Chapter 1
Introduction of Natural Pigments from Microorganisms

Siyuan Wang, Fuchao Xu and Jixun Zhan

Department of Biological Engineering, Utah State University, Logan, UT, USA

1.1 Introduction

Pigments are widely used in a variety of industries. In the food industry, one of the most important goals is to develop foods that have an attractive flavor and appearance. Artificial food coloring using synthetic dyes can make foods more appealing and desirable. However, the safety of these dyes has been questioned. Recent research has linked synthetic food dyes to a number of potential health problems, such as cancer in animals and attention-deficit disorder in children (Potera 2010). Synthetic colorants are criticized for having these problems, and consumers are showing more and more interest in products that do not include artificial coloring agents. Therefore, various natural sources of food-grade colorants are in high demand. The textile industry also uses millions of tons of dyes, pigments, and dye precursors every year, and almost all of them are manufactured synthetically (Chequer et al. 2013). Synthetic dyes have serious limitations in that their production involves the use of toxic chemicals and can generate hazardous wastes, which is unfriendly to the environment and to human health (Khan et al. 2013).

Biological pigments are substances from biological sources that have a particular color, corresponding to their structure. They are found in plants, animals, and microbial organisms. Natural pigments have been long studied, but they are receiving increasing attention from industry because of the potential health and environmental concerns around synthetic dyes. Biological pigments from microbial cells are termed "microbial pigments." In addition to their function as colorants, some microbial pigments are also used to promote human health, providing key nutrients or compounds required by the body. Some also have particular biological activities, such as anti-inflammatory, antibiotic, anticancer, and immunosuppressive properties (Soliev et al. 2011). Microbial pigments with fluorescence are used in laboratories to label antibodies (Mahmoudian et al. 2010). Some pigments can also be used to indicate the progress of specific reactions or to track pH changes through changes in their color (Venil et al. 2014). A large number of pigments are produced by various species of bacteria, yeasts, fungi, and algae, with colors including brown, black, red, orange, yellow, green, blue, and purple, and structures such as carotenoids, anthraquinones, flavonoids, and tetrapirroles. Different biosynthetic enzymes are involved in the biosynthesis of microbial pigments. For example, carotenoids are typically synthesized by terpene synthases, flavonoids are assembled by polyketide synthases (PKSs), and indigoidine - a bacterial blue pigment - is synthesized by a nonribosomal peptide synthetase. Microbial pigments are used for different purposes depending on their color property and biological function. This chapter covers a variety of microbial pigments from eukaryotic and prokaryotic sources and discusses their properties and applications.

1.2 Microbial Pigments from Eukaryotic Sources

The cells of eukaryotes such as plants, animals, and fungi contain a nucleus and other organelles. Eukaryotic microorganisms produce a lot of different pigments. Some representative pigments from these organisms are described in this section, categorized according to their source: algae, fungi, and yeasts.

1.2.1 Pigments from Algae

Algae produce a variety of pigments. The most commonly used in the industry is the carotenoid ß-carotene (Figure 1.1). Carotenoids belong to the family of tetraterpenoids and are found in the chloroplasts and chromoplasts of plants, algae, fungi, and some bacteria (Asker et al. 2007). They are yellow, orange, and red pigments that can be used for coloration. ß-carotene is a red-orange nonpolar pigment that can be obtained from Dunaliella salina, a kind of marine green microalga. The production of ß-carotene in D. salina is affected by high salinity, temperature, and light intensity. A high ß-carotene content in D. salina can help it protect itself from intense light and osmotic pressure in the ocean (Oren 2005). ß-carotene is well known for its antioxidant activity and for its use as food supplement (Stargrove et al. 2008). It is commercially produced across the world, due to its widespread use (Oren 2005). The first company to manufacture and sell natural ß-carotene, Betatene Ltd., was established in 1985 (Nelis and Deleenheer 1991). Production of ß-carotene from D. salina is often seen in large open ponds located in or near salt lakes in Australia, the United States, and China.

Figure 1.1 Structures of four representative carotenoids: ß-carotene, lutein, canthaxanthin, and astaxanthin.

Besides ß-carotene, many other carotenoids are produced by microalgae. For example, lutein (Figure 1.1) is obtained from different green algae, such as Chlorella, Chlorococcum, Chlamydomonas, and Spongiococcum. Lutein is a red-orange pigment that is generally insoluble in water. For some time, it was widely used in chicken feeds to improve the color of broiler chicken skin and egg yolks (Philip et al. 1976). In the human body, lutein is concentrated in the macula. Some research has revealed that lutein protects eyes against oxidation (Berendschot et al. 2000; Malinow et al. 1980). Canthaxanthin (Figure 1.1), a dark red food coloring agent, is another example of a cartenoid produced by algae. Dictyococcus cinnabarinus was reported to produce it canthaxanthin in 1970. The final concentration of cellular canthaxanthin in this organism is 1.0-1.2 mg/g (Tuttobelll and Ranciag 1970). Astaxanthin (Figure 1.1) is a red terpene that is biosynthesized by Haematococcus pluviais with up to 2% dry weight quantity (Nonomura 1990). This compound is a food coloring agent approved by the US Food and Drug Administration (FDA).

Algae produce many other microbial pigments, including water-soluble green chlorophyll, blue phycocyanins, and red phycoerythrins, from Rhodophta, Cyanophta, and Cryptophyta, respectively (Telford et al. 2001). Halobacterium spp. have been found to be responsible for the red color in the Great Salt Lake, Dead Sea, and Lake Magadi (Oren 2005).

1.2.2 Pigments from Fungi

Fungi comprise a diverse group of eukaryotic organisms, including yeasts, molds, and mushrooms. Some fungi are known to produce color compounds with particular biological properties. Many fungal pigments possess ecological functions varying from providing protection against environmental stress to preventing photo-oxidation. Some pigments, such as flavins, can even act as cofactors in enzyme catalysis (Mapari et al. 2010).

Riboflavin (vitamin B2) is a yellow food colorant that is approved for use in many countries. It is also used in the clinic to treat neonatal jaundice (Bailey et al. 1997) and it has been reported to prevent migraine (Sandor et al. 2000). Its structure is shown in Figure 1.2. Many molds can be used to produce riboflavin through fermentation (Jacobson and Wasileski 1994; Santos et al. 2005; Stahmann et al. 2000). Ashbya gossypi has been widely used in the production of riboflavin, as it provides a high yield and good genetic stability. Its final riboflavin level can reach 15 g/L (Broder and Koehler 1980).

Figure 1.2 Nine representative fungal pigments: riboflavin, monascin, ankaflavin, rubropunctatin, monascorubramine, atrovenetin, herqueinone, bikaverin, and catenarin.

A variety of color compounds have been discovered from fungi. The same genus may produce different pigments. This is exemplified by Monascus. Monascus can be classified into four different species: M. pilosus, M. purpureus, M. ruberand and M. froridanus. Different Monascus species produce many different industrially important pigments with three colors: red, orange, and yellowish. For example, M. purpureus 192F produces the yellow pigments monascin and ankaflavin, the orange pigment rubropunctatin, and the red pigment monascorubramine (Figure 1.2). Monascorubramine is the major product. The pH and nitrogen source in the fermentation broth affect the composition and yield of the pigments. Supplementation of Monascus pigments as a coloring agent into food can provide novel flavors (Chen and Johns 1993). These fungal metabolites have also shown interesting biological activities. For example, monascin and ankaflavin are natural 5´ adenosine monophosphate-activated protein kinase (AMPK) activators and have shown hypolipidemic and anti-inflammatory activities (Hsu et al. 2013, 2014). The two compounds have been found to improve memory and learning ability in amyloid ß-protein intracerebroventricular-infused rat by suppressing Alzheimer's disease risk factors (Lee et al. 2015). Anticancer, antiatherosclerotic, antiallergic, antioxidant, and antidiabetic properties have also been reported (Hsu and Pan 2014; Hsu et al. 2011, 2012, 2014; Lee et al. 2012).

While the most common method of pigment production from microbes on an industrial scale is submerged fermentation, an immobilized culture system or...