Characterization of Proteins Extracted from Ulva sp., Padina sp., and Laurencia sp. Macroalgae Using Green Technology: Effect of In Vitro Digestion on Antioxidant and ACE-I Inhibitory Activity

Macroalgal proteins were extracted from Ulva rigida (URPE) (green), Padina pavonica (PPPE) (brown), and Laurencia obtusa (LOPE) (red) using ultrasound-assisted enzymatic extraction, which is one of the green extraction technologies. Techno-functional, characteristic, and digestibility properties, and biological activities including antioxidant (AOA) and angiotensin-I converting enzyme (ACE-I) inhibitory activities were also investigated. According to the results, the extraction yield (EY) (94.74%) was detected in the extraction of L. obtusa, followed by U. rigida and P. pavonica. PPPE showed the highest ACE-I inhibitory activity before in vitro digestion. In contrast to PPPE, LOPE (20.90 ± 0.00%) and URPE (20.20 ± 0.00%) showed higher ACE-I inhibitory activity after in vitro digestion. The highest total phenolic content (TPC) (77.86 ± 1.00 mg GAE/g) was determined in LOPE. On the other hand, the highest AOACUPRAC (74.69 ± 1.78 mg TE/g) and AOAABTS (251.29 ± 5.0 mg TE/g) were detected in PPPE. After in vitro digestion, LOPE had the highest TPC (22.11 ± 2.18 mg GAE/g), AOACUPRAC (8.41 ± 0.06 mg TE/g), and AOAABTS (88.32 ± 0.65 mg TE/g) (p < 0.05). In vitro protein digestibility of three macroalgal protein extracts ranged from 84.35 ± 2.01% to 94.09 ± 0.00% (p < 0.05). Three macroalgae showed high oil holding capacity (OHC), especially PPPE (410.13 ± 16.37%) (p < 0.05), but they showed minimum foaming and emulsifying properties. The quality of the extracted macroalgal proteins was assessed using FTIR, SDS-PAGE, and DSC analyses. According to our findings, the method applied for macroalgal protein extraction could have a potential the promise of ultrasonication application as an environmentally friendly technology for food industry. Moreover, URPE, PPPE, and LOPE from sustainable sources may be attractive in terms of nourishment for people because of their digestibility, antioxidant properties, and ACE-I inhibitory activities.


INTRODUCTION
The world population is expected to reach 9.1 billion by 2050, a percent increase from now. 1 Therefore, global agricultural production must be increased by 70% from the current levels to meet the food requirements of the higher population in 2050. 2 Thereby, there is a growing development of emerging food technologies that promise to generate functional and bioactive ingredients, particularly protein-rich, for promoting human health. 3As protein demand increases with expanding populations, alternative protein sources are required for more environmentally friendly production.Compared to animalbased proteins, plant proteins appear to be the alternative to animal proteins due to their nutrient-rich composition (e.g., vitamins, minerals, fibers, proteins, and antioxidants) and their lower environmental effects, increasing sustainability. 4This has prompted scientists to look into new protein sources such as algae, legumes, fungi, and insects. 5acroalgae are a valuable and sustainable protein source, from a nutritional standpoint.Macroalgae present faster growth, low water consumption (or even growth in seawater), higher photosynthesis efficiency, and carbon storage ability compared to plant-based protein sources. 4Macroalgae can be the ideal choice for meeting a sizable portion of the world's food needs while having the least negative effects on the environment because they can absorb 10 to 50 times more CO 2 than land plants. 6In addition, protein yield of macroalgae is provided 2.5−7.5 tons/ha/year per unit of land, while soybean and wheat yield are obtained as 0.6−1.2tons/ha/year and 1.1 tons/ha/year, respectively.In this context, it should be stated that the world's macroalgae production reached 23.4 million tons and an economic value of 6.4 billion in 2013.Moreover, it is known that 75% of the world's freshwater resources should be used for protein production from plant and animal sources.Animal protein production requires 100 times more fresh water than is needed to produce an equivalent amount of vegetable protein.Macroalgae, on the other hand, do not require fresh water or arable land for their growth. 7he macroalgae have also gained widespread recognition for their significance as a source of functional components like minerals, polyphenols, carotenoids, proteins, and fibers due to their numerous health benefits.Hence, the isolation and research of novel components, such as proteins with biological activity derived from macroalgae, have received attention. 8part from their potential as a protein source, macroalgal proteins can also produce bioactive peptides and other proteinaceous compounds with biological value and positive effects on health.These beneficial effects include antioxidant, antiproliferative, anti-inflammatory, antihypertensive, antidiabetic, antiatherosclerotic, anticoagulant, and antimicrobial activity. 4ypertension (high blood pressure) is an important risk factor for many cardiovascular diseases that affects approximately 20% of adult population worldwide. 9,10Blood pressure is regulated by different biochemical reactions.One of them is the renin-angiotensin system, which is one of the main components of blood pressure regulation physiology.Blood pressure is regulated by angiotensin-converting enzymes (ACE).Angiotensinogen produces angiotensin I, which ACE transforms into angiotensin II, which raises blood pressure.−22 Padina pavonica is a classified brown macroalgae that contributes significantly to the total productivity of marine environments. 23Red macroalga Laurencia obtusa produces secondary metabolites having medicinal properties, 24 and U. rigida is a green macroalga that provides nutritional advantages while also assisting in the preservation of marine biodiversity.Furthermore, these species contain bioactive compounds with potential applications in the food industry, biomedicine, and cosmetics, demonstrating their ability to enhance both the environment and human health. 25hese algae are rich sources of protein and potential candidates for use in human and animal nutrition.Studies have shown that P. pavonica contains 5.2−7.8% of dry weight (dw) protein, 26 while L. obtusa contains 2.3−15.7%dw protein. 27,24. rigida has also been reported to contain high amounts of protein, up to 24% dw protein compared to terrestrial plants. 28,29The amino acid profiles of the proteins found in these macroalgae suggest that they are highly nutritious, with balanced amounts of essential amino acids. 30However, extracting these proteins is complex due to a strong cell wall. 7,31Improved extraction methods, such as cell disruption and specific chemical agents, can increase extraction efficiency while reducing drawbacks such as time, energy consumption, and protein integrity loss. 32,33−37 This study has applied a combination of pretreatments of osmotic shock and ultrasonication and a polysaccharidase enzyme to extract proteins from selected algae.In this context, the objectives of this study were to (i) perform protein extraction with high yield and characterize the protein extract of macroalgae, (ii) evaluate its techno-functional properties and the effect of in vitro gastrointestinal digestion on bioactivity, and (iii) comparison of protein extracts from U. rigida, P. pavonica, and L. obtusa in terms of bioactive, physicochemical, and techno-functional properties.
All of the solvents and chemicals used were of analytical or high-performance liquid chromatography grade.Hemicellulase enzyme (HSP 50000) was purchased from Bakezyme.

Protein Solubility and Surface Charge of Macroalgae.
The protein solubility assay of the powdered macroalgae was evaluated as a function of pH (2−13) and carried out according to the method of Morr et al. 38 Protein content in the supernatant was determined by the Lowry method 39 The protein solubility (%) of the powdered macroalgae was calculated using eq 1 protein solubility % protein content of supernatant (mg/mL) 50 / (weight of sample (mg) (protein content of sample (%)/100) 100 (1) The net surface charge (zeta potential) was measured as a function of pH using a Nano-ZS instrument (Zetasizer NanoZS90, Malvern Instruments, U.K.).

Ultrasound and Enzyme-Assisted Extraction of Macroalgal Proteins.
The combined ultrasound and enzyme-assisted extraction method was used to extract protein from three macroalgae. 40,41Briefly, 1 g of powdered macroalgae was mixed with 100 mL of citrate buffer solution (0.1 M, pH 4.5) and the mixture was kept at 4 °C overnight to induce cell lysis by osmotic shock.Then, the suspension was sonicated at a frequency of 53 kHz and 65% amplitude using an ultrasound homogenizer (Sonopuls HD 2200, Bandelin Electronic GmbH & Co. KG, Berlin, Germany).After the ultrasonication, hemicellulase enzyme was added to the suspension and kept in a shaking water bath (N-Biotek-303, Biotek Co., Ltd.) at different temperatures at 75 rpm for 24 h.Finally, the samples were kept in the shaking water bath at 85 °C for 10 min for enzyme inactivation.Then, the pH of the mixture was adjusted to the pH value where the protein solubility of macroalgae was the highest value determined based on the protein solubility assay, and the samples were again kept in the shaking water bath at 35 °C for a certain period for the second extraction.Then, the mixture was centrifuged at 18,782g (10,000 rpm in a Hettich 1720 Rotor, Hettich Rotina 380R, Germany) for 15 min.After centrifugation, the supernatant was collected and stored in the dark at −20 °C until analysis.

Determination of Protein Content and Extraction
Yield.The modified Lowry method, which includes precipitating the proteins from the samples with trichloroacetic acid (TCA) to remove any potentially interfering compounds, was used to measure the protein content (PC). 42To determine extraction yield, the crude protein of the macroalgae was obtained by Association of Official Analytical Chemists methods. 43Bovine serum albumin was used as the standard protein.Protein content was expressed as milligrams of bovine serum albumin equivalents per gram of dry weight (mg of BSA/g of sample dw).
The extraction yield was calculated using eq 2 (2) 2.5.Total Phenolic Content.The total phenolic content (TPC) of the macroalgal protein extract was determined according to the Folin-Ciocalteu method. 44Gallic acid was used as standard, and the results are expressed as milligrams of gallic acid equivalents per gram of dry weight (mg of GAE/g dw).
2.6.In Vitro Biological Activities.2.6.1.Angiotensin-I-Converting Enzyme (ACE-I) Inhibitory Activity.The in vitro ACE-I inhibitory activities of the macroalgal protein extracts were determined by the formation of hippuric acid.For the determination of ACE-I inhibitory activity, Marti ́nez-Alvarez et al.'s 45 method was revised and used.Briefly, 5 mM HHL, sample, and ACE (100 mU) were prepared in 100 mM sodium phosphate buffer (pH 8.3) containing 300 mM NaCl.Then, 200 μL of HHL and 50 μL of sample were mixed and incubated at 37 °C for 10 min.After 10 min, 20 μL of ACE enzyme was added to the mixture and incubated for 60 min at 37 °C in the shaking water bath.The enzymatic reaction was stopped by adding 250 μL of 1 M HCL.The released hippuric acid (HA) was then quantified by HPLC.ACE-I inhibitory activity was quantified by an HPLC system (SPD M20A, Shimadzu) on an analytical C18 column (4.6 mm × 150 mm × 5 μm).The sample was separated by passing 0.8 mL/min with an injection volume of 10 μL.Water containing 0.1% (v/v) TFA (eluent A) and acetonitrile containing 0.1% (v/v) TFA (eluent B) were used as the mobile phases.A linear gradient flow of 20% B was passed through the column for 5 min and then 60% B for the next 15 min.The elution was held isocratically at 60% B for 4 min and then returned to the initial eluent composition of 20% B. Elution peaks of hippuric acid and HHL were detected at 228 nm.
ACE inhibition (%) was calculated as follows where A sample and A control express the relative areas (A) of the HA peak of the assays performed with and without ACE inhibitors, respectively.2.6.2.Antioxidant Activity (AOA).2.6.2.1.Cupric Reducing Antioxidant Capacity Method.The cupric reducing anti-oxidant capacity (CUPRAC) assay was developed by Apak et al. 46 Trolox was used as the standard, and the results are expressed as milligrams of Trolox equivalent per gram of dry weight (mg TE/g dw).
2.7.Techno-Functional Properties.2.7.1.Water and Oil Holding Capacity.The water holding capacity (WHC) and oil holding capacity (OHC) were evaluated according to the method of Kumar et al. 48The WHC/OHC of samples was expressed as the weight of water/oil absorbed per gram of the tested samples according to eq 4 where W 0 is the weight of protein extract (g), W 1 is the weight of the tube containing protein extract (g), and W 2 is the weight of the tube after decantation of water and oil (g).2.7.2. Foaming Properties.The foaming properties were estimated using the method of Jarpa-Parra et al. 49 The foaming capability (FC) and foaming stability (FS) were calculated using eqs 5 and 6 FC (%) volume after whipping (mL) volume before whipping (mL) / volume before whipping (mL) 100 (5) FS (%) foam volume after 30 min (mL) volume before whipping (mL) / volume before whipping (mL) 100 2.7.3.Emulsifying Properties.The emulsifying activity (EA) and emulsion stability (ES) were evaluated with the method described by Tan et al. 50The emulsion activity of the samples was calculated using eq 7 height of emulsified layer height of contents of tube 100 The ES of the samples was calculated using eq 8 height of remaining emulsified layer height of original emulsified layer 100 2.8.Characterization of the Protein Extracts.

Fourier Transform Infrared (FTIR) Spectroscopy.
Organic groups in the macroalgal protein extracts were determined using FT-IR spectroscopy (Bruker Tensor II FTIR spectrometer equipped with the ATR diamond module (Bruker Optics, Germany)).All of the spectra were an average of 18 scans from 4000 to 400 cm −1 at a resolution of 4 cm −1 .

Differential Scanning Calorimetry (DSC).
The thermal properties of macroalgal protein extracts were determined by DSC (DSC 60 Plus, Shimadzu Instruments, Japan).Briefly, 20 mg of macroalgal protein extracts was placed in aluminum capsules.An empty aluminum capsule was taken as a reference.Run conditions were as follows: rate of heating, 10 °C/min; temperature range, 25−125 °C.
2.9.Simulated Gastrointestinal Digestion.In vitro gastrointestinal digestion of the macroalgal protein extracts was carried out according to the INFOGEST method. 51The collected samples taken after gastric and intestinal digestion were centrifuged at 10,000 rpm and 4 °C for 15 min and then stored at −80 °C until analysis.
2.10.Statistical Analysis.Statistical analysis was carried out using IBM SPSS Statistics 22 (Chicago) software.One-way ANOVA and the Tukey post hoc test were used to compare the treatments, and p < 0.05 was taken as a significant value.Microsoft Office Excel 2021 software (Microsoft Corporation) was used to calculate the correlation coefficients (R 2 ).

Protein Solubility and Surface Charge.
The point at which macroalgae were most effectively dissolved before extraction was determined by analyzing the protein solubility.The net surface charges and protein solubility of the macroalgae were examined in relation to pH using zeta potential measurements (Figure 1).According to the results, L. obtusa and P. pavonica had the minimum surface charges at pH 2 (−15.9 ± 2.53 and −12.5 ± 0.09 mV, respectively), while the maximum surface charges were recorded above pH 9 (−21.46± 0.38 and −24.93 ± 1.18, respectively).However, the surface charges of U. rigida were the lowest at pH 8 (−5.33 ± 0.25 mV) and the highest at pH 5 (−28.1 ± 2.39).As seen in Figure 1, the ζ potential values of three macroalgae were found to be negative due to the presence of polysaccharides with negative charges in the extracts. 52,53Similarly, Shao et al. 53 reported that the ζ potential of Ulva fasciata polysaccharides varied from −0.55 to −0.56 mV at pH values ranging from 5.0 to 10.0.Moreover, Wahlstrom et al. 54 stated that the ζ potential of Ulva spp.varied between −53 mV and −59 mV at a neutral pH value and Rosenhahn et al. 55 stated the ζ potential of Ulva linza as −19.3 mV at pH 8.2.Similar to these studies, Monsalve-Bustamante et al. 56 obtained the ζ potential of Gracilariopsis tenuifrons as −31.0 at pH 7, as well.In seawater, the phosphate group is negatively charged.Other groups, some of which have positive charges, such as choline, may be present, but these positive groups will be overwhelmed   Values are expressed as mean ± standard deviation for triplicate determinations.Different letters in the rows represent statistically significant differences (p < 0.05).Different superscript letters within the same column (a, b, c) and line (x, y, z) indicate significant difference (p < 0.05, Tukey).AOA ABTS : Antioxidant activity by ABTS method, AOA CUPRAC : Antioxidant activity by CUPRAC method.by the negative charges, thus; the surface charge is affected by negative charges, which might be significantly more frequent. 55e surface charge and pH values of proteins have an important effect on their solubility.Three macroalgae had the minimum solubility at pH 2, which corresponded to their isoelectric point.On the other hand, U. rigida (36.44 ± 3.64%) and L. obtusa (19.8 ± 1.16%) showed the highest solubility at pH 13, while P. pavonica (36.7 ± 0.09%) had the highest solubility at pH 10.It has been shown that protein solubility increases at alkaline pH values and decreases under acidic conditions by some studies in the literature. 57,58For instance, Juul et al. 59 reported that pH 2 was the isoelectric point of Ulva spp.and its solubility was the lowest level at this pH value.Harrysson et al. 60 also obtained the lowest solubility for Ulva lactula at pH 2.0 as ∼12%, while the highest solubility was recorded at pH 12 as 62.1 ± 5.1%.Similarly, Bozdemir et al. 61 reported that Gracilaria dura had maximum solubility (58.53 ± 4.26%) at pH 13.Likewise, Vilg and Undeland 58 stated that the solubility of brown macroalgae Saccharina latissima showed the minimum value (30%) at pH 2−3 and the maximum value (100%) at pH 12. Algal proteins typically seem to have a lower isoelectric point than proteins from other biomasses, but it appears that only marine species do not exhibit an increase in solubility at a lower pH.This is due to an effect of the salt concentration used in the experiments; the well-known process for lowering the isoelectric point is the interaction of anions with positively charged protein groups at a low pH. 58,62.2.Extraction Yield.The protein extraction yields for URPE, PPPE, and LOPE were determined to be 74.21,63.20, and 94.74%, respectively (p < 0.05).The results of the present study are consistent with the literature.Fleurence et al. 63 reported that the protein yield of U. rigida and U. rodundata doubled when the cellulase enzyme was used.Postma et al. 64 observed a 25−30% increase in protein yield for U. lactuca using cellulase and pectinase enzymes.Maehre et al. 40 found that the protein yield of Palmaria palmata increased by approximately 1.6-fold when both cellulase and xylanase enzymes were used.Vaśquez et al. 65 investigated enzymeassisted protein extraction from Macrocystis pyrifera and Chondracanthus chamissoi and found that the PC of the extract increased as a result of the breakdown of the cellulase-sensitive carbohydrate matrix.Harrysson et al. 60 determined the extraction yield for Ulva lactula with traditional methods as 19.6 ± 0.8%.The extraction yield is largely influenced by the type of enzyme utilized and extraction conditions performed on the algae. 4For instance, Fleurence et al. 63 studied the effect of polysaccharides on protein extraction using a combination of carrageenase and cellulase for Chondrus crispus, agarose, and cellulase for Gracilaria verrucose.They reported that these combinations increased the extraction yield 10-fold compared to that of untreated samples.

3.3.
In Vitro Biological Activities.3.3.1.Effect of In Vitro Digestion on Protein.In our previous study, the crude protein content of L. obtusa (red algea), U. rigida (green algae), and P. pavonica (brown algea) was found to be 116.5 ± 0.72 mg/g dw (11.65%), 74.15 ± 0.12 mg/g dw (7.41%), and 57.28 ± 0.12 mg/g dw (5.78%), respectively (p < 0.05). 66As observed in this study, it is known that the color of algae has an impact on their protein content. 67Red seaweeds are widely recognized to contain the highest protein content among macroalgae, whereas green macroalgae can have higher protein content than brown macroalgae. 68After ultrasound and enzymeassisted protein extraction, PC of URPE, PPPE, and LOPE was determined to be 160.21± 0.29 mg BSA/g dw, 205.09 ± 0.54 mg BSA/g dw, and 227 ± 0.01 mg BSA/g dw, respectively (Table 1).According to Saravanavel & Pillai, 69 the PC of macroalgae extracted by conventional methods was determined to be 15.08 mg/g dw for P. pavonica, 24.54 mg/g dw for Ulva fasciata, and 29.28 mg/g dw for L. obtusa.Compared with our results, the PC of these macroalgae extracted by traditional methods was found to be lower.Similarly, it is well established in the literature that ultrasound-assisted enzyme extraction has been shown to enhance protein content and extraction yield. 63,4he digestibility of macroalgal proteins in human gastrointestinal conditions is crucial for their utilization as human food. 70In vitro digestion analysis was performed on URPE, PPPE, and LOPE, and the results are given in Table 1.During in vitro gastric phase, 90.02 ± 0.23% of URPE, 98.65 ± 0.18% of PPPE, and 70.50 ± 1.64% of LOPE were hydrolyzed.After in vitro intestinal phase, the percentages of protein hydrolyzed were 84.35 ± 2.01% for URPE, 94.09 ± 0.00% for PPPE, and 90.26 ± 0.70% for LOPE.Similarly, Kazir et al. 70 reported that Ulva sp. and Gracilaria sp.proteins showed 47.8 ± 4.3 and 68.1 ± 0.7% digestion rates during in vitro gastric phase, respectively.Moreover, these proteins were highly digestible during the in vitro intestinal phase, with digestion rates of 89.4 ± 2.6 and 100% for Ulva sp. and Gracilaria sp.proteins, respectively.In vivo studies by Goni et al. 71 have shown that various macroalgae contain a significant proportion of indigestible protein, ranging from 2% to 24%.Based on these results, it appears that the macroalgal protein extracts can undergo hydrolysis by digestive enzymes, potentially enhancing their absorption in the intestine. 70

Effect of In Vitro Dilution on Phenolics.
In this study, a wide variation in the total phenolic content of the macroalgal protein extracts analyzed was obtained.The TPC for URPE, PPPE, and LOPE was found to be 19.32 ± 1.02 mg GAE/g dw, 49.92 ± 2.31 mg GAE/g dw, and 77.86 ± 1.0 mg GAE/g dw, respectively (Table 2).Red macroalgae L. obtusa has been found to possess the highest phenolic content compared to brown macroalgae P. pavonica and green macroalgae U. rigida (p < 0.05).Yuan et al. 72 stated that the TPC of conventional extracts from some brown macroalgae was between 0.38 ± 0.01 and 0.78 ± 0.05 mg GAE/g, while the TPC of microwave- assisted extracts was 0.73 ± 0.02 and 1.4 ± 0.1 mg GAE/g.Wang et al. 73 extracted TPC from Palmaria palmata using carbohydrase and protease enzymes and reported that TPC obtained by using the protease enzymes was found to be 3 times higher than the extract obtained without enzyme.In addition, the TPC of macroalgae might vary depending on location, environmental conditions, and seasonal fluctuations besides the novel extraction process, enzymes, and solvent. 74o investigate the effect of digestion on TPC, in vitro gastric and intestinal digestion assays were performed on macroalgae.In the present study, TPC of U. rigida, P. pavonica, and L. obtusa was 33.57± 1.92 mg GAE/g, 35.02 ± 2.13 mg GAE/g, and 77.47 ± 1.78 mg GAE/g, respectively, after in vitro gastric digestion.On the other hand, after in vitro intestinal digestion, TPC values were reduced to 8.74 ± 1.34 mg GAE/g, 19.22 ± 0.09 mg GAE/g, and 22.11 ± 2.18 mg GAE/g, respectively (Table 2).At the end of the gastric phase, an increase in TPC can be observed in U. rigida and L. obtusa.A low pH value might encourage the release of phenols after breaking bonds within the matrix, including those of polysaccharides and proteins. 75At the end of the intestinal phase, TPC decreases due to the instability of phenols at high pH values. 76Similar to these findings, Corona et al. 77 observed a significant reduction in TPC of brown macroalgae (Ascophyllum nodosum) after in vitro digestion, with a reduced level of 81.7%.In contrast, Huang et al. 78 reported increased TPC after in vitro gastric digestion of seven macroalgae, especially for Sargassum thunbergia (174.44%).They also observed that the bound phenolic content of macroalgae remained relatively stable at 22.14−69.61%after in vitro intestinal digestion. 78These findings suggest that the in vitro digestion process has a variable impact on the TPC of macroalgae, which may be related to the species and type of polyphenols present.−81

Effect of In Vitro Digestion on the Antioxidant Activity.
As seen in Table 3, AOA CUPRAC of protein extracts obtained from URPE, PPPE, and LOPE was 22.40 ± 0.10 mg TE/g dw, 74.69 ± 1.78 mg TE/g dw, and 28.95 ± 2.31 mg TE/g dw, respectively (p < 0.05).On the other hand, AOA ABTS of URPE, PPPE, and LOPE was 143.76 ± 3.2 mg TE/g dw, 251.29 ± 5.0 mg TE/g dw, and 187.34 ± 3.1 mg TE/g dw, respectively (p < 0.05) (Table 3).In our study, antioxidant activity assayed by CUPRAC and ABTS methods showed different trends since these methods have different mechanisms.They have different action modes in which CUPRAC allows the quantification of compounds capable of reducing the complex of Cu (II)-Neocuproine to Cu (I)-Neocuproine and ABTS allows the quantification of free radical scavenging capacity. 82,83On the other hand, CUPRAC and ABTS can test lipophilic and hydrophilic antioxidants simultaneously with the same precision due to the solubility of their single-charged chromophores in both aqueous and organic solvent environments. 84he protein extract from brown macroalgae P. pavonica has demonstrated higher antioxidant activity despite its low total phenolic content compared to the other samples.These findings suggest that coextracted bioactive compounds with antioxidant potencies, such as sulfated polysaccharides, tocopherols, proteins or peptides, and carotenoid pigments, may possess inherent antioxidant properties. 85Wang et al. 73 investigated the AOA of Palmaria palmata extract obtained by using carbohydrase and protease enzymes and reported that enzyme-assisted extract indicated higher AOA than conventional extract.Yuan et al. 72 reported that AOA ABTS of microwave-assisted extracts from some brown macroalgae species was higher than that of conventional extracts, and the highest AOA found was 0.95 ± 0.01 mg TE/g.The antioxidant activity of macroalgae is attributed to both amino acids with antioxidant properties and phenols.Besides, antioxidant activity and stability of macroalgal phenolic compounds are related to the type of algae, experimental temperature, and extraction conditions. 70,86In this work, after in vitro gastric digestion, L. obtusa showed the highest AOA ABTS and AOA CUPRAC , followed by U. rigida and P. pavonica (p < 0.05).After in vitro intestinal digestion, AOA was found to be highest in L. obtusa, which contained the highest PC and TPC, followed by P. pavonica and U. rigida (p < 0.05).Similarly, Huang et al. 78 stated that the AOA of six macroalgae markedly decreased after in vitro gastric digestion, while only the AOA of Undaria pinnatif ida increased.Goncalves et al. 87 investigated the effect of digestion on the antioxidant activity of four wild edible plants and reported that antioxidant activity values significantly decreased after the gastric phase for all of the extracts and after the intestinal phase only for P. major extract.Additionally, after being digested in vitro system, proteins have been shown to have increased antioxidant activity in several investigations. 88,89According to Senphan and Benjakul, 88 sea bass skin hydrolysate's ABTS radical scavenging activity and chelating activity both slightly increased during pepsin digestion.
Hydrolysis can increase the antioxidant activity of proteins by releasing amino acid side groups that contribute to the antioxidant activity.The accessibility of amino acid residues inside the protein's tertiary structure restricts its antioxidant activity prior to in vitro digestion.Antioxidant amino acids are exposed to more oxygen during the enzymatic hydrolysis process, which may increase their propensity to contribute hydrogen to the peroxyl radical. 90Moreover, antioxidant assay results may be impacted by the mode of action of antioxidants in various test systems and their localization in distinct food or biological system phases. 91

Effect of In Vitro Digestion on ACE-I Inhibitory Activity.
The ACE-I inhibition activity of macroalgal protein extracts before and after in vitro digestion is demonstrated in Table 3.Among macroalgal protein extracts, PPPE had the highest ACE-I inhibitory activity (13.1 ± 0.00%) followed by URPE and LOPE (p < 0.05).To our knowledge, there have been few studies on the ACE-I inhibitory properties of macroalgae protein extracts.Cermenõ et al. 92 reported that the ACE-I inhibitory activity of Porphyra dioica protein extract was 14.57 ± 1.1%.Kumagai et al. 93 stated that the Pyropia pseudolineariz protein inhibited ACE-I by 23.6%.Conversely, ACE-I inhibitory activity was found to be 79.87 ± 0.18% for Ulva sp. protein by Garcia-Vaquero et al. 94 Based on these results, it can be concluded that the ACE-I inhibitory activity of macroalgal protein extracts from three different macroalgae is less than or comparable to that of other macroalgae.The difference in ACE-I inhibitory activity seen may be related to the primary structure of the protein, chain length, amino acid composition and sequences, and also extraction conditions. 95,96fter in vitro digestion, the ACE-I inhibitory activity of macroalgae protein extracts increased.URPE (20.20 ± 0.00%) and LOPE (20.90 ± 0.00%) showed the highest ACE-I inhibitory activity after the partial hydrolyzed via pepsin and trypsin enzymes in the simulated gastrointestinal phase.According to Cermeno et al., 92 the ACE-I inhibitory activity of protein extract from Porphyra dioica after being hydrolyzed with alcalase and flavorzyme was determined to be 36.43± 3.4%.Garcia-Vaquero et al. 94 reported that Ulva sp. protein hydrolyzed with papain inhibited ACE-I by 82.37 ± 0.05%.Hydrolysate of the Pyropia pseudolineariz protein showed 67.7% ACE-I inhibitory activity. 93Similarly, Ulva intestinalis (48.72 ± 1.13%) and Gracilaria f isheri (36.43−62.56%)protein hydrolysate showed the greatest ACE-I inhibitory activity after the hydrolyzation. 15,97Biparva et al. 98 reported that the ACE-I inhibitory activity of Macrocystis pyrifera protein hydrolysate was 27.60 ± 0.005%.According to the literature, peptides are not active in the primary protein, but they can show their bioactive properties by being released by enzymecatalyzed proteolysis in vitro. 20Similar to this, Pripp et al. 99 reported that low-molecular-weight peptides exhibit stronger ACE-I inhibition activity compared to high-molecular-weight peptides.
3.4.1.2.Foaming and Emulsifying Properties.The foaming capacity (FC) and foaming stability (FS) of three macroalgal protein extracts are shown in Table 4. FC of three different macroalgae varied between 28.92% (URPE) and 5.61% (LOPE).The FC of macroalgae was comparable to or higher than E. linza (15.6 ± 0.9%) while lower than E. compressa (40.9 ± 2.9%), E. tubulosa (45.0 ± 2.0%), and K. alvarezii (38 ± 2.0% at pH 6.0, 53.33 ± 2.31% at pH 4.0). 101,48In addition, FC of macroalgae was lower than some plant proteins such as soybean protein (65.7 ± 0.5%) and whey protein (132%). 110,111Ragab et al. 112 emphasized that the solubility should be high for an effective foaming capability.Therefore, the FC of macroalgae can be explained as being related to their solubility profile of them.Moreover, Du et al. 113 reported that lower FC could be caused by high levels of hydrophobic amino acids.Also, it has been reported that the increase in the surface charge of the proteins due to the pH change can make proteins more flexible and reduce hydrophobic interactions, thus enhancing foam formation. 114,115The low and negative net surface charge of macroalgae may also be a reason for its low foaming capacity.The FS can be influenced by several variables, including extraction procedure, macroalgal genotype, temperature, pH, and specific protein characteristics. 116,117RPE and PPPE exhibited ∼11.00% foaming stability in the present study, whereas LOPE showed no foaming stability.These values were higher than the FS of E. linza (4.4 ± 2.0%) but lower than the FS of E. compressa (37.5 ± 2.0%), E. tubulosa (16.7 ± 1.5%), and K. alvarezii (45.33 ± 1.15%). 91,47he emulsifying activity (EA) and emulsion stability (ES) of the three macroalgae are exhibited in  118,119 According to the literature, the proteins' hydrophilic and hydrophobic qualities, net surface charge, and solubility may affect their EA and ES properties. 120,121.4.2.Structural Characterization.3.4.2.1.FT-IR.Proteins commonly contain a certain fraction of structural components such as α-helix, β-sheet, etc.In addition, determining protein secondary structures gives one of the most critical information for protein structure. 48Therefore, FT-IR spectrum has been used to estimate protein secondary structure (Figure 2).The FT-IR spectrum of the samples contained several typical bands for functional groups with variances in the absorption strength of some distinctive peaks.The broad band at ca. 3250−3350 cm −1 can be attributed to stretching vibrations of the O−H and N−H groups, stated Amide A. 122 The peaks at ∼2900 cm −1 are attributed to the stretching vibrations of C−H groups, indicating the existence of neutral proteins, carbohydrates, and lipids. 65The Amide I band (∼1645 cm −1 ) results from C�O stretching, and the Amide II band (∼1530 cm −1 ) is due to the presence of stretching of C−N and bending vibrations of N−H groups. 113The existence of these two bands is indicative of the presence of proteinaceous in the samples. 123Similarly, Murdock and Wetzel 124 indicated that cell walls of green algae contained abundant protein, implicating that strong amide I and amide II bands occurred at ∼1645 and 1530 cm −1 , respectively.Moreover, peaks at the wavelength ranging ca.1229−1301 cm −1 showed an amide III band stretching vibrations of C−N and N−H groups. 113The peaks ranging between 2400 and 2240 cm −1 for CO 2 and peaks observed at 1850−1600 cm −1 correspond to C � O stretching vibration that may be caused by ketones, aldehydes, carboxylic acids, primary amides, and esters. 125In addition, the peaks at ca. 1000−1100 cm −1 are attributed to the C−O−C band, showing the existence of polysaccharides in macroalgal cell walls. 126 127 also observed a limited number of protein bands in algae extract samples.Similarly, the electrophoresis pattern of U. rigida samples had low-molecular-weight bands (peptide bands <36 kDa, mainly peptides <12.3 kDa) close to those recorded in our study.In addition, the physical processing of algae might not alter the protein composition of algae.

Differential Scanning Calorimetry (DSC).
The thermal characteristics of macroalgal protein extracts are displayed in Figure 4.There were differences between the isolates in the denaturation enthalpy and temperature.The PPPE (69.51 °C) had the lowest denaturation temperature, while the URPE (94.58 °C) and LOPE (115.50 °C) showed noticeably greater denaturation temperatures.The denaturation enthalpy of PPPE, URPE, and LOPE was detected as 31.33,15.28, and 43.98 J/g, respectively.The denaturation enthalpy measures the energy liberated during the reaction. 128ll macroalgal proteins have different denaturation temperatures and enthalpies, which can be related to variations in the natural and chemical structures of the proteins.Rui et al. 129 obtained protein from Phaseolus vulgaris legume varieties and reported that their denaturation temperature and enthalpy were ∼90 °C and ∼11 J/g.Gundogan and Karaca 128 stated that various kinds of beans originating from Turkiye exhibited denaturation temperatures ranging from 90.5 to 152.4 °C, as well as corresponding denaturation enthalpies between 32.9 and 134 J/g.Compared with other vegetable proteins, the thermal stability of LOPE is similar to or higher than that of URPE and PPPE.The denaturation temperature and denaturation enthalpy can vary depending on the specific protein and the conditions of the experiment.A high denaturation temperature may indicate that the protein is heat-resistant.The homogeneity of the polypeptides, the type of bonding between the peptides, and the amino acid content of the protein are all factors that affect thermal stability. 130oreover, the interactions between proteins and residual salts may increase heat stability in addition to changes in protein structure. 121

CONCLUSIONS
In this study, we demonstrated that macroalgal protein could be considered an alternative source of protein, and we compared them to each other.Especially, L. obtusa had the highest protein content of 227 ± 0.01 mg BSA/g dw.The obtained macroalgal protein extracts have high antioxidant activity due to the presence of phenolic compounds.The utilization of macroalgae as a healthy food source for humans is supported by these antioxidant properties.Moreover, we developed/applied a novel extraction method including osmotic shock, enzyme, and ultrasound to improve the yield of macroalgal protein extraction.The highest extraction yield obtained for L. obtusa was 94.74%, following U. rigida (74.21%) and P. pavonica (63.20%).The three macroalgal protein extracts have similar functional properties to some commercial products in terms of water holding capacity, foaming capacity, stability, and emulsification activity, but they have a high oil holding capacity.Moreover, L. obtusa had higher thermal stability than U. rigida and P. pavonica.The structural conformation of macroalgal proteins had a significant impact on both their physicochemical and functional characteristics.In addition, during the in vitro intestinal phase, the digestibility of three macroalgal protein extracts was found to be remarkably high.The ACE-I inhibitory activity of LOPE and URPE was found to be 20.20 ± 0.00 and 20.90 ± 0.00% after in vitro gastrointestinal digestion, respectively.These results highlight the viability of employing macroalgae as a novel, renewable source of protein for human nutrition and commercial food processing.The antioxidant and ACE-I inhibiting peptides (compounds) in the macroalgal protein extract should be purified and identified in future pharmaceuticals or applications in food formulation.

■ AUTHOR INFORMATION
content of the extract after extraction) (the content of extract after extraction) / (The content of macroalgal protein before extraction) (the content of macroalgae before extraction) 100

Figure 1 .
Figure 1.Effect of pH on net surface charge (ζ potential) and solubility of protein extract from (A) U. rigida, (B) L. obtusa, and (C) P. pavonica.Results are given as mean ± standard deviation.

Table 1 .
Changes in Protein Content of Macroalgal Protein Extract before and after In Vitro Digestion aValues are expressed as mean ± standard deviation for triplicate determinations.Different letters in the rows represent statistically significant differences (p < 0.05).Different superscript letters within the same line (a, b, c) and column (x, y, z) indicate significant difference (p < 0.05, Tukey).

Table 2 .
Changes in Total Phenolic Content of Macroalgal Protein Extract before and after In Vitro Digestion aValues are expressed as mean ± standard deviation for triplicate determinations.Different letters in the rows represent statistically significant differences (p < 0.05).Different superscript letters within the same line (a, b, c) and column (x, y, z) indicate significant difference (p < 0.05, Tukey).

Table 3 .
Changes in Antioxidant Activity and Angiotensin-I-Converting Enzyme Inhibitory Activity of Macroalgal Protein Extract before and after In Vitro Digestion a

Table 4 .
Techno-Functional Properties of Macroalgal Protein Extracts aValues are expressed as mean ± standard deviation for triplicate determinations.Different letters in the rows represent statistically significant differences (p < 0.05).WHC: Water holding capacity, OHC: Oil holding capacity, EA: Emulsifying activity, ES: Emulsion stability, FC: Foaming capacity, FS: Foaming stability.
.4.2.2.Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE).The SDS-PAGE patterns of extracted macroalgae proteins showed slightly different banding patterns among the different species in Figure 3. Comparable band patterns at low-molecular-weight peptides (<17 kDa) were observed in Lane A, Lane B, and Lane C of PPPE, URPE, and LOPE, respectively.URPE and LOPE displayed a similar protein profile with two protein bands observed at 11 and 17 kDa, with the exception of the more intense 11 kDa protein band in LOPE.PPPE had additional protein bands at 35, 48, and 75 kDa compared to URPE and LOPE.Consistent with our findings, Rouxel et al.