ALGAE CAROTENOID PIGMENTS AS NEW SOURCES OF HALAL BIOACTIVE INGREDIENTS

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Introduction Microalgae, which are single-celled microorganisms, are considered to be a rich source of diverse bioactive molecules. They play a vital role in the aquatic food chain as primary producers and can store complex organic compounds in their bodies, which can be released with the help of sunlight (Chacon-Lee & Gonzalez-Marino, 2010; Batista et al., 2013). Microalgae have evolved distinctive metabolic pathways, resulting in the production of remarkable secondary metabolites and unique structures that differ from those found in superior plants. Due to the high structural diversity of pigments, microalgae have the potential to produce pharmacologically valuable compounds, making them a promising source of bioactive molecules (Rodrigues et al., 2015).
Some of these microorganisms produce two families of pigments, namely tetrapyrroles and carotenoids. Cyanobacteria, for example, contain closed tetrapyrroles such as chlorophyll a, and β-carotene, as well as two types of accessory pigments: open tetrapyrroles (phycobilin) and glycosylated xanthophylls (Chen & Blankenship, 2011). Because the compounds derived from microalgae are structurally and bioactively

A B S T R A C T
Halal Active Pharmaceutical Ingredient (API) is a substance used in a finished pharmaceutical product, intended to furnish pharmacological activity or contribute direct effect in the diagnosis, cure, mitigation, treatment or prevention of disease. It also provides a direct effect in restoring, correcting or modifying physiological functions in human beings. Microalgae, which are single-celled microorganisms, are considered to be a rich source of diverse bioactive molecules. They play a vital role in the aquatic food chain as primary producers and can store complex organic compounds in their bodies, which can be released with the help of sunlight. Microalgae have evolved distinctive metabolic pathways, resulting in the production of remarkable secondary metabolites and unique structures that differ from those found in superior plants. Due to the high structural diversity of pigments, microalgae have the potential to produce pharmacologically valuable compounds, making them a promising source of bioactive molecules. This study aimed to examine the carotenoids pigmentation profiling from selected microalgae species subjected to different culture conditions. The methods involve microalgae cell culture mass production, carotenoid sample extraction and HPLC analysis. The findings revealed that the composition and amount of carotenoids differ according to the microalgae species. Genetic factors appear to be a crucial factor in categorizing these species based on their individual carotenoids accumulation. In short, selecting the appropriate species with the ability to accumulate carotenoids is important in determining the sources of pigments for halal bioactive ingredients or for commercial purposes, particularly in the halal market. Keywords: Halal Bioactive Ingredient, Halal Market, Halal Pigment, Halal Product, Microalgae can be classified into four families based on their chemical structure: tetrapyrroles, carotenoids, polyphenolic compounds, and alkaloids, with chlorophyll, beta-carotene, anthocyanins, and betalains being examples of each family, respectively (Schoefs, 2004). Colour additives can be utilized for various purposes such as standardizing raw ingredient colours, giving colour identities to colourless foods, and substituting colour loss during processing or storage (Ronald and Catherine, 2012). Therefore, they can be used for specific purposes based on their colour.
Flavonoids known as anthocyanins are soluble in water and display pH-dependent colours that range from red to blue, and they possess bioactive properties such as antioxidant, anti-inflammatory, hypoglycaemic, and chemo-preventive effects (Chung et al., 2016). These anthocyanins can be found in various foods, including beverages, desserts, ice cream, and dairy products, and are susceptible to a range of pH-dependent colour gradients (Cortez et al., 2017;Meija et al., 2020). Fruits and vegetables are the primary sources of carotenoids, which are known for their red, orange, and yellow colours and contribute to the flavour of food and beverages (Rodriguez, 2019). The yellow, orange, and red shades of carotenoids provide additional health benefits, such as lycopene, a bioactive, red-coloured pigment found in red fruits that possesses antioxidant properties and has been linked to a lower risk of cancer, cardiovascular disease, and diabetes (Eletr et al., 2017).

Carotenoid and microalgae
Carotenoids are a group of pigments that are lipid-soluble and can be found in various organisms such as plants, algae, bacteria, fungi, and animals. They have different colours ranging from yellow to red and are essential for the health and growth of organisms (Tanaka et al., 2008;Ronald and Catherine, 2012). Unlike animals, plants and other organisms can synthesize carotenoids, and animals obtain them from their diet. Common sources of carotenoids include annatto, paprika, saffron, caramel, chlorophyll, and turmeric. Carotenoids are classified into two groups based on the presence or absence of an oxygen group in their carbon chain: xanthophylls (with oxygen group) such as lutein and zeaxanthin, and carotenes (without oxygen group) such as α-carotene, β-carotene, and lycopene. Carotenoids are composed of eight units of isoprenoids linked together in a symmetrical and linear molecule. The extraction of carotenoids from natural sources involves several pre-treatment steps, and organic solvents are used due to their hydrophobicity. However, the basic cyclic structure of carotenoids is susceptible to changes due to various factors such as hydrogenation, dehydrogenation, cyclization, and oxidation. Their conjugated double-bond structure makes them reactive, and they can be easily isomerized and oxidized (Meija et al., 2020;Oliver and Palou, 2000).
Carotenoids can serve as vitamin supplements since carotene is a precursor of vitamin A, which helps prevent night blindness caused by a vitamin A deficiency (Okafor et al., 2016). In addition to its role in growth and development, immunity, and vision, vitamin A is also a carotenoid with antioxidant and anti-tumour properties that protect against free radicals and singlet oxygen (Malendez-Martinez, 2019; Zeb and Mehmood, 2004). Certain carotenoids like lutein and zeaxanthin are stored in the retina and human lens, which can improve cognitive function in elderly individuals (Hammond et al., 2017;Thane and Reddy, 1997). Under stress conditions, microalgae can produce carotene, while Haematococcus pluvialis can produce astaxanthin (Jacob-Lopez et al., 2020). Fucoxanthin, one of the most abundant carotenoids, is primarily extracted from brown microalgae such as Undaria, Sargassum, Laminaria, Eisenia, Alaria, Cystoseira, and Hijikia, and possesses photoprotective, anti-obesity, anti-inflammatory, neuroprotective, anti-diabetic, and antioxidant properties when added to food (Lourenco-Lopez et al., 2020).
Carotenoids have been deemed safe by regulatory agencies, including the Food and Drug Administration (FDA) and the European Food Safety Authority (EFSA), and have been designated as Generally Recognized as Safe (GRAS). However, certain carotenoids have been recommended for acceptable daily intakes, such as lutein (1 mg/kg BW/day), lycopene (0.5 mg/kg BW/day), zeaxanthin (0.75 mg/kg BW/day), α-carotene and βcarotene (15 mg/kg BW/day), bixin (6 mg/kg BW/day), and norbixin (0.4 mg/kg BW/day) (Malendez-Martinez, 2019). Carotenoids are commonly used in meat products, vegetable oils, butter, and dairy products as colourants. However, their utilization as additives and functional ingredients is difficult due to their water insolubility, instability, and low bioavailability. Nevertheless, alternatives such as carotenoid delivery in water-dispersible products, colloidal suspensions, emulsions, and colloidal dispersions have been developed to overcome these limitations (Rodriguez Amaya, 2016).

Prospects of Carotenoid Derivatives as New and Halal Colorants Agent
Scientists, engineers, and investors are exploring microalgae biomass, insects, and mycoprotein as future food sources to tackle malnutrition. Habib et al. (2008) suggested that future foods should have high protein content, low reliance on chemicals, such as fertilizers and hormones, and a low carbon footprint. It is expected that future food will be in the form of powders, tablets, and capsules made from microalgae biomass. In recent years, there has been a doubling in the number of snacks and drinks containing microalgae, particularly in Western countries. The most popular new products introduced include bakery items, meals, and chocolate confectionery (Kratzer and Murkovic, 2021).
Experts anticipate that the cost of producing microalgae will decrease, and microalgae will be cultivated for protein production, with a range of newly developed microalgae products expected to enter the market soon (Kratzer and Murkovic, 2021). Biotechnological production of carotenoids has expanded due to the ability to use low-cost substrates, the designation of natural substances, and the minimal space required for bioproduction (Silva, 2004). Additionally, microalgae are unaffected by environmental factors such as climate, season, or soil composition, and controlled culture conditions further enhance the potential for carotenoid bio-production. Bioprospecting is used to identify the best-starting strain for improving the genotype of the microorganism in a bio-production system. Traditional breeding, mutagenesis, and selection can be used to enhance the strain (Torres-Tiji et al., 2019).
Carotenoids and anthocyanins are the most common colourants used in dairy products. However, the use of phycocyanobilins for blue pigmentation and betacyanins for pH-stable shades has expanded the range of chemical groups that can be incorporated (Luzardo-Ocampo et al., 2021). According to Torres-Tiji et al. (2019), there are numerous molecular tools accessible for modifying the genomes of microalgae. By strategically adjusting genetic information, these tools can be employed to enhance the production of a preferred product. Genetic tools are made up of various technologies needed for successful recombinant gene expression and endogenous gene expression regulation, such as promoters, untranslated regions (UTRs), enhancers, selection markers, and knock-out and knock-down technology. Kratzer and Murkovic (2021) affirm that the availability of suitable genetic tools is crucial for increasing the value of algae as a food source, as the degree and accuracy of genetic modification depend on their availability. Currently, genomic and gene expression data availability for specific algae species, physiological barriers like cell size or rigid cell walls, and site-directed mutagenesis tools for the desired algae species are limiting these techniques.
Two different violaxanthin or chlorophyll a-binding proteins, VCP1 and VCP2, were also effective in Nannochloropsis sp. for stable recombinant expression, according to Barrera et al., (2013) and Qin et al. (2012). Carotenoids are another example of natural products in algae that can be enhanced through metabolic engineering, as demonstrated by overexpression of the Chlorella zofingiensis phytoene synthase gene in transgenic Chlamydomonas reinhardtii (Cordero et al., 2011). There is tremendous potential to boost the nutritional quality of algae by increasing the accumulation of already present nutrients while also incorporating new ones into an edible and mass-producible system (Torres-Tiji et al., 2019). Because of its promising potential, research in this area has surged recently and is still ongoing.

Methodology
All the selected microalgae species were purchased from the Microalgae Bank of University Science Malaysia for further evaluation of the pigmentation profile. Several methods involved in this design experiment as follow: Table 1 presents a selection of four species of freshwater blue-green microalgae (cyanobacteria) and nine species of green microalgae (chlorophyta) that were chosen to analyse their carotenoid content.

Microalgae Cell Culture Mass Production
Blue-green microalgae were cultured in the standard growth medium, namely Bold Basal Medium (BBM) and BBM Modified, which contains two and three times the nitrate concentration. Microalgae Inoculation A volume of 1 ml of algae stock solution was transferred using a pipette into sterile jam jars filled with BBM medium at varying pH levels and mixed thoroughly by swirling.

Microalgae Biomass Freeze Dry
On day 14, the cell culture of microalgae and the growth medium were collected and centrifuged. The resulting microalgae biomass was then collected, weighed, and freezedried until it was completely dry and turned into a powder.

Measurement of Carotenoids Pigment Colour Intensity
Green and blue-green microalgae pigments colour intensity was measured by CIELAB.

High-Performance Liquid Chromatography (HPLC) Analysis
The carotenoids were analysed using HPLC, following the procedure outlined in previous studies (Othman, 2009;Radzali et al., 2016). An Agilent model 1200 series was used, which included a quaternary pump with an autosampler injector, micro-digesters, a column compartment equipped with a thermostat, and a diode array detector.

Carotenoid Sample Preparation
The carotenoid extract obtained from algae samples was re-suspended in 200 µl of ethyl acetate and inserted into HPLC for analysis.

Results and Discussion
Thirteen microalgae species were evaluated for their carotenoid content in terms of quantity and quality and nine were green algae and four were cyanobacteria. The carotenoid composition of these microalgae species can be classified into four groups, which are detailed in Table 2. Among the microalgae species tested, Synechococcus sp., a type of cyanobacteria, had the highest total carotenoid content (7751.87 ± 195.35 μg/g DW), which was significantly higher than all of the other species. In contrast, the lowest total carotenoid concentration was found in Selenastrum capricornutum, a type of green algae (40.39 ± 3.89 μg/g DW). Using HPLC analysis, at least six major carotenoid peaks were detected as shown in Figure 1 to Figure 13, including β-cryptoxanthin, β-carotene, zeaxanthin, neoxanthin, lutein, and violaxanthin. , and lutein and violaxanthin were highest in C. fusca at 220.14 µg/g DW and 307.94 µg/g DW, respectively. All 13 species can be categorized into several groups based on the accumulation of specific carotenoid pigments as explained in Table 2. Pandorina morum and Chlorococcum sp. green algae contained five and four individual carotenoid pigments, respectively, with relatively high concentrations of violaxanthin and lutein, compared to neoxanthin, β-carotene, and β-cryptoxanthin. However, six species, namely C. fusca, S. capricornutum, Phormidium sp., Scenedesmus sp., B. sudeticus, and C. vulgaris, were found to have three out of six carotenoid pigments, with relatively high concentrations of either β-carotene or violaxanthin. The remaining five species, namely B. braunii, Ankistodesmus sp., Pseudanabaena sp., Synechococcus sp., and Alkalinema sp., accumulated two carotenoid pigments, either β-carotene and zeaxanthin or lutein and violaxanthin. Generally, the highest carotenoid concentrations, either in total or individual carotenoid pigments, were detected in the four-carotenoid pigment group. It can be concluded that the total carotenoid concentration is highly associated with that of individual pigments, especially zeaxanthin. However, the relative distributions of individual carotenoids within each grouping did not necessarily correlate with the levels of total carotenoids.  According to Borowitzka (2013), out of 750 known carotenoids, 200 are derived from algae species. Algae carotenoids, like those in plants, act as light-harvesting pigments and antioxidants. Carotenoids of the xanthophylls class are abundant in algae and are useful in photosynthesis. For example, fucoxanthin, peridinin and vaucherioxanthin harvest light in the green region of the spectrum (500-550 nm) in algae. Other xanthophylls, such as zeaxanthin (found in green algae) and diatoxanthin + diadinoxanthin (found in chromophytic algae), are involved in non-photochemical quenching of the xanthophyll cycle. In addition to species, autotrophic growth factors such as temperature, light, nitrate, salinity, and iron play a crucial role in regulating the biosynthesis of carotenoids in algae (Minhas et al., 2016).

García-González et al. (2005) and Sanchez et al. (2008) conducted a study on
Dunaliella salina, which resulted in a higher accumulation of lutein as the temperature rose up to 30ºC. However, Minhas et al. (2016) observed that the autotrophic cultivation of microalgae under temperature stress, for astaxanthin or beta-carotene, is dependent on the species. The availability of light is also considered a crucial limiting factor in several carotenoid biomass production and fatty acids (FAs) (Del Campo et al., 2000). The growth, biomass, and other metabolites of interest in various microalgae species are governed by the light intensity and photoperiod (Abe et al., 2007;Lamers et al., 2012). Lamers et al. (2012) conducted a study on H. pluvialis, which indicated a threefold increase in astaxanthin under increasing light intensity.
According to Gomez et al. (2003), increasing light intensity from 100 to 1000 μmol photons/m²/s in D. salina led to an increase in beta-carotene content up to 3.1% of dry cell weight. Hadi et al. (2008) found that high light intensity (460 μmol photons/m²/s) caused Muriellopsis sp. lutein content to peak. Minhas et al. (2016) noted that astaxanthin, betacarotene, and lutein production in algae was increased by a combination of high light stress and nitrate. Nitrate was found to be a stress factor affecting not only lipid but also carotenoid accumulation in green algae (Minhas et al., 2016). According to Del Campo et al. (2000), lutein accumulation in microalgae is strongly influenced by nitrogen concentration in the medium. Under limited nitrogen conditions, the cells of aerial microalgae changed from green to red, and carotenoids such as β-carotene, astaxanthin, and canthaxanthin began to accumulate (Abe et al., 2007). Nitrogen-depleted conditions resulted in beta-carotene content rising up to 2.7% of dry cell weight in D. salina (Lamers et al., 2012). Lamers et al. (2012) also observed that under nitrogen-depleted conditions in H. pluvialis (mainly astaxanthin), carotenoid production was greater, whereas nitratedeficient conditions led to greater competition for carbon, which is a favourable condition for the synthesis of both carotenoids and proteins.

Conclusion
In essence, this investigation completed examines the production of carotenoids from selected microalgae subjected to different culture conditions. The findings reveal that the composition and amount of carotenoids differ according to the microalgae species. Genetic factors appear to be crucial in categorizing these species into four groups based on individual carotenoid accumulation. In addition, the genetic variations in carotenoid content among these microalgae species selection may be attributed to various regulatory factors in the carotenoid biosynthesis pathway. Selecting the appropriate species with the ability to accumulate carotenoids is important in determining the sources of pigments for halal food colourants or for commercial purposes, particularly in the halal market, health benefits, food products, and dye technology for huge production and commercialization.

Acknowledgment
The research has been financially reinforced by the International Islamic University of Malaysia (IIUM), under the Research Grant RMCG20-034-0034.