The escalating issue of meat inflation, the increasing global population and the need for a nutritionally balanced diet, while minimizing greenhouse gas emissions have all contributed to a surge in demand for protein from plant sources. Since the primary protein sources are derived from animals and the strain on current animal sources is deemed unsustainable, there is an urgent need for novel, sustainable protein sources that are safe and of high nutritional quality. Therefore, the food industry and researchers are tasked with finding alternative protein sources and production methods to satisfy consumer demand and anticipated global protein needs.

While plant-based proteins are nutritionally beneficial, they require land and water, which are becoming increasingly scarce. Moreover, the conversion of plant proteins into meat proteins is inefficient, requiring 6 kg of plant proteins to produce 1 kg of meat proteins (1). Single-cell proteins, mainly composed of dried cells (biomass) produced by algae, yeast, bacteria and fungi, present a viable alternative to traditional protein sources in this context (2). Microalgae, unicellular photosynthetic organisms, are considered promising alternative protein sources (3). They can produce biomass and oxygen by harnessing sunlight as an energy source, CO₂ as a carbon source and inorganic salts as a carbon supply (4).

Microalgae exist in freshwater, marine and edaphic habitats, with some species also found in humid soils (5). Unlike conventional food crops, microalgae can thrive in various conditions without requiring arable land, thus not competing with crops, such as soybeans and cereal grains (6).

The broad spectrum of microalgae includes >100,000 species divided into four distinct groups: Chlorophyceae (green algae), Bacillariophyceae (eukaryotic diatoms), Chrysophyceae (golden algae) and Cyanophyceae (blue-green algae) (7). Despite their vast diversity, microalgae remain under-researched and poorly understood (4). Researchers have found that microalgae are an attractive source for developing beneficial products with significant value in the cosmetics, food and nutrition, medicinal and pollution-prevention sectors (8).

Chlorella spp. was the first algae discovered and isolated in culture by Beijerinck (9). Of note >20 Chlorella species have been identified, with >100 strains documented (10). The exploration of the dietary benefit of Chlorella on human health began in the early 1950s when Chlorella was introduced as a food source during a global food crisis (11). It was first grown and consumed in Asia, mainly in Japan, before gaining popularity as a nutritional supplement worldwide (12).

Chlorella spp. contains protein levels similar to traditional protein sources such as meat, egg, soybean and milk (3). Research has shown that it contains 55-67% protein, 1-4% chlorophyll, 9-18% dietary fiber, and various minerals and vitamins on a dry matter basis (13). In particular, Chlorella vulgaris is known to contain up to 58% protein on a dry weight basis (14) and is considered safe for human consumption by the Food and Drug Administration (FDA) in the USA, along with other microalgae species, such as Arthrospira platensis (Spirulina) due to their long history of human consumption and nutrient profile (15). However, limited reports are available on the protein content and quality of Chlorella sorokiniana (C. sorokiniana) and its application in food. Thus, the present review aimed to evaluate C. sorokiniana biomass as an alternative ingredient for food. The present review aimed to enhance the understanding of the biomass characteristics, bioactive substances, functional components, biomass-based food products, and health advantages linked to the consumption of C. sorokiniana microalgae.

A systematic approach was utilized to select pertinent literature from databases such as Scopus, Web of Science, CrossRef, PubMed and Google Scholar to ensure a comprehensive review. The search for articles included in the present review involved utilizing specific key terms, such as ‘C. sorokiniana Characteristics’, ‘Macronutrients in C. sorokiniana’, ‘Protein Quality of C. sorokiniana’, ‘Micronutrients in C. sorokiniana’, ‘Bioactive compounds in C. sorokiniana’, and ‘C. sorokiniana Food Products’. Subsequently, the databases were used in conjunction with VOSviewer software to conduct a bibliometric analysis (16). Articles were selected based on various criteria, including their relevance to the topic and the presence of substantial reviews or experimental data on C. sorokiniana microalgae.

C. sorokiniana, a freshwater green microalga, is part of the Chlorophyta division. Initially, it was considered to be a thermotolerant mutant of Chlorella pyrenoidosa (17). However, chloroplast 16S rDNA and 18S rRNA profiling in the late 1980s and early 1990s identified C. sorokiniana as a separate species (10). This tiny, single-celled alga, ranging in size from 2 to 4.5 µm in diameter, can grow mixotrophically on various carbon and nitrogen sources, rendering it ideal for waste feedstock cultivation (18).

This chlorophyte has several advantages over other microalgae species. Previous studies have demonstrated that C. sorokiniana can achieve optimal growth at temperatures ranging from 35 to 40˚C (19), with phototrophic doubling times as short as 4-6 h (20). It can also grow under harsh conditions, including wastewater or heavy metal wastes (21). It has been reported to have a more rapid growth in mixotrophic and even heterotrophic conditions, with a preference for sugars, such as glucose or simple organic acids, such as acetate (22).

The typical physicochemical composition and biological value (BV) indicators of the dry biomass of C. sorokiniana, obtained by cultivation in a pilot bioreactor, are presented in Tables I and II. The dry biomass of C. sorokiniana contains an average of 48% protein, which is markedly higher than that of soy, peanut, wheat germ and other animal sources such as beef, fish and chicken (23,24). The lipid content of the biomass sample is 13-22%, comparable to or slightly higher than that of other aquacultures, such as Spirulina spp (25). The biomass is rich in physiologically active phytochemicals, the most abundant of which are chlorophylls (22.13 mg/g dry biomass) and carotenoids (6.04 mg/g dry biomass) (23) (Table III). C. sorokiniana consists of 30-38% carbohydrates on a dry weight basis (26), which is higher than that C. vulgaris, with ~15% carbohydrate content on a dry basis (27).

Factors such as temperature, nutritional composition and light availability can influence the quantities of biomass, macro- and micronutrients, and other useful bioactive substances, including antioxidants, in Chlorella cells (28).

The average macronutrient content of C. sorokiniana biomass based on previous studies (23,31,32) is summarized in Table IV. The analysis of C. sorokiniana dry biomass has revealed that it contains an average of 45% protein, which is much greater than the protein content of foods such as peanut (26%), wheat germ (27%) and other animal sources, such as beef (22%), fish (22%) and chicken (24%) (23,24,33).

The essential amino acid composition of C. sorokiniana biomass is presented in Table V. Notably, these amino acid profiles, which mammals do not synthesize, are present in notable concentrations in C. sorokiniana microalgae; thus, when compared to the recommendations of the World Health Organization for the use of essential amino acids in food, this microalga could be used in the food industry (32). The high protein content and favorable amino acid composition of C. sorokiniana microalgae suggest that this microalga may serve as a potential protein source.

According to Gouveia and Oliveira (25), the lipid content of C. sorokiniana biomass on a dry basis ranges from 11.24-21.6% dry matter. It is equivalent to or slightly higher than other aquacultures, such as Spirulina spp. (4-9% dry matter). As shown in Table VI, ~80% of the total fatty acid content of C. sorokiniana lipids is polyunsaturated fatty acids (PUFAs) (23). Since this microalga biomass contains sufficient amounts of omega-3 PUFAs (~25-28% of total fatty acids) and omega-6 PUFAs (~25-27% of total fatty acids), it can effectively prevent and treat several diseases. Indeed, omega-3 PUFAs, such as alpha-linolenic acid (ALA, C18:3), eicosapentaenoic acid (EPA, C20:5), docosapentaenoic acid (DPA, C22:5) and docosahexaenoic acid (DHA, C22:6) are effective in preventing or treating cardiovascular disorders (34), lowering hypertension (35) as well as preventing cancer (36,37), type 2 diabetes (38), inflammatory bowel disorder (39), asthma (13), arthritis (40), kidney and skin disorders (38,39), depression (13) and schizophrenia (41). Furthermore, due to its lipid composition, C. sorokiniana may also play an essential function in preventing atherosclerosis, hypercholesterolemia and tumors (40).

Another key class of macronutrient substances found in the hydrophilic fraction of C. sorokiniana microalgae biomass comprises carbohydrates. Effectively, 36-39% dry weight of carbohydrates found in C. sorokoniana microalgae biomass are found in the form of sucrose, glucose, xylose and fructose (23,31,42). Moreover, the cell wall of C. sorokinana is also composed of many insoluble polysaccharides, primarily mannose and glucose (43). Polysaccharides are polymeric carbohydrate structures commonly employed in the food industry (44). Owing to their high polysaccharide concentration, microorganisms such as Chlorella microalgae have been actively investigated in the search for novel natural antioxidants (44,45). In the study by Pugh et al (46), immurella polysaccharides, a high molecular weight polysaccharide, were isolated from C. sorokiniana, which presented a higher activity against cancer. Polysaccharides derived from C. sorokiniana microalgae have also been shown to exhibit substantial antioxidant, tumor-fighting, immunomodulatory characteristics, and the ability to remove superoxide and hydroxyl peroxide radicals (47).

The considerable protein content of C. sorokiniana has positioned it as a promising alternative protein source. The protein content in microalgae is typically determined by measuring the total nitrogen and multiplying it by the factor Nx6.25(48). Experimentally determined N-protein factors for specific microalgal species range from Nx3.06 to 5.95 (49,50). This variation in the N-protein factor is attributed to non-protein nitrogen, which comprises up to ~10% of the nitrogen in microalgae and includes substances, such as amines, glucosamines, nucleic acids and cell wall components (48).

Of note, four important quality parameters are considered to assess the nutritional value of algal protein: Protein efficiency ratio (PER), BV, digestibility coefficient (DC) or true digestibility, and net protein utilization (NPU) (14). PER is a fundamental method for evaluating protein quality, involving animal feeding trials with weanling rats throughout 3 to 4 weeks (48). It measures the weight gain per unit of protein consumed, expressed as PER=weight gain (g)/protein intake (g) (48).

BV, another metric for assessing protein quality, is based on the proportion of retained nitrogen to absorbed nitrogen (48). The calculation for BV is BV=[I-(F-F0)-(U-U0)]/[I-(F-F0)], where I represents nitrogen intake, F is fecal nitrogen, U is urinary nitrogen, and F0 and U0 are the amounts of fecal and urinary nitrogen excreted when animals are fed a nitrogen-free or low-nitrogen diet (51).

DC, also known as real digestibility, is another indicator of protein quality. It reflects the proportion of meal nitrogen absorbed by the animal and is calculated using the same parameters as BV: DC=[I-(F-F0)]/I (48). NPU, on the other hand, is determined by dividing nitrogen retained by nitrogen intake. The measure of both digestibility and BV is provided by the NPU, which is calculated using the formula NPU=(B-Bk)/I. Herein, B represents the body nitrogen assessed at the end of the test period in animals fed the test food. Bk represents the body nitrogen measured in another group of animals fed a protein-free or low-protein diet (48).

A comparison between Chlorella spp. microalgae and traditional protein sources based on four quality parameters (PER, BV, DC and NPU) is presented in Table III. The values in the table were compared to casein, the primary protein found in milk, which serves as a reference for scoring. Overall, the values presented in Table III indicate that the nutritional value of Chlorella spp. algal protein is lower than that of beef and casein. However, it is comparable to that of soy protein and superior to that of wheat gluten (52).

Chlorella is an excellent source of vitamins and minerals that adults and children can easily consume to meet their daily vitamin requirements (12). As demonstrated in Table VII, C. sorokiniana microalgae biomass contains the balanced mineral content required in humans. In acceptable amounts, iron, copper and zinc are trace elements necessary for the human body to function normally (74). It is important to note that C. sorokiniana biomass contains substantial amounts of iron (18 mg/100 g dry weight) and zinc (20.2 mg/100 g dry weight), of which adequate intake prevents anemia (75) and muscular weakness (76), respectively. Iron is required for respiration, energy production, DNA synthesis and cell proliferation (77). In previous a study conducted by Nakano et al (78), oral Chlorella supplementation (6 g/day) for 12-18 weeks lowered markers of anemia in a cohort of 32 women in their second and third trimesters of pregnancy compared to the control group. This suggests that Chlorella supplementation considerably reduces the risk of developing pregnancy-associated anemia (79).

Moreover, some of the vitamins found in notable quantities in Chlorella are vitamin A (in the form of β-carotene), vitamin C, vitamin E, Vitamins K and vitamin B, such as thiamine (B1), riboflavin (B2), niacin (B3), pantothenic acid (B5), pyridoxine (B6), folic acid (B9) and cobalamin (B12) (12,44). These vitamins fuel the body, detoxify and restore digestive function, activate the immune system and renew cells (44). Moreover, the vitamin E content of green microalgae Chlorella contributes to its high antioxidant activity (80,81).

Bioactive compounds are nutrients and non-nutrients found in food (both plant and animal sources) that have physiological effects in addition to their nutritional qualities (82). Foods high in bioactive chemicals that are routinely consumed positively impact human health by performing antioxidant activity, reducing the risk of developing diseases such as heart disease, diabetes, cancer, cataracts and stroke (83,84). Microalgae are highly diverse and contain significant bioactive compounds, including polyphenols, carotenoids and organic acids, as presented in Table VIII.

Pigments are essential for the metabolism of photosynthetic microalgae and have antioxidant, anti-carcinogenic, anti-inflammatory, anti-obesity, anti-angiogenic and neuroprotective properties in different metabolisms, including human metabolism (44). Chlorella contains substantial levels of chlorophyll, similar to higher plants, although in markedly higher concentrations than terrestrial plants, with the potential to collect chlorophyll to a concentration of 2.5% (86). Chlorophyll is the principal natural green pigment found in microalgae and plants, where it is needed for oxygenic photosynthesis (87). As presented in Table VIII, C. sorokiniana biomass contains a notable amount of biologically active phytochemicals, with chlorophylls (22.13 mg/g dry biomass) dominating (23). Azaman et al (88) also discovered that under photoautotrophic conditions, C. sorokiniana exhibited a total chlorophyll concentration of 24.37 µg/mg dry weight of the sample, which was more significant than that of Chromochloris zofingiensis.

Carotenoid is a pigment in algae, plants, fungi and bacteria that ranges in hue from yellow to red (84). It has health-promoting qualities are mostly linked to its antioxidant activity that scavenges singlet molecular oxygen and radicals (84). Previous research has shown that carotenoids significantly contribute to the total antioxidant capacity of microalgae (89,90). In particular, Diprat et al (91) identified the carotenoid types present in C. sorokiniana microalgae powder, which includes violaxanthin, lutein, zeaxanthin, β-carotene and α-carotene. Using high-performance liquid chromatography coupled to diode array and mass spectrometry detectors, Fernandes et al (92) also identified 11 carotenoids in C. sorokiniana, with the greatest significant quantities of all-trans-lutein (831.18±1.18 g/g dry weight), all-trans-β-carotene (156.21±0.22 g/g dry weight) and all-trans-α-carotene (71.47±0.10 g/g dry weight). Indeed, including C. sorokiniana biomass as a raw material in functional foods will allow goods to be enriched with essential nutritional components while obtaining the appropriate color range without synthetic dyes (23).

Phenolic compounds, or polyphenolics, are abundant secondary metabolites with antioxidant activity and numerous biological functions that can be derived from microalgae (93). Due to their importance as one of the most significant classes of natural antioxidants, phenolic compounds are gaining popularity among consumers and food manufacturers. They function as reducing agents and hydrogen donors, capable of scavenging free radicals (94). Phenolic compounds have two basic chemical structures formed by one or more aromatic rings with hydroxyl (-OH) groups (93). They are chemically classified into several classes, including phenolic acids (hydroxybenzoic acids, hydroxycinnamic acids), flavonoids (flavones, flavonols, flavanones, flavanonols, flavonols and anthocyanins), isoflavonoids (isoflavones and coumestans), stilbenes, lignans and phenolic polymers (proanthocyanidin) (95).

Based on the Folin-Ciocalteu method, Olasehinde et al (85) reported that C. sorokiniana dry biomass contains a total phenolic content of 12.27±2.31 gallic acid equivalent (GAE) mg/g, which is higher compared to C. minutissima (10.53±2.82 GAE mg/g dry weight) (Table VIII). Likewise, the same authors revealed that C. sorokiniana dry biomass contains a total flavonoid content of 15.05±3.83 quercetin equivalent (QE) mg/g dry weight (85). Furthermore, Safafar et al (96) also presented that C. sorokiniana dry biomass contains 5.84±0.04 GAE mg/g total phenolics and 2.45±0.04 QE mg/g total flavonoid content. Furthermore, they found that C. sorokiniana, grown in different light intensities, had the same phenolic acid profile (96). Still, the total detected phenolic acids in the sample grown in average light intensity was somewhat higher. Thus, generating phenolics and other antioxidant chemicals in microalgae depends on growth circumstances and challenges such as oxidative stress (96).

Apart from having antioxidant properties, phenolic compounds have therapeutic qualities such as anti-tumor and antibacterial activities (84). Chlorella phenolic chemicals may also prevent liver cell carcinogenesis by blocking lipid peroxidation on the cell membrane, neutralizing cellular free radicals and preventing DNA damage (84).

Given the abundance of nutrients and bioactive substances found in C. sorokiniana, it is no surprise that it can be regarded as one of the most promising new food and functional goods sources. C. sorokiniana has a well-balanced chemical composition and provides a source of beneficial bioactive chemicals (13). As a result of its potential, C. sorokiniana can represent an alternative and novel source of natural components that can be used as functional ingredients to improve the nutritional content of foods.

Chlorella sp. has been extensively utilized as a dietary supplement for an extended period of time. The microalgal market offers Chlorella sp. in powder and tablet forms, primarily due to its ability to effectively sequester heavy metals, such as mercury, recognized as toxic substances, and facilitate their elimination from the body (84). In recent times, there has been a surge in research efforts focused on advancing food products derived from C. sorokiniana biomass. Pasta (23) and gluten-free bread (91) have been incorporated with C. sorokiniana biomass for baked food products.

Bazarnova et al (23) examined the effects of incorporating the dry biomass of microalgae C. sorokiniana as a replacement for flour mixture to enrich pasta effectively. In their study, it was shown that flour replacement with C. sorokiniana should not be >5% due to the formation of a distinct fish flavor of the product. Moreover, substituting 5% C. sorokiniana biomass to pasta flour increased proteins to 15.7±0.50% and lipids to 4.1±0.06%. Furthermore, the addition of C. sorokiniana dry biomass to pasta has helped improve the polyunsaturated fatty acids, chlorophyll and carotenoid content of the product (23).

Diprat et al (91) also examined the partial replacement of pea flour with C. sorokiniana powder at 2.5 and 5% to increase the nutritional quality of gluten-free bread. The replacement of 5% C. sorokiniana powder vs. the control with pea flour increased the protein content (67 to 85 mg/g), lutein content (1.6 to 57.5 µg/g) and omega-3 content in the fatty acids (5.0-6.1%) of the gluten-free bread. Furthermore, it is interesting to note that compared with the pasta food product studied by Bazarnova et al (23), the sensory analysis of the gluten-free bread with 5% replacement of C. sorokiniana powder had an acceptance rate >70%, with no observed distinct fishy flavor being identified. Furthermore, the addition of C. sorokiniana powder had no effect on the texture and specific volume of the bread product.

The expanding number of studies on Chlorella as a food ingredient have revealed that Chlorella sp. has a significant potential to be an alternative protein source and future food. However, further product application of C. sorokiniana biomass in food needs to be conducted in order to assess its effects on the physicochemical, sensory, techno-functional and antioxidant properties as a food ingredient. Consumers also strongly demand food to prevent illness, improve mental health and increase life quality, as well as a desire to consume alternative proteins to be healthy and more sustainable. As a result, the worldwide market for Chlorella ingredients is predicted to expand and meet human nutritional requirements (84).

C. sorokiniana microalgae has shown promise as an alternative protein ingredient to meet the increasing demand for sustainable food products. There is a growing consumer interest and awareness regarding alternative protein sources, rendering the use of microalgal protein in developing healthier food products highly relevant. The high protein content, balanced amino acid profiles and bioactive substances in C. sorokiniana make it a potential dietary source with various health benefits. As numerous high-value microalgae products are intended for human consumption or other applications, they are subject to food laws and regulatory standards set by organizations such as the FDA. Collaborative efforts from researchers across disciplines are necessary to expand the market for microalgal food products further. These efforts should focus on identifying novel microalgae species and their unique characteristics, developing more cost-effective cultivation and biorefinery systems, improving the sensory properties of microalgal food products and assessing the availability of algal compounds.