Chapter 

Secondary Metabolism and Antimicrobial

Metabolites of Aspergillus
A.G. Rodrigues
Martin-Luther University Halle-Wittenberg, Halle, Germany

INTRODUCTION on enzymes, catalytic activity of their own, defense, and

interactions with other organisms (eg, pigments, odor-
Since the discovery of penicillin by Alexander Fleming the ants, and pheromones). They are divided into primary and
world has experienced a rise in wealth. The British researcher secondary metabolites. A primary metabolite is directly
was the first to observe and describe bacterial growth inhibi- involved in normal growth, development, and reproduction.
tion by what would become a medicine. However, even if It usually performs a physiological function in the organism
the compound was noticed by 1928, it took several years and is typically present in many organisms or cells.
until it could be actually produced in large scale and ben- On the other hand, a secondary metabolite is not directly
efit people. This work was accomplished due to the pres- involved in essential processes, but usually has an important
sure of the Second World War, in which many soldiers were ecological function. Secondary metabolites, also referred
losing their lives. To continue the work and open a new era to as natural products, are the products of metabolism not
in medicine it was necessary a great deal of work by several essential for normal growth, development, or reproduc-
scientists, among them Ernst Chain and Howard Florey at tion of an organism. Like primary metabolites, secondary
Oxford (Kardos and Demain, 2011, 2013). These achieve- metabolites are often used in industrial microbiology for
ments boosted the progress of discovery of biotechnologi- the production of food, amino acids, and antibiotics (Keller
cal compounds and presented decades of improvements et al., 2005). These compounds serve to meet the secondary
in industrial processes, from fermentation techniques requirements, as for defense against other microorganism.
to molecular tools, providing means by which products Empowering them to survive interspecies competition and
have been created and improved. Fungi, more specifically facilitate reproductive processes.
the Aspergilli, have been highly present and necessary in Well-known sources of secondary metabolites are plants,
this process, with their metabolites being discovered, bacteria, fungi, and marine organisms such as sponges,
explored, and optimized. Enzymes, organic acids, and many tunicates, corals, and snails. Many secondary metabolites
other molecules have brought a huge variety of products have proved invaluable as antibacterial or antifungal agents,
into the market and/or improved the existing ones to a level anticancer drugs, cholesterol-lowering agents, immunosup-
that was never before experienced. The effort of many pressants, antiparasitic agents, herbicides, diagnostics, and
research and industrial actors, as well as governmental pol- tools for research. Some of these have been found to play a
icies in some cases, helped all of us to benefit from such pivotal role in treatment or prevention of a multitude of bio-
results and evaluate the commitment and accomplishments logical disorders, many of which did not have any cure until
of science. these products were discovered (Vaishnav and Demain,
2010; Hansson, 2013).
Aspergilli have been successfully employed in the bio-
METABOLITES technology sector due to their great production of organic
Microorganisms produce several compounds that they use acids and extracellular enzymes (Khan et al., 2014). To fully
for their survival. These compounds are called metabolites understand key aspects of fungal secondary metabolites one
and are the intermediates and products of metabolism. The has to take into account the various factors governing such
term metabolite is usually restricted to low-molecular- events, including genetic information currently accessible,
weight molecules. Metabolites have various functions, as pointed out by Calvo et al. (2002) and Fox and Howlett
including cell signaling, stimulatory and inhibitory effects (2008). In this chapter important aspects of their role in

New and Future Developments in Microbial Biotechnology and Bioengineering. DOI: [Link]
© 2016 Elsevier B.V. All rights reserved. 81
82  SECTION | III  Secondary Metabolism

providing secondary metabolites will be described along composition, aeration conditions, fungal morphology, and
with their historical and biotechnological perspectives. broth rheology. More recently Osman et  al. (2011) and
Bizukojc et al. (2012) have explored the pH in the produc-
tion of lovastatin.
POLYKETIDES
In 1973 the group of Prof. Shuichi Seto in Japan discov-
The history of polyketides started when James Colie syn- ered pulvinones by a natural via. The scientists reported by
thesized orcinol at London University, in 1893 (Khan et al., that time the obtaining of such compounds from A. terreus
2014). In 1950 the Australian organic chemist Arthur Birch (Ojima et al., 1973). The name was then changed to aspulvi-
proved that polyketides are biosynthesized by acetate units none in view of existing pulvinones and pulvinones isolated
with the help of nuclear magnetic resonance (NMR), which from Suillus grevillei by Edwards and Gill at the University
was evolving in those years. In 1955 Birch published the of Bradford in the same year (Edwards and Gill, 1973). It
work on 6-methyl salicylic acid released by a fungus, presented anticoagulant or antiinflammatory and antibacte-
Penicillium griseofulvum (Birch et  al., 1955). Polyketides rial properties (Campbell et al., 1985; Rehse and Lehmke,
are the most abundant secondary metabolites in fungi, also 1985; Antane et al., 2006; Xu et al., 2013). In another recent
being produced by plants and bacteria. The compound is study, Gao et al. (2013) isolated aspulvinones from A. ter-
synthesized by the action of polyketide synthase (PKS), reus in a mangrove in Fujian, a Chinese province, with anti-
which is similar to fatty acid biosynthesis. These natural influenza A viral (H1N1) activity.
organic compounds have a complex chemical structure and In the fermentation of food, Aspergillus plays an impor-
have played important roles in the pharmaceutical field. tant role. Kim et al. (2013) described the fungal fermentation
Important antibiotics are polyketides, such as doxycycline, of tea using Aspergillus oryzae. The process permitted tea
clarithromycin, and erythromycin. Regarding the produc- products to be obtained with increased antioxidant content
tion of polyketides by Aspergillus, aflatoxin and lovastatin and activity, as the phenolic acid was elevated by the deg-
are among the more well-known and will be described here radation of flavonoids caused by the fungal fermentation.
in more detail (Keller et al., 2005). Ongoing research has Soybean is an important source of fermented food in Asian
also revealed more compounds that might be of interest countries and contains isoflavonones. It was studied with
(Fig. 6.1). the aim of enhancing the antioxidant capacity by metabolic
changes with Aspergillus sojae (John et al., 2013) (Fig. 6.2).
LOVASTATIN Besides products already on the market, researchers
have been making efforts to find compounds of interest.
Lovastatin is a potent 3-hydroxymethylglutaryl-CoA Several alkaloids compounds from the marine-derived fun-
(3-HMG-CoA) inhibitor, discovered in the late 1970s at gus Aspergillus carneus were described by a Russian group
Merck Research Laboratories in the fermented broth of and had their structures characterized for the first time
Aspergillus terreus, used in the treatment of hypercho- (Zhuravleva et al., 2012).
lesterolemia (Alberts et  al., 1980; Tobert, 2003). The
molecule was first named mevinolin entering the market
in 1987 and became a blockbuster, with annual sales of AFLATOXIN B1
over one billion dollars because of its efficacy in reduc- Several Aspergillus fungi have contributed to the field of
ing LDL cholesterol. In the following years other statins biotechnology. However, toxic metabolites are also pro-
were brought to market, including simvastatin, pravastatin, duced. It can impose a threat to other microorganisms
fluvastatin, atorvastatin, cerivastatin, and rosuvastatin, the
majority of which are fully synthesized. However, since
its discovery, research has been performed to optimize the
O
production of this polyketide metabolite such as medium
O
O
CH3 O OH
O
H
H3C OH

HO OH
Orcinol 6-Methyl salicilic acid Lovastatin

FIGURE 6.1  Chemical structure of orcinol and 6-methyl salicylic acid. FIGURE 6.2  Chemical structure of lovastatin.
 Secondary Metabolism and Antimicrobial Metabolites of Aspergillus  Chapter | 6  83

as well as for humans, among them Aspergillus flavus. Aspergillus niger, leading the authors to suggest that using
Although first described in 1809, the fungus that secretes real-time polymerization chain reaction (PCR) would allow
aflatoxin, came into the limelight in the 1960s, caus- early detection of expression of the gene before accumula-
ing the death of over 100,000 turkey poults in London tion of the toxin in food and the application of measures to
due to the contamination of the peanuts with which the tur- prevent its biosynthesis (Fig. 6.4).
keys had been fed (Keller et  al., 2005; Bhatnagar-Mathur
et al., 2015).
ENZYMES
Aflatoxins are nondigestible by animals and end up in
the meat. They are also heat- and freeze-stable and remain Fungi are great producers of enzymes and have contributed
indefinitely in the food. The toxin has a high impact on enormously to enable and facilitate industrial processes.
human health worldwide, causing aspergillosis and slow- From food to pharmaceutical products and chemical goods
ing the recovery rate from protein malnutrition (Amare and these enzymes have proven their importance in our every-
Keller, 2014). The endeavors to combat aflatoxin in crops day lives. The Aspergilli are specially required in the field,
with biotechnological tools have been recently reviewed by accounting for more than 200 species (Soares et al., 2012).
Bhatnagar-Mathur et al. (2015) (Fig. 6.3). Aspergillus oryzae and A. niger have fundamental impor-
tance as they are on the list of generally recognized as safe
(GRAS) of the Food and Drug Administration (FDA) in the
OCHRATOXIN
United States (Contesini et al., 2010).
Ochratoxin is a polyketide-derivative and is very important
in the fungal biotechnological process due to its properties.
Ochratoxin is a mycotoxin found in food and beverages that LIPASES
exhibits nephrotic effects and can, potentially, be associ- Lipases (triacylglycerol acyl hydrolases, EC [Link]) are
ated with human carcinogenesis. Ochratoxin is known for natural catalysts of the hydrolysis of triacylglycerol into
contaminating grapes and wines. Besides that, the com- di- and monoacylglycerols, fatty acids, and glycerol at an
pound has toxicological effects like nephrotoxicity and oil–water interface, a phenomenon known as interfacial
hepatotoxicity (Crespo-Sempere et  al., 2014). Because of activation. However, under certain conditions, they are also
its importance, work has still to be done in the direction able to catalyze synthetic reactions. The most reported of
of understanding better the gene expression and ochra- the reactions carried out by these enzymes are hydrolysis,
toxin production, as reported by Castellá et al. (2015), with acidolysis, alcoholysis, amylolysis, esterification and inter-
esterification. Currently, lipases are a popular choice as a
biocatalyst because they can be applied to chemo-, regio-,
O and enantioselective hydrolyses and also in the syntheses
O
of a broad range of compounds. These enzymes are con-
O sidered to have great potential as biocatalysts in numerous
H industrial processes, such as the synthesis of food ingre-
dients, their use as additives to detergents and to obtain
O enantiopure drugs and other refined products (Contesini
et al., 2010).
H O O Lipases occur in animals, plants, and microorganisms.
Microbial lipases show a broad spectrum of industrial appli-
Aflatoxin B1 cation due to their greater stability, substrate specificity and
FIGURE 6.3  Structure of aflatoxin B1. lower production costs, when compared to other sources.
In addition, the biodiversity of microorganisms highlights
lipase biotechnological importance and justifies the interest
on them. Filamentous fungi are recognized as the best lipase
HO O
producers and are currently the preferred sources since they
O OH O
produce extracellular lipases, facilitating the extraction
from fermentation media. The most reported species belong
N O to the genera Rhizopus sp., Mucor sp., Geotrichum sp.,
H Penicillium sp., and Aspergillus sp. Furthermore, the use of
directed evolution can be very helpful to optimize existing
lipases with respect to desired properties.
Ochratoxin A
According to Contesini et  al. (2010) the current main
FIGURE 6.4  Ochratoxin A structure. applications of lipase are in detergents (removal of oil stains
84  SECTION | III  Secondary Metabolism

from fabrics); in the food industry (attainment of functional of cellulose as a carbon source to circumvent this limitation
phenols and aroma ester synthesis); in the pharmaceutical (Kunamneni et al., 2007).
industry (Kinetic resolution of ketoprofen and Kinetic reso- The range of applications of this enzyme is broad and
lution of diltiazem intermediate) and in the fuel industry, as encompasses several industrial sectors, thereby being part
in biodiesel production. of many important processes. From ethanol production to
Lipases are of great importance in the extraction of drug analysis, wine clarification, trichlorophenol, bioreme-
olive oil with Spain holding 60% of the EU’s total produc- diation, herbicide degradation, decolonization of dies, in
tion, Italy with 22%, and Greece with 15%, as reported the paper industry and in the textile industry to name a few
by Salgado et  al. (2014). The authors screened filamen- (Mayer and Staples, 2002; Kunamneni et al., 2007).
tous fungi for the production of lipases in solid-state In the paper industry laccases offer a milder and cleaner
fermentation (SSF) using wastes of winery and olive oil delignification in the pretreatment of wood pulp and a more
mills as substrates. Aspergillus ibericus, A. niger, and ecological alternative method than that employing chlorine-
A. uvarum were selected as suitable microorganisms to based compounds and they have been examined as the cath-
produce lipase in SSF. The results, as suggested by the ode in enzymatic biofuel cells. These are some of the few
workers, may have potential application in the simultane- oxidoreductases commercialized as industrial catalysts. The
ous management and valorization of olive mill and winer- enzymes can also be used for textile dying/textile finish-
ies wastes. ing, wine cork making, teeth whitening, and many other
industrial, environmental, diagnostic, and synthetic uses
(Rodríguez-Couto, 2015).
LACCASES The hazing effect is a quality defect in beer, although
Laccase (EC [Link]), is a copper-containing oxidase many connoisseurs would disagree. It is characterized by
enzyme and was described for the first time by Hikorokuro “cloudiness” in the final product. Laccase can be added
Yoshida at the end of the 19th century (Yoshida, 1883). The to the wort or at the end of the process to remove the
Japanese researcher extracted the enzyme from the lac- polyphenols that may still remain in beer (Madhavi and
quer tree Rhus vernicifera. In 1885 Gabriel Bertrand then Lele, 2009). The polyphenol complexes, formed by laccases,
found that laccase is a metalloprotein. However, at that can be separated via filtration and remove the probability
time Prof. Bertrand pointed out manganese as the metal of the hazing effect occurring. Laccase can also remove
due to an insufficiently purified enzyme (Lehn et al., 1986). excess oxygen in beer and increase the storage life of the
The metal associate is in fact copper, as later reported by beverage. In fruit juices, such as apple and grape, excess
Keilin and Mann (1939). Laccase is predominantly pre- oxidation of phenolic compounds causes negative effects on
sent in plants and fungi, but is also found in insects and the taste, color, odor, and mouthfeel. Laccase has been pro-
bacteria (Kunamneni et al., 2007). Importantly, fungal lac- posed to delay the oxidation of polyphenols and stabilize
cases have higher redox potential than that from bacteria the juice.
or plant sources. They are involved in the degradation of Many Aspergilli, such as nidulans, oryzae, niger, and
lignin and removal of toxic phenolic compounds. Apart fumigatus have been reported to produce laccases (Thurston,
from that, laccases might also be involved in the synthesis 1994; Scherer and Fischer, 1998). Mander et  al. (2006)
of melanin (dark polymers produced against environmental explored A. niger and Aspergillus nidulans to produce lac-
stress) (Kunamneni et al., 2007). Laccases from fungi have case, and used the enzyme as a protein reporter. Studies like
been reviewed by Thurston (1994) and Mayer and Staples the one conducted by Ramos et al. (2011), are seeking to
(2002), who also pointed out the innumerous uses of the improve parameters and optimize the use of laccase pro-
enzyme, and Giardina et al. (2010), referring to the genetic duced by Aspergillus for biotechnological purposes.
regulation aspects. Because the use of laccase in the above-mentioned pro-
Regarding the molecular features, laccases have a broad cesses require a large amount of the enzyme at low cost,
range of substrate specificity towards aromatic molecules, researchers have made efforts to optimize the fermentation
which empowers the enzyme to react with polyphenolic process, thereby enabling the production of the enzyme in
groups found in lignin (Kunamneni et al., 2007). The sub- a more affordable industrial scale (Couto and Toca-Herrera,
strate also influences the pH optima of laccases, which is 2007). Concerning this issue, the lack of an efficient pro-
usually between 3 and 5. The optimal temperature also var- duction system was pointed out by Couto and Toca-Herrera
ies from one strain to another, found to be from 40°C to (2007) as one factor that is not favorable for large-scale
60°C. The carbon and nitrogen sources are another param- production. Different strategies have been used to overcome
eter of importance and influence when laccase produc- the drawbacks presented by the use of fungi in such pro-
tion is concerned, as well as the ratio of these compounds cesses, including solid submerged fermentation and immo-
employed. However, the use of a high concentration of glu- bilization of enzymes, either by entrapment or attachment.
cose appears to inhibit the enzyme production, thus the use However, the immobilization approach remains challenging
 Secondary Metabolism and Antimicrobial Metabolites of Aspergillus  Chapter | 6  85

and probably consumes more effort. SSF appears then as PECTINASES

a competitive technique with advantages over SmF due to
high production yields and less complex downstream pro- Pectinases are enzymes that break down pectin, a struc-
cess. But the need to develop new reactors, or redesign the tural heteropolysaccharide found in primary plant cell walls
existing ones, is a limitation for the use of this technique. of terrestrial plants, cereals, fibers, fruits, and vegetables.
However, Couto and Toca-Herrera (2007) reported the use They were first isolated and described in 1825 by Henri
of other systems such as trickle-film processing in the pro- Braconnot (Anisa et  al., 2013; Kohli and Gupta, 2015).
duction of fungal laccase, which could be examined in order Commonly referred to as pectic enzymes, they comprise
to ensure best process parameters according to the needs of pectin lyase, pectozyme, and polygalacturonase. One of
a specific situation. the most studied and widely used commercial pectinases is
polygalacturonase. It is useful because pectin is the jelly-
like matrix which helps cement plant cells together and in
TANNASES which other cell wall components, are embedded. Therefore
Tannin acyl hydrolase (EC [Link]) catalyzes the hydroly- pectinase enzymes are commonly used in processes involv-
sis of ester and depside bonds in hydrolyzable tannins such ing the degradation of plant materials, such as speeding up
as tannic acid, methyl gallate, ethyl gallate, n-propyl gal- the extraction of fruit juice, including apples and sapota.
late and isoamyl gallate, releasing glucose and gallic acid. Its first commercial use was in 1930 (Kertesz, 1930).
Gallic acid catalyzes the second step in the degradation Pectinases have also been used in wine production since
of tannic acid (Lal and Gardner, 2012). Tannase has been the 1960s. The function of pectinase in brewing is twofold;
reported to be produced in several fungi. Despite white first it helps break down the plant (typically fruit) mate-
rot fungi being good laccase producers, Aspergilli also rial and so helps the extraction of flavors from the mash.
present interest regarding the obtaining and application of Secondly, the presence of pectin in finished wine causes
the enzyme (Couto and Toca-Herrera, 2007; Kumar et al., a haze or slight cloudiness, pectinase is used to break this
2007; Paranthaman et al., 2008; Costa et al., 2013; George down and so clear the wine. Besides that the enzyme is also
and Ong, 2013). In particular A. niger has been used to used in the extraction of tomato pulp, prevent foaming of
produce tannase (Pinto et al., 2001) and compare the pro- instant tea powder and chocolate fermentation. In animal
duction in solid state and submerged fermentation (Aguilar feed the enzymes find their use as peptic oligosaccharides
et  al., 2001; Mata-Gomez et  al., 2009), using agricultural as functional and probiotic components. Added during the
residues as alternative substrates in different fermentation gridding of olives, pectinases easy the oil extraction, fol-
methods (Hamdy and Fawzy, 2012). lowed by separation procedures. Besides that, pectinases
As reviewed by Lal and Gardner (2012), fungal tannase are used in the pharmaceutical field, detergent, paper indus-
is used in many industrial applications including clarifica- try, in retting, textile processing, waste water pretreatment
tion of fruit juice (Shivastava and Kar, 2009), detannifica- and plant disease control (Soares et  al., 2012; Kohli and
tion of food (Boadi and Neufeld, 2001), preparation of food Gupta, 2015).
preservatives (Belmares et  al., 2004), high-grade leather The enzyme is present in plants, bacteria, yeast, and
tanning (Lehka and Lonsane, 1997), clarification of beer fungi. Fungi are preferred by the industry as a source of
and wines (Bajpai and Patil, 2008), manufacture of coffee- pectinase, since they secrete the enzyme in the culture
flavored drinks (Anwar and Imartika, 2007), manufacture medium, facilitating its recovery (Soares et al., 2012). They
of instant tea (Lehka and Lonsane, 1997), production of gal- can be extracted from fungi and the most popular fungus
lic acid, which is used for the synthesis of trimethoprim (Yu used to obtain pectinase is A. niger. The fungus produces
et al., 2004), treatment of green tea to inhibit the carcino- these enzymes to break down the middle lamella in plants
genic and mutagenic effects of N-nitrosamines, stabilization so that it can extract nutrients from the plant tissues and
of malt polyphenols (Lehka and Lonsane, 1997), improved insert fungal hyphae. If pectinase is boiled it is denatured
color stability and additional organoleptic properties. In (unfolded) making it harder to connect with the pectin at the
animal feeding, tannase is used to reduce the antinutritional active site and produce as much juice (Debing et al., 2006).
effects of tannins and improve animal digestibility. Nowadays the enzyme makes up a quarter of the global
Tannase is also utilized for bioremediation of effluents market of food enzymes and about 10% of the global mar-
from tanneries. In addition, tannase is used as a sensitive ket (Anisa et al., 2013; Kohli and Gupta, 2015). Therefore,
analytical probe for determining the structure of natu- studies such as those conducted by Sandri et al. (2011) and
rally occurring gallic acid ester. Ma et al. (2014) explored Anisa et al. (2013) still attempt to optimize the production
Aspergillus ficuum production of tannase in SSF. The of pectinases, exploring different Aspergilli and substrates
authors also performed studies to optimize the process, as well as comparing different fermentation methods. It has
obtaining relatively high yields of the enzyme using wheat been reported that SSF leads to higher enzymatic produc-
bran as substrate. tion than submerged fermentation (Maheshwari, 2003).
86  SECTION | III  Secondary Metabolism

Several substrates are known to be used in pectinase pro- substantially the production, indicating a route to obtain
duction, such as wheat bran, rice straw and Tween 80 have proteases at reduced costs.
also been applied to produce pectinase (Debing et  al., Further studies have explored agro-industrial wastes
2006). In this direction, Esawy et  al. (2013) studied the (De Castro and Sato, 2014) and potato pulp as substrates to
production of pectinase from A. niger using Egyptian citrus produce proteases from A. oryzae. Siala et al. (2012) studied
peels as the carbon source. In order to optimize its results the production of aspartic protease from A. niger. The work
the authors immobilized the enzyme in polyvinyl alcohol was based on maximizing the enzymatic production using
sponges. The researchers observed superiority in all prop- a low-cost fermentation medium since medium components
erties analyzed using pectinase immobilized over the free are of crucial importance when it comes to industrial enzy-
enzyme pointing out its suitability for orange juice clari- matic production. Rather than using methods such as “one-
fication. Regarding the substrates, wheat bran and potato variable-at-a-time” the authors used a statistical approach
starch have been employed with success. Another report by by employing Plackett–Burman designing for the screen-
Durairajan and Sankari (2014) described maximum produc- ing of the main factors involved in protease production and
tion of the enzyme from A. niger using banana peel rather subsequently Box–Behnken design to assess the optimal
than orange and pineapple peel. Other substrates are also region of the significant variables. The workers found that
commonly used for the production of pectinases, such as using shrimp wastes from the fish-processing industry as
sugar cane bagasse and citrus peels. substrate was a good option since it contains a great amount
of carbohydrates and proteins. The protease production was
increased more than fourfold while the production cost low-
PROTEASES ered, revealing other options for the production of proteases
Proteases have important roles in baking, brewing, in the at lower prices and with an important ecological appeal.
production of various Oriental foods such as soy sauce, Ongoing research to enhance the process parameters to
miso, meat tenderization and cheese manufacture. obtain protease from other Aspergilli have been conducted
The first contact of humans with protease activities as those employing Aspergillus clavatus (Tremacoldi et al.,
occurred when we started producing milk curd. Desert 2004), Aspergillus fumigatus, A. flavus (Oyeleke et  al.,
nomads from the East used to carry milk in bags made 2010), and Aspergillus foetidus (Souza et al., 2015).
of goat stomach. After long journeys, they realized that
the milk became denser and sour, without understand-
LACTASES
ing the process’s cause. Curds thus became a food source
and a delicacy. Renin, an animal-produced enzyme, is Lactases are β-galactosidases, enzymes that catalyze
the protease which caused the hydrolysis of milk protein the hydrolysis of lactose into galactose and glucose
(Soares et  al., 2012). The use of protease includes food (Maksimainen et  al., 2013). β-Galactosidase is highly
processing, detergents, dairy industry, animal nutrition, important in the dairy industry, in the hydrolysis of lac-
paper and pulp, textiles and leather making (Negi and tose into glucose and galactose with an improvement in the
Benerjee, 2006; Chutmanop et  al., 2008; Hamada et  al., solubility and digestibility of milk and its related products.
2013). Nowadays proteases account for nearly 60% of Food with low-lactose contents or lactose-free is thereby
the enzyme market, which raises interests in optimizing obtained (Soares et al., 2012). Thus, a relief for people who
its production and obtaining better-quality enzymes (Jinka suffer from lactose intolerance, estimated at 70% of adults
et al., 2009). worldwide, which is caused by lactase insufficiency or non-
The substrate is one of the most important parameters existence in the colon, resulting in abdominal pain, nausea,
in enzymatic production as it is related to the final cost of and diarrhea due to malabsorption of lactose (Ingram et al.,
the product. Taking this into account, Negi and Benerjee 2009; Maksimainen et  al., 2013; de Vrese et  al., 2015).
(2006) produced protease concomitantly with amylase. β-Galactosidases are also used in reverse hydrolysis to
Using wheat bran as substrate and SSF as the production obtain galacto-oligosaccharides (GOS), used as probiotics
method they reached good amounts of both enzymes from in food to stimulate the growth of beneficial bacteria in the
Aspergillus awamori in a single bioreactor. Chutmanop colon (Vera et al., 2012; Maksimainen et al., 2013).
et al. (2008) also used SSF to analyze protease production. Regarding its biotechnological production from fila-
In that case A. oryzae was the chosen fungus and rice bran mentous fungi A. oryzae has been especially studied for
was explored as a promising substrate due to its large avail- providing the enzyme in sufficient amounts, being commer-
ability in Asian countries, besides being cheaper than wheat cially available and used in the milk industry (Maksimainen
bran. The use of rice bran alone was shown to not be inter- et al., 2013). Research has been conducted to characterize
esting, as low porosity prevented sufficient oxygen penetra- and evaluate its production over the last decades (Friend
tion, resulting in low performance. Yet, the authors found and Shahani, 1982; Corazza et  al., 1992; de Vrese et  al.,
that a mix of rice bran with 25% wheat bran improved 2015). During last years researchers have also concentrated
 Secondary Metabolism and Antimicrobial Metabolites of Aspergillus  Chapter | 6  87

in revealing structural details of β-galactosidases, provid- industry, bakery, brewery, and as a subsidy for ethanol pro-
ing information to tune the application of this important duction; in the conversion of glucose into fructose, used in
enzyme (Ito et al., 2002; Cantarel et al., 2009; Maksimainen soft drinks, jams and yoghurts (Aquino et al., 2003; Nguyen
et al., 2013). et al., 2002).
Amylases provide better bread color, volume, and tex-
ture in the baking industry. The use of these enzymes in
CELLULASES
bread production retards its aging process and maintains
Cellulases comprise enzymes that break the glycosidic fresh bread for a longer period. Whereas fungal amylase
bonds of cellulose microfibrils, releasing oligosaccharides, provides greater fermentation potential, amyloglucosidase
cellobiose, and glucose. Cellulases from fungi have had improves flavor and taste and a better bread crust color
their properties and production process studied for dec- (Soares et al., 2012).
ades (Hurst et  al., 1977; Begum and Absar, 2009; Ncube Aspergillus oryzae has been a producer of amylase as
et al., 2012). exemplified by the work of Chang et al. (1995) and Kariya
These hydrolytic enzymes are not only used in food, et al. (2003), for the purification of amylase. Other groups
drug, cosmetics, detergents and textile industries, but also have explored amylase production in A. niger (Hernandéz
in the wood pulp and paper industry, in waste management, et  al., 2006; Rosés and Guerra, 2009) and A. fumiga-
and in the medical-pharmaceutical industry (Bhat and Bhat, tus (Ratnasri et  al., 2014). These works have explored
1997). In the food industry, cellulases are employed in the potential of alternative substrates such as sugar cane
the extraction of components from green tea, soy protein, bagasse, cereal flours, or brewery (supplemented by
essential oils, aromatic products, and sweet potato starch. casamino acids, peptone, and yeast extract) in the prodution
Coupled to hemicellulases and pectinases, they are used in of fungal amylases.
the extraction and clarification of fruit juices. After fruit
crushing, the enzymes are used to increase liquefaction
through the degradation of the solid phase.
ORGANIC ACIDS
Because of their importance cellulases are constantly Organic acids are the most common acids, such as car-
under study in order to improve the required parameters. boxylic acids. Fungal biotechnology is very important for
Sohail et  al. (2009), investigated the production of cel- the production of many organic acids. Although the con-
lulases from A. niger in an attempt to obtain a sufficient version of organic acids can reach as high as 80% in liv-
amount of β-glucosidase, which is produced in low levels ing cells, standing out in productivity terms, some of these
in species of Tricoderma, a well-studied system for enzy- compounds occupy a relevant place in industrial produc-
matic depolymerization of cellulosic material. The results tion due to economic reasons when using chemical routes
were promising, leading to a moderate to high production of for obtaining organic acids (Magnuson and Lasure, 2004;
endonuclease and β-glucosidase. The work was carried out Liaud et al., 2014). Examples of these latter are oxalic acid,
in different substrates, namely grass, corncob and bagasse. a dicarboxylic acid, is made by the oxidation of glucose
or carbohydrates by nitric acid among some other alterna-
tives; fumaric acid, obtained from catalytic isomerization of
AMYLASES
maleic acid, and malic acid, first isolated by Carl Wilhelm
Amylases are starch-degrading enzymes that started to be Scheele in 1785 from apple juice and now synthesized
produced during the 20th century due to their great indus- by double hydration of maleic anhydride (Magnuson and
trial importance, being responsible for approximately a Lasure, 2004).
quarter of the enzyme market (Ratnasri et  al., 2014). In Organic acids play an important economical role in our
fact, they are the most important industrial enzymes with contemporary society due to the wide variety of applica-
high biotechnological relevance. Their uses range from tions they are involved in. From food to pharmaceuticals
textiles, beer, liquor, bakery, infant feeding cereals, starch and chemical processes, moving markets and supporting
liquefaction-saccharification, animal feed industries to the advancement of technologies. The production of organic
chemical and pharmaceutical uses. The species Aspergillus acids benefited highly from the biotechnological knowledge
and Rhizopus are highly important among the filamentous and improvements made during the 20th century thanks to
fungus for the production of amylases (Pandey et al., 1999, many interdisciplinary teams involving biologists, chem-
2006). The food industry uses amylases for the conversion ists, pharmacists, and engineers, among other profession-
of starch into dextrin. The latter are employed in clinical als in projects such as the penicillin production. Although
formulas as stabilizers and thickeners; in the conversion of organic acids can be found in other microorganisms such as
starch into maltose, in confectioneries in the manufacture bacteria, it has been in filamentous fungi that they have been
of soft drinks, beer, jellies and ice cream; in the conversion produced for many decades due to the high yields and other
of starch into glucose with applications in the soft-drinks process advantages discussed in the following paragraphs.
88  SECTION | III  Secondary Metabolism

CITRIC ACID for citric acid production; however, researchers have also
reported the production of citric acid in SSF (Vandenberghe
Citric acid was discovered by Karls Scheels in England in et  al., 1999; Kareem et  al., 2010). Research groups have
1874 in lemon juice (Vandenberghe et al., 1999; Max et al., been making efforts to use waste residues and byproducts
2010). The production of citric acid is the oldest and most derived from the fruit-processing industry to produce citric
thoroughly studied filamentous fungal fermentation, dating acid from A. niger in both SSF and submerged fermenta-
back to 1917, when Currie optimized the conditions using tion (Kareem et  al., 2010; Dhillon et  al., 2011). Kareem,
a surface cultivation method (Currie, 1917) and nowadays Akpan, and Alebiowu explored the potential of pineapple
most of its production occurs via microbial processes (Max peel as a cheap medium to produce citric acid, resulting in a
et al., 2010). production of 60.6 mg/kg of pineapple in optimized condi-
Following that, submerged fermentation started being tions. Using apple pomace solid waste, citrus waste, brew-
used for producing citric acid. Many of the parameters ery spent grain, and sphagnum peat moss, Dhillon et  al.
important for a productive submerged citric acid fermenta- reported that the substrates were suitable for citric acid pro-
tion process were determined by Shu and Johnson (1947, duction by both methods and might offer significant social,
1948a,b). In the course of time genetic manipulation tech- economic, and environmental impact.
niques were introduced to boast the acid productivity. Later,
SSF was introduced, while the search for new and cheaper
raw materials appeared. The metabolic pathway is known,
ITACONIC ACID
as are the fermentation conditions that result in high yields First obtained from the distillation of citric acid, since 1960
in submerged culture. Research in the last 60 years has itaconic acid has been produced by fermentation of carbo-
revealed some of the answers as to why these parameters hydrates by A. terreus (Mitsuyasu et al., 2009; Hajian and
are important, but many questions about the physiological Yusoff, 2015). Itaconic acid has been applied in a numerous
and biochemical mechanisms underlying these empirically range of industries with the larger producers in the world
derived fermentation conditions remain unanswered. being the USA, Japan, Russia, and China (Global Industry
Citric acid has a GRAS nature and is extensively used Analysts Inc., 2011).
in food as a pH adjustment and flavor improvement agent, During the 1950s, itaconic acid was used in industrial
which is responsible for 70% of its application, and other adhesives. In that period, itaconic acid was used at an
uses such as in pharmaceuticals and cosmetics, making up industrial scale and large amounts of it were required. It has
the remaining 30%, for acidification and metal ion chela- been employed as a detergent and in shampoos, as well as
tion (Dhillon et al., 2011). The critical parameters for citric in plastics, elastomers, fiberglass, and in the coating pro-
acid production by A. niger were defined empirically and cess of carpets and book covers (Mitsuyasu et al., 2009; Jin
include: high carbohydrate concentration, low but finite et  al., 2010). Besides that itaconic acid may also be used
manganese concentrations, maintenance of high dissolved as artificial gems and synthetic glasses (Kin et al., 1998).
oxygen, constant agitation, and low pH (Schreferl et  al., Lately, the applications of the compound have reached the
1986; Zhang and Röhr, 2002). These physical and chemical biomedical fields, such as the ophthalmic, dental and drug
conditions are important for the adoption and maintenance delivery fields (Hajian and Yusoff, 2015).
of a pelleted morphology, which is also critical for citric Several studies have focused on improving and optimiz-
acid production. Knowledge of these factors has enabled the ing the production of itaconic acid from A. terreus in recent
development of highly efficient submerged fermentations years. The biotechnological aspects involved in the metabolic
 Secondary Metabolism and Antimicrobial Metabolites of Aspergillus  Chapter | 6  89

pathways of itaconic acid and the production process param- et  al., 2006). Being found later by Molliard (1922) in
eters have been reviewed by Klement and Büchs (2013). A. niger. Since then many researchers have studied the con-
Regarding the production, Amina et al. (2013) obtained ita- ditions and processes that would lead to better yields.
conic acid using oil byproduct jatropha curcas seed cake, while Gluconic acid production by fermentation of glucose
Li et al. (2011), Huang et al. (2014), and van der Straat et al. using A. niger is a mature bioprocess with literature report-
(2014) studied the itaconic acid production by using genetic ing highly efficient processes dating back to 1940 (Moyer
engineering techniques. In this process the relevant pathways et al., 1940).
have been revealed and new microbial production platforms Gluconic acid has applications in the food industry, as in
designed, contributing to an enhanced production of itaconic meat and dairy products, baked goods, flavoring agent, and
acid. Furthermore, the reduction of its production costs is an reducing fat absorption in doughnuts (Ramachandran et al.,
important aspect for itaconic acid producers, either by opti- 2006). Although with a market smaller than that of citric
mizing processes or by using cost-favorable raw materials. acid, gluconic acid finds its place, as well as its derivatives,
such as sodium, calcium, and iron gluconate, which is used
for dietary supplements, in the pharmaceutical and textile
KOJIC ACID industries (Ramachandran et al., 2006).
Kojic acid (5-hydroxy-2-(hydroxymethyl)-4-pyrone; KA) Nowadays, despite having access to a variety of methods
is an organic acid secreted by several microorganisms of to produce gluconic acid, microbial fermentation remains
Aspergillus genus such as A. oryzae, Aspergillus tamarri, the chosen approach since other methods are more expensive
Aspergillus parasiticus, and A. flavus (Bentley, 2006). The and less efficient compared to fermentation (Ramachandran
name KA was derived from “Koji,” a fungus or starter inoc- et  al., 2006). For that the microorganism most commonly
ulum used in Oriental food such as sake, shoyu, miso, and used is A. niger. Even though several factors influence
vinegar (Terabayashi et al., 2010; Chaudhary et al., 2014). microbial fermentation, it is believed that oxygen avail-
This crystalline substance was firstly isolated by Saito in ability and the pH of the medium are key parameters to be
1907, from the mycelia of A. oryzae grown on steamed rice addressed. Studies concentrate in exploring the fermentation
in solid state culture (Saito, 1907). The chemical structure processes, as well as alternatives such as cheaper raw mate-
was determined by Yabuta in 1924. It is multifunctional and rials, enzymatic immobilization, molecular biology tools, so
has weak acidic property. The natural origin of KA confirms that production can be optimal and the results the best possi-
its nonhazardous biodegradation, making it an attractive ble (Roukas, 2000; Ikeda et al., 2006; Ramachandran et al.,
and profitable skeleton for the development of biologically 2008; Lu et al., 2015; Shi et al., 2015).
active compounds by its derivation (Chaudhary et al., 2014).
KA is used as a food additive, antibiotic, antioxidant
FINAL REMARKS
(Bentley, 2006), a skin-whitening agent in the cosmetic
industry and in medicine, for the treatment of chloasma Filamentous fungi, especially Aspergilli, play a key role
(Terabayashi et al., 2010), antitumor agent (Tamura et al., in many industrial sectors nowadays from food to medi-
2006) and radioprotective agent (Emami et al., 2007). Due cines and chemicals. During the 20th century the incredible
to its wide range of applications, KA has been targeted by advancement in our knowledge about these microorgan-
researchers to discover the biosynthesis pathways in fila- isms has enabled the use of enzymes and other molecules
mentous fungi (Ariff et  al., 1996; Futamura et  al., 2001; favorably, saving lives and adding quality to many prod-
Rosfarizan et al., 2002). Terabayashi et al. (2010) disclosed ucts consumed and necessary for our existence. Despite the
information about the genes involved in KA biosynthesis in negative effects of molecules such as aflatoxin and ochra-
A. oryzae. Using DNA microarray technique the workers toxin, causing deaths and crop losses, resulting in economi-
found two genes that might be involved in the biosynthesis cal obstacles, much more can be celebrated. Fungi excrete
process, giving insights into the genetic regulation of KA many beneficial compounds that could be used in daily life
production. Other works have been related to the production through biotechnological exploration. Understanding the
methods of KA and bioreactors construction as described molecular relationship between these compounds and fun-
by Ogawa et al. (1995) and Wakisaka et al. (1998) in mem- gal development is essential to effectively take advantage of
brane-surface liquid culture (MSLC). the further progress yet to be made.
The advancement in the analytical techniques such as
mass spectrometry (MS) and hybrid equipments, LC/MS,
GLUCONIC ACID LC-NMR, along with the knowledge generated by metagen-
Gluconic acid is produced from glucose. In this glucose oxi- omics and the expertise gained in industrial microbiology
dase catalysis process, the dehydrogenation reaction leads in the last decades, provide us with great tools to direct
to its production (Ramachandran et al., 2006). It had already the search for secondary metabolites even though they are
been produced in 1870 (Röhr et al., 1983; Ramachandran not evident.
90  SECTION | III  Secondary Metabolism

ACKNOWLEDGMENTS Boadi, D.K., Neufeld, R.J., 2001. Encapsulation of tannase for the hydrol-
ysis of tea tannins. Enzyme Microbiol. Technol. 28, 590–595.
The author thanks Prof. V.K. Gupta for the invitation to write this Calvo, A.M., Wilson, R.A., Bok, J.W., Keller, N.P., 2002. Relationship
chapter. The author’s previous work in the field was supported by the between secondary metabolism and fungal development. Microbiol.
Coordination for the Improvement of Higher Level Personnel (CAPES) Mol. biol. Rev. 66, 447–459.
and São Paulo Research Foundation (FAPESP), Brazil. Igor and Hanne Campbell, A.C., Maidment, M.S., Pick, J.H., Stevenson, D.F.M.J., 1985.
Debergh are warmly thanked for the technical revision of the work. Synthesis of (E)- and (2)-pulvinones. Chem. Soc. Perkin Trans. 1,
1567–1576.
Cantarel, B.L., Coutinho, P.M., Rancurel, C., Bernard, T., Lombard, V.,
REFERENCES Henrissat, B., 2009. The Carbohydrate-Active EnZymes database
(CAZy): an expert resource for glycogenomics. Nucleic Acids Res.
Aguilar, C.N., Augur, C., Favela-Torres, E., Viniegra-González, G., 2001. 37, 233–238.
Production of tannase by Aspergillus niger Aa-20 in submerged and Castellá, G., Alborch, L., Bragulat, M.R., Cabañes, F.J., 2015. Real time
solid-state fermentation: influence of glucose and tannic acid. J. Ind. quantitative expression study of a polyketide synthase gene related
Microbiol. Biotechnol. 26 (5), 296–302. to ochratoxin a biosynthesis in Aspergillus niger. Food Control. 53,
Alberts, A.W., Chen, J., Kuron, G., Hunt, V., Huff, J., Hoffman, C., et al., 147–150.
1980. Mevinolin: a highly potent competitive inhibitor of hydroxym- Chang, C.T., Tang, M.S., Lin, C.F., 1995. Purification and properties of
ethylglutaryl-coenzyme A reductase and a cholesterol-lowering agent. alpha-amylase from Aspergillus oryzae ATCC 76080. Biochem. Mol.
Proc. Natl. Acad. Sci 77, 3957–3961. Biol. Int. 36 (1), 185–193.
Amare, M.G., Keller, N.P., 2014. Molecular mechanisms of Aspergillus Chaudhary, J., Pathak, A.N., Lakhawat, S., 2014. Production technology
flavus secondary metabolism and development. Fungal Genet. Biol. and applications of kojic acid. Annu. Res. Rev. Biol. 4 (21), 3165–3196.
66, 11–18. Chutmanop, J., Chuichulcherm, S., Chisti, Y., Srinophakun, P., 2008.
Anisa, S.K., Ashwini, S., Girish, K., 2013. Isolation and screening of Protease production by Aspergillus oryzae in solid-state fermentation
Aspergillus spp. for pectinolytic activity. Electr. J. Biol. 9 (2), 37–41. using agroindustrial substrates. J. Chem. Technol. Biotechnol. 83,
Antane, S., Caufield, C.E., Hu, W., Keeney, D., Labthavikul, P., Morris, K., 1012–1018.
et al., 2006. Pulvinones as bacterial cell wall biosynthesis inhibitors. Contesini, F.J., Lopes, D.B., Macedo, G.A., Nascimento, M.G., Carvalho,
Bioorg. Med. Chem. Lett. 16, 176–180. P.O., 2010. Aspergillus sp. lipase: potential biocatalyst for industrial
Anwar, Y.A.S., Imartika, H., 2007. The production of tannin acyl hydrolase use. J. Mol. Catal. B: Enzym. 67, 163–171.
from Aspergillus niger. Microbiol. Indonesia 1 (2), 91–94. Corazza, G.R., Benati, G., Sorge, M., Strocchi, A., Calza, G., Gasbarrini,
Aquino, A.C.M.M., Jorge, J.A., Terenzi, H.F., Polizeli, M.L.T.M., G., 1992. beta-galactosidase from Aspergillus niger in adult lactose
2003. Studies on a thermostable a-amylase from thermophilic malabsorption: a double-blind crossover study. Aliment. Pharmacol.
fungus Scytalidium thermophilum. Appl. Microbiol. Biotechnol. 61, Ther. 6 (1), 61–66.
323–328. Costa, A.M., Cristina Souza, G.M., Bracht, A., Kadowaki, M.K., de Souza,
Ariff, A.B., Salleh, M.S., Ghani, B., Hassan, M.A., Rusul, G., Karim, A.C.S., Oliveira, R.F., et al., 2013. Production of tannase and gallic
M.I.A., 1996. Aeration and yeast extract requirements for kojic acid acid by Aspergillus tamarii in submerged and solid state cultures. Afr.
production by Aspergillus flavus link. Enzyme Microb. Technol. 19 J. Biochem. Res. 7 (10), 197–202.
(7), 545–550. Couto, S.R., Toca-Herrera, J.L., 2007. Laccase production at reactor scale
Bajpai, B., Patil, S., 2008. A new approach to microbial production of gal- by filamentous fungi. Biotechnol. Adv. 25, 558–569.
lic acid. Braz. J. Microbiol. 39, 708–711. Crespo-Sempere, A., Martínez-Culebras, P.V., González-Candelas, L.,
Begum, M.F., Absar, N., 2009. Purification and characterization of intracel- 2014. The loss of the inducible Aspergillus carbonarius MFS trans-
lular cellulase from Aspergillus oryzae ITCC-4857.01. Mycobiology porter MfsA leads to ochratoxin A overproduction. Int. J. Food.
37 (2), 121–127. Microbiol. 181, 1–9.
Belmares, R., Contresras-Esquival, J.C., Rodriguez-Harerra, R., Coronel, Currie, J.N., 1917. The citric acid fermentationd of Aspergillus niger. J.
A.R., Aguilar, C.N., 2004. Lebensmittel-Wissenschaft Technologie. Biol. Chem. 31, 15–37.
Food Sci. Technol. 37 (8), 857–864. De Castro, R.J.S., Sato, H.H., 2014. Production and biochemical charac-
Bentley, R., 2006. From miso, sake and shoyu to cosmetics: a century of terization of protease from Aspergillus oryzae: an evaluation of the
science for kojic acid. Nat. Prod. Rep. 23 (1046), 1062. physical–chemical parameters using agroindustrial wastes as supports.
Bhat, M.K., Bhat, S., 1997. Cellulose degrading enzymes and their poten- Biocatal. Agric. Biotechnol. 3, 20–25.
tial industrial applications. Biotechnol. Adv. 15 (3-4), 583–620. De Vrese, M., Laue, C., Offick, B., Soeth, E., Repenning, F., Thoß, A.,
Bhatnagar-Mathur, P., Sunkara, S., Bhatnagar-Panwar, M., Waliyar, et  al., 2015. A combination of acid lactase from Aspergillus oryzae
F., Sharma, K.K., 2015. Biotechnological advances for combating and yogurt bacteria improves lactose digestion in lactose maldigest-
Aspergillus flavus and aflatoxin contamination in crops. Plant. Sci. ers synergistically: a randomized, controlled, double-blind cross-over
234, 119–132. trial. Clin. Nutr. 34 (3), 394–399.
Birch, A., Massywestropp, R., Moye, C., 1955. Studies in relation to bio- Debing, J., Peijun, L., Stagnitti, F., Xianzhe, X., Li, L., 2006. Pectinase
synthesis.7. 2-Hydroxy-6-methylbenzoic acid in Penicillium griseof- production by solid fermentation from Aspergillus niger by a new pre-
ulvum Dierckx. Aust. J. Chem., 539–544. scription experiment. Ecotox. Environ. Safe 64, 244–250.
Bizukojc, M., Pawlak, M., Boruta, T., Gonciarz, J., 2012. Effect of pH Dhillon, G.S., Brara, S.K., Verma, M., Tyagi, R.D., 2011. Utilization of
on biosynthesis of lovastatin and other secondary metabolites by different agro-industrial wastes for sustainable bioproduction of citric
Aspergillus terreus ATCC 20542. J. Biotechnol. 162, 253–261. acid by Aspergillus niger. Biochem. Eng. J. 54, 83–92.
 Secondary Metabolism and Antimicrobial Metabolites of Aspergillus  Chapter | 6  91

Durairajan, B., Sankari, P.S., 2014. Optimization of solid state fermenta- Ingram, C.J.E., Mulcare, C.A., Itan, Y., Thomas, M.G., Swallow, D.M.,
tion conditions for the production of pectinases by Aspergillus niger. 2009. Lactose digestion and the evolutionary genetics of lactase per-
J. Pharm. Biosci. 2, 50–57. sistence. Human Genetics. 124 (6), 579–591.
Edwards, R.L., Gill, M., 1973. Constituents of the higher fungi. Part X1V.l Ito, Y., Sasaki, T., Kitamoto, K., Kumagai, C., Takahashi, K., Gomi, K.,
3′,4′,4-trihydroxypulvin- one, thelephoric acid, and novel pyrandione et  al., 2002. Cloning, nucleotide sequencing, and expression of the
and furanone pigments from Suillus grevillei (Klotsch) Sing. [Boletus beta-galactosidase-encoding gene (lacA) from Aspergillus oryzae.
elegans (Schum. per Fries)]. J. Chem. Soc., Perkin Trans. 1, 1921–1929. J. Gen. App. Microbiol. 48 (3), 135–142.
Emami, S., Hosseinimehr, S.J., Taghdisi, S.M., Akhlaghpoor, S., 2007. Jin, H., Lei, H., Jianping, L., Zhinan, X., Peilin, C., 2010. Organic chemi-
Kojic acid and its manganese and zinc complexes as potential radio- cals from bioprocesses in China. Adv. Biochem. Eng. Biotechnol. 122,
protective agents. Bioorg. Med. Chem. Lett. 1 (1), 45–48. 17. 43–71.
Esawy, M.A., Gamala, A.A., Kamel, Z., Ismail, A.-M. S., Abdel-Fattah, Jinka, R., Ramakrishna, V., Rao, S., Rao, R.P., 2009. Purification and char-
A.F., 2013. Carbohydr. Polym. 92, 1463–1469. acterization of cysteine protease from germinating cotyledons of horse
Fox, E.M., Howlett, B.J., 2008. Secondary metabolism: regulation and role gram. BMC Biochem. 10, 1–11.
in fungal biology. Curr. Opin. Microbiol 11, 481–487. John, K.M.M., Jung, E.S., Lee, S., Kim, J.-S., Lee, C.H., 2013. Primary
Friend, B.A., Shahani, K.M., 1982. Characterization and evaluation and secondary metabolites variation of soybean contaminated with
of Aspergillus oryzae lactase coupled to a regenerable support. Aspergillus sojae. Food Res. Int. 54, 487–494.
Biotechnol. Bioeng. 24 (2), 329–345. Kardos, N., Demain, A.L., 2011. Penicillin: the medicine with the great-
Futamura, T., Okabe, M., Tamura, T., Toda, K., Matsunobu, T., Park, Y.S., est impact on therapeutic outcomes. Appl. Microbiol. Biotechnol. 92,
2001. Improvement of production of kojic acid by a mutant strain 677–687.
Aspergillus oryzae, MK107-39. J. Biosci. Bioeng. 93 (3), 272–276. Kardos, N., Demain, A.L., 2013. Ernst Chain: a great man of science.
Gao, H., Guo, W., Wang, Q., Zhang, L., Zhu, M., Zhu, T., et  al., 2013. Appl. Microbiol. Biotechnol. 97, 6613–6622.
Aspulvinones from a mangrove rhizosphere soil-derived fungus Kareem, S.O., Akpan, I., Alebiowu, O.O., 2010. Production of citric acid
Aspergillus terreus Gwq-48 with anti-influenza A viral (H1N1) activ- by Aspergillus niger using pineapple waste. Malays. J. Microbiol. 6
ity. Bioorg. Med. Chem. Lett. 23, 1776–1778. (2), 161–165.
George, D.S., Ong, C.-B., 2013. Improvement of tannase production under Kariya, M., Shigemi, Y., Yano, M., Konno, H., Takii, Y., 2003. Purification
submerged fermentation by Aspergillus niger FBT1 isolated from a and properties of α-amylase from Aspergillus oryzae MIBA316.
mangrove forest. BioTechnologia 94 (4), 451–456. J. Biol. Macromol. 3 (2), 57–60.
Giardina, P., Faraco, V., Pezzella, C., Piscitelli, A., Vanhulle, S., Sannia, Keilin, D., Mann, T., 1939. Laccase, a blue copper-protein oxidase from
G., 2010. Laccases: a never-ending story. Cell. Mol. Life. Sci. 67, the latex of Rhus succedanea. Nature 143, 23–24.
369–385. Keller, N.P., Turner, G., Bennett, J.W., 2005. Fungal secondary metabo-
Global Industry Analysts Inc., 2011. Global Itaconic Acid (IA) market to lism: from biochemistry to genomics. Nat. Rev. Microbiol. 3 (12),
reach US$398.3 million by 2017, according to a new report by Global 937–947.
Industry Analysts, Inc. In: Prweb. <[Link] Kertesz, Z. 1930. A new method for enzymic clarification of unfermented
itaconic_acid/renewable_chemicals/[Link]> (accessed apple juice, U.S. Patent No.1.932.833, New York State Agricultural
05.09.15). Experimentation Station (Geneva) Bill. No. 689.
Hajian, H., Yusoff, W.M.W., 2015. Itaconic acid production by microor- Khan, A.A., Bacha, N., Ahmad, B., Lutfullah, G., Farooq, U., Cox, R.J.,
ganisms: a review current research. J. Biol. Sci. 7 (2), 37–42. 2014. Fungi as chemical industries and genetic engineering for the
Hamada, S., Suzuki, K., Aoki, N., Suzuki, Y., 2013. Improvements in the production of biologically active secondary metabolites. Asian Pac. J.
qualities of gluten-free bread after using a protease obtained from Trop. Biomed. 4 (11), 859–870.
Aspergillus oryzae. J. Cereal sci. 57, 91–97. Kim, M.J., Maria John, K.M., Choi, J.N., Lee, S., Kim, A.J., Kim, Y.M.,
Hamdy, H.S., Fawzy, E.M., 2012. Economic production of tannase by et al., 2013. Changes in secondary metabolites of green tea during fer-
Aspergillus niger van tiegh adopting different fermentation proto- mentation by Aspergillus oryzae and its effect on antioxidant potential.
cols and possible applications. Romanian Biotechnol. Lett. 17 (4), Food Res. Int. 53, 670–677.
7441–7457. Kin, R., T. Sai and S. So, 1998. Itaconate copolymer with quadratic nonlin-
D Hansson. 2013. Structure and Biosynthesis of Fungal Secondary ear optical characteristic. JP Patent No. 10,293,331.
Metabolites: Studies of the Root Rot Pathogen Heterobasidion anno- Klement, T., Büchs, J., 2013. Itaconic acid—a biotechnological process in
sum s.l. and the Biocontrol Fungus Phlebiopsis gigantean. Thesis. change. Bioresour. Technol. 135, 422–431.
Hernández, M.S., Rodríguez, M.R., Guerra, N.P., Rosés, R.P., 2006. Kohli, P., Gupta, R., 2015. Alkaline pectinases: a review. Biocatal. Agric.
Amylase production by Aspergillus niger in submerged cultivation on Biotech. 4, 1–7.
two wastes from food industries. J. Food Eng. 73 (1), 93–100. Kumar, R., Sharma, J., Singh, R., 2007. Production of tannase from
Huang, X., Lu, X., Li, Y., Li, X., Li, J.-J., 2014. Improving itaconic acid Aspergillus ruber under solid-state fermentation using jamun
production through genetic engineering of an industrial Aspergillus (Syzygium cumini) leaves. Microbiol. Res. 162 (4), 384–390. 2(6).
terreus strain. Microb. Cell. Fact. 113 (119), 1–9. Kunamneni, A., Ballesteros, A., Plou, F.J., Alcalde, M., 2007. Fungal
Hurst, P.L., Nielsen, J., Sullivan, P.A., Shepherd, M.G., 1977. Purification laccase—a versatile enzyme for biotechnological applications.
and properties of a cellulase from Aspergillus niger. Biochem. J. 165 In: Méndez-Vilas, A. (Ed.), Communicating Current Research
(1), 33–41. Educational Topics Trends Applied Microbiology Formex, Spain,
Ikeda, Y., Park, E.Y., Okuda, N., 2006. Bioconversion of waste office paper pp. 233–245.
to gluconic acid in a turbine blade reactor by the filamentous fungus Lal, D., Gardner, J.J., 2012. Production, characterization and purification
Aspergillus niger. Bioresour. Technol. 97 (8), 1030–1035. of tannase from Aspergillus niger. Eur. J. Exp. Biol. 2 (5), 1430–1438.
92  SECTION | III  Secondary Metabolism

Lehn, J.-M., Malmström, B.G., Selin, E., Oblad, M., 1986. Metal analysis Nguyen, Q.D., Rezessy-Szabo, J.M., Claeyssens, M., Stals, I., Hoschke,
of the laccase of Gabriel Bertrand. Reflections Biochem, 228–230. A., 2002. Purification and characterisation of amylolytic enzymes
Lekha, P.K., Lonsane, B.K., 1997. Production and application of from thermophilic fungus Thermomyces lanuginosus strain ATCC
tannin acyl hydrolsase: state of the art. Adv. Appl. Microbiol. 44, 34626. Enzymes Microbial. Technol., 345–352.
215–260. Ogawa, A., Wakisaka, Y., Tanaka, T., Sakiyama, T., Nakanishi, K., 1995.
Li, A., van Luijk, N., ter Beek, M., Caspers, M., Punt, P., van der Werf, M., Production of kojic acid by membrane-surface liquid culture of
2011. A clone-based transcriptomics approach for the identification Aspergillus oryzae NRRL484. J. Ferment. Bioeng. 80 (1), 41–45.
of genes relevant for itaconic acid production in Aspergillus. Fungal. Ojima, N., Takenaka, S., Seto, S., 1973. New butenolides from Aspergillus
Genet. Biol. 3 (6), 602–611. terreus. Phytochemistry 12, 2527.
Liaud, N., Giniés, C., Navarro, D., Fabre, N., Crapart, S., Herpoël-Gimbert, Osman, M.E., Khattab, O.H., Zaghlol, G.M., Abd El-Hameed, R.M., 2011.
I., et al., 2014. Exploring fungal biodiversity: organic acid production Optimization of some physical and chemical factors for lovastatin pro-
by 66 strains of filamentous fungi. Fungal Biol. Biotech. 1 (1), 1–10. ductivity by local strain of Aspergillus terreus. Aust. J. Basic Appl.
Lu, F., Ping, K., Wen, L., Zhao, W., Wang, Z., Chu, J., et  al., 2015. Sci. 5 (6), 718–732.
Enhancing gluconic acid production by controlling the morphology Oyeleke, S.B., Egwim, E.C., Auta, S.H., 2010. Screening of Aspergillus
of Aspergillus niger in submerged fermentation. Process Biochem. 50 flavus and Aspergillus fumigatus strains for extracellular protease
(9), 1342–1348. enzyme production. J Microbiol. Antimicrob. 2 (7), 83–87.
Ma, W.-l, Zhao, F.-f, Ye, Q., Hu, Z.-X., Yan, D., Hou, J., et  al., 2014. Pandey, A., Benjamin, S., Soccol, C.R., Nigam, P., Kriger, N., Soccol, V.T.,
Production and partial purification of tannase from Aspergillus ficuum 1999. The realm of microbial lipases in biotechnology. Biotechnol.
Gim. 3.6. Prep. Biochem. Biotechnol. 45, 754–768. Appl. Biochem. 29, 119–131.
Madhavi, V., Lele, S.S., 2009. Laccase: properties and applications. Pandey, A., Webb, C., Soccol, C.R., Larroche, C. (Eds.), 2006. Enzyme
BioResources. 4, 1694–1717. Technology Springer. Asiatech Publishers, New Delhi.
Magnuson, J.K., Lasure, L.L., 2004. Organic acid production by fila- Paranthaman, R., Vidyalakshmi, R., Murugesh, S., Singaravadivel, K.,
mentous fungi. In: Jan, S., Lange, L. (Eds.), Advances in Fungal 2008. Optimisation of fermentation conditions for production of tan-
Biotechnology for Industry, Agriculture, and Medicine Kluwer nase enzyme by Aspergillus oryzae using sugarcane baggasse and rice
Academic/Plenum Publishers straw. Global J. Biotechnol. Biochem. 3 (2), 105–110.
Maheshwari, M. 2003. Microbial production of pectinases from coffee Pinto, G.A.S., Leite, S.G.F., Terzi, S.C., Couri, S., 2001. Selection of tannase-
pulp waste. Paper Presented at 44th Annual Conference of Association producing Aspergillus niger strains. Braz. J. Microbiol. 32 (1), 24–26.
of Microbiologists of India, Dharwad, India, pp. 12–14. Ramachandran, S., Fontanille, P., Pandey, A., Larroche, C., 2006. Gluconic
Maksimainen, M.M., Lampio, A., Mertanen, M., Turunen, O., Rouvinen, J., acid: properties, applications and microbial production. Food Technol.
2013. The crystal structure of acidic β-galactosidase from Aspergillus Biotechnol. 44 (2), 185–195.
oryzae. Int. J. Biol. Macromol. 60, 109–115. Ramachandran, S., Fontanille, P., Pandey, A., Larroche, C., 2008.
Mander, G.J., Wang, H., Bodie, E., Wagner, J., Vienken, K., Vinuesa, C., Permeabilization and inhibition of the germination of spores of
et al., 2006. Use of laccase as a novel, versatile reporter system in fila- Aspergillus niger for gluconic acid production from glucose.
mentous fungi. Appl. Environ. Microbiol. 72, 5020–5026. Bioresour. Technol. 99 (11), 4559–4565.
Mata-Gomez, M., Rodriguez, L.V., Ramos, E.L., Renovato, J., Cruz- Ramos, J.A.T., Barends, S., Verhaert, R. M.D., de Graaff, L.H., 2011.
Hernandez, M.A., Rodriguez, R., et al., 2009. A novel tannase from The Aspergillus niger multicopper oxidase family: analysis and
the xerophilic fungus Aspergillus niger GH1. J. Microbiol. Biotechnol. overexpression of laccase-like encoding genes. Microb. Cell. Fact. 10,
19 (9), 987–996. 78.
Max, B., Salgado, J.M., Rodríguez, N., Cortés, S., Converti, A., Ratnasri, P.V., Lakshmi, B.K.M., Ambika Devi, K., Hemalatha, K.P.J.,
Domínguez, J.M., 2010. Biotechnological production of citric acid. 2014. Isolation, characterization of Aspergillus fumigatus and optimi-
Braz. J. Microbiol. 41 (4), 862–875. zation of cultural conditions for amylase production. Int. J. Res. Eng.
Mayer, A.M., Staples, R.C., 2002. Laccase: new functions for an old Technol. 03 (02), 457–463.
enzyme. Phytochemistry 60, 551–565. Rehse, K., Lehmke, J., 1985. Anticoagulante 3-Aryl-5-
Mitsuyasu, O., Dwiarti, L., Shin, K., Enoch, P.Y., 2009. Biotechnological benzylidentetronäuren. Arch. Pharm. 318, 11.
production of itaconic acid and its biosynthesis in Aspergillus terreus. Rodríguez-Couto, S., 2015. Fungal laccase in the textile industry. In: Gupta,
Appl. Microbiol. Biotechnol. 84, 597–606. V.K., Mach, R.L., Sreenivasaprasad, S. (Eds.), Fungal Biomolecules:
Molliard, M., 1922. Sur une nouvelle fermentation acide produite par le Sources, Applications and Recent Developments, first ed. John Wiley
Sterigmatocystis nigra (A new acidic fermentation by Sterigmatocystis & Sons
nigra). CR Acad. Sci. 174, 881–883. Röhr, M., Kubicek, C.P., Kominek, J., 1983. Gluconic Acid In: Rehm,
Moyer, A.J., Umberger, E.J., Stubbs, J.J., 1940. Fermentation of concen- H.J. Reed, G. (Eds.), Biotechnology, 3 Verlag Chemie, Weinheim,
trated solutions of glucose to gluconic acid. Improved process. Ind. pp. 455–465.
Eng. -Chem., Ind. Ed 32, 1379–1383. Rosés, R.P., Guerra, N.P., 2009. Optimization of amylase production by
Ncube, T., Howard, R.L., Abotsi, E.K., van Rensburg, E.L.J., Ncube, I., Aspergillus niger in solid-state fermentation using sugarcane bagasse
2012. Jatropha curcas seed cake as substrate for production of xyla- as solid support material. World J. Microbiol. Biotechnol. 25 (11),
nase and cellulase by Aspergillus niger FGSCA733 in solid-state fer- 1929–1939.
mentation. Ind. Crops Products. 37 (1), 118–123. Rosfarizan, M., Arbakariya, A., Hassan, M.A., Karim, M.I.A., Hiroshi, S.,
Negi, S., Benerjee, R., 2006. Optimization of amylase and protease pro- Suteaki, S., 2002. Importance of carbon source feeding and pH con-
duction from Aspergillus awamori in single bioreactor through EVOP trol strategies for maximum kojic acid production from sago starch by
factorial design technique. Food Technol. Biotechnol. 44, 257–261. Aspergillus flavus. J. Biosci. Bioeng. 94 (2), 99–105.
 Secondary Metabolism and Antimicrobial Metabolites of Aspergillus  Chapter | 6  93

Roukas, T., 2000. Citric and gluconic acid production from fig by novel acid protease from Aspergillus foetidus. Int. J. Biol. Macromol.
Aspergillus niger using solid-state fermentation. J. Ind. Microbiol. 81, 17–21.
Biotechnol. 25 (6), 298–304. Terabayashi, Y., Sano, M., Yamane, N., Marui, J., Tamano, K., Sagara, J.,
Saito, K., 1907. Über die saurebildung bei Aspergillus oryzae. J. Botany et  al., 2010. Identification and characterization of genes responsible
21, 7–11. Deutsche. for biosynthesis of kojic acid, an industrially important compound
Salgado, J.M., Abrunhosa, L., Venâncio, A., Domínguez, J.M., Belo, I., from Aspergillus oryzae. Fungal Genet. Biol. 47 (12), 953–961.
2014. Integrated use of residues from olive mill and winery for lipase Thurston, C.F., 1994. The structure and function of fungal laccases.
production by solid state fermentation with Aspergillus sp. Appl. Microbiology 140, 19–26.
Biochem. Biotech. 172, 1832–1845. Tobert, J.A., 2003. Lovastatin and beyond: the history of the HMG-CoA
Sandri, I.G., Fontana, R.C., Barfknecht, D.M., da Silveira, M.M., 2011. reductase inhibitors. Nat. Rev. Drug Discov. 2, 517–526.
Clarification of fruit juices by fungal pectinases. LWT – Food Sci. Tremacoldi, C.R., Watanabe, N.K., Carmona, E.C., 2004. Production
Technol. 44, 2217–2222. of extracellular acid proteases by Aspergillus clavatus. World J.
Scheffler, R.J., Colmer, S., Tynan, H., Demain, A.L., Gullo, V.P., 2013. Microbiol. Biotechnol. 20, 639–642.
Antimicrobials, drug discovery, and genome mining. Appl. Microbiol. Vaishnav, P., Demain, A.L., 2010. Unexpected applications of secondary
Biotechnol. 97, 969–978. metabolites. Biotech. Adv. 29, 223–229.
Scherer, M., Fischer, R., 1998. Purification and characterization of laccase van der Straat, L., Vernooij, M., Lammers, M., van den Berg, W.,
II of Aspergillus nidulans. Arch. Microbiol. 170, 78–84. Schonewille, T., Cordewener, J., et  al., 2014. Expression of the
Schreferl, G., Kubicek, C.P., Röhr, M., 1986. Inhibition of citric acid Aspergillus terreus itaconic acid biosynthesis cluster in Aspergillus
accumulation by manganese ions in Aspergillus niger mutants with niger. Microb. Cell Fact. 13, 11.
reduced citrate control of phosphofructokinase. J. Bacteriol. 165 (3), Vandenberghe, L.P.S., Soccol, C.R., Pandey, A., Lebeault, J.-M., 1999.
1019–1022. Review: microbial production of citric acid. Braz. Arch. Biol. Technol.
Shi, F., Tan, J., Chu, J., Wang, Y., Zhuang, Y., Zhang, S., 2015. A qualita- 42, 1–14.
tive and quantitative high-throughput assay for screening of gluconate Vera, C., Guerrero, C., Conejeros, R., Illanes, A., 2012. Synthesis of
high-yield strains by Aspergillus niger. J. Microbiol. Method 109, galacto oligosaccharides by β-galactosidase from Aspergillus oryzae
134–139. using partially dissolved and supersaturated solution of lactose.
Shrivastava, A., Kar, K., 2009. Characterization and application of tannase Enzyme Microb. Technol. 50 (3), 188–194.
produced by Aspergillus Niger ITCC 6514.07 on pomegranate rind. Wakisaka, Y., Segawa, T., Imamura, K., Sakiyama, T., Nakanishi, K.,
Brazil J. Microbiol. 40, 782–789. 1998. Development of a cylindrical apparatus for membrane-surface
Shu, P., John, M.J., 1947. Effect of the composition of the sporulation liquid culture and production of kojic acid using Aspergillus oryzae
medium on citric acid production by Aspergillus niger in submerged NRRL484. J. Ferment. Bioeng. 85 (5), 488–494.
culture. J. Bact. 54, 161. Xu, H.-W., Xu, C., Fan, Z.-Q., Zhao, L.-J., Liu, H.-M., 2013. A facile syn-
Shu, P., Johnson, M.J., 1948a. The interdependence of medium constituents thesis, antibacterial activity of pulvinone and its derivatives. Bioorg.
in citric acid production by submerged fermentation. J. Bact. 56, 577. Med. Chem. Lett. 23, 737–739.
Shu, P., Johnson, M.J., 1948b. Citric acid: production by submerged fer- Yoshida, H., 1883. Chemistry of lacquer (Urushi). Part 1. J. Chem. Soc.
mentation with Aspergillus niger. Industr. Eng. Chem. 40, 1202. 43, 472–486.
Siala, R., Frikha, F., Mhamdi, S., Nasri, M., Kamoun, A.S., 2012. Yu, X., Li, Y., Wang, C., Wu, D., 2004. Immobilization of Aspergillus
Optimization of acid protease production by Aspergillus niger I1 niger tannase by microencapsulation and its kinetic characteristics.
on shrimp peptone using statistical experimental design. Biotechnol. Appl. Biochem. 40, 151–155.
ScientificWorldJournal 2012, 1–11. Zhang, A., Roehr, M., 2002. Citric acid fermentation and heavy metal
Soares, I., Távora, Z., Barcelos, R.P., Baroni, S., 2012. Microorganism- ions – II. The action of elevated manganese ion concentrations. Acta
produced enzymes in the food industry. In: Valdez, B. (Ed.), Scientific, Biotechnol. 22 (3-4), 375–382.
Health and Social Aspects of the Food Industry InTech Zhuravleva, O.I., Afiyatullov, S.S., Denisenko, V.A., Ermakova, S.P.,
Sohail, M., Siddiqi, R., Ahmad, A., Khan, S.A., 2009. Cellulase produc- Slinkina, N.N., Dmitrenok, P.S., et  al., 2012. Secondary metabo-
tion from Aspergillus niger MS82: effect of temperature and pH. lites from a marine-derived fungus Aspergillus carneus Blochwitz.
N. Biotechnol. 25 (6), 437–441. Phytochemistry. 80, 123–131.
Souza, P.M., Aliakbarian, B., Filho, E.X.F., Magalhães, P.O., Junior, A.P.,
Converti, A., et  al., 2015. Kinetic and thermodynamic studies of a