Review
Fungal polyketide azaphilone pigments
as future natural food colorants?
Sameer A.S. Mapari1, Ulf Thrane1 and Anne S. Meyer2
1
Center for Microbial Biotechnology, Department of Systems Biology, Building 221, Technical University of Denmark, DK-2800 Kgs.
Lyngby, Denmark
2
Bioprocess Engineering Section, Department of Chemical and Biochemical Engineering, Building 229, Technical University of
Denmark, DK-2800 Kgs. Lyngby, Denmark
The recent approval of fungal carotenoids as food colorants by the European Union has strengthened the prospects for fungal cell factories for the production of
polyketide pigments. Fungal production of colorants
has the main advantage of making the manufacturer
independent of the seasonal supply of raw materials,
thus minimizing batch-to-batch variations. Here, we
review the potential of polyketide pigments produced
from chemotaxonomically selected non-toxigenic fungal
strains (e.g. Penicillium and Epicoccum spp.) to serve as
food colorants. We argue that the production of polyketide azaphilone pigments from such potentially safe
hosts is advantageous over traditional processes that
involve Monascus spp., which risks co-production of the
mycotoxin citrinin. Thus, there is tremendous potential
for the development of robust fungal production systems for polyketide pigments, both to tailor functionality
and to expand the color palette of contemporary natural
food colorants.
Production of natural food colorants: the past, and the
current scenario
There is evidence that use of natural colorants, such as
plant extracts and wine, to color foods was adopted as early
as 1500 BC [1]. Following the industrial revolution, both
the food industry and processed foods developed rapidly.
The historical use of natural colorants was taken over by
chemically synthesized colors in the late 19th century, and
continued in the form of the coal-tar dyes of the 20th
century. This development was primarily governed by
the easier and more economical syntheses, as well as
superior coloring properties of chemically synthesized colors. However, safety concerns emerged with increasing
application of synthetic coloring agents, which led to
numerous regulations throughout the world and resulted
in the revival of the demand for natural food colorants.
Presently, consumer awareness has been increasing over
the link between diet and health; as a result, the trend is
towards clean label ingredients. The value of the international food colorant market was estimated at around
$1.15 billion USD in 2007, up 2.5% from $1.07 billion
USD in 2004. The natural food colorants accounted for
$465 million USD in 2007 – a 4.6% increase from 2004
(www.foodnavigator-usa.com). Natural colorants now comprise 31% of the colorant market, compared to 40% for
Corresponding author: Mapari, S.A.S. ([email protected]).
300
synthetics (data from Leatherhead Food International
LFI)(www.leatherheadfood.com). Furthermore, LFI asserts
that the market share for natural colorants is growing,
possibly indicating that natural colorants are poised to
surpass synthetic colorants in market value in the future.
Natural food colorants that are currently authorized in
the European Union (EU) are mostly derived from the raw
materials obtained from flowering plants and insects, as in
the case of carminic acid derived from scale insects (Box 1).
Typically, they are extracted by using food-grade solvents
and sold in the form of dried water- or oil-soluble powders,
or formulated into some kind of liquid forms or emulsions.
Thus, the current production of natural colorants is dependent on the external, seasonal supply of raw materials,
potentially resulting in batch-to-batch variations of the
extracted pigment profile [2]. As natural colorants are
extracted from natural sources, they are, in most cases,
mixtures of varying composition that depends upon the
cultivar and climatic conditions and, therefore, not easy to
characterize with respect to purity and contaminants. The
extracted plant pigment mixtures are not likely to be
produced as pure compounds or well-defined pigment compositions, unless they are produced in plant tissue cultures; nevertheless, obtaining high yields and longer
cultivation periods could be major bottlenecks. Moreover,
there is great variation in the stability and functionality of
different classes of existing natural food colorants [3].
Therefore, in many cases, the stability issues in relation
to heat, light and pH currently limit application to certain
types of products that fit the stability requirements of the
colorant. In the following sub-section, we discuss whether
the fungal production of natural food colorants would
tackle some of the above mentioned drawbacks associated
with the existing natural colorant production system.
Glossary
Pigment, dye and colorant: While these terms are often used interchangeably,
a pigment is insoluble in the given medium, whereas a dye is soluble. For
example, carotenoids are dyes in oil, but pigments in water. Pigments, dyes
and colorants are used in the text to mean colored substances in general.
Natural colorants: Pigments made by living organisms (i.e. they exist in
nature). Colorants such as caramel, vegetable carbon and Cu-chlorophyllin are
also considered natural, although they do not exist naturally.
Nature-identical colorants: Artificial pigments that are also found in nature.
Examples include b-carotene and riboflavin.
Synthetic colorants: Artificial colorants, such as azo-dyes, that are not found in
nature.
0167-7799/$ – see front matter ! 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibtech.2010.03.004 Trends in Biotechnology 28 (2010) 300–307
Review
Box 1. Scale insects as a source of polyketide natural
colorants
Scale insects (family Coccoidea) have had great importance since
ancient times as textile dyes. Various species have been used:
Kermes vermilio (kermes), Porphyrophora polonica (Polish cochineal), Porphyrophora hamelii (Armenian cochineal), Dactylopius
coccus (American cochineal or simply cochineal), and Kerria lacca
(lac, old name: Laccifer lacca). Of these, cochineal and lac are used
as food colorants. Cochineal, but not lac, is allowed in the EU and
the United States [56,57]. The anthraquinone-based pigments are
extracted from the bodies of dried female cochineal insects, which
feed on wild cacti, mainly in Peru, Mexico and the Canaries. The
major pigment carminic acid was first isolated in crystalline form in
1858, and its general structure was first established in 1913 [58]. Its
biosynthesis in insects is presumed to proceed through a five-stage
process, starting with an aliphatic heptaketide [59]. The color of
carminic acid in solution changes with pH; at low pH it is orange,
changing to red at slightly acidic and neutral pHs, and finally turns
violet in alkaline solution. However, its ability to form metal
chelates, particularly with aluminum and calcium, which results in
a complex called carmine, is of special interest because the color
shade is less dependent on pH than the color shade of non-chelated
carminic acid.
Fungi as readily available additional or alternative
sources of food colorants
Fungi, particularly ascomycetous and basidiomycetous
(most mushrooms), and lichens (symbiotic association of
a fungus with a photosynthetic partner, usually a green
alga or cyanobacterium), are known to synthesize and
secrete diverse classes of pigments naturally as secondary
metabolites of known or unknown function that possess an
extraordinary range of colors [4]. Thus, the potential for
exploring the vast fungal biodiversity for novel and safe
pigment producers using appropriate tools and techniques
remains untapped [5]. Mushrooms and lichens are difficult
to grow under laboratory conditions and therefore are not
suitable for large-scale industrial production. By contrast,
many ascomycetous fungi are better suited as production
hosts because they can be grown in a relatively easier way
to give high yields using the optimized cultivation technology [6]. Therefore, production of colorants by natural
fungal hosts would ensure that the colorant production is
accomplished under controlled conditions in bioreactors
that offer the colorant manufacturer independence from
the external, seasonal supply of raw materials, and potentially minimize batch-to-batch variations. As an added
advantage, colorant production from natural hosts avoids
genetic modifications.
Until a decade ago, use of fungi as a source of natural
food colorants was confined to the semi-fermentative production of riboflavin (vitamin B2). Riboflavin is an authorized natural yellow food colorant produced using the fungi
Eremothecium ashbyii and Ashbya gossypi [7,8]. However,
riboflavin is a colorant that is light-sensitive, which causes
color fading, thus limiting its application range [1].
Recently, industrial interest in fungi as sources of natural
colorants has been revived because the carotenoid b-carotene has been produced fermentatively from the fungus
Blakeslea trispora by DSMTM in the Netherlands and
approved for food use. Carotenoids are a class of orange–
red natural food colorants, with around 750 different
structures identified [9], which are usually derived from
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plant sources by the natural food colorant industry. Within
EU legislation, the B. trispora b-carotene has been
approved, as have some carotenes from plant sources
[10]. The otherwise-expensive carotenoid lycopene, for
which the only acceptable source has been tomatoes
(Lycopersicon esculentum), has also been approved recently
by EU food legislation, to be produced by B. trispora [11].
In contrast to this background of successful approval of
natural colorants from fungal sources, synthetic colorants
are either being banned because of their harmful effects or
facing newer tougher legislation regarding the labeling of
colorants (Supplementary Table 1). One example is the ban
on the import of red colorants of the Sudan series (Sudan I–
IV), for which there is evidence of carcinogenicity [12]. In
addition, under the new legislative package adopted by the
European Parliament, foods that contain synthetic colorants, such as tartrazine, quinoline yellow, sunset yellow
carmoisine, ponceau 4R, and allura red, will require the
label ‘may have an adverse effect on activity and attention
in children’. Together with the permission of the fungal
carotenoids, this could be viewed as a gradual change in
the outlook of EU legislation for the novel sources of
existing natural pigments, most likely in response to the
rising demand for safe and healthy alternatives to synthetic colorants. As such, we believe that the time is ripe to
capitalize on fungal sources of natural food colorants
because filamentous fungi hold enormous potential as
alternative or additional readily available sources for pigment production. Polyketide pigments from ascomycetous
fungi are relatively overlooked as potential food colorants,
thus warranting a review of recent research progress in
this novel area, as well as exploration of future prospects
and the research required for establishing filamentous
fungi as new and potentially safe cell factories for the
production of polyketide natural food colorants.
The fungal polyketide class of pigments as food
colorants
As mentioned earlier, fungal pigments are produced as
secondary metabolites of known or unknown function and
can be broadly classified chemically as carotenoids and
polyketides. Fungal polyketide pigments range in structure from tetraketides to octaketides, which have four or
eight C2 units that contribute to the polyketide chain.
Some of them are produced through mixed biosyntheses,
which means that they involve other pathways (i.e. amino
acid or terpenoid synthesis), in addition to the polyketide
pathway (Box 2). Representative classes include the
anthraquinones, hydroxyanthraquinones, naphthoquinones and azaphilone structures, each of which exhibits
an array of color hues (Figure 1).
In the past, polyketide pigments of ascomycetous fungi
have been used mainly for identification and species differentiation [13]; barring a couple of reports that also considered their applications with respect to food. For
instance, a water-soluble yellow pigment from Epicoccum
nigrum, with antioxidant power similar to that of natural
colorant curcumin and an absorption spectrum that
resembles that of the synthetic colorant tartrazine, was
reported as a potential food colorant by Stricker and
colleagues nearly 30 years ago [14].
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Box 2. Biosynthesis of fungal polyketides
Polyketides are formed by the condensation of an acetyl unit, or
other acyl unit with malonyl units, with simultaneous decarboxylation. This process is exemplified in fatty acid biosynthesis, without
obligatory reduction of the intermediate b-dicarbonyl system, which
results in the biosynthesis of a carbon chain, with alternate carbon
atoms donated by the methyl and carboxyl groups of the acyl
building blocks. The acetate origin of these compounds leads to
formation of even-numbered carbon chains. The resultant polycarbonyl compounds serve as substrates for various cyclases that
produce aromatic compounds that represent typical fungal metabolites [60]. Polyketides can be classified on the basis of their
biosynthesis, which indicates the number of C2 units that have
contributed to the polyketide chain and the type of cyclization that
the precursor has undergone. The terms triketide, tetraketide and
pentaketide denote compounds derived from three, four or five C2
units, respectively [61].
At this point, it is pertinent to mention that the polyketide pigments of commercially available Monascus pigments (azaphilone pigment mixtures of varying
composition) have been used as food colorants for hundreds
of years in the Orient [15]. Monascus purpureus has been
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used primarily in Southern China, Japan and Southeast
Asia for making red rice wine, red soybean cheese and
Anka (red rice) [16]. Monascus pigments typically comprise
six major azaphilone pigments: ankaflavin and monascin
(yellow); monascorubrin and rubropunctatin (orange); and
monascorubramine and rubropunctamine (purple–red). At
the same time, Monascus species has been also reported to
co-produce the mycotoxin citrinin, a hepato-nephrotoxic
compound in humans [17]. Pigment and citrinin biosyntheses in Monascus species have been shown to be independent of each other [18], but the exact factors by which
they are triggered are not yet clear. Thus, citrinin-producing ability is related to strain as well as media and
environmental conditions. In the case of M. purpureus –
the only authorized species for food use in Japan [19] – it has
been recently shown that the citrinin production is consistent with the presence of the functional citrinin biosynthetic
genes [20], and it is therefore very likely that it is produced
together with pigment under most of the current production
conditions. In addition to citrinin, other potential toxic
metabolites, such as monascopyridines [21–23], have also
been reported in Monascus fermented red rice.
Figure 1. Some example classes of fungal polyketide pigments exhibiting the color, typical structure skeletons, and their sources. An aminophilic moiety has been shown in
monascorubrin (from Monascus and Penicillium species) that results in a color change from orange to purple–red upon incorporation of amino acid; the resultant
compound is monascorubramine.
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There are a few reports on the toxicity of the specific
Monascus pigments in question. The yellow pigment ankaflavin has shown selective cytotoxicity to cancer cell lines
by an apoptosis-related mechanism, and has relatively low
toxicity for normal fibroblasts [24]. The structural
analogue monascin, however, has shown no cytotoxicity
to all cell lines tested [24]. This infers that both monascin
and ankaflavin are safe if consumed in appropriate doses,
and ankaflavin might even serve as a functional food
colorant. It has been reported that the red pigments monascorubramine and rubropunctamine, as well as the pigment derivatives of hydrophilic amino acids, have little
antimicrobial activity compared with that of orange pigments [25,26].
To the best of our knowledge, there have been no reports
of any acute health hazards on consumption of red rice or
the food products in which Monascus pigments have been
added in appropriate doses as food colorants. In a study in
Korea [27], two out of 10 commercial Monascus fermentation products were found to exceed the citrinin limit of
50 mg/kg set by the Korea Food and Drug Administration
(KFDA). The same study has provided information on the
levels of monacolin K, a cholesterol-lowering compound
often reported to be co-produced in Monascus fermented
products. Six out of 10 products were found to contain
monacolin K at levels lower than 500 mg/kg - the level
required by the KFDA to be considered as an functional
food. Altogether, these studies indicate the safe use of
certain Monascus pigments and their derivatives as food
colorants when selectively produced and appropriately
applied.
Nevertheless, the toxin issue, especially citrinin, has
certainly limited the food use of Monascus because of safety
concerns over its use directly in food or as an added
pigment. Meanwhile, much of the research in the past
20 years on Monascus pigments has focused on ways of
minimizing citrinin production or on developing strains
that are incapable of co-producing citrinin [28–30]. In the
EU and the United States, traditional food colorants from
the Orient (Monascus pigments) are still not allowed, and
there have been controversial views presented over their
safe use. This could be primarily because of the presence of
citrinin, and to some extent, the reluctance and lack of
awareness by the industry to invest in performing toxicological tests or to acquire the necessary expertise to produce safe pigments selectively in safe hosts.
Potential of ascomycetous fungi and their polyketide
pigments: beyond Monascus
Recent studies have highlighted the potential of using
polyketide-pigment-producing filamentous fungi as a
source of natural food colorants other than Monascus. At
the ‘Pigments in Food’ conference in France in 2004, the
polyketide anthraquinone-based natural food colorant
Arpink Red was first described. The colorant was claimed
to be produced by Penicillium oxalicum var. Armeniaca, a
nomen nudum (the variety was never formally described)
[31,32]. The colorant was awarded 2-year temporary
approval by the EU for distribution in the Czech Republic
from 2004–2006, and was manufactured by Ascolor Biotec
in that country. At present, the colorant is under evalu-
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ation by the European Food Safety Authority and its
current status is unknown. Based on the description in
the patent [31], the pigment-producing fungus is likely to
be misidentified as Penicillium oxalicum – a species that
produces the yellow toxic pigment secalonic acid D and
grows well at 378C [5]. This might indicate that the identity
of the production strain is unknown, and that there is no
information on potential mycotoxin production and pathogenicity towards humans, thereby posing a severe threat to
the safety of consumers and workers. This has led other
researchers to use a chemotaxonomic rationale to identify
potentially safe polyketide-pigment-producing fungi
[5,33,34] from the vast number of biologically and chemically diverse filamentous fungi. The chemotaxonomic
rationale was based on a priori knowledge of speciesspecific metabolite/pigment and/or mycotoxin profiles that
have been used successfully as fingerprints to differentiate
filamentous fungi at genus and species levels.
Polyketide azaphilone Monascus-like pigments and/or
their amino acid derivatives were discovered to be produced naturally by Penicillium aculeatum and P. pinophilum strains that belong to Penicillium subgenus
Biverticillium. These strains do not co-produce citrinin
or any other known mycotoxins, and are non-pathogenic
to humans [34]. More recently, ascomycetous fungi that
produce polyketide pigments have been evaluated based on
chemotaxonomy, and potentially safe genera have been
selected over those that are known to be human pathogens
or to produce mycotoxins [35]. This evaluation has highlighted species that belong to the two main genera, Penicillium and Epicoccum, as potential pigment producers.
Seven Penicillium strains that belong to Penicillium purpurogenum, P. aculeatum and Penicillium funiculosum
species have been reported [35] as novel producers of
Monascus-like azaphilone pigments and/or their amino
acid derivatives on solid media, in addition to the strains
reported previously [33,34]. Notably, four of these strains
can secrete extracellular pigments into the liquid media,
which suggests their potential for future adoption as cell
factories [35]. An international patent application has
already been filed on production of these polyketide azaphilone Monascus-like pigments, including their amino
acid derivatives, by potentially safe strains of Penicillium
species through the use of a combination of liquid and solid
cultivation techniques [36].
Many other pigment-producing ascomycetous fungi,
such as Penicillium herquei, show promise for future evaluation [35]. Cordyceps unilateralis strain BCC 1869 looks
promising as a source of the polyketide naphthoquinone
red pigments [37], which are of special interest because of
the chemical and structural similarities to those of the
commercial, plant-derived red pigments shikonin and
alkanin. The key component in the total C. unilateralis
BCC 1869 naphthoquinones has been identified as 3,5,8trihydroxy-6-methoxy-2-(5-oxohexa-1,3-dienyl)-1,4naphthoquinone, and has been found to be very stable
towards light, heat and acid and alkali solutions (0.1 M
each). Another potential pigment producer, E. nigrum, has
been evaluated for possible industrial-scale production of
natural colorants in liquid media and on a solid rice-based
medium [38]. By contrast, several species of Penicillium,
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such as P. crateriforme, P. islandicum, P. rugulosum, P.
variabile and P. marneffei, are not suitable as potential
pigment production strains because of their ability to
produce known toxic metabolites or pigments, or to exert
pathogenicity towards humans [35].
Application potential of fungal polyketide pigments as
food colorants
Traditionally, red and yellow hues have been the most
extensively used food colorants, although there is currently
a growing demand for blue [39]. In this regard, polyketide
pigments of ascomycetous fungi, including Monascus, hold
great potential because they produce a variety of red,
yellow, orange, green and blue hues [33,40–42]. A recent
study has indicated that the hues of the pigments produced
by selected Penicillium and Epicoccum species resemble
and/or complement the color palette of contemporary
natural food colorants in the red and yellow spectra [33].
Additionally, the same study also indicated the major color
component of the epicoccal extract to be the oxopolyene
orevactaene. The structure previously suggested [14] for
the unidentified water-soluble antioxidant compound
seems to match well with orevactaene, which indicates
the potential of orevactaene as a functional food colorant.
The primary orange azaphilone pigments of Monascus
have an aminophilic moiety, as shown in the exemplary
orange pigment monascorubrin in Figure 1, which can
react with amino acids provided in the medium to form
colorants of various red hues [40]. The resultant pigment
derivative might possess improved functionality, such as
light stability and water solubility [40,43]; the latter being
a property that could have a positive impact on
applications in many food products, such as beverages.
Thus, industrial application of the red Monascus pigment
derivatives and the typical purple–red pigments is
advantageous, provided that they are selectively produced
using citrinin-free strains of Monascus. Some of the
pigments and/or derivatives have lipase-inhibitory activity
as well as anti-atherogenic activity [44,45] and antioxidant
properties [46], which point toward their potential use
as nutraceuticals when added to specific food products.
Monascus pigments in solution are reported to be more
stable at near-neutral and/or alkaline pH [47,48]; yellow
pigments are more stable than the red ones in solution [47].
Water-soluble red complexes of Monascus pigments have
been reported to be considerably stable to pH changes but
cannot withstand the high temperatures used in thermal
processing [49].
Besides the disadvantage of citrinin co-production,
Monascus pigments (except for their amino acid derivatives) have also been criticized for their instability towards
ultraviolet and visible light [15,16,40]. Pigment extracts
from chemotaxonomically selected, potentially safe P. aculeatum (orange–red mixture of one major and 4–5 minor
components) and E. nigrum (yellow mixture, with orevactaene as major component and two minor components)
exhibit enhanced photostability over the commercially
available natural colorants Monascus Red (mixture of
several components) and turmeric (major component, curcumin) in liquid food model systems [50]. This highlights
the potential of these pigments as food colorants in terms of
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being derived from potentially safe, readily available
sources, as well as conferring an improved functionality
(here, photostability) in food systems.
In addition, several researchers have reported hitherto
unknown fungal pigments that might have potential as
food colorants. An unknown, light-stable red pigment produced by a submerged culture of the insect pathogen
Paecilomyces sinclairii has been reported [51]. More
recently, an unidentified species of Penicillium has been
reported to produce pigments with radical scavenging
properties, thus suggesting their potential to act as both
food colorants and nutraceuticals [52].
Complexity involved in the regulation of polyketide
pigment biosynthesis
There is only rudimentary knowledge about polyketide
pigments and their regulation at the genetic and molecular
levels. One of the primary reasons for this could be the
complex pathways involved in their biosynthesis. There
exists a unique structural and chemical diversity of polyketides, including pigments, because of the variations in
the domains of iterative type I fungal polyketide synthases
(PKSs). They contain 0-3 reductive domains, some a methyltransferase domain, and some a thioesterase domain.
Some PKSs, for unknown reasons, also contain tandem
acylcarrier protein motifs. Monascus is the most frequently
studied fungus for polyketide pigment production, and is
often seen as a model fungus for studying the regulation of
pigment biosynthesis. However, Monascus has only
recently been studied using systems biology approaches
to investigate the influence of phosphate limitation (via the
rice nitrate complex medium) on growth and pigment
production, as phosphate has been known to influence
secondary metabolite production [53].
There have been several hypotheses intended to explain
polyketide pigment production. One hypothesis is that
they are synthesized as intermediates for unutilized
branch pathway products. Another possible explanation
for pigment accumulation in the cell could be that they are
formed as excessive levels of intermediates that might be
needed to drive unfavorable biosynthetic steps by mass
action [54]. Unfortunately, some of the fully sequenced
genomes of filamentous fungi, including that of Monascus
(www.bcrc.firdi.org.tw/genome_project/index.jsp), are not
publicly available. However, even for the fully sequenced
fungal genomes that are publicly available, it is still a
difficult task to generate comprehensive information about
the function of the genes because of the complexity of the
regulation of fungal genomes to produce secondary metabolites. This problem is supported by a recent study that has
indicated that, in many cases, polyketide biosynthetic gene
clusters are silent under standard or optimal culture conditions, and that only under certain physiological conditions, such as stress conditions, are these genes
activated (i.e. upon intimate bacterial–fungal interactions)
[55].
Despite the fact that the cellular mechanisms underlying the effect of substrates on growth and polyketide
pigment production are still unknown, the currently available knowledge indicates that the synthesis of pigment
compounds is independently regulated by changing media
Review
and/or culture conditions, and can therefore be optimized.
It has been shown that manipulation of the concentration
of carbon and nitrogen sources results in enhanced pigment production [36]. A particular C:N ratio in the medium
has thus been shown to favor red pigment production over
biomass, and yellow pigments in the case of chemotaxonomically selected, potentially safe P. purpurogenum IBT
11181 [36]. It is also worth mentioning that fungi should be
maintained in a state of optimized physiology, thus avoiding any sort of stresses that could result in production of
other unwanted secondary metabolites such as mycotoxins. For example, for the production of mycelial food product QuornTM, for which the fungus Fusarium venenatum
is used in an air lift fermentor, the fungus has to be
maintained in a mycelial state to avoid any stresses that
could result in contamination problems with the tricothecene mycotoxins.
Recent research and commercial advances therefore
illustrate the potential for establishing potentially safe
natural fungal cell factories for polyketide azaphilone pigment production without genetic manipulations (Figure 2).
Conclusions and future perspectives
By keeping key limitations of existing natural food colorants in mind, such as dependence on the supply of seasonal
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raw materials and variation in pigment profile, and noting
the growing market for the natural food colorants, it can be
inferred that the future prospects of the industrial production of natural food colorants lie in three key areas:
robust production of colorants of uniform quality; discovery
of alternative sources of novel or known classes of pigments
and color hues; and improving functionality. The ability of
the polyketide class of natural pigments from ascomycetous fungi to serve as sustainable natural food colorants
has largely escaped the attention of food scientists, both in
academia and in industry, despite the tremendous
economic and marketing potential. The recent approval
of carotenoid food pigments derived from filamentous fungi
highlights the potential of the polyketide class of colorants,
which is still a relatively unexplored area of research, with
the exception of Monascus pigments that are popular in the
Asia. The fate of Monascus pigments in the EU and in the
United States will depend on a change of opinion by the
food authorities. To a large extent, this might be influenced
by developing a robust production process whereby selective and predictable production of safe colorants is
achieved using non-citrinin-producing wild-type strains.
In the case of genera other than Monascus, an initial
proof-of-concept has been demonstrated from the time it
was hypothesized [5] that filamentous fungi other than
Figure 2. Step-by-step representation of recent advances towards prospective fungal cell factories for the production of polyketide azaphilones as natural food colorants.
Step 1 highlights pre-selection of potentially safe pigment producers that were chosen over mycotoxigenic and human pathogenic strains based on metabolite profiles and
systematics. Step 2 depicts the identification of known or novel color leads using high-resolution LC/MS and colorimetric characterization of colorants. Step 3 illustrates a
study on enhanced photostability of orange–red fungal pigments by P. aculeatum IBT 14263 over commercially available Monascus pigments in liquid food model systems
[50]; color fading is illustrated before and after the light exposure. Step 4 shows ongoing research to enhance the production potential of potentially safe Penicillium strains
that produce Monascus-like azaphilone pigments. (a) Carbon sources are: L, lactose; PS, potato starch; RM, rice meal; S&G, starch and glucose; S&L, starch and lactose;
S&S, starch and sucrose. (b) Nitrogen sources are: AN, ammonium nitrate; AS, ammonium sulfate; CSL, corn steep liquor; MSG, monosodium glutamate; P, peptone; SM,
soybean meal. (c) Pigment production by P. purpurogenum IBT 11181 in the formulated medium comprising selected carbon and nitrogen sources using light-expanded
clay aggregates (LECA) as a solid support for mycelium [36].
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Monascus can naturally produce known or novel polyketide colorants with improved functionalities in food model
systems. In the future, it should be possible to secure
efficient and controlled production of polyketide pigments
in chemotaxonomically selected, potentially safe Penicillium strains using current knowledge, without genetic
manipulation. Future research should therefore include
systematic evaluation of the factors that influence polyketide azaphilone pigment production in potentially safe
fungal production strains. There is also a strong need to
develop reactor systems for large-scale (industrial) production; such bioreactors would need to capitalize on
unique fungal features with respect to the pigment-producing ability and scalability of biomass production, especially in liquid media.
Recent data indicate how filamentous fungi can be used
as cell factories for pigment production and could be developed to tailor functionality and expand the color palette of
existing natural food colorants, while taking advantage of
fungal biodiversity. However, there is clearly a significant
need to better understand how, why, and under which
circumstances filamentous fungi produce polyketide pigments. Similar to other novel natural colorants, fungal
polyketide colorants must be tested for toxicity before
approval. As far as acceptance by consumers and food
authorities is concerned, polyketide fungal natural colorants should be viewed as being no different than the
approved contemporary pigments of exotic origin, such
as carminic acid from cochineal insects or the recently
approved fungal carotenoids.
Acknowledgements
We acknowledge the Proof-of-Concept fund, Research and Innovation,
Technical University of Denmark (DTU), for financial support and the
patent application (WO 2009/026923 A2.) that we hold in relation to this
work. We declare no competing interests.
Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tibtech.2010.
03.004.
References
1 Downham, A. and Collins, P. (2000) Colouring our foods in the last and
next millennium. Int. J. Food Sci. Technol. 35, 5–22
2 Spears, K. (1988) Developments in food colourings: the natural
alternatives. Trends Biotechnol. 6, 283–288
3 Mortensen, A. (2006) Carotenoids and other pigments as natural
colorants. Pure Appl. Chem. 78, 1477–1491
4 Maldonado, M.C. and Ibarra, L.V. (2005) Organic dyes from fungi and
lichens. In Biodiversity of Fungi Their Role in Human Life (Deshmukh,
S.K. and Rai, M.K., eds), pp. 375–407, Oxford & IBH Publishing Co.
Pvt. Ltd
5 Mapari, S.A.S. et al. (2005) Exploring fungal biodiversity for the
production of water-soluble pigments as potential natural food
colorants. Curr. Opin. Biotechnol. 16, 231–238
6 Wissgott, U. and Bortlik, K. (1996) Prospects for new natural food
colorants. Trends Food Sci. Technol. 7, 298–302
7 Wickerham, L.J. et al. (1946) The production of riboflavin by Ashbya
gossypii. Arch. Biochem. 9, 95–98
8 Stahmann, K.P. et al. (2000) Three biotechnical processes using Ashbya
gossypii, Candida famata, or Bacillus subtilis compete with chemical
riboflavin production. Appl. Microbiol. Biotechnol. 53, 509–516
9 Britton, G. et al. (2004) Carotenoids – Handbook, Birkhauser ISBN 37643-6180-8
306
Trends in Biotechnology Vol.28 No.6
10 Report of the Joint FAO/WHO Expert Committee on Food Additives.
Evaluation of Certain Food Additives and Contaminants; 57th; WHO:
Geneva, Switzerland, 2002; p 909
11 Commission regulation (EC) No 721/2006 of 23 October 2006
authorising the placing on the market of lycopene from Blakeslea
trispora as a novel food ingredient under regulation (EC) No 258/97
of the European Parliament and of the council (notified under
document number C(2006) 4973) Official Journal of the European
Union, L 296, 2006
12 Europe Environment, 29 Jan 2004, (649), IV.5, Europe Information
Service sa, http://www.eis.be
13 Frisvad, J.C. and Samson, R.A. (2004) Polyphasic taxonomy of
Penicillium subgenus Penicillium. A guide to identification of food
and air-borne terverticillate Penicillia and their mycotoxins. Stud.
Mycol. 49, 1–173
14 Stricker, R. et al. (1981) Production and food applications of a hydro
soluble yellow pigment from mold Epicoccum nigrum. Lebensmitt.
Wissenschaft. Technol. LWT 14, 18–20
15 Dufosse, L. (2006) Microbial production of food grade pigments. Food
Technol. Biotechnol. 44, 313–321
16 Lin, Y-L. et al. (2008) Biologically active components and
neutraceuticals in the Monascus-fermented rice: a review. Appl.
Microbiol. Biotechnol. 77, 965–973
17 Blanc, P.J. et al. (1995) Characterization of monascidin-a from
Monascus as citrinin. Int. J. Food Microbiol. 27, 201–213
18 Pisareva, E. et al. (2005) Pigment and citrinin biosynthesis by fungi
belonging to genus Monascus. Z. Naturforsch. C-J. Biosci. 60, 116–120
19 Blanc, P.J. et al. (1994) Pigments of Monascus. J. Food Sci. 59, 862–865
20 Chen, Y-P. et al. (2008) Exploring the distribution of citrinin
biosynthesis related genes among Monascus species. J. Agric. Food
Chem. 56, 11767–11772
21 Wild, D. et al. (2003) New Monascus metabolites with a pyridine
structure in red fermented rice. J. Agric. Food Chem. 51, 5493–5496
22 Knecht, A. et al. (2006) New Monascus metabolites: structure
elucidation and toxicological properties studied with immortalized
human kidney epithelial cells. Mol. Nutr. Food Res. 50, 314–321
23 Knecht, A. and Humpf, H-U. (2006) Cytotoxic and antimitotic effects of
N-containing Monascus metabolites studied using immortalized
human kidney epithelial cells. Mol. Nutr. Food Res. 50, 406–412
24 Su, N.W. et al. (2005) Ankaflavin from Monascus-fermented red rice
exhibits selective cytotoxic effect and induces cell death on Hep G2
cells. J. Agric. Food Chem. 53, 1949–1954
25 Kim, C. et al. (2006) Effect of Monascus pigment derivatives on the
electrophoretic mobility of bacteria, and the cell adsorbtion and
antibacterial activities of pigments. Coll. Surfaces Biointerfaces 47,
153–159
26 Kim, C. et al. (2006) Antimicrobial activities of amino acid derivatives
of Monascus pigments. FEMS Microbiol. Lett. 264, 117–124
27 Kim, H.J. et al. (2007) Natural occurring levels of citrinin and
monacolin K in Korean Monascus fermentation products. Food Sci.
Biotechnol. 16, 142–145
28 Wang, J.J. et al. (2004) Modified mutation method for screening low
citrinin-producing strains of Monascus purpureus on rice culture. J.
Agric. Food Chem. 52, 6977–6982
29 Pereira, D.G. et al. (2008) Effect of dissolved oxygen concentration on
red pigment and citrinin production by Monascus purpureus ATCC
36928. Braz. J. Chem. Eng. 25, 247–253
30 Xu, M-J. et al. (2009) Construction of a Monascus purpureus mutant
showing lower citrinin and higher pigment production by replacement
of ctnA with pks 1 without using vector and resistance gene. J. Agric.
Food Chem. 57, 9764–9768
31 Sardaryan, E. (1999) Strain of the microorganism Penicillium oxalicum
var. Armeniaca and its application. WO 99/50434
32 Sardaryan, E. (2004) Food supplement. US 2004/ 0105864 A1
33 Mapari, S.A.S. et al. (2006) Colorimetric characterization for
comparative analysis of fungal pigments and natural food colorants.
J. Agric. Food Chem. 54, 7027–7035
34 Mapari, S.A.S. et al. (2008) Computerized screening for novel producers
of Monascus-like food pigments in Penicillium species. J. Agric. Food
Chem. 56, 9981–9989
35 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
Review
36 Mapari, S.A.S. et al. (2009) Production of Monascus-like azaphilone
pigment. WO 2009/026923 A2. (05.03.2009)
37 Unagul, P. et al. (2005) Production of red pigments by the insect
pathogenic fungus Cordyceps unilateralis BCC 1869. J. Ind.
Microbiol. Biotechnol. 32, 135–140
38 Mapari, S.A.S. et al. (2008) Evaluation of Epicoccum nigrum for growth
morphology and production of natural colorants in liquid media and on
a solid rice medium. Biotechnol. Lett. 30, 2183–2190
39 O’Carroll, P. (1999) Naturally exciting colours. World Ingred. 3/4,
39–42
40 Jung, H. et al. (2003) Color characteristics of Monascus pigments
derived by fermentation with various amino acids. J. Agric. Food
Chem. 51, 1302–1306
41 Bachmann, O. et al. (1986) The green pigment from the fungus
Roesleria hypogea. Liebigs Annalen. Chemie. 305–309
42 Robinson, N. et al. (1992) Blue pigments of Penicillium herquei. J. Nat.
Prod. 55, 814–817
43 Jung, H. et al. (2005) Enhanced photostability of Monascus pigments
derived with various amino acids via fermentation. J. Agric. Food
Chem. 53, 7108–7114
44 Kim, J.H. et al. (2007) Development of lipase inhibitors from various
derivatives of Monascus pigment produced by Monascus fermentation.
Food Chem. 101, 357–364
45 Jeun, J. et al. (2008) Effect of the Monascus pigment threonine
derivative on regulation of the cholesterol level in mice. Food Chem.
107, 1078–1085
46 Yang, J-H. et al. (2006) Antioxidant properties of methanolic extracts
from Monascal rice. LWT Food Sci. Technol. 39, 740–747
47 Fabre, C.E. et al. (1993) Production and food applications of the red
pigments of Monascus ruber. J. Food Sci. 58, 1099–1102 (1110)
48 Carvalho, J.C. et al. (2005) Biopigments from Monascus: strains
selection, citrinin production and color stability. Braz. Arch. Biol.
Technol. 48, 885–894
Trends in Biotechnology
Vol.28 No.6
49 Broder, C.U. and Koehler, P.E. (1980) Pigments produced by Monascus
purpureus with regard to quality and quantity. J. Food Sci. 45, 567–569
50 Mapari, S.A.S. et al. (2009) Photostability of natural orange-red and
yellow fungal pigments in liquid food model systems. J. Agric. Food
Chem. 57, 6253–6261
51 Cho, Y.J. et al. (2002) Production of red pigment by submerged culture
of Paecilomyces sinclaairii. Lett. Appl. Microbiol. 35, 195–202
52 Dhale, M.A. et al. (2009) Pigment and amylase production in
Penicillium sp NIOM-02 and its radical scavenging activity. Int. J.
Food Sci. Technol. 44, 2424–2430
53 Lin, W.Y. et al. (2007) Proteomic response to intracellular proteins of
Monascus pilosus grown under phosphate-limited complex medium
with different growth rates and pigment production. J. Agric. Food
Chem. 55, 467–474
54 Shier, W.T. et al. (2005) Yellow pigments used in rapid identification of
aflatoxin-producing Aspergillus strains are anthraquinones associated
with the aflatoxin biosynthetic pathway. Bioorg. Chem. 33, 426–438
55 Schroeckh, V. et al. (2009) Intimate bacterial–fungal interaction
triggers biosynthesis of archetypal polyketides in Aspergillus
nidulans. Proc. Natl. Acad. Sci. U. S. A. 106, 14558–14563
56 a) Off. J. Eur. Commun. L 226, 1 (1995); b) Off. J. Eur. Commun. L 206,
(1999), c) Off. J. Eur. Commun. L 190, 14 (2001)
57 Code of Federal Regulations – Title 21. Part 73: Listing of color
additives exempt from certification
58 Allevi, P. et al. (1998) Synthesis of carminic acid, the colorant principle
of cochineal. Trans. J. Chem. Soc., Perkin Trans. 1, 575–582
59 Dawson, T.L. (2009) Biosynthesis and synthesis of natural colours.
Color. Technol. 125, 61–73
60 Hanson, J.R. (2003) The classes of natural product and their isolation.
In Natural Products: The Secondary Metabolites. (Abel, E.W., ed.) p 2,
The Royal Soc. of Chem.
61 Turner, W.B. (1971) Polyketides. In Fungal Metabolites, pp 75–213,
Academic Press
307