Biotechnological Production of Carotenoids and Their Applications in Food and Pharmaceutical Products

Pigments can be divided into four categories: natural, nature-identical, synthetic, and inorganic colors. Artificial colorants are the most used in food and pharmaceutical industries because of their advantages related to color range, price, resistance to oxygen degradation, and solubility. However, many natural pigments present health-promot-ing activities that make them an interesting option for human use and consumption. Natural colorants are derived from sources such as plants, insects, and microorganisms. Carotenoids are natural pigments with important biological activities, such as antioxidant and pro-vitamin A activity, that can be either extracted from plants and algae or synthesized by various microorganisms, including bacteria, yeasts, filamentous fungi, and microalgae. Advantages of microbial production include the ability of microorgan- isms to use a wide variety of low cost substrates, the better control of cultivation, and the minimized production time. After fermentation, carotenoids are usually recovered by cell disruption, solvent extraction, and concentration. Subsequent purification steps are followed depending on the application. The most prominent industrial applications of carotenoids, considering their health benefits, are in the food, feed, and pharmaceutical industries.


Introduction
as life expectancy increases and the birth rate declines, the demand for solutions focusing on longevity and life quality increases too. The number of people aged >60 years is expected to account for approximately one-fifth of the world's population by 2050 [34].

Carotenoids diversity
Carotenoids are lipid-soluble pigments, colored from yellow to red, with a basic structure consisting in a tetraterpene with a series of conjugated double bonds. They can have only carbon and hydrogen in their structures or have one or more oxygen atoms, being classified as xanthophylls. The majority of carotenoids are C 40 terpenoids, which act as membrane-protective antioxidants scavenging O 2 and peroxyl radicals [35].
There are more than 700 types of carotenoids described and only about 50 are precursors of vitamin A. Carotenoids can reduce risks for degenerative diseases such as cancer, cardiovascular diseases, macular degeneration, and cataract. The biological activities, specially the antioxidant properties, depend on their chemical structure: number of conjugated double bonds, structural end-groups, and oxygen-containing substituents [36].
Carotenoids occur in photosynthetic systems of higher plants, algae, and phototrophic bacteria. In plants, carotenoids are embedded in the membranes of chloroplasts and chromoplasts. The colors of these pigments are masked by chlorophyll, but they contribute to the bright colors of many flowers and fruits [37].
Nonphotosynthetic organisms, as some bacteria and fungi, present carotenoids as protectors against photo-oxidative damage, a way of protection in growth conditions with light and abundant air. The main carotenoids produced by fungi are β-carotene, torulene, torularhodin, and astaxanthin [38]. Bacteria have been reported as producers of cantaxanthin mainly. The microalgae are producers of lutein, β-carotene, and astaxanthin [35].
Animals usually present carotenoids provenient from their diet. Marine animals that feed on algae or on products rich in carotenoids may exhibit the coloration of these pigments, as the salmon fish. The color of the feathers of some birds also comes from a diet rich in carotenoids, as flamingos [39].
The industrial production of carotenoids by plants is dependent on the season and geographic variability, and these cannot always be controlled. The chemical synthesis of carotenoids generates wastes that can cause damage to the environment and resistance by the consumers. Because of this, the biotechnological resources are becoming more interesting. The microbial production of carotenoids can be performed using low-cost substrates or substrates that are residues from industrial processes, like molasses, resulting in lower costs of production [40]. All conditions of this kind of production can be controlled and optimized, especially knowing the metabolic route of each microorganism utilized.
Carotenoids are intracellular products, and a process to increase their accessibility at the downstream stage is necessary. The techniques most used combine physical and chemical methods like maceration and contact with organic solvents [4].

Main carotenoid biosynthesis pathways
Carotenoids are usually produced from the building blocks geranyl geranyl diphosphate (GGPP) and farnesyl diphosphate (FPP), like other secondary metabolites such as sesquiterpenoids and steroids. The most common pathway is the condensation of 2 GGPP units into prephytoene diphosphate and then to phytoene, a 40-carbon polyunsaturated precursor which is colorless. This precursor is converted into lycopene and then into several derived carotenoids such as β-carotene and oxidized derivatives such as lutein. The condensation of two units of FPP leads to 30-carbon precursors that are converted to steroids or apocarotenoids such as staphyloxanthin [41,42]. Apocarotenoids can also be produced by oxidative cleavage of carotenoids. Figure 1 presents a simplified carotenoid biosynthesis pathway.
Most carotenoids present maximal absorption in the violet to green region of the visible spectrum, so these substances appear as red to yellow pigments. Table 1 shows the carotenoids with permitted food use according to the Food and Drug Administration (FDA) and the Food and Agriculture Organization (FAO).

Carotenoid sources
The most common sources for natural carotenoids for food and cosmetic use are plants, although microorganism biomass is becoming more common as a source for these substances. X max -maximum biomass concentration; P max -maximum carotenoids concentration; μ X -biomass production rate.

General downstream operations for carotenoid production
Carotenoids are nonpolar molecules that accumulate intracellularly in plant tissues and microorganisms. Therefore, the production usually consists in a biomass pretreatment that may accelerate the dissolution of these substances, followed by a solid-liquid extraction (leaching) with a suitable, low-polarity solvent. The resulting solution can be a final product, can be desolventized, and can be further purified, depending on the use intended for the extract. Figure 2 illustrates the main steps in the production of carotenoids.
The first step in carotenoid production is the pretreatment of the raw biomass, usually by drying and milling. Drying is convenient because it reduces the weight of the material to be processed, facilitates the access for solvents to the biomass, and reduces contaminants that could be extracted in water micelles with the solvent. The milling step is also important because it increases the surface area of the biomass matrix, facilitating contact with the solvent. In the case of tough-walled organisms, chemical or mechanical cell disruption may be done prior to drying. Fine milling of the dry biomass is less common.  The dry biomass is then extracted using a nonpolar solvent such as hexane or a vegetable oil, for the dissolution of carotenoids. A higher polarity solvent such as acetone can be used for the extraction of xanthophylls. In both cases, lipids are extracted in the mix. This extraction is an equilibrium operation; therefore, the final concentration in the solvent affects the extraction efficiency. Following extraction, the solution containing carotenoids must be concentrated and desolventized. This is why low boiling point solvents, which are easy to evaporate, are more common extractants than oils.
The carotenoids in the concentrated extract may be purified or not, depending on the intended use. For example, β-carotene that will be used as a vitamin A precursor must be purified, while paprika oleoresin is a mixture of carotenoids used mainly as a color and flavor additive and needs no further purification. In general, for carotenoids used as color additives, it is enough to concentrate the extract because (1) the tinctorial strength of the molecules is large-therefore, the additive is added at a low concentration to the formulated product and (2) the sources used are generally regarded as safe (GRAS), and the molecules extracted with the carotenoid are harmless in the concentrations used.
In the case of purified carotenoids, the operations to be used-adsorption, chromatography, crystallization, etc.-depend largely on the properties of the target molecule and the contaminants in the mixture, such as melting point, polarity, solubility, etc. All sorts of nonpolar compounds are extracted with the solvent, such as neutral and slightly polar lipids, steroids, and waxes. The differences in the properties of the carotenoid and the contaminants will be explored in the purification strategy.
Following extraction and purification, the carotenoid must be formulated for further application. This formulation will also depend on the intended use. The formulation may be as simple as adding an antioxidant such as butylated hydroxytoluene (BHT) or butylated hydroxyanisole (BHA) to the extract or may be more complex, such as emulsifying the carotenoid as an oil-in-water product for use in polar matrixes such as juices.

Industrial application of carotenoids as additives in food, feed and pharmaceutical products
Because of the rising of health concerns by consumers, the demand for carotenoids as natural coloring products is growing. Beta-carotene, astaxanthin, canthaxanthin, lycopen, and lutein are the most required and valuable carotenoids, and they are currently used by the food, feed, and cosmetic industries ( Table 3). The use of carotenoids is regulated by the legislation of each country that specifies the source, purity, product, and quantities of the colorant that can be used [59].
According to BBC Research [65], the carotenoid global market in 2014 was of US$ 1.5 billion, this value is increasing year by year and is expected to reach US$ 1.8 billion in 2019, with an annual growth rate of 3.9%. Beta-carotene, the carotenoid of highest value, had a global market of US$ 233 million in 2010, which is expected to reach US$ 309 million by 2018. Astaxanthin, due to its powerful antioxidant activity, is the third carotenoid in terms of high added value, with a global market size of US$ 225 million, estimated to increase to US$ 253 million by 2018.

Importance and use of carotenoids in food products
Commercial food products using carotenoids are expanding, and the greatest demand is in the Asian continent. The pigment is extracted from microalgae such as Chlorella, Dunaliella, Haematococcus [66,67], from the cyanobacterium Spirulina [68], and from the fungus Monascus [69].
In Asia, the production red koji dates of hundreds of years and uses the fermentation of rice by Monascus to produce the typical reddish color. These red pigments are also used as food colorants for wine, red soy cheese, meat, and by-products of meat and fish [26]. The French cheese named vieux-pan contains the carotenoid produced by Brevibacterium linens due to its orange-red-brown color that improves the sensory quality of the product [70]. In Russia, infant formulas are enriched with natural pigments such as lutein, which is present in breast milk, in order to improve children's health [71].
Nutraceutical food products have also been applied in bakery products and pasta. In Japan, Undaria pinnatifida (wakame), an edible seaweed rich in fucoxanthin, is commercialized as an ingredient for pasta [72]. In India, a pasta containing fucoxanthin as an ingredient to improve its biofunctional and nutritional qualities was developed [73].

Importance and applications of carotenoids in the pharmaceutical industry
Besides the use of nutraceutical foods as a form of prevention and treatment of diseases, the administration of the bioactive compounds in their concentrated form is also a possibility for promoting health. The transport of carotenoids occurs from the intestinal mucosa to the blood vessels carried by lipoproteins [74]. Carotenoids functional properties are related to reactions such as oxidation, reduction, hydrogen abstraction, and addition in biological membranes, and their antioxidant power is fundamental for cell protection against free radicals and singlet oxygen formed in tissues [75].  Some carotenoids are precursors of vitamins, and they also present activities such as antiinflammatory, antioxidant, immunomodulatory, anticancer, for cardiovascular therapy and neurodegenerative diseases [76], and anti-obesity [77]. The carotenoids included as pro-vitamin A are β-carotene, α-carotene, and cryptoxanthin. Vitamin A is an essential nutrient for operation and maintenance of biological functions including vision, reproduction, and immunity [78]. Beta-carotene is present in blood and tissues, which is associated with antioxidant activity and concomitantly with other carotenoids or antioxidants can enhance their activity against free radicals. However, it can bring health risk at high doses [79].
Carotenoids, acting as antioxidants eliminating free radicals, can modulate the risk of developing chronic diseases by inhibiting reactions mediated by reactive oxygen species (ROS). Reactive species are produced during cellular metabolism as a defense to infectious and chemical agents that may cause damage to DNA, proteins, and tissues, contributing to the development of chronic diseases such as diabetes, Parkinson's, Alzheimer's, cardiovascular diseases, and cancer [80].
In addition to the antioxidant properties, carotenoids exhibit anti-inflammatory activities owing to the protective effects of phytochemicals such as lutein and astaxanthin. Astaxanthin has been shown to inhibit the production of pro-inflammatory mediators such as nitric oxide (NO) in macrophages, to increase the level of inflammatory cytokines, and to reduce oxidative stress. Neuroprotective effect, reduced neuroinflammation, improvement of insulin signals, and reduction of lipid levels were also verified [81].
Inhibition of cell proliferation of colon cancer cells by the use of Neochloris oleoabundans carotenoids was observed, enabling its use as a functional food additive or nutraceutical with potential for the prevention of colon cancer [82]. Beta-carotene, astaxanthin, and capsanthin demonstrated antiproliferative effects on leukemic K562 cells [83]. Studies indicated that the simultaneous use of different carotenoids was efficient against liver cancer. Patients were administered with β-cryptoxanthin-enriched mandarin orange juice and capsules of a carotenoids mixture-containing lutein, β-cryptoxanthin, lycopene, zeaxanthin, and fucoxanthin. Analyses of DNA array and protein-antibody array showed that the carotenoids interferred in the induction of genes such as p16 and p73 [84].

Conclusion and final remarks
There are many advantages related to the use of carotenoids instead of artificial pigments in food products and for pharmaceutical applications. Their biological properties such as antioxidant, anti-inflammatory, antitumoral, and pro-vitamin A activities contribute to the quality of the product and to the consumer's health. Among the production strategies, microbial synthesis is considered advantageous, and the downstream techniques usually involve cell disruption, solvent extraction, concentration, and purification, when necessary. Several researches have proved the beneficial effects of carotenoids on health, so they can meet the demand for solutions focusing on longevity and life quality.

Author details
Ligia A. C. Cardoso