Isolation and Characterization of Natural Blue Pigments

The food and beverage industry is seeking to broaden the palette of naturally derived colorants. The natural food colorant market has grown to over $1 billion dollars annually. Many approved natural food colorants are available for replacing the red, orange, and yellow synthetic dyes. However, there is still a lack of available green, blue, and violet natural colorants. The overall goal of this project is to elucidate the structures of novel blue, green, and violet pigment compounds produced within the natural products world. New blue pigment compounds may serve as patentable lead candidate compounds for future use as natural blue food colorants. To provide a thorough and definitive framework from which to proceed with the discovery of new blue pigments, organic blue compounds from natural plant, animal, fungal, and microbial sources are first reviewed (Chapter 1). The scarcity of blue colored metabolites in the natural world relative to metabolites of other colors is discussed and structural trends common among natural blue compounds are identified. These compounds are grouped into seven structural classes and evaluated for their potential as new color additives. Although considerable effort has been devoted to the search for new blue colorants in fruits and vegetables, less attention has been directed towards blue compounds from other sources such as bacteria and fungi. Microorganisms, especially from the marine environment, continue to be a prolific source of previously uncharacterized secondary metabolites including pigments. Chapter 2 investigates pigments from marine-derived microbes for potentially novel lead compounds and led to the dereplication of a variety of known microbial pigments. Chapter 2 also discusses a variety of established extraction, fractionation, and chromatographic purification techniques used in this work. Known compound were dereplicated primarily using HPLC separation coupled with UV-Vis detection and high resolution tandem mass spectrometry. Chapter 3 covers the identification of the purple pigment of acidophilic Zygogonium sp. algae from Yellowstone National Park. The purple pigment, found to be a macromolecular ferric oligosaccharide gallate complex, was isolated and characterized by a great variety of chemical and spectroscopic techniques. Although the purple pigment is not suitable for use in commercial products due to low color intensity and iron content, the pigment is of ecological and natural products chemistry interest. The oligosaccharide gallate species detected in this work represent a new subclass of natural product molecules. There is also evidence that the pigment may represent one of the only known ferric biological sunscreens. The need for an accurate quantitation of bound galloyl groups in this work led to the development of an improved LC/MS method for the determination of bound galloyl groups was presented in Chapter 4. The structure elucidation of a series of novel trichotomine analogs (trichotomine G3, trichotomine G4, and trichotomine G5) extracted from Clerodendron trichotomum (Japanese kusagi berry) is presented in Chapter 5. The intense blue hue and low toxicity of the trichotomines make them compelling as natural blue colorant lead candidates. The novel analogs contained 6-O-malonyl attachments. Higher molecular weight trichotomine glycosides were identified by HRMSn analysis as well. The glycosides were found to be attached to the trichotomine carboxylic acid groups (COOGlu) as opposed to the indole rings (NGlu) as was previously reported. Unfortunately, color stability testing indicated that the acid and heat stability of the trichotomine analogs is not sufficient for their use as consumer colorants. Chapter 6 involves structural studies of the complex polymeric pigment “gardenia blue”. The pigment is derived from the reaction of the iridoid genipin (Gardenia jasminoides) with a primary amine. The blue nature-derived colorant is used in few food coloring applications due to its pseudo-natural status and limited regulatory approval. Chapter 6 includes the NMR analysis of a commercial “gardenia blue” product provided by San Ei Gen F.F.I., chromatographic and spectroscopic study of “gardenia blue” pigment generated fresh by the reaction of genipin with methylamine and other amines, and theoretical (DFT) calculations of 2-pyrindine dimers. The results suggested that the chromophoric portions of the “gardenia blue” pigment involve conjugated dimers of 2-pyrindine ring systems linked by an unsaturated C1 or C2 bridges. A natural blue colorant suitable for consumer beverage applications, termed the “holy grail” in the colorant field, has not yet been found. The work covered in this dissertation contributes substantially towards that goal. The pursuit of more promising research avenues, such as investigating avenues for increasing the stability of the trichotomine derivatives or searching for more heat stable versions of phycocyanin, may lead to a suitable natural blue consumer colorant in the future.