Bioactive substances and potentiality of marine microalgae

Abstract Microalgae is one of the most important components in the aquatic ecosystem, and they are increasingly used in food and medicine production for human consumption due to their rapid growth cycle and survival ability in the harsh environment. Now, the exploration of microalgae has been gradually deepening, mainly focused on the field of nutrition, medicine, and cosmetics. A great deal of studies has shown that microalgae have a variety of functions in regulating the body health and preventing disease, such as nitrogen fixation, antitumor, antivirus, antioxidation, anti‐inflammatory, and antithrombotic. Furthermore, microalgae can synthesize various high‐valued bioactive substances, such as proteins, lipids, polysaccharides, and pigments. In this paper, we have briefly reviewed the research progress of main bioactive components in microalgae, proteins, lipids, polysaccharides, pigments, and other nutrients included, as well as their present application situation. This paper can provide the guidance for research and development of industrial production of microalgae.

including pigments, polysaccharides, or polyunsaturated fatty acids (PUFAs), they have applications in many industries, food, health products, animal husbandry, aquatic feed, and cosmetics. Furthermore, there is growing interest in biomacromolecules as a potential drug, marine organisms being a great source. Hence, microalgae could be included in screening programs for promising bioactive substances.
Microalgae have much higher photosynthetic efficiency and are not sensitive to the seasons compared with higher plants, which allows for high yields of valuable molecules such as proteins, lipids, carbohydrates, and pigments, combined with the diversity of microalgae (Tarento et al., 2018;Welladsen et al., 2014;Yan et al., 2016). Next, microalgae can grow under less demanding conditions, such as wastewater or nonagricultural land. Moreover, microalgae can minimize associated environmental impacts by recycling atmospheric carbon dioxide.
In terms of food, resource, and environmental challenges, microalgae have a wide range of applications. At present, microalgae biotechnology is steadily rising, divided into four main research areas, including wastewater treatment, carbon dioxide sequestration, biofuel production, and high value-added active substance production. The first three areas have been well explored and reviewed over the past few decades, and now a recent shift occurs in the focus of microalgae applications, where producers focus primarily on the production of high value-added components rather than environmental applications. High value-added ingredients from microalgae had a very broad category, ranging from lipids, proteins, and carbohydrates with food and nutritional applications to dyes and sterols with cosmetic and pharmaceutical applications. Hence, the core content of this paper summarizes the production, application, and potentiality of high value-added active substances and points out the direction for future producers.
Moreover, marine microalgae are not only an essential part of the marine biological resources but also a major generator within the marine ecosystem. They play a crucial role in the material cycle and energy flow of the marine ecosystem and affect the productivity of the entire ocean system in direct or indirect ways, closely related to aquaculture, fishery resources, and geological and environmental protection.

| PROTEIN S
Marine microalgae have attracted attention because of its abundance, relatively inexpensive process, and a rich source of proteins and peptides with biological function (Rashidi et al., 2019;Zhang et al., 2019). Marine microalgae contain many active proteins with physiological functions. The main functions of these active substances include diazotrophic, antitumor, antioxidant, and immunestimulating activities. They can be used as components in medicinal cosmetics, and their bodies can be used as effective adsorbents for heavy metals in the ocean (Cornish & Garbary, 2010;Lee et al., 2013).
Generally, the main components in microalgae constitute of carbohydrates, proteins, and lipids. Due to the differences among microalgal species and cultivation conditions, the proportions of cellular components (carbohydrate, lipid, and protein) in microalgae can differ greatly. Marine microalgae are high in protein content, that is, 6%-70% of its dry weight (DW), and a majority having about 50%, as shown in Table 1. High protein content gives microalgae a high nutritional value. Thus, microalgal proteins can be used as a supplementary source of dietary protein, especially in some developing countries. Furthermore, the protein could be used as ingredients in healthy foods and even functional foods. With the continuous advancement in technology, active proteins from microalgae showed great potential and have become one of the research focuses.

| Phycobiliproteins
Phycobiliproteins, the water-soluble pigment-protein complex, exist in Cyanobacteria as well as some algae. The complex is formed based on apoprotein and covalently bound phycobilins serving as the chromophores. Phycobiliproteins are classified into four groups: phycocyanin (PC), phycoerythrin (PE), allophycocyanin (APC), and phycoerythrocyanin (PEC). PC was isolated from Spirulina and mainly exists in Cyanophyta, Rhodophyta,and Cryptophyta [80]. It is usually divided into C-phycocyanin (C-PC) and R-phycocyanin (R-PC), where the former is found in Cyanophyta, the latter in Rhodophyta, and both in Cryptophyta. PC is a protein-pigment complex that comprises of protein and nonprotein components, which participate in various biological effects, such as antioxidant, anti-free radical, and antitumor activities (Eriksen, 2008). PC can also prevent oxidative stress (OS) through scavenging reactive oxygen species (ROS) as well as reactive nitrogen species (RNS). Romay, Armesto et al. (1998) were the first to report that PC possessed antioxidant property and also confirmed that it could scavenge superoxide anion and hydroxyl, alkoxyl radical. Besides, the effect of PC on inflammation was first described by these authors (Romay, Ledón et al., 1998). In addition, Liu et al. (2015) investigated the recovery effects of PC on the oxidative damage in mice caused by X-ray radiation, and results proved that PC could effectively protect the body's antioxidant system and improve its antioxidant capacity. Many diseases have resulted from oxidative damage to the human body. PC could thus act as a nutraceutical in the clinical practice. Also, phycocyanin, as a natural blue pigment, has been widely utilized in cosmetics, such as lipstick, eyeliner, nail polish, or eye shadow (Jahan et al., 2017).

C-PC, a natural blue pigment, is frequently found in
Cyanobacteria, and R-PC is isolated from red algae (Glazer, 1989).

C-PC exhibits an antitumor effect. C-PC from Oscillatoria tenuis
showed antioxidation and antiproliferation activity on human tumor cells via inducing cell apoptosis, including the representative apoptotic characteristics, such as cell contraction, membrane blebbing, condensed nucleus, or DNA fragmentation (Thangam et al., 2013). In addition, C-PC suppressed HepG2 (human hepatoma cells) growth and multiplication at 7.0 µg/ml with an LC50 under 1.75 µg/ml (Basha et al., 2008). Cyclooxygenase 2 (COX-2) is one of the induced enzymes with high expression in inflammatory and cancer cells. COX-2 has been recently discovered to be related to colorectal cancer (CRC), breast cancer (BC), and gastric cancer (GC) (Cheng & Fan, 2013;Auberdiac et al., 2011;Liu et al., 2017). Saini and Sanyal (2014) suggested that C-PC, an inhibitor of COX-2, patients with asthma. Moreover, Liu et al. (2015) determined that R-PC had antiallergy potential after an experiment on mice or mast cells sensitized by antigens. It was also found that R-PC effectively reduced tropomyosin and histamine levels and lowered interleukin 4 (IL-4) and IL-13 levels in mice. In addition, this protein also inhibited pro-inflammatory factors and allergy markers, for example, suppressing the production of IL-4 together with tumor necrosis factorα (TNFα), decreasing the histamine, β-hexosaminidase, or ROS release in cells.

| Collagen-like protein
Collagen accounts for the major structural protein in the space outside cells of different animal connective tissues that showed extensive applications ranging from foods to medicines. It can also be adopted for burn or cosmetic operations (Jérome et al., 2015). There to form blooms covering the ocean extensively (Layton et al., 2008).
The collagen-like protein from marine microalgae could thus be potentially used for cosmetic and medical formulation.

| Diazotrophic protein
Some primary groups among nitrogen (N 2 ) fixation microorganisms were studied. The N 2 fixation microorganisms were less diverse based on nitrogenase gene (nifH) diversity. Most nifH sequences retrieved can be divided into two categories, including one constituted by Trichodesmium sequences and other by the α-proteobacterial group. At present, Trichodesmium is the highest abundant diazotroph, with as high as 6 × 10 5 nifH gene copies/L (Moisander et al., 2008).
N 2 fixation is catalyzed via the oxygen-sensitive nitrogenase.
However, there is no study on the mechanism of the nitrogenase from Trichodesmium.
Cyanobacteria represent the only diazotrophic microorganism producing O 2 as the photosynthetic by-product. Similar to eukaryotic algae and higher plants, they have chlorophyll, photolyze water, and release O 2 . It has been reported that one N 2 fixation cyanobacteria, Cyanothece 51142, contained the highest complete adjacent N 2 fixation-associated gene cluster (Welsh et al., 2008). In 1970, soluble nitrogenase was first observed in vegetative cells of the blue-green alga, Anabaena cylindrica (Smith & Evans, 1970). In 1979, the nitrogenase complex was originally separated by N-starving Anabaena cylindrica culture. The Mo-Fe protein having subunit composition, molecular weight (220,000 Da), isoelectric point (4.8), Mo (2 mol/ mol), S 2− (20 mol/mol), and Fe (20 mol/mol) contents, as well as amino acid (AA) composition similar to components that contained Mo and Fe, was subjected to homogeneous purification and separated based on additional bacteria (Hallenbeck et al., 1979). Furthermore, diazotrophic cyanobacteria contribute to human health in two main ways. On the one hand, diazotrophic cyanobacteria enhance crop productivity in an environmentally friendly and economically viable manner. On the other hand, diazotrophic cyanobacteria are widely used as biofertilizer and soil conditioner to improve grain output, plant growth, fruit quality, and nutritional characteristics.

| Bioactive peptides
Bioactive peptides (BP) as a specific protein fragment play a vital role in the physiological activity of most living organisms. BP was reported to have several therapeutic activities, including antioxidant, anti-inflammatory, antitumoral, antiproliferative, antihypertensive, and antimicrobial properties, as shown in Table 2. Peptides derived from microalgae are known to show high potential in the fields of functional food, medicine, and cosmetics on account of their selectivity, efficacy, safety, and good tolerance once consumed. Furthermore, the interest in the microalgae- Several studies have also demonstrated that peptides from microalgae had antitumoral and antihypertensive activity ( Table 2).

| LIPIDS
Microalgal oil has attracted much attention due to its short breeding cycle, its ability to capture carbon dioxide and accumulate a large Peptides from Vischeria helvetica KGU-Y001 had molecular weights lower than 400 Da and high ACE inhibitory activities Aburai et al. (2020) Peptides from Chlorella sorokiniana showed ACE inhibitory activity Lin et al. (2018) TA B L E 2 The biological activity of bioactive peptides from different strains amount of lipids, and its rich omega-3 long-chain polyunsaturated fatty acids. As shown in Table 3, the fatty acids of microalgae oil mainly include saturated fatty acids, monounsaturated fatty acids, and polyunsaturated fatty acids. Usually, polyunsaturated fatty acids in microalgae oil a large percentage of the total fatty acid content, especially the docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Polyunsaturated fatty acid (PUFA) is a fatty acid that contains more than two double bonds in its backbone. According to the position of the first unsaturated bond, it is divided into ω3, ω6, ω7, and ω9, from which ω3 and ω6 play a vital role in body physiological function modulation. ω3 fatty acids have anti-inflammatory, antithrombotic, antiarrhythmic, lowering blood lipid, and vasodilatation characteristics and are also essential for fetal and infant development, especially the brain and vision development. The main components of ω3 include alpha-linolenic acid, 20-carbon-5-enoic acid (EPA) along with 22-carbon-6-enoic acid (DHA). The human body does not synthesize these three PUFAs and thus must be absorbed from the diet (Dyerberg & Jørgensen, 1982;Garg et al., 2006). Fishes or microalgae are the primary sources of ω3 PUFAs; microalgaederived PUFAs being cheaper than fishes. Additionally, compared to fish oil, ω3 PUFA extracts from microalgae are odorless and do not contain cholesterol (Mimouni et al., 2012).
In the last few years, the study of microalgal PUFA has gathered interest increasingly. Jiang and Gao (2004) reported that reducing the growth temperature of a marine diatom, Phaeodactylum tricornutum, remarkably increased EPA and PUFAs yields. It indicated that P. tricornutum could be utilized as an aquacultural food organism as well as the candidate EPA source. Ryckebosch et al. (2014) assessed the nutrient contents of the total lipids isolated from the diverse PUFAs generated via microalgae. They used the microalgae, Isochrysis sp., Nannochloropsis sp., Phaeodactylum sp., Pavlova sp., and Thalassiosira sp. to produce ω3 PUFAs as a fish oil alternative in foods. Skeletonema costatum, Chaetoceros calcitrans, Porphyridium cruentum, as well as Nannochloropsis sp., showed high eicosapentaenoic acid (EPA) levels (Servel et al., 1994). High PUFA contents in these microalgae make them a good supplement for humans. In addition, Food and Drug Administration examined the safety of DHA and oils rich in AA after extraction from Crypthecodinium cohnii Javornicky single-cell organism, a heterotrophic marine microalga, and recommended their use as supplements for infants (Martins et al., 2013). Because fish oil cannot satisfy the growing requirements of purified PUFA, largescale cultivation of microalgae is an excellent alternative. Different microalgal strains (149), in particular diatoms, were constructed to be the stock cultures, among which 20 were screened for growth rate and EPA/DHA contents, indicating their potential use in largescale production (Steinrücken et al., 2017).

| Antioxidant activity
The recommended dietary intake of ω3 PUFAs for Chinese popula-

| Antimicrobial activity
Microalgae has the potential to limit microbial infection in aquaculture and has great application prospect as a natural antibiotic.

| Anti-inflammatory activity
Long-chain PUFA has therapeutic effects on diverse inflammatory diseases, like lupus, arthritis, or Alzheimer's disease (AD). Several

| P OLYSACCHARIDE S (PS)
Several species of microalgae produced PS, sulfated exopolysaccharides in particular (Table 4), containing sugars like glucose, an aldose, galactose, and xylose (Raposo et al., 2013). PS in microalgae includes fucoidans, carrageenans, alginates, and exopolysaccharides. They were detected in microalgae as cell wall components, one part in cells peripheral to glycocalyx or one of the polymers outside the cells (or exo-polysaccharides, EPS) (Table 4).

| Antitumor activity and immunoregulation
The potential activity of PS is that it prevents the growth of cancer microalgae, Gyrodinium impudicum strain KG03. p-KG03 showed immunostimulation activity and promoted the tumor-killing effects of natural killer cells and macrophages as well as suppressed tumor cell growth in vivo (Yim et al., 2005). Geresh et al. (2002) suggested that "Oversulfated" EPS (with >20% sulfate level) with a high molecular weight at a concentration of 200 µg/ml could suppress the growth of 80% mammalian cells. Gardeva et al. (2009)

| Antiviral and antibacterial activities
Antiviral activity of the sulfated polysaccharides may be the most extensively investigated quality of marine microalgae. A marine microalga, Cochlodinium polykrikoides, generated sulfated polysaccharides outside the cell (Hasui et al., 1995). These PS constituted of mannose, galactose, glucose, and uronic acid, as well as the sulfate groups (S = 7%-8% w/w). They suppressed cytopathic impacts of influenza virus types A and B on Madin-Darby canine kidney cells, as well as respiratory syncytial virus types A and B on HEp-2 cells, and human immune deficiency virus type 1 on MT-4 cells (Hasui et al., 1995).

| Digestive performance
Dvir and colleagues reported that Sprague-Dawley (SD) rats fed with PS from Porphyridium sp. caused a significant increase in feces bulk in animals, whereas decreased gastrointestinal transit time (Chayoth et al., 2000). Soluble EPS isolated from Porphyridium sp. may decrease the plasma and duodenal mucosal contents of blood lipid along with cholecystokinin, but increase fecal excretion of bile acid and neutral sterol. In addition, PS released by such red marine microalgae also induced morphological modifications within the colon and small intestine in SD rats. A notable decrease in goblet cell count in the mucosa layer was observed, along with increased fecal content viscosity. Moreover, the Jejunal tunica muscularis morphology showed enlargement. These morphological modifications could have taken place to overcome the nutrient and mineral malabsorption problems (de Jesus Raposo et al., 2016).

| PI G MENTS
Natural pigments play a vital role during algae photosynthetic metabolism and pigmentation. Microalgae account for the primary photosynthesizers that generate several essential pigments, including chlorophyll, β-carotene, phycobiliproteins, astaxanthin, and xanthophylls with applications in food, nutraceutical, pharmaceutical, aquaculture, and cosmetic industries (Table 5). Chlorophylls, carotenoids, together with phycobiliproteins, are the three primary classes of photosynthetic pigments of microalgae (Begum et al., 2016).
Most pigments are biologically active, with antiobesity, antiinflammatory, anticancer effects, mainly due to their potent antioxidant activity employed to protect the body from OS (Ciccone et al., 2013;Guedes et al., 2011). Phycobiliproteins are widely used in industrial and immunological research. As fluorescent markers, they are commonly used in immunoassays and as fluorescent dyes in molecular biology (Banskota, Roumiana et al., 2013;Banskota, Stefanova et al., 2013).
Chlorophyll is a fat-soluble pigment with a porphyrin ring, which accounts for 0.5%~1% of the dry weight of microalgae. Studies have found that chlorophylls have physiological effects like antimutagenesis and anticancer. Carotenoids, as essential nutrients, are mainly used in dietary supplements, fortified foods, food dyes, animal feeds, pharmaceuticals, and cosmetics (Vílchez et al., 2011). Carotenoids also show biological effects by affecting cell growth modulation, modulating gene level, or immune response (Kalra et al., 2020;Nascimento et al., 2020).
Epidemiological studies showed that increased carotenoid consumption and tissue level were related to the decreased risks of malignancies and cardiovascular diseases (Rock, 1997). And it has been widely used as a sophisticated health product due to its activity to protect the heart and liver. Carotenoids have also been used in creams and lotions for sun protection as stabilizers and preservatives (Del Campo et al., 2000;Xhauflaire-Uhoda et al., 2008). Humans and animals cannot synthesize carotenoids and rely on their diets for these essential nutrients. Thus, microalgae have been suggested to be a candidate source for carotenoids.

| Chlorophylls
There are five types of chlorophyll in microalgae, including chlorophyll a, b, c, d, and e. Chlorophyll a is the primary photosynthetic pigment, abundant in Cyanobacteria and Rhodophyta. Chlorophyll b exists in Chlorophyta and Euglenophyta, while chlorophylls c, d, and e are found in the freshwater diatoms (Begum et al., 2016). Spirulina sp. is the most important chlorophyll a source, producing 2-3 times more chlorophyll than other plants. In addition, the chlorophyll in Spirulina contains porphyrin, similar to the heme in human and animals. It is a direct supplement of the heme in humans and animals, and thus, chlorophyll a is called "green blood." Since Spirulina is rich in iron, the perfect combination of chlorophyll a and iron is the best treatment of iron deficiency anemia (Chamorro et al., 2002).
Recently, chlorophyll has drawn significant attention for being used as a cancer preventative agent. Ferruzi and Blakeslee (2007) reported that the biological effects of the chlorophyll derivatives conformed to cancer prevention, including antioxidant and antimutagenic activities, mutagen trapping, xenobiotic metabolic regulation, and apoptosis induction.

| Carotenoids
Carotenoids are the organic pigments produced by plants and algae, as well as several bacteria and fungi, but not synthesized in ani- There are over 1,100 carotenoids in nature, among which βcarotene is the most common (Yabuzaki, 2017).  (Rodrigues et al., 2012;Sachindra et al., 2007).
Astaxanthin is a kind of carotenoid not belonging to provitamin A. Haematococcus pluvialis is the primary source of astaxanthin. In addition, Chlorella zofingiensis, Chlorococcum sp., Neochloris wimmeri, Catenella repens, and Coelastrella striolata have also been reported to contain astaxanthin in lower content. The antioxidant performance of astaxanthin is better than that of β-carotene, zeaxanthin, canthaxanthin, vitamin C, and vitamin E. It can relieve photooxidative stress and inhibit photosensitive action. In addition, astaxanthin proved to have a strong anticancer effect, which could reduce the number and size of liver cancer and lung tumor lesions, and it also has an obvious inhibitory effect on bladder cancer, oral cancer, and colon cancer cells (Sathasivam & Ki, 2018).

| VITAMIN S AND MINER AL S
Microalgae are abundant sources for almost all important vitamins and minerals and rich in Cu, I, Fe, K, Zn, etc. (Christaki et al., 2011).

| CON CLUS ION
Previously, microalgae have attracted the interests of researchers because of their multiple sources and health benefits. This review highlights the bioactive substances and biological properties of marine microalgae and also illustrated the recent studies concerning biological activity and function. The bioactive substances include proteins, polysaccharides, pigments, and lipids. They demonstrate a series of physiological and pharmacological activities (e.g., nitrogen fixation, antitumor, antivirus, antioxidation, anti-inflammatory, and antithrombotic) with applications in some medical and industrial fields. These bioactive substances from marine microalgae provide new and vast sources for the development of human health care and industrial production.
Although there are many advantages in using microalgae to produce high-value products, its economic cost is too high due to a series of factors, such as low content of microalgae, low biomass, and much difficulty in harvesting, which greatly limited the utilization of microalgae resources. With the rapid development of synthetic biology, the use of this technology in microalgae artificially builds high-value biosynthetic pathways of natural compounds to realize the goal of synthetic products (Saini et al., 2020). This is a faster and more economical way of producing. For example, in terms of increasing the yield of natural biosynthetic vitamin E, scholars mainly focus on increasing the yield of plant plants through metabolic engineering (Sproles et al., 2021).
The combination of technological and economic factors allows us to analyze the current structure of the microalgae industry and make some predictions. In the nutraceutics perspective, proteins and peptides based on microalgal have a wide range of applications that can produce high value-added products and even use their properties to attract the cosmetic or pharmaceutical industries. The demand for DHA of consumers would greatly boost the production of PUFAs from microalgae. The market supply of PUFAs is still guaranteed by fish oil, but the PUFAs industry is likely to be one of the main drivers of microalgae to produce high value-added products in the future. Microalgae polysaccharides are also attractive to the cosmetics industry because of their properties, for example, pushing cosmetic sectors toward other texturing agents. In addition, microalgae can be considered to be used for dyeing in the food industry because microalgae are rich in pigments.
However, β-carotene and astaxanthin possess diverse bioactive activities and will yield a variety of health benefits, with potential applications mostly in the cosmetics and pharmaceutical industries. In the future, microalgae are likely to be used not only in the food and feed industries, but also in the nutrition, health care, cosmetics, and pharmaceutical industries. Whether focusing on nutrition/health products or cosmetics/ pharmaceuticals, the microalgae industry will be market-oriented. In order to conform to this market trend, it has some challenges, such as it needs to select relevant varieties in the early stage of cultivation, and producers will need to start from product development. Given the diversity of microalgae, this may not be an easy choice. Moreover, active substances of microalgae are extracted, produced and priced according to their different positive effects, and its industrialization development technology also needs further in-depth study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.