1. Cultivation Methods
Cyanobacteria can be cultivated in open systems (tanks/ponds or raceways) or closed systems (photobioreactors). The high risk of contamination is a critical issue that impedes the mass production of cyanobacterial-phycoerythrin (C-PE) in open systems
[1]. Enclosed photobioreactors are recommended as a preferable choice for pure, large-scale C-PE production since they allow for consistent light provision, temperature control, and carbon dioxide distribution as well as reduced contamination risk
[2].
1.1. Cultivation Mode
Cyanobacteria cultivation can be carried out in a variety of modes, including heterotrophic, photoautotrophic, and mixotrophic settings. Heterotrophic mode is usually performed in dark or harsh environments where cyanobacteria need to acclimate to the environment by consuming available organic compounds (i.e., acetate, glucose) as a carbon source
[3]. Although the organic compound promoted the growth of cyanobacteria, it also increased the potential for contamination, resulting in health risks within food industries
[4]. Photoautotrophic cyanobacteria use natural sunlight as an energy source and atmospheric air as a carbon dioxide supply. As a result, when the light intensity is too high (photo-inhibition) or too low (photo-limitation), the biomass is affected and reduced
[1]. Mixotrophic cultivation combines heterotrophic and photoautotrophic modes; both are performed in the presence of sunlight and carbon sources. Specifically, it is a dual-limiting system in which low light intensities or low organic carbon substrate concentrations can limit cell growth. At the same time, high light intensities or high carbon substrate concentrations can also suppress cell growth
[4]. Previous research has shown that cyanobacteria cultivated in a mixotrophic environment are more resistant to light stress than photoautotrophs
[5]. However, not all cyanobacteria can grow in mixotrophic or heterotrophic conditions
[1].
1.2. Light
Light condition is the major factor that affects C-PE accumulation. This condition involves not just light intensity but also light color and photoperiod. It has generally been documented that there is a preference for low to medium light intensity for C-PE production
[6]. This is related to the role of PE in increasing the range of light absorption in the photosynthetic system. Thus, in low light-intensity conditions, the cyanobacteria will stimulate PE production to capture more light and energy for growth.
In terms of light color, previous research has found that cyanobacteria cultivated under green light accumulate more C-PE compared to other light spectra
[7][8][9]. Under green light, the PSII overexcites and induces a transition to State II, resulting in more excitation energy transfer from the PSII to the PSI. This transfer enables PE to get the green light and use it to activate the photochemistry of both photosystems (PSI and PSII)
[10]. Hence, the light color changed the composition of phycobilisomes via CCA. Hsieh Lo et al. (2019) have discussed the mechanism of complementary chromatic adaptation (CCA), but further study is needed to explore it in detail
[1]. Further, Mishra et al. (2012) postulated that C-PE production could be stimulated under blue light since the light absorption PE ranges from 450 to 570 nm, allowing it to capture more blue light (460–475 mm)
[9][11]. On the other hand, photoperiod affects the balance of heterotrophic and photoautotrophic metabolisms. Although scientific literature has shown that a dark period is always required for phycobiliprotein production, the optimal photoperiod for C-PE accumulation has yet to be determined
[12][13]. Literature has shown that a photoperiod of 16 hours of light and 8 hours of darkness was the ideal condition for phycobiliprotein (PBP) synthesis
[6][13]. Nevertheless, most research on C-PE yield used a photoperiod of 12 hours of light and 12 hours of darkness in their study, despite the fact that this parameter has not been optimized for C-PE yet
[7][9][14][15][16].
1.3. Temperature, pH, and Salinity
Temperature is another major factor influencing cyanobacterial growth and C-PE productivity since it directly impacts cell membrane fluidity, nutrient availability, and absorption
[1][12]. The ideal temperature for target pigment synthesis varies with different cyanobacterial species or strains, depending on their adaptability and tolerance
[1]. Extremely high temperatures denature enzymes or proteins, inhibiting growth and decreasing C-PE production, whereas extremely low temperatures will cause certain cyanobacterial metabolism to malfunction
[17]. Previous studies reported that temperature in the range of 30–36 °C was ideal for the growth of most cyanobacteria
[12]. For example, the optimal temperature of
Anabaena sp. was reported at 30 °C
[7]. Studies to optimize temperature, focusing on C-PE production, still received little attention.
pH fluctuations affect nutrient solubility and bioavailability, the transportation of molecules across cytoplasmic membranes, enzyme activity, photosynthetic electron transport, and cell physiology
[18][19]. Most cyanobacteria have been found to prefer an alkaline environment for growth and phycobiliprotein production. Extreme pH levels will change the charge on protein, eventually leading to protein denaturation
[18]. The ideal pH for cyanobacteria growth is strain-dependent, ranging from 6.0 to 10.0 and never below a pH of 3.0 or exceeding a pH of 12.0
[6]. However, the study of the optimal pH for high C-PE production is still limited to a few strains. The neutral pH of 7.0 is reported to be ideal for high C-PE accumulation in
Nostoc sp. and
Spirulina platensis, while a pH of 8 is suitable for
Anabaena sp. and
Nodularia sphaerocarpa [11][18][19][20].
Salt stress decreases plant growth and metabolite production, which are usually related to lower photosynthesis
[21]. The optimal salinity depends on the strain. Halophilic cyanobacteria can thrive in hypersaline environments, but high salinity can inhibit the photosystem and electron transport chain in non-tolerant strains
[19][22]. Furthermore, excessive sodium ions can cause phycobilisome detachment from the thylakoid membrane, reducing photosynthetic activity and nutrient absorption
[12][23]. Pagels et al. (2019) compiled a few pieces of research that looked into the effect of salinity on the growth and phycocyanin production of marine cyanobacteria
[6]. Yet, there is a lack of research on the effect of salinity on C-PE production. Sharma et al. (2014) optimized the salinity level suitable for
Spirulina platensis and found that a 0.4 M salt concentration is optimal for increasing the C-PE content
[19].
1.4. Nutrients (Nitrogen, Carbon, and Phosphorus)
Nutrient uptake is crucial for cyanobacterial growth and pigment production. Nitrogen is one of the important macronutrients since it is required to synthesize nucleic acids and proteins
[24]. Furthermore, nitrogen, which serves as the primary nitrogen store within the cell, is essential for cell viability and can regulate the production of PBP
[25]. The nitrogen supply in the growth medium influences metabolite production, as it alters the nutrient assimilation mechanism within the cell
[6]. Nitrogen deprivation can impair cellular development rates and contribute to a phenomenon known as chlorosis, which provokes phycobiliprotein (PBP) degradation and leads to photosynthesis downregulation
[25][26]. The type of nitrogen source and the amounts required are species-dependent
[6]. Cyanobacteria have been demonstrated in studies to be capable of assimilating a wide range of nitrogen sources, including nitrate (NO
3−), ammonium (NH
4+), nitrite (NO
2−), and urea. A few research studies have compared various nitrogen sources on C-PE synthesis. Most cyanobacteria (including
Arthrospira platensis,
Fischerella sp.,
Nodularia sphaerocarpa, and
Spirulina maxima) prefer nitrate concentrations ranging from 0.2 to 2.5 g L
−1 [20][27][28][29].
Anabaena fertilissima supplemented with nitrite reported the best C-PE production, whereas an ammonium source was reflected in
Phormidium sp. and
Pseudoscillatoria sp.
[11][27]. The addition of an inappropriate nitrogen source (especially ammonium) will result in a significant influx of ammonium ions and a change in pH, which will be toxic to the cell and reduce C-PE production
[6][27]. Liotenberg et al. (1996), on the other hand, discovered that ammonium and nitrate sources may alter PBP proportion differentially depending on the cyanobacterial strain. The study revealed that the growth of
Calothrix sp. in the presence of ammonium, as compared to nitrate, increased the PC content by 46% but resulted in 35% lower intracellular levels of PE
[30]. Given that a PE monomer contains more tetrapyrrole chromophores than the other two phycobilin monomers, it may be assumed that fewer chromophores are required to construct phycobilisomes in ammonium-grown cells than in nitrate-grown cells
[30][31]. As phycobilisomes constitute a main cellular investment, with phycobiliproteins accounting for up to 50% of the cell’s soluble protein, a lower nitrogen level, per tetrapyrrole, might be more suitable in nutrient-limited conditions
[32]. This statement was consistent with the findings of Hemlata (2009) and Simeunovie et al. (2013) that
Anabaena sp. and
Nostoc sp. produced higher C-PE when no additional nitrogen source was supplied
[12][33]. Further, the nitrogen fixation capabilities of certain cyanobacteria (i.e.,
Nostoc sp.,
Oscillatoria sp., and
Trichodesmium sp.) can be driven when the inorganic nitrogen source is limited and inhibited when the inorganic nitrogen source is abundant
[19][34]. Non-nitrogen-fixing cyanobacteria are normally limited by nitrogen, but nitrogen-fixing cyanobacteria may use ubiquitous atmospheric nitrogen, giving them a competitive advantage in nitrogen-limiting conditions
[35]. Yet, although some cyanobacteria can fix atmospheric nitrogen, it is typically preferable to supply the medium with nitrogen
[6].
Phosphorus has major structural roles in nucleic acids and is functional in various metabolic processes
[25]. A phosphorus deficiency will affect respiration, photosynthesis, and the activities of ATP-dependent enzymes
[25][36]. However, phosphorus itself has little effect on growth and pigment content
[24]. Thus, only a few studies on the correlation between phosphorus and C-PE productivity have been published, and the main phosphorus source comes from K
2HPO
4 [28][37]. Instead, the effect of the nitrogen:phosphorus (N:P) ratio has received great attention
[25][38][39]. N
2-fixation is phosphorus-dependent, as the process needs a high amount of photosynthetically derived energy
[40]. Hence, an optimal N:P ratio is crucial. While the N:P ratio can reveal which of these two nutrients is the underlying limiting factor of cyanobacterial growth and pigment synthesis, it is also a better indicator than each nutrient’s absolute concentration
[38]. The extreme nutrient-limiting condition is usually encountered when the external N:P is less than 20:1
[39].
Carbon is another essential nutrient that contributes to all organic compounds, and a sufficient carbon source enables cyanobacteria to proliferate and accumulate metabolites of interest
[1]. Carbon, like nitrogen, can be utilized by cyanobacteria in both organic and inorganic sources. The flexibility of shifting toward different sources makes the mixotrophic cyanobacteria more independent of light, which does not limit growth as in autotrophic mode, and carbon assimilation can be supplemented by organic compounds, as in the heterotrophic mode. To be more specific, mixotrophic cultured cyanobacteria can use resources more efficiently and produce more biomass by taking advantage of both heterotrophic and photoautotrophic conditions. The higher biomass may cause cells to self-shade, boosting pigment synthesis, such as PE
[24]. The dose of carbon sources varied between strains and was highly dependent on light conditions and culture period
[6]. For example, upon pretreatment with either light or darkness,
Westiellopsis prolifica used exogenous organic carbon more quickly in the light than in the dark incubation
[41]. On the contrary,
Calothrix elenkenii utilizes glucose more quickly in the dark, especially after prolonged incubation
[42]. The culture media of cyanobacteria typically contained NaHCO
3 as the carbon source
[19][37][43]. Most research concluded that sucrose was the best organic carbon source. Sucrose (5 g L
−1)
−1 has been shown to enhance C-PE concentration in
Anabaena azollae,
Anabaena fertilissima, and
Nodularia sphaerocarpa (up to 90%) when compared to fructose and glucose
[11][20][44]. Sugarcane molasses, as an alternate source of pure sucrose, has been found to be the most promising substrate for producing C-PE in
Nostoc sp.
[45]. PBP synthesis may increase in the presence of sucrose due to the production of ATP and higher energy-related assimilation
[42]. The addition of glucose in the medium has also been shown to enhance growth and C-PE synthesis in
Calothrix sp. and
Nostoc sp.
[42][45][46][47]. Furthermore, glycerol can substitute glucose in enhancing PBP synthesis in
Nostoc sp.
[46] Lignite, a low-rank carbon byproduct of numerous carbon extractions, has an organic nature and benefits microbial nutrition
[6]. Hence, its use at a low concentration (0.06 g L
−1) positively affected growth and C-PE synthesis in
Spirulina platensis [48].
2. Downstream Processing of C-PE
2.1. Extraction of C-PE
C-PE downstream processes are typically multi-step and sophisticated in order to assure quantity, purity, and quality
[14]. A proper extraction should allow for the recovery of the greatest amount of C-PE with minimal contamination, which will be valuable for future analyses of its chemical structure and biological activity
[14][49].
The selection of the solvent is primordial for efficient C-PE extraction. The choice of extraction buffer must take into account not merely the polarity and solubility of C-PE, but also the economics, availability of the solvent, and simplicity of use
[6][14]. The phosphate buffer and tris chloride buffer are the common buffer types reported for the extraction of C-PE. These buffers have been widely used for PBP extraction in cyanobacteria and seaweed and have been proven effective
[50][51][52]. An acetate buffer at a pH of 5 is occasionally employed as a buffer for C-PE extraction
[53]. However, research by Julianti et al. (2019) and Zavrel et al. (2018) demonstrated that the C-PC yield and purity extracted with an acetate buffer are lower than that extracted with a phosphate buffer
[54][55]. A comparatively low-cost, double-distilled water was shown to extract the highest quantity of C-PC, with no significant difference in comparison to PC amounts extracted using a phosphate buffer
[56]. However, there is no information on utilizing double-distilled water for C-PE extraction. The pH solvent used in most C-PE extractions was between a pH of 7 and a pH of 8. Dilute or low-molarity buffers could provide better extraction than high-molarity buffers due to the salting-in effect
[14][57]. Since a pH of 8 is not suitable for other proteins, it can avoid undesirable contamination, making crude extract purification easier
[14].
Cyanobacteria cell walls are multi-layered and difficult to disrupt, yet cell rupture is needed to extract the C-PE. The crude extraction can be accomplished using several methods, including enzymatic digestion, high-pressure homogenization, ultrasonication, or continuous freezing and thawing of the biomass
[56]. The methods chosen must consider factors such as the physical strength of the cell wall, stability, and the composition of the cyanobacteria
[58]. Enzymatic extraction can be completed by using lysozyme to enzymatically digest the biomass. This enzymatic hydrolysis is particularly effective in seaweed, which has a strong cell covering or outer sheath to protect the cells
[59]. Nevertheless, as cyanobacterial cells lack special protection, purified enzymes may not be required. Mechanical methods, such as grinding, crushing, homogenization, or sonication, will generate heat transferred to the biomass solvent, causing the C-PE extracts to be denatured even before they are entirely extracted into the buffer
[14][50]. Instead, repeated freezing and thawing were recommended, as it is the gentler approach from the protein perspective and no heat was generated throughout the process
[14]. Thus, it is the most commonly used technique for C-PE extraction
[16][51][60]. Ghosh and Mishra (2020) also concluded that the freezing–thawing method was the best way to achieve the cell disruption of marine cyanobacteria during their screening of several potential C-PE extraction procedures
[14].
2.2. Purification of C-PE
Refined purification can increase the commercial value of C-PE extract. Purifying C-PE is time-consuming and labor-intensive since most cyanobacteria produce PC and allophycocyanin (APC) along with the target PE
[61]. Although the ultrafiltration process improves C-PE purity, it reduces C-PE concentration due to thermal and mechanical shear forces
[60]. Nowadays, PBP is usually purified from crude extract using at least one of the following methods: precipitation, centrifugation, or chromatography. According to Kamble et al. (2018), combining different methodologies is more beneficial for increasing the purity of C-PE while retaining a better yield
[53][60]. The first step can be completed by ammonium sulfate precipitation
[6]. Ammonium sulfate is water soluble at low temperatures, which inhibits bacterial growth and assists in protein concentration and purification
[62]. Different amounts and concentrations of ammonium sulfate were utilized as a salting-out agent to exclude unwanted proteins and boost the purity of C-PE
[60]. The precipitated C-PE was further purified based on size, color, and polarity using chromatography approaches, including size-exclusion and ion-exchange chromatography. Size-exclusion chromatography, also known as gel-permeation chromatography, commonly uses Sephadex as a gel filtration resin to remove high-molecular-weight proteins
[51][52]. Diethylaminoethyl cellulose (DEAE-C), a positively charged resin, was employed in ion-exchange chromatography to eliminate unwanted protein
[52][53][60]. A more developed, aqueous, two-phase system has been used effectively as a low-cost alternative to recover C-PC, but no studies have been conducted to assess its efficiency on C-PE
[63].
3. Strategies to Improve the Stability of C-PE
Another prerequisite for large-scale production and acceptability in the industry is the stability of C-PE
[1]. C-PE is unstable and susceptible from the time it is extracted from the biomass. As C-PE is a protein, pH, temperature, or light changes may modify its molecular structure. For these reasons, C-PE degradation is possible and can be significant during or after the extraction and purification process, resulting in its shorter shelf life
[64]. The pH level is the major factor influencing C-PE aggregation and dissociation, either in monomer, trimer, or hexamer forms in the solution. The most stable structure of C-PE, the hexameric form, predominates at a pH of 7 and easily dissociates at a higher or lower pH
[65][66]. C-PE extracted in the study completed by Ghosh et al. (2020) was stable within the pH range of 3–8. This broad pH range facilitates its use in the food and beverage industries. As most beverages have an acidic pH, a stable colorant under such conditions is beneficial
[14]. Further, it is recommended to keep extracted C-PE at a low temperature. Temperatures exceeding 40 °C reduce the number of alpha helices, resulting in a loss of stability and gradual degradation
[67]. It is also not suggested to keep C-PE at temperatures above room temperature since C-PE is more sensitive to deterioration by microorganisms
[65]. Sub-zero temperatures are favorable for long-term preservation since they restrict protein water activity to a minimum level
[14]. Furthermore, it is preferable to keep C-PE in the dark
[1]. Long-term exposure to high light intensity causes C-PE to lose its chromophores, resulting in color degradation and stability loss
[67].
Several stabilizing approaches or strategies have been introduced to increase C-PE stability, as outlined by Hsieh-Lo et al. (2019)
[1]. The addition of effective preservatives is the most popular and convenient method. Although dithiothreitol (DTT) and sodium azide (NaN
3) are commonly employed in C-PE for analytical purposes, both are poisonous and not suitable for use in food
[66]. Thus, only edible preservatives can be used to produce food-grade C-PE. According to Mishra et al. (2010), the stability of C-PE treated with citric acid is higher than that treated with sucrose, calcium chloride, or sodium chloride as preservatives
[66]. Benzoic acid is another useful edible additive. It has potent antioxidant properties that can preserve and enhance the stability of C-PE while also acting as an antibacterial agent that inhibits bacterial growth
[64].
Crosslinking is another non-additive technology used effectively on PE to increase stability
[68]. It is a method of reinforcing the folded structure of a protein by covalently joining two chemical groups on its surface using bifunctional reagents, either intermolecularly or intramolecularly
[69]. The intramolecular crosslinking of the protein molecule with silver nanoparticles, Ag
+, has been shown to prevent protein aggregation and enhance the thermal stability of PE
[68]. Other approaches, such as complex formation and microencapsulation, have been established on PC; however, more study is required to determine the efficacy of employing such techniques on PE
[70][71][72]. At present, most of the established strategies have focused on enhancing thermal stability. Hence, stability regarding other factors needs to be explored so that extracted C-PE may be widely marketed across various fields.
4. Application of C-PE
4.1. PE in Pharmaceuticals, Nutraceuticals, & Therapeutics
Several bioactive compounds, such as zeaxanthin, α-tocopherol, and caffeic acid, have already been employed in disease prevention and treatment. However, cyanobacterial bilin is more effective than other phytochemicals, attracting the attention of researchers
[73]. The study of PBP bioactivities has emerged in recent years, albeit such study is still restricted and mainly focused on PC
[6]. Indeed, PE displays a variety of bioactivities that allow it to be used in the nutraceutical, pharmaceutical, and therapeutic industries.
The accumulation of reactive oxygen species (ROS) causes oxidative stress, which stimulates organisms to produce bioactive compounds in response to their defensive mechanisms. An enzymatic or non-enzymatic antioxidant approach can neutralize these ROS. PBP is an example of a non-enzymatic antioxidant mechanism that scavenges ROS and reduces oxidation
[6]. Sonani et al. (2014) discovered that C-PE isolated from
Lyngbya sp. exhibited higher in vitro, dose-dependent, antioxidant activity than C-PC and C-APC
[52]. This might be because C-PE exhibits antioxidant activity through the main pathway, scavenging already produced ROS via a redox reaction, and has lower chelating and reducing ability
[74]. Other potential antioxidant properties of C-PE still require further investigation. In addition to its antioxidant capabilities, C-PE from
Lyngbya sp. also had anti-Alzheimer’s and anti-aging properties
[52][74]. Phycobilins, such as PCB, PEB, PUB, and PVB, have also been potent phytochemical inhibitors of SARS-CoV-2 Mpro and PLpro proteases
[75].
Anti-tumor activities and underlying mechanisms were primarily proven in the literature employing C-PC in medications
[6][76]. C-PE differs structurally and spectroscopically from C-PC, suggesting that it may potentially have unique anti-tumor properties
[77]. R-PE has been incorporated into photodynamic therapy (PDT), a treatment method for lung, stomach, skin, and oral tumors
[77][78]. Effective PDT can selectively destroy cancer or tumor cells by producing ROS-mediated damage, vascular damage, and immune system activation while causing no damage to normal cells
[77]. R-PE and its subunit had a stronger PDT effect as well as an inhibitory effect on human liver cancer cell SMC 7221 and mouse tumor cell S180. The smaller size of the β-subunit allows it to access the tumor cell easily. Furthermore, it emitted more significant fluorescence, which was employed as a fluorescent marker for detecting binding sites
[78]. In this regard, future research could focus on the role and dosage of C-PE in tumor therapy.
Oral treatment with C-PE as a hepatoprotective and neuroprotective drug has ameliorated kidney, redox, hepatobiliary, and hepatocellular biomarkers against CCl
4-induced toxicity in rats. It was determined that proteolytic enzymes might break down C-PE in the gastrointestinal system into bilirubin and low-molecular-weight proteins, mediating the pharmacological effects
[79]. Furthermore, C-PE was shown to alleviate diabetes complications by lowering oxidative stress and oxidized, low-density, lipoprotein-induced atherogenesis in streptozotocin-induced type 2 diabetic mice. C-PE administration decreased organ weights, food consumption, cholesterol concentrations, and serum concentrations of glucose and raised body weight, bilirubin, total protein, and the ferric-reducing capacity of plasma values
[80]. Also, hepatic and renal tissues showed substantial reductions in lipid hydroperoxide, thiobarbituric acid reactive substances (TBARS), and conjugated diene contents, with elevations in superoxide dismutase, catalase, and glutathione peroxidase, and reduced glutathione, vitamin C, and vitamin E levels
[77][80]. Although C-PE administration has become widely used, the detailed mode of action of C-PE in many diseases remains unknown. Further study is needed to elucidate the action point of C-PE in different metabolic pathways.
4.2. PE in Cosmetics
C-PE has long been recognized as an excellent quencher of various oxygen derivatives. Due to this capability, phytopigment of
Spirulina sp., such as C-PC, PCB, and PEB, are thought to be excellent antioxidant, anti-wrinkle, anti-melanogenic, and anti-aging agents, as well as natural, non-toxic colorants in eye shadows, eyeliners, and lipsticks
[81][82][83]. The majority of the PE content in commercial cosmetics and skin care products is in the form of B-PE or R-PE; however, there is no data on PE derived from cyanobacteria
[81][84][85]. For example, purified R-PE isolated from
Colaconema formosanum demonstrates anti-aging and anti-allergic properties without toxicity on several mammalian cell lines and epidermal tissues, indicating that this compound has potential for cosmetics usage
[84]. Various cyanobacteria strains are now commonly utilized in skin care products to treat various skin problems by functioning as anti-wrinkling agents, sunscreens, nourishing moisturizers, whitening agents, or texture enhancers
[83][86]. Hence, it is envisaged that C-PE, which has a range of beneficial bioactivities, will be included in cosmetics or skincare products.
4.3. PE in Food and Feed Industries
By appealing to consumers’ growing health awareness, PE with natural colorant properties has become an alternative to the use of synthetic dye
[2]. For example, the pink colorant exhibited by PE has been added as a dye into dairy products, such as milkshakes and yogurt, to make processed products more enticing and to provide color to otherwise colorless food
[87]. Red phycoerythrin has a unique yellow fluorescence. Transparent lollipops prepared from sugar solution, soft drinks, dried sugar-drop sweets for cake decorating (that fluoresce under UV light), and fluorescent alcoholic beverages that exploit the benefits of these spectral properties are still being researched
[88]. Another pivotal reason for using PE as a dye is that it has antioxidant properties that add value to processed food products. Phycoerythrin is famous as an antioxidant and anti-inflammatory drug, with features that are able to avert ROS-related abnormalities, protect against physiological changes caused by oxidative stress, and have anti-aging benefits
[6][89]. This substance also has fewer side effects than synthetic additives or chemical drugs. However, C-PE uses in food products need to be explored further and corroborated through toxicity testing. Further, more study on the bioavailability and interaction of diets and C-PE is necessary
[87].
Several studies have demonstrated the benefits of using PE as feed. For example, Lee et al. (2021) found that PE can variably increase the immunological response of whiteleg shrimp in vitro and in vivo and that it might be applied as an immunomodulator in shrimp production
[90]. Dietary PE supplementation may also modify the gut microbiota to improve intestinal nutrition and disease resistance in animals by increasing the beneficial bacteria and reducing the prevalence of harmful bacteria within the intestine
[91].
4.4. PE in Detection, Diagnosis, and Biotechnology
Compared to monomers, the high molar extinction coefficient of PE is ascribed to trimeric and hexameric packing, as well as the presence of open-chain tetrapyrrole chromophores covalently attached to them
[2][90]. The unique fluorescent features of PE have broadened its applications as a fluorescent label in immunoassays, flow cytometry, cell biology, and fluorescence microscopy for biomedical research and diagnostics
[92][93][94]. Furthermore, the staining of the PE-labelled monoclonal antibody probe was more clear and bright than the PC-labelled probe when detecting invasive amebiasis
[95]. PE can also be employed as a marker in electrofocusing or gel electrophoresis
[96] and as a photosensitizer in cancer therapy
[97].