Conjugation of Microalgal Phenolics and Protein for Bioactivity and Bioaccessibility Enhancement

TL;DR

This study shows that combining microalgal proteins and phenolics improves their stability, antioxidant activity, and bioaccessibility, making them more useful for food and nutraceutical products.

Contribution

The study demonstrates that conjugation enhances the bioactivity and bioaccessibility of microalgal compounds through structural changes and chemical reactions.

Findings

Conjugation significantly increased antioxidant activity by up to 644% and ACE inhibitory activity by up to 19.7%.

The 2.5% conjugate showed the highest bioaccessibility, 2.5 times that of free phenolics.

FTIR and NMR analyses confirmed covalent bond formation through Schiff base and Michael addition reactions.

Abstract

Microalgae are rich in protein and phenolics, thereby having great potential for production of functional foods and nutraceuticals. However, despite featuring high nutritional value, these compounds often suffer from low stability and bioaccessibility. In this study, phenolics and protein extracted from Chlorella vulgaris were conjugated at different ratios (2.5–10%) and the structure and bioactivity of the conjugates were comprehensively characterized. The fluorescence intensity of protein decreased from 340 to 130–98 a.u. after conjugation and the UV-vis absorbance dropped from 1.6 to 0.5 a.u., which confirms the alteration of the chromophore area. The FTIR spectra revealed shifts in the C=O, N-H, and C-N bands, and the 1H NMR spectra showed the broadening of signals and appearance of new peaks, indicating covalent bond formation through the Schiff base and Michael addition reactions. Conjugation significantly increased the antioxidant activities, in terms of ABTS inhibition by 644%, 257%, and 97%, as well as the ACE inhibitory activity, by 13.5%, 17.5%, and 19.7% for the 2.5%, 5% and 10% conjugates, respectively. The 2.5% conjugate showed the highest bioaccessibility (144%), which was 2.5 times that of free phenolics. Overall, this study proves that conjugation is an effective approach to enhancing the bioactivity and bioaccessibility of microalgae-derived compounds and unravel the structure–activity relationship of conjugates. The findings can promote the valorization of microalgae for product development in the food and nutraceutical industries.

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1. Introduction

Microalgae have become one of the most promising feedstocks for addressing food security concerns, owing to their resource-efficient production and high nutritional value [1]. Phenolic compounds derived from microalgae have diverse bioactivities such as antimicrobial, anticancer, antiviral, and immunomodulatory effects, making them emerging candidates for biopharmaceuticals and nutraceuticals [2]. For example, polyphenols extracted from green microalgae Nannochloropsis oculata were found to effectively inhibit the proliferation of the human breast cancer cell line [3].

Microalgae generally have very high protein contents, up to 70%, and hence have been utilized for protein enrichment in various sectors of the food industry. For example, in the bakery sector, marine microalgae such as Nannochloropsis sp. and Tetraselmis sp. have been incorporated into wheat tortillas to increase its protein content [4]. Moreover, in dairy processing, Spirulina sp. can be combined with cocoa powder to formulate high-protein chocolate milk powder [5]. Microalgal protein also features high nutritional quality. Chlorella and Arthrospira produce proteins containing well-balanced amino acid profiles that meet the WHO/FAO/UNU recommendations for human essential amino acids [6].

Although microalgae are a potent source of phenolic compounds for food formulation, the commercialization of microalgae-derived functional foods still faces challenges, primarily due to the inherent instability of phenolic compounds [7]. Techniques that have been applied to improve the stability of phenolic compounds include freeze-drying, spray drying, emulsification, coacervation, liposomes, and microgels [8,9]. Conjugation of phenolic compounds (especially phenolic acids and flavonoids) with proteins is another approach to increasing the stability, bioaccessibility and bioavailability of interacted phenolic compounds [10], while improving protein functionalities, such as modified water solubility by inducing protein cross-linking [11], improved foaming and emulsifying properties [12], and enhanced digestibility [13]. Although a variety of phenolic–protein conjugates derived from animal or plant proteins and plant phenolics have been synthesized and characterized [14], the conjugation of microalgal phenolic compounds and protein, especially sourced from a single microalgae species, and its benefits have not been explored.

This study aims to enhance the potential of microalgae-derived biocompounds for functional food applications, ultimately promoting the valorization of microalgal biomass. Phenolic compounds extracted from microalgae were conjugated with microalgal protein at different ratios. The structure, bioactivity and bioaccessibility of the synthesized conjugates were comprehensively characterized.

2. Materials and Methods

2.1. Microalgae

Chlorella vulgaris was cultivated with aquaculture wastewater collected from a local tilapia farm in Romney, Indiana. The cultivation was performed in 7-gallon photobioreactors (The Vintage Shop, Delta, BC, Canada) at an average temperature of 24.5 °C under a 16:8 light–dark cycle, which included 4 h of supplemental LED light (5000 K) for 14 days. Microalgal biomass was harvested using a continuous centrifuge (Algae Centrifuge, Sacramento, CA, USA). The microalgal paste collected was oven-dried at 60 °C for 48 h, then sifted through an 850-μm sieve to obtain microalgae powder. The powder was kept in a Ziploc bag and stored at 4 °C for experiments. The microalgae powder contained 39% (w/w) crude protein, 7% crude lipids, and 46% crude fiber, according to the proximate analysis performed by the A & L Greatlakes Laboratories (Fort Wayne, IN, USA).

2.2. Microalgal Phenolic Compound Extraction

Phenolic compounds were extracted from microalgae following Monteiro et al. (2020) [15] with modifications. One-half gram of microalgae powder was mixed with 10 mL of 50% (v/v) ethanol, followed by agitation at 220 rpm for 30 min. The sample was then centrifuged at 10,600× g and 4 °C for 15 min, and the supernatant was collected and stored at −20 °C for further analyses. According to the liquid chromatography mass spectrometry (LC-MS) analysis performed in our previous study [16], the major compounds of the phenolic extract such as phenolic acids (vanillic acid, protocatechuic acid, etc.), flavonoids, including quercetin-3-glucoside, quercetin, etc., are present in lower concentrations.

2.3. Microalgal Protein Isolation and Pretreatment

Microalgal protein was isolated from biomass using the method described by Biparva, Mirzapour-Kouhdasht, Valizadeh, and Garcia-Vaquero (2023) [17] with modifications. The microalgal biomass powder was mixed with distilled water at a ratio of 1:10 (w/v) and the pH of the suspension was adjusted to 12 using 1 M sodium hydroxide (NaOH) solution. After mixing for 4 h at 50 °C, the suspension was centrifuged at 10,000× g at 20 °C for 30 min. Subsequently, the pH of the supernatant was adjusted to 4.2 using 1 M hydrochloric acid (HCl) and stirred for 1 h, followed by centrifugation at 10,000× g at 20 °C for 30 min. The precipitated protein at the isoelectric point was resuspended in distilled water and neutralized by 1 M NaOH, followed by freeze-drying (Pharmaceutical Pro Freeze Dryer, Harvest Right, Salt Lake City, UT, USA).

The microalgal protein tested in this study was pretreated with cold plasma to improve its functionalities and digestibility. Two grams of dried protein isolate was dissolved in 25 mL of distilled water and treated with pin-to-plate cold plasma (Leap 100, Plasma Leap Technologies, Marrickville, Australia) at a voltage of 140 V and a frequency of 1000 Hz for 20 min. The sample was placed in a Petri dish (90 mm diameter) and 7 cm away from the pins. The treated sample was freeze-dried and then stored at −20 °C. The protein content of the sample was estimated to be 68%, based on the total nitrogen content determined by the A&L Great Lakes Laboratories (Fort Wayne, IN, USA).

2.4. Phenol–Protein Conjugation

The extracted phenolic compounds were conjugated with protein isolate using the method of Djuardi, Yuliana, Ogawa, Akazawa, and Suhartono (2020) [18] with modifications. One gram of protein powder was dissolved in 100 mL of deionized water and stirred at 150 rpm for 30 min to facilitate solubilization. Phenolic extract was added to the protein solution at different concentrations (w/w, based on the protein content): 0 (protein only), 2.5%, 5%, and 10%, and the pH was adjusted to 9.0 using 0.1 M NaOH. The mixture was covered with aluminum foil and incubated at ambient temperature under continuous agitation at 150 rpm for 24 h. After the incubation, the mixture was filtered using 10 kDa ultrafiltration membrane (Thermo Scientific, Waltham, MA, USA) and centrifuged at 5000× g for 15 min. The retentate was washed by adding an equal volume of deionized water and centrifuged again at 5000× g for 15 min. The collected retentate was freeze-dried for further analyses.

2.5. Conformational Analysis of Conjugate

2.5.1. Fluorescence Spectroscopy

The tertiary structure of the phenol–protein conjugate was characterized by measuring its fluorescence intensity using a multi-label plate reader (Biotek Synergy Neo2 HTS, Agilent Technologies, Winooski, VT, USA) following Li, Zhu, Wu, Chen, Wu, and Zhang (2024) [19] with modifications. The sample was prepared by dissolving 1 mg of conjugate into 50% ethanol (v/v). The fluorescence spectrum of the dissolved conjugate (20 μL) was measured at the excitation wavelength, and the emission fluorescence wavelength was recorded between 300 and 600 nm. The fluorescence spectra of microalgal phenolic compounds and protein were also separately measured for comparison.

2.5.2. Ultraviolet–Visible (UV-Vis) Spectroscopy

The samples of conjugate and phenolic extract were prepared by mixing each with 50% ethanol to reach the final phenolic concentration of 0.25, 0.5, or 1.0 µg/mL. The protein sample was dissolved in 50% ethanol at a concentration of 1 mg/mL. The UV-vis absorbance spectra of the prepared solutions were recorded over the range of 230–500 nm using a multi-label plate reader following the method of Karefyllakis, Salakou, Bitter, Van der Goot, and Nikiforidis (2018) [20] with minor modifications.

2.5.3. Fourier-Transform Infrared (FTIR) Spectroscopy

The secondary structure of conjugate was characterized using a Nicolet 6700 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) following Jiang et al. (2019) [21] with modifications. The spectrum was recorded over the range of 600–4000 cm^−1^ at the resolution of 4 cm^−1^. Both phenolic compounds and protein were also separately analyzed for comparison.

2.5.4. Nuclear Magnetic Resonance (NMR) Spectroscopy

Spectra of ^1^H NMR were obtained on a Bruker NEO NMR spectrometer (Rheinstetten, Germany) operating at 499.83 MHz, equipped with a 5 mm BBFO Prodigy cryoprobe and the TopSpin software v4.5.0. The sample temperature was set at 298 K. Data were acquired with suppression of the H_2_O peak using the Watergate technique via the pulse program zggpw5 in the TopSpin pulse program library. ^1^H NMR analysis of the phenolic extract was conducted in H_2_O/D_2_O (90:10, v/v), using a standard one-dimensional proton acquisition without water suppression (Watergate), due to the high ethanol content (~50%) in the extract. Acquisition parameters included a sweep width of approximately 7.1 kHz, 16,000 data points, a relaxation delay (D1) of 2 s, and 16 scans. Data processing included exponential multiplication (line broadening factor of 5), one zero-fill, manual phase correction, and baseline flattening using the TopSpin abs routine.

2.6. Antioxidant Capacity Determination

The antioxidant capacity of the conjugate was determined using the 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), ferric reducing ability of plasma (FRAP), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays.

The ABTS radical scavenging assay was conducted following Bao and Huang (2024) [22] and Re, Pellegrini, Proteggente, Pannala, Yang, and Rice-Evans (1999) [23] with slight modifications. The ABTS^•+^ solution (~7 mM) was prepared by mixing 95.2 mg of ABTS (Thermo Scientific, Waltham, MA, USA) with 25 mL of 2.45 mM potassium persulfate solution. The mixture was left at room temperature in the dark for 16 h, then further diluted until the initial absorbance at 734 nm reached 0.7. One microgram of phenol–protein conjugate was dissolved in 1 mL of 50% (v/v) ethanol, then mixed with the prepared ABTS^•+^ solution (294 µL) in a 96-well plate. The absorbance of 734 nm was measured after incubation at 30 °C for 10 min. The ABTS scavenging effect was determined by

$$[eqn]$$

where Ac,ABTS is the initial absorbance of ABTS radical in 50% ethanol and As,ABTS is the absorbance of ABTS radical in the test sample.

For the FRAP assay [24], 1 mg of phenol–protein conjugate was dissolved in 1 mL of 50% (v/v) ethanol. Fifty microliters of 0.1 M potassium phosphate buffer (pH 6.6) and 50 µL of 1% potassium ferricyanide were mixed with 20 µL of the dissolved conjugate in a 96-well plate and then incubated in a water bath at 50 °C for 20 min. After the incubation, 50 µL of 10% trichloroacetic acid was added to the mixture. Fifty microliters of the prepared solution were mixed with 50 µL of deionized water and 10 µL of 1% ferric chloride. The sample was left at room temperature for 30 min before measurement at the absorbance of 700 nm.

The DPPH radical scavenging activity was measured following Bao, Reddivari, and Huang (2020) [25]. One milligram of phenol–protein conjugate was dissolved in 1 mL of 50% (v/v) ethanol. Fifteen microliters of the dissolved conjugate were added into 285 μL of DPPH (Thermo Scientific, Waltham, MA, USA) solution and incubated at room temperature in the dark for 2 h. Decolorization of the solution was measured by optical absorbance at 515 nm. The DPPH scavenging effect was determined by

$$[eqn]$$

where Ac,DPPH is the initial absorbance of DPPH radical in 50% ethanol and As,DPPH is the absorbance of DPPH radical in the test sample.

2.7. Angiotensin-I Converting Enzyme (ACE-I) Inhibitory Activity Determination

The ACE-I inhibitory activity of the conjugate was analyzed at a concentration of 1 mg/mL using the kit-WST following the amended procedures of the manufacturer (Dojindo EU GmbH, Munich, Germany). Briefly, a mixture of 40 μL of deionized water and 20 μL of substrate buffer was added to the reagent blank 2 wells (no enzyme and inhibitor present, used for background correction). The wells designated for inhibitors were set up by mixing 20 μL of the conjugate or positive control (captopril solution, 20 ng/mL) with 20 μL of substrate buffer. Thereafter, 20 μL of enzyme working solution was added to each inhibitor and negative control (no inhibitor present) wells to initiate the enzymatic reaction. The microplate was incubated for 1 h at 37 °C in the dark, under continuous shaking at 50 rpm. Following the addition of 200 μL of indicator working solution to each well, the plate was left at room temperature for 10 min. The absorbance of the sample was recorded at 450 nm using a multimode microplate reader (Spark 10M, Tecan Trading AG, Männedorf, Switzerland). The ACE-I inhibitory activity of the conjugate was calculated by

$$[eqn]$$

where AB1, AI, and AB2 are the absorbances of the control, sample, and blank, respectively.

2.8. Phenolic Bioaccessibility Determination

Bioaccessibility refers to the fraction of a compound that becomes accessible for absorption after being released from the food matrix in the gastrointestinal tract [26]. The bioaccessibility of phenolic compounds in the conjugate was measured using the method of Zeng, Liu, Luo, Chen, and Gong (2016) [27], which has been commonly used for determining phenolic bioaccessibility [28,29,30], with modifications. Pepsin (250 U/mg, Sigma-Aldrich, St. Louis, MO, USA) and pancreatin (8 × USP, Sigma-Aldrich) were used to sequentially simulate the gastric and intestinal phases of digestion, respectively. Specifically, 32 mg of pepsin was dissolved in 10 mL of 0.1 M HCl, and 2.5 mg of pancreatin was thoroughly mixed with 0.1 M sodium bicarbonate solution. Both enzyme solutions were subsequently diluted 100-fold using their respective buffers before use in the experiment. Five milligrams of conjugate were added into the mixture of diluted pepsin solution (0.1 mL) and 0.1 M HCl (0.1 mL), then incubated at 37 °C for 2 h under continuous agitation. The mixture was then placed in a water bath at 95 °C for 10 min. Diluted pancreatin solution (0.15 mL) was added into the mixture, and the volume was adjusted by adding 0.1 M sodium bicarbonate solution to a total of 0.2 mL. After 2 h incubation at 37 °C under continuous agitation, the mixture was placed at 95 °C in a water bath for 10 min. The sample was then cooled to room temperature before centrifugation at 10,000× g for 10 min. The supernatant was collected as the bioaccessible fraction of the conjugate.

The total phenolic content (TPC) of the bioaccessible fraction obtained was determined following Clarke, Ting, Wiart, and Fry (2013) [31] with slight modifications. Ten microliters of the sample were mixed with 100 µL of tenfold diluted Folin–Ciocalteu (Spectrum Chemical Mfg. Corp., New Brunswick, NJ, USA) reagent. One hundred microliters of 7.5% (w/v) sodium carbonate were added into the mixture after 5 min of incubation at room temperature. The mixture was left at room temperature for 2 h before the measurement of absorbance at 700 nm. A standard curve of gallic acid was established with a concentration gradient from 40 to 200 μg/mL. The bioaccessibility of phenolic compounds was calculated by

$$[eqn]$$

2.9. Statistical Analysis

All the experiments were conducted in triplicate. Statistical analyses were performed using the GraphPad Prism softwarev9.5.1 (GraphPad Software Inc., Boston, MA, USA). A p value of less than 0.05 was considered statistically significant for all the tests. To separately evaluate the differences among phenolic extracts and the conjugates containing 2.5%, 5%, and 10% phenolics, an ordinary one-way analysis of variance (ANOVA) was conducted, followed by the Tukey multiple comparison test to identify groups with means significantly different from each other. For direct comparison between phenolic extract and corresponding conjugate, an unpaired, two-tailed t-test with Welch’s correction was used.

3. Results and Discussion

3.1. Conformational Characterization of Conjugates

3.1.1. Fluorescence Characteristics

Tryptophan (Trp) is the main fluorescent amino acid of protein; the fluorescence emission spectrum of protein changes when the microenvironment of Trp is altered. The change in the fluorescence intensity of Trp is widely used as a tool to monitor the structural changes in protein [32]. Figure 1a shows the fluorescence spectra of microalgal phenolic compounds and protein, as well as their conjugates at different phenol–protein ratios. Protein showed the highest fluorescence intensity at around 340 nm, corresponding to Lakowicz (2006) who discovered the maximum emission of Trp around 350 nm [33]. Conjugation with phenolic compounds reduced the fluorescence intensity, and the peak value became lower as the phenolic concentration increased, from 130 a.u. for 2.5% to 98 a.u. for 10% of phenolic compounds in the conjugate. The decreased fluorescence intensity is because free amino acids were consumed and interacted with phenolic compounds [34]. The decrease in fluorescence intensity with the phenolic concentration of conjugate was accompanied by a red shift in the maximum emission, from the wavelength of 335 nm for 2.5% to 350 nm for 10% phenolic compounds. The red shift indicates that the conjugation with phenolic compounds caused endogenous fluorescence quenching of protein [32]. Yang et al. (2019) associated red shift after conjugation with more side chains of protein exposed to solvent because of protein unfolding, which can make tryptophan transferred to a more hydrophilic environment [34]. Yan et al. (2021) combined purified phenolic extract and protein isolates from Cinnamomum camphora seed kernel and also found that the complexes with higher phenol concentration had a lower fluorescence intensity and a longer wavelength of maximum emission [35].

3.1.2. UV-Vis Spectroscopy

Figure 1b shows the UV-vis spectra of phenolic compounds, protein and their conjugates. At 400 nm, protein exhibited the highest absorbance of approximately 2.4 a.u., and phenolic extracts had the lowest values. Marked decreases in the absorbance were observed after protein conjugation with phenolic compounds, to approximately 1.6, 0.8, and 0.5 a.u. for the conjugates containing 2.5%, 5%, and 10% phenolic compounds, respectively. These results show that a chemical interaction occurred between phenolic compounds and protein, rather than simple physical mixing [19]. Similarly, Sęczyk, Świeca, Kapusta, and Gawlik-Dziki (2019) discovered that the interaction between pure catechin and white bean protein fractions (albumins and globulins) resulted in a significantly increased absorbance at 350–450 nm, with the maximum value detected at around 400 nm [36].

3.1.3. FTIR Spectroscopy

Figure 2 shows the FTIR spectra of phenolic compounds, protein, and their conjugates, in which the characteristic peak between 1600 and 1700 cm^−1^ (amide I region) represents stretching vibration of the C=O bond and the amide II region (1500–1600 cm^−1^) corresponds to N-H bending and C-N stretching in protein. No distinct amide peaks were observed in the spectra of the phenolic samples. Compared to the protein sample, the conjugates exhibited significant shifts in both characteristic peaks toward lower wavenumbers, implying alterations in the stretching and bending vibrations of C=O and N-H as well as C-N stretching. These differences indicate that the conjugation with phenolic compounds modified the secondary structure of the protein. Similar FTIR spectral red shifts were observed by Li, Zhu, Wu, and Zhang (2023) [32] for buckwheat protein–phenol complexes.

3.1.4. NMR Spectroscopy

Figure 3 shows the ^1^H NMR spectra of phenolic compounds, protein, and their conjugates. In the spectrum of phenolic extract (Figure 3a), the residual ethanol signals were fairly strong (δ ~ 1.2 and ~3.6 ppm), as were the water signals (δ ~ 4.7 ppm), whereas no significant signals of aromatic protons were observed in the regions generally expected (δ 6.0–8.0 ppm). This absence of observable signals for aromatic protons is consistent with the composition of the extract, which contains polyphenols such as gallic acid and rutin, according to our previous work [16]. These compounds are rather highly hydroxylated and/or glycosylated and thus have no free aromatic protons. Moreover, weak signals from less substituted phenolics might be obscured by solvent peaks or suppressed due to low concentration or rapid proton exchange between phenolic compounds and the NMR solvent. The spectrum of protein (Figure 3b) showed broad peaks mainly between 0.5 and 4.5 ppm, which are consistent with the signals of aliphatic protons and can be attributed to the amino acid side chains and backbone methylene/methine groups [37]. The breadth and overlap of the signals are indicative of the high molecular weight as well as the restricted mobility of protein chains in an aqueous environment [38]. Correia et al. (2022) also found wide signals arising from urinary proteins in ^1^H NMR metabolic profiles due to their size and complexity [39].

Conjugation with protein led to considerable changes in the spectrum of phenolic compounds. The two sharp signals (δ ~ 1.2 and ~3.6 ppm) disappeared, implying the loss (or structural alteration) of free phenolic moieties during conjugation. The spectrum became broader with increasing phenolic content from 2.5% to 10% (Figure 3c–e), especially in the 3.0–5.5 ppm range, confirming the occurrence of chemical bonding and increased molecular complexity [14,40]. The appearance of new and shifted peaks in the aliphatic and mid-field regions could indicate the formation of new covalent bonds, probably through the quinone-mediated conjugation pathways that include Schiff base formation (imine linkage) and Michael-type addition between oxidized phenolic rings and nucleophilic side chains of amino acids such as lysine, cysteine, or tyrosine [41,42]. The conjugates did not show signals in the aromatic region, indicating that the conjugated phenolics either were originally non-aromatic or underwent full substitution and/or oxidation that eliminated free aromatic protons. These findings support that phenolic compounds, even those without aromatic proton signals, were conjugated to the protein matrix.

3.2. Antioxidant Capacity of Conjugates

Figure 4a–c compare the antioxidant capacities of conjugates with microalgal phenolic compounds and protein. All the three assays used (ABTS, FRAP, and DPPH) demonstrated that the conjugates containing 2.5% and 5% phenolic compounds had considerably higher antioxidant capacities than their respective phenolic extracts, by 97–644% and 81–257%, respectively, with the ABTS results showing the largest increase. The enhanced antioxidant capacity by conjugation is because protein can stabilize phenolic compounds and protect their antioxidant properties against external impacts like processing, digestion, etc. [43]. Gu, Peng, Chang, McClements, Su, and Yang (2017) combined catechin polymers with egg white proteins through physical mixing and conjugation and examined their antioxidant capacities [44]. The authors found that while the mixture’s DPPH radical scavenging capacity and ABTS^•+^ scavenging activity were both 32%, the conjugate showed higher values of 79% and 97%, respectively. They attributed the difference to that conjugation facilitated covalent bonding of phenolic compounds to protein, making phenolic hydroxyl groups present in the conjugate backbone, but physical mixing only led to their non-covalent interactions. In the present study, the enhancing effect of conjugation on the antioxidant capacity did not statistically change as the phenolic concentration increased. This might be because the binding with protein involved more hydroxyl groups of phenolic compounds at higher concentration, masking the enhancing effect on the conjugate’s antioxidant capacity [45]. Wang, Li, Zhu, Liu, Liu, Yu, and Shao (2021) conjugated rice bran protein hydrolysate with ferulic acid of different concentrations and also reported enhanced antioxidant capacity determined by the DPPH, ABTS, and FRAP assays [46]. It should be noted that although the ABTS inhibition of 10% phenolic compounds increased by 97% after conjugation, its FRAP and DPPH results did not show significant changes. These different trends might be owing to the different mechanisms of the three assays, in terms of how the test compound stops the chain-breading reaction [47]. An opposite effect of conjugation was reported by Jiang et al. (2020) [48], who conjugated α-lactalbumin with three similar chalconoids (SYA, NHDC, and NGDC) and found that the FRAP values of the three conjugates were significantly lower than those of their respective chalconoids, and the reduction became more noticeable as the chalconoid concentration increased, especially for SYA, which features the most hydroxyl groups. Furthermore, Li et al. (2020) discovered that adding tea polyphenol in excessive concentrations (20, 50, and 100 µmol/g protein) to myofibrillar protein had pro-oxidative effects on oxidatively stressed protein, thereby leading to its denaturation and irregular aggregation [49].

The antioxidant capacity of the conjugates can be related to the profile of the phenolic extract identified in our previous study [16], as described in Section 2.2. Phenolic acids with hydroxyl and methoxy groups (e.g., vanillic acid and protocatechuic acid) are strong ABTS^•+^ radical scavengers because they can readily donate an electron and are stabilized by resonance. Therefore, these compounds may contribute substantially to the dramatic increases in ABTS inhibition after conjugation. In contrast, FRAP activity is primarily dependent on a compound’s capability of reducing ferric ions and, thus, the number of available hydroxyl groups. Flavonoids typically feature more available hydroxyl groups; however, they (e.g., quercetin) are present in the phenolic extract tested in this study with low concentrations.

3.3. ACE Inhibitory Activity

The ACE inhibitory activities of phenolic compounds, protein, and their conjugates were evaluated at a fixed concentration of 1 mg/mL, as shown in Figure 4d. The protein had an ACE inhibitory activity of 37%, while the 2.5%, 5%, and 10% phenolic extracts showed 33%, 40%, and 41% inhibition, respectively. Conjugation resulted in higher ACE inhibition activity, by 12–20%, compared to the phenolic extract, and the improvement was more significant as the phenolic concentration in the conjugate increased. The improved activity of the conjugate may be attributed to the Schiff base or Michael addition reactions between phenolic compounds and protein, which changed their conformations (Figure 1, Figure 2 and Figure 3) and exposed more functional groups that have stronger ACE inhibitory effects [50,51,52,53,54]. Similar enhancing effect of phenol–protein conjugation on enzyme-inhibitory activities has also been reported by previous studies [14,55]. However, excessive phenolic content could potentially inhibit the activity due to steric hindrance to chelate the active site of ACE or reduced peptide availability, which explains why the conjugates containing 5% and 10% phenolic compounds showed statistically similar ACE inhibitory activities. Li et al. (2015) investigated the structure–ACE inhibition relationship of phenolic compounds and found that more hydroxyl groups and less structural steric hindrance caused increased ACE inhibition of phenylethanoid glycosides [56].

3.4. Bioaccessibility of Phenolic Compounds

The bioaccessibilities of microalgal phenolic compounds before and after conjugation were measured after in vitro digestion. As shown in Figure 5, the sample’s bioaccessibility decreased with increasing phenolic concentration, regardless of conjugation, and conjugation significantly increased the bioaccessibility for all the cases. The improved bioaccessibility can be attributed to the better stability of phenolic compounds against degradation during digestion when conjugated with protein. Additionally, the interaction between phenolic compounds and protein may induce structural changes in protein to expose hindered nucleophilic regions [47]. However, when protein molecules are already saturated with bound phenolic compounds, the excessive phenolic compounds remain unconjugated and thus can be easily degraded [36,57], which explains the reduced bioaccessibility of conjugate with higher phenolic concentration. It is also possible that phenolic compounds aggregate at high concentration and interact with protein collectively, which could promote peptide dimerization and the formation of insoluble complexes [41]. The highest bioaccessibility was observed with the conjugate containing 2.5% phenolic compounds, of 144%, which was 2.5 times that of the corresponding phenolic extract. The bioaccessibility value surpassing 100% can be because the chemical treatment applied for in vitro digestion broke down polyphenols into smaller phenolic compounds, increasing the detectable concentration of specific phenolic compounds [58]. Milinčić, Stanisavljević, Pešić, Kostić, Similarly, Stanojević, and Pešić (2025) reported that the bioaccessibilities of procyanidin B2 and procyanidin B3 in grape seed extract were 109% and 125%, respectively, after gastric digestion [59]. For the samples containing 5% and 10% phenolic compounds, conjugation resulted in 135% and 75% increase in the bioaccessibility, which were lower than the enhancement shown by the 2.5% case. This trend is consistent with the results in Figure 4a–c that conjugation with phenolic compounds in lower concentration increased the antioxidant capacity by a larger extent. The phenolic compounds’ bioaccessibility determined in this study was comparable to Ashwar and Gani (2021) [60], who conjugated sea buckthorn polyphenol with whey protein and casein and recovered approximately 65% of the phenolic compounds after in vitro digestion.

4. Conclusions

In this study, phenolic compounds and protein extracted from microalgae were conjugated at different ratios, and the conformation, bioactivity, and bioaccessibility of the conjugates were analyzed. As confirmed by the fluorescence quenching, UV-vis absorbance shifts, and FTIR spectral changes detected, phenolic compounds and protein were successfully conjugated for all the phenolic concentrations tested. ^1^H NMR analysis evidenced that protein and phenolic compounds interacted at the molecular level which resulted in covalent bond formation, most probably through Schiff base and Michael addition mechanisms. These structural modifications account for the enhanced bioactivities of the conjugates. All the conjugates containing 2.5% and 5% phenolic compounds showed considerably higher antioxidant capacities than their respective phenolic extracts, particularly the ABTS inhibition, by up to 644%. Conjugation also greatly enhanced the ACE inhibitory activity, by 12–20% compared to the phenolic extracts, as well as the phenolic bioaccessibility by up to 249%; however, increasing the phenolic concentration appeared to mask the enhancing effects of conjugation.

This study advances the knowledge of phenol–protein conjugation by correlating conjugate structures with its bioactivities and bioaccessibility and further elucidating their relationships. The findings can largely contribute to the application of microalgae-derived bioactive compounds for development of functional foods and nutraceuticals. Further analyses, such as high-performance liquid chromatography (HPLC) and LC-MS, are needed to identify specific phenolic compounds that are conjugated with protein, which can provide a better understanding of the characteristics of individual compounds during and after conjugation, thereby guiding the optimization of conjugation efficiency and conjugate efficacy. Moreover, evaluating the relationship between conjugate concentration and bioactivities (i.e., dose–response) can help determine the corresponding half maximal inhibitory concentrations (IC50). In vivo digestion and absorption of the microalgal phenol–protein conjugates are also needed to further validate their bioaccessibility.