The demand for natural (rather than synthetic) coloring agents and food additives is growing rapidly. This is partly because consumers are increasingly looking for minimally processed foods that are free from synthetic chemical preservatives. It is also because they are associated with nutritional and health benefits.1 Some of the more well-known natural food colorants, including anthocyanins, carotenoids, betalains, chlorophylls, and curcumin, are considered beneficial for health due to their antioxidant activity.
Natural pigments can be derived from a variety of plants, animals, and microorganisms. Pigments from microorganisms offer many benefits over plant- and animal-derived ones, including high yields, better cost efficiencies, greater stability, and easier processing. Microalgae and fungi are particularly promising as they produce a vast range of water-soluble biopigments in an array of colors.2 These include metabolites such as azaphilones, melanins, carotenoids and quinones.
Industrial attention has turned to filamentous fungi, in particular, as they are straightforward to cultivate on a large scale.3 Colorants derived from the filamentous fungi Monascus sp., for example, ankaflavin and canthaxanthin, are already available commercially as well as Arpink red™ pigment (Natural red™) from the strain Penicillium oxalicum var. armeniaca. However, the natural pigments currently on the market still face some disadvantages, such as limited availability, instability around light, heat and pH, and low water solubility.
New Sources
With demand growing, researchers are seeking out new sources of natural colorants. To do this, they employ advanced analytical techniques to identify novel pigment components and to study their biosynthetic pathways.
A key area for further development is Monascus pigments, which were first identified in filamentous Monascus fungi but also found in Penicillium and Talaromyces fungi.4 These pigments are azaphilones, a group of secondary metabolites. They range in color from yellow, orange, and red and have historically been used as coloring agents in traditional foods (yogurt, red wines, tofu and meats) in East Asian countries; they are also used in Chinese traditional medicine.
However, there are concerns that Monascus pigments can be contaminated with mycotoxins, such as citrinin, and other compounds unsuitable for use in food, such as monacolin K (the active compound found in red yeast rice that is commonly extracted and used in cholesterol-lowering medications). That is why research has focused on the filamentous fungus Talaromyces atroroseus (previously Penicillium purpurgenum), as it secretes large amounts of red and yellow Monascus pigments without producing mycotoxins. The structure of most of these pigments remains unknown.
Now a team from the Technical University of Denmark has characterized one novel group of these pigments and elucidated their structure.5 They called them atrorosins.
Methods and Results
The team led by Thomas Ostenfeld Larsen fermented a liquid culture of T. atroroseus in a medium composed of sugars, salts, and a trace metal solution. Bioreactors were used to cultivate and grow pigments at a pH of 4.5. A Prima PRO Process Mass Spectrometer (Thermo Fisher Scientific) was used to sample l-liter bioreactors; it can measure concentrations of oxygen, carbon dioxide, ethanol, methanol, argon and nitrogen.
The resulting culture liquid was an intense red color. The researchers analyzed extracts of the liquid using ultra-high performance liquid chromatography coupled to diode array detection and high-resolution tandem mass spectrometry (UHPLC-DAD-MS/HRMS) and found an abundance of different pigments, including unknown pigment species and several known Monascus pigments.
Further analysis showed the presence of two isomers of the orange azaphilone PP-O. The team used reversed-phase HPLC to generate enough material for structural analysis and then turned to NMR to work out that the two PP-O isomers had either a cis- or a trans-double bond between C-2 and C-3. The trans-form of PP-O had not been reported before.
The researchers then investigated one of the major unknown red compounds, which had a UV-Vis absorption spectrum similar to that of the known red Monascus pigments. First, they purified it by reverse phase HPLC to give a dark red amorphous solid and then worked out its molecular formula (C26H29NO9) and determined its structure using 1D and 2D NMR.
From the NMR data, the team deduced that the compound was structurally very similar to the Monascus pigments PP-O and PP-V, apart from it contained the amino acid serine incorporated into the isochromene/isoquinoline system. They named the new compound atrorosin S.
Then, the researchers investigated whether T. atroroseus was also able to incorporate other amino acids into the azaphilone core. They found that when cultivation occurred with a single amino acid as the only source of nitrogen source, in most cases, the secondary metabolite profile contained mainly one single red compound, the atrorosin corresponding to the added amino acid. For a few (glycine, cysteine, threonine and tyrosine), other colored compounds were also detected.
Structure of the Novel Pigments
The team showed that atrorosins are red azaphilone pigments and are derivatives of the orange pigment cis-PP-O. The only difference is that they contain L-amino acids, incorporated randomly into the isochromene system during growth. They are unique because C-1 has been oxidized fully to a carboxylic acid group.
Significantly, the researchers were able to produce a wide variety of atrorosins by supplementing the growth media during fermentation with a range of amino acids. They found that proline was the only amino acid not incorporated into an atrorosin.
The orange precursor PP-O is naturally produced in both the cis- and trans-form, but the team was surprised to find that the atrorosins were almost entirely (99.5%) in cis-form. They speculate that this could be down to steric interactions with the incorporated amino acid. That is, repulsive forces between the C-1 carboxylic acid and the carboxylic acids on the larger amino acids encourage the isomerization of the trans-form almost completely to the cis-form.
It has been thought amino acids could potentially become incorporated into Monascus pigments via an imine (or Schiff base) intermediate that is formed under basic conditions. However, the Danish team found that the reaction also happens under acidic conditions. They suggest the acid-catalyzed reaction might happen via an enamine rather than the imine previously suggested.
Additionally, the team showed that they could produce atrorosins from other fungi, such as T. albobiverticillius (as well as both cis- and trans-PP-O); they are not exclusive to T. atroroseus.
References and Further Reading
- Tuli, H. S. et al. (2015). Microbial pigments as natural color sources: current trends and future perspectives. Int. J. Food Sci. Tech. 52, pp. 4669–4678. doi: 10.1007/s13197-014-1601-6
- Pombeiro-Sponchiado, S. R. et al, (2017). Production of melanin pigment by fungi and its biotechnological applications, in Melanin, ed M. Blumenberg (London, UK: InTech), pp. 47–74. doi: 10.5772/67375
- Mapari, S.A.S. et al. (2009). Identification of potentially safe promising fungal cell factories for the production of polyketide natural food colorants using chemotaxonomic rationale. Microb. Cell Fact. 8(24). doi: 10.1186/1475-2859-8-24
- Vendruscolo F. et al. (2016). Monascus: a Reality on the Production and Application of Microbial Pigments. Appl Biochem Biotechnol. 2016 Jan;178(2), pp. 211-223. doi: 10.1007/s12010-015-1880-z.
- Isbrandt T. et al. (2020)., Atrorosins: a new subgroup of Monascus pigments from Talaromyces atroroseus Applied Microbiology and Biotechnology, 104, pp. 615–622.
This information has been sourced, reviewed and adapted from materials provided by Thermo Fisher Scientific – Environmental and Process Monitoring Instruments.
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