SIKLUS BIOSINTESIS PATHWAY

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Comprehensive

Review of

Patulin Control

Methods in

Foods

Matthew M. Moake, Olga I. Padilla-Zakour,

and Randy W. Worobo

ABSTRACT: The mycotoxin, patulin (4-hydroxy-4H-furo [,!c" pyran-![#H"-one$, i% produced &y a num&er of fun'i com-mon to fruit-

and e'eta&le-&a%ed product%, mo%t nota&ly apple%) *e%pite patulin+% ori'inal di%coery a% an anti&iotic, it ha% come under heay

%crutiny for it% potential ne'atie health effect%) Studie% ine%ti'atin' the%e health effect% hae proed inconclu%ie, &ut there i% little

dou&t a% to the potential dan'er inherent in the contamination of food product% &y patulin) The dan'er po%ed &y patulin nece%%itate% it%

control and remoal from food product%, creatin' a demand for handlin' and proce%%in' techniue% capa&le of doin' %o, prefera&ly at

lo co%t to indu%try) .ith thi% &ein' the ca%e, much re%earch ha% &een deoted to under%tandin' the &a%ic chemical and &iolo'ical

nature of patulin, a% ell a% it% interaction ithin food% and food production) .hile pa%t re%earch ha% elucidated a 'reat deal, patulin

contamination continue% to &e a challen'e for the food indu%try) Here, e reie in depth the pa%t re%earch on patulin ith an empha%i%

upon it% influence ithin the food indu%try, includin' it% re'ulation, health effect%, &io%ynthe%i%, detection, uantification, di%tri&ution

ithin food%, and control, durin' the ariou% %ta'e% of apple /uice production) 0inally, 1ey area% here future patulin re%earch %hould

focu% to &e%t control the patulin contamination pro&lem ithin the food indu%try are addre%%ed)

Introduction

Patulin is a mycotoxin produced by a number of fungi common to

fruit- and vegetable-based products, most notably apples. Ap-ples

are the 3rd most important fruit crop in the United States after citrus

fruits and grapes, with !" of apples being used for #uice and other

processed products $US%A &!!&'. Patulin contamina-tion within

apple products poses a serious health ris( to consum-ers,

particularly children whom have been shown by a US%A sur-vey to

consume increased levels of apple products during the )st y of life

$*. g+(g body weight+d', compared with adults $) g+(g bw+d'

$Plun(ett and others )&', placing them at increased ris( for

patulin toxicity. he health ris(s posed by patulin necessitate its

control and removal from apple products, creating a demand for

food-processing techniues capable of doing so, preferably at low

cost to industry. /ere, we review past research devoted to the


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understanding and control of patulin, with an emphasis upon pat-

ulin0s influence within the food industry.

History and Regulation

Patulin $-hydroxy-/-furo 13,&c2 pyran-&1*/2-one', igure ), is

a water-soluble lactone )st isolated as an antibiotic during the

MS 20040335 Submitted 5/21/04, Revised 8/17/04, Accepted 10/18/04.

Authors are with Dept. of Food Science and Technology, New York State

Agricultural Experiment Station, Cornell Univ., Geneva, NY 14456-0462.

Direct inquiries to author Worobo (E-mail:[email protected]).

)!s $Stott and 4ullerman )56'. 7wing to co-discovery of the

compound by various groups, it has historically been (nown by

names such as clavacin $Anslow and others )3', expansine

$8an 9ui#( )3:', claviformin $;hain and others )&', clavatin

$4ergel and others )3', gigantic acid $Philpot )3', and

myo-cin ; $%e


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Patulin control in foods . . .

Table 1The health effects of patulin

Acute symptom

Source

Agitation, convulsions, dysponea, pulmonary congestion,

Escoula and others 1977; Hayes and others 1979

edema, hyperemia, GI tract distension

Nausea

Walker and Wiesner 1944

Epithelial cell degeneration, intestinal hemorrhage

Mahfoud and others 2002

Intestinal inflammation

McKinley and Carlton 1980a, 1980b; McKinley and others

1982; Mahfoud and others 2002

Ulceration

Escoula and others 1977; Hayes and others 1979; McKinley and Carlton 1980a,

1980b; McKinley and others 1982; Mahfoud and others 2002

Chronic symptom

Source

Genotoxic

Mayer and Legaror 1969; Korte 1980; Thust and others 1982; Lee and Roschenthaler

1986; Roll and others 1990; Hopkins 1993; Pfeiffer and others 1998


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Neurotoxic

Hopkins 1993

Immunotoxic

Hopkins 1993; Wichmann and others 2002

Immunosuppressive

Wichmann and others 2002

Teratogeneic

Ciegler and others 1976; Roll and others 1990

Carcinogenic

Dickens and Jones 1961; Oswald and others 1978

Cellular level effect

Source

Plasma membrane disruption

Riley and Showker 1991; Mahfoud and others 2002

Protein synthesis inhibition

Hatez and Gaye 1978; Miura and others 1993; Arafat and Musa 1995

Transcription disruption, translation disruption

Moule and Hatey 1977; Arafat and others 1985; Lee and Roschenthaler 1987

DNA synthesis inhibition

Cooray and others 1982

Na-coupled amino acid transport inhibition

Ueno and others 1976

Interferon- production inhibition

Wichmann and others 2002

RNA polymerase inhibition

Moule and Hatey 1977

Aminoacyl-tRNA synthetases inhibition

Arafat and others 1985

Na-K ATPase inhibition

Phillips and Hayes 1977, 1978; Riley and Showker 1991


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Muscle aldolase inhibition

Ashoor and Chu 1973

Urease inhibition

Reiss 1977

Loss of free glutathione

Burghardt and others 1992; Barhoumi and Burghardt 1996

Protein crosslink formation

Fliege and Metzler 1999

Protein prenylation inhibition

Miura and others 1993

Fhile 6! g+9 is now the norm for patulin regulation, several countries

have set even lower limits for patulin at &6 to 36 g+9 $van Dgmond

):'. =n con#unction with these maximum content standards, a #oint

ood and Agriculture 7rgani?ation-Forld /ealth 7rgani?ation

$F/7' expert committee established a provi-sional maximum daily

inta(e of !. g+(g body weight for patulin $F/7 )6'. he United

States has been much slower to set reg-ulation on patulin, but today

the U.S. ood and %rug Administra-tion limits patulin to 6! g+9 in

single-strength and reconstituted apple #uices $US%A &!!'. he

limitation of these regulations to apple #uice and apple #uice

concentrate was li(ely based upon the fact that, at the time, only

apple #uice and cider had been

Figure 1The structure of patulin !"#hydro$y#"H#furo %&'(c) pyran#

(%*H)#one+

found to be naturally contaminated by patulin $F/7 )!'.

Fhile this fact has since been disproved $see below, Patulin

Fith-in oods', apple #uice and cider remain the ma#or source of

hu-man patulin consumption.

Health ,ffects

Assessment of the health ris(s posed by patulin to humans is based

upon a wide number of studies during the past 6!-plus years that

implicate a number of acute, chronic, and cellular level health effects

as summari?ed in able ). inley and ;arlton ):!a, ):!bB

c>inley and others ):&B ahfoud and others &!!&'. ;hronic

health ris(s of patulin consumption can include neuro-toxic,

immunotoxic, immunosuppressive, genotoxic, teratogenic, and

carcinogenic effects $%ic(ens and Eones )*)B ayer and 9egaror

)*B ;iegler and others )5*B 7swald and others )5:B >orte

):!B hust and others ):&B 9ee and


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)56B 9ynen and others )5:'. he

gene encoding for *-SA has been

cloned and characteri?ed from P.

patulum and P. urticae $4ec( and

others )!B Fang and others ))'.

=nactivation of *-SA synthetase has

been shown to be the )st limitation on

patu-lin production $Ceway and

@aucher ):)'. *-SA synthetase loss

is a selective process because the

highly similar fatty acid synthetase of P.

urticae $9ynen and others )5:' is

stable under the same reaction

conditions that inactivate *-SA

synthetase $9am and others )::'.

his finding is further verified by

studies in which treatment of *-SA

synthetase reaction mixtures, contain-

ing Cicotanimide Adenine %inucleotide

Phosphate $CA%P/' co-factor, acetyl-

;oA, and malonyl ;oA, with the

reducing agent, dithiothreitol, and

proteinase inhibitor,

phenylmethylsulfonyl fluo-ride,

stabili?ed *-SA synthetase. his

suggests proteolysis and

conformational integrity play a role in

the regulation of *-SA synthetase

$9am and others )::'.

he next stage of patulin biosynthesis

involves the conversion of *-SA into m-

cresol via the activity of *-SA

decarboxylase $9am and others )::'.

-cresol is then converted into m-hy-

droxyben?yl alcohol by m-cresol &-

hydoxylase $urphy and 9ynen )56'.

he next step in patulin0s biosynthetic

pathway is debated among & main

mechanisms. 4oth agree that m-hydroxy-

ben?yl alcohol is eventually converted to

gentisaldehyde $orrest-er and @aucher

)5&B Lamir ):!', however the

intermediary be-tween these &

compounds is believed to be either

gentisyl alcohol $Se(iguchi and others

):3B =i#ima and others ):*' or m-hydrox-yben?aldehyde $Se(iguchi and

others ):3'. Some studies have

suggested that both are possible, with m-

hydroxyben?aldehyde being favorable

$@aucher )56', whereas others believe

that m-hydroxyben?aldehyde is not

converted to gentisaldehyde but rather to

m-hydroxyben?oic acid $urphy and

9ynen )56'. =n the &nd case, m-

hydroxyben?yl alcohol dehydrogenase

converts m-hydroxyben?yl alcohol into m-

hydroxyben?aldehyde $@auch-er )56B

urphy and 9ynen )56'. 4oth this

en?yme and m-cresol &-hydroxylase have

been shown to reuire oxygen and

CA%P/ to function $urphy and 9ynen

)56'.

7nce gentisaldehyde has been formed, it

is then converted to isoepoxydon,

phyllostine, neopatulin, D-ascladiol, andfinally to patulin $Se(iguchi and @aucher

)55, )5:B Se(iguchi and @au-cher

)5B Se(iguchi and others )5, ):3'.

he conversion of isoepoxydon to

phyllostine is accomplished via an CA%P-

depen-dent isoepoxydon dehydrogenase

$Se(iguchi and @aucher )5'.

;onversion of neopatulin to D-ascladiol is

accomplished through a reduction by

CA%P/. he product of this reaction, D-

ascladiol, is then either oxidi?ed to patulin

or nonen?ymatically transformed to its

isomer L-ascladiol $Se(iguchi and others

):3'. he biosynthetic pathway ofpatulin is summari?ed in igure &.

.etection and /uantification

he established limit of 6! g+9 in the

United States as the maxi-mum patulin

level allowed in fruit products has

provided an in-centive for the

development of faster and more specificanalytical methods with lower detection

limits. A comprehensive review of the

development of thin-layer gas and liuid

chromatographic methods for the

detection and confirmation of identity of

patulin has been previously published by

Shephard and 9eggott $&!!!'.

he most common method currently used

to uantify patulin in fruit products is high-

performance liuid chromatography

$/P9;' with ultraviolet $U8' detection.

his is the official method adopted by

A7A; =ntl. for apple #uice $method

6.)!' with a detection limit of 6 g+9. he

#uice is extracted 3 times with ethyl

acetate and cleaned up by liuid-liuid

extraction with a ).6" sodium car-bonate

solution. he ethyl acetate extract is dried

with anhydrous sodium sulfateB the

solvent is then evaporated, normally

under ni-trogen, and the dried residue is

dissolved with acidified water $p/


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by addition of

acetic acid'.

his prepared

extract is ready

for /P9;

analysis. he

recommendedliuid

chromatograph

y $9;' systems

include

analytical

reversed-phase

9; columns

such as

octadecylsilane

fully end-

capped with 6

m particle

stationaryphase, )& to &6

nm pore si?e,

carbon loading

of )&" to )5",

and a U8

detector set at

&5* nm,

although a

photo diode

array detector

is preferred to

aid in the

presumptiveidentification of

the patulin

pea(. he

system can be

run isocratically

at ) m9+min us-

ing 3" to )!"

acetonitrile in

acidified water

$!.!6 parts

per vol-ume

perchloric acid

*!"' as long

as patulin

separates from

6-hy-

droxymethylfurf

ural $/', a

common

compound

found in apple

#uice that elutes

#ust before

patulin. or

cloudy apple

#uice and ap-

ple puree, a

collaborative

study of )&

participants

from Duropean

countries was

recently

conducted tovalidate the

effectiveness of

this 9;

procedure for

patulin

determination

with a slight

modifica-tion.

Prior to the

ethyl acetate

extraction, the

samples were

treated withpectinase

en?ymes and

held overnight

at room

temperature or

for & h at ! M;

and then

centrifuged at

6!! N g for 6

min. 4ased on

the results, the

method is

recommendedfor patulin at

greater than 6!

g+9 in cloudy

apple #uice and

purees $ac-

%onald and

others &!!!'.

he

simultaneous

uantification ofpatulin and /

in apple #uice by

reversed-phase

/P9; has been

reported by

@o(men and

Acar $)'.

he method

developed uses

a 6- m ;):

analytical

column $)6! N

mm', a

photodiode


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array detector

operating at &6!

to 3!! nm, and

a mobile phase

of water-

acetonitrile $J)

v+ v' at ).!

m9+min. Sample

preparation

followed the

official method

describedearlier.

;omplete

separation of

/ and patulin

was achieved in

less than min

at detection

limits of less

than !.!) g+9

and less than 6

g+9 respectively.

he /P9;+U8

procedure is

routinely used

for uantitative

deter-mination

of patulin in

apple products,

but methods to

confirm the

presence of

patulin usually

include more

specificdetection tech-

niues such as

mass

spectrometry

$S' after 9; or

gas chroma-

tography $@;'

separations. or

@;-S

analysis, patulin

is detect-

ed as its

trimethyl silyl

derivative

$S-patulin'

with electron

ion-i?ation

$


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assays using

)3;-labeled

patulin as the

internal

standard. 7ne

method used

9;+S in neg-

ative

electrospray

ioni?ation mode

without

derivati?ation,

while the &nd

procedure

utili?ed high

resolution gas

chromatography

+ high resolution

mass

spectrometry

$/


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relevant en#2ymes involved in the

biosynthesis of patulin !3aucher

14567 Murphy and 8ynen 14567

e9iguchi and 3aucher 14547

e9iguchi and others 14:&7 Ii;ma

and others 14:*7 Priest and 8ight

14:4+0

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Patulin-production within fruits,

vegetables, and their products has been

investigated and often appears to be

dependent on such factors as water

activity $aw', temperature, p/, and other

chemical characteristics intrinsic to fruits

$Sommer and others )5B

Cortholt and others )5:B c;allum and

others &!!&'. p/ and patulin production

have been shown to be inversely related,

with patulin being unstable at high p/

$c;allum and others &!!&'.

emperature has been shown to affect

pathogen growth and, to a greater extent,

the production of patulin $c;allum and

others &!!&'. Patulin production has

been observed at all temperatures

permitting P. expansum growth,

encompassing an approximate range of !

to 3! M; $Sommer and others )5'. B.nivea has been shown to grow faster at

temperatures of 3! and 35 M; while patu-

lin production was highest at &) M;

$


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and Australia $Sommer and others

)5'.

Cumerous studies around the world

have examined the extent and degree

to which apple products have been

contaminated by

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patulin. =n

Fisconsin, &3

of ! roadside

apple #uices

were found to

contain

between )! to36! g patulin+9

$4rac(ett and

arth )5a'.

A &nd study

showed that :

of )3

commercial

apple #uices

tested

contained

between

and 3! g

patulin+9 #uice$Fare and

others )5'. A

ur(ish study

showed &)6 of

&)6 apple #uice

concentrates

examined had

between 5 and

356 ppb patulin

with 3" being

above the 6!-

ppb

internationalstandard

$@o(men and

Acar ):'.

inally, a )*

to ): study

in South Africa

showed that 6

of && #uices

sampled

contained

between )!

and 6 ppb

patulin, and

&" of infant

apple products

showed 6 to &!

ppb $4rown

and Shephard

)'. A

summary of

patulin con-

tamination

within foods is

given in able

&.

Control

.uring =pple

Harvest'

Processing'

and torage

=n Corth

America, apple

#uice is typically

a byproduct

pro-duced from

culled apples

unfit for other,

higher uality

and higher

profit, purposes

$


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efforts, and $'

adherence to

/a?ard

Analysis

;ritical ;ontrol

Points

principles

$9ope?-@arcia

and Par( ):B

Par( and

others )'.Dxisting and

developmental

preventive

measures

during

preproduction

are based upon

fruit uality and

facility

sanitation

measures. he

uality of fruit

resulting fromharvesting is

the )st step in

controlling

patulin levels.

Fith the

highest uality

hand-pic(ed

fruit being used

for direct-for-

retail sale,

processed

apple products

usually areproduced from

mechani-cal

harvest,

windfalls,

insect-

damaged, or

culled fruit.

4ruises, s(in

brea(s, and

other physical

damage within

these apples

pro-vide a

perfect entry for

P. expansum

and other

patulin-

producing

species into the

fruit. Studies

have examined

the effect of

fruit uality and

harvest method

on the patulin

content of the

result-ant

#uices. =n )

study, patulin

was

undetectable in

5 cultivars of

tree-pic(ed

cider, whereas

it was detected

between !.&

and 35 g+9 in cultivars of

ground-harvest

cider $Eac(son

and others

&!!3'. any of

the patulin

control

measures

suggested by

the Eoint

A7+F/7

ood

StandardsProgramme are

based upon the

careful

selection of

fruits as based

upon good

agricul-tural

practice

$;7%DQ &!!&'.

/owever, as

indicated by a

Cew Gor( State

/udson 8alley9ab study that

showed around

&!" of bagged,

in-store, retail

apples to

contain

consumer-

visible blue-rot

$


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processing,

including

washing,

sorting, and

pac(aging,

poses a &nd

means of both

fungal control

and

contamination

ali(e. Fashing

with high-pressure water

has been shown

to reduce patulin

levels within

apple #uice by

&)" to 6"

$Acar and others

):'. A &nd

study showed

that washing of

ground-

harvested

apples resulted

in a )!" to

)!!" patulin re-

duction,

depending on

initial patulin

level and

washing

treatment

$Eac(son and

others &!!3'.

/owever, these

same washes

can also serve

as a source ofcontamination.

;ontaminated

bins, storage

rooms, drencher

washes, drying

brushes after

apple wash, and

other steps

within the

processing cycle

can all provide a

source of fungal

inoculum cycling

in poorlysaniti?ed setups.

Prevention

methods aimed

at cleaning and

sterili?ing

storage and

processing

facilities

routinely and in

between

seasons are

being mapped

out, but have not

yet been fully

developed. Plus,

the older,

complicat-ed

design of many

pac(ing-house

and processing

euipment in-

hibits the ability

to effectively

saniti?e. Dven if

effective

strategies are

successfully

mapped out,

the inherent

variability in

apple han-dling

facilities will

reuire

customi?ation

of sanitation

methods for

each operation

$


20/58

stem-end in-

fected blue-rot

began to

appear with

increasing

freuency.

9ong-term,

controlled

atmosphere

storage has

now beenshown to allow

the slow growth

and stem-

based invasion

of fungi into

apples

$


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$


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their

effectiveness

against fungal

spores.

Vol. 1, 2005 — ;7P


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;


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Pierson and others 1971; Buchanan and others 1974; Sommer and others 1974

Apricots

Harvey and others 1972; Buchanan and others 1974; Sommer and others 1974

Persimmons

Sommer and others 1974

Strawberries

Andersson and others 1977; Frank and others 1977; Arici and others 2002

Nectarines

Harvey and others 1972

Raspberries

Andersson and others 1977; Arici and others 2002

Black mulberries

Arici and others 2002

White mulberries

Lingon berries

Andersson and others 1977

Peaches

Harvey and others 1972; Buchanan and others 1974; Andersson and others 1977; Frank

and others 1977

Plums

Harvey and others 1972; Buchanan and others 1974; Andersson and others 1977

Tomatoes

Greengages

Frank and others 1977

Bananas

Blueberries

Akerstrand and others 1976; Andersson and others 1977

Black currants

Andersson and others 1977

Almonds


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Pecans

Jiminez and others 1991

Peanuts

Hazelnuts

Foods contaminated with patulin Source

Apple juice

Lovett and others 1974; Sommer and others 1974; Frank and others 1977; Scott and others

1977;

Brackett and Marth 1979a; Prieta and others 1994; Rychlik and Schieberle 1999;

Leggott and Shephard 2001; Ritieni 2003

Apple-acerola juice

Rychlik and Schieberle 1999

Pear juice

Ehlers 1986

Grape juice

Harwig and others 1978; Rychlik and Schieberle 1999

Sour cherry juice

Blackcurrant juice

Rychlik and Schieberle 1999

Orange juice

Pineapple juice

Ake and others 2001

Passion fruit juice


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Apple cider

Brackett and Marth 1979; Wheeler and others 1987; Leggott and Shephard 2001

Apple puree

Leggott and Shephard 2001; Ritieni 2003

Corn

Lin and others 1993

Strawberry jam

Blackcurrant jam

Jelinek and others 1989

Blueberry jam

Baby food

Prieta and others 1994; Leggott and Shephard 2001; Ritieni 2003

Cheddar cheese

Bullerman and Olivigni 1974

Barley Malt

Lopez-Diaz and Flannigan 1997

Wheat Malt

Bread

Reiss 1972, 1976

Countries with apple and

juice contamination

Source

Canada

Scott and others 1972; Harwig and others 1973b; Sommer and others 1974

England


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Brian and others 1956; Sommer and others 1974

New Zealand

Walker 1969

United States

Sommer and others 1974; Ware and others 1974

South Africa

Leggott and Shephard 2001

Sweden

Josefsson and Andersson 1976

Turkey

Gokmen and Acar 1998

Brazil

de Sylos and Rodriguez-Amaya 1999

Austria

Steiner and others 1999a

Belgium

Tangi and others 2003

Australia

Sommer and others 1974

France

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28/58


standard apple

#uice production

steps are

centered on 3

areas. he )st

of these

involves theuality of the

fruit and

processing of

this fruit, prior

to pressing. As

previously

mentioned,

processed

apple products

often utili?e

lower uality

fruit that is

unsuitable fordirect mar(et

retail.


29/58

necessitating

the need to

re#ect entire

#uice loads of

apples. =f

reliant on this

method, small-

scale

producers who

cannot afford

these losseswill be forced to

sort by hand,

in-creasing

costs to the

point that many

may cease

operating

$


30/58

reduce patulin

levels by :"

and 55",

respec-tively.

/owever, these

methods ma(e

the removed

ca(e and+or fil-

ter potentially

highly toxic and

unfit for any

further use, suchas often is done

with #uice

sediments in

animal feed

$4issessur and

others &!!)'.

his could

represent a loss

of income for

many #uice

producers.

4atch

absorption with

synthetic

polymers has

also been

investigated

$;anas and

Aranda )*',

as has en?yme

treat-ment with

pectinase

en?ymes used

to brea( down

the pectin coat

surrounding

protein particles,allowing

aggregation and

sedimen-tation

of protein

particles and, in

the case of

patulin

contamina-tion,

their associated

patulin adducts.

his method has

resulted in a

53" decrease in

patulin contentwithin #uice

$4issessur and

oth-ers &!!&'.

he 3rd #uice

production

process

capable of

reducing

patulin

levels is the

pasteuri?ation

process. 7f the

3 processes

mentioned thus

far, this is by far

the least

effective.


31/58

to reduce patulin

levels by ):.:"

$Fheeler and

others ):5'.

Dv-idence also

shows that

patulin is

nonvolatile and,

upon distilla-tion

production of

apple aroma,

patulin remainswithin #uice con-

centrate $>ryger

&!!)'. inally,

not only is the

pasteuri?ation

pro-cess unable

to significantly

reduce patulin

levels, it often

fails to fully

remove heat

resistant patulin-

producing fungi,

such as B. nivea

and B. fulva

$7ugh and

;orison ):!',

allowing for

poten-tial

continued

production of

patulin within the

finished #uice.

As a final stageof the production

process, studies

have exam-ined

the effects of

storage on

patulin content.

ixed studies

show either a

decrease $Scott

and Somers

)*:B /arwig

and others

)53a' or no

change

$Pohland and

Allen )5!B

Legota and

others )::' of

patulin levels in

apple #uice with

refrigerated

storage. Similar

studies within

grape #uice have

shown stability

$7ugh and

;orison ):!' or

an approximate

6!" decrease

$Scott and Som-

ers )*:' of

spi(ed patulin

levels in grape

#uice after 6 w(.

Control

Postproducti

on

Filtering and

adsorption

A number of

studies have

been devoted to

the removal of

patu-lin from

#uice through the

use of

adsorption

filters, columns,

and agitation

treatments using

carbon-based

material.

Agitation with &!

mg+m9 activated

charcoal

followed byfiltration through

a !-or *!-mesh

charcoal column

reduced a 3!-

g+m9 patulin

solu-tion to

below

detectable

levels. urther,

use of 6 mg+m9

charcoal in

agitation was

able to reduce

patulin to belowdetectable levels

in naturally

contaminated

cider. /owever,

color loss was

mar(ed-ly

present in this

resulting #uice

$Sands and

others )5*'. =n

a &nd study, !,

!.6, ).!, ).6,

&.!, &.6, and 3

g+9 activated


32/58

charcoal was

added to

naturally

contaminated

apple #uice

containing *&.3

ppb patulin.

Samples were

mixed for !, 6,

)!, &!, and 3!

min. hree

grams+literactivated

charcoal was

found to be most

effective with a

time period of 6

min. ;learness

of #uice

increased, color

of #uice

decreased, and

small decreases

in fumaric acid,

p/, and M4rix

were also seen

$>ada(al and

Cas &!!&'. =n

another study,

ultrafine

activated carbon

was bound to

granular uart?

producing a

com-posite

carbon

adsorbent

$;;A'. ;olumns

with varyingamounts of ;;A

were prepared

and )! g+m9

patulin were

filtered through

at ) m9+min.

ifty percent

brea(through

values for

columns with

).!, !.6, and

!.&6 g ;;A

were )35.6,

3:.6, and ).

g, respectively

$/uebner and

others &!!!'.

=n a th study

designed to

compare theeffects of

different car-bon

activation

methods, steam

activated carbon

C7


33/58

;


34/58

through the addition of sulfur dioxide to

the hemiacetal ring of patulin, forming a

carbonyl hydroxysulfonate. he &nd

reaction is thought to be irreversible and

to occur due to an opening of the lactone

ring structure at one

of the double bonds $4urroughs )55'.

A th study found the sul-fur dioxide

inactivation of patulin to be reversibleor irreversible de-pending on p/

$Steiner and others )b'.

Sulfur compounds common to

biological systems and fre-uently

associated with patulin toxicity, such as

glutathione, cys-teine, and

thioglycolate, are believed by many to

produce biologi-cally inactive products

when reacted with patulin $;avallito

and 4ailey )B @eiger and ;onn

)6B en?ie and others )5'.

ur-ther, other mycotoxins such as

aflatoxins 4), @), 4&, and @& $aeba

)::' as well as ?earalonone $%oyle

and others ):&' have been shown to

be effectively degraded by o?one.

=n all of these cases, preliminary

evidence is promising. /owev-er,inadeuate research has been done to

examine the efficacy and full functional

range of applicability for the treatment.

urther-more, the reaction mechanisms

for few if any of the chemical

detoxification treatments are fully

understood, and both the reac-tion

products and their respective toxicities

are still un(nown and must be

determined prior to use. he regulatory

approval for some of the proposed

chemical treatments such as

ammoniation and potassium


35/58

permanganate must be sought prior to

use in the food industry.

-iological control

4iological methods of patulin control

result largely from the ob-

16 ;7P


36/58


servation that

patulin is almost

always

completely

degraded dur-

ing yeast

fermentation.

4esides being

uite successful,

this meth-od is

much better

understood

compared with

other

decontamina-

tion methods.

Approximately

!" of patulin

can be removed

during yeast

fermentation$4urroughs

)55'. =n )

study, * of :

yeast strains

reduced patulin

levels to below

detectable

levels, while all

: strains

resulted in a

" or better

decrease in total

pat-ulin content.

;ontrol #uice, onthe other hand,

stored for an

eual amount of

time $& wee(s',

had only a )!"

reduction

$Stinson and

others )5:'. =n

a &nd study,

yeast

fermentation

reduced patulin

levels

completely after

& w(. his same

study also

showed that

patulin levels

failed to

decrease

significantly in

#uices that had

been yeast

fermented and

then filter

sterili?ed to

remove yeast,

suggesting that

active yeast,

and not their

byproducts,

were re-uired

for the reduction

$/arwig and

others )53a'.

reatments of

patulin along

with

cyclohexamide,

a protein

synthesis

bloc(er of yeast,

completely

bloc(ed protein

synthesis and

prevented the

detoxification of

patulin. Addition

of

cyclohexamide 3h after pat-ulin

addition,

however,

resulted in a

reduced, but

continued, rate

of patulin

degradation,

suggesting that

the proteins

synthesi?ed

within the 3-h

window were

catalyticallyactive against

patulin and did

not #ust bind it

up in adduct

formation

$Sumbu and

others ):3'. A

later study

showed that 3

strains of

Saccharomyces

cere-visiae

reduced patulin

levels during

fermentive

growth but not

aerobic growth.

his reduction

resulted in the

production of &

ma-#or productsJ

D-ascladiol,

patulin0s

immediate

biosynthetic

precur-sor, and

its isomer L-


37/58

ascladiol. hese

& products were

also seen in the

treatment of

patulin with the

reducing agent

sodium borohy-

drate $oss and

9ong &!!&'. D-

ascladiol is itself

a mycotoxin

$Su-?u(i andothers )5)',

which has

reduced toxicity

compared with

patulin and also

reacts with

sulfhydryl-

containing

compounds

$Se(iguchi and

others ):3'.

Fhile effective,

biological

control with

yeast is limited

to prod-ucts

that can be

fermented.

urthermore,

yeast are

themselves

sensitive to

patulin, and atconcentrations

greater than

&!! g+ m9,

yeast have

been shown to

be completely

inhibited,

prevent-ing

fermentive

detoxification

$Sumbu and

others ):3'.

Co re-searchhas been done

to examine the

potential use of

other fer-

menting

microbes, such

as lactic acid

bacteria, in

decreasing pat-

ulin content

within #uices.

Similar

reducing

en?ymes and

environ-ments

produced by

these bacteria

may very well

be able to de-

grade patulin.

inally, no

research has

investigated the

direct en-?ymatic

degradation of

patulin.


38/58

been performed

specifically on

patulin $%oyle

and others

):&B 8alletrisco

and others

)!'.

Patulin

Production

in the Final

Product

9imited wor(

has been done

to examine the

potential growth

and production

of patulin by

fungi in stored,

finished product

#uices. =t is well

(nown that

many

Aspergillus and

Byssochlamys

spp. molds,

many of which

produce patulin,

are heat

resistant and

can survive the

pasteuri?ation

processes usedin #uice and ci-

der production.

urthermore,

many of the

same patulin-

produc-ing

molds have also

been shown to

grow and

produce patulin

at

standard cold

storage

temperature

down to !M;

$Sommer and

others )5'.

a(en together,

these & facts

suggest that

finished apple

#uice and cider

could very well

support the

continued pro-

duction of

patulin by fungi.

=f this is the

case, any

single

treatment

patulin control

method, such

as physical

adsorption,would po-

tentially be

moot due to

production of

patulin by

active fungi

postprocessing.

Studies must

be done to

examine this

potential within

#uices, and, if

possible, toprovide

correlates

between mold

numbers and

patulin levels

within #uice.

Fith the

possibility of

fungi actively

producingpatulin within

#uice, control

measures

should aim not

only at the

reduction of

patulin itself,

but the

inhibition of

both fungal

growth and

patulin

productionwithin #uice.

Some studies

have examined

the effects of

many patulin-

reducing

treatments on

live patulin-

producing

fungi. Sulfur

dioxide, sodium

ben?oate, and

potassium


39/58

sorbate have

been

investigated for

the affect on B.

nivea growth

and patu-lin

production

within #uice.

Seventy-five

ppm sulfur

dioxide, )6!ppm potassium

sorbate, and

6!! ppm

sodium

ben?oate all

signif-icantly

retarded B.

nivea growth

and patulin

production

$


40/58

P. expansum

growth $


41/58

;, Atindehou D. &!!).%Vtermination de la Patuline dans le Eus de ruits

;ommercialisVsen ;Wte d0=voire. Sci Aliment &)J)K

&!*.

A(erstrand >, olander A, Andersson A, Cilsson @.

)5*. 7ccurrence of moulds and mycotoxins in fro?en

blueberries. 8Xr Hda &:J)5K&!!.

Andersson AD, Eosefsson @, Cilsson @, A(erstrand >.

)55. Hgelsvampar och Patulin = ru(t 7ch 4Yr. 8Xr

Hda :J&&K:.

Annous 4A, Sapers @, attra??o A, ,


42/58

Proceedings of the &th ycotoxin For(shopB &!!&

Eun 3-*B 4erlin, @ermany.

Arti( C, Acar E, >abraman C, Poyra?oglu D. &!!).

Dffects of various clarification treatments on patulin,

phenolic compound, and organic acid compositions of

apple #uice. Dur ood lein


43/58

1;7%DQ2 ;ommittee on ood Additives and

;ontaminants. &!!&. Proposed draft code of practice

for the reduction of patulin contamination in apple #uice

and apple #uice ingredients in other beverages $at step

6 of procedure'. @eneva, Swit?erland. Eoint A7+F/7

ood Standards Programme. p )K5.

;onway FS, Eanisiewic? FE, >lein E%, Sams ;D.

). Strategy for combining heat

treatment, calcium infiltration, and biological control

to reduce postharvest de-

cay of @ala0 apples. /ort Sci 3$'J5!!K.

;ooray iessling >-/, 9indahl->iessling >. ):&.

he effects of patulin and pat-ulin-cysteine mixtures on

%CA synthesis and the freuency of sister-chromatid

exchanges in human lymphocytes. ood ;hem oxicol

&!J:3K:.

;orbett %. &!!3. PatulinRU.>. producers perspective

=nJ Patulin technical sympo-sium. ebruary ):K),

&!!3B >issimmee, la. Cational ;enter for ood safety

adn echnology. Summit, =ll.

%emirci , Arici , @umus . &!!3. Presence of

patulin in fruit and fruit #uices produced in ur(ey.

Drnaehrungs-Umschau 6!$5'J&*&K3.

%e Sylos ;,


44/58

18 ;7P


45/58


ran( />, 7rth ,

>ennedy 4P;, Scott

P. )53b.7ccurrence of patulin

and patulin-producing

strains of Penicillium

expansum in natural

rots of apple in

;anada. ;an. =nst.

ood Sci echnol E

*J&&K6.

/arwig E, 4lanchfield

4E, Scott P. )5:.

Patulin production by

Penicillium roque-forti

hom from grape.

;an =nst ood Sci

echnol E ))$3'J)K

6).


46/58

/ate? , @aye .

)5:. =nhibition of

translation in

reticulocyte by the

mycotoxin patulin.

D4S 9ett 6J&6&K*.

/ayes AF, Phillips

%, Filliams F9,

;iegler A. )5.

Acute toxicity of

patulin in mice and

rats. oxicology

)3J)K)!!.

/eatley C@, Philpot

E. )5. he routine

examination for

antibiotics produced

by molds. E @en

icrobiol )J&3&K5.

/offman /, int?laff

/E, Alperden =,

9eistner 9. )5).

Untersuchung uber

die =n-a(tiviering des

y(otoxins Patulin

durch

Suofydrylgruppen.

%ie leis-

chwirtschaft.

6)J)63K*, )63.

/op(ins E. )3. he

toxicological ha?ardsof patulin. 4r =nd 4iol

, Pallaroni

9, A(e ;9, 9em(e

S9, /errera P,

Phillips %.

&!!!. %evelopment

and characteri?ation

of a carbon-based

composite material

for reducing patulin

levels in apple #uice. E

ood Prot *3$)'J)!*K

)!.

=i#imia /, Dbi?u(a G,

San(awa U. ):*.

4iosynthesis ofpatulin, in vitro

conversion of gentisyl

alcohol into patulin by

microsomal

en?yme$s' and

retention of one of the

carbinol protons in

this reaction. ;hem

Pharm 4ull

3$:'J363K5.

=yengar , ;hirtel SE,

er(-

er


47/58

#uice. Cahrung+ood

*$)'J3)K3.

>orte A. ):!.

;hromosomal

analysis in bone

marrow cells of

;hinese hamsters

after treatment with

mycotoxins. utat

or?ybs(i ,

>ows?y(-@indifier L,

>urylowic? S. )*5.

Antibiotics origin,

nature, and

properties 8ol. ==.

Cew Gor(J Pergamon

Press. p )&&3K3!.

>ryger yria(ides . &!!).

%iffusion of patulin in

the flesh of pears

inoculated with four

post-harvest

pathogens. E Phy-

topathol

)J65K*).

9am >S, Ceway E7,

@aucher @. )::.

!n vitro

stabili?ation

of *-methylsalicyl-

ic acid

synthetase from

Penicillium urticae.

;an

E icrobiol

3J3!K5.

9arsen 7, risvad

E;, -S,

, Sung F4. )3.

Simultaneous thin

layer

chromatographic


48/58

determination of

?earalenone and

patulin in mai?e. E

Planar

;hromatography-

odern 9; *J&5K5.

9indroth S, von

Fright A. )!.

%etoxification of

patulin by adduct

formation with

cysteine. E Dnviron

Pathol oxicol 7ncol

)!$K6'J&6K.

9lovera , 8iladrich


49/58

sum by polymerase

chain reaction. =nt E

ood icrobiol

:J)3K.

ayer 8F, 9egaror

S. )*. Production

of petite mutants of

Saccharomyces cer-

evisiae by patulin. E

Agric ood ;hem

)5J6K*.

c;allum E9, sao


50/58

urphy @, 9ynen .

)56. Patulin

biosynthesisJ he

metabolism of m-

hydroxyben-?yl

alcohol and m-

hydroxyben?aldehyde

by particulate

preparations from

Peni-cillium patulum.

Dur E 4iochem

6:J*5K56.

utlu , /i?arcioglu

C, @o(men 8. )5.Patulin adsorption

(inetics on activated

carbon, activation

energy, and heat of

adsorption. E ood

Sci *&J)&:K3!.

Ceway E, @aucher

@. ):). =ntrinsic

limitations on the

continued production

of the antibiotic

patulin by Penicillium

urticae. ;an E

icrobiol &5J&!*K)6.

Corstadt A, c;alla

. )*3. Phytotoxic

substance from a

species of Penicilli-

um. Science

)!J)!K).

Corstadt A, c;alla

. )*:.

icrobiological

population in

stubblefield mulched

soil. Soil Sci )!5J)::.

Cortholt %, 8an

Dgmond /P, Paulsch

FD. )5. Patulin

production by some

fun-gal species in

relation to water

activity and

temperature. E ood

Prot )$))'J::6K !.

7ugh ;S, ;orison

;A. ):!.

easurement of

patulin in grapes and

wines. E ood Sci

6J5*K:.

7swald /, ran( />,

>omitows(i %, Finter

/. )5:. 9ong-term

testing of patulin

administered orally to

Sprague-%awley rats

and Swiss mice.

ood ;osmet oxi-col

)*J&3K5.

Par( %9, 9ee 9S,

Price


51/58

;


52/58

@;+S confirmation of patulin identity. =nJ ruc(sess

and others, editors. ycotoxins and food safety. Cew

Gor(J >luwer Academic+Plenum Pub-lishers. p )36K!.


53/58

Singh E. )*5. Patulin. =nJ @ottlieb %,

Shaw P%, editors. Antibiotics =J mechanics

of action. 4erlinJ Springer-8erlag. p

*&)K3!.

Sommer C, 4uchanan E


54/58

httpJ++www.usda.gov+nass+pubs+agr!&+!&[ch6.pdf .

Accessed

arch 6, &!!3.

1US%A2 U.S. ood and %rug Administration. &!!.

;ompliance policy guide. ;om-pliance policy guidance

for %A staff. Sec. 6)!.)6!. Apple #uice, apple #uice

concentrates,

and apple #uice productsRAdulteration with patulin.

Available

fromJ

httpJ++www.fda.gov+ora+compliance[ref+cpg+cpgfod+cpg

6)!-)6!.htm.

Ac-

cessed

arch

&, &!!.

8alletrisco S, ;asadio S, Stefanelli ;. )!. Use of

ultraviolet radiation to brea( down mycotoxins.

=ndustrie Alimentari &$&::'J))))K&.

van Dgmond /P. ):. ;urrent situation on

regulations for mycotoxins. 7verview of tolerances

and status of standard methods of sampling and

analysis. ood Addit ;ontam *J)3:K::.

8an 9ui#( A. )3:. Antagonism of Penicillium spp.

versus Pythium de"aryanum. ;hron 4ot J&)!.

Fal(er E, Fiesner 4P. ). Patulin and clavicin.

9ancet &*J&.

Fang =>, oehler PD. ):5. Presence

of patulin in pasteuri?ed apple cider. E ood Sci

6&J5K:!.

Fichmann @, /erbarth 7, 9ehmann =. &!!&. he

mycotoxins citrinin, gliotoxin, and patulin affect

interferon- rather than interleu(in- production in

human blood cells. Dnviron oxicol )5$3'J&))K:.

Fisniews(y A, @lat? 4A, @leason 9,


55/58


ood Prot *3J5!3K:.

1F/72 Forld /ealth

7rgani?ation. )!.

Dvaluation of certain

food additives and

contaminants. F/7

thirty-fifth report of

the #oint A7+F/7

expert committee on

food additives.

@enevaJ echnical


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57/58


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Vol. 1, 2005 — ;7P

SIKLUS BIOSINTESIS PATHWAY

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