Production and Characterization of Microbial Carotenoids as an Alternative to Synthetic Colors: a Review

Abstract

Carotenoids are important group of pigments with specific applications in food, pharmaceutical, and cosmetic industries owing to their biological properties like antioxidant, anti-carcinogenic, and immune-modulator. The demand of carotenoids as nutraceutical compounds has triggered the research to explore a commercially viable process for economic production of carotenoids. This article presents a review of carotenoids from microbial origin identifying the conditions used for their cultivation and applications.

Keywords:

• Carotenoids
• Microorganisms
• Astaxanthin
• Lycopene
• β-carotene
• Canthaxanthin
• Yeast

INTRODUCTION

In recent years, increasing evidences for toxicological effects of synthetic colors have prompted regulatory agencies, world over to drastically prune the list of permitted synthetic food colors. As a result of stringent rules and regulations applied to chemically synthesized/purified pigments, consumer preferences and growth of food industry, demand of carotenoids as safe and suitable coloring agents is on rise. A recent report predicts that global market demand for carotenoids has been growing at 2.9% per annum and is expected to reach at $ 1.02 billion by 2009 as consumers continue to look for natural ingredients.[1] In addition to acting as key precursors of vitamins and hormones, carotenoids fulfill a variety of biological and physiological roles in living organisms, ranging from light harvesting in photosynthesis to protection against light and oxidizing agents.[2–4] Another reason for the trend towards natural food colors is the reported correlation between consumption of certain carotenoids, like lycopene and its relation to prostate cancer.[5] More than 600 different carotenoids are produced by plants, algae, bacteria, and fungi.[3] However, only a few can be obtained in useful quantities by chemical synthesis, extraction from their natural sources or microbial fermentation.[4] Strategies employed to increase product yields from microorganisms include the use of overproducing strains, addition of bacterial and fungal enzymes that disrupt the yeast cell wall and the development of low cost culture media that diminishes production cost.[6 ,7] Microorganisms offer economical production of carotenoids through biotechnological methods and provide an alternative to chemical synthesis.[8] Although a wide variety of carotenoids occur in nature, only a few have been available commercially including β-carotene, lycopene, astaxanthin, canthaxanthin, and lutein.[4] The growing demand of carotenoids has triggered the development of carotenoids rich food products with nutraceutical properties for additional health benefits.

CHEMICAL CHARACTERISTICS

Carotenoids are a class of hydrocarbons consisting of eight isoprenoids units found in such a manner that the arrangement of isoprenoids units is reversed at the center of the molecule so that the two central methyl groups are in 1,6- positional relationship and remaining non-terminal methyl groups are in a 1,5-positional relationship.[9] Carotenoids constitute of large polyene chain with 35–40 carbon atoms, which is considered as backbone of the molecule. This polyene chain is also the feature mainly responsible for the chemical reactivity of carotenoids towards oxidizing agents and free radicals, and hence for any antioxidant role.[10] In general, the longer the polyene chain, the greater the peroxyl radical stabilizing ability. The basic skeleton of carotenoids may be modified in various manners, such as hydrogenation, dehydrogenation, cyclization, double bond migration, chain shortening or extension, rearrangement, isomerization, introduction of oxygen functions, or combinations of these processes, resulting in a great diversity of structures (Fig. 1).

Figure 1 Structures of common carotenoids.

HEALTH BENEFITS

Of 600 carotenoids from natural sources that have been characterized, fewer than 10% serve as precursors of vitamin A.[10] Recent interest in carotenoids has been stimulated by epidemiological studies that strongly suggest that consumption of carotenoid-rich foods reduces the incidence of several diseases such as cancers, cardiovascular diseases, age-related macular degeneration, cataracts, diseases related to low immune function, and other degenerative diseases.[11–14] Independent of the nutritional capacities, the carotenoids that contain nine or more conjugated double bonds can inactivate certain reactive oxygen species, such as singlet oxygen.[15] The long system of alternating double and single bonds common to all carotenoids allows them to absorb light in the visible range of the spectrum.[16] This feature has particular relevance to the eye, where lutein and zeaxanthin efficiently absorb blue light.[17] Reducing the amount of blue light that reaches the structures of the eye that are critical to vision may protect them from light-induced oxidative damage.[18] There have been many reports over the years of a positive effect of dietary or supplemental carotenoids on improving fertility or reproductive capacity in a number of animals.[19] Krinsky and Deneke[20] demonstrated that carotenoids including β-carotene were capable of inhibiting free radical-induced oxidation in liposomal lipids. However, involvement of carotenoids in preventing oxidation of low-density lipids is still controversial. Higher plasma carotenoids at baseline have been associated with significant reductions in cardiovascular disease risk in some prospective studies[21–24] but not in others.[25–28]

MICROBIAL SOURCES

Among microbial sources of carotenoids, besides algae like Dunaliella species, Eubacteria and yeasts belonging to the Basidiomycota are of special interest to carotenoids researchers.[29–31] These pigmented yeasts have an advantage over algae, fungi, and bacteria due to unicellular and relatively high growth rate with utilizing low-cost fermentation media.[32] Carotenoid biosynthesis is a specific feature of the Rhodotorula species,[33 ,34] Rhodosporidium,[35] and Phaffia genera.[33 ,36] The distinctive color of yeast cell is the result of pigments that yeast creates to block out certain wavelengths of light that would otherwise be damaging to the cell. Typical concentrations reported in literature for carotenoids produced from red yeasts range from 50–350 μg/g dry weight.[37 ,38] A number of carotenogenic genes have been cloned from microorganisms, thereby allowing the recombinant biosynthesis of different acyclic and cyclic carotenoids and oxo-carotenoids.[39] A much wider range of carotenoids can be produced by breeding biosynthetic genes and evolving new enzyme functions.[40] Most of the carotenogenic genes employed in recombinant biosynthesis are derived either from Rhodobacter or Erwinia species.[41 ,42] E. coli is a suitable host for carotenoid production because it has a powerful genetic tool system for metabolic engineering and is able to make various carotenoids such as lycopene, β-carotene, canthaxanthin, zeaxanthin and astaxanthin.[43]

Fermentation Conditions

Sucrose and glucose are the most common carbon sources used in the production of carotenoids. The use of glucose can lead to a higher efficiency in the specific production of carotenoids (1000 μg/g dry cell weight) by Rhodotorula spp.[8] A number of studies have been carried out in recent years on the fermentation of agricultural wastes (oats, wheat, barley, corn, rice, sugarcane molasses, grape must and cheese whey) to produce carotenoids by different strains in shake flask fermentation.[8,31,33,44] However, the types of carotenoids and their relative amount may vary depending on the cultivation medium, temperature, pH, rate of aeration and luminosity.[45] Carotenoid formation is triggered by depletion of nitrogen sources while sufficient carbon content is maintained. C/N ratio variation from 44–10 was reported for maximum carotenoid production for different types of yeast strains.[46 ,47]

Carotenoid-synthesizing yeasts are aerobes and the airflow rate in the culture is an essential factor to assimilate the substrate as well as for growth rate, cell mass and carotenoid synthesis. Intensive aeration stimulates β-carotene synthesis while proportion of torularhodin decreases with slight changes in torulene.[48] However, Davoli et al.[37] found that at higher aeration the concentration of total carotenoids increased but the composition of carotenoids (torulene > β-carotene > γ-carotene > torularhodin) remained unaltered for R glutinis, in contrast, Sporobolomyces roseus responded to enhanced aeration by a shift from the predominant β-carotene to torulene and torularhodin, indicating a biosynthetic switch at the γ-carotene branch point of carotenoid biosynthesis. Aeration could be beneficial to the growth and performance of microbial cells by improving the mass transfer characteristics with respect to substrate, product, and oxygen.[45] The pH of growth medium influenced not only biosynthetic activity of culture, but also culture growth rate. Aksu and Eren[45] reported that pH 7 yielded the highest biomass and carotenoid concentrations for R. mucilaginosa. Specific growth and carotenoid formation rates of the yeast increased with increase in temperature up to 30°C, however it decreased sharply above 30°C. This might be due to the denaturation of enzyme system of microorganism at higher temperatures. Many investigators[49–51] have tried the optimization of fermentative medium for carotenoids by Rhodotorula and Rhodobacter spp using trace metals like MgSO₄, FeSO₄, Na₂CO₃, MnSO₄, ZnSO₄, Al₂(SO₄)₃, and Co(NO₃)₂. Stimulatory effect of trace metal ions on carotenogenesis has been explained by hypothesizing a possible activation or inhibition mechanism on carotenogenic enzymes specifically desaturases involved in carotenoid biosynthesis.[3] Agents such as detergent additives, oils and surfactants have been suggested as activators for increasing carotenoid productivity.[52] Most of the above optimization efforts have relied on statistical experimental design and response surface analysis as these techniques cut down the development time and high experimental costs.

Extraction and Characterization of Carotenoids

The steps in determining microbial carotenoids are culture, cell disintegration, extraction, separation and quantitation. Cell disintegration is usually achieved by mechanical breakage and acid or alkaline hydrolysis, liquid or supercritical CO₂ extraction.[53–55] Enzymatic digestion of cells also appears as an attractive option, presumably resulting in higher recovery rates of carotenoids from the microbial matrix.[56] A combination of lysozyme/lyticase with synergistic sonication and freeze thawing provide a beneficial and robust alternative to other mechanical disruption methods.[57] Extracts prepared from A. luteus containing lyticase and other enzymes were found to be effective in lysis of different yeasts.[58 ,59] Lipase has been used to disintegrate lipid droplets containing carotenoids and to prevent capillary plugging of the HPLC system.[60 ,61] Most of the extraction processes employed acetone, ethyl acetate, cyclohexane, petroleum ether, and chloroform for recovery of carotenoids. Carotenoids have been isolated and analyzed using thin layer chromatography, open column chromatography and high performance liquid chromatography while for identification and structure elucidation, visible spectrophotometry, NMR, and mass spectrometry are being used.[62–64] Quantification of pigments is usually done based on calibration curves of available commercial standards or purified pigments; other peaks may be quantified using conversion formulae and values available in the literature.[54]

Analysis of pigment accumulation in Rhodotorula glutinis and Phaffia rhodozyma suggested biosynthesis of torulene from β-zeacarotene, the monocyclic product derived from neurosporene, through desaturation of the 7,8-dihydro-end group rather than cyclization of 3,4-didehydrolycopene.[57 ,65] However, the most frequently reported biosynthetic route is that involving the oxidation of γ-carotene via torulene to give torularhodin as the most polar final product.[66] Red yeasts are thought to synthesize only a limited range of carotenoids. Typical representatives of yeasts producing these carotenoids are species of Sporobolomyces and Rhodotorula.[66]

Commercially Significant Carotenoids

Astaxanthin

Astaxanthin (3,3'-dihydroxy-β, β-carotene-4,4'-dione), is an important and valuable ketocarotenoid accumulated by the fresh water microalga, Haematococcus pluvialis (Chlorophyceae),[67 ,68] Brevibacterium, Mycobacterium lacticola,[69] Agrobacterium auratium,[70] and Xanthophyllomyces dendrorhous.[71] Studies using low cost by-products and residues of agro-industrial origin have shown the possibility of astaxanthin production from several materials such as molasses,[6] grape juice,[55] hemicellulose hydrolysates of eucalyptus,[72] mustard waste hydrolysates,[73] and hydrolysates from Yucca fillifera.[74] Orosa et al.[75] reported strong effect of acetate and malonate compounds on the stimulation of astaxanthin synthesis and accumulation being four times higher in cultures with these compounds, although at higher concentrations these compounds inhibit growth. The availability of a carbon source during the stationary phase is important for astaxanthin synthesis, because cells suspended in medium or buffer in the absence of carbon do not exhibit increased astaxanthin concentration, but they do in media containing carbon or spent media from fermentation.[61]

Schroeder and Johnson[76] reported that astaxanthin can protect Xanthophyllomyces dendrorhous against oxidative stress. Under artificial cultivation conditions oxidative stress in Xanthophyllomyces/Phaffia and certain other yeasts results in a biosynthetic switch from β-carotene to oxygen containing carotenoids (xanthophylls). This can be triggered by a high degree of aeration in combination with ROS inducers such as duroquinone, H₂O₂, or ethanol.[37,77 ^– 79]

This orange red pigment is widely used as a food colorant, in cosmetics and medical applications due to its high antioxidant activity. These carotenoids are the main source of colors in fins, skin, and flesh of the rainbow trout as well as other kinds of salmonid fish.[80] In addition to its effect on color, one of the most important properties of astaxanthin, is its antioxidant properties which has been reported to surpass those of β-carotene or even α-tocopherols (100–500 fold high)[81] with extraordinary potential for protecting the organisms against a wide range of ailments such as cardiovascular problems, different types of cancer and immunological diseases. Various astaxanthin supplements consisting of injectable solutions, capsules or topical creams have been manufactured for sunburn prevention from UV exposure.[82]

Canthaxanthin

Canthaxanthin is a di-ketcarotenoid (β, β-carotene-4,4'-dione), occurring naturally in a wide variety of living organisms, where it plays important roles in animal displays of maturity and in the protection of tissues against oxidizing free radicals.[83] The low productivity of canthaxanthin by Brevibacterium KY-4313 and the slow growth (several weeks) of the microalgae Dictyococcus cinnabarinus together with the relatively low yield appeared to preclude biotechnological production of canthaxanthin by these microorganisms,[84] thus production of this ketocarotenoid is still dominated by chemical synthesis. However recent investigations have reported Haloferax alexandrinus as one of the most promising microorganisms for the commercial production of canthaxanthin. The ability of this archaeon, as a member of halophilc Archaea, to grow at high concentrations of NaCl and that its cells lyse readily in low ionic strength solution (fresh water) are remarkable advantages from the biotechnological point of view. Among the isolated strains, strain TM has been reported to produce remarkable amount of canthaxanthin (700 ug/g dry cells).[86] Canthaxanthin has been used as food and feed additive for egg-yolk, fish and crustacean farms, in cosmetics and as an orally administered pigmenting agent for human skin in pharmaceutical applications.[85]

Lycopene

Commercial lycopene preparations are formulated as oil suspensions or water dispersible powders. Among carotenoids with nine or more conjugated double bonds, lycopene is the most effective singlet oxygen quencher.[86] Lycopene, the strongest natural antioxidant, is a symmetrical tetraterpene assembled from 8 isoprene units. Fungi of the genera Phycomyces and Blakeslea are potential lycopene producers. Cyclase inhibitor 2-(4-chlorophenylthio)-triethylamine (CPTA)[87] and chemical stimulators such as pyridine, imidazole, and methylheptenone have been reported to stimulate lycopene accumulation in B. trispora and P. blakesleeanus.[88]The most important peculiarities of lycopene are its very high singlet oxygen-quenching activity and a manifest capacity to suppress the proliferation of MSF-7 tumor cells.[89] Lycopene finds applications in beverages, dairy foods, surimi, confectionery, soups, nutritional bars, breakfast cereals, pastas, chips, sauces, snacks, dips, and spreads.[90]

β-Carotene

It is produced predominantly by a number of fungi (Mucor, Phycomyces) and commercial production exploits Blakesea trispora with a record β-carotene content of 4-5 g/l,[89 ,91] while microalgae source includes Dunaliella which is the best carotenoid providing organism among the algae and other organisms. Commonly cultivated species are Dunaliella salina and D. bardawil.[92] Algal carotenoids are present in chloroplasts as a complex mixture characteristic of each class of algae. In its natural all-trans form, the molecule is long and straight, constrained by its eleven conjugated double bonds. β-carotene helps to mediate the harmful effects of free radicals, which are implied in over 60 life-threatening diseases including various forms of cancer, coronary heart disease, premature ageing and arthritis.[93] β-carotene has been applied to a range of food products including margarine, cheese, fruit juices, baked goods, dairy products, canned goods, confectionary and health condiments. It has also been used to improve the color of birds, fish and crustaceans as well as improving the appearance of pet food.[94] New food applications like the coloration of processed meats (sausage, ham), marine products like fish paste, surimi and tomato ketchup have also been described.[95]

CONCLUSION

Carotenoids are valuable bioactive compounds with nutraceutical properties. Yeasts such as Phaffia, Rhodotorula and algae like Dunaliella have been identified as commercially significant sources of carotenoids. However, the types of carotenoids and their relative amount may vary depending on the cultivation medium, presence of inhibitors and stimulators, temperature, pH, rate of aeration and luminosity. Although various physical, chemical, and enzymatic methods have been applied for extraction of carotenoids, recovery from cell matrix is still a costly process. Various carotenoids of microbial origin such as astaxanthin, lycopene, β-carotene, and canthaxanthin are being commercialized to some extent and finding applications in beverages, dairy foods, cereal products, meats, cosmetics, pharmaceutical, aquaculture and others. The growing demand of carotenoids has triggered the research to be more focused on the commercial production of carotenoids. Biotechnological development of new strains that can withstand robust industrial conditions and utilization of industrial waste as substrate would help to bring down the economics of the whole process. A vast biodiversity of microorganisms is still to be explored from various habitats for exploitation as potential carotenoid factories.