Production of Bioactive Peptides from Microalgae and Their Biological Properties Related to Cardiovascular Disease

Abstract

Microalgae are a substantial group of unicellular prokaryotic and eukaryotic marine organisms. Due to their high protein content of 50–70%, microalgae have the potential to become a sustainable alternative protein source, as well as aiding in the development of bioactive peptide-based nutraceuticals. A series of major steps are involved in the production of peptides from microalgae, which include the disruption of the microalgal cell wall, the hydrolysis of proteins, and the extraction or isolation of peptides derived from hydrolysis. Physical methods of cell wall disruptions are favored due to the ability to obtain high-quality protein fractions for peptide production. Bioactive peptides are protein fragments of two to twenty amino acid residues that have a beneficial impact on the physiological functions or conditions of human health. Strong scientific evidence exists for the in vitro antioxidant, antihypertensive, and anti-atherosclerotic properties of microalgal peptides. This review is aimed at summarizing the methods of producing microalgal peptides, and their role and mechanisms in improving cardiovascular health. The review reveals that the validation of the physiological benefits of the microalgal peptides in relation to cardiovascular disease, using human clinical trials, is required.

1. Introduction

Food-derived bioactive ingredients have been gaining the interest of both researchers and consumers owing to the increasing awareness of the impact of diet on health. Hence, dietary supplements and nutraceuticals have grown exponentially in the past decade [1,2]. Nevertheless, functional ingredients (bioactives) derived from primary food sources are resource-intensive and expensive. Hence, in order to achieve sustainable development, there is a need for innovative food ingredients that are affordable, sustainable, and have functional properties [3,4,5].

Microalgae are an unexploited natural resource for developing sustainable and innovative functional ingredients [6,7,8,9,10,11]. Several studies have shown that microalgae are a potentially significant source of nutrients and natural biologically active compounds (bioactives) [11]. Microalgae biomass is a good source of many bioactives, such as carotenoids, phycobilins, omega-3 fatty acids, polysaccharides, vitamins, sterols, and bioactive peptides [12,13]. Microalgae have the potential to be a sustainable, alternative protein source for functional ingredients, particularly bioactive peptides, with a protein content of 50–70% [14]. The use of microalgae as a source for extracting functional ingredients such as bioactive peptides is economically and environmentally sustainable, as well as requiring relatively low land area (<2.5 m² per kg of protein) [15].

The production of bioactive peptides requires processing to break down the microalgae cells and then hydrolyze the released proteins [16]. Generally, the processing of microalgae for the extraction of proteins requires a series of steps, which include the breakdown of the cell wall, the hydrolysis of the protein, and the isolation of the peptides. An emerging area of research involves exploring the bioactivities of microalgae-derived peptides and hydrolysates, such as the antioxidant, antihypertensive, immunomodulatory, antidiabetic, antibacterial/antimicrobial, and anticancer activities [17]. This review discusses the emerging technologies reported for protein extraction from microalgae, the hydrolysis of proteins, and their biological properties related to cardiovascular disease.

2. Microalgae as a Protein Source

Algae are a diverse group of photosynthetic organisms that live in oceans and freshwater bodies. The majority of the members of this group are capable of photosynthesis and producing biological compounds using carbon dioxide and other minerals. Algae are divided into two main categories, namely macroalgae and microalgae, depending on morphology, cellular division characteristics and their pigments [7]. Microalgae are single-cell microscopic organisms that are also now recognized as a sustainable source of dietary proteins. The most abundant microalgal divisions are Chlorophyta (green algae), Chrysophyta (golden algae), Bacillariophyta (diatoms), and Cyanophyta (blue-green algae) [18,19], have been cited more than 200,000 species of microalgae, making them an incredibly diverse group.

Microalgae can be easily cultured under laboratory conditions, but it is challenging to achieve high productivity on a large scale. Microalgae cultivation systems can be broadly classified into two categories: open ponds and photobioreactors. Open pond cultivation is widely used in the industry due to its relatively low construction, maintenance, and operational costs. In contrast, a photobioreactor is an enclosed system used to culture microalgae, preventing the direct exchange of materials between the culture and the environment [20]. There is a difference in the annual productivity of microalgae between these two methods. For example, the annual productivity of Chlorella vulgaris is estimated to be 5–7 kg/m² in open ponds and approximately 15 kg/m² in photobioreactors [21]. Harvesting microalgae accounts for 20–30% of the total cost of microalgae production due to high energy and capital demands. The commonly used harvesting methods include filtration, centrifugation, flocculation, and flotation [20,22,23]. Economic feasibility is crucial for the viability of microalgal production. The commercialization of microalgae is hindered by insufficient research funding, small-scale operations, and policy implementation challenges [24].

Microalgae-enriched food products are considered novel foods. They face challenges in consumer acceptance due to their fishy taste and smell, as well as consumers’ food neophobia and individual cultural backgrounds [21]. For example, a recent study assessed Spanish consumers’ knowledge and attitude towards microalgae as food. The results suggested that Spanish consumers perceive microalgae as sustainable, environmentally friendly, nutritious, healthy, and safe. The primary reason for not consuming microalgae as food is the lack of available information on microalgae-based food products [25]. Among the macronutrients of microalgae are 40–70% proteins, 12–30% carbohydrates, and 4–20% lipids. Microalgae are also abundant in micronutrients such as 8–14% carotene and considerable amounts of vitamins B1, B2, B3, B6, B12, E, K, and D [26,27]. The protein content of the microalgae differs based on species, growth phase, and light quality, which can be optimized through nutrient and environmental management [7].

Table 1. Macronutrient composition of different microalgae (% of dry matter).

Alga	Protein	Carbohydrates	Lipids
Anabaena cylindrica	43–56	25–30	4–7
Aphanizomenon flos-aquae	62	23	3
Arthrospira maxima	60–71	13–16	6–7
Chlamydomonas rheinhardii	48	17	21
Chlorella pyrenoidosa	57	26	2
Chlorella vulgaris	51–58	12–17	14–22
Dunaliella salina	57	32	6
Euglena gracilis	39–61	14–18	14–20
Porphyridium cruentum	28–39	40–57	9–14
Scenedesmus obliquus	50–56	10–17	12–14
Spirogyra sp.	6–20	33–64	11–21
Spirulina platensis	46–63	8–14	4–9
Synechococcus sp.	63	15	11

Source: [26].

shows the general composition of different microalgae that have been studied and reported [26].

Among these different species of microalgae, Arthrospira sp. contains the highest amount of proteins [28]. Among the investigated microalgae species, Athrospira sp., Chlorella sp., Scenedesmus obliquus, and Spirulina sp. have been selected for commercial production [26]. Chlorella species are considered in the literature as the most commonly exploited species due to their high protein content (51–58% DW) and the presence of essential amino acids [19].

The quality of the protein depends on the composition of the amino acids, primarily essential amino acids (EAAs) and digestibility [29]. In general, animal sources of protein are considered complete proteins since they are abundant in EAAs, which are not synthesized by humans [19]. Therefore, microalgae are considered a viable dietary protein with EAAs while meeting the FAO requirements and are equally as good as other major protein sources, such as soybean and egg [19]. However, it is also cited that microalgae proteins may have some deficiencies in terms of lower biological values, net protein utilization, low digestibility, and a poorer protein efficiency ratio than the protein standards considered in FAO/WHO references [26]. Also, microalgae can synthesize all 20 common amino acids, and they are capable of delivering EAAs as an unconventional source for human nutrition [30]. For example, Spirulina platensis is considered a food supplement due to its high protein content [31]. Also, cyanobacteria S. platensis has a protein content of 43–63% (dw) with the abundant amino acid profile of leucine, valine, isoleucine, phenylalanine, tyrosine, methionine, cysteine, and tyrosine [26].

Before being considered safe for human consumption, any novel food item is subjected to a series of toxicological tests to prove its harmlessness. Since microalgae are also grouped with unconventional protein sources, they also must go through this process. When considering the available information on algal protein toxicology, none of the algae tested so far have reported a negative effect [7,8,26,32,33,34,35]. However, it was identified that long-term consumption studies are lacking in this area of study.

3. Production of Bioactive Peptides from Microalgae

Microalgae-based extractions for intracellular constituents such as lipids, proteins, carbohydrates, and pigments for biofuel, food, and nutraceutical applications have attracted the attention of many researchers. Producing bioactive peptides from microalgae involves a series of steps that include cell wall disruption to release the intracellular proteins, the hydrolysis of proteins, and extraction or isolation, resulting in peptides (Figure 1).

3.1. Protein Isolate Hydrolysis

The generation of peptides using isolated proteins can be carried out using three methods [33]. These include enzymatic, fermentative, and chemical methods. The most widely used and effective method for generating bioactive peptides is enzymatic hydrolysis [36]. The enzymatic method is desired due to the high specificity for target peptide sequences and does not involve the use of toxic organic solvents. The enzymatic hydrolysis process can be scaled up for industrial processes [36,37]. However, controlling hydrolysis is essential, as otherwise, extensive hydrolysis can lead to the loss of biological activity in the produced bioactive peptides. One disadvantage of enzymatic hydrolysis is the high cost of the enzymes required [36]. The enzymes that are employed are gastrointestinal proteases such as pepsin, pancreatin, and trypsin, proteolytic enzymes from microbial production such as alcalase, neutrase, flavourzyme and plant-sourced proteases such as papain, ficin, bromelain. However, it is more effective to use combinations or cocktails of enzymes to generate bioactive peptides [37,38]. When developing an efficient process for bioactive peptide production, many factors are to be considered, including the nature of the protein, the enzyme–substrate ratio, the pre-treatment of protein before hydrolysis, the degree of hydrolysis, and protease specificity [37,39]. Recently, in silico enzymatic hydrolysis has been used to predict the production of peptide sequences from microalgae using Peptide Cutter. The potential bioactivities of these in silico-generated peptides can be predicted using PeptideRanker, and their novelty can be assessed against the reported peptide sequences in the BIOPEP-UWM database (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep; accessed on 10 September 2023) [40,41,42].

Fermentation is a metabolic process wherein sugars are converted to acids, gases, and alcohol through the action of fermenting microorganisms [6]. The most commonly used microorganisms are bacteria, yeasts, and fungi [6]. This technique is not widely applied when considering peptide production from microalgae. However, fermentation has been used for bioethanol production using microalgae hydrolysate [43]. Fermentation is a common approach to obtaining peptides from dairy products. Here, lactic acid bacteria (LAB) that possess cell wall-bound proteases and intracellular peptidases exert proteolytic activities on milk protein [38,44]. Htet and colleagues have used LAB fermentation to produce 2-pyrone 4, 6-dicarboxylic acid, using microalgae hydrolysate as a fermentation medium [45].

Chemical methods also involve using strong acids and bases to hydrolyze the protein sequence. This is a cheap and secure method. However, it has significant hindrances, such as a loss of functionality in the resulting hydrolysates, a lack of specificity, inferior nutrient quality, and undesirable products [46]. Acid hydrolysis has some disadvantages, such as the oxidation of cysteine and methionine, the deamination of asparagine and glutamine to aspartate and glutamate, and the breakdown of serine and threonine [37]. For these reasons, the hydrolysis of proteins using chemicals is not favored. In the literature, it is shown that the enzymatic method is the most commonly used and sought-after protein hydrolysis method, because of its specificity in cleavage positions in peptide bonds. It increases the reproducibility and predictability of generating peptides. The chemical method is less favored because of its inherent contamination problem and its inability to control hydrolysis, which leads to less reproducibility. The fermentative method lacks research on microalgae. However, since the fermentation method also uses exogenous and endogenous bacterial enzymes for hydrolysis, it also has specificity and, therefore, reproducibility. However, more research in this area is needed to draw a conclusion.

3.2. Isolation of Peptides

Cell disruption helps the release of cellular contents such as lipids, polysaccharides, nucleic acids, and phytochemicals. Hydrolysis helps the breakdown of protein molecules into peptides [47]. After the hydrolysis, the next step is to ensure the separation of peptides from other interfering compounds. Separation or fractionation techniques (centrifugation, precipitation, electrophoresis, ultrafiltration, and chromatographic techniques) help in obtaining pure protein/peptide fractions [48]. Some studies have used this step in between the cell wall disruption and protein hydrolysis step, as it helps to reduce the inferences from the other molecules in the solution [49,50]. However, it can be assumed that performing fractionation after cell wall disruption is better, since it provides an opportunity to obtain protein fractions with fewer interferences from the solutions.

Centrifugation is a technique that uses the gravitational field to separate proteins from other cell constituents, such as membranes, mitochondria, and nuclei, based on their different molecular weight and other physical characteristics [51]. The efficiency of centrifugation can be improved by a density gradient fractionation using sucrose. Precipitation involves the usage of organic solvents, organic polymers, and salts, or pH and temperature adjustments [52]. Electrophoresis is another technique where proteins can be separated based on size, shape, charge, or charge/mass ratio [48]. In electrophoresis, protein molecules are separated based on their charge due to the supplied electric field [53]. Electrophoresis in a free solution or macroporous gel (1–2% agarose) can be used to separate proteins according to net electric charge. Polyacrylamide gels can be used to separate proteins based on the molecular weight of proteins [52]. Ultrafiltration is a membrane separation technique that can be used to isolate macromolecules of 103–106 Da using a semi-permeable membrane. Chromatography is also a fractionation technique that can be used to separate proteins of varying properties. Size exclusion chromatography separates proteins based on size and shape; ion exchange chromatography is based on isoelectric points; reverse-phase chromatography is based on hydrophobicity, and affinity chromatography uses ligands for binding proteins. Even though these methods are extensively used for food-related studies, it is unusual to find these being used in microalgae-related protein extractions. These methods are used in combination with most of those from literature, as a result of the difficulty due to the interactions with the rest of the materials in the solution.

4. Peptides Derived from Microalgae

Bioactive peptides can be defined as protein fragments with health benefits beyond their nutritional values and have a positive impact on the body’s function or condition [40]. The size of the peptide may vary from 2 to 20 amino acid residues, and they possess multifunctional properties [54]. These peptides are not active when they are present within the sequence of the parent protein, but upon release through hydrolysis by proteolytic digestive enzymes or by proteolytic microorganisms, they provide several bioactivities within the human body [55]. Physiologically active peptides are naturally produced during the gastrointestinal digestion of proteins by lactic acid bacteria [55]. Enzymatic hydrolysis is the commonly used method of bioactive peptide production [16]. Gastrointestinal enzymes such as trypsin and pepsin are commonly used. When the hydrolysate contains adequate quantities of the active peptides, the product can be used as a therapeutic substance to manage a specific disease, or else hydrolysates can be used to isolate specific peptides and used in an available drug delivery system (e.g., encapsulation) to deliver to the body. Mass spectrophotometry is a commonly used technique for peptide identification.

Table 2. Examples of bioactive peptides derived from microalgae.

Amino Acid Sequence of Bioactive Peptide	Source of Peptide	Reported Bioactivity	Hydrolytic Method	Reference
Glu-, Asp-, Lys-, Arg-	Navicula incerta	Antioxidative	Enzymatic–Pepsin	[56]
Pro-Gly-Trp-Asn-Gln-Trp-Phe-Leu Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu, Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu	Navicula incerta	Hepatic fibrosis inhibitory effect (Cytotoxicity in HepG2/CYP2E1 cells)	Enzymatic–Papain	[56]
Val-Glu-Cys-Iyr-Gly-Pro-Asn-Arg-Pro-Glu-Phe	Chlorella vulgaris	Antioxidative	Enzymatic–Pepsin	[57]
Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Glu-Phe	Chlorella vulgaris	ACE inhibitory, Superoxide radical quenching	Enzymatic–Pepsin	[49]
Val-Glu-Cys-Iyr-Gly-Pro-Asn-Arg-Pro-Glu-Phe	Chlorella vulgaris	Anti-proliferation	Enzymatic–Pepsin	[58]
Ile-Val-Val-Glu	Chlorella vulgaris	ACE-I inhibitory	Enzymatic–Pepsin	[59]
Ile-Ala-Glu	Spirulina platensis	ACE-I inhibitory	Enzymatic–Pepsin	[59]
Ala-Ile-Tyr-Lys	Undaria pinnatifida	Antihypertensive	Enzymatic–Pepsin	[60]
Val-Tyr	Undaria pinnatifida	Antihypertensive	Enzymatic–Proteases	[61]
Tyr-His, Lys-Trp, Lys-Tyr, Lys-Phe, Phe-Tyr, Val-Trp, Val-Phe, Ile-Tyr, Ile-Trp, Val-Tyr	Undaria pinnatifida	Antihypertensive	Hot water extract	[62]
Ala-Lys-Tyr-Ser-Tyr	Porphyra yezoensis	Antihypertensive	Pepsin	[63]
Leu-Asn-Gly-Asp-Val-Trp	Chlorella ellipsiodea	Antioxidant activities	Pepsin	[64]
Pro-Gly-Trp-Asn-Gln-Trp-Phe-Leu and Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu	Navicula incerta	Antioxidant activities	Papain, pepsin, α-chymotrypsin, pronase-E, and neutrase	[65]
Peptide mixture	Chlorella pyrenoidosa	Antioxidant activities	-	[66]

illustrates the reported peptides identified from microalgae and their bioactivities.

The oral administration of protein hydrolysates or bioactive peptides may affect the major body systems, such as the cardiovascular, digestive, immune and nervous systems, depending on the amino acid sequence of the peptides [67]. Bioactive peptides are reported to exhibit antimicrobial, antioxidative, anti-inflammatory, antithrombotic, antihypertensive, and immunomodulatory activities in the human body [35,68,69,70,71]. Most of the body functions are modulated via the interaction of specific amino acid sequences of bioactive peptides, which can be used in a wide range of therapeutic applications in the future [67,69].

5. Cardiovascular Health Promotion from Microalgae Peptides

5.1. Antioxidant Biopeptides

As mentioned earlier, there are several peptides identified as possessing antioxidant capacity [16,72,73]. Antioxidant peptides contain 5–16 amino acid residues [72]. The advantages of the peptides generated from foods are that they are safe, have a low cost, are easy to absorb and have a relatively high stability [54,68]. Oxidative damage due to elevated levels of reactive oxygen species (ROS) has been implicated in many chronic disorders, such as certain cancers, diabetes mellitus, hypertension, and neurodegenerative and inflammatory diseases, as well as aging [74]. Therefore, the antioxidant activities of bioactive peptides have been shown to contribute to oxidative homeostasis.

The oxidation of proteins can cause modifications to the amino acid R-groups and the polymerization and/or fragmentation of proteins [75]. For example, the oxidation of bovine serum albumin with hydroxyl radicals in the absence of oxygen has resulted in extensive protein cross-linking while in the presence of oxygen, and some alteration of protein size has been observed [76]. The abstraction of alpha-hydrogen atoms to backbone carbons causes the breakage of peptide or amide bonds [76,77]. The oxidative damage to a given amino acid residue by free radical attack is determined in large by its R-group. However, solvent accessibility and the chemical properties of adjacent residues can also influence oxidation [78]. Although all 20 common amino acids are potentially oxidizable, the most vulnerable amino acids to oxidation are those containing either nucleophilic sulfur-containing side chains, i.e., cysteine and methionine, or aromatic side chains, i.e., tryptophan, tyrosine, and phenylalanine, from which hydrogen is easily abstracted. In addition, the imidazole-containing side chain of histidine is also oxidatively labile [77]. The cleavage of proteins, resulting in the peptides, improves the expression of amino acid R-groups, so the antioxidative properties of the peptides are comparatively higher than the proteins. Overall, the mode of the antioxidant function of proteins and peptides is due to their complex reactions of inactivating ROSs, scavenging free radicals, reducing hydroperoxides, chelating prooxidative transition metals, and enzymatically eliminating specific oxidants [77].

Marine microalgae species reported having high diversity and the ability to thrive in extreme environments, such as low light intensities and high oxygen concentrations, which make them more self-protective towards oxidative damage [79]. However, Chlorella sp., Navicula sp., and Spirulina sp. are among the widely studied microalgae species for antioxidative biopeptides (Figure 2). These species have been evaluated for potent antioxidant capacity in vitro and cell-based assays. Antioxidant peptides have been produced by the hydrolysis of Chlorella protothecoides using several extracellular bacterial proteases. The resulting hydrolysates showed high antioxidant activity: 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and a significant reduction of hydrogen peroxide and hydroxyl free radical. However, peptide sequences have not been identified in this study [80]. Ko and colleagues have reported the extraction of a hexapeptide (Leu-Asn-Gly-Asp-Val-Trp) from Chlorella ellipsiodea using peptic hydrolysis, which shows peroxyl radical and DPPH radical scavenging activities in vitro, and intracellular radical scavenging activity in monkey kidney cells [64]. Protein hydrolysates produced from C. ellipsiodea and Tetraselmis suecica using different microbial proteases were found to have moderate free radical scavenging capacity [65]. The neutrase and kojizyme hydrolysates of Tetrasel missuecica exhibited the ability to scavenge hydrogen peroxide in African green monkey kidney cells in vitro [65]. When papain, pepsin, α-chymotrypsin, pronase-E, and neutrase were used to hydrolyze Navicula incerta, antioxidant hydrolysates were yielded. For example, two peptides (Pro-Gly-Trp-Asn-Gln-Trp-Phe-Leu and Val-Glu-Val-Leu-Pro-Pro-Ala-Glu-Leu) generated using a papain reaction demonstrated antioxidant effects in HepG2/CYP2E1 cells [56,81]. Moreover, a Chlorella pyrenoidosa-derived peptide mixture showed antioxidant properties in skin fibroblasts. When UV irradiation-exposed skin cells were treated with the Chlorella-derived peptides (5 and 10 mg/L), 100% cytoprotection over a period of 72 h was observed [66]. However, it is not clear whether microalgal peptides were taken into cells to exhibit their antioxidant activity or only acted extracellularly and resulted in cytoprotection. Enzymatic hydrolysis is the most commonly used method of producing antioxidant peptides from microalgae. However, more evidence is required to validate the physiological antioxidant capacity of the microalgae-derived peptides, using in vivo studies of their applications in combating oxidative stress-associated disorders. The additional investigations will help to justify these unicellular organisms as an alternative source of dietary antioxidants to be used in the prevention of an array of human disorders.

5.2. Antihypertensive Biopeptides

Abnormally high blood pressure is one of the major risk factors for CVD [67]. Angiotensin I-converting enzyme (ACE) is one of the main enzymes that are responsible for the blood pressure regulating pathway called the renin–angiotensin system (RAS) and electrolyte homeostasis [82,83]. The RAS and kinin–arginine–nitric oxide system pathways are important for controlling blood pressure [84]. The enzyme renin, which is released by the circulation of the kidney, cleaves the inactive peptide angiotensinogen secreted from the liver, resulting in peptide angiotensin-I. Subsequently, ACE acts on angiotensin-I to produce angiotensin-II and inactivates the vasodilator bradykinin, leading to a rise in blood pressure. Therefore, blood pressure can be controlled by inhibiting either renin or ACE. Therefore, renin and ACE inhibitory factors have become primary targets in the treatment of hypertension. Synthetic ACE inhibitors such as Captopril have been used as antihypertensive drugs. Interestingly, some diseases, genetic or environmental factors, can stimulate the RAS and upregulate the levels of ACE in the human body, and high levels of angiotensin II result in blood vessel constriction, leading to hypertension [85,86]. Bioactive peptides have been shown to inhibit ACE in the renin–angiotensin–aldosterone system (RAAS) (Figure 2). Microalgal peptides play a significant role in the regulation of blood volume, blood pressure, and fluid balance regulation [87]. The most effective antihypertensive peptides seem to contain two to twenty amino acid residues and hydrophobic amino acid residues, such as proline [39,88,89]. The acting mechanisms of microalgae-derived peptides are related to ACE and rennin inhibitions.

In addition, the kinin–nitric oxide (NO) system, a complex network involving peptide kinins, plays a significant role in blood pressure regulation. Kinins, such as bradykinin and kallidin, are generated through the kallikrein–kinin system. Bradykinin activates Ca²⁺/calmodulin-dependent endothelial nitric oxide synthase (eNOS), which synthesizes nitric oxide (NO) and citrulline from arginine, resulting in vasodilation and a drop in blood pressure. It is shown that eNOS knockout and NO deficiency could result in clinical hypertension [90,91]. The regulation of the kinin–NO system by bioactive peptides is not well explored.

As in the antioxidant peptides, most antihypertensive peptides are also derived from a few microalgal sources, such as Chlorella vulgaris, Chlorella ellipsiodea, Spirulina platensis, and Nannochloropsis oculata. Many extracted peptides are illustrated in Table 2. These antihypertensive peptides have been used in vitro and in some animal studies to evaluate their antihypertensive properties. The peptides produced by the hydrolysis of Chlorella vulgaris using pepsin were found to possess in vitro and in vivo antihypertensive properties [57,59]. The by-products of Chlorella vulgaris processing have been used to produce an 11-amino acid residue peptide (Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe), which inhibited ACE activity in a dose-dependent manner [49]. The peptide has inhibited ACE activity non-competitively, which could be due to the non-specific binding of the peptide to the ACE protein. Pepsin hydrolysates of Chlorella vulgaris and Spirulina platensis yielded several short-chain peptides that were shown to be effective within 6 h after administration in reducing the high blood pressure of spontaneously hypertensive rats (SHRs) [59]. In addition, microalgal protein hydrolysates derived from Bellerochea malleus exhibited antihypertensive activity, resulting in a decrease of 17 mmHg after five days of oral administration to male SHRs [92]. Compared to the antioxidant activity of microalgae peptides, antihypertensive properties are widely studied. However, similar to antioxidant activity, further studies are needed to establish the physiological importance of the microalgal peptides, especially for the identified antihypertensive peptides in vitro, as in vitro activity does not necessarily translate into physiological antihypertensive effects. Recently, a bioactive hydrolysate was generated using Viscozyme^® and Alcalase from the red microalga Porphyridium species, and this hydrolysate inhibited in vitro COX-1 activity and exhibited antihypertensive activity in SHRs [93].

5.3. Anti-Atherosclerotic Biopeptides

Atherogenesis is the process leading to the formation of atherosclerotic plaques in the walls of arteries, which can result in CVDs, such as coronary artery disease, stroke, myocardial infarction and peripheral artery disease [94,95]. Atherosclerosis is a progressive inflammatory disorder of the arterial wall that is a common cause of cardiovascular morbidity and mortality [96]. Increased cardiovascular risk is associated with hypercholesterolemia, hypertension, diabetes mellitus, and cigarette smoking [97,98].

Bioactive peptides have been reported to have antithrombotic and antiatherosclerotic effects [33,99]. Numerous peptides generated from in vivo and in vitro hydrolysis and the enzymatic digestion of milk, soybean, and other primary food sources have shown cardiovascular benefits [100,101,102]. However, only a few studies have researched peptides extracted from microalgae and indicated positive associations with the above-mentioned conditions of atherogenesis and atherosclerosis. Shih and colleagues were able to isolate an undecapeptide (Val-Glu-Cys-Tyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe) from Chlorella pyrenoidosa, which can significantly suppress the levels of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), monocyte chemoattractant protein (MCP-1), and endothelin-1 (ET-1) gene expression (Figure 2) [103]. The suppression of these expressions has been shown to be a successful method of blocking the development of atherosclerosis [104]. Vo and Kim were able to isolate two peptides P1 (Leu-Asp-Ala-Val-Asn-Arg) and P2 (Met-Met-Leu-Asp) from the gastric enzymatic hydrolysate of Spirulina maxima [105]. P1 and P2 inhibited the production of adhesion molecules, such as P-selectin and E-selectin, through the down-regulation of the expression of early growth response factor 1 (Egr-1) via the histamine receptor and PKCδ-dependent MAPKs activation pathway. It was also found that P1 and P2 have anti-inflammatory activity, by reducing ROS production and inhibiting histamine release and IL-8 expression. These two pieces of research have depicted that micro algae-derived peptide plays a role in adhesion molecule reduction and anti-inflammation. A recent study isolated a novel nonapeptide, Glu-Met-Phe-Gly-Thr-Ser-Ser-Glu-Thr, from the microalgae Isochrysis zhanjiangensis, which demonstrated anti-apoptotic and anti-inflammatory effects in oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs), suggesting its potential to alleviate atherosclerosis. However, this study only employed the ox-LDL-mediated HUVEC model, lacking an animal model [106]. Reported studies related to the anti-atherosclerotic property of microalgal peptides are very scarce. Therefore, additional research in this field is required to identify the effects that microalgal peptides have on atherogenesis.

6. Concluding Remarks and Future Directions

Algae are divided mainly into two groups, microalgae, and macroalgae. Microalgae are unicellular, microscopic organisms. Microalgae are an incredibly diverse group with around 200,000 species. Microalgae consist of 40–70% proteins, 4–20% carbohydrates, and 8–14% lipids and other nutrients. The literature reveals that the composition of microalgae greatly varies depending on their species, growth phase, light, available nutrients, and environmental stress. For example, Anthrospira species are one of the sources with the highest protein content reported. Chlorella is found to be the genera that are mostly investigated. Interestingly, when looking at the protein content of microalgae, they consist of all essential amino acids; hence, the quality of the proteins meets the FAO/WHO reference values. Because of these factors, microalgae continued to be an untapped, underutilized, and sustainable protein source.

Peptide production comprises four main steps that involve the release of the intracellular protein from cells and the hydrolysis of the protein into smaller peptides. The complexity of the cell walls of microalgae requires vigorous treatments to break down the cell walls. Three types of approaches can be used to disrupt the cell walls, chemical, mechanical, and biological/biochemical. These different techniques have different advantages and disadvantages associated with their specificities. The main disadvantage associated with the chemical treatment is contamination or inability to efficiently remove the chemicals being used for the cell wall breakdown. Heat generation and high associated energy costs are the major drawbacks of the physical or mechanical treatments. Biological or biochemical methods suffer from the costs of commercially available enzymes and low yield compared to the time taken. Regarding the treatments to break down cell walls, the hydrolysis of the protein can be carried out using three methods—enzymatic, fermentative, and chemical methods. The most commonly used method to obtain bioactive peptides is enzymatic hydrolysis, due to its high specificity for the target peptide sequence. Commercially available enzymes have been widely used, such as pepsin, pancreatin, trypsin, alcalase, neutrase, flavourzyme, papain, ficin, and bromelain. Fermentation with microorganisms such as bacteria, yeasts, and fungi is a less costly method compared to enzymatic hydrolysis. The exogenous enzymes of the microorganisms used are utilized for the cleavage of the peptide bonds. Acid and base hydrolysis is the chemical method for protein hydrolysis, with the significant drawback of protein denaturation due to pH changes. The literature has cited many separation or isolation techniques for peptides, including centrifugation, precipitation, electrophoresis, ultrafiltration, and chromatographic techniques. However, many reported investigations have employed chromatographic techniques. Even though other methods are used in food-related research, their usage in the microalgae field is very uncommon.

Many multifunctional properties have been reported in bioactive peptides of sizes ranging from two to twenty amino acids. Among them, antimicrobial, antioxidative, antithrombotic, antihypertensive, and immunomodulatory activities are reported. These peptides have been used as hydrolysates and as isolated peptides to assess their bioactivities. There are several peptides identified as possessing antioxidant capacity, which has been widely studied. The antioxidant activity of peptides derived from microalgae has been investigated to mitigate oxidative stress created by ROSs. Chlorella sp., Navicula sp., and Spirulina sp. are among the widely studied microalgae species for antioxidative biopeptides. It is crucial to identify the amino acid sequences that possess the antioxidant capacity and then evaluate them to produce bulk hydrolysate. With more data regarding the specific sequences derived from microalgae, it is possible to conclude their use in combating oxidative stress-associated diseases. It is also identified that bioactive peptides inhibit ACE in the renin–angiotensin–aldosterone system, indicating their antihypertensive properties. It is also noted that only a few microalgae species have been studied for the extraction of antihypertensive peptides. Among them, Chlorella vulgaris, Chlorella ellipsiodea, Spirulina platensis, and Nannochloropsis oculate are the most prominent. Anti-inflammatory and adhesion molecule reduction properties have been reported in peptides extracted from Chlorella pyrenoidosa and Spirulina maxima.

Through this review, it is identified that long-term human consumption studies on microalgal-based protein and peptide products are lacking; even though they are considered safe due to their natural origin, future research in this area can facilitate the commercialization of microalgal peptide products as food additives or nutraceuticals. Currently, the use of algicidal methods for microalgae cell wall disruption is in the infant stage. Therefore, further research in this area towards safe and sustainable algicidal methods for cell wall disruption may shed light on efficient ways of developing bioactive peptides. For the hydrolysis of protein isolates, fermentation techniques have the potential to develop an economical method compared to the enzymatic method. Therefore, the continuation of research on fermentation applications will help develop new methods to break down microalgal cell walls to extract proteins for health applications. Microalgal peptides need to be further assessed for their bioavailability, antioxidant activity and cytoprotection in targeted tissues and organs. To date, only a few studies have identified the amino acid sequences of microalgal peptides with antioxidant properties. Microalgae peptides have been extensively investigated for their antihypertensive properties. However, further studies are needed to validate the antihypertensive peptides using placebo-controlled randomized human studies. The biostability, bioaccessibility, and bioavailability of microalgae-derived bioactive peptides need to be investigated in detail to establish their full potential in cardiovascular health. In future, the application of bioinformatics, in silico and artificial intelligence tools can be used collectively to predict the production of bioactive peptides using the knowledge derived from microalgae-derived peptides. To expand the market for microalgae peptide-based products, it is important to understand consumer perception toward functional food and nutraceutical products derived from microalgae in future.