynamics, consistent with previous reports on the effects of transition metals such as iron and copper on lipid metabolism (Gledhill and Buck, Reference Gledhill and Buck2012; Sunda, Reference Sunda2012).
PCA of the biochemical parameters of U. intestinalis provided an effective framework for visualizing seasonal patterns and the complex interrelationships among compositional variables. PC1 was characterized by high negative loadings for EAAs, SFA, and the ω-6/ω-3 ratio, and high positive loadings for NEAAs, the PUFA/SFA ratio, and PUFA, collectively representing a ‘lipid quality and amino acid balance’ axis. The separation of samples along PC1 illustrates how nutritional quality shifts seasonally: summer samples clustered in the negative region of PC1, reflecting the predominance of SFA and relatively higher EAA proportions, while spring samples positioned in the positive region were characterized by elevated PUFA and NEAA concentrations.
PC2 displayed positive loadings for MUFAs and trace elements and negative loadings for total fatty acids, the PUFA/SFA ratio, and PUFA, representing a ‘membrane adaptation and mineral accumulation’ axis. The clustering of winter samples in the positive region of PC2 reflects the elevated MUFA and trace element concentrations characteristic of this season; the high MUFA content underscores the role of MUFAs in maintaining membrane fluidity at low temperatures (Ratana-arporn and Chirapart, Reference Ratana-arporn and Chirapart2006), while trace element accumulation in winter may be associated with reduced growth rates or elevated environmental concentrations (Jacobsen et al., Reference Jacobsen, Boeckmann, Höper, Berge, Lock and Graeve2023). The positioning of spring samples in the negative region of PC2 corroborates the high total fatty acid and PUFA concentrations observed during this period.
PC3 accounted for 9.59% of the total variance and was dominated by macrominerals (loading: 0.692) and microelements (loading: 0.504), representing a ‘mineral nutrient uptake and cellular structure’ axis. The relatively modest explanatory power of this component indicates that mineral content is less seasonally variable than lipid and amino acid composition. Nevertheless, the co-loading of macro- and microelements on PC3 suggests coordinated uptake and shared regulatory mechanisms.
The biplot loading vectors visually confirmed the results of the correlation analysis: EAA and NEAA vectors projected in opposite directions along the same axis, consistent with their near-perfect inverse relationship (r = −1.000), while SFA and PUFA vectors also oriented in opposing directions, corroborating their strong negative correlation. These patterns collectively reinforce the biochemical antagonisms identified in the correlation analysis.
The PCA results carry meaningful practical implications for seasonal harvest optimization. Spring collection is favoured for functional food applications owing to high PUFA content and a favourable PUFA/SFA ratio, whereas winter harvest may be advantageous for applications requiring high MUFA concentrations and specific mineral contents. Summer biomass, while limited in total lipid content, offers a relatively balanced amino acid profile with an elevated EAA/NEAA ratio. Collectively, the PCA and correlation analyses demonstrate that the biochemical composition of U. intestinalis responds to seasonal environmental change in a highly coordinated and statistically coherent manner, with distinct seasonal windows offering distinct compositional advantages for targeted industrial and nutritional applications.
Conclusion
This comprehensive seasonal investigation of U. intestinalis from the Sinop coast of the Black Sea reveals significant temporal variation in its biochemical composition, offering valuable insights into its potential utilization as a functional food ingredient or feed supplement. The results demonstrate that seasonal environmental conditions substantially influence the nutritional profile of this green macroalga, with distinct patterns evident across fatty acid, amino acid, and mineral compositions. Spring collection of wild seaweed emerges as optimal for applications requiring high PUFA content, as spring samples exhibited the greatest total fatty acid concentration and the most favourable PUFA/SFA ratio. The consistently low ω-6/ω-3 ratio observed throughout the year further positions U. intestinalis as a nutritionally valuable ω-3 fatty acid source, in accordance with WHO dietary recommendations. Winter samples yielded the highest amino acid content, suggesting that this season is preferable for protein-oriented applications. Mineral analysis revealed pronounced calcium accumulation in spring, while the low concentrations of toxic elements such as cadmium and mercury indicate that this species is safe for human consumption. PCA effectively captured the multivariate relationships among biochemical parameters, confirming strong negative correlations between saturated and PUFA, as well as inverse relationships between EAA and NEAA. These patterns are consistent with the metabolic strategies employed by U. intestinalis in response to environmental stressors, particularly temperature fluctuations and variations in nutrient availability. Collectively, these findings contribute to the growing body of knowledge on Black Sea macroalgae and underscore the importance of region-specific investigations. The identification of optimal harvest windows based on targeted biochemical profiles enables strategic sourcing approaches that maximize nutritional value while supporting sustainable biomass management. Future research should examine the specific environmental drivers underlying these seasonal variations and assess bioactive compounds and functional properties to provide a more comprehensive understanding of the valorization potential of U. intestinalis. Taken together, the findings of the present study indicate that U. intestinalis from the Black Sea holds considerable promise as a sustainable source of nutritionally valuable compounds, with predictable seasonal dynamics that can be strategically leveraged for diverse industrial and nutritional applications.
Supplementary material
The supplementary material for this article can be found at https://doi.org/10.1017/S0025315426101453.
Acknowledgements
The authors thank Sinop University, Scientific and Technological Research Application and Research Center (SUBİTAM) for the analysis by gas chromatography–mass spectrometry (GC-MS).
Author contributions
Sampling, species identification, and analysis were conducted by G.Y. and A.K. Statistical analysis was performed by M.E.K. and G.Y. All authors made equal contributions to the writing, review, and approval of the final manuscript.
Declaration of interest
The authors declare that they have no conflict of interest.
Data availability statement
No datasets were generated or analysed during the current study.
Ethics and approach
All authors have read, understood, and have complied as applicable with the statement on ‘Ethical responsibilities of Authors’ as found in the Instructions for Authors.
Funding statement
Open access funding provided by the Scientific and Technological Research Council of Türkiye (TÜBİTAK) ULAKBİM and Sinop University.
Open access
