Microbial-Derived Carotenoids and Their Health Benefits

Abstract

Natural carotenoids (CARs) such as β-carotene, astaxanthin, lutein, norbixin, bixin, capsanthin, lycopene, β-Apo-8-carotenal, canthaxanthin, β-apo-8-carotenal-ester, and zeaxanthin are being explored for possible applications in feed, food, cosmeceuticals, and nutraceuticals. Three primary areas of carotenoid research are emerging: (1) encapsulations for improved chemical and physical properties; (2) natural source carotenoid manufacturing; and (3) preclinical, epidemiological, and clinical studies of carotenoids’ potential health benefits. The recent advancements in research on the chemistry and antioxidant activity, marketing strategies, dietary sources, bioavailability, and bioaccessibility, extraction, dietary consumption, encapsulating techniques, and health advantages of carotenoids are all extensively discussed in this review. Carotenoids are pigments found naturally in most fruits and vegetables, algae, plants, and photosynthetic bacteria. Carotenoids cannot be synthesized by humans and must be consumed in the form of food or supplements. There are several roles for carotenoids in human health. Although individual carotenoids may function in different ways, their main action is to act as antioxidants. There are validated techniques for separating and purifying carotenoids, yet, industrial production requires the development of economically viable techniques for larger-scale implementation. Carotenoids have been shown to boost cognitive performance and cardiovascular health, as well as help prevent some types of cancer. Despite evidence for carotenoids’ health benefits, major population-based supplementation trials have yielded conflicting outcomes for several carotenoids. This review includes recent developments in carotenoid metabolism and nutritional and health advantages. It also offers an outlook on future directions in these areas.

1. Introduction

A wide variety of lipid-soluble pigments known as carotenoids are found in red, orange, and yellow colours. A vast range of living organisms, such as bacteria, photosynthetic plants, microalgae, and, to a greater extent, yeasts and fungi, naturally contain them [1]. Certain carotenoids function as photoprotectors and precursors for the manufacture of vitamin A [2,3]. Approximately 700 different chemical compounds with unusual pigments and biological characteristics make up these pigments [4]. Carotenoids are lipophilic isoprenoid molecules with double bonds forming a chromophore that absorbs light, giving them their colouration properties [5]. Carotenoids are sensitive to heat, light, oxygen, and acids as well as processes like oxidation and isomerization, because of these double bonds [6,7,8]. An aggregate of 691 distinct organisms is the source of about 1158 naturally occurring carotenoids [9]. Only a limited number of carotenoids including canthaxanthin, lutein, astaxanthin, β-carotene, and lycopene are currently commercially accessible [2]. The market demand for different carotenoids is rising as a result of the growing number of uses in the animal feed, food processing, pharmaceutical, and cosmetics industries [2,10]. As an alternative to synthetic additives, the synthesis of carotenoids from microorganisms emerged to rival that of carotenoids produced by chemical techniques [11]. As evidenced by the rise in research on microbiological dyes, biotechnology has been viewed as the greatest substitute for the natural pigment market since different microorganisms can synthesise carotenoids [12]. Some benefits of biotechnological production are optimized control of cultivation, reduced production time, and the capacity of microorganisms to use inexpensive substrates [13,14]). Chemical synthesis is frequently used to produce carotenoids for commercial use. On the other hand, excessive amounts of carotenoids that are synthetic may be toxic to human health, and their production creates dangerous waste [15,16]. However, plant-based carotenoids for commercial use are somewhat costly, and their availability is restricted by unpredictable weather and geographic variations [10,17]. About 20 out of 700 carotenoids are detected in human blood and tissues, while approximately 50 are found in the human diet [18]. These include the most significant ones: α-Carotene, β-Carotene, Lycopene (LYC), Zeaxanthin, Lutein, β-Cryptoxanthin, γ-Carotene, α-Cryptoxanthin, Fucoxanthin (Fx), Phytofluene, Neurosporene, and Phytoen [19].

Several economically important carotenoids are currently being produced using microalgal platforms, but carotenoids produced using metabolic pathway engineering of bacteria and fungi are still in the early stages of development. Commercial production of carotenoids has been established using microalgae such as H. pluvialis (Astaxanthin), Dunaliella salina (β-carotene, open pond technology), Chlorella zofingiensis (Canthaxanthin), Botryococcus braunii (Echinenone), Phaeodactylum tricornutum (Fucoxanthin) and Scenedesmus spp. (Lutein) [20]. Carotenoids are currently produced on a large scale using the fungi M. circinelloides (β-carotene), bacterium Gordonia jacobea (canthaxanthin), B. trispora (lycopene, β-carotene), C. glutamicum (C-50 carotenoid Decaprenoxanthin), and yeast X. dendrorhous (astaxanthin, syn. Phaffia rhodozyma) [21]. Fungi such as Neurospora crassa, Fusarium sporotrichioides, the bacteria A. aurantiacum, Halobacterium salinarium, and Paracoccus carotinifaciens are also emerging as prospective sources of carotenoids. Several non-carotenogenic microbes, including Saccharomyces cerevisiae, Blakeslea trispora, Escherichia coli, and X. dendrorhous have been genetically engineered to produce commercially vital carotenoids like lycopene, β-carotene, canthaxanthin, and astaxanthin. Conversely, natural carotenoids have bioactive qualities that may enhance health, and many of them are found in foods consumed by human beings [22]. Having β-carotene-rich meals lowers the risk of cardiovascular/circulatory disease, the world’s leading cause of mortality. Additionally, meals enriched with β-carotene exhibited protection against esophagus cancer [23]. Numerous studies support many health benefits that a diet high in carotenoids may provide, from depression prevention to protection against various forms of cancer [24]. Consuming carotenoids, like lutein and zeaxanthin, was associated with a lower incidence of eye issues and a decreased risk of breast cancer [25]. Because of its strong antioxidant properties, lycopene consumption has been linked to a lower risk of prostate cancer and heart failure [26]. This review presents updates on a number of the latest emerging microbial carotenoid producers. The commercial uses and methods of producing these microbial carotenoids are also discussed.

2. Structure, Categorization and Synthesis of Natural Carotenoids

2.1. Chemistry and Types of Carotenoids

Carotenoids are lipophilic isoprenoids that are categorized based on their molecular and dietary properties. They belong to the chemical classes of xanthophylls and carotenes. The most well-known class of compounds are called carotenes, which include hydrogen and carbon atoms in their chemical structure. Examples of these include α-, β-, γ-, and δ-carotene, as well as torulene. Torularhodin, astaxanthin, and canthaxanthin are examples of the second class of xanthophylls, which also include hydrogen and carbon in their chemical structures [27,28,29]. Carotenoids are a category of hydrocarbons that are created when isoprene molecules with five carbons join together [3,30]. Differentiated structures of carotenoids can be produced by modifying the basic cyclic structure through dehydrogenation, hydrogenation, cyclization, chain shortening or extension, double bond migration, rearrangement, isomerization, oxidation, or combinations of these reactions [10]. Natural carotenoids are present in a broad variety of microorganisms, including cyanobacteria, microalgae, fungi, heterotrophic bacteria, and archaea, in addition to plant sources like tomatoes and carrots [31].

Isopentenyl pyrophosphate (IPP) and its allylic isoenzyme dimethylallyl diphosphate (DMAPP) are the two precursor molecules involved in the biosynthesis of carotenoids. The 2-C-methyl-D-erythritol 4-phosphate (MEP) and/or mevalonate (MVA) pathways produce the IPP and DMAPP. The MVA pathway is used by photosynthetic organisms like microalgae and plants, but the MEP pathway is widely used by most bacteria and fungi. Condensation of three IPP and one DMAPP molecule results in the production of a C-20 geranylgeranyl diphosphate (GGPP) molecule, an immediate precursor of carotenoid biosynthesis. Lycopene, a common intermediate for the biosynthesis of nearly all downstream C-40 carotenoids, is then produced [32]. Carotenoids can also be categorized into C-30, C-40, C-45, and C-50 groups based on how many carbons make up each group in their chemical structures. The carotenoids C-40 are among those that are abundant in bacteria, eukaryotic, and archaea [33].

Bacteria and archaea synthesize the C-30 and C-50 carotenoids. On the other hand, only certain types of bacteria can synthesize C-45 carotenoids, which are made up of nine isoprenoid units [9,34]. Figure 1 shows the chemical structures of the most prevalent carotenoids. The majority of carotenoids also have chirality because their molecules contain chiral centres. Carotenoids have chiral centres in their molecules, which contributes to their unique properties. Astaxanthin and zeaxanthin are examples of chiral carotenoids, containing two chiral centers at 3 and 30 carbons due to hydroxyl groups. These carotenoids have two different enantiomers: 3R,3′R and 3S,3′S, as well as 3R,3′S (optically inactive meso form) [35]. In the bioproduction of carotenoids, substrates consisting of sucrose and glucose were the most commonly reported carbon sources [36]. The type and composition of the substrate directly affect the yield of pigments and, consequently, the cost of biotechnological procedures. According to Marova et al., the optimal circumstances for maximum carotenoid yield include high cell growth rates and carbon source availability [37]. Byproducts and raw materials from food and agro-industries are promising substrates for microorganism growth and carotenoid production due to their high nutrient availability, low acquisition cost, and suitability for industrial biotechnological processes (Table 1). Polyhydroxyalkanoates are regarded as suitable replacements for petroleum-derived polymers due to their biocompatibility, biodegradability, and environmental friendliness. Despite these intriguing characteristics, higher manufacturing costs hindered commercial PHA yield. As a result, there is a need to look for alternate renewable feedstocks that can lower overall PHA costs. To make the procedure more cost-effective, research has been undertaken on PHA production using various affordable substrates such as defatted Chlorella biomass, molasses, waste cooking oil, coffee waste, and sugarcane bagasse [38,39,40]. Carotenoids are biological pigments that have numerous biological benefits, including anticancer and antioxidant characteristics. Several carotenoids were in high demand, with astaxanthin topping the list with a high market value. Astaxanthin is a lipophilic natural pigment that is produced by a variety of Paracoccus species. It has been reported that different members of the genus Paracoccus produce diverse carotenoids. Kumar and Kim proposed the co-production of PHA and other valuable metabolites, which could assist in reducing PHA’s high cost [41,42].

Table 1. Agro-industrial residues studied as substrates for carotene production.

Substrate	Species	Carotenoids	References
Triticum flour, Pennisetum glaucum seed flour	Phaffia rhodozyma	Astaxanthin	[43]
Camelina sativa meal hydrolysates	Rhodosporidium toruloides	Total carotenoids	[44]
Carob pulp syrup	Rhodosporidium toruloides	Total carotenoids	[45]
Water from rice parboiling	Sporidiobolus salmonicolo	β-carotene	[46]
Ultra-filtered whey	Rhodotorula acheniorum	β-carotene	[47]
sugar beet pulp hydrolysates	Rhodotorula mucilaginosa and Rhodotorula toruloides	Total carotenoids	[48]
Molasses	Rhodotorula mucilaginosa	Torulene, torularhodin and β-carotene	[49]
Potatoes	Rhodotorula mucilaginosa	β-carotene	[37]
Sugarcane broth	Rhodotorula rubra	Total carotenoids	[50]
Beetroot molasses	Rhodotorula glutinis	β-carotene, torulene, torularhodin	[51]
Residual effluent from potato starch	Rhodotorula glutinis	Torularhodin, torulene and β-carotene	[52]
Fermented radish brine	Rhodotorula glutinis	β-carotene	[53]
Corn extract	Rhodotorula glutinis	β-carotene, torulene, torularhodin	[51]

Table 1. Agro-industrial residues studied as substrates for carotene production.

Substrate	Species	Carotenoids	References
Triticum flour, Pennisetum glaucum seed flour	Phaffia rhodozyma	Astaxanthin	[43]
Camelina sativa meal hydrolysates	Rhodosporidium toruloides	Total carotenoids	[44]
Carob pulp syrup	Rhodosporidium toruloides	Total carotenoids	[45]
Water from rice parboiling	Sporidiobolus salmonicolo	β-carotene	[46]
Ultra-filtered whey	Rhodotorula acheniorum	β-carotene	[47]
sugar beet pulp hydrolysates	Rhodotorula mucilaginosa and Rhodotorula toruloides	Total carotenoids	[48]
Molasses	Rhodotorula mucilaginosa	Torulene, torularhodin and β-carotene	[49]
Potatoes	Rhodotorula mucilaginosa	β-carotene	[37]
Sugarcane broth	Rhodotorula rubra	Total carotenoids	[50]
Beetroot molasses	Rhodotorula glutinis	β-carotene, torulene, torularhodin	[51]
Residual effluent from potato starch	Rhodotorula glutinis	Torularhodin, torulene and β-carotene	[52]
Fermented radish brine	Rhodotorula glutinis	β-carotene	[53]
Corn extract	Rhodotorula glutinis	β-carotene, torulene, torularhodin	[51]

Figure 1. Carotenoids’ chemical structures [54].

Figure 1. Carotenoids’ chemical structures [54].

2.2. Natural Carotenoids Sources

The primary dietary source of carotenoids in the human diet is colourful fruits and vegetables [55]. Additionally, dietary supplements might raise the intake of carotenoids due to their health-promoting qualities. Nowadays, there are a plethora of dietary supplements based on provitamin A (like β-carotene) and non-provitamin A (like zeaxanthin and lutein for eye health) that are produced and marketed primarily by BASF (Ludwigshafen, Germany), OmniActive (Mumbai, India), DSM (Heerlen, The Netherlands), LycoRed Ltd. (Be’er Sheva, Israel), and Chrysantis Inc. (West Chicago, IL, USA) [56,57]. Plants, photosynthetic bacteria, algae, and certain heterotrophic bacteria and fungi are all natural sources of carotenoids [3]. Animals and humans get carotenoids from food because they cannot synthesize them; however, the carotenoids that are obtained can be modified with metabolic reactions for the synthesis of additional carotenoids or their byproducts. Figure 2 summarises the different roles of dietary carotenoids as exogenous antioxidants in the human body. β-carotene is a dominant carotenoid in carrots [58], capsicum pods, sweet potatoes, and green leafy vegetables [32,59,60]. The most prevalent carotenoids found in green leafy vegetables are β-carotene and lutein, followed by violaxanthin and neoxanthin [58,60]. Lactucaxanthin is a protein found only in lettuce (Lactuca sativa L.), one of the leafy green vegetables [61,62]. Citrus, persimmon, peach, papaya, and capsicum pods have substantial amounts of β-cryptoxanthin in the diet [63]. For all xanthophylls, esterified forms are primarily present in most fruits (≈50–99% of total xanthophylls) and include neoxanthin, lutein, zeaxanthin, and β-cryptoxanthin [64]. Capsicum pods contain mostly xanthophyll esters such as violaxanthin, lutein, zeaxanthin, and keto carotenoids (capsanthin, capsorubin diester) [64,65].

2.2.1. Microorganism

A number of microorganisms from the families Bacterium, Fungi, and Algae can accumulate various forms of carotenoids as metabolic byproducts within their cells.

The group of carotenoids belonging to non-photosynthetic microorganisms is linked to a generative reaction against photo-oxidative stress brought on by environments that are high in light and oxygen [66]. During the last decades, a large number of research groups have made the biotechnological production of carotenoids their top priority. The fact that over 600 of the 750 naturally occurring carotenoids can come from microbes is just suggestive [67]. Plant-derived pigments are challenging to characterize and standardize due to variations in climate conditions and cultivation. The stability and efficacy of these pigments are limited, especially when exposed to high temperatures, light, and pH changes [68].

2.2.2. Carotenoids Produced by Bacteria

Three types of bacteria are known to produce carotenoids: oxygenic, non-phototrophic, and phototrophic [69]. Photosynthetic oxygenic bacteria gather light using bacteriochlorophyll and other carotenoid pigments. It has been determined that the synthesis of more than one hundred distinct carotenoids is caused by over 50 genera and roughly 130 species of phototrophic anoxygenic bacteria. Cyanobacteria synthesize zeaxanthin, β-carotene, canthaxanthin, echinenone, and other carotenoids, making up the majority of oxidative phototrophic bacteria [70]. Nearly all forms of carotenoids, including C-50, C-45, C-40, and C-30 carotenoids, are produced by bacteria. The C-50 carotenoids decaprenoxanthin and its glycosylated derivatives are produced by the bacteria Corynebacterium glutamicum [71]. Gordonia alkanivorans strain IB generates canthaxanthin, astaxanthin, and lutein [72], while Gordonia terrae TWRH01 primarily produces echinenone [73]. To fulfill the enhanced demand for antioxidative protection, carotenoid production is up-regulated in environments with intense light and aerobic conditions [74]. For instance, Spirulina platensis’s ability to synthesize carotenoids was improved in light with high intensity [75], while Flavobacterium sp. produced zeaxanthin in response to high light [76]. Studies involving alphaproteobacteria, such as Rhodobacter species, demonstrated that light [77] and oxygen are the most important regulating factors. Oxygen has been shown to activate the transcription of the crtI-crtB operon [78]. Several regulatory cascades involving sigma factors manage carotenoid biosynthesis. For example, a cascade of RpoE2-RpoH2 and another of RpoE1-RpoH2 regulate the expression of geranylgeranyl pyrophosphate synthase, a crucial enzyme responsible for converting farnesyl pyrophosphate into GGPP [79]. The selection of carbon sources has a major impact on carotenogenesis. Even in the presence of oxygen, glucose, and other fermentable carbohydrates are metabolized by Xanthophyllomyces dendrorhous via the glycolytic pathway, which is followed by alcoholic fermentation [80]. Studies show that chemotrophic bacteria, including H. salinarum and Rhodopseudomonas spheroides, generate pigments to protect cells from photodamage [81]. Bacteria such as Halobacterium salinarum and Halococcus morrhuae, which produce orange and red colonies, have been explored for their potential biotechnological use in pigment manufacturing [82]. H. salinarum bacterioruberin is the most prevalent carotenoid [83]. Zeaxanthin is efficiently produced by Flavobacterium sp. which is a marine bacterium [84], whereas Haloferax alexandrinus has the potential to produce canthaxanthin in the industrial sector [85]. Due to their diverse colour tones and reduced media requirements, bacterial carotenoids have become a potentially useful source for industrial usage. Commercial applications of bacterial carotenoids are hampered by their manufacturing cost, which remains significantly higher than that of synthetic sources [86]. Improving the production approach of carotenoids-producing bacteria and discovering novel carotenoids result in higher yields and reduced prices.

2.2.3. Carotenoids Produced by Microalgae

Microalgae strains have been the subject of substantial research and are becoming increasingly popular due to the expanding industrial need for natural alternatives, particularly because, under stressed situations, they synthesize unique carotenoids [87]. The environmental variables that affect the production and composition of carotenoids in algae include salinity and nutrients in the growth medium [88,89]. The following carotenoids can be produced by green microalgae: lutein, β-carotene, α-carotene, violaxanthin, neoxanthin, and others. Chlorella contains 93% lutein, 2.6% α- and β-carotene, 1.3% zeaxanthin, 0.2% xanthophylls, and 0.2% β-cryptoxanthin [90]. Arthrospira (Spirulina), Chlorella, Dunaliella salina, and Aphanizomenon flos-aquae are the primary microalgae that are marketed [91]. Microalgae can yield 3–5% of biomass dry weight or 70% of prominent carotenoids, with minor carotenoids remaining in trace amounts [92]. For example, lutein, astaxanthin, β-cryptoxanthin, and other carotenoids comprise the minor sections, but β-carotene makes up 80% of the total carotenoids synthesized by Arthrospira. Although astaxanthin can make up as much as 3% of the dry weight of Haematococcus pluvialis, [93], Dunaliella salina has a high content of β-carotene, making up nearly 8% of its dry biomass [94]. Spirulina is a bacterial microalga, also known as cyanobacteria, produced in several countries, with China being the main producer. Because of its metabolic products, such as phycocyanin, which is utilized as a food ingredient, it is employed economically. Heterotrophic fermentation reactors with sugars in the absence of light are one potential process configuration for the production of spirulina [95]. In an autotrophic process, bicarbonate and air are combined in Chinese production to make CO₂, which is then used to produce Spirulina and Chlorella vulgaris [96]. Microalgae are especially regarded as an efficient cell factory for the production of carotenoids because of their strong potential to accumulate carotenoids under specific stress conditions. However, the naturally sluggish development, low cell yields, risk of bacterial and protozoal contamination, wide cultivation area, and high susceptibility to unfavorable weather conditions limited the production capacity. Therefore, enhancement of the microalgal carotenoid breakdown, culture process optimization, and revolt of extraction methods may require additional research [92,97].

2.2.4. Carotenoids Produced by Yeast

Yeasts are another prominent source of carotenoids in microbiology. According to Sanchez et al. (2013), astaxanthin is mostly formed by the yeast X. dendrorhous, while β-carotene is produced by fungi such as B. trispora and Mucor circinelloides [98]. Other carotenoid-producing yeasts such as Sporobolomyces, Sporidioblous, and Rhodoturula only synthesize torularhodin and torulene [2,99]. Several workers have reported about the production of carotenoids by yeasts like Rhodotorula spp. This yeast, which is extensively found in nature, can biosynthesize particular carotenoids in varying amounts, including β-carotene, torulene, and torularhodin [14,100]. The genus Rhodotorula produces different amounts of carotenoids depending on the medium ingredients and environmental factors. Many studies on astaxanthin production have been reported that use the yeast Xanthophyllomyces dendrorhous to develop viable biotechnological procedures. Yeasts are therefore trustworthy microbes for producing carotenoids. Knowledge of metabolic engineering tools is also essential for manipulating the biosynthetic pathway for carotenoid production by yeasts. A potential carotenoid synthesis biosynthetic pathway was reported by Simpson et al. in 1964 [101]. After revising the main pathways for carotenoid biosynthesis by yeasts, Goodwin came to the conclusion that there are three general phases in the carotenoid biosynthesis pathway [102,103].

(1)

HMG-CoA synthase catalyses the conversion of acetyl CoA to 3-hidroxy-3-methyl glutaryl-CoA (HMG-CoA), which is the first step in the synthesis process. Next, mevalonic acid (MVA), the first precursor of the terpenoid biosynthesis pathway, is produced by the conversion of HMG-CoA. MVA kinase and decarboxylation phosphorylate MVA, resulting in isopentenyl pyrophosphate.

(2)

Prenyl transferase catalyses the isomerization of IPP to dimethylallyl pyrophosphate (DMAPP), where three IPP molecules are added to create geranyl pyrophosphate (GGPP). The process begins with the condensation of two GGPP molecules to make phytoene, the pathway’s initial C40 carotene. Lycopene is next formed by desaturation of phytoene.

(3)

Lycopene undergoes several reactions that yield various cyclic carotenoids, such as β-carotene, γ-carotene, astaxanthin, torulahodin, and torulene.

Many studies show that utilising by-products as carbon sources can make carotenoid synthesis by yeasts industrially viable [104]. This also reduces environmental issues associated with waste and effluent emissions [105]. Research indicates that Rhodotorula glutinis 22P and Lactobacillus helveticus 12A produced carotenoids at a yield of approximately 8.4 mg/L [106]. Additionally, Phaffia rhodozyma displayed the highest yield of astaxanthin and β-carotene [107]. Many factors influence the synthesis of carotenoids in yeast. Certain red yeasts have β- and γ-carotene predominating at low temperatures, whereas torulene and torulularhodin develop more readily at higher temperatures [108]. The type of carbon and nitrogen sources in yeast has an unpredictable impact on the synthesis of carotenoids [109].

2.2.5. Carotenoids Produced by Fungi

Fungi inherited the ability to synthesise carotenoids from archaea as members of the Archaea-Eukarya lineage [110]. Thus, all fungal phyla contain species that contain carotenoids. Species that generate β-carotene [111,112] and resemble the C40-pathway in the class Halobacteria are common to all fungal groupings [113]. Furthermore, unique changes in β-carotene and its precursors through oxygenation have evolved in fungi. The crtYB fusion gene is characteristic of fungal carotenogenesis [114]. As demonstrated by Sulfolobus solfaticus [115], it originated from the co-transcribed overlapping genes of crtY and crtB in Archaea, where they are arranged in a gene cluster. Fungi have been producing pigments for hundreds of years [116]. Monascus purpureus, an ascomycete, is named after the reddish colour of rice tainted with the fungus [117]. Monascus can produce yellow, orange, or red pigments; the red pigments are more desirable for commercial uses [118]. In a submerged culture containing sugar and molasses, a Czech Republic-based business discovered a red-coloured Penicillium oxalicum [119]. A major barrier to the industrial application of fungi is the potential for co-production of mycotoxin. For instance, Fusarium species, which yield lycopene and β-carotene, have the potential to produce mycotoxins based on their growth conditions. In contrast, Neurospora intermedia pigment production does not result in the concurrent generation of mycotoxins, making it safe for human consumption [120]. The benefits of using fungi as sources include: (1) A wide range of nutrients, such as carbon sources (including potato extracts and maize stalk hydrolysate) and various inorganic and organic nitrogen sources. It complies with the current processing paradigm of high efficiency and environmental preservation. (2) The carotenoid process that fungi create is short-cycle, easily recyclable, and unaffected by climate. Furthermore, with high production efficiency, the fungus may continuously ferment carotenoids. (3) There are several by-products from the fungal fermentation broth, including protein, amino acids, fat, carbohydrates, and others, which can be used and developed further [121].

3. The Biological Activities of Carotenoid

Carotenoids are decisive structural components of the photosynthetic machinery that protect cells from photo-oxidative damage. They also have an ecological role by attracting pollinators and seed dispersers due to their different colours in fruits, flowers, and leaves. Certain carotenoids, such as violaxanthin and neoxanthin, serve as building blocks for the manufacture of the plant hormones strigolactone and abscisic acid (ABA) [122,123]. Provitamin A activity is the capacity of carotenoids to utilise carotene dioxygenase to produce vitamin A (retinal and retinol) [124]. To provide vitamin A activity, any carotenoid with at least one unaltered β-ionone ring can be cleaved down [125]. Provitamin A active carotenoids are β-carotene, α-carotene, γ-carotene, and β-cryptoxanthin. Because β-carotene has two β-ionone rings, it has 100% provitamin A activity, but lycopene has no β-ionone rings, hence it has no provitamin A activity. The biological characteristics of the carotenoid should ultimately be preserved, regardless of the architecture of the industrial facility, as biologically active carotenoids contain a variety of intriguing characteristics and roles that are vital to human health. Carotenoids and foods high in carotenoid content have been linked to several health advantages [126]. The prominent functions among these health benefits are retinoid precursors (such as vitamin A), antioxidant potential and free radical scavenging activity, immune system stimulation, and actions that prevent cancer and sunburn reactions [10]. Furthermore, because carotenoids protect against different reactive oxygen species (ROS), they are being proposed as preventive agents against diseases of the nervous system, the cardiovascular system, and the photosensitive, and neurological systems. The precise mechanisms underlying the bioavailability, absorption, transport, metabolism, and storage of carotenoids, as well as the variables that influence these processes, remain incompletely understood [18,127]. Out of all the known carotenoids, about 50 are precursors of retinoids, like vitamin A, with at least one unsubstituted β-ionone ring and a polyene side chain with at least 11 carbons. Ninety percent of vitamin A in the human body is found in the liver. About 40% of this is immediately utilized, with the remaining 40% being conserved. Thus, after consumption, provitamin A-containing carotenoids are absorbed and transformed in the gut into retinal, which is then transformed into retinol and transferred to the liver for storage [128]. Carotenoids have a wide range of antioxidant activity because they have numerous ways to counteract the effects of ROS. Naguib (2000) found that astaxanthin has stronger antioxidant activity than α-carotene, β-carotene, lutein, and lycopene [129]. Additionally, carotenoids are necessary for the maintenance and functioning of biological processes such as immunity, reproduction, and eyesight [130,131]. For instance, β-carotene enhances immune system performance and aids in the healthy operation of the reproductive system by shielding cells from free radicals [132]. The simultaneous pro-vitamin A and antioxidant properties are attributed to the structure of torulene, β-carotene, torulenehodin, and lycopene [52]. It is key to note, nevertheless, that carotenoids may not always be advantageous to humans. In particular, excessive and extended consumption of carotenoids, such as canthaxanthin, for cutaneous photoprotection (i.e., sunless tanning products) and cosmetic skin colouration can result in macular (eye) crystal deposition, according to reports from Geoffrey and Felix (1991) and Baker (2001) [133,134].

Table 2. Global commercial manufacturers of carotenoids and related products.

Carotenoid	Microbial Source	Name of Product/Company Names	Industrial Application	Country
β-carotene	Spirulina	Spirulina biomass (tablets, powder)/Tianjin Norland Biotech Co., Ltd.	Nutrition for animals, dietary supplements, and cosmetics	China
	Blakeslea trispora	Lyc-O-Beta, BetaBeads, and BetaCote (tablets and powder)/LycoRed Ltd.	Dietary supplement, food, and drink	Israel
	Dunaliella Bandawil	Dunaliella Hard Capsules/Nikken Sohonsha Corporation	Nutritional supplement	Japan
	Spirulina (Arthrospira platensis)	Hawaiian Spirulina (in tablet and powder form)/Cynotech Corporation	Dietary supplement	USA
Astaxanthin	Haematococcus pluvialis	ZestlifeTM (soft gels)/Zestlife Ltd.	Dietary supplement	United Kingdom
	Haematococcus pluvialis	Astalif® (oleoresin)/Algalif	Dietary supplements and cosmetics	Iceland
	Haematococcus pluvialis	AstaReal® (oleoresin, powder)/Co., Ltd. Fuji Chemical Industry	supplements, pharmaceuticals, and animal feed	Japan
	Haematococcus pluvialis	Astabio® (oil, powder, water soluble liquids) MC Biotech Sdn Bhd	Supplementary, medicinal, and cosmetic items	Brunei
	Haematococcus pluvialis	AstaFirst (powder)/Wefrst Biotechnology Co., Ltd.	Animal nutrition	China
	Spirulina	no name (powder)/E.I.D. Parry Ltd.	Dietary supplement	India
	Haematococcus pluvialis	CDX-°1 (powder)/Cardax, Inc.	Pharmaceutical and dietary supplement products	USA
Fucoxanthin	Phaeodactylum tricornutum	FucoVitalTM (powder)/Algatech International	Dietary supplement	Israel
	Laminaria japonica and Undaria pinnatifda Harvey	ThinOgen® (powder, softgel)/Beijing Gingko Group	Dietary supplements	China
Lutein	Chlorella sp.	Sun Chlorella Corporation	-	Japan
	Chlorella sp.	Maypro Industries Inc	-	USA
	Chlorella sp.	Far East Microalgae Ind Co., Ltd.	-	Taiwan
	Chlorella sp.	Roquette Klotze GmbH and Co. KG	-	Germany

lists several international commercial producers of microbial carotenoids along with their products.

4. Identification, Purification and Extraction of Carotenoids

Identification of any biosubstance can be done by physical methods (spectral) and/or chemical. Carotenoids can be identified by their ability to react with particular reactions in ways specific to their various functional groups. The functional groups ether (–O–), hydroxyl (–OH), carbonyl (C=O), ester (–COOR), carboxyl (–COOH), anhydride (–CO)₂O, nitrile (–C≡N), nitro (–NO₂), amide (–CONH₂), nitrozo (–N=O), azo (–N=N–), amine (–NH2), etc., can be identified through a series of procedures and chemical reactions. The primary attribute of carotenoids is their absorption spectrum, which is determined by their chromophore. Determining the absorption spectrum of carotenoids in visible light and various solvents is the most crucial step in the identification process. The absorption characteristics are investigated using TLC or column chromatography. Carotenoids can also be identified chemically via colour reactions, which identify the various functional groups in the pigment molecule. Methods for precisely determining some of the distinctive physical properties of carotenoids (such as NMR spectra, optical spectra, and electric spin resonance spectra) were refined through the advancement of experimental and theoretical physics and its various branches, such as magnetism, nuclear physics, atomic physics, optics, etc. Optical spectra, electric spin, NMR spectra, resonance spectra, and other precisely determined physical properties of carotenoids were made possible by advancements in theoretical and experimental physics and its various branches, such as optics, magnetism, atomic physics, nuclear physics, etc. Physical methods are utilized to study the various physical states of organic molecules in relation to their behaviour towards various physical agents used in the study, such as electromagnetic radiations, beams of elementary particles, magnetic fields, polarized light, etc. The basis of spectral approaches is the way electromagnetic radiation interacts with carotenoid molecules, which can absorb energy at various wavelengths. The majority of studies involving carotenoid extraction and purification were conducted on a laboratory scale [135]. A reliable, simple, and moderate saponification technique was created to quantify the carotenoids in plant material. Using a strongly basic resin, the chlorophylls and esterified fatty acids are selectively removed from the organic phase during the extraction process using acetone. HPLC was used to determine the primary carotenoid concentration of extracts from common plant material before and after the action of basic resin. Organic polar solvents such as acetone, dichloromethane, ethylic alcohol, methylic alcohol, dimethyl sulphoxide, and mixtures of solvents (diethyl ether/ethylic alcohol, hexane/acetone/dichloromethane/methylic alcohol/ethylic alcohol, acetone/methylic alcohol) are used for the extraction of carotenoids from the samples and their solubilization, regardless of the extraction method [136]. The fermentation process is followed by separation and purification methods to recover the pigments, and these usually represent the major production costs. After the extraction methods are set according to the characteristics of the sample, procedures to obtain the pure carotenoid are followed [137]. The technique of TLC is applied in the purification and isolation of carotenoids. When compared to column chromatography, this approach offers a higher degree of purification. The mobile phase, plate elution system, and stationary phase are selected based on the carotenoid’s characteristics [135]. Although the evolution of biotechnology contributed to the optimization of the synthesis of carotenoids, there is still a need for research to improve the process efficiency and commercial gain. Breaking the cell is required to release the intracellular carotenoids and extract their constituent parts [36]. Due to the molecule’s extreme sensitivity when removed from its surroundings, retrieving the extracted chemicals with the least amount of damage becomes difficult at this point [138]. Organic solvents are utilized in carotenoid extraction techniques in order to disperse the substances. Acetone, chloroform, dichloromethane, hexane, cyclohexane, methanol, ethanol, isopropanol, benzene, carbon disulfide, diethyl ether, and the recently published Supercritical Fluid Extraction (SFE) method with carbon dioxide are the most often used solvents. Conventional purification methods include differential extraction, countercurrent extraction, adsorption column chromatography, and differential crystallization [10]. Chromatography techniques, in conjunction with other techniques that enhance selectivity and separation efficiency, were employed in the majority of carotenoid research. Liquid chromatography coupled with mass spectrometry (LC-MS) is a viable method for the identification and purification of carotenoids [139,140,141]. This method involves comparing the mass spectra with standards or databases. If these are not accessible, the results can be improved by combining the methods of UV-VIS Photodiode Array (PDA) and Diode Array Detectors (DAD) with LC-MS/MS chromatography [142].

Three distinct coloured pigments are produced by Micrococcus bacteria: yellow, green, and red. The primary pigments of Micrococcus roseus have been identified through purification using an HPLC system, and mass spectra have been utilized to ascertain the molecular weight of the samples. Samples were subjected to controlled eluting with 80–100% ethanol at 470 nm using a photodiode detector on a C-18 column. β-carotene was the primary carotenoid found [143]. The pigments of the yeast R. glutinis and the bacteria Micrococcus luteus were purified using high-performance liquid chromatography (HPLC) with the aid of binary solvents, such as tert-butyl ether and ethyl-methyl ether. The samples were first filtered through a 0.2 µm cellulose filter and the reverse polymer phase C-30 at 10 °C. Using apo-CAR as an internal standard and a PDA, the compounds of interest were found and identified. Important carotenoids, such as cis and trans isomers, might be identified [144]. The composition of the carotenoids in a mutant strain of Paracoccus sp. called TSAO538 was examined using reverse phase HPLC, a Sherisorb ODS2 column, diode detectors, and a solvent that contained equal parts ethyl acetate, acetonitrile, and water. This strain is capable of selectively synthesising canthaxanthin. Kieselgel 60 F254 silica plates were subjected to TLC using diethyl ether, hexane, or ethyl acetate. Redistilled acetone and diethyl ether were used to record the absorption spectra using a UV/VIS spectrophotometer. A mass spectrometry analysis was then carried out [145]. Recombinant enzymes have been employed by researchers to release precursors of carotenoid pigments, which have been detected using Gas Chromatography-Flame Ionisation Detector (GC-FID) and Gas Chromatography-Mass Spectrometry (GC-MS) and further examined using HPLC-DAD and HPLC-MS [146]. Separation and purification procedures for analysing carotenoids are well known. However, future research should concentrate on developing cheaply scaled-up purifying techniques, as they are critical to industrial production [147].

5. Carotenoids’ Impact on Human Health

Carotenoids are known to be extremely significant substances for human health. Fruits, vegetables, and whole grains contain high levels of antioxidant compounds such as polyphenolic acids, ascorbic acid, carotenoids, and tocopherols which reduce the risk of many chronic diseases such as T2D, neurodegenerative disorders, CVDs, and various types of cancer [148]. Proinflammatory mediators such as circulating proinflammatory cytokines, oxidised phospholipids, tumour necrosis factor-alpha (TNF-α), inflammatory-stimulating prostaglandin E2, nuclear factor kappa-light-chain-enhancer of activated B cells, and C-reactive protein are generally associated with elevated levels of these chronic diseases. Carotenoids’ antioxidant characteristics allow them to modulate the amounts of these mediators by nuclear factor-erythroid 2-related factor 2 or oxidative stress modulation and peroxisome proliferator-activated receptor-mediated overexpression of antioxidant and cytoprotective Phase II enzymes [149,150,151,152]. Overexposure to oxidative stress promotes bone resorption and inhibits bone formation, increasing the risk of osteoporosis and bone loss. Carotenoids’ antioxidant qualities, however, might contribute to better bone health [153]. Age-related reductions in muscle mass and strength (attributed primarily to a lack of exercise, vitamin D, or dietary protein) were linked to poor physical function and other problems. A diet high in antioxidants, such as carotenoids, could mitigate the age-related loss of muscle and physical function. In the Framingham offspring prospective cohort study among elder participants (average age of 61 years), higher intakes of total carotenoids zeaxanthin/lutein, and lycopene were related to an enhanced annualized change in faster gait speed and grip strength [154]. The zeaxanthin, meso-zeaxanthin, and xanthophylls lutein, accumulate as macular pigment in the fovea and inner plexiform layer of the retina in humans, which protects the retinal membrane against the damaging effects of short-wavelength high-intensity light and enhances visual acuity. Remarkably, it has been demonstrated that low-density lipoproteins and high-density lipoproteins mediate the selective uptake of lutein and zeaxanthin in the human retina. Epidemiological and clinical studies have witnessed the critical role of dietary zeaxanthin/lutein in lowering the risk of age-related macular degeneration [155]. Carotenoids are frequently utilized in cosmetics, primarily for their UV-protective properties. Furthermore, Carotenoids have the potential to improve skin characteristics. According to a comprehensive evaluation of 11 clinical studies, supplementing with astaxanthin at a dose of 3 to 6 mg/d for 2 to 16 weeks enhanced the moisture content, texture, and appearance of wrinkles on the skin [156,157].

Carotenoids protect against oxidative stress and inflammation by quenching singlet oxygen, oxidising, isomerizing, and scavenging free radicals [158,159]. Beyond their antioxidant properties, carotenoids may also directly support the immune system [160], shield skin from UV ray damage [161], and fight against certain types of cancer [162,163] (Figure 3). They can avert a vitamin A deficiency, which is recognized as the necessary substance for fostering development, embryonic development, and visual function. Carotenoids’ subcellular distribution is determined by their lipophilicity; they are concentrated in lipid droplets and other lipophilic compartments, such as membranes [164]. Carotenoids are thought to act as antioxidants to shield membranes. Polar carotenoids also can control membrane fluidity [165,166]. The effectiveness of using carotenoids in the prevention and treatment of several chronic illnesses is well established. long-term illnesses. They have anti-inflammatory qualities and can stimulate an organism’s immune system.

Research has demonstrated that consuming foods high in lycopene may reduce the incidence of atherosclerosis and other cardiovascular conditions [167,168,169]. The potential of lycopene to modify high-density lipoprotein functioning and diminish systemic and high-density lipoprotein-associated inflammation is most likely responsible for these beneficial effects [170]. Additionally, studies have shown that astaxanthin can prevent atherosclerotic cardiovascular diseases by lowering oxidative stress and inflammation and improving glucose and lipid metabolism [171]. Providing the body with astaxanthin enables us to efficiently mitigate the adverse consequences arising from the oxidation and breakdown of cellular components. Lycopene may inhibit the release of chemokines and pro-inflammatory cytokines, according to a different study [172]. Carotenoids protect the retina by preventing cataracts and age-related macular degeneration [173,174]. There is concrete proof of lutein and zeaxanthin’s beneficial effects on eye health. In addition to improving visual performance and having good side effects like contrast sensitivity, glare tolerance, and photo-stress recovery, they may lower the incidence of age-related macular eye illnesses. β-Carotene and lutein have been shown to improve cognitive function [175]. Research has also been done on lycopene’s possible benefits for treating neurological illnesses like Alzheimer’s disease [170,176]. Due to its strong antioxidant activity, torularhominos can be employed as a neuroprotective drug against H₂O₂-induced oxidative stress. It was previously believed that lutein may be associated with the potential regulation of inflammation-related neurodegenerative illnesses [177]. Humans with amyotrophic lateral sclerosis disease showed protection from lycopene [169]. Carotenoids are being promoted by organizations like the International Carotenoid Society, but more studies and practical science on the health benefits of carotenoids in humans and animals need to be supported by all stakeholders involved.

6. Future Perspective

Carotenoids produced by microorganisms are popular in the commercial market due to their natural origin, vibrant hues, and ease of cultivation with shorter production times. The microorganism’s capacity to break down agro-industrial waste lowers the total cost of carotenoids’ synthesis. Low carotenoid content, safety issues with specific opportunistic infections that produce carotenoids, and consumer acceptance of microbial carotenoids frequently necessitate meticulous testing are further obstacles to microbial carotenoid production. Therefore, for the commercial production of microbial carotenoids, process optimization of the industrial-scale carotenoid production is required. The key elements of the production, extraction, and commercialization stages were covered in this broad review of the state-of-the-art microbial carotenoid technology. It is obvious that naturally occurring carotenoids obtained through biotechnological processes are becoming more and more popular due to their advantageous health effects and (bio)nature, progressively displacing the already used synthetic carotenoids. Thus, there is a lot of room for growth and potential for carotenoids produced by microorganisms in the global food and pharmaceutical industries. However, there are still processing industrial obstacles to be addressed, such as the high cost of the current technologies used to produce and extract carotenoids on an industrial scale or the need to use a lot of non-benign solvents as extract agents. Scientific research advancements have the potential to enhance the quality and value of microbial carotenoids, rendering this domain and market appealing to multiple biotechnological sectors.