Patulin

Patulin is a secondary metabolite of certain Penicillium, Aspergillus, and

Byssochlamys species but, more specifically, P. expansum appears to be the fungus
responsible for patulin production.

From: Encyclopedia of Food Sciences and Nutrition (Second Edition), 2003

Related terms:

Aflatoxin, Ochratoxin, Mycotoxins, Plant, Enzyme, Protein, Fungus, Cellulose, As-

pergillus, Apple

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Learn more about Patulin

MYCOTOXINS | Classifications
L.B. Bullerman, in Encyclopedia of Food Sciences and Nutrition (Second Edition),
2003

Patulin
Patulin is toxic to many biological systems, including bacteria, mammalian cell
cultures, higher plants, and animals, but its role in causing animal and human
disease is unclear. Patulin has a lactone structure and is carcinogenic when injected
intradermally into mice (Figure 4). Patulin is produced by numerous Penicillium and
Aspergillus species and Byssochlamys nivea. Penicillium expansum, which commonly
occurs in rotting apples, produces patulin. Patulin is of some public health concern
because of its potential carcinogenic properties, and because it has been found in
commercial apple juice and other apple products. Patulin appears to be unstable in
grains, cured meats, and cheese. When administered orally to rats, patulin showed
no toxicity or carcinogenicity.
Figure 4. Chemical structure of patulin.

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AFLATOXINS AND MYCOTOXINS |

Thin-Layer (Planar) Chromatography
M.E. Stack, in Encyclopedia of Separation Science, 2000

Patulin
Patulin (Figure 3) is a lactone metabolite of several moulds, including Penicillium
expansum, which causes brown rot in apples. Patulin is often found in apple juice,
especially juice from fallen apples. Patulin can be extracted from apple juice with eth-
yl acetate and cleaned up using silica gel column chromatography. After evaporation,
the extract is dissolved in chloroform and spotted on silica gel plates and developed
with toluene–ethyl acetate–formic acid (5:4:1 v/v). After drying, the plate is sprayed
with 3-methyl-2-benzothiazolinone hydrazone–HCl (MBTH) solution and heated
for 15 min in an oven at 130°. Under ultraviolet light at 365 nm, patulin (RF=0.5)
appears as a yellow-brown fluorescent spot. The amount of patulin in the sample
can be determined by comparing the intensity of fluorescence of the standard and
sample spots. Other TLC developers, such as hexane–anhydrous ether (1:3 v/v),
chloroform–methanol (95:5 v/v), and chloroform–acetone (9:1 v/v) can be used to
confirm the identity of the patulin. After development, plates are sprayed with MBTH
to reveal the patulin.

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Advances in the Analysis of Challenging

Food Contaminants
Lubinda Mbundi, ... Rosa Busquets, in Advances in Molecular Toxicology, 2014

4.3 Patulin
Patulin (structure in Fig. 2.4) is produced by a variety of molds, especially Aspergillus
and Penicillium, which are commonly detected in a variety of foodstuffs such as
cereals, vegetables, and fruits including apples and pears. Patulin is generally not
considered to be a potent toxin. However, the recent demonstration of its geno-
toxicity, which has yet to be confirmed, has led to fears of it being carcinogenic
[149]. Interestingly, patulin has shown antimicrobial properties against a number of
microbes [149]. The WHO-recommended maximum levels of patulin are 50 ng/mL
in apple juice and cider, 25 ng/g in solid apple products, and 10 ng/g in products
for infants and young children in the European Union [150]. In addition, the JECFA
has established a provisional maximum allowable daily intake of 0.4 μg/g of body
weight/day for patulin [151].

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MYCOTOXINS | Occurrence and Deter-

mination
A. Chrevatidis, in Encyclopedia of Food Sciences and Nutrition (Second Edition),
2003

Patulin
Patulin is a secondary metabolite of certain Penicillium, Aspergillus, and
Byssochlamys species but, more specifically, P. expansum appears to be the fungus
responsible for patulin production. It is a common contaminant of apples and apple
products but commodities such as grains and other fruits and vegetables are also
susceptible although to a lower extent. Even though the contamination incidence is
high, it is fortunate that the levels are usually low. Refrigeration temperatures are
shown to be ideal for the production of patulin by several fungi, thus long storage
periods must be avoided. The presence of patulin in apple juice is evidence that
rotten fruit were being used; however, it is fairly easy to avoid contamination as most
of the toxin is located in the rotten part of the fruit and once it has been removed,
the problem is minimized. Juice fermentation also results in a 99% destruction of
the toxin. Patulin has been a reason for concern due to its carcinogenic properties;
however, the IARC was unable to establish the severity of its effects on humans and
as a consequence no classification exists at present. Regulation levels set in several
countries vary from 30 to 50 μg l−1. The EC proposed limits of 50 p.p.b. in apple juice
and 25 p.p.b. in solid apple products.

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Systemic and Multi-Organ Diseases
In Veterinary Medicine (Eleventh Edition), 2017

Patulin
Patulin, an important toxin in human medicine, is produced by Aspergillus clavatus
and other fungi, including Byssochlamys nivea, Penicillium urticae, P. claviforme, and P.
patulum.3,4 Toxicity is most commonly associated with rotting apples or apple juice,
and poisoning may occur in pigs fed food waste containing rotten fruit.3

Cattle and sheep poisoned by patulin producing fungi develop brain hemorrhage,
pulmonary edema, or liver and kidney damage with abomasal hemorrhage. When
fed to piglets, it is associated with vomiting, salivation, anorexia, polypnea, weight
loss, leukocytosis, and anemia. Patulin may be the toxin associated with neurologic
problems in cattle ingesting malting by-products and sprouted grains.4,6 Affected
cattle develop neuromuscular signs, including salivation, ataxia, hind limb weakness,
muscle tremors, recumbency, and death.4-6

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Fruit development and ripening

Avtar K. Handa, ... Autar K. Mattoo, in Plant Biotechnology and Agriculture, 2012

Expansins
Expansins induce extension in isolated plant cell walls and are therefore implicated
in fruit textural changes (Rose et al., 1997). Expansins belong to a multi-gene family
with different members exhibiting differential expression during fruit development
and ripening (Choi et al., 2006). Antisense RNA inhibition of SlExp1, a ripen-
ing-specific expansin, resulted in firmer fruits that exhibited reduced polyuronide
depolymerization but were not impaired in the breakdown of structurally important
hemicelluloses, a major component of fruit softening. The transgenic fruit with
constitutive expression of SlExp1 had an opposite phenotype, showing enhanced
fruit softening correlated with the precocious and extensive depolymerization of
structural hemicelluloses (Brummell et al., 1999). However, polyuronide depolymer-
ization was not affected. It was proposed (Brummell et al., 1999) that Exp1 regulates
polyuronide depolymerization late in ripening by direct modulation of relaxation
of the cell walls, likely by controlling access of a pectinase to its substrate. The
depolymerization of hemicellulose occurs independent of or requires only very small
amounts of Exp1 protein. Simultaneous downregulation of Exp1 and PG resulted in
fruit that retained firmer texture and maintained cellular integrity for a longer period
compared to parental wild-type fruit (Powell et al., 2003).

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Chemical Ecology
Shohei Sakuda, Makoto Kimura, in Comprehensive Natural Products II, 2010

4.10.3.2 Miscellaneous Mycotoxins

Patulin (72 in Figure 15) is produced by a number of species of Penicillium and As-
pergillus. Among the patulin-producing Penicillium species, Penicillium expansum
is most important because it associates with spoilage and mycotoxin production in
apples and apple juices. Contamination of patulin in apple juices is now a significant
problem.147 Patulin shows cytotoxic activity, which is suggested to be caused by
forming covalent adducts between patulin and cellular thiols.148 The complicated
biosynthetic pathway of patulin has been confirmed as shown in Figure 15,149 but
there is still little information on the genes encoding the patulin biosynthetic en-
zymes. Only two genes encoding 6-methylsalicylic acid synthase and isoepoxydone
dehydrogenase have been identified in Penicillium urticae and P. expansum.150 The
former enzyme is a Type I PKS that produces 6-methylsalicylic acid (73) from four
acetate molecules. The latter one catalyzes the reaction from isoepoxydone (74) to
phyllostine (75). Two putative cytochrome P-450 monooxygenase genes, which may
be connected with the patulin biosynthesis, were also obtained from P. expansum.150
Figure 15. Biosynthesis of patulin.

Gliotoxin (76 in Figure 16) is a highly immunosuppressive compound produced by

a variety of fungi.151 Aspergillus fumigatus, a producer of gliotoxin, is a pathogenic
fungus causing the respiratory disease known as aspergillosis. Since gliotoxin pro-
duction is detected in infected animal tissues, it is thought that there is a relationship
between gliotoxin production by the fungus and pathogenesis of aspergillosis. The
gliotoxin biosynthetic gene cluster was found in the genome of A. fumigatus.152
Among the biosynthetic genes, the gene encoding a nonribosomal peptide synthase
was shown to be necessary for gliotoxin biosynthesis by a gene disruption experi-
ment.153,154 A nuclear protein, LaeA, is known as a regulator for the production of
various secondary metabolites including gliotoxin in A. fumigatus. It is thought that
LaeA is a key factor for the virulence of the fungus.155
Figure 16. Structures of gliotoxin, cyclopiazonic acid, citrinin, citreoviridin, lu-
teoskyrin, and cyclochlorotine.

Cyclopiazonic acid (77 in Figure 16) is produced by several species of Aspergillus and
Penicillium, and its contamination has been found in a variety of agricultural prod-
ucts. Since Aspergillus flavus often produces AF and cyclopiazonic acid concurrently,
it is speculated that mycotoxicosis caused by cyclopiazonic acid may be disguised in
the presence of aflatoxicosis.156 Cyclopiazonic acid is known as a potent inhibitor of
sarcoplasmic and endoplasmic reticulum Ca2+-activated ATPase. The indole-tetramic
acid skeleton of cyclopiazonic acid is biosynthesized from tryptophan, mevalonate,
and two molecules of acetate, but its biosynthetic genes are not obtained yet. VeA,
a global regulatory protein controlling AF production and sclerotial formation in A.
flavus, was also shown to regulate cyclopiazonic acid production by the fungus.157

Citrinin contamination in food is caused mainly by the infection of Penicillium

citrinum and P. verrucosum although a variety of fungi can produce citrinin.158,159
Citrinin (78 in Figure 16) is known as a nephrotoxin.160,161 Since P. verrucosum often
produces ochratoxin A, which also has a nephrotoxic effect, concurrently, citrinin
may interact synergistically with ochratoxin A.162 Monascus purpureus, a producer
of a useful red pigment, produces citrinin as an undesired side product. The gene
encoding PKS for citirinin biosynthesis in M. purpureus has been clarified.163

Citreoviridin (79), luteoskyrin (80), and cyclochlorotine (81) are historical mycotoxins
studied in Japan. Citreoviridin was isolated as a toxin from Penicillium citreoviride
associated with a disease called cardiac beriberi or ‘shoshin kakke’.164 Luteoskyrin
and cyclochlorotine were isolated from Penicillium islandicum, which was infected
into toxic yellowed rice.165 Genes responsible for biosynthesis of these compounds
are not obtained.

Penicillic acid (82 in Figure 17) is a toxic compound produced by many Penicillum
and some Aspergillus species.166 It is biosynthesized from orsellinic acid through the
pathway shown in Figure 17.167 A candidate of a gene encoding a PKS for penicillic
acid biosynthesis was obtained from A. ochraceus.168

Figure 17. Biosynthesis of penicillic acid.

Phomopsin A (83 in Figure 18) was isolated from Phomopsis leptostromiformis as

the hepatotoxic metabolite responsible for lupinosis, which is a disease in animals
caused by ingestion of Lupinus species infected with the fungus.169 Penicillium
species produce other significant mycotoxins such as penitrem A (84), PR toxin
(85), and rubratoxin B (86).170 Fusarin C (87) and fusaproliferin (88) are known
as mycotoxins produced by Fusarium species. A gene encoding a PKS fused with
an unusual nonribosomal peptide synthase module responsible for biosynthesis
of fusarin C was obtained.171 Fusaproliferin was discovered as a toxic metabolite
of Penicillium proliferatum in 1995,172 but it has become an important mycotoxin
because its contamination in corn samples has been detected in many countries.173
Figure 18. Structures of phomopsin A, penitrem A, PR toxin, rubratoxin B, fusarin
C, and fusaproliferin.

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New Aspects of Gravity Responses in

Plant Cells
Takayuki Hoson, Kouichi Soga, in International Review of Cytology, 2003

C Gravity-Induced Gene Expression

The expression of genes encoding expansins and induced by auxin has been exam-
ined in cucumber seedlings grown in space. Of seven -expansin genes, CsExp3 and
CsExp4 were more highly expressed in the peg and the root. However, there were no
detectable differences in expansin gene expression levels for the pegs of seedlings
grown in space or on the ground (Link et al., 2001). On the other hand, the expression
of an auxin-inducible gene, CS-IAA1, was greater on the lower side of the transition
zone in the horizontally placed seedlings on the ground just prior to and during the
initiation period of peg formation. In space, the accumulation of CS-IAA1 mRNA
occurred all around the transition zone (Kamada et al., 2000). Space-grown seedlings
of cucumber might develop two pegs symmetrically because the auxin level in the
entire transition zone is maintained above the threshold.

The transgenic Arabidopsis containing the alcohol dehydrogenase (Adh) gene pro-
moter linked to the -glucuronidase (GUS) reporter gene was developed to address
specifically whether hypoxia-induced responses occur in spaceflight and to assess
whether any spaceflight response was similar to hypoxia-induced gene expression
patterns in terrestrial controls (Paul et al., 2001). The Adh GUS reporter gene was
activated in roots during the flight. However, the patterns of expression were not
identical to terrestrial control inductions. Moreover, although terrestrial hypoxia
induces Adh GUS expression in the shoot apex, no apex staining was observed in
the spaceflight plants. This result indicates that either the normal hypoxia response
signaling is impaired in spaceflight or that spaceflight inappropriately induces Ad-
h GUS activity for reasons other than hypoxia.

The series of events leading to plant responses to the gravity signal have been
comprehensively analyzed by genomics approaches, such as the differential display
method, first developed by Liang and Pardee (1992), and the cDNA microarray
analysis. The changes in gene expression by hypergravity treatment have been
analyzed in Arabidopsis hypocotyls by the differential display method (Yoshioka et
al., 2003). Sixty-two cDNA clones were expressed differentially between control and
300 × g conditions: the expression levels of 39 clones increased, whereas those of
23 clones decreased under hypergravity conditions. The expression of these genes
was further analyzed by reverse transcription polymerase chain reaction (RT-PCR).
Finally, six genes were confirmed to be up-regulated by hypergravity. One gene en-
coded 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), which catalyzes a
reaction producing mevalonic acid, a key precursor of terpenoids such as membrane
sterols and several types of hormones. The expression of HMGR gene increased
within several hours after hypergravity treatment. Also, compactin, an inhibitor
of HMGR, prevented hypergravity-induced growth suppression, suggesting that
HMGR acts to suppress Arabidopsis hypocotyl growth under hypergravity conditions.
In addition, hypergravity increased the expression levels of genes encoding CCR1
(cold-regulated circadian rhythm 1), ERD15 (early response to dehydration 15), and
-tubulin. These genes may be involved in a series of cellular events leading to
growth suppression of stem organs under hypergravity conditions.

Modifications of cell wall metabolism are the typical cellular effects of hypergravity
stimuli. Nevertheless, no clear changes in the expression levels of genes involved
in cell wall metabolism were detected by the differential display method. One
explanation would be that most of the genes encoding the cell wall proteins are
constitutive and not inducible, limiting the variation in their expression levels to an
extent undetectable by the differential display method. Even in auxin-induced cell
wall loosening, the typical example of the cell wall modifications by hormones, the
elevation of the expression of wall protein genes was not so prominent (Caderas et
al., 2000; Kotake et al., 2000). In addition, the modification of gene expression is not
the only mechanism regulating cell wall properties. The cell wall contains various
types of molecules that regulate the activity of cell wall enzymes (Hoson, 1993). Also,
the wall metabolism is strongly influenced by the cell wall environment such as pH
(Hoson, 1993, 1998). Hypergravity has been shown to increase the apoplastic pH by
suppressing the activity of plasma membrane H+-ATPases, which acts to increase the
molecular size of matrix polysaccharides, leading to the increase in cell wall rigidity
(Soga et al., 2000a,b). Thus, cell wall properties may be regulated by the combination
of the rate of supply of various cell wall constituents and the cell wall environment
in gravity responses of plant seedlings.

Microarray analyses have been applied to Arabidopsis seedlings grown under hy-
pergravity and microgravity conditions. Transcripts of more than 200 genes were
significantly increased by hypergravity at 7 × g (Martzivanou et al., 2002). They
fall into several categories. Transcripts coding for enzymes of major pathways form
the largest group (25%), followed by gene products involved in cellular organiza-
tion and cell wall formation rearrangement (17%), signaling, phosphorylation de-
phosphorylation (12%), proteolysis and transport (10% each), hormone synthesis
plus related events (8%), defense (4%), stress response (2%), and gravisensing (2%).
Many of the alterations are part of a general stress response, but some changes
related to the synthesis rearrangement of cell wall components could be more
hypergravity specific. On the other hand, in space-grown Arabidopsis seedlings, 10
genes had the highest statistical change in expression level as well as the changes
for known gravitational mutants (Link et al., 2002). These genes are under analysis.
We have just started genomics approaches for analyzing the regulation by gravity of
gene expressions in plant seedlings, and they will provide important information on
plant responses to the gravity signal in the next decade.

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Trichoderma Secretome
Sunil S. Adav, Siu Kwan Sze, in Biotechnology and Biology of Trichoderma, 2014

New Candidates in Cellulose Degradation

Plant cell wall proteins named “expansins” disrupt hydrogen bonding between cell
wall polysaccharides without hydrolyzing them (Cosgrove, 2000). Similarly, in T.
reesei secretome, a protein with endoglucanase activity having sequence similarity
to expansin called “swollenin” has been iTRAQ quantified. This protein has N-ter-
minal fungal type cellulose-binding domain connected by a linker region to the
expansin-like domain. It has been well documented that the regulation of swollenin
gene resembles with regulation of T. reesei cellulase genes (Saloheimo et al., 2002).
The biological role of swollenin has been studied by disrupting the swo1 gene
from T. reesei (Saloheimo et al., 2002). Further, the swollenin gene was expressed
in yeast and Aspergillus niger var. awamori and also showed that the activities
of SWOI-containing yeast supernatant disrupts the structure of the cotton fibers
without detectable formation of reducing sugars (Saloheimo et al., 2002). It has been
presumed that these fungal strains encode three to four swollenin-like proteins
that vary in their mode of action, but contribute in polysaccharide hydrolysis. On
the contrary, plant parasitic roundworm, Globodera rostochiensis, can produce a
functional expansin (Gr-EXPB1) used to loosen cell walls when invading its host
plant (Qin et al., 2004). The upregulation of swollenin in the secretome of T. reesei
QM6a and Rut C30 during lignocellulose degradation indicated its possible role
in deconstructing lignocellulose structure (Adav et al., 2012a). It has also been
speculated that cell wall disruption by Trichoderma are more efficient due to
swollenin, which could facilitate the access to other cellulolytic enzymes at less
accessible areas of the substrate. It is quite fascinating that swollenin homologs are
found only in Trichoderma species, A. fumigatus, or its close relative Nassarius fischeri,
and not in other fungal phytopathogens.

Trichoderma reesei also secrete hydrophobins that are surface active proteins and
perform a wide variety of functions. Hydrophobins self-assembles in rodlet-like
structures on the outer surfaces of fungal cell walls, and mediate interactions
between the fungi and their environment, enabling aerial growth and conidiation,
recognition of the host surface, and also in symbiosis. Hydrophobin 1 and hy-
drophobin 2 were upregulated when T. reesei QM6a and Rut C30 cultured with
biomass. According to Kubicek et al. (Kubicek et al., 2008), T. virens and T. atroviride
had a much higher number of class II hydrophobin genes compared to other
ascomycetes. While, a novel set of hydrophobins from Trichoderma spp. that differs
in cysteine spacing and protein surface pattern from earlier reported protein have
also been reported (Seidl-Seiboth et al., 2011). Based on their characteristic surface
activity and capability to form amphiphilic protein films these proteins are also
treated as microbial surfactants. Due to high surface activity, hydrophobins reduces
the surface tension of the medium or the substratum in/on which fungi grow. This
further allows fungi to breach the air–water interface or preventing water logging
while maintaining permeability to gaseous exchange. Hydrophobins also play a
major role in masking the immunogenicity of airborne fungal spores (Bayry et al.,
 2012). By covering the spore surface, hydrophobins impart immunological inertness
to the spores and prevent activation of host immune system (Aimanianda et al.,
2009).

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Immunology
Helene Pendl •, Ian Tizard, in Current Therapy in Avian Medicine and Surgery, 2016

Aspergillus-related toxins.
Ochratoxin A (OTA), cyclopiazonic acid (CPA), gliotoxin, and patulin are mycotoxins
produced by several Aspergillus species. Lymphoid depletion of the thymus, bursa,
and spleen, resulting in leukopenia and lymphopenia, have been reported in poul-
try for both OTA151 and CPA.152 OTA is hepatotoxic and nephrotoxic and inhibits
phenylalanyl-transfer RNA synthetase activity by binding competitively at the site for
phenylalanine.149 Feed supplementation with l-phenylalanine in OTA-exposed laying
hens151 and broiler chicks153,154 revealed a protective effect against the toxic effects
of OTA. Similar positive effects may be seen with aqueous extracts of artichokes
and sesame seed as food additives in laying hens.151 Patulin impairs the function
of alveolar macrophages in rats in vitro155,156 and, like gliotoxin, may predispose to
allergies in humans. Human T-cell exposure to citrinin, gliotoxin, and patulin in vitro
resulted in a selective inhibition of IFN- –producing Th1 cells.157,158 This supports
the hypothesis that mycotoxins are responsible for the Th1 lymphopenia observed
in children exposed to molds. These children have a higher risk for the development
of allergic diseases,159 possibly caused by a shift of T-cell polarization toward the Th2
type under the influence of the mycotoxins.157 Similar pathophysiologic mechanisms
caused by secondary toxins may also apply to aspergillosis in noncommercial avian
species. Infections with Aspergillus sp. are frequently associated with signs of liver
and kidney disease and lymphopenia. To date, however, corresponding data for birds
other than poultry are not available.

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