1216

Journalof FoodProtection, Vol. 64, No. 8, 2001, Pages 1216– 1219 Copyright ,International Association forFood Protection

Reductionof Patulin during Apple Juice ClariŽcation

J.BISSESSUR, K.PERMAUL, AND B. ODHAV*

Departmentof Biological Sciences, M.L.SultanTechnikon, P.O. Box 1334, Durban 4001, South Africa

MS00-179:Received 9June2000/ Accepted 25September 2000

ABSTRACT

Patulinis a mycotoxinproduced by a numberof molds involved in fruit spoilage. This compound is carcinogenic and Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/8/1216/1674072/0362-028x-64_8_1216.pdf by guest on 25 September 2021 teratogenic.V ariousmethods are currently used to reduce the levels of patulin in apple juice, namely, charcoal treatment, chemicalpreser vation(sulfur dioxide), gamma irradiation, fermentation, and trimming of fungus-infected apples. Many of theseprocesses are expensive and time-consuming. Therefore, there is aneedto Ž nda convenientand economical process to controlpatulin levels. This study was undertaken to evaluatethe effectiveness of severalclariŽ cation processes for the reduction ofpatulin. ClariŽ cation was carried out on alaboratoryscale. Apple pulp was spiked with patulin, pressed, and clariŽ ed using fourdifferent processes, namely, Ž ningwith bentonite, enzyme (pectinase) treatment, paper Ž ltration,and centrifugation.Patulin wasrecovered from the clariŽ ed juiceby liquid-liquidextraction, and solid-phase chromatography was used for sample clean- upprior to analysis by high-performance liquid chromatography (HPLC). Theminimum detectable limit using HPLC was20 g/liter.Pressingfollowed by centrifugationresulted in an averagetoxin reduction of 89%.T otaltoxin reduction using Ž ltration, enzymetreatment, and Ž ningwere 70, 73, and 77%, respectively. Patulin reduction was due to the binding of the toxin to solidsubstrates that was veriŽ ed by analyzingthe clariŽ ed juiceas wellas the Ž ltercake, pellet, and sediment. The combined concentrationscorrelated to the spiked concentration. These results reveal that clariŽ cation was successful in the reduction of patulinlevels in apple juice. However ,clariŽcation resulted in high levels of patulin in the pressed pulp after Ž ltrationand centrifugation,and this could be harmful if they are used as animal feeds.

Patulin(4-hydroxy-4H-furo[3,2-c]p yran2[6H]-one),a pendedsolid material (19). Thiscan be accomplished in mycotoxin,is a secondarymetabolite produced by several oneof twoways: enzymatically and nonenzymatically. The fungi,primarily Aspergillus and Penicillium species.It was useof pectinase enzymes breaks down the pectin coat sur- isolatedby Birkinshaw et al. (1) from Penicilliumpatulum roundingprotein particulates in the emulsion, allowing the and Penicilliumexpansum. P. expansum isa well-known particlesto aggregate and sediment (17). Nonenzymeclar- postharvestpathogen that causes blue mold rot in apples iŽcation involves breaking the emulsion by other means, andalso produces the mycotoxin patulin (25). Patulin-pro- themost common of which is heat treatment. Other tech- ducingfungi occur in a varietyof foods, but only apple niquesinclude addition of bentonite,gelatin, casein, tannic juiceand apple cider have been found to be contaminated acid-proteincombinations (13), andchitosan (22). Mechan- naturallywith patulin (26). icalseparation techniques like decanting, centrifugation, Ž l- Thetoxicological properties of patulin have recently tration (27), pressureŽ ltration,and rotary vacuum Ž ltration beenreviewed, and patulin has been reported to be muta- andmore recently membrane (ultraŽ ltration) Ž ltration (16, genicand to cause neurotoxic, immunotoxic, genotoxic, 21) arealso used to separate the solid matter from thejuice andgastrointestinal effects in rodents, although little sci- inthe manufacture of apple juice (17, 19). entiŽc evidenceexists to support media contention that pa- Astudyon the effect of down-lineprocessing has em- tulinis carcinogenic to humans (12). Theinternational max- phasizedthe need to reduce the levels of patulin entering imumpermitted limit for patulincontamination is 50 ppb thefactory, as subsequent processes, other than the addition ofactivated carbon, have little or no inuence on thelevels (10). Asurveycarried out in South Africa for thepresence ofpatulin in the Ž nalproduct (4). Theuse of activated ofpatulin in apple products from 1996to 1998 revealed charcoal(while an effective agent for thereduction of pa- that5 outof 22 apple juice samples (23%) analyzed con- tulinlevels) is not feasible to apply on a commercialscale tainedpatulin levels between 10 and 45 ppb. Patulin was (4). Alternativemethods need to be foundto control patulin alsofound in 29% of infant apple products surveyed, with duringdown-line processing. toxinconcentration ranging between 5 and20 ppb (3). Pa- Thispaper describes the use of several conventional tulinwas detectedin all215 apple juice concentrates tested techniques(Ž ning with bentonite, paper Ž ltration,pectinase from threedifferent suppliers at concentrations ranging treatment,and centrifugation) to evaluate the removal of from 7to375 ppb and 43% of the samples exceeded the patulinduring the clariŽ cation processes involved in apple patulincontamination level of 50 ppb (10). juicemanufacture. Rawapple juice is normally clariŽ ed to remove sus- MATERIALS AND METHODS

*Authorfor correspondence. Tel: 12731308 5286; Fax: 12731 308 Standards. GoldenDelicious apples were bought from local 5351;E-mail: [email protected]. supermarketsin the Durban area. Apple juice clariŽ cation was J.FoodProt., Vol. 64, No. 8 CLARIFICATION PROCESSESFOR PATULINREDUCTION 1217 carriedout on a laboratoryscale. Patulin was purchased from 1),and 1 mlchloroform:ethyl acetate (1:1). T womilliliters of SigmaChemical Company (Atlasville, South Africa). Stock so- chloroform:ethylacetate (1:4) was used to elute patulin. The el- lutionsof 100 and 1,000 g/mlwere prepared in chloroform, uantwas evaporated under a gentlestream of nitrogen, and the dried,and stored at 2208C.Workingstandards of 0.1, 1.0, and driedresidue was dissolved in 1 mlwater:acetonitrile (9:1). 10 g/mlpatulinwere prepared by dilution of stock solutions with water:acetonitrile(9:1). Thin-layerchromatographic analysis. Themethod of Prie- ta et al. (18) wasused. T enmicroliters of the sample and patulin Apple juiceclariŽ cation. Approximately1 kgof apples was standardswere spotted on thin-layerchromatography plates (silica sliced,blanched (60 to 70 8C),and homogenized using a commercial gel 60 F254,Merck)and developed in a mobilephase consisting blender(W aringProducts Division, New Hartford,Conn.). The oftoluene:ethyl acetate:90% formic acid (5:4:1). The plates were crushedapple pulp was spiked with patulin to yield a concentration sprayedwith 0.5% 3-methyl-2-benzoth iazolinonehydrozone hy- of 2 g/g.Thepulp was pressed using a doublelayer of cheesecloth drochloridehydrate, heated in an oven at 130 8Cfor15 min, and andthe juice collected by vacuum extraction. In order to monitor examinedunder long-wave UV light.Patulin appeared as a yel- thetotal dissolved solids in the juice from different pressings, the low-brownspot with an Rf valueof 0.5. Once the toxin was de-

Brixwas determined by the method of Zubrick (28) and found to tected,their levels were quantiŽ ed by HPLC analysis. Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/8/1216/1674072/0362-028x-64_8_1216.pdf by guest on 25 September 2021 beapproximately 13 8 inall pressings. The pH of the pulp was determinedand ranged between 3.3 and 3.7. The pressed cake was HPLC analysis. AMerck-Hitachimodel D-7000 HPLC sys- removed,and 50-ml aliquots of the juice were further processed by temequipped with an L-7400 variable-wavelength UV detector eithercentrifugation, Ž ltration,enzyme treatment, and Ž ningor (Merck-Hitachi,T okyo,Japan) set at 276nm, an L-7200autosam- combinationsof these processes. Centrifugation of the apple juice pler(Merck-Hitachi), and an L-7100 pump (Merck-Hitachi) was wascarried out at 6,551 3 g for10 minin a Sigma3K30 centrifuge used.The mobile phase was water:acetonitrile (99:1) at a ow (SigmaLaborzentrifugen, Osterode am Harz,Germany). Paper Ž l- rateof 1 ml/min.T wenty-microliterinjections were separated in trationwas carried out using Whatman number one Ž lterpaper with aLichrosphere100 RP-18 analytical column (250 nm by 4 mm) 5gdiatomaceousearth as a Žlteraid (17). Enzymetreatment was atroomtemperature. A standardcur vewas constructed for patulin carriedout using pectinase from A. niger (Merck,Lindenplatz, Ger- concentration(concentration [ g/ml]of patulin versus peak area). many)that was added to a Žnalconcentration of 0.02% (wt/ vol). Patulinconcentration of samples was calculated with the follow- Enzymaticdigestion occurred at 40 to 45 8Cfor2 hinan orbital ingequation: shaker.Bentonite(Fluka, Steinham, Germany) was used as the Ž n- patulin ( g/liter) 5 (C 3 V )/V 3 1,000 ingagent and was added at aŽnalconcentration of 3% (wt/ vol)to t i 50ml juice. The suspension was maintained at room temperature where C isthe concentration of patulin in g/mlin the Ž nalso- for60 to 90 min until sedimentation was complete. The clariŽ ed lution, Vt isthe total volume of the Ž nalsolution (ml), and Vi is juicewas then decanted. After each clariŽ cation process, the clar- theinitial volume (ml) of the apple juice taken for extraction. All iŽed juice as well as the pellet, Ž ltercake, and sediment were an- experimentswere performed in triplicate, and mean and standard alyzedfor patulin by thin-layer chromatography and high-perfor- deviationof HPLC resultsfor patulin recovery were calculated. manceliquid chromatography (HPLC). RESULTS AND DISCUSSION Controls. Patulinlevels in the spiked pulp were determined tomeasure the efŽ ciency of the recovery rate from pulp. The ClariŽcation was carriedout on freshly prepared pulp recoverylevels from liquid-liquid extractions were established by thatwas spikedwith patulin to yield a Žnalconcentration spikingapple juice with a knownconcentration range and com- of 2 g/ml.Initial HPLC datawere erraticdue to thepres- paringconcentrations after extraction. Patulin levels in the pressed enceof a coextract(probably 5-hydroxymethylfur furyl juicebefore clariŽ cation treatments were determined to enable cal- [HMF]) thatpeaked at a similarretention time to patulin. culationof the patulin losses due to individual clariŽ cation treat- HMFisformed as a resultof dehydrationof ketopentoses, ments.A controlfor determining the loss of patulin due to the particularlyin acidic or high temperature environments extractionand puriŽ cation processes was performed by spiking (14). Thepulp pH rangedbetween 3.3 and 3.7 in ourstudy. theextraction solvents with patulin. An enzymetreatment control wasperformed to determine the temperature stability for patulin Thismay have contributed to the formation of HMF .Spa- atthe temperatures used for enzyme treatment. nos et al. (23) reportedincreasing levels of HMF inapple juiceduring storage. Combinationtreatments. Fourcombination treatments, viz., Forbitoand Babsky (9), whoused the same mobile Žningand centrifugation, enzyme treatment and centrifugation, phasefor HPLC analysesas that used initially in ourstudy, Žltrationand Ž ning,and Ž ltrationand enzyme treatment were test- alsoobserved the presence of an unknown peak overlap- edby performing the Ž rsttreatment followed by the second as describedabove. pingwith that corresponding to patulin.However ,Chudziak andT rojanowicz (6), Brauseet al. (2), andGokmen and PatulinpuriŽ cation. Sampleswere prepared for analysis by Acar (11) were ableto obtain effective separation of these themethod of Rovira et al. (20) withthe following changes: 50 twopeaks (HMF andpatulin) by reversed-phase liquid mlof clariŽ ed apple juice was extracted three times with equal chromatographyand the use of analternative mobile phase, volumesof ethyl acetate. The ethyl acetate fractions were com- viz.,water:acetonitrile (1:99). In our study, this mobile binedand evaporated to dryness under reduced pressure over ap- phasedid improve separation of thepeaks signiŽ cantly but proximately1 ganhydroussodium sulfate. The dried residue was redissolvedin 20 mlchloroform and dried for 1 h.Thechloroform nottotally. Complete separation was achievedwith the use ofthe drying agent, anhydrous sodium sulfate. Brause et solution(5 ml)was introduced into a Sep-PakSiO 2 cartridge(Wa- tersChromatography Division, Millipore Corp., Milford, Conn.), al. (2) reportedthat patulin may be destroyedwhen a ‘‘wet’’ previouslyconditioned with 5 mlchloroform. The cartridge was ethylactetate extract is evaporatedto dryness.The method washedwith 1 mlchloroform, 1 mlchloroform:ethyl acetate (4: initiallyused in this study required the ethyl acetate to be 1218 BISSESSURET AL. J.FoodProt., Vol. 64, No. 8

TABLE 1. Evaluationof single clariŽ cation treatments in patulin removal Patulinconcentration ( g/ml) % loss % loss % loss (minus (minus Process Recovered Lost (total) pressing) recovery)

Pressing 0.95 6 0.03 1.05 52.5 — — Centrifugation 0.22 6 0.02 1.78 89 36.5 20.5 Fining 0.46 6 0.05 1.54 77 24.5 8.5 Enzymetreatment 0.55 6 0.05 1.45 73 20.5 4.5 Filtration 0.68 6 0.08 1.32 70 17.5 3 Control 1.62 6 0.08 0.38 19 — —

driedcompletely immediately after extraction from the andclean-up steps. The patulin reduction achieved by cen- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/8/1216/1674072/0362-028x-64_8_1216.pdf by guest on 25 September 2021 aqueoussample without the use of a dryingagent. It is trifugationalone is therefore 20.5%. Fining was thesecond- possiblethat patulin may have degraded during the drying mosteffective clariŽ cation process, and it reduced the toxin stepand that the breakdown products in combination with levelby 8.5%. Filtration and enzyme treatment were lessef- HMFcausedpeak interference. Further studies will be re- fectivethan the two treatments already described. No patulin quiredto determine the actual breakdown products and reductionoccurred with Ž ltrationand only 4.5% with enzyme theirchemical properties. The addition of anhydrous sodi- treatment.Bullerman (5) reportedthat patulin may bind to umsulfate in the Ž rst dryingstep during sample preparation solidmaterial. The effectiveness of these clariŽ cation treat- improvedtoxin recovery and analysis because toxin and mentsin toxin removal can be attributed to the adherence of coextractpeaks were completelyresolved. patulinto solid particles because the patulin loss in the juice Thelimit of detection for patulinin this study was 20 canbe recovered in the centrifugation pellet and sediment g/liter.Theaverage patulin recovery rate from spikedapple from theŽ ningprocess. The combined concentrations corre- juicewas 84%for thethree patulin concentrations tested (re- latedwith the spiked concentration of 2 g/ml. sultsnot shown). The 16% loss of patulin may be due to Fourcombination treatments, viz., centrifugation and patulindamage or destruction during the drying stage of the Žning,centrifugation and enzyme treatment, Ž ningand Ž l- extractionas well as patulin loss in the cartridges. Pressing of tration,and Ž ltrationand enzyme treatment were tested(T a- theapple pulp to extract the juice was doneprior to testing ble2). Pressing resulted in a 48%loss of patulin. Surpris- theclariŽ cation procedures. The results obtained from thesin- ingly,the combination treatments resulted in a lowerper - gle-clariŽcation processes are summarized in T able1. An al- centageloss of patulin than the individual treatments. Cen- iquotof spiked (2 g/ml) pulp(unprocessed) was usedas a trifugationand Ž ningproduced the greatest loss of patulin controlin each experiment. Although the pulp was notpro- (20.5%),which was thevalue obtained for centrifugation cessed,a lossof patulin of 19% was observed.This value alone,followed by centrifugation and enzyme treatment reects the recovery potential of the extraction and clean-up (17%),Ž ningand Ž ltration(12%), and Ž ltrationand enzyme methodfrom applejuice. Most of the patulin was removed treatment(7.5%). The lower than expected values obtained duringthe pressing step. The apple pulp had been spiked with for thecombined treatments can be explained by the fact 2 g/mlpatulin, and approximately 0.95 g/mlwas recovered thatpatulin removal is due to the binding of the toxin to afterthe pressing step resulting in an average loss of 52.5%. particulatematter that is removed from thejuice. During After pressing,the juice was subjectedto individual clariŽ - centrifugation,patulin bound to particulate matter will be cationprocesses, viz., Ž ltration,Ž ning,centrifugation, and en- effectivelyremoved. This study has shown that most of the zymetreatment. Centrifugation was themost effective clari- toxinremoved is located in the press cake and the pellet Žcationprocedure with an average patulin removal of 89%. formedafter centrifugation. However ,whencentrifugation is Thisvalue is not the true reduction potential of this treatment combinedwith enzyme treatment, in this case pectinase, pec- onlybecause it includes the patulin already removed in the tinparticles present in the juice would be degraded by the pressingstep as well as the patulin levels lost in the extraction enzymeand centrifugation will no longer be as effective.

TABLE 2. Evaluationof combination clariŽ cation treatments in patulin removal

Patulinconcentration ( g/ml) % loss % loss % loss (minus (minus Process Recovered Lost (total) pressing) recovery)

Pressing 1.03 6 0.06 0.97 48.5 — — Centrifugationand Ž ning 0.34 6 0.04 1.66 83 34.5 20.5 Centrifugationand enzyme treatment 0.41 6 0.04 1.59 79.5 31 17 Finingand Ž ltration 0.51 6 0.03 1.49 74.5 26 12 Filtrationand enzyme treatment 0.60 6 0.05 1.4 70 21.5 7.5 Control 1.66 6 0.08 0.34 17 — — J.FoodProt., Vol. 64, No. 8 CLARIFICATION PROCESSESFOR PATULINREDUCTION 1219

Patulinwill be bound to smaller particles or remain unbound 6.Chudziak, A., andM. Trojanowicz.1994. HPLC determination of ifthere is noparticulatematter available and would therefore patulinin apple juice. J. Anal.Chem. 39:729. 7.Draughon, F .A.,and J. C.Ayres.1980. Insecticide inhibitionof notsediment during centrifugation. The combination of Ž n- growthand patulin production in Penicilliumexpansum, Penicillium ingwith centrifugation also resulted in lower toxin reduction urticae,Aspergillus clavatus, Aspergillus terreus and Byssocchlamys thancentrifugation alone. This could be due to loss of par- nivea. J.Agric.Food Chem. 28:1115– 1117. ticulatematter during the Ž ningstep. However ,thistreatment 8.Ellis. J. R.,T.M.McCalla, andF .A.Norstadt.1980. Soil effects on ismore effective than the Ž ningstep alone. patulindisappearance andthe effect ofammonia onpatulin phyto- toxicity.Soil Sci. 129:371– 375. Our resultsindicate that centrifugation is an effective clar - 9.Forbito, P .R.,andN. E.Babsky.1985. Rapid liquid chromatographic iŽcation process for thereduction of patulin. It also has the determinationof patulin in apple juice. J. Assoc. Off.Anal. Chem. potentialto be implemented on an industrial scale because 68:950–951. mostjuice-making plants have a pre-existingcentrifugation 10.Gokmen, V .,andJ. Acar.1998.Incidence of patulin in apple juice step.The centrifugal forces employed may have to be modi- concentrates producedin Turkey. J. Chromatogr.815:99–102. 11.Gokmen, V .,andJ. Acar.1999.Simultaneous determination of 5- Žedin order to remove as much of the particulate matter as hydroxymethylfurfuraland patulin in apple juice by reversed-phase Downloaded from http://meridian.allenpress.com/jfp/article-pdf/64/8/1216/1674072/0362-028x-64_8_1216.pdf by guest on 25 September 2021 possibleso as to obtain efŽ cient reduction in patulin levels. liquidchromatography. J. Chromatogr.847:69 –74. Patulinlosses obtained in our study (89% for centrifugation 12.Hopkins, J. 1993.The toxicological hazards ofpatulin. Br .Ind.Biol. and77% for Žning)are comparable to those obtained with Res. Assoc. Bull.32:3– 4. 13.Kilara, A.,and J. vanBuren. 1989. ClariŽ cation of apple juice. In activatedcharcoal treatment (45 to 64%) that has the disad- D.L.Downing(ed.), Processed appleproducts. V anNostrand Rein- vantageof being an expensive and time-consuming treatment hold,New York. (4). Trimming (15) andthe use of insecticides (7) result in 93 14.Lee, H.S.,R.L.Roussef,and S. Nagi.1986. HPLC determination to99% and 85 to 100% loss of patulin, respectively. However , offurfuryl and 5-hydroxymethyl furfural in citrus juices. J. Food theŽ rst methodis time consuming and labor intensive and Sci.51:1075. 15.Lovett, J., R.G.Thompson,and B. K.Boutin.1975. Trimming as thesecond has safety implications regarding the use of organ- ameans ofremoving patulin from fungus rotted apples. J. Assoc. ophosphates.Alcoholic fermentation (24) andammoniation Off.Anal. Chem. 58:909 –911. (8) produced 99%and 99.8% patulin loss but the former 16.Mangas, J. J.,B.Suarez,A. Picinelli,J. Moreno,and D. Blanco. canonly be used for theproduction of cider and not juices 1997.Differentiation by phenolic proŽ le ofapple juices prepared accordingto two membrane techniques.J. Agric.Food Chem. 45: andin the case of the latter the sample was notŽ tfor human 4777–4784. oranimal consumption and had to be discarded. The clariŽ - 17.McLellan, M. R.1996.Juice processing. In L.P.Somogyi,H. S. cationprocedures tested during this study are simple to im- Ramaswamy, andY .H.Hui(ed.), Processing fruits: science and plement,cost effective, neither time consuming nor labor in- technology.Biology principles and applications, vol. 1. Technomic PublishingCo., Basel, Switzerland. tensive.The only concern is for industriesthat use apple juice 18.Prieta, J., M. A.Moreno,J. L.Blanco,G. Suarez,and L. Dominguez. by-productslike the Ž lterand cake press. These products have 1992.Determination of patulin by diphasic dialysis extraction and thepotential of containing high levels of patulin, and caution thinlayer chromatography.J. FoodProt. 55:1001– 1002. willhave to be practiced to avoid health hazards to humans 19.Root, W .H.1996.Apples and apple processing. In L.P.Somogyi, andanimals. D.M.Barrett, andY .H.Hui(ed.), Processing fruits: science and technology.Major processed products, vol. 2. TechnomicPublishing ACKNOWLEDGMENTS Co.,Basel, Switzerland. 20.Rovira, R., F.Ribera,V .Sanchis,and R. Canela. 1993.Improvements inthe quantitation of patulin in apple juice by high-performance We are gratefulto the National Research Foundationfor their Ž nan- liquidchromatography. J. Agric.Food Chem. 41:214 –216. cial supportand thank R. Govindenfor preparing and proofreading the 21.Sheu, M. J.,R. C.Wiley,and D. V.Schlimme.1987. Solute and manuscript. enzyme recoveries inapplejuice clariŽ cation using ultraŽ ltration. J. REFERENCES FoodSci. 52:732– 736. 22.Soto-Peralta, N. V.,H.Muller,andD. Knorr.1989.Effects ofchi- tosantreatments onthe clarity and color of apple juice. J. FoodSci. 1.Birkinshaw, J. H.,A. Bracken,S. E.Michael,and H. Raistrick.1943. 54:495–496. Patulinin the common cold. Lancet 245:625–635. 23.Spanos, G., R.Wrolstad,and D. Heatherbell.1990. In uence ofpro- 2.Brause, A.R.,M.W.Trucksess,F .S.Thomas,and W .P.Samuel. cessingand storage on the phenolic composition of apple juice. J. 1996.Determination of patulin in apple juice by liquid chromatog- Agric.Food Chem. 38:1572– 1579. raphy:collaborative study. J. Assoc. Off.Anal. Chem. 79:451– 455. 24.Stinson, E. E.,S.F.Osman, C.N.Huntanen,and D. D.Bills.1978. 3.Brown, N. L.,andG. S.Shephard.1999. The incidence of patulin Disappearance ofpatulin during alcoholic fermentation of apple incommercial appleproducts manufactured and packaged in South juice.Appl. Environ. Microbiol. 36:620 –622. Africa. In The15th Biennial International Congress and Exhibition 25.Vismer, H.F.,E.W.Sydenham,M. Schlechter,N.L.Brown,A. D. oftheSouth African Associationfor Food Science andTechnology Hocking,J. P.Rheeder,andW .F.O.Marasas. 1996.Patulin-produc- inassociation with the Institute of Packaging (SA). 27– 29 Septem- ing Penicillium species isolatedfrom naturally infected apples in ber.Bellville Civic Centre, Cape Town,South Africa. SouthAfrica. S.Afr.J. Sci.92:530– 534. 4.Brown, N. L.,andG. S.Shephard.1999. The potential of charcoal 26.World Health Organization.1990. Evaluation of certain foodaddi- treatment forthe removal of themycotoxin, patulin, during the pro- tivesand contaminants. Thirty-Fifth Report of theJoint FAO/ WHO cessingof apple juice. In The15th Biennial International Congress ExpertCommittee onFood Additives. Technical Report Services andExhibition of the South African Associationfor Food Science 789,WHO, Geneva. andTechnology in association with the Institute of Packaging(SA). 27.Wu, M. L.,R. R.Zall,and W .C.Tzeng.1990. MicroŽ ltration and 27–29 September .BellvilleCivic Centre, Cape Town,South Africa. ultraŽltration comparison for apple juice clariŽ cation. J. FoodSci. 5.Bullerman, L. B.1978.Methods for detecting mycotoxins in foods 55:1162–1163. andbeverages, p. 447– 457. In L.R.Beuchat,Food and beverage 28.Zubrick, J. W.1988.The organic chem labsurvival manual: a stu- mycology.AVI PublishingCo. Inc., Westport, Conn. dent’s guideto techniques. Wiley, New York.