patulin is a secondary metabolite and know about its properties, physiological functions, importance of patulin and its biosynthesis . Types of patulin toxicity and its symptoms, detection and control measures of patulin.

1. Mrs.K.Sudha Rameshwari M.Sc., M.Phil.,PGDBI.,
Assistant professor,
Department of PG biochemistry,
V.V.Vanniaperumal college for women,
Virudhunagar
PATULIN- Secondary
Metabolite

2. Introduction
 Secondary metabolites are organic
compounds produced by bacteria, fungi, or
plants which are not directly involved in the
normal growth, development or reproduction
of the organism.
 Unlike primary metabolites, absence of
secondary metabolites does not result in
immediate death, but rather in long-term
impairment of the organism's survivability,
fecundity or aesthetics, or perhaps in no

3. Importance of secondary
metabolites
 Secondary metabolites often play an important role in plant
defense against herbivory and other interspecies defenses.
 Humans use secondary metabolites as medicines, flavorings,
pigments, and recreational drugs.
 Secondary metabolites aid a host in important functions such
as protection, competition and species interactions, but are
not necessary for survival.
 One important defining quality of secondary metabolites is
their specificity.
 Secondary metabolites are specific to an individual species.
 Secondary metabolites also have a strong impact on the food
humans eat.
For example, legumes use flavonoids to signal a symbiotic
relationship with nitrogen fixing bacteria (rhizobium) to
increase their nitrogen uptake.

4. PATULIN
 It is an example for secondary metabolite and mycotoxin.
 Patulin is produced by a large number of Pencillia including
P.claviforme, P.expansum , P.Patulum , by some aspergilli
(A.clavatus, A.terreus and others), and Byssochlamys nivea
and B.fulva.
 The main producer of patulin is Pencillium expansum which
contaminates mainly apple and apple products, and also
other fruits like cherry, blueberry, plums, strawberries
bananas,and grapes.
structure of Patulin

5. Properties
 Its biological properties are similar to those of
pencillic acid.
 Some patulin producing fungi can produce the
compound below 20C.
 This mycotoxin has been found in moldly bread,
sausage, fruits (including bananas, pears, pine-
apples, and grapes), apple juice and other products.
 In apple juice, levels as high as 440ug/liter a have
been found and in cider levels up to 45ppm.
Contd…..

6.  Minimum absorbance for growth of P.expansum and P.patulum
has been reported to be 0.83 and 0.81 respectiviely.
 In PDB incubated at 120c, patulin was produced after 10days
by P.patulum and P.roueforti.
 Patulin was produced in apple juice also at 120C by B.nivea but
the highest concentration was attained after 20days at 210C.
 After a 9days the highest amount was produced at 300C, with
much less at 370C.
 These investigators confirmed that patulin production is favored
at temperatures below the growth optimum.
 The latter investigators used P.expansum and found production
over the range 5◦C-20◦C, with only small amounts produced at
30◦C.
 Atmospheres of Co2 and N2 reduced production compared to
that in air.
 To inhibit production, SO2 was found more effective than

7. Physiological actions
 The LD50 for patulin in rats by the subcutaneous
route has been reported to be 15-25mg / Kg and it
induces subcutaneous sarcomas in some animals.
 When administered orally to rats, patulin showed no
toxicity or carcinogenicity.
 Both patulin and penicillic acid bind to –SH and –
NH2 groups, forming covalently linked adducts that
appear to abate their toxicities.
 Patulin causes chromosomal aberrvations in animal
and plant cells and is a carcinogen.

8. Biosynthesis
Its structure was elucidated by wood
word and sigh.
Bulock and Ryan showed that the 6-
methyl salicylic acid is converted to
patulin.
 The incorporation of radioactive 6-
methyl salicylicacid and other related
compounds indicate that patulin arises
by cleavage of an aromatic ring.
contd…..

9. Schematic representation of Patulin biosynthesis

10. Biosynthesis
 Patulin is a polyketide metabolite.
 The first step in the production of patulin is the formation
of 6MSA by the condensation of one acetyl-CoA and three
malonyl-CoA units.
 This formation is carried out by a single multifunctional
enzyme that has several enzymatic activities: acetyl and
malonyl transferase, ketoacyl synthase, ketoreductase and
dehydratase.
 The products generated from m-cresol and
gentisylaldehyde are structurally similar to 6-methylsalicylic
acid. 6MSA is modified to m-cresol by 6MSA
decarboxylase, then the methyl group of m-cresol is
oxidized to form an aldehyde group.
 This step is followed by a hydroxylation reaction that leads
to gentisaldehyde formation.

11.  The conversion of gentisaldehyde to a two
ring structure such as patulin needs the
opening of a ring by a mechanism mediated
either by a monooxygenase or by a
dioxygenase.: acetate, 6MSA, m-cresol, m-
hydroxybenzyl alcohol, m-
hydroxybenzaldehyde and gentisaldehyde,
a crude extract which catalyzed the
epoxidation of gentisyl alcohol to phyllostine
.
 Five of the enzymes involved in patulin
biosynthesis are 6-methylsalicylic acid
synthase, 6-methylsalicylic acid

12. Toxicity of Patulin
General toxicity
 Patulin has a strong affinity for sulfhydryl groups.
 Patulin has a lactone structure and is carcinogenic when injected
intradermally into mice
 Patulin adducts formed with cysteine are less toxic than the
unmodified compound in acute toxicity, teratogenicity, and
mutagenicity studies.
Acute toxicity
 In rodents, the oral LD50 of patulin ranges between 29 and 55
mg/kg body weight (b.w.) and Poultry with an oral LD50 of 170
mg/kg b.w.
 When administered by the intravenous, intraperitoneal or
subcutaneous routes, patulin is 3-6-times more toxic.
 Toxic signs consistently reported in all studies were agitation, in
some cases convulsions, dyspnea, pulmonary congestion, edema,
and ulceration, hyperemia and distension of the gastro intestinal
tract.
 Some compounds were able to modulate the toxicity of patulin.
 When a patulin/cysteine adduct was administered to mice
intraperitoneally, no acute toxicity was observed at levels up to 150

13. Sub-acute Toxicity
 The sub-acute administration of patulin has been mainly studied in
rats, where it was shown to induce weight loss, gastric and intestinal
changes and alterations in renal function.
 Repetitive doses lead to signs of neurotoxicity (tremors,
convulsions) as well as an inhibition of several enzymes (ATPase) in
the intestine and the brain, in particular, with consequences in terms
of lipid metabolism. Similar clinical signs were observed in mice,
hamsters and chickens.
 In monkeys, no sign of toxicity was observed after daily treatments
with 5 to 500 µg/kg b.w. for four weeks.
 Selmanoglu and Kockaya measured thyroid and testicular hormones
in rats receiving 0.1 mg patulin/kg b.w./day patulin by the oral route
for 60 or 90 days.
 A 60-day exposure increased the plasma level of testosterone and
decreased T4 hormone while there was no change in T3, TSH, LH
and GH. When the exposure lasted for 90 days, there was an
increase in testosterone and in LH without any other clinical signs.
 Histological examination of the thyroid showed lymphoid cell
infiltration and enlargement of interstitial tissue.
 At the tested level, edema, fibrosis, local Leydig cell hyperplasia and
disorganization of the seminiferous tubule epithelium were observed.

14. Immunotoxicity
 Patulin can alter the immune response of the host.
 Patulin inhibits several macrophage functions.
 In vitro exposure of alveolar rat macrophages to
patulin inhibited protein synthesis and altered
membrane functions.
 Patulin also significantly decreased the production of
O2−, phagosome-lysosome fusion, phagocythosis,
as well as lysosomal enzyme and microbiological
activity in mouse macrophages. In vivo studies using
mice indicate variable effects of patulin on the
immune system.
 These effects include an increased number of
splenic T lymphocytes and depressed serum
immunoglobulin concentrations, depressed delayed
hypersensitivity responses and increased neutrophil

15. Genotoxicity and embrotoxicity
 Genotoxicity of patulin by structural chromosomal
aberrations, genes mutations and chromosome
gaps and breaks.
 Patulin is known as embryos defective agent by
the proteins and DNA content reduction.
Anomalies include growth retardation, hypoplasia
and hyperplasia of embryos

16. Symptoms of toxicity
acute toxicity:
• Convulsions
• dyspnea
• pulmonary congestion
• edema
• ulceration gastrointestinal tract
subacute toxicity :
• Tremors
• convulsions
• changes in the hormones level
• histological dysfunctions

17. Detection
 Testing for the presence of patulin in food products
is not a simple or quick procedure.
 Current rapid test kits to detect the presence of
patulin are lacking throughout the global market.
 Most patulin testing occurs via the use of HPLC
(high performance liquid chromatography)-UV
and/or liquid chromatography coupled to tandem
mass spectrometry (LC/MS/MS) analyses within a
laboratory.
 Patulin does not employ fluorescent properties
and thus the use of UV detection is required.
 Often times the chromatography for patulin
analysis is complex.
 A compound known as HMF (5-
hydroxymethylfurfural) often times co-elutes or
presents close in retention time to the patulin peak of interest.

18.  Testing methods via HPLC-UV should include a HMF
standard to confirm retention time and proper
separation of this compound peak from the patulin
peak for quantitation purposes to avoid the potential
for false positives or elevated positive results.
 LC/MS/MS methods for detection of mycotoxins
continues to rise, more laboratories are turning to
this technology to accurately detect patulin
contamination at low levels of parts per billion (ppb).
 Many laboratory methods can detect patulin
contamination with limits of detection at 2 ppb.

19. Control measures
 Patulin is not particularly stable in an aqueous
environment except at reduced pH when it will even
survive elevated temperatures (Lovett and Peeler,
1973), hence its occurrence in pasteurised apple
juice.
 The removal of mouldy apples, or even the overtly
mouldy part of individual apples, is an effective
control measure
 Patulin is destroyed by the active fermentation of
apple juice to cider by Saccharomyces
cerevisiae (Moss and Long, 2002).
 It can also be removed with activated charcoal and
by treatment with sulphur dioxide.

20. Conclusion
Patulin was originally used as
an antibiotic against Gram-positive
and Gram negative bacteria causing the
common cold, but is no longer used for
that purpose due to its acute toxicity,
teratogenicity, embrotoxicity and
mutagenicity.